Effects of interferons and their interactions with other ligands in human aortic valve cells

Resumen

Introduction: Calcific aortic valve disease (CAVD) is the most common aetiology of acquired aortic valve disease. Initially considered a passive and degenerative process, the current view has redefined CAVD as an athero-inflammatory process in early stages that then progresses into a more complex condition. Supporting the role of inflammation-induced valve calcification are growing evidences on the induction of inflammation and subsequent pro-calcifying responses in valve interstitial (VIC) and endothelial cells (VEC) by immune mediators such as Toll-like receptor (TLR) ligands and tumor necrosis factor-α (TNF-α). Objectives: Prompted by evidences of a constitutive interferon (IFN) activity associated to ectopic calcification in a rare disease, the Singleton-Merten syndrome, and the infiltration of immune cells secreting IFN in diseased valves, we aimed to explore the role of these cytokines and Janus kinases (JAK)/Signal transducers and activators of transcription (STAT) pathways on CAVD pathogenesis. Materials and methods: Human aortic valve cells explanted from patients with no valve disease were used as a model for studying CAVD. Cells were exposed to recombinant IFN-α and IFN-γ combined or not with TLR ligands (in VIC) or TNF-α (in VEC). Inflammation, calcification and angiogenic responses as well as cell proliferation and apoptosis were studied using different cellular and molecular biology techniques such as Western Blot, ELISA, qPCR, flow cytometry, immunofluorescence and calcification assays. In addition, total protein and RNA were extracted from both non-mineralized and calcified valves for gene and protein expression analysis. Results: IFN activate several signalling pathways and promote inflammatory responses in VIC, characterized by adhesion molecule expression and nuclear factor-κB activation. In addition, IFN trigger VIC differentiation towards a pro-osteogenic phenotype and promote calcific nodule formation in high-phosphate conditions. Strikingly, we found an IFN-Lipopolysaccharide (a TLR4 ligand) interplay that further potentiates the pro-inflammatory, pro-angiogenic and pro-osteogenic responses. Significant findings include the blockade of the responses by Jakinibs, which are JAK inhibitors currently used in clinics. Additionally, this study provides several new insights about sex-differences in CAVD, with unexpected greater responses to IFN and LPS in male cells and identified some of the underlying molecular mechanisms such as larger protein kinase B/Akt activation in cells from females, and larger osteogenic signalling, hypoxia-inducible factor (HIF)-1α and extracellular signal-regulated kinases activation in male cells. We also found sex-differences in gene expression profile in calcified tissue that correlate with lower valve calcification in female patients. Furthermore, our work demonstrates a novel role for type I IFN signalling on TLR3-mediated effects in VIC. Finally, data unveiled differences on monocyte adhesion to side-specific VEC monolayers in response to IFN-γ and TNF-α with potential relevance in CAVD pathogenesis. Conclusions: Our findings highlight that IFN act as pro-inflammatory and pro-osteogenic cytokines in VIC and support the model of inflammation-induced calcification, the concept of additional pro-angiogenic mechanisms beyond valve thickening and hypoxia, and the notion of CAVD as a sex-divergent disease from the early inflammatory stages. Clinically relevant findings include the blockade of IFN-induced responses by Jakinibs, and by a HIF-1α inhibitor. JAK/STAT and HIF-1α pathways emerge as potential therapeutic targets for CAVD.