Michael Simons and 2Mark J Post

1The Angiogenesis Research Center, Section of Cardiology, Departments of Medicine and Pharmacology and Toxicology, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire 2Department of Physiology and Biomedical Technology, University of Maastricht, Maastricht, The Netherlands

Introduction

Disruption of blood flow to tissues leads to reduction in oxygenation and deterioration of function. In the myocardium this leads to reduction in myocardial contractility with subsequent development of heart failure. Similar functional consequences can be observed in other organs. Although the majority of tissues have the ability to partially compensate for the reduction in blood supply by stimulation of growth of new blood vessels, in most cases this response does not result in full compensation. Thus, in coronary arteries, chronic occlusions typically result in development of anginal symptoms and, frequently, in chronic myocardial stunning. To date, treatment of such an obstructive coronary disease relies either on the use of drugs designed to reduce, by various means, the oxygen requirement of the myocardium or on mechanical revascularization procedures including coronary bypass and various forms of percutaneous catheter-based approaches. Recent advances in our understanding of vessel growth have opened the possibility of therapeutic angiogenesis, that is, induction of neovascu-larization in the desired locations designed to restore blood flow to ischemic tissues. In this chapter we consider biological foundations of therapeutic angiogenesis and the current state of clinical research in this field.

Biology of Angiogenesis

The formation of mature vasculature in the course of embryonic development includes three distinct sequential steps—formation of primary capillary plexus from embryonic stem cells (vasculogenesis), sprouting of endothelium-lined vascular structures (angiogenesis), and remodeling of these structures into fully fledged vessels (arteriogenesis).

These series of events involve numerous growth factors and regulatory molecules that drive various parts of the pathway while preventing excessive vascular growth or formation of defective vasculature.

During vasculogenesis, primitive blood vessels originate from embryonic endothelial stem cells called angioblasts while the blood cells develop from hematopoietic cells. To date, little information is available with regard to the nature of these cells and the factors regulating their appearance and survival. They are thought to develop from a common blood cells/endothelial cell precursor referred to as heman-gioblast and express VEGF receptor flk-1 as well as fibroblast growth factor (FGF) receptors. The key growth factors involved in vasculogenesis include VEGF and TGF-P1. Disruptions of VEGF, flk-1, or TGF-P1 genes result in embryonic lethality with a failure of endothelial precursors to differentiate and to form the primary vascular plexus. In addition, FGF2, IGF-1, and GM-CSF have the ability to stimulate differentiation and mobilization of angioblasts from the bone marrow. Whereas vasculogenesis clearly plays a key role during embryogenesis, the occurrence of this process in adult tissues is fairly controversial. There appears to be a bone-marrow-derived population of endothelial precursor cells (EPCs) that under some circumstances can contribute to neovascularization.

Once formed, the primary plexus is then transformed by processes of branching angiogenesis into a primitive vascular system. The deletion of a single VEGF allele leads to a profound failure of branching, suggesting that this process is heavily VEGF-dependent. In addition, VEGF receptor flt-1 and the angiopoietin-1/tie-2 system are also critical for this series of events. At the same time, the extent of sprouting, that is, the total number of vascular branches and generations of branches, is likely regulated by FGFs. This is suggested by a marked reduction in branching of the trachea and bronchial tree in Drosophila following disruption of its single FGF gene and by increased arterial branching in mice overexpressing FGF1.

The differentiation of the primary vascular network into adult vasculature involves the envelopment of these structures by vascular smooth muscle and other mural cells, including pericytes, forming, in larger vessels, tunica media and tunica adventitia, a process in which platelet-derived growth factor (PDGF)-BB, TGF-b, Ang-1, and Ang-2 appear to play a role. The differentiation of the forming vas-culature into arterial and venous systems and the formation of connections between the two involves the ephrin family of proteins, whereas development of the lymphatic system may depend on a specific subset of VEGFs.

