Zheng Zhen Zhong, Tian Fu Xiao, Liang Jin, Bing Guo Zhi
Department of Cardiology, The First Afficiated Hospital of Nanchang University, Nanchang, Jiangxi, 330006, China.
Department of Electrocardiography, Hubei Provincial Maternal and Child Health Institute, Hubei, Wuhan, 430000, China.
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Dec;159(4):540-6. doi: 10.5507/bp.2015.009. Epub 2015 Mar 1.
Cardiotrophin-1 (CT-1), a member of the IL-6 superfamily, is elevated in the serum of patients with ischemic and valvular heart disease. In this study, we hypothesized that CT-1 induces endothelial cell angiogenesis and that the ADMA/DDAH pathway plays an important role in the process.
pEGFP-N1-CTF1-GFP and pEGFP-N1 were constructed and used to transiently transfect to HUVECs, mediated by LipofectamineTM 2000. After transfection, the expression of CT-1 was examined by qRT-PCR and western blotting. Endothelial cell proliferation assay was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) method. Migration assay was performed using transwell, tube formation test was examined on Matrigel, eNOSmRNA expression was assayed by qRT-PCR, DDAH I, DDAHII and VEGF expression were detected by western blotting, the level of ADMA and the activity of DDAH were measured by High Performance Liquid Chromatography, NOS activity and the concentration of NO were assayed by L-[3H] citrulline production from L-[3H]arginine.
Overexpression of CT-1, increased endothelial cell proliferation, migration and formation of blood vessels, upregulated the expression of eNOSmRNA, DDAHI, DDAHII and VEGF, elevated the activity of DDAH and NOS, decreased the level of ADMA and promoted NO synthesis. In contrast, ADMA partially inhibited the effects of CT-1 induction.
Overexpression of CT-1 increases cell proliferation, migration and formation of blood vessels. This result also suggests that CT-1 may regulate angiogenesis through the ADMA/DDAH pathway.
心肌营养素-1（CT-1）是白细胞介素-6超家族的成员之一，在缺血性和瓣膜性心脏病患者的血清中水平升高。在本研究中，我们假设CT-1可诱导内皮细胞血管生成，且不对称二甲基精氨酸/二甲基精氨酸二甲胺水解酶（ADMA/DDAH）途径在此过程中起重要作用。
构建pEGFP-N1-CTF1-GFP和pEGFP-N1，并通过LipofectamineTM 2000介导瞬时转染人脐静脉内皮细胞（HUVECs）。转染后，采用实时定量聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测CT-1的表达。使用3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐（MTT）法评估内皮细胞增殖试验。使用Transwell进行迁移试验，在基质胶上进行管形成试验，通过qRT-PCR检测内皮型一氧化氮合酶（eNOS）mRNA的表达，通过蛋白质免疫印迹法检测DDAH I、DDAHII和血管内皮生长因子（VEGF）的表达，采用高效液相色谱法测量ADMA水平和DDAH活性，通过L-[3H]精氨酸生成L-[3H]瓜氨酸来检测一氧化氮合酶（NOS）活性和一氧化氮（NO）浓度。
CT-1过表达可增加内皮细胞增殖、迁移和血管形成，上调eNOSmRNA、DDAHI、DDAHII和VEGF的表达，提高DDAH和NOS的活性，降低ADMA水平并促进NO合成。相反，ADMA部分抑制CT-1诱导的作用。
CT-1过表达可增加细胞增殖、迁移和血管形成。该结果还表明CT-1可能通过ADMA/DDAH途径调节血管生成。
