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Medicine & Science in Sports & Exercise:
doi: 10.1249/MSS.0b013e318279fb59
Basic Sciences

Aerobic Training Workload Affects Human Endothelial Cells Redox Homeostasis


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Author Information

1University of Salerno Medical School, Salerno, ITALY; 2Department of Medicine and Health Sciences, University of Molise, Campobasso, ITALY; 3Sport Medicine Service, Department of Experimental Medicine, Second University of Naples, Naples, ITALY; 4Department of Institutional Study and Territorial Systems, University of Naples “Parthenope,” Naples, ITALY; and 5“S. Maugeri Foundation,” Scientific Institute of Telese Terme, Telese Terme, ITALY

Address for correspondence: Graziamaria Corbi, M.D., Ph.D., via Giovanni Paolo II, Campobasso, 86100, Italy; E-mail:

Submitted for publication June 2012.

Accepted for publication October 2012.

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Purpose: Moderate aerobic exercise reduces oxidative stress, whereas intense physical activity may produce the opposite result. At present, the effects of different exercise loads on oxidative stress markers and the response of human cells to different exercise volumes have not been fully elucidated.

Methods: Human (Eahy-926) endothelial cells (EC), exposed or not exposed to oxidative stress, were conditioned with sera from two groups of triathletes practicing at different workloads.

Results: Although no differences in functional and hemodynamic variables were observed between the two groups of triathletes, significant changes in some markers for oxidative stress were found in their sera. Thiobarbituric acid reactive substances and superoxide dismutase activity were similar, but triathletes practicing the sport at lower volume (T1) had higher serum nitric oxide and lower catalase activity than triathletes performing the training at greater load (T2). The EC conditioned with serum from T1 (T1-EC) showed higher survival and proliferation rates and lower senescence levels than the EC supplemented with T2 (T2-EC) serum both before and after oxidative stress induction. These effects depended on catalase as demonstrated via enzyme activity inhibition using 3-amino-1,2,4-triazole. After oxidative stress induction, Sirt1 activity, a regulator of the oxidative stress response, was significantly increased in the T1-EC but not in the T2-EC. Moreover, the T1-EC required less catalase activity than the T2-EC to counteract an equal amount of oxidative stress after H2O2 administration.

Conclusion: This study demonstrates that the beneficial effects of aerobic exercise are eliminated when the training is performed at a greater workload. Moreover, we suggest an oxidative stress marker, serum catalase activity, as a valid tool to use in the supervision of changes to exercise volume.

The balance between the production of reactive oxygen species (ROS) and the activation of the antioxidant defense system is important to the human physiology and the control of cellular homeostasis. ROS play an important role in signaling processes, but their overproduction generates oxidative stress; this may in turn cause damage to cellular constituents, including DNA, proteins, and lipids, especially when ROS overproduction occurs with insufficient antioxidant enzyme activity (40).

Oxidative stress is an important aspect of cancer, diabetes, neurodegenerative, cardiovascular, and other diseases, and elevated ROS has been implicated in the mechanism of senescence and aging (1,14,23,42). Oxidant overproduction occurs in response to several stressors, including chemicals, drugs, pollutants, high-caloric diets, and exercise (24). Physical exercise can increase oxidative stress, eventually causing a perturbation of homeostasis that is dependent on training specificity and load.

It has been demonstrated that sirtuins, NAD+/NADH deacetylases, are involved in modulating the cellular stress response directly by deacetylation of some factors that are also implicated in endothelial function control (37,43).

Several studies have demonstrated that lifestyle interventions such as calorie restriction and exercise training induce enhancement of Sirt1 expression and activity (8,10). Many studies concurred to clarify the molecular calorie restriction effects, but the knowledge about long-term exercise training effects in humans is scarce. In particular, the influence of training volume on oxidative stress markers, the role of oxidative stress in training response, and the involvement of antioxidant enzymes, such as superoxide dismutase and catalase, have not been sufficiently investigated.

We recently demonstrated that different types of exercise training induce different molecular effects and that aerobic exercise was the most advantageous training protocol compared with both anaerobic and mixed exercises (9).

In the present study, we tested the hypothesis that slightly different loads of aerobic exercise should affect the expression of the beneficial effects of exercise by comparing athletes practicing the same type of aerobic sport but at different volumes.

