1Instituto de Investigaciones Médicas, Fundación Jiménez Díaz; 4Universidad Autónoma de Madrid, and 2Instituto Reina Sofía de Investigación Nefrológica, 28040 Madrid, Spain; and 3Molecular and Cell Biology Department, Imclone Systems Incorporated, New York, New York 10014
Submitted 17 July 2003 ; accepted in final form 23 December 2003
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ABSTRACT |
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vascular endothelial growth factor receptor 2; MG63-conditioned medium
Although vascular endothelial growth factor (VEGF) is a primum inter pares among all the agents involved in tumor vascularization, its specific role still has several major unknown aspects (12, 13). In a general view, VEGF and its tyrosine kinase receptors VEGFR1 (flt-1) and VEGFR2 (flk-1/KDR) are key mediators of physiological and pathological angiogenesis. They are expressed in most tissues during embryonic development but tend to be downregulated in the adult (12, 13). VEGFR2 mediates increased EC mitogenesis and permeability, whereas the role of VEGFR1 has not been yet sufficiently elucidated.
Upregulation of VEGF and VEGFRs has been observed during the induction of angiogenesis (12, 13). Such upregulation is particularly intense in hypoxic and neoplastic tissues. In the latter, current opinion maintains that tumor cells produce VEGF and stimulate VEGFR2 expression, therefore closing a loop of EC activation (18, 22). Although in this setting the pattern of expression of VEGFR2 appears to parallel VEGF expression, little is known about their mutual interaction and regulation. Moreover, tumor-produced VEGF can theoretically influence EC-produced, autologous endothelial VEGF; no data are yet available, however, to substantiate this hypothesis. Collectively, compelling evidence suggests that VEGF and its receptors are critical for tumor-associated angiogenesis and that they represent good targets for therapeutic intervention (22); however, data are still lacking for full understanding of the complete mechanisms of action of anti-VEGF strategies. In this regard, no description is currently available of the precise tumor-induced changes in confluent EC and the role of VEGF in each of these changes.
In light of the aforementioned rationale, the aim of the present study was to analyze the effect of tumors on monolayer, quiescent EC, with a focus on the role of VEGF and its receptors.
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MATERIALS AND METHODS |
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The conditions were modified when needed for the different experiments. Unless stated otherwise, all the experiments were done on confluent EC. Anti-VEGF (Sigma, St. Louis, MO) and anti-VEGFR2 (3.83; Imclone, New York) MAbs were used for blockade of VEGF actions. Both antibodies have been extensively tested for their blocking properties in our laboratory and by the manufacturers (1, 7, 8). Because of its scarce availability, the noncommercial MAb 3.83 was only used in selected experiments. Crystal violet, hematoxylin and eosin, Masson trichrome, and immunocytochemistry techniques were applied to the EC in different conditions. Both the number of nucleoli/chromatin condensations and the nucleus-to-cytosol diameter ratio were calculated on digitized microphotographs examined by three independent observers who were blinded for the experimental conditions.
MG63 culture and CM preparation. Human MG63, a line of malignant osteoblast-like cells (American Type Culture Collection), were maintained in Dulbecco's modified Eagle's medium (DMEM) + 10% FBS. For preparing CM, MG63 were cultured in MEM-D-valine without FBS for 24 h; medium was collected, filtered, and added to the EC without further maneuvers. When needed, a similar incubation was carried out with the hypoxia-mimicking agent CoCl2 (10 µM) added to the medium.
EC damage and proliferation. EC damage was addressed by flow cytometry (7). Lactate dehydrogenase (LDH) release was assessed in confluent EC maintained for 2448 h in growth factor-deficient conditions (MEM-D-valine without FBS) (7, 8). Two hundred micro-liters were sampled at different times, and percent LDH release was calculated. Cell counting was performed in a Neubauer chamber. Cell proliferation was quantified with a cell proliferation ELISA bromode-oxyuridine (BrdU) kit (Roche, Madrid, Spain). Experiments with a Transwell system were used for assessing the direct effect of the presence of MG63 on EC (see Fig. 1C for description).
