A Polymorphism in the Cyclooxygenase 2 Gene as an Inherited Protective Factor Against Myocardial Infarction and Stroke

CONTEXT
Myocardial infarction (MI) and ischemic stroke are thought to be caused by matrix digestion by metalloproteinases (MMPs) leading to rupture of atherosclerotic plaques. Production of macrophage MMP-2 and MMP-9 is induced by cyclooxygenase 2 (COX-2) and prostaglandin E(2) synthesis. Although COX-2 expression may be genetically determined, the relation between COX-2 polymorphisms and the risk of MI and stroke is unclear.


OBJECTIVE
To investigate the relationship between the -765G-->C polymorphism of the COX-2 gene and clinically evident plaque rupture.


DESIGN, SETTING, AND PARTICIPANTS
Prospective, matched case-control study conducted between March 2002 and October 2003 among 864 patients with first MI or atherothrombotic ischemic stroke and 864 hospitalized controls. The groups were matched for age, sex, body mass index, smoking, hypertension, hypercholesterolemia, and diabetes. The -765G-->C variant of the COX-2 gene was genotyped by restriction endonuclease digestion of polymerase chain reaction products.


MAIN OUTCOME MEASURES
Presence of the -765G-->C polymorphism of the COX-2 gene; COX-2, MMP-2, and MMP-9 expression and activity in plaques and in peripheral monocytes; urinary 6-keto PGF1alpha (marker of endothelial prostacyclin); and endothelium-dependent and -independent forearm blood flow vasodilation.


RESULTS
The prevalence of -765GC was 2.41 times higher among controls than among cases (43.3% vs 17.9%; P<.001). The prevalence of -765CC homozygosity was 5.81 times higher (6.4% vs 1.1%; P =.04). Among participants carrying the -765GC and -765CC genotypes, the prevalence ratios for MI or stroke were 0.48 (95% CI, 0.36-0.68) and 0.33 (95% CI, 0.24-0.55), respectively. Expression of COX-2 and MMPs was significantly lower in atherosclerotic plaques from participants carrying the -765C allele, while the -765G-->C polymorphism did not affect endothelial prostacyclin biosynthesis or endothelium-dependent vasodilation in vivo. In subgroup analyses (n = 224 cases), serum high-sensitivity C-reactive protein was significantly lower in patients carrying the -765C allele (mean [SD], 0.78 [0.1] vs 2.56 [0.4] mg/L; P =.04).


CONCLUSIONS
We found that the -765G-->C polymorphism of the COX-2 gene is associated with a decreased risk of MI and stroke. Detection of this genotype may be useful for predicting genetic risk of MI and stroke.

dividual's genetic makeup and environmental factors. 2,3 Increasing evidence supports a central role for inflammation in the cascade of events that results in plaque fissuring. 4,5 Lesion macrophages synthesize matrix metalloproteinases (MMPs); in particular, 72-kDa (MMP-2) and 92-kDa (MMP-9) gelatinase, proteolytic enzymes capable of degrading plaque constituents. 6 We recently reported that enhanced MMP-2 and MMP-9 production by macrophages in vulnerable plaques is due to enhancement in prostaglandin E 2 (PGE 2 ) synthesis as a result of the induction of COX-2. [6][7][8][9] Recently, Papafili et al 10 and Cipollone and Patrono 11 identified a new variant in the COX-2 promoter, a guanine to cytosine substitution at position −765 (−765G→C), and showed that this variant is located within a putative binding site for the transcription factor Sp1. Patients carrying the −765C allele had markedly (30%) lower promoter activity and lower plasma levels of C-reactive protein (CRP), a marker of low-grade inflammation, compared with patients homozygous for −765G.
Because lower COX-2 activity could reduce generation of culprit MMPs in plaques, and because CRP is closely re-lated to cardiovascular risk, 12 we hypothesized that −765G→C might be protective against plaque instability, leading to lower risk of MI and stroke. We therefore prospectively performed a case-control study in which we genotyped this locus in 1728 individuals at high risk of cardiovascular events (864 patients with well-characterized previous MI or stroke and 864 controls).

Study Participants
The study participants comprised 1728 unrelated Italian patients who were admitted to 4 participating hospitals (SS Annunziata University Hospital of Chieti, University Hospital of Palermo, S Chiara University Hospital of Pisa, and La Sapienza University Hospital of Rome, all in Italy) between March 2002 and October 2003 and who had at least 1 conventional risk factor for cardiovascular disease.
Cases (n = 864) were patients who had had a first-event MI or ischemic stroke. We specifically focused on ischemic strokes that were atherothrombotic and related to ulceration of a culprit internal carotid artery lesion. We made every effort to exclude patients with other subtypes of ischemic stroke by clinical examination and laboratory and imaging analyses. In particular, strokes had to be in the territory of the mid cerebral artery as documented by computed tomographic scan and cerebral angiography. All patients had Doppler evidence of an ulcerated culprit lesion in the internal carotid artery as well as preprocedural transcranial Doppler analysis of cerebral blood flow strongly indicative of active thromboembolism. Finally, all study participants were in sinus rhythm at the time of hospitalization and had no history of atrial fibrillation.
Controls (n=864) were high-risk patients prospectively matched for risk factors and concomitant therapy, as shown in The protocol was approved by local ethics review committees. Written informed consent was obtained from all patients prior to study enrollment and before each examination.

Data Collection and Analysis
Blood and urine samples were collected from all participants after an overnight fast, and multiple aliquots of whole blood, plasma, serum, and urine were immediately stored at −80°C until analysis.
Genomic DNA was prepared from frozen whole blood with the use of a blood DNA isolation kit (Helix Fast Blood DNA, DiaTech, Jesi, Italy). The −765G→C variant was genotyped by FAU I (Celbio, Milan, Italy) restriction endonuclease digestion of the polymerase chain reaction product (Figure 1). Genotypes were determined by independent investigators who were blinded to patients' identities and phenotypes. After carotid endarterectomy, plaque sections were prepared and analyzed by immunohistochemistry using a color image analysis system (AlphaEase 5.02, Alpha Innotech Corp, San Leandro, Calif), as previously described. 6 As previously described, 6 Western blot analysis on plaque extracts and plaque-derived macrophages was performed to detect COX-2, MMP-2, and MMP-9 expression; concurrent immunodetection of ␤-actin was performed to ensure equal gel loading. Bands were quantified by computer-assisted densitometry (AlphaEase 5.02) and expressed as densitometric units (DU).
Zymography on plaque extract and plaque-derived macrophages was performed to detect MMP-2 and MMP-9 gelatinolytic activity, as previously described. 6 Conditioned medium of human fibrosarcoma cell line HT1080 was used as a positive control with known gelatinolytic activity.
Sirius red polarization microscopy was used to detect type 1 and 3 interstitial collagen in plaque sections ac-cording to a previously described method. 9 Macrophages were selectively extracted from plaques as described by de Vries et al. 13 Flow cytometry analysis of purified cell preparations using a phycoerythrin-conjugated anti-CD68 monoclonal antibody (Clone Y1/ 82A, BD Biosciences PharMingen, San Diego, Calif ) showed that more than 98% of the selected cells were positive for CD68. Then, immunocytochemistry, Western blot, and zymography were performed. Results are representative of 3 different analyses (intrasample tests for in vitro reproducibility).
Peripheral blood monocytes were purified and cultured from 10 randomly selected patients carrying the −765GG genotype, 10 carrying the −765GC genotype, and 10 carrying the −765CC genotype. 6 In particular, to create 3 homogeneous groups, patients were selected not only on the basis of genotype but also on the basis of clinical features. Thus, for each genotype, 5 control patients and 5 case patients were selected (TABLE 2). Monocytes (20ϫ10 6 /4 mL of Dulbecco modified eagle's medium) were stimulated with lipopolysaccharide (1 µg/mL), oxidized low-density lipoprotein (LDL) (50 µg/mL), angiotensin II (10 −7 mol/L), and advanced glycation end products (800 µg/mL) as previously described. 6,9,14 At the end of the incuba-tion, COX-2 and MMP expression was evaluated by Western blot, MMP activity by zymography, and PGE 2 (Cayman Chemical, Ann Arbor, Mich) and MMP-9 (Amersham Biosciences, Piscataway, NJ) release by enzymelinked immunosorbent assay.
In vivo prostacyclin (PGI 2 ) production, a process largely dependent on COX-2 activity in endothelium, 15,16 was analyzed in 8 randomly selected participants with the −765GG genotype, 8 with −765GC, and 8 with −765CC by measuring urinary excretion of 6-keto PGF 1␣ , the major urinary metabolite of renal PGI 2 , according to a previously described radioimmunoassay. 17 Straingauge plethysmography was used to measure forearm blood flow changes induced by intrabrachial acetylcholine and sodium nitroprusside according to a previously validated method 18 in 6 randomly selected control participants with the −765GG genotype, 9 with −765GC, and 3 with −765CC to identify perturbations in endotheliumdependent vasodilation.

