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JOURNAL OF VIROLOGY , Feb. 2008, p. 1759–1776 Vol. 82, No. 4 0022-538X/08/$08.00 0 doi:10.1128/JVI.00873- Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Kaposi’s Sarcoma-Associated Herpesvirus Induces Sustained Levels of
Vascular Endothelial Growth Factors A and C Early during In Vitro
Infection of Human Microvascular Dermal Endothelial Cells:
Biological Implications
Ramu Sivakumar, Neelam Sharma-Walia, Hari Raghu, Mohanan Valiya Veettil, Sathish Sadagopan,
Virginie Bottero, Laszlo Varga, Rita Levine, and Bala Chandran*
Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois
Received 24 April 2007/Accepted 20 November 2007
Kaposi’s sarcoma (KS), a vascular tumor associated with human immunodeficiency virus type 1 infection, is characterized by spindle-shaped endothelial cells, inflammatory cells, cytokines, growth and angiogenic factors, and angiogenesis. KS spindle cells are believed to be of the lymphatic endothelial cell (LEC) type. Kaposi’s sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8) is etiologically linked to KS, and in vitro KSHV infection of primary human dermal microvascular endothelial cells (HMVEC-d) is character- ized by the induction of preexisting host signal cascades, sustained expression of latency-associated genes, transient expression of a limited number of lytic genes, sustained induction of NF- B and several cytokines, and growth and angiogenic factors. KSHV induced robust vascular endothelial growth factor A (VEGF-A) and VEGF-C gene expression as early as 30 min postinfection (p.i.) in serum-starved HMVEC-d, which was sustained throughout the observation period of 72 h p.i. Significant amounts of VEGF-A and -C were also detected in the culture supernatant of infected cells. VEGF-A and -C were also induced by UV-inactivated KSHV and envelope glycoprotein gpK8.1A, thus suggesting a role for virus entry stages in the early induction of VEGF and requirement of KSHV viral gene expression for sustained induction. Exogenous addition of VEGF-A and -C increased KSHV DNA entry into target cells and moderately increased latent ORF73 and lytic ORF50 promoter activation and gene expression. KSHV infection also induced the expression of lymphatic markers Prox-1 and podoplanin as early as 8 h p.i., and a paracrine effect was seen in the neighboring uninfected cells. Similar observations were also made in the pure blood endothelial cell (BEC)-TIME cells, thus suggesting that commitment to the LEC phenotype is induced early during KSHV infection of blood endothelial cells. Treatment with VEGF-C alone also induced Prox-1 expression in the BEC-TIME cells. Collectively, these studies show that the in vitro microenvironments of KSHV-infected endothelial cells are enriched, with VEGF-A and -C molecules playing key roles in KSHV biology, such as increased infection and gene expression, as well as in angiogenesis and lymphangiogenesis, thus recapitulating the microenvironment of early KS lesions.
Kaposi’s sarcoma (KS) is an AIDS-defining vascular tumor, and the pathogenesis of KS is under vigorous study. In the early stages, KS is characterized by inflammatory cell filtration, presence of cytokines and growth and angiogenic factors, en- dothelial cell activation, and angiogenesis. This is followed by the appearance of typical spindle-shaped cells that represent a heterogeneous population dominated by activated endothelial cells mixed with macrophages and dendritic cells. In advancing lesions, spindle cells tend to become the predominant cell type, and there is prominent angiogenesis (26, 33, 61). Available in vivo and in vitro evidence indicates that KS probably develops from nontumor cells (24, 44) that become characteristically “spindle”-shaped and induce angiogenesis under the influence of a variety of factors, including interleukin-1 (IL-1), IL-6,
gamma interferon, granulocyte-macrophage colony-stimulat- ing factor, tumor necrosis factor alpha, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), vascu- lar endothelial growth factor (VEGF), chemokines, and trans- forming growth factor (TGF-) (23). It is believed that a cell clone probably assumes neoplastic features during the course of development of KS, followed by genotypic alterations caus- ing KS hyperplastic lesions and transformation into sarcomas (4, 7, 42). KS-associated herpesvirus (KSHV, also called human her- pesvirus 8), first identified in an AIDS-KS lesion, is etiologi- cally associated with the four epidemiologically distinct forms of KS, primary effusion lymphoma (PEL), and some forms of multicentric Castleman’s disease (72). KSHV encodes more than 90 open reading frames (ORFs), which are designated as ORFs 4 to 75 by their homology to herpesvirus saimiri ORFs, and many of these KSHV-encoded proteins are homologs of host proteins (53, 67). These homologs include latency-associ- ated proteins K13 (v-FLICE inhibitory protein) and ORF (v-cyclin D), as well as lytic cycle proteins such as ORF (vBcl-2), K2 (viral IL-6 [vIL-6]), K4 (viral macrophage inhib-
- Corresponding author. Mailing address: Department of Microbi- ology 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@rosalindfranklin.edu. Published ahead of print on 5 December 2007.
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itory protein II), K3 and K5 (immunomodulatory proteins 1 and 2), K6 (viral macrophage inflammatory protein 1A), K (antiapoptotic protein), K9 (viral interferon regulatory factor [vIRF]), K11.1 (vIRF2), K14 (vOX-2), and ORF74 (viral G protein-coupled receptor). These viral proteins are believed to play roles in evading host intrinsic, innate, and adaptive im- mune defense mechanisms, blocking apoptosis and the induc- tion of neoplasia (53, 67, 72). In vivo, KSHV DNA and transcripts have been detected in human B cells, macrophages, keratinocytes, endothelial cells, and epithelial cells (11, 20, 72). In vitro, KSHV infects a variety of human cells, such as human B, macrophage, endothelial, fibroblast, keratinocyte, and epithelial cells (1–3, 10, 11, 19, 40, 63). In contrast to members of the alpha- and betaherpesvi- ruses, which initiate the lytic cycle soon after infection, the 2- KSHV infection of cultured cells results only in the establishment of latency (64). Infection of human dermal microvascular endo- thelial cells (HMVEC-d) and human foreskin fibroblast cells (HFF) is characterized by the expression of latency-associated ORF73, ORF72, and K13 genes as well as transient expression of a very limited number of early lytic genes, such as lytic cycle switch protein ORF50, K5, K8, and v-IRF2 (36, 38). However, infected cells do not support serial propagation of KSHV, and the viral genome is lost during successive passages (29, 63). The roles of KSHV genes and the potential interplay be- tween viral and host genes in endothelial cell transformation and establishment of KS angioproliferative lesions are under study. Angiogenesis is the outgrowth of new capillaries from preexisting vessels, and it is essential for embryonic develop- ment, organ formation, tissue regeneration, and remodeling (14, 27). Among angiogenic factors, VEGF-A is the best-char- acterized positive regulator, with its distinct specificity for vas- cular endothelial cells. VEGF-A is a proangiogenic molecule and has been reported to play a crucial role in the development of KSHV-associated diseases by acting as a mediator of angio- genesis and vascular permeability, factors that are central to the development of KS and PEL, and may potentially be in- volved in multicentric Castleman’s disease. VEGF-A stimu- lates endothelial cell proliferation, migration, differentiation, tube formation, increased vascular permeability, and mainte- nance of vascular integrity (12, 14, 25, 50). VEGF-A is a 46- kDa protein that dissociates on reduction into two apparently identical 23-kDa subunits. Alternative splicing of VEGF-A mRNA transcripts results in five different isoforms: VEGF 121 , VEGF 145 , VEGF 165 , VEGF 189 , and VEGF 206 (22, 54). Three of these isoforms of VEGF (VEGF 121 , VEGF 145 , and VEGF 165 ) are secreted by a broad variety of cells, including vascular smooth muscle cells, monocytes, mesangial cells, en- dothelial cells, and megakaryocytes (48, 65, 82). The angio- genic responses induced by VEGF-A are mediated by two structurally related tyrosine kinase receptors, VEGFR- (Flt-1) and VEGFR-2 (KDR/Flk-1), both of which are ex- pressed primarily on vascular cells of the endothelial lineage (18, 50, 77). VEGF-A is the prototype of an enlarging family of growth peptides that includes four other structurally related members. These are placenta growth factor (PlGF), VEGF-B, VEGF-C, and c- fos -induced growth factor (VEGF-D) (25, 55, 66). They show a similarity in their primary sequences, especially in the PDGF-like domain containing the conserved eight cysteine
residues. PlGF is predominantly expressed in the placenta and binds to VEGFR-1, but not to VEGFR-2. VEGF-B has been identified as a weak mitogen for endothelial cells, and robust expression is particularly detected in skeletal and cardiac mus- cle tissues. Both PlGF and VEGF-B modulate VEGF activity via formation of heterodimers. VEGF-C is a ligand for two receptors, VEGFR-2 and VEGFR-3 (Flt-4). The latter differs from VEGFR-1 and VEGFR-2 by being predominantly ex- pressed in lymphatic endothelial cells in adult tissues but at low levels in most other vascular endothelial cells (34, 39). VEGF-C recently has been characterized to be a fairly selec- tive growth factor for lymphatic vessels (32, 56). In addition, proteolytic processing is involved in the regulation of VEGF-C activity. VEGF-C also is shown to be involved in the regulation of physiological and pathological blood vessel growth (14). Several immunohistochemical studies have shown that the KS spindle cells are of endothelial origin, but it was not clear until recently whether these spindle cells are the blood endo- thelial cell (BEC) or lymphatic endothelial cell (LEC) type (60, 83). Based on the gene expression microarray profiles and expression of several lymphatic lineage-specific proteins, in- cluding VEGFR-3 and podoplanin, it has been proposed that KS spindle cells are of LEC origin (30). KSHV infection of BECs induces the expression of the homeobox gene Prox-1, a master gene that controls lymphatic vessel development and differentiation, and its expression leads to lymphatic endothe- lial reprogramming of blood endothelial cells (15, 30, 81). The lymphatic endothelium controls the fluid and lymphocyte up- take into lymph nodes and is also architecturally different from the blood vascular endothelium. Lymphangiogenesis is a dif- ferent process from blood or hemangiogenesis, utilizing VEGF-C and -D and their receptor, VEGFR-3 (5, 15, 58). The lymphatic vessels provide a major pathway for tumor metasta- sis in many types of cancers (58). KSHV-induced up-regulation of angiogenesis factors may potentially be involved in KS development and progression. Multiple KSHV lytic proteins, such as K1, vGPCR, and vIL-6, were shown to induce VEGF-A and have been postulated to play a role in KSHV pathogenesis (6, 7, 75, 82). However, since less than 1% of KS cells (inflammatory B cells/monocytes) express lytic cycle proteins (20, 68), we explored the other possibilities. We have shown that during in vitro infection of endothelial cells, KSHV induces the host cell preexisting FAK, Src, phosphatidylinositol 3-kinase, Rho-GTPases, Dia-2, Ezrin, protein kinase C , extracellular signal-regulated kinase 1/2, and NF-B signal pathways that are critical for virus entry, cytoplasmic transport, nuclear delivery of viral DNA, and ini- tiation of viral gene expression. In addition, we also observed the reprogramming of host transcriptional machinery regulat- ing a variety of cellular processes, including apoptosis, tran- scription, cell cycle regulation, signaling, the inflammatory re- sponse, and angiogenesis. Notable among the host cell genes modulated by KSHV is the strong induction of several proin- flammatory cytokines and growth factors, such as IL-1, IL-6, IL-8, prostaglandin E2 (PGE2), GRO, oncostatin M, bFGF, VEGF-A, VEGF-C, matrix metalloproteinases, pre-B-cell colo- ny-enhancing factor, macrophage-specific colony-stimulat- ing factor 1, etc. (52). This resemblance between the cytokines and growth and angiogenic factors detected during in vitro KSHV infection with that of KS lesions suggests that the early
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Luciferase reporter assays. The effects of VEGF-A and VEGF-C on the ORF73 promoter (pORF73-Luc) and ORF50 promoter (pORF50-Luc) were measured using the dual luciferase kit according to the manufacturer’s protocol (Promega). Briefly, 293 cells (1 10 5 ) 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 ex- periment. Transfection of 293 cells was performed with 0.5 g of ORF73 or ORF50 promoter luciferase constructs and with 50 ng of Renilla luciferase as a transfection efficiency control and using Lipofectamine 2000 (Invitrogen). After 24 h, cells were either uninfected or infected with KSHV at an MOI of 10 or treated with exogenous VEGF-A and -C (100 ng/ml and 250 ng/ml) for 4 h, 8 h, and 24 h. Cells were harvested at the indicated time points after KSHV infection in 1 passive lysis buffer, and luciferase assays were carried out in triplicate following the manufacturer’s procedure in a Synergy HT multidetection micro- plate reader (Bio-Tek Instruments, Inc., VT). Firefly luciferase activities for each time point were normalized to those of parallel controls for each time point. Alterations in promoter activities of ORF73 or ORF50 as a result of KSHV infection or VEGF-A or VEGF-C treatment were determined after normaliza- tion, using the Renilla luciferase activity as a control. Western blotting for LANA-1. The effects of VEGF-A and VEGF-C on the ORF73 promoter (pORF73-Luc) and ORF50 promoter (pORF50-Luc) were then confirmed by the detection of LANA-1 by Western blotting. HMVEC-d cells (80 to 90% confluence) were serum starved for 8 h, either untreated or treated with VEGF-A or VEGF-C (250 ng/ml) for 1 h at 37°C, and infected with KSHV at an MOI of 10. Expression of ORF73 protein was monitored at the indicated time points. Cells were harvested at the indicated time points in radioimmunoprecipitation assay lysis buffer with protease inhibitor cocktail. Cel- lular debris was removed by centrifugation at 13,000 g for 20 min at 4°C, and
equal amounts of protein samples were resolved by sodium dodecyl sulfate–7.5% polyacrylamide gel electrophoresis (SDS-PAGE), subjected to Western blotting, and reacted with rabbit polyclonal antibody against KSHV LANA-1. To confirm equal protein loading, blots were also reacted with antibodies against human -actin. Secondary antibodies conjugated either to horseradish peroxidase or to alkaline phosphatase were used for detection. Immunoreactive bands were de- veloped by either the enhanced chemiluminescence reaction (NEN Life Sciences Products, Boston, MA) or CDP-Star (Roche Diagnostics Corp., Indianapolis, IN) and quantified by following standard protocols (72). Western blotting for luciferase. The effects of VEGF-A and VEGF-C on the ORF73 promoter (pORF73-Luc) and ORF50 promoter (pORF50-Luc) were also confirmed by the detection of luciferase by Western blot assay. Briefly, 293 cells (4 10 5 ) seeded in a six-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 293 cells was performed with 1.0 g of ORF73 or ORF50 promoter luciferase constructs and with 100 ng of pcDNA-GFP as a transfection efficiency control, using Lipofectamine 2000 (Invitrogen). After 24 h, cells were either untreated or treated with exogenous VEGF-A and -C (250 ng/ml) for 4 h, 8 h, and 24 h. Cells were harvested at the indicated time points in radioimmunoprecipitation assay lysis buffer with protease inhibitor cocktail. Total cell lysates prepared were resolved on 10% SDS-polyacrylamide gels, and the resolved proteins were trans- ferred onto a nitrocellulose membrane and immunoblotted with antiluciferase primary antibodies. Transfection efficiency was detected by using anti-green fluorescent protein (GFP) mouse monoclonal antibodies. To confirm equal pro- tein loading, blots were also reacted with antibodies against human -actin. Secondary antibodies conjugated either to horseradish peroxidase or to alkaline phosphatase were used for detection. Immunoreactive bands were developed by
FIG. 1. Detection of VEGF-A and VEGF-C mRNA and protein in KSHV-infected HMVEC-d cells. HMVEC-d cells grown to 80 to 90% confluence were serum starved for 8 h and infected with KSHV at an MOI of 10. (A and C) Infected and uninfected cells were washed and lysed, and total RNA was prepared. DNase I-treated RNA (250 ng) was subjected to real-time RT-PCR with VEGF-A and VEGF-C gene-specific primers. Known concentrations of DNase I-treated, in vitro-transcribed VEGF-A and VEGF-C transcripts were used in a real-time RT-PCR to construct a standard graph from which the relative copy numbers of transcripts were calculated and normalized, with GAPDH used as the internal control. Each reaction was done in duplicate, and each bar represents the average standard deviation from three independent experiments. The VEGF-A and VEGF-C levels normalized to GAPDH in the uninfected cells were considered as 1 for comparison. (B and D) The levels of VEGF-A and VEGF-C proteins released in the cell-free culture supernatants were measured by enzyme-linked immunosorbent assay. The data were normalized to a 1-mg/ml total protein concentration in the supernatant. Each reaction was done in duplicate, and each point represents the average standard deviation from three independent experiments. VEGF-A and VEGF-C released from uninfected cells were considered as 1 for comparison, and the induction levels in infected cells are indicated.
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either an enhanced chemiluminescence reaction (NEN Life Sciences Products, Boston, MA) or CDP-Star (Roche Diagnostics Corp., Indianapolis, IN) and quantified by following standard protocols (72).
