Mariano Sentí

Effect of Simvastatin Therapy on Paraoxonase Activity and Related Lipoproteins in Familial Hypercholesterolemic Patients

Human paraoxonase (PON1) is a calcium-dependent esterase closely associated with high density lipoprotein (HDL)-containing apolipoprotein AI (apoAI), which has been shown to confer antioxidant properties to HDL. PON1 has been recently implicated in the pathogenesis of atherosclerosis. Low PON1 activities have been found in familial hypercholesterolemia (FH) and diabetes mellitus. We have undertaken a study of the effect of the lipid-lowering drug simvastatin on serum PON1 activity (in relation to paraoxon and arylesterase activity), on apoAI-containing and apolipoprotein B (apoB)-containing lipoproteins, and on lipid peroxide concentrations in 64 (39 women and 25 men) unrelated FH patients. We have also analyzed the influence of the PON1-192 and PON1-55 genetic polymorphisms on the response of PON1 activity to simvastatin therapy. A venous blood sample for a baseline analysis and another after 4 months of simvastatin therapy at a dosage of 20 mg per day were taken. The major effect of simvastatin on lipid traits was to decrease serum cholesterol, low density lipoprotein (LDL) cholesterol, and lipid peroxide concentrations by 19.9%, 26.3%, and 37.3%, respectively. There was also a significant decrease in serum apoB, LDL apoB, and triglyceride concentrations (20.5%, 21.1%, and 15.6%, respectively). Conversely, simvastatin had no significant influence on very low density lipoprotein–lipid content, HDL cholesterol, apoAI concentrations, and lipoprotein AI and AI:AII particles. Remarkably, serum PON1 activity toward paraoxon significantly increased during treatment with simvastatin (168.7±100.3 U/L before therapy versus 189.5±116.5 U/L after therapy, P =0.005). Arylesterase activity displayed only a nonsignificant trend to increase after therapy. Whereas PON1 activity levels were significantly lower in FH patients before simvastatin therapy compared with those of 124 normolipidemic subjects (168.7±100.3 versus 207.6±125.2 U/L, respectively;P <0.05), this difference disappeared after simvastatin therapy. After simvastatin therapy, a significantly negative correlation between PON1 activity and lipid peroxide concentration was observed (r =−0.35, P =0.028). The latter also strongly correlated with LDL cholesterol concentration (r =0.64, P <0.001). Serum PON1 activity levels were significantly lower in the low-activity PON1-192 QQ and PON1-55 M carriers than in R carriers and in LL carriers, respectively. No significant differences were found in the therapeutic response of PON1 activity between genotype groups (8.5% and 11.1% increase for QQ homozygous and R-carrier FH patients, respectively, and 12.7% and 9.5% increase for LL homozygotes and M carriers, respectively). We conclude that simvastatin may have important antioxidant properties through increasing serum PON1 activity, perhaps as a consequence of reducing oxidative stress, by a mechanism independent of apoAI-containing lipoprotein concentration and without the influence of PON1-192 and PON1-55 genetic polymorphisms. Further studies are clearly warranted to clarify the precise mechanism by which simvastatin therapy is associated with increased PON1 activity.

Gain-of-function mutation in the KCNMB1 potassium channel subunit is associated with low prevalence of diastolic hypertension

Hypertension is the most prevalent risk factor for cardiovascular diseases, present in almost 30% of adults. A key element in the control of vascular tone is the large-conductance, Ca(2+)-dependent K(+) (BK) channel. The BK channel in vascular smooth muscle is formed by an ion-conducting alpha subunit and a regulatory beta(1) subunit, which couples local increases in intracellular Ca(2+) to augmented channel activity and vascular relaxation. Our large population-based genetic epidemiological study has identified a new single-nucleotide substitution (G352A) in the beta(1) gene (KCNMB1), corresponding to an E65K mutation in the protein. This mutation results in a gain of function of the channel and is associated with low prevalence of moderate and severe diastolic hypertension. BK-beta(1E65K) channels showed increased Ca(2+) sensitivity, compared with wild-type channels, without changes in channel kinetics. In conclusion, the BK-beta(1E65K) channel might offer a more efficient negative-feedback effect on vascular smooth muscle contractility, consistent with a protective effect of the K allele against the severity of diastolic hypertension.

