adherence to lifestyle modifications after a cardiac rehabilitation program and endothelial...

© Schattauer 2014 Thrombosis and Haemostasis 112.1/2014

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Adherence to lifestyle modifications after a cardiac rehabilitation program and endothelial progenitor cellsA six-month follow-up study

Francesca Cesari1; Rossella Marcucci1; Anna Maria Gori2; Costanza Burgisser3; Sara Francini4; Anna Teresa Roberts3; Francesco Sofi2; Gian Franco Gensini2; Rosanna Abbate1; Francesco Fattirolli41Department of Clinical and Experimental Medicine, Thrombosis Centre, University of Florence, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy; 2Fondazione Don Carlo Gnocchi Italia, Florence, Italy; 3Geriatric Cardiology and Medicine, University of Florence, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy; 4Cardiac Rehabilitation Unit, University of Florence, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy

SummaryAn increase of endothelial progenitor cells (EPCs) among acute myo-cardial infarction (AMI) patients participating in a cardiac rehabili-tation (CR) program has been reported, but no data on the impact of adherence to lifestyle recommendations provided during a CR pro-gram on EPCs are available. It was our aim to investigate the effect of adherence to lifestyle recommendations on EPCs, inflammatory and functional parameters after six months of a CR program in AMI pa-tients. In 110 AMI patients (90 male/20 female; mean age 57.9 ± 9.4 years) EPCs, high sensitivity C-reactive protein (hsCRP), N-terminal pro-brain natriuretic peptide (NT-ProBNP) levels, and cardiopulmonary testings were determined at the end of the CR (T1) and at a six-month follow-up (T2). At T2 we administered a questionnaire assessing die-tary habits and physical activity. At T2, we observed a decrease of EPCs (p<0.05), of hsCRP (p=0.009) and of NT-ProBNP (p<0.0001). Pa-

tient population was divided into three categories by Healthy Lifestyle (HL) score (none/low, moderate and high adherence to lifestyle recom-mendations). We observed a significant association between adher-ence to lifestyle recommendations, increase in EPCs and exercise ca-pacity between T1 and T2 (Δ EPCs p for trend <0.05; ΔWatt max p for trend=0.004). In a multivariate logistic regression analyses, being in the highest tertile of HL score affected the likelihood of an increase of EPC levels at T2 [OR (95% confidence interval): 3.36 (1.0–10.72) p=0.04]. In conclusion, adherence to lifestyle recommendations pro-vided during a CR program positively influences EPC levels and exer-cise capacity.

KeywordsLifestyle modifications, endothelial progenitor cells, cardiac rehabili-tation, healthy lifestyle score

Correspondence to:Francesca CesariDepartment of Clinical and Experimental Medicine, Thrombosis Centre, University of FlorenceAzienda Ospedaliero-Universitaria CareggiLargo Brambilla 3, 50127 Florence, Italy Tel.: +39 055 7949420, Fax: +39 055 7949418 E-mail: [email protected]

Received: October 25, 2013Accepted after major revision: February 13, 2014Prepublished online: February 27, 2014

http://dx.doi.org/10.1160/TH13-10-0869Thromb Haemost 2014; 112: ■■■

Cardiovascular Biology and Cell Signalling

Introduction

Cardiac rehabilitation (CR) is widely accepted as a beneficial adjunct to medical therapy in patients with coronary artery disease, especially following myocardial infarction or coronary revascularisation (1, 2).

CR program includes strategies aimed at reducing modifiable risk factors for cardiovascular disease. Components of multidisci-plinary CR include patient assessment, nutritional, psychosocial and physical activity counselling, exercise training (ET), smoking cessation and risk factor management (3).

Randomised clinical trials and meta-analyses of CR following myocardial infarction revealed a significant reduction in both car-diac and total mortality upon completion of a CR program that in-cluded ET compared with usual medical care (4, 5).

Multiple factors directly or indirectly appear to contribute to this result. In particular, aerobic ET, as a key component of a CR

program, has anti-inflammatory, antiatherosclerotic, antithrom-botic, anti-ischaemic, antiarrhythmic, and positive psychological effects (6, 12).

