Vascular protection via alpha5beta1 integrin inhibition during neuroinflammation

 描述(申请人提供)：多发性硬化症(MS)是一种慢性炎症性疾病，以中枢神经系统(CNS)中的轴突脱髓鞘和变性为特征。令人信服的证据表明，血脑屏障(BBB)的破坏和血管新生重构发生在MS的早期阶段，在MS的动物模型实验性自身免疫性脑脊髓炎(EAE)中也是如此，并通过提供白细胞进入脑实质而在疾病发病机制的启动和维持中发挥核心作用。这种血管重塑又受到细胞外基质(ECM)蛋白及其内皮整合素细胞表面受体相互作用的强烈影响。特别是，在缺血性卒中和MS动物模型中，α5β1整合素及其配体纤维连接蛋白在脑血管重塑中强烈上调，内皮细胞中缺乏该受体的转基因小鼠(α5 EC-KO小鼠)对缺氧反应表现出延迟和减少的血管新生重塑。此外，我们最近在实验性中风模型中的研究表明，α5-EC-KO小鼠对血脑屏障的破坏有很强的抵抗力，从而显著缩小了缺血性脑梗塞的面积。药物阻断野生型(WT)小鼠的α-5-β-1整合素也有类似的保护作用。由于血脑屏障破坏和血管生成发生在MS和EAE的早期阶段，而且现有证据表明α5β1整合素是脑血管重塑的重要触发因素，我们假设抑制内皮细胞α5β1整合素可以稳定血脑屏障，减少脱髓鞘疾病中白细胞的浸润。支持这一假说的新的初步结果表明，药物阻断培养的WT脑内皮细胞中的α5β1整合素可以通过破坏屏障的炎性细胞因子TRAN-α来稳定其屏障特性。这些研究还表明，这种保护可能是由于claudin-5表达增加的结果，claudin-5是一种紧密连接蛋白，也是血脑屏障的关键组成部分。鉴于这些结果，我们提出了以下具体目标：(1)确定α5β1整合素基因缺失或药物阻断内皮细胞是否能稳定血脑屏障，从而减少EAE模型中的神经炎症和脱髓鞘；2)确定α5β1整合素抑制促进血脑屏障完整性和随后的EAE抵抗的分子机制。我们期望证明，抑制内皮细胞中的α5β1整合素通过稳定紧密连接蛋白和抑制白细胞归巢黏附分子ICAM-1和VCAM-1来增加血脑屏障的完整性，从而增强对EAE的抵抗力。这些研究的成功完成将进一步推动我们开发内皮细胞α5β1整合素作为一种新的MS治疗靶点的目标。

 DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a chronic inflammatory disease characterized by demyelination and degeneration of axons in the central nervous system (CNS). Compelling evidence suggests that breakdown of the blood-brain barrier (BBB) and angiogenic remodeling occur at an early stage of MS as well as in the animal model of MS, experimental autoimmune encephalomyelitis (EAE), and is central to the initiation and maintenance of disease pathogenesis by affording leukocyte infiltration into the brain parenchyma. This vascular remodeling, in turn, is strongly influenced by the interaction between extracellular matrix (ECM) proteins and their endothelial integrin cell surface receptors. In particular, the α5β1 integrin and its ligand fibronectin are strongly upregulated on remodeling cerebral vessels in animal models of ischemic stroke and MS, and transgenic mice lacking this receptor in endothelial cells (α5-EC-KO mice) show delayed and reduced angiogenic remodeling in response to hypoxia. Furthermore, our recent studies in an experimental stroke model showed that α5-EC-KO mice are profoundly resistant to BBB breakdown, resulting in dramatic reductions in size of ischemic infarct. Pharmacological blockade of α5β1 integrin in wild-type (WT) mice achieved similar protection. As BBB breakdown and angiogenesis occur at an early stage of MS and EAE, and current evidence suggests that the α5β1 integrin is an important trigger of cerebrovascular remodeling, we hypothesize that inhibition of endothelial α5β1 integrin stabilizes the BBB and reduces leukocyte infiltration in demyelinating disease. In support of this hypothesis, new preliminary results suggest that pharmacological blockade of α5β1 integrin in cultured WT brain endothelial cells stabilizes their barrier properties in presece of the barrier-disrupting inflammatory cytokine TNF-α. These studies also suggested that protection may be a result of increased expression of claudin-5, a tight junction protein and critical component of the BBB. Armed with these results, we propose the following specific aims: (1) determine whether genetic deletion or pharmacological blockade of α5β1 integrin on endothelial cells stabilizes the BBB, resulting in reduced neuroinflammation and demyelination in the EAE model, and 2) determine the molecular mechanism whereby α5β1 integrin inhibition promotes BBB integrity and subsequent resistance to EAE. We expect to demonstrate that inhibition of α5β1 integrin in endothelial cells confers robust resistance to EAE by increasing BBB integrity through stabilization of tight junction proteins and suppression of the leukocyte- homing adhesion molecules ICAM-1 and VCAM-1. Successful completion of these studies will further our goal of developing endothelial α5β1 integrin as a novel MS therapeutic target.