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Background— Elevated serum soluble E-selectin levels have been associated with a number of diseases. Although E-selectin levels are heritable, little is known about the specific genetic factors involved. E-selectin levels have been associated with the ABO blood group phenotype. Methods and Results— We performed a high-resolution genome-wide association study of serum soluble E-selectin levels in 685 white individuals with type 1 diabetes from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Intervention and Complications (EDIC) study to identify major loci influencing levels. Highly significant evidence for association ( P =10 −29 ) was observed for rs579459 near the ABO blood group gene, accounting for 19% of the variance in E-selectin levels. Levels of E-selectin were higher in O/O than O/A heterozygotes, which were likewise higher than A/A genotypes. Analysis of subgroups of A alleles reveals heterogeneity in the association, and even after this was accounted for, an intron 1 SNP remained significantly associated. We replicate the ABO association in nondiabetic individuals. Conclusion— ABO is a major locus for serum soluble E-selectin levels. We excluded population stratification, fine-mapped the association to sub-A alleles, and also document association with additional variation in the ABO region.

It has been shown that lactate induces insulin resistance. However, the underlying mechanisms have not been well understood. Based on our observation that lactate augments LPS-stimulated inflammatory gene expression, we proposed that lactate may enhance TLR4 signaling in macrophages, which has been shown to play an important role in insulin resistance in adipocytes. In this study, we demonstrated that lactate stimulated MD-2, a coreceptor for TLR4 signaling activation, NF-κB transcriptional activity, and the expression of inflammatory genes in human U937 histiocytes (resident macrophages). Similar enhancement of the inflammatory gene expression by lactate was also observed in human monocyte-derived macrophages. The essential role of MD-2 in lactate-augmented TLR4 signaling was confirmed by observation that the suppression of MD-2 expression by small interfering RNA led to significant inhibition of inflammatory gene expression. To further elucidate how lactate treatment enhances TLR4 activation, we showed that the augmentation of inflammatory gene expression by lactate was abrogated by antioxidant treatment, suggesting a critical role of reactive oxygen species in the enhancement of TLR4 activation by lactate. Finally, we showed that α-cyano-4-hydroxycinnamic acid, a classic inhibitor for monocarboxylate transporters, blocked lactate-augmented inflammatory gene expression and nuclear NF-κB activity, indicating that lactate transport through monocarboxylate transporters is required for lactate-enhanced TLR4 activation. Collectively, this study documents that lactate boosts TLR4 activation and NF-κB-dependent inflammatory gene expression via monocarboxylate transporters and MD-2 up-regulation.

Recent diabetes control and complications trial and epidemiology of diabetes interventions and complications (DCCT/EDIC) and other clinical studies have reported that glucose control in patients with diabetes leads to a significant reduction of cardiovascular events and atherosclerosis, indicating that hyperglycemia plays an essential role in cardiovascular disease in diabetic patients. Although several mechanisms by which hyperglycemia promotes atherosclerosis have been proposed, it remains unclear how hyperglycemia promotes atherosclerosis by interaction with inflammatory cytokines. To test our hypothesis that hyperglycemia interplays with interferon gamma (IFN gamma), a key factor involved in atherosclerosis, to up-regulate the expression of genes such as matrix metalloproteinases (MMPs) and cytokines that are involved in plaque destabilization, U937 macrophages cultured in medium containing either normal or high glucose were challenged with IFN gamma and the expression of MMPs and cytokines were then quantified by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Results showed that high glucose and IFN gamma had a synergistic effect on the expression of MMP-1, MMP-9 and IL-1 beta. High glucose also enhanced IFN gamma-induced priming effect on lipopolysaccharide (LPS)-stimulated MMP-1 secretion. Furthermore, high glucose and IFN gamma exert the synergistic effect on MMP-1 expression by enhancing STAT1 phosphorylation and STAT1 transcriptional activity. In summary, this study revealed a novel mechanism potentially involved in diabetes-promoted cardiovascular disease.

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-α and interleukin-1β 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-α and interleukin-1β 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-1β and tumor necrosis factor-α 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.

Patients with diabetes or metabolic syndrome frequently have higher triglycerides, lower high-density lipoprotein (HDL) cholesterol, and more particles containing apolipoprotein B (ApoB); this combination contributes significantly to their cardiovascular risk. Optimal management of dyslipidemia and increased atherosclerotic risk requires a fundamental understanding of diabetic dyslipidemia, the clinical evidence for different interventional strategies, and the potential benefit of achieving therapeutic targets. For this review, we considered guidelines, recent reviews, and clinical trial results. The features of dyslipidemia in type 2 diabetes and the metabolic syndrome are linked metabolically and are related to central adiposity and insulin resistance. Levels of ApoB and HDL cholesterol are particularly important markers of risk. Guidelines broadly agree that low-density lipoprotein (LDL) cholesterol should be reduced below population average levels. Additional or secondary strategies in patients with diabetes or the metabolic syndrome are to decrease non-HDL cholesterol, ApoB and/or LDL particle concentration, to increase HDL cholesterol, and to reduce triglycerides. Lifestyle changes and statins are the bedrock of treatment, although second-line treatment using fibrates or niacin will likely benefit many patients with residual risk. Ezetimibe, too, has a favorable effect on lipid profile and inflammatory biomarkers of risk. Dyslipidemia in type 2 diabetes and metabolic syndrome has a distinct profile, suggesting the need for a tailored therapy that targets the key features of lowered HDL cholesterol and raised triglycerides, in addition to the primary antiatherogenic strategy of lowering ApoB-containing lipoproteins, such as LDL. With the prominent failure of some recent intervention trials, new therapeutic strategies-particularly safe and effective means to raise HDL-are needed to manage dyslipidemia in this high-risk population.

Modified forms of LDL are immunogenic and activate both cell-mediated and humoral immune responses. Both types of responses are pro-inflammatory and are probably primary players in the perpetuation of the chronic inflammatory reaction characteristic of atherosclerosis. The immunologic response to modified LDL can be directed to MHC-II-associated peptides in the case of T helper cells, and to a variety of epitopes-modified lysine groups, modified phospholipids, proteins that become associated with oxidized LDL (such as beta2GP1)--in the case of B cell responses. T cell activation is likely to play a major role through cross-activation of macrophages. Humoral responses to modified LDL are pathogenic as a consequence of the formation of antigen-antibody complexes containing modified LDL and IgG antibodies. Those immune complexes induce cholesterol ester accumulation in macrophages and macrophage-like cells, and induce the release of pro-inflammatory cytokines, chemokines, oxygen active radicals, and matrix metalloproteinases from those cells. There is no conclusive evidence supporting a protective role for IgM antibodies in humans, possibly because autoantibodies to modified lipoproteins are predominantly of the IgG isotype.

