Human Herpesvirus-8-Transformed Endothelial Cells Have Functionally Activated Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor
2002; Elsevier BV; Volume: 160; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64344-1
ISSN1525-2191
AutoresRizwan Masood, Ethel Cesarman, D. Lynne Smith, Parkash S. Gill, Ornella Flore,
Tópico(s)Herpesvirus Infections and Treatments
ResumoKaposi's sarcoma is a vascular tumor commonly associated with human immunodeficiency virus (HIV)-1 and human herpesvirus (HHV-8) also known as Kaposi's sarcoma-associated herpesvirus. The principal features of this tumor are abnormal proliferation of vascular structures lined with spindle-shaped endothelial cells. HHV-8 may transform a subpopulation of endothelial cells in vitro via viral and cellular gene expression. We hypothesized that among the cellular genes, vascular endothelial growth factors (VEGFs) and their cognate receptors may be involved in viral-mediated transformation. We have shown that HHV-8-transformed endothelial cells (EC-HHV-8) express higher levels of VEGF, VEGF-C, VEGF-D, and PlGF in addition to VEGF receptors-1, −2, and −3. Furthermore, antibodies to VEGF receptor-2 inhibited cell proliferation and viability. Similarly, inhibition of VEGF gene expression with antisense oligonucleotides inhibited EC-HHV-8 cell proliferation/viability. The growth and viability of primary endothelial cells and a fibroblast cell line however were unaffected by either the VEGF receptor-2 antibody or the VEGF antisense oligodeoxynucleotides. VEGF and VEGF receptors are thus induced in EC-HHV-8 and participate in the transformation. Inhibitors of VEGF may thus modulate the disease process during development and progression. Kaposi's sarcoma is a vascular tumor commonly associated with human immunodeficiency virus (HIV)-1 and human herpesvirus (HHV-8) also known as Kaposi's sarcoma-associated herpesvirus. The principal features of this tumor are abnormal proliferation of vascular structures lined with spindle-shaped endothelial cells. HHV-8 may transform a subpopulation of endothelial cells in vitro via viral and cellular gene expression. We hypothesized that among the cellular genes, vascular endothelial growth factors (VEGFs) and their cognate receptors may be involved in viral-mediated transformation. We have shown that HHV-8-transformed endothelial cells (EC-HHV-8) express higher levels of VEGF, VEGF-C, VEGF-D, and PlGF in addition to VEGF receptors-1, −2, and −3. Furthermore, antibodies to VEGF receptor-2 inhibited cell proliferation and viability. Similarly, inhibition of VEGF gene expression with antisense oligonucleotides inhibited EC-HHV-8 cell proliferation/viability. The growth and viability of primary endothelial cells and a fibroblast cell line however were unaffected by either the VEGF receptor-2 antibody or the VEGF antisense oligodeoxynucleotides. VEGF and VEGF receptors are thus induced in EC-HHV-8 and participate in the transformation. Inhibitors of VEGF may thus modulate the disease process during development and progression. Kaposi's sarcoma (KS) is the most common tumor associated with human immunodeficiency virus (HIV)-1 infection.1Safai B Johnson KG Myskowski PL Cunningham-Randles S Godbold JH Dupont B The natural history of Kaposi's sarcoma in the acquired immunodeficiency syndrome.Ann Intern Med. 1985; 103: 744-750Crossref PubMed Scopus (218) Google Scholar, 2Haverkos HW Drotman DP Morgan M Prevalence of Kaposi's sarcoma among patients with AIDS.N Engl J Med. 1985; 312: 1518-1523PubMed Google Scholar, 3Ruszczak Z Silva AM Orfanos CE Kaposi's sarcoma in AIDS.Am J Dermatopathol. 1987; 9: 388-398Crossref PubMed Scopus (52) Google Scholar, 4Regezi JA Macphail LA Daniels TE De Souza YG Greenspan JS Greenspan D Human immunodeficiency virus-associated oral Kaposi's sarcoma: a heterogeneous cell population dominated by spindle-shaped endothelial cells.Am J Pathol. 