Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis

doi:10.1128/JVI.02052-08

J Virol. 2009 April; 83(7): 3342–3364.

Published online 2009 January 21. doi: 10.1128/JVI.02052-08

PMCID: PMC2655544

Kaposi's Sarcoma-Associated Herpesvirus Upregulates Angiogenin during Infection of Human Dermal Microvascular Endothelial Cells, Which Induces 45S rRNA Synthesis, Antiapoptosis, Cell Proliferation, Migration, and Angiogenesis

Sathish Sadagopan,¹ Neelam Sharma-Walia,¹ Mohanan Valiya Veettil,¹ Virginie Bottero,¹ Rita Levine,¹ Richard J. Vart,² and Bala Chandran¹^*

H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois,¹ Cancer Research U.K. Viral Oncology Group, Wolfson Institute for Biomedical Research, University College London, London, United Kingdom²

^*Corresponding author. Mailing address: Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064. Phone: (847) 578-8822. Fax: (847) 578-3349. E-mail: bala.chandran/at/rosalindfranklin.edu

Received September 29, 2008; Accepted January 13, 2009.

This article has been cited by other articles in PMC.

Abstract

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is associated with the angioproliferative KS lesions characterized by spindle-shaped endothelial cells, inflammatory cells, cytokines, growth factors, and angiogenic factors. De novo KSHV infection of human microvascular dermal endothelial cells results in increased secretion of several growth factors, cytokines, chemokines, and angiogenic factors, and the multifunctional angiogenic protein angiogenin is one of them. KS tissue sections were positive for angiogenin, highlighting the importance of angiogenin in KS pathogenesis. Examination of KSHV-mediated angiogenin upregulation and secretion and potential outcomes revealed that during infection of primary endothelial cells, KSHV induced a time- and dose-dependent increase in angiogenin gene expression and protein secretion beginning as early as 8 h postinfection and lasting until the fifth day of our observation period. TIVE latently transformed cells (TIVE-LTC) latently infected with KSHV secreted high levels of angiogenin. Angiogenin was also detected in BCBL-1 cells (human B cells) carrying KSHV in a latent state. Significant induction of angiogenin was observed in cells expressing KSHV ORF73 (LANA-1; latent) and ORF74 (lytic) genes alone, and moderate induction was seen with the lytic KSHV ORF50 gene. Angiogenin bound to surface actin, internalized in a microtubule-independent manner, and translocated into the nucleus and nucleolus of infected cells. In addition, it increased 45S rRNA gene transcription, antiapoptosis, and proliferation of infected cells, thus demonstrating the multifunctional nature of KSHV-induced angiogenin. These activities were dependent on angiogenin nuclear translocation, which was inhibited by neomycin. Upregulation of angiogenin led to increased activation of urokinase plasminogen activator and generation of active plasmin, which facilitated the migration of endothelial cells toward chemoattractants, including angiogenin, and chemotaxis was prevented by the inhibition of angiogenin nuclear translocation. Treatment of KSHV-infected cell supernatants with antiangiogenin antibodies significantly inhibited endothelial tube formation, and inhibition of nuclear translocation of angiogenin also blocked the expression of KSHV-induced vascular endothelial growth factor C. Collectively, these results strongly suggest that by increasing infected endothelial cell 45S rRNA synthesis, proliferation, migration, and angiogenesis, KSHV-induced angiogenin could be playing a pivotal role in the pathogenesis of KSHV infection, including a contribution to the angioproliferative nature of KS lesions. Our studies suggested that LANA-1 and vGPCR play roles in KSHV-induced angiogenesis and that the angiogenic potential of vGPCR might also be due to its ability to induce angiogenin.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is etiologically associated with the angioproliferative KS, a chronic inflammation-associated malignancy characterized by a heterogeneous population of spindle-shaped activated endothelial cells, inflammatory cells, cytokines, growth factors, and angiogenic factors (9, 11). In advanced lesions, spindle cells are the predominant cell type and are accompanied by elevated angiogenesis (10). Multiple results suggest that inflammatory cytokines, angiogenic factors, and chemokines such as gamma interferon, tumor necrosis factor alpha, beta interleukin-1 (IL-1β), IL-6, platelet-derived growth factor, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and prostaglandin E₂ expressed in KS lesions are critical elements of in vivo AIDS-KS pathogenesis, possibly operating by mediation of spindle cell viability and angioproliferation.

Our studies showed that the microenvironment induced during de novo KSHV infection of primary human microvascular dermal endothelial (HMVEC-d) cells resembled that of KS lesions (30). We have previously demonstrated that within minutes of infection, KSHV enters the adherent target cells, such as HMVEC-d and human foreskin fibroblast (HFF) cells, concomitant with the induction of preexisting host signal cascades such as those of focal adhesion kinase (FAK), Src, PI3K, AKT, PKCζ, MEK, extracellular signal-regulated kinase 1 or 2 (ERK1/2), and NF-κB (29, 32-34). KSHV infections of HMVEC-d and HFF cell infections are characterized by the transient expression of limited lytic cycle genes, persistent expression of latency-associated genes such as KSHV ORF71 (vFLIP), ORF72 (vCyclinD), and ORF73 (LANA-1 [latency-associated nuclear antigen]), and the establishment of latent infection (22, 29). KSHV-induced ERK1/2 and NF-κB were critical for the initiation and maintenance of viral gene expression (32, 33).

As an initial step toward understanding how KSHV establishes latent infection in vitro, we previously used oligonucleotide arrays to examine the modulation of HMVEC-d and HFF cell gene expression at 2 h and 4 h postinfection (p.i.) (30). These studies demonstrated that KSHV reprogrammed the elements of host cell transcriptional machinery that are involved in regulating a variety of processes such as apoptosis, cell cycle regulation, signaling, inflammatory response, and angiogenesis (30). Our subsequent cytokine array analysis showed that KSHV infection induced a significant increase in the secretion of several endothelial cell angiogenic molecules (VEGF, angiopoietin, and SDF-1), growth factors (platelet-derived growth factor, FGF, granulocyte-macrophage colony-stimulating factor, and insulin-like growth factor 1), chemokines (monocyte chemoattractant protein 2 [MCP-2], macrophage inflammatory protein [MIP], monokine inducible by gamma interferon [MIG], and eotaxin), and proinflammatory (IL-2, IL-3, IL-8, growth-regulated oncogene [GRO], and IL-16) and anti-inflammatory (IL-4, IL-5 and IL-15) cytokines and that many of these factors were induced in an NF-κB-dependent manner (32). The angiogenic factor angiogenin (ANG) was among the cytokines that were highly upregulated during de novo KSHV infection (32).

Under normal physiological conditions, angiostatic factors are more significant than angiogenic factors, whereas during the progression of diseases such as cancer, tissue regeneration and wound-healing angiogenic factors outweigh angiostatic factors, resulting in angiogenesis. KSHV-induced upregulation of angiogenesis factors may potentially be involved in KS development and progression. Angiogenesis is the growth of new blood vessels, which is the formation of new capillaries from existing ones. Normal angiogenesis is self-limiting and tightly regulated (40). An increase in the levels of angiogenesis is seen under several sets of pathological conditions during the progression of inflammatory arthritis, diabetic retinopathy, psoriasis, and cancer (40). Angiogenesis is initiated by degradation of the basement membrane and matrix, probably initiated by factors released from the endothelial cells. Subsequently, endothelial cells migrate chemotactically, proliferate, and form new capillary tubes.

ANG, a multifunctional 14-kDa angiogenic protein that performs several of the functions mentioned above, was first isolated from HT-29 human colon adenocarcinoma cell-conditioned media based on its angiogenic activity (37). ANG has been shown to play a role in tumor angiogenesis, and its expression is upregulated in several types of cancers, including pancreatic, breast, prostate, cervical, ovarian, colon, colorectal, gastric, urothelial, and endometrial cancers (37). Anti-ANG monoclonal antibodies used as antagonists inhibited the establishment, progression, and metastasis of human cancer cells inoculated into athymic mice (31).

Upon secretion from the cells, ANG is known to interact with a 42-kDa cell surface form of actin and forms an actin-ANG complex (19), which accelerates plasmin generation by triggering a plasminogen activator (18). Plasmin helps in extracellular matrix (ECM) degradation, which is required for the migration of endothelial cells. Once endothelial cells migrate to the region with reduced vessel density, the cells arrange in monolayers and form tube-like structures (20, 40).

ANG nuclear translocation is strictly dependent on cell type and cell density (28). It remains cytoplasmic in fibroblast cells, suggesting that the nuclear function of ANG is limited to endothelial cells (28). Nuclear translocation does not occur when the cells are confluent but occurs in cells of about 30% confluence (subconfluent cells) and in cells of >50 to 60% confluence (semiconfluent cells) (28). ANG can initiate proliferation of endothelial cells upon binding to a 170-kDa protein expressed on the cell surface (17). ANG is endocytosed; once inside the cell, it initiates signal transduction via ERK1/2. It also translocates into the nucleus in sub- and semiconfluent cells, moves into the nucleolus of subconfluent cells, and transcribes rRNA by binding to CT repeats that are abundant in the nontranscribed region of the rRNA gene (41). Nuclear translocation of ANG in endothelial cells has been shown to be necessary for the angiogenic potential of ANG (21). Mutating the nuclear localization signal (NLS) of ANG abolished the angiogenic activity (24).

ANG activity is relatively low compared with that of other growth factors such as VEGF and bFGF (21). However, nuclear translocation of ANG was reported to be necessary for that of VEGF and bFGF. The mechanism of action of ANG is still unclear despite a huge volume of work; this lack of clarify is due to the complexity of biological processes underlying the phenomenon of angiogenesis (7).

Here we demonstrate for the first time that KSHV infection of endothelial cells upregulates ANG in a time- and dose-dependent manner, resulting in the performance of angiogenic and proliferative functions such as an increase in synthesis of the infected endothelial cell 45S rRNA, proliferation, migration, and angiogenesis. KSHV infection-induced ANG upregulation and nuclear translocation were critical for VEGF-C transcription and for angiogenesis mediated by both ANG and VEGF, thus suggesting that ANG could be playing a critical role in influencing the angioproliferative nature of KS lesions.

MATERIALS AND METHODS

Cells.

