Reactive astrogliosis regulates blood-brain barrier permeability.

描述(申请人提供)：在多发性硬化症(MS)中，血脑屏障(BBB)的破坏发生在病变形成的早期，并与急性临床恶化相关。有对比剂增强斑块的患者更有可能有不可逆的损伤，如光谱和T1低信号所示。血脑屏障的破坏导致水肿，并允许炎症介质进入中枢神经系统，从而加剧神经病理，限制修复能力。通过限制致病细胞和炎症因子的进入，可以减少永久性损害。需要针对血脑屏障破裂的新疗法，因为目前只有大剂量的皮质类固醇有效地治疗病情恶化，而且它们在血脑屏障中的作用机制尚不清楚。了解血脑屏障的破坏可能导致限制MS急性加重的新疗法。血脑屏障存在于脑微血管内皮细胞(BMVEC)水平，它使用紧密的连接来限制细胞旁的通透性。导致紧密连接链的跨膜蛋白包括Claudins(CLN)和occludin(OCLN)。在BMVEC中，CLN-5在决定屏障性质方面起着关键作用，CLN5-/-小鼠表现出血脑屏障的破坏和围产期死亡。OCLN调节BBB的性质，但不是屏障形成所必需的。有趣的是，BBB的建立不是BMVEC固有的，而是依赖于星形胶质细胞和周细胞。这些细胞也强烈地参与了血脑屏障的破坏，但其潜在的机制尚不清楚。我们已经确定了星形胶质细胞的反应性和血脑屏障破坏之间的新联系。通过对人类星形胶质细胞的微阵列分析，我们发现在MS中表达的细胞因子诱导了导致内皮可塑性的基因。上调的转录本包括血管生成因子VEGF-A及其转录调节因子HIF-1a。血管内皮生长因子-A是血脑屏障破坏的有效诱导剂，并定位于MS病变中的反应性星形胶质细胞，但其影响血脑屏障的机制尚不清楚。我们的研究表明，在体外培养的BMVEC和中枢神经系统中，血管内皮生长因子-A干扰了CLN-5和OCLN的表达。这两种蛋白的下调伴随着诱导EAE中的VEGF-A和BBB的分解，EAE是一种广泛使用的MS动物模型。我们的体外救援实验表明，CLN-5的丢失是BBB开放的关键事件。重要的是，我们现在已经建立了条件基因敲除的GfapCre：Vegffl/fl和GfapCre：Hif1afl/fl小鼠，以确定VEGF-A和HIF-1a在血脑屏障破坏中的作用。我们的数据显示，血脑屏障的破坏在这两种基因型中都受到了显著的限制。在这里，我们将检验一种假设，即反应性星形胶质细胞中HIF-VEGF轴的激活通过破坏内皮细胞CLN-5和OCLN来促进血脑屏障的破坏。在目标1中，我们将确定血管内皮生长因子诱导中枢神经系统微血管内皮细胞通透性的机制。在目标2中，我们将使用GfapCre：Vegffl/fl和GfapCre：Hif1afl/fl小鼠来证实HIF-1a和VEGF-A在体内CLN-5和OCLN破坏和BBB破坏中的作用。我们还将研究拯救内皮细胞CLN-5或OCLN是否会限制血脑屏障的破坏。在目标3中，使用GfapCre：Vegffl/fl和GfapCre：Hif11fl/fl小鼠，我们将解决这些发现与MOG35-55 EAE疾病表现的相关性。这项建议旨在确定导致MS患者血脑屏障中断的途径。这项工作的长期目标是开发新的治疗方法，以限制MS患者的病变形成和急性加重的严重程度。 公共卫生相关性：在多发性硬化症(MS)中，转录因子HIF-1a及其下游效应因子VEGF-A与血脑屏障(BBB)的破坏和随后的中枢神经系统损伤密切相关，但其潜在机制仍不清楚。微血管内皮细胞水平的紧密连接是维持血脑屏障完整性的关键，最近我们发现，HIF-1a和VEGF-A通过破坏紧密连接蛋白CLN-5和OCLN来诱导BBB的破坏。在这项提案中，我们将使用条件基因敲除方法来确定HIF-1a、VEGF-A、CLN-5和OCLN在血脑屏障中断中的作用，目的是寻找新的治疗靶点来限制MS的病变形成。

DESCRIPTION (provided by applicant): In multiple sclerosis (MS), breakdown of the blood-brain barrier (BBB) occurs early in lesion formation and correlates with acute clinical exacerbation. Patients with contrast enhancing plaques are more likely to have irreversible injury, as marked by spectroscopy and T1 hypointensities. BBB disruption leads to edema, and allows CNS entry of inflammatory mediators that exacerbate neuropathology and restrict the capacity for repair. By limiting entrance of pathogenic cells and inflammatory factors, permanent damage can be reduced. New therapies directed at BBB breakdown are needed, as presently only high dose corticosteroids effectively treat exacerbations, and their mechanism of action at the BBB is poorly characterized. Understanding BBB disruption may lead to new therapies to restrict acute exacerbation in MS. The BBB exists at the level of brain microvessel endothelial cells (BMVEC), which use tight junctions to restrict paracellular permeability. Transmembrane proteins contributing to tight junction strands include claudins (CLN) and occludin (OCLN). In BMVEC, CLN-5 plays a key role in determining barrier properties, and CLN5-/- mice display BBB disruption and die perinatally. OCLN regulates properties of the BBB, but is not required for barrier