10th International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV 6-8 November 2008, London, UK

EX VIVO MODULATION OF MESENCHYMAL STEM CELL FUNCTION IN HIV-1 INFECTION

Antiviral Therapy 2008; 13(Suppl. 4):A13 (abstract no. O-18)

EJ Cotter¹, N Chew¹, WG Powderly² and PP Doran^1
1CRC, University College Dublin, Dublin, Ireland; ²School of Medicine and Medical Sciences, University College Dublin, Dublin, Ireland

INTRODUCTION: An increased incidence of bone and metabolic toxicities are associated with HIV-1 infection and its treatment. However, the exact mechanisms of these toxicities and the relative contribution of virus and treatment remains to be elucidated. Mesenchymal stem cells (MSC) are multipotent bone marrow-derived cells that can differentiate into a number of cell lines, including osteoblasts (OB) and adipocytes (AC). Herein, we hypothesize that perturbation of MSC function and differentiation might underpin the bone and fat abnormalities associated with HIV-1 infection and treatment.

METHODS: In this ex vivo study, human MSCs were treated with media supplemented with either HIV-negative (n=5), HIV-positive low (LVL; viral load 120–4,000, n=5) or high (HVL; viral load 100,000–500,000, n=4) viral load serum (5% concentration, HIV serum obtained from highly active antiretroviral therapy-naïve patients) and chemically induced to differentiate into either OBs or ACs. Calcium deposition and lipid levels, as measures of osteogenesis and adipogenesis, respectively, were assessed using established methods. To determine whether short-term exposure to serum induced changes in gene expression, non-differentiating cells were treated with serum from each group (5% serum, 24 h) and levels of mRNA for the differentiation markers RUNX-2, PPARγ and ß-catenin, as well as tumour necrosis factor (TNF)-α and CD4, were determined using real-time PCR with gene-specific primers. Finally, in order to determine if long-term exposure affected cell phenotype and function, non-differentiating MSCs were exposed to serum (5% serum) from each group over a 72 h time course. Cell activity and number were determined using MTS assay and ponceau red staining, respectively, and the levels of LPL protein and ALP activity were determined using whole cell ELISA and BCIP/NBT staining, respectively.

RESULTS: Serum from either high or low viral groups increased the degree of adipogenesis, an induction that was significant following exposure to HVL serum. Moreover, the degree of adipogenesis in HVL serum-treated cells was significantly greater than that observed in LVL serum-treated cells (P=0.01), suggesting the effect was dependent on viral concentration. Expression of PPARγ mRNA expression in non-differentiating cells was significantly upregulated by HVL serum (5%, 24h), whereas RUNX-2 and ß-catenin expression was unchanged. Treatment with serum from both groups also downregulated CD4 and TNF-α expression. Finally treatment of non-differentiating cells with HVL serum for 72 h, significantly increased cell activity, decreased cell number, decreased ALP activity and increased LPL protein levels.

CONCLUSIONS: Serum from HIV-1 patients both increased the degree of chemically induced adipogenesis from MSCs and drove the presentation of a pro-adipogenic phenotype in non-differentiating MSCs in a viral load-dependent manner. These findings underscore the hypothesis that perturbations in MSC cell differentiation are responsible, at least in part, for the observed toxicities seen in HIV patients in vivo.

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2008-11-06
O-18

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