Primary HMVEC-d cells (CC-2543; Clonetics, Walkersville, MD) and TIVE cells and TIVE-LTC were grown in endothelial basal medium 2 (EBM-2) with growth factors (Clonetics). HFF cells, HEK (human embryonic kidney) 293 cells, and HEK 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and antibiotics. BJAB cells and KSHV-carrying BCBL-1 cells were cultured in RPMI 1640 (Gibco BRL) medium with 10% heat-inactivated FBS (HyClone, Logan, UT), 2 mM l-glutamine, and antibiotics (1-3).

Antibodies, reagents, and growth factors.

Rabbit polyclonal antibodies against human ANG were from Santa Cruz Biotechnology Inc., Santa Cruz, CA. Phalloidin conjugated with Alexa 594, anti-goat, anti-rabbit, and anti-mouse antibodies linked to Alexa 488 or Alexa 594, and antifade DAPI (4′,6′-diamidino-2-phenylindole) were purchased from Molecular Probes, Eugene, OR. Antibodies against lamin B, tubulin, actin, recombinant human ANG, bovine serum albumin (BSA), tetradecanoyl phorbol acetate (TPA), Triton X-100, heparin, and paraformaldehyde were from Sigma, St. Louis, MO. Antifibrillarin and antiplasmin antibodies were from Abcam, Cambridge, MA.

Virus.

Procedures for induction of the KSHV lytic cycle in BCBL-1 cells, supernatant collection, and virus purification were described previously (22), and purity was assessed according to general guidelines established in our laboratory (2, 22, 29). KSHV DNA was extracted from the purified virus, and copy numbers were quantitated by real-time DNA PCR using primers amplifying the KSHV ORF73 gene (22). To prepare replication-defective virus, KSHV was irradiated with UV light (UV-KSHV) (365 nm) for 20 min at a 10-cm distance. KSHV DNA was extracted from the UV-irradiated virus, and the copy numbers were quantitated by real-time DNA PCR as described previously (22).

ANG protein quantitation.

The levels of ANG in the culture supernatant of uninfected or KSHV-infected HMVEC-d cells were quantitated using a human ANG enzyme-linked immunosorbent assay (ANG-ELISA) (R and D Systems, Minneapolis, MN). Briefly, ~30 to 40%-confluent HMVEC-d cells were serum starved for 8 h and infected with KSHV at a multiplicity of infection (MOI) of 10 (expressed in DNA copies per cell). Supernatants were collected at various times p.i., spun at 1,000 rpm for 10 min at 4°C to remove the particulates, and stored at −80°C until use. Total soluble protein was quantitated by a bicinchoninic acid protein assay (Pierce, Rockford, IL) to normalize the ELISA results. Since the concentration of protein present in the supernatant was the only variable for the various time periods and treatments, equal concentrations of protein made up to a volume of 200 μl were loaded onto each well and probed with anti-ANG antibody followed by secondary antibody, developed using the substrate, and read at 450 nm. IL-8 ELISAs were carried out in a similar fashion using an IL-8 immunoassay (Becton Dickinson, Bedford, MA) according to the instructions of the manufacturer.

For estimating ANG protein concentrations in BCBL cells, cells were left untreated or treated with TPA to induce a lytic cycle, supernatants were collected, and equal protein concentrations were used to assay the amount of ANG secretion. Supernatants from BJAB cells negative for KSHV were used as a control for comparisons. HEK 293T cells were transfected using increasing concentrations of LANA-1 expression plasmids. At 24 h posttransfection, the medium was changed to complete DMEM containing serum. Cells were allowed to grow for 24 h followed by serum starvation for 24 h, and the harvested supernatants were used for ANG-ELISA.

Quantitative real-time RT-PCR.

ANG expression, LANA expression, and 45S rRNA expression were detected by real-time reverse transcription-PCR (RT-PCR) using SYBR green chemistry. Two micrograms of total RNA was treated with DNase for 1 h and then reverse transcribed into cDNA by using random hexamer with a SuperScript first-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA). cDNA was used as a template with primers specific for ANG (forward, 5′-CCGTTTCTGCGGACTTGTTC-3′; reverse, 5′-GCCCATCACCATCTCTTCCA-3′), LANA (forward, 5′-CGCGAATACCGCTATGTACTCA-3′; reverse, 5′-GGAACGCGCCTCATACGA-3′), and 45S (forward, 5′-CGGGTTATTGCTGACACGC-3′; reverse, 5′-CAACCTCTCCAGCGACAGG-3′). Hypoxanthine phosphoribosyltransferase (HPRT) (forward primer, 5′-GGACAGGACTGAACGTCTTGC; reverse primer, 5′-CTTGAGCACACAGAGGGCTACA-3′) and tubulin (forward primer, 5′-TCCAGATTGGCAATGCCTG-3′; reverse primer, 5′-GGCCATCGGGCTGGAT-3′) were used as internal controls. PCR was performed using the Applied Biosystems 7500 real-time PCR system with SYBR green PCR master mix (Applied Biosystems, Foster City, CA). The standard amplification program included 40 cycles of two steps each of heating to 95°C and 60°C. The fluorescent product was detected at the last step of each cycle. The final mRNA levels of the genes studied were normalized using the comparative cycle threshold method.

293T transfection studies.

HEK 293T cells were transfected with increasing concentrations of pCI-neo full-length ORF73 gene constructs by use of the calcium phosphate precipitation method. The cells were incubated for 48 h at 3% CO₂, and total RNA was extracted using TRIzol reagent (Invitrogen) and subjected to DNase I digestion (Invitrogen). This RNA was subsequently used for cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen). Real-time quantitative RT-PCR (qRT-PCR) was done for ORF73 and ANG.

Dot blot analysis.

Increasing volumes with increasing concentrations of cell culture supernatant from serum-starved HMVEC-d cells infected with KSHV at indicated time points were adsorbed onto a presoaked nitrocellulose membrane by use of a dot blot apparatus. The membrane was blocked using 5% skim milk and immunoblotted with anti-ANG antibody (1:500). The spot density was calculated using Alpha Imager (Alpha Innotech, San Leandro, CA.), and the spot intensity in uninfected cell culture supernatant was assigned a value of 1 for comparisons.

Immunohistochemistry.

KS tissue sections obtained from the AIDS and Cancer Specimen Resource were deparaffinized with HistoChoice clearing reagent (Sigma) and hydrated with water before microwave treatment in 1 mM EDTA (pH 8) for 15 min for antigen retrieval and then blocked with blocking solution (10% normal horse serum, 5% BSA, 0.3% Triton X-100 in phosphate-buffered saline [PBS]). Sections were incubated with anti-ANG antibody overnight at 4°C followed by secondary antibody that was provided in a Vectastain avidin-biotin complex systems kit, and staining was done with a Vector NovaRED substrate kit (Vector Laboratories, Burlingame, CA) and hematoxylin.

Lentivirus production and infection of HMVEC-d cells.

Lentiviral infection was done as described by Vart et al. (38). Vesicular stomatitis virus-G envelope-pseudotyped lentivirus was produced using a four-plasmid transfection system as previously described (8, 26). Briefly, HEK 293T cells were transfected with lentiviral constructs expressing KSHV genes such as ORF71, ORF72, ORF73, ORF74, and K12 (38) and packaging plasmids by use of the calcium phosphate precipitation method. The cells were incubated overnight at 3% CO₂, and at 48 h and 72 h p.i., culture supernatants containing the lentiviral virions were harvested, cell debris was cleared by passage through a 0.45-μm-pore-size filter, and the preparation was either aliquoted directly or concentrated and stored at −80°C. Infections were carried out by incubating the desired amount of virus preparation with HMVEC-d cells in culture for 8 h, and the medium was replaced by complete growth medium for endothelial cells. Total RNA was extracted from lentiviral transduced HMVEC-d cells by the use of TRIzol reagent (Invitrogen) and subjected to DNase I digestion (Invitrogen). This RNA was subsequently used for cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen). Real-time qRT-PCR was done for all KSHV genes used in the current study and for ANG. The cells were split at 24 h after lentivirus infection. These cells were serum starved, and supernatants were collected at 48 h p.i., spun at 1,000 rpm for 10 min at 4°C to remove particulates, and used for measuring ANG levels by ELISA. Empty lentiviral vector (pSIN) was used as a control.

Ad infection of HMVEC-d cells.

Construction and characterization of adenovirus (Ad) constructs expressing ORF50 and green fluorescent protein (GFP) were carried out as described previously (6). Briefly, HEK 293 cells were transfected with adenoviral constructs expressing GFP or ORF50 by the use of Lipofectamine. Culture supernatants containing the Ad were harvested at 48 h p.i., cell debris was cleared by passage through a 0.45-μm-pore-size filter, and the preparation was aliquoted and stored at −80°C. Viral copy numbers were estimated by the use of real-time DNA PCR for GFP or ORF50 and of GFP and ORF50 standards. HMVEC-d cells were infected using AdGFP or AdORF50 at an MOI of 1 for 8 h, and the medium was replaced for 48 h with complete growth medium for endothelial cells. Total RNA was extracted from Ad-infected HMVEC-d cells by the use of TRIzol reagent (Invitrogen) and subjected to DNase I digestion (Invitrogen). This RNA was subsequently used for cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen). Real-time qRT-PCR was done for GFP, ORF50, and ANG. These cells were serum starved, and supernatants were collected at 48 h p.i., spun at 1,000 rpm for 10 min at 4°C to remove particulates, and used for measuring ANG levels by ELISA. ORF50 and GFP expression results were used for normalizing the efficiency of infection, and tubulin and 18S rRNA expression levels were measured and used as endogenous controls.

Immunofluorescence assays.

Confluent, semiconfluent, and subconfluent HMVEC-d cells in eight-well chamber slides (Nalge Nunc International, Naperville, IL) were either left uninfected or infected with KSHV (MOI of 10) at 37°C for 48 h. Immunofluorescence assays to determine ANG protein expression in target cells were done after fixing cells with 4% paraformaldehyde for 10 min at room temperature, permeabilizing with 0.4% Triton X-100 for 10 min at room temperature, and staining with primary antibody for 1 h at 37°C. Target cells were washed and incubated with an appropriate dilution of secondary antibodies for 1 h at room temperature. Nucleolar staining was done with antifibrillarin antibody. Nuclei were visualized by using DAPI (Molecular Probes, Eugene, OR) as a counterstain. Stained cells were washed and viewed with appropriate filters using a fluorescence microscope with a Nikon Metamorph digital imaging system.

Laser-scanning confocal immunofluorescence.

