Reactive astrogliosis regulates blood-brain barrier permeability.

描述（由申请人提供）：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，并与急性临床恶化有关。对比增强斑块的患者更有可能发生不可逆损伤，这可以通过光谱和T1低密度来标记。血脑屏障破坏导致水肿，并允许炎症介质进入中枢神经系统，从而加剧神经病理并限制修复能力。通过限制致病细胞和炎症因子的进入，可以减少永久性损伤。目前，只有高剂量的皮质类固醇能有效治疗急性加重，而且其对血脑屏障的作用机制尚不清楚，因此需要针对血脑屏障破裂的新疗法。了解血脑屏障的破坏可能会导致新的治疗方法来限制ms的急性加重。血脑屏障存在于脑微血管内皮细胞（BMVEC）水平，其使用紧密连接来限制细胞旁通透性。促进紧密连接链的跨膜蛋白包括clin （CLN）和occludin （OCLN）。在BMVEC中，CLN-5在决定屏障特性中起关键作用，CLN5-/-小鼠表现出血脑屏障破坏并在围产期死亡。OCLN调节血脑屏障的性质，但不是屏障形成所必需的。有趣的是，血脑屏障的建立并不是BMVEC固有的，而是依赖于星形胶质细胞和周细胞。这些细胞也与血脑屏障破坏密切相关，但其潜在机制尚不清楚。我们已经确定了星形胶质细胞反应性和血脑屏障分解之间的新联系。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细胞可塑性的基因。上调的转录物包括血管生成因子VEGF-A及其转录调节因子HIF-1a。VEGF-A是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，但其影响脑屏障的机制尚不清楚。我们的研究现在表明，VEGF-A破坏体外BMVEC和中枢神经系统中CLN-5和OCLN的表达。在广泛应用的多发性硬化症动物模型EAE中，这两种蛋白的下调伴随着VEGF-A和血脑屏障分解的诱导。我们的体外拯救实验表明，CLN-5的缺失是血脑屏障打开的关键事件。重要的是，我们现在已经产生了条件敲除GfapCre:Vegffl/fl和GfapCre:Hif1afl/fl小鼠，以确定VEGF-A和HIF-1a在血脑屏障分解中的作用。我们的数据显示，血脑屏障破坏在两种基因型中都明显受到限制。在这里，我们将验证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促进血脑屏障分解的假设。在Aim#1中，我们将定义vegf诱导中枢微血管内皮通透性的机制。在Aim#2中，我们将使用GfapCre:Vegffl/fl和GfapCre:Hif1afl/fl小鼠来确认HIF-1a和VEGF-A在体内CLN-5和OCLN破坏和血脑屏障分解中的作用。我们还将研究内皮细胞CLN-5或OCLN的拯救是否限制血脑屏障的破坏。在Aim#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疾病表达的相关性。这项工作的长期目标是开发新的治疗方法来限制MS患者的病变形成和限制急性加重的严重程度。

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.