Accumulation of cholesterol precursors and plant sterols in human stenotic aortic valves
2008; Elsevier BV; Volume: 49; Issue: 7 Linguagem: Inglês
10.1194/jlr.m800058-jlr200
ISSN1539-7262
AutoresSatu Helske, Tatu A. Miettinen, Helena Gylling, Mikko I. Mäyränpää, Jyri Lommi, Heikki Turto, Kalervo Werkkala, Markku Kupari, Petri T. Kovanen,
Tópico(s)Lipoproteins and Cardiovascular Health
ResumoThe pathogenesis of aortic valve stenosis (AS) is characterized by the accumulation of LDL-derived cholesterol in the diseased valves. Since LDL particles also contain plant sterols, we investigated whether plant sterols accumulate in aortic valve lesions. Serum samples were collected from 82 patients with severe AS and from 12 control subjects. Aortic valves were obtained from a subpopulation of 21 AS patients undergoing valve surgery and from 10 controls. Serum and valvular total cholesterol and noncholesterol sterols were measured by gas-liquid chromatography. Noncholesterol sterols, including both cholesterol precursors and sterols reflecting cholesterol absorption, were detected in serum samples and aortic valves. The higher the ratios to cholesterol of the cholesterol precursors and absorption markers in serum, the higher their ratios in the stenotic aortic valves (r = 0.74, P < 0.001 for lathosterol and r = 0.88, P < 0.001 for campesterol). The valvular ratio to cholesterol of lathosterol correlated negatively with the aortic valve area (r = −0.47, P = 0.045), suggesting attenuation of cholesterol synthesis with increasing severity of AS. The higher the absorption of cholesterol, the higher the plant sterol contents in stenotic aortic valves. These findings suggest that local accumulation of plant sterols and cholesterol precursors may participate in the pathobiology of aortic valve disease. The pathogenesis of aortic valve stenosis (AS) is characterized by the accumulation of LDL-derived cholesterol in the diseased valves. Since LDL particles also contain plant sterols, we investigated whether plant sterols accumulate in aortic valve lesions. Serum samples were collected from 82 patients with severe AS and from 12 control subjects. Aortic valves were obtained from a subpopulation of 21 AS patients undergoing valve surgery and from 10 controls. Serum and valvular total cholesterol and noncholesterol sterols were measured by gas-liquid chromatography. Noncholesterol sterols, including both cholesterol precursors and sterols reflecting cholesterol absorption, were detected in serum samples and aortic valves. The higher the ratios to cholesterol of the cholesterol precursors and absorption markers in serum, the higher their ratios in the stenotic aortic valves (r = 0.74, P < 0.001 for lathosterol and r = 0.88, P < 0.001 for campesterol). The valvular ratio to cholesterol of lathosterol correlated negatively with the aortic valve area (r = −0.47, P = 0.045), suggesting attenuation of cholesterol synthesis with increasing severity of AS. The higher the absorption of cholesterol, the higher the plant sterol contents in stenotic aortic valves. These findings suggest that local accumulation of plant sterols and cholesterol precursors may participate in the pathobiology of aortic valve disease. The prevalence of nonrheumatic aortic valve stenosis (AS) is rapidly increasing due to general aging of the population, with clinically significant AS being present in 2% and even in 5.5% of individuals over 65 and 85 years of age, respectively (1Stewart B.F. Siscovick D. Lind B.K. Gardin J.M. Gottdiener J.S. Smith V.E. Kitzman D.W. Otto C.M. Clinical factors associated with calcific aortic valve disease. Cardiovascular Health Study.J. Am. Coll. Cardiol. 1997; 29: 630-634Crossref PubMed Scopus (1621) Google Scholar, 2Lindroos M. Kupari M. Heikkila J. Tilvis R. Prevalence of aortic valve abnormalities in the elderly: an echocardiographic study of a random population sample.J. Am. Coll. Cardiol. 1993; 21: 1220-1225Crossref PubMed Scopus (955) Google Scholar). Epidemiological risk factors of AS resemble those of atherosclerosis, including elevated serum LDL cholesterol, hypertension, smoking, diabetes, and male sex (1Stewart B.F. Siscovick D. Lind B.K. Gardin J.M. Gottdiener J.S. 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A randomized trial of intensive lipid-lowering therapy in calcific aortic stenosis.N. Engl. J. Med. 2005; 352: 2389-2397Crossref PubMed Scopus (860) Google Scholar). In addition to cholesterol, LDL particles contain noncholesterol sterols, including cholesterol precursors reflecting hepatic cholesterol synthesis (i.e., cholestenol, desmosterol, lathosterol, and squalene) and sterols reflecting intestinal cholesterol absorption (i.e., cholestanol and the plant sterols campesterol, sitosterol, and avenasterol) (20Miettinen T.A. Strandberg T.E. Gylling H. Noncholesterol sterols and cholesterol lowering by long-term simvastatin treatment in coronary patients: relation to basal serum cholestanol.