Fig 2: Pharmacological blockade of vascular tissue resident macrophage cells attenuates JAK2V617F-mediated dissecting aortopathy.A Experimental protocol of Ki20227 treatment in C57BL/6 male mice. B Immunostaining of CD68 + LYVE-1+ macrophages (Yellow) in the aorta of Ki20227- or PBS-treated mice (N = 5/group), scale bar 50 µm. C Representative dot plot of CD45 + CD11b + Ly6G-Ly6C-CD64 + F4/80+ macrophage count in the aorta of Ki20227- or PBS-treated C57BL/6 mice. D Red blood cell, white blood cell and platelet count of Ki20227- or PBS-treated C57BL/6 mice. E experimental protocol of PBS or Ki20227 treatment in Jak2WT and Jak2V617F Myel mice. Jak2WT (blue, N = 8) and Jak2V617F Myel (Green, N = 8) mice received Ki20227 and a third group of Jak2V617F Myel (Red, N = 12) only received control diet without pharmacological drug. F survival curve of treated mice. G Immunostaining of CD68 + LYVE-1+ macrophages (Yellow) in the aorta of surviving Ki20227-treated Jak2WT (blue) and Jak2V617F Myel (green) mice (N = 8/group), scale bar 50 µm. H Quantification of Il6, Tnfa, Il1ß and Ccl2 mRNA expression by RT-qPCR in the aorta of surviving Ki20227-treated Jak2WT (blue) (N = 8) and Jak2V617F Myel (green) (N = 7) mice. I Quantitative analysis of the mean aortic diameter of surviving Ki20227-treated Jak2WT (blue) and Jak2V617F Myel (green) mice (N = 8/group) in the thoracic ascending (Asc.), thoracic descending (Desc.) and the abdominal (Abd.) aorta. J Experimental protocol describing ruxolitinib (60 mg/kg, daily during 5 weeks) or PBS treatment in Jak2V617F HC-EC mice. K quantification of Il6, Tnfa, Il1ß and Ccl2 mRNA expression by RT-qPCR in the aorta of Jak2V617F HC-EC mice treated with PBS (Red) (N = 10) or Ruxolitinib (Green) (N = 8). L Quantitative analysis of the mean aortic diameter of PBS (Red, N = 7) or ruxolitinib (Green, N = 8) -treated Jak2V617F HC-EC mice using ultrasonography in the thoracic ascending (Asc.), thoracic descending (Desc.) and the abdominal (Abd.) aorta. M experimental protocol describing Ruxolitinib (60 mg/kg, daily during 10 weeks) or PBS treatment in Jak2V617F Myel mice. N survival curve of treated mice (N = 11 PBS and N = 7 Ruxolitinib). Data are presented as mean values ± SD. [Two-tailed Mann-Whitney test or Kruskal-Wallis H, I, *P < 0.05, **P < 0.01, ***P < 0.001]. Difference in survival was evaluated using log-rank test. HC-EC, hematopoietic cells-endothelial cells; Il, interleukin; Tnf, tumor necrosis factor. Source data are provided as a Source Data file.

Fig 3: Validated flow cytometry gating strategy for murine myeloid cells, related to Figure 2(A) CITE-seq data from the murine myeloid cells in Figure 2A were exported as an FCS file and an in silico gating strategy identified in FlowJo software.(B) Application of the in silico gating strategy with a 21-color flow cytometry panel. Myeloid cells were pre-gated as live CD45+ lineage cells (Ly6G−CD19−NK1.1−B220−CD3−). Data are representative of 3 experiments with 3–6 mice per experiment.(C) cDC1s, cDC2s, migratory cDCs (Mig. cDCs), peritoneal macs (Peri. Macs), KCs, and non-KC macs (non-KCs) were FACS-purified using gating strategy in (B), mRNA was isolated and qPCR performed to examine expression of indicated genes defining each population to validate their identity. Data are representative of 2 experiments with n = 3–6.(D) Putative peritoneal macs were FACS-purified using gating strategy in (B) and expression of Gata6 was examined by qPCR compared with other hepatic myeloid populations. Data are from a single experiment with n = 6.(E) Peritoneal macs as a % of total macs recovered from the liver using different digestion techniques (in vivo, ex vivo, or capsule) or in supernatants in which livers were washed following removal from the mouse but prior to digestion (wash). Data are from a single experiment with n = 4. ∗p < 0.05, ∗∗p < 0.01 one-way ANOVA with Bonferroni post-test compared with wash data.(F) Expression of CD14 and CD207 within the non-KC mac population from (B) (left) and % of CD207+ and CD207− populations among total macs in livers digested using the ex vivo or in vivo protocols or in dissected and digested liver capsule (right). Data are representative of two experiments with n = 4–5 mice per experiment. ∗∗∗∗p < 0.0001 mixed effects analysis with Tukey’s multiple comparison test.(G) Expression of VSIG4, F4/80, GLUL, and DAPI by confocal microscopy. Insets represent zones featured in Figures 2E, 2G, and S3I.(H) Molecular Cartography of indicated genes and cell types. Insets represent zones featured in Figures 2F, 2H, and S3J.(I) Expression of VSIG4, F4/80, GLUL, and DAPI by confocal microscopy at the central vein. Scale bars, 50 μm.(J) Molecular Cartography of indicated genes and cell types at central vein.(K) Expression of F4/80, EPCAM, CCR2, GPNMB, and DAPI by confocal microscopy at a portal vein (top) or F4/80 or GPNMB alone (bottom). Scale bars, 25 μm.(L) Quantification of % of Gpnmb & Trem2 counts over Adgre1 counts in indicated regions of tissue as assessed using Molecular Cartography data. Each dot represents an individual region. ∗p < 0.05, ∗∗∗∗p > 0.0001 one-way ANOVA with Bonferroni post-test.(M) Expression of DESMIN and F4/80 at the liver capsule and underlying parenchyma (left) or EPCAM, DESMIN and F4/80 at the bile duct by confocal microscopy. PV, portal vein; CV, central vein; HA, hepatic artery; BD, bile duct. Arrows indicate specific cell types, where color corresponds to cell type/markers. All images are representative of 2–6 mice.

Fig 4: JAK2V167F mutation induces pro-inflammatory signature in the aortic wall.A Difference in gene expression in the aorta of 7-week-old Jak2WT (N = 4) and Jak2V617F HC-EC mice (N = 3). B Top Gene Ontology terms mostly enriched among genes upregulated in the aorta of Jak2V617F HC-EC mice when compared to Jak2WT mice. C Flow cytometry quantification of CD45 + CD11clowCMHIIlowCD11b + Ly6G-F4/80lowCD64lowLy6C + monocytes and CD45 + CD11clowCMHIIlowCD11b + Ly6G+ neutrophils in the aorta of 7-week-old Jak2WT (N = 10) and Jak2V617F HC-EC animals (N = 8). D Flow quantification and representative dot plot of CD45 + CD11clowCMHIIlowCD11b + Ly6G-F4/80highCD64high macrophage count in the aorta of 7-week-old Jak2WT (N = 10) and Jak2V617F HC-EC animals (N = 8). E Quantification and representative photomicrographs of CD68 positive macrophages in the aorta of 7-week-old mice (N = 11 in control group and N = 8 in Jak2V617F HC-EC group), scale bar 50 µm. F Quantification of Il6, Tnfa, Il1ß, and Il10 mRNA expression by RT-qPCR in the aorta of 7-week-old Jak2WT(N = 8) and Jak2V617F HC-EC animals (N = 9). G Immunofluorescence staining in the aortic wall of Jak2WT and Jak2V617F HC-EC mice, DAPI (Blue), CD68 (Red), LYVE-1 (Green), scale bar 50 µm. resident macrophages were defined as CD68 + LYVE-1+ cells (N = 5/group). H GSEA for genes related to a gene set signature for tissue resident macrophage19 in the aorta of 7-week-old Jak2WT and Jak2V617F HC-EC animals (N = 4/group). Data are presented as mean values ± SD. [Two-tailed Mann-Whitney test, *P < 0.05, **P < 0.01, ***P < 0.001]. HC-EC, hematopoietic cells-endothelial cells; Il, interleukin; Tnf, tumor necrosis factor; GSEA, gene set enrichment analysis. Source data are provided as a Source Data file.