p22^phox C242T SNP Inhibits Inflammatory Oxidative Damage to Endothelial Cells and Vessels

Abstract

Background

T NADPH oxidase, by generating reactive oxygen species, is involved in the pathophysiology of many cardiovascular diseases and represents a therapeutic target for the development of novel drugs. A single-nucleotide polymorphism (SNP) C242T of the p22^phox subunit of NADPH oxidase has been reported to be negatively associated with coronary heart disease (CHD) and may predict disease prevalence. However, the underlying mechanisms remain unknown.

Methods and Results

Using computer molecular modelling we discovered that C242T SNP causes significant structural changes in the extracellular loop of p22^phox and reduces its interaction stability with Nox2 subunit. Gene transfection of human pulmonary microvascular endothelial cells showed that C242T p22^phox reduced significantly Nox2 expression but had no significant effect on basal endothelial O₂^.- production or the expression of Nox1 and Nox4. When cells were stimulated with TNFα (or high glucose), C242T p22^phox inhibited significantly TNFα-induced Nox2 maturation, O₂^.- production, MAPK and NFκB activation and inflammation (all p<0.05). These C242T effects were further confirmed using p22^phox shRNA engineered HeLa cells and Nox2^-/- coronary microvascular endothelial cells. Clinical significance was investigated using saphenous vein segments from non CHD subjects after phlebectomies. TT (C242T) allele was common (prevalence of ~22%) and compared to CC, veins bearing TT allele had significantly lower levels of Nox2 expression and O₂^.- generation in response to high glucose challenge.

Conclusions

C242T SNP causes p22^phox structural changes that inhibit endothelial Nox2 activation and oxidative response to TNFα or high glucose stimulation. C242T SNP may represent a natural protective mechanism against inflammatory cardiovascular diseases.

Introduction

Single-nucleotide polymorphisms (SNP) are one base variations in DNA sequences that occur in at least 1% of the population¹. Although most SNPs have little or no effect on human health, some can predispose individuals to disease or have a major impact on the physiological response to environmental challenges or to drugs^{1, 2}. Vascular endothelial cells express constitutively a Nox2 (also called gp91^phox) containing NADPH oxidase, which by generating superoxide (O₂^.-) plays an important role in oxidative stress-related endothelial dysfunction and cardiovascular diseases (CVD)^3–7. The Nox2 catalytic subunit requires p22^phox in a 1:1 ratio to form the cytochrome b₅₅₈ complex to produce O₂^.-, and structural changes of p22^phox may affect its interaction with Nox2. In the context of Nox2-derived oxidative stress and CVD, SNPs of the p22^phox have attracted significant attention recently.

The p22^phox is encoded by the CYBA gene located on the long arm of chromosome 16 at position 24. It spans 8.5 kb and is composed of six exons and five introns encoding an open reading frame of approximately 600 bp⁸. The p22^phox has three structural domains: 1) a hydrophobic N-terminal domain consisting of three transmembrane helices; 2) a hydrophilic extracellular region; and 3) a cytosolic tail featuring a proline rich region (PRR)^{9, 10}. So far, seven SNPs of the p22^phox gene have been reported i.e. C242T, A640G; C549T; A930G; A675T; C852G and C536T¹¹, and only two of them (C242T and C549T) are translated into the protein. The C549T SNP changes an Ala¹⁷⁴ to Val¹⁷⁴ without any reported significant functional effect¹¹. In contrast, the C242T SNP changes His⁷² to Tyr⁷² that is located in the extracellular loop of the putative Nox2 binding region¹² and has been reported in several studies to be negatively associated with CVD, such as hypertension, atherosclerosis, and myocardial infarction and to reduce oxidative burst in neutrophils^11,13–15. However, others have found that the C242T variant had no effect on cardiovascular disease progression¹⁶ or even increased ROS production in CVD arteries^17,18. Given the contradictory evidence, it is important to elucidate the molecular mechanism responsible for the effects of p22^phox C242T SNP on inhibiting endothelial Nox2 activity and vascular oxidative stress in order to ascertain its effect on CVD.

In this study, we used computer structural modeling plus molecular and biochemical approaches to elucidate the mechanism of p22^phox C242T SNP inhibition of endothelial Nox2 activation and oxidative stress in response to TNFα or high glucose stimulation. Results were further confirmed using engineered p22^phox depleted (p22^phox-depl) HeLa cells, primary coronary microvascular endothelial cells (CMEC) isolated from Nox2^-/- mice and saphenous vein segments of patients without history of coronary heart diseases (CHD) after phlebectomies. We report for the first time that C242T SNP is linked to the p22^phox extracellular domain morphological change that interferes with Nox2 binding stability and inhibits endothelial oxidative stress and inflammatory response to TNFα or high glucose challenges.

