Human melanoma cells inhibit the earliest differentiation steps of human Langerhans cell precursors but failed to affect the functional maturation of epidermal Langerhans cells.

Tumour-derived factors suppress differentiation and function of in vitro generated DC. Here, we investigate the effect of two melanoma clones differing in their invasive and metastatic properties on the generation and/or functional maturation of human epidermal LC. LC were generated from CD34(+) cord blood progenitors under GM-CSF/TNF-alpha/TGF-beta 1. CD34(+) cells were co-cultured with or without melanoma cells using Transwell dishes. After 11 days of co-culture, CD34(+)-derived cells display a non-adherent undifferentiated morphology, a high level of monocytic CD14 marker, a down-regulated expression of LC markers (CD1a, E-cadherin) and DC markers (CD40, CD80, CD54, CD58, CD83, CD86, HLA-DR, HLA-class I). These cells were less potent than control LC in inducing allogeneic T cell proliferation. The generation of the CD14(+) population was correlated with a decrease in the CD1a(+) population, without any statistical differences between the two clones. Melanoma cells diverted the differentiation of CD34(+) cells towards a dominant CD14(+) population only if the progenitors were in an early growth phase. IL-10, TGF-beta 1 and VEGF were not responsible for these effects, as assessed by using blocking antibodies. By contrast, co-culture of fresh epidermal LC with melanoma cells did not affect their phenotype and function. Our data demonstrate that melanoma cells inhibit the earliest steps of LC differentiation, but failed to affect the functional maturation of epidermal LC. This suggests that melanoma cells participate in their own escape from immunosurveillance by preventing LC generation in the local cutaneous microenvironment.

Erythroblasts are a source of angiogenic factors.

In adult bone marrow, mature erythroblasts are produced within structures called erythroblastic islands and then cross the endothelial barrier to reach circulation. Erythroblastic islands are composed of a central macrophage surrounded by maturing erythroblasts. In this study, it is shown that erythroid cells, but not the other mature hematopoietic cells, coexpress 2 angiogenic factors, vascular endothelial growth factor A (VEGF-A) and placenta growth factor (PlGF). Secretion of both VEGF-A and PlGF increases during in vitro erythroid differentiation. Erythroblast-conditioned medium can induce both migration of monocytes and endothelial cells and the permeability of endothelial cells. These effects are inhibited by anti-PlGF and/or anti-VEGF antibodies. Finally, it is shown that VEGF-A and PlGF proteins are expressed by bone marrow erythroblasts in vivo. Angiogenic factors secreted by erythroblasts may promote interactions either with macrophages in erythroblastic islands or with endothelial cells that would facilitate the passage of erythroid cells through the endothelial barrier. (Blood. 2001;97:1968-1974)

Adsorption of vascular endothelial growth factor to two different apatitic materials and its release.

The aim of our study was to assess the ability of calcium phosphate powders to serve as growth factor carriers. Vascular endothelial growth factor (VEGF), in particular, is locally involved in the bone formation process throughout osteoblast differentiation. Two different apatitic substrates were tested: hydroxyapatite (HA), widely used as biomaterial, and nanocrystalline carbonated apatite (CA), which has a composition similar to bone mineral crystals. These materials have been compared for their VEGF adsorption and release properties. The adsorption of the growth factor was higher on CA than on HA probably due to differences of both the proteins and the powders involved. The specific activity of the VEGF released was also tested to determine the available activity for cells in contact with these materials. Interestingly, the bioactivity of the VEGF released from CA quantified on fetal bovine aortic endothelial cells (FBAE) by evaluating the proliferation activity, exhibited no marked difference compared to native VEGF. Qualitatively, VEGF adsorbed on CA material induced well-defined collagen type I immunostaining on osteoblast cells compared to the staining obtained after VEGF adsorption on HA.

[Anti-angiogenesis strategies in cancer]. / Stratégies anti-angiogéniques en cancérologie.

It is generally accepted that tumor development requires the secretion by cancer cells of soluble mediators which activate the formation of new vessels, called "Tumor Angiogenic Factors". The intense quest for identifying these factors has recently been confirmed by strong experimental data. The discovery in 1989 of the Vascular Endothelial Growth Factor VEGF and of new proteases had led to the identification of the key actors of tumor angiogenesis. The elucidation of their mechanisms of action allowed to design new therapeutic strategies already confirmed by preclinical trials. There are now almost twenty molecules which are under clinical investigation in man.

The mitogenic effect of 17beta-estradiol on in vitro endothelial cell proliferation and on in vivo reendothelialization are both dependent on vascular endothelial growth factor.

In addition to their actions on reproductive function, estrogens have important effects on endothelial cells. The present study was designed to evaluate the mechanism(s) by which 17beta-estradiol (E2) promotes endothelial cell proliferation. The potential involvement of vascular endothelial growth factor (VEGF) was investigated by the coadministration of polyclonal anti-VEGF antibody. First, the effect of E2 on the proliferation of cultured foetal bovine aortic endothelial cells (FBAEC) was studied. E2 stimulated this proliferation with an EC50 between 10(-11) and 10(-10) M and this effect was inhibited by the anti-VEGF antibody. The effect of a physiological dose of E2 was then studied in the rat model of carotid injury. After deendothelializing balloon injury, reendothelialization of the denuded surface may influence the growth of the underlying smooth muscle cells. Male Sprague-Dawley rats were castrated and then received E2 from subcutaneously implanted pellets that released 3.2 microg/kg/day. Endothelial regrowth (Evans blue staining) and neointimal thickening were evaluated 2 weeks after the carotid injury. In comparison to the placebo group, E2 increased the extent of reendothelialization (p = 0.0002) and reduced neointimal thickening (p = 0.0007). Anti-VEGF antibody abolished the effect of E2 on reendothelialization as well as on neointimal thickening. Thoracic aorta VEGF content was increased in E2-treated rats compared to control rats. In conclusion, the present study demonstrates that E2 increases endothelial cell proliferation in vitro and reendothelialization in vivo by means of a mechanism dependent on endogenous VEGF. This effect could contribute to the antiatherogenic effect of a physiological dose of E2.

Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis.

Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.

Neuropilin-1 is expressed on bone marrow stromal cells: a novel interaction with hematopoietic cells?

In adult bone marrow, hematopoietic stem cells are found in close association with distinctive stromal cell elements. This association is necessary for maintenance of hematopoiesis, but the precise mechanisms underlying the cross-talk between stromal cells and hematopoietic stem cells are poorly understood. In this study, we used a bone marrow stromal cell line (MS-5) that is able to support human long-term hematopoiesis. This hematopoietic-promoting activity cannot be related to expression of known cytokines and is abolished by addition of hydrocortisone. Using a gene trap strategy that selects genes encoding transmembrane or secreted proteins expressed by MS-5 cells, we obtained several insertions that produced fusion proteins. In one clone, fusion protein activity was downregulated in the presence of hydrocortisone, and we show that insertion of the trap vector has occurred into the neuropilin-1 gene. Neuropilin-1 is expressed in MS-5 cells, in other hematopoietic-supporting cell lines, and in primary stromal cells but not in primitive hematopoietic cells. We show that neuropilin-1 acts as a functional cell-surface receptor in MS-5 cells. Two neuropilin-1 ligands, semaphorin III and VEGF 165, can bind to these cells, and the addition of VEGF 165 to MS-5 cells increases expression of 2 cytokines known to regulate early hematopoiesis, Tpo and Flt3-L. Finally, we show that stromal cells and immature hematopoietic cells express different neuropilin-1 ligands. We propose that neuropilin-1 may act as a novel receptor on stromal cells by mediating interactions between stroma and primitive hematopoietic cells.

Inhibition of in vitro angiogenesis by platelet factor-4-derived peptides and mechanism of action.

In this study, we examined in detail the interaction of platelet factor-4 (PF-4) with fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) and the effect of PF-4-derived synthetic peptides. We show that a peptide between amino acids 47 and 70 that contains the heparin-binding lysine-rich site inhibits FGF-2 or VEGF function. This is based on the following observations: PF-4 peptide 47-70 inhibited FGF-2 or VEGF binding to endothelial cells; it inhibited FGF-2 or VEGF binding to FGFRs or VEGFRs in heparan sulfate-deficient CHO cells transfected with FGFR1 (CHOFGFR1) or VEGFR2 (CHOmVEGFR2) cDNA; it blocked proliferation or tube formation in three-dimensional angiogenesis assays; and, finally, it competed with the direct association of (125)I-PF-4 with FGF-2 or VEGF, respectively, and inhibited heparin-induced FGF-2 dimerization. A shorter C-terminal peptide (peptide 58-70), which still contained the heparin-binding lysin-rich site, had no effect. Peptide 17-58, which is located in the central part of the molecule, although it does not inhibit FGF-2 or VEGF binding or biologic activity in endothelial cells, inhibited heparin-dependent binding of (125)I-FGF-2 or (125)I-VEGF to CHOmFGFR1 or CHOmVEGFR2 cells, respectively. Shorter peptides (peptides 34-58 and 47-58) did not show any of these effects.

[Impact on tumor angiogenesis and tumor progression of expression of the 18 kd and 24 kd isoforms of FGF-2]. / Impact sur l'angiogenèse tumorale et la progression tumorale de l'expression des isoformes 18 kD et 24 kD du FGF-2.

