Biomedical subjects

M F Lopes-Virella

Publications and source records attributed to M F Lopes-Virella.

At least 19 recordsLinked to original sources

PubMed2007

High glucose-boosted inflammatory responses to lipopolysaccharide are suppressed by statin.

BACKGROUND AND OBJECTIVE: It has been established that periodontal diseases are more prevalent and of greater severity in diabetic patients than in nondiabetic patients. Recent studies have underscored the role of monocytes and macrophages in periodontal tissue inflammation and destruction in diabetic patients. Although it has been shown that monocytes isolated from diabetic patients produce more inflammatory cytokines and that gingival crevicular fluid collected from diabetic patients contains higher levels of inflammatory cytokines than that obtained from nondiabetic patients, the underlying mechanisms are not well understood. MATERIAL AND METHODS: U937 histiocytes cultured in medium containing either normal (5 mM) or high (25 mM) glucose were treated with 100 ng/ml of lipopolysaccharide for 24h. After the treatment, cytokines in the medium and cytokine mRNA in the cells were quantified using enzyme-linked immunosorbet assay and real-time polymerase chain reaction, respectively. RESULTS: In this study, we demonstrated that the pre-exposure of U937 histiocytes to high glucose concentrations markedly increased the lipopolysaccharide-induced secretion of pro-inflammatory cytokines and chemokines and the cellular inducible nitric oxide level compared with pre-exposure to normal glucose. Our data also showed that the increased secretion of cytokines was a result of increased mRNA expression. Furthermore, the effects of statin and peroxisome proliferators-activated receptor agonists on high glucose-enhanced secretion of cytokines were determined. The results showed that simvastatin, but not fenofibrate or pioglitazone, inhibited high glucose-enhanced cytokine release. CONCLUSION: This study has shown that high glucose concentrations and lipopolysaccharide act synergistically to stimulate the secretion of inflammatory mediators, and that statin is capable of suppressing the high glucose-boosted proinflammatory response. This study therefore delineates a novel mechanism by which hyperglycemia enhances the inflammatory responses of macrophages and suggests that statin may be useful in the treatment of periodontal disease in diabetic patients.

Chemokines↗

PubMed2004

The prospective association between adiponectin and coronary artery disease among individuals with type 1 diabetes. The Pittsburgh Epidemiology of Diabetes Complications Study.

AIMS/HYPOTHESIS: Recent findings suggest the potential involvement of adiponectin in obesity, diabetes and cardiovascular disease. We assessed the prospective association between adiponectin concentration and coronary artery disease in individuals with type 1 diabetes. METHODS: Participants were identified from the Pittsburgh Epidemiology of Diabetes Complications cohort, a prospective follow-up study of childhood-onset type 1 diabetes. At baseline, subjects had a mean age of 28 years, and a mean diabetes duration of 19 years. Cases (determined by physician-diagnosed angina, confirmed myocardial infraction, stenosis >or=50%, ischemic ECG or revascularization) were matched to the control subjects with respect to sex, age and diabetes duration. Samples and risk factors for analyses were identified from the earliest exam prior to incidence in cases. Sera and information on all covariates were available for 28 cases and 34 control subjects. Proportional hazards models were constructed including matching variables. RESULTS: Compared with those in men, adiponectin concentrations were elevated in females (p=0.009) and among individuals with macroalbuminuria (p=0.04). In multivariable analyses (adjusting for standard risk factors as well as lipoprotein measurements determined by nuclear magnetic resonance spectroscopy, E-selectin or antioxidants), adiponectin inversely predicted the incidence of coronary artery disease (hazard ratio=0.37 per 1 SD increase, 95% CI 0.19-0.73, p=0.004). CONCLUSIONS/INTERPRETATION: The results suggest that increased adiponectin concentration is prospectively associated with a lower risk of coronary artery disease type 1 diabetes. The potential of adiponectin determination as a useful marker of, and potential therapeutic target for, coronary artery disease prevention in type 1 diabetes should be further explored.

Adiponectin↗

PubMed2002

Oxidized LDL-anti-oxidized LDL immune complexes and diabetic nephropathy.

