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* Department of Oncology and IIT Network Research Unit of Molecular Neurosciences, and
Cardiovascular Department, DIBIT, San Raffaele H Scientific Institute, Milan, Italy;
Department of Biomedicine, University of Bergen, Bergen, Norway; and
Institute of General Pathology and Center for Studies on Cellular Pathology of the CNR, Milan, Italy
2Correspondence: DIBIT, San Raffaele H Scientific Institute, Via Olgettina 60, 20132 Milan, Italy. E-mail: elisabetta.ferrero{at}hsr.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: ERK/MAPK • capillary-like structures
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The vascular endothelial growth factor (VEGF) is one of the key proangiogenic factors, inducing critical steps in angiogenesis and tissue remodeling, such as endothelial permeability, proliferation, migration, invasion, and organization (2
3
4)
.
The mechanism of endothelial activation by VEGF involves activation of extracellular signal-regulated kinases (ERKs) and mitogen activated protein kinases (MAPKs) (5)
. We have recently shown that a recombinant fragment of chromogranin A (CgA) corresponding to residues 1–78 (VS-1) can inhibit tumor necrosis factor 
(TNF) and VEGF-induced changes of endothelial cell shape and barrier function in human umbilical vein endothelial cells (HUVEC) (6)
. This fragment contains the CgA1–76 sequence, also called vasostatin-I (VS-I), because of its inhibitory properties on vascular tension (7)
. Recombinant VS-1 may also inhibit TNF-induced gap formation and phosphorylation of p38MAPK by a pertussis toxin-sensitive mechanism in bovine pulmonary arterial endothelial cells, implicating a role for VS-I in protection of endothelial integrity via a G-protein regulation of the stress-activated MAPK pathway (8)
.
CgA, the precursor of VS-I, is a well-established member of the granin family of genetically distinct and uniquely acidic proteins that are ubiquitous in secretory cells of the nervous, endocrine, and immune system (7)
. This protein serves as a precursor of several regulatory peptides endowed with endocrine, paracrine, and autocrine effects, among them VS-I (9
10
11
12
13)
. CgA occurs as a conspicuous component in plasma (14)
, markedly elevated in a wide range of neuroendocrine tumors (11
, 15)
and in hepatic and renal failure (16)
. Moreover, patients suffering from chronic heart failure have elevated plasma CgA that correlates with TNF and soluble TNF-receptors and severity of the disease (17
18)
. However, despite the persistent use of circulating CgA as a diagnostic and prognostic marker of neuroendocrine tumors (11)
, a functional relevance for the tumor-derived CgA and VS-I has yet to be clarified.
The aim of the present study has been to assess the effects of VS-I on VEGF-induced processes in HUVEC, such as activation of ERK phosphorylation, cellular proliferation, motility, migration, sprouting, invasion, and capillary-like structure formation in vitro and in vivo. For this purpose we have used the recombinant VS-1 as a model of the naturally derived VS-I (19)
. The present results demonstrate that VS-1 antagonizes a wide number of VEGF-driven functions in HUVEC, suggesting that tumor released CgA may, via VS-I, serve as a potentially antiangiogenic factor of relevance for tumor progression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and cultured in 1% gelatin-coated flasks (Falcon; Becton Dickinson, Bedford, MA, USA) using endotoxin-free Medium 199 (BioWhittaker, Cambrex Bio Science Verviers, Belgium), containing 20% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 1% bovine retinal-derived growth factor, 90 µg/ml heparin, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Biochrom, Berlin, Germany), complete medium (CM). All experiments were carried out with HUVEC at passage 1–4. ECV 304 human endothelial cell line (ATCC, Manassas, VA, USA), was grown in medium 199 supplemented with 10% FBS. EA.hy926 endothelial cell line (ATCC) was cultured in DMEM (BioWhittaker) supplemented with 10% FBS and with 1% HAT Media Supplement (Sigma-Aldrich, S.r.l, Milano, Italy).
