Macrophages In Atherosclerotic Plaques Research Articles

Gadolinium(III)-Gold Nanorods for MRI and Photoacoustic Imaging Dual-Modality Detection of Macrophages in Atherosclerotic Inflammation

One of the features of high-risk atherosclerotic plaques is the preponderance of macrophages. Gadolinium(III)-gold nanorods (Gd(III)-GNRs) have been developed as a dual-modality probe for MRI and photoacoustic imaging (PAI) to trace macrophages for determining the degree of inflammation. Gd(III)-GNRs were utilized for MRI and PAI dual-modality detection of macrophages in living mice and ex vivo simulated macrophage-rich plaque. Gd(III)-GNRs were shown to be endocytosed by macrophages in vitro. Macrophages labeled with Gd(III)-GNRs were detected by both PAI and MRI. With Gd(III)-GNRs, it is possible to institute a multiscale complementary imaging protocol: MRI can screen to identify the location of the probe-phagocytosed macrophages, and intravascular PAI provides a subsequent precise morphology to quantify the infiltration area and invasion depth of macrophages in the arterial wall. This new dual-modality nanoparticle approach has promise for enabling quantitative detection of macrophages in atherosclerotic plaques.

Phage Display Identification of CD100 in Human Atherosclerotic Plaque Macrophages and Foam Cells

Atherosclerosis is a complex disease in which vessels develop plaques comprising dysfunctional endothelium, monocyte derived lipid laden foam cells and activated lymphocytes. Considering that humans and animal models of the disease develop quite distinct plaques, we used human plaques to search for proteins that could be used as markers of human atheromas. Phage display peptide libraries were probed to fresh human carotid plaques, and a bound phage homologous to plexin B1, a high affinity receptor for CD100, was identified. CD100 is a member of the semaphorin family expressed by most hematopoietic cells and particularly by activated T cells. CD100 expression was analyzed in human plaques and normal samples. CD100 mRNA and protein were analyzed in cultured monocytes, macrophages and foam cells. The effects of CD100 in oxLDL-induced foam cell formation and in CD36 mRNA abundance were evaluated. Human atherosclerotic plaques showed strong labeling of CD100/SEMA4D. CD100 expression was further demonstrated in peripheral blood monocytes and in in vitro differentiated macrophages and foam cells, with diminished CD100 transcript along the differentiation of these cells. Incubation of macrophages with CD100 led to a reduction in oxLDL-induced foam cell formation probably through a decrease of CD36 expression, suggesting for the first time an atheroprotective role for CD100 in the human disease. Given its differential expression in the numerous foam cells and macrophages of the plaques and its capacity to decrease oxLDL engulfment by macrophages we propose that CD100 may have a role in atherosclerotic plaque development, and may possibly be employed in targeted treatments of these atheromas.

Proliferating macrophages prevail in atherosclerosis

Macrophages accumulate in atherosclerotic lesions during the inflammation that is part of atherosclerosis development and progression. A new study in mice indicates that the accumulation of macrophages in atherosclerotic plaques depends on local macrophage proliferation rather than the recruitment of circulating monocytes.

Neovascularization in the carotid atherosclerotic plaque is not associated with inflammatory cell aggregation

Background Monocytes and macrophages in atherosclerotic plaque lead to plaque instability. The aim of the study was to determine if plaque neovascularization led to inflammation. Methods Patients were consecutively enrolled if their carotid intimal media thickness was >2 mm, as revealed by duplex ultrasound. The patients then underwent dynamic contrast enhanced magnetic resonance imaging (DCE MRI) and fluorine-18 fluorodeoxyglucose (18F-FDG) positron emission tomography combined with computed tomography (PET CT). A target to background ratio (TBR) of ≥1.25 or <1.25 served as the cutoff point for the presence and absence of inflammation, respectively. Results Twenty-six patients underwent bilateral carotid DCE MRI and 24 patients also underwent PET CT. One hundred and fifty-five plaques were evaluated by both DCE MRI and PET CT. There was no significant difference in plaque morphology between the TBR ≥1.25 (n=61) and TBR <1.25 (n=94) groups. No significant differences were found in plasma volume and transfer constant between the TBR ≥1.25 and TBR <1.25 groups. Conclusion Our study did not find a significant correlation between plaque neovascularization and the aggregation of inflammatory cells.

