Fig 2: AR is important for bone marrow monocytic development. BM of WT and MARKO male mice was analyzed by flow cytometry for monocytic and macrophage cell populations. (A) The upper plot represents UMAP analysis of live singlet lineage negative (CD3, Ly-6G, B220, Ter119) BM cells from WT and MARKO male mice. The lower plot in (A) is colored according to sample group. (B) UMAP graphs indicating intensities of c-Kit, CD16/32, Ly-6C, CD115, CD11b and F4/80 expression in WT and MARKO BM. (C–G) Quantification of BM cell populations utilizing gating schemes in Supplementary Figures 1A, D, F. Plots depict percentage and numbers of singlet live (C) oligopotent GMP (oGMP), (D) monocytic progenitors (MoPs), (E) CD11b+Ly6C+CD115+F4/80- cells, (F) CD11b+Ly6C-CD115-F4/80- cells, and (G) CD11b+Ly6C-CD115-F4/80+ cells. Graphs show pooled data from two to four experiments with two to three mice per group. Black filled squares denote WT, and red empty squares indicate MARKO BM. Comparisons were done with non-parametric t-test. ns, not significant; *p = 0.05, **p = 0.01.

Fig 3: Intratumoural injection of cGAMP strongly induces tumour vascular disruption in implanted LLC tumour.a-c Diagram depicting generation of implanted LLC tumour in B6 mice and treatment schedule of intratumoural (i.t.) PBS or cGAMP. Comparison of LLC tumour growths. n = 6 mice/group from two independent experiments. Dots and bars indicate mean ± SD. Plot indicates each individual tumour growth. d–f Diagram depicting generation of LLC tumour, i.t. PBS or cGAMP treatment, and tumour sampling at 24 h later. Representative images and comparisons of apoptosis in tumour ECs and whole tumour cells (whole cells). Dashed lines demarcate tumour vascular lining, while white arrowheads indicate apoptotic ECs. Scale bars, 1.0 mm (yellow bars) and 50 μm (white bars). Each dot indicates a value from one mouse and n = 8 mice/group from two independent experiments. Vertical bars indicate mean ± SD. g–k Representative images showing temporal responses of tumour ECs and whole tumour cells following i.t. cGAMP treatment in implanted LLC tumour. n = 6 mice/group. Note that progressive tumour EC apoptosis followed by extensive tumour cell apoptosis. Scale bars, 50 μm (g). Representative flow cytometry plots and comparisons showing gradual changes of live EC and dead cell populations. Each dot indicates a value from one mouse and n = 6 mice/group from three independent experiments. Vertical bars indicate mean ± SD. l, m Impaired vascular function 24 h after i.t. cGAMP treatment. Representative Images and comparisons of dextran leakage (n = 4 images/4 mice), TER119+ RBC leakage (n = 7 images/7 mice), lectin+ vascular perfusion (n = 5 images/5 mice) and GLUT1+ hypoxic area (n = 7 images/7 mice). Scale bars, 100 μm. Each dot indicates a value from one mouse from four independent experiments. Horizontal bars indicate mean ± SD. P values by two-tailed t-test (b, f, m), Kruskal–Wallis test followed by Dunn’s test (i) or Welch’s one-way ANOVA test followed by Dunnett’s T3 test (k). Source data are provided as a Source Data File.

Fig 4: AdvSca1-SM cells are distributed throughout the vascular wall and atherosclerotic plaque.A. Schematic of experimental approach. B. A subset of animals were injected intravenously with fluorescently labeled Griffonia simplicifolia lectin I (GSL I) isolectin B4 5 minutes prior to sacrifice to label functional vasculature in vivo. Representative immunofluorescence of aortic root sections from 24-week plaques stained for YFP (AdvSca1-SM and AdvSca1-SM-derived cells; green); lectin (red); DAPI for all cell nuclei (blue). Arrows indicate functional adventitial microvasculature (lectin labeled) surrounded by YFP+ cells; arrowheads indicate intraplaque YFP+ cells. C. Representative immunofluorescence image of 28-week aortic root plaque stained for aSMA (red) and YFP (green). Arrows indicate YFP+ medial cells. D. and E. Aortic root slides from 24-week plaques stained for YFP (green), aSMA (red), Ter-119 (magenta), and DAPI (blue). Low power and high-power insert (D) show YFP+ cells in the cap of the plaque. Panel E shows YFP+ cells in the plaque cap (arrows), YFP+/aSMA+ cells in the plaque cap (asterisk), and YFP+ cells in the body of the plaque (arrowheads). Scale bars for all immunofluorescence images are 50 µm, except low power panel D (100 µm).

Fig 5: AdvSca1-SM cells primarily differentiate into fibroblasts or remain in a stem-like state in atherosclerosis, but can also differentiate into SMCs.A. Feature plots showing the distribution of YFP+ and YFP- cells on the UMAP. B. Feature plots of major fibroblast (Col1a1, Col1a2, Col3a1, Dcn, Lum, and Tcf21) and stem cell markers (Ly6a/Sca1, Cd34, Scara5, Pi16) in YFP+ cells at all time points. C. Representative aortic root image from 24-week plaque stained for YFP (green), Sca1 (red), and DAPI (blue). Arrows indicate YFP+/Sca1+ cells in the adventitia; arrowheads indicate YFP+/Sca1- cells. * = cardiomyocyte autofluorescence. D. Aortic sinus, aortic arch, brachiocephalic artery, and carotid arteries from 16-week control or atherosclerotic mice were processed for FACS analysis. Representative image of a YFP and Sca1 density plot from an atherogenic animal. E. Double RNAscope and immunofluorescence image of an aortic root from 24-week atherogenic animals showing lumican (Lum; fibroblast cell marker; red) mRNA and YFP (green). Arrows indicate YFP+/lumican+ cells; arrowheads indicate YFP+/lumican- cells. F. Feature plots of SMC genes (Acta2, Cnn1, Mhy11, Tagln) in YFP+ cells at all time points. G. 24-week aortic root plaques were stained for YFP (green), aSMA (red), Ter-119 (erythroid cells; magenta), and DAPI (blue). Arrows indicate YFP+/aSMA+ cells forming the fibrous cap of the plaque. H. Stacked bar plot showing phenotypic shifts in YFP+ cells between 16-week control and atherogenic samples. Arrows indicate increases (red) or decreases (blue) in the cell population as a result of atherosclerosis. I. GO Biological Process between the Fib_2 cluster and all other cell clusters. Arrows indicate the top 4 processes positively associated with Fib_2. J. Violin plots demonstrating the stronger collagen/extracellular matrix gene signature (Col1a1, Col1a2, and Col3a1) in Fib_2 compared to Fib_3. All scale bars, 50µm.