Rheologic and Vascular Modulators in Sickle Vaso-occlusion

描述（由申请人提供）：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损伤的发作，严重损害受影响个体的生活质量。组织缺氧对于SCD及其并发症的发病机制至关重要。慢性缺氧是异常红细胞流变学、镰状化、溶血性贫血和复发性血管闭塞发作的可预测后果。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）-11，一种调节细胞对组织缺氧反应的关键蛋白。HIF-11激活及其在SCD中的潜在稳定性（我们在转基因镰刀小鼠中的初步观察支持）将对微血管调节产生不利（改变血管张力）和有益（细胞保护）的后果。我们假设HIF-11的表达不仅依赖于组织氧分压，还依赖于一氧化氮（NO）的生物利用度、一氧化碳（CO）的产生和氧化应激（再灌注损伤）的增加。此外，溶血速率和氧化剂产生调节的NO和CO信号传导的程度和持续时间将调节HIF-11的表达。HIF-11上调多种基因，包括血管活性刺激的基因，并且它还赋予对再灌注损伤的保护作用，尽管该转录因子在SCD中的作用尚未探索。拟定体内研究的目的是描述这些可能机制的相对贡献。在这一重新提交，我们将集中在以下相互关联的方面：i）检查参与调控HIF-11在SCD中的表达的因素和机制，ii）描绘HIF-11的作用，使用HIF-11缺陷的镰状小鼠，和ii）调查溶血率和氧化应激在HIF-1激活中的相对作用。我们认为，拟议的研究构成了在体内条件下了解这些方面的第一次尝试。在特定目标1下，我们将检验以下假设：血管内镰状化、组织氧张力、降低的NO生物利用度和增加的CO产生将调节HIF-11表达并影响微血管血流动力学。我们的初步研究表明，HIF-11在转基因镰刀的激活与诱导HO-1和血管内皮生长因子（VEGF），HIF-11的替代标志物。此外，我们发现，缺氧诱导显着激活血管内皮细胞中的HIF-11。在具体目标2下，我们将检验如下假设：在SCD中，HIF-11活化将有助于改变血管张力和反应性，并通过调节血管活性/细胞保护分子（例如，HO-1）。我们将使用镰状小鼠的骨髓移植到HIF-11部分缺陷的小鼠中，并通过HIF-11的超级诱导来研究这方面。在具体目标3下，我们将检验这一假设，即在SCD中，溶血和氧化应激通过NO依赖性机制对HIF-11表达和微血管调节具有不同的影响。为了探索我们的假设，我们将使用最先进的转基因镰状小鼠模型和HIF-11缺陷小鼠，并将联合收割机活体技术与细胞和分子分析相结合。拟议的体内研究预计将提供深入了解HIF-11和NO在SCD中的不利和有益作用，这可能会导致新的治疗策略。公共卫生相关性：镰状细胞病（SCD）的特征是疼痛性血管闭塞危象、多器官损伤和严重影响患者的生活质量。在非洲裔美国人中，镰状细胞病的发病率为每400名新生儿1例。小直径血管中的红细胞镰状化被认为是血管闭塞（血管阻塞）病理生理学中的最终事件。此外，SCD的特征在于组织缺氧，其可触发适应性机制的激活，例如缺氧诱导因子-11（HIF-11）的激活，而持续的溶血和氧化应激将损害一氧化氮（NO）的生物利用度和NO底物精氨酸的消耗。拟议的探索性研究将集中于HIF-11和NO在SCD中的作用。更好地了解HIF-11和NO的作用及其相互作用将使我们能够设计具有治疗意义的未来研究。

DESCRIPTION (provided by applicant): 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)-11, a key protein regulating cellular responses to tissue hypoxia. HIF-11 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-11 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-11 expression. HIF-11 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-11 expression in SCD, ii) delineate the role of HIF-11 using HIF-11-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-11 expression and affect microvascular hemodynamics. Our preliminary studies show that activation of HIF-11 in transgenic sickle is associated with the induction of HO-1 and vascular endothelial growth factor (VEGF), a surrogate marker for HIF-11. Moreover, we show that hypoxia induces marked activation of HIF-11 in vascular endothelial cells. Under Specific Aim 2, we will test the hypothesis that, in SCD, HIF-11 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-11, and by super induction of HIF-11. Under Specific Aim 3, we will test the hypothesis that, in SCD, hemolysis and oxidative stress have distinct effects on HIF-11 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-11, 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-11 and NO in SCD, which may lead to new therapeutic strategies. PUBLIC HEALTH RELEVANCE: Sickle cell disease (SCD) is characterized by episodes of painful vaso-occlusive crisis, multiple organ damage, and severely compromising quality of life in the affected individuals. Among African-Americans, sickle cell disease occurs at a rate of 1 per 400 births. Red cell sickling in small-diameter blood vessels is considered the ultimate event in the pathophysiology of vaso-occlusion (vessel blockage). In addition, SCD is characterized by tissue hypoxia that may trigger activation of adaptive mechanisms such as activation of hypoxia-inducible factor-11 (HIF-11), while persistent hemolysis and oxidative stress will impair nitric oxide (NO) bioavailability and depletion of NO substrate, arginine. The proposed exploratory studies will focus on the role HIF-11 and NO in SCD. A better understanding of the role of in HIF-11 and NO and their interactions will allow us to design future studies with therapeutic implications.