Poly (ADP-Ribose) synthetase in expression of endothelia

内皮损伤和中性粒细胞粘附于血管内膜是心肌缺血和再灌注损伤的早期病理事件。 中性粒细胞运输到损伤组织的精确分子机制尚不清楚。 实验证据表明，氧化应激是白细胞内皮粘附分子表达的重要信号转导事件。 在初步的体外和体内研究中，我们发现氧化应激通过由核酶聚（ADP核糖）合成酶（PARS）介导的途径引起细胞功能障碍和损伤。 在体外内皮细胞中，过氧亚硝酸盐和过氧化氢诱导DNA单链断裂并激活PARS，导致细胞高能磷酸盐耗尽，细胞功能障碍和死亡。 我们已经发现，在大鼠和小鼠心肌缺血和再灌注期间，PARS的体内药理学或遗传学抑制减少了梗死面积和中性粒细胞积聚到损伤组织中。本研究旨在探讨再灌注依赖性氧化应激诱导内皮细胞表达P-选择素、E-选择素、细胞间粘附分子-1（ICAM-1）和血管粘附分子-1（VCAM-1）的分子基础，特别关注PARS通路。 将在心肌缺血和再灌注损伤的体内实验啮齿动物模型中评价PARS介导的粘附分子内皮表达调节的病理生理学相关性。 具体而言，将在缺乏PARS基因的敲除动物和同窝野生型对照的心肌缺血和再灌注的鼠模型中进行实验。 对于临床相关性，将在心肌再灌注损伤的大鼠模型中研究PARS的药理学抑制对内皮损伤和中性粒细胞蓄积的影响。 此外，将在培养的人脐静脉内皮细胞中进行实验，这些细胞经受氧化和非氧化应激。 这些体外实验的目的是为了获得一个更精确的评估的作用，细胞内PARS对成人内皮功能障碍相关的人心肌梗死。 拟议项目的结果将提供重要的信息，PARS作为内皮屏障功能的调节剂的作用，并将有助于开发有效的治疗干预的心血管疾病的特点是内皮再灌注损伤。

Endothelial injury, and consequent adhesion of neutrophils to the vascular intima, is an early pathologic event in myocardial ischemia and reperfusion injury. The precise molecular mechanisms involved in neutrophil trafficking to injured tissue are not clear. Experimental evidence shows that oxidative stress is an important signal transduction event for the expression of endothelial adhesion molecules for leukocytes. In preliminary in vitro and in vivo studies we have found that oxidative stress causes cellular dysfunction and injury through a pathway mediated by the nuclear enzyme poly (ADP ribose) synthetase (PARS). In in vitro endothelial cells, peroxynitrite and hydrogen peroxide induce DNA single strand breaks and activate PARS, resulting in the depletion of cellular high-energy phosphates, cellular dysfunction and death. We have found that in vivo pharmacological or genetic inhibition of PARS reduces infract size and neutrophil accumulation into the injured tissue during myocardial ischemia and reperfusion in the rat and in the mouse. The present investigation is designed to evaluate the molecular basis of endothelial expressions of P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecular-1 (VCAM-1) induced by reperfusion-dependent oxidant stress, with special focus on the PARS pathway. The pathophysiological relevance of a PARS-mediated regulation of endothelial expression of adhesion molecules will be evaluated in in vivo experimental rodent models of myocardial ischemia and reperfusion injury. Specifically, experiments will be performed in a murine model of myocardial ischemia and reperfusion in knock-out animals lacking the gene of PARS and in littermate wild-type controls. For clinical relevance, the effect of pharmacological inhibition of PARS on endothelial injury and neutrophil accumulation will be studied in a rat model of myocardial reperfusion injury. Furthermore, experiments will be performed in cultured human umbilical vein endothelial cells subjected to oxidative and non oxidative stress. These in vitro experiments are proposed in order to obtain a more precise assessment of the role of intracellular PARS on the adult human endothelial dysfunction relevant to the human myocardial infarction. The results of the proposed project will provide important information on the role of PARS as a modulator of the endothelial barrier function and will contribute to the development of effective therapeutic interventions of cardiovascular diseases characterized by endothelial reperfusion injury.