Microparticles and microvascular dysfunction in diabetes

描述（由申请人提供）：糖尿病是美国主要的死亡原因之一，超过一半的糖尿病患者有心血管并发症。然而，糖尿病血管并发症发生的机制和关键因素仍然知之甚少。我们最近的研究发现，糖尿病大鼠微血管的基础通透性增加，对炎症介质的通透性反应明显增强。我们还发现，在糖尿病患者和糖尿病大鼠中，循环微粒（MPs），即活化后从细胞表面释放的小泡，都显著升高。最重要的是，我们新开发的实验结果表明，将糖尿病大鼠血浆输注到正常大鼠的血液循环中会立即引起白细胞粘附和微血管通透性增加，而从糖尿病血浆中去除MPs会消除这种效果，这表明糖尿病MPs在介导微血管炎症中发挥重要作用。当将分离的糖尿病MPs直接灌注到正常个体微血管中时，也得到了类似的观察结果。此外，糖尿病患者血浆MPs分析显示MPs水平与其血管并发症呈正相关。这些新颖的初步发现使我们假设糖尿病患者MPs水平的升高不仅仅是血管细胞活化和凋亡的结果，而且也是在整个血管系统中传播促炎和促凝介质的载体，加剧了血管功能障碍。本应用程序旨在通过三个具体目标来验证这一假设。目的1是表征细胞来源并确定糖尿病条件下MPs增加的机制。目的2是研究血浆MPs升高与糖尿病微血管炎症表现之间的因果关系，并确定MP介导的白细胞粘附、血栓形成和微血管通透性增加的机制。目的3：探讨MPs升高与糖尿病患者临床状况的相关性。我们将评估使用MP分析预测血管并发症的风险或严重程度的潜力，以及糖尿病治疗的疗效。目前关于MPs的知识主要来自体外研究。我们独特的实验方法将MPs的流式细胞术分析与单血管灌注、血浆输血、共聚焦成像、电子显微镜和完整微血管的定量渗透率测量相结合，将提供最需要的糖尿病MPs介导的微血管炎症的体内机制。实验动物研究结果与患者疾病状况之间的直接联系确保了从拟议研究中获得的知识将为糖尿病相关血管功能障碍的发病机制提供新的见解，并有利于靶向治疗。

DESCRIPTION (provided by applicant): Diabetes is one of the leading causes of mortality in the US and more than half of diabetic patients have cardiovascular complications. However, the mechanisms and the critical factors involved in the development of diabetic vascular complications remain poorly understood. Our recent studies found that microvessels in diabetic rats had increased basal permeability and a markedly augmented permeability response to inflammatory mediators. We also found that circulating microparticles (MPs), the small vesicles released from the cell surface upon activation, were significantly elevated in both diabetic patients and diabetic rats. Most importantly, results derived from our newly developed experiments showed that transfusion of diabetic rat plasma into the circulation of a normal rat causes immediate leukocyte adhesion and increased microvessel permeability, and that removal of MPs from the diabetic plasma abolished the effect, indicating an important role of diabetic MPs in mediating microvascular inflammation. Similar observations were replicated when isolated diabetic MPs were directly perfused into a normal individual microvessel. In addition, MP analysis of diabetic patients' plasma showed a positive correlation between the levels of MPs and their vascular complications. These novel preliminary findings led us to hypothesize that the increased levels of MPs under diabetic conditions are not simply the results of vascular cell activation and apoptosis, but are also vectors that disseminate pro- inflammatory and pro-coagulant mediators throughout the vascular system, exacerbating vascular dysfunction. This application aims to test this hypothesis with three specific aims. Aim 1 is to characterize the cellular sources and identify the mechanisms of increased MPs under diabetic conditions. Aim 2 is to investigate the causal relationship between the increased plasma MPs and the inflammatory manifestation of diabetic microvessels and to identify the mechanisms involved in MP- mediated leukocyte adhesion, thrombus formation, and increased microvessel permeability. Aim 3 is to investigate the correlations between the increased MPs and the clinical status of diabetic patients. We will evaluate the potential of using MP analysis to predict the ris or severity of vascular complications, as well as the efficacy of diabetic therapies. Present knowledge about MPs is largely derived from in vitro studies. Our unique experimental approaches that combine flow cytometry analysis of MPs with single vessel perfusion, plasma transfusion, confocal imaging, electron microscopy, and quantitative permeability measurements in intact microvessels, will provide the most needed in vivo mechanisms of diabetic MP- mediated microvascular inflammation. The direct linkage between laboratory animal findings with patient disease conditions assures that the knowledge gained from the proposed studies will provide new insight into the pathogenesis of diabetes-associated vascular dysfunction and benefit targeted therapies.