IADR Abstract Archives
The Effect of Intermittent Hypoxia on “Endothelial Cell—Colony Forming Units”
Objectives: Obstructive sleep apnea (OSA) is a highly prevalent sleep breathing disorder often associated with systemic pathological conditions, including craniofacial anomalies. OSA is characterized by intermittent hypoxia (IH) leading to blood hypoxemia, hypercapnia and sleep fragmentation. OSA associated IH is known to cause leukocyte activation, characterized by NADPH-oxidase reactive oxygen species production and increased oxidative stress (OS). Endothelial cell-colony forming units (EC-CFUs) are believed to reflect a reparatory response to vascular damage by leukocytes or tissue ischemia. This study aimed to examine the effect of IH on EC-CFUs formation.
Methods: EC-CFUs were produced by the Hill method using peripheral mononuclear cells (MNC) from healthy consenting volunteers. Starting the day of re-plating, the cells were exposed to 14 IH cycles (5-20% oxygen) during 6 hours for 3 consecutive days using the BioSpherix-OxyCycler-C42 system. Control cells were maintained in normoxic conditions (NRMOX) or in sustained hypoxia (SH) (5% oxygen) for the same duration. In separate experiments, the anti-oxidant agent N-acetylcysteine (NAC,1mM), NADPH-oxidase inhibitors apocynin (200µM) and diphenyleniodonium (DPI,5µM) were added. Nitro-blue-tetrazolium (NBT) test was performed. Confocal microscopy demonstrated NADPH-oxidase subunit NOX2 specific fluorescence.
Results: Under IH comparing to NRMOX and SH, EC-CFUs relative number was increased by 2.5±1.6 fold (p<0.05)(n=18). This increase was abolished by adding NAC or apocynin, while DPI treated cells failed to develop into EC-CFUs. EC-CFUs were positive for NBT test, whereas DPI abolished this effect. NOX2 specific fluorescence was increased by 4.6±1.7 fold (p<0.05) in EC-CFUs cultured under IH.
Conclusions: The number of EC-CFUs was increased under IH as compared to NRMOX and SH while the anti-oxidant NAC abolished this increase. This supports previous reports that EC-CFUs formation reflects leukocyte activation, probably through OS induction. NADPH-oxidase is active in the colonies, and is required for increased EC-CFUs formation. The above data can shed light on the reparatory mechanisms activated in OSA patients.
Methods: EC-CFUs were produced by the Hill method using peripheral mononuclear cells (MNC) from healthy consenting volunteers. Starting the day of re-plating, the cells were exposed to 14 IH cycles (5-20% oxygen) during 6 hours for 3 consecutive days using the BioSpherix-OxyCycler-C42 system. Control cells were maintained in normoxic conditions (NRMOX) or in sustained hypoxia (SH) (5% oxygen) for the same duration. In separate experiments, the anti-oxidant agent N-acetylcysteine (NAC,1mM), NADPH-oxidase inhibitors apocynin (200µM) and diphenyleniodonium (DPI,5µM) were added. Nitro-blue-tetrazolium (NBT) test was performed. Confocal microscopy demonstrated NADPH-oxidase subunit NOX2 specific fluorescence.
Results: Under IH comparing to NRMOX and SH, EC-CFUs relative number was increased by 2.5±1.6 fold (p<0.05)(n=18). This increase was abolished by adding NAC or apocynin, while DPI treated cells failed to develop into EC-CFUs. EC-CFUs were positive for NBT test, whereas DPI abolished this effect. NOX2 specific fluorescence was increased by 4.6±1.7 fold (p<0.05) in EC-CFUs cultured under IH.
Conclusions: The number of EC-CFUs was increased under IH as compared to NRMOX and SH while the anti-oxidant NAC abolished this increase. This supports previous reports that EC-CFUs formation reflects leukocyte activation, probably through OS induction. NADPH-oxidase is active in the colonies, and is required for increased EC-CFUs formation. The above data can shed light on the reparatory mechanisms activated in OSA patients.