Rheologic and Vascular Modulators in Sickle Vaso-occlusion

镰状血管闭塞中的流变学和血管调节剂 摘要 镰状细胞病（SCD）的特征是疼痛性血管闭塞危象和多器官功能障碍。 损害，严重影响受影响个人的生活质量。组织缺氧是主要的 重要性的发病机制的SCD及其伴随的并发症。慢性缺氧是可预测的 红细胞流变学异常、镰状化、溶血性贫血和复发性血管闭塞的后果 情节。在SCD中，慢性组织缺氧和氧化应激可能激活缺氧诱导因子 （HIF）-1？，一种调节细胞对组织缺氧反应的关键蛋白。HIF-1的激活及其潜力 SCD的稳定（我们在转基因镰状小鼠中的初步观察支持）将具有两种 对微血管调节的不利（血管张力改变）和有益（细胞保护）后果。 我们假设HIF-1 <$的表达不仅依赖于组织氧分压，而且还依赖于组织中的氧分压。 一氧化氮（NO）生物利用度、一氧化碳（CO）生成和氧化应激增加 （再灌注损伤）。此外，溶血率和溶血时间对NO和CO信号传导的调节程度和持续时间也有影响。 氧化剂的产生将调节HIF-1的表达。HIF-1上调多种基因，包括 对于血管活性刺激，它也赋予对再灌注损伤的保护，尽管这种作用 转录因子在SCD中的作用尚不清楚。拟定体内研究的目的是描述 这些可能机制的相对贡献。在这次重新提交中，我们将重点关注以下内容 相关方面：i）检查参与HIF-1表达调节的因素和机制 在SCD中，ii）使用HIF-1缺陷型镰状小鼠描述HIF-1的作用，ii）研究 在HIF-1活化中溶血率和氧化应激。我们认为，拟议的研究构成了 第一次尝试在体内条件下理解这些方面。在具体目标1下，我们将测试 假设血管内镰状化，组织氧张力，降低NO生物利用度和增加CO HIF-1的产生将调节HIF-1的表达并影响微血管血流动力学。我们的初步 研究表明，转基因镰刀中HIF-1的激活与HO-1的诱导有关， 血管内皮生长因子（VEGF），HIF-1的替代标记物。此外，我们表明，缺氧 诱导血管内皮细胞中HIF-1？的显著激活。在具体目标2下，我们将测试 假设在SCD中，HIF-1？激活将有助于改变血管张力和反应性， 通过调节血管活性/细胞保护分子的表达对再灌注损伤的细胞保护 (e.g., HO-1）。我们将利用镰状小鼠的骨髓移植来研究这方面的问题 部分缺乏HIF-1，并通过HIF-1的超诱导。在具体目标3下，我们将测试 假设在SCD中，溶血和氧化应激对HIF-1 <$的表达有不同的影响， 通过NO依赖机制的微血管调节。为了探索我们的假设，我们将使用 ART转基因镰状小鼠模型和HIF-1缺陷小鼠，并将联合收割机活体内技术与 细胞和分子分析。预期所提出的体内研究将提供对 HIF-1和NO在SCD中的不利和有益作用，这可能导致新的治疗策略。

Rheologic and Vascular Modulators in Sickle Vaso-occlusion ABSTRACT Sickle cell disease (SCD) is characterized by episodes of painful vaso-occlusive crisis and multiple organ damage, severely compromising the quality of life in the affected individuals. Tissue hypoxia is of primary importance to the pathogenesis of SCD and its attendant complications. Chronic hypoxia is a predictable consequence of abnormal red cell rheology, sickling, hemolytic anemia and recurring vaso-occlusive episodes. In SCD, chronic tissue hypoxia and oxidative stress are likely to activate hypoxia inducible factor (HIF)-1¿, a key protein regulating cellular responses to tissue hypoxia. HIF-1¿ activation and its potential stabilization in SCD (supported by our preliminary observation in transgenic sickle mice) will have both adverse (altered vascular tone) and beneficial (cytoprotective) consequences on microvascular regulation. We hypothesize that HIF-1¿ expression will be dependent not only on tissue oxygen tension, but also on nitric oxide (NO) bioavailability, carbon monoxide (CO) generation and increased oxidative stress (reperfusion injury). Also, the extent and duration of NO and CO signaling modulated by hemolytic rate and oxidant generation will regulate HIF-1¿ expression. HIF-1¿ up-regulates a variety of genes including those for vasoactive stimuli and it also confers protection against reperfusion injury although the role of this transcription factor remains unexplored in SCD. The objective of the proposed in vivo studies is to delineate the relative contribution of these likely mechanisms. In this resubmission, we will focus on the following interrelated aspects: i) examine the factors and mechanisms involved in the regulation of HIF-1¿ expression in SCD, ii) delineate the role of HIF-1¿ using HIF-1¿-deficient sickle mice, and ii) investigate relative roles of hemolytic rate and oxidative stress in HIF-1 activation. We believe that the proposed studies constitute the first attempt to understand these aspects under in vivo conditions. Under Specific Aim 1, we will test the hypothesis that intravascular sickling, tissue oxygen tension, reduced NO bioavailability and increased CO production will regulate HIF-1¿ expression and affect microvascular hemodynamics. Our preliminary studies show that activation of HIF-1¿ in transgenic sickle is associated with the induction of HO-1 and vascular endothelial growth factor (VEGF), a surrogate marker for HIF-1¿. Moreover, we show that hypoxia induces marked activation of HIF-1¿ in vascular endothelial cells. Under Specific Aim 2, we will test the hypothesis that, in SCD, HIF-1¿ activation will contribute to altered vascular tone and reactivity and confer cytoprotection against reperfusion injury by modulating expression of vasoactive/cytoprotective molecules (e.g., HO-1). We will investigate this aspect using bone marrow transplantation from sickle mice into mice partially deficient in HIF-1¿, and by super induction of HIF-1¿. Under Specific Aim 3, we will test the hypothesis that, in SCD, hemolysis and oxidative stress have distinct effects on HIF-1¿ expression and microvascular regulation via NO-dependent mechanism. To explore our hypotheses, we will use state-of-the art transgenic sickle mouse models and mice deficient in HIF-1¿, and combine intravital techniques with cellular and molecular analyses. The proposed in vivo studies are expected to provide insights into the adverse and beneficial effects of HIF-1¿ and NO in SCD, which may lead to new therapeutic strategies.