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Ethical approval

The study was approved by the Ethics Committee of the Second University of Naples, where participants were recruited and human experimentation was conducted according to the Declaration of Helsinki guidelines. The athletes gave their written informed consent after medical staff explained the purpose, possible risks, and stress associated with the study.

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Study population

Ten triathletes who trained for 14.80 ± 1.52 h·wk−1 were recruited from the Sport Medicine Service of the Second University of Naples and defined as “T1.” This group consisted of the same subjects defined as “triathletes (T)” group from our previous article (9), reenrolled for this study.

Eleven triathletes who trained for 18.37 ± 1.06 h·wk−1 were recruited from the Sport Medicine Service of the Federico II University of Naples and defined as “T2.” According to the International Triathlon Union (17) and the USA Triathlon (39), the T1 are classified as Olympic distance triathletes and the T2 as long-course triathletes. Olympic distance triathletes (T1) perform 1.5-km swim, 40-km ride, and 10-km run, whereas long-course triathletes (T2) perform 1.9-km swim, 90-km ride, and 21.1-km run.

The two groups differed for exercise volume calculated as the number of training sessions per week multiplied by the hours of training per day, which was significantly lower in T1 than that in T2 and also for the exercise workload performed in each session.

All subjects were men, had trained for triathlons for a minimum of 5 yr, and underwent a physical examination upon entering the study. All recruited athletes were healthy, were not receiving anti-inflammatory or anabolic drugs or antioxidant supplements, had normal thyroid, hepatic, and renal function, had no personal history of metabolic disorders, and had similar levels of LDL, HDL, triglycerides, and glucose (Table 1).

Table 1
Table 1
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Fifteen days before blood collection, all athletes received a food frequency questionnaire. Taking into account what was declared in their reports, dietary indications were given to the athletes a week before blood sampling to reflect similar distribution of carbohydrates, lipids, proteins, and fluids. Particular care was given to ensure that there were no significant differences between the two groups in the dietary habits (i.e., exposure to nitrate and nitrite) and other factors, which could influence serum antioxidant status. To avoid confounding factors related to the acute effects of exercise, athletes refrained from training for approximately 15 days and were subjected to examinations during the period of suspension from competitions.

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Blood samples

Subjects fasted for at least 12 h before blood collection. Blood samples were drawn from an antecubital vein before the treadmill stress test. Samples were collected in glass tubes to obtain serum for molecular data detection. After centrifugation at 1500g for 10 min, serum was transferred to clean tubes and stored at -80°C until the analysis.

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Spirometry and treadmill stress test

All tests were performed under the direct supervision of the Exercise Test Laboratory medical staff. The spirometry test was conducted with a spirometer (Flowscreen II Biasys, Germany). Spirometer calibration and measurements were carried out according to the ERS/ATS criteria (29).

For the treadmill stress test, subjects were connected to a treadmill stress test system (Cardiotread; Cardioline, Newark, NJ) and began symptom-limited exercise testing according to the standard Bruce and Hornsten (6) protocol. HR was continuously monitored, and blood pressure was measured at each exercise stage. The maximum predicted HR was calculated with the formula 220 bpm minus age. An adequate HR response to exercise was defined as ≥85% of the maximum predicted HR. The Bruce and Hornsten (6) protocol increases treadmill speed and slope incrementally at approximately 3-min intervals. After maximal exercise effort, the athletes were immediately seated and remained connected to the electrodes for at least 5 min, while continuous HR data were recorded by the treadmill and HR and blood pressure were manually recorded.

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Cell culture

We used the EA.hy926 cells because it was demonstrated that this immortalized endothelial cell (EC) line shows similarity in properties to those of isolated EC, such as HUVEC (4,5). In fact, EA.hy926 cells retain most of the characteristics of EC under baseline conditions as well as after treatment (3) and show morphological, phenotypic, and functional characteristics of human macrovascular EC (7,11,15,16,36).

To check that the obtained results between the cell line EA.hy926 and the HUVEC were similar, at the beginning we performed a pilot study confirming what we found in a previous study. In fact, the EA.hy926 cells produced results similar to those obtained with HUVEC at least for the present investigation (9).

