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Home , ADAM8, AGGF1, AGPAT5, AMOTL2, Angiogenin, ANGPT4

DATA-DRIVEN AND KNOWLEDGE-DRIVEN COMPUTATIONAL MODELS OF IN APPLICATION TO PERIPHERAL ARTERIAL

by Liang-Hui Chu

A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy

Baltimore, Maryland March, 2015

© 2015 Liang-Hui Chu All Rights Reserved

Abstract

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is involved in both physiological conditions (e.g. development, wound healing and exercise) and (e.g. , age-related macular degeneration, and ischemic diseases such as and peripheral arterial disease). Peripheral arterial disease

(PAD) affects approximately 8 to 12 million people in United States, especially those over the age of 50 and its prevalence is now comparable to that of coronary artery disease.

To date, all clinical trials that includes stimulation of VEGF (vascular endothelial ) and FGF () have failed. There is an unmet need to find novel and drug targets and predict potential therapeutics in PAD. We use the data-driven bioinformatic approach to identify angiogenesis-associated genes and predict new targets and repositioned drugs in PAD. We also formulate a mechanistic three-

compartment model that includes the anti-angiogenic isoform VEGF165b. The thesis can serve as a framework for computational and experimental validations of novel drug targets and drugs in PAD.

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Acknowledgements

I appreciate my advisor Dr. Aleksander S. Popel to guide my PhD studies for the five years at Johns Hopkins University. I also appreciate several professors on my thesis committee, Dr. Joel S. Bader, Dr. Feilim Mac Gabhann and Dr. Brian H. Annex, for the guidance and constructive suggestions in my thesis proposal and dissertation.

I especially thank many great scientists and post-docs in our labs or other labs with whom I have collaborated: Dr. Corban Rivera, Dr. Stacey Finley, Dr. Kerri-Ann Norton,

Dr. Niranjan Pandey, Dr. Esak Lee, Dr. Hojjat Bazzazi, Dr. Jacob Koskimaki, Dr. Vijay

Ganta and Dr. Yongjin Park. I also thank undergraduate and master students George Chen,

Conan Chen and Chen Zhou, who worked with me closely.

I also thank for Dr. Reza Shadmehr and Dr. David Yue who helped with my courses, rotations, annual BME retreats and talent shows. I must thank Hong Lan for her great help during my PhD studies at JHU. It has been enjoyable to study in our BME program at Hopkins.

Finally, I thank my family, previous advisors and Taiwanese friends who encouraged me to pursue the PhD degree in the United States.

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Table of Contents

Abstract ...... ii Acknowledgements ...... iii List of Tables ...... viii List of Figures ...... x I. INTRODUCTION ...... 1 1.1 Angiogenesis and diseases ...... 2 1.2 approaches in angiogenesis ...... 3 1.3 Biological networks of angiogenesis ...... 4 1.4 Microarray data on endothelial cells ...... 7 1.5 Peripheral arterial disease (PAD) ...... 8 1.6 Drug repositioning in PAD ...... 9

1.7 Anti-angiogenic isoform VEGF165b ...... 10 1.8 Mechanistic three-compartment model of VEGF ...... 12 II. CONSTRUCTING THE ANGIOME ...... 13 2.1 Methods...... 13 2.1.1 GeneHits: integration of heterogeneous data ...... 13 2.1.2 Construction and analysis of angiome ...... 14 2.1.3 Functional enrichment of in the network ...... 14 2.1.4 Analysis of microarray on endothelial cells...... 15 2.2 Results ...... 15 2.2.1 The set of angiogenesis-annotated genes ...... 15 2.2.2 Angiome, global -protein interaction network of angiogenesis 16 2.2.3 Structure and topological properties of angiome ...... 17 2.2.4 Functional Enrichment of proteins in angiome ...... 18 2.3 Discussion ...... 19 2.3.1 Regulators of angiogenesis ...... 19 2.3.2 Extension of angiome in the following chapters ...... 21 III. DYNAMIC ANGIOGENESIS INTERACTOME ...... 22 3.1 Methods...... 22 3.1.1 Proteins annotated as positive and negative regulation of angiogenesis ...... 22 3.1.2 Temporal activation pattern of proteins in dynamic angiogenesis interactome ...... 23 v

3.2 Results ...... 25 3.2.1 Constructing the networks of positive and negative regulation of angiogenesis ...... 25 3.2.2 Temporal expression pattern on endothelial cells ...... 25 3.2.3 Activation patterns of the protein tyrosine ...... 27 IV. CONSTRUCTING THE PADPIN ...... 30 4.1 Methods...... 30 4.1.1 Construction of PIN of immune response and arteriogenesis ...... 30 4.1.2 Microarray data in PAD mouse models ...... 31 4.1.3 microarray dataset in PAD ...... 32 4.2 Results ...... 33 4.2.1 Construction of the immunome and arteriome ...... 33 4.2.2 Differentially expressed genes in mouse PAD model ...... 34 4.2.3 Visualization of PADPIN ...... 43 4.2.4 Differentially expressed genes between two inbred mouse strains... 45 4.3 Discussion ...... 46 4.3.1 Prediction of potential drug targets in PAD ...... 46 4.3.2 Comparisons of between the two mouse strains .... 50 V. COMPUTATIONAL DRUG REPOSITIONING IN PAD ...... 51 5.1 Methods...... 51 5.1.1 Resources of drug-targets relations ...... 51 5.1.2 List of anti-angiogenic and pro-inflammatory genes ...... 52 5.2 Results ...... 53 5.2.1 Drug-targets relations in PADPIN ...... 53 5.2.2 Inhibition of anti-angiogenic and pro-inflammatory genes ...... 54 5.2.3 Drug-Target Network ...... 57 5.3 Discussion ...... 58

VI. COMPARTMENTAL MODEL OF VEGF165b ...... 59 6.1 Methods...... 59 6.1.1 Three-compartment models ...... 59

6.1.2 Secretion rate and secretion ratio of VEGF165b to total VEGF ...... 60 6.1.3 Molecular Interactions and kinetic equations of VEGF isoforms..... 60 6.1.4 Geometric parameters ...... 62 6.1.5 Transport parameters ...... 62 6.1.6 Sensitivity analysis...... 64 6.2 Results ...... 65 vi

6.2.1 Experimental measurements of VEGF165 and VEGF165b in human biopsies 65

6.2.2 Computational results of secretion rates of VEGF165b and secretion

ratio of VEGF165b over VEGF165 in three compartments ...... 66

6.2.3 Sensitivity analysis of VEGF165, VEGF165b and sVEGFR1 ...... 68 6.2.4 VEGF distribution and VEGFR occupancy in the three compartments 69 6.3 Discussion ...... 71 VII. Summary ...... 73 VIII. Bibliography ...... 76 Appendices ...... 86 Appendix A: 1,233 Proteins in the angiome ...... 86 Appendix B: Proteins in the positive and negative regulation of angiogenesis ...... 125 Appendix C: Differentially expressed genes between C57BL/6 versus BALB/c mouse of ischemic and nonischemic tissues ...... 145

Appendix D: 80 Equations of VEGF165b models ...... 166 Curriculum Vitae ...... 187

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List of Tables

Table 1.1 Network parameters for the biological networks……………………………..5

Table 1.2 Summary of the references on angiogenic biological network analyses……...7

Table 1.3 Five microarray datasets on three different lines treated with VEGFA…..8

Table 2.1 Measurement of network parameters in angiome with entire human interactome……………………………………………………………………………….18

Table 4.1 Summary of four microarray studies from PAD mouse model………………32

Table 4.2 Summary of three microarray studies from human PAD patients……………33

Table 4.3 Up-regulated genes in ischemic versus non-ischemic C57BL/6 mouse……...37

Table 4.4 Down-regulated genes in ischemic versus non-ischemic C57BL/6 mouse…..39

Table 4.5 Up-regulated genes in ischemic versus non-ischemic BALB/c mouse……….41

Table 4.6 Down-regulated genes in ischemic versus non-ischemic BALB/c mouse……43

Table 4.7 Comparison of the fold change of genome-wide differentially expressed genes between ischemic versus nonischemic muscles from mice with samples from human PAD patients…………………………………………………………………………………...47

Table 5.1 Predictions of pro-angiogenic FDA-approved drugs that target anti-angiogenic genes……………………………………………………………………………………..54

Table 5.2 Predictions of anti-inflammatory FDA-approved drugs that target viii pro-inflammatory genes………………………………………………………………….56

Table 6.1 Number of cell surface receptors VEGFR1, VEGFR2 and NRP1. Units of values: dimers/EC for VEGFR1 and VEGFR2 and monomer for NRP1……………….62

Table 6.2 Geometric parameters of diseased calf muscles for human patients…………62

Table 6.3 Transport parameters between the compartments and clearance, synthesis and degradation………………………………………………………………………………64

Table 6.4 Prediction of VEGF165b, VEGF165 and VEGF121……………………………..71

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List of Figures

Figure 2.1 Overlap of angiogenesis genes from SABiosciences, GeneCards and Gene

Ontology…………………………………………………………………………………16

Figure 2.2 Visualization of angiome …………………………………………………....17

Figure 2.3 Angiogenesis regulators and their receptors…………………………………20

Figure 2.4 Negative regulators, receptors, and factors of angiogenesis…..21

Figure 3.1 Flowchart of finding the protein complexes of angiome and merging time course gene expression data……………………………………………………………...24

Figure 3.2 Four statistically significant (adjusted p-value< 0.05 by Bonferroni correction)

(A) 2D and (B) 3D on TIME cells…………………………………27

Figure 3.3 Eighteen proteins in the GO category “transmembrane receptor protein activity”, merging with the time-course transcripts of type I 3D collagen on TIME cells……………………………………………………………………………29

Figure 4.1 Flowchart of construction of immunome, arteriome and PADPIN………….31

Figure 4.2 Venn diagram of angiome, immunome and arteriome……………………….34

Figure 4.3. Volcano plot of the fold-change and p-values from the ischemic versus nonischemic gastrocnemius muscle from C57BL/6 mice……………………………….35

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Figure 4.4 Visualization of PADPIN in BALB/c microarray datasets…………………..44

Figure 4.5 Visualization of PADPIN in C57BL/6 microarray datasets…………………44

Figure 5.1 Representative example of (A) pro-angiogenic and (B) anti-inflammatory repositioned drugs for PAD………………………………………………………………57

Figure 6.1 Three-compartment model of VEGF in peripheral arterial disease………….59

Figure 6.2 Molecular interactions of VEGF165, VEGF165b and VEGF121………………..61

Figure 6.3 Changing of the secretion ratio of VEGF165b to total VEGF165 isoforms in normal, blood and disease compartments………………………………………………..68

Figure 6.4 Sensitivity analysis of VEGF165, VEGF165b and VEGF121…………………...69

Figure 6.5 (A) VEGF distribution and (B) VEGFR distribution……………………...... 70

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I. INTRODUCTION

Angiogenesis is the formation of new blood vessels from preexisting microvessels.

One of the key important molecules in angiogenesis is VEGFA (vascular endothelial growth factor A). Excessive angiogenesis has been associated with various diseases such as cancer and age-related macular degeneration. Several angiogenesis inhibitors targeting

VEGF or receptors have been approved by the FDA (Food and Drug

Administration), such as VEGFA monoclonal and tyrosine kinase inhibitor . On the other hand, insufficient angiogenesis is associated with various diseases such as Alzheimer’s disease, ischemic heart and peripheral vascular diseases. So far the pro-angiogenic clinical trials including stimulation of VEGF have not yielded any

FDA-approved drugs (64). In my thesis, we focus on peripheral arterial disease (PAD), which is a disease characterized by insufficient angiogenesis. PAD affects approximately

8 to 12 million people in the United States, especially those over 50 years old (32).

We will apply both data-driven (bioinformatics) and knowledge-driven (mechanistic models) computational models to predict novel potential drug targets and drug repositioning in PAD. The research background will be introduced in this chapter.

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1.1 Angiogenesis and diseases

Angiogenesis is the formation of new blood vessels from preexisting microvessels.

Judah Folkman was the pioneer in discovering the role of tumor angiogenesis: without neovascularization, tumors would stop growing beyond a diameter of 2 to 3 mm (31). He proposed that tumors release a “tumor-angiogenesis factor” (TAF) that attracts the blood vessels supply to tumor to provide oxygen and nutrients for tumor growth (31).

Subsequently, important angiogenic molecules such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) were discovered (24, 25). The growth factor VEGF is a key molecule in angiogenesis (63). The discovery of angiogenic factors has a huge impact in therapeutic strategies to inhibit angiogenesis in cancer or eye disorders, and several angiogenesis inhibitors have been approved by FDA, such as anti-VEGF antibody bevacizumab and VEGF-Trap ().

Angiogenesis is involved in both physiological and pathological conditions. The imbalance between pro-angiogenic and anti-angiogenic molecules can cause excessive or insufficient angiogenesis in various diseases (11). Diseases characterized by excessive angiogenesis include cancer, obesity, rheumatoid arthritis, age-related macular degeneration, and diabetic retinopathy. Diseases characterized by insufficient

2 angiogenesis include Alzheimer’s disease, preeclampsia, ischemic heart and peripheral vascular diseases.

1.2 Bioinformatics approaches in angiogenesis

Large-scale datasets, which are often called “omics” data, are helpful for the biological insight into complex diseases and optimize therapeutic strategies in angiogenesis. For example, The Cancer Genome Atlas (TCGA) https://tcga-data.nci.nih.gov/tcga/ provides a platform for downloading and analyzing -scale datasets in various types of cancer, including breast cancer, and glioblastoma. Researchers can further utilize genomic information such as

DNA copy number, messenger RNA arrays and microRNA sequencing, define the subtypes of cancer such as breast cancer (10), and apply the data to angiogenesis research

(71).

The bioinformatics approach could also help to identify anti-angiogenic from the protein families containing anti-angiogenic fragments, based on multiple sequence alignment. Examples of these peptides include CXC chemokines, type IV , , and type 1 thrombospondin repeat-containing proteins (56). The activity of these peptides identified by bioinformatics has been tested in endothelial cell proliferation and migration assays (75). These computational methods for anti-angiogenic

3 cancer therapeutics have been reviewed by Finley et al (26) and Sharan et al. (81). In the next section 1.3, we will discuss the biological networks of angiogenesis, including the network parameters and related references.

1.3 Biological networks of angiogenesis

Biological networks play an important role in systems and its applications to drug discovery for various diseases. Protein–protein interaction networks (PINs) provide valuable information to understand the biological processes and cellular functions, and integrate diverse molecular events. The cellular networks are scale-free following the power-law distribution (7). In Table 1.1 we list some network parameters that are often used for the analysis of biological networks.

Terms Definition

Node A cellular entity that includes one or more of the following: genes,

proteins, and drugs

Edge A relationship connecting two nodes, which may involve a

physical protein-protein or drug-gene interaction

Degree The number of edges between a node and other nodes within a

network

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C (n)   (n)/ , where s and t are nodes in the Betweenness b snt  st st 

centrality network different from n ,  st denotes the number of shortest

paths from to t , and  st (n) is the number of shortest paths

from to that n lies on

Clustering Cn  2en kn (kn 1) , where en is the number of connected pairs

coefficient between all neighbors of n , and kn is the number of neighbors

of n

Table 1.1 Network parameters for the biological networks

Several studies use biological networks to study angiogenesis (73). We summarize these studies with brief descriptions in Table 1.2.

Reference Description of angiogenic networks

Abdollahi et Building the signaling networks by differentially al.2004 (2) expressed anti-angiogenic proteins on endothelial cells after

endostatin treatment

Abdollahi et al. Constructing the gene regulatory network related to the

2007 (3) angiogenic switch from the inversely regulated proangiogenic

genes in

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Chu et al. 2012 (16) Construction and analysis of the angiome, the global

angiogenesis PINs comprising 1,233 proteins and 5,726

interactions

Chu et al. 2014 (15) Using time course gene expression data on three different

types of endothelial cells to understand the protein complex

changing over time in angiome

Chen et al. 2008 Constructing the gene function association network by gene

(14) expression and simulating the network perturbation by

anti-angiogenic kinase inhibitors

Gu et al. 2010 (42) Developing a new method by recovering the gene modules in

and angiogenesis

Huang et al. 2010 Identifying characteristic sub pathway network by stimulation

(47) of -1 (IL-1) and α (TNF-α)

on human umbilical vein endothelial cells (HUVECs)

Rivera et al. 2011 Identifying crosstalk between three angiogenesis-modulating

(72) protein families: CXC chemokine ligands, type IV collagen

fibrils and type I thrombospondin domain-containing proteins

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Rivera et al. 2011 Integrating the human interactome with known

(74) angiogenesis-annotated proteins to identify a set of 202

angiogenesis-associated proteins

Table 1.2 Summary of the references on angiogenic biological network analyses

1.4 Microarray data on endothelial cells

Endothelial cells (EC) are widely used in angiogenesis research, such as HUVEC

(human umbilical vein EC), MEC (human microvascular EC) and TIME

(telomerase-immortalized human microvascular endothelial) cells. Several time course microarray studies have been conducted to identify the differentially expressed genes in

VEGFA-treated HUVEC (79), MEC (40) and TIME cells (67). Integration of time-course microarray data and angiogenic protein networks can reveal the protein complexes changing over time (15). We list the available microarray data resources on different endothelial cells in Table 1.3.

Treatment Cells Time Points Resource Ref

VEGFA HUVEC 0,0.5,1,2.5,6 GSE10778 Schweighofer et al. (79)

VEGFA MEC 0,0.5,1,2,4 hr GSE3891 Glesne et al. (40)

(proliferation)

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VEGFA MEC 0.5,1,2,4,8 hr GSE3891 Glesne et al. (40)

(tubulogenesis)

VEGFA TIME (3D 15min,1,3 6,9,12, Provided by Mellberg et al. (67)

collagen I) 18,24 hr authors

VEGFA TIME (2D 15min,1,3,6,9,12, Provided by Mellberg et al. (67)

fibronectin) 18,24 hr authors

Table 1.3 Five microarray datasets on three different cell lines treated with VEGFA

1.5 Peripheral arterial disease (PAD)

Peripheral arterial disease (PAD) is caused by atherosclerosis, which leads to narrowing of the peripheral arteries that supply the legs (4). PAD affects approximately 8 to 12 million people in the United States and over 200 million worldwide, especially those over the age of 50 (32). The vascular risk factors for lower extremity PAD include diabetes, smoking and (4). Numerous clinical trials have been conducted in the United States and Europe, including stimulation of VEGFA and bFGF; all the clinical trials of therapeutic angiogenesis in PAD have failed; as a result, no FDA approved drugs for PAD are available (64). This failure suggests a critical need to identify potential new targets that can improve perfusion recovery in PAD. One of the primary goals in my thesis is to use the “omics” approaches to identify important sets of genes and signaling 8 pathways in PAD, and predict new therapeutic targets.

In our bioinformatics studies on PAD, we include three major biological processes: angiogenesis, immune response and arteriogenesis. It was found that there are plenty of and T lymphocytes in the atherosclerotic lesions, and the pro-inflammatory

T cell (e.g. interleukin-2) were also found in a large proportion of the plaques

(34). Further studies suggest potential anti-inflammatory treatment in atherosclerosis (33).

Global gene expression analysis suggests that inflammatory and immune responses play a major role in PAD (36). Arteriogenesis is the growth or remodeling of collateral arteries from the preexisting arterioles in the upper leg, which is triggered by shear stress (78).

Bioinformatics approaches for cancer research have been applied widely, but applications to PAD have been few. We will use the “omics” approach to identify important sets of genes relevant to signaling pathways in angiogenesis, inflammation and arteriogenesis to predict new therapeutic targets. We will utilize four microarray datasets from PAD mouse models (45); details of these microarray datasets in PAD will be discussed in Methods.

1.6 Drug repositioning in PAD

The long-term outlook for new drug development has declined in the last ten years

(69). The average time of drug development is ~13.9 years and the cost of a new drug to

9 the market is estimated as 2.6 billion in 2015 (1). Drug repositioning, new use of old drugs, can shorten the development time and provide solutions for the high cost and low number of new successful drugs of the pharmaceutical companies (21).

There are several different methods in computational drug repositioning, including the drug-target network (99), disease network (101), transcriptional profiling from gene expression array (50), and side effects similarity (98). Currently all clinical trials for PAD including stimulation of VEGF have failed. The computational drug repositioning can guide new indications for old drugs in PAD. We propose two novel strategies to the PAD treatment: inhibiting anti-angiogenic proteins and inhibiting pro-inflammatory proteins.

For example, inhibition of thrombospondin-1, the anti-angiogenic gene involved in Nitric

Oxide (NO) pathway, enhances the survival of ischemic in the porcine model (53).

The combination of two peptides regulating angiogenesis and inflammation pathways independently in the mouse model of PAD improves the restoration of perfusion better than the treatment by either of the two peptides (100). We will make predictions for drugs repositioning in PAD based on the proteins annotated as anti-angiogenesis and pro-inflammation, respectively.

1.7 Anti-angiogenic isoform VEGF165b

Vascular Endothelial Growth Factor (VEGF) is a family of molecules regulating

10 angiogenesis. against VEGF or its receptors have been developed for treating cancer and age-related macular degeneration (AMD) as well as macular edema. The

VEGF family includes five ligands (VEGFA, VEGFB, VEGFC, VEGFD and PlGF), and five receptors (VEGFR1, VEGFR2, VEGFR3, NRP1 and NRP2). Different splice

isoforms of VEGFA are generated in two distinct forms: pro-angiogenic VEGFxxx (xxx

refers to the number of amino acids) and anti-angiogenic VEGFxxxb (these isoforms differ

from VEGFxxx by six amino acids at the C-terminal) (95). The balance between VEGF165

and VEGF165b may play a crucial role in many normal tissues and diseases. VEGF165b as a fraction of total VEGF is up-regulated in normal human tissues such as eye (60) and kidney (9), and down-regulated in various types of cancer such as colon cancer (93) and prostate cancer (95).

Understanding of the function of VEGF165b in PAD is still limited. Kikuchi et al.

measured the ratio of protein levels of VEGF165b versus VEGF165 in serum, and ratio of

mRNA level of VEGF165b versus VEGF165 in peripheral blood mononuclear cells

(PBMCs) in PAD patients, which are 4:1 and 8:1, respectively (57). Distribution of

VEGF165b complexes among receptors is still unknown. A computational model of

VEGF165b can help us understand the VEGF distribution and binding to different receptors.

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1.8 Mechanistic three-compartment model of VEGF

In this study, we use the previously developed three-compartment model in cancer

as the basis to develop the three-compartment models of VEGF165b and VEGF165 in

human PAD (28-30). Finley and Popel (30) predicted that targeting specific VEGF isoforms may be an effective treatment strategy in human cancer. In this model of mouse tumor xenograft, both human and mouse VEGF isoforms as well as soluble VEGFR1

(sFlt-1) and α-2-macroglobulin are included in the model. The secretion rates of tumor

VEGF are predicted in the range 0.007-0.023 molecules/cell/s by this optimized model

(29). These tumor models provide a framework to examine the effects of anti-VEGF agents in tumor. In a computational model of human PAD, Wu et al. provided the first three-compartment model in PAD that comprise blood, normal and diseased calf compartments (96). Details of geometric parameters in PAD calf compartment are fully described in (96).

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II. CONSTRUCTING THE ANGIOME

Missing gene annotations can be identified from heterogeneous resources of biological data. Proteins that physically interact are more likely to participate in the same biological process or pathway (e.g. angiogenesis). In addition, proteins that share similar domains are more likely to have similar molecular functions. We can improve the annotation of proteins by integration of these and other sources of data. In this chapter, we introduce the GeneHits algorithms to integrate these heterogeneous data and various bioinformatics tools (2.1), analysis of the protein-protein interaction network of angiogenesis, the angiome (2.2), and the application of angiome (2.3).

2.1 Methods

2.1.1 GeneHits: integration of heterogeneous data

We developed a method named GeneHits and implemented it as a to integrate the attributes of protein physical interactions and domain similarity (16). The link for GeneHits is http://sysbio.bme.jhu.edu/d/. We also provide the user’s tutorial in http://sysbio.bme.jhu.edu/s/tutorial.html and user’s help in http://sysbio.bme.jhu.edu/s/help.html. GeneHits website and the algorithms were developed by Dr. Corban Rivera in collaboration with Dr. Joel Bader and Dr. Aleksander

Popel. Details of the mathematical equations used in GeneHits are described in (16). 13

2.1.2 Construction and analysis of angiome

Using GeneHits, we combine the graph diffusion kernels from protein interactions and pairwise associations from protein domains to construct the angiome, the global protein-protein interaction network (PIN) of angiogenesis. The resource for the protein interaction is MiMI (Michigan Molecular Interactions), which integrates eleven protein interaction data sources (BIND, CCSB, DIP, GRID, HPRD, IntAct, KEGG, MDC,

MINT, PubMed and Reactome) (91). We plot the PIN using Cytoscape (84). We measure the network parameters of PIN using the NetworkAnalyzer (5) by Cytoscape (84). Details of these parameters are shown in Table 1.1 (Section 1.3).

2.1.3 Functional enrichment of proteins in the network

Gene Ontology (GO) http://geneontology.org/ is a common bioinformatics annotation resource for the description of gene products, associated biological processes, cellular components and molecular functions. We use several tools which have been well developed from other researchers for the functional enrichment of proteins in the angiome based on the GO annotations. We use BiNGO (Biological Networks Gene

Ontology tool) to identify the (GO) terms which are significantly overrepresented in the set of genes (65). Another resource is ToppGene (13) for gene list functional enrichment. In the PIN, the terms “gene” and “protein” are used

14 interchangeably. We use BiNGO (65) and ToppGene (13) to analyze the functional enrichment of proteins in the Angiome. We use Gene Set Enrichment Analysis (GSEA) software (87) to compute the q-value of the enrichment of angiogenesis-associated proteins in a ranked list of the most perturbed gene expression transcripts (16).

2.1.4 Analysis of microarray on endothelial cells

We used software packages in Bioconductor to analyze microarray data, including

Affy (39) and Limma (85). This package can help us extract microarray data from GEO

(Gene Expression Omnibus) databased in NCBI (National Center for Biotechnology

Information).

2.2 Results

2.2.1 The set of angiogenesis-annotated genes

We compare the list of angiogenesis-annotated genes from SABiosciences (84 genes),

Gene Ontology (GO) (370 genes) and GeneCards (1,244 genes) using the Venn diagram

(Figure 2.1). 82 of 84 proteins from SABiosciences overlap with GeneCards and Gene

Ontology. Because of the high overlap between SABiosciences and the two public databases, we used the list of 84 genes from SABiosciences as the seeds to construct the angiome.

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Figure 2.1. Overlap of angiogenesis genes from SABiosciences, GeneCards and Gene

Ontology

2.2.2 Angiome, global protein-protein interaction network of angiogenesis

We used the GeneHits method to expand the set of 84 genes (See Section 2.1.1). The angiome is composed of 478 proteins and 1,488 interactions as shown in Figure 2.2. The color from red to blue represents the degree of each node in descending order.

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Figure 2.2. Visualization of angiome

2.2.3 Structure and topological properties of angiome

By using the NetworkAnalyzer (5), we measured structural and topological parameters of angiome in Table 2.1. We also compare with the network parameter of angiome with the entire human interactome (55).

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Network Entire human Angiome

Parameters interactome

Number of nodes 13584 478

Number of edges 85083 1488

Average number of neighbors 11.27 6.226

Clustering coefficient 0.109 0.237

Average shortest path length 4.086 3.972

Table 2.1 Measurement of network parameters in angiome with entire human interactome

In the angiome, the proteins include all five VEGF ligands (VEGFA, VEGFB,

VEGFC, VEGFD and PIGF) and five VEGF receptors (VEGFR1, VEGFR2, VEGFR3,

NRP1 and NRP2). None of the VEGF ligands or receptors are in the top list with the high degree of nodes, which suggests that many proteins such as fibroblast growth factor (FGF) family, in addition to VEGF ligands and receptors play important roles in the development, maintenance and remodeling of the vasculature.

2.2.4 Functional Enrichment of proteins in angiome

To identify pathways and biological processes associated with angiogenesis, we computed the functional enrichment of genes in the angiome using BiNGO (65) to find 18 the significant Gene Ontology (Section 2.1.3). We identify 60 proteins in growth factor activity (adjusted p-value=9.505E-48), 34 proteins in binding (adjusted p-value=

1.014E-23), and 27 proteins in binding in the angiome (adjusted p-value=1.5269E-16).

As shown in Fig. 2.1, GeneCards and Gene Ontology contain 810 proteins that are absent from the angiome. We constructed an extended angiome by adding the original angiome and the genes in the union of the three databases shown in Figure 2.1, and all molecular interactions from MiMI (91). The extended angiome is composed of 1,233 proteins and 5,726 interactions. In the following section, we refer to the “angiome” as the

PIN of angiogenesis with 1,233 proteins and 5,726 interactions. The 1,233 proteins in the angiome are provided in the Appendix A.

2.3 Discussion

2.3.1 Regulators of angiogenesis

Hagedorn and Bikfalvi (43) summarized the receptor- interactions responsible for angiogenesis. We used BiNGO (65) to analyze functional enrichment of 171 genes enriched with growth factor-related annotations in angiome. In Figure 2.3, we present the major extracellular regulators of angiogenesis and their receptors: (A) Endothelial growth factor signaling; (B) Fibroblast growth factor signaling; (C)

19 signaling; (D) Transforming growth factor signaling; (E) -like growth factor signaling; (F) -derived growth factor signaling; (G) Inflammation and immune response; (H) Other cytokines; (I) Focal adhesion; (J) ; (K)

Others.

Figure 2.3 Angiogenesis regulators and their receptors

Angiogenesis inhibitors, particularly polypeptides or endogenous peptides, may become the safest and least toxic therapy for diseases associated with abnormal angiogenesis. These peptides have been derived from thrombospondin, collagens, chemokines, cascade proteins, growth factors, and other classes of proteins and target different receptors (75). We generally divided negative regulators of angiogenesis regulator proteins in Figure 2.4 into ten categories: (A) Chemokines; (B)

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Angiopoietin; (C) plasiminogen; (D) Collagen family; (E) Thrombospondin;

(F) Serpins; (G) ; (H) Brain-specific angiogenesis inhibitors; (I) activities; (J) Others. These proteins cover most of endogenous anti-angiogenesis proteins which were discussed in Rosca et al. (75).

Figure 2.4 Negative regulators, receptors, and transcription factors of angiogenesis

2.3.2 Extension of angiome in the following chapters

The methodology developed in this chapter will be continuously used in the following chapters. We will use time-course microarray datasets to investigate the dynamic changes of proteins complexes over time in the angiome (Chapter III). The methodology used in construction of angiome could be extended to other biological processes, such as immune response (Chapter IV). The pro- and anti-angiogenic proteins in the angiome could provide a novel prediction of therapeutic angiogenesis in disease

(Chapter V).

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III. DYNAMIC ANGIOGENESIS INTERACTOME

Time-course microarray datasets on various endothelial cells provide very useful information for understanding angiogenesis. The angiome network combined with time-course microarray datasets could reveal the protein complexes changing by the time-dependent manner. In this chapter, we will discuss the bioinformatics tool Short

Time-series Expression Miner (STEM) and flowchart in 3.1, and the temporal gene expression pattern of receptor protein tyrosine kinase in 3.2.

3.1 Methods

3.1.1 Proteins annotated as positive and negative regulation of angiogenesis

The major pro- and anti-angiogenic ligands and receptors were summarized by

Hagedorn and Bikfalvi in 2000 (43). Based on the proteins and interactions in angiome network, we further constructed two PINs of positive and negative regulation of angiogenesis (16). From the Gene Ontology (GO), we curate the list of genes in the angiome that are annotated as positive regulation of angiogenesis (GO: 0045766) and negative regulators of angiogenesis (GO: 0016525). Four genes are listed in both positive and negative regulators of angiogenesis: (THBS1), 4

(ANGPT4), chemokine receptor 1 (CX3CR1), and peptidase inhibitor member 1

(SERPINE1). We curate the four genes into a single category of GO using literature 22 search in NCBI: in the literature, THBS1 and SERPINE1 are identified as anti-angiogenic (92), ANGPT4 as pro-angiogenic (82) and Fractalkine (FKN) as pro-angiogenic (76).

After curation of the genes, 56 proteins annotated as positive regulators of angiogenesis include: ADM, AGGF1, ANGPT4, ANGPTL3, ANXA3, AQP1, BTG1, C3,

C3AR1, C5, CCL11, CCL24, CCL5, CCR3, CD34, CHRNA7, CTSH, CX3CR1, EPHA1,

ERAP1, F3, FGF1, FGF2, FLT1, GATA2, GATA4, GATA6, HDAC9, HIF1A, HIPK1,

HIPK2, HMOX1, IL1A, IL1B, KDR, MMP9, NOS3, PRKD1, PRKD2, PTGIS, PTGS2,

RAMP2, RAPGEF3, RHOB, RRAS, RUNX1, SFRP2, SPHK1, TEK, TNFRSF1A,

TNFSF12, TWIST1, UTS2R, VEGFA, VEGFB, WNT5A. 39 proteins annotated as negative regulators of angiogenesis include: AMOT, ANGPT2, APOH, BAI1, CCL2,

CCR2, COL4A2, COL4A3, CXCL10, FASLG, FOXO4, GHRL, GTF2I, HDAC5, HHEX,

HOXA5, HRG, , KLK3, KRIT1, LECT1, LIF, MAP2K5, NF1, NPPB, NPR1,

PDE3B, PF4, PML, PTPRM, ROCK1, ROCK2, SERPINE1, SERPINF1, STAB1,

THBS1, THBS2, THBS4, TIE1. We use the seeds in positive and negative regulation of angiogenesis to generate two PINs, respectively.

3.1.2 Temporal activation pattern of proteins in dynamic angiogenesis

23 interactome

We use the Short Time-series Expression Miner (STEM) (22) to identify the significant temporal expression profiles and gene ontology (GO) of these significant profiles from time course microarray experiments. The clustering method of gene expression profiles is based on the STEM clustering method (23). The model profiles starts at the beginning time point (e.g. 0 hr), and then the profile between the two time points can be unchanged, increase or decrease with an integer number of time units.

These model profiles are selected independently from the data. The STEM clustering algorithm assigns each gene to the model profile by the correlation coefficient.

We summarize the methods used in dynamic angiogenesis interactome in Figure 3.1.

The bioinformatics methods such as BiNGO (65), ToppGene (13) and GeneHits (16) in this Flowchart have been described in Section 3.1.1-3.1.6.

Figure 3.1 Flowchart of finding the protein complexes of angiome and merging time course gene expression data 24

3.2 Results

3.2.1 Constructing the networks of positive and negative regulation of angiogenesis

We constructed the two networks of positive and negative regulation of angiogenesis, respectively (see Section 3.1.1). The PIN of positive regulation of angiogenesis comprises 367 proteins and 1,972 interactions, and the PIN of negative regulation of angiogenesis comprises 245 proteins and 1,154 interactions (see Appendix B for the full list of proteins in the two networks). Some proteins in the positive regulation of angiogenesis are also connected to the proteins in the negative regulation of angiogenesis, by physical interactions present in the MiMI (91) and literature reports. For example, anti-angiogenic thrombospondin (THBS1) directly binds to angiogenic proteins

COL1A1 (collagen type I) (37) and MMP9 (matrix metallopeptidase 9) (8). We used

BiNGO 2.44 (Biological Networks Gene Ontology tool) (65) for the functional enrichment of genes in the two angiogenesis PINs.

3.2.2 Temporal gene expression pattern on endothelial cells

Among five time-course microarray datasets (Table 1.3), Mellberg's dataset on TIME cells (67) contains the most time points (15 min and 1, 3, 6, 9, 12, 18, and 24 h). We used

STEM (22) to identify significant temporal gene expression profiles and the genes

25 associated with these profiles integrated GO database (Section 3.1.2). We analyzed the temporal gene expression pattern of all the genes in the three raw microarray data separately. We normalized the microarray data to the first time point in Schweighofer et al. (79) and Glesne et al. (40); Mellberg's data (67) have been normalized to the untreated conditions. The maximum number of model profiles is set as the default value 20.

We show the four statistically significant (adjusted p-value< 0.05 by Bonferroni correction) temporal gene expression profiles of TIME cells on 2D fibronectin in Figure

3.2 A. The p-value was calculated by the number of genes assigned to the model profile, compared to the expected number of assigned genes. In Figure 3.2, the number on top left means the assigned profile number by STEM analysis and the number on bottom left means the significance level of p-value (before the Bonferroni correction). The black and red lines in the individual profile boxes indicate the assigned pattern, e.g. the sequence

(0,1,2,3,4,5,6,7,8) over the eight time points and initial points in profile #16, and the gene expression of genes assigned in that profile. We compare the four statistically significant profiles on 2D fibronectin in Figure 3.2 (A) with those in 3D type I collagen in Figure 3.2

(B). We found statistically significant patterns of continuous up- and down-regulation depicted by profiles #16 and #4 exist for both substrates on TIME cells.

26

Figure 3.2 Four statistically significant (adjusted p-value< 0.05 by Bonferroni correction)

(A) 2D fibronectin and (B) 3D collagen on TIME cells

3.2.3 Activation patterns of the receptor protein tyrosine kinase

We used the defined gene set of the 367 and 245 genes in the positive and negative regulation of angiogenesis by STEM clustering, respectively. We found 21 and 19 genes in the positive regulation of angiogenesis which were assigned to the increasing profile

#16 on 2D fibronectin and 3D collagen in Fig. 3.2A and B, respectively. We further used

Toppgene (13) to analyze the functional enrichment of proteins in the increasing profile

#16 of positive regulation of angiogenesis. One of the most significant GO molecular functions for 21 and 19 genes in the increasing profile #16 of positive regulation of angiogenesis on 2D fibronectin and 3D type I collagen is “protein tyrosine kinase activity”

27

(p-value=2.648E-3 and 9.568E-5, respectively).

We further look at the genes annotated as “protein tyrosine kinase activity” in the functional enrichment of proteins in the network of positive regulation of angiogenesis.

The GO category "transmembrane receptor protein tyrosine kinase activity" (adjusted p-value=1.64E-13) contains eighteen proteins ALK, EGFR, EPHA1, EPHB2, FGFR1,

FGFR2, FGFR3, FGFR4, FGFRL1, FLT1 (VEGFR1), FLT4 (VEGFR3), IGF1R, KDR

(VEGFR2), NRP1, NRP2, NTRK2, TEK and TIE1. We merge proteomic and genomic data in Figure 3.3 by Cytoscape (84). The proteins with gene transcripts on 3D type I collagen for TIME cells in Figure 3.3 show that VEGFR1 (FLT1) is activated consistently after 6 hr, VEGFR2 (KDR) only activated at 24 hr, and VEGFR3 (FLT4) decreased from

15 min to 9 hr then increased from 12 hr to 24 hr.

