Arq Bras Cardiol abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC HDvTrPsbBvgQsBskzfqm3DR S0066-782X2018001400162 S0066-782X2018005008102 10.5935/abc.20180108 Original Article Pioglitazone Induces Cardiomyocyte Apoptosis and Inhibits Cardiomyocyte Hypertrophy Via VEGFR-2 Signaling Pathway Zhong Wenliang 1 2 Jin Wen 3 Xu Shanghua 1 Wu Yanqing 1 Luo Shunxiang 1 Liang Minlie 1 Chen Lianglong 2 Fujian Medical University The First Hospital of Nanping City Department of Cardiology Nanping Fujian China Department of Cardiology, The First Hospital of Nanping City, affiliated to Fujian Medical University, Nanping, Fujian - China Fujian Medical University Union Hospital Department of Cardiology Fuzhou Fujian China Department of Cardiology, Union Hospital, Fujian Medical University, Fuzhou, Fujian - China Guangdong N°.2 Provincial People’s Hospital Cardiovascular Department Guangzhou Guangdong China Cardiovascular Department, Guangdong N°.2 Provincial People’s Hospital, Guangzhou, Guangdong - China Mailing Address: Wenliang Zhong, Department of Cardiology, The First Hospital of Nanping City, N°. 317, Zhongshan Road, Nanping, Fujian - China. E-mai: WL.Zhong@tom.com

Author contributions

Conception and design of the research: Zhong W, Chen L; Acquisition of data: Zhong W, Jin W, Wu Y, Luo S, Liang M; Analysis and interpretation of the data and statistical analysis: Zhong W, Jin W, Xu S, Wu Y, Luo S, Liang M;Obtaining financing: Zhong W; Writing of the manuscript: Zhong W, Jin W, Liang M; Critical revision of the manuscript for intellectual content: Zhong W, Xu S, Liang M.

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

 02 07 2018 08 2018 111 2 162 169 20 10 2017 23 01 2018 14 03 2018 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Abstract Background:

Pioglitazone has been widely used as an insulin-sensitizing agent for improving glycemic control in patients with type 2 diabetes mellitus. However, cardiovascular risk and protective effects of pioglitazone remain controversial.

 Objectives:

In this study, we investigated whether pioglitazone affects cardiomyocyte apoptosis and hypertrophy by regulating the VEGFR-2 signaling pathway.

 Methods:

Cardiomyocytes were enzymatically isolated from 1- to 3-day-old Sprague-Dawley rat ventricles. Effects of pioglitazone and the VEGFR-2-selective inhibitor apatinib on cardiomyocyte apoptotic rate was determined using flow cytometry, and hypertrophy was evaluated using [3H]-leucine incorporation. The protein expressions of unphosphorylated and phosphorylated VEGFR-2, Akt, P53, and mTOR were determined by Western-Blotting. Analysis of variance (ANOVA) was used to assess the differences between groups.

 Results:

Pioglitazone and VEGFR-2-selective inhibitor apatinib reduced rat cardiomyocyte viability and cardiomyocyte hypertrophy induced by angiotensin II in vitro. Furthermore, in the same in vitro model, pioglitazone and apatinib significantly increased the expression of Bax and phosphorylated P53 and decreased the expression of phosphorylated VEGFR-2, Akt, and mTOR, which promote cardiomyocyte hypertrophy.

 Conclusions:

These findings indicate that pioglitazone induces cardiomyocyte apoptosis and inhibits cardiomyocyte hypertrophy by modulating the VEGFR-2 signaling pathway.

 Keywords: Apoptosis Myocytes, Cardiac Cardiomegaly Heart Failure/physiopathology Antihypertensive Agents Thiazolidinediones Insulin Resistance Natural Science Foundation of Fujian Province Project 2010J01371 Introduction

Heart failure (HF) is the most common consequence of cardiovascular diseases and the leading cause of cardiovascular mortality worldwide.1 Basic pathophysiology of HF is cardiac remodeling, which involves a number of cellular changes including cardiomyocyte hypertrophy, loss of cardiomyocytes due to apoptosis, necrosis, fibroblast proliferation and fibrosis.2 Recent fundamental and clinical studies have demonstrated that diabetes mellitus (DM) drives cardiac remodeling, including myocardial hypertrophy and cardiomyocytes loss, via glucotoxicity and lipotoxicity, eventually resulting in HF.3 Intensive glucose control was shown to reduce the occurrence of major cardiovascular events including HF, but did not improve the overall survival rate in patients with type 2 DM, compared to patients receiving standard therapy.3 Thiazolidinediones, including pioglitazone, have been widely used as peroxisome proliferator-activated receptor (PPAR)-γ agonists and insulin-sensitizing agents for improving glycemic control in patients with type 2 DM. However, cardiovascular risks of pioglitazone remain controversial.

One view is that intensive glycemic control with pioglitazone or rosiglitazone increases the risk of HF (OR ≤ 2.1; 95% CI 1.08-4.08) based on meta-analyses of randomized clinical trials.4-6 Rosiglitazone was more likely to induce HF than pioglitazone.7 Additionally, animal experiments confirmed the increased risk of HF with pioglitazone treatment, as pioglitazone augmented cardiac damage in isoproterenol-induced HF rat model and induced rat ventricular hypertrophy in acute toxicity experiments.8,9 However, another point of view is that pioglitazone use does not significantly increase the risk of myocardial infarction or cardiac death, based on the PROspective pioglitAzone Clinical Trial In macroVascular Events (PROactive) data,10 and that pioglitazone can suppress overload-induced cardiac hypertrophy by inhibiting AKT/GSK3β and MAPK signaling pathways.11

Vascular endothelial growth factor receptors (VEGFR) are considered critical factors for cardiac hypertrophy and HF. Three different subtypes, VEGFR-1, -2, and -3 have been described. Recent studies showed that VEGFR-1 and VEGFR-2 are essential for regression and induction of cardiomyocyte hypertrophy, respectively,12 whereas VEGFR-3 was shown to be beneficial for the infarcted myocardium by promoting compensatory cardiomyocyte hypertrophy and improving survival.13 Additionally, VEGFR-2 is involved in the delayed phase of endothelial cell (pulmonary artery and human aortic endothelial cells) barrier dysfunction caused by high levels of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine oxidation products, contributes to stress fiber formation, and increases phosphorylation of myosin light chains.14 Pioglitazone decreased the expression of VEGFR-2 in splanchnic tissues and inhibited neoangiogenesis in a rat model of portal hypertension,15 indicating a possible direct effect on VEGFR-2 expression. Reverse screening approaches (reverse pharmacophore mapping and reverse docking) have been very important methods to discover new cardiovascular disease-related protein targets for pioglitazone. In this study, we used the PharmMapper for reverse pharmacophore mapping. The structure of pioglitazone in mol2 format was submitted to PharmMapper, obtaining 10 targets and poses, which were sorted by decreasing PharmMapper fit score. The top ten PharmMapper fit scores for potential pioglitazone targets showed that VEGFR-2 was the best-ranked potential target, which will be essential for understanding the interaction between pioglitazone and VEGFR-2.

Given the potential link between pioglitazone and VEGFR-2 and their function in cardiomyocyte hypertrophy and apoptosis in the pathophysiology of HF, we investigated, for the first time, whether pioglitazone affects cardiomyocyte hypertrophy and apoptosis by regulating the VEGFR-2 signaling pathway. Furthermore, it may be expected to explore a promising approach for clarifying the potential mechanism for the effect of pioglitazone on cardiovascular outcomes.

 Methods Ethics Statement

All animal experiments were approved by the Institutional Animal Care and Use Committee of the First Hospital of Nanping City.

