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 Figure 2 – 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, 20μM) and Apatinib (2μM) increased the apoptosis of the cardiomyocytes compared with the control group. E, Angiontensin II-induced [ 3 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 μM group, calculated by one-way ANOVA followed by the post hoc Bonferroni test for pairwise comparisons. Control Control Control Pioglitazone 10 µM Pioglitazone 10 µM Pioglitazone 10 µM Pioglitazone 20 µM Pioglitazone 20 µM Pioglitazone 20 µM Apatinib 2 µM Apatinib 2 µM Control Pioglitazone 10 µM Pioglitazone 20 µM Apatinib 2 µM Control Pioglitazone 10 µM Pioglitazone 20 µM Apatinib 2 µM Apatinib 2 µM PI Annexin V A B C D E * * * *# *# *# *# *# *# Cell counting (x 10 4 ) Apoptotic cells (%) 3 H-Leucine incorporation (cpm/well) 3 2 1 0 40 30 20 10 0 1000 800 600 400 200 0 hypertension. 24 Furthermore, pioglitazone protected from Ang II-induced cardiomyocyte hypertrophy by inhibiting AKT/ GSK3 β 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 166