Key words
exercise - humoral immunity - oxidized LDL - atherosclerosis - endothelium
Introduction
Beneficial effects of physical activity in cardiovascular health and disease
prevention are well known [1]
[2]. However, early atherosclerosis or acute coronary
syndrome in athletes or trained subjects remains not fully explored, and the
possible effects of long-term high-endurance exercise on the atherosclerosis process
are lacking or inconclusive [3]
[4]. In humans, data about the effects of exercise
intensity and volume on subclinical atherosclerosis is conflicting [5]
[6].
Exercise may have beneficial effects on atherosclerosis, including metabolic and
hormonal factors, due to increase in the enzymatic and non-enzymatic antioxidant
activities to reduce tissue damage. Furthermore, exercise may affect immunological
components, such as low-density lipoprotein (LDL) particle, as well as cellular and
humoral responses [7].
The humoral response to oxidized LDL and atherosclerosis progression has been poorly
investigated in athletes or high endurance-trained individuals [8]. The endurance training through metabolic and
immunological adaptation could impact vascular and atherosclerosis modulation [9]
[10].
Previously, we reported that postmenopausal hormone replacement therapy is associated
with lower autoantibody titers against apoB sequence and LDL oxidized, as compared
to controls without hormone therapy, and that this outcome may be associated with
vascular function [11]. However, these parameters
have not yet been evaluated in higher trained subjects.
Cytokine IL-8 acts via the stimulation of CXCR1 and CXCR2 receptors. CXRC2 is
expressed by vascular endothelial cells and is responsible for IL-8 induced
angiogenesis. It has been shown that exercise induces CXCR2 mRNA and protein
expression in the vascular endothelial cells, which may be related to vascular
function in trained subjects [12]. On the other hand,
IL-10 is a knowledge cytokine related to B cell regulations and humoral responses,
and chronic exercise may be modulating IL-10 expression [13]. However, there is a lack of evidence for higher trained subjects
that the expression of IL-8 and IL-10 could be associated with vascular health and
humoral immune response against autoantigens.
Given the limited evidence, this study aimed to investigate the possible differences
in immune-metabolic responses and vascular health parameters between high-endurance
trained and healthy non-trained subjects.
Materials and Methods
Subjects
We conducted an observational, cross-sectional study with nonrandom inclusions of
high endurance trained (HET) and healthy, active non-trained (HNT) subjects. The
individuals were matched for sex and age, and had no history of cardiovascular
disease or classical risk factors for atherosclerosis, inflammatory diseases,
and other comorbidities.
The HET group’s eligibility criteria were endurance-trained individuals
of both sexes age 20–40 years who had completed at least four
half-marathons or eight 10-km races in the previous two years. The HNT
group’s inclusion criteria were individuals of both sexes age
20–40 years who regularly exercised at a moderate or vigorous intensity
(no more than two days per week).
The exclusion criteria for both groups were history of diabetes mellitus,
hypertension, chronic kidney disease, heart disease, musculoskeletal disorders,
autoimmune diseases, dyslipidemias, or recent infectious disease.
Demographic, clinical data, and blood samples were collected at the inclusion
visit defined in the protocol.
This study was conducted according to the ethical standards of the institutional
committee on human experimentation, and the study protocol was approved by the
local ethics committee. Written informed consent was obtained from all
participants before the study was initiated.
Cardiopulmonary exercise stress testing
The subjects’ maximum functional capacity was assessed using a
cardiopulmonary test on a treadmill (Total Health Centurion 300; Micromed,
Brasilia, DF, Brazil) with a ramp protocol until exhaustion, starting at
5 km/h and a fixed slope (1%), with
1 km/h increments up to 18 km/h, and then the
slope was increased to 2% up to the maximal voluntary exhaustion. The
total exercise time varied between 8 and 17 min, as previously described
[14]. All assessments were performed in
temperature-controlled rooms (19–21°C), with a humidity of
50–60%. The athletes were advised about the diet before the test
and were told not to exercise within 24 h before the test.
