Kaposi's Sarcoma Spindle Cells Research Articles

Evidence for Kaposi Sarcoma Originating from Mesenchymal Stem Cell through KSHV-induced Mesenchymal-to-Endothelial Transition.

The major transmission route for Kaposi sarcoma-associated herpesvirus (KSHV) infection is the oral cavity through saliva. Kaposi sarcoma (KS) frequently occurs in the oral cavity in HIV-positive individuals and is often the first presenting sign of AIDS. However, the oral target cells for KSHV infection and the cellular origin of Kaposi sarcoma remain unknown. Here we present clinical and experimental evidences that Kaposi sarcoma spindle cells may originate from virally modified oral mesenchymal stem cells (MSC). AIDS-KS spindle cells expressed neuroectodermal stem cell marker (Nestin) and oral MSC marker CD29, suggesting an oral/craniofacial MSC lineage of AIDS-associated Kaposi sarcoma. Furthermore, oral MSCs were highly susceptible to KSHV infection, and infection promoted multilineage differentiation and mesenchymal-to-endothelial transition (MEndT). KSHV infection of oral MSCs resulted in expression of a large number of cytokines, a characteristic of Kaposi sarcoma, and upregulation of Kaposi sarcoma signature and MEndT-associated genes. These results suggest that Kaposi sarcoma may originate from pluripotent MSC and KSHV infection transforms MSC to Kaposi sarcoma-like cells through MEndT.Significance: These findings indicate that Kaposi sarcomas, which arise frequently in AIDS patients, originate from neural crest-derived mesenchymal stem cells, with possible implications for improving the clnical treatment of this malignancy. Cancer Res; 78(1); 230-45. ©2017 AACR.

Enhanced phosphorylation of sphingosine and ceramide sustains the exuberant proliferation of endothelial progenitors in Kaposi sarcoma.

Endothelial colony-forming cells (ECFCs), a unique endothelial stem cell population, are highly increased in the blood of Kaposi sarcoma (KS) patients. KS-derived ECFCs (KS-ECFCs) are also endowed with increased proliferative and vasculogenic potential, thus suggesting that they may be precursors of KS spindle cells. However, the mechanisms underlying the increased proliferative activity of KS-ECFCs remain poorly understood. Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are metabolically interconnected sphingoid mediators crucial to cell proliferation. Here, we investigated the metabolism, release, and proliferative effects of S1P and C1P in KS-ECFCs compared with control ECFCs (Ct-ECFCs). Metabolic studies by cell labeling, chromatographic analyses, and digital autoradiography revealed that S1P and C1P biosynthesis and S1P secretion are all efficient processes in KS-ECFCs, more efficient in KS-ECFCs than Ct-ECFCs. Quantitative PCR analyses demonstrated a significantly higher ceramide kinase and sphingosine kinase-2 expression in KS-ECFCs. Notably, also the expression of S1P1 and S1P3 receptors was augmented in KS-ECFCs. Accordingly, treatment with exogenous C1P or S1P induced a significant, concentration-dependent stimulation of KS-ECFC proliferation, but was almost completely ineffective in Ct-ECFCs. Hence, we identified C1P and S1P as autocrine/paracrine proliferative signals in KS-ECFCs. A better understanding of the mechanisms that enhance S1P/C1P formation in KS-ECFCs may yield effective therapeutic modalities.

Glycolysis, Glutaminolysis, and Fatty Acid Synthesis Are Required for Distinct Stages of Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS). KSHV infection induces and requires multiple metabolic pathways, including the glycolysis, glutaminolysis, and fatty acid synthesis (FAS) pathways, for the survival of latently infected endothelial cells. To determine the metabolic requirements for productive KSHV infection, we induced lytic replication in the presence of inhibitors of different metabolic pathways. We found that glycolysis, glutaminolysis, and FAS are all required for maximal KSHV virus production and that these pathways appear to participate in virus production at different stages of the viral life cycle. Glycolysis and glutaminolysis, but not FAS, inhibit viral genome replication and, interestingly, are required for different early steps of lytic gene expression. Glycolysis is necessary for early gene transcription, while glutaminolysis is necessary for early gene translation but not transcription. Inhibition of FAS resulted in decreased production of extracellular virions but did not reduce intracellular genome levels or block intracellular virion production. However, in the presence of FAS inhibitors, the intracellular virions are noninfectious, indicating that FAS is required for virion assembly or maturation. KS tumors support both latent and lytic KSHV replication. Previous work has shown that multiple cellular metabolic pathways are required for latency, and we now show that these metabolic pathways are required for efficient lytic replication, providing novel therapeutic avenues for KS tumors.IMPORTANCE KSHV is the etiologic agent of Kaposi's sarcoma, the most common tumor of AIDS patients. KS spindle cells, the main tumor cells, all contain KSHV, mostly in the latent state, during which there is limited viral gene expression. However, a percentage of spindle cells support lytic replication and production of virus and these cells are thought to contribute to overall tumor formation. Our previous findings showed that latently infected cells are sensitive to inhibitors of cellular metabolic pathways, including glycolysis, glutaminolysis, and fatty acid synthesis. Here we found that these same inhibitors block the production of infectious virus from lytically infected cells, each at a different stage of viral replication. Therefore, inhibition of specific cellular metabolic pathways can both eliminate latently infected cells and block lytic replication, thereby inhibiting infection of new cells. Inhibition of metabolic pathways provides novel therapeutic approaches for KS tumors.

Increased Frequency and Vasculogenic Potential of Endothelial Colony-Forming Cells in Patients with Kaposi’s Sarcoma

Kaposi's sarcoma (KS) is characterized by hyperproliferation of spindle cells that have an endothelial origin and assume their characteristic features upon infection with human herpesvirus-8, the causative agent for KS. The multifocal nature of KS suggests that spindle cells derive from circulating HHV8-infected precursors that yet lack identification. We investigated whether endothelial colony-forming cells (ECFCs) obtained from KS patients may be putative precursors of spindle cells by assessing whether their invitro behavior may evoke the invivo behavior of KS spindle cells. We isolated and cultured ECFCs from the blood of 83 patients with classic KS and compared them with ECFCs obtained from 86 healthy donors. ECFCs were highly increased in the blood of classic KS patients; they showed higher proliferative and vasculogenic potential and higher production of IL-6 than control ECFCs. Similarly to spindle cells in KS lesions, a variable proportion of cells within each ECFC colony expressed the human herpesvirus-8 latency-associated nuclear antigen. ECFCs obtained from classic KS patients evoked KS spindle cell behavior, thus supporting the hypothesis that ECFCs may be putative precursors of spindle cells. ECFCs can therefore represent a noninvasive tool for studying KS and screening drug activity, thus possibly guiding personalized care for KS patients.

Kaposi's sarcoma-associated herpesvirus infection of human bone-marrow-derived mesenchymal stem cells and their angiogenic potential.

Kaposi's sarcoma (KS) is a vascular tumor, and KS spindle cells express endothelial-cell-specific markers. Generally, it is believed that KS originates from endothelial cells. However, as various mesodermal-derived tissue markers are also expressed in KS spindle cells, the exact origin of KS still needs to be elucidated. Here, Kaposi's sarcoma-associated herpesvirus (KSHV) was used to infect human mesenchymal stem cells derived from bone marrow (hMSC-bm), and we investigated the angiogenic properties of these cells, which are one of the most important pathologic features of KS. KSHV-infected hMSC-bm showed latent infection and increased tube formation activity in vitro. In addition, the expression of endothelial-cell-specific markers and a growth factor that affects the angiogenesis of endothelial cells was induced in KSHV-infected cells. This study suggests that human mesenchymal stem cells might have important roles in KS pathogenesis.

