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You are here: Contents > 2013 > Volume 22 Number 5 September 2013 > AORTIC VALVE DISEASE > TGF-β1-Induced MAPK Activation Promotes Collagen Synthesis, Nodule Formation, Redox Stress and Cellular Senescence in Porcine Aortic Valve Interstitial Cells

TGF-β1-Induced MAPK Activation Promotes Collagen Synthesis, Nodule Formation, Redox Stress and Cellular Senescence in Porcine Aortic Valve Interstitial Cells

Dividutta Das1,2, Andrew Holmes1,2, Geraldine A. Murphy1, Kumaril Mishra1,3, Anke C. Rosenkranz1,4, John D. Horowitz1,2, Jennifer A. Kennedy1

1Cardiology Department, The Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, the Queen Elizabeth Hospital, Woodville South, South Australia, 2Department of Medicine, the University of Adelaide, Adelaide South Australia 3Present address: Cardiology Department, Lyell McEwin Hospital, Northern Adelaide Local Health Network, Elizabeth Vale, South Australia 4Present address: Department of Pharmacology and Clinical Pharmacology, University Hospital Dusseldorf, Germany

Background and aim of the study: Aortic valve stenosis is a major cause of valve replacement, particularly in the elderly. TGF-β1 is upregulated in stenotic valves and induces calcification and collagen synthesis in cultured valve interstitial cells. It has been shown previously that TGF-β1 increases reactive oxygen species (ROS) in these cells in association with calcifying nodule formation, but the cellular signaling pathways responsible for these TGF-β1-induced effects are not well defined.

Methods: Cultured porcine aortic valve interstitial cells were used to investigate the effects of inhibitors of TGF-β1 signaling pathways on 3H-proline incorporation into the extracellular matrix, the peak number of calcifying nodules formed, redox stress as dichlorofluorescein diacetate (DCF-DA) fluorescence, and senescence-associated β-galactosidase staining.

Results: Nodule formation and proline incorporation were inhibited by SB431542, implicating the Smad pathway, by SB203580, implicating the P38 MAPK pathway, and by U0126, implicating the Mek1/2/Erk1/2 pathway in both processes. Fasudil, an inhibitor of the Rho kinase pathway,

was selective in inhibiting nodule formation but not proline incorporation. It was verified that Smad 2 phosphorylation, Erk1/2 phosphorylation and p38 MAPK phosphorylation were all induced by TGF-β1, with Smad 2 phosphorylation peaking at 1-2 h and MAPK phosphorylation at 24-48 h. The effect of TGF-β1 on phosphorylation of Smad 2 was inhibited by SB431542, on the phosphorylation of p38 MAPK was inhibited by SB203580, and on the phosphorylation of Erk1/2 was inhibited by U0126. ROS generation in response to TGF-β1, measured as 2,7-dichlorofluorescein-diacetate fluorescence, was inhibited significantly by SB203580 and U0126, implicating both the p38 MAPK and Mek1/2/Erk1/2 signalling pathways. Both pathways also mediated TGF-β1-induced cellular senescence which was localized to cellular aggregates and mature nodules.

Conclusion: These data imply that the inhibition of either Smad or MAPK signalling pathways may have a therapeutic benefit in ameliorating the adverse pathological changes associated with aortic valve stenosis.

The Journal of Heart Valve Disease 2013;22:621-630

TGF-β1-Induced MAPK Activation Promotes Collagen Synthesis, Nodule Formation, Redox Stress and Cellular Senescence in Porcine Aortic Valve Interstitial Cells

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