LDL Immune Complexes Research Articles

445-P: Modified LDL-Immune Complexes (IC) and the Risk of CVD in Type 1 Diabetes

We have previously shown that high levels of oxidized LDL and AGE-LDL in circulating IC are strong predictors of atherosclerosis progression in 518 subjects with type 1 diabetes from the DCCT/EDIC cohort. In this study we investigated whether oxLDL, MDA-LDL and AGE-LDL in circulating IC measured in the same subjects at DCCT baseline were associated with acute CVD events. Three overlapping CVD outcomes were considered: any CVD events, major adverse cardiac and cerebrovascular events (MACE) and myocardial infarction (MI). Cox proportional hazards models assessed the association between IC biomarker values and the subsequent risk of CVD, using inverse probability weighting to obtain results representative of the full DCCT/EDIC cohort. Results: Levels of AGE-LDL, oxLDL and MDA-LDL in circulating IC, measured at baseline (Table) were significantly associated with the 3 CVD outcomes in unadjusted models and were minimally attenuated after adjustment for age and mean HbA1c. After adjustments for conventional CVD risk factors, including LDL-Chol, high levels of oxLDL-IC and MDA-LDL-IC remained independently associated with increased subsequent risk of any CVD, and higher oxLDL-IC with increased risk of MACE and MI events. Conclusions: High levels of oxLDL-IC measured more than one decade prior to the occurrence of CVD events identified patients at high risk, even after adjustment by LDL-C and conventional CVD risk factors. Disclosure M.F. Lopes-Virella: None. I. Bebu: None. K.J. Hunt: None. G. Virella: None. N.L. Baker: None. B. Braffett: None. J. Lachin: Board Member; Self; Tolerion, Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases

Modified LDL Immune Complexes and Cardiovascular Disease.

Modified forms of LDL, both spontaneously formed in the organism or prepared in the laboratory, are immunogenic. As a consequence, antigen-antibody complexes (immune complexes, IC) formed in vivo can be measured in the peripheral blood, and their levels are strong predictors of cardiovascular disease (CVD). It has been possible to generate antibodies that recognize different LDL modifications, allowing the analysis of circulating IC constitution. Clinical studies showed that the antigenic constitution of the IC has a modulating effect on the development of CVD. Patients whose IC react strongly with antibodies to copper oxidized LDL (oxLDL) show progressive development of atherosclerosis as demonstrated by increased intima-media thickness and increased coronary calcification scores. In contrast, patients whose IC react strongly with antibodies to the heavily oxidized malondialdehyde LDL prepared in vitro (MDA-LDL) are at a high risk of acute vascular events, mainly myocardial infarction. In vitro studies have shown that while oxLDL IC induce both cell proliferation and mild to moderate macrophage apoptosis, MDA-LDL IC induce a more marked macrophage apoptosis but not cell proliferation. In addition, MDA-LDL IC induce the release of higher levels of matrix metalloproteinases and TNF than oxLDL IC. High levels of TNF are likely to be a major factor leading to apoptosis and high levels of metalloproteinases are likely to play a role in the thinning of the fibrous cap of the atheromatous plaque. The combination of apoptosis and fibrous cap thinning is a well-known characteristic of vulnerable plaques, which are more prone to rupture and responsible for the majority of acute cardiovascular events.

Oxidized low density lipoprotein immune complexes act as a priming signal for the NLRP3 inflammasome (HUM1P.253)

Abstract Antibodies to oxidized LDL (oxLDL) and resulting immune complexes (ICs) are a prominent feature of atherosclerosis and autoimmune diseases associated with increased atherosclerosis. Although levels of oxLDL-ICs correlate with disease severity, it is unclear whether these ICs play an active role in disease pathogenesis. We hypothesize that oxLDL-ICs exacerbate atherosclerosis by signaling through multiple receptors on dendritic cells (DCs) resulting in an inflammatory adaptive immune response. To test this hypothesis, bone marrow derived dendritic cells (BMDC) were incubated with oxLDL or oxLDL-IC and cytokine levels in culture supernatants were measured by ELISA. Results indicated a 10-fold increase in IL-1β production from BMDC treated with oxLDL-IC compared to oxLDL, suggesting a role for the inflammasome in oxLDL-IC activation of DC. Pre-treatment of BMDC with a caspase 1 inhibitor and use of NLRP3-/- BMDC abolished IL-1β secretion in response to oxLDL-IC, further implicating the inflammasome. Additional investigation using Fab fragments and a TLR4 inhibitor uncovered a role for both FcγR and TLR4 in oxLDL-IC mediated IL-1β production. Finally, BMDC pre-treated with oxLDL-IC, but not oxLDL, and incubated with CD4+ OT-II T cells in the presence of OVA peptide skewed T cells towards a Th17 phenotype. Taken together, these data suggest a novel and pathologic role of oxLDL-ICs in activating the NLRP3 inflammasome and promoting an inflammatory T cell phenotype.

