The Role of GPNMB on Lymphangiogenesis

Thesis

Presented in partial fulfillment of the requirements for the degree Master of Science in the College of Graduate Studies of Northeast Ohio Medical University

Joshua Castor Bachelor of Science (B.S.) in Biomedical Engineering

Modern Anatomical Sciences Northeast Ohio Medical University

2021

Thesis Committee: Dr. Fayez Safadi (advisor) Dr. Erin Franks Dr. Chris Vinyard Dr. Michael Kelly

Abstract

Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a ubiquitous protein found in multiple tissue types in the body. While there is a novel understanding of its function in many areas of the body, namely its role in bone formation and resorption, how GPNMB affects the lymphatic system, and its lymphatic endothelial cells (LECs), is yet to be explored. Previous research of the effect of Rapamycin on LECs, an antibiotic used in treatment of lymphatic anomalies, when used as a treatment, stimulates autophagy. In addition, it showed that GPNMB was upregulated in addition to VEGF-C, a promoter of lymphangiogenesis, being downregulated and SEMA-3F, a suppressor of lymphangiogenesis, being upregulated. These findings suggest a possible link to GPNMB and autophagy and GPNMB and lymphangiogenesis. In this study, I investigated the functional role of GPNMB on LECs using C57BL/6

Primary Mouse Lymphatic Endothelial Cells through a wide range of assessments of cell functions when treated with different concentrations of recombinant osteoactivin (rOA), an orthologue of GPNMB, including cell proliferation, cell viability, gene expression of both autophagy and lymphangiogenesis genes, cell migration, and tube formation. There were observable and statistically significant differences in how lymphatic endothelial cells were affected by the addition of rOA. GPNMB appears to not affect proliferation at any concentration and appears to be cytotoxic at a minimum of 100ng/mL rOA. Based on the gene expression and tube formation experiments, GPNMB appears to stimulate lymphangiogenesis beginning at a minimum of 10ng/mL rOA and appears to stimulate the LC3 pathway in autophagy at

50ng/mL rOA. In addition, GPNMB appears to accelerate cell migration at 10ng/mL rOA but does not affect cell migration at 50ng/mL. Future studies may want to observe additional cell lines with overexpression of GPNMB, GPNMB knockouts, use of different media, modification or elimination of growth factor, repeat experiments on non-statistically significant data, and additional methods of measuring the cell functions listed above.

Acknowledgements

I would like to thank Dr. Fayez Safadi for his overwhelming support as I made my way through the entire previous year researching the material covered in this thesis. He has remained a massive help in ensuring that I steadily made my way through the thesis research and documentation during this previous difficult year in the pandemic. I would like to thank Dr. Chris Vinyard for his continued support throughout both years of the Modern Anatomical Sciences program and his constructive criticism as I made my way through the thesis. I look forward to his continued support next year as I transition from the college of graduate studies to the college of medicine. I would like to thank Dr. Dana Peterson for being my biggest cheerleader during her duration in the Modern Anatomical Sciences program and additionally helping me grow as a person professionally through the past two years. I would like to thank Dr. Erin

Franks for her support on the committee and being able to quickly step up after Dr. Peterson’s recent departure from NEOMED in addition to ensuring that the thesis was thorough. I would like to thank Dr.

Michael Kelly for his challenging questions and considerations he gave to me thanks to his expertise on the lymphatic system. His involvement in my thesis ensured that I thought more in-depth about the material I was researching and allowed me to get a detailed understanding. I would like to thank Ernesto

Solarzano Zepada, who I worked with and under this previous year in the Safadi lab. He remained patient with me even amongst his very busy schedule in the lab and was critical in all my experiments that were carried out in this study. Lastly, I would like to thank my parents for their support not just within the graduate program, but their unyielding support throughout my entire life. I would likely not be as successful as I am without their unconditional approval.

In addition, I would like to thank the Modern Anatomical Sciences program for allowing me to evolve and better myself for my professional career ahead of me. Through the program, I feel much more comfortable matriculating into medical school later this year and made connections that I hope to persist as I continue into medicine.

Vita

Graduate Researcher, Northeast Ohio Medical University ...... 2020-2021

Teaching Assistant, Northeast Ohio Medical University ...... 2020-2021

Fields of Study

Major Field: Modern Anatomical Sciences

iii

Table of Contents

Abstract ...... i

Acknowledgements ...... ii

Vita ...... iii

List of Tables and Figures ...... v

Chapter 1: Literature Review ...... 1

Chapter 2: Materials and Methods ...... 18

Chapter 3: Results ...... 24

Chapter 4: Conclusions and Discussion ...... 33

References ...... 37

List of Tables and Figures

Table 1 ...... 9

Table 2 ...... 21

Figure 1 ...... 3

Figure 2 ...... 4

Figure 3 ...... 6

Figure 4 ...... 8

Figure 5 ...... 12

Figure 6 ...... 24

Figure 7 ...... 25

Figure 8 ...... 26

Figure 9 ...... 27

Figure 10 ...... 28

Figure 11 ...... 29

Figure 12 ...... 30

Figure 13 ...... 31

Figure 14 ...... 32

Chapter 1: Literature Review/Introduction

Purpose

Currently, the effects and interactions of glycoprotein nonmetastatic melanoma protein B

(GPNMB) are unknown as it pertains directly to the lymphatic endothelial cells (LECs) within the lymphatic system. This study aims to address these interactions through multiple experiments documenting treatment on the effect of GPNMB on mouse LECs in vitro.

Lymphatic System: Development

Theories of origins of the lymphatic system have been up for debate throughout the years.

Development of the lymphatic system is largely agreed to be through lymphangiogenesis, the formation of lymphatic vessels from preexisting vessels. Some anatomists have theorized that primary lymphatic sacs bud off primitive veins and lymphatic vessels subsequently bud off these via lymphangiogenesis in a

“centrifugal” pattern. This theory was indicated by pre-molecular injection methods of dyes to characterize the earliest presence of lymphatic vessels and lymphatic sacs and was championed by

Florence Sabin (Sabin, 1902). In 1910, Huntington and McClure suggested that lymphatic organs originated from mesodermal clefts and mesenchymal spaces based on serial sectioning methods and instead develop along the course of embryonic veins, which then follows a “centripetal” model

(Huntington and McClure, 1910). More recent studies with quail somites marked by QH1, a specific endothelial antibody for identifying blood and lymphatic vessels, suggest that peripheral LECs originate from the paraxial mesoderm (Wilting et al., 2000). Further studies have suggested that LECs have a dual origin and may arise from both embryonic veins and lymphangioblasts derived from mesoderm (Wilting et al., 2006). These findings have best been characterized using animal models, including mice and zebrafish.

In mice, prospero-related homeobox 1 (PROX1) begins expression at the anterior cardinal vein approximately E9.5, which is found in isolated LECs and lymph sacs after, further suggesting a venous origin of LECs (Wigle and Oliver, 1999). Additional mice studies have suggested that LECs originate from mesenchymal cells with lymphatic and leukocytic properties that appear at approximately E10.5 to

1 later integrate into the lymphatic system (Buttler et al., 2006; Buttler et al., 2008). Zebrafish studies support a venous origin of LECs and have been shown to share multiple lymphatic characteristics like other vertebrates (Yaniv et al., 2006). Thoracic duct LECs were marked and run through an immunofluorescence, and a two-photon time-lapse was run that determined all the thoracic duct LECs marked arose from a posterior cardinal vein, providing more evidence favoring a venous origin of LECs.

The process of lymphangiogenesis will be described in specific detail in the next section.

Factors Regulating Lymphoangiogenesis/Signaling Pathways

In mice, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) is a gene previously found to have specific expression in LECs (Banerji et al., 1999). Expression of LYVE1 has been found in a subpopulation of cardinal vein venous endothelial cells, suggesting its role in lymphatic development

(Wigle et al., 2002). LYVE1, CD44 (a homolog of LYVE1), and vascular endothelial growth factor receptor 3 (VEGFR3) have all been found to play a significant role in lymphangiogenesis (Ozmen et al.,

2011; Luong et al., 2009). However, in animal models with an absence of LYVE1 and CD44, normal lymphatic development occurs, indicating that these components are not required for lymphatic differentiation or lymphangiogenesis (Luong et al., 2009). On the other hand, PROX1 is suggested to be required for lymphatic differentiation as previous experiments containing mice with a knockout gene for

PROX1 have led to no lymphatic vessel development (Wigle and Oliver, 1999). In blood endothelial cells

(BECs), PROX1 misexpression has shown that PROX1 can induce LEC-specific genes, including podoplanin (PDP) and VEGFR3 while reducing expression of genes associated with BECs (Hong et al.,

2002; Petrova et al.; 2002). Deletion of PROX1 in blood vessels causes lymphatic hypoplasia, indicating that it is mandatory for lymphatic development (Srinivasan et al., 2007). PROX1 is additionally required for maintaining mature LECs, as a previous study temporarily inactivating PROX1 caused LECs to lose identifying markers and revert to BECs (Johnson et al., 2008). The transcription of LECs can be seen in

Figure 1 below. LECs originate from the Raf-1 proto-oncogene serine/threonine kinase (RAF1)/MO15- related protein kinase (MRK)/extracellular-related kinase (ERK) axis (Wong et al., 2018). This cascade activates SRY-box transcription factor 18 (SOX18), which induces PROX1 expression by binding to its

2 promoter (Francois et al., 2008). Misexpression of PROX1 in human umbilical vein endothelial cells has led to increased cell motility, increased migration towards vascular endothelial growth factor C (VEGF-

C), and inhibition of endothelial cells sheet formation (Mishima et al., 2007).

