VAMs rapidly and efficiently uptake blood macromolecules. (A) Heat map of genes differentially expressed in VAMs displaying anti-inflammatory, insulin-sensitizing, repair, and detoxifying gene signatures. Each column represents consolidated data from three biological replicates. The z-score of the gene expression profiles gives a scale to measure the differential expression. Adult (average 16-wk-old, range 12–20) WT males were used. (B) Volcano plot comparing the top 7,000 genes expressed by VAM2 and CD11b⁺ DCs from eWAT. Signature genes are labeled in red. Consolidated data from at least three biological replicates. (C) VAM gene signature, analyzed by functional enrichment, reveals an association with endocytic activity. Consolidated data from at least three biological replicates. (D) VAMs are intimately associated with eWAT vessels. Representative confocal images of epididymal full-mount sections showing the distribution of VAMs (CD206⁺MHCII⁺DAPI⁺) in close contact to vessels labeled with isolectin. Bars, 10 µm. 60× magnification. For contextualization, the three upper panels display the following: VAM among adipocytes, which are shown translucently by the bright field channel (left); blood vessel labeled with isolectin (center); VAM labeled as CD206⁺MHCII⁺DAPI⁺ (right); n = 3, representative of five independent experiments. (E) VAMs are highly endocytic in vivo. Dextran-rhodamine 70 kD was injected i.v. 20 min after the injection, animals were anesthetized and perfused intracardially. Endocytic activity was evaluated by flow cytometry, which measured rhodamine fluorescence. Histograms display the fluorescence intensity for rhodamine of eWAT and splenic populations. Untr, untreated. CD11b⁻DC (CD90⁻CD19⁻CD11b⁻MHCII⁺CD11c⁺) Eos, eosinophils (CD90⁻CD19⁻CD11b⁺SiglecF⁺); Neu, neutrophils (CD90⁻CD19⁻CD11b⁺Ly6G⁺); CD11b⁺DC (CD90⁻CD19⁻CD11b⁺MHCII⁺CD11c⁺) Mon, monocytes (CD90⁻CD19⁻CD11b⁺SiglecF⁻Ly6G⁻MHCII⁻Ly6C^HIGH); MZ Mac, marginal zone macrophages (CD90⁻CD19⁻CD11b^INTCD64⁺CD206⁺MHCII⁺). Representative histogram; n = 4, representative of three independent experiments. (F) Fold increase of the median rhodamine fluorescence intensity after injection, normalized to the individual autofluorescence of each subpopulation (n = 4, representative of three independent experiments). (G) Intravital microscopy analysis of eWAT dextran-rhodamine uptake 5 min after i.v. injection (Video 1). qDots (Qtracker 655) and Hoechst were i.v. injected immediately before the beginning of the recording, while dextran-rhodamine was injected while the recording was taking place. 25× magnification. Bars, 10 µm. Representative image; n = 3, representative of two independent experiments. The arrows indicates a small field in which VAMs are uptaking dextran-rhodamine. (H) VAMs high endocytic capacity in vivo extends to proteins. Whole ovalbumin-A647 (Ova) was injected i.v., and 20 min after injection, animals were analyzed as described in the legend of B. Ptn, protein; MAC, macrophage. Adult (12–16-wk-old) WT males were used. Representative histogram; n = 3, representative of two independent experiments. (I) Light sheet microscopy of full epididymal fat pad after WAT clarification using Adipo-Clear technique (Chi et al., 2018; Video 2). CD31 marks blood vessels. Animals were injected with dextran-rhodamine and, after 1 h, submitted to sample preparation for Adipo-Clear. Panel shows VAMs around blood vessels from different z stacks. Bar, 50 µm. 4× magnification. Representative image; n = 3, representative of three independent experiments. (J) Analysis of VAM association with blood vessels was performed using Imaris/Bitplane software. Bar graph represent aggregated results of 2,234 macrophages analyzed distributed over 300 stacks of images acquired in I. (K) Representative confocal images of epididymal full-mount sections showing the distribution of VAMs (dextran-rhodamine⁺DAPI⁺) in close contact to blood vessels labeled with Isolectin. Bodipy labels lipid droplets, in macrophages and adipocytes. Bars, 5 µm. 63× magnification. Microscopy performed in a high-resolution Zeiss LSM 880 with Airyscan. Lipid droplets inside VAMs are indicated by arrows (Video 3). Representative image; n = 3, representative of two independent experiments. Bar graphs are shown as mean ± SEM.