Reactive astrogliosis regulates blood-brain barrier permeability.

反应性星形胶质细胞增生调节血脑屏障的通透性。

• 批准号: 8535219
• 负责人: GARETH R JOHN
• 金额: $ 28.05万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8535219
• 关键词: Acute; Address; Adrenal Cortex Hormones; Agonist; Angiogenic Factor; Animal Model; Antibodies; Astrocytes; Blood - brain barrier anatomy; Brain; Cells; Clinical; Data; Dextrans; Disease; Dose; Down-Regulation; Edema; Endothelial Cells; Endothelium; Event; Experimental Autoimmune Encephalomyelitis; Genes; Genotype; Goals; Human; In Vitro; Inflammation; Inflammation Mediators; Inflammatory; Injury; Integral Membrane Protein; Knock-out; Lead; Lesion; Link; Measures; Methylprednisolone; Microarray Analysis; Modeling; Multiple Sclerosis; Multiple Sclerosis Lesions; Mus; Nervous system structure; Pathway interactions; Patients; Pericytes; Permeability; Phenotype; Plasma Proteins; Play; Principal Investigator; Property; Proteins; Receptor Signaling; Recovery; Resistance; Rodent; Role; Severities; Signal Pathway; Signal Transduction; Signaling Molecule; Spectrum Analysis; Testing; Therapeutic; Tight Junctions; Transcript; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factors; Work; antibody inhibitor; astrogliosis; central nervous system injury; cytokine; defined contribution; dextran; experience; human NOS3 protein; improved; in vivo; neuropathology; new therapeutic target; novel; occludin; public health relevance; receptor; repaired; research study; small hairpin RNA; transcription factor; vector

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.

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，并且与急性临床恶化相关。具有对比增强斑块的患者更有可能出现不可逆损伤，如光谱和 T1 低信号所标志的那样。血脑屏障破坏会导致水肿，并使炎症介质进入中枢神经系统，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。需要针对血脑屏障破坏的新疗法，因为目前只有高剂量皮质类固醇才能有效治疗病情加重，并且其对血脑屏障的作用机制尚不清楚。了解血脑屏障破坏可能会带来限制多发性硬化症急性加重的新疗法。 BBB 存在于脑微血管内皮细胞 (BMVEC) 水平，利用紧密连接来限制细胞旁通透性。有助于紧密连接链的跨膜蛋白包括紧密连接蛋白 (CLN) 和闭合蛋白 (OCLN)。在 BMVEC 中，CLN-5 在决定屏障特性方面发挥着关键作用，CLN5-/- 小鼠表现出 BBB 破坏并在围产期死亡。 OCLN 调节 BBB 的特性，但不是屏障形成所必需的。有趣的是，BBB 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。我们发现星形胶质细胞反应性和血脑屏障破坏之间存在新的联系。通过对人星形胶质细胞的微阵列分析，我们发现 MS 中表达的细胞因子诱导导致内皮可塑性的基因。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 BBB 破坏的有效诱导剂，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。我们的研究现在表明，VEGF-A 会破坏体外 BMVEC 和 CNS 中 CLN-5 和 OCLN 的表达。在 EAE（一种广泛使用的 MS 动物模型）中，两种蛋白的下调伴随着 VEGF-A 和 BBB 分解的诱导。我们的体外救援实验表明 CLN-5 的丢失是血脑屏障开放的关键事件。重要的是，我们现在已经生成了条件敲除 GfapCre:Vegffl/fl 和 GfapCre:Hif1afl/fl 小鼠，以定义 VEGF-A 和 HIF-1a 在 BBB 破坏中的作用。我们的数据表明，两种基因型的血脑屏障破坏均受到显着限制。在这里，我们将测试以下假设：反应性星形胶质细胞中 HIF-VEGF 轴的激活通过破坏内皮 CLN-5 和 OCLN 促进 BBB 分解。在目标#1 中，我们将定义 VEGF 诱导中枢神经系统微血管内皮细胞通透性的机制。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 破坏以及 BBB 破坏中的作用。我们还将研究内皮 CLN-5 或 OCLN 的拯救是否会限制 BBB 破坏。在 Aim#3 中，我们将使用 GfapCre:Vegffl/fl 和 GfapCre:Hif11fl/fl 小鼠来探讨这些发现与 MOG35-55 EAE 中疾病表达的相关性。该提案旨在确定导致多发性硬化症 BBB 破坏的途径。这项工作的长期目标是开发新疗法来限制多发性硬化症患者病变的形成并限制急性加重的严重程度。 公共健康相关性：在多发性硬化症 (MS) 中，转录因子 HIF-1a 及其下游效应子 VEGF-A 与血脑屏障 (BBB) 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。微血管中枢神经系统内皮水平的紧密连接对于维持 BBB 完整性至关重要，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 诱导 BBB 破坏。在本提案中，我们将使用条件敲除方法确定 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来限制 MS 病变的形成。