WFA downregulates soluble vimentin and affects the skp2-p27^kip1 anti-fibrotic pathway in an experimental glaucoma filtration surgery model in rabbit.

<p>(A) Immunohistochemical analysis of vimentin expression (<i>red</i>) in Tenon's capsule tissues at surgical sites 28 days post-injury (30× magnification). Note the change in cell shape (<i>arrowhead</i>) in the injured sample (Veh) with abundant cytoplasmic vimentin staining (<i>red</i>) as compared to uninjured sample (Cont) and the downregulation and fragmentation of vimentin-IFs in the high dose WFA-treated sample. (B and E) Western blot analysis of representative tissues from Tenon's capsule tissues extracts at 28 days post-injury showing expression levels of vimentin, α-SMA, skp2 and p27^kip1 in low-salt buffer extracts (soluble fraction). Blots were re-probed consecutively with antibodies. (C–D and F–G) Densitometric quantification of soluble vimentin (<i>black</i>), α-SMA (<i>grey</i>), skp2 (<i>shaded grey</i>) and p27^kip1 (<i>shaded black</i>) normalized to GAPDH, using ImageJ software. Scale bars: 50 µm at 30× magnification. *P = 0.0012 Cont vs Veh; **P = 0.0005 Veh vs WFA high dose; ***P = 0.0069 Veh vs WFA low dose (<i>Panel C</i>). *P = 0.0001 Cont vs Veh; **P = 0.0001 Veh vs WFA high dose; ***P = 0.0001 Veh vs WFA low dose (<i>Panel D</i>). *P = 0.0001 Cont vs Veh; **P = 0.00005 Veh vs WFA high dose; ***P = 0.0012 Veh vs WFA low dose (<i>Panel F</i>). *P = 0.0357 Cont vs Veh; **P = 0.0005 Veh vs WFA high dose; ***P = 0.0012 Veh vs WFA low dose (<i>Panel G</i>).