CARP: Angiogenic Gene Therapy in Diabetic Wounds

描述（由申请人提供）：鼠伤口的基因表达谱显示心脏锚蛋白重复蛋白（CARP，ankrdl）的显著、持续诱导，在糖尿病小鼠中表达减少。通过腺病毒基因转移在伤口中过表达CARP产生新血管形成的显著增加，从而增强伤口愈合。根据先前的目标，我们在几种动物模型中建立了这些效应，并表明CARP诱导发生在微血管内皮细胞（MVEC）中，部分是通过TGF-1 S介导的p38激酶通路的激活。CARP还保护内皮细胞免于阿霉素介导的凋亡，并且它增强MVEC迁移，而不是增殖。为了避免重叠，拟议的研究将集中在CARP的功能获得方面。这些研究将通过描绘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。我们将检验高血糖、氧化应激与CARP诱导相关的假设。实验将确定TNF-α的影响和可能的会聚信号机制。将通过酵母双杂交、蛋白质微阵列和串联亲和纯化方法鉴定血管细胞中的结合配偶体。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。将扩大表达谱分析，以确定MVEC和SMC中的关键靶点。CARP被认为是心肌中机械应力的介质。实验将通过蛋白质的离散功能结构域的缺失和突变来测试SMC和MVEC中CARP的推定的核和胞质相互作用位点的意义，读出为靶细胞的迁移和应激反应。将确定这些网站的合作伙伴。CARP的临床相关性将在一系列糖尿病和缺血性伤口的大鼠愈合研究中使用新型基因递送系统进行研究。这些实验将确定修复的最佳剂量，并研究CARP在血管消退和稳定中的作用。如果这种形式的伤口治疗的益处和机制得到验证，它们可能导致CARP作为一种新型治疗剂的发展，并发现介导CARP增强的新血管形成的因子。

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.