The role of FGF-2 in tumor progression and tumor cell invasiveness was investigated using the rat bladder carcinoma cells NBT-II, which do not constitutively express FGF-2 or its membrane-spanning receptor. The NBT-II cells were transfected using expression vectors encoding either the 18 kD or the 24 kD isoform of FGF-2. The 24 kD isoform contains a nuclear localization signal. The transfected NBT-II cells that expressed 18 kD FGF-2 produced and secreted this factor as the biologically active form and retained an epithelial morphology. When injected to nude mice, the tumorigenic potential of these cells was not increased over that of non-transfected NBT-II cells; however, although the time to tumor development was long, the tumors were highly vascularized, indicating secretion of the angiogenic factor FGF-2. The transfected NBT-II cells that expressed 24 kD FGF-2 varied in their morphological appearance and did not secrete FGF-2; immunofluorescence and Western-blot studies showed that the FGF-2 was mainly intranuclear. When injected to nude mice, these cells produced tumors and migrated not only to the lymph nodes but also to the lungs where they produced metastases. In aggregate, these data indicate that stimulation of angiogenesis is not sufficient to increase tumor growth and that nuclear FGF-2 acts as a tumorigenic and metastasis-promoting factor in the NBT-II carcinoma model.

Signal relays in the VEGF system.

A considerable progress has been made during the past years in elucidating the molecular actors of angiogenesis. Vascular endothelial growth factor turned out to represent the major inducer of angiogenesis. Optional splicing of its pre messenger RNA generates various isoforms which differ not only by their storage in the extracellular matrix but also by their signaling pathways. VEGF binds and activates two tyrosine kinase receptors called VEGFR1 and VEGFR2 and neuropilin-1. The elucidation of the transduction pathways of each receptor suggests that VEGFR1 mediates cell migration whereas VEGFR2 mediates cell proliferation. The construction of internal images of VEGF by the anti-idiotypic antibody strategy allowed us to determine that quiescent endothelial cells need to be activated by so far unknown factors to become competent to respond to mitogenic signals and acquire an angiogenic phenotype. The discovery of the mechanisms of action of the VEGF system has allowed the design of promising drugs which already entered the pre-clinical or clinical assays.

Control of vascular endothelial growth factor angiogenic activity by the extracellular matrix.

In adult vessels the proliferation rate of differentiated endothelial cells is very low. In response to several environmental stimuli the expression of so-called 'angiogenic factors' is upregulated and the messenger RNAs are actively translated in secreted factors which induce the proliferation of endothelial cells; the digestion of their basement membrane then allows their migration and differentiation. Considerable progress has been made during the past years in elucidating the molecular actors of angiogenesis. Vascular endothelial growth factor turned out to represent the major inducer of angiogenesis. Optional splicing of its pre-messenger RNA generates various isoforms which differ not only by their storage in the extracellular matrix but also by their signaling pathways.

Vascular endothelial growth factor is up-regulated in vitro and in vivo by androgens.

Evidence from pathophysiological studies support the concept that embryonic development, tumor progression, and hormonally-regulated tissue masses such as adult prostate and corpus luteum are angiogenesis-dependent. We examined if the prostatic expression of vascular endothelial growth factor (VEGF), the major regulator of normal and pathological angiogenesis, was regulated by testosterone. Northern blot of VEGF messenger ribonucleic acid (mRNA) extracted from a human immortalized epithelial prostatic cell line (PNT1) showed that dihydrotestosterone (DHT) up-regulated VEGF mRNA at a level comparable to that observed upon exposure to growth factors. Polymerase chain reaction of reverse transcribed mRNA demonstrated that the ratio of the two splice variants encoding the 121 and 165 isoforms of VEGF were not affected by DHT. VEGF biological activity, measured in the conditioned medium by radio receptor assay, was increased by DHT. Injection of testosterone in adult rats induced a transient increase of the ventral lobe weight and the specific activity of prostatic VEGF, leading to a 7-fold increase in the prostate content of VEGF.

Systemic activation of the vascular endothelial growth factor receptor KDR/flk-1 selectively triggers endothelial cells with an angiogenic phenotype.

The hypothesis that tumor growth is angiogenesis dependent has been documented by a considerable body of direct and indirect experimental data. A prerequisite for the development of novel anti-angiogenic agents is the design of drugs that would be active only on those endothelial cells with an angiogenic phenotype. We took advantage of the anti-idiotypic strategy to obtain circulating agonists specific for the vascular endothelial growth factor receptor KDR/flk-1 (J-IgG). They induced in the absence of VEGF cell proliferation in vitro and angiogenesis in the corneal pocket assay either through local or systemic delivery. Intraperitoneal injections of J-IgG in nude mice grafted with a prostatic adenocarcinoma led to tumor enlargement associated with an increase in both tumor vascularization and proliferation. In contrast KDR/flk-1 overstimulation had no detectable effect on normal tissues. These data underline that KDR/flk-1 is a functional marker of the angiogenic phenotype of endothelial cells.