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Cell treatments

The EA.hy926 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U penicillin, and 100 μg·mL−1 streptomycin at 37°C with 5% CO2. The cells were cultured in medium supplemented with the athletes’ sera (10%) or FBS (10%) as a control and either exposed to oxidative stress or not. Specifically, the cells were seeded and cultured for 48 h in medium supplemented with triathletes’ sera (T1-conditioned EC [T1-EC] and T2-conditioned EC [T2-EC]). The culture medium was then aspirated, and the EC were exposed to 500 μM H2O2; 4 h after H2O2 exposure, the growth medium was replaced with fresh medium containing FBS, and the EC were maintained in culture for other 48 h. In a preliminary experiment, we evaluated the effect of oxidative stress 12, 24, 48, and 72 h after treatment of 500 μM of H2O2. Finally, we chose the time of 48 h as representative of the most relevant and persistent changes.

In this study, we used 500 μM H2O2 because as demonstrated by other studies, concentration levels lower than 1 mM and up to 35 μM H2O2 are physiological and nontoxic (12,20,26,42) and also because, as we previously demonstrated, 500 μM H2O2 represented the concentration of hydrogen peroxide that effectively induced significant results in terms of survival of control cells.

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Serum nitric oxide availability and lipid peroxidation

Serum nitric oxide (NO) availability was determined by the Griess method following the protocol previously described (9). All data are the mean ± SD of three independent experiments. Peroxidative damage to cellular lipid constituents was determined with the thiobarbituric acid reactive substances (TBARS) method. The assay was performed with 10 μL of serum and 10 μL of cell lysates; the amount of TBARS was expressed as nanomole per microgram of protein. All data are presented as mean ± SD of three independent experiments.

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Sirt1 activity and NAD+/NADH ratio quantitative determination

Sirt1 activity was determined in the nuclei extracted from the EC using the CycLex Sir2 Assay kit (Sir2 Assay Kit; CycLex, Ina, Nagano, Japan). The reaction was conducted by simultaneously mixing fluorescent-labeled acetylated peptide as substrate, 10 μL of sample, trichostatin A, NAD, and lysyl endopeptidase. The intensity of fluorescence at 440 nm was measured 60 min after reaction onset. Values are reported as relative fluorescence per microgram of protein (AU). All data are presented as mean ± SD of three independent experiments.

NAD+/NADH was quantified using the EnzyCromTM NAD+/NADH Assay Kit (BioAssay Systems, Hayward, CA). The optical density was read at 565 nm at time zero (OD0) and after incubation (15 min) at room temperature (OD15). ΔOD values were used to determine the sample NAD+/NADH concentration from the standard curve. All data are presented as mean ± SD of three independent experiments.

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Catalase and SOD activities

Catalase and SOD activities in the cell lysates and serum were determined using the Catalase Assay Kit and the SOD Assay Kit (Cayman Chemical, Ann Arbor, MI), respectively. For both assays, samples were first diluted with buffer (1:2 for cell lysates; 1:10 for serum). One unit of catalase activity was defined as the amount of enzyme that leads to the formation of 1.0 nmol of formaldehyde per min at 25°C. One unit of SOD activity was defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. The SOD assay measured all three types of SOD (Cu/Zn, Mn, and FeSOD). The values were reported as units per microgram of protein. All data are presented as mean ± SD of three independent experiments.

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RNA isolation and real-time quantitative PCR analysis

Catalase messenger RNA (mRNA) levels were quantified by SYBR Green real-time PCR (Roche Diagnostics, Indianapolis, IN). The following nucleotide sequences of the primers used for PCR were specific and lacked dimerization: catalase, forward 5′-CCAGAAGAAAGCGGTCAAGAA-3′, and reverse 5′-TGGATGTGGCTCCCGTAGTC-3′. The human housekeeping gene GAPDH was measured to control for the amount of input mRNA using the following primer sequences: forward 5′-CAGCCGCATCTTCTTTTGC-3′ and reverse 5′-CCATGGTGTCTGAGCGATGT-3′. The expression of genes was quantified by Sequence Detection 1.4 7500 System SDS software (Applied Biosystem, Sequence Detection 1.4 7500 System SDS software, Foster City, CA). The data were expressed as the relative mRNA expression. All data are presented as mean ± SD of three independent experiments.

