Exercise and hypoxia paradoxically modulate vascular thrombotic risks. The shedding of procoagulant-rich microparticles from monocytes may accelerate the pathogenesis of atherothrombosis. The present study explores the manner in which normoxic and hypoxic exercise regimens affect procoagulant monocyte-derived microparticle (MDMP) formation and monocyte-promoted thrombin generation (TG). Forty sedentary healthy males were randomized to perform either normoxic (NET; 21% O2, n=20) or hypoxic (HET; 15% O2, n=20) exercise training (60% VO2max) for 30 min/day, 5 days/week for 5 weeks. At rest and immediately after HET (100 W under 12% O2 for 30 min), the MDMP characteristics and dynamic TG were measured by flow cytometry and thrombinography respectively. The results demonstrated that acute 12% O2 exercise (i) increased the release of coagulant factor V (FV)/FVIII-rich, phosphatidylserine (PS)-exposed and tissue factor (TF)-expressed microparticles from monocytes, (ii) enhanced the peak height and rate of TG in monocyte-rich plasma (MRP) and (iii) elevated concentrations of norepinephrine/epinephrine, myeloperoxidase (MPO) and interleukin-6 (IL-6) in plasma. Following the 5-week intervention, HET exhibited higher enhancements of peak work-rate and cardiopulmonary fitness than NET did. Moreover, both NET and HET decreased the FV/FVIII-rich, PS-exposed and TF-expressed MDMP counts and the peak height and rate of TG in MRP following the HET. However, HET elicited more suppression for the HE (hypoxic exercise)-enhanced procoagulant MDMP formation and dynamic TG in MPR and catecholamine/peroxide/pro-inflammatory cytokine levels in plasma than NET. Hence, we conclude that HET is superior to NET for enhancing aerobic capacity. Furthermore, HET effectively suppresses procoagulant MDMP formation and monocyte-mediated TG under severe hypoxic stress, compared with NET.

CLINICAL PERSPECTIVES

  • HET (15% O2) is superior to NET (21% O2) for enhancing exercise performance and cardiopulmonary fitness in sedentary males.

  • Acute 12% O2 exercise (HE) (i) increased the release of FV/VIII-rich, PS-exposed and TF-expressed microparticles from monocytes (MDMPs), (ii) enhanced the peak height and rate of TG in MRP and (iii) elevated concentrations of norepinephrine/epinephrine, MPO and IL-6 in plasma. Compared with NET effects, HET elicits more suppression in the HE-promoted procoagulant MDMP formation and dynamic TG in MRP under an atherogenic stimulation.

  • In summary, HET can be considered an effective exercise strategy that improves aerobic capacity and simultaneously increases the resistance to monocyte-related thrombosis provoked by severe hypoxia.

INTRODUCTION

Atherosclerosis, a chronic inflammatory disease of the vessel wall, is characterized by archetypical chronic inflammatory cell infiltration of monocytes [1,2]. Atherosclerotic lesions may accumulate large numbers of procoagulant monocyte-derived microparticles (MDMPs) [3], consequently increasing the risk of atherothrombosis [4]. Exercise training in hypoxic environment provides substantial benefits over normoxic condition by increasing pulmonary ventilation, adaptation of the haemopoietic system and tissue utilization of O2 in healthy individuals [5,6] and patients with cardiovascular diseases [7]. Moreover, hypoxic pre-conditioning may reduce the loss of myocardial contractility [8] and the volume of cerebral infarction [9] immediately after sub-lethal ischaemic insult. However, extremely hypoxic environments are associated with increased incidence of vascular thromboembolic events [10]. To our knowledge, a ‘safe and effective’ strategy of exercise training combined with hypoxia that promotes superior aerobic fitness and simultaneously minimizes atherothrombotic risk has not yet been established.

Inflammation-related leucocyte vesiculation can disseminate membrane-associated procoagulant activities [11,12]. The negatively charged phosphatidylserine (PS) exposed on the microparticles can bind coagulation factors VIII (FVIII), FVa and FXa, providing a strongly catalytic surface for the assembly of prothrombinase and tenase [13]. Additionally, tissue factor (TF) acts as a receptor for the FVII/VIIa and the formation of this complex facilitates the cleavage of FX to FXa [14]. Activated prothrombinase complex catalyses the generation of thrombin (TG) from prothrombin, resulting in the formation of a fibrin clot [12]. The authors' previous study has demonstrated that an acute exposure to 12% O2, but not 15% O2, may promote blood coagulant FVIII-dependent TG by elevating circulatory oxidative stress [15]. Furthermore, intermittent exposure to 12% O2 for 4 weeks resulted in an impairment of vascular haemodynamics by suppressing vascular endothelial function. When the concentration of O2 was set to 15%, the risk of vascular complications became negligible [16]. However, no clear and comprehensive pictures of acute and chronic hypoxic exercise effects on monocyte-mediated atherothrombosis have become available. Hence, we further hypothesize that moderate hypoxic (15% O2) exercise training (HET) effectively suppresses procoagulant MDMP formation and monocyte-promoted TG in plasma under severe hypoxic (12% O2) stress.