Moreover, it has been demonstrated that ET is able to induce an improvement of endothelial function by increasing nitric oxide bioavailability and by modulating levels of endothelial progenitor cells (EPCs) from the bone marrow which enhance angiogenesis, promote vascular repair, and restore endothelial damage (13-17).

On this subject, we recently reported that, mobilization of EPCs and a significant decrease in pro-inflammatory biomarkers are among the benefits of a CR program for patients after an acute coronary syndrome (ACS) (18). Moreover, the global effect of a comprehensive CR program, ranging from cardiovascular risk re-duction to lifestyle modifications, was evidenced by a significant variation of some anthropometric and biochemical parameters, such as waist circumference, systolic blood pressure, high-density

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Thrombosis and Haemostasis 112.1/2014 © Schattauer 2014

2 Cesari et al. Adherence to lifestyles’ modifications and endothelial progenitor cells

lipoprotein (HDL)- cholesterol, glycosilated haemoglobin and triglycerides (18).

Lifestyle modifications can promote vascular health and have a direct effect on EPC levels and function (19). However, at present data evaluating the impact of global adherence to lifestyle recom-mendations provided during a CR program in ACS patients on EPC levels are lacking.

The aim of this study is to evaluate, in a population of ACS pa-tients who had previously attended a CR program, the effect of ad-herence to lifestyle recommendations on EPCs, inflammatory and cardiopulmonary parameters.

Materials and methodsStudy population and follow-up procedures

We conducted a prospective longitudinal study, evaluating each patients in three different moments: T0 at the beginning of the CR program, T1 at discharge from CR and T2 at the six-month fol-low-up. In each moment we performed a physical examination, a cardiopulmonary exercise test and a blood withdrawal to measure EPCs, high-sensitivity C-reactive protein (hsCRP) and N-terminal pro-brain natriuretic peptide (NT-ProBNP).

The study population, exclusion criteria and results relative to differences between T0 and T1 have been detailed elsewhere (18) while in the present paper we report results relative to T1 and T2.

Briefly, we recruited 112 patients (92 male [M]; 20 female [F], mean age 58.2 ± 9.5 years) admitted to the Cardiac Rehabilitation Unit of the University Hospital of Careggi after a hospitalisation for acute myocardial infarction (AMI). All patients took part in a 30 days CR program consisting of optimal pharmacological treat-ment according to available guidelines, serial clinical and instru-mental examinations, risk factors management, exercise program and counselling about dietary and lifestyle modification (18). One hundred ten out of 112 AMI (90 M/20 F; mean age 57.9 ± 9.4 years) patients were assessed at the six-month follow-up.

Healthy dietary and healthy lifestyle scores

In addition, at T2, all subjects were asked to report the frequency (times per week), duration (minutes [min] or hours [h] of exer-cise) and intensity of usual physical activity (in the previous 6 months, in terms of recreational and sporting activities). A physi-cal activity grade was assigned to each subject using qualitative terms such as absent or light (i.e. inactive or either occasional or strolling for 20 min once a week), moderate (i.e. regular walking for 30-60 min 2X per week or sporting exercise at least once a week) and intense (i.e. regular walking ≥ 60 min at least 3X per week , or frequent sporting activities only). The grade was not a measure of total time spent in physical activity but rather how much leisure time was dedicated to physical activity.

Dietary habits were also recorded through a semiquantitative food-frequency questionnaire, in order to assess the habitual con-sumption of 10 food items. Participants were asked to indicate the number of times a given item was consumed (daily, weekly,

monthly or yearly) from which the absolute frequency of con-sumption of each item was calculated.

For each of the seven potentially protective components (fruit, vegetables, cereals, legumes, red wine, fish and olive oil) partici-pants received one point if their intake was above the sex-specific median and one point if their intake was below the median for the three non-protective components (red and processed meat, dairy products, sweets). Ten points reflected maximum adherence and zero point reflected no adherence at all.

