Abstract
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.
References
1.
Lee MYK, Tse HF, Siu CW, Zhu SG, Man RYK, Vanhoutte PM: Genomic changes in regenerated porcine coronary arterial endothelial cells. Arterioscler Thromb Vasc Biol 2007;27:2443–2449.
2.
Fournet-Bourguignon MP, Castedo-Delrieu M, Bidouard JP, Leonce S, Saboureau D, Delescluse I, Vilaine JP, Vanhoutte PM: Phenotypic and functional changes in regenerated porcine coronary endothelial cells: increased uptake of modified LDL and reduced production of NO. Circ Res 2000;86:854–861.
3.
Vanhoutte PM, Fournet-Bourguignon MP, Vilaine JP: Dysfonction de la voie du monoxide d’azote au cours de la régénération de l’endothélium coronarien. Académie Nationale de Médecine 2002;186:1525–1541.
4.
Brandes RP, Fleming I, Busse R: Endothelial aging. Cardiovasc Res 2005;66:286–294.
5.
Erusalimsky JD, Kurz DJ: Cellular senescence in vivo: Its relevance in ageing and cardiovascular disease. Exp Gerontol 2005;40:634–642.
6.
Kudo FA, Warycha B, Juran PJ, Asada H, Teso D, Aziz F, Frattini J, Sumpio BE, Nishibe T, Cha C, Dardik A: Differential responsiveness of early- and late-passage endothelial cells to shear stress. Am J Surg. 2005;190:763–769.
7.
Minamino T, Komuro I: Vascular cell senescence: contribution to atherosclerosis. Circ Res 2007;100:15–26.
8.
Shi Q, Hubbard GB, Kushwaha RS, Rainwater D, Thomas CA, Leland MM, Vandeberg JL, Wang XL: Endothelial senescence after high-cholesterol, high-fat diet challenge in baboons. Am J Physiol Heart Circ Physiol 2007;292:H2913–H2920.
9.
Hayashi T, Matsui-Hirai H, Miyazaki-Akita A, Fukatsu A, Funami J, Ding QF, Kamalanathan S, Hattori Y, Ignarro LJ, Iguchi A: Endothelial cellular senescence is inhibited by nitric oxide: implications in atherosclerosis associated with menopause and diabetes. Proc Natl Acad Sci USA 2006;103:17018–17023.
10.
Shelton DN, Chang E, Whittier PS, Choi D, Funk WD: Microarray analysis of replicative senescence. Curr Biol 1999:9:939–945.
11.
Ota H, Eto M, Kano MR, Ogawa S, Iijima K, Akishita M, Ouchi Y: Cilostazol inhibits oxidative stress-induced premature senescence via upregulation of Sirt1 in human endothelial cells. Arterioscler Thromb Vasc Biol 2008;28:1634–1639.
12.
Shi Y, So KF, Man RY, Vanhoutte PM: Oxygen-derived free radicals mediate endothelium-dependent contractions in femoral arteries of rats with streptozotocin-induced di- abetes. Br J Pharmacol 2007;152:1033–1041.
13.
Kueng W, Silber E, Eppenberger U: Quantification of cells cultured on 96-well plates. Anal Biochem 1989;182:16–19.
14.
Haendeler J, Hoffmann J, Diehl JF, Vasa M, Spyridopoulos I, Zeiher AM, Dimmeler S: Antioxidants inhibit nuclear export of telomerase reverse transcriptase and delay replicative senescence of endothelial cells. Circ Res 2004;94:768–775.
15.
Kurz DJ, Hong Y, Trivier E, Huang HL, Decary S, Zang GH, Lüscher TF, Erusalimsky JD: Fibroblast growth factor-2, but not vascular endothelial growth factor, upregulates telomerase activity in human endothelial cells. Arterioscler Thromb Vasc Biol 2003;23:748–754.
16.
MacMillan-Crow LA, Crow JP, Kerby JD, Beckman JS, Thompson JA: Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc Natl Acad Sci USA 1996;93:11853–11858.
17.
Marumo T, Schini-Kerth VB, Busse R: Vascular endothelial growth factor activates nuclear factor-kappaB and induces monocyte chemoattractant protein-1 in bovine retinal endothelial cells. Diabetes 1999;48:1131–1137.
