P306	Fast versus slow release of exogenous VEGF: differential vessel formation and transcriptional regulation response.
1M.Ehrbar, 2G.Raeber, 2J.Hubbell, 4Chr.Schnell, 1A.Zisch
1Oral Biology, University, Zürich, CH; 2EPFL, Lausanne, CH; 4University Hospital, Zürich, CH.

Local, controlled induction of angiogenesis remains a challenge that limits tissue engineering approaches to replace or restore diseased tissues. Focusing on material development for clinics, we have established tissue engineering solutions for localized and prolonged delivery of recombinant VEGF protein. In nature, the principal form of this factor, VEGFG165, possesses a binding site for extracellular matrix components that may maintain it in the immobilized state until released by local cellular enzymatic activity. We created a mutant form of VEGF, a2PI1-8-VEGF121, that mimics this concept of matrix binding and cell-mediated release, working in the context of the surgically-relevant matrix fibrin. We demonstrate that this engineered VEGF formultion induces blood vessel formation more potently than conventional formulations of wild-type VEGF121 in fibrin, and that those vessels possess more normal morphologies at the microscopic and ultrastructural levels. Moreover, our measurements of levels of VEGF receptors 1 and 2 show that cellular proteolytic activity as a temporospatial release trigger for exogenously applied a2PI1-8-VEGF121 is compatible with cellular control of VEGF/VEGF receptor signaling. Further, we used non-invasive Xenogen biophotonic imaging method and a subcutaneous implant model in VEGFR2-luc-mice to examine over weeks the transcriptional regulation of angiogenesis in tissue exposed to a2PI1-8-VEGF121 versus wild-type VEGF121 release from fibrin implants. We show that both fast and slow release of VEGF from fibrin effectively promotes vessel growth. Fast release of VEGF was associated with hyperstimulation of VEGFR-2 transcription. In contrast, VEGFR-2 gene transcription appeared comparably unaffected in tissue exposed to slowly released a2PI1-8-VEGF121. In light of our studies of VEGF effects for vessel morphology and leakiness, rapid and strong upregulation of VEGFR-2 under conditions of fast VEGF release could be responsible for the aberrant, non-functional vessel growth. By contrast, cell-demanded release of a2PI1-8-VEGF121 has little effect on VEGFR-2 gene activation and produces more normal vessel structures.

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