Autoantibodies against oxidized LDL are associated with severe chest pain attacks in patients with coronary heart disease

Autoantibodies against oxidized low-density lipoprotein (oxLDL) predict the progression of atherosclerosis. Several studies have shown that oxLDL is present in atherosclerotic lesions and that several factors present in active atherosclerotic plaques can oxidatively modify LDL. Oxidation of LDL induces production of autoantibodies against oxLDL (oxLDLab) that can be measured using an EIA test. Our aim was to see whether oxLDLab are associated with severe chest pain attacks in coronary heart disease (CHD) patients. Patients having two- or three-vessel CHD, as assessed by coronary angiography, and their siblings were recruited into the study (n = 568, mean age 55.8 years, range 29.3–83.2 years). Nondiabetic patients having a history of severe chest pain attacks had significantly higher oxLDLab levels (0.611 ± 0.56) than those who did not have a history of severe chest pain attacks (0.487 ± 0.40) (p = 0.027), even though age, cholesterol level, body mass index, and blood pressure were similar in both groups. However, no difference was found in oxLDLab levels in diabetic patients with or without a history of severe chest pain attacks. Increased levels of oxLDL autoantibodies are associated with severe chest pain attacks in nondiabetic patients with CHD.     

Effects of alpha-tocopherol on superoxide production and plasma intercellular adhesion molecule-1 and antibodies to oxidized LDL in chronic smokers

Antioxidants have been postulated to exert beneficial effects in atherosclerosis. Atherosclerosis is associated with raised plasma levels of soluble intercellular adhesion molecule-1 (sICAM-1) and autoantibodies against oxidized low-density lipoprotein (oxLDL). It is not known whether antioxidants affect these plasma factors in chronic smokers. In a randomized double-blind placebo-controlled study involving 128 male normolipidemic chronic smokers the effect of a 2-year alpha-tocopherol treatment (400 IU dL-alpha-tocopherol daily) on plasma levels of sICAM-1 and autoantibodies against oxLDL was evaluated. In addition, we monitored production of superoxide by leukocytes ex vivo. It was found that compared to nonsmokers (n = 33) plasma levels of IgG but not IgM autoantibodies against oxLDL and concentrations of sICAM-1 in smokers were significantly elevated (30 and 42%, respectively). After supplementation with alpha-tocopherol concentration of TBARS in plasma and in vitro oxidizability of LDL had decreased, but autoantibodies and sICAM-1 had not changed. Production of superoxide was not different between alpha-tocopherol- and placebo-treated smokers. It is concluded that in chronic smokers, long-term treatment with alpha-tocopherol does not normalize the raised levels of sICAM-1 and autoantibodies against oxLDL, both risk factors for initiation or progression of cardiovascular disease, despite a decrease in in vitro oxidizability of LDL.     

The low-density lipoprotein (LDL)-associated PAF-acetylhydrolase activity and the extent of LDL oxidation are important determinants of the autoantibody titers against oxidized LDL in patients with coronary artery disease

