Changes in apolipoprotein B and oxidized low-density lipoprotein levels in gingival crevicular fluids as a result of periodontal tissue conditions.

BACKGROUND AND OBJECTIVE: Periodontal disease is a chronic inflammatory disease caused by bacterial infection that can lead to tooth loss. Gingival crevicular fluid can be collected easily and noninvasively. We previously discovered the presence of apolipoprotein B (apoB), the main constituent of low-density lipoprotein, and oxidized low-density lipoprotein (oxLDL) in the gingival crevicular fluid of healthy subjects. In this study, we investigated whether periodontal conditions affect the levels of apoB and oxLDL in gingival crevicular fluid. MATERIAL AND METHODS: The study population comprised 11 patients with chronic periodontitis. A pair of gingival crevicular fluid samples was collected from each patient at a healthy site and at a site with periodontitis (baseline samples). Thereafter, gingival crevicular fluid samples were collected from the same patients again at 4 and 8 wk after scaling and root planing (SRP). The levels of apoB, oxLDL, protein and cytokines in gingival crevicular fluid, in addition to gingival crevicular fluid volume, were measured. RESULTS: At baseline, the levels of apoB and oxLDL in gingival crevicular fluid were higher at the sites with periodontitis than at the healthy sites. The levels of apoB and oxLDL at periodontal sites decreased after SRP. The level of oxLDL in gingival crevicular fluid correlated well with the probing pocket depth. The oxLDL : apoB ratio in gingival crevicular fluid was significantly higher than that in plasma. CONCLUSIONS: The levels of apoB and oxLDL in gingival crevicular fluid change according to the periodontal tissue conditions.

Oxidized low-density lipoprotein increases interleukin-8 production in human gingival epithelial cell line Ca9-22.

BACKGROUND AND OBJECTIVE: Recent epidemiological studies have shown a correlation between periodontitis and hyperlipidemia. We have found high levels of oxidized low-density lipoprotein (OxLDL) in the gingival crevicular fluid of dental patients. In the present study, we tried to examine the possible role of OxLDL in periodontal inflammation in vitro. MATERIAL AND METHODS: Cells of the human gingival epithelial cell line Ca9-22 were cultured in media containing OxLDL, and the amounts of interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2)) produced were measured using ELISAs. RESULTS: Production of IL-8 by Ca9-22 cells was significantly increased when the cells were treated with OxLDL, but not with native LDL or acetylated LDL. Production of PGE(2) by Ca9-22 cells was enhanced by co-incubation with OxLDL and interleukin-1 beta (IL-1 beta). Scavenger receptor inhibitors, fucoidan and dextran sulfate, inhibited the OxLDL-induced IL-8 and PGE(2) production in the presence of IL-1 beta. The p(38) MAPK inhibitors SB203580 and SB202190 and the ERK inhibitor PD98059 inhibited the OxLDL-induced IL-8 production. Among oxidized lipids and chemically modified LDL, 7-ketocholesterol enhanced IL-8 production. CONCLUSION: This is the first report to show that OxLDL enhances IL-8 production in epithelial cells.

Detection of oxidized low-density lipoproteins in gingival crevicular fluid from dental patients.

BACKGROUND AND OBJECTIVE: Oxidative modification of low-density lipoprotein (LDL) occurs in various diseased tissues and sites of local inflammation. For example, an increased plasma oxidized low-density lipoprotein (OxLDL) level is a well-known risk marker for cardiovascular diseases. Gingival crevicular fluid, the exudate from gingival tissues into the sulci, can be easily collected in a non-invasive manner. However, the possible presence of OxLDL in gingival crevicular fluid has not been studied. In this study, we established a procedure to measure OxLDL in human gingival crevicular fluid. MATERIAL AND METHODS: Human gingival crevicular fluid was sampled with paper points or paper strips. The gingival crevicular fluid samples from healthy gingival sulci (pocket depth < 4 mm, n = 14) were subjected to western blot and/or sandwich ELISA. The amounts of OxLDL and LDL were measured by sandwich ELISA using an anti-oxidized phosphatidylcholine monoclonal antibody and two anti-apolipoprotein B antibodies. Venous blood samples were analyzed biochemically. RESULTS: We tested two methods of gingival crevicular fluid collection, namely paper points and paper strips. Gingival crevicular fluid could be collected very safely with paper points and they showed good recovery of LDL and OxLDL throughout the analysis. Apolipoprotein B, the major protein component in LDL, was detected in gingival crevicular fluid by western blot, and OxLDL was found to be present in gingival crevicular fluid by ELISA. The OxLDL/LDL ratio in gingival crevicular fluid was 17.0 times higher than that in plasma. CONCLUSION: This is the first report to show the presence of apolipoprotein B and apolipoprotein B- oxidized phosphatidylcholine complex, which correspond to LDL and OxLDL, respectively, in gingival crevicular fluid.

