Effects of deletion of macrophage ABCA7 on lipid metabolism and the development of atherosclerosis in the presence and absence of ABCA1

ABCA7, a close relative of ABCA1 which facilitates cholesterol efflux to lipid-poor apoproteins, has been implicated in macrophage lipid efflux and clearance of apoptotic cells in in vitro studies. In the current study, we investigated the in vivo effects of macrophage ABCA7 deficiency on lipid metabolism and atherosclerosis. Chimeras with dysfunctional ABCA7 in macrophages and other blood cells were generated by transplantation of bone marrow from ABCA7 knockout (KO) mice into irradiated low-density lipoprotein receptor (LDLr) KO mice. Unexpectedly, macrophage ABCA7 deficiency did not significantly affect atherosclerosis susceptibility of LDLr KO mice after 10 weeks Western-type diet feeding. However, ABCA7 deficiency was associated with 2-fold (p<0.05) higher macrophage ABCA1 mRNA expression levels. Combined disruption of ABCA1 and ABCA7 in bone-marrow-derived cells increased atherosclerotic lesion development (1.5-fold (p>0.05) as compared to wild type transplanted mice. However, single deletion of ABCA1 had a similar effect (1.8-fold, p<0.05). Macrophage foam cell accumulation in the peritoneal cavity was reduced in ABCA1/ABCA7 dKO transplanted animals as compared to single ABCA1 KO transplanted mice, which was associated with increased ABCG1 expression. Interestingly, spleens of ABCA1/ABCA7 double KO transplanted mice were significantly larger as compared to the other 3 groups and showed massive macrophage lipid accumulation, a reduction in CD3+ T-cells, and increased expression of key regulators of erythropoiesis. In conclusion, deletion of ABCA7 in bone marrow-derived cells does not affect atherogenesis in the arterial wall neither in the absence or presence of ABCA1. Interestingly, combined deletion of bone marrow ABCA1 and ABCA7 causes severe splenomegaly associated with cellular lipid accumulation, a reduction in splenic CD3+ T cells, and induced markers of erythropoeisis. Our data indicate that ABCA7 may play a role in T cell proliferation and erythropoeisis in spleen.     

Expanding expression of the 5-lipoxygenase/leukotriene B4 pathway in atherosclerotic lesions of diabetic patients promotes plaque instability

Emerging evidence now indicates that the 5-lipoxygenase (5-LO) pathway play a role in the pathogenesis of atherosclerosis and restenosis. The expression of 5-LO by activated macrophages in symptomatic plaques leads to leukotriene B(4) (LTB(4)) accumulation and enhanced synthesis and release of matrix metalloproteinases (MMPs) that can promote plaque rupture. However, the role of 5-LO pathway in diabetic vascular disease has not been previously reported. Thus, the present study was designed to analyze the expression of 5-LO in carotid plaques of diabetic patients and to investigate the possible role of 5-LO pathway in the pathogenesis and progression of diabetic atherosclerosis. Atherosclerotic plaques from 60 patients undergoing carotid endarterectomy were divided into non-diabetic and diabetic group. Plaques were analyzed for 5-LO, MMP-2 and MMP-9 by immunohistochemical, Western blot, and densitometric analyses, whereas zymography was used to detect MMP activity. Immunocytochemistry was also used to identify CD68+macrophages, CD3+T-lymphocytes, and HLA-DR+inflammatory cells. LTB(4) were quantified by enzyme-linked immunosorbent assay. 5-LO showed abundant immunoreactivity in human atherosclerotic carotid lesions, and was colocalized with macrophage infiltrates in atherosclerotic intima. 5-LO expression was higher in diabetic compared with non-diabetic plaques and was associated with increased MMP-2 and MMP-9 expression. Follow-up analyze with zymography assay revealed MMP activity was elevated in diabetic compared with non-diabetic plaques. Notably, in contrast to non-diabetic plaques, LTB(4) levels were significantly increased in diabetic plaques by enzyme-linked immunosorbent assay. These results suggest that overexpression of 5-LO and LTB(4) in atherosclerotic plaques possibly promote MMP-induced plaque rupture in diabetes. Hence, anti-LTs may be useful, not only in reducing atherogenesis, but also in the prevention and treatment of acute atherothrombotic events in diabetic patients.     

