Pathophysiology of Obstructive Sleep Apnea (OSA) - Blood Cells’ Reactive Oxygen Species and Inflammation Prevent Polycythemia
Authors: Song Jihyun , Sundar Krishna M , Robert Christensen , Horvathova Monika , Kralova Barbora , Divoky Vladimir , Ganz Tomas , Prchal Josef T. 
Published: October 2019

Abstract
Obstructive sleep apnea (OSA), characterized by intermittent hypoxia, causes cardiovascular, metabolic, neurocognitive and cancer complications. Hypoxia expands the red cell mass by stimulating erythropoietin (EPO) production; yet in our analysis of 527 OSA patients, <1% had OSA-related polycythemia (Gangaraju et al Blood 2016 128:2444). Typical hypoxia-induced red blood cells (RBCs) mass normalized upon return to normoxia by neocytolysis i.e. preferential destruction of young hypoxia-born RBCs. In an animal model, we demonstrated that neocytolysis is caused by excessive generation of reactive oxygen species (ROS) from increase of mitochondrial retention and accumulation of ROS. ROS increase resulted from decreased antioxidant enzyme catalase mediated by hypoxia-induced miR-21. We hypothesized that polycythemia in OSA was prevented by neocytolysis.
It is also well-known that OSA induces systemic inflammation markers including C-reactive protein, IL-6, TNF-α, IL-8, and NF-κb. Inflammation participates in the control of the number of RBCs by inducing hepcidin, the principal regulator of iron metabolism. Increased hepcidin suppresses erythropoiesis by inhibiting iron release from macrophages. Based on this evidence, we also hypothesized that the absence of polycythemia in OSA might be caused by an independent contribution of inflammation-mediated suppression of erythropoiesis.
We studied OSA patients before and after treatment with continuous positive airway pressure (CPAP). Increased erythropoiesis was evidenced by increased EPO and reticulocytosis. EPO levels correlated with time spent below sPO2 89 %, indicating that severe OSA patients had more augmented erythropoiesis. However, hematocrit levels were normal. Hemolysis was detected in some but not all OSA patients by end tidal carbon monoxide (a product from heme catabolism). After CPAP treatment, these changes diminished but hematocrits did not change. Conditions favoring neocytolysis were confirmed by increased ROS from expanded reticulocytes' mitochondria which correlated with time spent below sPO2 89 %. Downregulated catalase resulting from increased miR-21 was also detected. Also these changes normalized with CPAP. These results indicate that hemolysis of hypoxia-born RBCs prevents OSA patients from becoming polycythemic. Increased ROS was not only found in reticulocytes but also in leukocytes; these also normalized with CPAP.
Expression of inflammatory markers (NFKB1, TNF, and IL6) in granulocytes was higher in OSA compared to controls and normalized by CPAP; these levels correlated with apnea-hypopnea Index (AHI). OSA patients had higher hepcidin levels, correlating with inflammatory marker levels and inversely correlated with EPO. Iron and transferrin saturation levels were lower in OSA compared to controls, inversely correlating with high hepcidin levels. These data indicated that besides neocytolysis, coexistent suppression of erythropoiesis by inflammation contributed to the lack of polycythemia in OSA.
In OSA, inflammation mediated increase of ROS in leukocytes is a known causative factor of cardiovascular disease. We now report increase of both ROS and inflammatory markers in leukocytes.
We conclude that the absence of polycythemia in OSA is the result of hemolysis via neocytolysis and inflammation-mediated suppression of erythropoiesis. Increased ROS in blood cells and systemic inflammation from OSA-constitute mechanisms likely contributing to the pathophysiology of OSA.

Disclosures
Ganz: Vifor: Consultancy; Ablynx: Consultancy; Keryx Pharma: Consultancy, Research Funding; Silarus Pharma: Consultancy, Equity Ownership; La Jolla Pharma: Consultancy, Patents & Royalties: Patent licensed to La Jolla Pharma by UCLA; Akebia: Consultancy, Research Funding; Intrinsic LifeScience: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy.