Effects of Hypobaric Hypoxia on Endothelial Function and Adiponectin Levels in Airforce Aviators

Abstract

Background:

Hypobaric hypoxia (HH) increases the risk of high altitude-related illnesses (HARI). The pathophysiological mechanism(s) involved are still partially unknown. Altered vascular reactivity as consequence of endothelial dysfunction during HH might play a role in this phenomenon. Adiponectin exerts protective effect on cardiovascular system since it modulates NO release, antagonizing endothelial dysfunction. Aims of this study, performed in a selected population of airforce aviators, were (1) to investigate whether exposure to acute HH might be associated with endothelial dysfunction and (2) to evaluate whether adiponectin might be involved in modulating this phenomenon.

Methods:

Twenty aviators were exposed to acute HH in a hypobaric chamber by simulating altitude of 8000 and then 6000 m for 2 hours. Vascular reactivity was evaluated by the EndoPAT test immediately before and after the HH; salivary and blood adiponectin levels were measured.

Results:

EndoPAT performed immediately after HH divided pilots in two groups: 12 pilots with preserved vascular reactivity and 8 pilots with reduction of vascular reactivity, indicating that HH exposure might cause endothelial dysfunction. Salivary and blood adiponectin levels increased post-HH in a time-dependent manner in all aviators, but the significant increase was observed only in those with preserved vascular reactivity suggesting that HH stimulated release of adiponectin that, in turn, by exerting a protective effect, might reduce endothelial dysfunction.

Conclusions:

Acute HH may cause endothelial dysfunction due, at least in part, to reduced release of adiponectin. This phenomenon might be involved in pathophysiology of HARI.

Introduction

High altitude-related illnesses (HARI) include several diseases such as high altitude cerebral or pulmonary edema and acute mountain sickness, due to acute exposure to altitude, where a condition of hypobaric hypoxia (HH) exists (Bhagi et al., 2014). This condition seems to be due to fast ascent and, at the same time, to lack of previous acclimatization (Schneider et al., 2002; Richalet et al., 2012). The pathophysiological mechanism(s) involved are still partially unknown, but several evidences have indicated that one of the hypothetical mechanisms might be the endothelial dysfunction induced by HH (Berger et al., 2005; Ali et al., 2012). Although it has been demonstrated that patients developing HARI and, specifically, pulmonary edema have impairment of vascular reactivity as well as low levels of nitric oxide as compared with healthy controls, the effects of HH on vascular reactivity are still partially unknown (Souvannakitti et al., 2017).

In physiological conditions, vascular reactivity is modulated by endothelial cells that produce and release several substances to modulate vascular tone such as nitric oxide (Jiang et al., 2016). It has been demonstrated that many chemical mediators may be involved in modulating the endothelial-dependent vascular reactivity, including several adipocyte-derived substances known as adipocytokines (Berger et al., 2005). Many adipocytokines have been demonstrated to have deleterious effects on endothelial cells (De Rosa et al., 2009; Cirillo et al., 2010, 2012), finally leading to a dysfunctional endothelium and to altered vascular reactivity. On the contrary, the adipocytokine adiponectin seems to exert protective effects on cardiovascular system (Berger et al., 2005), probably by modulating NO release by endothelial cells (Chen et al., 2003). Thus, it has been suggested that levels of adiponectin might be considered as biomarker of endothelial function/dysfunction.

Aircraft military pilots represent a selected population of “healthy individuals,” potentially free of classical cardiovascular risk factors, who are routinely and repeatedly exposed to hypoxia awareness by hypobaric chamber training (Johnston et al., 2012). Thus, they represent an “ideal” experimental model to study the effects of acute HH such as that observed by fast ascending to altitude. In this study, in these selected individuals, first we have investigated the effects of acute HH on endothelial function, then we have studied whether adiponectin might be involved in modulating this phenomenon and, finally, we have evaluated the potential relationship between physical activity and endothelial function after HH.