 Molecule preparation

In order to characterize the binding sites in the predicted protein targets we used Genetic Optimization for Ligand Docking (GOLD) suite v5.3. VEGFR-2-inhibitor complex crystallographic structure (Protein Data Bank ID: 3CP9) was selected as the starting structure for predicting the binding site of pioglitazone at VEGFR-2. GOLD uses a genetic algorithm for docking ligands into the binding site of target proteins, with full conformational flexibility of the ligand and partial receptor flexibility. Ligand binding energy was predicted with the ChemScore scoring function and free energy of binding (ΔG) as implemented in GOLD.16 Solvent molecules were removed from the crystal structure and protein hydrogen atoms were added. Ligand binding site for docking was defined to include amino acids within 10 Å of the coordinates of the inhibitor in the crystal structure. Top 10 docking poses were obtained by terminating the simulation once root mean square deviation (RMSD) between any five ligand poses was reached < 1 Å PyMol v.1.3 and LigPlot+ v.1.4 were used to visualize the results.

 Isolation and culture of rat neonatal cardiomyocytes

Cardiomyocytes were enzymatically isolated from 1- to 3-day-old Sprague-Dawley rat ventricles as previously described.17 A total of twelve rats were used to perform twelve independent experiments in the study. Isolated cardiomyocytes were seeded onto cell culture plates precoated with 10 g/ml of fibronectin and cultured in a medium containing DMEMF-12 with HEPES (Invitrogen, Carlsbad, CA, USA), 5% heat-inactivated horse serum, 100 U/ml penicillin, 10 µg/ml streptomycin, 3 mM pyruvic acid, 2 mg/ml bovine serum albumin, 100 g/ml ampicillin, insulin-transferrin-sodium selenite media supplement (Sigma, St. Louis, MO, USA), 5 g/ml linoleic acid, and 100 M ascorbic acid at 37°C in a humidified atmosphere containing 5% CO2. For all experiments, cells were cultured at 5 × 104 cells/cm2 unless otherwise stated.

 Cell proliferation assay

Effects of pioglitazone and the VEGFR-2-selective inhibitor apatinib (Apexbio Technology LLC, Houston, TX, USA) on cardiomyocyte proliferation were measured by counting crystal violet-stained cells 24 h after treatment using an automated cell counter (BioRad). Briefly, cardiomyocytes (5 × 104 cells/well) were seeded in 96-well plates and cultured in standard medium for 24 h. After 24 h serum starvation, cardiomyocytes were treated with 0.1 µM angiotensin (Ang) II for 24 h. Pioglitazone (0, 10 or 20µM) and apatinib (2 µM) was added to the culture medium 2 h prior to Ang II administration. For crystal violet staining, the cells were washed twice with 1× phosphate-buffered saline, fixed with 20% methanol for 30 min, and stained with 0.2% crystal violet solution for 30 min at room temperature with gentle shaking. Stained cells were washed with water until a clear background was visible. Crystal violet dye was extracted using 1% SDS and the cells were counted using an automated cell counter.

 Detection of apoptosis by flow cytometry

Cardiomyocyte apoptotic rate was determined using flow cytometry with the annexin V-FITC (AV)/propidium iodide (PI) dual staining. Briefly, after treatment, the cells (1-5 × 105/ml) were collected, washed twice with phosphate-buffered saline, and resuspended in 500 µl of binding buffer. Next, the cells were incubated with 10 µL AV and 5 µL PI in the dark at room temperature for 15 min. The apoptotic cells were identified by FCM within 30 min.

 Cardiomyocyte hypertrophy

Cardiomyocytes were seeded at a density of 5 × 104 cells/well in 96-well plates and hypertrophy was evaluated using [3H]-leucine incorporation, as previously described.18 Briefly, 2 h after treatment with pioglitazone (0-20 µmol/l) and apatinib (2 µM), 0.1 µM Ang II was used to stimulate cardiomyocyte hypertrophy and 1 µCi [3H]-leucine was simultaneously added to each well. After stimulation with Ang II for 60 h, cells were harvested by precipitation with 10% trichloroacetic acid on ice for 30 min before being solubilized with 1 mol/l NaOH overnight at 4°C. The samples were neutralized with 1 mol/l HCl and [3H] levels were determined in scintillation fluid using a β counter to assess [3H]- leucine incorporation.

 Western Blot Analysis

Trypsinized cells were lysed in radioimmunoprecipitation assay buffer, homogenized on ice, and centrifuged. Supernatants were resolved via 10% SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were blocked with Tris-buffered saline (TBS) containing 5% non-fat milk and incubated with the following primary antibodies at 4°C overnight: VEGFR-2 (ab39256; 1:1000; Abcam), phospho-VEGFR-2 (Tyr1175) (#2478; 1:1000; Cell Signaling Technology), Akt (#9272; 1:1000; Cell Signaling Technology), phospho-Akt (Thr308) (#9275; 1:1000; Cell Signaling Technology), P53 (#9282; 1:1000; Cell Signaling Technology), phospho-P53 (Ser15) (#9284; 1:1000; Cell Signaling Technology), Bax (#14796; 1:1000; Cell Signaling Technology), rabbit anti-mTOR (#2972; 1:1000; Cell Signaling Technology), rabbit anti- phosphorylated-mTOR (Ser2448) (#5536; 1:1000; Cell Signaling Technology), and GAPDH (#2118, 1:1000; Cell Signaling Technology). On the following day, the membranes were washed three times with tris-buffered saline with tween (TBST) for 5 min at room temperature and subsequently incubated with anti-rabbit IgG secondary antibodies, for 1 h at room temperature. Following incubation, the membranes were washed with TBST and exposed to an X-ray film. Band intensities on the film were analyzed by densitometry, and the results were normalized to β-actin. GAPDH was used as the protein loading control.

 Statistical analysis

Statistical analysis was performed using the SPSS 13.0 software package. Kolmogorov Smirnov test was used to verify the normality of data distribution and Levene test was used to inspect the homogeneity of variance. All data satisfied normal distribution and homogeneity of variance were presented as mean ± SD. One-way ANOVA was used for comparisons between multiple groups, whereas post-hoc Bonferroni test was used for pairwise comparisons. P < 0.05 indicated statistical significance.

 Results VEGFR-2 was the optimal potential target for pioglitazone

Top ten PharmMapper fit scores of potential pioglitazone targets shown VEGFR-2 was the best-ranked potential target. To understand the interaction between pioglitazone and VEGFR-2 and assess ligand binding energy, we performed a docking study using GOLD. Optimal binding conformation of the pioglitazone-VEGFR-2 complex is presented in Figure 1A and 1B. ChemScore score and binding energy of pioglitazone-VEGFR-2 complex were comparable to the VEGFR-2-inhibitor complex crystallographic structure. Pioglitazone was predicted to form van der Waals interactions with Val363, Leu428, Cys454, Leu444, Leu310, Phe456, Gly387, Phe383, Val364, and Ile453 and bind to Cys384 and Asp455 with hydrogen bonds. Predicted hydrogen bonds are shown in Fig. 1B. Western blot was conducted to examine the effect of pioglitazone on expression of VEGFR-2 and phospho-VEGFR-2 in rat neonatal cardiomyocytes under hypertrophic stimuli. Pioglitazone treatment decreased VEGFR-2 phosphorylation in rat neonatal cardiomyocytes in a dose-dependent manner (Figure 1C).

VEGFR-2 is the potential target of pioglitazone. A, 3D molecular docking model of pioglitazone with VEGFR-2; green structure, the conformer of pioglitazone. B, 2D molecular docking model of pioglitazone with VEGFR-2; purple structure, the conformer of pioglitazone. H-bonding interactions between the pioglitazone and VEGFR-2 were indicated with green dashed lines. C and D, Representative western bolotting trace of VEGFR-2 and phospho-VEGFR-2 (Tyr1175) protein levels in rat neonatal cardiomyocytes under hypertrophic stimuli, and treated with 0, 10, 20 (µM) pioglitazone for 24 hours, and intensity of the bands in C normalized to β-actin (n = 12 in each group). All data shown are the mean ± SD. *p < 0.01 compared with control; #p < 0.01 compared with pioglitazone 10 µM group, calculated by one-way ANOVA followed by the post hoc Bonferroni test for pairwise comparisons.

