Erythroid lineage Jak2V617F expression promotes atherosclerosis through erythrophagocytosis and macrophage ferroptosis
Wenli Liu, … , Alan R. Tall, Nan Wang
Wenli Liu, … , Alan R. Tall, Nan Wang
Published May 19, 2022
Citation Information: J Clin Invest. 2022;132(13):e155724. https://doi.org/10.1172/JCI155724.
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Research Article Vascular biology

Erythroid lineage Jak2V617F expression promotes atherosclerosis through erythrophagocytosis and macrophage ferroptosis

Abstract

Elevated hematocrit is associated with cardiovascular risk; however, the causality and mechanisms are unclear. The JAK2V617F (Jak2VF) mutation increases cardiovascular risk in myeloproliferative disorders and in clonal hematopoiesis. Jak2VF mice with elevated WBCs, platelets, and RBCs display accelerated atherosclerosis and macrophage erythrophagocytosis. To investigate whether selective erythroid Jak2VF expression promotes atherosclerosis, we developed hyperlipidemic erythropoietin receptor Cre mice that express Jak2VF in the erythroid lineage (VFEpoR mice). VFEpoR mice without elevated blood cell counts showed increased atherosclerotic plaque necrosis, erythrophagocytosis, and ferroptosis. Selective induction of erythrocytosis with low-dose erythropoietin further exacerbated atherosclerosis with prominent ferroptosis, lipid peroxidation, and endothelial damage. VFEpoR RBCs had reduced antioxidant defenses and increased lipid hydroperoxides. Phagocytosis of human or murine WT or JAK2VF RBCs by WT macrophages induced ferroptosis, which was prevented by the ferroptosis inhibitor liproxstatin-1. Liproxstatin-1 reversed increased atherosclerosis, lipid peroxidation, ferroptosis, and endothelial damage in VFEpoR mice and in Jak2VF chimeric mice simulating clonal hematopoiesis, but had no impact in controls. Erythroid lineage Jak2VF expression led to qualitative and quantitative defects in RBCs that exacerbated atherosclerosis. Phagocytosis of RBCs by plaque macrophages promoted ferroptosis, suggesting a therapeutic target for reducing RBC-mediated cardiovascular risk.

Authors

Wenli Liu, Nataliya Östberg, Mustafa Yalcinkaya, Huijuan Dou, Kaori Endo-Umeda, Yang Tang, Xintong Hou, Tong Xiao, Trevor P. Fidler, Sandra Abramowicz, Yong-Guang Yang, Oliver Soehnlein, Alan R. Tall, Nan Wang

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Figure 3

VFEpoR RBCs, red pulp macrophages, and splenic CD11b+ cells show increased ROS and lipid peroxidation.

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VFEpoR RBCs, red pulp macrophages, and splenic CD11b+ cells show increas...
Control and VFEpoR mice were fed a Western diet and treated with LDLR ASO and EPO (3 times per week) for 12 weeks. (A) ROS in RBCs was assessed by H2 DCFDA staining and analyzed by flow cytometry as MFI. (B) Immunoblot and quantification of NOX2 expression in RBC lysates, which was normalized to β-actin. (C) RBC lipid peroxidation was assessed by C11 BODIPY staining and quantified by flow cytometry as MFI. (D) Splenic red pulp macrophages, CD11b+ cells, neutrophils, and Ly6Chi and Ly6Clo monocytes were stained with C11 BODIPY for lipid peroxidation and analyzed by flow cytometry. (E) Assessment of externalized phosphatidylserine level by annexin V staining in RBCs through flow cytometry. (F) Interaction network and clustering of upregulated proteins in VFEpoR RBCs. Bar graphs show relative abundance protein level of EIF2AK1 (also known as heme-regulated inhibitor, HRI) and EIF2A (eukaryotic translation initiation factor 2A). The lines between proteins represent interactions among proteins with confidence level; dashed line is the lowest and thick line is the highest confidence. (G) Proteins involved in cellular oxidant stress were analyzed and quantifications of relative abundance of glutamate cysteine ligase catalytic subunit (GCLC), glutathione transferase theta 1 (GSTT1), glutathione peroxidase 1 (GPX1) are shown. (H) The level of reduced and oxidized glutathione ratio in RBCs. Unpaired 2-tailed t test, *P < 0.05, **P < 0.01, ***P < 0.001.