Reagents and monoclonal antibodies
Recombinant STA-CgA1–78 (VS-1) was obtained by expression in E. coli and purified by reverse-phase HPLC using a SOURCE 15 RPC column (Pharmacia-Upjohn, Uppsala, Sweden) followed by gel-filtration chromatography on a Sephacryl S-200 HR column, as described previously (19)
. Human VEGF was purchased from PeproTech, INC. (Rocky Hill, NJ, USA). 4,6 diamidino-2-phenyindole dihydrochloride (DAPI) was from Sigma-Aldrich. The anti-CgA mAb (clone 5A8) was described previously (19)
. U-O126 ERK 1/2 inhibitor was from Calbiochem (EMD Chemicals Inc., Darmstadt, Germany).
ERK phosphorylation assay
Western blot analysis was performed as described (21)
, using the antiphospho-ERK mAb (clone E-4, dilution 1:100) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Briefly, HUVEC were grown to sub-confluence in 35 mm dishes and cultured in low serum (2%) for 24 h before treatments. The cells were then incubated with or without VEGF (10 ng/ml) and VS-1 (3 µg/ml) for 5 min in the presence or absence of mAb 5A8 as described previously. The cells were then lysed with a solution containing 50 mM Tris, 10 mM NaCl, 1 mM EDTA, 0.5% Nonidet-P40, PMSF and protease inhibitors ("complete" cocktail, Roche Diagnostics, Mannheim, Germany), and phosphatases inhibitors (100 mM NaF, 1 mM Na3VO4) (10 min, 4°C). The same amount of cell lysates was analyzed by 15% SDS-PAGE and blotted with antiphospho-ERK antibody. Anti ß-actin blotting (Sigma) was used to normalize for protein loading. Protein quantification was performed by densitometry using a molecular analyst software (Bio-Rad).
pERK was also assessed by FACS analysis as described (22)
. Briefly, after fixation using 2% paraformaldehyde, HUVEC were resuspended in 90% methanol and kept on ice for 30 min. After washing, samples were incubated with antiphospho-ERK mAb, followed by FITC-conjugated anti-mouse secondary antibody (Jackson Laboratories, Suffolk, UK). Cells were then analyzed using a FACScan instrument (Becton Dickinson, Mountain View, CA, USA).
Cell motility assay
Cell motility was analyzed by using Dunn chambers (Weber Scientific International, Teddington, UK) and by time-lapse video microscopy, as described (23)
. HUVEC were plated on 1% gelatin-coated coverslip for 24 h. Glass coverslips were then loaded onto the Dunn chamber. The effectors were added to the outer ring of the Dunn chamber. HUVEC were imaged using an inverted photomicroscope (DM IRB, Leica Microsystems AG, Wetzlar, Germany) for 1 h, and their motility was recorded with a digital CCD camera (ORCA, Hamamatsu Photonics Deutschland, Herrsching, Germany). Analysis was performed with ImageProPlus 4.0 software (Media Cybernetics, Leiden, The Netherlands). Cell paths and migration parameters were computed with Cell Buster software (gifted by A. Giacosi, Axess Logic). Cell speed was calculated as the mean of the total distance covered by 100 cells/h. Cell trajectories were plotted using Excel software.
Cell proliferation assay
HUVEC were seeded onto gelatin-coated 96-flat well plates (Costar, Cambridge, MA, USA) (5x103 cells/well) and allowed to attach for 24 h in CM. Cells were then incubated for 24 h with VEGF (10 ng/ml), alone or in combination with various concentrations of VS-1 and in the presence or absence of 5A8 mAb. HUVEC were fixed with 2.5% glutaraldehyde, then stained with 0.1% crystal violet and solubilized with 10% acetic acid. The absorbance at 590 nm of each well was then measured using a microplate reader (Bio-Rad, Milan, Italy). The number of cells in each well was calculated by interpolating the absorbance values on a calibration curve prepared in parallel with known amounts of cells.
