Erforschung der Grundlagen von Entzündungsprozessen
Final Report Abstract
In the course of a two year project at Queen’s University, Kingston, Canada, the implications of a dual lipoxygenase knockout were investigated in an in vitro model (peritoneal macrophages) as well as in vivo models (atherosclerosis, peritonitis) of apoE-deficient mice. These mice represent an accepted animal model for the human atherosclerotic disease. Shedding light on the mechanisms of atherosclerosis and the involvement of both lipoxygenases in this disease was one main goal of this study. The second goal comprised the analysis of in vitro functions of peritoneal macrophages, the only important inflammatory cell type that expresses both 5- and 12/15-lipoxygenase. Here, we were especially interested in how the arachidonic acid metabolism was altered under conditions where the major eicosaniod-producing enzymes were selectively (genetically or pharmacologically) inhibited. Lipoxygenase enzymes catalyze the conversion of arachidonic acid (AA) into biologically active lipid mediators. Two members, 12/15-LO and 5-LO, regulate inflammatory responses and have been studied for their roles in atherogenesis. Both 12/15-LO and 5-LO inhibitors have been suggested as potential therapy to limit the development of atherosclerotic lesions. In this study, we used 12/15-LO and 5-LO-deficient mice that were on an apolipoprotein E (apoE) background to study the impact of dual LO blockade in atherosclerosis and inflammation. Examination of AA conversion by stimulated macrophages from dual LO- deficient mice revealed extensive accumulation of AA, with virtually no diversion into the most common cyclooxygenase (COX) products measured (prostaglandin E2 and thromboxane B2). Instead, the COX-1 by-products 11-hydroxy-eicosatetraenoic acid (HETE) and 15-HETE were elevated. The interrelationship between the two LO pathways, in combination with COX-1 inhibition (SC-560), also revealed striking patterns of unique substrate utilization. 5-LO and dual LO-deficient mice exhibited an attenuated response to zymosan-induced peritoneal inflammation, emphasizing roles for 5-LO in regulating vascular permeability. We observed gender-specific attenuation of atheroma formation at 6 months of age at both the aortic root and throughout the entire aorta in chow-fed female dual LO- deficient mice. These results shed new light on the gender-specific implication of lipoxygenases in the pathophysiology of vascular diseases and will likely induce a series of future studies addressing the mechanisms of these effects. In summary, the most interesting in vitro results of this study are the effects of altered eicosanoid biosynthesis in cells where either the 12/15-LO, the 5-LO, or the COX pathway is inhibited. This simplistic experimental scenario could possibly model the situation of macrophages in vivo, when selective LO or COX inhibitors were given to patients. From our results, we can hypothesise how eicosanoid levels would be altered, and evaluate potential consequences of this effect. The most intriguing in vivo results refer to the gender dependency of LO effects on the development of atherosclerosis. Our data suggest that the application of LO inhibitors in atherosclerosis, which is still in discussion, would likely work only in female but not in male patients. Future studies will have to address the mechanisms of these sex-specific effects, but our results provide a starting point for these studies.