Recombinant Lectin-Like Domain of Thrombomodulin Suppresses Vascular Inflammation by Reducing Leukocyte Recruitment via Interacting With Lewis Y on Endothelial Cells

The N-terminal lectin-like domain (domain 1 [D1]) of thrombomodulin (TM) is known to have an anti-inflammatory function. We previously showed that recombinant TM domain 1 (rTMD1) interacts with a carbohydrate molecule, Lewis Y (Le(y)), which is found to be expressed on adhesion molecules and involved in cell adhesion. Here, we tested the effect of rTMD1/Le(y) interaction on leukocyte recruitment in inflammation. The expression of Le(y) on the surface of human umbilical vein endothelial cells was increased by tumor necrosis factor-α stimulation. Direct binding of rTMD1 to Le(y) on the cell surface was observed. rTMD1 inhibited Le(y)-mediated leukocyte adhesion on the Le(y)-immobilized flow chamber and activated endothelium under a shear flow. The following leukocyte transmigration to endothelium was also reduced by rTMD1 through binding Le(y). In vivo, treatment of rTMD1 reduced leukocyte recruitment to the inflammatory sites in carotid ligation injury and thioglycollate-induced peritonitis. rTMD1 administration in apolipoprotein E-deficient mice effectively suppressed atherosclerotic plaque formation and macrophage infiltration in atherosclerotic lesions. Increased Le(y) expression, as well as administered rTMD1, was observed in inflamed vessels. rTMD1 suppresses vascular inflammation by inhibiting leukocyte recruitment to endothelium through attenuating Le(y)-mediated adhesion and further protects against atherosclerosis progression. The present study provides a mechanism showing that rTMD1 can inhibit inflammation by binding to its carbohydrate ligand Le(y).

Abstract 458: Alternatively-Spliced Extra Domain A of Fibronectin Exacerbates Atherosclerosis via a Toll-Like Receptor 4- Dependent Mechanism

Rationale Fibronectin containing the alternatively-spliced extra domain A (EDA + -FN) is absent in healthy arteries, but expressed in the atherosclerotic arteries of humans and mice. The mechanistic role of EDA + -FN in the pathophysiology of atherosclerosis remains unclear. Recent studies by us and others suggest that EDA + -FN is a ligand for toll-like receptor 4 (TLR4). Hypothesis EDA + -FN exacerbates atherosclerosis via TLR4 pathway in ApoE -/- mice. Model and Methods We generated EDA +/+ /ApoE -/- (constitutively over express EDA + -FN) and EDA -/- /ApoE -/- mice (EDA-deleted allele of FN and express FN lacking EDA). ApoE -/- mice was used as control. To elucidate the role of TLR4 in EDA + -FN-mediated atherosclerosis, we generated EDA +/+ /TLR4 -/- /ApoE -/- and EDA -/- /TLR4 -/- /ApoE -/- mice. Male mice (n=12-14/group) were fed a high-fat Western diet (21% fat and 0.2% cholesterol) beginning at 6 weeks until they were sacrificed at 5 months of age (i.e., 14 weeks on high-fat Western diet). We compared the extent of atherosclerosis in whole aortae, stained with Oil Red O and en face lesion area measured by morphometry, and in the cross section area of the aortic sinus using the Verhoeff-Van Gieson stain. Macrophage infiltration was quantified by immunohistochemistry. Results We report that atherosclerotic plaque formation in the aortae and aortic sinus of EDA +/+ /ApoE -/- mice was increased by 2-fold, concomitant with significant increased macrophage infiltration (% of total plaque area) ( P <0.001 vs.ApoE -/- mice, ANOVA). Deletion of the EDA exon of FN gene in ApoE -/- mice significantly reduced atherosclerotic plaque formation (aorta and aortic sinus) and macrophage infiltration ( P <0.01 vs. ApoE -/- mice, ANOVA). Total cholesterol and triglycerides levels were similar among groups. TLR4 deficiency in EDA +/+ /ApoE -/- mice significantly reduced atherosclerotic plaque formation in the aorta and aortic sinus, concomitant with decreased macrophage infiltration in aortic sinus ( P <0.05 vs. EDA +/+ /ApoE -/- mice, Two way ANOVA), whereas TLR4 deficiency in EDA -/- /ApoE -/- mice had no effect compared to EDA -/- /ApoE -/- mice. Conclusion These findings reveal a mechanistic role of endogenous EDA + -FN in modulating atherosclerosis via a TLR4-dependent pathway.