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Figure 3.3. Eighteen proteins in the GO category “transmembrane receptor protein tyrosine kinase activity,” merging with the time-course transcripts of type I 3D collagen on TIME cells

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IV. CONSTRUCTING THE PADPIN

Peripheral arterial disease (PAD) is caused by atherosclerosis leading to narrowing of the peripheral arteries which supply the legs (Section 1.5). We use the term “PADPIN” as protein-protein interaction networks of angiogenesis, arteriogenesis, and inflammation in PAD. We will also use the mouse microarray data in two different strains to construct the PADPIN.

4.1 Methods

4.1.1 Construction of PIN of immune response and arteriogenesis

We constructed the global protein-protein interaction networks of immune response and arteriogenesis and refer to them as immunome (Fig. 4.1A) and arteriome (Fig. 4.1B), respectively. We use 162 immune response-associated proteins from SABiosciences as the query genes in GeneHits, the list of genes by searching the keyword “immune” in

GeneCards (77) and Gene Ontology (GO:0006955: immune system) to construct the immunome. There are no GO categories or SABiosciences annotations for arteriogenesis.

We searched the keyword "arteriogenic" and "arteriogenesis" in GeneCards. By combining the angiome, immunome and arteriome with the mouse microarray data (45), we are able to construct the PADPIN, i.e. PIN for peripheral arterial disease (Fig. 4.1 C).

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Figure 4.1 Flowchart of construction of immunome, arteriome and PADPIN

4.1.2 Microarray data in PAD mouse models

We use the four microarray gene expression datasets from ischemic and nonischemic gastrocnemius muscles at day 3 post hindlimb ischemia (HLI) in the genetically different inbred C57BL/6 and BALB/c mice (Table 4.1). Ischemic muscles are produced by unilateral femoral artery ligation. The resource of microarray datasets is from Hazarika et al (45). The C57BL/6 mouse strain recovers from hindlimb ischemia much better than the

BALB/c mouse strain (HLI). We hypothesize that identification of the differentially expressed genes in ischemic and nonischemic muscles in the two mouse strains will help the prediction of target genes in PAD. We will also identify the differential gene expression changes in the ischemic skeletal muscle between these two strains, as well as

31 the nonischemic muscle samples between the two strains. We will compare the differentially expressed genes in the PAD mouse model with the microarray datasets in human PAD patients.

Tissues of mRNA extraction Number of Description of Mouse Models samples Ischemic gastrocnemius muscle 3 C57BL/6 mice recover remarkably from C57BL/6 mice well from HLI Ischemic gastrocnemius muscle 3 The recovery after HLI in BALB/c from BALB/c mice is much poorer than C57BL/6 mice Nonischemic gastrocnemius muscle 3 C57BL/6 mice recover remarkably from C57BL/6 mice well from HLI Nonischemic gastrocnemius muscle 3 The recovery after HLI in BALB/c from BALB/c mice is much poorer than C57BL/6 mice

Table 4.1 Summary of four microarray studies from PAD mouse model (45)

4.1.3 Human microarray dataset in PAD

Available microarray datasets in PAD patients include gene expression analysis of human femoral arteries (17, 36) and peripheral blood mononuclear cells (66) in Table 4.2.

These cells or tissues are very different from the skeletal muscles that are the site of ischemia and the stimulus for vascular remodeling and growth. The study of Hazarika et al. (45) provides the only available gene transcriptional data comparing ischemic versus nonischemic gastrocnemius muscles from the two mouse strains. We will directly compare the fold change in the mouse model (45) with the fold change values listed in the three microarray datasets (17, 36, 66). 32

Species Samples of mRNA extraction Patient Descriptions Reference Human Thromboendarterectomy stage IIb-IV; n=20; three risk (17) specimens of the common factors: hypercholesterolemia femoral artery from 20 (n=11), smoking (n=5), and patients with PAD. 10 samples diabetes (n=6) of common femoral arteries and biopsies of the abdominal aorta from organ donors as controls. Human Primary femoral artery grade III, grade IV, grade V (36) specimens containing Risk factors include atherosclerotic lesions 30 hypertension, femoral artery samples hypercholesterolemia and diabetes Human Peripheral blood mononuclear Risk factors includes (66) cells by layering the blood hypertension, diabetes and samples over histopaque smoker Table 4.2 Summary of three microarray studies from human PAD patients

4.2 Results

4.2.1 Construction of the immunome and arteriome

We used the GeneHits to construct the global protein-protein interaction network of immune response and arteriogenesis as immunome and arteriome, respectively (see flowchart in Fig. 4.1A and B). We refer to “immune response” as inflammation. The immunome comprises 3,490 proteins and 21,164 interactions. The set of genes in immunome allows us to have a large pool of genes related to inflammation and overlap with angiogenesis and arteriogenesis in PAD. The arteriome comprises 289 proteins and

803 interactions. We compared the three sets in Venn diagram (Fig. 4.2). The high 33 overlap of genes between angiome and immunome might reflect the regulation by endothelial cells of various biological processes, such as regulation of both angiogenesis and immune response (18).

Figure 4.2. Venn diagram of angiome, immunome and arteriome

4.2.2 Differentially expressed genes in mouse PAD model

We identified the differentially expressed genes from the four microarray datasets provided in Table 4.1 by GenePattern 3.6.1 (61). First, we compared the gene expression transcripts from ischemic versus nonischemic gastrocnemius muscle from C57BL/6 mice.

Then we compared the gene transcripts from ischemic versus nonischemic gastrocnemius muscle from BALB/c mice. Next, we compared the differential gene expression of ischemic samples between C57BL/6 and BALB/c mice. Finally, we compared the differential gene expression of nonischemic samples between C57BL/6 and BALB/c mice.

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We sorted all the 21,747 genes in the raw data by the fold change in each experimental condition of the four comparisons. We also identify the differentially expressed genes which are included in the angiome, immunome and arteriome, with the absolute fold change ranked as top 5% of all the genes and p-value <0.05. We plot the volcano-plot of fold-change and p-values from the ischemic versus nonischemic gastrocnemius muscle from C57BL/6 mice in Fig. 4.3.

Figure 4.3. Volcano plot of the fold-change and p-values from the ischemic versus

nonischemic gastrocnemius muscle from C57BL/6 mice

The list of up- and down-regulated genes between ischemic versus nonischemic gastrocnemius muscle in C57BL/6 is provided in Table 4.3 and 4.4, respectively. Many of the differentially expressed genes in Table 4.3 and 4.4 are in the immunome, i.e. within the areas B, AB, BC or ABC in the Venn diagram in Fig. 4.2. For example, the anti-angiogenic protein thrombospondin-1 (THBS1) is identified by its up-regulated fold

35 change as 1.66 with the p-value 0.0418.

Name Description Fold Feature Venn Diagram Change P THBS1 thrombospondin 1 1.66 0.042 AB (angiome+immunome) Rap guanine nucleotide RAPGEF6 exchange factor (GEF) 6 1.524 0.035 B (immunome) TPP2 II 1.503 0.04 B (immunome) -conjugating UBE2D2 E2D 2 1.438 0.037 B (immunome) ataxia telangiectasia and ATR Rad3 related 1.432 0.046 B (immunome) complement component C8G 8, gamma polypeptide 1.407 0.009 B (immunome) minichromosome maintenance complex MCM3 component 3 1.405 0.015 B (immunome) baculoviral IAP repeat BIRC6 containing 6 1.398 0.047 B (immunome) nuclear factor of activated T-cells, cytoplasmic, -dependent 2 NFATC2IP interacting protein 1.381 0.039 B (immunome) mucosa associated lymphoid tissue lymphoma translocation MALT1 gene 1 1.374 0.002 B (immunome) kinase, 90kDa, RPS6KA1 polypeptide 1 1.372 0.038 B (immunome) TLR4 toll-like receptor 4 1.366 0.036 B (immunome) B-cell translocation gene BTG1 1, anti-proliferative 1.338 0.012 A (angiome) MYO1E myosin IE 1.334 0.037 B (immunome) WWTR1 WW domain containing 1.331 0.01 B (immunome)

36

transcription regulator 1 protein tyrosine , receptor PTPRJ type, J 1.326 0.011 B (immunome) MYO1F myosin IF 1.313 0.047 B (immunome) embryonal Fyn-associated EFS substrate 1.313 0.037 B (immunome) MYO1D myosin ID 1.299 0.04 B (immunome) Ras association (RalGDS/AF-6) domain RASSF2 family member 2 1.285 0.009 B (immunome) proprotein convertase PCSK5 /kexin type 5 1.285 0.044 B (immunome) Table 4.3 Up-regulated genes in ischemic versus non-ischemic C57BL/6 mouse

Name Description Fold Feature Venn Diagram Change P TSPAN7 7 -2.101 0.045 B (immunome) -activated protein MAP2K4 kinase kinase 4 -1.984 0.001 AB (angiome+immunome) family member KIF1C 1C -1.866 0.034 B (immunome) receptor, IL15RA alpha -1.824 0.023 B (immunome) retinoblastoma-like 2 RBL2 (p130) -1.762 0.045 B (immunome) phosphate cytidylyltransferase 1, PCYT1A choline, alpha -1.684 0.02 B (immunome) NCL -1.651 0.04 A (angiome) polymerase (DNA POLK directed) kappa -1.648 0.011 B (immunome) AB IDE insulin-degrading enzyme -1.611 0.009 (angiome+immunome)

37

catenin (cadherin-associated CTNNB1 protein), beta 1, 88kDa -1.58 0.038 AB (angiome+immunome) mitogen-activated protein MAPK8 kinase 8 -1.565 0.047 AB (angiome+immunome) tyrosine 3-monooxygenase/tryptop han 5-monooxygenase activation protein, gamma YWHAG polypeptide -1.548 0.021 B (immunome) IDE insulin-degrading enzyme -1.529 0.034 AB (angiome+immunome) microtubule-associated MAPT protein tau -1.525 0.007 B (immunome) inositol 1,4,5-trisphosphate ITPR1 receptor, type 1 -1.495 0.015 B (immunome) BCL10 B-cell CLL/lymphoma 10 -1.491 0.019 B (immunome) DTNA dystrobrevin, alpha -1.487 0.016 C (arteriome) SEC63 homolog (S. SEC63 cerevisiae) -1.482 0.019 B (immunome) oncogene, E3 MDM2 ubiquitin protein -1.47 0.03 AB (angiome+immunome) ABC fibroblast growth factor (angiome+immunome+arter FGFR1 receptor 1 -1.385 0.02 iome) CYCS , somatic -1.362 0.027 B (immunome) IL16 -1.354 0.032 AB (angiome+immunome) PTK2 protein -1.341 0.048 AB (angiome+immunome) O-linked N-acetylglucosamine OGT (GlcNAc) -1.307 0.048 B (immunome) AT rich interactive ARID4A domain 4A (RBP1-like) -1.299 0.024 B (immunome) platelet/endothelial cell ABC PECAM1 adhesion molecule 1 -1.298 0.044 (angiome+immunome+arter

38

iome) signal transducer and activator of transcription STAT5A 5A -1.296 0.011 B (immunome) ClpB caseinolytic peptidase B homolog (E. CLPB coli) -1.281 0.045 B (immunome) ARHGAP2 Rho GTPase activating 2 protein 22 -1.28 0.018 A (angiome) polymerase (RNA) III (DNA directed) POLR3D polypeptide D, 44kDa -1.275 0.006 B (immunome) signal transducer and activator of transcription STAT2 2, 113kDa -1.266 0.029 B (immunome) Table 4.4 Down-regulated genes in ischemic versus non-ischemic C57BL/6 mouse

We list the up- and down-regulated genes between ischemic and nonischemic

BALB/c mice in Table 4.5 and 4.6, respectively. For example, TLR4 (toll-like receptor 4) is identified with the up-regulation fold change 1.614 and p-value 0.004.

Name Description Fold Feature Venn Diagram Change P TLR4 toll-like receptor 4 1.615 0.004 B (immunome) potassium channel tetramerization domain KCTD12 containing 12 1.561 0.027 C (arteriome) solute carrier family 2 (facilitated glucose SLC2A3 transporter), member 3 1.526 0.009 AB (angiome+immunome) ABC (angiome+immunome+arter EPHA4 EPH receptor A4 1.522 0.012 iome)

39

BCL2-associated BAG4 athanogene 4 1.517 0.03 B (immunome) PDE10A 10A 1.506 0.004 B (immunome) NCOR1 corepressor 1 1.444 0.017 B (immunome) ABC CD44 molecule (Indian (angiome+immunome+arter CD44 blood group) 1.434 0.024 iome) CDKN2AI CDKN2A interacting P protein 1.412 0.045 B (immunome) NF2 neurofibromin 2 () 1.404 0.036 B (immunome) (prosome, macropain) 26S subunit, PSMD11 non-ATPase, 11 1.353 0.005 B (immunome) sprouty-related, EVH1 SPRED1 domain containing 1 1.342 0.006 B (immunome) Fc fragment of IgG, low affinity IIb, receptor FCGR2B (CD32) 1.338 0.006 B (immunome) EBF1 early B-cell factor 1 1.334 0.027 B (immunome) carcinoembryonic -related molecule 1 CEACAM1 (biliary glycoprotein) 1.329 0.012 AB (angiome+immunome) CD180 CD180 molecule 1.323 0.016 B (immunome) WDR12 WD repeat domain 12 1.318 0.029 C (arteriome) LAMB1 , beta 1 1.309 0.044 AC (angiome+arteriome) syntrophin, beta 2 (-associated protein A1, 59kDa, basic SNTB2 component 2) 1.304 0.021 C (arteriome) protein tyrosine phosphatase, receptor PTPRJ type, J 1.303 0.008 B (immunome) ESRRB -related receptor 1.298 0.002 B (immunome)

40

beta argininosuccinate ASS1 synthase 1 1.297 0.02 B (immunome) STEAP family member STEAP3 3, metalloreductase 1.297 0.006 B (immunome) CCAAT/enhancer binding protein (C/EBP), CEBPG gamma 1.29 0.009 B (immunome) homeodomain interacting HIPK2 2 1.284 0.049 A (angiome) COL1A2 collagen, type I, alpha 2 1.27 0.033 A (angiome) NOTCH-regulated NRARP repeat protein 1.266 0.05 A (angiome) embryonal EFS Fyn-associated substrate 1.263 0.045 B (immunome) hyaluronan-mediated ABC motility receptor (angiome+immunome+arter HMMR (RHAMM) 1.261 0.037 iome) CDC25A cycle 25A 1.25 0.025 AB (angiome+immunome) Table 4.5 Up-regulated genes in ischemic versus non-ischemic BALB/c mouse

Name Description Fold Feature Venn Diagram Change P solute carrier family 22 (organic cation/zwitterion SLC22A4 transporter), member 4 -1.529 0.024 B (immunome) TPP2 tripeptidyl peptidase II -1.431 0.038 B (immunome) F-box and WD repeat FBXW11 domain containing 11 -1.409 0.006 B (immunome) nuclear receptor subfamily 2, group C, NR2C1 member 1 -1.392 0.019 B (immunome) kinase suppressor of ras KSR1 1 -1.367 0.041 B (immunome)

41

Ras association (RalGDS/AF-6) domain RASSF5 family member 5 -1.365 0.018 B (immunome) homolog, transcriptional regulator SPEN () -1.349 0.011 B (immunome) CD72 CD72 molecule -1.339 0.044 B (immunome) heparin-binding HBEGF EGF-like growth factor -1.339 0.022 AB (angiome+immunome) ring-box 1, E3 ubiquitin RBX1 protein ligase -1.325 0.034 B (immunome) amyloid beta (A4) APLP2 precursor-like protein 2 -1.313 0.029 B (immunome) catenin, beta interacting CTNNBIP1 protein 1 -1.311 0.029 A (angiome) mindbomb E3 ubiquitin MIB2 protein ligase 2 -1.307 0.006 B (immunome) mitogen-activated protein kinase kinase MAP4K3 kinase kinase 3 -1.306 0.022 B (immunome) transient receptor potential cation channel, TRPV2 subfamily V, member 2 -1.285 0.024 B (immunome) guanine nucleotide GNL1 binding protein-like 1 -1.285 0.024 B (immunome) bone morphogenetic BMP6 protein 6 -1.28 0.008 AB (angiome+immunome) Ras association (RalGDS/AF-6) domain RASSF5 family member 5 -1.277 0.029 B (immunome) GBA glucosidase, beta, acid -1.254 0.001 B (immunome) SYT7 synaptotagmin VII -1.251 0.048 B (immunome) CHRD -1.251 0.021 AB (angiome+immunome) , alpha E ITGAE (antigen CD103, human -1.248 0.033 B (immunome)

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mucosal lymphocyte antigen 1; alpha polypeptide) NGFI-A binding protein 2 (EGR1 binding NAB2 protein 2) -1.248 0.013 AB (angiome+immunome)

Table 4.6 Down-regulated genes in ischemic versus non-ischemic BALB/c mouse

4.2.3 Visualization of PADPIN

To visualize the PADPIN, we select the ten most up-regulated and ten most down-regulated genes with the highest absolute fold change under the statistical significance p-value <0.05 between ischemic versus nonischemic gastrocnemius muscle in BALB/c mice. We select the proteins in the angiome, immunome and arteriome which are linked to the twenty proteins with the most differentially expressed gene expression in

BALB/c (TLR4, KCTD12, SLC2A3, EPHA4, BAG4, PDE10A, NCOR1, CD44,

CDKN2AIP, NF2, SLC22A4, TPP2, FBXW11, NR2C1, KSR1, RASSF5, SPEN, CD72,

HBEGF and RBX1) to construct the representative subnetwork of PADPIN in Fig. 4.4.

We select the proteins in the angiome, immunome and arteriome which are linked to the twenty proteins with the most differentially expressed gene expression in C57BL/6 mouse (THBS1, RAPGEF6, TPP2, UBE2D2, ATR, C8G, MCM3, BIRC6, NFATC2IP,

MALT1, TSPAN7, MAP2K4, KIF1C, IL15RA, RBL2, PCYT1A, NCL, POLK, IDE and

CTNNB1) to construct the representative subnetwork of PADPIN in Fig. 4.5. The color 43 of nodes from red to blue represents the gene expression level from high to low. The size of nodes from large to small represents the degree of nodes, i.e. the number of interactions, from high to low.

Figure 4.4. Visualization of PADPIN for BALB/c microarray datasets

Figure 4.5. Visualization of PADPIN for C57BL/6 microarray datasets

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The number of proteins in the PADPIN of BALB/c (Fig. 4.4) in Venn Diagram A-G

(Fig. 4.2) is 4, 112, 2, 70, 3, 2 and 22, respectively. The number of proteins in the

PADPIN of C57BL/6 (Fig. 4.5) in Venn Diagram A-G (Fig. 4.2) is 15, 207, 0, 146, 6, 3 and 18, respectively. The distinct visualization of PADPIN between the two PADPINs in

Fig. 4.4 and 4.5 clearly shows the diverse molecules involved in ischemic versus non-ischemic tissues between the two mouse strains.

4.2.4 Differentially expressed genes between two inbred mouse strains

We further compare the differential gene expression between C57BL/6 versus

BALB/c mice of ischemic and nonischemic muscles, respectively, with the same criteria as absolute fold change value ranked as top 5% of all the genes and p-value<0.05. There are 197 and 175 differentially expressed genes between the two mouse strains in ischemic and nonischemic muscles, respectively. Under the normal conditions (nonischemic muscles), the 69 up- and 106 down-regulated gene expressions strongly suggest the highly diverse gene expression profiles between the two mouse strains. The list of these differentially expressed genes between two mouse strains are provided in Appendix C.

We use Toppgene (13) for the gene list enrichment analysis of these differentially expressed genes between the two strains. The statistically significant GOs and pathway analysis between two mouse strains in ischemic tissues include cell proliferation

45

(GO:0008283, p-value=8.73E-18), positive regulation of immune system process

(GO:0002684, p-value=1.15E-09), negative regulation of apoptotic process (GO:0043066, p-value=1.29E-09), and PI3K-Akt signaling pathway (p-value=3.08E-06). The statistically significant GOs and pathway analysis between two mouse strains in nonischemic tissues include cell proliferation (GO:0008283, p-value=9.08E-19), apoptotic process (GO:0006915, p-value=1.90E-11), vasculature development

(GO:0001944, p-value=4.73E-11), pathways in cancer (p-value=4.49E-07), and

PI3K-Akt signaling pathway (p-value=2.02E-05). The GO and pathway analysis show the different regulation of cell proliferation, , vasculature and immune responses between the C57BL/6 and BALB/c mice.

4.3 Discussion

4.3.1 Prediction of potential drug targets in PAD

Currently microarray studies in human PAD are limited, and most tissue samples in human PAD are from diseases arteries (17, 36) and peripheral blood mononuclear cells

(66). We notice that all these tissues are very different from gastrocnemius muscles used in Hazarika et al. (45). In an attempt to overcome these limitations, we compared the differentially expressed genes between ischemic versus nonischemic C57BL/6 and

BALB/c mice with the three microarray datasets in PAD patients (17, 36, 66). Table 4.7

46 lists the fold change values for genes in angiome and immunome from each study.

Tissue of the This study: Fu (36): Croner (17): Masud (66): samples Mouse Human femoral Human Human gastrocnemius arteries common peripheral blood muscles femoral artery mononuclear Genes cells (A) Up-regulated genes of ischemic versus nonischemic C57BL/6 mice BTG1 1.337564 2.022 - - MYO1F 1.313371 2.558 - - THBS1 1.659983 4.575 - 2.12 TLR4 1.36603 - 40.571 1.82 (B) Down-regulated genes of ischemic versus nonischemic C57BL/6 mice CYCS -1.36222 -1.484 - - OGT -1.30688 -1.440 - - (C) Up-regulated genes of ischemic versus nonischemic BALB/c mice CEBPG 1.290306 -2.509 - - COL1A2 1.269932 2.342 - - EBF1 1.334283 -1.5 - - KCTD12 1.560756 1.623 - - LAMB1 1.30851 3.575 - - SLC2A3 1.526328 5.753 - - TLR4 1.614745 - 40.571 1.82 (D) Down-regulated genes of ischemic versus nonischemic BALB/c mice HBEGF -1.33892 2.032 - -

Table 4.7 Comparison of the fold change of genome-wide differentially expressed genes between ischemic versus nonischemic muscles from mice with samples from human PAD patients

We will discuss some potential drug targets in PAD in the following section:

47

Thrombospondin 1 (THBS1)

THBS1 is up-regulated in the ischemic versus nonsichemic muscle samples of

C57BL/6 mice (45), human femoral arteries (36), and human peripheral blood mononuclear cells (66). Inhibition of THBS1/CD47 signaling has also shown to promote tissue recovery and survival rate in aged and diet induced vasculopathy in experimental animal models of PAD (51). Blockage of THBS1-CD47 signaling pathways could alleviate tissue ischemia in the animal models, such as enhancement of ischemic tissue survival in a porcine model (53) and prevention of necrosis of full thickness skin grafts

(52). THBS1 has also been proposed as the potential biomarker of PAD (83). Thus, we propose that THBS1 and its receptors CD47 or CD36 can serve as therapeutic targets for

PAD.

Toll-like receptor-4 (TLR4)

TLR4 is listed in top 5% of up-regulated genes in ischemic versus nonischemic

C57BL/6 mice (Table 4.3) and in BALB/c mouse (Table 4.5), as well as in human PAD microarray studies from Croner (17) and Masud (66). TLR4 plays the pivotal role in inflammation and neuronal apoptosis of cerebral ischemia, and inhibition of TLR4 can reduce cerebral ischemia-induced shear stress (89).

Unknown protein functions in PAD

48

Several genes whose roles are still unknown in PAD could serve as potential targets in PAD. SLC22A4 (Solute carrier family 22 (organic cation/zwitterion transporter), member 4) is the top gene that is down-regulated in BALB/c ischemic muscle compared to non-ischemic. The role of SLC22A4 in vascular biology or ischemic diseases is not yet clear. PRKAA2a (AMPKa2) is the top gene that was up-regulated in C57BL/6 vs.

BALB/c ischemic and non-ischemic muscle comparison. PRKAA2a has been shown to have a great impact as a metabolic sensor in skeletal muscle (68). Activation of

PRKAA2a significantly improved the exercise performance and vascular insufficiency in high-fat diet-fed mice (6). EIF2S1 (Eukaryotic Initiation Factor 2, Subunit 1

Alpha, 35kDa) is the top gene that is down-regulated between C57BL/6 vs. BALB/c ischemic and nonischemic muscle. of EIF2S1 has been shown to play prominent roles in cerebral ischemia (38). EPH receptor A4 (EphA4) was the only gene at the intersect of arteriome, angiome and immunome in Table 4.5. family is a large family of receptor tyrosine that play critical roles in angiogenic remodeling of blood and lymphatic vessels (62) and tumor angiogenesis (70). Goldshmit et al. show that EphA4 is expressed on both small and large blood vessels (41), which is correlated with our angiome and arteriome intersect in bioinformatics analysis. Furthermore, EphA4 has also been shown to modulate adhesion of monocytes to endothelial cells in human

49 atherosclerosis plaques (54). Taken together, the functional and expression profiles of

EphA4 closely match the arteriome, angiome and immunome intersect in our bioinformatic analysis.

4.3.2 Comparisons of gene expression between the two mouse strains

Dokun et al. and Sealock et al. showed that the causative genetic variations to angiogenesis and arteriogenesis between the mouse strains were linked to Lsq1 and

Candq1 (now called determinant of collateral extent) quantitative trait loci on 7 (20, 80). We notice that the seven specific genes in Candq1 (Rabep2,

Sh2b1, Cln3, Apobr, Il27, MapK3, and Ppp4c) which are related to endothelial cells (80) are not listed as the differentially expressed genes. We have identified differentially expressed genes between the two mouse strains based on the microarray data analysis, and we correlated them with the differences in collateral blood flow.

50

V. COMPUTATIONAL DRUG REPOSITIONING IN PAD

In Chapter IV, we have predicted the potential drug targets in PADPIN, which include proteins involved in angiogenesis, arteriogenesis and immune response. However, there might be no approved drugs by FDA for these drug targets in PAD, such as

Thrombospondin 1. Considering the time and cost for developing a new drug, we are seeking to take advantage of two drug repositioning strategies for PAD, which are pro-angiogenic and anti-inflammatory (Section 1.6).

5.1 Methods

5.1.1 Resources of drug-targets relations

We use two major resources for drug information and drug-target combinations,

DrugBank 3.0 http://www.drugbank.ca/ (59) and Pharmacogenomics Knowledge Base

(PharmGKB) http://www.pharmgkb.org/ (94). DrugBank contains extensive omics data, such as pharmacogenomics and pharmacoproteomic data; We use DTome (Drug-Target interactome tool) (88) to compile all the drugs in DrugBank 3.0 (59) including the approved, experimental, nutraceutical, illicit, and withdrawn drugs. We mostly focus on

FDA-approved drugs in this chapter, based on the drug safety and development time. We download three binary relations from PharmGKB (94): gene-disease, gene-drug, and gene-gene interactions. This provides the interaction information for drug-target networks 51 in PAD.

In addition to FDA approved drugs from DrugBank and PharmGKB, we also include the 58 compounds and biologics that are listed in the library of industry-provided agents at National Institutes of Health (NIH) http://www.ncats.nih.gov/research/reengineering/rescue-repurpose/therapeutic-uses/direct ory.html. Pharmaceutical companies made these drugs available for potential repositioning.

5.1.2 List of anti-angiogenic and pro-inflammatory genes

We propose inhibition of anti-angiogenic and pro-inflammatory genes as a novel therapeutic approach for PAD (see Introduction for details). The 39 anti-angiogenic genes have been mentioned in Section 3.1.1. The 89 genes listed in positive regulation of inflammatory response (GO:0050729) are: ACE, ADAM8, ADORA2B, ADORA3,

AGER, AGT, AGTR1, ALOX5AP, AOC3, C3, CCL24, CCL3, CCL3L3, CCL5, CCR2,

CCR5, CCR7, CD28, CD47, CLOCK, CNR1, CTSS, CX3CL1, EDNRA, EGFR, FABP4,

FCER1A, FCER1G, FCGR1A, FCGR2A, FFAR3, GPRC5B, HSPD1, HYAL2, IDO1,

IL12B, IL15, IL18, IL1B, IL1RL1, IL2, IL21, IL23A, IL33, IL6, IL6ST, ITGA2, JAK2,

LBP, LTA, MAPK13, MIF, NLRP12, NPY5R, OSM, OSMR, PDE2A, PDE5A, PIK3CG,

52

PLA2G2A, PLA2G4A, PLA2G7, PRKCA, PTGER3, PTGER4, PTGS2, RPS19,

S100A12, , , SERPINE1, STAT5A, STAT5B, TAC1, TGM2, TLR2,

TLR3, TLR4, TLR7, TLR9, TLR10, TNF, TNFRSF11A, TNFRSF1A, TNFSF11,

TNFSF4, TNIP1, WNT5A and ZP3.

5.2 Results

5.2.1 Drug-targets relations in PADPIN

We collected 11,043 relations between the drug and drug targets from DrugBank 3.0

(59) and 3,138 binary relations between the drug and associated genes of that drug

(which may not be the direct targets) from PharmGKB (94). We match the genes in angiome, immunome and arteriome with the drug targets listed in the drug-gene binary relations from DrugBank and PharmGKB. We select the genes with at least one drug targeting that gene in the angiome, immunome and arteiome, and the genes without any drug-gene relations. We calculate the degree of nodes (i.e. number of links of the nodes) in angiome, immunome and arteriome, respectively. Hase et al. suggested that middle- to low-degree nodes are advantageous as drug targets (44), compared to high-degree nodes in the network. The rationale in Hase et al. (44) is based on the fewer side effects when targeting less prominent nodes. We agree with rationale that the genes with the high degree of nodes may not be suitable as drug targets. For example, the top

53

10 genes with the highest degree of nodes in immunome are , JUN, TP53, GRB2,

SRC, EGFR, CREBBP, FYN, MAPK1 and ESR1, and these drug targets are involved in multiple biological processes and pathways. The middle degree genes (with degree of nodes between 6 and 30, defined in (44)) are more specific to immune response, such as

TNF, TLR8 and TLR7.

5.2.2 Inhibition of anti-angiogenic and pro-inflammatory genes

We propose two strategies to the PAD treatment: pro-angiogenic and anti-inflammatory (Section 1.6). Starting from the 39 genes annotated in negative regulation of angiogenesis, we match the genes with drug targets and drugs listed, and only list the FDA-approved drugs from DrugBank in Table 5.1.

Gene name Drugbank

CCL2 Mimosine, Danazol

NPPB Carvedilol

Nitroprusside, Nitroglycerin, Isosorbide Dinitrate, Amyl Nitrite,

NPR1 Erythrityl Tetranitrate, Nesiritide

PF4 Drotrecogin alfa

Alteplase, Urokinase, Reteplase, Anistreplase, Tenecteplase,

SERPINE1 Drotrecogin alfa

Table 5.1 Predictions of pro-angiogenic FDA-approved drugs that target anti-angiogenic

genes 54

We match the 89 pro-inflammatory genes with drug targets and drugs listed in immunome and only list the FDA-approved drugs from DrugBank in Table 5.2.

Gene name Drugbank ACE Ramipril, Fosinopril, Trandolapril, Benazepril, Enalapril, Candoxatril, Moexipril, Lisinopril, Perindopril, Quinapril, Rescinnamine, Captopril, Cilazapril, Spirapril ADORA2B Theophylline, Adenosine, Enprofylline, Defibrotide AGTR1 Valsartan, Olmesartan, Losartan, Candesartan, Eprosartan, Telmisartan, Irbesartan, Forasartan, Saprisartan, Tasosartan AOC3 Phenelzine, Hydralazine C3 Intravenous Immunoglobulin CCR5 Maraviroc CNR1 Dronabinol, Nabilone, Rimonabant, Dronabinol EDNRA Bosentan, Sitaxentan EGFR , , Lidocaine, , , , FCER1A Omalizumab, Benzylpenicilloyl Polylysine FCER1G Benzylpenicilloyl Polylysine FCGR1A Cetuximab, Etanercept, Intravenous Immunoglobulin, Adalimumab, Abciximab, Gemtuzumab ozogamicin, Trastuzumab, Rituximab, Basiliximab, Muromonab, Ibritumomab, Tositumomab, Alemtuzumab, Alefacept, Efalizumab, Natalizumab, Palivizumab, Daclizumab, Bevacizumab, Porfimer FCGR2A Cetuximab, Etanercept, Intravenous Immunoglobulin, Adalimumab, Abciximab, Gemtuzumab ozogamicin, Trastuzumab, Rituximab, Basiliximab, Muromonab, Ibritumomab, Tositumomab, Alemtuzumab, Alefacept, Efalizumab, Natalizumab, Palivizumab, Daclizumab, Bevacizumab IL1B Minocycline, Gallium nitrate, Canakinumab IL6 Ginseng LTA Etanercept PDE5A Sildenafil, Theophylline, Pentoxifylline, Tadalafil, Vardenafil, Dipyridamole, Udenafil

55

PLA2G2A Indomethacin, Diclofenac, Ginkgo biloba, Suramin, Ginkgo biloba PLA2G4A Fluticasone Propionate, Quinacrine PRKCA Phosphatidylserine, Vitamin E PTGER3 Bimatoprost, Dinoprostone, Misoprostol PTGS2 gamma-Homolinolenic acid, Icosapent, Aminosalicylic Acid, Mesalazine, Acetaminophen, Indomethacin, Nabumetone, Ketorolac, Tenoxicam, Lenalidomide, Celecoxib, Tolmetin, Piroxicam, Fenoprofen, Diclofenac, Sulindac, Flurbiprofen, Etodolac, Mefenamic acid, Naproxen, Sulfasalazine, Phenylbutazone, Meloxicam, Carprofen, Diflunisal, Suprofen, Salicyclic acid, Meclofenamic acid, Acetylsalicylic acid, Bromfenac, Oxaprozin, Ketoprofen, Balsalazide, Thalidomide, Ibuprofen, Lumiracoxib, Magnesium salicylate, Salicylate-sodium, Salsalate, Trisalicylate-choline, Ginseng, Antrafenine, Antipyrine, Tiaprofenic acid, Etoricoxib, Niflumic Acid, Lornoxicam, Nepafenac, gamma-Homolinolenic acid, Icosapent, Ginseng, Thalidomide Olopatadine, Amlexanox SERPINE1 Alteplase, Urokinase, Reteplase, Anistreplase, Tenecteplase, Drotrecogin alfa STAT5B TLR2 OspA lipoprotein TLR7 Imiquimod, Hydroxychloroquine TLR9 Chloroquine, Hydroxychloroquine TNF Etanercept, Adalimumab, Infliximab, Chloroquine, Thalidomide, Glucosamine, Clenbuterol, Pranlukast, Amrinone, Thalidomide TNFSF11 Lenalidomide

Table 5.2 Predictions of anti-inflammatory FDA-approved drugs that target pro-inflammatory genes

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5.2.3 Drug-Target Network

We use the graph form to represent examples of pro-angiogenic and anti-inflammatory repositioned drugs for PAD in Figure 5.1 (A) and (B), respectively. We consider the FDA-approved drugs and industry-provided agents at the NIH that target the anti-angiogenic and pro-inflammatory proteins. Figure 5.1A shows seven compounds

Carvedilol, Drotrecogin alfa, Alteplase, Reteplase, Anistreplase, Urokinase and

Tenecteplase that target three proteins NPPB, PF4 and SERPINE1 annotated as negative regulation of angiogenesis. Figure 5.1B shows three compounds Sitaxentan, Bosentan and Zibotentan targeting receptor A1 (EDNRA). This drug-target network provides the drug target gene and the FDA-approved drugs, which are included in

DrugBank and PharmGKB.

Figure 5.1 Representative examples of (A) pro-angiogenic and (B) anti-inflammatory repositioned drugs for PAD

57

5.3 Discussion

Case Study of endothelin receptor antagonist

We select the endothelin receptor antagonists as a case study for computationally repositioned drugs for PAD. The endothelin receptor antagonists have been approved for use in pulmonary arterial hypertension (PAH) and have been assigned orphan drug status, such as Bosentan and Ambrisentan. Details of hepatotoxicity of Bosentan, Ambrisentan and Sitaxentan are reviewed in (19). Endothelin-1 is a powerful endogenous vasoconstrictor (35) and thus blocking endothelin could improve perfusion to the lower extremities in patients with PAD. De Haro Miralles et al. (19) examined plasma levels of endothelin and showed that endothelin levels were increased in patients with intermittent claudication compared to non-PAD controls.

The reuse of endothelin receptor antagonists in PAD patients with intermittent claudication is now in Phase II clinical trial without any new preclinical studies.

Importantly patients with the most severe form of PAD, critical limb ischemia (CLI), did not demonstrate elevated levels of endothelin which suggests that an elevation of endothelin is specific to the pathophysiology of intermittent claudication and not all forms of PAD. The details of clinical trial of Zibotentan are provided in

ClinicalTrials.gov with the identifier NCT01890135.

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VI. COMPARTMENTAL MODEL OF VEGF165b

6.1 Methods

6.1.1 Three-compartment models

The three-compartment model is comprised of normal tissue (representing all tissues and organs except the diseased calf), blood and diseased calf of PAD (Figure 6.1).

VEGF165, VEGF165b and VEGF121 are secreted by myocytes in the normal tissues and diseased calf muscles in PAD, and also by endothelial cells in all compartments. VEGF receptors (VEGFR1 and VEGFR2) and co-receptor -1 (NRP1) are localized on the surfaces of parenchymal and endothelial cells. We include

(GAG) and soluble VEGFR1 (sVEGFR1) in the interstitial space of normal and calf compartments, and alpha-2-macroglobulin (α2M) in the blood.

Figure 6.1 Three-compartment model of VEGF in peripheral arterial disease

59

6.1.2 Secretion rate and secretion ratio of VEGF165b to total VEGF

In our model, we change the secretion ratio of VEGF165b to total VEGF from 0 to 1,

and fix the secretion rate of VEGF121 as 10% of all VEGF isoforms. The secretion rate of

total VEGF including VEGF165, VEGF165b and VEGF121 is assumed as 0.02, 0.031 and

0.02 molecules/cell/s in normal, blood and PAD calf compartments, respectively. The value of secretion rate is based on the secretion rate of VEGF in the normal compartment in tumor models (29).

