M.Sc Thesis

M.Sc StudentDaniliuc Sharon
SubjectThe Effect of Hypoxia and Reoxygenation on the Expression
of Inducible Nitric Oxide Synthase (iNOS) in
Mouse Endothelial Cells and Macrophages
DepartmentDepartment of Medicine
Supervisors ASSOCIATE PROF. Nitza Lahat


Macrophages and endothelial cells are two key players in the inflammatory response, which accompanies ischemia and reperfusion (I/R). Hypoxia and Reoxygenation (H/R) are the main constituents of I/R and were used in this study to simulate the behavior of the two cell types in I/R.

Nitric oxide (NO) is a short-lived reactive nitrogen species (RNS) produced by three isoforms of nitric oxide synthase (NOS). It is produced in small amount by two constitutive isoforms in endothelial cells (ecNOS) and neurons (nNOS), and is induced (iNOS) by inflammatory mediators such as interferon g (IFNg) and lipopolysaccharide (LPS). iNOS may be induced in many cell types, including macrophages and endothelial cells. Tissue I/R alters the production of RNS, including NO. This may lead to changes in the inflammatory response that typicaly follows I/R. The molecular mechanisms causing the changes in the production of NO by the different isoforms in I/R and even more in H/R, have not been elucidated.

Our aim was to evaluate the effects of H/R on the expression and activity of iNOS in mouse endothelial cells and macrophages. We subjected the cells to normoxia (21% O2) or hypoxia  (<0.3% O2) for 2 or 24hr, without or with stimulation with IFNg (100 U/ml), LPS (1mg/ml) or their combination. Cells were reoxygenated by transferring them to normoxia for 2 or 24hr. Expression of iNOS was measured by western blot analysis, accumulation of nitrites, the stable product of NO, was determined by Griess reaction.

In the endothelial cell line bEND3 iNOS was induced in normoxia by the combined stimulation (x12-fold from control), while long hypoxia (24hr) reduced it’s induction by x4-fold from control, and reoxygenation restored it to normoxic levels. Hypoxia following the incubation in normoxia with the combined stimulation did not reduce iNOS expression. However, addition of the protein synthesis inhibitor cycloheximide, during prolonged hypoxia, prevented iNOS expression, suggesting that hypoxia inhibits its synthesis rather than enhances its degradation.  Supernatants removed from normoxic bEND3 cells showed basal nitrite secretion (20 mM), which was reduced during prolonged hypoxia and restored after reoxygenation. In rat primary aortic endothelial cells, the expression of iNOS was not reduced during hypoxia. Also,  nitrite levels in the supernatants were not detected in both basal and hypoxic conditions, although combined stimulation in normoxia resulted in its accumulation.

In the macrophage cell line RAW 264.7 and in primary rat macrophages, IFNg or the combined stimulation induced similar high levels of iNOS (x56-fold from control) in normoxia, hypoxia and hypoxia followed by reoxygenation. However, nitrite accumulation which amounted to 113 mM during normoxia in the presence of stimulator(s), was significantly reduced during hypoxia (p<0.01), and was restored only after prolonged reoxygenation. Since iNOS is active only as a homodimer, the effect of hypoxia on its dimerization was evaluated by gel filtration. No difference in the dimer percentage was observed among normoxic and hypoxic macrophages (33%, 36.5% respectively). However, the dimer separated from hypoxic cellular lysates did not produce NO despite addition of substrates (oxygen, arginine) and cofactores. Immunoprecipitation of iNOS-bound proteins revealed two protein bands in normoxia (130 kDA and 100 kDA), but only one in hypoxia (130 kDA). The bands were identified by mass spectroscopy as iNOS monomer (130 kDA) and a actinin 4 (100 kDA). It is suggested that in normoxia macrophage iNOS binds to a actinin 4, a protein known for its ability to anchor protein to the cytoskeleton. Lack of such binding, in hypoxia, may prevent critical compartmentalization of the enzyme, then leading to its inactivation. To summarize: iNOS expression and activity are differently regulated in endothelial cells and macrophages. In endothelial cells hypoxia inhibit iNOS production whereas in macrophages the enzyme is expressed but is inactive, possibly due to a defect in binding to a actinin 4.