טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHazan Brill Roni
SubjectAnalysis of Genes Involved in the Response to Hypoxia in
Human Brain Endothelial Cells
DepartmentDepartment of Medicine
Supervisor Professor Ariel Miller
Full Thesis textFull thesis text - English Version


Abstract

The blood-brain barrier (BBB) is composed of brain capillary endothelial cells (ECs) with tight junctions providing a critical barrier to maintain brain homeostasis. Brain ECs differ from other peripheral ECs in various structural properties. However, most studies on EC properties, and specifically their response to hypoxia, employ peripheral models and/or non-human tissue.

Dysregulation of the BBB occurs in a variety of central nervous system pathologies/diseases. A significant element of these diseases is hypoxia, a reduction in partial oxygen pressure. Following hypoxia, changes in ECs properties occur and determine the cellular fate towards survival or death. The post-hypoxia cascade of molecular events in human brain ECs is scarcely understood, and has relevance to different neurological disorders. 

In this study our aims were to identify human brain ECs genes that play a key role in the different stages of hypoxia, and to specifically investigate the differences between peripheral and brain ECs. We sought to investigate the brain EC response to different pharmacological interventions and to characterize the time period of hypoxia exposure following which the brain ECs can no longer be salvaged from cell death.

In order to achieve these goals Human Brain Microvascular Endothelial Cells (HBMEC) and human umbilical EAhy 926 ECs were exposed to hypoxia for various time periods. Assays to evaluate the effect of hypoxia included cell proliferation, wound assay and cell viability. Gene expression analysis of HBMEC following hypoxia was performed using Affymetrix GeneChip microarrays.

Significant changes in gene expression pattern were detected following exposure to hypoxia. Validation of 23 differentially expressed genes was performed by RT-PCR, 11 of them were new in the hypoxic response, and six genes were analyzed at the protein level using western blot analyses. Notably, a distinct pattern of response was observed following hypoxia in brain ECs versus peripheral ECs at the physiological level, with peripheral ECs being more resistant to the hypoxic damage than brain ECs.

Additionally, we found that cell death in response to hypoxia can be prevented by re-oxygenation of the cells, while cell proliferation was more difficult to restore. Orchestrating those different responses at the different time points might help in finding the truly optimal 'therapeutic window' for stroke patients. We also found that the drug Rasagiline, known for its neuroprotective properties, and its derivatives, can enhance the brain ECs migration capacity under hypoxia, suggesting they may aid restoration of the disrupted BBB in clinical conditions.