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.