Matrix metalloproteinase (MMP) plays a crucial role in periodontal disease and is up-regulated by oral Gram-negative, pathogen-derived LPS. In this study, we reported that simvastatin, a 3-hydroxyl-3-methylglutaryl-CoA reductase inhibitor, effectively inhibited LPS-stimulated MMP-1 as well as MMP-8 and MMP-9 expression by U937 mononuclear cells. Our studies showed that the geranylgeranyl transferase inhibitor inhibited LPS-stimulated MMP-1 expression, and addition of isoprenoid intermediate geranylgeranyl pyrophosphate (GGPP) reduced the inhibitory effect of simvastatin on LPS-stimulated MMP-1 expression. We also demonstrated that simvastatin inhibited the activation of Ras and Rac, and the inhibition was abolished by addition of GGPP. The above results indicate that protein isoprenylation is involved in the regulation of MMP-1 expression by LPS and simvastatin. Moreover, we showed that simvastatin inhibited LPS-stimulated nuclear AP-1, but not NF-κB activity, and the inhibition was reversed by addition of GGPP. Simvastatin also inhibited LPS-stimulated ERK but not p38 MAPK and JNK. Finally, we showed that the inhibition of LPS-stimulated ERK activation by simvastatin was reversed by GGPP. Taken together, this study showed that simvastatin suppresses LPS-induced MMP-1 expression in U937 mononuclear cells by targeting protein isoprenylation-mediated ERK activation.

Objective: We explored the relevance and significance of connective tissue growth factor (CTGF) as a determinant of renal and vascular complications among type 1 diabetic patients.Methods and Results: We measured the circulating and urinary levels of CTGF and CTGF N fragment in 1050 subjects with type 1 diabetes from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) Study cohort. We found that hypertensive diabetic subjects have significantly higher levels of plasma log CTGF N fragment relative to normotensive subjects (P = 0.0005). Multiple regression analysis showed a positive and independent association between CTGF N fragment levels and log albumin excretion rate (P < 0.0001). In categorical analysis, patients with macroalbuminuria had higher levels of CTGF N fragment than diabetic subjects with or without microalbuminuria (P < 0.0001). Univariate and multiple regression analyses demonstrated an independent and significant association of log CTGF N fragment with the common and internal carotid intima-media thickness. The relative risk for increased carotid intima-media thickness was higher in patients with concomitantly elevated plasma CTGF N fragment and macroalbuminuria relative to patients with normal plasma CTGF N fragment and normal albuminuria (relative risk = 4.76; 95% confidence interval, 2.21–10.25; P < 0.0001).Conclusion: These findings demonstrate that plasma CTGF is a risk marker of diabetic renal and vascular disease.

OBJECTIVE—Because endothelial cell dysfunction and inflammation are key contributors to the development of complications in type 1 diabetes, we studied risk factors related to endothelial dysfunction and inflammation (C-reactive protein and fibrinogen, soluble vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and E-selectin, and fibrinolytic markers) in a subgroup of patients from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Intervention and Complications (EDIC) study cohort. RESEARCH DESIGN AND METHODS—We determined which of these risk factors or clusters thereof are associated with the presence of and subsequent development of nephropathy and macrovascular complications (reflected by carotid intima-media thickness [IMT]). RESULTS—After adjustment for conventional risk factors (age, sex, DCCT treatment group, diabetes duration, A1C, systolic blood pressure, waist-to-hip ratio, total and HDL cholesterol, and smoking status), fibrinogen remained strongly associated with progression of internal and common carotid IMT (P < 0.01) and soluble E-selectin had a strong association with nephropathy (P < 0.01). CONCLUSIONS—The best predictor for IMT progression in the DCCT/EDIC cohort was plasma fibrinogen, and the levels of soluble E-selectin discriminate patients with albuminuria better than conventional risk factors.

We have demonstrated recently that high glucose augments lipopolysaccharide (LPS)-stimulated matrix metalloproteinase (MMP) and cytokine expression by U937 mononuclear cells and human monocyte-derived macrophages. Since CD14 is a receptor for LPS, one potential underlying mechanism is that high glucose enhances CD14 expression. In the present study, we determined the effect of high glucose on CD14 expression by U937 mononuclear cells. After being chronically exposed to normal or high glucose for 2 weeks or longer, cells were treated with LPS for 24 h. Real-time PCR showed that although high glucose by itself did not increase CD14 expression significantly, it augmented LPS-stimulated CD14 expression by 15-fold. Immunoassay showed a marked enhancement of both membrane-associated and soluble CD14 protein levels by high glucose. Further investigations using transcription factor activity assays and gel shift assays revealed that high glucose augmented LPS-stimulated CD14 expression by enhancing transcription factor nuclear factor κB (NFκB) and activator protein-1 (AP-1) activities. Finally, studies using anti-CD14 neutralizing antibody showed that CD14 expression is essential for the enhancement of LPS-stimulated MMP-1 expression by high glucose. Taken together, this study has demonstrated a robust augmentation by high glucose of LPS-stimulated CD14 expression through AP-1 and NFκB transcriptional activity enhancement, elucidating a new mechanism by which hyperglycemia boosts LPS-elicited gene expression involved in inflammation and tissue destruction.

Although a large number of studies have well documented a key role of toll-like receptor (TLR)4 in atherosclerosis, it remains undetermined if TLR4 antagonist attenuates atherogenesis in mouse model for type 2 diabetes. In this study, we induced type 2 diabetes in low-density lipoprotein receptor-deficient (LDLR(-/-)) mice by high-fat diet (HFD). At 8 weeks old, 20 mice were fed HFD and 20 mice fed regular chow (RC) for 24 weeks. In the last 10 weeks, half HFD-fed mice and half RC-fed mice were treated with Rhodobacter sphaeroides lipopolysaccharide (Rs-LPS), an established TLR4 antagonist. After the treatment, atherosclerotic lesions in aortas were analyzed. Results showed that the HFD significantly increased bodyweight, glucose, lipids including total cholesterol, triglycerides and free fatty acids, and insulin resistance, indicating that the HFD induced type 2 diabetes in LDLR(-/-) mice. Results also showed that Rs-LPS had no effect on HFD-increased metabolic parameters in both nondiabetic and diabetic mice. Lipid staining of aortas and histological analysis of cross-sections of aortic roots showed that diabetes increased atherosclerotic lesions, but Rs-LPS attenuated atherogenesis in diabetic mice. Furthermore, immunohistochemical studies showed that Rs-LPS reduced infiltration of monocytes/macrophages and expression of interleukin (IL)-6 and matrix metalloproteinase-9 in atherosclerotic lesions of diabetic mice. Finally, the antagonistic effect of Rs-LPS on TLR4 was demonstrated by our in vitro studies showing that Rs-LPS inhibited IL-6 secretion from macrophages and endothelial cells stimulated by LPS or LPS plus saturated fatty acid palmitate. Taken together, our study demonstrated that TLR4 antagonist was capable of attenuating vascular inflammation and atherogenesis in mice with HFD-induced type 2 diabetes.