Abstract 474: Atherosclerotic Macrophages Develop a Progressive Lysosomal Dysfunction that can be Rescued by Induction of a Lysosomal Biogenesis Program

Recent reports of the proatherogenic phenotype of mice with a macrophage-specific autophagy deficiency has renewed interest in the role of the autophagy-lysosomal system in atherosclerosis. Lysosomes have the unique role of serving to process both exogenous material including the excess of atherogenic lipids and endogenous cargo including dysfunctional proteins and organelles. Surprisingly, little is known about the effect of an atherogenic environment on macrophage lysosomes. To address this, we utilize a variety of complementary techniques to show that oxidized LDL and cholesterol crystals, two of the commonly encountered lipid species in the atherosclerotic plaque, create a profound lysosomal dysfunction in cultured peritoneal macrophages. Disruptions in lysosomal pH, enzyme activity, proteolytic capacity, membrane integrity, and morphology are readily seen when cells are incubated with such lipids. Using flow cytometry to isolate resident tissue macrophages, we show that atherosclerotic plaque macrophages show features of dysfunctional lysosomes, a process that appears to be progressive with advanced plaque formation. These observations lead us to investigate whether stimulation of lysosomal function can ameliorate some of these effects. TFEB is the only known transcription factor that acts as a master regulator of lysosomal biogenesis, although its role in macrophages has not been studied. We show that overexpression of TFEB in cultured macrophages initiates a robust prodegradative response including induction of lysosomal genes and the generation of nascent lysosomes. Interestingly, this response can rescue several deleterious effects seen with atherogenic lipid loading including reductions in the secretion of the proinflammatory cytokine IL-1β and reductions in foam cell formation. Taken together, these data demonstrate that lysosomal function is markedly impaired in atherosclerosis and suggest that induction of a lysosomal biogenesis program can have anti-atherogenic effects.

Eicosapentaenoic acid suppresses palmitate-induced cytokine production by modulating long-chain acyl-CoA synthetase 1 expression in human THP-1 macrophages

BackgroundChronic inflammation caused by macrophages may be associated with progression of arteriosclerosis or obesity, both risk factors for cardiovascular events. In the Japan EPA Lipid Intervention Study (JELIS), eicosapentaenoic acid (EPA), an n-3 polyunsaturated fatty acid, was found to reduce the incidence of cardiovascular events. MethodsThe effect of EPA on the expression of inflammatory factors induced by palmitate, a saturated fatty acid, was investigated using human THP-1 macrophages. ResultsPalmitate induced expression of inflammatory cytokines and activated NF-κB, similar to lipopolysaccharide (LPS). EPA strongly suppressed palmitate-induced up-regulation of inflammatory factors while slightly suppressing LPS-induced factors. Both palmitate and LPS up-regulated expression of long-chain acyl-CoA synthetase (ACSL) 1, while EPA preferentially suppressed palmitate-induced ACSL1 expression. Although an acyl-CoA synthetase inhibitor and ACSL1 siRNA both suppressed palmitate-induced tumor necrosis factor (TNF)-α expression, the former had no effect on LPS-induced TNF-α expression. Palmitate may therefore stimulate cytokine production through a different mechanism than LPS mediated through Toll-like receptor 4, at least partly, and ACSL1 may play an important role in this mechanism. Finally, palmitate induced expression of sterol regulatory element-binding protein-1a and ACSL1, while EPA suppressed the expression of these genes. ConclusionThe suppressive effects of EPA on palmitate-induced cytokine production may be mediated by the suppression of ACSL1 expression, at least partly. This anti-inflammatory effect of EPA may contribute to suppression of chronic inflammation caused by macrophages in atherosclerotic plaques.

Dual-Energy Computed Tomography Imaging of Atherosclerotic Plaques in a Mouse Model Using a Liposomal-Iodine Nanoparticle Contrast Agent

The accumulation of macrophages in inflamed atherosclerotic plaques has long been recognized. In an attempt to develop an imaging agent for detection of vulnerable plaques, we evaluated the feasibility of a liposomal-iodine nanoparticle contrast agent for computed tomography imaging of macrophage-rich atherosclerotic plaques in a mouse model. Liposomal-iodine formulations varying in particle size and polyethylene glycol coating were fabricated and shown to stably encapsulate the iodine compound. In vitro uptake studies using optical and computed tomography imaging in the RAW 264.7 macrophage cell line identified the formulation that promoted maximal uptake. Dual-energy computed tomography imaging using this formulation in apolipoprotein E-deficient (ApoE(-/-)) mice (n=8) and control C57BL/6 mice (n=6) followed by spectral decomposition of the dual-energy images enabled imaging of the liposomes localized in the plaque. Imaging cytometry confirmed the presence of liposomes in the plaque and their colocalization with a small fraction (≈2%) of the macrophages in the plaque. The results demonstrate the feasibility of imaging macrophage-rich atherosclerotic plaques using a liposomal-iodine nanoparticle contrast agent and dual-energy computed tomography.

Macrophage activation by heparanase is mediated by TLR-2 and TLR-4 and associates with plaque progression.

Factors and mechanisms that activate macrophages in atherosclerotic plaques are incompletely understood. We examined the capacity of heparanase to activate macrophages. Highly purified heparanase was added to mouse peritoneal macrophages and macrophage-like J774 cells, and the levels of tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 were evaluated by ELISA. Gene expression was determined by RT-PCR. Cells collected from Toll-like receptor-2 and Toll-like receptor-4 knockout mice were evaluated similarly. Heparanase levels in the plasma of patients with acute myocardial infarction, stable angina, and healthy subjects were determined by ELISA. Immunohistochemistry was applied to detect the expression of heparanase in control specimens and specimens of patients with stable angina or acute myocardial infarction. Addition or overexpression of heparanase variants resulted in marked increase in tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 levels. Mouse peritoneal macrophages harvested from Toll-like receptor-2 or Toll-like receptor-4 knockout mice were not activated by heparanase. Plasma heparanase level was higher in patients with acute myocardial infarction, compared with patients with stable angina and healthy subjects. Pathologic coronary specimens obtained from vulnerable plaques showed increased heparanase staining compared with specimens of stable plaque and controls. Heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression toward vulnerability.

TLR4 antagonist reduces early-stage atherosclerosis in diabetic apolipoprotein E-deficient mice

Although it has been reported that deficiency of toll-like receptor 4 (TLR4) is associated with reduced atherosclerosis in atherosclerosis-prone mice and attenuated pro-inflammatory state in diabetic mice, it remains undetermined whether treatment with a TLR4 antagonist reduces atherosclerosis in nondiabetic or diabetic mice that have TLR4 expression. In this study, we determined the effect of Rhodobacter sphaeroides lipopolysaccharide (Rs-LPS), an established TLR4 antagonist, on early-stage atherosclerosis in nondiabetic and streptozotocin-induced diabetic apolipoprotein E-deficient (Apoe(-/-)) mice. Analysis of atherosclerotic lesions of both en face aortas and cross sections of aortic roots showed that administration of Rs-LPS in 14-week-old diabetic Apoe(-/-) mice for 10 weeks significantly reduced atherosclerotic lesions. Although atherosclerotic lesions in nondiabetic Apoe(-/-) mice appeared to be decreased by Rs-LPS treatment, the difference was not statistically significant. Metabolic study showed that Rs-LPS significantly lowered serum levels of cholesterol and triglycerides in nondiabetic mice but not in diabetic mice. Furthermore, immunohistochemistry studies showed that Rs-LPS inhibited the expression of interleukin 6 and matrix metalloproteinase-9 and reduced the content of monocytes and macrophages in atherosclerotic plaques. Taken together, this study demonstrated for the first time that TLR4 antagonist inhibited vascular inflammation and atherogenesis in diabetic Apoe(-/-) mice and lowered serum cholesterol and triglyceride levels in nondiabetic Apoe(-/-) mice.

MicroRNA-155 promotes atherosclerosis by repressing Bcl6 in macrophages

Macrophages in atherosclerotic plaques drive inflammatory responses, degrade lipoproteins, and phagocytose dead cells. MicroRNAs (miRs) control the differentiation and activity of macrophages by regulating the signaling of key transcription factors. However, the functional role of macrophage-related miRs in the immune response during atherogenesis is unknown. Here, we report that miR-155 is specifically expressed in atherosclerotic plaques and proinflammatory macrophages, where it was induced by treatment with mildly oxidized LDL (moxLDL) and IFN-γ. Leukocyte-specific Mir155 deficiency reduced plaque size and number of lesional macrophages after partial carotid ligation in atherosclerotic (Apoe-/-) mice. In macrophages stimulated with moxLDL/IFN-γ in vitro, and in lesional macrophages, loss of Mir155 reduced the expression of the chemokine CCL2, which promotes the recruitment of monocytes to atherosclerotic plaques. Additionally, we found that miR-155 directly repressed expression of BCL6, a transcription factor that attenuates proinflammatory NF-κB signaling. Silencing of Bcl6 in mice harboring Mir155-/- macrophages enhanced plaque formation and CCL2 expression. Taken together, these data demonstrated that miR-155 plays a key role in atherogenic programming of macrophages to sustain and enhance vascular inflammation.

SONODYNAMIC EFFECT OF EMODIN ON MACROPHAGES

ObjectivesEmodin has been used as an anti-inflammatory agent, and inflammation is a crucial feature of atherosclerosis. Here, we investigated the sonodynamic effect of emodin on macrophages, the pivotal inflammatory cells...

APOLIPOPROTEIN E SUPPRESSES IL-12 PRODUCTION IN MACROPHAGES

ObjectivesAccumulation of T cells and macrophages in atherosclerotic plaques and the formation of antibodies directed against plaque proteins suggests that adaptive immunity contributes to the development of atherosclerosis. Apolipoprotein E...

5-AMINOLEVULINIC ACID-DERIVED PROTOPORPHYRIN IX MEDIATED FLUORESCENCE DIAGNOSIS AND PHOTODYNAMIC THERAPY OF MACROPHAGES WITHIN THE ATHEROSCLEROTIC PLAQUE

ObjectivesTo detect the accumulation of ALA-PpIX in the plaque macrophages and evaluate the effects of PDT on macrophages.MethodsThe fluorescence of PpIX was detected by fluorescence microscope in THP-1 macrophages and...

Elevated serum YKL-40 level predicts myocardial reperfusion and in-hospital MACE in patients with STEMI

Macrophages in atherosclerotic plaques secrete YKL-40, a new biomarker of acute and chronic inflammation in patients with stable CAD. We hypothesized that YKL-40 may be a specific marker reflecting the burden of localized inflammation in myocardium and a predictor in patients with STEMI. In this study, we investigated the relationship of YKL-40 to in-hospital major adverse cardiac events (MACE), reperfusion parameters and its predictors in patients with STEMI. In total, 80patients with STEMI and no history of prior coronary artery disease (CAD), who underwent primary percutaneous coronary intervention (p-PCI), were enrolled consecutively. In addition, 30patients with normal coronary arteries (NCA) were enrolled as a control group. Cardiac biomarker levels including creatinine kinase-MB fraction (CK-MB), troponin-I, admission glucose and inflammatory markers including leukocytes and YKL-40 levels were measured as admission values. In our study, YKL-40 levels correlated to high-sensitivity CRP levels (r = 0.333, p = 0.003), TIMI risk score (r = 0.445, p < 0.001), age (r = 0.477, p < 0.001), pain to balloon time (r = 0.432, p < 0.001), leukocyte and neutrophil count (r = 0.386, p < 0.001 and r = 0.430, p < 0.001, respectively), hemoglobin (r = - 0.345, p = 0.002), admission and fasting blood glucose (r = 0.388, p < 0.001 and r = 0.427, p < 0.001), creatinine levels (r = 0.395, p < 0.001) and myocardial blush grade (r = - 0.334, p = 0.004). When the patients were divided into two groups determined by presence or absence of MACE, the patients with MACE had significantly higher levels of YKL-40 in comparison to the patients without MACE and the control group (194 ± 104, 114 ± 61 and 110 ± 53μg/L, p < 0.001, respectively). In multivariate logistic regression analysis in STEMI patients, only YKL-40 level (OR: 1.011, 95%CI: 1.002-1.019, p = 0.011) and leukocyte count (OR: 1.264, 95%CI: 1.037-1.540, p = 0.020) were the independent predictors for MACE. Sensitivity and specificity of YKL-40 to predict MACE, when 125μg/l was accepted as a cut-off value, were 84% and 70%, respectively. We found that serum YKL-40 is related to older age, increased admission glucose levels, leukocyte counts and decreased hemoglobin levels; YKL-40 level and leukocyte count independently predicted MACE.