Pioglitazone promoted cardiomyocyte apoptosis and inhibited cardiomyocyte hypertrophy induced by Ang II

To validate the effects of pioglitazone, cardiomyocyte viability and Ang II-induced cardiomyocyte hypertrophy were evaluated. Crystal violet staining, a quick and versatile assay for screening cell viability under diverse stimulation conditions,19 was used to analyze cell viability. Pioglitazone inhibited cardiomyocyte viability in a dose-dependent manner (Figure 2A and 2B, p < 0.01), with effective concentrations ranging from 0 to 20 µmol/l. Apatinib also inhibited cardiomyocyte viability (Figure 2A and 2B, p < 0.01). Cardiomyocyte apoptotic rate was determined using FCM with AV/PI staining. Apoptotic rates in pioglitazone 20 µM, pioglitazone 10 µM, and apatinib 2 µM groups were significantly higher compared to control (Figure 2C and 2D, p < 0.01), with apoptotic rate in the pioglitazone 10 µM group significantly lower than rates in pioglitazone 20 µM and apatinib 2 µM groups (Figure 2C and 2D, p < 0.01). Ang II-induced [3H]-leucine incorporation was significantly decreased after pioglitazone or apatinib treatment, indicating that both inhibited cardiomyocyte hypertrophy.

Pioglitazone and apatinib induced apoptosis and inhibited hypertrophy of rat neonatal cardiomyocytes (n = 12 in each group). A, Crystal violet staining of rat neonatal cardiomyocytes in response to various concentrations of pioglitazone or apatinib. Both inhibited the viability of rat neonatal cardiomyocytes. B, Cell proliferation was determined using the automated cell counter. C and D, Pioglitazone and apatinib induced neonatal rat cardiomyocytes apoptosis, which was detected by flow cytometry with the annexin V (AV) / propidium iodide (PI) dual staining. Pioglitazone (10, 20<italic>µ</italic>M) and Apatinib (2<italic>µ</italic>M) increased the apoptosis of the cardiomyocytes compared with the control group. E, Angiontensin II-induced [<sup>3</sup>H]- leucine incorporation following various concentrations of Pioglitazone or Apatinib. All data represent the means ± SD Statistical comparison with control group: *p < 0.01 compared with controls; #p < 0.01 compared withi pioglitazone 10 <italic>µ</italic>M group, calculated by one-way ANOVA followed by the post hoc Bonferroni test for pairwise comparisons.

Pioglitazone inhibited cardiomyocyte hypertrophy and promoted cardiomyocyte apoptosis by suppressing VEGFR-2 signaling

Potential mechanisms of pioglitazone-induced inhibition of cardiomyocyte hypertrophy and promotion of cardiomyocyte apoptosis were assessed in vitro. Compared to cardiomyocytes under control conditions, pioglitazone significantly increased the expression of Bax and phospho-P53 and decreased the expression of phospho-VEGFR-2 in neonatal rat cardiomyocytes under hypertrophic stimuli (Figure 1C, Figure 3).

Pioglitazone and Apatinib regulate VEGFR-2 signaling in neonatal rat cardiomyocytes under hypertrophy induced by Angiotensin II (n = 12 in each group). A and B, Representative western bolotting trace of phospho-VEGFR-2, VEGFR-2, phospho-mTOR, mTOR, phospho-Akt, Akt, Bax, phospho-P53 and P53 protein levels in rat neonatal cardiomyocytes under hypertrophic stimuli, and treated with pioglitazone (20 µM) or apatinib (2 µM) for 24 hours, and intensity of the above bands in A normalized to GAPDH. All data represent the means ± SD. *p < 0.01 compared with controls; #p < 0.01 compared withi pioglitazone 20 µM group, calculated by one-way ANOVA followed by the post hoc Bonferroni test for pairwise comparisons.

To further investigate whether pioglitazone targets VEGFR-2, effects of pioglitazone on the VEGFR-2 expression and VEGFR-2-regulated intracellular signaling were determined in neonatal rat cardiomyocytes, under hypertrophy induced by Ang II. Compared to untreated hypertrophic cardiomyocytes, pioglitazone significantly decreased the expression of phospho-VEGFR-2, phospho-Akt, and phospho-mTOR, which may contribute to cardiomyocyte hypertrophy. Likewise, apatinib significantly decreased the expression of VEGFR-2, phospho-VEGFR-2, phospho-Akt, and phospho-mTOR (Figure 1C, Figure 3).

 Discussion

In the present study, we demonstrated that pioglitazone reduced cardiomyocyte viability and hypertrophy induced by Ang II in vitro. We further show that pioglitazone increased the expression of Bax and phosphorylated P53, and decreased the expression of phosphorylated VEGFR-2, Akt, and mTOR in vitro. These findings suggest that pioglitazone induces cardiomyocyte apoptosis and inhibits cardiomyocyte hypertrophy through effects on the VEGFR-2 signaling pathway.

Pioglitazone has been widely used to improve glycemic control in patients with type 2 DM. Additionally, the PROactive trial showed that pioglitazone reduced the main secondary composite outcome of cardiovascular death/myocardial infarction/stroke vs. placebo by 43 % in the trial population.10 These findings indicate that pioglitazone improves vascular function in diabetic patients and non-diabetic patients with insulin resistance and suggesting a possible beneficial effect of pioglitazone treatment on cardiovascular prognosis. Furthermore, a meta-analysis showed that supplementing insulin treatment with pioglitazone in type 2 DM patients with poorly controlled glucose levels could help decrease glucose levels and reduce the daily insulin dose without increasing the risks of myocardial infarction, HF, cardiac death and all-cause death, but at the cost of increasing total cholesterol levels and risks of hypoglycemia and edema.20

Given available evidence, pioglitazone treatment appears advantageous in patients with HF. However, pioglitazone was also reported to increase the risk of hospitalization for HF over a 30-day period, even though patients at high risk of HF were unlikely to be prescribed the drug.21 Furthermore, clinical studies indicated that low dose pioglitazone treatment does not reduce the rate of in-stent restenosis, neointima volume nor atheroma volume in DM patients who have undergone percutaneous coronary intervention with drug-eluting stents.22 Beyond different methodologies applied in the discussed studies, reasons for contradictory reports on effects of pioglitazone on HF remain unclear. Investigating cardiovascular targets of pioglitazone is a promising approach for clarifying the effect of the drug on cardiovascular outcomes.

Effects of pioglitazone on the cardiovascular system have been previously reported. Pioglitazone attenuated monocrotaline-induced rat right ventricular hypertrophy and fibrosis and decreased cardiomyocyte size.23 Pioglitazone (2.5 mg/kg) ameliorated systolic and diastolic cardiac dysfunction in a rat model of Ang II-induced hypertension.24 Furthermore, pioglitazone protected from Ang II-induced cardiomyocyte hypertrophy by inhibiting AKT/GSK3β and MAPK signaling pathways. However, pioglitazone (40 mg/kg) was observed to induce cardiac hypertrophy with increase in plasma volume, without compromising it effects on the metabolic switch in the heart and whole-body insulin sensitivity.25 These contradicting findings may be caused by differences in administered doses of pioglitazone, as treatment with pioglitazone at supratherapeutic doses was shown to induce cardiotoxicity.26,27 Chemical proteomics-based analysis of off-target binding profiles for pioglitazone suggested potential sources contributing to efficacy and cardiotoxicity: perturbations in mitochondrial function, cardiac ion channels, and disruption of the cardiac sympathetic signaling.28 In the present study, pioglitazone induced cardiomyocyte apoptosis and inhibits cardiomyocyte hypertrophy. We inferred that these findings indicate that pioglitazone treatment appears disadvantageous in patients with HF. It was reported increased number of apoptotic cells in the heart of spontaneously hypertensive rats, suggesting that apoptosis might be a mechanism involved in the reduction of myocyte mass that accompanies the transition from stable compensation to HF in this model.29 Furthermore, cardiac myocyte apoptosis is a more critical determinant during the transition from compensatory cardiac hypertrophy to HF.30 However, available studies on the mechanisms underlying possible cardiovascular risk effects of pioglitazone on cardiovascular risk factors have been conducted in vitro conditions and therefore, prospective cohort studies are needed to confirm these effects.

