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Research Communications |
a Human Genome Sciences, Inc., Rockville, Maryland 20850, USA
b Cytokine Research Laboratory, Department of Molecular Oncology, the University of Texas, M.D. Anderson Cancer, Houston, Texas 77030, USA
c Georgetown University Medical Center, Lombardi Cancer Center, Washington, D.C. 20007, USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: TNF • cytokine • anti-tumor • anti-angiogenesis • EST
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF)2 was first identified as a cytokine that caused the abrogation of tumors and has been investigated for clinical uses toward the treatment of various tumors (1–5). TNF is also implicated in pathological conditions such as septic shock, autoimmune disorders, and graft-versus-host disease. However, TNF is a highly pleiotropic cytokine whose biological activities include cell cytotoxicity, mediation of cell proliferation, and immune responses, depending on the target cells. These diverse functions result in general toxicity in vivo (1–5). The TNF ligand and receptor families are emerging gene families that play important roles in immune regulation and inflammation. In addition to TNF, lymphotoxin (LT or TNFß), lymphotoxin ß (LTß), and ligands for CD27, CD30, CD40, OX40, 4–1BB, and Fas, which were cloned by classical biochemical approaches and receptor–ligand interactions, new members of the TNF ligand family, TRAIL (APO-2L) (6, 7), TWEAK (8), TRANCE/RANK (9, 10), and LIGHT (11), have been identified from an expressed sequence tag (EST) database. In this report, we describe the identification of a new member of the TNF ligand family. Unlike other members of this family, vascular endothelial growth inhibitor (VEGI) is produced predominantly by the endothelial cells. We have shown that VEGI is a vascular endothelial growth inhibitor that suppresses the growth of colon carcinoma in mice via gene transfer. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning and sequencing of VEGI full-length cDNA
The cDNA clone containing the original EST was used to synthesize 32P-labeled probes. A lambda zap (Stratagene, San Diego, Calif.) cDNA library constructed using HUVEC cells was screened by conventional methods using nylon filter lifts. Six positive clones were isolated from approximately 106 plaques. The cDNA clones were converted into pBlueScript (SK+) plasmid (Stratagene) using helper phages by following a protocol provided by the manufacturer. The insert sizes of the cDNA clones were determined by polymerase chain reaction (PCR) using a vector-specific oligonucleotide (M13 reverse primer) and a gene-specific oligonucleotide. A cDNA clone containing the largest insert (4.5 kb) was sequenced using the Applied Biosystems automated sequencer, ABI 373 (Perkin-Elmer, Norwalk, Conn.). The partial sequence of the same gene was recently reported in which no function was identified (13).
Northern blot analyses
VEGI mRNA expression was detected on multiple tissue blots purchased from Clontech Laboratories, Inc. (Palo Alto, Calif.). Hybridization of the blots with radiolabeled VEGI probe was performed using manufacturer's suggested conditions. For RNA blots containing the RNA from cell lines, total RNA was purified using RNAzol (Qiagen, Chatsworth, Calif.) and electrophoresed on 1.2% formaldehyde agarose gel and blotted on a Nylon filter. Northern blot analysis was performed using standard procedure.
Transfection of VEGI into MC-38 cells
A fusion protein (VEGI/IL-6) consisting of the secretion signal of interleukin-6 (IL-6) and residues 23–174 of VEGI was constructed by PCR (5'-primer: 5'-GCG GGATCCG CCACCATGAA CTCCTTCTCC ACAAGCGCCT TCGGTCCAGT TGCCTTCTCC CTGGGGCTGC TCCTGGTGTT GCCTGCTGCC TTCCCTGCCC CAGTTGTGAG AC-3', containing a BamH I restriction endonuclease site, the first 84 bases of IL-6 coding sequence and 18 bases of VEGI starting from Pro23; 3'-primer: 5'-CGC GGATCCG ATATTTGCTC TCCTCCTCA-3', containing a BamH I restriction endonuclease site and a stop codon). For transfection of the full-length VEGI and IL-6/VEGI into MC-38 cells, the constructs were generated by cloning the inserts into the pcDNA3 expression vector (Invitrogen, Carlsbad, Calif.). Subsequent to transfection, G418 selection, and cloning, three clones for each constructs were picked for tumorigenicity studies. The expression of VEGI in MC-38 cells was confirmed by Northern blot analysis.
