VEGF expression is enhanced in the myocardium and in cardiomyocytes upon pressure overload-induced hypertrophy in vivo.

<p>Immunostaining for VEGF using a polyclonal anti-VEGF antibody in paraffin fixed heart tissue from a pressure-overload hypertrophy model or sham control at day 10. Box =  higher magnification inset. Representative of three independent experiments.</p

Cyclic mechanical stretch-induced VEGF secretion is independent of the MAPK/ERK1/2 or PI3K pathways.

<p>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 (<b>A</b>) 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>B</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 (<b>C</b>) 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>D</b>) 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. (<b>E</b>) 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. ***<i>P</i><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.</p

Cyclic mechanical stretch activates the NFkB, MAPK/ERK1/2 and PI3K pathways in ARCMs.

<p><b>A</b>) Isolated ARCMs attached to laminin were subjected to 24 h of cyclic mechanical stretch (10% stretch). Non-stretched control (0% stretch) ARCMs attached to laminin were incubated under identical conditions. Cells were then lysed, fractionated into cytosolic (C) and nuclear (N) fractions and immunoblotted with an anti-NFkB p65 antibody. In stretched ARCMs, the nuclear fraction contained significantly more of the NFkB p65 subunit compared to the non-stretched controls. Relative intensity of nuclear to cytoplasmic (N/C) fraction was determined. An antibody to the anti-TATA-binding protein (TBP) was used to determine fractionation efficiency. Cyclic mechanical stretch for 24 h induced a significant increase in MAPK/ERK1/2 and PI3K activity in isolated ARCMs attached to laminin. Isolated ARCMs were allowed to attach to laminin for 24 h and subsequently subjected to 24 h of cyclic mechanical stretch (10% stretch) or no stretch (0% stretch). Cells were lysed and immunoblotted for (<b>B</b>) ERK1/2 activity using an antibody against phospho-ERK1/2 (pERK) or a total ERK1/2 antibody (tERK) or for (<b>C</b>) PI3K activity via an antibody against phospho-AKT (pAKT) or total AKT (tAKT). Relative intensity of pERK/tERK or pAKT/tAKT was determined. *<i>P</i><0.05; ***<i>P</i><0.001; One-Way ANOVA with a Bonferroni post-test was used to determine the statistical significance of data. The values represent the average of three independent experiments.</p

Expression of a dominant negative mutant IkBα blocks cyclic mechanical stretch-induced VEGF secretion in a dose-dependent manner.

<p>Primary ARCMs transduced with a recombinant adenovirus encoding an IkBα dominant negative mutant (DN IkBα) were cultured on laminin for 24 h and then subjected to 24 h of cyclic mechanical stretch (10% stretch). Non-stretched control cells (0% stretch) were incubated under identical conditions. ELISA was used to determine VEGF levels secreted into the culture media at 24 h (<b>A</b>). (<b>B</b>) Expression of the IkBα dominant negative mutant blocks cyclic mechanical stretch-induced VEGF secretion in a dose-dependent manner. ELISA was used to analyze the concentration of VEGF in the media. (<b>C</b>) The IkBα dominant negative mutant was expressed in isolated ARCMs as IkBα levels were significantly increased in ARCMs expressing the mutant compared to control ARCMs. (<b>D</b>) The IkBα dominant negative mutant was active in isolated ARCMs as p65 levels were significantly decreased in the nucleus of ARCMs expressing the mutant compared to control ARCMs. ***<i>P</i><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. (<b>E</b>) Representative ChIP assay PCR showing hypertrophic stretch increases NFkB binding to the native VEGF promoter in ARCMs. Protein-DNA complexes were immunoprecipitated with an NFkB p65 antibody followed by DNA isolation and purification and PCR. Non-immunoprecipitated chromatin was used as an “input” control (-). ARMCs subjected to 24 h stretch (10%) resulted in increased binding of NFkB to the VEGF promoter over 0% stretch.</p

Cyclic mechanical stretch induces VEGF secretion in primary ARCMs.

<p><b>A</b>) Cyclic mechanical stretch for either 24 h or 48 h induces a significant increase in VEGF secretion in primary ARCMs attached to laminin compared to non-stretched controls. Stretched (10% stretch) and non-stretched control (0% stretch) cells were allowed to adhere to laminin for 24 h prior to initiating experiments. VEGF concentration in the conditioned media of non-stretched and stretched ARCMs was analyzed by ELISA. ***<i>P</i><0.001; One-Way ANOVA with a Bonferroni post-test was used to determine the statistical significance of data. The values represent the average of three independent experiments. <b>B</b>) Isolated ARCMs remain viable in culture when attached to laminin. Cell cultures were examined for their ability to reduce MTT after 24 h or 48 h in culture. Fold induction in relative mitochondrial activity represents the amount of viable cells at each time point. ***<i>P</i><0.001; One-Way ANOVA with a Bonferroni post-test was used to determine the statistical significance of data. <b>Insert:</b> The isolation procedure yielded a >90% pure ARCM population. Phase contrast image of isolated, primary ARCMs 24 h after binding to laminin demonstrate that ARCM differentiated morphology is maintained as indicated by the rod-shaped, branched striated cell examined at 40X magnification.</p