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Obstructive sleep apnea (OSA) is associated with increased cardiovascular morbidity and mortality. Free radicals and adhesion molecules were implicated in the pathogenesis of atherosclerosis leading to cardiovascular disorders. Therefore, we investigated the link between CD15, CD11c, CD11b, and CD64 expression on leukocytes and their ability to generate reactive oxygen species (ROS) in patients with OSA and control volunteers. We also studied the effects of hypoxia in vitro on monocytes from control subjects and the ability of monocytes from both groups to adhere to human endothelial cells in culture. The effect of nasal continuous positive airway pressure (nCPAP) treatment was studied as well. We found that OSA was associated with increased expression of adhesion molecules CD15 and CD11c on monocytes, increased adherence of monocytes in culture to human endothelial cells, increased intracellular ROS production in some monocyte and granulocyte subpopulations, and upregulation of CD15 expression due to hypoxia in vitro in monocytes of control subjects. Furthermore, nCPAP treatment was associated with downregulation of CD15 and CD11c monocyte expression and decreased basal ROS production in CD11c+ monocytes. Monocyte adherence to endothelial cells decreased as well. Our findings provide one of the possible mechanisms for explaining the high rate of cardiovascular morbidity in patients with sleep apnea.
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Keywords: adhesion molecules; monocytes; granulocytes; reactive oxygen species; nasal continuous positive airway pressure
Obstructive sleep apnea (OSA) syndrome has emerged in recent years as an important risk factor for cardiovascular morbidity. Association has been reported between sleep apnea and systemic hypertension (1, 2), pulmonary hypertension (3), ischemic heart disease (4, 5), and stroke (6, 7). Although the underlying mechanisms have not been fully elucidated, so far attempts to explain this relationship have mainly focused on hypoxia-related augmented sympathetic activation (8, 9) or on modification of basic cardiovascular physiologic regulatory mechanisms (10, 11).
Several lines of evidence implicate oxidative stress in the
pathogenesis of cardiovascular morbidity. Inflammatory leukocytes are one of the well-characterized pathways of free
radical formation. Leukocyte accumulation and adhesion via
appropriate receptors on the endothelium and initiation of
leukocyte/endothelial cell interactions may critically impair
endothelial cell function and propagate atherogenic processes
(12). Recent reports on endothelial dysfunction in OSA suggested that facilitated atherogenic processes may play a role in
the pathophysiology of cardiovascular morbidity in this syndrome. Thus, elevated plasma levels of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell
adhesion molecule-1 (VCAM-1), L-selectin, and sE-selectin, suggesting an activated endothelium, were shown in sleep apnea patients (13, 14). Also, the inflammatory cytokine tumor
necrosis factor-alpha (TNF-
) that was shown to greatly increase ICAM-1 and VCAM-1 synthesis, was increased in patients with OSA compared with normal control subjects (15).
Sleep apnea patients were reported to have higher levels of
platelet aggregation and activation, as measured by P-selectin
expression, than when treated with nasal continuous positive
airway pressure (nCPAP) (16). More recently, increased neutrophils' superoxide production was reported in OSA; this was
fully reversed by nCPAP therapy (17).
Because hypoxia per se enhances the production of adhesion molecules in a variety of cells, including endothelium and leukocytes (18), it is possible that the repetitive nocturnal hypoxemia in OSA may initiate atherogenic processes, which eventually lead to cardiovascular morbidity. In the present study, we characterized the cellular phenotypes of peripheral whole blood monocytes (PBMs) and granulocytes in patients with OSA in comparison with normal control subjects. These cellular phenotypes, which included identification of CD64, CD11c, CD11b, and CD15 positive cells, were correlated with oxidative metabolism and respiratory burst activity of monocytes and granulocytes. In addition, we compared the ability of monocytes of patients with OSA and normal control subjects to adhere to human coronary artery endothelial cells (HCAECs) or to human umbilical vein endothelial cells (HUVECs) in vitro. We also studied the effects of hypoxic conditions in vitro on the expression of CD15 by monocytes obtained from control subjects. Besides, we provide data demonstrating the effects of nCPAP treatment on some of these parameters. We used whole blood to minimize procedure-related changes in surface receptor expression (19).