6.1.3 Molecular Interactions and kinetic equations of VEGF isoforms

The molecular interactions between VEGF isoforms and their receptors are

illustrated in Figure 6.2. VEGF165, VEGF165b and VEGF121 bind to VEGFR1 and

VEGFR2. VEGF165b has the equivalent binding to VEGFR2 as VEGF165 (95) and

functions as a competitive inhibitor of the major downstream effects of VEGF165.

VEGF165 and VEGF165b are glycoproteins with heparin binding domain that can bind the

. VEGF121 is a freely diffusible protein without heparin binding

domain. VEGF165b can bind to VEGFR1 and VEGFR2 but cannot bind the co-receptor

NRP1 because it lacks 8a (12). VEGF165, VEGF165b and soluble VEGFR1

(sVEGFR1) can bind to glycosaminoglycan (GAG) chains in the interstitial space of normal and calf compartments.

60

Figure 6.2 Molecular interactions of VEGF165, VEGF165b and VEGF121

We also list the densities of cell-surface receptors VEGFR1, VEGFR2 and NRP1 in

Table 6.1, based on available in vivo and in vitro experimental data. We list the 80 ordinary differential equations (ODE) describing the system in Appendix D. We implement these equations in MATLAB (MathWorks, Natick, MA) SimBiology toolbox and solve them using the Sundial solver.

Receptors VEGFR1 VEGFR2 NRP1

Compartment

Luminal EC (normal) 550 350 17500

Abluminal EC (normal) 550 350 17500

Luminal EC (disease) 3750 300 20000

Abluminal EC (disease) 3750 300 20000

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Table 6.1 Number of cell surface receptors VEGFR1, VEGFR2 and NRP1. Units of

values: dimers/EC for VEGFR1 and VEGFR2 and monomer for NRP1

6.1.4 Geometric parameters

The geometric parameters in the normal and blood compartments are described in

(30). The geometric parameters in the diseased calf compartment are described in (96).

We list the details of geometric parameters of diseased calf muscles in our model in Table

6.2. These parameters correspond to human tissues.

Value Units

Compartment volume 738 cm3

Fluid volume in ECM 31.87% cm3/cm3 tissue

Fluid volume in EBM 0.45% cm3/cm3 tissue

Fluid volume in PBM 0.58% cm3/cm3 tissue

Muscle fiber cross-sectional area 417 cm2/cm3 tissue

Muscle fiber cell surface area 3,464 µm2

Table 6.2 Geometric parameters of diseased calf muscles for human patients

6.1.5 Transport parameters

Molecular species are transported between compartments via microvascular

permeability (kp) and lymphatic drainage (kL) as shown in Figure 6.1. All isoforms of 62 unbound VEGF and sVEGFR1 in the tissue compartments are subject to proteolytic

degradation (kdeg) and are removed from the blood via plasma clearance (cv). We list the transport parameters in Table 6.3.

Value unit Reference

Permeability between normal and blood compartments

4.0x10-8 cm/s Stefanini (86) VEGF

1.5x10-8 cm/s Wu (97) sVEGFR1

1.5x10-8 cm/s Wu (97) VEGF:sVEGFR1 complex

Permeability between disease and blood compartments

4.0x10-7 cm/s Assumed, based on high VEGF permeability in PAD 3.0x10-7 cm/s Assumed sVEGFR1

1.5x10-7 cm/s Assumed VEGF:sVEGFR1 complex

Clearance

1.1x10-3 s-1 Calculated, based on half-life VEGF

5.0x10-6 s-1 Wu (97) sVEGFR1

63

3.0x10-4 s-1 Wu (97) VEGF:sVEGFR1

3.9x10-5 s-1 Hudson (48) α2M

3.9x10-5 s-1 Assumed, based onα2M VEGF:α2M complex

3.9x10-3 s-1 Imber (49) α2Mfast

-3 -1 3.9x10 s Assumed, based onα2Mfast VEGF:α2Mfast complex

Degradation

1.9x10-4 s-1 Assumed, based on VEGF sVEGFR1

1.9x10-4 s-1 Assumed, based on VEGF VEGF:sVEGFR1 complex

Synthesis

3.5x1010 Molecules/ Calculated α2M cm3 tissue/s 1.9x1010 Molecules/ Calculated α2Mfast cm3 tissue/s

Table 6.3 Transport parameters between the compartments and clearance, synthesis

and degradation

6.1.6 Sensitivity analysis

The sensitivity analysis is implemented using Matlab SimBiology toolbox. The time-dependent sensitivities of the species states are calculated with respect to species 64 initial conditions and parameter values in the model. The sensitivity is calculated by d[Y]/d[X], where a species Y is the output with respect to each parameter value X.

6.2 Results

6.2.1 Experimental measurements of VEGF165 and VEGF165b in human biopsies

We hypothesize that the steady-state concentration of VEGF165b level in all the three

compartments is higher than the concentration of VEGF165 after 11 days, based on two experimental results from Dr. Brain Annex’s group (unpublished) and Kikuchi et al. (58).

Kikuchi et al. measured the ratio of mRNA level of VEGF165b versus VEGF165 in serum and peripheral blood mononuclear cells (PBMCs) in PAD patients, which were reported

as 4:1 and 8:1, respectively (58). We also assume that the concentration of VEGF165b and

VEGF165 should be no more than 10 pM in any of the three compartments to be consistent with the experimental data from Hoier et al. (46). Hoier et al. measured the interstitial VEGF protein concentration in PAD patients as 69±21 and 190±78 pg/ml in rest and active exercise, respectively; We convert these numbers as

69pg/ml*1000ml/l*1mole/46000g=1.5pM and 190pg/ml=4.1pM based on the molecular weight 46-kDa for VEGF homodimers (90).

65

6.2.2 Computational results of secretion rates of VEGF165b and secretion ratio of

VEGF165b over VEGF165 in three compartments

The secretion rate of total VEGF including VEGF165, VEGF165b and VEGF121 is assumed as 0.02, 0.031 and 0.02 molecules/cell/s in normal, blood and PAD calf compartments, respectively. The secretion rate of VEGF in normal and blood compartments is adopted from the secretion rate of VEGF in the normal and blood compartment of the tumor model, respectively (29). The secretion rate in PAD calf compartment is set up the same as the secretion rate in the normal compartment. The

secretion ratio of VEGF121 over total VEGF is 10%, i.e. 0.002, 0.0031 and 0.002 molecules/cell/s in normal, blood and disease compartments, respectively.

To determine the secretion ratio of VEGF165b over total VEGF165, we scan this ratio

in the three compartments from 0 and 1 in 0.1 increments. When the ratio of VEGF165b

over total VEGF165 is 0.5, i.e. secretion rate of VEGF165 and VEGF165b with equal values as 0.009, 0.01395 and 0.009 in normal and disease compartments, respectively, the

concentration of VEGF165b in the normal compartment reaches 31 pM. This is against our assumption based on the measurements of Hoier et al. (46) that the concentration of

VEGF165b and VEGF165 should be no more than 10 pM in any of the three compartments

(see Section 6.1.1). When the ratio of VEGF165b over all VEGF165 is 0.1, the

66 concentration of VEGF165b in the normal compartment is 6.5 pM, which is smaller than

10 pM in our model fitting. Therefore, we start with the assumption of the ratio of

VEGF165b over all VEGF165 as 0.1 in all the three compartments.

We plot the predictions for steady state concentrations of VEGF165b and VEGF165 by

varying the secretion ratio of VEGF165b over total VEGF165 in each of the three compartments in Figure 6.3. In Figure 6.3A, we change the secretion ratio of

VEGF165b/VEGF165 from 0 to 1 in 0.1 increments in the non-blood&non-PAD (normal)

compartment while fixing the secretion ratios of VEGF165b/VEGF165 in blood and disease compartments at 0.1. We follow the same strategy in Figures 6.3B and C, except we vary

the secretion ratio of VEGF165b/VEGF165 in the blood compartment for B and the disease compartment for C, while the total secretion rates are fixed and the ratios are fixed at 0.1 in the other compartments. The computational model which fits the experimental data is

under the condition when secretion ratio of VEGF165b/VEGF165 is 1 in the disease compartment, and 0.1 in the normal and blood compartments. This prediction gives us an

important biological insight that the PAD disease calf muscles secrete mostly VEGF165b

over all VEGF165. This finding could be further extended for the development of

VEGF165b antibody treatment in PAD.

67

Figure 6.3 Changing of the secretion ratio of VEGF165b to total VEGF165 isoforms in

non-blood&non-PAD (normal), blood and PAD calf compartments

6.2.3 Sensitivity analysis of VEGF165, VEGF165b and sVEGFR1

We use SimBiology to investigate the sensitivity d[Y]/d[X], where Y is the species

VEGF165, VEGF165b and VEGF121, and X is the three VEGF receptors VEGFR1,

VEGFR2 and NRP1 in the disease compartment. Figures 6.4 show that VEGF165 and

VEGF165b in the disease and blood compartment is more sensitive to VEGFR2 than

VEGFR1. VEGF121 in disease and blood compartment is sensitive to both VEGFR1 and

VEGFR2. Neither VEGF165, VEGF165b nor VEGF121 is sensitive to NRP1. The sensitivity

analysis in Figure 6.4 shows the importance of VEGF165b-VEGFR2 binding.

68

Figure 6.4 Sensitivity analysis of VEGF165, VEGF165b and VEGF121. “Normal”

represents non-PAD and non-blood compartment.

6.2.4 VEGF distribution and VEGFR occupancy in the three compartments

We calculate the distribution of VEGF and its receptors for each tissue in Figure 6.5

(A) and (B), respectively. The y-axis represents the percentage of each species in x-axis

for each VEGF ligand (VEGF165, VEGF165b and VEGF121) in Figure 6.5 (A) and each receptor (VEGFR1, VEGFR2, NRP1, and sVEGFR1) in Figure 6.5 (B). In PAD calf,

most VEGF165 is bound to the ECM and PBM (55% and 17%), as well as VEGF165b

bound to ECM and PBM (62% and 20%, respectively). Most VEGF121 isoform is bound

to VEGFR1 and NRP1 as the VEGF121:VEGFR1:NRP1 complex in the disease as 48% and normal (non-PAD & non-blood) as 51% compartment.

69

Figure 6.5 (A) VEGF distribution and (B) VEGFR distribution in the PAD disease

and non-PAD & non-blood (normal) compartments

In Figure 6.5B, the three receptors VEGFR1, VEGFR2 and NRP1 are in the free

states, except the VEGF121:VEGFR1:NRP1 (33%) and VEGF165:VEGFR2:NRP1 (49%) in the normal compartment. In the PAD calf compartment, most receptors are in the free

states, except the VEGF165:VEGFR2:NRP1 (20%) and VEGFR1:NRP1 (18%). Most soluble receptors sVEGFR1 are bound to ECM, EBM and PBM (62%, 15% and 19%, respectively). Our model in PAD resembles similar distribution of VEGF and VEGFR occupancy in tumor (86).

70

6.3 Discussion

We previously developed three-compartment models of VEGF in tumor (27-30).

The anti-angiogenic form of VEGF165b has not been included in any of the computational

models; this is the first study to include VEGF165b in our human PAD model. By changing the geometric parameters in human PAD calf muscles and adding the kinetics equations

of VEGF165b, we calculate the VEGF165b level in the PAD calf, blood and normal compartments. We summarize the results in Table 6.4. Our computational model shows

that the percentage of VEGF165b over total VEGF in PAD is 52%, 54% and 48% in disease, blood and normal compartments, respectively. This computational result is

consistent with the experimental measurements of VEGF165b as a major isoform of

VEGF165 in PAD (58). In many tissues, VEGFxxxb forms more or close to 50% of the total

VEGF protein in many of the tissues, including kidney (9), eye (60) and colon (93).

Compartments PAD calf Blood Normal (Total VEGF (Total VEGF (non-PAD&non-blood) secretion rate: secretion rate: (Total VEGF secretion Species/ratio 0.02 0.031 rate: 0.02 molecules/cell/s) molecules/cell/s) molecules/cell/s)

VEGF165 1.276 pM 0.886 pM 4.591 pM

VEGF165b 2.163 pM 1.863 pM 6.486 pM

VEGF121 0.701 pM 0.653 pM 2.416 pM

VEGF165b/ 52% 54% 48% total VEGF165*100%

Table 6.4 Computational estimation of VEGF165b, VEGF165 and VEGF121 71

This study is also important for computational modeling of pharmacokinetics and

pharmacodynamics (PK/PD) of administering a VEGF165b-antibody as a potential pro-angiogenic therapeutic. Current VEGF antibodies including bevacizumab bind both

VEGF165a and VEGF165b. We propose to introduce an antibody to VEGF165b as a PAD therapeutic to stimulate angiogenesis.

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VII. Summary

Angiogenesis is a key component of over 70 diseases, including excessive angiogenesis such as cancer and age-related macular degeneration and insufficient angiogenesis in coronary artery disease and peripheral arterial disease. In addition to pathophysiological, disease conditions angiogenesis plays fundamental role in physiological processes such as development, exercise, and aging. In many of these physiological and pathophysiological conditions, vast genomic and proteomic information is becoming available and accessible via public databases.

In Chapter II, we have constructed the angiome, the most complete network of angiogenesis-related proteins. The angiome provides us with a platform to identify those genes and proteins in the databases that are associated with angiogenesis. In Chapter III, we combine several gene expression data sets on endothelial cells and protein interactions from angiome to reveal the dynamics of positive and negative regulation of angiogenesis under different experimental conditions. Chapters II and III provide a foundation for bioinformatics research in angiogenesis in application to specific angiogenesis-related diseases such as cancer and ischemic disease.

In Chapters IV and V, we specifically focus on peripheral arterial disease (PAD). In chapter IV, we used bioinformatics tools to construct the global protein-protein 73 interaction networks of angiogenesis, immune response and arteriogenesis, identify novel genes and predict therapeutic targets that play critical roles in peripheral arterial disease.

Our bioinformatics analysis identified several genes that are differentially expressed between the two mouse strains with known functions in PAD including TLR4, THBS1, and PRKAA2, and several genes with unknown functions in PAD including EphA4,

TSPAN7, SLC22A4 and EIF2a that are the basis to play critical roles in the perfusion recovery of C57BL/6 and BALB/c mice by modulating angiogenesis, arteriogenesis, and immune responses in PAD. We provide bioinformatics predictions for future experimental validation in peripheral arterial disease. In Chapter V, we predict FDA-approved drugs whose targets are the proteins annotated as anti-angiogenic and pro-inflammatory by

Gene Ontology, respectively, for PAD. One example is Zibotentan, which is now in clinical trials. We provide the clinical insight of the identification of targets and prediction of repositioning drugs in PAD.

In addition to bioinformatics approaches, we applied the three-compartment model of VEGF distribution in the body in PAD. Experimental data in human biopsies

show that the expression level of VEGF165b is higher than VEGF165 and the computational model results are in agreement with these measurements. We provide a foundation for pharmacokinetics and pharmacodynamics models of the therapeutic inhibition of

74

VEGF165b in PAD in the future.

75

VIII. Bibliography

1. Drug development costs jump to $2.6 billion. Cancer discovery 5: OF2, 2015. 2. Abdollahi A, Hahnfeldt P, Maercker C, Grone HJ, Debus J, Ansorge W, Folkman J, Hlatky L, and Huber PE. Endostatin's antiangiogenic signaling network. Molecular cell 13: 649-663, 2004. 3. Abdollahi A, Schwager C, Kleeff J, Esposito I, Domhan S, Peschke P, Hauser K, Hahnfeldt P, Hlatky L, Debus J, Peters JM, Friess H, Folkman J, and Huber PE. Transcriptional network governing the angiogenic switch in human pancreatic cancer. Proceedings of the National Academy of Sciences of the United States of America 104: 12890-12895, 2007. 4. Annex BH. Therapeutic angiogenesis for critical limb ischaemia. Nature reviews Cardiology 10: 387-396, 2013. 5. Assenov Y, Ramirez F, Schelhorn SE, Lengauer T, and Albrecht M. Computing topological parameters of biological networks. Bioinformatics 24: 282-284, 2008. 6. Baltgalvis KA, White K, Li W, Claypool MD, Lang W, Alcantara ="tag">R, Singh BK, Friera AM, McLaughlin J, Hansen D, McCaughey K, Nguyen H, Smith IJ, Godinez G, Shaw SJ, Goff D, Singh R, Markovtsov V, Sun TQ, Jenkins Y, Uy G, Li Y, Pan A, Gururaja T, Lau D, Park G, Hitoshi Y, Payan DG, and Kinsella TM. Exercise performance and peripheral vascular insufficiency improve with AMPK activation in high-fat diet-fed mice. American journal of physiology Heart and circulatory physiology 306: H1128-1145, 2014. 7. Barabasi AL, and Oltvai ZN. Network biology: understanding the cell's functional organization. Nature reviews 5: 101-113, 2004. 8. Bein K, and Simons M. Thrombospondin type 1 repeats interact with matrix 2. Regulation of metalloproteinase activity. J Biol Chem 275: 32167-32173, 2000. 9. Bevan HS, van den Akker NM, Qiu Y, Polman JA, Foster RR, Yem J, Nishikawa A, Satchell SC, Harper SJ, Gittenberger-de Groot AC, and Bates DO. The alternatively spliced anti-angiogenic family of VEGF isoforms VEGFxxxb in human kidney development. Nephron Physiology 110: p57-67, 2008. 10. Cancer Genome Atlas N. Comprehensive molecular portraits of human breast tumours. Nature 490: 61-70, 2012. 11. Carmeliet P. Angiogenesis in life, disease and . Nature 438: 932-936,

76

2005. 12. Cebe Suarez S, Pieren M, Cariolato L, Arn S, Hoffmann U, Bogucki A, Manlius C, Wood J, and Ballmer-Hofer K. A VEGF-A splice variant defective for heparan sulfate and -1 binding shows attenuated signaling through VEGFR-2. Cellular and molecular life sciences : CMLS 63: 2067-2077, 2006. 13. Chen J, Bardes EE, Aronow BJ, and Jegga AG. ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic acids research 37: W305-311, 2009. 14. Chen Y, Wei T, Yan L, Lawrence F, Qian HR, Burkholder TP, Starling JJ, Yingling JM, and Shou J. Developing and applying a gene functional association network for anti-angiogenic kinase inhibitor activity assessment in an angiogenesis co-culture model. BMC 9: 264, 2008. 15. Chu LH, Lee E, Bader JS, and Popel AS. Angiogenesis interactome and time course microarray data reveal the distinct activation patterns in endothelial cells. PloS one 9: e110871, 2014. 16. Chu LH, Rivera CG, Popel AS, and Bader JS. Constructing the angiome: a global angiogenesis protein interaction network. Physiological genomics 44: 915-924, 2012. 17. Croner RS, Balzer K, Schellerer V, Muller V, Schlabrakowsi A, Sturzl M, Naschberger E, and Lang W. Molecular characterization of peripheral arterial disease in proximal extremity arteries. The Journal of surgical research 178: 1046-1058, 2012. 18. Danese S, Dejana E, and Fiocchi C. Immune regulation by microvascular endothelial cells: directing innate and adaptive immunity, coagulation, and inflammation. Journal of 178: 6017-6022, 2007. 19. de Haro Miralles J, Gonzalez AF, Varela Casariego C, and Garcia FA. Onset of peripheral arterial disease: role of endothelin in endothelial dysfunction. Interactive cardiovascular and thoracic surgery 10: 760-765, 2010. 20. Dokun AO, Keum S, Hazarika S, Li Y, Lamonte GM, Wheeler F, Marchuk DA, and Annex BH. A quantitative trait (LSq-1) on mouse is linked to the absence of tissue loss after surgical hindlimb ischemia. Circulation 117: 1207-1215, 2008. 21. Dudley JT, Deshpande T, and Butte AJ. Exploiting drug-disease relationships for computational drug repositioning. Briefings in bioinformatics 12: 303-311, 2011. 22. Ernst J, and Bar-Joseph Z. STEM: a tool for the analysis of short time series gene expression data. BMC bioinformatics 7: 191, 2006. 23. Ernst J, Nau GJ, and Bar-Joseph Z. Clustering short time series gene expression data. Bioinformatics 21 Suppl 1: i159-168, 2005. 77

24. Esch F, Baird A, Ling N, Ueno N, Hill F, Denoroy L, Klepper R, Gospodarowicz D, Bohlen P, and Guillemin R. Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proceedings of the National Academy of Sciences of the United States of America 82: 6507-6511, 1985. 25. Ferrara N, and Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochemical and biophysical research communications 161: 851-858, 1989. 26. Finley SD, Chu LH, and Popel AS. Computational systems biology approaches to anti-angiogenic cancer therapeutics. Drug discovery today 2014. 27. Finley SD, Dhar M, and Popel AS. Compartment model predicts VEGF secretion and investigates the effects of VEGF trap in tumor-bearing mice. Frontiers in oncology 3: 196, 2013. 28. Finley SD, Engel-Stefanini MO, Imoukhuede PI, and Popel AS. Pharmacokinetics and pharmacodynamics of VEGF-neutralizing antibodies. BMC systems biology 5: 193, 2011. 29. Finley SD, and Popel AS. Effect of tumor microenvironment on tumor VEGF during anti-VEGF treatment: systems biology predictions. Journal of the National Cancer Institute 105: 802-811, 2013. 30. Finley SD, and Popel AS. Predicting the effects of anti-angiogenic agents targeting specific VEGF isoforms. The AAPS journal 14: 500-509, 2012. 31. Folkman J. Tumor angiogenesis: therapeutic implications. The New England journal of medicine 285: 1182-1186, 1971. 32. Fowkes FG, Rudan D, Rudan I, Aboyans V, Denenberg JO, McDermott MM, Norman PE, Sampson UK, Williams LJ, Mensah GA, and Criqui MH. Comparison of global estimates of prevalence and risk factors for peripheral artery disease in 2000 and 2010: a systematic review and analysis. Lancet 382: 1329-1340, 2013. 33. Frostegard J. Immunity, atherosclerosis and . BMC medicine 11: 117, 2013. 34. Frostegard J, Ulfgren AK, Nyberg P, Hedin U, Swedenborg J, Andersson U, and Hansson GK. Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and -stimulating cytokines. Atherosclerosis 145: 33-43, 1999. 35. Frumkin LR. The pharmacological treatment of pulmonary arterial hypertension. Pharmacological reviews 64: 583-620, 2012. 36. Fu S, Zhao H, Shi J, Abzhanov A, Crawford K, Ohno-Machado L, Zhou J, Du 78

Y, Kuo WP, Zhang J, Jiang M, and Jin JG. Peripheral arterial occlusive disease: global gene expression analyses suggest a major role for immune and inflammatory responses. BMC Genomics 9: 369, 2008. 37. Galvin NJ, Vance PM, Dixit VM, Fink B, and Frazier WA. Interaction of human thrombospondin with types I-V collagen: direct binding and electron microscopy. The Journal of cell biology 104: 1413-1422, 1987. 38. Garcia-Bonilla L, Cid C, Alcazar A, Burda J, Ayuso I, and Salinas M. Regulatory proteins of eukaryotic initiation factor 2-alpha subunit (eIF2 alpha) phosphatase, under ischemic reperfusion and tolerance. Journal of neurochemistry 103: 1368-1380, 2007. 39. Gautier L, Cope L, Bolstad BM, and Irizarry RA. affy--analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20: 307-315, 2004. 40. Glesne DA, Zhang W, Mandava S, Ursos L, Buell ME, Makowski L, and Rodi DJ. Subtractive transcriptomics: establishing polarity drives in vitro human endothelial morphogenesis. Cancer research 66: 4030-4040, 2006. 41. Goldshmit Y, Galea MP, Bartlett PF, and Turnley AM. EphA4 regulates central nervous system vascular formation. The Journal of comparative neurology 497: 864-875, 2006. 42. Gu J, Chen Y, Li S, and Li Y. Identification of responsive gene modules by network-based gene clustering and extending: application to inflammation and angiogenesis. BMC systems biology 4: 47, 2010. 43. Hagedorn M, and Bikfalvi A. Target molecules for anti-angiogenic therapy: from basic research to clinical trials. Critical reviews in oncology/hematology 34: 89-110, 2000. 44. Hase T, Tanaka H, Suzuki Y, Nakagawa S, and Kitano H. Structure of protein interaction networks and their implications on drug design. PLoS computational biology 5: e1000550, 2009. 45. Hazarika S, Farber CR, Dokun AO, Pitsillides AN, Wang T, Lye RJ, and Annex BH. MicroRNA-93 controls perfusion recovery after hindlimb ischemia by modulating expression of multiple genes in the pathway. Circulation 127: 1818-1828, 2013. 46. Hoier B, Walker M, Passos M, Walker PJ, Green A, Bangsbo J, Askew CD, and Hellsten Y. Angiogenic response to passive movement and active exercise in individuals with peripheral arterial disease. Journal of applied physiology 115: 1777-1787, 2013. 47. Huang Y, and Li S. Detection of characteristic sub pathway network for angiogenesis based on the comprehensive pathway network. BMC bioinformatics 11 79

Suppl 1: S32, 2010. 48. Hudson NW, Kehoe JM, and Koo PH. Mouse alpha-macroglobulin. Structure, function and a molecular model. The Biochemical journal 248: 837-845, 1987. 49. Imber MJ, and Pizzo SV. Clearance and binding of two electrophoretic "fast" forms of human alpha 2-macroglobulin. The Journal of biological chemistry 256: 8134-8139, 1981. 50. Iorio F, Rittman T, Ge H, Menden M, and Saez-Rodriguez J. Transcriptional data: a new gateway to drug repositioning? Drug discovery today 18: 350-357, 2013. 51. Isenberg JS, Hyodo F, Pappan LK, Abu-Asab M, Tsokos M, Krishna MC, Frazier WA, and Roberts DD. Blocking thrombospondin-1/CD47 signaling alleviates deleterious effects of aging on tissue responses to ischemia. Arteriosclerosis, thrombosis, and vascular biology 27: 2582-2588, 2007. 52. Isenberg JS, Pappan LK, Romeo MJ, Abu-Asab M, Tsokos M, Wink DA, Frazier WA, and Roberts DD. Blockade of thrombospondin-1-CD47 interactions prevents necrosis of full thickness skin grafts. Annals of surgery 247: 180-190, 2008. 53. Isenberg JS, Romeo MJ, Maxhimer JB, Smedley J, Frazier WA, and Roberts DD. Gene silencing of CD47 and antibody ligation of thrombospondin-1 enhance ischemic tissue survival in a porcine model: implications for human disease. Annals of surgery 247: 860-868, 2008. 54. Jellinghaus S, Poitz DM, Ende G, Augstein A, Weinert S, Stutz B, Braun-Dullaeus RC, Pasquale EB, and Strasser RH. Ephrin-A1/EphA4-mediated adhesion of monocytes to endothelial cells. Biochimica et biophysica acta 1833: 2201-2211, 2013. 55. Kar G, Gursoy A, and Keskin O. Human cancer protein-protein interaction network: a structural perspective. PLoS computational biology 5: e1000601, 2009. 56. Karagiannis ED, and Popel AS. A systematic methodology for proteome-wide identification of peptides inhibiting the proliferation and migration of endothelial cells. Proceedings of the National Academy of Sciences of the United States of America 105: 13775-13780, 2008. 57. Kikuchi R, Nakamura K, MacLauchlan S, Ngo DT, Shimizu I, Fuster JJ, Katanasaka Y, Yoshida S, Qiu Y, Yamaguchi TP, Matsushita T, Murohara T, Gokce N, Bates DO, Hamburg NM, and Walsh K. An antiangiogenic isoform of VEGF-A contributes to impaired vascularization in peripheral artery disease. Nature medicine 20: 1464-1471, 2014. 58. Kikuchi R, Nakamura K, MacLauchlan S, Ngo DT, Shimizu I, Fuster JJ, Katanasaka Y, Yoshida S, Qiu Y, Yamaguchi TP, Matsushita T, Murohara T, Gokce 80

N, Bates DO, Hamburg NM, and Walsh K. An antiangiogenic isoform of VEGF-A contributes to impaired vascularization in peripheral artery disease. Nature medicine 2014. 59. Knox C, Law V, Jewison T, Liu P, Ly S, Frolkis A, Pon A, Banco K, Mak C, Neveu V, Djoumbou Y, Eisner R, Guo AC, and Wishart DS. DrugBank 3.0: a comprehensive resource for 'omics' research on drugs. Nucleic acids research 39: D1035-1041, 2011. 60. Konopatskaya O, Churchill AJ, Harper SJ, Bates DO, and Gardiner TA. VEGF165b, an endogenous C-terminal splice variant of VEGF, inhibits retinal neovascularization in mice. Molecular vision 12: 626-632, 2006. 61. Kuehn H, Liberzon A, Reich M, and Mesirov JP. Using GenePattern for gene expression analysis. Current protocols in bioinformatics / editoral board, Andreas D Baxevanis [et al] Chapter 7: Unit 7 12, 2008. 62. Kuijper S, Turner CJ, and Adams RH. Regulation of angiogenesis by Eph-ephrin interactions. Trends in cardiovascular medicine 17: 145-151, 2007. 63. Mac Gabhann F, and Popel AS. Systems biology of vascular endothelial growth factors. Microcirculation 15: 715-738, 2008. 64. Mac Gabhann F, Qutub AA, Annex BH, and Popel AS. Systems biology of pro-angiogenic therapies targeting the VEGF system. Wiley interdisciplinary reviews Systems biology and medicine 2: 694-707, 2010. 65. Maere S, Heymans K, and Kuiper M. BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 21: 3448-3449, 2005. 66. Masud R, Shameer K, Dhar A, Ding K, and Kullo IJ. Gene expression profiling of peripheral blood mononuclear cells in the setting of peripheral arterial disease. Journal of clinical bioinformatics 2: 6, 2012. 67. Mellberg S, Dimberg A, Bahram F, Hayashi M, Rennel E, Ameur A, Westholm JO, Larsson E, Lindahl P, Cross MJ, and Claesson-Welsh L. Transcriptional profiling reveals a critical role for tyrosine phosphatase VE-PTP in regulation of VEGFR2 activity and endothelial cell morphogenesis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 23: 1490-1502, 2009. 68. Mu J, Brozinick JT, Jr., Valladares O, Bucan M, and Birnbaum MJ. A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle. Molecular cell 7: 1085-1094, 2001. 69. Pammolli F, Magazzini L, and Riccaboni M. The productivity crisis in pharmaceutical R&D. Nature reviews Drug discovery 10: 428-438, 2011. 81

70. Pasquale EB. Eph receptors and in cancer: bidirectional signalling and beyond. Nature reviews Cancer 10: 165-180, 2010. 71. Pecot CV, Rupaimoole R, Yang D, Akbani R, Ivan C, Lu C, Wu S, Han HD, Shah MY, Rodriguez-Aguayo C, Bottsford-Miller J, Liu Y, Kim SB, Unruh A, Gonzalez-Villasana V, Huang L, Zand B, Moreno-Smith M, Mangala LS, Taylor M, Dalton HJ, Sehgal V, Wen Y, Kang Y, Baggerly KA, Lee JS, Ram PT, Ravoori MK, Kundra V, Zhang X, Ali-Fehmi R, Gonzalez-Angulo AM, Massion PP, Calin GA, Lopez-Berestein G, Zhang W, and Sood AK. Tumour angiogenesis regulation by the miR-200 family. Nature communications 4: 2427, 2013. 72. Rivera CG, Bader JS, and Popel AS. Angiogenesis-associated crosstalk between collagens, CXC chemokines, and thrombospondin domain-containing proteins. Annals of biomedical engineering 39: 2213-2222, 2011. 73. Rivera CG, Chu LH, Bader JS, and Popel AS. Applications of network bioinformatics to cancer angiogenesis. In: Systems Biology in Cancer Research and Drug Discovery, edited by Azmi ASSpringer Science+Business Media, Dordrecht 2012, p. 229-244. 74. Rivera CG, Mellberg S, Claesson-Welsh L, Bader JS, and Popel AS. Analysis of VEGF--a regulated gene expression in endothelial cells to identify genes linked to angiogenesis. PloS one 6: e24887, 2011. 75. Rosca EV, Koskimaki JE, Rivera CG, Pandey NB, Tamiz AP, and Popel AS. Anti-angiogenic peptides for cancer therapeutics. Current pharmaceutical biotechnology 12: 1101-1116, 2011. 76. Ryu J, Lee CW, Hong KH, Shin JA, Lim SH, Park CS, Shim J, Nam KB, Choi KJ, Kim YH, and Han KH. Activation of fractalkine/CX3CR1 by vascular endothelial cells induces angiogenesis through VEGF-A/KDR and reverses hindlimb ischaemia. Cardiovascular research 78: 333-340, 2008. 77. Safran M, Dalah I, Alexander J, Rosen N, Iny Stein T, Shmoish M, Nativ N, Bahir I, Doniger T, Krug H, Sirota-Madi A, Olender T, Golan Y, Stelzer G, Harel A, and Lancet D. GeneCards Version 3: the human gene integrator. Database : the journal of biological databases and curation 2010: baq020, 2010. 78. Schaper W, and Scholz D. Factors regulating arteriogenesis. Arteriosclerosis, thrombosis, and vascular biology 23: 1143-1151, 2003. 79. Schweighofer B, Testori J, Sturtzel C, Sattler S, Mayer H, Wagner O, Bilban M, and Hofer E. The VEGF-induced transcriptional response comprises gene clusters at the crossroad of angiogenesis and inflammation. Thrombosis and haemostasis 102: 544-554, 2009. 82

80. Sealock R, Zhang H, Lucitti JL, Moore SM, and Faber JE. Congenic fine-mapping identifies a major causal locus for variation in the native collateral circulation and ischemic injury in brain and lower extremity. Circulation research 114: 660-671, 2014. 81. Sharan S, and Woo S. Systems pharmacology approaches for optimization of antiangiogenic therapies: challenges and opportunities. Front Pharmacol 6: 1-7, 2015. 82. Sillen A, Brohede J, Lilius L, Forsell C, Andrade J, Odeberg J, Ebise H, Winblad B, and Graff C. Linkage to 20p13 including the ANGPT4 gene in families with mixed Alzheimer's disease and vascular dementia. Journal of human genetics 55: 649-655, 2010. 83. Smadja DM, d'Audigier C, Bieche I, Evrard S, Mauge L, Dias JV, Labreuche J, Laurendeau I, Marsac B, Dizier B, Wagner-Ballon O, Boisson-Vidal C, Morandi V, Duong-Van-Huyen JP, Bruneval P, Dignat-George F, Emmerich J, and Gaussem P. Thrombospondin-1 is a plasmatic marker of peripheral arterial disease that modulates endothelial progenitor cell angiogenic properties. Arteriosclerosis, thrombosis, and vascular biology 31: 551-559, 2011. 84. Smoot ME, Ono K, Ruscheinski J, Wang PL, and Ideker T. Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics 27: 431-432, 2011. 85. Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Statistical applications in genetics and molecular biology 3: Article3, 2004. 86. Stefanini MO, Wu FT, Mac Gabhann F, and Popel AS. A compartment model of VEGF distribution in blood, healthy and diseased tissues. BMC systems biology 2: 77, 2008. 87. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, and Mesirov JP. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proceedings of the National Academy of Sciences of the United States of America 102: 15545-15550, 2005. 88. Sun J, Wu Y, Xu H, and Zhao Z. DTome: a web-based tool for drug-target interactome construction. BMC bioinformatics 13 Suppl 9: S7, 2012. 89. Suzuki Y, Hattori K, Hamanaka J, Murase T, Egashira Y, Mishiro K, Ishiguro M, Tsuruma K, Hirose Y, Tanaka H, Yoshimura S, Shimazawa M, Inagaki N, Nagasawa H, Iwama T, and Hara H. Pharmacological inhibition of TLR4-NOX4 signal protects against neuronal death in transient focal ischemia. Scientific reports 2: 896, 2012. 83

90. Takahashi H, and Shibuya M. The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions. Clinical science 109: 227-241, 2005. 91. Tarcea VG, Weymouth T, Ade A, Bookvich A, Gao J, Mahavisno V, Wright Z, Chapman A, Jayapandian M, Ozgur A, Tian Y, Cavalcoli J, Mirel B, Patel J, Radev D, Athey B, States D, and Jagadish HV. Michigan molecular interactions r2: from interacting proteins to pathways. Nucleic acids research 37: D642-646, 2009. 92. Tolsma SS, Volpert OV, Good DJ, Frazier WA, Polverini PJ, and Bouck N. Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity. The Journal of cell biology 122: 497-511, 1993. 93. Varey AH, Rennel ES, Qiu Y, Bevan HS, Perrin RM, Raffy S, Dixon AR, Paraskeva C, Zaccheo O, Hassan AB, Harper SJ, and Bates DO. VEGF 165 b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy. Br J Cancer 98: 1366-1379, 2008. 94. Whirl-Carrillo M, McDonagh EM, Hebert JM, Gong L, Sangkuhl K, Thorn CF, Altman RB, and Klein TE. Pharmacogenomics knowledge for personalized medicine. Clinical pharmacology and therapeutics 92: 414-417, 2012. 95. Woolard J, Wang WY, Bevan HS, Qiu Y, Morbidelli L, Pritchard-Jones RO, Cui TG, Sugiono M, Waine E, Perrin R, Foster R, Digby-Bell J, Shields JD, Whittles CE, Mushens RE, Gillatt DA, Ziche M, Harper SJ, and Bates DO. VEGF165b, an inhibitory vascular endothelial growth factor splice variant: mechanism of action, in vivo effect on angiogenesis and endogenous protein expression. Cancer research 64: 7822-7835, 2004. 96. Wu FT, Stefanini MO, Mac Gabhann F, Kontos CD, Annex BH, and Popel AS. VEGF and soluble VEGF receptor-1 (sFlt-1) distributions in peripheral arterial disease: an in silico model. American journal of physiology Heart and circulatory physiology 298: H2174-2191, 2010. 97. Wu FT, Stefanini MO, Mac Gabhann F, and Popel AS. A compartment model of VEGF distribution in in the presence of soluble VEGF receptor-1 acting as a ligand trap. PloS one 4: e5108, 2009. 98. Yang L, and Agarwal P. Systematic drug repositioning based on clinical side-effects. PloS one 6: e28025, 2011. 99. Yildirim MA, Goh KI, Cusick ME, Barabasi AL, and Vidal M. Drug-target network. Nature biotechnology 25: 1119-1126, 2007. 100. Zachman AL, Wang X, Tucker-Schwartz JM, Fitzpatrick ST, Lee SH, Guelcher 84

SA, Skala MC, and Sung HJ. Uncoupling angiogenesis and inflammation in peripheral artery disease with therapeutic -loaded microgels. Biomaterials 35: 9635-9648, 2014. 101. Zhang M, Zhu C, Jacomy A, Lu LJ, and Jegga AG. The orphan disease networks. American journal of human genetics 88: 755-766, 2011.

