Inflammatory cytokines synergize with the HIV-1 Tat protein to promote angiogenesis and Kaposi's sarcoma via induction of basic fibroblast growth factor and the alpha v beta 3 integrin.

The Tat protein of HIV-1, a transactivator of viral gene expression, is released by acutely infected T cells and, in this form, exerts angiogenic activities. These have linked the protein to the pathogenesis of Kaposi's sarcoma (KS), a vascular tumor frequent and aggressive in HIV-1-infected individuals (AIDS-KS). In this study, we show that a combination of the same inflammatory cytokines increased in KS lesions, namely IL-1 beta, TNF-alpha, and IFN-gamma, synergizes with Tat to promote in nude mice the development of angioproliferative KS-like lesions that are not observed with each factor alone. Inflammatory cytokines induce the tissue expression of both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), two angiogenic molecules highly produced in primary KS lesions. However, bFGF, but not VEGF, synergizes with Tat in vivo and induces endothelial cells to migrate, to adhere, and to grow in response to Tat in vitro. Tat angiogenic effects correlate with the expression of the alpha v beta 3 integrin that is induced by bFGF and binds the arginine-glycine-aspartic acid (RGD) region of Tat. In contrast, no correlation is observed with the expression of alpha v beta 5, which is promoted by VEGF and binds Tat basic region. Finally, KS lesion formation induced by bFGF and Tat in nude mice is blocked by antagonists of RGD-binding integrins. Because alpha v beta 3 is an RGD-binding integrin that is highly expressed in primary KS lesions, where it colocalizes with extracellular Tat on vessels and spindle cells, these results suggest that alpha v beta 3 competitors may represent a new strategy for the treatment of AIDS-KS.     

Spindle cells and their role in Kaposi's sarcoma

Spindle cells represent the main cell type of the advanced final nodular stage of Kaposi's sarcoma lesions. Despite some clinical and epidemiological differences, the four Kaposi's sarcoma forms (classic, endemic, post-transplant and epidemic) display very similar histopathological features, with the proliferation of spindle cells (considered as the Kaposi's sarcoma tumor cells) associated with inflammation and neo-angiogenesis. Electron-microscopy and immuno-histochemistry studies have led to the consensus that the spindle cells originated from the endothelial lineage. However, only recently, studies that used specific lymphatic immunological markers (such as podoplanin) and molecular features (gene expression microarrays) strongly linked Kaposi's sarcoma spindle cells to the endothelium lymphatic cell lineage. Both hybridization and immuno-histochemistry techniques have demonstrated that human herpesvirus 8 also known as Kaposi's sarcoma associated herpesvirus was present in spindle cells at all stages of the disease (patch, plaque, nodule). Interestingly, while the human herpesvirus 8 latent genes are expressed in nearly all tumor spindle cells, only a small fraction of them expresses markers of viral lytic replication. Recent findings showing that nodular Kaposi's sarcoma lesions display all patterns of human herpesvirus 8 clonality support the model according to which this tumor begins as a polyclonal disease with a subsequent evolution to a mono/oligoclonal process involving infected spindle cells. Spindle cells appear to be the central masterpiece in KS tumorigenesis, however the exact respective role of each human herpesvirus 8 gene, in the initiation and the disease progression is still under investigation and the question of whether or not this tumor is a reactive process or a true malignant proliferation of spindle cells remains yet unclear.     

Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures

Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC. Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens. We conclude that de novo KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein. However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations.     

HIV-1 Tat regulates endothelial cell cycle progression via activation of the Ras/ERK MAPK signaling pathway

Tat, the transactivator of HIV-1 gene expression, is released by acutely HIV-1-infected T-cells and promotes adhesion, migration, and growth of inflammatory cytokine-activated endothelial and Kaposi's sarcoma cells. It has been previously demonstrated that these effects of Tat are due to its ability to bind through its arginine-glycine-aspartic (RGD) region to the alpha5beta1 and alphavbeta3 integrins. However, the signaling pathways linking Tat to the regulation of cellular functions are incompletely understood. Here, we report that Tat ligation on human endothelial cells results in the activation of the small GTPases Ras and Rac and the mitogen-activated protein kinase ERK, specifically through its RGD region. In addition, we demonstrated that Tat activation of Ras, but not of Rac, induces ERK phosphorylation. We also found that the receptor proximal events accompanying Tat-induced Ras activation are mediated by tyrosine phosphorylation of Shc and recruitment of Grb2. Moreover, Tat enabled endothelial cells to progress through the G1 phase in response to bFGF, and the process is linked to ERK activation. Taken together, these data provide novel evidence about the ability of Tat to activate the Ras-ERK cascade which may be relevant for endothelial cell proliferation and for Kaposi's sarcoma progression.     