In vivo tumorigenicity assay
Female C57BL/6 mice, 6–7 wk old, were purchased from Harlan Sprague Dawley (Indianapolis, Ind.). Various groups of MC-38 cells (2x105 cells/mouse) were injected into C57BL/6 mice. Mice were then ear tagged and randomized and tumors were measured twice weekly in a blinded fashion. The tumor size was assessed by measuring perpendicular diameters with a caliper and calculated by multiplying the measurements of diameters in two dimensions. Data are represented as the mean ± SD of nine mice in each group and the experiments were repeated with similar results.
Immunohistochemical analyses
Excised tumors were fixed in formalin and five-micron sections were cut and mounted on Superfrost Plus slides. Sections were stained with the following antibodies according to the manufacture's procedure: hematoxylin and eosin (H&E) or GR-1 (Ly-6G), a rat anti-mouse monoclonal antibody (mAb) that specifically recognizes neutrophils/granulocytes (PharMingen, San Diego, Calif.), or rabbit anti-human von Willebrand factor (vWF) mAb, which specifically recognizes the endothelial cells (DAKO Corp., Carpinteria, Calif.), or MMAC, a rabbit anti-mouse mAb that specifically recognizes monocytes and macrophages. The amounts of Gr-1+ neutrophils, vWF+ endothelial cells, or MMAC+ macrophages and the total tumor areas of each tumor were determined by the use of BioQuant-True image analysis system (R&M Biometrics, Nashville, Tenn.).
Cell proliferation assay
MC-38 tumor cells or the adult bovine aortic endothelial cells (ABAE) were harvested and seeded in wells of a 96-well plate at 3000–5000 cells/well in the appropriate growth medium containing 10% fetal bovine serum and 1 ng/ml of basic fibroblast growth factor (bFGF). The conditioned media collected from the soluble VEGI-expressing or the neo vector-transfected cells were then added at a final dilution 1:10 into each well. The cells were incubated in a final volume of 200 µl for a 4–5 days. Alamar blue was added to each well to a final concentration of 10%. The cells were incubated for 4 h. Cell viability was measured by reading in a CytoFluor fluorescence reader with excitation at 530 nm and emission at 590 nm. All assays were done in triplicates.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fig. 1A.
The cDNA encodes an open reading frame of 174 amino acids. There is an in-frame stop codon upstream of the predicted initiation codon, indicating that the translation cannot start further upstream. It is interesting that there are long untranslated regions at both ends of open reading frame. It is not clear whether these long untranslated regions play any functional role. The predicted protein VEGI exhibits 20–30% sequence homology to human TNF, TNF, and the Fas ligand ( Fig. 1B), similar to that among other TNF family members. In addition, two tryptophan (W42 and W132) and four tyrosine residues (Y70, Y73, Y111, and Y136) are conserved in all four sequences. Residues N59 and N152 may be potential glycosylation sites. A protein with a molecular mass of 22 kDa was produced in an in vitro transcription and translation experiment using the cDNA clone as a template (data not shown), consistent with the predicted open reading frame. Hydrophobicity analysis of the protein predicts a 13 amino acid hydrophobic region that follows the amino-terminal segment of 12 residues. This is characteristic of type II transmembrane proteins, with the carboxyl terminus on the exterior cell surface (residues 26–174), a single transmembrane domain, and a short cytoplasmic tail. Thus, this novel protein resembles other TNF family members, with the exception of TNFß (1). Since the new protein was subsequently found to be able to inhibit endothelial cell growth (see below), it is designated VEGI. The accession number for human VEGI cDNA in GenBank is AF039390.
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VEGI is specifically expressed in endothelial cells
The fluorescence in situ hybridization mapping procedure was used to investigate VEGI localization. Detailed analyses of seven individual chromosomes indicated that the VEGI gene is located within band 9q32. Using multiple tissue Northern blots, the VEGI transcript was found to be expressed in placenta, lung, kidney, skeletal muscle, pancreas, spleen, prostate, small intestine, and colon. Little VEGI signal was detected in heart, brain, liver, thymus, testis, ovary, and peripheral blood lymphocytes ( Fig. 2A).
