Arq Bras Cardiol abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC wVfZJ4SfZPkjKzsJYxfJGmh S0066-782X2017000300212 10.5935/abc.20170022 Original Articles Atherosclerosis/Endothelium/Vascular Effects of Chronic Exercise on Endothelial Progenitor Cells and Microparticles in Professional Runners Bittencourt Célia Regina de Oliveira 1 Izar Maria Cristina de Oliveira 1 França Carolina Nunes 2 Schwerz Valdir Lauro 1 Póvoa Rui Manuel dos Santos 1 Fonseca Francisco Antonio Helfenstein 1 Universidade Federal de São Paulo São Paulo SP Brazil Universidade Federal de São Paulo (UNIFESP),( )São Paulo, SP - Brazil Universidade Federal de São Paulo Universidade de Santo Amaro São Paulo SP Brazil Universidade de Santo Amaro, São Paulo, SP - Brazil Universidade de Santo Amaro Mailing Address: Francisco Antonio Helfenstein Fonseca • Rua Loefgren, 1350. Postal Code: 04040-00, V. Clementino, São Paulo, SP - Brazil. E-mail: ffonseca@cardiol.br; fahfonseca@terra.com.br

Author contributions

Conception and design of the research: Izar MCO, Fonseca FAH; Acquisition of data: Bittencourt CRO, França CN, Schwerz VL; Analysis and interpretation of the data and Critical revision of the manuscript for intellectual contente: Bittencourt CRO, Izar MCO, França CN, Schwerz VL, Póvoa RMS, Fonseca FAH; Statistical analysis: Bittencourt CRO, Izar MCO, França CN, Fonseca FAH; Obtaining financing: Fonseca FAH; Writing of the manuscript: Izar MCO, França CN, Póvoa RMS, Fonseca FAH.

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

 03 2017 108 3 212 216 11 05 2016 23 05 2016 10 11 2016 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background:

The effects of chronic exposure to exercise training on vascular biomarkers have been poorly explored.

 Objective:

Our study aimed to compare the amounts of endothelial progenitor cells (EPCs), and endothelial (EMP) and platelet (PMP) microparticles between professional runners and healthy controls.

 Methods:

Twenty-five half-marathon runners and 24 age- and gender-matched healthy controls were included in the study. EPCs (CD34+/KDR+, CD133+/KDR+, and CD34+/CD133+), EMP (CD51+) and PMP (CD42+/CD31+) were quantified by flow-cytometry. All blood samples were obtained after 12 h of fasting and the athletes were encouraged to perform their routine exercises on the day before.

 Results:

As compared with controls, the CD34+/KDR+ EPCs (p=0.038) and CD133+/KDR+ EPCs (p=0.018) were increased, whereas CD34+/CD133+ EPCs were not different (p=0.51) in athletes. In addition, there was no difference in MPs levels between the groups.

 Conclusion:

Chronic exposure to exercise in professional runners was associated with higher percentage of EPCs. Taking into account the similar number of MPs in athletes and controls, the study suggests a favorable effect of exercise on these vascular biomarkers.

 Keywords: Endothelial Progenitor Cells Biomarkers Athletes Sports Running Introduction

An appropriate number of circulating endothelial progenitor cells (EPCs) seems related with the maintenance of vascular homeostasis.1,2 In fact, decreased number of EPCs has been associated with cardiovascular risk factors, cardiovascular mortality, and recurrent cardiovascular events in subjects with coronary heart disease,3,4 despite some controversies regarding the measurement, characterization, origin and destiny of such cells.5,6

Microparticles (MPs) are small cell-derived anucleoid phospholipid particles (100-1000 nm) that can be identified by their origin from endothelium (EMP), platelets (PMP) or many other cells. Increased number of EMPs has been linked with endothelial injury or endothelial dysfunction.7,8 Interestingly, PMPs, initially considered markers of thrombosis, are now considered relevant for some transcriptional signaling, for the interaction with monocytes and activation of inflammatory responses.9

Regular exercise has been widely recommended for prevention of cardiovascular disease, but information regarding the effects of chronic and intense exposure to exercise on these vascular biomarkers is scarce.10,11 Thus, the objective of this study was to evaluate the effects of chronic exercise in professional runners on EPCs and MPs.

 Methods Study population

Professional half-marathon runners (n=25) and age and gender-matched controls (n=24) without known cardiovascular diseases were prospectively included. Subjects with cardiovascular risk factors such as hypertension, diabetes, obesity, smoking, or hypercholesterolemia were excluded. The local ethics committee approved the study (# 1808/08) and all participants have signed the informed consent prior to their inclusion in the study protocol.

Laboratory analysis

Blood samples were obtained after 12 hours of fasting and the analyses were performed at the central laboratory of our university. All athletes were allowed to maintain their daily exercises program even on the day before blood sample collection. The athletes had very similar exercise training programs, corresponding to two long-distance running sessions every day, 15 km in the morning and 10 km in the afternoon, and intensive training (100-1,000 meter shots, repeated many times)twice a week, on Tuesday and Thursday mornings. All blood samples were collected on Thursdays, before exercise.

Measurements of EPCs and MPs were performed as previously reported, using fresh blood samples in EDTA containing tubes.12-15 For determination of EPCs, a minimum of 500,000 events was acquired by flow-cytometry (FACSCalibur, BD Biosciences, USA). Fluorescently labeled mouse anti-human antibodies were used for EPCs (CD34 FITC, BD Biosciences, USA; CD133 APC, Miltenyi Biotec, USA; KDR PE, R&D Systems, USA), PMPs (CD42 FITC and CD31 PE, BD Biosciences, USA) and EMPs (CD51 FITC, BD Biosciences). Disposable containers (BD Biosciences) were used to quantify the number of microparticles per microliter of platelet-poor plasma (PPP).

 Statistical analysis

Results are presented as mean ± standard deviation (SD) or by median and interquartile range (IQR), for normal or non-Gaussian distributions, respectively. Categorical variables were compared by Pearson's Chi-square test. Kolmogorov-Smirnov and Shapiro-Wilk tests were used to assess normality of continuous variables. Between-group comparisons of continuous variables were made by unpaired t-test or Mann-Whitney test, when appropriate. Spearman's rank correlation test was used to evaluate correlations of EPCs and MPs with variables of ergospirometry. All analyses were performed using SPSS 17.0 for Windows (SPSS, Inc., Chicago, IL) and significance was set at p<0.05.

 Results

All athletes reported to have exercised on the day before (22.08 ± 2.67 km, mean ± SD), and the mean time between the last exercise session and blood collection was 16.5 ± 2.8 hours. Male and female athletes did not differ in both distance (124±25 vs. 128±29 km per week, p=0.88, respectively, mean ± SD, unpaired t test) and time spent in training (14±4 vs. 14±7 hours per week, mean±SD, p=0.53, respectively, unpaired t test). Despite exposure to the same training regimen, male athletes reported better mean time for 10,000 meters than female athletes (32.4±2.1 vs. 37.6±1.6 min, p<0.0001, mean±SD, unpaired t test). As compared with controls, athletes had lower weight, body mass index, abdominal circumference and percentage of body fat, lower heart rate, and higher body lean mass, but similar values of systolic and diastolic blood pressure. In addition, they presented lower serum levels of total cholesterol, LDL-C and triglycerides, and higher serum levels of HDL-C than controls.

Endothelial progenitor cells and microparticles

Compared to controls, the athletes presented higher percentage of two lineages of EPCs (CD34+/KDR+, and CD133+/KDR+) and similar percentage of CD34+/CD133+ cells (Figure 1).

Box-plots showing the percentage of circulating endothelial progenitor cells (EPCs) determined by flow-cytometry. Higher percentage of CD34+/KDR+ EPCs (A) (p=0.038 vs. controls, Mann-Whitney U test), as well as CD133+/KDR+ EPCs (p=0.018 vs. controls, Mann-Whitney U test) (B) were found in athletes. No differences were observed between groups for CD133+/CD34+ (p=0.51) (C).

The amount of EMPs and PMPs did not differ between the two groups (Figure 2).

No correlation between the percentage of EPCs or MPs with variables of ergospirometry was observed, including absolute and maximum rate of oxygen consumption (VO2max) (data not shown).

Box-plots representing the amount of circulating microparticles determined by flow-cytometry. The number of CD42+/CD31+ platelet microparticles (PMPs) (A) and CD51+ endothelial microparticles (EMPs) (B) was similar between the groups. (PMPs, p=0.695, Mann-Whitney U test; EMPs, p=0.496, Mann-Whitney U test). PPP - platelet-poor plasma.

Discussion

The present study revealed that the chronic exposure to exercise training among professional runners was associated with increased percentage of circulating EPCs without changes in the amount of EMPs or PMPs. These findings suggest that chronic exercise was not associated with endothelial cell apoptosis or thrombosis. In fact, it seemed to have a protective effect in these subjects, taking into account the observed increase in EPCs. In our athletes, blood samples were collected during their routine training program, since we wanted to evaluate EPCs and MPs in real-life context.

Several cardiovascular risk factors including diabetes,3 hypertension,16 smoking,17 hypercholesterolemia,18 and age.19 have been related to reduced function of circulating EPCs. Conversely, exercise has been recognized as a promise tool to increase EPCs.20,21 Early experimental and clinical studies22,23 reported increased number of EPCs after regular exercise, although the effects of exercise on EPCs seemed to be influenced by training regimen, age of subjects, and concomitant presence of cardiovascular disease, such as coronary heart disease or heart failure.20

Circulating EMPs have been linked to several stimuli, including the transcription of interleukins, chemokines and chemoattractants mediated by activation of nuclear factor-κB (NF-κB), and associated with oxidative stress.8,24 All these conditions have been long associated with classical cardiovascular risk factors, but more recently, new biological effects mediated by EMPs have been considered, including transport of mRNAs, microRNAs and other active molecules of physiologic relevance for angiogenesis and tissue repair.25

Cellular activation and apoptosis are linked to release of MPs. Of special interest, the amount of PMPs has been recognized as a possible marker of thrombosis, due to their high content of phospholipids and potential pro-thrombogenic roles because of thrombin generation.26 Besides, high shear stress triggers platelet aggregation and release of platelet derived MPs. 27 In addition, circulating PMPs may carry tissue factor (TF), which can also generate thrombin and platelet activation. However, it is also true that MPs may transport some inhibitors of coagulation, such as the TF pathway inhibitor (TFPI) that can neutralize, in part, the procoagulant properties of these MPs.28 More recently, interesting aspects linking PMPs to the signaling of inflammatory and immune responses have been proposed, considering the potential transcriptional factors in the platelets, that include nuclear factor kappa β (NF-κKB) and peroxisome proliferator-activated receptor gamma (PPARγ).29

