This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. There has been no change in the scope of this project. This project will develop a novel application for a recent technique within gene therapy in the field of reconstructive surgery. We propose to use adeno-associated viral vectors designed to cause infected cells to elaborate potent blood supply-building proteins, namely VEGF, PDGF, and FGF2. This enhanced vascular network appears to rescue ischemic tissue from death, allowing """"""""flaps"""""""" (tissue transferred from one anatomic location to another for the purpose of closing a wound or reconstructing parts of the body) to be constructed of longer length, greater size, or greater reliability. Statistics compiled by the American Society of Plastic Surgeons (www.plasticsurgery.org) tracked over 5.2 million reconstructive surgeries in the US last year alone. In addition, this project is germane to the overall mission of bettering wound healing, and may be applicable to any situation of tissue ischemia. It builds upon earlier, published work of the applicant (P Liu), who, though currently Chairman of Surgery at Roger Williams Hospital, Providence, RI, has never been the recipient of competitive Federal funding except a T32 training grant. It is not mentored, but will rely on the critical input from collaborators at Brown University and Roger Williams skilled in those techniques new to the applicant. The specific hypothesis tested is: Engineering tissue with AAV-delivered angiogenic genes can improve survival of ischemic flaps derived from that tissue via recruitment of endothelial progenitor cells. In addition to testing the effects of each of the transgenes, our approach will take advantage of the greater efficiency of viral-mediated gene transfer to assess the combination of VEGF + FGF2, which, when delivered via liposome in plasmid form, was more effective than single gene therapy delivered the same way. We propose the following specific aims: 1). Maximize tissue survival in a flap model by optimizing the timing and dosing of angiogenic gene transfers using AAV vectors, and assess the effects of combining VEGF and FGF2 gene therapy. 2). Develop a mechanism of action to account for enhanced tissue survival. We expect the approach to be both efficacious and clinically relevant. Addressing Aim 2 will help answer a controversial issue in vascular biology, namely, where does the new blood supply in injury repair come from? We will utilize siRNA methods of gene silencing to help get at that answer, as well as localization technology (IVIS) and adoptive transfer of endothelial progenitor cell-enriched populations into the ischemic tissue. Lastly, a new portable spectroscopic device, the ViOptix probe, measuring spectral shifts in the near infrared spectrum of oxygenated hemoglobin as a function of perfusion, will help determine real time tissue viability.
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