M.Sc Thesis

M.Sc StudentMarom Barak
SubjectThe Role of Hypoxia in the Regulation of Fibronectin
Fragmentation and MMPs Expression in Human
DepartmentDepartment of Medicine
Supervisors PROFESSOR EMERITUS Haim Bitterman


Macrophages are pivotal cells in the inflammatory process, that migrate from the blood vessel, through the extracellular matrix (ECM) into the inflammatory site by secreting proteases, such as the matrix metalloproteinases (MMPs), that degrade various components of the ECM. For example, MMP-2 and MMP-9 can degrade gelatin and fibronectin (FN). During their migration macrophages encounter gradients of various cytokines (e.g. TNFa and IL-4), chemokines and hypoxia, which make up the inflammatory microenvironment.  Our aim was to examine the effects of hypoxia on the secretion of MMPs, on FN fragmentation and on the relationship between FN fragments and MMPs. The U937 monocytic cells were incubated in normoxia or hypoxia for 48 hours in the presence or absence of TNFa, IL-4 or their combinations. TNFa induced the secretion of MMP-2 (by 3 fold), MMP-9 (by 10 fold) and FN fragmentation and IL-4 inhibited this induction. Hypoxia and native FN inhibited the TNFa induced MMP-9 secretion, while fragmented FN had an antagonistic effect. TNFa affected MMP-9 transcriptionally, while the effect of both hypoxia and FN was post-transcriptional. Use of inhibitors of signaling pathways demonstrated that TNFa mediated its effect through JNK, native FN mediated effects through the a4b1 and a5b1 integrins and PKC and hypoxia mediated effects through PKC and ERK1/2. Confocal microscopy revealed that hypoxia reduced microtubuli structure and inhibited the trafficking of secretory vesicles containing  MMP-9. We conclude that the migration of macrophages to the inflammatory site, which is supported by secretion of MMP-9, is regulated by the intricate relationship between cytokines, hypoxia and the ECM.