In our study, we found increased percentage of EPCs in athletes and similar number of EMPs and PMPs in comparison with healthy controls, despite the intensive training of these professional athletes. These promising findings are important because our understanding of the role of exercise on EPCs and MPs is mainly derived from acute exposure or in non-athletes.10,11,30,31 Intermittent and high-intensity exercise induces catecholamine release and decreases highly differentiated T cells, but does not increase the amount of EPCs compared with continuous exercise33. In other article, despite increase in white blood cells count, the amount of EPCs observed in advanced-aged marathon runners was not modified when collected in the early period after the race.33

In addition, among other biochemical variables, C-reactive protein levels were lower in athletes than in controls, and creatine phosphokinase levels modestly increased, even with the routine training on the day before blood sample collection, reinforcing protective properties of high-performance exercise.

Study limitations

Although this was a cross-sectional, case-control study, our results cannot be considered as hypothesis generating, since we do not have baseline laboratory values of the athletes. Finally, these results are applicable to marathon runners and cannot be extrapolated to other sports.

 Conclusions

Chronic exercise was associated with a favorable increase in EPCs, without affecting circulating levels of MPs in professional runners, suggesting a positive impact of prolonged exposure to chronic exercise on these vascular biomarkers.

Sources of Funding

This study was funded by FAPESP and was partially funded by CAPES.

Study Association

This article is part of the thesis of master submitted by José Henrique Cunha Figueiredo, from Universidade Federal do Rio de Janeiro.

