Hela Gitay-Goren

Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis

Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific mitogen. To evaluate whether VEGF may play a role in angiogenesis, we have determined the spatial and temporal patterns of expression of VEGF and VEGF receptors during natural angiogenic processes taking place within the female reproductive system. Four angiogenic processes were analyzed: neovascularization of ovarian follicles, neovascularization of the corpus luteum, repair of endometrial vessels, and angiogenesis in embryonic implantation sites. During all processes, VEGF mRNA was found to be expressed in cells surrounding the expanding vasculature. VEGF was predominantly produced in tissues that acquire new capillary networks (theca layers, lutein cells, endometrial stroma, and the maternal decidua, respectively). VEGF-binding activity, on the other hand, was found on endothelial cells of both quiescent and proliferating blood vessels. These findings are consistent with a role for VEGF in the targeting of angiogenic responses to specific areas. Using in situ hybridization, we show that VEGF is expressed in 10 different steroidogenic and/or steroid-responsive cell types (theca, cumulus, granulosa, lutein, oviductal epithelium, endometrial stroma, decidua, giant trophoblast cells, adrenal cortex, and Leydig cells). Furthermore, in some cells upregulation of VEGF expression is concurrent with the acquisition of steroidogenic activity, and expression in other cell types is restricted to a particular stage of the ovarian cycle. These findings suggest that expression of VEGF is hormonally regulated. We propose that excessive expression of VEGF during gonadotropin-induced ovulation may contribute to the development of ovarian hyperstimulation syndromes by virtue of the vascular permeabilization activity of this factor.

Vascular endothelial growth factor and its receptors.

Vascular endothelial growth factor (VEGF) is a highly specific mitogen for vascular endothelial cells and an angiogenic factor that is structurally related to platelet derived growth factor (PDGF). It is also known as the vascular permeability factor (VPF) because it efficiently potentiates the permeabilization of blood vessels. Five types of VEGF mRNA encoding VEGF species which differ in their molecular mass and in their biological properties are transcribed from a single gene as a result of alternative splicing. VEGFs are produced and secreted by several normal cell types including smooth muscle, luteal and adrenal cortex cells. VEGFs are also produced by different tumorigenic cells, and appear to play a major role in tumour angiogenesis. Antibodies directed against VEGF can inhibit the growth of a variety of VEGF producing tumours. Of the various VEGF species, the best characterized is the 165 amino acid long form (VEGF165). VEGF165 is a heparin binding growth factor, and its interaction with VEGF receptors on the cell surface of vascular endothelial cells depends on the presence of heparin-like molecules. Several cell types which do not proliferate in response to VEGF such as bovine corneal endothelial cells, HeLa cells and human melanoma cells also express cell surface VEGF receptors, but the function of the VEGF receptors in these cells is unclear. Recently, the tyrosine-kinase receptors encoded by the flt and KDRflk-1 genes were found to function as VEGF165 receptors.

Selective Binding of VEGF to One of the Three Vascular Endothelial Growth Factor Receptors of Vascular Endothelial Cells

VEGF and VEGF are vascular endothelial growth factor splice variants that promote the proliferation of endothelial cells and angiogenesis. VEGF contains the 44 additional amino acids encoded by exon 7 of the VEGF gene. These amino acids confer upon VEGF a heparin binding capability which VEGF lacks. I-VEGF bound to three vascular endothelial growth factor (VEGF) receptors on endothelial cells, while I-VEGF bound selectively only to the flk-1 VEGF receptor which corresponds to the larger of the three VEGF receptors. The binding of I-VEGF to flk-1 was not affected by the removal of cell surface heparan sulfates or by heparin. Both VEGF and VEGF inhibited the binding of I-VEGF to a soluble extracellular domain of the flk-1 VEGF receptor in the absence of heparin. However, heparin potentiated the inhibitory effect of VEGF by 2-3-fold. These results contrast with previous observations which have indicated that the binding of I-VEGF to the flk-1 receptor is strongly dependent on heparin-like molecules. Further experiments showed that the receptor binding ability of VEGF is susceptible to oxidative damage caused by oxidants such as HO or chloramine-T. VEGF was also damaged by oxidants but to a lesser extent. Heparin or cell surface heparan sulfates restored the flk-1 binding ability of damaged VEGF but not the receptor binding ability of damaged VEGF. These observations suggest that alternative splicing can generate a diversity in growth factor signaling by determining receptor recognition patterns. They also indicate that the heparin binding ability of VEGF may enable the restoration of damaged VEGF function in processes such as inflammation or wound healing.

