Monzack Elyssa L, Masters Kristyn S
Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA.
J Heart Valve Dis. 2011 Jul;20(4):449-63.
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. In the present study, VICs were compared against multiple osteoblastic cell types at different stages of differentiation. These findings may help to resolve whether VICs progress through a myofibroblastic phenotype before 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 harvested for qPCR, DNA, and protein analysis at time points ranging from one to eight days.
Culture of VICs in mineralization medium decreased the expression of alpha-smooth muscle actin (alpha-SMA; a myofibroblast marker), with no peak in alpha-SMA gene or protein expression in mineralization medium at any time point. The application of a 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 alpha-SMA increase at any point after the addition of mineralization medium to VICs indicated that these cells may not need to progress through a myofibroblastic stage before reaching an osteoblast-like gene expression profile.
主动脉瓣钙化被认为涉及瓣膜间质细胞（VICs）向肌成纤维细胞或成骨细胞样表型的分化。尽管病变的VICs和成骨细胞之间据称存在相似之处，但很少有研究在并排实验中直接比较VICs和成骨细胞。在本研究中，将VICs与处于不同分化阶段的多种成骨细胞类型进行了比较。这些发现可能有助于解决VICs是否在达到成骨细胞样阶段之前先经历肌成纤维细胞表型的问题。
使用三种代表一系列成骨细胞谱系定向和分化的细胞类型与VICs进行表型比较。具体而言，将VICs、胚胎成纤维细胞（C3H10T1/2）、前成骨细胞（MC3T3-E1）和成熟原代成骨细胞在组织培养聚苯乙烯上于对照或矿化培养基中培养，并在1至8天的时间点收获用于qPCR、DNA和蛋白质分析。
在矿化培养基中培养VICs会降低α-平滑肌肌动蛋白（α-SMA；一种肌成纤维细胞标志物）的表达，在任何时间点矿化培养基中α-SMA基因或蛋白质表达均无峰值。矿化培养基的应用导致所有细胞类型的碱性磷酸酶（ALP；一种早期矿化标志物）表达水平升高，尽管VICs中ALP升高的幅度远小于成骨细胞类型。只有成骨细胞类型显示骨钙素（后期矿化指标）有明显增加。
虽然添加矿化培养基通常会增加成骨标志物的表达并降低肌成纤维细胞标志物的表达，但VICs与用于比较的成骨细胞类型表现出不同的表达水平和模式。此外，在向VICs添加矿化培养基后的任何时间点α-SMA均未增加，这表明这些细胞在达到成骨细胞样基因表达谱之前可能不需要经历肌成纤维细胞阶段。
