Elevated ATP Levels Promote the Mineralization of Valvular Interstitial Cells
Elevated ATP Levels Promote the Mineralization of Valvular Interstitial Cells
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ATP 水平升高促进瓣膜间质细胞矿化
DOI：
10.1055/s-0037-1598848
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Akhyari P
中科院分区：
文献类型：
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作者：
Weber A;Dakaras K;Barth M;Baier K;Schrader J;Lichtenberg A;Akhyari P
目的：钙化性主动脉瓣疾病（CAVD）是一种伴有脂蛋白沉积、慢性炎症和进行性瓣叶矿化的活动性疾病过程。近年来的研究表明，调节三磷酸腺苷（ATP）及其代谢产物代谢的膜结合酶--外核苷酸酶和ATP及其代谢产物的细胞表面受体大家族的表达可能共同调节瓣膜间质细胞（VIC）的矿化过程。本研究的目的是探讨嘌呤能系统的酶在VIC钙化过程中的作用。在促变性条件下体外培养绵羊VIC（10 mM β-甘油磷酸盐，3 mM CaCl 2，补充有0.5%或10%FBS）并用外核苷三磷酸二磷酸水解酶-1（CD 39）或2′-（3′）-O-（4-苯甲酰基苯甲酰基）腺苷5′-三磷酸（BzATP），ATP受体（P2 X）的激动剂。实验在2D和3D细胞培养模型中进行。通过商业测定、茜素红和von Kossa染色评价钙沉积。在不同时间点对上清液进行碱性磷酸酶（ALP）、乳酸脱氢酶（LDH）、磷酸盐和基质金属蛋白酶（MMPs）的分析。半定量RT-PCR检测骨桥蛋白（OPN）、转化生长因子-β（TGF-β）、胶原1A 1（COL 1A 1）、外核苷酸焦磷酸酶/磷酸二酯酶1（ENPP 1）和肌动蛋白α 1（ACTA 1）的基因表达。2D培养液中LDH含量显著增加（p< 0.01），3D培养液中LDH含量无显著变化。在10%FBS条件下，ALP水平在2D（p< 0.05）和3D（p< 0.001）中均降低。3D组MMP-2表达明显高于正常对照组（P< 0.01）。在2D培养中，CD 39抑制后OPN（p< 0.01）、TGF-β和ENPP 1的表达增加，而COL 1A 1（p< 0.01）和CD 39的表达减少。结论：嘌呤能信号通路在主动脉瓣矿化过程中起重要作用。较高的ATP水平通过触发OPN和TGF-β等基因的表达来加速矿化。因此，有必要进一步研究嘌呤代谢在VIC矿化中的作用。
Objectives: Calcific aortic valve disease (CAVD) is an active disease process with lipoprotein deposition, chronic inflammation and progressive leaflet mineralization. Recent studies revealed that the expression of ecto-nucleotidases, a group of membrane-bound enzymes that regulate the metabolism of adenosine triphosphate (ATP) and its breakdown products and a large family of cell surface receptors for ATP and its metabolites may co-regulate the mineralization process of valvular interstitial cells (VICs). The aim of this study was to investigate the role of the enzymes of the purinergic system in the calcification process of VICs.Methods: Ovine VICs were cultivated in vitro under pro-degenerative conditions (10 mM β-glycerolphosphate, 3 mM CaCl2, supplemented with 0.5% or 10% FBS) and treated with an inhibitor of ectonucleoside triphosphate diphosphohydrolase-1 (CD39) or 2′-(3′)-O-(4-Benzoylbenzoyl) adenosine 5′-triphosphate (BzATP), an agonist of the ATP receptor (P2X). Experiments were performed both in 2D-and 3D-cell cultures models. Calcium deposition was evaluated by a commercial assay, alizarin red and von Kossa staining. Supernatant analysis of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), phosphate and matrix-metalloproteinase (MMPs) was implemented at different time points. Gene expression analyzes of osteopontin (OPN), TGF-β (TGF-β), collagen 1A1 (COL1A1), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and actin α 1 (ACTA1) were performed by semiquantitative RT-PCR.Results: CD39 inhibition and P2X activation significantly increased mineralization of VICs, both in 2D-and in 3D-cultures. LDH content in supernatants was increased in 2D (p< 0.01), but not in 3D. ALP levels were decreased at 10% FBS conditions, both in 2D (p< 0.05) and 3D (p< 0.001). Higher MMP-2 levels could be detected in 3D (p< 0.01). In 2D-cultures the expression of OPN (p< 0.01), TGF-β and ENPP1 was increased after CD39 inhibition, while COL1A1 (p< 0.01) and CD39 were decreased. In 3D-cultures, both treatments resulted in higher mRNA expression of OPN, Col1A1, TGF-β and ACTA1.Conclusion: Our findings suggest that the purinergic signaling pathways may play an important role in the mineralization process of aortic heart valves. The higher ATP levels accelerate the mineralization by triggering the expression of genes as OPN and TGF-β. Further studies focusing on the role of the purinergic metabolism on mineralization of VICs are necessary.