Heart rate was monitored throughout the test using a 12-lead electrocardiogram
(ErgoPC Elite; Micromed, Brasilia, DF, Brazil).
Breath-by-breath gas analyses were performed using silicone masks (Hans-Rudolph,
Shawnee, KS, USA) and gas analyzer (Cortex Metalyzer 3B with Metasoft software;
Micromed, Brasilia, DF, Brazil) before the test, during the exercise bout, and
up to the 6th min of recovery. The gas analyzer system was calibrated
before each test. Oxygen uptake (VO2) and carbon dioxide production
were determined using the respiratory gas exchange and a computerized system
(OxyScan; Micromed, Brasilia, DF, Brazil). Peak VO2 (VO2
peak) was defined as the maximum VO2 measured at the end of the
exercise bout, and anaerobic and ventilatory thresholds were determined [15].
Vascular health parameters assessment
Two methods were used to evaluate vascular health: endothelial-dependent
flow-mediated dilatation (FMD) and carotid intima-media thickness (cIMT).
Endothelial function and cIMT were measured using echocardiography with a SONOS
5500 Ultrasound System (Hewlett-Packard-Phillips; Palo Alto, CA, USA) equipped
with vascular software for two-dimensional imaging, color and spectral Doppler
ultrasound modes, internal electrocardiogram monitor, and linear-array
transducer (with a frequency range 7.5–12.0 MHz).
Endothelial function was evaluated based on the FMD of the brachial artery using
ultrasound [16]. The percent change in vessel
diameter from the baseline value was calculated to determine the FMD and
endothelium-independent dilatation. All assessments were performed blindly by
the ultrasonographers. The intra- and inter-sonographer variability values
were<1% and<2%, respectively.
The cIMT was assessed by ultrasonography of the carotid right and left arteries
using the protocol recommended by the American Society of Echocardiography:
Carotid Intima-Media Thickness in B-mode Ultrasound [17]. The ultrasonographers performing the measurements were blinded
to the study group.
Blood biochemistry analysis
Serum total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), and
triglycerides (TG) were determined enzymatically (Opera Bayer, Leverkusen,
Germany). LDL-cholesterol (LDL-C) was estimated using the Friedewald equation
when TG was<400 mg/dL. Analysis of apolipoproteins A-1
(ApoA-1) and B (ApoB) was performed using Siemens reagent on a Siemens BNI
nephelometer (Siemens Healthcare Diagnostics, Newark, DE, USA). Glucose was
measured using the glucose oxidase method (Hitachi-911; Boehringer Mannheim,
Mannheim, Germany).
Assessment of metabolic responses
Corticosterone and 17α-hydroxyprogesterone levels were evaluated using a
LC-MS/MS method. Plasma samples (0.2 mL) were spiked with
0.2 mL of internal standard (17α-hydroxyprogesterone-d8 in
methanol and zinc sulphate), vortexed and centrifuged. The supernatant was
filtered and 50 µL were injected into a 1260 LC system (Agilent
Technologies, Mississauga, ON, Canada). Chromatographic separation was performed
on a Kinetex C18 column (100×3.0 mm, 2.6 µm,
100ª; Phenomenex, Torrance, CA, USA) by gradient elution at a flow rate
of 0.55 mL/min, 45ºC, using water (mobile phase A) and
methanol (mobile phase B). LC system was coupled to a triple-quad mass
spectrometer (Sciex Qtrap 5500, Concord, ON, Canada) fitted with atmospheric
pressure chemical ionization (APCI) source in positive mode (height
5 mm; curtain gas 28 psi; medium collision gas; temperature 500⁰
C; nebulizer gas 50 psi; auxiliary gas 50 psi).
