Ph.D Thesis

Ph.D StudentEngelmayer Miri
SubjectEffects of Hypoxia on the Expression and Activity of TIMP-2
in Macrophages
DepartmentDepartment of Medicine
Supervisors ASSOCIATE PROF. Nitza Lahat


Tissue reduction in oxygen tensions (hypoxia) characterizing tumors, ischemia, and inflammation, has a role in recruiting monocytes/macrophages, immobilizing them at the hypoxic site and altering their function. We evaluated whether hypoxia, as a single stimulus, could activate monocytes and macrophages towards either a pro-inflammatory, classical activation or an anti-inflammatory, alternative activation. Specifically, we focused on the endogenous tissue inhibitor of matrix metalloproteinases-2 (TIMP-2), that is constitutively expressed and involved in cell migration. Peripheral blood monocytes or the human monocytic cell lines U937 and THP-1 were used, or subjected to PMA (20ng/ml) for 2-4 days to induce their maturation into macrophages, prior to their exposure to normoxia or hypoxia. Prolonged hypoxia (up to 72 h) did not affect cell viability, or induce secretion of either the classic molecules NO and TNFα, or the alternative cytokines IL-10 and AMAC-1. TIMP-2 accumulated in normoxic supernatants over time, but hypoxia inhibited this accumulation by 3- and 2-fold in monocytes and macrophages, respectively. In monocytes the hypoxia-induced reduction was regulated transcriptionally, as TIMP-2 mRNA steady-state levels were reduced, and binding of the transcription factor SP-1 to the TIMP-2 proximal promoter was abolished, probably due to its increased phosphorylation. In macrophages, hypoxia post-translationally increased the intracellular levels of TIMP-2 in the secretory vesicles, and prevented its secretion, probably by inhibiting their trafficking along the microtubule fibers. Whereas macrophages remained immobilized both in normoxia and hypoxia, TIMP-2 was not directly involved in monocyte migration, as hypoxia reduced their migration, anti-TIMP-2 had no additional effect and the recombinant protein further inhibited this migration. Thus, we speculate that the hypoxia-induced reduction of TIMP-2 levels may have an MMP-independent, anti-angiogenic role. In conclusion, this research demonstrates that monocytes and macrophage both reduce TIMP-2 secretion levels in hypoxia, but utilize different regulatory mechanisms to that end.