The Inflammatory Kinase MAP4K4 Promotes Reactivation of Kaposi's Sarcoma Herpesvirus and Enhances the Invasiveness of Infected Endothelial Cells

Kaposi's sarcoma (KS) is a mesenchymal tumour, which is caused by Kaposi's sarcoma herpesvirus (KSHV) and develops under inflammatory conditions. KSHV-infected endothelial spindle cells, the neoplastic cells in KS, show increased invasiveness, attributed to the elevated expression of metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). The majority of these spindle cells harbour latent KSHV genomes, while a minority undergoes lytic reactivation with subsequent production of new virions and viral or cellular chemo- and cytokines, which may promote tumour invasion and dissemination. In order to better understand KSHV pathogenesis, we investigated cellular mechanisms underlying the lytic reactivation of KSHV. Using a combination of small molecule library screening and siRNA silencing we found a STE20 kinase family member, MAP4K4, to be involved in KSHV reactivation from latency and to contribute to the invasive phenotype of KSHV-infected endothelial cells by regulating COX-2, MMP-7, and MMP-13 expression. This kinase is also highly expressed in KS spindle cells in vivo. These findings suggest that MAP4K4, a known mediator of inflammation, is involved in KS aetiology by regulating KSHV lytic reactivation, expression of MMPs and COX-2, and, thereby modulating invasiveness of KSHV-infected endothelial cells.

Cellular origin of Kaposi's sarcoma and Kaposi's sarcoma-associated herpesvirus-induced cell reprogramming

Kaposi's sarcoma (KS) is the most common malignancy in untreated HIV patients. KS is characterised by abnormal neoangiogenesis, inflammation, and proliferation of tumour cells [KS spindle cells (SCs)]. Kaposi's sarcoma-associated herpesvirus (KSHV) is the aetiological agent of KS. KS SCs are the predominant KSHV-infected cells in KS lesions. In this review, we report advances in understanding of the cellular origin of the KS SC, a contentious topic in KSHV research. KS SCs are now known to be of endothelial cell (EC) origin, phenotypically most similar to lymphatic ECs (LECs), but poorly differentiated. We focus on recent insights into KSHV's ability to exploit the normal differentiation pathway and intrinsic plasticity of ECs, through manipulation of EC-specific transcriptional regulators [i.e., prospero homeobox 1 (PROX1) and MAF] and discuss how this may contribute to viral persistence and KS sarcomagenesis.

Ets-1 Is Required for the Activation of VEGFR3 during Latent Kaposi's Sarcoma-Associated Herpesvirus Infection of Endothelial Cells

Kaposi's sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi's sarcoma (KS), is present in the predominant tumor cells of KS, the spindle cells. Spindle cells express markers of lymphatic endothelium and, interestingly, KSHV infection of blood endothelial cells reprograms them to a lymphatic endothelial cell phenotype. KSHV-induced reprogramming requires the activation of STAT3 and phosphatidylinositol 3 (PI3)/AKT through the activation of cellular receptor gp130. Importantly, KSHV-induced reprogramming is specific to endothelial cells, indicating that there are additional host genes that are differentially regulated during KSHV infection of endothelial cells that contribute to lymphatic reprogramming. We found that the transcription factor Ets-1 is highly expressed in KS spindle cells and is upregulated during KSHV infection of endothelial cells in culture. The KSHV latent vFLIP gene is sufficient to induce Ets-1 expression in an NF-κB-dependent fashion. Ets-1 is required for KSHV-induced expression of VEGFR3, a lymphatic endothelial-cell-specific receptor important for lymphangiogenesis, and Ets-1 activates the promoter of VEGFR3. Ets-1 knockdown does not alter the expression of another lymphatic-specific gene, the podoplanin gene, but does inhibit the expression of VEGFR3 in uninfected lymphatic endothelium, indicating that Ets-1 is a novel cellular regulator of VEGFR3 expression. Knockdown of Ets-1 affects the ability of KSHV-infected cells to display angiogenic phenotypes, indicating that Ets-1 plays a role in KSHV activation of endothelial cells during latent KSHV infection. Thus, Ets-1 is a novel regulator of VEGFR3 and is involved in the induction of angiogenic phenotypes by KSHV.

