Cell-Demanded Liberation of VEGF₁₂₁ from Fibrin Implants Induces Local and Controlled Blood Vessel Growth

Although vascular endothelial growth factor (VEGF) has been described as a potent angiogenic stimulus, its application in therapy remains difficult: Blood vessels formed by exposure to VEGF tend to be malformed and leaky. In nature, the principal form of VEGF possesses a binding site for ECM components that maintain it in the immobilized state until released by local cellular enzymatic activity. In this study, we present an engineered variant form of VEGF, α₂PI_1-8-VEGF₁₂₁, that mimics this concept of matrix-binding and cell-mediated release by local cell-associated enzymatic activity, working in the surgically-relevant biological matrix fibrin. We show that matrix-conjugated α₂PI_1-8-VEGF ₁₂₁ is protected from clearance, contrary to native VEGF ₁₂₁ mixed into fibrin, which was completely released as a passive diffusive burst. Grafting studies on the embryonic chicken chorioallantoic membrane (CAM) and in adult mice were performed to assess and compare the quantity and quality of neovasculature induced in response to fibrin implants formulated with matrix-bound α₂PI_1-8-VEGF ₁₂₁ or native diffusible VEGF₁₂₁. Our CAM measurements demonstrated that cell-demanded release of α2PI_1-8-VEGF ₁₂₁ increases the formation of new arterial and venous branches, whereas exposure to passively released wild-type VEGF₁₂₁ primarily induced chaotic changes within the capillary plexus. Specifically, our analyses at several levels, from endothelial cell morphology and endothelial interactions with periendothelial cells, to vessel branching and network organization, revealed that α₂PI_1-8-VEGF ₁₂₁ induces vessel formation more potently than native VEGF ₁₂₁ and that those vessels possess more normal morphologies at the light microscopic and ultrastructural level. Permeability studies in mice validated that vessels induced by α₂PI_1-8-VEGF ₁₂₁ do not leak. In conclusion, cell-demanded release of engineered VEGF₁₂₁ from fibrin implants may present a therapeutically safe and practical modality to induce local angiogenesis.