| IntroductionMatrix metalloproteinases ( MMPs) play a major role in atherosclerosis, restenosis after angioplasty and vein - graft stenosis by remodelling the extracellular matrix. MMPs together can catalyse the complete destruction of interstitial collagen, which is the main component of fibrous caps responsible for their tensile strength. Loss of collagen leads to structural weakness and less resistance to the mechanical stresses imposed during systole, which results in vulnerability to rupture. Expression of MMPs - 1, - 3 and - 9 is upregulated in cells present in atheromas, including VSMCs, and macrophages, but is low in the walls of normal arteries.Hydroxymethylglutaryl coenzyme A ( HMG - CoA) reductase inhibitors ( statins) are widely used to treat hyperlipidaemia; their use is associated with significant reduction of adverse coronary events, including myocardial infarction, and a marginal regression of plaque size. Furthermore, recent studies, both in vitro and in vivo, have suggested that the beneficial effects of statins may extend to mechanisms beyond cholesterol reduction. These pleiotropic effects of statins are mediated by their ability to block the synthesis of isoprenoid intermediates , which serve as lipid attachments for a variety of intracellular signalling molecules, especially Rho - family small GTP - binding proteins, whose proper membrane localization and function are dependent on isoprenylation.. Recently, studies demonstrated that statins reduced MMP -9 secretion by macrophages and MMP - 1 secretion from vascular endothelial cells. If these effects were more general to other MMP family members and other plaque resident cells they might have an important role in plaque stabilisation. We therefore investigated whetherstatins modulate MMP - 1, -2, -3 and -9 expression in cultured rabbit and human VSMCs and foam cells elicited in cholesterol - fed rabbits.MethodReagentsSheep polyclonal and - rabbit MMP - 1 and MMP - 3 antibodies were a generous gift from Dr G. Murphy, University of East Anglia, Norwich, UK. Mouse anti human MMP -1 antibody was purchased from CHEMICON INTERNATIONAL, sheep anti human MMP - 3 antibody was purchased from Binding Site company. Human recombinant IL - la and Human recombinant PDGFBB were purchased from R & D System. Cerivastatin from Bayer, Uk, Simvastatin from Merck Research Laboratories, USA. All other reagents were purchased from Sigma Chemical Company unless otherwise stated.Tissue CulturePrimary cultures of human saphenous vein and rabbit aortic smooth muscle cells were prepared by modifications of the explant technique, as previously described in detail. Explants were maintained in complete medium composed of DMEM containing penicillin -streptomycin (100 Units/ml and 100 jig/ml, respectively) , 8 mm L - glutamine and 15% foetal bovine serum (FBS, Advanced Protein Products, UK), cells were subcultured by trypsin/EDTA treatment. Cells between passage one to three were plated at a density of 2+10 cells/ well into 6 - well culture plates for zymography and western blotting or 1+ 106 cells/ 75 cm2 flasks for EMSA or UNA studies. For all experiments, sub - confluent cells were rendered quiescent by incubation in serum - free DMEM supplemented with 0. 25% (v/v) lactalbumin hydrolysate ( Gibco BRL, Paisley, UK) for 3 days. Cultures were then exposed to fresh serum -free medium containing the appropriate concentration of the agent under investigation for suitable hours.Rabbit experimental foam cells were isolated from subcutaneous granulomas of cholesterol - fed New Zealand White rabbits as previously described. Briefly, rabbits began a 1 % cholesterol diet 2 weeks before implantation of 2 to 6 polyu-rethane sponges (Baxter Scientific) under the dorsal skin. Sponges remained in place for 4 to 5 weeks to allow macrophage accumulation while the animal remained on a 1 % cholesterol diet throughout. The recovered sponges were gently squeezed over sterile test tubes, and the exudates were layered onto a discontinuous metrizamide...
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