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(Circulation. 2004;110:3335-3340.)
© 2004 American Heart Association, Inc.

Molecular Cardiology

Resistin Promotes Smooth Muscle Cell Proliferation Through Activation of Extracellular Signal–Regulated Kinase 1/2 and Phosphatidylinositol 3-Kinase Pathways

From the Research Center for Cardiovascular Diseases at the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston (P.C., E.T.H.Y.); Texas Heart Institute, St Luke’s Episcopal Hospital, Houston (P.C., J.T.W., E.T.H.Y.); Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station (I.S.); and Department of Cardiology, University of Texas M.D. Anderson Cancer Center, Houston (J.T.W., E.T.H.Y.).

Correspondence to Dr Edward T.H. Yeh, Department of Cardiology, Unit 449, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030. E-mail etyeh{at}mdanderson.org

Received March 6, 2004; de novo received May 11, 2004; revision received June 21, 2004; accepted June 25, 2004.

	Abstract

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Background— Resistin, a novel adipokine, is elevated in patients with type 2 diabetes and may play a role in the vascular complications of this disorder. One recent study has shown that resistin has a proinflammatory effect on endothelial cells. However, there is no information on whether resistin could also affect vascular smooth muscle cells (SMCs). Thus, the purpose of this study was to assess whether resistin could induce SMC proliferation and to study the mechanisms whereby resistin signals in SMCs.

Methods and Results— Human aortic smooth muscle cells (HASMCs) were stimulated with increasing concentrations of resistin for 48 hours. Cell proliferation was induced by resistin in a dose-dependent manner as assessed by direct cell counting. To gain more insights into the mechanism of action of resistin, we investigated the extracellular signal–regulated kinase (ERK) and/or phosphatidylinositol 3-kinase (PI3K) signaling pathways. Transient phosphorylation of the p42/44 mitogen-activated protein kinase (ERK 1/2) occurred after addition of resistin to HASMCs. U0126, a specific inhibitor of ERK phosphorylation, significantly inhibited ERK 1/2 phosphorylation and reduced resistin-simulated proliferation of HASMCs. LY294002, a specific PI3K inhibitor, also significantly inhibited HASMC proliferation after resistin stimulation.

Conclusions— Our results demonstrate that resistin induces HASMC proliferation through both ERK 1/2 and Akt signaling pathways. The proliferative action exerted by resistin on HASMCs may account in part for the increased incidence of restenosis in diabetes patients.

Key Words: diabetes mellitus • obesity • angioplasty • restenosis

	Introduction

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The prevalence of both type 2 diabetes mellitus and obesity has increased dramatically during the past decade.¹ Although cardiovascular mortality rates for the general population have been declining for several decades, mortality among people with diabetes has been increasing, and this increased mortality is primarily associated with cardiovascular causes.²

Patients with type 2 diabetes mellitus are indeed at increased risk for the development of atherosclerotic coronary artery disease.³ In addition, people with diabetes have a higher risk of postangioplasty restenosis, with or without implantation of coronary stents, than people who do not have diabetes.^4–6 Previously, adipose tissue was thought to be a passive depot for storing excess calories. More recently, however, studies have revealed that adipocytes synthesize and secrete a surprising number of biologically active molecules,⁷ generally called adipokines, including tumor necrosis factor-, leptin, interleukin-6, plasminogen activator inhibitor-1, adiponectin, and resistin.^8–11 Resistin is an adipocyte-derived peptide first identified during a search for targets of thiazolidinediones. Steppan et al¹¹ reported that serum concentrations of resistin are markedly elevated in obese mice and can be decreased by treatment with thiazolidinediones. They also found that administration of an anti-resistin antibody increases insulin-stimulated glucose uptake in obese mice and that treatment of normal mice with recombinant resistin impairs insulin action. Thus, resistin might link obesity with insulin resistance and diabetes in mice models. However, subsequent studies in rodent models^12,13 and in humans^14–16 have produced controversial findings on the role of resistin in obesity, insulin resistance, and diabetes. Recently, however, a specific ELISA method to measure the human resistin has been developed: compared with control individuals, plasma resistin concentrations of type 2 diabetic patients were increased 1.8 times in both men and women.¹⁷ Moreover, 2 independent studies recently showed direct vasoactive effects of resistin in cultured vascular endothelial cells.^18,19

