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Jian Qi, Bela Anand-Apte; Tissue Inhibitor of Metalloproteinase-3 (TIMP3) Induces Endothelial Apoptosis via MMP- and Caspase-Independent Mechanisms. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4592. doi: https://doi.org/.
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TIMP3 has been shown to induces apoptosis in certain non-endothelial cell types. Mutations in TIMP3 gene causes Sorsby fundus dystrophy (SFD, a rare, dominantly inherited, early onset macular degenerative disease. SFD has similar disease phenotypes to age-related macular degeneration (AMD) including increased accumulation of TIMP3 in Bruch’s membrane (BM) and an early phase of atrophy of the choriocapillaris, retinal pigment epithelium (RPE) and photoreceptor. To address the contribution of TIMP3 to the disease process, the apoptotic activity and pathways of TIMP3 were investigated in endothelial cells (ECs).
human RPE cells, monkey choroidal ECs, PAE/KDR cell line expressing wild type or SFD-S156C-TIMP3 and mouse aortic ECs derived from WT, heterozygous TIMP3+/S156 and homozygous TIMP3S156C/S156C mice were treated with recombinant adenoviruses carrying the wild-type (WT) TIMP3 or TIMP3 Cys1-Ser or serum deprivation. Apoptosis was analyzed by ApopTag direct kit, FACS or CasPASETM apoptosis assays. Immunoprecipitation and/or immunbotting were used to analyze FAK cleavage, levels of Bax and Bcl2 and FAK tyrosine phosphorylation and association with paxillin. Focal adhesion formation was determined by immunocytochemical staining.
Expression of TIMP3 promoted apoptosis in ECs, and to a lesser extent, in RPE cells. It appears that TIMP3-induced apoptosis is independent of MMP inhibition. This is because 1) TIMP3 Cys1-Ser, devoid of inhibitory activity towards MMP2, promoted apoptosis, and 2) S156C-TIMP3, which has attenuated MMP inhibitory activity, promoted or even enhanced apoptosis. TIMP3-induced apoptosis was associated with activation of caspases, up-regulation of bax/Bcl2 ratio and inhibition of tyrosine phosphorylation of FAK, association with paxillin and focal adhesion formation on matrix. However, cleavage of FAK, a prominent caspase substrate, was not increased by TIMP3. Furthermore, either a polycaspase inhibitor or a specific inhibitor of caspase 3 or 8 were ineffective against TIMP3-mediated apoptosis and inhibition of focal adhesion formation.
TIMP3 has a direct effect on ECs by triggering apoptosis via MMP- and caspase-independent pathways, which likely underlies atrophy of the choriocapillaris and subsequent RPE or photoreceptor in AMD and SFD disease processes.
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