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VEGF-A、-B、-C、- D和胎盘生长因子（PlGF）这五个成员组成，它们由包括心肌细胞在内的各种细胞类型合成。VEGF-B是相对研究不足，但正在成为一个主要的促生存因素。与PlGF类似，它选择性地结合受体VEGFR-1。我们最近发现VEGRF-1在心肌细胞中表达，并提供了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，并显着减弱起搏诱导的扩张型心肌病犬的心脏重塑和功能紊乱的第一个证据。另一方面，VEGF-A基因递送在体内没有产生类似的有益效果。VEGF 167的细胞保护/抗凋亡和最小的血管生成作用使该因子成为非缺血性扩张型心肌病基因治疗的有吸引力的候选者，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受益。本项目的总体目标是验证以下假设：VEGFR-1是VEGF诱导的非缺血性扩张型心肌病心脏细胞保护的主要介质。将在患有起搏诱导的扩张型心肌病的慢性器械犬中通过腺相关载体-9（AAV 9）进行VEGFR-1配体的心脏基因转移。将在体外进行平行实验以探索分子机制并优化转基因构建体。目的1是确定扩张型心肌病中VEGFR-1与VEGFR-2刺激不同作用的病理生理学和细胞机制。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（一种双重配体）在起搏心脏中进行比较。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。目的二是确定VEGF-B诱导扩张型心肌病起搏过程中心肌保护的最佳时机。在特定目标1中鉴定的最具心脏保护性的VEGFR-1配体将作为AAV携带的转基因在起搏方案期间的不同时间点递送至心脏。目的3是测试VEGF-B转基因诱导的内源性刺激，特别是发生在衰竭的心脏的心脏保护作用。未来临床应用的理想策略将是仅在与疾病相关的分子改变存在的情况下递送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以实现由衰竭心脏中发生的内源性刺激诱导的VEGF-B转基因表达：a）构建包含对氧化应激敏感的调节元件的人工启动子; B）利用心房利钠肽的天然启动子; c）在转基因的3' UTR区域中包含在健康心脏中表达但在衰竭心脏中下调的微小RNA的靶序列。 公共卫生相关性：本项目将探讨生长因子，即血管内皮生长因子（VEGF），在扩张型心肌病的病理条件下保护心脏细胞的作用。由于冠状动脉闭塞或功能障碍，这种心脏病的发生率低于其他心脏病，但它是非常恶性的，难以用可用的药理学药物治疗，并且在美国占心脏移植的50%以上。VEGF的管理可以是目前治疗扩张型心肌病的有效替代方案，但是，在实施这一新策略之前，有必要详细了解这些因素的功能和益处以及它们的有害影响。因此，我们将利用临床相关的扩张型心肌病犬模型，并将测试激活不同受体的VEGF家族的各种成员。我们的假设是，那些激活所谓的VEGF受体-1的VEGF成员更有可能显示出相关的有益作用，并被认为是扩张型心肌病的创新疗法。

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. PUBLIC HEALTH RELEVANCE: This project will explore the role of growth factors, named vascular endothelial growth factors (VEGF), in the protection of cardiac cells under a pathological condition known as dilated cardiomyopathy. This cardiac disease is less frequent that others due to coronary occlusion or dysfunction, however it is very malignant, difficult to treat with the available pharmacological agents, and is responsible for more than 50% of cardiac transplants in the US. The administration of VEGFs can be a valid alternative to the current therapies for dilated cardiomyopathy, but, before this new strategy can be implemented, it will be necessary to understand in detail the function and beneficial of these factors as well as their harmful effects. We will therefore utilize a clinically relevant dog model of dilated cardiomyopathy and will test various members of the VEGF family that activate different receptors. Our hypothesis is that those VEGF members that activate the so called VEGF receptor-1 are the ones more likely to display relevant beneficial effects and to be considered for an innovative therapy of dilated cardiomyopathy.