Protective effect of exenatide against efavirenz-mediated endothelial cell dysfunction.

P D'Orton-Gibson2, SL Dench1, R Lee2, J Mabley1. 1University of Brighton, Brighton, UK, 2Brighton and Sussex Medical School, Brighton, UK

Highly active anti-retroviral therapy (HAART) has transformed the management of HIV and the prevention of AIDS. HAART is comprised of a combination of anti-retroviral drugs selected from different classes including reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors, and integrase inhibitors. Efavirenz, a non-nucleoside reverse transcriptase inhibitor, is common to a lot of HAART regimens (Ford et al., 2013); however, it has been associated with a significant increased risk of cardiovascular disease (Durand et al., 2011). Efavirenz increases cellular oxidative stress via the inhibition of complex 1 of the electron transport chain and it is this mitochondrial toxicity that has been proposed to be the mechanism by which the drug causes cardiovascular cell dysfunction. Exenatide is an anti-diabetic drug used to treat hyperglycaemia by activation of the GLP-1 receptor leading to insulin release. Previous work has shown that activation of the GLP-1 receptor has protective effects on the cardiovascular system through mechanism that involve counteracting the effects of reactive oxygen species (Zhao et al., 2013). This study aimed to investigate if exenatide can protect against the endothelial cell dysfunction and inflammation induced by efavirenz.

The human endothelial cell line EA.hy926 was exposed to efavirenz (30 µM) alone or in combination with exenatide (3-30 nM) for 24 hours prior to measuring cell viability, apoptosis/necrosis levels, and inflammation as assessed by the MTT assay, Hoechst/Propidium iodide staining followed by morphological analysis, and IL-8 release. Ex vivo Sprague-Dawley male rat aortic rings were exposed to efavirenz (10 µM)±exenatide (30 nM) for 4h prior to measurement of acetylcholine-mediated NO-induced vasorelaxation. Data is expressed as mean±SEM and analysed using two-way ANOVA with Bonferroni’s correction.

Efavirenz (30 µM) reduced cell viability to 30±4% of untreated cells (p<0.05; n=3 with 6 replicates per experiment), exenatide protected against this loss of cell viability with the combination of efavirenz (30 µM) plus 3, 10 or 30 nM exenatide reducing viability to 51±4%, 56±5% and 61±7% respectively (p<0.05 vs. efavirenz alone; n=3 with 6 replicates per experiment). Exenatide alone had no effect on cell viability. Efavirenz (10 µM) increased endothelial cell apoptosis from 1.2±0.1% to 2.4±0.2% and necrosis from 5±0.7% to 9.4±1.3% (p<0.05 vs. untreated cells; n=4 with at least 300 nuclei analysed per treatment per experiment). Simultaneous application of exenatide at 3 nM or 10 nM protected against efavirenz (10 µM)-mediated increase in both apoptosis, reducing it to 1.2±0.2% and 0.9±0.1%, and necrosis, reducing it to 3.5±0.8% and 3.6±0.6% respectively (p<0.05 vs. efavirenz alone; n=4 with at least 300 nuclei analysed per treatment per experiment). Exenatide (3 or 10 nM) had no effect on apoptosis or necrosis levels as compared to untreated cells. Efavirenz (30µM) increased IL-8 release from 5.6±0.7 to 22.6±2.2 pg/ µg protein (p<0.05; n=3), an effect that was attenuated by simultaneous application of exenatide at 3 or 10 nM which reduced the release of IL-8 to 8.8±1 pg/µg protein and 7.8±1.1 pg/µg protein respectively (p<0.05 vs. efavirenz alone; n=3). Exenatide alone had no effect on IL-8 release. Efavirenz increased the EC50 for acetylcholine-mediated vasorelaxation from 0.05±0.004 µM to 0.13±0.05 µM (p<0.05; n=8), an effect that was attenuated by simultaneous addition of exenatide (30 nM), which reduced the EC50 to 0.049±0.005 µM (p<0.05 vs. efavirenz alone; n=8). Exenatide alone had no effect on the EC50 of acetylcholine (0.049±0.01 µM, n=5).

In conclusion, activation of GLP-1 receptors either via exenatide treatment or increasing endogenous GLP-1 levels may protect endothelial cells from efavirenz-mediated damage reducing the cardiovascular side effects.

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