In this study, reverse screening approaches (reverse docking and reverse pharmacophore mapping) were used to predict potential cardiovascular disease-related protein targets of pioglitazone. Pioglitazone was shown to bind strongly binding to VEGFR-2, suggesting that cardiovascular effects of pioglitazone may be related to the regulation of angiogenesis, neointima formation, and atherosclerosis associated with VEGFR-2-participating pathways.31-33 VEGFR-2 is a tyrosine kinase receptor that dimerizes upon ligand binding and is activated by trans-phosphorylation.33 VEGFR-2 activation stimulates downstream signaling, including activation of the c-Raf/MEK/ERK and PI3K/Akt pathways leading to increased cell proliferation, migration, and survival.33,34 VEGFR-2 is a critical factor in hypertrophic growth of cardiomyocytes.35,36 We found that pioglitazone directly targeted VEGFR-2 and inhibited phospho-VEGFR-2 expression, suggesting that pioglitazone induces cardiomyocyte apoptosis and inhibits cardiomyocyte hypertrophy in neonatal rats by inhibiting VEGFR-2 signaling. Downstream PI3K/Akt signaling pathway also participates in survival and hypertrophy of these cells by inhibiting P53-dependent pathways and activating mTOR-dependent pathways, respectively.37 In this study, pioglitazone and VEGFR-2 inhibitor apatinib increased phospho-P53 and Bax expression in cardiomyocytes and decreased phospho-Akt and phospho-mTOR expression in hypertrophic cardiomyocytes, indicating the connection between pioglitazone and the VEGFR-2 signaling pathway.

 Conclusion

In conclusion, these findings indicate that pioglitazone induces apoptosis and inhibits hypertrophy of cardiomyocytes in part by acting on the VEGFR-2 signaling pathway. These findings contribute to understanding cardiovascular risks of pioglitazone.

Sources of Funding

This study was funded by the Natural Science Foundation of Fujian Province Project (N°. 2010J01371).

Study Association

This article is part of the thesis of Post-Doctoral submitted by Wenliang Zhong, from Union Hospital of Fujian Medical University.

Ethics approval and consent to participate

This study was approved by the Ethics Committee on Animal Experiments of the First Hospital of Nanping City under the protocol number NP01371

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Yu L She T Li M Shi C Han L Cheng M Tetramethylpyrazine inhibits angiotensin II-induced cardiomyocyte hypertrophy and tumor necrosis factor-a secretion through an NF-kB-dependent mechanism Int J Mol Med 2013 32 3 717 722 19 Feoktistova M, Geserick P, Leverkus M. Crystal violet assay for determining viability of cultured cells. Cold Spring Harb Protoc. 2016;2016(4):pdb.prot087379. Feoktistova M Geserick P Leverkus M Crystal violet assay for determining viability of cultured cells Cold Spring Harb Protoc 2016 2016 4 pdb.prot087379 20 Tan A, Cao Y, Xia N, Mo Z, Gao F. The addition of pioglitazone in type 2 diabetics poorly controlled on insulin therapy: a meta-analysis. Eur J Intern Med. 2010;21(5):398-403. Tan A Cao Y Xia N Mo Z Gao F The addition of pioglitazone in type 2 diabetics poorly controlled on insulin therapy: a meta-analysis Eur J Intern Med 2010 21 5 398 403 21 Suh S, Seo GH, Jung CH, Kim MK, Jin SM, Hwang YC, et al. Increased risk of hospitalization for heart failure with newly prescribed dipeptidyl peptidase-4 inhibitors and pioglitazone using the korean health insurance claims database. Diabetes Metab J. 2015;39(3):247-52. Suh S Seo GH Jung CH Kim MK Jin SM Hwang YC Increased risk of hospitalization for heart failure with newly prescribed dipeptidyl peptidase-4 inhibitors and pioglitazone using the korean health insurance claims database Diabetes Metab J 2015 39 3 247 252 22 Lee HW, Lee HC, Kim BW, Yang MJ, Park JS, Oh JH, et al. Effects of low dose pioglitazone on restenosis and coronary atherosclerosis in diabetic patients undergoing drug eluting stent implantation. Yonsei Med J. 2013;54(6):1313-20. Lee HW Lee HC Kim BW Yang MJ Park JS Oh JH Effects of low dose pioglitazone on restenosis and coronary atherosclerosis in diabetic patients undergoing drug eluting stent implantation Yonsei Med J 2013 54 6 1313 1320 23 Behringer A, Trappiel M, Berghausen EM, Ten Freyhaus H, Wellnhofer E, Odenthal M, et al. Pioglitazone alleviates cardiac and vascular remodelling and improves survival in monocrotaline induced pulmonary arterial hypertension. Naunyn Schmiedebergs Arch Pharmacol. 2016;389(4):369-79. Behringer A Trappiel M Berghausen EM Ten Freyhaus H Wellnhofer E Odenthal M Pioglitazone alleviates cardiac and vascular remodelling and improves survival in monocrotaline induced pulmonary arterial hypertension Naunyn Schmiedebergs Arch Pharmacol 2016 389 4 369 379 24 Sakamoto A, Hongo M, Furuta K, Saito K, Nagai R, Ishizaka N. Pioglitazone ameliorates systolic and diastolic cardiac dysfunction in rat model of angiotensin II-induced hypertension. Int J Cardiol. 2013;167(2):409-15. Sakamoto A Hongo M Furuta K Saito K Nagai R Ishizaka N Pioglitazone ameliorates systolic and diastolic cardiac dysfunction in rat model of angiotensin II-induced hypertension Int J Cardiol 2013 167 2 409 415 25 Chang CS, Tsai PJ, Sung JM, Chen JY, Ho LC, Pandya K, et al. Diuretics prevent thiazolidinedione-induced cardiac hypertrophy without compromising insulin-sensitizing effects in mice. Am J Pathol. 2014;184(2):442-53. Chang CS Tsai PJ Sung JM Chen JY Ho LC Pandya K Diuretics prevent thiazolidinedione-induced cardiac hypertrophy without compromising insulin-sensitizing effects in mice Am J Pathol 2014 184 2 442 453 26 Elshama SS, El-Kenawy Ael-M, Osman HE. Toxicological evaluation of subchronic use of pioglitazone in mice. Iran J Basic Med Sci. 2016;19(7):712-9. Elshama SS El-Kenawy Ael-M Osman HE Toxicological evaluation of subchronic use of pioglitazone in mice Iran J Basic Med Sci 2016 19 7 712 719 27 Chinnam P, Mohsin M, Shafee LM. Evaluation of acute toxicity of pioglitazone in mice. Toxicol Int. 2012;19(3):250-4. Chinnam P Mohsin M Shafee LM Evaluation of acute toxicity of pioglitazone in mice Toxicol Int 2012 19 3 250 254 28 Hoffmann BR, El-Mansy MF, Sem DS, Greene AS. Chemical proteomics-based analysis of off-target binding profiles for rosiglitazone and pioglitazone: clues for assessing potential for cardiotoxicity. J Med Chem. 2012;55(19):8260-71. Hoffmann BR El-Mansy MF Sem DS Greene AS Chemical proteomics-based analysis of off-target binding profiles for rosiglitazone and pioglitazone: clues for assessing potential for cardiotoxicity J Med Chem 2012 55 19 8260 8271 29 Li Z, Bing OH, Long X, Robinson KG, Lakatta EG. Increased cardiomyocyte apoptosis during the transition to heart failure in the spontaneously hypertensive rat. Am J Physiol. 1997;272(5 Pt 2):H2313-9. Li Z Bing OH Long X Robinson KG Lakatta EG Increased cardiomyocyte apoptosis during the transition to heart failure in the spontaneously hypertensive rat Am J Physiol 1997 272 5 Pt 2 H2313 H2319 30 Hirota H, Chen J, Betz UA, Rajewsky K, Gu Y, Ross J Jr, et al. Loss of a gp130 cardiac muscle cell survival pathway is a critical event in the onset of heart failure during biomechanical stress. Cell. 1999;97(2):189-98. Hirota H Chen J Betz UA Rajewsky K Gu Y Ross J Jr Loss of a gp130 cardiac muscle cell survival pathway is a critical event in the onset of heart failure during biomechanical stress Cell 1999 97 2 189 198 31 Petrovan RJ, Kaplan CD, Reisfeld RA, Curtiss LK. DNA vaccination against VEGF receptor 2 reduces atherosclerosis in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol. 2007;27(5):1095-100. Petrovan RJ Kaplan CD Reisfeld RA Curtiss LK DNA vaccination against VEGF receptor 2 reduces atherosclerosis in LDL receptor-deficient mice Arterioscler Thromb Vasc Biol 2007 27 5 1095 1100 32 Bhardwaj S, Roy H, Babu M, Shibuya M, Yla-Herttuala S. Adventitial gene transfer of VEGFR-2 specific VEGF-E chimera induces MCP-1 expression in vascular smooth muscle cells and enhances neointimal formation. Atherosclerosis. 2011;219(1):84-91. Bhardwaj S Roy H Babu M Shibuya M Yla-Herttuala S Adventitial gene transfer of VEGFR-2 specific VEGF-E chimera induces MCP-1 expression in vascular smooth muscle cells and enhances neointimal formation Atherosclerosis 2011 219 1 84 91 33 Lohela M, Bry M, Tammela T, Alitalo K. VEGFs and receptors involved in angiogenesis versus lymphangiogenesis. Curr Opin Cell Biol. 2009;21(2):154-65. Lohela M Bry M Tammela T Alitalo K VEGFs and receptors involved in angiogenesis versus lymphangiogenesis Curr Opin Cell Biol 2009 21 2 154 165 34 Sarabipour S, Ballmer-Hofer K, Hristova K. VEGFR-2 conformational switch in response to ligand binding. Elife. 2016 Apr 7;5:e13876. Sarabipour S Ballmer-Hofer K Hristova K VEGFR-2 conformational switch in response to ligand binding Elife 2016 04 07 5 e13876 35 Masuda T, Muto S, Fujisawa G, Iwazu Y, Kimura M, Kobayashi T, et al. Heart angiotensin II-induced cardiomyocyte hypertrophy suppresses coronary angiogenesis and progresses diabetic cardiomyopathy. Am J Physiol Heart Circ Physiol. 2012;302(9):H1871-83. Masuda T Muto S Fujisawa G Iwazu Y Kimura M Kobayashi T Heart angiotensin II-induced cardiomyocyte hypertrophy suppresses coronary angiogenesis and progresses diabetic cardiomyopathy Am J Physiol Heart Circ Physiol 2012 302 9 H1871 H1883 36 Zheng L, Han P, Liu J, Li R, Yin W, Wang T, et al. Role of copper in regression of cardiac hypertrophy. Pharmacol Ther. 2015 Apr 18;148:66-84. Zheng L Han P Liu J Li R Yin W Wang T Role of copper in regression of cardiac hypertrophy Pharmacol Ther 2015 04 18 148 66 84 37 Song HK, Kim J, Lee JS, Nho KJ, Jeong HC, Kim J, et al. Pik3ip1 modulates cardiac hypertrophy by inhibiting PI3K pathway. PLoS One. 2015;10(3):e0122251. Song HK Kim J Lee JS Nho KJ Jeong HC Kim J Pik3ip1 modulates cardiac hypertrophy by inhibiting PI3K pathway PLoS One 2015 10 3 e0122251 Artigo Original Pioglitazona Induz Apoptose e Inibe Hipertrofia de Cardiomiócitos pela Via de Sinalização do VEGFR-2 Zhong Wenliang 1 2 Jin Wen 3 Xu Shanghua 1 Wu Yanqing 1 Luo Shunxiang 1 Liang Minlie 1 Chen Lianglong 2 Department of Cardiology, The First Hospital of Nanping City, affiliated to Fujian Medical University, Nanping, Fujian - China Department of Cardiology, Union Hospital, Fujian Medical University, Fuzhou, Fujian - China Cardiovascular Department, Guangdong N°.2 Provincial People’s Hospital, Guangzhou, Guangdong - China Correspondência: Wenliang Zhong, Department of Cardiology, The First Hospital of Nanping City, N°. 317 Zhongshan Road, Nanping, Fujian - China. E-mai: WL.Zhong@tom.com

