Arteriolar genesis and angiogenesis induced by endothelial nitric oxide synthase overexpression results in a mature vasculature

Background - Generation of physiologically active vascular beds by delivery of combinations of growth factors offers promise for vascular gene therapy. Methods and Results - In a mesenteric model of physiological angiogenesis, combining endothelial nitric oxide synthase ( eNOS) ( and hence NO production) with VEGF and angiopoietin-1 overexpression resulted in a more functional vascular phenotype than growth factor administration alone. eNOS gene delivery upregulated eNOS, VEGF, and Ang-1 to similar levels as gene transfer with VEGF or Ang-1. eNOS overexpression resulted in neovascularization to a similar extent as VEGF and Ang-1 combined, but not by sprouting angiogenesis. Whereas combining Ang-1 and VEGF increased both exchange vessels and conduit vessels, neither growth factor nor eNOS alone resulted in vessels with smooth muscle cell (SMC) coverage. In contrast, combining all three generated microvessels with SMCs (arteriolar genesis) and further increased functional vessels. Use of a vasodilator, prazosin, in combination with Ang1 and VEGF, but not alone, also generated SMC-positive vessels. Conclusion - Coexpression of eNOS, VEGF, and Ang-1 results in a more mature vascularization of connective tissue, and generates new arterioles as well as new capillaries, and provides a more physiological therapeutic approach than single growth factor administration, by combining hemodynamic forces with growth factors

Critical role of tissue kallikrein in vessel formation and maturation : Implications for therapeutic revascularization

OBJECTIVE : Human Tissue Kallikrein (hKLK1) overexpression promotes an enduring neovascularization of ischemic tissue, yet the cellular mechanisms of hKLK1-induced arteriogenesis remain unknown. Furthermore, no previous study has compared the angiogenic potency of hKLK1, with its loss of function polymorphic variant, rs5515 (R53H), which possesses reduced kinin-forming activity. METHODS AND RESULTS : Here, we demonstrate that tissue kallikrein knockout mice (KLK1) show impaired muscle neovascularization in response to hindlimb ischemia. Gene-transfer of wild-type Ad.hKLK1 but not Ad.R53H-hKLK1 was able to rescue this defect. Similarly, in the rat mesenteric assay, Ad.hKLK1 induced a mature neovasculature with increased vessel diameter through kinin-B2 receptor-mediated recruitment of pericytes and vascular smooth muscle cells, whereas Ad.R53H-hKLK1 was ineffective. Moreover, hKLK1 but not R53H-hKLK1 overexpression in the zebrafish induced endothelial precursor cell migration and vascular remodeling. Furthermore, Ad.hKLK1 activates metalloproteinase (MMP) activity in normoperfused muscle and fails to promote reparative neovascularization in ischemic MMP9 mice, whereas its proarteriogenic action was preserved in ApoE mice, an atherosclerotic model of impaired angiogenesis. CONCLUSIONS : These results demonstrate the fundamental role of endogenous Tissue Kallikrein in vascular repair and provide novel information on the cellular and molecular mechanisms responsible for the robust arterialization induced by hKLK1 overexpression. © 2009 American Heart Association, Inc

The maturation of vessels : a limitation to forced neovascularization?

Therapeutic angiogenesis is the induction of new blood vessels by the delivery of appropriate growth factors and is an attractive approach to the treatment of different ischemic conditions. The experience with initial clinical trials in the past decade has shown that this may be more complex than anticipated and highlights the need to incorporate current advancements in our understanding of the regulation of vessel growth in the design of novel strategies. The generation of new capillaries from neighboring microvasculature by angiogenesis can be represented as a two-step process: 1) tube formation, in which endothelial cells respond to gradients of angiogenic factors, proliferate and migrate towards areas where increased blood flow is needed, and 2) vascular maturation, in which pericytes are recruited to proliferating endothelium and induce quiescence and stabilization of the new capillaries through cell-cell contact and paracrine factors. The formation of a new vascular network with normal morphology and physiological function requires a proper balance between these two processes. Here we will review the current understanding of how the growth of normal or pathological blood vessels is determined by growth factor gradients in the microenvironment and what lessons can be learned to design more physiological strategies to achieve therapeutic angiogenesis for the treatment of ischemia. In particular, we will discuss the possibility to exploit vascular maturation as a target distinct from vessel induction, but capable of modulating the effects of angiogenic factors, and its implications for increasing safety and efficacy of therapeutic angiogenesis strategies

How Blood Vessel Networks Are Made and Measured

Tissue and organ viability depends on the proper systemic distribution of cells, nutrients, and oxygen through blood vessel networks. These networks arise in part via angiogenic sprouting. Vessel sprouting involves the precise coordination of several endothelial cell processes including cell-cell communication, cell migration, and proliferation. In this review, we discuss zebrafish and mammalian models of blood vessel sprouting and the quantification methods used to assess vessel sprouting and network formation in these models. We also review the mechanisms involved in angiogenic sprouting, and we propose that the process consists of distinct stages. Sprout initiation involves endothelial cell interactions with neighboring cells and the environment to establish a specialized tip cell responsible for leading the emerging sprout. Furthermore, local sprout guidance cues that spatially regulate this outward migration are discussed. We also examine subsequent events, such as sprout fusion and lumenization, that lead to maturation of a nascent sprout into a patent blood vessel