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(Circulation. 2004;110:571-578.)
© 2004 American Heart Association, Inc.

Original Articles

Role for Matrix Metalloproteinase-2 in Oxidized Low-Density Lipoprotein–Induced Activation of the Sphingomyelin/Ceramide Pathway and Smooth Muscle Cell Proliferation

Nathalie Augé, PhD; Françoise Maupas-Schwalm, MD; Meyer Elbaz, MD; Jean-Claude Thiers, MSc; Axel Waysbort, MD; Shigeyoshi Itohara, PhD; Hans-Willi Krell, PhD; Robert Salvayre, MD, PhD; Anne Nègre-Salvayre, PhD

From INSERM U-466, Department of Biochemistry (N.A., F.M.-S., M.E., J.-C.T., A.W., R.S., A.N.-S.) and Department of Cardiology (M.E.), CHU Rangueil, Toulouse, France; Roche Diagnostics GmbH, Penzberg, Germany (H.-W.K.); and RIKEN Brain Science Institute, Wako-Shi, Saitama, Japan (S.I.).

Correspondence to Dr A. Negre-Salvayre, Biochimie, INSERM U466, IFR-31, CHU Rangueil, 1, avenue Jean Poulhès, TSA-50032, 31059 Toulouse Cedex 9, France. E-mail anesalv{at}rangueil.inserm.fr or salvayre{at}rangueil.inserm.fr

Received July 8, 2003; de novo received December 26, 2003; revision received March 23, 2004; accepted March 30, 2004.

	Abstract

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Background— Oxidized LDLs (oxLDLs) and matrix metalloproteinases (MMPs) are present in atherosclerotic lesions. OxLDLs activate various signaling pathways potentially involved in atherogenesis. OxLDLs induce smooth muscle cell (SMC) proliferation mediated by the activation of the sphingomyelin/ceramide pathway and tyrosine kinase receptors. MMPs are also able to induce SMC migration and proliferation in addition to extracellular matrix degradation. The present study was designed to investigate whether MMPs play a role in the mitogenic effect of oxLDLs.

Methods and Results— OxLDLs induce the release of activated MMP-2 in SMC culture medium. MMP-2 was identified by its 65-kDa gelatinase activity on zymography and by using specific blocking antibodies and MMP-2^–/– cells. MMP inhibitors (batimastat and Ro28-2653) and the blocking antibodies anti–MMP-2 and anti–membrane type 1-MMP inhibited the oxLDL-induced sphingomyelin/ceramide pathway activation and subsequent activation of ERK1/2 and DNA synthesis but did not inhibit the oxLDL-induced epidermal growth factor receptor and platelet-derived growth factor receptor activation. Exogenously added activated MMP-2 or membrane type 1-MMP-1 triggered the activation of both sphingomyelin/ceramide and ERK1/2 pathways and DNA synthesis. Conversely, suppression of MMP-2 expression in MMP-2^–/– cells or in SMCs treated by small-interference RNA also blocked both sphingomyelin/ceramide signaling and DNA synthesis.

Conclusions— Together, these data demonstrate that MMP-2 plays a pivotal role in oxLDL-induced activation of the sphingomyelin/ceramide signaling pathway and subsequent SMC proliferation. These pathways may constitute a potential therapeutic target for modulating the oxLDL-induced proliferation of SMCs in atherosclerosis or restenosis.

Key Words: cells, muscle, smooth • lipoproteins • metalloproteases • sphingomyelins • ceramides

	Introduction

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Atherogenesis is a progressive process characterized by the accumulation of lipids and fibrous elements in the wall of large arteries.^1–3 Smooth muscle cell (SMC) migration, proliferation, and secretion of extracellular matrix play a major role in the formation of the fibroproliferative plaques. The fibrous cap contributes to plaque stability but also favors the development of occlusive lesions or restenosis.^1–4

LDLs are thought to become atherogenic after undergoing oxidative modifications.⁵ Oxidized LDLs (oxLDLs), present in atherosclerotic lesions, may induce changes in lipoprotein metabolism, gene expression (of adhesion molecules, growth factors, and cytokines), cell viability/apoptosis, migration, and proliferation.^2–6

