Correlations of DDAH1 transcript variants with human endothelial asymmetric dimethylarginine metabolizing activity.
Correlations of DDAH1 transcript variants with human endothelial asymmetric dimethylarginine metabolizing activity.
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DOI：
10.1093/ajh/hpt119
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发表时间：
2013-12
影响因子：
3.2
通讯作者：
T. Sun;Ji-Peng Zhou;Da-Bin Kuang;Mupeng Li;Y. Xiong;Jie Tang;J. Xia;Yongping Bai;Guo-ping Yang;Yuan‐jian Li;Xiaoping Chen
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文献类型：
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作者：
T. Sun;Ji-Peng Zhou;Da-Bin Kuang;Mupeng Li;Y. Xiong;Jie Tang;J. Xia;Yongping Bai;Guo-ping Yang;Yuan‐jian Li;Xiaoping Chen
二甲基精氨酸二甲氨基水解酶1（DDAH 1）是负责不对称二甲基精氨酸（ADMA）失活的主要酶。本研究旨在阐明人类DDAH 1转录变体mRNA表达水平与ADMA代谢活性之间的相关性。方法采用实时荧光定量聚合酶链反应（real-time polymerase chain reaction，RT-PCR）技术，检测正常人、急性缺血性脑卒中（acute ischemic stroke，AIS）和急性心肌梗死（acute myocardial infarction，AMI）患者外周血单个核细胞（peripheral blood mononuclear cells，PBMC）和原代培养的人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）中DDAH 1基因的mRNA表达水平。酶联免疫吸附法测定细胞裂解液的ADMA代谢活性。结果发现一个新的DDAH 1转录变体DDAH 1-V3。DDAH 1-V3 mRNA表达与-V2（R = 0.811; P = 0.000008）和-V1（R = 0.454; P = 0.04）显著相关。在健康受试者的PBMC中，仅在DDAH 1-V2和-V3之间观察到显著相关性（R = 0.571; P = 0.001; n = 36）。AIS患者外周血单个核细胞DDAH 1-V2和-V3转录本的Δ阈值循环（DCT）值均显著增加（P < 0.05）。偏相关分析显示，AIS和AMI患者外周血单个核细胞中DDAH 1 mRNA表达水平也呈正相关（P < 0.05）。在HUVECs中，只有DDAH 1-V1的mRNA表达水平与细胞内ADMA代谢活性显著相关（R = 0.805; P=0.002）。结论DDAH 1转录本变异体的mRNA表达水平之间存在正相关，但只有DDAH 1-V1转录本参与ADMA代谢，因此推荐使用转录本特异性引物检测DDAH 1 mRNA表达。
BACKGROUND Dimethylarginine dimethylaminohydrolases 1 (DDAH1) is the major enzyme responsible for inactivation of asymmetric dimethylarginine (ADMA). This study seeks to clarify the correlations between mRNA expression levels of DDAH1 transcript variants and the relationship with ADMA metabolizing activity in human. METHODS The mRNA expression levels of DDAH1 transcript variants in primarily cultured human umbilical vein endothelial cells (HUVECs) and peripheral blood mononuclear cells (PBMCs) from healthy control subjects and patients suffering from both acute ischemic stroke (AIS) and acute myocardial infarction (AMI) were determined by real-time polymerase chain reaction. ADMA metabolizing activity of the cell lysates from HUVECs was determined by enzyme-linked immunosorbent assay. RESULTS A novel DDAH1 transcript variant DDAH1-V3 was identified. DDAH1-V3 mRNA expression correlated significantly with that of both -V2 (R = 0.811; P = 0.000008) and -V1 (R = 0.454; P = 0.04) in HUVECs. In PBMCs from healthy subjects, significant correlation was observed only between DDAH1-V2 and -V3 (R = 0.571; P = 0.001; n = 36). Delta threshold cycle (DCT) values for both DDAH1-V2 and -V3 transcripts were increased significantly in PBMCs from AIS patients (P < 0.05, respectively). In PBMCs from patients suffering from both AIS and AMI, positive pairwise correlations between mRNA levels of DDAH1 transcripts were also observed as analyzed by partial correlation analysis (P < 0.05, respectively). However, only mRNA expression level of the DDAH1-V1 transcript correlated significantly with intracellular ADMA metabolizing activity in HUVECs (R = 0.805; P=0.002). CONCLUSIONS This study demonstrated that although there are positive correlations between mRNA expression levels of DDAH1 transcript variants, only the DDAH1-V1 transcript is responsible for ADMA metabolism, and transcript specific primers are recommended to determine DDAH1 mRNA expression.