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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: 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.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.

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

Effect of GGPP and FPP (A) and the ROCK inhibitor Y-27632 and Rac inhibitor NCS23766 (B) on simvastatin-induced fibulin-2 mRNA levels in human coronary artery SMCs.Cells were incubated with simvastatin and isoprenoids or with the inhibitors for 24 hours. The results are shown as the mean with the standard error of the mean (SEM) for twelve independent experiments in both A and B (2 with cell lot number 4C0915, 4 with cell lot number 4C1284, and 6 with cell lot number 886619). Comparisons were performed using ANOVA followed by Dunett post-test correction. FPP: farnesyl pyrophosphate, GGPP: geranylgeranyl pyrophosphate.
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pone.0133875.g004: Effect of GGPP and FPP (A) and the ROCK inhibitor Y-27632 and Rac inhibitor NCS23766 (B) on simvastatin-induced fibulin-2 mRNA levels in human coronary artery SMCs.Cells were incubated with simvastatin and isoprenoids or with the inhibitors for 24 hours. The results are shown as the mean with the standard error of the mean (SEM) for twelve independent experiments in both A and B (2 with cell lot number 4C0915, 4 with cell lot number 4C1284, and 6 with cell lot number 886619). Comparisons were performed using ANOVA followed by Dunett post-test correction. FPP: farnesyl pyrophosphate, GGPP: geranylgeranyl pyrophosphate.

Mentions: Mevalonate is a precursor of isoprenoid compounds such as FPP and GGPP in the cholesterol biosynthetic pathway. To evaluate which downstream isoprenoid was involved in fibulin 2 mRNA expression, we incubated human coronary artery SMCs with simvastatin alone or in combination with one of the isoprenoids. We found that GGPP (15 μM) but not FPP (15 μM) markedly reversed simvastatin-induced fibulin 2 mRNA expression, whereas the respective isoprenoids alone had no effect (Fig 4A). These results were specific for fibulin 2 because no change in the mRNA levels of fibulin-1, -4, or -5 was observed after incubation of cells with isoprenoids alone or in combination with simvastatin (S3 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)

Effect of GGPP and FPP (A) and the ROCK inhibitor Y-27632 and Rac inhibitor NCS23766 (B) on simvastatin-induced fibulin-2 mRNA levels in human coronary artery SMCs.Cells were incubated with simvastatin and isoprenoids or with the inhibitors for 24 hours. The results are shown as the mean with the standard error of the mean (SEM) for twelve independent experiments in both A and B (2 with cell lot number 4C0915, 4 with cell lot number 4C1284, and 6 with cell lot number 886619). Comparisons were performed using ANOVA followed by Dunett post-test correction. FPP: farnesyl pyrophosphate, GGPP: geranylgeranyl pyrophosphate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4514789&req=5

pone.0133875.g004: Effect of GGPP and FPP (A) and the ROCK inhibitor Y-27632 and Rac inhibitor NCS23766 (B) on simvastatin-induced fibulin-2 mRNA levels in human coronary artery SMCs.Cells were incubated with simvastatin and isoprenoids or with the inhibitors for 24 hours. The results are shown as the mean with the standard error of the mean (SEM) for twelve independent experiments in both A and B (2 with cell lot number 4C0915, 4 with cell lot number 4C1284, and 6 with cell lot number 886619). Comparisons were performed using ANOVA followed by Dunett post-test correction. FPP: farnesyl pyrophosphate, GGPP: geranylgeranyl pyrophosphate.
Mentions: Mevalonate is a precursor of isoprenoid compounds such as FPP and GGPP in the cholesterol biosynthetic pathway. To evaluate which downstream isoprenoid was involved in fibulin 2 mRNA expression, we incubated human coronary artery SMCs with simvastatin alone or in combination with one of the isoprenoids. We found that GGPP (15 μM) but not FPP (15 μM) markedly reversed simvastatin-induced fibulin 2 mRNA expression, whereas the respective isoprenoids alone had no effect (Fig 4A). These results were specific for fibulin 2 because no change in the mRNA levels of fibulin-1, -4, or -5 was observed after incubation of cells with isoprenoids alone or in combination with simvastatin (S3 Fig).

Bottom Line: 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.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.

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