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.

Implementation of multicenter and/or longitudinal studies requires an effective quality assurance program to identify trends, data inconsistencies and process variability of results over time. The Diabetes Control and Complications Trial (DCCT) and the follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) study represent over 30 years of data collection among a cohort of participants across 27 clinical centers. The quality assurance plan is overseen by the Data Coordinating Center and is implemented across the clinical centers and central reading units. Each central unit incorporates specific DCCT/EDIC quality monitoring activities into their routine quality assurance plan. The results are reviewed by a data quality assurance committee whose function is to identify variances in quality that may impact study results from the central units as well as within and across clinical centers, and to recommend implementation of corrective procedures when necessary. Over the 30-year period, changes to the methods, equipment, or clinical procedures have been required to keep procedures current and ensure continued collection of scientifically valid and clinically relevant results. Pilot testing to compare historic processes with contemporary alternatives is performed and comparability is validated prior to incorporation of new procedures into the study. Details of the quality assurance plan across and within the clinical and central reading units are described, and quality outcomes for core measures analyzed by the central reading units (e.g. biochemical samples, fundus photographs, ECGs) are presented.

Aims To relate nuclear magnetic resonance lipoprotein subclass profiles (NMR-LSP) and other lipoprotein-related factors with carotid intima-media thickness (IMT) in Type 1 diabetes. Methods Lipoprotein-related factors were determined in sera (obtained in 1997–1999) from 428 female [age 39 ± 7 years (mean ± sd)] and 540 male (age 40 ± 7 years) Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) participants. NMR quanti-fies chylomicrons, three very low-density lipoprotein (VLDL) subclasses, intermediate density lipoprotein (IDL), three low-density lipoprotein (LDL) subclasses, two high-density lipoprotein (HDL) subclasses, mean VLDL, LDL and HDL size, and LDL particle concentration. Conventional lipids, ApoA1, ApoB and Lp(a) and in vitro LDL oxidizibility were also measured. IMT was determined (in 1994–1995) using high-resolution B-mode ultrasound. Relationships between IMT and lipoproteins were analysed by multiple linear regression, controlling for age, diabetes-related factors, and cardiovascular disease (CVD) risk factors. Results IMT associations with lipoproteins were stronger for the internal than the common carotid artery, predominantly involving LDL. Internal carotid IMT was positively (P

The transformation of macrophages into foam cells is a critical event in the development of atherosclerosis. The most studied aspect of this process is the uptake of modified LDL through the scavenger receptors. Another salient aspect is the effect of modified LDL immune complexes on macrophages activation and foam cell formation. Macrophages internalize oxidized LDL immune complexes (oxLDL-IC) via the Fc-gamma receptor and transform into activated foam cells. In this study we examined the effect of oxLDL-IC on sphingosine kinase 1 (SK1), an enzyme implicated in mediating pro-survival and inflammatory responses through the generation of the signaling molecule sphingosine-1-phosphate (S1P). Intriguingly, oxLDL-IC, but not oxLDL alone, induced an immediate translocation and release of SK1 into the conditioned medium as evidenced by fluorescence confocal microscopy. Immunoblot analysis of cell lysates and conditioned medium revealed a decrease in intracellular SK1 protein levels accompanied by a concomitant increase in extracellular SK1 levels. Furthermore, measurement of S1P formation showed that the activity of cell-associated SK decreased in response to oxLDL-IC compared to oxLDL alone, whereas the activity of SK increased extracellularly. Blocking oxLDL-IC binding to Fc-gamma receptors resulted in decreased levels of extracellular S1P. The data also show that cell survival of human U937 cells exposed to oxLDL-IC increased compared to oxLDL alone. Exogenously added S1P further increased cell survival induced by oxLDL-IC. Taken together, these findings indicate that S1P may be generated extracellularly in response to modified LDL immune complexes and may therefore promote cell survival and prolong cytokine release by activated macrophages.

The plasma lactate concentration in patients with obesity and type 2 diabetes is often higher than that in nondiabetic individuals. Although it is known that increased lactate concentration is an independent risk factor for developing type 2 diabetes, the underlying mechanisms are not well understood. Because inflammation plays an important role in the development of type 2 diabetes, we postulated that increased lactate level might contribute to the pathogenesis of type 2 diabetes by enhancing inflammation. In the present study, we demonstrated that preexposure of U937 macrophage-like cells to sodium lactate increased LPS-stimulated matrix metalloproteinase (MMP)-1, IL-1beta, and IL-6 secretion. Augmentation of LPS-stimulated MMP-1 secretion was diminished when sodium lactate was replaced by lactic acid that reduced pH in the culture medium. Furthermore, quantitative real-time PCR indicated that the increased secretion of MMP-1, IL-1beta, and IL-6 was due to increased mRNA expression. To explore the underlying signaling mechanism, blocking studies using specific inhibitors for NF-kappaB and MAPK cascades were performed. Results showed that blocking of either NF-kappaB or MAPK pathways led to the inhibition of MMP-1, IL-1beta, and IL-6 expression stimulated by sodium lactate, LPS, or both. Finally, electrophoretic mobility shift assays showed a synergy between sodium lactate and LPS on AP-1 and NF-kappaB transcriptional activities. In conclusion, this study has demonstrated for the first time that sodium lactate and LPS exert synergistic effect on MMP and cytokine expression through NF-kappaB and MAPK pathways and revealed a novel mechanism potentially involved in the development of type 2 diabetes and its complications.

