Lipopolysaccharide and interferon-γ team up to activate HIF-1α via STAT1 in normoxia and exhibit sex differences in human aortic valve interstitial cells

In early stages of calcific aortic valve disease (CAVD), immune cells infiltrate into the valve leaflets and release cytokines such as interferon (IFN)-gamma. IFN-gamma has context-dependent direct effects, and also regulates other immune pathways. The purpose of this study was addressing the effects of IFN-gamma on human aortic valve interstitial cells (AVICs), focusing on the pathogenic processes underlying CAVD. Strikingly, under normoxic conditions, IFN-gamma induced hypoxia inducible factor (HIF)-1 alpha expression, an effect strongly potentiated by the Toll-like receptor (TLR)-4 ligand lipopolysaccharide (LPS). Immunodetection studies confirmed the nuclear translocation of HIFI a. Gene silencing showed that HIF-1 alpha expression is dependent on signal transducer and activator of transcription (STAT)-1 expression. Consistent with HIF-1 alpha induction, the secretion of the endothelial growth factor was detected by ELISA, and downregulation of the antiangiogenic factor chondromodulin-1 gene was observed by qPCR. Results also disclosed IFN-gamma as a proinflammatory cytokine that cooperates with LPS to induce the expression of adhesion molecules, prostaglandin E-2 and interleukins. Moreover, IFN-gamma induced an osteogenic phenotype and promoted in vitro calcification that were markedly potentiated by LPS. Pharmacological experiments disclosed the involvement of Janus Kinases (JAK)/STATs as well as ERK/HIF-1 alpha routes on the induction of calcification. Notably, IFN-gamma receptor I expression, as well as ERK/HIF-1 alpha activation, and the subsequent responses were more robust in male AVICs. This is the first report uncovering an immune and non-hypoxic activation of HIF-1 alpha via STAT1 in AVIC. The aforementioned results and the sex-differential responses may be potentially relevant to better understand CAVD pathogenesis.