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.

项目概要/摘要 脑室内出血（IVH）在任何年龄段都是一种使人衰弱的疾病，但尤其常见且 对早产儿来说是毁灭性的，因为它与大量的长期发病有关（例如脑瘫， 认知缺陷）和该人群的死亡率。在早产儿中，大多数 IVH 源于出血 生发基质 (GM)，是位于下方的高度血管化的神经元和神经胶质前体的集合 室管膜。周细胞 (PC) 和内皮细胞 (EC) 是血脑的关键组成部分 屏障（BBB）。在 GM 中，PC 数量和配体转化生长因子水平减少 与白质和皮质相比，(TGF)-β1 与 GM 的高倾向有关 出血。事实上，我们之前报道过编码激活素受体样激酶 5 的基因缺失 PC 中的 I 型 TGFβ 受体 (ALK5) 会导致胚胎小鼠出现严重 GM 出血 (GMH)-IVH (Dev Cell，44:665）主要是通过对 EC 的影响。然而，这些非细胞自主的机制 对 EC 的影响尚未完全确定。为了寻找可能向 EC 发出信号的 PC 衍生因子，我们 对人脑 PC 进行了批量 RNA 测序，有或没有 siRNA Alk5 敲低和 TGFβ1 治疗并鉴定出分泌因子血管生成素 (Angpt) 2。ANGPT2，广泛应用于 涉及 EC 管不稳定和/或重塑，主要由 EC 表达，但 PC 表达尚未见报道。此外，ANGPT2（当然还有 PC 衍生的 ANGPT2）在 GMH 中的作用 尚未研究 IVH。后续研究证实TGFβ1对人脑PC的治疗迅速且有效 强力下调 Angpt2 mRNA 水平（3 小时和 12 小时分别降低约 65% 和约 90%） 以 ALK5 依赖性方式表达蛋白质。 Angpt2 mRNA 的快速减少表明激动剂诱导的 转录不稳定，我们将在提议的实验中研究这一点。此外，染色质 免疫沉淀分析表明，TGFβ1 对 PC 的处理可促进表观遗传和转录 抑制 Angpt2，增加组蛋白脱乙酰酶 2 结合并减少组蛋白乙酰化（H3K9ac， H3K27ac) 和 RNA 聚合酶 II 与近端启动子结合。最后，在 PC 中 Alk5 缺失的小鼠中， 血管周围ANGPT2表达显着增加。因此，我们假设当 TGFβ-ALK5 PC 中的信号传导被破坏，ANGPT2 水平升高，破坏 EC 的稳定性并导致 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 筛选和去识别化的人脑样本以在两个特定目标上检验这一假设： 1) 确定 PC 中 TGFβ 介导的 Angpt2 调节机制以及对 EC 的影响； 2) 阐明 PC ANGPT2 在小鼠和人类 GMH-IVH 中的作用。该 R21 将提供有关 BBB 形成和 GMH-IVH 发病机制，并构成后续高影响 R01 资助的基础，该资助将提供 制定脑出血新治疗策略的具体步骤。