Pericyte angiopoietin2 and neonatal intracranial hemorrhage

周细胞血管生成素2与新生儿颅内出血

• 批准号: 10288547
• 负责人: Daniel Greif
• 金额: $ 46.06万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10288547
• 关键词: 3' Untranslated Regions; ANGPT2 gene; Age; Agonist; Angiopoietin-2; Angiopoietins; Apoptosis; Basement membrane; Binding; Biological Assay; Blood - brain barrier anatomy; Blood Vessels; Blood capillaries; Brain; Brain hemorrhage; CASP5 gene; Cell Proliferation; Cells; Cellular Morphology; Cerebral Palsy; Cerebral hemisphere hemorrhage; Cognitive deficits; Collection; Complication; Conditioned Culture Media; Embryo; Endothelial Cells; Ependyma; Epigenetic Process; Follow-Up Studies; Foundations; Future; Gene Deletion; Goals; Grant; HDAC2 gene; Hemorrhage; Histone Acetylation; Histone Deacetylase; Human; Immunohistochemistry; Intracranial Hemorrhages; Lead; Ligands; MADH2 gene; Mediating; Messenger RNA; MicroRNAs; Morbidity - disease rate; Mus; Neonatal; Neuraxis; Neurons; Pathogenesis; Pericytes; Perinatal subependymal hemorrhage; Permeability; Phenotype; Play; Population; Pregnancy; Premature Infant; Proteins; Proteomics; Publishing; RNA Polymerase II; Regulation; Reporting; Role; Sampling; Signal Transduction; Small Interfering RNA; TEK gene; TGF-beta type I receptor; TIMP3 gene; Testing; Tight Junctions; Tissues; Transcript; Transforming Growth Factor beta; Transforming Growth Factors; Transgenic Mice; Tube; Ventricular; blood-brain barrier disruption; blood-brain barrier permeabilization; brain endothelial cell; chromatin immunoprecipitation; experimental study; follow-up; gene repression; histone deacetylase 2; human RNA sequencing; insight; intraventricular hemorrhage; knock-down; migration; mortality; mutant; novel strategies; novel therapeutic intervention; novel therapeutics; prevent; promoter; receptor; stem; transcriptomics; white matter

Project Summary / Abstract Intraventricular hemorrhage (IVH) is a debilitating condition at any age but is especially common and devastating in premature infants as it is associated with substantial long-term morbidity (e.g., cerebral palsy, cognitive deficits) and mortality in this population. In premature infants, most IVH stems from hemorrhage in the germinal matrix (GM), a collection of highly vascularized neuronal and glial precursor located underneath the ventricular ependyma. Pericytes (PCs) and endothelial cells (ECs) are key components of the blood-brain barrier (BBB). In the GM, reduction in the numbers of PCs and levels of the ligand transforming growth factor (TGF)-β1 in comparison to the white matter and cortex has been implicated in the high propensity of the GM to hemorrhage. Indeed, we previously reported that deletion of the gene encoding activin receptor-like kinase 5 (ALK5), a type I TGFβ receptor, in PCs leads to gross GM hemorrhage (GMH)-IVH in embryonic mice (Dev Cell, 44:665) largely through effects on ECs. However, mechanisms underlying these non-cell autonomous effects on ECs are incompletely defined. To search for PC-derived factors that may signal to ECs, we conducted bulk RNA-sequencing of human brain PCs with and without both siRNA Alk5 knockdown and TGFβ1 treatment and identified the secreted factor angiopoietin (Angpt) 2. ANGPT2, which is broadly implicated in EC tube destabilization and/or remodeling, is predominantly expressed by ECs, but PC expression has not been reported. Moreover, the role ANGPT2, and certainly PC-derived ANGPT2, in GMH- IVH has not been studied. Follow-up studies confirmed that TGFβ1 treatment of human brain PCs rapidly and robustly down-regulates levels of Angpt2 mRNA (~65% and ~90% reduction in 3 h and 12 h, respectively) and protein in an ALK5-dependent manner. This rapid reduction in Angpt2 mRNA suggests agonist-induced transcript destabilization, which we will investigate in the proposed experiments. Furthermore, chromatin immunoprecipitation assays revealed that TGFβ1 treatment of PCs promotes epigenetic and transcriptional repression of Angpt2, increasing histone deacetylase-2 binding and reducing histone acetylation (H3K9ac, H3K27ac) and RNA polymerase II binding to the proximal promoter. Finally, in mice with Alk5 deletion in PCs, perivascular ANGPT2 expression is markedly increased. Thus, we hypothesize that when TGFβ-ALK5 signaling in PCs is disrupted, ANGPT2 levels are increased, destabilizing ECs and leading to disruption of the BBB and GMH-IVH. We will use cultured human brain vascular cells, transgenic mice, proteomic and 3’UTR/microRNA screens and de-identified human brain samples to test this hypothesis in two specific aims: 1) determine mechanisms of TGFβ-mediated regulation of Angpt2 in PCs and effects on ECs; and 2) elucidate role of PC ANGPT2 in GMH-IVH of mice and humans. This R21 will yield key insights into BBB formation and GMH-IVH pathogenesis and form the foundation of a subsequent high impact R01 grant that will provide concrete steps towards devising novel therapeutic strategies for brain hemorrhage.

