NME1 suppression of endometrial stromal cells promotes angiogenesis in the endometriotic milieu via stimulating the secretion of IL-8 and VEGF

Abstract

Nonmetastatic gene 23-H1 (NME1, also known as nm23-H1) is a wide-spectrum tumor metastasis suppressor gene that plays an important role in suppressing the proliferation, adhesion and invasion of endometrial stromal cells (ESCs). The present study is undertaken to explore the mechanism by which NME1 in ESCs from endometriosis modulates the angiogenesis and herein participates in the pathogenesis of endometriosis. The expression of NME1 in the primary ESCs from normal endometrium without endometriosis was higher than that from eutopic endometrium and ectopic lesion with endometriosis. Silencing NME1 stimulated the secretion of angiogenic factors interleukin-8 (IL-8) and vascular-endothelial growth factor (VEGF) of the eutopic ESCs from women with endometriosis, and these effects could be abrogated by MAPK/ERK1/2 or AKT inhibitor. In addition, the supernatant of NME1-silenced ESCs increased the expression of angiogenesis-relative molecules CD62E and CD105, and promoted angiogenesis of human umbilical vein endothelial cells (HUVECs). Anti-human IL-8 or VEGF neutralizing antibody reversed the effect on angiogenesis of HUVECs induced by NME1-silenced ESCs. Our current results suggest that the abnormal lower expression of NME1 in ESCs secrete more IL-8 and VEGF through activation of MAPK/ERK1/2 and AKT signal pathways, up-regulate the level of CD62E and CD105, and finally lead to numerous angiogenesis of vascular endothelial cells in the endometriotic milieu, which is beneficial to the origin and development of endometriosis.

Keywords: ESCs; HUVECs; NME1; angiogenesis; endometriosis.

Figure 1

The expression of NME1 is decreased in the eutopic and ectopic ESCs from women with endometriosis. The expression of NME1 in primary cultured ESCs from women with and without endometriosis was analyzed by immunocytochemistry. Here normal ESCs: ESCs from women without endometriosis; eutopic ESCs and ectopic ESC: ESCs of eutopic endometium and endometriotic peritoneal lesion from women with endometriosis. Original magnification: ×200.

Figure 2

Silencing NME1 of eutopic ESCs from women with endometriosis stimulates angiogenesis of HUVECs. A, B: We collected the ESCs supernatant of transfection for 72 h and added to HUVECs culture system, and then analyzed the pro-angiogenic activity in NME1-silenced ESCs by tube formation assay. Original magnification: ×200. Ctrl: the nontargeting siRNA oligonucleotides; si-NME1: NME1 is knocked down. Results were highly reproducible in three independent experiments. Data are mean±SD. **P<0.01 compared to the negative control.

Figure 3

NME1 silence of ESCs up-regulates the expression of CD62E and CD105 on HUVECs. A, B: Flow cytometry was performed to analysis the expression of CD62E and CD105 of HUVECs incubated with the supernatant of siRNA transfected ESCs for 48 h, which treated with or without U0126 and LY294002. Data are mean±SD. *P<0.05, **P<0.01 compared to the negative control. ##P<0.01 compared to NME1 silence. ΔP<0.05 compared to the negative control plus U0126 or LY294002 treatment.

Figure 4

Silencing NME1 stimulates the secretion of VEGF and IL-8 of ESCs. We used ELISA to detect the secretion level of VEGF, IL-6 and IL-8 of siRNA transfected ESCs. Data are mean±SD. *P<0.05 compared to the negative control. #P<0.05, ##P<0.01 compared to NME1 silence.

Figure 5

NME1 silence of ESCs stimulates angiogenesis of HUVECs through promoting the secretion of VEGF and IL-8 of ESCs. We added the supernatant of siRNA transfected ESCs to HUVECs culture system, which treated with or without VEGF or IL-8 neutralizing antibody for 48 h, and analyzed the tube formation of HUVECs by tube formation assay. Data are mean±SD. *P<0.05, **P<0.01 compared to the negative control. ##P<0.01 compared to NME1 silence. ΔP<0.05 compared to the negative control plus VEGF neutralizing antibody treatment.