Semiconfluent and subconfluent HMVEC-d cells infected with KSHV (MOI of 10) were fixed with 4% paraformaldehyde. The cells were then permeabilized with 0.2% Triton X-100 for 5 min, washed, and blocked with BSA for 30 min. For demonstration of nuclear localization of ANG, infected cells were stained with Topro and rabbit anti-ANG antibodies. For double staining, the infected semiconfluent and subconfluent HMVEC-d cells were stained with monoclonal antifibrillarin and rabbit anti-ANG antibodies, followed by staining with Alexa Fluor 594-labeled goat anti-rabbit secondary antibody and Alexa Fluor 488-labeled goat anti-mouse secondary antibody, respectively. An Olympus Fluoview 300 fluorescence confocal microscope was used for imaging, and analysis was performed using Fluoview software (Olympus, Melville, NY).

Luciferase reporter assays.

The effect of ANG on the ANG binding element (ABE) promoter (pGL3E-ABE) and on pGL3E was measured using a Promega dual luciferase kit according to the manufacturer's protocol. Briefly, HEK 293 cells (1 × 10⁵) seeded in a 24-well tissue culture plate were fed with antibiotic-free low-serum (0.5% FBS) DMEM for 12 to 18 h before transfection. Low-serum conditions were maintained throughout the experiment. Transfection of HEK 293 cells was performed with 1 μg of pGL3E or pGL3E-ABE promoter luciferase constructs and with 100 ng of p-Renilla luciferase as a transfection efficiency control by the use of Lipofectamine 2000 (Invitrogen). After 24 h, cells were either left uninfected or infected with KSHV at an MOI of 10 or treated with exogenous ANG (250 ng/ml) for 24 h. Cells were harvested at 48 h p.i. in 1× passive lysis buffer, and luciferase assays were carried out in triplicate experiments following the manufacturer's procedure and read using a Synergy HT multidetection microplate reader (Bio-Tek Instruments, Inc.). Firefly luciferase activities for each time point were normalized to those of parallel controls for each time point. Alterations in the promoter activities of pGL3E and pGL3E-ABE as a result of KSHV infection or ANG treatment were determined after normalization, using the Renilla luciferase activity as a control.

Mitotracker analysis.

HMVEC-d cells grown to 50% confluence in eight-well-chamber slides were serum starved for 6 to 8 h followed by infection with live KSHV, UV-KSHV, or ANG-treated KSHV (250 ng/ml) for 48 h. Cells were washed with Hanks balanced salt solution, added with Mitotracker (BD Transduction Laboratories, Los Angeles, CA) to EBM-2, incubated for 2 h at 37°C, and visualized using a Metamorph digital imaging system. Mitochondria of proliferating cells absorbed Mitotracker and fluoresced red. Since the nonproliferating cells were impermeable to Mitotracker, they fluoresced green.

Nuclear extract preparation.

HMVEC-d cells were infected with KSHV (MOI of 10) for 48 h. Nuclear extracts were prepared using a nuclear extract kit (Active Motif Corp, Carlsbad, CA) per the manufacturer's instructions. After protein concentrations were measured by the use of bicinchoninic acid protein assay reagent, extracts were stored at −70°C. The purity of the nuclear extracts was assessed by immunoblotting using anti-lamin B antibodies, and cytoskeletal contamination was checked by using anti-β-actin and anti-β-tubulin antibodies.

Western blotting.

Lysates or nuclear extracts (10 μg) were resolved on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, transferred to nitrocellulose membranes, blocked with 5% skim milk, and immunoblotted with rabbit polyclonal anti-ANG antibody (Santa Cruz), tubulin, and lamin B for nuclear extracts and with antiplasmin and actin for whole-cell lysates. Immunoreactive bands were developed by the use of enhanced chemiluminescence reactions (NEN Life Sciences Products, Boston, MA) and quantified following standard protocols (33).

EMSA.

Gel shift analysis was performed as described previously (32) using nuclear extracts from serum-starved HMVEC-d cells that were left uninfected or infected with KSHV or treated with ANG. Briefly, ABE double-stranded oligonucleotides were labeled at the 5′ end with [γ-³²P]ATP (Perkin Elmer) by the use of T4 polynucleotide kinase (Gibco BRL). Binding reaction mixtures were incubated on ice for 20 min, and reactions were performed in a 20-μl reaction volume containing 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM MgCl₂, 0.5 mM EDTA, 0.5 mM dithiothreitol, 9% (vol/vol) glycerol, 1 μg of poly(dI-dC), 5 μg of nuclear extract, and labeled probe (10,000 cpm). The resulting DNA-protein complexes were then size fractionated from the free DNA probe by electrophoresis at 200 V on a 5% native polyacrylamide gel. The gel was dried at 80°C and autoradiographed. Competition electrophoretic mobility shift assays (EMSA) were performed by adding a 100× molar excess of unlabeled double-stranded, ABE oligonucleotide probe. The nucleotide sequence of the annealed DNA probes for ABE consensus used was 5′-CTCTCTCTCCCTC-3′.

uPA assay.

Active and total uPA was assayed using a urokinase plasminogen activator (uPA) activity assay kit (Innovative Research, Southfield, MI) per the manufacturer's instructions. Briefly, HMVEC-d cells were lysed using NP-40 buffer with protease inhibitor cocktail, and 100 μg of the lysate was loaded onto each well and probed with anti-uPA antibody followed by secondary antibody, developed using the substrate, and read at 450 nm. For measuring active uPA levels, each well was precoated with biotinylated human PAI-1 before adding the lysate. Values obtained for active uPA were normalized to the values obtained using total uPA.

Chemotaxis assay.

Cell migration assays were done with an Innocyte cell migration assay kit (Calbiochem). The Innocyte cell migration assay kit is provided in a 96-well format with cell culture inserts containing a membrane with an 8-μm pore size that ensures migration of cells. HMVEC-d cell culture supernatant from uninfected or KSHV-infected cells with and without neomycin treatment was added to the lower chamber to serve as a chemoattractant. ANG at a 250 ng/ml concentration was used as a positive control. HMVEC-d cells were serum starved for 6 to 8 h and washed, the cell pellet was resuspended with serum-free cell culture medium to a final concentration of approximately 500,000 cells/ml with or without 100 μM neomycin or paromomycin, and 200 μl of cells was seeded onto the upper chamber (cell culture insert). The migration chamber was incubated for 4 h in a CO₂ incubator at 37°C, and cells in the upper chamber were removed using a cotton swab. The cells that moved to the bottom of the upper chamber due to chemotaxis were resuspended in 200 μl of labeling/cell detachment buffer with fluorescent dye and incubated for 30 min in a CO₂ incubator at 37°C, and the fluorescence was read using a Synergy fluorescent plate reader, with excitation at 485 nm and emission at 528 nm.

Matrigel assay.

Matrigel (Becton Dickinson, France) was thawed on ice overnight and spread evenly over each well (100 μl) of a 96-well plate. The plates were incubated for 30 min at 37°C to allow the Matrigel to gel. HMVEC-d cells (3 × 10⁴) from passage 7 or earlier were dispensed in 100 μl per well along with the indicated supernatants or EBM-2 with or without 250 ng/ml of recombinant ANG or VEGF (100 ng/ml) in the presence or absence of anti-ANG antibody, incubated for 16 h at 37°C, and visualized using a phase-contrast microscope with the Nikon Metamorph digital imaging system.

Flow cytometry.

HMVEC-d cells were infected with KSHV (MOI of 10) and harvested for flow cytometry at 48 h p.i. Cells were detached from the plate with 0.25% trypsin-EDTA, and viability was assessed by trypan blue exclusion. Samples were washed twice with 1× PBS, permeabilized using BD Pharmingen perm/wash buffer for 10 min, blocked for 30 min with 3% BSA in PBS, and treated with anti-VEGF antibody followed by secondary antibody. Flow cytometry was performed with a FACScalibur flow cytometer, and the results were analyzed with Cell Quest Pro software (Becton Dickinson, Bedford, MA) in the Rosalind Franklin University of Medicine and Science flow cytometry core facility. Prior to analysis, all samples were gated by forward and side scatter techniques to eliminate dead cells.

Cell survival (MTT) assay.

HMVEC-d cells serum starved for 12 h in EBM-2 were left untreated or pretreated with anti-ANG antibody (Santa Cruz) for 1 h, followed by incubation with serum-free, growth supplement-free EBM-2 either alone or in the presence of ANG (250 ng/ml) or VEGF (250 ng/ml) or serum and growth factors with EGM-2. For supernatant treatment studies, uninfected supernatants and supernatants from live or UV-KSHV-infected serum-starved HMVEC-d cells after 48 h were either pretreated with anti-ANG antibody or left untreated and added to serum-starved HMVEC-d cells. At 48 h posttreatment, 50 μl of MTT [(3-4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrasodium bromide)] reagent was added to each well, the mixture was incubated for 20 min, and the absorbance was read at 570 nm.

RESULTS

KSHV infection induces increased ANG secretion during the latent phase of the viral life cycle.

To assess the impact of the presence of ANG in KSHV pathogenesis, serum-starved HMVEC-d cells (~30 to 40% confluence) were infected with 10 MOI of KSHV, and supernatants were harvested at different hours p.i. and assayed for ANG secretion by ANG-ELISA. KSHV infection resulted in a time-dependent increase in ANG secretion into the supernatant (Fig. (Fig.1A).1A). A substantial amount of ANG secretion started at 8 h p.i. reached a peak at 72 h p.i., and remained high until 120 h p.i., the period of our observation. An appreciable amount of ANG secretion was also detected in cells induced with tumor necrosis factor alpha (20 ng/ml for 20 min) as a positive control (Fig. (Fig.1A).1A). Since our earlier microarray and cytokine array analyses had shown an increase in IL-8 secretion upon KSHV infection (30, 32), we compared the time courses for relative quantities of IL-8 secretion with ANG. There was a time-dependent increase in IL-8 secretion until 120 h p.i. that reached a peak at 72 h p.i. (Fig. (Fig.1A).1A). However, ANG secretion was twofold higher than IL-8 secretion at 48 h and 72 h, underscoring the impact of KSHV in inducing ANG secretion.

FIG. 1.

Increases in ANG protein and mRNA levels in KSHV-infected HMVEC-d cells. (A) HMVEC-d cells grown to ~30 to 40% confluence (subconfluent cells) were serum starved for 8 h and infected with KSHV at an MOI of 10. At various times p.i., the (more ...)

To demonstrate the specificity of ANG induction by KSHV, cells were infected with virus at increasing MOIs for 48 h and the supernatant was used for ANG-ELISA. ANG secretion increased with increasing MOIs of virus and peaked at an MOI of 20 (Fig. (Fig.1B).1B). KSHV binds to the adherent target heparan sulfate on the cell surface, and blocking this interaction with heparin, an analogue of heparan sulfate, prevents KSHV binding to the target cells and infection (3). To verify whether the ANG secretion was due to KSHV infection, KSHV (MOI of 10) pretreated with heparin was used for infection. There was a significant (>94%) reduction in ANG secretion seen with heparin-treated virus infection (Fig. (Fig.1B).1B). These results demonstrated that ANG secretion was indeed due to KSHV infection and not due to any host protein or lipopolysaccharide contamination.

To verify the time-dependent increase in ANG secretion, we next analyzed ANG gene expression by real-time RT-PCR. Compared to uninfected cell results, significant induction of ANG gene expression in subconfluent (~30% confluence) HMVEC-d cells was observed in infected cells, with an approximately 2-fold increase at 1 day p.i., peaking to 3.5-fold at 3 days p.i. and remaining at 3-fold on days 4 and 5 p.i. (Fig. (Fig.1C).1C). To further confirm the increase in ANG secretion upon KSHV infection, dot blot analysis was done using supernatants from subconfluent HMVEC-d cells infected with KSHV for different time periods. The dot blot assay results corroborated our ELISA results. ANG could be detected with 10 μg of supernatant at 24 h p.i. (Fig. (Fig.1D,1D, top panel), while it was undetectable with even 40 μg when the supernatant from uninfected cells was used. A time course analysis showed that there was a twofold increase in ANG secretion at 24 h p.i. compared to the results seen with uninfected cells and that this increase peaked to sixfold at 72 h p.i. when 10 μg of conditioned medium was used (Fig. (Fig.1D,1D, bottom panel).

To study the relevance of ANG induction and secretion in terms of in vivo KSHV infection, we used immunohistochemical staining to examine the tissue sections from KS patients. KS tissue sections stained strongly positive (brown) for ANG, whereas no significant staining was observed in normal tissue (Fig. (Fig.1E).1E). These results suggested that ANG could be playing an important role in KSHV infection and KS pathogenesis.

KSHV gene expression is essential for sustained ANG secretion during primary infection.

KSHV infection of adherent HMVEC-d, HFF, and 293 cells results in the transient expression of a limited number of lytic viral genes involved in antiapoptosis and immune modulation, sustained expression of latent genes, and secretion of several cytokines and growth factors (6, 22, 32). To elucidate whether viral gene expression is required for the observed secretion of ANG, serum-starved subconfluent HMVEC-d and HFF cells were infected with live KSHV and non-replication-competent UV-KSHV. RNA extracted at 48 h p.i. was analyzed for viral gene expression by quantitative real-time RT-PCR. As previously reported (33), more than 95% inhibition of viral latent and lytic genes was seen with UV-KSHV compared to live KSHV results (data not shown). Supernatants harvested from these cells were assayed using ANG-ELISA.

The level of ANG secretion by HMVEC-d cells seen with live KSHV was about 1,000 pg/ml; in contrast, the level of ANG secretion in HFF cells induced by live KSHV infection was only 600 pg/ml (Fig. (Fig.2A).2A). Interestingly, there was an approximately 50% reduction in ANG secretion for both cell types upon UV-KSHV infection compared to the induction observed with live KSHV (Fig. (Fig.2A).2A). Although the induction of ANG protein secretion by UV-KSHV was higher than that seen with uninfected cells, this induction was lower than the induction observed with live virus. These results suggested that, in similarity to ERK1/2, NF-κB, and COX-2 induction (32, 35), ANG induction was partially dependent upon the virus binding and entry stages. However, in contrast to ERK1/2 induction (33) but in similarity to NF-κB and COX-2 induction (32, 35), KSHV viral gene expression appears to be required for the augmented induction of ANG. Higher levels of ANG detected in live virus-infected cells suggested that viral gene expression early during infection, possibly together with viral gene-induced host cell gene expression, is probably essential for the increased and sustained ANG induction. These results suggested that viral gene expression could be necessary for the augmented and sustained induction and secretion of ANG.

FIG. 2.

Viral gene expression augments ANG secretion. (A) Supernatants from serum-starved HFF and HMVEC-d cells (\ [contains]

7ε30% confluence) infected with live KSHV or UV-KSHV (MOI of 10) for 48 h and supernatants from uninfected cells (more ...)

During KSHV infection of HMVEC-d cells, latency-associated ORF73 (LANA-1) gene expression was seen as early as 2 h p.i. and continued to be expressed in a sustained manner throughout the 5-day p.i. observation period (22). Since we observed a consistent increase in the levels of ANG secretion at 24 h and 48 h p.i. compared to that seen at earlier time points, we next examined the impact of latent gene expression in ANG secretion. An et al. (4) infected telomerase-immortalized human umbilical vein endothelial (TIVE) cells and isolated TIVE-LTC that were stably expressing LANA-1 and other latent genes (KSHV). When quantitative real-time PCR was done using mRNA extracted from TIVE cells and TIVE-LTC, there was an approximately fivefold increase in ANG mRNA expression levels in KSHV-positive TIVE-LTC compared to TIVE cell results (Fig. (Fig.2B).2B). ANG-ELISA performed using supernatants from TIVE cells and TIVE-LTC (KSHV) confirmed the increase in ANG levels in latently infected cells. We observed approximately sixfold-higher ANG levels in TIVE-LTC (KSHV) compared to the control TIVE cell results (Fig. (Fig.2C).2C). This further confirmed our finding that KSHV infection induced the secretion of ANG and that latent viral gene expression could be involved in this induction.

To substantiate these findings, supernatants harvested from uninduced and 48-h-TPA-induced BCBL cells were assayed by ANG-ELISA. Compared to the ANG levels seen in BJAB cell culture supernatant, there was an approximately twofold increase in ANG secretion from BCBL cells (Fig. (Fig.2D).2D). This was further upregulated to about 2.5-fold upon TPA induction (Fig. (Fig.2D).2D). These results demonstrated that ANG was induced by KSHV in B cells as well and suggested a role for both latent and lytic gene expression in inducing ANG secretion.

KSHV latency-associated ORF73 genes and lytic ORF74 and ORF50 genes play roles in ANG expression.

To identify the KSHV gene(s) that can induce ANG gene expression and secretion, we transduced the HMVEC-d cells with lentiviral vectors harboring different viral genes. Among the latency-associated KSHV genes tested, we observed a fourfold increase in ANG gene expression in ORF73 (LANA-1)-expressing cells compared to the results seen with cells transduced with pSIN (Fig. (Fig.3A).3A). KSHV ORF72 (vCyclinD) did not have any impact, while ORF71 (K13; vFLIP) and K12 increased ANG gene expression by twofold (Fig. (Fig.3A).3A). When we tested the lytic cycle-associated KSHV ORF74 (viral G protein-coupled receptor [vGPCR]), a powerful modulator of host cell signal cascades, we observed an approximately 4.7-fold increase in ANG mRNA levels (Fig. (Fig.3A).3A). When the supernatants of these transduced cells were examined for secreted ANG, we observed a significant (about 3.3-fold) increase in ORF73-transduced cell levels (Fig. (Fig.3B).3B). The lytic cycle ORF74 induced an increase in ANG secretion of about 3.4-fold (Fig. (Fig.3B3B).

FIG. 3.

KSHV latency-associated gene ORF73 and lytic genes ORF74 and ORF50 expression augments ANG secretion. (A) cDNA prepared from HMVEC-d cells transduced with lentivirus harboring viral genes was used for quantitative real-time RT-PCR to measure ANG gene (more ...)

Since we did not have the lentiviral constructs expressing the ORF50 gene, we used an Ad expressing ORF50 and compared it with an Ad expressing GFP. Serum-starved HMVEC-d cells were infected with AdORF50 or AdGFP at an MOI of 1 for 48 h, and mRNA was extracted and quantitated for ANG gene expression by real-time RT-PCR. ORF50 gene expression was normalized to GFP expression to overcome the difference in infectivity between AdORF50 and AdGFP. There was an approximately twofold increase in ANG gene expression in AdORF50-infected cells compared to AdGFP cell results (Fig. (Fig.3C).3C). An ANG-ELISA confirmed these findings, and an approximately twofold increase in ANG secretion was detected for AdORF50 cells (Fig. (Fig.3D).3D). This was approximately 50% less than the ANG induction observed upon ORF73 or ORF74 gene expression. These results further confirmed our finding that viral genes were indeed responsible for the increase in ANG expression and secretion.

Since LANA-1 was observed to induce ANG significantly, 293T cells transfected with increasing concentrations of LANA-1-expressing plasmids were assayed for ANG gene expression and secretion to validate this result. Transfection efficiency was confirmed by measuring LANA-1 gene expression (Fig. (Fig.4A)4A) and by Western blotting for ORF73 protein by the use of rabbit anti-ORF73 antibody (data not shown). ANG gene expression increased with increasing concentrations of LANA-1 (Fig. (Fig.4B)4B) by about 3-fold with 2 μg of LANA-1 plasmid; this increase reached a peak of 4.2-fold with a 5-μg plasmid concentration. Measuring the supernatant-associated ANG from serum-starved 293T cells transfected with increasing concentrations of LANA-1 by ANG-ELISA further confirmed these findings (Fig. (Fig.4C).4C). During primary infection of HMVEC-d cells, ORF74 is not expressed and ORF50 is only transiently expressed (22). Hence, the increase in ANG gene expression and secretion by ORF73 underscores the importance of the latency gene in regulating ANG gene expression during latency.

FIG. 4.

ANG expression and secretion is elevated upon LANA-1 expression. 293T cells were transfected with increasing concentrations of LANA-1 expression plasmids, and cDNA from these cells was used in quantitative real-time RT-PCRs to measure LANA-1 (A) and ANG (more ...)

KSHV-induced ANG localizes on the cell surface, cytoplasm, and nucleus of infected endothelial cells.