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mRNA isolation and reverse transcriptase-PCR. Total RNA was extracted as described previously with the TriPure isolation reagent (Boehringer Mannheim, Madrid, Spain) after different times of exposure (3, 9, 24 h) to the treatments (see below for further details) (1, 7). Reverse transcriptase (RT)-PCR was performed with oligonucleotide primers of bovine VEGF, VEGFR1, and VEGFR2, synthesized as described previously (7).
VEGF Western blot and ELISA. Samples were processed and analyzed by Western blotting as described previously (7). Anti-VEGF MAb (1:250; Sigma) was used as antibody. VEGF concentrations in MG63-CM were measured by a VEGF ELISA (Accucyte Human VEGF, Cytimmune Sciences).
p44/p42-mitogen-activated protein kinase and Akt Western blots. Western blots for phospho-p44/p42-mitogen-activated protein kinase (MAPK), phospho-Akt (Ser473), and their respective loading controls [total p44/p42-MAPK and protein kinase B (PKB)/Akt] were performed following the manufacturer's instructions (Cell Signaling Technology, Beverly, MA). The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY-294002 (20 µM; Sigma) and the MAPK kinase (MEK) inhibitor PD-98059 (50 µM; Calbiochem, La Jolla, CA) were used during the 45 min before addition of the medium.
Matrigel invasion assay. The activated state of EC was further assessed by their penetration into growth factor-reduced Matrigel (Becton Dickinson, Barcelona, Spain).
Immunocytochemistry of factor VIII expression. Cells grown to confluence on tissue culture chamber slides (Becton Dickinson USA) were submitted to the different experimental maneuvers, washed with PBS, incubated in MEM-D-valine-0.5% FBS, MG63-CM, and MG63-CM-CoCl2 for an additional 5 days, and then fixed with Merckofix (Merck). Immunostaining was performed by the alkaline phosphatase method, using factor VIII (1:1,000, polyclonal rabbit; Sigma) as primary antibody. Similarly fixed cultures were treated with nonimmunogenic rabbit IgG as negative control. After being washed with PBS, cells were incubated with biotinylated swine anti-rabbit IgG (Dako, Glostrup, Denmark) for 30 min, sequentially followed by incubation with avidin-biotin-peroxidase complex (Dako) and 3,3'-diaminobenzidine (Sigma).
Statistics. Results are expressed as means ± SE. Unless stated otherwise, each value corresponds to a minimum of five triplicate experiments. Comparisons were done by ANOVA or paired and unpaired Student's t-test, when appropriate. The Fisher and Scheffé tests for multiple comparisons were used to determine the P value, which was considered significant at <0.05. All statistic analyses were performed with the SPSS 8.0 package (Jandel, San Rafael, CA).
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RESULTS |
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In addition, we found that, in the absence of FBS, no cell proliferation, as assessed by percent BrdU incorporation, occurred in confluent, quiescent EC treated with MG63-CM or exogenous VEGF (FM: 68 ± 3%, MG63-CM: 66 ± 9%, MG63-CM + CoCl2: 53.8 ± 7%, 5 x 10-10 M VEGF: 68.8 ± 4%; n = 3 triplicate experiments). In these conditions, 20% FBS induced an increase in BrdU incorporation of 217 ± 43% (P < 0.001). Furthermore, no changes in either glucose or pH of the different media were detected after 24-h incubation (data not shown). Of importance, MG63-CM contained increased amounts of VEGF (FM: undetectable levels, MG63-CM: 13.5 ± 1.2 ng/ml; P < 0.01); VEGF levels were even higher in the presence of CoCl2 (MG63-CM + 10 µM CoCl2: 26.8 ± 1.5 ng/ml; P < 0.01 with respect to MG63-CM alone and P < 0.001 with respect to FM).
