CARP: Angiogenic Gene Therapy in Diabetic Wounds

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DESCRIPTION (provided by applicant): Gene expression profiling of murine wounds revealed marked, sustained induction of cardiac ankyrin repeat protein (CARP, ankrdl), with diminished expression in diabetic mice. Overexpression of CARP in wounds by adenoviral gene transfer produces a remarkable increase in neovascularization and thereby enhances wound healing. Under the previous aims, we established these effects in several animal models and showed that CARP induction occurs in microvascular endothelial cells (MVEC), in part, through TGF-IS mediated activation of a p38 kinase pathway. CARP also protects endothelial cells from doxorubicin-mediated apoptosis, and it enhances MVEC migration, not proliferation. To avoid overlap, the proposed studies will concentrate on gain-of-function aspects of CARP. The proposed studies will extend the original aims by delineating the CARP activation pathway as well as identifying its binding partners and direct transcriptional targets. We will test the hypothesis that hyperglycemic, oxidative stress is related to CARP induction. Experiments will determine the influence of TNF-a and possible convergent signal mechanisms. Binding partners in vascular cells will be identified by yeast two- hybrid, protein microarray, and tandem affinity purification methods. EMSA and ChIP analysis will be used to identify the transcriptional activators in MVEC and smooth muscle cells (SMC). Expression profiling will be expanded to identify key targets in both MVEC and SMC. CARP has been suggested to be a mediator of mechanical stress in cardiac muscle. Experiments will test the significance of CARP'S putative nuclear and cytoplasmic interaction sites in SMC and MVEC by deletion and mutation of discrete, functional domains of the protein, read out as migratory and stress responses of targeted cells. Partners for these sites will be identified. The clinical relevance of CARP will be investigated in a series of rat healing studies in diabetic and ischemic wounds with a novel gene delivery system. These experiments will define the optimal dose for repair, and investigate the role of CARP in vessel regression and stabilization. If the benefits and mechanisms of this form of wound therapy are validated, they could lead to the development of CARP as a novel therapeutic and to the discovery of factors that mediate CARP-enhanced neovascularization.