To test the hypotheses, the distinct effects of the normoxic exercise training (NET; 21% O2) and HET (15% O2) on (i) procoagulant MDMP formation and dynamic TG in monocyte-rich plasma (MRP) treaded without/with oxidized low-density lipoprotein (ox-LDL, an atherogenic lipoprotein) and (ii) plasma catecholamine/peroxide/pro-inflammatory cytokine concentrations during acute 12% O2 exercise were explored. The aim of the present study is to establish an effective hypoxic exercise strategy for improving individual aerobic capacity and simultaneously ameliorating the risk of atherothrombosis associated with severe hypoxic stress.

MATERIALS AND METHODS

Subjects

Forty healthy sedentary males who were non-smokers, non-users of medication/vitamins and free of any cardiopulmonary/haematological risk were recruited from Chang Gung University, Taiwan. No subject had engaged in any regular physical activity (exercise frequency ≤ once weekly, duration < 20 min) or had been exposed to high altitudes (≥ altitude of 3000 m) for at least 1 year before the experiment. Subjects were randomly divided into HET (n=20) and NET (n=20) groups. Anthropometric and cardiovascular characteristics of the two groups did not differ significantly before interventions (Table 1). The investigation was carried out according to the Helsinki declaration and the protocol was approved by the Institution Review Board of Chang Gung Memorial Hospital, Taiwan. All subjects provided informed consent after the experimental procedures had been explained.

Table 1
The effects of NET and HET on cardiopulmonary fitness

Values are means±S.E.M. BMI, body mass index; BP, blood pressure; Post, after the intervention; Pre, before the intervention. *P<0.05, Pre compared with Post; P<0.05, NET compared with HET.

NETHET
PrePostPrePost
Anthropometrics 
Age (y) 21.1±0.4 – 22.0±1.0 – 
Weight (kg) 70.7±2.9 70.2±2.7 70.5±3.3 70.1±3.1 
Height (cm) 175.1±1.2 175.1±1.2 172.0±1.4 172.0±1.4 
BMI (kg/m223.1±0.8 23.0±0.7 23.4±1.0 23.3±1.0 
Heart rate (bpm) 74±2 70±2* 73±2 68±2* 
Systolic BP (mmHg) 119±3 114±3* 120±3 115±2* 
Diastolic BP (mmHg) 74±2 73±3 73±3 72±2 
Peak exercise performance 
Work-rate (W) 193±7 210±8* 200±8 229±8*† 
Exercise time (min) 23.7±1.0 27.5±1.4* 24.5±1.3 29.3±1.1*† 
Heart rate (bpm) 194±4 196±3 195±3 196±3 
VE (l/min) 115±4 127±5* 112±5 133±4*† 
VO2 (ml/min/kg) 43.2±1.2 48.0±1.6* 42.6±1.4 53.6±1.6*† 
VCO2 (ml/min/kg) 52.1±1.2 55.2±0.3* 51.2±1.2 62.3±1.6*† 
NETHET
PrePostPrePost
Anthropometrics 
Age (y) 21.1±0.4 – 22.0±1.0 – 
Weight (kg) 70.7±2.9 70.2±2.7 70.5±3.3 70.1±3.1 
Height (cm) 175.1±1.2 175.1±1.2 172.0±1.4 172.0±1.4 
BMI (kg/m223.1±0.8 23.0±0.7 23.4±1.0 23.3±1.0 
Heart rate (bpm) 74±2 70±2* 73±2 68±2* 
Systolic BP (mmHg) 119±3 114±3* 120±3 115±2* 
Diastolic BP (mmHg) 74±2 73±3 73±3 72±2 
Peak exercise performance 
Work-rate (W) 193±7 210±8* 200±8 229±8*† 
Exercise time (min) 23.7±1.0 27.5±1.4* 24.5±1.3 29.3±1.1*† 
Heart rate (bpm) 194±4 196±3 195±3 196±3 
VE (l/min) 115±4 127±5* 112±5 133±4*† 
VO2 (ml/min/kg) 43.2±1.2 48.0±1.6* 42.6±1.4 53.6±1.6*† 
VCO2 (ml/min/kg) 52.1±1.2 55.2±0.3* 51.2±1.2 62.3±1.6*† 

Exercise training

The subjects were trained on a bicycle ergometer (Corvial 400, Lode, AN Groningen) at 60% of maximal O2 consumption (VO2max) under 15% O2 (HET) or 21% O2 (NET) conditions for 30 min daily, 5 days weekly for 5 weeks in an air-conditioned normobaric hypoxia chamber (Colorado Mountain RoomTM) [17]. The hypoxia chamber was maintained at a temperature of 22 (±0.5)°C and a relative humidity of 60 (±5)%; a CO2 scrubber eliminated CO2 from the air (≤3500 ppm). All subjects recorded their daily activity using a physical activity questionnaire, which was collected weekly until the end of the study. During the experiment, subjects were instructed to refrain from other regular physical activity. The rate of compliance with each intervention was 100%.

