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

描述(由申请人提供)：脑出血(ICH)是一种经常困扰早产儿的衰弱疾病。在这一人群中，ICH最常见的起源于生发基质，这是一组高度血管化的神经前体细胞，位于 靠近丘脑，通常延伸到脑室。ICH是血脑屏障(BBB)受损的结果，在小鼠胚胎脑中，血脑屏障由内皮细胞(ECs)、基底膜和称为周细胞(PC)的壁细胞组成。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，并设计新的策略来减少脑出血和脑室内出血(IVH)的发生率和发病率。对PC中调节血脑屏障并对脑出血有影响的信号通路知之甚少。PDGFR基因缺失的小鼠缺乏脑PC，血脑屏障通透性增加，但没有发生脑出血或血管破裂。与妊娠中期人脑的其他区域相比，生发基质中的转化生长因子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中的转化生长因子1信号会导致生发基质和丘脑以及IVH的大出血。携带PDGFR-CRE和转化生长因子受体1(R1)等位基因的胚胎，PC数量相对较少，但ECs的形态和组织明显异常。此外，最近有研究表明，神经血管蛋白水解酶在生发基质出血中起重要作用，而PC衍生的基质金属蛋白酶-9与阿尔茨海默病的血脑屏障破坏有关。我们在培养细胞和转基因胚胎小鼠中的初步结果表明，转化生长因子-R1降低的PC增加了具有血栓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，会破坏毛细血管内皮细胞的稳定。我们假设，壁细胞转化生长因子-R1介导的信号在完整的血脑屏障的形成中起关键作用，该信号通路的破坏刺激PC表达诱导内皮细胞异常极化、迁移和增殖的因子(如金属蛋白酶)，从而破坏血脑屏障并导致脑出血。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ECs与人脑组织的共同培养。我们的目标是：i)阐明转化生长因子β-R1诱导的PC信号在脑PC和EC发育中对血脑屏障形成至关重要的细胞过程中的作用；以及ii)确定金属蛋白酶，特别是ADAMTS1，在介导转化生长因子β-R1降低的PC对BBB形成和EC功能的有害影响中的作用。这个R21为随后的R01应用程序提供了基础，深入研究了脑出血背后的PC、ECS和/或周围组织的分子机制。综上所述，我们的研究有望对血脑屏障的形成和脑出血的发病机制产生开创性的见解，并提出预防和治疗脑出血的新的治疗策略。

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.