Macrophages in Atherosclerosis Regression

Macrophages play a central role in the development of atherosclerotic cardiovascular disease (ASCVD), which encompasses coronary artery disease, peripheral artery disease, cerebrovascular disease, and aortic atherosclerosis. In each vascular bed, macrophages contribute to the maintenance of the local inflammatory response, propagate plaque development, and promote thrombosis. These central roles, coupled with their plasticity, makes macrophages attractive therapeutic targets in stemming the development of and stabilizing existing atherosclerosis. In the context of ASCVD, classically activated M1 macrophages initiate and sustain inflammation, and alternatively activated M2 macrophages resolve inflammation. However, this classification is now considered an oversimplification, and a greater understanding of plaque macrophage physiology in ASCVD is required to aid in the development of therapeutics to promote ASCVD regression. Reviewed herein are the macrophage phenotypes and molecular regulators characteristic of ASCVD regression, and the current murine models of ASCVD regression.

407. The Effect of Streptococcus pneumoniae Pneumonia on Atherosclerosis

Background Clinical studies consistently find an increase in the risk of acute coronary syndrome (ACS) in the weeks following pneumonia, although the mechanisms underlying this finding are unknown. ACS most commonly occurs as a result of thrombosis at the site of ruptured atherosclerotic plaques. We hypothesized that the systemic inflammatory response to pneumococcal pneumonia leads to acute localized inflammatory changes within established atherosclerotic plaques, favoring plaque instability and rupture, thereby resulting in ACS. Methods Male ApoE-/- mice, a well-established model of atherosclerosis, were fed an atherogenic diet for 7–8 weeks before intranasal infection with Streptococcus pneumoniae or mock infection. Mice were sacrificed 2 or 8 weeks post-infection. Formalin-fixed, paraffin-embedded aortic sinus plaque sections were analyzed to assess markers of plaque vulnerability to rupture. To characterise post-pneumonic plaque macrophage phenotype, aortic sinus plaque cryosections 2 weeks post pneumonia/mock infection were immunostained for MAC-3 to identify macrophage-rich areas. These plaque regions were collected using laser capture microdissection and RNA extracted for microarray analysis. Results S. pneumoniae infection was associated with increased aortic sinus atherosclerotic plaque macrophage content (18.1 vs. 8.0%; P < 0.05) at 2 weeks post infection, but no significant difference in aortic sinus plaque burden, plaque smooth muscle or collagen content. There was no significant difference in any of these plaque vulnerability markers at 8 weeks post infection. Microarray analysis of laser capture micro-dissected plaque macrophages identified downregulation of the expression of three genes coding for specific E3 ubiquitin ligases following pneumonia. Pathway analysis identified a significant perturbation in the ubiquitin proteasome system pathway as a result. Conclusion In this murine model, pneumococcal pneumonia resulted in increased atherosclerotic plaque macrophage content, a marker of plaque instability, at 2 weeks post infection. Pneumonia may therefore lead to an increased propensity for atherosclerotic plaques to rupture soon after pneumonia, due to infiltration of macrophages into the plaque. Disclosures All authors: No reported disclosures.

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OBJECTIVES/SPECIFIC AIMS: Accumulation of cholesterol-laden macrophages in arterial walls leads to atherosclerosis. LXRs induce expression of genes that are atheroprotective in macrophages including CCR7, a chemokine receptor that promotes their emigration from the plaque. CCR7 expression has been shown to be negatively regulated by phosphorylation of LXRα at S198 and is reduced in diabetic mice that show impaired plaque regression. I hypothesized that LXRα phosphorylation at S198 diminishes macrophage emigration from atherosclerotic plaque and contributes to impaired regression in diabetes. METHODS/STUDY POPULATION: Inducible LXRα S198A phosphorylation deficient knock in mouse were used as donors for bone marrow transplantation into mice prone to develop atherosclerosis. Plaques were developed by placing mice on western diet; and regression was induced by lowering their lipid levels. Aortic plaques were then analyzed by using morphometric, histological, and molecular analyses in control and diabetic mice expressing either LXRα WT or LXRα S198A during regression. RESULTS/ANTICIPATED RESULTS: Surprisingly, lack of phosphorylation increased plaque macrophage content and impaired regression under normoglycemic condition; however, it did not exacerbate diabetic regression. Plaques in diabetic mice were associated with increased LXRα S198 phosphorylation. Consistent with this, LXRα phosphorylation is enhanced in macrophages cultured under hyperglycemic conditions indicating glucose-dependent regulation of LXRα phosphorylation. Monocyte trafficking studies reveal that lack of phosphorylation and diabetes independently increase recruitment of monocytes in the plaque that might contribute to increased macrophage content. Importantly, I found that diabetes also increases macrophage retention in the plaque, which is reversed in the absence of phosphorylation. We predict that this increased retention results from inhibition of emigration of plaque macrophages through enhanced phosphorylation in diabetes. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings suggest that inhibiting LXRα phosphorylation could be beneficial in diabetic atherosclerosis to reverse the accumulation of macrophages in the plaque. This study imparts insight on regulation of plaque macrophage trafficking through LXRα S198 phosphorylation.

