Brown remodeling of white adipose tissue protects against abdominal aortic aneurysm via batokine FSTL1

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  Figure 1. Browning of adipose tissue protects against AAA in vivo. (A) Representative immunofluorescent staining of uncoupling protein 1 (UCP-1, red) and Perilipin 1 (green) in perivascular adipose tissue (PVAT) in abdominal aortic aneurysm (AAA) patients or healthy control (n = 6). Scale bar = 50 μm. (B) Quantification of UCP-1 staining in (A). (C) Q-PCR analysis of UCP-1 mRNA level in PVAT of AAA patients and control (n = 6). (D) Western blot analysis of UCP-1 protein level in PVAT of AAA patients and control (n = 6). (E–H) Expression of browning-related genes in perivascular adipose tissues of dilated vs non-dilated AAA patients (GSE119717, n = 30). ND non-dilated, D dilated. (I) Experimental design. Twenty-week-old male ApoE−/− mice were injected with saline or CL316,243 and then infused with Ang II (1000 ng/kg/min) for 28 days. (J) AAA incidence. (K) Representative photographs of the abdominal aorta taken during sacrifice. (L) Representative ultrasound images of abdominal aortas 4 weeks after Ang II infusion. (M) Quantification of maximal abdominal aortic diameter (n = 6, 17, 18). (N) Hematoxylin and eosin (H&E) and Elastica Van Gieson (EVG) staining of abdominal aortic sections.…

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  Figure EV1. CL316,243 administration successfully induced adipose tissue browning. (A) Gross appearance of epididymal white adipose tissue (eWAT)and subcutaneous white adipose tissue (sWAT). (B) H&E staining. (C) Western blot analysis of Ucp-1 expression. (D) Immunohistochemical staining of Ucp-1 in perivascular adipose tissue (n = 3). Scale bar, 200 μm. Student’s t test was used to determine statistical difference.

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  Figure EV2. Characteristics of ApoE−/− mice after CL316,243 treatment. (A) Body weight (n = 10). (B) Blood glucose levels (n = 9). (C) Serum triglycerides (n = 10). (D) Serum cholesterol (n = 10). Student’s t test was used to determine statistical difference.

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  Figure EV3. CL316,243 administration did not significantly change systolic or diastolic blood pressures. (A) Systolic blood pressure. (B) Diastolic blood pressure (n = 4, 6). Two-way ANOVA was used to determine statistical difference.

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  Figure 2. Browning of adipose tissue inhibits VSMC apoptosis in AAA. (A) Heatmap of differentially expressed genes in aortic tissue. (B) Gene ontology (GO) analysis. (C) Representative immunofluorescent staining of alpha smooth muscle actin (α-SMA) in suprarenal abdominal aortic sections (scale bar, 50 μm). (D) Quantification of α-SMA positive area as in (C) (n = 6, 9, 9). (E) Representative immunohistochemical staining of Cleaved-Caspase 3 in suprarenal abdominal aortic sections (scale bar, 50 μm). (F) Quantification of Cleaved-caspase 3-positive area as in (E) (n = 6, 9, 9). (G) Representative terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and α-SMA staining of suprarenal aortic sections (scale bar, 50 μm). (H) Quantification of TUNEL-positive SMCs (n = 6, 9, 9). One-way ANOVA was used to determine statistical difference. Source data are available online for this figure.

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  Figure EV4. CL316,243 administration did not alter the expression of VSMC phenotypic switch markers. Immunofluorescence staining of Osteopontin (OPN) (A) and krüppel-like factor 4 (KLF4) (B) in aortic tissues from mice treated with saline or CL316,243 infused with Ang II (n = 5; scale bar, 25 μm). One-way ANOVA was used to determine statistical difference.

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  Figure 3. Secretome of browning adipocytes inhibits VSMC apoptosis. (A) Experimental design. Primary adipocytes were treated with PBS or CL316,243 for 24 h, and the culture medium (CM) was then collected and incubated with vascular smooth muscle cells (VSMCs), which further underwent H2O2 or TNFα plus cycloheximide (CHX) treatment to induce apoptosis. (B) Flow cytometric analysis of Annexin V-FITC staining in VSMCs treated with adipocyte CM and H2O2. (C) Quantification of apoptosis as in B (without H2O2, n = 3; with H2O2, n = 6). (D) Flow cytometric analysis of Annexin V-FITC staining in VSMCs treated with adipocyte CM and TNFα plus CHX. (E) Quantification of apoptosis as in (D) (without TNFα + CHX, n = 4; with TNFα + CHX, n = 5). (F) Western blot analysis of cleaved-PARP and cleaved-Caspase 3 in VSMCs treated with adipocyte CM and TNFα plus CHX. (G) Quantification of cleaved-PARP level (n = 3). (H) Quantification of cleaved-Caspase 3 level (n = 3). Two-way ANOVA was used to determine statistical difference. Source data are available online for this figure.

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  Figure EV5. CL316,243 did not have a direct effect on VSMC apoptosis. (A) Flow cytometric analysis of Annexin V-FITC staining in VSMCs treated with PBS or CL316,243 and then stimulated with H2O2 for 24 h. (B) Quantification of apoptosis as in (A) (n = 3). Two-way ANOVA was used to determine statistical difference.

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  Figure EV6. CL316,243 treatment robustly induced adipocyte browning. Primary adipocytes were treated with PBS or CL316,243 for 24 h. (A) Quantitative RT-PCR analysis of Ucp-1 mRNA level (n = 3). (B) Western blot analysis of Ucp-1 protein level. Student’s t test was used to determine statistical difference.

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  Figure 4. Identification of FSTL1 as a novel batokine inhibiting VSMC apoptosis. (A) Volcano plot of the proteins identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS) (n = 3). (B) Subcellular localization categorization of differentially expressed proteins based on subcellular localization. (C) Pie-chart of the number of upregulated and downregulated secreted proteins. (D) Heatmap of the upregulating secreted proteins. (E) Western blot analysis of FSTL1 in CM of adipocytes treated with PBS or CL316,243 for 24 h. Coomassie brilliant blue (CBB) staining was used as a loading control. (F) Western blot analysis of FSTL1 expression in epididymal white adipose tissue (eWAT) and subcutaneous white adipose tissue (sWAT) (n = 3). (G) ELISA analysis of FSTL1 in the plasma of control (n = 68) and AAA patients (n = 55). Student’s t test was used to determine statistical difference. (H, I) Flow cytometric analysis of Annexin V-FITC staining in VSMCs treated with recombinant FSTL1 (100 ng/mL) and stimulated with TNFα plus CHX (H) or H2O2 (I) (n = 3). (J, K) Western blot analysis of cleaved-PARP and cleaved-Caspase 3 in VSMCs treated with recombinant FSTL1 and stimulated with TNFα…