The question of whether this novel adipokine could have a direct effect on smooth muscle cells (SMCs), another cell population present in the arterial wall and often involved in atherosclerotic plaque formation and restenosis development, remained unanswered. In this study we investigated whether the adipokine resistin exerts direct effects to promote SMC proliferation, and we examined the possible molecular pathways involved in resistin action.

	Methods

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Cell Culture
Human aortic SMCs (HASMCs) and SMC growth supplement were purchased from Cascade Biologics. Penicillin, streptomycin, and medium 231 were obtained from Gibco BRL. Fetal bovine serum and gelatin were obtained from Sigma. HASMCs were plated onto 0.1% gelatin-coated culture dishes from Corning, Inc, and grown in 231 medium with growth supplement, 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 µg/mL) at 37°C in a humidified 95% air/5% CO₂ atmosphere. Cells were used at passages 2 through 6. Preliminary studies were performed to optimize incubation time (data not shown), and the human recombinant resistin (PeproTech, Inc) concentration was used according to a previous report.¹⁸ Endotoxin levels in the resistin preparation, as assessed with limulus assay, were <0.1 ng/µg.

Proliferation Assays
All experiments were performed on cells synchronized in the G₀ phase of the cell cycle. Initially, HASMC proliferation was evaluated in triplicate by a colorimetric assay.²⁰ This technique can determine cellular viability by measuring the metabolic conversion of a water-soluble tetrazolium salt, WST-1 (Roche), into formazan by mitochondrial dehydrogenases: it forms a dark red product that is soluble in tissue culture media. The amount of formazan produced is proportional to the number of live cells and is expressed as cellular viability. For this procedure, cells were seeded in 96-well plates and incubated in serum-free medium 231 for 24 hours. Cells were then stimulated with human recombinant resistin at 10, 25, 50, and 100 ng/mL, and assays were performed by adding WST-1 directly to the culture wells and incubating them for 120 minutes at 37°C. Plates were then read by a scanning multiwell spectrophotometer by measuring the absorbance of the dye with a wavelength of 450 nm and a reference wavelength of 630 nm. Three different experiments were performed for each experimental condition.

To better investigate the growth effect of resistin, cell proliferation was evaluated by direct cell counting. HASMCs were seeded at a density of 10 000 cells/cm² in 12-well plates and cultured overnight; the cells were then incubated in serum-free medium 231 for 24 hours. Cells were stimulated with 2.5% fetal bovine serum as positive control and with recombinant human resistin at 10, 25, 50, and 100 ng/mL, and after 48 hours they were washed with PBS, harvested by mild trypsinization, and counted with a hematocytometer. Experiments were performed in triplicate, and 3 different experiments were performed for each experimental condition.

The experiments were conducted in the absence or presence of pharmacological inhibitors of selected signaling pathways. Cells were treated with different inhibitors or vehicle for 60 minutes before and for the duration of the stimulation with resistin. The inhibitors used were phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (5 to 50 µmol/L), p38 inhibitor SB203580 (0.1 to 10 µmol/L), and MEK1 inhibitor U0126 (0.1 to 10 µmol/L).

