MMP-9 and Transport through Vascular Endothelium

描述：（由申请人提供）：拟定研究旨在测试 明胶酶B（MMP-9）表达增加的假说， 血管内皮细胞的单核细胞迁移导致 内皮对大分子的通透性。这种影响可能有助于 内皮屏障功能减弱和动脉粥样硬化起始 通过促进血浆的非特异性进入和积聚而损伤 包括循环脂蛋白。具体目标包括衡量 内皮细胞单层对大分子示踪剂的通透性 野生型和MMP-9敲除单核细胞的迁移，和 探讨单核细胞/内皮细胞介导MMP-9表达的机制 交互.我们建议研究在单核细胞 使用由小鼠骨组成的体外共培养系统的移植 骨髓单核细胞和小鼠主动脉内皮细胞在基质覆盖细胞上的生长 文化嵌入为了研究MMP-9在迁移中的具体作用， 我们将使用来源于野生型的单核细胞和内皮细胞， 来自MMP-9敲除小鼠。将使用以下方法测量内皮渗透性： 荧光标记的葡聚糖。将测量示踪剂浓度 荧光分析。MMP-9蛋白的表达和酶活性将是 分别通过Western印迹和SDS-PAGE酶谱法测定。 探讨MMP-9诱导单核细胞迁移的机制。 内皮，我们将研究单核细胞之间的特定相互作用的作用， 整合素和内皮细胞粘附分子。具体而言，我们建议 使用VCAM-1敲除评估单核细胞与内皮VCAM-1结合的作用 小鼠内皮细胞的迁移试验。这些研究应进一步 我们对有助于发展的早期事件的理解 动脉粥样硬化病变，并可以提供洞察MMP-9的使用 治疗内皮屏障功能障碍的抑制剂。

DESCRIPTION: (provided by applicant): The proposed research is designed to test the hypothesis that increased expression of gelatinase B (MMP-9) during monocyte transmigration of vascular endothelium results in an increase in endothelial permeability to macromolecules. Such an effect may contribute to diminished endothelial barrier function and initiation of atherosclerotic lesion through facilitating the non-specific entry and accumulation of plasma molecules, including circulating lipoproteins. Specific aims include measuring permeability of endothelial monolayers to macromolecular tracers during transmigration of both wild type and MMP-9 knockout monocytes, and investigating the mechanism of MMP-9 induction during monocyte/endothelial cell interactions. We propose to investigate permeability changes during monocyte transmigration using an in vitro coculture system consisting of mouse bone marrow monocytes and mouse aortic endothelium grown on matrix-covered cell culture inserts. To investigate the specific role of MMP-9 in transmigration, we will use monocytes and endothelial cells derived both from wild type and from MMP-9 knockout mice. Endothelial permeability will be measured using flourescently labeled dextrans. Tracer concentrations will be measured fluorimetrically. Expression of MMP-9 protein and enzymatic activity will be determined by means of Western blotting and SDS-PAGE zymography, respectively. To explore the mechanism of MMP-9 induction during monocyte transmigration of endothelium, we will examine the role of specific interactions between monocyte integrins and endothelial cell adhesion molecules. Specifically, we propose to assess the role of monocyte binding to endothelial VCAM-1 using VCAM-1 knockout mouse endothelial cells in transmigration assays. These studies should further our understanding of the early events that contribute to the development of athersclerotic lesions and could provide insight into the use of MMP-9 inhibitors to treat endothelial barrier dysfunction.