Poly (ADP-Ribose) synthetase in expression of endothelia

血管内皮细胞损伤和中性粒细胞与血管内膜的黏附是心肌缺血再灌注损伤的早期病理事件。中性粒细胞转运到受损组织的确切分子机制尚不清楚。实验证据表明，氧化应激是白细胞内皮细胞黏附分子表达的重要信号转导事件。在体外和体内的初步研究中，我们发现氧化应激通过核酶多(ADP核糖)合成酶(PARS)介导的途径导致细胞功能障碍和损伤。在体外培养的内皮细胞中，过氧亚硝酸盐和过氧化氢诱导DNA单链断裂，激活PARS，导致细胞高能磷酸盐的耗竭，细胞功能障碍和死亡。我们发现，在大鼠和小鼠的心肌缺血和再灌注期间，药物或遗传抑制PARS可以减少心肌梗死面积和中性粒细胞在受损组织中的积聚。本研究旨在探讨氧化应激诱导血管内皮细胞表达P-选择素、E-选择素、细胞间黏附分子-1(ICAM-1)和血管黏附分子-1(VCAM-1)的分子基础。在心肌缺血和再灌注损伤的实验啮齿动物模型中，将评估PARS介导的内皮细胞黏附分子表达调节的病理生理学相关性。具体地说，实验将在缺乏pars基因的敲除动物的心肌缺血和再灌注小鼠模型和小鼠野生型对照中进行。对于临床意义，将在大鼠心肌再灌注损伤模型上研究PARS的药理抑制对内皮损伤和中性粒细胞聚集的影响。此外，还将在氧化和非氧化应激下培养的人脐静脉内皮细胞上进行实验。这些体外实验的提出是为了更准确地评估细胞内PARs在与人类心肌梗死相关的成人内皮功能障碍中的作用。该项目的结果将提供有关PARs作为内皮屏障功能调节器的重要信息，并将有助于开发以内皮再灌注损伤为特征的心血管疾病的有效治疗干预措施。

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.