AIMS/HYPOTHESIS: Oxidized LDL is immunogenic and immune complexes formed by oxidized LDL and corresponding antibodies are pro-atherogenic and pro-inflammatory. Considering that macroalbuminuria is a risk factor for coronary heart disease and that common pathogenic factors for atherosclerosis and glomerulosclerosis exist, our aim was to determine whether the amount and/or characteristics of oxidized LDL- anti-oxidized LDL complexes correlated with the degree of albuminuria in patients with Type I (insulin-dependent) diabetes mellitus. METHODS: We studied 33 macroalbuminuric patients (albumin excretion rate >300 mg/24 h) and 29 microalbuminuric patients (albumin excretion rate >or=30 mg/24 h and <or=300 mg/24 h) who were matched for age, sex and HbA(1C) concentrations with 43 patients with normal albuminuria. In all patients we isolated and characterized oxidized LDL-anti-oxidized LDL complexes. A subgroup of 47 patients (22 with normal albuminuria, 11 with microalbuminuria, and 14 with macroalbuminuria) was randomly selected from the larger group for characterization of the antibody moiety of oxidized LDL immune complexes. RESULTS: The concentrations of oxidized LDL immune complexes were increased in patients with macroalbuminuria (total cholesterol in immune complexes isolated from patients with macroalbuminuria was 272+/-20 vs 204+/-18 micro g/ml in patients with normoalbuminuria, p<0.03). Also, the Kd of oxLDL antibodies isolated from the immune complexes was lower in patients with abnormal albuminuria than in patients with normoalbuminuria (0.86+/-0.1x10(-8) vs 1.13+/-0.1x10(-8) mol/l, p<0.05). CONCLUSION/INTERPRETATION: The data indicate that high concentrations of oxidized LDL-anti-oxidized LDL complexes, particularly when formed with antibodies of higher avidity, are associated with abnormal proteinuria.

Adult↗

PubMed2001

Quercetin inhibits matrix metalloproteinase-1 expression in human vascular endothelial cells through extracellular signal-regulated kinase.

Studies have shown that intake of quercetin was inversely associated with mortality from coronary heart disease. Since recent studies documented that disruption of atherosclerotic plaques is the key event triggering acute myocardial infarction, and vascular endothelium-derived matrix metalloproteinase-1 (MMP-1) contributes to plaque destabilization, we examined the effect of quercetin on MMP-1 expression in human vascular endothelial cells. Our results showed that quercetin significantly inhibited basal and oxidized LDL (oxLDL)-stimulated MMP-1 expression. Our data also indicated that extracellular signal-regulated kinase (ERK) mediated the basal and oxLDL-stimulated expression of MMP-1, and quercetin is a potent inhibitor of ERK, suggesting that quercetin may inhibit MMP-1 expression by blocking the ERK pathway. Finally, we showed that quercetin stimulated tissue inhibitor of metalloproteinase-1 expression in oxLDL- and PMA-treated cells. In conclusion, the present study demonstrated for the first time that quercetin inhibited MMP-1 expression in vascular endothelial cells, suggesting that quercetin might contribute to plaque stabilization.

Cells, Cultured↗

PubMed2001

Expression of adhesion molecules by human endothelial cells exposed to oxidized low density lipoprotein. Influences of degree of oxidation and location of oxidized LDL.

The main objective of this study was to determine the influence of the degree of low density lipoprotein (LDL) oxidation and the location of oxidized LDL (oxLDL) on expression of adhesion molecules on endothelial cells (EC). OxLDL preparations 1-4 with different degrees of oxidative modification were studied. All preparations of oxLDL, after addition to the medium, stimulated the expression of intercellular adhesion molecule-1 (ICAM-1) by human umbilical vein endothelial cells (HUVEC) as determined by cell-ELISA. Concentration-dependent studies examining ICAM-1 expression by HUVEC showed that the minimal concentration of oxLDL which significantly stimulated ICAM-1 expression was 5 microg/ml, suggesting that the predicted physiological concentration of oxLDL in plasma may be not high enough to elicit a substantial increase of ICAM-1 expression in EC. In contrast, very small amounts (0.15 microg/well) of oxLDL-3 and 4, the more heavily oxidized LDL preparations, stimulated effectively ICAM-1 expression by HUVEC when located below the endothelial cell monolayer by immobilizing to type I collagen. The results suggest that the increased expression of ICAM-1 induced by accumulated oxLDL may be one of the mechanisms by which oxLDL contributes to atherogenesis.

Cells, Cultured↗

PubMed2001

Oxidized LDL differentially regulates MMP-1 and TIMP-1 expression in vascular endothelial cells.

