Session: 113. Hemoglobinopathies, Excluding Thalassemia—New Genetic Approaches to Sickle Cell Disease: Fetal Hemoglobin Regulation And Reticulocyte Maturation In Sickle Cell Disease
Hematology Disease Topics & Pathways:
Diseases, Hemoglobinopathies, Biological Processes, erythropoiesis, pathways
Surprisingly, while global AT1R deficiency in WT mice (WT AT1R-/-) had no RBC phenotype, SCA AT1R-/- mice developed profound anemia, suggesting AT signaling may be important for the stress erythropoiesis (Stress-E) state, present in SCA. AT is known for its role as a renal erythropoietin (Epo) secretagogue, but its role in Stress-E is unknown. Induction of Stress-E in WT AT1R-/- mice (with phenylhydrazine or daily phlebotomies) resulted in higher anemia in WT AT1R-/- mice than in WT AT1R+/+ mice, suggesting AT1R signaling is important for Stress-E, not baseline erythropoiesis (Base-E). However, WT mice with erythroid-specific deficiency of AT signaling (WT eCre+), when stressed, were able to maintain hemoglobin comparable to their controls (WT eCre-), with an exponential increase in Epo level. Epo levels were high in SCA mice to begin with but were insufficient to compensate with loss of AT signaling in SCA AT1R-/- mice, resulting in development of anemia. RNAseq analysis of sorted Stress-E nucleated erythroid precursors and enucleated erythrocytes in WT AT1R-/- mice showed a remarkable downregulation of the Hedgehog, BMP4, KIT and WNT Stress-E signaling pathways, when compared to WT (AT1R+/+) mice, further confirming that AT signaling is critical, and conceivably upstream to these established Stress-E pathways.
However, AT-AT1R signaling in Stress-E states is a double-edged sword: on one hand it was essential to sustain erythropoiesis, while on the other hand it resulted in significant ROS production. This was seen in SCA, and in WT mice where Stress-E was associated with higher RBC ROS (Figure 1a). AT is known to activate NADPH oxidase (NOX), to generate ROS in other cell types. But pharmacological inhibition of NOX signaling, or genetic deficiency of Gp22phox (a common subunit of NOX isoforms) in SCA mice did not lower RBC ROS. However, AT-AT1R signaling in Stress-E increased the mitochondrial mass in erythroid precursors, and also resulted in retention of dysfunctional mitochondria with lower membrane potential in the enucleated erythrocytes, a source of high ROS (Figure 1b-c). Hence, AT signaling in Stress-E inhibited mitophagy, which is required for clearance of mitochondria after enucleation. The transcriptome profile corroborated these findings: With Stress-E, WT AT1R-/- mice had upregulation of genes involved in mitophagy, and genes involved in maintaining mitochondrial integrity/quality and cellular redox homeostasis under oxidative stress.
Taken together, our results show that AT signaling plays a critical role in increasing erythroid cell mass during Stress-E, but also downregulates mitophagy, antioxidant genes, and results in increased retention of dysfunctional mitochondria, which are the source of high RBC ROS that mediates end-organ injury.
Disclosures: Kalfa: Forma Therapeutics, Inc: Research Funding; Agios Pharmaceuticals, Inc: Consultancy, Research Funding. Malik: Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties.
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