Since this is the first report of ANG induction by KSHV infection, to determine the nature and functions of KSHV-induced ANG and their importance in KSHV pathogenesis we next carried out a series of morphological and functional assays. In similarity to the results seen with FGF, secreted ANG could act either in an autocrine fashion on the same cells or in a paracrine fashion on neighboring cells to exert its impact (13). Previous reports showed that once secreted into the milieu, ANG interacts with a cell surface 170-kDa receptor and actin is endocytosed and traffics through the cytoplasm in a microtubule-independent manner before moving into the nucleus (24). To determine the trafficking of KSHV-induced ANG, serum-starved semiconfluent (>50 to 60% confluence) HMVEC-d cells infected with KSHV for 48 h were stained for surface phalloidin and ANG without permeabilization. Barely detectable levels of ANG were observed on the uninfected cell surfaces (Fig. (Fig.5a).5a). In contrast, on the infected cells, significant levels of ANG colocalized with surface actin (Fig. (Fig.5b5b and inset).

FIG. 5.

Immunofluorescence analysis of ANG in KSHV-infected HMVEC-d and HFF cells. (a and b) Serum-starved HMVEC-d cells (>50 to 60% confluence) were either left uninfected (a) or infected with KSHV (MOI of 10) (b) for 48 h and stained for ANG (more ...)

To determine the fate of ANG following actin interaction, serum-starved HMVEC-d cells grown to various levels of confluence in eight-well chamber slides were infected with KSHV at an MOI of 10 for 48 h, permeabilized, and analyzed by immunofluorescence. There was a negligible amount of ANG in the uninfected confluent and semiconfluent HMVEC-d cells (Fig. 5a, c, and e). In 80-to-90%-confluent infected HMVEC-d cells, ANG remained cytoplasmic and did not colocalize with tubulin (Fig. (Fig.5d5d and inset), whereas in >50%-confluent (i.e., semiconfluent) infected HMVEC-d cells, ANG was detected both in the cytoplasm and the nucleus and the majority of ANG was associated with the cell nuclei (Fig. (Fig.5f5f and inset).

Nuclear localization of KSHV-induced ANG is dependent on the cell type.

There was a negligible amount of ANG in the uninfected semiconfluent HFF cells (Fig. (Fig.5g).5g). Even though we observed increased secretion of ANG in infected HFF cells (Fig. (Fig.2A),2A), ANG failed to move into the nucleus even in semiconfluent HFF cells (Fig. (Fig.5h).5h). It is unclear at the moment why there is nuclear translocation in HMVEC-d cells and not in HFF cells. This result is similar to the previously reported cell type-dependent movement of ANG into the nucleus (28). Since it is known that nuclear translocation of ANG is critical for its angiogenic function in endothelial cells, translocation could be due to HMVEC-d cell-specific factors that facilitate the transport of ANG and thus contribute to the higher angiogenic potential of HMVEC-d cells compared to HFF cells.

ANG could function in both an autocrine and a paracrine fashion.

When semiconfluent cells were examined at 48 h p.i., nuclear localization of ANG was observed in 80% of cells (Fig. (Fig.5i).5i). Examination with anti-LANA-1 antibody demonstrated the characteristic punctate nuclear staining of ORF73 protein in about 50% of cells (Fig. (Fig.5j).5j). ANG was detected both in infected LANA-positive cells and in uninfected LANA-1-negative cells, which indicates ANG could act both in an autocrine and in a paracrine fashion (Fig. 5i to l).

Nucleolus localization of KSHV-induced ANG is influenced by endothelial cell confluence.

Once ANG translocates into the nucleus, it enters the nucleolus, according to a previous report (42). Hence, we examined the localization of ANG in subconfluent and semiconfluent HMVEC-d cells by the use of fibrillarin (Nop1p) as a marker for nucleolar staining, since fibrillarin is a component of the nucleolar small nuclear ribonucleoprotein particle. After 48 h p.i., though we detected considerable amounts of ANG in the infected cell nuclei, we did not observe any noticeable nucleolar localization of ANG in the semiconfluent infected HMVEC-d cells (Fig. (Fig.6A,6A, panels a to e, and inset in panel e). In contrast, a distinct nucleolar localization of ANG was observed in the subconfluent infected cells (Fig. (Fig.6A,6A, panels f to j, and inset in panel j).

FIG. 6.

Localization of ANG in the nuclei and nucleoli of infected cells. (A) Serum-starved semiconfluent (50 to 60% confluence) (a to e) or subconfluent (30 to 40% confluence) (f to j) HMVEC-d cells were infected with KSHV for 48 h and stained (more ...)

To ascertain the differential nuclear and nucleolar localization characteristics of ANG in various states of cell confluence, semiconfluent and subconfluent HMVEC-d cells were stained for ANG along with the nuclear and nucleolar markers Topro and fibrillarin, respectively, and examined by confocal microscopy. Nuclear translocation of ANG in semiconfluent cells was clearly evident, as significant colocalization of ANG with Topro was observed (Fig. (Fig.6B,6B, panel a). ANG failed to colocalize with fibrillarin in semiconfluent cells (Fig. (Fig.6B,6B, panel b) but displayed significant colocalization with fibrillarin in subconfluent cells (Fig. (Fig.6B,6B, panel c). These are very interesting observations, since they demonstrate for the first time three distinct localizations for ANG during KSHV infection: cytoplasmic translocation in confluent cells, nuclear translocation in semiconfluent cells and subconfluent cells, and nucleolar translocation in subconfluent cells.

Neomycin blocks nuclear translocation of KSHV-induced ANG.

Nuclear translocation of ANG was reported to be critical for ANG to be functional, and blocking nuclear localization inhibits the angiogenic potential of ANG (15, 16). The aminoglycoside neomycin inhibits nuclear translocation of ANG by inhibiting PLC (phospholipase C) activation without affecting the viability of the cells (15, 16). Since HMVEC-d cells are poorly transfectable, instead of an ANG small interfering RNA approach, neomycin was used to determine the specificity of KSHV-induced ANG nuclear translocation. A dose-response study showed that neomycin, even at a 200 μM concentration, was noncytotoxic (data not shown). To test whether neomycin treatment could block nuclear translocation of ANG, subconfluent HMVEC-d cells (30 to 40% confluence) were pretreated with 100 μM neomycin for 1 h and infected with KSHV at an MOI of 10 for 48 h, and nuclear extracts were analyzed by Western blotting for the presence of ANG. The purity of the nuclear extracts was confirmed by the detection of lamin B in the nuclear extracts and by the absence of the cytoplasmic marker tubulin (Fig. (Fig.6C,6C, middle and lower panels). In the uninfected cells, ANG levels were undetectable in the nucleus (Fig. (Fig.6C,6C, top panel, lane 1). When uninfected cells were incubated with exogenous ANG for 48 h, ANG was detected in the nucleus (Fig. (Fig.6C,6C, top panel, lane 2). A significant amount of ANG accumulated in the nucleus of KSHV-infected cells (Fig. (Fig.6C,6C, top panel, lane 3); this accumulation was blocked by neomycin treatment (Fig. (Fig.6C,6C, lane 4). ANG-treated cells showed only an approximately 1.5-fold-higher nuclear accumulation of ANG compared to KSHV-infected cell results. This could have been due to that the fact that nuclear translocation of ANG is dependent on cell density and is probably controlled by multiple factors, such as signaling mediated by several growth factors in the mileu. Hence, it may be that all ANG that is either secreted or added extraneously might not end up in the nucleus, due to variations in the signaling involved in the two processes.

To determine the impact of neomycin treatment on ANG secretion, serum-starved HMVEC-d cells were pretreated with neomycin and infected with KSHV for 48 h and ANG secretion was assayed by ANG-ELISA. A fourfold increase in ANG secretion was observed in KSHV-infected cells compared to uninfected cell results (Fig. (Fig.6D).6D). Even though neomycin blocked the movement of ANG into the nucleus, it had only a moderate (threefold induction) inhibitory effect on ANG secretion (Fig. (Fig.6D).6D). The results shown in Fig. Fig.55 and and66 collectively suggest that upon secretion, KSHV-induced ANG binds to surface actin, traffics through the cytoplasm, translocates into the nucleus, and moves into the nucleolus in subconfluent infected and uninfected HMVEC-d cells.

KSHV-induced ANG increases 45S rRNA synthesis in infected cells.

The nucleolus is the site for 45S rRNA synthesis. After moving into the nucleolus, ANG binds to the CT-rich upstream sequences of 45S ribosomal DNA (rDNA) called ABE and upregulates 45S rRNA (41). To determine whether KSHV-induced ANG binds the 45S rDNA promoter and upregulates 45S rRNA transcription, a gel shift assay was done using the consensus ANG binding element sequences in 45S rDNA (41). Compared to uninfected cell results, in nuclear extracts prepared from subconfluent (30 to 40% confluence) HMVEC-d cells infected with KSHV for 72 h, we observed a higher intensity in the mobility-shifted bands (Fig. (Fig.7A,7A, lanes 1 and 3). This suggested that an interaction of KSHV-induced ANG with 45S rRNA transcription elements occurred during infection. ANG-treated HMVEC-d cells showed a much higher mobility-shifted band than was seen under KSHV infection conditions (Fig. (Fig.7A,7A, lane 4). The specificity of this reaction was demonstrated by the absence of ANG binding to the target DNA in the competition assay using a 100-fold molar excess of cold double-stranded ABE oligonucleotide probe (Fig. (Fig.7A,7A, lane 2).

FIG. 7.

KSHV-induced ANG binds to the 45S rRNA promoter and upregulates transcription of 45S rRNA. (A) Nuclear extracts prepared from subconfluent serum-starved HMVEC-d cells were either left uninfected (lane 1) or infected (lane 3) with KSHV (MOI of 10) for (more ...)

We next tested the ability of KSHV-induced ANG to activate the promoter (pGL3E-ABE) containing the ANG-binding element (CTCTCTCTCTCTCTCTCCCTC). HEK 293 cells transfected with pGL3E-ABE and control pGL3E luciferase constructs were infected with KSHV for 48 h or treated with exogenous ANG. In comparison to control plasmid cell or uninfected cell results, we observed a significant (>2-fold) increase in ABE promoter activation induced by KSHV infection and a >3-fold increase in activation induced by ANG (Fig. (Fig.7B).7B). To determine whether promoter activation could result in an increase in the levels of 45S rRNA, real-time qRT-PCR was carried out with RNA from 30 to 40% confluence HMVEC-d cells infected with KSHV for different time periods. We observed a 2-fold increase in 45S rRNA levels at 36 h p.i. and a >2.5-fold increase at 48 h p.i. for the infected cells (Fig. (Fig.7C).7C). To evaluate the importance of nuclear translocation of ANG in increasing 45S rRNA transcription, RNA extracted from HMVEC-d cells pretreated with neomycin was examined by real-time RT-PCR. As a positive control, serum-starved HMVEC-d cells treated with ANG, with and without neomycin, were assessed for the presence of 45S rRNA transcripts. We observed a significant inhibition in 45S rRNA transcript levels in HMVEC-d cells pretreated with neomycin compared to untreated cell results upon KSHV infection (Fig. (Fig.7D).7D). Similarly, the 3.3-fold increase in 45S rRNA levels observed with ANG treatment was also inhibited to basal levels with neomycin pretreatment (Fig. (Fig.7D).7D). These results suggested that KSHV-induced ANG moves into the nucleolus of subconfluent HMVEC-d cells and binds to the ANG-binding element in the 45S rDNA promoter, resulting in increased 45S rRNA transcription in infected cells.

