Novel Pathways in Ischemic Stroke in Sickle Cell Anemia

镰状细胞贫血症缺血性中风的新途径

• 批准号: 10200126
• 负责人: Jonathan Michael Flanagan
• 金额: $ 51.6万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10200126
• 关键词: Acute; Adhesions; Affect; Alleles; Animal Model; Biochemical; Biological; Biological Assay; Biomimetics; Blood Platelets; Blood Transfusion; Candidate Disease Gene; Caring; Cell Line; Chronic; Clinical; Code; Complex; Complication; Data; Development; Diphosphates; Disease; Endothelial Cells; Endothelium; Enzymes; Expression Profiling; Functional disorder; Funding; Genes; Genetic; Genetic Transcription; Goals; Golgi Apparatus; Heritability; Hydrolysis; In Vitro; Inflammatory; Injury; Insulin Receptor; Intervention; Ischemic Stroke; Kinetics; Link; Lipids; Liquid substance; Maintenance; Measures; Mediating; Mediator of activation protein; Microfluidics; Missense Mutation; Modeling; Modification; Molecular; Mutation; Output; Pathogenesis; Pathway interactions; Patients; Pediatric Hospitals; Physiological; Plasma; Process; Prognosis; Protein Activation Pathway; Protein Secretion; Proteins; Proteomics; Public Health; Receptor Signaling; Recurrence; Reporting; Research; Resources; Risk; Role; Sampling; Sickle Cell Anemia; Signal Transduction; Sorting - Cell Movement; Stenosis; Stroke; Stroke prevention; Structural Protein; Structure; System; Testing; Texas; Thrombosis; Thrombus; Variant; Vascular Diseases; Vascular Endothelium; calcification; cerebrovascular; cohort; exome sequencing; genetic variant; genome wide association study; high resolution imaging; hydroxyurea; improved; monocyte; novel; novel therapeutics; pediatric patients; protein expression; protein transport; response; shear stress; stroke risk; stroke therapy; targeted treatment; theories; thrombotic; von Willebrand Factor

Stroke is a devastating complication of sickle cell anemia (SCA) that will occur in ~11% of all patients without intervention. In pediatric patients with SCA, most of these strokes are ischemic and strongly linked with arterial vasculopathy. There is a strong heritable component that affects development of stroke in SCA patients. However, our understanding of the genes involved is very limited. Our group reported an unbiased genome wide association study of stroke in SCA using whole exome sequencing. We identified and validated two particular missense mutations in the GOLGB1 (Y1212C) and ENPP1 (K173Q) genes as being protective against stroke in SCA. Our study goal is to define the functional role of GOLGB1 and ENPP1 as modulators of vascular endothelial injury and ischemic stroke risk. We have previously observed that: a) GOLGB1 levels affect Golgi structure in monocytes and endothelial cells; b) The GOLGB1 Y1212C variant is associated with more dispersed Golgi in monocytes isolated from SCA patients; and c) ENPP1 enzymatic activity is higher in SCA patients with the protective K allele of the ENPP1 K173Q variant. From this preliminary data, we hypothesize that 1) GOLGB1 affects Golgi structure, which in turn regulates secretory output of pro-thrombotic factors such as von Willebrand factor (vWF) that can affect stroke in SCA; and 2) that ENPP1 affects risk of thrombosis either through endothelial cell signaling or by hydrolysis of the pro-inflammatory ATP inorganic pyrophosphate (PPi). We plan to test these hypotheses by analyzing the function of GOLGB1 and ENPP1 in samples from patients with SCA. We will isolate primary monocyte and endothelial cells from a large cohort of SCA patients receiving care at Texas Children’s Hospital.We will test our central hypotheses by pursuing three aims: Aim 1: Examine the role of GOLGB1 in the structure and function of Golgi complexes in sickle cell anemia - We will use high resolution imaging to determine the role of GOLGB1 in maintaining Golgi structure and protein trafficking in monocyte and endothelial cells from SCA patients; Aim 2: Determine the effect of ENPP1 activity in sickle cell anemia - We will measure plasma and endothelial activity of the ENPP1 enzyme.We will also test whether ENPP1 modulates vascular dysfunction in patients with SCA; Aim 3: Determine impact of GOLGB1 and ENPP1 on endothelial activation in a microfluidic arterial model - We will combine thrombotic protein assays and RNA expression profiling to identify endothelial activation pathways affected by our candidate genes. Our proposed studies will characterize the functional impact of our two genes and their genetic variants in SCA patient samples. This will benefit our understanding of the pathophysiological pathways of stroke and potentially provide targets for drug therapy to prevent or treat stroke in SCA patients.

中风是镰状细胞性贫血 (SCA) 的一种破坏性并发症，约 11% 的无症状患者会发生中风。 干涉。在患有 SCA 的儿科患者中，大多数中风是缺血性的，并且与动脉粥样硬化密切相关。 血管病变。有一个很强的遗传因素会影响 SCA 患者中风的发生。 然而，我们对所涉及基因的了解非常有限。我们的小组报告了全基因组的公正性 使用全外显子组测序进行 SCA 中风的关联研究。我们确定并验证了两个特定的 GOLGB1 (Y1212C) 和 ENPP1 (K173Q) 基因的错义突变可预防中风 斯卡。我们的研究目标是确定 GOLGB1 和 ENPP1 作为血管内皮细胞调节剂的功能作用 受伤和缺血性中风的风险。 我们之前观察到： a) GOLGB1 水平影响单核细胞和内皮细胞的高尔基体结构 细胞； b) GOLGB1 Y1212C 变体与从 SCA 分离的单核细胞中更分散的高尔基体相关 患者; c) 具有 ENPP1 保护性 K 等位基因的 SCA 患者中 ENPP1 酶活性较高 K173Q 变体。根据这些初步数据，我们假设 1) GOLGB1 影响高尔基体结构，进而影响高尔基体结构 调节促血栓形成因子的分泌输出，例如可影响中风的血管性血友病因子 (vWF) 在SCA； 2) ENPP1 通过内皮细胞信号传导或通过水解影响血栓形成的风险 促炎 ATP 无机焦磷酸盐 (PPi)。我们计划通过分析来检验这些假设 SCA 患者样本中 GOLGB1 和 ENPP1 的功能。我们将分离原代单核细胞和 来自德克萨斯儿童医院接受护理的一大群 SCA 患者的内皮细胞。我们将测试我们的 通过追求三个目标来提出中心假设： 目标 1：检查 GOLGB1 在结构和功能中的作用 高尔基复合体在镰状细胞性贫血中的作用 - 我们将使用高分辨率成像来确定 GOLGB1 在镰状细胞性贫血中的作用 维持 SCA 患者单核细胞和内皮细胞的高尔基体结构和蛋白质运输；目标 2： 确定 ENPP1 活性对镰状细胞性贫血的影响 - 我们将测量血浆和内皮活性 ENPP1酶。我们还将测试ENPP1是否调节SCA患者的血管功能障碍；目的 3：确定 GOLGB1 和 ENPP1 对微流体动脉模型中内皮激活的影响 - 我们将 结合血栓蛋白测定和 RNA 表达谱来识别内皮激活途径 受我们候选基因的影响。 我们提出的研究将描述我们的两个基因及其遗传变异的功能影响 SCA 患者样本中。这将有助于我们了解中风和中风的病理生理途径 可能为预防或治疗 SCA 患者中风的药物治疗提供目标。