טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentRosental Alex
SubjectThe Effect of Hypoxia and Reoxygenation on the Expression
of MMP-2 and MMP-9 and Their Inhibitors TIMP-1
and TIMP-2 in Mice
Macrophages
DepartmentDepartment of Medicine
Supervisors Professor Emeritus Haim Bitterman
Professor Nitza Lahat
Dr. Michal Rahat


Abstract

Hypoxia provokes inflammatory responses. Macrophages secrete matrix metalloproteinases (MMPs) that degrade the

extracellular matrix (ECM) and enable their recruitment into inflammatory areas. TIMP-2 and TIMP-1 inhibit MMP-2 and MMP-9, respectively, and the ratio of MMPs/TIMPs determines ECM metabolism. Macrophages also secrete inflammatory mediators, including nitric oxide (NO), which is produced by the inducible isoform of nitric oxide synthase (iNOS). We found in the macrophage cell line RAW 264.7 that normoxia (21% O2, 5% CO2, 74% N2), hypoxia (<0.3% O2, 5% CO2, 95% N2) or hypoxia and reoxygenation, with or without IFNg (100U/ml), LPS (1mg/ml) or their combination, did not change the secretion of MMP-2 or MMP-9. In contrast, TIMP-2 was elevated by 40-fold (p<0.01) after 48 hours in hypoxia or 24 hours in hypoxia followed by reoxygenation. Similarly, hypoxia and LPS synergistically elevated TIMP-1 levels by 10-fold after 48h (P<0.01). Hypoxia did not change the expression of iNOS induced by IFNg, but inhibited iNOS activity. To determine if NO is involved in regulation of TIMP-2 by hypoxia, the cells were incubated in hypoxia (48 hours) with NO donors (NOC-18, GlycoSNAP), NO scavenger (PTIO), iNOS inhibitor (L-NIL) and superoxide scavengers (MnTBAP and Tempol). Whereas addition of iNOS inhibitor and superoxide scavengers did not affect TIMP-2 expression, NO donors elevated TIMP-2 levels, with a positive correlation between exogenous NO and TIMP-2 (R2=0.63, p<0.004). We suggest that hypoxia inhibits ECM degradation by increasing TIMP-2 and TIMP-1. TIMP-2 is regulated by hypoxia in an NO-independent manner, but exogenous NO may contribute to the effects of hypoxia.