Inflammation status of rabbit carotid artery model endothelium during intermittent hypoxia exposure and its relationship with leptin.

OBJECTIVES: To develop an intermittent hypoxia/reoxygenation (IH/ROX) rabbit carotid artery model and then investigate the inflammation status of rabbit carotid artery endothelium after IH exposure and its relationship with leptin. MATERIALS AND METHODS: After anesthetization, rabbit's right common carotid artery was cleared of surrounding tissue with anatomic microscope, cannulated to its distal part and the proximal part was ligated. Preparations were challenged by changing the PO(2) of the gas mixture equilibrating the perfusate. Alternate perfusing (2 mL/min) of equilibrated perfusate bubbled with normoxia or hypoxia gas mixtures formed IH/ROX cycles in the right carotid common artery, simulating the pattern of hypoxic episodes seen in obstructive sleep apnea (OSA), or continuous perfusing of hypoxia perfusate to form continuous hypoxia (CH) modes. Sixty adult male New Zealand White rabbits (2.5-3.0 kg) were separated into six groups, ten per group. Groups were: A, intermittent normoxia (IN) group, perfused with perfusion equilibrated with 21% O(2) [PO(2) about 141 +/- 2.87 mmHg] for 15 s and 21% O(2) for 1 min 45 s, 60 cycles; B, severe IH group, 5% O(2) [PO(2) about 35.2 +/- 1.27 mmHg] 15 s and 21% O(2) 1 min 45 s, 60 cycles; C, mild IH group, 10% O(2) [PO(2) about 54.3 +/- 3.31 mmHg] 15 s and 21% O(2) 1 min 45 s, 60 cycles; D, severe IH+Lep group, protocol was the same with severe IH group; E, CH group, IN for 1 h 45 min and then 5% O(2) for 15 min; and F, Lep group, the same with IN group. Right common carotid artery parts distal to the cannula were harvested after exposure, and endothelial cell layers were gotten from longitudinal outspread vessels. Nuclear factor kappaB (NFkappaB) DNA binding activities of partial cell layers were measured with electrophoretic mobility shift assay in the IN group, severe IH group, mild IH group, and CH group nuclear extracts. The other part of the cell layers in the IN group, severe IH group, severe IH+Lep group, and Lep group were cultured for 2 h, and during the culture procedure, recombinated human leptin solutions were added to culture dishes of severe IH+Lep group and Lep group (resulted concentration, 10 ng/mL). Enzyme-linked immunosorbent assay was used to analyze medium interleukin-6 (IL-6) concentrations, reverse transcription polymerase chain reaction was used to analyze endothelial cell Ras homology A (RhoA) mRNA expression levels. Statistical analysis was done with SPSS 11.5 software package. RESULTS: NFkappaB DNA binding activities were significantly different between groups (F = 112.428, P < 0.001). This activity in the severe IH group (4.27 +/- 0.64) was higher than that in the mild IH group (2.33 +/- 0.45, P < 0.001), IN group (1.00 +/- 0.26, P < 0.001), and CH group (1.15 +/- 0.36, P < 0.001). RhoA mRNA expression levels were different in groups (F = 26.634, P < 0.001).This level in the severe IH+Lep group (2.54 +/- 0.53) was higher than that in the severe IH group (1.57 +/- 0.44, P = 0.002), IN group (1.00 +/- 0.31, P < 0.001), and Lep group (1.31 +/- 0.30, P < 0.001). IL-6 concentrations were different in groups (F = 79.922, P < 0.001). IL-6 concentration in the severe IH+Lep group (1591.50 +/- 179.57 pg/mL) was higher than that in the severe IH group (1217.20 +/- 320.62 pg/mL, P = 0.036), IN group (325.40 +/- 85.26 pg/mL, P < 0.001), and Lep group (517.40 +/- 183.09 pg/mL, P < 0.001). CONCLUSIONS: IH/ROX activated the inflammation pathway significantly in the endothelium, which was more intensive than CH and intensity-dependent. When exposed to both IH/ROX and leptin, inflammation occurs more dramatically. It means that synergic activating roles were performed by IH/ROX and leptin. This study may have a clinical implication that IH can cause endothelial damage through activated inflammation in OSA patients, and if the OSA patients have obesity at the same time, the endothelial damage or the inflammation would be more significant because of elevated leptin level as a synergic factor.