Abstract 152: Oxidized Low-Density Lipoprotein Immune Complexes Generate an Inflammatory Immune Response by Signaling Through Multiple Receptors on Dendritic Cells

Antibodies to oxidized LDL (oxLDL) and resulting immune complexes (ICs) are a prominent feature of atherosclerosis and diseases associated with increased atherosclerosis including Type 2-diabetes and rheumatoid arthritis. Although levels of oxLDL-ICs correlate with disease severity and associate with pro-inflammatory activation of macrophages in vitro , it is currently unclear whether these ICs are simply biomarkers or play an active role in disease pathogenesis. One possible mechanism by which oxLDL-ICs may regulate inflammation in atherosclerosis is by interacting with Fc gamma receptors (FcgRs) expressed on the surface of antigen presenting cells, such as dendritic cells (DCs). Not surprisingly, signaling through FcgRs is tightly regulated and dependent on their relative cell surface density. In addition, FcgR signaling has been shown to be linked to Toll like receptor-4 (TLR-4), a pattern recognition receptor. We hypothesize that oxLDL-ICs exacerbate atherosclerosis via signaling through FcgRs and TLR4 on DCs resulting in a pro-inflammatory T cell response. To test this hypothesis, bone marrow derived DCs (BMDCs) were treated with in vitro generated oxLDL ICs. Interestingly, BMDCs treated with oxLDL-ICs had increased expression of the activation markers MHC-II and CD40 and produced greater levels of the Th17 polarizing cytokines IL-1beta and IL-23 compared to cells treated with oxLDL alone. Secretion of pro-inflammatory cytokines was significantly decreased by pre-treatment of BMDCs with a TLR4 inhibitor and by blocking FcgR signaling. This suggests that responses to oxLDL-ICs involve both TLR-4 and FcgRs. Furthermore, incubation of OT-II T cells with BMDCs treated with oxLDL-IC prior to incubation with ovalbumin peptide displayed increased pro-inflammatory cytokine secretion compared to incubation with oxLDL alone. In conclusion, our studies provide new evidence that oxLDL-ICs signal through multiple receptors on DCs resulting in increased inflammatory potential. Because of their critical function in shaping the T cell response, we believe that DC signaling via FcgRs and oxLDL-ICs represents an important link between innate and adaptive immunity in atherosclerosis.

Immune complex formation in human diabetic retina enhances toxicity of oxidized LDL towards retinal capillary pericytes

Recently it has been shown that levels of circulating oxidized LDL immune complexes (ox-LDL-ICs) predict the development of diabetic retinopathy (DR). This study aimed to investigate whether ox-LDL-ICs are actually present in the diabetic retina, and to define their effects on human retinal pericytes versus ox-LDL. In retinal sections from people with type 2 diabetes, costaining for ox-LDL and IgG was present, proportionate to DR severity, and detectable even in the absence of clinical DR. In contrast, no such staining was observed in retinas from nondiabetic subjects. In vitro, human retinal pericytes were treated with native LDL, ox-LDL, and ox-LDL-IC (0-200 mg protein/l), and measures of viability, receptor expression, apoptosis, endoplasmic reticulum (ER) and oxidative stresses, and cytokine secretion were evaluated. Ox-LDL-IC exhibited greater cytotoxicity than ox-LDL toward retinal pericytes. Acting through the scavenger (CD36) and IgG (CD64) receptors, low concentrations of ox-LDL-IC triggered apoptosis mediated by oxidative and ER stresses, and enhanced inflammatory cytokine secretion. The data suggest that IC formation in the diabetic retina enhances the injurious effects of ox-LDL. These findings offer new insights into pathogenic mechanisms of DR, and may lead to new preventive measures and treatments.