Background. Predictors of diabetic nephropathy are only partly known and traditional risk factors do not adequately explain disease risk. We thus examined novel risk factors for overt nephropathy (ON) in type 1 diabetes. Methods. The EDC is a prospective study of childhood-onset type 1 diabetes. When first seen (1986–1988), mean age was 28 and diabetes duration 19 years. In the subsequent 10 years, 56 of 485 subjects without ON in 1986–88 developed ON. An age, duration (±3 years), and sex-matched control was identified for 47 cases. Forty-two matched pairs had available stored plasma samples obtained prior to ON onset in cases, and complete standard risk factor data. Results. Cases had a higher baseline albumin excretion rate (AER), HbA1, pulse rate, non-HDL cholesterol, fibrinogen, small LDL and lower eGDR and LDL particle size compared to controls (all P values

Epidemiological studies have established that a high level of iron body stores is associated with increased risk of acute coronary heart disease. To explain this association, it has been proposed that iron catalyzes the production of highly reactive forms of free oxygen species, and thus, promotes low-density lipoprotein (LDL) oxidation, a lipoprotein that plays a critical role in atherogenesis. However, few studies have provided evidence to support this hypothesis. In the present study, we determined the effect of iron loading of THP-1 mononuclear phagocytes on LDL metabolism. We demonstrated that iron loading of THP-1 cells stimulated conjugated diene formation in LDL in the culture medium. In addition, iron loading of THP-1 cells significantly increased cholesteryl ester accumulation in cells exposed to native LDL, suggesting that during the incubation of the cells with native LDL, the LDL became oxidized and was taken up by the cells. We further demonstrated that the degradation of 125I-oxidized LDL was significantly increased in iron-loaded THP-1 cells. Lastly, we demonstrated that iron loading of THP-1 cells stimulated scavenger receptor expression in these cells. In conclusion, this study demonstrates that loading of mononuclear phagocytes with iron leads to oxidization of LDL, increased cellular cholesterol accumulation and scavenger receptor expression, and supports the hypothesis that increased macrophage iron levels promote atherogenesis.

OBJECTIVE—To test the hypothesis that simvastatin reduces the levels of circulating immune complexes (ICs) containing modified lipoproteins (mLDLs; mLDL-ICs), which may represent an additional mechanism for the reduced incidence of cardiovascular events in patients treated with simvastatin. RESEARCH DESIGN AND METHODS—A total of 26 patients with type 2 diabetes and triglyceride levels RESULTS—Twenty-one patients completed the study. Their HbA1c remained within 1% of baseline levels. There was a highly significant decrease in mLDL-IC levels after 3 and 6 months of treatment with simvastatin, with a return to near baseline levels after discontinuation. CONCLUSIONS—Simvastatin significantly reduced the concentration of mLDL-IC, probably as a consequence of both a decrease in the formation of mLDL and to a reduction in the titers of mLDL antibodies. This effect is likely to have a beneficial impact in the inflammatory reaction associated with atherosclerosis.

Matrix metalloproteinases (MMPs) have been implicated in the disruption of atherosclerotic plaques that leads to acute coronary events. The present study investigates the effect of thiazolidinediones (TZDs), new antidiabetic drugs, on MMP-1 expression by human vascular endothelial cells. Results show that troglitazone, but not pioglitazone and rosiglitazone, stimulated MMP-1 secretion and mRNA expression in both human umbilical vein and aortic endothelial cells, but had no effect on TIMP-1 and TIMP-2 secretion. Interestingly, troglitazone at high concentrations (≥30 μmol/l) inhibited MMP-1 protein synthesis despite a marked stimulation on MMP-1 mRNA. Further studies revealed that troglitazone at higher concentrations inhibits de novo protein synthesis as determined by 35 S-methionine/cysteine incorporation, suggesting that the inhibition of MMP-1 synthesis by troglitazone is due to the suppression of total protein synthesis. Finally, our studies showed that high concentrations of troglitazone inhibited the translation initiation factor 4E (eIF4E), but not eIF4G. In summary, the present study demonstrates that insulin sensitizers have different effects on MMP-1 expression, and troglitazone stimulates MMP-1 mRNA expression and protein synthesis at the pharmacological concentrations, but inhibits MMP-1 synthesis at higher doses. This study also suggests that supra-pharmacological concentrations of troglitazone that could be attained in body tissues may inhibit protein synthesis and cause cytotoxicity.

We have previously shown that LDL-containing immune complexes (LDL-ICs) induce up-regulation of LDL receptor (LDLR) expression in human macrophages. The present study further investigated the molecular mechanisms leading to LDLR up-regulation by LDL-ICs as well as the signaling pathways involved. Results showed that treatment of U937 histiocytes with LDL-ICs did not increase the precursors and the cleaved forms of sterol-regulatory element binding proteins (SREBPs) 1a and 2, suggesting that SREBPs may not be involved in LDLR up-regulation by LDL-ICs. Promoter deletion and mutation studies showed that the AP-1 binding sites were essential for LDL-IC-stimulated LDLR expression. Electrophoretic mobility shift assays further demonstrated that LDL-ICs stimulated transcription factor AP-1 activity. Studies assessing the signaling pathways involved in LDLR up-regulation by LDL-ICs showed that the up-regulation of LDLR was extracellular signal-regulated kinase (ERK) dependent. In conclusion, the present study shows that LDL-ICs up-regulate LDLR expression via the ERK signaling pathway and the AP-1 motif-dependent transcriptional activation.

Two decades ago, well before the inflammatory nature of arteriosclerosis was generally accepted, several groups reported the finding of immunoglobulins and various complement components in atheromatous plaques of humans and rabbits ([12][1], [42][2], [69][3]). A decade later apolipoprotein B (ApoB)

Chlamydophila pneumoniaehas an epidemiological link with atherosclerosis and acute cardiovascular events. One mechanism that may explain such a link is the increased expression of intracellular adhesion molecule-1 (ICAM-1) inC pneumoniae–infected endothelial cells. Upregulation of ICAM-1 byC pneumoniaeis well recognized and has been extensively studied, but the signaling pathways involved are not yet defined. Because upregulation of ICAM-1 by cytokines and other stimuli has been shown to be mediated by either mitogen-activated protein kinase, protein kinase C (PKC), or nuclear factor-κB (NF-κB) pathways, we examined whether these pathways were involved in the ICAM-1 upregulation induced byC pneumoniae. Our data show a time-dependent phosphorylation of p44/p42 and SAPK/JNK pathways inC pneumoniae–infected cells. However, inhibition of the classic mitogen-activated protein kinase pathway using the PD98059 and U0126 inhibitors and inhibition of SAPK/JNK pathway did not suppressC pneumoniae–induced ICAM-1 expression.C pneumoniaealso activates the NF-κB pathway at 30 minutes after infection. Treatment of human aortic endothelial cells (HAECs) with the NF-κB inhibitors BAY117085 and caffeic acid phenethyl ester led to a concentration-dependent inhibition ofC pneumoniae–induced ICAM-1 upregulation. Finally,C pneumoniae–infected HAECs show membrane translocation of total PKC 30 minutes after cell infection. Calphostin C, a general PKC inhibitor, blocked bothC pneumoniae–induced ICAM-1 expression andC pneumoniae–induced NF-κB translocation. In conclusion, we demonstrated thatC pneumoniae–induced ICAM-1 expression in HAECs requires NF-κB and PKC activation and that NF-κB activation is PKC dependent.

