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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 26, 2002 as doi:10.1096/fj.01-0757fje. |
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Department of Oncology, Lombardi Cancer Center, Georgetown University Medical Center, Washington D.C., USA;
* Division of Rheumatology and Immunology, Medical University of South Carolina, Charleston, South Carolina, USA; and
School of Dentistry, University of California at Los Angeles, Los Angeles, California, USA
3Correspondence: Georgetown University Medical Center, Department of Oncology, 3970 Reservoir Rd. NW, RB/E301, Washington DC 20007, USA. E-mail: lilu{at}georgetown.edu
SPECIFIC AIMS
We address the hypothesis that alternate forms of vascular endothelial cell growth inhibitor (VEGI) are expressed in endothelial cells and describe the identification of two novel isoforms of VEGI. We demonstrate that one of the isoforms, VEGI-251, is secreted and that its overexpression results in the inhibition of endothelial cell growth and attenuation of human breast cancer xenograft tumors by interfering with tumor neovascularization.
PRINCIPAL FINDINGS
1. Detection of multiple VEGI transcripts in human tissue
The initially discovered VEGI (accession number AF039390; referred to here as VEGI-174) had no effect on the growth of xenograft tumors formed by the stable transfectants, unlike a recombinant secreted form, sVEGI (see section 5, below). Overexpression of the full-length VEGI-174 in cancer cells did not give rise to a VEGI peptide in cell-conditioned media. It is thus plausible that other VEGI isoforms exist in nature whose activity may be mimicked by sVEGI. Indeed, Northern blot analysis of human tissues with full-length VEGI-174 cDNA as a probe revealed multiple VEGI transcripts of 7.5, 2.0, and 1.5 kb in adult liver, kidney, and spleen, respectively.
2. Cloning and characterization of novel VEGI isoforms
We analyzed human arrayed cDNA library panels by using rapid amplification of cDNA ends (RACE) and identified two new VEGI isoforms. They encode peptides of 251 (VEGI-251) and 192 (VEGI-192) amino acid residues. The 5' regions of the open reading frames of these isoforms are unique whereas their 3' regions, including the untranslated regions, are identical. The NH2 terminus of VEGI-251 contains a stretch of 20 hydrophobic residues suggestive of a signal peptide. Using isoform-specific probes on Northern blots, differential expression of VEGI-174 and VEGI-251 was detected in human tissues. The 7.5 kb VEGI-251 transcript was found at high levels in adult placenta, kidney, lung, and liver; the 2 kb VEGI-174 was in liver, kidney, skeletal muscle, and heart. VEGI-251 was more abundant than VEGI-174 in fetal kidney and lung. Similar to VEGI-174, the novel isoforms exhibit endothelial cell-specific expression when examined by ribonuclease protection assays, with VEGI-251 being the most abundant and VEGI-192 marginally detectable.
3. Synthesis of VEGI isoform RNA by alternate splicing and parallel modulation of the isoforms by cytokines
We determined VEGI gene organization by using isoform-specific primers to analyze human DNA preparations, and found that the human VEGI gene comprises four exons and spans 
17 kb. The carboxyl-terminal 151 amino acid residues, an active domain shared by all the isoforms, is encoded by part of the fourth exon. VEGI-251 and VEGI-192 transcripts are generated by the use of cryptic splice sites and alternate exons, whereas VEGI-174 is encoded on a single exon. Although only VEGI-251 appears relatively abundant in cultured endothelial cells, the mRNA molecules of all isoforms are up-regulated in parallel when stimulated with tumor necrosis factor 
(TNF-) (2–90 ng/ml). Parallel down-regulation is also observed after treatment with interferon 
(IFN-, 20 U/ml). This indicates that isoform functions are not controlled primarily by differential transcriptional regulation. Instead, various NH2 termini of the isoforms suggest that divergent intracellular distribution may account for potential differences in function.
