Signaling Pathways Involved in Human Vascular Smooth Muscle Cell Proliferation and Matrix Metalloproteinase-2 Expression Induced by Leptin
Inhibitory Effect of Metformin
Ling Li1,
Jean-Claude Mamputu1,
Nicolas Wiernsperger2, and
Geneviève Renier1
1 CHUM Research Centre, Vascular Immunology Laboratory, Department of Medicine, Notre-Dame Hospital, University of Montreal, Quebec, Canada
2 INSERM U585, INSA Lyon, Batiment L. Pasteur, Villeurbanne, France
 |
ABSTRACT
|
---|
Accumulating evidence suggests that high concentrations of leptin observed in obesity and diabetes may contribute to their adverse effects on cardiovascular health. Metformin monotherapy is associated with reduced macrovascular complications in overweight patients with type 2 diabetes. It is uncertain whether such improvement in the cardiovascular outcome is related to specific vasculoprotective effects of this drug. In the present study, we determined the effect of leptin on human aortic smooth muscle cell (HASMC) proliferation and matrix metalloproteinase (MMP)-2 expression, the signaling pathways mediating these effects, and the modulatory effect of metformin on these parameters. Incubation of HASMCs with leptin enhanced the proliferation and MMP-2 expression in these cells and increased the generation of intracellular reactive oxygen species (ROS). These effects were abolished by vitamin E. Inhibition of NAD(P)H oxidase and protein kinase C (PKC) suppressed the effect of leptin on ROS production. In HASMCs, leptin induced PKC, extracellular signalregulated kinase (ERK)1/2, and nuclear factor-
B (NF-
B) activation and inhibition of these signaling pathways abrogated HASMC proliferation and MMP-2 expression induced by this hormone. Treatment of HASMCs with metformin decreased leptin-induced ROS production and activation of PKC, ERK1/2, and NF-
B. Metformin also inhibited the effect of leptin on HASMC proliferation and MMP-2 expression. Overall, these results demonstrate that leptin induced HASMC proliferation and MMP-2 expression through a PKC-dependent activation of NAD(P)H oxidase with subsequent activation of the ERK1/2/NF-
B pathways and that therapeutic metformin concentrations effectively inhibit these biological effects. These results suggest a new mechanism by which metformin may improve cardiovascular outcome in patients with diabetes.
Obesity is a strong risk factor for the development of type 2 diabetes and cardiovascular disease (1) and is associated with a marked increase in circulating leptin concentrations (2). In recent years, many but not all (3,4) studies have demonstrated positive associations between plasma leptin and clinical cardiovascular disease and leptin signaling has been implicated in the promotion of atherosclerosis. In vitro proatherogenic effects of leptin include endothelial cell activation, migration, and proliferation (5,6); smooth muscle cell proliferation, migration, and calcification (7,8); platelet agregation (9); activation of monocytes (10); and modulation of the immune response (11,12). In vivo, leptin receptors are expressed in vascular cells and atherosclerotic lesions (13), and leptin signaling promotes atherosclerosis in mice models (14,15).
The UKPDS (U.K. Prospective Diabetes Study) has demonstrated that metformin, a biguanide antidiabetic agent, reduces macrovascular events in overweight patients with type 2 diabetes (16). Its protective effects have been attributed in part to an improved lipid profile, a lack of weight gain, an antihyperinsulinemic effect, and an improved thrombolysis (17,18). Metformin exerts several intrinsic vasculoprotective effects that may account for its ability to reduce cardiovascular events (18). We have previously shown that in vitro, metformin inhibits early and critical key processes involved in the pathophysiology of atherogenesis, such as monocyte adhesion to endothelium and macrophage-derived foam cell formation (19). These effects may account for the antiatherosclerotic properties of this drug. The present study sought to determine whether this drug inhibits human aortic smooth muscle cell (HASMC) proliferation and matrix metalloproteinase (MMP)-2 expression in response to leptin and the molecular mechanisms involved in this effect.
 |
RESEARCH DESIGN AND METHODS
|
---|
Reagents.
Recombinant human leptin was purchased from R&D Systems (Minneapolis, MN). Vitamin E and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) were obtained from Sigma (St Louis, MO). PBS was obtained from Invitrogen (Burlington, ON, Canada). Smooth muscle cell basal medium (SmBM) and smooth muscle cell growth medium (SmGM-2) were purchased from BioWhittaker (Walkersville, MD). Fetal bovine serum (FBS) and trysin/EDTA were obtained from Wisent (St. Bruno, PQ, Canada). Apocynin, a selective NAD(P)H oxidase inhibitor (20); diphenyleneiodonium (DPI) chloride, an inhibitor of flavoprotein-containing enzymes, including NAD(P)H oxidase (21); thenoyltrifluoroacetone (TTFA), a mitochondrial complex II inhibitor (22); GF10923X, a selective protein kinase C (PKC) inhibitor (23); PD98059, a selective mitogen-activated protein kinase (MAPK) kinase inhibitor (24); and BAY 11-7085, a selective nuclear factor-
B (NF-
B) inhibitor (25) were obtained from Calbiochem (La Jolla, CA). Metformin was supplied by Merck-Santé (Lyon, France).