Graded exercise test

All subjects arrived at the testing centre at 9:00 hours to eliminate any possible diurnal effect. Participants were instructed to fast for at least 8 h and to refrain from strenuous physical exercise for at least 48 h before sampling. Each subject performed a graded exercise test (GXT) on a bicycle ergometer (Corival 400, Lode) to assess their aerobic fitness 4 days before and 4 days after the 5-week interventions [17]. The GXT comprised 2 min of unloaded pedalling followed by a continuous increase in work-rate of ∼20–30 W per 3 min until exhaustion (i.e., VO2max). Minute ventilation (VE), VO2 and carbon dioxide production (VCO2) were measured breath by breath using a computer-based system (MasterScreen CPX, Cardinal-health). Systolic and diastolic blood pressure were measured using an automatic blood pressure system (Tango, SunTech Medical) and arterial O2 saturation was monitored by finger pulse-oximetry (model 9500, Nonin Onyx) [17]. The VO2max was defined by the following criteria: (i) the level of VO2 increased less than 2 ml/kg/min over at least 2 min; (ii) heart rate exceeded its predicted maximum; (iii) the respiratory exchange ratio exceeded 1.2 and (iv) the venous lactate concentration exceeded 8 mM, consistent with the guidelines of American College of Sports Medicine for exercise testing [18].

Hypoxic exercise tests and blood collection

Each subject performed the HET 2 days before the intervention and 2 days after the intervention in an air-conditioned normobaric hypoxia chamber (Colorado Mountain Room), as mentioned in our previous studies [5,19]. The hypoxia chamber was maintained at a temperature of 22±0.5°C with a relative humidity of 60±5%; a CO2 scrubber eliminated CO2 in the air (<3500 ppm) [5,19]. The HET on the bicycle ergometer required 50 W of warm-up for 3 min, an increase in work rate to 100 W of continuous exercise for 30 min and then recovery to 50 W of cool-down for 3 min. During the test, the O2 concentration was set to 12%, which corresponds to altitudes of 4460 m.

At rest and immediately after the HET, 35 ml of blood samples were collected from an antecubital vein using a clean venipuncture (20-gauge needle) under controlled venous stasis at 40 torr (1 torr=0.133 kPa). The first 2 ml of blood was discarded and the remaining blood was used to measure haematological parameters. Blood cells were counted using a Sysmax SF-3000 cell counter (GMI) [19].

Procoagulant-rich MDMPs and monocytes

Thirty millilitre of the blood sample were transferred into a polypropylene tube that contained sodium citrate (3.8 g/dl, 1–9 vol blood; Sigma). Peripheral blood monocytes were isolated using a MACS-negative immunomagnetic selection method (Miltenyi Biotec), as described in our previous studies [20,21]. Monocyte purity, which was determined by flow cytometry using an anti-CD14 monoclonal antibody conjugated with FITC (eBioscience) and according to the cell distribution in forward and sideward scatter, was 90%. The cell-free plasma was obtained from citrated blood by centrifugation at 10000 g for 30 min at 4°C. Isolated monocytes were immediately resuspended in the cell free plasma (i.e., MRP), adjusted to 1×106 cells/ml and then maintained at 4°C for no more than 1 h before use [22].

The fresh MRP (1×106 cells/ml) was incubated in the absence or presence of ox-LDL (final concentration: 10 μg/ml; (Biomedical Technologies Inc.) for 30 min at 37°C. The aliquots were immediately re-incubated with a saturating concentration of FITC-conjugated monoclonal anti-human CD14 antibody (Serotec) combined with cyanine5 (Cy5)-conjugated monoclonal anti-human FV/FVa (Upstate), FVIII (American Diagnostica) or phycoerythrin (PE)-conjugated TF (BD Pharmingen) antibody, in darkness for 30 min at 4°C. The procoagulant-rich MDMP population was determined by FACSCalibur™ flow cytometry (Becton Dickinson) according to the events that had been labelled with CD14-FITC and their distribution in the light scatter. Standard size beads of 0.5, 0.9 and 3 μm (Megamix from Biocytex) were used to set the gating scale for the forward light scatter (FSC) parameter of microparticles, as described before [23,24]. In brief, the population of CD14+ MDMPs was gated separately from the CD14+ monocyte population on the basis of FSC, and the Cy5- or PE-stained events observed in the CD14+ MDMP gate were then expressed as definition FV/FVa-, FVIII- or TF-rich MDMPs (unit=events/ml). The exposure of PS on CD14+ MDMPs (unit=events/ml) was also detected by flow cytometry using a commercial Annexin V–Cy5 kit (Biovision) [23,24].