[Carotid body inflammation and carotid sinus nerve afferent activity after intermittent hypoxia exposure of various frequencies in rabbits].

OBJECTIVE: To explore the inflammatory reactions, endothelin level and carotid sinus nerve (CSN) afferent activity of carotid body (CB) after intermittent hypoxia/reoxygenation (IH/ROX) exposure of various frequencies in rabbits. METHODS: Forty-nine male adult New Zealand white rabbits (2.5-3.0 kg) were separated into 7 groups (n = 7 each). After anesthetization, the right carotid artery and CSN were cleared of surrounding tissues without touching the right CB and the left carotid region. The CSN was unenveloped to partially expose the myelin sheath, and electrodes were placed to the "single" chemoreceptor bundle of the CSN, with CSN afferent activity carefully monitored and recorded. Then the right common carotid artery was exposed, cannulated to distal part and its proximal part was ligated. Preparations were challenged by changing the PO2 of the gas mixture equilibrating the perfusate. Alternatively perfusion (2 ml/min) of equilibrated perfusate bubbled with normoxia or hypoxia gas mixtures formed IH/ROX cycles in right carotid common artery, simulating the pattern of hypoxic episodes seen in obstructive sleep apnea, or with continuously perfusing hypoxia perfusate to form continuous hypoxia (CH) modes. Groups were defined with different frequencies, and groups were: intermittent normoxia group (IN group) (21% O2, 15 s; 21% O2, 1 min 45 s), 10/hr group (5% O2, 15 s; 21% O2, 5 min 45 s), 30/hr group (5% O2, 15 s; 21% O2, 1 min 45 s), 50/hr group (5% O2, 15 s; 21% O2, 57 s), 60/hr group (5% O2, 15 s; 21% O2, 45 s) and 90/hr group (5% O2, 15 s; 21% O2, 25 s). All the above groups were exposed to 60 treatment cycles; continuous hypoxia group (CH group), IN for 1 h 45 min and then 5% O2 for 15 min. After exposure and 30 min of static placing, CSN afferent frequencies (Charge F) were recorded from chemoreceptor bundles, and the right CB was cleared of surrounding tissues and harvested. Interleukin-6 (IL-6), endothelin-1 (ET-1), hypoxia-inducible factor-1 (HIF-1), and vascular endothelial growth factor (VEGF) concentrations of the CB lysate were measured with enzyme linked immuno sorbent assay (ELISA) kits and standardized. Data were analyzed with SPSS 12.0 software package; and after one way analysis of variance (ANOVA) for whole difference, Tamhane's T2 was used for post hoc analysis. RESULTS: IL-6, ET-1 and Charge F increased but then decreased with increasing IH frequencies (F = 25,601.39, 2390.48, 6945.84, all P values < 0.01). IL-6, ET-1 and Charge F levels in 50/hr group were the highest among groups. Charge F levels correlated significantly with IL-6 or ET-1 (with IL-6: r = 0.736, P < 0.01; with ET-1: r = 0.757, P < 0.01, respectively). IL-6, ET-1 and Charge F levels between IN group and CH group were not statistically different (all P values > 0.05). HIF-1 levels elevated gradually (F = 5241.10, P < 0.01) with increasing exposure frequencies, and the CH group had the highest value (all P values < 0.01). VEGF level in CH group was the highest in all groups (all P values < 0.01). CONCLUSIONS: After IH/ROX exposure, afferent activity of CB CSN increases, which significantly correlates with inflammation and vasomotor mechanism of CB. CB inflammation comes not from IH phases but from ROX phases. Increased CB CSN activity results in elevated SNA tension, which plays a key role in the pathogenesis of systemic hypertension. This procedure influenced by IH/ROX frequencies. CH for 15 min causes no definitely damages. However, HIF-1 and VEGF can be considered as members of adaptive pathway during IH/ROX exposure.

[Effects of obstructive sleep apnea style intermittent hypoxia on endothelin-1, nitric oxide, and nitric oxide synthase in endothelium: experiment with human umbilical vein endothelial cells].

