DEFINING the ROLE of HYPOXIA and Hifs in VASCULAR SMOOTH MUSCLE CELLS

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DEFINING THE ROLE OF HYPOXIA AND HIFs IN VASCULAR SMOOTH MUSCLE CELLS

ANNA CLARK HENRY BORTON

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Department of Pathology

CASE WESTERN RESERVE UNIVERSITY

August, 2018

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Anna Clark Henry Borton

candidate for the PhD Degree*.

Committee Chair

Nicholas P. Ziats, Ph.D.

Committee Members

Diana L. Ramirez-Bergeron, Ph.D.

Aaron Proweller, M.D., Ph.D.

George R. Dubyak, Ph.D.

Clive R. Hamlin, Ph.D.

Date of Defense

June 25, 2018

*We also certify that written approval has been obtained

for any proprietary material contained therein.

Table of Contents

List of Tables……………………………………………………………………. 7

List of Figures…………………………………………………………………… 8

Acknowledgements…………………………………………………………….. 10

List of Abbreviations……………………………………………………………. 12

Abstract………………………………………………………………………….. 16

Chapter 1: Introduction…………………………………………………...... 18

Vascular Smooth Muscle ……………………………………………… 18

Structure of the vascular wall.....……………………………… 18

Diverse developmental origins of VSMCs……………………. 20

Vascular smooth muscle contractile function………………... 22

Phenotypic plasticity in VSMCs……………………………….. 27

Hypoxia-Inducible Factors……………………………………………... 29

Oxygen dependent regulation of HIFs………………………... 30

Alternative HIF stabilization pathways………………………... 32

HIF function in the vasculature………………………………... 33

HIF in pulmonary VSMCs……………………………………… 33

HIF in VSMCs of the systemic vasculature………………….. 35

Peripheral Vascular Disease…………………………………………... 37

Epidemiology and disease burden……………………………. 38

Clinical presentation and diagnosis…………………………… 38

Pathogenesis and vascular compensation…………………... 40

Therapeutic interventions……………………………………… 44

HIF in VSMCs of the peripheral vasculature………………………… 45

A murine model of PVD: HLI…………………………………... 45

HIFs in HLI model………………………………………………. 46

HIF as a therapeutic……………………………………………. 48

Summary and Hypothesis……………………………………………… 48

Chapter 2: Aryl Hydrocarbon Receptor Nuclear Translocator in

Vascular Smooth Muscle Cells is Required for Optimal Peripheral

Perfusion Recovery…………………………………………………………… 50

Authors…………………………………………………………………… 50

Summary………………………………………………………………… 51

Introduction……………………………………………………………… 53

Methods………………………………………………………………….. 56

Results…………………………………………………………………… 66

Generation of smooth muscle specific Arnt knockout mouse

model…………………………………………………………….. 66

ArntSMKO mice show impaired blood flow recovery in HLI

model…………………………………………………………….. 68

Limb ischemia stimulates collateral remodeling in ArntSMKO

and Arntlox/lox mice………………………………………………. 70

Early capillary angiogenesis does not depend on VSMC

Arnt in HLI……………………………………………………….. 73

ArntSMKO mice show increased hypoxia and damage in

gastrocnemius in response to ischemia……………………… 81

Altered vascular smooth muscle morphology in ArntSMKO

mice………………………………………………………………. 84

Loss of ARNT alters VSMC phenotype………………………. 87

Discussion……………………………………………………………….. 91

Chapter 3 Loss of Vascular Smooth Muscle Cell Aryl Hydrocarbon

Receptor Nuclear Translocator Impairs Vasoconstriction…………….. 97

Authors…………………………………………………………………… 97

Summary………………………………………………………………… 98

Introduction……………………………………………………………… 99

Methods………………………………………………………………….. 101

Results…………………………………………………………………… 105

Transcriptional expression of contractile elements are

reduced in VSMCs lacking ARNT…………………………….. 105

Large vessel structure and tunica media thickness is not

affected in ArntSMKO…………………………………………….. 107

Loss of Arnt in VSMCs impairs aortic vasoconstriction…….. 107

Loss of Arnt in VSMCs does not affect aortic vasorelaxation 109

Discussion……………………………………………………………….. 112

Conclusions……………………………………………………………… 115

Chapter 4: Discussion and Closing …………………………………...... 116

Discussion……………………………………………………………….. 116

Molecular dysregulation………………………………………... 116

Implications for other vascular pathologies………………….. 123

Potential for translation to human disease…………………… 129

Closing………………………….………………………………………... 135

Future Directions………………………………………………………... 136

References……………………………………………………………………... 139

List of Tables

Supplemental Table 1: qPCR primers………………………………………... 64

List of Figures

Figure 1.1. Layers of the vascular wall……………………………………….. 19

Figure 1.2. VSMCs and ECs of the vascular wall………………………..…. 21

Figure 1.3. VSMC myofilament components………………………………… 23

Figure 1.4. Regulation of VSMC contraction and relaxation……………….. 25

Figure 1.5. Canonical HIF regulation…………………………………………. 31

Figure 1.6. Vascular responses to flow limitation…………………………… 43

Figure 1.7. Hind limb ischemia: a murine model of PAD………………...... 47

Figure 2.1. Characterization of ArntSMKO mice……………………………….. 67

Figure 2.2. Bulk perfusion and functional recovery is reduced in ArntSMKO mice following femoral artery ligation…………………………………………. 69

Figure 2.3. VSMC ARNT is not required for ischemia induced collateralization………………………………………………………………….. 71

Supplemental Figure 2.1. Collateral vessel lumen cross sectional area vs limb perfusion…………………………………………………………………… 72

Supplemental Figure 2.2. Proximal HLI model………………………………. 75

Figure 2.4. Histological assessment of capillary number and perfusion status in gastrocnemius muscle (GC)………………………………………… 76

Supplemental Figure 2.3. Capillary density and smooth muscle cell colocalization in gastrocnemius (GC) at day 7………………………………. 78

Supplemental Figure 2.4. Regional assessment of capillary density and perfusion status in gastrocnemius muscle (GC)…………………………….. 79

Figure 2.5. Ischemic skeletal muscle regeneration and vessel integrity are impaired in ArntSMKO mice…………………………………………………. 83

Figure 2.6. Smooth muscle morphology and perimural wrapping of small arterioles…………………………………………………………………………. 85

Supplemental Figure 2.5. Additional images of arterioles in skeletal muscle……………………………………………………………………………. 86

Figure 2.7. Transcriptional expression of proliferation and migration regulators in response to hypoxia…………………………………………….. 89

Figure 2.8. Assessment of VSMC phenotype in vitro………………………. 90

Figure 3.1. Contractile gene expression is reduced in ArntSMKO VSMCs…. 106

Figure 3.2. Large vessel structure unchanged in ArntSMKO………………… 108

Figure 3.3. ArntSMKO vessels have impaired vasoconstriction……………… 110

Figure 3.4. Vasorelaxation is intact in ArntSMKO……………………………... 111

Figure 4.1. Effects of hypoxia exposure on human iliac artery VSMCs…... 130

Figure 4.2. Effects of hypoxia exposure on human iliac vein VSMCs…….. 133

Figure 4.3. Iliac vein smooth muscle cells are more susceptible to H2O2 induced apoptosis………………………………………………………………. 134

Acknowledgements

Numerous people have helped me reach this milestone, and for each of their

contributions, I am tremendously grateful.

It has been an outstanding privilege to train under Diana Ramirez-Bergeron. Her

enthusiasm and love of science are positively infectious, as is the commitment to

excellence, scientific rigor, and collaborative spirit she instills in her trainees. Her

mentorship inspired me to step outside my academic comfort zone, encouraged

me through challenges, and helped me develop skills I will use for the rest of my

career.

My thesis project, spanned the expertise of the Ramirez and Proweller

laboratories. I am incredibly grateful to Aaron Proweller for sharing his scientific

guidance, technical expertise, and perspective as a physician scientist.

Enormous thanks also to my thesis committee members, Nicholas Ziats, George

Dubyak, and Clive Hamlin for all their scientific insight and advice.

Science does not happen in a vacuum: I am grateful to have had the opportunity to work with and learn from all the past and present members of the Ramirez and

Proweller laboratories.

I would also like to thank Mukesh Jain, Xudong Liao, Lalitha Nayak, Yuan Lu,

Andrei Maiseyeu, Anne Hamik, Alex Huang, Rongli Zhang, Bryan Benson, Lee

Neilson, Iulia Barbur, Stephanie Lapping, Amer Alaiti, and Keiki Sugi for their technical assistance, professional and scientific advice, goodwill, and encouragement.

I have been fortunate to have a wonderful community of peers including the MSTP entering classes of 2011 and 2012 and my CVRI graduate student compatriots

Nelson, David, and Liyan.

I am grateful to the MSTP, our director Cliff Harding, the co-directors, and the administrative team for the opportunity to chase the dream of a career as a physician scientist. Thanks also to the CVRI and the Departments of Pathology and Medicine and their administrators for being my academic home during the

PhD.

Overwhelming thank yous are also due to my friends and extended family for graciously listening to the minutia of experimental success and failure, and cheering me through the celebratory moments and the frustrations.

I am so grateful to my family for fostering curiosity and dedication and for their love, support, and encouragement. Finally, a tremendous thank you to my wonderful husband Peter. He has been there every step of the way as my greatest supporter and the baker of thesis committee appeasing banana bread.

List of Abbreviations

ABI – Ankle-brachial index

ACC – American College of Cardiology Foundation

Ach – Acetylcholine

AHA – American Heart Association

AHR – Aryl hydrocarbon receptor

ANG1 – Angiopoietin

AngII – Angiotensin II

ARNT; HIF-1β – Aryl hydrocarbon receptor nuclear translocator

Arntlox/lox – Animals and cells with floxed ARNT gene

ArntSMKO – Smooth muscle specific ARNT deletion model

AT1 – Angiotensin receptor 1

BSA – Bovine serum albumin

Ca – Calcium

CaCm – Calcium calmodulin

CLI – Critical limb ischemia

Cnn1 – Calponin

DMOG – Dimethyloxalylglycine

DN- HIF-1α – Dominant negative HIF-1α

EC – Endothelial cell

EDHF – Endothelium-derived hyperpolarizing factors

EEL – External elastic lamina

eNOS – Endothelial nitric oxide synthase

ET-1 – Endothelin receptor 1

FGF2 – Fibroblast growth factor 2

FIH – Factor inhibiting HIF

GC – Gastrocnemius

Glut1 – Glucose transporter 1

HIFs – Hypoxia-inducible factors

HLI – Hind limb ischemia

HRE – Hypoxia response element

HXK2 – Hexokinase 2

IEL – Internal elastic lamina

IP – Intraperitoneal

IV – Intravenous

KLF4 – Krüppel-like factor 4

KO – Knockout

MAPK – Mitogen activated protein kinase

MEJ – Myoendothelial junctions

MLC; MYL9 – Regulatory myosin light chain

MLCK – Myosin light chain kinase

MLCP – Myosin light chain phosphatase

MMPs – Matrix metalloproteinases

O2 – Oxygen

OCT4 – Octamer-binding transcription factor 4

PAD – Peripheral arterial disease

PAH – Pulmonary artery hypertension

PAI-1 – Plasminogen activator inhibitor-1

PAS – Per-ARNT-Sim

PASMC – Pulmonary artery smooth muscle cells

PDGF-BB – Platelet derived growth factor BB

PE – Phenylephrine

PGI2 – Prostaglandin I2

PHD – Prolyl hydroxylase domain containing enzymes

PVD – Peripheral vascular disease

ROCK – Rho-associated protein kinase

ROS – Reactive oxygen species

Sca-1 – Stem cells antigen-1

SMA; Acta2 – Alpha smooth muscle actin

SMM – Smooth muscle myosin

SMMHC; MYH11 – Smooth muscle myosin heavy chain

SNP – Sodium nitroprusside

SRF – Serum response factor

THBS-1 – Thrombospondin 1

Thbs2 – Thrombospondin 2

Timp1 – TIMP metallopeptidase inhibitor 1

TNF-α – Tumor necrosis factor alpha

VEGFA – Vascular endothelial growth factor A

VEGFR-1 – Vascular endothelial growth factor receptor 1

VHL – von hippel-lindau protein

VSMCs – Vascular smooth muscle cells

WT – Wild type

Defining the Role of Hypoxia and HIFs in Vascular Smooth Muscle Cells

Abstract

ANNA CLARK HENRY BORTON

Vascular diseases are major causes of morbidity and mortality. Vascular smooth muscle cells (VSMCs), as integral constituents of the vascular wall, provide structural support and dynamically alter vessel shape and structure in response to environmental stimuli. While the role of their endothelial neighbors has been extensively examined in facilitation of ischemic responses and vascular remodeling, less is known about VSMCs. Hypoxia-inducible factors (HIFs) are master regulators of cellular responses to O2 tension. These heterodimeric transcription factors drive endothelial vascularization and are putative regulators of VSMC driven vascular remodeling in pulmonary artery hypertension. The studies presented in this dissertation examine the role of canonical HIF activity in modulating VSMC phenotype, vasoreactivity, and responses to peripheral ischemia. Mice with smooth muscle specific deletion of aryl hydrocarbon receptor nuclear translocator (ARNT; HIF-1β) (ArntSMKO), required for HIF transcriptional activity, feature impaired peripheral perfusion recovery following femoral artery ligation. Vessels from ArntSMKO mice display altered vasomotor function, increased hypoxic microvascular permeability, and disrupted arteriolar VSMC morphology.

Aberrant behavior and transcriptional profile in isolated ArntSMKO VSMCs indicate

16 phenotypic switching. Collectively, the data support a role for ARNT regulating

VSMC phenotype, essential for vasoreactivity and perfusion recovery in peripheral ischemia. As such, ARNT provides a therapeutic target for modulating VSMC phenotypic change, a feature of many vascular diseases.

Chapter 1: Introduction

In vertebrates, the vasculature is critical to sustaining life. Every cell in the body

resides no more than two hundred microns away from the nearest vessel1. At a

very simple level, blood vessels are conduits for the provision of nutrients and

oxygen and the removal of waste, but they also aid in endocrine signal delivery

and provide a network for the rapid transit of migratory cells from tissue to tissue.

Structure of the vascular wall

The vessel wall is composed of three layers, the intima, media, and adventitia

(Figure 1.1) 2.The innermost layer lining the vessel lumen is the intima. It is composed of a single layer of endothelial cells (ECs) that interact directly with blood and provide selectively permeable barrier function. Just deep to the intima, the internal elastic lamina marks the inner boundary of the tunica media. The media, much thicker than the intima especially in arteries, is formed by smooth muscle cells and connective tissue encircling the vessel in circumferential layers outward to the external elastic lamina. Vascular smooth muscle cells (VSMCs), the subject of this dissertation, provide structural support to the vessel, regulate the lumen diameter, and are integral to vessel physiology. Outside the external elastic lamina the tunica adventitia is composed primarily of connective tissue 3. In large

vessels, the adventitia may contain smaller neurovascular bundles. The vessels

within these bundles, namely vasovasorum, supply nutrients to the outer layers of the vessel wall too far from the lumen to be adequately supplied by diffusion 3. The

relative positions of the mural layers can be observed in Figure 1.2, a cross section

Figure 1.1 Layers of the vessel wall. The vascular wall can be divided into three distinct layers, the intima, the media, and the adventitia.

of popliteal artery, where the CD31+ endothelial cells of the intima are visible in

green and the α-smooth muscle actin+ (SMA) VSMCs of the medial layer are identified in red. Differences in tunica media thickness in the artery and vein are also apparent in this micrograph.

Diverse developmental origins of VSMCs

VSMCs can be seen wrapped around nearly every blood vessel larger than

capillaries. While they are typically referred to as a singular tissue type, the

developmental origins of these cells are diverse 4. Indeed, in the vertebrate

embryo, VSMCs are derived from at least 7 unique non-overlapping sources 5. The

majority of VSMCs differentiate from various mesodermal fields, though the

ectodermal neural crest population also participates. The contributors to the aorta

are an excellent example of this diversity. The root of the aorta, where it exits the

left ventricle, is formed from cells of the second heart field 6. Neighboring VSMCs

in the ascending aortic arch and pulmonic trunk are derived from neural crest cells

having migrated toward the developing heart 7, 8. As the aorta begins its descent

through the thorax, individual mesodermal somites that also form each vertebral

segment, contribute aortic VSMCs at their corresponding vertebral levels 9, 10.

Continuing distally to the abdominal aorta and vessels of the lower limbs reveals

VSMCs differentiated from the splanchnopleuric layer of the lateral plate

mesoderm 11. In total, VSMCs that wrap the aorta have at least 4 separate origins,

counting the somites collectively. Vessels and the VSMCs that invest them also

develop alongside the organ they perfuse. This pattern can be seen in pulmonary

Figure 1.2 VSMCs and ECs of the vascular wall. Micrograph of murine popliteal artery and vein cross section. CD31+ ECs of the tunica intima are labeled in green and SMA+ VSMCs of the tunica media are visible in red. The artery (right) features a thicker VSMC layer than the vein (left) supporting the open lumen.

VSMCs that differentiate from lung mesoderm, and in coronary VSMCs contributed by the pro epicardium 12,13, 14. The maintenance of VSMCs in the vascular wall is also supported by local populations. A population of resident progenitors, dubbed the medial side population, has been described within the tunica media 15 while a separate population of stem cells antigen-1 (Sca-1)+ cells in the adventitia can also differentiate into VSMCs when stimulated with PDGF-BB 16. These Sca-1+ cells are not committed along the VSMC lineage and can become endothelial cells in the presence of VEGFA 16. The diverse origins of VSMCs contribute to their tissue specific responses in vivo and in vitro, yet their expression of similar makers and overall function within the vessel wall underlie their classification as a single tissue type.

Vascular smooth muscle contractile function

A unifying characteristic of VSMCs is their contractile ability. Force generation, as in other muscle cells, is achieved by cyclical cross bridging of actin and myosin

(Figure 1.3) 17. In vascular smooth muscle, the primary myofilament interaction occurs between SMA and smooth muscle myosin (SMM). SMA is abundant in

VSMCs accounting for ~40% of total cellular protein 18. SMM is a type two myosin consisting of two intertwined heavy chains (SMMHC) and four light chains: two regulatory light chains (20kDa; MYL9, MLC herein) and two essential light chains

(17kDa). The structure of SMM is similar to myofilaments in striated muscle 19.The two sets of light chains associate with the neck region of the heavy chains. Unlike other muscle cells, SMA and SMM are not arranged in sarcomeres. Instead,

Figure 1.3. VSMC myofilament components. Actin and Myosin interact to generate contractile force in VSMCs. These interactions are physically regulated by MLC, calponin, tropomyosin, and caldesmon, among other proteins.

the number and structure of the myofilaments are dynamic 20. Indeed, studies show

increases in polymerized actin in response to stimulation by contractile agonist and

that short term inhibition of actin polymerization reduces contractile force 21, 22.