Dynamic TG assay

Dynamic TG in plasma or MRP treated with/without ox-LDL was measured by calibrated, automatic thrombinography (Synapse Thrombinoscope), as described in our previous studies [15,23,24]. Briefly, 80 μl of various plasma samples were allocated into the wells of round bottom 96-well microtitre plates (Nunc). Coagulation was started by adding 0.1 M CaCl2 (20 μl) in a fresh mixture of fluobuffer (containing 20 mM HEPES and 60 mg/ml BSA, pH 7.35) containing 2.5 mM Z-G-G-R-AMC (7-amino-4-methylcoumarin; the fluorogenic substrate; Synapse/Thrombinoscope). Upon cleaving by thrombin, the fluorescent AMC is released and measured with a 390-nm excitation and a 460-nm emission filter set in an Ascent Fluoroskan (Thermo Fisher Scientific). All reagents were warmed to 37°C before the experiment began. Fluorescence was recorded for 90 min. The fluorescence signal was corrected for substrate consumption, plasma colour variability and inner filter fluorescence effect by running in parallel calibrating wells where 80 μl of plasma samples were mixed with 20 μl of Thrombin Calibrator from Thrombinoscope BV.

The following parameters were recorded: (i) endogenous thrombin potential (ETP): area under the curve, which stands for the total amount of thrombin generated over the time; (ii) the lag time of TG: time to burst of TG, which roughly represents the clotting time; (iii) the peak height of TG: the highest thrombin concentration reached during the time course of thrombin formation and inhibition; and (iv) the rate of TG: peak height divided by the difference between time to peak and lag time, which represents the initial slope of TG [15,22,23].

Plasma catecholamine, peroxide and pro-inflammatory cytokine concentrations

From all subjects, an additional 5-ml of blood samples was obtained, placed in a cold centrifuge tube containing EDTA (final concentration, 4 mM; Sigma Chemical Co) and immediately centrifuged at 3000 g for 10 min at 4°C. The plasma samples were stored at −80°C until assay. Plasma norepinephrine/epinephrine (Labor Diagnostika Nord), myeloperoxidase (MPO; Immunology Consultants Laboratory) and interleukin-6 (IL-6; eBioscience) concentrations were quantified by commercially available ELISA kits.

Statistical analysis

Results are expressed as means±S.E.M. The statistical software package StatView was used for data analysis. Kolmogorov–Smirnov's goodness-of-fit test was used and normal distribution was observed in the present study. Experimental results were analysed by 2 (groups)× 4 (time sample points) repeated measures ANOVA and Bonferonni's post- hoc test to compare the monocyte count, MDMP characteristics and dynamic TG parameters before and immediately after HE at the beginning of the present study and after 5 weeks in various groups. In addition, the comparison of cardiopulmonary fitness during GXT at the beginning of the present study and 5 weeks later in various groups were analysed by 2 (groups) × 2 (time sample points) repeated measures ANOVA and Bonferroni's post-hoc test. The criterion for significance was P<0.05.

RESULTS

Cardiopulmonary fitness

Anthropometric variables did not significantly differ between NET and HET groups at the beginning of the study (Table 1). Both NET and HET for 5 weeks significantly decreased resting heart rate and systolic blood pressure, as well as, increased work-rate, exercise time, VE, VO2 and VCO2 at maximal exercise performance (Table 1, P<0.05). However, HET exhibited higher enhancements of pulmonary ventilation and aerobic capacity than NET did (Table 1, P<0.05).

Procoagulant MDMPs and monocytes

Acute bout of 12% O2 exercise significantly increased monocyte count in blood (Figures 1A and 1B, P<0.05). Following the 5-week intervention, HET significantly diminished the extent of increased monocyte count by HE (Figure 1B, P<0.05).

Exercise effect on monocyte-derived microparticles

Figure 1
Exercise effect on monocyte-derived microparticles

Comparisons of the effects of NET (A and C) and HET (B and D) on monocyte (A and B) and total MDMP (C and D) counts. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are mean±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-HE compared with Post-HE.

Figure 1
Exercise effect on monocyte-derived microparticles

Comparisons of the effects of NET (A and C) and HET (B and D) on monocyte (A and B) and total MDMP (C and D) counts. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are mean±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-HE compared with Post-HE.

