Microvascular Dysfunction: Impact Ischemia-Reperfusion Vascular Cell Interaction

缺血再灌注诱导毛细血管后区白细胞/内皮细胞黏附相互作用 上游小静脉和受损的内皮依赖性、一氧化氮介导的血管舒缩反应(EDD) 小动脉。即使白细胞不会沿小动脉内皮细胞滚动、黏附或迁移 肠缺血后，最近的工作表明L/R诱导的小静脉LECA与EDD有因果关系 小动脉。然而，L/R诱导的毛细血管后静脉LECA和EDD在上游的耦合机制 小动脉是未知的。我们的总体假设是，L/R诱导的小动脉EDD是通过一种 这一机制由LECA在毛细血管后小静脉中触发，并涉及在 由迁出的白细胞的蛋白分解活性引起的间质，它暴露了细胞内的基质部位 细胞外基质(ECM)与整合素avp3相互作用，诱导肥大细胞依赖的形成 血管紧张素II(Ang II)。NAD(P)H氧化酶的后续激活促进eNOS解偶联 血管壁并导致氧化剂的形成，使NO失活，导致小动脉EDD。我们的 具体目的是确定：目的A)静脉LECA是否在糖尿病的发生发展中起重要作用 目的B)白细胞衍生的蛋白水解酶在糖尿病的发生发展中的作用 目的C)avp3整合素在启动血管紧张素转换酶依赖的血管紧张素Ⅱ形成中的作用 D)依赖血管紧张素Ⅱ、NAD(P)H氧化酶和解偶联eNOS介导的氧化剂产生在 小动脉EDD。活体显微镜方法将被用来检查小动脉EDD，静脉LECA， 肥大细胞对I/R的反应将从非缺血区分离并暴露于 化疗后淋巴，作为检查由蛋白分解活性产生的间质信号的一种手段 渗出的白细胞。一种新型的带隔离空腔植入的三维ECM模型 在存在和不存在中性粒细胞的情况下，小动脉和种植肥大细胞也将被用来进一步探索我们的假设。从一些基因敲除和转基因动物模型中获得的分离的小动脉将被用来进一步探索小动脉EDD的机制。总而言之，这些目标解决了一种新的机制来解释缺血后组织中小动脉血管调节功能的深刻障碍。意义：这项工作将确定毛细血管后小静脉中的LECA、迁移的白细胞在间质中产生的信号以及小动脉中的EDD之间的新联系。鉴于内皮细胞在调节血管张力中的重要性，这些根本性的重要发现具有巨大的意义。 对我们理解以I/R为特征的血流失调的启示

Ischemia/reperfusion (I/R) induces leukoeyte/endothelial cell adhesive interactions (LECA) in postcapillary venules and impaired endothelium-dependent, NO-mediated vasodllatory responses (EDD) in upstream arterioles. Even though leukocytes do not roll along, adhere to, or emigrate across arteriolar endothelium in postischemic intestine, recent work indicates that l/R-induced venular LECA is causally linked to EDD in arterioles. However, the mechanisms coupling l/R-induced postcapillary venular LECA and EDD in upstream arterioles are unknown. Our overall hypothesis is that l/R-induced EDD in arterioles occurs by a mechanism that is triggered by LECA in postcapillary venules and involves the formation of signals in the interstitium elicited by the proteolytic activity of emigrated leukocytes which exposes matricryptic sites in the extracellular matrix (ECM) that interact with the integrin avp3 to induce mast cell-dependent formation of angiotensin II (Ang II). Subsequent activation of NAD(P)H oxidase promotes eNOS uncoupling in the vascular wall and leads to the formation of oxidants which inactivate NO, resulting in arteriolar EDD. Our Specific Aims are to determine: Aim A) whether venular LECA play an obligatory role in the development of EDD in upstream arterioles; Aim B) the contribution of leukocyte-derived proteases to the development of arteriolar EDD; Aim C) the role of avp3 integrin in initiating chymase-dependent formafion of Ang II; and Aim D) the role of Ang ll-dependent, NAD(P)H oxidase- and uncoupled eNOS-mediated oxidant production in arteriolar EDD. Intravital microscopic approaches will be used to examine arteriolar EDD, venular LECA, and mast cell responses to I/R. Arterioles will be isolated from non-ischemic intesfine and exposed to posfischemic lymph, as a means to examine interstitial signals that are generated by the proteolytic acfivity of extravasated leukocytes. A novel three-dimensional ECM model engrafted with isolated cannulated arterioles and seeded with mast cells in the presence and absence of neutrophils will also be used to to further explore our hypothesis. Isolated arterioles obtained from a number of gene knockout and transgenic animal models will be used to further explore the mechanisms of arteriolar EDD. Collectively, these aims address a novel mechanism to explain the profound disturbance in arteriolar vasoregulatory funcfion in postischemic tissues. Significance: This work will identify new links between LECA in postcapillary venules, signals generated in the interstitium by emigrated leukocytes, and EDD in arterioles. Given the importance of the endothelium in regulating vascular tone, these fundamentally important findings have enormous implications for our understanding of blood flow dysregulation in conditions characterized by I/R.