[1]顾秀莲 敬梅 樊济海 巢胜吾 李博 王玲 凌秋洋.Notch2高表达可使人主动脉平滑肌细胞骨形态发生蛋白4表达增加促进钙化[J].心血管病学进展,2020,(9):962-966.[doi:10.16806/j.cnki.issn.1004-3934.2020.09.019]
　GU Xiulian,JING Mei,FAN Jihai,et al.Over-expression of Notch2 Increases BMP-4 Expression and Promotes Calcification in Human Aortic Smooth Muscle Cells[J].Advances in Cardiovascular Diseases,2020,(9):962-966.[doi:10.16806/j.cnki.issn.1004-3934.2020.09.019]
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Notch2高表达可使人主动脉平滑肌细胞骨形态发生蛋白4表达增加促进钙化()
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《心血管病学进展》[ISSN:51-1187/R/CN:1004-3934]

卷:
期数:
2020年9期
页码:
962-966
栏目:
出版日期:
2020-09-25

文章信息/Info

Title:
Over-expression of Notch2 Increases BMP-4 Expression and Promotes Calcification in Human Aortic Smooth Muscle Cells
作者:
顾秀莲1 敬梅2 樊济海1 巢胜吾1 李博1 王玲1 凌秋洋3
（1.中国人民解放军海军特色医学中心心内科，上海 200052；2.中国人民解放军海军特色医学中心干部科，上海 200052；3.中国人民解放军联勤保障部队第九〇四医院心内科，江苏 无锡 214044）
Author(s):
GU Xiulian 1; JING Mei 2; FAN Jihai 1; CHAO Shengwu 1; LI Bo 1; WANG Ling1; LING Qiuyang 3
(1. Department of Cardiology, Naval Characteristic Medical Center of PLA, Shanghai 200052,China； 2. Department of Cadre, Naval Characteristic Medical Center of PLA, Shanghai 200052,China 3.Department of Cardiology, The 904th Hospital of PLA, Wuxi 214044,Jiangsu,China)
关键词:
人主动脉平滑肌细胞; Notch2; JAK-STAT3; 骨形态发生蛋白4
Keywords:
Human aortic smooth muscle cells ; Notch2; JAK-STAT3;  Bone morphogenetic protein-4
DOI:
10.16806/j.cnki.issn.1004-3934.2020.09.019
摘要:
目的 观察并验证人主动脉平滑肌细胞（HASMC）Notch2蛋白表达的变化对其细胞钙化的影响，并探讨其机制。方法 体外培养HASMC，将其分为两组，分别为正常对照组与诱导钙化组。前者常规培养，后者在常规培养的基础上加用钙化诱导剂β-甘油磷酸（5 mmol/L）、抗坏血酸（50 μg/mL）和地塞米松（100 nmol/L），两组细胞均培养7 d。另取上述方法诱导钙化的HASMC为钙化对照组，再设钙化抑制组，即在钙化对照组的基础上加入Notch特异性抑制剂γ-内分泌酶抑制剂（50 μmol/L），两组细胞均培养7 d。四组均用白介素-6诱导细胞的炎症反应。采用免疫印迹法检测各组细胞内Notch2、磷酸化核转录因子STAT3（p-STAT3）和骨形态发生蛋白4（BMP-4）的蛋白表达水平；酶联免疫法测定各组细胞培养上清液中BMP-4蛋白的含量；逆转录-聚合酶链反应检测诱导钙化组和正常对照组中HASMC Notch2 mRNA的表达；免疫荧光染色法观察钙化对照组与钙化抑制组中HASMC BMP-4蛋白的表达。结果 （1）诱导钙化组中HASMC Notch2、p-STAT3和BMP- 4蛋白表达水平明显高于正常对照组（P＜0.05）；诱导钙化组的培养液上清中BMP-4蛋白含量为（66.42±2.89）pg/mL明显高于正常对照组的（22.35±2.68）pg/mL（P＜0.05）；诱导钙化组中HASMC Notch2 mRNA的表达明显高于正常对照组（P＜0.05）。（2）钙化抑制组中HASMC Notch2、p-STAT3和BMP- 4蛋白的表达水平明显低于钙化对照组（P＜0.05）；钙化抑制组培养液上清中BMP-4蛋白的含量为（22.34±3.82）pg/mL明显低于钙化对照组的（62.89±3.26）pg/mL（P＜0.05）。免疫荧光染色显示钙化抑制组中HASMC BMP-4蛋白表达明显低于钙化对照组。结论 钙化的HASMC Notch2蛋白表达增高，促进了BMP-4蛋白表达增加，从而使HASMC发生成骨样改变，使血管发生钙化，这一机制可能与JAK-STAT3的活化有关。
Abstract:
