Estrogen and selective estrogen receptor modulators regulate vascular endothelial growth factor and soluble vascular endothelial growth factor receptor 1 in human endometrial stromal cells  Hidetaka Okada, M.D., Akihiro Tsutsumi, M.D., Miyuki Imai, B.A., Tatsuya Nakajima, M.D., Katsuhiko Yasuda, M.D., Hideharu Kanzaki, M.D.  Fertility and Sterility  Volume 93, Issue 8, Pages 2680-2686 (May 2010) DOI: 10.1016/j.fertnstert.2009.08.056 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 The effect of E2 on vascular endothelial growth factor (VEGF) and soluble VEGF receptor 1 (sVEGFR-1) in endometrial stromal cells (ESCs). The ESCs were cultured for 12 days with either vehicle (control) or E2 (10−8 mol/L) and analyzed for VEGF and sVEGFR-1 mRNA by real-time polymerase chain reaction (PCR) analysis. (A) Agarose gel electrophoresis of PCR products showing a pure single band in each amplication. Marker=molecular weight marker; RT(-)=reverse transcriptase negative control; EF=elongation factor-1α. (B) VEGF messenger RNA (mRNA) levels were calculated after normalization to EF-1α mRNA expression. (C) sVEGFR-1 mRNA levels were calculated after normalization to EF-1α mRNA expression. (D) Time course of VEGF secretion from ESCs by E2. Results are representative data of three experiments and each value represents mean±SEM in triplicate wells. (E) The dose-dependent effect of E2 on VEGF secretion from ESCs. The ESCs were grown for 12 days in medium containing with either vehicle (control, Con) or E2 (10−11 to 10−7 mol/ L) with medium changes every 3 days. The ELISA measured VEGF concentrations in the culture medium during the last 3 days of a 12-day culture period. The effect of vehicle is assigned a potency of 100%. Columns and vertical bars represent the mean±SEM of four separate experiments. Values significantly different (∗P<.05; ∗∗P<.01 vs. vehicle). Fertility and Sterility 2010 93, 2680-2686DOI: (10.1016/j.fertnstert.2009.08.056) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) The interference of coexisting recombinant (rec) human soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) to measured vascular endothelial growth factor (VEGF) value in culture media. Free VEGF levels are decreased in the culture media with sVEGFR-1. Human VEGF protein in three different culture media from endometrial stromal cells (754(▴), 1,343(♦), or 2,251.6(▪) pg/mL) was measured in the absence (control) or in the presence of various doses of sVEGFR-1 using the ELISA kit for VEGF, as described in the Materials and Methods section. (B) Free VEGF levels in culture media were decreased in the presence of exogenous sVEGFR-1. (C) The sVEGFR-1 levels in culture media were not changed in the presence of exogenous VEGF. The effect of vehicle (control) is assigned a potency of 100%. Columns and vertical bars represent the mean±SEM. Values significantly different (∗P<.01 vs. control). N.D=not detectable; NS=not significant. Fertility and Sterility 2010 93, 2680-2686DOI: (10.1016/j.fertnstert.2009.08.056) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 The effect of E2 on soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) secretion from endometrial stromal cells. (A) Endometrial stromal cells were grown for 12 days in medium containing with either vehicle (control; □) or E2 (10−8 mol/ L; E, ■) with medium changes every 3 days. The ELISA measured sVEGFR-1 concentrations in the culture medium during the last 3 days of a 12-day culture period. Data are given for the four individual cell preparations with treatments. (B) The effect of vehicle (control) is assigned a potency of 100%. Columns and vertical bars represent the mean±SEM for combined data of four separate experiments. Values significantly different (∗P<.01 vs. control). Fertility and Sterility 2010 93, 2680-2686DOI: (10.1016/j.fertnstert.2009.08.056) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effects of E2 or various selective estrogen receptor modulators on vascular endothelial growth factor (VEGF) (A) and soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) (B) secretion from endometrial stromal cells. Endometrial stromal cells were grown for 12 days in medium containing with vehicle (control), E2 (10−8 mol/L), 4-hydroxytamoxifen (OHT; 10−7 mol/ L), or raloxifene (Ral; 10−7 mol/ L) with medium changes every 3 days. The ELISA measured VEGF and sVEGFR-1 concentrations in the culture medium during the last 3 days of a 12-day culture period. The effect of vehicle is assigned a potency of 100%. Columns and vertical bars represent the mean±SEM of four separate experiments. Values significantly different (∗P<.01 vs. vehicle). Fertility and Sterility 2010 93, 2680-2686DOI: (10.1016/j.fertnstert.2009.08.056) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions