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Tumor Biology |
Departments of Cariology, Restorative Sciences, and Endodontics [J. E. N.], Oral Medicine/Pathology/Oncology [J. C., J. L., P. J. P.], and Biology and Materials Sciences [D. J. M.], School of Dentistry, and Department of Biomedical Engineering, School of Engineering [M. P., D. J. M.], University of Michigan, Ann Arbor, Michigan 48109; and Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California-Los Angeles School of Medicine, Los Angeles, California 90095 [R. M. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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IL-8 is the first member of a family of 8 structurally related chemokines that have been shown to induce angiogenesis in several physiological and pathological settings. IL-8 was initially discovered on the basis of its ability to induce chemotaxis of neutrophils (15, 16, 17) . IL-8 has been shown to be a key mediator of angiogenesis for several epithelial and mesenchymal tumors, including carcinoma of the lung and of the oral cavity and Kaposi’s sarcoma (17, 18, 19, 20, 21, 22) . IL-8 has also been implicated as a survival factor for endothelial cells. Molica et al. (23) have reported increased levels of IL-8 in the serum of patients with chronic B-cell lymphocytic leukemia that parallel increased intracellular levels of Bcl-2 in tumor cells. These investigators suggested that the survival effect of this chemokine might be exerted through a Bcl-2-dependent pathway. In contrast, Wakisaka et al. (24) failed to demonstrate a link between the expression of IL-8 and Bcl-2 in rheumatoid synovial cells, suggesting that the inhibition of apoptosis by IL-8 was independent of Bcl-2 in patients with rheumatoid arthritis.
We have previously reported that VEGF enhances endothelial survival and sustains angiogenesis (11) by inducing expression of the antiapoptotic protein Bcl-2. Using a SCID mouse model of human angiogenesis developed in our laboratory, we reported that HDMECs, seeded in PLLA sponges and implanted into SCID mice, organize into functional human and human/mouse chimeric microvessels. The implantation of endothelial cells that overexpress Bcl-2 (HDMEC-Bcl-2) resulted in a significant enhancement of neovascularization as compared with control implants (11) . Although the role of Bcl-2 in endothelial cell survival is well established, the mechanism by which this antiapoptotic protein promotes angiogenesis and its pathophysiological significance are unknown. The current study was undertaken to investigate how overexpression of Bcl-2 in endothelial cells modulates angiogenesis and affects tumor growth. In this report, we showed that cotransplantation of HDMEC-Bcl-2 and human tumor cells into SCID mice significantly enhances the growth of tumors and intratumoral microvascular density. Furthermore, we showed that Bcl-2 potentiates angiogenesis, in part, by enhancing expression of IL-8 in endothelial cells. These results demonstrate that overexpression of Bcl-2 in the endothelial cells that line tumor microvessels is sufficient to enhance tumor growth.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Endothelial Cell Proliferation and Capillary Tube Assays.
The ability of CM from cultures of HDMEC-Bcl-2 to induce HDMEC to proliferate and organize into capillary-like sprouts in vitro was examined in cultures grown on type I collagen gels (11)
. CM was collected for 24 h from 5 x 106 HDMEC-Bcl-2 or HDMEC, filtered, and concentrated. Total protein content was normalized for all of the samples. HDMECs were seeded in 60-mm tissue culture dishes (Corning Costar Corp., Cambridge, MA) coated with 1.5 ml of a gelled solution of type I collagen (Vitrogen 100, Cohesion Technologies Inc., Palo Alto, CA). Cells were fed every 2 days with concentrated HDMEC-Bcl-2 or HDMEC CM diluted in EGM-MV to a final concentration of 1x. One, 5, or 10 µg/ml antihuman IL-8 antibody (R&D Systems, Minneapolis, MN), or 10 µg/ml isotype-matched nonspecific immunoglobulin (Sigma), were added to HDMEC-Bcl-2 CM. At daily intervals, the number of cells in 10 random high power fields (x200) and the number of capillary-like sprouts (x100) were counted as described previously (11)
. The data were obtained from triplicate dishes per condition at each time point.
