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Macrophage Proteoglycans in Atherosclerosis

Final Report Summary - ATHEROGAG (Macrophage Proteoglycans in Atherosclerosis)

Cardiovascular diseases are the leading cause of death in Western societies. Cardiovascular-related deaths are primarily caused by complications of atherosclerosis, a disease initiated via focal infiltration and retention of lipoproteins in the subendothelial matrix of arteries due to a combination of aggregation and interaction with proteoglycans produced by the arteries. To get a more profound insight on the in vivo contribution of macrophage-derived and -associated proteoglycans during atherosclerosis development and progression we want to address two objectives: First, we want to determine the in vivo impact of reduced sulfation of macrophage proteoglycans on atherosclerosis using conditional mouse models lacking macrophage-specific Ndst1 functionality on an LDLR-deficient background. Atherosclerotic lesion quantity and quality will be analyzed in conjunction with macrophage lesion infiltration and lesion apoptosis and efferocytosis. Secondly, we want to evaluate the importance of proteoglycans for macrophage foam cell conversion, another key event in atherogenesis. Using macrophage cell cultures from wild-type or mutant mice we want to determine the importance of biosynthetic genes involved and the array of proteoglycans expressed before and after conversion. In addition the cells will be used to identify a specific proteoglycan important for conversion, which will be consequently evaluated for its impact on atherosclerosis in vivo. During his mobility, the applicant will receive didactic (glycobiology, management, intellectual property, grants) and hands-on (MS, macrophage cultures, qPCR, shRNA, flow assay) training as well as network opportunities.
In order to examine the role of macrophage heparan sulfate proteoglycans (HSPGs) in atherogenesis, we inactivated the biosynthetic gene GlcNAc N-deacetylase/N-sulfotransferase 1 (Ndst1) selectively in macrophages by crossing Ndst1f/f mice with LysMCre+ mice. Ndts1 inactivation reduced the overall sulfation of HSPG in macrophages by 30%. When bred onto an Ldlr-/- background and placed on an atherogenic diet, Ndst1f/fLysMCre+Ldlr-/- mice demonstrated a 2-fold increase in atherosclerosis compared to Ldlr-/- mice. Plaque analysis also revealed more advanced lesions and a significantly increased macrophage content in lesions from Ndst1f/fLysMCre+Ldlr-/- mice. Diminished HSPG sulfation in macrophages from Ndst1f/fLysMCre+ mice resulted in significantly increased expression of inflammatory genes such as CCL5, CCL7, CCL8 and ACAT2. Increased ACAT2 expression correlated with more ACAT enzyme activity and increased foam cell formation compared to wild-type macrophages. Motif analysis of promoters of up-regulated genes revealed increased Type-I interferon signaling in macrophages with reduced HSPG sulfation. Our data suggest that macrophage HSPGs are atheroprotective and act by maintaining Type-I interferon reception in a quiescent state through sequestration of IFN-beta.
Further analysis has revealed that Syndecan-4 plays an important role in the modulation of foam cell conversion and that variation of adipocyte HS has a profound impact on diet-induced obesity. Altogether our data imply that differences in macrophage and adipocyte HSPG sulfation can possibly predict and determine the outcome of atherosclerosis-related cardiovascular disease. Altogether, the generated data add to our understanding of macrophage proteoglycans and their importance for cardiovascular disease and provide the opportunity for developing prognostic markers, for identifying drug targets and for transfer of US collaborations and know-how to the EU.