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 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 [ 3 H] levels were determined in scintillation fluid using a β counter to assess [ 3 H]- 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). 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 FCMwith 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 [ 3 H]-leucine incorporation was significantly decreased after pioglitazone or apatinib treatment, indicating that both inhibited cardiomyocyte hypertrophy. 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). 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 164