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Appendices

Appendix A: 1,233 Proteins in the angiome

Gene Name Description Degree JUN jun oncogene 123 SRC v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) 94 MYC v-myc myelocytomatosis viral oncogene homolog (avian) 90 GRB2 -bound protein 2 87 TP53 tumor protein 86 epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) EGFR oncogene homolog, avian) 80 EP300 E1A binding protein p300 79 PIK3R1 phosphoinositide-3-kinase, regulatory subunit 1 (alpha) 70 MAPK1 mitogen-activated protein kinase 1 69 CTNNB1 catenin (cadherin-associated protein), beta 1, 88kDa 69 SHC1 SHC (Src 2 domain containing) transforming protein 1 66 ESR1 1 64 STAT3 signal transducer and activator of transcription 3 (acute-phase response factor) 64 FYN FYN oncogene related to SRC, FGR, YES 60 FN1 fibronectin 1 59 CREBBP CREB binding protein 59 AKT1 v-akt murine thymoma viral oncogene homolog 1 58 RELA v-rel reticuloendotheliosis viral oncogene homolog A (avian) 57 protein tyrosine phosphatase, non-receptor type 11; similar to protein tyrosine PTPN11 phosphatase, non-receptor type 11 57 MAPK3 hypothetical LOC100271831; mitogen-activated protein kinase 3 55 RB1 retinoblastoma 1 54 SMAD3 SMAD family member 3 54 casein kinase 2, alpha 1 polypeptide ; casein kinase 2, alpha 1 CSNK2A1 polypeptide 54 PXN 51 TGFBR1 transforming growth factor, beta receptor 1 50 AR 50

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STAT1 signal transducer and activator of transcription 1, 91kDa 50 PRKCA protein kinase C, alpha 49 CASP3 3, apoptosis-related cysteine peptidase 48 CBL Cas-Br-M (murine) ecotropic retroviral transforming sequence 48 SMAD2 SMAD family member 2 47 90kDa alpha (cytosolic), class A member 2; heat shock HSP90AA1 protein 90kDa alpha (cytosolic), class A member 1 47 RAF1 v-raf-1 murine leukemia viral oncogene homolog 1 46 PTK2 PTK2 protein tyrosine kinase 2 46 SP1 46 lymphocyte-specific protein tyrosine kinase 45 PLCG1 C, gamma 1 45 HDAC1 histone deacetylase 1 44 ABL1 c- oncogene 1, 43 TNFRSF1A tumor necrosis factor receptor superfamily, member 1A 42 NFKB1 nuclear factor of kappa polypeptide gene enhancer in B-cells 1 42 CAV1 , caveolae protein, 22kDa 42 PRKCD protein kinase C, delta 41 ITGB3 integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) 41 TSC2 tuberous sclerosis 2 41 PLG plasminogen 40 JAK2 2 40 transcription factor 1 40 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, ERBB2 neuro/glioblastoma derived oncogene homolog (avian) 40 SMAD4 SMAD family member 4 40 FGF2 fibroblast growth factor 2 (basic) 40 TRAF2 TNF receptor-associated factor 2 39 MAPK8 mitogen-activated protein kinase 8 39 PRKACA protein kinase, cAMP-dependent, catalytic, alpha 39 PTPN6 protein tyrosine phosphatase, non-receptor type 6 38 CDKN1A cyclin-dependent kinase inhibitor 1A (, Cip1) 38 integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes ITGB1 MDF2, MSK12) 37 APP amyloid beta (A4) precursor protein 37 87

RASA1 RAS p21 protein activator (GTPase activating protein) 1 37 TGFB1 transforming growth factor, beta 1 36 NCOR2 nuclear receptor co-repressor 2 35 ITGAV integrin, alpha V ( receptor, alpha polypeptide, antigen CD51) 35 KDR kinase insert domain receptor (a type III receptor tyrosine kinase) 35 THBS1 thrombospondin 1 35 CASP8 , apoptosis-related cysteine peptidase 34 matrix metallopeptidase 2 ( A, 72kDa gelatinase, 72kDa type IV MMP2 ) 34 HRAS v-Ha-ras Harvey rat sarcoma viral oncogene homolog 34 FGFR3 fibroblast growth factor receptor 3 34 SYK spleen tyrosine kinase 33 CHUK conserved helix-loop-helix ubiquitous kinase 33 TRAF6 TNF receptor-associated factor 6 33 INSR 32 PDGFRB platelet-derived growth factor receptor, beta polypeptide 32 GSK3B kinase 3 beta 32 F2 coagulation factor II () 32 BCL2 B-cell CLL/lymphoma 2 31 ras-related C3 substrate 1 (rho family, small GTP binding RAC1 protein Rac1) 31 PTK2B PTK2B protein tyrosine kinase 2 beta 31 GNB2L1 guanine nucleotide binding protein (), beta polypeptide 2-like 1 31 JAK1 31 FGFR1 fibroblast growth factor receptor 1 31 matrix metallopeptidase 9 (, 92kDa gelatinase, 92kDa type IV MMP9 collagenase) 30 FGF1 fibroblast growth factor 1 (acidic) 30 MAPK14 mitogen-activated protein kinase 14 30 hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription HIF1A factor) 30 IKBKB inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta 30 NR3C1 nuclear receptor subfamily 3, group C, member 1 () 29 cell division cycle 42 (GTP binding protein, 25kDa); cell division cycle 42 CDC42 pseudogene 2 29 88

EZR hypothetical protein LOC100129652; 29 PML promyelocytic leukemia; similar to promyelocytic leukemia 1 29 CDK2 cyclin-dependent kinase 2 29 CEBPB CCAAT/enhancer binding protein (C/EBP), beta 28 FOS v-fos FBJ murine osteosarcoma viral oncogene homolog 28 IKBKG inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma 28 VCL 28 CASP7 , apoptosis-related cysteine peptidase 27 FGFR2 fibroblast growth factor receptor 2 27 PSEN1 1 27 CCND1 27 ERBB3 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) 27 FGFR4 fibroblast growth factor receptor 4 26 CDH1 cadherin 1, type 1, E-cadherin (epithelial) 26 MYOD1 myogenic differentiation 1 26 RHOA ras homolog gene family, member A 25 VTN vitronectin 25 FAS Fas (TNF receptor superfamily, member 6) 25 nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, NFKBIA alpha 25 PTPN1 protein tyrosine phosphatase, non-receptor type 1 24 IGF1R insulin-like growth factor 1 receptor 24 FRS2 fibroblast growth factor receptor substrate 2 23 NTRK1 neurotrophic tyrosine kinase, receptor, type 1 23 IGFBP3 insulin-like growth factor binding protein 3 23 MDM2 Mdm2 p53 binding protein homolog (mouse) 23 FGF9 fibroblast growth factor 9 (glia-activating factor) 22 PCNA proliferating cell nuclear antigen 22 hypothetical gene supported by AF216292; NM_005347; heat shock 70kDa HSPA5 protein 5 (glucose-regulated protein, 78kDa) 22 FGF4 fibroblast growth factor 4 22 FASLG Fas ligand (TNF superfamily, member 6) 22 ADAM10 ADAM metallopeptidase domain 10 22 CDKN1B cyclin-dependent kinase inhibitor 1B (p27, Kip1) 22

89

FRS3 fibroblast growth factor receptor substrate 3 22 MAP3K1 mitogen-activated protein kinase kinase kinase 1 22 FGF6 fibroblast growth factor 6 22 NOTCH2 Notch homolog 2 (Drosophila) 22 TGFBR2 transforming growth factor, beta receptor II (70/80kDa) 22 TP73 tumor protein 21 MET met proto-oncogene ( receptor) 21 PLAT , tissue 21 ZBTB16 finger and BTB domain containing 16 21 PLAUR plasminogen activator, 21 ITGA5 integrin, alpha 5 (fibronectin receptor, alpha polypeptide) 21 JAG1 jagged 1 (Alagille syndrome) 21 CD44 CD44 molecule (Indian blood group) 20 FGF18 fibroblast growth factor 18 20 NOTCH1 Notch homolog 1, translocation-associated (Drosophila) 20 FGF5 fibroblast growth factor 5 20 HSPG2 heparan sulfate proteoglycan 2 20 (c-fos serum response element-binding transcription SRF factor) 20 PPP2CA 2 (formerly 2A), catalytic subunit, alpha isoform 20 DCN 20 similar to Mast/stem factor receptor precursor (SCFR) (Proto-oncogene tyrosine-protein kinase Kit) (c-) (CD117 antigen); v-kit KIT Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog 19 HMGA1 hypothetical LOC100130009; high mobility group AT-hook 1 19 NOTCH4 Notch homolog 4 (Drosophila) 19 SWI/SNF related, matrix associated, dependent regulator of chromatin, SMARCA4 subfamily a, member 4 19 tubulin, beta; similar to tubulin, beta 5; tubulin, beta pseudogene 2; tubulin, TUBB beta pseudogene 1 19 fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular FLT1 permeability factor receptor) 19 FGF23 fibroblast growth factor 23 19 SPP1 secreted phosphoprotein 1 19 MED1 mediator complex subunit 1 19 90

NRP1 neuropilin 1 19 PIN1 peptidylprolyl cis/trans , NIMA-interacting 1 19 PSEN2 presenilin 2 (Alzheimer disease 4) 19 ERBB4 v-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian) 19 IGF1 insulin-like growth factor 1 ( C) 19 PLAU plasminogen activator, urokinase 19 VEGFA vascular endothelial growth factor A 19 PECAM1 platelet/endothelial cell adhesion molecule 19 MAP3K7 mitogen-activated protein kinase kinase kinase 7 19 EPOR receptor 19 FGF8 fibroblast growth factor 8 (androgen-induced) 18 colony stimulating factor 2 receptor, beta, low-affinity CSF2RB (granulocyte-macrophage) 18 CDC25A cell division cycle 25 homolog A (S. pombe) 18 NOTCH3 Notch homolog 3 (Drosophila) 18 PSENEN presenilin enhancer 2 homolog (C. elegans) 18 FGF20 fibroblast growth factor 20 18 RHOD ras homolog gene family, member D 18 hypothetical LOC100132771; fibroblast growth factor 7 (keratinocyte growth FGF7 factor); fibroblast growth factor 7 pseudogene 2 18 FGF17 fibroblast growth factor 17 18 ZAP70 zeta-chain (TCR) associated protein kinase 70kDa 18 PARP1 poly (ADP-ribose) polymerase 1 17 VAV3 vav 3 guanine nucleotide exchange factor 17 TNF tumor necrosis factor (TNF superfamily, member 2) 17 MMP14 matrix metallopeptidase 14 (membrane-inserted) 17 FGF16 fibroblast growth factor 16 17 CTSG 17 CDH5 cadherin 5, type 2 (vascular ) 17 RAP1A RAP1A, member of RAS oncogene family 17 EPHB2 EPH receptor B2 17 CDK5 cyclin-dependent kinase 5 17 CDK4 cyclin-dependent kinase 4 17 FBLN2 fibulin 2 17

91

CCNA2 17 HMGB1 high-mobility group box 1; high-mobility group box 1-like 10 17 MAP2K1 mitogen-activated protein kinase kinase 1 17 COL4A3 collagen, type IV, alpha 3 (Goodpasture antigen) 17 CASP9 caspase 9, apoptosis-related cysteine peptidase 16 MMP3 matrix metallopeptidase 3 (stromelysin 1, progelatinase) 16 MMP7 matrix metallopeptidase 7 (matrilysin, uterine) 16 CALR 16 RET ret proto-oncogene 16 MUC1 mucin 1, cell surface associated 16 IL6ST signal transducer (gp130, receptor) 16 PDGFRA platelet-derived growth factor receptor, alpha polypeptide 16 JUP junction 16 HDAC4 histone deacetylase 4 16 COL1A2 collagen, type I, alpha 2 16 VCAN 16 EPHA2 EPH receptor A2 16 VAV2 vav 2 guanine nucleotide exchange factor 16 ID3 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein 16 ITGB4 integrin, beta 4 15 COL4A2 collagen, type IV, alpha 2 15 TEK TEK tyrosine kinase, endothelial 15 FGF19 fibroblast growth factor 19 15 KNG1 15 TNFRSF1B tumor necrosis factor receptor superfamily, member 1B 15 PDPK1 3-phosphoinositide dependent protein kinase-1 15 BCL2L1 BCL2-like 1 15 MBP basic protein 15 DLL4 delta-like 4 (Drosophila) 15 TGFB2 transforming growth factor, beta 2 15 SDC2 2 15 FOXO3 forkhead box O3; forkhead box O3B pseudogene 15 CREB1 cAMP responsive element binding protein 1 15 EWSR1 similar to Ewing sarcoma breakpoint region 1; Ewing sarcoma breakpoint 15

92

region 1 NCL nucleolin 15 NRAS RAS viral (v-ras) oncogene homolog 15 DLL1 delta-like 1 (Drosophila) 15 TYK2 tyrosine kinase 2 15 MMP1 matrix metallopeptidase 1 () 15 RBL1 retinoblastoma-like 1 (p107) 15 COL1A1 collagen, type I, alpha 1 15 TRAF1 TNF receptor-associated factor 1 15 CCL5 chemokine (C-C motif) ligand 5 15 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type SERPINE1 1), member 1 14 MAPK9 mitogen-activated protein kinase 9 14 NGF (beta polypeptide) 14 PIK3CA phosphoinositide-3-kinase, catalytic, alpha polypeptide 14 LRP1 low density lipoprotein-related protein 1 (alpha-2-macroglobulin receptor) 14 THBD 14 GAB1 GRB2-associated binding protein 1 14 ETS1 v-ets erythroblastosis virus E26 oncogene homolog 1 (avian) 14 CCL7 chemokine (C-C motif) ligand 7 14 RHOC ras homolog gene family, member C 14 CTSB 14 RARA receptor, alpha 14 fibroblast growth factor 3 (murine mammary tumor virus integration site FGF3 (v-int-2) oncogene homolog) 14 ITGA6 integrin, alpha 6 14 ITGB2 integrin, beta 2 ( receptor 3 and 4 subunit) 14 similar to -protein kinase ATM (Ataxia telangiectasia mutated) (A-T, ATM mutated); ataxia telangiectasia mutated 14 insulin-like growth factor 2 (somatomedin A); insulin; INS-IGF2 readthrough INS transcript 14 glyceraldehyde-3-phosphate dehydrogenase-like 6; hypothetical protein GAPDH LOC100133042; glyceraldehyde-3-phosphate dehydrogenase 14 F10 coagulation 14 IL8 14 93

PKD1 polycystic kidney disease 1 (autosomal dominant) 14 phosphatase and tensin homolog; phosphatase and tensin homolog PTEN pseudogene 1 14 FOXO1 forkhead box O1 14 CCR3 chemokine (C-C motif) receptor 3 13 BIRC3 baculoviral IAP repeat-containing 3 13 CDK6 cyclin-dependent kinase 6 13 RUNX2 runt-related transcription factor 2 13 SDC1 syndecan 1 13 CDKN2A cyclin-dependent kinase inhibitor 2A (melanoma, , inhibits CDK4) 13 CD36 CD36 molecule (thrombospondin receptor) 13 similar to Rho-associated, coiled-coil containing protein kinase 1; ROCK1 Rho-associated, coiled-coil containing protein kinase 1 13 TXN 13 homeodomain interacting protein kinase 2; similar to homeodomain HIPK2 interacting protein kinase 2 13 KRT8 pseudogene 9; similar to keratin 8; keratin 8 13 KRT18 ; keratin 18 pseudogene 26; keratin 18 pseudogene 19 13 FGF10 fibroblast growth factor 10 13 MSN 13 ANXA2 pseudogene 3; annexin A2; annexin A2 pseudogene 1 13 MST1R macrophage stimulating 1 receptor (c-met-related tyrosine kinase) 13 ESR2 estrogen receptor 2 (ER beta) 13 CDC6 cell division cycle 6 homolog (S. cerevisiae) 13 YES1 v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 13 HGF hepatocyte growth factor (hepapoietin A; scatter factor) 13 GNA13 guanine nucleotide binding protein (G protein), alpha 13 13 BRAF v-raf murine sarcoma viral oncogene homolog B1 13 VHL von Hippel-Lindau tumor suppressor 13 ENG 13 CDC25B cell division cycle 25 homolog B (S. pombe) 13 COL4A1 collagen, type IV, alpha 1 13 TNFSF11 tumor necrosis factor (ligand) superfamily, member 11 13 KRAS v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog 13 RHOB ras homolog gene family, member B 13 94

MEF2A myocyte enhancer factor 2A 13 ACVR2A activin A receptor, type IIA 12 ILK integrin-linked kinase 12 AGTR1 II receptor, type 1 12 JUND jun D proto-oncogene 12 AKT2 v-akt murine thymoma viral oncogene homolog 2 12 F5 coagulation (proaccelerin, labile factor) 12 NGFR nerve growth factor receptor (TNFR superfamily, member 16) 12 PPARG peroxisome proliferator-activated receptor gamma 12 MEF2C myocyte enhancer factor 2C 12 BMX BMX non-receptor tyrosine kinase 12 A2M alpha-2-macroglobulin 12 UBC 12 CTSD 12 KLK3 kallikrein-related peptidase 3 12 CFLAR CASP8 and FADD-like apoptosis regulator 12 CXCR4 chemokine (C-X-C motif) receptor 4 12 RPS6KB1 ribosomal protein S6 kinase, 70kDa, polypeptide 1 12 TBK1 TANK-binding kinase 1 12 SPARC secreted protein, acidic, cysteine-rich () 12 TGFB3 transforming growth factor, beta 3 12 ID1 inhibitor of DNA binding 1, dominant negative helix-loop-helix protein 12 EFNB2 ephrin-B2 12 PRKD1 12 ACAN 12 MDK (neurite growth-promoting factor 2) 12 ALK anaplastic lymphoma receptor tyrosine kinase 12 NCSTN 12 IQGAP1 IQ motif containing GTPase activating protein 1 12 CD40 CD40 molecule, TNF receptor superfamily member 5 12 TGFA transforming growth factor, alpha 12 CCL2 chemokine (C-C motif) ligand 2 12 EGF epidermal growth factor (beta-urogastrone) 12 EFNA1 ephrin-A1 12

95

MYH9 myosin, heavy chain 9, non-muscle 12 VDR vitamin D (1,25- dihydroxyvitamin D3) receptor 11 BIRC2 baculoviral IAP repeat-containing 2 11 ARRB1 , beta 1 11 FGF22 fibroblast growth factor 22 11 YBX1 11 JAG2 jagged 2 11 ADAM15 ADAM metallopeptidase domain 15 11 COL4A4 collagen, type IV, alpha 4 11 CTNNA1 catenin (cadherin-associated protein), alpha 1, 102kDa 11 CCR1 chemokine (C-C motif) receptor 1 11 FGA alpha chain 11 RUNX1 runt-related transcription factor 1 11 NID1 nidogen 1 11 F8 coagulation factor VIII, procoagulant component 11 BMP2 bone morphogenetic protein 2 11 TLR3 toll-like receptor 3 11 EPHA4 EPH receptor A4 11 PPARA peroxisome proliferator-activated receptor alpha 11 CCR5 chemokine (C-C motif) receptor 5 11 PTN 11 TGFBR3 transforming growth factor, beta receptor III 11 ARHGAP24 Rho GTPase activating protein 24 11 PRTN3 11 ARHGAP22 Rho GTPase activating protein 22 11 HAND2 heart and neural crest derivatives expressed 2 11 CLU 11 PROC (inactivator of coagulation factors Va and VIIIa) 11 LAMA5 laminin, alpha 5 11 GTF2I general transcription factor II, i; general transcription factor II, i, pseudogene 11 BAX BCL2-associated X protein 11 GHR growth 11 NTRK2 neurotrophic tyrosine kinase, receptor, type 2 11 EGR1 early growth response 1 11

96

PBX1 pre-B-cell leukemia 1 10 MME membrane metallo- 10 GATA2 GATA binding protein 2 10 APH1A anterior pharynx defective 1 homolog A (C. elegans) 10 TNFSF10 tumor necrosis factor (ligand) superfamily, member 10 10 YY1 YY1 transcription factor 10 platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) PDGFB oncogene homolog) 10 ACVR1 activin A receptor, type I 10 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), SERPINA5 member 5 10 similar to Complement C3 precursor; complement component 3; hypothetical C3 protein LOC100133511 10 SDCBP syndecan binding protein (syntenin) 10 CTGF connective tissue growth factor 10 IGFBP5 insulin-like growth factor binding protein 5 10 APH1B anterior pharynx defective 1 homolog B (C. elegans) 10 CTSL1 cathepsin 10 F3 coagulation factor III (thromboplastin, ) 10 VWF 10 HAND1 heart and neural crest derivatives expressed 1 10 ITGA4 integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) 10 CCNE1 10 ARNT aryl hydrocarbon receptor nuclear translocator 10 PF4 10 CDC25C cell division cycle 25 homolog C (S. pombe) 10 TSHR stimulating hormone receptor 10 FLT4 fms-related tyrosine kinase 4 10 furin (paired basic cleaving enzyme) 10 BMPR2 bone morphogenetic protein receptor, type II (serine/ kinase) 10 HDAC5 10 catenin (cadherin-associated protein), delta 2 (neural plakophilin-related CTNND2 arm-repeat protein) 10 ITGB5 integrin, beta 5 10 ADAM17 ADAM metallopeptidase domain 17 10 97

SHB Src homology 2 domain containing adaptor protein B 10 CANX 10 similar to (Protein AF-6); myeloid/lymphoid or mixed-lineage MLLT4 leukemia (trithorax homolog, Drosophila); translocated to, 4 10 AHR aryl hydrocarbon receptor 9 PTPRB protein tyrosine phosphatase, receptor type, B 9 transcription factor 3 (E2A immunoglobulin enhancer binding factors TCF3 E12/E47) 9 LEP 9 MAPK7 mitogen-activated protein kinase 7 9 L1CAM L1 cell adhesion molecule 9 EIF4EBP1 eukaryotic translation initiation factor 4E binding protein 1 9 NFATC2 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 9 DNAJA3 DnaJ (Hsp40) homolog, subfamily A, member 3 9 GJA1 gap junction protein, alpha 1, 43kDa 9 MEF2D myocyte enhancer factor 2D 9 IGFBP7 insulin-like growth factor binding protein 7 9 BCAN 9 AXL AXL receptor tyrosine kinase 9 TNC C 9 CCBP2 chemokine binding protein 2 9 BIRC5 baculoviral IAP repeat-containing 5 9 ACP1 1, soluble 9 APOA1 apolipoprotein A-I 9 PRNP protein 9 FOXO4 forkhead box O4 9 TIMP3 TIMP metallopeptidase inhibitor 3 9 PPARGC1A peroxisome proliferator-activated receptor gamma, coactivator 1 alpha 9 -related (biliary CEACAM1 glycoprotein) 9 ARHGEF4 Rho guanine nucleotide exchange factor (GEF) 4 9 GIPC1 GIPC PDZ domain containing family, member 1 9 APEX1 APEX (multifunctional DNA repair enzyme) 1 9 DPP4 dipeptidyl-peptidase 4 9 LEF1 lymphoid enhancer-binding factor 1 9 98

FBLN1 fibulin 1 9 CDH2 cadherin 2, type 1, N-cadherin (neuronal) 9 EPHB1 EPH receptor B1 9 COL4A6 collagen, type IV, alpha 6 9 NOS3 nitric oxide synthase 3 (endothelial cell) 9 HRG -rich glycoprotein 9 CXCR3 chemokine (C-X-C motif) receptor 3 9 CCL8 chemokine (C-C motif) ligand 8 9 PDGFA platelet-derived growth factor alpha polypeptide 8 CSK c-src tyrosine kinase 8 SLC2A4 solute carrier family 2 (facilitated glucose transporter), member 4 8 TIMP1 TIMP metallopeptidase inhibitor 1 8 MEIS1 Meis homeobox 1 8 IL8RB #N/A 8 OSM oncostatin M 8 ITGA3 integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor) 8 TFPI tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) 8 GATA4 GATA binding protein 4 8 TGIF1 TGFB-induced factor homeobox 1 8 MGMT O-6-methylguanine-DNA methyltransferase 8 NR4A1 nuclear receptor subfamily 4, group A, member 1 8 IL8RA #N/A 8 PRKG1 protein kinase, cGMP-dependent, type I 8 EFNA5 ephrin-A5 8 COL4A5 collagen, type IV, alpha 5 8 CCR2 chemokine (C-C motif) receptor 2 8 TERT telomerase reverse transcriptase 8 pleckstrin homology domain containing, family G (with RhoGef domain) PLEKHG5 member 5 8 NFE2L2 nuclear factor (erythroid-derived 2)-like 2 8 FGG 8 WT1 Wilms tumor 1 8 PREX1 phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1 8 TFAP2A transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha) 8

99

FES 8 MATN2 matrilin 2 8 MAP2K4 mitogen-activated protein kinase kinase 4 8 TGFBI transforming growth factor, beta-induced, 68kDa 8 F2R coagulation factor II (thrombin) receptor 8 CCL11 chemokine (C-C motif) ligand 11 8 CCL13 chemokine (C-C motif) ligand 13 8 STK11 serine/threonine kinase 11 8 CD47 CD47 molecule 8 APOE hypothetical LOC100129500; 8 TK1 , soluble 8 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), SERPINA1 member 1 8 NRP2 neuropilin 2 8 TSC1 tuberous sclerosis 1 8 TRAF3 TNF receptor-associated factor 3 8 insulin-like growth factor 2 (somatomedin A); insulin; INS-IGF2 readthrough IGF2 transcript 8 CXCL10 chemokine (C-X-C motif) ligand 10 8 MCL1 myeloid cell leukemia sequence 1 (BCL2-related) 8 eukaryotic translation initiation factor 4E; similar to hCG1777996; similar to EIF4E eukaryotic translation initiation factor 4E 7 ITGA2 integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor) 7 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type SERPINE2 1), member 2 7 RAN, member RAS oncogene family 7 THBS2 thrombospondin 2 7 HBEGF heparin-binding EGF-like growth factor 7 IL6R interleukin 6 receptor 7 CCL3 chemokine (C-C motif) ligand 3 7 NOS1 nitric oxide synthase 1 (neuronal) 7 ING1 inhibitor of growth family, member 1 7 HAPLN1 hyaluronan and proteoglycan link protein 1 7 COX5A cytochrome c oxidase subunit Va 7 FGFBP1 fibroblast growth factor binding protein 1 7 100

DARC Duffy blood group, chemokine receptor 7 RRAS related RAS viral (r-ras) oncogene homolog 7 SPHK1 7 TNFAIP3 tumor necrosis factor, alpha-induced protein 3 7 COL18A1 collagen, type XVIII, alpha 1 7 F2RL1 coagulation factor II (thrombin) receptor-like 1 7 CXCL12 chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) 7 INHBA inhibin, beta A 7 TLR2 toll-like receptor 2 7 PIK3CB phosphoinositide-3-kinase, catalytic, beta polypeptide 7 COL7A1 collagen, type VII, alpha 1 7 HOXA9 homeobox A9 7 ELN 7 CCRL1 chemokine (C-C motif) receptor-like 1 7 PIK3CG phosphoinositide-3-kinase, catalytic, gamma polypeptide 7 F12 coagulation factor XII (Hageman factor) 7 PPARD peroxisome proliferator-activated receptor delta 7 CBX5 chromobox homolog 5 (HP1 alpha homolog, Drosophila) 7 activating transcription factor 4 (tax-responsive enhancer element B67); ATF4 activating transcription factor 4C 7 ICAM1 intercellular adhesion molecule 1 7 KLF5 Kruppel-like factor 5 (intestinal) 7 AGT angiotensinogen (serpin peptidase inhibitor, clade A, member 8) 7 HSPB1 heat shock 27kDa protein-like 2 pseudogene; heat shock 27kDa protein 1 7 LEPR 7 MYOC , trabecular meshwork inducible glucocorticoid response 7 ID2 inhibitor of DNA binding 2, dominant negative helix-loop-helix protein 6 DUSP1 dual specificity phosphatase 1 6 EPHA5 EPH receptor A5 6 LIMK1 LIM domain kinase 1 6 IL1B interleukin 1, beta 6 SERPINB13 serpin peptidase inhibitor, clade B (), member 13 6 DOK4 docking protein 4 6 ATP5B ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide 6

101

VCAM1 vascular cell adhesion molecule 1 6 MMP13 matrix metallopeptidase 13 (collagenase 3) 6 MVP 6 BDKRB2 6 ACVRL1 activin A receptor type II-like 1 6 INHBB inhibin, beta B 6 IL6 interleukin 6 (, beta 2) 6 protein kinase CHK2-like; CHK2 checkpoint homolog (S. pombe); similar to CHEK2 hCG1983233 6 NRG1 1 6 PPBP pro-platelet basic protein (chemokine (C-X-C motif) ligand 7) 6 VEGFB vascular endothelial growth factor B 6 NCAM1 neural cell adhesion molecule 1 6 IRF1 interferon regulatory factor 1 6 SH2D2A SH2 domain protein 2A 6 PLXNA1 6 LAMC1 laminin, gamma 1 (formerly LAMB2) 6 HDAC9 histone deacetylase 9 6 PRKAA2 protein kinase, AMP-activated, alpha 2 catalytic subunit 6 TAL1 T-cell acute lymphocytic leukemia 1 6 ITGB6 integrin, beta 6 6 LAMA4 laminin, alpha 4 6 KLKB1 kallikrein B, plasma (Fletcher factor) 1 6 TIMP2 TIMP metallopeptidase inhibitor 2 6 SIRT1 (silent mating type information regulation 2 homolog) 1 (S. cerevisiae) 6 SERPINC1 serpin peptidase inhibitor, clade C (), member 1 6 ERN1 endoplasmic reticulum to nucleus signaling 1 6 GNAS GNAS complex locus 6 TUBA1B hypothetical gene supported by AF081484; NM_006082; tubulin, alpha 1b 6 TNFRSF11 B tumor necrosis factor receptor superfamily, member 11b 6 RAD50 homolog (S. cerevisiae) 6 membrane associated guanylate kinase, WW and PDZ domain containing 1; MAGI1 CNKSR family member 3 6 LGALS1 lectin, galactoside-binding, soluble, 1 6 102

EIF4G1 eukaryotic translation initiation factor 4 gamma, 1 6 MMP8 matrix metallopeptidase 8 () 6 KISS1 KiSS-1 -suppressor 6 EFNA4 ephrin-A4 6 TOP1 topoisomerase (DNA) I 6 SLC2A1 solute carrier family 2 (facilitated glucose transporter), member 1 6 MAPK11 mitogen-activated protein kinase 11 6 SELE E 6 non-metastatic cells 1, protein (NM23A) expressed in; NME1-NME2 NME1 readthrough transcript; non-metastatic cells 2, protein (NM23B) expressed in 6 FGB 6 BSG (Ok blood group) 6 BMP4 bone morphogenetic protein 4 6 MAGI2 membrane associated guanylate kinase, WW and PDZ domain containing 2 6 LMO2 LIM domain only 2 (rhombotin-like 1) 6 CCL21 chemokine (C-C motif) ligand 21 6 EDG1 #N/A 5 IFNAR2 interferon (alpha, beta and omega) receptor 2 5 IL3 (colony-stimulating factor, multiple) 5 ESRRA estrogen-related receptor alpha 5 RRM2 M2 polypeptide 5 S100 calcium binding protein P 5 IL2 5 CSF2 colony stimulating factor 2 (granulocyte-macrophage) 5 SHC3 SHC (Src homology 2 domain containing) transforming protein 3 5 EPHB3 EPH receptor B3 5 PARK2 Parkinson disease (autosomal recessive, juvenile) 2, 5 GFAP glial fibrillary acidic protein 5 hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A HADHB thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), beta subunit 5 SHBG sex hormone-binding globulin 5 GRB14 growth factor receptor-bound protein 14 5 AGTR2 angiotensin II receptor, type 2 5 PTPRZ1 protein tyrosine phosphatase, receptor-type, Z polypeptide 1 5

103

ACTC1 actin, alpha, cardiac muscle 1 5 RGS2 regulator of G-protein signaling 2, 24kDa 5 EPHA3 EPH receptor A3 5 PAPPA antisense RNA (non-protein coding); pregnancy-associated plasma PAPPA protein A, pappalysin 1 5 APOH (beta-2-glycoprotein I) 5 SHC2 SHC (Src homology 2 domain containing) transforming protein 2 5 SDC3 syndecan 3 5 ACVR1B activin A receptor, type IB 5 PDE3B phosphodiesterase 3B, cGMP-inhibited 5 IRF3 interferon regulatory factor 3 5 SLPI secretory leukocyte peptidase inhibitor 5 PAX2 paired box 2 5 MLXIPL MLX interacting protein-like 5 FOSL1 FOS-like antigen 1 5 HABP2 hyaluronan binding protein 2 5 ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 motif, 1 5 CRYAB , alpha B 5 TNFRSF25 tumor necrosis factor receptor superfamily, member 25 5 ST14 suppression of tumorigenicity 14 (colon carcinoma) 5 S100 calcium binding protein A4 5 PTPN3 protein tyrosine phosphatase, non-receptor type 3 5 ELK3 ELK3, ETS-domain protein (SRF accessory protein 2) 5 RUNX3 runt-related transcription factor 3 5 TCF12 transcription factor 12 5 PGR 5 FIGF c-fos induced growth factor (vascular endothelial growth factor D) 5 LAMC2 laminin, gamma 2 5 SERPINB2 serpin peptidase inhibitor, clade B (ovalbumin), member 2 5 PZP pregnancy-zone protein 5 LPA lipoprotein, Lp(a) 5 CCR10 chemokine (C-C motif) receptor 10 5 EPAS1 endothelial PAS domain protein 1 5 BAI1 brain-specific 1 5

104

NR1H2 nuclear receptor subfamily 1, group H, member 2 5 HES1 hairy and enhancer of split 1, (Drosophila) 5 CD151 CD151 molecule (Raph blood group) 5 RUNX1T1 runt-related transcription factor 1; translocated to, 1 (cyclin D-related) 5 NR2F2 nuclear receptor subfamily 2, group F, member 2 5 METAP2 methionyl 2 5 NOV nephroblastoma overexpressed gene 5 NF1 5 CYBA cytochrome b-245, alpha polypeptide 5 MTA1 metastasis associated 1 5 SPTBN1 , beta, non-erythrocytic 1 5 prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and PTGS2 cyclooxygenase) 5 PIK3CD phosphoinositide-3-kinase, catalytic, delta polypeptide 5 MAPK10 mitogen-activated protein kinase 10 5 TCF4 transcription factor 4 5 TF 5 TNFRSF12 A tumor necrosis factor receptor superfamily, member 12A 5 PPAP2B phosphatidic acid phosphatase type 2B 5 CDCP1 CUB domain containing protein 1 5 SMAD6 SMAD family member 6 5 CXCL13 chemokine (C-X-C motif) ligand 13 5 GPI glucose phosphate isomerase 5 EFNA3 ephrin-A3 5 EFNB1 ephrin-B1 5 PRKAB1 protein kinase, AMP-activated, beta 1 non-catalytic subunit 5 IFNA1 interferon, alpha 1 5 CD55 molecule, decay accelerating factor for complement (Cromer blood CD55 group) 4 EPHA8 EPH receptor A8 4 DSP 4 S100 calcium binding protein A13 4 RARB , beta 4 EDN1 4 105

EFNA2 ephrin-A2 4 FOXM1 forkhead box M1 4 BDNF brain-derived neurotrophic factor 4 serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium SERPINF2 derived factor), member 2 4 PGF 4 ANG , , RNase A family, 5 4 ID4 inhibitor of DNA binding 4, dominant negative helix-loop-helix protein 4 HOXA5 homeobox A5 4 VASP vasodilator-stimulated phosphoprotein 4 sema domain, immunoglobulin domain (Ig), short basic domain, secreted, SEMA3F () 3F 4 ADCYAP1 adenylate cyclase activating polypeptide 1 (pituitary) 4 NCOA4 nuclear receptor coactivator 4 4 THY1 Thy-1 cell surface antigen 4 IFNAR1 interferon (alpha, beta and omega) receptor 1 4 CSTA (stefin A) 4 KRIT1 KRIT1, containing 4 ACHE (Yt blood group) 4 PPP2R5D , regulatory subunit B', delta isoform 4 EIF2AK3 eukaryotic translation initiation factor 2-alpha kinase 3 4 GATA3 GATA binding protein 3 4 DUSP3 dual specificity phosphatase 3 4 EDNRB endothelin receptor type B 4 EPHA6 EPH receptor A6 4 TCTEX1D4 Tctex1 domain containing 4 4 IL1RAP interleukin 1 receptor accessory protein 4 PLXNB1 plexin B1 4 HIF1AN hypoxia inducible factor 1, alpha subunit inhibitor 4 ITGA9 integrin, alpha 9 4 CCL4 chemokine (C-C motif) ligand 4 4 TNNI3 troponin I type 3 (cardiac) 4 LTBP3 latent transforming growth factor beta binding protein 3 4 H2AFX H2A histone family, member X 4

106

PROX1 prospero homeobox 1 4 ADAM9 ADAM metallopeptidase domain 9 (meltrin gamma) 4 WASF2 WAS , member 2 4 PHYHIP phytanoyl-CoA 2-hydroxylase interacting protein 4 ITGB8 integrin, beta 8 4 IL2RA interleukin 2 receptor, alpha 4 serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium SERPINF1 derived factor), member 1 4 SAA1 4 HEY1 hairy/enhancer-of-split related with YRPW motif 1 4 TWIST1 twist homolog 1 (Drosophila) 4 TFRC (p90, CD71) 4 CALCRL receptor-like 4 VEGFC vascular endothelial growth factor C 4 TNIP2 TNFAIP3 interacting protein 2 4 HGFAC HGF activator 4 CTSK 4 CPB2 B2 (plasma) 4 INHBC inhibin, beta C 4 ZEB1 E-box binding homeobox 1 4 IGSF1 immunoglobulin superfamily, member 1 4 NCF2 neutrophil cytosolic factor 2 4 EPHA7 EPH receptor A7 4 similar to Pyruvate kinase, isozymes M1/M2 (Pyruvate kinase muscle isozyme) (Cytosolic thyroid hormone-binding protein) (CTHBP) (THBP1); PKM2 pyruvate kinase, muscle 4 HHEX hematopoietically expressed homeobox 4 LGALS3 lectin, galactoside-binding, soluble, 3 4 BAIAP2 BAI1-associated protein 2 4 natriuretic peptide receptor A/ A (atrionatriuretic peptide NPR1 receptor A) 4 BTG1 B-cell translocation gene 1, anti-proliferative 4 BTC 4 ING4 inhibitor of growth family, member 4 4 KITLG KIT ligand 4 107

