CD14loCD301b+ macrophages gathering as a proangiogenic marker in adipose tissues

Figures

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  Fig. 1. CD14 deletion alleviates HFD-induced obesity with more angiogenesis in adipose tissues. (A) Body weight and (B) epWAT mass of HFD- or chow-fed WT and Cd14−/− mice. (C) GTT analysis for WT and Cd14−/− mice, and the significant difference was shown between HFD-induced WT and Cd14−/− mice. (D) Representative H&E staining (left) and adipocytes size quantification (right) for epWAT (scare bar: 50 μm). (E) GO functional enrichment analysis for epWAT. The color of each bubble represents the P value, and bubble size represents the gene number. (F) Cd31 mRNA expression in epWATs of HFD-fed mice. (G) Immunohistochemical staining for CD31 and Endomucin in epWAT of HFD-fed mice (Scare bar: 50 μm). (H) The frequencies of CD14+ ATMs in overweight/obese (BMI＞25 kg/m2) and healthy weight (BMI＜25 kg/m2) individuals (left panel) and CD31 mRNA levels in CD14hi and CD14lo individuals (right panel), n = 4. (I) Energy expenditure curves and (J) ANCOVA analysis in HFD-fed mice. In A: +P < 0.05, ++P < 0.01 mean the significant difference between chow-induced WT and Cd14−/− mice, and other ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 mean the significant difference between groups in this figure. epWAT, epididymal…

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  Fig. 2. Loss of CD14 upregulates the frequency of CD301b+ macrophages in adipose tissue. (A) Representative images of tube formation (left) and total tube length (right) by bEND.3 cells conditioned with supernatant of epWAT-harvested SVFs. (B) Proliferation assay for bEND.3 cell. (C) The proportion of F4/80+CD11b+ macrophages in epWAT of HFD-fed mice. (D) Volcano plot and heatmap representing the differentially expressed gene level in Cd14−/− relative to WT mice. The data were generated from RNA deep sequencing as described. (E) RT-qPCR analysis for the relative mRNA abundance of CD301b in epWAT. Frequencies of (F) CD301b+, (G) CD206+, and CD11c+ ATMs in epWAT of HFD-fed mice. (H) The frequencies of CD301(CLEC10A)-positive ATMs in overweight/obese and healthy weight individuals. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 mean the significant difference between groups, and ns means not significant. epWAT, epididymal adipose tissue.

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  Fig. 3. The proangiogenic potential of CD14lowCD301+ ATMs. (A, B) UMPA presentation of six monocyte/macrophage clusters in (A) human and (B) mice adipose tissues. Clusters were annotated manually based on their respective protein and mRNA expression profiles. (C–F) Expression of marker genes in each Mono/Mac cluster and constitutive share of CD14low CD301+ ATMs from (C, D) human and (E, F) mice. (G) Representative immunofluorescent images showed the expressions of CD31, CD301(Clec10a), CD68 in CD14high, and CD14low human AT sections and (H) of CD31, CD301b, F4/80 in WT, and Cd14−/− mice sections.

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  Fig. 4. Cd14−/− ATMs facilitate angiogenesis by secreting IGF-1. (A) Pseudotime analysis reveals differentiation of CD301b+ ATM in vivo. Left: UMAP plots depicting the inferred diffusion pseudotime of CD14lowCD301bhigh Mac in ATMs. Right: relative expression of Cd301b, Igf1, Pdgf, and Vegf across pseudotime. (B) ELISA assay for the concentration of IGF-1 released by SVFs. (C) RT-qPCR analysis for Igf1 in epWAT (left), SVFs (middle), and adipocytes (right). (D) The concentration of IGF-1 in supernatants of WT and Cd14−/− ATMs. (E) Tube formation by bEND.3 cells conditioned with ATMs supernatant. Scare bar: 100 μm. ∗P < 0.05, ∗∗∗P < 0.001 mean the significant difference between groups, and ns means not significant. epWAT, epididymal adipose tissue.

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  Fig. 5. Cd14−/− BMDMs promote angiogenesis via secreting IGF-1. (A) Induction of BMDMs. Frequencies of (B) CD301b+ and (C) CD206+ cells in IL-4 (20 ng/ml) or LPS (100 ng/ml) conditioned BMDMs. (D) Cd301b mRNA responsiveness to IL-4. (E) IGF-1 concentration in supernatants of BMDMs. (F) Proliferation of bEND.3 cells conditioned with BMDMs supernatants. (G) Tube formation by bEND.3 cells conditioned with BMDMs supernatants. (H) Proliferation and (I) tube formation with or without IGF-1 Abs blockade. Scare bar: 100 μm. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 mean the significant difference between groups, and ns means not significant. LPS, lipopolysaccharide.