Advanced glycosylation end-products (AGEs) are believed to play a significant role in the development of vascular complications in diabetic patients. One such product, AGE-LDL, has been shown to be immunogenic. In this report, we describe the isolation and characterization of human AGE-LDL antibodies from the sera of seven patients with Type 1 diabetes by affinity chromatography using an immobilized AGE-LDL preparation that contained primarily the AGE N epsilon (carboxymethyl)lysine (CML, 14.6 mmol/mol lysine), and smaller amounts of N epsilon (carboxyethyl)lysine (CEL, 2.7 mmol/mol lysine). The isolated antibodies were predominantly IgG of subclasses 1 and 3, and considered proinflammatory because of their ability to promote Fc gamma R-mediated phagocytosis and to activate complement. We determined dissociation constants (Kd) for the purified antibodies. The average Kd values (4.76 +/- 2.52 x 10(-9) mol/l) indicated that AGE-LDL antibodies are of higher avidity than oxidized LDL antibodies measured previously (Kd = 1.53 +/- 07 x 10(-8) ml/l), but of lower avidity than rabbit polyclonal LDL antibodies (Kd = 9.34 x 10(-11)). Analysis of the apolipoprotein B-rich lipoproteins isolated with polyethylene glycol-precipitated antigen-antibody complexes from the same patients showed the presence of both CML and CEL, thus confirming that these two modifications are recognized by human autoantibodies. A comparative study of the reactivity of purified AGE-LDL antibodies with CML-LDL and CML-serum albumin showed no cross-reactivity.

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 ≥30 mg/24 h and ≤300 mg/24 h) who were matched for age, sex and HbA1C 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 µg/ml in patients with normoalbuminuria, p

Chronic endothelial infection is believed to be one of the factors able to cause endothelial cell damage and trigger the onset of human atherosclerosis. Chlamydophila pneumoniae infects endothelial cells and has received special attention because of both epidemiological and experimental evidence supporting its role as a risk factor for atherosclerosis. It is also possible that otherwise independent risk factors for atherosclerosis may have synergistic effects. Immune phenomena, such as the formation of circulating immune complexes (IC) containing modified LDL and corresponding antibodies, have been linked to the development of coronary artery disease. The antibodies involved in the immune response to modified lipoproteins are predominantly of the pro-inflammatory IgG1 and IgG3 subclasses. However, it is difficult to understand how circulating IC could cause endothelial damage and initiate the atherosclerotic process, unless they were formed in the subendothelial space or immobilized by endothelial cells. The last hypothesis would be possible if endothelial cells expressed Fcgamma receptors. Healthy endothelial cells do not express Fcgamma receptors, but endothelial cells infected by a variety of infectious agents do. Thus we decided to investigate whether infection of endothelial cells with C. pneumoniae is also able to cause the expression of Fcgamma receptors. The expression of Fcgamma receptors (CD64, 32, and 16) on human aortic endothelial cells infected with C. pneumoniae for 4, 24, 36, and 48 h was studied by flow cytometry. Twenty-four hours after infection 30-40% of the endothelial cells had detectable inclusion bodies, 8-9% of the total number of cells (approximately 25% of the infected cells) expressed FcgammaRII, and about 1.5-2% (5% of infected cells) expressed FcgammaRI and FcgammaRIII. Double-staining studies confirmed that the expression of FcgammaRII was limited to C. pneumoniae-infected endothelial cells. We conclude that C. pneumoniae infection induces primarily the expression of FcgammaRII by endothelial cells and this may be a significant link between two proposed pathogenic mechanisms involved in the pathogenesis of human atherosclerosis.

Interleukin-8 (IL-8) is one of cytokines detected at sites of inflammation and in macrophage-foam cells of atherosclerotic lesions. The expression of IL-8 gene can be induced in cholesterol loaded THP-1 macrophages by oxidized low density lipoprotein. We report for the first time that the expression of human IL-8 gene in THP-1 macrophages is upregulated in a time- and concentration-dependent manner by prostaglandin D2 metabolite 15-deoxy-delta12, 14 prostaglandin J2 (15d-PGJ2), which is a natural ligand for activation of peroxisome proliferator-activated receptor-gamma transcription factor. Studies to identify the signal transduction pathways involved showed that IL-8 upregulation-mediated by 15d-PGJ2 was markedly inhibited when the THP-1 macrophages were incubated with a highly selective and cell-permeable inhibitor of the mitogen-activated protein kinase (MAPK) signaling pathway, 2'-amino-3'-methoxyflavone (PD98059). This inhibition was concentration-dependent, suggesting that 15d-PGJ2 regulates the expression of IL-8 gene in THP-1 macrophages through a MAPK signaling pathway. In contrast, THP-1 macrophages when treated with pyrrolidine dithiocarbamate, an anti-oxidant and the selective inhibitor for nuclear factor kappaB, showed an enhanced 15d-PGJ2-mediated upregulation of IL-8 gene expression. The data presented in this report may contribute to unravel some of the mechanisms behind the inflammatory component of atherosclerosis.

Refractive error (RE) is a complex, multifactorial disorder characterized by a mismatch between the optical power of the eye and its axial length that causes object images to be focused off the retina. The two major subtypes of RE are myopia (nearsightedness) and hyperopia (farsightedness), which represent opposite ends of the distribution of the quantitative measure of spherical refraction. We performed a fixed effects meta-analysis of genome-wide association results of myopia and hyperopia from 9 studies of European-derived populations: AREDS, KORA, FES, OGP-Talana, MESA, RSI, RSII, RSIII and ERF. One genome-wide significant region was observed for myopia, corresponding to a previously identified myopia locus on 8q12 (p = 1.25×10-8), which has been reported by Kiefer et al. as significantly associated with myopia age at onset and Verhoeven et al. as significantly associated to mean spherical-equivalent (MSE) refractive error. We observed two genome-wide significant associations with hyperopia. These regions overlapped with loci on 15q14 (minimum p value = 9.11×10-11) and 8q12 (minimum p value 1.82×10-11) previously reported for MSE and myopia age at onset. We also used an intermarker linkage- disequilibrium-based method for calculating the effective number of tests in targeted regional replication analyses. We analyzed myopia (which represents the closest phenotype in our data to the one used by Kiefer et al.) and showed replication of 10 additional loci associated with myopia previously reported by Kiefer et al. This is the first replication of these loci using myopia as the trait under analysis. "Replication-level" association was also seen between hyperopia and 12 of Kiefer et al.'s published loci. For the loci that show evidence of association to both myopia and hyperopia, the estimated effect of the risk alleles were in opposite directions for the two traits. This suggests that these loci are important contributors to variation of refractive error across the distribution.

