CARP: Angiogenic Gene Therapy in Diabetic Wounds

描述（由申请人提供）：小鼠伤口的基因表达谱显示，心脏锚蛋白重复蛋白（CARP, ankrdl）在糖尿病小鼠中的表达降低。通过腺病毒基因转移在伤口中过表达CARP可显著增加新生血管，从而促进伤口愈合。在之前的目标下，我们在几种动物模型中建立了这些效应，并表明微血管内皮细胞（MVEC）中发生了CARP诱导，部分是通过TGF-IS介导的p38激酶途径的激活。CARP还可以保护内皮细胞免受阿霉素介导的凋亡，并增强MVEC的迁移，而不是增殖。为了避免重叠，拟议的研究将集中于鲤鱼的功能获得方面。拟议的研究将通过描绘CARP激活途径以及确定其结合伙伴和直接转录靶点来扩展最初的目标。我们将验证高血糖、氧化应激与鲤鱼诱导有关的假设。实验将确定TNF-a的影响和可能的收敛信号机制。血管细胞中的结合伙伴将通过酵母双杂交、蛋白质微阵列和串联亲和纯化方法进行鉴定。EMSA和ChIP分析将用于鉴定MVEC和平滑肌细胞（SMC）中的转录激活因子。表达谱分析将扩展到MVEC和SMC的关键靶点。CARP已被认为是心肌机械应力的介质。实验将通过删除和突变该蛋白的离散功能结构域来测试CARP在SMC和MVEC中假定的核和细胞质相互作用位点的意义，这些功能结构域被解读为靶细胞的迁移和应激反应。将确定这些站点的合作伙伴。研究人员将利用一种新的基因传递系统，在一系列大鼠糖尿病和缺血性伤口愈合研究中探讨CARP的临床意义。这些实验将确定修复的最佳剂量，并研究CARP在血管恢复和稳定中的作用。如果这种形式的伤口治疗的益处和机制得到验证，它们可能会导致鲤鱼作为一种新的治疗方法的发展，并发现介导鲤鱼增强新生血管的因素。

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.