M.Sc Thesis

M.Sc StudentHaghbi Pazit
SubjectThe Effect of Hypoxia on Gene and Protein Expression in
Human Brain Endothelial Cells
DepartmentDepartment of Medicine
Supervisor PROF. Ariel Miller
Full Thesis textFull thesis text - English Version


The blood-brain barrier (BBB) is composed of brain capillary endothelial cells (ECs), with tight junctions (TJs) between these cells providing a critical barrier to maintain brain homeostasis. Dysregulation of the BBB occurs in a variety of central nervous system (CNS) diseases such as stroke, multiple sclerosis and cancer. A significant element of these diseases is hypoxia, a reduction in partial oxygen (PO2) pressure. Following hypoxic conditions changes in cerebral microvascular ECs properties occur and determine the cellular fate towards survival or death.

Constituents of the barrier, like TJs proteins, and metalloproteinases (MMPs) that may modify the extracellular matrix (ECM) environment surrounding the barrier, may exhibit altered gene expression or protein activity levels and thus contribute to the overall dysfunction of the BBB. In this study our aims were to identify human brain ECs genes that play a key role in the different stages of hypoxia and may serve as potential therapeutic targets, and to specifically investigate the effect of hypoxia and reoxygenation on MMPs and TJs proteins.  In order to achieve these goals  primary human cerebral ECs (HCEC) and an EC line derived from human brain microvascular ECs (HBMEC), were exposed to hypoxia for various time periods (3-24 hours) followed by reoxygenation. Expression analysis was conducted using GeneChip microarrays combined with real time PCR and protein expression studies. Assessment of cellular response to hypoxia included cell viability and endothelial layer permeability. We observed increased cells monolayer permeability in response to hypoxia and a decrease in claudin-5 gene expression. However we were not able to detect protein levels of claudin-5 in both cells culture. In addition, MMPs gene expression profile was different in HCEC vs. HBMEC in response to hypoxia, while their corresponding endogenous inhibitors TIMP-2 and TIMP-1 gene expression did not appear to be affected by hypoxia in both cells culture. Genechip expression data analysis from both HBMEC and HCEC indicated that their overall gene expression patterns are distinct, at normoxia conditions as well as under hypoxia. Nevertheless, we identified a set of genes that appears to be similarly regulated by exposure to hypoxia, in both the primary brain ECs as well as the EC cell line. These genes include BCL2/adenovirus E1B 19kDa interacting protein 3 (BNIP3), vascular endothelial growth factor (VEGF), Adrenomedullin (ADM) and solute carrier family 2 (facilitated glucose transporter) member 3 (SLC2A3) also known as glucose transporter 3 (GLUT3).