The main causes of morbidity and mortality in diabetes are macrovascular and microvascular complications. The pathogenesis of these complications is multifactorial, but there is strong evidence implicating chronic, smoldering, and inflammation as a main pathogenic event in the development of diabetic complications (1). Although the mechanisms responsible for vascular inflammation in diabetes are similar to those involved in vascular disease in non-diabetics (2), chronic hyperglycemia and dysregulated immune responses in diabetes are responsible for the activation of inflammatory circuits, inducing oxidative stress and promoting insulin resistance (1, 3), thus creating conditions that lead to the development of diabetes and diabetic complications. Changes in gene expression associated with diabetes like increased ICAM-1 expression (4) may also play a role in inducing inflammation and the development of diabetic complications.

It is known that both saturated fatty acids (SFAs) and lipopolysaccharide (LPS) boost host chronic inflammatory response that contributes to inflammation-related diseases such as diabetes and atherosclerosis. However, it remains unclear how SFAs interact with LPS to promote inflammation. In this study, we compared the effects of LPS, palmitic acid (PA), a most abundant SFA, and the combination of LPS and PA on the expression of interleukin (IL)-6, a key pro-inflammatory cytokine, in human aortic endothelial cells (HAECs). Results showed that while LPS induced a quick but brief IL-6 mRNA expression that peaked at 12 h, PA stimulated a delayed but extremely high expression of IL-6 mRNA (22-fold of that stimulated by LPS) at 36 h (IL-6 mRNA/house-keeping gene mRNA ratios: control-0.21; LPS-0.4; PA-8.68). Results also showed that the combination of LPS and PA further increased IL-6 mRNA at 36 h (IL-6 mRNA/house-keeping gene mRNA ratios: PA-8.68; LPS plus PA-14.23). Consistently, quantification of IL-6 protein in culture medium showed that PA stimulated more IL-6 secretion than LPS and PA in the combination of LPS led to a much greater IL-6 secretion at 36 h (control-269; PA-2485; LPS-1258; LPS plus PA-5811 pg/ml). Since PA is known to affect ceramide biosynthesis, we determine if sphingolipid metabolism is involved in IL-6 upregulation by LPS, PA or LPS plus PA. Results showed that PA significantly increased sphingomyelin (SM) hydrolysis and PA in combination with LPS further increased it (cellular SM levels: control-18.5; LPS-16.5; PA-13.6; LPS plus PA-10.7 pmol/ng phosphate). Increased SM hydrolysis was associated with an increase in ceramide, a bioactive sphingolipid known to stimulate inflammatory cytokines. Moreover, using pharmacological inhibitor and RNA interfering, we found that neutral sphingomyelinase (nSMase), but not acid SMase, was involved in the SM hydrolysis. Finally, we found that inhibition of SM hydrolysis by nSMase inhibitor or nSMase siRNA markedly inhibited IL-6 expression stimulated by LPS, PA or LPS plus PA. In conclusion, this study demonstrated that PA not only stimulated a delayed but extremely high expression of IL-6, but also enhanced the effect of LPS in HAEC, and sphingolipid metabolism played a crucial role in the PA action.

Sphingolipid metabolism is altered in diabetes and we analyzed the plasma concentrations of sphingolipid species to investigate their association with the development of albuminuria in type 1 patients with diabetes.Samples were collected from 497 type 1 diabetic patients during their enrollment into the Diabetes Control and Complications Trial (DCCT). We determined plasma concentrations of multiple ceramide species and individual sphingoid bases and their phosphates using high performance liquid chromatography-tandem mass spectrometry and investigated their association with the development of albuminuria during 14-20 years of follow-up.Patients exhibited normal albumin excretion rates (AER40 mg/24h) at the time of plasma sampling. Although the majority of patients (N = 291; 59%) exhibited normal levels of albuminuria throughout follow-up, 141 patients (28%) progressed to microalbuminuria (40 mg/24h ≤ AER300 mg/24h), while 65 (13%) progressed to macroalbuminuria (AER ≥ 300 mg/24h). To test the association of log transformed plasma sphingolipid level with the development of albuminuria, generalized logistic regression models were used where normal, micro- and macroalbuminuria were the outcomes of interest. Models were adjusted for DCCT treatment group, baseline retinopathy, gender, baseline HbA1c %, age, AER, lipid levels, diabetes duration, and the use of ACE/ARB drugs. Increased plasma levels of very long, but not long chain ceramide species measured at DCCT baseline were associated with decreased odds to develop macroalbuminuria during the subsequent nineteen years (DCCT Baseline to EDIC year 8).These studies demonstrate, prospectively, that decreased plasma levels of select ceramide species are associated with the development of macroalbuminuria in type 1 diabetes.

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.

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.

Abstract —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-γ receptor I and II (FcγRI and FcγRII) 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 μmol/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 FcγRI and FcγRII 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.

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-κB (NF-κB), pyrrolidine dithiocarbamate (PDTC), and with protein kinase C (PKC) inhibitors bisindolylmaleimide I and Ro-31-8220. These results suggest that activation of both NF-κB 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.—Fu, Y., N. Luo, and M. F. Lopes-Virella. Oxidized LDL induces the expression of ALBP/aP2 mRNA and protein in human THP-1 macrophages. J. Lipid Res. 2000. 41: 2017–2023.

Abstract —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.

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.

Abstract —Our previous studies have shown that Fc gamma receptor (FcγR)-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 FcγR triggers activation of signal transduction pathways that alter gene expression in macrophages. In this study, we determined whether engagement of FcγR 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 FcγR 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.

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 μg 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%.

Peripheral arterial disease (PAD) is a significant contributor to the morbidity and mortality of >2 million Americans (1). In addition to its own specific disease risks, PAD has been shown to be a predictor of cardiovascular mortality and coronary artery disease, as well as a general marker for the atherosclerotic disease process (2,3). PAD complications, especially in the lower limb, are significantly greater in individuals with diabetes compared with those without diabetes, and the risk imparted by diabetes is similar or greater in magnitude to that seen for ischemic heart disease and stroke (4,5). The aortofemoral arteries are prime sites for PAD; hence, determination of claudication and measurement of blood flow in the lower extremities are the most common assessments (6). A relatively low ankle systolic blood pressure (ankle-to-brachial ratio index [ABI]) has been found to be an indicator of atherosclerosis/occlusion in this region (5). Conversely, in diabetes, a high-pressure ABI due to medial wall arterial calcification and noncompressible vessels may also be associated with adverse outcomes, including diabetic kidney disease (7). Sex differences have been reported in ABI measurements in type 1 diabetes, with women having a greater frequency of low ABI and men having a higher frequency of high ABI (7). Here, we extend these analyses by examining the effect of previous intensive diabetes management on the development of abnormally high and low ABIs in the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) cohort with the hypothesis that …