Treatment of MRP with ox-LDL significantly increased total MDMP counts (Figures 1C and 1D, P<0.05) and elevated the levels of PS-, TF-, FV- and FVIII-positive MDMPs (Figures 2 and 3, P<0.05) and monocytes (Figures 4 and 5, P<0.05).

Exercise effect on phosphatidylserine-/tissue factor-rich microparticles released from monocyte

Figure 2
Exercise effect on phosphatidylserine-/tissue factor-rich microparticles released from monocyte

Comparisons of the effects of NET (AC) and HET (DF) on (A and D) PS-, (B and E) TF- and (C and F) TF plus PS-positive MDMPs in MRP treated without (vehicle) or with ox-LDL. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are mean±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-R or Pre-HE compared with Post-R or Post-HE.

Figure 2
Exercise effect on phosphatidylserine-/tissue factor-rich microparticles released from monocyte

Comparisons of the effects of NET (AC) and HET (DF) on (A and D) PS-, (B and E) TF- and (C and F) TF plus PS-positive MDMPs in MRP treated without (vehicle) or with ox-LDL. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are mean±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-R or Pre-HE compared with Post-R or Post-HE.

Exercise effect on coagulant factor-rich microparticles released from monocyte

Figure 3
Exercise effect on coagulant factor-rich microparticles released from monocyte

Comparisons of the effects of NET (A and B) and HET (C and D) on (A and C) FV- and (B and D) FVIII-positive MDMPs in MRP treated without (vehicle) or with ox-LDL. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values were mean±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-R or Pre-HE compared with Post-R or Post-HE.

Figure 3
Exercise effect on coagulant factor-rich microparticles released from monocyte

Comparisons of the effects of NET (A and B) and HET (C and D) on (A and C) FV- and (B and D) FVIII-positive MDMPs in MRP treated without (vehicle) or with ox-LDL. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values were mean±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-R or Pre-HE compared with Post-R or Post-HE.

Exercise effect on phosphatidylserine-/tissue factor-rich monocyte

Figure 4
Exercise effect on phosphatidylserine-/tissue factor-rich monocyte

Comparisons of the effects of NET (AC) and HET (DF) on (A and D) PS-, (B and E) TF- and (C and F) TF plus PS-positive monocytes in MRP treated without (vehicle) or with ox-LDL. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are mean±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-R or Pre-HE compared with Post-R or Post-HE.

Figure 4
Exercise effect on phosphatidylserine-/tissue factor-rich monocyte

Comparisons of the effects of NET (AC) and HET (DF) on (A and D) PS-, (B and E) TF- and (C and F) TF plus PS-positive monocytes in MRP treated without (vehicle) or with ox-LDL. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are mean±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-R or Pre-HE compared with Post-R or Post-HE.

Exercise effect on coagulant factor-rich monocyte

Figure 5
Exercise effect on coagulant factor-rich monocyte

Comparisons of the effects of NET (A and B) and HET (C and D) on (A and C) FV- and (B and D) FVIII-positive monocytes in MRP treated without (vehicle) or with ox-LDL. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are mean±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-R or Pre-HE compared with Post-R or Post-HE.

Figure 5
Exercise effect on coagulant factor-rich monocyte

Comparisons of the effects of NET (A and B) and HET (C and D) on (A and C) FV- and (B and D) FVIII-positive monocytes in MRP treated without (vehicle) or with ox-LDL. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are mean±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-R or Pre-HE compared with Post-R or Post-HE.

At the beginning of the study, acute 12% O2 exercise markedly enhanced total MDMP count in untreated and ox-LDL-treated MRP (Figures 1C and 1D, P<0.05). Furthermore, the HET also increased the amounts of PS-, TF-, FV- and FVIII-positive microparticles released from monocytes (Figures 2 and 3, P<0.05) and the expression of these procoagulant factors on monocyte membranes (Figures 4 and 5, P<0.05) under the untreated and ox-LDL-treated conditions.

Both NET and HET decreased the amounts of total MDMPs and the TF-expressed, PS-exposed and FV/FVIII-rich MDMPs in untreated and ox-LDL-treated MRP following HE (Figures 2 and 3, P<0.05). Simultaneously, the two exercise regimens also decreased the HE-induced expression of these procoagulant factors on monocyte membranes under the untreated and ox-LDL-treated conditions (Figures 4 and 5, P<0.05). Moreover, HET had larger reductions in production of procoagulant MDMPs (Figures 2 and 3, P<0.05) and monocytes (Figures 4 and 5, P<0.05) induced by HE than those of NET.