CXCL5 chemokine (C-X-C motif) ligand 5 4 SDC4 syndecan 4 4 P2RY2 purinergic receptor P2Y, G-protein coupled, 2 4 THBS3 4 COL8A1 collagen, type VIII, alpha 1 4 chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, CXCL1 alpha) 4 hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A HADHA thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alpha subunit 4 PAX6 paired box 6 4 FIBP fibroblast growth factor (acidic) intracellular binding protein 4 TG 4 SERPINB6 serpin peptidase inhibitor, clade B (ovalbumin), member 6 4 CYBB cytochrome b-245, beta polypeptide 4 CAPN2 calpain 2, (m/II) large subunit 4 EPO erythropoietin 4 SHH homolog (Drosophila) 3 PTPRM protein tyrosine phosphatase, receptor type, M 3 JAM3 junctional adhesion molecule 3 3 NUMB homolog (Drosophila) 3 LCN2 lipocalin 2 3 SPOCK1 sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1 3 CXCL6 chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2) 3 CNTN2 (axonal) 3 TIE1 tyrosine kinase with immunoglobulin-like and EGF-like domains 1 3 MFNG MFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase 3 TMED10 transmembrane emp24-like trafficking protein 10 () 3 NPPB natriuretic peptide precursor B 3 PPP2R4 protein phosphatase 2A activator, regulatory subunit 4 3 AKT3 v-akt murine thymoma viral oncogene homolog 3 (, gamma) 3 C5 3 NFATC4 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4 3 CHRNA7 (cholinergic receptor, nicotinic, alpha 7, 5-10) and FAM7A (family with sequence similarity 7A, exons A-E) fusion; cholinergic receptor, CHRNA7 nicotinic, alpha 7 3 108

BMP6 bone morphogenetic protein 6 3 OS9 osteosarcoma amplified 9, endoplasmic reticulum associated protein 3 VASN vasorin 3 CLEC3B C-type lectin domain family 3, member B 3 PRKD2 protein kinase D2 3 CHRDL2 chordin-like 2 3 EZH2 enhancer of zeste homolog 2 (Drosophila) 3 MYOG (myogenic factor 4) 3 ribosomal protein L29 pseudogene 9; ribosomal protein L29 pseudogene 12; ribosomal protein L29 pseudogene 11; ribosomal protein L29; ribosomal RPL29 protein L29 pseudogene 26 3 CD59 CD59 molecule, complement regulatory protein 3 PPP2R2A protein phosphatase 2 (formerly 2A), regulatory subunit B, alpha isoform 3 CYLD cylindromatosis (turban tumor syndrome) 3 VLDLR very low density lipoprotein receptor 3 CA9 carbonic anhydrase IX 3 ACACA acetyl-Coenzyme A carboxylase alpha 3 LAMB1 laminin, beta 1 3 KLF4 Kruppel-like factor 4 (gut) 3 SFRP2 secreted frizzled-related protein 2 3 TNIK TRAF2 and NCK interacting kinase 3 CXCL2 chemokine (C-X-C motif) ligand 2 3 PKD2 polycystic kidney disease 2 (autosomal dominant) 3 TNR tenascin R (restrictin, janusin) 3 MAGI3 membrane associated guanylate kinase, WW and PDZ domain containing 3 3 RASIP1 Ras interacting protein 1 3 hypothetical LOC100129733; hairy/enhancer-of-split related with YRPW HEY2 motif 2 3 LAMB3 laminin, beta 3 3 CXCL9 chemokine (C-X-C motif) ligand 9 3 sema domain, immunoglobulin domain (Ig), short basic domain, secreted, SEMA3B (semaphorin) 3B 3 MYF5 myogenic factor 5 3 ADM 3 EPRS glutamyl-prolyl-tRNA synthetase 3 109

LTBP1 latent transforming growth factor beta binding protein 1 3 KHSRP KH-type splicing regulatory protein 3 nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 NFKB2 (p49/p100) 3 MAML3 mastermind-like 3 (Drosophila) 3 EFNB3 ephrin-B3 3 IFNGR1 receptor 1 3 RASGRP3 RAS guanyl releasing protein 3 (calcium and DAG-regulated) 3 MMP12 matrix metallopeptidase 12 () 3 heat shock protein 90kDa alpha (cytosolic), class A member 2; heat shock HSP90AA2 protein 90kDa alpha (cytosolic), class A member 1 3 TNFSF12-TNFSF13 readthrough transcript; tumor necrosis factor (ligand) superfamily, member 12; tumor necrosis factor (ligand) superfamily, member TNFSF12 13 3 BACE1 beta-site APP-cleaving enzyme 1 3 CXCL11 chemokine (C-X-C motif) ligand 11 3 HMMR hyaluronan-mediated motility receptor (RHAMM) 3 ANXA5 3 IFNG interferon, gamma 3 TAC1 tachykinin, precursor 1 3 SAT1 spermidine/spermine N1-acetyltransferase 1 3 STK4 serine/threonine kinase 4 3 MMP10 matrix metallopeptidase 10 () 3 PRKAA1 protein kinase, AMP-activated, alpha 1 catalytic subunit 3 MMP17 matrix metallopeptidase 17 (membrane-inserted) 3 PLA2G2A , group IIA (, synovial fluid) 3 PTPN14 protein tyrosine phosphatase, non-receptor type 14 3 serpin peptidase inhibitor, clade H (), member 1, SERPINH1 (collagen binding protein 1) 3 HMOX1 heme oxygenase (decycling) 1 3 MIF macrophage migration inhibitory factor (glycosylation-inhibiting factor) 3 EGLN1 egl nine homolog 1 (C. elegans) 3 ANTXR2 anthrax toxin receptor 2 3 PLA2G4A phospholipase A2, group IVA (cytosolic, calcium-dependent) 3 RAPGEF2 Rap guanine nucleotide exchange factor (GEF) 2; similar to RAPGEF2 3 110

protein CALCA calcitonin-related polypeptide alpha 3 FLT3 fms-related tyrosine kinase 3 3 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal CITED4 domain, 4 3 PTCH1 patched homolog 1 (Drosophila) 3 FMOD 3 ACE2 angiotensin I converting enzyme (peptidyl- A) 2 3 TRPC6 transient receptor potential cation channel, subfamily C, member 6 3 PDAP1 PDGFA associated protein 1; similar to PDGFA associated protein 1 3 IGFALS insulin-like growth factor binding protein, acid labile subunit 3 NPPA natriuretic peptide precursor A 3 ELK1 ELK1, member of ETS oncogene family 3 EFEMP1 EGF-containing fibulin-like extracellular matrix protein 1 3 GATA6 GATA binding protein 6 3 TPMT thiopurine S-methyltransferase 3 KLK1 kallikrein 1 3 MAPK12 mitogen-activated protein kinase 12 3 RTN4 3 MEP1A , alpha (PABA peptide ) 3 CCR4 chemokine (C-C motif) receptor 4 3 MPL myeloproliferative leukemia virus oncogene 3 VKORC1 vitamin K epoxide reductase complex, subunit 1 3 MAML2 mastermind-like 2 (Drosophila) 3 TBXA2R thromboxane A2 receptor 3 ADCY6 adenylate cyclase 6 3 POFUT1 protein O- 1 3 MAML1 mastermind-like 1 (Drosophila) 3 MAPK13 mitogen-activated protein kinase 13 3 S100 calcium binding protein A7 3 ELK4 ELK4, ETS-domain protein (SRF accessory protein 1) 3 AREG ; amphiregulin B 3 GHRL /obestatin prepropeptide 3 ANGPT1 3

111

LTA (TNF superfamily, member 1) 3 TXNIP thioredoxin interacting protein 3 XBP1 X-box binding protein 1 3 BMPER BMP binding endothelial regulator 3 GUK1 guanylate kinase 1 3 ANGPTL3 angiopoietin-like 3 3 C6 3 CFP complement factor 2 CEACAM3 carcinoembryonic antigen-related cell adhesion molecule 3 2 IRF9 interferon regulatory factor 9 2 CHRD chordin 2 GRP -releasing peptide 2 CD97 CD97 molecule 2 IFNGR2 interferon gamma receptor 2 (interferon gamma transducer 1) 2 roundabout, axon guidance receptor, homolog 1 (Drosophila); similar to ROBO1 roundabout 1 isoform b 2 AMOTL2 like 2 2 CTNNBIP1 catenin, beta interacting protein 1 2 RAMP1 receptor (G protein-coupled) activity modifying protein 1 2 POMC 2 PIGF phosphatidylinositol glycan anchor , class F 2 ADAM23 ADAM metallopeptidase domain 23 2 TNXB tenascin XB; tenascin XA pseudogene 2 ETV6 ets variant 6 2 FST 2 BMP3 bone morphogenetic protein 3 2 GPC4 4 2 SSTR2 receptor 2 2 MMP25 matrix metallopeptidase 25 2 LAMB2 laminin, beta 2 (laminin S) 2 VIP vasoactive intestinal peptide 2 RHBDL2 rhomboid, veinlet-like 2 (Drosophila) 2 RLN1 1 2 DTX1 deltex homolog 1 (Drosophila) 2

112

IL13 2 EBAG9 estrogen receptor associated, antigen, 9 2 MAGED1 melanoma antigen family D, 1 2 CRP C-reactive protein, pentraxin-related 2 PTK6 PTK6 protein tyrosine kinase 6 2 AMOT angiomotin 2 NFATC3 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 3 2 PKP4 plakophilin 4 2 PHYH phytanoyl-CoA 2-hydroxylase 2 POU4F1 POU class 4 homeobox 1 2 PPYR1 pancreatic polypeptide receptor 1 2 BECN1 beclin 1, related 2 IL16 interleukin 16 (lymphocyte chemoattractant factor) 2 ECM1 extracellular matrix protein 1 2 HIF3A hypoxia inducible factor 3, alpha subunit 2 EPHB4 EPH receptor B4 2 CD40LG CD40 ligand 2 S100 calcium binding protein A2 2 CDKN3 cyclin-dependent kinase inhibitor 3 2 ACE angiotensin I converting enzyme (peptidyl-dipeptidase A) 1 2 IHH Indian hedgehog homolog (Drosophila) 2 ICAM4 intercellular adhesion molecule 4 (Landsteiner-Wiener blood group) 2 UCN 2 NRG2 2 LIMS1 LIM and senescent cell antigen-like domains 1 2 FZD5 frizzled homolog 5 (Drosophila) 2 COL6A3 collagen, type VI, alpha 3 2 IFNB1 interferon, beta 1, fibroblast 2 RCAN1 regulator of calcineurin 1 2 PTGER2 prostaglandin E receptor 2 (subtype EP2), 53kDa 2 IRF7 interferon regulatory factor 7 2 IL1R1 interleukin 1 receptor, type I 2 SCARB1 scavenger receptor class B, member 1 2 PTP4A3 protein tyrosine phosphatase type IVA, member 3 2

113

serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), SERPINA4 member 4 2 FAP fibroblast activation protein, alpha 2 RAMP2 receptor (G protein-coupled) activity modifying protein 2 2 IL1R2 interleukin 1 receptor, type II 2 ADORA2B hypothetical LOC100131909; 2 ITGB1BP1 binding protein 1 2 MKI67 antigen identified by monoclonal antibody Ki-67 2 GRN 2 TIMP4 TIMP metallopeptidase inhibitor 4 2 RNF130 ring finger protein 130 2 SLIT2 slit homolog 2 (Drosophila) 2 GJA5 gap junction protein, alpha 5, 40kDa 2 NRCAM neuronal cell adhesion molecule 2 TP63 tumor protein p63 2 MMP16 matrix metallopeptidase 16 (membrane-inserted) 2 CHAT choline acetyltransferase 2 AGER advanced glycosylation end product-specific receptor 2 CAST 2 EPHB6 EPH receptor B6 2 GUCY1B3 guanylate cyclase 1, soluble, beta 3 2 TARS threonyl-tRNA synthetase 2 LIMK2 LIM domain kinase 2 2 MCAM melanoma cell adhesion molecule 2 SOX18 SRY (sex determining region Y)-box 18 2 ALOX12 arachidonate 12-lipoxygenase 2 GRIP1 glutamate receptor interacting protein 1 2 HHIP hedgehog interacting protein 2 TENC1 tensin like C1 domain containing phosphatase (tensin 2) 2 TFPI2 tissue factor pathway inhibitor 2 2 C5AR1 complement component 5a receptor 1 2 SLC6A4 solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 2 GPC1 2 SFRP1 secreted frizzled-related protein 1 2

114

THBS4 thrombospondin 4 2 BMP1 bone morphogenetic protein 1 2 ERAP1 endoplasmic reticulum aminopeptidase 1 2 PTTG1 pituitary tumor-transforming 1; pituitary tumor-transforming 2 2 BMP7 bone morphogenetic protein 7 2 MKKS McKusick-Kaufman syndrome 2 CGA glycoprotein hormones, alpha polypeptide 2 CCL26 chemokine (C-C motif) ligand 26 2 GC group-specific component (vitamin D binding protein) 2 ODC1 ornithine decarboxylase 1 2 MMP26 matrix metallopeptidase 26 2 TNFSF15 tumor necrosis factor (ligand) superfamily, member 15 2 HMGCR 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 2 CSPG4 chondroitin sulfate 2 SUFU suppressor of fused homolog (Drosophila) 2 TNFAIP6 tumor necrosis factor, alpha-induced protein 6 2 GPLD1 glycosylphosphatidylinositol specific 2 IL4 2 KRT10 2 KRT1 2 HTR1D 5-hydroxytryptamine (serotonin) receptor 1D 2 CXCL3 chemokine (C-X-C motif) ligand 3 2 NUDT6 nudix (nucleoside diphosphate linked moiety X)-type motif 6 2 RHO 2 PKD2L1 polycystic kidney disease 2-like 1 2 ARNT2 aryl-hydrocarbon receptor nuclear translocator 2 2 GULP1 GULP, engulfment adaptor PTB domain containing 1 2 HOXD10 homeobox D10 2 TNNI2 troponin I type 2 (skeletal, fast) 2 EPHA1 EPH receptor A1 2 MED28 mediator complex subunit 28 2 HOXB3 homeobox B3 2 EREG 2 TKT transketolase 2

115

CCL15 chemokine (C-C motif) ligand 14; chemokine (C-C motif) ligand 15 2 SELP selectin P (granule 140kDa, antigen CD62) 2 PKNOX1 PBX/knotted 1 homeobox 1 2 HEYL hairy/enhancer-of-split related with YRPW motif-like 2 NAB2 NGFI-A binding protein 2 (EGR1 binding protein 2) 2 sema domain, immunoglobulin domain (Ig), short basic domain, secreted, SEMA3A (semaphorin) 3A 2 HTR1A 5-hydroxytryptamine (serotonin) receptor 1A 2 KCNK18 potassium channel, subfamily K, member 18 2 IFI16 interferon, gamma-inducible protein 16 2 GPX1 glutathione peroxidase 1 2 NPY 2 DKK1 dickkopf homolog 1 (Xenopus laevis) 2 PON1 1 2 CHRDL1 chordin-like 1 2 ANGPT2 angiopoietin 2 2 TNFAIP1 tumor necrosis factor, alpha-induced protein 1 (endothelial) 2 MYF6 myogenic factor 6 (herculin) 2 SERPINI1 serpin peptidase inhibitor, clade I (), member 1 2 XDH 2 TSSK1B testis-specific serine kinase 1B 2 TYMP thymidine 2 sema domain, immunoglobulin domain (Ig), short basic domain, secreted, SEMA3C (semaphorin) 3C 2 PRSS3 , serine, 3 2 CYR61 cysteine-rich, angiogenic inducer, 61 2 CSF1 colony stimulating factor 1 (macrophage) 2 natriuretic peptide receptor C/guanylate cyclase C (atrionatriuretic peptide NPR3 receptor C) 2 tumor necrosis factor receptor superfamily, member 6b, decoy; regulator of TNFRSF6B telomere elongation helicase 1 2 DPT 2 PSMA7 proteasome (prosome, macropain) subunit, alpha type, 7 2 POLL polymerase (DNA directed), lambda 2 CD38 CD38 molecule 2 116

MERTK c-mer proto-oncogene tyrosine kinase 2 MAP2K5 mitogen-activated protein kinase kinase 5 2 BRMS1 breast cancer metastasis suppressor 1 2 SMG1 SMG1 homolog, phosphatidylinositol 3-kinase-related kinase (C. elegans) 2 RAPGEF3 Rap guanine nucleotide exchange factor (GEF) 3 1 ENPEP (aminopeptidase A) 1 CDKL1 cyclin-dependent kinase-like 1 (CDC2-related kinase) 1 TACR1 1 LIMA1 LIM domain and actin binding 1 1 SMA4 glucuronidase, beta pseudogene 1 IPO11 11 1 SOX17 SRY (sex determining region Y)-box 17 1 RHBDF1 rhomboid 5 homolog 1 (Drosophila) 1 PDZRN3 PDZ domain containing ring finger 3 1 ECH1 enoyl Coenzyme A hydratase 1, peroxisomal 1 TYR -like (pseudogene); tyrosinase (oculocutaneous albinism IA) 1 SPHKAP SPHK1 interactor, AKAP domain containing 1 GRPR gastrin-releasing peptide receptor 1 DEFA1 , alpha 1 1 ANGPT4 angiopoietin 4 1 CHID1 chitinase domain containing 1 1 TPI1 TPI1 pseudogene; triosephosphate isomerase 1 1 TSSK4 testis-specific serine kinase 4 1 RPS6KC1 ribosomal protein S6 kinase, 52kDa, polypeptide 1 1 PLXND1 plexin D1 1 SLC2A3 solute carrier family 2 (facilitated glucose transporter), member 3 1 PTGFR (FP) 1 PTGIS prostaglandin I2 (prostacyclin) synthase 1 ESM1 endothelial cell-specific molecule 1 1 CENPB , 80kDa 1 PCOLCE procollagen C-endopeptidase enhancer 1 CDH17 cadherin 17, LI cadherin (-intestine) 1 NTS 1 NRARP NOTCH-regulated ankyrin repeat protein 1

117

GH1 1 UTS2R urotensin 2 receptor 1 ADIPOQ , C1Q and collagen domain containing 1 DICER1 1, ribonuclease type III 1 ZMIZ1 zinc finger, MIZ-type containing 1 1 ALOX5 arachidonate 5-lipoxygenase 1 FBLN5 fibulin 5 1 PTGES prostaglandin E synthase 1 TRPC4 transient receptor potential cation channel, subfamily C, member 4 1 MAZ MYC-associated zinc finger protein (purine-binding transcription factor) 1 SENP1 SUMO1/sentrin specific peptidase 1 1 GPSM3 G-protein signaling modulator 3 (AGS3-like, C. elegans) 1 GPC3 1 PHLPP #N/A 1 ANPEP alanyl (membrane) aminopeptidase 1 EDNRA endothelin receptor type A 1 SLC16A1 solute carrier family 16, member 1 (monocarboxylic acid transporter 1) 1 CX3CR1 chemokine (C-X3-C motif) receptor 1 1 CHGA (parathyroid secretory protein 1) 1 sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and SEMA4F short cytoplasmic domain, (semaphorin) 4F 1 COL8A2 collagen, type VIII, alpha 2 1 CRHR2 corticotropin releasing hormone receptor 2 1 SAA4 serum amyloid A4, constitutive 1 SSX4 synovial sarcoma, X breakpoint 4; synovial sarcoma, X breakpoint 4B 1 alpha-2-glycoprotein 1, zinc-binding pseudogene 1; alpha-2-glycoprotein 1, AZGP1 zinc-binding 1 MMP24 matrix metallopeptidase 24 (membrane-inserted) 1 BRCC3 BRCA1/BRCA2-containing complex, subunit 3 1 GJB2 gap junction protein, beta 2, 26kDa 1 GLUD1 glutamate dehydrogenase 1 1 TCL1B T-cell leukemia/lymphoma 1B 1 FBXO34 F-box protein 34 1 HIPK1 homeodomain interacting protein kinase 1 1

118

RECQL5 RecQ protein-like 5 1 FOXC1 1 DTX3 deltex homolog 3 (Drosophila) 1 FGFRL1 fibroblast growth factor receptor-like 1 1 BCAM basal cell adhesion molecule (Lutheran blood group) 1 PTGER3 prostaglandin E receptor 3 (subtype EP3) 1 LECT1 leukocyte cell derived chemotaxin 1 1 IL32 1 TM4SF1 transmembrane 4 L six family member 1 1 MUC16 mucin 16, cell surface associated 1 APOD 1 EDIL3 EGF-like repeats and discoidin I-like domains 3 1 FCN1 ficolin (collagen/fibrinogen domain containing) 1 1 CCDC88A coiled-coil domain containing 88A 1 IL1A interleukin 1, alpha 1 CCDC17 coiled-coil domain containing 17 1 SCARB2 scavenger receptor class B, member 2 1 SNCG synuclein, gamma (breast cancer-specific protein 1) 1 WNT5A wingless-type MMTV integration site family, member 5A 1 ARD1B #N/A 1 LDLR low density lipoprotein receptor 1 LAP3 leucine aminopeptidase 3 1 CTSH 1 DYSF , limb girdle muscular dystrophy 2B (autosomal recessive) 1 1-acylglycerol-3-phosphate O-acyltransferase 5 (lysophosphatidic acid AGPAT5 acyltransferase, epsilon) 1 CNR1 cannabinoid receptor 1 (brain) 1 IDI2 isopentenyl-diphosphate delta isomerase 2 1 FLJ23356 #N/A 1 STAB1 stabilin 1 1 KLHL1 kelch-like 1 (Drosophila) 1 TBXAS1 thromboxane A synthase 1 (platelet) 1 PIGA phosphatidylinositol glycan anchor biosynthesis, class A 1 NOX1 NADPH oxidase 1 1

119

DEPDC2 #N/A 1 SERPINB5 serpin peptidase inhibitor, clade B (ovalbumin), member 5 1 CORO1A coronin, actin binding protein, 1A 1 KLF2 Kruppel-like factor 2 (lung) 1 TCL6 T-cell leukemia/lymphoma 6 1 LEFTY2 left-right determination factor 2 1 MFGE8 milk fat globule-EGF factor 8 protein 1 CEACAM5 carcinoembryonic antigen-related cell adhesion molecule 5 1 MPP1 membrane protein, palmitoylated 1, 55kDa 1 PDIK1L PDLIM1 interacting kinase 1 like 1 paternally expressed 3; PEG3 antisense RNA (non-protein coding); zinc PEG3 finger, imprinted 2 1 SAMD8 domain containing 8 1 DMP1 dentin matrix acidic phosphoprotein 1 1 KRT20 1 LIF leukemia inhibitory factor (cholinergic differentiation factor) 1 PDGFC platelet derived growth factor C 1 HAS1 hyaluronan synthase 1 1 BAIAP3 BAI1-associated protein 3 1 ADORA1 1 MFAP2 microfibrillar-associated protein 2 1 chorionic gonadotropin, beta polypeptide 5; chorionic gonadotropin, beta CGB5 polypeptide; chorionic gonadotropin, beta polypeptide 8 1 PIWIL1 piwi-like 1 (Drosophila) 1 PTAFR platelet-activating factor receptor 1 ZC3H12A zinc finger CCCH-type containing 12A 1 CDH13 cadherin 13, H-cadherin (heart) 1 NTN1 1 1 TMPRSS6 transmembrane protease, serine 6 1 LFNG LFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase 1 C3AR1 complement component 3a receptor 1 1 CD177 CD177 molecule 1 phosphodiesterase 4A, cAMP-specific (phosphodiesterase E2 dunce homolog, PDE4A Drosophila) 1 HTATIP2 HIV-1 Tat interactive protein 2, 30kDa 1 120

ENPP2 ectonucleotide pyrophosphatase/ 1 transglutaminase 1 (K polypeptide epidermal type I, TGM1 protein--gamma-glutamyltransferase) 1 HBZ hemoglobin, zeta 1 USP21 ubiquitin specific peptidase 21 1 IL15 interleukin 15 1 HOXA7 homeobox A7 1 RSF1 remodeling and spacing factor 1 1 HPN hepsin 1 GUCY1A2 guanylate cyclase 1, soluble, alpha 2 1 RAP1B RAP1B, member of RAS oncogene family 1 interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte IL12B maturation factor 2, p40) 1 C13orf34 open reading frame 34 1 C20orf121 #N/A 1 ROBO4 roundabout homolog 4, magic roundabout (Drosophila) 1 C6orf47 open reading frame 47 1 ANGPTL1 angiopoietin-like 1 1 CX3CL1 chemokine (C-X3-C motif) ligand 1 1 SMG7 Smg-7 homolog, nonsense mediated mRNA decay factor (C. elegans) 1 UTS2 urotensin 2 1 MYO3A myosin IIIA 1 SRGN 1 PROCR protein C receptor, endothelial (EPCR) 1 FZD6 frizzled homolog 6 (Drosophila) 1 HSD11B1 hydroxysteroid (11-beta) dehydrogenase 1 1 ACVR1C activin A receptor, type IC 1 REN 1 AQP1 (Colton blood group) 1 LOX 1 CTSE 1 RLN2 relaxin 2 1 ferritin, heavy polypeptide 1; ferritin, heavy polypeptide-like 16; similar to FTH1 ferritin, heavy polypeptide 1; ferritin, heavy polypeptide-like 3 pseudogene 1 PLP1 1 121

PENK 1 interleukin 12A (natural killer cell stimulatory factor 1, cytotoxic lymphocyte IL12A maturation factor 1, p35) 1 PREB regulatory element binding 1 BTBD10 BTB (POZ) domain containing 10 1 DRD2 receptor D2 1 TUSC2 tumor suppressor candidate 2 1 CD34 CD34 molecule 1 HTR2B 5-hydroxytryptamine (serotonin) receptor 2B 1 IGFBP4 insulin-like growth factor binding protein 4 1 PTHLH -like hormone 1 ERG v-ets erythroblastosis virus E26 oncogene homolog (avian) 1 PDCD10 programmed cell death 10 1 SIGLEC6 sialic acid binding Ig-like lectin 6 1 MMP11 matrix metallopeptidase 11 (stromelysin 3) 1 MEOX2 mesenchyme homeobox 2 1 PSG2 pregnancy specific beta-1-glycoprotein 2 1 sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and SEMA4D short cytoplasmic domain, (semaphorin) 4D 1 PRDX5 peroxiredoxin 5 1 SPRY1 sprouty homolog 1, antagonist of FGF signaling (Drosophila) 1 DCX 1 NES 1 EGLN3 egl nine homolog 3 (C. elegans) 1 HOXB13 homeobox B13 1 IGFBP6 insulin-like growth factor binding protein 6 1 SV2A synaptic vesicle glycoprotein 2A 1 DCUN1D1 DCN1, defective in neddylation 1, domain containing 1 (S. cerevisiae) 1 IL1RN interleukin 1 receptor antagonist 1 NRXN1 1 1 P2RX4 purinergic receptor P2X, ligand-gated channel, 4 1 RNH1 ribonuclease/angiogenin inhibitor 1 1 NTN4 netrin 4 1 SPON1 , extracellular matrix protein 1

122

ANXA3 1 CR2 complement component (3d/Epstein Barr virus) receptor 2 1 PDGFD platelet derived growth factor D 1 KRT17 ; keratin 17 pseudogene 3 1 CPM 1 DEFA3 defensin, alpha 3, neutrophil-specific 1 FGF13 fibroblast growth factor 13 1 HK1 hexokinase 1 1 NRXN3 neurexin 3 1 DNTT deoxynucleotidyltransferase, terminal 1 AGGF1 angiogenic factor with G patch and FHA domains 1 1 VHLL von Hippel-Lindau tumor suppressor-like 1 WNT2 wingless-type MMTV integration site family member 2 1 ACR acrosin 1 HRH1 histamine receptor H1 1 IDE insulin-degrading enzyme 1 IL18 (interferon-gamma-inducing factor) 1 FOXP3 forkhead box P3 1 GCLC glutamate-cysteine ligase, catalytic subunit 1 OXT , prepropeptide 1 B4GALT1 UDP-Gal:betaGlcNAc beta 1,4- , polypeptide 1 1 HPSE2 2 1 CCL23 chemokine (C-C motif) ligand 23 1 USH2A Usher syndrome 2A (autosomal recessive, mild) 1 TACR3 1 KISS1R KISS1 receptor 1 KLK6 kallikrein-related peptidase 6 1 SST somatostatin 1 RRH retinal pigment epithelium-derived rhodopsin homolog 1 NDRG1 N-myc downstream regulated 1 1 stonin 1; STON1-GTF2A1L readthrough transcript; general transcription GTF2A1L factor IIA, 1-like 1 NUMBL numb homolog (Drosophila)-like 1 EGR3 early growth response 3 1

123

NKD2 naked cuticle homolog 2 (Drosophila) 1 GAL prepropeptide 1 ROCK2 Rho-associated, coiled-coil containing protein kinase 2 1 IL22RA1 receptor, alpha 1 1 ADAMTSL 4 ADAMTS-like 4 1 P2RXL1 #N/A 1 COL4A3BP collagen, type IV, alpha 3 (Goodpasture antigen) binding protein 1 MMP15 matrix metallopeptidase 15 (membrane-inserted) 1 KLHL20 kelch-like 20 (Drosophila) 1 PHOX2A paired-like homeobox 2a 1 HP -related protein; haptoglobin 1 IRF5 interferon regulatory factor 5 1 RECK reversion-inducing-cysteine-rich protein with kazal motifs 1 HYAL2 hyaluronoglucosaminidase 2 1 teratocarcinoma-derived growth factor 3, pseudogene; TDGF1 teratocarcinoma-derived growth factor 1 1 LTBP4 latent transforming growth factor beta binding protein 4 1 GCLM glutamate-cysteine ligase, modifier subunit 1 CCL24 chemokine (C-C motif) ligand 24 1 HFE hemochromatosis 1 API5 API5-like 1; apoptosis inhibitor 5 1

124

Appendix B: Proteins in the positive and negative regulation of angiogenesis

367 proteins in positive regulation of angiogenesis

Gene Name Gene description Degree JUN jun oncogene 40 CBL Cas-Br-M (murine) ecotropic retroviral transforming sequence 39 EP300 E1A binding protein p300 37 SHC1 SHC (Src homology 2 domain containing) transforming protein 1 35 THBS1 thrombospondin 1 35 FRS2 fibroblast growth factor receptor substrate 2 30 hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription HIF1A factor) 30 FN1 fibronectin 1 29 PML promyelocytic leukemia 29 CREBBP CREB binding protein (Rubinstein-Taybi syndrome) 28 HSP90AA1 heat shock protein 90kDa alpha (cytosolic), class A member 1 28 PLCG1 , gamma 1 28 SP1 Sp1 transcription factor 28 androgen receptor (dihydrotestosterone receptor; testicular feminization; AR spinal and bulbar muscular atrophy; Kennedy disease) 27 FRS3 fibroblast growth factor receptor substrate 3 27 TP53 tumor protein p53 27 MYC v-myc myelocytomatosis viral oncogene homolog (avian) 26 CTNNB1 catenin (cadherin-associated protein), beta 1, 88kDa 25 SMAD3 SMAD family member 3 25 TRAF2 TNF receptor-associated factor 2 25 PTPN6 protein tyrosine phosphatase, non-receptor type 6 24 ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding RAC1 protein Rac1) 24 SMAD2 SMAD family member 2 24 STAT3 signal transducer and activator of transcription 3 (acute-phase response factor) 24 FGFR4 fibroblast growth factor receptor 4 23 125

HDAC1 histone deacetylase 1 23 RB1 retinoblastoma 1 (including osteosarcoma) 23 FASLG Fas ligand (TNF superfamily, member 6) 22 v-rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa RELA light polypeptide gene enhancer in B-cells 3, p65 (avian) 22 STAT1 signal transducer and activator of transcription 1, 91kDa 22 CAV1 caveolin 1, caveolae protein, 22kDa 21 CDC42 cell division cycle 42 (GTP binding protein, 25kDa) 21 CDKN1A cyclin-dependent kinase inhibitor 1A (p21, Cip1) 21 PIK3R1 phosphoinositide-3-kinase, regulatory subunit 1 (alpha) 21 RASA1 RAS p21 protein activator (GTPase activating protein) 1 21 TSC2 tuberous sclerosis 2 21 TRAF6 TNF receptor-associated factor 6 20 FAS Fas (TNF receptor superfamily, member 6) 19 GRB2 growth factor receptor-bound protein 2 19 PLG plasminogen 19 A2M alpha-2-macroglobulin 18 F2 coagulation factor II (thrombin) 18 TNFRSF1A tumor necrosis factor receptor superfamily, member 1A 18 COL4A3 collagen, type IV, alpha 3 (Goodpasture antigen) 17 IKBKG inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma 17 CASP7 caspase 7, apoptosis-related cysteine peptidase 16 CASP8 caspase 8, apoptosis-related cysteine peptidase 16 CEBPB CCAAT/enhancer binding protein (C/EBP), beta 16 EZR ezrin 16 FGF1 fibroblast growth factor 1 (acidic) 16 ITGB3 integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) 16 PTPN11 protein tyrosine phosphatase, non-receptor type 11 (Noonan syndrome 1) 16 SRC v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) 16 VTN vitronectin 16 CASP3 caspase 3, apoptosis-related cysteine peptidase 15 CCR5 chemokine (C-C motif) receptor 5 15 COL1A1 collagen, type I, alpha 1 15 COL4A1 collagen, type IV, alpha 1 15

126

COL4A2 collagen, type IV, alpha 2 15 FGF2 fibroblast growth factor 2 (basic) 15 GNB2L1 guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1 15 MYOD1 myogenic differentiation 1 15 NCOR2 nuclear receptor co-repressor 2 15 RHOD ras homolog gene family, member D 15 VAV2 vav 2 guanine nucleotide exchange factor 15 VAV3 vav 3 guanine nucleotide exchange factor 15 APP amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease) 14 MDM2 Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (mouse) 14 RHOB ras homolog gene family, member B 14 TP73 tumor protein p73 14 colony stimulating factor 2 receptor, beta, low-affinity CSF2RB (granulocyte-macrophage) 13 E2F1 E2F transcription factor 1 13 FGF23 fibroblast growth factor 23 13 FGF6 fibroblast growth factor 6 13 FGF9 fibroblast growth factor 9 (glia-activating factor) 13 ITGAV integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51) 13 BCL2 B-cell CLL/lymphoma 2 12 CCL5 chemokine (C-C motif) ligand 5 12 CCL7 chemokine (C-C motif) ligand 7 12 FGF16 fibroblast growth factor 16 12 FGF17 fibroblast growth factor 17 12 FGF18 fibroblast growth factor 18 12 FGF19 fibroblast growth factor 19 12 FGF20 fibroblast growth factor 20 12 fibroblast growth factor 4 (heparin secretory transforming protein 1, Kaposi FGF4 sarcoma oncogene) 12 FGF5 fibroblast growth factor 5 12 FGF8 fibroblast growth factor 8 (androgen-induced) 12 FOS v-fos FBJ murine osteosarcoma viral oncogene homolog 12 ITGA5 integrin, alpha 5 (fibronectin receptor, alpha polypeptide) 12 PPP2CA protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform 12

127

PTK2B PTK2B protein tyrosine kinase 2 beta 12 RHOA ras homolog gene family, member A 12 ARHGAP22 Rho GTPase activating protein 22 11 ARHGAP24 Rho GTPase activating protein 24 11 CD44 CD44 molecule (Indian blood group) 11 FYN FYN oncogene related to SRC, FGR, YES 11 matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV MMP9 collagenase) 11 PTK2 PTK2 protein tyrosine kinase 2 11 RUNX1 runt-related transcription factor 1 (acute myeloid leukemia 1; aml1 oncogene) 11 serum response factor (c-fos serum response element-binding transcription SRF factor) 11 ZBTB16 zinc finger and BTB domain containing 16 11 ARHGEF4 Rho guanine nucleotide exchange factor (GEF) 4 10 C3 complement component 3 10 epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) EGFR oncogene homolog, avian) 10 F10 coagulation factor X 10 F3 coagulation factor III (thromboplastin, tissue factor) 10 HDAC4 histone deacetylase 4 10 HDAC5 histone deacetylase 5 10 HSPA5 heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) 10 matrix metallopeptidase 2 (, 72kDa gelatinase, 72kDa type IV MMP2 collagenase) 10 PF4 platelet factor 4 (chemokine (C-X-C motif) ligand 4) 10 TRAF1 TNF receptor-associated factor 1 10 TUBB tubulin, beta 10 CCL3 chemokine (C-C motif) ligand 3 9 CCL8 chemokine (C-C motif) ligand 8 9 CDKN2A cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) 9 COL1A2 collagen, type I, alpha 2 9 HMGA1 high mobility group AT-hook 1 9 IQGAP1 IQ motif containing GTPase activating protein 1 9 MED1 mediator complex subunit 1 9 MMP7 matrix metallopeptidase 7 (matrilysin, uterine) 9 128

NOS3 nitric oxide synthase 3 (endothelial cell) 9 pleckstrin homology domain containing, family G (with RhoGef domain) PLEKHG5 member 5 9 PREX1 phosphatidylinositol 3,4,5-trisphosphate-dependent RAC exchanger 1 9 PRKCD protein kinase C, delta 9 RARA retinoic acid receptor, alpha 9 SYK spleen tyrosine kinase 9 TFPI tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) 9 THBD thrombomodulin 9 AGTR1 angiotensin II receptor, type 1 8 BIRC2 baculoviral IAP repeat-containing 2 8 CCL13 chemokine (C-C motif) ligand 13 8 CCR2 chemokine (C-C motif) receptor 2 8 CFLAR CASP8 and FADD-like apoptosis regulator 8 CXCL10 chemokine (C-X-C motif) ligand 10 8 GATA2 GATA binding protein 2 8 GATA4 GATA binding protein 4 8 MBP 8 MEF2A myocyte enhancer factor 2A 8 MEF2C myocyte enhancer factor 2C 8 MSN moesin 8 SDC2 syndecan 2 8 SPARC secreted protein, acidic, cysteine-rich (osteonectin) 8 ABL1 c-abl oncogene 1, receptor tyrosine kinase 7 CCL11 chemokine (C-C motif) ligand 11 7 CCNA2 cyclin A2 7 F5 coagulation factor V (proaccelerin, labile factor) 7 F8 coagulation factor VIII, procoagulant component (hemophilia A) 7 FGF22 fibroblast growth factor 22 7 FGFBP1 fibroblast growth factor binding protein 1 7 fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular FLT1 permeability factor receptor) 7 HSPG2 heparan sulfate proteoglycan 2 7 IL1RAP interleukin 1 receptor accessory protein 7

