Responses of Angiogenic Growth Factors to Exercise

Physiology & Biochemistry

Responses of Angiogenic Growth Factors to Exercise, to Hypoxia and to Exercise under Hypoxic Conditions

Affiliations

Key words ▶ angiogenesis ● ▶ VEGF ● ▶ EPO ● ▶ IL-6 ● ▶ IL-8 ● ▶ IGF-1 ●

P. Wahl1, 4, A. Schmidt2, M. deMarees1, S. Achtzehn1, W. Bloch3, J. Mester1, 4 1

Institute of Training Science and Sport Informatics, German Sport University Cologne, Cologne, Germany Institute of Pharmacology and Toxicology, German Armed Forces, Munich, Germany 3 Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Cologne, Germany 4 The German Research Centre of Elite Sport, German Sport University Cologne, Cologne, Germany 2

Abstract

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The purpose of the present study was to compare the acute hormonal response of angiogenic regulators to a short-term hypoxic exposure at different altitudes with and without exercise. 7 subjects participated in 5 experimental trials. 2 times subjects stayed in a sedentary position for 90 min at 2 000 m or 4 000 m, respectively. The same was carried out again in combination with exercise at the same relative intensity (2 mmol∙L − 1 of lactate). The fifth trial consisted of 90 min exercise at sea level. Venous blood samples were taken under resting conditions, 0

Introduction

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accepted after revision April 25, 2012 Bibliography DOI http://dx.doi.org/ 10.1055/s-0032-1314815 Published online: 2012 Int J Sports Med © Georg Thieme Verlag KG Stuttgart · New York ISSN 0172-4622 Correspondence Dr. rer. nat. Patrick Wahl Institute of Training Science and Sport Informatics German Sport University Cologne Am Sportpark Muengersdorf 6 50933 Cologne Germany Tel.: +49/221/4982 6071 Fax: +49/221/4982 3870 [email protected]

2 main stimuli were identified for the induction of skeletal muscle angiogenesis. On the one hand, a reduced oxygen tension, as it has been shown that there is an insufficient oxygen supply in skeletal muscle during exercise [26, 32]. On the other hand mechanical stimuli, as total skeletal muscle blood flow, and therefore shear stress and mechanical stretch, is elevated during physical activity [3, 8, 9]. One or both stimuli are present when hypoxia is induced or exercise/exercise under hypoxic conditions is performed. Therefore, training under hypoxic conditions might be beneficial to induce angiogenesis in working muscles. It was shown that high altitude training can improve the maintenance of oxygen to tissues in terms of an increased amount of red blood cells (RBC) and capillaries. In this context erythropoietin (EPO) and vascular endothelial growth factor (VEGF) are important hormones regulating angiogenesis and erythropoiesis. Both hormones are regulated by an oxygensensitive transcription factor (hypoxia-inducible factor; HIF-1). EPO receptors can be found on RBCprogenitor cells, as well as on endothelial cells,

and 180 min after each condition to determine VEGF, EPO, IL-6, IL-8 and IGF-1 serum concentrations. EPO, VEGF, and IL-8 showed increases only, when hypoxia was combined with exercise. IL-6 was increased after exercise, independent of altitude. IGF-1 showed no changes in any intervention. The present study suggests that short term hypoxic exposure combined with low intensity exercise is able to up-regulate angiogenic regulators, which might be beneficial to induce angiogenesis and to improve endurance performance. However, in some cases high altitudes are needed, or it can be speculated that exercise intensity needs to be increased.

showing also an angiogenic function. VEGF is the most potent cytokine involved in angiogenesis [8, 11] stimulating cell differentiation, proliferation, migration, and survival [35]. Interleukin-6 (IL-6) levels are increased in tissues undergoing angiogenesis. However, IL-6 is not directly involved in angiogenic processes, as IL-6 induces the expression of VEGF and has therefore an indirect effect [5]. IL-8 is known to be a potent angiogenic and mitogen factor, which induces the migration and proliferation of endothelial cells and smooth muscle cells, as well as neovascularisation [1, 33]. Growth hormone (GH) is the principal regulator of the hepatic synthesis of insulin-like growth factor I (IGF-1) and IGF-1 itself is the primary downstream mediator of GH actions. As it has been shown that oxygen availability also influences exercise evoked responses of hGH [38], we speculated IGF-1 levels to be influenced by hypoxia. IGF-1 has widespread anabolic and insulin-sensitizing effects [13], which might also be important for the activation of endothelial (progenitor) cells. According to the aforementioned facts, these factors might play a critical role in the induction and

Wahl P et al. Hypoxia/exercise & angiogenic regulators … Int J Sports Med

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Authors


Materials and Methods

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Subjects 7 healthy male non-smoking sport students (mean ± SD, age: 22.1 ± 1.9 years, weight: 73.4 ± 6.9 kg, height: 178.4 ± 6.3 cm, relative VO2max: 56.7 ± 7.7 mL∙min − 1∙kg − 1) participated in this study. All subjects gave written informed consent to the participation in the study. The study protocol was performed in accordance with the declaration of Helsinki, the standards of the Ethical Committee of the university and the ethical standards of the IJSM [14].

Exercise protocol and hypoxic exposure In order to test the influence of exercise, hypoxia and the combination of both on the acute hormonal response, subjects visited the laboratory on 5 separate occasions in a randomly chosen order with at least 1 week break between each session. At 3 visits, subjects performed constant load exercise for 90 min at sea level (E SL; 20.8 % O2), 2 000 m (E 2 000; 15.9 % O2) and 4 000 m (E 4 000; 13.2 % O2) at the same relative intensity (2 mmol∙L − 1 of lactate). At 2 other visits subjects stayed in a sedentary position at 2 000 m (S 2 000; 15.9 % O2) and 4 000 m (S 4 000; 13.2 % O2) for 90 min as well. The hypoxic conditions were induced by using a normobaric hypoxic-chamber (Hypoxic Training Systems, Hypoxico, New York). The O2 and CO2 concentrations were measured during the entire performance period with a Dräger Multiwarn O2 and CO2 Gas Analyser (Dräger, Lübeck, Germany). To keep the CO2 concentration within a physiologically tolerable range (0.03–0.3 %), a CS 2210 CO2 absorber (SK Engineering, Kiel, Germany) was used. During the 5 sessions SO2 [ %] was determined every 15 min. Therefore a capillary blood sample of 115 μL was collected from the earlobe and immediately analyzed (AVL Omni 6; Roche Diagnostics GmbH, Mannheim, Germany). All tests were carried out at the same time of day. Subjects were not allowed to perform strenuous exercise 24 h prior to testing. A light meal was allowed 2 h before each test. Food intake was partially standardized. That is, certain foods containing carbohydrates were recommended to the subjects and subjects were advised to ingest the same amount and composition of food before each test. Therefore, food intake was recorded before the first test and reproduced before the following.

Measurements At each of the 5 sessions venous blood samples were taken before (pre), directly after (0’) and 3 h after (180’) each intervention. Furthermore, in advance of the 5 sessions, baseline values of the hormonal status were determined over 2 weeks (7 measWahl P et al. Responses of Angiogenic Growth … Int J Sports Med

urements). These blood samples were taken in the morning in a fasting state. 9 and a half millilitres of blood was collected by the Vacutainer blood withdrawal system (Becton Dickinson). After storage at 7 °C for 30 min for deactivation of coagulation factors, the blood samples were centrifuged for 10 min at 1 861 g and 4 °C (Rotixa 50, Hettich Zentrifugen, Mühlheim, Germany). The serum was stored at − 80 °C. Serum levels of hormones were determined by using human ELISA kits: VEGF (Quantikine Human VEGF Immunoassay (Catalogue Number DVE00) R&D Systems GmbH, Wiesbaden-Nordenstadt Germany); EPO (Erythropoietin ELISA, DRG Instruments GmbH, Marburg Germany); IGF-1 (IGF-1 600 ELISA, DRG Instruments GmbH, Marburg Germany); Il-6 (Quantikine® HS High Sensitivity Human IL-6 Immunoassay, R&D Systems GmbH, Wiesbaden-Nordenstadt Germany); IL-8 (Quantikine® Human IL-8 Immunoassay, R&D Systems GmbH, Wiesbaden-Nordenstadt Germany). All samples for each parameter were analyzed in duplicate, and the mean was used for statistical analysis.

Determination of training intensity Before the start of the study, subjects visited the laboratory on 3 separate sessions in order to perform 3 incremental step tests on a cycle ergometer (Schoberer Rad Meßtechnik SRM GmbH, Juelich, Germany) until exhaustion, to determine the individual intensity for the 3 exercise sessions (E SL, E 2000, E 4000). One step test was carried out under normoxic conditions (SL; 20.8 % O2), and 2 step tests under normobaric hypoxic conditions (2 000 m; (15.9 % O2) and 4 000 m; (13.2 % O2)) in a randomised order. During each step test, peak pulmonary oxygen uptake (VO2peak) (nSpire, ZAN600USB, Oberthulba, Germany), heart rate (HR) (Polar S710, Polar Electro GmbH, Büttelborn, Germany) and lactate concentrations [LA] (EKF Diagnostic Sales, Magdeburg, Germany) were determined. The spirograph was calibrated prior to each test using calibration gas (15.8 % O2, 5 % O2 in N; Praxair Deutschland GmbH, Duesseldorf, Germany) comprised of the range of anticipated fractional gas concentrations. A 3 L syringe (nSpire, Oberthulba, Germany) was used for volume calibration. Heart rate was recorded in real time every 5 s during the tests using short-range telemetry. All respiratory and heart rate data were averaged every 30 s. A blood sample of 20 μL from the earlobe was collected at the end of 5 min intervals into a capillary tube (EBIOplus; EKF Diagnostic Sales, Magdeburg, Germany) and analyzed amperometric-enzymatically for the blood lactate concentration. The step test protocol started at 100 watts, thereafter the power was increased by 40 W every 5 min. After the 3 incremental tests, there was a break of one week before one of the main interventions was carried out.

Statistical analysis Statistical analyses of the data were performed by using a statistics software package (Statistica for Windows, 7.0, Statsoft, Tulsa, OK). Descriptive statistics of the data are presented as means ± SD. For the comparison of different terms, repeatedmeasures ANOVA with Fisher post-hoc test was used. Statistical differences were considered to be significant for p < 0.05. Furthermore, the effect size “partial η2” was calculated and is docu▶ Table 2. The thresholds for small, moderate, and mented in ● large effects were defined as 0.1, 0.25 and 0.4, respectively. Power was calculated post-hoc for ANOVA repeated measures using α, sample size and effect size with G*Power Version 3.1.3 ▶ Table 2). (Heinrich-Heine University Duesseldorf, Germany) (●


regulation of angiogenesis in physiological processes. A few studies have already investigated the effects of hypoxia or exercise under hypoxic conditions on single angiogenic growth factors [21, 29, 30, 32]. However, results are inconsistent and in most studies only one condition (hypoxia or exercise under hypoxic condition) was tested. Despite many years of research in high altitude training, little is known about the acute and longlasting effects on endurance performance. Therefore, the present study focused on the effects of hypoxia with and without exercise on circulating levels of VEGF, EPO, IL-6, IL-8 and IGF-1. We hypothesized that exercise under hypoxic conditions would result in a larger increase of angiogenic growth factors compared to exercise at sea level or hypoxia without exercise.


