Myeloproliferative neoplasm-driving Calr frameshift promotes the development of pulmonary hypertension in mice

Abstract

Frameshifts in the Calreticulin (CALR) exon 9 provide a recurrent driver mutation of essential thrombocythemia (ET) and primary myelofibrosis among myeloproliferative neoplasms (MPNs). Here, we generated knock-in mice with murine Calr exon 9 mimicking the human CALR mutations, using the CRISPR-Cas9 method. Knock-in mice with del10 [Calr^{del10/WT (wild-type)} mice] exhibited an ET phenotype with increases of peripheral blood (PB) platelets and leukocytes, and accumulation of megakaryocytes in bone marrow (BM), while those with ins2 (Calr^ins2/WT mice) showed a slight splenic enlargement. Phosphorylated STAT3 (pSTAT3) was upregulated in BM cells of both knock-in mice. In BM transplantation (BMT) recipients from Calr^del10/WT mice, although PB cell counts were not different from those in BMT recipients from Calr^WT/WT mice, Calr^del10/WT BM-derived macrophages exhibited elevations of pSTAT3 and Endothelin-1 levels. Strikingly, BMT recipients from Calr^del10/WT mice developed more severe pulmonary hypertension (PH)-which often arises as a comorbidity in patients with MPNs-than BMT recipients from Calr^WT/WT mice, with pulmonary arterial remodeling accompanied by an accumulation of donor-derived macrophages in response to chronic hypoxia. In conclusion, our murine model with the frameshifted murine Calr presented an ET phenotype analogous to human MPNs in molecular mechanisms and cardiovascular complications such as PH.

Keywords: CALR; Essential thrombocythemia; Macrophage; Myeloproliferative neoplasms; Pulmonary hypertension.

Fig. 1

Hematopoietic cells with Calr mutation exacerbate the development of pulmonary hypertension in response to chronic hypoxia. a The knock-in mice with C57BL/6 J background carrying frameshifted murine Calr, del10 (Calr^del10/WT mice) and ins2 (Calr^ins2/WT mice) were generated using the CRISPR-Cas9 method. Structure of wild-type (WT) and frameshifted murine CALR proteins are shown. Both generated mutant proteins with shortened calcium-buffering sites and absent KDEL sequence, which is the signal to retain the CALR protein in the endoplasmic reticulum. b Leukocyte (white blood cell) counts (WBC), red blood cell counts (RBC), and platelet counts (PLT) in WT mice (Calr^WT/WT mice, n = 21), Calr^ins2/WT mice (n = 17), and Calr^del10/WT mice (n = 16) in the peripheral blood. ^*P < 0.05 versus the WT group. c Schematic diagram of the experimental design of bone marrow (BM) transplantation (BMT). BM cells from control Calr^WT/WT mice or Calr^del10/WT mice were injected into the lethally irradiated WT mice (C57BL/6 J mice). Four weeks after BMT, the recipient mice transplanted with the BM cells from the Calr^WT/WT mice (WT-R) or Calr^del10/WT mice (del-R) were subjected to normoxia (21% O₂) or chronic hypoxia (10% O₂) for 3 weeks. d Allele frequency of the mutant Calr in the peripheral leukocytes of recipient mice at 4 weeks after BMT (n = 15, each). e Right ventricular (RV) systolic pressure (RVSP) and RV hypertrophy determined by dividing the RV weight by the left ventricular weight including the septum (RV/LV + S) (n = 6–8). f Representative hematoxylin–eosin (HE) staining and immunohistochemistry with antibodies to anti-α smooth muscle actin (αSMA) and anti-F4/80 images in the lung of BMT recipient mice from Calr^WT/WT or Calr^del10/WT mice. Scale bars, 50 µm. g Quantitative analysis of the percentage of muscularized distal pulmonary arteries in αSMA-immunostained sections (n = 3, each). h Quantitative analysis of the pulmonary perivascular macrophages determined as F4/80-positive cells, per 30 vessels (n = 5, each). Data are presented as means ± SEM. d, e, g, h ^*P < 0.05 versus the corresponding normoxia group and ^†P < 0.05 versus the corresponding BMT recipient mice from Calr^WT/WT mice. WT-R, recipient mice transplanted with BM cells from Calr^WT/WT mice; del-R, recipient mice transplanted with BM cells from Calr^del10/WT mice. Oligonucleotides and antibodies used are listed in Additional files 8, 9

Fig. 2

STAT3 phosphorylation and Endothelin-1 expression in the lung and macrophages from Calr^del10/WT mice. a Western blot of lung homogenates of the BMT recipients from Calr^WT/WT mice (WT-R) or Calr^del10/WT mice (del-R), immunoblotted with the indicated antibodies. b Phosphorylated STAT3 (p-STAT3) to total STAT3 (t-STAT3) or Endothelin-1 to β-actin ratios are shown in the graphs. The average value for WT-R under normoxia was set to 1 (n = 5, each). ^*P < 0.05 versus the corresponding normoxia group and ^†P < 0.05 versus the corresponding WT-R. c The lethally irradiated WT C57BL/6 J mice were transplanted with the BM cells from Calr^del10/WT/CAG-EGFP mice. These recipient mice were subjected to chronic hypoxia for 3 weeks, and then the lungs were fixed and stained with the indicated antibodies. Upper images show representative immunofluorescence of the lung sections stained with anti-GFP (green) and anti-αSMA (red) antibodies and DAPI (blue). Scale bars, 50 µm. Lower images show representative immunofluorescence of the lung sections stained with anti-GFP (green) and anti-F4/80 (red) antibodies and DAPI (blue). Scale bars, 10 µm. d-g BM mononuclear cells isolated from the Calr^WT/WT or Calr^del10/WT mice were cultured in the presence of 10 ng/mL of M-CSF for 6 days. d Representative immunofluorescence images of the cells stained with anti-F4/80 (green) and DAPI (blue) are shown. More than 90% of cells were macrophages expressing F4/80. Scale bars, 25 µm. e Dot plot of flow cytometry for cultured macrophages. Red, blue, and orange dots represent cells from Calr^WT/WT mice, Calr^del10/WT mice, and negative control (mixture of WT and Calr del10 cells), respectively. Over 90% WT and del10 cells were positive for both F4/80 and CD68. SSC indicates side scatter. f The cultured macrophages were then stimulated with 0.05 µg/mL of lipopolysaccharide (LPS), a potent activator of macrophages. The mRNA expression levels of Endothelin-1 (Edn1) were analyzed at the indicated time (n = 8, each). Actb was used for normalization. The average value for the macrophages from Calr^WT/WT mice at baseline was set to 1. g Left panels show western blots on STAT3, Endothelin-1, and β-actin in the macrophages stimulated with 0.05 µg/mL of LPS. Right graphs show phosphorylated STAT3 (p-STAT3) to total STAT3 (t-STAT3) or Endothelin-1 to β-actin ratios at the indicated time. The average value for the macrophages from Calr^WT/WT mice at the baseline was set to 1 (n = 4, each). All data are presented as means ± SEM. ^*P < 0.05 versus the corresponding WT group. WT, macrophages derived from the Calr^WT/WT mice; del10, macrophages from the Calr^del10/WT mice. Oligonucleotides and antibodies used are listed in Additional files 8, 9