Perilipin1 Deficiency in Whole Body or Bone Marrow-Derived Cells Attenuates Lesions in Atherosclerosis-Prone Mice

Aims

The objective of this study is to determine the role of perilipin 1 (Plin1) in whole body or bone marrow-derived cells on atherogenesis.

Methods and Results

Accumulated evidence have indicated the role of Plin1 in atherosclerosis, however, these findings are controversial. In this study, we showed that Plin1 was assembled and colocalized with CD68 in macrophages in atherosclerotic plaques of ApoE-/- mice. We further found 39% reduction of plaque size in the aortic roots of Plin1 and ApoE double knockout (Plin1-/-ApoE-/-) females compared with ApoE-/- female littermates. In order to verify whether this reduction was macrophage-specific, the bone marrow cells from wild-type or Plin1 deficient mice (Plin1-/-) were transplanted into LDL receptor deficient mice (LDLR-/-). Mice receiving Plin1-/- bone marrow cells showed also 49% reduction in aortic atherosclerotic lesions compared with LDLR-/- mice received wild-type bone marrow cells. In vitro experiments showed that Plin1-/- macrophages had decreased protein expression of CD36 translocase and an enhanced cholesterol ester hydrolysis upon aggregated-LDL loading, with unaltered expression of many other regulators of cholesterol metabolism, such as cellular lipases, and Plin2 and 3. Given the fundamental role of Plin1 in protecting LD lipids from lipase hydrolysis, it is reasonably speculated that the assembly of Plin1 in microphages might function to reduce lipolysis and hence increase lipid retention in ApoE-/- plaques, but this pro-atherosclerotic property would be abrogated on inactivation of Plin1.

Conclusion

Plin1 deficiency in bone marrow-derived cells may be responsible for reduced atherosclerotic lesions in the mice.     

Hematopoietic miR155 deficiency enhances atherosclerosis and decreases plaque stability in hyperlipidemic mice

microRNA-155 (miR155) is a central regulator of immune responses that is induced by inflammatory mediators. Although miR155 is considered to be a pro-inflammatory microRNA, in vitro reports show anti-inflammatory effects in lipid-loaded cells. In this study we examined the role of miR155 in atherosclerosis in vivo using bone marrow transplantation from miR155 deficient or wildtype mice to hyperlipidemic mice. Hematopoietic deficiency of miR155 enhanced atherosclerotic plaque development and decreased plaque stability, as evidenced by increased myeloid inflammatory cell recruitment to the plaque. The increased inflammatory state was mirrored by a decrease in circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells, and an increase in granulocytes (CD11b(+)Ly6G(+)) in blood of miR155(-/-) transplanted mice. Moreover, we show for the first time a crucial role of miR155 in monocyte subset differentiation, since hematopoietic deficiency of miR155 increases the 'inflammatory' monocyte subset (CD11b(+)Ly6G(-)Ly6C(hi)) and reduces 'resident' monocytes (CD11b(+)Ly6G(-)Ly6C(low)) in the circulation. Furthermore, cytokine production by resident peritoneal macrophages of miR155(-/-) transplanted hyperlipidemic mice was skewed towards a more pro-inflammatory state since anti-inflammatory IL-10 production was reduced. In conclusion, in this hyperlipidemic mouse model miR155 acts as an anti-inflammatory, atheroprotective microRNA. Additionally, besides a known role in lymphoid cell development, we show a crucial role of miR155 in myeloid lineage differentiation.     