Figure 1. Illustration of the proliferation of lymphatic endothelial cells (LECs). Black arrows indicate direct regulation, while red arrows with the flat ends indicate counter-regulation. Lymphatic differentiation is highlighted by the green arrow in the RAF1/MEK/ERK axis. (Figure obtained from Wong et al., 2018).

PROX1 knockout in mice is neonatal lethal at E14.5 (Escobedo and Oliver, 2018). Transforming growth factor-β (TGF-β) inhibits PROX1 expression, and, as a direct consequence, inhibits LEC differentiation

(Oka et al., 2008). NOTCH then works with PROX1 and chicken ovalbumin upstream provider transcription factor 2 (COUP-TFII) to produce VEGFR3 and fibroblast growth factor receptor (FGFR), which respectively play roles in LEC proliferation, migration, and sprouting when adhered with VEGF-C,

VEGF-D, and fibroblast growth factor (FGF) (Wong et al. 2018; Shin et al., 2006). Through phosphorylation, VEGFR3 affects both the PI3K/Akt and MAPK/ERK axes, which lead to LEC proliferation, migration, and spreading (Wong et al., 2018). VEGF-C controls both LEC migration and

LEC proliferation and is considered a major growth factor required for lymphangiogenesis (Lohela et al.,

2009). Transgenic and retroviral addition of VEGF-C and VEGF-D in areas of misexpression of mice

3 have been able to stimulate lymphangiogenesis (Byzova et al., 2002). The knockout gene for VEGF-C in mice leads to completely inhibited angiogenesis, which is neonatal lethal, while the knockdown gene for

VEGF-C causes hypoplasia of lymphatic vessels. (Haiko et al., 2008). However, differing from PROX1 knockout mice, LECs are fully differentiated as evidenced by the presence of common lymphatic markers in VEGF-C knockout mice (Karkkainen et al., 2004).

Figure 2. The process of lymphangiogenesis. LECs from the walls of existing lymphatic vessels migrate, and using VEGF-C and VEGF-D, are able to form primitive lymph sacs, which is induced with growth factors and proinflammatory cytokines IL-1β and TNF-α and inhibited by anti-inflammatory cytokines IL-4 and IL-13 (Figure obtained from Sáinz-Jaspeado and Claesson-Welsh, 2018).

COUP-TFII interacts with PROX1 physically to control transcription of Cyclin E1 (CCNE1), which maintains expression of VEGFR3 (Yamazaki, 2009). As VEGFR3 is activated, it forms homodimers or heterodimers with VEGFR2, which leads to receptor autophosphorylation (Dixelius et al., 2003).

VEGFR3 activation then permeates cell proliferation, migration, and survival through the PI3K and

MAPK pathways (Mäkinen et al., 2001). Mice with the knockdown gene for VEGFR3 are additionally neonatal lethal, dying at E10.5 (Haiko et al., 2008). Neuropilin-2 is another transmembrane receptor that

4 works as a coreceptor with VEGFR3 to promote lymphangiogenesis, causing abnormal lymphatic development in mice with mutations for the gene (Yuan et al., 2002). SEMA-3F plays a major role in lymphangiogenesis suppression via VEGF phosphorylation, while Neuropilin-2 acts as a coreceptor part of a holoreceptor of SEMA-3F (Ou et al., 2014). SEMA-3F knockouts lead to retarded growth, abnormal vascularization, and partially penetrant embryonic lethality (Regano et al., 2017). Neuropilin-2 knockouts leads to abnormal small lymphatic vasculature, which is either missing or significantly decreased (Yuan et al., 2002). PDP knockouts, another gene essential for lymphangiogenesis, is embryonic lethal as it prevents the formation of expanded alveolar sacs in the lungs due to its role in differentiation of type I alveolar cells (Quintanilla et al., 2019). Once LECs have been able to proliferate, they begin to undergo the process of lymphangiogenesis as seen in Figure 2 above. In addition to VEGFR3 and FGFR, angiopoietin 1 and angiopoietin 2 play a role in allowing for the growth of lymphatic vessels, which binds to Tie1 and Tie2 located on LECs (Tiechert et al., 2017). Lymphangiogenesis is modulated by cytokines, which are both pro-inflammatory and anti-inflammatory, which upregulate or downregulate lymphangiogenesis respectively (Jiang et al., 2018). Pro-inflammatory cytokines include IL-1β and TNF-

α and anti-inflammatory cytokines include IL-4 and IL-13 (Pepicelli et al., 2014; Savetsky et al., 2015).

Together, these are able to induce lymphangiogenesis from both existing venous structures and non- venous structures to build new lymphatic channels and develop a mature lymphatic system.

Lymphatic System: Structure and Function

The lymphatic system is composed of lymphatic vessels, blind-ended lymphatic capillaries, and lymphoid organs situated strategically throughout the body (Standring and Grey, 2016; Baluk et al.,

2007). Lymphoid organs include lymph nodes, tonsils, Peyer’s patches, spleen, bone marrow, and the thymus in children. Lymphatic tissue is found in varying amounts in the dermis, the dura mater of the brain, the eye, the heart, the lungs, the kidneys, the intestines, and the liver (Wong et al., 2018).

Lymphatic vessels are often found in close association with blood vessels. Lymph, or fluid contained within the lymphatic system, is predominated by lymphocytes and antigen-presenting cells meant to play a role in the body’s innate immunity and participates in the transfer of these cells from affected sites to

5 lymphoid organs. This is oftentimes instead referred to as the immune system, but the lymphatic system plays additional roles in fluid homeostasis and lipid absorption. Lymph additionally contains components within it that allow for autophagy to be carried out, a cell-mediated process that allows the body to clear itself of small bacteria and cellular debris (Standring and Grey, 2016). Lymphatic channels are found in the interstitial spaces of tissue and complement the circulatory system by assisting in maintaining homeostasis via fluid balance (Choi et al., 2012). The lymphatic system retrieves cells from extravasated from blood vessels to reduce build-up in bodily tissues. Lymphatics play a major role in several pathological processes, including participation in a pathway for tumor metastasis. Despite the significant role lymphatics play in homeostasis, lymphatics have historically remained unexplored relative to blood vasculature, though this has recently changed (Oliver and Alitalo, 2005).

LECs line the inside of lymphatic vessels, lymph nodes, and lymphatic channels. Structurally,

LECs are like that of BECs and originate from embryonic veins, however they differ by protein markers and location (Hirakawa et al., 2003). LECs are organized in discontinuous cell-cell junctions with leaflets, which open and close as needed for interstitial fluid pressure (Baluk et al., 2007).

Figure 3. Drawing demonstrating the details of the lymphatic system. (A) represents the rough gross anatomy of the lymphatic system. (B) represents the lymphatic vessels and shows the presence of anchoring filaments in red and valves in blue. (C) represents the surface appearance of the lymphatic vessels and shows the connections between LECs in cell-cell junctions highlighted in blue (Figure obtained from Butler et al., 2010).

Lymphatic capillaries lack a basement membrane or pericytes but are attached to the surrounding matrix and tissues by anchoring filaments. These anchoring filaments have a role in lymphatic vessel patency as interstitial pressure and permeability increases (Rossi et al., 2007). Lymphatic ducts have valves to prevent backflow of fluid. Like blood vessels, lymphatic capillaries empty into lymphatic vessels to drain into the main collecting trunks before returning to venous circulation. A visualization of the details of the lymphatic system can be seen in Figure 3 above.

