Peripheral-blood-derived-spindle cells (Pbsc) are found more frequently among human immunodeficiency virus (HIV)-infected (HIV+) KS patients and those HIV + individuals at high risk for developing KS. The purpose of this project is to further define the histogenesis of these mesenchymal cells, then examine the role of these cells in the initiation and progression of KS, Our studies show that Pbsc from KS patients are infected with a newly described human herpesvirus, HHV-8, which is suspect in the etiology of KS. Moreover, RNA transcripts for a HHV-8-encoded cyclin D2 gene homologue have been detected in the Pbsc and the spindle cells of KS lesions. Therefore, the current model for KS pathogenesis suggests that HHV-8-infection of a circulating mesenchymal progenitor is critical for KS initiation and progression. A systematic approach for the purification of Pbsc and characterization of their in vitro and in vivo function will lead to a better understanding of Pbsc biology and their role in KS pathogenesis. Furthermore, examination of HHV-8 cyclin function in the Pbsc may lead to an understanding of how this HHV-8 gene product contributes to KS pathogenesis. In the first aim, cells expressing CD34, FLK-1, ulex europaeus lectin I will be purified from peripheral blood mononuclear cells using antibody-coated magnetic beads. The isolated cells will be cultured in semi-solid medium (methylcellulose) and in liquid culture using a cocktail of hematopoietic and endothelial specific growth factors. Flow cytometry and immunohistochemistry will be used to examine endothelial specific cell surface maker expression before and during culture in vitro. Cell proliferation will be determined by BrdU incorporation followed by staining with anti-BrdU antibodies. PCR will be performed to access the presence of HHV-8. RT-PCR will be performed to examine the expression of HHV-8-encoded gene products in the Pbsc and the expression of lineage specific receptors e.g., FLK- 1, TIE-2, and CD34. In the second aim, the shall-less chick embryo culture (SLCEC) system will be used to examine the ability of the Pbsc to induce and/or differentiate into blood vessels ex ovo. Pbsc will be labeled prior to transplantation with di-I-C18 for identification in the developed blood vessels. The presence of HHV-8 within the developed lesion will be determined by immunohistochemical staining using specific antisera. The studies described will may lead to the development of an improved model for KS, but may also lead to the study of potential endothelial progenitor cells in other dermatological conditions associated with pathological wound healing and angiogenesis.

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Vanderbilt University Medical Center
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