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1340-1346Crossref PubMed Scopus (198) Google Scholar). While statin treatment attenuates cholesterol synthesis, it enhances the absorption of plant sterols from the intestine, thus elevating their concentrations in serum (20Miettinen T.A. Strandberg T.E. 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Indeed, the development of extremely early atherosclerotic disease, despite the presence of normal or only slightly elevated serum cholesterol levels but markedly increased phytosterol levels in these patients, suggests that plant sterols could be injurious to cardiovascular tissue (24Patel M.D. Thompson P.D. Phytosterols and vascular disease.Atherosclerosis. 2006; 186: 12-19Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Interestingly, early presentation of supravalvular aortic stenosis also has been described in a patient with phytosterolemia (28Watts G.F. Mitchell W.D. Clinical and metabolic findings in a patient with phytosterolaemia.Ann. Clin. Biochem. 1992; 29: 231-236Crossref PubMed Scopus (12) Google Scholar), suggesting that plant sterols could enter the supravalvular aortic tissue and perhaps even the aortic valve leaflets and participate in the pathogenesis of aortic valve disease in this rare metabolic condition. 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Lipid Res. 2007; 48: 139-144Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), rendering additional large-scale investigations in this area mandatory. In the present study, we investigated whether plant sterols accumulate in aortic valve leaflets and whether the degree of such accumulation would be related to circulating concentrations of the respective plant sterols. In the present study, we included 82 patients with clinically significant, symptomatic AS referred to the Helsinki University Central Hospital for valve replacement surgery. Only patients with isolated AS were included (i.e., those with more than mild aortic or mitral regurgitation or mitral stenosis were excluded). Other exclusion criteria included history of myocardial infarction or angiographically determined CAD (any proximal coronary artery stenosis > 50% of the luminal diameter), previous cardiac surgery, complicated diabetes, renal insufficiency (serum creatinine > 170 μmol/l), endocarditis, or malignancy. All patients underwent echocardiography and cardiac catheterization with coronary angiography. A more detailed description of the study population has been published elsewhere (36Kupari M. Turto H. Lommi J. Left ventricular hypertrophy in aortic valve stenosis: preventive or promotive of systolic dysfunction and heart failure?.Eur. Heart J. 2005; 26: 1790-1796Crossref PubMed Scopus (179) Google Scholar). The study protocol was approved by the Institutional Ethics Committee, and all participants signed an informed consent document. The investigation conformed with the principles outlined in the Declaration of Helsinki. The mean age of the patients was 67 ± 10 years, and their mean body mass index (BMI) was 27 ± 4.5 kg/m2. Mean (±SD) serum total cholesterol level was 5.15 ± 1.05 mmol/l, serum LDL was 3.14 ± 0.87 mmol/l, HDL was 1.44 ± 0.44 mmol/l, and triglycerides were 1.24 ± 0.44 mmol/l. Of the 82 patients, 20% received statin therapy (various agents), and 13 had consumed margarine or yogurt supplemented with plant stanol (Benecol®; n = 9) or plant sterol esters (Becel Pro-active®; n = 3) regularly on a daily basis prior to the valve replacement. Plant stanol or sterol supplements were not used by 58 patients, and reliable information was not available from 11 individuals. Control blood samples were obtained from 12 subjects undergoing electrophysiological studies for tachyarrythmias or unexplained syncope. The control subjects were free of structural heart disease and had normal echocardiographic findings. Characteristics of the patients and controls, from whom the blood samples were obtained, are shown in Table 1. Blood samples were obtained from the femoral vein of all AS patients and control subjects. Serum was separated from the blood by mild centrifugation and stored at −70°C until analysis.TABLE 1Characteristics of the patients with AS and control subjects (mean ± SD or number of patients)CharacteristicPatients with AS (n = 82)Control Subjects (n = 12)Age, years67 ± 1057 ± 4Sex, male/female39/437/5New York Heart Association class, 1/2/3/42/50/28/2All class 1Aortic valve area index, cm2/m20.36 ± 0.10Mean pressure gradient, mmHg49 ± 16Body mass index, kg/m226.8 ± 4.527.3 ± 3.1Prevalence of Left ventricular hypertrophyaEchocardiographic left ventricular mass index exceeding 110 g/m2 in women and 134 g/m2 in men.63 (77%)0 Hypertension27 (33%)2 (17%) Diabetes3 (4%)0 Bicuspid/tricuspid valve/four cusps12/69/10/12/0 Smoking, no/yes68/14 Plant stanol/plant sterol ester supplements (data were not available from 11 subjects)9 (11%)/3 (4%)UnknownMedication Angiotensin-converting enzyme inhibitor/AT1 blocker17 (21%)2 (17%) β-Blockers53 (65%)4 (33%) Diuretics28 (34%)0 Statins16 (20%)2 (17%) Digitalis5 (6%)1 (1.2%)AS, aortic valve stenosis.a Echocardiographic left ventricular mass index exceeding 110 g/m2 in women and 134 g/m2 in men. Open table in a new tab AS, aortic valve stenosis. Stenotic aortic valves removed at valve replacement surgery were collected from a random subpopulation of the AS patients (n = 21). Of these, four patients had used margarine or yogurt supplemented with plant stanol (Benecol®; n = 2) or plant sterol esters (Becel pro-active®; n = 2) regularly prior to the surgery. In this subgroup of 21 patients, statin therapy was used by 8 individuals. As control tissue samples, a separate subset of nonstenotic aortic valves (n = 10) was obtained from medicolegal autopsies. The stenotic and nonstenotic valves were snap-frozen in liquid nitrogen and stored at −70°C until analysis. Serum total and HDL cholesterol and triglycerides were quantified by routine methods used in our hospital. Serum noncholesterol sterols (cholestenol, desmosterol, lathosterol, campesterol, sitosterol, sitostanol, avenasterol, and cholestanol) and squalene were quantified by gas-liquid chromatography (GLC) using a 50 m long ULTRA-1 SE-30 column (Hewlett-Packard, Wilmington, DE) principally as shown elsewhere (37Miettinen T.A. Cholesterol metabolism during ketoconazole treatment in man.J. Lipid Res. 1988; 29: 43-51Abstract Full Text PDF PubMed Google Scholar). For this purpose, 0.2 ml of serum was saponified after addition of 5α-cholestane as an internal standard. The sterols were extracted and converted after evaporation of the solvent to TMS derivatives for the GLC running. The values for the noncholesterol sterols and squalene are expressed as μg/100 ml serum or as their ratios to the respective serum cholesterol values (102 × μg/mg cholesterol). To analyze the amount of noncholesterol sterols and squalene in valves, aortic valve tissue (100–360 mg) was weighted and the lipids were extracted by homogenizing the tissue with chloroform-methanol. Before extraction, 5α-cholestane and epicoprostanol were added as internal standards. Each homogenate was extracted three times, and the extract was evaporated and transferred in a small volume of ethyl ether onto a TLC plate coated with silica gel, and free and esterified sterol fractions were separated with hexane-ethyl ether (50:50, v/v). The fractions were extracted from the plate, and the ester fraction (including also 5α-cholestane and squalene) was saponified, after which the nonsaponifiable lipids were extracted with ethyl ether and the solvent was evaporated. The sterol fractions were silylated, and the noncholesterol sterols and squalene were quantified by GLC. The amounts of noncholesterol sterols and squalene in aortic valves are expressed as μg/100 g valvular tissue or as ratios of the sterols to the respective cholesterol values in the valves (102 × μg/mg cholesterol). For statistical calculations, SPSS software (version 11.0) was used. Differences between the groups were analyzed using Student's t-test or the Mann-Whitney U-test depending on data distribution. Normality of distribution was tested with the Kolmogorov-Smirnov method. The results are given as mean values and SD, or as medians and ranges. Differences were considered statistically significant at P < 0.05. Correlation coefficients were calculated with the Spearman rank correlation. The concentrations of cholesterol, noncholesterol sterols (cholestanol, lathosterol, desmosterol, campesterol, sitosterol, and avenasterol), sitostanol, and squalene in serum samples of AS patients and controls were in the same range (Table 2), the only statistically significant difference between patients and controls being the concentration and the ratio to cholesterol of cholestenol, which were higher in AS patients than in control subjects (Table 2; P < 0.001 for both). Serum concentrations of cholestenol, desmosterol, lathosterol, cholestanol, avenasterol, and squalene correlated positively with serum total cholesterol levels (r = 0.42–0.83, P = 0.02 to P < 0.001).TABLE 2Concentrations of cholesterol, noncholesterol sterols, and squalene in serum (S) and in aortic valves (AV) of patients with AS and control subjects (mean ± SD)VariablePatients with AS (n = 82)Controls (n = 12)aThe control group consisted of two subgroups. Serum samples were collected from the first subgroup (undergoing electrophysiological studies), and aortic valves were obtained from the second subgroup (from medicolegal autopsies).Total cholesterolbAnalyzed