项目总结/摘要 脑室内出血（IVH）在任何年龄都是一种使人衰弱的疾病，但特别常见， 在早产儿中是毁灭性的，因为它与大量的长期发病率有关（例如，脑瘫， 认知缺陷）和死亡率。在早产儿中，大多数IVH是由出血引起的， 生发基质（GM），位于下方的高度血管化的神经元和神经胶质前体的集合 心室室管膜周细胞和内皮细胞是血脑的重要组成部分 屏障（BBB）。在GM中，PC数量和配体转化生长因子水平的减少 与白色物质和皮质相比，（TGF）-β1与GM的高倾向性有关， 出血事实上，我们先前报道了编码激活素受体样激酶5的基因的缺失， （ALK 5），一种I型TGFβ受体，在PC中导致胚胎小鼠中的大体GM出血（GMH）-IVH（Dev Cell，44：665）主要通过对EC的作用。然而，这些非细胞自主的机制， 对EC的影响不完全确定。为了寻找可能向EC发出信号的PC衍生因子，我们 进行了具有和不具有siRNA Alk 5敲低的人脑PC的批量RNA测序， TGFβ1治疗并鉴定了分泌因子血管生成素（Angpt）2。ANGPT 2，它广泛地 与EC管不稳定和/或重塑有关的PC主要由EC表达，但PC 表达没有被报道。此外，ANGPT 2，当然还有PC衍生的ANGPT 2，在GMH-1中的作用， IVH尚未研究。后续研究证实，TGFβ1可快速且有效地治疗人脑PC 稳健下调Angpt 2 mRNA水平（3 h和12 h分别降低~65%和~90%）， 以ALK 5依赖的方式表达蛋白质。Angpt 2 mRNA的快速减少表明激动剂诱导的 转录不稳定，我们将在拟议的实验中调查。此外，染色质 免疫沉淀分析显示，TGFβ1处理PC促进表观遗传和转录， 抑制Angpt 2，增加组蛋白脱乙酰酶-2结合并减少组蛋白乙酰化（H3 K9 ac， H3 K27 ac）和RNA聚合酶II结合至近端启动子。最后，在PC中Alk 5缺失的小鼠中， 血管周围ANGPT 2表达显著增加。因此，我们假设当TGFβ-ALK 5 PC中的信号传导被破坏，ANGPT 2水平增加，使EC不稳定，并导致 BBB和GMH-IVH。我们将使用培养的人脑血管细胞，转基因小鼠，蛋白质组学和 3 'UTR/microRNA筛选和去识别人脑样品以在两个特定目标中测试该假设： 1)确定PC中TGFβ介导的Angpt 2调节机制和对EC的影响; 2）阐明 PC ANGPT 2在小鼠和人类GMH-IVH中的作用。这种R21将产生对BBB形成的关键见解， GMH-IVH发病机制，并形成随后的高影响R 01赠款的基础， 制定脑出血新治疗策略的具体步骤。