Objective To observe the effect of Notch2 protein expression on calcification of human aortic smooth muscle cells(HASMC) and explore its mechanism. Methods HASMC was cultured in vitro and divided into two groups: normal control group and induced calcification group. The former were cultured in conventional media. The latter?was supplemented with?calcification?inducers β-glycerophosphate (5 mmol/L),?ascorbic acid (50 μg/mL) and dexamethasone (100 nmol/L) on the basis of conventional culture. The cells of two group were cultured for 7 days. HASMC, which was induced calcification by the above-mentioned method, was taken as the calcification control group, and the calcification inhibition group was set up, in which Notch specific inhibitor-endocrine enzyme inhibitor (50 μmol/L) was added to the calcification control group. Interleukin-6 was used to induce the inflammatory reaction of the cells in all four groups. The expression levels of Notch2, phosphorylated nuclear transcription factor STAT3(p-STAT3) and bone morphogenetic protein-4(BMP-4) were detected by Western blotting and BMP-4 protein was detected by ELISA in all groups. The expression of Notch2 mRNA was detected by RT-PCR in induced calcification group and control group, and the expression of HASMC BMP-4 protein was detected by immunofluorescence staining in calcification inhibition group and calcified control group. Results (1)The expression of Notch2, p-STAT3, BMP-4 in induced calcification group is significantly?higher than that of the control group(P＜0.05). The expression of BMP-4 in the induced calcification group is (66.42±2.89) pg/mL, which is significantly?higher than that in control group[(22.35±2.68) pg/mL](P＜0.05). Notch2 mRNA expression in induced calcification group is significantly higher than that in normal control group (P＜0.05). (2)The expression of Notch2, p-STAT3 and BMP4 protein in the calcification inhibition group was significantly lower than that in the calcification control group(P＜0.05).The content of BMP-4 in supernatant of calcification inhibition group was (22.34±3.82) pg/mL, which was significantly lower than that of calcification control group(62.89±3.26) pg/mL(P＜0.05). Immunofluorescence staining showed that BMP-4 protein expression was significantly lower in calcification inhibition group than that in calcified control group. Conclusion The increased expression of calcified HASMC Notch2 promotes the expression of BMP-4, which leads to osteogenic changes of HASMC and calcification of blood vessels. This mechanism may be related to the activation of JAK-STAT3.

