Monounsaturated and Polyunsaturated n-6 Fatty Acid–Enriched Diets Modify LDL Oxidation and Decrease Human Coronary Smooth Muscle Cell DNA Synthesis
Abstract Proliferation of smooth muscle cells (SMCs) plays an important role in atherosclerotic lesion progression. The purpose of this investigation was to examine the effect of diets differing in fatty acid composition on human coronary SMC entry in the cell proliferation cycle. Twenty-four healthy men and women were placed on four consecutive diets lasting 5 weeks each: (1) saturated fatty acid (SFA)–rich diet with palm oil; (2) monounsaturated fatty acid (MUFA)–rich diet with olive oil; (3) polyunsaturated fatty acid (PUFA) n-6–rich diet with sunflower oil; and (4) PUFA n-3–rich diet (3.8 g/d). All diets supplied 35% of calories as fat. Compared with the SFA diet, all unsaturated diets reduced LDL cholesterol. Resistance of LDL to oxidative modification was significantly increased during the MUFA period (P<.05). Human coronary SMCs were cultured and induced by sera derived from the different groups. 3H-Thymidine incorporation into doubling DNA was significantly (P<.01) reduced during the MUFA and PUFA n-6 periods but not during the PUFA n-3 diet with respect to the SFA diet. This effect was more pronounced in women than in men. In conclusion, the MUFA-enriched diet reduced SMC DNA synthesis and LDL levels and protected LDL from oxidation. Therefore, these combined effects suggest that an oleic acid–rich Mediterranean diet could be better than PUFA (n-6)– or PUFA (n-3)–rich diets in the prevention of atherosclerosis.
- monounsaturated fatty acids
- polyunsaturated fatty acids
- lipid oxidation
- smooth muscle cells
- Received May 3, 1996.
- Accepted January 20, 1997.
Proliferation of smooth muscle cell (SMC) is a typical characteristic of atherosclerotic lesion progression1 and an important target for preventing the development of arterial lesions. Furthermore, SMC proliferation is a major limitation to the success of interventional revascularization procedures. The precise cellular nature of accelerated lesion progression is still debated but there is no doubt that intimal SMC proliferation plays a pivotal role in restenosis following angioplasty.2 3
The progression of coronary artery disease (CAD) is strongly influenced by intake of saturated fatty acids (SFA), an effect additionally mediated by mechanisms other than its influence on total and LDL cholesterol.4 Epidemiologic studies had suggested that intake of polyunsaturated fatty acids (PUFA) n-3 was associated with a low incidence of coronary events.5 A number of studies sought to determine whether dietary therapy with PUFA n-3 had beneficial effects on the inhibition of restenosis; however, the results have been contradictory, showing effects from beneficial to detrimental.6 7 8 9 10
Data from the Seven Countries Study11 suggested that the Mediterranean diet, which is rich in monounsaturated fatty acids (MUFA), primarily as olive oil, was associated with a low rate of CAD. Recently, a diet similar to the Mediterranean diet was successful in the secondary prevention of coronary events and death.12 However, there are few studies on the cellular and molecular mechanisms that operate to produce that clear preventive effect on cardiovascular disease. Several studies have documented the effects of PUFA n-3 on SMC, but to our knowledge there are no studies in humans comparing the effects of diets enriched in SFA, MUFA, PUFA n-6 and PUFA n-3 on SMC. The present study has been addressed to evaluate the effect of serum obtained from healthy subjects (men and women) on a dietary longitudinal study changing the four major classes of fatty acids on human coronary SMC de novo DNA synthesis by S-phase incorporation of 3H-thymidine.
The study consisted of a longitudinally designed protocol of four consecutive isocaloric diets, each lasting 5 weeks, each with increasing degrees of nonsaturation. The diets were consumed always in the same order (SFA, MUFA, PUFA n-6, and PUFA n-3) in all the study participants.13 Menus were prepared using similar food items to eliminate variations due to seasonal changes in food supply. Informed consent was obtained from all subjects, who provided their medical records and underwent physical examination. This protocol was approved by the Human Studies Committee of the Universidad Autónoma de Madrid.
The characteristics of the study population have been described.13 Briefly, volunteers from two religious communities were enrolled in the study. Their regular lifestyle contributed to our avoiding behavioral changes as possible variables during the study. None of them was on any drug treatment including vitamin supplements.
