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.

卒中是镰状细胞性贫血（SCA）的一种毁灭性并发症，约11%的患者会发生卒中， 干预在患有SCA的儿科患者中，大多数卒中是缺血性的，与动脉粥样硬化密切相关。 血管病变有一个很强的遗传成分，影响中风的发展，在SCA患者。 然而，我们对相关基因的了解非常有限。我们的小组报告了一个无偏见的全基因组 使用全外显子组测序进行SCA中卒中的关联研究。我们确定并验证了两个特定的 GOLGB 1（Y1212 C）和ENPP 1（K173 Q）基因的错义突变对中风有保护作用， SCA。我们的研究目标是确定GOLGB 1和ENPP 1作为血管内皮细胞生长因子的调节剂的功能作用。 损伤和缺血性中风风险。 我们以前观察到：a）GOLGB 1水平影响单核细胞和内皮细胞中的高尔基体结构， B）GOLGB 1 Y1212 C变体与分离自SCA的单核细胞中更分散的高尔基体相关 c）在具有ENPP 1的保护性K等位基因的SCA患者中，ENPP 1酶活性更高 K173 Q变种。根据这些初步数据，我们假设1）GOLGB 1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如血管性血友病因子（vWF）的分泌， 在SCA中;和2）ENPP 1通过内皮细胞信号传导或通过 促炎性ATP无机焦磷酸盐（PPi）。我们计划通过分析这些假设来验证这些假设。 在来自SCA患者的样品中GOLGB 1和ENPP 1的功能。我们将分离初级单核细胞， 内皮细胞从一个大队列的SCA患者接受照顾在得克萨斯州儿童医院。我们将测试我们的 通过追求三个目标的中心假设：目标1：检查GOLGB 1在结构和功能中的作用， 镰状细胞性贫血中的高尔基复合体-我们将使用高分辨率成像来确定GOLGB 1在镰状细胞性贫血中的作用。 维持来自SCA患者的单核细胞和内皮细胞中的高尔基体结构和蛋白质运输;目的2： 确定ENPP 1活性在镰状细胞性贫血中的作用-我们将测量ENPP 1的血浆和内皮活性。 我们还将测试ENPP 1是否调节SCA患者的血管功能障碍;目的 3：确定GOLGB 1和ENPP 1对微流体动脉模型中内皮活化的影响-我们将 联合收割机和RNA表达谱分析鉴定内皮活化途径 受候选基因的影响 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 在SCA患者样本中。这将有助于我们理解中风的病理生理学途径， 潜在地为预防或治疗SCA患者的中风的药物治疗提供靶点。