A novel non-hypoxic and sex-biased mechanism of HIF-1¿ in human aortic valve interstitial cells: crosstalk between JAK-STAT and TLR pathways

Abstract

[Introduction]: Immune cell infiltration is one of the earliest events in calcific aortic valve disease (CAVD). Recent data showed that infiltrated T lymphocytes secrete active interferon (IFN)­γ, the effects of which in resident valve cells remain unknown. In addition, angiogenesis has been pointed out as a key player in CAVD since new vessels formation and hypoxia­inducible factor (HIF)­1α have been detected in calcified aortic valves. However, its underlying molecular mechanisms are still poorly understood. Purpose: To elucidate the role of IFN­γ on inflammation, angiogenesis and calcification of human aortic valve interstitial cells (AVIC) isolated from male and female patients. Methods: AVIC were isolated from healthy valves by collagenase digestion and exposed to IFN­γ and/or lipopolysaccharide (LPS). Western Blot and ELISA were used to analyze pro­inflammatory and pro­angiogenic molecules. The osteogenic marker bone morphogenetic protein (BMP)­2 and the anti­angiogenic factor chondromodulin­1 (ChM­I) were analyzed by RT­qPCR. Immunofluorescence was used to evaluate HIF­1α nuclear translocation. Alizarin red staining and calcium deposits quantitation were performed to evaluate in vitro calcification of AVIC in high­phosphate conditions.

[Results]: Data showed that IFN­γ and LPS cooperated to promote nuclear factor (NF)­κB activation, and to induce adhesion molecule expression and interleukin (IL)­6 secretion. Moreover, IFN­γ and LPS combination promoted the induction of HIF­1α and the secretion of its target gene, vascular endothelial growth factor (VEGF)­A. The effect exhibited sex differences and was blocked with a HIF­1α inhibitor. Additionally, a decrease of ChM­I expression was observed. AVIC morphology markedly changed upon long­term stimulation with IFN­γ, exhibiting an osteogenic phenotype characterized by BMP­2 induction. Strikingly, marked sex differences were found in BMP­2 expression and IL­6 secretion. Finally, IFN­γ promoted AVIC calcification that was further potentiated by LPS, being male AVIC more prone to calcification. Remarkably, pre­treatment with the JAK1/2 inhibitor ruxolitinib abrogated IFN­γ effects.

[Conclusions]: IFN­γ promotes pro­inflammatory, pro­angiogenic and pro­osteogenic responses in AVIC, which are potentiated by LPS in a sex­specific manner. Also, IFN­γ combined with LPS induces HIF­1α by a hypoxia­independent mechanism. Our results point to JAK­STAT pathways as potential therapeutic targets for CAVD.