Tracking Adult Neovascularization during Ischemia and Inflammation Using Vegfr2-LacZ Reporter Mice

Abstract

The vascular endothelial growth factor/vascular endothelial growth factor receptor 2 (VEGF/VEGFR-2) signal transduction system plays a key role during embryonic vascular development and adult neovascularization. In contrast to many endothelial genes, VEGFR-2 is expressed at low levels in most adult vessels but is strongly upregulated during neovascularization, leading to a pro-angiogenic response. Here, we analyzed the activity of regulatory sequences of the murine Vegfr2 gene during neovessel formation in vivo under ischemic and inflammatory conditions. Hindlimb ischemia was induced in transgenic mice, expressing the LacZ reporter gene under the control of Vegfr2 promoter/enhancer elements. Most vessels in the ischemic muscle tissue showed strong endothelium-specific reporter gene expression, whereas nearly no LacZ-expressing capillaries were observed in untreated control tissue. Cutaneous punch wounds were created to induce angiogenesis under inflammatory conditions, leading to robust LacZ expression in the majority of the blood vessels in the wound tissue. Since the cornea is physiologically avascular, the functionality of these promoter/enhancer elements exclusively in newly formed vessels was confirmed using the cornea micropocket assay. Taken together, our results show that these Vegfr2 regulatory elements are active during adult neovessel formation in general. Therefore, these sequences may prove to be valuable targets for novel endothelium-specific anti-angiogenic as well as pro-angiogenic treatment strategies. They may especially allow directing therapeutic gene expression to sites of adult neovascularization. Moreover, the Vegfr2/LacZ reporter mice represent a powerful model to generally analyze the transcriptional control mechanisms involved in the induction of Vegfr2 expression during adult neovascularization.