Wnt3a/β-catenin increases proliferation in heart valve interstitial cells.
BACKGROUND
Valve interstitial cells (VICs), the most prevalent cells in the heart valve, mediate normal valve function and repair in valve injury and disease. The Wnt3a/β-catenin pathway, important for proliferation and endothelial-to-mesenchymal transition in endocardial cushion formation in valve development, is up-regulated in adult valves with calcific aortic stenosis. Therefore, we tested the hypothesis that Wnt3a/β-catenin signaling regulates proliferation in adult VICs.


METHODS
Porcine VICs were treated with 150 ng/ml of exogenous Wnt3a. To measure proliferation, cells were counted on day 4 posttreatment and stained for bromodeoxyuridine (BrdU) at 24 h posttreatment. β-Catenin small interfering RNA (siRNA) was used to knock down β-catenin expression. Apoptosis was measured with terminal deoxynucleotidyl transferase dUTP nick end labeling assay. To assess changes in β-catenin, cells were stained for β-catenin at days 1, 3, 6, and 9 posttreatment. Western blot for β-catenin was performed on whole cell, cytoplasmic, and nuclear extracts at day 4 posttreatment. To measure β-catenin-mediated transcription, TOPFLASH/FOPFLASH reporter assay was performed at 24 h posttreatment.


RESULTS
Wnt3a produced a significant increase in cell number at day 4 posttreatment and in the percentage of BrdU-positive nuclei at 24 h posttreatment. The increase in proliferation was abolished by β-catenin siRNA. Apoptosis was minimal in all conditions. Wnt3a produced progressively greater β-catenin staining as treatment length increased from 1 to 9 days. Wnt3a produced a significant increase in β-catenin protein in both whole cell and nuclear lysates after 4 days of treatment. Wnt3a significantly increased TOPFLASH/FOPFLASH reporter activity after 24 h of treatment.


CONCLUSIONS
Wnt3a/β-catenin signaling pathway is an important regulator of proliferation in adult VICs.
Department of Pathology, Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8.
Songyi
S
Xu
Avrum I
AI
Gotlieb
D000818:Animals; D017209:Apoptosis; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006351:Heart Valves / Q000166:cytology* / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D034421:Sus scrofa; D060509:Wnt3A Protein / Q000378:metabolism*; D051176:beta Catenin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D034741:RNA, Small Interfering; D060509:Wnt3A Protein; D051176:beta Catenin