We have reported recently that oxidized low-density lipoprotein (oxLDL) stimulates matrix metalloproteinase-1 (MMP-1) expression in human vascular endothelial cells. The present study was conducted to examine the effect of oxLDL on expression of Tissue inhibitor of metalloproteinase-1 (TIMP-1), an endogenous inhibitor of MMPs, in human vascular endothelial cells. Our enzyme-linked immunosorbent assay and Northern blot analysis showed that oxLDL inhibited TIMP-1 secretion and expression by human umbilical vein endothelial cells. In contrast, PMA stimulated TIMP-1 expression and secretion. Both oxLDL and PMA increased MMP-1 expression and secretion significantly as previously reported. Inhibition by oxLDL of TIMP-1 expression was also observed in human aortic endothelial cells. Collagenase activity as detected by an enzymatic activity assay demonstrated, as expected, an increase in collagenase activity in the culture medium from oxLDL-treated cells as compared with that from untreated cells. The presented data indicates that oxLDL differentially regulates TIMP-1 and MMP-1 expression, whereas PMA coordinately regulates TIMP-1 and MMP-1 in vascular endothelial cells. The lack of coordination in the secretion of MMP-1 and TIMP-1 induced by oxLDL leads to an increased collagen-degrading activity that may contribute to destabilization of atherosclerotic plaques.

Cells, Cultured↗

PubMed2001

Effect of inflammatory cytokines on the metabolism of low-density lipoproteins by human vascular endothelial cells.

Cytokines have been shown to activate multiple, varied metabolic pathways in endothelial cells. Little information is available concerning the effects of inflammatory cytokines on lipoprotein metabolism by vascular endothelial cells. Human umbilical vein endothelial cells (HuECs) and bovine aortic endothelial cells (BAECs) were incubated with the inflammatory cytokines recombinant human interleukin-1beta (IL-1), tumor necrosis factor alpha (TNF), interferon gamma (gamma-IF), and interferon beta (beta-IF) at increasing concentrations (0.1 to 1,000 U/mL), for increasing periods (6 to 72 hours). After the incubation period, the media were removed and replaced with serum-free media containing radiolabeled native or acetylated low-density lipoprotein (Ac-LDL) and the rates of degradation and accumulation of radiolabeled LDL were determined. The degradation and accumulation of 125I-LDL were significantly increased (P < .02) in HuECs preincubated with IL-1 (100 U/mL) compared with control incubations without the cytokine or incubations containing gamma-IF, beta-IF, or TNF. This resulted from a 38% increase in LDL receptor protein in cells incubated with IL-1. The increased rate of LDL catabolism by HuECs incubated with IL-1 was accompanied by a significant increase (P < .05) in the rate of cholesteryl ester synthesis in the cells. Cholesteryl ester synthesis rates in HuECs preincubated with gamma-IF, beta-IF, or TNF did not differ significantly from the rates in control incubations. The effect of preincubation with cytokine on the activity of the scavenger receptor was also determined. There were no significant differences in the rate of degradation or accumulation of radiolabeled Ac-LDL in control incubations compared with cultures preincubated with IL-1, gamma-IF, beta-IF, or TNF. There also were no significant differences in the rate of catabolism of native LDL or Ac-LDL in BAECs preincubated with cytokines. Although cytokines have been shown previously to alter the binding of monocytes to endothelial cells, there was no significant increase in the binding of monocytes to cultures incubated with IL-1 plus LDL compared with IL-1 alone. In summary, we now demonstrate that cytokines, specifically IL-1, may alter LDL metabolism by human vascular endothelial cells and alter endothelial cell cholesterol metabolism. These changes in endothelial cell metabolism provide additional evidence supporting the critical role of cytokines in atherogenesis.

Animals↗

PubMed2000

The preparation of copper-oxidized LDL for the measurement of oxidized LDL antibodies by EIA.

In the present study we try to define the optimal conditions for preparation of copper-oxidized low-density lipoprotein (oxLDL) to be used for the assay of oxLDL antibodies by enzyme immunoassay (EIA). Oxidation of LDL was monitored by measuring the formation of conjugated dienes at 234 nm and the generation of fluorescent products with emission at 430 nm when excitation is performed at 360 nm. The generation of immunogenic epitopes was evaluated by testing the reactivity of aliquots collected at different times during the oxidation process with human sera with high oxLDL antibody levels and with a purified human oxLDL antibody. The values of fluorescence emission at 430 nm correlated best with reactivity with oxLDL antibodies; strong reactivity was usually associated with values greater than 1.1 U. The time needed for fluorescence emission to reach maximum levels varied between 6 and 14 h for most LDL, but it was considerably longer in a few LDL preparations. The maximal reactivity of oxLDL with oxLDL antibodies was observed when the LDL oxidation reaction was stopped 4 or more hours after the fluorescence readings reached their peak. At this stage of the oxidation reaction, apolipoprotein B fragmentation and aggregation were observed as shown by Western blot analysis. The CV for 13 EIA runs of two reference oxLDL antibodies reacting with four different pools of standardized oxLDL prepared according to the stated guidelines was 14.5 and 3.9%, confirming the reproducibility of our oxidation conditions.