Rat Corneal Micropocket Assay.
The angiogenic activity of HDMEC-Bcl-2 CM was evaluated in the rat corneal micropocket assay (25)
. Briefly, 25 µl of HDMEC-Bcl-2 CM (10x), HDMEC-Bcl-2 CM (10x) supplemented with 0.2 µg or 1 µg of polyclonal antihuman IL-8 antibody (R&D Systems), or 25 µl of HDMEC-LXSN CM (10x) were combined with 25 µl of sterile hydron casting solution (IFN Sciences, New Brunswick, NJ), and 10-µl pellets were implanted into surgically created intracorneal pockets in the rat eye (Harlan Inc., Indianapolis, IN). After 7 days, rats were perfused with colloidal carbon, and eyes were enucleated, fixed in 10% buffered formalin overnight, flattened, and photographed. Positive controls consisted of hydron pellets containing 0.1 µg of recombinant human IL-8 (R&D Systems).
Transplantation of HDMECs and Human Tumor Cells into SCID Mice.
The effect of endothelial cell Bcl-2 on tumor angiogenesis and tumor growth was examined by seeding 0.9 x 106 HDMEC-Bcl-2, HDMEC-LXSN, or HDMECs along with 0.1 x 106 human oral squamous carcinoma cells (OSCC-3, a gift from M. Lingen) or a line of human Kaposi’s sarcoma cells (SLK, a gift from G. Nuñez) in PLLA sponges (11
, 26)
and implanting them in SCID mice (CB.17.SCID, Taconic, Germantown, NY). Each mouse was anesthetized using ketamine and xylazine, and two sponges were surgically implanted s.c. on the right and left flank region. After 21 days, mice were killed and the implants were retrieved, immediately measured with calipers, and weighed in an electronic balance. The care and treatment of experimental animals was in accordance with University of Michigan institutional guidelines.
Immunolocalization of Microvessels and in Situ TUNEL Assay.
Neovascularization and endothelial cell apoptosis were evaluated in human tumors grown in SCID mice (as described above). After retrieval, tumors were fixed in 10% buffered formalin overnight and processed for histology. Serial tissue sections were deparaffinized and antigen retrieval was achieved by microwaving for 14 min in 0.01 M citrate buffer (pH 6.0). Human microvessels were identified by incubating sections for 1 h with 2 µg/ml monoclonal mouse antihuman CD34 antibody (Serotec, Raleigh, NC; Ref. 11
). Sections were incubated with appropriate secondary antibodies, and visualization of the complex was accomplished using Vectastain Elite ABC (Vector Laboratories, Burlingame, CA). In situ TUNEL staining (Apoptag peroxidase in situ detection kit; Oncor, Gaithersburg, MD) was used according to the manufacturer’s instructions to identify apoptotic cells. All of the areas in which cells were stained with the endothelial cell marker CD34 (27)
were considered a microvessel, independently of their size or of the presence of a visible lumen or erythrocytes. Apoptotic microvessels were defined as blood vessels containing at least one ApopTag+ endothelial cell. The utilization of two consecutive tissue sections stained for CD34 or ApopTag allowed for the determination of the endothelial origin of ApopTag+ cells when small microvessels had no visible lumen or erythrocytes.
ELISA.
HDMEC-Bcl-2, HDMEC-LXSN, or parental HDMEC were cultured on collagen type I gels (Vitrogen 100) and fed with EGM-MV (Clonetics). Alternatively, HDMEC were cultured on Vitrogen 100 and fed with EGM-MV supplemented with 50 ng/ml rhVEGF (Intergen) to induce Bcl-2 expression (10
, 11)
. After 72 h in culture, HDMECs were retrieved from collagen with a solution of 2.5 mg/ml collagenase A, type IV (Sigma) in PBS. Cells were resuspended in 0.1% BSA in PBS, lysed by three sequential freeze/thaw cycles and centrifuged, and the supernatant used for ELISA. Expression levels of cell-associated IL-8, bFGF, and VEGF were examined by ELISA (Quantikine immunoassay kits; R&D Systems) according to the manufacturer’s instructions.