参考文献/References:

[1] Palit S,Kendrick J. Vascular calcification in chronic kidney disease:role of disordered mineral metabolism[J]. Curr Pharm Des,2014,20(37):5829-5833. [2] Valdivielso JM.Vascular calcification:types and mechanisms [J]. Nefrologia,2011,31(2):142-147. [3] Bäck M,Hansson GK. Anti-inflammatory therapies for atherosclerosis[J]. Nat Rev Cardiol,2015,12(4):199-211. [4] Park HJ,Kim MK,Kim Y,et al. Gastrin-releasing peptide promotes the migration of vascular smooth muscle cells through upregulation of matrix metalloproteinase-2 and -9[J]. BMB Rep,2017,50(12):628-633. [5] Li P,Li YL,Li ZY,et al. Cross talk between vascular smooth muscle cells and monocytes through interleukin-1β/interleukin-18 signaling promotes vein graft thickening[J]. Arterioscler Thromb Vasc Biol,2014,34(9):2001-2011. [6] Chen TC,Lin CT,Chien SJ,et al. Regulation of calcification in human aortic smooth muscle cells infected with high-glucose-treated porphyromonas gingivalis[J]. J Cell Physiol,2018,233(6):4759-4769. [7] Kim K,Kim S,Moh SH,et al. Kaempferol inhibits vascular smooth muscle cell migration by modulating BMP-mediated miR-21 expression[J]. Mol Cell Biochem,2015,407(1-2):143-149. [8] Cheng SL,Behrmann A,Shao JS,et al. Targeted reduction of vascular Msx1 and Msx2 mitigates arteriosclerotic calcification and aortic stiffness in LDLR- deficient mice fed diabetogenic diets[J]. Diabetes,2014,63(12):4326-4337. [9] Garg V,Muth AN,Ransom JF,et al. Mutations in NOTCH1 cause aortic valve disease[J]. Nature,2005,437(7056):270-274. [10]凌秋洋,刘洁,徐佰达,等. Notch1高表达可使钙化的人瓣膜间质细胞BMP-2/4表达增加[J].中华心血管病杂志,2016,44(3):255-259. [11]叶挺,程治源,凌秋洋,等. Notch1蛋白在人心脏瓣膜间质细胞凋亡与钙化关系中的作用[J].中国动脉硬化杂志,2017,25(2):122-128. [12]Cheng ZY,Ye T,Ling QY,et al. Parathyroid hormone promotes osteoblastic differentiation of endothelial cells via the extracellular signal-regulated protein kinase 1/2 and nuclear factor-κB signaling pathways[J]. Exp Ther Med, 2018,15(2):1754-1760. [13]Zhang L,Shao J,Zhou Y,et al. Inhibition of PDGF-BB-induced proliferation and migration in VSMCs by proanthocyanidin A2:involvement of KDR and Jak-2/ STAT-3/cPLA2 signaling pathways[J]. Biomed Pharmacother,2018,98:847-855. [14]Ben P,Hu M,Wu H,et al. L-theanine down-regulates the JAK/STAT3 pathway to attenuate the proliferation and migration of vascular smooth muscle cells induced by angiotensin Ⅱ[J]. Biol Pharm Bull,2018,41(11):1678-1684. [15]Tang X,Liu Y,Xiao Q,et al. Pathological cyclic strain promotes proliferation of vascular smooth muscle cells via the ACTH/ERK/STAT3 pathway[J]. J Cell Biochem,2018,119(10):8260-8270. [16]Rogers MA,Aikawa E. Cardiovascular calcification:artificial intelligence and big data accelerate mechanistic discovery[J]. Nat Rev Cardiol,2019,16(5):261-274. [17]石文坚,花蕾,孟详光,等. 环状RNA在动脉粥样硬化性心脏病中的研究进展[J].心血管病学进展,2019,40(9):1286-1289. [18]李雪鹤,李晓梅. 长链非编码RNA在急性心肌梗死发病中的研究进展[J].心血管病学进展,2019,40(9):1271-1274. [19]Durham AL,Speer MY,Scatena M,et al. Role of smooth muscle cells in vascular calcification:implications in atherosclerosis and arterial stiffness[J]. Cardiovasc Res,2018,114(4):590-600. [20]Borland SJ,Morris TG,Borland SC,et al. Regulation of vascular smooth muscle cell calcification by syndecan-4/FGF-2/PKCα signalling and cross-talk with TGFβ[J]. Cardiovasc Res,2017,113(13):1639-1652. [21]Jeong G,Kwon DH,Shin S,et al. Long noncoding RNAs in vascular smooth muscle cells regulate vascular calcification[J]. Sci Rep,2019,9(1):5848. [22]Lee GL,Yeh CC,Wu JY,et al. TLR2 promotes vascular smooth muscle cell chondrogenic differentiation and consequent calcification via the concerted actions of osteoprotegerin suppression and IL-6-mediated RANKL induction[J]. Arterioscler Thromb Vasc Biol,2019,39(3):432-445. [23]Kurozumi A,Nakano K,Yamagata K,et al. IL-6 and sIL-6R induces STAT3-dependent differentiation of human VSMCs into osteoblast-like cells through JMJD2B-mediated histone demethylation of RUNX2[J]. Bone,2019,124:53-61. [24]Moshapa FT,Riches-Suman K,Palmer TM. Therapeutic targeting of the proinflammatory IL-6-JAK/STAT signalling pathways responsible for vascular restenosis in type 2 diabetes mellitus[J]. Cardiol Res Pract,2019,2019:9846312.

备注/Memo

备注/Memo:
基金项目：江苏省青年医学重点人才培养(QNRC2016883) 通信作者：凌秋洋，E-mail：lqy_doctor@163.com 收稿日期：2020-02-03
更新日期/Last Update: 2020-12-03