PPARγ activation improves the microenvironment of perivascular adipose tissue and attenuates aortic stiffening in obesity

Figures

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  Fig. 1. Biochemical analysis, fat deposits and PVAT of obese mice after pioglitazone treatment. a Plasma glucose, b aortic pulse wave velocity (PWV), c heart rate, d mean BP, e body weight, the ratio of f gonadal fat weight/body weight, g inguinal fat weight/body weight, and h gonadal/inguinal fat weight were measured in 3-month-old male control, ob/ob, and pioglitazone-treated ob/ob (ob/ob + Piog) mice. i PVAT was visualized in 3-month-old male mice and j its weight was normalized to body weight. Numbers inside the bar = n in each group. k Histological examination of aorta and PVAT. l Expression of brown adipocyte markers in the PVAT (n = 3, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively). All data are resulted from 3-month-old male mice. ob/ob mice were treated with pioglitazone for 1 month starting at 2 months of age. *P < 0.05, **P < 0.01, and ***P < 0.001

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  Fig. 2. Expression of inflammatory mediators and reactive oxygen species-related enzymes in the aorta and PVAT. Expression of genes for chemokines, macrophage markers, and cytokines in the a aorta (n = 7, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively) and b PVAT (n = 3, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively). Expression of genes for ROS-scavenging enzymes and components of NADPH oxidase in the c aorta (n = 7, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively) and d PVAT (n = 3, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively). mRNA amount is expressed relative to the average expression in control mice. All data are resulted from 3-month-old male mice. ob/ob mice were treated with pioglitazone for 1 month starting at 2 months of age. *P < 0.05, **P < 0.01, and ***P < 0.001

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  Fig. 3. Elastin fiber fragmentation, collagen deposition, and elastolytic activity in the aorta. a Representative pictures for the elastic fiber network, and b quantification of elastin fiber breaks in the thoracic aortic media. Arrows indicate breaks in the elastin fiber. Each scale bar is 100 μm. c Collagen stain by the polarized view of picrosirius red-stained section and d quantification of collagen proportion of the aorta. Each scale bar is 1 mm. e Expression of collagen and elastic fiber components in the aorta (n = 7, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively). mRNA amount is expressed relative to the average expression in control mice. f Immunoblot analysis on type I collagen, tropoelastin, fibulin-4/5 in the aorta. Each band represents a tissue extract from a single mouse. g In situ zymography for elastolytic activity in the frozen section. Each scale bar is 200 μm, h Tissue lysates of aorta and PVAT for elastolytic activity assay. The elastolytic activity was demonstrated as the area under the curve (AUC) of fluorescence with normalization to protein concentration over time. All data are resulted from 3-month-old male mice. *P < 0.05 and **P < 0.01

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  Fig. 4. Expression and location of elastolytic enzymes in the aorta and PVAT. Expression of genes for cathepsins and MMPs in the a aorta (n = 7, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively) and b PVAT (n = 3, 5, and 6 for control, ob/ob, ob/ob + Piog groups respectively). mRNA amount is expressed relative to the average expression in control mice. *P < 0.05, **P < 0.01, and ***P < 0.001. ND, not detectable. c Immunofluorescence staining for CTSS (green), MMP-12 (red) and F4/80 (blue) in the thoracic aorta and PVAT. The DAPI nuclear counterstain appears light blue. Magnification of the square in (c) is shown in (d). The while arrow indicates the location of elastic fiber break with increased signals of MMP-12 and F4/80. e Plasma levels of CTSS, MMP-12, and MCP-1 by ELISA (n = 7, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively). All data are resulted from 3-month-old male mice. Original magnification × 400 for (c) and × 1200 for (d)

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  Fig. 5. Effect of PPARγ activation on expression of Ctss and Mmp12. a Raw 264.7 cells, b peritoneal macrophages, c 3T3-L1 undifferentiated cells, d 3T3-L1 differentiated cells, and e A7r5 cells were treated with various concentrations of pioglitazone for 20 h. Expression of Ctss and Mmp12 relative to the average expression of non-treatment control group. Data were collected from two independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001

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  Fig. 6. Binding of PPARγ in Ctss and Mmp12. ChIP assay in Raw 264.7 and 3T3-L1 undifferentiated cells treated with pioglitazone. a, b The schematic representation shows the location of primers flanking putative PPARγ-binding sites (grey rectangles) in Ctss and Mmp12. Sequences containing the potential PPARγ-binding sites in Ctss and Mmp12 loci were amplified by real-time polymerase chain reaction in (c, d) Raw 264.7 and (e, f) 3T3-L1 undifferentiated cells. Data were collected from three independent experiments

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  Fig. 7. The role of mitoNEET in attenuation of obesity-mediated arterial stiffness by pioglitazone. Expression of Cisd1 (coding for mitoNEET) in the a PVAT (n = 3, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively) and b aorta (n = 7, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively). *P < 0.05, **P < 0.01, and ***P < 0.001. c Immunofluorescence staining for mitoNEET (CISD1, red) and DAPI (blue) in the thoracic aorta and PVAT. Each scale bar is 200 μm. d The correlation between PWV and genes in the thoracic aorta (n = 5, 3, and 6 for control, ob/ob, and ob/ob + Piog groups respectively). Scatter plot illustrating the Spearman’s correlation of normalized reads per mouse between PWV and mitoNEET (Cisd1), CTSS (Ctss), and MMP-12 (Mmp12). Spearman’s rank correlation coefficients r and P value are provided in each plot. *P < 0.05. e ChIP assay in 3T3-L1 undifferentiated and Raw 264.7 cells treated with pioglitazone. The schematic representation shows the location of primers flanking putative PPARγ-binding sites (grey rectangles) in Cisd1. Sequence containing the potential PPARγ-binding sites in Cisd1 loci was amplified by real-time polymerase chain reaction in…

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  Fig. 8. The contribution of glucose and ECs in pioglitazone-mediated beneficial effect on improvement of arterial stiffness. a The correlation between PWV and plasma glucose level in mice (n = 5, 3, and 6 for control, ob/ob, and ob/ob + Piog groups respectively). Scatter plot illustrating the Spearman’s correlation of normalized reads per mouse between PWV and plasma glucose level. b Expression of genes for endothelial markers in the aorta (n = 7, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively). **P < 0.01. c The correlation between PWV and genes of endothelial markers in the thoracic aorta (n = 5, 3, and 6 for control, ob/ob, and ob/ob + Piog groups respectively). Scatter plot illustrating the Spearman’s correlation of normalized reads per mouse between PWV and VCAM (Vcam), P-selectin (Selp), vWF (Vwf), and eNOS (Nos3). Spearman’s rank correlation coefficients r and P value are provided in each plot. d Immunofluorescence staining for NF-κB p65 (red) and DAPI (blue) in the thoracic aorta. Each scale bar is 25 μm. Magnification of the square in upper panels is shown in lower panels. The while arrow indicates the location of positive nuclear staining for NF-κB. e…