The Roles of the Notch2 and Notch3 Receptors in Vascular Smooth Muscle Cells DISSERTATION Presented in Partial Fulfillment of Th

The Roles of the Notch2 and Notch3 Receptors in Vascular Smooth Muscle Cells

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Jeremy Todd Baeten

The Biomedical Sciences Graduate Program

The Ohio State University

2016

Dissertation Committee:

Dr. Brenda Lilly, Advisor

Dr. Joy Lincoln

Dr. Andrea Doseff

Dr. Aaron Trask

Copyrighted by

Jeremy Todd Baeten

2016

Abstract

The Notch signaling pathway has long been intricately linked with the development and function of the vasculature. In vascular smooth muscle cells (VSMCs), Notch signaling has a great influence on phenotype and is a strong promoter of differentiation and expression of contractile genes necessary to produce a functional vessel wall. However, the role of Notch signaling in VSMC proliferation and survival is less well defined, and some cases contradictory reports are given. Also, the contributions of each individual

Notch receptor have not been clearly described. Thus, to better understand Notch signaling in VSMC phenotype, we investigated the specific roles of the predominant

Notch receptors in VSMCs as they relate to differentiation, proliferation, and survival.

We found that Notch3 promotes Platelet-Derived Growth Factor (PDGF)-induced proliferation in VSMCs, while Notch2 inhibits it. We also found that Notch3 was able to promote cell survival in response to apoptosis cues, while Notch2 had no discernible effect. Interestingly, we also found the expression of Notch2 and Notch3 were changed in response to proliferation and apoptosis inducers. Notch2 mRNA was significantly decreased after PDGF-BB treatment, a proliferation inducer, and Notch3 protein was degraded rapidly in response to induction of apoptosis. Additionally, we demonstrated

ii that Notch3’s induction of cell survival genes required MEK/ERK signaling and Notch3 was capable of increasing levels of phosphorylated ERK. Altogether, these findings demonstrate that Notch2 and Notch3 have unique functions in regulating VSMC phenotype.

In a mouse model devoid of Notch2 and Notch3 in smooth muscle cells, we were able to show that Notch2 and Notch3 are required for normal closure of the ductus arteriosus.

Animals without Notch2 in VSMCs presented with patent ductus arteriosus with increasing incidence combined with the loss of Notch3. These mice died within one day of birth and also presented with aortic dilation. These phenotypes wee correlated with a significant loss of the smooth muscle contractile genes MYH11 and ACTA2, and it has been previously reported that defects in contractile differentiation can produce the PDA phenotype. This suggests that the Notch2 and Notch3 share an overlapping role in promoting VSMC differentiation.

The Notch-regulated microRNA 145 has been strongly implicated in VSMC differentiation and also recently shown to regulate cardiovascular fibrosis. In order to better understand these connections to differentiation and fibrosis, we created and characterized a transgenic mouse for conditional expression of miR145, miR145TG. We were able to select a line that faithfully expresses the transgene and produces mature miR145 transcript. This model will allow for further investigation into miR145’s roles in promoting VSMC differentiation and mediating fibrosis.

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This dissertation is dedicated to my wife, Rebekah, and our daughter, Jenny. Everything

I’ve done and everything I do is for you both.

Acknowledgments

I would like to express my gratitude to my advisor, Brenda Lilly, and all of my committee members for pushing me to become a better scientist. Without them I would not have made it this far.

In addition, I would like to acknowledge all of the past lab members who helped me; both scientifically and emotionally. I would like to thank Ning Zhao, Cho Hao Lin, and Qing

Qing Wang who were invaluable to me as I was just gaining my footing in the lab as well as Alyssa Dole, Caleb Priest, Jackie Metheny, and Megan Lowe who made my life much easier through their hard work.

I would also like to thank all of my center mates, especially Sara Koenig, Stephanie

LaHaye, and Lindsey Anstine, as well as the Center for Cardiovascular Research at NCH as a whole. It is truly an excellent environment to do good science and I appreciate all of the help and relationships I’ve obtained through my years here. I would also like to thank my collaborator Ashley Jackson for helping me master several protocols.

Finally I would like to acknowledge my family and friends who have supported me through the long hours and hard work, the culmination of which is this dissertation.

Vita

1988...... Born---Cincinnati, OH

2006...... Fort Zumwalt South High School

2010...... B.S. Cell and Molecular Biology, Missouri

State University

2011 to present ...... Graduate Research Associate, College of

Medicine, The Ohio State University

Publications

Baeten, J. T., and Lilly, B. (2016) “Notch Signaling in Vascular Smooth Muscle Cells.”

Adv Pharmacol, Vol. 78 (In Press)

Sachdeva, J., Mahajan, A., Cheng, J,. Baeten, J. T., Lilly, B., Kuivaniemi, H., Hans, C. P.

“SMC-specific Notch1 haploinsufficiency protects against the progression of AAA by modulating CTGF expression.” (In preparation)

Baeten, J. T., Jackson, A. R., McHugh, K. M., and Lilly, B. (2015) “Loss of Notch2 and

Notch3 in vascular smooth muscle causes patent ductus arteriosus.” Genesis, 53(12), 738-

748.

Baeten, J. T., and Lilly, B. (2015) “Differential Regulation of NOTCH2 and NOTCH3

Contribute to Their Unique Functions in Vascular Smooth Muscle Cells.” J Biol Chem,

290(26), 16226-16237.

Fields of Study

Major Field: The Biomedical Sciences Graduate Program

Research Emphasis: Molecular Basis of Disease

vii

Table of Contents

Abstract ...... ii

Acknowledgments...... v

Vita ...... vi

Publications ...... vii

Fields of Study ...... vii

Table of Contents ...... viii

List of Tables ...... xiv

List of Figures ...... xv

Chapter 1: Introduction and Background ...... 1

1.1 Introduction ...... 1

1.2 The Notch Signaling Pathway in Vascular Smooth Muscle ...... 4

1.2.1 The canonical Notch signaling pathway ...... 4

1.2.2 Interaction with other signaling pathways and non-canonical signaling ...... 6

1.3 Notch Signaling in VSMC Development ...... 10

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1.3.1 Constructing a vessel wall ...... 11

1.3.2 Arterial-venous specification ...... 13

1.4 Notch Signaling and VSMC Phenotype ...... 14

1.4.1 Differentiation ...... 14

1.4.2 Proliferation ...... 15

1.4.3 Survival ...... 16

1.4.4 Extracellular matrix synthesis ...... 17

1.4.5 Migration ...... 18

1.5 Notch Signaling in Vascular Disease ...... 19

1.5.1 CADASIL Syndrome ...... 19

1.5.2 Patent Ductus Arteriosus ...... 20

1.5.3 Alagille syndrome...... 21

1.5.4 Pulmonary arterial hypertension ...... 22

1.5.5 Infantile myofibromatosis...... 22

1.5.6 Vascular Injury ...... 23

1.6 Conclusion ...... 23

1.7 Figures and Tables ...... 25

References ...... 30

Chapter 2: Differential Regulation of NOTCH2 and NOTCH3 Contribute to their Unique

Functions in Vascular Smooth Muscle Cells ...... 42

2.1 Introduction ...... 42

2.2 Materials and Methods ...... 44

2.2.1 Cell culture ...... 44

2.2.2 Quantitative RT-PCR (qPCR) ...... 44

2.2.3 RNA interference ...... 46

2.2.4 Lentiviral transduction ...... 46

2.2.5 Western blotting ...... 47

2.2.6 Proliferation Assay ...... 48

2.2.7 UV-B irradiation ...... 48

2.2.8 CASPASE3 apoptosis assay ...... 48

2.2.9 Mouse lines, genotyping, and crosses ...... 49

2.2.10 Collection of mouse aortas and isolation of aortic smooth muscle cells ...... 50

2.2.11 Statistical Analysis ...... 50

2.3 Results ...... 50

2.3.1 Notch2 and Notch3 expression are uniquely regulated by PDGF-B ...... 50

2.3.2 Notch2 and Notch3 have opposing effects on smooth muscle cell proliferation

in response to PDGF-B ...... 51

2.3.3 Notch3 is uniquely regulated by inducers of apoptosis ...... 53

2.3.4 Notch3 protects VSMCs from induction of apoptosis ...... 54

2.3.5 Notch3 promotes transcription of cell survival genes via activation of MAPK55

2.3.6 Genetic deletion of Notch3, but not Notch2 has unique effects on cell survival

of primary aortic smooth muscle cells ...... 56

2.4 Discussion ...... 57

2.5 Figures ...... 62

References ...... 74

Chapter 3: Loss of Notch2 and Notch3 in Vascular Smooth Muscle Causes Patent Ductus

Arteriosus ...... 79

3.1 Introduction ...... 79

3.2 Materials and Methods ...... 81

3.2.1 Mouse lines, genotyping, and crosses ...... 81

3.2.2 Dissection, blue latex injections, aortic diameter measurements, and collection

of vessels...... 82

3.2.3 Quantitative RT-PCR (qPCR) ...... 82

3.2.4 Embryo collection and paraffin sectioning ...... 83

3.2.5 Immunohistochemistry and immunofluorescence ...... 84

3.2.6 Statistical analysis...... 85

3.3 Results ...... 85

3.3.1 Combined loss of Notch2 and Notch3 in vascular smooth muscle causes late

embryonic lethality...... 85

3.3.2 Loss of SMC-Notch2 causes patent ductus arteriosus, (PDA) phenotype and

Notch3 heterozygosity increases incidence...... 86

3.3.3 Loss of Notch2/3 presents with aortic dilation...... 87

3.3.4 PDA phenotype is associated with a decrease in contractile protein expression.

...... 88

3.4 Discussion ...... 89

3.5 Figures and Tables ...... 93

References ...... 103

Chapter 4: Conclusions and Future Directions ...... 107

4.1 Conclusions ...... 107

4.2 Future Directions ...... 109

4.2.1 Notch2 and Notch3 in proliferation and survival in vivo ...... 109

4.2.2 Notch3 proteasomal degradation by apoptosis induction ...... 110

4.2.3 Notch3 and a non-canonical interaction with MAPK ...... 111

4.2.4 Creating chimeric Notch receptors ...... 112

4.2.5 Clinical impact ...... 113

xii

References ...... 115

References ...... 119

Appendix A : Characterizing a Transgenic Mouse Model of Conditional MicroRNA145 expression ...... 138

A.1 Introduction and Background ...... 138

A.2 Creating the miR145TG Mouse ...... 140

A.3 Characterization of the miR145TG Mouse ...... 141

A.4 MiR145 as a Fibrotic Mediator ...... 142

A.5 Future Directions ...... 144

A.6 Figures and Tables...... 148

References ...... 156

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List of Tables

Table 1.1 Vascular diseases associated with mutations of Notch pathway components. . 25

Table 3.1 Combined deficiency of Notch2 in smooth muscle and Notch3 causes embryonic lethality...... 93

Table 3.2. Notch2-null mice display patent ductus arteriosus and incidence is increased with Notch3-heterozygosity...... 94

Table 3.3. Smooth muscle contractile associated genes with PDA phenotypes...... 95

Table A.1 Surviving progeny in miR145TG lines versus expected Mendelian ratios ... 148

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List of Figures

Figure 1.1 Notch family of ligands and receptors...... 1

Figure 1.2 Notch signaling pathway ...... 3

Figure 2.1. Notch2, but not Notch3 receptor expression is decreased by PDGF-B...... 62

Figure 2.2. Notch2 and Notch3 have opposing effects on PDGF-B dependent smooth muscle cell proliferation...... 63

Figure 2.3. Genetic deletion of Notch2 or Notch3 has unique effects on proliferation of mouse aortic smooth muscle cells...... 65

Figure 2.4. Notch3 is uniquely regulated by inducers of apoptosis...... 66

Figure 2.5. Notch3 protects smooth muscle cells from cell death...... 68

Figure 2.6. Notch3 promotes transcription of cell survival genes via activation of MAPK.