A Role for Virally Induced Reactive Oxygen Species in Kaposi's Sarcoma Herpesvirus Tumorigenesis

Kaposi's sarcoma (KS), caused by the Kaposi's sarcoma herpesvirus (KSHV), is an AIDS-associated cancer characterized by angiogenesis and proliferation of spindle cells. Rac1-activated reactive oxygen species (ROS) production has been implicated in KS tumorigenesis. We used an animal model of KSHV-induced Kaposi's sarcomagenesis (mECK36) to study the role of ROS in KS and the efficacy of N-acetyl l-cysteine (NAC) in inhibiting or preventing KS. Signaling by the KSHV early lytic gene viral G protein-coupled receptor (vGPCR) activated ROS production in mECK36 cells via a Rac1-NADPH oxidase pathway. Induction of the lytic cycle in KSHV-infected KS spindle cells upregulated ROS along with upregulation of vGPCR expression. We also found that expression of the major latent transcript in 293 cells increased ROS levels. ROS scavenging with NAC halted mECK36 tumor growth in a KSHV-specific manner. NAC inhibited KSHV latent gene expression as well as tumor angiogenesis and lymphangiogenesis. These effects correlated with the reduction of vascular endothelial growth factor (VEGF), c-myc, and cyclin D1, and could be explained on the basis of inhibition of STAT3 tyrosine phosphorylation. NAC prevented mECK36 de novo tumor formation. Molecular analysis of NAC-resistant tumors revealed a strong upregulation of Rac1 and p40(PHOX). Our results demonstrate that ROS-induction by KSHV plays a causal role in KS oncogenesis by promoting proliferation and angiogenesis. Our results show that both ROS and their molecular sources can be targeted therapeutically using NAC or other Food and Drug Administration (FDA)-approved inhibitors for prevention and treatment of AIDS-KS.

A20/TNFAIP3 inhibits NF-κB activation induced by the Kaposi's sarcoma-associated herpesvirus vFLIP oncoprotein.

Kaposi's sarcoma-associated herpesvirus (KSHV) K13/vFLIP (viral Flice-inhibitory protein) induces transcription of numerous genes through NF-κB activation, including pro-inflammatory cytokines, which contribute to the pathogenesis of Kaposi's sarcoma (KS). In this study, we report that KSHV vFLIP induces the expression of the NF-κB regulatory proteins A20, ABIN-1 and ABIN-3 (A20-binding NF-κB inhibitors) in primary human endothelial cells, and that KS spindle cells express A20 in KS tissue. In reporter assays, A20 strongly impaired vFLIP-induced NF-κB activation in 293T cells, but ABIN-1 and ABIN-3 did not. Mutational analysis established that the C-terminal domain (residues 427-790) is critical for A20 modulation of NF-κB, but the ubiquitin-editing OTU (ovarian tumor) domain is not. In functional assays, A20 inhibited vFLIP-induced expression of the chemokine IP-10, reduced vFLIP-induced cell proliferation and increased IKK1 protein levels. Thus, we demonstrate that A20 negatively regulates NF-κB activation directly induced by KSHV vFLIP. By attenuating excessive and prolonged vFLIP-induced NF-κB activation that could be harmful to KSHV-infected cells, A20 likely has an important role in the pathogenesis of KSHV-associated diseases, in which vFLIP is expressed.