formation. Interestingly, establishment of the BBB is not intrinsic to BMVEC, and depends on astrocytes and pericytes. These cells are also strongly implicated in BBB disruption, but the underlying mechanisms are not well understood. We have identified a novel link between astrocyte reactivity and BBB breakdown. Using microarray analysis of human astrocytes, we have found that cytokines expressed in MS induce genes that cause endothelial plasticity. Upregulated transcripts include the angiogenic factor VEGF-A and its transcriptional regulator, HIF-1a. VEGF-A is a potent inducer of BBB disruption and localizes to reactive astrocytes in MS lesions, but the mechanism underlying its effects on the BBB is unknown. Our studies now reveal that VEGF-A disrupts expression of both CLN-5 and OCLN in BMVEC in vitro, and in the CNS. Downregulation of both proteins accompanies induction of VEGF-A and BBB breakdown in EAE, a widely- used animal model of MS. Our rescue experiments in vitro implicate loss of CLN-5 as a key event in BBB opening. Importantly, we have now generated conditional knockout GfapCre:Vegffl/fl and GfapCre:Hif1afl/fl mice to define the roles of VEGF-A and HIF-1a in BBB breakdown. Our data show that BBB disruption is strikingly restricted in both genotypes. Here, we will test the hypothesis that activation of the HIF-VEGF axis in reactive astrocytes promotes BBB breakdown via disruption of endothelial CLN-5 and OCLN. In Aim#1, we will define the mechanism underlying VEGF-induced permeability of CNS microvascular endothelium. In Aim#2, we will use GfapCre:Vegffl/fl and GfapCre:Hif1afl/fl mice to confirm the roles of HIF-1a and VEGF-A in CLN-5 and OCLN disruption and BBB breakdown in vivo. We will also examine whether rescue of endothelial CLN-5 or OCLN restricts BBB disruption. In Aim#3, using GfapCre:Vegffl/fl and GfapCre:Hif11fl/fl mice we will address the relevance of these findings to disease expression in MOG35-55 EAE. This proposal aims to identify pathways responsible for BBB disruption in MS. The long-term goal of this work is to develop new therapies to restrict lesion formation and limit the severity of acute exacerbation in MS patients. PUBLIC HEALTH RELEVANCE: In multiple sclerosis (MS), the transcription factor HIF-1a and its downstream effector VEGF-A have been strongly linked to disruption of the blood-brain barrier (BBB) and subsequent damage to the CNS, but the underlying mechanisms have remained unknown. Tight junctions at the level of microvascular CNS endothelium are critical in maintaining BBB integrity, and recently we found that HIF-1a and VEGF-A induce BBB breakdown via disruption of the tight junction proteins CLN-5 and OCLN. In this proposal, using a conditional knockout approach we will determine the roles of HIF-1a, VEGF-A, CLN-5 and OCLN in BBB disruption, with the goal of identifying novel therapeutic targets to restrict lesion formation in MS.