1993; 143: 240-249PubMed Google Scholar Two features of AIDS-KS tumors include aberrant proliferation of vascular structures, proliferation of endothelial and spindle (tumor) cells, and enhanced vascular permeability. Endothelial cell (EC) growth factors are thus likely to play a central role in the development and progression of KS.5Nakamura S Salahuddin SZ Biberfeld P Ensoli B Markham PD Wong-Staal F Gallo RC Kaposi's sarcoma cells: long term culture with growth factor retrovirus-infected CD4+ T cells.Science. 1988; 242: 426-430Crossref PubMed Scopus (274) Google Scholar, 6Salahuddin SZ Nakamura S Biberfeld P Ensoli B Markham PD Larsson L Wong-Staal F Gallo RC Angiogenic properties of Kaposi's sarcoma-derived cells after long-term culture in vitro.Science. 1988; 242: 430-433Crossref PubMed Scopus (287) Google Scholar, 7Ensoli B Nakamura S Salahuddin SZ Biberfeld P Larsson L Beaver B Wong-Staal F Gallo RC AIDS Kaposi's sarcoma derived cells express cytokines with autocrine and paracrine growth effects.Science. 1989; 243: 223-226Crossref PubMed Scopus (418) Google Scholar, 8Barillari G Buonaguro L Fiorelli V Hoffman J Michaels F Gallo RC Ensoli B Effects of cytokines form activated immune cells on vascular cell growth and HIV-1 gene expression: implications for AIDS-Kaposi's sarcoma pathogenesis.J Immunol. 1992; 149: 3727-3734PubMed Google Scholar KS cells have previously been shown to produce several growth factors that have autocrine growth activity; these include basic fibroblast growth factor (bFGF), interleukin (IL)-1, IL-6, IL-8, and oncostatin-M. These factors are also shown to be expressed in the primary tumor tissue.7Ensoli B Nakamura S Salahuddin SZ Biberfeld P Larsson L Beaver B Wong-Staal F Gallo RC AIDS Kaposi's sarcoma derived cells express cytokines with autocrine and paracrine growth effects.Science. 1989; 243: 223-226Crossref PubMed Scopus (418) Google Scholar, 8Barillari G Buonaguro L Fiorelli V Hoffman J Michaels F Gallo RC Ensoli B Effects of cytokines form activated immune cells on vascular cell growth and HIV-1 gene expression: implications for AIDS-Kaposi's sarcoma pathogenesis.J Immunol. 1992; 149: 3727-3734PubMed Google Scholar, 9Miles SA Rezai AR Salazar-Gonzales JF Vander Meyden M Stevens RH Logan DM Mitsuyasu RT Taga T Hirano T Kishimoto T Martínez-Maza O AIDS-Kaposi's sarcoma-derived cells produce and respond to interleukin-6.Proc Natl Acad Sci USA. 1990; 87: 4068-4074Crossref PubMed Scopus (448) Google Scholar, 10Masood R Cai J Tulpule A Zheng T Hamilton A Sharma S Espina BM Smith DL Gill PS IL-8 is an autocrine growth factor and a surrogate marker for Kaposi's sarcoma.Clin Cancer Res. 2001; 7: 2693-2702PubMed Google Scholar, 11Nair BC DeVico AL Nakamura S Copeland TD Chen Y Patel A O'Neil T Oroszlan S Gallo RC Sarngadharan MG Identification of a major growth factor for AIDS-Kaposi's sarcoma cells as Oncostatin M.Science. 1992; 255: 1430-1432Crossref PubMed Scopus (216) Google Scholar, 12Li JJ Huang YQ Moscatelli D Friedman-Kien AE Expression of fibroblast growth factors and their receptors in acquired immunodeficiency syndrome-associated Kaposi's sarcoma tissue and derived cells.Cancer. 1993; 72: 2253-2258Crossref PubMed Scopus (36) Google Scholar, 13Ensoli B Gendelman R Markham P Fiorelli V Colombini S Raffeld M Cafaro A Chang HK Brady JN Gallo RC Synergy between basic fibroblast growth factor and human immunodeficiency virus type-1 Tat protein in induction of Kaposi's sarcoma.Nature. 1994; 371: 674-680Crossref PubMed Scopus (542) Google Scholar Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is an angiogenic factor that induces EC proliferation, angiogenesis, and enhances vascular permeability.14Leung DW Cachianes G Kuang W Goeddel DV Ferrara N Vascular endothelial growth factor is a secreted angiogenic mitogen.Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4466) Google Scholar, 15Tisher E Mitchell R Hartmann T Silva M Gospodarowicz D Fiddes JC Abraham JA The human gene for vascular endothelial growth factor.J Biol Chem. 1991; 266: 11947-11954PubMed Google Scholar, 16Dvorak HF Sioussat TM Brown LF Berse B Nagy JA Sotrel A Manseau EJ De Water LV Senger DR Distribution of vascular permeability factor (vascular endothelial growth factor) in tumors: concentration in tumor blood vessels.J Exp Med. 1991; 174: 1275-1278Crossref PubMed Scopus (468) Google Scholar VEGF/VPF receptors are localized primarily to the ECs. KS cells however also express VEGF and VEGF receptors (VEGFR) and use VEGF as an autocrine growth factor.17Masood R Cai J Zheng T Smith DL Naidu Y Gill PS Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma.Proc Natl Acad Sci USA. 1997; 94: 979-984Crossref PubMed Scopus (262) Google Scholar The role of VEGF in the pathogenesis of KS may thus be significant. Several VEGF-related proteins have been isolated by homology search and include VEGF-B, VEGF-C, VEGF-D, and PlGF (placental growth factor).18Enholm B Paavonen K Ristimaki A Kumar V Gunji Y Klefstrom J Kivinen L Laiho M Olofsson B Joukov V Eriksson U Alitalo K Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia.Oncogene. 1997; 14: 2475-2483Crossref PubMed Scopus (387) Google Scholar, 19Joukov V Pajusola K Kaipainen A Chilov D Lahtinen I Kukk E Saksela O Kalkkinen N Alitalo K A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt-4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar, 20Achen MG Jeltsch M Kukk E Makinen T Vitali A Wilks AF Alitalo K Staker SA Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk-1) and VEGF receptor 3 (Flt-4).Proc Natl Acad Sci USA. 1998; 95: 548-553Crossref PubMed Scopus (1020) Google Scholar, 21Maglione D Guerriero V Viglietto G Delli-Bovi P Persico MG Isolation of a human placental cDNA coding for a protein related to vascular permeability factor.Proc Natl Acad Sci USA. 1991; 88: 9267-9271Crossref PubMed Scopus (843) Google Scholar VEGF-C and VEGF-D are expressed primarily in lymphatic endothelium and bind to VEGFR-3 as homodimers and to VEGFR-2 and VEGFR-3 as heterodimers with VEGF. VEGF-C and VEGFR-3 expression in KS cells have also been shown.22Skobe M Brown LF Tongnazzi K Ganju RK Dezube BJ Alitalo K Detmar M Vascular endothelial growth factor-C (VEGF-C) and its receptors KDR and flt-4 are expressed in AIDS-associated Kaposi's sarcoma.J Invest Dermatol. 1999; 113: 1047-1053Crossref PubMed Scopus (108) Google Scholar These results are consistent with the consideration that KS may be derived from lymphatic endothelium. Isolation of Kaposi's sarcoma-associated herpesvirus/HHV-8 from KS tumor tissue and evidence for latent infection of KS spindle cells supports its role in KS pathogenesis.23Chang Y Cesarman E Pessin MS Lee F Culpepper J Knowles DM Moore PS Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.Science. 1994; 266: 1865-1869Crossref PubMed Scopus (5015) Google Scholar, 24Dupin N Fisher C Kellam P Ariad S Tulliez M Franck N Marck E Salmon D Gorin I Escande JP Distribution of human herpesvirus-8 latently infected cells in Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma.Proc Natl Acad Sci USA. 1999; 96: 4546-4551Crossref PubMed Scopus (611) Google Scholar Transformation of ECs with HHV-8 may suggest the role of this virus in the development of KS.25Flore O Rafii S Ely S O'Leary JJ Hyjek EM Cesarman E Transformation of primary human endothelial cells by Kaposi's sarcoma-associated herpesvirus.Nature. 1998; 394: 588-592Crossref PubMed Scopus (354) Google Scholar In this study, we examined the VEGFs in HHV-8-transformed cells. We show that expression of VEGF, VEGF-C, VEGF-D, PlGF, and their receptors is higher in transformed cells than in primary ECs. Furthermore, inhibition of VEGF binding to the cognate receptors or inhibition of VEGF expression reduces the proliferation and viability of HHV-8-transformed ECs. These studies indicate that induction of VEGF may be one of the ways that HHV-8 plays a role in KS pathogenesis. Human umbilical vein ECs and ECs transformed with HHV-8 (EC-HHV-8) were maintained as described.25Flore O Rafii S Ely S O'Leary JJ Hyjek EM Cesarman E Transformation of primary human endothelial cells by Kaposi's sarcoma-associated herpesvirus.Nature. 1998; 394: 588-592Crossref PubMed Scopus (354) Google Scholar The production of EC-HHV-8 has been described previously.25Flore O Rafii S Ely S O'Leary JJ Hyjek EM Cesarman E Transformation of primary human endothelial cells by Kaposi's sarcoma-associated herpesvirus.Nature. 