Surgical Intramyocardial Angiogenesis in a Swine Model of Endothelial Dysfunction

描述（由申请人提供）：我们实验室以及其他实验室最近的工作已经建立了外源性生长因子的治疗效用，如血管内皮生长因子（VEGF）和成纤维细胞生长因子-2 (FGF-2)，在慢性心肌缺血的大型动物模型中诱导心肌血流和左心室功能的改善。然而，临床试验迄今未能证明治疗性血管生成在病人身上与在正常猪和其他实验动物身上一样有效。由于VEGF、FGF-2和其他血管生成生长因子在很大程度上是通过酪氨酸激酶受体的激活释放内皮源性一氧化氮（NO）来起作用的，因此在冠状动脉疾病（CAD）、高胆固醇血症、糖尿病、高血压和其他内皮功能障碍危险因素患者中观察到的效果失效可能与生物可利用NO的刺激释放不足有关。它们的产生以及其他内皮衍生物质的产生由于这些疾病状态而显著改变。活性氧（ROS）在血管生成中起着至关重要的作用。体外和体内研究表明，血管组织中的血管生成反应部分是由ROS信号以高度协调的方式触发的。血管生成受氧化还原敏感转录因子（如核因子kb）、氧化剂（如过氧化氢）和自由基（如NO）的调控。这些药物可能在血管生成过程的途径中作为第二信使。此外，许多促血管生成基因，如VEGF、FGF-2及其受体的表达可能受到氧化还原信号的调节。因此，尽管已经发现ROS对缺血-再灌注损伤有不利影响，并且与高胆固醇血症相关的内皮功能障碍与基础氧化应激增加可能显著降低蛋白质生长因子诱导血管生成作用的潜力，但ROS也可能有助于长期血管生成反应。本应用的一个主要目的是确定高胆固醇血症引起的氧化应激增加及其对这些血管生成因子表达的调节的作用。尽管活性氧在调节缺血再灌注血管生成反应方面具有明显的双重作用，但我们假设，慢性减少氧化应激将对侧枝发育产生积极影响，重点关注侧枝依赖性血流、血管密度和微血管调节的功能变化。然而，这是否在慢性心肌缺血的临床相关模型中成立，还需要使用大型动物模型进行实验。为了验证这些假设，我们将用抗氧化剂（维生素E、普罗布考和白藜芦醇）治疗慢性高胆固醇血症猪，并确定它们对内源性和外源性（VEGF和FGF）血管生成反应的影响，以应对狭窄的环状冠状动脉引起的心肌缺血。在冠心病患者中使用血管生成生长因子（如血管内皮生长因子（VEGF）或成纤维细胞生长因子-2 （FGF-2）或编码这些因子的基因）的临床试验未能显著改善心肌灌注。由于高胆固醇血症诱导氧化应激，降低血管生成因子的有效性，我们将用抗氧化剂（维生素E、普罗布考和白藜芦醇）治疗高胆固醇血症猪，并检查参与侧枝发育的分子信号的变化，以及在内皮功能障碍和氧化应激增加的模型中，VEGF和FGF治疗是否可以改善心肌血流和左心室功能。

DESCRIPTION (provided by applicant): Recent work from our laboratory as well as that of others has established the therapeutic utility of exogenous growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), in inducing improvements in myocardial flow and left ventricular function in a large animal model of chronic myocardial ischemia. However, clinical trials have thus far failed to demonstrate that therapeutic angiogenesis is as effective in patients as it is in normal swine and other laboratory animals. Since VEGF, FGF-2, and other angiogenic growth factors operate in large part through the release of endothelial-derived nitric oxide (NO) via the activation of tyrosine kinase receptors, the failure of effect seen in patients with coronary artery disease (CAD), hypercholesterolemia, diabetes, hypertension, and other risk factors for endothelial dysfunction may be related to a deficiency in the stimulated release of bioavaiable NO, whose production as well as that of other endothelium-derived substances is significantly altered as a result of these disease states. Reactive oxygen species (ROS) have been found to play a critical role in vascular angiogenesis. In vitro and in vivo studies have demonstrated that the angiogenic response in vascular tissue is triggered in part by ROS signaling in a highly coordinated manner. Angiogenesis is regulated by redox-sensing transcription factors such as nuclear factor kb and oxidants such as hydrogen peroxide and free radicals such as NO. These agents may act as second messengers in the pathway of the angiogenic process. Furthermore, expression of many pro-angiogenic genes such as VEGF, FGF-2, and their receptors may be regulated by redox signaling. Thus, despite the fact that ROS have been found to contribute to the detrimental effects of ischemia- reperfusion injury, and that endothelial dysfunction associated with hypercholesterolemia with increased basal oxidant stress may markedly diminish the potential of protein growth factors to induce an angiogenic effect, ROS may also contribute to the long term angiogenic response. One overarching aim of this application is to determine the role of increased oxidant stress induced by hypercholesterolemia and its modulation of the expression of these angiogenic factors. Despite this apparent dual effect of ROS in regulating the angiogenic response to ischemia reperfusion, we hypothesize that a chronic reduction in oxidant stress will have a positive impact on collateral development with a focus on the functional changes in collateral-dependent blood flow, vascular density, and microvascular regulation. However, whether this is true in a clinically relevant model of chronic myocardial ischemia will require experiments using a large animal model. To test these hypotheses we will treat chronic hypercholesterolemic pigs with anti-oxidants (vitamin E, Probucol, and resveratrol) and determine their effects on the endogenous and exogenous (VEGF and FGF) angiogenic responses to myocardium ischemia induced by narrowing the circumflex coronary artery.Clinical trials involving patients with coronary disease using angiogenic growth factors such as vascular endothelial growth factor (VEGF) or fibroblast growth factor-2 (FGF-2) or genes encoding these factors have failed to improve myocardial perfusion to a significant degree. Since hypercholesterolemia induces oxidative stress that decreases the effectiveness of angiogenic factors, we will treat hypercholesterolemic pigs with anti- oxidants (Vitamin E, Probucol, and resveratrol) and examine the changes in the molecular signaling involved in collateral development, and whether myocardial flow and left ventricular function can be improved by VEGF and FGF treatment in this model of endothelial dysfunction and increased oxidative stress.