טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentRosenzweig Doron
SubjectThe Molecular Mechanisms Regulating the Decrease in Tissue
Inhibitor of Metrix Metalloproteinase 2 (TIMP 2)
by Hypoxia in Human Endothelial cells
DepartmentDepartment of Medicine
Supervisors Mr. Jacob Bornstein
Professor Nitza Lahat
Dr. Michal Rahat


Abstract

Hypoxia, or reduction in oxygen tension, is a strong inducer of angiogenesis, the process of sprouting of new blood vessels. During angiogenesis, endothelial cells (EC) secrete matrix-metalloproteinases (MMPs) which degrade the basal membrane and the extracellular matrix and allow EC movement. MMPs are tightly regulated, including by their endogenous inhibitors TIMPs, which bind to their catalytic site. Among them, TIMP-2 is considered the primary inhibitor of MMP-2.

The aim of this study was to evaluate the effects of hypoxia on the expression of TIMP-2 in a human endothelial cells. The endothelial line EaHy926 and primary endothelial cells (HUVEC) were exposed to normoxia (21% O2) or hypoxia (<0.3% O2) and the expression of gelatinases (MMP-2 and MMP-7) and TIMP-2 were assessed. After 48 hours hypoxia increased by 40% the accumulation of active MMP-2, but decreased by 2-fold MMP-7 and TIMP-2 protein and mRNA as determined by zymography, western and northern blot analysis. cAMP, which is involved in TIMP-2 regulation, did not mediate the effects of hypoxia, as it accumulated equally in normoxia and hypoxia. Furthermore, the phosphodiesterase inhibitor IBMIX, decreased TIMP-2 protein and mRNA accumulation equally in normoxia and hypoxia. Binding of transcription factors to TIMP-2 promoter was evaluated by EMSA and showed two complexes. While the heavier complex was similar in normoxia and hypoxia, the lighter complex accumulated 2-fold in hypoxia and was composed of AP-1, SP-1and NFkB/NF-IL6. In an in vitro wound assay, where a confluent endothelial cell culture was scratched in the absence or presence of TIMP-2 antibody or recombinant TIMP-2, hypoxia increased the number of cells per mm2, the migration distance, the length of the cells but not their area, and their orientation - all in a TIMP-2 dependent manner.

Thus, hypoxia is pro-angiogenic as it decreases MMP-7 and TIMP-2, induces active MMP-2 and controls morphological changes of endothelial cells.