Allograft inflammatory factor-1 augments macrophage phagocytotic activity and accelerates the progression of atherosclerosis in ApoE-/- mice

Allograft inflammatory factor (AIF)-1, originally cloned from a rat heart allograft under chronic rejection, is induced in various inflammatory conditions including atherosclerosis. Using mouse AIF-1 transfected macrophages and AIF-1 transgenic (AIF-1 Tg) mice, we analyzed the influence of AIF-1 overexpression on macrophage phagocytosis and the development of atherosclerosis. The AIF-1 transfectants showed significantly increased phagocytosis of latex beads and E. coli BioParticles as well as incorporation of acetylated low-density lipoprotein (LDL) compared to those of vector controls. Concordant results were obtained with elicited peritoneal exudate cells from AIF-1 Tg mice. When AIF-1 Tg mice were crossbred with apolipoprotein E knockout mice (ApoE-/-), these AIF-1 Tg ApoE-/- mice developed significantly increased atherosclerotic lesions compared to ApoE-/- mice. These results suggest that enhanced AIF-1 expression leads to augmented incorporation of degenerated LDL by macrophages and promotes development of atherosclerotic vasculopathy.     

In vitro incubation of low-density lipoproteins with inflammatory cells causes enhanced degradation by macrophages in culture

Utilizing the polyvinyl sponge-implant model, we have reported in vivo modification of low-density lipoproteins (LDL) isolated from interstitial inflammatory fluid (IF) of the rabbit. Further studies on the metabolism of IF-LDL by resident mouse peritoneal macrophages (MPM), demonstrated enhanced uptake and degradation of these modified lipoproteins by scavenger mechanisms. Based upon these studies, we attempted to examine the mechanisms of these observed in vivo modifications in IF-LDL by in vitro incubation of plasma LDL with inflammatory fluid subfractions. Incubation of LDL with inflammatory cells at 37°C resulted in an increased anodal electrophoretic mobility and alteration in apolipoprotein (APO) composition. Subsequent incubation of this modified plasma LDL with MPM resulted in a significant increase in cell surface binding and an increase in the appearance of degradation products in the medium. The formation of lipid peroxides, measured as thiobarbituric acid-reacting substances (T Bars), increased with the time of LDL incubation with inflammatory cells. Conversely, incubation of LDL with cell-free, lipoprotein-deficient IF (LPDIF,d>1.210 g/ml) significantly inhibited LDL degradation by MPM. LPDIF did not alter the electrophoretic mobility of LDL or result in the appearance of T Bars in the medium. These results implicate peroxidative reactions associated with an inflammatory response as mediators of the in vivo modifications in IF-LDL which facilitates enhanced uptake via the scavenger receptor in MPM.     

beta-Amyloid promotes accumulation of lipid peroxides by inhibiting CD36-mediated clearance of oxidized lipoproteins

BACKGROUND: Recent studies suggest that hypercholesterolemia, an established risk factor for atherosclerosis, is also a risk factor for Alzheimer's disease. The myeloid scavenger receptor CD36 binds oxidized lipoproteins that accumulate with hypercholesterolemia and mediates their clearance from the circulation and peripheral tissues. Recently, we demonstrated that CD36 also binds fibrillar beta-amyloid and initiates a signaling cascade that regulates microglial recruitment and activation. As increased lipoprotein oxidation and accumulation of lipid peroxidation products have been reported in Alzheimer's disease, we investigated whether beta-amyloid altered oxidized lipoprotein clearance via CD36. METHODS: The availability of mice genetically deficient in class A (SRAI & II) and class B (CD36) scavenger receptors has facilitated studies to discriminate their individual actions. Using primary microglia and macrophages, we assessed the impact of Abeta on: (a) cholesterol ester accumulation by GC-MS and neutral lipid staining, (b) binding, uptake and degradation of 125I-labeled oxidized lipoproteins via CD36, SR-A and CD36/SR-A-independent pathways, (c) expression of SR-A and CD36. In addition, using mice with targeted deletions in essential kinases in the CD36-signaling cascade, we investigated whether Abeta-CD36 signaling altered metabolism of oxidized lipoproteins. RESULTS: In primary microglia and macrophages, Abeta inhibited binding, uptake and degradation of oxidized low density lipoprotein (oxLDL) in a dose-dependent manner. While untreated cells accumulated abundant cholesterol ester in the presence of oxLDL, cells treated with Abeta were devoid of cholesterol ester. Pretreatment of cells with Abeta did not affect subsequent degradation of oxidized lipoproteins, indicating that lysosomal accumulation of Abeta did not disrupt this degradation pathway. Using mice with targeted deletions of the scavenger receptors, we demonstrated that Abeta inhibited oxidized lipoprotein binding and its subsequent degradation via CD36, but not SRA, and this was independent of Abeta-CD36-signaling. Furthermore, Abeta treatment decreased CD36, but not SRA, mRNA and protein, thereby reducing cell surface expression of this oxLDL receptor. CONCLUSIONS: Together, these data demonstrate that in the presence of beta-amyloid, CD36-mediated clearance of oxidized lipoproteins is abrogated, which would promote the extracellular accumulation of these pro-inflammatory lipids and perpetuate lipid peroxidation.     

