Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine
Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine
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DOI：
10.1159/000368524
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发表时间：
2015-01-01
影响因子：
2.8
通讯作者：
Lang, Florian
中科院分区：
文献类型：
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作者：
Alesutan, Ioana;Musculus, Katharina;Lang, Florian
背景/目标：过高的磷酸盐浓度会引发血管钙化，这是一个由血管平滑肌细胞（VSMC）的骨诱导促进的活性过程，其中转录因子RUNX 2（核心结合因子α 1，CBFA 1）、碱性磷酸酶（ALPL）、TGF β 1、转录因子NFAT 5和NFAT 5敏感性转录因子SOX 9的表达和活性增加。高磷血症klothohypomorphic小鼠的骨诱导信号传导和血管钙化可以通过NH 4Cl处理逆转，其作用涉及TGF α 1的减少和NFAT 5依赖性骨诱导信号传导的抑制。NH 4Cl的已知作用包括酸性细胞区室的碱化。本研究探讨了成骨/软骨形成信号传导是否会受到碱性化的酸性细胞隔室的液泡H+ ATP酶抑制与巴弗洛霉素A1或以下的耗散的pH梯度跨膜的酸性细胞隔室与甲胺。研究方法：原代人主动脉平滑肌细胞（HAoSMC）用高磷酸盐处理，以在存在或不存在巴夫洛霉素A1或甲胺的情况下触发骨/软骨形成信号传导和钙化。使用QuantiChrom钙测定法测定钙含量，通过比色测定法测定ALP活性，并通过定量RTPCR测定转录水平。结果如下：高磷可显著增加HAoSMCs钙沉积、CBFA 1和ALPL mRNA表达以及碱性磷酸酶活性，巴弗洛霉素A1和甲胺均可减轻上述作用。高磷酸盐进一步显著上调TGFB 1、NFAT 5和SOX 9的mRNA水平，通过用巴弗洛霉素A1或甲胺的额外处理显著减弱这种作用。用人TGF α处理HAoSMC的水平类似地高，不能通过高磷酸盐和TGF β 1联合处理进一步增加。巴弗洛霉素A1未能逆转由高磷酸盐与TGF β 1一起触发的骨/软骨形成信号传导。结论：液泡H+ ATP酶的抑制或酸性细胞区室膜上pH梯度的消散都破坏了VSMC中的骨/软骨形成信号和钙沉积，观察结果支持血管钙化需要酸性细胞区室的假设。1蛋白或高磷酸盐上调NFAT 5、SOX 9、CBFA 1和ALPL mRNA表达，
Background/Aims: Excessive phosphate concentrations trigger vascular calcification, an active process promoted by osteoinduction of vascular smooth muscle cells (VSMCs) with increased expression and activity of transcription factor RUNX2 (Core-binding factor alpha 1, CBFA1), alkaline phosphatase (ALPL), TGF beta 1, transcription factor NFAT5, and NFAT5-sensitive transcription factor SOX9. The osteoinductive signaling and vascular calcification of hyperphosphatemic klothohypomorphic mice could be reversed by treatment with NH4Cl, effects involving decrease of TGF alpha 1 and inhibition of NFAT5-dependent osteoinductive signaling. Known effects of NH4Cl include alkalinization of acidic cellular compartments. The present study explored whether osteo-/ chondrogenic signaling could be influenced by alkalinization of acidic cellular compartments following inhibition of the vacuolar H+ ATPase with bafilomycin A1 or following dissipation of the pH gradient across the membranes of acidic cellular compartments with methylamine. Methods: Primary human aortic smooth muscle cells (HAoSMCs) were treated with high phosphate to trigger osteo-/chondrogenic signaling and calcification in the absence or presence of bafilomycin A1 or methylamine. Calcium content was determined using a QuantiChrom Calcium assay, ALP activity by a colorimetric assay and transcript levels by quantitative RTPCR. Results: High phosphate increased significantly the calcium deposition, CBFA1 and ALPL mRNA expression as well as alkaline phosphatase activity in HAoSMCs, all effects ameliorated by both, bafilomycin A1 and methylamine. High phosphate further significantly up-regulated the mRNA levels of TGFB1, NFAT5 and SOX9, effects significantly blunted by additional treatment with bafilomycin A1 or methylamine. Treatment of HAoSMCs with human TGF alpha similarly high levels which could not be further increased by combined treatment with high phosphate and TGF beta 1. Bafilomycin A1 failed to reverse the osteo-/chondrogenic signaling triggered by high phosphate together with TGF beta 1. Conclusions: Inhibition of the vacuolar H+ ATPase or dissipation of the pH gradient across the membranes of acidic cellular compartments both disrupt osteo-/chondrogenic signaling and calcium deposition in VSMCs, observations supporting the hypothesis that vascular calcification requires acidic cellular compartments.1 protein or high phosphate up-regulated NFAT5, SOX9, CBFA1 and ALPL mRNA expression to