PPAR-Gamma Activation May Inhibit the In Vivo Degeneration of Bioprosthetic Aortic and Aortic Valve Grafts under Diabetic Conditions

Figures

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  Figure 1. Circulating adiponectin levels. Plasma probes (of n = 10 animals per group) were analyzed by enzyme-linked immunosorbent assay (ELISA) for the level of circulating adiponectin at 4 weeks (A) and 12 weeks (B), respectively. Animals with diabetic conditions (DM) showed no significant change in circulating adiponectin levels when compared to non-diabetic controls (Control) at either time point. The addition of pioglitazone (DM+Pio) led to a significant threefold increase in plasma adiponectin levels at 4 weeks and at 12 weeks, respectively. DM, diabetes mellitus; Pio, pioglitazone; * p < 0.05; ** p < 0.01; *** p < 0.001. Data were analyzed using Kruskal–Wallis with Dunn’s multiple comparisons test.

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  Figure 2. Aortic-valve-insufficiency operation, graft implantation. A graft was implanted into the recipient abdominal aorta. After explantation, the graft was divided into four parts, A1 (proximal ascending aorta and aortic valve), A2 (distal ascending aorta), B1 (proximal descending aorta), and B2 (distal descending aorta). AV, aortic valve.

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  Figure 3. Histological results of aortic region—media thickness. For each group, tissue of n = 4 animals was stained and evaluated. (A) A representative hematoxylin eosin-stained image for each group, including higher-magnification inserts. After 4 weeks, DM had a significant thickening of the media, but it was not statistically different between the control and the pioglitazone-administered group. After 12 weeks, thickening of the media with calcification was observed in the DM group. (B) Measurement result of the media after 4 weeks. There was a significant difference in medial thickening in the DM group. (C) Even after 12 weeks, the medial thickening of the DM group was significantly different. DM, diabetes mellitus; W, weeks; Pio, pioglitazone; * p < 0.05; ** p < 0.01; *** p < 0.001. Data were analyzed using Kruskal–Wallis with Dunn’s multiple comparisons test.

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  Figure 4. Histological results of aortic region—media calcification. For each group, tissue of n = 4 animals was stained and evaluated. (A) Representative von Kossa stainings after 12 weeks. The media was extensively calcified in the DM group. (B) Calcification scores after 12 weeks. Here, calcification was more advanced in the DM group. DM, diabetes mellitus; W, weeks; Pio, pioglitazone; * p < 0.05. Data were analyzed using Kruskal-Wallis with Dunn’s multiple comparisons test.

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  Figure 5. Histological results of aortic region –calcification of different regions. For each group, tissue of n = 4 animals was stained and evaluated. (A) Representative images of von Kossa staining of cross-sections at the level of the aortic valve after 12 weeks. Calcification of valve leaflets, annulus, and commissures was observed in all groups, with increased levels in DM and DM+Pio. (B) Semi-quantitative analysis for sub-segments of the aortic root as well as the entire cross-section. Highest calcification levels were observed in sub-segments of commissures. DM, diabetes mellitus; W, weeks; Pio, pioglitazone; * p < 0.05; ** p < 0.01; *** p < 0.001. Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test.

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  Figure 6. Gene-expression analysis of degenerative markers. Gene-expression analysis of aortic grafts after 4 and 12 weeks, respectively, revealed significant differences regarding key markers of calcific degeneration. For each group, tissue of n = 6 animals was analyzed. At the earlier time point of 4 weeks, bone morphogenic protein 2 and osteopontin expression were significantly reduced by pioglitazone (DM+Pio vs. DM and DM+Pio vs. control). This difference was lost at the later time point of 12 weeks. BMP2, Bone morphogenetic protein 2; OPN, osteopontin; Runx2, Runt-related transcription factor 2; * p < 0.05; ** p < 0.01. Data were analyzed using Kruskal–Wallis with Dunn’s multiple comparisons test.

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  Figure 7. Immunohistology. For each group, tissue of n = 4 animals was stained and evaluated. Representative images of cross-sections subjected to immunostaining for αSMA and vWF after 12 weeks in vivo demonstrates a myofibroblast activation (αSMA+) in the media of diabetic groups (DM, DM+Pio). Endothelial cells (vWF+) were detected in all groups at the luminal surface. DM, diabetes mellitus; W, weeks; Pio, pioglitazone; αSMA, alpha smooth muscle actin; vWF, von Willebrand factor; DAPI, 4′,6-diamidino-2-phenylindole.

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  Figure 8. Experimental protocol. Recipient Wistar rats (n = 60) were divided into three groups. A high-fat diet was given for 2 weeks, after which STZ was injected intraperitoneally. Aortic valve insufficiency was created 1 week after injection and the graft was transplanted 1 week later. Animals were sacrificed 4 and 12 weeks after transplantation. DM, diabetes mellitus; W, weeks; FBG, fasting blood glucose; Pio, pioglitazone; STZ, streptozotocin.