Effects of cytokines from activated immune cells on vascular cell growth and HIV-1 gene expression. Implications for AIDS-Kaposi's sarcoma pathogenesis.

Kaposi's sarcoma (KS) arises more frequently in homosexual and bisexual men than in other groups of HIV-1 infected individuals. Clinico-epidemiologic data indicate that homosexuals often are infected with multiple microbial agents and/or subjected to other antigenic stimuli, preceding or accompanying HIV-1 infection. Signs of immune activation, in fact, frequently have been detected in these individuals, and the onset of KS can precede any sign of immunodeficiency. These data have suggested that products from activated immune cells may affect the development of AIDS-KS. Here we report that conditioned media from activated or dysregulated T cells contain a variety of cytokines that promote the growth of spindle cells derived from KS lesions of AIDS patients (AIDS-KS cells) and induce normal vascular cells, potential cell progenitors of the AIDS-KS cells, to acquire features of the KS cell phenotype ("spindle" cell morphology and growth responsiveness to the mitogenic effect of extracellular HIV-1 Tat protein). The same conditioned media or cytokines promote HIV-1 gene expression and rescue defective HIV-1 proviruses, interrupting HIV-1 latency and increasing Tat production. The cellular and viral effects of cytokines are increased in an additive or synergistic manner by picomolar concentrations of extracellular Tat. These data suggest that cytokines produced by activated immune cells cooperate with HIV-1 infection in AIDS-KS pathogenesis.     

Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation.

During acute human immunodeficiency virus type 1 (HIV-1) infection or after transfection of the tat gene, Tat protein is released into the cell culture supernatant. In this extracellular form, Tat stimulates both HIV-1 gene expression and the growth of cells derived from Kaposi's sarcoma (KS) lesions of HIV-1-infected individuals (AIDS-KS cells). Tat protein and its biological activities appear in the cell supernatants at the peak of Tat expression, when the rate of cell death is low (infection) or cell death is undetectable (transfection) and increased levels of cytoplasmic Tat are present. Tat-containing supernatants stimulate maximal AIDS-KS cell growth but only low to moderate levels of HIV-1 gene expression. This is due to the different concentrations of exogenous Tat required for the two effects. The cell growth-promoting effects of Tat peak at between 0.1 and 1 ng of purified recombinant protein per ml in the cell growth medium and do not increase with concentration. In contrast, both the detection of nuclear-localized Tat taken up by cells and the induction of HIV-1 gene expression or replication require higher Tat concentrations (> or = 100 ng/ml), and all increase linearly with increasing amounts of the exogenous protein. These data suggest that Tat can be released by a mechanism(s) other than cell death and that the cell growth-promoting activity and the virus-transactivating effect of extracellular Tat are mediated by different pathways.     

Activation of matrix-metalloproteinase-2 and membrane-type-1-matrix-metalloproteinase in endothelial cells and induction of vascular permeability in vivo by human immunodeficiency virus-1 Tat protein and basic fibroblast growth factor