Macrophage Polarization Reflects T Cell Composition of Tumor Microenvironment in Pediatric Classical Hodgkin Lymphoma and Has Impact on Survival

Macrophages have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL) and have been suggested to have a negative impact on outcome. Most studies addressing the role of macrophages in cHL have relied on identification of macrophages by generic macrophage antigens, e.g., CD68. We have therefore conducted an in situ analysis of macrophage polarization in a series of 100 pediatric cHL (pcHL) cases using double staining immunohistochemistry, combining CD68 or CD163 with pSTAT1 (M1-like) or CMAF (M2-like). M1- or M2-polarised microenvironment was defined by an excess of one population over the other (>1.5). Expression of STAT1 and LYZ genes was also evaluated by RT-qPCR. Patients <14 years and EBV+ cases displayed higher numbers of CD68+pSTAT1+ cells than older children and EBV- cases, respectively (P=0.01 and P=0.02). A cytotoxic tumor microenvironment, defined by a CD8+/FOXP3+ ratio >1.5 was associated with higher numbers of CD68+pSTAT1+ (P=0.025) and CD163+pSTAT1+ macrophages (P<0.0005). Levels of STAT1 and LYZ expression were associated with the numbers of CD68+pSTAT1+ macrophages. EBV+ cHL cases disclosed a predominant M1 polarized microenvironment similar to Th1 mediated inflammatory disorders, while EBV- cHL showed a predominant M2 polarized microenvironment closer to Th2 mediated inflammatory diseases. Better overall-survival (OS) was observed in cases with higher numbers of CD163+pSTAT1+ macrophages (P=0.02) while larger numbers of CD163+CMAF+ macrophages were associated with worse progression-free survival (PFS) (P=0.02). Predominant M1-like polarization as disclosed by CD163+pSTAT1+/CD163+CMAF+ ratio > 1.5 was associated with better OS (P= 0.037). In conclusion, macrophage polarization in pcHL correlates with prevalent local T cell response and may be influenced by the EBV-status of neoplastic cells. Besides, M1-like and M2-like macrophages displayed differential effects on outcome in pcHL.     

小檗碱对CT26皮下移植瘤组织中肿瘤相关巨噬细胞的影响

目的观察小檗碱的抑瘤作用及其对肿瘤组织内肿瘤相关巨噬细胞数量的影响。方法 BABL/c小鼠40只随机分2组,全部皮下移植结肠癌细胞(CT26细胞系),次日进行药物干预。治疗组小鼠腹腔注射小檗碱,100mmol/L,200μl/d,14d;对照组腹腔注射等量生理盐水。肿瘤细胞接种7d后连续动态测定肿瘤体积,接种15d处死全部动物,取肿瘤组织、免疫组织化学显色检测肿瘤组织中M2型巨噬细胞标志物CD206及CD68的表达。结果小鼠皮下移植CT26后,小檗碱治疗组小鼠皮下移植瘤生长缓慢。与对照组比较,肿瘤体积及瘤重均显著减少(P<0.05);皮下移植15d肿瘤组织中可见大量CD206及CD68阳性细胞;肿瘤组织中CD206及CD68阳性细胞数量显著减少(P<0.05 orP<0.01)。结论小檗碱可能通过抑制肿瘤相关巨噬细胞的形成而发挥抗肿瘤作用。     

Neutrophil infiltration and oxidant-production in human atherosclerotic carotid plaques

To clarify the clinical implications of neutrophils in vulnerable plaques we evaluated the function and activity of infiltrated neutrophils in an atherosclerotic plaque, focusing on oxidant production. A histopathological investigation was performed using carotid arterial samples obtained from seven patients. The atherosclerotic plaques were examined cytochemically for naphthol-ASD-chloroacetate esterase activity and oxidant-production, and immunohistochemically using N-formyl peptide receptor-like 1 (fPRL1)-, CD66b-, CD68- or p22phox-specific antibodies. The cytoplasmic fPRL1 intensity value of the neutrophils in the plaque was estimated using an activity index. Naphthol-ASD-chloroacetate esterase activity was found in cells located in the atherosclerotic plaque, indicating that the cells were neutrophils. The cytoplasmic fPRL1 intensity value of the neutrophils in the plaque decreased to approximately 60% of the intensity observed in the capillary vessels. Oxidant-production was also detected in the plaques, and both neutrophils and macrophages were observed at the corresponding oxidant-production sites. p22phox expression was also located in the same areas in which oxidant-production was observed in these plaques. We could not directly evaluate how much ROS generated from the infiltrated neutrophils contributed the plaque vulnerability followed by its rupture. However, the infiltrated neutrophils in the atherosclerotic plaques morphologically appeared activated and were actively generating oxidant, implying that neutrophils, together with macrophages, infiltrate into atherosclerotic plaques and contribute to plaque vulnerability.     