1998; 394: 588-592Crossref PubMed Scopus (354) Google Scholar Briefly, purified HHV-8 virus particles were isolated from an EBV-negative primary effusion lymphoma cell line (BC-3). Human umbilical vein ECs were infected with 5 to 10 genome equivalents/cell and cultured in the presence of VEGF. These cultures have been continuously maintained for more than 4 years. ECs were grown on gelatin (1%)-coated flasks in Iscove-modified Dulbecco's media and F-12 Nutrient Mixture (Ham) (1:1) media supplemented with 15% fetal calf serum, 2 mmol/L glutamine, 30 μg/ml EC growth supplement (Boehringer Mannheim, Indianapolis, IN), 2 U/ml heparin, 100 U/ml penicillin, and 100 μg/ml streptomycin. No supplemental VEGF was added. T1 fibroblast cultures were obtained from Dr. Peter Jones, USC/Norris Comprehensive Cancer Center, and were grown in Dulbecco's minimal essential medium containing 10% fetal calf serum, penicillin, and streptomycin. KSC-10 is a long-term spindle cell isolate established from KS lesions of an AIDS-KS patient as previously described.26Masood R Husain SR Rhaman A Gill P Potentiation of cytotoxicity of Kaposi's sarcoma related to immunodeficiency syndrome (AIDS) by liposome-encapsulated doxorubicin.AIDS Res Hum Retroviruses. 1993; 9: 741-746Crossref PubMed Scopus (34) Google Scholar It has been maintained in RPMI 1640 medium supplemented with 15% fetal calf serum, 2 mmol/L glutamine, 0.5% essential amino acids, 0.5% nonessential amino acids, 1 mmol/L sodium pyruvate, and 1% Nutridoma HU (Boehringer Mannheim) in the absence of conditioned medium from transformed T cell lines. Neutralizing antibody to VEGFR-2 and polyclonal antibodies to VEGFR-1 and VEGFR-3 were obtained from R&D Systems (Minneapolis, MN). Phosphorothioate-modified oligonucleotides were synthesized and purified by Operon Technologies, Inc. (Alameda, CA). VEGF antisense (AS) oligonucleotides of the human VEGF-coding region shown previously to inhibit VEGF were used.17Masood R Cai J Zheng T Smith DL Naidu Y Gill PS Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma.Proc Natl Acad Sci USA. 1997; 94: 979-984Crossref PubMed Scopus (262) Google Scholar The sequence and location of AS-1 and AS-3 oligonucleotide are: AS-1, 5′-AGA CAG CAG AAA GTT CAT GGT-3′ (−3 to +18); AS-3, 5′-TGG CTT GAA GAT GTA CTC GAT-3′ (+261 to +281). An oligonucleotide consisting of a scrambled AS-3 sequence (S) 5′-TAC GTA GTA TGG TGT ACG ATC-3′ was used as a negative control. rhVEGF and VEGF enzyme-linked immunosorbent assay kits were purchased from R&D Systems. Cells (EC, EC-HHV-8) were seeded at a density of 1 × 104 per well in 24-well gelatin-coated plates on day 0. For experiments with antibodies, the cells were treated on day 1 at concentrations ranging from 10 to 1000 ng/ml and the cell viability was measured on day 3 by MTT assay. For experiments with oligonucleotides, cells were treated on days 1 and 3 at concentrations ranging from 1 to 10 μmol/L on, and the cell viability was measured on day 5 by MTT assay. The assays were performed in triplicate. Cells were plated as described, harvested, and total RNA was extracted. cDNAs were synthesized using a Superscript II kit (Life Technologies, Inc., Gaithersburg, MD) by standard protocols. Two μl of the cDNA reaction were amplified by RT-PCR for the VEGF family members, and as described earlier.17Masood R Cai J Zheng T Smith DL Naidu Y Gill PS Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma.Proc Natl Acad Sci USA. 1997; 94: 979-984Crossref PubMed Scopus (262) Google Scholar Amplification of the receptors was from 4 μl of cDNA. Primers for the amplification of VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF, and the receptors VEGFR-1, VEGFR-2, and VEGFR-3 are shown in Table 1. Each PCR cycle consisted of denaturation at 94°C for 1 minute, primer annealing at the temperatures indicated in Table 1 for 2 minutes, and extension at 72°C for 3 minutes. The samples were amplified for 30 cycles, 10-μl aliquots of PCR reaction mixtures were resolved by 1.5% agarose gel electrophoresis. The integrity and quantity of RNA was confirmed by RT-PCR for β-actin. RT-PCR reactions for the receptors were modified by increasing the primer concentration to 100 pmol.Table 1Sequences of Oligonucleotide