A cohort of 24 individuals (12 females and 12 males) were randomly selected for studying the effects of serum collected in the four dietary periods on human coronary SMC proliferation. Mean age was 42±2 years in men and 46±1 years in women.
All menus were prepared using conventional food items. Special emphasis was placed on using foodstuffs that were not substantially different from those habitually consumed by the communities, with seven daily menus rotating weekly. We used direct chemical analysis to evaluate the composition of the diets (see below), which was 15% protein, 50% carbohydrate, and 35% fat (percentage of total energy) and was maintained constant in all periods (Table 1⇓). This distribution reflects the regular diet of the participants, as well as the diet consumed in most Mediterranean countries. Dietary fiber (21-23 g) and cholesterol (<300 mg/d) were also maintained at constant levels between diet phases. Palm oil and butter constituted the major fats in the SFA period, representing about 55% of the total fat intake. Olive oil, rich in oleic acid in its natural cis-configuration, accounted for 55% of the fat intake during the MUFA period, whereas sunflower oil represented 50% of the PUFA n-6 and 37.5% of the PUFA n-3 diet periods. All diets, except for the PUFA n-3 period, included the following foods weekly: veal twice, chicken twice, ham or cheese twice, legumes twice, rice twice, pasta once, vegetables seven times, white fish four times a week, and three eggs. During the PUFA n-3 period, blue fish (mackerel, salmon, and sardines) were substituted for veal, chicken, and white fish, with one fish meal provided per day every day of the week. This regimen provided about 3.2 and 4.5 g/d of n-3 fatty acids in women and men, respectively. In addition, the subjects consumed daily a fixed amount of bread, cookies, fruit, green salad, jam, and milk.
Body weight was measured twice a week, and the individual carbohydrate intake was adjusted, if needed, to prevent weight changes of more than 2%. All meals were consumed in the dinning halls. Individualized and weighed portions were provided to each participant.
Duplicate samples of all meals from randomly selected individuals (one man and one woman) were collected every day during one week of each diet phase and stored at −20°C (Table 1⇑). Fatty acid composition of the dietary oils used in this study was determined by capillary gas-liquid chromatography at the Food Analysis Laboratory of the Spanish Ministry of Agriculture. Palm oil consisted mainly of (% by weight) 37% palmitic acid, 43.6% oleic acid, and 11.7% linoleic acid. The major fatty acids in olive oil were 9.2% palmitic acid, 80% oleic acid, and 4.7% linoleic acid. The sunflower oil contained 6.8% palmitic acid, 29% oleic acid, and 56% linoleic acid.
Blood Sampling and Analysis
Fasting (12-hour) blood samples were obtained at baseline and twice during the last week of each dietary period. Blood was collected and serum separated by low speed centrifugation (3000 rpm). When plasma was needed, blood was collected in EDTA-containing (4.0 mmol/L) vacutainers, which were immediately protected from exposure to light and chilled in ice. Plasma was separated by low-speed centrifugation and gentamicin and chloramphenicol (0.22 and 0.15 mmol/L, respectively) were added to the samples. Samples were kept at −70°C until used.
Lipoprotein separation and analysis were carried out as recommended by the Lipid Research Clinics manual of laboratory operations. Total cholesterol and triglyceride levels were measured by enzymatic methods (Boehringer Mannheim, Mannheim, Germany) on a Tecnikon RA-XT autoanalyzer. HDL was measured after precipitation of apo B-containing lipoproteins with phosphotungstic MgCl2 (Boehringer Mannheim).
Blood was collected in acid-citrate-dextrose after subjects fasted overnight and platelet rich plasma (PRP) was prepared by centrifugation (15 minutes, 200 g). Within 2 hours of PRP preparation, platelets (adjusted to 250×106/mL) were challenged with collagen (2 μg/mL), epinephrine (10 μmol/L), and ADP (2 μmol/L), and optical platelet aggregation was measured following standard techniques.14
LDL Isolation and Analysis of Oxidation-Related Variables
LDL was isolated by sequential ultracentrifugation immediately after separation of plasma. The LDL fraction was dialyzed for 24 hours at 4°C against phosphate-buffered saline (PBS, 10 mmol/L phosphate, 0.16 mol/L saline, pH 7.4) in the dark. The PBS was maintained in an oxygen-free state by purging with pure nitrogen. LDL protein content was determined by the method of Bradford.15
The content of thiobarbituric acid-reactive substances (TBARS) was assessed in freshly isolated LDL.16 The results are expressed as malondialdehyde (MDA) equivalents (nanomoles per MDA per milligram of LDL protein). LDL oxidation was initiated by incubation with 5 μmol/L of CuSO4 (100 μg of LDL protein/mL PBS). Kinetics of LDL oxidation were examined by monitoring changes in diene absorbance (at 234 nm, 37°C, for 4 hours at 2 minute intervals) (Beckman DU-8 B ultraviolet). Lag time, rate of oxidation, and total amount of conjugated dienes (normalized by milligrams of LDL protein) were evaluated.17
Plasma and LDL α-tocopherol were measured by high-performance liquid chromatography as previously described,18 using a reverse phase column (Resolve C18, 5 μm, Waters). Retinol acetate was used as internal standard, and α-tocopherol (Sigma) as external standard. Results are given as micromoles per liter of α-tocopherol for plasma values and in molecules per particle for LDL values.
Plasma Fatty Acid Composition
Fatty acids from cholesteryl esters and phospholipids were transmethylated and analyzed in a Perkin Elmer Autosystem chromatograph equipped with a capillary column (Supelco SP-2380; 60 m by 0.25 mm). Values were expressed as percentage of total fatty acid.19
Smooth Muscle Cell Culture
Primary coronary human SMCs were prepared by a modification of the explant technique.20 Segments of normal coronary arteries were obtained from excised hearts at transplantation (Heart Transplant Unit, Hospital San Pablo, Barcelona). Explants were incubated in M199 supplemented with 10% FCS, 1% l-glutamine, and antibiotics (Gibco BRL Life Technologies) in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Cells used in the experiments were from the second or third passages. SMC were identified morphologically by light microscopy and by their growth patterns. They were characterized by alpha-actin (+) and vWF (−) immunofluorescence.
3H-Thymidine Incorporation in Newly Synthesized DNA
Approximately 5×10 E4 cells per milliliter were plated in 24 well plates and grown to subconfluency (48 hours approximately). They were synchronized by incubation in serum-free medium with insulin, transferrin, and ascorbic acid for 48 hours. Serum (15% of final well volume) from the study subjects was incubated with the quiescent synchronized h-SMCc. Serum of each single subject from the four dietary periods was assayed in parallel without any other addition. In all experiments individual separated wells were incubated with a control pool of human serum (from nonparticipant healthy individuals) (HCS) and FCS, both in similar concentrations and used as external standards of h-SMCc activity. Labeled thymidine incorporation to newly formed DNA was measured as an index of cell entry into the proliferation cycle. After 24 hours of incubation with serum, cells were pulsed with [6-3H]-TdR (specific activity: 27 Ci/mmol; Amersham International plc, Buckinghamshire, United Kingdom) 0.5 μCi/mL. In a preliminary study with human coronary SMC, we compared the effect of adding 3H-TdR during the first 24 hours or from 24 to 48 hours after cell induction with human serum. The later time period had the peak of 3H-Td incorporation (2.5 times higher than the earlier period) and was selected for the study. Twenty-four hours later (48 hours after induction) cultures were washed with PBS, fixed with 95% methanol, treated with 10% TCA at 4°C, and dissolved in 0.3 N NaOH. Aliquots were counted in a β-counter (1217 RackBeta, Wallac, Pharmacia LKB). Results are expressed as cell-incorporated 3H-TdR dpm and normalized by counts incorporated into arrested Go cells. Protein content was measured in an aliquot of the cell extract by the method of Bradford.15
Similarly, cells were counted after 48 hours of induction by hemocytometry and in a cell counter (Coulter Multisizer II). Additionally, cell mitochondrial activity to test cellular survival was measured at the same time-period by the MTT method (reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide to blue formazan crystal in the cytoplasm of viable cells) as described.21
Results are expressed as mean±SEM unless otherwise stated. Statistical analysis for two-group tests was performed by Student’s t test for paired or unpaired observations when groups had equal variances (F test) and by Mann-Whitney U test for groups with unequal variances (F test). Multiple group means were compared by one or multiple factor ANOVA with factorial or repeated measures analysis as required and differences between groups analyzed by Fisher PLSD Scheffé F test for parametric data and Kruskal-Wallis test for nonparametric data. Simple regression analysis was performed between a dependent Y variable and an independent X variable (cell proliferation) in a one-X/one-Y statistic with ANOVA.