OxLDL-induced SMC proliferation involves various signaling pathways,⁷ including the sphingomyelin/ceramide/sphingosine-1-phosphate (Spm/Cer/S1P) pathway (which mediates DNA synthesis)⁸ and the epidermal growth factor receptor (EGFR)/PI3K/Akt pathway (which mediates cell survival).⁹

Matrix metalloproteinases (MMPs) are a broad family of zinc proteases, which participate in extracellular matrix turnover and vascular wall remodeling, angiogenesis, and atherosclerosis.^10–13 MMPs are regulated by gene expression and activation of latent proenzymes by cleavage of the N-terminal prosegment.^10,14 This partial proteolysis is mediated by various serine proteases, such as plasmin or thrombin,¹¹ or by cell surface–associated MMPs, such as membrane type 1 (MT-1)-MMP.¹² MMP activity is inhibited by specific natural tissue MMP inhibitors (TIMPs)¹⁴ and by selective chemical inhibitors such as batimastat.¹⁵ Because MMP activity is increased in atherosclerotic lesions, it has been hypothesized that MMPs may be involved in atherosclerotic processes, such as infiltration of inflammatory cells, SMC migration and proliferation, and plaque disruption.^11–13 OxLDLs and inflammatory cytokines, including interleukin-1 and tumor necrosis factor-, alter the expression and activity of MMPs and TIMPs.¹¹ For instance, oxLDLs enhance the expression and secretion of MMP-1 (interstitial collagenase) and MT1-MMP (activator of proMMP-2) in endothelial cells and that of MMP-9 (gelatinase B) in SMCs.^16–18

Because both oxLDLs and MMPs are present in atherosclerotic lesions and are able to trigger SMC proliferation, we investigated whether their mitogenic mechanisms were related. Because the MMP inhibitor batimastat inhibited the oxLDL-induced SMC proliferation, we investigated how MMPs mediate the oxLDL-induced mitogenic signaling. We report, for the first time, that the oxLDL-induced mitogenic signaling requires the activation of MT1-MMP and MMP-2, which in turn activates the Spm/Cer/S1P pathway.

	Methods

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SMC Culture
Rabbit femoral artery and human aortic SMCs (CRL 1999) (ATCC) were grown in RPMI-1640 supplemented with 10% FCS at 37°C, as described previously.⁹ MMP-2–deficient fibroblasts from MMP-2^–/– mice¹⁹ were grown in DMEM supplemented with 10% FCS. Twenty-four hours before each experiment, the medium was replaced with fresh RPMI medium containing 0.5% FCS.

Small-Interference RNA
MMP-2 expression was inhibited by small-interference RNA (siRNA) 5'-AGUUGGCAGUGCAAUACCUGA-3' (sense strand) (Dharmacon). SMCs were transfected with 20 µmol/L double-strand siRNA in Optimem medium (Gibco) mixed with oligofectamine, as described previously.²⁰ Three hours after transfection, SMCs were incubated in RPMI containing 10% FCS for 24 hours. Then SMCs were incubated for 12 hours in RPMI containing 0.5% FCS before treatment with oxLDLs (100 µg/mL). Alternatively, cells were treated with an irrelevant siRNA (p120 catenin siRNA) as negative control.

LDL Preparation and Oxidation
Human LDLs (1.019<d<1.063) were isolated from pooled fresh sera by sequential ultracentrifugation, dialyzed, sterilized by filtration, and oxidized by UV-C irradiation.⁹

Evaluation of DNA Synthesis and Cell Proliferation
DNA synthesis was evaluated by [³H]thymidine incorporation under the previously used conditions.⁸ Alternatively, proliferating cells were detected by immunocytochemistry using anti–proliferating cell nuclear antigen (PCNA) antibody. Cells grown on uncoated glass coverslips, fixed in 3% paraformaldehyde for 15 minutes and permeabilized with 0.1% Triton X-100, were incubated with PCNA primary antibody for 30 minutes and with a secondary FITC-conjugated anti-mouse IgG for 30 minutes and finally were examined by fluorescence microscopy.

