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Aortic valve calcification is believed to involve the differentiation of valvular interstitial cells (VICs) into either a myofibroblastic or an osteoblast-like phenotype. Despite purported similarities between diseased VICs and osteoblasts, few studies have directly compared VICs and osteoblasts in side-by-side experiments. The present work compares VICs against multiple osteoblastic cell types at different stages of differentiation, and may also help to resolve whether VICs progress through a myofibroblastic phenotype prior to reaching an osteoblast-like stage.
Three cell types representing a range of osteoblastic lineage commitment and differentiation were used in the phenotypic comparison against VICs. Specifically, VICs, embryonic fibroblasts (C3H10T1/2), pre-osteoblasts (MC3T3-E1), and mature primary osteoblasts were cultured on tissue culture polystyrene in control or mineralization medium, and were harvested for qPCR, DNA, and protein analysis at time points ranging from 1–8 days.
Culture of VICs in mineralization medium decreased expression of alpha-smooth muscle actin (α-SMA), a myofibroblast marker, with no peak in α-SMA gene or protein expression in mineralization medium at any time point. Application of mineralization medium led to increased expression levels of alkaline phosphatase (ALP), an early mineralization marker, for all cell types, although the magnitude of the increase in ALP was drastically smaller for VICs than for the osteogenic cell types. Only the osteogenic cell types demonstrated an appreciable increase in osteocalcin, an indicator of later-stage mineralization.
While the addition of mineralization medium generally increased the expression of osteogenic markers and decreased the expression of myofibroblastic markers, VICs displayed different levels and patterns of expression than the osteoblastic cell types used for comparison. Additionally, the lack of an α-SMA increase at any point after the addition of mineralization medium to VICs indicated that VICs may not need to progress through a myofibroblastic stage before reaching an osteoblast-like gene expression profile.
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