Regulation of soluble vascular endothelial growth factor receptor (sFlt-1/sVEGFR-1) expression and release in endothelial cells by human follicular fluid and granulosa cells

Abstract

Background: During the female reproductive cycle, follicular development and corpus luteum formation crucially depend on the fast generation of new blood vessels. The importance of granulosa cells and follicular fluid in controlling this angiogenesis is still not completely understood. Vascular endothelial growth factor (VEGF) produced by granulosa cells and secreted into the follicular fluid plays an essential role in this process. On the other hand, soluble VEGF receptor-1 (sFlt-1) produced by endothelial cells acts as a negative modulator for the bioavailability of VEGF. However, the regulation of sFlt-1 production remains to be determined.

Methods: We analyzed the influence of human follicular fluid obtained from FSH-stimulated women as well as of human granulosa cell conditioned medium on sFlt-1 production in and release from human umbilical vein endothelial cells (HUVEC) in vitro. Soluble Flt-1 gene expression was determined by RT-PCR analysis, amount of sFlt-1-protein was quantified by Sandwich-ELISA.

Results: Human follicular fluid as well as granulosa cell-conditioned medium significantly inhibit the production of sFlt-1 by endothelial cells on a posttranscriptional level. Treatment of cultured granulosa cells with either hCG or FSH had not impact on the production of sFlt-1 inhibiting factors. We further present data suggesting that this as yet unknown sFlt-1 regulating factor secreted by granulosa cells is not heat-sensitive, not steroidal, and it is of low molecular mass (< 1000 Da).

Conclusion: We provide strong support that follicular fluid and granulosa cells control VEGF availability by down regulation of the soluble antagonist sFlt-1 leading to an increase of free, bioactive VEGF for maximal induction of vessel growth in the ovary.

Figure 1

Proliferation of HUVECs incubated with culture medium containing 30% follicular fluid (+FF) or incubated with culture medium alone (control) for up to 4 days. From day 3 onwards a significant increase in cell number can be observed for those endothelial cells treated with follicular fluid compared to controls. * = p < 0.05.

Figure 2

Amount of sFlt-1 in the culture supernatant of HUVECs treated with medium only (grey bars) or with medium containing 30% human follicular fluid (FF, black bars) for up to 3 days. From day 2 onwards a significant increase in sFlt-1 content can be observed for those endothelial cells treated with medium only but not for those incubated with medium containing follicular fluid. * = p < 0.05.

Figure 3

Amount of sFlt-1 in the culture supernatant of HUVECs after 4 days of incubation with medium containing different concentrations of human follicular fluid. Amount of sFlt-1 decreases with increasing concentrations of follicular fluid showing a significant inhibition of sFlt-1 production at a concentration of 30%. * = p < 0.05.

Figure 4

Quantification of sFlt-1 in endothelial cell culture supernatant. Neither heat inactivation of culture medium containing FCS (C2) nor absence of FCS (C3) has a measurable effect on sFlt-1 production of endothelial cells compared to incubation with untreated control medium containing FCS (C1). The significant inhibitory effect of untreated human follicular fluid (FF) on sFlt-1 production is maintained after ultra filtration leaving only molecules smaller than 1000 Dalton. This inhibition is not prevented neither by heat inactivation nor by charcoal treatment of the follicular fluid-flow through. All follicular fluids have been added to the culture medium at a concentration of 30%. * = p < 0.05

Figure 5

RT-PCR of mRNA of HUVECs incubated with medium only (C) or with medium containing 30% follicular fluid (+FF). Densitometric evaluation revealed no significant difference in mRNA-expression of sFlt-1 between these two experimental groups at p < 0.05.

Figure 6

Amount of sFlt-1 in the culture supernatant of HUVECs incubated for 4 days with medium containing 30% granulosa cell (GC)-conditioned medium. Addition of GC-conditioned medium leads to a significant inhibition of sFlt-1 production of HUVECs (*). Treatment of granulosa cells with FCS or hCG during conditioning of medium has no influence on this inhibition. Incubation with follicular fluid (FF) of the same patients leads to a significant reduced production of sFlt-1 compared to controls as well as to treatment with GC-conditioned medium (**). p < 0.05.