Porras Ana M, van Engeland Nicole C A, Marchbanks Evelyn, McCormack Ann, Bouten Carlijn V C, Yacoub Magdi H, Latif Najma, Masters Kristyn S
Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI.
Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands.
J Am Heart Assoc. 2017 Mar 14;6(3):e005041. doi: 10.1161/JAHA.116.005041.
Valvular interstitial cells (VICs) in the healthy aortic valve leaflet exhibit a quiescent phenotype, with <5% of VICs exhibiting an activated phenotype. Yet, in vitro culture of VICs on tissue culture polystyrene surfaces in standard growth medium results in rapid transformation to an activated phenotype in >90% of cells. The inability to preserve a healthy VIC phenotype during in vitro studies has hampered the elucidation of mechanisms involved in calcific aortic valve disease. This study describes the generation of quiescent populations of porcine VICs in 2-dimensional in vitro culture and their utility in studying valve pathobiology.
Within 4 days of isolation from fresh porcine hearts, VICs cultured in standard growth conditions were predominantly myofibroblastic (activated VICs). This myofibroblastic phenotype was partially reversed within 4 days, and fully reversed within 9 days, following application of a combination of a fibroblast media formulation with culture on collagen coatings. Specifically, culture in this combination significantly reduced several markers of VIC activation, including proliferation, apoptosis, α-smooth muscle actin expression, and matrix production, relative to standard growth conditions. Moreover, VICs raised in a fibroblast media formulation with culture on collagen coatings exhibited dramatically increased sensitivity to treatment with transforming growth factor β1, a known pathological stimulus, compared with VICs raised in either standard culture or medium with a fibroblast media formulation.
The approach using a fibroblast media formulation with culture on collagen coatings generates quiescent VICs that more accurately mimic a healthy VIC population and thus has the potential to transform the study of the mechanisms of VIC activation and dysfunction involved in the early stages of calcific aortic valve disease.
健康主动脉瓣叶中的瓣膜间质细胞（VICs）表现出静止表型，只有不到5%的VICs表现出激活表型。然而，在标准生长培养基中，将VICs在组织培养聚苯乙烯表面进行体外培养会导致超过90%的细胞迅速转变为激活表型。在体外研究过程中无法保持健康的VIC表型，这阻碍了对钙化性主动脉瓣疾病相关机制的阐明。本研究描述了在二维体外培养中产生猪VICs静止群体的方法及其在研究瓣膜病理生物学中的应用。
从新鲜猪心脏分离出的VICs，在标准生长条件下培养4天内，主要为肌成纤维细胞（激活的VICs）。在应用成纤维细胞培养基配方并在胶原蛋白涂层上培养后，这种肌成纤维细胞表型在4天内部分逆转，在9天内完全逆转。具体而言，与标准生长条件相比，这种组合培养显著降低了VIC激活的几个标志物，包括增殖、凋亡、α-平滑肌肌动蛋白表达和基质产生。此外，与在标准培养或含成纤维细胞培养基配方的培养基中培养的VICs相比，在成纤维细胞培养基配方并在胶原蛋白涂层上培养的VICs对已知病理刺激物转化生长因子β1的处理表现出显著提高的敏感性。
使用成纤维细胞培养基配方并在胶原蛋白涂层上培养的方法可产生更准确模拟健康VIC群体的静止VICs，因此有可能改变对钙化性主动脉瓣疾病早期阶段VIC激活和功能障碍机制的研究。
