Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Cuevas, Rolando; Chu, Claire; Moorhead, William J.; Wong, Ryan; Sultan, Ibrahim; Hilaire, Cynthia St.

doi:10.3791/62439

Cited by 1 publication

(7 citation statements)

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“…We created patient-specific VIC lines isolated from CAVD and control valves. 23 At baseline, VICs freshly isolated from CAVD and control patients exhibited no morphological, proliferative, or migratory differences (Figures 2A, 2B, 2C, and Supplemental Figure 2A, respectively) while TERT and RUNX2 proteins were significantly elevated in CAVD VICs (Figure 2D). At baseline, we observed no differences between CAVD and control VICs in SM22 or αSMA — markers of an activated VIC phenotype (Supplemental Figure 2B)— but found significantly higher levels of the mineralizing enzyme tissue non-specific alkaline phosphatase (TNAP) in CAVD VICs (Supplemental Figure 2C).…”

Section: Resultsmentioning

confidence: 94%

“…Human aortic valves were collected after surgical aortic valve replacement procedures or from cadaveric tissue obtained via the Center for Organ Recovery and Education (CORE) of the University of Pittsburgh and processed as previously described. 23 Macroscopic examination and histological staining with Von Kossa were used to determine and confirm whether valves could be classified as a non-calcified control or as having calcific aortic valve disease (CAVD) (Figure 1A, Supplemental Figure 1A, Table 1 and 2 : Patient Information ). RUNX2 is the master transcription factor required for stem cells to differentiate into osteoblasts, 24 and is critical for the pathological osteogenic differentiation of vascular cells.…”

Section: Resultsmentioning

confidence: 99%

“…In our study, a portion of each patient valve was used for cell isolation while the rest was utilized for calcification assessment by histological staining, following a workflow that we developed that guarantee that cells isolated from surgically removed and postmortem tissues retain their proliferative capacity and endothelial and interstitial phenotypes in culture. 23…”

Section: Discussionmentioning

confidence: 99%

“…A detailed protocol describing tissue collection handling was published previously. 1 Briefly, valves were collected from valve replacement surgeries or recovered from cadaveric organs and stored in cold Belzer UW Cold Storage Transplant Solution (Bridge to Life) at 4°C for transporting. Aortic roots were excised and washed with a sterile rinsing solution (sterile PBS supplemented with 2.5 μg/mL of fungicide, 0.05 mg/mL of gentamicin, and 5 μg/mL of bactericide).…”

Section: Methodsmentioning

confidence: 99%

“…Human primary aortic valve interstitial cell (VIC) lines were generated from male and female subjects as previously described. 2 Briefly, leaflets were washed with PBS containing 10 mg/ml gentamicin (GIBCO) and 250 μg/ml fungizone (GIBCO) and dissociated with 0.1% collagenase II at 37 C and 5% of CO2 for 18 hours. Then, the tissue was further dissociated by gently mixing it by pipetting with a serological pipette to ensure the release of AVICs and then passed through a 0.70 μm filter to remove debris.…”

Section: Methodsmentioning

confidence: 99%

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

Cuevas

Hortells

Chu

et al. 2022

Preprint

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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.

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Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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