Materials and methods

The in silico p22^phox model was generated as reported previously by incorporating data from online prediction servers with the molecular operating environment (MOE; Chemical Computing Group Inc., Canada) and subjected to energy minimization using the AMBER99 forcefield^9,10. The docking of p22^phox with Nox2 peptides was performed in the MOE¹⁰. HapMap analysis of the CYBA gene was performed using the International HapMap Project (HapMap phase II+III; dbSNP b126) and six common CYBA SNPs were chosen from the National Centre for Biotechnology Information (NCBI) SNP database (dbSNP). Haplotype association was calculated using the Haplo-view analysis program (version 4.2) in the northern and western European population (CEU). Human pulmonary microvascular endothelial cells (HPMEC-ST1.6R) were a kind gift from Dr R. Unger (Johannes Gutenberg University, Germany)¹⁹. Mouse CMEC were isolated from the hearts of ~12 week old Nox2^-/- and WT mice and cultured as described previously^20,21. The shRNA p22^phox (p22^phox-depl) and shRNA scrambled control HeLa cells were generated as described previously²² in the laboratory of Professor Krönke (University of Cologne, Germany). Segments of human saphenous vein were collected from 36 patients (without history of CHD) undergoing phlebectomies at a specialist vein unit. Informed consents were obtained from the patients and the project was approved by the local NHS and the university ethical committees according to UK regulation. The IRB approval was obtained according to the guidelines noted in the Circulation Instruction to Authors. The C to T substitution at position 242 in the CYBA coding sequence was examined as described previously²³. ROS production was measured using four independent methods as described previously²⁴: 1) Lucigenin-chemiluminescence; 2) DHE fluorescence HPLC assay with or without superoxide dismutase–polyethylene glycol (PEG-SOD)^4,25; 3) DHE flow cytometry; and 4) DCF fluorescence.

See the online-only Data Supplement for a full description of materials and methods.

Statistics

Data were presented as means ± SD in figures. For cell culture experiments, results were taken from at least 3 independent cell cultures and gene transfections for each condition. In the case of CMEC isolation, 6 mice/per group were used for each isolation and the data presented were the means ± SD from at least 3 separate CMEC isolations. Comparisons were made by ANOVA with Bonferroni post hoc correction or as indicated in the figure legend. Values of P<0.05 were considered statistically significant.

Results

Structural changes in p22^phox associated with C242T SNP

Computer modeling was used to investigate potential protein structural changes linked to p22^phox C242T SNP⁹. Our consensus p22^phox model showed that His⁷² (WT p22^phox) is located within the extra-cellular region of the p22^phox (Figure 1A), which has been identified to be essential for interaction with Nox2^12,26. The histidine residue contains a polar hydrophilic imidazole in comparison to the aromatic and hydrophobic tyrosine residue. The substitution of His⁷² to Tyr⁷² alters the conformation of the extracellular region as shown by the backbone side-view (Figure 1A, left panels) and top-view (Figure 1A, right panels). Root mean squared deviation (RMSD) calculations of extracellular structures of WT and C242T p22^phox following energy minimization revealed significant difference between two structures (Figure 1C).

Figure 1. Computer modelling of p22^phox structural changes associated with C242T SNP.

(A) Morphological differences between WT and C242T p22^phox structures (ribbon presentation). (B) Docking of a Nox2-peptide (222-HGAERIVR-229) (skeleton in left panels and silver-space-fill in right panels) with p22^phox extracellular domain (ribbon). Nox2 peptide interacts and forms an H-bond with the N⁸⁶ (ball and stick presentation) of WT p22^phox but not with the N⁸⁶ of C242T p22^phox. (C) RMSD calculations of the extracellular domain structural differences between WT and C242T p22^phox following energy minimization. n=3 independent investigators. ^*p<0.05 for C242T values versus WT values (Mann-Whitney U-test). (D) Linkage disequilibrium (LD) plot from Haplo-view of common genotyped SNPs within the CYBA (p22^phox) gene. The darker a diamond appears, the greater the correlation (r² × 100) between the respective genotyped CYBA polymorphisms. The C242T SNP (rs4673) is outlined.