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Viability assay (MTT)

To evaluate cell viability in the presence or absence of stress induction, the EC were seeded (3.5 × 103 per well) in 96-well plates and cultured in the triathletes’ sera at 37°C. Cell viability was determined by a Cell Quanti-MTT assay (Bioassay Systems). The data were expressed as the percentage of viable cells relative to the FBS-EC (control). All data are presented as mean ± SD of three independent experiments.

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Senescence-associated β-galactosidase (SA-βgal) activity

Cultured cells were washed in PBS (pH 7.4) and fixed with 2% formaldehyde and 2% glutaraldehyde for 10 min at room temperature. After being washed twice with PBS, the cells were incubated at 37°C in freshly prepared staining buffer [40 mM citric acid/sodium phosphate (pH 6.0), 0.15 M NaCl, 2 mM MgCl2 5 mM potassium ferrocyanide, 1 mg·mL−1 X-gal (5-bromo-4 chloro-3-indolyl β-D-galactoside)]. At the end of 4 h of incubation, the SA-βgal rate was obtained by counting four random fields per dish and by assessing the percentage of SA-βgal-positive cells from 100 cells per field.

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BrdU incorporation assay

DNA synthesis was assessed using a BrdU (5-Bromo-2 deoxy-uridine) Labelling and Detection Kit (Roche Diagnostics, Milan, Italy). The assay was performed according to the manufacturer’s instructions. The results are expressed as the percentage of BrdU incorporation.

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Catalase activity inhibition

To investigate if catalase activity could influence changes in the survival and senescence of the cells conditioned with the triathletes’ sera, we inhibited catalase activity using 3-amino-1,2,4-triazole (ATZ) purchased by Sigma (Milan, Italy) at a concentration of 10 mM for 3 h.

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Statistical analysis

The results are expressed as the mean ± SD of three independent experiments. Differences between the two groups were analyzed using t-tests. Differences between multiple groups were analyzed using a one-way ANOVA with Bonferroni post hoc test. Multivariate analyzes were performed to assess correlations among variables. Statistical significance was determined as P < 0.05. All data were analyzed using the Statistical Package for the Social Sciences (Version 15.0; SPSS, Chicago, IL).

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Study population

The study population consisted of two groups of triathletes performing the same aerobic exercise but with significant differences in training volume. The groups were designated as T1 and T2 on the basis of the number of training sessions per week multiplied by the hours of training per day, which was significantly lower in T1 than that in T2 (P < 0.0001).

We evaluated biochemical and molecular variables in the sera of each group. The clinical and biochemical features, the functional data, determined by the treadmill stress and spirometry tests, and the information about the training durations are shown in Table 1.

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Serum oxidative stress markers and NO bioavailability

The same lipid peroxidation levels (assessed by TBARS) were found in T1 and T2 triathletes (Fig. 1A), whereas NO bioavailability was much higher in T1 than that in T2 serum (P < 0.0001) (Fig. 1B).

Figure 1
Figure 1
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The SOD activity levels (Fig. 1C) were similar in the two groups, but a large difference was found in catalase activity, with T2 showing higher levels than T1 (P < 0.0001) (Fig. 1D).

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In vitro cellular experiments

The EA.hy926 hybrid cell line resemble cultured human vascular EC in most aspects (i.e., secretion, increased synthesis, and enhanced production of t-PA and Pal type-1) and in other EC-specific functions (7,11,15,16,36).

We performed a pilot study to compare EA.hy926 versus HUVEC behavior. In particular, we carried out viability and BrdU incorporation assays and measured SA-βgal activity either on EA.hy926 cell line or HUVEC finding very similar results both at baseline and after 500 μM H2O2 oxidative stress induction (data not shown). Sirt1 activity and the NAD+/NADH ratio in EC conditioned with triathletes’ sera.

Before stress induction, Sirt1 activity was lower in the EC conditioned with T1 serum compared with the cells conditioned with T2 serum (P = 0.001) (Fig. 2A); similarly, T1-EC had a lower NAD+/NADH ratio than T2-EC (P = 0.003) (Fig. 2B). Interestingly, after stress induction, Sirt1 activity increased in T1-EC (P < 0.05) but not T2-EC compared with their baseline conditions (Fig. 2A).