Distinguished effects of antiphospholipid antibodies and anti-oxidized LDL antibodies on oxidized LDL uptake by macrophages.

Several interpretations have been made regarding the specificity of antiphospholipid antibodies and antibodies against oxidized low-density lipoprotein (oxLDL), but these are still controversial. In the present study, we delineated specificity of these two types of antibodies and analyzed their regulatory effect on oxLDL and/or beta( 2)-glycoprotein I (beta(2)GPI) binding to macrophages. Scavenger receptor-mediated binding of oxLDL (or its beta(2)GPI complexes) to macrophages was observed and the binding was partly prevented by beta( 2)GPI. The IgG monoclonal anti-beta(2)GPI antibody (WB-CAL-1), which was derived from NZW x BXSB F1 mouse (a model of antiphospholipid syndrome), significantly increased the oxLDL/beta(2)GPI binding to macrophages. In contrast, IgM anti-oxLDL natural antibody, EO6 (derived from apoe( -/-) mouse), prevented the binding. Different antigenic specificity of these antibodies to oxLDL and its beta(2)GPI complexes was also confirmed in TLC-ligand blot and ELISA. Thus, IgG anti-beta(2) GPI autoantibodies contribute to lipid metabolism (housekeeping of oxLDL by macrophages) whereas IgM natural anti-oxLDL antibodies may protect against atherogenesis. In addition, in vitro data suggest that relatively high dose of intravenous immunoglobulin preparations (mainly contain IgG anti-oxLDL antibodies) might also prevent atherogenesis by inhibiting the oxLDL binding to macrophages.

Possible involvement of the oxidized low-density lipoprotein/lectin-like oxidized low-density lipoprotein receptor-1 system in pathogenesis and progression of human osteoarthritis.

OBJECTIVE: Using human cartilage samples and cultured chondrocytes, to assess the possible involvement of oxidized low-density lipoprotein (ox-LDL) and lectin-like ox-LDL receptor-1 (LOX-1) in pathogenesis and progression of osteoarthritis (OA). METHODS: Thirty-two cartilage samples were obtained from 16 patients with knee OA, and 12 Control samples from six with femoral neck fracture. LOX-1 mRNA expressions in 12 OA and six Control samples were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemistry for ox-LDL and LOX-1 was performed in all samples. The histological OA grade was assessed with the modified Mankin score. The relative percentage of the ox-LDL and LOX-1 immunopositive chondrocytes was calculated in all samples. The effects of ox-LDL on cell viability in cultured human chondrocytes were investigated by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay and on proteoglycan synthesis by monitoring [35S] sulfate incorporation. RESULTS: There was a statistically significant difference between mean LOX-1/GAPDH (LOX-1/human glyceraldehyde-3-phosphate dehydrogenase) ratio of OA samples and that of Control samples (40.6%+/-10.3 and 11.9%+/-2.8, respectively, P<0.0001). The mean percentage of ox-LDL-positive cells was 23.0+/-15.7% in OA and 4.3+/-3.7% in Control cells (P=0.0002). The mean percentage of LOX-1-positive cells was 51.7+/-29.5% in OA and 10.0+/-8.1% in Control cells (P<0.0001). Both the ox-LDL immunoreactivity and the LOX-1 immunoreactivity were significantly correlated with the modified Mankin scores (R2=0.67 and 0.48, respectively; P<0.0001 for each). ox-LDL significantly reduced the human chondrocyte viability and proteoglycan synthesis, and pretreatment with anti-human LOX-1 monoclonal antibody reversed these effects. CONCLUSION: The ox-LDL/LOX-1 system may be involved in human OA.

