Severe Anemia Causes Chronic Hypoxia and Alters Hematopoiesis in Liver of Murine Neonates
Authors: Balamurugan Marie Amalie , Alnahhas Yasemin , Desiraju Suneetha , Chung Yerin , Guzman Pierre , Krishnan MohanKumar 
Published: May 2023

Background: Anemia is nearly universal in preterm infants and is associated with increased morbidity and mortality worldwide. We have recently described that the anemia associated “leaky gut” phenotype that leads to monocyte infiltration in intestine and RBC-transfusion activates these cells, resulting in necrotizing enterocolitis (NEC). In this study, we are investigating the effects of phlebotomy-induced severe anemia on hypoxia in the liver and the liver’s hematopoietic response. Methods: C57BL/6 mouse pups were studied in 3 groups (n=6 each): (1) naïve controls; (2) moderately anemic (hematocrit [hct] 27-32%); and (3) severely anemic (hct 20-24%). Pups were rendered severely anemic by facial vein phlebotomies on P2, 4, 6, 8, and 10. For moderately anemic, phlebotomized on P2, P6 and P10 only. Hypoxyprobe (pimonidazole hydrochloride) was administered i.p (60mg/kg body weight) to all groups of mice, 4 hours prior to sacrifice (P10) to evaluate hypoxia. On P11, the liver tissue was extracted and used for histology, protein analysis and flow cytometry. Immunostaining was performed on tissue sections of liver by using anti-GFP-pimonidazole. The liver protein samples were subjected to western immunoblotting for hypoxia-inducible factors (HIF-1α, HIF-2 and HIF-3). Single cell suspensions from liver tissue were stained with antibodies against SCA-1, cKit, CD16/32, CD34, CD135, CD48, CD41 and CD150, then these cells were acquired by BD LSRII-flow cytometry followed by FlowJo gating of hematopoietic progenitors (HSC), multipotent progenitors (MPP), common myeloid progenitors (CMP) and granulocyte macrophage progenitors (GMP). Results: Severely anemic liver sections showed stronger immunoreactivity for pimonidazole (fluorescence AFU, 74 ± 6.8; mean ± SE) than did moderately anemic liver, which showed minimal staining (32 ± 2.4; mean ± SE). No immunostaining was seen in controls (13 ± 0.9; mean ± SE), indicating that hypoxia was induced in the liver of anemic pups in a severity-dependent manner. In immunoblotting, we have detected an increased HIF-3 expression and decreased HIF-1α and HIF-2 expression in P10 livers of the severely anemic group, with opposite results seen in the livers of moderately anemic and control livers. We have also found that HIF-3 expression gradually increased after each phlebotomy, but HIF-1α and HIF-2 was suppressed in control mice. These findings indicate that progressive phlebotomy may induce chronic hypoxia in the liver, with gradually increased expression of HIF-3. Flow analysis showed higher frequencies (%) of stem cell progenitors, LSK cells, LT-HSC, MPP with increased GMP in severe anemia compared to moderate anemia and controls, showing that anemia-associated hypoxia skews the hematopoietic system toward granulocyte production. Conclusions: Severe anemia-associated chronic hypoxia state in the liver leads to increased expression of HIF-3 and alters the hematopoietic pool with increased production of granulocytes. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.