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ENDOCRINE AND DIABETES
/Activin Receptor-Like Kinase-4/5/7/Smad2 Signaling PathwayDepartment of Pharmacology, School of Medicine, Universidad Complutense, Madrid, Spain
Received October 3, 2006; accepted January 29, 2007.
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Abstract |
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1/Smad-2 pathway was involved. We isolated human internal mammary artery VSMC from patients who had undergone coronary-artery bypass graft. Pioglitazone (100 µM) induced apoptosis in human VSMC from diabetic and nondiabetic patients (NDP), analyzed by DNA fragmentation and by degradation of Bcl-2, in high-glucose-containing medium (15 and 25 mM). This apoptotic effect was inhibited by the activin receptor-like kinase-4/5/7/Smad2 inhibitor 4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide (SB-431542), denoting that the TGF-1/Smad-2 pathway was involved. Pioglitazone rapidly increased the extracellular TGF-1 levels and concomitantly induced phosphorylation of Smad2 in VSMC from DP and NDP. Thus, we demonstrated that pioglitazone induced apoptosis in human VSMC from DP, which are strongly resistant to the induced apoptosis. This effect of pioglitazone might contribute in the treatment of alterations of vascular remodeling in type 2 diabetes mellitus.
). This alteration has been postulated to be mediated by an imbalance in the ratio proliferation/apoptosis of vascular smooth muscle cells (VSMC) within the artery wall (Gibbons and Dzau, 1994). Moreover, we reported recently that arteries and isolated VSMC from diabetic patients showed increasing levels of the antiapoptotic protein Bcl-2 and increasing levels of cell proliferation (Ruiz et al., 2006). This alteration of tissue microarchitecture may be regarded as a "failure to die" of this cell type.
Thiazolidinediones constitute an emerging class of oral antidiabetic drugs that possess some direct vascular effects, independently of their hypoglycemic actions (Aizawa et al., 2001). In particular, pioglitazone (PIO) has been demonstrated to exert an additional benefit on the lipid profile compared with other thiazolidinediones (Goke and German Pioglitazone Study Group, 2002). In addition, one of the most remarkable direct vascular actions of PIO is the induction of VSMC apoptosis (Bishop-Bailey et al., 2002), which might be related to the clinical finding of a decreased intima/media thickness in patients treated with PIO (Takagi et al., 2003). In this context, the pleiotropic cytokine transforming growth factor (TGF-1) is thought to play a major role in vascular remodeling by decreasing the ratio proliferation/apoptosis (Ivanov et al., 1998; Kannan et al., 2003). The canonical pathway of TGF-1 in VSMC involves activation of ALK-4/5/7 receptor-related kinases, which phosphorylates Smad2 (Ten Dijke et al., 2002). Interestingly, the importance of the TGF-1/Smad2 pathway in PIO-mediated rat VSMC apoptosis has been demonstrated by our group (Redondo et al., 2005). We found that PIO increased TGF-1 release in rat VSMC and that this cytokine mediated the apoptosis of PIO through the Smad2 pathway. In this work, we aimed to determine whether PIO was able to overcome the human diabetic VSMC failure to die and whether TGF-1/ALK-4/5/7 was involved.
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) as fasting serum glucose concentration 
126 mgl/dl and use of antidiabetic oral drugs or insulin. Patient data included age, gender, active smoker, obesity, total cholesterol, cholesterol low-density lipoproteins, cholesterol high-density lipoproteins, triglycerides, glucose, and blood pressure (see patient details in Table 1). Internal mammary arteries were collected by the surgeons during the surgical procedure, labeled, and used within the next few minutes after the operations. The study was conducted according to the Declaration of Helsinki, and we obtained informed consent from all subjects before sampling took place.