Abstract 583: Receptor for Advanced Glycation End Products ( Ager ) and Diaphanous-1 ( Drf1) Suppress Macrophage Reverse Transendothelial Migration and Regression of Diabetic Atherosclerosis

Macrophages display complex trafficking properties in vivo , involving interaction with vascular endothelial cells. Macrophage migration is an important mechanism linked to the progression and regression of atherosclerosis. The underlying mechanisms involved in these processes are not fully defined. RAGE is a multiligand cell surface macromolecule, which binds distinct ligands. The RAGE cytoplasmic domain interacts with Diaphanous-1 ( Drf1 ), both in vitro and in vivo and this interaction is essential for RAGE ligand-mediated signaling in macrophages. We tested the hypothesis that RAGE and Diaphanous-1 suppress regression of diabetic atherosclerosis; at least in part by impaired reduction of plaque macrophage content in an AGE-enriched diabetic environment. In a aorta transplantation model, we examined the morphologic changes in LDLr-/- donor plaques found in Ager -/-, Drf1 -/- or WT recipient diabetic mice. After lipid normalization into Ager -/- recipient diabetic mice, we observed reduced plaque macrophage density (CD68 -46%;p<0.05), but increased plaque collagen (PSR+43%;p<0.06), compared to WT diabetic recipients. Oil Red O staining for fatty deposit suggests a decrease (-51%;p<0.05) in plaque area stained and lower AGE staining (-32%;p<0.05) in diabetic Drf1 -/- recipient mice compared to WT diabetic recipients. We employed a monocyte bead-tracking model in this in vivo aorta transplantation study. In WT recipient mice, when the donors were LDLr-/- mice fed a western diet, bead frequency per plaque section was reduced (22%;p< 0.05) on day 5 post-transplant compared with baseline. In contrast, bead frequency per plaque section was significantly reduced by (41%;p< 0.05) compared with baseline in the absence of Ager (gene encoding RAGE). In vitro, in a reverse transendothelial migration assay using primary aortic endothelial cells and Ager or Drf1 deficient bone marrow derived macrophages (BMDMs), revealed in the setting of treatment with RAGE ligand AGEs, deletion of Ager or Drf1 in BMDMs facilitated macrophage reverse migration vs. that observed in WT BMDMs. Therefore, we propose that deletion of Ager or Drf1 in recipient diabetic mice accelerates atherosclerotic plaque regression, via reduction in lesional macrophage content.

Inhibiting macrophage proliferation suppresses atherosclerotic plaque inflammation

Inflammation drives atherosclerotic plaque progression and rupture, and is a compelling therapeutic target. Consequently, attenuating inflammation by reducing local macrophage accumulation is an appealing approach. This can potentially be accomplished by either blocking blood monocyte recruitment to the plaque or increasing macrophage apoptosis and emigration. Because macrophage proliferation was recently shown to dominate macrophage accumulation in advanced plaques, locally inhibiting macrophage proliferation may reduce plaque inflammation and produce long-term therapeutic benefits. To test this hypothesis, we used nanoparticle-based delivery of simvastatin to inhibit plaque macrophage proliferation in apolipoprotein E–deficient mice (Apoe−/−) with advanced atherosclerotic plaques. This resulted in the rapid reduction of plaque inflammation and favorable phenotype remodeling. We then combined this short-term nanoparticle intervention with an 8-week oral statin treatment, and this regimen rapidly reduced and continuously suppressed plaque inflammation. Our results demonstrate that pharmacologically inhibiting local macrophage proliferation can effectively treat inflammation in atherosclerosis.