Subsequently, we computed a global Healthy Lifestyle (HL) score by adding lifestyle habits such as physical activity and absti-nence from smoking to the previously calculated score for adher-ence to the healthy diet (20). One point was given to subjects reporting at least moderate physical activity performed during the last six months and two points were given for intense physical ac-tivity. Moreover, one point was given to non-smokers.

Thus, the HL score ranged from 0 (null adherence) to 13 (maxi-mal adherence).

At follow-up, drug compliance and achievement of recom-mended target values of systolic blood pressure, low-density lipo-protein (LDL)- cholesterol, triglycerides and glucose levels were also assessed (21).

Current smoking status was determined at the time of blood collection. Subjects were classified as having hypertension accord-ing to the European Society of Hypertension/European Society of Cardiology guidelines (22). Diabetic subjects were defined by American Diabetes Association criteria (23) or on the basis of self-reported data (if confirmed by medication or chart review). Dys-lipidaemia was defined according to the Third report of the National Cholesterol Education Program (NCEP-III) (24). A posi-tive family history was defined as the presence of at least one first-degree relative who had developed coronary artery disease before the age of 55 years for men and 65 years for women. Body mass index (BMI) was calculated as weight (kg)/height (m2) and obesity was defined as BMI ≥ 30. All subjects gave informed consent; the study complies with the Declaration of Helsinki and was approved by the local ethic committee. Reporting of the study conforms to STROBE recommendations.

Cardiopulmonary exercise test

A Cardiopulmonary Exercise Testing (CPET) was performed on an electrically breaked cycle ergometer with a progressively in-creasing work rate, controlled by Stress Test System Esaote Bio-medica, which recorded a 12-lead exercise electrocardiogram (ECG) during the test. The exercise protocol was determined by the age and general fitness of the patient, and varied from 10 Watt work load increments every 1 min to 25 Watt every 2 min (25, 26).

Breath-by-breath measurements of oxygen uptake, carbon dioxide production, and respiratory �ow and volume parameters were obtained by applying a Hans Rudolph mask, connected to a pneumotach device. A CPX Ultima metabolic monitor was used to perform gas exchange analysis, linked into the BreezeSuiteTM soft-ware package (Medical Graphics Corporation). Oxygen, carbon dioxide and flow sensors were calibrated immediately before each




3Cesari et al. Adherence to lifestyles’ modifications and endothelial progenitor cells

test. Arterial pressure was measured manually every one or two minutes by cuff sphygmomanometer.

The following reasons for test termination were applied follow-ing standard guidelines: physical exhaustion, angina, ST-segment depression >3 mm, warning arrhythmias, hypertension (systolic blood pressure ≥ 250 mmHg), drop in systolic blood pressure (> 10 mmHg below the resting value).

Exercise capacity was expressed as maximal workload (Watt); peak oxygen uptake (VO2 peak) was averaged over the last 30 sec-onds of exercise, and expressed relative to body weight (ml/kg/min).

Blood collection

Two fasting blood samples were obtained for each patient: at time 1 (T1), at the end of the rehabilitation program (60 ± 5 days after the acute event) and at time 2 (T2), at the six-month follow-up. Samples were obtained at least 48 h after the last exercise program and at least 8-12 h from the last cigarette. Blood samples were col-lected from the antecubital vein into evacuated plastic tubes (Vacutainer) anticoagulated with ethylenediaminotetracetate (EDTA) 0.17 mol/l for obtaining plasma samples for EPCs’ evalu-ation and with no anticoagulant for obtaining sera samples for hsCRP and NT-ProBNP evaluation. The sera samples were centri-fuged at 2,000 g for 10 min a 4°C and then stored in aliquots at -80°C until analysis.

Flow cytometric analysis

EPCs number was assessed by using flow cytometry, as previously described (27, 28).