Overexpression of transferrin receptor and ferritin related to clinical symptoms and destabilization of human carotid plaques

Accumulation of tissue iron has been implicated in development of atherosclerotic lesions mainly because of increased iron-catalyzed oxidative injury. However, it remains unknown whether cellular iron import and storage in human atheroma are related to human atheroma development. We found that transferrin receptor 1 (TfR1), a major iron importer, is highly expressed in foamy macrophages and some smooth muscle cells in intimal lesions of human carotid atheroma, mainly in cytoplasmic accumulation patterns. In 52 human carotid atherosclerotic lesions, TfR1 expression was positively correlated with macrophage infiltration, ectopic lysosomal cathepsin L, and ferritin expression. Highly expressed TfR1 and ferritin in CD68-positive macrophages were significantly associated with development and severity of human carotid plaques, smoking, and patient's symptoms. The findings suggest that pathologic macrophage iron metabolism may contribute to vulnerability of human atheroma, established risk factors, and their clinical symptoms. The cytoplasmic overexpression of TfR1 may be the result of lysosomal dysfunction and ectopic accumulation of lysosomal cathepsin L caused by atheroma-relevant lipids in atherogenesis.     

Delivery of negatively charged liposomes into the atherosclerotic plaque of apolipoprotein E-deficient mouse aortic tissue

Liposomes have been used to diagnose and treat cancer and, to a lesser extent, cardiovascular disease. We previously showed the uptake of anionic liposomes into the atheromas of Watanabe heritable hyperlipidemic rabbits within lipid pools. However, the cellular distribution of anionic liposomes in atherosclerotic plaque remains undescribed. In addition, how anionic liposomes are absorbed into atherosclerotic plaque is unclear. We investigated the uptake and distribution of anionic liposomes in atherosclerotic plaque in aortic tissues from apolipoprotein E-deficient (ApoE^–/–) mice. To facilitate the tracking of liposomes, we used liposomes containing fluorescently labeled non-silencing small interfering RNA. Confocal microscopy analysis showed the uptake of anionic liposomes into atherosclerotic plaque and colocalization with macrophages. Transmission electron microscopy analysis revealed anionic liposomal accumulation in macrophages. To investigate how anionic liposomes cross the local endothelial barrier, we examined the role of clathrin-mediated endocytosis in human coronary artery endothelial cells (HCAECs) treated with or without the inflammatory cytokine tumor necrosis factor (TNF)-α. Pretreatment with amantadine, an inhibitor of clathrin-mediated endocytosis, significantly decreased liposomal uptake in HCAECs treated with or without TNF-α by 77% and 46%, respectively. Immunoblot analysis showed that endogenous clathrin expression was significantly increased in HCAECs stimulated with TNF-α but was inhibited by amantadine. These studies indicated that clathrin-mediated endocytosis is partly responsible for the uptake of liposomes by endothelial cells. Our results suggest that anionic liposomes target macrophage-rich areas of vulnerable plaque in ApoE^–/– mice; this finding may lead to the development of novel diagnostic and therapeutic strategies for treating vulnerable plaque in humans.     

Macrophage apoAI protects against dyslipidemia-induced dermatitis and atherosclerosis without affecting HDL

