| Objectives In the first part to construct a novel in vitro endothelial cell system for the simulation of intermittent hypoxia (IH) or continuous hypoxia (CH), and with the model,①to explore its inflammatory excretive protein levels in the cell medium of different hypoxia modes [Study 1, interleukin-6 (IL-6) and tumor necrosis factor-α(TNFα)],②to explore changes of nuclear factor and inflammatory surface protein of endothelial cells in this model [Study 2, nuclear factor-κB (NF-κB) and intercellular adhesion molecule-1 (ICAM-1)], and③to explore the translocation of NFkB from cytosol to nuclear and changes of inflammatory status associated (Study 3). In the second part to elucidate the process of developing a novel rabbit carotid body (CB) and carotid common artery model in vivo for the simulation of various intermittent hypoxia (IH) intensities, IH durations, IH reoxygenation (ROX) durations and continuous hypoxia (CH) modes, and with this novo-model,①to investigate the inflammation status of rabbit carotid artery endothelium during intermittent hypoxia exposure and its relationship with leptin (Study 1), and②to explore oxidative stress, inflammatory status, endothelin level and Carotid Sinus Nerve (CSN) afferent activity of CB after IH/ROX exposure of various frequencies (Study 2). Materials andmethods In the first part we①In study 1, treatment groups included Intermittent Normoxia (IN), IH, IH Hypercapnia, CH, CH Hypercapnia, CH added to IH, Different Intermittent Hypoxia Extent, Different Intermittent Hypoxia Frequency and Different Intermittent Hypoxia Duration. And then the culture media were analyzed for IL-6 and TNFa levels in this hypoxia model with enzyme-linked immunosorbent assay (ELISA).②In study 2, Cell samples were divided into the following groups according to IH duration/ROX duration. Group A (Intermittent Normoxia Group); Group B (Standard Culture Group); Group C: 1.5%O2 15 s/21%O2 3 min 45 s; Group D: 10%O2 15 s/21%O2 3 min 45 s; Fixed IH protocol as 1.5%O2 15s and ROX extent to 21%O2, 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%O2 30 s/21%O2 3 min 45 s; After the exposure of Group C, put the cell cultures back to standard incubation device for 60 min (Group J) and 120 min (Group K). Prepared cell lysates and cell monolayer were analyzed for NF-κB levels and ICAM-1 levels in this IH model with ELISA and cellular surface ELISA.③In study 3, prepared nuclear, cytoplasmic extraction and culture media were analyzed for standardized NFkB optical density levels and IL-6 concentrations in this IH/CH model with ELISA. In the second part, adult New Zealand rabbits (2.5-3.0 kg) were anesthetized with spontaneous breathing intact. In every rabbit the right carotid common artery and carotid sinus nerve (CSN) were cleared of surrounding tissue, and "single" chemoreceptor bundle of the CSN was placed suction electrodes with CSN afferent activity carefully monitored and recorded. 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 carotid common artery, simulating the pattern of hypoxic episodes seen in obstructive sleep apnea syndrome (OSAS), or with continuously perfusing hypoxia perfusate to form CH modes. After the systematic exposes, CB, carotid common artery part distal to cannula, and carotid bifurcation were harvested.①In Study 1, 60 rabbits were separated into 6 groups, 10 per group. Groups were: A. intermittent normoxia (IN) group. B. severe IH group. C. mild IH group. D. severe IH + leptin group (severe IH+Lep group). E. CH group. F. leptin culture group (Lep group). Right common carotid artery parts distal to cannula were harvested after systematic exposes, and endothelial cell layers were gotten from longitudinal outspreaded vessels. Nuclear factorκB (NFκB) DNA binding activities of partial cell layers were measured with Electrophoretic Mobility Shift Assay in IN group, severe IH group, mild IH group and CH group nuclear extracts. The other part of cell layers in IN group, severe IH group, severe IH+Lep group and Lep group were cultured for 2 hours, 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.