 References 1 Chan KH, Simpson PJ, Yong AS,Dunn LL, Chawantanpipat C, Hsu C, et al. The relationship between endothelial progenitor cell populations and epicardial and microvascular coronary disease-a cellular, angiographic and physiologic study. PLoS One. 2014;9(4):e93980. Chan KH Simpson PJ Yong AS Dunn LL Chawantanpipat C Hsu C The relationship between endothelial progenitor cell populations and epicardial and microvascular coronary disease-a cellular, angiographic and physiologic study PLoS One 2014 9 4 e93980 2 Madonna R, De Caterina R. Circulating endothelial progenitor cells: Do they live up to their name? Vascul Pharmacol. 2015 Apr-Jun.67-69:2-5. Madonna R De Caterina R Circulating endothelial progenitor cells: Do they live up to their name? Vascul Pharmacol 2015 Apr-Jun. 67-69 2 5 3 Saad MI, Abdelkhalek TM, Saleh MM, Kamel MA, Youssef M, Tawfik SH, et al.. Insights into the molecular mechanisms of diabetes-induced endothelial dysfunction: focus on oxidative stress and endothelial progenitor cells. Endocrine. 2015;50(3):537-67. Saad MI Abdelkhalek TM Saleh MM Kamel MA Youssef M Tawfik SH Insights into the molecular mechanisms of diabetes-induced endothelial dysfunction: focus on oxidative stress and endothelial progenitor cells Endocrine 2015 50 3 537 567 4 Peng J, Liu B, Ma QL, Luo XJ. Dysfunctional endothelial progenitor cells in cardiovascular diseases: role of NADPH oxidase. J Cardiovasc Pharmacol. 2015;65(1):80-7. Peng J Liu B Ma QL Luo XJ Dysfunctional endothelial progenitor cells in cardiovascular diseases: role of NADPH oxidase J Cardiovasc Pharmacol 2015 65 1 80 87 5 Werner N, Kosiol S, Schiegl T, Ahlers P, Walenta K, Link A, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med. 2005;353(10):999-1007. Werner N Kosiol S Schiegl T Ahlers P Walenta K Link A Circulating endothelial progenitor cells and cardiovascular outcomes N Engl J Med 2005 353 10 999 1007 6 Bakogiannis C, Tousoulis D, Androulakis E, Briasoulis A, Papageorgiou N, Vogiatzi G,et al. Circulating endothelial progenitor cells as biomarkers for prediction of cardiovascular outcomes. Curr Med Chem. 2012;19:2597-604. Bakogiannis C Tousoulis D Androulakis E Briasoulis A Papageorgiou N Vogiatzi G Circulating endothelial progenitor cells as biomarkers for prediction of cardiovascular outcomes Curr Med Chem 2012 19 2597 2604 7 Mause SF, Weber C. Microparticles: protagonists of a novel communication network for intercellular information exchange. Circ Res. 2010;107(9):1047-57. Mause SF Weber C Microparticles: protagonists of a novel communication network for intercellular information exchange Circ Res 2010 107 9 1047 1057 8 Berezin A, Zulli A, Kerrigan S, Petrovic D, Kruzliak P. Predictive role of circulating endothelial-derived microparticles in cardiovascular diseases. Clin Biochem. 2015;48(9):562-8. Berezin A Zulli A Kerrigan S Petrovic D Kruzliak P Predictive role of circulating endothelial-derived microparticles in cardiovascular diseases Clin Biochem 2015 48 9 562 568 9 Nomura S, Shimizu M. Clinical Significance of procoagulant microparticles. J Intensive Care. 2015;3(1):2. Nomura S Shimizu M Clinical Significance of procoagulant microparticles J Intensive Care 2015 3 1 2 2 10 Guiraud T, Gayda M, Juneau M, Bosquet L, Meyer P, Théberge-Julien G, et al. A single bout of high-intensity interval exercise does not increase endothelial or platelet microparticles in stable, physically fit men with coronary heart disease. Can J Cardiol. 2013;29(10):1285-91. Guiraud T Gayda M Juneau M Bosquet L Meyer P Théberge-Julien G A single bout of high-intensity interval exercise does not increase endothelial or platelet microparticles in stable, physically fit men with coronary heart disease Can J Cardiol 2013 29 10 1285 1291 11 Wahl P, Jansen F, Achtzehn S, Schmitz T, Bloch W, Mester J, et al. Effects of high intensity training and high volume training on endothelial microparticles and angiogenic growth factors. PLos One. 2014;9(4):e96024. Wahl P Jansen F Achtzehn S Schmitz T Bloch W Mester J Effects of high intensity training and high volume training on endothelial microparticles and angiogenic growth factors PLos One 2014 9 4 e96024 12 Camargo LM, França CN, Izar MC, Bianco HT, Lins LS, Barbosa SP, et al. Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor cells and platelet aggregation in subjects with coronary heart disease under platelet therapy. Braz J Med Biol Res. 2014;47(5):432-7. Camargo LM França CN Izar MC Bianco HT Lins LS Barbosa SP Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor cells and platelet aggregation in subjects with coronary heart disease under platelet therapy Braz J Med Biol Res 2014 47 5 432 437 13 Lins LC, França CN, Fonseca FA, Barbosa SP, Matos LN, Aguirre AC, et al. Effects of ezetimibe on endothelial progenitor cells and microparticles in high-risk patients. Cell Biochem Biophys. 2014;70(1):687-96. Lins LC França CN Fonseca FA Barbosa SP Matos LN Aguirre AC Effects of ezetimibe on endothelial progenitor cells and microparticles in high-risk patients Cell Biochem Biophys 2014 70 1 687 696 14 Pinheiro LF, França CN, Izar MC, Barbosa SP, Bianco HT, Kasmas SH,et al. Pharmacokinetic interactions between clopidogrel and rosuvastatin: effects on vascular protection in subjects with coronary heart disease. Int J Cardiol. 2012;158(1):1125-9. Pinheiro LF França CN Izar MC Barbosa SP Bianco HT Kasmas SH Pharmacokinetic interactions between clopidogrel and rosuvastatin: effects on vascular protection in subjects with coronary heart disease Int J Cardiol 2012 158 1 1125 1129 15 da Silva EF, Fonseca FA, França CN, Ferreira PR, Izar MC, Salomão R,et al. Imbalance between endothelial progenitor cells and microparticles in HIV-infected patients naive for antirretroviral therapy. AIDS. 2011;25(13):1595-601. da Silva EF Fonseca FA França CN Ferreira PR Izar MC Salomão R Imbalance between endothelial progenitor cells and microparticles in HIV-infected patients naive for antirretroviral therapy AIDS 2011 25 13 1595 1601 16 Pirro M, Schillaci G, Menecali C, Bagaglia F, Paltriccia R, Vaudo G, et al. Reduced number of circulating endothelial progenitors and HOXA9 expression in CD34+ cells of hypertensive patients. J Hypertens. 2007;25(10):2093-9. Pirro M Schillaci G Menecali C Bagaglia F Paltriccia R Vaudo G Reduced number of circulating endothelial progenitors and HOXA9 expression in CD34+ cells of hypertensive patients J Hypertens 2007 25 10 2093 2099 17 Lamirault G, Susen S, Forest V, Hemont C, Parini A, Le Corvoisier P, et al. Difference in mobilization of progenitor cells after myocardial infarction in smoking versus non-smoking patients: insights from the BONAMI trial. Stem Cell Res Ther. 2013;4(6):152. Lamirault G Susen S Forest V Hemont C Parini A Le Corvoisier P Difference in mobilization of progenitor cells after myocardial infarction in smoking versus non-smoking patients: insights from the BONAMI trial Stem Cell Res Ther 2013 4 6 152 152 18 Rodríguez C, Slevin M, Rodríguez-Calvo R, Kumar S, Krupinski J, Tejerina T, et al. Modulation of endothelium and endothelial progenitor cell function by low-density lipoproteins: implication for vascular repair, angiogenesis and vasculogenesis. Pathobiology. 