A true lymphatic vascular system is typically found only in vertebrates (Isogai et al., 2009). Fish, amphibians, and reptiles have anatomically similar lymphatic systems to mammals, but additionally contain striated muscle fibers in the lymphatic system that allow for propulsion of the lymph throughout the body. Mammals and adult birds lack these structures (Rusznyak et al., 1967). In amphibians, reptiles, and fish, the lymphatic network appears more interconnected sinuses and sacs as opposed to strictly tubular vessels. Mammalian vessels contain more valves and lymph nodes compared to other vertebrates.

Autophagy

Autophagy is an intracellular mechanism that involves the formation of autophagosomes from phagophores to promote self-degradation as needed in the body. Autophagy is initiated by the formation of a phagophore, also known as an isolation membrane (Axe et al., 2008). Once the phagophore encounters unwanted material, it engulfs and stores the material in what is then referred to as an autophagosome. The autophagosome then fuses with a lysosome to introduce proteases to the material, breaking it down and isolating amino acids for use elsewhere in the cell (Mizushima, 2005). The full process of autophagy/autophagosome formation can be observed in Figure 4 below.

Each gene used in this study, in the form of gene markers, identified either one step or multiple steps in autophagosome formation. Beclin-1 is one of the genes involved in regulation of the formation of the phagophore, which interacts with vesicular protein sorting 34 (VPS34) in the endoplasmic reticulum to regulate phagophore formation (Backer, 2008). Beclin-1 is mono-allelically deleted in various cancers, leading cancer biologists to suggest that autophagy may have tumor suppressor properties (Liang et al.,

1999). Beclin-1 knockout is embryonic lethal, while heterozygous mice are susceptible to various cancers

(Qu et al., 2007; Liang et al, 2006). Other researchers have argued that autophagy may play a role in promoting drug resistance and tumor cell adaptation (Amaravadi et al., 2007).

Figure 4. Diagram of autophagy/autophagosome formation. (a) demonstrates the control Beclin-2 and VPS34 have over phagophore formation in the endoplasmic reticulum due to stress signaling pathways. (b) demonstrates ATG-5 and ATG-12 conjugation with the help of ATG-7 and ATG-10 as enzymes. (c) demonstrates LC3-I being modified into LC3-II and being inserted into the membrane of the forming autophagosome. (d) demonstrates autophagosomes closing around targets for degradation. (e) demonstrates fusion of an autophagosome with a lysosome to degrade the captured materials (Figure obtained from Glick et al., 2010).

Autophagy related 7 (ATG-7) is additionally responsible for the initiation of autophagy as an activating enzyme that allows for conjugation of both autophagy related 12 (ATG-12) and microtubule associated protein light chain 3 (MAP1LC3, shortened to LC3) (Xiong, 2015). ATG-7 knockout mice die one day after birth (Komatsu et al., 2005). ATG-12 conjugates with autophagy related 5 (ATG-5) in a ubiquitin- like system, which later complexes with ATG16L, which is believed to introduce curvature to the isolation membrane. The complex formation allows for multimerization, or the combination of multiple small subunits, in the phagophore. Using ATG-7, LC3-I is converted into LC3-II, which is inserted into the membrane of the phagophore on both the internal and external sides of the membrane (Barth et al.,

2010). LC3-II works along with other autophagosome receptors in the selective uptake process (Johansen and Lamark, 2011). Beclin-1 knockdown mice are typically embryonic lethal due to neonatal starvation, and heterozygous mice have a higher risk for multiple cancers, including lymphoma. Mice with ATG-7 knockout are postnatal lethal, while knockdown mice lead to higher amounts of bacteria being sheltered, and lead to a higher severity of infection (Komatsu et al., 2005; Xiong, 2015). ATG-12 knockouts are autophagy-deficient, lack the formation of LC3-II, and are postnatal lethal (Malhotra et al., 2015). Once the autophagosome has engulfed the selected material, it fuses with a lysosome, which requires the presence of both Ras-related protein Rab-7a (RAB7) and Presenilin to break down the material inside, though this step of autophagy is relatively underdeveloped in research (Gutierrez et al., 2004; Eskelinen,

2005).

Diseases Associated with Lymphatic Anomalies

Disease Description Gorham-Stout Disease Referred to as vanishing bone disease, characterized by osteolysis/breakdown of bone and overgrowth of lymphatic vessels Hodgkin’s Lymphoma Lymphatic disease characterized by cancerous cell growth of lymphocytes; contains the presence of Reed-Sternberg cells Sub-types include: Chronic lymphocytic leukemia, cutaneous B-cell lymphoma, cutaneous T-cell lymphoma, lymphoma Non-Hodgkin’s Lymphatic disease characterized by cancerous cell growth of lymphocytes; Lymphoma absent of Reed-Sternberg cells Sub-types include: Chronic lymphocytic leukemia, cutaneous B-cell lymphoma, cutaneous T-cell lymphoma, follicular lymphoma, Waldenstrom macroglobulinemia Lymphadenitis Enlargement of one or several lymph nodes Lymphangitis Inflammation of one or several lymph nodes Lymphedema Significant swelling of the arms or legs, oftentimes caused by removal of lymph nodes for cancer treatment Lymphocytosis Increase in the number of lymphocytes in the body Milroy Disease Congenital lymphedema caused by lymphatic hypoplasia due to a mutation of VEGFR3 Generalized Lymphatic Lymphatic disease characterized by overgrowth of lymphatic vessels in the Anomaly (GLA)/ lungs, pleura, bone, and/or soft tissue Lymphangiomatosis Sub-types include: Kaposiform lymphangiomatosis Table 1. List of diseases associated with lymphatic anomalies.

Lymphatic anomalies are characterized by abnormal development of the lymphatic system. A brief list of various lymphatic anomalies can be seen in Table 1 above. Gorham-Stout disease (GSD),

9 sometimes referred to as vanishing bone diseases, is a rare malformation that leads to severe and progressive bone osteolysis and overgrowth of lymphatic vessels with a poor prognosis. It is a nonhereditary disease with unknown etiology mostly found in young adults under 40 years old and infrequently in the elderly. GSD tends to affect primarily the bones of the appendicular skeleton, ribs, cranium, clavicle, and cervical spine (Ozeki and Fukao, 2019). Oftentimes, the disease can be localized to one skeletal lesion. In osteoclasts in animals with GSD, osteoclastogenic factors are more sensitive, including interleukin-6 (IL‐6), interleukin-1β (IL‐1β), and tumor necrosis factor-α (TNF-α) (Colucci et al., 2005). Histologically, GSD is distinguishable from other bone malformations, including hereditary osteolysis. In later stages of the disease, lymphatic vessels assume hemangioma-like features, which is eventually replaced by fibrous tissue (Colucci et al., 2005).

TNF-α plays an important role in immune response as well as development of lymphatic organs as a pro-inflammatory cytokine (Sáinz-Jaspeado and Claesson-Welsh, 2018). For that reason, TNF-α deficient mouse models have been reviewed for immune responses within the lymphatic system. In mice with a TNF-α knockout, immune/lymphatic development proceeds as normal, indicating it is not required for survival. However, these mice showed a decreased contact hypersensitivity response. In addition,

TNF-α deficient mice lack B-cell follicles, which indicates that follicular dendritic cell networks are unable to form and humoral response is overall reduced humoral immune response (Pasparakis et al.,

1996). In the absence of TNF-α, fibroblast reticular cells in the spleen lose their function, and T cells lose their ability to home in on the spleen (Zhao et al., 2015).

GPNMB: Structure, Function, and Regulation

GPNMB is a type I transmembrane glycoprotein encoded from the GPNMB gene measuring 572 amino acids in length. It is referred to as osteoactivin (OA) within rat orthologues, which share 65% protein identity with human GPNMB, in addition to dendritic cell heparin integrin ligand (DC-HIL) in mouse orthologues, which share 71% protein identity (Weterman et al., 2006; Jin et al., 2018; Safadi et al., 2001). GPNMB is additionally referred to as hematopoietic growth factor inducible, neurokinin-1 type

(HGFIN). It is located on the small arm of chromosome 7 (Safadi et al., 2001). It was initially

10 characterized as a gene with tumor suppression properties and preferentially expressed in low-metastatic or non-metastatic cell lines (Rose and Siegel, 2007). Additionally, GPNMB is a lesser homologue of lysosome-associated membrane protein (LAMP-1) family members. It contains an N-terminal signaling peptide, an extracellular domain with an integrin-binding motif (RGD), a polycystic kidney disease domain (PKD), a transmembrane domain, and a tail of a cytoplasmic domain containing 53 amino acids

(aa) (Jin et al., 2018; Selim et al., 2009). A visual representation of this can be seen in Figure 5 below.

