Osteopontin Does not Inhibited Aortic Valve Fibrosis and Calcification in Three-Dimensional Ovine Aortic Valve Model
Osteopontin Does not Inhibited Aortic Valve Fibrosis and Calcification in Three-Dimensional Ovine Aortic Valve Model
复制标题
骨桥蛋白在三维羊主动脉瓣模型中不抑制主动脉瓣纤维化和钙化
DOI：
10.1055/s-0037-1598849
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Akhyari P
中科院分区：
文献类型：
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作者：
Raschke S;Lukic M;Nehrenheim L;Kistner J;Schlag F;Jenke A;Lichtenberg A;Akhyari P
目的：钙化性主动脉瓣疾病的特征是主动脉瓣纤维化并钙化，导致左心室流出道梗阻。骨桥蛋白 (OPN) 是一种多功能磷蛋白，据描述，根据 OPN 的磷酸化状态，参与抑制营养不良和异位部位（包括主动脉瓣组织）的生物矿化。在这里，我们研究了所描述的 OPN 对平滑肌细胞的影响是否可以转移到绵羊主动脉瓣细胞培养模型中。方法：在存在或不存在 100 ng/mL 牛乳 OPN 的情况下，在促纤维化和促钙化条件（5 ng/mL TGF-β、10 mM β-磷酸甘油/1.5 mM 氯化钙）下静态培养绵羊主动脉瓣外植体 (AVE) 14 天。使用磷酸化特异性抗体通过免疫印迹分析 OPN 的磷酸化状态。通过qPCR测量AVE中的基因表达，通过苏木精-伊红染色分析形态变化。通过 von Kossa 染色和碱性磷酸酶测定试剂盒评估钙化结果：促纤维化和促钙化培养诱导 AVE 的形态变化。除了 AVE 收缩和增厚之外，甚至细胞分布也受到干扰，细胞增殖略有增强，并且 AVE 内出现无细胞区域。同时，这些条件显着增强了 I 型纤维化标志物胶原蛋白（10.4 倍，n= 9，p< 0.05）和 α-平滑肌肌动蛋白（12.4 倍，n= 9，p< 0.05）和过程控制 Runt 相关转录因子（RUNX）2（2.53 倍，n= 9）的基因表达。与这些结果一致，AVE上清液中的胶原蛋白水平（4.8倍，n=3）和碱性磷酸酶（1.3倍，n=8，p<0.001）活性显着上调。仅在促纤维化/促钙化培养后才观察到钙沉积。虽然蛋白质印迹分析证明所用的 OPN 在酪氨酸、丝氨酸和苏氨酸磷酸化位点上被磷酸化，但 OPN 既不抑制对纤维化标志物的形态变化、基因和蛋白质表达的显着影响，也不抑制钙沉积和 ALP 活性。结论：我们的数据表明 OPN 不介导绵羊 AVE 的抗纤维化和抗钙化作用。因此，未来的研究应该评估磷酸化 OPN 在经典二维细胞培养模型中对平滑肌细胞显示的有希望的作用是否可以转移到 VIC 的三维细胞培养模型中。
Objectives: Calcific Aortic Valve Disease is characterized by aortic valve fibrosis followed calcification resulting in left ventricular outflow obstruction. Osteopontin (OPN), a multifunctional phosphoprotein, is described to be involved in the inhibition of biomineralization of dystrophic and ectopic sites, including aortic valve tissue, depending on the phosphorylation status of OPN. Here, we investigated whether the described effect of OPN on smooth muscle cells can be transferred to ovine aortic valve cell culture models.Methods: Ovine aortic valve explants (AVE) were cultivated statically under pro-fibrotic and pro-calcific conditions (5 ng/mL TGF-β, 10 mM β-glycerolphosphate/1.5 mM calcium chloride) for 14 days in presence or absence of 100 ng/mL bovine milk OPN. Phosphorylation status of OPN was analyzed by immunoblot using phospho-specific antibodies. Gene expression in AVE was measured by qPCR, morphological changes were analyzed by hematoxylin-eosin staining. Calcification was assessed by von Kossa staining and alkaline phosphatase assay kitResult: Pro-fibrotic and pro-calcific cultivation induced morphological changes in AVE. Beside shrinkage and thickening of AVE, even cell distribution was disturbed with enhanced cell proliferation marginally and cell-free regions within the AVE. Simultaneously, these conditions significantly enhanced gene expression of fibrosis markers collagen type I (10.4-fold, n= 9, p< 0.05) and α-smooth muscle actin (12.4-fold, n= 9, p< 0.05) and the process-controlling Runt-related transcription factor (RUNX) 2 (2.53-fold, n= 9). In line with these results, collagen protein level (4.8-fold, n= 3) and alkaline phosphatase (1.3-fold, n= 8, p< 0.001) activity in the supernatants of AVE was significantly up-regulated. Calcium deposits have been observed only after pro-fibrotic/pro-calcific cultivation. Although, western blot analysis has proven that the used OPN is phosphorylated on tyrosine, serine and threonine phosphorylation sites, OPN did neither inhibit the pronounced effects on morphological changes, gene and protein expression of fibrosis markers nor calcium deposition and ALP activity.Conclusion: Our data indicate that OPN does not mediate anti-fibrotic and anti-calcific effects in ovine AVE. Thus, future studies should assess whether the promising effects of phosphorylated OPN shown on smooth muscle cells in classical two-dimensional cell culture models can be transferred to three-dimensional cell culture models of VIC.