Transfection of HDMECs with Bcl-2 Antisense ODNs and Western Blot.
The construction and transfection of antisense bcl-2 were performed as described previously (28)
. The bcl-2 antisense phosphorothioate ODN (5'-AATCCTCCCCCAGTTCACCC-3'), or its scrambled sequence control (5'-ACACCCCAATTCTTCCGCCC-3'), were purified by high-pressure chromatography and transiently transfected into HDMECs with DOTAP (Boehringer Mannheim Corp., Indianapolis, IN). Ten or 20 µg of ODN were mixed with 50 or 100 µl of DOTAP and allowed to form complexes for 15 min at room temperature. The mixtures were diluted in 10 ml of EGM-MV and added to 1 x 106 HDMECs cultured in 100-mm culture dishes. Controls were treated with equivalent concentrations of DOTAP or EGM-MV alone. After a 48-h incubation at 37°C, cells were washed, trypsinized, seeded in Vitrogen 100-coated dishes, and cultured for an additional 48 h in EGM-MV that was supplemented or not with 50 ng/ml VEGF. Whole cell lysates (29)
were resolved by gel electrophoresis and membranes were probed overnight at 4°C with 0.1 µg/ml monoclonal hamster anti-Bcl-2 (15131A, PharMingen), or with 0.05 µg/ml monoclonal mouse anti-
-tubulin (CP06; Calbiochem-Novabiochem Corp., San Diego, CA) to control for equal loading. Blots were exposed to appropriate peroxidase-coupled secondary antibodies (Amersham Corp., Arlington Heights, IL) and washed, and the bound antibody was detected with ECL (Amersham). Relative band densities were measured using NIH Image software.
Passive Immunization with Anti-IL-8 Antibody.
Systemic administration of neutralizing anti-IL-8 antibody was performed as described previously (15)
. Briefly, PLLA sponges were seeded with 0.9 x 106 HDMECs and 0.1 x 106 SLK cells as described above. Two sponges were implanted into each of 12 SCID mice (Taconic). After implantation, 4 mice were injected i.p. every other day with 0.25 ml of 5 µg/ml of a polyclonal antihuman IL-8 antibody (R&D Systems); or with 5 µg/ml of an isotype-matched nonspecific immunoglobulin (R&D Systems) in PBS; or with PBS. Mice were killed 3 weeks after implantation, and the implants were retrieved and analyzed.
Incorporation of Anti-IL-8 Antibody into Sponge Implants.
The preparation of bioabsorbable polymer scaffolds for the slow release of antihuman IL-8 antibody were prepared as described (30)
. Briefly, 5.5 mg of poly-lactic-coglycolic acid polymer particles (Boehringer Ingleheim, Ingleheim, Germany) were combined with 250 µl of 0.1% alginate (MVM; ProNova, Oslo, Norway) and either 5 µg of polyclonal antihuman IL-8 antibody (R&D Systems) or 5 µg of isotype-matched nonspecific immunoglobulin (R&D Systems). These mixtures were vortexed for 10 s, flash-frozen in liquid nitrogen, and lyophilized. The resulting powder was mixed with 100 mg of NaCl (Sigma) and processed to yield discs in which the polymer particles fused to form a continuous matrix entrapping alginate, protein, and salt particles. After foaming, the alginate was gelled, and the NaCl particles were leached out yielding macropores within the protein-containing polymer matrix by placing each disc in 0.1 M CaCl2 for 24 h. Sponges were sterilized in 100% ethanol for 15 min and washed six times in sterile PBS. Sponges were then seeded with 0.9 x 106 HDMECs and 0.1 x 106 SLK cells. One sponge containing antihuman IL-8 antibody and one sponge containing the isotype-matched immunoglobulin were implanted dorsally into each of eight SCID mice (Taconic). Mice were killed after 3 weeks and the sponges retrieved for analysis.