Macrophage foam cell formation during early atherogenesis is determined by the balance between pro-oxidants and anti-oxidants in arterial cells and blood lipoproteins

Atherosclerosis is a multifactorial disease, where more than one mechanism, along more than one step, contributes to macrophage cholesterol accumulation and foam cell formation, the hallmark of early atherogenesis. Arterial macrophages take up oxidized low-density lipoproteins (Ox-LDL), leading to cellular accumulation of cholesterol and oxysterols. Atherogenic modifications of LDL include, in addition to oxidation, retention and aggregation. Intervention to inhibit LDL oxidation can affect the above additional LDL modifications. Indeed, we have demonstrated in the atherosclerotic apolipoprotein E-deficient mice that consumption of vitamin E or of flavonoids from red wine or licorice decreased LDL oxidation, LDL retention, and LDL aggregation and attenuated macrophage foam cell formation and atherosclerosis. The balance between pro-oxidants and anti-oxidants in the LDL particle (such as cholesteryl ester vs. vitamin E), as well as in arterial wall macrophages (such as NADPH oxidase vs. glutathione), determines the extent of LDL oxidation. Antioxidants can protect LDL from oxidation not only by their binding to the lipoprotein, but also following their accumulation in cells of the arterial wall. Whereas antioxidants can prevent the formation of Ox-LDL, human serum paraoxonase (PON 1), an HDL-associated esterase that hydrolyzes organophosphates, can eliminate oxidized LDL (by hydrolysis of its lipid peroxides), which is formed when antioxidant protection is not sufficient. Ox-LDL, in turn, can inactivate paraoxonase activity. Thus, the combination of antioxidants together with active paraoxonase decreases the formation of Ox-LDL and preserves PON1's ability to hydrolyze this atherogenic lipoprotein and hence, to attenuate atherosclerosis.     

小鼠腹腔巨噬细胞A型清道夫受体的研究

目的:研究A型清道夫受体在摄取氧化型低密度脂蛋白中的作用。方法:选用离体A型清道夫受体基因敲除和基因未敲除小鼠腹腔巨噬细胞作为实验对象,通过比较两组细胞对氧化型低密度脂蛋白结合和摄取的差异,来分析A型清道夫受体在摄取氧化型低密度脂蛋白中的作用。结果:基因敲除组小鼠腹腔巨噬细胞对氧化型低密度脂蛋白的摄取较基因未敲除组少35.8%,对氧化型低密度脂蛋白的降解较基因未敲除组少42%。结论:A型清道夫受体在摄取氧化型低密度脂蛋白中不占主导地位,有大约70%的氧化型低密度脂蛋白可能通过非A型清道夫受体途径被摄取。     

Induction of macrophage growth by the lipid moiety of lipoprotein and its augmentation by denaturation of the lipoproteins

