Supplementary Material for: Senescence of Cultured Porcine Coronary Arterial Endothelial Cells Is Associated with Accelerated Oxidative Stress and Activation of NFĸB

Aims: Endothelial dysfunction occurs following multiple passaging in vitro,but the molecular mechanisms involved remain unidentified. The present study defined the genomic changes related to dysfunction in cultured senescent endothelial cells. Methods and Results: Senescent cells were produced by multiple passaging of porcine coronary arterial endothelial cells for up to 4 weeks. Genomic and proteomic studies on cultured cells at the first passage (P1) and the fourth passage (P4) were performed. Senescence and decreased NO production were observed in cells and several signaling pathways – such as IFN/STAT, IGF, TGF-β, cytoskeleton rearrangement and lipid metabolism – were altered at P4, as judged from the microarray analysis. The basal and stimulated (by TNF-α) levels of NFĸB were augmented in senescent cells in electrophoretic mobility shift assays in association with increased oxidative stress, increased p53 protein stability, and activated apoptotic pathways. The increased oxidative stress was alleviated by treatment with the superoxide dismutase mimetic MnTMPyP. Conclusions: After multiple passaging in vitro, porcine coronary endothelial cells exhibited dysfunction and senescence associated with reduced proliferative capacity, increased oxidative stress, and activation of the NFĸB and p53 signaling pathways.