Wnt3a inhibits cell proliferation, but increases cell migration and contraction after 72 hour treatment.

<p>(A) Cell proliferation was measured after 72 hours of treatment with Wnt3a or vehicle. Wnt-treated cells grew at 77.4±4.5% the rate of vehicle treated cells (p<0.05). (B) Cells were treated for 72 hours with Wnt3a or vehicle, and then a scratch wound assay was performed to measure cell migration. Wnt-treated cells closed the scratch wound at a significantly faster rate than vehicle-treated cells, as measured 48 hours after injury (78.1±2.1% vs 61.9±3.8%, p<0.05). (C) Cells were treated for 72 hours with Wnt3a or vehicle and then a fibroblast-populated collagen lattice contraction assay was performed. Images of contracted gels taken at 24 hours are shown along with the quantified surface areas of contracted gels. Wnt3a treatment significantly increased the fibroblast-mediated contraction of collagen gels (16.1±0.6% vs 29.4±1.3% of initial surface area, p<0.05). (* denotes p<0.05)</p

Wnt3a induces canonical Wnt signaling in mouse fibroblasts.

<p>Confocal images of fibroblasts treated for 24 hours with vehicle (top panels) or 250 ng/mL Wnt3a (bottom panels) and immunonstained for β-catenin (green) and nuclei (blue). Wnt3a treatment induced clear nuclear accumulation of β-catenin in murine fibroblasts (arrows). (B) TOPFlash reporter assay demonstrated Wnt3a significantly increased luciferase activity 5.3±1.6 fold after a 24 hour treatment (p<0.05). (C) Wnt3a treatment induced a 255±71 fold increase in the mRNA expression of axin2, a target of classical Wnt signaling (p<0.05). (Scale bar = 23.00 µm in A, * denotes p<0.05)</p

Wnt3a-induced change in cell phenotype is dependent on β-catenin.

<p>(A) Western blot demonstrated β-catenin siRNA significantly decreased β-catenin expression in vehicle- and Wnt-treated fibroblasts when compared to a scrambled siRNA. (B) Western blot showed knock down of β-catenin expression significantly inhibited the Wnt3a-induced SMAD2 phosporylation (p<0.05). No difference in SMAD2 phosporylation was detected in vehicle treated cells (p = 0.25). (C) Western blot of smooth muscle α-actin expression demonstrated that β-catenin siRNA significantly decreased smooth muscle α-actin expression in Wnt3a-treated fibroblasts (p<0.05). No significant difference was seen in the vehicle-treated cells (p = 0.27). (D) Immunohistochemistry showed Wnt3a promoted smooth muscle α-actin stress fibre formation in control siRNA transfected cells (green, arrows), but β-catenin siRNA completely inhibited the Wnt3a-induced smooth muscle α-actin expression. Cell nuclei are stained blue with DAPI. (Scale bar = 23.00 µm in D, * denotes p<0.05)</p

Wnt3a-induced change in cell phenotype is dependent on TGF-β expression.

<p>(A) Representative Western blots of vehicle- and Wnt3a-treated fibroblasts showing TGF-β expression, SMAD2 phosphorylation, and smooth muscle α-actin expression at 12, 24, 48, and 72 hours of treatment. (B) Graphical representation of the densitometry results for the blots in A shows, in a sequential manner, that TGF-β expression peaks between 12 and 24 hours, followed by SMAD2 phosphorylation peaking between 24 and 48 hours, which is then followed by smooth muscle α-actin expression peaking after 72 hours of treatment. (C) Western blot of SMAD2 phosphorylation in fibroblasts treated with or without Wnt3a and a TGF-β neutralizing antibody. Densitometry demonstrated the TGF-β neutralizing antibody significantly inhibited Wnt3a-induced SMAD2 phosphorylation (p<0.05). No change was seen in the vehicle-treated cells (p = 0.74). (D) Western blot of smooth muscle α-actin expression in fibroblasts treated with or without Wnt3a and the TGF-β neutralizing antibody. Densitometry confirmed TGF-β neutralization significantly inhibited the Wnt3a-induced smooth muscle α-actin expression (p<0.05). No change was seen in vehicle-treated cells (p = 0.71). (* denotes p<0.05)</p

Wnt3a induces a spindle-like morphology with increased stress fibre formation after 72 hours of treatment.