Primers Used for RT-PCRGenePrimer sequences (forward and reverse 5′ to 3′)PositionSize (bp)Annealing temperature (°C)VEGF-A5′-CGA AGT GGT GAA GTT CAT GGA TG-3′+170/+192607, 535, 403605′-TTC TGT ATC AGT CTT TCC TGG TGA G-3′+681/+705VEGF-B5′-TGG CCA AAC AGC TGG TGC-3′+191/+208411555′-GAG GAA GCT GCG GCG TCG-3′+585/+602VEGF-C5′-GAT CTG GAG GAG CAG TTA CGG TC-3′+263/+285294605′-TTA AG AAG CTG TTT GTC GCG ACT-3′+535/+557VEGF-D5′-TTG TAC GTC CAG CTG GTG CAG′+40/+60320605′-CTC CAC GCA CGT TTC TCT AGG-3′+337/+357PlGF5′-ATG AGG CTG TCC CCT TGC TTC-3′+10/+30388605′-AGA GGC CGG CAT TCG CAG CGA A-3′+326/+398VEGFR-15′-CAA GTG GCC AGA GGC ATG GAG TT-3′+3262/+3284498625′-GAT GTA GTC TTT ACC ATC CTG TTG-3′+3736/+3759VEGFR-25′-GAG GGC CTC TCA TGG TGA TTG T-3′+2954/+2975709625′-TGC CAG CAG TCC AGC ATG GTC TG-3′+3640/+3662VEGFR-35′-GTG ACA GCC TGT CCA TCT CCT-3′+131/+151320605′-GGT TGA CCA CGT TGA GGT G-3′+431/+451β-actin5′-GTG GGG CGC CCC AGG CAC CA-3′546555′-CTC CTT AAT GTC ACG CAC GAT TTC-3′ Open table in a new tab Cells were collected onto glass slides using a Cytospin centrifuge (Shandon, Astmoor, UK) and fixed in acetone for 5 minutes. Slides were incubated with the primary rabbit antibodies against either VEGFR-1 or VEGFR-2 (1:100) at 4°C overnight. Isotype-specific rabbit IgG was used as control. The immunoreactivity for these receptors was revealed using an avidin-biotin kit from Vector Laboratories (Burlingame, CA). Peroxidase activity was revealed by the diaminobenzidine (Sigma-Aldrich, St. Louis, MO) cytochemical reaction. The slides were then counterstained with 0.12% methylene blue or hematoxylin and eosin. Supernatants from equal numbers of EC-HHV-8, ECs, KS primary isolate (KSC-10), and fibroblast (T1) (1 × 106 cells per six-well plate) were cultured for 24 hours in the absence of VEGF or other growth factors and the supernatant VEGF levels were measured by enzyme-linked immunosorbent assay. The levels of VEGF protein were substantially higher in EC-HHV-8 cells than in ECs or the T1 fibroblast cell line but were comparable to a KS isolate, KSC-10 (Figure 1A). We then examined the gene expression of various VEGF family members by RT-PCR (Figure 1B). VEGF, VEGF-C, VEGF-D, and PlGF were expressed in EC-HHV-8 but not seen in ECs. VEGF-B expression was not observed in either cell type (Figure 1B). Low input in this cDNA may explain the discrepancy between these results and our earlier findings of VEGF expression detected by RT-PCR in ECs.17Masood R Cai J Zheng T Smith DL Naidu Y Gill PS Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma.Proc Natl Acad Sci USA. 1997; 94: 979-984Crossref PubMed Scopus (262) Google Scholar However, induction of most of the VEGF family member gene expression is observed in the presence of HHV-8. These results suggest that HHV-8 induces the expression of VEGF, VEGF-C, and VEGF-D and PlGF in ECs. We have previously shown that HHV-8 viral G-protein coupled receptor induces VEGF expression in fibroblasts.27Bais C Santomasso B Coso O Arvanitakis L Geras-Raaka E Gutkind JS Arch AS Cesarman E Gershengorn MC Mesri E G-protein coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator.Nature. 1998; 391: 86-89Crossref PubMed Scopus (752) Google Scholar However, these data cannot rule out the role of latency associated gene regulation of VEGFs. Because the EC-HHV-8 cells express most VEGF family members, and ECs express the VEGF receptors, we considered the possibility that VEGF is an autocrine growth factor for EC-HHV-8 cells. The expression of VEGF receptors was examined. Because receptors are typically low-copy number mRNAs, twice the input cDNA was used in these PCR reactions than was used for the VEGF ligand molecules. By RT-PCR the receptor tyrosine kinases VEGFR-1, VEGFR-2, and VEGFR-3 were strongly expressed in EC-HHV-8 cells. Although the gene expression for all three receptors was detected in ECs, the level of expression was lower in all cases (Figure 1B). The integrity of the mRNA was confirmed by the amplification of β-actin. Expression of VEGF receptors was also evaluated by immunocytochemistry. Expression of all three receptors was detected in the primary