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.

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

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.

Altered steroidogenic activity of human granulosa-lutein cells at different cell densities in culture.

In the present study, the relationship between human granulosa-lutein cell (hGLC)-plating density and steroidogenic activity was evaluated. Increasing hGLC-plating density 32-fold, from 0.25 x 10(4) to 8 x 10(4) cells/well, was associated with a concomitant increase in the total amount of progesterone (P4), testosterone (T), and estradiol-17 beta (E2) secretion. The daily amount of each steroid (P4, T, and E2) secreted by hGLC at different cell-plating densities was further normalized per 10(3) cells. Thus, an increase in hGLC-plating density from 0.25 x 10(4) to 1 x 10(4) cells/well was associated with approximate increases of 1.3-fold in P4 and 3-fold in T and a 50% decrease in E2 secretion, per 10(3) cells. A further increase in hGLC-plating density, from 1 x 10(4) to 8 x 10(4) cells/well, was associated with a significant decrease of approximately 3.7-fold in P4 and 6-fold in T per 10(3) cells. A similar increase in hGLC-plating density was associated with no change or a 2-fold decrease, per 10(3) cells, in E2 secretion during days 0-3 or days 3-5 of culture, respectively. The P4/E2 ratio was increased and the E2/T ratio decreased with extension of the culture period. These two ratios had a tendency to be altered inversely, concurrent with the increase in cell-plating densities. At 1-2 x 10(4) cells/well, P4/E2 was maximal, whereas E2/T was minimal.(ABSTRACT TRUNCATED AT 250 WORDS)

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 3 beta-hydroxysteroid dehydrogenase activity in human cumulus cells by pregnenolone is limited to the high steroidogenic active cumuli.

PURPOSE: Several reports imply that lower progesterone secretion by cumulus-oocyte complexes (COCs) is associated with lower fertilization in the corresponding oocyte. The possible role of progesterone in oocyte fertilization in humans was studied using two approaches: (a) increasing the total progesterone secretion by culturing more than one COC per dish; and (b) increasing the cumulus cell progesterone secretion by providing pregnenolone as a substrate. METHODS: Mature COCs were cultured individually or cocultured in groups. Oocyte fertilization and progesterone secretion were tested after 20 hr and 3 days in culture, respectively. The cumuli from individually plated COCs were cultured in the absence of oocyte for an additional 3 days in order to test the effects of pregnenolone on progesterone secretion and the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. A comparable study with pregnenolone was performed on the corresponding granulosa-lutein cells. RESULTS: Increasing the number of COC to two instead of one led to a significant increase in both fertilization rate and progesterone secretion. The addition of pregnenolone during days 3-6 increased significantly both progesterone secretion and 3 beta-HSD activity. Comparable results were observed in granulosa-lutein cells subjected to pregnenolone treatment. Following the first 3 days culture, cumulus masses were categorized as secreting high or low progesterone levels. Adding pregnenolone had a greater effect on both progesterone secretion and 3 beta-HSD activity in the high-progesterone-secreting cumuli. CONCLUSIONS: Addition of pregnenolone increased progesterone secretion and 3 beta-HSD more efficiently in the higher-progesterone-secreting cumuli. Coculture of two COCs instead of one led to a higher fertilization rate and greater progesterone secretion.

Effects of prolactin on the morphology of cultured rat granulosa cells.

Morphological changes were correlated with biochemical data induced by prolactin (PRL) in cultured rat granulosa cells from large preovulatory follicles. Biochemical results indicated that PRL exerted a significant dose-dependent inhibition in gonadotrophin-induced secretion of progesterone and 17 beta-oestradiol. PRL alone failed to affect basal steroidogenic secretion. In parallel morphological experiments, using phase-contrast microscopy, untreated and 100 ng/ml PRL-treated cells appeared as a monolayer of flattened, fibroblast-like cells. Upon exposure to 0.4 IU/ml human chorionic gonadotrophin (hCG), aggregates of rounded, epithelioid-shaped cells were formed. The addition of PRL to hCG in the same doses minimized the changes induced by hCG. Similarly, electron microscopy of untreated and PRL-treated cultures revealed flat cells devoid of microvilli, with evenly dispersed microfilaments. The addition of hCG caused rounding of the cells and was accompanied by the appearance of microvilli and by pronounced steroid-producing organelles. Bundles of microfilaments were noted at the cell periphery. PRL added to hCG caused a reduction of the hCG effects, and the cell morphology was intermediate to that seen in untreated and hCG-treated cultures. The finding that PRL can prevent or minimize morphological changes caused by hCG in rat cultured granulosa cells correlates with the biochemical changes induced by PRL, and supports the concept that PRL is a modulator of gonadotrophic action in the ovary.

