Abstract 15570: A Novel Ex Vivo Test System to Assess Cardiovascular Toxicity of HIV-Nef in PBMC From HIV Positive Donors

Abstract

Background: To address how cardiovascular disease is increased in HIV patients with and without therapy, we have tested the effects of HIV-infected T cell when in contact to human coronary endothelial cells (HCAEC). We found that these T cells could induce endothelial dysfunction and cell death at a much larger extent than free virus itself. We could identify HIV-Nef protein, a major pathogenic factor in HIV disease progression and HIV-induced end organ disease, as the mechanism for this finding. Because of our finding that HIV-Nef is transferred from T cells to HCAEC to induce cell death, we asked whether PBMC form HIV-infected patients can recapitulate these findings. Methods: PBMC were isolated from either healthy donors or from untreated HIV patients or from those on ART using standard Ficoll purification, and intracellular Nef was determined by flow cytometry and ELISA. PBMC were co-cultivated with HCAEC for 24 hours, washed off and HCAEC were analyzed for Nef by immunofluorescence microscopy representation and ELISA. Further, HCAEC were analyzed for apoptosis by TUNEL. Results: HIV-Nef protein was found in PBMC of untreated HIV-positive and surprisingly also in patients receiving virally suppressive anti-retroviral therapy (ART). Importantly, we could demonstrate in untreated HIV patients Nef protein transfer to HCAEC and increased induction of endothelial apoptosis (see figure, ** p<0.01). Currently, equivalent studies with PBMC from patients on ART are in progress. Conclusion: This study demonstrates that Nef-positive PBMC at least in untreated HIV patients can be transferred to HCAEC to cause endothelial cell apoptosis. This will be the basis for future studies analyzing the role of NADPH oxidases and reactive oxygen species in this toxic activity of Nef. Based on this assay system we can address the hypothesis that HIV-Nef plays a pivotal role in the development of cardiovascular diseases in HIV patients with or without ART.