Statistical Analysis.
Statistical significance was determined using one-way ANOVA or the Student-Newman-Keuls test.
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Overexpression of Bcl-2 in Microvascular Endothelial Cells Enhances Tumor Angiogenesis and Tumor Growth.
We have previously demonstrated that human functional microvessels can be engineered in SCID mice by implanting HDMECs seeded in biodegradable sponges s.c. (11
, 12)
. These cells organize into microvessels that anastomose with the host vasculature and transport mouse blood cells (11)
. Here we used this model system to evaluate the effect of endothelial cell Bcl-2 on tumor neovascularization and growth. We implanted transduced HDMECs along with human oral squamous cell carcinoma (OSCC-3) in SCID mice, and observed the development of tumors (Fig. 1C and D)
populated with human CD34 positive microvessels (Fig. 2B)
. Tumors that developed in implants populated with HDMEC-Bcl-2 and OSCC-3 were significantly larger in volume and weight (P < 0.01) as compared with tumors in implants containing HDMEC-LXSN and OSCC-3 (Fig. 1, A–D)
. Similar results were observed in implants containing SLK and endothelial cells (Fig. 1, A and B)
, suggesting that the effect of endothelial cell Bcl-2 on tumor growth was independent of tumor type.
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but did not demonstrate any features indicative of a transformed phenotype (11)
. In vitro, HDMEC-Bcl-2 cells were neither anchorage independent nor did they exhibit immortalized life span (data not shown). Coimplantation of endothelial cells and OSCC-3 uniformly resulted in larger tumors than implantation of OSCC-3 alone. This finding underlines the role of enhanced angiogenesis in the tumor growth (Fig. 1)
.
We observed that implants populated with HDMEC-Bcl-2 and tumor cells were more vascularized (P < 0.01) than implants containing HDMEC-LXSN and tumor cells (Fig. 2A)
. In addition to enhanced neovascularization, implants containing HDMEC-Bcl-2 exhibited a marked reduction in the number of endothelial cells undergoing apoptosis (Fig. 2, A and D)
. In contrast, most tumors populated with control HDMECs showed a substantial number of apoptotic endothelial cells (Fig. 2, A and C)
. Apoptotic tumor microvessels showed frequent signs of aggregation of erythrocytes (Fig. 2C)
.
Neutralizing IL-8 Antibody Attenuates the Angiogenic Phenotype of Endothelial Cells That Overexpress Bcl-2.
We have recently reported that endothelial cells overexpressing Bcl-2 not only show enhanced survival, but they also engage in more vigorous angiogenic responses in vitro and in vivo (11)
. Interestingly, HDMEC-Bcl-2 have the ability to spontaneously develop capillary-like sprouts on collagen gels in vitro, a phenomenon not observed with control HDMECs (11)
. We, therefore, reasoned that HDMEC-Bcl-2 cells produced a soluble factor that promoted angiogenesis and perhaps tumor growth. We thus assayed HDMEC-Bcl-2 cell lysates by ELISA for production of endogenous mediators of angiogenesis. Among the proangiogenic mediators detected, the chemokine IL-8 was increased 15-fold in HDMEC-Bcl-2 cells as compared with control HDMECs (Fig. 3A)
. In contrast, the expression level of other angiogenic factors such as bFGF and VEGF was not altered in HDMEC-Bcl-2 as compared with controls (data not shown). HDMECs were exposed to VEGF to determine whether this growth factor, known to induce Bcl-2 expression, would also affect the expression of IL-8. We found a 4-fold increase in cell-associated IL-8 expression in HDMECs exposed to VEGF (Fig. 3A)
. Western blot analysis of the same cells revealed that Bcl-2 expression in HDMECs exposed to VEGF and HDMEC-Bcl-2 was increased 3-fold and 8-fold, respectively, as compared with levels of Bcl-2 expression in untreated HDMECs (Fig. 3B)
. Increasing levels of Bcl-2 expression in endothelial cells (Fig. 3B)
correlated with up-regulation of IL-8 expression (Fig. 3A)
.
