Ribonucleotide Reductase Inhibitors Reduce Atherosclerosis in a Double-Injury Rabbit Model

Atheroproliferative disorders such as atherosclerosis are an important health problem and one of the leading causes of morbidity and mortality in the United States. Minimally invasive therapeutic procedures, including angioplasty with stent deployment, are used frequently for obstructive coronary artery disease. However, restenosis, a proliferative vascular response, is a common sequela to this procedure. The current study investigated the effect of inhibiting ribonucleotide reductase (RR), an enzyme necessary for cellular proliferation, in an attempt to ameliorate the proliferative response. Two RR inhibitors, didox and hydroxyurea, were chosen for their potent antiproliferative properties. Studies were carried out by using a double-injury rabbit model, in which endothelial denudation was followed by the administration of a high-fat diet. At 4 wk after initial endothelial denudation, the developing atherosclerotic lesion was subjected to transluminal balloon dilation to simulate clinical intervention with percutaneous transluminal angioplasty. The degree of restenosis and atheroproliferation was assessed at 8 wk. Histologic evaluation of the lesion demonstrated that treatment with didox and hydroxyurea significantly decreased lesion area and lumen loss. These results suggest that RR inhibition may be an effective new tool for the treatment of atheroproliferative disorders.Abbreviations: RR, ribonucleotide reductaseCoronary artery disease and atherosclerosis, in particular, are multifactorial processes that include numerous molecular and cellular cascades culminating in inflammatory and proliferative vascular responses involving the endothelium, vascular smooth muscle cells, and leukocytes. Among these responses, impaired endothelial function, manifested as impaired nitric oxide production, and increased signaling through reactive oxygen species have been recognized as critical components in the pathogenesis of atherosclerosis. The complex nature of the atherogenic process has hindered the development of animal models that mimic human atherosclerosis.²⁷Rabbits typically are used for studies of atherosclerosis because they are one of the few species that can develop atheromatous foci with many of the characteristics of human atherosclerotic lesions. Primary rabbit models of atherosclerosis include both genetically altered strains and models that are diet-induced. The lesions in atherosclerotic rabbits whose disease is induced through consumption of high-cholesterol diet exhibit several pathologic features associated with human lesions, including fatty streaks, accumulation of foam cells, and fibrous plaque formation. The atheromatous changes in this model manifest quickly, with marked lesion formation within 4 to 12 wk of initiation of a high-cholesterol diet.^19,28 Prolonged (8 mo or more) consumption of high-cholesterol diet causes lesions that are rich in smooth muscle cells and closely resemble human lesions. To induce more advanced lesions in these rabbits, consumption of a high-cholesterol diet can be combined with vascular endothelial denudation by using a single- or double-balloon injury.^16,26 Moreover, the double-balloon injury model closely approximates the clinical setting of balloon dilatation of a diseased atheromatous vessel.Currently, although percutaneous transluminal coronary angioplasty with stent deployment is the mainstay of treatment of obstructive coronary artery disease therapy, this procedure is plagued by a high incidence of restenosis, or vessel renarrowing, which is responsible for 30% to 40% of long-term failures.^1,2 Drug-eluting stents recently have come to the forefront as a promising treatment modality for restenosis, but some evidence suggests that the clinical benefits may be overestimated, given that drug-eluting stents have been implicated in causing late-developing fatal thrombosis.^14,17,18 Therefore, prevention of restenosis after successful percutaneous transluminal coronary angioplasty remains one of the most challenging tasks in the treatment of obstructive coronary artery disease, and alternative pharmacologic approaches are currently being pursued.Through a cascade of molecular events, the vascular trauma associated with percutaneous transluminal coronary angioplasty initiates vascular smooth muscle cells to undergo modulation from a contractile to a synthetic phenotype. Vascular smooth muscle cells proliferate in the tunica media and migrate to the tunica intima, resulting in intimal hyperplasia referred to as ‘neointimal formation.’^7,8,25,33 The result of neointimal formation constitutes restenosis. Pharmacologic agents that impede the proliferation and migration of vascular smooth muscle cells are being investigated to ameliorate this response.Ribonucleotide reductase (RR) is an enzyme that, when activated by a free-radical intermediate, catalyzes the conversion of ribonucleotides to deoxyribonucleotides. This reductive reaction is a rate-limiting step in the biochemical pathway leading to DNA synthesis and cell replication.^6,30,31 Hydroxyurea is a commercially available RR inhibitor that has been used for the treatment of various cancers. Didox is a more potent RR inhibitor than is hydroxyurea and has additional antiinflammatory and antioxidant properties.^{4,10,21,29,32} Recently the use of the RR inhibitor didox in a rat model of balloon-mediated carotid artery injury led to reduction of restenosis and cell cycle arrest.¹² Because cellular proliferation and migration are involved in the formation of atherosclerotic plaques, the present study uses histologic analysis and the measurement of vascular reactivity to investigate the role of the RR inhibitors hydroxyurea and didox in preventing the development of atherosclerotic lesions.