Flow-induced coronary vasospasm in diabetic patients

描述（由申请人提供）：2型糖尿病（T2-DM）患者有较高的无回流现象发生率，无回流现象是经皮冠状动脉介入治疗（PCI）的一种尚不清楚且不可预测的并发症，即尽管成功治疗了心外膜冠状动脉闭塞病变，但流向远端微血管床的血流量仍然减少。目前预防无血流的治疗干预措施是无效的。与此应用相关的初步观察导致了我的主要假设，即糖尿病患者的小冠状动脉在血流突然增加时表现出矛盾的收缩，这是一种改变，导致无血流。我认为rhoa依赖性精氨酸酶I和eNOS的共定位导致T2-DM血流反应中NO合成减少和NO介导的扩张减弱。我还假设，随着腔内血流的增加，刺激血小板内皮细胞粘附分子-1 （Pecam-1，被称为内皮初级血流传感器）会升高内皮[Ca2+] I，内皮[Ca2+] I通过诱导磷脂酶A2和花生四烯酸的释放导致T2-DM患者冠状血管中血栓素A2的产生增加。为了验证这些假设，我的目标是从接受心脏手术的T2-DM患者的（废弃的）心房附件中分离出小冠状动脉。使用小血管压力肌图和视频显微镜，在特定信号通路抑制剂存在的情况下，测量孤立的冠状动脉（< 100 5m）暴露于腔内流量突然增加时的直径变化。为了研究eNOS和精氨酸酶I的（co）定位，以及Pecam-1激光扫描共聚焦显微镜和荧光共振能量转移方法在分离的、加压的冠状动脉和培养的冠状动脉内皮细胞中的空间分布和相互作用。此外，血流诱导的内皮[Ca2+]i的变化将通过Fura-2荧光在完整的加压冠状动脉中测量，以揭示亚细胞水平[Ca2+]i升高的空间差异。如果在项目过程中获得的结果支持我的假设，这将是人类冠状动脉中pecam -1偶联收缩前列腺素产生的第一次描述。研究结果还将提供一种新的机制，Pecam-1的空间分布决定了释放血管活性介质的性质，从而增加T2-DM的血流。该项目获得的数据也将有助于开发有效治疗策略的新途径，例如在PCI时使用前列腺素抑制剂，以防止T2-DM患者无再流。

DESCRIPTION (provided by applicant): Patients with type 2 diabetes mellitus (T2-DM) have higher prevalence of no-reflow phenomenon - a poorly understood and unpredictable complication of percutaneous coronary intervention (PCI) in which diminished blood flow to distal microvascular beds persists despite the successful treatment of the occlusive lesion of the epicardial coronary artery. Current therapeutic interventions to prevent no reflow are ineffective. Preliminary observations related to this application led to my main hypothesis that small coronary arteries of diabetic patients exhibit a paradoxical constriction to sudden increases in flow, an alteration, which contributes to no reflow. I propose that RhoA-dependent co-localization of arginase I and eNOS leads to reduced NO synthesis and diminished NO-mediated dilatation in response to flow in T2-DM. I also hypothesize that stimulation of platelet endothelium cell adhesion molecule -1 (Pecam-1, known as primary flow sensor in endothelium) with increases in intraluminal flow elevates endothelial [Ca2+]i, which via inducing phospholipase A2 and arachidonic acid release leads to enhanced production of thromboxane A2 in coronary vessels of T2-DM patients. To test these hypotheses, I aim to isolate small coronary vessels from the (discarded) atrial appendages of patients with T2-DM undergoing cardiac surgery. Using small vessel pressure myography and videomicroscopy, diameter changes of the isolated, coronary arteriole (< 100 5m) exposed to sudden increase in intraluminal flow will be measured in the presence of inhibitors of specific signaling pathways. To investigate (co)localization of eNOS and arginase I as well as to detect spatial distribution and interaction of Pecam-1 laser scanning confocal microscopy and fluorescence resonance energy transfer approaches will be used in isolated, pressurized coronary arteries and coronary endothelial cells in culture. Moreover, flow-induced changes in endothelial [Ca2+]i will be measured with Fura-2 fluorescence in intact, pressurized coronary arterioles to reveal spatial differences of [Ca2+]i elevations at subcellular level. Should the results obtained in the course of the project support my hypothesis this will be the first description of Pecam-1-coupled constrictor prostanoid production in human coronary arteries. Results will also provide a novel mechanism by which spatial distribution of Pecam-1 determines the nature of vasoactive mediators released to increase in flow in T2-DM. Data obtained in this project will also help to develop novel avenues for effective therapeutic strategies, such as the use of prostanoid inhibitors at the time of PCI, to prevent no reflow in patients with T2-DM. PUBLIC HEALTH RELEVANCE: This proposal seeks to support research to elucidate mechanism(s), which may contribute to coronary no- reflow, a serious complication of percutaneous coronary intervention in having higher prevalence in diabetic patients. I aim to isolate small coronary vessels from the (discarded) atrial appendages of diabetic patients undergoing cardiac surgery. Using this approach the proposal aims to provide a rationale for effective therapeutic intervention to prevent no reflow in diabetic patients.