Regulation of contraction centers on the phosphorylation status of the regulatory

MLC as determined by opposing regulatory enzymes, myosin light chain kinase

(MLCK) and myosin light chain phosphatase (MLCP) 23. Phosphorylation of MLC

by MCLK releases the myosin head to interact with actin and increase contraction,

while dephosphorylation by MLCP promotes relaxation. Many upstream factors

and second messengers further regulate the function of these enzymes (Figure

1.4).

Depolarization of the cell opens L type calcium channels increasing the

concentration of cytosolic free calcium (Ca) though influx of extracellular Ca and

release of intracellular Ca reservoirs in the sarcoplasmic reticulum 24. Increased

calcium activates calcium calmodulin (CaCm), which subsequently activates

MCLK. MLCK in turn phosphorylates MLC promoting constriction. In addition to

direct neuromodulation of the membrane potential, increased intraluminal tension

can stimulate myogenic vasoconstriction as initially described by Bayliss in the

early 1900s 25. L type calcium channels also participate as mediators of myogenic tone 26. Contraction can also be stimulated by receptor dependent vasoconstrictors

which activate G protein coupled receptors and, via signaling cascades, converge

to increase activity of MLCK and/or decrease activity of MLCP 24, 27.

Figure 1.4. Regulation of VSMC contraction and relaxation. Signaling, including from a variety of G protein coupled receptors and ion channels, converges on enzymes, namely MLCK and MLCP, which regulate phosphorylation and de-phosphorylation of MLC to control myosin activity and thus contraction. Pathways colored green promote contraction while pathways colored red inhibit contraction and promote relaxation.

Angiotensin II (AngII), a key factor in renin-angiotensin mediated hypertension,

28 binds to the angiotensin receptor 1 (AT1) on the cell surface . AT1, a Gq coupled receptor increases intracellular IP3 and Rho-associated protein kinase (ROCK).

IP3 facilitates calcium release from the sarcoplasmic reticulum thus increasing

MLCK activity through CaCm. ROCK in contrast inhibits MLCP activity to further increase the population of phosphorylated MLC and intensify contraction. Other vasoconstrictors that act through Gq coupled receptors include norepinephrine and phenylephrine (PE) which bind α1-adrenergic receptors, endothelin which binds endothelin ETA receptor, vasopressin which binds vasopressin 1 receptor, and serotonin which binds 5-HT2 receptor. Adrenergic stimulation also acts synergistically through α2-adrenergic receptors, which are Gi G-protein coupled receptors and whose activity decreases cAMP to further disinhibit MLCK.

Vasorelaxation is regulated by NO, prostaglandin (PGI2), and other factors collectively known as endothelium-derived hyperpolarizing factors (EDHF). NO in the smooth muscle increases cGMP activating MLCP to dephosphorylate MLC thus decreasing contractile force. The source of NO is thought to be endothelial cells where stimulation of muscarinic receptors by acetylcholine activates endothelial nitric oxide synthase (eNOS) to produce NO 29, 30. Vasorelaxation can also be achieved by activation of Gs coupled receptors that increase cAMP to inhibit MLCK. This pathway is activated by prostaglandin binding to IP, epinephrine binding to β2 adrenergic receptors, and adenosine binding to A2. The primary

relaxing substance differs by artery size, with NO as the primary mediator of large

artery relaxation and EHDF modalities more common in smaller vessels 31, 32.

Phenotypic plasticity in VSMCs

Mature VSMCs in the vascular wall typically exhibit a contractile phenotype and

rarely divide; however, they retain a considerable amount of phenotypic plasticity.

When stressed these cells can take on a more synthetic phenotype with increased

proliferation, migration, and secreted protein production. Markers of VSMC

contractile phenotype include many contractile apparatus proteins the most

prominent of which are, SMA33, 34, SMMHC35, calponin36, 37, and SM22α36, 38.

Correspondingly, a shift away from contractile phenotype is marked by reduced

expression of those same myofilaments and others 39. Presence of a synthetic

phenotype in VSMCs is a feature in many pathologies, including pulmonary

hypertension, systemic hypertension, atherosclerosis, graft stenosis, aneurysm,

and dissection 39, 40.

Through the study of these pathologies and VSMCs in culture, multiple factors

have been identified as initiators or mediators of phenotypic changes. Indeed,

VSMC phenotype as identified by expression of contractile marker genes can be

influenced by mechanical forces, contractile agonists, extracellular matrix components such as laminin, elastin, and collagens, neuronal factors, reactive oxygen species, oxygen homeostasis, endothelial-VSMC interactions, and growth factors including, PDGF-BB, thrombin, TGF-B1, and FGF-2 39. PDGF-BB is a

particularly good example of a driver of synthetic phenotype as it down regulates

expression of nearly every VSMC marker gene and promotes increased

proliferation and migration of VSMCs 41-45.

In searching for a master regulator of VSMC phenotype, Wang et. al. identified myocardin, which is exclusively expressed in VSMCs and cardiomyocytes 46. At the molecular level, it is a transcriptional co-activator that cooperates with the ubiquitously expressed transcription factor serum response factor (SRF) to promote VSMC development and contractile phenotype. CArG elements which allow for SRF binding and thus regulation have been identified in the promoter regions of many genes that mark smooth muscle identity including: SMA,

SMMHC, SM22α, calponin, smoothelin, caldesmon, and telokin among others 47.

Indeed, inhibition of myocardin through use of a dominant negative myocardin or by siRNA mediated suppression decreased the expression of VSMC marker genes

48, 49. However, not all marker genes are activated by myocardin, suggesting that other regulatory pathways also contribute to phenotypic determination 50.

Many well-known transcription factors have been implicated as contributors to

VSMC phenotype modulation. Krüppel-like factor 4 (KLF4) and octamer-binding

transcription factor 4 (OCT4), key regulators of embryonic stem cell differentiation,

are among that group. OCT4 expression is increased in synthetic VSMCs within

atherosclerotic plaques, and cultures of VSMCs with lentiviral overexpression of

OCT4 feature increased migration though proliferation is reduced 51. KLF4 levels

are also increased in VSMCs associated with atherosclerotic plaques and vascular

injury; however the effects of its induction are less clear. One study demonstrated

reduced proliferation attributable to KLF4 expression 52 while several more identify

KLF4 as a driver of synthetic phenotypes with the ability to repress myocardin

activity and contractile gene expression 53, 54. Yes-associated protein 1 (YAP1) of the Hippo-YAP/TAZ pathway is also increased by PDGF-BB stimulation and promotes increased proliferation 55. This is particularly interesting as YAP is known to be responsive to mechanical forces in ECs, specifically having increased expression in areas of disturbed blood flow 56. The Notch family, well known for

mediators of direct cell to cell signaling and cell fate decision making, has been

implicated in promoting both the contractile and synthetic phenotypes. Direct

action by intracellular Notch in cooperation with myocardin/SRF promotes SMA

expression while increased expression of Notch target genes, HEY1 and HEY2, support a synthetic phenotype either by inhibiting myocardin/SRF binding or through an independent pathway 57-59. Furthermore the receptor for mitogen

PDGF-BB, namely PDGFR-β, is also an independent Notch target 60. Finally,

hypoxia driven activation of hypoxia-inducible factors (HIFs) play a role in VSMC

phenotypic modulation in the pulmonary circulation and aorta, and as they are the

topic of this dissertation project are discussed at length below.

Hypoxia-Inducible Factors

Tissues require oxygen (O2) for survival. Hypoxia occurs when the O2 supply is

below the tissue demand. Hypoxia-inducible factors are activated by low oxygen

and are master regulators of cellular responses to hypoxia. HIF-1 was initially

discovered as a driver of hypoxia induced erythropoietin expression and has since

been shown to be involved in hypoxia driven gene expression changes in multiple

cell types 61-63. In the subsequent decade, additional family members HIF-2 and

HIF-3 were also identified 64, 65.

Oxygen dependent regulation of HIFs

HIFs are heterodimers formed by the complexation of a HIF-α subunit with aryl

hydrocarbon nuclear translocator (ARNT; HIF-1β). Both the HIF-α subunits and

ARNT are basic helix-loop-helix proteins that contain Per-ARNT-Sim (PAS)

domains. ARNT and HIF-1α are ubiquitously expressed while HIF-2α and -3α have

more tissue restricted expression patterns 66. HIF-α subunits are the limiting factors of HIF activity and are rarely detectable in normoxia due to post translational regulation illustrated in Figure 1.5 and reviewed by Giaccia et al 67. Under normoxic

conditions HIF-1α is hydroxylated at prolyl residues Pro 402 and Pro 564 by

oxygen dependent prolyl hydroxylase domain containing enzymes (PHDs), most

prominently PHD2. Hydroxylated HIF-αs are ubiquitinated through a von Hippel-

Lindau protein (VHL) dependent pathway and targeted to the proteasome for

degradation. HIF-1’s transcriptional activity can also be inhibited through

hydroxylation of an asparagine residue on the C-terminal transactivation domain

of HIF-1α by an oxygen dependent asparagine hydroxylase enzyme also known

as factor inhibiting HIF (FIH) that prevents complex formation with transcriptional

binding partners in the nucleus 68. Under hypoxic conditions, PHD enzymes are

Figure 1.5. Canonical HIF regulation. In normoxia, HIF-α is hydroxylated and degraded. In hypoxia, PHD is inhibited and HIF-α accumulates in the cell. It binds ARNT to form HIF and complexes with CBP/P300 to alter the transcription of a variety of genes regulated by HREs including those involved with vascular responses.

inhibited and the HIF-1α subunit is stabilized in the non-hydroxylated form allowing

it to accumulate in the cell and dimerize with ARNT. In the nucleus, HIF-1

complexes with CBP/ P300 and binds to hypoxia response elements (HRE) found

in the regulatory elements of genes. HIF-1 individually has been implicated in the

transcriptional regulation of hundreds of genes.

Alternative HIF stabilization pathways

In addition to hypoxic HIF-1α accumulation, there is evidence that HIF-1α plays a

role in mediating the intracellular effects of growth factors and mechanical

stimulation even in normoxic conditions. Cytokines implicated in the regulation of

cell growth including tumor necrosis factor alpha (TNF-α), PDGF, AngII, thrombin,

endothelin-1 (ET-1), and fibroblast growth factor 2 (FGF2) have been shown to

increase HIF-1α protein levels in VSMCs independent of the oxygen status. Major

signaling pathways play a role in stabilization, as evidenced by thrombin, PDGF-

BB, AngII and others signaling through mitogen activated protein kinase (MAPK)

and increased reactive oxygen species (ROS) to trigger HIF-1α accumulation. ET-

1 may rely on changes in calcium in addition to the previously listed pathways 69.

Many of the growth factors implicated are also known to be regulated by HRE containing promoters. Thus the activation of the HIF-1 pathway can establish a positive feedback system in which growth factors activate HIF-1 inducing growth factor production and release that in turn further increases HIF-1α stabilization.

Experiments conducted by Black et al. support this pattern of regulation in the

FGF2/HIF axis 70. Cyclic stretch has also been shown to stabilize HIF-1α through

MAPK signaling 71. The diversity of mechanisms involving HIF-1 effects supports

its importance in regulating cellular function and response to environmental

changes.

HIF function in the vasculature

HIF-1 is essential for vascular development. As embryos grow they become too

large to receive oxygen by diffusion and the developing circulatory system

ultimately alleviates this relative hypoxia. Correspondingly, global Hif-1α or Arnt

knockouts die in utero by 11 days gestation and display malformations of the heart

and vasculature and decreased erythropoiesis 72-74. Genes regulated by HREs include many that have been implicated in vascular growth and remodeling including those that contribute to endothelial cell proliferation and migration, circulating progenitor cell homing, and differentiation, and vascular smooth muscle proliferation and migration 75.

HIF in pulmonary VSMCs

The importance of HIF in the pulmonary smooth muscle has been demonstrated

in a mouse model of chronic hypoxia where mice are placed in conditions of 10%

O2 in inhaled air to induce pulmonary artery hypertension (PAH). Smooth muscle

specific Hif-1α knockouts experienced less pulmonary vascular remodeling and

hypertension than their wild type (WT) littermates when treated with low levels of

76, 77 inhaled O2 . Furthermore, pharmacologic HIF-1 pathway inhibition has

reduced pathologic PAH remodeling in the same model 78. Examining isolated

VSMCs, Pak and colleagues reviewed the available studies of pulmonary artery

smooth muscle cells (PASMCs) in hypoxia in 2007 and found disagreement

regarding whether hypoxia increases (12 publications) or decreases (12

publications) proliferation of PASMCs 79. Debate is also ongoing regarding whether hypoxia is a direct stimulus or augments other stimuli 79. Variations between studies may be explained in part by variations in level of hypoxia, source of particular isolates selected, and serum levels in media 79. The role of HIF-1, downstream of hypoxia in the proliferative phenotype, is equally murky. For example, Schultz et. al. reported in 2006 that increases in HIF-1α promoted a proliferative response after stimulation by hypoxia and growth factors, by demonstrating knockdown of HIF-1α decreased proliferation of PASMCs 80. In

contrast, the same author reported in 2009 that HIF negatively impacted

proliferation, when increased HIF-1α levels following PHD inhibition decreased

proliferation that was rescued by HIF-1α depletion 81. While there may be debate about the effects of HIF-1, there is consistency that HIF-1 levels are increased in

the lung by hypoxia. In ex-vivo testing this increase is seen in as little as thirty

minutes of exposure but returns to baseline after 4 hours of exposure and is even

less than baseline by 16 hours of exposure 82. This apparent periodicity of HIF-1

exposure may contribute to the inconsistencies in observed responses in vitro. The

downstream effects of HIF-1 observed in PASMCs are diverse and consistent with the many HIF-1 target genes. HIF-1 is also required for thrombin induced proliferation 83. In addition to hypoxic regulation of growth factors, HIF-1 has effects on ion channels and cellular pH that contribute to the vascular pathology 78, 84. HIF-

1 activity in the pulmonary system may have effects in the systemic vasculature

through inducing expression of angiotensin converting enzymes that increase

levels of AngII 85.

HIF in VSMCs of the systemic vasculature

Differences in the response to hypoxia on the level of gene regulation emerge

when comparing systemic and pulmonary VSMCs 86. Consideration of the systemic vascular network includes both arteries and veins. The normoxic proliferation and apoptosis characteristics differ between arterial and venous

VSMCs87. Saphenous vein VSMCs have reduced migration toward PDGF compared to mammary artery VSMCs due to differences in receptor expression88.

There is additional evidence that aortic and umbilical vein VSMCs express different levels of vascular endothelial growth factor receptor 2 (VEGFR-2) 89. The role of

these receptors in proliferation and migration and their status as HIF-1 targets make it important to examine arteries and veins independently. As with the pulmonary system, the proliferative response of systemic arterial and venous

VSMCs to hypoxia is under debate. HIF involvement is confirmed by HIF-1α knock down resulting in decreased proliferation, decreased migration, and increased apoptosis 90, 91. Downstream HIF-1 targets including Thrombospondin 1 (THBS-

1), hexokinase 2 (HXK2), plasminogen activator inhibitor-1 (PAI-1), matrix

metalloproteinases (MMPs), and adrenergic receptors have all been proposed as

important in systemic VSMC physiology and response to hypoxic stimuli. Hypoxic

coronary artery SMCs feature increased migration and increased expression of

THBS-1 and its receptors, integrin-β1,-β3, CD36, which were mediated by HIF-1 activity and inhibited by receptor inhibition 90. In the functional regulation of THBS-

1, HIF-1 is also essential for the protein’s extracellular release 90. In the aorta, HIF-

1 has been implicated in the expression of PAI-1, collagen I, and multiple matrix

metalloproteinases, including MMP-9,-2,-7 and -1, that contribute to the pathologic vascular remodeling in aneurysm and hypertension92, 93. Aortic vasoreactivity is also regulated by hypoxia through control of expression of α1badrenergic receptors, again differentiating it from the vena cava that does not experience such regulation94. HIF-1 is also implicated in mitochondrial regulation through HXK2.

Induction of HXK2 by HIF-1 triggers mitochondrial hyperpolarization resulting in apoptotic responses 91. In addition to direct effects of HIF-1 regulated genes, there is also evidence that HIF-1 action promotes autocrine signaling. In coronary artery smooth muscle cells, hypoxic accumulation of HIF-1α promotes expression of vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor receptor-1 (VEGFR-1) 95. Intrinsic increases in both receptor and ligand promote proliferation through autocrine stimulation of VEGFR-1 by VEGFA, inhibited with a VEGF neutralizing antibody95. The role of HIF-1 in systemic vascular remodeling in the vascular wall as a whole was examined in a model of

HIF-1 inhibition by local infusion of a dominant negative HIF-1α (DN- HIF-1α) adenovirus. Decreased carotid remodeling and neointimal formation in response to ligation was seen in mice related with the DN-HIF-1α compared to WT 91. This

implicates HIF-1 in the proliferative remodeling response, but does not help to

clarify which cells of the vascular wall are affected by the inhibition, and thus

responsible for the phenotypes.

In smooth muscle specific studies, the interplay between AngII and HIF-1α has

been a focus of several studies published on VSMC Hif-1α knockout in the

systemic circulation. Previously, AngII has been shown to increase cell proliferation and migration96. AngII stabilizes HIF-1α through induction of PI3k,

MAPK, and ROS signaling pathways 69. However the regulation is not unidirectional, as VSMC specific Hif-1α knockouts express higher levels of ATR1

indicating HIF dependent regulation of the angiotensin pathway 97. Phenotypically,

AngII induced aortic remodeling was decreased in Hif-1α knockouts, which in WT

animals occurs independently of vasoconstriction 93. HIF-1α has also been shown

to regulate blood pressure. In one study a VSMC specific Hif-1α knockout

increased systolic, diastolic, and mean arterial pressure ultimately resulting in

systemic hypertension independent of cardiac function, but this finding was not

replicated in a subsequent study 93, 97. The role of VSMC HIF-1 mediated vessel

remodeling and contractility in the periphery is unknown.

Peripheral Vascular Disease

Peripheral vascular disease is an umbrella classification that describes both

arterial and venous pathologies. Peripheral arterial disease (PAD), only slightly

smaller in scope, includes an array of non-coronary arterial syndromes caused by

the altered structure and function of the arteries that supply the brain, visceral

organs, and the limbs. I will focus here on lower-extremity PAD, where occlusive

lesions limit perfusion to distal tissues in the limb.

Epidemiology and disease burden

PAD is a major cause of morbidity affecting 8-12 million patients in the United

States and more than 200 million worldwide 98, 99. Prevalence increases with age from 5.28% in the 45-49 year old population to 29% in those over 70 98, 99. The

distribution between men and women is nearly equal, though the presentation

differs between genders 100. Risk factors for PAD are similar to those of other

cardiovascular diseases, and include smoking, diabetes, hypertension, and hypercholesterolemia 98. In fact, smoking has a higher correlation with PAD than with coronary disease 101, 102. Consequences of PAD can include pain, mobility

limitation, tissue damage, and in severe cases can necessitate amputation of the

limb. PAD also correlates with increased mortality, such that the likelihood of death

over a 10 year period is increased 6 fold in a patient newly diagnosed with PAD

compared to a patient without PAD 103.

