ABSTRACT
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 exerts its 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.
Calcific aortic valve disease (CAVD) is the most prevalent form of aortic valve pathology. CAVD strongly correlates with age and leads to heart failure and a high risk of stroke. Currently, the only therapeutic option is valve replacement, which comes with significant healthcare costs and additional risks to patients.
Runt-related transcription factor 2 (RUNX2) is the master transcription factor required for osteogenic differentiation of stem cells to osteoblasts and osteogenic reprogramming of cardiovascular cells. Yet, the early events driving its transcription in aortic valve cells are poorly defined.
In addition to its reverse transcriptase enzymatic activity, TERT exhibits non-canonical transcriptional regulatory functions and overexpression of TERT primes mesenchymal stem cells to differentiate down the osteoblast lineage.
TERT protein levels in calcified aortic leaflets and valve interstitial cells, and its non-canonical osteogenic activity are independent of changes in telomere length and cell senescence.
Genetic loss or depletion of TERT blocks calcification in valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells.
Early in the osteogenic reprogramming of VICs TERT co-localizes with STAT5, and this TERT-tethered STAT5 binds to the RUNX2 gene promoter, the master regulator of osteogenic transcriptional programs.
STAT5 depletion and pharmacological inhibition prevent calcification of human valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells.
We have identified TERT-STAT5 as a novel signaling axis that orchestrates the early steps in the osteogenic reprogramming of aortic valve cells. Inhibiting TERT and STAT5 activity, or their interaction, may be leveraged to develop pharmacological or biological therapeutic strategies to halt or prevent calcification in the aortic valve, bioprosthetic valves, and perhaps other cardiovascular tissues.
Invasive and expensive surgical procedures are currently the only treatment option for patients with CAVD. The discovery and defining of the early events driving vascular calcification identifies novel and druggable targets for developing non-surgical therapies.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
We have provided new in vivo and ex vivo data in Tert+/+ and Tert-/-mice that illustrate a requirement for TERT in osteogenic reprogramming; performed CUT&Tag sequencing to unbiasedly demonstrate a requirement for TERT in the recruitment of STAT5 to gene promoters during osteogenic reprogramming; and created a tool that enabled the visualization and further confirmation of TERT-STAT5 protein-protein interaction in live cells (splitFAST).
ABBREVIATIONS
- αSMA
- alpha 2 smooth muscle actin
- αTUB
- alpha tubulin
- hVICs
- human aortic valve interstitial cells
- CAVD
- calcific aortic valve disease
- Ch-IP
- chromatin immunoprecipitation
- ECM
- extracellular matrix
- γ-H2AX
- phosphorylated gamma histone 2AX
- LPS
- lipopolysaccharide
- BMMSCs
- bone marrow mesenchymal stem cells
- MSCs
- Mesenchymal stem cells
- NT
- no treatment
- mVICs
- mouse aortic valve interstitial cells
- OPN
- osteopontin
- OST
- osteogenic treatment
- PCNA
- proliferating cell nuclear antigen
- RUNX2
- Runt-elated transcription factor 2
- STAT5
- Signal Transducer and Activator of Transcription 5A/B
- TERC
- telomerase RNA component
- TERT
- telomerase reverse transcriptase catalytic subunit
- TL
- telomere length.