Compounds were monitored with selected-reaction monitoring at transitions
347.1/12.1 for corticosterone, 331.0/97.0 for
17α-hydroxyprogesterone and 339.2/100.1 for internal
standard.
Measurements of cytokines
Plasma samples were stored at -70 ºC until analyzed. Concentrations of
Interleukin (IL)-8 and IL-10 were determined using commercially available
enzyme-linked immunosorbent assay (ELISA) kits according to the information
provided by the manufacturer (R&D Systems, Minneapolis, MN, USA).
Assessment of humoral immune responses against LDL particle
To determine the autoantibodies (Abs) to copper-oxidized LDL, we used an
established enzyme-linked immunosorbent assay (ELISA) method as previously
described [18]
[19]. The evaluation of titers of anti-oxLDL Abs, isotype IgG and IgM
(0.1 µg/ml and 10 µg/ml;
purified human IgG and IgM, KPL, Kirkegaard & Perry Laboratories,
Gaithersburg, Maryland, USA) were performed by a protocol previously established
in our laboratory [20]. The buffer blank
(phosphate-buffered saline (PBS) was used as control to compensate for
intra-plate variation. Inter-plate imprecision was minimized by processing all
the samples in the same time period. To minimize false positive results due to
cross-reactivity with antigen naïve epitopes, antibody titers were
expressed as the reactivity index (RI), calculated as RI=[(ODsample–ODsample blank)/(ODIgG or IgM-ODIgG
or IgM blank)], where IgG or IgM were used as controls. Samples
were run in duplicate and the variation within the duplicates did not exceed
5% of the mean.
Assessment of humoral immune responses against apolipoprotein B
Quantification of anti-ApoB-D autoantibodies (Abs) was assessed in total plasma
by ELISA method, as previously described [21]. To
evaluate the immune response to the ApoB-D peptide, we also used ELISA. Briefly,
we coated the plaques overnight with 10 µg/mL of the
peptide (ApoB-D, is apoliprotein B-peptide fragment with 22 amino acids of
conserved I region of LDL particle). After three cycles of wash, the plate of
samples of volunteers was added (1/1000 in PBS). Three more cycles of
wash were performed in sequence, and we added IgG or IgM antibodies to evaluate
titers of anti-ApoB-D peptide Abs respectively
(0.1 µg/ml and 10 µg/ml;
purified goat anti-human IgG and IgM, KPL, Kirkegaard & Perry
Laboratories, Gaithersburg, Maryland, USA). After two hours of incubation, the
plaques were washed, and 3,3′,5,5′-tetramethylbenzidine
(6.5% in dimethyl sulfoxide; Sigma, St Louis, MO) and H2O2 (Sigma)
diluted in citrate/phosphate buffer (0.1 mol/l;
250 μl; pH 5.5) were added (at room temperature) as enzyme
substrate. The reaction was stopped by addition of H2SO4
(2 mol/l). The optical density (OD) of samples was measured at
450 nm. Autoantibody (Abs) titers were expressed as the
reactivity index (RI), calculated as RI=[(OD sample–OD
sample blank)/(OD IgG or IgM–OD IgG or IgM
blank)] where the IgG or IgM antibodies were used as controls.
Samples were run in duplicate and the variation within the duplicates did not
exceed 5% of the mean.
Statistical analyses
Categorical variables are expressed as n (%) and compared using
Chi-square test. Numerical variables are expressed as the mean with standard
error (SE) or median with interquartile range (IQR). The normality of their
distribution was assessed using the Kolmogorov-Smirnoff test. Since the
distributions of IL-8 and IL-10 concentrations were non-Gaussian, their values
were log-transformed prior analysis. The numerical variables were compared
between HET and HNT subjects using the independent-samples t-test or
Mann-Whitney U test. Correlations between autoantibodies, interleukins, and
hormones were assessed using Spearman or Pearson correlation coefficients. All
analyses were performed using the Statistical Package for Social Science 17.0
software package (SPSS Inc., Chicago, IL, USA). Two-sided
P-values<0.05 were considered significant.