Tissue cholesterol accumulation, macrophage infiltration, and inflammation are features of atherosclerosis and some forms of dermatitis. HDL and its main protein, apoAI, are acceptors of excess cholesterol from macrophages; this process inhibits tissue inflammation. Recent epidemiologic and clinical trial evidence questions the role of HDL and its manipulation in cardiovascular disease. We investigated the effect of ectopic macrophage apoAI expression on atherosclerosis and dermatitis induced by the combination of hypercholesterolemia and absence of HDL in mice. Hematopoietic progenitor cells were transduced to express human apoAI and transplanted into lethally irradiated LDL receptor^−/−/apoAI^−/− mice, which were then placed on a high-fat diet for 16 weeks. Macrophage apoAI expression reduced aortic CD4⁺ T-cell levels (−39.8%), lesion size (−25%), and necrotic core area (−31.6%), without affecting serum HDL or aortic macrophage levels. Macrophage apoAI reduced skin cholesterol by 39.8%, restored skin morphology, and reduced skin CD4⁺ T-cell levels. Macrophage apoAI also reduced CD4⁺ T-cell levels (−32.9%) in skin-draining lymph nodes but had no effect on other T cells, B cells, dendritic cells, or macrophages compared with control transplanted mice. Thus, macrophage apoAI expression protects against atherosclerosis and dermatitis by reducing cholesterol accumulation and regulating CD4⁺ T-cell levels, without affecting serum HDL or tissue macrophage levels.     

Overexpression of TGF-ß1 in macrophages reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice

Although macrophages represent the hallmark of both human and murine atherosclerotic lesions and have been shown to express TGF-ß1 (transforming growth factor β1) and its receptors, it has so far not been experimentally addressed whether the pleiotropic cytokine TGF-ß1 may influence atherogenesis by a macrophage specific mechanism. We developed transgenic mice with macrophage specific TGF-ß1 overexpression, crossed the transgenics to the atherosclerotic ApoE (apolipoprotein E) knock-out strain and quantitatively analyzed both atherosclerotic lesion development and composition of the resulting double mutants. Compared with control ApoE^−/− mice, animals with macrophage specific TGF-ß1 overexpression developed significantly less atherosclerosis after 24 weeks on the WTD (Western type diet) as indicated by aortic plaque area en face (p<0.05). Reduced atherosclerotic lesion development was associated with significantly less macrophages (p<0.05 after both 8 and 24 weeks on the WTD), significantly more smooth muscle cells (SMCs; p<0.01 after 24 weeks on the WTD), significantly more collagen (p<0.01 and p<0.05 after 16 and 24 weeks on the WTD, respectively) without significant differences of inner aortic arch intima thickness or the number of total macrophages in the mice pointing to a plaque stabilizing effect of macrophage-specific TGF-ß1 overexpression. Our data shows that macrophage specific TGF-ß1 overexpression reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice.     

P‐7P16 ANTIBODY AS AN ADJUNCT TO ROUTINE CERVICAL SCREENING IN PROBLEMATIC CASES: A COMPARISON OF TWO COMMERCIALLY AVAILABLE ANTIBODIES

Introduction: Despite the cervix having the largest concentration of lymphocytes and macrophages in the lower genital tract, there is limited knowledge of these cell types in CIN. This study was undertaken to compare T‐cell and macrophage populations in women with and without low‐grade CIN. Methods: Control group: 10 women with a normal recent cervical smear result, mean age 30 years. Study group: 10 women with low‐grade CIN, mean age 33.2 years. Colposcopically directed cervical biopsies were taken from control women and from areas of CIN in the study group, and studied using immunoperoxidase and immunofluorescence staining. Results: T‐cell numbers remained constant. In CIN, there were increased proportions of recruited (CD8+CD5+), activated (CD8+HLA‐DR+) and cytolytic (CD8+TIA‐1+) CD8 T‐cells. There were more activated CD4+HLA‐DR+ T‐cells in CIN. Memory T‐cell (CD8+CD45RO+ and CD4+CD45RO+) proportions were reduced. Total macrophage numbers (CD68+) remained constant, but activated macrophages (CD68+HLA‐DR+) rose. Phagocytic (RFD7+) macrophage proportions decreased with a concomitant increase in suppresser macrophages (RFD1+RFD7+). Discussion: Despite cellular numbers remaining unchanged, there were significant changes in T‐cell and macrophage populations in women with low‐grade CIN. There is recruitment of activated and cytolytic CD8 T‐cells. The swing to a predominantly suppressive macrophage phenotype may predispose to CIN. Carcinoma of the cervix occurs more frequently in women who are immunosuppressed. Our data suggests that local and systemic immune mechanisms may be relevant in the response to HPV‐induced neoplasia.     