Oxidized phosphatidylcholine in alveolar macrophages in idiopathic interstitial pneumonias.

It has been suggested that oxidative stress plays a pathogenic role in idiopathic interstitial pneumonias. Macrophage- or neutrophil-derived oxidants seem to be important sources of oxidative stress in this group of inflammatory disorders. Recent experimental studies have revealed that oxidative injury during inflammation or apoptosis can change phosphatidylcholine of cell membrane into its oxidized form, which serves as a ligand for macrophage scavenger receptor CD36. Recently, we developed a monoclonal antibody against oxidized phosphatidylcholine. Using this novel antibody, we performed an immunohistochemical investigation to clarify the localization of oxidized phosphatidylcholine in lung tissues of idiopathic interstitial pneumonias and a relationship between oxidized phosphatidylcholine localization and CD36 expression. Lung specimens obtained from patients with desquamative (n = 8) or usual interstitial pneumonia (n = 15) were studied. Thirteen normal lung tissues were also examined as controls. Antibodies against oxidized phosphatidylcholine, CD36, epithelial cells, macrophages, and neutrophils were used as primary antibodies. The positive cell number was counted by computer-aided morphometry. While there were no oxidized phosphatidylcholine-positive cells in normal lungs, lungs of desquamative or usual interstitial pneumonia contained large numbers of oxidized phosphatidylcholine-positive cells in the alveolar spaces. Double-staining analysis revealed that most oxidized phosphatidylcholine-positive cells were macrophages. The oxidized phosphatidylcholine-positive cells were increased in association with the increase in the densities of macrophages (Rs = 0.87, p < 0.0001) and neutrophils (Rs = 0.89, p < 0.0001). Accumulated macrophages also showed distinct CD36 expression. These findings suggest that oxidative stress and the related product, oxidized phosphatidylcholine, play an important role in the pathophysiology of idiopathic interstitial pneumonias.

Raised plasma oxidised LDL in acute cerebral infarction.

BACKGROUND: The association between oxidised low density lipoprotein (OxLDL) and cerebral infarction is suspected but not established. OBJECTIVES: To determine whether plasma OxLDL is a useful marker for monitoring oxidative stress in stroke patients. METHODS: Plasma OxLDL concentrations were determined in 56 stroke patients with cerebral infarction (n = 45) or cerebral haemorrhage (n = 11), and in 19 age matched controls, using a novel sandwich enzyme linked immunosorbent assay. RESULTS: Compared with the controls (0.130 (0.007) ng/ micro g LDL, mean (SEM)), OxLDL was significantly raised in patients with cerebral infarction (0.245 (0.022); p < 0.0001) but not in those with haemorrhage (0.179 (0.023)). Patients with cortical ischaemic infarcts (n = 22) had higher OxLDL levels than either the controls (p < 0.0001) or the patients with non-cortical ischaemic infarcts (n = 23) (p < 0.001). Increased OxLDL concentrations in patients with cortical infarcts persisted until the third day after stroke onset. The National Institutes of Health stroke scales in patients with cortical infarction were higher than in those with non-cortical infarction (p < 0.01). CONCLUSIONS: There is a significant association between raised plasma OxLDL and acute cerebral infarction, especially cortical infarction. Plasma OxLDL may reflect oxidative stress in stroke patients.

Foam cell formation containing lipid droplets enriched with free cholesterol by hyperlipidemic serum.