18.
Shi Q, Aida K, Vandeberg JL, Wang XL: Passage-dependent changes in baboon endothelial cells: relevance to in vitro aging. DNA Cell Biol 2004;23:502–509.
19.
Maniatis NA, Brovkovych V, Allen SE, John TA, Shajahan AN, Tiruppathi C, Vogel SM, Skidgel RA, Malik AB, Minshall RD: Novel mechanism of endothelial nitric oxide synthase activation mediated by caveolae internalization in endothelial cells. Circ Res 2006;99:870–877.
20.
Saliez J, Bouzin C, Rath G, Ghisdal P, Desjardins F, Rezzani R, Rodella LF, Vriens J, Nilius B, Feron O, Balligand JL, Dessy C: Role of caveolar compartmentation in endothelium-derived hyperpolarizing factor-mediated relaxation: Ca2+ signals and gap junction function are regulated by caveolin in endothelial cells. Circulation 2008;117:1065–1074.
21.
Hong Y, Quintero M, Frakich NM, Trivier E, Erusalimsky JD: Evidence against the involvement of nitric oxide in the modulation of telomerase activity or replicative capacity of human endothelial cells. Exp Gerontol 2007;42:904–910.
22.
Shimokawa H, Flavahan NA, Vanhoutte PM: Natural course of the impairment of endothelium-dependent relaxations after balloon endothelium removal in porcine coronary arteries: possible dysfunction of a pertussis toxin-sensitive G protein. Circ Res 1989;65:740–753.
23.
Gorgoulis VG, Pratsinis H, Zacharatos P, Demoliou C, Sigala F, Asimacopoulos PJ, Papavassiliou AG, Kletsas D: p53-dependent ICAM-1 overexpression in senescent human cells identified in atherosclerotic lesions. Lab Invest 2005;85:502–511.
24.
Moiseeva O, Mallette FA, Mukhopadhyay UK, Moores A, Ferbeyre G: DNA damage signaling and p53-dependent senescence after prolonged beta-interferon stimulation. Mol Biol Cell 2006;17:1583–1592.
25.
Romzova M, Hohenadel D, Kolostova K, Pinterova D, Fojtikova M, Ruzickova S, Dostal C, Bosak V, Rychlik I, Cerna M: NFkappaB and its inhibitor IkappaB in relation to type 2 diabetes and its microvascular and atherosclerotic complications. Hum Immunol 2006;67:706–713.
26.
Donato AJ, Eskurza I, Silver AE, Levy AS, Pierce GL, Gates PE, Seals DR: Direct evidence of endothelial oxidative stress with aging in humans: relation to impaired endothelium-dependent dilation and upregulation of nuclear factor-kappaB. Circ Res 2007;100:1659–1666.
27.
Delafontaine P, Song YH, Li Y: Expression, regulation, and function of IGF-1, IGF-1R, and IGF-1 binding proteins in blood vessels. Arterioscler Thromb Vasc Biol 2004;24:435–444.
28.
Thum T, Hoeber S, Froese S, Klink I, Stichtenoth DO, Galuppo P, Jakob M, Tsikas D, Anker SD, Poole-Wilson PA, Borlak J, Ertl G, Bauersachs J: Age-dependent impairment of endothelial progenitor cells is corrected by growth-hormone-mediated increase of insulin-like growth-factor-1. Circ Res 2007;100:434–443.
29.
Sakurai H, Suzuki S, Kawasaki N, Nakano H, Okazaki T, Chino A, Doi T, Saiki I: Tumor Necrosis Factor-alpha-induced IKK phosphorylation of NF-kappaB p65 on serine 536 is mediated through the TRAF2, TRAF5, and TAK1 signaling pathway. J Biol Chem 2003;278:36916–36923.
30.
Zirlik A, Bavendiek U, Libby P, MacFarlane L, Gerdes N, Jagielska J, Ernst S, Aikawa M, Nakano H, Tsitsikov E, Schönbeck U: TRAF-1, -2, -3, -5, and -6 are induced in atherosclerotic plaques and differentially mediate proinflammatory functions of CD40L in endothelial cells. Arterioscler Thromb Vasc Biol 2007;27:1101–1107.
31.