ECs, however, only VEGFR-3 expression was apparent in the majority of ECs with very low signal for VEGFR-1 and VEGFR-2 (Figure 1C, left-hand column). Arrows point to representative staining for VEGFR-1 and VEGFR-2 in the primary ECs. Note that although not quantitative, the RT-PCR results indicating mRNA levels agree with the relative levels of expression of the receptor proteins obtained by immunocytochemistry. For the EC-HHV-8 cells, in contrast, strong staining for all three VEGF receptors was evident (Figure 1C, right-hand column). Both RT-PCR and immunocytochemistry confirm the robust expression of VEGF receptors in HHV-8-transformed ECs. It should be noted that HHV-8 was present in only 1 to 5% of the total cell population.25Flore O Rafii S Ely S O'Leary JJ Hyjek EM Cesarman E Transformation of primary human endothelial cells by Kaposi's sarcoma-associated herpesvirus.Nature. 1998; 394: 588-592Crossref PubMed Scopus (354) Google Scholar We next wished to determine whether the endogenous production of VEGF can induce proliferation of HHV-8-transformed ECs through VEGFR-2. A dose-dependent inhibition of EC-HHV-8 cell proliferation was observed in response to treatment with neutralizing antibody to VEGFR-2 (Figure 2A). Similarly, a KS cell line (KS-SLK) showed significant inhibition of cell proliferation consistent with autocrine growth factor activity of VEGF in KS. However, the effect of the antibody was minimal in T1 fibroblasts and modest in primary ECs (Figure 2A). Antibody to another EC-specific receptor tyrosine kinase, tie-2, had no effect on HHV-8-transformed ECs (Figure 2B). These results strongly support the consideration that induction of VEGF in EC-HHV-8 cell cultures plays an important role in cell proliferation and viability. VEGF AS oligonucleotides (AS-1 and AS-3) have previously been shown to specifically inhibit VEGF expression and in turn inhibit proliferation of a number of cell types that express VEGF and VEGF receptors.17Masood R Cai J Zheng T Smith DL Naidu Y Gill PS Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma.Proc Natl Acad Sci USA. 1997; 94: 979-984Crossref PubMed Scopus (262) Google Scholar, 28Masood R Cai J Zheng T Smith DL Hinton DR Gill PS Vascular endothelial growth factor (VEGF) is an autocrine growth factor for VEGF receptor positive human tumors.Blood. 2001; 98: 1904-1913Crossref PubMed Scopus (310) Google Scholar These include KS, melanoma, and ovarian carcinoma cell lines. We thus wished to determine whether inhibition of VEGF expression would similarly inhibit the proliferation of HHV-8-transformed ECs. Treatment with AS-1 and AS-3 led to a dose-dependent inhibition of EC-HHV-8 with minimal effect on ECs (Figure 3). The IC50 of AS-1 and AS-3 oligonucleotides were less than 5 μmol/L. The scrambled oligonucleotide (base composition corresponds to AS-3 had minimal inhibitory effect on primary ECs or EC-HHV-8 (Figure 3). VEGF is a mitogen for ECs that is required for both vasculogenesis and angiogenesis.29Ferrara N Carver-Moore K Chen H Dowd M Lu L O'Shea KS Powell-Braxton L Hilan KJ Moore MW Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene.Nature. 1996; 380: 438-442Crossref Scopus (3062) Google Scholar The closely related VEGF-C and VEGF-D molecules have also been shown to be mitogenic for ECs, especially lymphatic ECs.30Ristimaki A Narko K Enholm B Joukov V Alitalo K Proinflammatory cytokines regulate expression of the lymphatic endothelial mitogen vascular endothelial growth factor-C.J Biol Chem. 1998; 273: 8413-8418Crossref PubMed Scopus (343) Google Scholar VEGF and VEGF-C expression has been detected in KS spindle cells in tumor samples.22Skobe M Brown LF Tongnazzi K Ganju RK Dezube BJ Alitalo K Detmar M Vascular endothelial growth factor-C (VEGF-C) and its receptors KDR and flt-4 are expressed in AIDS-associated Kaposi's sarcoma.J Invest Dermatol. 1999; 113: 1047-1053Crossref PubMed Scopus (108) Google Scholar Now we show that ECs infected with HHV-8 express VEGF, VEGF-C, VEGF-D, and PlGF, in contrast to untransformed ECs that did not express the VEGF family mRNAs in our hands. VEGF receptors are highly restricted in their expression to ECs.31DeVries C Escobedo JA Ueno H Houck KA Ferrara N Williams LT The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor.Science. 