Impact of plasma from patients with severe aortic stenosis on valvular endothelial cells

Abstract Background The natural history of aortic stenosis (AS) encompasses a spectrum of pathophysiological stages, including endothelial dysfunction, inflammation, fibrosis and calcification. Accumulating data underscore the detrimental impact of the haemostatic system on AS, contributing to disease progression and the development of a prothrombotic phenotype within the valve. Understanding these mechanisms is pivotal for the exploration of novel therapeutic avenues, particularly in the context of transcatheter aortic valve replacement (TAVR). Following TAVR, the native diseased aortic valve persists, maintaining its pathological activity and thrombogenic effects on the bioprosthetic valve, potentially influencing its durability and patient outcomes. The precise effects of plasma and its various components on valvular endothelial cells (VECs) dysfunction remains inadequately explored. Purpose To evaluate the influence of plasma derived from individuals with severe AS on aortic VECs. Methods Plasma samples obtained from patients with severe AS undergoing TAVR (n=100) were collected immediately before the procedure, alongside plasma samples from healthy individuals. Factor (F)Xa activity within the plasma samples was quantified using fluorometer-based method. Porcine VECs were then exposed to either plasma (at 10% concentration) or isolated FXa (1 nM), with or without different pharmacological modulators. Protein expression levels were assessed by Western blot analyses. The level of reactive oxygen species (ROS) was evaluated by dihydroethidium staining. Results Exposure of VECs to plasma from severe AS patients promoted ROS formation compared to plasma from healthy individuals (1402 ± 438 AU vs 732 ± 137 AU, p=0.001) (Figure 1). The heightened oxidative stress elicited by plasma from severe AS patients was attenuated by empagliflozin, perindoprilat, losartan, neutralizing antibodies directed against IL-1, IL-6 and TNF-α. Plasma from severe AS patients showed elevated FXa activity compared to plasma from healthy individuals (179.4 ± 101.3 ng/mL vs 63.2 ± 87.2 ng/mL, p=0.02). Stimulation of VECs with isolated FXa resulted in a sustained formation of ROS, which was blunted by empagliflozin, perindoprilat, losartan, rivaroxaban, dabigatran and PAR-1, 2 and 4 antagonists. Quantification of protein expression in VECs stimulated by isolated FXa revealed an endothelial dysfunction phenotype characterized by increased expression of SGLT2, VCAM-1 and COX-1, and decreased expression of eNOS. Conclusions Plasma from patients with severe AS exerts a detrimental effect on aortic VECs, characterized by a pronounced oxidative stress response. This adverse impact is mediated by multiple mechanisms, including activation of the AT1R/NADPH oxidase/SGLT2 pathway, upregulation of pro-inflammatory cytokines, and activation of PAR. FXa plays a pivotal role by promoting oxidative stress in VECs and contributing to valvular endothelial dysfunction.Pro-oxidant state in VECs.