Non-canonical Telomerase Reverse Transcriptase Controls Osteogenic Differentiation of Aortic Valve Cells Through STAT5

Abstract

BackgroundCalcific aortic valve disease (CAVD) is the pathological remodeling of the valve leaflets which leads to heart failure and high stroke risk. While several mechanisms are known to drive cardiovascular calcification, the initial steps orchestrating the osteogenic reprogramming of cells are not fully understood. Non-canonical functions of telomerase reverse transcriptase (TERT) include service as a cofactor to stimulate gene transcription, and TERT overexpression primes mesenchymal stem cells to differentiate into osteoblasts. We investigated whether TERT contributes to osteogenic reprogramming of valve interstitial cells.MethodsBaseline transcription of TERT and osteogenic markers, senescence, DNA damage, and telomere length in valve tissue and primary aortic valve interstitial cells (VICs) from control and CAVD patients were assessed. TERT expression was depleted in cells using lentiviral vectors. Cells from Tert+/+ and Tert-/- mice were used to validate human findings. Immunofluorescence staining, proximity ligation assay, and chromatin immunoprecipitation assay were used in mechanistic experiments.ResultsTERT protein was highly expressed in calcified valve leaflets, without changes in telomere length, DNA damage, or senescence. These phenotypic features were retained in primary VICs isolated and cultured from those diseased tissues. TERT levels were increased with osteogenic or inflammatory stimuli, and genetic deletion or reduction of TERT prevented calcification of VICs isolated from humans and mice. Similar results were seen in smooth muscle cells (SMCs) and mesenchymal stem cells (MSCs). TERT and Signal Transducer and Activator of Transcription 5A/B (STAT5) colocalize and bind to the Runt-Related Transcription Factor 2 (RUNX2) gene promoter, and TERT and STAT5 co-localized in calcified valve tissues. Pharmacological inhibition of STAT5A prevented calcification in vitro.ConclusionsThese data show that non-canonical TERT activity is required for the calcification of VICs. TERT partners with STAT5A to bind to and activate the RUNX2 gene promoter. These data identify a novel therapeutic target to abate vascular calcification.Novelty and SignificanceWhat Is Known?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 osteoblasts and osteogenic reprogramming of vascular cells, yet the early events driving its transcription in valve cells are not well defined.Overexpression of TERT primes mesenchymal stem cells to differentiate down the osteoblast lineage, suggesting that TERT signaling plays an important role in cell differentiation and phenotype.What New Information Does This Article Contribute?TERT protein is highly expressed in calcified aortic leaflets and valve interstitial cells, independent of changes in telomere length.Genetic loss or depletion of TERT blocks calcification in valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells.TERT co-localizes with STAT5 in the cytosol and on the RUNX2 gene promoter, the master regulator of osteogenic transcriptional programs.Pharmacological inhibition of STAT5 prevents calcification of human valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells.What are the clinical implications?We have identified TERT/STAT5 as novel signaling axis that promotes the early transcriptional reprogramming in cardiovascular cells. Inhibiting TERT and STAT5 interaction and activity can be leveraged for the development of pharmacological or biological therapeutic strategies to halt or prevent calcification in the aortic valve and perhaps other cardiovascular tissues.Surgical procedures are currently the only treatment option for patients with CAVD. Discovering the early events driving vascular calcification identifies novel and druggable targets for the development of non-surgical therapies.

Unraveling the Mechanisms of Valvular Heart Disease to Identify Medical Therapy Targets: A Scientific Statement From the American Heart Association

“…However, multiple clinical trials of traditional preventive therapies for cardiovascular disease, including statins, have failed when applied to AS, 32,56 and no medically efficacious treatments remain. Robust prior genetic and experimental studies in CAVD, as described in this scientific statement, have generated numerous molecular targets and pathways (Table 1), [1][2][3]6,13,21,23,26,35,38,[42][43][44][45][46]52,[56][57][58][59][60][61][62][63][64][67][68][69][70] which may, we hope, provide an opportunity for targeted drug development (reviewed in depth elsewhere 9,96 ). A handful of ongoing trials of medical therapy are targeting the progression of AS (Table 2).…”