Whereas the process of vascular growth in the embryo is fairly well understood, the process of neovascularization in the adult is much less clear. There appear to be at least two and possibly three distinct processes—capillary growth in ischemic tissues (angiogenesis); growth and remodeling of preexisting vessels into arterioles and arteries; or de novo formation of such vessels (arteriogenesis) and formation of vessels, capillaries, or larger vascular structures from circulating EPCs (vasculogenesis). Although it is superficially similar to embryonic development, neovascularization in mature tissues is not well understood. Furthermore, the relative physiological importance of various forms of arterialization under various pathological conditions is not established.

The primary stimulus driving angiogenesis is tissue ischemia, which results in activation of hypoxia-inducible factor (HIF)-la that in turn stimulates expression of VEGF, VEGF receptor flt-1, PDGF-BB, Ang-2, iNOS, and a number of other genes. Although it is certainly effective in increasing capillary numbers in ischemic tissues, the contribution of this process to the restoration of perfusion is not clear.

In contrast to angiogenesis taking place in the ischemic myocardium (in the case of myocardial ischemia), the formation of epicardial collaterals by the process of arteriogen-esis occurs in nonischemic areas. Furthermore, this process has the potential to fully restore the blood flow to the distal coronary bed, resulting in an effective "biological bypass." Therefore, arteriogenesis, and not angiogenesis, is the most appealing target for a therapeutic intervention. Remarkably, there is very little understanding of the arteriogenesis process itself or of the factors that regulate it. For example, it is not clear whether arteriogenesis proceeds by the enlargement of preexisting vessels that remodel into arteri-oles large enough to carry significant volume of blood flow or by the de novo growth of these vessels. This is a fundamental distinction since agents that might affect vascular remodeling are likely to be very different from agents that primarily stimulate endothelial cell proliferation and migration, although the latter is definitely required for remodeling as well. The distinction will also have consequences for the preferred site and route of administration of any such agent. Furthermore, we have only a vague notion of the stimuli that initiate arteriogenesis and of the growth factors involved.

Accumulation of blood-derived monocytes/macrophages appears to be critical to this process, as these cells secrete a number of growth factors and cytokines involved in endothelial, pericyte, and smooth muscle cell growth and differentiation, including VEGF, FGF2, TGF-b, Il-8, and MCP-1. Animal studies have demonstrated that activation of monocyte/macrophage accumulation at the arterial occlusion sites when stimulated by MCP-1 leads to a robust arteriogenic response that in turn can lead to significant restoration of tissue perfusion. At the same time, deficiency of the VEGF-related growth factor PLGF prevents collateral growth by impairing monocyte recruitment. Furthermore, monocyte ability to respond to hypoxic stress by increasing their HIF-1a protein levels appears to correlate with the extent of coronary collateral development in patients with advanced coronary artery disease (CAD). The factors responsible for monocyte recruitment to sites of arterial narrowing have not been defined, although shear stress has been suggested as a potential contributor.

Angiogenic Growth Factors

A large number of genes can influence a process as complex as angiogenesis. At the same time, a smaller number of proteins have the ability to directly stimulate endothelial cell proliferation. Traditionally, such proteins have been referred to as angiogenic growth factors. Among these, VEGFs, PDGFs, and FGFs have received the greatest attention and are discussed briefly here.

The VEGF family includes five VEGFs (A-E) and a closely related protein PlGF (placental growth factor). VEGF-A (commonly referred to as simply VEGF) was first isolated as a vascular permeability factor (VPF) and was subsequently shown to have endothelial cell growth stimulatory properties. Four different isoforms are generated by alternative splicing from a single VEGF-A gene: VEGF206, VEGF189, VEGF165, and VEGF121. Other rare splice variants such as VEGF145 probably also exist. VEGF189 and VEGF205 isoforms possess high avidity for heparan sulfates and as a result are tightly bound to the cell surface. This property likely severely limits their therapeutic utility. VEGF165 and VEGF145 also demonstrate heparan sulfate binding (significantly less than the other two), whereas VEGF121 completely lacks the heparan sulfate binding site.

Angiogenic activity of VEGF in large measure is dependent on release of nitric oxide (NO) as blockade of NO generation markedly reduces VEGF activity. Whereas VEGF plays a crucial role in embryonic vascular development, its role in adult tissues is less clear, as deactivation of the VEGF-A gene in mature animals does not lead to significant vascular defects. This may not be the case, however, for newly forming vasculature in adult tissues where the loss of VEGF leads to a rapid involution of newly formed vasculature.

