Microparticles and microvascular dysfunction in diabetes

描述(申请人提供)：糖尿病是美国死亡的主要原因之一，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，糖尿病大鼠的微血管基础通透性增加，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，循环微粒子(MPS)，即激活时从细胞表面释放的小囊泡，都显著增加。最重要的是，我们新开发的实验结果表明，将糖尿病大鼠的血浆输入正常大鼠的循环中会立即引起白细胞黏附和微血管通透性增加，而从糖尿病大鼠血浆中去除MPS则取消了这一作用，表明糖尿病大鼠MPS在介导微血管炎症中发挥了重要作用。当分离的糖尿病MPS直接灌流到正常的个体微血管中时，类似的观察结果也重复了。此外，对糖尿病患者血浆进行的MP分析显示，MPS水平与其血管并发症呈正相关。这些新的初步发现使我们假设，糖尿病条件下MPS水平的增加不仅仅是血管细胞激活和凋亡的结果，也是在血管系统中传播促炎和促凝介质的媒介，加剧了血管功能障碍。这项申请旨在通过三个具体目标来检验这一假设。目的1是确定糖尿病条件下MPS升高的细胞来源和机制。目的2探讨血浆MPS升高与糖尿病微血管炎症表现的因果关系，明确MP介导的白细胞黏附、血栓形成和微血管通透性增加的机制。目的3探讨糖尿病患者MPS升高与临床状态的关系。我们将评估使用MP分析来预测RIS或血管并发症严重程度的潜力，以及糖尿病治疗的有效性。目前有关MPS的知识主要来源于体外研究。我们独特的实验方法将MPS的流式细胞术分析与单血管灌流、血浆输注、共聚焦成像、电子显微镜和完整微血管的定量通透性测量相结合，将为糖尿病MP介导的微血管炎症提供最需要的体内机制。实验室动物发现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.