Response to Comment on Lopes-Virella et al. Baseline Markers of Inflammation Are Associated With Progression to Macroalbuminuria in Type 1 Diabetic Subjects. Diabetes Care 2013;36:2317–2323

We appreciate the comments of Drs. Jialal and Devaraj (1) on our article (2). Our study was run concomitantly with the study reported by Joslin investigators (3) and reached similar conclusions concerning the predictive value of soluble tumor necrosis factor (TNF) receptors in nephropathy progression. We mentioned their results in our discussion. We did not mention the possible role of increased Toll-like receptor (TLR) activity in triggering the TNF pathway as other alternative explanations could better explain our findings. We previously published data showing that oxidized LDL immune complexes (oxLDL-ICs) are associated with and predict the development of proteinuria (4) and that oxLDL-ICs are much stronger activators of the TNF pathway than oxLDL, which activates innate immunity through the interaction with TLR-4 (5). The concern over the relationship between soluble vascular cell adhesion …

Pathogenic Role of Modified LDL Antibodies and Immune Complexes in Atherosclerosis

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.

Differential regulation of acid sphingomyelinase in macrophages stimulated with oxidized low‐density lipoprotein (LDL) and oxidized LDL immune complexes: role in phagocytosis and cytokine release

Oxidized low-density lipoprotein (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to the formation of lipid-laden macrophages (foam cells). Fcγ receptors mediate uptake of oxLDL-IC, whereas scavenger receptors internalize oxLDL. We have previously reported that oxLDL-IC, but not free oxLDL, activate macrophages and prolong their survival. Sphingomyelin is a major constituent of cell membranes and lipoprotein particles and acid sphingomyelinase (ASMase) hydrolyses sphingomyelin to generate the bioactive lipid ceramide. ASMase exists in two forms: lysosomal (L-ASMase) and secretory (S-ASMase). In this study we examined whether oxLDL and oxLDL-IC regulate ASMase differently, and whether ASMase mediates monocyte/macrophage activation and cytokine release. The oxLDL-IC, but not oxLDL, induced early and consistent release of catalytically active S-ASMase. The oxLDL-IC also consistently stimulated L-ASMase activity, whereas oxLDL induced a rapid transient increase in L-ASMase activity before it steadily declined below baseline. Prolonged exposure to oxLDL increased L-ASMase activity; however, activity remained significantly lower than that induced by oxLDL-IC. Further studies were aimed at defining the function of the activated ASMase. In response to oxLDL-IC, heat-shock protein 70B' (HSP70B') was up-regulated and localized with redistributed ASMase in the endosomal compartment outside the lysosome. Treatment with oxLDL-IC induced the formation and release of HSP70-containing and IL-1β-containing exosomes via an ASMase-dependent mechanism. Taken together, the results suggest that oxLDL and oxLDL-IC differentially regulate ASMase activity, and the pro-inflammatory responses to oxLDL-IC are mediated by prolonged activation of ASMase. These findings may contribute to increased understanding of mechanisms mediating macrophage involvement in atherosclerosis.

Abstract 019: Oxidized LDL and AGE-LDL in Circulating Immune Complexes Strongly Predict Progression of Carotid IMT in Type 1 Diabetes

Many studies have demonstrated a relationship between modified LDL and cardiovascular disease. Nonetheless, few studies have examined the relationship between modified LDL immune complexes (IC) and cardiovascular disease, even though the majority of modified LDL in circulation is bound to IC. 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 individuals with type 1 diabetes. OxLDL-IC, AGE-LDL-IC and MDA-LDL-IC levels were measured in a subgroup of 459 patients participating in the Diabetes Control and Complications Trial (DCCT) and it’s follow up study the Epidemiology of Diabetes Interventions and Complications (EDIC). Internal carotid intima-medial thickness (IMT) was measured at EDIC follow-up years 1, 6 and 12. Levels of oxLDL-IC, AGE-LDL-IC and MDA-LDL-IC were moderately correlated with lipid levels [i.e., rho<0.32], but not with age. Modified LDL-IC significantly predicted having elevated internal carotid IMT (i.e., IMT ≥1.00 mm) as well as progression of IMT defined as being in the upper quintile of change from EDIC year 1 to 12 after adjusting for DCCT treatment group [intensive vs. conventional], retinopathy cohort [primary prevention vs. secondary intervention], age, gender, diabetes duration, albumin excretion rate, LDL, HDL, SBP, smoking status and IMT reader (Table). Relative to those in the lowest quartile, individuals in the upper quartile of oxLDL-IC had a 3.7-fold increased odds (CI: 1.72, 7.94) of having IMT ≥ 1.00 mm and had a 6.1-fold increased odds (CI: 2.57, 14.6) of having significant IMT progression. Parallel odds ratios for AGE-LDL-IC were 3.26 (CI: 1.57, 6.79) and 5.31 (CI: 2.23, 12.6), while results for MDA-LDL-IC were not as strong. 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.