Briefly, 200 μl of peripheral venous blood were incubated for 20 min in the dark with:• Fluoresceine isothiocyanate (FITC)-labelled monoclonal anti-

bodies against human CD34 (BD Pharmingen, San Diego, CA, USA)

• Allophycocyanin (APC)-labelled monoclonal antibodies against human AC133 (Miltenyi Biotec, Bergisch Gladbach, Germany)

• Phycoerythrin (PE)-labelled monoclonal antibodies against human VEGFR2-KDR (R&D Systems Inc, Minneapolis, MN, USA)

• Allophycocyanin-Cyanin7(APC-Cy7)-labelled monoclonal antibodies against human CD45 (Becton Dickinson, San Jose, CA, USA)

• LDS751, a nucleic acid dye, (Molecular Probes, Invitrogen, Eu-gene, OR, USA)

Mouse isotype-identical antibodies served as controls (Becton Dickinson, San Jose, CA, USA). Red blood cells and platelets were subsequently lysed by NH4Cl lysing solution (Autolyse solution; BioSource International, Camarillo, CA, USA). For analysis, 300,000 cells within the leukocyte gate were acquired using a FACSCanto analyzer (Becton Dickinson, San Jose, USA) and data were processed using BD FacsDiva software. Circulating EPCs

were identified through their expression of CD34, KDR, and CD133 and were considered as EPCs cells CD34+/KDR+/CD45dim, CD133+/KDR+/CD45dim and CD34+/CD133+/KDR+/CD45 dim.

The intra- and inter-observer variations of our method showed an intraclass correlation coefficient of 0.97 and 0.92, respectively (29). The intra-assay coefficient of variation of the EPC measure-ment was 7.8%. When EPCs were detectable, it was possible to rec-ord at least 1 cell/300,000 gated cells

hsCRP determination

hsCRP was assessed with a high-sensitivity assay on a BN II nep-helometer (Dade Behring, Marburg, Germany), allowing detection of values as low as 0.11 mg/l with a CV intra-assay of 3.9% and inter-assay of 5.5%.

NT-pro-BNP determination

NT-pro-BNP was measured with a chemiluminescent immunoas-say kit (Roche Diagnostic Laboratory, Indianapolis, IN, USA) on an Elecsys 2010 analyzer allowing detection of values as low as 3 pg/ml with a total CV of 0.8% (30).

Statistical analysis

Statistical analysis was performed using the SPSS (Statistical Pack-age for Social Sciences, Chicago, IL, USA) software for Windows (Version 18.0). Values are presented as median and range or by mean and standard deviation (SD) as appropriate.

The Chi-square test was used to identify statistically significant differences between categorical variable. The continuous variables were analysed by using nonparametric tests, as they did not follow a normal distribution. The Mann-Whitney test for unpaired data was used for comparison between two groups.

The Wilcoxon test for related data was used to evaluate differ-ences between two time-points and the Kruskal-Wallis test was used for comparison between groups (i.e. 1st tertile n=25, 2nd ter-tile n=40, 3rd tertile n=45). The Spearman’s test was used to ident-ify significant correlations between two numerical variables.

Post-hoc sample size calculation indicated that the number of 110 patients has a sufficient statistical power (Beta=0.80) to detect a significant difference in EPC number between T1 and T2, with an alpha coefficient of 0.05.

In order to analyse EPCs’, Watt max and VO2 peak variations (Δ= follow up – end of the rehabilitation) in relation to the adherence to lifestyle recommendations we divided our study population into tertiles of HL scores (1st tertile HL score ≤ 6 no/low adherence; 2nd tertile HL score 7-9 moderate adherence; 3rd tertile HL score ≥ 10 high adherence).

A multiple logistic regression analysis was used to test the inde-pendent association between HL tertiles and the likelihood of EPCs’ increase at six-month follow-up. All odds ratios (OR) are given with their 95% confidence interval (CI). A p-value <0.05 was considered to be statistically significant.





ResultsBaseline characteristics of the study populationDemographic and clinical characteristics of the study population are summarised in ▶ Table 1.

No significant differences of baseline EPC levels in relation to traditional cardiovascular risk factors, medications, comorbidities, age or gender were observed.