1992; 255: 989-991Crossref PubMed Scopus (1896) Google Scholar, 32Terman BI Dougher-Vermazen M Carrion ME Dimitrov D Armellino DC Gospodarowicz D Bohlen P Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.Biochem Biophys Res Commun. 1992; 187: 1579-1586Crossref PubMed Scopus (1405) Google Scholar Although VEGF binds both VEGFR-1 and VEGFR-2, only binding to VEGFR-2 generates mitogenic signal in ECs.33Millauer B Wizigmann-Voos S Schnurch H Martinez R Moller NP Risau W Ullrich A High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis.Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1764) Google Scholar, 34Waltenberger J Claesson-Welsh L Siegbahn A Shibuya M Heldin CH Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor.J Biol Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar However, binding of VEGF to VEGFR-1 seems to be necessary for high-affinity binding to VEGFR-2. The expression of both of these receptors was markedly increased in EC-HHV-8 compared to ECs. Furthermore, VEGF-C and VEGF-D bind to VEGFR-3 on lymphatic ECs. Expression of VEGFR-3 was also strongly up-regulated in EC-HHV-8 compared to primary ECs. Thus HHV-8 seems to induce the expression of both vascular and lymphatic EC-specific growth factors and their receptors. We determined that the VEGFs secreted from the HHV-8-infected cells could act in an autocrine/paracrine manner. Blocking the binding site for the secreted VEGF (VEGFR-2) using neutralizing antibody resulted in a decrease in cell viability. In addition, blocking expression of VEGF in the EC-HHV-8 culture system by the AS-1 and AS-3 VEGF-specific AS oligonucleotides also resulted in decreased cell viability. Removal of either ligand (VEGF) or receptor (VEGFR-2) activity resulted in reduced viability thus demonstrating a functional autocrine loop in this culture system. From the gene expression studies we conducted it is clear that HHV-8-mediated EC transformation involves the regulation of VEGF family proteins that are ligands for receptor tyrosine kinases restricted to the ECs. In support of this, several recent reports indicate that some viral genes can regulate the expression of VEGF. The first of these is the vGPCR, which is a constitutively active broad specificity CXC chemokine receptor. Activity of this receptor can be induced by IL-8 and growth-related oncogene-α and repressed by interferon inducible protein (IP)-10 and stromal cell-derived factor-1α.35Gershengorn MC Geras-Raaka E Verma A Clark-Lewis I Chemokines activate Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor in mammalian cells in culture.J Clin Invest. 1998; 102: 1469-1472Crossref PubMed Scopus (145) Google Scholar, 36Rosenkilde MM Kledal TN Brauner-Osborne H Schwartz TW Agonists and inverse agonists for the herpesvirus 8-encoded constitutively active seven-transmembrane oncogenes product, ORF-74.J Biol Chem. 1998; 274: 956-961Crossref Scopus (173) Google Scholar The vGPCR can transform fibroblasts, which were also tumorigenic in nude mice.27Bais C Santomasso B Coso O Arvanitakis L Geras-Raaka E Gutkind JS Arch AS Cesarman E Gershengorn MC Mesri E G-protein coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator.Nature. 1998; 391: 86-89Crossref PubMed Scopus (752) Google Scholar, 37Arvanitakis L Geras-Raaka E Gershengorn MC Cerarman E Human herpesvirus KSHV encodes a constitutively active G protein-coupled receptor linked to cell proliferation.Nature. 