Macrophage‐mediated cholesterol handling in atherosclerosis

Formation of foam cells is a hallmark at the initial stages of atherosclerosis. Monocytes attracted by pro‐inflammatory stimuli attach to the inflamed vascular endothelium and penetrate to the arterial intima where they differentiate to macrophages. Intimal macrophages phagocytize oxidized low‐density lipoproteins (oxLDL). Several scavenger receptors (SR), including CD36, SR‐A1 and lectin‐like oxLDL receptor‐1 (LOX‐1), mediate oxLDL uptake. In late endosomes/lysosomes of macrophages, oxLDL are catabolysed. Lysosomal acid lipase (LAL) hydrolyses cholesterol esters that are enriched in LDL to free cholesterol and free fatty acids. In the endoplasmic reticulum (ER), acyl coenzyme A: cholesterol acyltransferase‐1 (ACAT1) in turn catalyses esterification of cholesterol to store cholesterol esters as lipid droplets in the ER of macrophages. Neutral cholesteryl ester hydrolases nCEH and NCEH1 are involved in a secondary hydrolysis of cholesterol esters to liberate free cholesterol that could be then out‐flowed from macrophages by cholesterol ATP‐binding cassette (ABC) transporters ABCA1 and ABCG1 and SR‐BI. In atherosclerosis, disruption of lipid homoeostasis in macrophages leads to cholesterol accumulation and formation of foam cells.     

Lysosomal cholesterol accumulation in macrophages leading to coronary atherosclerosis in CD38−/− mice

The disruption in transportation of oxLDL‐derived cholesterol and the subsequent lipid accumulation in macrophages are the hallmark events in atherogenesis. Our recent studies demonstrated that lysosomal Ca²⁺ messenger of nicotinic acid adenine dinucleotide phosphate (NAADP), an enzymatic product of CD38 ADP‐ribosylcyclase (CD38), promoted lipid endocytic trafficking in human fibroblast cells. The current studies are designed to examine the functional role of CD38/NAADP pathway in the regulation of lysosomal cholesterol efflux in atherosclerosis. Oil red O staining showed that oxLDL concentration‐dependently increased lipid buildup in bone marrow‐derived macrophages from both wild type and CD38^?/?, but to a significant higher extent with CD38 gene deletion. Bodipy 493/503 fluorescence staining found that the deposited lipid in macrophages was mainly enclosed in lysosomal organelles and largely enhanced with the blockade of CD38/NAADP pathway. Filipin staining and direct measurement of lysosome fraction further revealed that the free cholesterol constituted a major portion of the total cholesterol segregated in lysosomes. Moreover, in situ assay disclosed that both lysosomal lumen acidity and the acid lipase activity were reduced upon cholesterol buildup in lysosomes. In CD38^?/? mice, treatment with Western diet (12 weeks) produced atherosclerotic damage in coronary artery with striking lysosomal cholesterol sequestration in macrophages. These data provide the first experimental evidence that the proper function of CD38/NAADP pathway plays an essential role in promoting free cholesterol efflux from lysosomes and that a defection of this signalling leads to lysosomal cholesterol accumulation in macrophages and results in coronary atherosclerosis in CD38^?/? mice.     

Expression of lipoprotein receptors in the aortic walls of diabetic APA hamsters.

Syrian hamsters of the APA strain (APA hamsters) have recently been demonstrated to develop atheromatous lesions in the aortic arches under the diabetic condition induced by a single injection of streptozotocin (SZ). Various lipoprotein receptors are reported to play important roles in atherogenesis mainly in vitro, while there are few reports on the relative expressions of these receptors in vivo. In this study, we therefore examined messenger RNA (mRNA) expressions of several lipoprotein receptors on the aortic arches of diabetic APA hamsters at 6, 14 and 26 weeks after the injection (WAI) of SZ. In semi-quantitative RT-PCR, scavenger receptor (SR)-AI, macrosialin (MS)/CD68, and receptor for advanced glycation end-products (RAGE) mRNAs showed significant increases at 6 WAI of SZ, and SR-AI and CD36 mRNA obviously increased until 26 WAI, as compared with the control. Low-density lipoprotein receptor mRNA showed a significant decrease at 14 and 26 WAI, and SR-BI mRNA significantly decreased at 6 and 14 WAI, as compared with the control. Very low-density lipoprotein receptor mRNA was at the same level as the control. By means of in situ hybridization, SR-AI, MS/CD68 and RAGE mRNA were detected in the foam cells of the fatty streaks at 6 WAI, which suggested that SR-AI, MS/CD68 and RAGE play crucial roles in the formation of the fatty streaks, the initial lesions of atherogenesis in diabetic APA hamsters. SR-AI and CD36 were also believed to be related to the progression of atherogenesis in this model.     