Sphingolipid Quantification and Neutral Sphingomyelinase Determination
SMCs were prelabeled with [methyl-³H]choline or [³H]palmitic acid (0.5 µCi/mL), then were incubated with oxLDLs (100 µg apolipoprotein B/mL) with or without MMP inhibitors, and radiolabeled sphingolipids were quantified as described previously.⁹

Neutral sphingomyelinase activity was determined in cell extracts (100 µg protein) in the presence of [choline-methyl-¹⁴C]sphingomyelin (120 000 dpm/assay) as reported.⁸

Zymography and Determination of MMP Activity
Gelatinase activity of SMC culture media was assayed by zymography analysis according to Xu et al.¹⁷ Briefly, culture media (concentrated 10 times on columns 10 to 300 kDa, Pall Gelman Laboratory) were run on 10% SDS-PAGE gel containing 1 mg/mL gelatin. The gel was washed twice in 2.5% Triton X-100 solution and 5 times with water and was incubated overnight at 37°C in developing buffer (Tris-HCl 50 mmol/L, pH 7.4, CaCl₂ 10 mmol/L, ZnCl₂ 5 mmol/L, and 0.05% Brij-35), then stained with 0.5% Coomassie blue and destained in a 5% methanol/7% acetic acid solution.

MMP activity was determined on concentrated SMC media by use of the DNP-pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH₂ fluorogenic substrate (Calbiochem-WWR) as described previously.²¹ After overnight incubation (37°C), 1 mL Tris-HCl buffer, pH 7, was added, and the fluorescence was read (excitation and emission wavelengths, 280 and 360 nm, respectively).

Western Blots
Western blots were performed as described previously.²²

Statistical Analysis
Data are presented as mean±SEM. Estimates of statistical significance were performed by ANOVA (Tukey test; SigmaStat software), values of P<0.05 being considered significant.

Chemicals
[³H]Thymidine was purchased from Amersham; [methyl-³H]choline chloride, [choline-methyl-¹⁴C]sphingomyelin, and [9,10-³H]palmitic acid from DuPont NEN; antibodies were from Santa Cruz, except anti-(activated) phosphoMAPK from Promega; blocking anti–MMP-2 monoclonal antibody (CA-4001) was from Chemicon; and monoclonal (directed to the catalytic domain of) anti–MT1-MMP (114-6G6) from Oncogene-WWR. Pure recombinant rMT1-MMP, rMMP-1, and rMMP-2 proenzymes were from Calbiochem and were activated for 2 hours of incubation with 10 mmol/L APMA (manufacturer’s instructions). Batimastat and Ro28-2653 were a generous gift from H.W. Krell (Roche Diagnostics, Penzberg, Germany).

	Results

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MMP Inhibitors Inhibit SMC Proliferation Induced by oxLDL
OxLDLs (100 µg/mL) are mitogenic and induce both ERK1/ERK2 activation and DNA synthesis (ie, [³H]thymidine incorporation) (Figure 1) in cultured rabbit and human SMCs. It may be noted that, under the conditions used (nontoxic concentrations of UV-oxLDLs), we observed a significant increase in SMC numbers after 7 days of cell culture.⁶ The MMP inhibitors, 10 nmol/L batimastat or Ro28-2653, strongly inhibited MAPK activation and SMC proliferation induced by oxLDL. The same results were obtained by silencing MMP-2 with siRNA and in MMP-2^–/– fibroblasts (optimal mitogenic effect at 10 µg/mL oxLDLs for fibroblasts) (Figure 1, D and E). In contrast, MMP inhibitors did not inhibit growth factor–induced DNA synthesis triggered by FCS (data not shown). OxLDLs induce MMP activation.

View larger version (50K): [in this window] [in a new window]   Figure 1. Effect of MMP inhibitors on oxLDL-induced proliferation and ERK1/ERK2 phosphorylation. A through C, RFA-SMCs (A and B) and human SMCs (C) were starved for 48 hours in RPMI containing 0.5% FCS, previous incubation with MMP inhibitors (A, Ro28-2653; B, batimastat), and oxLDLs (100 µg/mL). After 48 hours, cell proliferation was evaluated by [3H]thymidine uptake. Values are mean±SEM of 5 separate experiments. D and E, Silencing MMP-2 blocks oxLDL proliferation. RFA-SMCs transiently transfected with siRNA (D) and MMP-2–/– or MMP-2+/+ fibroblasts (E) were incubated with oxLDL (100 µg/mL) as described in A. Top, MMP-2 expression visualized by Western blots of extracts from siRNA-treated SMCs and from MMP-2–/– and MMP-2+/+ fibroblasts (revealed with anti–MMP-2 antibody). F, Western blots of extracts from SMCs incubated for 3 hours with oxLDL in presence of 10 nmol/L batimastat (Bati), Ro28-2653 (Ro), or PD98059 (PD) (revealed with anti–phospho-ERK and anti–total ERK1/2 antibodies). Values are representative of 4 separate experiments. *P<0.01 (comparison between oxLDL treated without and with inhibitor).

