Translational Studies of Endothelial Progenitor Cells as a Novel Cardiovascular D

描述（由申请人提供）：心血管疾病（CVD）是美国死亡的主要原因，这导致了对CVD危险因素的强烈研究。最近的努力集中在新的心血管疾病危险因素上，因为传统的危险因素可以解释50%以下的心血管疾病。内皮祖细胞（EPCs）是一种新的心血管疾病危险因子。耐力运动训练增加EPC数；然而，大多数先前的研究只评估了EPC数量和集落形成单位，这代表了EPC功能的最小特征。本研究扩展了我们之前的研究工作，研究训练相关的离体EPC功能变化的细胞/分子机制，采用横断面比较，结合久坐老年人的运动训练和老年耐力训练运动员的停止训练。我们还将确定这些训练诱导的变化是否转化为体内内皮功能的改善。为了更好地表征离体EPC功能，我们将测量NADPH氧化酶活性和表达、一氧化氮（NO）和活性氧（ROS）水平，以及CD34+衍生的推定EPCs在标准和致动脉粥样硬化培养环境下的管状形成。我们的假设1是，在横断面基础上，老年运动员在标准培养条件下和对动脉粥样硬化培养环境的反应比匹配的老年久坐个体具有更好的离体EPC功能。我们对“更好”EPC功能的定义是更低的体外EPC NADPH氧化酶表达/活性和ROS水平，更高的NO水平和管形成。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，久坐不动的个体进行运动训练会改善，而老年运动员停止训练会恶化，标准培养条件下的体外EPC功能以及对动脉粥样硬化环境和体内内皮功能的反应。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的NADPH氧化酶活性和NO水平来进行机制研究，以评估通过模拟运动员的细胞内EPC环境，久坐受试者的EPC功能可以改善到何种程度。我们的假设3是，抑制NADPH氧化酶和增加久坐不动的人和停止训练后的运动员的EPCs的细胞内NO，将使体外EPC功能提高到与训练后的运动员相似的水平。这种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EPCs作为一种新的CVD危险因素与运动训练之间的相互作用，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型心血管疾病风险因素的益处的机制的理解。

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.