ANG increases the survival of KSHV-infected endothelial cells.

Cancer is characterized by uncontrolled proliferation of cells, requiring continuous protein synthesis that depends on a steady supply of ribosomes. rRNA synthesis is a rate-limiting step during ribosome biogenesis (21). Since ANG upregulated 45S rRNA synthesis in KSHV-infected endothelial cells, we examined whether KSHV-induced ANG could increase the survival of serum-starved endothelial cells. HMVEC-d cells were serum starved for 12 h and then treated with anti-ANG antibody before treatment with growth factors or with supernatants from HMVEC-d cells. MTT (Sigma) is a pale yellow substrate that is cleaved by living cells to yield a dark blue formazan product. This process requires active mitochondria, and even freshly dead cells do not cleave significant amounts of MTT. The cell survival assay was done using MTT assay reagent, and values obtained with endothelial growth media containing serum and growth supplement were defined as representing a result of 100% and compared with values obtained using various treatments. ANG and VEGF treatments resulted in about 61% and 82% cell survival, respectively, while pretreatment of these supernatants with anti-ANG antibody reduced the survival significantly to about 21% and 43%, respectively (Fig. (Fig.8A).8A). Uninfected cell culture supernatants did not increase cell survival, whereas supernatants from live KSHV infections showed 50% cell survival and those from UV-KSHV infections showed only about 20% cell survival (Fig. (Fig.8A).8A). Anti-ANG antibody treatment significantly blocked the increase in cell survival mediated by both live and UV-KSHV-infected cell culture supernatants, indicating the important role played by ANG in augmenting the survival of KSHV-infected endothelial cells (Fig. (Fig.8A8A).

FIG. 8.

KSHV infection and the presence of ANG increase HMVEC-d cell survival and block apoptosis. (A) HMVEC-d cells were serum starved using EBM-2, left untreated or pretreated with anti-ANG antibody for 1 h followed by incubation for 48 h with growth factors (more ...)

ANG blocks apoptosis in KSHV-infected endothelial cells.

We next examined whether KSHV-induced ANG could block apoptosis and increase the KSHV-infected HMVEC-d cell survival, which is an important prerequisite for an increase in the proliferation of cells. Following the induction of apoptosis signals in a cell, the transmembrane potential of mitochondria undergoes changes that results in altered membrane permeability, and the MitoSensor dye in the MitoTracker dye kit is sensitive to these changes in permeability. MitoSensor is a cationic dye that fluoresces differently in apoptotic and nonapoptotic cells. In live cells, the dye is taken up in the mitochondria, where it forms aggregates and exhibits intense red fluorescence. In contrast, because of the altered mitochondrial membrane permeability in the apoptotic cells, the dye cannot accumulate in the mitochondria; thus, it remains in monomeric form in the cytoplasm and fluoresces green. The fluorescence signals are easily distinguished by fluorescence microscopy or flow cytometry.

HMVEC-d cells were serum starved for 8 h and were then either left uninfected or infected with live KSHV or UV-KSHV for 48 h or treated with ANG in the absence of serum and were stained with the MitoSensor dye. The percentage of cells that fluoresced green or red was calculated by counting the total number of cells in a field by the use of phase-contrast microscopy and dividing the number of green or red cells by the total number of cells. Since serum withdrawal triggers apoptosis, serum-starved (for 8 h and 48 h) 30-to-40%-subconfluent uninfected HMVEC-d cells exhibited increased apoptosis, as evidenced by the cells fluorescing predominantly green (Fig. (Fig.8B,8B, panel a). In contrast, >80% of serum-starved (for 8 h and 48 h) subconfluent HMVEC-d cells infected with KSHV stained red due to dye uptake in the intact mitochondria, thus indicating a block in apoptosis and cell survival (Fig. (Fig.8B,8B, panel b). Similarly, >90% of ANG-treated serum-starved (for 8 h and 48 h) subconfluent cells fluoresced red, confirming the antiapoptotic survival and proliferative capacity of ANG (Fig. (Fig.8B,8B, panel c). When serum-starved (for 8 h and 48 h) subconfluent cells were infected with non-replication-competent UV-treated virus, only about 10% of UV-KSHV-infected cells fluoresced red (Fig. (Fig.8B,8B, panel d). These results highlighted the importance of viral gene expression in ANG secretion, antiapoptosis, and survival of infected endothelial cells (Fig. (Fig.8B8B).

Next, we analyzed the importance of nuclear translocation of ANG in antiapoptosis and cell survival of KSHV-infected cells by pretreating serum-starved cells with neomycin before KSHV infection for 48 h or treatment with ANG for 48 h in the absence of serum. More than 90% of the serum-starved uninfected HMVEC-d cells fluoresced green, indicating the triggering of apoptosis in these cells (Fig. (Fig.8C,8C, panel a). The antiapoptosis seen with KSHV infection or ANG treatment (Fig. (Fig.8B)8B) was greatly reduced or abolished by neomycin treatment (Fig. (Fig.8C,8C, panels b and c). Paromomycin is an aminoglycoside that does not inhibit nuclear translocation of ANG (15). When serum-starved cells were pretreated with paromomycin and infected, more than 90% of cells fluoresced red (Fig. (Fig.8C,8C, panel d). Taken together, these results suggested that KSHV-induced ANG plays critical roles in antiapoptosis and cell survival of KSHV-infected cells and thus could be contributing to the eventual proliferation of endothelial cells in vivo and in KSHV-mediated KS pathogenesis.

KSHV infection of HMVEC-d cells increases uPA activation and plasmin generation in infected endothelial cells.

During angiogenesis, endothelial cells proliferate and migrate to areas of lesser density, thus inducing neovasculogenesis (40). Degradation of the basal lamina is an essential first step for endothelial cell migration (40). Plasmin is a serine protease that degrades the basement membrane fibrin (18). Previous studies have reported that the ANG-actin complex formation on the cell membrane can accelerate plasmin generation by activating the plasminogen activator in a more potent manner than actin or ANG alone. Since we observed a significant colocalization of ANG with surface actin in KSHV-infected HMVEC-d cells (Fig. (Fig.5b)5b) and our earlier gene array studies demonstrated a significant upregulation of genes encoding uPA receptor precursor and tissue plasminogen activator in KSHV-infected cells (30), we next examined whether actin interaction with KSHV-induced ANG accelerates the plasmin generation required for the degradation of basal lamina through the activation of plasminogen activator in infected endothelial cells.

For this experiment, lysates from serum-starved 30-to-40%-confluent HMVEC-d cells infected with KSHV for various times were harvested, assayed for uPA activation, and normalized with total uPA to estimate the extent of uPA activation. As shown in Fig. Fig.9A,9A, there was a time-dependent increase in uPA activation in KSHV-infected HMVEC-d cells that correlated well with the observed increase in ANG secretion upon KSHV infection shown in Fig. Fig.1.1. We observed an approximately threefold increase in uPA activation in HMVEC-d cells treated with exogenous ANG (Fig. (Fig.9B).9B). KSHV infection also induced a >2-fold increase in uPA activation which was inhibited by neomycin but not by paromomycin pretreatment (Fig. (Fig.9B),9B), thus supporting the idea of a role played by ANG during uPA activation in KSHV-infected endothelial cells.

FIG. 9.

Increases in uPA activation and cell migration upon KSHV infection. (A) Cell lysates from serum-starved HMVEC-d cells (30 to 40% confluence) left uninfected or infected with KSHV (MOI of 10) for the indicated time periods were prepared. Lysates (more ...)

The outcome of uPA activation is the generation of plasmin from plasminogen. We next analyzed the generation of active plasmin from uncleaved plasminogen at 48 h post-KSHV infection. Western blot analysis of uninfected HMVEC-d cell lysates showed only the uncleaved 120-kDa form of plasmin (Fig. (Fig.9C,9C, lane 1). In contrast, KSHV infection appears to result in the activation of uPA and the conversion of plasminogen to plasmin, since we observed the cleaved 60-kDa plasmin heavy chain and a small amount of 23-kDa plasmin light chain (Fig. (Fig.9C,9C, lanes 2 and 3). Similarly, the cleaved form of plasmin was also observed in the cells added with exogenous ANG (Fig. (Fig.9C,9C, lane 5) but not in untreated cells (Fig. (Fig.9C,9C, lane 4). Neomycin pretreatment inhibited plasmin generation during KSHV infection (Fig. (Fig.9C,9C, lanes 6 and 7); this result could have been due to moderate inhibition of ANG secretion by neomycin (Fig. (Fig.6C6C).

ANG plays a role in KSHV infection-induced endothelial cell migration.

The results detailed above demonstrated uPA activation and subsequent plasmin generation during KSHV infection and suggested that the ANG-actin complex could lead to proteolytic degradation of the basement membrane and ECM by plasmin, thereby aiding in infected and uninfected endothelial cell migration and invasion processes. This led us to ask whether KSHV infection induces the migration of HMVEC-d cells and whether ANG plays a role in this migration. In the cell migration assay, the chemoattractant is added to the lower chamber and HMVEC-d cells with or without aminoglycosides are added to the upper chamber. After 4 h of incubation, the cells that had migrated to the bottom of the chamber were dislodged and assayed fluorimetrically. Compared to the results seen with supernatant from uninfected cells, when KSHV-infected cell culture supernatant was used as a chemoattractant, we observed an approximately twofold increase in the number of cells that migrated to the bottom of the upper chamber (Fig. (Fig.9D).9D). Supernatant with ANG added showed about 2.5-fold-higher migration compared to uninfected cell results (Fig. (Fig.9D).9D). Interestingly, when cells in the upper chambers were pretreated with neomycin, there was a significant inhibition in migration with all three supernatant categories (Fig. (Fig.9D).9D). Paramomycin did not show any inhibitory effect (Fig. (Fig.9D).9D). These data suggested that KSHV infection of endothelial cells induced factors that aid in cell migration, that ANG is one of the major factors, and that ANG transport to the nucleus might play additional roles in the modulation of factors associated with the migration of endothelial cells.