A significant negative relationship between VO2 peak and base-line hsCRP, NT-ProBNP and age was detected (VO2 peak and hsCRP r= -0.26 p=0.01; VO2 peak and NT-ProBNP r= -0.33 p=0.001; VO2 peak and age r= -0.43 p<0.001).

Patients with hypertension and diabetes showed lower values of VO2 peak compared to patients without these CV risk factors (data not reported).

As regarding baseline Watt max, significant negative correlations with age and NT-ProBNP levels were observed (Watt max and age r=-0.46 p<0.0001; Watt max and NT-ProBNP r= -0.32 p=0.001).

Modifications of VO2 peak, Watt max, EPC number, hsCRP and NT-ProBNP after six months of follow-up

Adherence to secondary preventive medications at the 6-month follow up are reported in ▶ Table 2.

At the six-month follow-up eight patients had presented a car-diovascular (CV) event (8/110; 7.3%): two patients experienced a percutaneous coronary intervention, one patient had coronary ar-tery bypass grafting, one patient presented instable angina, one pa-tient a ventricular fibrillation, one patient an acute heart failure and two patients an acute pulmonary oedema. No differences in EPCs, hsCRP and NT-ProBNP levels, Watt max or VO2 peak at T1 or in the Δ between T2 and T1 were observed between patients who experienced a new CV event compared to patients who did not (data not shown).

In addition, at six months of follow-up, target values had been reached for systolic blood pressure in 84/110 (76.4%), LDL choles-terol in 90/110 (81.8%), triglycerides in 97/110 (88.2) and glucose in 98/110 (89%) of the patients. Eight out of 110 (7.3%) patients continued to smoke.

No significant differences in the Δ of EPCs, hsCRP, NT-proBNP and Watt max were recorded in relation to cardiovascular risk factors control, while VO2 peak variations were significantly lower in patients who continued to smoke with respect to non smokers (Δ VO2 peak: -4.38 ± 6.80 vs 0.09 ± 3.74 p=0.003).

No significant modifications of VO2 peak, Watt max, BMI and heart rate were detected at the six month follow-up (▶ Table 3). On the contrary a significant increase in waist circumference and systolic blood pressure was reported (▶ Table 3).

With regard to biomarkers, after six months of follow-up, a sig-nificant (p<0.05) decrease of EPCs’ numbers and of hsCRP and NT-proBNP levels were observed (▶ Table 3).

In fifty-two out of 110 patients (47.3%) EPC levels had in-creased or not decreased, while in 58 (52.7%) they had decreased. No differences regarding age, sex, drug therapy, control of risk fac-tors, Watt max, VO2 peak, hsCRP and NT-ProBNP levels were ob-

Table 1: Demographic and clinical characteristics of the study popu-lation (n=110). STEMI, ST-elevation myocardial infarction; NSTEMI, no ST-elevation myocardial infarction; BMS, bare metal stent; DES, drug-eluting stent; POBA, plain-old balloon angioplasty; LVEF, left ventricular ejection frac-tion; BMI, body mass index; CHD, coronary heart disease; POAD, peripheral obstructive arterial disease; TIA, transient ischaemic attack; COPD, chronic obstructive pulmonary disease.

Age (years)

Males, n (%)

STEMI, n (%)

NSTEMI, n (%)

BMS, n (%)

DES, n (%)

POBA, n (%)

Cardiovascular risk factors

Hypertension, n (%)

Smoking habit, n (%)

Dyslipidaemia, n (%)

Diabetes, n (%)

Family history of CHD, n (%)

Obesity, n (%)

Depression , n (%)

Comorbidities

POAD n (%)

TIA/Stroke, n (%)

COPD, n (%)

Atrial fibrillation, n (%)

57.9 ± 9.4

90 (81.8)

81 (73.6)

29 (26.4)

29 (26.4)

77 (70.0)

4 (3.6)

54 (49.1)

58 (52.3)

56 (50.9)

18 (16.4)

35 (31.8)

15 (13.6)

12 (10.9)

2 (1.8)

2 (1.8)

11 (10.0)

3 (2.7)

Table 2: Pharmacological treatment at the end of the rehabilitation period (T1) and at six-month follow-up (T2) (n=110).

