Ribozyme-Mediated Inhibition of Expression of Leukocyte-type 12-Lipoxygenase in Porcine Aortic Vascular Smooth Muscle Cells

Abstract

Activation of a leukocyte-type 12-lipoxygenase (12-LO) has been proposed to be an important mechanism for angiotensin II– and glucose-induced vascular smooth muscle cell growth. Currently, no specific pharmacological inhibitors for the leukocyte-type 12-LO are available to test this hypothesis. We have therefore designed a chimeric DNA-RNA hammerhead ribozyme to produce cleavage at the first GUC sequence at nucleotide 7 of porcine leukocyte 12-LO mRNA. The ribozyme was tested in vitro with a 206-base 12-LO mRNA as substrate. We observed that the ribozyme specifically and dose-dependently cleaved porcine leukocyte 12-LO mRNA at the predicted site under physiological temperature. Furthermore, we also efficiently delivered the ribozyme into porcine aortic vascular smooth muscle cells by transfection with cationic liposomes. The ribozyme caused a dose-dependent decrease in levels of porcine leukocyte-type 12-LO mRNA in these cells and was more potent than an antisense oligonucleotide directed against porcine leukocyte 12-LO. The 12-LO ribozyme also attenuated 12-LO protein levels in the cells. The action of the ribozyme was primarily a result of its catalytic activity, since a modified ribozyme that lacks catalytic activity showed reduced effects. This represents the first ribozyme directed against a mammalian LO pathway. These results demonstrate the potential utility of new ribozyme technology to generate novel agents for gene modulation experiments to modify the development or progression of vascular disease in humans.

Role of 12-lipoxygenase and oxidant stress in hyperglycaemia-induced acceleration of atherosclerosis in a diabetic pig model

“…Thus 12-LO mRNA and protein expression were markedly increased in the neointima of balloon-injured rat-carotid arteries [17]. Furthermore, treatment of these injured rat-carotid arteries with a pharmacological LO inhibitor [17,35], or a specific molecular LO inhibitor, namely a 12-LO ribozyme [36,37] could significantly reduce the extent of neointimal thickening in this model of restenosis. The direct in vivo significance of 12-LO activation in atherosclerosis was recently demonstrated by studies showing that cross-breeding of a leukocyte 12-LO knock-out mouse with the atherosclerosis-susceptible apoE-deficient mouse dramatically reduced lesion development in the latter [38].…”

Specific monocyte adhesion to endothelial cells induced by oxidized phospholipids involves activation of cPLA2 and lipoxygenase

“…The structure of the 12-LOX-hammerhead ribozyme has been described previously (22)(23)(24). Transfections of porcine aortic endothelial cells with ribozyme and monocyte adhesion assays were performed according to Patricia and coworkers (25).…”

Adenoviral Delivery of a Leukocyte-Type 12 Lipoxygenase Ribozyme Inhibits Effects of Glucose and Platelet-Derived Growth Factor in Vascular Endothelial and Smooth Muscle Cells

“…15 In the present study, we have packaged the ribozyme in an adenoviral vector to allow for enhanced transfer of the ribozyme into cells and to achieve longer expression compared with naked oligonucleotides. Previous studies have shown successful use of adenovirus to deliver functionally expressed ribozymes in vitro 16 and in vivo.…”