Cells.
HASMCs were obtained from BioWhittaker (Walkersville, MD). The cells were grown in SmGM-2 at 37°C in a 5% CO2/95% air atmosphere. At confluence, cells were trypsinized and subcultured in 24- or 96-well culture plates or 100-mm tissue culture dishes depending on assay conditions. Cells were used in all experiments between passages 3 and 6. Leptin treatment was carried out in cells cultured in SmBM supplemented with 2% FBS and 1% (vol/vol) penicillin-streptomycin.
HASMC proliferation assay.
Subconfluent HASMCs were serum starved in SmBM supplemented with 2% FBS for 48 h, preincubated for 1 h with metformin or other appropriate agents, and then treated with leptin for an additional 72 h at 37°C. HASMC proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-diphenyltetrazolium bromide (MTT) assay (Promega, Madison, WI). This assay is a colorimetric method based on reduction of the tetrazolium salt, MTT, by actively growing cells to produce a blue formazan product (26).
Determination of reactive oxygen species production.
Subconfluent HASMCs were pretreated or not pretreated for 1 h with metformin or other appropriate agents prior to exposure to leptin for a further 24-h period at 37°C, with the cell-permeable fluorogenic probe DCF-DA (20 µg/ml) added during the final 20-min incubation period. At the end of this incubation period, cells were washed and trypsinized. Intracellular reactive oxygen species (ROS) production was monitored by measuring fluorescence in an LS50B luminescence spectrophotometer (Perkin Elmer) using excitation and emission wavelengths of 498 and 522 nm, respectively.
Measurement of MMP-2 protein expression.
Subconfluent HASMCs were pretreated or not pretreated for 1 h with metformin or other appropriate agents and then incubation was pursued in the presence of leptin for an additional 72 h at 37°C. The amount of MMP-2 secreted in the culture medium was measured using a double-sandwich enzyme-linked immunosorbent assay (ELISA) (Amersham Biosciences, Baie dUrfé, QC, Canada) (27). The minimum detectable concentration of MMP-2 with this assay is 1.5 ng/ml. The intra- and interassay coefficients of variation of this assay were <5.3 and 8.3, respectively. Levels of MMP-2 in the supernatants were normalized to the levels of total cell proteins.
Determination of PKC activation.
Confluent HASMCs were treated or not treated with leptin in 100-mm tissue culture dishes in the presence or absence of metformin for 30 min at 37°C. At the end of this incubation period, cells were washed three times with cold PBS and harvested. Cell pellets were suspended in 1 ml cold sample preparation buffer (50 mmol/l Tris-HCl, pH 7.5, 5 mmol/l EDTA, 10 mmol/l ethylene glycol-bis tetraacetic acid, 50 mmol/l 2-mercaptoethanol, 1 mmol/l phenylmethylsulfonyl fluoride, and 10 mmol/l benzamidine) and sonicated. Membrane and cytosol fractions were separated by centrifugation at 100,000g for 1 h at 4°C. Cytosolic and membrane PKC activities were measured using the MESACUP protein kinase assay kit (MBL, Naka-ku Nagoya, Japan) (28).
Determination of extracellular signalregulated kinase 1/2 activation.
Confluent HASMCs were treated or not treated with leptin in 96-well plates in the presence or absence of metformin or other appropriate agents for 30 min at 37°C. At the end of this incubation period, cells were rapidly fixed with 4% formaldehyde. Total and phosphorylated extracellular signalregulated kinase (ERK)1/2 activities were measured using a fast-activated cell-based ELISA (FACE) kit (MJS Biolynx, Brockville, ON, Canada) (29). Levels of total and phosphorylated ERK1/2 were normalized to cell number as determined by crystal violet cell staining.
Measurement of NF-
B activation.
Confluent HASMCs were treated or not treated with leptin in 100-mm tissue culture dishes in the presence or absence of metformin or other appropriate agents for 30 min at 37°C. At the end of this incubation period, cells were washed twice with cold PBS/phosphate inhibitor buffer (125 mmol/l sodium fluoride, 250 mmol/l ß-glycerophosphate, 250 mmol/l paranitrophenyl phosphate, and 25 mmol/l sodium orthovanadate), scraped, and centrifuged. Cell pellets were resuspended in 1 ml ice-cold hypotonic buffer (20 mmol/l HEPES, pH 7.5, 5 mmol/l sodium fluoride, 10 µmol/l sodium molybdate, and 0.1 mmol/l EDTA), and incubation was pursued on ice for 15 min. At the end of this incubation period, 10% nonidet P-40 was added to the lysed cells. After centrifugation, nuclear pellets were resuspended in Complete Lysis Buffer (MJS Biolynx) and nuclear extracts were collected by centrifugation at 9,000g for 10 min. Aliquots of the supernatants were kept at 70°C, and protein concentration was determined. Levels of NF-
B activation in 3-µg nuclear extracts were determined using the TransAM NF-
B family transcription factor assay kit (MJS Biolynx) (30).
Determination of total protein concentrations.