We next performed protein/protein docking of p22^phox with a known Nox2 peptide (222-HGAERIVR-229) that corresponds to the Nox2 extracellular region involved in binding to p22^phox in a previous report²⁷. We found that the Nox2 peptide interacted successfully with the WT p22^phox extracellular domain and formed a hydrogen-bond between Arg²²⁶ of the Nox2 and Asn⁸⁶ of the p22^phox (Figure 1B, upper panels). However, this interaction was lost in the C242T model (Figure 1B, lower panels). Further docking experiments were performed using two Nox2 peptides (peptide 1: 154-NFARKRIKNPEGGLY-168; peptide 2: 222-HGAERIVRG-230), which are located in the Nox2 extracellular domain and have been reported previously to be recognized by a Nox2 antibody (7D5)²⁷ (Figure 2). We found several important hydrogen bonds (labeled by H) formed between the Nox2 peptides and the WT p22^phox configuration, which further illustrated the importance of Nox2 Arg²²⁶ in stabilizing the interaction with WT p22^phox. However, these links were disrupted by the C242T p22^phox configuration. We also performed HapMap analysis of the CEU (European) population, and found that the C242T SNP is only in strong correlation (r² = 0.99) with one SNP located within a non-coding region of intron 4 (rs12933505; Figure 1D). Moreover, the genotyped allele frequency of the CC (WT) and the CT and TT variants is 53% and 47%, respectively. This confirms the functional effect of C242T SNP is independent of other known p22^phox SNPs at the protein level.

Figure 2. Docking of two Nox2 peptides with p22^phox extracellular domain.

Images are shown as a tiled 45 degrees side-view/top-view. Two Nox2 peptides (peptide 1: 154-NFARKRIKNPEGGLY-168; peptide 2: 222-HGAERIVRG-230) used for the docking have been reported previously to be the extracellular epitopes of Nox2 recognised by an antibody (7D5) to Nox2. WT p22^phox configuration forms several hydrogen bonds (short grey dashed lines labelled by H) with Nox2 peptides. The location of Nox2 Arg²²⁶ is indicated. However, these interactions were lost in the C242T p22^phox configuration. Red ribbons represent alpha helix.

Effect of C242T SNP on endothelial Nox2 activity

The functional effect of p22^phox C242T SNP on human endothelial Nox2 activity was firstly investigated by site-directed mutagenesis followed by gene transfection of human pulmonary microvascular endothelial cells (HPMECs) with or without acute TNFα (100 unit/ml; 30 min) stimulation. HPMECs displayed low levels of O₂^.- production at basal conditions (without TNFα stimulation) whereas TNFα increased significantly the levels of O₂^.- production in cells either transfected with an empty vector or with WT p22^phox, without significant differences between them (Figure 3A). However, compared to WT p22^phox, C242T p22^phox had no significant effect on endothelial basal O₂^.- production, but did significantly inhibit TNFα-induced O₂^.- production (Figure 3A; right panel). The enzymatic sources of TNFα-induced O₂^.- production in vector or WT p22^phox transfected cells was confirmed using human Nox2ds-tat peptide (a specific inhibitor of Nox2)²⁸ and different enzyme inhibitors (Figure 3B). TNFα-induced O₂^.- production was inhibited significantly by Nox2ds-tat but not by NoxA1ds peptide (a specific inhibitor of Nox1, see Supplemental Figure 1) and was completely abolished by an O₂^.- scavenger (Tiron; 10mmol/L) which confirmed the detection of O₂^.-. It was significantly inhibited by apocynin (100µmol/L, a Nox2 inhibitor) and diphenyliodium (DPI 20µmol/L, a flavoprotein inhibitor), but not by L-Nitroarginine-methyl-ester (L-NAME 100µmol/L, an eNOS inhibitor), oxypurinol (100µmol/L, a xanthine oxidase inhibitor) or rotenone (50µmol/L, a mitochondrial electron transport chain inhibitor) (Figure 3B). Compared to WT p22^phox transfected cells, C242T p22^phox had no significant effect on Nox4 activity (H₂O₂ production) as shown using HEK293 cells (no endogenous Nox4 expression) co-transfected with Nox4 plus p22^phox (Supplemental Figure 2). These data further confirmed a crucial role of Nox2-containing NADPH oxidase in TNFα-induced endothelial O₂^.- production.

Figure 3. The effect of p22^phox C242T SNP on HPMEC ROS production.

(A) Kinetic measurement of O₂^.- production. Area under curve (AUC) was calculated for each sample. ^*p<0.05 for TNFα AUC values verses vehicle AUC values analyzed by unpaired t-test with Welch's correction. (B) Inhibition of O₂^.- production measured by lucigenin-chemiluminescence. Left panel: Nox2ds-tat peptide; Right panel: Effects of different enzyme inhibitors presented as percentages to TNFα values without inhibitor (filled bar). ^*p<0.05 for indicated values versus vehicle values under the same gene transfection. †p<0.05 for indicated values verses TNFα values (filled bar) under the same gene transfection. (C) O₂^.- production (2-OH-E) measured by DHE-HPLC. The amount of 2-OH-E was quantified against a standard curve and expressed as nmol/mg protein. (D) Intracellular ROS production examined by DCF fluorescence microscopy. Tiron was used to confirm the detection of O₂^.-. (C-D) ^*p<0.05 for TNFα values verses vehicle values under the same gene transfection. †p<0.05 for indicated values versus WTp22 TNFα values. Sample size (A-C): n=4 independent experiments; (D): n=3 independent experiments.

