The present study is the first comprehensive study detailing the up-regulation of VEGF-A and VEGF-C upon primary 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 heparin (13
). The smallest isoform, VEGF121
, does not bind heparin and is freely soluble upon secretion, whereas the higher-molecular-weight isoforms bind to the cell surface/extracellular matrix (ECM) HS proteoglycans (31
). 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 degradation of ECM components necessary for neovascularization (31
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. ). 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 expression appears to be unnecessary for the expression of VEGF-A by HCMV (62
). Sustained VEGF-A and -C expression requires 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 reportedly stabilizes the cytokines which have this adenylate-uridylate-rich element in their 3′ untranslated region (47
VEGF-A expression is regulated by a variety of external factors. Angiogenic cytokines, growth factors, and gonadotropins that do not stimulate angiogenesis directly also can modulate angiogenesis by modulating VEGF-A expression in specific 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-β, keratinocyte growth factor, insulin-like growth factor I, IL-1β, IL-6 (54
), and PGE2. Among these, FGF-4, PDGF, and TGF-β are up-regulated following KSHV infection of HMVEC-d cells (72
). Furthermore, COX-2, an inducible component of the inflammatory prostaglandin synthesis pathway, is also up-regulated by KSHV infection in HMVEC-d cells (52
). VEGF-A expression was reduced by COX-2 inhibition (N. Sharma-Walia, B. Chandran, et al., unpublished data), indicating that COX-2 partially regulates VEGF-A expression. It is interesting that KSHV also induces the expression 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 multiple 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 factor 1 (HIF1), a heterodimeric protein that binds to hypoxia response elements in the promoter/regulatory regions of hypoxia-inducible genes, including the VEGF-A gene, and initiates transcription by recruitment of transcriptional activators such as CREB/p300 (21
). Since latent KSHV infection of HMVEC-d cells leads to increased expression of HIF1α and HIF2α (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 on their affinity for VEGFR-2 and VEGFR-3 indicate that the enzymes carrying out this processing are important regulators of lymphangiogenesis and angiogenesis. The fibrinolytic serine protease plasmin was recently shown to generate the fully processed, mature forms of the VHD with greatly enhanced capacities to activate both VEGFR-2 and VEGFR-3. Plasmin can also release ECM-bound VEGF and activates both angiogenic and lymphangiogenic growth factors. It is interesting that the two enzymes that regulate plasmin activation, tissue plasminogen activator and plasminogen activator inhibitor 2, are also up-regulated during the in vitro infection of HMVEC-d cells by KSHV (52
). COX-2 has also been shown to up-regulate VEGF-C expression in human lung adenocarcinoma cells (76
). In addition to VEGF-C, COX-2 activity and some prostaglandins produced by COX-2 (59
) also elevate angiopoietin-2, another protein required for lymphangiogenesis. COX-2 is therefore an inducer of two proteins integral to lymphangiogenesis, and induction of sustained COX-2 and PGE2 in KSHV-infected HMVEC-d cells may also be responsible for the sustained VEGF-C induction observed here.
The lymphatic vessels develop subsequent to the formation of the blood vasculature, leading to the suggestion that they are derived from the blood vessels. Recent advances in the understanding of lymphatic development have shown that expression of the homeobox transcription factor Prox-1 is a defining characteristic of differentiation to the lymphatic phenotype. Subsequent budding and sprouting of Prox-1-positive cells gives rise to the lymphatic vasculature (84
). Two members of the VEGF family, VEGF-C and VEGF-D, have been shown to act as lymphangiogenic growth factors. Disruption of the VEGF-C gene demonstrates that the growth factor is indispensable for embryonic lymphangiogenesis (35
). Prox-1 expression was detected in endothelial cells of VEGF-C-deficient embryos, but the Prox-1-positive cells failed to migrate from the cardinal vein (35
), indicating that VEGF-C is required for migration of the endothelial cells which go on to form the lymphatic system. IL-3 has been known to induce the lymphatic markers Prox-1 and podoplanin in HMVEC-d cells (28
), and induction of IL-3 by KSHV infection of HMVEC-d cells (68
) suggests that besides VEGF-C, KSHV-induced IL-3 could also be one of the factors playing a role in Prox-1 expression.
