IADR Abstract Archives
“Endothelial Cell—Colony Forming Units”: A Possible Protective Role in Obstructive Sleep Apnea
Objectives: Obstructive sleep apnea (OSA) is a highly prevalent sleep breathing disorder frequently associated with various pathological conditions, including craniofacial anomalies. OSA is characterized by intermittent hypoxia (IH) leading to blood hypoxemia and sleep fragmentation. IH induces leukocyte activation, and increases NADPH-oxidase dependent reactive oxygen species (ROS) and oxidative stress (OS). However, IH and OS may also stimulate adaptive-protective mechanisms such as Endothelial Cell-Colony Forming-Units (EC-CFUs), which reflect a reparatory response to vascular damage. This study aimed to examine the effect of IH on EC-CFUs paracrine function.
Methods: EC-CFUs were cultured using peripheral mononuclear cells from 15 healthy consenting volunteers. 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 normoxia (NRMOX) or sustained hypoxia (SH) (5% oxygen) for the same duration. EC-CFUs conditioned media from NRMOX, IH and SH treatments were collected and used to determine endothelial tube formation using EA.hy926 endothelial cells grown on Extracellular Matrix Gel. Additionally, the effect of ROS scavenger N-acetylcysteine (NAC 1mM) and two NADPH-oxidase inhibitors apocynin (100 μM) and DPI (5 µM) was assessed. Generation of networks of vessel-like structures was determined by net length calculation.
Results: Conditioned media from IH and SH-treated cells significantly increased endothelial tube formation as compared to NRMOX (2388±359 and 2586±196 vs. 855±287 μm/field, respectively, p<0.0017 for both). Separately, added inhibitors caused a significant decrease in tube length compared to IH-only and SH-only treatments, respectively (both p<0.05).
Conclusions: The significant increase in endothelial tube formation after treating with IH and SH-conditioned media may indicate a significantly augmented EC-CFUs angiogenic capacities compared to normoxia. NAC addition abolished this effect signifying the role of ROS in promoting these capacities. NADPH-oxidase inhibition also prevented tube formation, emphasizing its role in vascular function through ROS formation.
Methods: EC-CFUs were cultured using peripheral mononuclear cells from 15 healthy consenting volunteers. 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 normoxia (NRMOX) or sustained hypoxia (SH) (5% oxygen) for the same duration. EC-CFUs conditioned media from NRMOX, IH and SH treatments were collected and used to determine endothelial tube formation using EA.hy926 endothelial cells grown on Extracellular Matrix Gel. Additionally, the effect of ROS scavenger N-acetylcysteine (NAC 1mM) and two NADPH-oxidase inhibitors apocynin (100 μM) and DPI (5 µM) was assessed. Generation of networks of vessel-like structures was determined by net length calculation.
Results: Conditioned media from IH and SH-treated cells significantly increased endothelial tube formation as compared to NRMOX (2388±359 and 2586±196 vs. 855±287 μm/field, respectively, p<0.0017 for both). Separately, added inhibitors caused a significant decrease in tube length compared to IH-only and SH-only treatments, respectively (both p<0.05).
Conclusions: The significant increase in endothelial tube formation after treating with IH and SH-conditioned media may indicate a significantly augmented EC-CFUs angiogenic capacities compared to normoxia. NAC addition abolished this effect signifying the role of ROS in promoting these capacities. NADPH-oxidase inhibition also prevented tube formation, emphasizing its role in vascular function through ROS formation.