Lipoprotein (d less than 1.21) isolated from mouse tumor ascitic fluid or mouse-serum induced growth of peritoneal macrophages in vitro. Lipoprotein fractions that stimulated macrophage growth were the chylomicron, very-low-density lipoprotein (VLDL), and low-density lipoprotein (LDL), whereas the high-density lipoprotein (HDL) fraction did not. Lipids extracted from total lipoprotein also showed significant macrophage-growth-stimulating activity and lost this activity when hydrolyzed. The macrophage-growth-stimulating activity of the lipoprotein (d less than 1.21) was increased about ten times by heat treatment of the lipoprotein (100 degrees C, 30 min). The HDL fraction that had no activity in the native form also showed activity after heat treatment. Lipoprotein-depleted ascitic fluid and simple proteins such as bovine serum albumin (BSA) had no activity even after heat treatment. These results show that the lipid moiety of lipoproteins caused proliferation of macrophages and that denatured lipoproteins were more effective than native ones.     

A comparative microscopic and biochemical study of the uptake of fluorescent and 125I-labeled lipoproteins by skin fibroblasts, smooth muscle cells, and peritoneal macrophages in culture.

Uptake of low density lipoprotein (LDL) and of acetyl LDL was compared in skin fibroblasts, smooth muscle cells, and peritoneal macrophages with the use of lipoproteins labeled with either 125I or the fluorescent probe 3,3'-dioctadecylindocarbocyanine (DiI). The uptake of DiI-labeled lipoproteins was assessed by quantitative spectrofluorometry and by fluorescence microscopy. The DiI was quantitatively retained by the cells, while the 125I-LDL was degraded and 125I-labeled degradation products were excreted from the cells. In smooth muscle cells and fibroblasts the uptake of LDL was virtually the same whether measured with the use of the DiI or 125I-label (sum of cell-associated and degraded 125I). The labeling of acetyl LDL with DiI enhanced its uptake in peritoneal macrophages by an average of 18%. With the DiI label, lipoprotein uptake (DiI-LDL for smooth muscle cells and skin fibroblasts and DiI-acetyl-LDL for mouse peritoneal macrophages) could be determined after as little as 10 minutes of incubation at 37 C. The pattern of uptake of the DiI-labeled lipoproteins was consistent with binding to specific receptors, because no DiI could be detected in mutant cells without LDL receptors, and uptake was competitively inhibited by addition of excess unlabeled lipoprotein. When the DiI-labeled lipoproteins were removed from the medium, there was a 5-15% loss of DiI from all cell types studied over the first 24 hours. Thereafter, DiI loss from cells was dependent on cell type and culture medium. No further loss of DiI occurred from skin fibroblasts for up to 96 hours of incubation in medium supplemented with either lipoprotein-deficient serum (LPDS) or 10% fetal bovine serum. During this same time period there was a 40-60% loss of DiI from smooth muscle cells and macrophages incubated in medium supplemented with LPDS. Most of the DiI lost from the cells (60-70%) could be recovered in the culture medium but was not the result of cell death, as was indicated by the relatively constant protein concentrations per dish. The loss of DiI was markedly reduced in smooth muscle cells and macrophages when 10% fetal bovine serum was substituted for the LPDS in the culture medium. This suggests that some cells incubated with LPDS undergo changes, perhaps in the plasma membrane, that alter their ability to retain the DiI. In the presence of 10% fetal bovine serum, however, the DiI label is quantitatively retained by all cells tested for up to 96 hours.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Modulatory Effect of Lipoproteins on the Release of Interleukin 1 by Human Peritoneal Macrophages Stimulated with β-1,3-d-Polyglucose Derivatives

Human peritoneal macrophages were stimulated in vitro with beta-1,3-D-polyglucose-derivatized microbeads (GDM) or soluble aminated beta-1,3-D-polyglucose (AG) in combination with lipoproteins. The release of interleukin 1 (IL-1) was analysed in cell supernatants in a thymocyte proliferation assay. We report that the release of IL-1 is markedly enhanced in macrophages stimulated with polyglucose in either form in combination with native low-density lipoprotein (LDL) or acetyl LDL at a concentration of 100 micrograms/ml. By increasing the amount of lipoproteins up to 10-fold, the IL-1 release decreased sharply. There was only a slight increase in activity when high-density lipoprotein (HDL) or very low-density lipoprotein (VLDL) were added. Other stimulatory agents, such as gamma interferon (IFN-gamma) and lipopolysaccharide (LPS) showed about half the activity of polyglucose. There was no significant difference between native LDL and acetyl LDL in potentiating effect. Our observations also suggest that the potentiating effect of LDL or acetyl LDL is not dependent on binding to their specific receptors. These findings provide a connection between macrophages, lipoproteins, and cytokines with regard to their role in the inflammatory response.     

