Endocrine gland–derived vascular endothelial growth factor stimulates proliferation and tube formation in human uterine microvascular endothelial cell.

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Presentation transcript:

Endocrine gland–derived vascular endothelial growth factor stimulates proliferation and tube formation in human uterine microvascular endothelial cell through the mitogen- activated protein kinase but not through the Akt pathway Yin-Lau Lee, Ph.D., Yuk-Ling Chan, B.Sc., Wan-Ngai Chow, M.Phil., Ernest Hung-Yu Ng, M.D., Kai-Fai Lee, Ph.D., William S.B. Yeung, Ph.D., Pak-Chung Ho, M.D. Fertility and Sterility Volume 91, Issue 5, Pages 2163-2171 (May 2009) DOI: 10.1016/j.fertnstert.2008.03.076 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Reverse transcriptase–mediated real-time PCR of the EG-VEGF, PKR1, and PKR2 mRNA expression. The expression of EG-VEGF (A), PKR1 (B), and PKR2 (C) in UtMVEC-Myo at passage 5 (column 1), 9 (column 2), and 12 (column 3), endometrial stromal cells (column 4), endometrial glandular epithelial cell (column 5), and secretory-phase endometrial biopsy (column 6) were studied. The relative expressions of the transcripts were normalized with 18S. Fertility and Sterility 2009 91, 2163-2171DOI: (10.1016/j.fertnstert.2008.03.076) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effects of EG-VEGF, VEGF, and FGF-2 on UtMVEC-Myo cell proliferation. UtMVEC-Myo at 10,000 cells/well in 24-well plate was treated with 1, 10, and 100 ng/mL of EG-VEGF, VEGF, and FGF-2 for 48 hours. The cells were trypsinized, and total cell numbers were counted by hematocytometer. Data represent mean ± SEM of five individual experiments. ∗Significant increase in total cell number counts compared with controls (P<.05). Fertility and Sterility 2009 91, 2163-2171DOI: (10.1016/j.fertnstert.2008.03.076) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effects of EG-VEGF, VEGF, and FGF-2 on UtMVEC-Myo tube formation using Fibrin Gel In Vitro Angiogenesis Assay under phase-contrast microscopy. (A) UtMVEC-Myo was cultured to confluence on plastic culture flask (inset a) and subcultured to a fibrin gel–coated 96-well plate (inset b). The cells were treated with 100 ng/mL of VEGF (inset c), 100 ng/mL of FGF-2 (inset d), 100 ng/mL of EG-VEGF (e), and combined EG-VEGF/VEGF/FGF-2 treatment (f). (B) The angiogenic score was evaluated as described in the Materials and Methods section using a 5-step scoring system. ∗Significant increase in the angiogenic score when compared with controls (P<.05). Fertility and Sterility 2009 91, 2163-2171DOI: (10.1016/j.fertnstert.2008.03.076) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effects of EG-VEGF on MAPK and Akt cell signaling pathways in UtMVEC-Myo cells. (A) Exposures of UtMVEC-Myo for 15 minutes to 10 ng/mL of VEGF induced a prominent phosphorylation of p44/42 MAPK (P-p44/42 MAPK). EG-VEGF at 25, 50, and 100 ng/mL also activated the p44/42 MAPK pathway, although to a lesser extent. The specificity of EG-VEGF-induced phosphorylation of p44/42 MAPK was confirmed by the dose-dependent inhibitory effects of PD98059. (B) Time-course effects of EG-VEGF-induced phosphorylation of p44/42 MAPK. The response of UtMVEC-Myo to EG-VEGF treatment peaked after 15 minutes but declined thereafter. (C) Dependency of EG-VEGF induced p44/42 MAPK phosphorylation on VEGF. UtMVEC-Myo was preincubated with 4 μg/mL of antibodies (Ab) against VEGF or EG-VEGF for 2 hours, followed by EG-VEGF (100 ng/mL) and VEGF (10 ng/mL) treatment for 15 minutes. EG-VEGF-induced p44/42 MAPK phosphorylation was independent of VEGF. (D) Exposures of UtMVEC-Myo to 10 ng/mL VEGF or 25, 50, and 100 ng/mL of EG-VEGF at different time intervals did not significantly induce Akt phosphorylation (both Thr308 and Ser473). (E) Phosphorylation of Akt (P-Akt) was found in a human breast cancer cell (MCF-7) line treated with 10 ng/mL IGF-1 for 15 and 60 minutes. (F) Effect of PD98059 on EG-VEGF induced cell proliferation. UtMVEC-Myo was treated with 100 ng/mL of EG-VEGF with or without preincubation of 10 μM PD98059 for 30 minutes. VEGF was used as a control. The cell proliferation assay XTT was performed, and the absorbances were recorded. Data represent mean ± SEM of three replicates of three individual experiments. ∗Significant increase in the cell proliferation when compared with controls (P<.05). Fertility and Sterility 2009 91, 2163-2171DOI: (10.1016/j.fertnstert.2008.03.076) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions