MODULATION OF VASCULAR FUNCTION BY HYPOXEMIA/ISCHEMIA

低氧血症/组织缺氧(H)长期以来一直与促凝剂事件有关，特别是静脉血栓形成，这是发病率和死亡率的主要原因。实验性肢体固定导致血氧张力快速下降，同时纤维蛋白沉积在静脉瓣膜袋中，从而为血栓形成提供了一个中心。在一个常压缺氧模型中，我们提供了缺氧触发凝血的途径的第一个轮廓：缺氧上调单核吞噬细胞（MPs）中的组织因子和纤溶酶原激活物抑制剂(PAI)-1，最终导致血管纤维蛋白沉积。我们假设在这个h触发的通路中有两个关键的和意想不到的事件，它们都独立于缺氧诱导因子(HIF)-1：蛋白激酶C异构体β aii （PKCbetaII）的激活和转录因子Egr-1的激活。在缺氧MPs中，组织因子诱导是由于启动子中Egr-1位点的转录增加，Egr-1缺失小鼠在缺氧条件下表现出缺乏组织因子诱导和血管纤维蛋白沉积。我们的体外研究已经将h相关通路最终导致Egr-1转录追溯到PKCbetaII：缺氧激活PKCbetaII，后者触发raf和mek依赖通路激活MAP激酶和Elk-1；激活的Elk-1与血清反应因子协同作用，诱导Egr-1的转录。我们的基本概念是，在缺氧的最早阶段发生的事件，PKCbetaII和Egr-1激活（在H后几分钟内出现），对低氧血症血管中血栓性灶的形成至关重要。我们的第一个目标是利用pkcβ O小鼠来评估pkcβ在h相关血管纤维蛋白形成中的作用，并通过分析pkcβ O和Egr-10小鼠的反应，将我们的h诱导血管扰动的概念扩展到肺缺血模型。我们的第二个目标是通过制造和分析MPs中PKCbetaII靶向抑制或激活的转基因小鼠，解决MPs中PKCbetaII激活是驱动促凝机制的关键事件的假设，以及导致缺血性组织损伤的其他途径。我们的总体目标是确定PKCbetaII和Egr-1是否在低氧血症和缺血性应激反应中作为维持血管稳态的潜在治疗靶点。

Hypoxemia/tissue hypoxia (H) has long been associated with procoagulant events, especially venous thrombosis, a major cause of morbidity and mortality. Experimental limb immobilization leads to a rapid fall in blood oxygen tension in parallel with fibrin deposition in venous valve pockets, thereby providing a nidus for thrombus formation. In a model of normobaric hypoxia, we have provided the first outline of a pathway through which oxygen deprivation triggers coagulation: hypoxia upregulates tissue factor and plasminogen activator inhibitor (PAI)-1 in mononuclear phagocytes (MPs), ultimately causing vascular fibrin deposition. We hypothesize that there are two pivotal and unexpected events in this H-triggered pathway, both of which are independent of hypoxia-inducible factor (HIF)-1: activation of protein kinase C isoform betaII (PKCbetaII) and activation of the transcription factor Egr-1. Tissue factor induction in hypoxic MPs is due to increased transcription at Egr-1 sites in the promoter, and Egr-1 null mice subject to oxygen deprivation display absence of tissue factor induction and vascular fibrin deposition. Our in vitro studies have traced the H-associated pathway culminating in Egr-1 transcription back to PKCbetaII: hypoxia activates PKCbetaII, the latter triggers a raf- and MEK-dependent pathway activating MAP kinases and Elk-1; and, activated Elk-1, in concert with Serum Response Factor, induces transcription of Egr-1. Our underlying concept is that events occurring at the earliest stage of oxygen deprivation, PKCbetaII and Egr-1 activation (each seen within minutes of H), are critical to the formation of thrombogenic foci within hypoxemic vasculature. Our first aim is to evaluate the role of PKCbeta in H-associated vascular fibrin formation using PKCbeta O mice, and to extend our concept of H-induced vascular perturbation to a model of lung ischemia by analyzing the response of PKCbeta O and Egr-10 mice. Our second aim addresses the hypothesis that activation of PKCbetaII in MPs is a key event driving the procoagulant mechanism, as well as other pathways contributing to ischemic tissue injury, by making and analyzing transgenic mice with targeted suppression or activation of PKCbetaII in Mps. Our overall goal is to determine if PKCbetaII and Egr-1 are potential therapeutic targets for preserving vascular homeostasis in response to hypoxemic and ischemic stress.