Kaposi’s sarcoma (KS)-associated herpesvirus or human herpes virus 8 is considered the etiological agent of KS, a highly vascularized neoplasm that is the most common tumor affecting HIV/AIDS patients. The KS-associated herpesvirus/human herpes virus 8 open reading frame 74 encodes a constitutively active G protein-coupled receptor known as vGPCR that binds CXC chemokines with high affinity. In this study, we show that conditional transgenic expression of vGPCR by cells of endothelial origin triggers an angiogenic program in vivo, leading to development of an angioproliferative disease that resembles KS. This angiogenic program consists partly in the expression of the angiogenic factors placental growth factor, platelet-derived growth factor B, and inducible NO synthase by the vGPCR-expressing cells. Finally, we show that continued vGPCR expression is essential for progression of the KS-like phenotype and that down-regulation of vGPCR expression results in reduced expression of angiogenic factors and regression of the lesions. Together, these findings implicate vGPCR as a key element in KS pathogenesis and suggest that strategies to block its function may represent a novel approach for the treatment of KS.
The open reading frame (ORF)3 74 of the human γ-2-herpes virus, Kaposi’s sarcoma (KS)-associated herpesvirus or human herpes virus 8 (KSHV/HHV8), encodes a constitutively active G protein-coupled receptor viral G protein-coupled receptor (vGPCR) with tumorigenic properties. KSHV/HHV8 was discovered during systematic DNA screening of KS lesions (1). Epidemiological and experimental studies have since established a strong link between this virus and KS (2, 3, 4).
KS is a multifocal highly vascularized neoplasm characterized by the presence of spindle-shaped tumor cells (spindle cells), angiogenesis, extravasated erythrocytes, and inflammatory infiltrates dominated by mononuclear cells. The pathogenesis of KS is complex. In addition to the proposed involvement of KSHV/HHV8, immune dysregulation and increased expression of inflammatory cytokines and growth factors have been suggested to play key roles in KS pathogenesis (5, 6). However, how these factors interact to initiate KS is poorly understood. Currently, no effective therapy for KS is available.
vGPCR is expressed within KS lesions, but its role in the pathogenesis is not clear (7, 8). We, and others, have proposed that vGPCR contributes to KS pathogenesis by triggering expression of paracrine factors (8, 9, 10, 11). Indeed, transfection of vGPCR in many cell lines leads to expression of vascular endothelial growth factor-A (VEGF-A); the NF-κB-dependent inflammatory cytokines IL-6, IL-1β, TNF-α, and GM-CSF; adhesion molecules (VCAM-1, ICAM-1, and E-selectin); and chemokines CCL2 (MCP-1), CCL5 (RANTES), and CXCL8 (IL-8) (9, 11, 12, 13). Some of these factors are implicated in controlling angiogenesis and cell growth, and have been detected in KS lesions (14, 15, 16, 17, 18, 19, 20). We have previously reported that mice expressing vGPCR from a segment of the human CD2 promoter develop angioproliferative lesions and tumors on the ears, tail, nose, and paws that strongly resemble, macroscopically and microscopically, those seen in KS. Moreover, few cells in the angioproliferative lesions expressed vGPCR, suggesting that vGPCR may induce disease via a paracrine mechanism (21). These observations have been confirmed by recent studies (22, 23).
To further define the role of vGPCR in KS pathogenesis, we devised a conditional transgenic system for the expression of vGPCR. The use of a conditional approach for transgenic expression of vGPCR offers several advantages. First, vGPCR expression can be induced after birth, bypassing embryonic development and generating a better correlate of KSHV/HHV8 infection. Second, the temporal effects of vGPCR expression can be closely monitored, and precise gene expression analysis in lesional areas and vGPCR-expressing cells can be performed. Third, the relevance of vGPCR to disease progression can be assessed by discontinuing transgene expression. Using this model, we show that vGPCR expression triggers transcription of proinflammatory and angiogenic genes in vivo. Induction of these genes leads to the development of KS-like disease. Finally, we show that continued vGPCR expression is required for progression of the KS-like phenotype, and that cessation of the vGPCR stimulus results in partial regression of the lesions. Together, these findings implicate vGPCR as a key element in KS pathogenesis and suggest that strategies to block its function may represent a novel approach for the treatment of KS.
