A vasculo-protective role for beta4 integrin in neuroinflammation

项目总结/摘要 多发性硬化症（MS）是一种慢性炎症性疾病，其特征在于脱髓鞘， 中枢神经系统（CNS）中轴突的变性。早期MS和实验MS（实验 自身免疫性脑脊髓炎（EAE）导致血脑屏障（BBB）破坏和血管生成重塑 通过提供白细胞浸润到CNS中来启动和维持发病机制。这种血管重塑， 反过来，受到细胞外基质（ECM）蛋白及其内皮整合素受体的强烈影响。我们 假设一种特定的整合素，即层粘连蛋白受体，在血管保护中起重要作用， 通过促进BBB完整性和抑制内皮炎症在神经炎症过程中发挥作用。 这一假设得到了若干观察结果的支持。首先，层粘连蛋白是细胞膜的主要成分， 血管基底层，星形胶质细胞和周细胞层粘连蛋白缺陷的转基因小鼠显示BBB缺陷 完整第二，虽然正常大脑中的大多数血管仅表达低水平的多巴胺整合素， 所有血管中的内皮细胞在炎症期间强烈上调该整联蛋白。第三，我们最近 发现缺乏内皮细胞整合素表达的转基因小鼠（EC-KO小鼠）表现出更差的临床表现， EAE中的疾病和神经炎症增加。第四，我们对脑内皮细胞的体外研究表明， β 4整联蛋白通过维持血管完整性（通过密蛋白-5稳定）和减少血管完整性来保护血管。 血管激活（通过减少ICAM-1表达）。综上所述，这些数据表明， 上调可能是在应激条件下稳定BBB的诱导性保护机制。 此外，我们发现降胆固醇药物阿托伐他汀上调内皮细胞凋亡， 整合素在体内的表达，这表明这可能提供一种有效的方法来治疗操纵整合素， 作为这些研究的一部分，我们还将产生新的转基因小鼠品系，其中 在内皮细胞中特异性地过表达整合素（EC-EC-high），并测试这些小鼠是否 防止神经炎症。体外研究将确定哪些炎症介质诱导 内皮细胞整合素4的表达，并研究他汀类药物诱导内皮细胞整合素表达的机制。 整联蛋白最后，我们将确定三种信号通路在介导整合素介导的血管内皮细胞增殖中的作用。 保护：（i）TGF-β ALK 1-Smad 1/5/8，（ii）MAP激酶，和（iii）肌球蛋白轻链（MLC）激酶。 我们的假设将在三个特定的目标进行测试：（1）机械地定义β 4整合素如何保护 抗神经炎性疾病，（2）证实内皮细胞α 4整合素的上调保护了 抗EAE和（3）确定介导血管保护作用的细胞内信号通路 的整合素。该方法是创新的，因为它将测试新的概念，即β-整合素促进BBB 完整性和抑制血管炎症过程中EAE。这项研究意义重大，因为成功 这些研究的完成将评价内皮细胞粘附分子整合素作为新的MS治疗靶点。

PROJECT SUMMARY/ABSTRACT Multiple sclerosis (MS) is a chronic inflammatory disease characterized by demyelination and degeneration of axons in the central nervous system (CNS). Early in MS and experimental MS (experimental autoimmune encephalomyelitis, EAE) breakdown of the blood-brain barrier (BBB) and angiogenic remodeling initiate and maintain pathogenesis by affording leukocyte infiltration into the CNS. This vascular remodeling, in turn, is strongly influenced by extracellular matrix (ECM) proteins and their endothelial integrin receptors. We hypothesize that one specific integrin, the laminin receptor, plays an important vasculo-protective role during neuroinflammation by promoting BBB integrity and suppressing endothelial inflammation. This hypothesis is supported by several observations. First, laminin is a major component of the vascular basal lamina, and transgenic mice deficient in astrocyte and pericyte laminin show defective BBB integrity. Second, while most blood vessels in the normal brain express only low levels of  integrin, endothelial cells in all vessels strongly upregulate this integrin during inflammation. Third, we have recently found that transgenic mice lacking endothelial  integrin expression (-EC-KO mice) show worse clinical disease and increased neuroinflammation in EAE. Fourth, our in vitro studies of brain endothelial cells suggest that 4 integrin protects both by maintaining vascular integrity (via claudin-5 stabilization) and by reducing vascular activation (via reduced ICAM-1 expression). Taken together, these data suggest that  integrin upregulation may be an inducible protective mechanism that stabilizes the BBB under conditions of stress. Furthermore, we have found that the cholesterol-lowering drug atorvastatin upregulates endothelial  integrin expression in vivo, suggesting that this could provide an effective way to therapeutically manipulate  integrin levels in MS. As part of these studies we will also generate a novel transgenic mouse strain in which  integrin is over-expressed specifically in endothelial cells (-EC-high) and test whether these mice are protected against neuroinflammation. In vitro studies will define which inflammatory mediators induce endothelial 4 integrin expression and examine the mechanisms underlying statin-induction of endothelial integrin. Lastly, we will define the roles of three signaling pathways in mediating  integrin-mediated vasculo- protection: (i) TGF- ALK1-Smad 1/5/8, (ii) MAP kinase, and (iii) myosin light chain (MLC) kinase. Our hypothesis will be tested in three specific aims: (1) define mechanistically how 4 integrin protects against neuroinflammatory disease, (2) demonstrate that upregulation of endothelial 4 integrin protects against EAE and (3) define the intracellularsignaling pathways that mediate the vasculo-protective influence of integrin. The approach is innovative because it will test the novel concept that  integrin promotes BBB integrity and suppresses vascular inflammation during EAE. This research is significant because successful completion of these studies will evaluate endothelial  integrin as a novel MS therapeutic target.