Mechanisms of Collateral Development and Collateral Growth in Ischemia

描述（由申请人提供）：心脏、大脑和周围肢体的闭塞性血管疾病是美国发病率和死亡率的主要原因。血管生成和侧支血管的生长（重塑）是限制终末器官损伤的主要适应。然而，与血管生成相比，我们对侧枝生长的机制知之甚少。没有研究确定COLs是如何或何时发展的。侧枝密度和重塑在不同物种和人类之间差异很大，这表明了遗传基础。然而，人们对这种变化的来源一无所知。我们的初步研究表明，COLs在胚胎晚期到出生后早期发育。我们还发现，与C57BL6 （B6）小鼠相比，BALB/c （BC）小鼠的COL形成受损——在BC基本缺乏COL的脑循环中最为明显。这些发现为确定COL形成的具体因素创造了极好的机会。虽然可能涉及多个基因，但我们的初步工作表明，BC缺乏VEGF-A，并且在其位点处/附近具有多态性。这一点，加上来自过表达VEGF的突变小鼠的数据，提出了VEGF信号在COL形成中至关重要的新假设。这是第一个被确定的调节正常组织COL密度的因素。许多被认为在成人缺血性疾病中调节COL生长的信号在血管生成中是重要的，特别是VEGF。然而，VEGF的作用是有争议的，因为在以往的研究中很难定位VEGF的操作。此外，还没有研究确定VEGF在COL生长中的来源。为了解决这些问题，我们开发了新的方法，允许1)无创的COL灌注成像和2)在COL形成部位局部操纵VEGF信号。我们在小鼠后肢模型中的初步结果表明，VEGF是缺血中COL重塑的关键决定因素。本研究将探讨COLs在围产期形成的总体假设，VEGF信号传导是这一过程的核心，VEGF也是成人缺血性疾病中COLs扩大的关键决定因素。目的：我将决定如何以及何时开发抵押品。我们对这个基本过程一无所知。我们还将检验VEGF在指定侧支形成中起关键作用的假设。我们将描述围产期COL的发展特征，并验证VEGF信号是关键决定因素的假设，并确定VEGF- r1和-R2的参与。Aim II将使用阵列和基因组定位来鉴定多态性和vegf依赖性和vegf非依赖性候选基因指导COL的形成。Aim III将使用小鼠后肢模型验证VEGF在成人缺血性疾病侧支重塑中起重要作用的假设，并将寻求确定相关机制。我们将使用VEGF突变体，改变VEGF及其受体，并确定COL生长过程中VEGF的来源。我们希望我们的工作将导致治疗方法刺激形成新的COLs和增加原有COLs的生长闭塞性血管疾病。公共卫生相关性：心脏、大脑和周围肢体的缺血性疾病是西方国家发病率和死亡的最常见原因，在世界范围内正在增加，许多受影响的个体不适合经皮或旁路血运重建术，由于多血管疾病、弥漫性疾病或不利的先决条件，被认为“没有选择”（或面部截肢）。增强血管生成（毛细血管密度增加）和动脉生成（络扩大）的疗法在治疗缺血性疾病方面具有巨大的潜力。虽然几项“第二代”血管生成试验正在进行中，但针对动脉生成的治疗方法的发展，涉及到不同的机制，人们对这些机制知之甚少，如果没有更好地理解正常组织中侧支形成的遗传和生理机制，以及在缺血性疾病中调节其扩大的机制，就无法实现。

DESCRIPTION (provided by applicant): Occlusive vascular disease of the heart, brain and peripheral limbs is the primary cause of morbidity and mortality in the US. Angiogenesis and growth (remodeling) of collateral vessels are major adaptations that limit end-organ damage. Yet, compared to angiogenesis, much less is known about the mechanisms directing collateral growth. And no studies have determined how or when COLs develop. Collateral density and remodeling vary widely among species and humans, suggesting a genetic basis. Yet nothing is known about the source of this variation. Our preliminary studies show that COLs develop during the late embryonic-to-early postnatal period. We also find that compared to C57BL6 (B6) mice, COL formation in BALB/c (BC) mice is impaired --most dramatically in the cerebral circulation where BC essentially lack COLs. These findings create an excellent opportunity to identify factors specifying COL formation. Although multiple genes are likely involved, our preliminary work shows that BC are deficient in VEGF-A and are polymorphic at/near its locus. This, plus data from mutant mice overexpressing VEGF, suggest the novel hypothesis that VEGF signaling is crucial in COL formation. This is the first factor to be identified that regulates COL density in normal tissues. Many signals postulated to regulate COL growth in adult ischemic disease are known to be important in angiogenesis, in particular VEGF. However, VEGF's role is controversial because of difficulties in previous studies in localizing manipulation of VEGF. Also, no studies have identified the source (s) of VEGF in COL growth. To address these problems, we have developed new methods permitting 1) non-invasive imaging of COL perfusion and 2) local manipulation of VEGF signaling at the site of COL formation. Our preliminary results in the mouse hindlimb model suggest VEGF is a critical determinant of COL remodeling in ischemia. This proposal will investigate the overall hypothesis that COLs form perinatally, that VEGF signaling is central to this process, and that VEGF is also a key determinant of COL enlargement in adult ischemic disease. Aim I will determine how and when collaterals develop. Nothing is known about this fundamental process. We will also test the hypothesis that VEGF is critical in specifying collateral formation. We will characterize perinatal COL development and test the hypothesis that VEGF signaling is a key determinant and identify VEGF-R1 and -R2 involvement. Aim II will use array and genomic mapping to identify polymorphisms and VEGF-dependent and VEGF-independent candidate genes directing COL formation. Aim III will test the hypothesis that VEGF is important in collateral remodeling in adult ischemic disease, using the mouse hindlimb model, and will seek to identify the responsible mechanisms. We will use VEGF mutants, alter VEGF and its receptors, and identify the source of VEGF during COL growth. We hope our work will lead to therapies to stimulate formation of new COLs and augment growth of pre-existing COLs in occlusive vascular diseases. PUBLIC HEALTH RELEVANCE: Ischemic disease of the heart, brain and peripheral limbs, which is the most common cause of morbidity and death in western countries, is increasing worldwide, and many affected individuals are not candidates for percutaneous or bypass revascularization and are deemed "no option" (or face amputation) because of multiple vessel disease, diffuse disease or adverse preconditions. Therapies to augment angiogenesis (increase in capillary density) and arteriogenesis (enlargement of collaterals) have tremendous potential to treat ischemic disease. While several "second generation" angiogenesis trials are underway, development of therapies directed at arteriogenesis, which involves different mechanisms that are much less well understood, cannot come without a better understanding of the genetic and physiological mechanisms that specify collateral formation in normal tissue and that regulate their enlargement in ischemic disease - the specific aims of this proposal.