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E. Deindl and C. Kupatt (eds.), Therapeutic Neovascularization — Quo Vadis?, 123-137. © 2007 Springer.

PHD, prolyl-hydroxylase domain-containing protein; ODD, oxygen-dependent degradation domain; Siah: seven in absentia homologue; ROS, reactive oxygen species; FIH, factor inhibiting HIF; VHL, von-Hippel Lindau protein; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein; NO, nitric oxide; HUVEC, human umbilical vein endothelial cells; BNIP3, Bcl-2/adenovirus E1B 19 kDa interacting protein; SDF-1, stromal-derived factor; MSC: mesenchymal stem cells

Angiogenesis, the growth of new blood vessels by branching from pre-existing vessels, is a complex, coordinated process involving activation, proliferation, migration and maturation of endothelial cells, as well as mural cells such as pericytes and smooth muscle cells. In the adult, angiogenesis is limited, occurring normally only as part of the female reproductive system. However the process can be initiated in other tissues as part of the physiological response to ischemia (which implies hypoxic stress) and also as an essential component of the response to tissue injury leading to wound repair. Angiogenesis is one component of the response leading to vessel growth in a low oxygen environment. Subsequent to vascular occlusion, the process of arteriogenesis-the remodeling of pre-existing vessels into larger arterioles and arteries - also contributes to the restoration of blood flow to areas of ischemic tissue. However, arteriogenesis is not dependent on ischemia but is instead stimulated by fluid shear stress and resulting endothelial activation [1].

Angiogenesis and arteriogenesis were, until recently, thought to be the primary mechanisms of new blood vessel growth in the adult. However, the discovery of endothelial progenitor cells (EPC's) derived from the bone marrow of adults [2] has led to the proposal that vasculogenesis, the de novo formation of new vessels via the recruitment and differentiation of bone marrow-derived endothelial progenitor cells (EPC) into mature endothelial cells, might also play a role in post-natal neovascularization. However, the exact nature of the contribution of EPC's to neovascularization remains undefined as the extent of incorporation of EPC's into new blood vessels as documented in the literature is highly variable (see [3] and references therein). For a review of EPC biology, the reader is referred to several recent publications [4, 5, 6].

While there are many factors involved in vascular growth, the fundamental roles of specific pro-angiogenic cytokines, such as the vascular endothelial growth factor (VEGF) proteins, are demonstrated by the observation that loss of a single VEGF allele leads to defective vascular development and early embryonic lethality [7, 8]. VEGF isoforms promote the proliferation and migration of endothelial cells and likely also smooth muscle cells, as sustained expression of VEGF is required for stabilization of nascent vessels [9]. The angiopoietins, Ang-1 and Ang-2, as well as platelet derived growth factor (PDGF), are also involved in the maturation of the newly formed vascular network via the recruitment of peri-endothelial cells such as pericytes and smooth muscle cells [10, 11]. Members of the fibroblast growth factor (FGF) family are associated with arteriogenesis (reviewed in [1]). Other likely mediators of vascular growth include placental growth factor (PlGF), hepatocyte growth factor (HGF), monocyte chemotactic protein-1 (MCP-1), granulocyte-monocyte colony- stimulating factor (GM-CSF), insulin-like growth factor (IGF) and transforming growth factor —p (TGFP) (reviewed in [12]).

Therapeutic angiogenesis, the induction of blood vessel growth via the delivery of angiogenic growth factor proteins or genes, is an attractive treatment strategy for ischemic diseases. Most of the earliest therapeutic angiogenesis studies employed recombinant human protein formulations of angiogenic growth factors, such as members of the VEGF-A and FGF families of proteins. However, despite a substantial and growing body of supportive in vitro and experimental animal research in the field of therapeutic angiogenesis, data from clinical trials using recombinant protein therapeutics consisting of single growth factors have been disappointing. Though there has been only modest evidence of efficacy, there has also been little evidence of any clinically significant toxicity to date.

Similarly modest evidence for clinical efficacy has been obtained when these growth factors (e.g. VEGF-A isoforms, such as VEGF-A165, and VEGF-A121, as well as FGF4) were administered as gene therapies. As recently reviewed by Yla-Herttuala and colleagues, there are a number of possible explanations for the lack of success with this approach [13]. These include the use of suboptimal delivery methods (e.g., an insufficiently low concentration and/or duration of expression of the targeted pro-angiogenic cytokine) and a failure to rigorously characterize both local and systemic biodistribution of vector and transgene. In addition, the widespread use of young, physiologically normal animals as proof-of-concept for efficacy in coronary artery or peripheral arterial disease models may not be appropriate for modeling the pro-angiogenic response in aged, hypercholesterolemic humans. This is an important issue as the kinetics and extent of transgene expression and biologic responsiveness in normal young animals differs from that in older humans who often have multiple co-morbidities, such as advanced atherosclerosis and diabetes.

An additional potential explanation for the lack of efficacy in many of the angio-genesis clinical trials to date is the use of a single angiogenic cytokine. It has been hypothesized that delivery of a combination of pro-angiogenic cytokines may act synergistically to not only initiate the process of endothelial cell growth and sprouting, but also to promote vessel wall maturation. In theory, a more robust and durable biological response should result from stimulating the recruitment of smooth muscle cell precursors and inflammatory cells to contribute to the process of vascular remodeling and enlargement [14]. This strategy has been tested in animal models using a combination of cytokines such as Ang-1 and a VEGF-A isoform [15, 16] with either PDGF-BB and FGF-2 [17], or PDGF-BB and VEGF-A [18]. Alternatively, genetically modified zinc-finger transcription factors may also be used to induce expression of multiple normal splice variants of the VEGF-A gene [19]. Another strategy is the induction of multiple pro-angiogenic signaling cascades through the administration of a single gene. One example of such a pleiotropic agent is hepatocyte growth factor (HGF). HGF promotes neovascularization directly through effects on endothelial cell proliferation, migration and survival as well as indirectly by up-regulating of the expression of a variety of pro-angiogenic factors (reviewed in [20]).

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