Medications

Statins, n (%)

Antiplatelets , n (%)

Angiotensin converting enzyme inhibitors, n (%)

β-blockers, n (%)

Diuretics, n (%)

Hypoglicaemics n (%)

Anticoagulants, n (%)

Antiarrhythmics, n (%)

Calcium-antagonists, n (%)

Antidepressants n (%)

Ansiolytics, n (%)

T1

110 (100.0)

110(100.0)

107 (97.3)

102 (92.7)

25 (22.7)

19 (17.3)

1 (0.9)

2 (1.8)

14 (12.7)

22 (20)

25 (22.7)

T2

110 (100)

106 (96.4)

105 (95.5)

101 (91.8)

23 (20.9)

17 (15.5)

2 (1.8)

2 (1.8)

16 (14.5)

15 (13.6)

16 (14.5)





served between patients with an increase or a decrease in EPCs number (data not shown).

A significant and positive correlation was observed between Δ VO2 peak and Δ Watt max (Δ Watt max and Δ VO2 peak r=0.43 p<0.0001) and between Δ CRP and Δ NT-ProBNP (Δ CRP and Δ NT-ProBNP r=0.38 p<0.0001).

Modifications of VO2 peak, Watt max, EPC number, hsCRP and NT-ProBNP in relation with adherence to lifestyle’s modifications

After dividing our population into tertiles of HL score, a signifi-cant increase in Watt variations at increasing HL score was re-ported (▶ Table 4).

On the contrary, VO2 peak, hsCRP and NTproBNP variations were not significantly affected by HL score (▶ Table 4). Albeit not significant, VO2 peak levels and Watt max at T2 were progressively higher as HL score increased (VO2 peak levels 19.54 ± 4.85, 20.44 ± 6.72, 22.86 ± 6.98 ml/kg/min p=0.07; Watt max 132 ± 38, 138 ± 48, 152 ± 45; p=0.21).

With regard to EPCs, we observed a significant increase in the Δ of all the three types of EPCs and in the number of EPCs at T2, as HL score increases (▶ Figure 1 and ▶ Figure 2).

In addition, a significantly higher HL score was evidenced in patients with an increase or stable levels of EPCs at T2 compared to patients with a decrease [10 (4-13) vs7 (2-12) p=0.04].

In a logistic regression analysis the highest tertile of HL score significantly affected the likelihood of having an increase of EPCs at the six-month follow-up [OR (95%CI): 2.92 (1.06-8.07) p=0.03], even after adjustment for age, sex, control of cardiovascular risk factors and VO2 peak and Watt max at T1 [OR (95%CI): 3.36 (1.0-10.72) p=0.04].

Discussion

In this study we report for the first time, in a population of AMI patients, an association between adherence to lifestyle recommen-dations provided during a CR program and an increase of EPCs and functional parameters at six-month follow-up.

EPCs have been described as a backup system which con-tribute to maintain endothelial integrity, prevent endothelial dysfunction and enhance revascularisation (31). After incorpor-ation into vasculature, they differentiate into mature endothelial cells by releasing angiogenic growth factors such as VEGF and stromal derived factor-1, that act in a paracrine manner to sup-

Variable

EPCs

CD34+/KDR+, (cells/106 events)*

CD133+/KDR+, (cells/106 events)*

CD34+/CD133+/KDR+, (cells/106 events)*

Biochemical parameters

hsCRP, (mg/l)*

NT-ProBNP, (pg/ml)*

Cardiopulmonary and anthropometric parameters

Watt max

VO2 peak, ml/kg/min

Heart rate, bpm

Systolic Blood Pressure (mmHg)

Waist Circumference (cm)

Body Mass Index

T1

10 (0–37)

10 (0–33)

10 (0–33)

2.1 (0.3–10.1)

290 (19–3799)