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Compared to SL (56.7 ± 7.7 mL∙kg − 1∙min − 1), VO2peak significantly decreased by − 14.8 % at 2 000 m (48.3 ± 7.2 mL∙kg − 1∙min − 1) and by − 24.9 % at 4 000 m (42.6 ± 7.9 mL∙kg − 1∙min − 1) respectively. Power output at 2 mmol∙L − 1 of lactate was about − 10 and − 22 % lower at ▶ Table 1). Power 2 000 and 4 000 m respectively compared to SL (● output at 4 mmol∙L − 1 of lactate was about − 8 and − 17 % lower at ▶ Table 1). 2 000 and 4 000 m, respectively, compared to SL (● Under resting conditions SO2 [ %] significantly decreased under hypoxic conditions by − 3.5 % (94.8 ± 2.2 %; 2 000 m) and − 11.0 % (87.2 ± 3.8 %; 4 000 m) compared to SL (98.2 ± 1.0 %). Under exercise conditions SO2 [ %] significantly decreased under hypoxic conditions by − 8.1 % (88.5 ± 4.4 %; 2 000 m) and − 20.9 % (76.2 ± 7.1 %; 4 000 m) compared to SL (96.3 ± 2.5 %).

Table 1 Power output (PO) in Watt [W] at 2 mmol∙L-1 and 4 mmol∙L-1 of lactate at different altitudes. Altitude

PO [W] at 2 mmol∙L-1 of lactate

E SL E 2 000 m E 4 000 m

169 ± 35 152 ± 39 132 ± 27

Altitude

PO [W] at 4 mmol∙L − 1 of lactate

E SL E 2 000 m E 4 000 m

205 ± 42 189 ± 31 170 ± 28

Values are shown as means ± SD. E SL: exercise at sea level; E 2 000: exercise at 2000 m; E 4000: exercise at 4000 m

VEGF: VEGF showed great inter-individual differences ranging from 96 pg∙mL − 1 up to 1 130 pg∙mL − 1. The mean VEGF concentration during baseline was 361 ± 279 pg∙mL − 1 (mean over all subjects and the 7 baseline measurements; 95 % confidence interval [CI] 89–627 pg∙mL − 1). Pre-values before each intervention were not significantly different from baseline values ▶ Table 2). Overall ANOVA showed significant differences for (● different points in time of one condition (p = 0.006; F = 7.98) but not for different conditions (p = 0.52; F = 0.82). Post-hoc analysis revealed that E 4000 was the only intervention, which significantly increased VEGF levels (p = 0.02) compared to pre-values. However, VEGF levels decreased back to pre-values 180’ after the exercise. 180’ after E SL VEGF levels significantly decreased ▶ Table 2). below pre values (● EPO: The mean EPO concentration during baseline was 6.6 ± 3.4 mIU∙mL − 1 (mean for all subjects and the 7 baseline measurements; 95 % CI 3.9–9.4 mIU∙mL − 1). The pre-values of E SL and S ▶ Table 2). Over4000 were significantly higher compared to BL (● all ANOVA showed significant differences for different points in time of one condition ((p < 0.001); F = 22.68) but not for different conditions (p = 0.18; F = 1.67). Post-hoc analysis revealed that only the 2 exercise interventions under hypoxic conditions significantly increased EPO levels compared to pre-values (p < 0.001). However, S 4000 nearly reached statistical signifi▶ Table 2). cance (p = 0.06) (● IGF-1: The mean IGF-1 concentration during baseline was 411 ± 137 μg∙L − 1 (mean for all subjects and the 7 baseline measurements; 95 % CI 302–528 μg∙L − 1). Pre-values before each intervention were not significantly different from baseline values

Table 2 Changes in hormone concentrations under different conditions. Parameter

BL

EPO [mlU∙mL − 1]

6.6 ± 3.4

VEGF [pg∙mL − 1]

361 ± 279

IGF-1 [μg∙L − 1]

411 ± 137

IL-6 [pg∙mL − 1]

0.92 ± 0.32

IL-8 [pg∙mL − 1]