Different morphological and steroidogenic patterns in oocyte/cumulus-corona cell complexes aspirated at in vitro fertilization.

We attempted to correlate distinct morphological data on cumulus cells to oocyte and cumulus cell activity. Oocyte/cumulus-corona cell complexes, which were mature 4 h after aspiration, were divided into four subgroups designated according to the type of cumulus culture morphology after 3 days of culture: type A, compact clumps; type B, partially spread clumps; type C, nonhomogeneously spread cells; and type D, homogeneously spread cells. Fertilization and cleavage rates of mature oocytes appeared to differ according to their prospective cumulus culture morphology. Fertilization and cleavage rates were 81.5 and 62.6%, respectively, in oocyte/cumulus-corona cell complexes yielding type D cumulus cells, versus 54 and 34%, respectively, in those yielding type A cumulus cells. Basal secretion of progesterone in type A cumulus cells was 105.2 +/- 10.3 ng/ml compared to 231.8 +/- 22.5 ng/ml in type D cumulus cells (p less than 0.001). Testosterone and estradiol secretion exhibited a significant difference as well: testosterone was 293 +/- 10 pg/ml in type A cumulus cells versus 224 +/- 11 pg/ml in type D cumulus cells (p less than 0.001), and estradiol was 4.6 +/- 0.4 ng/ml in type A cumulus cells versus 3.5 +/- 0.3 ng/ml in type D cumulus cells (p less than 0.05). The present study demonstrated by indirect means that oocyte/cumulus-corona cell complexes, characterized as mature a few hours after aspiration, are composed of a heterogeneous population and differ in their potential for fertilization and consequent cleavage.

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-17 beta secretion was significantly higher in type A than type C/D cumulus. PURPOSE: Our purpose was to assess whether the decreased estradiol-17 beta 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-17 beta, 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-17 beta 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-17 beta/ testosterone and progesterone/estradiol-17 beta 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-17 beta secretion in the presence of various androgens suggests that, under basal conditions, androgen is less available for estradiol-17 beta 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.

Altered steroidogenic pattern of human granulosa-lutein cells in relation to cumulus cell culture morphology.

It has been reported that human-oocyte complexes (COC) retrieved at a stimulated cycle manifest an asynchrony between oocyte meiotic maturation and cumulus mucification. However, when mature COC were subdivided into subtypes marked by the culture morphology of their cumulus cells following 3 days' culture, successful fertilization and cleavage were approximately 1.5-fold lower in mature COC yielding cumulus cells aggregated into clumps (type A and B COC) than in mature COC yielding homogeneously spread cells (type C-D COC). To determine whether the existing relationship between cumulus culture morphology and oocyte functionality in the various COC types (A-D) could be extended to another follicular compartment--the granulosa-lutein (G-L) cells--basal steroid secretion by the corresponding G-L cells was evaluated within 5 days of culture. Over the first 3 days of culture, secretion of progesterone was 3-fold lower and secretion of testosterone (T) was 2.5-fold higher in cultures of G-L cells from follicles yielding type A COC than in type C-D COC. During days 4 and 5 of culture, G-L cells were incubated with or without 10(-7) M 3 beta-hydroxy-5-pregnen-20-one (pregnenolone), dehydroepiandrosterone (DHA), 4-androstene-3,17-dione (androstenedione), or T. The pattern of progesterone level noted over the first 3 days of culture was not altered in the presence of pregnanolone. DHA, androstenedione, or T. Addition of pregnenolone, DHA, androstenedione, or T increased T level 2.5-, 5.6-, 7.3-, and 17.7-fold, respectively, in cultures of G-L cells from follicles yielding type A COC, but did not significantly alter T level in cultures of G-L cells from follicles yielding type C-D COC. In cultures of G-L cells from follicles yielding type A COC, addition of androgens unsaturated at position 4 preferentially increased oestradiol-17 beta (E2) level, whereas in cultures of G-L cells of type C-D COC, DHA and androstenedione preferentially increased E2 level. Taken together, the asynchrony between oocyte and cumulus activity could be diminished when the activity of various follicular cell compartments is evaluated according to cumulus culture morphology rather than cumulus expansion and mucification. The present study suggests that follicles yielding mature COC represent a non-homogenous population in which G-L cells from follicles yielding type A-B COC manifest a less luteinized state than those from follicles yielding type C-D COC.