RESULTS
In vitro infection of HMVEC-d cells by KSHV induces VEGF-A and VEGF-C gene expression. We have previously analyzed the transcriptional reprogramming of 22,283 host genes from KSHV-infected HMVEC-d and HFF cells at 2 and 4 h p.i. With a criterion of twofold gene induction as signifi- cant, transcriptional modulation of several genes was detected, including VEGF-A and VEGF-C genes. In HMVEC-d cells, we observed 2.59- and 2.62-fold up-regulation of VEGF-A RNA at 2 h and 4 h p.i., respectively, and 3.4- and 2.15-fold induction of the VEGF-C gene was observed at 2 h and 4 h p.i., respectively (52). Here, we used real-time RT-PCR to examine the kinetics of VEGF-A and VEGF-C mRNA induction in serum-starved HMVEC-d cells infected with KSHV (10 DNA copies/cell). A significant level of VEGF-A mRNA over unin- fected cells was detected as early as 0.5 h p.i., which increased steadily with about 6.2-, 5.6-, 3.4-, 8.2-, 2.7-, 5.5-, 2.9-, and 15.1-fold induction at 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, and 72 h p.i., respectively (Fig. 1A). Peak induction (15-fold) was observed at 72 h p.i. When VEGF-A protein expression in the culture supernatant was quantitated by ELISA, compared to VEGF-A protein levels in the uninfected HMVEC-d cells (Fig. 1B), VEGF-A levels in KSHV-infected cells increased by about 2.5-, 1.5-, 2.8-, 2.0-, 2.3-, 4.3-, 4.8-, 4.3-, and 10-fold at 0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, 48 h, and 72 h p.i., respectively. The RNA samples and culture supernatants from the above experiments were also used for quantification of VEGF-C mRNA and protein expression levels. Like VEGF-A, signifi- cant levels of VEGF-C mRNA induction were observed as early as 0.5 h p.i. (1.6-fold), which increased to about 2.2-, 1.4-, 3.8-, 1.8-, 2.3-, and 1.7-fold at 1 h, 2 h, 4 h, 8 h, 48 h, and 72 h p.i., respectively (Fig. 1C). In contrast to VEGF-A, maximum induction in VEGF-C expression (3.8-fold) was observed at 4 h p.i. Similarly, VEGF-C protein levels were elevated by about 1.2-, 1.9-, 1.6-, 1.9-, 1.4-, 2.1-, 1.9-, 1.9-, and 1.8-fold at 0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, 48 h, and 72 h p.i., respectively (Fig. 1D). Specificity of KSHV-induced VEGF expression. Incubation of KSHV with 100 g/ml of heparin reduced the VEGF-A expression to the basal level (Fig. 2A). In a parallel experi- ment, serum-starved HMVEC-d cells were incubated for var- ious durations with and without KSHV infection, and VEGF-A protein levels in the culture supernatants were then measured. As shown in the Fig. 2B, unlike KSHV-infected cells, VEGF-A levels in uninfected cells did not increase sig- nificantly with an increase in time, indicating that KSHV in- fection up-regulates VEGF-A expression. Further, a Limulus amebocyte lysate assay confirmed that all of the reagents used in the viral stock preparation were free of bacterial LPS (data not shown). These results demonstrated that VEGF-A and -C were specifically induced by KSHV infection and not by con- taminating host cell factors and/or LPS, or any other factor(s). These results suggested that KSHV strongly up-regulates VEGF-A and VEGF-C mRNA and protein expression at sus-
FIG. 2. Blocking KSHV binding by heparin inhibits VEGF-A expres- sion. (A) KSHV was incubated at 37°C for 1 h with DMEM containing 100 g/ml of soluble heparin. This mixture was then added to a serum- starved (8 h) HMVEC-d cell monolayer and incubated for 4 h at 37°C. The levels of VEGF-A released in the cell-free culture supernatants were measured by enzyme-linked immunosorbent assay. (^) ****** , statistically signif- icant ( P 0.02). (B) HMVEC-d cells grown to 80 to 90% confluence were serum starved for 8 h and either uninfected (control) or infected with KSHV at an MOI of 10. The levels of VEGF-A proteins released in the cell-free culture supernatants collected at the indicated time points were measured by ELISA. The data were normalized to a 1-mg/ml total protein concentration in the supernatant. Each reaction was done in duplicate, and each point represents the average standard deviation from three independent experiments. (C) VEGF-A and VEGF-C enhance KSHV entry. HMVEC-d cells were serum starved for 8 h and either untreated or treated with VEGF-A or VEGF-C (100 and 250 ng/ml) or with human EGF or BSA (250 ng/ml) for 1 h at 37°C in serum-free EBM2 medium and infected (10 DNA copies per cell) for 2 h. Cells were washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) for 5 min, washed, and collected, and total DNA was prepared. The KSHV ORF73 gene in 100 ng of DNA was amplified by real-time DNA PCR, and the copy numbers were calculated from the standard graph generated by the real- time PCR using known concentrations of a cloned ORF73 gene. Each reaction was done in duplicate, and each point represents the average standard deviation of three experiments.
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during infection (Fig. 5A). In contrast, ORF73 promoter ac- tivity increased by about 1.8-, 2.0-, and 1.9-fold at 4 h, 8 h, and 24 h post-KSHV infection, respectively, compared to the ac- tivities in the uninfected cells transfected with pORF73-Luc (Fig. 5A). VEGF-A treatment alone increased ORF73 pro- moter activity by 1.3-, 1.5-, and 1.9-fold at 100 ng/ml and 1.2-, 1.5-, and 1.9-fold at 250 ng/ml at 4, 8, and 24 h, respectively. The addition of 250 ng/ml VEGF-A did not have any added effect over 100 ng/ml on ORF73 promoter activity (Fig. 5B). Similarly, VEGF-C treatment increased ORF73 promoter ac- tivity by 1.2-, 1.6-, and 1.9-fold at 100 ng/ml at 4 h, 8 h, and 24 h,
respectively, and 1.7-, 1.6-, and 2.3-fold at 250 ng/ml at the corresponding time points. Unlike VEGF-A, an increase in ORF73 promoter activity by VEGF-C was dose dependent (Fig. 5C). The luciferase activity from the basic reporter pGL3- Luc construct was not affected by VEGF-A or VEGF-C addi- tion (Fig. 5B and C). To further confirm the effect of VEGF-A or VEGF-C on KSHV ORF73 promoter activation, real-time RT-PCR was carried out to quantify KSHV ORF73 mRNA (36) from RNA isolated from cells that were either untreated or pretreated with VEGF-A or VEGF-C (100 ng/ml; 37°C, 1 h) and then
FIG. 3. Immunofluorescent detection of KSHV infection-induced VEGF-A and -C protein expression in HMVEC-d cells. (A and B) Unin- fected HMVEC-d cells (panels a, b, and c) or cells infected with KSHV (MOI, 10) for 72 h (panels d, e, and f) were permeabilized and stained with anti-VEGF-A mouse monoclonal or VEGF-C goat polyclonal antibody, respectively, and detected by Alexa-594-coupled anti-mouse antibody or Alexa-488-coupled anti-goat antibody. Infected HMVEC-d cells were permeabilized and also stained with anti-ORF73 rabbit polyclonal antibody (A, panel f). Nuclei were counterstained with DAPI (b and e). The inset in panel g indicates nuclear staining for ORF73 in a cell with cytoplasmic staining for VEGF-A. KSHV-infected HMVEC-d cells express both VEGF-C (B, panel g) and VEGF-A (B, panel h). The inset demonstrates coexpression of VEGF-C and VEGF-A in the cytoplasm (i) of infected HMVEC-d cells. Magnification, 40.