OBJECTIVE: To investigate the effects of intermittent hypoxia (IH) on the expression of endothelin-1 (ET-1), nitric oxide (NO), and nitric oxide synthase (NOS) in endothelium, and to evaluate the role of functional disorder of endothelium in the mechanism of obstructive sleep apnea syndrome (OSAS) induced hypertension. METHODS: Human umbilical vein endothelial cells (HUVECs) of the line ECV304 were cultured and divided into 4 groups: IH group (exposed to 1.5% O(2) for 15 s and 21% O(2) for 1 min 15 s, 3 min 45 s, 5 min 15 s, or 8 min 15 s respectively alternatively with 60 episodes), intermittent normal oxygen group (exposed to 21% O(2) for 15 s and 225 s alternatively with 60 episodes), continuous hypoxia group (exposed to 1.5% for 15 min), and blank control group. Then the culture fluid was collected. The levels of activity of ET-1 (EIA) NO, and NOS were detected by enzyme immune assay, nitric acid reductase method, and chemical colorimetric analysis respectively. RT-PCR was used to detect the mRNA expression of endothelial NOS (eNOS). RESULTS: Compared with those of the intermittent normal oxygen group and blank control groups the ET-1 levels of the 4 IH sub groups were significantly higher (F = 28.453, P = 0.000), the NO levels ere significantly lower F = 65.252, P = 0.000), the NOS activity levels were significantly lower (F = 5.969, P = 0.008), and eNOS mRNA was significantly down-regulated (F = 16.630, P = 0.000). Compared with continuous hypoxia group with the exposure time equivalent to the accumulated hypoxia time (15 s with 60 episodes) of the IH group, the ET-1 level was significantly higher (t = 2.742, P = 0.024), the NO level was significantly lower (t = 3.347, P = 0.004), the NOS activity level was significantly lower (t = 3.989, P = 0.004), and the eNOS mRNA expression was down-regulated (t = 5.045, P = 0.000). In the different IH group along with the prolongation of the re-oxygenation time from 105 s to 495 s, while maintaining the duration of hypoxic episodes at 15 s, the ET-1 level increased (F = 213.254, P = 0.000), the NO level decreased (F = 15.747, P = 0.000), NOS activity level decreased (F = 2.933, P = 0.048), and eNOS mRNA expression was down-regulated (F = 5.954, P = 0.002). However, there was a tendency for ET-1 to decrease and the NO system indicators when the re-oxygenation time was 8 min 15 s. CONCLUSION: Hypoxia and re-oxygenation both cause endothelium dysfunction and unbalance of NO/ET-1, which is important to OSAS with hypertension.

[Role of sympathetic nerve activity and arterial endothelial function in pathogenesis of hypertension in patients with obstructive sleep apnea-hypopnea syndrome].

OBJECTIVE: To explore the role of sympathetic nerve activity and vessel endothelial function in the pathogenesis of hypertension in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS). METHODS: Based on polysomnography (PSG), blood pressure (BP) and disease history, 93 subjects were divided into four groups: OSAHS with hypertension (OH), OSAHS without hypertension (O), hypertension without OSAHS (H), normal control (N). In addition to the blood pressure measurement, blood samples were collected before and after sleep during the PSG testing night to measure norepinephrine, endothelin, and NO levels. Urine samples were also collected during this time to test the level of vanillyl mandalic acid (VMA). RESULTS: Patients in OH group and O group had significantly increased plasma NE value (P < 0.05) in the next morning compared with those before sleep and the change was more significant in OH group compared to O group (P < 0.01). Pre-and after-sleep urine VMA levels in all groups showed no significant differences. Plasma NE and ET levels in OSAHS with and without hypertension after sleep were positively correlated with mean arterial pressure (MAP), apnea-hypopnea index (AHI), number of oxygen desaturation >or= 4% per hour (ODI(4)), percentage of time of oxygen saturation lower than 90% (T90) and correlated negatively with minimum arterial oxygen saturation (minSaO(2)) and mean arterial oxygen saturation (MSaO(2)). Moreover, plasma ET also correlated positively with MAP, AHI, maximum apnea time, total apnea time. Compared with other groups plasma ET value increased significantly and serum NO value decreased in the next morning in both O and OH group. Serum NO value after one night sleep in both hypertensive and norhypertensive OSAHS patients was negatively correlated with MAP, AHI, maximum apnea time, total apnea time, ODI(4), T90, and positively with minSaO(2) and MSaO(2). CONCLUSIONS: Sympathetic nerve activation and endothelial dysfuntion characterized by an imbalance of endothelium-derived systolic and diastolic factors may play an important role in the development of transient and sustained increase of blood pressure in patients with OSAHS.