Clinical presentation and diagnosis

Symptomatic PAD classically presents with lower extremity pain exacerbated by

exercise and relieved by rest; however, symptoms vary substantially. Indeed,

intermittent claudication, defined as aching pain, cramping, weakness, numbness,

or heaviness of the leg, only affects 10-35% of patients. 40-50% of patients present

with atypical leg pain involving the calf, thigh, or buttock, and an additional 20-50%

are asymptomatic 104, 105. Overall, men are more likely to present with claudication

(50%) than women (25%) 100. The most severe cases, categorized as critical limb

ischemia (CLI), affect 1-2% of patients 104, 106. Findings in CLI include pain, aching or burning at rest and many also present with tissue loss characterized by non- healing ulcers, necrosis, or gangrene that can threaten the limb. Overall, any indication of exertional limitation in lower extremity muscles, history of walking impairment particularly with claudication, presence of a poorly healing or non- healing wound on the legs or feet, or pain at rest localized to the lower leg or foot particularly if it varies with upright or recumbent position should raise suspicion for

PAD 106. Physical exam findings for any PAD patient may also include diminished

pulses, skin changes such as cool temperature to touch and pallor or rubor, loss

of hair, brittle nails, muscle atrophy, and/or bruits auscultated in the distal aorta,

iliacs or femoral arteries 107, 108.

Severity of PAD can be classified by the Fontaine staging system which stratifies

patients based on symptom severity 109. A stage 1 patient is asymptomatic. Stage

2 includes those with intermittent claudication subdivided into IIa where

claudication only presents after more than 200m of walking and IIb where

claudication arises in less than 200m of walking. In stage III, patients have pain at

rest and/or overnight. Finally, stage IV, the most severe, is assigned when necrosis

or gangrene is involved. The level of arterial obstruction can also be assessed and

stratified non-invasively using the ankle-brachial index (ABI) which is a ratio of the

highest systolic pressure of the dorsalis pedis and posterior tibial arteries and the

systolic brachial artery pressure 110. The American Heart Association (AHA) and

American College of Cardiology Foundation (ACC) defines an ABI of ≤0.9 as

abnormal with 0.71-0.90 as mild obstruction, 0.41-0.70 as moderate obstruction,

and 0.00-0.40 as severe obstruction. Above 0.9, 0.9-0.99 is borderline, 1.0-1.4 is

normal, and >1.40 as indicative of noncompressible, stiff vessels 104, 111. The AHA

and ACC recommends that all patients ≥65 years of age be screened for PAD with

an ABI assessment 105. At diagnosis, angiography, through conventional contrast

radiography (the gold standard), ultrasound, computed tomography, or magnetic

resonance, can provide visualization of location and degree of vessel occlusion

and pressure gradients across lesions 112.

Pathogenesis and vascular compensation

The most common etiology of PAD is atherosclerosis, characterized by subintimal accumulation of lipid and cholesterol that form lesion which encroach on the vessel lumen limiting blood flow to distal tissue. These lesions are most commonly found in the femoral and popliteal arteries, but can also occur in the abdominal aorta, iliac arteries, and distal vessels including tibial and fibular arteries 113. The vasculature responds to flow limitations and ischemic sequelae through arteriogenic and angiogenic processes promoting restored perfusion.

Arteriogenesis

Arteriogenesis, defined by dilation and arterialization of a smaller pre-existing vessel, allows for increased blood flow. Adaptive arteriogenesis, often called collateral remodeling, describes the lumen diameter enlargement of smaller

collateral vessels to compensate for blockage of a larger vessel while de novo

arteriogenesis refers to the arterialization of a newly formed capillary 114. Collateral

vessels connect artery to artery or arteriole to arteriole in a redundant circuit. If flow through the primary conduit vessel is restricted, collaterals provides a pathway for blood to be detoured around the occlusion and renter the vascular tree at a distal point (Figure 1.6). In the context of peripheral ischemia, small diameter collateral vessels at baseline, engorge shortly after blockage 114. Over the subsequent days

to weeks a remodeling process occurs thickening the vessel wall and further

improving their carrying capacity. The drivers of this process remain the subject of

debate. Shear stress from rapidly increased blood flow has been implicated in

collateral remodeling 115-117. However, shear stress is not the only mediator, as evidenced by collateral growth in ischemia without the presence of a pressure gradient 118, 119. A proposed pathway relies on NO such that activation of eNOS by

shear stress on the endothelium or another means, triggers vessel dilation. The

increased vessel diameter corresponds to increased circumferential wall stress

promoting VSMC proliferation 115. NO appears to be critical to the process as demonstrated by eNOS global KO mice having reduced arteriogenesis 120. Cells outside the vessel wall have also been identified as contributors. Macrophages

particularly appear to play an important role through secretion of vasoactive

cytokines including FGFs and VEGFA 121, 122. HIF has also been identified as a

regulator of these outcomes with HIF stabilization through PHD2 deletion in

macrophages increasing arteriogenesis in mouse models of PAD 123.

Angiogenesis

Angiogenesis in the adult is an adaptive process through which new vessels are formed at the capillary level. These new vessels can replaced damaged vessels, provide alternative pathways if the capillary network is blocked, or expand the network to supply growing tissue. In the setting of ischemia, hypoxic tissue initiates an angiogenic cascade by secreting and array of cytokines, most notably VEGFA.

Indeed, hypoxia increases VEGF expression up to 30 fold within a matter of

minutes 124. This milieu of VEGF and other angiogenic cytokines stimulate

endothelial cells in the existing vasculature to initiate vessel sprouting 124, 125. In each sprout, a single endothelial cell acquires tip cell identity and neighboring cells follow behind forming the stalk 125. The newly formed vessel tube then elongates along the cytokine gradient toward the ischemic tissue 126. Further support by a

mural layer of pericytes or VSMCs marks vessel maturation and enhances vessel

stability. Vessels with associated pericytes persist even after reoxygenation of the

tissue, while those without regress 127. HIFs are important drivers of angiogenesis in hypoxia, and are well known to increase VEGF expression 128-130. Indeed, loss of ARNT disrupts angiogenesis 73. VEGFA is necessary for effective angiogenesis

but not sufficient to complete the process alone 131 as VEGFA treatment in isolation

also detrimentally increases vascular permeability. Angiopoietin signaling through

Tie2 receptor helps to balance these effects as such co-expression of the ANG1 and VEGF result in increased vessel numbers without excessive permeability 131,

132. When effective, vessel growth restores blood flow, alleviating hypoxia and thus

inhibiting further angiogenic stimulation.

Figure 1.6. Vascular responses to flow limitation. Obstruction of a main artery generates hypoxia in the distal tissue. The vasculature responds through (A) arteriogenesis and (B) angiogenesis to alleviate the ischemia and hypoxia. Arteriogenesis is characterized by lumen diameter enlargement and remodeling of existing vessels and collaterals to detour blood around the obstructed segment. Angiogenesis involves the growth of new vessels at the capillary level to expand local delivery and reperfuse ischemic tissue.

Therapeutic interventions

Treatment of PAD can include modifying underlying systemic disease, addressing risk factors for progression, and direct mechanical intervention to restore blood flow. Many factors that contribute to PAD progression are also risk factors for other cardiovascular diseases thus systemic intervention can not only impact PAD outcomes but also improve or prevent disease in other vascular beds. Such systemic interventions include smoking cessation, dietary adjustment, exercise therapy, lipid lowering, hypertension management, blood sugar control in diabetic patients, and antiplatelet therapy 104, 105. With focus on the affected area, proper foot care can prevent or treat wounds that are slow to heal or non-healing in the context of PAD. Symptomatic relief of claudication can be achieved with pharmacologic therapy to promote vasodilation and inhibit platelet aggregation and exercise therapy, which in some cases can have improvements comparable to stent revascularization 133, 134.

In severe presentations such as critical limb ischemia (CLI), direct and rapid revascularization can limit tissue damage and prevent limb loss. Endovascular interventions can include plain balloon angioplasty, drug-coated balloons, bare- metal or drug-eluting stents, covered stents, and plaque removal 104-106, 135.

These interventions have better outcomes for aortoiliac disease than those of the smaller vessels and for lesions that are stenotic rather than occlusive 104, 136.

Open surgical interventions can also be beneficial allowing for direct plaque removal by endarterectomy. Bypass grafting using autologous veins, typically

saphenous, or prosthetic polymer grafts provide a new conduit for blood flow

circumventing the lesion. Duration of graft patency varies by both graft type and

location of bypass placement 105, 137.

HIF in VSMCs of the peripheral vasculature

While headway is being made in understanding the regulation of pulmonary and

aortic VSMC responses to hypoxic stress, the findings may not extend to the

peripheral vasculature. In rabbits, aortic and carotid artery VSMCs have greater

capacity for PDGF-BB induced proliferation and migration than femoral artery

VSMCs 138. This may be explained in part by increased PDGF induction of MAPK in aortic VSMCs 138. Thus, studies of the periphery specifically are necessary to shed light on the functions of VSMCs in PAD.

Model of PVD: HLI

Flow limiting lesions in PAD most frequently localize to the iliac and femoral

arteries. Mouse models of PAD, referred to as hind limb ischemia (HLI), are

generated by ligature occlusion of vessels at locations corresponding to the sites

of human disease 113, 139, 140. Occlusions are made in the iliac, femoral, or superficial femoral arteries generating ischemia in the distal tissue. Selection of occlusion site determines severity of ischemia and also affects the ease of study of angiogenic and arteriogenic processes 139, 141. Ligation of the superficial femoral artery occurs distal to the branching of the deep femoral artery (Figure 1.7), leaving intact the collaterals fed by the deep femoral artery and allowing for assessment

of arteriogenic remodeling of those vessels in addition to angiogenic

vascularization of the gastrocnemius. Occlusion of the femoral artery or iliac artery

above the branching of the collaterals, generates a more profound ischemia and

limits the contributions of the deep femoral artery collaterals to reperfusion.

HIFs in HLI model

A considerable body of literature has implicated the involvement of HIF-1 in the

response to peripheral ischemia. Levels of stabilized HIF-1α affect the response

to femoral artery ligation. Global Hif+/- mice show decreased reperfusion following

ligation 142. Non-specific HIF-1 activity is known to promote angiogenesis and arteriogenesis. Indeed, rabbits injected with a mutant, constitutively stabilized,

HIF-1α in the ischemic thigh after artery ligation showed increased angiogenesis and arteriogenesis 143, 144. Systemic treatment with dimethyloxalylglycine (DMOG)

to inhibit hydroxylation and thus stabilize HIF-1α resulted in increased

angiogenesis in the hind limbs of mice following femoral artery ligation 145.

Arteriogenesis was not examined in this model. With regard to the cells of the vascular wall, a reliance on HIFs has been shown in endothelial knockouts of HIF-

2α that display decreased reperfusion compared to wild type controls following induction of hind limb ischemia 146. Likewise, a recent paper demonstrated that

HIF-1α deletion in VSMCs reduced perfusion in a severe model of PAD, but did not examine mechanism for this deficit 147. HIFs are clearly important factors in promoting reperfusion responses to peripheral ischemia and a promising therapeutic target worthy of further study.

Figure 1.7. Hind limb ischemia: a murine model of PAD. Permanent ligatures occlude the superficial femoral artery proximal to the popliteal bifurcation and distal to the branching of the deep femoral artery to model large peripheral vessel blockage.

HIF as a therapeutic

In the last decade, gene therapy employing HIF-1α has been trialed in patients with CLI or intermittent claudication 148. Injections of stabilized HIF-1α in replication-deficient adenoviral vectors into ischemic limbs proved safe in phase 1; however, later stage testing in patients with intermittent claudication found no benefit over placebo in claudication onset time, ABI, or quality-of-life measurements 149, 150. This lack of response may in part be explained by lack of increased HIF-1 activity on a functional level as no change was seen in expression of HIF target, VEGF, or in EC progenitor recruitment 149.

Summary and Hypothesis

Although a variety of factors present during an ischemic episode may influence the accumulation of HIF-1α, the best recognized and singularly important is hypoxia.

HIF target genes have been implicated in regulation of vascular responses to peripheral ischemia through observations of global or endothelial deficiencies of

HIF-1α. VSMCs are an integral part of the mature vascular wall, yet their role in regulating these responses is not well understood. Hypoxia influences VSMC phenotype and contributes to remodeling of the pulmonary and central systemic vasculature, but its impact may be highly dependent on the specifics of the vessel type, the microenvironment, and pathology. This dissertation centers on the hypothesis that HIFs are essential regulators of smooth muscle cell activation and phenotype in the systemic vasculature. The following studies will evaluate the consequences of loss of canonical HIF activity in VSMCs on vascular remodeling,

48 including angiogenic and arteriogenic responses to peripheral ischemia, and on vasomotor function. The results obtained will offer important insights on HIF’s mechanistic regulation of VSMCs’ contributions to vascular responses challenging the paradigm that endothelial cells are the sole regulators of vascular function.

Chapter 2: Aryl Hydrocarbon Receptor Nuclear Translocator in Vascular

Smooth Muscle Cells is Required for Optimal Peripheral Perfusion

Recovery

Author List:

Anna Henry Borton, Bryan L. Benson, Lee E. Neilson, Ashley Saunders, M. Amer

Alaiti, Alex Y. Huang, Mukesh K. Jain, Aaron Proweller, Diana L. Ramirez-

Bergeron

Affiliations:

From the Department of Pathology (A.H.B., B.L.B., A.Y.H.), Case Cardiovascular

Research Institute (A.H.B., A.S.,M.A.A., M.K.J., A.P., D.L.R.-B.), Division of

Pediatric Hematology-Oncology, Department of Pediatrics (A.Y.H.), and Case

Comprehensive Cancer Center (A.Y.H.) at Case Western Reserve University

School of Medicine Cleveland OH 44106, USA, and Harrington Heart & Vascular

Institute (A.H.B., A.S., M.A.A., M.K.J., A.P., D.L.R.-B.), Neurological Institute

(L.E.N.), Angie Fowler Adolescent and Young Adult Cancer Institute and University

Hospitals Rainbow Babies and Children's Hospital (A.Y.H.) at University Hospitals,

Cleveland, OH 44106, USA.

Portions of this chapter are published in Journal of the American Heart Association

2018; 7. DOI:10.1161/jaha.118.009205

Summary

Background- Limb ischemia resulting from peripheral vascular disease (PVD) is a common cause of morbidity. Vessel occlusion limits blood flow creating a hypoxic environment that damages distal tissue requiring therapeutic revascularization.

Hypoxia-inducible factors (HIFs) are key transcriptional regulators of hypoxic vascular responses including angiogenesis and arteriogenesis. Despite vascular smooth muscle cells’ (VSMCs) importance in vessel integrity, little is known about their functional responses to hypoxia in PVD. This study investigated the role of

VSMC HIF in mediating peripheral ischemic responses.

Methods and Results- We utilized ArntSMKO mice with smooth muscle specific deletion of Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT, HIF-1β), required for HIF transcriptional activity, in a femoral artery ligation model of PVD.

ArntSMKO mice exhibit impaired perfusion recovery despite normal collateral vessel dilation and angiogenic capillary responses. Decreased blood flow manifests in extensive tissue damage and hypoxia in ligated limbs of ArntSMKO mice.

Furthermore, loss of ARNT changes the proliferation, migration, and transcriptional profile of cultured VSMCs. ArntSMKO mice display disrupted VSMC morphology and wrapping around arterioles, and increased vascular permeability linked to decreased local blood flow.

Conclusions- Our data demonstrate that traditional vascular remodeling responses are insufficient to provide robust peripheral tissue reperfusion in

ArntSMKO mice. In all, this study highlights HIF responses to hypoxia in arteriole

VSMCs critical for the phenotypic and functional stability of vessels that aid in the recovery of blood flow in ischemic peripheral tissues.

Introduction

Limb ischemia is a sequela of peripheral vascular disease, a major cause of

morbidity affecting millions of people worldwide 98. Arterial occlusion limits blood

flow creating a hypoxic environment and damaging tissue, which in severe cases

can necessitate amputation. Vascular responses to episodes of ischemia attempt

to restore perfusion through arteriogenic processes of redirecting blood flow

through extant collateral circulation and angiogenic processes initiating de novo

growth of vessels to regenerate the downstream vascular network 151-153. While the role of endothelial cells (ECs) in initiating and effectuating these responses has been extensively examined, far less is known about the contributions and regulators of neighboring vascular smooth muscle cells (VSMCs).

In response to various physiological stresses, VSMC undergo phenotypic switching, permitting them to proliferate and migrate, contributing to the remodeling of the vascular wall in pulmonary hypertension (PAH), systemic hypertension, and atherosclerosis among others 154, 155. For many of these conditions hypoxia is an

important pathologic trigger. Hypoxia-inducible factors (HIFs) are heterodimeric

transcription factors, composed of HIF-α and aryl hydrocarbon receptor nuclear

translocator (ARNT, HIF-β) subunits, essential for cellular responses to hypoxia 67,

72, 73, 156-159 . In the presence of oxygen (O2), HIF-1α and -2α are hydroxylated and

targeted for proteasomal degradation. Hypoxic conditions inhibit the hydroxylation

of α- subunits which dimerize with ubiquitously expressed ARNT to form active

HIF-1 or HIF-2 functioning as master regulators of oxygen homeostasis by binding to and activating gene promoters containing hypoxia response elements 159. While

HIF-2’s expression and activity are more restricted, HIF-1 has been implicated in

the transcriptional regulation of hundreds of genes including many involved in

vascular growth and remodeling 75, 160, 161.

Hypoxia and HIFs have been studied extensively in VSMCs of the pulmonary

vasculature. In the context of hypoxia driven PAH, HIF-1 is essential for VSMC

proliferation in vivo 76-78. However, the pulmonary vasculature differs from the

systemic vasculature in hemodynamic forces and O2 status suggesting that these

results may not be directly translatable to other arterial beds. Studies of the role of

HIF in VSMCs outside of the pulmonary circulation are limited and have primarily

focused on examining the interplay between HIF-1 and ANGII signaling in vascular

remodeling of the aorta 93, 97.

A considerable body of literature demonstrates the involvement of HIF in response

to peripheral ischemia. Human patients with critical limb ischemia exhibit changes

in HIF-1α protein levels and vascular density 162. Increased levels of stabilized HIF-

1α are also seen in mouse models of hind limb ischemia (HLI) induced by femoral

artery ligation 142. While global Hif-1α+/- mice show decreased blood reperfusion following ligation, injection of stabilized HIF-1α in ischemic muscle promotes angiogenesis and arteriogenesis thereby increasing reperfusion 142-145. Specific to vascular cells, EC responses are known to be dependent on HIFs, with EC-specific knockouts of HIF-2α displaying decreased blood flow following induction of hind limb ischemia due to aberrant arteriogenic and angiogenic responses 146. Most

recently, VSMC HIF-1α knockout mice were characterized with decreased limb

reperfusion following femoral artery ligation due to the enhanced production of

thrombospondin-2 (THBS-2), known to inhibit angiogenesis 147. However, while

HIFs’ regenerative vascular properties have been well described in the non- targeted and EC contexts, the specific mechanisms of revascularization dependent on HIF driven VSMC responses to peripheral ischemia are not well described.