Dynamic TG in plasma and MRP

With respect to the analytical parameters of dynamic TG, the HET increased the ETP, peak height and rate of TG (P<0.05) but did not change the lag time of TG in plasma or MRP treated without/with ox-LDL (Figure 6).

Exercise effect on monocyte-mediated dynamic thrombin generation

Figure 6
Exercise effect on monocyte-mediated dynamic thrombin generation

Comparisons of the effects of NET (AD) and HET (EH) on dynamic TG [i.e., (A and E) lag time, (B and F) ETP, (C and G) peak height and (D and H) rate] in plasma as well as MRP treated without (vehicle) or with ox-LDL. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are mean±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-R or Pre-HE compared with Post-R or Post-HE.

Figure 6
Exercise effect on monocyte-mediated dynamic thrombin generation

Comparisons of the effects of NET (AD) and HET (EH) on dynamic TG [i.e., (A and E) lag time, (B and F) ETP, (C and G) peak height and (D and H) rate] in plasma as well as MRP treated without (vehicle) or with ox-LDL. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are mean±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-R or Pre-HE compared with Post-R or Post-HE.

NET significantly decreased the extent of HE-promoted ETP, peak height and rate of TG in untreated MRP rather than ox-LDL-treated MRP (Figure 6). However, HET markedly depressed both resting and HE-mediated dynamic TG under treatment of MRP without or with ox-LDL (Figure 6, P<0.05).

Plasma catecholamine, peroxide and pro-inflammatory cytokine concentrations

At the beginning of the study, acute HE increased concentrations of norepinephrine/epinephrine, MPO and IL-6 in plasma (Table 2, P<0.05). Following the 5-week intervention, both NET and HET markedly decreased plasma norepinephrine/epinephrine, MPO and IL-6 levels following HE (Table 2, P<0.05). However, HET had lower resting MPO level and the extent of HE-enhanced elevation of norepinephrine/epinephrine and MPO than those of NET (Table 2, P<0.05).

Table 2
The effects of NET and HET on plasma catecholamine, peroxide and pro-inflammatory cytokine concentrations

Values are means±S.E.M. Post, after the intervention; Pre, before the intervention; R, resting. *P<0.05, R compared with HE; P<0.05, Pre compared with Post; P<0.05, NET compared with HET.

NETHET
PrePostPrePost
Norepinephrine (pg/ml) 
284.5±39.2 253.5±37.7 292.3±29.5 234.2±39.5 
HE 823.6±99.7* 572.5±78.2*† 784.2±72.7* 484.5±67.2*†‡ 
Epinephrine (pg/ml) 
72.8±9.8 67.8±9.3 70.9±10.9 50.2±9.8 
HE 154.2±19.2* 111.8±16.4*† 150.8±18.9* 79.2±12.1*†‡ 
Myeloperoxidase (ng/ml) 
15.4±2.1 12.3±3.7 17.8±4.2 7.2±1.5†‡ 
HE 30.3±5.4* 21.3±3.1*† 29.4±5.1* 13.2±3.1*†‡ 
IL-6 (pg/ml) 
2.7±0.8 2.2±0.5 2.5±0.7 2.1±0.7 
HE 5.2±1.2* 3.4±0.9 5.8±1.9* 2.4±0.6 
NETHET
PrePostPrePost
Norepinephrine (pg/ml) 
284.5±39.2 253.5±37.7 292.3±29.5 234.2±39.5 
HE 823.6±99.7* 572.5±78.2*† 784.2±72.7* 484.5±67.2*†‡ 
Epinephrine (pg/ml) 
72.8±9.8 67.8±9.3 70.9±10.9 50.2±9.8 
HE 154.2±19.2* 111.8±16.4*† 150.8±18.9* 79.2±12.1*†‡ 
Myeloperoxidase (ng/ml) 
15.4±2.1 12.3±3.7 17.8±4.2 7.2±1.5†‡ 
HE 30.3±5.4* 21.3±3.1*† 29.4±5.1* 13.2±3.1*†‡ 
IL-6 (pg/ml) 
2.7±0.8 2.2±0.5 2.5±0.7 2.1±0.7 
HE 5.2±1.2* 3.4±0.9 5.8±1.9* 2.4±0.6 

DISCUSSION

The present study was first to demonstrate that an acute bout of 12% O2 exercise substantially accelerated ox-LDL-promoted TG in MRP, which was associated with enhanced TF expression, PS exposure and FV/FVIII binding on MDMPs and monocytes. Notably, HET for 5 weeks significantly attenuated the enhancement of procoagulant MDMP formation and monocyte-related dynamic TG caused by HE, compared with NET. Moreover, the HET effect on monocyte-induced coagulation was accompanied by a depressed increases in plasma norepinephrine/epinephrine, MPO and IL-6 concentrations following HE. Hence, the HET regimen effectively ameliorates the HE-promoted dynamic TG in MRP via depressing the production of procoagulant MDMPs and monocytes, which may reduce the risk of atherothrombosis evoked by severe hypoxia.