The oxidation of low-density lipoprotein (LDL) induces immunogenic epitopes, many of which are due to oxidatively modified phospholipids (oxPL). Lysophosphatidylcholine (lyso-PC) which is generated during LDL oxidation through the hydrolysis of oxPL by LDL-associated PAF-acetylhydrolase (PAF-AH) is also immunogenic. We investigated whether the LDL-associated PAF-AH and the extent of LDL oxidation influence the autoantibody titers against oxidized LDL (oxLDL) in patients with stable angina as well as in apparently healthy volunteers. Three types of copper-oxidized LDL, were prepared at the end of the lag, propagation or decomposition phase (oxLDL(L), oxLDL(P) and oxLDL(D), respectively). Similar types of oxidized LDL were prepared after previous inactivation of endogenous PAF-AH [oxLDL(-)]. All these types of oxLDL as well as malondialdehyde-modified LDL (MDA-LDL) were used as antigens. Antibody titers against the above antigens were measured with an ELISA method in the serum of 65 patients with stable angina and 47 apparently healthy volunteers. Both groups exhibited higher autoantibody titers against each type of oxLDL(-) compared to the respective type of oxLDL (P<0.00001). In both groups autoantibody titers were higher when the oxLDL(P) and oxLDL(D) or oxLDL(-)(P) and oxLDL(-)(D) were used as antigens compared to oxLDL(L) (P<0.04) or to oxLDL(-)(L), respectively (P<0.0001 for all comparisons). Patients had significantly higher titers against all types of oxLDL (enriched in lyso-PC) and oxLDL(-) (enriched in intact oxPL) compared to controls. Autoantibody titers against MDA-LDL did not differ between patients and controls. Multivariate logistic regression analysis showed that among the autoantibody titers measured only those towards oxLDL(P) are associated with a significantly higher risk for coronary artery disease. LDL-associated PAF-AH activity may play an important role in decreasing the overall immunogenicity of oxLDL, whereas the extent of LDL oxidation seems to modulate the epitopes formed on oxLDL. Lyso-PC, a major component of oxLDL(P), could be mainly responsible for the elevated autoantibody titers against oxLDL in patients with stable angina.     

Paraoxonase genotype, LDL-oxidation and carotid atherosclerosis in male life-long smokers

Paraoxonase (PON-1) is a high-density lipoprotein (HDL) associated enzyme that hydrolyzes lipid peroxides in vitro, which may therefore protect against the onset of atherosclerosis. Heavy smokers are more exposed to oxidative stress and hence at high-risk for oxidative modification of LDL. Our hypothesis is that the anti-oxidative properties of PON-1 inhibit LDL oxidation, especially in populations exposed to high oxidative stress. We have studied the effects of PON-1 genotype and smoking to variation in oxidative status parameters and intima-media thickness (IMT), a surrogate marker of atherosclerosis. The contribution of two common polymorphisms in the PON-1 gene (Q192R and L55M) to LDL oxidizability, autoantibodies directed against oxLDL and IMT were studied in 207 male life-long smokers. Smokers were classified into average, heavy and excessive smokers based on pack years of cigarettes smoked. PON-1 genotype was not associated with autoantibodies to oxLDL, LDL oxidizability or IMT. Smoking was associated with IMT in subgroups with the high levels of LDL, but not in the population at large. The lack of association of PON-1 genotype with oxidative status parameters and IMT suggests that PON-1 is not a major inhibitor of LDL oxidation in a population of life-long smokers.     

Evaluation of low density lipoprotein oxidation in a course of hypothyroidism

INTRODUCTION: The aim of this study was to evaluate the influence of hypothyroidism on oxidative modification of low density lipoprotein (LDL). MATERIAL AND METHODS: 24 patients with overt hypothyroidism and 10 patients with mild hypothyroidism were enrolled to the study. The control group consisted of 24 healthy subjects with normal serum TSH. Plasma level of oxidized LDL (oxLDL) and serum level of antibodies against oxidized LDL (anti-oxLDL) determined lipoprotein oxidation. RESULTS: Significantly increased plasma oxLDL levels were found in patients with overt hypothyroidism in comparison to patients with mild hypothyroidism and control group. Anti-oxLDL levels in patients with overt or mild hypothyroidism and in the control group showed no significant differences. OxLDL plasma levels in patients with hypothyroidism inversely correlated with FT(4) levels and positively correlated with TSH, total cholesterol, LDL cholesterol and triglycerides levels. CONCLUSIONS: The presented study indicates increased lipoprotein oxidation in patients with hypothyroidism which depends on the degree of hypothyroidism and changes in lipid profile. Elevated cholesterol and triglycerides levels are the factors increasing lipoprotein oxidation. Plasma oxLDL levels may constitute a useful marker indicating the risk for atherosclerosis in hypothyroidism.     