The inhibitory effect of p22^phox C242T on TNFα-induced endothelial O₂^.- production was further confirmed by two independent methods: DHE high-performance liquid chromatography (HPLC) detection of 2-OH-E⁺ (Figure 3C) and the tiron-inhibitable DCF-fluorescence microscopy using intact adherent endothelial cells (Figure 3D).

Effects of C242T p22^phox on endothelial Nox subunit expression and binding to p22^phox

The effects of p22^phox C242T SNP on the basal levels of NADPH oxidase subunit expression was examined by Western blot. Compared to vector-transfected cells, the levels of p22^phox expression were significantly increased in cells transfected with WT or C242T p22^phox, which confirmed the success of gene transfection (Figure 4A). Nox1 expression was nearly undetectable. We found that C242T p22^phox reduced significantly Nox2 expression without significant effect on Nox4 or Nox2 regulatory subunits i.e. p47^phox, p67^phox, p40^phox and Rac1/2 and Nox4 as compared to cells transfected with vector or WT p22^phox (Figure 4A).

Figure 4. NADPH oxidase subunit expression, Nox2 maturation and binding to p22^phox in HPMEC.

A) Western blots. Optical densities (OD) of protein bands of p22^phox, Nox1, Nox2, Nox4, p47^phox, p67^phox, p40^phox and Rac1/2 were quantified and normalized to the levels of α-tubulin (loading control) detected in the same samples. (B) Flow cytometry for the binding of Nox2 antibody (7D5). ^*p<0.05 for indicated values versus to vector values. †p<0.05 for indicated values versus WT values. (C) Left panel: p22^phox was immunoprecipited (IP) followed by immunoblotting (IB) of Nox1, Nox2 and Nox4. Right panels: Optical densities (OD) of protein bands were quantified and normalized to the levels of total p22^phox detected in the same samples. ^*p<0.05 for indicated values versus vehicle values under the same gene transfection. †P<0.05 for indicated values versus WT TNFα values. n=4 independent experiments.

Nox2 antibody 7D5 recognizes specifically an extracellular domain of human Nox2 and the levels of antibody 7D5 binding have been successfully used previously to indicate the levels of Nox2 maturation (cell surface membrane expression and stable cytochrome b formation) in cells^27,29,30. Using flow cytometry, we found that TNFα increased significantly antibody 7D5 binding to endothelial cells, and C242T p22^phox reduced significantly the antibody 7D5 binding at both basal and in response to TNFα stimulation as compared to cells transfected with vector or WT p22^phox (Figure 4B). We then examined the effects of C242T p22^phox on p22^phox binding to Nox1, Nox2 and Nox4 by immuno-pull-down assay using p22^phox antibody coated beads (Figure 4C). Supplemental Figure 3 showed the molecular weights on full gel presentation, and the protein levels detected in the whole cell homogenates were shown in Supplemental Figure 4. The levels of Nox1 pulled-down by p22^phox were barely detectable, and we could not see a significant effect of C242T p22^phox on Nox1. Under basal condition (vehicle stimulated), there was no significant difference in the levels of Nox2 or Nox4 pulled-down by p22^phox beads between cells transfected with vector or WT or C242T p22^phox (Figure 4C). TNFα stimulation (24 h) increased significantly the levels of Nox2 and reduced the levels of Nox4 pulled-down by p22^phox beads in vector or WT p22^phox transfected cells. However, in cells transfected with C242T p22^phox, TNFα-induced Nox2 binding to p22^phox was significantly reduced, while the levels of Nox4 binding to p22^phox remained at vehicle levels as compared to vector or WT p22^phox transfected cells. Taken together our results strongly suggested that p22^phox C242T morphology inhibits specifically the interaction stability between Nox2 and p22^phox and reduced TNFα-induced Nox2 activation in endothelial cells.

Effects of C242T p22^phox on TNFα-induced p22^phox/p47^phox binding and redox signaling through MAPKs and NF-κB in endothelial cells

p22^phox, through its intracellular C-terminal domain, anchors p47^phox to Nox2 in the membrane to activate NADPH oxidase. In our computer model, C242T SNP was predicted not to affect p47^phox membrane translocation and binding to p22^phox. To confirm this, we prepared endothelial cell membrane fraction and examined for the membrane expression of p47^phox by immunoblot (Figure 5A). Compared to vehicle control cells, TNFα stimulation significantly increased the levels of p47^phox membrane expression without significant difference between vector and WT or C242T p22^phox transfected cells. We then immuno-precipitated p47^phox and examined the levels of p22^phox pulled down by p47^phox beads and the levels of p47^phox phosphorylation (Figure 5B). Compared to vehicle control cells, TNFα stimulation increased the levels of p22^phox/p47^phox complex formation and p47^phox phosphorylation without significant difference between cells transfected with vector and WT or C242T p22^phox. Membrane ROS production was shown in the Supplemental Figure 5. Once again, our data confirmed that the C242T p22^phox inhibition of TNFα-induced endothelial O₂^.- production is specific to the Nox2 catalytic subunit.

