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Keywords:

  • heat-shock protein 70;
  • monocytes;
  • oxidative stress;
  • sleep apnoea

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. References

Obstructive sleep apnoea (OSA) is associated with a variety of nightly stresses, including intermittent hypoxaemia, oxidative stress and sleep fragmentation. Heat-shock proteins (HSPs) are upregulated in response to an array of environmental and metabolic stresses. We hypothesized that the OSA-related stresses would affect the expression of HSP70 in monocytes. Basal (30 min, at 37 °C), heat stress-induced HSP70 (30 min, at 43 °C) and basal tumour necrosis factor-α (TNF-α) were determined by flow cytometry in monocytes of 10 patients with OSA and 10 controls matched by age, gender and body mass index. Oxidative stress was determined by thiobarbituric acid-reactive substances (TBARS) and antioxidant paraoxonase-1 activity. Basal HSP70 expression was 1.8-fold higher in patients with OSA as compared with controls (< 0.0005) and was significantly positively correlated with TBARS (= 0.56, < 0.009). However, induction of HSP70 in response to heat stress treatment was lower by 40% in OSA monocytes as compared with controls (< 0.0003). Furthermore, heat stress-induced HSP70 expression was significantly negatively correlated with basal HSP70 expression independently of apnoea severity (= −0.69, < 0.0006). Also, basal intracellular TNF-α expression was inversely correlated with heat-shock-induced HSP70 (= −0.78, < 0.015) in OSA monocytes but not in controls. In conclusion, basal HSP70 overexpression that is a protective mechanism indicative of disease-associated stress was significantly higher in patients with OSA and was correlated with oxidative stress. On the other hand, in response to a defined heat-stress treatment, the induction of HSP70 was lower in patients with OSA, indicative of a possible maladaptive response to an acute stress. Correlations with oxidative stress and TNF-α further support this conclusion.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. References

Obstructive sleep apnoea (OSA) is characterized by a cyclic occurrence of apnoeic events during sleep that are associated with intermittent hypoxaemia and terminated by brief electroencephalographic and autonomic arousals (Malhotra and White, 2002). A large body of evidence demonstrates an association between OSA and cardiovascular morbidity, particularly with hypertension (Quan and Gersh, 2004; Shamsuzzaman et al., 2003). Although the underlying mechanisms are not fully elucidated, cumulative evidence indicates that oxidative stress (Lavie, 2003, 2005; Suzuki et al., 2006) and activated inflammatory cells play a major role in this association (Dyugovskaya et al., 2002, 2003, 2005a,b).

Heat-shock proteins (HSPs) or stress proteins constitute a large family of proteins that protect cells from a variety of stresses and environmental challenges. Members of the HSP family are divided into subgroups according to their molecular weight, ranging from low 10 to 110 kD, yet the most studied by far is the HSP70 family (Minowada and Welch, 1995). Under normal physiological conditions, HSPs are expressed in a constitutive manner at low levels, and participate in growth and regulatory functions (Kiang and Tsokos, 1998). However, a diverse array of metabolic and environmental insults upregulates their expression. Such upregulated HSP expression was shown in response to heat (King et al., 2002), ischaemia (Mestril et al., 1994; Patel et al., 2004), oxidative stress (Bachelet et al., 2002; Fratelli et al., 2005; Marini et al., 1996), infections, heavy metals and toxins (Kiang and Tsokos, 1998; Westerheide and Morimoto, 2005). Likewise, HSP70 was upregulated in response to acute psychological stress (Isosaki and Nakashima, 1998), exercise (Fehrenbach et al., 2000), catecholamines (Matz et al., 1996), sleep deprivation in mice (Terao et al., 2003), intermittent hypoxia in rats (Zhong et al., 2000) and in patients with OSA (Ghandour et al., 1999; Noguchi et al., 1997). The diverse stresses that induce increased HSP expression make it a universal response to stress and, accordingly, the HSPs are now regarded as stress proteins. Their importance is emphasized by their ubiquitous presence in all organisms and cells, and their highly conserved structure across the evolutionary scale. Thus, upregulated expression of HSPs denotes a universally conserved cellular defence programme (Kiang and Tsokos, 1998; Minowada and Welch, 1995; Westerheide and Morimoto, 2005).

