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Cardiovascular Research 2004 63(2):323-330; doi:10.1016/j.cardiores.2004.03.018
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Copyright © 2004, European Society of Cardiology

Role of NADPH oxidase-mediated superoxide production in the regulation of E-selectin expression by endothelial cells subjected to anoxia/reoxygenation

Alain Rupin, Jérôme Paysant, Patricia Sansilvestri-Morel, Nathalie Lembrez, Jean-Michel Lacoste, Alex Cordi and Tony J Verbeuren*

Division of Angiology, Servier Research Institute, 11 rue des Moulineaux, Suresnes 92150, France

* Corresponding author. Tel.: +33-1-55-72-2518; fax: +33-1-55-72-2430. Email address: tony.verbeuren{at}fr.netgrs.com

Received 22 September 2003; revised 12 March 2004; accepted 23 March 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Anoxia followed by reoxygenation (A/R) increases endothelial cell superoxide (O2–) generation which is implicated in E-selectin overexpression. The mechanisms which govern these processes are not fully understood and therefore the goal of our study was to determine the functional importance of NADPH oxidase in the regulation of E-selectin expression in human umbilical veins endothelial cells (HUVECs) submitted to A/R. Methods: O2– production was estimated using lucigenin chemiluminescence and formazan accumulation. NADPH oxidase expression in HUVECs was studied by RT-PCR and Western blot and E-selectin by Northern blot analysis. NF{kappa}B activation was assessed by electrophoretic mobility shift assay. Results: A/R caused an increased O2– production which was inhibited by the superoxide dismutase mimetic M40403 (50 µmol/l), the protein kinase C inhibitor chelerythrine (10 µmol/l), the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 µmol/l) and the NADPH oxidase assembly blocker apocynin (600 µmol/l). At the end of the anoxic period, the mRNA expression and the protein p47phox was increased as compared to normoxic HUVECs. NF{kappa}B activation of anoxic HUVECs was maximal after 1 h of reoxygenation and returned to basal normoxic levels after 2 h of reoxygenation. Apocynin reduced the NF{kappa}B activation at 1 h of reoxygenation. E-selectin mRNA expression was increased after 3 h of reoxygenation of anoxic HUVECs and the SOD mimetic M40403 as well as apocynin prevented this overexpression. Conclusions: Activated NADPH oxidase is a critical enzyme in E-selectin overexpression after A/R of HUVECs. Moreover, A/R increased expression of membranous and cytosolic NADPH oxidase subunits as well as the protein p47phox. Strategies aimed at preventing endothelial NADPH oxidase activation and/or activity may be useful in controlling leukocyte adhesion during ischemia/reperfusion.

KEYWORDS NADPH oxidase; Human endothelial cells; Anoxia/reoxygenation; E-selectin; NF{kappa}B; Apocynin


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Oxidant generation in the endothelium may have an important role in signal transduction, gene expression, cell proliferation, apoptosis, and in the pathophysiology of various diseases including tissue injury from ischemia/reperfusion [1–6]. Exposure of endothelial cells to anoxia/reoxygenation induces leukocyte adhesion by increasing the surface expression of various endothelial cell adhesion molecules [7]. After a transient peak of neutrophil adhesion at 20–30 min of reoxygenation mediated via P-selectin, a second long-lasting phase of adhesion occurs, essentially through E-selectin expression [7]. Xanthine oxidase might be involved in the first transient neutrophil adhesion [7,8] while a NAD(P)H oxidase distinct from the phagocytic NAD(P)H oxidase was suggested to be involved in the second long-lasting period of E-selectin mediated neutrophil adhesion [7–9]. During this second phase, E-selectin expression is believed to be mediated by redox-sensitive kinases which signal the rapid activation of NF{kappa}B [10]. However, a direct relationship between E-selectin expression, NF{kappa}B activation and NADPH oxidase has never been clearly demonstrated.