Our demonstration of KSHV infection induction of the LEC markers and switching from the BEC to the LEC phenotype confirms other earlier studies (15
). More importantly, we extend these studies by the following important observations: (i) the in vitro microenvironment of KSHV-infected HMVEC-d and TIME cells is rich with VEGF-A and -C, which are known to play key roles in angiogenesis and lymphangiogenesis. (ii) Induction of Prox-1 occurs early during infection (as early as 8 h p.i.), thus suggesting that commitment to the LEC phenotype is induced early during KSHV infection of HMVEC-d cells. This is in contrast to other studies, where Prox-1 induction was examined only at 48 h p.i. in TIME cells, at 7 days p.i. in HMVEC-d cells, and at 2 and 7 days p.i. of BEC and LEC in studies by Carroll et al. (15
), Hong et al. (30
), and Wang et al. (81
), respectively. VEGF-C, a key molecule of the BEC-to-LEC switch, is induced by KSHV infection early during infection of HMVEC-d cells, which was sustained throughout our observation period of 72 h p.i. We made similar observations in pure BEC-TIME cells. This finding is further strengthened by the induction of Prox-1 mRNA and protein expression in BEC-TIME cells. This is in contrast to other studies, where VEGF-C induction was examined only at 48 h p.i. in TIME cells (15
). Significant amounts of VEGF-C mRNA and protein expression were observed in vivo in blood endothelial cells adjacent to KS lesions (73
), demonstrating a paracrine function of VEGF-C. Our observations of VEGF detection in LANA-1-positive cells as well as the majority of the neighboring uninfected cells suggest a similar paracrine effect during the in vitro KSHV infection. These results suggest that the in vitro microenvironments of KSHV-infected HMVEC-d and TIME cells, which are enriched with VEGF-A and -C, recapitulate the microenvironment of early KS lesions.
KS is considered a chronic inflammation-associated malignancy due to the presence of spindle-shaped endothelial cells, slit-like neovascular structures, variable quantities of infiltrating inflammatory cells, growth and angiogenic factors, and inflammatory cytokines, such as VEGF, PDGF, bFGF, PGE2, TGF-β, IL-1β, IL-6, and gamma interferon. The driving force behind the initiation and maintenance of the cytokine and growth factor cascade remains to be elucidated thoroughly. VEGF-A has been identified in KS lesions, and VEGF-C has gained attention because of the presence of its receptor VEGFR-3 in KS lesions and its ability to induce lymphangiogenesis (44
). Skobe et al. (73
) reported the expression of flt-4, the receptor for VEGF-C in KS cells, and strong expression of VEGF-C in KS blood vessels. Coexpression of VEGF-A and bFGF has been shown in AIDS-KS and classic KS lesions, and the production of these factors is believed to be induced synergistically by inflammatory cytokines (69
). Masood et al. (45
) reported that AIDS-KS cell lines express higher levels of VEGF-A and its receptor than normal HUVEC, and AIDS-KS cell growth was inhibited by blocking VEGF-A expression with antisense oligonucleotides. However, KSHV's role in VEGF-A up-regulation was not clear, since these cells were devoid of KSHV. VEGF-A has been shown to be induced by KSHV lytic gene products such as vGPCR (a homolog of the human chemokine IL-8) (7
), vIL-6 (6
), and K1 (82
). While these findings suggest that KSHV has devised multiple ways to induce VEGF-A, the actual scenario in KS lesions appears to be different, since KS lesion endothelial cells express latent gene products and only <1% of inflammatory cells have been shown to express KSHV lytic cycle proteins (20
Demonstration of a moderate increase in viral entry and viral gene expression in VEGF-pretreated endothelial cells suggests that besides the induction of BEC to LEC, VEGF-A and -C must be playing important roles in KSHV biology. The increase in viral entry could possibly be due to induction of host signal molecules, such as FAK, Src, phosphatidylinositol 3-kinase, and Rho GTPases, which have been shown to be essential for KSHV entry (37
). In the microenvironment of KS lesions, the presence of VEGF-A and -C may potentially facilitate an increase in infection of uninfected cells by KSHV released from the B cells and monocytes in the inflammatory cell pool. These effects, though moderate, suggest that besides playing a major role in the lymphatic lineage switch through activation of prox-1, VEGF induced by KSHV may also be contributing an active role in an increase of KSHV infection in neighboring cells.
Several facts, such as requirements of lytic KSHV infection for KS lesion formation and KSHV reactivation leading to increased virus load under immunosuppression conditions (20
), together with our studies demonstrating a robust induction of cytokines, growth factors, and angiogenic factors including COX-2 by KSHV at 4 h, 8 h, and 24 h p.i. of endothelial cells (72
), and sustained activation of NF-κB, a key inflammatory induction molecule (68
), suggest that primary infection of endothelial cells could create the microenvironment observed in early KS lesions and could be the initiating factor for KS lesion formation. This notion is further strengthened 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 microenvironment 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 formation of the highly angiogenic and hyperplastic multifocal KS lesions (Fig. ). 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 lymphatic reprogramming, all of which will lead to a better understanding of KSHV biology and KS pathogenesis.
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 immunosuppression conditions, resulting in increased circulating virus. (more ...)