CARP: Angiogenic Gene Therapy in Diabetic Wounds

描述(由申请人提供)： 糖尿病患者的伤口愈合缺陷是导致发病率和死亡率的主要原因。其中一个因素是组织损伤后血液循环不良和新的毛细血管形成减少。虽然动脉供血不足可以通过手术纠正，但需要医学方法来刺激新血管的形成(血管生成)。因此，一些已知的血管生成生长因子，如成纤维细胞生长因子、血管生成素-1和血管内皮生长因子，已经在临床前甚至临床开发中被用作促进各种组织中新生血管的药物。只有有限的，临床前的成功已经在伤口愈合应用中取得。作为对创伤愈合相关新基因的功能基因组研究的一部分，人们发现，迄今为止仅与心血管发育和心肌肥大相关的核因子--心脏锚蛋白重复蛋白(CARP)在创伤愈合的早期被急剧诱导，并在肉芽组织形成的整个阶段保持高表达。为了确定这种分子在创伤部位的作用和潜力，通过基因枪法和腺病毒法在多种创伤模型中表达了这种细胞内蛋白。新生血管的显著增加立即明显，这表明鲤鱼的过度表达要么诱导已知的血管生成因子的活性，要么作用于共同的下游途径，刺激内皮生长、迁移和募集等过程。此外，还可能对支持和稳定新生血管的血管平滑肌细胞或周细胞产生影响。为了解决这些观察，这个项目将寻求一系列相互关联的目标：(1)在正常和糖尿病模型系统中，确定腺病毒鲤鱼的表达是否以及以何种剂量导致皮肤伤口愈合的改善；(2)了解鲤鱼的表达如何改变血管内皮细胞和血管平滑肌细胞的生理；(3)确定鲤鱼在伤口处的内源性来源，并测试鲤鱼是否处于生长因子信号的下游；(4)检验鲤鱼本身负责血管生成因子的诱导以及是否通过促进内皮祖细胞的募集而发挥作用的假设；(5)利用基因芯片分析，确定鲤鱼伤口和肉芽组织间充质细胞中哪些基因被激活。这项研究具有重要意义，因为它确定了一种改善关键医疗领域伤口愈合的新策略。这是独一无二的，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CARAP是如何工作的，以及它是否可以用于糖尿病患者慢性伤口修复的改进治疗。

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.