In human endothelial cells, recent evidence illustrates that the NADPH oxidase complex is present and functional [6,11–19]. NADPH oxidase is a highly regulated membrane-bound enzyme complex that catalyzes the one-electron reduction of oxygen to superoxide anion with the simultaneous oxidation of cytosolic NADPH. All subunits of a phagocyte-type NADPH oxidase are expressed in endothelial cells and include the membrane-bound cytochrome b558, composed of two subunits, p22phox and gp91phox, and four cytosolic regulatory subunits, p47phox, p67phox, p40phox, and the small GTP-binding protein Rac1/Rac2. Assembly of the active NAD(P)H oxidase complex requires a protein kinase C-dependent phosphorylation of p47phox followed by the translocation of the cytosolic factors p47phox, p67phox, and Rac1/Rac2 to the plasma membrane where these components interact with cytochrome b558 [15–18]. In contrast to the neutrophil enzyme, the oxidase in endothelial cells is constitutively active at a low level even in unstimulated cells, and may be present as already fully preassembled complexes [16–18]. After stimulation by various agonists such as angiotensin II or TNF-{alpha}, serine phosphorylation of p47phox is followed by its translocation and stable binding to membrane-bound cytochrome b558. These events initiate NADPH oxidase activation [15,18].

In the present paper, we provide evidence indicating that in anoxic endothelial cells subjected to reoxygenation, superoxide anions are increased and produced by activated NADPH oxidase during the reoxygenation period and may be responsible for NF{kappa}B activation and E-selectin overexpression.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Endothelial cell culture
Human umbilical vein endothelial cells (HUVECs, Clonetics, San Diego, CA, USA) were cultured in endothelial cell basal medium (EBM-2, Clonetics) supplemented with 2% of fetal calf serum (FCS, Clonetics) and endothelial growth medium (EGM-2, Clonetics) at 37 °C and 5% CO2 in a fully humidified incubator. For experiments, HUVECs were subcultured in 6- or 96-well tissue culture plates (Costar) at second or third passage with 5000 cells/cm2. After 4 days, cells were at confluence and the culture medium was removed and replaced by a specific medium (140 mmol/l NaCl, 5.5 mmol/l KCl, 2.5 mmol/l CaCl2, 2.5 mmol/l MgSO4, 20 mmol/l Hepes/Tris pH 7.4) containing 10% FCS. HUVEC monolayers were exposed to anoxia through incubation in a chamber that was continuously purged with 100% N2. After a 3 h period of anoxia, reoxygenation was initiated by exposing the endothelial cells to the incubator atmosphere (5% CO2 and 95% air, 37 °C) during a time indicated for each experiment [9]. Separate experiments from each study were performed with HUVECs from different human sources.

2.2 Production of O2–
The lucigenin-enhanced chemiluminescence was used. Endothelial cells at confluence in 96-well culture plate were incubated in the specific medium described before. After a 3 h period of anoxia, reoxygenation was performed for 15 min. Ten minutes before recording luminescence, NADPH (200 µmol/l, Sigma, St. Louis, MO) and lucigenin (25 µmol/l, Sigma) were added to the cells. After 10 min of dark adaptation, light emission was recorded during 5 s by a Microbetajet (Perkin-Elmer) and was expressed as photons/second/well. Experiments were performed in triplicate. In experiments with O2– scavengers, i.e. superoxide dismutase mimetic M40403 (50 µmol/l, Institut de Recherches Servier) or with inhibitors, i.e. DPI (10 µmol/l, Sigma) and chelerythrine (10 µmol/l, Sigma), these were incubated at the start of anoxic period.

Conversion of nitro blue tetrazolium (NBT, Sigma) to insoluble formazan was used as a measurement of O2– generation as described by Patterson et al. [20]. Just before the start of the 3 h period of reoxygenation of anoxic HUVECs and during the last 3 h of normoxia of normoxic HUVECs, NBT was added to the specific medium at a final concentration of 100 µmol/l. HUVECs were washed two times in Hepes buffered saline solution (HBSS, Clonetics) and trypsinized. The enzymatic reaction was stopped by adding culture medium containing 2% FCS. After centrifugation (2000 x g, 5 min) the cells were washed in cold PBS and formazan was dissolved with 100% pyridine during 3 h at 80 °C. The absorbance was read at 540 nm in a spectrophotometer. Results were expressed in pmol of formazan generated using extinction coefficient {varepsilon}=7.2 x 10–7 ml pmol–1 mm–1 and were corrected for the number of HUVECs per well. Where indicated, apocynin (600 µmol/l, Sigma), diphenyleneiodonium (DPI, 10 µmol/l, Sigma), M40403 (50 µmol/l) or DMSO vehicle were diluted in the specific medium and applied to the HUVECs either before anoxia or before reoxygenation. HUVECs under normoxia were also treated with apocynin and DPI (3 or 6 h before cell collection).