Immunofluorescence
HUVEC were plated on 15 mm2 glass coverslips, fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton-X100, and rinsed in Dulbecco’s PBS. Proliferating cells were incubated with mouse-anti-Ki67 mAb (clone MIB-1, DAKO Carpinteria CA, USA), diluted 1:100, for 1 h at room temperature (RT), followed by rabbit-anti-mouse-IgG-AlexaFluor 594 (Molecular Probes, Eugene, OR, USA) diluted 1:500 (45 min, RT). Cell nuclei were stained with 0.2 nM 4'-6-diamidino-2-phenylindole (DAPI) (Sigma Immunochemicals, St. Louis, MO, USA) (20 min, RT). Slides were mounted with Fluorsave (Calbiochem VWR International s.r.l., Milano, Italy) and staining was evaluated by confocal microscope (Leica TCS SP2, Laser Ar/Kr, He/Ne at 50% of power, laser UV at 30% of power, Pinhole [airy] 1.000769, objective HCX PL APO CS 40.0x1.25 OIL; free projection max were obtained from single channel acquired Z-series). Proliferation rate with Ki-67 marker was calculated under fluorescence microscope (Eclipse 55i, Nikon, Tokyo, Japan) on 3 fields randomly chosen (objective PLAN APO 20x0.75; 400 to 1700 cells/slide counted). Single channel images were collected and superposed by Adobe Photoshop software.
Nuclei count and size
Nuclei of HUVEC grown on Matrigel were stained with DAPI. Images from 3 to 4 fields/specimen randomly chosen were acquired using a fluorescence microscope (Eclipse 55i, objective PLAN 10x0.30, equipped with a DS-L1 camera) and analyzed with the support of LUCIA 4.82 morphometry software (Nikon). The nuclear area of 500 nuclei was measured; nuclei were grouped in categories on the basis of their areas.
Cell apoptosis assay
Apoptosis was evaluated by FACS analysis, after staining with annexin-V FITC-conjugate (Bender Medical Systems, Prodotti Gianni, Milan, Italy) to show the exposure of phosphatidyl serine on the external side of plasma membranes, and with propidium iodide (PI). HUVEC were cultured in 24-well flat-bottomed plates and treated with or without VEGF and/or VS-1 for 24 h. The cells were then trypsinized, washed, and stained. We analyzed 10,000 cells/sample and calculated the percentage of annexin V/PI positive cells.
Sprouting assay
HUVEC (4000/ml) were suspended in culture medium containing 20% (v/v) methocel, seeded into nonadherent round-bottom 96-well plates (Greiner, Frickenhausen, Germany), and cultured overnight at 37°C (5% CO2). The methocel used was diluted from a stock solution obtained by dissolving 6 g of carboxymethylcellulose (Sigma-Aldrich) in 500 ml of medium 199. The spheroids were harvested by gently pipetting, centrifuged at 300 g for 15 min, and embedded into collagen gels. Collagen gels were prepared from a rat tail collagen solution (Boehringer Ingelheim, Italy) in medium 199, pH 7.4. This solution (0.5 ml) was mixed with 0.5 ml of complete medium (CM) containing 1.2% methylcellulose, HUVEC spheroids, and the substances to test. The spheroids-containing gels were rapidly transferred into 96-well plates and left to incubate overnight at 37°C (5% CO2). Phase-contrast images were captured using a camera (Sony DSC-S70, Japan) linked to an inverted microscope (Labovert, Leitz, Leica Microsystems AG, Wetzlar, Germany).
In vitro morphogenesis and tube formation assay
The tube formation assay was performed as described (24)
. Matrigel (Collaborative Biomedical Products, BD Biosciences) was diluted to 4 mg/ml in Dulbecco’s PBS (DPBS, Biowhittaker, Bergamo, Italy), added to 24-well plate, and incubated at 37°C for 30 min to allow gel formation. HUVEC (2x104/well) in Medium 199 containing 1% FCS with or without VEGF and/or VS-1 were then plated on Matrigel. After an overnight incubation, the cell 2-dimensional organization and the network growth area were examined using an inverted phase contrast photomicroscope (DM-IRB, Leica Microsystems AG, Wetzlar, Germany) and photographed. The assay was repeated at least three times.