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Cell Cultures and Treatments. Human internal mammary artery vascular smooth muscle cells were cultured from explants in RPMI 1640 medium (Life Technologies, Barcelona, Spain) containing 10% fetal calf serum. The cells exhibited typical "hill and valley" smooth muscle morphology observed by phase-contrast microscopy, and the cultures were stained positively with a monoclonal anti-
-actin antibody. Experiments were performed with VSMC between passages 3 and 5. For the analysis of cell death by apoptosis induced by PIO, cells (diabetic and nondiabetic) were treated with different concentrations of PIO (10–100 µM) for 24 h in a normoglycemic cellular medium (5 mM glucose). Alternatively, cells from nondiabetic and diabetic patients were pretreated with 15 and 25 mM glucose (or 25 mM mannitol) for 48 h before treating the cells with 100 µM PIO for 24 h. because PIO induced apoptosis under hyperglycemic conditions, the following experiments were performed in VSMC pretreated with 15 mM glucose for 48 h. To determine the role of TGF1/ALK-4/5/7 in the apoptotic effect of PIO, cells from nondiabetic and diabetic patients were pretreated with the ALK-4/5/7 blocker (inhibitor and antagonist) SB-431542 at 10 µM 30 min before PIO treatment. For the analysis of TGF-1 release and Smad2 phosphorylation, cells were pretreated with 15 mM glucose or RPMI 1640 medium containing 10 ng/ml PDGF and 0.2% BSA 48 h before PIO treatment to avoid the interference of serum-contained TGF-1.
Measurement of Cellular DNA Fragmentation. Vascular smooth muscle cells from nondiabetic and diabetic patients were plated on 96-well plates at a density of 7000 cells/well, and the cells were allowed to attach for 24 h. Cellular DNA fragmentation was measured with a commercially available cellular DNA fragmentation enzyme-linked immunosorbent assay kit (Roche-Boehringer, Madrid, Spain) following the manufacturer's instructions. DNA fragmentation was expressed as -fold increase of the control values.
Analysis of Caspase-3 Activity. Human vascular smooth muscle cells (nondiabetic and diabetic) were plated on 90-mm Petri dishes and allowed to attach for 24 h. The cells were then treated with glucose 15 mM for 48 h and then with 100 µM PIO for 12 h. Caspase-3 activity was measured spectrophotometrically using a commercially available kit (Calbiochem, San Diego, CA) following the manufacturer's instructions. Data are represented as caspase-3 activity (picomoles per minute per milligram of protein).
Western Blotting. To determine the expression of Bcl-2, cells from diabetic and nondiabetic patients were plated onto 60-mm Petri dishes and allowed to attach for 24 h. The cells were pretreated with 5 or 15 mM glucose for 48 h before PIO treatment in the presence or absence of the ALK-4/5/7 blocker SB-431452. At the time of harvest, the cells were washed with ice-cold PBS, lysed on ice with 200 µl of lysis buffer (10% glycerol, 2.3% SDS, 62.5 mM Tris-HCl, pH 6.8, 150 mM NaCl, 10 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 5 µg/ml chymostatin, 1 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride), and boiled for 5 min. Equal amounts of protein were run on 12.5% SDS-polyacrylamide gel electrophoresis. The proteins were then transferred to polyvinylidene difluoride membranes (Immobilon-P; GE Healthcare, Madrid, Spain) and blocked overnight at 4°C in blocking solution [5% skimmed milk in TBS-T: 25 mM Tris-HCl, 75 mM NaCl, pH 7.4, and 0.1% (v/v) Tween 20]. For analysis of Bcl-2, the blots were incubated for 2 h with agitation at room temperature in the presence of a specific mouse monoclonal anti-Bcl-2 (Neomarker, Bionova Científica, Madrid, Spain) at 1.5 µg/ml in 0.3% bovine serum albumin in TBS-T. After washing in TBS-T solution, the blots were further incubated for 1 h at room temperature with a horseradish peroxidase conjugated anti-mouse secondary antibody diluted 1:3000 (Promega, Madison, WI) in blocking solution. The blots were then washed five times in TBS-T, and antibody-bound protein was visualized with an enhanced chemiluminescence kit (GE Healthcare). Smooth muscle -actin was used as a housekeeping protein, and it was determined following the same procedure as mentioned above, using a specific anti--actin mouse monoclonal antibody (Sigma-Aldrich, Madrid, Spain), at 1:1000 in TBS-T.
