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.2013 Jan;33(1):114-20.
doi: 10.1161/ATVBAHA.112.300278. Epub 2012 Nov 15.

Cadherin-11 regulates cell-cell tension necessary for calcific nodule formation by valvular myofibroblasts

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Cadherin-11 regulates cell-cell tension necessary for calcific nodule formation by valvular myofibroblasts

Joshua D Hutcheson et al. Arterioscler Thromb Vasc Biol.2013 Jan.

Abstract

Objective: Dystrophic calcific nodule formation in vitro involves differentiation of aortic valve interstitial cells (AVICs) into a myofibroblast phenotype. Interestingly, inhibition of the kinase MAPK Erk kinase (MEK)1/2 prevents calcific nodule formation despite leading to myofibroblast activation of AVICs, indicating the presence of an additional mechanotransductive component required for calcific nodule morphogenesis. In this study, we assess the role of transforming growth factor β1-induced cadherin-11 expression in calcific nodule formation.

Methods and results: As shown previously, porcine AVICs treated with transforming growth factor β1 before cyclic strain exhibit increased myofibroblast activation and significant calcific nodule formation. In addition to an increase in contractile myofibroblast markers, transforming growth factor β1-treated AVICs exhibit significantly increased expression of cadherin-11. This expression is inhibited by the addition of U0126, a specific MEK1/2 inhibitor. The role of increased cadherin-11 is revealed through a wound assay, which demonstrates increased intercellular tension in transforming growth factor β1-treated AVICs possessing cadherin-11. Furthermore, when small interfering RNA is used to knockdown cadherin-11, calcific nodule formation is abrogated, indicating that robust cell-cell connections are necessary in generating tension for calcific nodule morphogenesis. Finally, we demonstrate enrichment of cadherin-11 in human calcified leaflets.

Conclusions: These results indicate the necessity of cadherin-11 for dystrophic calcific nodule formation, which proceeds through an Erk1/2-dependent pathway.

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Figures

Fig. 1
Fig. 1
The effect of MEK1/2 inhibition on hallmarks of dystrophic calcification. AVICs were treated TGF-β1, U0126, U0126 + TGF-β1 and assayed for αSMA and calcific nodule formation. MEK1/2 inhibition causes αSMA expression (A) but suppresses dystrophic calcific formation (B) in AVICs, both in the presence and absence of TGF-β1. Inset in B shows calcific nodules with bright field and fluorescence microscopy, demonstrating an apoptotic outer ring surround a necrotic core. Scalebar = 250 µm. All error bars indicate standard error of the mean. * indicates significant difference (p< 0.005) versus control.
Fig. 2
Fig. 2
U0126 does not interfere with canonical TGF-β1 signaling. U0126 is a specific MEK1/2 inhibitor as indicated by the complete inhibition of pErk1/2 (A) but no change in pSmad3 (B) or pp38 (C). Inset in A shows representative blots and A-C are average densitometry from three independent experiments. Similar to the pSmad3 results, U0126 does not inhibit PAI-1 promoter activity following TGF-β1 treatment (D). All error bars indicate standard error of the mean. * indicates significant difference (p< 0.005) versus control.
Fig. 3
Fig. 3
TGF-β1 incubation for 24 h increases cadherin-11 expression in AVICs. (A) pPCR reveals a 1.58 fold increase in cadherin-11 mRNA in samples treated with TGF-β1 and a decrease in cadherin-11 mRNA with inhibition of MEK1/2. (B) Immunostaining shows cadherin-11 in TGF-β1 groups with minimal to no stain in other treatment groups. Scalebar = 10 µm. All error bars indicate standard error of the mean. * indicates significant difference (p< 0.005) versus control.
Fig. 4
Fig. 4
Cadherin-11 generates intercellular tension through αSMA that enables calcific nodule morphogenesis. (A) Wound assay reveals strength of intercellular connection which correlates to wound area. At normal Ca2+ levels, with all cadherins functional, TGF-β1, U0126, and U0126 + TGF-β1 treatments all increased wound area size due to increased αSMA expression. However, at low Ca2+ levels, where cadherin-11 is still functional and others are not, TGF-β1 treated cells created a large wound. (B) siRNA knockdown of cadherin-11 in physiologic Ca2+ media decreases TGF-β1 initiated wound area to control levels. (C) siRNA knockdown of cadherin-11 prevents calcific nodules. Inset Western blots show that cadherin-11 is knocked down at the time of the wound assay and calcific nodule experiments. All error bars indicate standard error of the mean. * indicates significant difference (p < 0.005) versus control. # indicates significant difference (p < 0.05) versus Scramble. ** indicates significant difference (p < 0.005) versus Sham and Scramble.
Fig. 5
Fig. 5
Cadherin-11 and αSMA expression are increased in calcified human aortic valve leaflets. (A) Immunostaining of a non-calcified leaflet reveals cadherin-11 expression along the periphery of the leaflet, sparse αSMA staining, and very little calcification indicated by the von Kossa stain. (B) Calcified leaflet shows enriched cadherin-11 and αSMA co-localization in areas of significant calcification (panels a and b), as seen in the von Kossa stain, but not in areas where calcification is less intense (panel c). (C) mRNA for cadherin-11 and αSMA are increased in the calcified leaflet (n=1) compared to the non-calcified leaflets (n=2). Scalebar = 500 µm for von Kossa; = 100 µm for immunofluorescence.
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References

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