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infected with KSHV for 12 and 24 h p.i. (Fig. 5D). Significant increases of about 3.1- and 1.5-fold in ORF73 gene expression at 12 and 24 h p.i., respectively, were observed in VEGF-A- pretreated cells. Similarly, VEGF-C induced about 2.6- and 1.3-fold increases in ORF73 gene expression (Fig. 5D). These results suggested that KSHV infection-induced VEGF-A and VEGF-C play a moderate role(s) in viral latent gene expres- sion. Exogenous VEGF-A and VEGF-C modulate KSHV lytic ORF50 promoter and gene expression. We next examined the role of VEGF-A and VEGF-C in ORF50 (RTA) expression. 293 cells transfected with pLuc or ORF50 promoter construct (pORF50-Luc) were either uninfected or infected with KSHV at an MOI of 10 or treated with exogenous VEGF-A and VEGF-C at 100 and 250 ng/ml for 4 and 8 h. The luciferase activity from the basic reporter pLuc construct was not affected by KSHV infection at any time after infection (Fig. 6A). In contrast, ORF50 promoter activity increased by about 1.4- and 1.6-fold at 4 h and 8 h p.i., respectively, compared to the uninfected cells transfected with pORF50-Luc (Fig. 6A). VEGF-A treatment increased ORF50 promoter activity by about 1.5- and 1.7-fold at 100 ng/ml and about 1.2- and 1.4-fold at 250 ng/ml (Fig. 6B). The addition of 250 ng/ml VEGF-A did not have any added effect on ORF50 promoter activity (Fig. 6B). VEGF-C increased ORF50 promoter activity by about 1.4- and 1.7-fold at 100 ng/ml at 4 and 8 h, respectively, and about 1.4- and 1.9-fold at 250 ng/ml (Fig. 6C). The luciferase
activity from the basic reporter pLuc construct was not affected by VEGF-A or VEGF-C addition (Fig. 6B and C). When ORF50 gene transcripts were examined by real-time RT-PCR in HMVEC-d cells pretreated with VEGF-A and infected with KSHV, a significant increase of about 2.3- and 1.2-fold in ORF50 gene expression at 12 and 24 h p.i., respectively, was observed (Fig. 6D), whereas a 5.8- and 2.8-fold increase in ORF50 gene expression was observed in VEGF-C (100 ng/ml)- treated cells (Fig. 6D), thus suggesting a potential role of VEGF-A and VEGF-C in KSHV lytic gene expression. We next examined the serum-starved untreated and VEGF-A- or -C-treated (250 ng/ml) and KSHV-infected HMVEC-d cells for the expression of LANA-1 by flow cytometry and by Western blot reactions (Fig. 6E and F). In VEGF-A- and VEGF-C-treated cells infected with KSHV, we observed a shift in the geometric mean fluorescent index (GMFI, which is the test mean fluores- cence divided by the mean fluorescence of the isotype control). At 48 h p.i., we observed a LANA GMFI of 10.54 in untreated infected cells (Fig. 6E, panel a) which increased to statistically significant GMFI levels of 12.24 ( P 0.03) and 12.39 ( P 0.02) in VEGF-A- and VEGF-C-treated cells, respectively (Fig. 6E, panels b and c). These results suggested that VEGF treatment increases ORF73 expression. When HMVEC-d cells untreated or VEGF treated and KSHV infected were lysed, resolved by SDS– 7.5% PAGE, and subjected to Western blotting using rabbit poly- clonal antibody against KSHV LANA-1, at 72 h p.i. we observed about 1.8-fold and 1.7-fold increases in LANA-1 expression in
FIG. 4. Induction of VEGF-A by UV-inactivated KSHV and viral glycoproteins gB and gpK8.1A. (A) HMVEC-d cells (80 to 90% confluence) serum starved for 8 h were uninfected or infected with either live KSHV or UV-irradiated KSHV for the indicated times at an MOI of 10 per cell. (B) Serum-starved HMVEC-d cells were uninfected, infected with KSHV (MOI, 10), or induced with KSHV glycoproteins gB and gpK8.1A alone or together for the indicated times. (C) Serum-starved HMVEC-d cells were uninfected, infected with KSHV (MOI, 10), or induced with KSHV glycoprotein gpK8.1A at the indicated concentrations for 0.5 and 1 h. (D) Serum-starved HMVEC-d cells were uninfected, infected with KSHV (MOI, 10), or induced with KSHV glycoproteins gB and gpK8.1A together at the indicated concentrations for 0.5 and 1 h. The levels of VEGF-A released in the cell-free culture supernatants were measured by ELISA. Each reaction was done in duplicate, and each point represents the average standard deviation from three independent experiments.
