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

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 VEGF₁₂₁, TG-VEGF₁₂₁ 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-VEGF₁₂₁ versus passively released, diffusible wild-type VEGF₁₂₁ 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 VEGF₁₂₁, but no detectable elevation over fibrin alone by TG-VEGF₁₂₁. Histology at 3 weeks, however, demonstrated that TG-VEGF₁₂₁ promoted vessel growth significantly more effectively and reliably than wild-type VEGF₁₂₁. 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-VEGF₁₂₁ elicited lower VEGFR2-luc activation than wild-type VEGF₁₂₁ yet significantly more vascularization. In the absence of true physiological demand, even stabilized vessels are ultimately regressed.