Apoptotic macrophage-derived foam cells of human atheromas are rich in iron and ferritin, suggesting iron-catalysed reactions to be involved in apoptosis

We investigated the presence of low-molecular-weight iron and ferritin in human atheromas, and their possible relation to the apoptotic process. Arterial wall segments with fatty streaks were collected from coronary arteries and thoracic aortas of 12 clinical autopsy cases with general atherosclerosis. Normal appearing regions from the same cases together with normal coronary arteries from seven young forensic autopsy cases, without any sign of atherosclerosis, were used for comparison. Anti-CD68 (macrophage marker) and anti-ferritin antibodies were applied to serial sections of the arterial wall segments, fixed in formadehyde and embedded in paraffin wax, using an avidin-biotin complex (ABC) technique. Similarly, apoptotic cells were assayed by the TUNEL technique, while low-molecular-weight iron was cytochemically detected by autometallography. Cell counting and computerised image analysis were performed to compare the distribution of macrophages, ferritin- and iron-rich cells, and apoptotic cells in the intima, media, and adventitia of the arteries.

Pronounced ferritin accumulation, occurrence of lysosomal low-molecular-weight iron, and apoptosis mainly concerned CD68-positive cells (macrophages) in the atherosclerotic lesions. No ferritin- or CD68-positivity was found in normal coronary arteries from the young forensic-autopsy cases, while a moderate number of such cells were observed in the intima of normal looking vessel areas from the control cases. In the intima, cytosolic ferritin and low-molecular-weight iron with a lysosomal type distribution were found in many CD68-positive macrophages which frequently were surrounded by erythrocytes. A substantial number of apoptotic cells within the intima, media, and adventitia were registered in all atherosclerotic lesions examined, although mainly in the vulnerable macrophage-enriched areas of the atheroma shoulder.

We suggest that iron may occur within the cytosol, mainly bound in ferritin, but also in low-molecular weight, redox-active form within the acidic vacuolar apparatus of macrophages and macrophage-derived foam cells following erythrophagocytosis or phagocytosis of apoptotic cells. Low-molecular-weight iron within lysosomes, present due to degradation of iron-containing structures, such as ferritin, may partially become exocytosed and contribute to cell-mediated LDL-oxidation. Moreover, such lysosomal iron may also sensitise lysosomes to oxidative stress and induce apoptosis of macrophage/foam-cells that may result in instability and rupture of atherosclerotic plaques.     

Monocytes treated with ciprofloxacin and oxyLDL express myristate,priming atherosclerosis

Antibiotics are essential in many life‐threatening diseases. On the other hand, improper use of antibiotics can be disastrous. Cell morphological changes were observed in the ciprofloxacin‐treated cells starting at 48 hours. Changes in cell morphology were continuously observed up to 14 days, which showed gradual morphological changes from monocyte to plaque‐like cells at day 12, and foam cell, which is an intermediate stage in atherosclerosis was observed at day 8, which was confirmed with Oil Red O staining. Flow cytometry data revealed that oxidized LDL (oxyLDL)‐induced cells showed 60.16% of CD64 (proinflammatory macrophage markers) and no expression of CD23 (anti‐inflammatory macrophage markers), whereas ciprofloxacin‐treated cells expressed 67.97% of CD64 and 13.78% of CD23. Chemokine antibody array analysis revealed that ciprofloxacin exposed cells showed a proinflammatory role (ENA78, Eotaxin1, Eotaxin2, IP‐10, MIG, MIP‐3β, SDF‐1β, TECK, CXCL16, and Fractalkine). Liquid chromatography with tandem mass spectrometry (LC‐MS/MS) revealed that myristic acid was incorporated into a protein with 68 kDa molecular mass in exposing oxyLDL‐induced monocytes with ciprofloxacin, which could be a reason for the observed foam cells and in vitro plaque formation. As myristic acid primes atherosclerosis, it is better to limit the intake of antibiotics like ciprofloxacin for common illness, specifically the high‐risk patients, which may contribute to atherosclerosis.     

Suppression of FAT/CD36 mRNA by human growth hormone in pancreatic β-cells

Fatty acid-induced damage in pancreatic β-cells is assumed to play an important role in the development of type 2 diabetes. Lactogens (prolactin, placental lactogen and growth hormone) improve β-cell survival via STAT5 activation but the molecular targets are incompletely characterized. The aim of this study was to examine the effect of human growth hormone (hGH) on mRNAs of fatty acid transport and binding proteins expressed in pancreatic β-cells, and to examine this in relation to β-cell survival after exposure to fatty acids. hGH decreased mRNA levels of FAT/CD36, whereas mRNAs of GPR40, FASN, FABP2, FATP1 and FATP4 were unchanged. RNAi against FAT/CD36 decreased fatty acid-induced apoptosis. Over-expression of constitutively active STAT5 was able to mimic hGH’s suppression of FAT/CD36 expression, whereas dominant negative STAT5 was unable to block the effect of hGH indicating that STAT5 did not bind directly to the FAT/CD36 promoter. The hGH-mediated suppression of FAT/CD36 mRNA was associated with a decrease in palmitate uptake and fatty acid-induced basal hyper-secretion of insulin resulting in improved glucose-stimulated insulin secretion. This study suggests that hGH can protect β-cells against fatty acid-induced damages.     

长江刀鲚生殖洄游期间脂肪酸组成及含量变化分析

为探究长江刀鲚生殖洄游过程中脂肪酸组成及其含量变化规律,研究选择洄游距离、卵巢发育和规格大小3个影响因子设置梯度,对长江刀鲚肝胰腺、肌肉和卵巢的脂肪酸组成及含量进行实验分析。实验结果显示, 62尾雌性刀鲚3个组织均检测出28种脂肪酸,以单不饱和脂肪酸(MUFA)含量最高,大于各组织总脂肪酸含量的56.23%,各类脂肪酸中的C18:1、C16:0、C16:1、DHA和EPA含量较高,为主要脂肪酸。在生殖洄游过程中,刀鲚肝胰腺总脂肪含量随洄游距离的延长呈上升趋势,从崇明江段的(526.61±38.50) mg/g增加至安庆江段的(587.21±124.72) mg/g,而肌肉和卵巢总脂肪酸含量呈显著下降趋势,分别下降了33.03%和57.09%(P<0.05)。在各体长组中,肌肉总脂肪酸、SFA、MUFA和PUFA含量与体长呈正相关(P<0.05),而肝胰腺和卵巢总脂肪酸及各类脂肪酸含量与体长无显著相关性(P>0.05)。在卵巢发育过程中,刀鲚肝胰腺和肌肉总脂肪酸、MUFA和PUFA含量随卵巢由Ⅱ期发育至Ⅳ期均呈下降趋势,总脂肪酸含量分别减少了47.56%和22.40%,...     