Materials and Methods
Transgene construction and generation of transgenic mice
Mice expressing vGPCR conditionally (iORF74 mice) were generated by coinjection of a doxycycline (DOX)-dependent activator and responder transgene. The responder transgene was constructed by cloning the cDNA for vGPCR (21) into the pTRE-HA plasmid (BD Clontech). The activator transgene was constructed by cloning reverse tetracycline-controlled trans activator (rtTA) obtained from the pTet-On vector (BD Clontech) into an expression vector containing the human CD2 enhancer/promoter and locus control region (24). The LacZ transgene was generated by excision of the tetracycline-responsive element promoter element and the LacZ gene from the pBI-G plasmid (BD Clontech). Transgenes were microinjected into (C57BL/6J × DBA/2)F2 (Charles River Laboratories) eggs. The microinjected eggs were transferred into oviducts of ICR (Charles River Laboratories) foster mothers, according to published procedures (25). Identification of the transgenic mice was accomplished by PCR amplification of tail DNA. Transgenic mice were kept under pathogen-free conditions. All experiments involving animals were performed following the guidelines of the Mount Sinai School of Medicine Animal Care and Use Committee.
Histological analysis and whole mount vessel staining
Fresh frozen sections were stained with anti-CD31 (MEC13.3) and anti-CD45 (30-F11) (BD Pharmingen), followed by incubation with Alexa Fluor 488-labeled goat anti-rat Ab (Molecular Probes). β-Galactosidase histochemistry was done, as previously described (26).
For whole mount vessel staining, male mice were injected i.v. with 100 μg of biotinylated Lycopersicon esculentum lectin (Vector Laboratories), followed by fixation perfusion. The ear skin was incubated overnight in 0.3% Triton X-100 in PBS at room temperature, then incubated with an avidin-biotin-HRP-peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories) overnight at room temperature, followed by staining with diaminobenzidine peroxidase substrate kit (Vector Laboratories).
Gene expression analysis
Total RNA from ears was extracted using the RNeasy Maxi Kit (Qiagen), according to the manufacturer’s instructions. For cDNA syntheses, 3 μg of RNA was DNase treated and then incubated with 0.1 mg/ml oligo(dT) (Invitrogen Life Technologies) and 0.01 mg/ml random hexamers (Promega) for 10 min at 70°C, then with 400 U/sample Superscript II reverse transcriptase, 0.5 mM dNTPs, 3 μM DTT, and 1× reverse-transcriptase buffer (all Invitrogen Life Technologies) at 42°C, 50 min. Samples without Superscript II reverse transcriptase were used as negative controls in the quantitative real-time PCR (Q-PCR).
Q-PCR was conducted in duplicates with 30 μl reaction volumes of 1× SYBR Green PCR Master Mix (Applied Biosystems), 0.4 μM each primer, and 25 ng of cDNA (SYBR Green), or with 25 μl reaction volumes of 1× TaqMan Universal PCR Master Mix (Applied Biosystems), 0.9 μM each primer, 200 nM probe, 1× internal control mix (rRNA) (Applied Biosystems), and 35 ng of cDNA using the ABI PRISM 7700 instrument. A standard curve for ORF74 mRNA was generated using a plasmid containing the ORF74 cDNA.
PCR cycling conditions were: 50°C for 2 min, 95°C for 15 min, and 40 cycles of 95°C for 15 s, 60°C for 1 min. Relative expression levels were calculated as 2Ct ribosomal RNA − Ct gene (for details, see ABI PRISM 7700, User Bulletin 2) using 18S ribosomal RNA as endogenous control.
Four samples collected by 20 days of DOX treatment were pooled and screened for the expression of angiogenic factors using specific primer sets and SYBR Green dye. Targets showing >3-fold up- or down-regulation compared with uninduced mice were further evaluated by analyzing the individual samples from each time point using target-specific primer sets and a fluorescent probe, except for inducible NO synthase (iNOS), matrix metalloproteinase-9 (MMP-9), MMP-13, KC, and integrin β3, which were analyzed using specific primer sets and SYBR Green dye.