Figure 5. TNFα-induced p47^phox membrane translocation and binding to p22^phox, redox signaling and cell apoptosis in HMEC.

(A) Western blot. The optical densities (OD) of protein bands were quantified and normalized to the levels of CD31 (an endothelial membrane marker) detected in the same samples. (B) p47^phox was immunoprecipitated (IP) and detected by Western blot for the presence of p22^phox and p47^phox phosphorylation (P-p47). The optical densities (OD) of protein bands were quantified and normalized to the levels of total p47^phox (T-p47) detected in the same samples. (C) Western blots. The phospho-bands were quantified and normalized to the total levels of the same protein detected in the same samples. (D) Right panels: Annexin-V/propidium iodide detection of cell apoptosis (upper right square) by flow cytometry. Apoptotic cells were presented as a percentage of total cells (20,000). ^*p<0.05 for indicated value versus vehicle values under the same gene transfection. †p<0.05 for indicated values versus WTp22 TNFα values. n=4 independent experiments..

Next, we examined the effect of C242T p22^phox on TNF_α-induced redox activation of extracellular regulated kinase-1/2 (ERK1/2), p38^MAPK and NFκB in endothelial cells (Figure 5C). Compared to vehicle treated cells, TNFα stimulation increased significantly the levels of ERK1/2, p38^MAPK and NFκB^Ser311 phosphorylation in WT p22^phox transfected cells (Figure 5C), and these were accompanied by significant increases in endothelial apoptosis detected by annexin-V flow cytometry (Figure 5D). Interestingly, these TNFα effects were significantly inhibited in cells transfected with C242T p22^phox. The levels of JNK phosphorylation was very low with no significant changes being detected after TNFα stimulation (data not shown).

p22^phox C242T inhibition of TNFα-induced endothelial vascular cell adhesion molecule-1 (VCAM-1) expression and inflammation

TNFα plays a key role in the pathogenesis of atherosclerosis and many other cardiovascular disorders. In order to explore the clinical significance of C242T p22^phox in inhibiting inflammatory endothelial dysfunction, we examined TNFα (24 h)-induced endothelial expression of VCAM-1 and NFĸB nuclear translocation by immunofluorescence (Figure 6A). Compared to vehicle treated cells, TNFα increased significantly the levels of NFκB (green) in the nuclei and VCAM-1 expression (red) mainly around the plasma membrane in cells transfected with vector or WT p22^phox, but not with C242T p22^phox (Figure 6A). The inhibitory effect of p22^phox C242T on TNFα-induced endothelial VCAM-1 expression was further confirmed by flow cytometry (Figure 6B).

Figure 6. TNFα-induced NFκB nuclear translocation, VCAM1 expression and monocyte adherence in HPMECs.

(A) Immunofluorescence microscopy detection of NFĸB (green) nuclear translocation and VCAM1 (red) expression. Nuclei were labelled with DAPI (blue) to visualize cells. The fluorescence intensity was quantified from 20 images/per group. n=3 independent experiments. (B) Flow cytometry detection of VCAM-1 expression. (C) U937 monocytes were labeled with FITC (green) and the number of monocytes adhering to the HPMEC monolayer was counted for quantification. ^*p<0.05 for indicated value versus vehicle values under the same gene transfection. †p<0.05 for indicated values versus WTp22 TNFα values. n=4 independent experiments (B-C) .

We examined also TNFα-induced monocyte adhesion to endothelial cells using FITC-labelled U937 cells, and found that 24 h of TNFα stimulation increased significantly the number of monocytes attached to the endothelial monolayer in vector or WT p22^phox transfected HPMEC but not in C242T p22^phox transfected cells (Figure 6C). The inhibitory effect of C242T p22^phox on endothelial Nox2 activation was further confirmed using cells stimulated with high level of glucose (25 mmol/L) (Supplemental Figure 6)

