Mural cell TGF-beta-mediated signaling and neonatal intracerebral hemorrhage

描述（由申请人提供）：脑出血（ICH）是一种使人衰弱的疾病，经常折磨早产儿。在这一人群中，ICH最常见的起源于脑胚基质，一组高度血管化的神经前体细胞，位于脑胚基质中。 靠近丘脑，并经常延伸到脑室。脑出血是由血脑屏障（BBB）受损引起的，血脑屏障在小鼠胚胎脑中由内皮细胞（EC）、基底膜和壁细胞（称为周细胞（PC））组成。我们项目的总体目标是对血脑屏障的形成产生关键的见解，并设计新的策略来降低ICH和脑室内出血（IVH）的发生率和发病率。 对PC中调节BBB并对ICH有影响的任何信号通路知之甚少。PDGFR基因敲除小鼠缺乏脑前体细胞，血脑屏障通透性增加，但没有脑出血或血管破裂。与妊娠中期人类大脑的其他区域相比，生殖基质中的转化生长因子（TGF）<$1蛋白水平降低，我们的初步结果首次表明，减弱小鼠胚胎PC中的TGF <$1信号传导导致生殖基质和丘脑和IVH中的严重出血。携带PDGFR的胚胎TGF β受体1（R1）的Cre和floxed等位基因，具有PC数量相对适度的减少，但EC的形态和组织明显异常。此外，最近有人提出，神经血管蛋白酶是重要的球员在germinal基质出血，和PC-衍生的基质金属蛋白酶-9被牵连作为促成BBB崩溃阿尔茨海默氏病。我们在培养细胞和转基因胚胎小鼠中的初步结果表明，TGF β-R1减少的PC具有增加的具有血小板反应蛋白基序的去整合素和金属蛋白酶（ADAMTS-1）的表达，ADAMTS-1是一种分泌的基质金属蛋白酶，使毛细血管EC管不稳定。 我们假设壁细胞TGF β-R1介导的信号传导在完整BBB的发育中起关键作用，并且该信号传导途径的破坏刺激PC表达因子（例如，在某些实施方案中，脑出血是由诱导异常EC极化、迁移和增殖的蛋白酶（金属蛋白酶）引起的，从而破坏BBB并引起ICH。为了验证这一假设，我们的研究将利用转基因小鼠，人脑PC和EC和人脑组织的共培养物。我们的目标是：i）阐明PC中TGF β-R1诱导的信号传导对脑PC和EC发育中的细胞过程的影响，这些细胞过程对BBB形成至关重要;和ii）确定金属蛋白酶，特别是ADAMTS 1，在介导TGF β-R1降低的PC对BBB形成和EC功能的有害作用中的作用。该R21为随后的R 01应用提供了基础，该应用深入研究了ICH背后的PC、EC和/或周围组织中的分子机制。综上所述，我们的研究有望对BBB形成和ICH发病机制产生开创性的见解，并提出预防和治疗ICH的新治疗策略。

DESCRIPTION (provided by applicant): Intracerebral hemorrhage (ICH) is a debilitating disease that frequently afflicts premature infants. In this population, ICH most commonly originates in the germinal matrix, a collection of highly vascularized neural precursors located in proximity to the thalamus, and often extends into the ventricles. ICH results from compromise of the blood brain barrier (BBB) which in the mouse embryonic brain is composed of endothelial cells (ECs), basement membrane and mural cells known as pericytes (PCs). The overall goal of our project is to yield key insights into BBB formation and devise novel strategies to reduce the incidence and morbidity of ICH and intraventricular hemorrhage (IVH). Little is known about any signaling pathways in PCs that modulate the BBB and have implications for ICH. PDGFR-¿ null mice lack brain PCs and have increased BBB permeability, but no brain hemorrhage or vessel rupture ensues. Transforming growth factor (TGF)¿1 protein levels are reduced in the germinal matrix compared to other regions of the midgestation human brain, and our preliminary results are the first to suggest that attenuating TGF¿ signaling in PCs in the mouse embryo results in gross hemorrhage in the germinal matrix and thalamus and IVH. Embryos carrying PDGFR-¿-Cre and floxed alleles for the TGF¿-receptor 1 (R1), have a relatively modest reduction in PC number but a markedly aberrant morphology and organization of ECs. In addition, it was recently suggested that neurovascular proteases are important players in germinal matrix hemorrhage, and PC-derived matrix metalloproteinase-9 is implicated as contributing to BBB breakdown in Alzheimer's disease. Our initial results in cultured cells and transgenic embryonic mice indicate that PCs with reduced TGF¿-R1 have increased expression of a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS-1), a secreted matrix metalloproteinase that destabilizes capillary EC tubes. We hypothesize that mural cell TGF¿-R1-mediated signaling plays a key role in the development of an intact BBB and that disruption of this signaling pathway stimulates PC expression of factors (e.g., metalloproteinases) that induce aberrant EC polarization, migration and proliferation, thereby disrupting the BBB and causing ICH. To test this hypothesis, our studies will utilize transgenic mice, co-cultures of human brain PCs and ECs and human brain tissue. We aim to: i) elucidate the effects of TGF¿-R1-induced signaling in PCs on cellular processes in brain PC and EC development that are critical for BBB formation; and ii) determine the roles of metalloproteinases, and specifically ADAMTS1, in mediating the deleterious effects of PCs with reduced TGF¿-R1 on BBB formation and EC function. This R21 provides the foundation for a subsequent R01 application investigating in-depth the molecular mechanisms in PCs, ECs and/or surrounding tissues which underlie ICH. Taken together, our studies promise to yield seminal insights into BBB formation and ICH pathogenesis and suggest novel therapeutic strategies to prevent and treat ICH.