Molecular Medicine Hyperhomocysteinemia Activates Nuclear Factor-B in Endothelial Cells via Oxidative Stress Kathy K.W. Au-Yeung, Connie W.H. Woo, Fion L. Sung, Johnny C.W. Yip, Yaw L. Siow, Karmin O Abstract—Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases. Our previous studies demonstrated an important interaction between nuclear factor-B (NF-B) activation and homocysteine (Hcy)-induced chemokine expression in vascular smooth muscle cells and macrophages. The objective of the present study was to investigate the in vivo effect of hyperhomocysteinemia on NF-B activation and the underlying mechanism of Hcy-induced NF-B activation in endothelial cells. Hyperhomocysteinemia was induced in Sprague-Dawley rats after 4 weeks of a high-methionine diet. The activated form of NF-B and increased level of superoxide anions were detected in the endothelium of aortas isolated from hyperhomocysteinemic rats. The underlying mechanism of Hcy-induced NF-B activation was investigated in human umbilical cord vein endothelial cells and in human aortic endothelial cells. Incubation of cells with Hcy (100 mol/L) activated IB kinases (IKK␣ and IKK), leading to phosphorylation and subsequent degradation of IB␣. As a consequence, NF-B nuclear translocation, enhanced NF-B/DNA binding activity, and increased transcriptional activity occurred. Additional analysis revealed a marked elevation of superoxide anion levels in Hcy-treated cells. Treatment of cells with a superoxide anion scavenger (polyethylene glycol-superoxide dismutase) or IB kinase inhibitor (prostaglandin A1) could prevent Hcy-induced activation of IKK kinases and NF-B in endothelial cells. In conclusion, these results suggest that Hcy-induced superoxide anion production may play a potential role for NF-B activation in the early stages of atherosclerosis in the vascular wall via activation of IB kinases. (Circ Res. 2004;94:28-36.) Key Words: homocysteine 䡲 nuclear factor-B 䡲 IB␣ 䡲 IB kinase 䡲 superoxide
H
yperhomocysteinemia is a common and independent risk factor for cardiovascular disorders.1– 4 Although the precise mechanisms of homocysteine (Hcy)-induced atherosclerosis remain to be additionally investigated, endothelial dysfunction/injury is considered to be one of the leading mechanisms contributing to atherogenesis.5– 8 It has been proposed that Hcy-caused endothelial injury may be mediated by oxidative stress, attenuation of NO-mediated vasodilatation, and disturbance in the antithrombotic activities of the endothelium.9 During the early stages of atherosclerosis, stimulation of endothelial cells results in the secretion of various chemokines and adhesion molecules, leading to the recruitment of leukocytes including monocytes to the vascular wall.10 We reported previously that the expression of monocyte chemoattractant protein-1 (MCP-1), a potent chemokine for monocytes, was markedly elevated in Hcy-treated endothelial cells,11 vascular smooth muscle cells (VSMCs),12 and macrophages.13 The activation of a transcription factor, nuclear factor kappa-B (NF-B), has been linked to the expression of inflammatory factors during the onset of atherosclerosis.14 –16
Hcy treatment caused an activation of NF-B, leading to increased chemokine expression in VSMCs and macrophages.12,13 NF-B is normally present in the cytoplasm in an inactive form that is associated with an inhibitory protein named IB.14 In the presence of various NF-B stimuli, IB␣ (a well-studied IB protein) is rapidly phosphorylated, leading to ubiquitination and subsequent degradation of IB␣ as well as translocation of NF-B into the nucleus (activated NF-B). Phosphorylation of IB is mediated by enzymes called IB kinases (IKKs).15 After dissociation from IB, the active NF-B is translocated into the nucleus, where it binds to the B-binding motifs in the promoters or enhancers of the genes encoding cytokines. Recently, 2 IB kinases, IKK␣ and IKK, have been identified, and both are serine/threonine kinases that can phosphorylate IB proteins.16 The third IB kinase (IKK␥) lacks kinase activity but is required for the activation of the other 2 IB kinases in vivo.15 Activated NF-B has been detected in macrophages, endothelial cells, and vascular smooth muscle cells in human atherosclerotic lesions17,18 and in animal models.19,20 Welch et al21 first demonstrated that Hcy-induced expression of inducible NO
Original received April 28, 2003; revision received October 15, 2003; accepted November 3, 2003. From the Department of Pharmacology (F.L.S., J.C.W.Y., Y.L.S., K.O), Faculty of Medicine, The University of Hong Kong, and National Centre for Agri-Food Research in Medicine (K.K.W.A-Y., C.W.H.W., Y.L.S., K.O.), Departments of Animal Science and Physiology, University of Manitoba, Canada. Correspondence to Dr Karmin O, MD, PhD, Laboratory of Integrative Biology, NCARM, St Boniface Hospital Research Centre, R4032, 351 Tache Ave, Winnipeg, Manitoba R2H 2A6, Canada. E-mail