Other VEGF genes include VEGF-C (also known as VEGF-2), VEGF-B (also known as VEGF-3), VEGF-D, and VEGF-E. VEGF-C is predominantly involved in lymphan-giogenesis, whereas VEGF-B may play a role in development of coronary capillaries. The functions of VEGF-D and E isoforms have not been fully established.

VEGF levels are very low in normal tissues and are exquisitely sensitive to hypoxia and ischemia with even a transient episode resulting in rapid and substantial increases in expression. This sensitivity is driven by the hypoxia-inducible factor (HIF)-la, a transcription factor with very short half-life in normal cells that gets rapidly and profoundly extended by the onset of cellular hypoxia. In addition, VEGF expression in the myocardium is regulated by tissue stretch, with higher intraventricular pressures inducing significant increases in VEGF expression in a TNFa-dependent manner.

Although all VEGFs are able to stimulate endothelial cell proliferation in vitro and in vivo, they are, by themselves, very weak mitogens. This suggests that biological effects of these molecules may have a lot more to do with activities other than direct stimulation of endothelial cell growth. One such activity is the ability to induce a local inflammatory response by increasing vascular permeability that leads to chemoattraction and accumulation of blood-derived mono-cytes. Other biological activities include stimulation of local production of nitric oxide, enhancement of monocyte/leuko-cyte adhesion to the endothelium, activation of tissue digesting enzymes such as matrix metalloproteinases (MMPs), and stimulation of expression of growth factors such as FGF2. Finally, VEGF may also play an important role in stimulation of bone marrow release of endothelial precursor cells.

platelet-derived growth factors constitute a four-gene family. Whereas PDGF A and B have been extensively studied, comparatively little is known about PDGF C and D. The A and B chains can form homodimers (AA or BB) or a heterodimer (AB), which have distinctly different properties and biological activities. PDGFs act by binding to high-affinity tyrosine kinase receptors (PDGF-R) that are composed of two chains, PDGF-Ra and PDGF-Rb, which can also form either homodimers (aa or bb) or a heterodimer (ab). PDGF-AA binds only to PDGF-Raa and PDGF-BB binds to all three PDGF receptors, whereas PDGF-AB is limited to aa or ab. Homozygous disruptions of either PDGF-B or PDGF-b receptor have shown that PDGF-B is responsible for vascular maturation. Respective knockout mice die late in gestation with abnormal kidney glomeruli and vascular wall abnormalities that are particularly prominent in the brain and the heart and are related to insufficient recruitment and organization of pericytes and smooth muscle cells. The PDGF-A and PDGF-a knockouts show less overlapping phenotypes with less specific vascular pathology.

PDGF-BB appears to be a potent angiogenic growth factor with the ability to induce formation of both capillaries and larger vessels. However, in addition to its angiogenic activity, PDGF-BB has also been implicated in the formation of intimal hyperplasia following arterial injury and in progression of atherosclerosis. These aspects of PDGF-BB's activity profile raise concerns that therapeutic benefits of application of this growth factor in the setting of atherosclerotic cardiovascular disease might be offset by stimulation of restenosis and atherosclerosis.

Fibroblast growth factors are a 23-member family of closely related proteins. One of the key differences between various FGFs is the presence or absence of the leader sequence required for conventional peptide secretion (absent in FGF 1 and 2 but present in FGF 4 and 5 and most other FGFs) and the difference in affinity for various iso-forms of FGF receptors. All FGFs demonstrate high affinity for heparan sulfates recognizing specific sequences in gly-cosaminoglycan chains. For most FGFs, their ability to bind cell surface and matrix heparan sulfates serves both to prolong effective tissue half-life and to facilitate binding to corresponding high-affinity receptors. A pattern of specific FGF-FGF receptor and FGF-heparan sulfate interactions probably accounts for differences in activity of various FGFs.

