Differential toxicity of two murine endothelial cells to ROS duress: understanding oxidative stress-induced blood-brain barrier dysfunction

Abstract

The blood-brain barrier (BBB) is a critical interface between the blood circulation and brain tissue which performs critical selection of circulating molecules that gain access to the brain tissue. Its unique ability to adjust to changes in the constituents of the blood circulation confer in the BBB a dynamic nature enabling changes in its properties to suit the homeostatic needs of the brain. Dysfunction of the BBB has been established to be pivotal to the initiation and/or maintenance of an array of neurological disorders, most of which involve the production of excess reactive oxygen species (ROS) and oxidative stress in their pathophysiology. Thus, clinical trials of exogenous antioxidant agents have been proposed and initiated, with most results being inconclusive. Extensive studies of the impact, capacity and plasticity of endogenous antioxidants in the cells that constitute the blood-brain barrier, especially the brain endothelial cells, therefore, became necessary for the rational choice, timing, and the mode of application of antioxidants in the management of oxidative stress-mediated neurological diseases. In the present study, incremental hydrogen peroxide (H2O2) concentrations were dosed in cell culture medium to simulate OS and gauge the capacity of the brain endothelial cell (BECs) models of the BBB to resist ROS toxicity, the contribution of endogenous antioxidants to this resistance and to study the morphological changes in ROS-mediated BBB dysfunction. Two types of mammalian endothelial cells, b.End5 and bEnd.3, commonly used for in vitro studies of the blood-brain barrier, were selected for use in this study. A combination of spectrophotometry, fluorescent microscopy, trypan blue exclusion method and flow cytometry were used to assess cell viability, cellular glutathione (GSH) content, cell cycle changes, cellular death by apoptosis and necrosis in BECs exposed to incremental H2O2 concentrations for 24hr. Exogenous antioxidants were variably administered to study the effects of externally incident antioxidants when the cells were under H2O2 exposure. Results showed that b.End5 cell line significantly tolerated higher concentrations of H2O2 than the bEnd.3 cell line. GSH contents for both cell lines were fairly similar under physiological conditions but after exposure to H2O2, b.End5 cells demonstrated higher resistance to GSH depletion than the bEnd.3 cell line, although the two cell lines were obtained from the same animal species. Along incremental concentrations of H2O2, increased cell proliferation, cell necrosis and apoptosis and cell cycle arrest were observed concurrently. At H2O2 concentrations that defined OS, live cells were depleted in b.End5 cells used while there was significant increases in apoptotic and necrotic cells with apoptotic cells as the significant majority comparatively. Cell cycle studies showed arrest of cell division at the G2/M phase of the cell cycle at higher concentrations of H2O2. Application of exogenous antioxidants ameliorated the H2O2-induced cellular depletion as well as improved recovery in cellular viability following withdrawal of H2O2 after 24hr exposure. It was conclusive that apoptotic pathway of cellular death is a major pathway of BECs response to OS. Also, there was differential H2O2 toxicity and GSH de novo synthesis capacity between the b.End5 and bEnd.3 cell lines despite their common origin from the same animal species and their possession of similar contents of endogenous antioxidant GSH under physiological conditions. This finding calls for more caution for the choice of cellular models for specific studies of the BBB to ensure that results obtained are reproducible, reliable and sufficiently conclusive. Furthermore, our results tend to suggest that the processes responsible for the endothelial component of BBB dysfunction under conditions of oxidative stress occur concurrently and include increased proliferation, necrotic and apoptotic cell death as well as cell cycle arrest. Additionally, study suggests that the clinical administration of antioxidants could be an appropriate intervention for the alleviation of neurological diseases.