OxLDLs triggered an increase of MMP activity released in the SMC culture medium that peaked after 90 minutes of pulse (Figure 2A). No significant MMP activity was detected in cell- and serum-free medium, suggesting that oxLDL-activated MMPs originated from SMCs. SDS-PAGE zymography showed a rise of the gelatinolytic activity of 65-kDa after 1 hour of pulse with oxLDLs (Figure 2B), suggesting that oxLDLs trigger an early activation of MMP-2 in the culture medium. Both batimastat and Ro28-2653 inhibited this oxLDL-induced MMP activation. Note that the (1 hour) oxLDL-induced MMP activation in the culture medium did not require de novo protein synthesis, because cycloheximide was ineffective to block this activation (Table) and because the level of MT1-MMP and MMP-2 protein expression remained unchanged in SMCs treated for 1 hour with oxLDL (Figure 2C). This suggests that increased MMP-2 visualized by zymography in the culture medium probably results from the processing of pro-MMP-2 (see below).

View larger version (29K): [in this window] [in a new window]   Figure 2. OxLDLs induce MMP-2 activation. A, SMCs starved for 48 hours were incubated with oxLDL (100 µg/mL). Culture media were concentrated 10 times, and MMP activity was determined by use of fluorogenic substrate DNP-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2. Data are expressed as % of untreated control. Values are mean±SEM of 5 separate experiments. *P<0.01. B, Zymography of culture media from SMCs incubated with or without oxLDLs (100 µg/mL for 1 hour), with or without 10 nmol/L batimastat (Bati) or Ro28-2653 (Ro), and with activated MMP-2 (1 nmol/L for 1 hour) as described in Methods. C, Western blots of MT1-MMP and MMP-2 in extracts from SMCs incubated (or not) for 1 hour with oxLDL (using EGFR as control). Values are representative of 4 separate experiments.

View this table: [in this window] [in a new window]   Activity of MMPs in SMC Culture Medium and Sphingomyelinase in SMC Homogenates and Effect of Diverse Agonists

Inhibition of MMP-2 Blocks the oxLDL-Induced Activation of the Sphingomyelin/Ceramide Pathway
As we previously described, oxLDL-induced SMC proliferation is mediated by the sphingomyelin/ceramide pathway.^8,9 We investigated whether a link exists between MMP-2 activation and these signaling pathways activated by oxLDLs. The oxLDL-induced activation of the sphingomyelin/ceramide pathway (ie, neutral sphingomyelinase activation, sphingomyelin hydrolysis, and ceramide generation) was inhibited by 10 nmol/L batimastat and was abolished in MMP-2–silenced cells (SMCs treated with siRNA) and in MMP-2^–/– fibroblasts (in contrast to MMP-2^+/+ wild-type cells, which exhibited sphingomyelinase activation) (Figure 3). These data suggest that MMP-2 is implicated in the oxLDL-induced activation of the sphingomyelin/ceramide pathway.

View larger version (38K): [in this window] [in a new window]   Figure 3. MMP-2 inhibition blocks oxLDL-induced activation of sphingomyelin/ceramide pathway. Cells were incubated with oxLDLs (100 µg/mL) with or without batimastat (10 nmol/L). A, Hydrolysis of cellular sphingomyelin (prelabeled with [methyl-3H]choline). B, Ceramide generation determined after labeling SMCs with [3H]palmitic acid. C, Sphingomyelinase activity. *P<0.01. D and E, Effect of oxLDLs (100 µg/mL for 1 hour) on sphingomyelinase activity in SMCs treated or not with anti–MMP-2 siRNA (D) and in MMP-2–/– and MMP-2+/+ fibroblasts (E). Results are expressed as % of unstimulated control. Values are mean±SEM of 4 separate experiments. *P<0.05.

Interestingly, the mitogenic effect of oxLDL was inhibited by anti–MMP-2 and anti–MT1-MMP blocking antibodies, as assessed by the inhibition of ERK1/2 activation, DNA synthesis, and nuclear translocation of PCNA (Figure 4, A through C). This suggests that MMP-2 activation is mediated by MT1-MMP.