Lack of nitric oxide synthases increases lipoprotein immune complex deposition in the aorta and elevates plasma sphingolipid levels in lupus

Systemic lupus erythematosus (SLE) patients display impaired endothelial nitric oxide synthase (eNOS) function required for normal vasodilatation. SLE patients express increased compensatory activity of inducible nitric oxide synthase (iNOS) generating excess nitric oxide that may result in inflammation. We examined the effects of genetic deletion of NOS2 and NOS3, encoding iNOS and eNOS respectively, on accelerated vascular disease in MRL/lpr lupus mouse model. NOS2 and NOS3 knockout (KO) MRL/lpr mice had higher plasma levels of triglycerides (23% and 35%, respectively), ceramide (45% and 21%, respectively), and sphingosine 1-phosphate (S1P) (21%) compared to counterpart MRL/lpr controls. Plasma levels of the anti-inflammatory cytokine interleukin 10 (IL-10) in NOS2 and NOS3 KO MRL/lpr mice were lower (53% and 80%, respectively) than counterpart controls. Nodule-like lesions in the adventitia were detected in aortas from both NOS2 and NOS3 KO MRL/lpr mice. Immunohistochemical evaluation of the lesions revealed activated endothelial cells and lipid-laden macrophages (foam cells), elevated sphingosine kinase 1 expression, and oxidized low-density lipoprotein immune complexes (oxLDL-IC). The findings suggest that advanced vascular disease in NOS2 and NOS3 KO MRL/lpr mice maybe mediated by increased plasma triglycerides, ceramide and S1P; decreased plasma IL-10; and accumulation of oxLDL-IC in the vessel wall. The results expose possible new targets to mitigate lupus-associated complications.

Development of immunoassays for anti-electronegative LDL autoantibodies and immune complexes

BackgroundElectronegative low-density lipoprotein (LDL−) promotes atherosclerosis through inflammatory and immunologic mechanisms that lead to the production of anti-LDL(−) autoantibodies and to the subsequent formation of immune complexes (IC) and macrophage foam cells. We described the development and validation of an ELISA for the quantification of free anti-LDL(−) autoantibodies and an ELISA for the quantification of IC consisting of LDL(−)-bound IgG in human plasma. MethodsLDL(−) purified from human plasma, and anti-LDL(−) monoclonal antibody Fab fragments were adsorbed onto ELISA plates to capture anti-LDL(−) autoantibodies and IC-LDL(−), respectively. The performance characteristics of both ELISAs, including the limits of detection and quantification, accuracy and inter- and intra-assay precision were evaluated. Linearity, interference and stability tests were also performed. ResultsThe calibration range of the anti-LDL(−) assay was 0.004–0.125mU/l and plasma demonstrated a dilutional linearity when diluted 1:100, 1:200, 1:400 and 1:800. The calibration range of the IC-LDL(−) assay was 0.06–4U/l, and plasma demonstrated a dilutional linearity when diluted 1:12.5, 1:25, 1:50 and 1:100. Both ELISAs showed intra- and inter-assay precision and recovery within the required limits for immunoassays. ConclusionThese ELISAs can be used in clinical studies and for the biochemical investigation of atherosclerosis. In addition, they will enable the comprehensive evaluation of the importance of bound or free autoantibodies against LDL(−) in this disease.

If oxidized LDL immune complexes are relevant in diabetic atherosclerosis, shouldn't they also be relevant in diabetic nephropathy?

