Pericyte angiopoietin2 and neonatal intracranial hemorrhage

项目摘要/摘要 脑室出血(IVH)在任何年龄都是一种衰弱的情况，但尤其常见和 对早产儿是毁灭性的，因为它与大量的长期发病率有关(例如，脑性瘫痪， 认知缺陷)和死亡率。在早产儿中，大多数IVH源于 生发基质(GM)是位于其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(ECs)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 (转化生长因子)-β-1与白质和皮质相比，与GM的高倾向有关 大出血。事实上，我们之前曾报道，编码激活素受体样激酶5的基因缺失 I型转化生长因子β受体(ALK5)在PCs中的表达可导致胎鼠GM大出血 细胞，44：665)主要通过对内皮细胞的影响。然而，这些非细胞自主的机制 对ECs的影响还没有完全定义。为了寻找可能向EC发出信号的PC衍生因子，我们 对具有和不具有siRNA Alk5基因敲除和siRNA Alk5基因敲除的人脑PC进行了批量RNA测序 转化生长因子β_1治疗和鉴定的分泌因子血管生成素(Angpt)_2。 与EC管失稳和/或重塑有关，主要由ECs表达，但PC 目前还没有关于表达的报道。此外，ANGPT2，当然还有PC衍生的ANGPT2，在GMH- IVH尚未被研究过。随访研究证实，转化生长因子β-1对人脑PC的治疗作用迅速， 强势下调Angpt2mRNA水平(3h和12h分别降低~65%和~90%)和 蛋白质以依赖于Alk5的方式。这种Angpt2 mRNA的快速下降表明激动剂诱导了 转录失稳，我们将在拟议的实验中进行研究。此外，染色质 免疫沉淀分析显示转化生长因子β-1促进PC的表观遗传和转录 抑制Angpt2，增加组蛋白脱乙酰酶-2结合，减少组蛋白乙酰化(H3K9ac， H3K27ac)和与近端启动子结合的RNA聚合酶II。最后，在PC中有Alk5缺失的小鼠中， 血管周围ANGPT2表达明显增加。因此，我们假设当转化生长因子β-ALK5 PC中的信号转导中断，ANGPT2水平增加，使内皮细胞不稳定，并导致 BBB和GMH-IVH。我们将使用培养的人类脑血管细胞，转基因小鼠，蛋白质组和 3‘非编码区/microRNA筛选和未识别的人脑样本在两个特定目标上验证这一假说： 1)确定转化生长因子β介导的血管紧张素原2的调节机制及对内皮细胞的影响；2)阐明 PC-ANGPT2在小鼠和人GMH-IVH中的作用此R21将对血脑屏障的形成和 GMH-IVH发病机制，并形成后续高影响力R01赠款的基础，该基金将提供 为脑出血设计新的治疗策略的具体步骤。

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.