Abstract
Over expression of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in vascular smooth muscle cells (VSMCs) induces apoptosis and reduces neointima formation occurring after saphenous vein interposition grafting or coronary stenting. In studies to address the mechanism of TIMP-3-driven apoptosis in human VSMCs we find that TIMP-3 increased activation of caspase-8 and apoptosis was inhibited by expression of Cytokine response modifier A (CrmA) and dominant negative FAS-Associated protein with Death Domain (FADD). TIMP-3 induced apoptosis did not cause mitochondrial depolarisation, increase activation of caspase-9 and was not inhibited by over-expression of B-cell Lymphoma 2 (Bcl2), indicating a mitochondrial independent/type-I death receptor pathway. TIMP-3 increased levels of the First Apoptosis Signal receptor (FAS) and depletion of FAS with shRNA showed TIMP-3-induced apoptosis was FAS dependent. TIMP-3 induced formation of the Death-Inducing Signalling Complex (DISC), as detected by immunoprecipitation and by immunofluorescence. Cellular-FADD-like IL-1 converting enzyme-Like Inhibitory Protein (c-FLIP) localised with FAS at the cell periphery in the absence of TIMP-3 and this localisation was lost on TIMP-3 expression with c-FLIP adopting a perinuclear localisation. Although TIMP-3 inhibited FAS shedding, this did not increase total surface levels of FAS but instead increased FAS levels within localised regions at the cell surface. A Disintegrin And Metalloproteinase 17 (ADAM17) is inhibited by TIMP-3 and depletion of ADAM17 with shRNA significantly decreased FAS shedding. However ADAM17 depletion did not induce apoptosis or replicate the effects of TIMP-3 by increasing localised clustering of cell surface FAS. ADAM17-depleted cells could activate caspase-3 when expressing levels of TIMP-3 that were otherwise sub-apoptotic, suggesting a partial role for ADAM17 mediated ectodomain shedding in TIMP-3 mediated apoptosis. We conclude that TIMP-3 induced apoptosis in VSMCs is highly dependent on FAS and is associated with changes in FAS and c-FLIP localisation, but is not solely dependent on shedding of the FAS ectodomain.
Highlights
The mechanisms that regulate vascular smooth muscle cell (VSMC) proliferation, migration and extra-cellular matrix (ECM) invasion are key to understanding their role in the development of vascular pathology including atherosclerosis, neointima formation and restenosis [1, 2]
Apoptosis of human VSMCs (hVSMCs) was significantly inhibited by DN-First Apoptosis Signal receptor (FAS)-Associated protein with Death Domain (FADD) or Cytokine response modifier A (CrmA), but not B-cell Lymphoma 2 (Bcl2) (Fig 2B and 2C)
Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) overexpression shows potential as a therapeutic agent in human vascular disease [6], no studies have documented the mechanism of apoptosis in human vascular smooth muscle cells
Summary
The mechanisms that regulate vascular smooth muscle cell (VSMC) proliferation, migration and extra-cellular matrix (ECM) invasion are key to understanding their role in the development of vascular pathology including atherosclerosis, neointima formation and restenosis [1, 2]. TIMP-3’s effectiveness is likely due to its combined ability to inhibit metalloproteinases (MPs), cause apoptosis and decrease proliferation in VSMCs [12, 13] and a variety of tumour-derived cell lines [9, 10, 14, 15]. The type II pathway is death receptor dependent, yet subsequent caspase-8 activation cannot efficiently activate effector caspases. Inhibition of TIMP-3 induced apoptosis with adenoviral expression of dominant-negative (DN) FAS-Associated protein with Death Domain (FADD), Bcl or Cytokine response modifier A (CrmA) blocked caspase-8 cleavage, subsequent mitochondrial depolarisation and caspase-9 activation in these cells [13]. We show that TIMP-3-induced apoptosis in human VSMCs (hVSMCs) is via a type-I death receptor pathway that is highly dependent on FAS (CD95/Apo-1/ TNFRSf6). Depletion of A Disintegrin And Metalloproteinase 17 (ADAM17), which is potently inhibited by TIMP-3 [18, 19] and is required for FAS shedding, does not replicate the apoptotic effects of TIMP-3, implicating ADAM17-independent effects of TIMP-3 on cellular apoptosis
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