Proteomic analysis of liver tissue from HBx‐transgenic mice at early stages of hepatocarcinogenesis

The hepatitis B virus X‐protein (HBx), a multifunctional viral regulator, participates in the viral life cycle and in the development of hepatocellular carcinoma (HCC). We previously reported a high incidence of HCC in transgenic mice expressing HBx. In this study, proteomic analysis was performed to identify proteins that may be involved in hepatocarcinogenesis and/or that could be utilized as early detection biomarkers for HCC. Proteins from the liver tissue of HBx‐transgenic mice at early stages of carcinogenesis (dysplasia and hepatocellular adenoma) were separated by 2‐DE, and quantitative changes were analyzed. A total of 22 spots displaying significant quantitative changes were identified using LC‐MS/MS. In particular, several proteins involved in glucose and fatty acid metabolism, such as mitochondrial 3‐ketoacyl‐CoA thiolase, intestinal fatty acid‐binding protein 2 and cytoplasmic malate dehydrogenase, were differentially expressed, implying that significant metabolic alterations occurred during the early stages of hepatocarcinogenesis. The results of this proteomic analysis provide insights into the mechanism of HBx‐mediated hepatocarcinogenesis. Additionally, this study identifies possible therapeutic targets for HCC diagnosis and novel drug development for treatment of the disease.     

Content and composition of phosphoglycerols and neutral lipids at different developmental stages of the eggs of the codling moth,Cydia pomonella (Lepidoptera: Tortricidae)

Phosphoglycerol, triacylglycerol, diacylglycerol, and free fatty acid content was studied in eggs of the codling moth Cydia pomonella at the white, red ring, and black head developmental stages. The composition of total phosphoglycerols and of the three classes of neutral lipids was also analyzed. The highest total lipid content was found in eggs at the white stage, the amount decreasing during development mainly as a result of a diminution in the quantity of phosphoglycerols, which account for approximately 50% of total content at all stages of egg development. The amount of triacylglycerols and free fatty acids changes significantly during development, whereas only minor changes were found in diacyglycerol levels. The total phosphoglycerol acyl composition of eggs at the white and red ring stages is similar, whereas differences are evident at the black head stage of development. Triacylglycerols and free fatty acids are enriched in saturated fatty acids in all analyzed stages. The acyl profile of diacylglycerols is different at each stage. The unsaturation index decreases in diacylglycerols and free fatty acids as a function of egg development. The results of the present paper suggest that triacylglycerols may constitute an important source of energy during the final period of egg development while phosphoglycerols may function as fuel during the beginning. Phosphoglycerols could be precursors for the triacylglycerol biosynthesis that takes place between white and red ring stages.     

Degradation of yolk in the brine shrimp Artemia. Biochemical and morphological studies on the involvement of the lysosomal system

The degradation of yolk granules during the development of Artemia was studied. The results obtained suggest that lysosomes are involved in the process. In homogenates of embryos and larvae at different stages of development, the distribution of 2 lysosomal markers, acid phosphatase and cathepsin B, was studied by sucrose isopycnic gradient centrifugation. Three peaks of enzyme activity of densities > 1.3 and around 1.25 and 1.18 were observed. As revealed by electron microscope analysis, the 3 peaks were found to be associated with increasingly degraded yolk structures which stained for acid phosphatase. The process can be mimicked in vitro by incubating isolated yolk granules and lysosomes. The enzyme activity levels of the 3 peaks observed during development presented an oscillatory pattern, suggesting that degradation of yolk is cyclic. Five cycles of degradation were observed during the initial 60 hr of development.     

Morphofunctional state of the digestive tract in the early stages of atherosclerosis]

Cholesterol, alpha- and beta-lipoproteids, serotonin were determined in the blood serum of dogs which were on atherogenic diet for 2 months. The serotonin content was examined in various parts of the gastrointestinal tract (GIT). Parallel studies of the structural changes were carried out in the vascular system (VS) and various parts of the GIT. There was found a direct correlation between a rise in the cholesterol and serotonin level in the blood and serotonin in the GIT tissues. The initial stages of atherosclerotic changes were revealed in the vascular system. Along with compensatory-adaptive changes detected in the duodenum and the upper protions of the small intestine, initial stages of dystrophic-atrophic processes were observed in the lower portions. Comparative analysis of biochemical and morphological data indicated that disturbances of the morphofunctional state in the GIT played an important role in the genesis of the early stages of atherosclerosis.     

Requirement for CD154 in the progression of atherosclerosis.

