Characterization and assessment of potential microRNAs involved in phosphate-induced aortic calcification
Characterization and assessment of potential microRNAs involved in phosphate-induced aortic calcification
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DOI：
10.1002/jcp.26121
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发表时间：
2018-05-01
影响因子：
5.6
通讯作者：
Badran, Bassam
中科院分区：
文献类型：
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作者：
Fakhry, Maya;Skafi, Najwa;Badran, Bassam
内侧动脉钙化是2型糖尿病和慢性肾脏疾病(CKD)的标志，被认为是心血管死亡和发病率的独立危险因素。与CKD相关的高磷血症是血管钙化的强烈刺激因素，但调控这一过程的分子机制仍不完全清楚。我们用茜素红和Von Kossa染色显示，将SD-Dawley大鼠的主动脉外植体暴露于高水平的无机磷(P-I，6 mM)后，可诱导钙化。这种钙化与高组织非特异性碱性磷酸酶(TNAP)活性、血管平滑肌细胞去分化有关，通过免疫印迹分析、免疫荧光检测平滑肌22α(SM22)蛋白的表达下调，以及通过实时定量PCR检测Run相关转录因子2(Runx2)、TNAP、骨钙素和骨桥蛋白的mRNA水平上调而向骨软骨细胞样细胞转化。为了揭示这一过程的可能机制(S)，微RNA(MiR)的表达谱显示10个miR，5个miR在P-I治疗后第3天和6天与对照未治疗的主动脉差异表达。在第3天，miR-200C、-155、322表达上调，miR-708、331表达下调。治疗6天后，miR-328、-546、-301a表达上调，miR-409、miR-542表达下调。我们的结果表明，高P-I水平会触发主动脉钙化和某些MIR的调节。这些观察表明，调节主动脉钙化的机制可能涉及MIR，这值得在未来的研究中进行进一步的研究。
Medial artery calcification, a hallmark of type 2 diabetes mellitus and chronic kidney disease (CKD), is known as an independent risk factor for cardiovascular mortality and morbidity. Hyperphosphatemia associated with CKD is a strong stimulator of vascular calcification but the molecular mechanisms regulating this process remain not fully understood. We showed that calcification was induced after exposing Sprague-Dawley rat aortic explants to high inorganic phosphate level (P-i, 6mM) as examined by Alizarin red and Von Kossa staining. This calcification was associated with high Tissue-Nonspecific Alkaline Phosphatase (TNAP) activity, vascular smooth muscle cells de-differentiation, manifested by downregulation of smooth muscle 22 alpha (SM22) protein expression which was assessed by immunoblot analysis, immunofluorescence, and trans-differentiation into osteo-chondrocyte-like cells revealed by upregulation of Runt related transcription factor 2 (Runx2), TNAP, osteocalcin, and osteopontin mRNA levels which were determined by quantitative real-time PCR. To unravel the possible mechanism(s) involved in this process, microRNA (miR) expression profile, which was assessed using TLDA technique and thereafter confirmed by individual qRT-PCR, revealed differential expression 10 miRs, five at day 3 and 5 at day 6 post P-i treatment versus control untreated aortas. At day 3, miR-200c, -155, 322 were upregulated and miR-708 and 331 were downregulated. After 6 days of treatment, miR-328, -546, -301a were upregulated while miR-409 and miR-542 were downregulated. Our results indicate that high P-i levels trigger aortic calcification and modulation of certain miRs. These observations suggest that mechanisms regulating aortic calcification might involve miRs, which warrant further investigations in future studies.