doi: 10.1161/01.ATV.0000218504.71680.b5
© 2006 American Heart Association, Inc.
Letters to the Editor |
Nonpharmacological Treatment of Hypercholesterolemia Increases Circulating Endothelial Progenitor Cell Population in Adults
Department of Internal Medicine and Public Health, University of L’Aquila, Italy
To the Editor:
Hypercholesterolemia represents a major cardiovascular risk factor because of its ability to promote and sustain proatherogenic inflammation of vascular wall.1 A reduction of number and activity of bone marrow–derived endothelial progenitor cells (EPCs) could participate in the development of vascular damage in hypercholesterolemic patients.2 Indeed, EPCs serve as a cellular reservoir to replace dysfunctional endothelium and to form a cellular patch at the site of denuding injury.3 According to this, the level of circulating EPCs predicts the occurrence of cardiovascular events and death from cardiovascular causes.4 Nonpharmacological treatment represents the first-line approach to primary prevention in hypercholesterolemia because of its effects on lipid profile and cardiovascular outcomes.5 Despite the wealth of evidence derived from epidemiological and interventional trials, there is limited understanding of the underlying molecular mechanisms. To clarify this topic, we evaluated whether or not changes in dietary habits, alone or in association with regular physical activity, were able to affect the number of circulating EPCs in patients with isolated hypercholesterolemia.
We studied 38 never-treated hypercholesterolemic patients (LDL cholesterol between 4.1 and 4.9 mmol/L) without additional cardiovascular risk factors and/or concomitant diseases, including clinical conditions in which neovascularization might be present, such as cardiovascular disease, retinopathy, wound healing, or cancer. Patients were consecutively recruited among those who met the above criteria and referred to our Outpatient Unit for Cardiovascular Prevention between October 2004 and June 2005. After enrollment, all patients were randomly assigned to a 4-week treatment period based either on diet alone (10F/10M, 46.8±8.3 years) or on diet+physical training (8F/10M, 47.8±6.2 years). For this purpose, dietitians provided individualized dietary counseling aiming to indicate acceptable substitutions for favorite foods contributing to increase LDL cholesterol, according to Adult Treatment Panel III – Therapeutic Lifestyle Changes.5 For physical training, patients were asked to exercise daily for 30 minutes (including 5-minute warm-up and cool-down periods during each session, respectively) on a bicycle ergometer at 50% to 70% of their maximum heart rate.6
At baseline and after the intervention period blood samples were taken for routine hematochemical check and assessment of circulating number of EPCs. For this purpose, mononuclear cells were isolated using Ficoll density-gradient centrifugation from 20 mL of peripheral blood, washed three times in PBS, resuspended in EGM-2 Bullet kit (Cambrex, Milan, Italy). Then, 106 mononuclear cells per cm2 were seeded on fibronectin-coated culture dishes (Becton & Dickinson). After 4 days of culture, nonadherent cells were discarded by washing with PBS while adherent cells were maintained in culture for further 3 days and then underwent cytochemical analysis. To confirm the EPC phenotype, adherent cells were incubated with DiI-labeled acLDL (Molecular Probes), at a concentration of 2.4 µg/mL for 1 hour at 37°C. Cells were then fixed with 1% paraformaldehyde for 10 minutes and incubated with fluorescein isothiocyanate (FITC)-labeled Ulex europaeus agglutinin I (Ulex-lectin; Sigma) at a concentration of 10 µg/mL for 1 hour. Dual-staining cells positive for both DiI-acLDL and FITC-labeled Ulex-lectin were judged as EPCs.7 EPCs were counted manually in 10 randomly selected microscopic fields by two independent investigators with an inverted fluorescent microscope (x20).
