Similar risk factors and mediators are involved in calcific aortic stenosis (CAS) and atherosclerosis. Since normal valves harbour a low percentage of smooth muscle cells (SMCs), we hypothesize that the SMC phenotype participates in the pathogenesis of CAS. We analysed 12 normal and 22 calcified aortic valves for SMC markers and the expression of co-activators of SMC gene expression, myocardin and myocardin-related transcription factors (MRTF-A/B). Transforming growth factor β (TGFβ1) was used to upregulate SMC markers and co-activators in valve interstitial cells (VICs) and transmission electron microscopy (TEM) was used to detect the presence of SMC in atypical regions of the valve leaflets. Smooth muscle cell markers and co-activators, myocardin, MRTF-A, and MRTF-B, demonstrated an increased incidence and aberrant expression around calcified nodules in all 22 calcified valves as well as in surface and microvessel endothelial cells. Smooth muscle cell markers and MRTF-A were significantly increased in calcified valves. Transforming growth factor β1 (TGFβ1) (10 ng/mL) was able to significantly upregulate the expression of some SMC markers and MRTF-A in VICs. Transmission electron microscopy of the fibrosa layer of calcified valves demonstrated the presence of bundles of SMCs and smooth muscle-derived foam cells. Smooth muscle cell markers and co-activators, myocardin and MRTFs, were aberrantly expressed in calcified valves. Transforming growth factor β1 was able to significantly upregulate SMC markers and MRTF-A in VICs. Transmission electron microscopy unequivocally identified the presence of SMCs in calcified regions of valve leaflets. These findings provide evidence that the SMC phenotype plays a role in the development of CAS.

Valve interstitial cells (VICs) are fibroblastic in nature however in culture it is widely accepted that they differentiate into a myofibroblastic phenotype. This study assessed a fibroblast culture media formulation for its ability to maintain the phenotype and function of VICs as in the intact healthy valve. Normal human VICs were cultured separately in standard DMEM and in fibroblast media consisting of FGF2 (10ng/ml), insulin (50ng/ml) and 2% FCS for at least a week. Cell morphology, aspect ratio, size, levels and distribution of protein expression, proliferation, cell cycle, contraction and migration were assessed. Some VICs and some valve endothelial cells expressed FGF2 in valve tissue and this expression was increased in calcified valves. VICs in DMEM exhibited large, spread cells whereas VICs in fibroblast media were smaller, elongated and spindly. Aspect ratio and size were both significantly higher in DMEM (p<0.01). The level of expression of α-SMA was significantly reduced in fibroblast media at day 2 after isolation (p<0.01) and the expression of α-SMA, SM22 and EDA-fibronectin was significantly reduced in fibroblast media at days 7 and 12 post-isolation (p<0.01). Expression of cytoskeletal proteins, bone marker proteins and extracellular matrix proteins was reduced in fibroblast media. Proliferation of VICs in fibroblast media was significantly reduced at weeks 1 (p<0.05) and 2 (p<0.01). Collagen gel contraction was significantly reduced in fibroblast media (p<0.05). VICs were found to have significantly fewer and smaller focal adhesions in fibroblast media (p<0.01) with significantly fewer supermature focal adhesions in fibroblast media (p<0.001). Ultrastructurally, VICs in fibroblast media resembled native VICs from intact valves. VICs in fibroblast media demonstrated a slower migratory ability after wounding at 72 hours (p<0.01). Treatment of human VICs with this fibroblast media formulation has the ability to maintain and to dedifferentiate the VICs back to a fibroblastic phenotype with phenotypic and functional characteristics ascribed to cells in the intact valve. This methodology is fundamental in the study of normal valve biology, pathology and in the field of tissue engineering.

Background: Valvular interstitial cells ( VIC s) in the healthy aortic valve leaflet exhibit a quiescent phenotype, with <5% of VIC s exhibiting an activated phenotype. Yet, in vitro culture of VIC s on tissue culture polystyrene surfaces in standard growth medium results in rapid transformation to an activated phenotype in >90% of cells. The inability to preserve a healthy VIC phenotype during in vitro studies has hampered the elucidation of mechanisms involved in calcific aortic valve disease. This study describes the generation of quiescent populations of porcine VIC s in 2‐dimensional in vitro culture and their utility in studying valve pathobiology. Methods and Results: Within 4 days of isolation from fresh porcine hearts, VIC s cultured in standard growth conditions were predominantly myofibroblastic (activated VIC s). This myofibroblastic phenotype was partially reversed within 4 days, and fully reversed within 9 days, following application of a combination of a fibroblast media formulation with culture on collagen coatings. Specifically, culture in this combination significantly reduced several markers of VIC activation, including proliferation, apoptosis, α‐smooth muscle actin expression, and matrix production, relative to standard growth conditions. Moreover, VIC s raised in a fibroblast media formulation with culture on collagen coatings exhibited dramatically increased sensitivity to treatment with transforming growth factor β1, a known pathological stimulus, compared with VIC s raised in either standard culture or medium with a fibroblast media formulation. Conclusions: The approach using a fibroblast media formulation with culture on collagen coatings generates quiescent VIC s that more accurately mimic a healthy VIC population and thus has the potential to transform the study of the mechanisms of VIC activation and dysfunction involved in the early stages of calcific aortic valve disease.