...... 70

Figure 2.7. Genetic deletion of Notch3 has unique effects on the survival of mouse aortic smooth muscle cells...... 72

Figure 2.8. Notch2 and Notch3 have unique roles in proliferation and cell survival related to their expression...... 73

Figure 3.1. Combined loss of Notch2 and Notch3 causes perinatal lethality...... 96

Figure 3.2. Patent ductus arteriosus is present in postnatal mice with smooth muscle deletion of Notch2 combined with a Notch3 mutation...... 97

Figure 3.3. Patent ductus arteriosus phenotype is accompanied by aortic dilation...... 98

Figure 3.4. Loss of Notch2 and Notch3 causes decreased expression of contractile markers...... 99

Figure 3.5. Expression of contractile proteins in ductus arteriosus...... 101

Figure 3.6. Excision of floxed Notch2 gene with Myocardin-Cre driven recombination.

...... 102

Figure A.1 Expression of transgene and mature miR145 transcript in whole embryos normalized to Cre...... 149

Figure A.2 Comparison of RFP expression in embryonic hearts of lethal lines ...... 150

Figure A.3 Expression of transgene in whole embryos from BB line with MyoCardin-

Cre...... 152

Figure A.4 Lethal vascular phenotypes in miR145TG/+; MCC/+ embryos...... 153

Figure A.5 Predicted miR145 targets in the MAPK signaling pathway with miR145 mimic in HDFNs...... 154

Figure A.6 Predicted miR145 targets in the aortas of miR145 null mice...... 155

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Chapter 1: Introduction and Background

1.1 Introduction

The Notch signaling pathway is a highly conserved pathway involved in cell fate determination in embryonic development and also functions in the regulation of physiological processes in several systems. It plays an especially important role in vascular development and physiology by influencing angiogenesis, vessel patterning, arterial/venous specification, and vascular smooth muscle biology. Aberrant or dysregulated Notch signaling is the cause of or a contributing factor to many vascular disorders, including inherited vascular diseases, such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), associated with degeneration of the smooth muscle layer in cerebral arteries. Like most signaling pathways, the Notch signaling axis is influenced by complex interactions with mediators of other signaling pathways. This complexity is also compounded by different members of the Notch family having both overlapping and unique functions. Thus, it is vital to fully understand the roles and interactions of each Notch family member in order to effectively and specifically target their exact contributions to vascular disease. Notch2 and Notch3 are the predominant Notch receptors in vascular smooth muscle cells

(VSMCs), and in this document I will present the work I have done over the course of my

1 graduate career in delineating the distinct and overlapping roles of Notch2 and Notch3 in

VSMCs, both in vitro and in vivo.

The Notch signaling pathway is an evolutionarily-conserved pathway that plays a critical role in cell fate decisions for numerous systems in vertebrates [1, 2]. Over the past ~15 years, many studies have demonstrated the importance of Notch signaling in regulating vascular development and physiology through control of endothelial cells and smooth muscle cells and their interactions with each other. The overall contribution of Notch signaling to the vasculature and especially the role of endothelial cell-derived Notch signaling has been well-studied and previously covered in several exceptional reviews [3-

9].

Originally discovered in Drosophila (and named for the “notched” wings that resulted from its mutation)[10], the canonical Notch signaling pathway is relatively straightforward, consisting of a membrane-bound ligand and receptor engagement on neighboring cells, leading to cleavage of the intracellular domain of the receptor, which shuttles to the nucleus and acts as a transcriptional co-factor. This series of events allows for a signal to be passed directly from receptor activation to transcriptional modulation while bypassing the signaling intermediates and kinase cascades necessary for many other signaling pathways. This linear system, coupled with its inherent feedback mechanisms, allows for the creation of signal-sending: signal-receiving binary cell fate determinations in early developmental processes, such as those seen in the lateral

2 specification of neural and epidermal cells in Drosophila [11]. However, beyond its role initially described in lower species, this basic signaling pathway has been adapted and utilized by nearly every cell type and organ system to enact a myriad of functions.

The cardiovascular system is the first organ system to develop in embryogenesis and is made up of the heart and an expansive network of vessels of different types and sizes

[12]. Early in development, angioblasts differentiate into endothelial cells and aggregate into a nascent vascular network in a process called vasculogenesis. This network is extended, pruned, and remodeled over the course of development to meet the needs of the developing embryo. To support the growing network of endothelial cell tubes, mural cells are recruited and differentiate into VSMCs or pericytes. These mural cells are vital to the maturation of vessels into functional arteries, veins, and capillaries. VSMCs and pericytes play countless roles in these vessels, from controlling blood flow to regulating permeability, and those roles shift depending upon the state of the vessel, embryological origin, and the environmental context. VSMCs are not “terminally differentiated” cells, and are capable of adapting their activities in response to molecular and physiological cues. This ability of VSMCs to radically alter their functions in different contexts is known as VSMC phenotypic switching or plasticity. This concept has been well-studied and reviewed [13-15] and attributed to many of the activities of VSMCs in vascular diseases.

In VSMCs, the Notch receptors Notch2 and Notch3 and the ligand Jagged1 predominate

[16, 17]. Notch1 and Notch4 are more highly expressed in endothelial cells, although

Notch1 has been shown to play a role in VSMCs in the context of vascular injury [18] and in pericytes [19]. Over the past few decades, extensive work has been performed to ascertain the role of these receptors and Notch signaling as a whole in VSMCs. While significant progress has been made and strong connections have linked the Notch pathway to vascular development, VSMC differentiation and phenotype, and vascular disease, there is still much that is not understood about the precise mechanisms involved.

1.2 The Notch Signaling Pathway in Vascular Smooth Muscle

1.2.1 The canonical Notch signaling pathway

In mammals, there are four Notch receptors (Notch1-4) and 5 ligands (Jagged1 and 2,

Delta-like 1, 3, and 4) which are all Type I transmembrane proteins (Figure 1.1). Both the ligands and receptors have long extracellular domains (ECD) made up of epidermal growth factor-like (EGF) repeats which serve as the binding platform for the receptor- ligand interaction. The receptor arrives at the cell membrane as a heterodimer after it is cleaved by furin at the site 1 (S1). After a receptor is bound by ligand, the negative regulatory region (NRR) is displaced, exposing the site 2 (S2) for cleavage by one of the

ADAM metalloproteases (a disintegrin and metalloproteinase domain-containing protein), and the site 3 (S3) for cleavage by the γ-secretase complex, releasing the Notch intracellular domain (NICD) fragment. After cleavage at the S3 site, the NICD’s nuclear

4 localization signal triggers translocation to the nucleus. The NICD acts as a transcriptional cofactor by forming a complex with the CSL (CBF1/SuH/Lag1) transcription factor named for its homologs in mammals, drosophila, and C. elegans, though it is now designated RBPJ-κ (Recombination signal binding protein for immunoglobulin kappa J region) in mammals. This complex binds to its target DNA sequence, and Mastermind-like (MAML), which acts a transcriptional activator. The formation of this complex displaces transcriptional repressors including SKIP (Ski- interacting protein), CIR (CBF1 interacting corepressor), KyoT2, and SHARP (SMRT and HDAC associated repressor) and histone deactylases (HDACs) from RBPJ-κ, leading to expression of Notch target genes [20]. Among the most highly activated genes are a family of basic helix-loop-helix (bHLH) proteins Hairy/Enhancer of Split (Hes) and Hes- related proteins (Hey/HRT/HERP) which act as transcriptional repressors.

Though the transduction of the Notch signal via NICD cleavage and nuclear translocation is well established, there are many opportunities along its path for regulation to introduce signaling diversity. One such mechanism is the Fringe family of glycosyltransferases,

Lunatic Fringe, Manic Fringe and Radical Fringe [21]. Both Notch ligands and receptors have attached sugar moieties on the EGF repeats of the ECDs, and Fringe glycosyltransferases are able to elongate N- and O-linked glycans at certain positions during post-translational processing in the Golgi. The length and position of these glycans can determine whether ligands and receptors interact between neighboring cells

(trans activation) or within the same cell (cis inhibition) [22]. The glycosylation state can

5 also influence the binding affinities for the different receptor-ligand pairs [23, 24]. This influence on which receptor and ligand interact can then control ultimate effect of Notch signaling, as the different Notch ligands and Notch receptors are capable of producing different signaling outcomes. For instance, the ligands Jag1 and DLL4 have opposing effects on angiogenesis and their activity is regulated by their glycosylation [25], and

DLL4, but not DLL1, can induce pericyte differentiation in myoblasts [26]. Also, the

NICDs of the different Notch receptors can activate transcription of their target genes with varying efficacy depending on the orientation and number of CSL binding sites in the promoter [27]. Another potential mechanism of Notch signal diversity is the control of the intensity and duration of the Notch signal. It has been demonstrated that some

Notch target genes can be activated by low levels of active NICD, while others require high levels of activity [27]. The number of NICD molecules present in the nucleus is dependent on both the influx of NICD from receptor activation and the turnover or degradation of NICD. The degradation of NICDs is regulated by the E3 ubiquitin ligases

[28] which ubiquitylate NICDs in their PEST domain (rich in proline [P], glutamic acid

[E], serine [S], and threonine [T]) and target them for degradation by the proteasome.

1.2.2 Interaction with other signaling pathways and non-canonical signaling

In addition to the long understood canonical Notch pathway, there is increasing evidence that the Notch pathway can exert effects outside of its prominent role as a transcriptional cofactor [29-32]. An example of this is the ability of the NICD to bind active β-catenin, leading to its degradation by the lysosome [33]. Though non-canonical activities of Notch

6 have been shown in many different cell types and systems, evidence for these activities in

VSMCs is relatively sparse and the mechanisms are not well defined [34]. However, based on the ubiquity of non-canonical signaling in other contexts, it seems likely that some of the effects of Notch signaling in VSMCs are due to undiscovered non-canonical interactions.

Most of the observed non-canonical signaling involves interactions of Notch components with members of other signaling pathways, but the overlap of Notch signaling and other pathways is not limited to the non-canonical pathway. Signals from outside of the Notch pathway can regulate Notch expression and activity, as well as alter the transcription factors recruited to Notch target genes. Notch signaling can also in turn regulate the expression and activity of other signaling pathways. Much of the diversity in Notch signaling outcomes can be attributed to its interaction with other signaling pathways.

Here, we will discuss some of the known interactions with other signaling pathways in

VSMCs.

1.2.2.1 PDGF-B

Platelet-Derived Growth Factor B (PDGF-B) is a major mitogen and chemoattractant in

VSMCs that acts through its receptor, PDGFRβ [35]. It has been shown to interact with

Notch signaling in VSMCs in two major capacities. First, it has been shown to directly promote the transcription of PDGFRβ and enhance the signaling effects of PDGF-B [35,

36]. This relationship is especially apparent in pericytes, as both PDGFRβ and Notch3 are considered markers of pericytes and their dysfunction in animal models and humans can produce similar phenotypes [37-39]. Second, it has been shown that PDGF-B signaling can regulate the expression of Notch components (Chapter 2) [40, 41]. Overall, it is apparent that the PDGF and Notch signaling pathways are strongly intertwined, with significant functional overlap in pericytes and co-regulation in VSMCs.

1.2.2.2 TGFβ

The transforming growth factor β (TGFβ) pathway is a vital contributor to the differentiation of VSMCs [42]. The Notch and TGFβ signaling pathways cooperatively regulate the differentiation of VSMCs through direct interaction of NICD and SMAD2/3

(a transcription factor within the TGFβ pathway) and transcription of smooth muscle contractile genes [16]. This cooperative induction of VSMC differentiation has also been shown in progenitor cell types [43, 44] and the direct interaction of NICD and SMAD3 as transcriptional activators has been shown in other systems [45]. Interestingly, it has also been shown that these two pathways can negatively regulate each other’s expression.

This is seen in fibroblasts where TGFβ signaling decreases expression of Notch3 [46] and in VSMCs where Notch signaling promotes the transcription of miR145 which inhibits the TGFβ pathway by targeting the TGFβ receptor 2 [47, 48]. Taken together, the Notch and TGFβ pathways are able to both cooperatively promote transcription of their

8 downstream genes as well as inhibit each other’s expression, suggesting a very complex relationship and potentially a mechanism for negative feedback within each pathway.

1.2.2.3 MAPK

The mitogen-activated protein kinase (MAPK) pathway is one of the major signaling pathways controlling cell growth, survival, and many other processes and is a main transducer of growth factor signals [49]. In cultured VSMCs, Notch3 is capable of activating this pathway by phosphorylation of the MAPK component ERK (Chapter 2)

[41, 50], through a mechanism that is not yet known. Physical interaction of Notch and

MAPK components has previously been shown in other models [51]. However, it may simply be a result of upregulation of growth factor ligands or receptors by Notch signaling, such as PDGFRβ. Regardless of how it is enacting this effect, it is clear that at least in some contexts, Notch signaling is capable of promoting MAPK pathway activity.

The Notch regulated microRNA, miR145, is another potential intersection of the Notch and MAPK pathways. Several predicted targets of miR145 lie within the MAPK pathway, and phosphorylation of p38 MAPK is increased in miR145 null nice

(Appendix)[48]. These studies are investigating the ability of miR145 to mediate fibrosis, but targeting the MAPK pathway in VSMCs could be another avenue by which Notch and miR145 promote VSMC differentiation, as the ETS transcription factors are activated by MAPK signaling and directly oppose VSMC differentiation [52].

1.2.2.4 Wnt

The Wnt (Wingless-related integration site) signaling pathway is an important developmental pathway that regulates smooth muscle proliferation, migration, and survival [53]. The Wnt and Notch pathways have significant crosstalk in many systems, both by direct effect on each other’s pathway components and shared regulation of developmental processes [32, 54-57]. These interactions have been shown to extend to

VSMCs as well, as early mesoderm cells in chick embryos are pushed towards a VSMC lineage through cooperation of the Notch and Wnt pathway [58]. While evidence for

Wnt/Notch pathway interaction in VSMCs is limited, the results in mesodermal smooth muscle progenitors coupled with the known crosstalk between Notch and Wnt signaling in other systems suggests that these pathways cooperate to drive early VSMC differentiation.

1.3 Notch Signaling in VSMC Development

Notch signaling plays an essential role in the development of the cardiovascular system, from hematopoiesis to cardiac development to endothelial cell sprouting and vasculogenesis. In the development and differentiation of VSMCs, the functions of Notch signaling can be categorized into two main roles: construction of the vessel wall and arterial-venous specification.

1.3.1 Constructing a vessel wall

Following the initial stages of vasculogenesis, nascent vessels are composed of an endothelial tube surrounded by perivascular cells. For a vessel to mature into a conduit capable of withstanding increasing pressure and volume demands, it must recruit mural cells to encompass it and differentiate into one or more layers of vascular smooth muscle.

This recruitment is largely driven by a PDGF-B gradient secreted by the endothelial cells

[59]. Once recruited to the nascent vessel, endothelial cell-derived Notch ligands can activate Notch signaling in these mural cells, which induces integrin adhesion to the endothelial basement membrane and initiates maturation and differentiation [60]. We have learned a great deal about how endothelial-derived Notch signals affect VSMCs and mural cells from mutant mouse models and in vitro systems. In the great vessels of the outflow tract, cardiac neural crest progenitors give rise to the VSMCs within the vessel walls. With the complete abrogation of Notch signaling by “knocking in” of a dominant negative MAML in cardiac neural crest cells, mice display various defects of the outflow tract and aortic arch associated with decreased differentiation and expression of smooth muscle markers [17]. Mice with an endothelial deletion of Jagged1 also display decreased

VSMC differentiation and expression of smooth muscle markers [61]. Through a combination of in vitro co-culture experiments [62] and the cardiac neural crest-specific

Jagged1 knockout mice [63], it was demonstrated that induction of Notch signaling in

VSMCs by endothelial-expressed Jagged1 leads to the increased expression of both

Notch3 and Jagged1. This allows for lateral induction of Notch signaling by homotypic

VSMC-VSMC interactions through multiple layers of smooth muscle to promote VSMC

11 differentiation after the initial signal is received from endothelial-derived Jagged1 [64].

This model has been further supported by smooth muscle deletion of Jagged1 , Rbpj-κ , and a smooth muscle deletion of Notch2 coupled with heterozygous deletion of Notch3

(Chapter 3) [65-67]. All three of these models develop patent ductus arteriosus (PDA) due to contractile deficits of the VSMCs, and present decreased expression of smooth muscle markers. An interesting parallel exists between this model and one proposed from experiments performed by the Krasnow lab [68], where they demonstrate that the expression of PDGFRβ and ACTA2 (alpha smooth muscle actin) move through layers of smooth muscle like a wave. They describe a major role for endothelial-derived PDGF-B in the migration, orientation, and proliferation of these smooth muscle cell layers, and suggest that there may be another signaling pathway contributing to the radial pattern of construction. Indeed, Notch signaling would elegantly fit into this proposed model, as it is known to induce PDGFRβ and ACTA2, and could be passed through the layers of smooth muscle via cell-cell contact [36, 69].

An interesting finding from comparing the knockout phenotypes of mice deficient in

Notch2 and Notch3 is their unique roles in different vascular beds. Notch2 is mainly expressed in the outflow tract and other large caliber vessels, while Notch3 is expressed in all mural cells [17, 70]. Likewise, in large caliber vessels, Notch2 signaling is indispensable, with loss of Notch3 only acting to amplify defects in Notch2-null mice

[71, 72]. This demonstrates that, while there may be some functional redundancy of

Notch2/3 in large caliber vessels, Notch3 cannot completely replace Notch2 function.

However, in pericytes and VSMCs in smaller caliber vessels where Notch2 expression is significantly diminished, Notch3 plays a major role, especially in the cerebral vasculature

[37, 73-75].

1.3.2 Arterial-venous specification

Another important role for Notch signaling in VSMCs in the developing vasculature is the specialization of vessels into arteries and veins. Arteries and veins have distinct structural properties and express distinct protein markers that contribute to their unique functions in the cardiovascular system [76]. In endothelial cells, Notch signaling regulates arterial specification in cooperation with VEGF [77, 78]. Similarly, Notch3 regulates arterial specification in VSMCs. In a mouse model with global knockout of

Notch3, arteries acquired a more venous phenotype, with enlarged vessels, thinner and disorganized smooth muscle layers, and less festooned elastic lamina [79]. These changes were not observed in the outflow tract or aorta where Notch2 is highly expressed, likely suggesting a functional redundancy for Notch2 and Notch3 in this context. The expression of the arterial marker smoothelin and a LacZ reporter driven by an arterial specific promoter element were also reduced in these Notch3-null mice, showing both structural and morphological defects in arterial specification. In zebrafish, Notch3 expression was found to be restricted to arteries, and inhibition of Notch signaling in zebrafish leads to decreased arterial specification and similar phenotypes compared to the mouse model [80].

1.4 Notch Signaling and VSMC Phenotype

The ability of VSMCs to display various phenotypes and enact disparate functions depending on their cellular context, known as phenotypic switching or plasticity, is central to their changing roles from early developmental vasculogenesis to vascular homeostasis and remodeling in adult animals. Notch signaling has been closely tied to the many possible phenotypic endpoints of VSMCs. While there are still disparities in the literature about some of the phenotypic effects of Notch signaling in VSMCs, a clear picture emerges of how Notch activation drives VSMCs to different endpoints and the contributions from the different Notch receptors.

1.4.1 Differentiation

A “differentiated” VSMC is generally described as a quiescent cell expressing contractile proteins (such as ACTA2, smooth muscle myosin heavy chain [MYH11], transgelin

[SM22α], and calponin [CNN1]) and primed to provide contractile force to constrict (or dilate) the vessel in which it resides in response to external cues. This is the state necessary for mature vessels to maintain physiological homeostasis. Notch signaling has been tied to regulation of VSMC differentiation and expression of contractile proteins for some time. While initial studies in vitro and in injury models showed that Notch activated genes of the HRT (hairy-related transcription factors) family could repress myocardin- induced contractile protein expression [81-83], there were also several studies showing that Notch activity promoted VSMC differentiation and contractile marker expression

[17, 79, 84]. Although the discrepancy has not been conclusively explained, it is likely

14 that the observed repression of contractile genes by the HRT family is negative feedback, as this family of repressors interferes with the transcription of many Notch target genes, including the HRT genes themselves [85-87]. Notch activity has since been shown to directly promote the transcription of ACTA2 [69] and Notch-induced transcription of

ACTA2 was repressed by HRT transcription factors [88]. Notch signaling also promotes the expression of miR143/145, co-transcribed microRNAs that drive differentiation by targeting KLF4/5, a repressor of Myocardin/SRF regulated contractile genes [47, 89].

Overall, the preponderance of data supports the hypothesis that Notch signaling in

VSMCs promotes contractile differentiation [16, 17, 61, 65, 69, 72, 73, 79, 84, 90-93], which fits with our understanding of its role in development of the vessel wall and response to contact-induced signaling with endothelial cells [61-65, 72, 93-97].