143.6 ± 44.4

21.7 ± 6.0

61 ± 9

120 ± 15

95 ± 11

27.5 ± 4.3

T2

7 (0–43)

7 (0–43)

7 (0–43)

1.5 (0.2–12.0)

143 (9.0–6835)

142.4 ± 45.4

21.2 ± 6.6

61 ± 10

126 ± 16

97 ± 13

27.5 ± 4.5

P-value

0.01

0.03

0.03

0.009

<0.0001

0.75

0.80

0.45

<0.0001

0.0001

0.75

Table 3: Levels of circulating endothelial progenitor cells (EPCs), and biochemical, an-thropometric and car-diopulmonary parame-ters at the end of the rehabilitation period (T1) and at six-month follow-up (T2). CPCs, circulating progenitor cells; EPCs, endothelial progenitor cells; hsCRP, high sensitivity C reactive protein; NT-ProBNP, N-ter-minal pro brain natriu-retic peptide. *Values are presented as median and (range).

Δ

Δ VO2 peak (ml/kg/min)

Δ Watt max

Δ hsCRP (mg/l)*

Δ NT-ProBNP (pg/ml)*

1st tertile(HL score ≤ 6)

-1.05 ± 2.84

-9.79 ± 14.10

0 (-8.0; +5.9)

-77.5 (-1039; +456.3)

2nd tertile(HL score 7–9)

-0.65 ± 5.25

1.02 ± 26.91

-0.60 (-8.10; +7.0)

-61.45 (-2595; 702.20)

3rd tertile(HL score ≥ 10)

0.56 ± 3.65

4.66 ± 17.30

-0.30 (-7.80;+5.0)

-123.1 (-1497.2; 2252)

P for trend

0.19

0.004

0.06

0.76

Table 4: Variations of VO2peak, Watt max, hsCRP and NT-ProBNP levels (Δ) according to tertiles of HL score. hsCRP, high sensitivity C reactive pro-tein; NT-ProBNP, N-terminal pro brain natriuretic pep-tide. *Values are presented as median and (range).





Figure 1: Variations in EPCs’ number between T1 and T2 according to adherence to life-styles’ recommen-dations.

Figure 2: EPCs’ number at T2 according to ad-herence to lifestyles’ recommendations.





port local angiogenesis and mobilise tissue residing progenitor cells (32, 34).

At present different methods to determine EPCs (flow cyto-metry or in vitro cell culture assays) have been carried out and there is a considerable debate on technical aspects related to EPC enumeration.

Flow cytometry offers the advantage of a multimarker approach involving the concurrent use of endothelial and stem cell markers such as CD34, CD133 and KDR. However, the lack of an estab-lished and generally accepted marker combination and of a stan-dardised protocol with regards to gating strategies may account for the widespread inter-laboratory variations in quantification of EPC (35).

On the other hand, in vitro cell culture assays allow to test the in vivo relevance of EPCs’ population but require multiple steps and is liable to variability during the culture process.

To date, the effect of a healthy lifestyle on EPC levels and func-tion is well recognised. Various studies demonstrated an influence of physical activity and exercise training in determining EPCs’ mo-bilisation from the bone marrow, both in healthy subjects and in patients with vascular disease (14-18), probably mediated by a shear stress-induced up-regulation of endothelial nitric oxide syn-thase and by the subsequent increase in nitric oxide bioavailability (36).

Alongside physical exercise, a role for a dietary approach in augmenting the number of EPCs has also been observed. Two studies conducted among obese subjects reported that a period of dietary caloric restriction with a subsequent weight loss is able to increase the number of circulating progenitor cells and to deter-mine a beneficial effect on the functional properties of early out-growth EPCs (37, 38).

In addition, an elegant study conducted in animal models dem-onstrated that different types of diet have different effects on vas-cular health, with a low-carbohydrate high-protein diet being proven to have adverse vascular effects on the number of endothe-lial progenitors, in both the bone marrow and in the peripheral blood (39).