Cutaneous Colesional Acquired Immunodeficiency Syndrome Associated Kaposi Sarcoma and Cryptococcosis

The clinicopathologic features of 4 AIDS patients with cutaneous colesional Kaposi sarcoma (KS) and cryptococcosis, a rare phenomenon, are described. Biopsies from 3 patients who were highly active antiretroviral therapy (HAART)-naive demonstrated predominant KS with a conspicuous spindle cell component and small aggregates of cryptococcal yeasts in 2 biopsies and predominant gelatinous cryptococcosis with attenuated KS spindle cells in 1 biopsy. One patient was HAART exposed. He had childhood pulmonary tuberculosis, was treated for disseminated cutaneous cryptococcosis 18 months earlier and presented with cutaneous lesions, odynophagia and massive cervical lymphadenopathy in the eighth week of HAART, after achieving viral suppression and a CD4 cell increase from 28 to 184 cells/μL. His skin biopsy demonstrated a dense lymphoplasmacytic infiltrate, neutrophils, and granulomas with admixed aggregates and single Cryptococcus neoformans and focal aggregation of human herpes virus 8-immunopositive spindle cells. Acid fast bacilli were not identified and mycobacterial molecular studies were negative. The features were compatible with cutaneous cryptococcal immune reconstitution inflammatory syndrome. His nodal and oropharyngeal biopsies demonstrated dense mixed, including granulomatous, inflammation with few cryptococcal yeasts and acid fast bacilli, confirmed to be Mycobacterium tuberculosis on polymerase chain reaction testing, without KS. These features were also compatible with immune reconstitution inflammatory syndrome, but the exact role of each infection in the extracutaneous sites was unconfirmed. Colesional KS and cryptococcosis served as the sentinel lesion of AIDS in 3 patients and of immune reconstitution inflammatory syndrome in 1 patient.

ID2-VEGF-related Pathways in the Pathogenesis of Kaposi’s Sarcoma: A Link Disrupted by Rapamycin

The Id-proteins are a family of four related proteins implicated in the control of differentiation and cell-cycle progression. Down-regulation of Id-gene expression is essential for the differentiation of several cell types. In addition, deregulated Id2 activity inhibits the Rb tumor suppressor pathway and promotes the expression of vascular endothelial growth factor (VEGF). Several members of VEGF family could be involved in Kaposi's sarcoma (KS) development and progression. Lymphatic vascular endothelial hyaluronan receptor-1 (LYVE-1) is the first marker of lymphatic endothelial competence during development in the mature vasculature, and is also expressed on KS spindle cells. Rapamycin (RAPA), an immunosuppressive drug, has been shown to reverse KS growth and to reduce tumor angiogenesis. We evaluate, in transplantation-associated KS and in cultured KS-cells the RAPA effect on Id2 and on de novo lymphangiogenesis. Markers of lymphatic-endothelial-cells (VEGFR-3, LYVE-1) and Id2, expressed at low levels within the normal skin, were up-regulated in KS and returned to normal levels after RAPA introduction. The association between Id2 and lymphangiogenesis is suggested by co-localization of Id2, VEGFR-3 and LYVE-1. RAPA inhibition on Id2 expression was confirmed in vitro in KS-cells, both in basal conditions and upon stimulation with VEGF. In conclusion, our data would suggest a novel molecular mechanism for the antineoplastic effects of RAPA in posttransplant KS.

Peripheral Blood Endothelial Progenitors as Potential Reservoirs of Kaposi's Sarcoma-Associated Herpesvirus

BackgroundThe cellular reservoirs of Kaposi's sarcoma-associated herpesvirus (KSHV) and the exact nature of the putative KSHV-infected circulating precursor of spindle cells of Kaposi's sarcoma (KS) still remain poorly defined. Because KS spindle cells are thought to be of endothelial origin, and because mature endothelial cells do not sustain persistent KSHV-infection, our attention was focalized on circulating hematopoietic precursors able to differentiate into endothelial lineage.Methods and FindingsLate endothelial progenitor cells (late-EPCs) were cultured from the peripheral blood mononuclear cells of 16 patients with classic KS. The presence and load of KSHV genomes were analyzed by real-time polymerase chain reaction in DNA extracted from cells and supernatants of late-EPC cultures obtained from 7 patients. Endothelial colonies cultured from the peripheral blood of KS patients were found to satisfy all requisites to be defined late-EPCs: they appeared from the CD14-negative fraction of adherent cells after 11–26 days of culture, could be serially expanded in vitro, expressed high levels of endothelial antigens but lacked leukocyte markers. Late-EPC cultures were found to harbor KSHV-DNA at variable levels and to retain the virus after multiple passages in cells as well as in supernatants, suggesting that a quote of KSHV lytic infection may spontaneously occur. Lytic phase induction or hypoxia could amplify virus release in supernatants.ConclusionOur results suggest that circulating endothelial progenitors from KS patients are KSHV-infected and support viral productive replication and may therefore represent potential virus reservoirs and putative precursors of KS spindle cells.