Statistical Analyses
The significance of differences in biochemical marker expression and inflammatory cell infiltration among the 3 genotypes was analyzed by unpaired t test. Discrete data (including prevalence of the −765C allele in cases and controls) were analyzed by the 2 test,  2  3  3  3  2  2  Hypertension  4  3  3  3  3  3  Diabetes  2  2  2  1  2  2 Cigarette smoking 2 1 2 1 1 3 Body mass index, mean (SD) † 26 (7) 27 (5) 26 (4) 28 (6) 28 (5)  with the allele frequency predicted on the basis of the Hardy-Weinberg equilibrium. We used logistic regression modeling to test the association of the COX-2 polymorphisms on the risk of MI and ischemic stroke 19 after adjusting for age, body mass index, smoking status, hypertension, diabetes mellitus, and hypercholesterolemia. All analyses were performed using SPSS software, version 11.0.1 (SPSS Inc, Chicago, Ill). 20 PϽ.05 was considered statistically significant.

Characteristics of the Study Participants
The characteristics of the 1728 study participants are shown in Table 1. There were no significant differences between cases and controls in age, body mass index, or prevalence of conventional risk factors for cardiovascular disease. To investigate the association of the COX-2 polymorphism with plaque growth, we analyzed the atherosclerotic burden as a function of the −765C allele in patients who had accurate quantification of atherosclerosis by carotid Doppler or coronary angiography. We did not find any association between the −765C allele and severity of atherosclerosis at either the carotid or the coronary level (Table 1)

COX-2 Expression in Carotid Plaques of Patients With the −765G→C Polymorphism
COX-2 staining was significantly more abundant in −765GG carotid plaques (FIGURE 1)  *The prevalence ratio is for the cumulative risk of MI and atherothrombotic ischemic stroke among patients who were heterozygous or homozygous for the −765G→C polymorphism compared with those who were not, calculated based on the odds ratio from logistic regression after adjustment for confounders. 20 [117] DU for −765GG, −765GC, and −765CC, respectively; PϽ.001) (FIGURE 2). These observations remained consistent when separate analyses for the case and control groups were performed. To exclude the possibility that observed discrepancies in COX-2 expression were merely a secondary effect of differences in inflammatory infiltration, we repeated immunocytochemistry and Western blot analyses on macrophages that had been selectively extracted from plaques. We again noted that plaque macrophages from patients carrying the −765G→C polymorphism had a lower COX-2 expression.

MMP Expression and Activity in Carotid Plaques of Patients With the −765G→C Polymorphism
Staining for MMP-2 and MMP-9 was significantly more abundant in −765GG lesions (n = 145) than in −765GC (n = 78)

Inflammatory and Proteolytic Response in Monocytes/ Macrophages of Patients With the −765G→C Polymorphism
In monocytes isolated from patients carrying the −765GG genotype, lipopolysaccharide, oxidized low-density lipoprotein, angiotensin II, and advanced glycation end products induced significant increases in COX-2, MMP-2, and  . Similarly, no differences in endotheliumd e p e n d e n t a n d e n d o t h e l i u mindependent (sodium nitroprusside) vasodilation were observed among participants carrying the 3 genotypes (FIGURE 4).

