Kaposi sarcoma herpesvirus–induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma

The biology of Kaposi sarcoma is poorly understood because the dominant cell type in Kaposi sarcoma lesions is not known. We show by gene expression microarrays that neoplastic cells of Kaposi sarcoma are closely related to lymphatic endothelial cells (LECs) and that Kaposi sarcoma herpesvirus (KSHV) infects both LECs and blood vascular endothelial cells (BECs) in vitro. The gene expression microarray profiles of infected LECs and BECs show that KSHV induces transcriptional reprogramming of both cell types. The lymphangiogenic molecules VEGF-D and angiopoietin-2 were elevated in the plasma of individuals with acquired immune deficiency syndrome and Kaposi sarcoma. These data show that the gene expression profile of Kaposi sarcoma resembles that of LECs, that KSHV induces a transcriptional drift in both LECs and BECs and that lymphangiogenic molecules are involved in the pathogenesis of Kaposi sarcoma.</p

Endothelial Cells Support Persistent Gammaherpesvirus 68 Infection

A variety of human diseases are associated with gammaherpesviruses, including neoplasms of lymphocytes (e.g. Burkitt's lymphoma) and endothelial cells (e.g. Kaposi's sarcoma). Gammaherpesvirus infections usually result in either a productive lytic infection, characterized by expression of all viral genes and rapid cell lysis, or latent infection, characterized by limited viral gene expression and no cell lysis. Here, we report characterization of endothelial cell infection with murine gammaherpesvirus 68 (γHV68), a virus phylogenetically related and biologically similar to the human gammaherpesviruses. Endothelial cells supported γHV68 replication in vitro, but were unique in that a significant proportion of the cells escaped lysis, proliferated, and remained viable in culture for an extended time. Upon infection, endothelial cells became non-adherent and altered in size, complexity, and cell-surface protein expression. These cells were uniformly infected and expressed the lytic transcription program based on detection of abundant viral gene transcripts, GFP fluorescence from the viral genome, and viral surface protein expression. Additionally, endothelial cells continued to produce new infectious virions as late as 30 days post-infection. The outcome of this long-term infection was promoted by the γHV68 v-cyclin, because in the absence of the v-cyclin, viability was significantly reduced following infection. Importantly, infected primary endothelial cells also demonstrated increased viability relative to infected primary fibroblasts, and this increased viability was dependent on the v-cyclin. Finally, we provide evidence for infection of endothelial cells in vivo in immune-deficient mice. The extended viability and virus production of infected endothelial cells indicated that endothelial cells provided a source of prolonged virus production and identify a cell-type specific adaptation of gammaherpesvirus replication. While infected endothelial cells would likely be cleared in a healthy individual, persistently infected endothelial cells could provide a source of continued virus replication in immune-compromised individuals, a context in which gammaherpesvirus-associated pathology frequently occurs

Pre-Micro RNA Signatures Delineate Stages of Endothelial Cell Transformation in Kaposi Sarcoma

MicroRNAs (miRNA) have emerged as key regulators of cell lineage differentiation and cancer. We used precursor miRNA profiling by a novel real-time QPCR method (i) to define progressive stages of endothelial cell transformation cumulating in Kaposi sarcoma (KS) and (ii) to identify specific miRNAs that serve as biomarkers for tumor progression. We were able to compare primary patient biopsies to well-established culture and mouse tumor models. Loss of mir-221 and gain of mir-15 expression demarked the transition from merely immortalized to fully tumorigenic endothelial cells. Mir-140 and Kaposi sarcoma–associated herpesvirus viral miRNAs increased linearly with the degree of transformation. Mir-24 emerged as a biomarker specific for KS

Integrated microarray and multiplex cytokine analyses of Kaposi's Sarcoma Associated Herpesvirus viral FLICE Inhibitory Protein K13 affected genes and cytokines in human blood vascular endothelial cells

<p>Abstract</p> <p>Background</p> <p>Kaposi's sarcoma (KS) associated herpesvirus (KSHV) is the etiological agent of KS, a neoplasm characterized by proliferating spindle cells, extensive neoangiogenesis and a prominent inflammatory infiltrate. Infection of blood vascular endothelial cells with KSHV in vitro results in their spindle cell transformation, which is accompanied by increased expression of inflammatory chemokines and cytokines, and acquisition of lymphatic endothelial markers. Mimicking the effect of viral infection, ectopic expression of KSHV-encoded latent protein vFLIP K13 is sufficient to induce spindle transformation of vascular endothelial cells. However, the effect of K13 expression on global gene expression and induction of lymphatic endothelial markers in vascular endothelial cells has not been studied.</p> <p>Methods</p> <p>We used gene array analysis to determine change in global gene expression induced by K13 in human vascular endothelial cells (HUVECs). Results of microarray analysis were validated by quantitative RT-PCR, immunoblotting and a multiplex cytokine array.</p> <p>Results</p> <p>K13 affected the expression of several genes whose expression is known to be modulated by KSHV infection, including genes involved in immune and inflammatory responses, anti-apoptosis, stress response, and angiogenesis. The NF-κB pathway was the major signaling pathway affected by K13 expression, and genetic and pharmacological inhibitors of this pathway effectively blocked K13-induced transcriptional activation of the promoter of CXCL10, one of the chemokines whose expression was highly upregulated by K13. However, K13, failed to induce expression of lymphatic markers in blood vascular endothelial cells.</p> <p>Conclusion</p> <p>While K13 may account for change in the expression of a majority of genes observed following KSHV infection, it is not sufficient for inducing lymphatic reprogramming of blood vascular endothelial cells.</p