View larger version (52K): [in this window] [in a new window]   Figure 4. Involvement of MT1-MMP and MMP-2 in SMC proliferation via sphingomyelin/ceramide pathway. A through C, Effect of blocking anti–MMP-2 and anti–MT1-MMP antibodies on ERK1/2 phosphorylation (A), PCNA (visualized by immunocytochemistry) (B), and DNA synthesis (C) in SMCs treated with oxLDLs (100 µg/mL for 1 hour). D through G, Effect of 1 nmol/L of exogenous APMA-activated rMT1-MMP, rMMP-2, and rMMP-1 on sphingomyelinase activity (D), sphingomyelin hydrolysis (E), ERK1/2 phosphorylation (F), and DNA synthesis (G). Values are mean±SEM of 3 separate experiments. *P<0.01.

To confirm the role of MMP-2 and MT1-MMP, SMCs were treated by exogenous recombinant rMMP-2 and rMT1-MMP. As shown in Figure 4, D through G, rMT1-MMP and rMMP-2 triggered the activation of the neutral sphingomyelinase, leading to sphingomyelin hydrolysis, ERK1/2 phosphorylation, and DNA synthesis. These data strongly support the hypothesis that MT1-MMP and, subsequently, MMP-2 are able to activate the sphingomyelin/ceramide pathway. It may be noted that MMP-1 was unable to activate sphingomyelinase and DNA synthesis, thus suggesting that the effect of MMP-2 in cell signaling is not a trivial property of all the MMPs.

Together, these data suggest that the oxLDL-induced activation of the sphingomyelin/ceramide pathway involved in SMC proliferation is mediated by an MT1-MMP/MMP-2–dependent mechanism.

MMPs Are Not Involved in the oxLDL-Induced Receptor Tyrosine Kinase Activation
Tyrosine kinase receptors (TKRs), such as EGFR and platelet-derived growth factor receptor (PDGFR), are activated by oxLDLs through adduct formation by lipid oxidation products and generation of reactive oxygen species.²² For instance, PDGF stimulates SMC migration through mechanisms involving MMP-2 and MMP-9,²³ and a decreased MMP-2 expression is associated with PDGFR downregulation.²⁴ PDGFR and reactive oxygen species trigger MMP activation in various cellular systems and in animal models of atherosclerosis through mechanisms involving gene induction, mRNA stabilization, and pro-MMP activation.^11,12 This led us to investigate whether a relationship exists between oxLDL-induced TKR signaling and MMPs.

Because batimastat did not inhibit PDGFR and EGFR activation induced by mitogenic concentrations of oxLDL (data not shown), it is suggested that the oxLDL-induced activation of the 2 TKRs did not require MMPs. Conversely, the EGFR and PDGFR inhibitors AG1478 and AG1295, which effectively blocked the oxLDL-dependent autophosphorylation of these TKRs, did not inhibit sphingomyelin hydrolysis or sphingomyelinase activation triggered by oxLDLs (Table). Conversely, EGF and PDGF did not trigger any sphingomyelinase activation under the experimental conditions used here (Table).

Together, MMPs are required for the oxLDL-induced activation of the sphingomyelin/ceramide pathway but not for the oxLDL-induced TKR (PDGFR and EGFR) activation.

	Discussion

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MMPs play a complex role in vascular remodeling, because they are able to degrade the extracellular matrix and to promote SMC proliferation^11,12 and may thereby participate in generating unstable plaques (in case of prominent proteolysis) or stenosis (in case of prominent SMC proliferation).^11,12 Focal activation of MMP-2 may be elicited by partial proteolysis by thrombin or MT1-MMP in response to cytokines and oxLDLs.²⁵ MMP-2 may also induce cell proliferation of rat mesangial cells²⁵ and SMCs,^26,27 but the mechanism of the mitogenic effect of MMPs is largely unknown.

In preliminary experiments, we observed that batimastat, a broad-specificity MMP inhibitor, inhibited the oxLDL-induced proliferation of SMCs. Because oxLDL-induced SMC proliferation involves 2 major mitogenic signaling pathways, the sphingomyelin/ceramide and the TKR/PI3K/Akt pathways,^8,9 we investigated whether MMPs were implicated in the activation of these pathways and subsequent SMC proliferation.

