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165 In situ Examination of Plaque Macrophage Populations Using Multicolour Florescence Microscopy Reveals Critical Differences between Murine Models of Experimental Atherosclerosis
  1. Jessica Johnston,
  2. Endre Kiss-Toth,
  3. Sheila E Francis
  1. University of Sheffield

Abstract

Macrophages play a central role in the development of atherosclerosis. They are highly plastic and heterogeneous cells that exhibit a spectrum of phenotypes, from pro-inflammatory or ‘classically activated’ to anti-inflammatory or ‘alternatively activated’. Plaque macrophages in particular have roles in the pathogenesis of lesion stability and rupture, the most common cause of cardiac associated mortality. Despite considerable work aiming to characterise these phagocytic cells in disease, 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 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 (R2=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 (R2=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.

  • Macrophage
  • Plaque
  • Polarisation

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