Antigen-Antibody Complex↗

PubMed2000

Fc-gamma receptor cross-linking by immune complexes induces matrix metalloproteinase-1 in U937 cells via mitogen-activated protein kinase.

Matrix metalloproteinase-1 (MMP-1) secreted by macrophages potentially contributes to plaque rupture. Because large quantities of immunoglobulin G and ICs (ICs), including low density lipoprotein-containing ICs (LDL-ICs), are present in atherosclerotic lesions, we examined the effect of LDL-ICs on macrophage MMP-1 expression. With the use of ICs prepared with human LDL and rabbit anti-LDL antiserum, our enzyme-linked immunosorbent assays and Northern blots showed that MMP-1 secretion and expression by U937 histiocytes were induced by LDL-ICs. Furthermore, our results showed that blocking of Fc-gamma receptor I and II (FcgammaRI and FcgammaRII) inhibited 70% and 55%, respectively, of the LDL-IC-induced secretion of MMP-1. Finally, our data showed that both PD98059, an inhibitor of the mitogen-activated protein kinase pathway, and Ro-31-2880, an inhibitor of protein kinase C, inhibited LDL-IC-stimulated MMP-1 secretion in a dose-dependent manner, with 96% and 95% inhibition, respectively, at the respective doses of 50 micromol/L and 80 nmol/L. In conclusion, this study demonstrated for the first time that LDL-ICs induce macrophage MMP-1 secretion by cocross-linking FcgammaRI and FcgammaRII and triggering a protein kinase C-dependent mitogen-activated protein kinase pathway. These results suggest that LDL-ICs and other ICs localized in atherosclerotic plaques may be potent stimulators for macrophage MMP-1 expression and may contribute to plaque rupture and acute coronary events.

Antibodies, Anti-Idiotypic↗

PubMed2000

Use of fenofibrate in the management of protease inhibitor-associated lipid abnormalities.

Human immunodeficiency virus (HIV) protease inhibitors are associated with several metabolic abnormalities including hypercholesterolemia and hypertriglyceridemia. Fenofibrate is a new lipid-lowering agent for adults with very high triglyceride levels that was administered to two HIV-positive patients who were taking protease inhibitors and developed hypertriglyceridemia. Starting dosages were 134 and 201 mg/day, and were increased to 268 mg/day in both patients. Triglyceride levels decreased from 1450 to 337 mg/dl (76.8%) and from 1985 to 322 mg/dl (83.8%), respectively, after 10 months of therapy. High-density lipoprotein levels increased in both patients.

Adult↗

PubMed2000

Anti-modified LDL antibodies, LDL-containing immune complexes, and susceptibility of LDL to in vitro oxidation in patients with type 2 diabetes.

We investigated the hypothesis that modified lipoproteins trigger an immune response leading to the production of autoantibodies and subsequently to the formation of atherogenic immune complexes (IC). We recruited 20 type 2 diabetic patients with macrovascular disease, 14 nondiabetic patients with coronary artery disease (CAD), and 34 healthy control subjects matched for age, sex, and race. Serum antibodies to oxidized and glycated LDL did not differ significantly among the 3 groups. Serum IC contained variable, but not statistically different, amounts of IgG, IgM, and IgA. In contrast, the content of cholesterol in IC isolated from diabetic patients was significantly higher than that in IC isolated from control subjects, and the content of apolipoprotein (apo)-B was significantly higher than that in IC isolated from control subjects and patients with CAD. Cholesteryl ester accumulation in human monocyte-derived macrophages incubated with IC, a measure of the atherogenic potential of IC, was significantly higher in macrophages incubated with red blood cell-adsorbed IC isolated from diabetic patients compared with IC isolated from control subjects (P < 0.03) or from patients with CAD (P < 0.04) and was strongly correlated with the content of apoB (r = 0.68, P < 0.001) and cholesterol (r = 0.61, P < 0.001) in IC. LDL from diabetic patients was more susceptible to oxidation in vitro, was significantly smaller, and contained significantly less alpha-tocopherol than LDL isolated from subjects in the other groups. In addition, the n-3 polyunsaturated fatty acid content of phospholipids and cholesteryl esters in LDL isolated from diabetic patients was significantly increased (P < 0.05) compared with that from patients with CAD or from control subjects. We postulate that LDL size, susceptibility to oxidation, and lipid fatty acid composition may play a critical role in the production of antibodies to oxidized LDL and consequently in the formation of LDL-containing IC in patients with type 2 diabetes.

