Functional Analysis of Vascular NTPDases

描述(由申请人提供)： 细胞外核苷酸(如ATP、ADP、UTP)为在血管系统内工作的血小板和细胞提供重要的环境信号。这些介体激活血小板、内皮、血管平滑肌和白细胞上的2型嘌呤能/嘧啶能(P2Y和P2X)受体。这一过程调节了血小板的活化和黏附、细胞代谢、一氧化氮(NO)的释放、内皮的激活、增殖和凋亡。胞外核苷酸酶水解胞外核苷酸，最终形成相应的核苷。这种胞外酶的CD39家族成员似乎在血管系统中有高水平的差异表达。血管内皮细胞(EC)和血管平滑肌细胞(VSMC)表达CD39/NTPDase1，CD39L(IKE)1/NTPDase2与CD39L(IKE)1/NTPDase2相关，CD39L(IKE)1/NTPDase2与CD39L(IKE)1/NTPDase2有关。在小鼠中，CD39的全局缺失会导致止血缺陷、炎症变化和血栓调节紊乱，这些紊乱会扰乱血管稳态，并阻止移植物的长期存活。由于这些突变小鼠出人意料地表现出胰岛素抵抗，胰岛素(和其他生长因子)的作用可能也受到细胞外核苷酸的调节。这项应用解决了这样一种可能性，即CD39和CD39L1在血管系统中的差异表达对P2介导的血小板和血管细胞反应的时间和空间调节具有重要影响。我们认为，在血管炎症部位，NTPDase可能调节血栓形成或血管重塑，并调节局部的“代谢环境”，例如在再灌流的器官、损伤的血管或血管化的移植物中。具体目的1：利用CD39和/或CD39L1缺失突变小鼠，通过共聚焦显微镜观察动脉血栓的急性发展，探讨CD39/NTPDase1和CD39L1/NTPDase2对急性和慢性血管损伤的影响。我们还将研究这些血管NTPDase如何在体内改变血管壁损伤更持久的VSMC反应。特异性目的2：确定CD39/NTPDase1调节急性血管损伤和移植物存活的机制。我们将研究CD39的表达如何直接和间接地影响EC和VSMC的细胞激活/凋亡与胰岛素反应性，以探索这一现象的相关性。具体目的3：评估NTPDase如何与内皮型一氧化氮合酶(ENOS)相互作用。我们将在上面提出的实验模型中，通过调节CD39和/或eNOS缺陷的野生型和突变小鼠的NTPDase和NO的作用，来确定它们是相加的还是协同的。了解两种主要的血管NTPDase和eNOS各自的功能将有助于深入了解急性血管损伤和局限性血管病变的机制。从这些研究中收集的信息可能会指导炎症性血管疾病的新治疗策略，包括动脉粥样硬化性心血管疾病。

DESCRIPTION (provided by applicant): Extracellular nucleotides (e.g. ATP, ADP, UTP) provide important environmental signals to platelets and cells, that areoperative within the vasculature. These mediators activate type-2 purinergic/ pyrimidinergic (P2Y and P2X) receptors on platelets, endothelium, vascular smooth muscle, and leukocytes. This process modulates platelet activation andadhesion, cellular metabolism, nitric oxide (NO) release, endothelial activation, proliferation and apoptosis. Ectonucleotidases hydrolyze extra-cellular nucleotides, ultimately to the respective nucleosides. Members of the CD39 family of such ectoenzymes appear to be differentially expressed at high levels within the vasculature. Endothelial cells (EC) and vascular smooth muscle cells (VSMC) express CD39/NTPDase1, which has both ecto-ATPase and -ADPase activities, while pericytes are associated with CD39L(ike)1/NTPDase2, a preferential ecto-ATPase. Global deletion of cd39 in mice results in hemostatic defects, inflammatory changes with thromboregulatory disturbances that perturb vascular homeostasis and preclude long term transplant graft survival. As these mutant mice unexpectedly exhibit insulin resistance, actions of insulin (and other growth factors) may be also modulated by extracellular nucleotides. This application addresses the possibility that the differential expression of CD39 and CD39L1 by the vasculature has important consequences for the temporal and spatial modulation of P2-mediated platelet and vascular cellular reactions. We propose that at sites of vascular inflammation, NTPDases may regulate thrombogenesis or vascular remodeling and modulate the local "metabolic milieu" e.g. in reperfused organs, injured vessels or vascularized grafts. SPECIFIC AIM 1: Investigate how CD39/NTPDase1 and CD39L1/NTPDase2 influence acute and chronic vascular injury by studying the acute development of arterial thrombi by confocal microscopy using mutant mice null for cd39 and/or cd39L1. We will also study how more protracted VSMC responses to vascular wall injury are altered by these vascular NTPDases in vivo. SPECIFIC AIM 2: Determine the mechanisms whereby CD39/NTPDase1 modulates acute vascular injury and graft survival. We will examine how CD39 expression both directly and indirectly influences cell activation/apoptosis with insulin responsiveness in EC and VSMC to explore the relevance of this phenomenon. SPECIFIC AIM 3: Evaluate how NTPDases interact with endothelial nitric oxide synthases (eNOS). We will determine whether effects of NTPDases and NO are additive or synergistic by regulating each in wild type and mutant mice, deficient in cd39 and/or eNOS, in the experimental models proposed above. An understanding of the respective functions of the two major vascular NTPDases and eNOS should provide insights into mechanisms of acute vascular injury and localized vasculopathy. Information gleaned from these studies may direct new therapeutic strategies for inflammatory vascular disorders, including atherosclerotic cardiovascular disease.