This project was funded by the University of the
This project was funded by the University of the Philippines Diliman through the Natural Sciences Research Institute with grant No. CHE-09-2-02.
Introduction There is compelling evidence for an anti-inflammatory effect of 12- and 15-lipoxygenases through the generation of lipid mediators involved in the resolution of inflammation . On the other hand, there is compelling evidence for a pro-atherosclerotic effect through the formation of oxLDL which accelerates foam cell formation and through its role in signalling of angiotensin II mediated mechanisms and vascular smooth muscle cell proliferation . Hence, the 12/15-lipoxygenases seem to be two-faced enzymes with an anti-inflammatory effect through lipid mediator production, and a pro-inflammatory and atherogenic effect through oxLDL formation and participation in signalling pathways . Animal models of atherosclerosis did not solve the question of whether the 12/15-lipoxygenase activity is pro- or anti-atherogenic because different animal models showed contrasting results , , , , , . Monocyte specific 15-lipoxygenase expression in transgenic rabbits reduced atherosclerosis and supported the anti-inflammatory role of the 15-lipoxygenase , . Similarly, an extensive mouse study applying several overexpressing and knockout mouse lines showed an atheroprotective effect of the 15-lipoxygenase under a normal diet . However, conditional macrophage-specific and general disruption of the mouse homolog 12-lipoxygenase gene reduced atherosclerosis , , while overexpression of human 15-lipoxygenase in vascular endothelium enhanced atherosclerosis in other mouse strains on a cholesterol rich diet . The discrepancies between the different animal models have been explained by the different positional selectivities of the mammalian 12- and 15-lipoxygenase iso-enzymes which oxidize arachidonic p0035 australia at the carbon atoms 12 and 15 and which have different expression patterns, and by the composition of the food used in these animal studies , . To investigate the role of the 12/15-lipoxygenases in human atherosclerosis, genetic studies have been carried out which investigated the association of the human ALOX15 gene with coronary artery disease and myocardial infarction , , . Although there is currently more support for a neutral or an atheroprotective role of ALOX15 than for the contrary, these human genetic studies did not consistently show an association of functional variants in ALOX15 with clinical end points of atherosclerosis . The lack of consistent associations may be explained by the lack of power of the studies due to the low frequency of the two functional polymorphisms . However, another explanation may be redundancy for the 12/15-lipoxygenase activity in human macrophages. Recently a second 15-lipoxygenase isoform, ALOX15B, was detected in human atherosclerotic plaques , . Immunohistochemical analyses showed abundant ALOX15B expression in macrophage-rich areas of carotid lesions, and lipidomic analyses demonstrated the presence of typical ALOX15B products in plaque tissue . These findings suggest that eventually more than one 12/15-lipoxygenase isoform may play a role in human atherosclerosis. Humans have two 12-lipoxygenases and two 15-lipoxygenases which show different expression patterns, substrate specificities and stereo-selective metabolism . However, in cells involved in atherosclerosis only the 12-lipoxygenase, ALOX12, and the two 15-lipoxygenases, ALOX15 and ALOX15B, seem to be expressed . To identify the major 12/15-lipoxygenases in human macrophages and to better understand their role in human atherosclerosis, we investigated the basal and stimulated expression of these three 12/15-lipoxygenase isoforms ALOX12, ALOX15 and ALOX15B in human primary macrophages.
Materials and methods
Discussion In addition to the differentiation of monocytes into resident macrophages, macrophages can be polarized into at least two subpopulations, identified as M1 and M2 macrophages, analogous to the well characterized Th1 and Th2 subpopulations of T-helper cells . Stimulation of macrophages with INF-γ (released by Th1 cells) and LPS will lead to the classically activated pro-inflammatory M1 macrophages, which secrete IL-1β, IL-15, IL-18, TNF-α and IL-12 . Such M1 cells are characterized by enhanced endocytic functions and enhanced ability to kill intracellular pathogens. In contrast, stimulation of macrophages with IL-4, IL-10 (both released by Th2 cells), IL-13, glucocorticoids and TGF-β will lead to an anti-phlogistic macrophage phenotype M2 involved in tissue regeneration and homeostasis , . Both types of activated macrophages have been detected in atherosclerotic plaques, although the role of the differently activated macrophages in human atherosclerosis has not been fully elucidated , . Classically activated M1 macrophages predominate in the lipid core of human carotid atherosclerotic lesions, whereas anti-phlogistic M2 macrophages prevail in the shoulder region as well as in the periphery of the plaque . There is some indication from the secreted cytokine profile of polarized macrophages that classically activated M1 macrophages are pro-atherogenic, while the anti-phlogistic M2 macrophages are atheroprotective .