BMP4-mediated browning of perivascular adipose tissue governs an anti-inflammatory program and prevents atherosclerosis

Figures

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  Fig. 1. Interaction between BMP4, PVAT metabolism and CAD. (A)Western blot analysis and quantification (B) of UCP1 and BMP4 expression in different adipose tissues (n = 3). (C) Relative mRNA levels of UCP1 and BMP4 were measured by qPCR. (n = 9). (D) Representative images corresponding to en face Oil Red O staining of the atherosclerotic lesion of whole aortas from WT and ApoE−/− mice. (E) Western blot analysis and quantification (F) of UCP1, PGC1α and BMP4 in PVAT of WT or ApoE−/− mice. (G) Relative mRNA levels of BMP4 UCP1, Cidea and PGC1α were measured by qPCR. (n = 3). (H) Correlation analysis of co-expression of BMP4 and UCP1 in human SAT, EAT and PAT. (I) Correlation analysis of co-expression of BMP4 and brown-related genes (PGC1α, Cidea, PRDM16) in human SAT, EAT and PAT. (J) Relative mRNA levels of BMP4 were determined in PAT and EAT of patients with coronary artery diseases (CAD) or those without. Values are means ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student's t-test (F, G and J) or one-way analysis of variance (ANOVA) (B and C). Pearson's correlations between BMP4, and brown-related genes were calculated. (For interpretation of the references to colour in…

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  Fig. 2. Absence of BMP4 in PVAT produces a white-like phenotype. (A) Oil Red O staining of adipocytes differentiated from brown preadipocytes on day 8. (B) Western blot analysis of UCP1, Cidea, PGC1α, aP2, PPARγ and C/EBPβ in brown adipocytes with LacZ knockdown or BMP4 knockdown. (C) Relative mRNA levels of BMP4, UCP1, Cidea, PGC1α, aP2, PPARγ and C/EBPα in brown adipocytes with LacZ knockdown or BMP4 knockdown. (D)Schema of FABP4/aP2-Cre-BMP4flox/flox mouse models. (E) Gross picture of adult mouse thoracic aortic PVAT. (F) Representative HE and IHC(UCP1) microscopic picture of PVAT isolated from BMP4fl/fl ApoE−/− and BMP4ΔaP2 ApoE−/− mice fed with WD for 16 weeks. (G) Western blot analysis and quantification (H) of BMP4, UCP1 PGC1α, aP2, PPARγ and C/EBPβ in PVAT of BMP4fl/fl ApoE−/− and BMP4ΔaP2 ApoE−/− mice fed with WD for 16 weeks (n = 6–9). (I) Relative mRNA levels of BMP4, UCP1, Cidea, PGC1α, aP2, PPARγ and C/EBPα in PVAT of BMP4fl/fl ApoE−/− and BMP4ΔaP2 ApoE−/− mice fed with WD for 16 weeks (n = 7). (J) Schema of UCP1-Cre-BMP4flox/flox mouse models. (K) Representative HE and IHC(UCP1) microscopic picture of PVAT isolated from BMP4fl/fl ApoE−/− and BMP4ΔUCP1 ApoE−/− mice fed with…

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  Fig. 3. Absence of BMP4 in PVAT decreases the fatty acid metabolism. (A) Pie graph of RNA sequencing comparing PVAT of BMP4fl/fl ApoE−/− with BMP4ΔaP2 ApoE−/− mice. (B) Volcano plot of RNA sequencing comparing PVAT of BMP4fl/fl ApoE−/− with BMP4ΔaP2 ApoE−/− mice. (C) Gene ontology analysis for downregulated genes in KEGG pathways, pathways with p value < 0.00015 were listed. (D) Down-regulated genes in PVAT of BMP4ΔaP2 ApoE−/− mice identified by RNA sequencing are marked in red. (E) qPCR validation of genes selected from the pathways in (D). (F) Western blot analysis and quantification (G) of FASN, SCD1, HSL and ATGL in PVAT of BMP4fl/fl ApoE−/− and BMP4ΔaP2 ApoE−/− mice fed with WD for 16 weeks (n = 9). (H) Western blot analysis and quantification (I) of FASN, SCD1, HSL and ATGL in PVAT of BMP4fl/fl ApoE−/− and BMP4ΔUCP1 ApoE−/− mice fed with WD for 16 weeks (n = 3). (J) Western blot analysis of FASN, SCD1, HSL and ATGL in brown adipocytes with LacZ knockdown or BMP4 knockdown. (K) Relative mRNA levels of FASN, SCD1, HSL and ATGL in brown adipocytes with LacZ knockdown or BMP4 knockdown. Values are means ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student's t-test (E, G, I,…

