PROGENITORI ENDOTELIALI CIRCOLANTI: CARATTERIZZAZIONE IN SOGGETTI CON SARCOMA DI KAPOSI CLASSICO AD EFFETTI DELL'INFEZIONE IN VITRO DA HHV-8

Background Kaposi’s sarcoma (KS) is an angioproliferative disorder associated with human herpesvirus-8 (HHV-8) infection. Spindle cells are the predominant cell type in the KS lesions and are endothelial in origin. We previously demonstrated that endothelial progenitor cells (EPCs) cultured ex vivo from KS classic (cKS) patients as late-EPCs are HHV-8-infected and support viral productive replication. In this study we investigated whether late-EPCs from KS patients showed a different in vitro behaviour compared to late-EPCs from healthy controls, in terms of efficiency of ex vivo isolation, and biological features. Moreover we infected in vitro control late-EPCs to investigate whether late EPCs can be infectable with HHV-8 and whether they support viral replication. We also analyzed the effects of HHV-8 infection on multiple aspects of late-EPCs biology. Methods Late-EPC were cultured from peripheral blood mononuclear cells (PBMCs) of 69 cKS patients (cKS-late-EPC) and 40 sex and age-matched healthy HHV-8 seronegative controls (ctr-late-EPC). Briefly, PBMCs were seeded on collagen-coated or fibronectin-coated plates in EGM-2 and late-EPCs were identified as cobblestone-like colonies. Cultures from all subjects were observed for initial late-EPCs colony appearance and number of colonies, cell viability, cell proliferation and cytokine production. ctr-late-EPC was infected in vitro with HHV-8 and the effects of HHV-8 infection on late-EPC biology were investigated by analyzing the following parameters: cell morphology, proliferation, immunophenotype and angiogenesis. Results In our study we demonstrated that the frequency of late EPCs isolation depends on substrate used to seed PBMCs. The average time of initial late-EPCs colony appearance was significantly lower in cKS-late-EPC than ctr-late-EPC (P<0.001). The number of late-EPC colonies per 20x106 seeded PBMCs was significantly higher in cKS-late-EPC than ctr-late-EPC (P<0.001). Senescence appear later time in cKS-late-EPC than ctr-late-EPC. Morover, cell proliferation and IL-6 production was higher in cKS patients than healthy controls. This data suggest that the presence of HHV-8 infection in cKS-late-EPC may induce late-EPCs to acquire several feature that resemble spindle cells. HHV-8 in vitro infection induced ctr-late-EPC to acquire a spindle shape similar to the morphology of KS spindle cells. HHV-8 induced ctr-late-EPC to reduce proliferation and promoted the activation of late-EPCs inducing the up-regulation of VCAM-1, ICAM-1, e-selectin. HHV-8-infection also induced the up-regulation of CD49c and αvβ3 integrins. Conclusions In this study we provide evidence that late-EPCs obtained from patients with cKS are characterized by strikingly higher ex vivo expansion and proliferation rate than late-EPCs from healthy controls. Moreover, upon HHV-8 in vitro infection, EPCs support persistent viral productive replication and acquire morphologic and functional features of KS spindle cells. These novel findings may be particularly relevant to the comprehension of KS pathogenesis, because late-EPCs may represent putative precursors of spindle cells that may home to permissive sites and propagate to produce KS lesions.