[email protected] © 2004 American Heart Association, Inc. Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000108264.67601.2C
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synthase in VSMCs was mediated via NF-B activation. In contrast, little or no activation of NF-B is found in normal aorta or arteries. We previously reported that the expression of endothelial MCP-1 and adhesion molecules was significantly elevated in vitro11 and in vivo.22 In rats with dietinduced hyperhomocysteinemia, the expressions of MCP-1, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in the aortic endothelium were significantly increased, leading to enhanced monocyte binding to the endothelium.22 We hypothesize that the activation of NF-B might play an important role in the expression of these inflammatory factors induced by Hcy in endothelial cells. Results from the in vitro studies suggest that Hcy at pathophysiological concentrations can activate NF-B. However, it remains unclear whether Hcy can initiate similar changes in vivo. Furthermore, it is unclear how Hcy regulates NF-B activity in vascular cells. In the present study, we aimed to investigate the in vivo effect of dietary-induced hyperhomocysteinemia on NF-B activation in the rat aorta and to elucidate the underlying mechanism of Hcy-induced NF-B activation in human vascular endothelial cells.
Materials and Methods Induction of Hyperhomocysteinemia in an Animal Model Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, Del) aged 8 weeks were divided into three groups (n⫽20 for each group) and maintained for 4 weeks on the following diets before experiments: (1) regular diet (control): PMI Rodent Diet 5001 (PMI Nutrition International); (2) high-methionine diet: regular diet plus 1.7% methionine (wt/wt); or (3) high-cysteine diet: regular diet plus 1.2% cysteine (wt/wt). Results from our previous study suggested that a high-methionine diet for 4 weeks was sufficient to induce hyperhomocysteinemia in rats.22 A high-cysteine diet group was included to test the specificity of Hcy effect on NF-B activation in the animal model. The plasma Hcy levels were measured with the IMx Hcy Assay based on the fluorescence polarization immunoassay technology (Abbott Diagnostics Division).22 All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals by the National Research Council.
In Situ Detection of Superoxide in the Rat Aortic Endothelium The in situ detection of superoxide anions in the aorta was performed as previously described.23 The thoracic aorta was isolated, and the aortic segments were immediately frozen in Tissue-Tec OTC embedding medium. Sequential cross sections (6 m) were prepared, and the oxidative fluorescent dye hydroethidium (oxidative dye) was used to evaluate the endothelial levels of superoxide anions. Hydroethidium is cell membrane permeable and oxidized to ethidium bromide by superoxide (turns to red fluorescence with excitation at 488 nm and emission at 610 nm). An increase in the fluorescence intensity under a confocal microscopy reflected an increase in superoxide anions in the endothelium. To differentiate the endothelium of the aorta from the media and adventitia, the same section was double-stained with a fluorescent marker for endothelial cells. In brief, the aortic section was stained with anti-von Willebrand (anti-vWf) factor (1:800, Abcam Ltd) primary antibodies followed by fluorescein-isothiocyanate–labeled anti-rabbit IgG secondary antibody (FITC-goat anti-rabbit IgG 1:50, Zymed Laboratories).24
In Situ Detection of Activated Form of NF-B The thoracic aorta was isolated, and cryosections were prepared and then fixed in acetone. The fixed sections were incubated with primary antibody against the activated p65 subunit of NF-B
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(Chemicon International). This antibody is specific for the activated form of NF-B, thus allowing identification of active NF-B.17,19 The secondary antibody for immunofluorescent staining was tetramethylrhodamine-isothiocyanate– bound rabbit anti-mouse IgG (1:25, Zymed Laboratories). Because the activated NF-B was located in the nuclei, the aortic cryosections were also double-stained with 1 mmol/L bis-benzamide (Hoechst 33258, Molecular Probes) to identify cell nuclei.25 The activated NF-B and the nuclei were visualized under Zeiss fluorescent microscope (Axioskop2 MOT).