②In Study 2, 49 rabbits were separated into 7 groups, 7 per group. Groups were: A. intermittent normoxia group (IN group); B. IH/ROX frequency equals to 10 episodes per hour group (10·hr-1 group); C. IH/ROX frequency equals to 30 episodes per hour group (30·hr-1 group); D. IH/ROX frequency equals to 50 episodes per hour group (50·hr-1 group); E. IH/ROX frequency equals to 60 episodes per hour group (60·hr-1 group); F. IH/ROX frequency equals to 90 episodes per hour group (90·hr-1 group); G. continuous hypoxia group (CH group). After exposure, CSN afferent frequencies (Charge F) were collected from chemoreceptor bundles, and 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 ImmunoSorbent Assay kit and standardized. SPSS software package was used to analyze statistical characteristics.Results In the first part, this cell system model resulted in cyclic fluctuations of cellular PO2 values, between (76.28±1.2930) mmHg and (54.94±1.0502) mmHg, or PCO2 values, between (38.26±1.4943) mmHg and (88.64±1.5027) mmHg, during each hypoxic episode [PO2, from 158 mmHg (about 21%O2) to 11 mmHg (about 1.5%O2)] or hypercapnic hypoxia [PCO2, from 152 mmHg (about 20%CO2) to 38 mmHg (about 5%CO2)]challenge as designed.①In study 1, IL-6 and TNFαlevels in IH [(770.40±21.60) and (126.93±2.58) pg·ml-1·100mg protein-1] were higher than those in IN [(374.06±38.10) and (31.96±13.64) pg·ml-1·100mg protein-1 ](U=0.000, P=0.002) but lower than those in IH Hypercapnia [(829.27±7.16) and (78.77±4.00) pg·ml-1·100mg protein-1)(U=0.000, P=0.002), and IL-6 levels of CH Hypercapnia were higher than values of corresponded CH (U=0.000, P=0.002). IL-6 and TNFαlevels in CH added to IH [(536.74±14.97) and (51.10±6.80) pg·ml-1·100mg protein-1)were higher than those in IN but lower than those in IH (X2 =23.4, P<0.05). The levelsof IL-6 and TNFαincreased with lowered IH extent (X2 =23.4, P<0.05). Those levels in Different Intermittent Hypoxia Frequency and in Different Intermittent Hypoxia Duration were complicated and considerable.②In study 2, NF-κB and ICAM-1 levels in Group C were (0.8218±0.2760) and (1561.86±55.81) pg/ml, and those in Group A were (0.3668±0.0743) and (768.11±79.46) pg/ml, which showed statistical significance when compared with Group C (Z)=225, P<0.01 and D=176.04, P<0.05, respectively). Those levels in Group D were (0.6574±0.2207) and (1112.81±75.99) pg/ml, which were also lower than Group C significantly (U=25.000 and U=0.000, both P<0.01). NF-kB and ICAM-1 levels in Group I were (0.4450±0.1621) and (1154.88±19.00) pg/ml, which were statistically significant compared with Group C (U=27.000 and U=0.000, both P<0.01). In the same time, IH Group had the highestNF-kB and ICAM-1 levels amongst groups with different IH frequencies (X2 =35.632and 56.893, both P<0.01). NF-κB levels in Group J [(0.6233±0.0534)] did not differentiate from Group C significantly (D=36, P>0.05) and NF-κB levels in Group K [(0.3050++++++0.0013)] were lower than Group C (D=234, P<0.01).③In study 3, Standardized nuclear NFκB optical density levels in IH Group (6.2980±0.2754) were higher than those in correlated CH Group (1.1680±0.3626) which was the same in hypoxic extent and collected hypoxic duration. Both the nuclear NFκB and IL-6 levels in IH Group were higher than those in CH added to IH Group, (3.5077±0.3497) vs. (6.2980±0.2754) and (536.74±14.28) vs. (770.40±20.59) pg/mL/100mg protein respectively. With rehabilitation of 2 hrs from IH/ROX exposure, IH Group still had higher nuclear NFκB levels than Control Group, (2.7683±0.3968) vs. (1.0000±0.4855), whereas those in CH Group did not change throughout the procedure, (1.1680±0.3626) vs. (1.0000±0.4855). Cytoplasmic NFκB levels did not have obvious difference in all these groups. In the second part,①In Study 1, NFκB DNA binding activities were significantly different between groups (F=112.428, P<0.001); this activity in severe IH group (4.27±0.64) was higher than that in mild