Helenius M, Kyrylenko S, Vehviläinen P, Salminen A: Characterization of aging-associated up-regulation of constitutive nuclear factor-kappa B binding activity. Antioxid Redox Signal 2001;3:147–156.
32.
Han ES, Muller FL, Perez VI, Qi W, Liang HY, Xi L, Fu C, Doyle E, Hickey M, Cornell J, Epstein CJ, Roberts LJ, Van Remmen H, Richardson A: The in vivo gene expression signature of oxidative stress. Physiol Genomics 2008;34:112–126.
33.
Trougakos IP, Gonos ES: Regulation of clusterin/apolipoprotein J, a functional homologue to the small heat shock proteins, by oxidative stress in ageing and age-related diseases. Free Radic Res 2006;40:1324–1334.
34.
Campisi J, d’Adda di Fagagna F: Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol 2007;8:729–740.
35.
Marfella R, Di Filippo C, Laieta MT, Vestini R, Barbieri M, Sangiulo P, Crescenzi B, Ferraraccio F, Rossi F, D’Amico M, Paolisso G: Effects of ubiquitin-proteasome system deregulation on the vascular senescence and atherosclerosis process in elderly patients. J A Biol Sci Med Sci 2008;63:200–203.
36.
Lee MYK, Sørensen GL, Holmskov U, Vanhoutte PM: The presence and activity of SP-D in porcine coronary endothelial cells depend on Akt/PI3K, Erk and nitric oxide and decrease after multiple passaging. Mol Immunol 2009;46:1050–1057.
37.
Ohashi M, Runge MS, Faraci FM, Heistad DD: MnSOD deficiency increases endothelial dysfunction in ApoE-deficient mice. Arterioscler Thromb Vasc Biol 2006;26:2331–2336.
38.
Zhang LN, Velichko S, Vincelette J, Fitch RM, Vergona R, Sullivan ME, Croze E, Wang YX: Interferon-beta attenuates angiotensin II-accelerated atherosclerosis and vascular remodeling in apolipoprotein E deficient mice. Atherosclerosis 2008;197:204–211.
39.
Busuttil RA, Rubio M, Dolle ME, Campisi J, Vijg J: Oxygen accelerates the accumulation of mutations during the senescence and immortalization of murine cells in culture. Aging Cell 2003;2:287–605.
40.
Fu Y, Luo N, Lopes-Virella MF, Garvey WT: The adipocyte lipid binding protein (ALBP/aP2) gene facilitates foam cell formation in human THP-1 macrophages. Atherosclerosis 2002;165:259–269.
41.
Uysal KT, Scheja L, Wiesbrock SM, Bonner-Weir S, Hotamisligil GS: Improved glucose and lipid metabolism in genetically obese mice lacking aP2. Endocrinology 2000;141:3388–3396.
42.
Boord JB, Maeda K, Makowski L, Babaev VR, Fazio S, Linton MF, Hotamisligil GS: Adipocyte fatty acid-binding protein, aP2, alters late atherosclerotic lesion formation in severe hypercholesterolemia. Arterioscler Thromb Vasc Biol 2002;22:1686–1691.
43.
Furuhashi M, Tuncman G, Gorgun CZ, Makowski L, Atsumi G, Vaillancourt E, Kono K, Babaev VR, Fazio S, Linton M, Sulsky R, Robl JA, Parker RA, Hotamisligil GS: Treatment of diabetes and atherosclerosis by inhibiting fatty-acid-binding protein aP2. Nature 2007;447:959–965.
44.
Exil VJ, Roberts RL, Sims H, McLaughlin JE, Malkin RA, Gardner CD, Ni G, Rottman JN, Strauss AW: Very-long-chain acyl-coenzyme a dehydrogenase deficiency in mice. Circ Res 2003;93:448–455.
45.
Catar RA, Müller G, Heidler J, Schmitz G, Bornstein SR, Morawietz H: Low-density lipoproteins induce the renin-angiotensin system and their receptors in human endothelial cells. Horm Metab Res 2007;39:801–805.
46.
Webb NR, Connell PM, Graf GA, Smart EJ, de Villiers WJ, de Beer FC, van der Westhuyzen DR: SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmic tail, mediates lipid transfer between high density lipoprotein and cells. J Biol Chem 1998;273:15241–15248.
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