2009;76(1):11-22. Rodríguez C Slevin M Rodríguez-Calvo R Kumar S Krupinski J Tejerina T Modulation of endothelium and endothelial progenitor cell function by low-density lipoprotein: simplication for vascular repair, angiogenesis and vasculogenesis Pathobiology 2009 76 1 11 22 19 Goligorsky MS. Endothelial progenitor cells: from senescence to rejuvenation. Semin Nephrol. 2014;34(4):365-73. Goligorsky MS Endothelial progenitor cells: from senescence to rejuvenation Semin Nephrol 2014 34 4 365 373 20 Koutroumpi M, Dimopoulos S, Psarra K, Kyprianou T, Nanas S. Circulating endothelial and progenitor cells: Evidence from acute and long-term exercise effects. World J Cardiol. 2012;4(12):312-26. Koutroumpi M Dimopoulos S Psarra K Kyprianou T Nanas S Circulating endothelial and progenitor cells: Evidence from acute and long-term exercise effects World J Cardiol 2012 4 12 312 326 De Biase C, De Rosa R, Luciano R, De Luca S, Capuano E, Trimarco B,et al. Effects of physical activity on endothelial progenitor cells (EPCs). Front Physiol. 2014; Feb 3;4:414. De Biase C De Rosa R Luciano R De Luca S Capuano E Trimarco B Effects of physical activity on endothelial progenitor cells (EPCs) Front Physiol. 03 02 2014 4 414 22 Laufs U, Werner N, Link A, Endres M, Wassmann S, Jürgens K, et al. Physical training increases endothelial progenitor cells, inhibits neointima formation, and enhances angiogenesis. Circulation. 2004;109(2):220-6. Laufs U Werner N Link A Endres M Wassmann S Jürgens K Physical training increases endothelial progenitor cells, inhibits neointima formation, and enhances angiogenesis Circulation 2004 109 2 220 226 23 Hoetzer GL, Van Guilder GP, Irmiger HM, Keith RS, Stauffer BL, DeSouza CA. Aging, exercise, and endothelial progenitor cell clonogenic and migratory capacity in men. J Appl Physiol. 1985; (1985). 2007;102(3):847-52. Hoetzer GL Van Guilder GP Irmiger HM Keith RS Stauffer BL DeSouza CA Aging, exercise, and endothelial progenitor cell clonogenic and migratory capacity in men J Appl Physiol. 1985; (1985) 2007 102 3 847 852 24 Lee SK, Yang SH, Kwon I, Lee OH, Heo JH. Role of tumour necrosis fator receptor-1 and nuclear fator-kB in productionof TNF-α-induced pro-inflammatory microparticles in endothelial cells. Thromb Haemost. 2014;112(3):580-8. Lee SK Yang SH Kwon I Lee OH Heo JH Role of tumour necrosis fator receptor-1 and nuclear fator-kB in productionof TNF-α-induced pro-inflammatory microparticles in endothelial cells Thromb Haemost 2014 112 3 580 588 25 Enjeti AK, Lincz LF, Seldon M. Microparticles in health and disease. Semin Thromb Hemost. 2008;34(7):683-91. Enjeti AK Lincz LF Seldon M Microparticles in health and disease Semin Thromb Hemost 2008 34 7 683 691 26 Berckmans RJ, Nieuwland R, Boing AN, Romijn FP, Hack CE, Sturk A. Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation. Thromb Haemost. 2001;85(4):639-46. Berckmans RJ Nieuwland R Boing AN Romijn FP Hack CE Sturk A Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation Thromb Haemost 2001 85 4 639 646 27 Nomura S. Function and clinical significance of platelet-derived microparticles. Int J Hematol. 2001;74(4):397-404. Nomura S Function and clinical significance of platelet-derived microparticles Int J Hematol 2001 74 4 397 404 28 Keuren FJ, Magdeleyns EJ, Govers-Riemslag JW, Lindhout T, Curvers J. Effects of storage-induced platelet microparticles on the initiation and propagation phase of blood coagulation. Br J Haematol. 2006;134(3):307-13. Keuren FJ Magdeleyns EJ Govers-Riemslag JW Lindhout T Curvers J Effects of storage-induced platelet microparticles on the initiation and propagation phase of blood coagulation Br J Haematol 2006 134 3 307 313 29 Lannan KL, Sahler J, Kim N, Spinelli SL, Maggirwar SB, Garraud O, et al. Breaking the mold: transcription factors in the anucleate platelet and platelet-derived microparticles. Front Immunol. 2015;Feb 6:48. Lannan KL Sahler J Kim N Spinelli SL Maggirwar SB Garraud O Breaking the mold: transcription factors in the anucleate platelet and platelet-derived microparticles Front Immunol 2015 02 06 48 30 Fernandes JM, Rosado-Alvares D, Da Silva Grigoletto ME, Rangel-Zuniga OA, Landaeta-Diaz LL, et al. Moderate-to-high-intensity training and a hypocaloric Mediterranean diet enhance endotelial progenitor cells and fitness in subjects with the metabolic syndrome. Clin Sci (Lond). 2012 ; 123(6):361-73. Fernandes JM Rosado-Alvares D Da Silva Grigoletto ME Rangel-Zuniga AO Landaeta-Diaz LL Moderate-to-high-intensity training and a hypocaloric Mediterranean diet enhance endotelial progenitor cells and fitness in subjects with the metabolic syndrome Clin Sci (Lond) 2012 123 6 361 373 31 Rakobowchuk M, Harris E, Taylor A, Baliga V, Cubbon RM, Rossiter HB, et al. Heavy and moderate interval exercise training alters low-flow-mediated constriction but does not increase circulation progenitor cells in healthy humans. Exp Physiol. 2013;97(3):375-85. Rakobowchuk M Harris E Taylor A Baliga V Cubbon RM Rossiter HB Heavy and moderate interval exercise training alters low-flow-mediated constriction but does not increase circulation progenitor cells in healthy humans Exp Physiol 2013 97 3 375 385 32 Kruger K, Alack K, Ringseis R, Mink L, Pfeifer E, Schinle M, et al. Apoptois of T-Cell subsets after acute high-intensity interval exercise. Med Sci Sports Exerc. 2016;48(10):2021-9. Kruger K Alack K Ringseis R Mink L Pfeifer E Schinle M Apoptois of T-Cell subsets after acute high-intensity interval exercise Med Sci Sports Exerc 2016 48 10 2021 2029 33 Adams V, Linke A, Breuckmann F, Leineweber K, Erbs S, Kränkel N, et al. Circulating progenitor cells decrease immediately after marathon race in advanced-age marathon runners. Eur J Cardiovasc Prev Rehabil. 2008;15(5):602-7. Adams V Linke A Breuckmann F Leineweber K Erbs S Kränkel N Circulating progenitor cells decrease immediately after marathon race in advanced-age marathon runners Eur J Cardiovasc Prev Rehabil 2008 15 5 602 607 Artigos Originais Aterosclerose/Endotélio/Vascular Efeitos de Exercício Crônico Sobre Células Progenitoras Endoteliais e Micropartículas em Corredores Profissionais Bittencourt Célia Regina de Oliveira 1 Izar Maria Cristina de Oliveira 1 França Carolina Nunes 2 Schwerz Valdir Lauro 1 Póvoa Rui Manuel dos Santos 1 Fonseca Francisco Antonio Helfenstein 1 Universidade Federal de São Paulo (UNIFESP),( ) São Paulo, SP - Brasil. Universidade de Santo Amaro, São Paulo, SP - Brasil. Correspondência: Francisco Antonio Helfenstein Fonseca • Rua Loefgren, 1350. CEP 04040-00, V. Clementino, São Paulo, SP - Brasil. E-mail: ffonseca@cardiol.br; fahfonseca@terra.com.br