Essential roles of lipoxygenases in LDL oxidation and development of atherosclerosis

Oxidative modification of low-density lipoprotein (LDL) is one of the critical steps for the development of atherosclerosis. Accumulating studies have indicated that 12/15-lipoxygenase highly expressed in macrophages plays an essential role in the oxidation of circulating LDL. It has been demonstrated that LDL needs to bind the LDL receptor-related protein (LRP), a cell-surface receptor, prior to its oxidation by 12/15-lipoxygenase expressed in macrophages. LRP is suggested to mediate the selective transfer of cholesteryl ester in LDL to the plasma membrane of macrophages without endocytosis and degradation of the LDL particle. At the same time, binding of LDL to LRP translocates the 12/15-lipoxygenase from the cytosol to the plasma membrane. It is also demonstrated that 5-lipoxygenase localized in macrophages generates leukotrienes, which exhibit strong proinflammatory activities in cardiovascular tissues and contribute to lesion development. Therefore, the inhibition of these lipoxygenases may be effective in the prevention and treatment of the inflammatory diseases.     

Chlamydia pneumoniae augments the oxidized low-density lipoprotein-induced death of mouse macrophages by a caspase-independent pathway

Chlamydia pneumoniae is a common respiratory pathogen that is associated with an increased risk of cardiovascular disease. However, the mechanisms by which C. pneumoniae contributes to cardiovascular disease have not been determined yet. C. pneumoniae infection may accelerate the death of cells within atherosclerotic lesions and contribute to the formation of unstable lesions. To test this hypothesis, the impact of C. pneumoniae infection on the death of lipid-loaded mouse macrophages was investigated. It was observed that RAW 264.7 cells are highly susceptible to the toxic effects of oxidized low-density lipoprotein (LDL) and exhibit markers of cell death within 24 h of treatment with as little as 5 microg/ml oxidized LDL. Subsequent infection with either live C. pneumoniae or heat-killed or UV-inactivated C. pneumoniae at a low multiplicity of infection for 24 to 72 h stimulated both additional binding of annexin V and the uptake of propidium iodide. Thus, C. pneumoniae augments the effects of oxidized LDL on cell death independent of a sustained infection. However, unlike oxidized LDL, C. pneumoniae infection does not activate caspase 3 or induce formation of the mitochondrial transition pore or the fragmentation of DNA, all of which are classical markers of apoptosis. Furthermore, primary bone marrow macrophages isolated from mice deficient in Toll-like receptor 2 (TLR-2) but not TLR-4 are resistant to C. pneumoniae-induced death. These data suggest that C. pneumoniae kills cells by a caspase-independent pathway and that the process is potentially mediated by activation of TLR-2.     

The effect of allicin on blood and tissue lead content in mice

Previous experimental results have revealed that garlic (Alium sativum) can reduce lead toxicity and tissue lead content in lead-exposed rodents. In the present study, the effects of different doses of allicin, the main active constituent of garlic, in reducing of organ and blood lead levels were evaluated in mice exposed to 1,000 ppm of lead acetate in drinking water. Three groups of mice received allicin at doses of 12, 24, and 48 μg/kg orally (twice daily) during ongoing 14-day lead exposure. Mice were killed on experimental day 15. Allicin treatment reduced lead retention in blood and tissues. Reduction of lead concentration in blood and tissues was dose dependent. With the highest dose of allicin, the greatest rate of reduction of lead concentrations were observed in liver (73.7%), kidney (45%), brain (45%), and bone (44.4%), respectively. Liver zinc concentration was significantly reduced in all treated groups. It was concluded that allicin administered during lead exposure in mice can reduce tissue lead retention and, therefore, might have some therapeutic effect on lead poisoning.     

