Impact de l'indoxyl sulfate sur la calcification des cellules interstitielles de la valve aortique humaine

Background. Calcific aortic valve disease (CAVD) results from fibro-calcific degeneration of the aortic valve leaflets. In chronic kidney disease (CKD) patients, this pathological remodelling is more frequent, appears earlier, progresses faster and increases the mortality rate. The uremic toxin indoxyl sulfate (IS) is a powerful predictor of aortic calcification and cardiovascular mortality in CKD patients. IS is also a powerful promoter of macrophage (MФ) inflammation, which is a key driver for valvular calcification. To date, the role of IS in aortic valve mineralization remains unknown. Objective. This work aimed to evaluate whether IS can influence primary human valvular interstitial cells (hVICs) mineralization, either directly (Task 1) or indirectly by modulating MФ inflammation (Task 2). Methods. Primary hVICs isolated from aortic tricuspid valves were exposed for 14 days to increasing concentrations of IS in the presence of an osteogenic medium containing 2.2 mmol/L calcium and 2.2 mmol/L phosphate. Their osteogenic transition was assessed by qPCR and western blot following Bmp2 and Runx2 expression, and their mineralization was quantified by the o-cresolphthalein complexone method. Inflammation was assessed following IL-1β, IL-6 and TNF-α expression and secretion by qPCR and ELISA. Activation of the NFΚB pathway was assessed by monitoring the phosphorylation of P65 via western blot. A siRNA approach was used to determine the signaling pathway involved in the effects of IS. In task 2, THP1-derived MФ were exposed for 48 hours to increasing concentrations of IS. Their inflammatory potential was monitored by qPCR and ELISA prior to evaluate the impact of conditioned media (CM) of these MФ on osteogenic differentiation and mineralization of hVICs. The expression and activity of Enhancer of Zeste Homolog 2 (EZH2, a key modulator of MФ inflammation) was assessed by western blot. To confirm the implication of EZH2 in IS-induced MФ inflammation, experiments were repeated in the presence of an EZH2 inhibitor called GSK-343 (5µM). Results. In task 1, we showed that IS increased hVICs osteogenic transition and calcification in a concentration-dependent manner, an effect blocked in hVICs transfected with a siRNA targeting the expression of IS receptor AhR (aryl hydrocarbon receptor). Exposure to IS promoted P65 phosphorylation in an AhR dependent manner. Blockade of NFкB pathway with a siRNA targeting P65 inhibited IS-induced expression of Bmp2 and RunX2 as well as hVICs mineralization, suggesting that hVICs inflammation plays a key role in the procalcific effects of IS. Exposure to IS increased the expression of IL-1β, IL-6 and TNF-α transcripts, an effect abolished by silencing AhR and P65. Interestingly, hVICs did not secrete TNF-α and produced very low quantity of IL-1β in response to IS. By contrast, the exposure to IS induced an important release of IL-6, which was blocked when AhR and P65 were silenced. The use of an antibody neutralizing IL-6 completely abolished IS-induced hVICs osteogenic transition and calcification. In task 2, we showed that CM from IS-polarized MФ contained high levels of IL-1β, IL-6 and TNF-α and promoted hVICs osteogenic transition and calcification. Exposure to IS upregulated MФ expression of EZH2, the blockade of which inhibited IS-induced MФ secretion of IL-6 and subsequent induction of runx2 in hVICs. HVICs do not express the receptor of IL-6, but their exposure to CM from IS-MФ promoted the secretion of its soluble form (IL-6Rs). Blockade of IL-6/IL-6Rs signaling using gp130Fc inhibited the mineralization of hVICs induced by CM from IS-MФ. Conclusion. Indoxyl sulfate-induced secretion of IL-6 either by VICs or MФ drives hVICs osteogenic transition and mineralization. The fact that GSK-343, gp130Fc and antibodies neutralizing IL-6 can block this mechanism sheds light on IL-6 as a new therapeutic target to prevent CAVD in CKD patients