Multigene Therapy by Ultrasound-mediated Plasmid Delivery:

Transcription

1 Multigene Therapy by Ultrasound-mediated Plasmid Delivery: Temporally separated delivery of vascular endothelial growth factor and angiopoietin-1 promotes sustained angiogenesis in chronically ischemic skeletal muscle by Alexandra Helen Smith A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto Copyright by Alexandra Helen Smith 2010

2 Multigene Therapy by Ultrasound-mediated Plasmid Delivery: Temporally separated delivery of vascular endothelial growth factor and angiopoietin-1 promotes sustained angiogenesis in chronically ischemic skeletal muscle Alexandra Helen Smith Master of Science Institute of Medical Science University of Toronto 2010 ABSTRACT Endogenously, VEGF initiates angiogenesis, then later Angiopoietin (Ang)-1 matures vessels. We hypothesized that multigene therapy of VEGF before Ang1 to ischemic hindlimb tissue would result in persistent angiogenesis. At 2, 4 and 8 wks after inducing ischemia, blood flow was assessed by contrast-enhanced ultrasound. Animals were treated with VEGF at 2 wks, VEGF/Ang1 at 2 wks, or VEGF at 2 wks and Ang1 at 4 wks. In untreated controls, blood flow remained reduced. After VEGF delivery, resting flow and vessel density increased; however, flow reserve remained reduced, and vasculature was capillary-rich and eventually regressed. After VEGF/Ang1 co-delivery, flow increased marginally, flow reserve improved and vascular architecture remained normal. After separated VEGF and Ang1 delivery, flow, vessel density and flow reserve increased and were sustained, while vascular architecture remained normal. In conclusion, temporally separated VEGF and Ang1 delivery promotes sustained angiogenesis and improved vessel functionality. ii

3 Acknowledgments and Contributions Dr. Howard Leong-Poi s support during my graduate studies has motivated me to aim for excellence. His cheerful temperament created a friendly and trusting environment in the lab. He taught me directly and gave me the freedom to learn independently. Dr. Leong- Poi s mentorship has contributed to my growth as a researcher and a person. My program advisory committee, Dr. Tom Parker and Dr. Dan Dumont, posed important questions and offered thoughtful, helpful direction during this study. I am grateful for all that I have learned from them. The members of the Leong-Poi lab, Michael Kuliszewski, Christine Liao, Dmitriy Rudenko, Bryan Ross, Raymond Wong, Paul Lee and Hiroko Fujii, provided continual assistance and encouragement. I also appreciate the guidance I received from the rest of the research community in the Terrence Donnelley Heart Centre and research vivarium at St. Michael s Hospital. This study was made possible by funding from the Canadian Institutes of Health Research and from the Ontario Government Ministry of Research and Innovation s Early Researcher Award. iii

4 Table of Contents List of Abbreviations... vii List of Figures... viii List of Tables... viii Chapter 1 Literature Review and Introduction Angiogenesis and Disease Coronary and peripheral artery diseases Current options for CAD and PAD patients Process of Angiogenesis Stages of angiogenesis Vascular endothelial growth factor Angiopoietin-1 and Temporal regulation Therapeutic Angiogenesis in Clinical Trials Target growth factor Protein transfer Gene therapy vectors Gene therapy administration New Technology: Ultrasound-mediated Plasmid Delivery Contrast-enhanced ultrasound Targeted gene transfection UMPD for therapeutic angiogenesis New Goal: Mature Vasculature Arteriogenesis Mature vasculature Clinical trials aiming for vessel maturation Multigene therapy Remaining Challenges / Unresolved Issues Delivery technique Translational endpoints in animal studies Target genes Timeline of gene delivery Chapter 2 Rationale and Objectives Rationale and Novelty Ultrasound-mediated plasmid delivery Multigene delivery of VEGF and Ang Temporal separation iv

5 2.2 Overall Objective and Hypothesis Objective I Microvascular Blood Flow Hypothesis Approach: contrast-enhanced ultrasound Objective II Hindlimb Function Hypothesis Approach: treadmill test Objective III Molecular and Cellular Mechanisms Hypothesis Approach: FMA, Evan s Blue, Immunofluorescence and RT-PCR. 34 Chapter 3 Methods Experimental Protocol Animal Preparation Ultrasound-mediated Plasmid Delivery Microbubble and DNA preparation Plasmid delivery Perfusion Imaging Contrast-enhanced Ultrasound Microbubble Preparation Contrast-enhanced ultrasound Resting blood flow Blood flow reserve Exercise Tolerance Treadmill Test Optimizing the protocol Final treadmill test protocol Vascular Architecture Fluorescent Microangiography Adductor muscle tissue collection and preparation Confocal microscopy and 3D analysis Vascular Permeability Evan s Blue Supporting Cells Immunofluorescence Gene Expression Real Time PCR Statistical Analysis Chapter 4 Results Objective I Microvascular Blood Flow Sustained increase in blood flow after VEGF + Ang1 late delivery Flow Reserve improved after co-delivery of VEGF and Ang Objective II Hindlimb Function Objective III Molecular and Cellular Mechanisms Sustained increase in vessel density after VEGF + Ang1 late v

6 4.3.2 Normal vascular architecture after VEGF + Ang1 late therapy Vascular permeability not quantified accurately Mural cell coverage supports flow reserve and stability Transient increase in transgene expression after gene delivery Chapter 5 Discussion Specific Objectives Objective I Microvascular blood flow Objective II Hindlimb function Objective III Molecular and cellular mechanisms Overall Objective Unresolved Issues Stated in Introduction Delivery technique: UMPD Translational endpoints: treadmill test and flow reserve Target genes: VEGF and Ang Timeline: amplifying endogenous expression Future Directions Chapter 6 Conclusion References vi

7 List of Abbreviations 3D 3 dimensional SMC Smooth muscle cell!-sma!-smooth muscle actin TGF-"1 Transforming growth factor-"1 ABI Ankle brachial index TNF-! Tumor necrosis factor-! Ad Adenoviral UMD Ultrasound-mediated delivery Ang1 Angiopoietin-1 UMPD Ultrasound-mediated plasmid delivery Ang2 Angiopoietin-2 US United States or ultrasound Bcl-2 B-cell lymphoma 2 VE-cadherin Vascular endothelial cadherin bfgf Basic fibroblast growth factor VEGF Vascular endothelial growth factor CAD Coronary artery disease VEGFR1 Vascular endothelial growth factor receptor 1 CEU Contrast-enhanced ultrasound VEGFR2 Vascular endothelial growth factor receptor 2 CMV Cytomegalovirus CVD Cardiovascular disease ECM Extracellular matrix ELISA Enzyme-linked immunosorbent assay FMA Fluorescent microangiography GFP Green fluorescent protein GM-CSF Granulocyte-macrophage colony-stimulating factor IC Intra-coronary IGF-1 Insulin-like growth factor 1 IHC Immunohistochemistry IM Intra-muscular / intra-myocardial IV Intravenous MCP-1 Monocyte chemotactic protein 1 MEF2C Monocyte enhancer binding factor 2C MI Myocardial infarction N Sample size N/A Not applicable NO Nitric oxide NS Not significant PAD Peripheral arterial disease PDGF-B Platelet-derived growth factor B PDGFR-" Platelet-derived growth factor receptor " PECAM-1 Platelet endothelial cell adhesion molecule-1 PlGF Placental growth factor po2 Oxygen tension RT-PCR Real time polymerase chain reaction SD Standard deviation SEM Standard error of the mean vii

8 Introduction List of Figures Figure 1: Overall process of angiogenesis... 5 Figure 2: Timeline of endogenous VEGF and angiopoietin expression Methods Figure 3: Gene therapy timeline in four treatment groups Figure 4: Detailed flow chart of study design Figure 5: Microbubble/DNA complex for UMPD Figure 6: CEU in ischemic and non-ischemic hindlimb skeletal muscle Figure 7: Modified rodent treadmill Figure 8: FMA imaging and analysis Figure 9: Branch order Results Figure 10: Resting blood flow Figure 11: Resting blood volume and velocity Figure 12: Blood flow reserve Figure 13: Exercise tolerance Figure 14: Vascular density by FMA Figure 15: Vascular architecture Figure 16: Evan s Blue assay Figure 17: Smooth muscle cell coverage Figure 18: Pericyte coverage Figure 19: Gene expression by RT-PCR Introduction List of Tables Table 1: Therapeutic angiogenesis clinical trials Table 2: Multi-gene therapy animal studies Methods Table 3: Power analysis viii

9 Chapter 1 Literature Review and Introduction 1.1 Angiogenesis and Disease Coronary and peripheral artery diseases The increased sedentary lifestyle that has developed in North America and Europe has led to an increase in the morbidity of type II diabetes, obesity and hypertension conditions which result in atherosclerosis and cardiovascular complications such as angina, claudication and skin ulcers 1, 2. In patients with coronary artery disease (CAD), significant atherosclerosis results in stenoses leading to symptoms of angina and myocardial infarction (MI) 3. Repeated ischemia and MI induce endogenous angiogenesis and collateral formation in attempts to restore blood flow to the area of myocardium distal to the stenosis 3. When the endogenous angiogenesis has been insufficient in restoring flow to the area, however, symptomatic ischemia and angina result. In patients with peripheral arterial disease (PAD), occlusion of major arteries due to atherosclerotic lesions leads to hypoxia which also results in collateral formation that is often insufficient for even resting metabolic requirements 4-7. If left untreated, PAD progresses to refractory symptoms and critical limb ischemia, requiring amputation 4. The symptoms experienced by CAD and PAD patients can be painful and debilitating and the affect that these diseases have on our population is extensive. Cardiovascular disease (CVD) represents the leading cause of death globally, accounting for 36% of the deaths in Europe and the US 1, Angina pectoris caused by CAD is a major cause of disability, affecting 7 million people in the United States 3. Critical limb ischemia develops in 500 to 1000 people/million/year 11. Overall, 27 million people in North America and Europe are affected by PAD and 25 to over 50% of these patients frequently experience painful symptoms 12, 13. Due to its overwhelming affect on public health, CVD incurs the most cost in Europe (#169 billion) and the US ($403 billion) compared to any other disease 9, 14. The prevalence of major CVD risk factors have increased (such as diabetes and obesity) or stayed the same (smoking) in recent years 15, 1

10 16. Age is another noteworthy risk factor for CVD, so with our aging population the social and economic burden due to CVD will worsen in years to come Current options for CAD and PAD patients Current therapies for patients with ischemic vascular diseases aim to reduce plaque formation or increase blood flow 1. The severity of CAD/PAD dictates the therapeutic approach. Mild and less symptomatic stenoses can be treated with risk factor modification and pharmacological therapy. Severe and symptomatic stenoses and occlusions can be treated with either percutaneous transluminal coronary angioplasty and stenting or with coronary arterial bypass grafting surgery. Developments in pharmacological, interventional and surgical therapies have reduced mortality and morbidity due to CVD over the last decades (e.g. congestive heart disease mortality decreased by 47% from 1980 to 2000) and increased overall life span by 4.5 years 9, 16. A fourth and relatively large category of ischemic vascular disease patients have severe diffuse ischemia. The ischemia is severe enough that there is no effective pharmacological treatment and the stenoses are so diffuse and extensive that patients are not eligible for percutaneous or surgical interventions 7. These patients eventually become candidates for cardiac transplant surgery (CAD) or limb amputation (PAD). CAD/PAD patients with severe diffuse ischemia that is untreatable by available options are in need of a safe, effective, localized and long-lasting strategy for therapeutic angiogenesis. 1.2 Process of Angiogenesis Neovascularization, or new blood vessel growth, can occur by angiogenesis, vasculogenesis or arteriogenesis 7. Angiogenesis describes hypoxia-induced blood vessel formation from existing vascular endothelial cells for general growth and remodelling of a primitive network or for post-ischemic recovery and wound healing 17. Vasculogenesis occurs in adult tissue or in embryo when precursor cells differentiate into endothelial cells to form a primitive network 17. Adult vasculogenesis, triggered by ischemia or a wound, describes neovascularization by bone marrow-derived endothelial progenitor cells. Embryonic vasculogenesis progresses as hemangioblasts differentiate into 2

11 endothelial cells 17. Arteriogenesis describes the shear stress-induced interconnection of the arterial system (collateralization) and development of neovessels into more muscular arterioles or arteries, and accordingly involves the movement of smooth muscle cells Stages of angiogenesis I. Initiation. Initiation of angiogenesis begins with nitric oxide (NO)-induced vasodilation and a vascular endothelial growth factor (VEGF)-induced increase in vascular permeability to allow for the extravasation of plasma proteins that will provide a transitional scaffold for migrating endothelial cells. Permeability is increased by the formation of fenestrations and vesiculo-vacuolar organelles and by the redistribution of platelet endothelial cell adhesion molecule (PECAM)-1 and vascular endothelial (VE)-cadherin II. Matrix degradation. Urokinase plasminogen activator converts plasminogen to plasmin and contributes to inflammation. Inflammatory cells release matrix metalloproteases which, along with VEGF, chymase proteinases and heparanase proteinases stimulate extracellular matrix degradation. Angiopoietin-2 (Ang2) may bind to Tie-2 to block the stabilizing influence of angiopoietin-1 (Ang1). Endothelial cell mutual contact is interrupted, peri-endothelial cell support loosens and the basement membrane disintegrates. Matrix degradation and vessel destabilization allows for endothelial cell migration and liberation of angiogenic growth factors such as VEGF, basic fibroblast growth factor (bfgf) and insulin-like growth factor 1 (IGF-1) III. Cell migration and proliferation. The growth factors VEGF, bfgf and Ang2 are released from endothelial cells and/or inflammatory cells to promote endothelial cell migration and proliferation. IV. Tube formation. VEGF, bfgf, Ang1 and NO promote cell differentiation and tube formation. New and recruited endothelial cells intercalate into existing vessels or line up to form solid chords before acquiring a lumen. Lumen formation is directed by VEGF, integrins and the 18, 19 3

12 monocyte enhancer binding factor 2C (MEF2C) transcription factor. Endothelial cells of existing vessels thin to increase vessel length and diameter in response to VEGF. Ang1 also promotes increased vessel diameter. Enlarged venules sprout or become divided into individual capillaries (intussusception) V. Maturation. Transforming growth factor B (TGF-B) slows proliferation and angiogenic growth. Ang1, platelet-derived growth factor (PDGF) and NO promote vessel stabilization and spatial cues that guide the endothelial cells into the organized patterns and three dimensional (3D) networks. Endothelial cells express integrins that mediate signal transduction with the extracellular matrix for reconstruction of the basement membrane and matrix deposition. Ephrin-B2 is required to distinguish arterial and venous vessels. Disorganized, uniformly sized neovessels are remodelled and pruned to develop structured vascular networks VI. Regression. Ideally, when perfusion matches metabolic need, unnecessary microvessels will destabilize and regress because excess branching or bends can contribute to resistance to flow 21. This phase is promoted by thrombospondins, inhibitory Per-Arnt-Sim domain proteins, C-reactive proteins, chemokines bound to CXCR3, VEGF soluble receptors Flt1 and Tie-2, proteinase products, and by Ang2 at low VEGF levels , 19 VII. Survival. Once assembled, endothelial cells quiesce and survive for years. Reduced endothelial survival results in vascular regression 23. Apoptosis can be induced by a vessel obstruction, reduced nutrient supply, Ang2 (in the absence of other angiogenic growth factors), NO, reactive oxygen species, angiostatin, TSP-1, interferon-$, TNF-!, tissue factor pathway inhibitor or VEGF inhibitor. Endothelial survival is maintained by hemodynamic forces (shear stress) or by VEGF, Ang1 or!!"3 expression that leads to activation of the following survival pathways: PI3-kinase/Akt, p42/44 mitogen-activated protein kinase, Bcl-2, A1 or survivin. 17 4

13 Figure 1: Overall process of angiogenesis. Stages I to V are illustrated with endothelial cells (EC), pericytes, smooth muscle cells (SMC) and inflammatory cells (Inflam. cell) Vascular endothelial growth factor VEGF (also known as vascular permeability factor) is secreted as a homodimer and is part of the cystine knot growth factor family of proteins, which includes placental growth factor (PlGF), VEGF-A, -B, -C and -D 18, 24, 25. The most common splice variants of VEGF-A are VEGF165 and VEGF121; less common variants include VEGF145, VEGF189, VEGF206 18, 24, 26. The VEGF proteins influence developing and adult endothelial growth and function of vascular and lymphatic systems 18. VEGF-A interacts with tyrosine kinase receptors VEGFR-1 (flt-1) and VEGFR-2 (flk-1 or KDR) to promote blood vessel growth 27. VEGF-C and -D regulate lymphatic angiogenesis 25. PlGF and VEGF-B bind to VEGFR-1 but not VEGFR-2 25, 28, 29. Neuropilin-1 is a non-tyrosine kinase co-receptor expressed on endothelial cells that modulates VEGF/VEGFR-2 binding to regulate VEGF bioavailability and VEGF-induced angiogenesis 18. VEGF (from now on referring to VEGF-A) promotes both vessel growth and permeability increasing vascular endothelial cell proliferation and vessel number, inducing vasodilation through NO and increasing vascular permeability 1, 20, 25, 30. VEGF is 5

14 the most important catalyst for vascular formation, initiating the production of immature vessels by vasculogenesis or angiogenic sprouting in adult tissue 20. VEGF and one of its receptors are critical for mouse embryonic vascular development, loss of even one VEGF allele leads to embryonic defects and lethality 25, Mice lacking VEGF exhibit abnormal endothelial cell differentiation and proliferation and vessel formation A 25% reduction in VEGF expression, due to an altered promoter sequence, leads to spinal cord hypoperfusion and progressive motor neuron degeneration in mice 35. VEGF does not seem to have a continuous maintenance role, as a lack of VEGF is less traumatic in older animals 20. VEGF expression is very responsive to environmental cues, mainly regulated by oxygen tension 18. Tissue hypoxia causes hypoxia-inducible transcription factor (HIF) to increase VEGF gene transcription and causes stabilization of VEGF mrna, upregulating levels of VEGF protein by up to 30-fold, quickly and reversibly 18, 22. Normoxia down-regulates VEGF expression and promotes vascular regression 18. VEGF binds to VEGFR-2 on endothelial cells and VEGFR-1 on endothelial cells or other cell types The two receptors share 44% amino acid homology but differ in their distinct roles 24. VEGFR-2 mediates cell proliferation (via the Raf pathway), differentiation, migration (p38mapk pathway), permeability and survival (PI3K/Akt pathway) actions 18, 20, 33, 36, Mice lacking VEGFR-2 fail to develop vasculature, have very few endothelial cells, and show defects in vasculogenesis, angiogenesis and hematopoiesis 20, 33, 36. VEGFR-1 has an opposing role, upregulated by hypoxia to act as a decoy receptor to suppress VEGFR-2 signalling and may be involved in vascular remodelling 20, 25, 36, 45, 46. Mice with reduced VEGFR-1 expression have an excess of endothelial cells that integrate into disorganized tubules and abnormal vascular channels and mice lacking both alleles die in utero due to uncontrolled angioblast proliferation20, 25, 45, 47. VEGF also improves endothelial cell survival and resistance to death signals by increasing expression of survival proteins, such as B-cell lymphoma 2 (Bcl-2) and A

15 Up-regulation of VEGF expression on its own promotes formation of leaky, unstable and immature capillaries, limiting its clinical usefulness 20, 48. An excess of VEGF in adult tissue has been shown to lead to leaky and hemorrhagic vessels, often associated with an inflammatory response leading to swelling and edema 20, Due to the risks associated with either under- or over-expression of VEGF, this growth factor must be regulated spatially, temporally and quantitatively Angiopoietin-1 and -2 Angiopoietin-1 (Ang1) is a glycoprotein released from pericytes that binds to and phosphorylates the tyrosine kinase receptor, Tie-2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), expressed on vascular endothelial and hematopoietic cells The Ang1 protein is a multimer held by coiledcoil structures and disulfide cross links 54. Angiopoietin-2 (Ang2) has a high affinity for Tie-2 and acts as a partial agonist to either activate or antagonize it, depending on its concentration and on the other growth factors present 20, 56. Once Tie-2 is activated, ligands are released from the endothelial cell surface while Tie-2 is rapidly internalized and degraded 56. Embryos lacking Ang1 or Tie-2 develop normal primary vasculature but no further remodelling occurs and endothelial cells fail to associate with any underlying support cells and fail to form normal vascular networks 20, 32, 57, 58. Mice lacking Ang2 lack regression of hyaloid vasculature and vascularization of the retina, as well as proper aortic wall development demonstrating Ang2 s role as angiogenic agonist or antagonist 20. Ang1 is primarily expressed in peri-endothelial support cells (mural cells) of quiescent vasculature 7, 59. Ang1 plays a permissive role in angiogenesis, optimizing the interaction between endothelial cells and supporting cells to allow them to receive critical signals from the micro-environment 20, 57. Tie-2 phosphorylation by Ang1 inhibits NF-%Bdependent expression of inflammatory genes (e.g. intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and E-selectin) 60. Ang1 is only involved in cell 7

16 migration and tube formation stages of angiogenesis in the presence of NADPH oxidase, but its Tie-2 mediated functions are NADPH oxidase-independent 61. Ang1 recruits and sustains mural cell support and stimulates endothelial cell/pericyte interaction, a critical step required to stabilize vascular networks initiated by VEGF 20, 53, 62, 63. Indirectly, Ang1 expression leads to the repair of damaged and leaky vessels and vascular network maturation 20, 53, 64. Once vessels mature, constitutive Ang1/Tie-2 interaction maintains vascular homeostasis, quiescence and stability (via PI3K/Akt survival pathway) 7, 18, 20, 60. Ang2 is expressed and released from activated endothelium of vessels undergoing remodelling as a dynamically regulated antagonizing autocrine signal that helps initiate sprouting or regression 7, 20, 59. Ang2 expression is upregulated by hypoxia or VEGF expression to disrupt the Ang1/Tie-2 interaction and its constitutive stabilization influence, leading to vessel destabilization 18. Specifically, endothelial cell intercellular junctions are disassembled (via Rho kinase pathway) and endothelial cells are no longer surrounded by pericytes or the extracellular matrix 18, 60. Ang2 renders quiescent capillaries plastic and destabilized and therefore more responsive to VEGF, but in absence of angiogenic stimuli, Ang2 is associated with vessel destabilization and regression 22, 59, 65. Individually, neither Ang1 nor Ang2 promote significant neovascularization 53. Administration of Ang1 will protect adult vasculature from VEGF-induced permeability or inflammatory changes by mural cell recruitment 20, 54, 64. Transgenic over-expression of Ang1 in mice promotes a substantial increase in vessel size and leakage resistance, with a minor increase in vessel number 20, 63. Ang1 over-expression in murine skin has also been shown to lead to hypervascularity, more so than VEGF over-expression, promoting vascular remodelling events and decreasing normal vascular pruning 32, 63. Myocardialspecific over-expression of Ang1 in double transgenic mice leads to cardiac hemorrhage and embryonic lethality 66. An excess of Ang2 disrupts blood vessel formation in the embryo by antagonizing the effects of Ang1 and Tie Ang2 over-expression in embryonic endothelium lethally disrupts vascular development 20, 32, 59. In ocular microvessels, increased Ang2 increases capillary diameter and endothelial cell 8

17 proliferation and migration if VEGF is present; however, if VEGF is inhibited, increased Ang2 causes endothelial cell apoptosis and vessel regression 65. Ang1 promotes vessel stability and Ang2 can support angiogenesis or lead to regression Temporal regulation In embryo, VEGF acts before Ang1, then Ang2 recaptures the destabilized state by making vessels plastic enough to respond to VEGF again 20. After periods of hypoxia, there is greater Ang2 than Ang1 initially, and then later there is greater Ang1 than Ang2 67. The same pattern of temporal regulation can be found in adult ischemia-induced angiogenesis. A 2003 canine study examined the time course of expression of these three growth factors during angiogenesis induced by repeated myocardial occlusions 68. At day 3 of occlusions, VEGF and Ang2 expression in myocardial interstitial fluid peaks, while Ang1 levels remain constant 68. At 1 wk, capillary density and diffusion distance are optimal, then gradually return to baseline 68. Although Tie-2 expression levels remain constant, Tie-2 phosphorylation is greatly reduced at 1 wk and elevated at 3 wks, at which point Ang1 mrna expression increases 68. The change in capillary density parallels the VEGF/Ang2 expression and is inversely related to Tie-2 phosphorylation 68. The Ang1:Ang2 ratio is lowest at day 1 and highest at 3 wks 68. Consistent with these results, in rat models of myocardial infarction (MI), within 1 wk post-mi VEGF expression is elevated by 3 to 4-fold and Ang2 is doubled 69, 70. By the second week, Ang1 expression increases 70. In a rat subcutaneous polymer disc, at 1 wk VEGF expression is high and Ang1 expression is low, then at 2 to 3 wks VEGF is low and Ang1 is high 71. In a rat hindlimb ischemia model, VEGF expression increases by 1.8-fold and Ang2 expression increases by 1.5-fold after 1 day; both return to normal by 1 wk 72. Ang1 expression is unchanged during the first week and is elevated at 4 wks 72. All of these studies agree that during endogenous angiogenesis, VEGF and Ang2 expression are increased acutely while Ang1 expression is elevated later (Figure 2). 9