The protective role of HSPs as protein chaperones has been widely documented, particularly that of the HSP70 family that was shown to restore aberrantly misfolded proteins (Westerheide and Morimoto, 2005) and to protect cells from death, by inhibiting multiple apoptotic cell death pathways (Giffard and Yenari, 2004; Steel et al., 2004). Specifically, the HSP70 family of proteins, which ranges from a molecular weight of 66 to 78 kD, includes a constitutive (HSC70) and a highly stress-induced form (HSP70) (Tavaria et al., 1996; Westerheide and Morimoto, 2005). The induced form is present in human monocytes and various tissues, at low levels, and is further increased after stress (Hunter-Lavin et al., 2004).

In recent years, it has become increasingly evident that oxidative stress plays a major role both in the control of HSP synthesis (Ahn and Thiele, 2003; Kalmar and Greensmith, 2009) and in activation of inflammatory pathways via nuclear factor kappa B (NFκB). Conversely, HSP70 was shown to attenuate inflammation via inhibition of NFκB, and downstream gene transcription of cytokines and adhesion molecules (Cahill et al., 1996; Ding et al., 2001; Ianaro et al., 2001; Sun et al., 2005). Such inhibition confers protection from the deleterious effects of tumour necrosis factor-α (TNF-α) and other proinflammatory cytokines (Chen et al., 2005; Sun et al., 2005). Also, HSP70 was shown to regulate the cellular redox status by modulating glutathione-related enzyme activities (Guo et al., 2007).

Overexpression of HSP70 was noted in human atherosclerotic plaques (Berberian et al., 1990; Johnson et al., 1995) and in rat aorta after acute hypertension (Xu et al., 1995). Its protective role from ischaemic stroke is well documented in mice and rats overexpressing HSP70 (Rajdev et al., 2000; Sun et al., 2006). Conversely, in mice deficient in HSP70, infarct size was increased and the outcome after experimental stroke was worsened (Lee et al., 2001). Collectively, these data suggest that HSP70 confers protection against the progression of atherosclerosis (Xu, 2002), ischaemic injury (Sun et al., 2006) and stroke (Yenari et al., 2005).

This study was designed to determine the levels of basal HSP70 expression as well as the levels of the induced HSP70 in response to a well-defined heat stress (HS) stimulus in monocytes of patients with OSA and in controls. Given that the expression of HSP70 is redox-regulated and similarly to OSA is also associated with oxidative stress, expression of basal as well as HS-induced HSP70 was correlated with circulating oxidative stress markers and with each other. Additionally, the involvement of the intracellular levels of the inflammatory cytokine TNF-α and their effects on HS-induced HSP70 expression were also investigated in monocytes.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. References

Subjects

Patients with sleep apnoea and controls were recruited from the patients’ population of the Technion Sleep Medicine Center. Details regarding the diagnostic procedure in our centre have been published before (Lavie et al., 2004). Ten consecutive newly diagnosed patients with OSA were recruited using the following inclusion criteria: a diagnosis of OSA based on whole-night polysomnography finding of apnoea–hypopnoea index (AHI) >10 associated with characteristic symptoms, and age between 20 and 60 years. Patients with sleep apnoea were matched with individuals who were referred to a sleep study because of complaints about snoring and were found to have an AHI ≤10. Patients and controls were matched by gender, age (±5 years) and body mass index (BMI; ±2 units). Exclusion criteria for both groups were: fasting glucose level >126 mg dL−1, diabetes, cardio/cerebrovascular or other chronic disease, sickness during the 2 weeks before the study, and regularly using medications, vitamins or antioxidants. The following sleep apnoea severity measures were determined for all participants: AHI, sleep time spent with oxygen saturation below 90% and oxygen desaturation index (ODI3%) defined as the total number of decreases in oxygen saturation of at least 3% divided by hours of sleep.

The study was approved by the local ethical committee and all participants signed an informed consent before being enrolled.