Contribuição dos autores

Concepção e desenho da pesquisa: Zhong W, Chen L; Obtenção de dados: Zhong W, Jin W, Wu Y, Luo S, Liang M; Análise e interpretação dos dados e análise estatística: Zhong W, Jin W, Xu S, Wu Y, Luo S, Liang M; Obtenção de financiamento: Zhong W; Redação do manuscrito: Zhong W, Jin W, Liang M; Revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Zhong W, Xu S, Liang M.

Potencial conflito de interesses

Declaro não haver conflito de interesses pertinentes.

 Resumo Fundamento:

A pioglitazona tem sido amplamente utilizada como droga sensibilizadora da insulina para melhorar o controle glicêmico em pacientes com diabetes mellitus tipo 2. No entanto, o risco cardiovascular bem como os efeitos protetores da pioglitazona ainda são controversos.

 Objetivos:

Neste estudo, investigamos se os efeitos da pioglitazona sobre a apoptose e a hipertrofia de cardiomiócitos ocorrem via regulação da via de sinalização do VEGFR-2.

 Métodos:

os cardiomiócitos foram isolados enzimaticamente dos ventrículos de ratos Sprague-Dawley de 1-3 anos de vida. Os efeitos da pioglitazona e do inibidor seletivo de VEGFR-2 apatinib sobre a taxa de apoptose dos cardiomiócitos foram avaliados por citometria de fluxo, e a hipertrofia avaliada por incorporação de leucina-[3H]. As expressões de VEGFR-2, Akt, P53, e mTOR fosforiladas e não fosforiladas foram determinadas por Western Blotting. Análise de variância (ANOVA) foi usada para avaliar diferenças entre os grupos.

 Resultados:

a pioglitazona e o apatinib reduziram a viabilidade e a hipertrofia de cardiomiócitos induzida por angiotensina II in vitro. Além disso, no mesmo modelo in vitro, a pioglitazona e o apatinib aumentaram significativamente a expressão de Bax e P53 fosforilada, e diminuiu a expressão de VEGFR-2, Akt, e mTOR, que promove hipertrofia de cardiomiócitos.

 Conclusões:

Esses resultados indicam que a pioglitazona induz a apoptose e inibe a hipertrofia de cardiomiócitos pela modulação da via de sinalização de VEGFR-2.

 Palavras-chave: Apoptose Miócitos Cardíaco Cardiomegalia Insuficiência Cardíaca/fisiopatologia Anti-Hipertensivos Tiazolidinedionas Resistência à Insulina Introdução

A insuficiência cardíaca (IC) é a consequência mais comum das doenças cardiovasculares e a principal causa de mortalidade em todo o mundo.1 A base da fisiopatologia da IC é o remodelamento cardíaco, que envolve um número de mudanças celulares incluindo hipertrofia de cardiomiócitos, perda de cardiomiócitos por apoptose, necrose, proliferação de fibroblasto e fibrose.2 Estudos clínicos recentes demonstraram que o diabetes mellitus (DM) provoca remodelamento cardíaco, incluindo hipertrofia do miocárdio e perda de cardiomiócitos via glicotoxicidade e lipotoxicidade, resultando em IC.3 Foi demonstrado que um controle rigoroso da glicemia reduz a ocorrência de eventos cardiovasculares importantes incluindo a IC, mas não melhora a taxa de sobrevida global de pacientes com DM tipo 2 em comparação a pacientes que recebem tratamento padrão.3 As tiazolidinedionas, incluindo a pioglitazona, são amplamente utilizadas no controle glicêmico de pacientes com DM tipo 2 como agonistas de receptores ativados por proliferador de peroxissomo (PPAR)-γ e como agentes que melhoram a sensibilidade à insulina. No entanto, os riscos cardiovasculares da pioglitazona ainda são controversos.