If oxidized LDL immune complexes are relevant in diabetic atherosclerosis, shouldn't they also be relevant in diabetic nephropathy?

Oxidized LDL immune complexes stimulate collagen IV production in mesangial cells via Fc gamma receptors I and III

Diabetic nephropathy is characterized by progressive mesangial expansion. Although we have reported that circulating oxidized LDL-containing immune complexes (oxLDL-IC) are associated with abnormal levels of albuminuria, the underlying mechanisms have not been investigated. In this study, we have studied the effect of oxLDL-IC on collagen IV expression by mesangial cells. We found that oxLDL-IC markedly stimulated collagen IV expression in a concentration- and time-dependent fashion while oxLDL only had moderate effect. We also found that oxLDL-IC stimulated collagen IV expression by engaging Fc gamma receptor (FcγR) I and III, but not FcγRII, and that p38 MAPK, JNK and PKC pathways were involved in collagen IV expression. Furthermore, we found that oxLDL-IC stimulated FcγRI expression, suggesting a positive feedback mechanism involved in oxLDL-IC-stimulated collagen IV expression. Taken together, this study showed that oxLDL-IC stimulated collagen IV in mesangial cells via FcγRI and FcγRIII, and the expression of FcγRI was increased by oxLDL-IC.

Oxidized LDL immune complexes and coronary artery calcification in type 1 diabetes

ObjectiveOxidized LDL (oxLDL) and oxLDL antibodies form immune complexes (IC) that reflect essential components in the development of atherosclerosis: dyslipidemia, oxidative stress and induction of a pro-inflammatory humoral immune response. We measured oxLDL in IC (oxLDL-IC) isolated from patients with type 1 diabetes to assess the relationship between oxLDL-IC and coronary artery calcification (CAC). MethodsOxLDL was measured in IC isolated from baseline samples from a subgroup of 476 patients of the Diabetes Control and Complications Trial (DCCT). CAC was determined by computed tomography (CT) 11–20 years later. Multivariable log-binomial regression models were used to estimate the risk ratios associated with having a high CAC score with an increase of 1 standard deviation (SD) of the natural logarithm of oxLDL-IC. ResultsMultivariable regression models indicate that a 1 SD increase in the levels of oxLDL-IC was associated with a 37% increase in the risk of having high CAC score (RR=1.36; 95% CI: 1.12–1.67) at follow-up after adjustment for DCCT treatment group, retinopathy/AER groups, gender and CT scanning site as well as baseline age, diabetes duration and HbA1C %. Further adjustment for smoking status, blood pressure and LDL resulted in a risk ratio of 1.23 (95% CI: 1.01–1.50) which remained statistically significant indicating that baseline oxLDL-IC is independently associated with the development of CAC. DiscussionIncreased levels of oxLDL-IC are associated with the development of coronary calcification. This observation reinforces previously published clinical and experimental data demonstrating that oxLD-IC has pro-inflammatory and proatherogenic properties.

Differential Trafficking of Oxidized LDL and Oxidized LDL Immune Complexes in Macrophages: Impact on Oxidative Stress

BackgroundOxidized low-density lipoproteins (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to formation of lipid-laden macrophages (foam cells). It has been shown that oxLDL-IC are considerably more efficient than oxLDL in induction of foam cell formation, inflammatory cytokines secretion, and cell survival promotion. Whereas oxLDL is taken up by several scavenger receptors, oxLDL-IC are predominantly internalized through the FCγ receptor I (FCγ RI). This study examined differences in intracellular trafficking of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC and the impact on oxidative stress.Methodology/FindingsFluorescently labeled lipid and protein moieties of oxLDL co-localized within endosomal and lysosomal compartments in U937 human monocytic cells. In contrast, the lipid moiety of oxLDL-IC was detected in the endosomal compartment, whereas its apolipoprotein moiety advanced to the lysosomal compartment. Cells treated with oxLDL-IC prior to oxLDL demonstrated co-localization of internalized lipid moieties from both oxLDL and oxLDL-IC in the endosomal compartment. This sequential treatment likely inhibited oxLDL lipid moieties from trafficking to the lysosomal compartment. In RAW 264.7 macrophages, oxLDL-IC but not oxLDL induced GFP-tagged heat shock protein 70 (HSP70) and HSP70B', which co-localized with the lipid moiety of oxLDL-IC in the endosomal compartment. This suggests that HSP70 family members might prevent the degradation of the internalized lipid moiety of oxLDL-IC by delaying its advancement to the lysosome. The data also showed that mitochondrial membrane potential was decreased and generation of reactive oxygen and nitrogen species was increased in U937 cell treated with oxLDL compared to oxLDL-IC.Conclusions/SignificanceFindings suggest that lipid and apolipoprotein moieties of oxLDL-IC traffic to separate cellular compartments, and that HSP70/70B' might sequester the lipid moiety of oxLDL-IC in the endosomal compartment. This mechanism could ultimately influence macrophage function and survival. Furthermore, oxLDL-IC might regulate the intracellular trafficking of free oxLDL possibly through the induction of HSP70/70B'.

Heat Shock Protein 70B′ (HSP70B′) Expression and Release in Response to Human Oxidized Low Density Lipoprotein Immune Complexes in Macrophages

Heat shock proteins (HSPs) have been implicated in the activation and survival of macrophages. This study examined the role of HSP70B', a poorly characterized member of the HSP70 family, in response to oxidatively modified LDL (oxLDL) and immune complexes prepared with human oxLDL and purified human antibodies to oxLDL (oxLDL-IC) in monocytic and macrophage cell lines. Immunoblot analysis of cell lysates and conditioned medium from U937 cells treated with oxLDL alone revealed an increase in intracellular HSP70B' protein levels accompanied by a concomitant increase in HSP70B' extracellular levels. Fluorescence immunohistochemistry and confocal microscopy, however, demonstrated that oxLDL-IC stimulated the release of HSP70B', which co-localized with cell-associated oxLDL-IC. In HSP70B'-green fluorescent protein-transfected mouse RAW 264.7 cells, oxLDL-IC-induced HSP70B' co-localized with membrane-associated oxLDL-IC as well as the lipid moiety of internalized oxLDL-IC. Furthermore, the data demonstrated that HSP70B' is involved in cell survival, and this effect could be mediated by sphingosine kinase 1 (SK1) activation. An examination of regularly implicated cytokines revealed a significant relationship between HSP70B' and the release of the anti-inflammatory cytokine interleukin-10 (IL-10). Small interfering RNA knockdown of HSP70B' resulted in a corresponding decrease in SK1 mRNA levels and SK1 phosphorylation as well as increased release of IL-10. In conclusion, these findings suggest that oxLDL-IC induce the synthesis and release of HSP70B', and once stimulated, HSP70B' binds to the cell-associated and internalized lipid moiety of oxLDL-IC. The data also implicate HSP70B' in key cellular functions, such as regulation of SK1 activity and release of IL-10, which influence macrophage activation and survival.

The level of malondialdehyde-modified LDL and LDL immune complexes in patients with rheumatoid arthritis

Objectives To explore possible associations of malondialdehyde-modified low-density lipoprotein (MDA-LDL) and LDL-immune complexes (LDL-IC) with atherosclerosis in rheumatoid arthritis (RA). Design and methods Plasma MDA-LDL, LDL-IC levels and mechanisms of the changes were investigated in RA patients with or without coronary artery disease (CAD), simple CAD patients and control. Results MDA-LDL and LDL-IC levels were found increased in all the studied patients, the RA patients with CAD exhibited the most significant changes. MDA-LDL levels were higher in the RA patients with CAD than those both in the simple RA and CAD patients. Multiple linear regression analysis showed that CAD, LDL-IC and erythrocyte sedimentation rate accounted for 36.5% of the variation in MDA-LDL levels; and age, activity, MDA-LDL and rheumatoid factors accounted for 34.5% of the variation in LDL-IC. Conclusions High levels of MDA-LDL and LDL-IC are risk factors for increased risk of atherosclerosis in RA patients and are associated with inflammation.