In cell culture as well as in vivo studies, FGF-1,2,4 and 5 are potent mitogens for cells of mesenchymal, neural, and epithelial origin, including all cell types found in the vascular wall (endothelial cells, smooth muscle cells, and peri-cytes). FGF2, and probably other FGFs, also is able to activate nitric oxide release, to induce synthesis of plas-minogen activator and matrix metalloproteinases, and to stimulate smooth muscle cell and monocyte chemotaxis. One interesting aspect of FGF2 biology is the synergy of its biological activity with VEGF. A combination of FGF2 and VEGF is far more potent in inducing angiogenesis in vitro and in vivo than either growth factor alone. Furthermore, FGF2 induces VEGF expression in smooth muscle and endothelial cells.

Both FGF1 and FGF2 are present in the normal myocardium (as well as other tissues) where their expression is not significantly affected by hypoxia, tissue ischemia, or hemodynamic stress. However, despite significant levels of FGFs in normal tissues, the growth factors do not appear to be biologically active. In part this lack of activity may be due to their sequestration in the extracellular matrix by virtue of binding to the heparan sulfate-carrying proteoglycan perlecan that would make them unavailable to cell surface receptors. In addition, normal tissues demonstrate low levels of expression of FGF receptors FGF-R1, R2, and syndecan-4. Thus, unlike VEGF, where biological activity appears to be driven by the amount of the ligand present, FGF activity is likely controlled by the level of expression of FGF receptors and their ability to bind the ligand.

Among other angiogenic growth factors, angiopoietins and hepatocyte growth factor (HGF) may also play particularly important biologic and therapeutic roles. The angiopoietin family consists of four members. Overexpression of Ang-1 in the skin leads to a striking increase in vascularization that is characterized by a pronounced increase in the vessel size with only a modest increase in the vessel number. A combined VEGF/Ang-1 overexpression leads to a very pronounced increase in vascularity that, in contrast to overexpression of VEGF alone, does not exhibit increased vascular permeability. These findings suggest that Ang-1 plays a role in stabilizing the existing vessel in a yet undefined manner.

The role played by Ang-2 in angiogenesis in adult tissues is not clear. Whereas its overexpression during embryonic development leads to early mortality with morphological defects resembling those of Ang-1 and Tie-2 knockouts, induction of ischemia in adult hearts led to a rapid initial rise in Ang-2 expression that equally rapidly declines while Ang-1 expression shows a gradual increase. These observations suggest that Ang-2 might provide a destabilizing signal necessary for the initiation of angiogenic response.

HGF, also known as the scatter factor, is a powerful endothelial cell mitogen. HGF and its receptor c-met are essential for the normal embryonic development, but are not primarily involved in the development of cardiovascular system. However, the HGF/c-met expression is markedly upregulated in ischemic hearts, and the lack of this increase in expression correlates with poor native collateral development. HGF has a robust angiogenic effect in vivo, which may at least in part be mediated by stimulation of VEGF expression. For use as a therapeutic agent, the definite pro-oncogenic properties of HGF and c-met are a major concern. Transgenic overexpression of HGF leads to a variety of tumors of mesenchymal and epithelial origin, and mutations in the tyrosine kinase domain of MET that lead to its constitutive activation produce papillary renal carcinomas.

Therapeutic Applications of Angiogenesis Concepts

The concept of using angiogenic growth factors as a treatment for occlusive coronary artery and peripheral vascular disease has been extensively tested in both animal and clinical studies. Although in principle the concept appears to be correct, a number of significant challenges prevented an easy transition from successful animal to human usage. In chronic-ischemia large-animal models of myocardial angio-genesis, single-bolus intracoronary, periadventitial, or intrapericardial administration of several growth factors including FGF1, FGF2, VEGF165, and PDGF-BB enhanced neovascularization and restored blood flow in the ischemic territory to essentially normal levels. Likewise, gene therapy approaches have been used with equal success including intracoronary injections of FGF-4, FGF-5, VEGF121, and VEGF165 adenoviruses and VEGF165 plasmid.

Similar success has been seen in studies that utilized a hind limb ischemia model. In these studies, an occlusion of the femoral artery is typically associated with a severe reduction in blood flow to the ipsilateral limb. Treatment with angiogenic growth factors accelerates recovery and, in some cases, preserves tissue loss in the foot and the distal ankle.