The following studies tested the hypothesis that endogenous HIF in VSMCs contributes to peripheral perfusion recovery. Canonical HIF transcriptional activity was ablated in VSMCs in vivo through the tissue specific deletion of ARNT, the required HIF-β subunit. Our findings reveal impaired reperfusion following femoral artery ligation despite signs of arteriogenesis and angiogenesis. Evidence of disrupted VSMC organization at the arteriolar level implicate VSMCs as central to this deficit. Moreover, this study finds hypoxia’s capacity to regulate the molecular markers of VSMC structure, homeostasis, proliferation, and migration is ARNT- dependent. Our findings underscore the essential role of HIF-dependent hypoxia driven responses by VSMCs to achieve optimal peripheral perfusion recovery.

Methods

The data, analytic methods, and study materials will be made available upon

reasonable request to other researchers for purposes of reproducing the results or

replicating the procedure.

Mouse generation

Previously described SM22α-Cre+/- and Arntlox/lox mice were crossed and

maintained in a C57Bl/6j background 163-167. The progeny of SM22-Cre+/- Arnt+/lox

x Arntlox/lox were utilized for experiments. Arnt deletion was verified by protein and mRNA levels in aortic VSMCs isolated and cultured as described below. Sex matched and litter matched, or if unavailable, aged matched Cre- Arntlox/lox or Cre-

Arntwt/lox mice were selected as controls. All animal studies were performed with

the approval of the Case Western Reserve University Institutional Animal Care and

Use Committee.

Echocardiography

For transthoracic echocardiography, mice were anaesthetized by inhalation of 1%

isoflurane with O2. All data were recorded and analyzed by the VEVO 770 High

Resolution Imaging System (Fujifilm Visual Sonics Inc.) and the RMV-707B 30

MHz probe. In M-mode short-axis images, ejection fraction, and fractional

shortening were measured at the papillary muscle level. Measurements were

acquired at heart rates above 500 bpm.

HLI model

HLI procedure, tissue harvest for frozen preparation, and pigment perfusion were

performed as previously described with minor modifications139. Briefly, 8-12 week old mice, assigned a number for blinding purposes, were anesthetized by intraperitoneal (IP) injection of ketamine (80mg/kg; IP) and xylazine (7mg/kg; IP).

Surgical depth of anesthesia was verified by toe pinch. Following hair removal, pre-operative perfusion assessment, and preparation of the surgical field, an incision was made in the left inner thigh. Blunt dissection was used to visualize and separate the femoral artery from the neighboring vein and nerve. Two ligatures of 7-0 braided silk suture were placed on the superficial femoral artery distal to the branching of the deep femoral artery and proximal to the popliteal bifurcation, and the intervening artery was cut. Alternatively, a more severe model of HLI was achieved by placing both ligatures proximal to the branching of the deep femoral artery. The epigastric artery was dissected and cauterized (Bovie). Wound clips

(7.5mm, Michel) and 6-0 polypropylene suture were used to close the incision.

Post-operative analgesia with buprenorphine (0.06 mg/kg; IP; every 12 hours) was administered for 3 days. Male mice were utilized for these studies due to previously described improved HLI recovery in male vs female mice168, allowing more

dynamic range and increasing power to detect recovery deficits.

To assess perfusion, mice were anesthetized as described above and placed on

37°C heating pad for 5 minutes prior to scanning. Infrared laser Doppler scans of

the foot pad were performed at 4ms/pix in triplicate on a MOORLDI2-IR (Moore

Instruments Ltd). Mean blood flow on flux images was assessed by moorLDI v5.0

software package. Perfusions heat maps for visual display are presented with

palate limits of 0 and 2000.

Where indicated, 594-conjugated lycopersicon esculentum lectin (100μl, Dylight,

DL-1177) was administered via the jugular vein and allowed to circulate for 5

minutes at 7 days following HLI. For vascular permeability assessments, 2mg of

2,000 kDa FITC-Dextran (Sigma, FD2000S) was administered via tail vein, 2 hours

before euthanasia. In anesthetized mice, the chest was opened and the left

ventricle was cannulated and infused with vasodilation solution (10U/ml heparin

[Sigma], 100μM adenosine [Sigma], 100μM papaverine hydrochloride [Sigma],

0.05% wt/vol bovine serum albumin [BSA, fraction V, Fisher] in Ca+ Mg+ DPBS

[Invitrogen]) at 10ml/min followed by warm 10% neutral buffered formalin, or

pigmented fixation solution (8g gouache [Winsor & Newton, 470] in 50ml 4%

paraformaldehyde). Some mice were injected IP with the hypoxic marker

pimonidazole [1-[(2-hydroxy-3-piperidinyl)propyl]-2-nitroimidazole] hydrochloride

(60mg/kg, HPI) 30 minutes before euthanasia to visualize hypoxic regions.

Collateral assessment

Limbs perfused with pigmented fixation solution were removed at the hip. After removing the skin, limbs were dehydrated to 100% methanol and the tissues cleared with 1:1 benzyl alcohol: benzyl benzoate. Vessels were imaged with Leica

MZ 16 FA at 10x, without a filter, and 65x, with a GFP filter for improved contrast.

Average diameter was quantified in Image J as the dividend of the vessel profile

58 area and the vessel length from tiled 65x images segmented in Photoshop

(Adobe).

Histology and immunohistochemistry

Gastrocnemius (GC) muscles for histological assessment were harvested, cryoprotected in 15% sucrose then 30% sucrose overnight, embedded in OCT compound (Tissue Tek), frozen and stored at -80°C. 8-μm serial transverse sections of GC muscle were obtained with a cryostat (Leica CM 1850 UV) and post-fixed in 2% paraformaldehyde. For damage assessment, sections were stained with hematoxylin & eosin and imaged with Leica DM 2000LED (40x objective). For capillary quantification, sections were blocked with 2% BSA and 5% normal goat serum in PBS and subsequently incubated with anti-CD31 (1:50, BD-

Pharmingen, 550274) followed by goat anti-rat IgG-488 (1:200, Invitrogen, A-

11006) and, where indicated, Cy3-conjugated SMA (1:400, Sigma, C6198). To detect areas of hypoxia, 0.1% tritonX-100 was added to the blocking buffer above followed by FITC-conjugated mouse anti-pimonidazole (1:200, Hypoxyprobe).

Tissues were counterstained with DAPI in mounting medium (Vector). Images were captured on a Leica DMI 6000 B with a 10x objective. For myocyte enumeration, GC sections were labeled with 5μg/ml wheat germ agglutinin,

Oregon green-488 (Invitrogen, W7024) in PBS. Damage and hypoxic areas were quantified as a percentage of GC cross sectional area in image J. CD31+ capillaries, Lectin+ vessels, SMA+ VSMCs, and skeletal myocytes were

enumerated in blinded raw images collected at the same exposure. For each limb,

6, 301um2 regions of 2 GC sections were quantified.

Arteriole imaging in spinotrapezius whole mount

Collection and visualization of vessels in the spinotrapezius has been previously

169 described . Briefly, following euthanasia with CO2 and left ventricular infusion of

warm PBS, spinotrapezius muscles were collected and fixed in 4%

paraformaldehyde in PBS for 20 minutes at room temperature. Tissues were

blocked with 2% BSA and 0.3% Triton-X 100 in PBS ON at 4°C. VSMCs were

identified by FITC-conjugated anti-SMA (1:400, Sigma, F3777). Confocal images

were taken on a Leica SP5 DMI 6000B using argon 488 nm and helium-neon 633

nm laser lines with a Leica 506192 HCX PL APO lambda blue 63x/1.4 oil objective

with 0.17 mm glass correction. For detection, 12-bit photomultiplier tubes were

used with Leica LAS AF acquisition software.

Vascular imaging in thick sections of GCs

Following post HLI tissue collection as described above, 400μm coronal sections

of GC were cut by vibratome (Leica VT 1200). Sections were blocked with 5%

normal goat serum, 2% BSA, and 0.3% Triton-X 100 in PBS. In permeability

studies, vessels were labeled with anti-CD31 (1:50, BD-Pharmingen, 550274)

followed by goat anti-rat IgG-647 (1:200, Invitrogen, A21247). VSMCs were

labeled with FITC-conjugated anti-SMA (1:400, Sigma, F3777). A Leica SP5

confocal microscope equipped with a 20x water immersion lens (Leica HCX-APO-

L, N.A. 1.0) and a tunable 16W Ti/Sapphire IR laser tuned to 800 nm (Chameleon

Coherent, Inc.) was used for two-photon laser scanning microscopy imaging using

non-descanned detectors set to capture Alexa Fluor 647 CD31 and/or FITC

(Dextran or SMA) fluorescence. For perfusion imaging, XYZ images with an XY

dimension of 775 x 775 µm were obtained at 512x512 pixels in 5 µm z stacks. For

SMA imaging of GC, XYZ images with an XY dimension of 310 x 310 µm were

obtained at 1024x1024 pixels in 1.95 µm z stacks.

Confocal and multiphoton image processing

High resolution confocal and multi photon images were deconvolved by Huygens

Professional 16.10 using Classic Maximum Likelihood Estimation at an estimated

signal-to-noise ratio of 5. Point spread functions were estimated a priori in Huygens

Professional using the wavelengths of excitation and emission light, position of the cover slip and orientation of the lens, lens immersion and specimen mounting media, and pinhole radius. These parameters were held identical within sets of images. Following restoration, data were imported to Imaris (BitPlane, Inc.) to

generate figures. Low resolution confocal images were not deconvolved, and a

median filter of 3x3 voxels was applied in Imaris. Due to inhomogeneities in SMA

staining intensity between and within tissues, brightness and contrast were

adjusted to facilitate comparison of smooth muscle cell architecture.

Cell culture

Isolation and culture of primary aortic VSMCs were performed as previously

described 164. Briefly, explants of thoracic aorta were divided longitudinally and plated lumen side down in 1mm2 pieces on 2 tissue culture dishes and covered

with glass cover slips. Plates were incubated in DMEM/F12 supplemented with

GlutaMAX-1 (Gibco, 10565-018) and 20% FBS (Atlanta or Gemini bio-products).

After 2 weeks of growth at 37°C and 5% CO2, explants and cover slips were

removed, and media was reduced to 10% FBS. For transcription analysis, the

following day, cells were starved in DMEM/F12+0.5% FBS for 16 hours, then

exposed to 2% O2 (hypoxia) or 21% O2 (normoxia) for 24 hours in

DMEM/F12+10% FBS.

Proliferation assays

20,000 cells per well of VSMC cultures were plated in 12- well plates. Following 16

hours starvation in serum free DMEM/F12, cells were exposed to hypoxia or

normoxia in DMEM/F12 +5% FBS. At 24 hours exposure, BrdU (5-bromo-2'-

deoxyuridine) was added to 10μM and incubated for an additional 8 hours in normoxia or hypoxia. Cells were fixed in 4% paraformaldehyde, followed by antigen retrieval with 1M HCl, and blocking with 0.75% BSA and 0.1% Triton-X

100. BrdU positive cells were labeled with Anti-BrdU (1.25ug/ml, BD biosciences,

B44) and counter stained with DAPI (Vectashield, H-1200). Assays were performed in triplicate. 6 representative images from each well were quantified.

Migration assay

Cells were plated at confluence and starved for 16 hours in serum free DMEM/F12.

Following scratch with 200- µl standard pipette tip, cultures were placed in normoxia or hypoxia in DMEM/F12+0.5% FBS+ 25ng/ml PDGF-BB (PeproTech,

315-18). Migration distance was quantified from phase contrast images taken at 0 and 6.5 hours and analyzed using NIH Image J software.

Real-time RT-PCR

Total RNA was isolated from cultured cells using TRIzol reagent (Ambion). RNA was reversed transcribed to cDNA with QuantiTect reverse transcription kit

(Qiagen, 205311). Relative expression was quantified in technical triplicates by real-time quantitative RT-PCR using the FastStart Universal SYBR Green Master

(ROX) Mix (Roche, 04913850001) on a StepOnePlus system (Applied

Biosystems). Target gene expression was analyzed using the 2-ΔΔCt method

(threshold values) with normalization to 18S ribosomal rRNA 170. Results are

reported as gene expression relative to Arntlox/lox in normoxia. Primers utilized for

target genes are presented in supplemental table 1.

Western blotting

Cell extraction buffer (Invitrogen, FNN0011) supplemented with cOmplete

protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail (Roche,

04693159001 and 04906845001) was used to collect protein either from samples

of GC muscle through bead homogenization (Qiagen TissueLyserII) or from

VSMCs in culture. Protein was quantified by BCA. 40μg of cell lysate or 100μg of

GC lysate in SDS sample buffer (Boston Bioproducts) were run on 8%

polyacrylamide gels in tris-gylcine-SDS running buffer. Semi dry transfer to nitrocellulose membrane was conducted in transfer buffer with 20% methanol.

Western blots of total protein isolates were probed overnight at 4°C with rabbit anti-

ARNT (1:800, Cell signaling, 5537S) diluted in Pierce protein-free T20 (TBS) blocking buffer (Thermo Scientific, 37571). To detect the protein bands, blots were washed and probed with secondary HRP linked, anti-rabbit IgG (1:1000 or 1:2000,

Cell Signaling, 7074S) and detected by enhanced chemiluminescence according to the manufacturer's instructions (Pierce, 32106). Membranes were stripped

(Thermo Scientific, 21059) and reprobed with β-actin rabbit antibody (1:5000, Cell

Signaling, 4967S).

Statistical analysis

Results are reported as the mean ± SEM. Statistical analyses were performed as

identified in each figure legend. Normality and homoscedasticity were evaluated

with Shapiro-Wilks test and F-test or Brown-Forsythe. Post testing utilized Tukey’s

multiple comparisons test for one-way and two-way ANOVAs, Bonferroni’s multiple

comparisons test for repeated measures two-way ANOVA, and two-stage linear

step-up procedure of Benjamini, Krieger and Yekutieli for Kruskal-Wallis test

(GraphPad). Significance was defined as p <0.05.

Results

Generation of smooth muscle specific Arnt knockout mouse model

To examine the role of HIF signaling in vascular smooth muscle, we generated a

model of Arnt deletion to disrupt HIF canonical transcriptional activity, and avoid compensatory changes in other HIF family members which can confound single α- subunit deletion models 171, 172. The smooth muscle specificity of this deletion was

accomplished by crossing well characterized SM22α-Cre mice 163-165 with Arntlox/lox

166. Primary aortic VSMCs isolated from SM22α-Cre+/-/Arntlox/lox mice (ArntSMKO)

show loss of Arnt expression at the mRNA and protein levels (Figure 2.1A, B).

Maintenance of ARNT expression in skeletal muscle was assessed in bulk tissue

samples of GC. Similar ARNT mRNA and protein levels were present in GCs of

ArntSMKO and Arntlox/lox mice (Figure 2.1C, D). ArntSMKO mice display no overt phenotypes with similar weight, appearance, and longevity to littermate controls

(Arntlox/lox) (Figure 2.1E and data not shown). Since SM22α-Cre has demonstrated

activity in cardiac tissue 163, cardiac function was assessed by echocardiography

173. No differences were seen in heart rate, ejection fraction, fractional shortening,

or left ventricle mass of ArntSMKO compared to Arntlox/lox littermate controls (Figure

2.1F-I). These results indicate that mice with tissue specific deletion of Arnt in the smooth muscle appear healthy overall without compromise of cardiac function.

Figure 2.1. Characterization of ArntSMKO mice. Isolated aortic VSMCs from ArntSMKO mice show loss of ARNT expression in (A) mRNA; n=5, and (B) total protein; n=3. Tissue samples from gastrocnemius muscle show no differences in bulk ARNT expression in (C) mRNA; n=5, and (D) total protein; n=4. (E) Bodyweights of male mice are similar in ArntSMKO; n=8, and Arntlox/lox littermates; n=11. Smooth muscle specific ARNT deletion does not compromise cardiac function assessed by (F) heart rate, (G) ejection fraction, (H) fractional shortening, and (I) left ventricular mass on echocardiogram; n=5, Unpaired 2-tailed t-test (A- F,H,I) with Welch’s correction (H), or Mann-Whitney test (G): *p<0.05.

ArntSMKO mice show impaired blood flow recovery in HLI model

While atherosclerotic obstruction of peripheral arteries ordinarily contributes to

PVD in humans, we employed inducible hindlimb ischemia (HLI), the most

common mouse model of PVD. The superficial femoral artery was surgically

ligated distal to the deep femoral artery restricting downstream blood flow to the

limb. Peripheral perfusion recovery in the foot pad was examined by infrared laser

Doppler at 3, 7, 14, and 21 days after ligation (Figure 2.2A). Results, expressed as

ratio of flow in ligated/ unligated limb, show decreased perfusion recovery in the

ligated limbs of ArntSMKO mice compared to Arntlox/lox littermate controls beginning

at day 7 with continued separation through day 21 after femoral artery ligation

(Figure 2.2B). Recovery of blood flow in ArntSMKO mice is not only slower, but also plateaus at a lower level than controls. By day 7 a gap in perfusion of nearly 20% emerges, closing by only an additional 5% over the subsequent 2 weeks.

Functional motor recovery, evaluated using a 0-3 point scale from full function of foot flexion and toe grasp to dragging of the ligated limb, was delayed in ArntSMKO

mice, though did not achieve statistical significance (Figure 2.2C). Collectively,

mice lacking ARNT in vascular smooth muscle demonstrate a significant and

persistent disruption in perfusion recovery after femoral artery ligation.

Figure 2.2. Bulk perfusion and functional recovery is reduced in ArntSMKO mice following femoral artery ligation. Ligations distal to the deep femoral artery branch point were performed in the left limbs of age- and gender- matched adult ArntSMKO and Arntlox/lox mice. (A) Representative images of perfusion measured using infrared laser Doppler scanning for ArntSMKO and Arntlox/lox mice over 21-day recovery. (B) Perfusion, reported as a ratio of ligated to unligated limb, was significantly lower in ArntSMKO compared to Arntlox/lox mice from days 7 through 21; n≥10, repeated-measures ANOVA *p<0.05 ArntSMKO vs. Arntlox/lox (C) Functional scoring on days 3 and 7 show impairment trend in functional recovery in ArntSMKO mice; n=11.

Limb ischemia stimulates collateral remodeling in ArntSMKO and Arntlox/lox mice

Appearance of perfusion deficits in the first week after ligation and previously described roles for smooth muscle in arteriogenic responses prompted assessment of collateral vessel dilation and remodeling. Luminal diameters of collateral blood vessels branching proximal to the ligation and traveling through the adductor muscles were visualized by pigment perfusion and subsequent tissue clearance which revealed similar collateral patterning and diameter in unligated limbs of Arntlox/lox and ArntSMKO mice (Figure 2.3A-C). Seven days post ligation, the time point with the largest perfusion deficit in ArntSMKO, we observed significant collateral vessel dilation in the ligated limbs of either mouse genotype (Figure 2.3A-

C). Surprisingly, despite lower levels of distal limb perfusion, collateral vessels in

ArntSMKO have significantly larger lumen diameters than those in control animals

(Figure 2.3C). Resistance to blood flow through a vessel is inversely proportional to the its cross-sectional area, indicating that collaterals in ArntSMKO have on average 2.5- fold lower resistance to flow than the analogous vessels in Arntlox/lox mice (Figure 2.3D). The collateral cross-sectional area is inversely correlated with perfusion of the limb; mice with the lowest perfusion had the largest diameter collaterals (Supplemental Figure 2.1). These results suggest that the observed deficit perfusion in ArntSMKO is not due to inadequate collateral vessel dilation.