Culture of Endothelial Cells Human umbilical cord vein endothelial cells (HUVECs) were purchased from American Type Culture Collection, whereas human aortic endothelial cells (HAECs) were purchased from Clonetics. Cells were cultured in F-12K Nutrient medium (GIBCO BRL) containing endothelial cell growth supplement (Sigma), 10% FBS, and antibiotics (100 U/mL penicillin, 10 g/mL streptomycin, and 20 g/mL neomycin).
Immunofluorescence Staining for NF-B in Endothelial Cells Cells (2⫻105) were cultured on cover slips in 6-well plates in F-12K Nutrient medium in the absence or presence of Hcy. After incubation, cells were fixed in methanol/acetone (1:1, vol/vol) for 10 minutes at ⫺20°C. Cell slides were then incubated with antibodies against NF-B (anti-p65) (Santa Cruz Biotechnology) as primary antibodies at 4°C overnight. After washing with PBS, cells were incubated with fluorescein (FITC)-conjugated goat anti-rabbit IgG (Zymed Laboratories) as secondary antibodies at 37°C for 45 minutes. Bound antibodies were viewed under confocal microscopy (Bio-Rad MRC-1024).
Electrophoretic Mobility Shift Assay Nuclear proteins were isolated from endothelial cells, and electrophoretic mobility shift assay (EMSA) was performed to determine NF-B/DNA binding activity. Nuclear proteins (10 g) were incubated with the reaction buffer for 15 minutes followed by incubation with 32P end-labeled oligonucleotide containing a sequence for NF-B/DNA binding site (5⬘-AGAGTGGGAATTTCCACTCA-3⬘) (Promega Corporation). The reaction mixture was separated in a nondenaturing 6% polyacrylamide gel that was later exposed to radiograph film at ⫺70°C. The binding of labeled oligonucleotide to nuclear proteins was blocked by adding unlabeled oligonucleotide to the reaction mixture to confirm that binding of 32P end-labeled oligonucleotide to NF-B was sequence specific.
Transient Transfection and Luciferase Reporter Assay Endothelial cells were transiently transfected with NF-B–Luc plasmid and SV--galactosidase control plasmid (pSV-gal). Cells growing in F-12K Nutrient medium were transfected by using the PathDetect In Vivo Signal Transduction Pathway cis-Reporting Systems (Promega Corporation). Twenty-four hours after transfection, cells were incubated with Hcy for various time periods and then harvested. Cellular proteins were prepared and assayed for luciferase activity as well as -galactosidase activity according to the manufacturer’s instruction (Promega Corporation). The ratio of luciferase activity to -galactosidase activity served to normalize the luciferase activity to correct for any differences in transfection efficiencies.26
Western Immunoblotting Analysis The cellular levels of IB␣, IKK␣, and IKK proteins were determined by Western immunoblotting analysis.12,13,27 For IB␣ protein analysis, cellular proteins were separated by SDS 12.5% polyacrylamide gel electrophoresis followed by electrophoretic transfer of proteins from the gel onto nitrocellulose membrane. The membrane was then probed with either rabbit anti-IB␣ or anti-phosphorylated IB␣ (Ser32) polyclonal antibodies (New England Biolabs). For IKK␣ and IKK protein analysis, cellular proteins were
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separated by SDS 10% polyacrylamide gel electrophoresis. After electrophoretic transfer of proteins from the gel to a nitrocellulose membrane, the membrane was probed with rabbit anti-IKK␣ or anti-IKK antibodies (Cell Signaling Technology). Bands corresponding to IB␣, serine phosphorylated IB␣ proteins, IKK␣, or IKK proteins were visualized using enhanced chemiluminescence reagents (Amersham Bioscience) and analyzed with a gel documentation system (Bio-Rad Gel Doc1000 and Multi-Analyst version 1.1).