Compared with baseline, two factor analysis of variance for repeated measures on one factor demonstrated a significant increase of EPC number in both treatment groups (Figure, panel A). Increments of EPCs were more evident in patients on diet+physical activity than in those on diet alone (Figure, panel A). LDL cholesterol levels significantly decreased after 4 weeks under both nonpharmacological treatment strategies being the decrement more evident in patients on diet+physical activity than in those on diet alone (Figure, panel B). Serum HDL cholesterol levels increased (P<0.04) while serum triglyceride concentrations decreased (P<0.004) in both treatment groups. Spearman nonparametric correlation showed a significant inverse relationship between treatment-induced changes in serum LDL cholesterol concentrations and circulating EPC number in the whole hypercholesterolemic population (Figure, panel C). In a multivariate regression analysis with a stepwise approach the reduction of serum LDL cholesterol concentrations independently predicted changes in EPC number (r2=0.19, P=0.0056).
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), on the number of circulating endothelial progenitor cells (A) and serum LDL cholesterol concentrations (B) in 38 hypercholesterolemic patients (symbols indicate means, vertical bars indicate standard deviation). Panel C shows the relationship between treatment-induced changes in circulating endothelial progenitor cell number and serum LDL cholesterol levels in the combined two patient groups.Our study provides the first evidence that a nonpharmacological approach to hypercholesterolemia is associated with a significant increase of circulating EPC number in adults. This effect is more evident if dietary intervention is combined with physical training and mainly depends on LDL cholesterol reduction. In this regard, it is worth mentioning the recent demonstration that oxidized LDLs interfere with differentiation8 while increasing senescence9 of EPCs, thus potentially reducing their circulating pool. Because hypercholesterolemia is associated with increased lipid peroxidation,10,11 it is intriguing to speculate that a reduction of oxidative stress after nonpharmacological correction of hypercholesterolemia could have played a role in the observed increase in EPC population.
Considering the suggested critical role of EPCs in restoring and maintaining multiple endothelial functions and counteracting atherogenesis and acute vascular complications of atherosclerosis,3 our findings shed new light on the mechanisms underlying the observed benefits deriving from a healthy lifestyle in hypercholesterolemic patients.5
References
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3. Urbich C, Dimmeler S. Endothelial progenitor cells. Characterization and vascular biology. Circ Res. 2004; 95: 343–353.
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5. Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) Final Report. Circulation. 2002; 106: 3143–3421.
6. Fletcher GF, Balady GJ, Amsterdam EA, Chaitman B, Eckel R, Fleg J, Froelicher VF, Leon AS, Pina IL, Rodney R, Simons-Morton DA, Williams MA, Bazzarre T. Exercise standards for testing and training. A statement for healthcare professionals from the American Heart Association. Circulation. 2001; 104: 1694–1740.
7. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, Li T, Isner JM, Asahara T. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A. 2000; 97: 3422–3427.
8. Imanishi T, Hano T, Matsuo Y, Nishio I. Oxidized low-density lipoprotein inhibits vascular endothelial growth factor-induced endothelial progenitor cell differentiation. Clin Exp Pharmacol Physiol. 2003; 30: 665–670.[CrossRef][Medline] [Order article via Infotrieve]
9. Imanishi T, Hano T, Sawamura T, Nishio I. Oxidized low-density lipoprotein induces endothelial progenitor cell senescence, leading to cellular dysfunction. Clin Exp Pharmacol Physiol. 2004; 31: 407–413.[CrossRef][Medline] [Order article via Infotrieve]
10. Davì G, Alessandrini P, Mezzetti A, Minotti G, Bucciarelli T, Costantini F, Cipollone F, Bon GB, Ciabattoni G, Patrono C. In vivo formation of 8-Epi-prostaglandin F2 alpha is increased in hypercholesterolemia. Arterioscler Thromb Vasc Biol. 1997; 17: 3230–3235.
11. Desideri G, Croce G, Tucci M, Passacquale G, Broccoletti S, Valeri L, Santucci A, Ferri C. Effects of bezafibrate and simvastatin on endothelial activation and lipid peroxidation in hypercholesterolemia: evidence of different vascular protection by different lipid-lowering treatments. J Clin Endocrinol Metab. 2003; 88: 5341–5347.
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