Endothelial Apoptosis Induced by Oxidative Stress Through Activation of NF-&kgr;B: Antiapoptotic Effect of Antioxidant Agents on Endothelial Cells

Injury of endothelial cells has been assumed to be an initial trigger of the development of atherosclerosis. In this study, we investigated the molecular mechanisms of endothelial cell death induced by hypoxia, which leads to oxidative stress. To study the relation between hypoxia-induced cell death and activation of nuclear factor-&kgr;B (NF-&kgr;B) in a hypoxic state, we evaluated the effect of 2 antioxidant drugs, probucol and pyrrolidine dithiocarbamate (PDTC), on human endothelial apoptosis. Although hypoxic treatment of human aortic endothelial cells resulted in a significant decrease in cell number and a significant increase in apoptotic cells compared with that of cells under normoxia (P <0.01), treatment with probucol (50 &mgr;mol/L) or PDTC (100 &mgr;mol/L) significantly attenuated the decrease in cell number (P <0.01) and was accompanied by inhibition of NF-&kgr;B activation. Furthermore, downregulation of bcl-2 caused by hypoxia was inhibited by these drugs. We further investigated the translocation of bax protein from the cytoplasm to the mitochondrial heavy fraction membrane, as translocation of bax protein is considered to be a determinant of apoptosis. Interestingly, we found that antioxidant treatment inhibited the translocation of bax protein caused by hypoxia. Moreover, upregulation of p53, a proapoptotic molecule, was observed in hypoxia, whereas treatment with probucol attenuated the expression of p53 accompanied by suppression of NF-&kgr;B activation. These data suggest functional links between p53 and endothelial apoptosis through the activation of NF-&kgr;B. Overall, the current study demonstrated that oxidative stress induced apoptosis in human aortic endothelial cells through the downregulation of bcl-2, translocation of bax, and upregulation of p53, probably through NF-&kgr;B activation. Oxidative stress may play an important role in endothelial apoptosis mediated by hypoxia, through the activation of NF-&kgr;B.

[1]  D. Lane,et al.  p53, guardian of the genome , 1992, Nature.

[2]  S. Ylä-Herttuala,et al.  Cardiovascular gene therapy , 2000, The Lancet.

[3]  G. Gibbons,et al.  The emerging concept of vascular remodeling. , 1994, The New England journal of medicine.

[4]  J. Mehta,et al.  Ox-LDL induces apoptosis in human coronary artery endothelial cells: role of PKC, PTK, bcl-2, and Fas. , 1998, American journal of physiology. Heart and circulatory physiology.

[5]  Y. Taniyama,et al.  Hypoxia-Induced Endothelial Apoptosis Through Nuclear Factor-κB (NF-κB)–Mediated bcl-2 Suppression In Vivo Evidence of the Importance of NF-κB in Endothelial Cell Regulation , 2000 .

[6]  Y. Tsujimoto,et al.  Involvement of the bcl-2 gene in human follicular lymphoma. , 1985, Science.

[7]  M. Aoki,et al.  Inhibition of the p53 tumor suppressor gene results in growth of human aortic vascular smooth muscle cells. Potential role of p53 in regulation of vascular smooth muscle cell growth. , 1999, Hypertension.

[8]  C. Cordon-Cardo,et al.  Lipopolysaccharide Induces Disseminated Endothelial Apoptosis Requiring Ceramide Generation , 1997, The Journal of experimental medicine.

[9]  S. Lowe,et al.  p53-Dependent apoptosis suppresses tumor growth and progression in vivo , 1994, Cell.

[10]  M. Narita,et al.  Apoptotic cytosol facilitates Bax translocation to mitochondria that involves cytosolic factor regulated by Bcl-2. , 1999, Cancer research.

[11]  G. Remuzzi,et al.  Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-kB-dependent fashion. , 1998, The Journal of clinical investigation.

[12]  D. Morel,et al.  Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid. , 1983, Journal of lipid research.

[13]  J. Trent,et al.  WAF1, a potential mediator of p53 tumor suppression , 1993, Cell.

