The role of actively released fibrin-conjugated VEGF for VEGF receptor 2 gene activation and the enhancement of angiogenesis

Abstract

A major challenge for therapeutic delivery of angiogenic agents such as vascular endothelial growth factor (VEGF) is to achieve sustained, low dose signaling leading to durable neovessel formation. To this end, we recently created a variant of VEGF 121, TG-VEGF 121 that directly binds to fibrin and gets released locally in proteolysis-triggered manner. Here we combined noninvasive biophotonic monitoring of VEGF receptor 2 gene activation in transgenic VEGFR2-luc mice and histomorphometry to compare endothelial activation and long-term neovascularization by actively released TG-VEGF 121 versus passively released, diffusible wild-type VEGF 121 in subcutaneous fibrin implants. Monitoring in real-time over 3 weeks of luciferase signal driven by the VEGFR2 promoter revealed endothelial activation in skin exposed to wild-type VEGF 121, but no detectable elevation over fibrin alone by TG-VEGF 121. Histology at 3 weeks, however, demonstrated that TG-VEGF 121 promoted vessel growth significantly more effectively and reliably than wild-type VEGF 121. The majority of vessels surviving to 3 weeks contained stabilizing smooth muscle cells. Yet, by 6 weeks, no extra vessels induced by exogenous VEGF were left. In conclusion, release of fibrin-conjugated variant TG-VEGF 121 elicited lower VEGFR2-luc activation than wild-type VEGF 121 yet significantly more vascularization. In the absence of true physiological demand, even stabilized vessels are ultimately regressed.