Pulmonary arterial remodelling by deficiency of peroxisome proliferator-activated receptor-γ in murine vascular smooth muscle cells occurs independently of obesity-related pulmonary hypertension

Fig. 1. a Immunofluorescent stainings (magnification × 400) of isolated PASMCs derived from WT and SmPparγ−/− mice. Cells from SmPparγ−/− mice displayed lack of PPARγ immunoreactivity. b Immunoblotting analyses of isolated aortae from WT and SmPparγ−/− mice also demonstrates marked reduction in PPARγ expression in vasculature from SmPparγ−/− animals. c Weight gain curve of WT and SmPparγ−/− mice on low fat diet (LFD) or high fat diet (HFD). Significant differences (p < 0.05) were detectable between LFD- and HFD-fed mice, regardless of the genotype, starting after 2 weeks of feeding (n = 10–17 per group). For better visualization of weight curves, significances between LFD and HFD of the same genotypes are not depicted

Fig. 2. Glucose tolerance tests (GTT) were conducted after fasting a) at week 0, b) after 12 weeks of LFD and HFD, and c) after 24 weeks of LFD and HFD. The corresponding AUC (right) was calculated (n = 43–44) (a), n = 10–12 for LFD and n = 30–32 for HFD (b), n = 10–17 mice per genotype (WT and SmPparγ−/−) and diet (c). For better visualization of glucose curves, significances (p < 0.05) between LFD and HFD of the same genotypes are not depicted. A linear two factor repeated measurement model with interaction was performed. The intervention (GTT at 12 weeks) as within-subject factor was highly significant (p < 0.001). The interaction of GTT at 12 weeks with diet was significant with p < 0.001, whereas the interaction of GTT with genotype was not significant (p = 0.262). There was no three-way interaction of the GTT challenge with diet and genotype (p = 0.780). For further testing the with-in subject effects at 12 weeks testing for sphericity was performed by a Mauchly test (p = 0.057). The Huynh Feldt correction was used for the within-subject effects. The intervention (GTT at 24 weeks) as within-subject factor was significant (p < 0.001). The interaction of GTT at 24 weeks with diet was…

Fig. 3. Insulin tolerance tests (ITT) were conducted after fasting a) at week 0, b) after 12 weeks of LFD and HFD, and c) after 24 weeks of LFD and HFD. The corresponding AUC (right) was calculated (n = 43) (a), n = 10–12 for LFD and n = 31–32 for HFD (b), n = 10–17 per genotype (WT and SmPparγ−/−) and diet (c). Values were normalized to baseline, fasting glucose levels, which were arbitrarily set as 100%. For better visualization of glucose curves, significances (p < 0.05) between LFD and HFD of the same genotypes are not depicted. A linear two factor repeated measurement model with interaction was performed. The intervention (ITT at 12 weeks) as within-subject factor was significant (p < 0.001). The interaction of ITT at 12 weeks with diet was significant with p = 0.043, whereas the interaction with genotype achieved a p = 0.072/p = 0.94. The three-way interaction of the ITT challenge with diet and genotype was borderline with p = 0.057/p = 0.061. For further testing the with-in subject effects at 12 weeks testing for sphericity was performed by a Mauchly test (p = 0.039). The Huynh Feldt correction was used for the within-subject effects. The intervention (ITT at 24 weeks) as…

Fig. 4. a Right ventricular systolic pressure (RVSP) was determined as stated in the Materials and Methods section in mice after 24 weeks in WT and SmPparγ−/− mice subjected to LFD or HFD. In a two-factor ANOVA with interaction the diet had the predominant impact with p = 0.01. The effect of genotype did not reach significance (p = 0.143), and there was no interaction (p = 0.754). b Systemic arterial pressure (SAP, mmHg) in mice after 24 weeks in WT and SmPparγ−/− mice subjected to LFD or HFD is shown. c Heart rate (beats per minute, bpm) in mice after 24 weeks in WT and SmPparγ−/− mice subjected to LFD or HFD (n = 8–15 per group). d Analyses of correlation between right ventricular systolic pressure (RVSP) and area under the curve (AUC) of absolute glucose levels in ITT determined at the 24 week time point for all analyzed animal groups. Pearson r = 0.45, p < 0.01

Fig. 5. Effect of genotype and diet on pulmonary vascular remodelling (arteries sized 20–70 μm) in monitored study groups: a Representative pictures from immunohistochemical stainings of pulmonary tissues in all analyzed animal groups using α-smooth muscle actin antibodies for visualization of vascular muscularization. b Quantification of vascular muscularization was performed in lung arteries. Shown are the ratios of non- and muscularized pulmonary arteries (n = 23–27 arteries / N = 5 animals). There was a borderline effect (p = 0.06) of genotype without interaction with diet (a two-factor linear model with interaction and a non-parametric Kruskal-Wallis test was performed). c Shown are quantifications of lung immunohistochemical stainings with antibodies against osteopontin for detection of extracellular matrix remodelling in each study group (n = 5). A univariate general two factor model with interactions was used for testing. A logarithmic transformation was used to avoid violation of homodekasticity. Additional non-parametric tests (Kruskal-Wallis, Jonckheere-Terpstra) were performed. The p-values were significant for genotype (p < 0.001) and diet (p = 0.021) with significant…

Fig. 6. Impact of genotype (WT and SmPparγ−/−) and diet on the right (RV) and left ventricle (LV) remodelling and hypertrophy: a RV to LV + septum (S) ratios (= Fulton’s Index) from mice of each study group are shown at the 24 week time point (n = 8–15 per group). b Shown are representative hematoxylin-eosin stainings of the RV and the LV derived from all analyzed animal groups (scale bar = 50 μm). Large pictures were digitalized at × 40, insets are shown for better visualization. c Quantifications of the myocyte cross section area (in μm2) in the RV and LV in each study group (n = 16–50 myocytes / N = 4–5 animals). For a, c and d a two-factor linear model with interaction and a non-parametric Kruskal-Wallis test was performed

Fig. 7. a Relative logarithmized gene expression of brain natriuretic peptide (BNP) in the right ventricle (RV) in each study group. Transcript levels were determined by qPCR analysis, and were normalized to the expression of 18S and to the expression of LFD WT mice (n = 6–8 per group). b Relative logarithmized gene expression analyses of osteopontin in the RV in each study group. Transcript levels were determined by qPCR analysis, and were normalized to the expression of 18S and to the expression of LFD WT (n = 4–6 per group). By a two-factor linear model with interaction and a non-parametric Kruskal-Wallis test we detected that diet had a significant effect on osteopontin (p = 0.01) with borderline interaction with genotype (p = 0.06). c Relative gene expression analyses of Egr-1 in the RV in each study group. Transcript levels were determined by qPCR analysis, and were normalized to the expression of 18S. The expression of LFD WT mice was arbitrarily set as 1 (n = 6–7 per group). By a two-factor linear model with interaction and a non-parametric Kruskal-Wallis test we detected that the genotype had a significant effect on Egr-1 (p = 0.019)