Functional Analysis of Vascular NTPDases

描述（由申请人提供）： 细胞外核苷酸（如ATP、ADP、UTP）为血小板和细胞提供重要的环境信号，这些信号在血管系统中起作用。这些介质激活血小板、内皮、血管平滑肌和白细胞上的2型嘌呤能/嘧啶能（P2 Y和P2 X）受体。该过程调节血小板活化和粘附、细胞代谢、一氧化氮（NO）释放、内皮活化、增殖和凋亡。外核苷酸酶水解细胞外核苷酸，最终水解成相应的核苷。这些胞外酶的CD 39家族成员似乎在血管系统内以高水平差异表达。内皮细胞（EC）和血管平滑肌细胞（VSMC）表达CD 39/NTPDase 1，该酶具有胞外ATP酶和-ADP酶活性，而周细胞则与CD 39 L（ike）1/NTPDase 2（一种优先的胞外ATP酶）相关。小鼠cd 39的整体缺失导致止血缺陷、炎症变化和血栓调节紊乱，扰乱血管稳态并妨碍移植物的长期存活。由于这些突变小鼠出乎意料地表现出胰岛素抵抗，胰岛素（和其他生长因子）的作用也可能受到细胞外核苷酸的调节。本申请提出了这样的可能性，即血管系统的CD 39和CD 39 L1的差异表达对于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的后果。我们提出，在血管炎症部位，NTPD酶可以调节血栓形成或血管重塑，并调节局部“代谢环境”，例如在再灌注器官、受损血管或血管化移植物中。具体目标1：通过使用cd 39和/或cd 39 L1缺失的突变小鼠，通过共聚焦显微镜研究动脉血栓的急性发展，研究CD 39/NTPDase 1和CD 39 L1/NTPDase 2如何影响急性和慢性血管损伤。我们还将研究如何更持久的VSMC血管壁损伤的反应是由这些血管NTPDases在体内改变。具体目的2：确定CD 39/NTPD酶1调节急性血管损伤和移植物存活的机制。我们将研究CD 39表达如何直接和间接影响EC和VSMC中胰岛素反应性的细胞活化/凋亡，以探讨这种现象的相关性。具体目的3：评价NTPD酶如何与内皮型一氧化氮合酶（eNOS）相互作用。我们将确定NTPDases和NO的作用是否是相加的或协同的，通过调节野生型和突变型小鼠，在cd 39和/或eNOS缺陷，在上述提出的实验模型。了解这两种主要的血管NTPDases和eNOS的各自功能，将有助于深入了解急性血管损伤和局部血管病变的机制。从这些研究中收集的信息可能会指导炎症性血管疾病，包括动脉粥样硬化性心血管疾病的新的治疗策略。

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.