129

JAK2 (a protein tyrosine kinase) 7 KDR kinase insert domain receptor (a type III receptor tyrosine kinase) 7 LCK lymphocyte-specific protein tyrosine kinase 7 PRKD1 protein kinase D1 7 RRAS related RAS viral (r-ras) oncogene homolog 7 SHB Src homology 2 domain containing adaptor protein B 7 SPHK1 sphingosine kinase 1 7 THBS2 thrombospondin 2 7 TSC1 tuberous sclerosis 1 7 VCAN versican 7 VHL von Hippel-Lindau tumor suppressor 7 APEX1 APEX nuclease (multifunctional DNA repair enzyme) 1 6 CCBP2 chemokine binding protein 2 6 CCL4 chemokine (C-C motif) ligand 4 6 CCR1 chemokine (C-C motif) receptor 1 6 CCR3 chemokine (C-C motif) receptor 3 6 CCRL1 chemokine (C-C motif) receptor-like 1 6 CDH5 cadherin 5, type 2, VE-cadherin (vascular epithelium) 6 CHUK conserved helix-loop-helix ubiquitous kinase 6 CTSG cathepsin G 6 CXCR3 chemokine (C-X-C motif) receptor 3 6 DOK4 docking protein 4 6 FGF10 fibroblast growth factor 10 6 HDAC9 histone deacetylase 9 6 IL1B interleukin 1, beta 6 IL8 interleukin 8 6 MAPK1 mitogen-activated protein kinase 1 6 MME membrane metallo-endopeptidase 6 RAP1A RAP1A, member of RAS oncogene family 6 SHC2 SHC (Src homology 2 domain containing) transforming protein 2 6 ACP1 acid phosphatase 1, soluble 5 AKT1 v-akt murine thymoma viral oncogene homolog 1 5 ARNT aryl hydrocarbon receptor nuclear translocator 5 BDKRB2 bradykinin receptor B2 5

130

COL18A1 collagen, type XVIII, alpha 1 5 catenin (cadherin-associated protein), delta 2 (neural plakophilin-related CTNND2 arm-repeat protein) 5 DARC Duffy blood group, chemokine receptor 5 fibroblast growth factor 3 (murine mammary tumor virus integration site FGF3 (v-int-2) oncogene homolog) 5 FGF7 fibroblast growth factor 7 (keratinocyte growth factor) 5 HAPLN1 hyaluronan and proteoglycan link protein 1 5 IGF1R insulin-like growth factor 1 receptor 5 IL1R1 interleukin 1 receptor, type I 5 LEF1 lymphoid enhancer-binding factor 1 5 MAP3K7 mitogen-activated protein kinase kinase kinase 7 5 MAPK3 mitogen-activated protein kinase 3 5 MYH9 myosin, heavy chain 9, non-muscle 5 prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and PTGS2 cyclooxygenase) 5 SDC1 syndecan 1 5 SH2D2A SH2 domain protein 2A 5 BMX BMX non-receptor tyrosine kinase 4 BTG1 B-cell translocation gene 1, anti-proliferative 4 DNAJA3 DnaJ (Hsp40) homolog, subfamily A, member 3 4 ERN1 endoplasmic reticulum to nucleus signaling 1 4 fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer FGFR2 syndrome, Jackson-Weiss syndrome) 4 HEY1 hairy/enhancer-of-split related with YRPW motif 1 4 HHEX hematopoietically expressed homeobox 4 HIF1AN hypoxia-inducible factor 1, alpha subunit inhibitor 4 HOXA5 homeobox A5 4 IKBKB inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta 4 IL1A interleukin 1, alpha 4 IL6R interleukin 6 receptor 4 JAK1 Janus kinase 1 (a protein tyrosine kinase) 4 MAPK8 mitogen-activated protein kinase 8 4 MLLT4 myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, 4 131

Drosophila); translocated to, 4 MTA1 metastasis associated 1 4 NOS1 nitric oxide synthase 1 (neuronal) 4 NRP1 neuropilin 1 4 OS9 amplified in osteosarcoma 4 P2RY2 purinergic receptor P2Y, G-protein coupled, 2 4 PAPPA pregnancy-associated plasma protein A, pappalysin 1 4 PLXNA1 plexin A1 4 PPP2R5D protein phosphatase 2, regulatory subunit B', delta isoform 4 proteinase 3 (serine proteinase, neutrophil, Wegener granulomatosis PRTN3 autoantigen) 4 RARB retinoic acid receptor, beta 4 TIMP metallopeptidase inhibitor 3 (Sorsby fundus dystrophy, TIMP3 pseudoinflammatory) 4 TNF tumor necrosis factor (TNF superfamily, member 2) 4 TNFRSF12A tumor necrosis factor receptor superfamily, member 12A 4 TNFRSF25 tumor necrosis factor receptor superfamily, member 25 4 TNIP2 TNFAIP3 interacting protein 2 4 VEGFA vascular endothelial growth factor A 4 ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 motif, 1 3 ADM adrenomedullin 3 ANGPTL3 angiopoietin-like 3 3 C5 complement component 5 3 C6 complement component 6 3 CALCRL -like 3 CANX calnexin 3 CCND1 cyclin D1 3 CHRNA7 cholinergic receptor, nicotinic, alpha 7 3 COL4A5 collagen, type IV, alpha 5 (Alport syndrome) 3 COL4A6 collagen, type IV, alpha 6 3 CTSB cathepsin B 3 CXCL11 chemokine (C-X-C motif) ligand 11 3 CXCL13 chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant) 3 CXCL9 chemokine (C-X-C motif) ligand 9 3

132

DPP4 dipeptidyl-peptidase 4 (CD26, complexing protein 2) 3 EDN1 endothelin 1 3 EDNRB endothelin receptor type B 3 EPHB2 EPH receptor B2 3 EPO erythropoietin 3 FIBP fibroblast growth factor (acidic) intracellular binding protein 3 FIGF c-fos induced growth factor (vascular endothelial growth factor D) 3 GATA6 GATA binding protein 6 3 GPC1 glypican 1 3 GRB14 growth factor receptor-bound protein 14 3 HAND2 heart and neural crest derivatives expressed 2 3 HMOX1 heme oxygenase (decycling) 1 3 IGFBP7 insulin-like growth factor binding protein 7 3 IL1R2 interleukin 1 receptor, type II 3 PAX2 paired box 2 3 PRKD2 protein kinase D2 3 PTPRB protein tyrosine phosphatase, receptor type, B 3 PZP pregnancy-zone protein 3 SDC3 syndecan 3 3 SDC4 syndecan 4 3 sema domain, immunoglobulin domain (Ig), short basic domain, secreted, SEMA3F (semaphorin) 3F 3 SFRP2 secreted frizzled-related protein 2 3 TEK tyrosine kinase, endothelial (venous malformations, multiple cutaneous TEK and mucosal) 3 TNFSF12 tumor necrosis factor (ligand) superfamily, member 12 3 twist homolog 1 (acrocephalosyndactyly 3; Saethre-Chotzen syndrome) TWIST1 (Drosophila) 3 VEGFC vascular endothelial growth factor C 3 YES1 v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 3 AKT2 v-akt murine thymoma viral oncogene homolog 2 2 ANGPT1 angiopoietin 1 2 ANGPT2 angiopoietin 2 2 AREG amphiregulin (schwannoma-derived growth factor) 2 ARNT2 aryl-hydrocarbon receptor nuclear translocator 2 2 133

BDNF brain-derived neurotrophic factor 2 BTC betacellulin 2 CALCA calcitonin-related polypeptide alpha 2 CBX5 chromobox homolog 5 (HP1 alpha homolog, Drosophila) 2 CCL15 chemokine (C-C motif) ligand 15 2 CCL26 chemokine (C-C motif) ligand 26 2 CCR4 chemokine (C-C motif) receptor 4 2 COL4A4 collagen, type IV, alpha 4 2 COX5A cytochrome c oxidase subunit Va 2 CSTA cystatin A (stefin A) 2 CTGF connective tissue growth factor 2 chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, CXCL1 alpha) 2 CXCL5 chemokine (C-X-C motif) ligand 5 2 EGLN1 egl nine homolog 1 (C. elegans) 2 EGLN3 egl nine homolog 3 (C. elegans) 2 ERAP1 endoplasmic reticulum aminopeptidase 1 2 ETV6 ets variant gene 6 (TEL oncogene) 2 fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2, Pfeiffer FGFR1 syndrome) 2 FGFR3 fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism) 2 FLT4 fms-related tyrosine kinase 4 2 FZD5 frizzled homolog 5 (Drosophila) 2 GPC4 2 hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A HADHA thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alpha subunit 2 HIF3A hypoxia inducible factor 3, alpha subunit 2 KISS1 KiSS-1 metastasis-suppressor 2 LCN2 lipocalin 2 2 LTA lymphotoxin alpha (TNF superfamily, member 1) 2 MMP10 matrix metallopeptidase 10 (stromelysin 2) 2 NFATC4 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4 2 NRP2 neuropilin 2 2 PAX6 paired box 6 2 RAMP2 receptor (G protein-coupled) activity modifying protein 2 2 134

RASIP1 Ras interacting protein 1 2 RHOC ras homolog gene family, member C 2 S100A13 S100 calcium binding protein A13 2 TIMP1 TIMP metallopeptidase inhibitor 1 2 TNXB tenascin XB 2 TPI1 triosephosphate isomerase 1 2 ADCY6 adenylate cyclase 6 1 AGGF1 angiogenic factor with G patch and FHA domains 1 1 ALK anaplastic lymphoma receptor tyrosine kinase 1 ANGPT4 angiopoietin 4 1 ANGPTL1 angiopoietin-like 1 1 ANXA3 annexin A3 1 ANXA5 annexin A5 1 API5 apoptosis inhibitor 5 1 AQP1 aquaporin 1 (Colton blood group) 1 ARD1B ARD1 homolog B (S. cerevisiae) 1 C3AR1 complement component 3a receptor 1 1 C5AR1 complement component 5a receptor 1 1 CCL24 chemokine (C-C motif) ligand 24 1 CD34 CD34 molecule 1 CDK6 cyclin-dependent kinase 6 1 CFP complement factor properdin 1 CSNK2A1 casein kinase 2, alpha 1 polypeptide 1 CTSH cathepsin H 1 CX3CL1 chemokine (C-X3-C motif) ligand 1 1 CX3CR1 chemokine (C-X3-C motif) receptor 1 1 CXCL6 chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2) 1 DLL4 delta-like 4 (Drosophila) 1 EFNA1 ephrin-A1 1 EPHA1 EPH receptor A1 1 FGFRL1 fibroblast growth factor receptor-like 1 1 GC group-specific component (vitamin D binding protein) 1 GPC3 glypican 3 1 HIPK1 homeodomain interacting protein kinase 1 1

135

HIPK2 homeodomain interacting protein kinase 2 1 MAPK9 mitogen-activated protein kinase 9 1 MMP26 matrix metallopeptidase 26 1 NTRK2 neurotrophic tyrosine kinase, receptor, type 2 1 PGF placental growth factor, vascular endothelial growth factor-related protein 1 PSMA7 proteasome (prosome, macropain) subunit, alpha type, 7 1 PTCH1 patched homolog 1 (Drosophila) 1 PTGIS prostaglandin I2 (prostacyclin) synthase 1 PTPN3 protein tyrosine phosphatase, non-receptor type 3 1 RAPGEF3 Rap guanine nucleotide exchange factor (GEF) 3 1 RECK reversion-inducing-cysteine-rich protein with kazal motifs 1 RPS6KC1 ribosomal protein S6 kinase, 52kDa, polypeptide 1 1 SPHKAP SPHK1 interactor, AKAP domain containing 1 SSX4 synovial sarcoma, X breakpoint 4 1 TGFB1 transforming growth factor, beta 1 1 TIE1 tyrosine kinase with immunoglobulin-like and EGF-like domains 1 1 UTS2 urotensin 2 1 UTS2R urotensin 2 receptor 1 VEGFB vascular endothelial growth factor B 1 VHLL von Hippel-Lindau tumor suppressor-like 1 WNT5A wingless-type MMTV integration site family, member 5A 1

245 proteins in positive regulation of angiogenesis Gene Name Gene description Degree JUN jun oncogene 40 EP300 E1A binding protein p300 37 THBS1 thrombospondin 1 35 ESR1 estrogen receptor 1 34 FN1 fibronectin 1 29 PML promyelocytic leukemia 29 CREBBP CREB binding protein (Rubinstein-Taybi syndrome) 28 SP1 Sp1 transcription factor 28 androgen receptor (dihydrotestosterone receptor; testicular feminization; AR spinal and bulbar muscular atrophy; Kennedy disease) 27

136

TP53 tumor protein p53 27 MYC v-myc myelocytomatosis viral oncogene homolog (avian) 26 CTNNB1 catenin (cadherin-associated protein), beta 1, 88kDa 25 SMAD3 SMAD family member 3 25 SMAD2 SMAD family member 2 24 STAT3 signal transducer and activator of transcription 3 (acute-phase response factor) 24 HDAC1 histone deacetylase 1 23 RB1 retinoblastoma 1 (including osteosarcoma) 23 FASLG Fas ligand (TNF superfamily, member 6) 22 v-rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa RELA light polypeptide gene enhancer in B-cells 3, p65 (avian) 22 STAT1 signal transducer and activator of transcription 1, 91kDa 22 CDKN1A cyclin-dependent kinase inhibitor 1A (p21, Cip1) 21 PIK3R1 phosphoinositide-3-kinase, regulatory subunit 1 (alpha) 21 TSC2 tuberous sclerosis 2 21 PRKCA protein kinase C, alpha 20 SMAD4 SMAD family member 4 20 FAS Fas (TNF receptor superfamily, member 6) 19 GRB2 growth factor receptor-bound protein 2 19 PLG plasminogen 19 F2 coagulation factor II (thrombin) 18 TNFRSF1A tumor necrosis factor receptor superfamily, member 1A 18 COL4A3 collagen, type IV, alpha 3 (Goodpasture antigen) 17 CASP8 caspase 8, apoptosis-related cysteine peptidase 16 EZR ezrin 16 integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes ITGB1 MDF2, MSK12) 16 ITGB3 integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) 16 PRKACA protein kinase, cAMP-dependent, catalytic, alpha 16 PTPN11 protein tyrosine phosphatase, non-receptor type 11 (Noonan syndrome 1) 16 SRC v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) 16 VTN vitronectin 16 CASP3 caspase 3, apoptosis-related cysteine peptidase 15 CCR5 chemokine (C-C motif) receptor 5 15

137

COL1A1 collagen, type I, alpha 1 15 COL4A1 collagen, type IV, alpha 1 15 COL4A2 collagen, type IV, alpha 2 15 FGF2 fibroblast growth factor 2 (basic) 15 GNB2L1 guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1 15 NCOR2 nuclear receptor co-repressor 2 15 APP amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease) 14 MDM2 Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (mouse) 14 PLAT plasminogen activator, tissue 14 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type SERPINE1 1), member 1 14 TP73 tumor protein p73 14 ITGAV integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51) 13 LRP1 low density lipoprotein-related protein 1 (alpha-2-macroglobulin receptor) 13 MAPK14 mitogen-activated protein kinase 14 13 PLAUR plasminogen activator, urokinase receptor 13 ROCK1 Rho-associated, coiled-coil containing protein kinase 1 13 secreted phosphoprotein 1 (, I, early SPP1 T-lymphocyte activation 1) 13 CCL2 chemokine (C-C motif) ligand 2 12 CCL5 chemokine (C-C motif) ligand 5 12 CCL7 chemokine (C-C motif) ligand 7 12 DCN decorin 12 FOS v-fos FBJ murine osteosarcoma viral oncogene homolog 12 KLK3 kallikrein-related peptidase 3 12 RHOA ras homolog gene family, member A 12 transforming growth factor, beta receptor I (activin A receptor type II-like TGFBR1 kinase, 53kDa) 12 FYN FYN oncogene related to SRC, FGR, YES 11 GTF2I general transcription factor II, i 11 HRAS v-Ha-ras Harvey rat sarcoma viral oncogene homolog 11 IGFBP3 insulin-like growth factor binding protein 3 11 matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV MMP9 collagenase) 11 PLAU plasminogen activator, urokinase 11 138

PTK2 PTK2 protein tyrosine kinase 2 11 serum response factor (c-fos serum response element-binding transcription SRF factor) 11 ZBTB16 zinc finger and BTB domain containing 16 11 F10 coagulation factor X 10 FOXO1 forkhead box O1 10 HDAC5 histone deacetylase 5 10 matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV MMP2 collagenase) 10 PF4 platelet factor 4 (chemokine (C-X-C motif) ligand 4) 10 CALR calreticulin 9 CCL8 chemokine (C-C motif) ligand 8 9 CD36 CD36 molecule (thrombospondin receptor) 9 FOXO4 forkhead box O4 9 HRG histidine-rich glycoprotein 9 KNG1 kininogen 1 9 MED1 mediator complex subunit 1 9 MMP3 matrix metallopeptidase 3 (stromelysin 1, progelatinase) 9 MMP7 matrix metallopeptidase 7 (matrilysin, uterine) 9 RARA retinoic acid receptor, alpha 9 TFPI tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) 9 TGFBR2 transforming growth factor, beta receptor II (70/80kDa) 9 THBD thrombomodulin 9 CCL13 chemokine (C-C motif) ligand 13 8 CCR2 chemokine (C-C motif) receptor 2 8 CDH1 cadherin 1, type 1, E-cadherin (epithelial) 8 CXCL10 chemokine (C-X-C motif) ligand 10 8 FGA 8 GATA2 GATA binding protein 2 8 MBP myelin basic protein 8 MEF2A myocyte enhancer factor 2A 8 MEF2C myocyte enhancer factor 2C 8 MMP1 matrix metallopeptidase 1 (interstitial collagenase) 8 MSN moesin 8

139

PTEN phosphatase and tensin homolog (mutated in multiple advanced 1) 8 SDC2 syndecan 2 8 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), SERPINA5 member 5 8 SPARC secreted protein, acidic, cysteine-rich (osteonectin) 8 TNFSF10 tumor necrosis factor (ligand) superfamily, member 10 8 VWF von Willebrand factor 8 ANXA2 annexin A2 7 CCL11 chemokine (C-C motif) ligand 11 7 EPOR 7 FBLN2 fibulin 2 7 FURIN furin (paired basic amino acid cleaving enzyme) 7 HSPG2 heparan sulfate proteoglycan 2 7 JAK2 Janus kinase 2 (a protein tyrosine kinase) 7 LCK lymphocyte-specific protein tyrosine kinase 7 PDPK1 3-phosphoinositide dependent protein kinase-1 7 PIN1 protein (peptidylprolyl cis/trans isomerase) NIMA-interacting 1 7 PRKD1 protein kinase D1 7 SERPINB2 serpin peptidase inhibitor, clade B (ovalbumin), member 2 7 THBS2 thrombospondin 2 7 TP63 tumor protein p63 7 VCAN versican 7 ARRB1 arrestin, beta 1 6 CCBP2 chemokine binding protein 2 6 CCR1 chemokine (C-C motif) receptor 1 6 CCR3 chemokine (C-C motif) receptor 3 6 CCRL1 chemokine (C-C motif) receptor-like 1 6 CD47 CD47 molecule 6 collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and COL7A1 recessive) 6 CTSG cathepsin G 6 CXCR3 chemokine (C-X-C motif) receptor 3 6 EWSR1 Ewing sarcoma breakpoint region 1 6 F2R coagulation factor II (thrombin) receptor 6

140

IGFBP5 insulin-like growth factor binding protein 5 6 IL6ST interleukin 6 signal transducer (gp130, ) 6 IL8 interleukin 8 6 INS insulin 6 IRF3 interferon regulatory factor 3 6 ITGA4 integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) 6 MEF2D myocyte enhancer factor 2D 6 NR4A1 nuclear receptor subfamily 4, group A, member 1 6 RAP1A RAP1A, member of RAS oncogene family 6 YY1 YY1 transcription factor 6 AKT1 v-akt murine thymoma viral oncogene homolog 1 5 APOH apolipoprotein H (beta-2-glycoprotein I) 5 ARNT aryl hydrocarbon receptor nuclear translocator 5 BAI1 brain-specific angiogenesis inhibitor 1 5 CDC25A cell division cycle 25 homolog A (S. pombe) 5 DARC Duffy blood group, chemokine receptor 5 MAPK3 mitogen-activated protein kinase 3 5 MAPK7 mitogen-activated protein kinase 7 5 MATN2 matrilin 2 5 MYOC myocilin, trabecular meshwork inducible glucocorticoid response 5 neurofibromin 1 (neurofibromatosis, von Recklinghausen disease, Watson NF1 disease) 5 PDE3B phosphodiesterase 3B, cGMP-inhibited 5 platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) PDGFB oncogene homolog) 5 PROC protein C (inactivator of coagulation factors Va and VIIIa) 5 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), SERPINA1 member 1 5 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type SERPINE2 1), member 2 5 TGFBI transforming growth factor, beta-induced, 68kDa 5 TNFRSF11B tumor necrosis factor receptor superfamily, member 11b () 5 EIF4E eukaryotic translation initiation factor 4E 4 F12 coagulation factor XII (Hageman factor) 4 GFAP glial fibrillary acidic protein 4 141

HABP2 hyaluronan binding protein 2 4 HHEX hematopoietically expressed homeobox 4 HOXA5 homeobox A5 4 ITGA3 integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor) 4 KRIT1 KRIT1, ankyrin repeat containing 4 MAPK11 mitogen-activated protein kinase 11 4 MAPK8 mitogen-activated protein kinase 8 4 natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide NPR1 receptor A) 4 OSM oncostatin M 4 PRKG1 protein kinase, cGMP-dependent, type I 4 RUNX3 runt-related transcription factor 3 4 serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium SERPINF1 derived factor), member 1 4 TNF tumor necrosis factor (TNF superfamily, member 2) 4 ANTXR2 anthrax toxin receptor 2 3 BMP3 bone morphogenetic protein 3 (osteogenic) 3 CCND1 cyclin D1 3 DPP4 dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexing protein 2) 3 GATA6 GATA binding protein 6 3 GHRL ghrelin/obestatin preprohormone 3 IGFBP7 insulin-like growth factor binding protein 7 3 ITGB1BP1 integrin beta 1 binding protein 1 3 JAG1 jagged 1 (Alagille syndrome) 3 KLF4 Kruppel-like factor 4 (gut) 3 LIMK1 LIM domain kinase 1 3 MAGI1 membrane associated guanylate kinase, WW and PDZ domain containing 1 3 MEIS1 Meis homeobox 1 3 MMP8 matrix metallopeptidase 8 (neutrophil collagenase) 3 NPPB natriuretic peptide precursor B 3 PBX1 pre-B-cell leukemia homeobox 1 3 PTPRM protein tyrosine phosphatase, receptor type, M 3 SAA1 serum amyloid A1 3 TEK tyrosine kinase, endothelial (venous malformations, multiple cutaneous TEK and mucosal) 3 142

THBS3 thrombospondin 3 3 twist homolog 1 (acrocephalosyndactyly 3; Saethre-Chotzen syndrome) TWIST1 (Drosophila) 3 AKT2 v-akt murine thymoma viral oncogene homolog 2 2 AMOT angiomotin 2 ANGPT1 angiopoietin 1 2 ANGPT2 angiopoietin 2 2 CBX5 chromobox homolog 5 (HP1 alpha homolog, Drosophila) 2 CCR10 chemokine (C-C motif) receptor 10 2 LAMB3 laminin, beta 3 2 LEP leptin (obesity homolog, mouse) 2 LIMK2 LIM domain kinase 2 2 LPA lipoprotein, Lp(a) 2 MAGI3 membrane associated guanylate kinase, WW and PDZ domain containing 3 2 MAP2K5 mitogen-activated protein kinase kinase 5 2 NPPA natriuretic peptide precursor A 2 natriuretic peptide receptor C/guanylate cyclase C (atrionatriuretic peptide NPR3 receptor C) 2 RHOC ras homolog gene family, member C 2 THBS4 thrombospondin 4 2 VLDLR very low density lipoprotein receptor 2 ACE2 angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 1 1-acylglycerol-3-phosphate O-acyltransferase 5 (lysophosphatidic acid AGPAT5 acyltransferase, epsilon) 1 BAIAP2 BAI1-associated protein 2 1 BAIAP3 BAI1-associated protein 3 1 CHID1 chitinase domain containing 1 1 COL4A3BP collagen, type IV, alpha 3 (Goodpasture antigen) binding protein 1 CORO1A coronin, actin binding protein, 1A 1 CSNK2A1 casein kinase 2, alpha 1 polypeptide 1 EGR3 early growth response 3 1 LDLR low density lipoprotein receptor (familial hypercholesterolemia) 1 LECT1 leukocyte cell derived chemotaxin 1 1 LIF leukemia inhibitory factor (cholinergic differentiation factor) 1 MFAP2 microfibrillar-associated protein 2 1 143

NPY neuropeptide Y 1 phosphodiesterase 4A, cAMP-specific (phosphodiesterase E2 dunce homolog, PDE4A Drosophila) 1 PHYHIP phytanoyl-CoA 2-hydroxylase interacting protein 1 PTP4A3 protein tyrosine phosphatase type IVA, member 3 1 ROCK2 Rho-associated, coiled-coil containing protein kinase 2 1 SCARB2 scavenger receptor class B, member 2 1 SLPI secretory leukocyte peptidase inhibitor 1 SRGN serglycin 1 STAB1 stabilin 1 1 TGFB1 transforming growth factor, beta 1 1 TIE1 tyrosine kinase with immunoglobulin-like and EGF-like domains 1 1 TNFAIP6 tumor necrosis factor, alpha-induced protein 6 1 TNFRSF6B tumor necrosis factor receptor superfamily, member 6b, decoy 1 USH2A Usher syndrome 2A (autosomal recessive, mild) 1

144

Appendix C: Differentially expressed genes between C57BL/6 versus

BALB/c mouse of ischemic and nonischemic tissues

Fold Feature Name Description Change P Venn Diagram PRKAA protein kinase, AMP-activated, 2 alpha 2 catalytic subunit 43.246 0.006 A (angiome) fibroblast growth factor binding FGFBP1 protein 1 40.621 0.011 F (angiome+arteriome) methyl-CpG binding domain protein MBD2 2 40.569 0.004 B (immunome) D PTTG1 pituitary tumor-transforming 1 33.334 0.001 (angiome+immunome) RAB6B, member RAS oncogene RAB6B family 33.078 0.027 B (immunome) D PTTG1 pituitary tumor-transforming 1 21.124 0.001 (angiome+immunome) PLA2G5 phospholipase A2, group V 15.03 0.013 B (immunome) D HFE hemochromatosis 14.081 0.016 (angiome+immunome) eukaryotic translation initiation EIF4G3 factor 4 gamma, 3 12.013 0.023 B (immunome) D HFE hemochromatosis 11.754 <0.001 (angiome+immunome) D ACTC1 actin, alpha, cardiac muscle 1 8.838 0.002 (angiome+immunome) D PTTG1 pituitary tumor-transforming 1 6.803 0.003 (angiome+immunome) G (angiome+immunome+ NOS1 nitric oxide synthase 1 (neuronal) 5.173 0.038 arteriome) succinate dehydrogenase complex, SDHA subunit A, flavoprotein (Fp) 4.826 0.003 B (immunome) ALDH2 aldehyde dehydrogenase 2 family 4.213 0.012 B (immunome)

145

(mitochondrial) E2F6 E2F transcription factor 6 4.189 0.033 B (immunome) LMAN1 lectin, mannose-binding, 1 3.941 <0.001 B (immunome) CCL25 chemokine (C-C motif) ligand 25 3.702 0.005 B (immunome) COL9A1 collagen, type IX, alpha 1 3.557 0.022 B (immunome) PRDX2 3.524 0.004 B (immunome) D CLU clusterin 3.461 0.001 (angiome+immunome) G (angiome+immunome+ NOS1 nitric oxide synthase 1 (neuronal) 3.352 0.004 arteriome) phosphatidylinositol-4,5-bisphospha D PIK3CD te 3-kinase, catalytic subunit delta 3.126 0.011 (angiome+immunome) growth factor, augmenter of liver GFER regeneration 3.035 0.007 B (immunome) APOD apolipoprotein D 3.009 0.001 A (angiome) FOXP2 forkhead box P2 2.825 0.043 B (immunome) ATPase, Cu++ transporting, alpha ATP7A polypeptide 2.796 0.003 B (immunome) excision repair cross-complementing rodent repair deficiency, complementation group 1 (includes overlapping antisense ERCC1 sequence) 2.773 <0.001 B (immunome) D CLU clusterin 2.732 0.025 (angiome+immunome) D CLU clusterin 2.713 0.003 (angiome+immunome) D PTEN phosphatase and tensin homolog 2.691 <0.001 (angiome+immunome) SORBS1 sorbin and SH3 domain containing 1 2.641 0.006 B (immunome) E2F transcription factor 2 2.624 <0.001 B (immunome) D CLU clusterin 2.613 0.012 (angiome+immunome) TLN2 2 2.39 0.003 B (immunome) 146

FBXW1 F-box and WD repeat domain 1 containing 11 2.387 0.02 B (immunome) G (angiome+immunome+ EFNB1 ephrin-B1 2.332 0.001 arteriome) natriuretic peptide receptor C/guanylate cyclase C D NPR3 (atrionatriuretic peptide receptor C) 2.318 0.028 (angiome+immunome) TLN2 talin 2 2.274 0.008 B (immunome) ubiquitin protein ligase E3 UBR1 component n-recognin 1 2.256 0.044 B (immunome) neural precursor cell expressed, NEDD4 developmentally down-regulated L 4-like, E3 ubiquitin protein ligase 2.222 0.002 B (immunome) RPS6KA ribosomal protein S6 kinase, 90kDa, 1 polypeptide 1 2.209 0.005 B (immunome) solute carrier family 2 (facilitated D SLC2A3 glucose transporter), member 3 2.199 0.002 (angiome+immunome) MAP3K mitogen-activated protein kinase D 7 kinase kinase 7 2.185 <0.001 (angiome+immunome) DNA fragmentation factor, 45kDa, DFFA alpha polypeptide 2.178 0.015 B (immunome) excision repair cross-complementing rodent repair deficiency, complementation group 1 (includes overlapping antisense ERCC1 sequence) 2.172 0.001 B (immunome) nuclear receptor subfamily 6, group NR6A1 A, member 1 2.128 0.032 B (immunome) MAP3K mitogen-activated protein kinase D 7 kinase kinase 7 2.046 0.001 (angiome+immunome) RSF1 remodeling and spacing factor 1 2.034 0.015 F (angiome+arteriome) v-akt murine thymoma viral D AKT2 oncogene homolog 2 2.028 0.027 (angiome+immunome) LTBP3 latent transforming growth factor 2.026 0.025 D

147

beta binding protein 3 (angiome+immunome) c-fos induced growth factor (vascular endothelial growth factor D FIGF D) 2.017 0.006 (angiome+immunome) E2F2 E2F transcription factor 2 1.993 0.003 B (immunome) GTF3A general transcription factor IIIA 1.974 <0.001 B (immunome) RAD23 B RAD23 homolog B (S. cerevisiae) 1.968 <0.001 B (immunome) NES nestin 1.961 0.044 A (angiome) uncoupling protein 3 UCP3 (mitochondrial, proton carrier) 1.961 0.008 B (immunome) D RUNX1 runt-related transcription factor 1 1.96 0.004 (angiome+immunome) phosphoinositide-3-kinase, PIK3R4 regulatory subunit 4 1.922 0.048 B (immunome) glutamyl aminopeptidase D ENPEP (aminopeptidase A) 1.921 0.007 (angiome+immunome) RPS6KA ribosomal protein S6 kinase, 90kDa, 2 polypeptide 2 1.912 0.004 B (immunome) RPTOR independent companion of RICTOR MTOR, complex 2 1.905 0.027 B (immunome) GAA glucosidase, alpha; acid 1.897 <0.001 B (immunome) dopa decarboxylase (aromatic DDC L-amino acid decarboxylase) 1.888 0.04 B (immunome) growth factor, augmenter of liver GFER regeneration 1.876 <0.001 B (immunome) guanine nucleotide binding GNL1 protein-like 1 1.852 0.03 B (immunome) BIRC6 baculoviral IAP repeat containing 6 1.844 0.034 B (immunome)

148

Down-regulated genes in C57BL/6 versus BALB/c mouse of ischemic Fold Feature Name Description Change P Venn Diagram eukaryotic translation initiation EIF2S1 factor 2, subunit 1 alpha, 35kDa -32.648 0.001 B (immunome) guanylate binding protein 1, GBP1 interferon-inducible -15.79 0.03 B (immunome) D PLAU plasminogen activator, urokinase -12.674 0.001 (angiome+immunome) ATPase, Na+/K+ transporting, ATP1A2 alpha 2 polypeptide -10.27 0.004 B (immunome) spondin 1, extracellular matrix D SPON1 protein -4.244 0.007 (angiome+immunome) interleukin 6 signal transducer D IL6ST (gp130, oncostatin M receptor) -4.18 0.005 (angiome+immunome) D EZR ezrin -3.94 0.029 (angiome+immunome) NT5E 5'-, ecto (CD73) -3.894 0.007 B (immunome) AURKA aurora kinase A -3.667 0.001 B (immunome) D PLAU plasminogen activator, urokinase -3.611 0.004 (angiome+immunome) TAF7 RNA polymerase II, TATA box binding protein TAF7 (TBP)-associated factor, 55kDa -3.536 <0.001 B (immunome) D ITGA9 integrin, alpha 9 -3.524 <0.001 (angiome+immunome) D EGR1 early growth response 1 -3.357 0.01 (angiome+immunome) TPMT thiopurine S-methyltransferase -3.209 0.001 A (angiome) D FMOD fibromodulin -3.19 0.018 (angiome+immunome) D FMOD fibromodulin -3.183 0.017 (angiome+immunome) ZFP36L1 ZFP36 ring finger protein-like 1 -3.173 0.015 B (immunome) ANXA4 -3.093 0.004 B (immunome)

149

regulator of G-protein signaling 2, D RGS2 24kDa -3.028 0.037 (angiome+immunome) D FMOD fibromodulin -2.92 0.03 (angiome+immunome) guanine nucleotide binding D GNA13 protein (G protein), alpha 13 -2.9 0.006 (angiome+immunome) D FMOD fibromodulin -2.879 0.018 (angiome+immunome) CCNDBP1 cyclin D-type binding-protein 1 -2.777 0.008 B (immunome) D RARB retinoic acid receptor, beta -2.752 0.003 (angiome+immunome) PYGL phosphorylase, glycogen, liver -2.727 0.016 B (immunome) D TXNIP thioredoxin interacting protein -2.661 0.01 (angiome+immunome) ITCH itchy E3 ubiquitin protein ligase -2.641 0.028 B (immunome) CDC28 protein kinase regulatory CKS1B subunit 1B -2.639 0.014 B (immunome) JAM2 junctional adhesion molecule 2 -2.634 <0.001 B (immunome) CYB5R3 cytochrome b5 reductase 3 -2.626 0.025 B (immunome) serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), D SERPINF1 member 1 -2.62 0.003 (angiome+immunome) CUB and zona pellucida-like CUZD1 domains 1 -2.568 0.002 B (immunome) HCFC2 host cell factor C2 -2.567 0.011 B (immunome) EFNA1 ephrin-A1 -2.541 0.016 A (angiome) G (angiome+immunome+ DLL1 delta-like 1 (Drosophila) -2.537 0.015 arteriome) IL2RG interleukin 2 receptor, gamma -2.526 0.035 B (immunome) TAGLN -2.506 0.042 B (immunome) proteasome (prosome, macropain) PSMB8 subunit, beta type, 8 -2.493 0.04 B (immunome) IL15RA interleukin 15 receptor, alpha -2.468 0.004 B (immunome) 150

G Duffy blood group, atypical (angiome+immunome+ DARC chemokine receptor -2.467 0.001 arteriome) D FMOD fibromodulin -2.448 0.02 (angiome+immunome) COL8A1 collagen, type VIII, alpha 1 -2.404 0.007 A (angiome) regulator of G-protein signaling 2, D RGS2 24kDa -2.36 0.002 (angiome+immunome) GPC4 glypican 4 -2.351 0.007 F (angiome+arteriome) sterile alpha motif domain SAMD8 containing 8 -2.318 0.031 A (angiome) D NTS neurotensin -2.308 <0.001 (angiome+immunome) anti-Mullerian hormone receptor, AMHR2 type II -2.306 0.001 B (immunome) D TNC -2.306 0.01 (angiome+immunome) GCH1 GTP cyclohydrolase 1 -2.296 0.033 B (immunome) D TIMP3 TIMP metallopeptidase inhibitor 3 -2.283 0.004 (angiome+immunome) CRLF2 cytokine receptor-like factor 2 -2.253 <0.001 B (immunome) D LEPR leptin receptor -2.242 0.042 (angiome+immunome) G (angiome+immunome+ ACP1 acid phosphatase 1, soluble -2.232 <0.001 arteriome) 1-acylglycerol-3-phosphate AGPAT2 O-acyltransferase 2 -2.23 0.035 B (immunome) insulin-like growth factor 1 D IGF1 (somatomedin C) -2.219 0.016 (angiome+immunome) FADS1 fatty acid desaturase 1 -2.218 0.008 B (immunome) phosphatidylinositol glycan PIGF anchor biosynthesis, class F -2.217 0.002 A (angiome) D FMOD fibromodulin -2.215 0.026 (angiome+immunome)