Results
Subjects characteristics
This study enrolled 96 subjects, 44 in the HET group and 52 in the HNT group.
[Table 1] shows the demographic
characteristic of the HET and HNT subjects. The age distribution did not differ
significantly between groups. As expected, oxygen consumption by
VO2max analysis and body composition differed significantly between
groups (P<0.001).
Table 1 Demographic, biochemistry and cardiovascular
characteristics of subjects.
|
Variables
|
Overall (N=96)
|
Healthy non-trained (N=52)
|
High endurance-trained (N=44)
|
P-values
|
|
Demographic characteristics
|
|
Sex (M/F)
|
43/53
|
21/31
|
22/22
|
|
|
Age (years), mean, SE
|
32.0 (7.5)
|
33.5 (8.0)
|
31.0 (6.5)
|
0.176
|
|
Fat mass (%), mean, SE
|
20.0 (9.0)
|
27.3 (6.0)
|
12.8 (4.5)
|
<0.001
|
|
VO2 max (ml/Kg.min-1), mean,
SE
|
41.5 (6.5)
|
31.0 (6.5)
|
54.5 (6.5)
|
<0.001
|
|
Biochemistry parameters
|
|
Glucose (mg/dL), median, IQR
|
87 (82–92)
|
89 (85–93)
|
85 (80–89)
|
0.003
|
|
Total cholesterol (mg/dL), median, IQR
|
165 (148–191)
|
176 (149–210)
|
156 (144–153)
|
0.004
|
|
LDL-c (mg/dL), median, IQR
|
93 (76–113)
|
105 (84–131)
|
81 (71–97)
|
<0.001
|
|
HDL-c (mg/dL), median, IQR
|
57 (48–70)
|
50 (46–61)
|
62 (55–72)
|
<0.001
|
|
Triglycerides (mg/dL), median, IQR
|
66 (49–81)
|
77 (61–102)
|
54 (42–66)
|
<0.001
|
|
ApoB/ApoA, median, IQR
|
0.50 (0.42–0.62)
|
0.58 (0.50–0.77)
|
0.45 (0.37–0.52)
|
<0.001
|
|
Creatinokinase (mg/dL), median, IQR
|
140 (90–248)
|
99 (72–140)
|
232 (158–334)
|
<0.001
|
|
Vascular health markers
|
|
Carotid Intima-Media Thickness (mm), mean, SE
|
0.76 (0.11)
|
0.76 (0.12)
|
0.75 (0.10)
|
0.547
|
|
Flow-mediated dilatation (%),mean, SE
|
12.8 (9.5)
|
13.05 (8.5)
|
12.60 (10.35)
|
0.831
|
Abbreviations: SE, standard error; 95% IQR, interquartile
range; ApoB/ApoA, apolipoprotein B/apolipoprotein A.
Vascular heath parameters
The FMD measurements did not differ significantly between the HET and HNT groups
(P=0.831; [Table 1]). In
addition, they did not differ significantly between the HET and HNT subjects
among males (12.2±1.7 and 10.9±1.9, respectively;
P=0.624) or females (12.9±2.8 and 14.2±1.7,
respectively; P=0.701; [Fig.
1a]).
Fig. 1
a. Box plots representing the median and interquartile ranges of
the flow-mediated dilation (FMD) of the brachial artery expressed in
percentual values, by sex and group. b. Boxplots representing the
median and interquartile ranges of the carotid intimal medial-thickness
(cIMT) expressed in millimeters, evaluated by non-invasive
echocardiography method, by sex and group.; The sex groups are male
(dark gray) and female (light gray) HET, high
endurance trained subjects; HNT, health non-trained
subjects.