Macrophage uptake of low-density lipoprotein modified by 4-hydroxynonenal. An ultrastructural study

We have documented the ultrastructural characteristics of the uptake and processing by mouse peritoneal macrophages (MPM) of low-density lipoprotein (LDL) modified with 4-hydroxynonenal (HNE), an intermediate of lipid peroxidation. This was performed as part of a larger biochemical study assessing the role of LDL oxidation in lipid loading of macrophages during atherogenesis. Gold-labeled LDL that was modified with HNE leading to particle aggregation represented the morphologic probe used. When incubated with MPM, the probe became associated with short segments of cell membrane, probably derived from blebs or from lysed cells. At 37 degrees C there was a time-dependent increase in uptake by MPM, and at 4 hours the increase paralleled the degradation by MPM of 125I-labeled HNE-LDL-cAu. Clathrin-coated pits on the cell surface were consistently associated with probe. Uptake of probe appeared to occur via phagocytosis, because pseudopods frequently surrounded probe, and cytochalasin D quantitatively prevented probe uptake. A time-dependent increase was found in the number of gold particles per unit area within vacuoles, some of which were secondary lysosomes, based on acid phosphatase-positive staining. Thus, HNE-induced aggregation of LDL during oxidation, binding of aggregates to clathrin-coated pits on MPM, and subsequent phagocytosis may represent one of the ways lipid-laden foam cells are formed in vivo.     

Insoluble low-density lipoprotein-proteoglycan complexes enhance cholesteryl ester accumulation in macrophages.

The interaction of arterial proteoglycans (PGs) and low-density lipoproteins (LDLs) has been postulated to be an important factor in extracellular cholesterol accumulation in the arterial wall. In the present study, insoluble complexes of LDL and PG (LDL-PG) were prepared and their effects on cholesteryl ester accumulation in mouse peritoneal macrophages was tested. The cholesteryl ester content of macrophages incubated with LDL-PG for 3 days was greater than 20 times that observed in cells incubated with LDL alone. The uptake of 125I-LDL by macrophages was markedly stimulated if LDL was incorporated into a complex with PG. However, in contrast to either LDL or acetylated LDL (ALDL), the extent of subsequent degradation of LDL-PG by the cells was reduced. The uptake and degradation of LDL-PG complexes stimulated macrophage incorporation of 14C-oleic acid into cholesteryl oleate 4- to 5-fold over LDL alone; however, esterification was significantly less than that observed with ALDL, even though more LDL-PG was degraded. Ultrastructurally, macrophages incubated with LDL-PG complexes contained lipid droplets as well as numerous phagocytic vacuoles often containing material similar in appearance to insoluble complexes. These results demonstrate that components of the extracellular matrix, such as PG, can modify the catabolism of LDL by scavenger cells. This phenomenon may be potentially important with respect to foam-cell genesis from macrophages in the arterial wall.     

Allicin stimulates lymphocytes and elicits an antitumor effect: a possible role of p21ras

Allicin, the main organic allyl sulfur component in garlic, exhibits immune-stimulatory and antitumor properties. Allicin stimulated [(3)H]thymidine incorporation in mouse splenocytes and enhanced cell-mediated cytotoxicity in human peripheral mononuclear cells. Multiple administration (i.p.) of allicin elicited a marked antitumor effect in mice inoculated with B-16 melanoma and MCA-105 fibrosarcoma. The immune-stimulatory and antitumor effects of allicin are characterized by a bell-shaped curve, i.e. allicin at high, supra-optimal concentrations is less effective or inhibitory. Allicin induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in human peripheral mononuclear cells, and also in wild-type Jurkat T-cells. Allicin failed to activate ERK1/2 in Jurkat T cells that express p21(ras), in which Cys118 was replaced by Ser. These cells are not susceptible to redox-stress modification and activation. We postulate that the immune stimulatory effect of allicin is mediated by redox-sensitive signaling such as activation of p21(ras). It is suggested that the antitumor effect of allicin is related to its immune-stimulatory properties.     

In vitro glycated low-density lipoprotein interaction with human monocyte-derived macrophages.

