Endothelial Progenitor Cells following exercise training in haemodialysis patients

Endothelial progenitor cells (EPCs) are correlated to the risk factors and to the cardiovascular outcome. EPCs were found increased in animals and humans following training. Haemodialysis patients have poor exercise capacity, high cardiovascular mortality and a low number of EPCs, correlated to their exercise capacity. No data are available that address variation in the levels of EPCs following training. Objectives This study aimed to evaluate the response of EPCs to a six-month exercise program conducted at moderate intensity in haemodialysis patients.Methods Thirty dialysis patients (20 males, age 67±12) were included into two groups: prescribed exercise (E), n=16; and control (C), n=14. Patients were evaluated upon entry and after six-months. 1)Haematological evaluation We studied peripheral blood CD34+ cells and EPCs, assessed both as CD34+ cells coexpressing AC133 and vascular-endothelial-growth-factor (VEGF) receptor-2 and as endothelial-colony-forming-units (e-CFU). a) Quantification of CD34+ cells: performed with double-labeling, with fluorescein Isothiocyanate-(FITC)-anti-CD45 and phycoerythrin-(PE)-anti-CD34 monoclonal antibodies (Becton Dickinson) on a FACSCalibur flow cytometer (Becton Dickinson) according to standardized procedures. b) Enumeration of EPC or CD34+ cells coexpressing AC133 and VEGFR-2: performed by flow cytometry on immunomagnetically purified PB CD34+ cells (Miltenyi Biotech) by triple labeling with Peridinin Chlorophyll Protein-conjugated anti-CD34 (Becton Dickinson), PE-conjugated anti-AC133 (Miltenyi Biotech), unconjugated anti-VEGFR-2 (Santa Cruz Biotechnology) followed by FITC-conjugated swine anti-rabbit (Dako) as secondary reagent. Enumeration of endothelial colony forming units (e-CFUs) was performed as described by Hill et al. Density gradient isolated peripheral blood mononuclear cells were resuspended in Medium 199 (GIBCO BRL Life Technologies) supplemented with 20% fetal calf serum, penicillin and streptomycin and plated on dishes coated with human fibronectin (Becton Dickinson). After 48 hours, non-adherent cells were collected and replated. Growth medium was changed every 3 days and colonies were counted after 7 days. Confirmation of endothelial-cell lineage was performed with antibodies against CD31 (Becton Dickinson), VEGFR-2 and factor VIII (Dako). 2)Exercise capacity evaluation Assessed using the six-minute walking test. Exercise program: Two daily home walking sessions (10-min each), conducted at moderate intensity (50% of the individual maximal treadmill speed) and updated during monthly visits at the hospital, were prescribed to the E-group on the non-dialysis day. No exercise was prescribed to the C-group. Results Twenty-two patients (E=14, C=8) completed the study. E-group: a different training load in relation to present or intercurrent comorbidities was performed (walking speed=69±11 steps/min; total time=2571±2233 min). Patients significantly increased 6MWD (308±101 to 347 ±114m, P=0.001). No significant changes of CD34+ or CD34+AC133+VEGFR2+ cells were observed, whereas e-CFUs significantly increased (P<0.05). The e-CFU variation was found to be directly correlated to the reported training time and intensity (r=0.75, P=0.002; r=0.74, P=0.003 respectively). C-group: no significant variation of performance or of CD34+ and EPCs levels was observed. Conclusions In haemodialysis patients, exercise training conducted at moderate-intensity selectively increased e-CFUs, suggesting an improved capacity of EPCs to proliferate.