151

GSR -2.21 0.004 B (immunome) PRPF31 pre-mRNA processing factor 31 -2.21 0.005 B (immunome) LIM and senescent cell LIMS1 antigen-like domains 1 -2.189 0.004 A (angiome) insulin-like growth factor 2 D IGF2 (somatomedin A) -2.182 0.006 (angiome+immunome) RGS4 regulator of G-protein signaling 4 -2.181 0.045 B (immunome) toll-interleukin 1 receptor () TIRAP domain containing adaptor protein -2.168 0.027 B (immunome) nuclear receptor subfamily 2, D NR2F2 group F, member 2 -2.167 0.027 (angiome+immunome) cysteine-rich, angiogenic inducer, CYR61 61 -2.162 0.021 A (angiome) D THBS2 thrombospondin 2 -2.152 0.045 (angiome+immunome) COL1A2 collagen, type I, alpha 2 -2.143 0.026 A (angiome) TLR4 toll-like receptor 4 -2.137 0.005 B (immunome) interferon regulatory factor 2 IRF2BP2 binding protein 2 -2.121 0.025 B (immunome) NF2 neurofibromin 2 (merlin) -2.096 0.007 B (immunome) D CTSK cathepsin K -2.091 0.035 (angiome+immunome) hematopoietically expressed D HHEX homeobox -2.084 0.039 (angiome+immunome) amyloid beta (A4) precursor D APP protein -2.08 0.006 (angiome+immunome) BTLA B and T lymphocyte associated -2.048 0.024 B (immunome) spondin 1, extracellular matrix D SPON1 protein -2.044 0.026 (angiome+immunome) interferon regulatory factor 2 IRF2BP2 binding protein 2 -2.043 0.013 B (immunome) methylenetetrahydrofolate MTHFR reductase (NAD(P)H) -2.041 0.006 B (immunome) coagulation factor II (thrombin) D F2R receptor -2.037 0.011 (angiome+immunome) 152

G (angiome+immunome+ ACP1 acid phosphatase 1, soluble -2.036 <0.001 arteriome) phosphodiesterase 5A, PDE5A cGMP-specific -2.034 0.05 B (immunome) guanine nucleotide binding GNG10 protein (G protein), gamma 10 -2.027 0.015 B (immunome) cysteine-rich, angiogenic inducer, CYR61 61 -2.01 0.022 A (angiome) nuclear factor of activated T-cells, cytoplasmic, D NFATC2 calcineurin-dependent 2 -2.01 0.013 (angiome+immunome) pellino E3 ubiquitin protein ligase PELI2 family member 2 -2.007 0.024 B (immunome) SH3-domain kinase binding SH3KBP1 protein 1 -1.999 0.005 B (immunome) COL8A1 collagen, type VIII, alpha 1 -1.987 <0.001 A (angiome) coagulation factor II (thrombin) D F2R receptor -1.981 0.002 (angiome+immunome) matrix metallopeptidase 11 D MMP11 (stromelysin 3) -1.979 0.018 (angiome+immunome) toll-interleukin 1 receptor (TIR) TIRAP domain containing adaptor protein -1.978 0.007 B (immunome) RGS5 regulator of G-protein signaling 5 -1.977 0.012 B (immunome) G ADAM metallopeptidase domain (angiome+immunome+ ADAM10 10 -1.972 0.036 arteriome) anti-Mullerian hormone receptor, AMHR2 type II -1.966 0.003 B (immunome) COL1A2 collagen, type I, alpha 2 -1.961 0.025 A (angiome) phosphoinositide-3-kinase, D PIK3R1 regulatory subunit 1 (alpha) -1.957 0.045 (angiome+immunome) single-stranded DNA binding SSBP2 protein 2 -1.948 0.007 B (immunome) CRIP1 cysteine-rich protein 1 (intestinal) -1.948 <0.001 B (immunome)

153

mitogen-activated protein kinase MAP3K5 kinase kinase 5 -1.947 0.007 B (immunome) GOLM1 golgi membrane protein 1 -1.942 0.002 B (immunome) EFNA1 ephrin-A1 -1.933 0.002 A (angiome) TBKBP1 TBK1 binding protein 1 -1.931 0.036 B (immunome) BCL6 B-cell CLL/lymphoma 6 -1.928 0.022 B (immunome) eukaryotic translation initiation EIF2AK2 factor 2-alpha kinase 2 -1.926 0.036 B (immunome) UTS2R urotensin 2 receptor -1.919 0.039 A (angiome) MYOG myogenin (myogenic factor 4) -1.916 0.031 A (angiome) COL11A1 collagen, type XI, alpha 1 -1.915 0.036 B (immunome) anti-Mullerian hormone receptor, AMHR2 type II -1.912 0.001 B (immunome) protein tyrosine phosphatase, PTPRM receptor type, M -1.901 0.032 A (angiome) solute carrier family 2 (facilitated D SLC2A1 glucose transporter), member 1 -1.9 0.011 (angiome+immunome) D ANGPTL1 angiopoietin-like 1 -1.892 0.012 (angiome+immunome) ZFP36L1 ZFP36 ring finger protein-like 1 -1.888 0.001 B (immunome) HOMER1 homer homolog 1 (Drosophila) -1.878 0.002 B (immunome) JUNB jun B proto-oncogene -1.874 0.038 B (immunome) vesicle-associated membrane VAMP3 protein 3 -1.873 0.031 B (immunome) insulin-like growth factor 1 D IGF1 (somatomedin C) -1.867 0.019 (angiome+immunome) C-type lectin domain family 3, CLEC3B member B -1.865 0.003 A (angiome) inner membrane protein, IMMT mitochondrial -1.865 0.029 B (immunome) NUP98 nucleoporin 98kDa -1.862 0.006 B (immunome) regulator of G-protein signaling 2, D RGS2 24kDa -1.861 0.002 (angiome+immunome) LUM -1.858 0.014 B (immunome) dedicator of cytokinesis 7 -1.856 0.012 B (immunome) 154

ANXA4 annexin A4 -1.854 0.001 B (immunome) 1-acylglycerol-3-phosphate AGPAT5 O-acyltransferase 5 -1.847 0.009 A (angiome) PDLIM5 PDZ and LIM domain 5 -1.845 <0.001 B (immunome) protein phosphatase 1, catalytic PPP1CC subunit, gamma isozyme -1.843 0.049 B (immunome) PBX2 pre-B-cell leukemia homeobox 2 -1.842 0.005 B (immunome) glutamic-pyruvate transaminase GPT (alanine aminotransferase) -1.841 0.027 B (immunome) ERBB receptor feedback inhibitor ERRFI1 1 -1.84 0.004 B (immunome) FBLN2 fibulin 2 -1.837 0.027 A (angiome) nuclear factor of kappa light polypeptide gene enhancer in E NFKBIZ B-cells inhibitor, zeta -1.837 0.012 (immunome_arteriome)

155

Up-regulated genes in C57BL/6 versus BALB/c mouse of nonischemic Fold Feature Name Description Change P Venn Diagram protein kinase, AMP-activated, PRKAA2 alpha 2 catalytic subunit 73.065 0.012 A (angiome) methyl-CpG binding domain MBD2 protein 2 50.154 0.003 B (immunome) fibroblast growth factor binding FGFBP1 protein 1 43.533 0.003 F (angiome+arteriome) D PTTG1 pituitary tumor-transforming 1 28.31 0.002 (angiome+immunome) RAB6B, member RAS oncogene RAB6B family 27.805 0.008 B (immunome) D PTTG1 pituitary tumor-transforming 1 24.017 0.002 (angiome+immunome) PLA2G5 phospholipase A2, group V 21.938 0.04 B (immunome) D HFE hemochromatosis 15.247 0.032 (angiome+immunome) D HFE hemochromatosis 11.614 0.002 (angiome+immunome) CD28 CD28 molecule 9.161 0.03 B (immunome) D ACTC1 actin, alpha, cardiac muscle 1 8.139 0.013 (angiome+immunome) D PTTG1 pituitary tumor-transforming 1 6.843 0.012 (angiome+immunome) E2F6 E2F transcription factor 6 4.584 0.034 B (immunome) succinate dehydrogenase complex, subunit A, flavoprotein SDHA (Fp) 4.429 0.046 B (immunome) LMAN1 lectin, mannose-binding, 1 4.263 0.03 B (immunome) D CLU clusterin 3.984 0.002 (angiome+immunome) COL9A1 collagen, type IX, alpha 1 3.823 0.029 B (immunome) G NOS1 nitric oxide synthase 1 (neuronal) 3.81 <0.001 (angiome+immunome+

156

arteriome) aldehyde dehydrogenase 2 family ALDH2 (mitochondrial) 3.78 0.035 B (immunome) G (angiome+immunome+ NOS1 nitric oxide synthase 1 (neuronal) 3.564 0.019 arteriome) PRDX2 peroxiredoxin 2 3.462 0.003 B (immunome) APOD apolipoprotein D 3.039 0.036 A (angiome) BLNK B-cell linker 3.015 0.015 B (immunome) sorbin and SH3 domain SORBS1 containing 1 3.014 0.001 B (immunome) CCL25 chemokine (C-C motif) ligand 25 2.838 0.001 B (immunome) ribosomal protein S6 kinase, RPS6KA2 90kDa, polypeptide 2 2.826 0.004 B (immunome) D CLU clusterin 2.747 0.009 (angiome+immunome) D CLU clusterin 2.699 0.009 (angiome+immunome) FZD4 frizzled family receptor 4 2.682 0.023 B (immunome) growth factor, augmenter of liver GFER regeneration 2.657 <0.001 B (immunome) D CLU clusterin 2.6 0.002 (angiome+immunome) D PTEN phosphatase and tensin homolog 2.594 0.018 (angiome+immunome) ATPase, Cu++ transporting, alpha ATP7A polypeptide 2.535 0.02 B (immunome) D IDE insulin-degrading enzyme 2.381 0.017 (angiome+immunome) E2F2 E2F transcription factor 2 2.309 0.004 B (immunome) peroxisome proliferator-activated D PPARA receptor alpha 2.303 0.017 (angiome+immunome) v-akt murine thymoma viral D AKT2 oncogene homolog 2 2.299 0.028 (angiome+immunome) DAPP1 dual adaptor of phosphotyrosine 2.242 0.011 B (immunome) 157

and 3-phosphoinositides excision repair cross-complementing rodent repair deficiency, complementation group 1 (includes overlapping antisense ERCC1 sequence) 2.229 <0.001 B (immunome) c-fos induced growth factor (vascular endothelial growth D FIGF factor D) 2.215 0.002 (angiome+immunome) TLN2 talin 2 2.184 0.005 B (immunome) c-fos induced growth factor (vascular endothelial growth D FIGF factor D) 2.16 0.007 (angiome+immunome) CCND2 2.146 0.025 B (immunome) F-box and WD repeat domain FBXW11 containing 11 2.145 0.012 B (immunome) RSF1 remodeling and spacing factor 1 2.142 0.03 F (angiome+arteriome) nuclear receptor subfamily 6, NR6A1 group A, member 1 2.12 0.034 B (immunome) DNA fragmentation factor, DFFA 45kDa, alpha polypeptide 2.109 0.003 B (immunome) TPD52 tumor protein D52 2.088 0.007 B (immunome) mitogen-activated protein kinase D MAP3K7 kinase kinase 7 2.072 0.008 (angiome+immunome) phosphatidylinositol 3-kinase, PIK3C3 catalytic subunit type 3 2.038 0.022 B (immunome) TLN2 talin 2 2.032 0.003 B (immunome) mitogen-activated protein kinase D MAP3K7 kinase kinase 7 2.008 0.008 (angiome+immunome) RAD23 homolog B (S. RAD23B cerevisiae) 1.961 0.005 B (immunome) D COL4A5 collagen, type IV, alpha 5 1.961 0.023 (angiome+immunome) FOXP2 forkhead box P2 1.93 0.008 B (immunome)

158

excision repair cross-complementing rodent repair deficiency, complementation group 1 (includes overlapping antisense ERCC1 sequence) 1.921 0.013 B (immunome) KIF1C kinesin family member 1C 1.919 0.014 B (immunome) GTF3A general transcription factor IIIA 1.889 0.003 B (immunome) growth factor, augmenter of liver GFER regeneration 1.88 0.002 B (immunome) solute carrier family 2 (facilitated D SLC2A3 glucose transporter), member 3 1.87 0.018 (angiome+immunome) G insulin-like growth factor 1 (angiome+immunome+ IGF1R receptor 1.87 0.027 arteriome) tissue factor pathway inhibitor (lipoprotein-associated D TFPI coagulation inhibitor) 1.857 0.001 (angiome+immunome) tumor necrosis factor receptor TNFRSF21 superfamily, member 21 1.841 0.011 B (immunome) dedicator of cytokinesis 8 1.836 0.023 B (immunome) transient receptor potential cation TRPM7 channel, subfamily M, member 7 1.83 0.007 B (immunome) zinc finger and BTB domain D ZBTB16 containing 16 1.825 0.02 (angiome+immunome) ATPase, Na+/K+ transporting, ATP1A2 alpha 2 polypeptide 1.823 0.008 B (immunome) D HDAC1 histone deacetylase 1 1.808 0.019 (angiome+immunome) ribosomal protein S6 kinase, RPS6KC1 52kDa, polypeptide 1 1.807 0.002 A (angiome)

159

Down-regulated genes in C57BL/6 versus BALB/c mouse of nonischemic Fold Feature Name Description Change P Venn Diagram eukaryotic translation initiation -22.26 0.041 factor 2, subunit 1 alpha, EIF2S1 35kDa B (immunome) guanylate binding protein 1, -15.15 0.042 GBP1 interferon-inducible B (immunome) plasminogen activator, -14.248 0.003 D PLAU urokinase (angiome+immunome) dentin matrix acidic -12.464 0.042 DMP1 phosphoprotein 1 F (angiome+arteriome) ATPase, Na+/K+ transporting, -10.203 0.001 ATP1A2 alpha 2 polypeptide B (immunome) interleukin 6 signal transducer -4.927 0.001 D IL6ST (gp130, oncostatin M receptor) (angiome+immunome) -4.79 0.001 D EZR ezrin (angiome+immunome) NT5E 5'-nucleotidase, ecto (CD73) -4.094 0.002 B (immunome) -4.087 <0.001 D ITGA9 integrin, alpha 9 (angiome+immunome) anti-Mullerian hormone -3.597 0.034 AMHR2 receptor, type II B (immunome) CUB and zona pellucida-like -3.577 0.001 CUZD1 domains 1 B (immunome) -3.526 0.027 D EGR1 early growth response 1 (angiome+immunome) AURKA aurora kinase A -3.453 0.009 B (immunome) GPC4 glypican 4 -3.12 0.001 F (angiome+arteriome) -3.099 0.03 D TKT transketolase (angiome+immunome) JAM2 junctional adhesion molecule 2 -3.042 0.001 B (immunome) mucosa associated lymphoid -2.93 0.032 tissue lymphoma translocation MALT1 gene 1 B (immunome)

160

-2.835 0.011 G Duffy blood group, atypical (angiome+immunome+ DARC chemokine receptor arteriome) ZFP36 ring finger protein-like -2.811 0.004 ZFP36L1 1 B (immunome) cyclin D-type binding-protein -2.775 0.014 CCNDBP1 1 B (immunome) itchy E3 ubiquitin protein -2.74 0.045 ITCH ligase B (immunome) BTLA B and T lymphocyte associated -2.722 0.035 B (immunome) FADS1 fatty acid desaturase 1 -2.718 0.031 B (immunome) guanine nucleotide binding -2.695 0.032 D GNA13 protein (G protein), alpha 13 (angiome+immunome) ANXA4 annexin A4 -2.644 0.05 B (immunome) proteasome (prosome, -2.626 0.007 macropain) subunit, beta type, PSMB9 9 B (immunome) CRLF2 cytokine receptor-like factor 2 -2.615 0.003 B (immunome) toll-interleukin 1 receptor -2.587 0.003 (TIR) domain containing TIRAP adaptor protein B (immunome) pre-mRNA processing factor -2.585 0.027 PRPF31 31 B (immunome) toll-interleukin 1 receptor -2.56 0.015 (TIR) domain containing TIRAP adaptor protein B (immunome) GOLM1 golgi membrane protein 1 -2.541 0.003 B (immunome) spondin 1, extracellular matrix -2.514 0.015 D SPON1 protein (angiome+immunome) anti-Mullerian hormone -2.456 0.012 AMHR2 receptor, type II B (immunome) cysteine-rich, angiogenic -2.452 0.013 CYR61 inducer, 61 A (angiome) nuclear factor of activated -2.426 0.003 D NFATC2 T-cells, cytoplasmic, (angiome+immunome)

161

calcineurin-dependent 2 TIMP metallopeptidase -2.404 0.001 D TIMP3 inhibitor 3 (angiome+immunome) TPMT thiopurine S-methyltransferase -2.385 0.007 A (angiome) insulin-like growth factor 1 -2.343 0.038 D IGF1 (somatomedin C) (angiome+immunome) human immunodeficiency -2.305 0.009 virus type I enhancer binding E HIVEP3 protein 3 (immunome+arteriome) single-stranded DNA binding -2.265 0.003 SSBP2 protein 2 B (immunome) MYOD1 myogenic differentiation 1 -2.258 0.008 A (angiome) matrix metallopeptidase 2 -2.25 0.019 (gelatinase A, 72kDa gelatinase, 72kDa type IV D MMP2 collagenase) (angiome+immunome) cysteine-rich, angiogenic -2.238 0.015 CYR61 inducer, 61 A (angiome) CDC7 cell division cycle 7 -2.206 0.011 B (immunome) -2.184 0.042 D APOE apolipoprotein E (angiome+immunome) nuclear factor of activated -2.173 0.048 T-cells, cytoplasmic, D NFATC2 calcineurin-dependent 2 (angiome+immunome) NUP98 nucleoporin 98kDa -2.173 0.018 B (immunome) fumarylacetoacetate hydrolase -2.156 0.02 FAH (fumarylacetoacetase) B (immunome) -2.146 0.013 G (angiome+immunome+ ACP1 acid phosphatase 1, soluble arteriome) -2.144 0.04 G interferon (alpha, beta and (angiome+immunome+ IFNAR2 omega) receptor 2 arteriome) protein tyrosine phosphatase, -2.133 0.001 PTPRM receptor type, M A (angiome)

162

solute carrier family 2 -2.124 0.005 (facilitated glucose D SLC2A1 transporter), member 1 (angiome+immunome) -2.113 0.032 D BCL2 B-cell CLL/lymphoma 2 (angiome+immunome) CDC28 protein kinase -2.11 0.001 CKS1B regulatory subunit 1B B (immunome) TAGLN transgelin -2.076 0.04 B (immunome) anti-Mullerian hormone -2.072 0.012 AMHR2 receptor, type II B (immunome) solute carrier family 9, -2.066 <0.001 subfamily A (NHE3, cation SLC9A3R proton antiporter 3), member 3 1 regulator 1 B (immunome) AHNAK AHNAK nucleoprotein -2.065 0.003 B (immunome) phosphatidylinositol glycan -2.052 0.004 PIGF anchor biosynthesis, class F A (angiome) coagulation factor II -2.045 0.023 D F2R (thrombin) receptor (angiome+immunome) -2.045 0.03 D ANGPTL1 angiopoietin-like 1 (angiome+immunome) -2.04 0.033 D LEPR leptin receptor (angiome+immunome) JUNB jun B proto-oncogene -2.031 0.047 B (immunome) pellino E3 ubiquitin protein -2.023 0.007 PELI2 ligase family member 2 B (immunome) MMRN1 -1.994 0.02 B (immunome) insulin-like growth factor 1 -1.985 0.024 D IGF1 (somatomedin C) (angiome+immunome) cysteine-rich, angiogenic -1.979 0.021 CYR61 inducer, 61 A (angiome) AHNAK AHNAK nucleoprotein -1.976 0.007 B (immunome) inhibitor of DNA binding 4, -1.973 0.05 dominant negative ID4 helix-loop-helix protein A (angiome)

163

-1.971 0.046 D THBS4 thrombospondin 4 (angiome+immunome) MYOG myogenin (myogenic factor 4) -1.968 0.025 A (angiome) PLCE1 phospholipase C, epsilon 1 -1.963 0.004 B (immunome) -1.959 0.005 G (angiome+immunome+ ACP1 acid phosphatase 1, soluble arteriome) LIM and senescent cell -1.954 0.023 LIMS1 antigen-like domains 1 A (angiome) EFNA1 ephrin-A1 -1.942 0.001 A (angiome) zinc finger, MIZ-type -1.941 0.002 D ZMIZ1 containing 1 (angiome+immunome) -1.94 0.008 D HDAC9 histone deacetylase 9 (angiome+immunome) ANXA4 annexin A4 -1.938 0.029 B (immunome) cysteine-rich protein 1 -1.935 0.01 CRIP1 (intestinal) B (immunome) telomeric repeat binding factor -1.926 0.009 TERF1 (NIMA-interacting) 1 B (immunome) PLCB4 phospholipase C, beta 4 -1.923 0.01 B (immunome) CDC28 protein kinase -1.92 0.024 CKS1B regulatory subunit 1B B (immunome) LIM domain only 2 -1.919 0.021 D LMO2 (rhombotin-like 1) (angiome+immunome) matrix metallopeptidase 11 -1.917 <0.001 D MMP11 (stromelysin 3) (angiome+immunome) regulator of G-protein -1.91 0.014 D RGS2 signaling 2, 24kDa (angiome+immunome) FBLN2 fibulin 2 -1.902 0.01 A (angiome) cysteine-rich, angiogenic -1.891 0.014 CYR61 inducer, 61 A (angiome) v- avian -1.876 0.048 musculoaponeurotic fibrosarcoma oncogene MAF homolog B (immunome)

164

-1.869 0.042 D CDK6 cyclin-dependent kinase 6 (angiome+immunome) transforming growth factor, -1.868 0.023 D TGFBR2 beta receptor II (70/80kDa) (angiome+immunome) phosphodiesterase 4B, -1.863 0.012 PDE4B cAMP-specific B (immunome) pre-B-cell leukemia homeobox -1.861 0.003 PBX2 2 B (immunome) phosphodiesterase 5A, -1.858 0.017 PDE5A cGMP-specific B (immunome) hematopoietically expressed -1.857 0.041 D HHEX homeobox (angiome+immunome) matrix metallopeptidase 2 -1.854 0.042 (gelatinase A, 72kDa gelatinase, 72kDa type IV D MMP2 collagenase) (angiome+immunome) IL33 -1.85 0.014 B (immunome) CTSC -1.84 0.017 B (immunome) TPP2 tripeptidyl peptidase II -1.833 0.01 B (immunome) NUBP2 nucleotide binding protein 2 -1.828 0.006 B (immunome) Cbl proto-oncogene B, E3 -1.826 0.024 CBLB ubiquitin protein ligase B (immunome) mitogen-activated protein -1.826 0.009 MAP3K5 kinase kinase kinase 5 B (immunome) -1.815 0.016 G (angiome+immunome+ EFNB2 ephrin-B2 arteriome) -1.814 0.036 D TKT transketolase (angiome+immunome) GULP, engulfment adaptor -1.804 0.008 GULP1 PTB domain containing 1 A (angiome) cysteine and glycine-rich -1.798 0.016 CSRP2 protein 2 B (immunome) single-stranded DNA binding -1.798 <0.001 SSBP2 protein 2 B (immunome)

165

Appendix D: 80 Equations of VEGF165b models

I. Chemical reactions

A systematic of 80 ordinary differential equations describes the temporal change of each molecular species' tissue and blood concentrations as a function of interactions and transport processes. The relevant chemical reactions are presented here (molecular species and parameters are defined in the glossary):

kon, V 165, MECM VMVM165 ECM 165 ECM koff, V 165 MECM kon, V 165, MEBM VMVM165 EBM 165 EBM koff, V 165 MEBM kon, V 165, MPBM VMVM165 PBM 165 PBM koff, V 165 MPBM

kon, V 165 b , MECM VMVM165b ECM 165 b ECM koff, V 165 b , MECM

kon, V 165 b , MEBM VMVM165b EBM 165 b EBM koff, V 165 b , MEBM

kon, V 165 b , MPBM VMVM165b PBM 165 b PBM koff, V 165 b , MPBM

kon,sR1, MECM sR11 MECM sR M ECM koff,sR1, MECM

kon,sR1, MEBM sR11 MEBM sR M EBM koff,sR1, MEBM

kon,sR1, MPBM sR11 MPBM sR M PBM koff,sR1, MPBM

kon, V 165, R 1 VRVR165 1 165 1 koff, V 165, R 1

kon, V 165, R 2 VRVR165 2  165 2 koff, V 165 R 2 166

kon, V 165, N 1 VNVN165 1 165 1 koff, V 165 N 1 kc, V 165 N 1, R 2 VNRRVN165 1 2 2 165 1 koff, V 165 N 1 R 2

kon, V 165, sR 1 V165 sR 1 V 165 sR 1 koff, V 165 sR 1

kon, V 165 b , R 1 VRVR165b  1 165b 1 koff, V 165 bR 1

k V + R ¾¾on,V 165¾b,R¾2 ®V R 165b 2 ¬¾k ¾¾¾ 165b 2 off ,V 165bR 2 k V + sR ¾¾on,V 165¾b,s¾R1®V sR 165b 1 ¬¾k ¾¾¾ 165b 1 off ,V 165bsR1

kon, V 121, R 1 VRVR121 1 121 1 koff, V 121 R 1 kon, V 121, R 2 VRVR121 2 121 2 koff, V 121 R 2

kon, V 121, R 1 N 1 VRNVRN121 1 1 121 1 1 koff, V 121 R 1 N 1

kc, V 121 R 1, N 1 VRNVRN121 1 1 121 1 1 kdissoc, V 121 R 1 N 1

kon, V 121, sR 1 V121 sR 1 V 121 sR 1 koff, V 121 sR 1

kon, V 121, sR 1 N 1 V121 sR 1 N 1 V 121 sR 1 N 1 koff, V 121 sR 1 N 1

kon, V 121 sR 1, N 1 V121 sR 1 N 1 V 121 sR 1 N 1 koff, V 121 sR 1 N 1

kon, V 165, 2 VV16522   165 koff, V 165 2

kon, V 165 b , 2 VV165bb22   165 koff, V 165 b 2

kon, V 121, 2 VV12122   121 koff, V 121 2 167

kon, 2 V 165, R 1 22VRVR165  1  165 1 koff, 2 V 165 R 1

kon, 2 V 165, R 2 22VRVR165 2   165 2 koff, 2 V 165 R 2

kon, 2 V 165, N 1 22VNVN165  1  165 1 koff, 2 V 165 N 1

kc, 2 V 165 N 1, R 2 22VNRRVN165 1  2  2 165 1 koff, 2 V 165 N 1 R 2

kc, 2 V 165 R 2, N 1 22VRNRVN1652 1   2 165 1 koff, 2 V 165 R 2 N 1

kon, 2 V 165 b , R 1 22VRVR165b  1  165b 1 koff, 2 V 165 bR 1

kon, 2 V 165 b , R 2 22VRVR165b 2   165b 2 koff, 2 V 165 bR 2

kon, 2 V 121, R 1 22VRVR121  1  121 1 koff, 2 V 121 R 1

kon, 2 V 121, R 2 22VRVR121  2  121 2 koff, 2 V 121 R 2

kon, 2 V 121, R 1 N 1 22VRNVRN121  1 1  121 1 1 koff, 2 V 121 R 1 N 1

kc, 2 V 121 R 1, N 1 22VRNVRN121 1  1  121 1 1 kdissoc, 2 V 121 R 1 N 1

kon, V 165, 2 fast VV16522 fast  fast  165 koff, V 165 2 fast

kon, V 165 b , 2 fast VV165bb22 fast  fast  165 koff, V 165 b 2 fast

kon, V 121, 2 fast VV12122 fast  fast  121 koff, V 121 2 fast

kon, 2 VR 165, 1 22 VRVR fast   fast165 1k fast 165 1 off,1 2 fast V 65R 1

kon, 2 V 165, R 2 22 VRVR fast   fast1652 k fast 1 65 2 off, 2 fas t V 165R 2

kon, 2 V 165, N 1 22 VNVN fast   fast165 1k fast 165 1 off, 2 fas t V 165N 1 168

kc, 2 V 165 N 1, R 2 22 VNRRVN fast   fast165 1 2k fast 2 165 1 off , 2 fastVNR 165 1 2

kc, 2 V 165 R 2, N 1 22 VRNRVN fast   fast16521k fast 2 165 1 off, 2 fast V 165 R 2N 1

kon, 2 V 165 b , R 1 22 VRVR fast   fast165bb 1k fast 165 1 off, 2 fast V 165 bR 1

kon, 2 V 165 b , R 2 22 VRVR fast   fast165bb2 k fast 165 2 off, 2 fast V 165 bR 2

kon, 2 VR 121, 1 22 VRVR fast   fast121 1k fast 121 1 off,1 2 fast V 21R 1

kon, 2 V 121, R 2 22 VRVR fast   fast121 2k fast 121 2 off, 2 fas t V 121R 2

kon, 2 V 121, R 1 N 1 22 VRNVRN fast   fast121 1 1k fast 121 1 1 off, 2 fast V 121 R 1 N 1

kc, 2 V 121 R 1, N1 22 VRNVRN fast   fast121 1 1k fast 121 1 1 dissoc, 22 fastV 1 1 R11 N

Receptor coupling

kc, R 1, N 1 RNRN1 1 1 1 kdissoc, R 1 N 1

Soluble receptor

kon, sR 1, MECM sR11 MECM sR M ECM koff,1 sR MECM

kon, sR 1, MEBM sR11 MEBM sR M EBM koff,1 sR MEBM

kon, sR 1, MPBM sR11 MPBM sR M PBM koff,1 sR MPBM

kon, sR 1, N 1 sR1 N 1 sR 1 N 1 koff, sR 1 N 1

II. Equations for molecular species

The complete list of ordinary differential equations is presented below:

Normal Body and Calf Muscle PAD Tissue Equations

169

I. Interstitial Matrix

[MX] denotes the concentration of unoccupied matrix binding sites in the ECM, EBM

or PBM as specified by the subscript X . []V165 and []V165b are the concentration of

interstitial free VEGF165 and VEGF165b , respectively. [sR1 ] is the concentration of interstitial free sVEGFR1. i=N for normal ECs and i=D for diseased calf muscle ECs. dM[ ] EBM i  ki [][][]V M  ki V M dt on, V 165, MEBM 165i EBM i off , V 165 MEBM 165 EBM i

i i kon, V 165 b , MEBM[][][]V 165 b i M EBM i k off , V 165 bMEBM V 165b M EBM i (S. 1)

i i kon, sR 1, MEBM[][][sR1 i M EBM ikM off , sR11 MEBM sR EBM] i dM[] PBM i  ki [][][]VM  ki V M dt on, V 165, MPBM165 iPBM i off , V 165 MPBM 165 PBM i

i i kon, V 165 b , MPBM[]VM165bP i[][]BM i k off , V 165 bMPBM V165 b M PBM i (S. 2)

ii kon, sR 1, MPBM[][][]sR1 i MPBM i k off , sR 1 MPBM sR 1 M PBM i dM[] ECM i  ki [][][]V M  ki V M dt on, V 165, MECM165 i ECM i off , V 165 MECM 165 ECM i

ii kon, V 165 b , MECM[][][]V165b i M ECM i k off , V 165 bMECM V165 b M ECM i (S. 3)

ii kon, sR 1, MECM[][][sR1 i MECM ikM off , sR 1 MECM sR 1 ECM] i d[] V M 165 EBM i kii[]][V [] M k V M (S. 4) dt on, V 165, MEBM165 i EBM i off , V 165 MEBM 16 5 EBM i d[] V M 165 PBM i kii[]][V [] M k V M (S. 5) dt on, V 165, MPBM165 i PBM i off , V 165 MPBM 16 5 PBM i d[] V M 165 ECM i kii[]][V [] M k V M (S. 6) dt on, V 165, MECM165 i ECM i off , V 165 MECM 16 5 ECM i d[ V M ] 165b EBM i kii[]]V [ M][ k V M (S. 7) dt on, V 165 b , MEBM165b i EBM i off , V 165 bMEBM 16 5 b EBMi d[ V M ] 165b PBM i kii[]]V [ M][ k V M (S. 8) dt on, V 165 b , MPBM165b i PBM i off , V 165 bMPBM 16 5 b PBMi

170 d[ V M ] 165b ECM i kii[]]V [ M][ k V M (S. 9) dt on, V 165 b , MECM165b i ECM i off , V 165 bMECM 16 5 b ECMi d[ sR M ] 1 EBM i  kkii[sR ][][]M sR M (S. 10) dt on, sR1, MEBM1 iEBM i off , sR 1 MEBM 1 EBM i d[ sR M ] 1 PBM i  kkii[sR ][][]M sR M (S. 11) dt on, sR1, MPBM1 iPBM i off , sR 1 MPBM 1 PBM i d[ sR M ] 1 ECM i  kkii[sR ][][]M sR M (S.12) dt on, sR1, MECM1 iECM i off , sR 1 MECM 1 ECM i

II. Abluminal Endothelial Cell Surface

[R1 ], [R2 ] and [N1 ] denote unoccupied VEGFR1, VEGFR2 and NRP1. [V R1 ] ,

[V R2 ] and [V N1 ] represent VEGF-bound VEGFR1, VEGFR2 and NRP1,

respectively. [R VN ] and [V RN ] are NRP1-coupled VEGF-ligated VEGFRs. kc

denoted the coupling rate between a VEGFR and the co-receptor NRP1. kdissoc denotes the

direct decoupling of VEGFR and NRP1. kint is the internalization of free or bound

receptors, and sR is the insertion rate of free receptors back in the . dR[] 1 i si  k i[][V [] R  k i V R ] dt R1 onVR , 165, 1165 i 1 i offVR , 165 1 165 1 i

ii konVbR, 165 , 1[][]V165b i[] R 1 i k offVbR , 165 1 V 165 b R 1 i (S. 13) i i kon, V 121, R 1[][] V 121ii[] R 1 k off , V 121 R 1 V 121 R 1 i i i kc, R 1, N 1[][][] N1ii R 1 k dissoc , R 1 N1 R 1N 1 i dR[] 2 i si  k i[][][][] R  k i V R  k N V R dt R2 intR , 2 2 i onVR , 121, 2 121 i 2 i offVR , 121 2 121 2 i i i konVR, 165, 2[][][] V 165 i R 2 i k offVR , 165 2 V 165 R 2 i (S. 14) i i konVbR, 165 , 2[][][] V 165 bi R 2 i k offVbR , 165 2 V 165 b R 2 i i i kc, V 165 N 1, R 2[][] V 165 N 1 i R 2 i  koff, V 165 N 1, R 2[] R 2 V 165 N 1 i

171 dN[ ] 1 i skNki  i[]]]]  i[[[ VRNk  i VRN dt N1 int , N 1 1 i c , V 121 R 1, N 1 121 1 i 1 i dissoc , V 121 R 1 N 1 121 1 1 i iN kon, V 165, N 1[[[ V 165]]] i N 1i k off , V 165 N 1 V 165 N 1 i (S. 15) ii kc, V 165 R 2, N 1[[[ V 165 R 2] i N 1]] i k off ,V 165 R 2 N 1 V 165 R 2 N 1 i i i kNc, R 1, N 1[] 1 i[][]R1i k dissoc , R 1 N 1 R 1 N 1 i d[] V R 165 1 i  ki [[[ V R]]]]  kii V R  k[ V R (S. 16) dt int, V 165 R 1 165 1i on , V 165, R 1 165 i 1 i off , V 165R 1 165 1 i d[] V R 165 2 i  ki[][][][] V R  k i V R  k i V R dt intVR, 165 2 165 2 i onVR , 165, 2 165 i 2 i offVR , 165 2 165 2 i (S. 17) ii kcVRN, 165 2, 1[][][] V 165 R 2 i N 1 i k offVRN , 165 2 1 R 2 V 165 N 1 i d[] V R 165bi 1  ki[[ V R]  k i V]]][ R  k i [ V R (S. 18) dt intV, 165 bR 1 165 b 1 i onV , 165 bR , 1 165 bi 1 i offV , 165 bR 1 165 b 1 i d[] V R 165bi 2  ki[][][][] V R  k i V R  k i V R (S. 19) dt intV, 165 bR 2 165 b 2 i onV , 165 bR , 2 165 bi 2 i offV , 165 bR 2 165 b 2 i d[] V N 165 1 i  ki[][][][] V N  k i V N  k i V N dt intVN, 165 1 165 1 i onVN , 165, 1 165 i 1 i offVN , 165 1 165 1 i (S. 20) ii kcVNR, 165 1, 2[][][] V 165 N 1 i R 2 i k offVNR , 165 1 2 R 2 V 165 N 1 i d[] R V N 2 165 1 i ki [] R V N dt int, V 165 R 2 N 1 2 165 1 i i i kcVRN, 165 2, 1[][][] V 165 R 2ii N 1 k offVRN , 165 2 1 R 2 V 165 N 1 i (S. 21) i i kc, V 165 N 1, R 2[][][] V 165 N 1ii R 2 k off , V 165 N 1 R 2 R 2 V 165 N 1 i d[] V R 121 1 i ki [] V R dt int, V 121 R 1 121 1 i i i konVR, 121, 1[][][] V 121 i R 1 i k offVR , 121 1 V 121 R 1 i (S. 22) i i kc,1,11211 R N[][][] V R i N 1 i k dissoci ,1112111RN V R N d[] V R 121 2 i  ki[][][][] V R  k i V R  k i V R (S. 23) dt int,V121R2 121 2i onVR , 121, 2 121 i 2 i offVR , 121 2 121 2? i d[] V R N 121 1 1 i ki [] V R N dt int, V 121 R 1 N 1 121 1 1 i ii kc, V 121 R 1, N 1[][][] V 121 R 1 i N 1 i k dissoc , V 121 R 1 N 1 V 121 R 1 N 1 i (S. 24) ii kon, V 121, R 1 N 1[][][] V 121 i R 1 N 1i k off ,VR 121 1Ni 1 V 121 R 1 N 1

172 d[] R N 11i ki [] R N dt int, R 1 N 1 1 1 i i i kc,1,11 R N[][][] R i N 1 i k dissoc ,1111 R N R N i (S. 25) i i kon, V 121, R 1[][][] V 121 i R 1 N 1 i k off , V 121Ri 1 V 121 R 1 N 1 d[] Vs R N 121 1 1 i ki [] Vs R N dt int, V 121 sR 1 N 1 121 1 1 i i i kon,1,11211 sR N[][][] V R i N 1 i k off ,11121 sR N Vs R 11N i (S. 26) ii kon, V 121 sR 1 N 1[][][] V 121 i R 1 N 1 i k off , V 121 sR 1 N 1 V 121s R 1 N 1 i d[] sR N 11i  ki [][][][] sRN  kii sRN  k sRN dt int,11 sR N 11 i on ,1,1 sR N 1 i 1 i off ,11 sR N 11 i (S. 27) i i kon, V 121, sR 1 N 1[][][] V 121 i sR 1 N 1 i k off , V 121, sR 1 N 1 V 121 sR 1 N 1 i