Contribuição dos autores

Concepção e desenho da pesquisa: Izar MCO, Fonseca FAH; Obtenção de dados: Bittencourt CRO, França CN, Schwerz VL; Análise e interpretação dos dados e Revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Bittencourt CRO, Izar MCO, França CN, Schwerz VL, Póvoa RMS, Fonseca FAH; Análise estatística: Bittencourt CRO, Izar MCO, França CN, Fonseca FAH; Obtenção de financiamento: Fonseca FAH; Redação do manuscrito: Izar MCO, França CN, Póvoa RMS, Fonseca FAH.

Potencial conflito de interesses

Declaro não haver conflito de interesses pertinentes.

 Resumo Fundamento:

Os efeitos da exposição crônica ao exercício sobre biomarcadores vasculares foram pouco estudados.

 Objetivo:

Nosso estudo teve como objetivo comparar as quantidades de células progenitoras endoteliais (CPEs), e de micropartículas endoteliais (MPEs) e plequetárias (MPPs) de corredores profissionais com controles sadios.

 Métodos:

Vinte e cinco corredores de meia maratona e 24 controles pareados quanto à idade e ao sexo foram incluídos no estudo. CPEs (CD34+/KDR+, CD133+/KDR+ e CD34+/CD133+), MPE (CD51+) e MPPs (CD42+/CD31+) foram quantificadas por citometria de fluxo. Todas as amostras de sangue foram obtidas após 12 horas de jejum, e os atletas foram incentivados a realizar seus exercícios de rotina no dia anterior à coleta.

 Resultados:

Em comparação aos controles, CPEs CD34+/KDR+ (p=0,038) e CD133+/KDR+ (p=0,018) estavam aumentados, e CPEs CD34+/CD133+ não foram diferentes (p=0,51) nos atletas. As concentrações de MP não diferiram entre os grupos.

 Conclusão:

A exposição crônica ao exercício em corredores profissionais associou-se a uma maior porcentagem de CPEs. Considerando o número similar de MPs entre atletas e controles, o estudo sugere um efeito favorável do exercício sobre esses biomarcadores vasculares.

 Palavras-chave: Células Progenitoras Endoteliais Exercício Biomarcadores Atletas Esportes Corrida Introdução

Um número adequado de células progenitoras endoteliais (CPEs) circulantes parece estar relacionado com a manutenção da homeostase vascular.1,2 De fato, número reduzido de CPEs foi associado com fatores de risco e mortalidade cardiovasculares, e recorrência de eventos cardiovasculares em indivíduos com doença coronariana,3,4 apesar de algumas controvérsias em relação à forma de medida, caracterização, origem e destino das células.5,6

Micropartículas (MPs) são pequenas partículas (100-1000nm) anucleadas de fosfolipídio, que podem ser identificadas por sua origem - MPs endoteliais (MPEs), plaquetárias (MPPs), ou de várias outras células. Número aumentado de MPEs foi associado com dano e disfunção endotelial.7,8 As MPPs, inicialmente consideradas como marcadores de trombose, são atualmente consideradas importantes para a sinalização transcricional, pela interação com monócitos, e ativação de resposta inflamatória.9

A prática de exercícios regulares tem sido amplamente recomendada para a prevenção de doença cardiovascular. Porém, informações sobre os efeitos da exposição crônica e intensa sobre esses biomarcardores vasculares são ainda escassos.10,11 Assim, o objetivo deste estudo foi avaliar os efeitos do exercício crônico sobre CPEs e MPs em corredores profissionais.