The cIMT measurements did not differ significantly between the HET and HNT groups
(P=0.547). However, these values were significantly lower in
male HET subjects (0.72±0.09) than in male HNT subjects
(0.81±0.09; P=0.009). However, they were similar between
female HET (0.78±0.10) and HNT (0.73±0.13) subjects
(P=0.265; [Fig. 1b]).
Immune and metabolic assessment
Regarding humoral immune responses, titers of immunoglobulin G (IgG)
autoantibodies against oxidized LDL (oxLDL) were significantly lower in the HET
group than in the HNT group (P=0.020). Nonetheless, titers of
immunoglobulin (IgM) autoantibodies against oxLDL were similar among groups
(P=0.530). Titers of IgG autoantibodies against the ApoB-D
peptide were similar between groups, while titers of IgM autoantibodies against
the ApoB-D were higher in the HET group than in the HNT group
(P<0.001) ([Table 2]).
Table 2 Autoantibodies anti-oxLDL, anti-ApoB-D peptide,
immunological and hormonal measurements of subjects.
|
Variables
|
Healthy non-trained (52)
|
High endurance-trained (44)
|
P-values
|
|
Autoantibodies
|
|
IgG anti-oxLDL Abs, RI, median, (IQR)
|
4.80 (3.50–8.30)
|
4.32 (2.85–6.00)
|
0.029
|
|
IgM anti-oxLDL Abs, RI, median, (IQR)
|
0.55 (0.30–0.80)
|
0.47 (0.36–0.47)
|
0.530
|
|
IgG anti-ApoB-D Abs, RI, median, (IQR)
|
1.85 (1.30–2.70)
|
1.71 (1.20–1.65)
|
0.682
|
|
IgM anti-ApoB-D Abs, RI, median, (IQR)
|
0.25 (0.17–0.35)
|
0.46 (0.28–0.62)
|
<0.001
|
|
Cytokines
|
|
IL-8, pg/mL, median, (IQR)
|
6.62 (3.44–20.40)
|
5.00 (3.72–17.50)
|
0.781
|
|
IL-10, pg/mL, median, (IQR)
|
2.12 (1.05–2.20)
|
2.25 (2.05–2.38)
|
0.014
|
|
Immune cells
|
|
Leucocytes, cell/mm³, median,
(IQR)
|
5.97 (5.31–7.00)
|
5.59 (5.70–6.30)
|
0.041
|
|
Basophils, cell/mm³, median, (IQR)
|
0.05 (0.03–0.06)
|
0.03 (0.02–0.05)
|
0.011
|
|
Monocytes, cell/mm³, median, (IQR)
|
0.31 (0.25–0.36)
|
0.23 (0.20–0.28)
|
<0.001
|
|
Hormones
|
|
Corticosterone, ng/dL, median, (IQR)
|
168.4 (92.0–246.2)
|
237.5 (167.3–382.7)
|
0.028
|
|
17 α-hydroxyprogesterone, ng/dL, median,
(IQR)
|
57.0 (33.5–76.9)
|
86.2 (45.4–145.1)
|
0.047
|
Abbreviations: Abs, autoantibodies; RI, reactivity index;
95% IQR, interquartile range.
Sex-based analyses of immune responses showed that titers of IgM autoantibodies
against the ApoB-D peptide were higher in male HET subjects (0.53,
IQR=0.25–0.66) than in male HNT subjects (0.23,
IQR=0.15–0.31; P=0.003). Similarly, titers of IgM
autoantibodies against the ApoB-D peptide were higher in female HET subjects
(0.43, IQR=0.29–0.58) as compared to female HNT subjects (0.25,
IQR=0.18–0.46; P=0.025). In addition, in the
female group, lower titers of IgG autoantibodies against oxLDL were found in HET
compared with the HTN groups (P=0.040). Likewise, titers of IgM
autoantibodies against oxLDL did not differ between male and female HET and HNT
subjects ([Fig. 2]).
