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Simvastatin inhibits MMP-9 secretion from human saphenous vein smooth muscle cells by inhibiting the RhoA/ROCK pathway and reducing MMP-9 mRNA levels Neil A. Turner,* David J. O’Regan,† Stephen G. Ball,* and Karen E. Porter*,1 *Institute for Cardiovascular Research, University of Leeds, Leeds, UK; and †Department of Cardiac Surgery, The Yorkshire Heart Centre, Leeds General Infirmary, Leeds, UK To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2852fje; doi: 10.1096/fj.04-2852fje SPECIFIC AIMS Coronary artery bypass grafting (CABG) using the autologous saphenous vein (SV) is routinely used to revascularize atherosclerotic coronary arteries, although subsequent occlusions are frequent in such grafts. Recent evidence suggests that statins, widely prescribed cholesterol-lowering drugs, can reduce SV graft occlusions and improve cardiovascular outcome after CABG, independent of effects on cholesterol. As increased matrix metalloproteinase-9 (MMP-9) expression is associated with intimal hyperplasia in SV bypass grafts, the aim of the present study was to investigate the intracellular mechanisms underlying the inhibition of MMP-9 secretion by simvastatin in cultured human SV smooth muscle cells (SMC).
PRINCIPAL FINDINGS 1. Simvastatin inhibits MMP-9 secretion via inhibition of the RhoA/ROCK pathway Using gelatin zymography, we demonstrated that the phorbol ester TPA (100 nM) or a combination of 15 ng/mL platelet-derived growth factor-BB (PDGF) ⫹ 20 ng/mL interleukin-1␣ (IL-1) significantly increased MMP-9 (but not MMP-2) secretion from cultured human SV-SMC. Simvastatin (0.5–10 M) reduced MMP-9 (but not MMP-2) secretion induced by either stimulus in a concentration-dependent manner; maximal inhibition (⬎75%) observed with 10 M simvastatin. Similar results were obtained with atorvastatin, but not lovastatin or pravastatin. The effects of statins are attributable to inhibition of HMG-CoA reductase, resulting in decreased synthesis of intracellular mevalonate. Mevalonate is also a precursor of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), isoprenoids 804
required for prenylation of Ras and Rho-family GTPases, respectively. This ability of statins to inhibit the prenylation and resultant activity of small GTPases may underlie many of their cholesterol-independent effects. Coincubation of simvastatin-treated SV-SMC cells with mevalonate (100 M) or GGPP (10 M), but not FPP (10 M), fully prevented its inhibitory effects on TPA- or PDGF/IL-1-induced MMP-9 secretion, indicating that simvastatin was reducing MMP-9 secretion via inhibition of Rho geranylgeranylation. To confirm that geranylgeranylation of Rho-family GTPases was essential for MMP-9 secretion, we investigated the effects of GGTI-286, a specific inhibitor of geranylgeranyl transferase-I (GGTase-I). GGTI-286 (10 M) mimicked the inhibitory effect of simvastatin on MMP-9 secretion (58% inhibition), indicating a role for inhibition of Rho-family GTPases. Although the Rho-family of GTPases comprises three main sub-families (Rho, Rac, cdc42), it is only RhoA that activates Rho-kinase (ROCK). Consistent with the effects of both simvastatin and GGTI286, the ROCK inhibitor Y27632 (10 M) also reduced MMP-9 secretion (59% inhibition). These data suggest that inhibition of MMP-9 secretion by simvastatin occurs via inhibition of the RhoA/ROCK pathway in human SV-SMC. 2. Simvastatin reduces MMP-9 mRNA levels via inhibition of the RhoA/ROCK pathway Using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) we demonstrated that MMP-9 mRNA levels were increased after TPA or PDGF/IL-1 treatment. Simvastatin (10 M) markedly 1 Correspondence. Institute for Cardiovascular Research, Worsley Building, University of Leeds, Leeds LS2 9JT, UK. E-mail:
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0892-6638/05/0019-0804 © FASEB
3. Simvastatin does not inhibit signal transduction pathways required for MMP-9 expression MMP-9 gene expression can be activated via a number of signal transduction pathways, including those involving extracellular signal-regulated kinases (ERK-1/2), p38 mitogen-activated protein kinase (p38 MAPK), c-Jun NH2-terminal kinase ( JNK), phosphoinositide 3-kinase (PI3K)/Akt and nuclear factor-B (NF-B). Immunoblotting studies with phospho-specific antibodies revealed that PDGF/IL-1 treatment activated the ERK, p38 MAPK, PI3K/Akt, and NFB pathways over 2–30 min but did not activate the JNK pathway. In contrast, TPA activated only the ERK pathway. MMP-9 secretion induced by PDGF/IL-1 was inhibited by selective pharmacological inhibitors of the ERK (30 M PD98059), p38 MAPK (20 M SB203580) and NF-B (5 M MG-132) pathways, but not by inhibition of the PI3K/Akt pathway (10 M LY294002). In contrast, TPA-induced MMP-9 secretion required activation of only the ERK pathway. We then investigated whether simvastatin was reducing MMP-9 secretion by preventing activation of the ERK, p38 MAPK, PI3K/Akt, or NFB pathways. Surprisingly, simvastatin had no inhibitory effect on any of the signaling pathways studied (Fig. 2). However, in keeping with this observation, neither GGTase-I inhibition (GGTI-286) nor ROCK inhibition (Y27632) had any effect (Fig. 2). RhoA activity was not affected by PDGF/ IL-1 or TPA treatment. Thus, although the RhoA/
Figure 1. Simvastatin reduces MMP-9 mRNA levels in parallel with secretion. Cells were treated with TPA with or without 10 M simvastatin for 16 – 40 h. A) RT-PCR. Line graph depicts MMP-9/GAPDH ratio after densitometric analysis (n⫽4). B) Gelatin zymography. Line graph depicts densitometric analysis of MMP-9 band intensity (n⫽4).
