Using Na3PO4 to Enhance In vitro Animal Models of Aortic Valve Calcification.
Using Na3PO4 to Enhance In vitro Animal Models of Aortic Valve Calcification.
复制标题
使用 Na3PO4 增强主动脉瓣钙化的体外动物模型。
DOI：
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
N. Alotti
中科院分区：
文献类型：
--
作者：
D. Lerman;S. Prasad;N. Alotti
背景/目标
钙化性主动脉瓣病(CAVD)的发病机制涉及瓣膜间质细胞(VICS)的活跃炎症过程，其特征是特定的成骨信号通路激活和细胞凋亡。可以通过分析自发性钙化的某些分子标记和基因表达途径来研究这一过程。本研究的目的是研究磷酸钠(Na3PO4)作为钙化促进剂的作用，目的是改进体外动物模型以测试潜在的钙化抑制剂。
材料和方法
用连续胶原酶消化法从6个月龄健康的新鲜猪心中提取VIC。定量聚合酶链式反应(QPCR)被用来定量VICS自发性钙化过程中感兴趣基因的反式分化。加入Na3PO4(3 mM，pH 7.4)可增加VICS的自发性钙化。钙沉积采用茜素红染色，胶原染色采用天狼星红染色。用比色法定量测定钙和胶原沉积。用动力学方法测定碱性磷酸酶(ALP)的活性。为了进行统计分析，我们使用了SPSS和Microsoft Office Excel 2013。
结果
猪VIC自发钙化，可见钙质和胶原沉积。在这项研究中，我们观察到从第0天到第14天，钙和胶原沉积增加(钙：376%；P<；0.001，胶原：3553%；P<；0.001)。第14天的定量聚合酶链式反应分析结果显示：作为成肌细胞表型标志的α-肌动蛋白增加了1.6倍；P<；0.001。成骨细胞标志物Runx2上升到1.30倍；P<；0.05，成骨促进因子β上升到3.2倍；P<；0.001；成肌细胞结节形成调节因子RhoA上升到0天的4.5倍；P<；0.001。RANKL mRNA和Calponin无明显变化。用Na3PO4(3 mM，pH7.4)处理猪VIC后，第14天钙沉积显著增加(522%；P<；0.001)，第7天碱性磷酸酶活性显著增加(228%；P<；0.05)。到第14天，两组之间的ALP活性没有显著变化。
结论
本研究证实，在自发性钙化过程中，主动脉内皮细胞某些特定分子表达上调，并伴有钙、胶原和碱性磷酸酶活性的活跃增加。在这个体外模型中，可以用Na3PO4(3 mM，pH 7.4)增加自发的VICS钙化到可以测试钙化抑制剂的水平，以确定一种新的潜在的治疗钙化性主动脉狭窄的策略。
BACKGROUND/OBJECTIVES
The pathogenesis of calcific aortic valvular disease (CAVD) involves an active inflammatory process of valvular interstitial cells (VICs) characterized by the activation of specific osteogenic signaling pathways and apoptosis. This process can be studied by analyzing certain molecular markers and gene expression pathways of spontaneous calcification. The purpose of our study is to investigate the role of sodium phosphate (Na3PO4) as a calcification promoter, with the aim of improving in vitro animal models for testing potential calcification inhibitors.
MATERIALS AND METHODS
VICs were extracted from 6 healthy 6-month-old fresh porcine hearts by serial collagenase digestion. Quantitative polymerase chain reaction (qPCR) was used to quantify trans-differentiation of genes of interest during spontaneous calcification of VICs. Spontaneous calcification of VICs was increased by adding Na3PO4 (3 mM, pH 7.4). The degree of calcification was estimated by Alizarin Red staining for calcium deposition, and Sirius Red staining for collagen. Colorimetric techniques were used to determine calcium and collagen deposition quantitatively. Additionally, the enzymatic activity of alkaline phosphatase (ALP) was measured by a kinetic assay. For statistical analysis we used SPSS and Microsoft Office Excel 2013.
RESULTS
Porcine VICs calcify spontaneously with demonstrable calcium and collagen deposition. In this study we observed an increase of calcium and collagen deposition from day 0 to day 14 (calcium: 376%; P<0.001, collagen: 3553%; P<0.001). qPCR analysis of mRNA by day 14 showed the following results: α-actin, a marker of myoblast phenotype, was increased to 1.6-fold; P<0.001. Runx2, an osteoblast marker, rose to 1.3 fold; P<0.05, TGF-β, a promoter of osteogenesis, increased to 3.2-fold; P<0.001, and RhoA, a regulator of nodular formation in myoblasts, increased to 4.5-fold; P<0.001, compared to their levels at day 0. RANKL mRNA and calponin did not change significantly. Treatment of porcine VICs with Na3PO4 (3 mM, pH 7.4) led to a marked increase in calcium deposition by day 14 (522%; P<0.001), and a significant increase in ALP activity by day 7 (228%; P<0.05). There were no significant changes in ALP activity between the groups by day 14.
CONCLUSION
This study has demonstrated the upregulation of some specific molecules during spontaneous calcification of aortic VICs with an active increase of calcium, collagen and ALP activity. In this in vitro model it was possible to increase spontaneous VICs calcification with Na3PO4 (3 mM, pH 7.4) to a level in which inhibitors of calcification could be tested to identify a novel potential therapeutic strategy against calcific aortic stenosis.
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