Figure 2
Figure 2
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Moreover, the NAD+/NADH ratio remained the same before and after stress in T1-EC, whereas it greatly increased in the EC conditioned with T2 serum (P < 0.01) (Fig. 2B).

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Oxidants and antioxidants in EC conditioned with triathletes’ sera

As shown in Figure 3, the TBARS amount was lower in the EC conditioned with T1 serum than the cells supplemented with T2 serum (P < 0.0001). After stress induction, the TBARS increased only in the T1-EC compared with basal conditions (P < 0.0001) (Fig. 3A).

Figure 3
Figure 3
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Before and after stress, mRNA levels of catalase were the same in the two groups (Fig. 3B), whereas catalase activity was higher in the T1-EC than that in the T2-EC (P < 0.0001) and increased significantly after stress induction (P < 0.0001 and P < 0.005, respectively) (Fig. 3C).

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Viability markers in EC conditioned with triathletes’ sera

EC conditioned with T1 serum showed higher survival (MTT assay) compared with that measured in the T2-EC (P < 0.0001). After H2O2 stress induction, survival decreased in both the T1-EC and T2-EC compared with baseline (P < 0.0001) (Fig. 4A).

Figure 4
Figure 4
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The proliferation rate (assessed by the BrdU incorporation assay) was higher in the T1-EC than that in the T2-EC (P < 0.0001) (Fig. 4B). After stress, the proliferation rate decreased in both the T1-EC (P = 0.005) and the T2-EC (P < 0.02) compared with the starting condition. Fluorescence images of the cells conditioned with triathletes’ sera, with and without stress induction, are shown in Figure 4C.

The senescence level (measured by the SA-βgal assay) was lower in the T1-EC than that in the T2-EC (P < 0.0001). Stress induction caused a significant increase in both the T1-EC (P < 0.02) and the T2-EC (P = 0.001) compared with their respective baselines (Fig. 5A). In Figure 5B, representative images of SA-βgal staining in EC conditioned with triathletes’ sera are shown.

Figure 5
Figure 5
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Catalase effects on viability markers in the EC conditioned with triathletes’ sera

Because serum catalase was very different between T1 and T2 triathletes, we decided to investigate a possible role for catalase activity in determining the beneficial effects of aerobic exercise. In particular, to investigate the effect of catalase on viability biomarkers (survival, proliferation, and senescence) in the EC conditioned with triathletes’ sera, we inhibited catalase activity with ATZ. ATZ caused a significant decrease in survival in the T1-EC (P < 0.0001), but conversely, it produced an increase in survival in the T2-EC (P < 0.0001) compared with their respective baselines. The T1-EC showed a lower proliferation rate compared with the T2-EC (P < 0.01) in the presence of ATZ. The inhibition of catalase produced a decline in the cellular proliferation rate in the T1-EC (P < 0.0001) and an increase in T2-EC (P < 0.05) compared with their initial conditions (Fig. 4B).

Moreover, catalase was strongly involved in controlling the senescence rate. At baseline, the T1-EC had lower senescence levels than the T2-EC (P < 0.0001) (Fig. 5A), and ATZ doubled the senescence rate in the T1-EC compared with baseline conditions (P < 0.0001). In contrast, the T2-EC treated with ATZ demonstrated the opposite, showing a significant senescence reduction compared with baseline conditions (P = 0.01) (Fig. 5A).

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The effects of both acute and chronic physical activity on the redox system are well known to depend strongly on physical activity specificity (2,25,31).

In a recent study, we investigated the consequences of oxidative stress on EC treated with various athletes’ sera (9). Despite no difference in the functional and hemodynamic variables, we found significant changes in serum oxidative stress markers between the athletes. In particular, subjects performing aerobic training (triathletes) had higher nitric oxide (NO) serum levels and lower serum catalase activity than those others practicing anaerobic and mixed exercises. We concluded that the high NO and low catalase activity were characteristics of the triathletes’ serum that lead to positive cellular adaptations.