III. Interstitial Fluid

The interstitial concentrations of the complexes formed between the VEGF isoforms and

sVEGFR1 are denoted []V sR1 N . Free VEGF isoforms are secreted at constant rates of

qV from myocytes, while the free sVEGFR1 was secreted abluminally by endothelial cells

at a constant rate of qsR1 . All interstitial soluble species were subjected to lymphatic

drainage into the blood at a rate of kL . The bidirectional vascular permeability flow at rates

ij of k pV, represent the microvascular permeability of VEGF from compartment i to

compartment j (N=normal tissue, B=blood, D=PAD calf). Si B denotes the total abluminal

endothelial surface area exposed to the interstitial space of tissue i. Ui is the volume of compartment i (N=normal tissue, B=blood, D=PAD calf). The geometric conversion

factors ( SiB ,Ui and K AV ) confine the macromolecular exchange volumes to the available

173 interstitial fluid volume in the tissue compartments and the plasma volume in the blood compartment. dV[] 165 i  qi  kV[] dt V165 deg,Vi 165

ii kon, V 165, MEBM[][][] V 165 i M EBM i k off , V 165, MEBM V 165M EBM i ii kVon, V 165, MPBM[][][] 165 iM PBM i k off , V 165, MPBM V 165M PBM i ii kon, V 165, MECM[][][ V 165 i M ECM ikV off , V 165, MECM 165M ECM] i i i (S. 28) kon, V 165, R 1[][][] V 165 i R 1 i k off , V 165 R 1 V 165R 1 i i i kon, V 165, R 2[][][] V 165 i R 2 i k off , V 165 R 2 V 165R 2 i ii kon, V 165, N 1[][][] V 165 i N 1 i k off , V 165 N 1V 165N 1 i k kiB S []VSU  L p, V iB 165 i kVBi iB B [] p, V 165 B UKi AV,i UUi P dV[ ] 165bi qi  kV[] dt Vb165 deg,V 165 b i

i i kon, V 165 b , MEBM[][][] V 165 b i M EBM i k off , V 165 b , MEBM V 165 bM EBM i ii kon, V 165 b , MPBM[][][] V 165 b i M PBM i k off , V 165 b , MPBM V 165 bM PBM i i i kon, V 165 b , MECM[]] V 165 b i[ M ECM i k off , V 165 b ,MECM[]V165 bM ECM i (S. 29) ii konV, 165 bR , 1[][][] V 165 bi R 1 i k offV , 165 bR 1V 165 bR 1 i ii konVbR, 165 , 2[][ V 165 bNR 2][ N k offVbR , 165 2V 165 bR 2 ] i iB kL k p, V S iB []VSU165b iBi iB B    k p, V[]V 165 b B UUiK AV,i U i P dV[ ] 121 i  qRiik[][][][] V  ki V R  k V dt Vi121 deg,V 121 ionVR , 121, 1 121 1 ioffVR , 121 1 121 1 i i i kVonVRN, 121, 1 1[][][] 121 iRN 1 1 offVRN , 121 1 1 V 121R 1 N 1 i i i (S. 30) kon,V 121, R 2[][] V 12 i R 2 i k off, V 121 R 2[] V 121R 2 i iB kL k p,ViS B []VSU121 iBi iB B  kVp, V[] 121 B  Ui  KUUAV,i i P

174 d[] sR 1 i qi ki [s R ] dt sR1 deg, sR 1 1 i

ii kon, sR 1, MEBM[][][] sR 1 i M EBM N k off , sR 1, MEBMsR 1M EBM i ii kon, sR 1, MPBM[][][] sR 1 i M PBM N koff , sR 1, MPBM sR 1M PBM i ii kon, sR 1, MECM[][][] sR 1 i M ECM N k off , sR 1, MECM sR 1M ECM i i i kon,VR 165,1 s [][][]V165i sR 1i  k off , V 165 sR 1 V 165 sR 1 i (S. 31) ii kon, V 165 b , sR 1[][][] V 165 b i sR 1 i k off , V 165 bsR 1 V 165 b sR 1 i ii kon, V 121, sR 1[][][] V 121 i sR 1 i k off , V 121 sR 1 V 121 sR 1 i i i kon,1,1 sR N[][][] sR 1 i N 1 i k off ,11 sR NsR 11N i iB kL k p,1 sR SiB []sR SU 1 i  k Bi iB B []sR UKUUp, sR 1 1 B i AV,i i P d[] Vs R 165 1 i  ki [[[[ Vss R]]]]  kii V sR  k V R dt deg,VsR 1 165 1 i on , V 165, sR 1 165 i 1 i off , V 165 sR 1 165 1 i iB (S. 32) kL k p,1 VsR S iB []VSU165sR 1 iBi iB B  kVp, VsR 1[] 165sR 1 B UKUiAP V,i i U d[] Vs R 165bi 1  ki [[[[ Vss R]]  kii V sR]]  k V R dt deg,VsR 1 165 b 1 i on , V 165 b , sR 1 165 b i 1 i off , V 165 bsR 1 165 b 1 i iB (S. 33) kL k p,1 VsR S iB []VSU165bsR 1 iBi iB B  kVp, VsR 1[ 165 bsR 1] B UKUUi AV, i i P d[] Vs R 121 1 i ki [] Vs R dt deg,VsR 1 121 1 i ii kon, V 121, sR 1[][][] V 121 i sR 1 i k off , V 121 sR 1 V 121s R 1 i (S. 34) ii kon,1,1121 sR N[][][] Vss R 1 i N 1 i k off ,11121 R sN V R 11 N i iB kkL p,1 VsRS iB []VSU121sR 1 i Bi iB B  kVp, VsR 1[ 121sR1]B UKUUiAVP,i i

Blood compartment equations

We denote the luminal receptors and ligand-receptor complexes on endothelial cells (ECs) by the subscript i (i=N for normal ECs; i=D for diseased calf muscle ECs).

I. Luminal side of normal/ calf endothelium

175 dR[ ] 1,Bi sBBBB  k[]] R  k [[[ V R]]  k V R dt R1 int , R 1 1 B , i on , V 165, R 1 165 B 1 B , i off , V 165 R 1 165 1 B , i BB kon, V 165 b , R 1 [[[ V 165 b]]] B R 1 B , i k off , V 165 bR 1 V 165 b R 1 B , i BB kon, V 121, R 1[ V 121]] B[ R 1 B , i k off , V 121 R 1[ V 121 R 1] B , i B B kc, R 1, N 1[[[N1]]B , i R 1 B , i k dissoc , R 1 N 1 R N N 1] B , i BB kon, V 165, R 1 [ 2   V 165]]] B [ R 1 B , i  k off , V 165 R 1 [ 2   V 165 R 1 B , i BB kon, V 165 b , R 1 [ 2   V 165 b]]] B [ R 1 B , i  k off , V 165 bR 1 [ 2   V 165 b R 1 B , i B B kon, V 121, R 1[ 2[   V 121]] B R 1 B , i  k off , V 121R 1[2 VR 121 1] B , i BB kc,1,1 R N[ 2   N 1,1,]]] B i [ R B i  k dissoc ,11 R N [ 2   R1 N 1, B i BB kon, V 165, R 1 [ 2  fast  V 165]]] B [ R 1 B , i  k off , V 165 R 1 [ 2  fast  V 165 R 1 B , i BB kon, V 165 b , R 1 [2  fast  V 165 b] B [ R 1] B , i  k off , V 165 bR 1 [ 2  fastVR165 b 1] B , i BB kon, V 121, R 1[ 2  fast  V 121]]] B [ R 1 B , i  k off , V 121 R 1 [ 2  fast  V 121 R 1 B , i BB kc, R 1, N 1[ 2  fast  N 1] B , i [ R 1]] B , i  k dissoc, R 1 N 1 [ 2  fast  R1 N 1 B , i (S. 35) dR[ ] 2,Bi sBBBB  k [[[[ R]]]]  k V R  k V R dt R2 int , R 2 2 B , i on , V 165, R 2 165 B 2 B , i off , V 165 R 2 165 2 B , i BB kon, V 165 b , R 2[[[ V 165 b]]] B R 2 B , i k off , V 165 bR 2 V 165 b R 2 B , i BB kon, V 121, R 2[[[ V 121]]] B R 2 B , i k off , V 121 R 2 V 121 R 2 B , i BB kc, V 165 N 1,R2[[[V 165 N 1]]] B R 2 B , i k off , V 165 N 1, R 2 R 2 V 165 N 1 B , i BB kon, V 165, R 2[ 2   V 165]]] B [ R 2 B , i  k off , V 165 R 2 [ 2   V 165 R 2 B , i BB kon, V 165 b , R 2[2   V 165 b]]] B[ R 2 B , i  k off , V 165 bR 2 [ 2   V 165 b R 2 B , i B B kon, V 121, R 2[ 2   V 121]] B [ R 2 B ,ik off, V 121 R 2[2   V 121 R 2] B , i BB kc, V 165 N 1, R 2[ 2   V 165 N 1]]] B [ R 2 B , i  k off , V 165 N 1, R 2 [ 2   R 2 V 165 N 1 B , i BB kon, V 121, R 2[ 2  fast  V 121]]] B [ R 2 B , i  k off , V 121 R 2 [ 2  fast  V 121 R 2 B , i B B kon, V 165, R 2[ 2  fast  V 165] B[ R 2] B , i  koff, V 165 R 2[2  fastVR 165 2] B , i BB kon, V 165 b , R 2[ 2  fast  V 165 b]] B [ R 2 B , i  k off , V 165 bR 2 [ 2  fast  V 165 b R 2] B , i BB kc, V 165 N 1, R 2[2[  fast  V 165 N 1] B R 2]] B , i  k off , V 165 N 1, R 2[ 2  fast  R 2 V 165 N 1 B , i (S. 36)

176 dN[ ] 1,Bi sBBBB  k[[[[ N]]]]  k V N  k V N dt N1 int , N 1 1 B , i on , V 165, N 1 165 B 1 B , i off , V 165 N 1 165 1 B , i BB kc, V 121 R 1, N 1[[[ V 121 R 1]]] B N 1 B , i k dissoc , R 1 N 1 V 121 R 1 N 1 B , i BB kc, V 165 R 2, N 1[[ V 165 R 2]] B , i[ N 1 B , i k off , V 165 R 2, N 1 R 2 V 165 N 1] B , i BB kc,1,1 R N[[[ N 1,]]] B i R 1, B i k dissoc ,1111, R N R N B i BB kc, V 121 R 1, N 1[ 2   V 121 R 1]]] B [ N 1 B , i  k dissoc ,V121 R 1 N 1 [ 2   V 121 R 1 N 1 B , i (S. 37) BB kon, V 165, N 1[ 2   V 165]]] B [ N 1 B , i  k off , V 165 N 1 [ 2   V 165 N 1 B , i B B kc, V 165 R 2, N 1[ 2   V 165 R 2 ]]]B, i[N 1 B , i k off , V 165 R 2 N 1 [ 2   R 2 V 165 N 1 B , i BB kc, V 121 R 1, N 1[ 2  fast  V 121 R 1] B [ N 1]] B , i  k dissoc ,V121 R 1 N 1 [ 2  fast  V 121 R 1 N 1 B , i BB kon, V 165, N 1[ 2  fast  V 165]] B [ N 1] B , i  k off , V 165 N 1[ 2  fast  V 165 N 1 B , i BB kc, V 165 R 2, N 1[2[fast V165 R 2] B , i N 1]] B , i  k off , V 165 R 2 N 1 [ 2  fast  R 2 V 165 N 1 B , i d[] V R 165 1Bi ,  kBBB[][][][] V R  k V R  k V R (S. 38) dt intVR, 165 1 165 1 Bi , onV , 165, R 1 165 B 1 Bi , offVR , 165 1 165 1 Bi , d[] V R 165 2Bi ,  kBBB[][][][] V R  k V R  k V R (S. 39) dt intVR, 165 2 165 2 Bi , onV , 165, R 2 165 B 2 Bi , offVR , 165 2 165 2 Bi , d[] V R 165b 1 B , i k B[][][][] V R  k B V R  k B V R (S. 40) dt intV, 165 bR 1 165 b 1 Bi , onV , 165 bR , 1 165 bB 1 Bi , offV , 165 bR 1 165 b 1 Bi , d[] V R 165b 2 B , i k B[][][][] V R  k B V R  k B V R (S. 41) dt intV, 165 bR 2 165 b 2 Bi , onV , 165 bR , 2 165 bB 2 Bi , offV , 165 bR 2 165 b 2 Bi , d[] V N 165 1Bi ,  kBBB[][][][] V N  k V N  k V N dt int, V 165 N 1 165 1 B , i on , V 165, N 1 165 B 1 B , i off , V 165 N 1 165 1 B , i (S. 42) BB kc, V 165 N 1, R 2[][][] V 165 N 1 B , i R 2 B , i k off , V 165 N 1 R 2 R 2 V 165 N 1 B , i d[] R V N 2 165 1Bi , kB [] R V N dt int, V 165 R 2 N 1 2 165 1 B , i BB kc,1652,1 V R N[][][] V 1652 R B , i N 1 B , i k off ,16521 V R N R 2165 V N 1 B , i (S. 43) BB kc, V 165 N 1, R 2[][][] V 165 N 1 B , i R 2 B , i k off , V 165 N 1 R 2 R 2 V 165 N 1 B , i d[] V R 121 1Bi , kB [] V R dt int, V 121 R 1 121 1 B , i BB kon, V 121, R 1[][][] V 121 B , i R 1 B , i k off , V 121 R 1 V 121 R 1 B , i (S. 44) BB kc,1,11211, R N[][][] V R B i N 1, B i k dissoc ,1112111, R N V R N B i d[] V R 121 2Bi ,  kBBB[][][][] V R  k V R  k V R (S. 45) dt intVR, 121 2 121 2 Bi , onVR , 121, 2 121 B 2 Bi , offVR , 121 2 121 2 Bi , 177 d[] V R N 121 1 1Bi , kB []? V R N dt intV121 R 1 N 1 121 1 1 B , i BB kc, V 121 R 1, N 1[][][] V 121 R 1 B , i N 1 B , i k dissoc , V 121 N 1 V 121 R 1 N 1 B , i (S. 46) BB kon, V 121 R 1 N 1[][][] V 121 B R 1 N 1 B , i k off , V 121 R 1 N 1 V 121 R 1 N 1 B , i d[] R N 1 1B , ik B [] R N dt int, R 1 N 1 1 1 B , i BB kcRN,1,11,[][][] R Bi N 1, Bi k dissocRN ,1111, R N Bi (S. 47) BB konVR, 121, 1[][][] V 121 B R 1 N 1 Bi , k offVR , 121 1 V 121 R 1 N 1 Bi , d[] sR N 1 1B , i k B[][][][] sRN  k B sRN  k B sRN dt int,11 sR N 11, B i on ,1,1 sR N 1 B 1, B i off ,11 sR N 11, B i (S. 48) BB konVsRN, 121, 1 1[][][] V 121 B sR 1 N 1 Bi , k offVsRN , 121, 1 1 V 121 sR 1 N 1 Bi , d[] Vs R N 121 1 1B , ik B [] Vs R N dt int, V 121 sR 1 N 1 121 1 1 B , i BB kon,1,11211 sR N[][][] V R B N 1, B i k off ,11121 sR N Vs R 11, N B i (S. 49) BB konVsRN, 121 1 1[][][] V 121 B R 1 N 1 Bi , k offVsRN , 121 1 1 V 121s R 1 N 1 Bi , d[ 2 V R ] 165 1Bi , kBB[ 2  V ] [ R ]  k [ 2  V R ] (S. 50) dt onVR, 165, 1 165 B 1 BioffVR , , 165 1 165 1 Bi , d[ 2 V R ] 165 2Bi , kBB[ 2  V ] [ R ]  k [ 2  V R ] (S. 51) dt onVR, 165, 2 165 B 2 BioffVR , , 165 2 165 2 Bi , d[ 2 V R ] 165b 1 B , i kBB[ 2  V ] [ R ]  k [ 2  V R ] (S. 52) dt onVbR, 165 , 1 165 bB 1 Bi , offVbR , 165 1 165 b 1 Bi , d[ 2 V R ] 165b 2 B , i kBB[ 2  V ] [ R ]  k [ 2  V R ] (S. 53) dt onVbR, 165 , 2 165 bB 2 Bi , offVbR , 165 2 165 b 2 Bi , d[ 2 V N ] 165 1Bi , kBB[ 2   V ] [ N ]  k [ 2   V N ] dt on, V 165, N 1 165 B 1 B , i off , V 165 N 1 165 1 B , i (S. 54) BB kc, V 165 N 1, R 2[ 2   V 165 N 1 ] B , i [ R 2 ] B , i  k off , V 165 N 1 R 2 [ 2   R 2 V 165 N 1 ] B , i d[ 2 R V N ] 2 165 1Bi , kBB[ 2   V R ] [ N ]  k [ 2   R V N ] dt c,1652,1 V R N 1652, B i 1, B i off ,16521 V R N 21651, B i (S. 55) BB kc, V 165 N 1, R 2[ 2   V 165 N 1 ] B , i [ R 2 ] B , i  k off , V 165 N 1 R 2 [ 2   R 2 V 165 N 1 ] B , i d[ 2 V R ] 121 1Bi , kBB[ 2   V ] [ R ]  k [ 2   V R ] dt on, V 121, R 1 121 B , i 1 B , i off , V 121 R 1 121 1 B , i (S. 56) BB kc,1,1 R N[ 2   V 1211, R ] B i [ N 1, ] B i  k dissoc ,11 R N [ 2   V 12111, R N ] B i

178 d[ 2 V R ] 121 2Bi , kBB[ 2  V ] [ R ]  k [ 2  V R ] (S. 57) dt onVR, 121, 2 121 B 2 BioffVR , , 121 2 121 2 Bi , d[ 2 V R N ] 121 1 1Bi , kBB[ 2   V R ] [ N ]  k [ 2   V R N ] dt c, V 121 R 1, N 1 121 1 B , i 1 B , i dissoc , V 12 N 1 121 1 1 B , i (S. BB kon, V 121 R 1 N 1[ 2   V 121 ] B [ R 1 N 1 ] B , i  k off , V 121 R 1 N 1 [ 2   V 121 R 1 N 1 ] B , i

58) d[ 2 V R ] fast165 1 B , i kBB[ 2   V ] [ R ]  k [ 2   V R ] (S. 59) dt on, V 165, R 1 fast 165 B 1 B , i off , V 165 R 1 fast 165 1 B , i d[ 2 V R ] fast165 b 1 B , i kBB[ 2   V ] [ R ]  k [ 2   V R ] (S. 60) dt on, V 165 b , R 1 fast 165 b B 1 B , i off , V 165 bR 1 fast 165 b 1 B , i d[ 2 V N ] fast165 1 B , i kBB[ 2   V ] [ N ]  k [ 2   V N ] dt on, V 165, N 1 fast 165 B 1 B , i off , V 165 N 1 fast 165 1 B , i (S. 61) BB kc, V 165 N 1, R 2[ 2  fast  V 165 N 1 ] B , i [ R 2 ] B , i  k off , V 165 N 1 R 2 [ 2  fast  R 2 V 165 N 1 ] B , i d[ 2 R V N ] fast2 165 1 B , i kBB[ 2   V R ] [ N ]  k [ 2   R V N ] dt c,1652,1 V R N fast 1652, B i 1, B i off ,16521 V R N fast 21651, B i (S. 62) BB kc, V 165 N 1, R 2[ 2  fast  V 165 N 1 ] B , i [ R 2 ] B , i  k off , V 165 N 1 R 2 [ 2  fast  R 2 V 165 N 1 ] B , i d[ 2 V R ] fast121 1 B , i kBB[ 2   V ] [ R ]  k [ 2   V R ] dt on, V 121, R 1 fast 121 B , i 1 B , i off , V 121 R 1 fast 121 1 B , i (S. 63) BB kc,1,1 R N[ 2  fast  V 1211, R ] B i [ N 1, ] B i  k dissoc ,11 R N [ 2  fast  V 12111, R N ] B i d[ 2 V R ] fast121 2 B , i kBB[ 2   V ] [ R ]  k [ 2   V R ] (S. 64) dt on, V 121, R 2 fast 121 B 2 B , i off , V 121 R 2 fast 121 2 B , i d[ 2 V R N ] fast121 1 1 B , i kBB[ 2   V R ] [ N ]  k [ 2   V R N ] dt c, V 121 R 1, N 1 fast 121 1 B , i 1 B , i dissoc , V 121 N 1 fast 121 1 1 B , i BB kon, V 121 R 1 N 1[ 2  fast  V 121 ] B [ R 1 N 1 ] B , i  k off , V 121 R 1 N 1 [ 2  fast  V 121 R 1 N 1 ] B , i

(S. 65)

II. Plasma

179 dV[ ] 165 B  qBBB c[] Vk[][][] V R k V R dt V165 V 165 165 B on , V 165, R 1 165 B 1 B , i off , V 165 R 1 165 1 B , i BB kon, V 165, R 2[][][] V 165 B R 2 B , i k off , V 165 R 2 V 165R 2 B , i BB kon, V 165, N 1[] V 165 B[][] N 1 B , i k off , V 165 N 1 V 165N 1 B , i B B kon, V 165, 2 [V 165][][]B22 B kV off , V 165 2   165 B (S. 66) BB kon, V 165, 2 fast[][ V 165 B22 fast][] B k off , V 165 2 fast  fast  V 165 B kBN S k k NB S []V p,, V NB[]V L p V NB 165 N 165 B  U pU BK AV, N kBD S k k DB S [V ] p,, V DB[V ] L p V DB 165 D 165 B  UUpBK AV ,D dV[] 165bBqB c[] V dt V165 b V 165 b 165 b B

BB kon, V 165 b , R 1[][][] V 165 b B R 1 B , i k off , V 165 bR 1 V 165 bR 1 B , i BB kon, V 165 b , R 2[][][] V 165 b B R 2 B , i k off , V 165 bR 2V 165 bR 2 B , i BB kon, V 165 b , 2[][] V 165 b B22 B k off , V 165 b 2[] V 165 b B (S. 67) BB kon, V 165 b , 2 fast[][][] V 165 b B22 fast B k off , V 165 b 2 fast  fast  V 165 b B kBN S k k NB S []V p,, V NB[]V L p V NB 165bN 165bB  UUp BK AV, N kBD S k k DB S []V p,, V DB[]V L p V DB 165bD 165bB  UUp BK AV ,D dV[] 121 B qBBB c[][][][]V k V R k V R dt V121 V 121 121 B on , V 121, R 1 121 B 1 B , i off , V 121 R 1 121 1 B , i BB kon, V 121, R 1 N 1[][][] V 121 B R 1 N 1 B , i k off , V 121 R 1 N 1 V 121R 1 N 1 B , i BB kon, V 121, R 2[][][] V 121 B R 2 B , i k off , V 121 R 2 V 121R 2 B , i BB kon, V 121,2[V 121 ]B [ 2 ] B  k off , V 1212 [ 2   V 121 ] B (S. 68) BB kon, V 121,2fast[ V 121 ] B [ 2  fast ] B  k off , V 1212 fast [ 2]   V 121 B kSBNkS k NB []V p, V NB[V ] L p, V NB 121 N 121 B  UUp BK AV, N kBD S k k DB S []V p,, V DB[]V L p V DB 121 D 121 B   UUpB  K AV ,D 180 d[] sR1 B B qsR1  c sR 1 k deg, sR 1[]sR 1 B dt

BB kon, V 165, sR 1[][][] V 165 B sR 1 B k off , V 165 sR 1Vs 165R 1 B

BB kon, V 165 b , sR 1[][][] V 165 b B sR 1 B k off , V 165 bsR 1 V 165 b sR 1 B BB kon, V 121, sR 1[][[] V 121 B sR 1] B k off , V 121 sR 1 V 121 sR 1 B (S. 69) BB kon,1 sR ,11N[][][sR B N 1, B i k off ,1111, sR N sR N ] B i Sk kNB S [] sR kBN NB[ sR ] L p,1 sR NB 1 N p, sR 1 1 B  UUKp B AV, N Sk kDB S [] sR kBD DB[ sR ] L p DB 1 D p, sR 1UUK 1 B  p B AV ,D d[] V165s R 1 B  csR1  k deg, VsR 1[ V 165s R 1 ] B dt BB kon, V 165, sR 1[[[ V 165]]] B sR 1 B k off , V 165 sR 1 V 165s R 1 B SVRk kNB S []s kBN NB[ Vs R ] L p,1 sR NB 165 1 N (S. 70) p, sR 1 165 1 B  UUKp B AV, N S k kDB S [VRs ] kBD DB [ Vs R ] L p DB 165 1 D p, sR 1 165 1 B  U pBU K AV ,D d[] V165bBs R 1  csR1  k deg, VsR 1[ V 165 bs R 1] B dt BB kon, V 165 b , sR 1[[[ V 165 b]]] B sR 1 B k off , V 165 bsR 1 V 165 bs R 1 B SVRk kNB S []s kBN NB[ Vs R ] L p,1 sR NB 165 b 1 N (S. 71) p, sR 1 165 b 1 B  UUKp B AV, N S k kDB S []VRs kBD DB [ Vs R ] L p DB 165bD 1 p, sR 1 165 b 1 B  UUpBK AV ,D d[] V121s R 1 B  csR1  k deg, VsR 1[] V 121s R 1 B dt BB kon, V 121, sR 1[][][] V 121 B sR 1 B k off , V 121 sR 1 V 121s R 1 B BB kon,1,1121 sR N[ Vss R 1][][] B N 1, B i k off ,11121 R sN V R 11, N B i (S. 72) NB BN SVRNB kL kS p,1 sR NB [ 121s 1]N kp, sR 1[ V 121s R 1] B  UUpBK AV, N DB BD SVRDBkL k p S DB []121s 1 D kp, sR 1[ V 121s R 1] B  UUKp B AV ,D

181 d[ 2 ] B kc [ 2  ] dt syn, 2 2 B kBB[]2 ] [ V k[] 2   V on, 2 , V 165 B 165 B off, 2 V 165 165 B (S. 73) BB kVon, 2 , V 165 b[][2 ] B [ 165 b BkV off , 2 V 165 b 2   165 b] B

BB kon, 2 , V 121[][]2 ] B [ V 121 B k off , 2 V 121 2   V121 B d[ 2 ] fast B kc [ 2  ] dt syn, 2 fast 2 fast fast B kBB[][]2] [ V k 2   V on, 2 , V 165 fast B 165 B off , 2 V 165 fast 165 B (S. 74) BB kon, 2 , V 165 b[]2 fast ] B [ V 165 b B k off , 2 V 165 b[] 2  fast  V 165 b B

B B kVon, 2 , V 121[2 fast] B[ 121] B  kVoff, 2 V1 21[2 fast 121] B dV[ 2 ] 165 B  c [ 2  V ] dt  2VB 165 165 kBB[][][]22  V R k   V R on, V 165, R 1 165 B 1 B , i off , V 165 R 1 165 1 B , i (S. 75)

BB kon, V 165, R 2[][][22 V 165 B R 2 B , i k off , V 165 R 2V 165R 2] B , i BB kon, V 165, N 1[2V 165][[ BN 1] B , i k off , V 165 N 1 2V165N 1]Bi , dV[ 2 ] 165bB cV[ 2   ] dt  2V 165 b 165 b B

BB kon, V 165 b , R 1[][][]22  V 165 b B R 1 B , i k off , V 165 bR 1   V 165 bR 1 B , i (S. 76)

BB kon, V 165 b , R 2[][][]22  V 165 b B R 2 B , i k off , V 165 bR 2  V 165 bR 2 B , i dV[ 2 ] 121 B  c []2  V dt  2VB 121 121

BB kon, V 121, R 1[][][]22  V 121 B R 1 B , i k off , V 121 R 1   V 121R 1 B , i (S. 77) BB kon, V 121, R 1 N 1[][][]22 V 121 B R 1 N 1 B , i k off , V 121 R 1 N 1 V 121R 1 N 1 B , i B B kon, V 121, R 2[][]2  V 121 B R 2 B , i k off , V 121R 2[]2V 121R 2 B , i

182 dV[ 2 ] fast165 B  cV[ 2   ] dt  2fastV 165 fast 165 B

BB kon, V 165, R 1[][][]22 fast  V 165 B R 1 B , i k off , V 165 R 1  fast  V 165R 1 B , i (S. 78)

BB kVon, V 165, R 2[][]22 fast 165 BR 2 B , i k off , V 165 R 2[] fast V 165R 2 B , i B B kon, V 165, N 1[ 2 fastV165]]] B[[ N 1 B , i k off , V 165 N 12 fast V 165N 1 B , i dV[ 2 ] fast165 b B  cV[2]   dt  2fastV 165 b fast 165 b B

BB kon, V 165 b , R 1[][][]22 fast  V 165 b B R 1 B , ik off , V 165 bR 1  fast V 165 bR 1 B , i (S. 79)

BB kon, V 165 b , R 2[][][2 fast V 165 b B R 2 B , i k off , V 165 bR 22 fast V 165 bR 2] B , i dV[ 2 ] fast121 B  c []2  V dt  2fastV 121 fast 121 B

BB kon, V 121, R 1[][][]22 fastV 121 B R 1 B , i k off , V 121 R 1 fast V 121R 1 B , i BB (S. 80) kon, V 121, R 1 N 1[][]22 fast  V 121 B R 1 N 1 B , i k off , V 121 R 1 N 1[]  fast V 121R 1 N 1 B , i BB kon, V 121, R2[][][]22fast V 121 B R 2 B , i k off , V 121 R 2  fast  V 121R 2 B , i BB kon, V 121, A[][[]2 fast V 121 B A] B k off , V 121 A2  fast V 121 A B

III. Glossary

A. Concentrations

[V165], [V165b], [V121] Concentration of unbound VEGF165,

VEGF165b and VEGF121

[MECM], [MEBM], [MPBM] Concentration of VEGF binding sites in the ECM, EBM and PBM

[ViMECM], [ViMEBM], [ViMPBM] Concentration of VEGF isoform i bound to the ECM, EBM and PBM, i=165, 165b

[R1], [R2] Concentration of un-occupied VEGFR1 and VEGFR2

[N1] Concentration of un-occupied NRP1

[R1N1] Concentration of the VEGFR1-NRP1 complex

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[ViRj] Concentration of VEGF isoform i bound to VEGFR j

[ViN1] Concentration of VEGF isoform i bound to NRP1, i=165 and 121

[R2V165N1] Concentration of the

VEGFR2-VEGF165-NRP1 ternary complex

[V121R1N1] Concentration of the

VEGF121-VEGFR1-NRP1 ternary complex

[sR1] Concentration of sVEGFR1

[sR1MECM], [sR1MEBM], [sR1MPBM] Concentration of sVEGFR1bound to the ECM, EBM, and PBM

[sR1N1] Concentration of sVEGFR1bound to NRP1

[Vi sR1] Concentration of the VEGF isoform i bound to sVEGFR1, i=165, 165b, 121

[V121sR1N1] Concentration of the

VEGF121-sVEGFR1-NRP1 ternary complex [2M] Concentration of alpha-2-macroglobulin (2M)

[2M·Vi] Concentration of 2M bound to VEGF isoform i, i=165, 165b, 121

[2M·ViR1] Concentration of 2M bound to VEGF isoform i-VEGFR1, i=165, 165b, 121

[2M·ViR2] Concentration of 2M bound to VEGF isoform i-VEGFR2, i=165, 165b, 121

[2M·R2V165N1] Concentration of 2M bound to the

VEGFR2-VEGF165-NRP1 ternary complex

[2M·V121R1N1] Concentration of 2M bound to the

VEGF121-VEGFR1-NRP1 ternary complex

[2Mfast] Concentration of activated

alpha-2-macroglobulin (2Mfast)

[2Mfast ·Vi] Concentration of 2Mfast bound to VEGF 184

isoform i, i=165, 165b, 121

[2Mfast ·ViR1] Concentration of 2Mfast bound to VEGF isoform i-VEGFR1, i=165, 165b, 121

[2Mfast ·ViR2] Concentration of 2Mfast bound to VEGF isoform i-VEGFR2, i=165, 165b, 121

[2Mfast ·ViN1] Concentration of 2Mfast bound to VEGF isoform i-NRP1, i=165, 121

[2Mfast ·V121R1N1] Concentration of 2Mfast bound to the VEGF isoform i-VEGFR1-NRP1 ternary complex, i=165, 121

[2Mfast ·ViR2N1] Concentration of 2Mfast bound to the VEGF isoform i-VEGFR2-NRP1 ternary complex, i=165, 121 B. Geometric parameters

Ui Volume of compartment i (N=normal tissue, B=blood, P=plasma, D=calf muscle)

SiB Total surface area of endothelial cells at the interface of compartment i and blood (N=normal tissue, T=tumor)

KAV,i Available volume fraction in the tissue, i.e., ratio of available fluid volume to

total tissue volume Ui C. Kinetic parameters qV165, qV165b, qV121 Secretion rate of VEGF165, VEGF165b and

VEGF121 qsR1 Secretion rate of sVEGFR1 kon Kinetic binding rate koff Kinetic unbinding rate kc Kinetic coupling rate for receptors kint Internalization rate of receptors

ij k pV, Microvascular permeability of VEGF from

compartment i to compartment j (N=normal tissue, B=blood, D=PAD calf)

185 kL Lymphatic drainage rate cV165, cV165b, cV121 Rate of plasma clearance of VEGF165,

VEGF165b and VEGF121 kdeg,V Rate of degradation of VEGF isoforms

ij k p,1 sR Microvascular permeability of sVEGFR1

from compartment i to compartment j (N=normal tissue, B=blood, D=PAD calf)

ij k p,1 VsR Microvascular permeability of

VEGF-sVEGFR1 complex from compartment i to compartment j (N=normal tissue, B=blood, D=PAD calf) kdeg,sR1 Rate of degradation of sVEGFR1 kdeg,VsR1 Rate of degradation of VEGF-sVEGFR1 complex k syn,2M Rate of synthesis of 2M k syn,2Mfast Rate of synthesis of 2Mfast c2M Rate of plasma clearance of 2M c2MV Rate of plasma clearance of 2M-VEGF complex c2Mfast Rate of plasma clearance of 2Mfast c2MfastV Rate of plasma clearance of 2Mfast-VEGF complex

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Curriculum Vitae

Name: Liang-Hui Chu Date of Birth: Sep 30, 1983 City of Birth: Taipei, Taiwan

Educational History Ph.D. 2015 Biomedical Engineering Johns Hopkins University Mentors: Aleksander S. Popel, PhD M.S. 2007 Electrical Engineering National Tsing Hua University B.S. 2005 Electrical Engineering National Tsing Hua University

Professional Experience Intern 2014 summer Clinical Pharmacokinetics, MedImmune, United State Research Rotation 2010 Lab of Aleksander Popel, Johns Hopkins University Research Rotation 2009-2010 Lab of Sridevi Sarma, Johns Hopkins University Research Rotation 2009 Lab of Joel Bader, Johns Hopkins University

Scholarships Studying Abroad Scholarship from Taiwanese Ministry of

Journal Publications Chu LH, Vijay CG, Annex BH, Popel AS. Construction of PADPIN: Protein-Protein Interaction Networks of Angiogenesis, Arteriogenesis and Inflammation in Peripheral Artery Disease. (In revision) Finley S, Chu LH, Popel AS. (2014) Computational systems biology approaches to anti-angiogenic cancer drug. Drug Discovery Today, Oct 5. pii: S1359-6446(14)00395-X. Chu LH, Lee E, Bader JS, Popel AS. (2014) Angiogenesis interactome and time course microarray data reveal the distinct activation patterns in endothelial cells. PLoS One. Oct 16;9(10):e110871. Chu LH*, Rivera CG*, Popel AS, Bader JS. (2012) Constructing the Angiome - a Global Angiogenesis Protein Interaction Network. Physiol Genomics. Oct 2;44(19):915-24. *Contributed equally. Wang CY, Hsiao TH, Chu LH, Lin YL, Huang JL, Chen CH, Peck K. (2012) Unraveling virus identity by detection of depleted probes with capillary electrophoresis. Anal Chim

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Acta Jul 13;734:88-92. Chu LH, Chen BS. (2008) Construction of a cancer-perturbed protein-protein interaction network for discovery of apoptosis drug targets. BMC Syst Biol. Jun 30;2:56. Chu LH, Chen BS. (2008) Comparisons of Robustness and Sensitivity between Cancer and Normal Cells by Microarray Data. Cancer Inform. 6:165-81.

Book Chapters Rivera CG*, Chu LH*, Popel AS, Bader JS. (2012) Applications of Network Bioinformatics for Cancer Angiogenesis (from "Systems Biology in Cancer Research and Drug Discovery") *Contributed equally Chu LH, Chen BS. (2010) Chapter 24: Construction of Cancer-Perturbed Protein-Protein Interaction Network of Apoptosis for Drug Target Discovery, in Systems Biology for Signaling Networks, Springer. Chu LH, Chen BS. (2010) Chapter 12: A Systems Biology Approach to Construct a Cancer-Perturbed Protein–Protein Interaction Network for Apoptosis by Means of Microarray and Database Mining, in Medical Biostatistics for Complex Diseases, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.

Posters Chu LH, Annex BH, Bader JS, Popel AS. (2014) PADPIN: Protein-Protein Interaction Networks of Angiogenesis, Arteriogenesis, and Inflammation in Peripheral Arterial Disease. BMES Annual Meeting. Chu LH, Annex BH, Bader JS, Popel AS. (2014) PADPIN: Protein-Protein Interaction Networks of Angiogenesis, Arteriogenesis, and Inflammation in Peripheral Arterial Disease. IMAG Multiscale Modeling. Chu LH, Sarma SV. (2010) Encoding and Decoding Finger and Wrist Movements from M1 Neurons in a Primate using Point Process Models. Symposium on Control and Modeling of Biomedical Systems.

Service and Leadership 2013-2014 Director of Career Fair, BME EDGE (Extramural Development in Graduate Education) 2012 Spring Teaching Assistant in “EN.580.223 Models and Simulations” 2011 Fall Teaching Assistant in “EN.580.111 Modeling and Design”

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