Antigen-Antibody Complex↗

PubMed2000

Oxidized LDL induces the expression of ALBP/aP2 mRNA and protein in human THP-1 macrophages.

The adipocyte lipid-binding protein (ALBP/aP2) belongs to a multigene family of fatty acid and retinoid transport proteins. This protein is abundantly expressed in the cytoplasm and nuclear region of adipocytes and is postulated to serve as a lipid shuttle, solubilizing hydrophobic fatty acids and delivering them to the appropriate metabolic system for utilization. This report demonstrates that human cholesterol-loaded THP-1 macrophages express ALBP/aP2 and that its expression can be stimulated by oxidized low density lipoprotein (oxLDL). The increase in mRNA expression was paralleled by a similar increase in ALBP/aP2 protein. The increase in ALBP/aP2 mRNA and protein in oxLDL-stimulated THP-1 macrophages is concentration and time dependent and is inhibited by treatment of the cells with an antioxidant inhibitor of nuclear factor-kappaB (NF-kappaB), pyrrolidine dithiocarbamate (PDTC), and with protein kinase C (PKC) inhibitors bisindolylmaleimide I and Ro-31-8220. These results suggest that activation of both NF-kappaB and PKC signaling pathways is necessary for oxLDL-induced ALBP/aP2 gene expression in THP-1 macrophages and that the upregulation of the fatty acid carrier may be a necessary event in foam cell formation.

Carrier Proteins↗

PubMed1999

Antibodies to oxidized LDL and LDL-containing immune complexes as risk factors for coronary artery disease in diabetes mellitus.

Several groups have published results from clinical studies supporting the involvement of anti-modified LDL antibodies as risk factors for the initiation or progression of cardiovascular disease. However, the data published so far are judged inconclusive because of several contradictory observations concerning the correlation between clinical evidence of arteriosclerosis and the levels of antibodies to oxidized LDL (oxLDL Ab). We have previously reported that oxLDL Ab exist both in free form and as antigen-antibody complexes (LDL-IC) in patients with insulin-dependent diabetes mellitus (IDDM). The presence of LDL-IC in IDDM patients has important implications: it may interfere with the assay of oxLDL antibodies and the levels of LDL-IC may correlate better with the development of arteriosclerosis than the levels of free oxLDL antibodies. To clarify these questions baseline samples collected from 49 IDDM patients, who subsequently developed coronary artery disease (CAD) during an 8-year follow-up period, were compared to baseline samples from 49 age-, sex-, and duration-matched control IDDM subjects who remained free of clinical CAD during an identical follow-up period. The levels of free oxLDL antibody were significantly lower in the patients who developed CAD. The same patients had significantly higher concentrations of total cholesterol, apolipoprotein B, and IgA in immune complex-enriched polyethylene glycol (PEG) precipitates. The concentration of IgG was also higher in PEG precipitates from patients who developed CAD, but did not reach statistical significance. This indicates that patients who develop CAD had higher levels of circulating LDL-IC, a fact that could not be deduced from the measurement of free oxLDL antibody concentrations. A linear regression analysis of the correlation between the concentrations of total cholesterol in PEG precipitates, taken as a surrogate measurement of PEG-precipitated oxLDL-IC, and the concentration of free oxLDL antibody in serum showed a statistically significant negative correlation (r = -0.229, P = 0. 024). Our results support the conclusion that oxLDL-IC may be a risk factor for the development of macrovascular disease in IDDM patients. We also have demonstrated that circulating oxLDL-IC interfere with the assay of free oxLDL antibodies.

Adult↗

PubMed1999

Oxidized LDL stimulates matrix metalloproteinase-1 expression in human vascular endothelial cells.

It has been well documented that acute myocardial infarction is triggered by disruption of atherosclerotic plaques. Immunocytochemistry studies have shown that matrix metalloproteinase-1 (MMP-1) is specifically expressed by cells present in atherosclerotic plaques, including luminal and neovascular endothelial cells. Since MMP-1 degrades type I collagen, a major type of collagen in atherosclerotic lesions, it is likely that MMP-1 is involved in promoting destabilization of plaques. To date, however, the stimulatory factors that induce MMP-1 expression in endothelial cells have not been well defined. In the present study, we found that oxidized low density lipoprotein (LDL) stimulated MMP-1 release from both human umbilical vein and aortic endothelial cells. We also found that oxidized LDL markedly stimulated MMP-1 expression in these cells and that the degree of LDL oxidation was positively correlated with the level of MMP-1 mRNA expression. Furthermore, our data showed that stimulated MMP-1 secretion was inhibited by actinomycin D and that the nascent MMP-1 mRNA synthesis was stimulated by oxidized LDL, indicating that oxidized LDL activated transcription of the MMP-1 gene. Finally, both zymography and activity assays demonstrated that collagenase activity in conditioned medium was stimulated by oxidized LDL. Taken together, these results have shown for the first time that oxidized LDL stimulates MMP-1 transcription and secretion by vascular endothelial cells, suggesting that oxidized LDL may be a potent stimulator for MMP-1 expression in atherosclerotic plaques, thus promoting plaque rupture.

Arteriosclerosis↗

PubMed1999

Oxidized LDL-containing immune complexes induce Fc gamma receptor I-mediated mitogen-activated protein kinase activation in THP-1 macrophages.

Our previous studies have shown that Fc gamma receptor (FcgammaR)-mediated uptake of LDL-containing immune complexes (oxLDL-ICs) by human monocyte-derived macrophages leads to not only transformation of macrophages into foam cells but also macrophage activation and release of cytokines. It has been shown that cross-linking of FcgammaR triggers activation of signal transduction pathways that alter gene expression in macrophages. In this study, we determined whether engagement of FcgammaR by oxLDL-ICs leads to activation of mitogen-activated protein (MAP) kinase pathway, a signaling cascade serving many important functions, including the regulation of gene expression, in THP-1 macrophage-like cells. Our results from immunoblotting, using specific anti-phosphorylated MAP kinase antibodies, showed that oxLDL-ICs induced extracellular signal regulated kinase 2 (ERK2) MAP kinase phosphorylation in THP-1 macrophage-like cells in time- and dose-dependent manners. Cholesterol loading before stimulation led to a longer phosphorylation of ERK2. Nuclear translocation of phosphorylated ERK was markedly increased after the stimulation. Moreover, our data showed that oxLDL-IC induction of MAP kinase was prevented by human monomeric IgG1, suggesting that the specific engagement of type I FcgammaR by oxLDL-IC is responsible for the MAP kinase activation. Finally, we showed that human anti-oxLDL autoantibody-containing immune complexes immobilized on type I collagen induced MAP kinase activation in THP-1 cells. These results strongly suggest that oxLDL-IC, which has been detected in atherosclerotic plaques, may play an important role in macrophage activation and atherogenesis.

Animals↗

PubMed1999

Antibodies to oxidized LDL predict coronary artery disease in type 1 diabetes: a nested case-control study from the Pittsburgh Epidemiology of Diabetes Complications Study.

The pathogenesis of excess cardiovascular risk in type 1 diabetes is unclear. LDL cholesterol is only weakly predictive, and its concentration is often normal in type 1 diabetes. We therefore examined whether markers of LDL oxidation such as antibodies to oxidized LDL (Ab-OxLDL) and LDL-containing immune complexes, rather than LDL concentration, were predictive of coronary artery disease (CAD) in type 1 diabetes. This nested case-control study from an epidemiologic cohort study included 49 incident cases of myocardial infarction (MI), angina, or CAD death and 49 age-, sex-, and duration-matched control subjects. Ab-OxLDL was measured by enzyme immunoassay and the apolipoprotein B (ApoB) content of immune complexes (ApoB-IC) precipitated by polyethylene glycol by immunoelectrophoresis in baseline stored samples. Ab-OxLDL was inversely, and ApoB-IC directly, related to subsequent CAD. In multivariate analyses, Ab-OxLDL remained a significant independent predictor along with previously recognized predictors, hypertension and Beck depression score. In conclusion, oxidation of LDL and the immune response it elicits may play a role in predicting the development of CAD in type 1 diabetes and explain at least some of the enhanced CAD risk in type I diabetes.