The incubation of human macrophages with antigen antibody complexes prepared with rabbit anti-LDL and human LDL (LDL-IC) is followed by ingestion of those immune complexes (IC), massive cholesterol ester accumulation, cytokine release and overexpression of the LDL receptor. The massive accumulation of cholesterol esters and overexpression of the native LDL receptor are specifically induced by immune complexes containing native or modified LDL, but not by any other type of IC. We report the results of a series of experiments aimed at defining the receptor preferentially involved in LDL-IC uptake. Flow cytometry studies using CD16, CD32 and CD64 monoclonal antibodies showed a sharp reduction on the expression of CD64 (Fc gamma RI) both by human monocyte-derived macrophages and THP-1 cells after incubation with LDL-IC, suggesting preferential engagement of this type of Fc receptor. Blocking experiments with aggregate-free IgG1 and CD32 monoclonal antibody confirmed that blocking Fc gamma RI prevented both LDL-IC uptake and the upregulation of LDL receptors on THP-1 cells. In contrast, blocking Fc gamma RII did not affect either the uptake of LDL-IC or the expression of LDL receptors on the same cells. The preferential engagement of Fc gamma R-I by LDL-IC suggests a biological difference of LDL-IC relative to other types of IC and opsonized particles. The precise molecular mechanism(s) responsible for the paradoxical upregulation of LDL receptor after the uptake of LDL-IC remain to be elucidated.

We have previously shown that uptake of low density lipoprotein-containing immune complexes (LDL-IC) by human monocyte-derived macrophages led to the transformation of these cells into foam cells and induced a paradoxical increase in receptor-mediated binding of 125I-labeled LDL due to an increase in the number of LDL receptors (LDL-R). The same metabolic changes are also observed in PMA-treated THP-1 cells after incubation for 2 h with 150 microg/ml of immune complexes containing either native, oxidized (ox), or malondialdehyde (mda) LDL. After stimulation, PMA-treated THP-1 cells showed not only a 40-fold increase in 125I-labeled LDL binding but also a 40-fold increase in the immunoreactive LDL-R protein, confirming that the increase in LDL binding is due to an increase LDL-R number. In this study we have investigated, in PMA-treated THP-1 cells, the regulatory mechanism(s) responsible for the increased receptor-mediated binding of LDL induced by LDL-IC. By Northern blot and nuclear run-on analysis we have shown transcriptional activation of the LDL-R gene with a 7-fold increase in the LDL-R mRNA level in LDL-IC stimulated cells. Due to the marked difference between the increase in LDL-R mRNA and LDL-R protein, we estimated LDL-R mRNA stability using a inhibitor chase method and have shown that LDL-IC did not alter the LDL-R mRNA stability in THP-1 cells. We have also demonstrated, using cycloheximide as a inhibitor of protein synthesis, that the marked increase in LDL-R protein observed in LDL-IC-stimulated THP-1 cells resulted from de novo synthesis of LDL-R protein. To determine whether the increase in transcriptional activity of the LDL-R gene was secondary to changes in the cholesterol regulatory pool we performed experiments in which the cell cholesterol content was modified by the addition of either 25-hydroxycholesterol and mevalonate or inhibitors of ACAT activity (SA-58035 and progesterone). These experiments showed that the enhanced LDL-R expression was not affected by the addition of any of the above compounds. In conclusion, LDL-IC induced both transcriptional and post-transcriptional activation of the LDL-R gene in PMA-treated THP-1 cells and this induction was independent of the free cholesterol content of these cells.

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.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 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.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.

There is strong evidence supporting a key role of the adaptive immune response in atherosclerosis, given that both activated Th cells producing predominantly interferon-γ and oxidized LDL (oxLDL) and the corresponding antibodies have been isolated from atheromatous plaques. Studies carried out using immune complexes (IC) prepared with human LDL and rabbit antibodies have demonstrated proatherogenic and pro-inflammatory properties, mostly dependent on the engagement of Fcγ receptors Ⅰ and Ⅱ in macrophages and macrophage-like cell lines. Following the development of a methodology for isolating modified LDL (mLDL) antibodies from serum and isolated IC, it was confirmed that antibodies reacting with oxLDL and advanced glycation end product-modified LDL are predominantly IgG of subtypes 1 and 3 and that mLDL IC prepared with human reagents possesses pro-inflammatory and proatherogenic properties. In previous studies, LDL separated from isolated IC has been analyzed for its modifications, and the reactivity of antibodies isolated from the same IC with different LDL modifications has been tested. Recently, we obtained strong evidence suggesting that the effects of mLDL IC on phagocytic cells are modulated by the composition of the mLDL. Clinical studies have shown that the level of mLDL in circulating IC is a strong predictor of cardiovascular disease (CVD) and, in diabetic patients, other significant complications, such as nephropathy and retinopathy. In conclusion, there is convincing ex vivo and clinical data supporting the hypothesis that, in humans, the humoral immune response to mLDL is pathogenic rather than protective.

Over 90% of modified LDL in circulation is associated to specific antibodies circulating as part of immune complexes (IC); however, few studies have examined their relationship with cardiovascular disease.We report the relationship between circulating concentrations of IC of oxidized LDL (oxLDL-IC), malondialdehyde-LDL (MDA-LDL-IC) and advanced glycation end products-LDL (AGE-LDL-IC) and progression of atherosclerosis over a 12 year period in 467 individuals with type 1 diabetes who participated in the Diabetes Control and Complications Trial (DCCT) and the Epidemiology of Diabetes Interventions and Complications (EDIC) study. OxLDL-IC, AGE-LDL-IC and MDA-LDL-IC levels were measured at DCCT closeout. Internal carotid intima-medial thickness (IMT) was measured at EDIC follow-up years 1, 6 and 12.OxLDL-IC, AGE-LDL-IC and MDA-LDL-IC levels were significantly correlated with age, lipid levels, blood pressure levels and albumin excretion rates. Levels of oxLDL, AGE-LDL and MDA-LDL in isolated LDL-IC were highly inter-correlated (r = 0.66-0.84, P0.0001). After adjusting for cardiovascular risk factors individuals in the upper quartile of oxLDL-IC had a 2.98-fold increased odds (CI: 1.34, 6.62) of having IMT ≥ 1.00 mm and had a 5.13-fold increased odds (CI: 1.98, 13.3) of having significant IMT progression, relative to those in the lowest quartile. Parallel odds ratios for AGE-LDL-IC were 2.95 (CI: 1.37, 6.34) and 3.50 (CI: 1.38, 8.86), while results for MDA-LDL-IC were 1.76 (0.87, 3.56) and 2.86 (1.20, 6.81).Our study indicates that high levels of oxLDL-IC and AGE-LDL-IC are important predictors of carotid intima-medial thickening in patients with type 1 diabetes.