Uma linha afirma que o uso de pioglitazona ou rosiglitazona para controle glicêmico aumenta o risco de IC (OR ≤ 2,1; IC95% 1,08-4,08) com base em metanálises de ensaios clínicos randomizados.4-6 A rosiglitazona apresentou maior chance de induzir à IC em comparação à pioglitazona.7 Além disso, estudos experimentais confirmaram o risco aumentado de IC com tratamento com pioglitazona, uma vez que seu uso aumentou os danos cardíacos em um modelo de IC com ratos induzida por isoproterenol e levou à hipertrofia ventricular nos animais em experimentos sobre toxicidade aguda.8,9 Uma outra linha defende que o uso de pioglitazona não aumenta significativamente o risco de infarto do miocárdio ou mortalidade por doença cardíaca, baseando-se em dados do estudo PROspective pioglitAzone Clinical Trial In macroVascular Events (PROactive),10 e que a pioglitazone é capaz de suprimir a hipertrofia cardíaca induzida por sobrecarga por meio da inibição da AKT/GSK3β e de vias de sinalização da MAPK.11

Receptores do fator de crescimento endotelial vascular (VEGF) são considerados críticos para hipertrofia cardíaca e IC. Três diferentes subtipos foram descritos- VEGFR-1, VEGFR-2, e VEGFR-3. Estudos recentes mostraram que o VEGFR-1 e o VEGFR-2 são essenciais na regressão e na indução de hipertrofia dos cardiomiócitos, respectivamente,12 ao passo que o VEGFR-3 mostrou-se benéfico no miocárdio infartado por promover hipertrofia dos cardiomiócitos e melhor sobrevida.13 Ainda, o VEGFR-2 está envolvido na fase tardia da ruptura da barreira endotelial (células endoteliais da artéria pulmonar e células endoteliais da aorta) causada por altos níveis de produtos de oxidação do 1-palmitoil-2-araquidonil-sn-glicero-3-fosfatidilcolina, além de contribuir para a formação de fibras de estresse e aumentar a fosforilação de cadeias leves de miosina.14 A pioglitazona reduziu a expressão de VEGFR-2 em órgãos esplâncnicos e inibiu a neoangiogênese em um modelo de hipertensão portal com ratos,15 sugerindo um efeito direto sobre a expressão de VEGFR-2. Abordagens com rastreamento reverso - mapeamento farmacofórico reverso e docagem (docking) reversa são métodos importantes para identificar novas proteínas alvo para pioglitazona, relacionadas a doenças cardiovasculares. Em nosso estudo, utilizamos o PharmMapper para mapeamento farmacofórico reverso. A estrutura da pioglitazona em formato mol2 foi submetida ao PharmMapper, obtendo-se dez alvos e poses, classificados em ordem decrescente do escore de ajuste (fit score). Entre os dez compostos com maiores escores, o VEGFR-2 foi o alvo potencial mais bem ranqueado, dado que será essencial para se compreender a interação entre a droga e o VEGFR-2.

Dada a potencial ligação entre a pioglitazona e o VEGFR-2 e seus papeis na hipertrofia e apoptose de cardiomiócitos e na fisiopatologia da IC, nós investigamos, pela primeira vez, se a pioglitazona afeta a hipertrofia e apoptose de cardiomiócitos por regulação da via de sinalização do VEGFR-2. Além disso, espera-se que essa seja uma abordagem promissora para esclarecer o mecanismo potencial do efeito da pioglitazona sobre desfechos cardiovasculares.

 Métodos

Todos os experimentos envolvendo animais foram aprovados pelo comitê para uso e cuidado de animais (Institutional Animal Care and Use Committee, IACU) do Primeiro Hospital da cidade de Nanping.

Preparação das moléculas

Para caracterizar os sítios de ligação na identificação de proteínas-alvo, utilizamos o programa Genetic Optimization for Ligand Docking (GOLD) versão 5.3. A estrutura cristalográfica do inibidor do VEGFR-2 (Banco de Dados de Proteínas, PDB, ID:3CP9) foi selecionada como a estrutura inicial 2. O GOLD utiliza um algoritmo genético para ancorar os ligantes no sódio de ligação de proteínas alvo, com flexibilidade conformacional total do ligante e flexibilidade parcial do receptor. A energia de ligação do ligante foi predita pela função ChemScore e energia livre de ligação (ΔG) do GOLD.16 As moléculas solventes foram removidas da estrutura cristalina e átomos de hidrogênio da proteína foram adicionados. Para a definição dos sítios de ligação, estabeleceu-se que a inclusão de aminoácidos localizados dentro de 10 Å das coordenadas do inibidor na estrutura cristalina. As dez principais poses ancoradas foram obtidas finalizando a simulação uma vez que a raiz do erro médio quadrático (do inglês Root Mean Square Deviation, RMSD) entre qualquer pose de ligante fosse atingido. Os resultados foram visualizados utilizando-se o PyMol versão 1.3 e o LigPlot+ versão 1.4.

 Isolamento e cultura de cardiomiócitos de ratos neonatos

Os cardiomiócitos foram isolados enzimaticamente de ventrículos de ratos Sprague-Dawley com 1 a 3 dias de idade conforme descrito previamente.17 Doze ratos foram utilizados para a realização de 12 experimentos independentes. Os cardiomiócitos isolados foram semeados em placas de culturas celulares cobertas com 10 g/mL de fibronectina e cultivadas em um meio contendo DMEMF-12 com HEPES (Invitrogen, Carlsbad, CA, EUA), 5% de soro de cavalo inativado pelo calor, 100 U/mL de penicilina, 10 µg/mL de estreptomicina, 3 mM de ácido pirúvico, 2 mg/mL de albumina sérica bovina, 100 g/mL de ampicilina, meio insulina-transferrina-selênio (Sigma, St. Louis, MO, EUA), 5 g/mL ácido linoleico, e 100 M de ácido ascórbico a 37°C em uma atmosfera umidificada com 5 % CO2. Para todos os experimentos, as células foram cultivadas a 5 × 104 células/cm2 (ou outro valor especificado, se o caso).

 Ensaio de proliferação celular

Efeitos de pioglitazona e do inibidor seletivo de VEGFR-2 apatinib (Apexbio Technology LLC, Houston, TX, EUA) sobre a proliferação celular foram medidos por contagem de células coradas por cristal violeta 24 horas após o tratamento, por um contador automático (BioRad). Resumidamente, os cardiomiócitos (5x104 células/poço) foram semeados em placas de 96 poços e cultivados em um meio padrão por 24 horas. Após 24 horas sem soro, os cardiomiócitos foram tratados com angiotensina 0,1 µM (Ang) II por 24 h. Pioglitazona (0, 10 ou 20µM) e inibidor seletivo de VEGFR-2 apatinib (2 µM) foi adicionado ao meio de cultura 2 horas antes da administração de Ang II. Para a marcação com cristal violeta, as células foram lavadas duas vezes com tampão fosfato salina, fixados com metanol 20% por 30 minutos, e corados com solução de cristal violeta 0,2% por 30 minutos à temperatura ambiente sob leve agitação. As células coradas foram lavadas até clara visualização do fundo. O corante cristal violeta foi extraído utilizando-se SDS 1% e as células foram contadas utilizando-se um contador automático.

 Detecção de apoptose por citometria de fluxo

A taxa de apoptose foi determinada por citometria de fluxo com corante anexina V-FITC (AV) combinada com iodeto de propídio (IP). Em resumo, após o tratamento, as células (1-5 × 105/ml) foram coletadas, lavadas duas vezes com tampão salina fostato, ressuspendidas em 500 µL de tampão de ligação. Em seguida, as células foram incubadas com 10 µL de AV e 5 µL de PI em sala escura e temperatura ambiente por 15 minutos. As células apoptóticas foram identificadas por CMF dentro de 30 minutos.

 Hipertrofia dos cardiomiócitos

Os cardiomiócitos foram semeados a 5 × 104 células por poço em placas de 96 poços e a hipertrofia foi avaliada por incorporação de [3H]-leucina, conforme descrito anteriormente.18 Em resumo, 2 horas após o tratamento com pioglitazona (0-20 µmoL/L) e apatinib (2 µM), Ang II 0,1 µM foi usada para estimular a hipertrofia dos cardiomiócitos, e 1 µCi [3H]-leucina foi simultaneamente adicionada a cada poço. Após estimulação com AngII por 60 horas, as células foram colhidas por precipitação com ácido tricloroacético a 10% em gelo por 30 minutos, antes de ser solubilizado com NaOH 1 moL/L de um dia para outro a 4ºC. As amostras foram neutralizas com HCl 1 moL/L e os níveis de [3H] foram determinados em solução de cintilação usando um contador (beta) para avaliar a incorporação de [3H]-leucina.

