ABC | Volume 111, Nº2, August 2018

Original Article Zhong et al Pioglitazone and VEGFR-2 signaling pathway Arq Bras Cardiol. 2018; 111(2):162-169 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% CO 2 . For all experiments, cells were cultured at 5 × 10 4 cells/cm 2 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 × 10 4 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 × 10 5 /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 5×10 4 cells/well in 96-well plates and hypertrophy was evaluated using [ 3 H]‑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 [ 3 H]-leucine was simultaneously added 163