Fig. 2
a. Box plots representing the distribution of IgM anti-oxLDL;
b. IgG anti-oxLDL; c. IgG anti-ApoB-D-peptide;
d. IgM anti-ApoB-D autoantibody titers in the total plasma,
expressed as RI, by sex and group. The sex groups are male (dark
gray), and female (light gray). HET, high endurance-
trained subjects; HNT, health non-trained subjects. RI,
reactivity index.
Regarding hormones, both plasma corticosterone (P=0.028) and 17
α-hydroxyprogesterone (P=0.047) concentrations were
higher in the HET group than in the HNT group ([Table
2]). Similarly, 17 α-hydroxyprogesterone
(P=0.050; [Fig. 3a]) and
corticosterone (P=0.029; [Fig.
3b]) plasma concentrations were higher in male HET subjects than in
male HNT subjects, whereas corticosterone (P=0.019; [Fig. 3b]) plasma concentrations were higher in
female HET subjects than in female HET subjects.
Fig. 3
a. Boxplots representing the median and interquartile range of
the 17 α-hydroxyprogesterone plasma concentrations, expressed as
ng/dL, by sex and group. b. Boxplots representing the median and
interquartile range of corticosterone plasma concentrations, expressed
as ng/dL, by sex and group. Sex groups are male (dark gray) and
female (light gray). HET, high endurance trained subjects;
HNT, health non-trained subjects.
Correlations between immunoendocrine responses and vascular health
parameters
Correlations between immunoendocrine parameters and vascular health parameters
were examined. Measurements of cIMT were inversely correlated with 17
α-hydroxyprogesterone hormone concentrations
(r=− 0.37; P=0.006). In addition,
FMD measurements were directly correlated with the IL-10 concentrations
(r=0.22; P=0.049; [Table
3]). Titers of autoantibodies against oxLDL and the ApoB-D peptide
were not correlated with the vascular health parameters, FMD and cIMT.
Table 3 Correlations between immune-endocrine parameters
with FMD and cIMT.
|
Concentrations
|
cIMT
|
FMD
|
|
r
|
P-value
|
r
|
P-value
|
|
IgG anti-oxLDL Abs
|
− 0.02
|
0.854
|
− 0.07
|
0.492
|
|
IgM anti-oxLDL Abs
|
0.05
|
0.610
|
− 0.06
|
0.561
|
|
IgG anti-ApoB-D Abs
|
0.05
|
0.612
|
0.04
|
0.677
|
|
IgM anti-ApoB-D Abs
|
− 0.03
|
0.722
|
− 0.04
|
0.245
|
|
IL-8
|
− 0.18
|
0.094
|
− 0.04
|
0.724
|
|
IL-10
|
− 0.09
|
0.391
|
0.22*
|
0.049
|
|
Corticosterone
|
− 0.12
|
0.364
|
0.08
|
0.577
|
|
17 α-hydroxyprogesterone
|
0.37*
|
0.006
|
− 0.04
|
0.757
|
Abbreviations: cIMT, carotid intimal medial-thickness; FMD,
flow-mediated dilatation, Abs, autoantibodies; r, Pearson correlation;
*Significant, P<0.05.
Discussion
In this study, we demonstrated that long-term high-endurance training might affect
the humoral responses against LDL particles and the immune dominant region of ApoB,
concomitant with changes in metabolic pathways associated with health vascular
parameters, in a sex-dependent manner.
The sex differences in the humoral immune response findings might be due to sex
hormones exerting suppressive effects on both humoral and cellular responses. A
previous study showed that female high trained subjects presented higher levels of
these sex hormones than female HNT subjects [22].
However, our findings did not show sex-based differences in hormone concentrations
associated with immune responses, contrasting with previous results in non-trained
subjects [23].
The high-intensity exercise might induce LDL particle modifications leading to
epitopes recognized by autoantibodies [24]. Recent
studies have shown that low to moderate exercise can elevate autoantibodies to virus
protein-derivate [25] and improve influenza and
COVID-19 vaccines [26]
[27], indicating that the exercise intensity balance can improve
antibodies against autoantigens and external agents.
