C242T Polymorphism of NADPH Oxidase p22phox and Recurrence of Cardiovascular Events in Coronary Artery Disease
Objectives— The common C242T polymorphism in the gene for the p22phox subunit of NADPH oxidase has been reported to be negatively associated with oxidative stress, but whether it confers prognostic information is not yet clear.
Methods and Results— The incidence of major adverse cardiovascular events (MACE) were determined in 237 patients with coronary stenosis during a median follow-up of 7.8 years. The p22phox genotypes were evaluated in 213 patients (89.9%) by polymerase chain reaction and RsaI. digestion. Plasma levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of oxidative stress, were also measured. In the univariate analysis, patients with CT/TT genotypes showed reduced recurrence of cardiovascular deaths, nonfatal MI, and revascularization procedures compared with homozygous carriers of the C allele. After controlling for confounders, a significantly lower risk of new revascularization procedures (HR=0.31, 95% CI 0.12 to 0.70; P=0.014) remained associated with the T allele. The Kaplan–Meier analysis showed a longer survival free from fatal and nonfatal MI in carriers of T allele (P<0.001). The presence of the 242T allele was associated with significantly reduced plasma concentrations of 8-OHdG.
Conclusions— The 242T allele was a predictor of lower risk of recurrence of cardiovascular events in high-risk patients and was associated with reduced systemic oxidative stress.
The beneficial effects of LDL cholesterol–lowering therapy in the prevention of coronary artery disease (CAD) are well established.1 However, despite intensive LDL cholesterol (LDL-C) reduction, a significant proportion of coronary patients still develop cardiovascular events.2 Therefore, factors in addition to LDL-C that may contribute to the residual cardiovascular risk have received greater attention in recent years.
Reactive oxygen species (ROS) play a prominent role in the pathophysiology of atherosclerosis.3,4 Compelling evidence indicates that the major source of ROS in the artery wall is related to the activity of NADPH oxidase.5 NADPH oxidase is composed of membrane (gp22 phox and gp91phox) and cytosolic (gp40 phox, gp47 phox, gp67 phox and rac1) subunits that assemble on the cellular surface to generate superoxide anions.3 NADPH oxidase is expressed in human atherosclerotic plaque where it promotes oxidative stress and, eventually, plaque progression and instability.6 Experimental studies demonstrated reduced formation of atherosclerotic lesions in animals knocked-out for gp47 phox compared to wild-type animals.7 The role of NADPH oxidase in vascular disease is less evident in humans.
The identification of the functional C242T polymorphism of the p22phox subunit, in which the T allele is associated with lower production of superoxide anions,8–11 allowed further analysis of the role of NADPH oxidase in the atherogenic process. Subjects carrying the T allele (CT and TT genotypes) were hypothesized to exhibit less atherosclerotic disease compared to those carrying the wild-type allele. This hypothesis has been intensely investigated in cross-sectional studies that, however, generated divergent results.12–19 Few studies have evaluated whether this genetic variant carries prognostic information,20–21 and, more importantly, none of these studies evaluated whether carriers of the C242T genotypes showed different levels of systemic oxidative stress.
Based on these considerations, we undertook a study of the relationship between the C242T polymorphism and the recurrence of cardiovascular events in a cohort of patients with angiographic evidence of coronary atherosclerosis. We also investigated whether patients in this population carrying the T allele were characterized by systemic signs of lower oxidative stress.
Materials and Methods
A cohort of 530 consecutive patients selected based on angiographic evidence of coronary atherosclerosis (1 or more stenoses >50% in at least 1 epicardial vessel) was enrolled between December 1993 and August 1997. Patients underwent coronary angiography because of myocardial infarction (MI; n=220) or angina pectoris (n=310), and were enrolled 3 months after the acute coronary event. Those with concurrent thyroid, liver, or renal disease and those already on lipid-lowering medication were excluded. After angiographic evaluation, 383 patients (72.3%) received revascularization procedures (256 with coronary bypass [CABG] and 127 with percutaneous coronary intervention [PCI]), and 147 (27.7%) received medical therapy alone.