Measurements of TGF-1 Levels. To determine TGF-1 levels (total, acid-activable) in the culture medium of control and treated samples, we used a solid-phase TGF-1-specific enzyme-linked immunosorbent assay, following the manufacturer's instructions (R&D Systems, Minneapolis, MN). Cells were seeded onto 24-well plates and allowed to attach for 24 h. The cells were pretreated with 15 mM glucose for 48 h in normal medium and then treated with pioglitazone at 100 µM or control for a defined period (30–180 min) in RPMI 1640 medium containing 10 ng/ml PDGF and 0.2% BSA.
Immunofluorescence Staining. Subcellular location of phosphoSmad2 protein was also analyzed by confocal images of immunofluorescence-stained samples. The cells were plated onto coverslips, allowed to attach for 24 h, and then treated with 15 mM glucose 48 h. The cells then were cultured in the presence or absence of pioglitazone at 100 µM from 30 to 180 min. In some experiments, 2 µM GW9662 or 10 µM SB-431542 was added 30 min before treatment with PIO. Cells were washed with PBS and fixed for 20 min in 4% paraformaldehyde in PBS and permeabilized with 0.4% Triton X-100 for 30 min at room temperature. After blocking with 3% BSA in PBS, the cells were then incubated with goat polyclonal anti-phosphoSmad2 (1:100) for 1 h. Excess primary antibody was removed by washing with blocking solution, followed by incubation with donkey anti-goat Alexa 568 (1:100; Invitrogen, Carlsbad, CA) for 1 h. The cells were washed four times with blocking buffer every 5 min. Images were captured using a Leica TCS SP2 inverted microscope (Leica, Wetzlar, Germany). Intensity of staining was analyzed by NIH ImageJ software.
Materials. Pioglitazone was a generous gift from Takeda Chemical Industries (Osaka, Japan) and was directly diluted in cell culture media. SB-431542 and GW9662 were purchased from Tocris Cookson Inc. (Bristol, UK) and dissolved in dimethyl sulfoxide at 1000x concentrated stock solution. Fluvastatin was kindly donated by Novartis Pharmaceutical Ltd. (Madrid, Spain). All other reagents were obtained from Sigma-Aldrich unless otherwise stated.
Statistical Analysis. The results are expressed as the mean ± S.D. and accompanied by the number of observations. A statistical analysis of the data was carried out by a Student's t test or by a one-way analysis of variance when necessary, followed by Dunnett's post test where significance was detected. Differences with a P value of less than 0.05 were considered statistically significant.
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). According to this, we also determined the effect of PIO on the levels of Bcl-2 measured by Western blotting. Because the apoptotic effect of PIO was observed in high-glucose conditions, independently of the osmotic effect, we performed the following experiments in a 15 mM glucose-containing medium, a serum glucose concentration typically achieved by diabetic patients. Figure 3A shows that basal levels of Bcl-2 were highly expressed in cells from diabetic patients, as already seen. Similarly to DNA fragmentation, cells pretreated with PIO exhibited reduced Bcl-2 protein levels, which denote cell death by apoptosis. Interestingly, cells from diabetic patients also showed degradation of Bcl-2 when treated with PIO.
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Role of ALK-4/5/7 on the Apoptosis Induced by Pioglitazone. Because the TGF-1/Smad2 pathway has been shown to be involved in the apoptotic effect of PIO in rat VSMC, we determined whether the intermediate pathway ALK-4/5/7 was also involved in human VSMC from nondiabetic and diabetic patients. Figure 3A shows that pretreatment with 10 µM SB-431542 for 30 min before PIO treatment abolished the effect of PIO on Bcl-2 protein levels; moreover, this inhibitor blocked the DNA fragmentation induced by PIO in cells from both nondiabetic and diabetic patients (Fig. 3B).