1.4.2 Proliferation

The proliferation of VSMCs is inversely tied to their differentiation status, so much so that they are often discussed as polar opposites, where mitotically-active cells are considered “dedifferentiated” or “synthetic” and differentiated VSMCs are assumed to be quiescent, though there is likely a range of intermediate phenotypes between these extremes [15]. Since Notch signaling in VSMCs promotes contractile differentiation, one would assume cells with active Notch signaling would not be actively proliferating.

However, the initial data suggested the opposite, with several papers showing that Notch signaling promoted proliferation in vitro [40, 98, 99] and in the context of vascular injury in vivo [18, 50]. In 2013, findings from Lucy Liaw’s lab demonstrated that the Notch2

15 receptor specifically inhibited proliferation via cell cycle arrest and Notch2 was localized in non-proliferating regions of injured vessels suggesting a receptor-specific regulation of

VSMC proliferation [100]. Indeed, the previous data demonstrating promotion of proliferation was largely focused on the activity of Notch1 and/or Notch3 [18, 50, 98,

99]. Delving further into receptor specific roles, our own lab demonstrated that manipulation of Notch2 and Notch3 had opposite effects on PDGF-B dependent proliferation, and that PDGF-BB treatment decreased expression of Notch2, but increased expression of Notch3 (Chapter 2) [41]. Interestingly, while Notch2 and Notch3 have disparate roles in regulating VSMC proliferation, they both promote contractile differentiation (Chapter 3) [65]. Together these results indicate that regulation of the expression of the different Notch receptors in VSMCs could have a large impact on phenotypic outcome.

1.4.3 Survival

The role of Notch signaling in VSMC survival and apoptosis has drawn significant attention due to the human disease CADASIL, which involves the loss of VSMCs and pericytes from cerebral vessels and is caused by mutation of the Notch3 receptor (which will be discussed further in the next section). Notch signaling has been implicated in suppression of apoptosis in a number of cell types [101-104], especially in many forms of cancer [105]. This role as an inhibitor of apoptosis and promoter of cell survival appears to be expressed in VSMCs and pericytes as well [18, 34, 37, 41, 50, 73, 74, 98, 106].

Interestingly, the Notch receptors responsible for promotion of cell survival in mural cells

16 are also those implicated in the promotion of proliferation, with Notch1 and Notch3 promoting survival, but with no known role for Notch2 (Chapter 2) [18, 34, 37, 41, 50,

73, 74, 98]. Direct comparison of the effects of Notch2 and Notch3 manipulation in vitro on VSMC survival revealed that Notch3 promoted survival, but Notch2 had no effect

(Chapter 2) [41]. Notch3 activity correlates with MAPK/ERK pathway activation and with survival gene expression, which could be abrogated by inhibition of the

MAPK/ERK pathway. This suggests that Notch3 may be promoting VSMC survival through a non-canonical association with the MAPK pathway, which had previously been suggested [34]. Overall, Notch activity promotes survival in VSMCs, but this effect is receptor specific and involves a non-canonical interaction with the MAPK pathway.

1.4.4 Extracellular matrix synthesis

VSMCs and their precursors are predominant contributors to the production of the extracellular matrix (ECM) that comprises the basement membrane and supports tensile strength and elasticity in the vessel [107-109]. The ability of Notch signaling to promote

ECM synthesis has been demonstrated in fibroblasts, where it induces collagen production [96, 110], and in renal podocytes, where inhibition of Notch signaling reduced expression of collagen IV and laminin, but increased expression of MMP-2 and MMP-9

[111]. This relationship is maintained in VSMCs, where co-culture studies identified that endothelial-derived Notch signaling in VSMCs promotes collagen synthesis and other markers of a synthetic phenotype [93, 96]. This may be a recapitulation of the developmental program in early vessel development, wherein Notch and other synthetic

17 signaling pathways (such as TGF-β) induce ECM synthesis in perivascular cells to produce the framework of the vessel wall.

1.4.5 Migration

The migration of VSMCs is primarily important in two situations: recruitment of mural cells to the developing vessel and in response to vessel injury [59]. The main chemoattractant for VSMCs is PDGF-B secreted by endothelial cells as well as platelets and macrophages [59, 112]. Because Notch signaling has been shown to increase expression of the PDGF receptor β [36], it would follow that active Notch signaling in

VSMCs would increase migration in response to PDGF-B. Indeed, much of the work done on Notch and VSMC migration has shown an induction of migratory ability. Similar to their roles in proliferation, Notch1 and Notch3 were shown to promote VSMC migration in vitro [98] and in a mouse injury model, Notch1 was shown to be important for migration, proliferation, and formation of the neointimal layer [18]. The Notch responsive gene Hey2 was also shown to promote proliferation, migration and neointimal formation [113], and Notch signaling has been shown to regulate expression of MMP-2 and MMP-9, which are important in the matrix remodeling necessary for migration through the vessel wall rich in ECM [114]. Notch signaling has also been shown to regulate cellular adhesions between cells via N-cadherin [115, 116] and to the surrounding matrix via integrins [60]. These results support the idea that Notch signaling is important for recruitment of mural cells to the vessel wall in response to a PDGF-B gradient, and maintaining their position via cellular adhesions.

1.5 Notch Signaling in Vascular Disease

The various roles of Notch signaling in regulating smooth muscle development and phenotypic modulation make it an obvious player in many vascular diseases associated with VSMCs. These include both inherited genetic diseases and ailments brought on by defective physiological homeostasis.

1.5.1 CADASIL Syndrome

Perhaps the most studied Notch-related disease in VSMCs, Cerebral Autosomal

Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is caused by mutations in the NOTCH3 gene through a mechanism we still do not fully understand [117]. This disorder presents in the patient as migraines, mood disorders, ischemic attacks, cognitive impairment, and stroke and is marked by lesions within the cerebral and systemic vasculature characterized by accumulation of granular osmiophilic material (GOM) at smooth muscle basement membrane and progressive loss of VSMCs

[70, 118, 119]. These GOM deposits appear to be comprised of oligomerized Notch3

ECD, and the mutations found in human patients lead to a gain or loss of a cysteine residue within the EGF-like repeats of the ECD [119]. It is not clear what exactly causes the disease progression of CADASIL, whether it is impaired function of Notch3, aberrant effects of GOM deposition, or a combination of both. Mouse models expressing Notch3 receptors containing disease causing mutations have had mixed results in recreating

CADASIL disease states. A knock-in model of a CADASIL Notch3 mutation did not develop disease phenotypes or GOM deposits [120], but another mouse with ectopically over-expressed mutant Notch3 protein did produce GOM deposits and mirrored many of the CADASIL disease symptoms [121]. Notch3-null mutant mice are viable and fertile and do not show any overt symptoms associated with CADASIL [122], though they do exhibit some disruption of the VSMCs in cerebral arteries [74]. It has also been shown that hypomorphic mutations of Notch3 in humans do not cause CADASIL [123]. These results would seem to suggest that it is the gained aberrant function of GOM deposits that causes CADASIL phenotypes. However, a homozygous-null Notch3 mutation has been shown to produce a recessive early-onset arteriopathy and cavitating leukoencephalopathy very similar to CADASIL without the deposition of GOM [124], and a mouse model with combined deficiency of Notch1 and Notch3 exhibits pericyte dysfunction and models some phenotypes of CADASIL [19]. While it is still difficult at this point to definitively rule out any of the possible explanations, it seems likely that impaired Notch3 signaling plays at least some role in the CADASIL phenotypes, based on the known function of Notch3 in regulating cell survival in VSMCs and pericytes [41,

73].

1.5.2 Patent Ductus Arteriosus

Patent ductus arteriosus (PDA) results from the failure of the ductus arteriosus (DA), which diverts blood around the fetal lungs, to close shortly after the first breath and inflation of the lungs [125]. There are several signaling pathways involved in regulating

20 the timing of this closure, but ultimately it relies on the VSMCs of the DA to contract and functionally close the vessel. Because of this, defects in contractile proteins or VSMC differentiation often result in PDA [126-128]. Since Notch signaling promotes VSMC differentiation, it is not surprising that certain mouse models of Notch deficiency also present with the PDA phenotype (Chapter 3) [65-67]. This connection is also seen in human disease, where a subset of patients afflicted with Alagille, Adams-Oliver or

Hajdu-Cheney syndromes, all caused by mutation of Notch family members, present with

PDA [129-131].

1.5.3 Alagille syndrome

Alagille syndrome is an autosomal dominant disorder caused by mutation of NOTCH2 or

JAGGED1 and is characterized by many developmental defects, including those of the liver, heart, skeleton, eye, face, and kidney [130, 132]. While many of these defects are caused by the roles of Notch signaling in other cell types, there are some interesting vascular phenotypes that could be related to dysfunctional Notch signaling in VSMCs. In addition to the PDA previously discussed, Alagille syndrome patients often present with defects of the cardiac outflow tract and great vessels [133]. These defects are similarly modeled in mice with abrogated Notch signaling in the cardiac neural crest lineage that gives rise to the VSMCs of the outflow tract and great vessels [17].

1.5.4 Pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is characterized by a persistent elevation of pulmonary arterial pressure, pulmonary arterial remodeling, and right ventricular hypertrophy [134]. This pulmonary arterial remodeling presents as increased VSMC proliferation, survival, and expression of contractile proteins such as ACTA2. Notch signaling has been proposed to regulate the pathogenesis of PAH, as elevated Notch3 expression is one of the hallmarks of the disease [134, 135], and this would fit with the proliferation, survival, and differentiation roles that have been shown for Notch3. Two

NOTCH3 mutations were identified in cases of childhood PAH, and in vitro experiments using the mutant proteins demonstrated increased proliferation [136].This role for Notch3 in PAH is further confirmed by the discovery that Notch3-null mice do not develop PAH in a hypoxia-induced PAH mouse model and that treatment with the Notch inhibitor

DAPT [137], or with a soluble Jagged1 ligand, which functions as a Notch antagonist, could prevent disease progression [138].

1.5.5 Infantile myofibromatosis

Infantile myofibromatosis is an unusual mesenchymal malignancy that is seen at or near birth and typically spontaneously regresses in early childhood [139]. Although the tumors rarely present any significant harm to the patient, they are of interest to us because of their causative mutations: PDGFRβ and NOTCH3 [38, 39]. These mutations are predicted to create constitutively active forms of the proteins [38], which fits our current

22 understanding of the relationship between Notch3 and PDGFRβ in promotion of VSMC proliferation (Chapter 2) [36, 41].

1.5.6 Vascular Injury

In situations of vascular injury, many signaling pathways are activated to repair and remodel the vessel. However, these changes can become pathological if not properly regulated and can lead to neointimal hyperplasia, an aggressive migration and proliferation of VSMCs into the lumen of the vessel [140]. Expression of members of the

Notch signaling pathway, including Notch1, Notch3, Hey1, Hey2, and Jag1, are regulated in response to vascular injury, with an initial downregulation, followed by upregulation after several days [8, 18, 115]. Several of these members have been specifically shown to regulate migration and proliferation of the neointima in injury models, including Notch1,

Notch3, and Hey2 [18, 50, 113]. Overall, the general consensus is that the Notch signaling pathway drives neointimal formation after vascular injury and is therefore a potential pharmacological target in situations of restenosis or other vascular injuries.