Total protein content was measured according to the Bradford method using a colorimetric assay (Bio-Rad, Mississauga, ON, Canada).
Determination of cell viability.
To exclude the possibility that experimental agents at maximal concentration used in the study may exert cytotoxic effects, cell viability was determined by trypan blue exclusion. It was consistently found to be
90% (data not shown).
Statistical analysis.
Statistical analysis of the results was performed by one-way ANOVA followed by the Student-Newman-Keuls test. Differences were considered statistically significant at P < 0.05. Results are expressed as means ± SE.
 |
RESULTS
|
---|
Stimulatory effect of leptin on HASMC proliferation, MMP-2 expression, and ROS production.
Incubation of HASMCs with leptin (0200 ng/ml) increased the proliferation of these cells in a concentration-dependent manner. Maximal effect was observed after 72 h at a concentration of 100 ng/ml leptin (Fig. 1A). Leptin also enhanced, in a dose-dependent manner, MMP-2 production (Fig. 1B) and intracellular accumulation of ROS (Fig. 1C) in HASMCs, with a maximal effect at 100 ng/ml. Pretreatment of HASMCs with vitamin E abolished the stimulatory effects of leptin on HASMC proliferation, MMP-2 expression, and ROS production (Fig. 1AC).

View larger version (25K):
[in this window]
[in a new window]
|
FIG. 1. Stimulatory effect of leptin on HASMC proliferation and MMP-2 expression. Role of oxidative stress. A: Serum-starved HASMCs were incubated for 72 h with increasing concentrations of leptin in the presence or absence of vitamin E (50 µmol/l). Cell proliferation was assessed by the MTT assay. B: Subconfluent HASMCs were incubated for 72 h with increasing concentrations of leptin in the presence or absence of vitamin E (50 µmol/l). The amounts of MMP-2 secreted in the culture medium were determined by ELISA. C: Subconfluent HASMCs were incubated for 24 h with increasing concentrations of leptin in the presence or absence of vitamin E (50 µmol/l), with 20 µg/ml DCF-DA added during the final 20-min incubation period. Intracellular ROS generation was quantified by measuring fluorescence. Data represent the means ± SE of four independent experiments. *P < 0.05, **P < 0.01 vs. medium.
|
|
Generation of ROS in leptin-treated HASMCs arises from the NAD(P)H oxidase and is PKC dependent.
To evaluate the role of NAD(P)H oxidase and mitochondria in leptin-induced intracellular ROS production, HASMCs were preincubated with the NAD(P)H oxidase inhibitors apocynin and DPI chloride or with the inhibitor of the mitochondrial electron transport, TTFA, prior exposure to leptin. As shown in Fig. 2, apocynin and DPI chloride prevented the effect of leptin on intracellular ROS production, whereas TTFA was ineffective, thus identifying NAD(P)H oxidase as the source of ROS in leptin-treated HASMC. Like TTFA, the mitochondrial superoxide dismutase mimetic, MnTBAP [Mn(III)tetrakis (4-benzoic acid) pophyrin chloride] failed to inhibit leptin-induced ROS production in HASMCs (ROS production [percent of control values]: control 100 ± 2, leptin 156 ± 11, P < 0.05 vs. control; MnTBAP + leptin 135 ± 6). To determine whether PKC is involved in the stimulation of ROS by leptin, PKC activation in leptin-treated HASMCs was assessed and the effect of PKC inhibition on leptin-induced ROS generation determined. As shown in Fig. 3, treatment of HASMCs with 100 ng/ml leptin for 30 min induced PKC activation, as assessed by the translocation of this kinase to the membrane fraction (Fig. 3A). Pretreatment of HASMCs with the specific classic PKC inhibitor GF10923X totally prevented leptin-induced ROS generation (Fig. 3B). Apocynin and GF10923X also inhibited the effect of leptin on HASMC proliferation (Fig. 4A) and MMP-2 expression (Fig. 4B).

View larger version (16K):
[in this window]
[in a new window]
|
FIG. 2. Effect of NAD(P)H oxidase and mitochondrial electron transport inhibitors on leptin-induced ROS generation in HASMCs. HASMCs were pretreated for 1 h with the NAD(P)H oxidase inhibitors apocynin (Apo; 10 µmol/l) and DPI (10 µmol/l) or with the mitochondrial electron transport inhibitor TTFA (10 µmol/l) and then exposed for 24 h to 100 ng/ml leptin (Lep), with 20 µg/ml DCF-DA added during the final 20-min incubation period. Intracellular ROS generation was quantified by measuring fluorescence. Data represent the means ± SE of four independent experiments. *P < 0.05, ***P < 0.001 vs. medium (Med).
|
|

View larger version (17K):
[in this window]
[in a new window]
|
FIG. 3. Role of PKC in leptin-induced ROS generation. A: HASMCs were incubated with leptin (Lep) (100 ng/ml) for 30 min. Cytosolic and membrane PKC activities were measured using the MESACUP protein kinase assay kit. B: HASMCs were pretreated for 1 h with the specific PKC inhibitor GF10923X (GF; 20 nmol/l) and then exposed to leptin (Lep) for 24 h, with 20 µg/ml DCF-DA added during the final 20-min incubation period. Intracellular ROS generation was quantified by measuring fluorescence. Data represent the means ± SE of four independent experiments. *P < 0.05 vs. medium (Med).