18 Figure 2: Timeline of endogenous VEGF and angiopoietin expression. Simplified illustration of timeline of endogenous VEGF and angiopoietin expression after ischemic injury To summarize, for development of dense, stable and mature vasculature, an early increase in VEGF and Ang2 expression followed by a later increase in Ang1 expression is ideal. Given the importance of this temporal relationship during endogenous angiogenesis and the repercussions of over-expressing VEGF and Ang1 simultaneously, it will likely be worthwhile to consider the timeline of transfection during therapeutic angiogenesis. 1.3 Therapeutic Angiogenesis in Clinical Trials Therapeutic angiogenesis describes the delivery of angiogenic growth factors to ischemic tissue in order to potentiate the natural reparative mechanisms that stimulate blood vessel growth and restore flow 7, 19. A selection of clinical trials testing protein and gene therapy strategies for therapeutic angiogenesis are listed in Table 1 (page 14-15). This section will review common target growth factors, protein therapy, and gene therapy vectors and administration Target growth factor The most common growth factors used in angiogenic trials include VEGF and fibroblast growth factor (FGF). VEGF is the prototype angiogenic growth factor for gene therapy, where isoform 165 is the most promising because it is nearly 100-fold more potent than isoform The fibroblast growth factors (FGF) are able to stimulate mesenchymal and endothelial cells 74. FGF-4 and -5 are popular candidates for gene therapy because they are efficiently secreted 74, 80, 81. Target growth factors for vessel maturation are outlined in section

19 1.3.2 Protein transfer Using growth factor protein for therapeutic angiogenesis bypasses barriers associated with gene therapy, such as transcription, translation and post-translational modifications 9. Protein therapy is most often delivered by direct intramuscular injection because the halflife of proteins is too short to administer orally or even intravascularly and untargeted systemic administration could result in unwanted side effects 3, 9. Despite these limitations, early animal studies showed some success; for example, intra-arterial injection of VEGF led to collateral vessel formation and capillary development in a rabbit model of hindlimb ischemia 82. The clinical trials were not as successful, though. After intracoronary infusion of recombinant VEGF protein in the VIVA trial, angina symptoms improved compared to the placebo group but exercise tolerance did not 75. Direct injection of FGF protein after coronary artery bypass grafting led to angiogenesis with no improvement in exercise tolerance Gene therapy vectors Gene therapy, the transfer of DNA to somatic cells, has also been used for therapeutic angiogenesis 19. Gene transfer is preferred over recombinant protein therapy due to the potential for longer-term growth factor expression by incorporation into the endothelium for sustained protein production, compared to the short-lived growth factor bolus after protein transfer 3, 11. A longer half-life lessens the need for repeated administration. One strategy for gene transfer is to take advantage of the high transduction efficiency of viruses devoid of immunogenic or replicative genes 83. Viral vectors provide increased transduction through efficient cell uptake (by binding to surface receptors) and intracellular transport of packaged DNA (protected from lysosomal degradation) to a nucleus Adenoviral vectors can lead to gene transfer in non-target organs or limited transfection efficiency when an immune response mounts against the viral particles and clears transduced cells 83, Third generation adenoviral vectors lacked the entire genome and produced no viral proteins 83. Adeno-associated viruses, non-pathogenic parvoviruses, result in transgene expression that lasts for months in animal studies 83,

20 Regardless, when adenoviral VEGF DNA was administered to patients with PAD vascularity improved but clinical outcomes were uneffected 83, In three studies delivering FGF4 DNA by adenoviral gene transfer, only one study showed benefit compared to placebo: symptoms improved in female patients at 1 yr, but exercise tolerance improved only temporarily 3, 81, 99. Viral gene transfer is a highly efficient method for gene transfection, but has unsafe, immunogenic side effects 11. Use of naked plasmid DNA has some advantages over viral vectors, such as no immunogenic effects, low cost, low toxicity and the option for high specificity 83. Plasmid DNA is water soluble, heat stable, easily cleaned and can carry large genes 9, 83. The expression does not last as long as after viral gene transfer; however, gene therapy for angiogenesis does not require perpetual transgene expression 83. Transfection efficiency is much lower than viral gene transfer because cell uptake of plasmid DNA is poor 83. Intramuscular injection of naked plasmid DNA is an inefficient method for transfection because expression is limited to the injection site 9, 11. Use of liposome complexes or cationic polymers to improve cellular uptake does not significantly increase efficacy of plasmid DNA in vivo 74, 100, 101. Plasmid DNA vectors have shown success in Phase I PAD clinical trials but neither the NORTHERN nor the EUROINJECT ONE Phase II CAD trials demonstrated improved exercise tolerance or angina relief after VEGF plasmid injection 79, Gene therapy administration Gene therapy (viral or plasmid DNA) is most commonly administered by intramyocardial/muscular (IM) injection, intracoronary (IC), intravenous (IV) or intrapericardial injection. IM injection is invasive and associated with procedural risks but is organ specific and bypasses the endothelial barrier of the heart 9. In a phase I study, VEGF plasmid IM injection (with no control group) improved CAD symptoms in 5 patients and the procedure was considered safe 106. Several phase I trials in patients with PAD also indicated that other than some edema the procedure is relatively safe and has shown signs of improved blood flow compared to pre-treatment , 107, 108. Doubts 12

21 were cast on the strategy after placebo-controlled studies using catheter-based or IM injection showed improved symptoms, but no effect on perfusion or exercise tolerance 83, 105, 109. Although IV injection is less invasive than IM, systemic administration of angiogenic proteins or gene therapy vectors introduces the risk of increasing angiogenesis and potentially tumor growth in other organs 9, 76. As well, the endothelial barrier of capillaries impedes transfection efficiency 83. Intracoronary (IC) administration (or intra-arterial for PAD) is less invasive than IM injection and more site-specific than IV injection. IC delivery of VEGF plasmid DNA in a small (n=6) Phase I trial showed reduced angina at 1 year 110. Additionally, IC adenoviral FGF-4 was used in the AGENT trials to treat stable angina pectoris patients 3, 81, 99. The Phase III trial did not achieve the primary endpoint, exercise tolerance at 3 months, but improved exercise tolerance was noted in females at 6 months 81. IC administration requires an open coronary vessel and the use of contrast dye, of which not all patients are tolerant. The ideal delivery system would be as non-invasive as IV injection and as targeted as IM injection, without the use of contrast dyes and their toxicities. 13

22 Title, Author, Year, (Ref #) Seiler et al., 2001 (156) FIRST Simons et al., 2002 (153) Zbinden et al (157) Phase, # of patients Phase I, 21 patients Phase II, 337 patients Phase I/II, 14 patients Placebocontrolled Therapy Udelson et al., 2000 (187) 59 patients! Protein - IC or IV - FGF-2 VIVA Henry et al., 2003 (75) AGENT Grines et al., 2002 (3) AGENT-2 Grines et al., 2003 (99) REVASC Stewart et al., 2006 (78) AGENT-3, -4 Henry et al., 2007 (81) KAT Hedman et al., 2003 (77) EUROINJECT ONE Kastrup et al., 2005 (105) NORTHERN Stewart et al., 2009 (79) Losordo et al., 1998 (106) Phase II, 178 patients Phase I/IIa, 79 patients Phase II, 52 patients Phase II, 67 patients Significant improvement in perfusion or cardiac function at end of study Protein - IC, SC - GM-CSF Collateral flow:! at 2 wks N/A Significant improvement in symptoms or exercise tolerance at end of study Protein - IC - FGF-2! Perfusion: no change at 6 months! Exercise: no change at 6 months Protein - SC - GM-CSF Collateral flow:! at 2 wks N/A Protein - IC + IV - VEGF N/A Ischemia: " at 6 months! Perfusion:! at 2 but not 6 months N/A Angina: " with high dose at 4 months! Exercise: trend at 4 months Gene - IC - adenoviral FGF4! Stress echo: no diff.! Exercise: trend at 1 & 3 months Gene - IC - adenoviral FGF4 Ischemic defect size: " at 2 months! Perfusion defect size: " at 1 but not 2 months N/A! Gene - IM - adenoviral VEGF! Perfusion: no diff. at 6.5 months Phase IIb/III Gene - IA - adenoviral FGF4 Phase II, 103 patients Phase II, 80 patients Phase II/III, 93 patients Phase I, 5 patients Gene - IC - adenoviral or plasmid VEGF Gene - IM - plasmid VEGF CCS class: " to 1 yr in females! in males Exercise:! at 6.5 months (not at 3 months) Angina: " at 6.5 months! Adverse events: no diff. at 6.5 months Exercise:! at 6 months in females! Exercise: no diff. in males Perfusion defect: " at 6 months! Exercise: no diff. at 6 months Local wall motion:! at 3 months! Perfusion: no diff.! Angina: no diff. Gene - IM - plasmid VEGF! Perfusion: no diff. at 6 months! Exercise: no diff. at 6 months! Gene - IM - plasmid VEGF Perfusion:! at 2 months Angina: " Reilly et al., 2005 (76) 29 patients! Gene - IM - plasmid VEGF N/A Angina: " at 2 yrs! 2 / 30 patients diagnosed w/ cancer Vale et al., 2001 (110) 6 patients Gene - IC - plasmid VEGF Perfusion! & Ischemia " at 3 months Angina: " at 1 yr Losordo et al., 2002 (109) Phase I/II, 19 patients Gene - IM - plasmid VEGF! Wall motion: no diff. Angina: " at 3 months! Exercise: trend at 3 months Table 1a: Therapeutic angiogenesis CAD clinical trials. For each trial reference details, phase & size, placebo, therapy (protein/gene, administration, vector & growth factor) and results are included. Studies are arranged according to therapy (protein therapy, viral gene therapy, then plasmid gene therapy) and success. Increases (!) or decreases (") are compared to placebo (or baseline if no placebo). Trend refers to non-significant improvement. N/A (not applicable) indicates that a study did not report results for this category. Studies with positive results for exercise tolerance or angina at the latest time point are in grey (light grey for non-placebo controlled studies, dark grey for controlled studies) 14

23 ! Gene - IM - plasmid VEGF Collaterals & flow:! at 2 months ABI:! at 2 months Title, Author, Year, (Ref #) TRAFFIC Lederman et al., 2002 (152) START van Royen et al., 2005 (155) VEGF PVD trial Makinen et al., 2002 (96) RAVE Rajagopalan et al., 2003 (97) Baumgartner et al., 1998 (102) Isner et al., 1998 (103) Kim et al., 2004 (104) Morishita et al., 2004 (108) Kusumanto et al., 2006 (98) Phase, # of patients Phase II, 190 patients Phase, 40 patients Phase II, 54 patients Phase II, 105 patients Phase I, 9 patients Phase I, 6 patients Phase I, 9 patients Phase I/IIa, 6 patients Phase I/II, 54 patients Placebocontrolled Therapy Significant improvement in perfusion at end of study Significant improvement in symptoms or exercise tolerance at end of study Protein - IA - FGF-2 N/A! Exercise: no diff. at 3 months Protein - SC - GM-CSF Flow: prevented decrease at 3 months Gene - IA - adenoviral or plasmid VEGF Gene - IM - adenoviral VEGF Vascularity:! distal to treated site at 3 months N/A! Exercise: no diff. at 3 months! ABI: no diff. at 3 months! ABI & Grade: no diff.! Gene - IM - plasmid VEGF Collaterals & flow:! ABI! & Symptoms "! Gene - IM - plasmid VEGF N/A ABI:! at 9 months! Ankle brachial index (ABI): no diff. at 6 months! Exercise: no diff. at 3 or 6.5 months! Gene - IM - plasmid HGF N/A Pain " & ABI! at 3 months Gene - IM - plasmid VEGF! Amputation rate: no diff. at 3 months ABI! & Symptoms " at 3 months Table 1b: Therapeutic angiogenesis PAD clinical trials. For each trial reference details, phase & size, placebo, therapy (protein/gene, administration, vector & growth factor) and results are included. Studies are arranged according to therapy (protein therapy, viral gene therapy, then plasmid gene therapy) and success. Increases (!) or decreases (") are compared to placebo (or baseline if no placebo). Trend refers to non-significant improvement. ABI refers to ankle brachial index. N/A (not applicable) indicates that a study did not report results for this category. Studies with positive results for exercise tolerance or angina at the latest time point are in grey (light grey for non-placebo controlled studies, dark grey for controlled studies). 15