Western Immunoblotting
Confluent HASMCs were growth-arrested in serum-free medium for 48 hours. Cells were treated for 60 minutes with different inhibitors or vehicle as described above, followed by stimulation with resistin at 100 ng/mL for 10, 15, and 60 minutes. In another set of experiments, cells were treated with an increasing dose of human recombinant resistin (10 to 100 ng/mL) for 15 minutes. Cells were then lysed in 50 mmol/L Tris-HCl/150 mmol/L NaCl (pH 7.5) ice-cold buffer containing 1% Nonidet P40, 0.5% sodium deoxycholate, 100 mmol/L NaF, 2 mmol/L Na₃VO₄, 10 mmol/L phenylmethylsulfonylfluoride, 500 µmol/L 4-(2-aminoethyl)-benzenesulfonylfluoride, 150 nmol/L aprotinin, and 1 µmol/L leupeptin. Protein concentration was determined with the use of the BioRad DC protein assay (BioRad Laboratories). Equal amounts of protein (30 µg) were electrophoresed in 12% sodium dodecyl sulfate polyacrylamide gel and transferred to nitrocellulose membranes. Bands were visualized by reaction with specific anti-phosphoprotein antibodies directed against phospho–p44/p42 mitogen-activated protein kinase (MAPK) (thr202/thr204), total p44/42 MAPK, phospho–p38 MAPK, total p38 MAPK, phospho-Akt (ser 473), and total Akt (all at 1:1000 dilution; Cell Signaling). In brief, membranes were blocked in 5% bovine serum albumin for 2 hours at room temperature and probed with anti-phosphoprotein antibody for 1 hour at room temperature. After secondary incubation in horseradish peroxidase–conjugated goat anti-mouse or goat anti-rabbit IgG antibody (1:2000 dilution; Sigma), the immunocomplexes were visualized with an enhanced chemiluminescence detection kit according to the manufacturer’s instructions (Amersham Pharmacia Biotech) and quantified by densitometry.

Statistical Analysis
All data are presented as mean±SD. The data were analyzed with the use of 1-way ANOVA followed by the Scheffé test for multiple comparisons. A probability value <0.05 was considered statistically significant.

	Results

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Effect of Resistin on HASMC Proliferation
To evaluate the direct effects of resistin on HASMC proliferation, in a first set of experiments the colorimetric assay (WST-1) for nonradioactive quantification of cell proliferation was used. After 2 days of treatment with increasing concentrations (10 to 100 ng/mL) of resistin, a dose-dependent proliferative effect was detected in the treated sample (maximum effect at 100 ng/mL 1.7-fold compared with the control; Figure 1A; P<0.05).

View larger version (16K): [in this window] [in a new window]   Figure 1. A, Effects of resistin on SMC proliferation and effects of U0126, a specific MEK1 inhibitor assessed by the colorimetric assay (WST-1) for the nonradioactive quantification of cell proliferation. Serum-starved HASMCs were treated with the MEK1 inhibitor U0126 (10 µmol/L) for 60 minutes before and for the duration of the stimulation with resistin (10 to 100 ng/mL). Results are expressed as percent change in WST-1 U vs control cells 48 hours after treatment with resistin, and each bar represents the mean±SD of triplicate determinations. *P<0.05 vs untreated cells, **P<0.05 vs resistin treatment. B, In parallel experiments, direct cell counting was used. Serum-starved HASMCs were treated with 2.5% fetal bovine serum (FBS) as positive control and with increased concentrations (10 to 100 ng/mL) of resistin, and cell counts were determined after 48 hours. Values are expressed as percentage of control, and each bar represents the mean±SD of triplicate determinations. *P<0.05 vs untreated cells. C, Effects of U0126 on resistin-induced HASMC proliferation assessed by direct cell counting were also evaluated. Serum-starved HASMCs were pretreated with U0126 at different concentrations (0.1 to 10 µmol/L) for 1 hour before resistin treatment (100 ng/mL), and the cell counts were determined after 48 hours. Values are expressed as percentage of untreated cells, and each bar represents the mean±SD of triplicate determinations. *P<0.05 vs resistin-treated cells.

