Abstract
Calcific aortic valve stenosis (CAS) is the most frequent heart valve disease in the elderly, accompanied by valve calcification. Tumor necrosis factor-α (TNF-α), a pleiotropic cytokine secreted mainly from macrophages, has been detected in human calcified valves. However, the role of TNF-α in valve calcification remains unclear. To clarify whether TNF-α accelerates the calcification of aortic valves, we investigated the effect of TNF-α on human aortic valve interstitial cells (HAVICs) obtained from patients with CAS (CAS group) and with aortic regurgitation or aortic dissection having a noncalcified aortic valve (control group). HAVICs (2 × 104) were cultured in a 12-well dish in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The medium containing TNF-α (30 ng/ml) was replenished every 3 days after the cells reached confluence. TNF-α significantly accelerated the calcification and alkaline phosphatase (ALP) activity of HAVICs from CAS but not the control group after 12 days of culture. Furthermore, gene expression of calcigenic markers, ALP, bone morphogenetic protein 2 (BMP2), and distal-less homeobox 5 (Dlx5) were significantly increased after 6 days of TNF-α treatment in the CAS group but not the control group. Dorsomorphin, an inhibitor of mothers against decapentaplegic homologs (Smads) 1/5/8 phosphorylation, significantly inhibited the enhancement of TNF-α-induced calcification, ALP activity, Smad phosphorylation, and Dlx5 gene expression of HAVICs from the CAS group. These results suggest that HAVICs from the CAS group have greater sensitivity to TNF-α, which accelerates the calcification of aortic valves via the BMP2-Dlx5 pathway.
Footnotes
This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan [Grant-in-Aid 20590245]; and Hirosaki University Educational Improvement Promotional Aid. K.-I.F. received supported from the Research Foundation for Pharmaceutical Science (Japan).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.177915.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- CAS
- calcific aortic valve stenosis
- TNF-α
- tumor necrosis factor-α
- HAVIC
- human aortic valve interstitial cell
- ALP
- alkaline phosphatase
- FBS
- fetal bovine serum
- BMP2
- bone morphogenetic protein 2
- Dlx5
- distal-less homeobox 5
- Smad
- mothers against decapentaplegic homologs
- IL
- interleukin
- ERK
- extracellular signal-regulated kinase
- MAP
- p38 mitogen-activated protein
- NF-κB
- nuclear factor-κB
- Msx2
- muscle segment homeobox 2
- Runx2
- runt-related gene 2
- DMEM
- Dulbecco's modified Eagle's medium
- FBS
- fetal bovine serum
- TNFRSF1A
- tumor necrosis factor receptor superfamily
- G3PDH
- glyceraldehyde 3-phosphate dehydrogenase
- TTBS
- Tween-Tris buffered saline
- PCR
- polymerase chain reaction
- Wnt
- wingless/Int
- BMS-345541
- 4-(2′-aminoethyl)amino-1,8-dimethylimidazo[1,2-a]quinoxaline
- U-0126
- 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene
- SB239063
- trans-1-(4-hydroxy-cyclohexyl)4-(fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazole.
- Received December 3, 2010.
- Accepted December 28, 2010.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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