Ears were homogenized on ice in lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS, 50 mM Tris, pH 8.0, and 0.5 mM EDTA; Sigma-Aldrich) containing 1× complete mini protease inhibitor (Roche Applied Science). VEGF-A, placental growth factor (PlGF), monokine induced by IFN-γ (MIG), and KC were quantified in 3–6 mg of total protein using Quantikine M kits (R&D Systems)
Ears were cut into small pieces and incubated with 4 ml of RPMI 1640 medium containing 0.25 mg/ml Liberase Blendzyme 3 (Roche Applied Science) at 37°C, 90 min, and then passed through a 100-micron nylon cell strainer (BD Discovery Labware). Cells (5 × 106) were loaded with fluorescein di-β-d-galactopyranoside (FDG; Molecular Probes), according to the manufacturer’s instructions. For phenotypical analysis, 106 FDG-loaded cells were stained with the directly conjugated mAbs: CD31 (MEC13.3), CD34 (RAM34), CD45 (30-F11), CD11b (M1/70), and IAb (25-9-17) (BD Pharmingen). Detection of ORF74 expression in thymocytes was done according to a previously described method (27). Events (2–3 × 105) were acquired on a FACScan (BD Immunocytometry Systems), and sorting was done on a Mo-Flo sorter (DakoCytomation) according to standard protocols. Data were analyzed using the FlowJo software (Tree Star).
Statistical and graphical analyses were performed using Prism software (GraphPad).
Transgenic mice expressing vGPCR conditionally develop KS-like disease
To express vGPCR conditionally in vivo, we used the tet-on variant of the tetracycline-dependent gene expression system. In this system, vGPCR expression is positively controlled by DOX, a tetracycline analog. Two transgenes were generated: the activator transgene, which encodes the rtTA under transcriptional control of a segment of the human CD2 promoter, and the responder transgene, which encodes vGPCR under the control of a tetracycline-responsive promoter (Fig. 1 A). We used the human CD2 promoter to control rtTA expression, because we have previously reported that transgenic mice expressing vGPCR under control of this promoter develop angioproliferative lesions in vivo (21, 27).
The two transgenes were coinjected into fertilized eggs, and we derived seven transgenic founder mice, which carried both the activator and responder transgenes. These transgenic mice will be referred to as iORF74 mice (inducible ORF74). To test whether DOX treatment resulted in vGPCR expression, all founders and their progeny were fed a diet containing 1000 parts per million of DOX. The human CD2 (hCD2) promoter is known to target transgenic expression to thymocytes. Therefore, we isolated thymocytes from DOX-treated and untreated transgenic mice. Thymocytes derived from untreated mice did not express vGPCR, whereas four of the seven lines were found to express vGPCR with expression level ranging from 1 to 4% of the thymocytes (data not shown). Importantly, two founders and their progeny developed visible angiogenic lesions in the ears when treated with DOX. The incidence of the phenotype was 100% in both lines. Wild-type littermates did not develop angiogenic lesions upon DOX treatment. Within 20–40 days of treatment, iORF74 mice developed highly vascularized lesions in the ears (Fig. 1,B, upper panel). Longer treatment (>100 days) led to development of tumors in the tail and paws (Fig. 1,B, lower panels). By 60 days of DOX treatment, histological analysis of the lesions in ear skin revealed the presence in the dermis of multiple clusters of spindle-shaped cells (Fig. 1,C) and of many vascular spaces containing erythrocytes (Fig. 1,C, inset). The majority of cells in the lesions stained positively for the endothelial cell marker CD31, whereas only blood vessels in control sections were CD31 positive (Fig. 1, D and E), suggesting that the spindle cells in the lesions were of endothelial origin.
To characterize the early angiogenic events in the ear skin, we visualized the vasculature of iORF74 mice with biotinylated lectin from Lycopersicon esculentum, a molecule that binds specifically to the endothelium. Analysis of whole mount preparations showed that the vasculature of the ears of untreated iORF74 mice was well organized and consisted of few high caliber vessels with branching capillaries supplying the hair follicles (Fig. 1,F). In contrast, the vascular tree of iORF74 mice treated with DOX for only 20 days displayed a marked increase in both the number and caliber of blood vessel (Fig. 1 G).
KS is characterized by the presence of inflammatory infiltrates dominated by mononuclear cells (15). CD45 immunostaining showed that the KS-like lesions in iORF74 mice treated with DOX for 60 days contained a large number of inflammatory cells (Fig. 2, A and B), including CD11b-positive cells (Fig. 2,C). These CD11b-positive cells expressed higher levels of MHC class II than CD11b cells from untreated mice (Fig. 2 C), indicating that angioproliferation was associated with a significant inflammatory component.