At the same time, clinical trials have been less successful in demonstrating benefits of therapeutic angio-genesis. Whereas the initial series of open label studies testing VEGF165, FGF1, and FGF2 all demonstrated significant improvement in every parameter measured, including measures of myocardial perfusion and ventricular function, larger randomized double-blind trials have shown similarly significant improvements in both placebo and treatment groups. These results suggest the presence of a large placebo effect as well as a potential for a significant improvement in the control group.

The efficacy of VEGF165 treatment was tested in the VIVA trial in which the protein was given by a combination of an intracoronary and intravenous infusion. The trial enrolled 178 "no-option" patients with stable exertional angina, who were randomized to receive placebo, low-dose recombinant human VEGF (17ng/kg/min), or high-dose rhVEGF (50 ng/kg/min) by intracoronary infusion on day 0, followed by intravenous infusions on days 3, 6, and 9. Assessment of exercise capacity, angina class, quality of life, and myocardial perfusion were performed 60 and 120 days later. The primary end point, change in exercise treadmill test (ETT) time from baseline to day 60, showed equally significant improvements in all three. Angina class and quality of life were also significantly improved within each group, with no difference among groups.

The therapeutic efficacy of FGF2 was studied in several trials. A single bolus protein intracoronary administration of FGF2 protein was tested in the double-blind, randomized, placebo-controlled trial (FIRST). The 337 patients were randomized to an intracoronary injection of rFGF2 (0.3, 3, or 30 mg/kg) or placebo. Efficacy was evaluated at 90 and 180 days by exercise tolerance test, SPECT imaging, and quality-of-life questionnaires. Similar to the VIVA finding, exercise tolerance was increased at 90 days in all groups but was not significantly different between the placebo and FGF2-treated groups. rFGF2 treatment reduced angina symptoms with the differences more pronounced in highly symptomatic patients. However, none of the differences were significant at 180 days because of continued improvement in the placebo group.

The concept that the prolonged presence of FGF2 in tissues would be superior to a single bolus therapy was tested in a small trial of a heparin alginate sustained-release FGF2 formulation implanted into the unrevascularizable but viable myocardium at the time of coronary artery bypass grafting. Twenty-four patients were randomized to implantations of 10 heparin-alginate capsules containing 0, 10, or 100 |mg of FGF2. At 3-month follow-up, there was a significant reduction in the occurrence of angina in the high-dose rFGF2-treated patients compared to other groups. Remarkably, this benefit was preserved after 3 years of follow-up.

An alternative to achieving sustained local levels of a growth factor involves a gene transfer. This approach was used in the AGENT trial that examined the therapeutic efficacy of adenoviral FGF-4 therapy. Seventy-nine patients were randomized to a single IC infusion of placebo (n = 19) or Ad5-FGF4 (n = 60). Overall, patients receiving Ad5-FGF4 tended to have greater improvements in exercise time at 4 weeks. Improvement was the greatest in patients with more severe baseline impairment in exercise capacity. Interestingly, patients with low baseline anti-adenoviral antibody titers showed a significantly better response than patients with high levels of antibodies. The safety and feasibility of catheter-based local intracoronary VEGF165 gene transfer in the prevention of in-stent restenosis and in the treatment of chronic myocardial ischemia were recently also studied. Although this procedure had no effect on the extent of restenosis, adenoviral but not plasmid-based gene transfer resulted in increased myocardial perfusion as determined by SPECT imaging 6 months later, after percutaneous interventions. The significance of this finding is not clear, as the patients were not randomized to assess this specific end point. Furthermore, there was no difference in nitrates requirement and exercise time in all study groups. Two other gene transfer trials, one involving adenoviral VEGF121 (REVASC trial) and one plasmid-based VEGF165 (Euroin-ject One trial), reported essentially negative results. Yet another trial examined the utility of plasmid-based gene transfer by direct intramyocardial injections of VEGF2 plas-mid in 27 inoperable patients with class III or IV angina. Twelve weeks after injection, there was a significant improvement in the angina class in the VEGF2 versus placebo-treated patients. However, the small size of the trial with a particularly small control group and the absence of the usual placebo effect (likely another reflection of the small trial size) make efficacy conclusions tenuous.

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