Paraoxonase Genotype,LDL-oxidation and Carotid Atherosclerosis in Male Life-long Smokers

Paraoxonase (PON-1) is a high-density lipoprotein (HDL) associated enzyme that hydrolyzes lipid peroxides in vitro, which may therefore protect against the onset of atherosclerosis. Heavy smokers are more exposed to oxidative stress and hence at high-risk for oxidative modification of LDL.

Our hypothesis is that the anti-oxidative properties of PON-1 inhibit LDL oxidation, especially in populations exposed to high oxidative stress.

We have studied the effects of PON-1 genotype and smoking to variation in oxidative status parameters and intima-media thickness (IMT), a surrogate marker of atherosclerosis. The contribution of two common polymorphisms in the PON-1 gene (Q192R and L55M) to LDL oxidizability, autoantibodies directed against oxLDL and IMT were studied in 207 male life-long smokers. Smokers were classified into average, heavy and excessive smokers based on pack years of cigarettes smoked.

PON-1 genotype was not associated with autoantibodies to oxLDL, LDL oxidizability or IMT. Smoking was associated with IMT in subgroups with the high levels of LDL, but not in the population at large.

The lack of association of PON-1 genotype with oxidative status parameters and IMT suggests that PON-1 is not a major inhibitor of LDL oxidation in a population of life-long smokers.     

Fatty acids bound to human serum albumin and its structural variants modulate apolipoprotein B secretion in HepG2 cells

Epidemiologic studies have shown an inverse relationship between human serum albumin (HSA) levels and coronary heart disease (CHD). However, no mechanisms have been identified to explain this relationship. We hypothesized that this relationship is due to differences in binding affinity of fatty acids to HSA and subsequent atherogenic lipoprotein synthesis and secretion from hepatocytes. To test our hypothesis we undertook the current study. Using HepG2 cells, we demonstrated that oleic acid (OA) bound to HSA in a molar ratio of 4:1 and after incubation for 24 h stimulated apolipoprotein B (apoB) secretion. We also tested whether mutant forms of HSA could alter the binding affinity for fatty acids and change the availability of substrate for lipoprotein secretion. Based on the results obtained in this study using 11 HSA mutant proteins complexed with OA, we were able to classify into three major mutant groups based on their effects on apoB secretion. One group in particular (R410Q/Y411W, R410A/Y411A, and W214L/Y411W) showed a significantly diminished effect on apoB secretion when compared to the wild type HSA/OA complex. Furthermore, the amount of free OA internalized in HepG2 cells in the presence of HSA mutant proteins was in good agreement with the effects seen on apoB secretion by the various HSA mutants. This suggests that some mutant forms of HSA might potentially bind fatty acids with a much higher binding affinity and thus deprive fatty acids available for lipoprotein assembly in hepatocytes. In conclusion, our data illustrate that certain HSA polymorphic forms may be protective against the development of CHD and warrants further investigation.     

LDL受体对清道夫受体活性的影响

应用经ＰＭＡ诱导衍生的ＴＨＰ－１巨噬细胞为模型,以单克隆抗体Ｃ７Ｂ封闭ｏｘＬＤＬ上的ＬＤＬ受体结合位点,结果发现,正常细胞在摄取ｏｘＬＤＬ时ＬＤＬ受体与清道夫受体起协同作用;但Ｃ７Ｂ作用于蓄积了脂质的ＴＨＰ－１巨噬细胞时,对细胞脂质蓄积程度无明显影响,清道夫受体活性不但不降低反而有所升高,说明由于脂质蓄积ＬＤＬ受体的作用减弱．     

Oxidative modification of low-density lipoprotein and immune regulation of atherosclerosis

