Objective: We have previously reported that human calcified aortic cusps have abundant expression of smooth muscle (SM) markers and co-activators. We hypothesised that cells in bicuspid aortic valve (BAV) cusps and those affected by rheumatic heart valve (RHV) disease may follow a similar phenotypic transition into smooth muscle cells, a process that could be regulated by transforming growth factors (TGFs). Aims: Cusps from 8 patients with BAV and 7 patients with RHV were analysed for ealy and late SM markers and regulators of SM gene expression by immunocytochemistry and compared to healthy aortic valves from 12 unused heart valve donors. The ability of TGFs to induce these markers in valve endothelial cells (VECs) on two substrates was assessed. Results: 7 out of 8 BAVs and all the RHVs showed an increased and atypical expression of early and late SM markers α-SMA, calponin, SM22 and SM-myosin. The SM marker coactivators were aberrantly expressed in 6 of the BAV and 6 of the RHV, in a similar regional pattern to the expression of SM markers. Additionally, regions of VECs, and endothelial cells lining the vessels within the cusps were found to be positive for SM markers and coactivators in 3 BAV and 6 RHV. Both BAVs and RHVs were significantly thickened and HIF1α expression was prominent in 4 BAVs and 1 RHV. The ability of TGFβs to induce the expression of SM markers and myocardin was greater in VECs cultured on fibronectin than on gelatin. Fibronectin was shown to be upregulated in BAVs and RHVs, within the cusps as well as in the basement membrane. Conclusion: BAVs and RHVs expressed increased numbers of SM marker-positive VICs and VECs. Concomittantly, these cells expressed MRTF-A and myocardin, key regulators of SM gene expression. TGFβ1 was able to preferentially upregulate SM markers and myocardin in VECs on fibronectin, and fibronectin was found to be upregulated in BAVs and RHVs. These findings suggest a role of VEC as a source of cells that express SM cell markers in BAVs and RHVs. The similarity between SM marker expression in BAVs and RHVs with our previous study with cusps from patients with aortic stenosis suggests the existance of a common pathological pathway between these different pathologies. Abbreviations: BAV, bicuspid aortic valve; RHV, rheumatic heart valve; SM, smooth muscle; TGF, transforming growth factor; MRTF-A, myocardin related transcription factor A; VIC, valve interstitial cell; VEC, valve endothelial cell; PCNA, proliferating nuclear cell antigen; HIF1α, hypoxia inducible factor 1α
