CARP: Angiogenic Gene Therapy in Diabetic Wounds

描述（由申请人提供）： 伤口愈合缺陷是糖尿病患者发病和死亡的主要原因。其中一个促成因素是组织损伤后循环不良和新毛细血管形成减少。虽然动脉功能不全可以通过手术纠正，但需要医学方法来刺激新血管的形成（血管生成）。因此，几种已知的血管生成生长因子如FGF、血管生成素-1和VEGF已经通过临床前甚至临床开发作为促进多种组织中新血管形成的试剂。在伤口愈合应用中仅取得了有限的临床前成功。作为参与伤口愈合的新基因的功能基因组搜索的一部分，发现心脏锚蛋白重复蛋白（CARP），一种迄今为止仅与心血管发育和心脏肥大相关的核因子，在早期伤口愈合中被急剧诱导，并且在肉芽组织形成的整个阶段保持高表达。为了确定该分子在伤口部位的作用和潜力，通过质粒cDNA的基因枪递送和腺病毒递送在多种伤口模型中表达该细胞内蛋白。新血管形成的急剧增加是立即明显的，表明CARP过表达诱导已知的血管生成因子的活性，或者它在刺激内皮生长、迁移和募集等过程的常见下游途径中起作用。此外，可能对支持和稳定新血管形成的血管平滑肌细胞或周细胞有影响。为了解决这些问题，本项目将寻求一系列相互关联的目标：（1）确定腺病毒CARP表达是否以及在多大剂量下导致正常和糖尿病模型系统中皮肤伤口愈合的改善;（2）了解CARP表达如何改变血管内皮细胞和血管平滑肌细胞的生理学;（3）鉴定伤口部位CARP的内源性来源，并测试CARP是否在生长因子信号传导的下游途径中;（四）检验CARP本身负责诱导血管生成因子的假设，以及它是否通过增强内皮祖细胞的募集而起作用。单元格;（5）利用微阵列分析来确定哪些基因在伤口和肉芽组织的间充质细胞中被CARP激活。这一系列的调查是重要的，因为它确定了一个新的战略，以改善伤口愈合的关键医疗领域。它是独一无二的，因为基因治疗方法允许发现一种新型的血管生成剂，具有揭示新的作用机制的潜力。这是具有挑战性的，因为需要了解很多关于CARP如何工作，以及它是否可以用作糖尿病患者慢性伤口修复的改进疗法的一部分。

DESCRIPTION (provided by applicant): Wound healing defects in the diabetic are a major cause of morbidity and mortality. One of the contributing factors is poor circulation and reduced formation of new capillaries after tissue injury. While arterial insufficiency may be correctable by surgery, medical approaches are required to stimulate formation of new blood vessels (angiogenesis). Thus, several of the known angiogenic growth factors such as FGF, angiopoietin-1, and VEGF have been taken through preclinical and even clinical development as agents to promote neovascularization in a variety of tissues. Only limited, preclinical success has been achieved in the wound healing applications. As part of a functional genomic search of new genes involved in wound healing, it was discovered that cardiac ankyrin repeat protein (CARP), a nuclear factor heretofore only associated with cardiovascular development and cardiac hypertrophy, was sharply induced in early wound healing, and high expression was maintained throughout the phase of granulation tissue formation. To determine the role and potential of this molecule at the wound site, this intracellular protein was expressed in a variety of wound models by both gene gun delivery of plasmid cDNA and by adenoviral delivery. Dramatic increases in neovascularization were immediately evident, suggesting that CARP overexpression either induces the activity of known angiogenic factors, or it acts in a common, downstream pathway that stimulates such processes as endothelial growth, migration, and recruitment. In addition, there may be effects on vascular smooth muscle cells or pericytes that support and stabilize neovascularization. To address these observations, this project will seek a series of interrelated objectives: (1) determine whether and at what dose adenoviral CARP expression leads to improved cutaneous wound healing in normal and diabetic model systems; (2) learn how CARP expression alters the physiology of the vascular endothelial cell and the vascular smooth muscle cell; (3) identify the endogenous sources of CARP at the wound site and test whether CARP is in a pathway downstream of growth factor signaling; (4) test the hypothesis that CARP itself is responsible for the induction of angiogenic factors and if it works by enhancing recruitment of endothelial progenitor cells; (5) utilize microarray analysis to determine which genes are activated by CARP in wounds and in the mesenchymal cells of granulation tissue. This line of investigation is significant because it identifies a novel strategy for improving wound healing in a critical medical area. It is unique since the gene therapy approach has permitted the discovery of a new type of angiogenic agent with the potential to reveal new mechanisms of action. It is challenging because much needs to be learned about how CARP works and whether it can be used as part of an improved therapy for chronic wound repair in the diabetic patient.