Regulated Therapeutic Angiogenesis: for Ischemic Disease

描述（由申请人提供）：治疗性血管生成涉及将血管生成生长因子转移到缺血组织以促进新血管的生长。它对外周肢体缺血和心肌缺血的治疗都有良好的前景。目前的方案包括所有正在进行的临床试验，使用质粒或腺病毒载体与强不调节的病毒启动子来传递和表达血管内皮生长因子（VEGF）或成纤维细胞生长因子基因。由于载体的短暂寿命和对高水平短期表达的要求，需要使用强大的启动子。缺点包括缺乏血管生长的方向性线索，以及对局部剂量和暴露于生长因子的时间缺乏控制。最近的证据表明，这可能导致混乱不稳定的血管渗漏和容易形成血管瘤。我们推测刺激侧支血管生长的信号应该来自缺血组织，刺激的持续时间应该与缺血的持续时间有关。在成人病理相关的血管生成过程中，缺血区域为新血管的生长提供方向和时间线索。我们已经开发了一种新的基因开关，为将促血管生成生长因子基因传递到缺血肌肉提供了这样的线索。该开关包括由缺氧激活的启动子元件和在正常灌注条件下有条件地沉默基因表达的抑制元件。我们已经证实了这种开关在腺病毒载体转移到缺血骨骼和心脏组织后的功能。本文建议研究在半永久性AAV载体的开关引导下传递VEGF基因的pad和CAD模型中的血管生成特征。我们建议表征血管生成途径，并确定这些模型中侧支血管的特性、功能和稳定性。我们将在Matrigel塞和完整肌肉中分析侧支血管生长、稳定性和内皮祖细胞募集的空间线索。这些研究将为评估腺病毒与调节载体的疗效提供基础，并解决一些涉及治疗性血管生成的突出机制问题。

DESCRIPTION (provided by applicant): Therapeutic angiogenesis involves the transfer of angiogenic growth factors to ischemic tissues to promote the growth of new blood vessels. It shows promise for the treatment of both peripheral limb ischemia andl myocardial ischemia. Current protocols including all of the clinical trials in progress use plasmid or adenoviral vectors with strong unregulated viral promoters to deliver and express vascular endothelial growth factor: (VEGF) or fibroblast growth factor genes. Powerful promoters are used because of the transient lifetime of the: vectors and the requirement for high-level short-term expression. Disadvantages include an absence of directional cues for vessel growth and lack of control over the local dose and time of exposure to the growth factors. Recent evidence suggests that this may lead to disorganized unstable vessels that are leaky and prone to form hemangiomas. We hypothesize that the signals for stimulating the growth of collateral vessels should emanate from the ischemic tissue and the duration of the stimulus should be related to the persistence of ischemia. This is the case during pathology-related angiogenesis in the adult where ischemic regions provide the directional and temporal cues for the growth of new vessels. We have developed a novel gene switch that provides such cues for pro-angiogenic growth factor genes delivered to ischemic muscle. The switch includes promoter elements that are activated by hypoxia and represser elements that conditionally silence gene expression under conditions of normal perfusion. We have confirmed the function of this switch in adenoviral vectors after transfer to ischemic skeletal and cardiac tissues. Studies are proposed here to characterize angiogenesis in models of PAOD and CAD in which the VEGF gene is delivered under the direction of the switch in semi-permanent AAV vectors. We propose to characterize the angiogenic pathway and determine the properties, function, and stability of the collateral vessels in these models. The spatial cues for collateral vessel growth, stability, and recruitment of endothelial progenitor cells will be analyzed in Matrigel plugs and intact muscle. These studies will provide a basis for evaluating the efficacy of adenoviral versus regulated vectors and address some of the outstanding mechanistic questions involved in therapeutic angiogenesis.