Matrigel plug assay
All animal experiments were performed in accordance with institutional animal care guidelines. This assay was performed as described (25)
. Briefly, 500 µl of Matrigel (growth factor free) with or without VEGF and/or VS-1 was injected subcutaneously (s.c.) into the dorsal tissue of three 6-week-old Fischer 344 rats (160–180 g; Charles River Laboratories, Calco, Italy). After one week, rats were killed by cervical dislocation and plugs were excised. Specimens were fixed with 4% paraformaldehyde, embedded in Tissue-Tek medium (Killik, BioOptica, Milano, Italy), and snap-frozen in liquid nitrogen. Serial sections (5 µm thick) were submitted to hematoxylin/eosin and to immunohistochemistry with mAb anti-CD31/PECAM-1 (clone M89D3) and with polyclonal Ab anti-von Willebrandt factor (DAKO, Carpinteria, CA, USA). Binding was detected with the ABC method (ABC Elite kit, Vector Laboratories Inc., Burlingame, CA, USA) and 3,3'-diaminobenzidine (Sigma-Aldrich). Two pathologists working blindly counted the capillary structures (each area surrounded by few contiguous cells forming a patent lumen was considered a capillary structure) present on three different sections for every staining method (objective 10x).
Statistical analysis
Data are presented as means ± SD. Statistical differences were determined using the unpaired two-tailed Student‘s t test and, when indicated, the Wilcoxon/Kruskall Wallis test. Differences were considered statistically significant at a P-value < 0.05. All experiments were repeated at least three times unless otherwise indicated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Therefore, we investigated whether VS-1 could affect VEGF-induced ERK phosphorylation (p-ERK) of human umbilical vein endothelial cells (HUVEC) by Western blotting of HUVEC lysates and also by FACS analysis of cells (Fig. 1
). The intensity of p-ERK band (Fig. 1A, B
) and the mean fluorescence (Fig. 1C
) were increased by VEGF (10 ng/ml). This effect was inhibited by cotreatment with VS-1 (3 µg/ml) in both experimental settings. The inhibitory effect of VS-1 was significantly neutralized by the anti-VS-I antibody 5A8, suggesting that the inhibitory mechanism was specific.
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VS-1 inhibits VEGF-induced endothelial cell motility
Cell motility was studied using HUVEC seeded on a Dunn chamber and monitored by time-lapse videomicroscopy. VEGF (10 ng/ml) and/or VS-1 (3 µg/ml) were added to the outer ring of the Dunn chamber and locomotion of individual HUVEC was recorded during a period of 1 h. The videos were then analyzed by cell-tracking software to quantify the distance and the speed of migrating cells. The mean distance spontaneously covered by untreated cells was used as an arbitrarythreshold value to compare the effects of various treatments on cell motility. As illustrated in Fig. 2
A, 
46% of untreated cells covered a distance greater than the threshold value. In contrast, cell motility dropped to 19% when the cells were treated with VS-1 alone. The number of HUVEC having migrating beyond the threshold markedly increased (85%) when cells were treated with VEGF. This value was significantly lower (56.5%) after treatment with VEGF+VS-1. Individual cell trajectories and migration speed were also calculated by computer-assisted analysis (Fig. 2B
). VEGF induced an increase in the length of tracks, as expected, and this effect was inhibited by VS-1. Cells treated with VS-1 alone exhibited a lower speed than the untreated controls (9.3 µm/h vs. 16.2 µm/h, Fig. 2C
) and the cells treated with VEGF+VS-1 revealed a lower mean migration speed than that of VEGF alone (16.8 µm/h vs. 34.6 µm/h). These results suggest that VS-1 inhibits both spontaneous and VEGF-induced HUVEC locomotion.
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VS-1 inhibits VEGF-induced endothelial cell proliferation
The effects of VS-1 and VEGF were investigated on endothelial cell proliferation. The effect of various doses of VS-1 alone on HUVEC was first investigated. VS-1 could inhibit basal cell proliferation, i.e., the spontaneous proliferation in the absence of VEGF, with a "bell-shaped" dose-response curve, 3 µg/ml being the most effective dose (Fig. 3
A). VS-1 (3 µg/ml) could also inhibit VEGF-induced cell proliferation (Fig. 3A
). In both cases the inhibitory activity of VS-1 was reverted by cotreatment with mAb 5A8, pointing to a specific mechanism. Similar experiments were performed using immortalized ECV 304 and EAhy926 cell lines. Also in these cases VS-1 could inhibit VEGF-induced cell proliferation to an extent similar to that obtained with U-0126 a specific inhibitor of ERK 1/2 (Fig. 3A
).