NOS1-mediated macrophage and endothelial cell interaction in the progression of atherosclerosis

Atherosclerosis is a chronic inflammatory disease arising due to an imbalance in lipid metabolism and maladaptive immune response driven by the accumulation of cholesterol-laden macrophages in the artery wall. Interactions between monocytes/macrophages and endothelial cells play an essential role in the pathogenesis of atherosclerosis. In our current study, nitric oxide synthase 1 (NOS1)-derived nitric oxide (NO) has been identified as a regulator of macrophage and endothelial cell interaction. Oxidized LDL (OxLDL) activates NOS1, which results in the expression of CD40 ligand in macrophages. OxLDL-stimulated macrophages produce some soluble factors which increase the CD40 receptor expression in endothelial cells. This increases the interaction between the macrophages and endothelial cells, which leads to an increase in the inflammatory response. Inhibition of NOS1-derived NO might serve as an effective strategy to reduce foam cell formation and limit the extent of atherosclerotic plaque expansion.     

Increased uptake of folate conjugates by activated macrophages in experimental hyperlipemia

In the pathogenesis of atherosclerosis, macrophages become activated and play a crucial role in plaque formation. Activated synovial macrophages have recently been shown to express receptors for folic acid. We have determined whether activated macrophages also over-express folate receptor (FR) in atherosclerosis. Most normal cells express little or no FR, and, if FR is present on activated macrophages, folate-linked compounds and drugs could be selectively targeted to those cells that do express FR. To evaluate the FR on macrophages of atherosclerotic animals, golden Syrian hamsters were maintained on a hyperlipidemic diet until extensive vascular lesions had developed. Uptake of folic acid conjugated to fluorescent tags was then examined in tissue fragments from lesion-prone areas, and peritoneal activated macrophages were harvested from the same animals. Spectrofluorimetric and fluorescence microscopic analyses showed a significantly greater uptake of folate-conjugates by peritoneal macrophages of hyperlipidemic hamsters compared with those of hamsters fed a normal or folate-deficient diet. Systemically administered folate-fluorescent conjugates were found to accumulate as bright spots in protrusions of atherosclerotic plaques populated by macrophages, whereas a low level of fluorescence was detected uniformly dispersed across the lesion. The uptake of the folate conjugate by U937 macrophage cells grown in a high-lipid culture medium was significantly higher than in controls. Our data thus indicate that hyperlipidemic conditions induce an increased uptake of folate attributable to the over-expression of FRs on activated macrophages. This increase in FR expression can be exploited to deliver folate-linked compounds selectively to atherosclerotic lesions. This work was supported by a grant from the Romanian Academy and Ministry of Education, Research and Technology, Bucharest, Romania, and partially by a grant from Endocyte and the Indiana 21st Century Fund.     

Lipoteichoic acid isolated from Lactobacillus plantarum suppresses LPS-mediated atherosclerotic plaque inflammation

Chronic inflammation plays an important role in atherogenesis. Experimental studies have demonstrated the accumulation of monocytes/macrophages in atherosclerotic plaques caused by inflammation. Here, we report the inhibitory effects of lipoteichoic acid (LTA) from Lactobacillus plantarum (pLTA) on atherosclerotic inflammation. pLTA inhibited the production of proinflammatory cytokines and nitric oxide in lipopolysaccharide (LPS)-stimulated cells and alleviated THP-1 cell adhesion to HUVEC by down-regulation of adhesion molecules such as intracellular adhesion molecule-1 (ICAM-I), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. The inhibitory effect of pLTA was mediated by inhibition of NF-κB and activation of MAP kinases. Inhibition of monocyte/macrophage infiltration to the arterial lumen was shown in pLTA-injected ApoE^−/− mice, which was concurrent with inhibition of MMP-9 and preservation of CD31 production. The anti-inflammatory effect mediated by pLTA decreased expression of atherosclerotic markers such as COX-2, Bax, and HSP27 and also cell surface receptors such as TLR4 and CCR7. Together, these results underscore the role of pLTA in suppressing atherosclerotic plaque inflammation and will help in identifying targets with therapeutic potential against pathogen-mediated atherogenesis.