Methods

Twenty male pilots were enrolled in the study (age 32 ± 5 years, waist circumference 89 ± 9 cm, height 177 ± 9 cm, body mass index 25 ± 8, negative cardiovascular clinical history, and drugs free), which was conducted with respect to the principles of the Declaration of Helsinki and approved in advance by the Italian Air Force Aerospace Medicine Department Review Board (M_D ARM0017/2016). Subjects were volunteers from our institution who signed a written informed consent before participating in the study. Confirmation of the health status was undertaken considering that they underwent health checkup every 6 months.

Experimental setup

The hypobaric chamber was a model developed by AMST Systemtechnik GmbH–Ranshofen (Austria). It was operated according to the instructions released by the factory and in compliance with the safety requirements regarding pressure-driven systems laid down by the European Union and Italy. Of the four training protocols described (Morgagni et al., 2010), type 1 is the most commonly used and it was chosen in our study. It normally includes the following items: (1) ascent to 25,000 ft (7620 m) for acute hypoxia demonstration; (2) descent to 18,000 ft (5486 m) for slow hypoxia demonstration; and (3) return to ground level.

In our practice, the hypoxia demonstrations take 2–5 minutes at highest altitude and 12–15 minutes at lowest altitude with blood saturation close to 55%–60% in both exposures. The usual safety procedures, including a preliminary sinus check and a 30-minute period of preoxygenation, were carried out to minimize the risk of barotrauma and decompression sickness. Each exposure took about 90 minutes, during which all volunteers received pure oxygen except for the periods of hypoxia.

At the desired altitude, the mask was taken off, and participants were forced to breathe ambient air while performing various psychomotor activities until symptoms of hypoxia occurred. Thereafter, they refitted the mask, switching to an emergency supply of 100% oxygen: this was the common challenge for the cognitively impaired participant. Pilots were monitored with cardiopulmonary telemetric (Fukuta Denshi), including continuous electrocardiogram and blood oxygen saturation [SpO2] monitoring during the test.

Endothelial function assessment

The endothelial function was assessed by the EndoPAT2000 system (Itamar Medical Ltd., Caesarea, Israel) that consists of a fingertip plethysmograph. The device includes two finger probes, each placed on a fingertip on each hand. The probes are connected to a computer and permit the parallel evaluation of vascular reactivity. The probe consists of a rigid external cap around an air-filled chamber with a sensor. When the chamber is filled with air, a uniform pressure is provided, which prevents the venoarteriolar vasoconstrictive reflex. The probe detects changes in volume in relation to the arterial pulsation. A cuff is placed on the arm in which the measurement was performed. Measurements obtained by the other probe serve as a control. Each measurement consists of three phases: baseline, occlusion, and reactive hyperemia.

For baseline measurements, the probe is set to inflate to 10 mmHg below diastolic pressure. For occlusion, the test arm is occluded to suprasystolic pressure for 5 minutes (Faizi et al., 2009). The subsequent increase in blood flow leads to a flow-mediated dilatation, manifesting as reactive hyperemia, which is measured by the device as an increase in the pulse-signal amplitude. The increase is directly related to the endothelial dysfunction, which is systemic. Therefore, a direct relationship has been demonstrated between the peripheral and coronary circulations (Kuvin et al., 2003; Bonetti et al., 2004; Nohria et al., 2006).

All pilots were asked to avoid food for 8 hours before HH and evaluation of vascular reactivity, whereas smoking and drugs were forbidden for 24 hours before performing each test. They were evaluated by EndoPAT immediately before and after HH. Results obtained by the EndoPAT software were expressed as the natural logarithm of the reactive hyperemia index (LnRHI) that represents the logarithm of the post-/preocclusion ratio value.

Salivary and blood levels of adiponectin

Pilots were recommended not to smoke, eat, or tooth brush for the last 60 minutes, as shown in Thanakun et al. (2014). Saliva samples were drawn at baseline, immediately after HH (0 hour) at 3, 6, and 24 hours. Similarly, blood samples were obtained at baseline and at 24 hours. The assessment of adiponectin was made by a specific ELISA kit (Human Adiponectin ELISA Kit ab108786 from Abcam^®, United Kingdom tested for cell culture supernatant, saliva, milk, urine, serum, and plasma. Sensitivity = 0.7 ng/mL, range 0.781–50 ng/mL), as described by the manufacturer.

Evaluation of physical activity

Since it has been described that endothelial function is significantly improved in individuals who perform regular physical activity, that means “any bodily movement due to contraction of the skeletal musculature and associated with the consumption of energy” (Di Francescomarino et al., 2009), we have evaluated the relationship between endothelial function, adiponectin levels, and the cardiorespiratory fitness in the aviators enrolled in the study. Specifically, all pilots were asked to perform a 2 km walking test to assess the cardiorespiratory fitness as previously described (Oja et al., 1991).

Statistical analysis

Variable distribution was assessed by Shapiro–Wilk's test. Data were expressed as mean ± SD or median (interquartile range) for parametric and nonparametric distribution, respectively. Pre- and post-HH paired differences were evaluated by the t-test. Paired differences were expressed as mean (95% confidence interval). Correlations were calculated employing Pearson (r) and Spearman (ρ) correlation test for parametric and nonparametric values, respectively. p-Values ≤0.05 were considered significant in the bilateral situation. All the statistical analyses were performed with SPSS^® 24.0 (IBM, IL).

Results

No biochemical characteristics were taken since military pilots are considered healthy by default and, it was not permitted to access to their data for military security reasons. During the Hyperbaric Chamber Tender, no serious adverse events took place. Importantly, heart and respiratory-rate assessment, electrocardiogram and O₂ arterial saturation showed no data of relevance to acute deterioration on health.

At baseline, EndoPAT evaluation of endothelial function in the whole pilot group showed a normal value of 0.67 ± 0.02 LnRHI. Interestingly, at the end of HH, pilots could be divided in two groups because endothelial function increased in 12 pilots (LnRHI = 0.92 ± 0.03; Fig. 1A) and decreased in other 8 (LnRHI = 0.49 ± 0.03; Fig. 1B), indicating that HH caused a different vascular response.

FIG. 1.

EndoPAT evaluation of endothelial function. (A) Immediately after HH, 12 pilots showed increased endothelial function expressed as LnHRI value measured by EndoPAT (*p < 0.005 vs. base, data are expressed as mean ± SD). (B) On the contrary, other eight pilots showed reduced endothelial function (*p < 0.005 vs. base, data are expressed as mean ± SD), indicating a divergent vascular response to HH. HH, hypobaric hypoxia; LnHRI, natural logarithm of reactive hyperemia index; SD, standard deviation.

Adiponectin levels measured in the saliva increased in a time-dependent manner. Interestingly, the time-dependent increase of adiponectin levels was more pronounced in pilots belonging to the group with higher value of LnRHI, and with increased endothelial function (Fig. 2). On the contrary, adiponectin levels did not significantly increased in pilots in whom value of LnRHI decreased after HH exposure (Fig. 2). Similarly, a significant increase of plasma adiponectin levels was observed at 24 hours in those pilots with increased endothelial function after HH (Fig. 3).

FIG. 2.

Salivary adiponectin levels. Adiponectin levels measured in the saliva increased in a time-dependent manner. This increase of adiponectin levels was more pronounced in pilots who had increased endothelial function expressed as LnRHI value >0.51 as compared with pilots in whom the endothelial function was decreased after HH exposure expressed as LnRHI value <0.51. *0.005 versus base; ^#p < 0.005 versus LnRHI <0.51 at the same time point. Data are expressed as mean.

FIG. 3.

Plasma adiponectin levels. Adiponectin levels measured at 24 hours in the plasma increased in pilots who had increased endothelial function expressed as LnRHI value >0.51 as compared with pilots in whom the endothelial function was decreased after HH exposure expressed as LnRHI value <0.51. *0.005 versus base; ^#p < 0.005 versus LnRHI <0.51 at the same time point. Data are expressed as mean.

Since it has been shown that endothelial function improves with regular physical activity, relationship among endothelial function, adiponectin levels, and cardiorespiratory performance was studied. As shown in Figure 4, we found that, those pilots who showed a good performance at 2-km walking test had elevated salivary levels of adiponectin and increased endothelial function after HH. Indeed, these pilots followed a fitness program based on a daily aerobic activity.

FIG. 4.

Correlation between cardiorespiratory performance, salivary adiponectin, and endothelial function. In pilots who showed a good performance at 2-km walking test (VO2max), elevated salivary levels of adiponectin and increased endothelial function after HH were observed. On the contrary, pilots with a poor performance had lower levels of adiponectin as well as reduced endothelial function.

Discussion

HH is a pathophysiological condition characterized by low air pressure associated with reduced oxygen levels (Coppel et al., 2015). This condition may be observed in cases of acute exposure to altitude with fast ascent and lack of previous acclimatization, or during aviation and space activities (Temme et al., 2010; Johansson et al., 2014). In fact, several reports have pointed their attention on the impact of HH on different aspects of flight operations such as cognitive response (Neuhaus and Hinkelbein, 2014), flight performance (Temme et al., 2010), or heart rate (Vigo et al., 2010). However, the hypothetical relationship between this acute condition and vascular reactivity has been poorly investigated obtaining contrasting results (Boos et al., 2012; Iglesias et al., 2015).

In this pathophysiological context, endothelial function has been studied without obtaining an univocal point of view. Some studies have demonstrated that endothelial function assessed by flow-mediated vasodilation was not altered in healthy subjects exposed to acute HH (Iglesias et al., 2015), and have indicated that acute HH did not cause any change in arterial stiffness (Boos et al., 2012). On the contrary, other experimental and clinical studies have indicated that HH might be associated with endothelial dysfunction (Boos et al., 2012; Johansson et al., 2014) and cardiovascular events (Hurtado et al., 2012; Stub et al., 2015). Specifically, in rabbits exposed to HH but resistant to hypoxia, upregulation of NO synthase and increase of NO levels were measured, whereas in rabbits not resistant to hypoxia, oxidative stress and elevated plasma levels of several markers of endothelial dysfunction were measured (Kaur et al., 2005; Fradette et al., 2007). Moreover, Natah et al. (2009) showed the correlation between hypoxia and vasogenic cerebral edema in rats. Finally, Kaur et al. (2005) recorded suffering Purkinje cells following to HH along with motor impairment in rats: endothelial NO synthase (eNOS) boosted acutely from 3 to 24 hours, suggesting the endothelial action.

These experimental observations were confirmed by some clinical studies (Johansson et al., 2014; Iglesias et al., 2015). In fact, Johansson et al. (2014) registered a reduction in reactive hyperemia index after acute HH and Iglesias et al. (2015) recorded a peak in endothelial dysfunction markers in 10 healthy subjects exposed to HH for 4 hours. In line with those contrasting results, we have observed that HH can be associated with a divergent vascular response in a selected population of military pilots who were acutely exposed to HH since they underwent to hypobaric chamber training. In fact, endothelial function post-HH showed that a group of eight pilots had abnormal/reduced endothelial function (LnRHI <0.51). On the contrary, a second group showed increase of endothelial function. In this study, endothelial function has been evaluated by EndoPAT.