A monoclonal antibody, ASH1a/256C (256C), which binds to atherosclerotic lesions in Watanabe heritable hyperlipidemic rabbit (WHHL) aorta in vivo, recognizes complex structures of phosphatidylcholine mixed with neutral lipids. In the present study, a cell culture system is described in which foam cells express 256C-positive lipid droplets. J774.1 macrophages were incubated in the presence of a small volume of WHHL serum for 24 h to produce foam cells, which were then incubated without the WHHL serum for 3 days. Oil red O-positive lipid droplets appeared on day 1, and were present in the cells during the whole incubation period. The lipid droplets in the cells were positively immunostained with antibody 256C on day 4, although they were negative on day 1. Expression of the antigenic lipid droplets was also induced by the addition of acetylated LDL or sera from patients with hyperlipidemia. When foam cells were induced by the addition of WHHL serum, cellular content of cholesteryl ester was greatly increased but then decreased to near basal levels by day 4. Concomitantly, cellular free cholesterol increased during the culture period, indicating that the cholesteryl ester changes to free cholesterol by day 4. The lipid droplets in the foam cells on day 4 were positively stained with filipin, a fluorescent probe for free cholesterol, as well as with 256C antibody, indicating that free cholesterol is enriched in antigenic lipid droplets. These observations suggest that hydrolysis and rearrangement of cellular cholesterol take place in foam cells to form complex structures of phosphatidylcholine and free cholesterol in lipid droplets.

Heme oxygenase-1 inhibits atherogenesis in Watanabe heritable hyperlipidemic rabbits.

BACKGROUND: Heme oxygenase-1 (HO-1) is proposed to have a variety of adaptive responses against oxidative stress. To examine the function of HO-1 against atherogenesis in vivo, we observed the effects of HO-1 inhibition on atherosclerotic lesion formation in Watanabe heritable hyperlipidemic rabbits (WHHL). Methods and Results- During 4 weeks of a 1% cholesterol diet, intravenous injections of Sn-protoporphyrin IX to inhibit HO-1 (S group, n=10) and saline as a control (C group, n=10) were given to 3-month-old WHHL rabbits. The percentages of en face atherosclerotic lesion areas in total descending aorta by Sudan IV staining (EFA) and the ratio of intima to media in microscopic atherosclerotic lesions in the ascending aortas (I/M) were calculated. Two different quantitative methods revealed significantly greater atherosclerotic lesions in the S group than the C group (EFA, P<0.001; I/M, P<0.005). HO-1 expression in atherosclerotic lesions was confirmed by Northern blot and immunohistochemical analyses. The dominant cell types expressing HO-1 were macrophages and foam cells, in which oxidized phospholipids were also accumulated. HO inhibition increased plasma and tissue lipid peroxide levels without affecting plasma lipid co osition. CONCLUSIONS: These results suggest the possibilities that HO-1 has antiatherogenic properties in vivo and that the antiatherogenic properties of HO-1 are conducted through the prevention of lipid peroxidation.

The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells.

Collectins are a family of C-type lectins that have collagen-like sequences and carbohydrate recognition domains (CRD). They are involved in host defense through their ability to bind to carbohydrate antigens of microorganisms. The scavenger receptors type A and MARCO are classical type scavenger receptors that have internal collagen-like domains. Here we describe a new scavenger receptor that is a membrane-type collectin from placenta (collectin placenta 1 (CL-P1)), which has a typical collectin collagen-like domain and a CRD. The cDNA has an insert of about 2.2 kilobases coding for a protein containing 742 amino acid residues. The deduced amino acid sequence shows that CL-P1 is a type II membrane protein, has a coiled-coil region, a collagen-like domain, and a CRD. It resembles type A scavenger receptors because the scavenger receptor cysteine-rich domain is replaced by a CRD. Northern analyses, reverse transcription-polymerase chain reaction, and immunohistochemistry show that CL-P1 is expressed in vascular endothelial cells but not in macrophages. By immunoblotting and flow cytometry CL-P1 appears to be a membrane glycoprotein of about 140 kDa in human umbilical vein or arterial endothelial cells, placental membrane extracts, and CL-P1 transfected Chinese hamster ovary cells. We found that CL-P1 can bind and phagocytose not only bacteria (Escherichia coli and Staphylococcus aureus) but also yeast (Saccharomyces cerevisiae). Furthermore, it reacts with oxidized low density lipoprotein (OxLDL) but not with acetylated LDL (AcLDL). These binding activities are inhibited by polyanionic ligands (polyinosinic acid, polyguanylic acid, dextran sulfate) and OxLDL but not by polycationic ligands (polyadenylic acid or polycytidylic acid), LDL, or AcLDL. These results indicate that CL-P1 might play important roles in host defenses that are different from those of soluble collectins in innate immunity.

Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes.

BACKGROUND: There is accumulating data that acute coronary syndromes relate to recent onset activation of inflammation affecting atherosclerotic plaques. Increased blood levels of oxidized low density lipoprotein (ox-LDL) could play a role in these circumstances. METHODS AND RESULTS: Ox-LDL levels were measured in 135 patients with acute myocardial infarction (AMI; n=45), unstable angina pectoris (UAP; n=45), and stable angina pectoris (SAP; n=45) and in 46 control subjects using a sandwich ELISA method. In addition, 33 atherectomy specimens obtained from a different cohort of patients with SAP (n=10) and UAP (n=23) were studied immunohistochemically for ox-LDL. In AMI patients, ox-LDL levels were significantly higher than in patients with UAP (P<0.0005) or SAP (P<0.0001) or in controls (P<0.0001) (AMI, 1.95+/-1.42 ng/5 microgram LDL protein; UAP, 1.19+/-0.74 ng/5 microgram LDL protein; SAP, 0.89+/-0.48 ng/5 microgram LDL protein; control, 0.58+/-0.23 ng/5 microgram LDL protein). Serum levels of total, HDL, and LDL cholesterol did not differ among these patient groups. In the atherectomy specimens, the surface area containing ox-LDL-positive macrophages was significantly higher in patients with UAP than in those with SAP (P<0.0001). CONCLUSIONS: This study demonstrates that ox-LDL levels show a significant positive correlation with the severity of acute coronary syndromes and that the more severe lesions also contain a significantly higher percentage of ox-LDL-positive macrophages. These observations suggest that increased levels of ox-LDL relate to plaque instability in human coronary atherosclerotic lesions.

Heme oxygenase-1 inhibits atherosclerotic lesion formation in ldl-receptor knockout mice.

Heme oxygenase-1 (HO-1) is induced by a variety of conditions associated with oxidative stress. We demonstrated that mildly oxidized LDL markedly induces HO-1 in human aortic endothelial and smooth muscle cell cocultures and that its induction results in the attenuation of monocyte chemotaxis resulting from treatment with mildly oxidized LDL in vitro. To elucidate the role of HO-1 in the development of atherosclerotic lesions in vivo, we modulated HO-1 expression in LDL-receptor knockout mice fed high-fat diets. During 6-week high-fat diet trials, intraperitoneal injections of hemin (H group) or hemin and desferrioxamine (HD group) to induce HO-1, Sn-protoporphyrin IX to inhibit HO-1 (Sn group), and saline as control (C group) were performed. Both the H and HD groups showed significantly less mean atherosclerotic lesions in the proximal aorta compared with the C group, whereas the Sn group showed larger lesion compared with the C group. Modulation of HO expression and HO activities were confirmed by Northern blot analysis and HO activity assay. Immunohistochemical studies revealed significant HO-1 expression in atherosclerotic lesions, where oxidized phospholipids also localized. Major cell types expressing HO-1 were macrophages and foam cells in the lesions. HO modulations affected plasma lipid hydroperoxide (LPO) levels and nitrite/nitrate levels. These results suggest that HO-1, induced under hyperlipidemia, functioned as an intrinsic protective factor against atherosclerotic lesion formation, possibly by inhibiting lipid peroxidation and influencing the nitric oxide pathway.

Glycoxidation and lipid peroxidation of low-density lipoprotein can synergistically enhance atherogenesis.

