Novel Regulatory Mechanisms in the Human Microcirculation

项目摘要 微血管系统在多种肿瘤的发生和后果中起着关键作用。 心血管疾病微血管功能的主要评估是通过内皮依赖性NO- 介导的扩张，其作为冠状动脉疾病（CAD）和心肌病的前兆而减少。在 来自患有CAD的受试者的人小动脉的NO介导的血流介导的扩张（FMD）的损失通过以下方式补偿： 内皮线粒体过氧化氢（H2 O2）。虽然两者都是扩张剂，但NO和H2 O2具有 对血管健康的相反作用，NO促进静止，H2 O2促进血管和 实质炎症导致动脉粥样硬化。了解负责这种转换的机制， 介质可能是关键，以尽量减少组织应力或损伤血管旁分泌氧化还原毒性。 本研究的目的是确定基本的细胞通路调节这种开关从NO到H2 O2。 我们提出，两个系统，最近被证明是剪切敏感和基本的细胞功能是相连的 作为人小动脉（HA）中FMD的关键。第一种是自噬，我们认为它是控制开关 调节剪切诱导的NO或H2 O2的产生。阻断自噬通量可减少NO， 活性氧（ROS）。第二种途径涉及脂质磷酸磷酸酶3（LPP 3）， 通过抑制溶血磷脂酸（LPA）、降低ROS和促进NO来响应剪切。 在80%的人群中发现的该基因的核苷酸多态性与CAD的高风险相关。 我们认为，剪切诱导的LPP 3激活是维持NO介导的FMD在HA中所必需的。 LPP 3和自噬都与FMD的介质无关。我们将研究新鲜的人类冠状动脉 和脂肪小动脉在人微血管内皮细胞体外使用刺激剂和抑制剂， 自噬和LPA以确定它们在FMD中的作用。局部组织的影响可能是深远的， NO与H_2O_2对心血管功能的影响。我们将检验以下假设： 假设1.自噬在维持NO作为人冠状动脉FMD介质中的关键作用 微循环减少自噬导致切换到H2 O2作为FMD的介质。 假设2. LPP 3通过微血管内皮剪切上调，导致LPA水解、衰减 内皮ROS，与NOS依赖性FMD的维持。如果LPP 3在机制上与 微血管功能障碍，这可能是一个重要的目标，无论是直接或通过LPA，为减少 血管炎症在大量的遗传CAD易感个体。

Project Abstract The microvasculature plays a critical role in the development and consequences of a broad range of cardiovascular diseases. The main assessment of microvascular function is via endothelium-dependent NO- mediated dilation which is reduced as a precursor to coronary artery disease (CAD) and cardiomyopathy. In human arterioles from subjects with CAD loss of NO-mediated flow-mediated dilation (FMD) is compensated by hydrogen peroxide (H2O2) from endothelial mitochondria. Although both are dilators, NO and H2O2 have opposing effects on vascular health, with NO promoting quiescence and H2O2 promoting vascular and parenchymal inflammation leading to atherosclerosis. Understanding mechanisms responsible for this switch in mediator may be key to minimizing tissue stress or injury from vascular paracrine redox toxicity. The goal of this study is to determine fundamental cellular pathways regulating this switch from NO to H2O2. We propose that two systems, recently shown to be shear sensitive and fundamental to cell function are linked as critical for FMD in human arterioles (HA). The first is autophagy which we propose is the controlling switch that regulates shear-induced production of NO or H2O2. Blocking autophagic flux reduces NO and enhances reactive oxygen species (ROS). The second pathway involves lipid phosphate phosphatase 3 (LPP3), which responds to shear by inhibiting lysophosphatidic acid (LPA), lowering ROS and promoting NO. A single nucleotide polymorphism of this gene, seen in 80% of the population is associated with heightened risk for CAD. We propose that shear-induced activation of LPP3 is needed to maintain NO-mediated FMD in HA. Neither LPP3 nor autophagy has been linked to the mediator of FMD. We will study fresh human coronary and adipose arterioles in human microvascular endothelial cells in vitro using stimulators and inhibitors of autophagy and LPA to determine their role in FMD. The local tissue impact can be profound given the different effect of NO vs. H2O2 on cardiovascular function. We will test the following hypotheses: Hypothesis 1. Autophagy is critical in maintaining NO as the mediator of FMD in the human coronary microcirculation. Reduced autophagy leads to a switch to H2O2 as the mediator of FMD. Hypothesis 2. LPP3 is upregulated by microvascular endothelial shear resulting in LPA hydrolysis, attenuation of endothelial ROS, with maintenance of NOS-dependent FMD. If LPP3 is mechanistically linked to microvascular dysfunction, this could be an important target, either directly or through LPA, for reducing the vascular inflammation in a large number of genetically CAD-susceptible individuals.