The size of VEGI mRNA is approximately 6.5 kb. It is unusual for a human gene of 6.5 kb to contain only a small open reading frame of 522 nucleotides. Unlike other members of the TNF family, VEGI is specifically expressed in endothelial cells. Northern analyses of total RNA preparations from 23 cell lines and primary cell cultures showed that VEGI mRNA was detected only in HUVEC and human venous endothelial (VE) cells ( Fig. 2B). The mRNA of VEGI was not detected in a later passage of the venous endothelial (VE-2) and human artery endothelial cells. Analysis of a panel of primary endothelial cells derived from different organ origins showed that VEGI is expressed in subset of endothelial cells such as HUVEC and HMMVEC but not in HPAEC, HIAEC, or HCAEC (data not shown). These results indicate that only a subset of endothelial cells express the gene. The observation that VEGI is highly expressed in some vascularized tissues such as kidney but not in other vascularized tissues, such as heart, could be explained by the fact that VEGI is only detected in a subset of endothelial cells.
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Potent in vivo anti-tumor activities of the soluble VEGI via gene therapy
The amino acid sequence of VEGI homologous to the TNF family prompted us to examine whether the novel gene has potential anti-cancer activity. In addition to the full-length VEGI, a secreted form of VEGI (VEGI/IL-6) was constructed by replacing the amino-terminal hydrophobic segment of VEGI (residues 1–22) with the IL-6 signal peptide and was cloned into the eukaryotic expression vector pcDNA3. The murine colon carcinoma MC-38 cells were transfected with expression vectors expressing either full-length (VEGI) or the secreted form (VEGI/IL-6) of VEGI. Several stable cell lines expressing either the full-length VEGI or IL-6/VEGI were selected. Expression of VEGI on these tumor cells was confirmed by Northern blot analysis. VEGI transfection did not alter the in vitro proliferation rates of these tumor clones. These tumor cells were then subcutaneously injected into syngeneic C57BL/6 mice to test their abilities to form tumors. The two MC-38 stable cell lines expressing the full-length VEGI gene (VEGI-C1 and VEGI-C2) grew normally and caused rapid growth of tumors at a rate similar to that of wild-type MC-38 and cells transfected with the vector alone, MC-38/vector. On the other hand, all three cell lines expressing the secreted form of VEGI, including VEGI/IL-6-C1, VEGI/IL-6-C2, and VEGI/IL-6-C3, showed potent inhibition of the growth of MC-38 colon cancers in syngeneic C57BL/6 mice ( Fig. 3).
Thus, the replacement of the amino-terminal VEGI sequence with the IL-6 signal peptide, which make the VEGI protein soluble, led to suppression of tumor growth in vivo under the experimental setting. Our data indicated that the deletion of amino-terminal cytoplasmic and transmembrane domains of VEGI (residues 1–23) from the full-length protein appear to be critical for the inhibition of tumor growth in vivo.
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The soluble VEGI reduces the microvessel density in tumors
Immunohistochemical analyses were performed to investigate the potential mechanisms of action for VEGI-mediated tumor rejection. As shown in Fig. 4,
immunohistochemical staining with an antibody against vWF, a specific marker for the endothelial cells, revealed a significant reduction of the intratumoral microvessel density in the soluble VEGI/IL-6-expressing tumors ( Fig. 4f), as compared with the neo control tumors ( Fig. 4b) and tumors expressing the membrane-bound VEGI ( Fig. 4j). Conversely, tumors with neither soluble nor membrane-bound VEGI recruit neutrophils and macrophages into the transfected tumors, as compared with the neo control tumors ( Fig. 4c, d, g, h, k, l). Consistently, there was a significant decrease of microvessels in VEGI/IL-6-expressing tumors: the average numbers of vWF+ endothelial cells (mean ±SD) per mm2 tumor size in neo control, VEGI, and VEGI/IL-6-transduced MC-38 tumors were 241 ±34, 356 ±49, and 117 ±10, respectively (P<0.03), based on the immunohistological staining using the vWF mAb. In contrast, the mean values ±SD of Gr-1-positive neutrophils per mm2 tumor size in neo control, VEGI, and VEGI/IL-6-transduced MC-38 tumors were 182 ±88, 160 ±104, and 102 ±81, respectively (P>0.05). These data suggest that the soluble form of VEGI, but not the membrane-bound VEGI, produced locally through transfection into tumor cells inhibits tumor-induced neovascularization in vivo. On the other hand, injection of VEGI-expressing tumor cells did not cause gross abnormalities in the syngeneic C57BL/6 mice, such as weight loss during the experiment period. Our data suggest that locally producing the soluble VEGI via gene transfer might exert a potent anti-tumor effect without inducing systemic toxicity.
