MOUSE MODEL OF ANGIOGENESIS

Neovascularization of ischemic muscle may be sufficient to preserve ti ssue integrity and/or function and may thus be considered to be therap eutic. The regulatory role of vascular endothelial growth factor (VEGF ) in therapeutic angiogenesis was suggested by experiments in which ex ogenously administered VEGF was shown to augment collateral blood flow in animals and patients with experimentally induced hindlimb or myoca rdial ischemia, To address the possible contribution of postnatal endo genous VEGF expression to collateral vessel development in ischemia ti ssues, we developed a mouse model of hindlimb ischemia, The femoral ar tery of one hindlimb was ligated and excised. Laser Doppler perfusion imaging (LDPI) was employed to document the consequent reduction in hi ndlimb blood flow, which typically persisted for up to 7 days. Serial in vivo examinations by LDPI disclosed that hindlimb blood flow was pr ogressively augmented over the course of 14 days, ultimately reaching a plateau between 21 and 28 days. Morphometric analysis of capillary d ensity performed at the same time points selected for in vivo analysis of blood flow by LDPI confirmed that the histological sequence of neo vascularization corresponded temporally to blood flow recovery detecte d in vivo. Endothelial cell proliferation was documented by immunostai ning for bromodeoxyuridine injected 24 hours before each of these time points, providing additional evidence that angiogenesis constitutes t he basis for improved collateral-dependent flow in this animal model. Neovascularization was shown to develop in association with augmented expression of VEGF mRNA and protein from skeletal myocytes as well as endothelial cells in the Ischemic hindlimb; that such reparative angio genesis is indeed dependent upon VEGF up-regulation was confirmed by i mpaired neovascularization after administration of a neutralizing VEGF antibody. Sequential characterization of the in vivo, histological, a nd molecular findings in this novel animal model thus document the rol e of VEGF as endogenous regulator of angiogenesis in the setting of ti ssue ischemia, Moreover, this murine model represents a potential mean s for studying the effects of gene targeting on nutrient angiogenesis in vivo.