reduced TPA-induced MMP-9 mRNA levels over a 16 – 40 h period (Fig. 1A). Gelatin zymography of CM from the same experiments revealed a similar inhibitory profile on MMP-9 secretion (Fig. 1B). Comparison of the areas under the curves (Fig. 1A, B) showed that simvastatin inhibited the total amount of TPA-induced MMP-9 mRNA and MMP-9 secretion over a 40 h period by 52.6 ⫾ 11.6% (P⬍0.05, n⫽4) and 57.8 ⫾ 3.7% (P⬍0.001, n⫽4), respectively. The inhibitory effects of simvastatin on MMP-9 mRNA levels could be mimicked with the GGTase-I inhibitor GGTI-286 or the ROCK inhibitor Y27632. These data indicate that the inhibitory effect of simvastatin on MMP-9 secretion occurs via the RhoA/ROCK pathway and at the level of mRNA. STATINS INHIBIT HUMAN SV-SMC MMP-9 SECRETION
Figure 2. Effects of simvastatin, GGTI-286, and Y27632 on signal transduction pathways. Cells were pretreated with 10 M simvastatin (5 h), GGTI-286, or Y27632 (1 h) before incubation with PDGF/IL-1 or TPA for 5 min. Immunoblotting was performed with antibodies that recognize the phosphorylated forms of ERK-1/2, p38 MAPK, JNK, Akt, and IB-␣ to determine activation of ERK, p38 MAPK, PI3K/Akt, and NFB pathways, respectively. Equal loading was confirmed with ␣-smooth muscle actin (SMA) antibody. Blots represent results from 4 patients. 805
Figure 3. Schematic diagram depicting the mechanism by which simvastatin inhibits MMP-9 secretion in human SV-SMC. PDGF/IL-1 stimulates MMP-9 secretion via activation of the ERK, p38 MAPK, and NF-B pathways whereas TPA stimulates MMP-9 secretion solely via the ERK pathway. The RhoA/ROCK pathway regulates MMP-9 expression independent of effects on the ERK, p38 MAPK, and NF-B pathways. Simvastatin inhibits MMP-9 secretion by preventing geranylgeranylation of RhoA, leading to decreased ROCK activation and reduced MMP-9 mRNA levels.
ROCK pathway is inhibited by simvastatin in human SV-SMC, it is not required for activation of the signal transduction pathways coupled to MMP-9 secretion. 4. Simvastatin does not reduce MMP-9 mRNA stability In a number of other cell types, MMP-9 mRNA levels have been shown to be regulated post-transcriptionally at the level of mRNA stability. We therefore investigated whether such a mechanism may underlie the ability of simvastatin to reduce MMP-9 mRNA levels in human SV-SMC. Using RT-PCR we demonstrated that simvastatin did not decrease MMP-9 mRNA levels in the presence of the transcription inhibitor actinomycin D, indicating that the suppressive effect of simvastatin on MMP-9 mRNA levels was not the result of increased mRNA degradation.
CONCLUSIONS AND SIGNIFICANCE In summary, simvastatin reduces MMP-9 secretion from human SV-SMC by inhibiting the RhoA/ROCK pathway and decreasing MMP-9 mRNA levels independent of effects on the signal transduction pathways required for MMP-9 expression (Fig. 3). Since SMC invasion requires the activity of MMP-9, this property of simvastatin may explain our previous report of its ability to reduce neointima formation in cultured human SV. Although studies in other cell types have shown that statins can reduce MMP-9 secretion, they have reported divergent effects (increased, decreased or unchanged) on MMP-9 mRNA levels. A recent study using rabbit vascular SMC observed no change in MMP-9 mRNA levels, but did report that statin treatment also inhibited secretion of several other MMPs (MMPs 1, 2, 3) as
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well as MMP-9, indicating a nonselective post-translational mechanism. These results are clearly different from our own that suggest a selective effect on MMP-9. One mechanism by which statins may reduce MMP-9 mRNA levels is to inhibit the signal transduction pathways coupled to MMP-9 gene expression. For example, recent studies in vascular SMC have shown that statins can inhibit the ERK and NF-B pathways. In the present study in SV-SMC, simvastatin did not inhibit any of the signaling pathways necessary for MMP-9 expression. RhoA activity was not modified by PDGF/IL-1 or TPA treatment, and neither did inhibition of the RhoA/ ROCK pathway affect these signaling pathways, indicating that RhoA and ROCK are not upstream activators of these pathways. Our results suggest that the mechanism whereby RhoA/ROCK inhibition leads to a reduction in MMP-9 mRNA levels is likely to be at the transcriptional level, either as a result of reduced transcription factor binding or increased binding of a transcriptional repressor. It is apparent that the mechanism of MMP-9 inhibition by statins is species and cell type dependent, so our present study using human SV-derived SMC, together with our previous study of the effects of simvastatin on neointima formation and MMP-9 expression in organcultured human SV, are particularly relevant. Although the precise mechanism by which simvastatin inhibits MMP-9 secretion from human SV-SMC requires further study, we clearly demonstrate here that simvastatin reduces MMP-9 secretion by inhibiting the RhoA/ROCK pathway and decreasing MMP-9 mRNA levels independent of effects on the signal transduction pathways required for MMP-9 expression. The ability of simvastatin to reduce MMP-9 secretion from human SV-SMC is likely to contribute to its cholesterol-independent clinical benefits in reducing the incidence of SV graft failure in humans.
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TURNER ET AL.