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The inhibitory activity of VS-1 on VEGF-induced HUVEC proliferation was also assessed by immunostaining for Ki67, a proliferation marker. The percentage of Ki67-positive nuclei/treatment, calculated on a total of 500 nuclei stained with DAPI, was maximal in VEGF-treated cells and significantly lower in the presence of VS-1 and in control (Fig. 3B,C
).
To determine whether the inhibitory effect of VS-1 was related to cell apoptosis induction, HUVEC were treated with VS-1 ± VEGF, stained with annexin V/propidium iodide, and then analyzed by FACS (Fig. 3D
). The percentage of living cells (double negative for annexin V/propidium iodide) was similar in treated and untreated cells, suggesting that the treatments did not induce apoptosis and that the inhibition by VS-1 of VEGF-induced proliferation was not due to a decrease in cell viability. Notably, the addition of mAb 5A8 did not affect HUVEC viability/apoptosis.
VS-1 affects endothelial cell morphology in vitro
It is well known that VEGF can induce morphological changes in endothelial cells and formation of capillary-like structures in vitro (26)
. To establish the effect of VS-1 on capillary-like structure formation, HUVEC were plated on a layer of growth factor deprived Matrigel and incubated overnight in the presence or absence of effectors. While HUVEC formed interconnected networks in the presence of VEGF (10 ng/ml), incomplete structures were formed when cells were treated with VEGF +VS-1 (Fig. 4
A). Similarly, when HUVEC were plated on Matrigel containing growth factors, almost all cells formed a network in the presence of VEGF (Fig. 4B
). In contrast most cells tended to form monolayers in the presence of VEGF plus VS-1, or VS-1 alone, or in the absence of both effectors (Fig. 4B
). These findings suggest that VS-1 could inhibit, at least in part, the effect of VEGF on cell morphology and organization by stabilizing the monolayer modus.
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The effects of VS-1 and VEGF on HUVEC cell nuclear size were also investigated. HUVEC were seeded on Matrigel for 24 h in the absence or presence of effectors and stained with DAPI. Again, in the presence of VEGF, cells tended to form organized, tubule-like structures that were abolished by VS-1 (Fig. 4C
). For each treatment, nuclear sizes were grouped in arbitrary categories; we found differences exclusively in the two extreme categories containing small (50–100 nm2) and large nuclei (601–1500 nm2). The results showed that 24% of cells treated with VEGF had small-sized nuclei (Fig. 4D
), and less than 5% of large size nuclei, in line with the high proliferation pattern showed in Fig. 3A
. Conversely, in cells treated with VS-1 alone predominated large nuclei (33% of large vs. 5% of small), corresponding to the low cell number in Fig. 3A
. Cells treated with VEGF+VS-1 revealed an intermediate nuclear pattern (8% of large nuclei vs. 17% of small), indicating that VS-1 could affect changes in nuclear size induced by VEGF, most likely related to inhibition of proliferation.
VS-1 inhibits VEGF-induced three-dimensional organization of HUVEC and invasion of collagen gel
The effect of VS-1 on VEGF-induced three-dimensional endothelial cell organization and morphogenesis was further investigated using HUVEC spheroids, i.e., cell aggregates, embedded in a collagen gel. In the presence of VEGF, HUVEC spheroid sprouted and invaded the surrounding collagen gel forming capillary-like structures (Fig. 5
and Table 1
). VS-1 inhibited VEGF-induced sprout formation in a significant manner without inhibiting sprouting by itself.