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. Conversely, VEGF failed to induce Bcl-2 expression in HDMECs that were transfected with antisense constructs, particularly [HDMEC-AS-Bcl-2(b)] (Fig. 3B)
. ELISA demonstrated that VEGF also failed to induce IL-8 expression in HDMEC-AS-Bcl-2(b), whereas in HDMEC-SC/AS-Bcl-2, the expression of IL-8 was increased 5-fold as compared with untreated HDMEC (Fig. 3A)
.
Cell proliferation and sprouting assays were performed to examine whether HDMEC-Bcl-2 CM was angiogenic in vitro, and to evaluate the role of IL-8 in this process. We observed that HDMEC-Bcl-2 CM added to HDMEC cultures induced cell proliferation and sprouting, and that anti-IL-8 antibody partially blocked these effects in a dose-dependent manner (Fig. 3, C and D)
. The rat corneal micropocket assay confirmed these results in vivo. HDMEC-Bcl-2 CM induced potent neovascularization in corneas (Fig. 4A)
, which resembled the angiogenic response seen with purified IL-8 (Fig. 4D)
. However, the angiogenic activity of HDMEC-Bcl-2 CM was consistently attenuated on addition of anti-IL-8 antibody to the implants (Fig. 4B)
. HDMEC CM consistently failed to induce corneal neovascularization (Fig. 4C)
. Taken together, these results demonstrate that IL-8 is a key mediator of the neovascularization induced by endothelial cells overexpressing Bcl-2.
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50% reduction (P < 0.01) in tumor weight (Fig. 5A)
and 33% reduction (P < 0.01) in tumor volume (Fig. 5B)
. In contrast, tumors treated with control IgG grew vigorously throughout the time period evaluated. A similar trend was observed when HDMECs and SLK cells were implanted in sponges containing anti-IL-8 antibody. We observed a 50% reduction in both tumor weight (P < 0.01) and volume (P < 0.01) as compared with controls (Fig. 5, A and B)
. The inhibitory effect of neutralizing IL-8 was also reflected in the intensity of tumor neovascularization (Fig. 5C)
. Tumors treated with the control antibody were rapidly and extensively vascularized, forming numerous branching vascular channels (Fig. 5, C–E)
. In contrast, tumors treated with anti-IL-8 antibody showed a dramatic reduction (P < 0.01) in intratumoral microvascular density (Fig. 5, C and F)
. Areas of the tumor that were deficient in vascularization correlated with an apparent decrease in tumor cell density (Fig. 5F)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the NIH (to P. J. P.) and Coordenaç
o de Aperfeiçoamento de Pessoal de Nível Superior (to J. E. N.). 
2 To whom requests for reprints should be addressed, at University of Michigan School of Dentistry, 1011 North University, Room 5211, Ann Arbor, MI 48109-1078. Phone: (734) 936-9300; Fax: (734) 936-1597; E-mail: jenor{at}umich.edu
3 The abbreviations used are: VEGF, vascular endothelial growth factor; IL, interleukin; SCID, severe combined immunodeficient; HDMEC, human dermal microvascular endothelial cell; PLLA, poly(L-lactic acid); CM, conditioned medium/media; bFGF, basic fibroblast growth factor; ODN, oligodeoxynucleotide.
Received 7/ 5/00. Accepted 1/ 2/01.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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), 1 µg/ml anti-IL-8 (
), 5 µg/ml anti-IL-8 (
), or 10 µg/ml anti-IL-8 (
); or to HDMEC CM (
). Data represent mean values ± SE of daily counts in 10 random fields from three independent experiments.