Previous studies indicated that the Tat protein of human immunodeficiency virus type-1 (HIV-1) is a progression factor for Kaposi's sarcoma (KS). Specifically, extracellular Tat cooperates with basic fibroblast growth factor (bFGF) in promoting KS and endothelial cell growth and locomotion and in inducing KS-like lesions in vivo. Here we show that Tat and bFGF combined increase matrix-metalloproteinase-2 (MMP-2) secretion and activation in endothelial cells in an additive/synergistic manner. These effects are due to the activation of the membrane-type-1-matrix-metalloproteinase and to the induction of the membrane-bound tissue inhibitor of metalloproteinase-2 (TIMP-2) by Tat and bFGF combined, but also to Tat-mediated inhibition of both basal or bFGF-induced TIMP-1 and -2 secretion. Consistent with this, Tat and bFGF promote vascular permeability and edema in vivo that are blocked by a synthetic MMP inhibitor. Finally, high MMP-2 expression is detected in acquired immunodeficiency virus syndrome (AIDS)-KS lesions, and increased levels of MMP-2 are found in plasma from patients with AIDS-KS compared with HIV-uninfected individuals with classic KS, indicating that these mechanisms are operative in AIDS-KS. This suggests a novel pathway by which Tat can increase KS aggressiveness or induce vasculopathy in the setting of HIV-1 infection.     

Mechanism of paclitaxel activity in Kaposi's sarcoma

Kaposi's sarcoma (KS) is an angioproliferative disease characterized by proliferation of spindle-shaped cells predominantly of endothelial cell origin, neoangiogenesis, inflammatory cell infiltration, and edema. At least in early stage, KS behaves as a reactive lesion sustained by the action of inflammatory cytokines and growth factors, has a polyclonal nature, and can regress. However, in time it can become monoclonal, especially in the nodular stage, evolving into a true sarcoma, likely in association with the increased expression of antiapoptotic oncogenes. We have recently demonstrated by immunohistochemical analysis that Bcl-2, a proto-oncogene known to prolong cellular viability and to antagonize apoptosis, is highly expressed in spindle cells and vessels of both AIDS-KS and classical KS lesions and that its expression increases with lesion stage. Paclitaxel, a microtubule-stabilizing drug known to inhibit Bcl-2 antiapoptotic activity and to be highly effective in the treatment of certain neoplasms, has recently been found to be active also in patients with advanced HIV-associated KS. In this report we investigated the mechanism(s) of paclitaxel activity in KS. By using a model of experimental KS induced by the inoculation of KS-derived spindle cells in nude mice and primary cultures of KS spindle cells, we found that paclitaxel promotes regression of KS lesions in vivo and that it blocks the growth, migration, and invasion of KS cells in vitro. Furthermore, paclitaxel treatment promoted apoptosis and down-regulated Bcl-2 protein expression in KS cells in vitro and in KS-like lesions in mice. Our results suggest that paclitaxel interferes with KS by down-regulating Bcl-2 antiapoptotic effect.     

Differential expression of viral Bcl-2 encoded by Kaposi's sarcoma-associated herpesvirus and human Bcl-2 in primary effusion lymphoma cells and Kaposi's sarcoma lesions

Expression of human herpesvirus 8 viral Bcl-2 protein was demonstrated in spindle cells of late-stage Kaposi's sarcoma lesions but not in primary effusion lymphoma cell lines. In contrast, strong expression of human Bcl-2 was found in stimulated primary effusion lymphoma cells, whereas in Kaposi's sarcoma lesions preferential mononuclear cells, and to a lesser extent spindle cells, stained positive.     

Tumorigenesis by human herpesvirus 8 vGPCR is accelerated by human immunodeficiency virus type 1 Tat

Human herpesvirus 8 (HHV-8), also called Kaposi's sarcoma (KS) herpesvirus, can cause KS but is inefficient. Untreated human immunodeficiency virus type 1 (HIV-1) coinfection is a powerful risk factor. The HHV-8 chemokine receptor, vGPCR (ORF74), activates NF-kappaB and NF-AT, and their levels of activation are synergistically increased by HIV-1 Tat. Transgenic vGPCR mice develop KS-like tumors. A cell line derived from one such tumor expresses vGPCR and forms tumors in nude mice. Here we show that transfection of DNA encoding HIV-1 tat (but not a transactivation-defective mutant) into these tumor cells increases NF-kappaB and NF-AT activation levels and accelerates tumor formation. Tumorigenesis was also accelerated when Tat DNA was transfected into normal cells and the transfected cells were mixed with the tumor cells and injected into a single site. Tumorigenesis was also increased when the two cell types were injected at separate sites, suggesting that tumorigenesis is accelerated by Tat through soluble factors.     

Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice

We report the first endothelial lineage-specific transgenic mouse allowing live imaging at subcellular resolution. We generated an H2B-EYFP fusion protein which can be used for fluorescent labeling of nucleosomes and used it to specifically label endothelial cells in mice and in differentiating embryonic stem (ES) cells. A fusion cDNA encoding a human histone H2B tagged at its C-terminus with enhanced yellow fluorescent protein (EYFP) was expressed under the control of an Flk1 promoter and intronic enhancer. The Flk1::H2B-EYFP transgenic mice are viable and high levels of chromatin-localized reporter expression are maintained in endothelial cells of developing embryos and in adult animals upon breeding. The onset of fluorescence in differentiating ES cells and in embryos corresponds with the beginning of endothelial cell specification. These transgenic lines permit real-time imaging in normal and pathological vasculogenesis and angiogenesis to track individual cells and mitotic events at a level of detail that is unprecedented in the mouse.     

HIV-1 Tat protein-induced VCAM-1 expression in human pulmonary artery endothelial cells and its signaling

Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-kappaB, facilitated nuclear translocation of NF-kappaB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-kappaB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-kappaB translocation, which are the characteristics of pulmonary endothelial cell activation.     

HIV-1-Tat protein activates phosphatidylinositol 3-kinase/ AKT-dependent survival pathways in Kaposi's sarcoma cells

In this study we found that Tat protected vincristine-treated Kaposi's sarcoma cells from apoptosis and from down-regulation of several anti-apoptotic genes such as AKT-1, AKT-2, BCL2, BCL-XL, and insulin-like growth factor I and induced the de novo expression of the interleukin-3 gene. Moreover, we found that Tat enhanced phosphorylation of AKT and BAD proteins. The inhibition of phosphatidylinositol 3-kinase with two unrelated pharmacological inhibitors, wortmannin and LY294002, abrogated both the anti-apoptotic effect and the phosphorylation of AKT induced by Tat. After treatment with Tat, the AKT enzymatic activity showed a biphasic increase: an early activation (15 min), independent from protein synthesis; and a delayed activation (24 h), which was significantly decreased upon blockage of protein synthesis. Experiments with a function blocking anti-vascular endothelial cell growth factor receptor-2 antibody suggested that both the early and delayed AKT activation and the protection from apoptosis were triggered by the interaction of Tat with vascular endothelial cell growth factor receptor-2. Moreover, experiments with function-blocking antibodies directed against insulin-like growth factor I/insulin-like growth factor I receptor or interleukin-3 indicated their involvement in the delayed activation of AKT and their contribution to the anti-apoptotic effect of Tat on vincristine-treated Kaposi's sarcoma cells.     

Transgenic pigs expressing human decay-accelerating factor regulated by porcine MCP gene promoter

Porcine membrane cofactor protein (pMCP) is abundantly expressed throughout the body with particularly strong expression on the vascular endothelia. Previous studies demonstrated that the promoter of the pMCP gene induced efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55), in transgenic mice. In the present study, we tried to produce transgenic pigs with two hybrid genes, 0.9/hDAF and 5.4/hDAF, which were composed of human DAF (hDAF) gene regulated under pMCP promoters of different lengths (0.9 and 5.4 kb). Five live founder transgenic pigs were obtained only with the 0.9/hDAF construct. Although, four founder pigs transmitted the transgene to the second generation, the transmission rates varied among founders. We examined the expression of hDAF in tissues of descendants of two lines (Dm1 and Dm4). Human DAF specific RNAs were confirmed by an RT-PCR analysis in all organs examined. Levels of hDAF protein in the organs from the descendants of Dm1 line were higher than those in the corresponding human organs as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies showed that the tissue distribution of hDAF in the descendants of both lines was similar to that of endogenous pMCP. The expression level of hDAF on the vascular endothelial cells in Dm1 line was twice that on the corresponding human cells. We tested whether proinflammatory cytokines upregulate an efficiency of pMCP promoter on hDAF expression in transgenic pigs. Although the expression of hDAF on the human endothelial cells increased with a combination of cytokines, tumor necrosis factor alpha and interferon-gamma, no cytokine-induced upregulation was seen in the cells of transgenic pigs. The endothelial cells from transgenic pigs exhibited high resistance to the human serum-mediated cytolysis.