Pioglitazone Induces the Release of TGF-1. Because TGF-1 seems to be involved in the apoptotic effect of PIO, we studied whether this drug was able to increase the release of TGF-1 from cultured human VSMC obtained from nondiabetic and diabetic patients pretreated with 15 mM glucose for 48 h before PIO treatment. Figure 4A shows a rapid and transient increase in the release of total TGF-1 induced by 100 µM pioglitazone as early as 30 min after treatment in VSMC from nondiabetic patients, as reported in rat VSMC (Redondo et al., 2005). However, VSMC from diabetic patients showed a different pattern of TGF-1 release. Thus, TGF-1 levels reached a peak after 60-min treatment with PIO, although levels of TGF-1 progressively increased after 30-min treatment. Although none of the patients were taking pioglitazone, we also analyzed the concentration of TGF-1 in sera from nondiabetic and diabetic patients, and we found no differences (Table 1).
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1 itself induces apoptosis in this cell model, VSMC from nondiabetic and diabetic patients were pretreated with 15 mM glucose for 48 h and then with 400 pg/ml TGF-1 for 60 min. Twenty-four hours after the exposure to TGF-1, we analyzed the DNA fragmentation and Bcl-2 expression. Figure 4B shows that treatment with TGF-1 increased DNA fragmentation in VSMC from both nondiabetic and diabetic patients in approximately 1.5-fold increase, a value comparable with the effect of PIO. We also found that protein levels of Bcl-2 (analyzed by Western blotting) diminished in cells from diabetic and nondiabetic patients (Fig. 4B) treated with TGF-1.
Pioglitazone Enhanced the Phosphorylation of Smad2. We showed previously that pioglitazone at 100 µM rapidly increased the nuclear recruitment of phospho-Smad2 in rat VSMC. In human VSMC from nondiabetic and diabetic patients pretreated with 15 mM glucose, 100 µM PIO increased both cytosolic and nuclear staining of phosphoSmad2, although we did not observe a clear nuclear recruitment of phospho-Smad2 as seen in rat VSMC (Fig. 5). Increase in phosphorylation of Smad2 was induced after 30-min stimulation with PIO in both nondiabetic and diabetic isolated cells. However, the level of Smad2 phosphorylation in the cytosol was higher in cells from diabetic patients. This effect of PIO was blocked by the ALK-4/5/7 blocker SB-431542 and by the peroxisome proliferator-activated receptor (PPAR)
antagonist GW9662 (Fig. 6).
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; Guan et al., 2002; Boitier et al., 2003). Upon activation, PPARs form heterodimers with retinoid X receptors and bind the peroxisome proliferator-response elements in the promotor regions of responsive genes to modify gene transcription (Corton et al., 2000). Pharmaceutical ligands of PPARs are used clinically to treat chronic diseases such as diabetes and hyperlipidemia, and they are being investigated for treatment of cancer, atherosclerosis, and other diseases (Dunaif et al., 1996; Fajas et al., 2001). Pioglitazone is a PPAR agonist that is used for treatment of type 2 diabetes mellitus. In this study, we have demonstrated that the PPAR agonist pioglitazone induced apoptosis in vascular smooth muscle cells isolated from nondiabetic and diabetic patients under hyperglycemic conditions. This effect is likely to be mediated through the PPAR receptor, because apoptotic trigger in vascular smooth muscle cell cultures induced by thiazolidinediones has been related to PPAR by previous reports by our group (Redondo et al., 2005) and other groups (Bishop-Bailey et al., 2002). Other PPAR agonists, such as troglitazone, also induced apoptosis in vascular smooth muscle cells (Gouni-Berthold et al., 2001; Lin et al., 2004) and in several tumor cell lines by a mechanism of action involving the decrease in the expression of the antiapoptotic protein Bcl-2 (Yoshizawa et al., 2002; Shiau et al., 2005). In this context, we also observed that PIO treatment decreased protein level of Bcl-2 in cells from both nondiabetic and diabetic patients analyzed by Western blotting.
