Fig 1: Cyclic mechanical stretch-induced VEGF secretion is independent of the MAPK/ERK1/2 or PI3K pathways.Inhibition of either the MAPK/ERK1/2 or PI3K signaling pathways in isolated ARCMs did not block VEGF secretion induced by cyclic mechanical stretch. Isolated ARCMs attached to laminin for 24 h were treated with the ERK1/2 kinase inhibitor (U0126; 0.5 µM) or DMSO-carrier control and subjected to (A) 24 h or 48 h of cyclic mechanical stretch (10% stretch) or no stretch (0% stretch). ELISA was used to determine the amount of VEGF secreted into the media. (B) The ERK1/2 kinase inhibitor was active in isolated primary ARCMs as pERK1/2 levels decreased in cells treated with the ERK1/2 kinase inhibitor (U0126; 0.5 µM) compared to the DMSO-carrier control. Stretch-activated VEGF secretion is independent of PI3K. ARCMs attached to laminin for 24 h were treated with the PI3K inhibitor (LY294002; 0.5 µM) or DMSO-carrier control and subjected to 24 h or 48 h (C) of cyclic mechanical stretch (10% stretch) or no stretch (0% stretch). ELISA was used to determine the amount of VEGF secreted into the media. (D) The PI3K inhibitor (LY294002; 0.5 µM) was active in isolated ARCMs as pAKT levels decreased in cells treated with the PI3K inhibitor (LY294002; 0.5 µM) compared to the DMSO-carrier control. Relative intensity of phospho-ERK1/2/total ERK1/2 (pERK/tERK) or phospho-AKT/total AKT (pAKT/tAKT) was determined. (E) ELISA was used to determine the amount of BNP secreted into the media. Treatment with the either ERK1/2 (U0126) or PI3K (LY294002) inhibitors blocked BNP secretion at 24 hours compared to DMSO-carrier control. ***P<0.001; One-Way ANOVA with a Bonferroni post-test was used to determine the statistical significance of data. n.s. = not significant. The values represent the average of three independent experiments.