[Interleukin-6 and tumor necrosis factor-alpha levels of endothelial cells in different hypoxia modes: in vitro experiment].

OBJECTIVE: To investigate the damage of different patterns of intermittent hypoxia (IH) and continuous hypoxia (CH) on endothelial cells. METHODS: Human umbilical vein endothelial cells of the line ECV304 were cultured in a program-controlled gas delivery system newly developed and divided into 8 groups to undergo different IH/reoxygenation (ROX) cycles so as to simulate the patterns of hypoxic episode seen in recurrent apnea and chronic obstructive pulmonary disease: intermittent normoxia (IN) group (exposed to 21% O2 15 s/21% O2 225 s for 60 cycles), IH group (exposed to 1.5% O2 15 s/21% O2 225 s for 30 or 60 cycles), IH hypercapnia group (exposed to 1.5% O2 and 20% CO2 15 s/21% O2 and 5% CO2 225 s, for 60 cycles), continuous hypoxia (CH group, exposed to 1.5% or 10% O2 for 15, 30 or 60 min), CH hypercapnia group (exposed to 10% O2 and 10% CO2 for 15, 30 or 60 min), CH added to IH group (exposed to 1.5% O2 15 s/10% O2 225 s for 60 cycles), different intermittent hypoxia extent group (exposed to 1.5% or 10% O2 15 s/21% O2 225 s for 60 cycles), different intermittent hypoxia frequency group (exposed to 1.5% O2 15 s/21% O2 225 s 315 s, 495 s or 105 s for 60 cycles), and different intermittent hypoxia duration group (exposed to 1.5% O2 15 s or 30 s/21% O2 225 s for 60 cycles). Then ELISA was conducted to examine the levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFalpha) Bicinchoninic acid method was used to standardize the cell total protein level. RESULTS: The levels of IL-6 and TNFalpha levels in the IH group were (770.40 +/- 21.60) and (126.93 +/- 2.58) pg.ml(-1).100 mg protein(-1) respectively, both significantly higher than those in the IN group [(374.06 +/- 38.10) and (31.96 +/- 13.64) pg.ml(-1).100 mg protein(-1) respectively, U = 0.000, P = 0.002], but significantly lower than those in the IH hypercapnia group [(829.27 +/- 7.16) and (78.77 +/- 4.00) pg.ml(-1).100 mg protein(-1) respectively, U = 0.000, P = 0.002]. The IL-6 levels of the CH hypercapnia 15, 30, and 60 min subgroups were all significantly higher than those of the corresponding CH subgroup (U = 0.000, P = 0.002). The IL-6 and TNFalpha levels of the CH added to IH group were (536.74 +/- 14.97) and (51.10 +/- 6.80) pg.ml(-1).100 mg protein(-1) respectively, both were significantly higher than those of the IN group, but significantly lower than those of the IH group (chi(2) = 23.4, P < 0.05). The levels of IL-6 and TNFalpha increased along with the increase of the IH degree (chi(2) = 23.4, P < 0.05). The level changes of IL-6 and TNFalpha of the groups with different intermittent hypoxia frequency and with different intermittent hypoxia duration were complicated. CONCLUSION: IH and CH significantly damage the endothelial cells dose-dependently, especially combined with hypercapnia. In IH/ROX, the inflammatory damage comes from ROX phase but not IH phase. Hypoxia duration and hypoxia frequency are also important parameters in the activation of inflammation.

[Changes of nuclear factor-kappaB and intercellular adhesion molecule-1 in endothelial cells exposed to various intermittent hypoxia].