2.3 Quantification of p22phox, p67phox and p47phox mRNA expression by RT-PCR and Southern blot analysis
HUVECs subjected to A/R or normoxia were lysed in 7 mol/l guanidinium isothyocyanate, 0.97% 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS) and 0.01 mol/l Tris–HCl at pH 7.5 (Fluka, Buchs, Switzerland). Total RNA was then isolated by the SV total RNA Isolation System (Promega, Madison, WI). Reverse transcription (RT) was performed with 0.2 and 0.5 µg of total RNA for each condition with first-strand cDNA synthesis kit (Amersham, Buckinghamshire, UK). Two microliters of first-strand cDNA was amplified by using human-specific primers for p22phox (5'-TGG-GCG-GCT-GCT-TGA-TGG-T-3' and 5'-GTT-TGT-GTG-CCT-GCT-GGA-GT-3', revealing a fragment of 316 bp), p67phox (5'-ATG-CCT-TCA-GTG-CCG-TCC-AG-3' and 5'-TGC-TTC-CAG-ACA-CAC-TCC-ATC-G-3', revealing a fragment of 400 bp), p47phox (5'-ACC-TTC-ATC-CGT-CAC-ATC-G-3' and 5'-TCA-AAC-CAC-TTG-GGA-GCT-G-3'revealing a fragment of 250 bp). PCR was performed with pfu polymerase (Stratagene, La Jolla, CA) by using a protocol of amplification of 40 s at 95 °C, 40 s at 57 °C for p22phox, 61 °C for p67phox and 52 °C for p47phox, 90 s at 72 °C during 30 cycles in a PCR system 9700 (Applied Bioscience, Boston, MA). Semi-quantitative PCR was carried out by normalizing all cDNA to β-actin (human β-actin control amplimer set, Clontech, Palo Alto, CA). The products of amplification were resolved on 1.5% agarose gels and denatured in 1.5 mol/l NaCl, 0.5 mol/l NaOH and then neutralized in 1.5 mol/l NaCl, 0.5 mol/l Tris pH 7.2. Gels were then transferred to Hybond membranes (Amersham). The membranes were denatured in 0.4 mol/l NaOH, prehybridized and hybridized at 42 °C in 6 x saline sodium citrate (SSC, 0.1 mol/l sodium citrate, 1 mol/l NaCl, pH 7.0), 5 x Denhardt (0.1% BSA, 0.1% Ficoll and 0.1% polyvinylpyrolidone), 0.1% SDS and 100 µg/ml denatured salmon sperm DNA. The probes used for hybridizations were human-specific oligonucleotides hybridizing inside the amplified region (p22phox: 5'-GTT-TGT-GTG-CCT-GCT-GGA-GT-3', p67phox: 5'-AGG-AAT-TAC-TAT-GTT-CGG-GCG-3', p47phox: 5'-AAA-TGG-CAG-GAC-CTG-TCG-GA-3' and β-actin oligonucleotides, Clontech). The DNA oligonucleotides were labeled by 3' tailing with [{alpha}-32P]-dCTP (Amersham). The membranes were washed and exposed to Biomax film. Autoradiographic bands were quantified by a gel analysis software (Imager, Appligene Oncor, Illkirch, France). The results were normalized with β-actin and expressed as relative area of amplification bands between p22phox, p67phox, p47phox and β-actin bands.

