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，在糖尿病小鼠中，心脏强直蛋白重复蛋白(CARP，ankrdl)显著持续诱导，表达减弱。通过腺病毒基因转移在伤口中过表达鲤鱼可显着增加新生血管，从而促进伤口愈合。在前面的目标下，我们在几个动物模型中建立了这些效应，并表明鲤鱼的诱导发生在微血管内皮细胞(MVEC)中，部分是通过转化生长因子-IS介导的p38激酶通路的激活。CARP还可保护内皮细胞免受阿霉素诱导的细胞凋亡，并促进MVEC迁移，而不是增殖。为了避免重叠，拟议的研究将集中在鲤鱼的功能获得方面。拟议的研究将通过描绘鲤鱼激活途径以及确定其结合伙伴和直接转录靶标来扩展最初的目标。我们将检验这一假设，即高血糖、氧化应激与鲤鱼诱导有关。实验将确定肿瘤坏死因子-α的影响和可能的汇聚信号机制。将通过酵母双杂交、蛋白质芯片和串联亲和纯化等方法鉴定血管细胞中的结合伙伴。EMSA和芯片分析将用于鉴定MVEC和SMC中的转录激活子。表达谱分析将扩大以确定MVEC和SMC中的关键靶点。鲤鱼被认为是心肌机械应激的中介体。实验将通过缺失和突变蛋白质的离散功能域来测试鲤鱼在SMC和MVEC中假定的核和细胞质相互作用部位的重要性，这些功能域被读出为目标细胞的迁移和应激反应。这些网站的合作伙伴将被确定。在一系列糖尿病和缺血创面的老鼠愈合研究中，将使用一种新的基因输送系统来研究鲤鱼的临床相关性。这些实验将确定修复的最佳剂量，并研究鲤鱼在血管退化和稳定中的作用。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.