|
|

View larger version (19K):
[in this window]
[in a new window]
|
FIG. 4. Effect of NAD(P)H oxidase and PKC inhibitors on leptin-induced HASMC proliferation and MMP-2 expression. HASMCs were pretreated for 1 h with the NAD(P)H oxidase inhibitor apocynin (Apo; 10 µmol/l) or the specific PKC inhibitor GF10923X (GF; 20 nmol/l) and then exposed for 72 h to 100 ng/ml leptin (Lep). At the end of this incubation period, HASMC proliferation (A) and levels of MMP-2 in the supernatants (B) were determined. Data represent the means ± SE of four independent experiments. ***P < 0.001 vs. medium (Med).
|
|
Role of ERK1/2 and NF-
B in leptin-induced HASMC proliferation and MMP-2 production.
Incubation of HASMCs for 30 min with 100 ng/ml leptin resulted in a marked increase in phophospecific ERK1/2 expression (Fig. 5A) and nuclear levels of NF-
B activation (Fig. 5B). Leptin-induced ERK1/2 activation was abolished by GF10923X, apocynin, and PD98059 (Fig. 5A). These compounds, as well as BAY 11-7085, also prevented the induction of NF-
B in response to leptin (Fig. 5B). Inhibition of the ERK1/2 and NF-
B signaling pathways totally abrogated the stimulatory effect of leptin on HASMC proliferation (Fig. 5C) and MMP-2 production (Fig. 5D).
Inhibitory effect of metformin on leptin-induced HASMC proliferation and MMP-2 production.
Preincubation of HASMCs with metformin (15 µg/ml) inhibited the stimulatory effect of leptin (100 ng/ml) on HASMC proliferation (Fig. 6A) and MMP-2 expression (Fig. 6B) with maximal effect being observed at concentrations ranging from 2.5 to 5 µg/ml metformin (Fig. 6B). Metformin (5 µg/ml) also abolished leptin-induced intracellular accumulation of ROS in HASMCs (Fig. 7A) and inhibited PKC (Fig. 7B), ERK1/2 (Fig. 7C), and NF-
B (Fig. 7D) activation in these cells.

View larger version (20K):
[in this window]
[in a new window]
|
FIG. 6. Inhibitory effect of metformin on leptin-induced HASMC proliferation and MMP-2 production. Serum-starved (A) or subconfluent (B) HASMCs were preincubated for 1 h with metformin (15 µg/ml) and then incubated for 72 h in the presence of leptin (Lep; 100 ng/ml). At the end of this incubation period, HASMC proliferation (A) and levels of MMP2 in the supernatants (B) were measured as previously described. Data represent the means ± SE of four independent experiments. *P < 0.05 vs. medium (Med).
|
|
 |
DISCUSSION
|
---|
Metformin has multiple biological effects, among which vasculoprotective properties have recently been identified (18). In experimental models of atherosclerosis, administration of metformin reduces the development and extent of atherosclerotic lesion formation (31). In patients with diabetes, this drug reduces the risk for developing macrovascular complications (16). The biological mechanisms responsible for the antiatherosclerotic properties of metformin are poorly understood but may include a suppressive effect of this drug on early key cellular events involved in atherogenesis, such as arterial lipid accumulation and metabolism (32,33), leukocyte-endothelium interaction, foam cell formation (19), and smooth muscle cell proliferation (34,35). It is well established that proliferative growth and migration of vascular smooth muscle cells (VSMCs) contribute to the arterial neointimal thickening observed in atherosclerosis, and recent in vivo and in vitro studies have demonstrated that leptin promotes neointimal growth in mice (36), VSMC proliferation (7), and endothelial cell MMP expression (6), thus supporting a role for this hormone in vascular lesion growth and matrix remodeling. Evidence that plasma leptin concentrations are associated with coronary atherosclerosis in patients with type 2 diabetes (37) further supports the possibility that leptin signaling may represent a therapeutic target for the prevention of atherosclerotic disease in these subjects. In smooth muscle cell, ROS are key mediators of the proliferative effect of many growth factors. Evidence that leptin stimulates smooth muscle cell growth and induces oxidative stress both in vivo and in vitro (5,3840) supports the possibility that ROS may act as second messengers in leptin-induced smooth muscle cell proliferation. In line with this possibility, our results demonstrate that leptin induces intracellular ROS production in HASMCs and that vitamin E inhibits the proliferative effect of leptin on these cells. Leptin-induced ROS generation in HASMCs may arise from the NAD(P)H oxidase. Indeed, this enzyme is the major source of ROS in VSMCs, and redox signaling through NAD(P)H oxidase induces smooth muscle cell proliferation (41,42). Our observations that inhibitors of NAD(P)H oxidase totally abolish leptin-induced ROS production and proliferation in HASMCs indicate that ROS generation in these cells arises from the NAD(P)H oxidase system and mediates the proliferative effect of leptin in HASMCs. Previous studies have demonstrated that metformin has antioxidant properties (18,4345). Consistent with these observations, we found that metformin suppressed the intracellular accumulation of ROS in leptin-treated HASMCs. These results, together with our observations that metformin inhibits the effect of leptin on smooth muscle cell growth to a similar extent as vitamin