[14]  N. Makino,et al.  Interactions of nitric oxide and oxygen in cytotoxicity: Proliferation and antioxidant enzyme activities of endothelial cells in culture , 2000, Free radical research.

[15]  T. Ogihara,et al.  In vivo transfection of cis element “decoy” against nuclear factor- κB binding site prevents myocardial infarction , 1997, Nature Medicine.

[16]  M. Kibbe,et al.  Gene therapy for restenosis. , 2000, Circulation research.

[17]  G. Chisolm,et al.  Oxidized low density lipoprotein suppresses the expression of tumor necrosis factor-alpha mRNA in stimulated murine peritoneal macrophages. , 1990, Journal of immunology.

[18]  H. Daida,et al.  Effectiveness of an antioxidant in preventing restenosis after percutaneous transluminal coronary angioplasty: the Probucol Angioplasty Restenosis Trial. , 1997, Journal of the American College of Cardiology.

[19]  X. Sun,et al.  Identification of a novel p53 promoter element involved in genotoxic stress-inducible p53 gene expression , 1995, Molecular and cellular biology.

[20]  A. Koong,et al.  Hypoxia Causes the Activation of Nuclear Factor κB through the Phosphorylation of IκBα on Tyrosine Residues , 1994 .

[21]  C. Y. Wang,et al.  NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. , 1998, Science.

[22]  P. Libby.  Gene therapy of restenosis: promise and perils. , 1998, Circulation research.

[23]  P. Libby,et al.  Culture of quiescent arterial smooth muscle cells in a defined serum‐free medium , 1983, Journal of cellular physiology.

[24]  P. Reaven,et al.  Effect of dietary antioxidant combinations in humans. Protection of LDL by vitamin E but not by beta-carotene. , 1993, Arteriosclerosis and thrombosis : a journal of vascular biology.

[25]  A. Koong,et al.  Hypoxic activation of nuclear factor-kappa B is mediated by a Ras and Raf signaling pathway and does not involve MAP kinase (ERK1 or ERK2). , 1994, Cancer research.

[26]  M. Aoki,et al.  Transcription factor decoy for nuclear factor‐κB inhibits tumor necrosis factor‐α‐induced expression of interleukin‐6 and intracellular adhesion molecule‐1 in endothelial cells , 1998, Journal of hypertension.

[27]  John Calvin Reed.  Bcl-2 and the regulation of programmed cell death , 1994, The Journal of cell biology.

[28]  G. Farrell,et al.  Role of oxidative stress in hypoxia‐reoxygenation injury to cultured rat hepatic sinusoidal endothelial cells , 2000, Hepatology.

[29]  H. Wu,et al.  NF-kappa B activation of p53. A potential mechanism for suppressing cell growth in response to stress. , 1994, The Journal of biological chemistry.

[30]  T. Chase,et al.  Nuclear Factor-κB Contributes to Excitotoxin-Induced Apoptosis in Rat Striatum , 1998 .

[31]  S. Young,et al.  Probucol inhibits oxidative modification of low density lipoprotein. , 1986, The Journal of clinical investigation.

[32]  R. Muschel,et al.  Induction of apoptosis at different oxygen tensions: evidence that oxygen radicals do not mediate apoptotic signaling. , 1995, Cancer research.

[33]  A. Dembińska-kieć,et al.  Gene transfer of vascular endothelial growth factor and endothelial nitric oxide synthase--implications for gene therapy in cardiovascular diseases. , 1999, Polish journal of pharmacology.

[34]  Peter J. Smith.  Earthquakes without precursors? , 1975, Nature.

[35]  Y. Kaneda,et al.  Development and characterization of cationic liposomes conjugated with HVJ (Sendai virus): reciprocal effect of cationic lipid for in vitro and in vivo gene transfer. , 1997, Human gene therapy.

[36]  K. Roebuck.  Oxidant stress regulation of IL-8 and ICAM-1 gene expression: differential activation and binding of the transcription factors AP-1 and NF-kappaB (Review). , 1999, International journal of molecular medicine.

[37]  John Calvin Reed,et al.  Identification of a p53-dependent negative response element in the bcl-2 gene. , 1994, Cancer research.

