Fig 7. TNFα induces early calcification and changes in aortic valve extracellular matrix composition and structure, rescued differentially by BH4 or peg-SOD.
A, TNFα increased collagen and decreased GAG and elastin (arrows). BH4 maintains GAG and elastin at control levels, but does not block increases in collagen. Peg-SOD significantly reduced collagen compared to TNFα and maintained control levels of GAG and elastin (Blue: glycosaminoglycans, Red: mucins, Yellow: collagen, Black, elastin). B. TNFα increased synthesis of COL1A1 and COL3A1 mRNA, rescued by peg-SOD but not by BH4. COL2A1 was decreased in all treatment conditions. * indicates p < 0.05 versus control, # indicates p < 0.05 versus TNFα. N = 6. One-way ANOVA with Tukey’s post hoc test were used to compare means. C, TNFα increased calcium deposition (Alizarin Red S stain) that is rescued by BH4 or peg-SOD. D, TNFα increased mineralization (von Kossa stain), rescued by BH4 or peg-SOD. E, TNFα increased alkaline phosphatase, decreased Sox9, and increased MMP-9 mRNA, rescued differentially by BH4 or peg-SOD. Representative images from n = 6 leaflets per condition. Scale bar = 200μm. * indicates p < 0.05 versus control, # indicates p < 0.05 versus TNFα, ^ indicates p < 0.05 versus TNFα+BH4. N = 6.