This finding has important medical relevance, because we showed previously that VSMC from diabetic patients are resistant to induced apoptosis due, at least in part, to an increase in the protein levels of Bcl-2 within the artery wall, and, in addition, that this resistance of apoptosis may be acquired in vitro by cells from nondiabetic patients when cultured in a high-glucose medium (Ruiz et al., 2006). The apoptotic effect of PIO does not seem to be an unspecific effect, because other drugs, such as fluvastatin, did not induce apoptosis in cells from diabetic patients even under high-glucose conditions (Fig. 2).
The current study was undertaken in an effort to determine whether pioglitazone might induce apoptosis in VSMC from diabetic patients and therefore overcome the resistance to induced apoptosis reported in those patients. We show here that the apoptotic effect of pioglitazone (measured as DNA fragmentation and Bcl-2 expression) was significantly decreased by pretreatment with the ALK-4/5/7 inhibitor SB-431542 (Fig. 3), which demonstrates the role of the TGF-1/Smad2 pathway in the apoptotic effect of pioglitazone in these cells. The compound SB-431542 that we have used in the present study has been demonstrated to act as a selective pharmacological inhibitor of ALK-4/5/7 (Inman et al., 2002). Nevertheless, since ALK-5 has been highlighted as the most important ALK isoform for TGF-1-mediated growth arrest (Ten Dijke and Hill, 2004), it may be considered that the apoptotic effect of pioglitazone described in the experimental conditions of the present study is likely mediated by ALK-5.
In our previous work, we also showed that short-time exposure to pioglitazone, as well as the physiological PPAR ligand 15-deoxy-prostaglandin J2, induced the release of TGF-1 into the cellular medium (Redondo et al., 2005) and that TGF-1 was able to stimulate the phosphorylation of Smad2 and therefore its nuclear recruitment. In the present work, we also observed that pioglitazone induced the release of TGF-1 in human VSMC from nondiabetic and diabetic patients and that TGF-1 added exogenously also induced apoptosis in VSMC from diabetic and nondiabetic patients. It is important to point out that rat VSMC are normally grown in high-glucose medium, and we found that pioglitazone only induced apoptosis in human VSMC pretreated with 15 or 25 mM glucose (but not with 25 mM mannitol, which rules out an osmotic effect). This is important, since diabetic patients, although well controlled, possess abnormal (usually increased) blood levels of glucose, such as those found in our cohort of patients, and therefore the observed apoptotic effect of pioglitazone only would take place in a pathological situation of hyperglycemia. It is noteworthy that the serum TGF-1 concentration in our diabetic patients (whose mean glucose concentration is about 10 mM) does not exert any variation compared with nondiabetics (Table 1). Thus, in vitro glucose concentration was increased up to 15 and 25 mM to assess whether variations in the TGF-1 pathway took place. Our approach is based on the conclusion from the UKPDS study, which establishes that future complications in type 2 diabetes mellitus (such as macrovascular disease) are related to a poor glycemic control (United Kingdom Prospective Diabetes Study Group, 1998), where postprandial peaks of 15 mM glucose can be achieved.
The role of TGF-1 in atherosclerosis and type 2 diabetes mellitus is controversial. Although an increase in the expression of TGF-1 in atherosclerotic clinical specimens has been found (Panutsopulos et al., 2005) and the serum levels of TGF-1 were reported to be increased in diabetic patients (Pfeiffer et al., 1996), we analyzed the serum concentration of TGF-1 in our diabetic and nondiabetic patients, and we found no statistical differences in both groups of patients (39.94 pg/ml, confidence limit95% = 35.87–44.01 in nondiabetic patients and 44.91 pg/ml, confidence limit95% = 41.11– 48.71 in diabetic patients; P = 0.07; n = 96). Moreover, the bioavailability of TGF-1 is thought to be reduced in atherosclerotic patients (Byrne et al., 1998; O'Neil et al., 2004) as well as Smad2/3 signaling (Kalinina et al., 2004). Moreover, current cardiovascular risk factors such as the antifibrinolytic plasminogen activator inhibitor-1 or a high-fat diet have been reported to sequester active TGF-1 (Ten Dijke and Hill, 2004).
