Flow-induced coronary vasospasm in diabetic patients

描述(申请人提供)：2型糖尿病(T2-DM)患者无复流现象的发生率更高--一种鲜为人知且不可预测的经皮冠状动脉介入治疗(PCI)并发症，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仍然减少。目前防止无复流的治疗干预措施无效。与这一应用相关的初步观察导致了我的主要假设：糖尿病患者的小冠状动脉表现出一种矛盾的收缩，导致血流突然增加，这是一种改变，有助于无复流。我认为在T2-DM中，依赖于RhoA的精氨酸酶I和eNOS的共定位导致NO合成减少，并减少了NO介导的血管扩张。我还假设，随着血管内流量的增加，刺激血小板内皮细胞黏附分子-1(Pecam-1，在血管内的主要流量感受器)会升高内皮细胞[Ca~(2+)]i，这是通过诱导磷脂酶A2和花生四烯酸释放而导致T2-DM患者冠状动脉内血栓素A2产生增加的。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)心耳中分离出微小的冠状动脉。使用小血管压力肌成像术和视频显微镜，在存在特定信号通路的抑制剂的情况下，将测量暴露于腔内流量突然增加的分离的冠状动脉小动脉(100 5米)的直径变化。为了研究eNOS和精氨酸酶I的共同定位，以及检测Pecam-1激光扫描共聚焦显微镜和荧光共振能量转移技术在体外培养的冠状动脉和冠状动脉内皮细胞中的空间分布和相互作用。此外，将用Fura-2荧光法在完整、加压的冠状动脉小动脉中测量血流诱导的内皮细胞[Ca~(2+)]i的变化，以揭示亚细胞水平上[Ca~(2+)]i升高的空间差异。如果项目过程中获得的结果支持我的假设，这将是第一次描述Pecam-1偶联的收缩前列腺素在人类冠状动脉中的产生。研究结果还将提供一种新的机制，即Pecam-1的空间分布决定了T2-DM中释放的血管活性介质的性质，从而增加了血流。该项目中获得的数据还将有助于开发有效治疗策略的新途径，例如在经皮冠状动脉介入治疗时使用前列腺素抑制剂，以防止T2-DM患者无复流。 公共卫生相关性：这项建议旨在支持研究以阐明机制(S)，这可能导致冠状动脉无复流，这是经皮冠状动脉介入治疗的一种严重并发症，在糖尿病患者中发病率较高。我的目标是从接受心脏手术的糖尿病患者的(废弃的)心耳中分离出微小的冠状动脉。使用这一方法，该提案旨在为有效的治疗干预提供理论基础，以防止糖尿病患者的无复流。

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.