Aerobic capacity

In the present study, HET exhibited higher values of VE and VO2 at peak performance than NET did, as reflected in a superior improvement of aerobic capacity following HET. Recently, the authors' investigation revealed that HET simultaneously enhanced cardiac and muscular haemodynamic functions by increasing production of nitric oxide (NO) and facilitating mobilization and differentiation of circulating progenitor cells (CPCs) [17]. Since the lack of CPC functional response triggered by angiogenic factors, the improvement of muscular haemodynamics caused by NET mainly depended upon enhancing the NO-dependent vasodilatation [17]. Additionally, muscular adaptations, including increased citrate synthase activity, mitochondrial density, capillary-to-fibre ratio and fibre cross-sectional area, have been reported in sedentary humans subjected to HET [25,26]. Accordingly, the superior ventilatory, haemodynamic and metabolic adaptations induced by HET may extensively improve exercise performance in healthy or diseased individuals.

MDMP formation and TG promoted by HE

Reactive oxygen species (ROS) derived from infiltrated monocytes contribute to oxidative stress at inflammatory sites, thereby promoting oxidation of LDL [27]. Elevated ox-LDL induces expression of the procoagulant proteins in monocytes, thereby producing a procoagulant state in blood [28]. High circulating levels of microparticles have been found in the blood of patients with atherosclerosis [29], whereas the MDMPs retained TF activity in atherosclerotic plaques [30]. The present study also revealed that the ox-LDL treatment increased the amounts of FV/FVIII-rich, PS-exposed and TF-expressed microparticles released from monocytes, which may promote prothrombinase complex assembly and subsequently facilitate TG in MRP.

Blood is subjected to oxidative stress during extremely hypoxic exposure [15]. A previous study has shown that acute exposure to 12% O2 promoted the oxidative burst activity of leucocytes through increasing lipid peroxidation and decreasing antioxidative capacity [31]. Moreover, the hypoxia stress also increased plasma FVIII level/activity and accelerated endogenous TG, which was accompanied by increased urinary peroxide level and decreased plasma total antioxidant content and superoxide dismutase activity [15]. The results of the present study further demonstrated that the 12% O2 exercise markedly increased the release of procoagulant microparticles from monocytes and the expression of procoagulant factors on monocytes, which were accompanied by elevated peak height and rate of TG in ox-LDL-treated MRP. Hence, acute HE may accelerate endogenous TG in MRP via increasing the levels of procoagulant MDMPs and monocytes under an atherogenic stimulation.

Effects of exercise training on HE-mediated coagulant MDMP formation and TG in MRP

After 5 weeks of interventions, both NET and HET diminished the enhancement effects of acute 12% O2 exercise on ox-LDL-mediated procoagulant MDMP formation and dynamic TG in MRP. Moreover, HET was superior to NET for suppressing the monocyte-dependent coagulation activation following the HET. Compared with NET effects, HET also significantly eliminated the elevations in plasma catecholamine, peroxide and pro-inflammatory cytokine caused by HE.

Several mechanisms potentially explain why HET effectively depressed HE-induced coagulation activation. First, the β-adrenergic pathway has been implicated as a pathway potentially mediating an exercise/hypoxia-induced increase in FVIII levels as β-blockade blunts this increase [32,33]. Hence, blunted adrenergic-induced coagulation activation may partially contribute to the HET effect on the blood coagulation system. Second, elevated MPO in plasma can react with the polyunsaturated fatty acids of lipid membranes, resulting in peroxidation of lipid [34]. Severe hypoxic exposure may promote FVIII-dependent TG by enhancing lipid peroxidation [2]. Therefore, HET reduces HE-promoted TG, probably by depressing circulatory oxidative stress. Finally, growing evidence indicates that IL-6 enhances TF production and microparticle formation on monocytes, thus triggering the prothrombotic state [35]. At the beginning of exercise training, acute HE significantly increased IL-6 level in plasma, as described in the present study. This result agrees with the previous literature [36]. In 2011, the authors' investigation further found that HET decreased plasma IL-6 level at rest and after strenuous exercise [37]. The previous findings [37] were similar to the present results. Accordingly, HET diminishes the extents of enhanced procoagulant MDMP formation and TG by HE, which may be partially attributed to depressing HE-induced IL-6 production.