4. Secretion of VEGI causes endothelial cell apoptosis
The highly hydrophobic nature of the putative signal peptide region suggests that VEGI-251 may be secreted from the cell. To test this hypothesis, we analyzed conditioned media of HUVE cells and VEGI-251-transfected human breast cancer MDA-MB-231 cells by immunoprecipitation and Western blotting. A VEGI peptide of 25 kDa was detectable (Fig. 1
) and was absent from the conditioned media of empty vector-transfected cells (Fig. 1A
). The VEGI peptide was not detected within total cell lysate of HUVE cells by Western blotting (Fig. 1B
). These results indicate that VEGI-251 can be secreted when overexpressed in mammalian cells and that HUVE cells produce a secreted form of VEGI likely to have been derived from VEGI-251. We also determined whether VEGI-251 possesses the same activity observed for a truncated recombinant VEGI, which consists of 146 amino acid residues common to the carboxyl-terminal segment of all isoforms. By overexpressing various VEGI constructs in HUVE cells with a lentiviral vector, we found that only secreted forms of VEGI exhibit inhibitory activity on endothelial cell growth. Thus, lentiviral transduction of HUVE cells with VEGI-251 or with a secreted fusion protein, sVEGI, consisting of a 28 residue secretion signal peptide derived from interleukin-6 and the VEGI common domain led to a dose-dependent decrease in the number of endothelial cells (Fig. 1C
). The VEGI-251 transduced cells were found to undergo apoptosis as determined by TUNEL assay (Fig. 1F, G
). In contrast, overexpression of VEGI-174 had no effect on the growth of these cells.
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5. VEGI-251 overexpression inhibits tumor neovascularization and progression
The effect of VEGI-251 overexpression was further examined in a mouse xenograft tumor model. Athymic nude mice were inoculated with MDA-MB-231 cells stably transfected with either pcDNA3 vector, VEGI-174, VEGI-251, or sVEGI. Tumors from VEGI-251 and sVEGI groups showed significantly reduced growth rates (Fig. 2
A). Microvessel densities in the tumors were markedly decreased in these two groups (Fig. 2B
).
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CONCLUSIONS AND SIGNIFICANCE
VEGI is an endothelial cell-specific gene and an inhibitor of endothelial cell growth. Previous studies have shown that overexpression of a recombinant secreted fusion protein, sVEGI, by cancer cells retarded xenograft tumor growth, demonstrating the importance of the secretory process for VEGI function. A soluble, active peptide could not have been derived from the initially discovered VEGI-174, however, because VEGI-174 overexpression had no effect on tumor growth. The discovery of VEGI-251 as a secretable protein provides evidence for a physiologically significant autocrine inhibitor of endothelial cell proliferation. Detection of transcripts for all three isoforms in endothelial cells suggests possibly different functions for the isoforms. Alternatively, the presence of several isoforms in the same cells may represent a mechanism of VEGI activity regulation.
Using a paradigm of endothelial cell activation with TNF-, we observed a parallel increase in mRNA levels for all VEGI isoforms, with VEGI-251 RNA remaining the most abundant. This indicates a common mode of transcriptional regulation for the isoforms, at least in the primary cultures of adult endothelial cells; mechanisms underlying developmental regulation of the synthesis of the isoforms and their respective functional role remain to be investigated. The data indicate that VEGI may mediate some of the actions of TNF- on endothelial cells, including endothelial cell growth inhibition. Contrary to known synergistic effects with TNF on endothelial cell apoptosis, another inflammatory cytokine, IFN-, elicited an opposite response in VEGI expression. Additional studies are needed to address the complex relationship between cytokines and VEGI action.
Detection of a VEGI-derived peptide in the conditioned media of VEGI-251-transfected cells indicates that VEGI-251 is a secreted protein. Inhibition of HUVE cell growth by VEGI-251 (but not VEGI-174) overexpression suggests secretion is critical for its effects and that secreted VEGI may act through an autocrine mechanism (Fig. 3
). Inhibition of tumor growth and the reduction in tumor microvessel density by VEGI-251 or sVEGI overexpression strongly support the view that a soluble VEGI is able to act on the endothelial cells surrounding the cancer cells to inhibit tumor angiogenesis. Intact VEGI-174 is not secreted, nor is it able to induce endothelial cell death when overexpressed. It is unclear, however, whether this isoform possesses other as yet uncharacterized activities. Clarification of distinct functions exerted by the various isoforms of VEGI will aid greatly in understanding the angiogenic process and in assessing the potential utility of VEGI peptides as a therapeutic agent.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0757fje; to cite this article, use FASEB J. (March 26, 2002) 10.1096/fj.01-0757fje 
2 These authors contributed equally to this work.
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