Macrophage Content In Atherosclerotic Plaques Research Articles

Transient increase in atherosclerotic plaque macrophage content following Streptococcus pneumoniae pneumonia in ApoE-deficient mice.

Despite epidemiological associations between community acquired pneumonia (CAP) and myocardial infarction, mechanisms that modify cardiovascular disease during CAP are not well defined. In particular, largely due to a lack of relevant experimental models, the effect of pneumonia on atherosclerotic plaques is unclear. We describe the development of a murine model of the commonest cause of CAP, Streptococcus pneumoniae pneumonia, on a background of established atherosclerosis. We go on to use our model to investigate the effects of pneumococcal pneumonia on atherosclerosis. C57BL/6J and ApoE-/- mice were fed a high fat diet to promote atherosclerotic plaque formation. Mice were then infected with a range of S. pneumoniae serotypes (1, 4 or 14) with the aim of establishing a model to study atherosclerotic plaque evolution after pneumonia and bacteremia. Laser capture microdissection of plaque macrophages enabled transcriptomic analysis. Intratracheal instillation of S. pneumoniae in mice fed a cholate containing diet resulted in low survival rates following infection, suggestive of increased susceptibility to severe infection. Optimization steps resulted in a final model of male ApoE-/- mice fed a Western diet then infected by intranasal instillation of serotype 4 (TIGR4) S. pneumoniae followed by antibiotic administration. This protocol resulted in high rates of bacteremia (88.9%) and survival (88.5%). Pneumonia resulted in increased aortic sinus plaque macrophage content 2 weeks post pneumonia but not at 8 weeks, and no difference in plaque burden or other plaque vulnerability markers were found at either time point. Microarray and qPCR analysis of plaque macrophages identified downregulation of two E3 ubiquitin ligases, Huwe1 and Itch, following pneumonia. Treatment with atorvastatin failed to alter plaque macrophage content or other plaque features. Without antibiotics, ApoE-/- mice fed a high fat diet were highly susceptible to mortality following S. pneumoniae infection. The major infection associated change in plaque morphology was an early increase in plaque macrophages. Our results also hint at a role for the ubiquitin proteasome system in the response to pneumococcal infection in the plaque microenvironment.

407. The Effect of Streptococcus pneumoniae Pneumonia on Atherosclerosis

BackgroundClinical 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.MethodsMale 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.ConclusionIn 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.

AIBP reduces atherosclerosis by promoting reverse cholesterol transport and ameliorating inflammation in apoE−/− mice

Background and aimsApoA-1 binding protein (AIBP) is a secreted protein that interacts with apoA-I and accelerates cholesterol efflux from cells. We have recently reported that AIBP promotes apoA-1 binding to ABCA1 in the macrophage cell membrane, partially through 115–123 amino acids. However, the effects of AIBP on the development of atherosclerosis in vivo remain unknown. MethodsApoE−/− mice with established atherosclerotic plaques were infected with rAAV-AIBP or rAAV-AIBP(Δ115-123), respectively. ResultsAIBP-treated mice showed reduction of atherosclerotic lesion formation, increase in circulating HDL levels and enhancement of reverse cholesterol transport to the plasma, liver, and feces. AIBP increased ABCA1 protein levels in aorta and peritoneal macrophages. Furthermore, AIBP could diminish atherosclerotic plaque macrophage content and the expression of chemotaxis-related factors. In addition, AIBP prevented macrophage inflammation by inactivating NF-κB and promoted the expression of M2 markers like Mrc-1 and Arg-1. However, lack of 115–123 amino acids of AIBP(Δ115-123) had no such preventive effects on the progression of atherosclerosis. ConclusionsOur observations demonstrate that AIBP inhibits atherosclerosis progression and suggest that it may be an effective target for prevention of atherosclerosis.

Myeloid Deletion of α1AMPK Exacerbates Atherosclerosis in LDL Receptor Knockout (LDLRKO) Mice.

