P6504NETosis is associated with the severity of aortic stenosis

Abstract

Abstract Background High hemodynamic forces similar to those observed in aortic stenosis (AS) can trigger neutrophil extracellular traps (NETs) formation. Purpose An involvement of NETosis in the AS pathogenesis is unknown. Methods We enrolled 25 patients, median age 64.9 [range, 58–69] years with isolated severe AS (transvalvular pressure gradient, PGmean: 53.6±11.3 mm Hg, PGmax: 85.1±17.6 mmHg), after aortic valve replacement and 15 healthy sex/age-matched controls. Autopsy-derived aortic valves from 5 healthy donors served as negative control. Transverse valve sections were taken from the mid and commissural areas of the leaflet and cryosectioned by a Leica CM 1520 cryostat. Valvular expression of citrullinated histone H3 (citH3), together with myeloperoxidase (MPO), and neutrophil elastase (NE) as NETs biomarkers and macrophages (CD68) were evaluated by single- and double- immunostaining. Plasma concentrations of citH3 and interleukin 6 (IL-6) were also determined. Results All stenotic and healthy valves expressed citH3 in the leaflets' endothelial and sub-endothelial layers at the aortic side. The in loco expression of citH3-positive cells was higher in AS patients compared with controls (42.3±8.7% vs. 7.2±4.8%, p<0.05) and correlated with disease severity measured as aortic valve area (AVA; r=−0.84, p<0.0001), as well as PGmean (r=0.62, p<0.001), and PGmax (r=0.52, p>0.05). Double-staining revealed that within stenotic leaflets 28.3±8.4% of cells were citH3/MPO- and 25.2±7.1% citH3/NE-positive. None of control valves showed MPO or NE-positivity. Moreover, 6.6±1.9% of valvular cells (17.2±5.4% of citH3-positive cells) showed citH3/CD68 double-positivity and were identified as macrophages. Plasma levels of citH3 were 59% higher in AS patients then in controls (p<0.05), and the concentrations of citH3 correlated with IL-6 levels (r=0.44, p<0.05) and AVA (r=−0.48, p<0.05). Conclusions The presence of NETs in stenotic valves and association with AS severity might suggest novel mechanisms involved in the disease progression. This work was supported by the grant from the Polish National Science Center (DEC-2017/01/X/NZ5/02006 to R.K-T.). Acknowledgement/Funding This work was supported by the grant from the Polish National Science Center (DEC-2017/01/X/NZ5/02006 to R.K-T.).