KSHV-induced ANG increases endothelial cell tube formations.

During the process of angiogenesis, the effect of stimuli induced by angiogenic factors such as VEGF, ANG, bFGF, and EGF that promote proliferation of endothelial cells outweighs the effect of angiostatic factors. The proliferating endothelial cells migrate along the gradient through the disintegrated basement membrane into the remodeled and softened perivascular space. Once the endothelial cells reach the area with reduced vessel density, the endothelial cells arrange in a monolayer and form tube-like structures. Since the ultimate function of an angiogenic factor is blood vessel formation, we used a Matrigel assay to investigate the role of KSHV-induced ANG in endothelial cell tube formation.

Serum-starved subconfluent HMVEC-d cells treated with ANG for 16 h displayed an increase in the angiogenic index, which was calculated based on the number of branch points formed from each node (Fig. 10A, panel a, and 10D). When cells were pretreated with anti-ANG antibody (200 μg/ml) for 1 h, we observed a significant (70%) reduction in tube formation (Fig. 10A, panel b, and 10D), thus indicating a role for ANG in endothelial cell tube formation.

FIG. 10.

ANG secretion induces increased tube formation. (A) Serum-starved HMVEC-d cells (30 to 40% confluence) incubated with or without anti-ANG antibody (200 μg/ml) were mixed with EBM-2 containing ANG (250 ng/ml) (a and b) or VEGF (100 ng/ml) (more ...)

Previous reports have shown that nuclear translocation of ANG is critical for VEGF- and bFGF-mediated angiogenic activity (21). Our recent studies have shown that in addition to ANG, KSHV also induced sustained VEGF-A and VEGF-C gene expression in the infected HMVEC-d cells and that this induction was accompanied by significant secretion of VEGF-A and VEGF-C into the infected cell milieu (36). To determine whether blocking ANG could affect tube formation mediated by KSHV-induced VEGF, serum-free media mixed with 100 ng of VEGF with and without anti-ANG antibody was used in a Matrigel assay. VEGF treatment induced tube formation of HMVEC-d cells (Fig. 10A, panel c, and 10D); this induction was significantly (>80%) inhibited by anti-ANG treatment (Fig. 10A, panel d, and 10D). Culture supernatant from KSHV-infected cells induced a higher level of tube formation than supernatants from uninfected cells (Fig. 10A, panels e and g, and 10D), while anti-ANG antibody treatment significantly inhibited about 80% of the tube-forming ability of these supernatants (Fig. 10A, panels f and h, and 10D).

While anti-ANG antibody inhibited tube formation significantly, similar concentrations of normal rabbit immunoglobulin G (IgG) or rabbit anti-ERK2 IgG antibodies used as a control did not block tube formation mediated by infected HMVEC-d cell culture supernatant (Fig. 10B, panels a and b). Serum-free EBM-2 used as a control displayed negligible tube formation (Fig. 10B, panel c). These results clearly suggested that ANG plays a pivotal role in KSHV infection-induced endothelial cell tube formation. Neomycin treatment blocked tube formation mediated by exogenous ANG, VEGF, and infected HMVEC-d cell culture supernatant (Fig. 10C, panels a to f, and 10D), indicating a role for nuclear translocation of ANG in VEGF- and ANG-mediated endothelial cell tube formation.

It is known that blocking nuclear translocation of ANG inhibits the angiogenic potential of VEGF and FGF (21). Since we observed that both anti-ANG antibody and neomycin blocked VEGF- and infected cell culture supernatant-mediated tube formation, we hypothesized that the block in tube formation upon VEGF treatment and HMVEC-d cell supernatant treatment could be due to feedback control by ANG. To check whether ANG treatment could influence VEGF-C induction, HMVEC-d cells pretreated with neomycin for 1 h were subjected to ANG treatment or KSHV infection for 48 h and VEGF-C messages were quantitated by real-time RT-PCR. We observed approximately two- and threefold increases in VEGF-C expression induced by KSHV infection and by exogenous ANG, respectively (Fig. 10E). VEGF-C gene expression induced by KSHV was reduced to 1.5-fold by neomycin treatment, while paromomycin did not have any influence (Fig. 10E). VEGF-C induction by ANG was also significantly reduced by neomycin treatment (Fig. 10E). These results clearly suggested that VEGF-C expression could be induced by ANG treatment; however, the reduction due to neomycin treatment was more significant with ANG treatment than with KSHV infection, thus indicating that in addition to ANG, other factors such as viral gene expression and\or KSHV-induced cytokines and growth factors (32) play a role in VEGF induction.

To authenticate these findings, we used fluorescence-activated cell sorter (FACS) analysis to detect VEGF-C after neomycin treatment. There was an increase in VEGF-C expression induced by ANG treatment (Fig. 10F, panel a) as well as by KSHV infection (Fig. 10F, panel b). A significant reduction in VEGF-C expression was observed when cells pretreated with neomycin were infected with KSHV (Fig. 10F, panel b). These results suggest that KSHV-induced ANG has multiple roles to play in angiogenesis. As an angiogenic molecule, ANG could influence tube formation directly, by increasing 45S rRNA synthesis and cell proliferation and activating plasminogen activator, resulting in increased cell migration, as well as indirectly, by increasing VEGF-C expression, which is a key factor involved in tube formation during KSHV infection.

DISCUSSION

The hallmark of the KSHV infection-associated KS lesion is the extensive angioproliferation of spindle-shaped endothelial cells, infiltrating inflammatory cells, cytokines, angiogenic factors, and growth factors (9). The presence of a disorganized collection of blood-filled vascular slits in KS lesions composed of spindle-shaped endothelial cells is suggestive of angiogenesis in the lesion. Angiogenesis is the formation of new blood vessels from existing blood vessels. Under normal physiological conditions, angiostatic factors are more significant than angiogenic factors; however, under conditions such as wound healing, tumorigenesis, and tissue repair, angiogenic factors outweigh angiostatic factors, resulting in angiogenesis. The various angiogenic factors such VEGF, FGF, SDF-1, and angiopoietin that are secreted upon de novo KSHV infection of endothelial cells (30, 32, 36, 43) could induce angiogenesis by promoting endothelial cell migration, and the combination of multiple cytokines, growth factors, and chemokines modulated by KSHV (30, 32, 36, 43) could be responsible for promoting and spreading the infection. Here, we report for the first time the upregulation of the important angiogenic factor ANG by in vitro KSHV infection, and our comprehensive study clearly demonstrated that ANG induction results in several consequences in the infected endothelial cells (Fig. (Fig.11)11) that are highly relevant to KSHV biology, KS biology, and pathogenesis. Detection of ANG in KS tissue not only underscores the importance of ANG in KS pathogenesis but also suggests the possibility of ANG-mediated consequences during in vivo infection by KSHV.

FIG. 11.

Schematic representation of the potential consequences of the presence of KSHV-induced ANG for the infected endothelial cell population. KSHV infection induces signal pathway activation leading to viral entry, nuclear delivery of the genome, and viral (more ...)

For clarity and better understanding, we have summarized in the following sections the potential implications of the multiple roles that ANG could play in KSHV biology and KS pathogenesis in overlapping categories.

ANG and KSHV genes.

Significant ANG gene expression and protein secretion from 24 h p.i. and its continued production coinciding with sustained KSHV latent gene expression during primary infection of endothelial cells (26, 38), together with the detection in TIVE-LTC and BCBL-1 cells, clearly demonstrate that latent gene expression plays roles in ANG gene expression. Among the latent genes tested, KSHV ORF73 appears to have the more profound effect on ANG induction. During primary infection of HMVEC-d cells, ORF74 is not expressed and ORF50 is only transiently expressed (22). Hence, the increase in ANG gene expression and secretion by ORF73 underscores its importance in regulating ANG gene expression during latency.

During de novo infection of endothelial cells, KSHV establishes latency, and <3 to 5% of cells spontaneously undergo lytic switching after 72 h p.i. Similarly, 3 to 5% of BCBL-1 cells spontaneously enter the lytic cycle, and the lytic cycle is detected in the inflammatory cells of KS lesions (22). The ability of lytic ORF50 and ORF74 to induce ANG expression not only clearly indicated that ANG must play a major role in KSHV pathogenesis and angiogenesis of KS lesions but also indicated that the lytic cycle may also contribute to angiogenesis. This is the first report showing that vGPCR induces ANG, a key molecule in angiogenesis. Since the KSHV vGPCR (ORF74) gene that has been shown to play a role in the induction of angiogenesis and KS-like lesions induced a significant amount of ANG, we speculate that the angiogenic potential hitherto attributed to vGPCR might be due its ability to induce ANG. Further studies using LANA-1- and vGPCR-deleted bacterial artificial chromosome-KSHV need to be performed to fully evaluate the role of these genes in ANG induction and the mechanism(s) behind ANG induction.

We reported previously that blocking of NF-κB by the use of Bay 11-7082 could partially inhibit ANG secretion (32). Since vGPCR is a known inducer of the NF-κB pathway, it could possibly induce ANG secretion via the NF-κB pathway. The latent ORF71 (vFLIP) gene is also a known inducer of NF-κB; however, we observed only moderate induction of ANG by the lentivirus transduction method. This could be due to the efficiency of the expression system used and/or to the involvement of several transcription factors such as AP-1, CREB, NFκB, and Nrf2 that have binding sites on the ANG promoter/enhancer elements. An et al. (5) demonstrated the role played by LANA-1 in upregulating IL-6 via the AP-1 pathway. Studies are in progress to determine the involvement of NF-κB and the AP-1 family of transcription factors in LANA-1-mediated ANG induction.

Implications of 45S rRNA synthesis and nuclear and nucleolar ANG.