Measurement of IKK Activity After cells were incubated with Hcy, the activities of IB kinases (IKK␣ and IKK) were detected by an in vitro immune complex kinase assay with IB␣ (amino acids 1 to 317) fusion protein as substrate (Santa Cruz Biotechnology). In brief, cell lysates were incubated with rabbit antibodies against IKK␣ or IKK (Cell Signaling Technology) followed by precipitation with agarose-immobilized protein-A (Oncogene Research Products). The immunoprecipitated IKK␣ or IKK was incubated with IB␣ fusion protein. The reaction was initiated with [␥-32P]ATP, and the phosphorylation (IB␣) reaction was carried out at 37°C for 45 minutes. The reaction product was separated and analyzed by SDS-10% polyacrylamide gel electrophoresis and autoradiography.27
Measurement of Intracellular Superoxide Levels The content of intracellular superoxide anion radicals was determined by the nitro blue tetrazolium (NBT) reduction assay. After incubation for various time periods in the absence or presence of Hcy, cells were incubated for another 2 hours in Krebs-Henseleit buffer containing NBT (1 mg/mL). Formazan was generated by reduction of NBT, which was proportional to the amount of superoxide formed in cultured cells. The absorbance of the supernatant at 540 and 450 nm was determined.
Statistical Analysis The results were analyzed using two-tailed independent Student’s t test. The level of statistical significance was set at P⬍0.05.
Results NF-B Activation and Superoxide Detection in the Aorta Hyperhomocysteinemia was induced in a group of rats after 4 weeks of dietary treatment. The high-methionine diet resulted in a significant increase in the plasma Hcy levels (23.28⫾2.46 versus 3.08⫾0.25 mol/L in control rats). There was no significant elevation of serum Hcy levels in rats fed a high-cysteine diet (2.29⫾0.28 versus 3.08⫾0.25 mol/L in control rats). Immunofluorescent staining was carried out to detect NF-B activation in the aorta. Using a monoclonal antibody that specifically recognized active NFB, the activated form of NF-B was found in the endothelial cell nuclei of the aorta isolated from hyperhomocysteinemic rat (fed with high-methionine diet), whereas little was detected in the aortas isolated from rats fed a regular diet (control) or a high-cysteine diet (Figure 1, left). To confirm that the activated form of NF-B was present in the endothelial nuclei, the identical cross sections were double-stained with a nucleus marker, Hoechst 33258 (Figure 1, middle). The fluorescence image demonstrated that the endothelial cell nuclei were located outside the internal elastic laminae. The superimposed image revealed that the activated form of NF-B was colocalized in the endothelial nuclei of the aorta isolated from hyperhomocysteinemic rat after 4 weeks of high-methionine diet (Figure 1, right). Next, the presence of superoxide anions in the aortic wall was detected. The cross
cryosections of the aortas were incubated with hydroethidium to detect superoxide anions (Figures 2a, 2f, and 2k, magnification ⫻200; Figures 2c, 2h, and 2m, magnification ⫻400). The cross sections of aortas isolated from hyperhomocysteinemic rats displayed a marked increase in fluorescence intensity, reflecting an increase in superoxide anion in these aortas (Figures 2f and 2h). Such increase was observed in the endothelium as well as in the media and adventitia. To verify that the level of superoxide anions was indeed elevated in the endothelium of aorta isolated from hyperhomocysteinemic rats, double staining with an endothelial cell marker (vWf) was performed (Figures 2b, 2g, and 2l, magnification ⫻200; Figures 2d, 2i, and 2n, magnification ⫻400). There was a marked increase in fluorescence intensity in the cross section of the aorta isolated from hyperhomocysteinemic rat that was colocalized with the endothelium, indicating that the level of superoxide anions was indeed elevated in the endothelium of the aorta isolated from hyperhomocysteinemic rats (Figure 2j). In contrast, few superoxide anions were detected in the aortas isolated from the rats fed with a regular diet (control) or a high-cysteine diet (Figures 2e and 2o). Taken together, these results indicated an activation of NF-B as well as an increase in the level of superoxide anions in the aortic endothelium during hyperhomocysteinemia. No atherosclerotic lesion was found in the aortic endothelium of hyperhomocysteinemic rats after 4 weeks of high-methionine diet.