Our previous studies showed that increased IgM anti-oxLDL titers correlate with
better cardiovascular health associated with lower coronary artery disease burden
and subclinical atherosclerosis [28]
[29]. Interestingly, our results do not show
differences in the natural immune response to LDL particles between groups. However,
trained subjects showed higher IgM anti-ApoB-D peptide autoantibody titers compared
to non-trained subjects. Recent studies have shown that higher titers of IgM
autoantibodies against ApoB-derived peptides are associated with lower cIMT and less
atherosclerosis severity in the general population [30]
[31]. Our group has demonstrated that
apoB-peptides can lead to endothelial dysfunction by altering endothelium
permeability [32]. In addition, individuals with
elevated titers of IgM autoantibodies against the ApoB-D peptide showed improved
endothelial function, revealing associations between the natural immune response to
the ApoB-D peptide and endothelium function [28].
This study did not demonstrate better endothelial function in HET subjects compared
to HNT subjects. Interestingly, exercise promoted improved endothelial function via
nitric oxide availability, reduced inflammation, and other neural activations [33]. However, high-intensity exercise might reduce the
FMD with an increase of dilation between 1–24 h post-exercise, and
normalization can occur 24–48 h after exercise cessation. These
effects are intensity-, bout-, and exercise mode-dependent, with higher intensity
leading to more sustained FMD reduction [34]. In this
study, the HET subjects had trained with a 10–20 km run the day
before recruitment for ultrasonography analysis, which might have impacted the FMD
results.
Measurements of cIMT were similar between groups, although they differed
significantly between male HET and HNT subjects. Nevertheless, studies on the
long-term effects of exercise on subclinical atherosclerosis have reported
conflicting data [35]
[36]. We believe exercise induces different morphological modifications
inside atherosclerosis plaques that cannot be evaluated by measuring cIMT with
ultrasonography techniques.
Interestingly, our study showed that the natural immune response was associated with
hydroxyprogesterone in the HET subjects. This finding is consistent with previous
studies showing that sex hormones modulate the synthesis of different immunoglobulin
isotypes in B cells differently [37]
[38]. However, the functional role of exercise in B
cells and its relevance in cardiovascular disease is poorly investigated. Further
studies on interrelation among the exercise, humoral response, and cardiovascular
disease need to comprehensively examine the immunoendocrine pathways to support new
therapeutics and future prevention.
Limitations
Our trial has other limitations that also merit consideration. First, due to its
cross-sectional design, further randomized clinical trials are needed to
reinforce our findings. Exercise could modulate humoral immune responses and
provide improvements in vascular health. Second, previous exercise training
might have affected FMD measurements and endocrine responses, impacting our
results. Third, the open-label design might have led to reporting bias, although
we attempted to control for ascertainment bias by masking all laboratories
analyses, and the vascular health by endothelial-dependent flow-mediated
dilatation and carotid intimal-medial thickness were measured by clinicians
blinded to the study group subjects. Fourth, we included only
high-endurance-trained subjects, without comparative moderate and sedentary
groups. Highly trained subjects are frequently examined for performance, and
health status may be in the gray zone related to risks for
lesions/disease.
Conclusion
In conclusion, our results demonstrated that long-term high-endurance exercise
subjects showed a sex-dependent immune-endocrine modulation compared to active
non-trained subjects, reflecting substantial effects on vascular health
parameters.
Author Contribuition
HARF, FAF, MCOI, AMFN: Concept and design of the study; CRB, AMM, LRS, CEFS:
Performed the clinical and laboratory analysis; HARF: Performed statistical
analysis; HARF, MG, FAF, and MCOI: Data analysis and interpretation the data; HARF
and MG: Drafted the manuscript; all authors approved the final manuscript.
