Antiretroviral Tenofovir Induces Senescence-Associated β-Galactosidase Activity in Primary Human Brain Vascular Cells in Multi-Layer Three-Dimensional Co-Culture

Abstract

IntroductionIn the current context of early diagnosis of HIV infection, immediate initiation of antiretroviral (ARV) therapy, and lifelong chronic treatment, the potential ARV toxicity is of particular concern. Emtricitabine (FTC) and tenofovir (TFV) are commonly used as backbone drugs in ARV regimens recommended for initial therapy of HIV infection. Here we assessed the effects of FTC and TFV exposure on senescence-associated β-galactosidase (SA-β-Gal) activity, a marker of cellular senescence, in human brain vascular cells.DesignMulti-layer three-dimensional cell co-cultures and in vitro assays.MethodsTo mimic the small vessel wall structure in vivo, three types of primary human brain vascular cells (endothelial cells, smooth muscle cells, and pericytes) were co-cultured on three Alvetex Scaffold disks placed on top of each other in order (three-layer three-dimensional cell co-cultures) and exposed to clinically relevant concentrations of ARV drugs (FTC, TFV, or FTC+TFV combination) or vehicle for eight days (four or five biological replicates per condition, 18 replicates totally). The SA-β-Gal activity was quantitatively assayed in vitro by using the chemiluminescent Galacto-Star System (T1012; Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) in 54 protein lysates extracted from individual cell-culture disks. Three-factor analysis of variance (cell type, FTC, TFV) was used to assess differences in the SA-β-Gal activity levels normalized by the corresponding total protein concentrations.ResultsThere was a trend for the FTC by TFV interaction effect on SA-β-Gal activity (P = 0.058). The effects of FTC and TFV were not significantly different among the three cell types. The overall effect of FTC was not significant when controlling for TFV and cell type. The overall effect of TFV was significant when controlling for FTC and cell type (F(1,48) = 30.61, P < 0.001, partial η2 = 0.389). In the absence of FTC, TFV raised SA-β-Gal activity by 0.631 units on average, regardless of cell type (P < 0.001, partial η2 = 0.368). In the presence of FTC, TFV raised SA-β-Gal activity by 0.303 units on average, regardless of cell type (P = 0.015, partial η2 = 0.118).ConclusionOur preliminary findings suggest that primary human brain vascular cells exposed to TFV at clinically relevant concentrations undergo cellular senescence. This potential adverse effect of TFV should be further studied in animal models of HIV infection.