24 1.4 New Technology: Ultrasound-mediated Plasmid Delivery Ultrasound-mediated plasmid delivery (UMPD) is a non-invasive method for gene therapy with relatively high gene transfection efficiency and high organ specificity Contrast-enhanced ultrasound Ultrasound is a safe, inexpensive, widely available and common imaging modality to visualize ventricular function and blood flow 24, 111. The temporal and spatial resolution available by ultrasound surpasses that of nuclear imaging 24. To conduct ultrasound imaging, short pulses of pressure waves at high frequencies are sent into a patient s body. Since the speed of sound in soft tissue is known, the time delay of reflected sound signals is used to obtain information about internal organs, tissues and blood vessels 111. Biological tissues (except bone and lung) consist primarily of water, which has a low compressibility allowing sound waves to propagate with little scatter or reflection 111. For contrast-enhanced ultrasound (CEU), a contrast agent is used to visualize blood flow. Gas-filled lipid microbubbles infused intravenously oscillate when sonified by ultrasound, due to rapid pressure variations that cause the volume of gas in the microbubble to compress and expand by up to 100-fold 111, 112. The change in microbubble volume causes ultrasound signal scattering that is detected and visualized 111, 112. CEU was first conducted with agitated saline (containing tiny air bubbles); later, a surfactant was used to create lipid microbubbles which allowed for transpulmonary passage 111, 113. With a diameter under 4.5 &m, the microbubbles could easily transit through the microcirculation 111. Use of water-insoluble fluorinated gases instead of air allows microbubbles to circulate for several minutes 111. Careful examination of the safety of modern contrast agents, such as Definity (lipid-shelled with perfluoropropane gas), in animal models has provided evidence that intravenous injection results in negligible and transient entrapment in the pulmonary circulation and that after pulmonary passage the microvascular rheology of these microbubbles is similar to that of red blood cells 114. Anionic microbubbles are used if myocardial or pulmonary retention and persistent opacification (opaque, visible contrast) is desirable, by complement-mediated 16

25 attachment 115. CEU is routinely used to monitor patients blood flow in large and small vessels Targeted gene transfection At high ultrasound energies (ultrasound pressure or mechanical index > 0.4), oscillations lead to microbubble destruction 83, 111. To achieve gene transfection, microbubbles loaded with plasmid DNA are destroyed in a target organ by high power ultrasound, propelling DNA into the vascular endothelium 83. Secondary mechanical effects facilitate transfer across cell membranes 83. High amplitude microbubble oscillations cause cavitation and form microjets that create micropores in the cell membrane and facilitate membrane and capillary permeability 9, 116, 117. In 1998, it was shown that ultrasound-mediated delivery (UMD) transfers particles past the endothelial barrier into surrounding interstitium by insonifying microbubbles and causing microvessel rupture 118. UMD is minimally invasive. Intravital microscopy of rat skeletal muscle revealed that microbubble destruction after exposure to ultrasound creates enough microvessel rupture for red blood cell extravasation, but cell and tissue damage is limited to the rupture itself 119. While many studies have established diagnostic ultrasound to be safe, a few mouse studies reported hemorrhage at high energy doses 9, After further investigation, it is understood that hemolysis is possible under diagnostically relevant conditions but the effect is not likely to be clinically significant 9. In 2002, in vitro and in vivo studies showed that low intensity ultrasound (US) with inert Optison microbubbles and plasmid DNA is a safe and efficient method of gene therapy 11. For this method, protein-based bubbles enwrap the plasmid and the microbubble/dna complexes are injected intramuscularly 11. The ultrasound creates small holes that return to normal after 1 day 11. Transfection efficiency with Optison/DNA/US exceeds that after DNA and after DNA/US by and 10-fold, respectively 11. Exceptional organ specificity can be accomplished by UMD because successful transfection requires spatial and temporal proximity of microbubbles, ultrasound and DNA 83. When UMD is used to deliver adenoviral or plasmid DNA to rat myocardium, 17

26 adenoviral DNA is found in the liver and the heart, but plasmid DNA transfection is specific to the heart 125. Another study targeting UMD to the heart showed that luciferase activity peaks at 4 days and transfection is limited to the target organ 126. UMD has also been used to deliver interference RNA, recombinant proteins and small molecules9, 83, ; however, plasmid DNA is the best candidate for therapeutic angiogenesis balancing safety, targeted delivery and transfection duration. Transfection efficiency by UMD has improved substantially over the last several years. In 2002, UMD of microspheres was performed in a rat spinotrapezius muscle to optimize the ultrasound parameters for maximal transfection efficiency 136. Results showed that the highest number of extravasation points and greatest microsphere delivery is achieved with a pulsing interval of 5 or 10 sec 136. No difference was found between IV and intra-arterial microsphere infusion 136. Higher microvascular pressure corresponds to increased delivery with no change in the number of extravasation points 136. By comparison, a separate study that used UMD of luciferase plasmid demonstrated that intra-arterial microbubble infusion creates more microporations and greater perivascular deposition of fluorescent plasmid than IV infusion 137. The ultrasound parameters were further optimized in a study that delivered adenoviral or plasmid DNA to rat myocardium 125. Low transmission frequency with maximum mechanical index were deemed optimal 125. Transfection efficiency is much higher with triggered delivery as opposed to continuous, presumably because a pause between ultrasound pulses allows the capillary bed to replenish 125. Approximately 15% of the plasmid DNA adhere to the microbubbles 125. When a longer duration of transgene expression is needed, expression can be easily re-elevated by repeating the UMD, as was done by Bekeredjian et al. rat myocardium 126. Transfection efficiency improves with cationic microbubble phospholipids, as was shown in rat hindlimb muscle and canine myocardium 9, 137, 138. A criticism of UMD is the low transfection efficiency compared to direct intramuscular (IM) injection. Recently, our lab compared UMD (with cationic microbubbles) to direct IM injection for transfection of VEGF165/GFP (green fluorescent protein) plasmid DNA in a rat model of hindlimb ischemia 2 wks post-ligation 139. By 4 18

27 wks, blood flow improves in both groups 139. Although exogenous mrna expression is greater after IM injection, ultrasound-mediated plasmid delivery (UMPD) increases blood flow more 139. Imaging of the GFP fluorescence revealed that transgene expression after IM injection is concentrated to focal perivascular regions and myocytes; by contrast, transgene expression after UMPD is spread evenly throughout the tissue in the vascular endothelium of arterioles and capillaries 139. UMPD promoted angiogenesis more efficiently than direct IM injection despite low transfection efficiency, likely due to the more diffuse delivery UMPD for therapeutic angiogenesis UMPD has been used for therapeutic angiogenesis in models of hindlimb ischemia (rat, mouse, rabbit) and myocardial infarction (rat). When hepatocyte growth factor DNA (HGF, involved in cardiomyocyte survival) is delivered by UMPD (using Optison microbubbles) in a rabbit hindlimb ischemia model, more angiogenesis is stimulated after DNA/ultrasound/Optison therapy than after DNA or DNA/ultrasound, with no acute toxicity 11. In a rat model of myocardial infarction, Kondo et al. performed UMPD of HGF 140. Control groups included HGF plasmid alone, HGF plasmid with ultrasound, or control plasmid with ultrasound and microbubbles 140. The study established that 3 wks after UMPD of HGF, left ventricle weight and scar size are reduced, while capillary and arterial densities are increased compared to all three control groups 140. Following those studies, Yoshida et al. enhanced endogenous angiogenesis by UMD without growth factor DNA 1 wk after femoral artery excision in mice 141. Increased capillary density, flow and exercise tolerance compared to untreated controls 3 wks after treatment were attributed to recruitment of VEGF-releasing inflammatory cells 141. In a 2007 study, Leong-Poi et al. presented UMPD as an effective strategy of gene therapy for therapeutic angiogenesis in the setting of severe chronic ischemia 142. Two weeks after induction of unilateral hindlimb ischemia, once endogenous angiogenesis was complete, rats were treated with delivery of plasmid DNA containing either GFP 19

28 alone or GFP with VEGF 142. There was also a group of untreated controls 142. Microvascular blood flow and volume were measured by CEU and vessel density was measured by fluorescent microangiography. The study showed that blood flow remains reduced in the control and GFP groups 142. Blood volume and vessel density are elevated at 4 wks post-ligation in the GFP group but not at 8 wks, demonstrating that the angiogenic effect of ultrasound and microbubbles without DNA, observed also by Yoshida et al., is only transient 141, 142. VEGF/GFP therapy results in significantly increased blood flow, blood volume and vessel density at 4 and 8 wks after delivery; however, some vascular regression occurs by 8 wks 142. More recently, Fujii et al. delivered either VEGF, stem cell factor (SCF) or GFP by UMPD to rat myocardium 1 wk after coronary artery ligation 143. Analysis 3 wks later showed an increase in capillary and arteriolar density, myocardial perfusion and cardiac function in the VEGF and SCF groups New Goal: Mature Vasculature A shift in therapeutic angiogenesis research has taken place; the goal is to create not just more vessels, but mature vasculature. The theory is that clinical trials were unsuccessful because neovessels lacked stability and organization. With the understanding that robust coronary collateral circulation improves post-mi survival rates and reduces the incidence of cardiovascular events in CAD, strategies for therapeutic arteriogenesis have recently been explored 1, Arteriogenesis Recall that angiogenesis describes sprouting capillary growth from pre-existing vessels in response to hypoxia, as mentioned in section By contrast, arteriogenesis is the enlargement of pre-existing arteriolar anastomoses into larger collateral vessels in response to changes in shear stress 1, 145, 146. Broadly, an occlusion in a main artery increases the pressure gradient in downstream anastamoses, which causes small vessels to proliferate and remodel to increase lumen size and blood flow, in an attempt to preserve limb or organ function 1. 20

29 According to Heil and Schaper, successful remodelling depends on the existing arteriolar network that will connect pre- and post-occlusive vessels, shear stress-induced activation of endothelium, bone marrow-derived cell incorporation and endothelial and smooth muscle cell proliferation 145. Increased fluid shear stress causes endothelium activation, transforming a quiescent vessel layer with low adhesion tendency into one that supports attraction, activation and adhesion of leukocytes 145. Endothelial activation leads to monocyte invasion and matrix degradation 145. Granulocyte macrophage colonystimulating factor (GM-CSF) decreases macrophage apoptosis and supports mobilization of mononuclear cells from the bone marrow, which are crucial during early arteriogenesis 145, 147, 148. FGF-induced proliferation of smooth muscle cells, adventitial fibroblasts and endothelial cells results in collateral vessel growth 145. Increased circumferential wall stress causes smooth muscle cell migration and proliferation, enlarging vessel diameter and wall thickness 145. Once fluid stresses return to normal, growth stops 145. Collateral vessels are as muscular as regular arteries but can be excessively tortuous (corkscrew path rather than straight path) and may join to distal part of occluded artery at non-physiological angles, characteristics which can create resistance to flow 145, 149. Therefore, paradoxically, excessive collateral growth can impair blood flow conductance 145. Accordingly, therapeutic production of mature vessels depends on more than arteriogenic wall thickening and collateral growth Mature vasculature In order to create stable and mature vasculature therapeutically, it is important to understand the factors that enable stability and maturity. Numerous characteristics of mature vasculature have been described, in addition to arteriogenic growth. The key characteristics include vessel wall specialization, appropriate network organization and minimal regression 150. Firstly, vessel wall structural support and functional regulation are specialized to the need of the organ. In hindlimb tissue, minimal vascular permeability improves flow reserve, so increased intercellular adhesion and pericyte support is ideal. 21

30 To support abrupt increases in oxygen demand, skeletal muscle vasculature will have substantial smooth muscle cell coverage. Secondly, network organization can be improved with modest collateral growth, minimal tortuosity and increased vessel diameter to support appropriate pattern formation. Peirce et al. define pattern formation as vessel assembly into a functional network of arterioles, venules and capillaries at suitable length and diameter distributions 151. Network function stimulates different patterns for different organs; conversely, the pattern of a particular network can reveal the extent to which that function is served 151. Finally, to minimize vascular regression, vessels can be stabilized with increased interaction between endothelial cells and the surrounding mural cells (pericytes and smooth muscle cells) and extracellular matrix Clinical trials aiming for vessel maturation Recent clinical trials have attempted to use arteriogenic growth factors to restore perfusion to ischemic tissue. Three months after a single intra-arterial injection of bfgf, walking times improved in patients with PAD 152. In a separate study, an intracoronary bolus of bfgf resulted in improved symptoms in patients with coronary heart disease at 3 months, but not at 6 months 153. Both of these therapies came with side effects, such as hypotension 1. Cytokines such as monocyte chemotactic protein 1 (MCP-1) and GM-CSF are indirect vascular growth factors that act via circulating monocytes and endothelial progenitor cells 74, 154. MCP-1 gene or protein therapy has not made it to clinical trial because of this chemokine s ability to increase atherosclerotic plaque formation 155. Although intracoronary and subcutaneous injection GM-CSF improved coronary collateral blood flow in CAD patients after 2 wks; in a separate study, this therapy also led to acute coronary syndrome with coronary artery occlusion, possibly due to leukocyte accumulation in the atherosclerotic lesions and plaque rupture 156, 157. In PAD patients, subcutaneous administration of GM-CSF failed to improve exercise tolerance 158. Vessel maturation by therapeutic arteriogenesis has not yet been demonstrated. 22