2.4 Quantification of p47phox protein by Western blot analysis
HUVECs submitted to A/R or to normoxia were lysed in PBS supplemented with 1% triton and a protease inhibitor cocktail (Roche). Protein concentration was determined using the Bio-Rad DC protein Assay (Biorad, Hercules, CA) derived from the Lowry procedure. Twenty-five micrograms of reduced and denatured proteins were loaded in 10% polyacrylamide gels and then transferred on Hybond P membranes (Amersham). Membranes were saturated in 40 mM Tris–HCl pH 7.6, 300 mM NaCl, 6% dry mil overnight at 4 °C. An anti-p47phox monoclonal antibody (Transduction Laboratories) was added to the membranes (dilution 1:1000) in 10 mM Tris–HCl pH 7.5, 100 mM NaCl, 2% BSA, 0.1% Tween 20 for 90 min at room temperature. Membranes were washed four times in the same solution. Membranes were then incubated with an anti-mouse IgG conjugated with peroxidase (Jackson, West Grove, PA) for 60 min at room temperature and washed. The bands were revealed by ECL detection system (Amersham). Autoradiographic bands were quantified as described before.

2.5 Immunocytochemical staining of p47phox
HUVECs submitted to normoxia or 3 h anoxia followed by 15 min reoxygenation were fixed in methanol at –20 °C and air-dried. Cells were washed in PBS and incubated for 90 min at room temperature with a mouse anti-human p47phox (1/25, Transduction Laboratories) or a non-relevant mouse IgG in PBS +0.1% BSA. Cells were washed four times in PBS and then incubated with an anti-mouse IgG conjugated with FITC (Jackson) for 45 min at room temperature. After washes, cells were photographed on a Leitz DM-IRB inverted microscope with an epifluorescence system using a numeric camera.

2.6. Electrophoretic mobility shift assay (EMSA)
HUVECs submitted to A/R or HUVECs submitted to normoxia in the presence or not of 0.1 U/ml TNF-{alpha} were scraped in a hypotonic buffer containing 10 mM Hepes pH 7.6, 10 mmol/l KCl, 0.1 mmol/l EDTA, 1 mmol/l 1,4-dithio-DL-threitol (DTT) and 0.5 mmol/l phenylmethanesulfonyl fluoride (PMSF) and incubated for 10 min on ice. The samples were then centrifuged for 1 min at 12,000 x g and the supernatants containing cytosolic proteins were eliminated. The pellets were diluted in 20 mmol/l Hepes pH 7.6, 25% glycerol, 0.4 mol/l NaCl, 1 mmol/l EDTA, 1 mmol/l DTT and 0.5 mmol/l PMSF and incubated for 30 min on ice. After centrifugation, the supernatants containing nuclear proteins were recovered. Protein concentrations were determined using the Bio-Rad DC protein assay.

EMSA was performed with the Gel Shift Assay Systems (Promega). Briefly, 10 µg of nuclear protein extract were incubated in 20% glycerol, 5 mmol/l MgCl2, 2.5 mmol/l EDTA, 2.5 mmol/l DTT, 250 mmol/l NaCl, 50 mmol/l Tris–HCl pH 7.5, 0.25 mg/ml poly(dI–dC) with 10,000 counts per minute (cpm) of NF{kappa}B or AP-1 consensus oligonucleotides labeled with [{gamma}-32P]-dATP (3000 Ci/mmol, Amersham) by 5' tailing with T4 polynucleotide kinase (Amersham). For controls of specificity, samples were incubated with an excess of specific and non-specific unlabeled oligonucleotides. The samples were electrophoresed in a 4% polyacrylamide gel in Tris borate EDTA (TBE) and autoradiographed. Autoradiographic bands were quantified as described above.

2.7. Quantification of E-selectin mRNA expression by Northern blot analysis
Total RNA from HUVECs submitted to A/R or normoxia were isolated as described above. Fifteen micrograms of total RNAs/condition were submitted to electrophoresis through agarose gel containing 2.2 mol/l formaldehyde (Sigma), transferred to Biodyne B membranes (Pall, Portsmouth, UK) and fixed by ultraviolet irradiation. The membranes were prehybridized and hybridized at 42 °C in 6 x SSC, 5 x Denhardt solution, 0.1% SDS and 100 µg/ml denatured salmon sperm DNA. E-selectin hybridization was performed with a cocktail of 30-mer oligonucleotides complementary to human E-selectin cDNA (R&D Systems, Abingdon, UK), labeled with [{alpha}-32P]-dCTP (3000 Ci/mmol, Amersham) by 3' tailing with terminal deoxyribonucleotidyl transferase (Amersham) to a specific activity greater than 108 cpm/ng DNA and used at 2 x 105 cpm/ml. The membranes were washed in 2 x SSC, 0.1% SDS at room temperature and 42 °C to increase stringency and exposed to Biomax film at –70 °C. The results were normalized with a β-actin hybridization performed with a DNA probe of a 2-kb full human β-actin cDNA (Clontech) labeled with [{alpha}-32P]-dCTP by random priming (Amersham). Autoradiographic bands were quantified as described above. The results are expressed as relative area of mRNA bands between E-selectin and β-actin mRNA bands.

2.8. Statistical analysis
Statistical analysis (Student's paired or unpaired t-test and one-way ANOVA with Dunnett) was performed using Prism software. P<0.05 was considered to be significant for the two tests.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Detection of O2– in HUVECs subjected to A/R
Using lucigenin-enhanced chemiluminescence in the presence of exogenous NADPH, O2– production by HUVECs subjected to normoxia was decreased by 78±9% in the presence of the flavin protein inhibitor DPI (10 µmol/l), by 58±21% in the presence of the cell-permeable SOD mimetic M40403 (50 µmol/l) but not modified (12±7%) by a broad spectrum inhibitor of protein kinase C chelerythrine (10 µmol/l). HUVECs subjected to A/R produced significantly more O2– than normoxic cells (Fig. 1). Superoxide production by HUVECs subjected to A/R was strongly decreased by 84±5%, 83±4% and 75±5% with DPI, M40403 and chelerythrine, respectively (Fig. 1, p<0.05 paired t-test, n=5).


Figure 1
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  		Fig. 1 NADPH-dependent superoxide anion production by HUVECs submitted to normoxia or anoxia/reoxygenation (A/R) measured by lucigenin-enhanced chemiluminescence and expressed as mean photons/second/well. The flavoprotein inhibitor DPI (10 µmol/l), the protein kinase C inhibitor chelerythrine (CHE, 10 µmol/l) and the superoxide dismutase mimetic M40403 (50 µmol/l) inhibit superoxide generation by HUVECs submitted to A/R (°p<0.05 compared with normoxia, *p<0.05 compared with A/R, paired t-test, n=5).

 
To evaluate the A/R-induced oxidative stress, the production of O2– was also assessed using the NBT reduction assay which quantified the formazan accumulation during a 3 h period of reoxygenation. Normoxic HUVECs generated formazan at the rate of 6.9±1.7 pmol/min/106 cells. When HUVECs were submitted to anoxia, the rate of formazan generation during reoxygenation significantly increased to 11.8±4.0 pmol/min/106 cells (p<0.05, Student's paired t-test, n=6). To assess the specificity of the assay, the cell-permeable superoxide dismutase mimetic M40403 at 50 µmol/l was used. In these conditions, formazan accumulation was strongly decreased to 2.5±0.9 and 3.0±1.1 pmol/min/106 cells in HUVECs subjected to 6 h normoxia and 3 h of anoxia followed by 3 h of reoxygenation, respectively (p<0.05, Student's paired t-test, n=4). DPI at 10 µmol/l decreased formazan accumulation by 71±8% and 63±3% in normoxic HUVECs treated for 3 h (Fig. 2A) and 6 h (Fig. 2B), respectively. In contrast apocynin at 600 µmol/l had no effect whatever the duration of its administration. In HUVECs submitted to A/R, DPI added before anoxia (6 h before cell collection) or before reoxygenation (3 h before cell collection) strongly reduced formazan accumulation by 67±5% and 82±4%, respectively. Under similar conditions, apocynin added before anoxia or reoxygenation of HUVECs also significantly decreased formazan accumulation by 30±6% and 49±9%, respectively (P<0.05, Student's paired t-test).


Figure 2
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  		Fig. 2 Superoxide anion generation evaluated by formazan accumulation is increased in HUVECs submitted to anoxia/reoxygenation (A/R). The flavoprotein inhibitor DPI at 10 µmol/l inhibits superoxide generation in HUVECs submitted to normoxia and A/R whatever its time of introduction at 6 h (panel A) or 3 h (panel B) before cell collection. Apocynin at 600 µmol/l inhibits superoxide generation only after A/R, at 6 h (panel A) or 3 h (panel B) before cell collection. The results are expressed as percentage of the rate of formazan generation in control conditions when HUVECs are submitted to normoxia (100%=6.9+1.7 pmol/min/106 cells). Values are mean±SEM of six experiments for panel A and four experiments for panel B. *p<0.05 vs. control anoxia/reoxygenation, paired t-test. °p<0.05 vs. control normoxia, paired t-test.