Diet composition, fatty acid profile, and cholesterol content data are shown in Table 1⇑. Protein, carbohydrate, and fat contents of the four diets were practically identical. Body weight was not changed by the dietary interventions.
Compliance to the diets was assessed by analysis of fatty acids in plasma phospholipids and cholesteryl esters (Table 2⇓). Consumption of the SFA diet resulted in slight but statistically significant increases in C16:0 as compared with the three other dietary periods. C18:1 was significantly increased during the MUFA diet compared with the other diet periods. C18:2 was significantly elevated during both PUFA diets compared with the SFA and MUFA diets; additionally, the PUFA n-3 phase was significantly enriched in the long-chain PUFA (C20:5 and C22:6) compared with the three other diet phases.
Plasma Lipid Levels
Plasma cholesterol concentrations were significantly elevated during the SFA period (227±5 mg/dL, P<.02) relative to the MUFA (205±5), PUFA n-6 (193±4), and PUFA n-3 (186±4) periods (Table 3⇓). When data were analyzed by gender, men displayed significantly higher cholesterol levels during the SFA period (217±6 mg/dL, P<.05 versus the three other diets), whereas similar cholesterol levels were observed during the MUFA (197±6 mg/dL), PUFA n-6 (191±6 mg/dL), and PUFA n-3 (183±6 mg/dL) periods. In women, as described for the entire group, the highest cholesterol level was associated with the consumption of the SFA diet (240±7 mg/dL). A significant decrease was observed during the MUFA (216±6 mg/dL), PUFA n-6 (196±6 mg/dL), and PUFA n-3 (190±7 mg/dL) diets, respectively, P<.05. The difference between the latter two diets was not statistically significant, but they were significantly reduced from the MUFA diet. Diet effects on LDL cholesterol levels paralleled those described for total cholesterol. HDL cholesterol levels were lower in men than in women. For all subjects, no difference in HDL cholesterol was observed between the SFA (53±2 mg/dL) and the others. In women during the PUFA n-6 and PUFA n-3 periods, however, HDL cholesterol levels (56±2 mg/dL, P<.05) were lower than during the SFA and MUFA periods. When men were analyzed separately, dietary fat saturation did not have any significant effect on HDL cholesterol levels. There were no changes in diet-induced plasma triglycerides.
Vitamin E content of the LDL particle (molecules of alpha-tocopherol per LDL particle) was measured at the end of each diet period. No differences were noted between the SFA (9.8±0.5) and the MUFA (11.0±0.3) periods; however, these values were significantly lower (P<.05) than those obtained during both the PUFA n-6 (13.2±0.7) and the PUFA n-3 (13.9±0.6) periods. No differences in vitamin E content in LDL were noted between the latter two periods. Plasma vitamin E levels were also measured in micromoles per liter, which showed a profile similar to that of LDL. SFA (34.5±1.4) and MUFA (34.9±1.6) periods gave significantly lower values than PUFA n-6 (41.8±2.9) and PUFA n-3 (45.3±2.8) periods.
TBARS (expressed as nanomoles per milligram of LDL protein) were determined in freshly isolated LDL. Identical values were observed during the SFA (0.89±0.05) and the MUFA (1.06±0.04) periods, but they were significantly higher during the PUFA n-6 (1.56±0.08) or the PUFA n-3 (1.70±0.07) periods. Resistance to Cu2+-induced oxidative modification was increased during the MUFA period (P<.05) with respect to all others, and conjugated diene formation was significantly increased only with PUFA (n-6 and n-3) diets (Table 4⇓).
SMC Entry in the Cell Proliferation Cycle
Human coronary smooth muscle cell DNA synthesis was significantly (P<.05) reduced during the MUFA and PUFA n-6 periods but not during the PUFA n-3 diet with respect to the SFA period (Fig 1A⇓). Protein content per well was not significantly modified (range 55-57 μg per well; P=.58 ANOVA among the four dietary periods); therefore, there were no hypertrophic changes. Cells counted at 48 hours after induction did not show significant changes among the four dietary periods (range 7×104−10×104 cells/mL, P=.851 ANOVA among the four dietary periods). Cellular survival measured by mitochondrial activity (MTT method) was similar at this time point among the four periods (range from 1.46 to 1.89, P=.1573). Cell premitotic DNA synthesis induced by FCS was significantly lower (3.2±0.2 times over arrested cells; P=.0001) than that induced by HCS (8.2±0.5) and from that induced by sera from the different fatty acid–enriched diets (Fig 1A⇓).