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.

Tissue–Specific Insulin Signaling, Metabolic Syndrome, and Cardiovascular Disease

Impaired insulin signaling is central to development of the metabolic syndrome and can promote cardiovascular disease indirectly through development of abnormal glucose and lipid metabolism, hypertension, and a proinflammatory state. However, insulin's action directly on vascular endothelium, atherosclerotic plaque macrophages, and in the heart, kidney, and retina has now been described, and impaired insulin signaling in these locations can alter progression of cardiovascular disease in the metabolic syndrome and affect development of microvascular complications of diabetes mellitus. Recent advances in our understanding of the complex pathophysiology of insulin's effects on vascular tissues offer new opportunities for preventing these cardiovascular disorders.

Human Adipose Tissue Macrophages Display Activation of Cancer-related Pathways

Obesity is associated with a significantly increased risk for cancer suggesting that adipose tissue dysfunctions might play a crucial role therein. Macrophages play important roles in adipose tissue as well as in cancers. Here, we studied whether human adipose tissue macrophages (ATM) modulate cancer cell function. Therefore, ATM were isolated and compared with monocyte-derived macrophages (MDM) from the same obese patients. ATM, but not MDM, were found to secrete factors inducing inflammation and lipid accumulation in human T47D and HT-29 cancer cells. Gene expression profile comparison of ATM and MDM revealed overexpression of functional clusters, such as cytokine-cytokine receptor interaction (especially CXC-chemokine) signaling as well as cancer-related pathways, in ATM. Comparison with gene expression profiles of human tumor-associated macrophages showed that ATM, but not MDM resemble tumor-associated macrophages. Indirect co-culture experiments demonstrated that factors secreted by preadipocytes, but not mature adipocytes, confer an ATM-like phenotype to MDM. Finally, the concentrations of ATM-secreted factors related to cancer are elevated in serum of obese subjects. In conclusion, ATM may thus modulate the cancer cell phenotype.

Abstract 193: First Demonstration that Hepatic Apob100 and Intestinal Apob48 Colocalize with Macrophages in Human Carotid Atherosclerotic Plaques

Background: We previously demonstrated the presence and localization of hepatic apoB100 and intestinal apoB48 in human carotid atherosclerotic plaques by immunoperoxidase, immunofluorescence (IF), immunoelectron microscopy, and western blot; however, co-localization of apoB48 and apoB100 with macrophages has not been shown. Methods: In 3 patients undergoing carotid endarterectomy (CEA), plaques were stained for apoB100/CD68 and apoB48/CD68 dual stain (DS)-IF. CD68 was used to localize macrophages. Slides were processed for antigen retrieval and incubated with primary antibodies(ApoB100: ab50069-100 rabbit polyclonal antibody recognizing the C-terminal; apoB48:Shibayagi AKHB48E goat monoclonal antibody recognizing the C-terminal; CD68: Santa Cruz Biotechnologies SC-20060 mouse monoclonal antibody) and species-specific secondary antibodies (Invitrogen;A21447,A21235,A21244, or A11010 ).Confocal microscopy was used to visualize DS-IF. Results: There is intense CD68 staining throughout the plaques, demonstrating the presence of macrophages(B,E). As previously demonstrated, both apoB100 and apoB48 are present in plaques(A,D). Importantly, while CD68 staining is present throughout the plaque and in areas independent of apoB100 and apoB48, both apoB100 and apoB48 are always co-localized with CD68(C,F). Conclusions: This is the first demonstration that both hepatic apoB100 and intestinal apoB48 co-localize with macrophages in human carotid atherosclerotic plaques.