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.

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.

Melatonin receptor signaling in pregnant and nonpregnant rat uterine myocytes as probed by large conductance Ca2+-activated K+ channel activity.

The mRNAs of MT1 and MT2 melatonin receptors are present in cells from nonpregnant (NPM) and pregnant (PM) rat myometrium. To investigate the coupling of melatonin receptors to Gq- and Gi-type of heterotrimeric G proteins, we analyzed the activity of large-conductance Ca2+-activated K+ (BKCa) channels, the expression of which in the uterus is confined to smooth muscle cells. The melatonin receptor agonist 2-iodomelatonin induced a pertussis toxin (PTX)-insensitive increase in channel open probability that was blocked by the nonselective antagonist luzindole. The 2-iodomelatonin effect on channel open probability was suppressed by overexpression of the Gqalpha-inactivating protein RGS16 and the phospholipase C inhibitor U-73122. The activity of BKCa channels is differentially regulated by protein kinase A (PKA) in NPM and PM cells. Thus, the beta-adrenoceptor agonist isoprenaline inhibited the BKCa channel conducted whole-cell outward current (Iout) in NPM cells and enhanced Iout in PM cells. Additional application of 2-iodomelatonin antagonized the isoprenaline effect on Iout in NPM cells but enhanced Iout in PM cells. All 2-iodomelatonin effects on Iout were sensitive to PTX treatment and the PKA inhibitor H-89. We therefore conclude that melatonin activates both the PTX-insensitive Gq/phospholipase C/Ca2+ and the PTX-sensitive Gi/cAMP/PKA signaling pathway in rat myometrium.

Smoking impairs angiogenesis during maturation of human oocytes.

OBJECTIVE: This study determines whether smoking influences ovarian vascularization which thus may impair follicular development. DESIGN: Prospective laboratory study of follicular fluids and granulosa cells from patients undergoing in vitro fertilization. SETTING: University Hospital Aachen, Germany. PATIENT(S): Fifty smoking women and 50 nonsmoking women. INTERVENTION(S): Cultivation of human granulosa cells. Cultivation of human umbilical vein endothelial cells (HUVECs) with either granulosa cell-conditioned medium or follicular fluid. Determination of clinical parameters. MAIN OUTCOME MEASURE(S): Quantification of soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) and cotinine. RESULT(S): Mean sVEGFR-1 concentration in follicular fluid of smokers was 499.6 pg/mL compared with 159.2 pg/mL in nonsmokers. Correspondingly, supernatant of HUVECs cultured with follicular fluid from smoking and nonsmoking women showed, respectively, 1,174.1 pg/mL versus 794.2 pg/mL sVEGFR-1. The HUVECs incubated with conditioned medium from smokers' granulosa cells at culturing days 5, 9, 13, and 17 secreted, respectively, 1,712.4, 1,560.6, 1,619.0, and 1,635.0 pg/mL sVEGFR-1, whereas nonsmokers showed, respectively, 1,147.6, 1,067.2, 1,135.9, and 1,206.3 pg/mL sVEGFR-1. Mean cotinine concentration in smoking women was 83.9 ng/mL and in nonsmoking was 2.8 ng/mL. In all four comparisons, differences between groups reached statistical significance. CONCLUSION(S): This study showed that smokers secrete significantly higher amounts of sVEGFR-1 than nonsmokers, which may result in decreased ovarian vascularization and reduced oocyte maturation.

beta8 integrins are required for vascular morphogenesis in mouse embryos.

In order to assess the in vivo function of integrins containing the beta8 subunit, we have generated integrin beta8-deficient mice. Ablation of beta8 results in embryonic or perinatal lethality with profound defects in vascular development. Sixty-five percent of integrin beta8-deficient embryos die at midgestation, with evidence of insufficient vascularization of the placenta and yolk sac. The remaining 35% die shortly after birth with extensive intracerebral hemorrhage. Examination of brain tissue from integrin beta8-deficient embryos reveals abnormal vascular morphogenesis resulting in distended and leaky capillary vessels, as well as aberrant brain capillary patterning. In addition, endothelial cell hyperplasia is found in these mutant brains. Expression studies show that integrin beta8 transcripts are localized in endodermal cells surrounding endothelium in the yolk sac and in periventricular cells of the neuroepithelium in the brain. We propose that integrin beta8 is required for vascular morphogenesis by providing proper cues for capillary growth in both yolk sac and embryonic brain. This study thus identifies a molecule crucial for vascular patterning in embryonic yolk sac and brain.