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Patients and Control Subjects
Five milliliters of blood were withdrawn in the morning after overnight fasting from a total of 26 moderate-to-severe patients with OSA and 31 healthy volunteers. Of these, in 18 patients with OSA and 26 control subjects we studied the expression of adhesion molecules and oxidative metabolism in monocytes. Eight patients with OSA were studied more than 6 mo after the initiation of nCPAP treatment for two consecutive days, with and without nCPAP. Eleven normal control subjects participated only in the study on the effects of hypoxia in vitro on whole blood CD15 expression in monocytes. The clinical and demographic data of each of the groups are summarized in Table 1. Twelve of the 26 control subjects who participated in the adhesion molecules and oxidative metabolism experiments were recorded in the sleep laboratory and were found to be free of any significant breathing disorders in sleep (mean respiratory disturbance index [RDI] = 6.5). In the other 14, as well as the additional control subjects participating in the hypoxia in vitro experiment, OSA was ruled out by a detailed clinical interview. Because there were no significant differences between control subjects who were recorded in the laboratory and those who were only interviewed, their data were combined for statistical analysis. Except for one participant with a history of ischemic heart disease, none of the control individuals had any history of cardiovascular, pulmonary, or metabolic diseases. Comparison of the demographic data between patients with OSA and control subjects revealed that patients had significantly higher body mass index (BMI) (p < 0.01). The protocol was approved by the local human rights committee and all participants signed an informed consent before their enrollment.
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Detection of Reactive Oxygen Species and Fluorescence Staining by Flow Cytometry
An expanded version of the technical details of the methodologies used can be found in the online data supplement.
Oxidative metabolism was assessed by flow cytometry (20-22). For all patients with OSA and control subjects, blood samples were processed for reactive oxygen species (ROS) production within 30 min of blood collection. Each of the experiments was conducted on groups of 8 to 15 patients with OSA and 9 to 17 control subjects.
Cultured Endothelial Cell Monolayers
Adherence of leukocytes was studied either on HCAECs or on HUVECs. Cryopreserved HCAEC 5279 were obtained from Clonetics (Normal Human Cell Systems; BioWhittaker, A Cambrex Company, Walkersville, MD) and cultivated according to the manufacturer's instructions and used from passage 2 to 5. Freshly prepared HUVECs were kindly provided by Dr. N. Lanir from Rambam Medical Center and used only in the first and second passages. Endothelial cells (HCAECs or HUVECs) were pretreated for 1 h at 37° C with anti-CD62E/CD62P or anti-CD54 antibodies (Serotec, Oxford, UK), to inhibit adhesion to endothelium via E-selectin/P-selectin or ICAM-1, respectively, after which leukocytes derived from patients with OSA and control subjects were added.
Adherence Assay
Mononuclear cells (MNCs) were isolated from peripheral blood of patients with OSA and control subjects using gradient 1.077 (Sigma Diagnostics, Israel) and radiolabeled by incubation with 51Cr. Radioactivity in the adherent cells was determined by an auto-gamma scintillation counter (LKB-Wallac, Turku, Finland) in triplicates. The percentage of cells adhering to HCAECs or to HUVECs was calculated as follows: Adherence = (Mean cpm in Lysate)/(Mean cpm in Overlaid cells) × 100% (23).
CD15 Expression under Hypoxic Conditions In Vitro
Freshly obtained blood from 11 healthy control subjects was exposed to hypoxic conditions in a Modular Incubator Gas Chamber (Hot Box system, Del Mar, CA). Blood samples were exposed to hypoxia by purging with 1% O2, 5% CO2, 94% N2, and placed in a 37° C incubator for an overnight period or for an overnight period of hypoxia followed by 6 h reoxygenation (21% O2, 5% CO2). Control tubes were identically treated, except that they were kept under normoxic conditions (21% O2, 5% CO2) for the same durations.
The percentage of CD15+ monocytes in each individual was determined before and after exposure to hypoxic and normoxic conditions.
nCPAP Treatment
The following parameters were compared between the nCPAP and no nCPAP conditions: expression of %CD15+ (n = 8) and %CD11c+ (n = 8), %CD11c+ monocytes participating in basal ROS production (n = 8), and monocytes' ability to adhere to endothelial cells in culture (n = 7). Three of the patients were studied with HCAECs and four with HUVECs. Generally, monocytes adhered more avidly to HUVECs than to HCAECs, but this did not affect the differences between patients with OSA and control subjects.
