Mechanisms of Aortic Fibrosis

通常处于静止状态的中膜血管平滑肌细胞(VSMC)的激活、迁移、增殖和丰富的基质合成是动脉粥样硬化病变发展的中心事件。我们最近的发现表明，赖氨酰氧化酶(LO)是一种细胞外酶，它氧化多肽赖氨酸，启动弹性蛋白和胶原的共价交联和不溶解，对VSMC的行为产生了以前意想不到的强烈影响。我们发现LO对VSMC有很强的趋化作用，并且它与这些细胞的表面受体结合，初步结果表明它含有一个β1整合素亚基。在与VSMC表面结合后，LO进入细胞，然后在细胞核内浓缩。我们还发现，组蛋白H1在VSMC细胞核内被内源性LO氧化。伴随着这些事件的是胶原蛋白的产生和细胞内信号蛋白的磷酸化。我们的指导性假设是，LO可能是VSMC功能的重要激活物，作为细胞外和细胞内的信号刺激VSMC的表型变化，就像在动脉疾病中一样。本项目的具体目标是：(1)阐明LO与质膜受体结合的机制和意义。我们将鉴定作为LO的全受体(S)的VSMC膜蛋白，并评估它们在VSMC对LO结合、摄取和激活信号转导通路的反应中的作用。(2)阐明LO催化的核修饰的性质及其对基因表达的潜在影响。我们将鉴定可被核LO氧化的核蛋白，并评估LO对VSMC特异性胶原基因转录的影响。(3)研究血管损伤后LO在VSMC中的表达、细胞内和细胞外定位及丰度。免疫细胞化学和原位转录分析将用来探测LO在正常血管组织和诱导的血管病变中的表达和细胞内外定位。 在大鼠股动脉损伤模型上。综上所述，这些研究将探索LO可能影响动脉粥样硬化中VSMC活动的机制。

The activation, migration, proliferation and abundant matrix synthesis by the normally quiescent medial vascular smooth muscle cell (VSMC) are central events in the development of atherosclerotic lesions. Our recent findings have revealed that lysyl oxidase (LO), the extracellular enzyme which oxidizes peptidyl lysine to initiate the covalent crosslinking and insolubilization of soluble forms of elastin and collagen, plays previously unexpected roles strongly influencing the behavior of VSMC. We have found that LO is strongly chemotactic for VSMC and that it binds to a surface receptor of these cells indicated by our preliminary results to contain a beta1 integrin subunit. Following binding to the VSMC surface, LO enters the cell and then concentrates within the cell nucleus. We have also found that histone H1 is oxidized within VSMC nuclei by endogenous LO. These events are accompanied by increased production of collagen and phosphorylation of intracellular signaling proteins. Our guiding hypothesis is that LO may serve as an important activator of VSMC functions, acting both as an extracellular and intracellular signal stimulating phenotypic changes in the VSMC as in arterial disease. Specific Aims of this project are: (1) Elucidate the mechanism and significance of LO binding to plasma membrane receptors. We will identity the VSMC membrane proteins acting as holoreceptor(s) of LO and assess their roles in the responses of VSMC to LO binding, uptake, and activation of signal transduction pathways. (2) Elucidate the nature of the nuclear modifications catalyzed by LO and the potential consequences on gene expression. We will identify nuclear proteins which can be oxidized by nuclear LO and assess for the effect of LO on the transcription of specific collagen genes of VSMC. (3) Characterize the expression, intraeellular and extracellular localization and abundance of LO in VSMC in response to vascular injury. Immunocytochemistry and in situ transcription assays will be used to probe for the expression and intra- and extracellular localization of LO within normal vascular tissue and in vascular lesions induced in a rat femoral artery injury model. Taken together, these studies will explore mechanisms by which LO may influence activities of the VSMC that occur in atherosclerosis.