A vasculo-protective role for beta4 integrin in neuroinflammation

项目摘要/摘要 多发性硬化症(MS)是一种慢性炎症性疾病，以脱髓鞘和 中枢神经系统(CNS)的轴突变性。早期多发性硬化症和实验性多发性硬化症(实验性 自身免疫性脑脊髓炎(EAE)血脑屏障(BBB)破坏与血管新生重塑 通过使白细胞渗入中枢神经系统来启动和维持发病机制。这种血管重塑，在 反过来，细胞外基质(ECM)蛋白及其内皮整合素受体对其影响很大。我们 假设一种特殊的整合素，层粘连蛋白受体，起着重要的血管保护作用 通过促进血脑屏障完整性和抑制内皮细胞炎症，在神经炎症过程中发挥作用。 这一假设得到了几个观察结果的支持。首先，层粘连蛋白是 血管基膜，缺乏星形胶质细胞和周细胞层粘连蛋白的转基因小鼠显示出血脑屏障缺陷 正直。第二，虽然正常大脑中的大多数血管只表达低水平的整合素， 在炎症过程中，所有血管中的内皮细胞都会强烈上调这种整合素。第三，我们最近 发现缺乏内皮整合素表达的转基因小鼠(-EC-KO小鼠)临床表现较差 疾病和增加的神经炎症在EAE中。第四，我们对脑内皮细胞的体外研究表明 4整合素通过保持血管完整性(通过Claudin-5稳定)和通过减少 血管激活(通过减少ICAM-1的表达)。综上所述，这些数据表明整合蛋白 上调可能是一种诱导的保护机制，在应激条件下稳定血脑屏障。 此外，我们还发现降胆固醇药物阿托伐他汀上调内皮细胞 整合素在体内的表达，提示这可能为治疗操作提供一种有效的方法 作为这些研究的一部分，我们还将产生一种新的转基因小鼠品系，在该品系中 整合素在内皮细胞中高表达(-EC-HIGH)，并测试这些小鼠是否 防止神经发炎。体外研究将确定哪些炎症介质可以诱导 内皮-4整合素的表达及他汀类药物对内皮细胞诱导机制的研究 整合素。最后，我们将确定三个信号通路在介导整合素介导的血管生成中的作用。 保护：(I)转化生长因子-ALK1-Smad1/5/8，(Ii)MAP激酶，和(Iii)肌球蛋白轻链(MLC)激酶。 我们的假设将在三个具体目标中得到验证：(1)机械地定义4整合素如何保护 抗神经炎性疾病，(2)证明内皮4整合素上调具有保护作用 和(3)确定介导血管保护影响的细胞内信号通路 整合素。这种方法是创新的，因为它将检验整合素促进血脑屏障这一新概念 在EAE期间具有完整性和抑制血管炎症的作用。这项研究之所以意义重大，是因为成功了 这些研究的完成将评估内皮整合素作为一种新的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.