8.4 ± 3.5

Condition E SL E 2000 E 4000 S 2000 S 4000 E SL E 2000 E 4000 S 2000 S 4000 E SL E 2000 E 4000 S 2000 S 4000 E SL E 2000 E 4000 S 2000 S 4000 E SL E 2000 E 4000 S 2000 S 4000

pre $

9.3 ± 3.0 6.6 ± 3.6 7.1 ± 3.8 7.0 ± 3.7 9.4 ± 4.3$ 339 ± 284 310 ± 268 347 ± 321 349 ± 265 344 ± 377 441 ± 151 437 ± 167 417 ± 120 474 ± 179 549 ± 82 1.27 ± 0.71 1.07 ± 0.72 1.00 ± 0.74 1.05 ± 0.48 1.07 ± 0.53 12.2 ± 8.8 7.5 ± 7.0 4.6 ± 1.4 9.7 ± 7.6 7.5 ± 4.8

0’

180’

Partial η2

Power (1−β)

8.5 ± 3.1 7.1 ± 2.6 7.0 ± 3.0 7.6 ± 4.2 10.0 ± 5.3 330 ± 266 316 ± 224 401 ± 350# 373 ± 281 357 ± 350 396 ± 115 478 ± 113 497 ± 127 420 ± 191 457 ± 113 2.42 ± 0.59# 1.99 ± 0.97# 2.11 ± 0.98# 1.07 ± 0.53 1.04 ± 0.32 15.1 ± 9.9 7.8 ± 6.7 8.4 ± 4.5# 8.1 ± 7.8 9.8 ± 6.2

9.3 ± 3.3 10.6 ± 2.8* 10.4 ± 3.1* 7.3 ± 2.3 10.8 ± 4.8(#) 282 ± 261# 344 ± 301 338 ± 329+ 346 ± 276 344 ± 308 384 ± 140 421 ± 155 421 ± 149 481 ± 177 467 ± 109 2.2 ± 1.36# 1.46 ± 0.53+ 0.93 ± 0.46+ 1.04 ± 0.35 1.11 ± 0.49 12.2 ± 9.7 5.1 ± 2.9 9.6 ± 8.1# 9.4 ± 4.7 12.1 ± 7.7#

0.22 0.83 0.64 0.05 0.18 0.41 0.14 0.60 0.32 0.03 0.09 0.09 0.24 0.12 0.36 0.56 0.64 0.63 0.00 0.02 0.22 0.19 0.33 0.09 0.45

0.78 1.0 0.99 0.19 0.66 0.99 0.51 0.99 0.94 0.13 0.33 0.36 0.83 0.42 0.98 0.99 0.99 0.99 0.06 0.09 0.78 0.71 0.96 0.35 0.99

BL: Baseline; E SL: exercise at sea level; E 2 000: exercise at 2 000 m; E 4 000: exercise at 4 000 m; S 2 000: sitting at 2 000 m; S 4 000: sitting at 4 000 m. * significantly different from “pre” and “0’”; # significantly different from “pre”;+significantly different from “0’”; $ significantly different from BL. Values are shown as means ± SD. Partial η2: The thresholds for the effect size “partial η2” for small, moderate, and large effects were defined as 0.1, 0.25 and 0.4, respectively. Power (1- β) was calculated post-hoc for ANOVA repeated measures



Results

▶ Table 2). Overall ANOVA showed no significant differences (● neither for different time points of one condition (p = 0.35; F = 1.14) nor for different conditions (p = 0.39; F = 1.08). None of the 5 interventions caused significant changes in IGF-1 levels ▶ Table 2). (●

