167 The Mechanism of P22PHOX C242T SNP Inhibition of TNF Alpha-Induced Nox2 Activation in Human Endothelial Cells and Vessels

Daniel Meijles, Lampson Fan, Maziah Ghazaly, Jian-Mei Li
2016 Heart  
our knowledge of their complexity in vivo is only partial. Here we wished to characterise plaque macrophages from two of the most common murine models; ApoE -/-(Apolipoprotein E null) and LDLR -/-(low density lipoprotein receptor null) and a relatively new and less characterised model; PCKS9-AAV8 (Adeno-associated virus serotype 8-proprotein convertase subtilisin kexin type 9) induced hyperlipidaemia. Using immunohistochemistry, plaques from high-fat diet fed ApoE -/-, LDLR -/and PCSK9-AAV8
more » ... /and PCSK9-AAV8 mice were simultaneously stained for the pan macrophage marker Mac-3 and the pro-/ anti-inflammatory markers inducible nitric oxide synthase (iNOS) and Arginase I (ArgI). Plaques were imaged using fluorescence microscopy and analysed by ImageJ. Individual Mac-3 + cells were selected as the region of interest and corresponding iNOS and ArgI staining was quantified. The analysis allowed consideration for the spectrum of marker co-expression and characterisation of individual cells based on staining intensity. Using this approach, complex populations of plaque macrophagesincluding single+ ArgI, double positive, double negative and single+ iNOS were quantified. To understand the roles of these populations in atherosclerosis further, we correlated macrophage quality and quantities with lesion size and collagen content. We show that ApoE -/plaque macrophages are significantly more pro-inflammatory than LDLR -/and PCSK9 plaque macrophages (p < 0.05 and p < 0.0001). The population responsible for the pro-inflammatory phenotype of ApoE -/macrophages were single+ iNOS cells (p < 0.0001). We also show that the abundance of these cells significantly correlates (R 2 =0.4791, p = 0.0183) with lesion size in the aortic sinus. In addition, the frequency of double negative macrophages correlated with lesion collagen content (R 2 =0.4451, p = 0.0178). For the first time, plaque macrophages from three murine atherosclerosis models have been comprehensively characterised using a multi-colour image analysis strategy and suggest that plaque macrophages from ApoE -/mice are significantly more pro-inflammatory than LDLR -/and PCSK9 macrophages. We show that single iNOS+ cells may have a role in promoting lesion formation and double negative cells may also have a role in lesion stability. We envisage our platform provides a novel tool to gain a further, in-depth understanding of macrophage phenotype in atherosclerosis and will use it to elucidate the action of modulators of macrophage polarisation in vivo.
doi:10.1136/heartjnl-2016-309890.167 fatcat:o4i4j4m24rfwzer6j2upjtk7na