Simvastatin modulates chemokine and chemokine receptor expression by geranylgeranyl isoprenoid pathway in human endothelial cells and macrophages  Niels R. Veillard, Vincent Braunersreuther, Claire Arnaud, Fabienne Burger, Graziano Pelli, Sabine Steffens, François Mach  Atherosclerosis  Volume 188, Issue 1, Pages 51-58 (September 2006) DOI: 10.1016/j.atherosclerosis.2005.10.015 Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Dose-dependent inhibition of CCR4 and MCP-1 by simvastatin. RNase protection assays performed on ECs for CCR4 (A). Real-time semi-quantitative PCR performed on macrophages for MCP-1 (B). ECs and macrophages under unstimulated conditions (control), treated with TNF-α (10ng/ml, 4h) or IFN-γ (5000U/ml, 4h) alone or in the presence of simvastatin (10μM–20nM). Results (n=3 from different donors) are expressed as percentage compared to TNF-α or IFN-γ stimulated cells (4h). Data were normalized to GAPDH or 18S. *P<0.05 vs. TNF-α or IFN-γ. Atherosclerosis 2006 188, 51-58DOI: (10.1016/j.atherosclerosis.2005.10.015) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Simvastatin reduces MCP-1 secretion. ELISA for RANTES and MCP-1 within supernatants of the different cell stimulations performed with ECs (A) and macrophages (B). Stimulation abbreviations—T: TNF-α, I: IFN-γ, Simva: simvastatin, Meva: mevalonate. Results (n=5 from different donors) are expressed as percentage of mean OD value of TNF-α or IFN-γ stimulated cells (4h). *P<0.05 vs. TNF-α or IFN-γ. Atherosclerosis 2006 188, 51-58DOI: (10.1016/j.atherosclerosis.2005.10.015) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Simvastatin inhibits chemokine mRNA expression. Real-time quantitative PCR performed on ECs (A) and macrophages (B) for the chemokines RANTES, MCP-1, MIP-1α and MIP-1β. Stimulation abbreviations—Simva: simvastatin, Meva: mevalonate. Results (n=5 from different donors) are expressed as percentage compared to TNF-α or IFN-γ stimulated cells (4h). Data were normalized to 18S. *P<0.05 vs. TNF-α or IFN-γ. Atherosclerosis 2006 188, 51-58DOI: (10.1016/j.atherosclerosis.2005.10.015) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Simvastatin inhibits chemokine receptor mRNA expression. RNase protection assays performed on ECs for CCR4 (A) and macrophages for the chemokine receptors CCR1, CCR2 and CCR5 (B). Stimulation abbreviations—S or Simva: simvastatin, M or Meva: mevalonate, T: TNF-α. Data were normalized to GAPDH and L32. Results (n=5 from different donors) are expressed as percentage compared to TNF-α or IFN-γ stimulated cells (4h). *P<0.05 vs. TNF-α or IFN-γ. Atherosclerosis 2006 188, 51-58DOI: (10.1016/j.atherosclerosis.2005.10.015) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Effect of simvastatin on chemokine mRNA expression is GGPP-dependent. Real-time quantitative PCR performed on ECs (A) and macrophages (B) for the chemokines RANTES, MCP-1, MIP-1α and MIP-1β. Stimulation abbreviations, GGTI: geranylgeranyl transferase inhibitor, FTI: farnesyl transferase inhibitor. Results (n=5 from different donors) are expressed as percentage compared to TNF-α or IFN-γ stimulated cells (4h). Data were normalized to 18S. *P<0.05 vs. TNF-α or IFN-γ. Atherosclerosis 2006 188, 51-58DOI: (10.1016/j.atherosclerosis.2005.10.015) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 6 Effect of simvastatin on chemokine receptor mRNA expression is GGPP-dependent. RNase protection assays performed on ECs for CCR4 (A) and macrophages for the chemokine receptors CCR1, CCR2 and CCR5 (B). Stimulation abbreviations, T: TNF-α, GGTI: geranylgeranyl transferase inhibitor, FTI: farnesyl transferase inhibitor. Results (n=5 from different donors) are expressed as percentage compared to TNF-α or IFN-γ stimulated cells (4h). Data were normalized to GAPDH or 18S. *P<0.05 vs. TNF-α or IFN-γ. Atherosclerosis 2006 188, 51-58DOI: (10.1016/j.atherosclerosis.2005.10.015) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 7 Hypothesized regulation pathway of chemokines and chemokine receptors by statins. The effect of statin on chemokine and chemokine receptor expression could be mediated via two mechanisms. (1) Statins inhibit geranylgeranylation of RhoA and thus, NF-κB, leading to a downregulation of MCP-1, IL-8 and IP-10. (2) By increasing the activity of the transcriptional repressor Oct-1, statins regulate CCR2 and CCR5 expression via geranylgeranyl pathway. Atherosclerosis 2006 188, 51-58DOI: (10.1016/j.atherosclerosis.2005.10.015) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions