Fig. 2: Topology of intimal myeloid cells in relation to the endothelium.

a, Cdh5^CreERT2;R26^tdTomato construct of the double transgenic mice used for 3D surface rendering of adult aortae to visualize the spatial location of intimal CD45⁺ cells (green) relative to the endothelium (tdTomato (tdTom)). VE-Cad is shown in white. Scale bars, 8 μm; n = 3 mice. Max-int. proj., maximum-intensity projection. b, Confocal and scanning electron microscopy (SEM) images of the same aorta were overlaid to determine the location of intimal CD45⁺ cells (green in the confocal view) in relation to the endothelium. ERG was used to visualize endothelial nuclei in confocal images. High-vac., high-vaccume. Scale bar, 10 μm; n = 3 mice. c, Sm22^Cre;R26^tdTomato construct of the double transgenic mice used for 3D rendering. Side view of a 3D surface image of adult aortae to visualize the spatial location of intimal CD45⁺ cells (green) relative to vascular smooth muscle cells (tdTom). Endothelial cells were visualized with anti-VE-Cad antibodies (white). Note that the green CD45⁺ cell is embedded in an area with white staining (VE-Cad). Scale bars, 5 μm; n = 3 mice. d, Schema of the experimental design related to e,g. Inj., injection; i.v., intravenous. e, Tail vein injection of rat anti-CD45 antibody (detected in green) was followed by euthanasia and fixation of the aorta 30 min later. Subsequent permeabilization and staining with an additional, distinct anti-CD45 antibody (detected using far red, labeled in white). The illustration was created using https://biorender.com/. e, En face images of the experiment described. Yellow arrowheads indicate the portions of immune cells exposed to the lumen (green). Mouse anti-CD45 biotinylated antibody labeled with streptavidin–A647 used in immunostaining after permeabilization is shown in white. PECAM1, red; 4,6-diamidino-2-phenylindole (DAPI), blue. Scale bar, 5 μm; n = 3 mice. f, En face images of the lesser curvature, staining for PECAM1 (green). Yellow arrowheads show faint PECAM1 positivity in CD45⁺ cells residing in the intima. Endothelial cells were detected in green with PECAM1. Scale bar, 5 μm; n = 6 mice. g, C57BL/6 mice were injected intravenously with a non-blocking PECAM1-specific (390) antibody to further examine exposure of cell bodies to the lumen. Mice were killed and harvested 15–30 min post-injection. 15 min after injection. CD45 (in green) was used to identify immune cells, and ERG (red) was used to identify endothelial cells. Yellow arrowheads indicate PECAM1⁺ regions of intimal immune cells. Scale bar, 4 μm; n = 3 mice.