This cytoplasmic tail contains a half immunoreceptor tyrosine-based activator motif (hemITAM) and a dileucine motif prior to the C-terminal. The RGD domain plays a role in integrin binding as well as cell- cell adhesion, the PKD domain plays a role in protein-protein interactions and protein-carbohydrate interactions, and the hemITAM motif and the dileucine motif both play roles in cell signaling. It contains several glycosylation sites for N- and O- glycosylation (Kuan et al., 2006). GPNMB is a part of the

Pmel17/NMB family, Pmel17 participating in the pigment biosynthesis of melanocytes (Yamaguchi and

Hearing, 2009). It contains two isoforms, a short 560aa and a longer 572aa splice variant, however there is no known difference in function between the two isoforms (Rose et al., 2010; Kuan et al., 2006). CD44 is a receptor for hyaluronic acid, but additionally has been found to bind GPNMB in osteoclast precursor cells, mesenchymal stem cells, and astrocytes. Mice with the knockout gene for CD44 have inhibition of

GPNMB’s anti-inflammatory effects in astrocytes (Neal et al., 2018).

GPNMB is often found overexpressed in various metastatic cancers, including breast cancer, prostate cancer, osteosarcoma, hepatocellular carcinoma, lung cancer, cutaneous melanoma, glioblastomas, and lymphangioleiomyomatosis (Maric et al., 2013; Kuan et al., 2006; Jin et al., 2018). As it is associated with metastatic cancers, GPNMB has become a target gene for multiple therapeutic cancer treatments (Maric et al., 2013). GPNMB appears to accelerate apoptosis and necrosis in some cancers while it appears to be protumor in others (Li et al., 2019). However, upon overexpression of GPNMB in mammary epithelium, it does not have oncogenic properties, but rather accelerates tumor onset in mouse mammary tumor virus (MMTV)/Wnt-1 mice (Maric et al., 2019).

GPNMB mRNA has been found to be expressed in long bones, calvaria, bone marrow, adipose tissue, thymus, skin, placenta, heart, kidney, lung, liver, and skeletal muscle (Bandari et al., 2003; Safadi et al., 2001; Shikano et al., 2001). To that extent, it demonstrates multiple physiological roles dependent on its location in the body and functions on more than one tissue type in an organ system. Previous research indicates that GPNMB is upregulated with injury and tissue repair (Li et al., 2010; Chung et al.,

2019; L. Lin et al., 2018; Neal et al., 1996; Frara et al., 2016). GPNMB is associated with both internal and external injuries, mainly being expressed through macrophages at the site of injury, performing tasks within the body such as promoting a balance between fibrosis and fibrolysis in the liver and promoting

M2 macrophage polarization in the kidneys during acute kidney injury (AKI) (Kumagai et al., 2015; Zhou

Figure 5. A visual representation of the GPNMB protein. Displayed are distinct sections of the protein as well as roles (Figure obtained from Maric et al., 2013). et al., 2017; Li et al., 2010). In addition, GPNtaMB has been found to be upregulated in the brain tissue of rats after a stroke, being an important factor in maintaining motor neuron stability. Degrading glycosylated GPNMB through ubiquitin in these rats led to motor neuron death (Tanaka et al., 2012).

In the musculoskeletal system, GPNMB is expressed in osteoblasts, osteoclasts, and dendritic cells (Loftus et al., 2008; Safadi et al., 2001; Sheng et al., 2008). GPNMB plays an essential role in

12 upregulating osteoblasts, downregulating osteoclasts, and upregulating autophagy (Abdelmagid et al.,

2010). A previous study has shown that inhibition of GPNMB impairs osteoblast differentiation and decreases bone matrix formation (Abdelmagid et al., 2006). GPNMB has high expression during chondrogenesis and osteogenesis involved in fracture repair after traumatic injury, the formation of new cartilage and bone respectively (Abdelmagid et al., 2010). GPNMB plays a role in mediating cell fusion for the formation of osteoclasts, physically associating with β1 or β3 integrins in osteoclasts that additionally mediates differentiation. Neutralization of GPNMB with antibodies additionally showed a decrease in osteoclast numbers and size (Sheng et al., 2008).

Within the skin, the effects of GPNMB are thoroughly understood. GPNMB is heavily involved with melanocytes, involved in development through expression in melanoblasts, and are found in late- stage melanosomes, strongly implicating a role in the overall melanosome life cycle (Loftus et al., 2008;

Hoashi et al., 2010; Tomihari et al., 2009). Ultraviolet A (UVA) irradiation, ultraviolet B (UVB) radiation, α-melanocyte-stimulating hormone (αMSH), interferon γ (IFNγ), and TNFα stimulation have all lead to upregulation of GPNMB in melanocytes (Tomihari et al., 2009). Through its RGD domain,

GPNMB can adhere melanocytes to keratinocytes while adhered to the cell surface but can additionally be found in endosomal and lysosomal parts of skin cells (Hoashi et al., 2010; Tomihari et al., 2009).

GPNMB’s PKD domain, upon sufficient glycosylation, plays a role in melanocyte differentiation and localization in conjunction with Pmel, a homologue of GPNMB (Theos et al., 2013).

In the lymphatic and immune system, GPNMB has been detected in macrophages and dendritic cells, playing a role in certain cell-cell interactions. GPNMB can mediate the adhesion of dendritic cells through its RGD domain and can suppress T-cell proliferation and activation by binding to syndecan-4 with its PKD domain. Therefore, GPNMB has an ability to modulate adaptive immunity (Huang et al.,

2012; Shikano et al., 2001). In addition to the ability of GPNMB to affect dendritic cell adhesion,

GPNMB activation in dendritic cells has been found to induce innate immune response against fungal agents through phosphorylation of the hemITAM tyrosine residue. This leads to an increase in gene and protein expression, namely increased cytokine secretion of TNFα and IL- β (Chung et al., 2009).

GPNMB Signaling/Receptor

GPNMB expression is regulated by microphthalmia-associated transcription factor (MITF) activation, as inhibition of MITF significantly reduced the expression of GPNMB as both mRNA and as a protein (Li et al., 2019). GPNMB was previously found to be able to bind to CD44, a widely expressed hyaluronan receptor with roles in cell adhesion and migration, which demonstrated reduced nuclear factor-κ B ligand (NFκB) activation and inflammatory response in macrophages (Neal et al., 2018).

GPNMB was found to additionally bind to CD44 receptors in mesenchymal stem cells and peripheral stem cells in the immune system. GPNMB is able to inhibit RANKL-induced osteolysis, which is CD44 dependent. This is mediated directly through the CD44-ERK signaling axis (Sondag et al., 2016).

GPNMB inhibition has been shown to block the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of Rapamycin (mTOR) signaling pathway, which is responsible for the formation of osteosarcoma cells (Jin et al., 2018).

Animal Models for GPNMB

GPNMB has previously been studied in rat and mouse models. In a rat model with osteopetrosis,

OA has shown overexpression by over three-fold compared to wild-type control osteocytes (Frara et al.,

2016). Overexpression of GPNMB in mice additionally leads to an increase in bone mass, enhances bone formation, stimulates proliferation and differentiation of osteoprogenitor cells, and enhances TGF-β1 expression in osteoblasts (Frara et al., 2016). In the liver, transgenic GPNMB has been shown to play a role in hepatic fibrosis attenuation alongside suppression of other genes, leading to an overall decrease in hepatic fibrosis compared to wild-type rats (Abe et al., 2007). Transgenic GPNMB additionally has been studied in mice in the nervous system. A previous study has shown that transgenic GPNMB prevents dopaminergic neurodegeneration in Parkinson’s disease mice models, reduces gliosis and microglial changes after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment, and attenuates lipopolysaccharide (LPS)-induced inflammation in microglia (Budge et al., 2020).

DBA/2J mice animal models have additionally been studied for mutations in GPNMB, as its phenotype is determined by the genes for GPNMB and tyrosine-related protein 1 (Tyrp1). These animal

14 models have shown hereditary development of glaucoma comparable to humans, however mutations in

GPNMB have been shown to prevent the development of glaucoma if deficient in bone marrow derived cells (Anderson et al., 2008). In addition, DBA/2J mice with homozygous GPNMB and Tyrp1 do not lead to glaucoma (Howell et al., 2007).

Bridging Lymphangiogenesis and GPNMB

Rapamycin, an antibiotic typically used to treat various bacterial infections including lymphatic anomalies, has recently been found to play a role in autophagy (X. Lin et al., 2018). These findings suggest that Rapamycin may mimic a role in immune system (Li et al., 2010). When applied to LECs,

Rapamycin appears to inhibit lymphangiogenesis by downregulation of VEGFR3 protein expression while additionally inhibiting LEC motility and proliferation (Luo et al., 2012). GPNMB has been previously found to stimulate autophagy via the LC3 pathway (Li et al., 2010). When Rapamycin was previously tested in LECs in the Safadi laboratory, there was a significant upregulation of autophagy and expression of GPNMB. In addition, there was a downregulation in VEGF-C markers and an upregulation of SEMA-3F markers. Based on previous research, GPNMB plays an important role in bone homeostasis via regulation of osteoblasts and osteoclasts, however, as seen in lymphatic anomalies such as GSD,

GPNMB can be linked to overall loss of bone and over-proliferation of lymphatics (Safadi et al., 2001;

Abdelmagid et al., 2010; Ozeki and Fukao, 2019). The cellular mechanism that leads to the overall loss of bone is additionally unknown. Overall, these findings suggest a possibility that GPNMB may be directly or indirectly linked to lymphangiogenesis.

As further evidence for a potential link between GPNMB and lymphangiogenesis, Dr. Mike

Dellinger from Texas Tech University (per personal communication with Dr. Fayez Safadi) did an unpublished study that showed upregulation of GPNMB in patients with lymphatic anomalies.

Specifically, when LECs were obtained from patients with lymphatic anomalies and compared tissues obtained from these patients with a control group composed of LECs from tissues of healthy patients without lymphatic anomalies. Using RNA-Seq., a real-time method for determining the presence and quantity of RNA as a sample, he determined that some genes were upregulated, and some were

15 downregulated within the LEC samples. He noted that GPNMB was upregulated four-fold in the patients with lymphatic anomalies compared to the control group’s LECs. Based on Dr. Dellinger’s work, he has previously suggested that the upregulation of GPNMB may be related to causation of the anomaly.

However, there is a possibility that GPNMB upregulation may not cause the disease, but rather be a response to the lymphatic anomaly. It is additionally possible that GPNMB may play a role directly or indirectly in lymphangiogenesis of the LECs, however it should also be considered that it may act solely as a generalized repair factor in the body and perform routine maintenance.

Hypothesis

There currently exists no research that links GPNMB to LECs when applied in vitro. Our hypothesis proposes a link between GPNMB and lymphangiogenesis. This study intends to assess the effects of recombinant GPNMB being directly applied to mouse LECs in a cell culture in vitro. It is predicted this application will lead an increase in proliferation, no change in viability, an upregulation in autophagy and lymphatic marker gene expression, an upregulated rate at which tubes are able to form in vitro, and an upregulated rate of cell migration.

Specific Aims

The study aims to assess the effects of recombinant GPNMB directly on mouse LECs in vitro. To test for these effects in a multifaceted manner, we ran various cell culture experiments to test for the effects of GPNMB when added to C57BL/6 Mouse Primary LECs in treated media as it relates to proliferation, viability, gene expression, tube formation of a lymphatic channel, and cell migration.

Multiple repeats were performed of each experiment to emphasize statistical power in the results, with multiple replicates in each experiment that was analyzed through one-way ANOVAs.

Implications of Research

This study aims to further uncover the significance of GPNMB as a transmembrane protein.

Primarily, this study focuses on the lymphatic system to define how GPNMB interacts with this system as these potential interactions are currently unknown. While a baseline of knowledge for GPNMB exists, namely in its role within the musculoskeletal system, this study aims to objectively determine if there is a

16 definitive link between GPNMB levels and LEC modulation. If our cell culture results show statistically significant differences in LEC parameters with application of GPNMB from the control, GPNMB can be suggested to play a role in lymphangiogenesis. Alternatively, if there is no or few statistically significant differences, GPNMB can be suggested to not play a role in lymphatic modulation/lymphangiogenesis.

Future work then should focus on exploring GPNMB’s role as a repair factor. Overall, if the study suggests that GPNMB plays an important role in lymphangiogenesis, then it will aid in furthering general knowledge of the physiology and potentially pathophysiology of the lymphatic system.

Chapter 2: Materials and Methods

Cell Culture

The cells used through the experiments were C57BL/6 Mouse Primary Dermal LECs (obtained from Cell Biologics). These were grown in a 37°C incubator with 5% CO2 atmosphere (obtained from

Panasonic) on 10cm cell culture plates in α-MEM (obtained from Corning Inc.) with heat inactivated 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were regularly passaged every 2-3 days using

1X PBS to wash the plate and 0.05% trypsin (obtained from Gibco Laboratories) to lift the cells off the plate.

Cell Proliferation

C57BL/6 mouse LECs were obtained from cell culture and centrifuged to be diluted in 9mL α-

MEM with heat inactivated 2% fetal bovine serum and 1% penicillin/streptomycin. The mixture of media and cells was then split into five 10mL tubes, one being used for control, and others being used for treatment with 10ng/mL, 25ng/mL, 50ng/mL, or 100ng/mL of recombinant mouse osteoactivin/GPNMB

Fc Chimera (obtained from R&D Systems; shortened to recombinant osteoactivin (rOA)). These concentration values were determined as they are all within physiological range of the body. Cells were seeded in three 96-well plates at a density of 1x104 cells and distributed in 150μL of cell media per well.

Five replicates were made for the control and each treatment in columns on the 96-well plate, and in addition a column of only cell media was added for plate blank calibration. The three 96-well plates were then incubated in a 37°C incubator with 5% CO2 atmosphere and run at 24 hours, 48 hours, and 72 hours once for each plate.

The proliferation labeling and detection kit was obtained from Thermo Fischer Scientific. 1X

HBSS buffer was prepared by diluting 0.65mL 5X HBSS buffer in 2.6mL deionized water. 1X dye binding solution was prepared by diluting 6.5μL of CyQUANT® NF dye reagent to the 1X HBSS buffer.

The cell media was aspirated off each well and 100μL of dye binding reagent was added. The plate was then incubated for 60 minutes in a 37°C incubator with 5% CO2 atmosphere and read under a fluorescence plate reader for a fluorescent signal signifying DNA synthesis at an excitation of 485nm and

18 emission detection of 530nm. The values generated for each of the wells by the plate reader were normalized in Excel by dividing each value by the average of the control values, which was then transferred to Prism, run through a one-way ANOVA with Dunnett’s test to determine statistical significance, and graphed. To give the values statistical power, the experiment was repeated three times and three one-way ANOVAs with Dunnett’s test were generated.

Cell Viability

® The same protocol as the first paragraph of cell proliferation was followed. CellTiter 96 AQueous

One Solution Reagent was thawed and 20μL was added to each well onto the cell media. The plate was then incubated for an hour in a 37°C incubator with 5% CO2 atmosphere. After incubation was complete, the cells were then read under a colorimetric plate reader with a color signifying mitochondrial activity at

490nm. The values generated for each of the wells by the plate reader were normalized in Excel by dividing each value by the average of the control values, which was then transferred to Prism, run through a one-way ANOVA with Dunnett’s test to determine statistical significance, and graphed. To give the values statistical power, the experiment was repeated three times and three one-way ANOVAs with

Dunnett’s test were generated.

Gene Expression

To isolate RNA from the cells, cells were seeded in six wells of a 6-well plate in 3mL cell media

4 at a density of 7.5x10 cells per well. These were grown overnight in a 37°C incubator with 5% CO2 atmosphere. The next morning, the cell media was aspirated off and replaced with a control cell media as well as treated cell media for 10ng/mL and 50ng/mL recombinant osteoactivin in two replicates for each.

These cells were incubated for 24 hours prior to RNA isolation.

After 24 hours had passed, the cell media was aspirated off the wells and 1mL of Trizol was added. Cells were scraped off the well plates using pipette tips and added to 1.5mL RNase free microcentrifuge tubes. 200μL of chloroform (obtained from Sigma Aldrich) was added to the Trizol and vortexed for 10 seconds, which was then centrifuged at 12,000g for 15 minutes at 4°C in an Eppendorf

5424R centrifuge. The aqueous phase was then carefully transferred off the tube and placed in a new

1.5mL tube. Isopropanol (obtained from Fischer Bioreagents) was then added to each tube in a 1:1 volume (~400μL), the tube was inverted several times, then incubated for 10 minutes at room temperature. After incubating, each tube was centrifuged at 12,000g for 10 minutes at 4°C to allow an

RNA pellet to form. The supernatant was removed by tipping each tube slowly over a waste tube. The pellet was then washed with 1mL 70% ethanol, vortexed briefly, and centrifuged at 12,000g for 5 minutes at 4°C. The supernatant was removed again as mentioned previously and the pellet was air dried for 10 minutes at room temperature. The RNA pellet was then resuspended in 20μL of RNase free water, which was vortexed and centrifuged down prior to storage in a -80°C freezer.

