Figure 1.
Pyrvinium inhibits Wnt signaling.
(A) Pyrvinium inhibits TOPflash activation with an EC50 of ∼10 nM in contrast to the structurally related compound VU-WS211. HEK 293 STF (TOPflash) luciferase reporter cells incubated with Wnt3a-conditioned media were treated as indicated. Graph represents mean ± SEM of TOPflash signal normalized to cell number (performed in quadruplicate). (B) Pyrvinium (100 nM) decreases transcript levels of endogenous Wnt target genes Myc, Dkk-1, and Axin2 as assessed by real-time PCR. GAPDH is control. (C) Pyrvinium binds CK1α in vitro. CK1α and GSK3 (0.5 µg each, in duplicates) were spotted on nitrocellulose, incubated with pyrvinium (10 nM), and bound pyrvinium detected based on its fluorescent property. (D) Pyrvinium stimulates CK1α activity. CK1α (100 nM) was incubated with recombinant casein (100 nM) plus or minus pyrvinium (10 nM) in a kinase reaction containing [γ32P]ATP followed by SDS-PAGE and exposure to PhosphoImager screen. (E) Pyrvinium decreases and increases intracellular β-catenin and Axin levels, respectively. HEK 293 cells were treated for 16 hours as indicated, and cytoplasmic preparations were immunoblotted for β-catenin and Axin. β-tubulin is loading control. (F) Pyrvinium decrease steady-state levels of Pygopus. HEK 293 STF cells expressing HA-tagged human Pygopus-2 were treated with pyrvinium as indicated. Lysates were immunoblotted for HA. β-galactosidase (β-gal) is transfection control.
Figure 2.
Pyrvinium increases granulation tissue organization, proliferation, and vascularity in the sponge model of tissue repair.
(A) Representative images of the pyrvinium- and compd 211-treated sponges stained with H&E to assess organization of the granulation tissue, anti-Ki-67 to analyze proliferation, and anti-PECAM-1 to assess vascular density of the sponge granulation tissue. SP = sponge matrix, arrows point at positive stain. (B) Proliferation graphed as number of Ki-67 positive cells/total tissue area and (C) vascular density graphed as percentage of immunopositive PECAM-1 area/total tissue area in histologic sections from granulation tissue. Data represents averages of multiple 40x fields from unpaired samples (n = 6). The statistical significance between experimental groups and control was determined by Mann Whitney Test, p<0.05 was considered statistically significant.
Figure 3.
Pyrvinium prevents adverse myocardial remodeling.
(A) Representative Masson trichrome-stained sections of hearts from mice 30 days after experimental infarct and treatment with pyrvinium and/or compd 211. Arrows point at the scarred ventricular wall. L = lumen. (B) The infarct size was quantified as the percentage of the left ventricular wall that exhibited myocyte replacement by scar. (C) LVIDD and LVIDS to represent cardiac remodeling, and (D) fractional shortening, as a measurement of cardiac function, were determined by echocardiography and are plotted as percentage difference values (mean +/− SEM) between 7 and 30 days after infarct. The statistical significance between experimental groups and control was determined by unpaired t-test (n = 6 for compd 211 and n = 7 for pyrvinium).
Table 1.
Pyrvinium improves cardiac remodeling.
Figure 4.
Pyrvinium promotes proliferation of myocytes in the peri-infarct and distal areas of the injured heart.
(A and B) Representative images of anti-Ki-67 stained sections of compd 211- and pyrvinium-treated myocardium and the distal myocardium following MI. (A and B) Proliferation graphed as percentage of immunopositive Ki-67 positive cells/20x field in histologic sections from the proximal and the distal infarcted heart. (C) Representative images of anti-PECAM-1 staining to designate vascular density among experimental cohorts. Arrows point towards positive stain. Graph demonstrating the difference in PECAM-1 positive area/40x field. Data represents averages of multiple fields from unpaired samples (n = 6). (D) Representative immunostained confocal images of the sections of remote myocardium co-stained with anti-pH3 (1∶50; red) for cells undergoing mitosis, anti-alpha sarcomeric actin (1∶500; green) for cardiac muscles, and DAPI (blue) for the nuclei; yz axis designates anti-pH3 and anti-alpha sarcomeric actin co-staining. The statistical significance between experimental groups and control was determined by one way ANOVA with Newman-keuls post-test.