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