High Levels of Biologically Active Vascular Endothelial Growth Factor (VEGF) are Produced by the Baculovirus Expression System

Vascular endothelial growth factor (VEGF) is a recently discovered mitogen for endothelial cells in vitro, and a potent angiogenesis promoting factor in vivo. VEGF is secreted from producing cells as a homodimer, binds to specific receptors on the cell surface of endothelial cells, and is produced in four forms as a result of alternative splicing. We have expressed the cDNA encoding the 165 amino-acid long isoform of VEGF in insect cells using the baculovirus based expression vector. We show that infected insect cells secrete large amounts of VEGF. Antibodies directed against a synthetic peptide prepared from human VEGF identify the secreted factor. The baculovirus derived VEGF expressed in insect cells (inVEGF) binds directly to the VEGF receptors inVEGF competes with pure mammalian cells derived [125I]-VEGF for binding to the VEGF receptors that are present on the cell surface of endothelial cells. Furthermore, inVEGF is biologically active and induces the proliferation of human umbilical vein derived endothelial cells.

Glycosylation of vascular endothelial growth factor is not required for its mitogenic activity

We have stably expressed the cDNA encoding the 165 amino-acid long form of human vascular endothelial growth factor (VEGF) in BHK-21 cells. VEGF was partially purified from the conditioned medium of transfected cells using heparin-sepharose affinity chromatography. The partially purified VEGF was mitogenic for various types of endothelial cells and inhibited the binding of pure [125I]VEGF to its receptors. Western blot analysis, using anti-VEGF antibodies, revealed a 47 kDa VEGF homodimer in the partially purified VEGF fraction. Preincubation of the transfected cells with the N-glycosylation inhibitor tunicamycin resulted in the conversion of the 47 kDa VEGF homodimer into a smaller, deglycosylated form of 42 kDa. Partially purified preparations of the deglycosylated VEGF displayed a mitogenic activity that was similar to that of the glycosylated form and efficiently inhibited the binding of native [125I]VEGF to the VEGF receptors of bovine aortic arch derived endothelial cells.

Growth factors, receptor kinases, and protein tyrosine phosphatases in normal and malignant melanocytes

Summary: Normal human melanocyte proliferation and differentiation is dependent on stimulation of one of three growth factor/receptor systems. They are fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and mast cell growth factor (MGF), which activate the FGF receptor, c-Met, and c-Kit, respectively, known to be receptor tyrosine kinases. In contrast, human melanoma cells from primary nodular and metastatic lesions grow autonomously partially because of inappropriate production of basic FGF (bFGF) and continuous activation of the bFGF-receptor kinase. Activation of transmembrane receptor tyrosine kinases in melanocytes stimulates not only proliferation but also the expression of pigmentation. Melanoma cells constitutively express several tyrosyl-phosphorylated proteins that in normal melanocytes are stimulated in response to growth factors. This high level of phosphorylation was not due to either the presence of constitutively active Kit kinase and Met kinase nor to the absence of any of several known protein tyrosine phosphatases. Because bFGF by itself does not transform melanocytes to melanomas, there must be additional cooperating factors that confer the malignant phenotype to pigment cells.

Prolactin inhibits hCG-stimulated steroidogenesis and cAMP accumulation, possibly by increasing phosphodiesterase activity, in rat granulosa cell cultures.

The effects of prolactin (PRL), alone and together with human chorionic gonadotropin (hCG), on steroidogenesis and cAMP accumulation in the preovulatory ovary were studied. Cultured granulosa cells obtained from large preovulatory follicles of pregnant mare serum gonadotropin (PMSG)-treated immature rats were used. The results indicated that PRL inhibited, in a dose-dependent manner, hCG-induced cAMP accumulation and 17 beta-estradiol (E2) secretion. When added to 0.4 IU/ml hCG (designated as 100% activity), 1, 10 and 100 ng/ml PRL decreased cAMP accumulation to 86, 64 and 59%, respectively, following 1 h incubation and to 87, 81 and 66% E2 secretion, respectively, following 48 h incubation. PRL alone failed to cause any significant change in cAMP or E2 concentrations. The inhibition of PRL was apparently not at the hCG receptor level, since a similar inhibitory effect was observed in prostaglandin E1 (PGE1)-induced cAMP accumulation. Nor was the inhibitory pathway of adenylate cyclase involved, since pertussis toxin--an inactivator of the Gi regulatory protein--failed to abolish the suppressive effect of PRL on hCG-induced cAMP accumulation. The phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methyl-xanthine, abolished the inhibitory effect of PRL on hCG- and PGE1-induced cAMP accumulation and on hCG-induced E2 secretion, indicating that PRL might be inhibiting cAMP accumulation and steroidogenesis in preovulatory granulosa cells by enhancement of PDE activity.

