P584Smooth muscle cell-cytoskeleton dynamics in lipid-loaded human vascular smooth muscle cells is dependent on ROCK-signaling

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Atherosclerotic plaques with a large lipid-necrotic core and a thin fibrous cap are the most prone to rupture. It is known that vascular smooth muscle cells (VSMC) show impaired vascular repair function due to lipid-induced changes in cytoskeleton proteins involved in cell migration and we have previously suggested that plaque-stabilizing effects of statins relate with an increase in VSMC-migration. However, mechanisms involved are poorly understood. Here, we investigated whether the changes induced by atherogenic LDL in the cytoskeleton organization of human artery derived VSMC are dependent on ROCK signaling.


Human coronary VSMC, treated with/without an HMGCoA-Reductase inhibitor (rosuvastatin) to block protein isoprenylation, were incubated (24 hours) in the presence/absence of 100µg/mL aggregated LDL (agLDL). Cytoskeleton regulatory proteins were analyzed by western blot and activity assays. Protein subcellular localization and actin fiber formation during cell adhesion and migration were assessed by confocal microscopy.


Rosuvastatin significantly increased in a dose-dependent manner (2.5-10uM) myosin regulatory light chain (MRLC) phosphorylation in lipid-loaded VSMC. Western blot analysis demonstrated that agLDL increased > 2fold (p < 0.05) the phosphorylated form of PAK-1 (p21-activated protein kinase), protein that downregulates MRLC-phosphorylation. This effect was not reversed by rosuvastatin, which in turn increased cytosolic ROCK levels (2.5fold, p < 0.05) and MRLC-phosphorylation by a ROCK-mediated pathway in agLDL-VSMC. In addition, rosuvastatin strongly promoted filamentous actin organization in lipid-loaded cells, whereas co-incubation with the ROCK-inhibitor Y-27632 prevented this effect of rosuvastatin. The ROCK-mediated effects were related to a strong increase in the level of RhoA-GTP (active RhoA) in the cytosol of agLDL-VSMC, as demonstrated by a pull-down assay. In contrast, rosuvastatin did not increase active Rac-1 in these cells. Furthermore, confocal analysis showed that rosuvastatin increased > 2.5fold RhoA labeling (p < 0.05) at the front edge of migrating cells.


Isoprenylation blockade improves MRLC-phosphorylation and actin-cytoskeleton organization in VSMC exposed to atherogenic LDL through a mechanism that mainly depends on the increase of cytosolic RhoA-GTP and its target ROCK. These processes improving VSMC function may contribute, to the effect of statins in plaque-stabilization.

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