After completion of the RNA isolation, the samples were thawed and 1μL of purified RNA was added to a Nanodrop 2000c machine and read until a consistent value of nucleic acids with sufficient purity >1.60 280/260 was added. The nucleic acid values were then divided under 600 to determine how much RNA was needed for cDNA conversion. This was added in addition to 2μL 5x qRT SuperMix and diluted in dH2O in qPCR tubes to total 10μL in each tube. The mixture was then transferred to a Biometra

Tadvanced Analytikjena thermocycler and run for 45 minutes. Once this was completed, 32μL of RNase free water was added to the cDNA and was stored in a -20°C freezer.

Once the cDNA conversion had been completed, the forward and reverse primers for Beclin-1,

ATG-7, ATG-12, LC3-I, LC3-II, VEGF-C, VEGFR3, SEMA-3F, Neuropilin-2, PROX-I, GPNMB,

CD44, PDP, and RPL13A (all obtained from Integrated DNA Technologies) were obtained from a -20°C freezer and slowly thawed. The sequence of these primers can be seen in Table 2 below. cDNA mixtures were prepared for each primer being tested by adding 56μL RNase free water, 80μL cold SYBR® Green

Master Mix (obtained from Applied Biosystems), 8μL of the forward primer, and 8μL of the reverse primer in that order to each tube. These were vortexed for 10 seconds each and centrifuged briefly. 18μL of each primer mixture was transferred into seven wells of a qPCR mount, two for control, 10ng/mL, and

50ng/mL recombinant osteoactivin, and one for a blank with 2μL RNase free water. Two replicates of

2μL cDNA were added to each primer for each treatment and the qPCR mount was sealed. The qPCR mount was then centrifuged at 1000rpm for one minute and placed in a qPCR machine. Using StepOne

Plus software, the qPCR was run for two hours and melt curves were monitored to ensure validity of the primers. Once the qPCR was done running, data was interpreted using the 2ΔΔCT method to get relative values for gene expression. These values were then transferred to Prism, run through a one-way ANOVA with Dunnett’s test to determine statistical significance, and graphed. To give the values statistical power, each gene was repeated two times and two one-way ANOVAs with Dunnett’s test were generated for each respective gene.

Primer Forward and Reverse Sequences Beclin-1 5’-AGG CGG AGA GAT TGG ACC A-3’ 5’-AAG CGA CCC AGT CTG AAA TTA-3’ ATG-7 5’-CAT TTC AGG GGT GTC CCT TCA-3’ 5’-GCT GTG GAG CTG ATG GTC TC-3’ ATG-12 5’-TCC GAG CCA GCG GCC TAA CT-3’ 5’-AAG GAG GCG CCG GAG TAG GG-3’ LC3-I 5’-TTG GTC AAG ATC ATC CGG C-3’ 5’-GCT CAC CAT GCT GTG CTG G-3’ LC3-II 5’-CGG AGC TTT GAA CAA AGA GTG-3’ 5’-TCT CTC ACT CTC GTA CAC TTC-3’ VEGF-C 5’-AGC CAA CAG GGA ATT TGA TG-3’ 5’-CAC AGC GGC ATA CTT CTT CA-3’ VEGFR3 5’-CTG GCA AAT GGT TAC TCC ATG A-3’ 5’-ACA ACC CGT GTG TCT TCA CTG-3’ PROX-I 5’-TAC CAG GTC TAC GAC AGC ACC G-3’ 5’-GTC TTC AGA CAG GTC ATC-3’ PDP 5’-TTT CTG GAA GGT TCT CGC CC-3’ 5’-TGG GCA TTC CAT GGG TCA TC-3’ GPNMB 5’-AAT GGG TCT GGC ACC TAC TG-3’ 5’-GGC TTG TAC GCC TTG TGT TT-3’ CD44 5’-GAA TTC TGC GCC CTC GGT T-3’ 5’-CTG CCT CAG TCC GGG AGA TA-3’ SEMA-3F 5’-AGC AGA CCC AGG ACG TGA G-3’ 5’-AAG ACC ATG CGA ATA TCA GCC-3’ Neuropilin-2 5’-GCT GGC TAC ATC ACT TCC CC-3’ 5’-CAA TCC ACT CAC AGT TCT GGT G-3’ Table 2. Sequence of forward and reverse primers. Sequences are listed in order of forward and reverse primers, respectively.

Tube Formation

Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix was aliquoted and thawed from a -20°C freezer to a 4°C fridge overnight. Using cooled pipette tips, 40μL of the

Matrigel was transferred into a total of 9 wells of a pre-cooled 96-well plate. The LECs were seeded in a

96-well plate at a density of 2x104 cells per well. Cells were distributed in 2.4mL cell media and split into

21 three 1.5mL tubes for control, treatment for 10ng/mL, and 50ng/mL recombinant osteoactivin. The cells were added in 200μL of media in replicates of three for the control and each treatment. Next, the cells were monitored for qualitative changes initially every half hour for the first two hours, followed by every hour until five hours had passed, followed by images taken at 11 hours, 19 hours, and 24 hours, during which they were qualitatively observed.

Once 24 hours had passed, the cells in each well were imaged thoroughly under a microscope at both 10X and 40X, the cell media was aspirated off each well, and the cells were fixed in 2% paraformaldehyde and incubated at room temperature for 15 minutes. The completed tubes, which were surrounded by a noticeable amount of cell bodies branching out on all sides, were then manually counted in each well. These values were then run through a one-way ANOVA with Tukey’s post hoc analysis to determine statistical significance in tube length, and the values were graphed.

Cell Migration

Chamber slides (obtained from Falcon) were prepared by seeding each well with 1.25x104 cells per well in 500μL. These cells were grown in a 37°C incubator with 5% CO2 atmosphere until fully confluent. A negative control that was not scratched, a control, and a 10 ng/mL and 50 ng/mL treatment with recombinant osteoactivin was prepared and added just prior to scratching. Using a 100μL pipette tip, the control and treatments were mechanically scratched vertically down the middle of each chamber slide and checked every two hours for changes in cell migration over the course of 14 hours. Once one of the gaps in the samples showed a significant difference in gap closure compared to the initial scratch, the cell media was delicately aspirated off, the cells were fixed in 4% formalin for 15 minutes, and the cells were placed in a 4°C fridge in 1X PBS.

The chamber slides were later washed three times with 500μL 1X PBS at increments of five minutes at a time and stained with actin staining solution. The staining solution was aspirated after incubating at room temperature for 30 minutes and again washed with 500μL 1X PBS at increments of five minutes at a time. The chambers of the chamber slides were then removed to isolate the stained cells and coverslips with DAPI were added. The slides were then imaged using immunofluorescence under a

22 confocal microscope and each scratch was imaged in three different areas along the area of the scratch.

The images were added to ImageJ Analysis Software and the dark area missing cells in each image was quantified. These values were placed in a one-way ANOVA with Tukey’s post hoc analysis in Prism to determine statistical significance in between scratches, and the values were graphed. This experiment was repeated a second time on a separate chamber slide for statistical power and two one-way ANOVAs with

Dunnett’s test were generated.

Chapter 3: Results

Effects of Osteoactivin on LEC Proliferation

Proliferation was one of the first two cell functions assessed to determine how recombinant osteoactivin affected LEC growth. Running this experiment as one of the first was important as it was able to assess if rOA affected cell growth positively or adversely at a high or low concentration, which was needed for consideration in follow-up experiments.

The results of this experiment can be seen in Figure 6 below. Based on the lack of statistical significance in any of the graphs, it was determined that cell proliferation was not affected by the treatment of osteoactivin at those times at any concentration. As rOA was determined to not affect cell proliferation, all four concentrations were deemed viable to use in subsequent experiments barring that viability was not significantly affected by varying concentrations of rOA.

Figure 6. Effects of rOA on LEC Proliferation. LEC samples were treated with 10, 25, 50, and 100ng/mL recombinant osteoactivin (rOA) at (A) 24 hours, (B) 48 hours, and (C) 72 hours compared to a control. Five replicates were used for each sample. Values were normalized to relative proliferation by diving each raw data value by the average of the control. Statistical significance was determined using a one-way ANOVA with Dunnett’s test.

Effects of Osteoactivin on LEC Viability

In addition to proliferation, viability was the other one of the first two concurrent cell functions assessed to determine how gradually increasing concentrations of OA affected LEC growth. Like proliferation, viability was analyzed to assess ideal concentration parameters by identifying if cell sustainability was affected at concentrations of rOA that were too low or too high.