1.6 Conclusion

Our understanding of the molecular mechanisms of Notch signaling in VSMCs is constantly expanding, and in the last few years we have made great strides in uncovering some of the unique functions of the different Notch family members. There is still much we do not know, however, both in what roles each Notch component has in VSMC

23 development and disease, and also the precise mechanisms that allow for functional divergence of the Notch signal. In this document we detail our efforts to address these topics through examination of the roles of the Notch2 and Notch3 receptors in VSMCs both in vitro and in vivo. We hypothesize that the vital role of promoting smooth muscle differentiation is conserved between the Notch2 and Notch3 receptors. However, based upon the pertinent literature and our own preliminary observations, we hypothesize that

Notch2 and Notch3 have unique roles in regards to smooth muscle proliferation and survival. We aim to characterize these overlapping and distinct roles and define the mechanisms by which they are effected. A clear understanding of these functional differences between receptors will allow us to understand the specific contributions of

Notch2 and Notch3 to development and disease states, and inform the future use of receptor-specific therapies to treat Notch-related vascular diseases.

1.7 Figures and Tables

Table 1.1 Vascular diseases associated with mutations of Notch pathway components.

Notch Mutation site Notch signaling effect Disease outcome Component Jagged1 Identified in every exon Alagille syndrome Loss of function and several splice sites [141] Tetralogy of Fallot EGF-like repeat 2 Not known [142] DLL4 DSL domain, N-terminal Predicted loss of Adams-Oliver domain, and EGF-like function syndrome [143] repeats Notch1 Haploinsufficiency, EGF- Predicted loss of Adams-Oliver like repeat 11, LNR 2, function syndrome [129] ANK domain 3

Haploinsufficiency, EGF- Predicted loss of Bicuspid aortic like repeat 29 function valve [144]

Notch2 Predicted loss of Alagille syndrome Truncated NICD function [132] Predicted gain of Hadju-Cheney PEST domain function [145] Notch3 CADASIL [70, EGF-like repeats 1-32 Likely loss of function 117-119] Predicted gain of Lehman syndrome PEST domain function [146] Infantile Heterodimerization Predicted gain of myofibromatosis domain function [38] EGF-like repeats 21 and Childhood PAH Not known 23 [136]

RBPJ-κ Adams-Oliver DNA binding domain Loss of Function syndrome [147] EOGT Predicted loss of Adams-Oliver Domains not defined function syndrome [148]

Figure 1.1 Notch family of ligands and receptors (Continued)

Figure 1.1 (cont.) Abbreviations: Cys-rich = Cysteine-rich region; DOS = Delta and OSM11-like proteins motif; DSL = Delta/Serrate/Lag2 motif; NECD = Notch Extracellular Domain; LNR = Lin12-Notch repeats; NRR = Negative Regulatory Region; S2 = cleavage site 2; S3 = cleavage site 3; NICD = Notch Intracellular Domain; RAM = RBPJ-κ Association Module; NLS = Nuclear Localization Signal; ANK = Ankyrin repeats; TAD = Transactivation Domain; PEST = Proline/glutamic acid/serine/threonine-rich motifs.

Figure 1.2 Notch signaling pathway (Continued)

Figure 1.2 (cont.) (1) Notch receptor and ligand genes are transcribed and translated into protein. (2) During post-translational processing in the golgi, Notch receptors are cleaved by furin to create heterodimers; both ligands and receptors are glycosylated on their EGF-like repeats by Fringe family enzymes. (3) Trans-activation of Notch receptor by Notch ligand from a neighboring cell, triggering cleavage by ADAM10 at S2 and γ-secretase at S3, releasing the NICD. (4) Cis-inhibition of Notch receptor by Notch ligand on the same cell, preventing activation. (5) Endocytosis of Notch ligand with associated NECD, can be degraded by the proteasome or recycled back to the cell surface. (6) NICD is shuttled to the nucleus where it binds to CSL, displacing HDACs and corepressors such as CIR and recruiting MAML to activate transcription of Notch target genes. (7) NICD can both directly interact with Ras and promote ERK phosphorylation, though the mechanism is not yet known. (8) NICD can bind to the Wnt signaling mediator β-catenin and target it for degradation in the lysosome. (9) Transcription of some genes, including smooth muscle contractile genes, are co-regulated by NICD/CSL and the TGFβ transcription factor SMAD3. (10) Many signaling pathways can regulate expression of Notch components, including PDGFRβ signaling via the MAPK pathway.

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115. Lindner, V., et al., Members of the Jagged/Notch gene families are expressed in injured arteries and regulate cell phenotype via alterations in cell matrix and cell-cell interaction. Am J Pathol, 2001. 159(3): p. 875-83.

116. Li, F., et al., Endothelial Smad4 maintains cerebrovascular integrity by activating N-cadherin through cooperation with Notch. Dev Cell, 2011. 20(3): p. 291-302.

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129. Stittrich, A.B., et al., Mutations in NOTCH1 cause Adams-Oliver syndrome. Am J Hum Genet, 2014. 95(3): p. 275-84.

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133. McElhinney, D.B., et al., Analysis of cardiovascular phenotype and genotype- phenotype correlation in individuals with a JAG1 mutation and/or Alagille syndrome. Circulation, 2002. 106(20): p. 2567-74.

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136. Chida, A., et al., Mutations of NOTCH3 in childhood pulmonary arterial hypertension. Mol Genet Genomic Med, 2014. 2(3): p. 229-39.

137. Li, X., et al., Notch3 signaling promotes the development of pulmonary arterial hypertension. Nat Med, 2009. 15(11): p. 1289-97.

138. Xiao, Y., D. Gong, and W. Wang, Soluble JAGGED1 inhibits pulmonary hypertension by attenuating notch signaling. Arterioscler Thromb Vasc Biol, 2013. 33(12): p. 2733-9.

139. Hatzidaki, E., et al., Infantile myofibromatosis with visceral involvement and complete spontaneous regression. J Dermatol, 2001. 28(7): p. 379-82.

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142. Eldadah, Z.A., et al., Familial Tetralogy of Fallot caused by mutation in the jagged1 gene. Hum Mol Genet, 2001. 10(2): p. 163-9.

143. Meester, J.A., et al., Heterozygous Loss-of-Function Mutations in DLL4 Cause Adams-Oliver Syndrome. Am J Hum Genet, 2015. 97(3): p. 475-82.

144. Garg, V., et al., Mutations in NOTCH1 cause aortic valve disease. Nature, 2005. 437(7056): p. 270-4.

145. Simpson, M.A., et al., Mutations in NOTCH2 cause Hajdu-Cheney syndrome, a disorder of severe and progressive bone loss. Nat Genet, 2011. 43(4): p. 303-5.

146. Gripp, K.W., et al., Truncating mutations in the last exon of NOTCH3 cause lateral meningocele syndrome. Am J Med Genet A, 2015. 167A(2): p. 271-81.

147. Hassed, S.J., et al., RBPJ mutations identified in two families affected by Adams- Oliver syndrome. Am J Hum Genet, 2012. 91(2): p. 391-5.

148. Shaheen, R., et al., Mutations in EOGT confirm the genetic heterogeneity of autosomal-recessive Adams-Oliver syndrome. Am J Hum Genet, 2013. 92(4): p. 598-604.

149. Luo, B., et al., Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor. Mol Cell Biol, 1997. 17(10): p. 6057-67.

150. Beckers, J., et al., Expression of the mouse Delta1 gene during organogenesis and fetal development. Mech Dev, 1999. 84(1-2): p. 165-8.

151. Villa, N., et al., Vascular expression of Notch pathway receptors and ligands is restricted to arterial vessels. Mech Dev, 2001. 108(1-2): p. 161-4.

152. Shutter, J.R., et al., Dll4, a novel Notch ligand expressed in arterial endothelium. Genes Dev, 2000. 14(11): p. 1313-8.

153. Wu, J., et al., Molecular determinants of NOTCH4 transcription in vascular endothelium. Mol Cell Biol, 2005. 25(4): p. 1458-74.

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Chapter 2: Differential Regulation of NOTCH2 and NOTCH3 Contribute to their Unique Functions in Vascular Smooth Muscle Cells

2.1 Introduction

Notch receptors belong to an evolutionarily conserved family of cell surface receptors

(Notch1-4) that transduce signals between neighboring cells via cell-to-cell contact [1,

155]. Notch receptor activation is triggered when a ligand (Jagged-1,-2 or Delta-like-1,-

3,-4 in mammals) expressed on an adjacent cell surface binds to one of the receptors. The

Notch receptor then undergoes cleavage, which allows the intracellular domain (NICD) to translocate to the nucleus. In the canonical pathway, the NICD binds with the transcription factor CSL (named after a trio of related proteins across species: CBF,

Suppressor of Hairless, Lag-1) to regulate downstream gene expression [156, 157]. There is also evidence of non-canonical signaling by Notch receptors through direct interaction of the receptors or NICDs with members of other signaling pathways such as TGFβ [45,

158], Wnt [57, 159, 160], and MAPK [34, 161]. In vascular smooth muscle cells

(VSMCs), the Notch2 and Notch3 receptors predominate [16-18, 61]. Notch signaling is critical for growth regulation and cell fate determination in many cell types, including vascular cells [3, 5, 80]. The regulation of contractile differentiation and extracellular matrix synthesis by Notch activity has been well defined [16, 61, 72, 73, 84, 91, 93, 97], but what is less clear is the exact role of Notch receptors in VSMC proliferation and cell

42 survival. Notch signaling has been shown to both increase and decrease VSMC proliferation, suggesting context dependence and/or receptor-selective functions [18, 93,

98, 99]. A recent paper provided some clarity on this issue, by identifying a specific and unique role for Notch2 in inhibiting VSMC proliferation [100]. Thus, individual Notch receptors may possess unique roles in regulating VSMC proliferation, which could be linked to distinct signaling cues that control cell fate.

Notch signaling has also been closely associated with apoptosis and cell survival, especially in cancer cells [160, 162-170] and smooth muscle cells [18, 34, 74, 98, 171].

Notch3 specifically has been shown to promote cell survival in several different cell types [34, 74, 98, 160, 167-169] and is a poor prognostic biomarker in several cancers

[163, 164, 172]. The role of Notch2 in cell survival is less clear, but has been shown to enhance apoptosis in certain cellular contexts [165, 173], in contrast to the effect of

Notch3. There is also some evidence that Notch3 can promote cell survival by enhancing or inducing MAP kinase signaling, though the exact mechanism and role for Notch3 in regulating VSMC survival is unclear [34, 167].

In this study, we sought to better define the specific roles of Notch2 and Notch3 in the regulation of proliferation and cell survival in smooth muscle cells. Additionally, we investigated the expression profiles of these two receptors in response to proliferation and apoptosis cues.