 Análise por Western Blotting

Células tripsinizadas foram lisadas em tampão de radioimunoprecipitação, homogeneizadas em gelo, e centrifugadas. O sobrenadante foi diluído com SDS-PAGE 10% e transferido para membranas PVDF (Millipore). As membranas foram bloqueadas com salina tamponada (TBS) contendo 5% de leite desnatado e incubadas com os seguintes anticorpos primários a 4ºC de um dia para o outro: VEGFR-2 (ab39256; 1:1000; Abcam), fosfo-VEGFR-2 (Tyr1175) (#2478; 1:1000; Cell Signaling Technology), Akt (#9272; 1:1000; Cell Signaling Technology), fosfo-Akt (Thr308) (#9275; 1:1000; Cell Signaling Technology), P53 (#9282; 1:1000; Cell Signaling Technology), fosfo-P53 (Ser15) (#9284; 1:1000; Cell Signaling Technology), Bax (#14796; 1:1000; Cell Signaling Technology), anti-mTOR de coelho (#2972; 1:1000; Cell Signaling Technology), anti-mTOR fosforilado de coelho (Ser2448) (#5536; 1:1000; Cell Signaling Technology), e GAPDH (#2118, 1:1000; Cell Signaling Technology). No dia seguinte, as membranas foram lavadas três vezes com TBS com tween (TBST) por 5 minutos em temperatura ambiente e depois incubadas com anticorpos secundários anti-IgHG de coelhos por 1 hora à temperatura ambiente. Após a incubação, as membranas foram lavadas com TBST e expostas a um filme de raio X. As intensidades da banda no filme foram analisadas por densitometria, e os resultados normalizados para β actina. A proteína GAPDH foi usada como controle de carga.

 Análise estatística

A análise estatística foi realizada utilizando-se o programa SPSS 13.0. O teste Kolmogorov Smirnov foi usado para se testar a normalidade da distribuição dos dados e o teste de Levene para avaliar a homogeneidade da variância. Todos os dados apresentaram distribuição normal e a homogeneidade da variância foi apresentada como média ± DP. O teste ANOVA de um fator (one-way ANOVA) foi usado para comparações entre vários grupos, e o teste de Bonferroni usado para comparações pareadas. Um valor de p < 0,05 indicou significância estatística.

 Resultados VEGFR-2 como melhor alvo potencial para pioglitazona

Entre os dez compostos que apresentaram melhor pontuação do PharmMaper, o VEGFR-2 foi o que apresentou maior pontuação. Para entender a interação entre o pioglitazona e o VEGFR-2 e avaliar a energia de ligação, realizamos um estudo de docagem (docking) utilizando o GOLD. A conformação ótima de ligação do complexo pioglitazona-VEGFR-2 está apresentada nas Figuras 1A e 1B. O escore ChemScore e a energia de ligação do complexo pioglitazona-VEGFR-2 foram comparáveis à complexa estrutura cristalográfica do inibidor de VEGFR-2. Foram previstas interações de van der Waals da pioglitazona com Val363, Leu428, Cys454, Leu444, Leu310, Phe456, Gly387, Phe383, Val364, e Ile453, e sua ligação com Cys384 e Asp455 por ligações de hidrogênio (Figura 1B). A técnica de Western Blotting foi realizada para avaliar o efeito da pioglitazona sobre a expressão do VEGFR-2 e do fosfo-VEGFR-2 em cardiomiócitos de ratos neonatos de modo dose-dependente (Figura 1 C).

O VEGFR-2 é o alvo potencial para a pioglitazona. (A) modelo de docagem (docking) molecular 3D da pioglitazona com VEGFR-2; estrutura verde: conformador da pioglitazona. (B) modelo de docking molecular 2D da pioglitazona com VEGFR-2. Ligações de hidrogênio entre a pioglitazona e o VEGFR-2 estão indicadas com linha verde. (C e D) Western Blot dos níveis de VEGFR-2 e fosfo-VEGFR-2 (Tyr1175) de cardiomiócitos de ratos neonatais sob estímulo hipertrófico, tratados com 0, 10, 20 (µM) de pioglitazona por 24 houras, e (C) intensidade das bandas normalizada para β-actina (n = 12 em cada grupo). Todos os dados são apresentados em média ± DP. Comparações com o grupo controle: *p < 0,01 com grupo pioglitazona 10 µM; # p < 0,01, calculado por one-way ANOVA e teste post hoc de Bonferroni para comparações pareadas.

A pioglitazona promoveu apoptose e inibiu a hipertrofia dos cardiomiócitos induzida pela Ang II

Para validar os efeitos da pioglitazona, foram avaliadas a viabilidade do cardiomiócitos e da hipertrofia induzida pela Ang II. O corante cristal violeta, um teste rápido e versátil para se detectar a viabilidade celular sob diversas condições de estímulo,19 foi usado para avaliar a viabilidade celular. A pioglitazona inibiu a viabilidade dos cardiomiócitos de modo dose dependente (Figuras 2A e 2B, p < 0,01), com concentrações efetivas variando de 0 a 20 µmoL/L. O inibidor seletivo para VEGFR-2 apatinib também inibiu a viabilidade dos cardiomiócitos - (Figuras 2A e 2B, p < 0,01). A taxa de apoptose dos cardiomiócitos foi determinada pela técnica de marcação com corante AV/PI. As taxas apoptóticas nos grupos pioglitazona 20 µM, pioglitazona 10 µM, e apatinib 2 µM foram significativas maiores que no grupo controle (Figuras 2C e 2D, p < 0,01). A incorporação de [3H]-leucina induzida por Ang II foi significativamente menor após o tratamento com pioglitazona ou com apatinib, indicando que ambos inibiram a hipertrofia de cardiomiócitos.

A pioglitazona e o apatinib induziram hipertrofia de cardiomiócitos de ratos neonatais (n = 12 em cada grupo). (A) Coloração com cristal violeta em resposta a várias concentrações de pioglitazona e apatinib. Ambos inibiram a viabilidade de cardiomiócitos de ratos neonatais. (B) A proliferação celular apatinib foi determinada usando contador celular automático. (C e D) A pioglitazona e o apatinib induziram apoptose de cardiomiócitos de ratos neonatais, detectado por citometria de fluxo com coloração com anexina V (AV) combinada com iodeto de propídio. A pioglitazona (10, 20 µM) e o apatinib (2 µM) aumentaram a apoptose de cardiomiócitos em comparação ao grupo c ontrole. (E) Incorporação de leucina-[<sup>3</sup>H] induzida por angiotensina II em reposta a diferentes concentrações de pioglitazona ou apatinib. Todos os dados são apresentados em média ± DP. *p < 0,01 em comparação ao grupo controle; # p < 0,01, em comparação à pioglitazona 10 µM, calculados por one-way ANOVA seguido do teste de Bonferroni para comparações pareadas.

A pioglitazona inibiu a hipertrofia e promoveu a apoptose de cardiomiócitos pela supressão da sinalização do VEGFR-2

Os mecanismos potenciais da inibição induzida da hipertrofia dos cardiomiócitos pela pioglitazona e a promoção de apoptose dessas células foram avaliados in vitro. Em comparação aos cardiomiócitos sob condições controle, a pioglitazona aumentou significativamente a expressão do Bax e fosfo-P53 e diminuiu a expressão do fosfo-VEGFR-2 em cardiomiócitos de ratos neonatos sob estímulo hipertrófico (Figuras 1C e 3).

