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Simvastatin Increases Fibulin-2 Expression in Human Coronary Artery Smooth Muscle Cells via RhoA/Rho-Kinase Signaling Pathway Inhibition.

Serra N, Rosales R, Masana L, Vallvé JC - PLoS ONE (2015)

Bottom Line: Simvastatin induced a significant increase in mRNA and protein levels of fibulin-2 at 24 hours of incubation (p<0.05), but it did not affect fibulin-1, -4, and -5 expression.Mevalonate and GGPP were able to reverse simvastatin's effect, while FPP did not.Furthermore, activation of the RhoA/ROCK pathway with arachidonic acid decreased fibulin-2 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Facultat de Medicina, Unitat de Recerca en Lípids i Arteriosclerosi, Universitat Rovira i Virgili, CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Institut Investigació Sanitària Pere Virgili (IISPV), Reus, Catalonia, Spain.

ABSTRACT
The composition and structure of the extracellular matrix (ECM) in the vascular wall and in the atherosclerotic plaque are important factors that determine plaque stability. Statins can stabilize atherosclerotic plaques by modulating ECM protein expression. Fibulins are important components of the ECM. We evaluated the in vitro effect of simvastatin on the expression of fibulin-1, -2, -4 and -5 in human coronary artery smooth muscle cells (SMCs) and the mechanisms involved. Cells were incubated with simvastatin (0.05-1 μM), mevalonate (100 and 200 μM), geranylgeranyl pyrophosphate (GGPP) (15 μM), farnesyl pyrophosphate (FPP) (15 μM), the Rho kinase (ROCK) inhibitor Y-27632 (15 and 20 μM), the Rac-1 inhibitor (another member of Rho family) NSC23766 (100 μM), arachidonic acid (a RhoA/ROCK activator, 25-100 μM) and other fatty acids that are not activators of RhoA/ROCK (25-100 μM). Gene expression was analyzed by quantitative real-time PCR, and fibulin protein levels were analyzed by western blotting and ELISA. Simvastatin induced a significant increase in mRNA and protein levels of fibulin-2 at 24 hours of incubation (p<0.05), but it did not affect fibulin-1, -4, and -5 expression. Mevalonate and GGPP were able to reverse simvastatin's effect, while FPP did not. In addition, Y-27632, but not NSC23766, significantly increased fibulin-2 expression. Furthermore, activation of the RhoA/ROCK pathway with arachidonic acid decreased fibulin-2 mRNA. Simvastatin increased mRNA levels and protein expression of the ECM protein fibulin-2 through a RhoA and Rho-Kinase-mediated pathway. This increase could affect the composition and structure of the ECM.

No MeSH data available.


Related in: MedlinePlus

A: Effect of mevalonate on simvastatin-induced fibulin-2 mRNA levels in human coronary artery SMCs.Cells were preincubated for 2 hours with mevalonate (100, 200 μM) and treated with simvastatin for 24 hours. B: Effect of mevalonate on simvastatin-induced fibulin-2 protein levels in human coronary artery SMCs. Cells were preincubated for 2 hours with mevalonate (200 μM) and treated with simvastatin for 24 hours. Blot bands were normalized to actin and quantified with Quantity One Analysis Software version 4.6.2. A representative Western blot is shown. The results are shown as the mean with the standard error of the mean (SEM) for twelve independent experiments in 2A (2 with cell lot number 4C0915, 4 with cell lot number 4C1284, and 6 with cell lot number 886619) and six independent experiments in 2B (2 with cell lot number 4C0915, 2 with cell lot number 4C1284, and 2 with cell lot number 886619). Comparisons were performed using ANOVA followed by Dunett post-test correction.
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pone.0133875.g002: A: Effect of mevalonate on simvastatin-induced fibulin-2 mRNA levels in human coronary artery SMCs.Cells were preincubated for 2 hours with mevalonate (100, 200 μM) and treated with simvastatin for 24 hours. B: Effect of mevalonate on simvastatin-induced fibulin-2 protein levels in human coronary artery SMCs. Cells were preincubated for 2 hours with mevalonate (200 μM) and treated with simvastatin for 24 hours. Blot bands were normalized to actin and quantified with Quantity One Analysis Software version 4.6.2. A representative Western blot is shown. The results are shown as the mean with the standard error of the mean (SEM) for twelve independent experiments in 2A (2 with cell lot number 4C0915, 4 with cell lot number 4C1284, and 6 with cell lot number 886619) and six independent experiments in 2B (2 with cell lot number 4C0915, 2 with cell lot number 4C1284, and 2 with cell lot number 886619). Comparisons were performed using ANOVA followed by Dunett post-test correction.

Mentions: By inhibiting HMG-CoA reductase, statins cause a depletion of mevalonate in the cells. To determine whether this depletion was involved in simvastatin increase of fibulin 2 expression, we incubated human coronary artery SMCs with simvastatin alone (1 μM) or in combination with mevalonate. We found that pre-incubating the cells for 2 hours with increasing concentrations of mevalonate (100, 200 μM), completely and significantly reversed the simvastatin-dependent induction of fibulin 2 mRNA and protein (Fig 2), confirming the specificity of simvastatin’s effect. Mevalonate alone did not affect fibulin 2 mRNA or protein expression. Furthermore, we showed that mevalonate alone or in combination with simvastatin did not affect fibulin -1, -4, or -5 gene expression (S2 Fig).