DOX triggers the expression of vGPCR and angiogenic molecules
The development of a KS-like disease in DOX-treated iORF74 mice led us to analyze lesions at different stages by gene expression analysis to identify factors involved in the pathogenesis of KS-like disease (Table I). We isolated RNA from ear skin at 0 (uninduced), 7, 20, 60, and 120 days of DOX treatment (n = 4 each time point), and measured mRNA expression levels by Q-PCR. Upon DOX treatment, expression of vGPCR mRNA was readily detectable and increased over time (Fig. 3,A). Furthermore, several potent angiogenic factors were also significantly up-regulated in response to induction of vGPCR expression, including PlGF, iNOS, platelet-derived growth factor B (PDGF-B), TNF-α, angiopoietin-2, and the metalloproteases (MMP)-9 and MMP-13 (Fig. 3,B). In contrast, VEGF-A and VEGF-B were significantly down-regulated (Fig. 3,B). mRNA for angiogenic factor receptors such as VEGF receptors (VEGFR) 1 and 2, the angiopoietin receptors Tie1 and Tie2, and the PDGF receptor β (PDGFRβ) were also up-regulated upon vGPCR induction (Fig. 3 C). CXCR4, a chemokine receptor expressed by endothelial cells and its ligand CXCL12 (stromal cell-derived factor), and vascular endothelial cadherin, an endothelial cell marker involved in VEGFR-2 signaling (28), were also significantly up-regulated. Importantly, we identified a group of up-regulated ligand/receptor pairs, including PlGF/VEGFR-1, CXCR4/CXCL12, Ang2/Tie2, and PDGF-B/PDGFRβ, suggesting a functional relevance for these receptor/ligand pairs in the development of KS-like disease.
|Target .||Fold Induction .||Target .||Fold Induction .||Target .||Fold Induction .||Target .||Fold Induction .||Target .||Fold Induction .|
|Ephrin A1||1.5||Integrin β3||13.9||TIMP-3||1.8||INOS-2||14.4|
|Ephrin B1||1.4||Integrin β4||1.0||TIMP-4||1.2||COX-1||1.2|
|Target .||Fold Induction .||Target .||Fold Induction .||Target .||Fold Induction .||Target .||Fold Induction .||Target .||Fold Induction .|
|Ephrin A1||1.5||Integrin β3||13.9||TIMP-3||1.8||INOS-2||14.4|
|Ephrin B1||1.4||Integrin β4||1.0||TIMP-4||1.2||COX-1||1.2|
RNA was isolated from ear tissues of uninduced iORF74 mice and iORF74 mice treated 20 days with DOX and converted to cDNA. Expression levels in a pool of four cDNA samples were determined by Q-PCR using specific primers and SYBR green dye. Ubiquitin was used as endogenous control. Data is shown as fold expression after 20 days of DOX treatment relative to uninduced. Molecules shown in bold were further analyzed by determining expression levels in individual samples at different time points by Q-PCR. ANG, angiopoietin; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; HGF, hepatocyte growth factor; OPN, osteopontin; VE-cadherin, vascular endothelial-cadherin; TSP, thrompospondin; TIMP, tissue inhibitor of metalloproteinase; MMP, matrix metalloproteinase; iNOS, inducible nitric oxide synthase; COX, cyclooxygenase.
Many investigators have ascribed the angiogenic activity of vGPCR to its ability to up-regulate the expression of VEGF-A (9, 11, 13, 29). Therefore, it was surprising to find that VEGF-A mRNA was down-regulated. To further investigate this finding, we analyzed the expression of VEGF-A and PlGF protein by quantitative ELISA. VEGF-A was transiently down-regulated and PlGF was significantly up-regulated after induction of vGPCR, confirming the mRNA data (Fig. 4).
Chemokines of the CXC family regulate angiogenesis and bind to and modulate the signaling activity of vGPCR. In addition to CXCL12, we found that KC, a CXCL8 (IL-8) homologue and a vGPCR neutral agonist (27), was significantly induced at the transcriptional level (Table I) and at the protein level (2.1 ± 0.2 pg/mg uninduced mice vs 4.9 ± 2.2 pg/mg after 20 days of DOX treatment, p < 0.05). Finally, expression of the IFN-γ-inducible CXC chemokines CXCL9 (MIG) and CXCL10 (IFN-γ-inducible protein-10 (IP-10); which are vGPCR antagonists), was also increased (Table I, and MIG protein, 8.1 ± 3.7 pg/mg uninduced mice vs 61.5 ± 26.4 pg/mg after 20 days of DOX treatment, p < 0.01).