High oxidative stress in patients with stable coronary heart disease

Oxidized low density lipoprotein (oxLDL) plays a pivotal role in the development of atherosclerosis. The aim of the study was to investigate the relationship between oxLDL and other oxidative stress biomarkers with stable coronary heart disease (CHD). We compared the degree of oxidative stress in patients with CHD and sex-matched healthy control subjects in a case-control study. The study included 64 male subjects: 32 patients with stable CHD and 32 normal control subjects. Levels of circulating oxLDL were measured by a monoclonal antibody 4E6-based competition ELISA. Comparison of oxidative stress marker levels between cases and controls, adjusted for age, revealed significantly higher plasma oxLDL levels (63.32+/-25.49 vs. 37.73+/-20.58 U/l, P=0.001), lower serum levels of autoantibodies against oxLDL (341.53+/-350.46 vs. 796.45+/-1034.2 mU/ml, P=0.021), higher activities of the antioxidant enzymes superoxide dismutase in erythrocytes (951+/-70.2 vs. 771.6+/-191.2 U/g, P=0.032) and glutathione peroxidase in whole blood (GSH-Px: 10714.4+/-3705.4 vs. 5512.2+/-1498.1 U/l, P<0.001). The risk of having CHD was 20.6-fold greater (95% CI, 1.86-228.44, P=0.014) in the highest tertile of the oxLDL distribution than in the lowest, determined by logistic regression analysis on the combined study population after adjustment for age and other potential confounding factors. When the risk associated with GSH-Px levels was calculated, the odds ratio was 305.3 (95% CI, 5.07-18369.95, P=0.006) in the highest tertile compared with the lowest. Our results showed that an oxidative stress occurs in patients with CHD despite being clinically stable and under medical treatment. The combination of oxLDL levels and GSH-Px activity may be useful for the identification of patients with stable CHD.

Protective effect of the KCNMB1 E65K genetic polymorphism against diastolic hypertension in aging women and its relevance to cardiovascular risk.

The E65K polymorphism in the &bgr;1-subunit of the large-conductance, Ca2+-dependent K+ (BK) channel, a key element in the control of arterial tone, has recently been associated with low prevalence of diastolic hypertension. We now report the modulatory effect of sex and age on the association of the E65K polymorphism with low prevalence of diastolic hypertension and the protective role of E65K polymorphism against cardiovascular disease. We analyzed the genotype frequency of the E65K polymorphism in 3924 participants selected randomly in two cross-sectional studies. A five-year follow-up of the cohort was performed to determine whether cardiovascular events had occurred since inclusion. Estrogen modulation of wild-type and mutant ion channel activity was assessed after heterologous expression and electrophysiological studies. Multivariate regression analyses showed that increasing age upmodulates the protective effect of the K allele against moderate-to-severe diastolic hypertension in the overall group of participants (odds ratio [OR], 0.35; P=0.006). The results remained significant when analyses were restricted to women (OR, 0.18; P=0.02) but not men (OR, 0.46; P=0.09). This effect was independent of the reported acute modulation of BK channels by estrogen. A five-year follow-up study also demonstrated a reduced age- and sex-adjusted hazard ratio of 0.11, 95% CI, 0.01 to 0.79 of K-carriers for “combined cardiovascular disease” (myocardial infarction and stroke) compared with EE homozygotes. Our study provides the first genetic evidence for the different impact of the BK channel in the control of human blood pressure in men and women, with particular relevance in aging women, and highlights the E65K polymorphism as one of the strongest genetic factors associated thus far to protection against myocardial infarction and stroke.

Relationship of age-related myocardial infarction risk and Gln/Arg 192 variants of the human paraoxonase1 gene: the REGICOR study

Paraoxonase1 (PON1) seems to exert a major antioxidant effect by removing lipid-peroxidation products. A common polymorphism of the PON1 gene, the PON1-192 genetic polymorphism, modulates PON1 activity and has been related in some studies to coronary heart disease. Oxidized LDL is believed to play a crucial role in the pathogenesis of atherosclerosis and there are studies providing support for the oxidative stress theory of aging. We have conducted a case-control study to determine whether PON1 activity and PON1-192 genetic variants have a different impact on myocardial infarction (MI) risk among individuals stratified by tertiles of age distribution. PON1-192 genotypes and PON1 activity were determined in 280 consecutive MI patients and 396 control subjects. Serum PON1 activity levels were significantly higher in controls than in MI patients [226 U/l (159-351) vs. 216 U/l (146-298), median (interquartile range), P=0.005]. A decline of PON1 activity levels with advancing age in subjects carrying the low-activity QQ genotype was observed, particularly in MI patients. PON1 activity and age negatively correlated in MI patients but not in controls. In the entire population, middle-aged and older subjects showed MI risks of 1.89 (P=0.012) and 2.69 (P<0.001) respectively, compared with young subjects. These risks increased to 2.41 (P=0.016) and 4.39 (P<0.001), respectively, in QQ homozygotes in comparison with younger QQ homozygotes, decreased to 1.53 (P=0.314) and 2.08 (P=0.112), respectively, in QR heterozygotes, and also lowered to 1.95 (P=0.410) and 0.51 (P=0.508) in RR homozygotes who were middle-aged and older, respectively, compared with younger RR carriers. The effect of PON1-192 genotypes on the association of the older age-category and MI risk was gene-dosage related. PON1 activity decreases as a function of age in subjects homozygous for the Q allele. Age may also act on MI risk as a function of PON1-192 alleles. The risk of MI increases with advancing age, principally among subjects carrying the low-activity QQ genotype.