Low-density lipoproteinimmune complexes in patients with type 2 diabetes

：This study was designedto observe the clinical value of low-density lipoprotein immune complexes (LDL-ICs) inpatients with type 2 diabetes mellitus (T2DM). Fifty-four patients with type 2 diabetes +macroangiopathy (DM-MA group), 48 patients with type 2 diabetes (DM group), 45 patientswith macroangiopathy ( MA group), and 30 normal controls ( NC group) were enrolled.LDL-ICs levels in the DM-MA group were significantly higher than those in the other threegroups. The levels of LDL-ICs in the MA group were higher than those in the NC and DMgroups. There was a significant difference in LDL-ICs between the DM and NC groups.LDL-ICs levels were positively related with body mass index (BMI), glycesylated hemoglobinAlc(HbAlc), blood pressure, total cholesterol, and triglyceride. There was no significantrelationship between low-density lipsprotcin cholesterol and LDL-ICs. A significantlynegative correlation was recorded between high-density lipoprotein cholesterol andLDL-ICs. Our study suggests that LDL-ICs, blood pressure, and HbAlc may be risk factors ofmacroangiopathy in type 2 diabetic patients.

Oxidized LDL immune complexes and oxidized LDL differentially affect the expression of genes involved with inflammation and survival in human U937 monocytic cells

Objective To compare the global effects of oxidized LDL (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) on gene expression in human monocytic cells and to identify differentially expressed genes involved with inflammation and survival. Methods and results U937 cells were treated with oxLDL-IC, oxLDL, Keyhole limpet hemocyanin immune complexes (KLH-IC), or vehicle for 4 h. Transcriptome profiling was performed using DNA microarrays. oxLDL-IC uniquely affected the expression of genes involved with pro-survival ( RAD54B, RUFY3, SNRPB2, and ZBTB24). oxLDL-IC also regulated many genes in a manner similar to KLH-IC. Functional categorization of these genes revealed that 39% are involved with stress responses, including the unfolded protein response which impacts cell survival, 19% with regulation of transcription, 10% with endocytosis and intracellular transport of protein and lipid, and 16% with inflammatory responses including regulation of I-κB /NF-κB cascade and cytokine activity. One gene in particular, HSPA6, greatly up-regulated by oxLDL-IC, was found to be required for the process by which oxLDL-IC augments IL1-β secretion. The study also revealed genes uniquely up-regulated by oxLDL, including genes involved with growth inhibition ( OKL38, NEK3, and FTH1), oxidoreductase activity ( SPXN1 and HMOX1), and transport of amino acids and fatty acids ( SLC7A11 and ADFP). Conclusions These findings highlight early transcriptional responses elicited by oxLDL-IC that may underlie its cytoprotective and pro-inflammatory effects. Cross-linking of Fcγ receptors appears to be the trigger for most of the transcriptional responses to oxLDL-IC. The findings further strengthen the hypothesis that oxLDL and oxLDL-IC elicit disparate inflammatory responses and play distinct roles in the process of atherosclerosis.

OxLDL immune complexes activate complement and induce cytokine production by MonoMac 6 cells and human macrophages

Oxidized low density lipoprotein (OxLDL) is immunogenic and induces autoimmune responses in humans. OxLDL antibodies are predominantly of the proinflammatory IgG1 and IgG3 isotypes. We tested the capacity of immune complexes prepared with copper-oxidized human LDL and affinity chromatography-purified human OxLDL antibodies [OxLDL-immune complexes (ICs)] to activate complement and to induce cytokine release by MonoMac 6 (MM6) cells and by primary human macrophages. The levels of C4d and C3a were significantly higher in human serum incubated with OxLDL-ICs than after incubation with OxLDL or OxLDL antibody, indicating complement activation by the classical pathway. MM6 cells and primary human macrophages were incubated with OxLDL-ICs, with or without prior conditioning with interferon-gamma. After 18 h of incubation, both MM6 cells and primary human macrophages released significantly higher levels of proinflammatory cytokines after incubation with OxLDL-ICs than after incubation with OxLDL or with OxLDL antibody, both in primed and unprimed cells. OxLDL-ICs were more potent activators of MM6 cells than keyhole limpet hemocyanin-ICs. Blocking Fc gamma receptor I (FcgammaRI) with monomeric IgG1 significantly depressed the response of MM6 cells to OxLDL-ICs. In conclusion, human OxLDL-ICs have proinflammatory properties, as reflected by their capacity to activate the classical pathway of complement and to induce proinflammatory cytokine release from MM6 cells and primary human macrophages.