vGPCR+ cells present within the lesions express endothelial cell markers, PlGF, PDGF-B, and iNOS
Having confirmed the expression of vGPCR within the lesions, we asked whether vGPCR cells expressing could contribute to the disease by producing some of the angioproliferative factors. As we have been unable to sort vGPCR+ cells using available Abs, we crossed iORF74 mice to transgenic mice carrying a tetracycline-dependent responder transgene encoding the LacZ reporter gene (Fig. 5,A). When treated with DOX, the iORF74/LacZ animals develop an angioproliferative disease that is identical with the one shown by the iORF74 animals (data not shown). Mice containing all three transgenes (iORF74/LacZ mice) were treated with DOX for 60 days, and ear specimens were obtained. Single cell suspensions were incubated with FDG, a substrate for β-galactosidase. In the presence of β-galactosidase, FDG is processed to release a fluorescent moiety. Quantification of LacZ+ cells by flow cytometry demonstrated that by 60 days of DOX treatment, 2.1 ± 0.3% (n = 4) of all cells in the ears expressed the LacZ marker (Fig. 5,B). Q-PCR analysis of cells sorted based on LacZ expression showed that vGPCR was expressed predominantly by LacZ+ cells (Fig. 5,C). Surprisingly, we also found that sorted LacZ+ cells expressed the endothelial cell markers CD34, CD31, vascular endothelial cadherin, and VEGFR-1 and 2 (data not shown). To further verify the phenotype of the cells, we examined expression of the endothelial markers CD31 and CD34 by flow cytometry. In six independent experiments, >75% of the LacZ+ cells expressed the endothelial markers CD31 and CD34 (Fig. 5 D). These results indicate that the majority of cells expressing vGPCR in the skin lesions were of endothelial origin.
To test whether some of the angioproliferative factors expressed in the ear lesions originated from vGPCR-expressing cells, we sorted LacZ+ and LacZ− cells and performed Q-PCR. LacZ+ cells expressed higher levels of PDGF-B, iNOS, and PlGF than LacZ− cells (Fig. 5 E), suggesting that vGPCR expression directly triggered the expression of these angiogenic molecules.
Finally, we examined the contribution of lymphocytes to development of angioproliferation in our model. To determine whether lymphocytes were involved in the development of KS-like disease in the skin, we crossed the iORF74 mice to RAG2−/− mice, which lack mature T and B cells. iORF74 mice lacking both RAG2 alleles developed an angioproliferative disease indistinguishable from iORF74 mice (Fig. 5, F and G). These results indicate that T and B cells are not required for the development of the KS phenotype.
Taken together, these results suggest that endothelial cells expressing vGPCR within the lesions, but not lymphocytes, are inducing disease via secretion of paracrine factors.
Reduced vGPCR expression impairs growth of KS-like lesions
The ability of vGPCR to trigger KS-like disease in mouse models strongly suggests a role of vGPCR in KS pathogenesis. However, it has not been established whether progression of KS requires persistent expression of vGPCR. To determine whether continuing DOX treatment is necessary to sustain the angioproliferative lesions, we examined lesion development and vGPCR expression in two groups of iORF74 mice. Initially, both cohorts were treated with DOX for 60 days. At this point, lesions were well established and the average ear thickness was similar in the two groups (Fig. 6,A). In one group, the DOX treatment was discontinued (n = 24), whereas the other continued to receive DOX (n = 20). In two independent experiments, sustained DOX treatment resulted in a further increase in ear thickness, whereas withdrawal of the DOX resulted in regression of the ear lesions (Fig. 6,A). Histological analysis of the ears at 60 days of DOX treatment (n = 6) showed the presence of clusters of spindle-shaped cells and many blood vessels (Fig. 6,B). After 120 days of DOX treatment, the number and size of clusters of cells were markedly increased (Fig. 6,C). In contrast, there was a clear reduction in both number and size of cell clusters in sections of ear skin from mice after DOX withdrawal for 60 days (Fig. 6 D).
To investigate whether these marked changes were associated with changes in vGPCR expression, we compared the expression level of vGPCR in ear samples from the two groups. DOX withdrawal reduced vGPCR expression significantly, although residual expression was detected (Fig. 7,A). We also found that the number of LacZ+ cells was markedly reduced in iORF74/LacZ mice upon discontinuation of the DOX treatment (Fig. 7 B). Furthermore, the relative and absolute numbers of endothelial cells (CD31+/CD34+ positive cells) and leukocytes (CD45+ cells) were significantly reduced after 60 days of DOX withdrawal (CD31+/CD34+, 8.2 E5 ± 3.5 E5 cells (+DOX) vs 2.3 E5 ± 1.1 E5 cells (−DOX), p < 0.05; CD45+, 10.4 E5 ± 4.5 E5 cells (+DOX) vs 2.3 E5 ± 0.6 E5 cells (−DOX), p < 0.01), suggesting that these cells either died or exited the tissue upon DOX discontinuation.
Next, we examined whether expression of angiogenic factors was affected by DOX withdrawal. Strikingly, expression of the angiogenic ligands and receptors that were up-regulated by 120 days of DOX treatment was significantly reduced in iORF74 mice that no longer received DOX (Fig. 7 C). Together, these data demonstrate that continued vGPCR expression is required for the KS-like lesions to progress and strongly suggest that vGPCR-dependent activation of the angiogenic program is the driving force responsible for growth of KS-like lesions.
Despite intensive investigation, the pathogenesis of KS is not well understood and there is no effective treatment available for patients affected with KS. Compelling evidence shown in this study and elsewhere in the literature suggests that a KSHV/HHV8-encoded chemokine receptor, vGPCR, has a key role in initiating KS disease (21, 22, 23, 27). However, the molecular mechanisms associated with the development of this KS-like phenotype in vivo and the dependence of this phenotype on sustained expression of vGPCR have not been elucidated. To address these questions, we developed a conditional expression system using the tetracycline-inducible system. In this study, we demonstrate that vGPCR expression induces expression of proangiogenic molecules and development of KS-like disease. Importantly, we show that reducing vGPCR expression reverses the overexpression of these molecules and results in regression of the KS-like lesions.
In the transgenic model described in this study, DOX treatment leads to the induction of vGPCR expression and to the development of a KS-like phenotype characterized by ear swelling, redness, and angiogenesis after 20–40 days of DOX treatment. The ear lesions are mostly formed by spindle-shaped cells of endothelial origin, by many blood vessels at different stages of development, and by inflammatory cells. The development of a KS-like phenotype in response to induction of vGPCR expression in mature animals is significant because it rules out that the phenotype is due to effects of vGPCR during embryogenesis.
To investigate the molecular mechanism by which vGPCR induces KS-like disease, we examined the expression of 77 angiogenic factors, cytokines, and chemokines in angioproliferative lesions at different stages and in vGPCR-expressing cells. We found that several potent angiogenic factors and receptors were significantly up-regulated in the vGPCR-induced lesions. These included PlGF/VEGFR-1, angiopoietin-2/Tie2, PDGF-B/PDGFRβ, TNF-α, and iNOS. Interestingly, this gene profile is strikingly similar to that reported in biopsies from KS patients (6, 20, 30, 31, 32). Thus, the inducible vGPCR model resembles KS not only at the gross and histological level, but also at the molecular level.
There was a strong correlation between the expression level of vGPCR and some of the factors that were up-regulated in the lesions as a whole. We expanded on this observation by determining which angiogenic factors were expressed specifically by sorted cells expressing vGPCR and LacZ. We found that expression of PlGF, iNOS, and PDGF-B was markedly elevated in cells expressing vGPCR relative to other cells in the lesions. These molecules are involved in different aspects of angiogenesis. iNOS is a known vasodilator, and its expression in the lesions might contribute to the observed increase in vessel density and the extravasation of erythrocytes. PDGF-B is a mitogenic factor and has been shown to induce angiogenesis in vivo and to induce chemotaxis of endothelial cells in vitro (33), and is implicated in pathological proliferation of endothelial cells in KS and other cancer types (32, 34).
PlGF was also up-regulated within the lesions and preferentially expressed in cells expressing vGPCR. Ectopic expression of PlGF in skin is sufficient to initiate angiogenesis in a quiescent vasculature (35). Also, PlGF expression seems to be required for angiogenesis during pathological conditions such as ischemia and for angiogenesis in certain tumor models (36). Importantly, elevated systemic levels of PlGF mobilize hemopoietic stem cells from the bone marrow, which could contribute to the inflammatory and angiogenic processes taking place in the skin of vGPCR-expressing mice (37).
A number of investigators have ascribed the ability of vGPCR to induce KS-like disease in vivo to up-regulation of VEGF-A (9, 22). In our study, except for a transient decrease, the levels of VEGF-A protein remained unaltered in lesional areas. These results do not rule out a role for VEGF-A in our system, as it remains possible that VEGF-A bioavailability may be increased. It is important to notice, however, that while overexpression of VEGF-A leads to angiogenesis, it does not necessarily induce a KS phenotype (21, 38). Furthermore, gene profiling of KS biopsies suggests that VEGF-A is not up-regulated in skin affected by KS, whereas, for example, PlGF and PDGF-B are significantly induced (20). Based on the results presented in this work, we suggest that induction of PlGF and PDGF-B in KS lesions is promoted by vGPCR expressed by HHV8-infected endothelial cells.