Experiments using engineered p22^phox-depl HeLa cells

In order to confirm that our results of C242T p22^phox would not be affected by the intrinsic p22^phox protein expressed in HPMECs, we used engineered HeLa cells after p22^phox depletion using a specific short-hairpin RNA (shRNA). A scrambled shRNA was used as controls in all experiments. The absence of p22^phox protein in p22^phox-depl cells was confirmed by immunoblotting (Figure 7A) and immunofluorescence (Figure 7B and Supplemental Figure 7). The p22^phox protein was detected in scrambled shRNA treated cells and in p22^phox-depl cells after gene transfection of WT or C242T p22^phox (Figure 7A and B). Compared to scrambled shRNA treated control cells, the levels of both Nox2 and Nox4 were low in p22^phox-depl cells, and increased to control levels after p22^phox gene transfection. However, compared to WT p22^phox transfected cells, the levels of Nox2 (but not Nox4) remained significantly lower in C242T p22^phox transfected cells (Figure 7A). The levels of Nox1 expression was unaffected by the absence of p22^phox. We then examined Nox2 maturation recognized by Nox2 antibody 7D5 and analyzed by flow cytometry (Figure 7C). Compared to scrambled shRNA control cells, p22^phox-depl cells had significantly lower (19.5±2.8%) levels of Nox2 maturation, and this was restored to the control levels after WT p22^phox transfection. However, the levels of Nox2 maturation remained significantly lower (58.7±2.3%) in C242T p22^phox transfected cells.

Figure 7. Experiments using p22^phoxdepl HeLa cells.

(A) Western blots. Optical densities (OD) of protein bands were quantified and normalized to the levels of α-tubulin detected in the same sample. (B) Immunofluorescence detection of p22^phox expression. The color images and quantification of p22^phox fluorescence intensities are presented in the Supplemental Figure 7. (C) Flow cytometry detection of anti-Nox2 antibody (7D5) binding to HeLa cells. Shaded area represents control cells without Nox2 antibody labelling. (D) O₂^.- production. SCR: scrambled shRNA. ^*p<0.05 for indicated values versus SCR values under the same treatment. †p<0.05 for indicated values versus WTp22 values under the same treatment. n=4 independent experiment.

The levels of O₂^.- production by these cells were examined by lucigenin chemiluminescence in cell homogenates (Figure 7D, left panel) and the tiron-inhibitable DHE fluorescence flow cytometry (Figure 7D, right panel). Compared to scrambled shRNA treated control cells, p22^phox-depl cells had significantly lower levels of O₂^.- production under basal condition and lost completely the O₂^.- response to TNFα stimulation. TNFα-induced O₂^.- production was restored to the scrambled shRNA control levels after gene transfection of WT p22^phox. However, gene transfection of C242T p22^phox only restored the basal but not the TNFα-induced O₂^.- production (Figure 7D). Reduced interaction between C242T p22^phox and Nox2 (but not Nox4) after TNFα stimulation was further confirmed in p22^phox-depl cells (Supplemental Figure 8).

Experiments using Nox2^-/- coronary microvascular endothelial cells (CMEC)

In order to further confirm if C242T p22^phox affected only Nox2 (but not Nox4 and/or Nox1), we isolated primary CMEC from wild-type and Nox2 knockout mice. Cells were then subjected to gene transfection and examined for O₂^.- production with or without TNFα stimulation (Supplemental Figure 9). Under basal conditions, Nox2^-/- cells produced less O₂^.- compared to WT cells without significant differences between cells transfected with vector or WT p22^phox or C242T p22^phox. TNFα stimulation greatly increased the levels of O₂^.- production in WT CMEC transfected with vector or WT p22^phox. Compared to WT p22^phox transfected cells, p22^phox C242T inhibited significantly the levels of TNFα-induced O₂^.- production (40±14% reduction) (Supplemental Figure 9). In contrast, TNFα increased only slightly (but still statistically significant) the levels of O₂^.- production by Nox2^-/- CMEC and there was no significant difference between cells transfected with vector, WT or C242T p22^phox. It was clear that C242T p22^phox had no significant effect on TNFα-induced endothelial O₂^.- production in the absence of Nox2.

C242T SNP frequency and inhibition of Nox2 expression and high-glucose induced ROS production in human saphenous veins

In order to investigate the clinical significance, we genotyped 36 saphenous vein samples collected from patients after phlebectomies who had no history of coronary heart disease. We found that 50% samples were wild-type (CC), ~27.8% were heterozygotes (CT) and ~22.2% had p22^phox C242T SNP (TT) with ~36.1% frequency of T allele appearance (Figure 8A). Dot-blot showed a significant reduction of Nox2 expression in TT vessels in comparison to CC vessels (Figure 8B). Immunofluorescence images revealed that the reduction of Nox2 was mainly in the endothelium (Figure 8D). It is well known that superoxide (O₂^.-) reacts with nitric oxide (NO) to form peroxynitrite which modifies tyrosine residue to form 3-nitrotyrosine (3NT), a biomarker of tissue oxidative damage. We found significantly lower levels of 3-nitrotyrosine (3NT) detected in TT samples as compared to CC samples (Figure 8C). When the vessels were challenged ex vivo with high level of glucose (25 mmol/L) for 24 h, TT samples had significantly less ROS production in comparison to CC sample (Figure 8E). Superoxidase dismutase was used to confirm the detection of O₂^.-.