31 1.5.4 Multigene therapy In recognition of the complexity of angiogenesis and vessel maturation, an emerging therapeutic approach is to use multiple angiogenic growth factor genes. Several combinations have been explored in animal models of CAD and PAD (Table 2, page 26). The most common objective in multigene therapy studies for vessel maturation is to quantify arteriole formation by measuring smooth muscle cell or smooth muscle!- actin coverage. VEGF/Ang1 adenoviral (rat) or plasmid (mouse) co-delivery immediately after myocardial infarction (MI) improves smooth muscle coverage more than VEGF single delivery when measured within 2 wks of treatment 159, 160. In diabetic and nondiabetic mice with MI,!-actin coverage increases after VEGF/Ang1 viral gene therapy compared to control 7 wks after treatment (single delivery not measured) 161. VEGF/FGF plasmid co-delivery improves!-actin coverage 2 wks after treatment in mice and 8 wks after treatment in rabbits with hindlimb ischemia 162, 163. FGF-2/PDGF-BB protein injection into porcine heart increases!-actin coverage more than single delivery up to 14 wks after treatment 164. A second objective has been to increase vessel diameter. A simultaneous increase in VEGF and Ang1 in murine cornea micropocket improves perfusion of corneal neovasculature, increases capillary density and increases luminal diameter of arteries, but does not affect the length of circumferential neovascularization 53. Ang2 with VEGF results in longer vessels, more circumferential neovascularity and more frequent sprouting cells than after VEGF alone or VEGF/Ang1, but no change in luminal diameter 53. In rat mesentery fat pad, vessel diameter increases after adenoviral gene transfer of Ang1 alone or VEGF/Ang1, but not after transfer VEGF alone 165. A third common objective in vessel maturation studies is to increase the number of collateral vessels. In mouse hindlimb ischemia, PlGF/VEGF co-delivery 1 wk postligation increases perfusion and collaterals (measured semi-quantitatively) more than either growth factor alone 166. In a rabbit hindlimb ischemia model, collateral conductance 23

32 1 month after delivery of VEGF/bFGF-transfected fibroblasts is greater than after delivery of VEGF- or bfgf-transfected fibroblasts 167. Vessels covered by mural cells are protected from vascular regression 168, 169. Supporting cell coverage is assessed by identifying pericyte markers, such as PDGFR-", surrounding vessels. Interaction between PDGF-B (released from endothelial cells) and its receptor, PDGFR-", is critical for pericyte recruitment and proliferation along a growing vessel 170, 171. Endothelial cell/mural cell interaction is an important aspect of vessel maturation and is facilitated in large part by Ang1 20. The rat mesentery fat pad study also showed that pericyte coverage increases after gene transfer of Ang1 alone but not after VEGF alone or VEGF/Ang1 together 165. Another objective has been to reduce vascular permeability. Angiotensin converting enzyme (ACE) expression and activity increases in response to VEGF and decreases in response to Ang Angiotensin II type I receptor blocker (ARB) treatment prevents VEGF-induced reduction in VE-cadherin and increase in edema 172. In mouse hindlimb ischemia, capillary density increases in response to VEGF gene delivery with and without ARB pre-treatment 172. Adenoviral delivery of Ang1 to adult mice protects vasculature from leakage when injected prior to intradermal injection of VEGF protein or exposure to mustard oil 64. Pattern formation was quantified by Peirce et al. in a study examining temporal and spatial growth factor regulation using rat dorsal skin window chambers 151. Branch order ratios were measured (ratio of most distal vessels of a vascular tree to the second most distal, or capillary/arteriole ratio), as well as vessel diameters and smooth muscle!- actin coverage 151. They found that increased VEGF leads to increased branch order ratio and that the order ratio returned to normal when Ang1 levels were increased 1 wk after VEGF 151. A recent objective is to generate sustained angiogenesis, as the positive results of many therapeutic angiogenesis trials have been only transient. In a rabbit ear ischemia model by Zhou et al., adenoviral (Ad) gene therapy was performed 8 wks after ischemia 24

33 was induced 49. According to this study, AdVEGF-induced angiogenesis is transient, while AdAng1-induced angiogenesis is sustained 49. Although AdAng1-induced angiogenesis requires elevated levels of endogenous VEGF, AdVEGF/AdAng1 co-delivery results in angiogenesis as transient as AdVEGF therapy alone

34 Title, Author, Year, (Ref #) Benest et al., 2006 (165) Siddiqui et al., 2003 (160) Liu et al., 2007 (159) Samuel et al., 2010 (161) Lu et al., 2007 (164) Babiak et al., 2004 (166) Sano et al., 2006 (172) Lee et al., 2007 (162) Kondoh et al., 2004 (167) Zhou et al., 2004 (49) Chen et al., 2007 (163) Chae et al., 2000 (62) Disease Model Mesentery (rat) CAD (mice) Time btwn Ischemia & Delivery (wks) CAD (rat) 0 CAD (rat) diabetic CAD (pigs) PAD (mice) PAD (mice) PAD (mice) PAD (rabbit) PAD (rabbit) PAD (rabbit) PAD (rabbit) Growth Factors 0 VEGF + Ang1 0 VEGF + Ang1 VEGF + Ang1 + Bromodexyuridine 0 VEGF + Ang1 Delivery Method Gene viral Gene - IM plasmid Gene - IC viral Gene - IM viral Capillary Density or Perfusion at end of study ( if Combo! Single delivery) Evidence of Arteriogenesis or Maturation ( if Combo! Single delivery) Vessel area: Combo = V, A1 (> control)! Pericyte ensheathment: Combo = V, < A1 1 Time after delivery (wks)! Cap density: Combo = A1, < V Arteriole/cap ratio: Combo > V, A1 alone 1.5 Cap density: Combo = V, > A1 Cap density: Combo > control (didn t measure single delivery) 0 FGF-2 + PDGF-BB Protein Vessel count & flow: Combo > PDGF, FGF 1 VEGF + PlGF 0 VEGF + ARB Angiotensin II rec blocker) 0 VEGF + bfgf 3 VEGF + bfgf in fibroblasts 8 VEGF + Ang1 10 VEGF + Ang1 + EPCs 1.5 VEGF + Ang1 Protein - IM repeated Gene - IM plasmid Gene plasmid Gene - IA viral Gene - SC viral Gene viral Gene - IM plasmid Arterioles: Combo = A1, > V Cardiac function: Combo > control at 1 wk Arterioles: Combo > control 7 "-SMA: Combo > PDGF, FGF LVEF: Combo > control Perfusion: Combo = VEGF, > PlGF Collateral # & diameter: Combo > control 1 Cap density: Combo = VEGF (> control) Permeability: Combo < VEGF Cap density: Combo = V, > FGF "-SMA: Combo = FGF, > V 2 Angiographic score: Combo > FGF > VEGF Flow: Combo > V or FGF Cap density: Combo = V & FGF (> control) Arterial diameter: Combo > V or FGF "-SMA: Combo > FGF > V Collateral conductance: Combo > additive! Flow: Combo = V & control, < A1 N/A 4 Flow: V/A1/EPCs > V/A1 or EPC Cap density: V/A1/EPCs = V/A1 (> control) Cap density: Combo > V or A almost 2 "-SMA: V/A1/EPC > V/A1 or EPC 8 Resting & max flow: Combo > V or A1 Larger vessels: Combo = A1, > V 4 10 Table 2: Multi-gene therapy animal studies. Study details, disease model, time between ischemia and treatment, delivery method, angiogenesis results, arteriogenesis results and time after delivery are arranged from left to right. Differences between treatment groups are expressed with greater than (>), less than (<) or same as (=) symbols. Grey shading highlights studies that used VEGF and Ang1. Combo refers to combination, or both genes. Cap refers to capillary. N/A (not applicable) indicates that a study did not report results for this category. Studies arranged according to animal model (CAD at the top, PAD at the bottom) and latest timepoint after delivery (far right column). 26

35 1.6 Remaining Challenges / Unresolved Issues The negative results of therapeutic angiogenesis clinical trials can likely be improved by enhancing targeted delivery of genetic material and altering the candidate gene selection Delivery technique Protein transfer is too short-lived. Viral gene transfer has immunogenic side effects. Direct intra-muscular injection of plasmid DNA was too focal and intravenous injection not targeted enough. Moving forward, strategies for therapeutic angiogenesis will be most effective with the use of UMPD. This method results in transient (but not shortlived), non-invasive and targeted (but not overly focal) gene delivery. Use of cationic microbubbles and high power ultrasound improves transfection efficiency over that of intravenous injection of naked plasmid DNA Translational endpoints in animal studies Given that many therapeutic angiogenesis clinical trials were unsuccessful, despite positive animal studies, we need to question the endpoints used. To be clinically relevant, future animal studies should involve more robust, functional endpoints. For optimal skeletal muscle function during exercise, hyperemia (an increase in flow to active tissue) is accomplished by increased cardiac output, increased vascular resistance in the inactive tissues (e.g. viscera, skin) and decreased vascular resistance in the active skeletal muscle 173, 174. The primary determinants of hyperemia in exercising skeletal muscle are local vascular control mechanisms, especially the skeletal muscle pump which supports increased cardiac output and metabolic vasodilation which contributes to reduced vascular resistance in the active muscle 173, 175, 176. Greater vasodilation is accomplished in vascular beds that have higher vasomotor tone and lower flow at rest, as relaxation of the smooth muscle layer of arterioles allows for rapid shunting of blood to areas of high demand 173. A vascular bed s ability to induce hyperemia in response to increased oxygen demand can be quantified by measuring flow reserve, the ratio of exercise flow to resting 27

36 flow. Dr. Lindner measured flow reserve in a canine model of peripheral arterial stenosis by conducting CEU during electrically stimulated muscle contraction 177. The study showed that normal flow reserve induced by adenosine or by a pulse generator is 2.5 to 3.5 and that stenosis causes complete loss of flow reserve by blunting changes in volume and velocity 177. In patients with PAD, skeletal muscle flow reserve (as measured by CEU) is impaired and correlates with severity of symptoms 178. Recent research indicates that measuring perfusion flow reserve may reveal dysfunction not evident at rest 179. Exercise tolerance has been measured by a treadmill test in rat models of hindlimb ischemia with inconsistent results. Orito et al. showed a correlation between severity of unilateral hindlimb ischemia to exercise tolerance at 7 wks post-ligation 180. In another model of rat unilateral hindlimb ischemia, signs of critical limb ischemia (including limping) and oxygen tension are nearly all resolved by 40 days after ligation of the common iliac and femoral arteries and their branches 181, Target genes Therapeutic angiogenesis clinical trials have demonstrated that VEGF is not the ideal candidate, due in part to unstable neovessels, hypotension and edema 1, 9, 79, 91, FGF and hepatocyte growth factor have also been studied in patients, but with less than compelling results 9, 99, 108, 186, 187. In fact, promoting angiogenesis by single gene therapy has not been sufficient for formation of mature and functional vasculature and the early therapeutic arteriogenesis studies have shown only some promise 1, 144. Most likely a combination of both processes would be ideal for improved flow and exercise tolerance. Ang1 has been the candidate for promoting vessel maturation in several animal studies. In a murine model of myocardial infarction (MI), smooth muscle coverage and arteriolar density in diabetic MI mice rises to levels of wild-type MI mice after adenoviral Ang1 gene therapy 55, Timeline of gene delivery Coordinated spatial and temporal administration of a combination of angiogenic factors would be ideal for formation of mature, functional vascular networks 7. Endogenously, an 28