In accordance with our previous work, pioglitazone also induced Smad2 phosphorylation. This effect was due to the effect of pioglitazone on the PPAR receptor, because it was blocked by the PPAR antagonist GW9662 (Fig. 5). Concerning the timing of Smad2 phosphorylation, in both nondiabetic and diabetic patients pioglitazone induced phosphorylation of Smad2 early after 30-min treatment (which correlates with an increase of TGF-1 in both types of patients).
In relation with the concentration of pioglitazone used in this study, we have found that pioglitazone induces VSMC apoptosis when incubated at a concentration of 100 µM. A clinical study on the pharmacokinetics of pioglitazone reported average peak serum concentrations of 3.38 µM for 45-mg dose (Budde et al., 2003). However, the existence of active liver metabolites (Budde et al., 2003) makes the relationship between the concentrations found in vivo and the relationship used in our cell culture studies an interesting topic.
Apoptotic effects that have been related to pioglitazone incubation in the present study may be related to some in vivo and clinical results. This drug has been proven to decrease experimental balloon angioplasty in animal models (Aizawa et al., 2001). In addition, a pioglitazone-mediated decrease of intima-media thickness has been reported in retrospective studies (Koshiyama et al., 2001). Moreover, in type 2 diabetics, pioglitazone has been reported to decrease cardiovascular risk markers independently of glycemic control at the dose of 45 mg (Pfutzner et al., 2005) and to prevent macrovascular complications in doses ranging from 15 to 45 mg (Dormandy et al., 2005).
An increased PPAR expression in neointimal VSMC has been described previously (Bishop-Bailey et al., 2002). It is noteworthy that the decisive role of high glucose and PPAR for the apoptotic effect of pioglitazone might mediate a molecular correlation for pioglitazone selectivity.
The clinical relevance of these findings concerns the findings that pioglitazone induced apoptosis in VSMC from diabetic patients and under high-glucose concentrations. That pioglitazone exhibits this effect is important, because this drug is extensively used in the treatment of type 2 diabetes mellitus. It is also important to point out that the effect only occurred under abnormal glucose conditions, which are comparable with those found in diabetic patients. Although this is an in vitro study, we can argue that treatment with pioglitazone might ameliorate the vascular remodeling and abnormal intima/media thickening after angioplastia that occurs in arteries from diabetic patients, which seems to be due to an imbalance between proliferation and apoptosis of vascular smooth muscle cells.
Interestingly, Smad-dependent pathways have been described to mediate TGF-1-induced apoptosis of VSMC, an effect exerted by high concentrations of this cytokine (Ten Dijke et al., 2002). According to the present study, high concentrations of TGF-1 would activate these mediators and trigger a subsequent VSMC apoptosis.
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Acknowledgements |
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Footnotes |
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E.R. and S.R. contributed equally to this work.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: VSMC, vascular smooth muscle cell(s); PIO, pioglitazone; TGF, transforming growth factor; ALK, activin receptor-like kinase; PGDF, platelet-derived growth factor; SB-431542, 4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TBS-T, Tris-buffered saline/Tween 20; GW9662, 2-chloro-5-nitro-N-phenylbenzamide; PPAR, peroxisome proliferator-activated receptor.
Address correspondence to: Dr. Teresa Tejerina, Department of Pharmacology, School of Medicine, Universidad Complutense, 28040 Madrid, Spain. E-mail: teje{at}med.ucm.es
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