Obstructive sleep apnea (OSA) is a prevalent disease affecting both children and adults. This sleep breathing disorder, caused by an abnormal increase in upper airway collapsibility, is characterized by recurrent events of airway obstruction, each finishing with the patient's unconscious arousal. These repetitive respiratory disturbances, which could appear more than once every minute in patients with severe OSA, induce increases in sympathetic activation, large negative intrathoracic pressure swings, hypoxia/reoxygenation events and disruption of sleep architecture. Extensive data in the literature prove that, in addition to immediate symptoms such as abnormal diurnal somnolence, OSA increases the mid- and long-term risk of metabolic dysfunctions and cardiovascular diseases 12. Systemic inflammation and endothelial dysfunction triggered by recurrent hypoxia/reoxygenation have proved to be relevant processes as regards determining the consequences of OSA 345. The susceptibility of distinct OSA patients to these consequences would, however, depend on the effectiveness of the individual homeostatic response to the challenges posed by the syndrome.

The main physiological response to the intermittent hypoxia and increased inspiratory efforts characteristic of OSA consists of the upregulation of well known signalling cascades that counteract oxidative stress and inflammation. Interestingly, data recently published on diseases distinct from OSA suggest that bone marrow-derived mesenchymal stem cells (MSC) circulating in peripheral blood could also contribute to the homeostatic response in OSA. Indeed, it has been shown that these stem cells play anti-inflammatory, anti-oxidative stress and endothelium-repairing roles via paracrine secretion of soluble factors 67. The recent finding that the number of circulating MSC was acutely increased in a rat model of recurrent obstructive apneas 8 adds support to the hypothesis that MSC could be involved in the response to the injurious stimuli in OSA. In order to shed light on the potential role of MSC in this sleep breathing disorder, this study sought to investigate whether the blood serum of rats subjected to recurrent obstructive apneas mimicking OSA modulates basic mechanisms in the response to inflammation and endothelial damage: MSC migration and adhesion to endothelial cells and endothelial wound healing.

The serum of apneic rats increased the motility of MSC. As shown by the examples of transwell membrane images in Figure 2 (top), more cells migrated in the Apnea/Control transwell than in the Control/Control transwell. The bottom panel of this figure shows that the chemotaxis index for the serum of the apneic rats (2.20 ± 0.58) was significantly higher than that for the control serum (1.00 ± 0.26; p < 0.05). In contrast, the increase observed in the chemokinesis index when comparing the serum from apneic rats and the control serum was not statistically significant.

MSC exhibited significantly more adhesion to the monolayer of cultured endothelial cells when incubated in apneic rat serum as compared to control rat serum. Figure 3 (top) illustrates that more labelled-MSC adhered to the endothelial monolayer in the case of the apneic serum. This figure's bottom panel shows that the MSC-endothelial adhesion index was significantly higher in apneic serum than in control serum: 1.75 ± 0.14 and 1.00 ± 0.06, respectively (p < 0.01).

As shown in Figure 4, endothelial wound healing was significantly increased when the injured monolayer was cultured with serum from rats subjected to recurrent apneas as compared with culture in control serum (2.01 ± 0.24 vs 1.00 ± 0.34; p < 0.05). Fig. 4 also shows that preconditioning the control and apneic rat sera with MSC resulted in an increase in the endothelial wound closure index (3.11 ± 0.39 and 2.99 ± 0.41, respectively; p < 0.01 in both cases).

In this work we have assessed whether MSC could play a role in the physiological response to the injurious stimuli that characterize OSA and cause the cardiovascular consequences of this sleep breathing disorder. We focused our attention on basic mechanisms that potentially contribute to the repair of endothelial damage. The results obtained in this acute animal model study show that short-term recurrent obstructive apneas mimicking OSA triggered an early activation of MSC: specifically, an increase in the mobility of these stem cells and in their adhesion to endothelial cells. Moreover, it was also found that the serum of apneic rats improved endothelial wound healing and that MSC could contribute to this enhanced repair.

The potential protective or therapeutic role of MSC in various human diseases 10 and animal models has been extensively investigated for non-respiratory diseases 111213, as well as for several respiratory pathologies 14151617. It has been proven that, in addition to their capacity for tissue regeneration by homing at the injured tissue and differentiating onto damaged cell phenotypes, MSC secrete soluble mediators which can immuno-modulate inflammation and anti-oxidative cascades improving repair of vascular endothelium 67. Only a very few studies, however, have focused on the potential role of stem cells in OSA, although there have been recent reports of the detection of circulating endothelial progenitor cells in patients 181920 and circulating MSC in a rat model 8. Accordingly, to our knowledge this is the first work studying how the stimuli characterizing OSA activate MSC responses and thus potentially contribute to the response to inflammation and the enhancement of vascular repair.

The methods used in the present study consisted of an in vivo rat model to obtain serum from control rats and from rats subjected to apneas and in vitro techniques to compare the effects of these sera on MSC and endothelial cell properties. One advantage of the animal model used in this study is that by non-invasively applying recurrent obstructive apneas in healthy animals the rats were subjected to periodic stimuli very similar (in magnitude, duration and frequency) to the ones experienced by patients with OSA: strenuous breathing efforts against a closed airway and hypoxemic events 2122. This acute model allowed us to study the early effects of these injurious stimuli while avoiding other confounding factors found in OSA patients that also cause inflammation and endothelial dysfunction (metabolic syndrome, obesity, hypertension, etc) 12345. The methodology employed in this short-term study may be also useful to investigate the chronic effects of long-term recurrent obstructive apneas in a chronic animal model 21. Specifically, to investigate potential adaptation mechanisms in response to a long-term challenge as in OSA patients and to study the role of endothelial progenitor cells in vascular repair. The in vitro methodology used to investigate the effects of control and apneic rat sera on MSC and endothelial cells is widely reported in the literature. Indeed, the transwell setting for assessing chemotaxis and chemokinesis has previously been used to study MSC migration in response to different biochemical stimuli 23 and the fluorescent-staining method employed for assessing the adhesion of MSC to endothelial cell monolayers has been used in both in vivo and in vitro studies 24. Moreover, the endothelial wound healing assay used in the present study is frequently encountered in research on endothelial repair mechanisms in vitro 2526. Interestingly, these in vitro methods are readily applicable in future studies to investigate whether the serum of OSA patients, before and after CPAP therapy, activates MSC and enhances endothelial repair when compared with serum from healthy controls.