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VS-1 inhibits the VEGF-dependent formation of capillary-like structures in Matrigel plugs in vivo
The effect of VS-1 on endothelial cell invasion and organization into capillary-like structures was investigated using the in vivo Matrigel plugs assay, performed in rats. Each rat was injected subcutaneously in the dorsal midline in four different positions with Matrigel containing a) VS-1 (3 µg/ml), b) VEGF (10 ng/ml), c) VEGF+VS-1, or d) none. This assay takes advantage from the fact that after injection, the Matrigel solution forms gel plugs, which can be subsequently colonized by host endothelial cells. After 1 wk, plugs were excised and analyzed to assess the presence of endothelial cells and neoformation of vessels. Hematoxylin and eosin (H&E) staining demonstrated that the maximal localization of cells organized in capillary-like structures occurred in plugs containing VEGF (Fig. 6
A). The number of such structures, i.e., areas surrounded by few adjacent cells, was counted on the same sections and also on the immunostained sections (Fig. 6D
) and confirmed this indication. Immunohistochemistry with anti-CD31 and anti-vWF antibodies stated that infiltrating cells were endothelial cells and that VEGF, with respect to control, enhanced angiogenesis (Fig. 6B, C
). Red blood cells were seen in some of these capillaries, suggesting that they were functional. VEGF-induced increase of capillary-like structures formation was significantly inhibited by cotreatment with VEGF+VS-1 and was not observed in specimens treated with VS-1 alone. These findings suggest that VS-1 can inhibit VEGF activity on endothelial cells migration and invasion also in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, our findings suggest that CgA-derived VS-I may serve as significant inhibitor of these VEGF-induced processes.
The naturally derived N-terminal domain CgA1–76 was named vasostatin I (VS-I) in 1992 (27)
on basis of its inhibitory effects on blood vessel contractility. A wide range of inhibitory activities has since been assigned to the CgA-derived VS-I (7)
, which is structurally characterized by its amphipathic properties (28)
and interactions with phospholipids (29)
, mammalian (30)
and microbial membranes (28)
. The CgA-derived VS-I is structurally unrelated to other known peptide inhibitors of blood vessel integrity and angiogenesis (31)
, such as plasminogen-derived angiostatin, collagen-derived endostatin, and calreticulin-derived vasostatin, first reported in 1998 (32)
.
What is the mechanism of VEGF inhibition? Activated MAPK cascades are involved in a range of cellular responses such as proliferation, inflammatory apoptosis, and stress responses (33)
. In the present study we have shown that recombinant VS-1 can inhibit VEGF-induced phosphorylation of ERK. The protein tyrosine receptor VEGR-2 is characteristic of endothelial cells and mediates VEGF-induced proliferation and migration during angiogenesis via ERK signaling (34)
. In retinal microvascular endothelial cells VEGF stimulates cell proliferation and activation of ERK in a "bell-shaped", concentration-dependent manner, between 1–200 ng/ml (35)
. A 50% reduction in phosphorylation of VEGFR-2 and ERK was reported to be associated with an increase in the protein tyrosine phosphatase (SH-PTP) and endothelial nitric oxide synthase (eNOS) at 10–20 ng/ml VEGF. Throughout the present study we have used 10 ng/ml VEGF and observed enhanced cell proliferation, motility, migration, sprouting, and ERK phosphorylation in various in vitro assays as well as invasion and capillary-like structure formation in an in vivo assay. VS-1 could inhibit all these effects. Experiments on the time course of ERK activation showed that phosphorylation occurs very rapidly (5 min after exposure to VEGF) and transiently, as a decrease was observed after 1 h (data not shown and, 36
). Due to the length of proliferation/migration assays, lasting several hours, it is difficult to speculate whether inhibition of ERK and inhibition of proliferation/migration are functionally linked. However, other investigators have shown that activation of ERK is crucial for the induction of a cascade of events leading to increased cell proliferation and migration (37
38
39
40)
. Accordingly, we observed that addition of U-0126, a specific ERK 1/2 enzyme inhibitor (41)
, inhibited endothelial cell proliferation. Although we cannot exclude that other important kinases or signaling molecules, activated at later stages, are also inhibited by VS-1, this suggest that inhibition of ERK contributes to the overall effect of this CgA fragment.