E, suggest that the suppressive effect of metformin on leptin-induced HASMC proliferation may be related to its antioxidant effects. Accumulating data indicate that stimulation of ROS through PKC-dependent activation of NAD(P)H oxidase and subsequent ERK activation may be critical mechanisms responsible for smooth muscle cell proliferation (4648). Previous studies have shown that leptin induces PKC and MAPK activation in vascular cells (5,49,50) and stimulates VSMC proliferation through the MAPK pathway (7). In agreement with these data, we found that leptin induces PKC and MAPK activation in HASMCs and that inhibition of these signaling events abrogates the proliferative effect of leptin on these cells. Our findings that inhibition of PKC suppresses ROS production in leptin-treated HASMCs and that apocynin blocks MAPK activation in these cells further support the notion that generation of ROS in leptin-stimulated HASMCs is PKC dependent and that leptin-induced MAPK activation involves NAD(P)H oxidasegenerated ROS. Interestingly, our data demonstrate that metformin inhibits PKC and MAPK activation in leptin-treated HASMCs. On the basis of previous observations showing that inhibition of VSMCs by vitamin E correlates with PKC inhibition (51,52), it is tempting to speculate that metformin, by reducing PKC activity, may reduce ROS generation, thereby leading to decreased MAPK activation and smooth muscle cell growth. Because metformin activates AMP kinase (AMPK) and that activation of this kinase appears to reduce diacylglycerol synthesis (5355), metformins inhibitory effect on PKC activation may involve AMPK activation, with consequent inhibition of diacylglycerol synthesis.
Proliferation of VSMCs is regulated by nuclear transcription factors including NF-
B. NF-
B activation is redox sensitive and is induced through PKC- and MAPK-dependent pathways (56,57). Previous data have proposed a role for ROS-dependent activation of NF-
B in leptin-induced activation of endothelial cells (38). The present study, which demonstrates that leptin induces HASMC proliferation through NF-
B activation, further stresses the crucial role of this transcription factor in the regulation of vascular cell function by leptin. In accordance with previous observations (38,56,57), we found that activation of NF-
B by leptin was linked to ROS generation and PKC/ERK1/2 activation. We further demonstrated that metformin inhibits leptin-induced NF-
B activation. Because AMPK appears to inhibit NF-
B activation in endothelial cells (58), metformins inhibitory effect on NF-
B activation in HASMCs may involve AMPK activation.
MMP expression is associated with smooth muscle cell proliferation and migration. MMP-2 is expressed abundantly in atherosclerotic lesions and plays an important role in increasing VSMC migration to the intima (59). Evidence that MMP-2 expression is upregulated by leptin in vascular cells (6) suggests that this effect may account, at least partly, for the neointimal growthpromoting property of leptin (36). Induction of MMP-2 is redox sensitive and occurs, in angiotensin-treated smooth muscle cell, in a NAD(P)H oxidasedependent manner (60). In accordance with these data, the present study demonstrates that leptin induces, through a PKC-dependent NAD(P)H oxidase activation, MMP-2 expression in HASMCs. Furthermore, it demonstrates that this effect involves activation of the MAPK/NF-
B pathway. These findings are in line with previous observations showing a role for PKC, ERK1/2, and NF-
B in the regulation of MMP secretion (61,62). Our observation that metformin abrogates the induction of MMP-2 by leptin in HASMCs further supports the possibility that this drug may reduce the development of atherosclerotic lesions by regulating intimal thickening. Supporting this possibility, metformin treatment of cholesterol-fed rabbits has been shown to inhibit smooth muscle cell proliferation (35). In conclusion, this study demonstrates that metformin inhibits, by interfering with the PKC/MAPK/NF-
B pathways, leptin-induced HASMC proliferation and MMP-2 expression in vitro. These data suggest a new mechanism by which metformin may reduce cardiovascular risk in type 2 diabetic subjects.
 |
ACKNOWLEDGMENTS
|
---|
This study was supported by Merck-Santé (Lyon, France).
 |
FOOTNOTES
|
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Address correspondence and reprint requests to Geneviève Renier, MD, PhD, CHUM Research Centre, Notre-Dame Hospital, 1560 Sherbrooke St. East, Room Y-3622, Montreal, Quebec, Canada H2L 4M1. E-mail: genevieve.renier{at}umontreal.ca
Received for publication February 21, 2005
and accepted in revised form April 18, 2005
AMPK, AMP kinase; DCF-DA, 2',7'-dichlorodihydrofluorescein diacetate; DPI, diphenyleneiodonium; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signalregulated kinase; FBS, fetal bovine serum; HASMC, human aortic smooth muscle cell; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-5-diphenyltetrazolium bromide; NF-
B, nuclear factor-
B; PKC, protein kinase C; ROS, reactive oxygen species; TTFA, thenoyltrifluoroacetone
 |
REFERENCES
|
---|
- Abate N: Obesity and cardiovascular disease: pathogenic role of the metabolic syndrome and therapeutic implications.