Figure 2.3. VSMC ARNT is not required for ischemia induced collateralization. Collateral vessel responses in adductor muscles visualized with pigment perfusion angiography at day 7. (A) Lumen diameter enlargement visible in ligated compared to respective unligated limbs of ArntSMKO and Arntlox/lox mice. 10x images in top rows illustrate similar patterning; arrowheads indicate collaterals of interest. Higher magnification inset outlined by dashed rectangle. Scale bar, 1mm. 65x images in bottom rows captured through GFP filter for improved contrast. Scale bar, 250μm. (B) Tracings of skeletonized images across entire length of collaterals. Scale bar, 1mm. (C) Average collateral diameters show dilation in ligated limbs of both ArntSMKO and Arntlox/lox mice. Collaterals in ArntSMKO ligated limbs are significantly larger than in Arntlox/lox; n=3. (D) Cross sectional area calculations reflect approximation of flow resistance differences and projected flow rate. Measurements indicate 2.5-fold larger collateral vessels in ArntSMKO relative to Arntlox/lox mice; n=3, one-way ANOVA: *p<0.05 ArntSMKO vs. Arntlox/lox, ^p<0.05 ligated vs. unligated.

Supplemental Figure 2.1. Collateral vessel lumen cross sectional area vs limb perfusion. (A) An inverse correlation is present between ratio of cross sectional area of collateral vessel lumens in ligated/unligated limbs and foot pad perfusion, reported as LDI ratio ligated/unligated; R2=0.848; n=6.

Early capillary angiogenesis does not depend on VSMC Arnt in HLI

With sufficient collateralization observed, we next evaluated the angiogenic

response to induced ischemia in the downstream vascular beds. A more severe

ischemia ligation model eliminating collateralization of the deep femoral artery

showed decreased reperfusion in ArntSMKO mice implicating dysregulation of

smaller vessels in impaired perfusion recovery (Supplemental Figure 2.2). To

assess for differences in angiogenesis, gastrocnemius (GC) sections were

collected seven days post ligation. Capillary number was evaluated by staining for

CD31+ ECs, and the presence or absence of blood flow in each vessel was

assessed by intravenous (IV) lectin injection prior to tissue collection (Figure 2.4A).

At baseline, the unligated limbs of ArntSMKO and Arntlox/lox show equivalent capillary

densities and fractional perfusion (Figure 2.4B-D). At day 7, similar capillary and

perfused vessel densities in ligated limbs of Arntlox/lox and ArntSMKO mice were also

observed (Figure 2.4B-D). In the ligated limbs, total number of capillaries in a

301μm2 area are not significantly different between Arntlox/lox and ArntSMKO (Figure

2.4B). While there is a decrease in the number of CD31+ capillaries/myocyte in the

ligated limb of Arntlox/lox and ArntSMKO relative to their counterpart unligated limb, it

is only statistically significant in ArntSMKO mice (Figure 2.4C). In contrast, fractional

perfusion of capillaries is significantly decreased in the ligated relative to unligated

limbs in both ArntSMKO and Arntlox/lox mice (Figure 2.4D). Sections from unligated

and ligated limbs were stained with SMA to identify smooth muscle cells

associated with any identified CD31+ capillaries. Unligated and day 7 sections

exhibited little co-localization of SMA with capillaries (Supplemental Figure 2.3).

As the bulk of angiogenic responses are known to occur between days 7 and 28

post femoral artery ligation, capillary density was also measured at day 28 to

thoroughly assess for effects of Arnt deficiency on angiogenesis. Quantified by

average capillaries/301μm2, both Arntlox/lox and ArntSMKO have increased numbers

of capillaries in the day 28 ligated GCs relative to unligated GCs (Figure 2.4F); however, when capillaries per myocyte were quantified, the smaller myocytes of

ArntSMKO ligated GCs reveal a deficiency in late angiogenic responses. While

Arntlox/lox shows recovery in the number of capillaries per myocyte by day 28,

ArntSMKO recovery is limited with significantly reduced capillary to myocyte ratios in

ligated limbs (Figure 2.4E, G). No difference was apparent at day 28 in frequency

of SMA+ CD31+ vessels between ligated ArntSMKO and Arntlox/lox limbs (Figure 2.4E,

H). To comprehensively evaluate for variability in regional vascularization as recently shown by Schaad et. al. 174, we assessed vascularity in 6 distinct regions

across the GC muscle. Numbers of capillaries and perfused vessels between

ArntSMKO and Arntlox/lox were comparable in all 6 regions at days 7 and 28

(Supplemental Figure 2.4). In summary, while angiogenic deficiencies in ArntSMKO

may contribute to late (day 14-28) perfusion plateau, similar angiogenic patterns

and utilization of capillary beds are seen in both ArntSMKO and Arntlox/lox mice at day

7 following femoral artery ligation.

Supplemental Figure 2.2. Proximal HLI model. The left femoral artery was ligated proximal to the deep femoral artery branch point in age- and gender- matched adult ArntSMKO and Arntlox/lox mice. (A) Reduced perfusion, reported as a ratio of ligated/unligated limbs, was observed at days 3 and 14 in ArntSMKO mice; n=7, repeated-measures ANOVA: *p<0.05 ArntSMKO vs. Arntlox/lox.

Figure 2.4. Histological assessment of capillary number and perfusion status in gastrocnemius muscle (GC). (A) Representative micrographs of perfused vessels assessed by staining sections for CD31 (green) from GCs processed after mice were infused with endothelium binding IV lectin at day 7 following FAL (red). While, (B) the number of CD31+ capillaries per field at day 7 is not significantly different, ligated limbs of ArntSMKO and Arntlox/lox mice show decreased (C) CD31+ capillaries/myocyte, and (D) fraction of perfused, double positive, vessels compared to unligated limbs at day 7; n=4. No significant differences were seen between ArntSMKO vs. Arntlox/lox in any of the above metrics at day 7. (E) Representative images of immunostained sections for CD31 labeled vessels (green) and α-smooth muscle actin (SMA, smooth muscle cells, red) from day 28 post ligation GCs. (F) Numbers of CD31+ capillaries per field in day 28 ligated limbs of ArntSMKO and Arntlox/lox are similar to if not increased over unligated limbs; n=3. (G) Ligated limbs of ArntSMKO mice show decreased CD31+ capillaries/myocyte compared to ligated limbs of Arntlox/lox mice at day 28; n=3. Scale bars, 50μm. (H)

Day 28 ligated limbs of ArntSMKO and Arntlox/lox have similar numbers of CD31+ SMA+ vessels; n=3, One-way ANOVA (B), Kruskal-Wallis test (C,D,F,G), or unpaired 2-tailed t-test (H): *p<0.05 ArntSMKO vs. Arntlox/lox, ^p<0.05 ligated vs. unligated.

Supplemental Figure 2.3. Capillary density and smooth muscle cell colocalization in gastrocnemius (GC) at day 7. Representative images of immunostained sections for CD31 labeled vessels (green) and α-smooth muscle actin (SMA, smooth muscle cells, red) from (A) unligated and (B) day 7 post ligation GCs. No apparent difference in number of SMA+ vessels between ArntSMKO and Arntlox/lox in (A) unligated or (B) ligated day 7 limbs; n=3.

Supplemental Figure 2.4. Regional assessment of capillary density and perfusion status in gastrocnemius muscle (GC). (A) A diagram illustrates relative locations of evaluated regions in GC. (B) At day 7, assessment of CD31+ vessels shows similar capillary densities all regions ArntSMKO and Arntlox/lox GCs from both ligated and unligated limbs. Reductions in capillary density are seen in regions 1, 2, and

5 of ligated limbs relative to unligated while capillary density increases in region 3 of ligated limbs. (C) Comparable density of lectin+ vessels are also present in ArntSMKO and Arntlox/lox across the GC. Reductions in number of perfused vessels are seen in regions 1, 2, and 5 of ligated limbs. (D) Likewise, reductions in fraction of capillaries perfused are observed in regions 1, 2, 3, 5, and 6 of ligated limbs; n=4. (E) At day 28, GCs from ligated limbs of ArntSMKO and Arntlox/lox have similar CD31+ capillary densities; n=3, two-way ANOVA: ^p<0.05 ligated vs. unligated.

ArntSMKO mice show increased hypoxia and damage in gastrocnemius in

response to ischemia

To assess the anatomical consequences of perfusion deficit, GC muscle cross-

sections were stained with H&E. While ArntSMKO and Arntlox/lox unligated limbs

displayed normal skeletal muscle histology, striking differences were observed

between ligated limbs at day 7 (Figure 2.5A, B). Disruption of the muscular

structure, defined by centrally located nuclei and decreased myocyte diameter and

abundant infiltrating cells were prominent features in ArntSMKO while Arntlox/lox

tissues were relatively unaffected (Figure 2.5A). At day 28 post femoral artery

ligation, centralized nuclei and evidence of regenerating myocytes were still visible

in ArntSMKO GC sections (Figure 2.5A). Across the GC, these pathologic features extend over more than 60% of the ligated limbs of ArntSMKO mice compared to less

than 30% of the area of Arntlox/lox GC muscle (Figure 2.5B, C). Overall, the histology

reflects a maladaptive injury response consistent with chronically reduced blood

flow recovery in ArntSMKO mice.

At the tissue level, hypoxia identifies areas of limited perfusion; thus, to quantify the extent of hypoxic GC muscle following femoral artery ligation, pimonidazole intraperitoneal injection was used to label hypoxic tissues. Pimonidazole adducts, which identify tissue with <10mmHg O2, were neither found in GC sections from

unligated limbs of ArntSMKO and Arntlox/lox (Figure 2.5D), nor were they present in

Arntlox/lox sections at day 7 175, 176. However, large hypoxic pimonidazole+ areas

were present in ligated limbs of ArntSMKO mice 7 days post ligation (Figure 2.5D-

E). Together, significantly increased tissue damage and hypoxic areas

demonstrate that loss of VSMC ARNT leads to substantial skeletal muscle injury

following induced HLI.

Next, vascular integrity was examined by evaluating permeability to IV infused high

molecular weight FITC-Dextran. In both GCs of Arntlox/lox and the unligated limb of

ArntSMKO, Dextran was limited to CD31+ vascular structures in thick coronal sections imaged by multi-photon microscopy; however, extravascular Dextran infiltrates were diffusely present in GCs of ArntSMKO ligated limbs (Figure 2.5F).

Collectively, increased tissue damage, hypoxic areas, and vascular permeability suggest that compromised vasculature in ArntSMKO underlie impaired perfusion and

skeletal muscle injury following induced HLI.

Figure 2.5. Ischemic skeletal muscle regeneration and vessel integrity are impaired in ArntSMKO mice (A) Representative images of H&E stained GC sections show comparable muscle phenotype in unligated limbs. Day 7 assessment illustrates widespread atrophic myocytes and infiltrating cells in ArntSMKO while present only in small, well defined areas of Arntlox/lox GCs. Signs of damage and delayed regeneration, including centralized nuclei, persist to day 28 in ArntSMKO mice; n=3. Scale bars, 50μm. (B,C) Whole GC cross-sections show increased tissue damage in ArntSMKO compared to Arntlox/lox in ligated limbs at day 7; n=5. Scale bar, 500μm. Histological analysis (D) and quantification (E) of ArntSMKO ligated limbs show an increase in hypoxic regions identified by pimonidazole (Hypoxyprobe, green) staining at day 7 that is absent from unligated limbs of ArntSMKO and either Arntlox/lox limb, DAPI (blue, nuclei counter stain); n≥5. Scale bars: 50μm. (F) Representative multi-photon images of fluorescein-conjugated dextran (yellow) administered intravenously show increased permeability in ligated limbs of ArntSMKO mice at day 7. CD31+ vessels (purple); n=2. Scale bars: 100μm. Unpaired 2-tailed t-test (C) or Mann-Whitney test (E): *p<0.05 ArntSMKO vs. Arntlox/lox.

Altered vascular smooth muscle morphology in ArntSMKO mice

Despite substantial collateralization and similar capillary responses in the first week after ligation, the presence of significant and persistent perfusion deficits and tissue disruption point to a smooth muscle effect outside of these classically endothelial driven perfusion restoration mechanisms. In light of recent reports of aberrant arteriolar VSMC wrapping in dysfunctional vasculature 177, we examined

the cellular architecture of arterioles using whole mount confocal fluorescent

microscopy imaging of skeletal muscle samples. Visualization of α-SMA+ arterioles

revealed striking differences in VSMC morphology and conduit coverage in native

spinotrapezius muscle tissue from ArntSMKO versus Arntlox/lox mice (Figure 2.6A).

While the Arntlox/lox VSMC appear well organized and tightly wrapped with smooth coverage along the length of the vessel, the ArntSMKO VSMCs appeared more rounded, clearly delineated and detached (Figure 2.6A and Supplemental Figure

2.5). Similarly sized vessels were also examined deep in the GCs by multi-photon microscopy. Morphologic disruption of SMA+ VSMCs affected arterioles in ligated

and unligated limbs of ArntSMKO mice (Figure 2.6B). The aberrant perimural morphology of arterioles in ArntSMKO mice implicate a dysregulated VSMC

phenotype at the cellular level linked to impaired perfusion recovery.

Figure 2.6. Smooth muscle morphology and perimural wrapping of small arterioles. (A) Representative confocal images of SMA+ (cyan) VSMCs around small arterioles in spinotrapezius muscle illustrate disruption of organization and VSMC morphology in ArntSMKO mice; n=3. Top row scale bars: 60μm. Bottom row scale bars: 10μm. (B) Representative peripheral arterioles in GCs of ArntSMKO mice also display aberrant morphology and organization of SMA+ VSMCs visualized by multi-photon microscopy; n=3. Scale bars: 60μm, inserts 3x magnification.

Supplemental Figure 2.5. Additional images of arterioles in skeletal muscle. (A, B) Representative confocal images of SMA+ VSMCs around small arterioles in spinotrapezius muscle illustrate disruption of organization and VSMC morphology in ArntSMKO. (A) Scale bars: 60μm. (B) Scale bars: 10μm.

Loss of ARNT alters VSMC phenotype

To assess ARNT’s role in VSMC phenotype and responses to hypoxic stress in

ischemic limbs, cultured aortic VSMCs from ArntSMKO and Arntlox/lox mice were challenged with 24-hour hypoxic exposure at 2% oxygen 178, 179. Evaluation of transcripts revealed divergent changes in drivers of hypoxia induced phenotype modulation well described in pulmonary artery and aortic VSMCs (Figure 2.7) 80,

90, 92, 93, 180-182. Expression of HIF targets, vascular endothelial growth factor A

(Vegfa) and glucose transporter 1 (Glut1), whose levels rise in hypoxia and are

attenuated by ARNT deletion in ECs, responded as expected 167, 183-185. Hypoxia induced expression of Vegfa and Glut1 in Arntlox/lox VSMCs, and loss of ARNT

prevented induction of expression in hypoxia (Figure 2.7A, B). Transcriptional

changes were also observed in critical proliferative and migratory genes. ArntSMKO

VSMCs displayed reduced expression of serpin family E member 1 (Serpine1,

Pai1), fibroblast growth factor 2 (Fgf2), platelet derived growth factor receptor beta

(Pdgfrβ), and TIMP metallopeptidase inhibitor 1 (Timp1) mRNA levels relative to

hypoxic Arntlox/lox VSMCs (Figure 2.7C-E, H). Furthermore, differences in gene expression were observed under normoxia in ArntSMKO VSMCs including increases in matrix metalloproteinase 3 (Mmp3) and decreases in Vegfa, Glut1, Fgf2, Pdgfrβ, and thrombospondin-2 (Thbs2) relative to Arntlox/lox VSMCs (Figure 2.7A, B, D, E,

H, J), While hypoxic treatment of control VSMCs lead to reduced expression of

thrombospondin-1 and -2 (Thbs1 and 2), ArntSMKO VSMCs failed to mitigate Thbs2 reduction (Figure 2.7I, J). To examine the phenotypic consequences of transcriptional dysregulation, migration and proliferation were evaluated. Migration

assessed by 2-dimensional scratch assay in the presence of platelet derived

growth factor B (PdgfB) showed ArntSMKO cells are less migratory in hypoxia than

Arntlox/lox cultures (Figure 2.8A). Proliferation, quantified by percentage of BrdU+

cells, increased when Arntlox/lox cells were exposed to hypoxia for 24hours (Figure

2.8B). ArntSMKO VSMCs show no increased proliferation in hypoxia over normoxia,

but notably have greater percentage of replicating cells than Arntlox/lox under both conditions. Collectively, these findings suggest that gene expression differences

in hypoxia treated ArntSMKO VSMCs underlie cellular derangements responsible for vascular reperfusion impairment.

Figure 2.7. Transcriptional expression of proliferation and migration regulators in response to hypoxia. (A-J) mRNA from ArntSMKO and Arntlox/lox mouse aortic VSMC cultures following 24 hours hypoxia (2% O2) exposure were analyzed by qPCR, normalized to 18s rRNA, and compared to normoxic (21% O2) control samples. Transcriptional profile of VSMCs from ArntSMKO differs from control Arntlox/lox samples: (A) Vegfa, (B) Glut1, (C) Pai1 (Serpine1), (D) Fgf2, (E) Pdgfrβ, (F) Pdgfb, (G) Timp1, (H) Mmp3, (I) Thbs1, (J) Thbs2; n=3, one-way ANOVA: *p<0.05 ArntSMKO vs. Arntlox/lox, ^p<0.05 hypoxia vs. normoxia.

Figure 2.8. Assessment of VSMC phenotype in vitro. (A) Migration, detected in a 2-dimensional scratch assay, was measured after 6.5 hours in normoxic (21% O2) SMKO or hypoxic (2% O2) conditions. Arnt VSMCs show decreased migration in hypoxia compared to Arntlox/lox cultures; n=9. (B) Quantitative analyses of immunopositive BrdU cells reveal increased proliferation of ArntSMKO VSMCs in normoxia and hypoxia; n=3, one-way ANOVA: *p<0.05 ArntSMKO vs. Arntlox/lox, ^p<0.05 hypoxia vs. normoxia.

Discussion

The present study illustrates the importance of HIF orchestrated VSMC responses

in peripheral perfusion recovery in a model of induced HLI. Specifically, knockout

of ARNT in smooth muscle impairs perfusion restoration following femoral artery

ligation. While insufficient blood flow manifests in increased hypoxic tissue and

skeletal muscle damage in the ischemic limbs of ArntSMKO mice, interestingly there

were no signs of limitation in collateral dilation or of disruption of perfused capillary

density. However, loss of ARNT led to morphologic disorganization of VSMC

coverage of small arterioles and increased vascular permeability in ligated limbs.

