Polycythemia of Mice with Human Gain-of-Function EPOR (mtHEPOR) Is Transiently Corrected in Perinatal Life in Association with Low Epo and Increased Erythrocyte Phosphatidylserine Exposure

Konference: 2015 57th ASH Annual Meeting - účast ČR

Kategorie: Nádorová biologie/imunologie/genetika a buněčná terapie

Téma: 101. Red Cells and Erythropoiesis, Structure and Function, Metabolism, and Survival, Excluding Iron: Poster III

Číslo abstraktu: 3335

Autoři: Jihyun Song, MS; Mgr. Barbora Kráľová; Mgr. Monika Horváthová, Ph.D.; Donghoon Yoon; MD Josef T. Prchal; Doc.RNDr. Vladimír Divoký, Ph.D.

Gain-of-function mutations of erythropoietin receptor (EPOR) resulting from truncation of the cytoplasmic domain of EPOR are associated with primary familial and congenital polycythemia (PFCP). We generated and previously reported a PFCP mouse model where the murine EpoR gene (mEpoR) was replaced with wild-type human EPOR (wtHEPOR) or mutant human EPOR (mtHEPOR) genes. We showed that mtHEPOR mice become polycythemic at 3-6 weeks of age, but not at birth, similar to the polycythemia phenotype of affected humans, while the wtHEPOR mice were anemic (Divoky et al, Proc Natl Acad Sci U S A 2001;98:986). In addition, we previously reported (Yoon et al, ASH Annual Meeting Abstracts 2005 106: Abstract 567) that mtHEPOR fetuses are polycythemic at the mid- and late-gestation stages (embryonic days, E12.4-18.5), while wtHEPOR fetuses are anemic, similar to what we observed in adult mice. The mtHEPOR fetal liver cells (FLCs) revealed augmented and sustained activation of Stat5, while wtHEPOR FLCs had significantly reduced Stat5 activation. We also previously presented (Yoon and Prchal, ASH Annual Meeting Abstracts 2007 110: Abstract 3664) that the fetal polycythemia of mtHEPOR mice is associated with a delayed switch from primitive to definitive erythropoiesis, and that polycythemia in mtHEPOR mice is transiently corrected in perinatal life and reappears at 3 weeks after birth. Here, we hypothesized that the change of oxygen tension at delivery (from the uterus to ambient atmosphere) and differences in Epo levels may account for the inexplicable perinatal absence of polycythemia in mtHEPORmice.

Epo levels in adult mice were substantially elevated in wtHEPOR (1271±240 pg/mL) and significantly reduced in mtHEPOR (98±51 pg/mL) when compared to mEpoR mice (265±62 pg/mL). We then proposed that destruction of young red blood cells (RBCs), termed neocytolysis (a mechanism that overcorrects increased RBC mass generated during chronic hypoxemia after restoration of normoxic conditions) contributes to the anemia of newborns (Song et al, J Mol Med 2015;93:857). When adult mice were challenged by rapid conversion from 10 days of hypoxia (12% of O2) to normoxia, the mtHEPOR mice had greater changes of hematocrit (drop of 16.9%) than other genotypes (drop of 14.8% for mEpoR and 13.8% for wtHEPOR). Similarly, the most pronounced decrease of hematocrit after birth, at perinatal day 7, (PN7) was detected in mtHEPOR mice (drop of 56%) followed by mEpoR(drop of 48%) and only a relatively moderate decrease of hematocrit in wtHEPOR neonates (drop of 24%). In parallel, PN7 mtHEPOR newborns had the lowest levels of Epo (310±102 pg/mL), while PN7 wtHEPOR neonates had Epo levels markedly increased (2757±912 pg/mL); the Epo levels of control mEpoR at PN7 littermates were 494±102 pg/mL. This suggests that Epo protects from neocytolysis. These changes corresponded to exposure of phosphatidylserine; a stimulus for RBC removal from circulation. There was a gradual increase of phosphatidylserine-positive RBCs in mtHEPOR neonates and, to a lower extent, also in mEpoR between PN0 and PN7 with a maximum positivity at PN7 (mean fluorescence: 6.5±2.8 and 6.2±1.7 for mtHEPOR and mEpoR, respectively). This correlated with a maximal decrease of hematocrit and lowest Epo levels at PN7 in mtHEPOR. The proportion of phosphatidylserine-positive RBCs then declined with age (mean fluorescence of 0.6 in adult mtHEPOR mice). In contrast, wtHEPOR mice had the lowest numbers of phosphatidylserine-positive RBCs, which were comparable at all analyzed time-points (mean fluorescence at PN0=0.7±0.1; PN7=1.3±0.3; in adults=0.6±0.04), consistent with the lowest reduction in perinatal hematocrit and very high Epo levels in these mice.

In conclusion, our study describes that transient correction of polycythemia of the mtHEPOR PFCP mouse model in the perinatal period is associated with low Epo levels and increased erythrocyte phosphatidylserine exposure. Increased phosphatidylserine exposure on the membrane of mtHEPOR RBCs is consistent with accelerated destruction of these cells by macrophages, thus contributing to the decrease of hematocrit in mtHEPOR neonatal mice.

Authorship: JS and BK: equal credit as first authors

Acknowledgment: VD and MH were supported by the Czech Science Foundation (project P301/12/1503) and by the Ministry of Health Czech Republic (project NT13587).

Disclosures: No relevant conflicts of interest to declare.



Datum přednesení příspěvku: 7. 12. 2015