Rheologic and Vascular Modulators in Sickle Vaso-occlusion

镰状细胞病（SCD）以痛苦的血管闭塞危象和多器官损伤发作为特征，严重影响患者的生活质量。组织缺氧是SCD发病机制及其并发症的重要因素。慢性缺氧是红细胞流变异常、镰状细胞、溶血性贫血和反复发作的血管闭塞发作的可预见后果。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，这是调节细胞对组织缺氧反应的关键蛋白。HIF-11的激活及其在SCD中的潜在稳定性（我们在转基因镰状小鼠中的初步观察支持）将对微血管调节产生不利（血管张力改变）和有益（细胞保护）的后果。我们假设HIF-11的表达不仅取决于组织氧张力，还取决于一氧化氮（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。溶血率和氧化剂生成调节NO和CO信号的程度和持续时间也会调节HIF-11的表达。HIF-11上调多种基因，包括那些血管活性刺激基因，它也提供了对再灌注损伤的保护，尽管该转录因子在SCD中的作用尚未被探索。拟议的体内研究的目的是描述这些可能机制的相对贡献。在本次再提交中，我们将重点关注以下相关方面：1)检查与SCD中HIF-11表达调节相关的因素和机制，2)用HIF-11缺陷的镰状小鼠描述HIF-11的作用，以及2)研究溶血率和氧化应激在HIF-1激活中的相对作用。我们认为，拟议的研究是在体内条件下了解这些方面的第一次尝试。在Specific Aim 1中，我们将验证血管内镰状细胞增生、组织氧张力、一氧化氮生物利用度降低和一氧化碳生成增加会调节HIF-11表达并影响微血管血流动力学的假设。我们的初步研究表明，HIF-11在转基因镰状细胞中的激活与HO-1和血管内皮生长因子（VEGF）的诱导有关，VEGF是HIF-11的替代标志物。此外，我们发现缺氧诱导血管内皮细胞中HIF-11的显著激活。在Specific Aim 2中，我们将验证以下假设：在SCD中，HIF-11的激活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（如HO-1）的表达，赋予细胞保护作用，防止再灌注损伤。我们将利用镰状小鼠的骨髓移植到部分缺乏HIF-11的小鼠体内，并通过对HIF-11的超诱导来研究这方面的问题。在Specific Aim 3中，我们将检验假设，在SCD中，溶血和氧化应激通过no依赖机制对HIF-11表达和微血管调节有明显影响。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缺乏HIF-11的小鼠，并将活体技术与细胞和分子分析相结合。这些体内研究有望深入了解HIF-11和NO在SCD中的利弊作用，从而产生新的治疗策略。公共卫生相关性：镰状细胞病（SCD）的特点是发作痛苦的血管闭塞危象，多器官损害，严重影响患者的生活质量。在非裔美国人中，镰状细胞病的发病率为每400个新生儿中有1个。小直径血管中的红细胞镰状细胞被认为是血管闭塞（血管阻塞）病理生理学的最终事件。此外，SCD的特征是组织缺氧，可能会触发缺氧诱导因子-11 （HIF-11）激活等适应性机制的激活，而持续的溶血和氧化应激会损害一氧化氮（NO）的生物利用度和一氧化氮底物精氨酸的消耗。拟开展的探索性研究将重点关注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.