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  Fig. 4. BMP4 knockout adipocyte increase endothelial inflammation in vitro. (A) Relative mRNA levels of BMP4 in brown adipocytes with BMP4 knockdown and the controls. (B) Relative mRNA levels of VCAM-1, ICAM-1, MCP1, IL-6 and IL1β in TNFα pretreated HUVECs incubated with conditioned medium from brown adipocytes with BMP4 knockdown and the controls. (C) Western blot analysis of VCAM-1 expression in TNFα pre-treated HUVECs incubated with conditioned medium from brown adipocytes with BMP4 knockdown and the controls. (D) Relative mRNA expression levels of BMP4 in brown adipocytes with BMP4 overexpression and the controls. (E) Relative mRNA levels of VCAM-1, ICAM-1, MCP1, IL-6 and IL1β in TNFα pre-treated HUVECs incubated with conditioned medium from brown adipocytes with BMP4 overexpression and the controls. (F) Western blot analysis of VCAM-1 expression in TNFα pre-treated HUVECs incubated with conditioned medium from brown adipocytes with BMP4 overexpression and the controls. (G)Schematic of the transwell coculture chamber. (H) Relative mRNA levels of VCAM-1, ICAM-1, MCP1, IL-6 and IL1β in TNFα pre-treated HUVECs that were co-cultured for 24 h with PVAT isolated from BMP4fl/fl ApoE−/− and…

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  Fig. 5. BMP4 deficiency accelerates atherosclerotic plaque formation in ApoE−/−mice. (A) En face Oil Red O staining of whole aortas from BMP4fl/fl ApoE−/−, BMP4ΔaP2 ApoE−/− mice fed with WD for 16 weeks. (B) Quantification of atherosclerotic lesion size in the whole aortas (n = 7). (C) Representative oil red O staining of cross-sections of aortic sinus. (D) Quantification of positive area for oil red O staining (n = 8–9). (E) Quantification of average lesion size in aortic sinus (n = 8–9). (F) Western blot analysis and quantification (G) of VCAM-1 expression (n = 3). (H) The levels of plasma total triglyceride (TG), cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C) in mice (n = 17). (I) En face Oil Red O staining of whole aortas from BMP4fl/fl ApoE−/−, BMP4ΔUCP1 ApoE−/−mice fed with WD for 16 weeks. (J) Quantification of atherosclerotic lesion size in the whole aortas (n = 7). (K) Representative oil red O staining of cross-sections of aortic sinus. (L) Quantification of positive area for oil red O staining (n = 5–6). (M) Quantification of average lesion size in aortic sinus (n = 5–6). (N) Western blot analysis and…

