Late adulthood mortality among African-American and white American people with Type 1 diabetes according to age at diabetes diagnosis.

AIMS: To estimate the overall and cause-specific mortality in a population of African-Americans and white Americans with a low socio-economic status who had young-onset insulin-treated diabetes and had survived beyond the age of 40 years, and to examine whether any excess risk varied according to age at diabetes onset. METHODS: Using the Southern Community Cohort Study, we conducted a mortality follow-up of a cohort of mostly low-income participants aged 40-79 years (mean 50 years) at cohort entry with insulin-treated diabetes diagnosed before age 30 years (n=475) and without diabetes (n=62 266). Childhood onset was defined as diabetes diagnosed before age 20 years (n=162), while young-adulthood onset was defined as diabetes diagnosed between ages 20 and 29 years (n=313). Cause-specific mortality was based on both underlying and contributing causes of death, obtained from death certificates. Multivariable Cox analysis was performed. RESULTS: During follow-up (mean 9.5 years), 38.7% of those with and 12.9% of those without diabetes died. Compared with those without diabetes, increases in mortality rate were generally similar among those with childhood- and young-adulthood-onset diabetes for deaths from: all causes (childhood: hazard ratio 4.3, CI 3.3-5.5; young adulthood: hazard ratio 4.9, CI 4.0-5.8); ischaemic heart disease (childhood: hazard ratio 5.7, CI 3.5-9.4; young adulthood: hazard ratio 7.9, CI 5.6-11.0); heart failure (childhood: hazard ratio 7.3, CI 4.2-12.7; young adulthood: hazard ratio 5.4, CI 3.3-8.9); sepsis (childhood: hazard ratio 10.3, CI 6.1-17.3; young adulthood: hazard ratio 8.8, CI 5.7-13.5); renal failure (childhood: hazard ratio 15.1, CI 8.6-26.5; young adulthood: hazard ratio 18.2, CI 12.3-27.1); respiratory disorders (childhood: hazard ratio 3.9, CI 2.3-6.7; young adulthood: hazard ratio 5.3, CI 3.7-7.7); suicide/homicide/accidents (childhood: hazard ratio 2.3, CI 0.72-7.0; young adulthood: hazard ratio 5.8, CI 3.4-10.2); and cancer (childhood: hazard ratio 2.1, CI 0.98-4.4; young adulthood: hazard ratio 1.2, CI 0.55-2.5). CONCLUSIONS: We observed high excess long-term mortality for all-cause, renal failure, ischemic heart disease and heart failure mortality in African-American and white American people with early-onset insulin-treated diabetes.

CD36 is upregulated in mice with periodontitis and metabolic syndrome and involved in macrophage gene upregulation by palmitate.

BACKGROUND: We reported that high-fat diet (HFD)-induced metabolic syndrome (MetS) exacerbates lipopolysaccharide (LPS)-stimulated periodontitis and palmitate, the major saturated fatty acid in the HFD, amplified LPS-stimulated gene expression in vitro. As CD36 is a major receptor for fatty acids, we investigated periodontal CD36 expression in mice with periodontitis and MetS, and the role of CD36 in inflammatory gene expression in macrophages stimulated by palmitate. METHODS: MetS and periodontitis were induced in mice by HFD and periodontal injection of LPS, respectively. The periodontal CD36 expression and its relationship with alveolar bone loss were studied using immunohistochemistry, real-time PCR, and correlation analysis. The role of CD36 in upregulation of inflammatory mediators by LPS and palmitate in macrophages was assessed using pharmacological inhibitor and small interfering RNA. RESULTS: Periodontal CD36 expression was higher in mice with both MetS and periodontitis than that in mice with periodontitis or MetS alone and was correlated with osteoclastogenesis and alveolar bone loss. In vitro studies showed that CD36 expression in macrophages was upregulated by LPS and palmitate, and targeting CD36 attenuated palmitate-enhanced gene expression. CONCLUSION: CD36 expression is upregulated in mice with periodontitis and MetS and involved in gene expression in macrophages stimulated by palmitate and LPS.

Metabolic syndrome exacerbates inflammation and bone loss in periodontitis.

Clinical studies have shown that metabolic syndrome (MetS) is associated with increased risk of developing periodontitis. However, the underlying mechanisms remain largely unknown. Since it is known that lipopolysaccharide (LPS)-activated toll-like receptor 4 signaling pathways play a crucial role in periodontitis, we hypothesized that MetS enhances LPS-induced periodontal inflammation and alveolar bone loss. In this study, we induced MetS in C57BL/6 mice by feeding them high-fat diet (HFD), and we induced periodontitis by periodontal injection of Aggregatibacter actinomycetemcomitans LPS. We found that mice fed a HFD had significantly increased body weight, plasma lipids, insulin, and insulin resistance when compared with mice fed regular chow, indicating that the mice developed MetS. We also found that a HFD markedly increased LPS-induced alveolar bone loss, osteoclastogenesis, and inflammatory infiltration. Analysis of gene expression in periodontal tissue revealed that HFD and LPS injection cooperatively stimulated expression of cytokines that are known to be involved in periodontal tissue inflammation and osteoclastogenesis-such as interleukin 6, monocyte-chemotactic protein 1, receptor activator of nuclear factor kappa-B ligand, and macrophage colony-stimulating factor. To further understand the potential mechanisms involved in MetS-boosted tissue inflammation, our in vitro studies showed that palmitic acid-the most abundant saturated fatty acid (SFA) and the major SFA in the HFD used in our animal study-potently enhanced LPS-induced proinflammatory gene expression in macrophages. In sum, this study demonstrated that MetS was associated with increased periodontal inflammation and alveolar bone loss in an LPS-induced periodontitis animal model. This study also suggests that SFA palmitic acid may play an important role in MetS-associated periodontitis by enhancing LPS-induced expression of inflammatory cytokines in macrophages.