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FIG. 6. Exogenous addition of VEGF-A or VEGF-C activates KSHV ORF50 promoter and gene expression. 293 cells were transfected with control p-Luc or ORF50 promoter-Luciferase constructs and after 24 h, cells were either uninfected or infected with KSHV at an MOI of 10 for 4 h and 8 h (A) or treated with exogenous VEGF-A (B) and C (C) (100 ng/ml and 250 ng/ml). At the indicated time points cells were harvested, lysed, and assayed for firefly luciferase activity. The data represent the mean relative luciferase units after normalizing with the cotransfected Renilla luciferase activity. (D) HMVEC-d cells (80 to 90% confluence) were serum starved for 8 h, either untreated or treated with VEGF-A or VEGF-C (100 ng/ml) for 1 h at 37°C, and infected with KSHV at an MOI of 10. Expression of ORF50 mRNA was monitored at the indicated time points. DNase I-treated RNA (250 ng) was subjected to real-time RT-PCR with an ORF50 gene-specific primer. Known concentrations of DNase I-treated, in vitro-transcribed ORF50 transcripts were used to construct a standard graph from which the relative copy numbers of transcripts were calculated and normalized, with GAPDH used as the internal control. Each reaction was done in duplicate, and each bar represents the average standard deviation from three independent experiments. **, ***, ****, -statistically significant at P 0.02, P 0.01, and P 0.001, respectively. (E) Exogenous addition of VEGF-A or VEGF-C induces KSHV LANA-1 expression at the protein level. HMVEC-d cells (80 to 90% confluence) were serum starved for 8 h, either untreated or treated with VEGF-A or VEGF-C (250 ng/ml) for 1 h at 37°C, and infected with KSHV at an MOI of 10. Cells were then harvested for flow cytometry after 48 h postinfection and detached from the plate with 0.25% trypsin–EDTA, and viability was assessed by trypan blue exclusion. Nuclear staining to identify KSHV-infected cells was performed using rat monoclonal antibody (ABI) to the KSHV LANA-1 protein. Flow cytometry was performed with a FACSCalibur flow cytometer and analyzed with CellQuest Pro software. Expression of LANA-1 at 48 h p.i. is shown as a green line for KSHV alone (a), a red line for VEGF-A-treated and KSHV-infected cells (b), a black line for VEGF-C-treated and KSHV-infected cells (c) compared to the isotype control, which is shown in a shaded purple histogram. (F)
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LEC cells (70:30 ratio). In contrast, after 72 h post-KSHV infection, 80% cells stained positive for Prox-1 and podopla- nin (Fig. 7B panels a and d, respectively). The expression levels of BEC markers like CD34 in the same cells were not tested in this study. The infected cells were also stained for KSHV ORF73/LANA-1, and colocalization of Prox-1 and podoplanin with ORF73 is shown in Fig. 7B (panels c and f). Most of the LANA-1-positive cells were also positive for Prox-1 and podo- planin (Fig. 7B, panels c and f). Interestingly, Prox-1 was de- tected in many cells that did not exhibit LANA-1 (Fig. 7B, panel c). Since there was no increase in the number of cells on the infected cover slides, these findings suggest that KSHV infects both BEC and LEC and up-regulates the expression of LEC markers in BEC, thus changing the cell lineage. These observations are in concordance with previous reports (15, 30,
- and demonstrate that KSHV infection induces the LEC markers, switching the BEC phenotype to LEC. Kinetics of KSHV-induced Prox-1 expression in HMVEC-d cells. Prox-1 is a homeobox gene that controls lymphatic vessel development and differentiation. It is the earliest lymphatic marker expressed, and its expression indicates the onset of lymphatic lineage transformation (30). We next examined the kinetics of Prox-1 mRNA expression following KSHV infec- tion by real-time RT-PCR. Prox-1 expression increased to about fourfold over uninfected cells at 2 h p.i., reached a peak of about eightfold at 24 h p.i., and slowly declined to about sevenfold at 48 h and about sixfold level at 72 h p.i. (Fig. 7C). This is the first report showing the kinetics of Prox-1 expression at earlier time points immediately after KSHV infection and demonstrated that KSHV induces the blood endothelial-to- lymphatic endothelial lineage switch early during de novo in- fection. KSHV infection of TIME cells induces the expression VEGF-C and lymphatic markers. To further confirm the above findings, serum-starved TIME cells, pure blood vascular endo- thelial cells (15), were infected with KSHV, and the kinetics of VEGF-C mRNA induction was examined (Fig. 8A). A signif- icant increase in VEGF-C mRNA was detected as early as 0.5 h p.i., and this was maintained relatively steadily with about 15.9-, 8.5-, 6.6-, 4.9-, 5.0-, 8.9-, 7.3-, and 3.7-fold increases at 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h p.i., respectively (Fig. 8A). By IFA, only a few of the uninfected TIME cells were positive for VEGF-C expression (data not shown). In contrast, a sig- nificant increase in VEGF-C-positive cells expressing high lev- els of VEGF-C was readily detected after 48 h post-KSHV infection (Fig. 8B, panels a to d). We next examined the kinetics of Prox-1 mRNA expression
in TIME cells following KSHV infection (Fig. 8C). Prox- expression increased to about 2.5-fold over uninfected cells as early as 0.5 h p.i., reached a peak of 11- to 12-fold at 12 and 24 h p.i., and declined to 2-fold at 48 h p.i. (Fig. 8C). By IFA, all uninfected TIME cells were positive for the pan-endothelial cell marker CD31 (Fig. 8D, panels a to c), and only very low background staining for the LEC marker podoplanin was ob- served (Fig. 8D, panels d and e). In contrast, KSHV-infected TIME cells showed clear membranous expression of podopla- nin in about 60% of cells (Fig. 8D, panels f to h). Most of the LANA-1-positive cells were also positive for podoplanin (Fig. 8D, panel h). Since there was no increase in the number of cells, these findings demonstrated that KSHV infection of BEC and VEGF-C induction lead to the expression of LEC mark- ers, thus changing the cell lineage. To further confirm the expression of lymphatic marker ex- pression early during infection, TIME cells were infected with KSHV (MOI, 10) and harvested at 24 h and 48 h postinfection for flow cytometry analysis. As shown in Fig. 9A, panel a, about 7.2% of infected cells showed expression of podoplanin at 24 h p.i. and about 52.2% (Fig. 9A, panel b) after 48 h p.i. compared to the isotype control. Nuclear staining to identify KSHV- infected cells was performed using a rat monoclonal antibody to the KSHV LANA-1 protein, and about 25.0% of the cells were positive for LANA-1 at 24 h p.i. (Fig. 9A, panel c). Exogenous VEGF-C induces Prox-1 mRNA in TIME cells. Since VEGF-C is a lymphangiogenic growth factor and was up-regulated by KSHV infection in TIME cells (BEC), we next examined Prox-1 expression by incubating cells for 24 and 48 h in medium containing VEGF-C. Minimal Prox-1 induction was observed after 24 h of treatment; however, after 48 h with VEGF-C, about fivefold higher Prox-1 expression was ob- served (Fig. 9B) by real-time PCR. Prox-1 induction by VEGF-C was not dose dependent. EGF, used as a control, had a moderate induction of Prox-1 (Fig. 9B). It is interesting that the level of Prox-1 expression in KSHV-infected TIME cells at 24 h p.i. (Fig. 8C) was much higher than with VEGF-C treat- ment alone (Fig. 9B). By IFA, about 50% of TIME cells were positive for Prox-1 after 48 h of treatment with 250 ng/ml of VEGF-C (Fig. 9C, panels d to f). These studies further suggest that KSHV infection-induced VEGF-C in the culture super- natant probably plays an important role in lymphangiogenesis.