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  Fig. 6. Overexpression of BMP4 in adipocyte promotes browning of PVAT and inhibits atherosclerosis in ApoE−/−mice. Male ApoE−/− and BMP4-Tg ApoE−/− mice were fed with WD for 20 weeks. (A) Representative HE and IHC(UCP1) microscopic picture of PVAT isolated from ApoE−/− and BMP4-Tg ApoE−/−mice. (B) Western blot analysis and quantification (C) of BMP4, UCP1 PGC1α, aP2, PPARγ and C/EBPβ in PVAT of ApoE−/− and BMP4-Tg ApoE−/− mice (n = 4). (D) Relative mRNA levels of UCP1, Cidea PGC1α, aP2, PPARγ and C/EBPα in PVAT of ApoE−/− and BMP4-Tg ApoE−/− mice (n = 6). (E) En face Oil Red O staining of whole aortas from ApoE−/− and BMP4-Tg ApoE−/− mice. (F) Quantification of atherosclerotic lesion size in the whole aortas (n = 7). (G) Representative Oil Red O staining of cross-sections of aortic sinus. (H) Quantification of positive area for oil red O staining (n = 3–5). (I) Quantification of average lesion size in aortic sinus (n = 3–5). (J) Western blot analysis and quantification (K) of VCAM-1 expression (n = 3). (L)The levels of plasma TG, TC, LDL-C and HDL-C in mice (n = 14–16). Values are means ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student's t-test (C, D, F, H, I, K and L).…

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  Fig. 7. BMP4 knockdown in brown adipocytes increase adipocyte inflammation and pro-inflammatory factors secretion. (A) Gene ontology analysis for up-regulated genes in KEGG pathways, pathways with p value < 0.00015 were listed. (B) qPCR validation of genes selected from the T cell activation pathways. (C) Immunohistochemical staining of F4/80 expression in PVAT of BMP4fl/fl ApoE−/− and BMP4ΔaP2 ApoE−/− mice. The right graph shows the quantification of F4/80 positive cells in C (n = 5). (D) Relative mRNA levels of BMP4, UCP1, Cidea, PGC1α, aP2, PPARγ and C/EBPα in PVAT of BMP4fl/fl ApoE−/− and BMP4ΔaP2 ApoE−/− mice fed with WD for 16 weeks (n = 5). (E) Immunohistochemical staining of F4/80 expression in PVAT of BMP4fl/fl ApoE−/− and BMP4ΔUCP1 ApoE−/− mice. The right graph shows the quantification of F4/80 positive cells in C (n = 5). (F) Relative mRNA levels of BMP4, UCP1, Cidea, PGC1α, aP2, PPARγ and C/EBPα in PVAT of BMP4fl/fl ApoE−/− and BMP4ΔUCP1 ApoE−/− mice fed with WD for 16 weeks (n = 5). (G) Immunohistochemical staining of F4/80 expression in PVAT of ApoE−/− and BMP4-Tg ApoE−/− mice. The right graph shows the quantification of F4/80 positive cells in C (n = 3–4). (H) Relative…

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  Fig. 8. BMP4 knockdown impair PGC1α mediated fatty acid metabolism and activate NF-κB signaling in brown adipocytes.(A)Relative mRNA levels of PGC1α in brown adipocytes with PGC1α knockdown or lacZ knockdown. (B) Relative mRNA levels of TNFα，IL1β, IL-6 and MCP1 in brown adipocytes with PGC1α knockdown or lacZ knockdown. (C) Relative mRNA levels of VCAM-1, ICAM-1, MCP1, IL-6 and IL1β were determined in TNFα pre-treated HUVEC incubated with conditioned medium from brown adipocyte with PGC1α knockdown or lacZ knockdown. (D) Oil Red O staining of brown adipocytes differentiated from SVF on day 8. (E) Relative mRNA levels of PGC1α in brown adipocytes differentiated from SVF with PGC1α knockdown or lacZ knockdown. (F) Relative mRNA levels of TNFα，IL1β, IL-6 and MCP1 brown adipocytes differentiated from SVF with PGC1α knockdown or lacZ knockdown. (G) Relative mRNA levels of VCAM-1, ICAM-1, MCP1, IL-6 and IL1β were determined in TNFα pre-treated HUVEC incubated with conditioned medium from brown adipocytes differentiated from SVF with PGC1α knockdown or lacZ knockdown. (H) Western blot analysis of VCAM-1 expression in HUVECs incubated with conditioned medium from brown adipocytes differentiated…