 Métodos População do estudo

Vinte e cinco corredores profissionais de meia maratona e 24 controles pareados por sexo e idade, sem diagnóstico de doenças cardiovasculares foram incluídos prospectivamente. Foram excluídos indivíduos com fatores de risco cardiovasculares tais como hipertensão, diabetes, obesidade, tabagismo, ou hipercolesterolemia. O comitê de ética local aprovou o estudo (#1808/08) e todos os participantes assinaram o termo de consentimento antes de serem incluídos no protocolo do estudo.

Análises laboratoriais

As amostras de sangue foram obtidas após 12 horas de jejum, e as análises foram realizadas no laboratório central de nossa universidade. Todos os atletas mantiveram seus programas de exercício diário até mesmo no dia anterior ao da coleta de amostra de sangue. Os programas de treinamento dos atletas eram muito semelhantes entre si, correspondendo a duas sessões de corrida de longa distância diariamente, 15 km de manhã e 10 km à tarde, e treinamentos intensivos (tiros de 100 a 1000 m de distância, repetidos várias vezes) duas vezes por semana, nas terças e quintas de manhã. Todas as amostras de sangue foram colhidas nas quintas-feiras, antes do exercício.

As medidas de CPEs e MPs foram realizadas conforme descrito anteriormente, utilizando amostras de sangue fresco colhido em tubos contendo EDTA.12-15 Para a determinação de CPEs, foram adquiridos um mínimo de 500 000 eventos for citometria de fluxo (FACSCalibur, BD Biosciences, USA). Foram usados anticorpos de camundongos anti-humanos marcados por fluorescência para CPEs (CD34 FITC, BD Biosciences, USA; CD133 APC, Miltenyi Biotec, USA; KDR PE, R&D Systems, USA), MPPs (CD42 FITC e CD31 PE, BD Biosciences, USA), e MPEs (CD51 FITC, BD Biosciences). Foram usados recipientes descartáveis para quantificar o número de micropartículas por microlitro de plasma pobre em plaquetas (PPP).

 Análise estatística

Os resultados são apresentados em média ± desvio padrão (DP) ou mediana e intervalo interquartil para distribuições normais e não normais respectivamente. As variáveis categóricas foram comparadas por teste de qui-quadrado de Pearson. Os testes de Kolmogorov-Smirnov e Shapiro-Wilk foram usados para avaliar a normalidade das variáveis contínuas. Comparações entre grupos das variáveis contínuas foram feitas pelo teste t não pareado ou pelo teste de Mann-Whitney quando apropriado. O teste de correlação de Spearman foi usado para avaliar correlações de CPEs e MPs com variáveis de ergoespirometria. Todas as análises foram realizadas pelo programa SPSS para Windows, versão 17.0 (SPSS, Inc., Chicago, IL), e um valor de p<0.05 foi considerado significativo.

 Resultados

Todos os atletas relataram haver feito exercício no dia anterior à coleta de sangue (22,08±2,67 km, média ± DP), e a média de tempo entre a última sessão de exercício e a coleta de sangue foi de 16,5 ± 2,8 horas. Não houve diferença entre homens e mulheres quanto à distância percorrida (124 ± 25 vs. 128 ± 29 km por semana, p=0,88, respectivamente, média ± DP, teste t não pareado) ou tempo de treinamento (14±4 vs. 14±7 horas por semana, média ± DP, p=0,53, respectivamente, teste t não pareado). Apesar de haverem recebido o mesmo treinamento, atletas do sexo masculino relataram um melhor tempo médio para a distância de 10 000 metros em comparação a atletas do sexo feminino (32,4 ± 2,1 vs. 37,6±1,6 min, p<0,0001, média ± DP, teste t não pareado). Em comparação aos controles, os atletas apresentaram menor peso, índice de massa corporal, circunferência abdominal, e porcentagem de gordura corporal, menor frequência cardíaca, maior porcentagem de gordura corporal e valores similares de pressão sistólica e diastólica. Apresentaram ainda menores níveis de colesterol total, LDL-c e triglicerídeos, e valores mais elevados de HDL-c.

Células progenitoras endoteliais e micropartículas

Em comparação aos controles, os atletas apresentaram maior porcentagem de duas linhagens de CPEs (CD34+/KDR+ e CD133+/KDR+) e porcentagem similar de células CD34+/CD133+ (Figura 1).

Box-plots mostrando a porcentagem de células progenitoras endoteliais (CPEs) determinadas por citometria de fluxo. Porcentagens mais altas de CD34+/KDR+ EPCs (A) (p=0,038 vs. controles, teste de Mann-Whitney) e de CD133+/KDR+ EPCs (p=0,018 vs. controles, teste de Mann-Whitney) (B) foram encontradas em atletas. Não foi observada diferença entre os grupos para CD133+/CD34+ (p=0,51) (C).

A quantidade de MPP não foi diferente entre os grupos (Figura 2).