The binding of vascular endothelial growth factor to its receptors is dependent on cell surface-associated heparin-like molecules.

Vascular endothelial growth factor (VEGF) induces the proliferation of endothelial cells and is a potent angiogenic factor that binds to heparin. We have therefore studied the effect of heparin upon the interaction of VEGF with its receptors. Heparin, at concentrations ranging from 0.1 to 10 micrograms/ml, strongly potentiated the binding of 125I-VEGF to its receptors on endothelial cells. Scatchard analysis of 125I-VEGF binding indicates that 1 microgram/ml heparin induces an 8-fold increase in the apparent density of high affinity binding sites for VEGF, but does not significantly affect the dissociation constant of VEGF. Cross-linking experiments showed that heparin strongly potentiates the formation of the 170-, 195- and 225-kDa 125I-VEGF-receptor complexes on endothelial cells. At high 125I-VEGF concentrations (4 ng/ml), heparin preferentially enhanced the formation of the 170- and 195-kDa complexes. Preincubation of the cells with heparin, followed by extensive washes, produced a similar enhancement of subsequent 125I-VEGF binding. The binding of 125I-VEGF was completely inhibited following digestion of endothelial cells with heparinase and could be restored by the addition of exogenous heparin to the digested cells. The enhancing effect of heparin facilitated the detection of VEGF receptors on cell types that were not known previously to express such receptors. Our results suggest that cell surface-associated heparin-like molecules are required for the interaction of VEGF with its cell surface receptors.

Human melanoma cells but not normal melanocytes express vascular endothelial growth factor receptors.

Vascular endothelial growth factor (VEGF) is a specific mitogen for endothelial cells in vitro and an angiogenic factor in vivo. Its role in other cell types is not yet clear. To explore its possible involvement in malignant transformation, we studied the expression of its receptors in normal and malignant melanocytes. Binding and cross-linking experiments showed that human melanoma cells but not normal melanocytes express VEGF receptors. Separation of reaction products by SDS-PAGE demonstrated the presence of 125I-VEGF/receptor complexes of 180 and 165 kDa in the melanoma cells. A diffuse complex with a mass of approximately 235 kDa was also detected in some experiments. Heparin enhanced the binding of the radioactive ligand to the receptors of the WW94 and SW1614 melanoma cell lines. This binding was completely abolished by heparinase digestion and was restored by the addition of exogenous heparin, indicating that heparin-like molecules are necessary for ligand/receptor interaction. This study suggests that the aberrant expression of VEGF receptors is one of the phenotypic changes occurring in melanoma cells during malignant transformation.

Release of cell surface-associated basic fibroblast growth factor by glycosylphosphatidylinositol-specific phospholipase C.