Furthermore, the transcriptional dysregulation of multiple genes involved in VSMC

function affect proliferation and migration of isolated ArntSMKO cells. In all, this study

identifies hypoxia mediated responses in VSMCs critical to maintaining VSMC

phenotype, cellular organization around arterioles, and vessel integrity, and to

achieving optimal reperfusion of ischemic peripheral tissues.

Our observation of impaired limb recovery with diminished reperfusion is striking

in a mouse genetic model targeting smooth muscle cells. ECs have been

described as the primary regulators of blood flow and orchestrators of reperfusion;

however, the degree of impairment observed in ArntSMKO is more profound than that seen in global Hif-1α+/- mice 142, and on par with endothelial specific deletions of Hif-2α 146. Furthermore, our findings are supported by a recent report of

decreased reperfusion with Hif-1α deletion in VSMCs using a severe form of HLI

147. Our genetic model conditionally deleting VSMC-ARNT permits efficient study of all HIF- canonical transcriptional function, while HIF-α subunit activities involving

non-canonical binding partners remain undisrupted 186. In context, our results

demonstrate the importance of smooth muscle cell canonical HIF- dependent responses in perfusion recovery necessitated by regional peripheral ischemia.

Perfusion deficits in HLI models are often explained by impaired angiogenic and/or arteriogenic responses 187. Classically, hemodynamic changes prompt

arteriogenesis assessed by number and dilation of collaterals, while hypoxia drives

angiogenesis visualized by increased capillary density; however, local stabilization

of HIF-1α through introduction of a constitutively active variant or by inhibition of

its degradation pathway increases both angiogenic and arteriogenic responses 123,

144, 182, 188. The impacts on reperfusion mechanisms are not explicitly described in

Hif-1α+/- or tissue specific HIF-1α models, but endothelial specific deletion of Hif-

2α impairs both benchmark vascular remodeling processes 146. Lower capillary

density in day 28 ArntSMKO GCs help to explain the persisting perfusion limitations after recovery has plateaued. Yet, at day 7 when the perfusion deficit is largest, measurements of arteriogenesis and angiogenesis in response to HLI are similar in ArntSMKO and Arntlox/lox mice. Indeed, ligated limbs of ArntSMKO mice have larger

diameter collateral vessels at day 7 than their littermate controls. The inverse

correlation between collateral cross-sectional area and bulk perfusion in ArntSMKO

and Arntlox/lox limbs suggests that collateral vessel diameter is responsive to

changes in blood flow and compensating for elevated ischemia in mutant mice. In

a model that spares HIF signaling in the endothelium, traditionally viewed as the

primary driver of vascular ischemic responses, it may therefore be unremarkable

that these processes appear intact 189. Despite substantial arteriogenic and

angiogenic responses, these compensatory changes are insufficient to restore

perfusion to the affected tissue in the ArntSMKO mice. Whether due to greater extent of ischemic insult, injury, and/or impaired recovery, substantial areas of hypoxia remain at least 7 days post ligation and signs of tissue damage persist across the

GC muscle after 28 days of recovery. These findings suggest that established responses to ischemia through well-defined revascularization mechanisms do not adequately explain the perfusion deficit at day 7. Indeed, while increases in microvessel number have also been documented in affected peripheral tissues from patients with chronic limb ischemia, these responses also do not appear to be sufficient to alleviate ischemia162.

Perfusion, as measured by laser Doppler, can be described as a function of RBC concentration and flowrate 190. In the absence of differences in cardiovascular function, global volumetric flowrates should be comparable in ArntSMKO and

Arntlox/lox mice. Thus, the perfusion deficit can be attributed to local changes in

vascular flowrate in the hindlimb. Recent reports of pathologic vessel patterning in

regenerated vascular networks following HLI could clarify the ArntSMKO reperfusion

deficit. Arpino et al. implicated disordered vascular smooth muscle cell phenotype

and wrapping around small arterioles in limiting RBC transit through the

microvasculature of regenerating skeletal muscle 177. Similarly, in skeletal muscle

of ArntSMKO mice, we detect aberrant smooth muscle cell morphology and

investment of the small arterioles consistent with these novel reports. Furthermore,

the integrity of the microvasculature is compromised as evidenced by increased

permeability. Vascular leak affects pressure gradients and contributes to disruption

of local blood flow. Thus, while the hallmark revascularization measurements namely, collateral diameter, and capillary number and fractional perfusion appear normal, disruption in VSMC patterning in vessels of ArntSMKO mice likely

contributes to reduced reperfusion and muscle recovery following HLI.

In the pulmonary circulation, HIF-1 is a well described mediator of hypoxia

triggered vascular remodeling and VSMC phenotype modulation. Mouse models

of chronic hypoxia induced PAH with reduced HIF-1α activity through either global

haplodeficiency or SMC specific deletion demonstrate decreased vascular

remodeling 76, 191, 192. Mature VSMCs retain a remarkable amount of plasticity and

can exhibit a phenotypic spectrum ranging from principally contractile and rarely

dividing to highly synthetic, proliferative, and migratory 39. Early debate in the field regarding VSMC responses to hypoxia emerged in studies utilizing pulmonary artery SMC (PASMC) cultures 79. However, recent reports of PASMCs consistently

show HIF mediated hypoxic responses increase proliferation, survival, and

migration, and stimulate growth factor production including VEGFA 80, 180-182. HIF

targets including PDGFs and FGFs are key promoters of VSMC proliferation and

migration; their inhibition impairs proliferation of PASMCs in vitro and prevents

vascular remodeling in models of PAH193, 194. Expression of multiple HIF targets in

ArntSMKO VSMCs diverge from levels seen in control cells. Failure to maintain

expression levels of Pdgfrβ, Pdgfb, and Fgf2 indicates dysregulation of pathways

central to proliferative and migratory responses to hypoxia. Indeed, ArntSMKO cells do not show proliferation increase with hypoxic exposure over normoxia and are less migratory than Arntlox/lox in hypoxia.

While these effects of HIF dependent phenotypic modulation are varied in differing

oxygenation and hemodynamic environments outside of the pulmonary circulation,

several recent reports have identified HIF-1 as central to maintaining the structure

and function of the arterial wall 93, 97, 195. Downstream HIF-1 targets have been

identified as important in systemic VSMC physiology and response to hypoxic

stimuli 90, 92, 93. HIF targets, PDGF and THBS-2 regulate VSMC attachment to extracellular matrix; impaired expression of Pdgfrβ and Thbs2 in ArntSMKO help explain disrupted VSMC morphology observed around arterioles 196, 197. As

supporting cells in the vascular wall, it is well recognized that VSMCs are involved

in physiologic responses to mechanical and biochemical changes in blood vessels.

Changes in VSMC phenotype have consequences for vasoreactivity. Tissue

specific deletion of HIF-1α has been shown to increase contractility, a putative

marker of mature VSMC phenotype, in studies of aortic and pulmonary artery

VSMCs 77, 97, 198. The morphologic changes in VSMCs around small arterioles in

ArntSMKO mice taken together with the abnormal proliferative and migratory

behavior and the altered gene expression observed in isolated cells are consistent

with phenotypic dysregulation of VSMCs.

In summary, the present study indicates that loss of hypoxic signals in VSMCs

limits the ability of mice to recover from inducible hindlimb ischemia, a classic

model for PVD. We provide evidence that despite conventional compensatory

arteriogenic and angiogenic responses in the first week after ligation, dysregulated

smooth muscle cell function in ArntSMKO arterioles manifested by morphologic

disruption, aberrant expression of key phenotypic regulators, and altered

proliferation and migration is sufficient to compromise vascular integrity and

ultimately impair limb reperfusion. Our results therefore underscore a critical role

for VSMC HIF in peripheral perfusion recovery and reiterate the importance of

understanding the regulation of VSMC function in arteriolar vessels in supporting optimal blood flow.

Acknowledgements

We thank Alla Gomer, Keiki Sugi, Stephanie Lapping, and Alice Jo for their technical assistance.

Funding Sources

Funding for this work was provided by the NIH F30 HL127985 (AHB), RO1

HL128281 (AP & DLR-B), RO1 HL096597 (DLR-B), T32 HL105338 (AHB, MAA),

T32 GM7250 (AHB, BLB), and TL1 RR024991 (AHB, BLB), F31 NS096857 (BLB),

T32 NS077888 (BLB), R25 HL103152 (AS).

Conflict of Interest Disclosures

None.

Chapter 3: Loss of Vascular Smooth Muscle Cell Aryl Hydrocarbon

Receptor Nuclear Translocator Impairs Vasoconstriction

Authors:

Anna Henry Borton, Iulia Barbur, Alice Jo, Nicholas P Ziats, Aaron Proweller,

Diana L. Ramirez-Bergeron

Affiliations:

From the Department of Pathology (A.H.B., N.P.Z.), Case Cardiovascular

Research Institute (A.H.B., I.B., A.J., A.P., D.L.R.-B.) at Case Western Reserve

University School of Medicine Cleveland, OH, USA, and Harrington Heart &

Vascular Institute (A.H.B., I.B., A.J., A.P., D.L.R.-B.), at University Hospitals,

Cleveland, OH, USA.

Summary

Vasoreactivity mediated by vascular smooth muscle cells (VSMCs) is critical for

cardiovascular homeostasis. Mature VSMCs retain phenotypic plasticity, and can

shift under stress from contractile to synthetic states. We previously identified a

requirement for smooth muscle Aryl hydrocarbon receptor nuclear translocator

(ARNT; HIF-1β), in hypoxia-induced arterial reperfusion prompting an interrogation

of the consequences of smooth muscle specific loss of ARNT (ArntSMKO) on

vasomotor function. Isolated primary ArntSMKO VSMCs display reduced myofilament expression consistent with a shift away from a contractile phenotype.

Moreover, though morphologically indistinguishable from control arteries, ArntSMKO

arteries exhibit impaired vasoconstriction with preserved vasorelaxation assessed by ex-vivo wire myography. Taken together, our data implicate ARNT as a key regulator of the contractile phenotype in VSMCs and arterial vasoconstriction.

Introduction

Vascular reactivity is essential for maintaining stable blood pressure and

appropriate distribution of blood flow. Vascular smooth muscle cells (VSMCs)

residing in the tunica media of the arterial wall are responsible for constricting and

dilating the luminal diameter in response to physiologic and pathologic stimuli.

Alterations in vasoconstriction and contractile dynamics are features of many

pathologies, including hypertension, pulmonary hypertension, sepsis, and

ischemia199-202.

Hypoxia can both precipitate and be a sequela of alterations in blood flow.

Hypoxia-inducible factors (HIFs) are master regulators of oxygen homeostasis.

This family of transcription factors are heterodimers formed when HIF-1α, -2α, or

-3α bind to a common β-subunit, aryl hydrocarbon nuclear translocator (HIF-1β,

ARNT) 67, 72, 73, 156-159. HIF-1α is widely expressed with HIF -2α and -3α more cell type restricted 75, 160, 161. In the presence of oxygen, the regulatory α-subunits are

hydroxylated and rapidly targeted for proteasomal degradation. In hypoxia, HIF

transcriptional activity is induced through the stabilization of the α-subunits and

their dimerization with constitutively expressed ARNT.

While investigating the role of HIFs in regulating the functional responses of

VSMCs in the systemic circulation, we reported that loss of ARNT led to decreased

reperfusion recovery with altered VSMC phenotype using a tissue specific

knockout mouse model 203. In vivo, VSMCs featured aberrant periarteriole

wrapping and morphology, while in vitro mutant cells displayed altered proliferation

and migration. Collectively these findings implicated ARNT as essential for

99 phenotype stability. VSMC phenotype is classically described as a spectrum between contractile and synthetic cell states39. Contractile cells are quiescent and rarely proliferate while synthetic cells proliferate and migrate with greater frequency. At baseline most cells are contractile, however stress or other pathologic triggers can prompt a phenotypic change toward a more synthetic state.

Our previous observations indicating a shift toward the synthetic phenotype in

VSMCs lacking ARNT prompted closer examination of contractile function. The following studies tested the hypothesis that loss of ARNT in VSMCs disrupts vasoreactivity. At the cellular level, ARNT deleted VSMCs display significant reductions in expression of contractile myofilaments. Vessels lacking VSMC ARNT feature impaired vasoconstriction with intact vasorelaxation and preserved dimensional structure of the vascular wall. Together, our findings describe an important role for VSMC canonical HIF activity in maintaining functional vasoreactivity.

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Methods

Mouse model

All animal studies were carried out under the approval of the Case Western

Reserve University Institutional Animal Care and Use Committee. A smooth

muscle specific mouse genetic model of Arnt deletion was generated as previously

described and characterized. Briefly, SM22-Cre+/- Arnt+/lox male mice were crossed with Arntlox/lox females yielding SM22-Cre+/- Arntlox/lox experimental mice and Cre-

Arntlox/lox or Cre- Arntwt/lox controls. Pairs of experimental and control mice used for experiments were sex matched and age, if not litter, matched.

Cell culture

Cells were isolated and cultured as previously described. Briefly, thoracic aortas were explanted, cleaned of adipose tissue, divided longitudinally into 2 tissue culture dishes, and plated in 1mm2 pieces, endothelial side down, under glass cover slips. Explants were incubated for 2 weeks in DMEM/F12 supplemented with

GlutaMAX-1 (Gibco, 10565-018) and 20% FBS (Atlanta or Gemini bio-products) at

37°C and 5% CO2. Thereafter, cover slips and explanted tissue pieces were

removed and FBS supplementation was reduced to 10%. Cells were starved for

16hours in DMEM/F12+0.5% FBS, then returned to DMEM/F12+10% FBS and

placed in hypoxia (2% O2, 5% CO2) or normoxia (atmospheric O2, 5% CO2) for 24

hours.

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Real-time RT-PCR

At collection, cells were lysed and total RNA collected with Triazol reagent.

Following RNA isolation and quantification, cDNA was reverse transcribed from

500ng of RNA using the QuantiTect reverse transcription kit (Qiagen, 205311).

Relative expression was quantified by real-time quantitative RT-PCR in technical

triplicates on a StepOnePlus system (Applied Biosystems) using the FastStart

Universal SYBR Green Master (ROX) Mix (Roche, 04913850001). Target gene

expression was analyzed using the 2-ΔΔCt method (threshold values) with normalization to 18S rRNA 170and reported as gene expression relative to

normoxic Arntlox/lox sample. Primers for each target gene are as follows:

18s Forward: 5'-GAATTCCCAGTAAGTGCGGG-3', Reverse: 5'-

GGGCAGGGACTTAATCAACG-3'; Cnn1 Forward: 5'-

GGCCAAGACAAAAGGAAAC-3', Reverse: 5'-CCATCTGCAGTCCAATGATG-3';

Myh11 Forward: 5'-GGCTTCATTTGTTCCTTCCA-3', Reverse: 5'-

CGAGCGTCCATTTCTTCTTC-3'; Acta2 Forward: 5'-

TGTGCTGGACTCTGGAGAT-3', Reverse: 5'-GAAGGAATAGCCACGCTCA-3'.

Histology

Carotid arteries were collected from adult mice and fixed in 4% paraformaldehyde overnight. Tissues specimens were dehydrated to 100% ethanol followed by embedding in paraffin wax. 8μm sections affixed to slides were deparaffinized, rehydrated, and stained with hematoxylin & eosin or Verhoeff–Van Gieson elastin stain. Images were acquired with Leica DM 2000LED microscope (20x objective).

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To quantify the average tunica media thickness, the areas enclosed by the external

elastic lamina (EEL) and internal elastic lamina (IEL) were manually outlined using

the free-hand selection tool (ImageJ 1.50, National Institutes of Health, USA). The

area was then used to calculate the radius of a circle of equivalent circumference.

In each image, the average radius of the IEL was subtracted from the average

radius of the EEL to obtain the average tunica media thickness.

Isometric tension measurement

Vasomotor function studies were performed as previously described 204. Briefly, thoracic aortas from 10-19 week mice were cleaned of adipose tissue and cut into

2mm rings. Two rings from the proximal thoracic aorta were suspended in an

isometric tension myograph (610M, Danish Myo Technology), in physiological

saline solution equilibrated with a gas mixture of 95% O2 and 5% CO2 at 37 °C.

All vessel segments were pretensed to 6mN and allowed to equilibrate. For depolarization-induced vasoconstriction studies, potassium chloride was added to a final concentration of 60mM. To assess agonist-based constriction, dose- response curves for phenylephrine (PE) and angiotensin II (AngII) over a range of

10-9 to 10-5 M were acquired in triplicate. Relaxation responses to acetylcholine

(ACh), and sodium nitroprusside (SNP) were assessed over a range of 10-9 to 10-

5 M in triplicate following a 10-2 M dose of PE. Data are presented as active tension in millinewtons for depolarization and agonist-induced contraction and as percentage of maximum PE-induced tension for ACh- and SNP-mediated relaxation.

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Statistical analysis

Results are reported as mean ± standard error of the mean. Normality and homogeneity of variance were evaluated with Shapiro-Wilks test and F-test or

Brown-Forsythe. Statistical tests were performed as described in figures legends with post testing by Tukey’s multiple comparisons test for one-way ANOVAs, or

Bonferroni’s multiple comparisons test for repeated measures two-way ANOVA,

(GraphPad). Significance was defined as p <0.05.

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Results

Transcriptional expression of contractile elements are reduced in VSMCs lacking ARNT

Phenotypic shifts in VSMCs are often marked by changes in expression of contractile genes 39. To determine whether ablation of canonical HIF activity impacted the contractile apparatus, tissue specific ARNT deletion in VSMCs was accomplished in a murine model, as previously described, by intercrossing a

SM22α driven CRE transgenic mouse line 163-165 with Arnt floxed mice 166

(Arntlox/lox) to generate SM22α-Cre Arntlox/lox (ArntSMKO). Primary aortic smooth

muscle cells were isolated from ArntSMKO and Arntlox/lox mice and mRNA expression was examined under normoxia (21% O2) and after 24- hour exposure to hypoxia

(2% O2). Results report expression normalized to 18srRNA and relative to

normoxic Arntlox/lox cell cultures. ArntSMKO VSMCs displayed reduced expression of alpha smooth muscle actin (Acta2), smooth muscle myosin heavy chain (Myh11), and Calponin (Cnn1) in normoxia with Myh11 and Cnn1 expression also significantly lower than Arntlox/lox cells in hypoxic conditions (Figure 3.1A-C).

Moreover, relative to normoxia, hypoxia exposure reduced expression of Acta2,

Myh11 and Cnn1 in controls but resulted in no further decline in transcript levels in

ArntSMKO cultures (Figure 3.1A-C). Collectively, absence of response to hypoxia and overall reduction in contractile genes indicated a shift away from contractile phenotype in ArntSMKO VSMCs.

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Figure 3.1. Contractile gene expression is reduced in ArntSMKO VSMCs. mRNA from ArntSMKO and Arntlox/lox primary aortic VSMC cultures exposed to normoxia (21% O2) or hypoxia (2% O2) were analyzed by qPCR, normalized to 18s rRNA. Results are reported relative to expression in normoxic Arntlox/lox cells. (A) Alpha smooth muscle actin (Acta2), (B) smooth muscle myosin heavy chain (Myh11), and (C) Calponin (Cnn1) mRNA expression was reduced in SMKO VSMCs relative to Arntlox/lox in normoxia and hypoxia. Relative to normoxia Arntlox/lox VSMCs decreased expression of Acta2, Myh11, or Cnn1 expression under hypoxia, while ArntSMKO cells have similar levels in normoxia and hypoxia; n=3. One-way ANOVA: *p<0.05 ArntSMKO vs. Arntlox/lox, ^p<0.05 hypoxia vs. normoxia.

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Large vessel structure and tunica media thickness is not affected in ArntSMKO

To assess for baseline changes in structure of the vessel wall, cross sections of

carotid artery were stained with Hematoxylin and Eosin (H&E) or Elastin as

displayed in Figures 3.2A and 3.2B, respectively. Vessels from Arntlox/lox and

ArntSMKO mice exhibit similar structure without disruption of the elastic lamina or

visible alterations in the intima or medial layers (Figure 3.2A, B). The average thickness of the tunica media composed of vascular smooth muscle cells was quantified and no difference was found between Arntlox/lox and ArntSMKO,

25.7±8.6μm and 22.9±6.7μm, respectively (Figure 3.2C). These data indicate

similar baseline vascular structure in large arteries of Arntlox/lox and ArntSMKO mice.

Loss of Arnt in VSMCs impairs aortic vasoconstriction

Consequences of ArntSMKO on constrictor function of aortic segments were determined ex vivo via wire myography. When constriction was induced through electromechanical coupling by depolarization with potassium chloride (KCl),

ArntSMKO aortas demonstrated significantly reduced force generation compared to

Arntlox/lox aortas (Figure 3.3A). Similarly, contractile responses to stimulation through pharmacomechanical coupling by receptor dependent agonists, phenylephrine (PE) and angiotensin II, were also significantly reduced in ArntSMKO

aortas (Figure 3.3B, C). Contractile force generation in response to PE was

reduced by more than 50% on average between 10-7-10-5M, with statistical

significance at 10-5M. Responses to AngII were significantly reduced at concentrations from 10-7-10-5M by an average of more than 60%. Collectively,

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Figure 3.2. Large vessel structure unchanged in ArntSMKO. Histologic assessment by (A) H&E and (B) elastin staining and (C) quantification of tunica media thickness show similar structure of the vascular wall in cross sectional views of Arntlox/lox and ArntSMKO carotid arteries. Scale bars 50μm; n=8. Unpaired t-test.

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reduced force generation to multiple contractile agonists indicates vasoconstriction deficits in ArntSMKO aortas.

Loss of Arnt in VSMCs does not affect aortic vasorelaxation

Impaired contraction can, in some cases, be explained by changes in structure or coordination that would also affect vasorelaxation. Endothelial dependent relaxation in response to acetylcholine (ACh) and smooth muscle intrinsic relaxation by sodium nitroprusside (SNP) were both evaluated in aortic segments ex vivo by wire myography and reported as percent of maximal constriction to PE

(Figure 3.4A, B). ArntSMKO and Arntlox/lox aortas showed similar relaxation responses to ACh or SNP. Maintenance of normal relaxation suggests functional endothelial responses and myo-endothelial coordination and implicates impaired smooth muscle rather than structural limitations as responsible for contractile deficits.

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Figure 3.3. ArntSMKO vessels have impaired vasoconstriction. Assessed by ex vivo wire myography, ArntSMKO aortic rings show decreased contractile force generation when stimulated by depolarization with (A) potassium chloride (KCl); n=7. Dose dependent vasoconstriction in response to receptor dependent agonists (B) phenylephrine (PE); n=4, and (C) angiotensin II (AngII); n=3, were also significantly reduce in ArntSMKO aortas. (A) Unpaired t-test or (B, C) Repeated measures ANOVA: *p<0.05 ArntSMKO vs. Arntlox/lox.

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Figure 3.4. Vasorelaxation is intact in ArntSMKO. Relaxation responses to (A) acetylcholine (ACh) and (B) sodium nitroprusside (SNP) are similar in ArntSMKO and Arntlox/lox aortic segments. Results from myography are reported as a percentage of maximal phenylephrine stimulated constriction; n=3. Repeated measures ANOVA.

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Discussion

Our current study highlights the importance of canonical HIF regulation of VSMCs

in maintaining functional vasoconstriction. Isolated primary VSMCs from ArntSMKO

mice have reduced expression of myofilament components of the contractile

machinery consistent with previously described phenotypic alteration 203.

Moreover, deletion of ARNT in VSMCs impairs aortic contractile responses to multiple agonists without affecting tunica media thickness or vasorelaxation.

Vasoregulation by contraction and relaxation is a primary function of VSMCs.

Mature VSMCs are phenotypically contractile and quiescent; however, when stimulated, VSMCs display a surprising amount of phenotypic plasticity. Increased proliferation and migration as well as decreased expression of components of the contractile apparatus are well-described markers of a phenotypic shift 39. Hypoxia

through HIF is a known driver of synthetic phenotypes in VSMCs80. Likewise, in control aortic VSMCs we see reductions in Acta2, Myh11, and Cnn1 with 24- hour

exposure to hypoxia. ArntSMKO VSMCs not only fail to demonstrate similar reductions to hypoxia, but also have significantly decreased expression of Acta2,

Myh11, and Cnn1 in normoxia relative to Arntlox/lox. These findings are consistent

with our previous report of increased proliferation and altered transcriptional profile

in ArntSMKO VSMCs. Collectively, these findings underscore the classification of

ArntSMKO VSMCs as less phenotypically contractile and more synthetic than

Arntlox/lox control VSMCs regardless of oxygen tension.

As in other muscle cells, contractile force is generated in VSMCs by the cyclic formation of actin and myosin cross bridges. Mutations in either ACTA2 or MYH11

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have been shown to impair vasoconstriction 205. Likewise, the reduction of

contractile force generation in ArntSMKO aortas in response to membrane

depolarization and to receptor dependent agonists occurs in the context of reduced

VSMC expression of contractile genes. Maintenance of the tunica media structure

and intact vasorelaxation further support that VSMC dysfunction at the contractile

level underlies impaired vasoconstriction rather than endothelial dysregulation or

structural limitations. In a similar pattern, systemic in vivo exposure to 48 hr of

hypoxia has been shown to decrease aortic VSMC contractility due to changes in

aortic smooth muscle function206. Consistent with these findings, we now report

decreases in contractile gene expression in control VSMCs exposed to hypoxia.

Vascular remodeling driven by hypoxia and HIF mediated responses in VSMCs

have been implicated in a number of pathologies. The muscularization of

pulmonary arteries in response to chronic hypoxia resulting in pulmonary artery

hypertension is well described. Here, HIF-1α global heterozygosity or tissue

specific homozygous deletion of HIF-1α in mature VSMCs inhibits vascular

remodeling 76. In the systemic circulation, VSMC HIF-1α is necessary to maintain mural structure and function in the aorta and mesenteric arteries and mediates aortic remodeling in response to AngII 93, 97, 195. While wall structure does not

appear to be affected in ArntSMKO carotid arteries, the small vessels of the peripheral circulation are modified. In our initial characterization we reported disrupted VSMC morphology and wrapping around 25 μm feeder vessels in skeletal muscle. Moreover, when ArntSMKO mice were subjected to ischemia induced by femoral artery ligation microvascular integrity was lost in the mutant

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mice allowing for extravasation of high molecular weight dextran into the

perivascular space. Our findings of limited vasoconstrictor activity may hinder the

redistribution of blood flow and further contribute to impairment of perfusion

recovery in this model.

Interestingly, while evidence supports roles for both ARNT and HIF-1α subunits as

regulators of VSMC contractility, functional outcomes with deletion of either subunit

differ. Huang et al. reported increased vasoconstriction in response to AngII in

mesenteric vessels from VSMC-HIF-1α KO mice while responses to other

contractile agonists were unchanged 97. When challenged with hypoxia inhalation,

Kim et al. also found evidence of increased vasoconstriction in VSMC-HIF-1α KO

mice, which featured increased right systolic ventricular pressure and increased

phosphorylation of the regulatory myosin light chain77, 198.

The ablation of HIF transcriptional activity by ARNT deletion model differs from

HIF-1α deletion in several key areas. First, loss of ARNT eliminates the

transcriptional function of both HIF-1 and HIF-2. The loss of HIF-2 activity may

contribute to the divergent contractile behavior 207. Second, ARNT deletion also disrupts any non-HIF related complexes in which ARNT participates208, 209. Finally,

HIF-1α is also known to have an array of alternative binding partners186. Deletion

models that ablate HIF activity though the α-subunit could also affect these non-

canonical pathways. These subunit specific observations help clarify the

functionality of HIF in the vasculature. As such, potential therapeutics that target

HIF functionality for vasomotor modulation should consider subunit specificity.

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Conclusions

Collectively, our data implicate ARNT and thus canonical HIF regulation in VSMCs

as necessary for optimal vasoconstrictor function. Phenotypic changes in ArntSMKO

VSMCs characterized by reduced expression of myofilaments impair vessel

contractility. Altered vasoreactivity can have consequences for blood flow

distribution and vessel homeostasis, supporting the consideration of ARNT (in

addition to HIF-1α) as a therapeutic target.

Sources of Funding

Funding for this work was provided by the NIH F30 HL127985 (AHB), RO1

HL128281 (AP & DLR-B), RO1 HL096597 (DLR-B), T32 HL105338 (AHB), T32

GM7250 (AHB), and TL1 RR024991 (AHB).

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Chapter 4: Discussion and Conclusions

In the preceding chapters, we have demonstrated that loss of ARNT disrupts

VSMC phenotype, changing the morphologic appearance of VSMCs around small arterioles, reducing the expression of contractile phenotype markers in isolated cells in normoxia and hypoxia, and altering proliferation and migration.

Functionally, loss of VSMC ARNT impairs vasoconstriction to multiple agonists without affecting vasorelaxation. While displaying no baseline differences in vessel number, diameter, or wall thickness, faced with an ischemic challenge in the form of femoral artery ligation, ArntSMKO mice have impaired perfusion recovery and

increased hypoxia, tissue damage, and microvascular permeability. The

consequences of ARNT deletion provide insight into the regulation of VSMCs, yet

the direct mechanism by which loss of ARNT disrupts phenotype and contractility

at the molecular level remains to be delineated.

Determinants of contraction

Fundamentally, changes in cellular contraction are attributable to alterations in the

contractile apparatus or its regulators. Reduced levels of myofilament components

could limit the number of actin-myosin cross bridges, and thus decrease force

generation. Impaired vasoconstriction in ArntSMKO vessels occurs in the context of reduced mRNA expression of both SMA and SMMHC myofilaments in VSMCs isolated from these vessels. Additional support for this explanation would be bolstered by measurements of protein content in intact vessels; however, myofilament content does not thoroughly characterize the functional state of the

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contractile apparatus. The organization of the actin and myosin network across the

cell is precise and dynamic 20. Upon stimulation with a contractile agonist, non-

filamentous actin polymerizes, through elongation or branch addition to existing

filaments or generation of new filaments, to increases the number of available locations for cross bridging 21, 210. Organization of actin alignment improves

contractility while depolymerization of actin filaments with cytochalasin D leads to

relaxation 22, 211, 212. Overall, actin polymerization or depolymerization can be quantified by changes in ratios of F (filamentous) to G (globular) actin and in the number and organization of stress fibers visualized microscopically. Assessing changes in myofilament quantity and organization could further characterize the cause of contractile deficits. At the regulatory level, RhoA and ROCK abundance and activity are also worthy of consideration. Not only does RhoA activity contribute to maintaining phosphorylated MLC, but also the pathway participates in the polymerization of actin 213, 214. Furthermore in developmental models,

RhoA/ROCK promote VSMC differentiation and expression of contractile genes downstream or HIF activity 214, 215. These alterations in contractile apparatus

structure and function could also contribute to the morphologic changes of VSMCs

associated with arterioles.

The contractile apparatus is tethered to the cell membrane and by extension to the

extracellular matrix at focal adhesions. Without this tethering, shortening of the contractile apparatus fails to generate force 216, 217. Focal adhesions are complex structures forming physical connections between transmembrane integrins and the

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cytoskeleton20, 218. Integrins, and thus focal adhesions, are modulated by hypoxia

and HIF. For example, hypoxia increases integrin β1 expression in mouse ES cells

219 and HIF and ARNT regulate the expression of αvβ3 integrin in focal adhesions

of trophoblast stem cells 220. This link is far less studied in VSMCs. In bovine aortic and coronary VSMCs, HIF stabilization with cobalt chloride or constitutively active

HIF-1α decreased attachment of cells in culture to a variety of extracellular matrix materials 221. No changes were seen in focal adhesion number or location, but

focal adhesion kinase phosphorylation was decreased 221. Interestingly, unlike

other isolates these VSMCs did not show increased proliferation and had

decreased migration despite increased HIF activity, raising questions about the

broad applicability of these findings. Focal adhesions serve as points of integration

for matrix to cell connections such that alterations in the matrix affect VSMC

function. Indeed, VSMCs cultured on matrix with higher Young’s modulus

generated more force without changes in phenotypic markers 222. Not only does the matrix composition impact VSMC function, but VSMCs can also impact the matrix through regulation of matrix metalloproteinases and synthesis of matrix components 223-225. All told, visual indicators of morphologic detachment seen in

ArntSMKO arteriolar VSMCs could point to changes in cell-matrix interaction either

contributing to or as a result of phenotypic changes.

Disrupted transcriptional regulation

Much of the literature describing HIFs in VSMCs centers on the function of HIF-1α

and reports the consequences of HIF-1α deletion as a model of functional HIF

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ablation. In our studies, VSMC-ARNT-KO phenocopied reports of VSMC-HIF-1α

deletion in impairing hindlimb reperfusion after femoral artery ligation 147. However,

the phenotypes of these deletion models diverge in studies of AngII induced

vasoconstriction where VSMC-Hif-1α- KO vessels are hypercontractile while

VSMC-Arnt-KO vessels are hypocontractile compared to controls 97. Deletion of

ARNT, the common binding partner of HIF-1α, -2α, and -3α, disrupts canonical

transcriptional activity for all members of the HIF family simultaneously while the

alpha subunits, and thus any non-canonical pathways, remain intact.

HIF-1 and HIF-2 are differentially expressed and, while sharing many target genes, also have their own unique transcriptional signatures and subset of targets. Thus, individual loss of either HIF-1 or HIF-2 may differ in consequence from their

simultaneous deletion. In VSMCs, HIF-1 is thought to be the primary driver of

hypoxic responses, as indicated by proliferation; however, loss of HIF-1α could

expose underlying contributions from HIF-2 207, 226. Indeed HIF-2 has been

specifically implicated in increased vasoconstriction in PAH, which is consistent

with reports of hypercontractility in VSMC-Hif-1α deletion 77, 207. The retained function of HIF-2 in models of Hif-1α deletion could thus help explain inconsistencies in phenotype between Arnt vs Hif-1α KO models.

Non-canonical subunit activity may also contribute to phenotypic divergence.

While canonical HIF-1 (HIF-1α/HIF-1β dimer) is a master regulator of cellular

responses, interactions of HIF-1α with other proteins have also been described 186.

119

Most of these interacting proteins serve to regulate the stability of HIF-1α, but

some, NOTCH, BCL2, NEMO, and STAT3 to name but a few, are independently

described cellular regulators 227-230. ARNT’s presence for or participation in these interactions is not routinely determined. If these interactions produce meaningful changes independent of ARNT, they could help to explain the divergent phenotypes in HIF-1α and ARNT knockout models. Along similar lines, the effects of ARNT dimerization with proteins other than the HIFα subunits should also be

considered 209. ARNT was initially described, and thus named, based on its dimerization with aryl hydrocarbon receptor (AHR) 231, 232. AHR is classically activated by environmental pollutants including dioxins, polycyclic aromatic hydrocarbons, and polychlorinated biphenyls; however, naturally occurring compounds including derivatives of tryptophan, biliverdin, bilirubin, prostaglandins, and some modified low-density lipoproteins have also been identified as AHR ligands in mouse hepatoma cell lines 233-236. AHR deletion in hematopoietic stem cells leads to hyper proliferation followed by bone marrow exhaustion 237-239, and

modulation of AHR activity in endothelial and immune cells has been described in

vascular pathologies including atherosclerosis and hypertension 233, 240, 241. While

reports in VSMCs are limited, there is some indication that AHR activity plays a

role in responses to high concentrations of a uremic toxin 242-244. The impact of

AHR activity under physiologic or ischemic conditions remains to be determined; however, it seems unlikely that loss of AHR/ARNT would outweigh the effects of lost HIF activity in an ARNT deletion model of ischemia pathologies.

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Altered intercellular communication

While originally thought to be physically isolated from each other, ECs and VSMCs are in fact physically connected by cellular structures that reach through the internal elastic lamina termed myoendothelial junctions (MEJ) 245, 246. MEJs are known to allow communication between the cells via gap junctions and NO, provided locally via eNOS 247, 248. While their contributions are yet undefined, the presence of endoplasmic reticulum and ribosomes within the MEJ provides an opportunity for local protein synthesis that may also contribute to signaling 246.

Along an arterial tree, MEJs are present with greater frequency in smaller vessels

249, 250. Correspondingly, the disruption of VSMCs in ArntSMKO skeletal muscle in

localizes around small feeder arterioles, suggesting that MEJs may be

compromised by the aberrant VSMC morphology or that disrupted MEJs may

contribute to VSMC dysfunction. Indeed, PAI-1 is necessary for MEJ formation,

and ArntSMKO VSMCs have decreased Pai-1 mRNA compared to controls 251.

Communication through MEJs occurs both from ECs to VSMCs and vice versa.

Altered signaling from VSMCs to ECs could also underlie the increased vascular permeability, typically a sign of EC changes, in ligated limbs of ArntSMKO mice.

Intercellular signaling that requires physical cell to cell contact also characterizes the Notch family of transcription factors. Anchored within the plasma membranes of neighboring cells, interactions between Notch ligands (Delta-like 1, 3, and 4,

Jagged 1 and 2) and receptors (Notch 1-4) trigger the cleavage of the transcriptionally active intracellular domain of the Notch receptor by gamma

121

secretase 252. Suppression of Notch activity in VSMCs by introduction of a dominant negative cofactor, alters developmental blood vessel patterning impairs vasoconstriction and vasorelaxation, and compromises angiogenesis 164, 204, 253,

254. Notch has also been implicated in VSMC phenotype switching 58. Several reports show interaction between the Notch and HIF pathways 255-259. These reports describe interactions between HIF-1α or HIF-2α and the Notch intracellular domain, which would remain intact in our ARNT deletion model. In contrast, disruption of Notch signaling by physical separation of VSMCs and/or loss of function in regulatory pathways that require both HIF and Notch could contribute to the ArntSMKO phenotype.

