Figure legends
FIGURE 1 Inactivation of SERCA2 C674 suppresses PPARγ by activation of calcineurin/NFAT/NF-κB pathways. (a) The main signaling pathways from the Kyoto encyclopedia of genes and genomes pathway enrichment analysis in duplicate aorta samples of WT and SKI mice in LDLR−/−background. (b) The mRNA levels of PPARγ2 in aortas of WT and SKI mice in LDLR−/− background. (c) Representative Western blots of PPARγ from the aorta of WT and SKI mice in LDLR−/− background, and quantification of band intensities in graph. (d) The mouse PPARγ2 promoter activity in aortic SMCs. (e) Effects of CsA or PDTC on PPARγ2 promoter activity in SKI aortic SMCs. (f) Representative Western blots of PPARγ from aortic SMCs and the quantification of band intensities in graph. (b)-(f), mean ± SEM, unpaired Student’s t test, *P < .05, significantly different as indicated, n = 5
FIGURE 2 Activation of NF-κB accounts for the phenotypic modulation of SKI aortic SMCs. (a) Representative Western blots from SKI aortic SMCs treated with PDTC and quantification of band intensities in graph. Mean ± SEM, unpaired Student’s t test, *P< .05, significantly different as indicated, n = 5. (b) Cell proliferation. (c) Cell migration. (d) Macrophage adhesion to aortic SMCs. (b)-(d), mean ± SEM, unpaired Student’s t test, *P < .05, significantly different as indicated,n = 6
FIGURE 3 The down-regulated PPARγ2 accounts for the phenotypic modulation of SKI SMCs. (a) Representative Western blots from SKI aortic SMCs transfected with PPARγ2 plasmids and quantification of band intensities in graph. Mean ± SEM, unpaired Student’s t test, *P < .05, significantly different as indicated,n = 5. (b) Cell proliferation. (c) Cell migration. (d) Macrophage adhesion to aortic SMCs. (b)-(d), mean ± SEM, unpaired Student’st test, *P < .05, significantly different as indicated, n = 6
FIGURE 4 Activation of PPARγ by pioglitazone prevents the phenotypic modulation of SKI SMCs. (a) Representative Western blots from SKI aortic SMCs treated with pioglitazone and quantification of band intensities in graph. Ctrl, solvent control; PIOG, pioglitazone. Mean ± SEM, unpaired Student’s t test, *P < .05, significantly different as indicated, n = 5. (b) Cell proliferation. (c) Cell migration. (d) Macrophage adhesion to aortic SMCs. (b)-(d), mean ± SEM, unpaired Student’s t test, *P< .05, significantly different as indicated, n = 6
FIGURE 5 Activation of PPARγ represses the activation of NFAT/NF-κB in SKI aortic SMCs. Representative Western blots of NFAT4 and p65NF-κB from SKI aortic SMCs treated with pioglitazone and quantification of band intensities in graph. Histone H3 is the load control of nuclear protein and GAPDH is the load control of total protein. Ctrl, solvent control; PIOG, pioglitazone. Mean ± SEM, unpaired Student’s t test, *P < .05, significantly different as indicated, n = 5
FIGURE 6 Activation of PPARγ ameliorates angiotensin II-induced aortic aneurysm in SKI mice. (a) The flow chart of animal procedure in LDLR−/− background mice. (b) Survival rate. SKI mice treated with solvent control (SKI/Ctrl) vs. WT mice treated with solvent control (WT/Ctrl) or SKI mice treated with pioglitazone (SKI/PIOG), *P < .05, Kaplan-Meier analysis and comparisons are performed using the log-rank test. (c) Incidence. (d) Representative photographs of aortic aneurysms and the summary of their composition in graph. A red arrow indicates the location of the aneurysm. Ruptured aneurysm, SKI/Ctrl vs. WT/Ctrl or SKI/PIOG, *P < .05, Chi-square test. (b)-(d), n = 11. (e) Representative photographs of elastin degradation and the percentage of different grades of elastin degradation summarized in graph. Grade IV, SKI/Ctrl vs. WT/Ctrl or SKI/PIOG, *P < .05, Chi-square test. (f) Representative photographs of collagen deposition and its score summary in graph. (e)-(f), scale = 500 μm. Mean ± SEM, WT/Ctrl, n = 8; SKI/Ctrl,n = 7; SKI/PIOG, n = 9
FIGURE 7 Activation of PPARγ decreased the size and number of angiotensin II-induced aortic aneurysm in SKI mice. (a) Representative photographs of HE staining of aortic aneurysms and the diameter and area of aneurysm summarized in graph. Scale = 500 μm. (b) The length of aneurysm. (c) The average number of aortic aneurysms in each group. Mean ± SEM, One-way ANOVA, *P < .05, significantly different as indicated, WT/Ctrl, n = 8; SKI/Ctrl, n = 7; SKI/PIOG,n = 9
FIGURE 8 Pioglitazone therapy inhibited the expression of aneurysm related proteins in angiotensin II-infused SKI aorta. (a) Representative Western blots from the aorta of SKI mice in LDLR−/− background infused with angiotensin II, and quantification of band intensities in graph. Ang II, angiotensin II; Ctrl, solvent control; PIOG, pioglitazone. Mean ± SEM, unpaired Student’s t test, *P < .05, significantly different as indicated, n = 5