Oxidized low-density lipoprotein (oxLDL) is thought to promote atherosclerosis through complex inflammatory and immunologic mechanisms that lead to lipid dysregulation and foam cell formation. Recent findings suggested that oxLDL forms complexes with β₂-glycoprotein I (β₂GPI) and/or C-reactive protein (CRP) in the intima of atherosclerotic lesions. Autoantibodies against oxLDL/β₂GPI complexes occur in patients with systemic lupus erythematosus (SLE) and/or antiphospholipid syndrome (APS) and significantly correlate with arterial thrombosis. IgG autoantibodies having similar specificity emerged spontaneously in non-immunized NZW × BXSB F1 mice, an animal model of APS, and a monoclonal autoantibody (WB-CAL-1; IgG2a) against complexed β₂GPI (oxLDL/β₂GPI complexes) was derived from the same mice. WB-CAL-1 significantly increased the in vitro uptake of oxLDL/β₂GPI complexes by macrophages. This observation strongly suggests that such IgG autoantibodies are pro-atherogenic. In contrast, IgM anti-oxLDL natural antibodies found in the atherosclerosis-prone mice (ApoE^-/- and LDL-R^-/- mice) have been proposed to be anti-atherogenic (protective). The presence of IgG anti-oxLDL antibodies in humans has been documented in many publications but their exact clinical significance remains unclear. In this article, we review recent progress in our understanding of the mechanisms involved in oxidation of LDL, formation of oxLDL complexes, and antibody mediated-immune regulation of atherogenesis.     

Reactive oxygen species damaged human serum albumin in patients with type 1 diabetes mellitus: biochemical and immunological studies

The role of hydroxyl radical (.OH) damaged human serum albumin (HSA) in type 1 diabetes has been investigated in the present study. Hydroxyl radical induced modification on HSA has been studied by UV absorption spectroscopy, ANS fluorescence and carbonyl estimation. Hydroxyl radical modified HSA was found to be highly immunogenic in rabbits as compared to native HSA. The binding characteristics of circulating autoantibodies in type 1 diabetes patients against native and modified HSA were assessed. Diabetes patients (n=31) were examined by direct binding ELISA and the results were compared with healthy age-matched controls (n=22). High degree of specific binding by 54.8% of patients sera towards .OH modified HSA, in comparison to its native analogue (p<0.05) was observed. Sera from those type 1 diabetes patients having smoking history, high aging with high degree of disease showed substantially stronger binding to .OH modified HSA over native HSA in particular. Normal human sera showed negligible binding with either antigen. Competitive inhibition ELISA reiterates the direct binding results. Gel retardation assay further substantiated the enhanced recognition of modified HSA by circulating autoantibodies in diabetes patients. The increase in total serum protein carbonyl levels in the diabetes patients was largely due to an increase in oxidized albumin. HSA of diabetes mellitus patients (DM-HSA) and normal subjects (normal-HSA) were purified on a Sephacryl S-200 HR column. Spectroscopic analysis confirmed that the DM-HSA samples contained higher levels of carbonyls than normal-HSA (p<0.001). DM-HSA was conformationally altered, with more exposure of its hydrophobic regions. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in type 1 diabetes mellitus patients.     

Generation and characterization of chicken monoclonal antibodies against human LOX-1

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is the major receptor for oxidized LDL (oxLDL), and plays a key role in the pathogenesis of atherosclerosis and cardiovascular diseases. Monoclonal antibodies (mAbs) specific for human LOX-1 (hLOX-1) were generated by a phage display technique using chickens immunized with recombinant hLOX-1 (rhLOX-1). A total of 53 independent scFv clones reactive for rhLOX-1 were obtained. Of the 53 clones, 49 recognized the C-type lectin-like domain (CTL domain), which contributes to the binding of oxLDL. Of these, 45 clones inhibited oxLDL-binding with LOX-1. Furthermore, some of these clones cross-reacted with rabbit, pig and/or mouse LOX-1. For possible application as therapeutic agents in the future, two cross-reactive mAbs were re-constructed as chicken-human chimeric antibodies. The chimeric antibodies showed similar characteristics compared to the original antibodies, and inhibited oxLDL binding to LOX-1 expressed on CHO cells. The results obtained in this study indicate that anti-LOX-1 mAbs might be useful tools for functional analyses and development of therapeutic agents for cardiovascular indications such as atherosclerosis.Key words: LOX-1, oxLDL, chicken monoclonal antibody, chimeric antibody, neutralizing antibody     