A pioglitazona e o apatinib regulam a sinalização de VEGFR-2 em cardiomiócitos de ratos neonatais após hipertrofia induzida por Angiotensina II (n = 12 em cada grupo). (A e B) Western blot dos níveis de fosfo-VEGFR-2, VEGFR-2, fosfo-mTOR, mTOR, fosfo-Akt, Akt, Bax, fosfo-P53 e P53 em cardiomiócitos de ratos neonatais sob estímulo hipertrófico e tratados com pioglitazona (20 µM) ou apatinib (2 µM) por 24 horas, e (A) intensidade das bandas normalizadas para GAPDH. Todos os dados são apresentados em médias ± DP. *p < 0,01 em comparação ao grupo controle; # p < 0,01, em comparação à pioglitazona 20 µM, calculados por one-way ANOVA seguido do teste de Bonferroni para comparações pareadas.

Para avaliar se o VEGFR-2 é alvo da pioglitazona, os efeitos da droga sobre a expressão de VEGFR-2 e sinalização intracelular regulada pelo VEGFR-2 foram determinados em cardiomiócitos de ratos neonatos sob indução de hipertrofia pela Ang II. Em comparação aos cardiomiócitos hipertróficos não tratados, a pioglitazona diminuiu significativamente a expressão de fosfo-VEGFR-2, fosfo-Akt, e fosfo-mTOR, o que pode contribuir para a hipertrofia dos cardiomiócitos. Além disso, o apatinib reduziu significativamente a expressão de VEGFR-2, fosfo-VEGFR-2, fosfo-Akt, e fosfo-mTOR (Figura 1C, Figura 3).

 Discussão

No presente estudo, mostramos que a pioglitazona reduziu a viabilidade dos cardiomiócitos e a hipertrofia induzida por Ang II, aumentou a expressão de Bax e P53 fosforilada, e diminuiu a expressão de VEGFR-2, Akt, e mTOR fosforilados in vitro. Esses achados sugerem que a pioglitazona induz a apoptose e inibe a hipertrofia de cardiomiócitos por seus efeitos na via de sinalização de VEGFR-2.

A pioglitazona tem sido amplamente utilizada no controle glicêmico em pacientes com DM tipo 2. O estudo PROactive mostrou que a pioglitazona reduziu o principal desfecho secundário composto, o qual englobou morte por doença cardiovascular, infarto do miocárdio e acidente vascular cerebral em comparação a placebo em 43% da população do estudo.10 Esses achados indicam que a pioglitazona melhora a função vascular em pacientes diabéticos e em pacientes não diabéticos com resistência à insulina, e sugerem um possível efeito benéfico do tratamento com pioglitazona no prognóstico desses pacientes. Além disso, uma metanálise mostrou que a suplementação da terapia insulínica com pioglitazona em pacientes com DM tipo 2 e controle deficiente dos níveis glicêmicos contribuiria para a redução desses níveis bem como da dosagem de insulina, sem aumentar os riscos de infarto do miocárdio, IC, morte por doença cardíaca e morte por todas as causas, porém, com aumento nos níveis de colesterol total e nos riscos de ocorrência de hipoglicemia e edema.20

Com base nas evidências disponíveis, o tratamento com pioglitazona parece ser vantajoso em pacientes com IC. Contudo, também foi relatado que a pioglitazona aumenta o risco de hospitalização por IC por uma período maior que 30 dias, apesar de a probabilidade de se prescrever pioglitazona a pacientes com alto risco de IC ser menor.21 Além disso, estudos clínicos indicaram que o tratamento com baixas doses de pioglitazona não reduz a taxa de reestenose intra-stent, volume neointimal ou volume do ateroma em pacientes com DM que foram submetidos à intervenção coronária com stents liberadores de droga.22 Além das diferentes metodologias aplicadas nesses estudos, as razões para os resultados controversos sobre o efeito da pioglitazona na IC ainda não claras. Por isso, a investigação de alvos cardiovasculares da pioglitazona é uma abordagem promissora para esclarecer o efeito da droga sobre desfechos cardiovasculares.

Os efeitos da pioglitazona sobre o sistema cardiovascular foram previamente publicados. Em ratos, a pioglitazona atenuou a hipertrofia ventricular direita induzida por monocrotalina e fibrose, e diminuiu o tamanho dos cardiomiócitos.23 A pioglitazona (2,5 mg/Kg) melhorou a disfunção cardíaca sistólica e diastólica em um modelo de hipertensão induzida por Ang II em ratos.24 Além disso, a pioglitazona preveniu a hipertrofia de cardiomiócitos induzida por Ang II inibindo-se as vias de sinalização de AKT/GSK3β e MAPK. No entanto, a pioglitazona (40 mg/Kg) induziu a hipertrofia cardíaca com aumento do volume plasmático, sem afetar seus efeitos cardíacos metabólicos e sobre a sensibilidade insulínica em todo o corpo.25 Esses achados contrastantes podem ser causados por diferenças nas doses de pioglitazona utilizadas, uma vez que o tratamento com essa droga em doses supraterapêuticas induz cardiotoxicidade.26,27 Análise proteômica dos perfis de ligação off-target para a pioglitazona sugeriu fontes potenciais de eficácia e de cardiotoxicidade - distúrbios na função mitocondrial e nos canais de íons cardíacos, e ruptura na sinalização simpática cardíaca.28 No presente estudo, a pioglitazona induziu a apoptose e inibiu a hipertrofia dos cardiomiócitos, o que indica, a nosso ver, que o tratamento com pioglitazona não é vantajoso em pacientes com IC. Foi relatado um número aumentado de células apoptóticas no coração de ratos espontaneamente hipertensos, sugerindo que a apoptose pode ser um mecanismo envolvido na redução da massa cardíaca, que acompanha a transição de um estado de compensação estável para IC nesse modelo.29 Ainda, a apoptose de cardiomiócitos é um fator determinante ainda mais crítico na transição de uma hipertrofia cardíaca compensatória à IC.30 Contudo, estudos sobre os possíveis mecanismos da pioglitazona sobre fatores de risco cardiovasculares foram conduzidos, até o momento, em condições in vitro e, por isso, estudos coortes prospectivos são necessários para confirmar tais efeitos.

Neste estudo, abordagens de rastreamento reverso (docking e mapeamento farmacofórico) foram utilizadas para identificar potenciais proteínas alvo da pioglitazona, relacionadas com doença cardiovascular. A pioglitazona pode estar relacionada com a regulação da angiogênese, formação de neoíntima, e aterosclerose associadas com vias que envolvem o VEGFR-2.31-33 O VEGFR-2 é um receptor de tirosina quinase que se dimeriza ao se ligar ao composto ligante e é ativado por trans-fosforilação.33 A ativação do VEGFR-2 estimula sinalização downstream, incluindo a ativação de vias de c-Raf/MEK/ERK e PI3K/Akt, levando ao aumento de proliferação, migração e sobrevida das células.34,35 Em nosso estudo, identificamos que a pioglitazona atuou diretamente no VEGFR-2 e inibiu a expressão de fosfo-VEGFR-2, indicando que a pioglitazona induz a apoptose e inibe a hipertrofia de cardiomiócitos35,36 de ratos neonatais por meio da inibição da sinalização do VEGFR-2. A via de sinalização downstream do PI3/Akt também tem participação na sobrevida e hipertrofia dessas células, por inibição de vias dependentes de P53 e ativação de vias dependentes de m-TOR, respectivamente.37 Neste estudo, a pioglitazona e o inibidor de VEGFR-2 apatinib aumentaram a expressão de fosfo-P53 e Bax nos cardiomiócitos e diminuíram a expressão de cardiomiócitos hipertróficos, indicando a ligação entre a pioglitazona e a via de sinalização do VEGFR-2.

 Conclusão

Os achados aqui apresentados indicam que a pioglitazona induz a apoptose e inibe a hipertrofia de cardiomiócitos, em parte por ativação da via de sinalização do VEGFR-2. Esses achados contribuem para o entendimento dos riscos cardiovasculares da pioglitazona.

Fontes de financiamento

O presente estudo foi financiado pelo Natural Science Foundation of Fujian Province Project (No. 2010J01371).

Vinculação acadêmica

Este artigo é parte de tese de Pós-Doutorado de Wenliang Zhong pela Union Hospital of Fujian Medical University.

Aprovação ética e consentimento informado

Este estudo foi aprovado pela Comissão de Ética em Experimentação Animal do First Hospital of Nanping City sob o número de protocolo NP01371.