The patients’ medical history, known CAD risk factors, and present medications were recorded via a structured questionnaire at the time of enrolment. Traditional risk factors were diagnosed as previously described.22 The study protocol was approved by the institutional Ethics Committee, and written informed consent was obtained from each participant.
Assessment of Outcomes
The follow-up for the occurrence of major adverse cardiovascular events (MACEs) (cardiovascular deaths, nonfatal MI, nonfatal ischemic stroke, unstable angina [UA], and revascularization procedures) as well as noncardiovascular events was performed by an annual telephone interview with patients or family members. Interviews were conducted by a trained research cardiologist using a structured questionnaire. In a few cases, the questionnaire was mailed to the patients. When possible, information was verified through the patient’s physician or hospital records. Of the original cohort, 200 (37%) patients missed the follow-up, 38 (7.0%) were unwilling to participate, and 8 (1.5%) provided incomplete information. Thus, reliable follow-up data were obtained for 284 patients (53.6%), and this group represented the study patients.
As reported,23 the diagnosis of MI was based on prolonged chest pain either with persistent ST elevation or without persistent ST elevation but increased plasma troponin or CPK MB levels; diagnosis of UA was based on history of angina at rest or with minimal exertion without clear-cut ECG and cardiac enzyme changes. The diagnosis of ischemic stroke was confirmed by CT scan. Cardiovascular death was defined according to reported criteria.24 The median length of follow-up was 7.8 years (range 1 to 9.8 years) with a total cohort follow-up of 2307.5 person per year.
DNA analysis, measurement of routinary laboratory variables, and serum 8-0HdG, a marker of oxidative stress,25 are reported in supplemental data (available online at http://atvb.ahajournals.org).
Continuous variables are presented as mean±SD, and differences were evaluated by the Student t or Mann-Whitney U test, depending on the shape of the distribution curve. Categorical variables were compared by χ2 or Fisher’s exact tests.
The association between the p22phox genotypes and the risk of cardiovascular events was estimated by Kaplan–Meier survival and Cox regression analyses. In these analyses, the composite of MACEs (defined as cardiovascular deaths, nonfatal MI, nonfatal ischemic stroke, UA, and revascularization procedures) as well as the individual components of MACE were considered primary end points. All calculations were carried out by combining patients with CT and TT genotypes based on the assumption that the 242T allele has a protective effect. Data from patients who died from causes unrelated to cardiovascular disease were censored at the time of death, and survival curves were compared using log-rank test. Hazard ratios (HR) and the 95% confidence intervals were estimated by Cox regression. We included age, sex, BMI, smoking, hypertension, hypercholesterolemia, revascularization procedures, history of MI, and plasma levels of HDL-C, total triglycerides, uric acid, insulin, and 8-OHdG as possible confounders in the model. The probability values are 2-sided; a probability value of less than 0.05 was considered to indicate statistical significance. All computations were carried out with SPSS software 13.0 (SPSS Inc).
Clinical Characteristics of Study Population
At baseline, clinical characteristics of patients with and without follow-up were similar with the exception of revascularization procedures, that tended to be higher in patients with follow-up compared to those without follow-up (P=0.046; see supplemental Table) In addition, when we compared the clinical characteristics of those with (n=237) and without DNA analysis (n=47), no differences emerged, with the exception of a tendency toward increased prevalence of diabetes mellitus in the former group (P=0.032; data not shown).
Among the coronary patients with DNA analysis (n=237), 21 patients (8.8%) died because of cardiovascular disease during the follow-up, 15 (6.3%) had nonfatal MI and 5 (2.1%) experienced a nonfatal stroke; 39 (16.9%) complained of new episodes of UA, and 48 (16.9%) underwent new revascularization. As 14 patients had both unstable angina and nonfatal MI, and 15 with UA and 14 with nonfatal MI underwent revascularization, the resulting incidence of total first new MACEs was 85 (35.9%). Clinical records were available for 12 patients with nonfatal MI, and 10 showed ST elevation. One patient was diagnosed with stable angina, therefore this was not included as a new event. Eighteen (7.6%) noncardiovascular deaths were also recorded, and most were attributable to cancer. During the follow-up, 80 patients (31.6%) initiated lipid-lowering medications, mostly statins (90%).