In the present study, we reenrolled the same group of triathletes previously evaluated (9) (herein T1) and showed that serum from T1 (trained for 14.80 ± 1.52 h·wk−1) had higher NO levels and lower catalase activity than T2 triathletes (trained for 18.37 ± 1.06 h·wk−1). Hence, by comparing athletes practicing the same aerobic exercise (triathlon) at different loads, we emphasize that the volume, in addition to the exercise type, is crucial for determining the effects of the training.

Although regular aerobic exercise reduces oxidative stress, intense physical activity may produce the opposite result, favoring free radical build-up (18,33). The effects of different exercise loads on oxidative stress markers and the role of oxidative stress in training response to various exercise volumes have not previously been thoroughly clarified.

To our knowledge, few studies on humans (28,38) have indicated oxidative stress markers as possible tools to determine a dose–response relationship with different exercise volumes. In particular, by examining the changes in athletes’ redox status in response to a progressively increased and decreased exercise workload, Margonis et al. (28) proposed using stress biomarkers to diagnose overtraining. These authors showed that some ROS and antioxidant enzymes increased proportionally to exercise dose in the serum of trained subjects.

In this study, we show that serum TBARS and SOD activity in the two groups were the same, whereas T1 athletes had lower serum catalase activity and higher NO levels than T2, suggesting that the latter two parameters were influenced by changes in training volume.

It is important to consider that the present investigation substantially differs from others. Although studies are usually conducted on subjects enrolled to perform a defined training protocol, we carried out an observational study on athletes that had been performing an aerobic sport continuously for at least 5 yr.

As suggested by other authors (25,28,39), we propose serum catalase activity as a tool to identify exercise-associated redox status variations in a training load-dependent manner. However, this is the first study that demonstrates a very large difference in serum catalase between two groups of athletes conducting the same type of sport.

Using an in vivoin vitro technique, we recently demonstrated that conditioning EC with athletes’ sera-induced changes in redox homeostasis through circulating factors released during training (9). Compared with other athletes (soccer players and sprinters), the triathletes had the best results. In particular, the conditioning of EC with triathletes’ serum enhanced cellular survival and improved the action of molecules crucial to preserving cellular homeostasis, especially after inducing oxidative stress. Indeed, the triathletes’ serum also increased Sirt1 activity, a well-known regulator of the oxidative stress response, after H2O2 addition without any change in the levels of its cofactor NAD+.

The NAD+/NADH ratio contributes to the control of the intracellular redox homeostasis, and it fluctuates in response to changes in cellular metabolism (27). Sirt1 activity dependence on NAD+ implies that Sirt1 effectiveness is strongly linked to the cellular metabolic state. It has been suggested that H2O2 accelerates cellular senescence by depletion of NAD+, causing a consequent reduction in the function of Sirt1 (19).

By using the same in vitroin vivo technique in the present investigation, we show that even small changes in exercise volume might strongly influence training effects and precisely that the triathletes’ serum benefits on EC could be abrogated when the same training is performed at a greater load.

Finally, the positive adaptations associated with chronic, aerobic exercise are limited in T2-EC in comparison with T1-EC. After the H2O2 addition, in the EC conditioned with T1 serum (T1-EC), Sirt1 activity increased with no change in the NAD+/NADH ratio. In contrast, in the cells supplemented with T2 serum (T2-EC), there was a dramatic increase in the NAD+/NADH ratio and no change in Sirt1 activity. This result indicates better adaptation of Sirt1 in the T1-EC than that in T2-EC according to what previously observed when comparing aerobic with mixed and anaerobic exercises (9).

Similarly to Sirt1, we observed an optimal adaptation of catalase in T1-EC but not in T2-EC. Actually, with catalase mRNA levels that did not vary between the two groups, an adaptive change in the catalase activity was reached in the EC conditioned with T1 serum. After stress induction, the T1-EC required less enzyme activity than the T2-EC to counteract the same amount of oxidative stress. We observed that the T1-EC, which were cultured in medium poor in catalase, were guided to activate this antioxidant enzyme especially in presence of H2O2 stress induction.

We hypothesized that T1 serum, containing lower levels of catalase activity than T2 serum, elicited the enzyme activity in the EC. On the contrary, the T2-EC did not need to activate the enzyme as much as the T1-EC because the T2-EC were treated with serum already containing a sufficient amount of active catalase.

