Senescence of cultured porcine coronary arterial endothelial cells is associated with accelerated oxidative stress and activation of NFkB.
OBJECTIVE
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.


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-beta, cytoskeleton rearrangement and lipid metabolism - were altered at P4, as judged from the microarray analysis. The basal and stimulated (by TNF-alpha) levels of NFkappaB 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 NFkappaB and p53 signaling pathways.
Lee
Mary Y K
MY
Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, SAR, China.
Wang
Yu
Y
Vanhoutte
Paul M
PM
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects / Q000235:genetics; D003331:Coronary Vessels / Q000187:drug effects / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D008665:Metalloporphyrins / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics; D015398:Signal Transduction; D034421:Sus scrofa; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000975:Antioxidants; D008665:Metalloporphyrins; C502230:Mn(III) 5,10,15,20-tetrakis(N-methylpyridinium-2-yl)porphyrin; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; D009569:Nitric Oxide