Simvastatin inhibits lipopolysaccharide-induced osteoclastogenesis and reduces alveolar bone loss in experimental periodontal disease.

BACKGROUND AND OBJECTIVE: Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase and have anti-inflammatory effects independent of cholesterol lowering. Recent clinical studies have indicated that statin intake has a beneficial effect on periodontal disease. However, the underlying mechanisms have not been well understood. In the current study, we employed a rat model with lipopolysaccharide (LPS)-induced periodontal disease and determined the effect of simvastatin, a commonly prescribed statin, on osteoclastogenesis, gingival inflammation and alveolar bone loss. MATERIAL AND METHODS: Sprague-Dawley rats were injected with Aggregatibacter actinomycetemcomitans LPS in periodontal tissue three times per week for 8 wk and part of the rats with LPS injection were also given simvastatin via gavage. After the treatments, the rat maxillae were scanned by microcomputed tomography and the images were analyzed to determine alveolar bone loss. To explore the underlying mechanisms, the effect of simvastatin on osteoclastogenesis and gingival expression of proinflammatory cytokines were also determined by tartrate-resistant acid phosphatase staining and real-time polymerase chain reaction assays, respectively. RESULTS: Results showed that LPS treatment markedly increased bone loss, but administration of simvastatin significantly alleviated the bone loss. Results also showed that LPS treatment stimulated osteoclastogenesis and the expression of inflammatory cytokines, but simvastatin significantly modulates the stimulatory effect of LPS on osteoclastogenesis and cytokine expression. CONCLUSION: This study demonstrated that simvastatin treatment inhibits LPS-induced osteoclastogenesis and gingival inflammation and reduces alveolar bone loss, indicating that the intake of simvastatin may hinder the progression of periodontal disease.

Simvastatin inhibits LPS-induced alveolar bone loss during metabolic syndrome.

Studies in recent years have shown a positive relationship between metabolic syndrome (MS) and periodontal disease (PD). Given that patients with MS take statins to reduce cholesterol, and statins also have anti-inflammatory effects, it is important to determine if statin intake hinders the progression of MS-associated PD. In this study, PD was induced in Zucker fat rats (ZFRs), an animal model for MS, and in control lean rats by periodontal injection of Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS), while simvastatin was given to some of the rats via gavage. After 4 wk of treatment, alveolar bone loss was determined by micro-computed tomography. To explore the underlying mechanisms, we determined the effect of simvastatin on tissue inflammation and the expression of molecules involved in osteoclastogenesis. Results showed that while bone loss was increased by LPS in both ZFRs and the control lean rats, it was significantly more in the former than the latter. Simvastatin effectively alleviated bone loss in both ZFRs and the control rats. Results also showed that LPS stimulated leukocyte tissue infiltration and expression of molecules for osteoclastogenesis, but simvastatin significantly modulated the stimulation. This study demonstrated that simvastatin inhibited LPS-induced alveolar bone loss and periodontal tissue inflammation in rats with MS.

A trend of increase in periodontal interleukin-6 expression across patients with neither diabetes nor periodontal disease, patients with periodontal disease alone, and patients with both diseases.

BACKGROUND AND OBJECTIVE: Epidemiological studies have established that patients with diabetes have increased prevalence and severity of periodontal disease. However, the periodontal expression of inflammatory cytokines and matrix metalloproteinases (MMPs) in diabetic patients has not been well characterized. The objective of this study was to determine the difference in the periodontal expression of MMP-1, MMP-8, interleukin-6, tumor necrosis factor-alpha and interleukin-1beta between diabetic and nondiabetic patients. MATERIAL AND METHODS: Periodontal tissue specimens were collected from nine nondiabetic patients without periodontal disease (group 1), from 11 nondiabetic patients with periodontal disease (group 2) and from seven diabetic patients with periodontal disease (group 3). The expression of MMP-1, MMP-8, interleukin-6, tumor necrosis factor-alpha and interleukin-1beta was quantified using real-time polymerase chain reaction. RESULTS: The nonparametric Kruskal-Wallis test showed that the difference in interleukin-6 expression among the groups was statistically significant (p = 0.04). Furthermore, the generalized Kruskal-Wallis nonparametric linear-by-linear association test showed a statistically significant trend of increase in the expression of interleukin-6 from group 1 to group 2 to group 3 (p = 0.02) and a suggestion of such a trend for MMP-1 (p = 0.05). No increase in MMP-8 expression was observed in patients in group 3 compared to patients in groups 1 and 2. Although the average expression levels of MMP-1, interleukin-1beta and tumor necrosis factor-alpha were increased from group 1 to group 3, the differences were not statistically significant. CONCLUSION: A trend of increased interleukin-6 expression in periodontal tissues was observed across patients with neither diabetes nor periodontal disease, patients with periodontal disease alone, and patients with both diseases.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.