Profibrotic commitment of aortic valve interstitial cell via tissue factor expression and signalling

Purpose: Aortic valve stenosis (AVS) is an inflammatory atherosclerosis-like process characterized by valve interstitial cell (VIC) proliferation and commitment to fibrocalcification. No effective medical treatment is currently able to alter AVS clinical course. Tissue factor (TF) and thrombin expressions were previously shown to be significantly associated with fibrocalcification in human stenosed aortic valves. We investigated differential TF expression in normal and pathological valves and VIC and further described its signalling pathways in profibrosis commitment of normal VIC. Methods: We collected 50 fibrocalcific and 6 normal human aortic valves from which tissue lysates and VIC were isolated. VIC phenotype was analysed by flow cytometry. Normal VIC were stimulated or not with IL-1beta (major inflammatory cytokine involved in AVS at 1 ng/mL). In valve lysates and in VIC, TF and PAR-2 were quantified by qPCR for relative mRNA expression, and by flow cytometry/ELISA for protein and activity quantification. Downstream signalling pathways ERK (cell activation and proliferation) and Smad2 (profibrosis transcription factor) of VIIa/TF in TF-overexpressing VIC were assessed by westernblotting (incubation with FVIIa at 50 ng/mL for 30 min) in presence or absence of anti-TF antibody. Results are expressed as median (interquartile range). Results: VIC were defined as CD31(-) and CD68(-). TF and PAR-2 relative mRNA expressions were significantly higher in fibrocalcific vs normal valves (3.2 (1.5-.8) vs 0.18 (0.02-0.29), p<0.05; 1.15 (0.48-1.62) vs 0.16 (0.02-0.22), p<0.05; respectively). Normal VIC constitutively expressed TF (relative mRNA 4.1 (2.1-21.2); activity 36.2 (13.2-101.3) pg/mL) as well as PAR-2 (mRNA and protein by flow cytometry). TF expression was significantly increased in fibrocalcific VIC (relative mRNA 22.8 (9.8-33.8), p=0.05; activity 153 (71.5-247.0) pg/mL, p<0.05) when compared to normal VIC. Following IL-1beta stimulation of VIC, TF expression was significantly upregulated compared to unstimulated cells (relative mRNA 15.4 (12.9-28.2) fold increase, p<0.05; activity 2.7 (2.2-5.5) fold increase, p<0.05). ERK and Smad2 signalling pathways were upregulated in VIIa stimulated TF-overexpressing VIC (pERK/ERK 3.5 (2.9-4.8) fold increase; pSmad2/Smad2/3 1.8 (1.2-1.8) fold increase), an upregulation blunted by VIC preincubation with an anti-TF antibody. Conclusions: Our results demonstrate the implication of TF/VIIa/PAR-2 axis in VIC commitment to fibrocalcific aortic valve disease. Modulation of this pathway may represent a new therapeutic target for early medical treatment of AVS.