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

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

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

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

Monitoring cGMP levels after single perturbation of oxidatively impaired NO˙-cGMP pathway.

<p>(A) The effect of H₂O₂ on cGMP dynamics. H₂O₂ caused ~6-fold reduction in cGMP levels. (B and C) Reducing either k₃ or k₁₀ during oxidative stress was not an effective perturbation for restoring cGMP to the normal level. The value of k₃ or k₁₀ was 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>), with perturbation of k₃ or k₁₀ denoted as ρk₃ and ρk₁₀, respectively. While these perturbations can be effective strategies in enhancing cGMP levels in the absence of H₂O₂, they are not effective in restoring cGMP levels to normal in the presence of H₂O₂. (D) All single perturbations of the NO˙-cGMP pathway in the presence of H₂O₂. The cGMP levels were monitored in the absence of H₂O₂. Subsequently, in the presence of H₂O₂, the system was perturbed by reducing one of thirteen rate constants (k₁‒k₁₃) to 10% of their original values, denoted as ρks, and then the cGMP levels calculated. Note that ρk₁, ρk₃, ρk₁₀, and ρk₁₂ markedly increased the cGMP levels beyond other perturbations.</p

Pairwise perturbation of optimal rate constants increased cGMP levels beyond that predicted by the Bliss model.

<p>Comparison of cGMP levels after individual and pairwise perturbation of (A) k₁ + k₃, (B) k₁ + k₁₀, and (C) k₃ + k₁₀ with the Bliss model [generated using the perturbation of corresponding individual rate constants and applying <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004822#pcbi.1004822.e016" target="_blank">eq (15)</a> in the Supplement]. Single perturbations were used to predict paired perturbation signatures. The simulated combination produced cGMP levels that were greater than those predicted by the Bliss model.</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

PKI (inhibitor of PKA) prevented the high glucose-induced decrease of G6PD activity, prevented the high glucose-mediated increase in NOX activity, and prevented colocalization of G6PD and gp91.

<p>A: Inhibition of PKA rescues the high glucose-induced decrease in G6PD activity. B: Inhibition of PKA prevents the high glucose-induced increase in NADPH oxidase activity. C: Left hand panel shows highly significant colocalization of G6PD and gp91 caused by high glucose and the right hand panel shows that inhibition of PKA by PKI prevents the colocalization by PKI. *, P<0.05 compared with 25 mM. #, P<0.05 compared with 5.6 mM. n = 5.</p

Pharmacologiic Inhibition of protein kinase A improved antioxidant activities in endothelial cells.

<p>High glucose increases cAMP, at least in part by activation of adenylate cyclase, which leads to activation of PKA (see text) and subsequent inhibition of G6PD. To inhibit PKA, endothelial cells were treated with a specific cell-permeable PKA inhibitor 14–22 amide (10 µmol/l) for the last 24 hours. Addition of PKI to cells exposed to high glucose led to: A: Glutathione reductase activity increase. B: SOD activity increase. C: Catalase activity increase. D: ROS level decrease. E: TBARs level decrease. *, p<0.05 compared with 25 mM condition. #, p<0.05 compared with 5.6 mM condition. n = 8.</p

Overexpression of G6PD rescued the high glucose-indueed decrease in the antioxidant enzymes and reduced ROS level in endothelial cells.

<p>Adenovirus vector inserted with human G6PD cDNA was constructed and purified as described in the Methods. Endothelial cells were infected with either Ad2-G6PD (MOI: 5) or empty vector control (Laz). A: G6PD protein was significantly increased with adenovirus infection in endothelial cells exposed to high glucose. Overexpression of G6PD led to the following changes in cells exposed to high glucose as compared to cells exposed to high glucose with wild type G6PD activity: B: G6PD activity was increased. C: ROS level was decreased. D: NADPH level was increased. E: GSH/GSSG level was increased. F: Catalase activity was increased. *, p<0.05 compared with 25 mM conditions. #, p<0.05 compared with 5.6 mM condition. n = 8.</p