组织蛋白酶Cathepsin L/V促进血管内膜新生的机制研究

Restenosis is the major complication of percutaneous coronary interventions (PCIs). New mechanistic insights of the pathogenesis underlying restenosis are essential for improving clinical outcome in interventional cardiology. A hallmark of restenotic lesion is neointimal hyperplasia (NH), characterized by extensive vascular smooth muscle cell (SMC) proliferation and extracellular matrix deposition. Recent studies including ours demonstrate that sterile inflammation critically contributes to neointimal formation, resulting from the activation of pattern recognition receptors (PRRs) (e.g. toll-like receptors, TLRs). Inasmuch, damage-associated molecular pattern (DAMP) molecules (e.g. high mobility group box-1, HMGB1) can serve as potent stimulators to activate innate immune system...Our latest cell-based screening has identified that saquinavir (SQV), a FDA-approved HIV protease inhibitor, inhibits TLR4 signaling-mediated cytokine release in macrophage. Further high-throughput screening for inhibitory activity of SQV against 58 known human proteases showed that SQV exhibits high selectivity on cathepsin V, one of the 11 cysteine cathepsin family members. Cathepsins are primarily lysosomal proteases that not only function in intracellular protein degradation, but also in extracellular space when secreted. Striking new findings from our preliminary studies indicate that 1) SQV markedly inhibited neointimal formation, 2) neointimal lesion was retarded in mice deficient of Cat L (an analog of human Cat V, with markedly reduced SMC growth, and 3) the activity of mTORC1, a master cell growth signaling molecule, was significantly decreased in parallel. These intriguing findings form the basis for our central hypothesis that Cat L critically promotes neointimal hyperplasia through both extracellular and intracellular cell signaling pathways, converging on extensive SMC growth. The objective of this proposal is to determine the cellular mechanisms by which Cat L promotes neointimal hyperplasia. To test our hypothesis and achieve our objective, we plan to pursue the following 3 specific aims:..Aim 1: To delineate the mechanisms by which intracellular Cat L stimulates SMC growth. Our preliminary studies showed that 1) Cat L expression is dramatically increased in injured artery; 2) Cat L promotes SMC proliferation; and 3) Cat L deficiency in SMC attenuates lysosomal degradation capacity, with decreased mTORC1 activity. Based on these findings, we will pursue the hypothesis that intracellular Cat L stimulates SMC growth through lysosome-centered mTORC1 pathway..Aim 2: To determine how extracellular Cat L signaling pathway contributes to SMC proliferation. Our preliminary studies revealed that 1) both SQV and Cat L KO mice exhibited marked NH reductions; 2) SQV inhibits both TLR4-mediated inflammatory response and the activity of Cat V (an analog of mouse Cat L); 3) Cat L was constitutively secreted into extracellular space; and 4) Cat L interacted with TLR4 receptors. Based on these findings, we hypothesize that Cat L initiates extracellular TLR4 receptors to promote SMC growth..Aim 3: To determine how Cal L affects neointimal lesion development in vivo. Our preliminary studies demonstrate that 1) Cat L KO mice exhibited marked NH reductions; and 2) Cat L interacted with TLR4 receptors. Based on these findings, we will validate whether Cat L activates TLR4 receptor via its MyD88 and/or TRIF signaling pathways, and whether blocking Cat L results in reduced neointimal lesion in vivo...This project, will for the first time, identify key new mechanisms of protease Cat L regulation of neointimal formation, which will uncover a previously unrecognized role of intracellular and extracellular Cat L on restenosis. These findings may enable the design of new therapeutic strategies to combat restenosis. In addition, the implications of this work will likely extend to a variety of other proliferative vascular diseases.

血管再狭窄是影响支架植入术远期疗效的最主要原因。以血管SMCs大量增殖为主要病理特征的内膜新生是血管再狭窄的显著标志。TLR4介导的免疫炎症反应，以及mTORC1调控的细胞生长均可促进SMCs增殖。我们在蛋白酶活性抑制筛选实验中意外发现，沙奎那韦（SQV）作为一种能抑制TLR4信号通路的蛋白酶抑制剂对Cat V（系小鼠Ca L同源物）具有高度选择性。我们预实验显示：1）SQV在抑制Cat L活性的同时延缓野生型小鼠内膜新生；2）Cat L-/-小鼠的内膜新生明显减少，且SMCs增殖显著降低； 3）分泌到细胞外的Cat L能与TLR4相互作用，而TLR4-/-小鼠的内膜新生减少；4）SMCs的Cat L水平与mTORC1活性呈正相关。因此本项目将深入探讨Cat L通过膜上TLR4及胞内mTORC1信号通路促进SMCs增殖从而导致血管内膜新生的机制，为介入术后血管再狭窄的防治提供新的理论依据。

血管再狭窄是影响支架植入术远期疗效的最主要原因。以血管平滑肌细胞大量增殖为主要病理特征的内膜新生是血管再狭窄的显著标志。TLR4介导的免疫炎症反应，以及mTORC1调控的细胞生长均可促进血管平滑肌细胞增殖。我们预实验提示Cathepsin L（Cat L）在血管再狭窄中发挥关键调控作用。本项目深入探讨Cat L通过膜上TLR4及胞内mTORC1信号通路促进血管平滑肌细胞增殖从而导致血管内膜新生的机制。研究发现，敲除Cat L后，血管平滑肌细胞增殖显著减少，同时，Cat L表达和溶酶体中细胞生长和代谢的关键调控因子mTORC1的活性呈正相关；Cat L敲除后原代血管平滑肌细胞中溶酶体降解能力降低。发现TLR4与细胞外Cat L而不是与溶酶体中的Cat L形成蛋白复合物，Cat L 可作为一个接头蛋白在细胞外与TLR4发生相互作用；发现HMGB1通过巨噬细胞中TLR4信号通路促进内膜新生，内源性免疫反应在动脉损伤后导致的炎症及内膜新生过程中发挥关键作用。与对照组相比，颈动脉损伤组小鼠颈动脉内膜新生组织中Cat L 表达升高；高通量测序分析发现，HIV 蛋白酶抑制剂沙奎那韦（SQV）可抑制巨噬细胞中TLR4信号通路介导的炎症因子释放；受到SQV 影响的11 种人组织蛋白酶中，Cat V表现出了高选择性；SQV干预降低颈动脉损伤小鼠Cat L活性，明显抑制动脉损伤后的内膜新生；全身敲除Cat L 也可明显抑制动脉内膜新生。 综上，本项目研究证实：细胞外的Cathepsin L激活TLR4-MyD88/TRIF 信号通路，细胞内的Cathepsin L 正向调节溶酶体mTORC1信号通路，共同促进血管平滑肌细胞增殖从而导致血管内膜新生。这一研究结果将为介入术后血管再狭窄的防治提供重要理论依据。

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