DISCUSSION
The present study is the first comprehensive study detailing the up-regulation of VEGF-A and VEGF-C upon primary
HMVEC-d cells (80 to 90% confluence) were serum starved for 8 h, either untreated or treated with VEGF-A or VEGF-C (250 ng/ml) for 1 h at 37°C, and infected with KSHV at an MOI of 10. Expression of LANA-1 protein was monitored at the indicated time points by Western blotting. Equal amounts of protein samples were resolved by SDS-7.5% PAGE, subjected to Western blotting, and reacted with rabbit polyclonal antibody against KSHV LANA-1. To confirm equal protein loading, blots were also reacted with antibodies against human -actin. Each blot is representative of at least three independent experiments. The LANA-1 level in the untreated but KSHV-infected cells was considered as 1 for comparison. (G) Exogenous addition of VEGF-A or VEGF-C activates KSHV ORF73-luciferase expression. 293 cells were transfected with control pGL3-Luc or the ORF73 promoter-luciferase construct along with pcDNA-GFP as a transfection efficiency control. After 24 h, cells were either untreated (NT) or treated with exogenous VEGF-A or VEGF-C (250 ng/ml). At the indicated time points, cells were harvested and lysed, and total cell lysates were immunoblotted with antiluciferase primary antibodies. Transfection efficiency was detected by using anti-GFP mouse monoclonal antibody. To confirm equal protein loading, blots were also reacted with antibodies against human -actin. Each blot is representative of at least three independent experiments. The luciferase level in the untreated cells was considered as 1 for comparison.
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KSHV infection of endothelial cells, a mixture of BEC and LEC, and the roles of these molecules in KSHV latent and lytic gene expression in addition to induction of a lymphatic lineage switch. Further study is required to understand how latency is favored despite the induction of the lytic ORF50 gene by both VEGF-A and VEGF-C. VEGF-A exists as a number of isoforms derived from mRNA splice variants. The isoforms differ at the C terminus, with the larger variants exhibiting substantial affinity for hep- arin (13, 66). The smallest isoform, VEGF 121 , does not bind heparin and is freely soluble upon secretion, whereas the higher- molecular-weight isoforms bind to the cell surface/extracel- lular matrix (ECM) HS proteoglycans (31, 57). It has been proposed that the ECM-bound forms of VEGF represent a pool of available growth factors, which can be activated in the course of tissue remodeling in conjunction with the degrada- tion of ECM components necessary for neovascularization (31, 54, 57).
The earlier onset of VEGF-A and -C expression could be due to binding and entry of KSHV, as evidenced from the induction by UV-inactivated virus (Fig. 4A). Since target cell infection by KSHV starts with its binding to HS molecules, VEGF induction at the entry stage could also be attributed to the release of sequestered, extracellular bound VEGF into a soluble form by viral glycoproteins competing for HS binding sites of VEGF-A (8). In contrast to KSHV, viral gene expres- sion appears to be unnecessary for the expression of VEGF-A by HCMV (62). Sustained VEGF-A and -C expression re- quires KSHV gene expression. Available evidence suggests that this induction must be occurring in conjunction with the many cytokines produced during primary infection by KSHV (68). In addition to that, the VEGF mRNAs possess adenylate- uridylate-rich elements in their 3 untranslated regions (17), and so it is very much possible that the VEGF-A mRNA is stabilized by kaposin B, a latent protein of KSHV, as it report-
FIG. 8. KSHV infection of TIME cells induced the expression VEGF-C and lymphatic markers. (A) TIME cells grown to 80 to 90% confluence were serum starved for 8 h and infected with KSHV at an MOI of 10, and the kinetics of VEGF-C mRNA induction was quantitated as per procedures described in the Fig. 1 legend. The VEGF-C level normalized to GAPDH in the uninfected cells was considered 1 for comparison. (B) Cells infected with KSHV (MOI, 10) for 48 h (a, b, c, and d) were permeabilized and stained with goat anti-VEGF-C antibodies and detected by Alexa 594-coupled anti-goat antibodies. Infected cells were also permeabilized and stained with anti-ORF73 polyclonal antibody and detected by Alexa 488-coupled anti-rabbit antibodies (c). Nuclei were counterstained with DAPI (b). The solid arrow indicates cytoplasmic staining for VEGF-C in cells infected with KSHV (d). Magnification, 40. (C) Prox-1 mRNA from uninfected and KSHV-infected TIME cells was quantified by real-time RT-PCR as per the procedures described in the Fig. 7C legend. The Prox-1 level normalized to GAPDH in the uninfected cells was considered 1 for comparison. (D) Uninfected TIME cells (a to e) or cells infected with KSHV (MOI, 10) for 48 h (f to h) were permeabilized, stained with anti-CD31, and detected by Alexa 488- or Alexa 594-coupled anti-mouse antibodies. Magnification, 20 (a to c) or 40 (d and e). Infected cells were permeabilized and also stained with anti-ORF73 polyclonal antibody and detected by Alexa 488-coupled anti-rabbit antibodies (g). Nuclei were counterstained with DAPI (b). The inset in panel h indicates membrane staining for podoplanin in infected TIME cells. Magnification, 40.
