Aggregated low‐density lipoprotein induce impairment of the cytoskeleton dynamics through urokinase‐type plasminogen activator/urokinase‐type plasminogen activator receptor in human vascular smooth muscle cell

Abstract

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.