Circulating oxidized low density lipoprotein, autoantibodies against them and homocysteine serum levels in diagnosis and estimation of severity of coronary artery disease

The oxidative hypothesis of atherosclerosis proposes that oxidative modification of low density lipoprotein (LDL) plays a critical role in atherogenesis. The evaluation of LDL oxidation in vivo is therefore very important. However, data concerning the evaluation of the above biochemical marker is very limited in clinical practice. This study was conducted to test the hypothesis that plasma levels of ox-LDL reflect atherosclerosis and determine the clinical significance in the measurement of circulating ox-LDL and autoantibodies against them as well as their correlation with homocysteine and lipid parameters in the diagnosis and severity of coronary heart disease. A total of 273 individuals were examined: 41 suffering from unstable angina pectoris (UAP), 62 from stable angina pectoris (SAP) and 170 healthy control subjects. We used a sensitive method for detecting ox-LDL that is based on a direct sandwich technique (ELISA) in which two monoclonal antibodies are directed against separate antigenic determinants on the oxidized apolipoprotein-B molecule along with another enzyme immunoassay designed to determine human antibodies to oxidized LDL (anti-oxLDL) directly in serum. Total homocysteine (HCY) was evaluated by means of a fully automated fluorescence polarization immunoassay. Patients with UAP exhibited marked elevations in oxLDL levels as compared to patients with SAP (161.2 +/- 28.4 vs. 119.2 +/- 26.6, p < 0.001) and the control subjects (67 +/- 18.8, p < 0.001). The difference in oxLDL levels between patients with SAP and the control group was also statistically significant. Similarly, patients with UAP showed marked elevations in anti-oxLDL antibodies compared to both patients with SAP (602.2 +/- 62.2 vs. 510.8 +/- 50.3,p < 0.001) and control subjects (368 +/- 79.6, p < 0.001). The difference in anti-oxLDL levels between patients with SAP and the controls was also statistically significant. OxLDL levels were not correlated with age in any of the groups studied. Triglycerides, LDL-cholesterol and total cholesterol were elevated in patients with UAP as opposed to patients with SAP and the control subjects, while HDL levels were elevated in the control subjects when compared to patients with SAP and UAP. Homocysteine levels were elevated in patients suffering from UAP and SAP when compared to healthy subjects. Patients with UAP or SAP did not differ on homocysteine levels. Our findings demonstrate the presence of oxLDL in vivo, its strong association with coronary artery disease as well as with the severity of the clinical presentation.     

No upregulation of lectin-like oxidized low-density lipoprotein receptor-1 in serum-deprived EA.hy926 endothelial cells under oxLDL exposure, but increase in autophagy

The oxidized low-density lipoprotein (oxLDL)-dependent activation of the lectin-like oxLDL receptor-1 (LOX-1) triggers apoptosis in vascular cells and appears to be involved in atherosclerosis. Autophagy might be an alternate to apoptosis in endothelial cells. The EA.hy926 endothelial cell line has been reported to undergo necrosis under oxLDL stimulation. For this reason, we studied the expression of LOX-1 and its oxLDL-dependent function in EA.hy926 cells under serum starvation. Untreated and oxLDL-treated cells expressed the LOX-1 protein at similar levels 6h after starvation. After 24h without oxLDL and with native LDL (nLDL), statistically significant higher levels were found in LOX-1 than in the oxLDL-treated probes. The oxLDL cultures with low LOX-1 expression displayed stronger features of autophagy than those with nLDL as there were remodelling of actin filaments, disrupture of adherens junctions (immunofluorescence staining), and autophagosomes with the characteristic double membrane at the ultrastructural level. For the advanced oxLDL exposure times (18 and 24 h), autophagic vacuoles/autophagolysosomes were morphologically identified accompanied by a decrease in lysosomes. The autophagosome marker protein MAP LC3-II (Western blotting) was significantly augmented 6 and 18 h after oxLDL treatment compared with cultures treated with nLDL and medium alone. Signs of apoptosis were undetectable in cultures under oxLDL exposure, yet present under staurosporin (apoptosis inducer), i.e. presence of apoptotic bodies and cleaved caspase 3. We conclude that serum starvation upregulates LOX-1 in EA.hy926 cells, whereas the additional oxLDL treatment downregulates the receptor and intensifies autophagy probably by increase in oxidative stress.     

Binding and uptake of differently oxidized low density lipoprotein in mouse peritoneal macrophages and THP-1 macrophages: involvement of negative charges as well as oxidation-specific epitopes

Oxidatively modified low-density lipoprotein (LDL) has been found in vivo, and oxidized LDL (oxLDL) could bind to scavenger receptors, leading to foam cell formation. Macrophages bear a number of different scavenger receptors for oxLDL, and macrophages of different origins may have a different scavenger receptor repertoire. In addition, LDL oxidized to different degrees may differ in the ability to bind macrophage scavenger receptors. In this study, we characterized the patterns of the binding and uptake of differently oxidized LDL in mouse peritoneal macrophages (MPM) and human THP-1 macrophages, and the influence of negative charge and oxidation-specific epitopes in oxLDL on these processes. Thresholds of increased binding and uptake in MPM were found when LDL was oxidized to the degrees with a relative electrophoretic mobility (REM) of 2.6 (minor threshold) and 3.0 (major threshold), corresponding to 49 and 57%, respectively, of the loss of free amino groups in these oxLDL. There was no threshold for the binding of oxLDL to THP-1 macrophages, while for uptake, a major threshold with REM of 3.0 (57% free amino groups lost) was found. The presence of the F(ab')(2) fragments of the monoclonal antibody OB/04, which was raised against copper-oxidized LDL, led to the reduction of the binding and uptake, respectively, of Eu(3+)-oxLDL (REM:3.6) in MPM by 31 and 29%, and by 19 and 22% in THP-1 macrophages. It is concluded that LDL oxidized to different degrees binds differently to macrophages, and the patterns of binding and uptake are different for MPM and human THP-1 macrophages. Both, the negative charge and the oxidation-specific epitopes of oxLDL are involved in these processes.     

Low-density lipoproteins induce the renin-angiotensin system and their receptors in human endothelial cells.

Increased levels of low-density lipoproteins are well-established risk factors of endothelial dysfunction and the metabolic syndrome. In this study, we evaluated the effect of native low-density lipoprotein (nLDL) and oxidized LDL (oxLDL) on the expression of genes of the renin-angiotensin system (angiotensin-converting enzyme, ACE; angiotensin II type 1 receptor, AT(1)) and their receptors (low-density lipoprotein receptor: LDLR; lectin-like oxLDL receptor: LOX-1; toll-like receptor 4: TLR4) in primary cultures of human umbilical vein endothelial cells. ACE and AT(1) expressions were significantly increased after stimulation with nLDL and oxLDL. OxLDL receptor LOX-1 showed a maximum induction after 7 hours. Increased LOX-1 protein expression in response to oxLDL could be blocked by a LOX-1-specific antibody. TLR4 expression was increased by nLDL and oxLDL as well. We conclude that LDL and oxLDL can activate the renin-angiotensin system and their receptors LDLR, LOX-1, and TLR4 in human endothelial cells. These data suggest a novel link between hypercholesterolemia and hypertension in patients with the metabolic syndrome.     