Simvastatin Increases Fibulin-2 Expression in Human Coronary Artery Smooth Muscle Cells via RhoA/Rho-Kinase Signaling Pathway Inhibition.

Serra N, Rosales R, Masana L, Vallvé JC - PLoS ONE (2015)

A: Effect of mevalonate on simvastatin-induced fibulin-2 mRNA levels in human coronary artery SMCs.Cells were preincubated for 2 hours with mevalonate (100, 200 μM) and treated with simvastatin for 24 hours. B: Effect of mevalonate on simvastatin-induced fibulin-2 protein levels in human coronary artery SMCs. Cells were preincubated for 2 hours with mevalonate (200 μM) and treated with simvastatin for 24 hours. Blot bands were normalized to actin and quantified with Quantity One Analysis Software version 4.6.2. A representative Western blot is shown. The results are shown as the mean with the standard error of the mean (SEM) for twelve independent experiments in 2A (2 with cell lot number 4C0915, 4 with cell lot number 4C1284, and 6 with cell lot number 886619) and six independent experiments in 2B (2 with cell lot number 4C0915, 2 with cell lot number 4C1284, and 2 with cell lot number 886619). Comparisons were performed using ANOVA followed by Dunett post-test correction.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone.0133875.g002: A: Effect of mevalonate on simvastatin-induced fibulin-2 mRNA levels in human coronary artery SMCs.Cells were preincubated for 2 hours with mevalonate (100, 200 μM) and treated with simvastatin for 24 hours. B: Effect of mevalonate on simvastatin-induced fibulin-2 protein levels in human coronary artery SMCs. Cells were preincubated for 2 hours with mevalonate (200 μM) and treated with simvastatin for 24 hours. Blot bands were normalized to actin and quantified with Quantity One Analysis Software version 4.6.2. A representative Western blot is shown. The results are shown as the mean with the standard error of the mean (SEM) for twelve independent experiments in 2A (2 with cell lot number 4C0915, 4 with cell lot number 4C1284, and 6 with cell lot number 886619) and six independent experiments in 2B (2 with cell lot number 4C0915, 2 with cell lot number 4C1284, and 2 with cell lot number 886619). Comparisons were performed using ANOVA followed by Dunett post-test correction.
Mentions: By inhibiting HMG-CoA reductase, statins cause a depletion of mevalonate in the cells. To determine whether this depletion was involved in simvastatin increase of fibulin 2 expression, we incubated human coronary artery SMCs with simvastatin alone (1 μM) or in combination with mevalonate. We found that pre-incubating the cells for 2 hours with increasing concentrations of mevalonate (100, 200 μM), completely and significantly reversed the simvastatin-dependent induction of fibulin 2 mRNA and protein (Fig 2), confirming the specificity of simvastatin’s effect. Mevalonate alone did not affect fibulin 2 mRNA or protein expression. Furthermore, we showed that mevalonate alone or in combination with simvastatin did not affect fibulin -1, -4, or -5 gene expression (S2 Fig).

Bottom Line: Simvastatin induced a significant increase in mRNA and protein levels of fibulin-2 at 24 hours of incubation (p<0.05), but it did not affect fibulin-1, -4, and -5 expression.Mevalonate and GGPP were able to reverse simvastatin's effect, while FPP did not.Furthermore, activation of the RhoA/ROCK pathway with arachidonic acid decreased fibulin-2 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Facultat de Medicina, Unitat de Recerca en Lípids i Arteriosclerosi, Universitat Rovira i Virgili, CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Institut Investigació Sanitària Pere Virgili (IISPV), Reus, Catalonia, Spain.

ABSTRACT
The composition and structure of the extracellular matrix (ECM) in the vascular wall and in the atherosclerotic plaque are important factors that determine plaque stability. Statins can stabilize atherosclerotic plaques by modulating ECM protein expression. Fibulins are important components of the ECM. We evaluated the in vitro effect of simvastatin on the expression of fibulin-1, -2, -4 and -5 in human coronary artery smooth muscle cells (SMCs) and the mechanisms involved. Cells were incubated with simvastatin (0.05-1 μM), mevalonate (100 and 200 μM), geranylgeranyl pyrophosphate (GGPP) (15 μM), farnesyl pyrophosphate (FPP) (15 μM), the Rho kinase (ROCK) inhibitor Y-27632 (15 and 20 μM), the Rac-1 inhibitor (another member of Rho family) NSC23766 (100 μM), arachidonic acid (a RhoA/ROCK activator, 25-100 μM) and other fatty acids that are not activators of RhoA/ROCK (25-100 μM). Gene expression was analyzed by quantitative real-time PCR, and fibulin protein levels were analyzed by western blotting and ELISA. Simvastatin induced a significant increase in mRNA and protein levels of fibulin-2 at 24 hours of incubation (p<0.05), but it did not affect fibulin-1, -4, and -5 expression. Mevalonate and GGPP were able to reverse simvastatin's effect, while FPP did not. In addition, Y-27632, but not NSC23766, significantly increased fibulin-2 expression. Furthermore, activation of the RhoA/ROCK pathway with arachidonic acid decreased fibulin-2 mRNA. Simvastatin increased mRNA levels and protein expression of the ECM protein fibulin-2 through a RhoA and Rho-Kinase-mediated pathway. This increase could affect the composition and structure of the ECM.

No MeSH data available.


Related in: MedlinePlus