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，并与急性临床加重相关。对比增强斑块的患者更可能有不可逆的损伤，如光谱和T1低信号所示。BBB破坏导致水肿，并允许炎性介质进入CNS，从而加剧神经病理学并限制修复能力。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。需要针对BBB破坏的新疗法，因为目前仅高剂量皮质类固醇有效地治疗恶化，并且它们在BBB的作用机制的特征很差。了解血脑屏障破坏可能会导致新的治疗方法，以限制急性加重MS。血脑屏障存在于脑微血管内皮细胞（BMVEC）的水平，使用紧密连接，以限制细胞旁通透性。有助于紧密连接链的跨膜蛋白包括claudins（CLN）和occludin（OCLN）。在BMVEC中，CLN-5在确定屏障特性中起关键作用，并且CLN 5-/-小鼠显示BBB破坏并在围产期死亡。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。这些细胞也与BBB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破坏之间的新联系。使用人类星形胶质细胞的微阵列分析，我们发现，细胞因子在MS中表达诱导基因，导致内皮可塑性。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BBB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，但其对BBB影响的机制尚不清楚。我们的研究现在揭示，VEGF-A在体外和CNS中破坏BMVEC中CLN-5和OCLN的表达。两种蛋白质的下调伴随着EAE（广泛使用的MS动物模型）中VEGF-A和BBB破坏的诱导。我们的体外拯救实验表明CLN-5的丢失是BBB开放的关键事件。重要的是，我们现在已经产生了条件性敲除GfapCre：Vegffl/fl和GfapCre：Hif 1afl/fl小鼠，以确定VEGF-A和HIF-1a在BBB破坏中的作用。我们的数据表明，血脑屏障破坏是惊人的限制在两种基因型。在这里，我们将测试这一假设，即在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促进血脑屏障破裂。在目标1中，我们将确定VEGF诱导CNS微血管内皮通透性的机制。在目标#2中，我们将使用GfapCre：Vegffl/fl和GfapCre：Hiflafl/fl小鼠来确认HIF-1a和VEGF-A在体内CLN-5和OCLN破坏和BBB破坏中的作用。我们还将研究是否拯救内皮细胞CLN-5或OCLN限制血脑屏障破坏。在目标#3中，使用GfapCre：Vegffl/fl和GfapCre：Hifllfl/fl小鼠，我们将解决这些发现与MOG 35 -55 EAE中疾病表达的相关性。这项工作的长期目标是开发新的治疗方法，以限制病变的形成和限制MS患者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及其下游效应物VEGF-A与血脑屏障（BBB）的破坏和随后的CNS损伤密切相关，但其潜在机制仍不清楚。微血管CNS内皮水平的紧密连接在维持BBB完整性方面至关重要，最近我们发现HIF-1 a和VEGF-A通过破坏紧密连接蛋白CLN-5和OCLN诱导BBB破裂。在这项提议中，我们将使用条件性敲除方法确定HIF-1a，VEGF-A，CLN-5和OCLN在BBB破坏中的作用，目的是确定新的治疗靶点以限制MS中的病变形成。

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.