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 2

Increased lesion area, plaque instability, and iron deposition in VFEpoR mice after EPO injection.

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Increased lesion area, plaque instability, and iron deposition in VFEpoR...
Control and VFEpoR mice were fed a Western diet and treated with LDLR ASO and EPO (3 times per week) for 12 weeks. (A) H&E staining of aortic root sections and quantification of absolute lesion and necrotic core area. Necrotic core regions indicated by broken lines. Scale bar: 200 μm. (B) Aortic root sections were stained with Masson’s trichrome staining for fibrous cap (red, outlined by broken lines) and collagen (blue) content area, and then quantified as the ratio of total lesion area. Scale bar: 100 μm. (C) Iron (Perl’s blue) and redox-active iron deposition (Perl’s blue + DAB), IHC staining of RBCs (anti-Ter119) and macrophages (anti-Mac2) in aortic roots. Bar graph shows quantification of iron (II + III)–positive and erythrophagocytosis (Ter119+Mac2+) cell counts per section. Scale bar: 100 μm. (D) TUNEL and immunofluorescence staining of macrophage (anti-Mac2) in aortic roots and quantification of TUNEL-positive cell counts per section. Scale bar: 50 μm. (E) Lipid peroxidation product 4-HNE staining, quantified as the percentage of total lesion area. Scale bar: 100 μm. (F) Immunofluorescence staining of TfR1 and macrophage (anti-Mac2), and quantification of TfR1 and macrophage costaining cell counts per section. Scale bar: 50 μm. (G) Aortic root sections were immunostained for Mac2 and quantified as absolute Mac2-positive area. Scale bar: 250 μm. (H) Lesions were stained for citrullinated histones (H3Cit) and activated neutrophils using myeloperoxidase (MPO); the overlap H3cit and MPO (NETs) cell counts were quantified. Scale bar: 50 μm. Unpaired 2-tailed t test or Mann-Whitney U test, *P < 0.05, **P < 0.01, ***P < 0.001.