J Diabetes Complications14
:154
174,2000[Medline]
- Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL, Caro JF: Serum immunoreactive-leptin concentrations in normal-weight and obese humans.
N Engl J Med334
:292
295,1996[Abstract/Free Full Text]
- Couillard C, Lamarche B, Mauriege P, Cantin B, Dagenais GR, Moorjani S, Lupien PJ, Despres JP: Leptinemia is not a risk factor for ischemic heart disease in men: prospective results from the Quebec Cardiovascular Study.
Diabetes Care21
:782
786,1998[Abstract]
- Piemonti L, Calori G, Mercalli A, Lattuada G, Monti P, Garancini MP, Costantino F, Ruotolo G, Luzi L, Perseghin G: Fasting plasma leptin, tumor necrosis factor-
receptor 2, and monocyte chemoattracting protein 1 concentration in a population of glucose-tolerant and glucose-intolerant women: impact on cardiovascular mortality.
Diabetes Care26
:2883
2889,2003[Abstract/Free Full Text]
- Yamagishi SI, Edelstein D, Du XL, Kaneda Y, Guzman M, Brownlee M: Leptin induces mitochondrial superoxide production and monocyte chemoattractant protein-1 expression in aortic endothelial cells by increasing fatty acid oxidation via protein kinase A.
J Biol Chem276
:25096
25100,2001[Abstract/Free Full Text]
- Park HY, Kwon HM, Lim HJ, Hong BK, Lee JY, Park BE, Jang Y, Cho SY, Kim HS: Potential role of leptin in angiogenesis: leptin induces endothelial cell proliferation and expression of matrix metalloproteinases in vivo and in vitro.
Exp Mol Med33
:95
102,2001[Medline]
- Oda A, Taniguchi T, Yokoyama M: Leptin stimulates rat aortic smooth muscle cell proliferation and migration.
Kobe J Med Sci47
:141
150,2001[Medline]
- Parhami F, Tintut Y, Ballard A, Fogelman AM, Demer LL: Leptin enhances the calcification of vascular cells: artery wall as a target of leptin.
Circ Res88
:954
960,2001
- Nakata M, Yada T, Soejima N, Maruyama I: Leptin promotes aggregation of human platelets via the long form of its receptor.
Diabetes48
:426
429,1999[Abstract/Free Full Text]
- Santos-Alvarez J, Goberna R, Sanchez-Margalet V: Human leptin stimulates proliferation and activation of human circulating monocytes.
Cell Immunol194
:6
11,1999[Medline]
- Faggioni R, Feingold KR, Grunfeld C: Leptin regulation of the immune response and the immunodeficiency of malnutrition.
FASEB J15
:2565
2571,2001[Abstract/Free Full Text]
- Lord GM, Matarese G, Howard JK, Baker RJ, Bloom SR, Lechler RI: Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression.
Nature394
:897
901,1998[Medline]
- Kang SM, Kwon HM, Hong BK, Kim D, Kim IJ, Choi EY, Jang Y, Kim HS, Kim MS, Kwon HC: Expression of leptin receptor (Ob-R) in human atherosclerotic lesions: potential role in intimal neovascularization.
Yonsei Med J41
:68
75,2000[Medline]
- Yen TT, Allan JA, Pearson DV, Schinitsky MR: Dissociation of obesity, hypercholesterolemia, and diabetes from atherosclerosis in ob/ob mice.
Experientia33
:995
996,1977[Medline]
- Hasty AH, Shimano H, Osuga J, Namatame I, Takahashi A, Yahagi N, Perrey S, Iizuka Y, Tamura Y, Amemiya-Kudo M, Yoshikawa T, Okazaki H, Ohashi K, Harada K, Matsuzaka T, Sone H, Gotoda T, Nagai R, Ishibashi S, Yamada N: Severe hypercholesterolemia, hypertriglyceridemia, and atherosclerosis in mice lacking both leptin and the low density lipoprotein receptor.
J Biol Chem276
:37402
37408,2001[Abstract/Free Full Text]
- UK Prospective Diabetes Study (UKPDS) Group: Effect of intensive blood glucose control with metformin on complications in overweight patients with type 2 diabetes (UKPDS 34).
Lancet352
:854
865,1998[Medline]
- Howlett HCS, Bailey CJ: A risk-benefit assessment of metformin in type 2 diabetes mellitus.
Drug Saf20
:489
503,1999[Medline]
- Wiensperger NF: Metformin: intrinsic vasculoprotective properties.
Diabetes Tech Therap2
:259
272,2000
- Mamputu JC, Wiernsperger N, Renier G: Metformin inhibits monocyte adhesion to endothelial cells and foam cell formation.
Br J Diabetes Vasc Dis3
:302
310,2003
- Hamilton CA, Brosnan MJ, Al-Benna S, Berg G, Dominiczak AF: NAD(P)H oxidase inhibition improves endothelial function in rat and human blood vessels.