2.2 Materials and Methods

2.2.1 Cell culture

Primary cultures of human aortic smooth muscle cells (HAoSMCs) were purchased from

Vasculife and maintained in SMC media: DMEM (Thermo Fisher Scientific) supplemented with 5% FBS (HyClone), insulin (4 ng/ml), EGF (5 ng/ml), ascorbic acid

(50 ng/ml), 2 mM glutamine, 1 mM sodium pyruvate, and 100 units/ml penicillin/streptomycin. Primary cells between passages 6 and 8 were used for all experiments. All cultures were maintained in humidified 5% CO2 at 37 °C. Serum- starved cells were cultured in DMEM supplemented with 0.2% FBS, 2 mM glutamine, 1 mM sodium pyruvate, and 100 units/ml penicillin/streptomycin for 48 hours before treatment. PDGF-B (20ng/ml, Peprotech) was added to this media where specified.

Inhibitors used were all dissolved in DMSO and used at the following concentrations: 5

µM PDGF-receptor Tyrosine Kinase Inhibitor I (Fisher), 10 µM U0126 (Calbiochem), 10

µM JNK Inhibitor II (Calbiochem), and 10 µM MG-132 proteasome inhibitor (ApexBio).

Hydrogen peroxide (Kroger) was added at concentrations indicated. For phospho-ERK experiments, cells were serum-starved for 24 hours, and then cultured in 10% FBS

DMEM for 1 hour before collection.

2.2.2 Quantitative RT-PCR (qPCR)

Total RNA was isolated from cells using RiboZol reagent (Amersco) according to the manufacturer's instructions and reverse transcribed with M-MLV reverse transcriptase

(Promega) to generate cDNA. Real-time PCR was performed using a StepOne PCR

44 system (Applied Biosystems) with SYBR Green and 50 ng of cDNA as template. The fold difference in target gene mRNA levels was calculated using the ΔΔCT method and normalized to GAPDH mRNA from the same sample. Primer sequences were as follows:

NOTCH3, 5′- GAG CCA ATG CCA ACT GAA GAG (forward) and 5′- GGC AGA

TCA GGT CGG AGA TG (reverse); NOTCH2, 5′ - ACA GTT GTG TCT GCT CAC

CAG GAT (forward) and 5′ - GCG GAA ACC ATT CAC ACC GTT GAT (reverse);

GAPDH, 5′- ATG GAA ATC CCA TCA CCA TCT T (forward) and 5′- CGC CCC ACT

TGA TTT TGG (reverse); BCL2, 5′- TCC CTC GCT GCA CAA ATA CTC (forward) and 5′- ACG ACC CGA TGG CCA TAG A (reverse); BIRC5, 5′- GCC AAG AAC AAA

ATT GCA AAG G (forward) and 5′- TTT CTC CGC AGT TTC CTC AAA (reverse);

CFLAR, 5′- GCT GGC AGC TGA TTA GAT GGT (forward) and 5′- TTT GAG TCA

GTG GAC TGG GAA A (reverse). Mouse primers: Gapdh, 5′- GAC GGC CGC ATC

TTC TTG T (forward) and 5′- CAC ACC GAC CTT CAC CAT TTT (reverse); Notch2,

5′- CGG ACC AGC CTG AGA ACC T (forward) and 5′- CCT CAA GAA GCT TCG

CGA AT (reverse); Notch3, 5′- TTG TCT GGA TGG AAG CCC ATG T (forward) and

5′- ACT GAA CTC TGG CAA ACG CCT (reverse); Bcl2, 5′- TGC CTG CTT TAT

GGA GAC CAA (forward) and 5′- GCT GCC CAG GCC AAG TT (reverse); Birc5, 5′-

CAC TGC CCT ACC GAG AAC GA (forward) and 5′- AGC CTT CCA ATT CCT TAA

AGC A (reverse); Cflar, 5′- TGG ACA AAG TGT ATG CGT GGA A (forward) and 5′-

TGC AGG CTG AGG CTG TAT TTC (reverse).

2.2.3 RNA interference

HAoSMCs were plated in a 6-well plate at 1 × 105 cells/well. After 24 hours, cells were transfected with siRNA using RNAiMAX (Invitrogen). NOTCH2 siRNA was purchased from QIAGEN (GS4853) and NOTCH3 siRNA was synthesized by IDT as follows: 5′-

AAC UGC GAA GUG AAC AUU G. Control siRNA was purchased from Invitrogen.

All siRNAs were transfected at 20 nM. 24 hours post-transfection, cells were used in assays as described. Efficacy of knockdown was confirmed by western blot, as shown in

Figure 2.5.

2.2.4 Lentiviral transduction

The lentivirus plasmids containing GFP, NICD2-FLAG, and NICD3-FLAG constructs were made as described previously [72]. The lentivirus plasmids were transfected into

TN-293 cells using PolyJet (SignaGen), and the viral particles were amplified and purified as described previously [62], with the following changes. Virus-containing supernatant collected from a 10 cm plate was concentrated by adding 10% Polyethylene glycol (PEG) and 150 mM NaCL, rocking overnight at 4ºC, pelleted by centrifugation at

3000 rcf, and resuspended in 150 µl DMEM. For transduction of GFP or NICDs,

HAoSMCs were seeded in a six-well plate at a density of 1×105 cells per well, 24 h before viral infection. 20 µl of lentivirus suspension diluted in 2 mL DMEM with 5%

FBS was added to each well. Polybrene was supplemented at a final concentration of

6 μg/mL. Twenty-four hours later, cells were transferred to fresh DMEM containing 5%

FBS for additional 24 h incubation. Cells were then collected for mRNA, protein, or used

46 in assays as described. The efficiency of transduction was evaluated using GFP expression and qPCR. Viral particles were titrated to achieve 90% to 100% transduction.

Expression of cDNAs was confirmed using qPCR (not shown) and western blot analysis using a FLAG antibody (Figure 2.4 and Figure 2.5).

2.2.5 Western blotting

Cells were homogenized in RIPA buffer containing 50 mM Tris-Cl, pH 7.4, 150 mM

NaCl, 1% NP40, 0.25% SDS, and protease inhibitors (Sigma). For detection of phosphorylated protiens, 1 mM Na3VO4 and 1 mM NaN3 phosphotase inhibitors were added. Protein concentrations were determined by the Bradford assay (BioRad).

Equivalent amounts of protein were run on 10% SDS-polyacrylamide gels and proteins were transferred to nitrocellulose membranes (GE Healthcare). The membranes were incubated for 1 hour at room temperature with 5% nonfat dry milk, then with primary antibodies against NOTCH3 (1:1,000, sc-5593, Santa Cruz Biotechnology), NOTCH2

(1:200, C651.6DbHN, Developmental Studies Hybridoma Bank), TUBULIN (1:20,000,

T7816; Sigma), MKI67 (1:1000, ab66155, ABCAM), FLAG (1:1000, M2, Fisher),

CASPASE3 (1:1000, 8G10, Cell Signaling Technology, Inc.), ERK1/2 (1:2000 ERK1, sc-93, and 1:2000 ERK2, sc-154, Santa Cruz Biotechnology), p-ERK (1:1000, 4377S,

Cell Signaling Technology, Inc.) in 5% nonfat dry milk overnight at 4°C. Secondary antibodies conjugated to the horseradish peroxidase (HRP), (1:5,000; Amersham

Biosciences) were incubated for 1 hour at room temperature. Proteins were detected by enhanced chemiluminescence (Thermo Fisher Scientific) and films were either digitally

47 scanned or chemiluminescence was directly measured using the Bio-Rad ChemiDoc

XRS+. Protein was quantified using ImageLab software (Bio-Rad). Relative protein expression was calculated by normalization to TUBULIN.

2.2.6 Proliferation Assay

Relative proliferation was determined by methylene blue assay as described previously

[174], using 3000 HAoSMCs per well of a 96-well collagen coated plate.

2.2.7 UV-B irradiation

HAoSMCs were plated in a 6-well plate at 1 × 105 cells/well the day before irradiation.

Media was removed and the cells were washed in PBS, before irradiation with 25-100

µJ/cm2 with the Spectrolinker XL-1500 (Spectronics Corporation), with bulbs producing

UV-B radiation at the wavelength 254 nm. Cells were collected 9 hours-post irradiation.

2.2.8 CASPASE3 apoptosis assay

Lysates were prepared by suspension of cells in KPM buffer [50 mM KCl, 50 mM

PIPES, 10 mM EGTA, 1.92 mM MgCL2 pH 7.0, 1mM DTT, 1 mM PMSF, 10 µg/ml cytochalasin B (Acros Organics), 2 µg/ml protease inhibitors (Sigma)] and 5-10 freeze/thaw cycles. Lysates were then used in a CASPASE3 enzymatic activity assay using AFC-DEVD substrate (MP Biomedicals) as described previously [175].

Fluorescence was measured by Spectramax M5 (Molecular Devices, filters: excitation,

400 nm; emission, 508 nm). Vmax values were calculated by Softmax Pro software

(Molecular Devices).

2.2.9 Mouse lines, genotyping, and crosses

All strains were maintained in C57Bl/6 background. Notch3-/- (B6;129S1-Notch3tm1Grid/J)

[122] mutant mice were generated and generously provided by Dr. Thomas Gridley.

Notch2fl/fl (B6.129S-Notch2tm3Grid/J)(JAX 010525)[176] and Myocardin-Cre

(Myocdtm1(cre)Jomm/J)(JAX 014180)[177] were purchased from the Jackson Laboratory.

Notch2fl/fl and Myocardin-Cre mice were crossed to produce smooth muscle-deficient

Notch2 mice. Genotyping of mice and embryos was carried out by PCR with the following primers: Notch3wt1: 5′- CCA TGA GGA TGC TAT CTG TGA C, Notch3wt2:

5′- CAC ATT GGC ACA AGA ATG AGC C, Notch3dl1: 5′- GGT ACT GAG AAC

CAA ACT CAG C, Notch3dl2: 5′- CCT TCT ATC GCC TTC TTG, Notch2flox1: 5’-

TAG GAA GCA GCT CAG CTC ACA G, Notch2flox2: 5’-ATA ACG CTA AAC GTG

CAC TGG AG, Myocardin-Cre forward: 5′-TCC TGC CCT GCT GGT TAA TTA GCC

TCG, Wild-type reverse: 5′-TCA GCA AAG AGT GCA GAC CCC AGG AG,

Myocardin-Cre reverse: 5′-AAC CTC ATC ACT CTT GCA TCG ACC GG. All mouse studies were carried out in accordance with protocols approved by the Institutional

Animal Care and Use Committee (IACUC) at the Research Institute at Nationwide

Children’s Hospital. The Nationwide Children’s Hospital Research Institute IACUC specifically approved this study.

2.2.10 Collection of mouse aortas and isolation of aortic smooth muscle cells

Aortas were removed from euthanized mice and stripped of their adventitia and endothelial cells. For RNA isolation, aortic tissue was placed in RiboZol and lysed using the Tissuelyser II (Qiagen). For isolation of primary aortic smooth muscle cells, aortic tissue was digested in HBSS (CellGro) with 175 U/ml Collagenase II (Sigma), 175 U/ml

Elastase (Sigma), and 1% Antibiotic-Antimycotic Solution (CellGro). After 45 minutes at

37ºC, aortas were homogenized by pipetting, pelleted, and grown in SMC media supplemented with 1% Antibiotic-Antimycotic Solution.