IH group (2.33±0.45) (standard error = 0.2481, P<0.001), IN group (1.00±0.26) (standard error = 0.2191, P<0.001) and CH group (1.15±0.36) (standard error = 0.2329, P<0.001). RhoA mRNA expression levels were different in groups (F=26.634, P<0.001); this level in severe IH+Lep group (2.54±0.53) was higher than that in severe IH group (1.57±0.44) (standard error = 0.2183, P=0.002), IN group (1.00±0.31) (standard error = 0.1937, P<0.001) and Lep group (1.31±0.30) (standard error = 0.1930, P<0.001). IL-6 concentrations were different in groups (F=79.922, P<0.001); IL-6 concentration in severe IH+Lep group (1591.50±179.57 pg/ml) was higher than that in severe IH group (1217.20±320.62 pg/ml) (standard error = 0.3572, P=0.036), IN group (325.40±85.26 pg/ml) (standard error = 0.1931, P<0.001) and Lep group (517.40±183.09 pg/ml) (standard error = 0.2491, P<0.001).②In Study 2, Tendencies of IL-6, ET-1 and Charge F means were going up and then turning down with the increase of IH frequencies. IL-6, ET-1 and Charge F levels in 50·hr-1 group were the highest among groups (Compared with IN, 10·hr-1, 30·hr-1, 60·hr-1, 90·hr-1, and CH group, standard errors and P values were: IL-6: 5.582, 7.760, 6.396, 6.636, 7.748 and 7.066, all P values<0.001; ET-1: 1.445, 1.637, 1.771, 1.771, 1.710 and 1.692, all P values<0.001; Charge F: 0.006788, 0.008099, 0.006819, 0.007601, 0.006732 and 0.008433, all P values<0.001, respectively.). 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, Spearman's nonparametric correlations.). HIF-1 levels elevated gradually, with the CH group had the highest one (Compared with IN, 10·hr-1, 30·hr-1, 50·hr-1, 60·hr-1, and 90·hr-1 group, standard errors and P values were: 7.173, 7.127, 7.179, 7.629, 8.923 and 8.481, all P values<0.001, respectively.). VEGF level in CH group was the highest (Compared with IN, 10·hr-1, 30·hr-1, 50·hr-1, 60·hr-1, and 90·hr-1 group, standard errors and P values were: 9.630, 10.081, 10.512, 9.783, 10.318 and 11.773, all P values<0.001, respectively.). Conclusions In the first part, our data indicated a dose-dependent activation of inflammatory pathways on ECV304 cells by IH/ROX and CH, and both could be aggravated by hypercapnia. In IH/ROX, the inflammatory damage comes from ROX phase but not IH phase. In the same time, different hypoxia durations and different hypoxia frequencies were also important parameters in the activation of inflammation. The inflammatory pathways were activated selectively on ECV304 cells by IH/ROX cycles with moderate frequencies, and a long time was needed for the cell rehabilitation from IH/ROX exposure. Our data also indicated a selective activation of nuclear factor-κB translocation from cytosol to nuclear department by IH in human umbilical vein endothelial cells, which had greater extent than by CH, and the translocation should have to cause dramatic inflammatory reaction on cellular level. Though long after the termination of IH, ECV304 cells still had a substantial residual inflammation. In the second part, IH/ROX activated inflammation pathway significantly in endothelium, as was more intensive than CH and intensity dependent. When exposed to both IH/ROX and leptin, endothelial cell layers inflammation were activated synergistically and had more permeability. After IH/ROX exposure, afferent activity of CB CSN increases obviously, which correlates significantly with inflammation status and vasomotor mechanism of CB, and CB inflammation comes from not IH phase but the ROX phase. Increased CB CSN activity will result in elevated SNA tension, which plays a key role in the pathogenesis of systemic hypertension. This procedure is interfered powerfully with IH/ROX frequencies, and the inflammatory status, ET-1 and CSN long-term facilitation of CB go up to the peak levels and then turn down to the minimum with the IH/ROX frequencies increasing continuously. However, HIF-1 and VEGF can be considered as members of adaptive pathway during IH/ROX.
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