The results of this experiment can be seen in Figure 7 below. Based on the large error bars, none of these values were determined to be statistically significant from one another. However, it should be noted that across all graphs, 100ng/mL rOA is markedly decreased, suggesting the possibility of cytotoxicity, though a cytotoxic value may exist between 50ng/mL and 100ng/mL rOA. This finding is consistent across all three experiments. Follow up studies on this may find results that are statistically significant at 100ng/mL rOA. As a result of this finding, 100ng/mL rOA was not used as a dose for any of the following experiments, and instead a low-dose concentration of 10ng/mL rOA and a high-dose concentration of 50ng/mL rOA were used.

Figure 7. Effects of rOA on LEC viability. LEC samples were treated with 10, 25, 50, and 100ng/mL recombinant osteoactivin at (A) 24 hours, (B) 48 hours, and (C) 72 hours compared to a control. Five replicates were used for each sample. Values were normalized to relative viability by diving each raw data value by the average of the control. Statistical significance was determined using a one-way ANOVA with Dunnett’s test. Due to large error bars, no values were statistically significant.

Effects of Osteoactivin on LEC Gene Expression

To examine OA’s role in autophagy, genes were run through a qPCR to determine if genes were upregulated or downregulated in the presence of varied concentrations. The first set of genes used all focused on different stages of autophagy, which included the genes for Beclin-1, autophagy related 7

(ATG-7), autophagy related 12 (ATG-12), microtubule associated protein light chain 3 (LC3-I), and LC3-

II. Beclin-1 represents the earliest step of autophagy as it is a regulator, ATG-7 is present in intermediate steps as an enzyme, ATG-12 is needed for multimerization in an intermediate step, and LC3-I and LC3-II are needed for insertion of receptors into the phagophore relatively later. The results for this experiment can be seen in Figure 8 below. Beclin-1, ATG-7, and ATG-12 show that these values are not significantly

25 affected by the introduction of recombinant osteoactivin. However, it should be noted that these graphs are the combination of graphs across multiple experiments, which showed fluctuations in relative gene expression. LC3-I and LC3-II both consistently showed an increase at 50ng/mL rOA, however only LC3-

II showed a statistically significant increase by about 1.7-fold, which was consistent across two runs. The autophagy genes run overall suggest that OA seems to affect autophagy only through the LC3 pathway, but future qPCR runs are needed to confirm this data.

Figure 8. Relative gene expression of all genes involved in autophagy. LECs were treated with 10 and 50ng/mL recombinant osteoactivin, including (A) Beclin-1, (B) ATG-7, (C) ATG-12, (D) LC3-I, and (E) LC3-II. Statistical significance for LC3-II can be seen at 50ng/mL rOA. ** indicates a p-value <0.01.

The second set of genes included VEGF-C and its receptor, VEGFR3, both major key promoters of lymphangiogenesis. The results of these genes can be seen in Figure 9 below. Based on the data,

VEGF-C was found to be significantly increased at 50ng/mL by about three-fold, but not at 10ng/mL rOA. This indicates that at a relatively higher concentration dose, OA can upregulate the VEGF-C gene.

While VEGFR3 does show an increase, it is not significantly so, due to the wide error bars. The relative

26 gene expression values additionally do not rise between 10ng/mL and 50ng/mL of rOA, indicating that the gene may reach a point of saturation at 10ng/mL rOA or a lower concentration. Future studies may want to observe gene expression of VEGFR3 at lower concentrations to determine when this point of saturation is.

Figure 9. Relative gene expression of major promoters of lymphangiogenesis (A) VEGF-C and (B) VEGFR3. Statistical significance for VEGF-C can be seen at 50ng/mL rOA. ** indicates a p-value <0.01.

The third set of genes included PROX1 and PDP, two major genes responsible for the regulation and differentiation of LECs. The results for these genes can be seen in Figure 10 below. As seen in the graphs, both PROX1 and PDP are significantly elevated at 10ng/mL rOA and drastically elevated at

50ng/mL, a consistent finding across two different runs of both genes. Both PROX1 and PDP have an approximate five-fold increase at 10ng/mL rOA, while PROX1 has a 60-fold increase and PDP has a 25- fold increase. These findings indicate that in the presence of OA, PROX1 and PDP are major factors leading in the promotion of lymphangiogenesis as their genes are significantly upregulated, which agrees with the other lymphangiogenesis genes tested additionally showing upregulation, VEGF-C and

VEGFR3. Based on the elevation at 10ng/mL rOA, PROX1 and PDP appear to play a larger role in lymphangiogenesis in the presence of OA compared to the former genes. To determine if this increase is exponential or gradual, intermediate doses between the two, such as 25ng/mL rOA, can be run in follow- up experiments.

Figure 10. Relative gene expression of major lymphatic differentiators, (A) PROX1 and (B) PDP. Statistical significance can be seen for both PROX1 and PDP at 10ng/mL and 50ng/mL rOA. * indicates a p-value <0.05. *** indicates a p-value <0.001.

The fourth set of genes included GPNMB and its receptor, CD44, GPNMB being the treatment for this experiment while CD44 plays a role in lymphangiogenesis promotion. The results for these genes can be seen in Figure 11 below. As seen in the graph, GPNMB is increased at 10ng/mL rOA by about 50-fold and drastically increased at 50ng/mL rOA by about 500-fold. CD44 is additionally drastically increased at

50ng/mL rOA by about 40-fold. These results indicate that in the presence of a high enough concentration of GPNMB, the GPNMB gene is highly upregulated and may follow an autoregulatory loop along with

CD44. Similarly to PROX1 and PDP, intermediate doses should be run for GPNMB and CD44 to determine if these gene expression values follow a linear increase or if this suddenly increases at a specific dosage.

Figure 11. Relative gene expression of (A) GPNMB and (B) CD-44. Statistical significance can be seen for GPNMB and CD-44 at 50ng/mL rOA. ** indicates a p-value <0.01. *** indicates a p-value <0.001.

The final set of genes included SEMA-3F and its receptor, Neuropilin-2, responsible for inhibition of lymphangiogenesis. Neuropilin-2 allows for binding of VEGF-C, additionally making it a lymphangiogenesis promoter. The results for these genes can be seen in Figure 12 below. The results show a significant increase of the SEMA-3F gene at 50ng/mL rOA by about 70-fold while Neuropilin-2 shows a significant increase at both 10ng/mL by about three-fold and 50ng/mL rOA by about six-fold. As several of the lymphangiogenesis promoting genes were elevated, it may seem contradictive that lymphangiogenesis suppressing genes were additionally elevated. However, it may be possible that the introduction of a large concentration of OA could drastically raise both lymphangiogenesis promotion and lymphangiogenesis suppression to overall lead to attenuation of lymphangiogenesis. It is alternatively possible that these processes may be occurring at different points in the lymphangiogenesis process.

Figure 12. Relative gene expression of (A) SEMA-3F and (B) Neuropilin-2. Statistical significance can be seen for Neuropilin-2 at 10ng/mL and for both SEMA-3F and Neuropilin-2 at 50ng/mL rOA. * indicates a p-value <0.05. ** indicates a p-value <0.01. *** indicates a p-value <0.001.

Effects of GPNMB on LEC Cell Migration

Along with cell proliferation, cell migration is an important cell function to test as it is an important function within lymphangiogenesis as it is a critical step after LEC proliferation and prior to

LEC sprouting. By running the scratch assay experiment and allowing cells to migrate over time, we were able to tell if the addition of rOA affected cell migration rate. The results for the cell migration can be seen in Figure 13 below. Based on the data calculated from the values obtained from ImageJ, the

50ng/mL rOA group did not show a significant difference when compared to the control. The 10ng/mL rOA treatment group did show a statistically significant decrease on the first chamber slide, indicating that GPNMB appears to increase the rate of cell migration at a concentration of 10ng/mL rOA, though an ideal concentration may be present that can be further analyzed in future studies. It is possible that at

50ng/mL rOA or lower concentrations, cells that were on the periphery of the scratch die, potentially due to apoptosis caused by autophagy.

Figure 13. Results of cell migration. Immunofluorescence of first group of chambers on chamber slides, (A) negative control, (B) control, (C) 10ng/mL rOA, and (D) 50ng/mL rOA. Based on three separate pictures of the same scratch, values were calculated using ImageJ, normalized by diving each value by the average of the control, and graphed for statistical significance using a one-way ANOVA with Tukey’s post hoc analysis. (E) Chamber slide 1 shows a statistically significant decrease at 10ng/mL rOA but shows no change at 50ng/mL rOA. * indicates a p- value <0.05.

