The Small Molecule Drug Pirfenidone Attenuates Fibrosis and Calcification in Cell and Tissue Culture Models of Calcific Aortic Valve Disease

Objectives: Calcific aortic valve disease (CAVD) is the most frequently acquired heart valve disease. However, because of its complex pathogenesis, there is currently no drug-based CAVD therapy available. Pirfenidone is a small molecule drug with well-known antifibrotic impact, which is already approved for the treatment of idiopathic pulmonary fibrosis. Here, employing cell culture of valvular interstitial cells (VICs) and tissue culture of aortic valve leaflets as CAVD models, we investigate effects of pirfenidone on fibrosis and calcification.

Methods: Ovine VICs and leaflets of aortic valves were stimulated with TGF-β1 or β-GP and CaCl2 to induce fibrosis and calcification as phenotypical characteristics of CAVD, respectively. Collagen protein secretion, mothers against decapentaplegic homolog (SMAD)3 activation as well as expression of vimentin, and α-smooth muscle actin (α-SMA) were quantified by immunoblot. Calcification was analyzed by alizarin-red staining and Ca²⁺ assay. Alkaline phosphatase (ALP) activity was determined by nitro-blue tetrazolium/indolylphosphate (NBT/BCIP) staining. Toxicity was assessed by measuring lactate dehydrogenase activity in culture supernatants.

Results: Pirfenidone treatment dose-dependently inhibited the calcification of VICs without mediating toxic effects in the analyzed concentration range. Correspondingly, pirfenidone also attenuated the calcification of valvular tissue. The anticalcific effects of pirfenidone were accompanied by a significant inhibition of degenerative VIC differentiation as evidenced by reduced ALP activity, decreased α-SMA, and increased vimentin expression. With regard to fibrosis, pirfenidone dose-dependently inhibited the TGF-β1-induced upregulation of collagen type 1 secretion in VICs. Moreover, pirfenidone also attenuated the TGF-β1-induced upregulation of collagen type 1 secretion in valvular tissue and blocked the associated activation of transcription factor SMAD3.

Conclusion: Our observations in cell and tissue culture models indicate that pirfenidone attenuates the emergence of fibrosis and calcification as phenotypical characteristics of CAVD by mechanisms involving inhibition of SMAD3 activation and degenerative VIC differentiation.