Interestingly, the role of diet in determining a beneficial effect on the endothelium, has also been confirmed in a study conducted among healthy subjects, which reported that a Mediterranean style diet is associated with an improvement in the regenerative capacity of the endothelium compared to other diets, probably mediated by the quantity of antioxidants (40). In addition, it was reported that a therapeutic model of hypocaloric Mediterranean diet improves endothelium regenerative capacity, in particular the number of CD34+/KDR+ EPCs, and cardiometabolic risk factors in meta-bolic syndrome patients without manifest cardiovascular disease (41).

The deleterious effects of cardiovascular risk factors on EPC number and functionality have been extensively investigated. In particular cigarette smoking is associated with a reduced number of circulating EPCs and with an increased EPCs’ senescence (42). By analysing lifestyle recommendations through the use of a global healthy lifestyle score that includes dietary habits, duration and in-tensity of physical activity and smoking cessation we were able to

demonstrate a relationship between a higher adherence to life-styles recommendations and increased EPCs and exercise toler-ance at the six-month follow-up.

In addition the highest tertile of HL score, which indicates a high adherence to healthy lifestyle recommendations, is able to de-termine EPC increase, independently from age, sex and achiev-ement of recommended target values of systolic blood pressure, LDL- cholesterol, triglycerides and glucose levels. This datum in-dicates that, despite the importance of a pharmacological ap-proach in preventing endothelial dysfunction, this is not enough to provide a sufficient endothelial homeostasis and there is a need to put strong emphasis on the lifestyle modification.

The long term care of ACS patients represents a huge challenge for healthcare professionals. As the results of the EUROASPIRE surveys showed, there is a continuous gap between the standards set by cardiovascular disease prevention guidelines and clinical practice (43).

In our study population, nearly 20% of the patients did not re-port, at the six-month follow-up, target values of systolic blood pressure or LDL cholesterol, 7% continued to smoke, the majority of patients did not adhere to lifestyle recommendations in terms of dietary profile and physical activity. In addition a significant in-crease in systolic blood pressure and waist circumference and a significant decrease of EPCs’ number was reported.

These findings confirm how difficult it is for patients to modify their habits in the long term, even after a recent acute coronary event. In fact, it has been demonstrated that the benefits of physi-cal activity on EPC number are reversible and that eight weeks after cessation of training, progenitor cells number returned to baseline, indicating that a sustained physical training is necessary for long-term preservation of improved endothelial function (44). An optimal management of risk factors related to lifestyle habits

What is known about this topic?• Lifestyle modifications can promote vascular health and have a

direct effect on endothelial progenitor cells (EPCs) number and function.

• An increase of EPCs among acute coronary syndrome (ACS) pa-tients participating in a cardiac rehabilitation (CR) program has been reported, but no data on the impact of adherence to lifestyle recommendations provided during a CR program on EPCs are available.

What does this paper add?• By analysing lifestyle recommendations through the use of a glo-

bal healthy lifestyle, we demonstrate a relationship between a higher adherence to lifestyles recommendations and EPCs and ex-ercise tolerance increase at the six-month follow-up in a popu-lation of ACS patients.

• A high adherence to healthy lifestyle recommendations, is able to determine EPC increase, independently from age, sex and achiev-ement of recommended target values of systolic blood pressure, LDL-cholesterol, triglycerides and glucose levels.





needs a sustained professional support by physicians and the health-care system, as it cannot rely on patients’ self-management. This has been clearly confirmed by a recent multicentre study con-ducted across several European countries that demonstrated the beneficial role of a structured cardiac rehabilitation program after ACS, including counseling, emotional support and education on management of cardiovascular risk factors, to prevent new cardio-vascular events (45). Endothelial progenitor cells represent a key component in the maintaining of a good endothelial homeostasis and our data indirectly show a compelling need for more effective lifestyle management of patients with coronary artery disease by investing in articulated programs for secondary prevention.

Study limitations

A possible limitation of our study is that dietary habits and physi-cal activity were assessed by self-reported questionnaires, and are therefore based on subjective information given by the patients.

Conflict of interestNone declared

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