OBJECTIVE: To construct a novel in vitro endothelial cell system to explore the changes of nuclear factor-kappaB (NF-kappaB) and intercellular adhesion molecule-1 (ICAM-1) levels after exposure to various intermittent hypoxia (IH) and re-oxygenation, an IH/reoxygenation (ROX) model. METHODS: We developed a gas control delivery system that permitted the exposure of ECV304 cell cultures, immortalized endothelial cell strain cultures of human umbilical vein endothelial cells (HUVEC), to IH/ROX cycles, simulating the pattern of hypoxic episodes seen in recurrent apnea. Cell samples were divided into the following groups according to IH duration/ROX duration. Group A (Intermittent Normoxia Group): 21% O(2) 15 s/21% O(2) 3 min 45 s; Group B (Standard Culture Group): no exposure; Group C: 1.5% O(2) 15 s/21% O(2) 3 min 45 s; Group D: 10% O(2) 15 s/21% O(2) 3 min 45 s; Fixed IH protocol as 1.5% O(2) 15s and ROX extent to 21% O(2), IH/ROX frequencies varied as 12 (Group C), 9.23 (Group E), 6.32 (Group F), 20 (Group G) and 40 (Group H) episodes per hour; Group I: 1.5% O(2) 30 s/21% O(2) 3 min 45 s; and after the exposure of Group C, the cell cultures were exposed to standard incubation device for 60 min (Group J) and 120 min (Group K). Prepared cell lysates and cell monolayers were analyzed for NF-kappaB levels and ICAM-1 levels in this IH model with enzyme-linked immunosorbent assay (ELISA) and cellular surface ELISA, and the cell total protein levels were measured with the method of bicinchoninic acid for standardization. SPSS 11.5 software package was used for statistical analysis. RESULTS: NF-kappaB and ICAM-1 levels in Group C were (0.82 +/- 0.28) and (1562 +/- 56) pg/ml, and those in Group A were (0.37 +/- 0.07) and (768 +/- 80) pg/ml, which showed statistical significance as compared with Group C (D = 225.00, 176.04, P < 0.01, < 0.05, respectively). Their levels in Group D were (0.66 +/- 0.22) and (1113 +/- 76) pg/ml, which were also significantly lower than Group C (U = 25.00, 0.00, all P < 0.01). NF-kappaB and ICAM-1 levels in Group I were (0.45 +/- 0.16) and (1155 +/- 19) pg/ml, which were statistically significant compared with Group C (U = 27.00, 0.00, all P < 0.01). In the same time, IH group had the relatively highest NF-kappaB and ICAM-1 levels amongst groups C, E, F, G and H. Which had different IH frequencies (chi(2) = 35.63, 56.89, all P < 0.01). NF-kappaB levels in Group J [(0.6233 +/- 0.0534)] did not differ significantly from Group C (D = 36.00, P > 0.05) and NF-kappaB levels in Group K [(0.3050 +/- 0.0013)] were lower than Group C (D = 234.00, P < 0.01). CONCLUSIONS: Our data indicated a selective and dose-dependent activation of inflammatory pathways on ECV304 cells by IH/ROX cycles with moderate frequencies, and a long time was needed for the cell rehabilitation from IH/ROX exposure.

[The relationship between arousal and nocturnal heart rate variability in patients with obstructive sleep apnea-hypopnea syndrome].

OBJECTIVE: To investigate the relationship between arousal and nocturnal heart rate variability (HRV) in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS). METHODS: Twenty-seven patients with moderate to severe OSAHS who underwent overnight polysomnography (PSG) were enrolled, excluding patients with cardiopulmonary and nervous system diseases. An hour continuous PSG monitoring in non-rapid eye movement (NREM) was recorded for all patients. The mean heart rate (HR) in 10 s before each arousal and the peak HR in 10 s after arousal were compared, and the breathing disorder related arousal index (B-ArI) and pulse rate rise index (PRRI) were calculated at this time, and then a correlation analysis was performed. Furthermore, according to minimal oxygen saturation (minSaO(2)), 18 patients with moderate-to-severe OSAHS were matched into 10 events with and without EEG arousal in NREM, and then DeltaHR at apnea termination between these events were compared. RESULTS: The peak HR in 10 s after arousal onset [(81.6 +/- 9.4) beats/min] was significantly higher than the mean HR in 10 s before arousal onset [(69.6 +/- 7.3) beats/min, t = -14.87, P < 0.01], and B-ArI was positively correlated to PRRI (r = 0.97, P < 0.01). The DeltaHR of events with EEG arousal at apnea termination [(11.1 +/- 2.8) beats/min] was higher than those without EEG arousal [(7.0 +/- 2.4) beats/min, t = 4.702, P < 0.01]. CONCLUSIONS: The results indicate that arousal can influence the function of cardiovascular regulation. Frequent arousal is one of the main causes of the cardiovascular consequences in OSAHS patients.