Prostaglandin E2 and vascular endothelial growth factor A mediate angiogenesis of human ovarian follicular endothelial cells

Abstract

Study question: Which receptors for prostaglandin E2 (PGE2) and vascular endothelial growth factor A (VEGFA) mediate angiogenesis in the human follicle around the time of ovulation?

Summary answer: PGE2 and VEGFA act via multiple PGE2 receptors (PTGERs) and VEGF receptors (VEGFRs) to play complementary roles in follicular angiogenesis.

What is known already: Production of PGE2 and VEGFA by the follicle are prerequisites for ovulation. PGE2 is an emerging regulator of angiogenesis and has not been examined in the context of the human ovulatory follicle. VEGFA is an established regulator of follicular angiogenesis.

Study design, size, duration: Ovarian biopsies containing the ovulatory follicle were obtained from 11 women of reproductive age (30-45 years) undergoing surgery for laparoscopic sterilization. In some cases, women received hCG to substitute for the ovulatory LH surge before ovarian biopsy. In addition, aspirates from four women of reproductive age (18-31 years) undergoing gonadotrophin stimulation for oocyte donation were obtained for isolation of human ovarian microvascular endothelial cells (hOMECs).

Participants/materials, setting, methods: Ovarian biopsies were utilized for immunocytochemical detection of von Willebrand factor to identify endothelial cells. hOMECs were cultured with PGE2, PTGER receptor selective agonists, VEGFA, or VEGFR selective agonists. hOMECs were assessed for proliferation by Ki67 immunocytochemistry. hOMEC migration was determined by counting cells which migrated through a porous membrane in vitro. Sprout formation was quantified by determining sprout number and length from photographs take after culture of hOMECs in a 3-dimensional matrix.

Main results and the role of chance: Endothelial cells were not observed within the granulosa cell layer of human ovulatory follicles prior to an ovulatory dose of hCG and were first seen amongst granulosa cells 18-34 h after hCG. In vitro, PGE2 enhanced migration and sprout formation but did not alter hOMEC proliferation. Agonists selective for each PTGER increased migration with no change in proliferation. PTGER1 and PTGER2 agonists increased the number of sprouts, while only PTGER1 affected sprout length. VEGFA increased hOMEC proliferation, migration, and formation of structures resembling capillary sprouts. Signaling through VEGFR1 promoted hOMEC migration, proliferation, and the formation of few, long endothelial cell sprouts, while VEGFR2 stimulation promoted hOMEC migration and the formation of many, short sprouts. All effects of treatments in vitro were considered significant at P < 0.05.

Limitations, reasons for caution: While primary cultures of hOMECs respond to PGE2 and VEGFA differently than other cultured endothelial cells, hOMECs may not respond to PGE2 and VEGFA in vivo as they do in vitro.

Wider implications of the findings: Agonists and antagonists selective for PTGER1, PTGER2, VEGFR1, or VEGFR2 may have therapeutic value to promote or prevent ovulation in women.

Study funding/competing interests: This research was supported by grant funding from the Eunice Kennedy Shriver National Institutes of Child Health and Human Development (HD071875 to D.M.D., T.E.C., M.B.). The authors have no conflicts of interest to disclose.

Keywords: PGE2 receptor; VEGF receptor; endothelial cell; follicle; ovary; ovulation; prostaglandin; vascular endothelial growth factor.

Figure 1

Endothelial cells of the ovulatory follicle are present in the granulosa cell layer prior to ovulation and express prostaglandin E2 (PGE2) and vascular endothelial growth factor A (VEGFA) receptors. (A–H) Human follicular biopsies obtained prior to (no hCG, A) or 12–18 h (C and D), 18–34 h (E and F), or 44–70 h (G and H) after hCG were immunostained for von Willebrand Factor (VWF) (brown); nuclei are blue. VWF+ cells in the stroma (arrows), VWF+ cells in large stromal vessels (large arrowheads), and VWF+ cells in the granulosa cell layer (small arrowheads) are indicated. (A)–(H) are oriented with antrum (an) in upper right, granulosa cells (gc) central, and stroma (st) in lower left. (B) shows staining when primary antibody was omitted and bar =50 µm. Images are representative of biopsies obtained prior to hCG (n = 2), 12–18 h after hCG (n = 4), 18–34 h after hCG (n = 4), or 44–70 h after hCG (n = 1). (I–P) Immunodetection (green) of PTGER1 (I), PTGER2 (J), PTGER3 (K), PTGER4 (L), VEGFR1 (M) and VEGFR2 (N) in human ovarian microvascular endothelial cells (hOMECs) in vitro; nuclei are blue. For (I), upper shows merged image and lower shows PTGER1 only. No staining was seen when primary antibody was omitted under conditions used for PTGER (P) and VEGFR (O) immunodetection. Representative of n = 4 hOMEC lines. (PTGER, PGE2 receptor; VEGFR, VEGF receptor.)

Figure 2

Prostaglandin E2 (PGE2) and vascular endothelial growth factor A (VEGFA) stimulate human ovarian microvascular endothelial cell (hOMEC) migration. (A) Migration during culture with basal medium alone or containing PGE2, VEGFA, or both PGE2 and VEGFA. (B) Migration during culture with PGE2 and PTGER selective agonists. (C) Migration during culture with VEGFA and VEGFR receptor selective agonists. Representative migration in basal (D), PGE2 (E), or VEGFA (F) treatment groups; representative pore (arrowhead) and migrated cell (arrow) are indicated in (D). For each Panel, groups with different superscripts are different by repeated measures ANOVA and Duncan's post hoc test, P < 0.05, n = 4 hOMEC lines.

Figure 3

Vascular endothelial growth factor A (VEGFA), but not prostaglandin E2 (PGE2), stimulates human ovarian microvascular endothelial cell (hOMEC) proliferation. (A) Percentage of cells expressing Ki67 after culture with basal media alone or with addition of PGE2, VEGFA, or both PGE2 and VEGFA. (B) Proliferation after culture with PGE2 or PTGER selective agonists. (C) Proliferation in response to VEGFA and VEGFR selective agonists. Representative Ki67 staining in basal (D), PGE2 (E), or VEGFA (F) treatment groups are shown; representative Ki67+ (arrowhead) and Ki67− (arrow) nuclei are indicated in (D). For each Panel, groups with different superscripts are different by repeated measures ANOVA and Duncan's post hoc test, P < 0.05, n = 4 hOMEC lines.

Figure 4

Prostaglandin E2 (PGE2) and vascular endothelial growth factor A (VEGFA) promote sprout formation by human ovarian microvascular endothelial cell (hOMECs). (A–F) Sprout counts (A) and sprout lengths (C) after culture with basal media with or without PGE2 at 1 and 2 days in vitro. Sprout number (B) and length (D) after 1 day in vitro in response to PGE2 and PTGER agonists are also shown. Images show hOMEC sprouting in response to basal (E) and PGE2 (F). (G–L) Sprout counts (G) and sprout lengths (I) after culture with basal media with or without VEGFA at 1 and 2 days in vitro. Sprout number (H) and length (J) after 2 days in vitro in response to VEGFA and VEGFR agonists are also shown. Images show hOMEC sprouting in response to basal (K) and VEGFA (L). For (A, C, G and I) treatment effects were determined by 2-tailed paired t-test; asterisk (*) indicates P < 0.05, n = 4 hOMEC lines. For (B, D, H and J) groups with different superscripts are different by repeated measures ANOVA and Duncan's post hoc test, P < 0.05. n = 4 hOMEC line. Images in (E), (F), (K) and (L) are representative of four hOMEC lines.