Cellular and Molecular Mechanisms of Coronary Endothelial Dysfunction in Diabetes

描述(申请人提供)：冠心病和缺血性中风是糖尿病患者发病率和死亡率的主要原因。血管内皮细胞损伤和功能障碍是糖尿病血管病变的常见起点。因此，研究糖尿病冠脉血管内皮细胞功能障碍的细胞和分子机制具有重要意义。内皮细胞(ECs)胞浆内钙离子浓度([Ca~(2+)]Cyt)的变化对内皮功能起着调节作用。[Ca~(2+)]Cyt受细胞内钙离子动员的控制 再加上钙离子从外部介质中内流。在内皮细胞中，内质网(ER)约占细胞内总钙存储的75%，内质网中的[Ca~(2+)]决定着重要的Ca~(2+)信号的产生。本研究的目的是探讨糖尿病血管内皮细胞内钙离子内质网是否发生改变，以及[钙离子]内质网异常如何导致糖尿病心脏血管内皮细胞功能障碍。在目标1中，我们将研究从糖尿病小鼠分离的冠状动脉内皮细胞是否以及如何减弱内质网钙释放引起的[Ca~(2+)]Cyt升高(指示[Ca~(2+)]ER的水平)和[Ca~(2+)]ER的水平。内质网膜由钙泵(SERCA)和几类细胞内钙释放通道组成。近年来，基质相互作用分子(STIM)被发现是钙离子进入内质网(SOCE)所必需的蛋白质，并通过与内质网膜上的SERCA相互作用，促进钙离子重新进入内质网。在目标2中，我们将探讨STIM1在糖尿病冠脉内皮细胞[Ca~(2+)]ER降低中的潜在作用。我们的初步数据显示，SERCA3和STIM1蛋白在糖尿病冠脉内皮细胞中的表达明显减少，而STIM1在糖尿病冠脉内皮细胞中的过度表达显著增加了内质网钙离子的泄漏。此外，内质网转运到线粒体的钙离子在细胞凋亡中起着至关重要的作用；线粒体钙离子([Ca2+]MIT)的增加可以在许多细胞类型中引发线粒体碎裂、线粒体完整性丧失和细胞死亡。结果表明，糖尿病患者冠脉内皮细胞内游离钙离子浓度显著升高。在目标3中，我们将确定糖尿病冠脉内皮细胞中钙离子从内质网到线粒体的转运是否增加，并研究内质网和线粒体之间的钙离子通讯在糖尿病心脏内皮功能障碍中的作用。研究内质网对糖尿病冠脉内皮细胞的钙调节作用将有助于我们进一步了解糖尿病冠脉血管并发症的病理生理学方面。这项研究的完成将为开发糖尿病心脏缺血的新治疗干预措施提供重要的见解。

DESCRIPTION (provided by applicant): Coronary heart disease and ischemic stroke are the main causes of morbidity and mortality in diabetic patients. Endothelial injury and dysfunction are common starting points for diabetic angiopathy. It is thus important to investigate the cellula and molecular mechanisms involved in coronary vascular endothelial dysfunction in diabetes. Endothelial function is regulated by changes in cytosolic Ca2+ concentration ([Ca2+]cyt) in endothelial cells (ECs). [Ca2+]cyt is controlled by the Ca2+ mobilization from intracellular stores coupled to Ca2+ influx from the external medium. In ECs, the endoplasmic reticulum (ER) accounts for approximately 75% of the total intracellular Ca2+ stores and the [Ca2+] in the ER ([Ca2+]ER) determines in great part the generation of important Ca2+ signals. The objective of this study is to examine whether [Ca2+]ER is altered in diabetic ECs and how abnormal [Ca2+]ER leads to vascular endothelial dysfunction in diabetic heart. In Aim 1, we will examine whether and how the rise in [Ca2+]cyt due to Ca2+ release from the ER (indicative of the level of [Ca2+]ER) and [Ca2+]ER are attenuated in coronary ECs isolated from diabetic mice. The ER membrane constitutes Ca2+ pumps (sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)) and several classes of intracellular Ca2+ releasing channels. Recently, stromal interaction molecule (STIM) was identified as an essential protein for the store-operated Ca2+ entry (SOCE) and a contributor to the Ca2+ refilling into the ER by interacting with SERCA on the ER membrane. In Aim 2, we will investigate the potential role of STIM1 in decreased [Ca2+]ER in diabetic coronary ECs. Our preliminary data show that SERCA3 and STIM1 protein expressions are markedly decreased in diabetic coronary ECs and STIM1 overexpression in diabetic coronary ECs significantly increases Ca2+ leak from the ER. In addition, Ca2+ transported to mitochondria from the ER plays a crucial role in cell apoptosis; increased mitochondrial Ca2+ ([Ca2+]mit) can trigger mitochondrial fragmentation, loss of mitochondrial integrity and cell death in many cell types. Our results show that [Ca2+]mit is significantly increased in coronary ECs in diabetes. In Aim 3, we will define whether Ca2+ transportation from the ER to mitochondria is increased in coronary ECs in diabetes and examine the role of the Ca2+ communication between the ER and mitochondria in endothelial dysfunction in diabetic heart. To examine Ca2+ handling by the ER in diabetic coronary ECs will further our understanding of pathophysiological aspects of coronary vascular complications in diabetes. Completion of this study will provide important insights into developing new therapeutic interventions for cardiac ischemia in diabetes.