Triptolide inhibits angiogenesis in microvascular endothelial cells through regulation of miR-92a
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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.
KeywordsAtherosclerosis Angiogenesis Triptolide miR-92a ERK PI3K/AKT
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Conflict of interest
The authors declare that there are no conflicts of interest.
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