Human low-density lipoprotein (LDL) was glycated in vitro (5 days, glucose 50 mmol/l), labelled with 125I, and its binding and uptake by human monocyte-derived macrophages studied. Glycation produced lower binding and lower uptake. Competition experiments using unlabelled LDL (control, glycated, and acetyl-LDL) showed that most glycated LDL was taken up by the apolipoprotein-B100: E receptor pathway. Results suggest that less of the glycated LDL may enter the cells via scavenger receptors, and very minute amount via non-saturable receptor-independent pathways.     

Isolation of the intracellular stage of Trypanosoma cruzi and its interaction with mouse macrophages in vitro.

Interaction between Trypanosoma cruzi spheromastigotes (amastigotes) and mouse macrophages was studied. The spheromastigotes, isolated from the spleens of infected mice, were incorporated and digested by the macrophages. By use of horseradish peroxidase labeling of the macrophage lysosomes we showed that fusion of lysosomes with phagocytic vacuoles containing T. cruzi occurred. Parasites showing alterations indicative of digestion were seen inside the phagocytic vacuoles. Our results suggest that intracellular sphermastigotes of T. cruzi, isolated from the spleens of infected mice, and not able to induce a protective infection in mouse macrophages maintained in vitro.     

The phenomenon of atherosclerosis reversal and regression: Lessons from animal models

Studies in non-rodent and murine models showed that atherosclerosis can be reversed. Atherosclerosis progression induced by high-fat or cholesterol-rich diet can be reduced and reversed to plaque regression after switching to a normal diet or through administration of lipid-lowering agents. The similar process should exist in humans after implementation of lipid-lowering therapy and as a result of targeting of small rupture-prone plaques that are major contributors for acute atherosclerotic complications. Lowering of low density lipoprotein (LDL) cholesterol and the activation of reverse cholesterol transport lead to a decline in foam cell content, to the depletion of plaque lipid reservoirs, a decrease in lesional macrophage numbers through the activation of macrophage emigration and, probably, apoptosis, dampening plaque inflammation, and the induction of anti-inflammatory macrophages involved in clearance of the necrotic core and plaque healing. By contrast, plaque regression is characterized by opposite events, leading to the retention of atherogenic LDL and oxidized LDL particles in the plaque, an increased flux of monocytes, the immobilization of macrophages in the intimal vascular tissues, and the propagation of intraplaque inflammation. Transfer of various apolipoprotein (apo) genes to spontaneously hypercholesterolemic mice deficient for either apoE or LDL receptor and, especially, the implementation of the transplantation murine model allowed studying molecular mechanisms of atherosclerotic regression, associated with the depletion of atherogenic lipids in the plaque, egress of macrophages and phenotypic switch of macrophages from the proinflammatory M1 to the anti-inflammatory M2.     

The pharmacological effects of allicin,a constituent of garlic oil

Garlic has been used in herbal medicine for thousands of years. While garlic oil contains many components and has been widely studied, the pharmacology of pure allicin, a constituent of garlic oil, is not well understood. We report that allicin inhibits human platelet aggregationin vitro without affecting cyclooxygenase or thromboxane synthase activity or cyclic adenosine monophosphate (AMP) levels. Allicin does not alter the activity of vascular prostacyclin synthase. However, it inhibits ionophore A23187-stimulated human neutrophil lysosomal enzyme release.In vivo allicin dilates the mesenteric circulation of the cat independent of prostaglandin release or a beta adrenergic mechanism.     

The effect of a high-fat diet on murine macrophage activity

The phagocytic, oxygen free radical generating and cytotoxic activities of macrophages from C57BL/6J mice fed either a normal or an atherogenic high-fat diet have been investigated. Phagocytosis of aggregated low density lipoprotein (LDL) was only slightly inhibited by the high-fat diet although phorbol myristate acetate (PMA)-induced hydrogen peroxide (H2O2) and superoxide anion (O2-) production was significantly reduced. Activation of tumoricidal activity against L929 target cells by lipopolysaccharide (LPS) or lymphocyte-derived macrophage-activating factor (MAF), but not N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), was also significantly reduced in macrophages from mice fed the high-fat diet. These results indicate that an atherogenic diet is capable of significantly affecting the responsiveness of macrophages to a number of stimulatory agents which act via specific membrane receptors.