Statistical Analysis
All data were expressed as mean ± SD. All differences between OSA and control groups were evaluated by nonparametric analysis of covariance (ANCOVA) after adjusting for BMI. Paired t tests were used to compare between variables before and after hypoxia or normoxia treatments and before and after nCPAP treatment. A nonparametric matched pairs Wilcoxon test was used for variables not normally distributed.
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Expression of CD64 and Adhesion Molecules CD11b, CD11c, and CD15 on Monocytes and Granulocytes
Monocyte, granulocyte, and lymphocyte subgroups were distinguished by side scatter and fluorescence characteristics (24). For each subpopulation, percentage of positive cells was obtained using a gate that included < 1% of cells of the mouse
isotypic negative control. The percentages of CD64, CD11b,
CD11c, and CD15 positive cells in PBMs of patients with OSA
and control subjects are depicted in Figure 1. Patients with OSA
exhibited a 1.9-fold increase in the percentage of CD11c+
monocytes (p < 0.004) and a 9-fold increase in the percentage
of CD15+ (p < 0.0005) monocytes, but percentage of CD64+
(Fc
RI) monocytes was significantly lower than control subjects (p < 0.03). The value for CD64+ monocytes in healthy
control subjects was reported to be 81.3 ± 6.8%, which is in
agreement with our results (25). There was no significant difference in the percentage of CD11b between groups. Similar values (83.7 ± 8%) were reported by Sacks and coworkers for
CD11b+ monocytes in healthy individuals (25). Also, the expression of CD14 markers, used to identify monocytes, did not
differ significantly between patients with OSA and controls,
55.2 ± 6.8% (48 to 64%) versus 67.7 ± 14.9% (51 to 82%).
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Control; 
OSA.
Nonactivated granulocytes of patients with OSA and control subjects were practically devoid of CD64 and CD11c expression (ranging from 0.8 to 8.0%). On the other hand, CD11b+ expression was very high in granulocytes of both groups (from 92 to 99.5%), but no differences between the groups were observed.
Measurement of ROS Production in Whole Blood Monocytes
ROS production was determined in monocytes and granulocytes from the same preparation, without further purification or manipulation of the cells in order to avoid activation (22, 24). By using monoclonal antibodies (mAbs) for identification of CD64, CD11b, CD11c, and CD15 subpopulations of monocytes and granulocytes, we could specifically analyze the relationship between each subpopulation of cells bearing a specific surface marker and its ability to produce ROS either under nonactivated state representing basal ROS production or in response to phorbol myristate acetate (PMA). The intensity of ROS production by each individual cell, represented by the mean fluorescence intensity (MFI)/cell and the percentage of cells that express a specific surface molecule and oxidize dihydroethidium (DHE), was combined to an index ([MFI/ cell] × % cells that oxidize DHE) representing total ROS production of a specific subpopulation.
Significant differences were detected in CD11c+ monocytes between patients with OSA and control subjects. The percentage of CD11c+ cells participating in basal oxidation of DHE was higher in patients with OSA than in control subjects (38.8 ± 17.2% versus 24.1 ± 10.6%, p < 0.05). Likewise, basal ROS production in OSA patients was 2.5-fold higher than in control individuals (106.2 ± 98.7 versus 42.7 ± 11 MFI/cell, p < 0.0003). Similarly, in response to PMA stimulation, both the percentage of CD11c+ monocytes producing ROS and the intensity of ROS production in MFI/cell were significantly higher in patients with OSA (73.8 ± 12.5% versus 57.8 ± 10.8%, p < 0.03 and 385.5 ± 158.8 versus 184 ± 112.7 MFI/ cell, p < 0.01, respectively). Total ROS production, represented by index, of nonactivated and PMA-activated CD11c+ monocytes is depicted in Figure 2A. As expected, the ROS production indices by CD11c+ monocytes were significantly higher in patients with OSA than control subjects (p < 0.003 and p < 0.0005 for nonactivated and PMA-activated, respectively, Figure 2A).
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In contrast, ROS production of the small subpopulation of CD15+ monocytes did not differ significantly between patients with OSA and control subjects neither in basal conditions nor after PMA stimulation, but MFI/cell was high in control as well as in OSA (data not shown).
Analysis of the covariance revealed borderline statistically significant differences between groups in the percentages of nonactivated CD64+ monocytes participating in the DHE oxidation (52 ± 18.4% versus 42.8 ± 16.4%, p < 0.08, and 82.8 ± 46.3 versus 53.9 ± 30.9 MFI/cell, p < 0.08 in OSA and control, respectively). However, after PMA stimulation the percentage of CD64+ monocytes participating in DHE oxidation increased by 1.6-fold in comparison with basal conditions in patients with OSA (52.0 ± 18.4% versus 82.03 ± 16.6%, p < 0.003) but did not change significantly in control subjects (42.8 ± 16.4% versus 50.2 ± 15.3%). Although the percentage of nonactivated CD64+ monocytes did not differ significantly between patients and control subjects, percentage of activated CD64+ monocytes as well as ROS production (MFI/cell) by PMA-activated monocytes were significantly higher in patients with OSA (p < 0.002 and p < 0.009, respectively). Thus, index for total ROS production (Figure 2B) by PMA-activated CD64+ monocytes of patients with OSA was increased by more than 5-fold as compared with control subjects (p < 0.0004).
Despite the high level of CD11b expression on monocytes, only 2 to 7% of CD11b+ monocytes participated in DHE oxidation at rest. Also, the intensity of ROS production (MFI/cell) did not vary between OSA and control groups, and no differences were detected in this subpopulation due to PMA activation. Neither percentages of monocytes participating in DHE oxidation nor the MFI/cell varied between the groups (36.8 ± 10.5% versus 40.0 ± 9.4% and 113.8 ± 30 versus 112 ± 40.8 MFI/cell in patients with OSA and control subjects, respectively).
Measurement of ROS Production in Whole Blood Granulocytes
As mentioned previously, the percentage of CD11c+ and CD64+ nonactivated granulocytes was low (0.8 to 8%) and similar in patients and control individuals. Basal ROS production in CD11c+ and CD64+ granulocytes was variable and significantly different between groups (p < 0.05) only in CD64+ cells (ROS index: 610 ± 528 versus 446 ± 462, for patients with OSA and control subjects, respectively). Thus, PMA stimulation of whole blood granulocytes increased both the number of CD11c+ and CD64+ participating granulocytes (85 to 96%) and fluorescence intensity. Generally, PMA-stimulated granulocytes of patients with OSA exhibited higher ROS production as compared with control subjects. This is illustrated in Figure 3 by depicting the index ([MFI/cell] × % cells) for CD11c+ (p = 0.04) and CD64+ granulocytes (p < 0.08).
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Adhesion of Monocytes from Patients with OSA and Control Subjects to HCAECs and HUVECs in Culture
The ability of monocytes from patients with OSA (n = 8) and control subjects (n = 8) to adhere to endothelial cells in culture was assessed. In each experiment, patients and control subjects were compared by using the same batches of endothelial cell monolayers under the same experimental conditions. Monocytes from patients with OSA adhered significantly more avidly to both HCAEC and HUVEC monolayers than monocytes of control subjects (in both at least p < 0.05) as summarized in Table 2. Monocyte adhesion of normal subjects to HCAEC or HUVEC in culture was unaffected by antibodies to CD62E/CD62P, that block binding through selectins. Conversely, in OSA monocytes, adhesion to HCAECs and HUVECs was inhibited by 31% and 38.8% (in both at least p < 0.05), respectively. Antibodies to CD54 (binding ICAM-1 molecules) slightly and nonsignificantly inhibited monocyte HCAECs (by 19% and 14.4% for control subjects and patients with OSA, respectively) (Table 2A). However, adhesion of OSA monocytes to HUVECs was significantly decreased by 26% (p < 0.05, Table 2B) after treatment with Abs to CD54.
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Effect of Hypoxia In Vitro on CD15 Expression in Monocytes of Control Subjects
To investigate the effects of hypoxemia on the expression of CD15 molecules, whole blood monocytes of 11 control subjects were exposed in vitro to hypoxia (1% O2, 5% CO2, 94% N2) or normoxia (21% O2, 5% CO2), for an overnight period. In five of the control subjects, hypoxia was followed by 6 h of reoxygenation. As shown in Table 3, the initial values obtained for %CD15+ monocytes were low as shown earlier in control subjects (Figure 1). However, after overnight hypoxia, percentage of CD15+ monocytes increased to comparable values observed in patients with OSA (12.8 ± 5.7% versus 13.9 ± 7.2%, respectively). This increase was significantly higher than the increase under normoxic conditions (12.8 ± 5.7% versus 4.0 ± 1.4%, p < 0.001). The expression of CD15 was only slightly increased in control monocytes kept overnight under normoxia. Moreover, hypoxic treatment followed by 6 h of reoxygenation resulted in a further increase in percentage of CD15+ monocytes, without any further increase in the expression under normoxia for the comparable period (17.1 ± 3.0% versus 3.8 ± 1.6%, p < 0.02). Similar results were obtained with the index (MFI × % cells expressing CD15). Figure 4 illustrates the expression of CD15+ monocytes of a control subject by fluorescent-activated cell sorter (FACS) analysis under the various treatments described.