In another set of experiments, the effect of resistin on HASMC proliferation was evaluated by direct cell counting. Cells were stimulated for 48 hours with increasing concentrations (10 to 100 ng/mL) of resistin, and the assays were performed. Fetal bovine serum (2.5%) was used as positive control. We demonstrated that resistin induced HASMC proliferation in a dose-dependent manner (Figure 1B; P<0.05), with a significant increase observed at a concentration of 10 ng/mL and the maximum effect at 100 ng/mL (1.5-fold over medium alone). Thus, both methods supported the conclusion that resistin can induce HASMC proliferation.

U0126 and LY294002 Inhibit Resistin-Induced HASMC Proliferation
To better understand the molecular mechanisms involved in resistin-induced HASMC proliferation, we investigated the possible involvement of MAPK and PI3K signaling pathways. To determine whether extracellular signal–regulated kinase (ERK) 1/2 activation is involved in resistin-induced HASMC proliferation, cells were treated with the MEK1 inhibitor U0126 (10 µmol/L) for 60 minutes before and for the duration of the stimulation, and proliferation assays were performed with the use of the WST-1 assay. Although UO126 alone did not significantly affect the growth of HASMCs, ERK 1/2 inhibition led to a complete block of resistin-induced HASMC proliferation (Figure 1A; P<0.05 versus resistin treatment). In another set of experiments, ERK 1/2 inhibition significantly reduced resistin-mediated SMC proliferation in a dose-dependent manner, as evaluated by direct cell counting (Figure 1C; P<0.05).

Additional signaling molecules, including PI3K and p38, have been shown to be involved in SMC proliferation. To determine the role of PI3K and p38 in resistin-mediated HASMC proliferation, HASMC proliferation assays, in which direct cell counting was used, were performed with cells treated as described above with the PI3K inhibitor LY294002 (5 to 50 µmol/L) or the p38 inhibitor SB203580 (0.1 to 10 µmol/L). PI3K pathway inhibition by LY294002 significantly reduced HASMC proliferation in a dose-dependent manner after resistin stimulation (Figure 2; P<0.05 versus resistin treatment). In contrast, inhibition of p38 did not affect the HASMC proliferation induced by resistin (Figure 3).

View larger version (12K): [in this window] [in a new window]   Figure 2. Effects of LY294002, a specific PI3K inhibitor, on resistin-induced HASMC proliferation assessed by direct cell counting. Serum-starved HASMCs were pretreated with LY294002 at different concentrations (5 to 50 µmol/L) for 1 hour before resistin treatment (100 ng/mL), and the cell counts were determined after 48 hours. Values are expressed as percentage of untreated cells, and each bar represents the mean±SD of triplicate determinations. *P<0.05 vs resistin-treated cells.

View larger version (11K): [in this window] [in a new window]   Figure 3. Effects of SB203580, a specific p38 inhibitor, on resistin-induced HASMC proliferation assessed by direct cell counting. Serum-starved HASMCs were pretreated with SB203580 at different concentrations (0.1 to 10 µmol/L) for 1 hour before resistin treatment (100 ng/mL), and the cell counts were determined after 48 hours. Values are expressed as percentage of untreated cells, and each bar represents the mean±SD of triplicate determinations. *P<0.05 vs resistin-treated cells.

Resistin-Activated ERK 1/2 and Akt Phosphorylation Are Inhibited by U0126 and LY294002
The previous experimental finding prompted us to further define whether resistin activates 2 major signaling cascades such as ERK 1/2 and PI3K pathways. To address this issue, the cell lysates were probed for ERK 1/2 and Akt, a downstream substrate of PI3K, protein levels and their phosphorylation forms. Western blot analysis showed that increased ERK 1/2 phosphorylation in SMCs treated with resistin 100 ng/mL was detected after 10 minutes, with maximal increase occurring after 15 minutes of treatment and decreasing after 1 hour (Figure 4A). In the dose-response experiments, the increase in ERK 1/2 phosphorylation after 15 minutes was detected also at 10, 25, and 50 ng/mL of resistin, reaching a maximum at 100 ng/mL (Figure 4B). Next, we examined the action of the specific inhibitor U0126 on the ERK 1/2 pathway activation. Pretreatment with U0126 completely abrogated resistin-induced (10- and 15-minute) ERK 1/2 phosphorylation (Figure 4A). Western blot analysis for Akt also showed that the phosphorylation of Akt was affected by resistin. In a manner similar to that for ERK 1/2 activation, resistin induced an increase of Akt activation after 10 and 15 minutes and a slight decrease at 1 hour (Figure 5).