Our experimental setting was designed to assess the early effects on MSC and endothelial cells induced by the serum of rats subjected to recurrent airway obstructions. Accordingly, the changes observed in migration, adhesion and wound healing were exclusively caused by the soluble factors released into the animals' serum as a result of subjecting them to the breathing stimulus mimicking OSA. This approach allowed us to identify the effects of soluble factors in blood from the effects of the other potentially important stimuli also experienced by these cells in OSA patients, such as intermittent hypoxia due to the recurrent changes in arterial oxygen saturation. The specific effects of intermittent hypoxia on cultured MSC and endothelial cells in OSA remain mostly unknown, given the technical difficulty of adequately applying, at the cell level, the high-rate of oxygen pressure changes mimicking OSA 27. As regards the design of this study, it should also be pointed out that the experiments to assess the effects of rat serum on MSC-endothelial adhesion and on endothelial wound healing did not allow us to differentiate between the specific serum effects on each type of cell separately. This limitation does not, however, affect the aim of this study as these two types of cells are simultaneously exposed to the same blood serum in patients.

The blood serum of rats acutely subjected to recurrent obstructive apneas was chemoattractant for MSC (Figure 2). This could explain the finding in a previous study on a similar rat model that the number of MSC circulating in peripheral blood increased in apneic rats when compared with controls 8. Accordingly, the soluble factors released into the serum as a rat's physiological response to the obstructive stimulus would be sensed by the MSC in the bone marrow (and in other tissues which are also reservoirs of these cells) and would induce their mobilization from their original niche to the bloodstream. In fact, this interpretation is consistent with the generally accepted hypothesis that a gradient of soluble factors secreted into the bloodstream is one of the mechanisms by which injured tissue induces the mobilization and recruitment of MSC. Although the exact mechanism triggering MSC mobilization is not known in detail, there is evidence to suggest that various growth factors and inflammatory cytokines characterizing inflammation in OSA (e.g. IL1-β and TNF-α) contribute to MSC migration 23.

Once released into the blood, circulating MSC migrate and home at the injured organ via a dynamic process similar to that of neutrophils in response to inflammation 28: transient adhesion to endothelial cells, rolling, firm adhesion to the endothelium and transmigration into the tissues where MSC participate in the repair of the damaged tissue 29. The enhancement observed in the adhesion of MSC on to endothelial cells (Figure 3) when cultured with apneic serum could be caused by the higher levels of pro-inflammatory cytokines in apneic serum. Effectively, IL1-β and TNF-α, which increase in the serum of animals in OSA models and in that of patients with this sleep disorder, have proved to increase the adhesion of MSC to cardiac endothelial cells both in vivo and in vitro 24. Increased MSC-endothelial adhesion, together with the increase in MSC migration capacity (Figure 2) observed when mesenchymal stem cells were acutely exposed to the serum of rats subjected to recurrent apneas, suggests that OSA could facilitate a crucial step in tissue repair: the homing of MSC on the endothelium and injured sub-endothelial tissues.

A remarkable finding in this study was that, when compared with controls, the serum of rats subjected to recurrent obstructive apneas enhanced the wound closure of endothelial cell monolayers (Figure 4). Accordingly, the injurious stimuli in OSA (namely, intermittent hypoxia and strenuous breathing) would also trigger a response to enhance the repair of the injured endothelium. Whereas most of the published literature on vascular dysfunction in OSA is focused on the deleterious effects caused by blood soluble factors on the endothelium, little is known about the potential repair mechanisms triggered by injurious OSA stimuli. As endothelial wound healing is strongly enhanced by vascular endothelial growth factor (VEGF) 26 and the stimulus of OSA promotes an increase in blood VEGF 18, the observed increase in wound closure when culturing endothelial cells with apneic serum could be attributed to VEGF. In this respect, it is interesting to note that one potential source of the VEGF in the blood of apneic rats could be the circulating MSC induced by recurrent obstructive apneas 8. Indeed, it has been shown that MSC express VEGF in response to a hypoxic stimulus 25. It should be noted, however, that, regardless of the specific potential role of VEGF, MSC per se secrete factors that promote endothelial repair 30. In line with this interpretation, we found that acutely preconditioning rat serum with MSC was sufficient to increase wound closure in both apneic and control sera (Figure 4). The fact that this increase was higher when the sera were preconditioned with MSC than when using apneic serum with no MSC preconditioning could be explained by the fact that the concentration of MSC in MSC-preconditioned sera was lower than in the circulating blood of apneic rats 8.

This animal model study suggests that bone marrow-derived MSC could play a role in the physiological response to counterbalance the pro-inflammatory, oxidative stress and endothelial dysfunction mechanisms that lead to the middle- and long-term consequences of OSA.

The authors declare that they have no competing interests.

The conception and scientific direction of this work was undertaken by RF. Animal experimentation was carried out by AC and TS. Data processing and statistical analysis was undertaken by AC, TS, and JMM. AC, MR, TS, JMM, and DN participated in the discussion of the results and contributed to the manuscript draft. All authors read and gave critical input to this manuscript.