M.Sc Thesis

M.Sc StudentPinchevsky Sigalit
SubjectThe Effects of Hypoxia on Macrophage Activation via
Trafficking of Secreted Proteins: Involvement
of the Cytoskeleton
DepartmentDepartment of Medicine
Supervisors ASSOCIATE PROF. Nitza Lahat
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Areas of ischemia and hypoxia (low oxygen tension) characterize inflamed tissues, due to the occlusion of the local blood supply, extensive vasodilatation or the inability of local vessel growth to keep pace with the increased metabolic demands of cells in that area. Monocytes and macrophages play a central role in this process. In order to enter the inflammatory site in the tissue; monocytes/macrophages have to degrade the ECM, a process which is mediated by various proteases, including matrix metalloprteinases (MMPs). MMPs are secreted from different cells, including monocytes/macrophages. Two of the MMPs, MMP-2 and MMP-9, degrade collagen IV, a main component of the ECM, and its denatured form gelatin and therefore called gelatinases. MMPs are tightly regulated by their secreted endogenous inhibitors, (tissue inhibitor of MMPs). TIMP-2 directly modulates the activity of MMP-2, and indirectly that of other MMPs, such as MMP-9.

Our first aim was to evaluate the effect of hypoxia on the secretion of pro-inflammatory (e.g TNF, NO) and migration regulatory (MMP-2, MMP-9 and TIMP-2) factors from monocytes/macrophages cell lines. Based on the results obtained in the first part of the study, our second aim was to explore the effect of hypoxia on the traffic and secretion of TNF, in a complex inflammatory environment, which includes the pre- activation of the cells.

Human U937 and THP-1 monocytes and mouse RAW 264.7 macrophage were incubated in normoxia or hypoxia in the presence or absence of LPS, which stimulates gram negative bacteria infection and accumulation of MMP-2, MMP-9, TIMP-2, NO and TNF in the supernatants was measured.

In the second part, human monocyte cell lines were exposed to prior activation by LPS or IFN in normoxia followed by incubation in normoxia or hypoxia in the presence or absence of LPS.

Our results show that hypoxia, in the context of inflammatory microenvironment (LPS), can restrain the pro-inflammatory response and partially direct it towards pro-angiogenic and/or anti-inflammatory pathways. In this study, this is demonstrated by the hypoxia-induced reduction of NO and TNF, which in low concentrations are not cytotoxic, but rather pro-angiogenic. Moreover, hypoxia, via down regulation of MMP-2 and MMP-9 secretion from monocytes/ macrophages may lead to autocrine arresting effects of these cells in the inflammatory/hypoxic site.

In addition to its known effects on gene transcription, hypoxia influences the post- translational regulation of proteins. In this study this effect is demonstrated by altered intracellular accumulation, trafficking and lysosomal degradation of TNF.