View larger version (20K): [in this window] [in a new window]   Figure 4. Time course and dose response of ERK activation in HASMCs stimulated with resistin and inhibition with U0126. HASMCs treated with resistin (100 ng/mL) for 10, 15, and 60 minutes (A) or treated with increasing does of resistin (10 to 100 ng/mL) for 15 minutes (B) were analyzed by Western blot for phosphorylated ERK 1/2 (p ERK 1/2). The blots were stripped and incubated with ERK 1/2 antibody to demonstrate protein loading. For the time course experiment, HASMCs were pretreated with U0126 (10 µmol/L) for 1 hour, treated with resistin (100 ng/mL) for 10 and 15 minutes, and then analyzed by Western blot for phosphorylated ERK 1/2. The blot was stripped and incubated with ERK 1/2 antibody to demonstrate protein loading. C indicates control; t ERK 1/2, total ERK 1/2. *P<0.05 vs untreated cells, **P<0.05 vs resistin treatment.

View larger version (18K): [in this window] [in a new window]   Figure 5. Time course of Akt activation in HASMCs stimulated with resistin. HASMCs treated with resistin (100 ng/mL) for 10, 15, and 60 minutes were analyzed by Western blot for phosphorylated Akt (p AKT). The blot was stripped and incubated with Akt antibody to demonstrate protein loading. C indicates control; t AKT, total Akt. *P<0.05 vs untreated cells.

	Discussion

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Traditionally, adipose tissue was thought to be an inert organ, which functions to protect the body from temperature and trauma and to store excess energy in the form of triglycerides. Recent studies indicate that fat cells have the ability to actively produce a surprising number of substances, generally called adipokines, including tumor necrosis factor-, leptin, interleukin-6, plasminogen activator inhibitor-1, adiponectin, and resistin.^8–11 The capability of these molecules to not only contribute to the pathogenesis of the insulin-resistant syndrome but also to participate in determining the vascular inflammatory process leading to atherosclerosis is the object of numerous studies.

Resistin (FIZZ3/ADSF) is the most recently described adipocyte-derived peptide, and it has been initially suggested to play a role in the development of insulin resistance and obesity.¹¹ Resistin was reported to antagonize insulin action in cells in vitro as well as in vivo, and circulating levels were increased in obese (ob/ob) and diabetic (db/db) mice. Its receptor has not yet been identified and cloned, and little is known about this adipokine signaling. However, the initial excitement over the putative role of resistin as a hormone that links obesity to diabetes abated considerably in response to the results of recent studies.^14–16 In humans, the expression of resistin in adipocytes is low compared with that in rodents, but resistin mRNA is readily detectable in circulating mononuclear cells, which suggests that human resistin may be regulated by a different mechanism or may have a different role than that in rodents.¹⁴ Furthermore, the expression of resistin in adipocytes does not differ among normal, insulin-resistant, and type 2 diabetic individuals.^15,16 Nevertheless, some genetic case-control studies have demonstrated that polymorphisms of the human resistin gene have been linked to insulin resistance in certain populations.^21–23 Moreover, resistin expression is positively correlated with insulin resistance in humans,^24,25 and serum resistin levels are elevated in human obesity²⁶ and type 2 diabetes.¹⁷ The plasma concentrations of resistin in patients with insulin resistance remain to be carefully defined, although preliminary studies suggest that mean circulating resistin levels may be 40 ng/mL in diabetes (versus 15 ng/mL in lean nondiabetic patients).^27,28 In the study of Shalev et al,²⁹ the average serum resistin level in healthy control subjects was 30.7 ng/mL, and serum resistin levels were significantly elevated in patients with diabetes (49.7 ng/mL). The differences in these results may also be due to different ELISAs used in different studies. The range of concentrations (10 to 100 ng/mL) of resistin used in the present study were therefore within the physiological range. Additionally, Banerjee at al,³⁰ investigating the role of resistin in glucose metabolism, reported that mice lacking resistin exhibit low blood glucose levels after fasting as a result of reduced hepatic glucose production. It is therefore attractive to hypothesize that human and mouse resistin have similar metabolic functions despite their divergent sites of production.

