Search

Objective: 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., Methods: 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., Results: 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, P < 0.0001). 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)., Conclusion: 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., (Published by Elsevier Ireland Ltd.)

Objective: Circulating immune complexes (IC) containing modified forms of LDL (mLDL) are strongly pro-inflammatory and strong predictors of cardiovascular disease (CVD) progression in type 1 diabetes. The present study was undertaken to determine whether the levels of oxidized LDL (oxLDL), malondialdehyde-LDL (MDA-LDL) and advanced glycation end products-LDL (AGE-LDL) in IC predict incident CVD events in type 2 diabetes (VADT cohort)., Methods and Results: Levels of mLDL in IC were measured in 907 patients of the VADT cohort, a median of two years after entry into the study. Participants were followed for an average of 3.7 years for vascular outcomes. Hazard ratios (HRs) for CV endpoints in relation to mLDL-IC quartiles were calculated by Cox proportional hazard models. The primary composite CVD endpoint included documented myocardial infarction (MI); stroke; death from CVD; congestive heart failure; cardiac, cerebrovascular, or peripheral VD surgical intervention; inoperable CVD; and amputation for ischemic gangrene. During follow-up, 4.7% and 16.8% of participants had an MI or a composite endpoint, respectively. After adjustments by conventional risk factors, individuals in the highest quartile of MDA-LDL-IC were at higher risk of MI [HR = 2.44 (95% CI: 1.03, 5.77)] and composite endpoint [HR = 1.71 (95% CI: 1.04, 2.80)], relative to individuals in the lowest quartile. Similar comparisons for oxLDL and AGE-LDL levels yielded HR values of 1.08 and 1.31 for MI and 0.91 and 1.34 for composite endpoint., Conclusions: Our study indicates that high levels of MDA-LDL in isolated IC predict future MI and acute CV events in patients with type 2 diabetes., (Published by Elsevier Ireland Ltd.)

Objective: Oxidized 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)., Methods: OxLDL 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., Results: Multivariable 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., Discussion: Increased 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., (Published by Elsevier Ireland Ltd.)

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 4h. 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-kappaB /NF-kappaB 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-beta 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 Fc gamma 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.

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.

Modified lipoproteins induce autoimmune responses including the synthesis of autoantibodies with pro-inflammatory characteristics. Circulating modified lipoprotein autoantibodies combine with circulating antigens and form immune complexes (IC). We now report the results of a study investigating the role of circulating IC containing modified lipoproteins in the progression of carotid intima-media thickness (IMT) in patients enrolled in the Epidemiology of Diabetes Interventions and Complications (EDIC) Trial, a follow-up study of the Diabetes Control and Complications Trial (DCCT). This cohort includes 1229 patients with type 1 diabetes in whom B-mode ultrasonography of internal and common carotid arteries was performed in 1994-1996 and in 1998-2000. Conventional CHD risk factors, antibodies against modified forms of LDL and modified lipoprotein IC were determined in 1050 of these patients from blood collected in 1996-1998. Cholesterol and apolipoprotein B content of IC (surrogate markers of modified ApoB-rich lipoproteins) were significantly higher in patients who showed progression of the internal carotid IMT than in those showing no progression, regression or mild progression. Multivariate linear and logistic regression modeling using conventional and non-conventional risk factors showed that the cholesterol content of IC was a significant positive predictor of internal carotid IMT progression. In conclusion these data demonstrate that increased levels of modified ApoB-rich IC are associated with increased progression of internal carotid IMT in the DCCT/EDIC cohort of type 1 diabetes.

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

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.