Activation of NF-B in Vascular Endothelial Cells
The effect of Hcy on NF-B activation was additionally examined in cultured endothelial cells. First, the activity of luciferase reporter gene for NF-B was determined in endothelial cells 24 hours after its transient transfection simultaneously with pSV-gal. Hcy treatment significantly enhanced the transcriptional activity of NF-B, as indicated by an increase in the luciferase activity (Figure 3A). Next, EMSA was performed to determine the activity of NF-B binding to DNA. The NF-B/DNA binding activity was also significantly increased in cells incubated with Hcy (Figure 3B). These results confirmed an activation of NF-B in endothelial cells.
Effect of Hcy on Phosphorylation of IB␣ Protein
To investigate whether NF-B activation was a result of increased phosphorylation of IB␣ protein, Western immunoblotting analysis was performed. The level of serine phospho-IB␣ was elevated in cells incubated with Hcy for 15 to 60 minutes and returned to the basal level after 2 hours of incubation (Figure 4A). In accordance with these results, there was a marked reduction in the levels of IB␣ protein in these cells treated with Hcy (Figure 4B). Taken together, increased phosphorylation might contribute to a significant reduction in IB␣ protein levels in Hcy-treated cells, leading to dissociation and subsequent activation of NF-B.
Effect of Hcy on IB Kinase Activities and Protein Levels
To determine the upstream regulation of IB protein phosphorylation, the effect of Hcy treatment on IB kinases was examined. The activities of IKK␣ and IKK were markedly
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Figure 1. Immunofluorescence staining of activated form of NF-B and cell nuclei. Thoracic aortas were isolated from rats fed a regular diet (control), high-methionine diet (methionine), or high-cysteine diet (cysteine). The aortic frozen sections were prepared. Left, Activated form of NF-B was identified as intensive red fluorescence (arrows). Middle, Endothelial cell nuclei of the identical sections were visualized as blue fluorescence (arrows). Right, Superimposed image of NF-B staining with a nuclear marker. Hoechst staining revealed the localization of the activated NF-B in aortic endothelial nuclei of the hyperhomocysteinemic rat (purple fluorescence, arrows). Representative photos were obtained from 5 separate experiments (magnification ⫻400).
elevated in cells incubated with Hcy for 5 to 30 minutes (Figure 5A). Such activation could result in phosphorylation of IB␣ protein in endothelial cells, leading to NF-B activation. Levels of IKK proteins (IKK␣ and IKK) were not elevated in Hcy-treated cells (Figure 5B). To additionally demonstrate the link of IKK activity with Hcy-induced NF-B activation, the effect of IKK inhibitor, prostaglandin A1 (PGA1, Cayman Chemical), on NF-B activation was examined in HUVECs and HAECs.28 Treatment of cells with
PGA1 not only inhibited the activities of IKK␣ and IKK but also abolished Hcy-induced IB␣ phosphorylation and degradation and eventually prevented Hcy-induced NF-B activation in HUVECs (Figure 6). Similar results were obtained from experiments performed in another type of endothelial cells, HAECs (Figure 6). These results suggested that Hcyinduced NF-B activation in endothelial cells was mediated via IKK kinases. Endothelial cells derived from vein or aortic endothelium displayed similar response on Hcy treatment.