The transcription of ribosomal proteins is reported to be regulated via the AKT-PI3K-mTORS6K pathway (37), while ANG is critical for rRNA transcription and thus plays an important role in regulating the 45S rRNA required for increased ribosome biogenesis. There are no reports on the regulation of ribosome biogenesis during KSHV infection so far, and this is the first report on increased 45S rRNA regulation during de novo KSHV infection. The nucleolus is a non-membrane-bound subnuclear organelle where ribosome biogenesis takes place (12). ANG contains an NLS at its N terminus, in similarity to other NLS peptides found in other nuclear proteins (23). Nucleolar localization of ANG is implicated in 45S rRNA synthesis, and association of ANG with the nucleoli of infected cell populations was predominately observed in the subconfluent cells. This could be due to the fact that increased 45S rRNA synthesis and proliferation is required only when the cells are less confluent, and nucleolar trafficking was reported to be critical for both the proliferation and angiogenic functions of ANG (37). Proteins translocate into the nucleolus via a process involving protein-protein, protein-RNA, and protein-DNA interactions. ANG could be associating with either host or viral proteins and could be shuttling between the nucleolus and nucleoplasm in a process that could be energy dependent. Studies are under way to determine whether ANG interacts with proteins associated with GTP-binding motifs and utilizes the energy obtained from these proteins for its translocation.

Implications of ANG blockage of apoptosis and cell proliferation.

KSHV latent genes such as LANA-1, vFLIP, and vCyclin D have all been shown to play a role in blocking apoptosis in primary-effusion lymphoma cells (PEL/BCBL cells) by a variety of mechanisms, including blocking of p53 function and activation of NF-κB. One of the most important findings in our study is that of the ability of ANG to block apoptosis. Serum starvation of endothelial cells is known to trigger apoptosis, and we witnessed a decrease in apoptosis upon live KSHV infection compared to serum-starved uninfected cell results. UV-KSHV infection resulted in increased cell death, indicating the necessity for viral gene expression in inducing antiapoptosis, including augmented ANG secretion. Our finding that negation of the block in apoptosis by the prevention of the nuclear translocation of ANG by neomycin in endothelial cells is very interesting and suggests that ANG must be playing a major role in KSHV-induced antiapoptosis in endothelial cells. The mechanism behind ANG-mediated apoptosis in KSHV-infected cells is not known, and whether it involves the induction of antiapoptotic genes and/or other pathways need to be evaluated further.

Proliferation of KSHV-infected endothelial cells depends on the growth factors available in the infected cell environment. Multiple growth factors were upregulated upon KSHV infection (30, 32), and all these factors could cumulatively increase the proliferation of endothelial cells. Nuclear ANG assumes an essential role in endothelial cell proliferation and is necessary for angiogenesis induced by other angiogenic molecules. Kishimoto et al. (21) reported that FGF, EGF, and VEGF stimulation increased nuclear accumulation of ANG. Silencing VEGF was reported to reduce the expression of several angiogenic factors, including ANG, MCP-1, IL-6, IL-8, and TGF β (14). We observed sustained VEGF expression during de novo KSHV infection, indicating the existence of a loop in the regulation mechanism mediated by these growth factors. VEGF induces nuclear translocation of ANG (21), which is necessary for the angiogenic potentials of VEGF, and by inducing expression of both VEGF and ANG KSHV probably ensures an increase in proliferation.

Implications of actin-ANG interactions, induction of plasmin generation, and cell migration.

Multiple proteins can bind to actin and function in the nucleating, capping, stabilizing, severing, bundling, and mechanical movement of actin filaments. Through the interactions with these proteins, actin filaments participate in several essential cellular functions, including cell motility, cytokinesis, and maintenance of cell structure and organelle movement (27). KSHV-induced ANG bound to endothelial cell surface actin, and the plasmin generation induced by uPA and seen in our study was reminiscent of the results of the earlier studies reporting the acceleration of this phenomenon by means of an actin-ANG complex (18). The actin-ANG association has been observed under even confluent cell conditions in our studies, and such an association in vivo could play roles in endothelial cell migration and tube formation.

Angiogenesis is a complex process involving endothelial cell migration, proliferation, and differentiation. Repetitive cycling of these steps ultimately forms new capillaries. Under normal circumstances, endothelial cells are quiescent and are surrounded by a basement membrane which is normally devoid of pores that would allow passive migration of cells. It is a prerequisite of angiogenesis that endothelial cells acquire an invasive phenotype so that they can penetrate the basement membrane and interstitial matrix. This involves proteolytic degradation and can be accomplished by activation of cellular proteases. Hu and Riordan (18) demonstrated the role played by ANG in mediating the angiogenic potential of endothelial cells and their capacity to degrade the ECM. The mechanism by which endothelial cells migrate for vasculogenesis during KSHV infection is not well established. Here we report for the first time the formation of an actin-ANG complex upon KSHV infection that could potentially be responsible for the activation of uPA, resulting in the generation of plasmin from plasminogen necessary for ECM degradation and cell migration. It is interesting to note that the plasminogen activator, plasminogen activator inhibitor, and tissue plasminogen activator were upregulated during in vitro infection of HMVEC-d cells by KSHV (30).

The cleavage of ECM by plasmin promotes endothelial cell migration toward chemoattractants. Our chemotaxis assay showed an increase in migration of endothelial cells when ANG was used as a chemoattractant. Nuclear translocation of ANG is critical for proliferation and angiogenesis induced by ANG (28). Neomycin acts by inhibiting PLC, which is required for nuclear translocation of ANG but not for internalization (15). Evidence obtained in these studies also suggests that inhibition of PLC might lead to accumulation of the protein in the cytosol, with a simultaneous decrease in nuclear ANG. This indicates that importing of ANG from the cytosol to the nucleus is regulated by ANG itself, since it activates PLC by binding the endothelial cell surface. Hence, if ANG accumulates in the cytosol, there should be a reduction in the amount of ANG that is secreted into the supernatant, resulting in reduced actin-ANG interaction. This might lead to a decrease in plasmin generation and migration. Concordantly, in our study, we observed a reduction in ANG secretion upon neomycin treatment. ANG treatment was reported to increase ERK, AKT, and PKB phosphorylation. It is not known whether ANG could activate NF-κB and thus whether it has feedback control over its own secretion. VEGF is known to activate NF-κB and, in consequence, to regulate several process, including proliferation, migration, and tubulogenesis. Further studies are needed to examine the pathway(s) that ANG could trigger in KSHV-infected endothelial cells for modulating the multiple cellular functions it controls.

Angiogenesis control by ANG.

Once the endothelial cells migrate along the gradient through the disintegrated basement membrane into the remodeled and softened perivascular space and reach the area with reduced blood vessel density, the endothelial cells arrange in a monolayer and form tube-like structures. Multiple growth factors, including VEGF, angiopoietin, and MCP-1, are implicated in KSHV-induced tube formation. Even though in vitro assays show the importance of a single factor in controlling vasculogenesis activity, in vivo angiogenesis could be the result of an interplay between multiple factors. Not all angiogenic molecules are secreted at the same time at the same level. Hence, KSHV has probably evolved to induce multiple growth factors to ensure angiogenesis.

ANG was originally isolated in conditioned culture media from human colon adenocarcinoma cells as a potent inducer of new blood vessel formation in the chicken embryo chorioallantoic membrane and rabbit cornea (40). In addition, results of studies of anti-ANG experimental therapy have shown that it is an in vivo angiogenic factor (40). It was reported that ANG could easily stimulate neoangiogenesis in the tumor secreting it. Matrigel assays using KSHV-infected supernatants demonstrated an increase in tube formation, and there was a significant reduction in both ANG-mediated and VEGF-mediated tube formation with anti-ANG treatment, indicating the importance of ANG in angiogenesis. Neomycin treatment that blocked nuclear ANG also blocked VEGF- and ANG-mediated tube formation in our studies. Kishimoto et al. (21) reported that endogenous ANG was required for proliferation and angiogenesis induced by growth factors, including VEGF and FGF. Even though KSHV induces expression of several angiogenic factors such as VEGF, FGF, and EGF, ANG appears to be necessary for their functions. Plasmin that is generated as a result of ANG action is known to cleave propeptide forms of the lymphangiogenic growth factors VEGF-C and VEGF-D to generate a mature active form of VEGF (25). Our previous studies showed that KSHV infection induced VEGF-C secretion that is critical for lymphangiogenesis (36, 39).

In the present study, we observed an upregulation of VEGF-C expression by the use of both quantitative PCR and FACS analysis with KSHV infection and ANG treatment. Neomycin treatment significantly inhibited ANG-mediated VEGF-C induction, while only moderate inhibition was observed when cells pretreated with neomycin were infected with KSHV. Hence, there could be multiple reasons for the reduction in tube formation by anti-ANG and neomycin treatment during VEGF-C and KSHV infection: (i) ANG binding and entry or nuclear translocation could be activating signal pathways and downstream transcription factors required for host gene upregulation, which could be inhibited as a result; (ii) there could be a block in the interaction between ANG and host transcriptional factors required for upregulation of VEGF-C and other growth factors; and (iii) plasmin generated due to ANG upregulation could be increasing the expression of active forms of VEGF-C, resulting in increased angiogenesis. Since we observed a reduction in plasmin levels after neomycin treatment, this could also be affecting VEGF-C levels and thus negatively influencing angiogenesis. Although these data clearly show a role played by ANG in modulating VEGF-C induction, they emphasize the importance of viral gene expression in regulating VEGF-C levels. This suggests that ANG might possibly have an additional, yet-unexplored role.

Results presented here clearly suggested a role for nuclear translocation of ANG in VEGF- and ANG-mediated endothelial cell tube formation. ANG must be influencing tube formation directly, by increasing 45S rRNA synthesis and cell proliferation and activating the plasminogen activator, resulting in increased cell migration, as well as indirectly, by increasing VEGF-C expression, which is a key factor involved in tube formation during KSHV infection. The mechanism of VEGF-C induction by ANG is under investigation.

In summary, our findings suggest that KSHV-induced ANG brings about multiple consequences for the infected endothelial cell population, such as increases in 45S rRNA synthesis, antiapoptosis, cell proliferation, cell migration, and angiogenesis (Fig. (Fig.11),11), all of which could be contributing immensely to KSHV biology and KS pathogenesis. Further studies as discussed above are under way to fully elucidate the role of ANG in KS pathogenesis, which is one of the first steps toward the development of effective control of ANG and of treatments for KSHV infection and KS.

Acknowledgments

This study was supported in part by Public Health Service grant CA 099925 and the Rosalind Franklin University of Medicine and Science H. M. Bligh Cancer Research Fund to B.C.

We thank Keith Philibert for critically reading the manuscript. We thank Rolf Renne (University of Florida), Don Ganem (Howard Hughes Medical Institute, University of California, San Francisco), and Guo-Fu Hu (Harvard Medical School) for providing valuable reagents.

Footnotes

Published ahead of print on 21 January 2009.

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