Interaction between the Gln-Arg 192 variants of the paraoxonase gene and oleic acid intake as a determinant of high-density lipoprotein cholesterol and paraoxonase activity.

Olive oil, rich in oleic acid, could play a particular beneficial role in the anti-atherogenic effects attributed to the Mediterranean diet. Paraoxonase (PON1) has emerged as the component of high-density lipoproteins (HDL) most likely to explain its ability to attenuate the oxidation of low-density lipoproteins. We hypothesised that oleic acid intake might be associated with changes in PON1-HDL associated particles, and investigated the impact, if any, on this association of the PON1-192 polymorphism, a common polymorphism that strongly modulates PON1 activity. Six hundred and fifty-four men randomly selected from the census were studied. Oleic acid intake was calculated from a 72-h recall questionnaire with specific software. Oleic acid intake groups (low vs. high) were created by stratifying the population according the median value as a cut-point. After adjusting for confounding variables, high oleic acid intake was associated with increased HDL cholesterol levels and PON1 activity only in subjects with the QR and the RR genotypes, respectively. Analyses of the variance showed a statistically significant interaction between PON1-192 genotypes and oleic acid intake for log PON1 activity (P=0.005) and a marginally significant interaction for HDL cholesterol (P=0.066). These results suggest that the beneficial effect of increasing oleic acid intake on HDL and PON1 activity at population level is especially observed in subjects carrying the R allele of the PON1-192 polymorphism.

Interrelationship of smoking, paraoxonase activity, and leisure time physical activity: a population-based study.

METHODS: We determined the effect of smoking on PON1 activity levels in a population-based sample of 1380 subjects and examined the possibility of regular physical activity modulating the effect of cigarette smoking on PON1 activity levels at a population level. RESULTS: Mean PON1 activity was significantly lower in smokers than in non- or ex-smokers (237.6+/-8.4 vs. 276.7+/-5.3 U/l, mean+/-S.E.M.; P= 0.001). PON1 activity levels were significantly higher in physically active subjects than in those who were inactive (296.1+/-10.4 vs. 260.1+/-9.2 U/l; P=0.002). Inactive smokers showed significantly lower PON1 activity levels than inactive nonsmokers (P=0.002). These differences disappeared when active nonsmokers were compared with active smokers. Serum PON1 activity levels were found to be significantly increased in active smokers compared to those in inactive smokers. A statistically significant interaction (P=0.003) between smoking and physical activity on PON1 activity was observed. CONCLUSION: Smoking appears to have a deleterious effect on PON1 activity levels. Being physically active clearly attenuates this effect.

Assessment of the value of a genetic risk score in improving the estimation of coronary risk

BACKGROUND The American Heart Association has established criteria for the evaluation of novel markers of cardiovascular risk. In accordance with these criteria, we assessed the association between a multi-locus genetic risk score (GRS) and incident coronary heart disease (CHD), and evaluated whether this GRS improves the predictive capacity of the Framingham risk function. METHODS AND RESULTS Using eight genetic variants associated with CHD but not with classical cardiovascular risk factors (CVRFs), we generated a multi-locus GRS, and found it to be linearly associated with CHD in two population based cohorts: The REGICOR Study (n=2351) and The Framingham Heart Study (n=3537) (meta-analyzed HR [95%CI]: ~1.13 [1.01-1.27], per unit). Inclusion of the GRS in the Framingham risk function improved its discriminative capacity in the Framingham sample (c-statistic: 72.81 vs.72.37, p=0.042) but not in the REGICOR sample. According to both the net reclassification improvement (NRI) index and the integrated discrimination index (IDI), the GRS improved re-classification among individuals with intermediate coronary risk (meta-analysis NRI [95%CI]: 17.44 [8.04; 26.83]), but not overall. CONCLUSIONS A multi-locus GRS based on genetic variants unrelated to CVRFs was associated with a linear increase in risk of CHD events in two distinct populations. This GRS improves risk reclassification particularly in the population at intermediate coronary risk. These results indicate the potential value of the inclusion of genetic information in classical functions for risk assessment in the intermediate risk population group.