Incubation with MG63-CM induced relevant phenotypic changes in confluent EC, which were consistent with an activated state. Of specific importance, no phenotypic changes were detected by treating the EC with exogenous VEGF (5 x 10-10 M) or with EC-CM (with or without CoCl2; images not shown). The changes produced by MG63-CM are depicted in Fig. 2 and include the following. 1) The number of nucleoli/chromatin condensations (EC + MG63-CM 5.3 ± 0.7 vs. EC-FM 3.4 ± 0.4 nucleoli/cell; P < 0.01) and the nucleus-to-cytosol ratio (FM 0.41 ± 0.09 control; MG63-CM-CoCl2 0.68 ± 0.08; P < 0.01) were increased. 2) Acellular areas appeared, consistent with attempted capillarization (Fig. 2B,f). 3) Vacuolated, seal-shaped EC (Fig. 2, A,c and B,d) appeared; these structures were also consistent with attempted capillarization in bidimensional growth conditions and resemble those found in malignant angiomas (17). The aforementioned changes were more marked when the EC were incubated with MG63-CM + CoCl2 (10 µM). 4) Migration of the EC within Matrigel occurred; no significant migration occurred by incubating the EC with FM (Table 1). 5) Treatment with MG63-CM and MG63-CM-CoCl2 induced a marked increment in factor VIII, as assessed by immunocytochemistry. Moreover, factor VIII distribution was different in MG63-CM-treated EC with respect to nonconditioned media or EC-CM-treated EC (Fig. 2C). Changes induced by the addition of anti-VEGF antibody are commented on below. 6) Pretreatment of EC with MG63-CM induced growth advantage in reseeding in FBS-poor conditions. This growth advantage was more marked when the CM was obtained from MG63 + CoCl2 (Table 2).
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Additional experiments were performed with HMEC to analyze whether the findings in EC originated in a great vessel were also valid in EC of microvascular type. HMEC growth rate and phenotype were rather different than those of EC (Fig. 2D); however, a significant protective effect on HMEC was also found in the presence of MG63-CM (Figs. 1B and 2D).
Role of VEGF and VEGFR2-mediated pathways in changes induced by MG63-CM on confluent, quiescent EC. After the aforementioned alterations were identified, we analyzed whether VEGF was a critical factor in the protective effect of MG63-CM. These experiments revealed a remarkably important fact, i.e., that the inhibition of VEGF effects by a specific anti-VEGF antibody not only blocks the protection induced by MG63-CM but also unmasks a deleterious effect of MG63-CM on EC (Fig. 3, A and B). In HMEC, however, both anti-VEGF and anti-VEGFR2 induced a complete reversal of the MG63-CM protective effect, but without inducing a significant increase in cell death over the baseline (Fig. 3C).
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Treatment with the anti-VEGF antibody completely blocked the effect of MG63-CM on EC phenotype (Fig. 2) and migration within Matrigel (Table 1). In the same regard, the number of nucleoli, as well as the nucleus-to-cytosol ratio, were significantly reduced in the presence of anti-VEGF MAb (MG63-CM-CoCl2 + anti-VEGF MAb: 3.9 ± 0.6 nucleoli/cell and nucleus-to-cytosol ratio 0.41 ± 0.06, both P < 0.01 with respect to MG63-CM-CoCl2 without anti-VEGF MAb; for both measurements, see above for comparison). As shown in Fig. 2, acellular spaces were markedly reduced when the anti-VEGF MAb was present in the media. Moreover, the formation of vacuolated capillary-like structures was almost abolished in the presence of the anti-VEGF MAb (2 ring shaped/402 EC; P < 0.001; Fig. 2) compared with EC treated with MG63-CoCl2 (98 ring shaped/394 EC) or MG63-CoCl2 + nonspecific IgG (72 ring shaped/376 EC). In addition, treatment with anti-VEGF MAb significantly decreased the MG63-CM-induced increase in factor VIII expression; an identical image was observed by using anti-VEGFR2 (MAb 3.83). All the experiments using anti-VEGF or anti-VEGFR2 antibodies included controls incubated in identical conditions but exposed to nonspecific IgG of the same type of anti-VEGF and 3.83 antibodies. No changes were detected in these controls with respect to MG63-CM or FM without antibodies (data not shown).