KSHV/HHV-8 and HIV infection in Kaposi's sarcoma development

Kaposi's sarcoma (KS) is a highly and abnormally vascularized tumor-like lesion affecting the skin, lymphnodes and viscera, which develops from early inflammatory stages of patch/plaque to late, nodular tumors composed predominant of spindle cells (SC). These SC are infected with the Kaposi's sarcoma-associated herpesvirus or human herpesvirus-8 (KSHV/HHV-8). KS is promoted during HIV infection by various angiogenic and pro-inflammatory factors including HIV-Tat. The latency associated nuclear antigen type 1 (LANA-1) protein is well expressed in SC, highly immunogenic and considered important in the generation and maintenance of HHV-8 associated malignancies. Various studies favour an endothelial origin of the KS SC, expressing "mixed" lymphatic and vascular endothelial cell markers, possibly representing hybrid phenotypes of endothelial cells (EC). A significant number of SC during KS development are apparently not HHV8 infected, which heterogeneity in viral permissiveness may indicate that non-infected SC may continuously be recruited in to the lesion from progenitor cells and locally triggered to develop permissiveness to HHV8 infection. In the present study various aspects of KS pathogenesis are discussed, focusing on the histopathological as well as cytogenetic and molecular genetic changes in KS.

Lymphatic and vascular origin of Kaposi's sarcoma spindle cells during tumor development

The histogenesis of Kaposi's sarcoma (KS) tumor spindle cells (SC) remains controversial but several immunohistochemical studies favor a lymphatic origin. Twenty KS surgical biopsies were analyzed for the coexpression of LANA, CD34, LYVE-1, D2-40, VEGFR-2, VEGFR3 by using double or triple immunostaining. Most of the SC in both early and late KS expressed the lymphatic markers LYVE-1, D2-40 and VEGFR-3 and the blood vascular endothelial/endothelial precursor cell markers CD34 and endothelial stem cell marker VEGFR-2. All the LANA+ SC in early and late KS were LYVE-1+, but only 75% of these LANA+ cells were CD34(+). The CD34(+)/LANA+ cells increased from early- (68.8%) to late-stage KS (82.2%). However, approximately 18% of the LANA+ SC in early KS were CD34(-) but were LYVE-1+, suggesting that resident lymphatic endothelial cells (LEC) are targeted for primary infection by human herpesvirus-8. This LANA+/LYVE-1+/CD34(-) (resident LEC) cell population clearly decreased during the development of KS from early (18.7%) to late KS (2.9%). Thus, in late stages of KS, most SC were LANA+/CD34(+)/LYVE-1+. However, in both early- and late-stage KS, approximately 18% of the SC were CD34(+)/LANA-/LYVE-1 -- and could represent newly recruited endothelial precursor cells, which become infected in the lesion and eventually undergo a phenotype switch expressing LEC markers. Our study apparently indicates that KS represents a unique variant of tumor growth with continues recruitment of tumor precursor cells as well as proliferation and decreased apoptosis of SC.

The Potential Role of Delta(9)-Tetrahydrocannabinol in the Pathogenesis of Kaposi's Sarcoma.

