Inhibition of Late Vein Graft Neointima Formation in Human and Porcine Models by Adenovirus-Mediated Overexpression of Tissue Inhibitor of Metalloproteinase-3

Abstract

Background-Autologous saphenous vein coronary artery bypass graft surgery is complicated by late graft failure due to neointima formation and subsequent atherosclerosis. Growth factors and metalloproteinases (MMPs) act in concert to promote neointima formation. Tissue inhibitor of metalloproteinase-3 (TIMP-3), an extracellular matrix-associated MMP inhibitor, uniquely promotes apoptosis of isolated vascular smooth muscle cells. Here, we overexpressed TIMP-3 at the luminal surface of human saphenous veins before organ culture and in pig saphenous veins before interposition grafting into carotid arteries in vivo to assess neointima formation. Methods and Results-In both models, high TIMP-3 immunoreactivity occurred in the luminal and upper medial extracellular matrix after adenovirus delivery. MMP activity measured by in situ zymography was reduced throughout the veins, confirming a bystander effect. By use of 3 independent techniques, apoptosis levels in the neointima and medial layer were significantly elevated by TIMP-3 overexpression. Neointima formation was reduced by 84% in 14-day human organ cultures and by 58% in 28-day pig vein grafts (both PϽ0.05). In contrast, TIMP-2 overexpression had no effect on neointima formation in vivo. Conclusions-Our results highlight the potential therapeutic benefit for TIMP-3 overexpression to reduce neointima formation associated with late vein graft failure. (Circulation. 2000;101:296-304.)

Ultrasound-mediated delivery of TIMP-3 plasmid DNA into saphenous vein leads to increased lumen size in a porcine interposition graft model

“…It has been reported that transient overexpression of TIMP-3 using an adenoviral vector reduces adverse SVG remodelling, focusing upon effects on intimal thickness, whereas our study showed particularly striking effects upon lumen and total vessel areas. 6 TIMP-3 is particularly suited to SVG gene therapy as it is a secreted protein that binds extracellular matrix (and is thereby maintained locally) and exhibits a prolonged bystander effect to induce apoptosis of surrounding VSMC. 6 These properties make TIMP-3 a particularly attractive gene for nonviral approaches that are generally less efficient than viral vectors.…”

“…6 TIMP-3 is particularly suited to SVG gene therapy as it is a secreted protein that binds extracellular matrix (and is thereby maintained locally) and exhibits a prolonged bystander effect to induce apoptosis of surrounding VSMC. 6 These properties make TIMP-3 a particularly attractive gene for nonviral approaches that are generally less efficient than viral vectors. It has recently been confirmed that inducing VSMC apoptosis immediately after arterialization has beneficial effects on SVG remodelling measured later at 28 d, as adenoviral overexpression of another proapoptotic gene, p53, also increased lumen size in arterialized SVG, and these phenotypic changes were sustained well beyond the duration of exogenous transgene expression.…”

“…4,5 It has previously been shown that ex vivo adenoviral gene transfer of tissue inhibitor of metalloproteinase 3 (TIMP-3) reduces adverse remodelling in porcine SVG. 6 Successful nonviral delivery of TIMP-3 is therefore highly appealing. The present study investigates whether ex vivo gene transfer of TIMP-3 plasmid using microbubble-enhanced ultrasound also influences arterialization-induced remodelling of porcine SVG.…”

“…Porcine VSMC were transfected with pCI-lacZ or pCI-TIMP-3 (pCI backbone from Promega) with or without USE as described above. TIMP-3 expression was assayed after 48 h in control and plasmidtransfected cultures by Western blotting 20 mg total cell protein using a mouse monoclonal anti-human TIMP-3 primary antibody that detects human and porcine TIMP-3 6 (1:500; TCS Cellworks Ltd, Buckingham, UK) with a goat anti-mouse IgG-horseradish peroxidase secondary antibody (1:1000; Dako, High Wycombe, UK). Lane a, recombinant human TIMP-3 (23 kDa, Sigma); b, untransfected VSMC; c, USE+BR14 (50% (v/v)); d, lacZ plasmid; e, lacZ plasmid+USE+BR14 (50% (v/v)); f, TIMP-3 plasmid; g, TIMP-3 plasmid+USE+BR14 (50% (v/v)).…”

Post-intervention vessel remodeling

“…Vein graft stenosis has similar features to neointimal hyperplasia in arteries, and thus the same therapeutic approaches may apply. Suppressing SMC migration and proliferation by inhibiting MMPs with TIMP-1, TIMP-2 or TIMP-3 40 has been shown to inhibit neointima formation. TIMP-2 reduces neointima formation in human saphenous veins and TIMP-3 decreased stenosis in human and porcine veins.…”