One of the consequences of oxidative stress, which is enhanced in diabetes due to hyperglycemia, is protein modification, which affects LDL. Oxidized LDL (oxLDL), malondialdehyde-modified LDL (MDA-LDL), and advanced glycation end-products LDL (AGE-LDL) are major LDL modifications, which elicit the formation of autoantibodies and immune complexes (IC) in humans, are pro-inflammatory and play a role in the pathogenesis of atherosclerosis. The proinflammatory potential of modified LDL (mLDL) is enhanced several fold when mLDL combines with specific IgG antibodies. Clinical studies by our group have shown that the levels of mLDL in circulating IC significantly predict the development of diabetic complications. Furthermore, the composition of mLDL in IC modulates the pattern of cell activation. In the case of macrovascular disease, when the predominant modifications in IC are oxLDL and AGE LDL there is a strong association with intima-media thickening, but not with acute events. In contrast, high levels of MDA-LDL in IC are associated with acute CVD events. In vitro studies suggest that the reason for these differences result from different effects of mLDL-IC in macrophages and other cells.

We reported recently that treatment of diabetic apolipoprotein E-deficient mice with the Toll-like receptor 4 (TLR4) antagonist Rs-LPS, a lipopolysaccharide isolated from Rhodobacter sphaeroides, inhibited atherosclerosis. Since it is known that Rs-LPS antagonizes TLR4 by targeting TLR4 co-receptor MD-2, this finding indicates that MD-2 is a potential target for the treatment of atherosclerosis. In this study, we determined if MD-2 is involved in the gene expression regulated by signalling pathways independent of TLR4. Given that interferon-γ (IFNγ) and hyperglycaemia play key roles in atherosclerosis, we determined if MD-2 is involved in IFN-γ and high-glucose-regulated gene expression in mononuclear cells. Results showed that IFN-γ and high glucose synergistically stimulated matrix metalloproteinase 1 (MMP-1), a proteinase essential for vascular tissue remodelling and atherosclerosis, in U937 mononuclear cells, but Rs-LPS inhibited the MMP-1 stimulation. To provide more evidence for a role of MD-2 in IFN-γ-stimulated MMP-1, studies using antibodies and small interfering RNA demonstrated that MD-2 blockade or knockdown attenuated the effect of IFN-γ on MMP-1. Furthermore, studies using PCR arrays showed that MD-2 blockade had a similar effect as IFN-γ receptor blockade on the inhibition of IFN-γ-stimulated pro-inflammatory molecules. Although these findings indicate the involvement of MD-2 in IFN-γ signalling, we also observed that MD-2 was up-regulated by IFN-γ and high glucose. We found that MD-2 up-regulation by IFN-γ played an essential role in the synergistic effect of IFN-γ and LPS on MMP-1 expression. Taken together, these findings indicate that MD-2 is involved in IFN-γ signalling and IFN-γ-augmented MMP-1 up-regulation by LPS.

Aim To determine if serum pigment epithelium-derived factor (PEDF) levels in Type 2 diabetes are related to vascular risk factors and renal function. Methods PEDF was quantified by ELISA in a cross-sectional study of 857 male Veterans Affairs Diabetes Trial (VADT) subjects, and associations with cardiovascular risk factors and renal function were determined. In a subset (n=246) in whom serum was obtained early in the VADT (2.0±0.3years post-randomization), PEDF was related to longitudinal changes in renal function over 3.1years. Results Cross-sectional study: In multivariate regression models, PEDF was positively associated with serum triglycerides, waist-to-hip ratio, serum creatinine, use of ACE inhibitors or angiotensin receptor blockers, and use of lipid-lowering agents; it was negatively associated with HDL-C (all p Longitudinal study: PEDF was not associated with changes in renal function over 3.1years (p>0.09). Conclusions Serum PEDF in Type 2 diabetic men was cross-sectionally associated with dyslipidemia, body habitus, use of common drugs for blood pressure and dyslipidemia, and indices of renal function; however, PEDF was not associated with renal decline over 3.1years.

Diabetic nephropathy is the leading cause of end-stage renal disease. The urinary albumin to creatinine ratio is used as a predictor for the development of nephropathy but it is neither sensitive nor specific. Here we used liquid chromatography/mass spectrometry on urine of eight normoalbuminuric patients with type 2 diabetes from the VA Diabetes Trial to identify candidate markers for loss of renal function. Initial verification of seven markers (agrin, haptoglobin, mannan-binding lectin serine protease 2, LAMP-2, angiotensinogen, NGAL, and uromodulin) in the urine of an additional 30 patients showed that haptoglobin was the best predictor of early renal functional decline. We then measured this in the urine of 204 patients with type 2 diabetes who did not yet have significant kidney disease (estimated glomerular filtration rate stage 2 or better and an albumin to creatinine ratio300 mg/g). In comparing the highest to lowest tertiles, the odds ratio for having early renal function decline was 2.70 (CI: 1.15, 6.32) using the haptoglobin to creatinine ratio compared with 2.50 (CI 1.14, 5.48) using the albumin to creatinine ratio after adjusting for treatment group and use of ACE inhibitors. Addition of the haptoglobin to creatinine ratio to a model using the albumin to creatinine ratio to predict early renal function decline resulted in improved predictive performance. Thus, the haptoglobin to creatinine ratio may be useful to predict patients with type 2 diabetes at risk of nephropathy before the development of macroalbuminuria or reduced glomerular filtration rate.

Modified lipoproteins are able to induce inflammatory reactions through innate immunity pathways and are immunogenic, leading to an autoimmune response that results in the formation of proinflammatory immune complexes. The measurement of circulating oxidized lipoproteins and corresponding antibodies has, therefore, been proposed as an approach to assess the risk for complications in patients with diabetes and for the risk of cardiovascular disease in the general population. However, the majority of modified low density lipoprotein (LDL) in the peripheral circulation exists in the form of immune complexes, and this is a significant obstacle for the measurement of modified LDL and the corresponding antibodies. In this manuscript, we describe in detail the methodology developed by our group for isolation and fractionation of circulating immune complexes (IC), allowing the accurate assay of different LDL modifications. This approach has resulted in several studies showing that the levels of modified LDL are risk factors with a stronger association to diabetic retinopathy, nephropathy, and macrovascular disease. Ongoing research is focused on evaluating the predictive power of modified LDL levels for the development or progression of atherosclerotic cardiovascular disease in other patient populations and on the simplification of the assay to make it more applicable to diagnostic laboratories.