Project description:Aims Calcific aortic valve disease (CAVD) is a significant cause of illness and death worldwide. The disease mechanism involves age-associated immuno-osteogenic processes but developmental origins and early disease mechanisms are not fully characterized. Identification of early predictive markers could help optimize therapy decisions and patient follow-up. Methods and Results RNA-sequencing was carried out on human fetal aortic valves at weeks 9, 13, and 22, and adult control, calcified bicuspid (BAV) and tricuspid aortic valves (TAV). K-means clustering was implemented to identify co-expressed gene patterns simultaneously up- and down-regulated over the different conditions. Canonical pathway analysis revealed a predominant immune-metabolic gene signature indicative of ongoing innate and adaptive immune responses, including lymphocyte T-cell metabolic adaptation. Specifically, cytokine and chemokine signalling, cellular migration and proliferation, were all increased in CAVD, while oxidative phosphorylation and protein translation were decreased. Discrete immune-metabolic gene signatures were present at foetal stages and adult controls, indicating that inflammation initiates as soon as the valves are formed and is maintained and amplified during post-natal life. Multiple pathways involved in cellular stress response and neurodegenerative disease were also aberrantly expressed, suggesting that chronic inflammation drives metabolic stress- and age-associated valve pathology in CAVD. Comparing the valve RNA-sequencing dataset with whole blood transcriptomes from asymptomatic individuals with early aortic valve calcification identified a gene signature including the CD28 pathway, highly predictive of patients with CAVD and healthy individuals with moderate levels of aortic valve calcification. Conclusion These data deepen and broaden our understanding of the molecular basis of CAVD, and identify a gene signature for the early detection of aortic valve calcification. Keywords Human foetal valve, CAVD, BAV, inflammation, biomarker, gene signature,PESA

Project description:Background: Calcific aortic valve disease (CAVD) is the pathological remodeling of valve leaflets. The initial steps in the osteogenic reprogramming of the leaflet are not fully understood. Studies have shown that TERT overexpression primes mesenchymal stem cells to differentiate into osteoblasts, and we investigated whether TERT contributes to the osteogenic reprogramming of valve interstitial cells (VICs). Methods: Human control and CAVD aortic valve leaflets and patient-specific hVICs were used in in vivo and in vitro calcification assays. Loss of function experiments in hVICs and cells isolated from Tert-/- and Terc-/- mice were used for mechanistic studies. In silico modeling, proximity ligation and co-immunoprecipitation assays defined novel TERT interacting partners. Chromatin immunoprecipitation and CUT&TAG sequencing defined protein-DNA interactions. Results: TERT protein was highly expressed in calcified valve leaflets without changes in telomere length, DNA damage, or senescence markers, and these features were retained in isolated primary hVICs. TERT expression increased with osteogenic stimuli, while knock-down or genetic deletion of TERT prevented calcification. Mechanistically, TERT was required to initiate osteogenic reprogramming independent of its canonical telomere-extending activity and the lncRNA TERC. TERT’s osteogenic functions via binding with Signal Transducer and Activator of Transcription 5 (STAT5). Depletion and inhibition of STAT5 prevented calcification. STAT5 was found to bind the promoter region of Runt-Related Transcription Factor 2 (RUNX2), the master regulator of osteogenic reprogramming. Finally, we demonstrate that TERT and STAT5 are upregulated and colocalized in CAVD tissue. Conclusions: TERT's non-canonical activity is required to initiate VIC calcification. TERT partners with STAT5 to bind the RUNX2 gene promoter. These data identify a novel mechanism and potential therapeutic target to decrease vascular calcification.

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A novel long noncoding RNA SNHG3 promotes osteoblast differentiation through BMP2 upregulation in calcific aortic valve disease

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