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Updated: Apr 5, 2026
Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro
Published on: May 10, 2021
Osteopontin alters endothelial and valvular interstitial cell behaviour in calcific aortic valve stenosis through
Margaret Passmore1, Maria Nataatmadja2, Yoke L Fung3
1Critical Care Research Group, University of Queensland, Brisbane, Australia m.passmore@uq.edu.au.
Insights
Osteopontin (OPN) alters high-mobility group box 1 (HMGB1) function in calcific aortic valve stenosis (CAVS). This suggests OPN regulates CAVS progression by affecting HMGB1, impacting inflammation and fibrosis.
Area of Science:
- Cardiovascular Biology
- Immunology
- Molecular Medicine
Background:
- Calcific aortic valve stenosis (CAVS) is a prevalent condition in the elderly.
- CAVS progression involves valve endothelial injury, inflammation, fibrosis, and calcification.
- Osteopontin (OPN) and high-mobility group box 1 (HMGB1) are implicated in inflammatory and fibrotic processes.
Purpose of the Study:
- To investigate the regulatory role of OPN on HMGB1 function in CAVS.
- To elucidate the impact of OPN on inflammatory and fibrotic responses in CAVS.
- To determine the cellular mechanisms underlying OPN and HMGB1 interaction in CAVS.
Main Methods:
- Aortic valve leaflets from CAVS patients (n=40) and controls (n=15) were analyzed.
- Quantitative PCR, immunohistochemistry, and Western blot were used to assess gene and protein expression.
- Primary cell cultures (endothelial cells, valvular interstitial cells) were treated with OPN or anti-OPN antibodies to evaluate proliferation and HMGB1 expression.
Main Results:
- CAVS valves showed increased OPN, TNF-alpha, and fibrosis markers, with elevated plasma OPN.
- HMGB1 was detected in secretory granules of endothelial cells and VICs from CAVS valves.
- OPN inhibited proliferation and promoted extracellular HMGB1 release in cultured cells, while OPN neutralization had opposite effects.
Conclusions:
- Altered OPN expression in CAVS influences HMGB1 cellular function.
- OPN induces cytoplasmic translocation and secretion of HMGB1 in endothelial cells and VICs.
- OPN plays a regulatory role in CAVS progression via modulation of HMGB1 function.
Objectives:
Calcific aortic valve stenosis (CAVS) is an important clinical problem predominantly affecting elderly individuals. Studies suggest that the progression of CAVS is actively regulated with valve endothelial injury leading to inflammation, fibrosis and calcification. The aim of this study was to delineate the possible regulatory role of osteopontin (OPN) on high-mobility group box 1 (HMGB1) function and the associated inflammatory and fibrotic response in CAVS.
Methods:
Aortic valve leaflets were collected from CAVS patients undergoing aortic valve replacement (n = 40), and control aortic valve leaflets were obtained from heart transplant recipients (n = 15). Valves and plasma were analysed by quantitative real-time polymerase chain reaction (PCR), immunohistochemical staining and Western blot. Recombinant OPN or neutralizing OPN antibody was added to cultured endothelial and valvular interstitial cells (VICs), and cell proliferation scores and HMGB1 expression were assessed.
Results:
CAVS valves had a decreased total percentage of VICs but increased numbers of infiltrating macrophages relative to control valves. RT-PCR studies showed higher expression of OPN, the inflammatory cytokine tumour necrosis factor-alpha as well as markers of fibrosis, tissue inhibitor of matrix metalloproteinase 1 and matrix metalloproteinase 2 in CAVS valves. Elevated expression of OPN was also observed in plasma of CAVS patients compared with controls. HMGB1 was detected in the secretory granules of cultured valve endothelial and VICs derived from CAVS valves. The addition of exogenous OPN inhibited the proliferation of cultured endothelial and VICs from CAVS valves and was associated with the extracellular expression of HMGB1, whereas neutralizing OPN had the opposite effect.
Conclusions:
We conclude that altered OPN expression in CAVS affects cellular HMGB1 function inducing cytoplasmic translocation and secretion of HMGB1 in endothelial cells and VICs, thus indicating a regulatory role for OPN in the progression of CAVS through alteration of HMGB1 function.
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