Comparison of baseline characteristics of patients with and without MACEs (Table 1) revealed no differences, with the exception of significantly lower HDL-C (P=0.02) in patients with MACEs. Baseline plasma levels of 8-OHdG tended to be higher in patients with MACEs compared to those without MACEs, but the difference did not reach statistical significance. The percent of patients using statins during the follow-up was not different between the groups (32% in patients with MACEs and 29% in those without MACEs, data not shown).
Association of NADPH p22phox Genotypes and Recurrence of Cardiovascular Events
The distribution of C242T genotypes among patients with follow-up is reported in supplemental data (see Table). The frequency of the rare (T) allele was 0.458, which was slightly higher than previous reports in other White populations.13,14,20 The distribution of genotypes agreed with the Hardy-Weinberg equilibrium (P=0.2).
After preliminary analysis demonstrated no difference in baseline characteristics between CT and TT carriers (data not shown), these patients were combined and compared with CC carriers (Table 2). Higher prevalence of hypercholesterolemia (P=0.018) and history of MI (P=0.045) and increased plasma levels of uric acid (P=0.004) were noted in CC homozygotes compared to 242T carriers.
Table 3 lists the distribution of MACEs according to p22phox genotypes. Overall, MACEs occurred slightly less frequently in patients with CT/TT that in those with CC genotypes (P=0.049). Among the individual components of MACEs, a significantly reduced incidence of cardiovascular deaths (P=0.026), nonfatal MI (P=0.027), and new revascularization procedures (P=0.007) was noted in the CT/TT patients compared to CC patients, whereas no difference in new episodes of UA was observed. Interestingly, post-hoc analysis revealed a strikingly lower incidence of fatal MI among CT/TT carriers (8.2% versus 1.3%, P=0.022); thus, when fatal and nonfatal MI events were combined, a highly significant difference between patients with TC/TT and CC genotypes was observed (5.3% versus 21.3%, respectively; P<0.001; data not shown). No significant difference in noncardiovascular events was detected between CC and CT/TT carriers (3.3.% versus 5.0%, respectively; P=0.07; data not shown).
After correction for confounders, the 242T allele was found to be associated with lower risk of MACEs as well as the individual components, even though only the new revascularization procedures reached statistical significance (P=0.014; Table 3). Based on the univariate association between C242T genotypes and the recurrence of both fatal and nonfatal MI, we retested this association after controlling for other risk factors and found that the T allele predicted a 79% lower risk of MI recurrence (P=0.012). This protective effect was also confirmed by the Kaplan–Meier analysis that demonstrated a longer MI-free survival in CT/TT than in CC individuals (Figure 1). At the 8-year follow-up time point, the MI-free survival rate in patients with the T allele was 93% compared to 65% in those homozygous for the C allele. The estimated median interval time for a new MI was 7.3 years (95% CI 6.7 to 7.9 years) and 6.6 years (95% CI 5.6 to 7.5 years) in patients with and without the T allele, respectively. This difference was highly statistically significant (log-rank test P<0.001).
Association of NADPH p22phox Genotypes and Marker of Oxidative Stress
Baseline plasma levels of 8-OHdG were significantly higher in CC compared to CT/TT carriers (Table 2). Furthermore, as shown in Figure 2, a clear gene-dosage effect was observed in the association of the p22phox genotype with plasma 8-OHdG levels, where CC homozygotes showed the highest concentrations (n=42; 5.6±0.9 ng/mL), TT homozygotes the lowest (n=29; 3.2±1.4 ng/mL) and CT heterozygotes showed intermediate levels (n=79, 4.6±0.8 ng/mL). All these differences were highly statistically significant (P<0.0001). The differences between the CC and CT/TT genotypes were independent of the number of diseased vessels, indicating that they were not secondary to the severity of vascular damage.
This study provides evidence that the C242T polymorphism in the p22phox gene predicts the risk of recurrence of cardiovascular events in patients with established coronary disease and significantly affects the systemic level of oxidative stress.