Figure 8. The effects of C242T p22^phox SNP on Nox2 expression and high-glucose induced ROS production in human saphenous veins.

A) Genotyping of C242T p22^phox SNP (left panel) and calculated frequency in a total of 36 samples. WT p22^phox appeared as a single band of 348 bp and C242T SNP had two bands at 188 and 160 bp. B) Nox2 expression detected by dot-blotting. C) 3NT formation detected by dot-blotting. ^*p<0.05 for indicated values versus CC values. D) Nox2 (red color) expression detected by Immunofluorescence. Lumen was labeled for the location of the endothelium. Vessel structure was shown by H&E staining (right panel). E) Left panels: Representative images of vessel ROS production with or without high-glucose (H-glucose) challenge (24 h) detected by DHE fluorescence on vessel sections. Superoxide dismutase (SOD) was used to confirm the detection of O₂^.-. ^*p<0.05 for indicated values versus control values in the same genetic group. †p<0.05 for indicated values versus CT values under H-glucose.

Discussion

The NADPH oxidase, by generating O₂^.-, plays a crucial role in the inflammatory response involved in the pathogenesis of many cardiovascular diseases. The p22^phox is an integral membrane-associated subunit of NADPH oxidase responsible for assembling and stabilising Nox subunits into an active enzyme complex for ROS production⁸. A number of clinical studies have reported that individuals bearing a naturally-occurring p22^phox C242T SNP have diminished O₂^.- production and are less susceptible to inflammatory cardiovascular diseases^11,23,31,32. However, little progress has been made to clarify the underlying molecular mechanisms. For the first time, our study provides scientific evidence that p22^phox C242T SNP causes p22^phox structural changes in the extracellular domain that are unfavorable for Nox2 maturation and activation, and inhibits endothelial O₂^.- production in response to TNFα or high glucose stimulation.

A number of findings have been made by the current study in understanding the mechanism of p22^phox C242T SNP inhibition of Nox2 activation: 1) There is a significant structural difference between WT and the C242T p22^phox protein extracellular region that is important for Nox2 maturation^12,33; 2) C242T p22^phox structure compromises its binding stability with Nox2. The complex formed between Nox2 and C242T p22^phox is less stable due to the lacking of crucial hydrogen bonds to bind them together. Unstable binding may led to Nox2 protein degradation and this may explain the low levels of Nox2 detected in C242T p22^phox transfected cells and in vein samples of TT individuals.

The C242T SNP does not affect Nox1 because the Arg²²⁶ (crucial for the hydrogen bond formation to stabilize the interaction between Nox2 and WT p22^phox) is replaced by Gly in Nox1. Furthermore, the putative epitope 1 (160-IKNP-163) that is required to interact with p22^phox27 is absent in Nox1 (Supplemental Table 1). Nevertheless, Nox1 expression is extremely low in endothelial cells and is not a major source of endothelial ROS production in inflammation. Nox4 is highly expressed in endothelial cells. Nox4 generates H₂O₂ and has been found to be involved mainly in cellular growth and normal function, and is protective to cardiovascular function^34,35. Using immunoblotting, real-time PCR (Supplemental Figure 10) and co-immunoprecipitation we have shown that C242T p22^phox does not inhibit Nox4 expression or its complex formation with p22^phox. Furthermore, using HEK293 cells that do not express endogenous Nox4, we have demonstrated that (after Nox4 gene transfection) C242T p22^phox has no effect on Nox4 activity (Supplemental Figure 2). Under the basal condition (without TNFα), C242T p22^phox has no effect on endothelial Nox2 mRNA expression (Supplemental Figure 10) but reduces the protein levels of Nox2 expression and Nox2 maturation. These results strongly suggest that the effect of C242T p22^phox on Nox2 expression is post-translational. When the cells were challenged by TNFα, the inhibitory effect of C242T p22^phox on TNFα-induced Nox2 mRNA expression (Supplemental Figure 10) is more likely due to reduced levels of oxidative stress and redox signaling (such as NFκB) that impair Nox2 transcription in C242T p22^phox transfected cells.

