Both accelerating the reendothelialization of the vessel after balloon injury and restoring its function by replacing nitric oxide have been used to ameliorate restenosis. VEGF is a potent and specific m itogen for endothelial cells. The recom binant VEGF protein has been applied to rat carotid arteries after injury and been show n to reduce neointim al form ation and accelerate the restoration of endothelial co n tin u ity (Asahara et sL 1995). Transfer of the gene encoding VEGF has not how ever been used in this setting, but the results obtained in the rat model have contributed to the gene transfer w o rk w ith VEGF currently being applied to peripheral vascular disease both in angiogenesis and restenosis (Isner e t al. 1996a; Isner e t ai. 1996b). Restenosis is likely to be triggered by the early release of m itogens and chem otactic factors after endothelial denudation. It has been suggested th a t under normal conditions the endothelium may supply inhibitory factors to keep this process in check (von der Leyden e t ai.
1995). The ability of nitric oxide (NO) to inhibit platelet aggregation and VSMC m igration and proliferation in vitro implicates it in this process. The firs t dem onstration of ability to transfer genes w ith potential therapeutic use to the vasculature used the HVJ-liposome com plex of the gene for endothelial cell nitric oxide synthase (ecNOS) to overexpress nitric oxide synthase and hence nitric oxide after balloon injury in the rat (von der Leyden e t ai. 1995). The investigators were able to dem onstrate restored NO production and vascular reactivity, in addition to a 70 % reduction in neointim al form ation.
Both antisense and gene transfer techniques have been used w ith success to suppress neointim al form ation in animal models of angioplasty restenosis in normal and atherosclerotic vessels. A fte r the failure of pharmacological agents such as angiotensin converting enzyme inhibitors to alter restenosis in man, despite favorable results in small animals, more groups are now testing gene transfer methods in porcine peripheral and coronary models. A t present, no one gene or antisense molecule has been show n to be
superior to another, and apart from marker gene studies com paring vectors or delivery devices there is a paucity of com parative data. It seems possible th a t a com bination of several strategies may be needed to overcom e the considerable biological redundancy in the restenotic cascade. I have therefore chosen to investigate w hether containm ent of smooth muscle cells and g ro w th factors w ithin the m atrix of the arterial media by the overexpression of TIMP1 w ill alter the response to vascular injury.
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h e s is.
M atrix m etalloproteinase enzymes are know n to be upregulated after vascular injury and may be an essential step in liberating sm ooth muscle cells from the arterial media to migrate and proliferate form ing a neointima and narrowing the vessel. The aim of this project was to study the effects of inhibiting m atrix metalloproteinase a c tiv ity in vitro in sm ooth muscle cells and in vivo at the site of balloon catheter injury (utilizing a rat model of angioplasty) by system ic adm inistration of a pharm acological inhibitor and by local overexpression of tissue inhibitor of metalloproteinases 1 (TIMP1) using a replication deficient adenoviral vector. These studies were expected to give insights into the possible physiological functions of the MMPs and TIMPs in vivo in the arterial w all. The principcu^ aim was to investigate the potential for MMP inhibition, via effects on m igration and proliferation of sm ooth muscle cells, to prevent the developm ent of a neointim al lesion after vascular injury.
2 .1 Hy p o t h e s is
The hypothesis investigated in this thesis is th a t inhibition of MMP a ctivity by pharm acological means or by gene transfer of human TIMP1 w ill inhibit intimai hyperplasia after vascular injury.
2 . 2 Sp e c if ic a im s
1) Assessm ent of the pharm acokinetics and effects on lesion developm ent of a pharm acological inhibitor of the metalloproteinases in vivo in the rat carotid balloon injury model.
2) Assessm ent of the efficiency of gene transfer using a firs t generation adenoviral vecto r encoding a marker gene, 15-Galactosidase, in primary
and SV40 transform ed sm ooth muscle cells and optim ization of in vivo
gene delivery techniques.
3) Assessm ent of the efficie n cy of gene transfer using fo u r herpes virus vectors, encoding 15-Galactosidase, in prim ary and SV40 transform ed sm ooth muscle cells and investigation of the effects of in vivo gene delivery of one of the vectors.
4) Developm ent of a novel gene transfer vecto r encoding human tissue inhibitor of m etalloproteinase 1 (A v I .TIMP1 ) and assessment of its ability to produce fun ction al protein in vitro.
5) Investigation o f the e ffe ct of overexpression of TIMP1 using the vector, Av1 .TIM P1, on sm ooth muscle cell m igration and proliferation in vitro.
6) Investigation of the ability of the vecto r A v I.T IM P I to express TIMP1
in vivo in the injured rat carotid and assessment of its effects on neointim al developm ent, sm ooth muscle cell proliferation and m igration, medial cell numbers, and m a trix com position in com parison w ith a vector encoding 8-Galactosidase.