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Effects of nCPAP Treatment
Treatment with nCPAP decreased RDI (55.6 ± 13.2 versus 15.1 ± 9.4, p < 0.008) and the percent time below 90% arterial oxygen saturation (20.4 ± 18.7% versus 0.75 ± 1.2%, p < 0.02). The percentage of CD15+ (n = 8) and CD11c+ (n = 8) monocytes was significantly decreased after treatment (20.9 ± 10.4% versus 7.0 ± 4.0%, p < 0.02 and 71.2 ± 22.7% versus 58.6 ± 20.2%, p < 0.04, respectively). Of note, %CD15+ did not decrease in one patient who remained hypoxic during treatment. Also the percentage of CD11c+ monocytes participating in basal ROS production (n = 8) was decreased after treatment (72.6 ± 19.9% versus 36.2 ± 23.9%, p < 0.02). Monocytes' adhesion index to HCAECs or HUVECs was significantly decreased after treatment as well (n = 3, 4.3 ± 0.8 versus 1.4 ± 7, p < 0.01 and n = 4, 9.8 ± 4.8 versus 5.6 ± 4.1, p < 0.01, respectively).
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This study provides strong support to the relevance of monocyte adhesive properties and oxidative metabolism to the pathogenesis of cardiovascular morbidity in sleep apnea syndrome. The following is a summary of our major findings. We showed that: (1) Sleep apnea syndrome is associated with increased expression of two adhesion molecules, CD15 and CD11c in monocytes. (2) Monocytes of patients with OSA adhered significantly more avidly to endothelial cells in culture than those of control subjects. (3) Treatment with hypoxia in vitro increased CD15 expression in monocytes of control subjects to percentages comparable to those of patients. (4) Patients with OSA had increased intracellular ROS production in some monocyte and granulocyte subpopulations. (5) nCPAP treatment downregulated adhesion molecule expression (CD15 and CD11c on monocytes), lowered basal ROS production by CD11+ monocytes, and decreased monocyte adherence to human endothelial cells in culture.
Activated leukocytes play a key role in the inflammatory response to injury caused by hypoxia/reoxygenation that may initiate atherogenic processes. Their interaction with the endothelium is mediated and closely regulated by adhesion molecules (26). The selectin binding CD15, which is primarily expressed on neutrophils and on a small subgroup of monocytes, and CD11c, which binds to ICAM-1 on activated or dysfunctional endothelial cells, were increased in patients with OSA. These observations clearly indicate that monocytes of patients with OSA are activated (19, 27). Upregulation of CD15 expression was shown in lipopolysaccharide-activated monocytes (22). In addition, hypoxia/reoxygenation stimulated the expression of ICAM-1 and VCAM-1 on human endothelial cells (28), upregulated CD15 expression, and activated leukocytes in vivo and in vitro (29, 30). Moreover, exposing HUVECs to anoxia/reoxygenation in culture, increased neutrophil/HUVECs adherence (18). Soluble selectin and ICAM-1 in sera of patients with OSA were also shown to increase, which was attributed to surges in apneas' related sympathetic activity (13, 14). However, in our study, exposure of control monocytes to hypoxia in vitro increased the percentage of CD15+ monocytes to comparable levels of patients with OSA. Subsequent exposure of these cells to reoxygenation further increased CD15 expression. Although the duration and magnitude of our in vitro hypoxemia are different than the intermittent nocturnal hypoxemia experienced by patients with OSA, our findings provide preliminary support to the notion that the apnea's related hypoxia is largely responsible for the increased CD15 expression. Further in vitro studies using gradually increasing hypoxemia levels are needed to conclusively prove this association.
The functional significance of the increased expression of these molecules was evident from the findings that monocytes of patients with OSA adhered significantly more avidly to HCAECs or HUVECs as compared with controls. Because mAbs against CD62E/CD62P (anti-E-selectin/P-selectin) inhibited the binding of OSA monocytes, this suggests that selectins may be primarily involved in this process (31, 32). Employing mAbs against CD54 (anti-ICAM-1) significantly inhibited the binding of OSA monocytes only to endothelial cells from umbilical vein origin, emphasizing the unique characteristics of endothelial cells from different origins.