Cell release of bioactive fibroblast growth factor 2 by exon 6-encoded sequence of vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor which is synthesized and secreted by many differentiated cells in response to various stimuli including hypoxia and growth factor exposure. Alternative splicing of vascular endothelial growth factor mRNA results in three distinct molecular forms: V189 and V165 or V121 which lack the exons 6 or 6 and 7, respectively. To clarify the functions of the 24-amino acid insertion, the biological activity of V165 was compared with that exerted by purified recombinant V189 and a synthetic peptide designed on the sequence encoded by exon 6 (Ex6P). V189 and Ex6P, but not V165, induced cell proliferation on corneal endothelial cells cultured in vitro. These effects were due to the release of fibroblast growth factor 2 (FGF2) stored in the extracellular matrix but not to direct interactions with FGF receptors since V189 was inefficient on heparan sulfate-deficient cells expressing constitutively FGF-R1. Moreover corneas incubated ex vivo with Ex6P solubilized 10-fold more FGF2 than a isocationic peptide containing a scrambled sequence. Ex6P elicited an angiogenic response in a corneal pocket assay which was totally inhibited by addition of anti-FGF2 IgG. Moreover the angiogenic response to V189, but not to V165, was inhibited by FGF2 immunoneutralization. These findings demonstrate that the presence of the exon 6-encoded sequence confers VEGF with the ability to exert its biological effects through FGF2 signaling pathways.

Extracellular cleavage of the vascular endothelial growth factor 189-amino acid form by urokinase is required for its mitogenic effect.

Alternative splicing of vascular endothelial growth factor (VEGF) mRNA results in three distinct molecular forms of 121 or 165 (V165) amino acids that are released in the conditioned medium of cultured cells and one longer isoform of 189 amino acids (V189) that remains cell-associated. V189 has been expressed in wild type CHO-K1 cells and in glycosaminoglycan-deficient pgsA-745 Chinese hamster ovary (CHO) mutant cells. It could be released from CHO-K1 cell membranes by heparin or a synthetic peptide designed on the sequence encoded by exon 6 but was freely released from CHO mutant cells. In both cases, the immunoreactive V189 was mainly released as a 40-kDa cleaved form, provided that the serine protease urokinase, but not plasmin, was active. Recombinant V189 was purified from insect cells infected with a recombinant baculovirus as a nonmitogenic 50-kDa precursor that binds to the receptor Flt-1 but not to Flk-1. It could be matured by urokinase as a 38-kDa fragment able to bind to Flk-1 and to trigger cell proliferation. V165 and V189, however, could be cleaved by plasmin as 34-kDa fragments that exhibit a decreased mitogenic activity. These findings indicate that the carboxyl-terminal domain of V189 masks its binding domain to Flk-1.

[Tumoral vascularization: physiopathology and therapeutic prospects]. / Vascularisation tumorale: physiopathologie et perspectives thérapeutiques.

The Folkman's hypothesis postulates that tumor or metastase development is angiogenesis dependent. Since 1971, several lines of experimental data have supported this hypothesis in particular during the past months. A better understanding of the functional balance existing between positive and negative regulators of endothelial cell proliferation has paved the way to new therapeutic prospects.

Vascular endothelial growth factor confers a growth advantage in vitro and in vivo to stromal cells cultured from neonatal hemangiomas.

Neonatal hemangioma is a common benign proliferation of unorganized structures containing stromal and capillary endothelial cells. We tested the hypothesis that such cell proliferation might result from the release by stromal cells of endothelial cell mitogens. Stromal cells cultured from biopsies of surgically removed life-threatening hemangiomas released an endothelial cell mitogen in vitro that was indistinguishable from vascular endothelial growth factor (VEGF) based on independent criteria such as affinity chromatography for heparin or anti-VEGF IgG and radioreceptor assay. A functional product of the KDR gene encoding a cognate VEGF receptor was also expressed by these stromal cells. Transient transfection with antisense oligonucleotides targeted on the translation initiation codon of KDR abolished its tyrosine phosphorylation and mitogenic response of neonatal hemangioma cells to VEGF, confirming the existence of an autocrine loop of proliferation. When grafted in nude mice, these stromal cells elicited an angiogenic response that was blocked by neutralizing anti-VEGF IgG. These results might provide a clue to the importance of stromal cells in the pathogeny of neonatal hemangiomas.