Triptolide inhibits angiogenesis in microvascular endothelial cells through regulation of miR-92a

  • Xiaomeng Xu
  • Li Tian
  • Zhimian ZhangEmail author
Original Article


Atherosclerosis is one common chronic inflammatory disease in which angiogenesis is involved. Here we established an in vitro cell model of angiogenesis made by human dermal microvascular endothelial cells (HMEC-1) and work to investigate the role of triptolide (TPL) in this model. To induce angiogenesis, HMEC-1 cells were cultured in Matrigel-conditioned medium. The ratio of tubes to nucleus was detected. To evaluate angiogenesis, Western blot assay was carried out to detect endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor receptor-2 (VEGFR2) and VEGF. Cell counting kit-8 was utilized to estimate the viability of HMEC-1 cells. microRNA (miR)-92a was analyzed by qRT-PCR. The targeting relationship between integrin subunit alpha 5 (ITGA5) and miR-92a was verified through luciferase activity assay. The effects of ITGA5 on signaling transducers (ERK, PI3K, and AKT) in a phosphorylated form were valued using Western blot method. After stimulated by TPL, LY294002 and PD98059, the alteration in phosphorylation of the signaling transducers was evaluated by Western blot assay. The ratio of tubes to nucleus and angiogenesis related factors were increased with the delaying of culture time. TPL decreased the expression of angiogenesis factors. Furthermore, miR-92a was upregulated by TPL and miR-92a silence upregulated angiogenesis factors. In addition, TPL decreased ITGA5 which was proved as a target of miR-92a. ITGA5 overexpression resulted in the abundance of angiogenesis factors while ITGA5 silence led to the opposite results. Meanwhile, ITGA5 overexpression increased phosphorylation of ERK, PI3K and AKT while ITGA5 silence reversed the trend. TPL (as an anti-angiogenesis agent) suppressed angiogenesis by upregulating miR-92a, and miR-92a-mediated down-regulation of ITGA5 blocked the signaling transduction of ERK and PI3K/AKT pathways.


Atherosclerosis Angiogenesis Triptolide miR-92a ERK PI3K/AKT 


Funding information

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.


  1. 1.
    Andreou I, Sun X, Stone PH, Edelman ER, Feinberg MW (2015) miRNAs in atherosclerotic plaque initiation, progression, and rupture. Trends Mol Med 21:307–318CrossRefGoogle Scholar
  2. 2.
    Bonauer A, Carmona G, Iwasaki M, Mione M, Koyanagi M, Fischer A, Burchfield J, Fox H, Doebele C, Ohtani K, Chavakis E, Potente M, Tjwa M, Urbich C, Zeiher AM, Dimmeler S (2009) MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice. Science 324:1710–1713CrossRefGoogle Scholar
  3. 3.
    Cai J, Jiang WG, Ahmed A, Boulton M (2006) Vascular endothelial growth factor-induced endothelial cell proliferation is regulated by interaction between VEGFR-2, SH-PTP1 and eNOS. Microvasc Res 71:20–31CrossRefGoogle Scholar
  4. 4.
    Chen J, Qiao Y, Tang B, Chen G, Liu X, Yang B, Wei J, Zhang X, Cheng X, Du P, Jiang W, Hu Q, Hua ZC (2017) Modulation of Salmonella tumor-colonization and Intratumoral anti-angiogenesis by Triptolide and its mechanism. Theranostics 7:2250–2260CrossRefGoogle Scholar
  5. 5.
    Dai J, Peng L, Fan K, Wang H, Wei R, Ji G, Cai J, Lu B, Li B, Zhang D, Kang Y, Tan M, Qian W, Guo Y (2009) Osteopontin induces angiogenesis through activation of PI3K/AKT and ERK1/2 in endothelial cells. Oncogene 28:3412–3422CrossRefGoogle Scholar
  6. 6.
    Efimenko A, Sagaradze G, Akopyan Z, Lopatina T, Kalinina N (2016) Data supporting that miR-92a suppresses angiogenic activity of adipose-derived mesenchymal stromal cells by down-regulating hepatocyte growth factor. Data Brief 6:295–310CrossRefGoogle Scholar
  7. 7.
    Gallant-Behm CL, Piper J, Dickinson BA, Dalby CM, Pestano LA, Jackson AL (2018) A synthetic microRNA-92a inhibitor (MRG-110) accelerates angiogenesis and wound healing in diabetic and nondiabetic wounds. Wound Repair Regen 26:311–323CrossRefGoogle Scholar
  8. 8.
    Gimbrone MA Jr, Garcia-Cardena G (2016) Endothelial cell dysfunction and the pathobiology of atherosclerosis. Circ Res 118:620–636CrossRefGoogle Scholar
  9. 9.
    Giral H, Kratzer A, Landmesser U (2016) MicroRNAs in lipid metabolism and atherosclerosis. Best Pract Res Clin Endocrinol Metab 30:665–676CrossRefGoogle Scholar
  10. 10.
    Hu H, Luo L, Liu F, Zou D, Zhu S, Tan B, Chen T (2016) Anti-cancer and Sensibilisation effect of Triptolide on human epithelial ovarian Cancer. J Cancer 7:2093–2099CrossRefGoogle Scholar
  11. 11.
    Huang M, Qiu Q, Xiao Y, Zeng S, Zhan M, Shi M, Zou Y, Ye Y, Liang L, Yang X, Xu H (2016) BET Bromodomain suppression inhibits VEGF-induced angiogenesis and vascular permeability by blocking VEGFR2-mediated activation of PAK1 and eNOS. Sci Rep 6:23770CrossRefGoogle Scholar
  12. 12.
    Huang Y, Tang S, Ji-Yan C, Huang C, Li J, Cai AP, Feng YQ (2017) Circulating miR-92a expression level in patients with essential hypertension: a potential marker of atherosclerosis. J Hum Hypertens 31:200–205CrossRefGoogle Scholar
  13. 13.
    Insull W Jr (2009) The pathology of atherosclerosis: plaque development and plaque responses to medical treatment. Am J Med 122:S3–s14CrossRefGoogle Scholar
  14. 14.
    Kalinina N, Klink G, Glukhanyuk E, Lopatina T, Efimenko A, Akopyan Z, Tkachuk V (2015) miR-92a regulates angiogenic activity of adipose-derived mesenchymal stromal cells. Exp Cell Res 339:61–66CrossRefGoogle Scholar
  15. 15.
    Lee SJ, Namkoong S, Kim YM, Kim CK, Lee H, Ha KS, Chung HT, Kwon YG, Kim YM (2006) Fractalkine stimulates angiogenesis by activating the Raf-1/MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways. Am J Physiol Heart Circ Physiol 291:H2836–H2846CrossRefGoogle Scholar
  16. 16.
    Loyer X, Potteaux S, Vion AC, Guerin CL, Boulkroun S, Rautou PE, Ramkhelawon B, Esposito B, Dalloz M, Paul JL, Julia P, Maccario J, Boulanger CM, Mallat Z, Tedgui A (2014) Inhibition of microRNA-92a prevents endothelial dysfunction and atherosclerosis in mice. Circ Res 114:434–443CrossRefGoogle Scholar
  17. 17.
    Lu F, Zhao LY, Zhang ZM, Zou Q, Yu XL, Wei CY (2018) The intervention of enalapril maleate and folic acid tablet on the expressions of the GRP78 and CHOP and vascular remodeling in the vascular smooth muscle cells of H-hypertensive rats with homocysteine. Eur Rev Med Pharmacol Sci 22:2160–2168PubMedGoogle Scholar
  18. 18.
    Luo L, Yang T (2016) Triptolide inhibits the progression of atherosclerosis in apolipoprotein E(−)/(−) mice. Exp Ther Med 12:2307–2313CrossRefGoogle Scholar
  19. 19.
    Michel JB, Martin-Ventura JL, Nicoletti A, Ho-Tin-Noe B (2014) Pathology of human plaque vulnerability: mechanisms and consequences of intraplaque haemorrhages. Atherosclerosis 234:311–319CrossRefGoogle Scholar
  20. 20.
