Abstract
Intermittent hypoxia (IH)—the hallmark of obstructive sleep apnea (OSA)—increases leukocyte activation, production of NADPH-oxidase dependent reactive oxygen species (ROS) and oxidative stress, affecting endothelial function. However, IH and oxidative stress can also stimulate adaptive-protective mechanisms by inducing the development of Endothelial Cell-Colony Forming Units (EC-CFUs), which are considered as a good surrogate marker for endothelial progenitor cells (EPCs), and likely reflect a reparatory response to vascular damage or tissue ischemia by leukocytes. Blood samples were obtained from 15 healthy consenting volunteers to evaluate the effects of IH and sustained hypoxia (SH) in vitro on EC-CFUs development and functions. The variables measured included: their numbers, the area, the proliferative capacity and ROS production. Additionally, NADPH-oxidase, VEGF and nuclear factor-erythroid 2 related factor 2 (Nrf2) expression, as well as their paracrine effects on endothelial tube formation were determined. The involvement of ROS was probed using the anti-oxidant N-acetylcysteine (NAC) and NADPH-oxidase inhibitors apocynin and diphenyl-iodide. Compared to normoxia, IH-dependent increases in EC-CFUs numbers were observed, showing an individual donor-dependent trait. Also, the expression of VEGF and gp91phox, a subunit of NADPH-oxidase, were significantly increased. ROS production and oxidative stress markers were also significantly increased, but Nrf2 expression and colony size were unaffected by IH. Additionally, conditioned media harvested from IH- and SH-treated mature EC-CFUs, significantly increased endothelial tube formation. These effects were markedly attenuated or diminished by the ROS and NADPH-oxidase inhibitors employed. In conclusion, we show here for the first time that IH-associated oxidative stress promotes EC-CFUs' vascular and paracrine capacities through ROS. However, the large inter-individual variability expressed in EC-CFUs numbers and functions to a given IH stimulus, may represent an individual trait with a potential clinical significance.
Highlights
Intermittent hypoxia (IH) is a common denominator in a wide range of pathological conditions including ischemic heart attacks and obstructive sleep apnea (OSA)
The number of Endothelial Cell-Colony Forming Units (EC-CFUs) developed in culture was assessed after treating with Norm, IH and sustained hypoxia (SH)
Non-adherent cells (1 × 106) from 15 healthy donors were re-plated after 48 h on fibronectincoated 24 well plates and exposed to 14 IH cycles or to 6 h of SH daily, for 3 consecutive days in culture after replating, as described in Methods
Summary
Intermittent hypoxia (IH) is a common denominator in a wide range of pathological conditions including ischemic heart attacks and obstructive sleep apnea (OSA). The nightly IH associated with OSA leads to intermittent blood oxygen desaturation and sleep fragmentation inducing oxidative stress and inflammation. OSA has an established association with various systemic pathological conditions. OSA patients are at an increased risk for cardiovascular diseases such as systemic hypertension, pulmonary hypertension, ischemic heart disease (IHD) and stroke [2, 3]. Leukocytes exposed to IH or in OSA are activated and their NADPH oxidasedependent ROS production is increased [5]. At the same time, OSA-associated IH may stimulate adaptive-protective mechanisms [4]
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