Only 2 prospective studies have examined the C242T polymorphism as a predictor of recurrence of cardiovascular events in patients with atherosclerosis. In the first study by Cahilly et al,20 313 patients with proven CAD were assigned to placebo or fluvastatin. In the placebo group (n=152), the risk of recurrence of cardiovascular events after 2.5 years of follow-up was similar across C242T genotypes. Opposite findings were observed in a more recent study in which the prevalence of p22phox genotypes was analyzed in a cohort of 663 post-MI patients over a 26-month follow-up.21 In patients defined with a high risk for recurrences (n=103), based on high cholesterol and C-reactive protein levels, the prevalence of the T allele was significantly higher in those who did not experience cardiovascular complications during the follow-up.
Our results showed a significantly lower recurrence of cardiovascular deaths, nonfatal MI, and revascularization procedures in patients carrying the T allele (CT and TT genotypes) compared to those carrying the CC genotype. However, after controlling for classical risk factors, only the risk of revascularization procedures remained significantly associated with the T allele. This suggests that the protection conferred by the T allele becomes apparent only in the background of a high-risk status.
An interesting finding of our study was the protective effect of the T allele against the recurrence of fatal and nonfatal MI that was independent from the coexistence of other risk factors. This might reinforce the notion that the C242T polymorphism has a role in plaque instability6 and supports the results from a recent study showing that animals deficient for functional NADPH oxidase with reduced systemic oxidative stress had less atherosclerotic lesions as well as plaque inflammation.26 However, because of the retrospective nature of our analysis, a prospective study in a larger population should be performed to finally address this hypothesis.
Our data brings up the possibility that the influence of the C242T polymorphism on cardiovascular risk may be exerted via its modulation of oxidative stress. This has been, at least in part, corroborated by data showing that the 242T allele was associated with a 25% reduction of plasma concentration of 8-OHdG, which is a marker of systemic oxidative stress.25 We previously reported that patients with a hereditary deficiency of the NADPH oxidase who are characterized by low cellular production of ROS showed very low serum levels of 8-OHdG.27 The link between plasma levels of 8-OHdG and NADPH oxidase activity was further confirmed by the observation that the C242T genotypes showed a dose-dependent relationship with plasma 8-OHdG levels. Taken together, these data provide the first evidence that the p22phox polymorphism is associated with changes of oxidative stress not only within the cells,8,10,11 but also at systemic levels. A potential limitation of such an assertion is that we used only plasma levels of 8-OHdG as marker of oxidative stress; however, we have previously reported that changes in this marker parallel those of urinary isoprostanes, a well-established marker of whole body oxidative stress.27
In interpreting these results, several limitations should be considered. In general, the present study shares the limitations of nonrandomized observational studies, but has the advantage of being longitudinal in design with a large person per year follow-up. In addition, a relatively large number of new events were registered, thus making statistical estimations more robust. Follow-up data were available only for a subset of the original cohort. Nevertheless, when we compared the baseline characteristics of patients with and without follow-up, no appreciable differences were detected, indicating a “random effect” in the selection of patients included in the follow-up analysis. Moreover, the lack of association between classical risk factors and recurrence of new events might have inflated the association between new events and the p22phox genotypes. However, it is not an unusual finding that classical risk factors show a low predictive power in patients with clinical manifestation of atherosclerotic disease.28 Finally, several potential confounders (ie, left ventricular ejection fraction) have not been measured, leaving the possibility that the independent predictive power of the C242T polymorphism in CAD patients could be smaller.
In conclusion, our findings indicate that patients carrying the 242T allele and showing lower oxidative stress had less cardiovascular events compared to those carrying the 242C allele. These findings may help to identify patients at risk of atherosclerotic progression beyond the classic risk factors for atherosclerosis.
We thank Dr Bruno Mazzarella, Dr Roberto Carnevale, and Renzo Cantini for technical support.
Sources of Funding
This study was supported by grants from La Sapienza University of Rome (Progetto Ateneo 2004 and 2006) to M.A. and from Fondi Ateneo Università La Sapienza to F.V.
Original received August 29, 2007; final version accepted January 23, 2008.
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