Translational Studies of Endothelial Progenitor Cells as a Novel Cardiovascular D

描述(申请人提供)：心血管疾病(CVD)是美国的主要死亡原因，这导致了对CVD危险因素的深入研究。最近的努力集中在新的心血管疾病风险因素上，因为传统的风险因素可能解释50%的心血管疾病。内皮祖细胞是一种新的心血管危险因素。耐力运动训练增加了EPC数量；然而，以前的大多数研究只评估了EPC数量和集落形成单位，这代表了EPC功能的最小特征。这项建议扩展了我们之前的工作，研究了与训练相关的体外EPC功能变化背后的细胞/分子机制，使用横断面比较，结合久坐不动的老年人的运动训练和接受耐力训练的老年运动员的训练停止。我们还将确定这些训练诱导的变化是否会转化为体内内皮功能的改善。为了更好地表征体外EPC的功能，我们将在标准和致动脉粥样硬化的培养环境下，测量CD34+来源的EPC的NADPH氧化酶活性和表达，一氧化氮(NO)和活性氧(ROS)水平，以及管状形成。我们的假设#1是，在横截面的基础上，年龄较大的运动员在标准培养条件下和对致动脉粥样硬化的文化环境的反应中，将比匹配的久坐不动的老年人具有更好的体外EPC功能。我们对“较好的”EPC功能的定义是较低的体外EPC NADPH氧化酶表达/活性和ROS水平，以及较高的NO水平和管状形成。我们还将评估体内内皮功能，以确定它是否可以作为体外EPC功能的临床指标。我们的假设2是，在标准培养条件下，在动脉粥样硬化环境和体内内皮功能的反应下，久坐不动的人的运动训练将会改善，而老年运动员的训练停止将会恶化体外EPC功能。这些结果将使我们能够有效地得出结论，运动训练可以改善体外EPC和体内内皮功能。然后，我们将通过操纵培养物中NADPH氧化酶活性和NO水平来进行机制研究，以评估通过模仿运动员明显的细胞内EPC环境，久坐受试者的EPC功能可以得到改善的程度。我们的假设#3是，抑制NADPH氧化酶和增加停止训练后运动员体内假定的EPC中的NADPH氧化酶和增加细胞内NO都将改善体外EPC功能，使其达到与训练有素运动员相似的水平。这一机制方法将使我们能够量化运动训练对EPC功能的影响程度是EPC NADPH氧化酶活性变化的函数。很少有研究涉及作为一种新的心血管疾病危险因素的内皮祖细胞与运动训练之间的相互作用，而且几乎没有研究利用人类受试者并将研究扩展到细胞、代谢和机制水平。因此，我们处于一个独特的位置，可以极大地扩展我们对运动训练对这种新的心血管疾病危险因素的益处的机制的理解。 与公共健康相关：在美国非常流行的许多心血管疾病都是血管内膜--内皮细胞受损的结果。血管内皮细胞是由存在于血液中的血管内皮祖细胞修复的。该项目建议确定运动训练如何影响这些细胞的功能，并研究任何观察到的变化背后的潜在细胞和分子机制。

DESCRIPTION (provided by applicant): Cardiovascular disease (CVD) is the major cause of death in the US, which has resulted in an intense search for CVD risk factors. Recent efforts have focused on novel CVD risk factors, as conventional risk factors may explain <50% of CVD. Endothelial progenitor cells (EPCs) are one such novel CVD risk factor. Endurance exercise training increases EPC number; however, most previous studies assessed only EPC numbers and colony forming units, which represent a minimal characterization of EPC function. This proposal expands on our previous work to study the cellular/molecular mechanisms underlying training-related changes in ex vivo EPC function, using a cross-sectional comparison combined with exercise training in sedentary older adults and training cessation in older endurance-trained athletes. We also will determine whether these training- induced changes translate to improved in vivo endothelial function. To better characterize ex vivo EPC function we will measure NADPH oxidase activity and expression, nitric oxide (NO) and reactive oxygen species (ROS) levels, and tube formation in CD34+derived putative EPCs, under both standard and atherogenic culture environments. Our Hypothesis #1 is, that on a cross-sectional basis, older athletes will have better ex vivo EPC function under standard culture conditions and in response to an atherogenic culture environment than matched older sedentary individuals. Our definition of "better" EPC function is lower ex vivo EPC NADPH oxidase expression/activity and ROS levels, and higher NO levels and tube formation. We will also assess in vivo endothelial function to determine if it can be used as a clinical index of ex vivo EPC function. Our Hypothesis #2 is that exercise training in sedentary individuals will improve, and training cessation in older athletes will worsen, ex vivo EPC function under standard culture conditions and in response to an atherogenic environment and in vivo endothelial function. These results will allow us to validly conclude that exercise training improves ex vivo EPC and in vivo endothelial function. We will then perform mechanistic studies by manipulating NADPH oxidase activity and NO levels in cultures to assess the degree to which EPC function in sedentary subjects can be improved by mimicking the intracellular EPC milieu evident in athletes. Our Hypothesis #3 is that both inhibiting NADPH oxidase and increasing intracellular NO in putative EPCs from sedentary individuals and from athletes after stopping training will improve ex vivo EPC function to levels similar to those of trained athletes. This mechanistic approach will allow us to quantify the extent to which exercise training effects on EPC function are a function of changes in EPC NADPH oxidase activity. Very few studies have addressed the interaction between EPCs, as a novel CVD risk factor, and exercise training, and virtually no studies have utilized human subjects and extended the studies to the cellular, metabolic, and mechanistic levels. Thus, we are in a unique position to expand dramatically our understanding of the mechanisms that underlie the benefits of exercise training on this novel CVD risk factor. PUBLIC HEALTH RELEVANCE: Many of the cardiovascular diseases that are so prevalent in the US are the result of damage to the internal lining of blood vessels, the endothelium. The endothelium is repaired by endothelial progenitor cells that are present in our blood. This project proposes to determine how exercise training affects the function of these cells, as well as examining the potential cellular and molecular mechanisms underlying any observed changes.