Adult↗

PubMed1998

Preparation of a human standard for determination of the levels of antibodies to oxidatively modified low-density lipoproteins.

As part of our ongoing effort to develop a standardized competitive enzyme immunoassay for human autoantibodies to oxidized low-density lipoprotein (oxLDL), we have generated a reference human antibody standard and revised some of the conditions of the assay. The preparation of the standard involved purification of human anti-oxLDL antibodies by affinity chromatography using immobilized oxLDL. The total concentration of antibody in this purified human oxLDL antibody was established by adding the concentrations of immunoglobulin G (IgG), IgA, and IgM determined in the standard by radial immunodiffusion. The isolated antibody was used to calibrate a whole human serum standard, which was used to calibrate the assays to detect antibody in serum samples. We also revisited the general conditions for performance of our competitive assay. We determined that 1/20 was the ideal dilution for performing the absorption step, and that 1/20 and 1/40 were optimal dilutions to assay oxLDL antibody in unknown serum samples. We also established that the optimal concentration of oxLDL for absorption of free antibody in serum samples was 200 microgram of oxLDL/ml; no significant decrease in the reactivity of samples with immobilized oxLDL was observed when higher concentrations of oxLDL were used for absorption. The minimum detection level of the assay is 0.65 mg/liter. Because serum samples are diluted 1/20 and 1/40 for the assay, the minimal concentration of antibody detectable in serum is 20-fold higher, i.e., 13 mg/liter. The intraassay coefficient of variation calculated from seven determinations of three samples containing antibody concentrations of 240, 340, and 920 mg/liter ranged from 8 to 6.1%. The interassay coefficients of variation for sera with antibody levels of 100 to 594 mg/liter varied from 9.2 to 7.0%, and for isolated antibodies with concentrations of 52 to 111 mg/liter, the coefficients varied from 5.8 to 3.9%.

Autoantibodies↗

PubMed1997

Anti-modified LDL antibodies and LDL-containing immune complexes in IDDM patients and healthy controls.

Antibodies to oxidized LDL (ox-LDL) and LDL-containing immune complexes (LDL-IC) have been reported to be associated with the presence or progression of arteriosclerosis. We screened for anti-modified LDL antibodies and isolated soluble IC by precipitation with 3.5% (w/v) polyethylene glycol (PEG) 6000 in two groups. The patient group was constituted by 16 insulin-dependent diabetes mellitus subjects free of macrovascular complications. The control group was constituted by 16 healthy, age-, gender-, race-, and body mass index-matched nondiabetic subjects. We detected anti-ox-LDL antibodies and anti-malondialdehyde-modified LDL antibodies with similar levels in patients and controls, while the levels of anti-glycated LDL antibodies were very low, but slightly higher in diabetics than in healthy controls. Isolated LDL-IC were adsorbed to red blood cells (RBC) and incubated with human macrophages for 18 hr at 37 degrees C. Under those experimental conditions, RBC-adsorbed IC are taken up by macrophages but the RBC remain intact and are not ingested. Slightly higher levels of cholesteryl ester (CE) accumulation were measured in macrophages incubated with RBC to which we adsorbed IC isolated from diabetics (15.4 +/- 2.5 micrograms/mg of protein, mean +/- SEM) than in macrophages incubated with IC isolated from controls (12.5 +/- 1.6 micrograms/mg of protein, mean +/- SEM), but the difference did not reach statistical significance. PEG-precipitable IC isolated from both normal and diabetic subjects led, in some instances, to the transformation of macrophages into foam cells. Significant correlations were observed between CE accumulation and the content of apo B (P < 0.0001), total cholesterol (P = 0.0004), IgG (P = 0.015), and IgA (P = 0.015) in the isolated IC. The correlation between CE accumulation and the content of apo B in isolated IC was stronger in diabetics than in the control group (r = 0.759 vs r = 0.500). Fractionation of isolated IC in immobilized protein A/G yielded immunoglobulin-rich fractions which contained cholesterol and IgG anti-ox-LDL antibodies. The cholesterol content of these fractions was significantly correlated (P = 0.001) with CE accumulation. In conclusion, both diabetics and normal individuals have circulating IC whose atherogenic potential appears to be related to the presence of LDL and antibodies of the IgG and IgA isotypes.

Adult↗