Fluvastatin upregulates the α 1C subunit of CaV1.2 channel expression in vascular smooth muscle cells via RhoA and ERK/p38 MAPK pathways

Abstract

Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis. And this process has been related to remodeling of L-type calcium channel (LTCC). We attempted to investigate whether fluvastatin has any effect on VSMC proliferation and LTCCα 1C subunit (LTCCα 1C) expression as well as the potential mechanisms involved. The VSMCs proliferation was assayed by osteopontin immunofluorescent staining and [(3)H]-thymidine incorporation. The cell cycle was detected by flow cytometric analysis. The activity of RhoA was determined with pull-down assay. MAPK activity and LTCCα 1C expression were assessed by western blotting. We demonstrated fluvastatin prevented the VSMCs dedifferentiating into a proliferative phenotype and induced cell cycle arrest in the G0/G1 phase in response to PDGF-BB stimulation. Fluvastatin dose-dependently reversed the downregulation of LTCCα 1C expression induced by PDGF-BB. Inhibition of ROCK, ERK, or p38 MAPK activation largely enhanced the upregulation effect of fluvastatin (P < 0.01). However, blockade of JNK pathway had no effect on LTCCα 1C expression. We concluded LTCCα 1C was a VSMC contractile phenotype marker gene. Fluvastatin upregulated LTCCα 1C expression, at least in part, by inhibiting ROCK, ERK1/2, and p38 MAPK activation. Fluvastatin may be a potential candidate for preventing or treating vascular diseases.

Figure 1

Effects of Flu on VSMCs proliferation analyzed by immunofluorescent staining and [³H]-thymidine uptake. Primary cell culture (a); expression of α-smooth muscle-actin in VSMCs as depicted by immunofluorescent staining (b). Enhanced osteopontin expression, nuclear division (arrow), and increased cell density were noted in VSMCs treated with PDGF (c). Reduced osteopontin fluorescence, spindle-shape appearance was observed in VSMCs by coincubation of fluvastatin and PDGF (d). Flu inhibited [³H]-thymidine incorporation in a time- and concentration-dependent manner (e, f). Results represented as mean ± SEM of 3 independent experiments in triplicate. ^* P < 0.05 versus blank control and ^# P < 0.05 versus cells incubation with PDGF. Flu: fluvastatin; PDGF: platelet derived growth factor; and M: mol/L.

Figure 2

Fluvastatin inhibits cell cycle progression as detected by flow cytometry. VSMCs were incubated for 24 hrs with vehicle (a), PDGF (b), or Flu and PDGF (c). Bar graph illustrating the percentage of cells in G0/G1, S, and G2/M phase by the indicated medication (d). Results represented as mean ± SEM of 3 independent experiments in triplicate. ^* P < 0.05 versus blank control. ^# P < 0.05 versus PDGF stimulated cells. Flu: fluvastatin; PDGF: platelet derived growth factor.

Figure 3

Effects of Flu on the levels of LTCCα _1C protein determined by western blot analysis. PDGF time-dependently inhibited LTCCα _1C expression, which could be prevented by fluvastatin. (a) Dose-effect response of PDGF stimulation on the LTCCα _1C expression in VSMCs. (b) Time-effect response of Flu on the LTCCα _1C expression incubated by PDGF. β-actin served as the loading control. Data was represented as means ± SE of 3 separate experiments conducted in triplicate. ^* P < 0.05 versus blank control and ^# P < 0.05 versus cells incubation with PDGF. LTCCα _1C: L-type calcium channel α _1C subunit; Flu: fluvastatin; and PDGF: platelet derived growth factor.

Figure 4

Effects of MAPK or ROCK inhibition on LTCCα _1C expression after PDGF stimulation in VSMCs. LTCCα _1C protein expression was evaluated by western blot analysis. Data was described as means ± SEM from three experiments performed in triplicate. ^* P < 0.05 versus blank control and ^# P < 0.05 versus PDGF stimulated cells. MAPK: mitogen activated protein kinase; ROCK: Rho associated protein kinase; LTCCα _1C: L-type calcium channel α _1C subunit; Flu: fluvastatin; and PDGF: platelet derived growth factor.

Figure 5

Effect of Flu on PDGF-induced phosphorylation of ERK1/2, p38 MAPK, and JNK. Phosphorylation of ERK1/2, p38 MAPK, and JNK was detected by western blotting using specific antibodies. Total ERK1/2, p38 MAPK, and JNK proteins were used as internal controls. Quantification of band intensities from three independent experiments was determined by densitometry. Data was described as means ± SEM from three experiments performed in triplicate. ^* P < 0.05 versus blank control and ^# P < 0.05 versus cells incubation with PDGF. Flu: fluvastatin; PDGF: platelet derived growth factor. ERK1/2: extracellular signal regulated kinase; MAPK: mitogen activated protein kinase; and JNK: c-Jun N-terminal kinase.

Figure 6

Regulation of membrane-associated RhoA by PDGF in the absence and presence of fluvastatin. (a) Localization of RhoA was determined by immunofluorescent staining. Distribution of RhoA (green) in VSMCs. Nuclei were stained with DAPI (blue). (b) RhoA activation by PDGF in the absence and presence of fluvastatin. RhoA activation was evaluated by rate of GTP-Rho (active form) and total RhoA. Quantification of band intensities from three independent experiments was determined by densitometry. Data was described as means ± SEM from three experiments performed in triplicate. ^* P < 0.05 versus blank control and ^# P < 0.05 versus cells incubation with PDGF. RhoA: ras homolog family member A; Flu: fluvastatin; and PDGF: platelet derived growth factor.