PROTEOGLYCANS, GLYCOSAMINOGLYCANS AND ATHEROSCLEROSIS

摘要：以前的工作表明，多能蛋白聚糖，主要的血管间质硫酸软骨素蛋白聚糖（CSPG），和糖胺聚糖，透明质酸（HA），它与多能蛋白聚糖相互作用，积累在细胞外基质（ECM）在早期动脉粥样硬化和再狭窄病变。我们还表明，这两种分子的合成受到特定生长因子和细胞因子的高度调节，并在动脉平滑肌细胞（ASMC）受到刺激迁移和增殖时进行调节。抑制多功能蛋白聚糖和HA与细胞表面受体的相互作用可阻断ASMC增殖和迁移。多功能蛋白聚糖在ECM中积累的一个结果是捕获和保留脂蛋白。我们已经表明，多功能蛋白聚糖与载脂蛋白B和载脂蛋白E的脂蛋白相互作用，这种相互作用增强的因素，影响硫酸软骨素链的生物合成。这些观察结果使我们继续探索ECM中多功能蛋白聚糖/HA复合物在ASMC表型调节和脂蛋白在血管壁中的保留中的重要性。我们假设ASMC增殖和迁移需要多功能蛋白聚糖/HA合成的修饰，并且这些修饰影响ASMC的其他特性，例如在动脉粥样硬化形成的不同阶段重塑ECM的能力。我们进一步假设，ECM内多功能蛋白聚糖的积累是部分负责脂蛋白的细胞外积累。我们建议在四个目标来检验这些假设。第一个目标将解决有关多功能蛋白聚糖和多功能蛋白聚糖亚型表达在ASMC表型和基质重塑的调节中的作用的问题。在这项工作中，我们将表达多功能蛋白聚糖异构体和含有多功能蛋白聚糖不同结构域的小基因，并研究对细胞增殖，迁移和基质蛋白沉积的影响。第二个目标包括旨在检查ASMC的HA酶表达的实验，以及HA作为基质的细胞外组分和作为新发现的细胞内分子在ASMC增殖、迁移和ECM重塑中的作用。第三个目的是扩展我们的研究有关脂蛋白与多能蛋白聚糖硫酸软骨素侧链的相互作用。我们建议确定负责载脂蛋白B和载脂蛋白E的脂蛋白多能蛋白聚糖的结合的特定寡糖序列，并确定诱导硫酸软骨素链结构变化的因素，从而产生具有不同亲和力的脂蛋白多能蛋白聚糖形式。在第四个目标中，我们将使用新的试剂来检测天然存在的动脉粥样硬化病变中多功能蛋白聚糖变体和蛋白水解裂解产物的存在，通过细胞介导的基因转移方法在损伤的大鼠颈动脉新生内膜模型中研究多功能蛋白聚糖变体和HA酶的表达改变对病变进展的影响，并在脂肪喂养的转基因小鼠模型中使用多功能蛋白聚糖变体的靶向过表达来检查多功能蛋白聚糖表达对体内血管病变中脂蛋白积累的作用。多功能蛋白聚糖和HA代谢的操作应影响动脉粥样硬化病变发展的进展和/或消退。

Abstract: Previous work indicates the versican, the major vascular interstitial chondroitin sulfate proteoglycan (CSPG), and the glycosaminoglycan, hyaluronan (HA), which interacts with versican, accumulate in the extracellular matrix (ECM) in early atherosclerotic and restenotic lesions. We have also shown that the synthesis of these two molecules is highly regulated by specific growth factors and cytokines and is modulated when arterial smooth muscle cells (ASMC) are stimulated to migrate and proliferate. Inhibition of the interaction of versican and HA with receptors at the cell surface blocks ASMC proliferation and migration. One consequence of versican accumulation in the ECM is the trapping and retention of lipoproteins. We have shown that versican interacts with apoB and apoE-containing lipoproteins and this interaction is enhanced by factors that influence chondroitin sulfate chain biosynthesis. These observations have led us to continue to explore the importance of versican/HA complexes in the ECM in the regulation of ASMC phenotype, and in the retention of lipoproteins in the vascular wall. We hypothesize that modifications in versican/HA synthesis are required for ASMC proliferation and migration, and that these modifications influence other properties of ASMC, such as the ability to remodel the ECM during different phases of atherogenesis. We further postulate that the accumulation of versican within the ECM is partly responsible for the extracellular accumulation of lipoproteins. We propose to examine these hypotheses in four aims. The first aim will address questions concerning the role of versican and versican isoform expression in the regulation of ASMC phenotype and matrix remodeling. In this work we will express versican isoforms and mini-genes that contain various domains of versican, and examine the effect on cell proliferation, migration and matrix protein deposition. The second aim includes experiments designed to examine the expression of HA synthases by ASMC, and the role of HA, both as an extracellular component of the matrix and as a newly discovered intracellular molecule, in ASMC proliferation, migration and ECM remodeling. The third aim is designed to extend our studies concerning the interaction of lipoproteins with versican chondroitin sulfate side chains. We propose to determine the specific oligosaccharide sequences that are responsible for the binding of ApoB and ApoE-contain lipoproteins to versican, and determine factors that induce changes in chondroitin sulfate chain structure that give rise to versican forms with differing affinities for lipoproteins. In the fourth aim, we will use new reagents to examine the presence of versican variants and proteolytic cleavage products in naturally-occurring atherosclerotic lesions, investigate the effect of altered expression of versican variants and HA synthases on lesion progression by a cell-mediated gene transfer approach in the injured rat carotid neointimal model, and use targeted over-expression of versican variants in a fat-fed transgenic mouse model to examine the role of versican expression on the accumulation of lipoproteins in vascular lesions in vivo. Manipulation of versican and HA metabolism should influence the progression and/or regression of atherosclerotic lesion development.