Flow variation recorded by EndoPAT is indicative of microvascular functions and resistance of arteries. Some studies have shown that the endothelial function evaluated by EndoPAT method correlates with microvascular function of the coronaries in patients in early atherosclerosis stages (Bonetti et al., 2004) and is a predictor of vascular events (Rubinshtein et al., 2010). EndoPAT is easy to perform and operator independent. However, digital probes are expensive and reproducibility is not uniform, and predictive cardiovascular value is not well established (Rubinshtein et al., 2010; Poredos and Jezovnik, 2013; Matsuzawa et al., 2015). Based on other studies, evaluation of endothelial function by EndoPAT seems to be better for younger individuals in whom microvascular function predicts early cardiovascular events (Flammer et al., 2012).

Another finding of this study was that HH caused the time-dependent increase of the adipocytokine adiponectin levels measured in saliva and in blood collected at different time points. Interestingly, the adiponectin levels were significantly elevated only in those pilots in whom endothelial function post-HH was increased, suggesting that this adipocytokine might exert a protective action on endothelium.

A growing body of evidence indicates that endothelial function correlates with adiponectin levels (Wang and Scherer, 2008; Rojas et al., 2014). Preclinical studies clearly showed that administration of adiponectin increases NO production in aortic endothelial cells (Chen et al., 2003). Moreover, adiponectin-deficiency impairs endothelium-dependent vasodilation and NO production in animals (Ouchi et al., 2003; Ouedraogo et al., 2007). These effects are the result of several molecular mechanisms that involve eNOS expression, phosphatidylinositol 3-kinase-dependent phosphorylation, and adenosine monophosphate activated protein kinase activity (Wang and Scherer, 2008).

Several reports clearly indicated that regular physical exercise, which is known to promote a favorable cardiovascular state even in the elderly (Siasos et al., 2013), may improve endothelial function through several mechanisms (Di Francescomarino et al., 2009). It has been suggested that one of these mechanisms might be the increase of plasma adiponectin levels (Simpson and Singh, 2008). Despite controversial data actually available on the relationship between physical activity, adiponectin levels, and endothelial function (Kozakova et al., 2013; Nurnazahiah et al., 2016), several evidences suggest that exercise training improves endothelial function through adiponectin-dependent and -independent pathways, especially in some high-risk populations, such as diabetic and obese patients (Lee et al., 2011; Jeon et al., 2013). In line with those observations, we found that pilots who performed physical exercise, witnessed by a good performance in the 2 km walking test, had higher adiponectin levels and enhanced endothelial function after HH as compared with those who did not follow a fitness program. Thus, it might be speculated that exercise exerts a protective effect after HH since it might play a favorable effect in modulating adiponectin-mediated endothelial response.

Some limitations should be considered for this study: (1) endothelial dysfunction may be evaluated by measuring plasma concentration of several markers such as soluble adhesion molecules, nitric oxide, or others. However, in our study we have evaluated only adiponectin levels and these levels have been measured mainly in saliva. We were “obliged” to measure time course only of salivary adiponectin concentrations since it was extremely complicated to perform a time curve by obtaining blood samples from the selected study population represented by military pilots. (2) Moreover, another potential limitation might be represented by the small population of the study, but this limitation goes with the first one. (3) Finally, the described fitness test represents a very indirect method for assessing the VO2max; thus, it may represent another potential limitation.

In conclusion, this study indicates that acute HH might cause divergent vascular response due to a variable endothelial function and that adiponectin, a “cardioprotective” adipocytokine, might play a pivotal role in modulating this phenomenon. Specifically, our results permit to speculate that exposure to HH, an event that occurs in acute exposure to altitude with fast ascent and lack of previous acclimatization, induces activation of a cardioprotective mechanism by increasing the release of adiponectin. This adipocytokine, in turn, modulates endothelial function/response to HH, probably leading to vasorelaxation. In those patients in whom a less prompt adiponectin release occurs, HH causes an abnormal endothelial response. Interestingly, this phenomenon seems significantly mitigated by performing physical activity.

Taken together, these results highlight the importance of exercise before exposure to all those conditions that are potentially associated with HH such as exposure to altitude, especially while fast ascending.

Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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