Paraoxonase1-192 Polymorphism Modulates the Nonfatal Myocardial Infarction Risk Associated With Decreased HDLs

Abstract —Serum paraoxonase1 (PON1), a high density lipoprotein (HDL)-linked enzyme, appears to have a role in the protection of low density lipoproteins from oxidative stress. PON1 enzyme activity for paraoxon as a substrate is modulated, along with others at the PON1 locus, by the PON1-192 polymorphism, which contains low paraoxon–activity and high paraoxon–activity alleles (Q and R, respectively). The association of PON1 with HDL suggests that impaired serum concentrations of the lipoprotein could have consequences for the susceptibility to oxidative stress. Because PON1-192 polymorphism strongly influences PON1 activity toward paraoxon, we tested the hypothesis that this polymorphism may modulate the myocardial infarction (MI) risk associated with low HDL cholesterol concentrations. Two hundred eighty consecutive MI patients and 396 control subjects were studied. When considered as a whole, PON1-192 genetic polymorphism was not associated with higher MI risk. In the entire population, decreased HDL cholesterol concentration (<0.90 mmol/L in men and <1.11 mmol/L in women) conferred an MI risk of 2.56 (P =0.0001) compared with normal HDL levels. The risk increased to 4.51 (P <0.0001) in QQ homozygous HDL-deficient subjects relative to QQ homozygotes with normal HDL levels, decreased to 1.83 (P =0.1046) in QR heterozygote HDL-deficient subjects, and also decreased (to 1.41, P =0.6243) in RR homozygote HDL-deficient individuals compared with RR carriers with normal HDL cholesterol concentration. The effect of PON1-192 genotypes on the association of low HDL cholesterol levels and MI was related to gene dosage. A significantly decreased enzyme activity was found in HDL-deficient MI patients compared with HDL-deficient control subjects (median 208 U/L [interquartile range 136 to 298 U/L] versus median 235 U/L [interquartile range 163 to 350 U/L], respectively;P =0.025]. QQ homozygous MI patients showed the greatest difference of PON1 activity levels between normal and HDL-deficiency state groups (14.9%, P =0.002). Our observations raise the question of whether the decrease in PON1 activity and the MI risk associated with HDL deficiency are more evident in the low paraoxon–activity QQ genotype. It can be argued that the low paraoxon–activity QQ genotype, which may be adequate to prevent lipid peroxidation in normolipidemic subjects, may be insufficient when an HDL-deficiency state and low PON1 activity reflecting oxidative stress coexist. The risk of nonfatal MI is increased in HDL-deficiency states, principally among subjects carrying the low paraoxon–activity QQ genotype.

The Antioxidant Function of High Density Lipoproteins: A New Paradigm in Atherosclerosis

The one disease associated with the greatest morbidity and mortality in industrialized countries is coronary heart disease (CHD). High density lipoprotein (HDL) is one of the most important independent protective factors for the arteriosclerosis which underlies CHD. Paraoxonase 1 (PON1) is an enzyme that confers antioxidant properties to HDL. In vitro, PON1 hydrolyzes a large variety of endogenous or exogenous substrates, some of which are clearly involved in the progression of arteriosclerosis. A close relationship between PON1 deficiency and accelerated progression of arteriosclerosis has been found in animal models. Moreover, PON1 activity is reduced in high oxidative stress diseases such as CHD, dyslipoproteinemias, inflammatory processes, diabetes and certain neuropathies. Reduced PON1 enzyme activity is associated with several arteriosclerosis-related diseases. The most thoroughly studied genetic variant of PON1 is PON1-192, in which the R allele is associated with elevated paraoxonase activity. This allele, present in 24.8% of the Italian population, is found in up to 78.9% of the population of Ecuadorian Cayapa Indians. A metaanalysis of studies on the relationship between CHD and the R allele showed the latter to be an independent risk factor for this disease, with an odds ratio of 1.18 (95% confidence interval, 1.10-1.27). The PON1 enzyme is a potentially useful new qualitative indicator in addition to the well known reverse cholesterol transport capacity associated with high plasma levels of HDL.