OSAS对冠心病患者的血糖和血脂水平的影响

目的:探讨阻塞性睡眠呼吸暂停综合征(Obstructive sleep apnea syndrome,OSAS)对冠心病患者血糖和血脂的影响。方法:纳入我科住院的明确诊断冠心病患者共168人,根据呼吸暂停低通气指数(Apnea-hypopnea index,AHI)共分2组:冠心病组133例,冠心病合并OSAS组45例(轻度OSAS组16例;中度OSAS组18例,重度OSAS组12例)。对比分析冠心病组和冠心病合并OSAS组患者血脂及血糖水平及其相关性。结果:BMI、LDL、HDL在四组间差异有统计学意义(P0.05)。冠心病合并OSAS的BMI、LDL、HDL明显高于冠心病组,各组间BMI差异有统计学意义(P0.05)。FPG、HOMA-I、RHb A1、OX-LDL、AHI在冠心病组和冠心病合并轻度OSAS组之间差异无统计学意义(P0.05)。FPG、HOMA-I、RHb A1、OX-LDL、AHI在OSAS轻、中、重各组间差异有统计学意义(P0.05)。冠心病合并OSAS患者AHI和BMI、LDL、FPG、HOMA-I、OX-LDL呈正相关关系(P0.05)。结论:OSAS增加了冠心病患者血脂水平和糖尿病的风险,及机制可能与胰岛素抵抗和OX-LDL水平增高有关。     

Oxidized but not acetylated low-density lipoprotein reduces preproinsulin mRNA expression and secretion of insulin from HIT-T15 cells

We examined the effect of oxidized low-density lipoprotein (oxLDL) on the insulin secretion in the culture of HIT-T15 cell line, an islet beta-cell line derived from a hamster pancreatic tumor. In order to check the uptake of modified LDL by HIT-T15 cells, we prepared DiI-labeled native LDL (nLDL), acetylated LDL (AcLDL), and oxLDL. After the addition of each LDL into the cultures of HIT-T15 cells, fluorescence microscopic study was done. It was suggested that AcLDL and oxLDL were taken up by HIT-T15 cells, as well as nLDL. mRNA expression of the LDL receptor, CD36, and SR-B1 was detected in HIT-T15 by RT-PCR. The medium insulin level was measured in the culture of HIT-T15 cells with each LDL. oxLDL significantly reduced the insulin secretion stimulated by various concentrations of glucose, the intracellular content of insulin, and the expression of preproinsulin mRNA compared to the control cultures without LDL addition. In contrast, nLDL and AcLDL had no effect on the insulin secretion, the intracellular insulin level, or the expression of preproinsulin mRNA. MTT assay findings (reflecting cell numbers) were not different between cultures with and without LDLs. These results indicated that oxLDL disturbed the insulin metabolism of HIT-T15 cells.     

Generation and characterization of chicken monoclonal antibodies against human LOX-1

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is the major receptor for oxidized LDL (oxLDL), and plays a key role in the pathogenesis of atherosclerosis and cardiovascular diseases. Monoclonal antibodies (mAbs) specific for human LOX-1 (hLOX-1) were generated by a phage display technique using chickens immunized with recombinant hLOX-1 (rhLOX-1). A total of 53 independent scFv clones reactive for rhLOX-1 were obtained. Of the 53 clones, 49 recognized the C-type lectin-like domain (CTL domain), which contributes to the binding of oxLDL. Of these, 45 clones inhibited oxLDL-binding with LOX-1. Furthermore, some of these clones cross-reacted with rabbit, pig and/or mouse LOX-1. For possible application as therapeutic agents in the future, two cross-reactive mAbs were re-constructed as chicken-human chimeric antibodies. The chimeric antibodies showed similar characteristics compared to the original antibodies, and inhibited oxLDL binding to LOX-1 expressed on CHO cells. The results obtained in this study indicate that anti-LOX-1 mAbs might be useful tools for functional analyses and development of therapeutic agents for cardiovascular indications such as atherosclerosis.