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VEGI inhibits the in vitro proliferation of the endothelial cells
To further investigate whether suppression of the in vivo tumor growth by the soluble VEGI was due to an anti-angiogenic effect or direct anti-tumor activity, the conditioned media were collected from cells expressing membrane-bound VEGI or the soluble VEGI/IL-6 and tested for their ability to inhibit the proliferation of MC-38 tumor cells or ABAE bovine aortic endothelial cells in vitro. As shown in Fig. 5,
proliferation of ABAE endothelial cells stimulated by 1 ng/ml of bFGF was significantly inhibited by the conditioned media derived from the VEGI/IL-6-expressing cells. In contrast, no inhibitory activities on ABAE in vitro growth were observed in the conditioned media from the vector control or the membrane-bound VEGI-expressing cells. The data are consistent with the immunohistological studies demonstrating that the soluble VEGI/IL-6 inhibited intratumoral microvessel formation in vivo. However, overexpression of VEGI or VEGI/IL-6 in MC-38 tumor lines did not alter tumor cell growth rates in vitro. Thus, the soluble VEGI-mediated tumor rejection via gene transfer probably is at least in part due to its anti-angiogenic activity in inhibiting the endothelial cell proliferation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The recent identification of the genes encoding TNF-like ligands and receptors has led to a better understanding of the roles and mechanisms of action of these molecules (1–5). To identify endothelial cell expression-specific gene(s), we have constructed 8 cDNA libraries using RNA derived from various endothelial cells, and generated approximately 30,000 ESTs. The ESTs were compared with ESTs in the human genome databases derived from a wide variety of types of cells. The ESTs unique to endothelial cells were further analyzed for sequence homology to the TNF family. VEGI was identified from an HUVEC cDNA library by EST database substraction. Like other TNF-related ligands, VEGI is a type-II transmembrane protein with the carboxyl terminus on the exterior cell surface, a single transmembrane domain, and a short cytoplasmic tail. It is a novel protein that exhibits 20–30% sequence homology to human TNF, TNFß, and the Fas ligand.
It is well documented that members of the TNF family display a wide variety of biological activities (1–5). Despite the functional redundancy of this family, specificity may be accomplished through restricted spatial and temporal expression of the ligands and their receptors to specific cell types. In this study, we described the identification of VEGI, a novel ligand of the TNF family, derived from the HUVEC endothelial cell cDNA library. Remarkably, VEGI expression is restricted to the endothelial cells according to Northern blot analysis and EST database search, which suggests that VEGI may play a role in the function of the normal vasculature.
TNF is a potent cytokine that evokes multiple biological responses: it alters the proliferation, differentiation, and metabolism of a wide variety of cells (2) and activates the immune responses by activated macrophages, cytotoxic T cells, natural killer cells, and neutrophils (1, 14). The anti-tumor effects of TNF have been well demonstrated by systemic administrations of the protein as well as by local production via gene transfer (14–16) . The homology of VEGI to the TNF family prompted us to examine whether the novel gene has potential anti-cancer activity. We approached this by using gene transfer in a syngeneic tumor model, since gene therapy has been valuable in assessing the anti-tumor effects of various cytokines (11, 17–19). As shown in 
Fig. 3, transgenic overexpression of the soluble VEGI completely suppressed the growth of MC-38 colon cancers in syngeneic C57BL/6 mice. However, no significant inhibitory effect on in vitro cell proliferation was observed in murine colon cancer MC-38 cells stably transfected with VEGI, suggesting that the expression of VEGI did not directly cause cytotoxicity in these tumor cells in vitro.
A number of possible mechanisms may account for the anti-tumor effects of VEGI observed in the syngeneic tumor model. The endothelial cell-specific expression of VEGI suggests of a possible mechanism by which the anti-tumor effect observed is mediated through the control of tumor angiogenesis. This working hypothesis of VEGI was further supported by our observation that immunohistologic staining with the endothelial-specific marker vWF reveals a significant reduction in the intra-tumoral microvessel density in VEGI/IL-6-transduced tumors. Consistently, the conditioned media obtained from cells expressing soluble but not the membrane-bound-VEGI specifically inhibit in vitro proliferation of ABAE endothelial cells, but not proliferation of MC-38 tumor cells. Thus, local production of the soluble VEGI via gene therapy shifts the net balance between angiogenic stimulators and inhibitors, thus inhibiting tumor growth in vivo. It remains to be seen whether other mechanisms, such as activation of tumor-specific or nonspecific B or T lymphocytes or induction of cytokines, would also be involved in the soluble VEGI-mediated tumor rejection.
We have recently demonstrated that local production of LIGHT, a newly discovered TNF-like cytokine, caused marked infiltration of neutrophils into tumors (11). Our results are consistent with those from other laboratories in which CD95L-mediated neutrophil recruitment was responsible for the primary tumor rejection (17). However, such a phenomenon was not observed for VEGI-transfected tumors under our experimental setting. Whether the VEGI protein possesses similar activities under other conditions remains to be investigated. Regardless of the exact mechanisms of VEGI action, our results showed that a soluble version of VEGI, which correspond to the putative extracellular domain consisting of residue 23–174, is capable of inhibiting the growth of endothelial cells in vitro, and in vivo. Thus, VEGI may function as an angiogenesis inhibitor.
Angiogenesis is required for a variety of physiological processes such as organogenesis during fetal development, wound healing, and organ regeneration. Abnormal neovascularization leads to progression of many diseases such as cancers and diabetic retinopathy. Recent studies have also demonstrated that tumor growth and metastasis are angiogenesis dependent (20–22), and result from up-regulation of angiogenic stimulators such as VEGF or bFGF and/or down-regulation of angiogenesis inhibitors like endostain (20).
The in vivo anti-tumor effects of VEGI-gene therapy may be mediated by potentiating both anti-angiogenic and other nonspecific immune responses. Since the anti-tumor activity of VEGI was demonstrated by local production of the protein via gene transfer, additional studies including the administration of VEGI protein in vivo (which is not currently available) would be necessary to prove this conclusion. The generation of recombinant VEGI protein, development of antibodies against VEGI, and further identification of its natural forms as well as its receptor(s) should provide tools to understand the biological functions of VEGI.
In summary, we have identified and functionally characterized a novel member of the TNF superfamily. Our major findings are: 1) VEGI is specifically expressed by the endothelial cells as demonstrated by the Northern blot analyses and supported by its unique presence in the expression tags isolated only from the endothelial cells; 2) Local production via in vivo gene transfer of the secreted form, but not the membrane-bound form of VEGI, inhibits the growth of colon carcinomas in C57BL/6 mice; 3) Histological examination of the tumors showed marked reduction of vascularization in MC-38 tumors that express soluble but not membrane-bound VEGI or when transfected with only the control vector. On the other hand, VEGI expression does not attract the neutrophils and macrophages to infiltrate into the tumor mass. 4) The conditioned media from cells expressing soluble VEGI drastically inhibit in vitro proliferation of the ABAE cells. These results indicate that VEGI is a novel angiogenesis inhibitor of the TNF family and functions at least in part by directly inhibiting the proliferation of endothelial cells. Thus, this novel gene will be a valuable tool for future study of the molecular mechanism of vascular biology and will also serve as a potential target in the development of angiogenesis-based cancer therapy.
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FOOTNOTES |
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2 Abbreviations: ABAE, adult bovine aortic endothelial cells; bFGF, basic fibroblast growth factor; EST, expression sequence tag; HUVEC, human umbilical vein endothelial cells; HMMVEC, human myometrialuterine microvascular endothelial cells; HPAEC, human pulmonary artery endothelial cells; HIAEC, human iliac artery endothelial cells; HCAEC, human coronary artery endothelial cells; IL, interleukin; LT, lymphotoxin; mAb, monoclonal antibody; PCR, polymerase chain reaction; TNF, tumor necrosis factor; VE, venous endothelial; VEGI, vascular endothelial growth inhibitor; vWF, von Willebrand factor.
Received for publication August 12, 1998.
Revision received September 28, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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