COMMENT
We found that the −765G→C polymorphism of the COX-2 gene is associated with a reduction in the risk of MI and stroke, suggesting that this allele may offer protection against clinical events related to atherosclerotic plaque rupture. The prevalence distribution of this polymorphism in our control group was significantly higher than that recently reported by Papafili et al 10 in a smaller sample in the United Kingdom. Since the −765G→C polymorphism is associated with lower expression and activity of the COX-2 gene in plaque macrophages, the finding of a lower frequency of this polymorphism in the United Kingdom is consistent with the higher incidence of MI and stroke in that country compared with in Italy. In addition, it could explain, at least in part, the higher risk of MI observed in northern Europe compared with Mediterranean populations with similar levels of cholesterol and blood pressure. 21 Both the −765GC and −765CC genotypes were significantly related to COX-2 expression and activity in circulating blood monocytes and carotid plaque macrophages, suggesting that the association between COX-2 genotype and MI and stroke may occur as a result of the modulation of COX-2 activity in inflammatory cells. Indeed, patients with the −765CC genotype, who had the lowest risk of MI and stroke, also had the lowest level of COX-2 expression and activity in plaque  sections, in plaque-derived macrophages, and in circulating blood monocytes. Furthermore, the presence of at least one −765C allele was associated with a significant reduction in serum CRP, thus suggesting that this polymorphism may also influence systemic inflammatory status. The precise mechanism by which the COX-2 polymorphism may affect the risk of MI and stroke is unclear. Both MI and atherothrombotic stroke are often triggered by rupture of vulnerable atherosclerotic plaques, with the propensity of rupture enhanced by PGE 2dependent MMPs. 22 Thus, the marked reduction in plaque MMPs observed in patients carrying the −765G→C polymorphism supports our hypothesis that reduction of MMP biosynthesis as a consequence of lower PGE 2 generation may explain the risk reduction we observed. The lower COX-2 induction observed in monocytes and macrophages of patients carrying the −765C allele after stimulation with oxidized low-density lipoprotein, angiotensin II, and advanced glycation end products (stimuli upregulated in the setting of hypercholesterolemia, 23 hypertension, 24 and diabetes, 14 respectively) is also consistent with the possibility that the −765G→C polymorphism may be protective against MI and stroke. However, we cannot exclude that other important mechanisms-for example, the reduction of aspirin-insensitive thromboxane biosynthesis in circulating monocytes 25 -may also contribute to the risk reduction associated with the −765C allele. [26][27][28] Furthermore, because adjustment for baseline risk factors was not performed in these in vitro substudies, we cannot exclude a potential influence of selection bias.
We did not find differences in the severity of atherosclerotic lesions among patients carrying the 3 different genotypes. This observation confirms previous studies suggesting that COX-2 is involved in the evolution of atherosclerotic plaque toward instability 6,9,14 rather than plaque growth. 29,30 We noted that the −765G→C polymorphism was not associated with en-dothelial generation of PGI 2 and endothelium-dependent vasodilation. This is consistent with previous observations in epithelial cells 11 and suggests that transcription factor Sp1 has differing roles regarding induction of COX-2 in monocytes and macrophages compared with endothelial and epithelial cells. Because PGI 2 is considered a potent antiatherogenic mediator, 31 these data strengthen support for the protective role of the −765G→C polymorphism and point out a major difference with respect to COX-2 inhibition through COX-2 inhibitors (coxibs), which also inhibit COX-2 at the endothelial level. 15,16 Our study also raises a number of provocative questions regarding the potential clinical importance of this genotype in asymptomatic patients taking selective coxibs. The individual pharmacodynamic response to COX-2 inhibitors may vary, 32 as responders and nonresponders to coxibs have been described. 33,34 Thus, patients with the −765GC and −765CC genotypes may represent subgroups of patients with different drug responses.
Further research will be needed to determine whether genotyping patients for the COX-2 polymorphism leads to better treatment outcomes.
In conclusion, consistent with the presence of genetic determinants of clinical atherosclerotic risk, we found that the −765G→C polymorphism of the COX-2 gene is associated with a lower risk of MI and ischemic stroke. The reduction in risk may be related to modification of COX-2 expression in plaque macrophages. Further studies are necessary to identify additional polymorphisms in the arachidonic acid pathway that are associated with risk of MI and stroke. Mean increase in the forearm blood flow induced by acetylcholine in participants carrying the −765 GG, −765 GC, and −765 CC genotypes. No differences in endothelium-dependent and -independent (sodium nitroprusside) vasodilation were observed among participants carrying the 3 genotypes. Error bars indicate SD.