Hypertension40
:755
762,2002[Abstract/Free Full Text]
- Holland PC, Clark MG, Bloxham DP, Lardy HA: Mechanism of action of the hypoglycemic agent diphenyleneiodonium.
J Biol Chem248
:6050
6056,1973[Abstract/Free Full Text]
- Kimura C, Oike M, Ito Y: Hypoxia-induced alterations in Ca(2+) mobilization in brain microvascular endothelial cells.
Am J Physiol Heart Circ Physiol279
:H2310
H2318,2000[Abstract/Free Full Text]
- Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F, et al: The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C.
J Biol Chem266
:15771
15781,1991[Abstract/Free Full Text]
- Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR: A synthetic inhibitor of the mitogen-activated protein kinase cascade.
Proc Natl Acad Sci U S A92
:7686
7689,1995[Abstract/Free Full Text]
- Pierce JW, Schoenleber R, Jesmok G, Best J, Moore SA, Collins T, Gerritsen ME: Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo.
J Biol Chem272
:21096
21103,1997[Abstract/Free Full Text]
- Berridge MV, Tan AS: Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction.
Arch Biochem Biophys303
:474
482,1993[Medline]
- Fujimoto N, Mouri N, Iwata K, Ohuchi E, Okada Y, Hayakawa T: A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) using monoclonal antibodies.
Clin Chim Acta221
:91
103,1993[Medline]
- Yano T, Taura C, Shibata M, Hirono Y, Ando S, Kusubata M, Takahashi T, Inagaki M: A monoclonal antibody to the phosphorylated form of glial fibrillary acidic protein: application to a non-radioactive method for measuring protein kinase activities.
Biochem Biophys Res Commun175
:1144
1151,1991[Medline]
- Versteeg HH, Nijhuis E, van den Brink GR, Evertzen M, Pynaert GN, van Deventer SJ, Coffer PJ, Peppelenbosch MP: A new phosphospecific cell-based ELISA for p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and cAMP-response-element-binding protein.
Biochem J350
:717
722,2000[Medline]
- Renard P, Ernest I, Houbion A, Art M, Le Calvez H, Raes M, Remacle J: Development of a sensitive multi-well colorimetric assay for active NFkappaB.
Nucleic Acid Res29
:E21
E25,2001
- Mamputu JC, Wiernsperger NF, Renier G: Antiatherogenic properties of metformin: the experimental evidence.
Diabetes Metab29
:71
76,2003
- Marquié G: Metformin action on lipid metabolism in lesions of experimental aortic atherosclerosis of rabbits.
Atherosclerosis47
:7
17,1983[Medline]
- Marquié G: Comparative effects of metformin and phenformin on the progression and regression of cholesterol induced atherosclerosis in rabbits.
Paroi Arterielle5
:209
218,1973
- Bünting CE, Koschinsky T, Rütter R, Gries FA: Metformin inhibits the growth of human vascular cells: a new potentially antiatherogenic drug effect (Abstract).
Diabetologia29
:523A
,1986
- Weber G, Catapano A, Ghiselli G, Sirtori CR: Experimental studies on the antiatherosclerotic effect of metformin. In
Proceedings of the International Conference on Atherosclerosis. Carlson LA, Paoletti R, Sirtori CR, Weber G, Eds. New York, Raven Press,1978
, p.318
325
- Schäfer K, Halle M, Goeschen C, Dellas C, Pynn M, Loskutoff DJ, Konstantinides S: Leptin promotes vascular remodeling and neointimal growth in mice.
Arterioscler Thomb Vasc Biol24
:112
117,2004[Abstract/Free Full Text]
- Reilly MP, Iqbal N, Schutta M, Wolfe ML, Scally M, Localio AR, Rader DJ, Kimmel SE: Plasma leptin levels are associated with coronary atherosclerosis in type 2 diabetes.
J Clin Endocrinol Metab89
:3872
3878,2004[Abstract/Free Full Text]
- Beltowski J, Wojccicka G, Jamroz A: Leptin decreases plasma paraoxonase 1 (PON 1) activity and induces oxidative stress: the possible novel mechanism for proatherogenic effect of chronic hyperleptinemia.
Atherosclerosis170
:21
29,2003[Medline]
- Bouloumie A, Marumo T, Lafontan M, Busse R: Leptin induces oxidative stress in human endothelial cells.
FASEB J13
:1231
1238,1999[Abstract/Free Full Text]
- Maingrette F, Renier G: Leptin increases lipoprotein lipase secretion by macrophages: involvement of oxidative stress and protein kinase C.
Diabetes52
:2121
2128,2003[Abstract/Free Full Text]
- Lee HS, Son SM, Kim YK, Hong KW, Kim CD: NAD(P)H oxidase participates in the signaling events in high-glucose-induced proliferation of vascular smooth muscle cells.
Life Sci72
:2719
2730,2003[Medline]
- Gorlach A, Kietzmann T, Hess J: Redox signaling through NADPH oxidases: involvement in vascular proliferation and coagulation.
