isolation-characterization-primary-rat-valve-interstitial-cells-new

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

University of Edinburgh

The importance of matrix vesicles (MVs) has been repeatedly highlighted in the formation of cartilage, bone, and dentin since their discovery in 1967. These nano-vesicular structures, which are found in the extracellular matrix, are believed to be one of the sites of mineral nucleation that occurs in the organic matrix of the skeletal tissues. In the more recent years, there have been numerous reports on the observation of MV-like particles in calcified vascular tissues that could be playing a similar role. Therefore, here, we review the characteristics MVs possess that enable them to participate in mineral deposition. Additionally, we outline the content of skeletal tissue- and soft tissue-derived MVs, and discuss their key mineralisation mediators that could be targeted for future therapeutic use.

Characterisation of matrix vesicles in skeletal and soft tissue mineralisation.

Patients with end-stage renal disease (ESRD) have elevated circulating calcium (Ca) and phosphate (Pi), and exhibit accelerated progression of calcific aortic valve disease (CAVD). We hypothesized that matrix vesicles (MVs) initiate the calcification process in CAVD. Ca induced rat valve interstitial cells (VICs) calcification at 4.5 mM (16.4-fold; p < 0.05) whereas Pi treatment alone had no effect. Ca (2.7 mM) and Pi (2.5 mM) synergistically induced calcium deposition (10.8-fold; p < 0.001) in VICs. Ca treatment increased the mRNA of the osteogenic markers Msx2, Runx2, and Alpl (p < 0.01). MVs were harvested by ultracentrifugation from VICs cultured with control or calcification media (containing 2.7 mM Ca and 2.5 mM Pi) for 16 hr. Proteomics analysis revealed the marked enrichment of exosomal proteins, including CD9, CD63, LAMP-1, and LAMP-2 and a concomitant up-regulation of the Annexin family of calcium-binding proteins. Of particular note Annexin VI was shown to be enriched in calcifying VIC-derived MVs (51.9-fold; p < 0.05). Through bioinformatic analysis using Ingenuity Pathway Analysis (IPA), the up-regulation of canonical signaling pathways relevant to cardiovascular function were identified in calcifying VIC-derived MVs, including aldosterone, Rho kinase, and metal binding. Further studies using human calcified valve tissue revealed the co-localization of Annexin VI with areas of MVs in the extracellular matrix by transmission electron microscopy (TEM). Together these findings highlight a critical role for VIC-derived MVs in CAVD. Furthermore, we identify calcium as a key driver of aortic valve calcification, which may directly underpin the increased susceptibility of ESRD patients to accelerated development of CAVD.

End stage renal disease-induced hypercalcemia may promote aortic valve calcification via Annexin VI enrichment of valve interstitial cell derived-matrix vesicles.

Cui Lin

Developmental Biology,
The Roslin Institute and R(D)SVS,
Developmental Biology, The Roslin Institute and R(D)SVS

University of Edinburgh

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Cui Lin

Developmental Biology,The Roslin Institute and R(D)SVS,Developmental Biology, The Roslin Institute and R(D)SVS

University of Edinburgh

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Developmental Biology,
The Roslin Institute and R(D)SVS,
Developmental Biology, The Roslin Institute and R(D)SVS