2.2.11 Statistical Analysis

Data analyses were performed using GraphPad Prism, and comparisons between data sets were made using Student’s t test, one-way ANOVA, or two-way ANOVA as noted.

Differences were considered significant if p<0.05. Data are presented as mean + S.D. unless otherwise noted. Data shown are representative of at least three independent experiments.

2.3 Results

2.3.1 Notch2 and Notch3 expression are uniquely regulated by PDGF-B

To identify potential functional differences between Notch2 and Notch3 in VSMCs, we first sought to detect expression changes in response to a well-known mediator of VSMC phenotypes, PDGF-B. Treatment of serum-starved human aortic smooth muscle cells

(HAoSMCs) with PDGF-B produced a progressive loss of NOTCH2 mRNA and protein,

50 but did not significantly decrease Notch3 expression (Figure 2.1A, B). NOTCH2 transcript expression measured by quantitative PCR (qPCR) showed significant decrease at 12 hours and beyond, while the protein decrease by western analysis lagged, exhibiting a significant reduction at the 24 and 48-hour time points. In contrast, although NOTCH3 mRNA expression was not affected, protein levels were increased by approximately two- fold at 48 hours. To determine which pathway(s) downstream of PDGF-B might be responsible for Notch2 downregulation, we co-treated HAoSMCs with PDGF-B and select pathway inhibitors. PDGF-B-associated Notch2 decrease was ablated by both a

PDGF receptor inhibitor (PDGFRI) and the MEK/ERK inhibitor U0126, but not by a

JNK inhibitor (JNKII) (Figure 2.1C). Overall, these data demonstrate that Notch2 is specifically down regulated by PDGF-B at the RNA and protein level, while NOTCH3 transcript expression remains unaffected, and protein levels were increased in marked contrast to Notch2.

2.3.2 Notch2 and Notch3 have opposing effects on smooth muscle cell proliferation in response to PDGF-B

Given the distinct effect of PDGF-B on Notch2 and Notch3 receptor expression, we next wanted to determine how manipulating Notch2 and Notch3 would affect PDGF-B- dependent proliferation. We over-expressed and ablated Notch2 or Notch3 in HAoSMCs and measured the cells ability to proliferate in response to PDGF-B using a methylene blue proliferation assay. For receptor ablation, cells were transfected with siRNA directed against each receptor to knockdown expression and compared to control siRNA in the

51 presence or absence of PDGF-B. For over-expression studies, the Notch2 (NICD2) and

Notch3 (NICD3) intracellular domains were transduced via a lentivirus and compared to control GFP-expressing virus with and without PDGF-B treatment.

In the absence of Notch2 (siNOTCH2), smooth muscle cells without PDGF-B exhibited increased proliferation compared to control siRNA-transfected cells. In the presence of

20 ng/ml of PDGF-B, while there was an overall increase in the rate of proliferation, no difference was observed between control and siNotch2 cells (Figure 2.2A). This lack of a difference is likely due to the ability of PDGF-B to decrease Notch2 expression similar to the effects of siRNA knockdown. Over-expression of NICD2 produced complementary results, as NICD2 over-expression inhibited PDGF-B associated proliferation (Figure

2.2B). In complete contrast to the inhibitory effects of Notch2 on cell proliferation, manipulation of Notch3 revealed a pro-proliferation activity. In response to 20 ng/ml

PDGF-B, cells treated with siNOTCH3 had a reduced proliferation rate (Figure 2.2C), and NICD3-expressing cells showed increased proliferation (Figure 2.2D). These data were further supported by western blots measuring the expression of the proliferation marker, MKI67 (Ki67), which had stronger induction in siNotch2 cells, and weaker in siNOTCH3 samples, compared to control siRNA-treated cells (Figure 2.2E).

Consistently, NICD2 overexpression caused reduced MKI67 expression, while NICD3 promoted its expression in PDGF-B treated smooth muscle cells (Figure 2.2F).

Collectively, these data demonstrate opposite functions of the Notch2 and Notch3 receptors on smooth muscle proliferation.

To further validate our proliferation results, we isolated aortic smooth muscle cells from mice with a global knockout of Notch3 (N3-/-), and a smooth muscle-specific knockout of

Notch2 (N2fl/fl; MCC+/-). These mouse primary cells were utilized in our proliferation assays as described for the HAoSMCs and compared to wild-type (WT) cells derived from control mice. Similar to the siRNA knockdown findings with human smooth muscle cells, Notch3 null smooth muscle cells exhibited reduced proliferation, and Notch2- deleted cells exhibited enhanced proliferation (Figure 2.3). However, in these Notch- deficient cells both the untreated and PDGF-B treated cells had significantly different proliferation rates compared to wild-type cells. Thus, in both human and mouse aortic smooth muscle cells, Notch2 is anti-proliferative and Notch3 acts in a pro-proliferative manner. Overall, these results demonstrate that Notch2 and Notch3 have different roles in regulating VSMC proliferation in response to PDGF-B.

2.3.3 Notch3 is uniquely regulated by inducers of apoptosis

Notch3 has been strongly linked to cell survival, thus we asked if Notch3 might be regulated by cell death signals, similar to how PDGF-B down regulates Notch2. To test this, cells were treated with hydrogen peroxide or ultraviolet (UV) irradiation to promote apoptosis, followed by RNA and protein isolation to measure Notch receptor expression.

At the transcript level there was no significant changes in either receptor’s expression

(Figure 2.4A, B). However, western analysis of Notch3 protein revealed a precipitous decline when HAoSMCs are treated with either apoptosis inducer, while Notch2 protein

53 expression remained unchanged (Figure 2.4C, D). Given that Notch3 protein, but not mRNA expression was decreased by two different apoptosis inducers, we hypothesized it was due to a common mechanism involving protein degradation. Indeed, when we treated cells with the proteasomal inhibitor MG-132 the UV-induced decrease of Notch3 protein was ablated (Figure 2.4E). The loss of Notch3 by UV irradiation could also be seen using the over-expressed Flag-tagged-NICD3, whose degradation was similarly blocked by

MG-132 (Figure 2.4F). In contrast, Flag-tagged-NICD2 was not affected by UV irradiation or MG-132 treatment. Taken together, these data indicate that the intracellular domain of Notch3 is specifically targeted for proteasomal degradation following apoptosis induction.

2.3.4 Notch3 protects VSMCs from induction of apoptosis

Since Notch3 protein is targeted for degradation after apoptosis stimulation we hypothesized that Notch3 downregulation is induced to allow VSMCs to enter apoptosis.

If this is true, Notch3 signaling should be protective against apoptosis induction. To test this, we utilized the same siRNA knockdown and overexpression strategies as described for proliferation assays to measure the effects of Notch2 and Notch3 on UV-induced apoptosis. Apoptosis was monitored by western analysis to measure activated cleaved

CASPASE3, and an enzymatic Caspase assay utilizing the fluorescent CASPASE3 substrate, AFC-DEVD. Overexpression of NICD3 in the presence of UV irradiation showed a protective effect by decreasing CASPASE3 activity (Figure 2.5A, C).

Accordingly, siRNA knockdown of Notch3 resulted in an increase in cleaved

CASPASE3 protein and CASPASE3 activity (Figure 2.5B, D). Conversely, Notch2 overexpression or knockdown had no effect on smooth muscle cell apoptosis as measured by CASPASE3 activity. These data reveal that, as with proliferation, Notch2 and Notch3 have differing effects on cell survival, with Notch3 acting as a protective factor against

UV-induced apoptosis.

2.3.5 Notch3 promotes transcription of cell survival genes via activation of MAPK

To determine the mechanism by which Notch3 inhibits apoptosis, we measured the expression of known pro-survival genes in HAoSMCs with altered Notch receptor expression. From this we identified three pro-survival genes that were positively associated with Notch3 expression. Pro-survival genes, BCL2 [178], BIRC5 (Survivin)

[179], and CFLAR (cFLIP) [180] showed a significant increase in transcript expression in the presence of lentivirally-expressed NICD3, and a comparable reduction in expression in cells transfected with NOTCH3-specific siRNA (Figure 2.6A).

Overexpression or deletion of Notch2 had no significant effect on these genes expression.

These pro-survival genes are known to be regulated by the MAPK pathway [181-183], therefore we tested to see if Notch3 induction of these genes was through MAPK.

HAoSMCs over-expressing control GFP, NICD2, or NICD3 were treated with the MEK inhibitor, U0126 and RNA was collected for expression analysis. As predicted, the MEK inhibitor caused a general decrease in the pro-survival genes mRNA expression in all conditions, but importantly completely blocked the induction of these genes by NICD3

(Figure 2.6B).

Notch receptors have been previously reported to interact with other signaling pathways both directly and via their transcriptional targets [34, 45, 57, 158-161]. To test if Notch3 could activate the MAPK pathway in smooth muscle cells, we measured the phosphorylation of the MAPK signaling mediator ERK in HAoSMCs with over- expressed or knocked down Notch2 or Notch3 (Figure 2.6C, D). Knockdown of Notch3 decreased ERK phosphorylation (phospho-ERK), while over-expression of NICD3 caused an increase. Interestingly, although knockdown of Notch2 did not affect phospho-

ERK levels, over-expression of NICD2 caused a strong decrease. From these results we demonstrate a causal link between Notch3’s ability to induce pro-survival gene expression and VSMC survival. The data indicate that Notch3 acts through the

MEK/ERK signaling pathway to up-regulate these pro-survival genes.

2.3.6 Genetic deletion of Notch3, but not Notch2 has unique effects on cell survival of primary aortic smooth muscle cells

In order to validate the regulation of pro-survival genes by Notch3, we measured the mRNA expression of the three identified pro-survival genes in the aortas of Notch- deficient mice. We found that transcript expression of all three genes was significantly decreased in the aortas of Notch3-null (Notch3-/-) mice, but not the Notch2-mutant

(Notch2fl/fl; MCC+/-) mice (Figure 2.7A). Accordingly, we found that primary aortic smooth muscle cells with genetic deletion of Notch3 mirrored the Notch3 siRNA results in HAoSMC (Figure 2.4D), showing increased CASPASE3 activity in response to

56 apoptosis induction (Figure 2.7B). Additionally, these mouse aortic smooth muscle cells exhibited a similar phospho-ERK profile to their human counterpart (Figure 2.5C), where

Notch3-deficient cells had dramatically reduced ERK phosphorylation (Figure 2.7C).

These results reinforce our hypothesis that Notch3 promotes VSMC survival by inducing

ERK signaling and promoting expression of pro-survival genes.

Overall, the data presented herein illustrate unique aspects of Notch2 and Notch3 function and expression in VSMC. Our results demonstrate a direct interaction between their expression and function in governing proliferation and cell survival. Moreover, these findings reveal receptor-specific relationships between the Notch pathway and ERK signaling, which suggests complexities that contribute their unique functions.