Macrophage inflammation marks all stages of atherogenesis, and AMPK is a regulator of macrophage inflammation. We therefore generated myeloid α1AMPK knockout (MAKO) mice on the LDL receptor knockout (LDLRKO) background to investigate whether myeloid deletion of α1AMPK exacerbates atherosclerosis. When fed an atherogenic diet, MAKO/LDLRKO mice displayed exacerbated atherosclerosis compared with LDLRKO mice. To determine the underlying pathophysiological pathways, we characterized macrophage inflammation/chemotaxis and lipid/cholesterol metabolism in MAKO/LDLRKO mice. Myeloid deletion of α1AMPK increased macrophage inflammatory gene expression and enhanced macrophage migration and adhesion to endothelial cells. Remarkably, MAKO/LDLRKO mice also displayed higher composition of circulating chemotaxically active Ly-6Chigh monocytes, enhanced atherosclerotic plaque chemokine expression, and monocyte recruitment into plaques, leading to increased atherosclerotic plaque macrophage content and inflammation. MAKO/LDLRKO mice also exhibited higher plasma LDL and VLDL cholesterol content, increased circulating apolipoprotein B (apoB) levels, and higher liver apoB expression. We conclude that macrophage α1AMPK deficiency promotes atherogenesis in LDLRKO mice and is associated with enhanced macrophage inflammation and hypercholesterolemia and that macrophage α1AMPK may serve as a therapeutic target for prevention and treatment of atherosclerosis.

215 Increased Atherosclerotic Plaque Macrophage Content following Streptococcus Pneumoniae Pneumonia

Background Clinical studies consistently find an increase in the risk of acute coronary syndrome (ACS) within 1–2 weeks after a respiratory infection. It has yet to be established whether a causal relationship exists. Post mortem studies have revealed that the majority of ACS events occur as a result of atherosclerotic plaque disruption. We hypothesise that the systemic inflammatory response to pneumonia leads to acute localised inflammatory changes within established atherosclerotic plaques, favouring plaque instability, thereby resulting in ACS. Our objective was to investigate the effect of resolving pneumonia on atherosclerotic plaque composition. Materials and methods 11–13 week old male ApoE -/- mice, a well-established model of atherosclerosis, were fed an atherogenic diet for 7–8 weeks before intranasal infection with 5 × 10 5 cfu type 4 Streptococcus pneumoniae or mock infection. All mice received 3 doses of 100 mg/kg subcutaneous amoxicillin, 12 hly, the first dose given at 24 h post infection. Mice were sacrificed 2 weeks post infection and the heart and brachiocephalic artery excised. Formalin fixed, paraffin embedded aortic sinus and brachiocephalic artery atherosclerotic plaque sections were analysed by histological and immunohistochemical staining. Results This model of atherosclerotic plaque development following pneumonia resulted in high rates of bacteraemia at 24 h post infection and high survival rates at 5 days post infection (92% and 83% respectively). Streptococcus pneumoniae infection was associated with significantly increased aortic sinus atherosclerotic plaque macrophage content (18.1 vs. 8.0%; p Discussion In this murine model, pneumococcal pneumonia resulted in increased atherosclerotic plaque macrophage content, a marker of plaque instability. Pneumonia may therefore lead to an increased propensity for atherosclerotic plaques to rupture, although further work is required to assess the polarisation of macrophages in atherosclerotic plaques post pneumonia.

LDL-receptor-deficient mice lacking microRNA-143/145 have less atherosclerosis.

MicroRNAs (miRNAs, miRs) have emerged as important post-transcriptional regulators of gene expression. In vascular pathologies, the miR-143/145 cluster has attracted particular attention. The miR-143/145 cluster is encoded by a bicistronic transcript, and regulates the differentiation, plasticity and contractile function of vascular smooth muscle cells (VSMCs). VSMCs from miR143/145–deficient mice were locked in a synthetic state, which favoured neointimal lesion development despite normal levels of serum cholesterol and lipoproteins (1). In contrast, deficiency of miR-143/145 on the low-density lipoprotein receptor (Ldlr-/-) background reduced VLDL and LDL cholesterol (2). When Sala et al. crossed miR-143/145-/mice with Ldlr-/mice, the double knock-out mice had less atherosclerosis. Bioinformatic prediction algorithms identified the ATP-binding cassette transporter ABCA1 as a potential target of miR-145. Functional and luciferase experiments confirmed a direct interaction of miR-145 with the 3’UTR of ABCA1. In tissues from Ldlr-/mice, miR-145 levels were significantly higher in the aorta than in liver or macrophages. In the double knockout mice, ABCA1 expression was markedly increased in the aorta and the liver, but there was no change in HDL cholesterol although ABCA1 promotes cellular cholesterol efflux. The macrophage content in atherosclerotic plaques, however, was decreased. Based on an in vitro approach, the authors postulated that miR-145 may be transferred from VSMCs to macrophages. A similar transfer of miR-143/145 had previously been reported for endothelial cells: miR-143/145 secreted in exosomes of endothelial cells could apparently regulate VSMC function (3). It is currently unclear whether such a miRNA transfer between vascular cell types is just an in vitro phenomenon or also occurs in vivo to modify gene expression. In summary, therapeutic strategies targeting miR-145 to maintain a contractile VSMC phenotype have to be reevaluated in the context of dyslipidaemia.