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Figure 2. In situ detection of superoxide anions in rat aortas. Thoracic aortas were isolated from rats fed a regular diet (control), high-methionine diet (methionine), or high-cysteine diet (cysteine). In a, f, and k (magnification ⫻200) and c, h, and m (magnification ⫻400), cross cryosections of the aortas were incubated with hydroethidium. Superoxide anions labeled with red fluorescence in the aortas were observed (E indicates endothelium, arrows). In b, g, and l (magnification ⫻200) and d, i, and n (magnification ⫻400), the identical sections of the aorta were stained with anti-vWf antibodies (green fluorescence) to identify the location of endothelium. In e, j, and o, double-fluorescence staining of superoxide with endothelial marker (vWf) demonstrates that endothelium layer (green) produces superoxide anions (red) in j. Representative photos were obtained from 5 separate experiments.
Involvement of Superoxide Anions in Hcy-Induced NF-B Activation To determine whether Hcy treatment resulted in an increase in oxidative stress in endothelial cells leading to NF-B activation, the level of intracellular superoxide anions was measured. There was a significant elevation of superoxide anion levels in Hcy-treated cells (Figure 7A). Pretreatment of these cells with cell-permeable polyethylene glycol-bound superoxide dismutase (PEG-SOD, Sigma), a known superox-
ide scavenger,29 reversed Hcy-induced elevation of superoxide anion levels in these cells. The PEG-SOD treatment also abolished Hcy-induced IKK␣ and IKK activation (Figure 7B) and, hence, reversed Hcy-induced NF-B activation in these cells (Figure 7C). Finally, immunofluorescence imaging of NF-B in endothelial cells was performed after incubation of cells with Hcy for 30 minutes. Cytoplasmic localization of NF-B (p65 subunit) was found in control cells (Figure 7D). Hcy treatment caused a marked increase in
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Figure 3. Activation of NF-B in vascular endothelial cells. A, HUVECs were transiently cotransfected with NF-B–Luc plasmid and pSV-gal followed by incubation with Hcy (100 mol/L). The extent of activation of NF-B luciferase construct was determined. Results were expressed as fold induction of luciferase activity over that of mock-transfected endothelial cells (control), with the average activity of mock-transfected cells arbitrarily given as 1. Results are expressed as mean⫾SD (error bar) of 3 separate experiments. B, HUVECs were incubated in the absence or presence of Hcy (100 mol/L) for various time periods. Nuclear proteins were isolated, and EMSA was performed to determine NF-B/DNA binding activity. The results were analyzed as integrated intensity units and expressed as percentage of control⫾SD (error bar) of 5 separate experiments. The control value was expressed as 100% (28.91⫾5.53 densitometric unit). *P⬍0.05 compared with control values.
the nuclear staining intensity for NF-B, indicating the translocation of NF-B into the nucleus (Figure 7D). On the other hand, addition of PEG-SOD prevented Hcy-induced translocation of NF-B from cytosol into the nucleus (Figure 7D). These results suggested that Hcy-induced elevation of superoxide level in endothelial cells contributed to NF-B activation.
Discussion The present study has clearly demonstrated an elevation of superoxide anion level and the presence of activated NF-B in the aortic endothelium of diet-induced hyperhomocysteinemic rats. These findings were confirmed in cultured endothelial cells, and the underlying mechanism was investigated. Our results indicate that oxidative stress and subsequent activation of IB kinases (IKK␣ and IKK) are essential for Hcy-induced activation of NF-B in endothelial cells.
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Figure 4. Effect of Hcy on IB␣ protein levels in vascular endothelial cells. HUVECs were incubated in the absence or presence of Hcy (100 mol/L) for various time periods. A, Intracellular levels of phospho-IB␣ protein were determined by Western immunoblotting analysis with anti-phosphorylated IB␣ antibodies. B, Intracellular levels of total IB␣ were analyzed with antiIB␣ antibodies. The immunoblots were analyzed by densitometry, and the data were generated as integrated intensity units. The results were expressed as percentage of control⫾SD (error bar) of 3 separate experiments each performed in duplicate. The control value was expressed as 100% (0.66⫾0.05 densitometric unit for phosphor-IB␣, 38.76⫾1.68 densitometric unit for IB␣). *P⬍0.05 compared with control values.