Heparan sulfate proteoglycans (HSPG) are ubiquitous constituents of mammalian cell surfaces and most extracellular matrices. A portion of the cell surface HSPG is anchored via a covalently linked glycosyl-phosphatidylinositol (Pl) residue, which can be released by treatment with a glycosyl-Pl specific phospholipase C (Pl-PLC). We report that exposure of bovine aortic endothelial and smooth muscle cells to Pl-PLC resulted in release of cell surface-associated, growth-promoting activity that was neutralized by antibasic fibroblast growth factor (bFGF) antibodies. Active bFGF was also released by treating the cells with bacterial heparitinase. Under the same conditions there was no release of mitogenic activity from cells (BHK-21, NIH/3T3, PF-HR9) that expressed little or no bFGF, as opposed to Pl-PLC-mediated release of active bFGF from the same cells transfected with the bFGF gene. The released bFGF competed with recombinant bFGF in a radioreceptor assay. Addition of Pl-PLC to sparsely seeded vascular endothelial cells resulted in a marked stimulation of cell proliferation, but there was no mitogenic effect of Pl-PLC on 3T3 fibroblasts. Studies with exogenously added 125I-bFGF revealed that about 6.5% and 20% of the cell surface-bound bFGF were released by treatment with Pl-PLC and heparitinase, respectively. Both enzymes also released sulfate-labeled heparan sulfate from metabolically labeled 3T3 fibroblasts. Pl-PLC failed to release 125I-bFGF from the subendothelial extracellular matrix (ECM), as compared to release of 60% of the ECM-bound bFGF by heparitinase. Our results indicate that 3-8% of the total cellular content of bFGF is associated with glycosyl-Pl anchored cell surface HSPG. This FGF may exert both autocrine and paracrine effects, provided that it is released by Pl-PLC and adequately presented to high affinity bFGF cell surface receptor sites.

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.

Transforming growth factor beta modulates gonadotropin receptor expression in porcine and rat granulosa cells differently.

Almost without exception, the studies to date describing the effects of transforming growth factor beta (TGF beta) upon various ovarian cell types have utilized the subtype TGF beta 1. Since TGF beta 1 and TGF beta 2 have been demonstrated to influence major developmental processes differentially during hematopoiesis and embryogenesis, we investigated in two species (rat and pig) whether they might also differentially modulate principal regulatory processes of ovarian cellular differentiation, specifically FSH and/or LH receptor (FSHR, LHR) expression. TGF beta 1 plus FSH significantly stimulated LHR binding levels by cultured granulosa cells (GC) from prepubertal diethylstilbestrol-treated rats. TGF beta 2 produced the same effect. In porcine GC from 1-3-mm antral follicles, TGF beta 1 and TGF beta 2 again acted similarly, but the direction of the response was opposite to that in the rat GC system. This difference could not be ascribed to the fact that the GC utilized represented different stages of follicular development in vivo because TGF beta 1 also potentiated FSH-dependent LHR induction in GC from antral follicles of cycling rats at all stages of the estrous cycle. The major effect of TGF beta 1 on FSHR expression in the rat system was to increase binding by attenuating the down-regulatory action of cholera toxin (CTX) or FSH. In the porcine system, TGF beta reduced FSHR binding at FSH or CTX concentrations that enhanced expression, and it did not attenuate the down-regulatory effect of FSH or CTX at higher doses. In summary, TGF beta up- or down-regulated LHR and FSHR binding coordinately within species.(ABSTRACT TRUNCATED AT 250 WORDS)

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 KDR/flk-1 genes were found to function as VEGF165 receptors.

VEGF121, a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells.

Four vascular endothelial growth factor (VEGF) splice variants containing 121, 165, 189, and 206 amino acids are produced from a single human gene as a result of alternative splicing. VEGF121 is not a heparin-binding protein, while the other VEGF species possess heparin binding ability. YU-ZAZ6 human melanoma cells expressed the mRNA encoding the VEGF receptor flt-1, but not the mRNA encoding the VEGF receptor KDR/flk-1. Both VEGF121 and VEGF165 bound to the VEGF receptors of these cells. Unexpectedly, heparin inhibited the binding of VEGF121 as well as the binding of VEGF165 to the VEGF receptors of the melanoma cells. Digestion of the cells with heparinase also inhibited the binding of both VEGF variants. The VEGF165 binding ability of heparinase-digested cells could be partially restored by the addition of exogenous heparin to the binding reaction. In contrast, the addition of heparin to heparinase-digested cells did not restore VEGF121 binding. These results suggest that cell-surface heparan sulfates may regulate the binding ability of the VEGF receptors of the melanoma cells. They also indicate that heparin is not able to fully substitute for cell surface-associated heparan sulfates since VEGF121 binding to the VEGF receptors of heparinase-treated cells is not restored by heparin. These data suggest that changes in the composition of cell-surface heparin-like molecules may differentially affect the interaction of various VEGF isoforms with VEGF receptors.

