Hypoxic Ischemic Injury Of The Blood-brain Barrier Differential Proteomics

The blood-brain barrier (BBB) is an impermeable barrier that physically separates the blood from the interstices of the brain. Its main function is to prohibit the free exchange between the blood and the brain. However, the research of the past decades showed the functions of BBB are far more than previous known. BBB is the in-and-out passage of nutrients, metabolism products, and many signal molecules. It plays important roles in the supply of energy substrate, synthesis and clearance of the neurotransmitters for maintaining the homeostasis of brain. Previous reports have shown that BBB participates the regulation of profound ischemia/hypoxia mechanism, however progress of this field is slow because of the multi-cells' component of BBB and the complexity on the mechanism of ischemia/hypoxia. According to which, we aimed to systemically study the ischemia/hypoxia related proteins expressed in BBB using proteomics approaches for better understanding the functions of BBB.To explore the hypoxia-related proteins in the endothelial cells, the ECV304 with endothelial properties induced by chemical and physical hypoxic insults respectively and analyzed the differential by expressed proteins by two dimensional gel electrophoresis (2-DE) followed with mass spectrometry (MS). Eight proteins were identified. Among them, 5 proteins (Proteasome 26S non-ATPase, vimentin, tropomyosin 4 and Translationally controlled tumor protein (TCTP)) were up-regulated, and 3 proteins (annexin IV , hnRNP H2 and vascular ATP synthase) were down-regulated in both of the two hypoxic models (chemical and physical hypoxia). Some differentially expressed proteins were further confirmed by RT-PCR (at mRNA level) and Western blot.To evaluate the changes of the hypoxia related proteins in the brain microvascular endothelial cell which is a component of the BBB, the differential protein expression profile in the mouse brain endothelial cell line bEnd.3 between the normal and oxygen-glucose deprivation (OGD) was further analyzed. Results demonstrated that 45 proteins were identified. Among them, 41 proteins increased and 4 decreased in cells treated with OGD. The result of GO-based functional analysis of the data showed that proteins involved in signal transduction, carbohydrate metabolism, alternative splicing and cytoskeleton remodeling account for a larger proportion (about 60%). Data mining results suggested that about 1/4 of the total proteins have been reported in previous reports.Take together, a number of proteins differentially expressed in ischemia/hypoxia insults have been detected in two endothelial cell lines using proteomics methods. The data might help us to understand the regulations of ischemia responses in BBB.