Ehrbar Martin, Djonov Valentin G, Schnell Christian, Tschanz Stefan A, Martiny-Baron Georg, Schenk Ursula, Wood Jeanette, Burri Peter H, Hubbell Jeffrey A, Zisch Andreas H
Institute for Biomedical Engineering, and University of Zurich, Zurich, Switzerland.
Circ Res. 2004 Apr 30;94(8):1124-32. doi: 10.1161/01.RES.0000126411.29641.08. Epub 2004 Mar 25.
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, alpha2PI1-8-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 alpha2PI1-8-VEGF121 is protected from clearance, contrary to native VEGF121 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 alpha2PI1-8-VEGF121 or native diffusible VEGF121. Our CAM measurements demonstrated that cell-demanded release of alpha2PI1-8-VEGF121 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 alpha2PI1-8-VEGF121 induces vessel formation more potently than native VEGF121 and that those vessels possess more normal morphologies at the light microscopic and ultrastructural level. Permeability studies in mice validated that vessels induced by alpha2PI1-8-VEGF121 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.
尽管血管内皮生长因子（VEGF）已被描述为一种强大的血管生成刺激因子，但其在治疗中的应用仍然困难：暴露于VEGF形成的血管往往畸形且有渗漏。在自然界中，VEGF的主要形式具有与细胞外基质（ECM）成分结合的位点，该位点将其保持在固定状态，直到通过局部细胞酶活性释放。在本研究中，我们展示了一种工程化的VEGF变体形式，α2PI1-8-VEGF121，它通过局部细胞相关酶活性模拟这种基质结合和细胞介导释放的概念，在手术相关的生物基质纤维蛋白中起作用。我们表明，与天然VEGF121混入纤维蛋白中不同，基质结合的α2PI1-8-VEGF121受到保护不被清除，天然VEGF121作为被动扩散爆发完全释放。对胚胎鸡绒毛尿囊膜（CAM）和成年小鼠进行了移植研究，以评估和比较用基质结合的α2PI1-8-VEGF1-8-VEGF121或天然可扩散的VEGF121配制的纤维蛋白植入物诱导的新生血管的数量和质量。我们对CAM的测量表明，α2PI1-8-VEGF121的细胞需求释放增加了新的动脉和静脉分支的形成，而暴露于被动释放的野生型VEGF121主要诱导毛细血管丛内的混乱变化。具体而言我们从内皮细胞形态、内皮细胞与周内皮细胞的相互作用、到血管分支和网络组织等多个层面的分析表明，α2PI1-8-VEGF121比天然VEGF121更有效地诱导血管形成，并且这些血管在光学显微镜和超微结构水平上具有更正常的形态。小鼠的通透性研究证实，由α2PI1-8-VEGF121诱导的血管不会渗漏。总之，从纤维蛋白植入物中细胞需求释放工程化的VEGF121可能是一种治疗上安全且实用的诱导局部血管生成的方式。