The increased expression of adhesion molecules on circulating monocytes from patients with OSA was accompanied by increased intracellular ROS production. This was evident both at rest and after PMA stimulation. Total basal ROS production of CD11c+ monocytes was increased by 2.5-fold in patients with OSA. Similarly, after PMA stimulation, total ROS production of CD11+ monocytes was 2.7-fold greater in OSA patients. In contrast to the increased expression of CD11c monocytes in OSA, in inflammatory conditions CD11b expression was shown to be upregulated (25, 33, 34). In our study, monocytes' expression of CD11b did not differ between patients with OSA and healthy control subjects.
Although basal total ROS production of CD64+ monocytes did not differ significantly between patients and control
subjects, it was 1.9-fold higher than control subjects. Thus, PMA
stimulation of this subpopulation resulted in more than 5-fold
increase in total ROS production as compared with control individuals. These results as well point to the possibility that
CD64+ monocytes of OSA patients are preactivated. FcRI
(CD64) mediates many cellular responses crucial in host defense, including phagocytosis, cell cytotoxicity, and production
and secretion of proinflammatory cytokines such as interleukin (IL)-1
, IL-6, and TNF- (35, 36). A finding of high concentrations of TNF- was reported in sleep apnea patients,
which is in agreement with these results (15).
In granulocytes, there were no significant between-group differences in total basal ROS production of CD11c+ cells. In CD64+ granulocytes, however, basal ROS production was significantly higher in patients with OSA. In the PMA-activated cells, ROS production of CD11c+ and CD64+ granulocytes was significantly and nearly significantly higher in patients with OSA, respectively. These results are in agreement with Schulz and coworkers (17) who reported on an enhanced release of superoxide from polymorphonuclear neutrophils of patients with OSA.
The effects of nCPAP treatment on adhesion molecule expression and ROS production were studied in monocytes, by omitting nCPAP for a single night in patients treated for more than 6 mo. The %CD11c+ and %CD15+ monocytes were relatively low on treatment night and were significantly increased after omitting treatment. %CD11c monocytes participating in basal ROS production was significantly increased as well. In seven out of eight patients, CD15+ monocyte expression was decreased by at least 3-fold. In one patient, in whom %CD15 cells remained elevated, nCPAP treatment was not effective. Most importantly, in seven out of seven patients tested, treatment decreased monocytes adhesion index to endothelial cells. Collectively these data implicate hypoxemia in the upregulation of CD15 and CD11c expression.
Our in vitro observations on increased adherence of monocytes from patients with OSA to endothelial cells and the decreased adherence of OSA monocytes as a result of nCPAP treatment clearly indicate that patients with OSA are exposed to atherogenic insult nightly. In vivo, this may cause endothelial dysfunction, which constitutes the initial step in the chain of events leading to cardiovascular morbidity. Indeed, there is recent evidence of endothelial dysfunction in normotensive patients with OSA free of any known cardiovascular disease (37, 38).
In conclusion, we postulate that the repeated apneic-related hypoxic events, in a similar manner to hypoxia/reoxygenation injury, result in endothelial and monocyte activation. This was evident by the increased expression of CD15 and CD11c receptors and the increased oxidative metabolism. These primed monocytes adhere to the endothelium and become fully activated under hypoxic conditions. Subsequently, they emigrate into the subendothelial space and release lytic enzymes and oxygen radicals that injure the endothelium. The functionally impaired endothelium may initiate pathogenesis of cardiovascular morbidity in patients with sleep apnea.
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Correspondence and requests for reprints should be addressed to Lena Lavie, Unit of Anatomy and Cell Biology, The Bruce Rappaport Faculty of Medicine, Technion, POB 4649, 31096, Haifa, Israel. E-mail: lenal{at}techunix.technion.ac.il
(Received in original form April 26, 2001 and accepted in revised form December 17, 2001).
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.orgAcknowledgments: This study was supported in part by a grant from the Israeli Academy of Sciences to L. Lavie and P. Lavie and, in part, by a joint grant from the Center for Absorption in Science of the Ministry of Immigration Absorption and the Committee for Planning and Budgeting of the Council for Higher Education under the framework of the KAMEA program.
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References |
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