Contributions of other potentially affected cell types

VSMCs wrap vessels from the aorta to small arterioles; however, the smallest vessels, namely pre-capillary arterioles, capillaries, and post capillary venules, are not in contact with VSMCs. Instead, the mural cell populations around these

vessels are provided by pericytes. These cells have been most extensively studied

in the blood brain barrier where loss of pericytes correlates with increased

permeability 260, 261. In the coronary circulation, VSMCs differentiate from cells with

pericytes markers 262. While SM22α is typically thought to be SMC restricted,

SM22α positive pericytes have been identified in the developing retina 263. The

SM22α-Cre driver we employ is not expressed identically with native SM22α 163;

however, closer examination of Cre activity in the pericyte population is warranted.

Given their role in maintaining endothelial monolayer integrity in the blood brain

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barrier, pericyte dysregulation could contribute to the increased vascular

permeability seen in ligated limbs of ArntSMKO mice.

Cells of the myeloid lineage also warrant assessment for off target Cre activity.

Two SM22α-Cre lines from The Jackson Laboratory have evidence of Cre mediated deletion in neutrophils, monocytes, and macrophages 264. HIF activity in

macrophages can stimulate arteriogenic remodeling of collaterals in the hind limb

123. Thus, dysregulation of macrophage contributions could confound the

mechanistic interpretation of impaired perfusion phenotype. As stated above, the

expression of specific SM22α-Cre drivers can vary from native expression and

between different lines. Cre activity in blood cells was not examined in Lepore et.

al.’s initial characterization of the SM22α-Cre used in our studies. While worthy of

consideration, arteriogenesis is not the factor limiting perfusion recovery in

ArntSMKO mice subjected to HLI, thus reducing the likelihood that this could

compromise our findings.

Implications for other vascular pathologies

In the previous chapters, we examined the consequences of VSMC-ARNT-KO in

peripheral ischemia and aortic reactivity, but VSMC dysfunction contributes to

many other pathologies.

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Pulmonary Artery Hypertension

Pulmonary artery hypertension (PAH), for example, has various distinct etiologies

all of which manifest in vascular remodeling and sustained vasoconstriction 78.

Several of these pathogenic conditions are associated with hypoxic environmental changes including chronic obstructive pulmonary disease and high altitude exposure. Furthermore, mouse models of exposure to less than atmospheric levels of O2 in inhaled air reveal pulmonary changes that phenotypically approximate

PAH 265. With such a strong hypoxic link, it is no surprise that HIFs have been

implicated in the pathogenesis of PAH. Global heterozygosity of either HIF-1α or

HIF-2α protects against hypoxia induced PAH in mice 191, 192, and the levels of HIF-

1α correspond with the extent of remodeling and severity of disease in rats 266.

Interestingly, the role of HIF-1α in VSMCs remains controversial, with pulmonary artery pressure reported to either increase or decrease with VSMC-HIF-1α deletion

198, 267. Accordingly, these findings would suggest that HIF-1α has a regulatory role

in promoting or limiting vasoconstriction and/or remodeling. As for the role of

ARNT, only the increased proliferation of ArntSMKO VSMCs would be consistent

with an increased remodeling phenotype, while decreased vasoconstriction in

ArntSMKO mice, reduced hypoxic migration of isolated ArntSMKO VSMCs, and the

preponderance of data supporting HIF activity as a driver of increased vascular

remodeling suggest that ArntSMKO mice would be protected from hypoxia induced

PAH. ArntSMKO mice subjected to models of induced PAH would be an important extension of our analysis relevant to clarifying mechanism of action.

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Hypertension

Changes in vasoconstriction with hypoxia exposure are key pathologic findings in

PAH, and, as such, many of the pharmacologic interventions stimulate vasodilation

to relieve the increased vascular resistance 201. In the systemic circulation,

changes in vasoreactivity and peripheral resistance contribute to hypertension.

HIF-1α has also been implicated in regulating systemic vasoreactivity and thus

blood pressure. VSMC specific HIF-1α deletion increased systolic blood pressure

and vasoconstriction of mesenteric arteries stimulated by AngII 97. This is

consistent with the paradigm that hypoxia stimulates vasodilation in the systemic

vasculature, though interestingly, measurement of blood pressure by tail cuff,

rather than surgically placed aortic probe, reported no difference in systolic blood

pressure in either inducible or developmental models of VSMC HIF-1α deletion 267.

Our data demonstrate changes in systemic vasoreactivity with ARNT deletion, though notably, we report reduced rather than augmented vasoconstriction.

Arguably, our observations were made an elastic vessel, the aorta, rather than a resistance vessel, the mesenteric artery. Indeed conduit and resistance vessels have different structural compositions and physiologic functions 268. However, a

trend toward increased or decreased contractility in the aorta is often observed in

the resistance vessels 204, 269.

Aneurysm and dissection

In larger elastic vessels, alterations in VSMC phenotype are associated aneurysm and dissection 270-272. In Marfan syndrome, where a genetic mutation in fibrillin-1

125

underlies aneurysmal dilation of the vessel wall, aortic VSMCs display a

hypercontractile phenotype. Increased stress fibers, focal adhesions, and

exaggerated expression of contractile phenotype markers including SMA,

smoothelin, SM22α, and calponin-1 in addition to collagen I and myocardin were

identified in VSMCs from dilated vessel segments and in non-dilated areas, though

to a lesser degree 273. In contrast, shifts away from the contractile phenotype in

VSMCs are more common in aneurysm and dissection. Mutations in components of the VSMC contractile apparatus increase risk for both aortic aneurysm and dissection 271. Correspondingly, VSMCs isolated from human aortic aneurysms

had decreased staining for SM22α and SMA and increased matrix

metalloproteinase (MMP) -2 and -9 274, 275. In a mouse aneurysm model using

intraluminal elastase treatment, VSMCs associated with aortic dilations also had

decreased SM22 and SMA and increased MMPs 275. Moreover, VSMCs bordering

aortic dissections also featured decreased expression of contractile proteins and

markers and when isolated, have an increased proliferative phenotype 276, 277. HIF

has been implicated in the progression of aneurysm. Expression of HIF-1α and

MMPs are increased in the VSMCs of abdominal aortic aneurysms 92.

Correspondingly, chemical stabilization of HIF increased the incidence of

abdominal aortic aneurysm while global chemical inhibition of HIF activity

decreased aneurysm formation in a mouse model of AngII induced aortic

aneurysm 278. Seemingly in contrast, SMC specific HIF-1α knockout augmented

aneurysm formation and disruption of elastin fibers compared with controls when

challenged in vivo with β-aminopropionitrile and AngII 195. Yet studies by the same

126

group reported that VSMC Hif-1α knockout decreased AngII induced aortic

remodeling 93. In all, it would be reasonable to suggest that ArntSMKO mice may

exhibit an altered response in an aneurysm model. While the lack of consensus in

the HIF data complicates projections, synthetic phenotype and impaired

vasoconstriction in ArntSMKO would suggest that loss of ARNT may increase

susceptibility to aneurysm.

Atherosclerosis

Atherosclerotic disease affects the structure and composition of the vascular wall.

Not only does it underlie PAD, but also its stenotic and thrombotic consequences in coronary and carotid artery diseases can precipitate myocardial infarction and stroke respectively. While a complex disease involving many cell types, modulation of VSMC phenotype plays an important role in atherosclerotic lesion development and stability. Lesion formation is characterized by lipid accumulation in the sub intimal layer of the vessel wall. VSMCs of the tunica media proliferate and migrate into the intima forming a fibrous cap between the lipid pocket and the vessel lumen

279. The cells of this neointima display significant characteristics of synthetic VSMC phenotype including reduced expression of contractile proteins, SMMHC, SMA, and calponin, loss of myofilaments, alterations in contractility, increased DNA synthesis, and altered cell morphology 39, 280-287. VSMC migration into the fibrous cap is beneficial, as cap disruption is associated with plaque instability, which often precedes plaque rupture and thrombus formation 288, 289. VSMCs that remain within the tunica media are also affected by lipid accumulation. In the lipid core of the

127

plaque, VSMCs can take up LDL and become foam cells losing their VSMC

markers and acquiring monocyte markers 279. HIFs in VSMCs have been

implicated in the progression of atherosclerosis. Samples of lesions from human

carotid arteries show signs of hypoxia and stabilized HIF-1α localized to cells

bordering the necrotic core, including VSMCs 290, 291. HIF also appears to play a

role in lipid uptake by VSMCs. Hypoxia through HIF-1 activity increases expression

of low density lipoprotein receptor related protein 1 that allows for internalization

of aggregated LDL 292. The effects of VSMC HIF-1 inhibition on disease

progression are less clear. In a mouse model, VSMC specific knockout of Hif-1α in an ApoE null background reduced deposition of lipid and lesion size on a high fat diet 293. However, loss of the HIF target OCT4, a regulator of pluripotency in

embryonic stem cells, in VSMCs increased lesion size 51. In a non-targeted model,

IV injection of stabilized HIF-1α decreased lesion size in the ApoE null background

294. This lack of consensus once again makes predicting effects of VSMC-ARNT

difficult. The prevalent synthetic phenotype of ArntSMKO VSMCs could promote the

growth of larger plaques due to increased proliferation. Alternatively, the lack of

further changes in proliferation or migration of ArntSMKO VSMCs in response to

hypoxia, suggests they may not dramatically respond to the pathologic plaque

microenvironment, thus potentially resulting in decreased lesion size, consistent

with VSMC-Hif-1α-KO models.

128

Potential for translation to human disease

Arterial circulation

To determine the translatability of our findings to human disease we obtained

primary cultures of human iliac artery VSMCs from a single apparently healthy

donor through the National Institute on Aging. Exposure of these cells to hypoxia

(2% O2) for 24 hours altered transcriptional expression of phenotypic modulators.

Relative to normoxia and normalized to 18s, expression of HIF1A decreased in hypoxia while VEGFA increased consistent with increased HIF1 activity (Figure

4.1 A, B). Expression of PAI1 and THBS1 also increased, PAI1 significantly so, while no change was seen in PLAUR (Figure 4.1C-E). Changes in transcription of

Thbs1 and Pai1 were also seen in the murine aortic VSMCs (Chapter 2), though interestingly they were decreased rather than increased in response to hypoxia.

Some of these differing responses may be attributable to the diverse vascular origins of the VSMCs (aortic vs iliac), different durations in culture (direct isolation vs passage >25) and species specificity (mouse vs human). Functionally, 48 hours of hypoxia increased proliferation in human iliac artery VSMCs mirroring the proliferative response to hypoxia in murine VSMCs, although not sufficient for statistical significance (p < 0.07) (Figure 4.1F). Collectively, similar phenotypic responses to hypoxia, though not necessarily identical on a gene-by-gene basis inspire interest to further interrogate translatable correlations with human disease.

A more complete assessment of transcriptional and protein changes including

VSMC marker genes, examination of isolated VSMCs from additional individuals,

129

Figure 4.1. Effects of hypoxia exposure on human iliac artery VSMCs. Following 24 hour exposure to hypoxia (2% O2) human iliac artery VSMCs have decreased mRNA expression of (A) HIF1A, increased expression of (B)VEGFA, (C)PAI1, (E)THBS1, and no change in (D) PLAUR. (F) Proliferation measured by MTT assay was also increased by 48 hour hypoxia exposure though not to a statistically significant degree. n=3; 2 tailed t-test: ^p<0.05 hypoxia vs. normoxia.

130

and evaluation of cells with less duration in culture (<25 passages), would

considerably bolster findings and provide a framework for translation.

Venous circulation

Diseases of the peripheral vasculature are not limited to the arterial side of the

network. Venous pathologies, including varicosities and vein graft stenosis, are also thought to have VSMC alterations and hypoxic contributions295-300. Isolated

primary iliac vein VSMCs, from the same donor as the iliac artery cells, were also

exposed to 24 hours of hypoxia. They too had significant decreases in HIF1A

expression and increases in VEGFA indicative of HIF1 activity (Figure 4.2A, B).

Changes were also seen in PAI1, THBS1, and PLAUR, though interestingly all

were decreased relative to normoxia, PLAUR significantly so (Figure 4.2C-E).

Compared to increases seen in arterial cells, differential responses in venous

VSMCs could be attributable to the different baseline hemodynamic and oxygen

environment of the venous circulation. Venous cells also show increased

proliferation after 48hours in hypoxia relative to normoxia (p < 0.06) (Figure 4.2F).

While peripheral artery and vein VSMCs both show increased proliferation in

hypoxia, they differ in their ability to survive oxidative stress. When challenged with

600μM hydrogen peroxide in DMEM-F12 +5% FBS media for 24 hours, venous

VSMCs showed complete loss of cell viability by MTT assay in both normoxia and

hypoxia (Figure 4.3A). In contrast, arterial VSMCs under normoxia showed 30%

survival after the same stress, and further increased to 70% under hypoxic

conditions (Figure 4.3A). Interestingly, the loss of viability could be rescued in both

131

cell types under normoxia and hypoxia by culturing in EGM2-MV media rather than

DMEMF12 (Figure 4.3B). EGM2-MV is supplemented with hydrocortisone, ascorbic acid, and growth factors, namely VEGF, FGF-2, IGF-1, and EGF in addition to FBS. This suggests that in the right growth factor milieu, both iliac artery and vein VSMCs can survive high levels of oxidative stress. Taken together, this data demonstrates that iliac vein VSMCs also respond to hypoxia by increasing proliferation and altering transcription of phenotypic modulators, though in a pattern different from iliac artery VSMCs. Moreover, venous cells are more susceptible to oxidative stress when not in the presence of environmental enrichment than their arterial counterparts. Thus, venous VSMCs and their associated pathologies, would benefit from separate consideration in light of their deviation from arterial responses.

132

Figure 4.2. Effects of hypoxia exposure on human iliac vein VSMCs. mRNA collected from human iliac vein VSMCs following 24 hour exposure to normoxia or hypoxia (2%O2) and normalized to 18s, displayed decreased expression of (A) HIF1A, and increased expression of (B) VEGFA. Expression of (C) PAI1, (D) PLAUR, and (E) THBS1 were all reduced, PLAUR to a significant degree. (F) Exposure to hypoxia for 48 hours increases proliferation measured by MTT assay but not to a significant degree. n=3; 2 tailed t-test: ^p<0.05 hypoxia vs. normoxia.

133

Figure 4.3. Iliac vein smooth muscle cells are more susceptible to H2O2 induced apoptosis. ILA and ILV VSMC were treated with 600µM H2O2 in (a) DMEM-F12 +5% FBS or (b) EGM2-MV and incubated for 24hours in normoxia or hypoxia (2%O2). Cell numbers were measured using MTT assay and reported as ratios of optical density at 24 hours / time 0. (A) In minimal media, hypoxia increased the survival of ILA cells while no viable cells were detectable in venous cultures under either normoxia or hypoxia. (B) Culture in media enriched with growth factors removed the benefit of hypoxia in arterial cells and rescued venous cultures. n=3, one-way ANOVA: *p<0.05 arterial vs. venous VSMCs, ^p<0.05 hypoxia vs. normoxia.

134

Closing

HIFs are integrally connected master regulators of cellular responses to oxygen tension. Novel insights into the role of canonical HIF modulation shed light not only on the importance of HIFs in VSMCs but also on how crucial appropriate VSMC responses are to maintaining vascular function. These findings have implications for PAD, a major cause of morbidity, and moreover, for vascular diseases affecting

nearly every organ system. Further examination of the interplay between cellular

participants, including VSMCs, ECs, and non-vascular populations, and the molecular mechanisms driving these responses would improve understanding of pathophysiology and thus therapeutic design. Harnessing HIFs’ regulatory role could allow for improved vessel regulation and tissue perfusion, ameliorating diseases that take the lives of millions worldwide.

135

Future Directions

While there are many potential avenues for continued investigation, the most

immediate focus of future studies would be to determine the molecular

underpinnings of impaired organization and contractility in ArntSMKO vessels and

the implications of these changes on vascular homeostasis. The data reported in

Chapter 3 demonstrate reduced mRNA expression of contractile genes in isolated

ArntSMKO VSMCs. Further assessment of myofilament abundance at the protein

level in these isolated cells and in intact vessels would provide valuable insights.

Alterations in myosin, actin, and/or their regulators may contribute to the observed

phenotype. Contractile regulation of myosin activity converges on the

phosphorylation and dephosphorylation of MLC; quantifying the relative

abundance of MLC-P in ArntSMKO and Arntlox/lox vessels after contractile agonist stimulation would assess for altered activity in regulatory pathways. Further upstream, relative abundance of MLCK, MLCP, and their regulators, including

CaCm and ROCK respectively, as well as quantification of their individual contributions through the use of enzyme specific inhibitors on contractile agonist stimulated vessels or even isolated cells, could help determine the pathways implicated in any altered MLC phosphorylation status. Abnormalities in VSMC actin could also result in impaired vasoconstriction. While challenging to assess in intact vessels, actin organization could be evaluated microscopically in isolated cells by labeling SMA. Studies would assess for disparities in actin structure at baseline and following agonist stimulation in ArntSMKO and Arntlox/lox VSMCs. In a similar

assay, the polymerization status of actin could also be determined though

136

comparisons of F/G actin ratios. The contributions of altered RhoA/ROCK activity

to any aberrant actin structure should also be assessed in the Arnt deficient

VSMCs. Evaluation of relative abundance of phosphorylated and total RhoA and

ROCK as well as responses to pathway inhibition by small molecule ROCK inhibitors or dominant negative RhoA would quantify the importance of the

RhoA/ROCK pathway to VSMC phenotype and function.

Additional studies of vessel function could also strengthen conclusions. Regulation of perfusion distribution and blood pressure principally occurs at the level of resistance vessels, thus additional myographic studies in the muscular arteries of the mesentery would more directly evaluate physiologically relevant vasoreactivity.

In light of altered peripheral perfusion, myographic assessment of vessels from the skeletal muscle vasculature would also be of interest. Given the requirement of

ARNT for the hypoxic mediated responses by HIFs, stabilization of HIF-alpha subunits with PHD inhibitors or sparging buffers with oxygen depleted gas mixtures, could provide novel insights to the intrinsic role of vasoreactivity by vessels in light of hypoxic conditions. These studies would be particularly interesting when comparing the responses of pulmonary arteries, which vasoconstrict in response to hypoxia, to arteries of the systemic vasculature, which feature hypoxic vasodilation. Meaningfully, the aberrant normoxic phenotype in

ArntSMKO VSMCs and vessels could be confounded during embryonic

development. Utilization of a SMC specific inducible Cre-recombinase driver such

as SMMHC-CreERT2 mouse line would allow comparison of phenotypes in

developmental vs adult deletion. Finally, alterations in vasoreactivity can impact

137 blood pressure homeostasis, thus catheter based analysis of intravascular blood pressure at baseline and in response to vasoconstrictors would assess for systemic in vivo sequela of altered vasoconstriction as a consequence of VSMC-

ARNT deletion.

138

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