OBJECTIVE: The purpose of this study was to clarify the role of glycoxidation and lipid peroxidation of low-density lipoprotein (LDL) in atherogenesis. METHODS AND RESULTS: We examined the formation of N(epsilon)-(carboxymethyl) lysine (CML), a glycoxidation product, and malondialdehyde (MDA), a lipid peroxidation product, in vitro and their co-localization in human atherosclerotic lesions. Immunochemical analysis revealed that CML was formed in a time-dependent manner by human LDL incubated with copper ions and glucose, i.e. an in vitro model of glycoxidation of LDL. When LDL was exposed to copper ions alone, a small amount of CML was formed, however this was significantly less in oxidized LDL than glycoxidative LDL. In contrast, MDA formation was observed in both oxidation and glycoxidation of LDL, but not in glycation of LDL. Hexitol-lysine (HL), an Amadori product, was formed by both glycation and glycoxidation of LDL, but not by oxidation of LDL. Immunohistochemical analysis showed that CML and MDA accumulated mainly in macrophage/foam cells, while pyrraline, a non-oxidative product of glycation, and apolipoprotein B were localized in the extracellular matrix in atherosclerotic lesions. Atheromas were positive for CML and MDA, but negative for pyrraline. Macrophage/foam cells in atherosclerotic lesions exhibited co-localization of macrophage scavenger receptor-A with CML and MDA, but not with pyrraline. CONCLUSION: Our results suggest that glycoxidation and lipid peroxidation of LDL synergistically promote the development of atherosclerotic lesions through interaction with macrophage scavenger receptor-A.

Oxidized low-density lipoprotein is associated with apoptosis of vascular smooth muscle cells in human atherosclerotic plaques.

BACKGROUND: Cytotoxic oxidized LDL (oxLDL) has been shown to promote apoptosis in cultured vascular smooth muscle cells (VSMCs). We investigated the localization of oxLDL and its association with apoptosis and the expression of apoptosis-related proteins in early and advanced atherosclerotic lesions. METHODS AND RESULTS: Atherosclerotic plaques (n=23) from patients undergoing aortic, carotid, or femoral arterial surgery were studied. In early lesions, oxLDL was located predominantly in the superficial intima and in the media just beneath the internal elastic lamina. Medial VSMCs staining positive for oxLDL showed expression of BAX, a proapoptotic protein of the BCL-2 family. Apoptosis, as detected by DNA in situ terminal deoxynucleotidyl transferase end-labeling (TUNEL), was not present in these early lesions. In advanced plaques, areas of the intima positive for oxLDL showed lower alpha-smooth muscle actin immunoreactivity (P<0.01) and higher BAX immunoreactivity (P<0.05). Furthermore, these areas showed an increased number of apoptotic VSMCs (P<0.01). Western blot analysis revealed that oxLDL increases BAX expression in cultured human coronary VSMCs. CONCLUSIONS: We conclude that in early atherosclerotic lesions, oxLDL-positive VSMCs express BAX, which increases the susceptibility of these cells to undergo apoptosis. This could be important in our understanding of the transition of early lesions into advanced atherosclerotic plaques, which are characterized by regions of cell death. In advanced plaques, oxLDL-positive areas of the intima show higher BAX immunoreactivity and TUNEL-positive VSMCs, and this may contribute to plaque instability and rupture.

Lysosomal accumulation of oxidized phosphatidylcholine-apolipoprotein B complex in macrophages: intracellular fate of oxidized low density lipoprotein.

Oxidized phosphatidylcholine (OxPC) formed in oxidized low density lipoprotein (OxLDL) is thought to be involved in the development of atherosclerosis. OxPC has been found in foam cells in atherosclerotic lesions and suggested to be the epitope for OxLDL recognition by macrophages. OxPC is present as a complex with apolipoprotein B (apoB) in OxLDL, since some OxPC can bind with proteins. In the current study, the intracellular fate of OxPC-apoB complexes after internalization of OxLDL by macrophages was investigated. Murine macrophage cell line J774.1 was incubated with either OxLDL or acetylated LDL for 24 h, then the cells were further incubated for up to 24 h in new medium without lipoprotein. Modified apoB in the cells was quantitated by sandwich ELISA using monoclonal antibodies against OxPC and apoB. Intracellular OxLDL decreased rapidly for the first 4 h to approx. 20% of that before medium change, with the apparent metabolism of OxPC-apoB complex ceasing. OxPC-apoB complexes that remained in the cells after 24 h chasing increased as the period of OxLDL loading in macrophages prolongs. Acetylated LDL in the cells decreased quickly and disappeared after 4 h of chasing. Subcellular fractionation using sucrose density gradient ultracentrifugation of macrophages, which had already accumulated OxPC-apoB complexes by 24 h of incubation with OxLDL and further 24 h chasing, showed that the complex was co-localized with endosomal and lysosomal markers. Immunohistochemical double staining studies demonstrated that OxPC and apoB co-localize in foam cells in early atherosclerotic lesions obtained from human coronary artery. These results suggest that OxPC-apoB complexes originating from OxLDL accumulate in foam cells in human atherosclerotic lesions as well as in macrophages in vitro.