Role of tumor-CM on autologous VEGF and VEGFR2 mRNA expression in EC. Incubation with MG63-CM induced major changes in the expression of VEGF by EC. MG63-CM markedly inhibited autologous VEGF mRNA compared with the expression detected in the presence of FM (Fig. 4A); a similar result was obtained for VEGF protein (Fig. 4B). The fact that the addition of anti-VEGF MAb was not capable of blocking the MG63-CM-induced abolition of autologous VEGF expression indicates that this abolition was not related to the presence of VEGF in the MG63-CM. Therefore, molecules different from VEGF contained in the MG63-CM are probably involved, e.g., other growth factors.
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To further analyze the mechanisms involved in the effect of MG63-CM, the expression of VEGFR2 mRNA was assessed by RT-PCR. As can be seen in Fig. 5, MG63-CM induced an increase in VEGFR2 mRNA expression in samples studied at 0, 3, 9, and 24 h. In terms of mechanism, the VEGFR2 increase was critically related to a paracrine effect secondary to VEGF present in the MG63-CM. This VEGF acts through the VEGFR2 receptor, as judged by the marked decrease in the stimulation of VEGFR2 expression in the presence of anti-VEGF antibody and anti-VEGFR2 antibodies (Fig. 5). With respect to the VEGFR1/flt-1 receptor, no differences in mRNA expression were detected in the same samples used for the study of VEGFR2 expression (Fig. 5).
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Effect of MG63-CM on Akt and MAPK phosphorylation. Additional experiments were performed to analyze the putative pathways involved in EC activation by MG63-CM. The pathways analyzed were chosen on basis of the known signaling routes of VEGF. Incubation of confluent EC in the presence of MG63-CM induced a significant increase in Akt and MAPK phosphorylation (Fig. 6). Both increased phosphorylations were blocked in the presence of the PI3-kinase inhibitor LY-294002 and the MEK inhibitor PD-98059, respectively (Fig. 6).
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DISCUSSION |
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The literature on the effects of tumor-CM on EC was mostly generated in the 1970s and 1980s. The studies were mainly focused on two major subjects, namely, the effect of tumors on EC proliferation and tumor-induced endothelial permeability (6, 15). Rather unexpectedly, the aforementioned studies contained almost no information on the actions of tumor cells and/or tumor cell-CM on confluent EC in the contact-inhibited state.
Our study has detected significant effects of tumor-CM on EC phenotype, indicating activation. To our knowledge, the pattern of EC activation in the presence of tumor-CM and tumor cells, namely, MG63-CM, is described for the first time, and it includes marked increases in nuclear size and number of nucleoli/cromatin condensations, as well as formation of capillary-like structures. The changes found in our experiments with MG63-CM are particularly illustrative of the magnitude of the activation of normal EC in the tumor microenvironment and resemble those described in vivo as an angiogenic phenotype (21). The effects on factor VIII expression are of special interest regarding the potential role of VEGF in the procoagulating activity of tumors and deserve further attention.
In addition to the morphological changes, our data demonstrate that soluble factors present in MG63-CM have protective effects against FBS deprivation. Of utmost importance, for its significance in terms of putative therapeutic applications, this protective response of MG63-CM became deleterious when VEGF was blocked. This finding reveals, for the first time, that on blockade of the key protective mediator VEGF, EC damage is significantly potentiated by tumor-borne products and therefore highlights the importance of autologous VEGF.
The anti-EC effects of MG63-CM in the presence of VEGF or VEGFR2 blockade are most probably conveyed through soluble factors. Several antiangiogenic agents have been described in tumors, e.g., angiostatin, endostatin, and thrombospondin (10). However, the precise identification of the deleterious factors contained in the MG63-CM, albeit of interest, is beyond the specific aims of the present research.
Our results also show that the effects of tumor-CM and VEGF blockade are relevant in EC of microvascular origin. The existence of some differences between the findings in EC and HMEC, e.g., death rate, phenotype, and time to confluence, can be traced to the fact that HMEC are a continuous cellular line with a markedly different growth rate and, more importantly, with metabolic modifications aimed to ensure a more efficient survival. In this regard, the results obtained in the EC in primary culture are probably closer to the actual properties of EC in vivo.