The first major finding is the crucial role that MMP-2 plays in the oxLDL-induced proliferation of rabbit and human SMCs, as assessed by the use MMP inhibitors (batimastat and Ro28-2653) and MMP-2–deficient cells.²⁸ In our experimental system, the mitogenic MMP was clearly identified as MMP-2 on the basis of zymography (65-kDa activated form), of the inhibitory effect of anti–MMP-2 antibodies, of anti–MMP-2 siRNA, and by the lack of mitogenic response in MMP-2^–/– fibroblasts. Interestingly, in our model system, in agreement with Kuzuya et al,²⁹ MMP-2 inhibitors did not reduce cell proliferation induced by PDGF and EGFR, thus suggesting that MMP-2 is not required for growth factor–induced growth factors.

This oxLDL-induced MMP-2 activation is mediated by MT1-MMP, as assessed by the effects of anti–MT1-MMP antibody and of exogenous rMT1-MMP. This is consistent with the general role of MT1-MMP in the proteolytic activation of pro-MMP-2.³⁰ The mechanism of MT1-MMP activation by oxLDLs is independent of any MT1-MMP overexpression and may result from a regulation of MT1-MMP activity by (1) clathrin-dependent endocytosis,³¹ (2) activation of integrins,³² and (3) serine proteases.^11,13 This latter hypothesis is consistent with our previous results that show that TPCK, a synthetic serine protease inhibitor, efficiently inhibited oxLDL-induced MMP activation (data not shown), sphingomyelin/ceramide pathway activation, and SMC proliferation.⁸

The second major finding is the role of MMP-2 in sphingomyelin/ceramide pathway activation. The precise molecular mechanism of regulation of the sphingomyelin/ceramide pathway remains poorly understood.³³ Our data strongly support a crucial role of MMP-2 in triggering sphingomyelinase activation, because Ro28-2653 (specific to MMP-2 and MMP-9), blocking anti–MMP-2 antibodies, and MMP-2 defect (MMP-2^–/– cells or cells depleted by anti–MMP-2 siRNA) inhibit sphingomyelinase activation, sphingomyelin hydrolysis, and ceramide production. This is also supported by the effect of exogenous MMP-2 (but not MMP-1), which triggered both the activation of the sphingomyelin/ceramide pathway and DNA synthesis. This suggests that the ability of MMP-2 to activate sphingomyelinase is not a trivial property of all MMPs. Our data suggest that MMP-2 may be a missing link between lipoproteins and the cell signaling machinery involved in SMC proliferation.³⁵

In conclusion, our data shed light on a novel cell signaling mechanism involved in oxLDL-induced SMC proliferation, in which MMP-2 plays a central role in the activation of the sphingomyelin/ceramide pathway and subsequent DNA synthesis (Figure 5). Interestingly, MMP-2 is not implicated in cell proliferation induced by PDGF, in agreement with the observations of Kuzuya et al.²⁹ This suggests that the mechanism reported here might be of pathophysiological importance in atherosclerotic lesions in which oxLDLs are present^2,5,7 and in which MMP-2 is overexpressed.^11,12 Finally, because the mechanism described here is triggered by exogenously added MMP-2, it might be of importance under conditions leading to high levels of activated MMP-2 in the vascular wall.^25–27

View larger version (68K): [in this window] [in a new window]   Figure 5. Schema of signaling pathways activated by mitogenic concentrations of oxLDL and role of MMP-2 in activation of sphingomyelin/ceramide pathway.

In addition to the well-known role of MMPs in extracellular matrix turnover and vascular remodeling, this novel role of MMP-2 in the regulation of the sphingomyelin/ceramide pathway may be of importance in various cellular responses, including proliferation, migration, and apoptosis.^34–36 These cellular events may participate not only in intimal hyperplasia²⁹ but also in the formation of the atherosclerotic plaques.

From a pathophysiological point of view, our data provide a new mechanistic explanation and a molecular basis to the potential clinical use of MMP inhibitors to prevent excessive SMC proliferation such as observed in coronary artery disease, carotid stenosis, and peripheral artery disease. However, drugs that reduce SMC proliferation must be used cautiously, because a strong fibroproliferative cap may prevent the plaque instability and reduce the risk of atherothrombotic events.

	Acknowledgments

 
The authors acknowledge INSERM and Université Paul Sabatier for financial support; J. Dumoulin and C. Mora for the technical assistance; and Dr V. Gallet (SNCF Laboratory, Toulouse) for providing human serum.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Stary HC. The sequence of cell and matrix changes in atherosclerotic lesions of coronary arteries in the forty years of life. Eur Heart J. 1990; 11: 3–19.[Free Full Text]
   
2. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362: 801–809.[CrossRef][Medline] [Order article via Infotrieve]
   
3. Lusis AJ. Atherosclerosis. Nature. 2000; 407: 233–241.[CrossRef][Medline] [Order article via Infotrieve]
   
4. Libby P. Changing concepts of atherogenesis. J Intern Med. 2000; 247: 349–358.[CrossRef][Medline] [Order article via Infotrieve]
   
5. Witztum JL, Steinberg D. The oxidative modification hypothesis of atherosclerosis: does it hold for humans? Trends Cardiovasc Med. 2001; 11: 93–103.[CrossRef][Medline] [Order article via Infotrieve]
   
6. Auge N, Pieraggi MT, Thiers JC, et al. Proliferative and cytotoxic effects of mildly oxidized LDL on vascular smooth-muscle cells. Biochem J. 1995; 309: 1015–1020.[Medline] [Order article via Infotrieve]
   
7. Chisolm GM III, Chai Y. Regulation of cell growth by oxidized LDL. Free Radic Biol Med. 2000; 28: 1697–1707.[CrossRef][Medline] [Order article via Infotrieve]
   
8. Auge N, Escargueil-Blanc I, Lajoie-Mazenc I, et al. Potential role for ceramide in MAPK activation and proliferation of vascular smooth muscle cells induced by oxidized LDL. J Biol Chem. 1998; 273: 12893–12900.[Abstract/Free Full Text]
   
9. Auge N, Garcia V, Maupas-Schwalm F, et al. Oxidized LDL-induced smooth muscle cell proliferation involves the EGF receptor/PI-3 kinase/Akt and the sphingolipid signaling pathways. Arterioscler Thromb Vasc Biol. 2002; 22: 1990–1995.[Abstract/Free Full Text]
   
10. Chang C, Werb Z. The many faces of metalloproteases: cell growth, invasion, angiogenesis and metastasis. Trends Cell Biol. 2001; 11: S37–S43.[Medline] [Order article via Infotrieve]
    
11. Galis ZS, Khatri JJ. Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ Res. 2002; 90: 251–262.[Abstract/Free Full Text]
    
12. Ikeda U, Shimada K. Matrix metalloproteinases and coronary artery diseases. Clin Cardiol. 2003; 26: 55–59.[Medline] [Order article via Infotrieve]
    
13. Lijnen HR. Extracellular proteolysis in the development and progression of atherosclerosis. Biochem Soc Trans. 2002; 30: 163–167.[CrossRef][Medline] [Order article via Infotrieve]
    
14. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res. 2003; 92: 827–839.[Abstract/Free Full Text]
    
15. Skiles JW, Gonnella NC, Jeng AY. The design, structure, and therapeutic application of matrix metalloproteinase inhibitors. Curr Med Chem. 2001; 8: 425–474.[Medline] [Order article via Infotrieve]
    
16. Huang Y, Song L, Wu S, et al. Oxidized LDL differentially regulates MMP-1 and TIMP-1 expression in vascular endothelial cells. Atherosclerosis. 2001; 156: 119–125.[CrossRef][Medline] [Order article via Infotrieve]
    
17. Xu XP, Meisel SR, Ong JM, et al. Oxidized LDL regulates matrix metalloproteinase-9 and its tissue inhibitor in human monocyte-derived macrophages. Circulation. 1999; 99: 993–998.[Abstract/Free Full Text]
    
18. Rajavashisth TB, Liao JK, Galis ZS, et al. Inflammatory cytokines and oxidized LDL increase endothelial cell expression of membrane type 1-matrix metalloproteinase. J Biol Chem. 1999; 274: 11924–11929.[Abstract/Free Full Text]
    
19. Itoh T, Ikeda T, Gomi H, et al. Unaltered secretion of beta-amyloid precursor protein in gelatinase A (matrix metalloproteinase 2)-deficient mice. J Biol Chem. 1997; 272: 22389–22392.[Abstract/Free Full Text]
    