    Munoz-Vega M, Masso F, Paez A, Carreon-Torres E, Cabrera-Fuentes HA, Fragoso JM, Perez-Hernandez N, Martinez LO, Najib S, Vargas-Alarcon G, Perez-Mendez O (2018) Characterization of immortalized human dermal microvascular endothelial cells (HMEC-1) for the study of HDL functionality. Lipids Health Dis 17:44CrossRefGoogle Scholar
  21. 21.
    Murata K, Ito H, Yoshitomi H, Yamamoto K, Fukuda A, Yoshikawa J, Furu M, Ishikawa M, Shibuya H, Matsuda S (2014) Inhibition of miR-92a enhances fracture healing via promoting angiogenesis in a model of stabilized fracture in young mice. J Bone Miner Res 29:316–326CrossRefGoogle Scholar
  22. 22.
    Natarajan R, Fisher BJ, Fowler AA 3rd (2003) Regulation of hypoxia inducible factor-1 by nitric oxide in contrast to hypoxia in microvascular endothelium. FEBS Lett 549:99–104CrossRefGoogle Scholar
  23. 23.
    Nazari-Jahantigh M, Wei Y, Noels H, Akhtar S, Zhou Z, Koenen RR, Heyll K, Gremse F, Kiessling F, Grommes J, Weber C, Schober A (2012) MicroRNA-155 promotes atherosclerosis by repressing Bcl6 in macrophages. J Clin Invest 122:4190–4202CrossRefGoogle Scholar
  24. 24.
    Nguyen LTH, Muktabar A, Tang J, Dravid VP, Thaxton CS, Venkatraman S, Ng KW (2017) Engineered nanoparticles for the detection, treatment and prevention of atherosclerosis: how close are we? Drug Discov Today 22:1438–1446CrossRefGoogle Scholar
  25. 25.
    Ohyagi-Hara C, Sawada K, Kamiura S, Tomita Y, Isobe A, Hashimoto K, Kinose Y, Mabuchi S, Hisamatsu T, Takahashi T, Kumasawa K, Nagata S, Morishige K, Lengyel E, Kurachi H, Kimura T (2013) miR-92a inhibits peritoneal dissemination of ovarian cancer cells by inhibiting integrin alpha5 expression. Am J Pathol 182:1876–1889CrossRefGoogle Scholar
  26. 26.
    Otsuka F, Yasuda S, Noguchi T, Ishibashi-Ueda H (2016) Pathology of coronary atherosclerosis and thrombosis. Cardiovasc Diagn Ther 6:396–408CrossRefGoogle Scholar
  27. 27.
    Ridker PM, Everett BM, Thuren T, MacFadyen JG, Chang WH, Ballantyne C, Fonseca F, Nicolau J, Koenig W, Anker SD, Kastelein JJP, Cornel JH, Pais P, Pella D, Genest J, Cifkova R, Lorenzatti A, Forster T, Kobalava Z, Vida-Simiti L, Flather M, Shimokawa H, Ogawa H, Dellborg M, Rossi PRF, Troquay RPT, Libby P, Glynn RJ (2017) Antiinflammatory therapy with Canakinumab for atherosclerotic disease. N Engl J Med 377:1119–1131CrossRefGoogle Scholar
  28. 28.
    Santulli G (2015) microRNAs distinctively regulate vascular smooth muscle and endothelial cells: functional implications in angiogenesis, atherosclerosis, and in-stent restenosis. Adv Exp Med Biol 887:53–77CrossRefGoogle Scholar
  29. 29.
    Song JM, Molla K, Anandharaj A, Cornax I, MG OS, Kirtane AR, Panyam J, Kassie F (2017) Triptolide suppresses the in vitro and in vivo growth of lung cancer cells by targeting hyaluronan-CD44/RHAMM signaling. Oncotarget 8:26927–26940PubMedPubMedCentralGoogle Scholar
  30. 30.
    Wei Y, Nazari-Jahantigh M, Chan L, Zhu M, Heyll K, Corbalan-Campos J, Hartmann P, Thiemann A, Weber C, Schober A (2013) The microRNA-342-5p fosters inflammatory macrophage activation through an Akt1- and microRNA-155-dependent pathway during atherosclerosis. Circulation 127:1609–1619CrossRefGoogle Scholar
  31. 31.
    WHO. 2014. Global staus report on noncommunicable diseases 2014.Editor^editors: World Health OrganizationGoogle Scholar
  32. 32.
    Yin YL, Zhu ML, Wan J, Zhang C, Pan GP, Lu JX, Ping S, Chen Y, Zhao FR, Yu HY, Guo T, Jian X, Liu LY, Zhang JN, Wan GR, Wang SX, Li P (2017) Traditional Chinese medicine xin-mai-jia recouples endothelial nitric oxide synthase to prevent atherosclerosis in vivo. Sci Rep 7:43508CrossRefGoogle Scholar
  33. 33.
    Zhang Q, Kandic I, Kutryk MJ (2011) Dysregulation of angiogenesis-related microRNAs in endothelial progenitor cells from patients with coronary artery disease. Biochem Biophys Res Commun 405:42–46CrossRefGoogle Scholar
  34. 34.
    Zhou HF, Liu XY, Niu DB, Li FQ, He QH, Wang XM (2005) Triptolide protects dopaminergic neurons from inflammation-mediated damage induced by lipopolysaccharide intranigral injection. Neurobiol Dis 18:441–449CrossRefGoogle Scholar
  35. 35.
    Ziaei S, Halaby R (2016) Immunosuppressive, anti-inflammatory and anti-cancer properties of triptolide: a mini review. Avicenna J Phytomed 6:149–164PubMedPubMedCentralGoogle Scholar

Copyright information

© University of Navarra 2019

Authors and Affiliations

  1. 1.Medical Examination Center of Qilu Hospital of Shandong UniversityJinanChina
  2. 2.Department of Health ManagementJining NO.1 People’s HospitalJiningChina
  3. 3.Department of Critical Care MedicineJining NO.1 People’s HospitalJiningChina

Personalised recommendations