VEGF receptor-1-mediated protection in dilated cardiomyopathy

描述（由申请人提供）：在哺乳动物中，血管内皮生长因子（VEGF）家族由5个成员组成，分别是VEGF- a、- b、- c、- D和胎盘生长因子（PlGF），由各种细胞类型合成，包括心肌细胞。VEGF-B的研究相对较少，但它正在成为一个主要的促生存因子。与PlGF类似，它选择性地结合受体VEGFR-1。我们最近发现vegf -1在心肌细胞中表达，并首次提供了VEGF-B167基因转移减少心肌细胞凋亡和血管紧张素ii诱导的氧化应激，并显著减轻起搏诱导的扩张型心肌病狗的心脏重塑和功能紊乱的证据。另一方面，VEGF-A基因传递在体内并没有产生类似的有益效果。VEGF167的细胞保护/抗凋亡和最小血管生成作用使其成为非缺血性扩张型心肌病基因治疗的有吸引力的候选因子，非缺血性扩张型心肌病不是由冠状动脉疾病引起的，因此不会从血管生成中获益。本项目的总体目标是验证VEGFR-1是非缺血性扩张型心肌病心脏中vegf诱导的细胞保护的主要介质这一假设。将通过腺相关载体-9 （AAV9）在患有起搏诱导扩张型心肌病的慢性仪器狗中进行VEGFR-1配体的心脏基因转移。平行实验将在体外进行，以探索分子机制和优化转基因结构。目的1是确定VEGFR-1与VEGFR-2刺激在扩张型心肌病中不同作用的病理生理和细胞机制。我们将首先确定最具心脏保护作用的VEGFR-1配体，然后将其与VEGF-E（一种选择性VEGFR-2配体）和VEGF-A（一种双配体）进行比较。将在培养的心肌细胞中研究VEGFR-1和VEGFR-2介导的心肌保护的分子基础。目的2是确定在起搏诱导的扩张型心肌病过程中vegf - b诱导的心脏保护的最佳时机。在特异性靶1中鉴定的最具心脏保护作用的VEGFR-1配体将作为aav携带的转基因在起搏方案的不同时间点递送到心脏。目的3是测试内源性刺激诱导的VEGF-B转基因在衰竭心脏中的心脏保护作用。未来临床应用的一个理想策略是提供仅在与疾病相关的分子改变存在的情况下表达的治疗性转基因。为了实现这一目标，我们将开发以下创新策略来实现衰竭心脏内源性刺激诱导VEGF-B的转基因表达：a)构建包含对氧化应激敏感的调控元件的人工启动子；B)心房利钠肽天然启动子的利用；c)在转基因的3' UTR区包含在健康心脏中表达但在衰竭心脏中下调的microrna的靶序列。

DESCRIPTION (provided by applicant): In mammals, the vascular endothelial growth factor (VEGF) family consists of five members, VEGF-A, -B, -C, - D and placenta growth factor (PlGF), synthesized by various cell types, including cardiomyocytes. VEGF-B is relatively understudied and yet is emerging as a major pro-survival factor. Similar to PlGF, it binds selectively the receptor VEGFR-1. We have recently found that VEGRF-1 is expressed in cardiomyocytes and provided the first evidence that VEGF-B167 gene transfer reduces myocyte apoptosis and angiotensin II-induced oxidative stress and markedly attenuates cardiac remodeling and functional derangement in dogs with pacing- induced dilated cardiomyopathy. On the other hand, VEGF-A gene delivery did not yield similar beneficial effects, in vivo. The cytoprotective/antiapoptotic and minimally angiogenic effects of VEGF167 render this factor an appealing candidate for gene therapy of non-ischemic dilated cardiomyopathy, which is not caused by coronary artery disease, hence would not benefit from angiogenesis. The overall goal of the present project is to test the hypothesis that VEGFR-1 is the main mediator of VEGF-induced cytoprotection in hearts with non-ischemic dilated cardiomyopathy. Cardiac gene transfer of VEGFR-1 ligands will be performed via adeno-associated vector-9 (AAV9) in chronically instrumented dogs with pacing-induced dilated cardiomyopathy. Parallel experiments will be conducted in vitro to explore molecular mechanisms and to optimize transgene constructs. Aim 1 is to determine the pathophysiological and cellular mechanisms underlying the different effects of VEGFR-1 versus VEGFR-2 stimulation in dilated cardiomyopathy. We will first identify the most cardioprotective VEGFR-1 ligand and then compare it to VEGF-E, a selective VEGFR-2 ligand, and to VEGF-A, a dual ligand, in paced hearts. The molecular basis of VEGFR-1 and VEGFR-2- mediated myocardial protection will be studied in cultured cardiomyocytes. Aim 2 is to determine the optimal timing for VEGF-B-induced cardioprotection during the course of pacing-induced dilated cardiomyopathy. The most cardioprotective VEGFR-1 ligand identified in specific aim 1 will be delivered as an AAV-carried transgene to the heart at different time points during the pacing protocol. Aim 3 is to test the cardioprotective effects of VEGF-B transgenes inducible by endogenous stimuli specifically occurring in the failing heart. An ideal strategy for future clinical applications would be to deliver therapeutic transgenes expressed only in the presence of molecular alterations relevant to the disease. Toward this goal, we will develop the following innovative strategies to achieve VEGF-B transgene expression induced by endogenous stimuli occurring in the failing heart: a) construction of artificial promoters comprising regulatory elements sensitive to oxidative stress; b) utilization of the natural promoter of the atrial natriuretic peptide; c) inclusion, in the 3' UTR region of the transgene, of target sequences for microRNAs that are expressed in the healthy heart, but downregulated in the failing heart.