 
3.2 Immunocytochemical staining of p47phox
To assess whether a change in p47phox distribution could be detected in HUVECs after A/R, we undertook immunocytochemical staining. In normoxic HUVECs, p47phox had a mainly perinuclear and rather punctuate distribution (Fig. 3B). After A/R, the perinuclear p47phox labeling appeared more intense and organized consistent with a redistribution of p47phox (Fig. 3C) while the non-specific labeling showed a very slight staining (Fig. 3A).


Figure 3
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  		Fig. 3 Immunocytochemical staining for p47phox protein in HUVECS submitted to normoxia (panel B) and A/R (panel C). In normoxic HUVECs (panel B), p47phox had a mainly perinuclear and rather punctuate distribution. After A/R, the perinuclear p47phox labeling is more intense and organized consistent with a redistribution of p47phox (panel C). The non-specific binding performed with a non-relevant primary antibody is shown on panel A with cells submitted to A/R.

 
3.3 Induction of NADPH oxidase p22phox, p47phox and p67phox subunits by A/R in HUVECs
Using a semi-quantitative RT-PCR analysis, a comparison of the NADPH oxidase subunit mRNA expression in normoxic HUVECs and in HUVECs submitted to 3 h of anoxia followed by 0, 30 or 180 min of reoxygenation was performed (Figs. 4A,B,C). A significant mRNA overexpression of the three subunits of NADPH oxidase p22phox, p47phox and p67phox was noted after 3 h of anoxia without reoxygenation (P<0.05, one-way ANOVA with Dunnett). After 3 h of reoxygenation, a significant mRNA overexpression of p47phox and p67phox was observed (p<0.05, one-way ANOVA with Dunnett). In comparison to normoxic HUVECs p47phox protein quantified by Western blot was significantly increased after 3 h of anoxia (P<0.05, one-way ANOVA with Dunnett, Fig. 4D).


Figure 4
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  		Fig. 4 Quantification of p22phox mRNA (panel A), p67phox mRNA (panel B) p47phox mRNA (panel C) and p47phox protein (panel D). The mRNA expression of the 3 subunits of NADPH oxidase is quantified by RT-PCR and significantly increased after 3 h of anoxia (A) in comparison to normoxia (N) (p<0.05, one-way ANOVA with Dunnett, n=5). The expression of p47phox and p67phox mRNA is also significantly increased after 3 h of reoxygenation (*p<0.05, one-way ANOVA with Dunnett, n=5). The p47phox protein of the NADPH oxidase is quantified by Western blot and is significantly increased after 3 h of anoxia (A) in comparison to normoxia (N) (*p<0.05, one-way ANOVA with Dunnett, n=5).

 
3.4. NF{kappa}B activation in HUVECs submitted to A/R: effect of apocynin
To investigate the kinetics of A/R-induced NF{kappa}B activation, anoxic HUVEC monolayers were exposed to varying durations of reoxygenation and EMSAs were performed with nuclear extracts. Control experiments were performed with normoxic HUVECs treated with 0.1 U/ml TNF-{alpha} for 4 h. Under these control conditions, one specific NF{kappa}B nucleoprotein adduct appeared which was completely abolished by an excess of specific unlabeled oligonucleotides (data not shown). Nuclear NF{kappa}B DNA binding in anoxic HUVECs reached a maximal value after 1 h of reoxygenation and returned to control levels 2 h after the onset of reoxygenation (Fig. 5A). Apocynin at 600 µmol/l added before anoxia significantly decreased nuclear NF{kappa}B DNA binding in anoxic HUVECs submitted to a 1 h reoxygenation period to a level similar to that noted in normoxic HUVECs (Fig. 5B). EMSA were also performed with the same nuclear extracts but with consensus oligonucleotides of the AP-1 region. No band shift was observed under any condition of HUVEC culture (data not shown).