[38]  Bert Vogelstein,et al.  p53 function and dysfunction , 1992, Cell.

[39]  P. Baeuerle,et al.  Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF‐kappa B transcription factor and HIV‐1. , 1991, The EMBO journal.

[40]  M. Aoki,et al.  Serum deprivation-induced apoptosis accompanied by up-regulation of p53 and bax in human aortic vascular smooth muscle cells. , 1997, Heart and vessels.

[41]  R. Jennings,et al.  Ischemic tissue injury. , 1975, The American journal of pathology.

[42]  K. Kugiyama,et al.  Biphasic regulation of transcription factor nuclear factor-kappaB activity in human endothelial cells by lysophosphatidylcholine through protein kinase C-mediated pathway. , 1998, Arteriosclerosis, thrombosis, and vascular biology.

[43]  A. Axon,et al.  Clinical and histological associations of cagA and vacA genotypes in Helicobacter pylori gastritis. , 1998, Journal of clinical pathology.

[44]  S. Egginton,et al.  Angiogenesis in skeletal and cardiac muscle. , 1992, Physiological reviews.

[45]  B. Herman,et al.  Role of ICE-like proteases in endothelial cell hypoxic and reperfusion injury. , 1997, Biochemical and biophysical research communications.

[46]  A. Yeung,et al.  The effect of cholesterol-lowering and antioxidant therapy on endothelium-dependent coronary vasomotion. , 1995, The New England journal of medicine.

[47]  A. Ooshima,et al.  Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[48]  T. Imamura,et al.  Induction of apoptosis in murine clonal osteoblasts expressed by human T‐cell leukemia virus type I tax by NF‐κPB and TNF‐α , 1996 .

[49]  A. Alayash,et al.  Effects of hypoxia and glutathione depletion on hemoglobin- and myoglobin-mediated oxidative stress toward endothelium. , 2000, Biochimica et biophysica acta.

[50]  A. Chait,et al.  The influence of oxidatively modified low density lipoproteins on expression of platelet-derived growth factor by human monocyte-derived macrophages. , 1991, The Journal of biological chemistry.

[51]  S. Nattel,et al.  Probucol and multivitamins in the prevention of restenosis after coronary angioplasty. Multivitamins and Probucol Study Group. , 1997, The New England journal of medicine.

[52]  R. von Harsdorf,et al.  p53 regulates mitochondrial membrane potential through reactive oxygen species and induces cytochrome c‐independent apoptosis blocked by Bcl‐2 , 1999, The EMBO journal.

[53]  Hirofumiyasue,et al.  Biphasic Regulation of Transcription Factor Nuclear Factor-κB Activity in Human Endothelial Cells by Lysophosphatidylcholine Through Protein Kinase C–Mediated Pathway , 1998 .

[54]  S. Dower,et al.  Response of tumour cells to hypoxia: role of p53 and NFkB. , 1998, Molecular pathology : MP.

[55]  T. Gardner,et al.  Inhibition of Neutrophil Adherence Improves Postischemic Ventricular Performance of the Neonatal Heart , 1993, Circulation.

[56]  S. Jay,et al.  Probucol and multivitamins in the prevention of restenosis after coronary angioplasty. , 1997, The New England journal of medicine.

[57]  Laboratorium farMolekulare.  Reactive oxygen intermediates as apparently widely usedmessengers intheactivation oftheNF-xB transcription factor andHIV-1 , 1991 .

[58]  R. Foresti,et al.  Curcumin, an antioxidant and anti-inflammatory agent, induces heme oxygenase-1 and protects endothelial cells against oxidative stress. , 2000, Free radical biology & medicine.

[59]  P. Bonin,et al.  Growth factor-induced modulation of endothelin-1 binding to human smooth-muscle cells. , 1993, Journal of cardiovascular pharmacology.

[60]  P. Gallagher,et al.  Dietary vitamin E and the attenuation of early lesion development in modified Watanabe rabbits. , 1992, Atherosclerosis.

[61]  K Sugimachi,et al.  HVJ (Sendai virus)-cationic liposomes: a novel and potentially effective liposome-mediated technique for gene transfer to the airway epithelium , 1997, Gene Therapy.