When results were analyzed by gender a significant effect was observed between male and female data for SFA, PUFA n-6, and PUFA n-3 periods, with a higher proliferative response systematically induced by female sera (Fig 1B⇑). Interestingly, the lowest proliferative effects were found during the PUFA n-6 period in male sera and the second lowest for the MUFA period in female sera. Simple regression of dependent variables (Y) on cell DNA synthesis data was analyzed (Table 5⇓). Only statistically significant regressions are presented in Table 5⇓. Total cholesterol, LDL cholesterol, HDL cholesterol, and TGL did not show a significant regression on induction of DNA synthesis. Oxidation of LDL, within the physiologic ranges of the study, did not show any relation to premitotic cell DNA synthesis either.
The female subset of data was reanalyzed in terms of pre- or postmenopausal condition. Sera from postmenopausal women (n=8) induced a significantly higher proliferative index than sera from premenopausal women (n=4) in all dietary periods (Fig 2⇓). Cell DNA synthesis induced by premenopausal female sera was significantly higher than that induced by male sera in all dietary interventions.
Platelet aggregation did not show significant changes during the four dietary periods (Table 6⇓). Neither ADP, collagen, nor epinephrine showed evidence of changes in platelet function during the interventions.
This controlled diet study in healthy men and women demonstrates that changes in fatty acid composition without extreme variations in their regular solid food diet produce significant effects on human coronary SMC DNA doubling measured as 3H-thymidine incorporation into de novo synthesized DNA.
The isocaloric substitution of SFA by MUFA, PUFA n-6, or PUFA n-3 resulted in a significant decrease in total plasma and LDL cholesterol levels, as reported in other diet studies.22 23 24 The reduction in total cholesterol achieved with either type of PUFA exceeded that obtained with the MUFA diet. This finding probably is related to the degree of nonsaturation.25 HDL cholesterol levels in the total population were similar during the four dietary phases. However, HDL cholesterol levels in women were significantly lower during the PUFA n-6 diet compared with the other three diets, a finding in agreement with results of previously reported studies.19 22
In the present study the susceptibility of plasma LDL to undergo oxidative modification, as measured by several parameters, was significantly reduced during the MUFA-enriched diet. Other studies have shown an increased resistance of LDL to oxidation when comparing MUFA with PUFA-enriched diets.26 27 28 The increase in MUFA content of the diet and the associated increase in oleic acid and decrease in linoleic acid (Table 2⇑) seem to regulate the resistance of LDL to oxidative changes. LDL fatty acid distribution (data not shown) shows a similar profile to that found in plasma (Table 2⇑).13 Although lag time was decreased during the SFA diet compared with MUFA diet, conjugated diene formation was similar in both periods. This suggests that differences in fatty acid content (18:1/18:2) may influence the lag time before diene formation is initiated.
Dietary fat saturation may be involved in additional biologic effects beyond those seen in plasma lipoprotein. We tested platelet aggregation to show changes in platelet function. There was no difference among the four dietary periods in platelet response to diverse agonists (ADP, epinephrine, collagen). However, this does not preclude other possible effects of platelets and platelet-released products on SMC function. Serum, used in this study, is a multifactorial universal mitogen, with agonists derived also from blood cells and coagulation. Just because of that multichanneled mitogenic capacity of serum, the reported changes in SMC DNA synthesis by dietary intake are most important. The odds were against finding differences in such a mitogen-rich milieu. Our results demonstrate that human SMC incubated with serum isolated during the MUFA and PUFA n-6 diets have the lowest rate of entry in the S phase of the cell cycle. Conversely, serum obtained during the SFA and PUFA n-3 phases induced the highest DNA synthetic activity. The effect was more pronounced in women than in men, and postmenopausal women show higher SMC proliferation than premenopausal women.
In this study there was no association between any of the lipid parameters, LDL oxidation, and human SMC premitotic DNA synthesis. The relationship between lipoproteins and smooth muscle cell growth has been repeatedly studied, with controversial results.29 30 31 In in vitro–supplemented SMC cultures, lipoproteins have not been shown to be direct growth factors but in some circumstances may potentiate growth in mitogen-stimulated cells.29 30 Our data, with in vivo–modified LDL levels within a physiologic span of oxidation, do not show any correlation between LDL and human SMC entry in the cell cycle. However, fatty acids may have a regulatory effect in cell cycle cell entry, as they have in the regulation of gene expression and cell signaling pathways.32 33 Phospholipid and cholesteryl ester fatty acid 20:5 showed a highly significant positive correlation with SMC premitotic DNA synthesis. Cell viability measured by mitochondrial activity at the same time point after induction (48 hours) was unaffected by the different sera.
A limitation of this study is that at the selected time point (48 hours of SMC induction with the human sera) we cannot measure modification in human SMC numbers. In fact, we investigated this point further with HCS and coronary SMC to find that more than 3 days are needed to be able to show a significant difference (data not shown). Changes in 3H-TdR incorporation measure cell entry in the cell cycle by premitotic DNA synthesis but do not exclude changes in cell ploidy and size. Therefore further studies are needed to measure changes in cell number and actual proliferation.
Recent studies have shown that the procedural outcome of coronary angioplasty for postmyocardial infarction ischemia is similar in women and men, but in long-term follow-up data women experience an increased incidence of recurrent angina, an outcome also reported after bypass surgery.34 Interestingly, we have found that sera derived from female individuals, regardless of the dietary period, induced higher rates of premitotic DNA doubling than male sera.
With controversial results, several studies on restenosis have addressed the potential role of a diet supplemented with n-3 fatty acids obtained from fish oil.6 7 8 9 10 The largest trial to date on n-3 fatty acids failed to prevent restenosis after angioplasty.10 Furthermore, a remarkable reduction in recurrent myocardial infarction and death has been found among subjects consuming a diet similar to the usual Mediterranean diet.12 Our results obtained with an enriched olive oil diet could provide, in part, a candidate mechanism to explain the beneficial effects found in secondary prevention of coronary artery disease (CAD) with diets similar to the Mediterranean diet.
This study correlating fatty acid changes in the same individuals with vascular-mediated events provides a unique tool to measure vascular effects of nutrition. Our study comparing four diets has provided detailed information on the effects of fatty acid changes not shown in studies with only two interventions. Direct comparison of SFA with MUFA or PUFA n-6 could have provided evidence of a relation between SMC induction and the lowering effect on LDL, whereas direct comparison of MUFA with PUFA n-3 could have provided evidence of a relation to oxidation.
In summary, our results demonstrate that MUFA and PUFA n-6–enriched diets produce the lowest induction of SMC entry in the cell cycle when compared with SFA- and PUFA n-3–enriched diets. Whether or not this effect influences cell division and proliferation remains to be elucidated. In addition, a diet rich in MUFA, when substituted for saturates, lowers total and LDL cholesterol without altering HDL cholesterol and without impairing LDL oxidation. These combined effects suggest that a feasible dietary intervention with MUFA could be beneficial and one of the most valuable interventions against atherosclerosis and its complications.
Selected Abbreviations and Acronyms
|MUFA||=||monounsaturated fatty acid(s)|
|PUFA||=||polyunsaturated fatty acid(s)|
|SFA||=||saturated fatty acid(s)|
|SMC||=||smooth muscle cell(s)|
|TBARS||=||thiobarbituric acid–reactive substances|
This study has been supported in part by grants FIS 93/0428, FIS 95/0917, FIS 95/0838, CYCIT SAF 94/0712, and the Cardiovascular Research Foundation–Cátedra Catalana Occidente, Barcelona. We are indebted to the participants in the study for their great cooperation and enthusiasm. We thank José R. García-Hierro and the Analytical Laboratory of the Spanish Ministry of Agriculture for the analysis of the diets. Olive and sunflower oils were kindly supplied by Aceites Toledo, S.A., Spain, and palm oil by Agra, S.A., Spain. We also thank the Cardiac Transplant Team of the Department of Cardiology of Hospital de la Sta. Cruz y San Pablo, Barcelona, for their cooperation.
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