Figure 5
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  		Fig. 5 Effect of normoxia (N) and anoxia followed by different periods of reoxygenation (A/R, 0.5, 1, 2 and 4 h) on NF{kappa}B activation in HUVECs (panel A). The effect of apocynin (600 µmol/l) is shown on normoxic cells and cells subjected to 1 h reoxygenation and is quantified by a gel analysis software (panel B). The specifically shifted band which represent activated NF{kappa}B is shown (panel A). Cells were harvested for preparation of nuclear extracts. Nuclear extracts (10 µg) were used for electrophoretic mobility shift assays (EMSA) using labeled double-stranded consensus NF{kappa}B as described in Methods. The picture represents one typical experiment out of five (panel A). Gel analysis results (panel B) are expressed as percentage of the peak height of NF{kappa}B band found under normoxic conditions. Values are the mean±SEM of five experiments. *p<0.05 for anoxia/reoxygenation vs. normoxia, paired t-test. $p<0.05 for anoxia/reoxygenation vs. anoxia/reoxygenation plus apocynin, paired t-test.

 
3.5. E-selectin expression in HUVECs submitted to A/R: effect of apocynin and M40403
E-selectin mRNA expression in HUVECs was quantified using Northern blot analysis. When compared to normoxic HUVECs, E-selectin mRNA expression significantly increased in anoxic HUVECs reoxygenated for 3 h (Fig. 6). Apocynin at 600 µmol/l added before anoxia strongly decreased E-selectin mRNA overexpression induced by A/R but had no significant effect on E-selectin mRNA expression in normoxic HUVECs (Fig. 6). The SOD mimetic M40403 at 50 µmol/l, added 6 h before collection, also decreased significantly E-selectin mRNA overexpression induced by A/R but had no effect during normoxia (Fig. 6).


Figure 6
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  		Fig. 6 E-selectin mRNA expression is increased in HUVECs submitted to anoxia/reoxygenation (A/R). This effect is decreased by the presence of apocynin and M40403. Total RNA of HUVECs submitted to A/R or submitted to normoxia were extracted and hybridized to human E-selectin and β-actin probes. All results were normalized to β-actin and are expressed as percentage of the ratio found with HUVECs subjected to A/R (100% corresponded to a ratio of 0.65±0.18). Values are mean±SEM of five experiments. ***P<0.001 for A/R vs. normoxia, unpaired t-test. °°°P<0.001 for A/R plus apocynin or A/R plus M40403 vs. A/R, unpaired t-test, n=5).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
A/R induced neutrophil adhesion on HUVECs through E-selectin overexpression and NF{kappa}B activation [7,10]. In endothelial cells A/R is associated with an increased generation of reactive oxygen species produced at least in part by NADPH oxidase [16,21]. The results of the present study show that reoxygenation of anoxic HUVECs produces an increased level of O2– inhibited by the SOD mimetic M40403 and the NADPH oxidase assembly blocker apocynin. Both compounds also normalize E-selectin expression suggesting that oxidant stress generated by NADPH oxidase activated at reoxygenation is responsible for E-selectin overexpression.

Using lucigenin-enhanced chemiluminescence in the presence of NADPH, we found an increased O2– production at the reoxygenation of anoxic HUVECs in comparison to normoxic HUVECs. This production was inhibited by the SOD mimetic M40403 as well as the NADPH oxidase inhibitor DPI. M40403 is a manganese-based superoxide dismutase that is a true mimetic of the native enzyme; the compound is very selective, cell-permeable and does not react with other biologically relevant oxidizing species [22].