Little is known about resistin and vascular inflammation. Under in vitro experimental conditions, Verma et al¹⁸ showed a direct effect of human recombinant resistin in promoting endothelial cell activation, suggesting possible direct vascular effects of this adipokine.

In the present study we demonstrated a direct effect of resistin on vascular SMCs. Indeed, we demonstrated, using 2 different approaches (WST-1 proliferation assay and direct cell counting), that resistin was able to induce, in a dose-dependent manner, HASMC growth after 2 days of stimulation.

The proliferation of vascular SMCs is a crucial process in the pathogenesis of restenosis,³¹ and serial intravascular ultrasonographic studies have indicated that increased restenosis associated with type 2 diabetes results from excessive intimal hyperplasia of vascular SMCs.⁵ SMCs proliferate minimally in the intact artery but are stimulated to divide after arterial de-endothelialization owing in large measure to the local accumulation of growth factors and cytokines at the injury site,³² activating different pathways. In particular, p42/p44 MAPK has been thought to play a pivotal role in controlling SMC proliferation.³³ Moreover, specific inhibitors of MAPK phosphorylation markedly inhibit SMC growth in vitro.³⁴ This important role of MAPK activation in SMC proliferation has also been evaluated in several in vivo studies, demonstrating a marked increase in p42/p44 MAPK activation after balloon catheter injury in animal arteries.^35,36 In our study we found that the proliferation of HASMCs induced by resistin treatment was associated with an induction of MAPK activation. Moreover, the inhibition of this signaling pathway, with the use of a specific MEK inhibitor, significantly reduced resistin-induced HASMC proliferation.

Additional signaling molecules reported to play important roles in SMC migration and proliferation are PI3K and p38. The PI3K/Akt pathway is an important mediator of cell growth and survival in response to growth factors and other signals.³⁷ PI3K activates the Akt serine/threonine kinase by generating specific inositol phospholipids, which recruit Akt to the cell membrane and enable its activation. Akt mediates cell survival and growth signals by phosphorylating and inactivating proapoptotic proteins.³⁸ In a recent study, Akt was found to influence cell-cycle progression of SMCs both in vitro and in vivo.³⁹ In the present study we also found that the PI3K-Akt pathway was involved in resistin-induced HASMC proliferation, and we demonstrated that the use of a specific inhibitor of this pathway, LY294002, dramatically reduced the proliferation after resistin stimulation.

Finally, p38, a subfamily of the MAPKs, is strongly activated by environmental stress factors,⁴⁰ including diabetic conditions⁴¹ and proinflammatory cytokines.⁴² In our study, however, the p38 pathway seemed not to be involved in HASMC proliferation induced by resistin stimulation. Inhibition of this signaling pathway did not result in any modulation of the proliferative phenomenon.

In summary, we have demonstrated for the first time that resistin, a novel adipokine, exerts direct effects on SMC proliferation; moreover, in HASMCs, resistin causes activation of both the ERK 1/2 and PI3K pathways. These data support the hypothesis that the resistin molecule can play a role in the development of the atherosclerotic process, accounting in part for the increased incidence of restenosis in diabetes patients.

	Footnotes

 
Guest Editor for this article was Prediman K. Shah.

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