These results agree with the concept of hormesis, which indicates that an increase in ROS production eventually induces endogenous defense, culminating in increased stress resistance (32).

In the previous study on exercise specificity (aerobic versus mixed and anaerobic exercises), we measured cellular survival, proliferation, and senescence, demonstrating that survival and proliferation rates were higher and senescence levels were lower in EC supplemented with triathletes’ serum than that in the others.

It has been suggested that cell vitality is related to NO bioavailability and NO production is strongly limited in senescent EC (21,34). Here, we observed that the beneficial effects of aerobic exercise, such as higher cellular vitality and lower senescence levels, are mainly dependent on workload of the training. In fact, survival and proliferation rates were higher in the EC conditioned with T1 serum, which had higher levels of NO than that in the EC supplemented with T2 serum with lower NO bioavailability (Fig. 4), and senescence was lower in the T1-EC than that in T2-EC both with and without stress induction (Fig. 5).

Endothelial cellular senescence is involved in endothelial dysfunction and atherogenesis, and it was shown that vascular senescent EC are present in human atherosclerotic lesions (30). Moreover, it is suggested that enhancement of Sirt1 expression tightly contributes to inhibit a senescent phenotype in human EC and that NO is a crucial for up-regulation of Sirt1 (22).

In this study, we demonstrate a crucial role for catalase activity in determining the beneficial effects of aerobic exercise. Catalase inhibition induced a reduction in cell survival and proliferation and an increase in senescence levels in the T1-EC (Figs. 4 and 5).

This finding is in agreement with other studies showing that the increased hydrogen peroxide generation guides to an increase in endothelial senescence, which could be attenuated by catalase treatment in a dose-dependent manner (44).

Indeed, ATZ, a catalase activity inhibitor, caused the abrogation of T1 serum protective effect as demonstrated by decrease in cellular survival and proliferation and by increase in senescence levels. Therefore, indicators of function and health in the cells conditioned with serum of athletes undergoing aerobic training could be influenced by catalase activity in reason of both training specificity and workload. In fact, we found that triathletes performing the sport at lower volume (T1) had lower serum catalase activity than triathletes performing the training at greater load (T2). In our opinion, these results demonstrate a correlation between training volume and serum catalase activity. Therefore, we suggested that the catalase could be a marker of changes in exercise volume on the basis of its activity levels in the sera, related to the volume of training sustained by the T1 and T2 groups.

A possible limitation of this study is that we did not investigate the effect on the EC of other circulating factors (such as NO levels) that, besides the catalase, can justify our cellular data and the better performance exhibited by the T1-EC. Moreover, the EC used in this study were not exposed to shear stress as in vivo.

Another possible limitation of the study is the absence of data on endothelial function in vivo. It has been demonstrated that while strenuous exercise increases oxidative metabolism and produces a pro-oxidant environment, regular moderate physical activity promotes an antioxidant state and preserves endothelial function (13). It would be interesting to perform further studies to verify the correlation between endothelial function in vivo and the data obtained through this in vivo/in vitro technique.

The adaptive human responses to training dose represent an important issue. After hard training, many athletes report symptoms, which often evolve to clinical conditions, such as chronic fatigue syndrome (35).

Although the exercise is an integral part of cardiac rehabilitation programs, the training protocol to obtain the best results still remains to define. In fact, the choice of optimal exercise workload could be very important to improve human physiological functions, avoiding the accumulation of oxidative damage and subsequently fostering a preventive effect against many diseases.

In conclusion, our data suggest the potential utility of oxidative stress markers, such as systemic catalase activity, to supervise changes in the exercise workloads. Moreover, these findings indicate catalase activity as valid indicator of different exercise volumes and offer new insights into understanding the molecular basis of the systemic effects of physical activity in humans.

The authors thank native English speaker Dr. Anna D’Angelo for her language revisions of the manuscript. No funding was received for this study. The results on this study do not constitute endorsement by the American College of Sports Medicine.

All authors report no conflict of interest of any financial and personal relationships with other people or organizations that inappropriately influence the work of this study.

Conti and Russomanno contributed equally to this work.

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