In TNF-{alpha} induced activation of NADPH oxidase in endothelial cells, translocation of cytosolic subunits to the membrane is initiated by phosphorylation of p47phox by protein kinase C{xi} [15]. Thus, we looked at the effect of a broad spectrum inhibitor of protein kinase C, chelerythrine, on oxidant generation by HUVECs. Chelerythrine prevented the O2– overproduction by reoxygenated HUVECs suggesting that oxidant stress induced by reoxygenation necessitates phosphorylation of p47phox by protein kinase C. When anoxic HUVECs were reoxygenated, a significant increase in formazan accumulation was found in comparison to normoxic HUVECs. Under these conditions, the SOD mimetic M40403 strongly inhibited the formazan accumulation suggesting that our assay is sensitive for oxidant generation. Apocynin also decreased formazan accumulation to the normoxic level while it has no effect during normoxia. Apocynin reacts with intracellular peroxidases and may form a symmetrical dimer that impedes assembly of the p47phox subunit with the cytochrome b558 [19,23]. Moreover, apocynin added before anoxia or before reoxygenation, decreases O2– production to comparable levels suggesting that activation of NADPH oxidase occurs during the reoxygenation. Thus, similarly to endothelial cells stimulated by angiotensin II or TNF-{alpha}, reoxygenation of anoxic HUVECs seems to involve p47phox phosphorylation, its translocation and assembly to the membrane-bound cytochrome b558 followed by an increase oxidant generation [15,18]. Immunocytochemical staining of p47phox distribution in normoxic and reoxygenated HUVECs is in accordance with these conclusions. Indeed it shows on endothelial cells subjected to anoxia followed by 15 min of reoxygenation, a more organized reticular labeling in the perinuclear region of the cell as described with endothelial cells stimulated by angiotensin II [18].

To address the effects of A/R on mRNA expression of NADPH oxidase subunits in HUVECs, mRNA transcripts of p22phox, p47phox, p67phox were analyzed using RT-PCR. At reoxygenation after 3 h of anoxia, transcripts for the three subunits were increased. P47phox protein being also increased, our results show that 3 h of HUVECs anoxia induced an increased synthesis of the p47phox subunit at reoxygenation. Thus, A/R activates NADPH oxidase in endothelial cells but also upregulates its subunit expression. These results suggest that A/R not only activates NADPH oxidase but also induces a slight but significant increase of the subunits which may help to sustain a long-term superoxide production.

A major goal of our study was to determine whether the increased superoxide anion production by NADPH oxidase during the reoxygenation of HUVECs was responsible for NF{kappa}B activation and E-selectin overexpression. We first studied NF{kappa}B activation using the electrophoretic mobility shift assay [24]. By analysis of the kinetics, we detected maximal NF{kappa}B activation and translocation at 1 h after reoxygenation of anoxic HUVECs; these parameters decreased rapidly and returned to baseline levels at 2 h. Similar results were found by Kokura et al. [10] who reported that 30 min of reoxygenation induces a degradation of I{kappa}B followed by a rise at 60 min. Interestingly, apocynin abolished the NF{kappa}B activation noted at 1 h of reoxygenation suggesting that oxidant stress generated by activated NADPH oxidase may participate to the presence of active NF{kappa}B in the nucleus.

In our study, we demonstrated that E-selectin mRNA was increased after 3 h of reoxygenation, this increase was fully inhibitable by the SOD mimetic M40403 as well as apocynin. These results strongly suggest that the oxidant stress generated by activated NADPH oxidase at reoxygenation of anoxic HUVECs is responsible for E-selectin overexpression. The SOD mimetic M40403 has shown protective and beneficial roles in experimental models of ischemia/reperfusion injuries in the heart, liver, kidneys and brain [22]. In these pathophysiological conditions, leukocyte rolling through P- and E-selectin in post-capillary venules are an essential prerequisite before firm leukocyte adhesion which induces the reperfusion injury. Our findings that both M40403 and apocynin could decrease the long-lasting E-selectin expression on endothelial cells through the blockade of the oxidative stress generated by NADPH activation at reoxygenation explain at least in part the mechanism of action of these agents in these experimental models.

In conclusion, the present study implicates activated NADPH oxidase as the critical enzyme that increases A/R-induced oxidant generation in endothelial cells. A/R also increased the mRNA expression of different NADPH oxidase subunits and specifically the p47phox protein. Dismutation of O2– or inhibition of NADPH oxidase assembly prevented A/R-induced E-selectin overexpression through NF{kappa}B activation. Thus, strategies aimed at preventing endothelial NADPH oxidase activation or activity such as apocynin and M40403 may be useful in controlling leukocyte adhesion in human diseases where ischemia/reperfusion plays an important role.


   Notes
 
Time for primary review 20 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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