Withaferin A Effectively Targets Soluble Vimentin in the Glaucoma Filtration Surgical Model of Fibrosis

Withaferin A (WFA) is a natural product that binds to soluble forms of the type III intermediate filament (IF) vimentin. Currently, it is unknown under what pathophysiological contexts vimentin is druggable, as cytoskeltal vimentin-IFs are abundantly expressed. To investigate druggability of vimentin, we exploited rabbit Tenon\u27s capsule fibroblast (RbTCF) cell cultures and the rabbit glaucoma filtration surgical (GFS) model of fibrosis. WFA potently caused G₀/G₁ cell cycle inhibition (IC₅₀ 25 nM) in RbTCFs, downregulating ubiquitin E3 ligase skp2 and inducing p27(Kip1) expression. Transforming growth factor (TGF)-ß-induced myofibroblast transformation caused development of cell spheroids with numerous elongated invadopodia, which WFA blocked potently by downregulating soluble vimentin and α-smooth muscle actin (SMA) expression. In the pilot proof-of-concept study using the GFS model, subconjunctival injections of a low WFA dose reduced skp2 expression in Tenon\u27s capsule and increased p27(Kip1) expression without significant alteration to vimentin-IFs. This treatment maintains significant nanomolar WFA concentrations in anterior segment tissues that correspond to WFA\u27s cell cycle targeting activity. A ten-fold higher WFA dose caused potent downregulation of soluble vimentin and skp2 expression, but as found in cell cultures, no further increase in p27(Kip1) expression was observed. Instead, this high WFA dose potently induced vimentin-IF disruption and downregulated α-SMA expression that mimicked WFA activity in TGF-ß-treated RbTCFs that blocked cell contractile activity at submicromolar concentrations. These findings illuminate that localized WFA injection to ocular tissues exerts pharmacological control over the skp2-p27(Kip1) pathway by targeting of soluble vimentin in a model of surgical fibrosis

Bleb appearance in representative rabbit eyes subjected to GFS.

<p>(A) WFA treated eyes (top panels a–c; high dose) and vehicle treated eyes (bottom panels d–f) at weeks 1, 2 and 3 post-surgery. The arrows indicate the presence of the bleb and arrowheads point to the cannula. Note the increased vascularization at two weeks post surgery (e). (B) Kaplan-Meier bleb survival plot of rabbit eyes treated with vehicle (solid line), low dose WFA (dotted line) and high dose WFA (dashed line).</p

WFA prevents myofibroblast transformation and collagen gel contraction in RbTCF cells.

<p>Immunohistochemical analysis of vimentin (<i>green</i>) and α-SMA (<i>red</i>) in RbTCF cells treated with TGF-β1 (2 ng/ml) for 3 days in presence and absence of WFA. (A) TGF-β1 treatment induces formation of spheroid-like agglomerates (upper panels, 10× magnification) with sprouting extensions positive for vimentin (<i>green</i>) and α-SMA (<i>red</i>) (lower panels, 30× magnification). (B–C) WFA causes a dose-dependent downregulation of both vimentin (<i>green</i>) and α-SMA (<i>red</i>) expression. Upon treatment with 500 nM WFA, cells form spheroids poorly (C, insert panels) and those that do form spheroids have shorter and thinner extensions (C, <i>arrowheads</i>). Scale bars: 70 µm at 10× magnification; 30 µm at 30× magnification. (D) Western blot analysis of soluble vimentin and α-SMA expression in RbTCF cells (left panel) and RbCF cells (right panel). (E) Densitometric quantification of vimentin (<i>black and orange</i>) and α-SMA (<i>grey and blue</i>) in RbTCF cells and RbCF cells, respectively, normalized to GAPDH using ImageJ software. (F) Representative images of polymerized collagen gels containing RbTCF cells (n = 6 gels per treatment group) treated with TGF-β1 (2 ng/ml) in presence and absence of different doses of WFA for 3 days (10× magnification). (G) Quantification of percentage of gel contraction normalized to the initial size of the gel. Data are the mean of 2 independent experiments; Dotted circles represent the well's area. *P<0.05 TGF-β vs WFA treated cells.</p

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>).</p

WFA inhibits cell migration in RbTCF cells.

<p>Scratch wound assay <i>in vitro</i>. (A) Representative phase-contrast images of RbTCF cells immediately after a cross-stripe scrape injury on a confluent cell layer (0 h), 24 h post-wounding (Veh), and after treatment with different concentrations of WFA. Mitomycin-C (MMC; 15 µM) was used as positive control. (B) Arbitrary values of wound gap at 24 h normalized to size of initial wound gap and calculated from two independent experiments (n = 3 for each treatment group). *P<0.05 vehicle vs drug treated; ns = non-significant. (C) Quantification of total numbers of DAPI nuclei per wound area at 24 h post-scratch injury (**P<0.0001 vehicle vs drug treated) and Ki-67 positive cells (blue bars; ^#P<0.0001 vehicle vs drug treated). (D) Fraction of Ki67 positive nuclei in the wound area as a percentage of DAPI nuclei at 24 h post-scratch injury. *** P<0.0001 vehicle vs drug treated.</p

WFA's cell cycle effects are regulated by the skp2-p27^kip1 pathway in RbTCF cells.

<p>(A) Representative images of RbTCFs stimulated to enter the cell cycle after 48 h serum starvation. Cells were stained with vimentin (<i>green</i>) and phalloidin (<i>red</i>) at 24 h post-WFA treatment. WFA's low concentration cell cycle activity does not affect the vimentin-IF network (<i>green</i>). Vimentin-IFs become affected only at higher concentrations (1 µM, <i>arrow heads</i>) and leads to dismantling of the actin fibers (<i>red</i>) that are observed at the periphery of cytoplasm (<i>arrows</i>). (B) Western blot analysis of skp2 and p27^kip1 expression in RbTCF cells at 24 h post-WFA treatment. C) Densitometric quantification of skp2 (<i>black</i>) and p27^kip1 (<i>grey</i>) normalized to β-actin, using ImageJ software. Scale bars: 30 µm at 30× magnification. * P<0.05 WFA vs control.</p

WFA potently arrests RbTCFs in G₀/G₁ phase.

<p># mean of two experiments.</p

WFA distribution in anterior segment tissues.

<p>WFA distribution in anterior segment tissues.</p