NF-B has been implicated to play an important role in the initiation and development of atherosclerosis. This transcription factor can be activated by diverse pathogenic signals. The activated form of NF-B has been detected in the atherosclerotic lesions. In the present study, although there was no atherosclerotic lesion observed, the activated form of NF-B was detected in the aortic endothelium of hyperhomocysteinemic rats. In addition, several lines of evidence indicated that NF-B was activated in endothelial cells on Hcy treatment. First, nuclear translocation of NF-B occurred in endothelial cells after incubation with Hcy for 15 to 30 minutes. Second, the results from EMSA demonstrated that Hcy treatment caused a significant increase in the NF-B/ DNA binding activity. Third, results from transient transfection demonstrated an enhanced NF-B–regulated transcriptional activity in Hcy-treated cells. We previously reported that Hcy treatment stimulated MCP-1 expression in smooth muscle cells,12 macrophages,13 and endothelial cells.11 Pretreatment of cells with NF-B inhibitors could alleviate the
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Figure 5. Effect of Hcy on IB kinase activities and protein levels in vascular endothelial cells. HUVECs were incubated in the absence or presence of Hcy (100 mol/L) for various time periods. A, Cytoplasmic extracts were prepared and immunoprecipitated with anti-IKK␣ or anti-IKK antibodies. The activities of IB kinase (IKK␣ or IKK) were determined by an in vitro immune complex kinase assay with IB␣ protein as substrate. The results were expressed as percentage of control⫾SD (error bar) of 3 separate experiments, each performed in duplicate. The control activities were expressed as 100% (4.15⫾0.40 densitometric unit for IKK␣, ▫; 5.48⫾0.34 densitometric unit for IKK, 䡲). B, Levels of IKK␣ or IKK proteins were determined by Western immunoblotting analysis with specific antibodies against these 2 kinases. The control levels were expressed as 100% (22.53⫾5.02 densitometric unit for IKK␣, ▫; 25.35⫾1.63 densitometric unit for IKK, 䡲). *P⬍0.05 compared with control values.
stimulatory effect of Hcy on MCP-1 expression, supporting the notion that Hcy-stimulated chemokine expression was mediated via NF-B activation.12,13 In the present study, we additionally investigated the underlying mechanism by which Hcy activated NF-B in endothelial cells. Hcy treatment resulted in a rapid and transient increase in the level of serine phosphorylated IB␣ protein in these cells, followed by a decrease in total IB␣ protein levels, which preceded NF-B activation. The activities of IKK␣ and IKK in endothelial cells were rapidly increased within 5 to 30 minutes of Hcy treatment and then reduced to the basal level. Furthermore, treatment of cells with IB kinase inhibitor could prevent Hcy-induced IB␣ phosphorylation and NF-B activation. These results suggested that the control of IB␣ phosphorylation in response to Hcy treatment was mediated via the IB
Figure 6. Effect of IB kinase inhibitor on NF-B activation in vascular endothelial cells. Endothelial cells (HUVECs or HAECs) were incubated with Hcy (100 mol/L) in the absence or presence of PGA1 (30 mol/L). The activities of IKK␣ and IKK were determined by an in vitro immune complex kinase assay (A). Intracellular levels of phospho-IB␣ protein (B) and total IB␣ protein (C) were determined by Western immunoblotting analysis. EMSA was performed to determine NF-B/DNA binding activity (D). Representative results were obtained from 3 separate experiments, each performed in duplicate. Experimental differences were ⬍10%.