Nox2 has low basal activity in endothelial cells and generates a small quantity of O₂^.- mainly used for redox-signaling under physiological conditions. Another important finding by the current study is that C242T p22^phox configuration can still allow Nox2 to bind and support a low basal level of O₂^.- production. However, when the cells face challenge by TNFα C242T p22^phox could not support a full Nox2 activation and thereby inhibits endothelial oxidative response to TNFα and protects endothelial cells from inflammation and cell apoptosis. The inhibitory effect of C242T p22^phox on TNFα-induced endothelial ROS production by Nox2 was examined using four independent complementary methods: lucigenin-chemiluminecscence; DHE-HPLC; DHE-flow cytometry and DCF fluorescence microscopy and further confirmed by experiments using Nox2ds-tat, several enzyme inhibitors and Nox2^-/- CMEC.

Previously, C242T SNP was reported to form a haplotype correlated with two other p22^phox SNPs: namely the C521T (rs1049254) and A24G (rs1049255) that reduced Nox2-activity in Epstein-Barr virus-transformed B-lymphocytes isolated from 50 healthy individuals of an Utah pedigree³³. However, our HapMap analysis could not identify a close correlation of C242T with any other SNPs of p22^phox currently identified within the HapMap database. Our results strongly suggest that the functional effects of C242T SNP on NADPH oxidase are independent of any other known protein-translated p22^phox SNPs.

Using engineered p22phox-depl HeLa cells we further demonstrated that in the absence of p22^phox, both Nox2 and Nox4 expressions were reduced, and could be restored after gene transfection of WT p22^phox. However, gene transfection of C242T p22^phox into p22^phox-depl cells only fully restored the expression of Nox4 (but not Nox2). When cells were subjected to TNFα challenge, C242T p22^phox transfected cells failed to increase O₂^.- production. C242T p22^phox had no significant effect on its interaction with p47^phox or on TNFα-induced p47^phox phosphorylation. However, due to the inhibitory effect of C242T p22^phox on Nox2 activation, TNFα-induced ROS production and redox-signaling through MAPK and NFκB were compromised in endothelial cells, which protected endothelial cells from oxidative damage and inflammation.

Increased VCAM-1 expression and monocyte adherence to endothelial cells are crucial steps in the pathogenesis of inflammatory cardiovascular diseases. The clinical significance of C242T p22^phox was demonstrated by showing that TNFα-induced endothelial VCAM-1 expression and monocyte adherence to endothelial cells were significantly inhibited in C242T p22^phox transfected cells. Saphenous veins are commonly used as conduits for bypass surgery to treat coronary artery diseases. Information on the p22^phox genetic variation can predict graft oxidative response to environment challenge and help clinical management of vein grafts. We found a genetic frequency of 22.2% for TT genotype (36.1% for T allele) in our samples, which is in accordance to those reported previously for control samples or for the general population^36–38. Using both dot-blotting and immunofluorescence, we confirmed that p22^phox C242T SNP is indeed associated with reduced Nox2 expression mainly in the endothelium, and TT vessels had significantly less ROS generation in response to high glucose challenge as compared to CC vessels.

Limitations need to be considered in the interpretation of our p22^phox molecular models. Although our computer models are supported by extensive cell and molecular experimental data, the structural models still need to be confirmed by X-ray crystallography or nuclear magnetic resonance spectroscopy.

In summary, this is the first report to describe the mechanism of p22^phox C242T SNP in inhibiting Nox2 activity and protecting endothelial cells from TNFα-induced oxidative damage and inflammation. p22^phox C242T SNP represents a natural protective mechanism against inflammatory cardiovascular diseases. The molecular mechanism reported here can be further explored for novel drug development.

Clinical Perspective.

NADPH oxidase, by generating reactive oxygen species, is involved in the pathophysiology of many cardiovascular diseases and represents a therapeutic target for the development of novel drugs. A number of clinic studies have reported that individuals bearing a naturally-occurring p22^phox C242T single-nucleotide polymorphism (SNP) have diminished ROS production in the cardiovascular system and are less susceptible to inflammatory cardiovascular diseases. However, little progress has been made to clarify the underlying molecular mechanisms. The current study using computer molecular modelling plus cell and molecular techniques provides scientific evidence that p22^phox C242T SNP causes p22^phox structural changes in the extracellular domain that compromises its binding stability with Nox2 (the catalytic subunit of the NADPH oxidase) and reduces the levels of Nox2 protein expression and maturation. In response to TNFα challenge, C242T p22^phox inhibits endothelial NADPH oxidase O₂^.- production, inflammatory VCAM-1 expression and monocyte adherence to endothelial cells. Clinical significance was investigated using saphenous vein segments from non-coronary heart disease subjects after phlebectomies. TT (C242T) allele was common (prevalence of ~22%) and compared to CC, veins bearing TT allele had significantly lower levels of Nox2 expression and O₂^.- generation in response to high glucose challenge. C242T SNP may represent a natural protective mechanism against inflammatory cardiovascular diseases. The molecular mechanism reported in this study can be further explored for novel drug development.