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.