<p>(A) Light microscope images of mouse fibroblasts that had been treated for 72 hours with vehicle (left panel) or 250 ng/mL Wnt3a (right panel). Wnt3a treatment induced a spindle-like morphology in fibroblasts. (B) Confocal images of vehicle-treated (left panel) or Wnt3a-treated (right panel) fibroblasts immunostained for f-actin (red) and nuclei (blue) showing the increased formation and parallel organization of stress fibres following 72 hours Wnt3a treatment. (C) Low density culture of vehicle-treated (left panel) or Wnt3a-treated (right panel) fibroblasts highlights the increased formation of stress fibres seen after Wnt3a treatment. (Scale bars = 47.00 µm in B, 23.00 µm in C)</p

Wnt3a increases TGF-β expression, SMAD2 phosphorylation and smooth muscle α-actin expression.

<p>(A) Representative Western blot of TGF-β expression in vehicle-treated and Wnt3a-treated fibroblasts after 72 hours. Densitometry showed TGF-β expression to be significantly increased after Wnt3a treatment (p<0.05). (B) Western blot of SMAD2 phosphorylation after 72 hours of vehicle or Wnt3a treatment. Densitometry showed Wnt3a significantly increased SMAD2 phosphorylation at 72 hours. (C) Western blot of smooth muscle α-actin expression in vehicle-treated or Wnt3a-treated cells. Wnt3a-treatment significantly increased the expression of smooth muscle α-actin expression in mouse fibroblasts, as measured by densitometry (p<0.05). (D) Confocal images of fibroblasts immunostained for smooth muscle α-actin (green) and nuclei (blue). Wnt3a-treated fibroblasts had clearly visible smooth muscle α-actin positive stress fibres while the vehicle-treated cells did not display expression of smooth muscle α-actin in their stress fibres. (Scale bar = 47.00 µm in D, * denotes p<0.05)</p

Granzyme B and perforin contribute to plaque development in the descending aorta of apolipoprotein Eknockout mice.

<p>(A) Representative images on the descending aorta from high fat diet-fed apolipoprotein E knockout (ApoE KO), granzyme B (GzmB)/ApoE double knockout (DKO) and perforin (Prf1)/ApoE DKO mice stained en face with sudan IV. (B) When plaque area was quantified, GzmB/ApoE DKO mice (n = 22) had significantly reduced plaque area compared to ApoE KO mice (n = 16). Prf1/ApoE DKO mice (n = 14) had significantly less plaque than both the ApoE KO mice and the GzmB/ApoE DKO mice. *<i>P</i><0.05, ***<i>P</i><0.005 (One-way ANOVA with bonferonnipost test). Error bars represent SEM.</p

Plaque area in aortic roots from granzyme B or perforin deficient apolipoprotein E knockout mice.

<p>(A) Representative images of aortic root cross sections from high fat diet-fed wild type (WT), apolipoprotein E knockout (ApoE KO), granzyme B (GzmB)/ApoE double knockout (DKO) and perforin (Prf1)/ApoE DKO mice stained with Movat’spentachrome. Scale bars = 500 µm. No significant difference in the size of plaque was observed in GzmB/ApoE DKO (n = 9) or Prf1/ApoE DKO mice (n = 8) compared to ApoE KO mice (n = 10). (B) Example images of plaques from aortic roots stained with Movat’spentachrome. The same number of animals were used for these measurements as in panel A. Arrows indicate boundaries of the intimal plaque. Scale bars = 100 µm. No significant difference was detected in the ratio of intimal/medial thickness. ns = not significant (One-way ANOVA with bonferronipost test). Error bars represent SEM.</p

Granzyme B and perforin are present in atherosclerotic plaques from apolipoprotein E knockout mice.

<p>(A)Representative images of aorta cross sections from high fat diet-fed wild type (WT),apolipoprotein E knockout(ApoE KO), Granzyme B (GzmB)/ApoE double knockout (DKO) and perforin (Prf1)/ApoE DKOmice stained for GzmB and Prf1. Black scale bars = 400 µm, white scale bars = 100 µm.(B)Neither GzmB nor Prf1 deficiency resulted in a significant difference in circulating levels of cholesterol (n = 4) and triglycerides (n = 7) in ApoE KO mice when fed a high fat diet for 30 weeks.</p

Increased decorin in plaques from granzyme B and perforin deficient apolipoprotein Eknockout mice.

<p>Representative images of aortic root sections fromapolipoprotein E knockout (ApoE KO), granzyme B (GzmB)/ApoE double knockout (DKO) and perforin (Prf1)/ApoE DKO mice stained for decorin. Decorin in the GzmB deficient animals was observed near the surface of the plaque in concentrated pockets (black arrowheads) while decorin in Prf1 deficient animals stained more diffusely throughout the plaque (white arrowheads). White scale bars = 50 µm, black scale bars = 500 µm.</p