Reagents and antibodies

Culture medium (RPMI 1640), phosphate-buffered saline, foetal calf serum and bovine serum were from Biological Industries (Kibbutz Beit Haemek, Israel). Saponin, Ficoll-Histopaque-1077 (Sigma, St Louis, MO, USA), HSP70-FITC SPA-810 (StressGen, Victoria, Canada), rat isotypic negative control, rat anti-human TNF-α, CD14-PE, CD14-FITC (Serotec, Kidlington, UK), monoclonal antibodies and human monocyte enrichment cocktail (RosetteSep; StemCell Technologies, Vancouver, Canada) were used.

Cell preparation and HS treatment

Blood was withdrawn under fasting conditions at the end of the sleep study just after awakening at 6:00–6:30 h. Peripheral blood mononuclear cells were recovered by Ficoll-density gradient centrifugation and were heat-stressed at 43 °C for 30 min, or maintained at 37 °C for a comparable time (basal expression). After a 4-h recovery period at 37 °C, cells were permeabilized, stained and analysed for intracellular HSP70 expression. All in all, cells were maintained in culture for a period of 4.5 h until flow cytometry determination. Therefore, basal expression of HSP70 was also determined immediately after harvesting the cells. No differences were observed between the two time points at 37 °C (data not shown).

Determination of intracellular HSP70 and TNF-α expression

Intracellular HSP70 expression was determined by flow cytometry (FACS Calibour; Becton Dickinson, Lincoln Park, NJ, USA) in CD14+ monocytes with and without HS treatment in the presence of 0.1% saponin using dual-staining protocol (Bachelet et al., 1998; Durand et al., 2000). Data are presented as the percentage of positive cells containing HSP70, and by mean fluorescence intensity, which is indicative of the intensity of expression. Data were corrected for background fluorescence with the corresponding isotype controls.

Intracellular TNF-α was detected in non-stimulated CD14+ enriched monocytes by membrane permeabilization with 0.1% saponin. The results are expressed as percentage of CD14+ cells containing TNF-α. In one of the OSA patients, the TNF-α values were lost during the analysis.

Measure of oxidative stress markers

Thiobarbituric acid-reactive substances (TBARS) and the antioxidant enzyme paraoxonase-1 (PON1) were determined as previously described (Lavie et al., 2004).

Statistical analysis

Data are expressed as mean ± SD. Differences between the OSA and control groups were evaluated by t-tests for independent groups for variables normally distributed and by the Mann-Whitney test for variables not normally distributed. Pearson correlations were used to determine the relationships between basal HSP70 and HS-induced HSP70 levels, and between HSP70 levels and oxidative stress markers and TNF-α. Partial correlations were used to determine if these correlations were independent of sleep apnoea severity as determined by AHI. Two-tailed tests were used, and < 0.05 was considered significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. References

Basal and HS-induced HSP70 expression in monocytes

Table 1 presents the demographic, clinical and blood chemistry data of patients with OSA and controls. As expected, patients with OSA had significantly higher AHI (< 0.001), per cent time <90% saturation (< 0.02) and ODI3% (P < 0.01), but there were no significant differences between groups in age, BMI, % men, % current smokers, triglycerides, high-density lipoprotein, glucose, and systolic and diastolic blood pressures. Controls had significantly higher levels of cholesterol (< 0.04) and low-density lipoprotein (< 0.01) than patients with OSA. Nonetheless, as previously noted, the oxidative stress marker TBARS was significantly higher in patients with OSA (< 0.009), while the antioxidant enzyme PON1 was lower (< 0.0004) then controls.

Table 1.   Demographic, sleep and blood chemistry data for control and obstructive sleep apnoea subjects
DemographicsControls (n = 10)OSA (n = 10)P
  1. AHI, apnoea–hypopnoea index; BMI, body mass index; BP, blood pressure; HDL, high-density lipoproteins; HSP, heat-shock protein; LDL, low-density lipoproteins; MFI, mean fluorescence intensity; NS, non-significant; ODI, 3% oxygen desaturation index; PON1, paraoxonase-1; TBARS, thiobarbituric acid-reactive substances; OSA, obstructive sleep apnoea.

Age (years)42.9 ± 9.644.9 ± 9.5NS
Gender (F/M)2/82/8NS
BMI (kg m−2)28.0 ± 2.628.8 ± 2.2NS
AHI (events h−1)6.5 ± 3.728.7 ± 18.9<0.001
ODI 3% (events h−1)3.1 ± 3.119.7 ± 18.4<0.01
% Time <90%0.39 ± 0.814.18 ± 4.81<0.02
Current smokers (%)30%40%NS
Systolic BP119.6 ± 13.0124.5 ± 10.4NS
Diastolic BP78.7 ± 8.277.5 ± 7.3NS
Blood chemistry
 Creatinine (mg dL−1)0.73 ± 0.170.69 ± 0.07NS
 Cholesterol (mg dL−1)201.4 ± 34.8174.8 ± 16.8<0.04
 Triglycerides (mg dL−1)132.8 ± 77.5146.2 ± 78.8NS
 HDL (mg dL−1)37.0 ± 13.733.4 ± 11.9NS
 LDL (mg dL−1)137.9 ± 21.5112.1 ± 18.9<0.01
 Glucose (mg dL−1)89.8 ± 7.392.3 ± 4.3NS
Oxidative stress markers and HSP values
 TBARS13.0 ± 2.617.9 ± 4.6<0.009
 PON199.9 ± 13.776.6 ± 10.5<0.0004
 HSP70 basal (MFI)50.7 ± 12.993.2 ± 29.0<0.0005
 HSP70 inducible (MFI)488.7 ± 112.9292.4 ± 81.2<0.0003

Ninety to hundred per cent of monocytes from both study groups expressed basal HSP70. However, the intensity of its expression in each cell, representing the intracellular content, was significantly higher in patients with OSA as compared with the control group (Table 1; < 0.0005). The individual values are presented in Fig. 1a that also denotes which of the individuals smoked. Of note, obesity was unrelated to basal and HS levels of HSP70.

image

Figure 1.  Expression of intracellular basal and heat-stress induced heat-shock protein (HSP)70 expression in monocytes of 10 controls and 10 patients with obstructive sleep apnoea (OSA). Peripheral blood mononuclear cell suspensions were incubated either at 37 °C for basal expression (a) or at 43 °C for 30 min (b), followed by a 4-h recovery period at 37 °C. The cells were permeabilized by 0.1% saponin, stained and analysed for intracellular HSP70 expression using flow cytometry, as described under Materials and methods section. Data are presented as mean fluorescence intensity (MFI). HS, heat stress.

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In order to evaluate the ability of monocytes of patients with OSA to respond to a given stress, we exposed cells to a controlled HS treatment in vitro. Exposure for 30 min at 43 °C resulted in an impressive induction in the levels of HSP70 compared with values of monocytes kept at 37 °C, both in controls and in patients with OSA as previously reported (Bachelet et al., 1998; Durand et al., 2000). A 9.6-fold increase was noted in the expression of HSP70 in monocytes of control subjects after HS treatment as compared with basal levels. But in patients with OSA, only a threefold increase from basal level was noted. The difference in the induced amount of HSP70 between the groups was statistically significant (Table 1; < 0.0003). The individual data for HS-induced HSP70 expression of each study group are depicted in Fig. 1b, that also denotes which of the individuals smoked.

The correlation between basal and HS-induced HSP70 expression calculated across all subjects shows a strong negative correlation (= −0.69, < 0.0006), as depicted in Fig. 2. Controlling for apnoea severity (using AHI as an index of severity) by partial correlation did not affect the magnitude of the correlation (= −0.65, < 0.002).

image

Figure 2.  Correlation between intracellular basal heat-shock protein (HSP)70 expression and HSP70 expression in response to heat stress (HS) in control and obstructive sleep apnoea (OSA) monocytes. Basal and HS-induced HSP70 expression were determined in monocytes treated at 37 °C and at 43 °C for 30 min respectively, followed by 4 h recovery at 37 °C. Data are presented as mean fluorescence intensity (MFI).

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Basal HSP70 was significantly positively correlated with AHI (r = 0.45; < 0.05), with per cent time below 90% saturation (r = 0.62; < 0.004), and positively but not significantly with ODI3% (r = 0.38; < 0.1). Induced HSP70 expression was significantly negatively correlated with all three variables (AHI: r = −0.57; < 0.01, per cent time <90% saturation r = −0.58; < 0.008; ODI3%: r = −0.45; < 0.05).

