Figure 6

Methylglyoxal increased HO-1 expression, but diminished TNF-α induced VCAM-1 expression. HUVECs were stimulated with different combinations of methylglyoxal (MGO, 0 µM, 400 µM and 800 µM), TNF-α (12.5 ng/ml) and carnosine (CN, 20 mM). Western blotting and qPCR regarding HO-1 and VCAM-1 were performed after protein and RNA isolation. (A) HO-1 Western blotting demonstrates dose-dependent increase of HO-1 expression by MGO, which was abrogated by CN. (B) VCAM-1 Western blotting shows a clear TNF-α induced increase of the negligible baseline VCAM-1 signal, which was dose-dependently reduced by MGO. Equal protein loading was demonstrated by staining for β-actin. Displayed are the cropped blots. (A,B) show two independent experiments. The black surrounding lines demarcate individual blots. The scanned full-length blots are provided in Supplementary Figs 1 and 2. (C) HO-1 densitometry (figure to the left) and gene expression (figure to the right) show a significant and dose dependent increase of HO-1 quantity, which was significantly reversed by CN. Protein and mRNA expression were normalized to β-actin. (D) VCAM-1 densitometry (figure to the left) and gene expression (figure to the right) demonstrate a significant, dose-dependent reduction of VCAM-1 by MGO at the protein, but not at the mRNA level. Carnosine did not significantly affect VCAM-1 quantity. Protein and mRNA expression were normalized to β-actin. For C and D data were analyzed using two-way ANOVA followed by Turkey’s multiple correction test. A p-value < 0.05 was considered to be significant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.