IL-6: The mean IL-6 concentration during baseline was 0.92 ± 0.32 pg∙mL − 1 (mean for all subjects and the 7 baseline measurements; 95 % CI 0.7–1.1 pg∙mL − 1). Pre-values before each intervention were not significantly different from baseline val▶ Table 2). Overall ANOVA showed significant differences ues (● for different points in time of one condition (p < 0.001; F = 14.7) and for different conditions (p < 0.001; F = 5.9). Post-hoc analysis revealed that all exercise interventions significantly increased IL-6 levels (p < 0.001). However, 180’ after E SL IL-6 remained elevated, whereas 180’ after E 2000 and E 4000 IL-6 levels sig▶ Table 2). nificantly decreased back to pre-values (● IL-8: The mean IL-8 concentration during baseline was 8.4 ± 4.6 pg∙mL − 1 (mean for all subjects and the 7 baseline measurements; 95 % CI 5.2–11.6 pg∙mL − 1). The pre-value of E 4000 ▶ Table 2). Overall was significantly lower compared to BL (● ANOVA showed significant differences for different time points of one condition ((p = 0.049); F = 3.7) but not for different conditions (p = 0.08; F = 2.3). Post-hoc analysis showed significant increases in IL-8 levels (p < 0.001 & p = 0.01) for the 2 interven▶ Table 2). tions at 4000m (E 4000 and S 4000) only (●

Discussion

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The major finding of the present study was that angiogenic regulators can be increased by moderate acute short term exercise under hypoxic conditions, but not by hypoxia or exercise alone. As all 3 exercise protocols were performed at the same relative intensity (2 mmol∙L − 1 lactate), the effects of both exercise interventions under hypoxic conditions on the acute hormonal response cannot be attributed to higher intensities. However, SO2 [ %] was significantly lower under hypoxic conditions compared to SL. For most subjects and most parameters BL values were quite constant and except for the values of EPO (E SL & S 4 000), mean BL values did not differ from pre-values of the 5 conditions. However, in the case of EPO and IL-8 2 subjects showed greater intra-individual variations. Therefore, for some parameters it might be necessary to determine baseline values. Furthermore, some parameters showed great inter-individual variations (VEGF, EPO, IGF-1). As EPO is regulated by oxygen-sensitive transcription factor (HIF-1; hypoxia-inducible factor) [10], it was not surprising, that hypoxia combined with exercise increased circulating levels. Similar to our results, Schmidt et al. found that only exercise under hypoxic conditions (60 min, 60 % PPO, PIO2 92 mmHg), but not under normoxic conditions, increases EPO levels 3 h post exercise to a similar amount (+5,5 mU∙mL − 1) [30] as in the present study. As exercise was carried out at the same relative intensity, EPO levels showed similar responses at 2 000 and 4 000 m. Furthermore, they found that 90 min of hypoxia alone also increases EPO levels (+ 5,0 mU∙mL − 1) 3 h post [30], which is in contrast to our data, as 90 min at 2 000 and 4 000 m caused no significant increase in EPO levels. However, levels nearly reached statistical significance for 4 000 m. In their study, EPO levels Wahl P et al. Responses of Angiogenic Growth … Int J Sports Med