37 early increase in VEGF expression precedes a destabilized environment (e.g. matrix degradation) that allows for cell migration and proliferation. During this phase, Ang2 binds to Tie2 to block Ang1-induced quiescence and allow for further vessel destabilization. After adequate tube formation, a late increase in Ang1 expression restabilizes vasculature (e.g. matrix deposition). Altering this inverse relationship of expression may create a situation where Ang1-induced stabilization interferes with VEGF-induced cell migration and tube formation. Supporting this theory, in transgenic mice over-expressing VEGF and Ang1, the VEGF-induced angiogenesis was inhibited 189. Visconti et al. studied transgenic mice over-expressing VEGF, Ang1, Ang2 or a combination of these 189. Ang2 and VEGF collaborate synergistically to induce angiogenesis 189. In animals over-expressing VEGF and Ang1, the VEGF-induced edema is reduced; however, the VEGF-induced angiogenesis is also inhibited 189. This study revealed the role of Ang1 as a negative regulator of VEGF in vivo. Furthermore, Babaei et al. discovered that individually VEGF and Ang1 could each induce neovascularization in Matrigel, but when both growth factors are present vessel growth is less than additive 190. Adeno-associated co-delivery of Ang1 with VEGF to rat hindlimb muscle prevents VEGF-induced angiogenesis and permeability 191. Peirce et al. examined the spatial and temporal regulation of angiogenesis by VEGF and Ang1 in normal subcutaneous tissue, using protein-releasing beads to increase the concentration of these growth factors in controlled locations 151. This group was particularly interested in the effects on pattern formation. They reported that increased VEGF results in a short term increase in capillary number and density that regresses within 3 wks, with no change in smooth muscle!-actin coverage. An abnormally high proportion of the new vessels are small capillaries. When the concentration of Ang1 is increased 6 days after VEGF, the increase in capillary number and density is maintained to 3 wks and smooth muscle!-actin coverage is elevated. The new vasculature in the VEGF with Ang1 late group maintains a normal ratio of the smallest capillaries to those one level upstream (i.e. branch order ratio). It was concluded that increased Ang1 after 29

38 increased VEGF induces vascular growth, while maintaining native vascular network patterning. To definitively clarify whether temporal separation of VEGF and Ang1 gene delivery is beneficial, a study that compares VEGF/Ang1 co-delivery to temporally separated delivery in a model of ischemia is required. 30

39 Chapter 2 Rationale and Objectives 2.1 Rationale and Novelty Ultrasound-mediated plasmid delivery Ultrasound-mediated plasmid delivery (UMPD) is a safe method for targeted and diffuse gene transfection. Plasmid (rather than viral) delivery results in transient transfection. Since ultrasound settings and probe placement determine the location of microbubble destruction, gene transfection can be localized to a specific site of injury. Transfection efficiency is lower than intramuscular injection (even with cationic microbubbles and optimal ultrasound parameters); however, the diffuse transfection by UMPD targeted specifically to the vascular endothelium induces a more robust biological effect than focal transfection after intramuscular injection 192. Future strategies for therapeutic angiogenesis may be more successful with the use of targeted, effective UMPD Multigene delivery of VEGF and Ang1 Increased vessel growth has been achieved in clinical trials but not peak walking time, possibly because resultant vasculature lacks stability and functionality. Initiating angiogenesis by gene delivery of VEGF alone is not sufficient for a sustained improvement in blood flow and the early vessel maturation studies have shown only limited promise 75, 79, 96, 152, 156. We propose that therapeutic angiogenesis with vessel maturation may achieve the desired functionality, improving both flow and exercise tolerance. Increased Ang1 expression is important for vessel maturation, repairing leaky vessels, organizing vascular networks and maintaining vascular homeostasis 18, 20, 52, 58, 193. Ang1 over-expression or single gene delivery induces far less vessel growth than VEGF, though, and is not a strong trigger for angiogenesis 159, 160, 189. Accordingly, Ang1 is a promising candidate to complement VEGF gene delivery but not on its own Temporal separation Mimicking and amplifying the endogenous timeline of VEGF and Ang1 expression will optimize the microenvironment for each. Research has shown that an increase in Ang1 31

40 protein 6 days after increased VEGF induces vessel growth while maintaining the native network pattern 151. If instead VEGF and Ang1 levels increase simultaneously, Ang1- induced stabilization may interfere with VEGF-induced cell migration and proliferation. In transgenic mice over-expressing VEGF and Ang1 and after viral VEGF/Ang1 codelivery to rat hindlimb muscle, VEGF-induced angiogenesis is inhibited 189, 191. Ang1 gene delivery before VEGF would stabilize vessels before trying to induce angiogenesis, which would be illogical. To clarify whether VEGF and Ang1 delivery for therapeutic angiogenesis should follow the endogenous timeline, the current study conducted simultaneous and temporally-separated delivery in a rat model of chronic hindlimb ischemia. During endogenous angiogenesis (as described in section 1.2.4), approximately 2-3 wks after VEGF increases, Ang1 expression increases. According to our lab s previous study, 2 wks after UMPD of VEGF, transgene expression is low and blood flow is high (pre-regression). A temporal separation of 2 wks between VEGF and Ang1 delivery was chosen to parallel endogenous angiogenesis, to avoid competition between exogenous VEGF and Ang1, and to capture vessels for maturation before vascular regression. 2.2 Overall Objective and Hypothesis The overall objective of this study was to compare concurrent and temporally separated transfection of VEGF and Ang1 genes by UMPD for improvement of skeletal muscle perfusion in a rat model of severe chronic ischemia. Treatment groups included a group treated with VEGF alone, a group treated with VEGF and Ang1 at the same time point, a group treated with VEGF early and Ang1 two weeks later, and a control group that received no therapy. The main hypothesis was as follows: temporally separated delivery of VEGF and Ang1 will result in sustained improvement in skeletal muscle perfusion, as compared to VEGF delivery or concurrent VEGF/Ang1 delivery. 2.3 Objective I Microvascular Blood Flow The primary objective was to measure resting blood flow and flow reserve in response to UMPD of VEGF alone, VEGF/Ang1 together, or VEGF with Ang1 late or no treatment. 32

41 2.3.1 Hypothesis After VEGF single delivery or after co-delivery of VEGF/Ang1, resting blood flow will increase transiently, with a higher peak in the VEGF alone group. VEGF with Ang1 late will induce a sustained increased in resting blood flow and improved flow reserve Approach: contrast-enhanced ultrasound To conduct contrast-enhanced ultrasound (CEU), ultrasound pulsing intervals are gradually increased, allowing contrast agent to perfuse deeper into the vasculature. CEU was performed at rest and during electrically-stimulated skeletal muscle contraction to measure blood flow reserve, the ratio of exercise flow to resting flow. Dr. Lindner s lab established this method to study peripheral arterial stenosis in a canine model 177. According to Dr. Lindner, perfusion imaging provides the most accurate information regarding the severity of PAD by assessing all sources of flow, including nutritive and non-nutritive pathways Objective II Hindlimb Function The secondary objective was to determine the effect on hindlimb function and exercise tolerance Hypothesis Exercise tolerance will be recovered after VEGF with late Ang1 therapy, related to flow reserve Approach: treadmill test With a translational research perspective, a rodent-sized treadmill was used to test exercise tolerance. In the clinical management of PAD, the goal is to improve peak walking time; however, few therapeutic angiogenesis pre-clinical studies measure exercise tolerance. 33

42 2.5 Objective III Molecular and Cellular Mechanisms The final objective was to examine mechanisms for changes in blood flow; such as, changes in network architecture, vascular permeability, peri-endothelial support cells, and gene expression Hypothesis Transgene expression will peak 3 days post-delivery and dissipate thereafter. VEGF delivery will lead to temporarily increased vessel density with poor network organization and few supporting cells. Late Ang1 delivery will lead to sustained improvements in vascular density, network organization and supporting cell recruitment, preventing late vascular regression. After VEGF/Ang1 co-delivery, their respective effects will be suppressed Approach: FMA, Evan s Blue, Immunofluorescence and RT-PCR According to Visconti et al., thorough evaluation of angiogenesis should quantify of vessel density, vascular permeability and supporting cell recruitment 189. Fluorescent microangiography forms an arterial cast of hindlimb vasculature, visible by confocal microscopy for vessel density and patterning analysis. Evan s Blue assays measure vascular permeability. Immunofluorescence reveals smooth muscle cells and pericytes. Real time polymerase chain reaction (RT-PCR) quantifies growth factor expression. 34

43 Chapter 3 Methods 3.1 Experimental Protocol Unilateral hindlimb ischemia was induced by ligation of the left common iliac artery in 168 rats on day 0. This model of ischemia induces an immediate 65-70% loss of blood flow and skeletal muscle oxygen tension 194. After 2 wks of endogenous neovascularization, muscle flow and oxygenation plateau at approximately 50% of normal 194. To study chronic ischemia, baseline blood flow was measured in the left and right adductor muscles after 2 wks, once endogenous angiogenesis was complete. Animals were divided into 4 treatment groups (Figure 3). In the control group, animals were not treated. In the VEGF alone group, animals were treated with ultrasoundmediated plasmid delivery (UMPD) of VEGF 2 wks after ligation. In the VEGF/Ang1 early group, animals were treated with UMPD of VEGF and Ang1 together at 2 wks. In the VEGF + Ang1 late group, animals were treated with UMPD of VEGF at 2 wks and Ang1 at 4 wks. Blood flow was reassessed at 4 and 8 wks post-ligation. As outlined in Figure 4, animals were sacrificed at 4 and 8 wks post-ligation as well as 3 days postdelivery and tissue from the adductor muscles was collected for histological, permeability and gene expression analysis. Figure 3: Gene therapy timeline in four groups. Iliac artery ligation at day 0. Baseline flow measured at 2 wks. Controls were untreated. Other groups were treated with UMPD of VEGF at 2 wks (VEGF alone), VEGF and Ang1 at 2 wks (VEGF/Ang1 early), or VEGF at 2 wks and Ang1 at 4 wks (VEGF + Ang1 late). Flow was measured again at 4 and 8 wks post-ligation. 35

44 Figure 4: Detailed flow chart of study design. Ligation and gene therapy are shown as described in Figure 3. Baseline blood flow was measured by contrast-enhanced imaging (CEU) 2 wks after ligation. Blood flow was measured again at 4 and 8 wks post-ligation. Animals were sacrificed at 4 and 8 wks post-ligation as well as 3 days post-delivery for histological, permeability and gene expression analysis. 3.2 Animal Preparation The study protocol was approved by the Animal Care and Use Committee at St. Michael s Hospital Health Sciences Research Centre, University of Toronto. To induce unilateral proximal hindlimb ischemia, 168 male Sprague-Dawley rats (Charles River) were anaesthetized by intraperitoneal injection of ketamine hydrochloride (10 mg/kg) and xylazine (8 mg/kg) and the left common iliac artery was exposed and ligated with 4-0 suture, using aseptic technique. The incision was closed in layers and animals were recovered with one dose of buprenex (0.025 mg/kg). For perfusion analysis and gene delivery, animals were anaesthetized in the same manner and the jugular vein was cannulated for intravenous infusion of the microbubble contrast agent. Animals were recovered with one dose of anafen (5 mg/kg). 36

45 3.3 Ultrasound-mediated Plasmid Delivery Microbubble and DNA preparation For gene delivery, cationic lipid microbubbles (1x10 9 ) were charge-coupled to 500 &g of plasmid DNA, as previously described 137, 142. Microbubbles with a cationic lipid shell (zeta potential of +60mV) were incubated with plasmid DNA, resulting in approximately 6700 plasmids covering each microbubble 137, 195. Plasmid vectors were constructed by incorporating the gene for human VEGF165 into the vector pcdna3.1(+) and for human Ang1 into the vector pflag-cmv-1 (Addgene). Both vectors contained a cytomegalovirus (CMV) promoter (Figure 5). Figure 5: Microbubble/DNA complex for UMPD. Cationic lipid microbubbles were chargecoupled to plasmid DNA. The VEGF gene was encoded into a pcdna3.1 plasmid vector and the Ang1 gene was encoded into a pflag-cmv-1 plasmid vector, both with CMV promoters Plasmid delivery UMPD was conducted with a Sonos 5500 ultrasound machine (Philips Healthcare, Andover, Massachusetts) using ultraharmonic imaging (single pulse technique). The S3 probe was positioned to target a transverse section of the proximal hindlimb adductor muscle and release single frames of high power (120 V, mechanical index > 1.6) ultrasound every 5 sec. At 1.3 MHz and a transmit power of 0.9 W, peak negative acoustic pressure by needle hydrophone was 2100 kpa (assuming tissue attenuation coefficient of 0.3 db/cm/mhz, at tissue depth of 1 cm) 139. A 0.5 ml bolus of the microbubble/dna solution was injected, then 1 ml was continuously infused over 10 min (infusion pump Model AS50, Baxter). The cannula and stopcock were then flushed 37