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edly stabilizes the cytokines which have this adenylate-uridy- late-rich element in their 3 untranslated region (47). VEGF-A expression is regulated by a variety of external factors. Angiogenic cytokines, growth factors, and gonadotro- pins that do not stimulate angiogenesis directly also can mod- ulate angiogenesis by modulating VEGF-A expression in spe- cific cell types and thus exert an indirect angiogenic or antiangiogenic effect. For example, factors that can potentiate VEGF-A production include FGF-4, PDGF, tumor necrosis factor, TGF-, keratinocyte growth factor, insulin-like growth factor I, IL-1, IL-6 (54), and PGE2. Among these, FGF-4, PDGF, and TGF- are up-regulated following KSHV infec- tion of HMVEC-d cells (72). Furthermore, COX-2, an induc- ible component of the inflammatory prostaglandin synthesis pathway, is also up-regulated by KSHV infection in HMVEC-d cells (52, 72). VEGF-A expression was reduced by COX- inhibition (N. Sharma-Walia, B. Chandran, et al., unpublished data), indicating that COX-2 partially regulates VEGF-A ex- pression. It is interesting that KSHV also induces the expres- sion of cytokines such as IL-10 and IL-13 in HMVEC-d cells (68), and these cytokines can inhibit the release of VEGF (46). These results indicate that KSHV has evolved to utilize mul-
tiple pathways and inflammatory responses for the regulation of VEGF-A levels in target cells. The cellular response to hypoxia is mediated by the hypoxia-inducible transcription fac- tor 1 (HIF1), a heterodimeric protein that binds to hypoxia response elements in the promoter/regulatory regions of hy- poxia-inducible genes, including the VEGF-A gene, and ini- tiates transcription by recruitment of transcriptional activators such as CREB/p300 (21). Since latent KSHV infection of HMVEC-d cells leads to increased expression of HIF1 and HIF2 (16), hypoxia could also be one of the factors inducing VEGF-A in the infected target cells. Compared to VEGF-A, we observed a moderate VEGF-C expression level. Several mechanisms have been reported for the regulation of VEGF-C expression, and KSHV probably utilizes a few or all of these. VEGF-C is synthesized as a proprotein, with the central receptor binding VEGF homology domain (VHD) flanked by N- and C-terminal propeptides. The propeptides are cleaved, yielding the mature VHD, and VEGF-C acquires the capacity to bind to VEGFR-2. Full- length forms of the growth factors bind to VEGFR-3 but do so with greater affinity after proteolytic maturation. The marked effects of the proteolytic activation of VEGF-C and VEGF-D
FIG. 9. Exogenous VEGF-C induces Prox-1 mRNA in TIME cells. (A) TIME cells were infected with KSHV (MOI, 10) and harvested for flow cytometry at 24 h (a) and 48 h postinfection (b). Cells were detached from the plate with 0.25% trypsin–EDTA, and viability was assessed by trypan blue exclusion. For podoplanin, cell surface staining was carried out for 1 h at 4°C. Nuclear staining to identify KSHV-infected cells was performed using rat monoclonal antibody to the KSHV LANA-1 protein (c). Flow cytometry was performed with a FACSCalibur flow cytometer and analyzed with CellQuest Pro software. Expression of podoplanin and LANA-1 at 24 h and 48 h p.i. is shown by the green line and compared to the isotype control, which is shown in the shaded purple histogram. (B) TIME cells were incubated with serum-free EBM2 containing different concentrations (100 and 250 ng/ml) of recombinant VEGF-C for 24 h and 48 h at 37°C. As a control, cells were treated for corresponding times with 250 ng/ml human EGF. Prox-1 mRNA was then quantified by real-time RT-PCR as per the procedures described in the Fig. 7C legend. The Prox-1 level normalized to GAPDH in the untreated cells was considered 1 for comparison. (C) Untreated (a to c) or VEGF-C-treated (250 ng/ml for 48 h) TIME cells (d to f) were permeabilized and stained with Prox-1 antibodies and detected with Alexa 488-coupled anti-mouse antibodies. Nuclei were counterstained with DAPI. Magnification, 20 (a to c).
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for KS lesion formation and KSHV reactivation leading to increased virus load under immunosuppression conditions (20), together with our studies demonstrating a robust induc- tion of cytokines, growth factors, and angiogenic factors in- cluding COX-2 by KSHV at 4 h, 8 h, and 24 h p.i. of endothe- lial cells (72), and sustained activation of NF-B, a key inflammatory induction molecule (68), suggest that primary infection of endothelial cells could create the microenviron- ment observed in early KS lesions and could be the initiating factor for KS lesion formation. This notion is further strength- ened by the present study, which demonstrated that KSHV infection stimulates the transcription of VEGF-A and VEGF-C early during infection, resulting in the subsequent secretion of the protein in culture supernatants. In the micro- environment of KS lesions, sustained low levels of VEGF-A and VEGF-C expression could potentially increase entry of newly arriving KSHV into additional new target endothelial cells, increase virus gene expression, increase cytokine and growth factor induction including VEGF-C and -A, and induce lymphangiogenesis. The repeated cycle of the above processes in an immunosuppressed individual could lead to the forma-
tion of the highly angiogenic and hyperplastic multifocal KS lesions (Fig. 10). In the absence of a robust immune system response, such as that seen in human immunodeficiency virus type 1-infected individuals, reduced host immune regulation, inability to control inflammation, and elimination of infected cells could lead to clinical manifestations of Kaposi’s sarcoma. Further studies are essential to determine the significance of the BEC-to-LEC switch in KSHV biology and the role of various host and viral factors involved in KSHV-induced lym- phatic reprogramming, all of which will lead to a better under- standing of KSHV biology and KS pathogenesis.
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. REFERENCES
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FIG. 10. Schematic model depicting the potential implications of VEGF-A and VEGF-C induction during in vitro KSHV infection of endothelial cells. KSHV has been shown to be reactivated under im- munosuppression conditions, resulting in increased circulating virus. Similar to in vitro infection of HMVEC-d cells, primary infection of endothelial cells in vivo could also result in the induction of preexisting host signal cascades, sustained expression of latency-associated genes, transient expression of a limited number of lytic genes, sustained induction of NF-B and several cytokines and growth and angiogenic factors, including VEGF-A and -C (step 1). These factors when re- leased to the extracellular environment of infected cells could act in an autocrine or paracrine fashion on the infected cells as well as neigh- boring cells (step 2). In the microenvironment of KS lesions, the presence of VEGF-A and -C may potentially be facilitating (i) an increase in infection of uninfected neighboring cells by KSHV released from the B cells and monocytes in the inflammatory cell pool, (ii) modulation of viral gene expression, and (iii) induction of lymphangio- genesis early during infection (step 3). Continual repetitions of these steps in an immunosuppressed individual could result in an increase in new infection of endothelial cells and increased autocrine actions of VEGF and other factors, along with viral gene expression, probably contributing to the maintenance of latency, angiogenesis, lymphatic lineage switch, neovascularization, inflammation, and growth stimula- tion. Reduced host immune regulation, an inability to control inflam- mation, and elimination of infected cells could ultimately lead to the formation of multifocal KS lesions (step 4).
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