Maximally effective, inhibitory concentration of VS-1 on proliferation of HUVEC was 3 µg/ml. At this concentration VS-1 consistently inhibited cell mobility and proliferation even in absence of VEGF, indicating interaction with VEGF-independent signaling. Furthermore, VS-1 at the same concentration could also inhibit the increase of permeability induced by VEGF, TNF, or thrombin in HUVEC monolayers (6)
. Thus, it seems unlikely that the inhibitory activities of VS-1 on VEGF-activated processes are related to a specific interaction with the VEGFR-2. A specific, enhanced fluidity exerted by low nanomolar concentrations of VS-1 in monolayers of phosphatidylserine suggests that VS-1 may engage in receptor-independent interactions with other "classical" receptors in phosphatidyl serine rich microdomains in the outer leaflet of the plasma membrane (29)
. Such a mechanism for modulation of intracellular signaling pathways has been proposed for the VS-1 protection against disruption of the barrier and the pertussis sensitive phosphorylation of p38MAPK by TNF- in bovine pulmonary endothelial cells (8)
. It is possible that VS-1 may perturb VEGF-signaling by a similar, receptor-independent mechanism. Concerning the fate of VS-1 after interaction with endothelial cells, we demonstrated in a previous work that FITC-labeled VS-1 enters into HUVEC and colocalizes with transferrin, a marker of endocytotic vesicles (6)
.
It is well known that tumors cannot grow beyond 2–3 mm without an adequate vascular supply, and for this reason tumor growth requires angiogenesis. Considering the well known role of VEGF in tumor angiogenesis and progression and the notion that CgA is released in large amounts by neuroendocrine tumors (11
, 14)
the possibility exists that CgA affects tumor growth. According to this view, previous work has shown that overexpression of CgA in cancer cells can inhibit tumor growth and morphogenesis in mouse models (42
, 43)
. Tumor-released CgA is assumed to be distributed in a ratio of 24:1 between the extravascular and intravascular compartments (44)
. The question rises as to whether the compartmentalized CgA is processed to VS-I in sufficient concentrations during angiogenesis or vascular remodeling. Angiogenic factors induce expression of integrins on endothelial cells; production of plasminogen activators; and activation of plasmin, metalloproteases, and collagenases (45)
. These proteases are critical for cell migration, tissue invasion, and vascular remodeling (46)
. Interestingly, VS-I can be cleaved from CgA by plasmin (47)
. Furthermore, several works have shown that the plasminogen/plasmin system is also involved not only in endothelial cell migration but also in tumor cell migration, invasion, and tissue remodeling (48)
. Thus, considering the large amounts of CgA locally released in neuroendocrine tumors and the presence of active plasmin, it seems likely that CgA-derived N-terminal fragments, such as VS-I, are generated in certain areas of growing tumors, and that these fragments may affect endothelial cells by paracrine mechanisms. Taking into account that the recombinant VS-1 can also inhibit VEGF- and TNF-induced barrier-disrupting effects of the endothelium (6)
and that VS-1 promotes adhesion and spreading to solid-phases of fibroblast and smooth muscle cells (47
, 49)
, tumor-derived CgA and VS-I may inhibit tumor growth a) by inhibiting endothelial proliferation and migration, which are critical steps in angiogenesis and vascular remodeling; and b) by decreasing endothelial transport of nutrients and growth factors essential for tumor cell proliferation. The possibility that exogenous VS-1 may be used as an antiangiogenic agent in cancer deserves future investigations.
In conclusion, inhibitory activity of recombinant VS-1 has been demonstrated on a wide range of VEGF stimulated functions in HUVEC in vitro, without inhibiting cell viability, and in the subcutaneous Matrigel plugs implanted in rats in vivo. These findings strongly suggest that the CgA-derived VS-I may serve as an inhibitor not only of VEGF-stimulated ERK phosphorylation, cell proliferation, mobility, migration, and sprouting but also of invasion and formation of capillary-like structures. Hence, CgA-derived VS-I emerges as a novel antiangiogenic agent originating from the stress-activated diffuse neuroendocrine system and related neuroendocrine tumors, presumably acting via suppression of activities in the MAPK pathway.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication July 21, 2006. Accepted for publication April 19, 2007.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mean in (C) is calculated from three independent experiments.
0.01 (by non-parametric Wilcoxon/Kruskal-Wallis test). D) Apoptotic cells were quantified by FACS analysis using annexin V and propidium iodide staining: in each quadrant the percentage of positive cells is indicated; one experiment, representative of three, is shown.