kinases. Taken together, results obtained from in vivo and in vitro experiments suggest that Hcy-induced NF-B activation in endothelial cells may represent one important mechanism by which Hcy causes atherosclerosis. Multiple pathways mediating NF-B activation have been proposed. Reactive oxygen species have been implicated to stimulate IB␣ degradation and NF-B activation in vascular cells.30 It was demonstrated that oxidative stress contributed to vascular dysfunction in animal models with hypercholesterolemia-induced atherosclerosis.23 Superoxide levels were significantly increased in the vascular wall, causing impaired vessel relaxation in hypercholesterolemic rabbits.23 It was reported that hydrogen peroxide stimulated NF-B activity via activation of IKK␣ and IKK in HeLa cells.31 Antioxidants were shown to be able to block IB␣ degradation and NF-B activation.32–34 Oxidative stress has been proposed to be an important mechanism of Hcy-induced endothelial dysfunction.34,35 Administration of antioxidants such as vitamins were shown to alleviate the adverse effect of Hcy.34 In the present study, the level of superoxide anions was increased in the aortas of hyperhomocysteinemic rats. Furthermore, the superoxide levels were significantly elevated in Hcy-treated endothelial cells, which preceded the activation of IKK and the degradation of IB␣. Pretreatment of endothelial cells with PEG-SOD not only prevented the elevation of superoxide levels in endothelial cells but also abolished Hcy-induced activation of IKK␣ and IKK. Taken together, these results suggest that oxidative stress might contribute to
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other hand, elevation of plasma homocysteine induced by high-methionine diet resulted in NF-B activation in the endothelium, representing a plausible mechanism of homocysteine-mediated atherosclerosis. In conclusion, the present study has clearly demonstrated that superoxide formation and NF-B activation occur in the aortic endothelium of hyperhomocysteinemic rats. Hcyinduced oxidative stress involving IKK activation may play an important role in NF-B activation in endothelial cells. Such mechanism may regulate the inflammatory response in the vascular wall in the early stages of atherosclerosis in hyperhomocysteinemia.
Acknowledgments This study was supported by a grant (HKU7249/02 mol/L) from the Research Grant Council of Hong Kong and by an Operating Grant from the Manitoba Health Research Council to K.O.
References
Figure 7. Effect of Hcy on superoxide level and NF-B activation in vascular endothelial cells. A, HUVECs were incubated for 15 minutes in the absence (control) or presence of Hcy (100 mol/L). One set of cells was preincubated with PEG-SOD (300 U/mL) for 15 minutes followed by incubation with Hcy (100 mol/L) for another 15 minutes (Hcy⫹PEG-SOD). At the end of incubation, the cellular superoxide levels were determined by NBT reduction assay. Cells cultured in the medium alone were used as control. Results are expressed as mean⫾SD (error bar) of 3 separate experiments. *P⬍0.05 compared with control values. B, After cells were treated in the same manner as stated in A, cytoplasmic extracts were prepared and immunoprecipitated with anti-IKK␣ or anti-IKK antibodies. The activity of IKK␣ or anti-IKK was determined by an in vitro immune complex kinase assay with IB␣ protein as substrate. C, Cells were incubated for 30 minutes in the absence (control) or presence of Hcy (100 mol/L). One set of cells was preincubated with PEGSOD (300 U/mL) for 15 minutes followed by incubation with Hcy (100 mol/L) for another 30 minutes (Hcy⫹PEG-SOD). Nuclear proteins were isolated and EMSA was performed to determine NF-B/DNA binding activity. D, After cells were treated in the same manner as stated in (C), immunofluorescent staining was performed to detect intracellular NF-B. Endothelial cells were incubated with Hcy (100 mol/L) in the absence or presence of PEG-SOD (300 U/mL) for 30 minutes. NF-B protein (in green) was identified by use of confocal microscopy at a magnification of ⫻400. Photomicrographs are representatives from 5 separate experiments.
the activation of IKK kinase via superoxide formation, leading to NF-B activation in Hcy-treated endothelial cells. Although cysteine also contains a thiol group, the failure of 4-week high-cysteine diet to induce NF-B activation and increase superoxide anion levels in the aortic endothelium suggests that elevation of this amino acid in the body may not play a significant role in the activation of NF-B. On the
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Hyperhomocysteinemia Activates Nuclear Factor-κB in Endothelial Cells via Oxidative Stress Kathy K.W. Au-Yeung, Connie W.H. Woo, Fion L. Sung, Johnny C.W. Yip, Yaw L. Siow and Karmin O Circ Res. 2004;94:28-36; originally published online November 20, 2003; doi: 10.1161/01.RES.0000108264.67601.2C Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2003 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
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