Selective binding of VEGF121 to one of the three vascular endothelial growth factor receptors of vascular endothelial cells.

VEGF121 and VEGF165 are vascular endothelial growth factor splice variants that promote the proliferation of endothelial cells and angiogenesis. VEGF165 contains the 44 additional amino acids encoded by exon 7 of the VEGF gene. These amino acids confer upon VEGF165 a heparin binding capability which VEGF121 lacks. 125I-VEGF165 bound to three vascular endothelial growth factor (VEGF) receptors on endothelial cells, while 125I-VEGF121 bound selectively only to the flk-1 VEGF receptor which corresponds to the larger of the three VEGF receptors. The binding of 125I-VEGF121 to flk-1 was not affected by the removal of cell surface heparan sulfates or by heparin. Both VEGF165 and VEGF121 inhibited the binding of 125I-VEGF121 to a soluble extracellular domain of the flk-1 VEGF receptor in the absence of heparin. However, heparin potentiated the inhibitory effect of VEGF165 by 2-3-fold. These results contrast with previous observations which have indicated that the binding of 125I-VEGF165 to the flk-1 receptor is strongly dependent on heparin-like molecules. Further experiments showed that the receptor binding ability of VEGF165 is susceptible to oxidative damage caused by oxidants such as H2O2 or chloramine-T. VEGF121 was also damaged by oxidants but to a lesser extent. Heparin or cell surface heparan sulfates restored the flk-1 binding ability of damaged VEGF165 but not the receptor binding ability of damaged VEGF121. 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 VEGF165 may enable the restoration of damaged VEGF165 function in processes such as inflammation or wound healing.

Platelet factor-4 inhibits the mitogenic activity of VEGF121 and VEGF165 using several concurrent mechanisms.

The 121-amino acid form of vascular endothelial growth factor (VEGF121) and the 165-amino acid form (VEGF165) are mitogenic for vascular endothelial cells and induce angiogenesis in vivo. VEGF165 possesses a heparin binding ability and in the absence of heparin-like molecules does not bind efficiently to the VEGF receptors of vascular endothelial cells. The binding of 125I-VEGF165 to the VEGF receptors of endothelial cells, and the heparin-dependent binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1, were inhibited by the angiogenesis inhibitor platelet factor-4 (PF4). In contrast, PF4 was not able to inhibit the binding of VEGF121, a VEGF isoform which lacks a heparin binding capacity, to the VEGF receptors of the cells or to KDR/flk-1. These results indicate that PF4 may inhibit VEGF165 binding to VEGF receptors by disrupting the interaction of VEGF165 with cell surface heparan sulfates. Since PF4 mutants lacking a heparin binding ability retain their anti-angiogenic activity, alternative inhibitory mechanisms were also examined. 125I-PF4 bound with high affinity (Kd 5 x 10(-9) M) to VEGF165-coated wells. The binding of 125I-PF4 to the VEGF165-coated wells was inhibited by several types of heparin binding proteins, including unlabeled PF4 and unlabeled VEGF165. The binding was not inhibited by proteins which lack a heparin binding capacity, nor was it inhibited by VEGF121. Heparinase did not inhibit the binding of 125I-PF4 to VEGF165, indicating that heparin-like molecules are not required. These experiments suggest that PF4 can bind to heparin binding proteins such as VEGF165 leading to an inhibition of their receptor binding ability. In agreement with these results, we have observed that PF4 inhibits efficiently the VEGF165 induced proliferation of vascular endothelial cells. Unexpectedly, PF4 also inhibited efficiently the VEGF121-induced proliferation of the cells, indicating that PF4 can disrupt VEGF receptor mediated signal transduction using an unknown mechanism which does not interfere with VEGF121 binding.