Correlations with systemic oxidative stress markers

The oxidative stress marker TBARS and the level of the antioxidant enzyme PON1 were measured in the circulation. These values are presented in Table 1. Basal HSP70 was significantly positively correlated with the oxidative marker TBARS (= 0.56, < 0.009), as illustrated in Fig. 3a, but was not correlated with the antioxidant enzyme PON1 (= −0.21, NS). Controlling for the effect of apnoea severity by partial correlation using AHI as an index of severity reduced the correlation between basal HSP70 and TBARS, but it remained significant (= 0.49, < 0.03). In contrast with basal HSP70, HS-induced HSP70 was significantly negatively correlated with TBARS (= −0.51, < 0.02), as depicted in Fig. 3b, and tended to be positively correlated with PON1 (= 0.39, < 0.09). Controlling for apnoea severity reduced both correlations to non-significant levels (= −0.29, NS; = 0.16, NS respectively). Similar results were obtained when per cent time below 90% arterial oxygen saturation was used, suggesting that these correlations were mediated by apnoea severity.

image

Figure 3.  Correlations between heat-shock protein (HSP)70 expression and lipid peroxidation. (a) Basal HSP70 expression versus thiobarbituric acid-reactive substances (TBARS). (b) Heat stress (HS)-induced HSP70 expression versus TBARS. MFI, mean fluorescence intensity.

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TNF-α-dependent HSP70 expression in monocytes

A negative correlation was previously reported between serum levels of TNF-α and HSP70 levels in monocytes after exposure to HS (Njemini et al., 2002). We therefore correlated basal TNF-α expression of monocytes, prior to exposing them to HS treatment, with their ability to express HSP70 after HS treatment, separately for patients and controls. As illustrated in Fig. 4, a significant negative correlation was noted in patients with OSA (= −0.78, < 0.015), but not in controls (= −0.21, NS). This correlation increased after controlling by partial correlation for apnoea severity as indexed by AHI (= −0.87, < 0.005). Neither patients nor controls showed significant correlations between basal TNF-α and basal HSP70 levels (OSA: = 0.53, NS; controls: = 0.40, NS).

image

Figure 4.  Correlation between basal intracellular tumour necrosis factor (TNF)-α expression before heat-stress (HS) treatment and the expression of heat-shock protein (HSP)70 after heat-stress treatment in monocytes of controls (a) and patients with obstructive sleep apnoea (OSA) (b). Intracellular TNF-α expression was detected in CD14+ monocytes before heat-stress treatment. Data are presented as mean fluorescence intensity (MFI) in CD14+ monocytes, which contain TNF-α. The expression of HSP70 was determined in heat-treated monocytes, at 43 °C for 30 min, followed by 4 h recovery at 37 °C.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. References

To our knowledge, this study provides novel findings on the expression of basal as well as stress-induced HSP70 in monocytes of patients with OSA, regarding the associations with each other, with systemic oxidative stress status and with the expression of TNF-α. Our major findings are summarized as follows:

  • 1
    Monocytes of patients with OSA expressed significantly higher amounts of basal HSP70 than controls. Basal HSP70 was significantly positively correlated with AHI and per cent time below 90% arterial oxygen saturation.
  • 2
    In contrast, HSP70 expression in monocytes of patients with OSA was lower than in controls after treatment with a well-defined HS stimulus. Heat-stress-induced HSP70 was significantly negatively correlated with AHI and per cent time below 90% arterial oxygen saturation and ODI3%.
  • 3
    Expressions of basal and HS-induced HSP70 were inversely correlated.
  • 4
    The systemic oxidative stress marker, TBARS, was positively correlated with basal HSP70 expression independently of OSA severity. The relationships between induced HSP70 and oxidative markers appeared to be mediated by the apnoea severity.
  • 5
    The expression of basal TNF-α in monocytes was inversely correlated with their ability to respond to HS by synthesizing de novo-induced HSP70. This was shown in patients but not in controls.

It should be acknowledged, however, that we did not perform post-continuous positive airway pressure (CPAP) measurements, insofar as it is not clear if the changes observed in HSP70 expression are reversible after CPAP use.

