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Mobilisation of mesenchymal cells in cardiac patients: is intense exercise necessary?
  1. A Lucia1,
  2. A De La Rosa2,
  3. M Avila Silván1,
  4. L M López-Mojares1,
  5. A Boraita3,
  6. M Pérez1,
  7. C Foster4,
  8. J García-Castro5,
  9. M Ramirez6
  1. 1
    Universidad Europea de Madrid, Spain
  2. 2
    Hospital Universitario La Laguna, Spain
  3. 3
    Consejo Superior de Deportes, Madrid, Spain
  4. 4
    University of Wisconsin-La Crosse, USA
  5. 5
    Centro andaluz de Células Madre, Granada, Spain
  6. 6
    Hospital Infantil Universitario Niño Jesús, Madrid, Spain
  1. Alejandro Lucia, MD PhD, Universidad Europea de Madrid, 28670 Villaviciosa de Odón; alejandro.lucia{at}uem.es

Abstract

Circulating mesenchymal cells (cMCs) have a potential for regenerating damaged tissue, e.g., ischaemic myocardium. In patients (age range: 53–76 years) with stable coronary artery disease cMCs were determined before and after dynamic exercise of moderate (< respiratory compensation threshold (RCT)) (n = 9 patients) or high intensity (>RCT) (n = 11). Only high-intensity exercise (i.e., provoking signs of myocardial ischaemia in 3 patients and ventricular extrasystoles in another) induced a significant increase in cMCs (p = 0.009). These results support the hypothesis that intense exercise (near or at the point of myocardial ischaemia) is a potent stimulus for MC mobilisation.

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In patients with stable coronary artery disease (CAD), regular “aerobic” exercise training improves myocardial perfusion and delays disease progression.14 The biological mechanisms behind this effect remain to be fully elucidated, though it was recently shown that exertional myocardial ischaemia is a potent stimulus for the release of endothelial progenitor cells (EPCs) from the bone marrow to the peripheral blood.5 This finding is clinically relevant given the potential of EPCs to form entirely new vessels in ischaemic tissues (vasculogenesis).69 Although more research is necessary, this finding also gives support for the possibility that some bouts of intense dynamic exercise (i.e., potentially inducing myocardial ischaemia or at least approaching the “ischaemic threshold”) could be included in the training programmes of patients with stable CAD to maximise the possibility of vasculogenesis.

Mesenchymal cells (MCs) are involved in the repair of damaged tissues and thus play an important role in regenerative medicine.10 Several groups are developing therapies with MCs for cardiac, bone, muscle, cartilage and inflammatory diseases.11 MCs reside in several adult tissues such as bone marrow, adipose tissue, cartilage and skin.12 Under several stimuli, MCs migrate towards organs different from where they normally reside.13 For instance, bone marrow MCs have been shown to migrate to (and repair) infarcted myocardium in animal models.14 In humans, direct infusion of bone marrow mononuclear cells into the coronary arteries after myocardial infarction can improve left ventricular function.15 The potential therapeutic contribution of MCs to myocardial repair is attributable to several factors including: direct differentiation into cardiomyocytes and vascular endothelial cells, spontaneous cell fusion or stimulation of endogenous repair.10

The clinical use of MCs first requires that the cells are mobilised into the blood and directed to damaged tissues.10 We recently showed increased mobilisation of MCs into the blood of healthy individuals, peaking 2 h after an acute bout of prolonged exercise which induced skeletal muscle damage.16 We were interested in whether intense exercise (i.e., near the ischaemic threshold) might stimulate MC migration into the blood of patients with stable CAD as has previously been shown with EPCs.5

The purpose of this study was to assess the effects on MC mobilisation induced by two protocols of cycle-ergometer exercise: i) an acute bout (40–50 min) of moderate intensity exercise (55–90% peak heart rate (HRpeak)), below the threshold that provokes ischaemia, significant arrhythmias or symptoms of exercise intolerance, as recommended by reference guidelines for cardiac rehabilitation programmes17; and ii) an exercise bout of shorter duration but of very high intensity, i.e., stress test until exhaustion with attainment of HRpeak and clinical abnormalities.

METHODS

Written informed consent was obtained from each subject. The study was approved by the Institutional Ethics Committee and adhered to the principles of the Declaration of Helsinki. Eleven male patients (mean (SEM) age: 64 (2) years; range: 53–76) with stable CAD (clinically asymptomatic) who were participating in a cardiac rehabilitation programme were selected as subjects. Six patients were taking β blockers at the time of the study.

Peripheral venous blood was collected from subjects (see below) before and 2 h15 after each of two cycle-ergometer exercise protocols (involving no skeletal muscle damage): i) a graded (ramp-like) stress test (10 W+10 W/min until exhaustion) preceded by a 5 min warm-up (n = 11 patients); and ii) a “typical” training session of their cardiac rehabilitation programme, which was performed by nine of the 11 patients a few weeks after the stress test. During the stress test, gas exchange data were collected (Vmax 29C; Sensormedics; California, USA) to determine peak oxygen uptake (VO2peak), and the ventilatory (VT) and respiratory compensation thresholds (RCT).18 Capillary blood lactate (YSI 1500; Yellow Springs, USA) and pH (ABL77 Radiometer; Copenhagen, Denmark) were determined at end-exercise. Twelve-lead ECG tracings and HR were monitored during each exercise protocol. The incremental tests were supervised by an experienced cardiologist and a venous catheter was inserted in all patients in case medication administration was needed.