Kaposi's sarcoma (KS) is a frequent neoplastic manifestation of immuno-compromised conditions such as AIDS and organ transplantation. The etiologic agent of the tumor is the KS-associated herpesvirus (KSHV), also known as human herpesvirus 8. Other factors, including HIV-1 Tat protein, may also contribute to the development and progression of KS in people with AIDS. Marijuana is the most commonly used illicit drug in the United States. In addition, its major active component, THC (Tetrahydrocannabinol), is prescribed for medicinal use. This includes treatment of HIV-related cachexia as well as pain and wasting in other immuno-compromised patients. However, relatively limited knowledge exists about the effects of cannabinoids on KS growth and spread. To investigate how cannabinoids may affect KS from the initiation of KSHV infection to proliferation of KS spindle cells, we first assessed whether KS spindle cells expressed cannabinoid receptors. The CB1 and CB2 receptors were detected on KS cells by flow cytometry. These receptors were found to be functional based on calcium flux upon ligand stimulation. We then sought to determine whether cannabinoids modulated viral entry. KSHV infection was quantitated in primary microvascular endothelial cells in the presence of a dose range of THC. We observed that low concentrations of Δ9-THC (less than 1 ng/ml) increased KSHV infection by about 30%. Higher concentrations of Δ9-THC induced significant cell death following KSHV exposure. Blocking of the CB1 and CB2 receptors had no effect on THC-modulated KSHV infection. We then studied the functional and biochemical changes in KS spindle cells after exposure to cannabinoids. We observed a dose-dependent inhibition of growth in these spindle cells in the presence of Δ9-THC. To elucidate the molecular mechanism of growth inhibition, we assayed signaling molecules involved in cell survival and proliferation. We found that Δ9-THC activated ERK1/2 MAP kinase and Akt, as well as VEGFR-2 and EGFR in KS spindle cells. High concentrations of Δ9-THC significantly inhibited VEGF-C and VEGF-D secretion but not VEGF-A production. Since KS is a vascular neoplasm and blockade of the CB1 and CB2 receptors did not antagonize the effects of Δ9-THC in KS, we investigated the activity of abn-cbd [(−)-4-(3-3, 4-trans-p-menthadien-[1, 8]-yl)-olivetol], a novel selective cannabinoid-like agonist specific for endothelium. Similar inhibitory effects in response to abn-cbd were observed in the KS spindle cells. These inhibitory effects were reversed by ARA-S (N-Arachidonoyl Serine) and O-1918 [(−)-4-(3-3, 4-trans-p-menthadien-(1, 8)-yl)-orcinol], specific antagonists of abn-cbd. These antagonists also reversed the reduction in VEGF-C and VEGF-D production. Taken together, our study indicates a biphasic effect of Δ9-THC, in that the cannabinoid may enhance KSHV infection at low concentrations, but inhibit KS spindle cell growth at higher concentrations. This modulation of KS cell function may involve a third putative receptor other than CB1 and CB2. Further knowledge regarding cannabinoid modulation of KS biology should allow for a greater understanding of the appropriate use of marijuana and its active constituents in immuno-suppressed hosts at risk for 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.

Kaposi’s sarcoma in Morocco: a pathological study with immunostaining for human herpesvirus-8 LNA-1

The Maghreb region is considered to be an endemic area for classical Kaposi's sarcoma (KS) but the few published reports have focused on clinical descriptions. We report here the clinical, virological and pathological features of 26 KS cases, including 17 classic forms, eight epidemic forms and one case of post-transplant KS. Evidence of human herpesvirus (HHV)-8 infection was detected by serology, immunohistochemistry and polymerase chain reaction. Antibodies directed against HHV-8 latent nuclear antigen (LNA-1/ORF73) were present in sera from all 26 KS cases and the ORF26 HHV-8 gene fragment was amplified from the tumour DNA of all patients. KS lesions that met the classical criteria for KS (spindle cells, positive endothelial markers) and were immunostained with the anti-LNA-1 marker, demonstrated typical granular intranuclear labelling of the spindle cells in all but one case of KS, in which very few spindle cells were present. Furthermore, whereas LNA-1 staining was largely confined to spindle cells in the late nodular stage, it was also observed in endothelial cells forming the walls of slit-like vessels in early patch-stage lesions. This study confirms that Morocco is an endemic area for classical KS, which remains the most frequent type of KS in this country, and emphasises the value of the LNA-1 marker for the early diagnosis of KS lesions.

Salivary Lactoferrin Is Recognized by the Human Herpesvirus-8

Human herpesvirus-8 (HHV-8) is commonly detected in all epidemiologic forms of Kaposi's sarcoma. Despite the broad cellular tropism of HHV-8, studies on mucosal shedding of HHV-8 have shown that infectious particles are restricted to saliva isolated from the oropharynx. We used biotinylated purified HHV-8 particles in a direct binding assay to whole clarified human salivary samples isolated from HHV-8-infected and uninfected individuals. We found that the major binding activity was carried out by a protein of 78-kDa size, which was further characterized as human lactoferrin (hLf) using 2-D electrophoresis and MALDI-ToF analysis. Preliminary comparison of HHV-8 binding activity of 76 salivary samples from HHV-8-infected and uninfected individuals showed that 7.8% of the uninfected population exhibited a form of Lf not recognized by HHV-8. Deglycosylation of hLf by PNGase F did not reduce HHV-8 binding activity, whereas endoproteinase cleavage of native hLf generated a non-glycosylated 8-kDa peptide recognized by HHV-8 particles and was located at the position Ala606-Tyr679 in the native hLf amino acid sequence, corresponding to the C-terminal region of the glycoprotein. This work identify the lactoferrin in saliva as a ligand for HHV-8 and suggests that this glycoprotein could be used as a carrier for the viral particles.

Human herpesvirus 8/Kaposi sarcoma herpesvirus cell association during evolution of Kaposi sarcoma.

Kaposi sarcoma (KS) is associated with a herpesvirus (HHV-8/KSHV), which expresses a latency-associated nuclear antigen (LANA). The histopathology of KS is characterized by angiogenesis, inflammatory cells, and the development of CD34+ tumor spindle cells (SCs). However, the cellular basis for the recruitment and dissemination of HHV-8 during the development of KS lesions is not clear. Twenty-nine KS biopsies with AIDS (AKS, n=22) and without HIV infection (endemic KS or EKS, n=7) were immunostained by a triple antibody method to characterize HHV-8-infected and noninfected (LANA+/-) CD34+ SCs, infiltrating CD3+, CD68+, CD20+, and CD45+ leukocytes as well as proliferating (Ki67+) cells. The CD34+/LANA+ SCs were more frequent in late (nodular) as compared with early (patch/plaque) KS stages. However, in late AKS 36.0% of SCs (median of 11 cases) were CD34+/LANA- compared with 20.7% in early cases (median of 11 cases). Furthermore, both AKS and EKS showed, at all stages, a small (4.1-6.5%) population of LANA+/CD34- cells. Proliferating Ki67+ cells were seen (4.5-11.5%) at all KS stages, and were usually more frequent in early AKS, but no significant difference was observed between nodular AKS and EKS. Most of the proliferating cells in the KS lesions were LANA+/CD34+ but a small fraction was LANA+/CD34-. Lesional CD68+ and CD3+ cells varied between AKS (7.3 and 5.2%, respectively) and EKS (4.9 and 3.1%, respectively) but were not clearly stage related. No LANA+ cells were CD3+, CD20+, or CD45+ and very few (<0.5%) were CD68+. These results indicate that not all CD34+ KS SCs were LANA+, suggesting recruitment of noninfected SCs to the lesions. Cell proliferation in general was much higher in early as compared with the late AKS stages. LANA+ SCs could have a proliferative advantage as suggested by higher frequency of cycling (Ki67+) LANA+ SCs. Few macrophages but no lymphocytes are LANA+.