Basal and HS-induced HSP70 expression

A large body of evidence demonstrates that the ubiquitous HS response is induced under a variety of stresses as well as under certain pathological conditions, such as hypertension, ischaemia, stroke, cardiovascular morbidities, diabetes and inflammation (Minowada and Welch, 1995; Rajdev et al., 2000; Sun et al., 2006; Xu, 2002; Xu et al., 1995). In OSA, patients are nightly exposed to intermittent hypoxic and hypercapnic insults, sleep fragmentation, deep-sleep deprivation, surges in sympathetic nerve activity and blood pressure elevations, swings in intrathoracic pressures as well as oxidative stress and systemic inflammation, all of which constitute severe stresses with clinical manifestations. Thus far, only two studies addressed HSP production in OSA monocytes. Noguchi et al. (1997) demonstrated a decrease in basal HSP70 (HSP72) expression of OSA monocytes during sleep that was higher than in controls before sleep. Additionally, in agreement with the present findings, in a preliminary study we reported that basal HSP70 levels were increased in OSA monocytes and, conversely, after HS treatment HSP70 levels were lower than in controls (Ghandour et al., 1999). In addition, plasma and serum levels were also shown to increase in patients with OSA as compared with controls (Hayashi et al., 2006; Lavie, 2003). In accord with these findings and the current study, in rats exposed to intermittent hypoxia, the levels of HSP70 mRNA expression increased progressively in the myocardium along with the duration of intermittent hypoxia. Moreover, the tolerance of the rat heart to ischaemia/reperfusion injury was increased along with the duration of pretreatment with intermittent hypoxia (Zhong et al., 2000).

The HS response is a sequential and ordered genetic response to a variety of metabolic and environmental stressors that results in an immediate induction of sets of genes encoding molecular chaperones, proteases and other proteins essential for protection and recovery from cellular damage (Westerheide and Morimoto, 2005). The HSP70 family, particularly, confers protection against a variety of noxious stresses, such as heat, hypoxia and ischaemia/reperfusion, by restoring conformational changes in aberrantly misfolded proteins and preventing their aggregation. Because HSP70 is expressed in conditions such as hypoxic/oxidative stress (Mestril et al., 1994), it is not surprising that basal HSP70 levels were significantly elevated in patients with OSA. Similarly, basal HSP70 overexpression was found in epithelial and alveolar macrophages of asthmatic patients (Vignola et al., 1995) and in acute respiratory distress syndrome (Bromberg et al., 2005). These studies demonstrated that such increases may prevent cellular and organ damage by denatured or abnormal proteins (Bromberg et al., 2005). It is likely that in OSA as well, aberrant proteins may accumulate due to increased oxidative stress, causing protein modifications and structural changes (Zhan et al., 2005). Additionally, basal HSP70 overexpression could also result from the overall OSA-associated stresses, such as elevated sympathetic nerve activity (Somers et al., 1995), arousals as well as surges in blood pressure (Shamsuzzaman et al., 2003). But apart from preserving protein structure and conformation, functionally, HSP70 was also shown to prevent heat-induced apoptosis (Mosser et al., 1997), to downregulate inflammation by attenuating the expression of inflammatory cytokines (Yoo et al., 2000) and to act as an antioxidant that improves cardiac preservation during oxidative insult (Su et al., 1998).

Unlike the overexpression of basal HSP70 in OSA monocytes, the response to HS treatment, which is an acute and severe stress, was lower as compared with controls. All in all, this response in OSA constituted only 60% of that of controls (Table 1). Moreover, in response to HS, the expression of HSP70 was inversely correlated with basal HSP70 expression. Such an inverse correlation was previously documented in young healthy individuals demonstrating a negative feedback control. Apparently, basal HSP70 regulates the stress-induced HSP70 production through inhibition at the transcriptional level, thus suppressing its own synthesis (Boshoff et al., 2000). In addition, because HSP70 synthesis is redox-regulated, the oxidant/antioxidant status of individuals, which is determined by genetic/environmental factors/disease states, may affect its expression. Therefore, inter-individual differences in basal HSP70 expression could be an important determinant in its potential inducibility under severe stress conditions (Boshoff et al., 2000), as observed in the current study for OSA monocytes exposed to HS.