20. Caplen NJ, Parrish S, Imani F, et al. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci U S A. 2001; 98: 9742–9747.[Abstract/Free Full Text]
    
21. Netzel-Arnett S, Mallya SK, Nagase H, et al. Continuously recording fluorescent assays optimized for five human matrix metalloproteinases. Anal Biochem. 1991; 195: 86–92.[CrossRef][Medline] [Order article via Infotrieve]
    
22. Escargueil-Blanc I, Salvayre R, Vacaresse N, et al. Mildly oxidized LDLs induce activation of PDGF ß-receptor pathway. Circulation. 2001; 104: 1814–1821.[Abstract/Free Full Text]
    
23. Kenagy RD, Clowes AW. A possible role for MMP-2 and MMP-9 in the migration of primate arterial smooth muscle cells through native matrix. Ann N Y Acad Sci. 1994; 732: 462–465.[Medline] [Order article via Infotrieve]
    
24. Palumbo R, Gaetano C, Melillo G, et al. Shear stress downregulation of PDGF-ß and matrix metalloprotease-2 is associated with inhibition of smooth muscle cell invasion and migration. Circulation. 2000; 102: 225–230.[Abstract/Free Full Text]
    
25. Rajavashisth TB, Xu XP, Jovinge S, et al. Membrane type 1 matrix metalloproteinase expression in human atherosclerotic plaques: evidence for activation by proinflammatory mediators. Circulation. 1999; 99: 3103–3109.[Abstract/Free Full Text]
    
26. Johnson S, Knox A. Autocrine production of matrix metalloproteinase-2 is required for human airway smooth muscle proliferation. Am J Physiol. 1999; 277: L1109–L1117.[Medline] [Order article via Infotrieve]
    
27. Wang H, Keiser JA. Expression of membrane-type matrix metalloproteinase in rabbit neointimal tissue and its correlation with matrix-metalloproteinase-2 activation. J Vasc Res. 1998; 35: 45–54.[CrossRef][Medline] [Order article via Infotrieve]
    
28. Lein M, Jung K, Ortel B, et al. The new synthetic matrix metalloproteinase inhibitor (Roche 28-2653) reduces tumor growth and prolongs survival in a prostate cancer standard rat model. Oncogene. 2002; 21: 2089–2096.[CrossRef][Medline] [Order article via Infotrieve]
    
29. Kuzuya M, Kanda S, Sasaki T, et al. Deficiency of gelatinase a suppresses smooth muscle cell invasion and development of experimental intimal hyperplasia. Circulation. 2003; 108: 1375–1381.[Abstract/Free Full Text]
    
30. Seiki M. The cell surface: the stage for matrix metalloproteinase regulation of migration. Curr Opin Cell Biol. 2002; 14: 624–632.[CrossRef][Medline] [Order article via Infotrieve]
    
31. Jiang A, Lehti K, Wang X, et al. Regulation of membrane-type matrix metalloproteinase 1 activity by dynamin-mediated endocytosis. Proc Natl Acad Sci U S A. 2001; 98: 13693–13698.[Abstract/Free Full Text]
    
32. Bendeck MP, Irvin C, Reidy M, et al. Smooth muscle cell matrix metalloproteinase production is stimulated via _vß₃ integrin. Arterioscler Thromb Vasc Biol. 2000; 20: 1467–1472.[Abstract/Free Full Text]
    
33. Auge N, Negre-Salvayre A, Salvayre R, et al. Sphingomyelin metabolites in vascular cell signaling and atherogenesis. Prog Lipid Res. 2000; 39: 207–229.[CrossRef][Medline] [Order article via Infotrieve]
    
34. Gouni-Berthold I, Sachinidis A. Does the coronary risk factor low density lipoprotein alter growth and signaling in vascular smooth muscle cells? FASEB J. 2002; 16: 1477–1487.[Abstract/Free Full Text]
    
35. Argraves KM, Obeid LM, Hannun YA. Sphingolipids in vascular biology. Adv Exp Med Biol. 2002; 507: 439–444.[Medline] [Order article via Infotrieve]
    
36. Payne SG, Milstien S, Spiegel S. Sphingosine-1-phosphate: dual messenger functions. FEBS Lett. 2002; 531: 54–57.[CrossRef][Medline] [Order article via Infotrieve]
    

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