Effects of Osteoactivin on LEC Tube Formation A straightforward method of measuring how introduction of treatment may affect lymphangiogenesis is by inclusion of a tube formation assay. Through the formation of completed tubes in each well, we were able to assess if rOA affected the rate of lymphangiogenesis. The results for the tube formation experiment can be seen in Figure 14 below. Based on the number of tubes that were counted in each well, there was a statistically significant increase in tube formation/lymphangiogenesis at

50ng/mL rOA, but no significant increase at 10ng/mL rOA. However, it should be noted that qualitatively, the 10ng/mL rOA wells had more branching of forming tubes relative to the control wells.

These findings overall support that GPNMB upregulates the rate of tube formation/lymphangiogenesis at

50ng/mL rOA, but this may be increased further at higher or lower concentrations that can be tested in the future.

Figure 14. Results of tube formation. (A) control, (B) 10ng/mL rOA, and (C) 50ng/mL rOA. These wells were measured for statistical significance via hand counted tubes using a way one-ANOVA with Tukey’s post hoc analysis and graphed (D). There is statistical significance at 50ng/mL rOA, but no statistical significance at 10ng/mL rOA. * indicates a p-value <0.05.

Chapter 4: Conclusions and Discussion

Results Summary

Overall, the goal of this study was to observe the effects that recombinant OA/GPNMB has when used as a treatment for C57BL/6 Mouse Primary Dermal LECs as it pertains to cell proliferation, cell viability, gene expression of autophagy and lymphatic markers, cell migration, and tube formation. We anticipated that GPNMB would increase proliferation, have no effect on viability, upregulate autophagy and lymphatic marker gene expression, upregulate the rate at which tubes are able to form in vitro, and an upregulate the rate of cell migration. Based on the results of the experiments, OA has no effect on proliferation, and affects viability at a minimum concentration of 100ng/mL rOA, which appears to be a cytotoxic dosage. OA overall affects autophagy through LC3 pathway, a step in autophagy important for inserting receptors into the phagophore, as the LC3-I and LC3-II genes were upregulated at 50ng/mL rOA, but one of them only significantly, while values for Beclin-1, ATG-7, and ATG-12 fluctuated across different qPCR runs. Additionally, it appears to be a lymphangiogenesis promoter at 10ng/mL and

50ng/mL rOA based on the elevated VEGF-C, PROX-I, and PDP. The tube formation experiment additionally suggests that GPNMB is a statistically significant lymphangiogenesis promoter at 50ng/mL rOA. It appears to be only attenuated at 50ng/mL rOA based on the high expression of the gene SEMA-

3F. Based on these findings, GPNMB may have a direct or indirect role in promoting lymphangiogenesis via one of these genes. GPNMB itself seems to be autoregulated by binding to CD44 at a minimum of

50ng/mL rOA. In addition, at 10ng/mL rOA, GPNMB appears to increase the rate of cell migration, but does not appear to at 50ng/mL rOA. Based on these findings, the addition of recombinant OA/GPNMB may have a therapeutic use in promoting lymphangiogenesis while additionally stimulating autophagy.

These findings differ from Rapamycin, as Rapamycin inhibits lymphangiogenesis while stimulating autophagy. This conclusion introduces the possibility of using GPNMB to stimulate autophagy without inhibition of lymphangiogenesis, which may find a niche use in lymphatic anomalies where autophagy is affected but lymphangiogenesis is not.

Future Studies

As the proliferation and viability experiments were run with α-MEM with heat inactivated 2% fetal bovine serum and 1% penicillin/streptomycin, there are a few changes that can be considered for future experiments. Future experiments may use a non-heat inactivated serum to determine if the effects of recombinant osteoactivin on LECs are altered in the presence of additional growth factors from the α-

MEM. The use of 2% FBS can be lowered further or completely omitted, and cells can be allowed to grow on their own to determine if the effects of recombinant osteoactivin is not dependent on the percentage of FBS. Additional media that support the LECs growth can be substituted in these experiments.

LECs obtained from mice with the knockout gene for GPNMB/OA can be isolated in addition to mice overexpressing the GPNMB gene to observe the effects of introducing endogenous GPNMB. These in vitro studies can be further observed in in vivo studies in various areas of the body at high concentrations of known lymphatic tissue, including in the dura mater of the brain, liver, and kidneys. In the future, one may want to observe the effects of GPNMB on LECs in mice with GPNMB knockout gene and transgenic mice overexpressing the GPNMB gene. Based on the results of this experiment, mice overexpressing GPNMB is estimated have a normal phenotype as lymphangiogenesis would be attenuated. The Safadi laboratory is planning to isolate LECs from transgenic GPNMB mice ears and repeat lymphatic functions with recombinant GPNMB/OA observed in these experiments in the near future. In addition, the peptide for GPNMB can be applied in place of recombinant GPNMB/OA to see if significant changes take place after posttranslational modifications of each gene. An intermediate concentration of 25ng/mL rOA could be applied to determine how cell migration functions more gradually, whether it acts more similarly to 10ng/mL or 50ng/mL rOA.

Alternative methods of obtaining proliferation results, namely measuring proliferation of LECs through qPCR, can be used to support or challenge these results. Additionally, how GPNMB affects LEC viability can be clarified with further experiments examining the apoptotic markers after being given a cytotoxic dose. Additional concentrations of GPNMB can be administered to LECs to determine what

34 concentration cytotoxicity begins to occur. The results from this may find importance in patients containing chronic higher than baseline levels of GPNMB, as it can be indicative of cell destruction and potentially lymphatic anomalies that involve cell destruction.

As GPNMB’s role in autophagy appeared to fluctuate with each experiment, repeat experiments using ATG-7, ATG-12, and Beclin-1 can be run as these markers are critical to determining if GPNMB has a significant role in autophagy beyond the LC3 pathway. Should future experiments produce similar fluctuating results, then it may indicate that the cells when harvested for RNA were at different steps of autophagy/autophagosome formation. Additional genes that promote lymphangiogenesis can be tested as well to validate or challenge the results of this study. Seeing as GPNMB appeared to be highly upregulated along with its receptor CD44 at 50ng/mL rOA, a block of CD44 in the form of antibodies or knockouts can be used to further understand how GPNMB plays a role in lymphangiogenesis. Other genes that regulate or are regulated by the genes tested within this study that were found to have statistically significant changes can be tested to observe the potential links that GPNMB may have. To bring further clarification to the autophagy and lymphatic genes that were tested, a Western blot of the protein produced by these genes could be run for a potentially more accurate representation of GPNMB’s role in autophagy and lymphangiogenesis.

As the tube formation experiment was not run more than once with a separate one-way ANOVA, repeat experiments will be needed to either support or challenge the current results. Further experiments exploring tube formation of lymphatic channels can use a different type of Matrigel, including LDEV-free and high concentration Matrigel cultures, to determine if tube formation in the presence of recombinant

GPNMB is dependent on type of Matrigel used. In addition, tube formation can be observed by mixing cells in a 3D cell culture as opposed to the on-top culture that was performed in this study. Alternative methods of examining cell migration, including running a trans-well migration assay, can be run to support or challenge the results of this study.

Lastly, GPNMB as it plays a role in lymphatic anomalies can be explored further. To accomplish exploring this role, tissues obtained from living patients with lymphatic anomalies can be isolated, de-

35 identified, processed, embedded in paraffin, sectioned, and placed on glass slides. These glass slides can then be stained with a GPNMB antibody in an immunohistochemistry and compared to a control group possessing no lymphatic anomalies, which will also be stained with a GPNMB antibody. The tissues can be observed for staining intensity, which will signify if GPNMB is upregulated relatively more in patients with lymphatic anomalies compared to that of a control group.

Challenges Encountered

Throughout the study, there were a few sources of difficulty within the individual experiments.

The gene expression experiments were challenging because the graphs did not produce consistent results during repeat experiments, namely within the autophagy genes Beclin-1, ATG-7, and ATG-12. These results concluded that GPNMB did not overall affect autophagy in these genes, but it could be argued that these genes were at different points of autophagy when the RNA was isolated from the LECs. Within the tube formation experiments, there was oftentimes ambiguity when counting live cells under the hemacytometer, which affected cell density when seeding out the wells. Due to cell density being off, two runs of the tube formation assay were unable to proceed to the fixation process. In addition, the fixation process itself was inconsistent as cells seemed to easily lift off the Matrigel during aspiration of the cell media or the 2% paraformaldehyde. While attempting to reinforce the Matrigel matrix membrane with

1% glutaraldehyde, Matrigel was instantly polymerized during the mixing process and resources ended up being not used. Careful aspiration of the media off the cells lead to much more consistent results.

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