Microvascular Dysfunction: Impact Ischemia-Reperfusion Vascular Cell Interaction

缺血/再灌注（I/R）诱导毛细血管后白细胞/内皮细胞粘附相互作用（LECA） 小静脉和受损的内皮依赖性，NO介导的血管舒张反应（EDD）在上游 小动脉即使白细胞不会沿着滚动，粘附或迁移穿过动脉内皮， 最近的研究表明，I/R诱导的小静脉LECA与缺血后肠的EDD有因果关系， 小动脉然而，I/R诱导的毛细血管后微静脉LECA和EDD在上游的耦合机制可能是一个未知的问题。 小动脉未知。我们的总体假设是，I/R诱导的小动脉EDD是通过以下途径发生的 在毛细血管后微静脉中由LECA触发的机制，涉及在微静脉中信号的形成。 迁移的白细胞的蛋白水解活性引起的白细胞增多， 细胞外基质（ECM）与整合素avp 3相互作用，诱导肥大细胞依赖性的 血管紧张素II（Ang II）。随后NAD（P）H氧化酶的激活促进了eNOS的解偶联。 血管壁，并导致形成氧化剂，氧化剂使NO，导致小动脉EDD。我们 具体目的是确定：目的A）小静脉LECA是否在发展中发挥强制性作用， 目的B）白细胞衍生的蛋白酶对血管内皮细胞的发育的贡献。 C）avp 3整联蛋白在启动血管紧张素II的凝乳酶依赖性形成中的作用;以及 D）血管紧张素II依赖性、NAD（P）H氧化酶介导的和未偶联的eNOS介导的氧化剂产生在血管紧张素II依赖性、NAD（P）H氧化酶介导的氧化剂产生中的作用。 小动脉EDD将使用活体显微镜方法检查小动脉EDD、小静脉LECA， 和肥大细胞对I/R的反应。将从非缺血性肠中分离小动脉，并暴露于 缺血后淋巴，作为检查由蛋白水解活性产生的间质信号的手段 白细胞外渗一种新的三维ECM模型， 在存在和不存在中性粒细胞的情况下，也将使用小动脉和接种肥大细胞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离体小动脉将用于进一步探索小动脉EDD的机制。总的来说，这些目标提出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严重紊乱。重要性：这项工作将确定新的联系LECA毛细血管后微静脉，信号产生的迁移白细胞，和EDD在小动脉的血小板。考虑到内皮细胞在调节血管张力中的重要性，这些重要的发现具有巨大的意义。 对我们理解I/R条件下血流失调的意义。

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.