Blood samples were collected from the anti-cubital vein.16 In order to remove the potentially confounding effect of increased MC mobilisation into the blood in response to skeletal muscle injury,16 serum total creatine kinase (CK) activity (a marker for skeletal muscle injury)19 was measured in all samples with an automated analyser (Hitachi 911, Boehringer Mannheim, Mannheim, Germany).

Flow cytometry analysis (EPICS XL-MCL cytometer, Coulter Electronics, Hialeah, FL) was used for detecting blood circulating MCs as previously described in detail.16 Briefly, peripheral blood was stained with anti-human-CD45, CD13 and CD29 (Immunotech, Marseille, France) and red blood cells were lysed by lysis solution (Quick Lysis, VITRO, Spain). One hundred μl containing Flow-count TM fluorospheres (Beckman-Coulter, Brea, CA) was added before flow cytometer acquisition. In parallel, endothelial cells were detected using the CellQuant FF-CD146 kit (Biocytex, Marseille, France). This population did not overlap with CD45-CD13+CD29+ cells, further supporting the mesenchymal origin of the latter. For each analysis, at least 100 000 cells were collected.

Comparison between pre and post-exercise values of cMCs in each of the two exercise bouts was performed with the Wilcoxon test. Individual results of cMCs were normalised to the pre-exercise values (each of which was considered as 100%) obtained in the stress test and training session, respectively, and expressed as mean (SEM). The level of significance was set at p⩽0.05.

RESULTS

Stress tests

Three patients showed ECG evidence of ischaemia (ST depression >1.5 mm in one or more leads) at peak exercise (n = 2) or immediately after exercise (n = 1, together with chest discomfort). One additional patient had monomorphic ventricular extrasystoles with complete left bundle block during exercise and stopped the test before reaching RCT. All ischaemic signs disappeared shortly after exercise and the patients recovered satisfactorily. Their mean ((SEM)) values of HRpeak, VO2peak, peak workload, peak respiratory exchange ratio, lactate and blood pH at end-exercise and systolic/diastolic blood pressure at end-exercise were: 137 (5) beats/min, 25.3 (1.4) mlO2/kg/min, 137 (5.3) W, 1.02 (0.00), 4.4 (0.4) mmol/l, 7.27 (0.05) and 205 (7)/85 (2) mm Hg, indicating the attainment of very high relative exercise intensities. All but the aforementioned patient surpassed the workload eliciting the RCT. Exercise duration (warm-up + test) averaged approximately 18 min.

Training session

Total exercise duration averaged approximately 49 min. Individual HR data were consistently below the HR at the RCT in the previous stress test (total time spent at ⩽55% HRpeak and between 55–90% HRpeak averaged 13 (3) min and 36 (3) min).

Mesenchymal cell determination

Mean values of cMCs significantly increased after the stress test (p = 0.009) whereas no significant change was observed after the training session (p>0.05) (fig 1). Three of the four patients with greatest increases in cMCs after the stress test were those demonstrating clinical/ECG abnormalities during exercise testing. All individual CK levels were within the reference limits of our laboratory (<250 U/l), suggesting that significant skeletal muscle damage could not account for changes in cMCs.

Figure 1

Mean (SD) normalised values of circulating mesenchymal cells (cMCs) before and after the two exercise bouts (upper figure) and individual responses (lower figure). *p = 0.009. Discontinuous lines represent data of patients with cardiac signs/symptoms during stress tests.

DISCUSSION

The present data suggest that only when exercise intensity surpasses a certain intensity threshold (i.e., ⩾RCT, ⩾90% HRpeak, or perhaps the ischaemic threshold) does it represent a stimulus for mobilisation of cMCs into the blood. This finding is of clinical relevance as these cells have the potential for regeneration of damaged tissues, e.g., infarcted myocardium.10 Although it is commonly recommended that patients with CAD engage in exercise of moderate intensity (55–90% of HRpeak and below the ischaemic threshold), our data, together with previous studies on EPCs,5 support the need for research trials designed to determine whether controlled bouts of carefully and professionally supervised, intense dynamic exercise (i.e., maybe inducing repeated episodes of myocardial ischaemia) should be included in the training sessions of patients with stable CAD to maximise possibility of improved myocardial perfusion as a clinical outcome.

What is already known on the topic

Mesenchymal cells (MCs) are involved in the repair of damaged tissues (e.g., particularly ischaemic myocardium).

What this study adds

In patients with stable coronary artery disease, MCs are mobilised into blood in response to intense (⩾90% peak heart rate) dynamic exercise. Although more research is needed, our finding, together with previous research on endothelial progenitor cells, supports the notion that intense exercise might be useful to maximise gains in cardiac rehabilitation programmes.

Acknowledgments

This study was supported by a grant from Fondo de Investigaciones Sanitarias (FIs, ref. # PI061183).

REFERENCES

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Footnotes

  • Competing interests: None.

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