46 with saline (0.4 M) and ultrasound delivery continued for an additional 10 min to destroy any residual circulating microbubble/dna complexes. During delivery, the probe was adjusted, scanning slightly proximally and distally along the limb to distribute transfection throughout the proximal hindlimb muscle. For VEGF/Ang1 early animals, Ang1 was delivered 5 min after VEGF. 3.4 Perfusion Imaging Contrast-enhanced Ultrasound Microbubble Preparation For contrast-enhanced ultrasound (CEU) perfusion imaging, inert lipid-shelled perfluoropropane microbubbles were used (Definity). A Coulter Multisizer IIe (Beckman- Coulter) was used to measure the microbubble concentration by electrozone sensing Contrast-enhanced ultrasound CEU was conducted to measure blood flow in the proximal hindlimb adductor muscles with gated pulse inversion imaging (HDI 5000, Philips Ultrasound), using a linear array transducer (L7-4), a mechanical index of 1.0 and a transmission frequency of 3.3 MHz. Gain settings were optimized and held constant. Data were recorded on magnetic-optical disks then transferred to a computer for analysis using HDI Lab, DigiLink1.7.2 and MCE2.9.4 software programs Resting blood flow Background images were acquired before microbubble infusion for subtraction of tissue signal. During continuous intravenous infusion of microbubbles (1x10 7 min -1 ), triggered imaging was then performed while the time between ultrasound pulses was increased (pulsing intervals, PI), specifically 0.2, 0.5, 1, 2, 3, 5, 7, 10, 20 and 40 sec. Five averaged background frames were digitally subtracted from averaged contrast-enhanced frames at each PI. PI versus signal intensity (SI) data were fit to the function y=a(1 e "t ), (Figure 6). Y is the signal intensity at pulsing interval t. A is the plateau signal intensity representing an index of maximum microvascular blood volume. " is the rate constant, or the time to reach plateau, used as a measure of microvascular blood velocity

47 Microvascular blood flow was calculated by the product of A and ". Flow in the ischemic left leg was normalized to the normal leg to account for small differences in body temperature, microbubble concentration, volume of distribution and ultrasound imaging parameters between animals. The ratio of ischemic limb flow to normal limb flow was calculated and compared between groups. Figure 6: CEU in ischemic and non-ischemic hindlimb. (Top) As pulsing interval is gradually increased, contrast agent perfuses deeper into vasculature, creating a brighter image. (Bottom) Background-subtracted signal intensity was plotted against pulsing interval and fit to the function y=a(1-e -"t ), where y represents signal intensity, t represents pulsing interval, A reflects blood volume, and " reflects blood velocity Blood flow reserve To measure flow reserve (ratio of exercise flow to resting flow), CEU was performed during electrically stimulated contraction induced by a pulse generator (AV Sequential Demand Pulse Generator, Medtronic 5330), immediately after measuring resting flow. This technique was previously demonstrated in a canine model of peripheral arterial stenosis by Dr. Lindner 177. To modify the protocol for a rat model of hindlimb ischemia, the voltage was reduced to 3 ma and the pulse frequency was increased to 2 Hz (matching approximate number of steps taken at velocity of 20 cm/s or 12 m/min). As in 39

48 Dr. Lindner s study, a modest level of exercise was induced rather than maximal exercise to mimic the low exercise tolerance expected in patients with PAD (normal limb flow reserve of 3, rather than 10). In protocol optimization, the importance of certain technical details became clear; namely, carefully securing the hindlimb, precise and consistent positioning of needle electrodes, collecting more frames when the ultrasound PI and pulse generator fell out of sync, and performing protocol quickly to improve recovery. CEU perfusion images were collected as described for resting flow. Flow reserve of the ischemic limbs were compared between groups. 3.5 Exercise Tolerance Treadmill Test Optimizing the protocol In gene therapy clinical trials for PAD patients, the primary endpoint is typically exercise tolerance (peak walking time). In this animal study, exercise tolerance was tested to measure hindlimb skeletal muscle function and demonstrate clinical significance. The process of attempting to optimize the protocol is described here, although an ideal protocol was not achieved. Hindlimb ischemia in rodents is most often studied with motor-driven treadmills 180, Orito et al. demonstrated that treadmill test results correlate to the severity of hindlimb ischemia 180. We purchased a treadmill with adjustable speed and incline (Panlab, Harvard Apparatus). Some groups use electric shock or poking as stimuli to walk. Instead, we blocked access to the back stage entirely (Figure 7). When a rat stopped walking, it was pushed up against the barrier while the treadmill belt still ran beneath it, which likely created discomfort and pain without trauma. We also incorporated positive auditory and odorous stimuli at the front of the treadmill. Figure 7: Modified rodent treadmill. Physical barrier was designed to block rat from back stage. (Left) Original treadmill design. (Right) Modified treadmill with rat walking (top) and dragging (bottom). 40

49 The main elements of the protocol were habituation, incline, speed and endpoint. Animals were habituated to the treadmill frequently enough to become accustomed to it, yet mildly enough to avoid exercise-induced angiogenesis (3 min, 15 cm/s) 197. The treadmill was set at a 5 incline to focus the work on the hindlimbs. An increasing speed gradient was incorporated because at a constant speed some rats walked for over 1 hr. The test endpoint was fatigue, defined as limbs not moving for 7 s. Ligated control rats were compared to age- and weight-matched normal rats to find the upper and lower limits. Tests were performed at only 2 and 8 wks post-ligation, with the assumption that rats would forget the discomfort between tests. Data collection included detailed qualitative observations during each habituation session and tolerance test regarding breaks, hopping (an indicator of fatigue), and ability to keep up with speed. Some animals walked continuously while others took short pauses, doing less work in the same total time. To account for this, a second timer was used to measure the time spent moving all 4 limbs. A third group of animals took almost no steps and were excluded from the data. After optimizing habituation, incline, speed, endpoint, number of tests and walking vs. dragging time, a final test protocol was carried out in control and normal rats Final treadmill test protocol Age-matched normal and control rats were habituated to the treadmill for 2 wks leading up to each test at a 5 incline. During the first minute speed gradually increased from 5 cm/s to 15 cm/s, where it remained for 2 min more. Rats were tested 2 and 8 wks after ligation. At a 5 incline, speed gradually increased from 5 to 15 cm/s during the first minute, where it stayed for 4 min. Every 5 min, the speed was increased by 5 cm/s, until 35 cm/s. Walking time and distance, rat weight vs. time, and percent walking time for before and after treatment were examined. 41

50 3.6 Vascular Architecture Fluorescent Microangiography Adductor muscle tissue collection and preparation Fluorescent microangiography was performed as previously described 142. After sacrifice, the lower limbs were pressure perfused (140 mmhg) with 100 ml of heparinized saline via the abdominal aorta. Before limbs were removed, FluoSpheres carboxylate-modified microspheres (10% solution, 0.2 &m and 0.02&m diameter; Invitrogen) were mixed with low melting point agarose (1% solution; Sigma) at 45 C and slowly injected. Limbs were covered with ice to rapidly cool and solidify the solution, forming an arterial cast. The right and left adductor muscles were removed and stored in 10% buffered formalin (VWR) at 4 C. Tissue was sectioned (200 &m; Leica VT1000S) and mounted onto a slide with Vectashield Mounting Medium (Vector Laboratories) Confocal microscopy and 3D analysis Confocal microscopy (Biorad, Radiance 2100) was used to visualize the fluorescent casting agent (Figure 8 and 9, left). A series of 51 stacked images (2 &m apart, total 100 &m) was taken and uploaded on to NeuroLucida Software to create a 3D tracing (Figure 8 middle, right). The tracing was analyzed using Neuroexplorer. These software programs were originally designed to analyze neural dendrites and had not previously been used by our lab 202, 203. In each 3D section, total length of vessels per tissue volume, length of vascular trees (continuity), branch order ratios, segment length, tortuosity, diameter and volume were recorded. Maximum length of a vascular tree was used to quantify branching between vessels (i.e. collateral growth), referred to as continuity. Tracings colour-coded by vascular tree are shown in Figure 8 (middle, right) and Figure 9 (middle). Branch order ratios were calculated to indicate vessel network patterning 151, comparing capillaries to arterioles (order 0 to order 1) according to Neuroexplorer s Microvascular ordering system (Figure 9, right). This is similar to Strahler s ordering scheme, with 0 at the most distal branches and increasing order number as branches converge 151, 204, 205 ; however, the Microvascular system considers branch diameter in addition to the number of converging branches. Non-ischemic limbs were analyzed to 42

51 determine the normal values for each measure. Ischemic limb results were averaged per group at 4 and 8 wks post-ligation. Figure 8: FMA imaging and analysis. Representative images from non-ischemic muscle (top) and ischemic muscle (bottom). (Left) Confocal microscopy was used to visualize vasculature. (Middle and Right) Neurolucida was used to create 3D reconstruction of vasculature. Each vascular tree is colour-coded. Figure 9: Branch order. (Left) Original confocal image from 20#m thick. (Middle) Tracing colour-coded by vascular tree. (Right) Tracing numbered and colour-coded by branch order. White arrow indicates direction of flow. 43

52 3.7 Vascular Permeability Evan s Blue Vascular permeability was measured to indicate vascular stability and maturity, which may contribute to functionality and flow reserve. Increased vascular permeability is characterized by an increase in transport of large macromolecules, such as albumin, across vascular endothelium. An Evan s Blue assay measures the amount of dye (bound to albumin protein) that leaks from the circulation to skeletal muscle tissue. The initial protocol was designed based on several studies 49, 80, 206, 207. An anaesthetized rat was injected with Evans Blue dye (20 mg/kg) dissolved in PBS and heparin intravenously. After 1 hr, tissue was pressure perfused with heparinized saline (100 ml, 140 mmhg), isolated, minced and heated in formamide (Sigma-Aldrich, 48 hrs, 65 C). Absorbance was measured at 620 nm (normalized to wet weight). Each ischemic limb was normalized to the contralateral normal limb. The protocol was first tested in Control and VEGF-treated tissue, the two groups that were expected to show the greatest difference in permeability based on previous research 20, 48, 49. Preliminary data was promising; however, further assays became inconsistent. Evan s Blue dye concentration was increased to 30 mg/kg. Other variables adjusted included light exposure, incubation time, temperature, volume, mechanical homogenizer mincing vs. cutting and accurate wet weight. The final protocol produced quantitative data that correlated to qualitative observations. 3.8 Supporting Cells Immunofluorescence Recruitment and proliferation of supporting cells is an important part of the maturation phase of angiogenesis. Hindlimb muscle was cryosectioned (20&m) and fixed in 4% paraformaldehyde. Endothelial cells were identified with a goat polyclonal antibody targeting von Willebrand Factor (Santa Cruz) 208 with the secondary antibody, donkey anti-goat Cy3 (red, AffiniPure). Von Willebrand Factor is synthesized by endothelial cells, localized to storage granules and secreted to promote platelet adhesion to sites of vascular injury. Supporting cells, including smooth muscle cells and pericytes, were examined by immunofluorescence. Smooth muscle cells were stained with mouse 44

53 monoclonal antibody targeting!-smooth muscle actin (!-SMA, Abbiotec). Pericytes were stained with a mouse monoclonal antibody targeting platelet-derived growth factor receptor beta (PDGFR-", Abcam) 209. The secondary antibody for both was goat antimouse Cy5 (green, Abcam). Nuclei were counter-stained with Topro-3 (blue). 3.9 Gene Expression Real Time PCR Primers specific to total (rat and human) VEGF and exogenous (human) Ang1, were designed to quantify mrna expression by real time PCR (RT-PCR). Exogenous VEGF was not measured separately from endogenous because much of the sequence is conserved between species. At 3 days post-delivery and 8 wks post-ligation, animals were sacrificed and adductor muscle tissue was isolated, snap frozen and stored at -80 C until RNA isolation. Hindlimb tissue was sonicated in Trizol (1 ml) using an ultrasonic homogenizer and RNA was extracted using an Aurum Total RNA mini kit (Bio-Rad). RNA (1&g) was reverse transcribed and the cdna was PCR amplified. Cyclophilin, a housekeeping gene, was quantified as an internal control for variations in amount of total cdna. To verify that all DNA was removed during RNA isolation, a non-reverse transcribed sample of RNA was run through real-time PCR. Exogenous Ang1 mrna levels in ischemic limbs of Ang1-treated animals were expressed relative to cyclophilin. Mean Ang1 expression at 3 days post-delivery was compared to 8 wks post-ligation. Total VEGF mrna levels in the ischemic limbs of all groups were quantified by the delta-delta method and expressed relative to cyclophilin and to the normal limbs. Mean VEGF levels in treated groups were compared to untreated Controls Statistical Analysis Data are expressed as mean ± standard error of the mean (SEM), unless otherwise indicated. Comparisons between treatment groups and time points were made with a twoway analysis of variance. When differences in the means were found, Bonferroni correction was performed. Differences were considered significant when P < Resting blood velocity was the only paired comparison. Sample sizes for each endpoint were chosen aiming for a statistical power ' 80%. A power analysis was done at the 45

54 beginning of the study with estimated values for mean and SD and repeated at study completion. The final analysis of the key endpoints is summarized in Table 3, which includes sample size, standard deviation (SD) and detectable difference at 80% power. Sample Size SD Detectable Difference with 80 % Power Resting flow Flow reserve FMA - density Table 3: Power analysis using minimum and maximum sample sizes for each of the primary endpoints: resting flow, flow reserve and FMA vessel density. These endpoints were all analyzed by 2-way ANOVA with Bonferroni correction (unpaired). 46