The use of anti-VEGF antibodies or drugs as antiangiogenic agents is generally supposed to occur because of the blockade of EC proliferation but not the facilitation of EC damage by tumor-related factors. Therefore, our data extend the scope of putative effects of anti-VEGF therapy in cancer to antiprotective actions on EC. Accordingly, our results add further evidence for the interpretation of the mechanisms of the antiangiogenic effect of VEGF antagonism in tumors and provide a source of explanation for the vessel regression observed on VEGF blockade (2, 3).
The data demonstrating that MG63-CM suppress autologous VEGF gene expression in EC are potentially relevant in terms of interpreting the deleterious effects of VEGF withdrawal or antagonism (2, 3, 11, 16, 25). Tumor-MG63-CM suppresses autologous VEGF, therefore rendering the EC dependent on exogenous, tumor-generated VEGF. Our results agree with findings in human tumors showing the absence of VEGF mRNA in EC from tumors in which VEGF is produced by the neoplastic cells (4, 5, 9, 19). In these, intratumoral hypoxia should have stimulated production of VEGF by the EC within the tumor (25); in fact, the absence of such production suggests that it may actually be suppressed by the VEGF present in the tumor microenvironment. These particular results add new information to previously published work by Wang et al. (24). These authors reported that tumor-CM regulates the expression of VEGF receptors on EC surface, but they did not analyze the fate of autologous VEGF expression by EC (24).
Several of the effects found with the anti-VEGF and anti-VEGFR2 blocking MAbs deserve special comment. First, the induction of EC growth advantage in reseeding, as well as the induction of Matrigel penetration, indicate that VEGF is a critical factor in triggering properties of the EC that are relevant for the mechanism of metastatic seeding. Second, the different degree of blockade of diverse effects by the anti-VEGF antibody compared with the anti-VEGFR2 antibody suggests either that a more relevant role of VEGFR1 may exist in some of the MG63-CM actions or that other types of VEGF, e.g., VEGF-C or VEGF-D, are involved.
In terms of mechanism, the present results reveal that MG63-CM activates the two main survival pathways, i.e., PI3-kinase/Akt and p44/p42-MAPK. These pathways are involved in the intracellular signaling routes of several growth factors, including VEGF (23). A more precise definition of these routes is beyond the scope of the present study. However, experiments are presently being performed in our laboratory to specifically characterize precise mechanisms involved in the Akt and p44/p42-MAPK activation by MG63-CM.
The findings described herein provide new information for understanding anti-VEGF strategies in the therapy of tumors. The presence of high amounts of exogenous VEGF appears to be critical in allowing the EC to withstand the challenge of deleterious factors produced by the tumor cells. Furthermore, the fact that tumor medium dramatically downregulates autologous VEGF expression in EC may have potential relevance in terms of the response of these EC to forthcoming aggressions. The finding that the interference with VEGF- or VEGFR2-related effects determines a growth disadvantage on reseeding, which interferes with matrix penetration as well, is potentially important for therapeutic approaches against tumor metastatization and local tissue invasion.
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ACKNOWLEDGMENTS |
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GRANTS
This study was financed by grants of Fondo de Investigaciones Sanitarias (FIS 01/0345, FIS 01/3131, and PI 020486), Red Cardiovascular (Instituto de Salud Carlos III), Comunidad Autónoma de Madrid (CAM 08.3/0010/2000.1 and 0010/2002.3), Fundación Mapfre Medicina, and Instituto Reina Sofía de Investigacíon Nefrológica (IRSIN). M. A. Castilla is a fellow of PEN.SA-IRSIN; F. Neria is a fellow of Fundación C. Rábago; F. R. González-Pacheco and S. Jiménez are fellows from CAM and MVAA; S. Yagüe and J. J. P. Deudero are fellows of Instituto de Salud Carlos III.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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