Our early studies using human [38] and animal [39] models demonstrated that exercise training decreased ox-LDL-potentiated platelet activation through enhancing NO release and depressing lipid peroxidation. Recently, the authors' study showed that HET increased plasma nitrite/nitrate concentrations at rest and after strenuous exercise, whereas NET increased only resting NO metabolites in plasma [17]. As with decreasing ox-LDL-potentiated platelet activation [38,39], exercise training in normoxic or hypoxic environment depresses the HE-promoted TG in MRP under an atherogenic condition, possibly by heightening NO production and subsequently eliminating lipid peroxidation.

Limitations of study

A limitation of the present study is the lack of a control group that undergoes hypoxic (15% O2) intervention alone. The authors' previous investigation demonstrated that 15% O2 exposure alone for 4 weeks did not suffice to change haemodynamic responses to 12% O2 exercise [16]. Moreover, plasma redox status, pro-inflammatory cytokine level [40] or dynamic TG [15] remained constant in response to 15% O2 exposure without exercise. Based on these previous findings [15,16,40], the present study does not include a control group with 15% O2 intervention alone. However, exactly how chronic 15% O2 intervention influences monocyte characteristics and dynamic TG remains unclear and demands further investigation.

In the present study, 5-week HET suppressed procoagulant MDMP formation and monocyte-mediated TG and was accompanied by decreased catecholamine, peroxide and pro-inflammatory cytokine levels in plasma following HE. Whether HET depressed HE-promoted TG in MRP is due to (i) change of plasma composition or/and (ii) down-regulation of procoagulant factors on monocytes to diminish the enhancement of plasma TG by HE remains unresolved. Hence, the potential confounding effects of the different plasma compositions at various experimental conditions need further study.

Additionally, the present study is limited in that only young and sedentary males are analysed. Further clinical evidence is required to extrapolate these results to subjects who have been acclimatized at high altitude and to patients with cardiovascular disorders.

Conclusion

HET is superior to NET for enhancing the capacities of systemic O2 delivery and utilization during exercise. Compared with NET effects, HET also elicits more suppression in the HE-promoted TG in MRP under an atherogenic stimulation, which is associated with reduced procoagulant microparticles released from monocytes and down-regulated procoagulant factor expression on monocytes. Additionally, HET also diminishes the elevations of circulatory catecholamines, peroxide and pro-inflammatory cytokines caused by HE. Therefore, HET can be considered an effective exercise strategy that improves aerobic capacity and simultaneously increases the resistance to monocyte-related thrombosis provoked by severe hypoxia. These findings provide new insights into how exercise training under a hypoxic condition influences the risk of thrombosis associated with atherosclerosis.

AUTHOR CONTRIBUTION

Jong-Shyan Wang and Ya-Lun Chang were involved in conception and design of research. Ya-Lun Chang and Yi-Ching Chen performed experiments. Jong-Shyan Wang and Ya-Lun Chang analysed data, interpreted results of experiments, prepared the figures and drafted the paper. Jong-Shyan Wang, Ya-Lun Chang, Yi-Ching Chen, Hsing-Hua Tsai and Tieh-Cheng Fu edited and revised the paper. Jong-Shyan Wang, Ya-Lun Chang, Yi-Ching Chen, Hsing-Hua Tsai and Tieh-Cheng Fu approved the final version of paper.

The authors would like to thank the volunteers for their enthusiastic participation in the present study.

FUNDING

This work was supported by the National Science Council of Taiwan [grant numbers 102–2628-B-182–018-MY3 and 103–2314-B-182–005-MY3]; the Chang Gung Medical Research Program [grant numbers CMRPD1A0132, 1A0302, 2C0161, 1C0531, 1B0331 and 1B0332]; and the Healthy Aging Research Center, Chang Gung University [grant number EMRPD1A0841].

Abbreviations

     
  • AMC

    7-amino-4-methylcoumarin

  •  
  • CPC

    circulating progenitor cell

  •  
  • Cy5

    cyanine5

  •  
  • ETP

    endogenous thrombin potential

  •  
  • FSC

    forward light scatter

  •  
  • FV

    coagulant factor V

  •  
  • GXT

    graded exercise test

  •  
  • HE

    hypoxic exercise

  •  
  • HET

    hypoxic exercise training

  •  
  • IL-6

    interleukin-6

  •  
  • MDMP

    monocyte-derived microparticle

  •  
  • MPO

    myeloperoxidase

  •  
  • MRP

    monocyte-rich plasma

  •  
  • NET

    normoxic exercise training

  •  
  • NO

    nitric oxide

  •  
  • ox-LDL

    oxidized low-density lipoprotein

  •  
  • PE

    phycoerythrin

  •  
  • PS

    phosphatidylserine

  •  
  • TF

    tissue factor

  •  
  • TG

    thrombin generation

  •  
  • VCO2

    carbon dioxide production

  •  
  • VE

    minute ventilation

  •  
  • VO2max

    maximal O2 consumption

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