Effects of prolactin on steroidogenesis and cAMP accumulation in rat luteal cell cultures

The effects of prolactin (PRL), alone and as a modulator of human chorionic gonadotropin (hCG) action, on steroidogenesis and cAMP accumulation in rat luteal cell cultures were examined. Cultured rat luteal cells were prepared from immature rats primed with pregnant mare serum gonadotropin and hCG. In vitro treatment was performed with 0.1 and 0.2 IU/ml hCG and 1-100 ng/ml PRL. Cultures were incubated for 48 h for evaluation of progesterone (P4) secretion and for 1 h for measurement of cAMP accumulation. The same doses of hormones and incubation periods were also used in preovulatory rat granulosa cell cultures and found to cause a significant, dose-dependent inhibition in estradiol, P4 and cAMP accumulation. In luteal cell cultures, on the other hand, P4 secretion was significantly elevated, in a dose-dependent manner, by PRL. Moreover, identical doses of PRL caused a significant, dose-dependent stimulation of cAMP accumulation. Basal levels of P4 were also significantly elevated by PRL alone, but no such stimulation by PRL was detected in basal levels of cAMP. Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, increased the stimulation of P4 and cAMP by hCG + PRL in a manner dependent on PRL concentrations. The overall data therefore demonstrate divergent effects of PRL on cAMP accumulation and steroidogenesis in the ovary: inhibitory in the preovulatory and stimulatory in the postovulatory state. Moreover, these findings suggest a possible common mechanism linking the effects of PRL before and after ovulation: inhibition of cAMP accumulation via enhanced breakdown of the nucleotide.

Increased Progesterone Secretion and 3β-Hydroxysteroid Dehydrogenase Activity in Human Cumulus Cells by Pregnenolone Is Limited to the High Steroidogenic Active Cumuli

AbstractPurpose: Several reports imply that lower progesteronesecretion by cumulus-oocyte complexes (COCs) isassociated with lower fertilization in the corresponding oocyte.The possible role of progesterone in oocyte fertilization inhumans was studied using two approaches: (a) increasingthe total progesterone secretion by culturing more than oneCOC per dish; and (b) increasing the cumulus cell progesterone secretion by providing pregnenolone as a substrate. Methods: Mature COCs were cultured individually orcocultured in groups. Oocyte fertilization and progesteronesecretion were tested after 20 hr and 3 days in culture, respectively.The cumuli from individually plated COCs were cultured inthe absence of oocyte for an additional 3 days in order totest the effects of pregnenolone on progesterone secretionand the 3β-hydroxysteroid dehydrogenase (3β-HSD)activity. A comparable study with pregnenolone was performedon the corresponding granulosa-lutein cells. Results: Increasing the number of COC to two instead ofone led to a significant increase in both fertilization rateand progesterone secretion. The addition of pregnenoloneduring days 3–6 increased significantly both progesteronesecretion and 3β-HSD activity. Comparable results wereobserved in granulosa-lutein cells subjected to pregnenolonetreatment. Following the first 3 days culture, cumulus masseswere categorized as secreting high or low progesteronelevels. Adding pregnenolone had a greater effect on bothprogesterone secretion and 3β-HSD activity in thehigh-progesterone-secreting cumuli. Conclusions: Addition of pregnenolone increased progesterone secretion and 3β-HSD more efficiently in thehigher-progesterone-secreting cumuli. Coculture of two COCsinstead of one led to a higher fertilization rate and greaterprogesterone secretion.

Effect of androgen substrates on the steroidogenic pattern of cumulus cells: Correlation with cumulus culture morphology

Background: In previous studies, higher progesterone secretion was observed in mature versus immature cumulusoocyte complexes. In mature cumulus mass that become homogeneously spread in culture (type C/D) progesterone secretion was higher than in partially (type B) or totally (type A) aggregated morphology. In sharp contrast, estradiol-17β secretion was significantly higher in type A than type C/D cumulus.Purpose: Our purpose was to assess whether the decreased estradiol-17β level in type C/D cumulus culture is caused by deficiency of substrates.Methods: The different cumulus types were incubated with or without 10−7 M dehydroepiandrosterone, 4-androstane-3, 17-dione, or testosterone. The levels of estradiol-17β, testosterone, and progesterone, were measured after 24 hr of culture.Results: The addition of dehydroepiandrosterone or 4-androstane-3,17-dione significantly increased the estradiol-17β levels in all types of cumulus cells, whereas the addition of testosterone was less effective. In all types of cumulus cells the testosterone levels increased significantly on adding these androgen substrates. In the type C/D cumulus, the testosterone increased to lower levels compared to type A cumulus cells. In the presence of these androgens progesterone secretion is significantly reduced in type A cumulus cells. In type C/D cumulus cells, however, progesterone levels were significantly higher than in type A. The estradiol-17β/testosterone and progesterone/estradiol-17β ratios, which partially resemble the degree of aromatase activity and the degree of selectivity for progesterone secretion, respectively, were higher in type C/D than in type A cumulus cells.Conclusions: In type C/D cumulus the significant increase in estradiol-17β secretion in the presence of various androgens suggests that, under basal conditions, androgen is less available for estradiol-17β biosynthesis compared to type A cumulus. Furthermore, the higher progesterone secretion in type C/D cumulus may suggest that the follicles yielding type C/D cumulus cells are more mature than the follicles yielding type A cumulus.