1997; 385: 347-350Crossref PubMed Scopus (577) Google Scholar This transformation was also associated with activation of VEGF. vGPCR seems to enhance the expression of VEGF through phosphorylation of hypoxia inducible factor (HIF)-1α to in turn activate transcription of VEGF.38Sodhi A Montaner S Patel V Zohar M Bais C Mesri EA Gutkind JS The Kaposi's sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion through mitogen-activated protein kinase and p38 pathways acting on hypoxia-inducible factor 1α.Cancer Res. 2000; 60: 4873-4880PubMed Google Scholar Phosphorylation of HIF-1α by both the p38 and MAPK pathways was found to be involved in the vGPCR-mediated induction of VEGF expression in this system. Further, ectopic expression of vGPCR has been found to protect human umbilical vein ECs against apoptosis induced by serum starvation; however, this was independent of VEGF.39Montaner S Sodhi A Pece S Mesri EA Gutkind JS The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor promotes endothelial cell survival through the activation of Akt/protein kinase B.Cancer Res. 2001; 61: 2641-2648PubMed Google Scholar In addition to its effects on VEGF expression, the vGPCR has been shown to be involved in the pathogenesis of KS because vGPCR transgenic mice develop angioproliferative lesions with the hallmarks of KS.40Yang T-Y Chen S-C Leach MW Manfra D Homey B Wiekowski M Sullivan L Jenh C-H Narula SK Chensue SW Lira SA Transgenic expression of the chemokine receptor encoded by human herpesvirus 8 induces an angioproliferative disease resembling Kaposi's sarcoma.J Exp Med. 2000; 191: 445-453Crossref PubMed Scopus (360) Google Scholar Another virally encoded gene, vIL-6, up-regulates VEGF expression when expressed ectopically in murine fibroblasts.41Aoki Y Jaffe ES Chang Y Jones K Teruya-Feldstein J Moore PS Tosato G Angiogenesis and hematopoiesis induced by Kaposi's sarcoma-associated herpesvirus-encoded interleukin-6.Blood. 1999; 93: 4034-4043Crossref PubMed Google Scholar The vIL-6 has 62.2% sequence similarity to the human protein, and retains the four conserved cysteines found in all IL-6 proteins.42Moore PS Boshoff C Weiss RA Chang Y Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV.Science. 1996; 274: 1739-1744Crossref PubMed Scopus (826) Google Scholar, 43Nicholas J Ruvolo VR Burns WH Sandford G Wan X Ciufo D Hendrickson SB Guo HG Hayward GS Reitz MS Kaposi's sarcoma-associated herpesvirus-8 encodes homologues of macrophage inflammatory protein-1 and interleukin-6.Nat Med. 1997; 3: 287-292Crossref PubMed Scopus (337) Google Scholar One important difference between the actions of vIL-6 and cellular IL-6 is that the vIL-6 signals directly through the gp130 subunit of the IL-6 receptor complex and does not first bind to the IL-6Rα subunit, which is a prerequisite of cellular IL-6 signaling through gp130.44Molden J Chang Y You Y Moore PS Goldsmith MA A Kaposi's sarcoma-associated herpesvirus-encoded cytokine homolog (vIL-6) activates signaling through the shared gp130 receptor subunit.J Biol Chem. 1997; 272: 19625-19631Crossref PubMed Scopus (249) Google Scholar Both the vGPCR and vIL-6 transcripts are present in only a small number of cells in KS lesions compared to the widespread distribution of the latent transcripts for Kaposin or LANA.42Moore PS Boshoff C Weiss RA Chang Y Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV.Science. 1996; 274: 1739-1744Crossref PubMed Scopus (826) Google Scholar, 45Kirshner JR Staskus K Haase A Lagunoff M Ganem D Expression of the open reading frame 74 (G-protein-coupled receptor) gene of Kaposi's sarcoma (KS)-associated herpesvirus: implications for KS pathogenesis.J Virol. 1999; 73: 6006-6014PubMed Google Scholar, 46Sarid R Wiezorek JS Moore PS Chang Y Characterization and cell cycle regulation of the major Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) latent genes and their promoter.J Virol. 1999; 73: 1438-1446Crossref PubMed Google Scholar Therefore, it is unlikely that either vGPCR or vIL-6 directly transform spindle cells in vivo because expression as lytic genes would be transient and occurs in a cell population destined for imminent death. However, both of these viral genes could affect KS pathogenesis through contributions to angiogenesis and inflammatory cell infiltration. Our demonstration that HHV-8 induces the VEGF family proteins and their receptors in ECs, and that a functional autocrine pathway is present, underscores the importance of VEGF in this disease. It is clear that the VEGFs and VEGF receptors are unique targets for the treatment of KS. As a result, various inhibitors of VEGF and VEGF receptors are under clinical investigation.
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