The constitutive activity of vGPCR is regulated by a broad spectrum of CXC chemokines that bind this receptor with high affinity (39, 40). In this study, we found the expression of several CXC chemokines to be turned on upon expression of vGPCR in the lesions, including MIP-2 and the ELR− angiostatic chemokines CXCL9 (MIG) and CXCL10 (IP-10). The murine chemokine ligand, IP-10, binds vGPCR and functions as an inverse agonist of the constitutive activity of this receptor, whereas MIP-2 is an agonist (27).
Chemokines may affect development of disease by several mechanisms. First, they may directly regulate vGPCR signaling in vivo, and thus control the production of paracrine factors. Support for this hypothesis derives from studies that show that deletion of the far N-terminal segment of vGPCR, which deprives the receptor of its ability to bind any chemokine (41, 42), prevents the formation of the KS-like tumors in the transgenic animals (27). Second, chemokines may act on endogenous receptors to alter angiogenic and immune mechanisms. Angiogenic and angiostatic responses are thought to be mediated by CXCR2 and CXCR3, respectively (43). Third, chemokines may affect cell survival, migration, or proliferation of nonendothelial cells. Both CXCL12 and its receptor CXCR4 were induced in the angioproliferative lesions upon DOX treatment. CXCR4 and CXCL12 are implicated in mobilization of a number of cells, including hemopoietic stem cells and metastatic cancer cells (44, 45). Thus, it is possible that vGPCR+ cells expressing CXCR4 are mobilized to the lesional areas in response to increasing levels of CXCL12. In addition, CXCR4 and CXCL12 could stimulate proliferation and survival of vGPCR-expressing cells, as has been reported for a number of cancer cells (46).
The expression of angiostatic chemokines in an environment of dramatic angiogenesis is somewhat puzzling. Interestingly, expression of both angiogenic and angiostatic factors has also been documented within KS lesions (20). We suspect that angiostatic chemokines may be produced as part of a counterregulatory mechanism to the pronounced angioproliferative process underway. In this setting, they may act negatively on vGPCR, turning down its constitutive activity, and/or on their endogenous receptor, CXCR3, inhibiting angiogenesis. We speculate that the angiogenic phenotype associated with expression of vGPCR would progress even faster and more aggressively in the absence of the angiostatic chemokines.
To date, there are no models for the HHV-8 viral infection in vivo, and thus it has been difficult to assess the direct contribution of HHV8 genes to pathogenesis. Our results and those of other groups (21, 22, 23, 27) have suggested a role for vGPCR in angioproliferation and tumorigenesis in vivo. In this study, we show that vGPCR+ cells present within the angioproliferative lesions of iORF74 are the source of some of the angioproliferative factors detected within the lesions, providing strong support to the hypothesis that vGPCR contributes to disease via paracrine mechanisms. However, it remains unclear how exactly vGPCR expression contributes to angioproliferation in KS. Few tumor cells express vGPCR, and the expression of vGPCR has been documented during the lytic phase of the viral cycle, when there is accelerated global mRNA turnover (47). This could be interpreted as evidence that the paracrine contribution of vGPCR would be minimal, or not mediated by the genes identified in this study. It is clear, however, that many of the target genes identified in this work are indeed expressed within KS lesions (20), and no other genes in the HHV8 genome have been shown to affect their expression to date. So, how to explain these apparent discrepancies? One explanation would be that regulation of vGPCR expression in vivo might occur outside the lytic phase and initiate a cascade of events that would lead to development of angioproliferation. Indeed, studies done by Liang and Ganem (48) indicate that the vGPCR promoter can be activated by the transcription factor RBP-J, a downstream effector of the Notch pathway. Dysregulation of vGPCR expression could then provide for initiation of angioproliferation, as shown in this study and elsewhere (23, 49). The resolution of these issues will have to wait for development of better viral infection models.
