Suppression of COUP-TFII upregulates angiogenin and promotes angiogenesis in endometriosis

Abstract

Study question: How does hypoxia-mediated downregulation of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) promote angiogenesis in endometriosis?

Summary answer: Suppression of COUP-TFII by hypoxia stimulates angiogenesis through induction of angiogenin (ANG).

What is known already: The level of COUP-TFII is downregulated in endometriotic tissues, and downregulation of COUP-TFII contributes to the development of endometriosis.

Study design, size, duration: Twenty-seven patients of reproductive age with endometriosis were recruited in this study. Eutopic endometrial and ectopic endometriotic stromal cells were isolated, cultured and subjected to various treatments.

Participants/materials, setting, methods: Microarray hybridization, quantitative RT-PCR, and Western blot were used to detect gene expression in normal and endometriotic samples. A luciferase reporter assay and chromatin immunoprecipitation in normoxia- or hypoxia-treated primary cultures of human endometrial stromal cells were performed. Tube formation analysis was performed using primary human umbilical vein endothelial cells (HUVECs).

Main results and the role of chance: Protein level of COUP-TFII was downregulated by hypoxia (P < 0.05, normoxia versus hypoxia). Loss of COUP-TFII increased the angiogenic capacity of endometrial stromal cells (P < 0.05, COUP-TFII knockdown versus knockdown control). A novel COUP-TFII target gene, ANG, was identified through microarray analysis. Chromatin immunoprecipitation and promoter activity assays demonstrated that the ANG promoter was bound and suppressed by COUP-TFII (P < 0.05, COUP-TFII overexpression versus empty vector). The levels of ANG mRNA and protein were elevated in ectopic endometriotic stromal cells and negatively correlated with COUP-TFII (P < 0.05, endometrial versus endometriotic tissues/stromal cells). Both knockdown and forced-expression of COUP-TFII further demonstrated that ANG expression and ANG-mediated angiogenic activity were negatively regulated by COUP-TFII (P < 0.05, COUP-TFII knockdown versus knockdown control, and COUP-TFII overexpression versus empty vector).

Limitations, reasons for caution: This study was conducted in primary human endometrial stromal cell cultures and HUVECs, therefore, may not fully reflect the situation in vivo.

Large scale data: The raw data were submitted to Gene Expression Omnibus (GSE107469).

Wider implications of the findings: This is the first study to highlight that the aberrant expression of ANG in endometriotic lesions is mediated by hypoxia-suppressed COUP-TFII expression, which reveals an as yet unidentified molecular pathogenesis of endometriosis.

Study funding/competing interest(s): This work was supported by research grants (MOST 105-2314-B-006-059-MY3 to M.H.W. and MOST 104-2320-B-006-036-MY3 to S.J.T.) from the Ministry of Science and Technology, Taiwan. The authors declare that there is no conflict of interest.

Keywords: COUP-TFII; angiogenesis; angiogenin; chromatin immunoprecipitation PCR; endometriosis; hypoxia; nuclear receptor subfamily 2 group F member 2; promoter activity assay; transcriptional repression.