Ann N Y Acad Sci973
:505
507,2002[Abstract/Free Full Text]
- Pavlovic D, Kocic R, Kocic G, Jevtovic T, Radenkovic S, Mikic D, Stojanovic M, Djordjevic PB: Effect of four-week metformin treatment on plasma and erythrocyte antioxidative defense enzymes in newly diagnosed obese patients with type 2 diabetes.
Diabetes Obes Metab2
:251
256,2000[Medline]
- Bonnefont-Rousselot D, Raji B, Walrand S, Gardes-Albert M, Jore D, Legrand A, Peynet J, Vasson MP: An intracellular modulation of free radical production could contribute to the beneficial effects of metformin towards oxidative stress.
Metabolism52
:586
589,2003[Medline]
- Gargiulo P, Caccese D, Pignatelli P, Brufani C, De Vito F, Marino R, Lauro R, Violi F, Di Mario U, Sanguigni V: Metformin decreases platelet superoxide anion production in diabetic patients.
Diabetes Metab Res Rev18
:156
159,2002[Medline]
- Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T, Naruse M, Sano H, Utsumi H, Nawata H: High glucose level and free fatty acids stimulate reactive oxygen species production through protein kinase Cdependent activation of NAD(P)H oxidase in cultured vascular cells.
Diabetes49
:1939
1945,2000[Abstract]
- Lu G, Greene EL, Nagai T, Egan BM: Reactive oxygen species are critical in the oleic-acid-mediated mitogenic signaling pathway in vascular smooth muscle cells.
Hypertension32
:1003
1010,1998[Abstract/Free Full Text]
- Hattori Y, Kakishita H, Akimoto K, Matsumura M, Kasai K: Glycated serum albumin-induced vascular smooth muscle cell proliferation through activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by protein kinase C.
Biochem Biophys Res Commun281
:891
896,2001[Medline]
- Bouloumie A, Drexler HCA, Lafontan M, Busse R: Leptin, the product of Ob gene, promotes angiogenesis.
Circ Res83
:1059
1066,1998[Abstract/Free Full Text]
- Goetze S, Bungenstock A, Czupalla C, Eilers F, Stawowy P, Kintscher U, Spencer-Hänsch C, Graf K, Nürnberg B, Law RE, Fleck E, Gräfe M: Leptin induces endothelial cell migration through Akt which is inhibited by PPAR
ligands.
Hypertension40
:748
754,2002[Abstract/Free Full Text]
- Tasinato A, Boscoboinik D, Bartoli GM, Maroni P, Azzi A: d-alpha tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties.
Proc Natl Acad Sci U S A92
:12190
12194,1995[Abstract/Free Full Text]
- Azzi A, Boscoboinik D, Clement S, Marilley D, Ozer NK, Ricciarelli R, Tasinato A: Alpha-tocopherol as a modulator of smooth muscle cell proliferation.
Prostaglandins Leukot Essent Fatty Acids57
:507
514,1997[Medline]
- Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear LJ, Moller DE: Role of AMP-activated protein kinase in mechanism of metformin action.
J Clin Invest108
:1167
1174,2001
- Ido Y, Carling D, Ruderman N: Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells: inhibition by the AMP-activated protein kinase activation.
Diabetes51
:159
167,2002[Abstract/Free Full Text]
- Muoio DM, Seefeld K, Witters LA, Coleman RA: AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target.
Biochem J338
:783
791,1999[Medline]
- Yerneni KK, Bai W, Khan BV, Medford RM, Natarajan R: Hyperglycemia-induced activation of transcription factor
B in vascular smooth muscle cells.
Diabetes48
:855
864,1999[Abstract]
- Hoshi S, Goto M, Koyama N, Nomoto K, Tanaka H: Regulation of vascular smooth muscle cell proliferation by nuclear factor-
B and its inhibitor, I-
B.
J Biol Chem275
:883
889,2000[Abstract/Free Full Text]
- Cacicedo JM, Yagihashi N, Keaney JF, Ruderman NB, Ido Y: AMPK inhibits fatty acid-induced increases in NF-
B transactivation in cultured human umbilical vein endothelial cells.
Biochem Biophys Res Comm324
:1204
1209,2004[Medline]
- Newby AC, Southgate KM, Davies M: Extracellular matrix degrading metalloproteinases in the pathogenesis of arteriosclerosis.
Basic Res Cardiol89
:59
70,1994[Medline]
- Luchtefeld M, Grote K, Grothusen C, Bley S, Bandlow N, Selle T, Struber M, Haverich A, Bavendiek U, Drexler H, Schieffer B: Angiotensin II induces MMP-2 in a p47phox-dependent manner.
Biochem Biophys Res Commun328
:183
188,2005[Medline]
- Hussain S, Assender JW, Bond M, Wong L-F, Murphy D, Newby AC: Activation of protein kinase C zeta is essential for cytokine-induced metalloproteinase-1, -3, and -9 secretion from rabbit smooth muscle cells and inhibits proliferation.
J Biol Chem277
:27345
27352,2002[Abstract/Free Full Text]
- Bond M, Fabunmi RP, Baker AH, Newby AC: Synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor NF-kappa B.
FEBS Lett435
:29
34,1998[Medline]