2.4 Discussion

Data presented here provide new insight into how the Notch signaling pathway is regulated within VSMCs, and exerts its effects to influence phenotypic properties. Our results demonstrate that the Notch2 and Notch3 receptors have different roles in VSMC proliferation and survival (Figure 2.8). Notch3 acts via the MEK/ERK pathway to promote cell survival, and its protein is targeted for proteasomal degradation in response to apoptosis cues. The growth factor PDGF-B increases Notch3 protein expression, which in turn promotes proliferation, possibly through MEK/ERK signaling. In contrast,

Notch2 acts to inhibit proliferation, and PDGF-B signals through the MEK/ERK pathway

57 to remove this inhibition by down regulating the Notch2 receptor. Overall, these findings identify Notch2 as anti-proliferative and Notch3 as pro-proliferative and pro-survival in smooth muscle cells, with MEK/ERK signaling serving a pivotal role in these functional outcomes.

One interesting conclusion from our results is that the expression of the two receptors is linked to the phenotypes they promote. Notch2 is anti-proliferative and a proliferation inducer, PDGF-B, reduces its expression. Likewise, Notch3 is pro-survival and apoptosis inducers decrease its protein expression. This reciprocal regulation of Notch2 and Notch3 by mediators of the very processes they influence suggests that these inducers exert their effects, at least in part, through controlling Notch receptor expression. In fact, one could envision Notch2 as a brake that must be removed before proliferation can proceed and a similar role for Notch3 as an impediment to the progression of apoptosis. Indeed, in an report demonstrating Notch2’s inhibitory role in smooth muscle proliferation, the Liaw lab showed NOTCH2 mRNA expression is sharply decreased after S-phase allowing the cell to undergo mitosis [100]. Our data showing rapid proteasomal degradation of Notch3 protein by apoptosis mediators suggests a similar strategy, in which Notch3 must be decreased to allow apoptosis to proceed. Thus, in mature smooth muscle cells, where both Notch receptors are expressed, they not only contribute to a contractile differentiated phenotype, but also maintain a quiescent cell that is protected from apoptosis.

Our results identify MAPK signaling as being intricately connected to the Notch2 and

Notch3 receptors. We show that MEK/ERK signaling acts upstream of Notch2 and contributes to its down regulation by PDGF-B. Conversely, we demonstrate that the

MEK/ERK pathway is downstream of Notch3, and its activation is critical for Notch3- dependent up regulation of pro-survival genes. MAPK signaling has long been linked to cell survival [184, 185], but Notch3’s ability to activate it to enhance survival is a novel finding. Additionally, the large disparity in phospho-ERK levels between NICD2 and

NICD3 over-expression reveal significant differences in how Notch2 and Notch3 interact with the MAPK pathway. Since the MAPK pathway is also a strong promoter of cell growth and proliferation, it is likely that the regulation of MEK/ERK signaling by Notch2 and Notch3 is at least partially responsible for their observed effects on proliferation. One possible mechanism for Notch3’s regulation of proliferation and/or MEK/ERK signaling is by influencing growth factor receptors. Notch signaling has been shown to increase expression of PDGFRβ [36], the receptor for PDGF-B. However, we did not see any difference in PDGFRβ expression between Notch2 and Notch3 in our over-expression or knockdown studies (data not shown), suggesting additional mechanisms contribute to their unique effects on proliferation.

VSMC phenotypes are tightly regulated by a number of factors that contribute to how these cells function and respond to stimuli. With this in mind, the varied expression of these two receptors in different phases of vascular development, injury, and disease [6,

18] implicates the balance of Notch2 and Notch3 signaling as a key determinant of

VSMC phenotypes. One could imagine that an overabundance of Notch3 activity leads to rapid expansion and maintenance of a smooth muscle population, while a surplus of

Notch2 activity leads to quiescence. The classic understanding of VSMC phenotype holds that a differentiated contractile cell is quiescent, and a proliferating cell is dedifferentiated. Given that Notch signaling is a known promoter of differentiation [61,

79, 84, 91], and we have shown that both Notch2 and Notch3 stimulate expression of contractile genes, this indicates a more complex picture of these phenotypes. Our data suggests that Notch3 may contribute to an intermediate VSMC phenotype capable of both proliferating and expressing contractile genes. This distinct function is likely important for development, but also might contribute to disease. Pulmonary arterial hypertension exemplifies this concept, where VSMCs have excessive proliferation, resist apoptosis, and have increased expression of contractile genes, all correlated with increased Notch3 expression. Interestingly, these disease phenotypes are ameliorated with a reduction of

Notch3 signaling [137].

The advent of CRISPR-Cas9 technology is quickly making traditional knockdown methods obsolete. The recent discovery that knockdowns by traditional methods do not always match the phenotypes seen by deletion with CRISPR-Cas9 systems [186], suggests it may be a better alternative to avoid off-target effects [187, 188]. While we are confident in the efficiency of our siRNA knockdowns, given our results are corroborated by data using both over-expression and genetic deletion systems, in moving forward one must consider deletion of Notch2/3 using CRISPR-Cas9. Additionally, an exciting

60 avenue of investigation made easier by CRISPR-Cas9 is the potential identification of the structural domains of the Notch receptors that harbor their unique effects. It is now relatively simple to create chimeric proteins composed of different regions of individual proteins. Introduction of Notch2/Notch3 chimeric receptors into cells and animal models would significantly advance our understanding of modularity and distinct functions of the

Notch family of receptors.

Taken together, our results show for the first time that Notch2 and Notch3 can uniquely regulate VSMC phenotypes and demonstrate that their expression is closely related to their functions. These results clearly show that Notch signaling is an important mediator of smooth muscle biology and can exert unique forces through expression of different receptor combinations.

2.5 Figures

Figure 2.1. Notch2, but not Notch3 receptor expression is decreased by PDGF-B.

(A,B) HAoSMCs were serum-starved for 48 hours, then treated with 20 ng/ml PDGF-B and collected to isolate mRNA for qPCR (A), or protein for western blot analysis (B), at designated timepoints. qPCR shown as expression relative to GAPDH, n=5. Notch2 and Notch3 band intensity quantified relative to TUBULIN, n=6. (C) Serum starved HAoSMCs were treated with/without 20 ng/ml PDGF-B, and vehicle control, 5 µM PDGF-receptor inhibitor (PDGFRI), 10 µM MEK inhibitor (U0126), or 10 µM JNK inhibitor (JNKII) for 48 hours. qPCR shown as expression relative to GAPDH, n=4. Significance determined by one-way ANOVA ,*p<0.05. n.s.=Not significant.

Figure 2.2. Notch2 and Notch3 have opposing effects on PDGF-B dependent smooth muscle cell proliferation. (Continued)

Figure 2.2 (cont.) HAoSMCs with siRNA knockdown of (si)NOTCH2 and (si)NOTCH3, or lentiviral over-expression of Notch2 (NICD2) and Notch3 (NICD3) intracellular domains. (A-D) Cells were serum starved for 48 hours and treated with 20 ng/ml PDGF- B for proliferation assays, n=3. “*” indicates significant difference compared to GFP or siRNA (siControl) controls. Significance determined by two-way ANOVA, p<0.05, +/- SEM. (E, F) Protein extracts collected for western blots after 24 hours to measure proliferation marker MKI67 expression. Significance determined by one-way ANOVA, p<0.05.

Figure 2.3. Genetic deletion of Notch2 or Notch3 has unique effects on proliferation of mouse aortic smooth muscle cells.

SMCs were isolated from aortas of wild-type (WT), Notch2 (Notch2fl/fl; MCC+/-), and Notch3 (Notch3-/-) deficient mice and used in methylene blue proliferation assays. (A) Comparison of WT and Notch2-null cells. (B) Comparison of WT and Notch3-null cells, n=4. “*” indicates significant difference compared to WT in the same PDGF-B treatment group determined by two-way ANOVA, p<0.05, +/- SEM.

Figure 2.4. Notch3 is uniquely regulated by inducers of apoptosis. (Continued)

Figure 2.4 (cont.) HAoSMCs were treated with increasing concentrations of H2O2 for 24 hours or exposed to indicated levels of UV radiation and collected 12 hours later for RNA and protein expression analysis. (A, B) qPCR expression analysis of NOTCH2 and NOTCH3, shown relative to GAPDH, n=3. (C, D) Western blots of Notch2 and Notch3 expression with TUBULIN as a loading control. (E) HAoSMCs were exposed to UV radiation and cultured for 12 hours with 10 µM MG-132 proteasome inhibitor or with vehicle control. Western blots of Notch3 expression with TUBULIN as a loading control, n=3. (F) HAoSMCs with lentivirally over-expressed FLAG-tagged NICD2 or NICD3 were UV-irradiated and cultured for 9 hours with 10 µM MG-132. Western blots to detect FLAG-tagged proteins, relative to TUBULIN, n=3. Significance determined by one-way ANOVA , *p<0.05, n.s.=Not significant.

Figure 2.5. Notch3 protects smooth muscle cells from cell death. (Continued)

Figure 2.5 (cont.) HAoSMCs with lentivirally over-expressed NICD2, NICD3, and GFP, or siRNA knockdown of NOTCH2 (siN2), NOTCH3 (siN3), and control (siCTL) were exposed to 50 µJ/cm2 UV radiation and collected for protein 9 hours later. (A, B) Western blots to detect Notch2 and Notch3 expression, and level of total and cleaved CASPASE3 with TUBULIN used as loading control, n=3. (C, D) CASPASE3 enzymatic activity assay using fluorescent caspase substrate AFC-DEVD, n=4. Significance determined by one-way ANOVA , *p<0.05.

Figure 2.6. Notch3 promotes transcription of cell survival genes via activation of MAPK. (Continued)

Figure 2.6 (cont.) HAoSMCs with lentiviral over-expression of GFP (control), NICD2 and NICD3, or siRNA knockdown of (si)NOTCH2, (si)NOTCH3 and control (siCTL). (A) RNA expression of putative pro-survival genes were measured by qPCR with relative expression compared to GAPDH, n=3. (B) HAoSMCs with lentiviral over-expressed NICDs were treated with 10 µM U0126 or vehicle control for 24 hours, and RNA was isolated to measure pro-survival gene expression, n=3. (C, D) Western blots to measure total and phosphorylated (phospho)-ERK, with TUBULIN as loading control, n=3. Significance determined by one-way ANOVA,*p<0.05.

Figure 2.7. Genetic deletion of Notch3 has unique effects on the survival of mouse aortic smooth muscle cells.

(A) mRNA expression of pro-survival genes in aortas of wild-type (WT), Notch2- deficient (Notch2fl/fl; MCC+/-), and Notch3-mutant (Notch3-/-) adult mice. qPCR with relative expression compared to GAPDH, n=4. (B) SMCs were isolated and cultured from aortas of wild-type, Notch2, and Notch3 deficient mice and used in enzymatic CASPASE3 activity assays, n=4. (C) Total and phosphorylated (phospho)-ERK was measured in cultured smooth muscle cells following serum challenge, with TUBULIN used as loading control, n=4. Significance determined by one-way ANOVA, *p<0.05.

Figure 2.8. Notch2 and Notch3 have unique roles in proliferation and cell survival related to their expression.