Oxidative stress and HSP70

HSP70 is regulated by the heat-shock factor-1 (HSF-1) that controls its transcription and consequently its synthesis. Because HSF-1 transcription factor is redox-regulated, the expression of HSP70 largely depends on the cellular redox state and oxidative stress (Ahn and Thiele, 2003; Kalmar and Greensmith, 2009). Oxidative stress was also shown to be a fundamental component of OSA pathology (Lavie et al., 2004). Thus, we correlated oxidative stress markers with HSP70 expression. The lipid peroxidation marker TBARS was positively correlated with the expression of basal and negatively with the HS-induced HSP70 form (Fig. 3). However, controlling apnoea severity by partial correlation analysis revealed that TBARS was independently correlated only with basal HSP70 expression. The significant negative correlation between HS-induced HSP70 and TBARS and the antioxidant enzyme PON1 were reduced to non-significant levels once apnoea severity was controlled, thus suggesting that these relationships were mediated by apnoea severity. Indeed, the levels of the induced HSP70 were significantly negatively correlated with AHI, with per cent time oxygen saturation below 90% and with ODI3%. Although the correlation between basal HSP70 and oxidative stress supports previous results on the role of oxidative stress in mediating the stress proteins response, the independent significant negative correlation between basal and induced forms of HSP suggests that the elevated basal levels of HSP70 negatively control its own synthesis to a subsequent stress, as also demonstrated previously (Boshoff et al., 2000). This is further supported by the lack of correlation between HS-induced HSP and oxidative stress markers independently of apnoea severity.

TNF-α and the HS response

Recent data indicate that the interrelations between HSPs and proinflammatory cytokines are complex in nature. Although HSPs inhibit TNF-α production (Cahill et al., 1996; Ding et al., 2001; Ianaro et al., 2001) through inhibition of NFκB (Chen et al., 2005; Sun et al., 2005), basal levels of serum TNF-α were negatively correlated with the expression of the induced HSP70 after treatment with HS (Njemini et al., 2002). In agreement with this report, basal TNF-α expression (at 37 °C) in monocytes was negatively correlated with their ability to synthesize induced HSP70 in response to HS treatment at 43 °C. This was shown for OSA (Fig. 4b) but not in controls (Fig. 4a). In contrast to the induced HSP70, no correlations were found between basal levels of HSP70 and TNF-α, as previously reported for healthy individuals (Njemini et al., 2002).

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. References

The data presented in this study demonstrate the participation of the cellular stress response in OSA and its association with oxidative stress and inflammatory markers as TNF-α. The mechanism proposed is illustrated in Fig. 5. Intermittent hypoxia acts as the primary challenge in OSA, activates monocytes, increases reactive oxygen species production (Dyugovskaya et al., 2002) and induces overexpression of basal HSP70 that may act as a ‘protective’ mechanism against the intermittent hypoxia and its consequences. However, exposure to a second challenge in the form of HS results in a reduced expression of the induced HSP70 in patients with OSA as compared with controls. Attenuation in the ability of OSA monocytes to synthesize induced HSP70 as compared with controls could result from the increased basal HSP70 through transcriptional inhibition (Boshoff et al., 2000) or through the levels of basal TNF-α (Njemini et al., 2002), as demonstrated previously. Because the induction of HSP70 in response to stress is indicative of the ‘adaptive ability’ to a given harsh stress, it may indicate impairment in adaptive responses in OSA.

image

Figure 5.  A schematic illustration of the proposed interactions between heat-shock proteins (HSPs), reactive oxygen species (ROS) formation and tumour necrosis factor (TNF)-α in monocytes of patients with obstructive sleep apnoea. Increase in function (+); dashed arrows and (−) indicate inhibition of HSP70 induction.

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Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. References

This study was supported by a grant from the US-Israel Binational Science Foundation (BSF) Grant No. 2005265 and by the Committee for Planning and Budgeting of the Council for Higher Education under the framework of the KAMEA programme. The authors would like to thank the staff of The Lloyed Rigler Sleep Apnea Research Laboratory, the staff of the Sleep Medicine Center, Rambam Hospital, Haifa, Israel, and Dr A. Polyakov for their invaluable help.

Conflict of interest

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. References

P. Lavie is a board member and consultant of Itamar Medical Ltd that manufactures equipment for sleep apnoea diagnosis. L. Lavie, L. Dyugovskaya and O. Golan-Shany do not have any conflict of interest.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Conflict of interest
  10. References
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