The addition of the LacZ reporter gene, which permitted the sorting of the vGPCR+ cells from the lesions, helped us to begin investigating the phenotype of the cell expressing vGPCR within the lesions. We found that the vast majority of cells expressing vGPCR within the lesions displayed the endothelial markers CD31 and CD34. These results were surprising, considering that the CD2 promoter targets transgene expression primarily to thymocytes and bone marrow cells (21, 50). The observation that the hCD2 promoter cassette used in this study targeted, in addition to thymocytes and bone marrow cells, a cell in the endothelial lineage helps to explain an apparent paradox raised by our original use of the CD2 promoter. The predominant tumor cell type in KS seems to be an endothelial cell, and to our knowledge, there is no evidence to date linking T cells or thymocytes with angioproliferation in KS. The mechanisms favoring transcription of a hCD2-vGPCR transgene in cells of endothelial origin are unclear at present. They do not seem to reflect fortuitous integration events, as several independent transgenic lines present the same angioproliferative phenotype (21, 27) (current study). An alternative explanation would be that a combination of specific regulatory elements present in the promoters, coding regions and polyadenylation sequences of the hCD2 transgenes described in our studies, could have generated an ectopic pattern of expression, as observed in other studies (51).
Our observations strongly suggest that the ability of vGPCR to induce angioproliferation in vivo is dependent on a cellular context. When expressed by thymocytes, vGPCR does not cause angioproliferative disease in the thymus, but will do so when expressed in endothelial cell compartment, as shown in this work and by Montaner et al. (22). Furthermore, iORF74 mice lacking T and B cells develop angioproliferative disease and tumors, indicating that these cells are not relevant for disease induction in our models. We hypothesize that vGPCR activates a specific genetic program in a specific subset of endothelial cells. This hypothesis is based on our observations and on the fact that vGPCR triggers different gene programs when expressed in different cell types in vitro (9, 11, 12, 13).
Our studies show that continuous vGPCR activity is required to sustain the expression of paracrine growth factors and to support growth of KS-like lesions. The angioproliferative disease observed at 60 days progressed in animals treated with DOX, whereas there was a significant disease regression in animals in which DOX was discontinued. This was associated with a significant decrease in the number of endothelial and inflammatory cells, as well as vGPCR-expressing cells within the lesional areas. Current studies are aimed at defining whether discontinued vGPCR expression causes cell death, reduced immigration, or migration of cells out of the lesions.
We should note that despite the dramatic changes caused by DOX withdrawal, the lesions did not disappear completely. We suspect that this is due to residual or leaky expression of vGPCR. vGPCR leakage is not observed spontaneously in young mice (<30 days of age), but we have observed low level expression of vGPCR and angioproliferative lesions in the ears of older mice, even in the absence of DOX. Residual expression of vGPCR would thus explain the low level expression of some angioproliferative factors 60 days after DOX discontinuation.
In summary, our findings provide a strong link between vGPCR expression and KS pathogenesis. We show that expression of vGPCR within cells of the endothelial lineage, but not lymphocytes, is associated with development of an angioproliferative disease that closely resembles KS at the gross histological and molecular level. In addition, we show that multiple angiogenic factors are up-regulated upon vGPCR expression in vivo. Based on these results, we hypothesize that expression of vGPCR within human KS lesional areas may provide the stimulus for expression of cytokines and growth factors that drive the angioproliferative and inflammatory processes. The generation of the animal model reported in this work paves the way for future mechanistic studies on the role of these factors in KS pathogenesis. Finally, our results conclusively demonstrate that vGPCR is not only important for lesion development, but it is required for disease progression in this animal model. Thus, we suggest that therapeutics targeting vGPCR expression or function may affect the angioproliferative component of KS and prevent further progression of disease.
The authors have no financial conflict of interest.
We thank C. Canasto, C. Pinzon, L. Johnson, B. Wilburn, A. Golovko, and P. Zalamea for expert technical assistance. We are grateful to M. Wiekowski, J. Unkeless, M. Lipp, and P. Frenette for their contributions to the manuscript.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work is supported by a grant from the Irene Diamond Fund and by National Institutes of Health Grant CA109259.
Abbreviations used in this paper: ORF, open reading frame; DOX, doxycycline; FDG, fluorescein di-β-d-galactopyranoside; hCD2, human CD2; HHV8, human herpes virus 8; iNOS, inducible NO synthase; iORF, inducible ORF; IP-10, IFN-γ-inducible protein-10; KS, Kaposi’s sarcoma; KSHV, KS-associated herpesvirus; MIG, monokine induced by IFN-γ; MMP, matrix metalloproteinase; PDGF-B, platelet-derived growth factor B; PDGFRβ, PDGF receptor β; PlGF, placental growth factor; Q-PCR, quantitative real-time PCR; rtTA, reverse tetracycline-controlled trans activator; VEGF, vascular endothelial growth factor; vGPCR, viral G protein-coupled receptor.