55 Chapter 4 Results 4.1 Objective I Microvascular Blood Flow Sustained increase in blood flow after VEGF + Ang1 late delivery Contrast-enhanced ultrasound data at 2, 4 and 8 wks post-ligation are expressed as a ratio of resting flow in the ischemic leg to that of the contra-lateral normal leg (Figure 10). Flow in the ischemic leg of the control group remained at approximately 55-60% of normal flow for all 8 wks. Delivery of VEGF at 2 wks post-ligation resulted in increased flow at 4 wks (0.58 ± 0.26 to 0.86 ± 0.23, P < 0.001), but flow regressed at 8 wks (0.67 ± 0.19, NS vs. 2 wks). Flow increased slightly after simultaneous VEGF/Ang1 delivery (0.69 ± 0.13 at 4 wks, NS vs. 2 wks, P < 0.05 vs. Control at 4 wks). In the VEGF with Ang1 late group, flow increased 2 wks after VEGF delivery (0.55 ± 0.18 to 0.87 ± 0.29, P < 0.001) and remained elevated after late Ang1 delivery (0.90 ± 0.21, P < vs. 2 wks and vs. all other groups at 8 wks). As predicted, temporally separated transfection of VEGF and Ang1 results in a sustained improvement in skeletal muscle blood flow. 47

56 A) B) Normalized Blood Flow wks 4 wks 8 wks *** *** *** N= Control VEGF alone VEGF/Ang1 early VEGF + Ang1 late # ### Figure 10: Resting blood flow. (A) Representative CEU images at pulsing intervals 1s, 7s and 20s (top to bottom) for four groups (left to right) at 8 wks post-ligation. (B) Resting blood flow in ischemic limb normalized to contra-lateral normal limb for four treatment groups at 2, 4 and 8 wks post-ligation. Data plotted as mean ± SEM. Sample size indicated at bottom of each column. *** P < vs. 2 wks and Control. # P < 0.05 vs. Control. ### P < vs. all other groups at 8 wks. 48

57 Data for blood volume and velocity used to calculate flow are presented in Figure 11. Blood volume increased after VEGF single delivery in the VEGF alone (0.94 ± 0.33 at 2 wks to 1.21 ± 0.33 at 4 wks, P < 0.01) and VEGF with Ang1 late groups (0.82 ± 0.20 at 2 wks to 1.30 ± 0.43 at 4 wks, P < 0.001). Blood velocity increased after Ang1 delivery in the VEGF with Ang1 late group (0.64 ± 0.22 at 2 wks to 0.82 ± 0.27 at 8 wks, P < 0.05). After co-delivery of VEGF and Ang1, the minor increases in volume and velocity were not significant. VEGF induces changes in blood volume while Ang1 induces changes in blood velocity. Normalized Blood Volume Normalized Blood Velocity wks 4 wks 8 wks ** N= Control VEGF alone V/A1 early V + A1 late N= Control VEGF alone V/A1 early V + A1 late *** *** * Figure 11: Resting blood volume (top) and velocity (bottom) in ischemic limb normalized to normal limb for all groups at 2, 4 and 8 wks post-ligation. Dotted line highlights normal volume. Mean ± SEM. Sample size indicated at bottom of columns. Volume not paired, velocity paired. * P < 0.05, ** P < 0.01 and *** P < vs. corresponding 2 wk time point. 49

58 4.1.2 Flow Reserve improved after co-delivery of VEGF and Ang1 Blood flow reserve was calculated in the ischemic hindlimbs from the ratio of exercise flow to resting flow measured by CEU (Figure 12). Mean flow reserve in normal limbs was 2.41 ± In the Control and VEGF alone groups, flow reserve remained reduced at all time points, near By comparison, flow reserve did improve in the ischemic legs after simultaneous co-delivery of VEGF/Ang1 (2.33 ± 0.70 at 4 wks, P < 0.01 vs. 2 wks; 2.42 ± 0.60 at 8 wks, P < vs. 2 wks) and after temporally separated delivery of VEGF and Ang1 (2.41 ± 0.52 at 8 wks, P < 0.01 vs. 2 wks). Whether separated or simultaneous, co-delivery of VEGF and Ang1 improves flow reserve. 50

59 A) B) Flow Reserve wks 4 wks 8 wks # ** *** # # ** Normal N= Control VEGF alone VEGF/Ang1 early VEGF + Ang1 late Figure 12: Blood flow reserve. (A) Representative CEU images during electrically stimulated contraction at pulsing intervals 1s, 7s and 20s (top to bottom) at 8 wks post-ligation. (B) Flow reserve in ischemic limb at 2, 4 and 8 wks post-ligation. Dotted line highlights normal flow reserve (2.41). Mean ± SEM. Sample size indicated at bottom of columns. Recall that in the VEGF + Ang1 late group, 4 wks is after VEGF delivery and before Ang1 delivery. ** P < 0.01 and *** P < vs. 2 wks. # P < 0.05 vs. Control and VEGF. 51

60 4.2 Objective II Hindlimb Function Exercise tolerance in ischemic rats was reduced compared to normal rats at 2 wks (P < 0.05), but there was no difference at 8 wks (Figure 13A). By measuring the walking time at each speed interval, walking distance was calculated but no significant difference was found (Figure 13B). Rat weight was plotted against walking time and no significant correlation was found (Figure 13C). Percent walking time was calculated but no significant trends were observed (Figure 13D). With no difference between normal and control rats at 8 wks, the test was not pursued in the treated groups. A) B) 20 Normal Control 250 Normal Control Walking time to fatigue (min) * Walking Distance (m) wks 8 wks Time after Ligation 20 C) D) 100 Walking Time (min) % Walking Time Normal Control 2 wks 8 wks Time after Ligation Body Weight (g) Normal at 2wks ( r 2 = ) Normal at 8wks (r 2 = ) Ischemic at 2wks (r2 = ) Ischemic at 8wks (r2 = ) 60 2 wks 8 wks Time after Ligation Figure 13: Exercise tolerance for normal (non-ischemic, black) and untreated control (green) rats at 2 and 8 wks. (A) Walking time, excluding dragging time. (B) Distance travelled, excluding distance spent dragging. (C) Rat weight vs. walking time. (D) Percent walking time. Mean ± SEM. * P <

61 4.3 Objective III Molecular and Cellular Mechanisms Sustained increase in vessel density after VEGF + Ang1 late Vascular density, analyzed by fluorescent microangiography (FMA), indicated changes in vessel length per volume of muscle tissue (Figure 14). Normal vessel density in rat hindlimb skeletal muscle was 1073 ± 186 mm/mm 3. After VEGF delivery alone, vessel density improved compared to Control (1307 ± 104 mm/mm 3 at 4 wks vs. 881 ± 122 mm/ mm 3 in Control, P < 0.05 vs. Control, NS vs. Normal); however, these vessels later regressed (741 ± 321 mm/mm 3 at 8 wks, NS vs. Control, P < 0.01 vs. Normal). After VEGF/Ang1 co-delivery, vessel density reached a value not significantly different from Normal, nor from Control (1004 ± 297 mm/mm 3 at 8 wks, NS vs. Normal or vs. Control). After VEGF with Ang1 late delivery, vessel density was elevated compared to Control (1132 ± 196 mm/mm 3 at 8 wks, P < 0.01 vs. Control, NS vs. Normal). Temporallyseparated delivery of VEGF and Ang1 induces a sustained increase in vessel density. 53

62 A) B) Vessel Density (mm vessel/mm 3 tissue) wks 8 wks * ** Normal N= Control VEGF alone V/A1 early V+A1 late Figure 14: Vascular density. (A) Representative FMA images at 4 and 8 wks post-ligation. (B) Ischemic limb vessel density represented by total vessel length (mm) per tissue volume (mm 3 ). Dotted line highlights normal vessel density. Mean ± SEM. Sample size at bottom of columns. Animals were not sacrificed at 4 wks in the V+A1 late group because this tissue would be identical to the VEGF alone animals at 4 wks. * P < 0.05, ** P < 0.01 vs. Control Normal vascular architecture after VEGF + Ang1 late therapy Arteriogenesis describes collateral vessel growth and development of capillaries into arterioles, both of which were quantified by further analysis of FMA images. Maximum length of a vascular tree indicated vessel continuity and collateral formation (Figure 15, left). Normal continuity in hindlimb skeletal muscle tissue was 21 ± 3.5 cm. After VEGF single delivery, continuity was elevated compared to Control (35 ± 2.0 cm vs. 20 ± 2.3 cm, P < 0.05) but was also significantly greater than Normal (P < 0.05). New collaterals regressed by 8 wks (16 ± 4.0 cm, NS vs. Control). No significant change in continuity 54

63 was detected after VEGF/Ang1 co-delivery. In the VEGF with Ang1 late group, continuity was elevated at 8 wks compared to Control and was not significantly higher than Normal (29 ± 2.6 cm, P < 0.01 vs. Control, NS vs. Normal). VEGF delivery with late Ang1 delivery induces collateral formation without surpassing normal continuity. Branch order ratios (order 0 : order 1) indicated whether the ideal capillary to arteriole ratio was conserved or altered (Figure 15, right). Normal capillary/arteriole ratio was 1.9 ± After VEGF delivery, the capillary/arteriole ratio increased compared to Control and compared to Normal (2.8 ± 0.46 at 4 wks, P < 0.01 vs. Control, P < vs. Normal). After simultaneous or temporally separated delivery of VEGF and Ang1, capillary/arteriole ratio was not significantly elevated compared to Control or Normal. VEGF-induced neovasculature is more capillary-rich than normal tissue. Ang1 delivered with or after VEGF, prevents changes in the ratio of capillaries to arterioles. VEGF with Ang1 late treatment induces arteriogenesis that conserves normal vascular architecture. Continuity (cm per vascular tree) wks 8 wks # * ** Capillaries/Arterioles (Branch order 0 : order 1) wks 8 wks ### ** 0 Control VEGF alone V/A1 early V+A1 late Control VEGF alone V/A1 early V+A1 late Figure 15: Vascular architecture. (Left) Continuity presented as maximum length of vascular tree in cm 3 of tissue. (Right) Capillary:arteriole ratio, represented by branch order ratio (order 0:1). Mean ± SEM. N values same as Figure 14. Animals were not sacrificed at 4 wks in the V+A1 late group because this tissue would be identical to the VEGF alone animals at 4 wks. * P < 0.05, ** P < 0.01 vs. Control. # P < 0.05, ### P < vs. Normal Vascular permeability not quantified accurately As shown in Figure 16, the VEGF-induced increase in permeability was not significant compared to Control tissue 3 days post-delivery so the assay was not continued in the other treatment groups. 55

64 Normalized Vascular Permeability wks + 3 days 4 wks 8 wks N= Control VEGF alone Figure 16: Evan s Blue assay for vascular permeability in ischemic limb normalized to non-ischemic limb for Control and VEGF alone groups 3 days post-delivery, and 4 and 8 wks post-ligation. Dotted line highlights normal permeability. Mean ± SEM. Sample size indicated at bottom of columns Mural cell coverage supports flow reserve and stability Qualitative observation of the immunofluorescent-stained skeletal muscle sections at 8 wks post-ligation revealed increased supporting cell coverage of microvessels in the Ang1-treated groups. Increased!-SMA-expressing smooth muscle cells in the VEGF/ Ang1 early and VEGF with Ang1 late groups indicated greater arteriole development compared to the Control and VEGF alone groups (Figure 17). PDGFR-" expressing cells also increased in the VEGF/Ang1 early and VEGF + Ang1 late groups compared to Control and VEGF groups, with the greatest pericyte coverage after temporally separated VEGF + Ang1 delivery (Figure 18). Co-localization of vwf and PDGFR-" was especially evident in the VEGF + Ang1 late group. Increased smooth muscle cell coverage corresponded to improved flow reserve and improved pericyte coverage corresponded to improved vessel stability. 56

65 vwf $-SMA Topro-3 Figure 17: Smooth muscle cell coverage in normal skeletal muscle and in skeletal muscle from 4 treatment groups at 8 wks post-ligation. Scale bar = 100#m. vwf endothelial cells (red), $-smooth muscle actin (green) and Topro-3 nuclei (blue). vwf PDGFR-" Topro-3 Figure 18: Pericyte coverage in normal skeletal muscle and in skeletal muscle from 4 treatment groups at 8 wks post-ligation. Scale bar = 20# m. vwf endothelial cells (red), PDGFR-" (green) and Topro-3 nuclei (blue). Yellow indicates colocalization of vwf and PDGFR-". 57