Atherosclerosis is a systemic disease of the large arteries, and activation of inflammatory pathways is important in its pathogenesis. Increasing evidence supports the importance of CD40-CD154 interactions in atherosclerosis, interactions originally known to be essential in major immune reactions and autoimmune diseases. CD40 is present on atheroma-derived cells in vitro and in human atheromata in situ. Ligation of CD40 on atheroma-associated cells in vitro activates the production of chemokines, cytokines, matrix metalloproteinases, adhesion molecules and tissue factor, substances responsible for lesion progression and plaque destabilization. Administration of antibody against CD154 to low-density lipoprotein receptor-deficient mice has been shown to reduce atherosclerosis and decrease T-lymphocyte and macrophage content; however, only initial lesions were studied. Here, we determined the effect of genetic disruption of CD154 in ApoE-/- mice in both initial and advanced atherosclerotic lesions. Plaque area was reduced 550%. In contrast to previous reports, initial lesion development was not affected. Advanced plaques in CD154-/-ApoE-/- mice had a less-lipid-containing, collagen-rich, stable plaque phenotype, with a reduced T-lymphocyte/macrophage content. These data indicate that CD40-CD154 signaling is important in late atherosclerotic changes, such as lipid core formation and plaque destabilization.     

DropArray™, a Wall‐Less 96‐Well Plate for Uptake and Immunofluorescence Microscopy,Confirms CD22 Recycles

CD22 is a cell surface glycoprotein restricted to normal and malignant B‐cells and is the target of several anti‐CD22 antibody‐based cancer therapies. For therapeutic antibody‐payload conjugates, it is important to understand the subcellular trafficking of anti‐CD22 antibodies to optimize antibody and/or linker–drug properties to maximize antitumor efficacy. It is agreed that anti‐CD22 antibodies rapidly internalize, but controversial whether they recycle or are degraded in lysosomes, and it is unclear if trafficking is antibody or cell‐type dependent. No studies examined anti‐CD22 trafficking to either pathway in B‐cells over time by dual immunofluorescence microscopy, likely partly because multiple samples of suspension cells are tedious to stain. We overcame this by using DropArray?, a novel wall‐less 96‐well plate technology allowing rapid simultaneous staining of suspension or adherent cells in small (10–20 μL) volumes. We examined the time‐course of trafficking of five different anti‐CD22 antibodies in eight B‐cell lines representing four B‐cell cancer types and show that in all cases antibodies internalize within 5 min and recycle, with only small amounts eventually trafficking to lysosomes. CD22 also localizes to recycling endosomes at steady state in the absence of antibody. Our data may help explain the differential efficacies of anti‐CD22 antibodies conjugated to different therapeutic payloads.      

Changes in fatty acid transport and transporters are related to the severity of insulin deficiency

We have examined the effects of streptozotocin (STZ)-induced diabetes (moderate and severe) on fatty acid transport and fatty acid transporter (FAT/CD36) and plasma membrane-bound fatty acid binding protein (FABPpm) expression, at the mRNA and protein level, as well as their plasmalemmal localization. These studies have shown that, with STZ-induced diabetes, 1) fatty acid transport across the plasma membrane is increased in heart, skeletal muscle, and adipose tissue and is reduced in liver; 2) changes in fatty acid transport are generally not associated with changes in fatty acid transporter mRNAs, except in the heart; 3) increases in fatty acid transport in heart and skeletal muscle occurred with concomitant increases in plasma membrane FAT/CD36, whereas in contrast, the increase and decrease in fatty acid transport in adipose tissue and liver, respectively, were accompanied by concomitant increments and reductions in plasma membrane FABPpm; and finally, 4) the increases in plasma membrane transporters (FAT/CD36 in heart and skeletal muscle; FABPpm in adipose tissue) were attributable to their increased expression, whereas in liver, the reduced plasma membrane FABPpm appeared to be due to its relocation within the cell in the face of slightly increased expression. Taken together, STZ-induced changes in fatty acid uptake demonstrate a complex and tissue-specific pattern, involving different fatty acid transporters in different tissues, in combination with different underlying mechanisms to alter their surface abundance.     

Characterization of macrophage-like cells in the external layers of human small and large intestine

Summary In the external layers of human small and large intestine macrophage-like cells were characterized by immunohistochemical, histochemical and electronmicroscopical methods. Using immunohistochemistry and a number of monoclonal antibodies, the presence and distribution of phenotypic subpopulations of macrophages were evaluated. In all locations macrophage-like cells were identified with antibody EBM11, which recognizes CD68 antigen, C3bi which recognizes CD11b, and partly with an antibody which recognizes protein 150,95 (CD11c). Macrophage-like cells in the external muscle layer were HLA-DR-positive (expressing the MHC class-II antigen), in contrast to macrophage-like cells in the subserosa and submucosa. Macrophage-like cells in the external muscle layer were mostly acid phosphatase-negative, and at the electron-microscopic level they were found to have features of macrophages: primary lysosomes, coated vesicles and pits. However, very few secondary lysosomes were present. Birbeck granules were not observed. It is concluded that in the external muscle layer of human small and large intestine numerous macrophages of a special type are present. It is discussed whether this cell type plays a role in gastrointestinal motility and/or has an immunological function.     

Autophagy maturation associated with CD38‐mediated regulation of lysosome function in mouse glomerular podocytes

Podocytes are highly differentiated glomerular epithelial cells that contribute to the glomerular barrier function of kidney. A role for autophagy has been proposed in maintenance of their cellular integrity, but the mechanisms controlling autophagy in podocytes are not clear. The present study tested whether CD38‐mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes. Podocytes were found to exhibit a high constitutive level of LC3‐II, a robust marker of autophagosomes (APs), suggesting a high basal level of autophagic activity. Treatment with the mTOR inhibitor, rapamycin, increased LC3‐II and the content of both APs detected by Cyto‐ID Green staining and autophagolysosomes (APLs) measured by acridine orange staining and colocalization of LC3 and Lamp1. Lysosome function inhibitor bafilomycin A1 increased APs, but decreased APLs content under both basal and rapamycin‐induced conditions. Inhibition of CD38 activity by nicotinamide or silencing of CD38 gene produced the similar effects to that bafilomycin A1 did in podocytes. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, the fusion of APs with lysosomes in living podocytes was observed by co‐transfection of GFP‐LC3B and RFP‐Lamp1 expression vectors. A colocalization of GFP‐LC3B and RFP‐Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 shRNA, and bafilomycin. Moreover, blockade of the CD38‐mediated regulation by PPADS completely abolished rapamycin‐induced fusion of APs with lysosomes. These results indicate that CD38 importantly control lysosomal function and influence autophagy at the maturation step in podocytes.     

Inflammation in atherosclerosis: a cause or a result of vascular disorders?

Sound data support the concept that in atherosclerosis, inflammation and dyslipidemia intersect each other and that irrespective of the initiator, both participate from the early stages to the ultimate fate of the atheromatous plaque. The two partakers manoeuvre a vicious circle in atheroma formation: dyslipidaemia triggers an inflammatory process and inflammation elicits dyslipidaemia. Independent of the initial cause, the atherosclerotic lesions occur focally, in particular arterial-susceptible sites, by a process that, although continuous, can be arbitrarily divided into a sequence of consecutive stages that lead from fatty streak to the fibro-lipid plaque and ultimately to plaque rupture and thrombosis. In the process, the initial event is a change in endothelial cells (EC) constitutive properties. Then, the molecular alarm signals send by dysfunctional EC are decoded by specific blood immune cells (monocytes, T lymphocytes, neutrophils, mast cells) and by the resident vascular cells, that respond by initiating a robust inflammatory process, in which the cells and the factors they secrete hasten the atheroma development. Direct and indirect crosstalk between the cells housed within the nascent plaque, complemented by the increase in risk factors of atherosclerosis lead to atheroma development and outcome. The initial inflammatory response can be regarded as a defense/protective reaction mechanism, but its further amplification, speeds up atherosclerosis. In this review, we provide an overview on the role of inflammation and dyslipidaemia and their intersection in atherogenesis. The data may add to the foundation of a novel attitude in the diagnosis and treatment of atherosclerosis.