Onion-bulb formation after a single compression injury in the macrophage scavenger receptor knockout mice.

Onion-bulb (OB) formation is often encountered in acquired neuropathies such as chronic inflammatory demyelinating polyradiculoneuropathy and diabetic neuropathy and is believed to require repeated injuries to peripheral nerves. Although this suggests that remaining damaged cell membranes, including myelin debris, might trigger OB formation, the molecular mechanism remains unclear. In this study, we were successful in producing many small OBs after a single compression injury to peripheral nerves of the knockout mice deficient of macrophage scavenger receptor class A (MSR-A). Although morphometry showed no difference in the average densities of the remaining myelinating fibers between wild-type and MSR-A knockout mice after the compression injury, there were more macrophages and myelin debris positive for oxidized-phosphatidylcholine in the nerves from the MSR-A knockout mice. We believe that OB formation was induced after a single compression injury as the result of delayed phagocytosis of myelin debris possessing oxidized lipids by MSR-A deficient macrophages. The present work shed light on the molecular mechanism of OB formation seen in chronic neuropathies and provided a model for further investigation.

Circulating oxidized low density lipoprotein levels. A biochemical risk marker for coronary heart disease.

Recent studies have established oxidative modification of low density lipoprotein (LDL) as an important atherogenic factor. We examined the clinical relevance of circulating oxidized LDL (OxLDL) levels in atherosclerotic disease by an enzyme immunoassay with use of specific antibodies against OxLDL (FOH1a/DLH3) and apolipoprotein B. Plasma OxLDL levels were significantly higher in patients with coronary heart disease (n=65) than in control subjects (n=181; 201. 3+/-11.2 versus 112.4+/-3.3 U/dL, respectively; P<0.01). OxLDL levels were not associated with age, sex, total cholesterol, or apolipoprotein B levels in normal control subjects. Our results suggest that circulating OxLDL may be a possible biochemical risk marker for coronary heart disease.

Preparation of radioactive aldehyde-containing phosphatidylcholine.

Short-chain, aldehyde-containing phosphatidylcholine (PC), formed during the oxidation of PC, is thought to be involved in cellular responses in atherosclerosis and inflammation. Here we report a convenient procedure for a small-scale preparation of aldehyde-containing PC. PC containing an unsaturated fatty acyl chain was treated with osmium tetroxide followed by sodium periodate at room temperature. The reaction product was purified by TLC. This preparation showed a single peak on reverse-phase HPLC, and its identity was confirmed by fast atom bombardment mass spectrometry. This procedure does not require special equipment and is easily applicable for preparation of radioactive materials.

Successful generation of peripheral neuropathy with onion-bulb formation in the macrophage scavenger receptor classA knockout mouse treated with isoniazid.

We previously reported successful generation of many onion-bulbs (OBs) and formation of oxidized phosphatidylcholine after compression injury to the peripheral nerve of mice deficient of macrophage scavenger receptor classA (MSR-A). In the present study, we employed chemical injury with isoniazid to the peripheral nerve of the MSR-A knockout mice to investigate the role of the MSR-A in toxic neuropathy. Peripheral neuropathy has not previously been generated with isoniazid in mice. In the present study, we also noted little histological change after isoniazid administration not only to control littermates but also to the A/J strain mice known to be slow acetylators. Surprisingly, however, we were successful in generating peripheral neuropathy with isoniazid in the MSR-A knockout mice. Histologically, the predominant feature was the presence of many thinly myelinated fibers with some OBs, which have not been observed in rats with isoniazid neuropathy. Deficiency of the MSR-A appears to have played an important role in generation of peripheral neuropathy with isoniazid in mice.