Box-plots representando a quantidade de micropartículas circulantes determinada por citometria de fluxo. O número de micropartículas plaquetárias (MPPs) de CD42+/CD31+ (A) e micropartículas endoteliais (MPEs) de CD51+ (B) foi similar entre os grupos (MPPs, p=0,695, teste de Mann-Whitney; MPEs, p=0,496, teste de Mann-Whitney). PPP: plasma pobre em plaquetas.

Não houve correlação entre a porcentagem de CPEs ou MPs com variáveis de ergospirometria, incluindo taxa absoluta e máxima de consumo de oxigênio (VO2 max) (dados não apresentados).

 Discussão

O presente estudo revelou que a exposição crônica ao treinamento em corredores profissionais esteve associada com porcentagens aumentadas de CPEs circulantes sem alterações na quantidade de MPEs ou MPPs. Esses achados sugerem que o exercício crônico não se associou com apoptose de células endoteliais ou trombose nesses indivíduos. De fato, parece haver exercido um papel protetor, considerando-se o aumento nas CPEs. Em nossos atletas, as amostras de sangue foram coletadas durante seus programas de treinamento de rotina, uma vez que nosso objetivo foi avaliar CPEs e MPs no contexto da vida real desses indivíduos.

Vários fatores de risco cardiovasculares, incluindo diabetes,3 hipertensão,16 tabagismo,17 hipercolesterolemia,18 e idade19 foram relacionados à disfunção de CPEs. Por outro lado, o exercício foi reconhecido como uma ferramenta promissora para aumentar os níveis de MPEs.20,21 Estudos experimentais e clínicos iniciais22,23 relataram CPEs aumentadas após a prática regular de exercício, apesar de os efeitos dos exercícios sobre CPEs parecerem ser influenciados pelo regime de treinamento, idade dos indivíduos, e presença de doença cardiovascular, tais como doença arterial coronariana e insuficiência cardíaca.20

MPEs circulantes foram associadas a vários estímulos, incluindo a transcrição de interleucinas, quimiocinas e quimioatraentes, e ao estresse oxidativo.8,24 Todas essas condições estão associadas a fatores de risco cardiovascular clássicos mas, mais recentemente, novos efeitos biológicos mediados pelas MPEs foram considerados, incluindo transporte de RNAm, micro-RNAs e outras moléculas ativas, de importância fisiológica para a angiogênese e reparo tecidual.25

A ativação celular e a apoptose estão associadas à liberação de MPs. Particularmente, a quantidade de MPPs foi considerada como um possível marcador de trombose, dada à quantidade de fosfolipídios dessas partículas e potencial papel pró-trombogênico pela produção de trombina.26 Além disso, uma tensão de cisalhamento elevada leva à agregação plaquetária e liberação de MPs derivadas de plaquetas.27 Além disso, MPPs podem carregar fator tissular, o qual também pode produzir trombina e ativação plaquetária. No entanto, também é verdade que as MPs podem transportar alguns inibidores da coagulação, tais como inibidor da via do fator tecidual (TFPI) que pode neutralizar, em parte, as propriedades pró-coagulantes dessas MPs.28 Mais recentemente, foram propostos aspectos interessantes que ligam MPPs à sinalização de resposta imune e inflamatória, considerando fatores transcricionais potenciais nas plaquetas, que incluem o fator nuclear kappa B (NF-kB) e receptor ativado por proliferadores de peroxissoma gama (PPAR-γ).29

Em nosso estudo, encontramos porcentagens aumentadas de CPEs nos atletas, e quantidades semelhantes de MPEs e MPPs em comparação a indivíduos controles, apesar do treinamento intensivo desses indivíduos. Esses resultados promissores são importantes, uma vez que nosso entendimento sobre o papel do exercício sobre CPEs e MPs deriva-se principalmente da exposição aguda ao exercício ou em indivíduos não atletas.10,11,30,31 O exercício intermitente e de alta intensidade induz a liberação de catecolaminas e reduz células T altamente diferenciadas, mas não aumenta a quantidade de CPEs quando comparado ao exercício contínuo.33 Em outro artigo, apesar do aumento na contagem global de leucócitos, não houve alteração no número de CPEs em corredores maratonistas de idade avançada, quando coletados no período precoce após a corrida.33

Além disso, entre outras variáveis bioquímicas, os níveis de proteína C reativa foram mais baixos nos atletas que nos controles, e os níveis de creatinofosfoquinase um pouco aumentados, apesar do treino de rotina no dia anterior à coleta de amostra, o que reforça as propriedades protetoras do exercício de alto desempenho.

Limitações do estudo

Apesar de ser um estudo transversal, de caso controle, nossos resultados não podem ser considerados como geradores de hipótese, uma vez que não temos dados laboratoriais basais dos atletas. Além disso, esses resultados são aplicáveis aos maratonistas, e não podem ser extrapolados a outros esportes.

 Conclusões

O exercício crônico foi associado com um aumento favorável nos níveis de CPEs, sem afetar os níveis de MPs circulantes em corredores profissionais, sugerindo um impacto positivo da exposição prolongada ao exercício crônico nesses biomarcadores vasculares.

Fontes de financiamento

O presente estudo foi financiado pela FAPESP e parcialmente financiado pela CAPES.

Vinculação acadêmica

Este artigo é parte de dissertação de Mestrado de Célia Regina de Oliveira Bittencourt pela Universidade Federal do Rio de Janeiro.