Haploinsufficiency of E-selectin ligand-1 is associated with reduced atherosclerotic plaque macrophage content while complete deficiency leads to early embryonic lethality in mice

E-selectin-1 (ESL-1), also known as golgi complex-localized glycoprotein-1 (GLG1), homocysteine-rich fibroblast growth factor receptor (CGR-1), and latent transforming growth factor-β complex protein 1 (LTCP-1), is a multifunctional protein with widespread tissue distribution. To determine the functional consequences of ESL-1 deficiency, mice were generated carrying an ESL-1 gene trap. After backcrossing to C57BL6/J for 6 generations, mice heterozygous for the gene trap (ESL-1+/−) were intercrossed to produce ESL-1−/− mice, however ESL-1−/− mice were not viable, even at embryonic day E10.5. To determine the effect of heterozygous ESL-1 deficiency on atherosclerosis, apolipoprotein E deficient (ApoE−/−), ESL-1+/− mice were generated and fed western diet. Compared to ApoE−/−, ESL-1+/+ mice, atherosclerotic lesions from ApoE−/−, ESL-1+/− contained more collagen and fewer macrophages, suggesting increased plaque stability. In conclusion, heterozygous deficiency of ESL-1 is associated with features of increased atherosclerotic plaque stability while complete deficiency of ESL-1 leads to embryonic lethality.

Detecting and assessing macrophages in vivo to evaluate atherosclerosis noninvasively using molecular MRI.

We investigated the ability of targeted immunomicelles to detect and assess macrophages in atherosclerotic plaque using MRI in vivo. There is a large clinical need for a noninvasive tool to assess atherosclerosis from a molecular and cellular standpoint. Macrophages play a central role in atherosclerosis and are associated with plaques vulnerable to rupture. Therefore, macrophage scavenger receptor (MSR) was chosen as a target for molecular MRI. MSR-targeted immunomicelles, micelles, and gadolinium-diethyltriaminepentaacetic acid (DTPA) were tested in ApoE-/- and WT mice by using in vivo MRI. Confocal laser-scanning microscopy colocalization, macrophage immunostaining and MRI correlation, competitive inhibition, and various other analyses were performed. In vivo MRI revealed that at 24 h postinjection, immunomicelles provided a 79% increase in signal intensity of atherosclerotic aortas in ApoE-/- mice compared with only 34% using untargeted micelles and no enhancement using gadolinium-DTPA. Confocal laser-scanning microscopy revealed colocalization between fluorescent immunomicelles and macrophages in plaques. There was a strong correlation between macrophage content in atherosclerotic plaques and the matched in vivo MRI results as measured by the percent normalized enhancement ratio. Monoclonal antibodies to MSR were able to significantly hinder immunomicelles from providing contrast enhancement of atherosclerotic vessels in vivo. Immunomicelles provided excellent validated in vivo enhancement of atherosclerotic plaques. The enhancement seen is related to the macrophage content of the atherosclerotic vessel areas imaged. Immunomicelles may aid in the detection of high macrophage content associated with plaques vulnerable to rupture.

Atheroprotective immunization with MDA-modified apo B-100 peptide sequences is associated with activation of Th2 specific antibody expression

Objective: The objective of this study was to evaluate if immunization with MDA-modified human apo B-100 fragments is associated with a shift in the Th1/Th2 balance.Methods and Results: Apo E deficient mice were immunized with one of the peptides (P45; amino acids 688–707, P74; amino acids 1123–1142 or P240; amino acids 3613–3632) at 6, 9 and 11 weeks of age and compared to controls given carrier alone. Immunization with P45 and P74 reduced atherosclerosis in the aorta of 25-week-old mice by 48% (p=0.02) and 31% (p=0.06) and macrophage content in atherosclerotic plaques by 33% (p=0.02) and 39% (p=0.02), respectively. The levels of Th2-specific IgG1 against each peptide increased more than 50-fold in response to immunization, whereas the levels of specific IgM and Th1-associated IgG2a were only marginally affected. However, there was an increase in the plaque expression of both Th1 and Th2 cytokines as assessed by real time PCR. Immunization with P240, a non-homologous peptide used as control, induced a 10-fold increase of specific IgG1 but did not influence atherosclerosis or plaque content.Conclusions: Immunization with MDA apo B-100 fragments induce a shift from Th1 to a Th2 specific oxidized LDL antibody expression, but without a concomitant downregulation of plaque IFN-γ expression.