Cardiovascular Research Center (CSIC-ICCC), Biomedical Research Institute Sant- Pau (IIB-Sant Pau), Barcelona, Spain.
J Thromb Haemost. 2012 Oct;10(10):2158-67. doi: 10.1111/j.1538-7836.2012.04896.x.
Urokinase-type plasminogen activator (UPA) regulates vascular smooth muscle cell (VSMC) functions relevant in vascular remodeling by facilitating proteolysis at the cell surface and inducing cell signaling pathways. Our previous results demonstrated that aggregated low-density lipoprotein (agLDL) impair cytoskeleton dynamics, a key event contributing to VSMC behavior during progression of atherosclerotic plaques.
To investigate whether mechanisms underlying inhibition of cytoskeleton dynamics in lipid-loaded VSMC occurs through a UPA-mediated process.
Adhesion assay was performed in lipid-loaded human VSMC after 16-h exposition to agLDL (100 μg mL(-1)). Protein subcellular localization and actin-fiber formation were assessed by confocal microscopy. For analysis of protein expression western blots were carried out. Co-immunoprecipitates of UPAR were examined by one-dimensional- or two-dimensional electrophoresis (1-DE or 2-DE), mass spectrometry MALDI-TOF and western blot.
agLDL induced UPA subcellular delocalization and significantly decreased UPA levels during attachment of VSMC. UPA (enhanced endogenous-expression or exogenous added) acting as a urokinase-type plasminogen activator receptor (UPAR)-ligand restored actin-cytoskeleton organization and adhesion capacity of lipid-loaded cells to control levels. UPAR co-immunoprecipitated with the unphosphorylated form of myosin regulatory light chain (MRLC) in lipid-loaded cells. The detrimental effects of agLDL on MRLC phosphorylation were reversed by high levels of UPA. The UPA effects on VSMC exposed to agLDL involved FAK phosphorylation.
The detrimental effects of atherogenic LDL on VSMC are mediated by a decrease and delocalization of the UPA-UPAR interaction that result in an impairment of cytoskeleton dynamics and adhesion capacity affecting cell phenotype and function.
尿激酶型纤溶酶原激活物（uPA）通过促进细胞表面的蛋白水解和诱导细胞信号通路，调节血管平滑肌细胞（VSMC）功能，这些功能与血管重塑有关。我们之前的研究结果表明，聚集的低密度脂蛋白（agLDL）会损害细胞骨架动力学，这是导致动脉粥样硬化斑块进展过程中 VSMC 行为改变的关键事件。
研究脂质负载的 VSMC 中细胞骨架动力学的抑制机制是否通过 uPA 介导的过程发生。
在 agLDL（100μg/ml）孵育 16 小时后，对脂质负载的人 VSMC 进行黏附实验。通过共聚焦显微镜评估蛋白亚细胞定位和肌动蛋白纤维形成。通过 Western blot 分析蛋白表达。通过一维或二维电泳（1-DE 或 2-DE）、MALDI-TOF 质谱和 Western blot 检查 UPAR 的免疫沉淀。
agLDL 诱导 uPA 亚细胞移位，并在 VSMC 黏附时显著降低 uPA 水平。uPA（增强的内源性表达或外源性添加）作为尿激酶型纤溶酶原激活物受体（UPAR）配体，恢复了脂质负载细胞的肌动蛋白细胞骨架组织和黏附能力，使其达到对照水平。在脂质负载的细胞中，UPAR 与非磷酸化形式的肌球蛋白调节轻链（MRLC）共同免疫沉淀。agLDL 对 MRLC 磷酸化的有害影响被高水平的 uPA 逆转。uPA 对暴露于 agLDL 的 VSMC 的作用涉及 FAK 磷酸化。
致动脉粥样硬化 LDL 对 VSMC 的有害影响是通过 uPA-UPAR 相互作用的减少和移位介导的，这导致细胞骨架动力学和黏附能力受损，影响细胞表型和功能。