with gas-liquid chromatography. S (mg/100 ml) (n = 82)183 ± 37190 ± 38 AV (mg/100 g tissue) (n = 21)813 ± 385787 ± 948Squalene S (μg/100 ml)28 ± 1336 ± 19 AV (μg/100 g tissue)693 ± 1,0991,247 ± 752Cholestenol S (μg/100 ml)94 ± 4936 ± 16cP < 0.001. AV (μg/100 g tissue)146 ± 64129 ± 90Lathosterol S (μg/100 ml)206 ± 98221 ± 121 AV (μg/100 g tissue)590 ± 354508 ± 397Desmosterol S (μg/100 ml)187 ± 70172 ± 60 AV (μg/100 g tissue)918 ± 460831 ± 784Campesterol S (μg/100 ml)483 ± 271507 ± 169 AV (μg/100 g tissue)2,407 ± 1,4801,887 ± 2108Sitosterol S (μg/100 ml)227 ± 116241 ± 79 AV (μg/100 g tissue)1,193 ± 662926 ± 1017Sitostanol S (μg/100 ml)14 ± 713 ± 5 AV (μg/100 g tissue)122 ± 70132 ± 60Avenasterol S (μg/100 ml)71 ± 2374 ± 15 AV (μg/100 g tissue)365 ± 149420 ± 237Cholestanol S (μg/100 ml)284 ± 76302 ± 76 AV (μg/100 g tissue)1,654 ± 8722,263 ± 3151a The control group consisted of two subgroups. Serum samples were collected from the first subgroup (undergoing electrophysiological studies), and aortic valves were obtained from the second subgroup (from medicolegal autopsies).b Analyzed with gas-liquid chromatography.c P < 0.001. Open table in a new tab Comparison of the ratios to cholesterol of the noncholesterol sterols revealed that the ratios, which reflect cholesterol synthesis correlated positively with each other (e.g., r = 0.62, P < 0.001 for desmosterol and lathosterol). Similarly, the serum ratios to cholesterol of the plant sterols (avenasterol, sitosterol, and campesterol) and cholestanol correlated positively with each other (e.g., r = 0.84, P < 0.001 for campesterol and sitosterol). Furthermore, the serum ratios to cholesterol of lathosterol correlated negatively with those of campesterol (r = −0.56, P = 0.001) and sitosterol (r = −0.58, P < 0.001). The patients who were treated with statins (n = 16) had lower serum total cholesterol levels (P < 0.003) than the subjects not receiving statins (n = 66). Similarly, the ratios to cholesterol of the cholesterol precursors desmosterol and lathosterol were lower in subjects receiving statin therapy (P = 0.01 and P < 0.001, respectively). In contrast, the serum ratios to cholesterol of the plant sterols campesterol and sitosterol were higher in patients treated with statins compared with individuals without statin therapy (P = 0.03 and P = 0.006, respectively). Serum concentrations of cholesterol and noncholesterol sterols did not differ significantly between patients with and without having consumed margarine or yogurt supplemented with plant stanol (Benecol®; n = 9) or plant sterol esters (Becel pro-active®; n = 3). However, data concerning the use of plant stanol or plant sterol ester supplements were not available from 11 patients. Mean concentrations and the variations of cholesterol, noncholesterol sterols, and squalene (μg/100 g tissue ± SD) in aortic valves are shown in Table 2. Analysis of aortic valves with GLC revealed that, besides cholesterol, the cholesterol precursors cholestenol, desmosterol, lathosterol, and squalene were present in aortic valvular tissue. Furthermore, cholestanol, plant sterols including campesterol, sitosterol, and avenasterol, and the plant stanol sitostanol were detected in the valves. The concentrations of all cholesterol precursors (except squalene), cholestanol, and plant sterols in the valves correlated with those of cholesterol in the valves (r values ranged from 0.53 for sitostanol to 0.95 for desmosterol, P < 0.001 for all). The correlation between valvular cholesterol and sitosterol is shown in Fig. 1A. Furthermore, the concentrations of noncholesterol sterols (including cholesterol precursors and plant sterols) in aortic valves correlated positively with each other (r values ranged from 0.38 for cholestenol-sitostanol, P = 0.037, to 0.98 for sitosterol-campesterol, P < 0.001). Squalene, in contrast, correlated positively only with valvular sitostanol concentrations (r = 0.48, P = 0.007). Patients using statin therapy (n = 8) had a trend toward higher levels of plant sterols (campesterol, sitosterol, and avenasterol) and cholestanol in their aortic valves, although these differences were not statistically significant. The concentration of sitostanol, instead, was significantly higher in subjects receiving statins compared with that of subjects not receiving statins (P = 0.048). When valvular noncholesterol sterols and squalene concentrations were proportioned to valvular cholesterol concentrations, a positive correlation appeared between the cholesterol precursors cholestenol, desmosterol, lathosterol, and squalene (r = 0.37–0.84, P < 0.05 to P < 0.001). Similarly, the ratios of plant sterols (campesterol, sitosterol, and avenasterol) to cholesterol correlated positively with each other (r = 0.38–0.84, P < 0.05 to P
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