The effects of linear reduction of values for three individual rate constants on cGMP dynamics.

<p>Using the linear reduction of values for k₁, k₃, and k₁₀, we created a vector of eleven different values for each of these rate constants. We next generated three linearly spaced vectors for each of the rate constants by fractionally reducing each decrementally. Using these vectors of rate constants, cGMP dynamics were calculated. The cGMP levels after serial reduction of (A) k₁, (B) k₂, and (C) k₃. (D) The relative cGMP levels. Data are shown as mean (dashed lines) ± S.E.M (shaded lines) of 11 simulated replicates. Note: k₃ was the most sensitive parameter in cGMP accumulation as compared with k₁ and k₁₀. In addition, the highest cGMP levels were achieved at ~40 seconds.</p

Pairwise or triple perturbations of three agents additively enhance the cGMP_T levels.

(A-C) The cGMPT dose matrix-responses to all possible combined perturbations of three rate constants. We iteratively reduced the rate constants by fractional decrements with ρ defined as the (normalized) perturbation ratio with values either 0.3 or 0.5 for pairwise or triple perturbations, respectively. For pairwise perturbations, two vectors of rate constants were combined in 11×11 matrices in which the value of each rate constant was fractionally reduced by linear decrements along each axis. The combined effects of (A) k1 plus k3, (B) k1 plus k10, and (C) k3 plus k10 on cGMPT levels. (D-E) The contour plots illustrate that the pairwise perturbations exerted additive effects on cGMPT levels. (G-I) The cGMP dose matrix responses to the triple perturbation of k1, k3, and k10. Three vectors of rate constants were combined in 11×11×11 matrices in which the value of each rate constant was linearly reduced along each axis. (J-L) The contour plots reveal additive augmentation of cGMPT levels.</p

Increases in cGMP in PAVSM cells in the presence and absence of pharmacological agents that affect the cGMP pathway.

<p>In (A) and (B), PAVSM cells were pre-incubated for 90 min with buffer, 10 mM NAC (N-acetylcysteine), or 100 nM sildenafil (sild), followed by 30 min incubation with 500 μM hydrogen peroxide. Cells were then incubated with 100 μM SNAP for 10 min prior to harvesting for cGMP determination. Panel (A) shows a representative experiment, with three biological replicates under each condition. Shown in (B) are the averages of 3–4 separate experiments with each condition tested in duplicate or triplicate. Sildenafil, NAC, H₂O₂, plus SNAP condition was not significantly different from the sildenafil plus SNAP condition; these two conditions (* indicates p<0.05 by ANOVA followed by a Newman-Keuls test) were significantly different from all other conditions.</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

Relative cGMP_T levels after single, paired, and triple kinetic perturbations of the oxidatively impaired NO˙-cGMP pathway.

<p>The relative level of cGMP_T as a function of all possible single (13 brown bars), paired (78 gray bars), and triple (286 green bars) perturbations is shown. Values of the rate constants were reduced to 10% of their original values (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004822#pcbi.1004822.s002" target="_blank">S1 Table</a>). Each bar shows the relative integrated cGMP levels over the period of simulation, or . Note that some of the optimal perturbations are highlighted in this figure.</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-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 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

Steps for modeling NO˙-cGMP signaling pathway.

<p>The reaction schema: NO˙ is synthesized in a generator cell and freely diffuses, either within the same cell or to a target cell, to activate sGC; sGC is oxidized by H₂O₂ and inactivated. Either sGC or NO˙-sGC can convert GTP to cGMP, which is degraded by PDE to GMP. Note that oxidative stress drives the system toward the oxidative limb, and that the goal of pharmacological modulation of this pathway is to reverse the adverse effects of oxidative stress and to minimize PDE inhibition in order to optimize cGMP levels. NAC can be used as an antioxidant (impairing hydrogen peroxide-dependent oxidation of SGC and, perhaps, oxidation of NO˙), sildenafil as a PDE5 inhibitor, and SNAP as an NO˙ donor to modulate this pathway experimentally. Abbreviations: cGMP, cyclic guanosine 3', 5'-monophosphate; GMP, guanosine-5'-monophosphate; GTP, guanosine-5'-triphosphate; H₂O₂, hydrogen peroxide; NAC, N-acetylcysteine; NO˙, nitric oxide; NO_x, oxidized (inactive) nitrogen oxides; PDE, phosphodiesterase; SNAP, S-Nitroso-n-nacetylpenicillamine; sGC, soluble guanylyl cyclase.</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