remained elevated even 2 days after [30]. Eckardt et al. showed first significant increases in EPO levels after 114 min (3 000 m) and 84 min (4 000 m) of hypoxia [7]. Our results show that a short term hypoxic exposure combined with exercise can increase EPO levels, however to induce an EPO response with hypoxia exclusively, high altitudes are needed. We can only speculate if these increases lead to an increased angiogenesis. Previous studies showed that EPO is capable of eliciting a proangiogenesis program e. g. in human mesenchymal stem cells. However, much higher concentrations (0–100 U∙mL − 1) were used in this in-vitro study [41]. Just like EPO, VEGF is also regulated by HIF-1 [12]. Therefore, one major stimulus for the synthesis/release of VEGF is a reduced oxygen tension which is existent during exercise or hypoxic conditions. A second potent stimulus is an increased shear stress/ mechanical stretch which occurs during exercise due to an increased blood flow [26]. Furthermore, hypoxia has been associated with increased levels of reactive oxygen species. Attempts to restore normal oxygen levels after episodes of hypoxia result in the generation of various types of reactive oxygen species. Previous studies have shown that in-vivo oxidative stress caused by hypoxia increases VEGF protein expression and may trigger angiogenesis [28]. In a previous study, we have already been able to show that high intensity interval exercise for 90 min at 2 500 m increases VEGF levels [37]. This increase might also be attributed to the higher intensity chosen in this study, as in the present study VEGF significantly increased only after exercise at 4 000 m, but not at 2 000 m. This may indicate that the sole exercise-induced alterations in SO2 in the circulating blood and skeletal muscle fibres, as probably the greatest source of VEGF, do not affect VEGF release at SL or at lower altitudes. It seems that for low intensity exercise, higher altitudes are needed or exercise intensity has to be increased at lower altitudes in order to increase VEGF release. Similar to previous results [37], we found high inter-individual differences in VEGF levels even for BL values. Subjects with low basal VEGF levels tended to show lower absolute increases in VEGF levels, whereas subjects with high basal VEGF levels showed major absolute increases in VEGF in response to hypoxia and exercise at 4 000 m. However, changes in percent showed no major differences between subjects. Except for one subject (slight decrease), all subjects showed increases for this condition. The major source of serum IL-6 levels during exercise is the skeletal muscle [18, 34], as IL-6 is important for the maintenance of glucose homeostasis. But IL-6 also induces the expression of VEGF [5] and is produced by endothelial cells as well [40]. Low intracellular glycogen concentrations stimulate IL-6 production and release [16, 25, 34]. Therefore, the intensity and duration of exercise influence the increase in serum IL-6 levels [24, 25]. As all 3 exercise conditions were carried out at the same relative intensity in the present study, IL-6 levels increased to a similar extent. In contrast to EPO levels, hypoxia had no influence on IL-6 concentrations. Similar to our results, Lundby et al. showed that 60 min of cycling at the same relative intensity at SL and 4 100 m caused similar IL-6 increases [21]. In contrast, the same absolute intensity caused higher increases under hypoxic conditions [21]. Therefore, it can be concluded that the IL-6 response is mainly dependent on the intensity of exercise and not on hypoxia. However, hypoxia alone has been associated with increases in IL-6 levels as well [15, 19, 22].



IL-8, also belonging to the cytokines, induces the migration and proliferation of endothelial cells [1, 33]. However, the exact mechanisms or stimuli leading to an alteration are not known. IL-8 significantly increased only after both hypoxic conditions at 4 000 m, which could lead to the assumption that severe hypoxia is a major stimulus for the release of IL-8. Mucci et al. showed that an exercise induced-hypoxemia is associated with an increase in circulating IL-8 levels [23]. Furthermore, Karakurum et al. showed that hypoxia induces an increase in IL-8 mRNA expression and protein secretion in endothelial cells [17]. In contrast, cycling exercise at normoxic conditions for 60 min and 180 min did not increase circulating IL-8 levels but did increase IL-8 expression in skeletal muscle [1, 4]. IGF-1 has widespread anabolic and insulin-sensitizing effects, and plays a critical role in formation, maintenance, and regeneration of skeletal muscles [13]. The quantification of (total) IGF-1 levels in serum or plasma has yielded inconsistent results, with levels being reported to decline [20], to increase [2, 6, 31], or to remain unchanged [36, 39] after the onset of exercise. Little is known about the effects of hypoxia in combination with exercise on IGF-1 levels [29]. In the present study, none of the interventions caused significant changes in IGF-1 levels. Even after a prolonged exposure (3–4 days) to an altitude of 4 350 m, no change was observed in levels of IGF-1, and IGFBP-3 (which was not measured in the present study) [27]. Therefore, it seems that IGF-1 is insensitive to hypoxia or low intensity exercise under hypoxic conditions.

Conclusion

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In conclusion, the present study showed that short term hypoxic exposure combined with low intensity exercise is able to upregulate angiogenic regulators, which might be beneficial to induce angiogenesis and to improve endurance performance. However, in some cases high altitudes are needed, or it can be speculated that exercise intensity needs to be increased at lower altitudes, in order to cause changes. With respect to limitations of our present study it must be considered that angiogenesis was not directly measured. Nevertheless the findings indicate changes in growth factors and might contribute to a better understanding of the effects of short-term high altitude training interventions.

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