Antibodies against malondialdehyde among 60-year-olds: prediction of cardiovascular disease.

Malondialdehyde (MDA) is generated in oxidized LDL. It forms covalent protein adducts, and is recognized by antibodies (anti-MDA). We previously studied IgM anti-MDA, and here we focus on IgG, IgG1 and IgG2 anti-MDA in predicting cardiovascular disease (CVD). We determined, by ELISA, anti-MDA in a 7-year follow-up of 60-year-old men and women from Stockholm County (2039 men, 2193 women). We identified 210 incident CVD cases (defined as new events of myocardial infarction (MI), and hospitalization for angina pectoris) and ischemic stroke, and 620 age- and sex-matched controls. IgG anti-MDA was not associated with CVD. Median values only differed significantly for IgG1 anti-MDA among men, with lower levels among cases than controls (p = 0.039). High IgG1 anti-MDA (above 75th percentile) was inversely associated with CVD risk after adjustment for smoking, body mass index, type 2 diabetes, hyperlipidemia, and hypertension, (OR and 95% CI: 0.59; 0.40-0.89). After stratification by sex, this association emerged in men (OR and 95% CI: 0.46; 0.27-0.77), but not in women. IgG2 anti-MDA were associated with protection in the whole group and among men though weaker than IgG1 anti-MDA. IgG2 anti-MDA above the 75th percentile was associated with an increased risk of MI/angina in women (OR and 95% CI: 2.57; (1.08-6.16)). IgG1 and less so IgG2 anti-MDA are protection markers for CVD and MI/angina in the whole group and among men. However, IgG2 anti-MDA was a risk marker for MI/angina among women. These findings could have implications for both prediction and therapy.

Antibodies against phosphorylcholine in hospitalized versus non-hospitalized obese subjects.

Obesity associates with reduced life expectancy, type 2 diabetes, hypertension and cardiovascular disease, and is characterized by chronic inflammation. Phosphorylcholine (PC) is an epitope on oxidized low-density lipoprotein, dead cells and some microorganisms. Antibodies against PC (anti-PC) have anti-inflammatory properties. Here, we explored the role of anti-PC in hospitalized versus non-hospitalized obese. One-hundred-and-twenty-eight obese (BMI ≥ 30 kg/m2) individuals (59.8 (± 5.5) years, 53.9% women) from the Malmö Diet and Cancer Cardiovascular Cohort were examined and IgM, IgG1 and IgG2 anti-PC were analyzed by ELISA. Individuals with at least one recorded history of hospitalization prior to study baseline were considered hospitalized obese (HO). Associations between IgM, IgG1 and IgG2 anti-PC and HO (n = 32)/non-hospitalized obese (NHO) (n = 96), but also with metabolic syndrome and diabetes were analysed using logistic regressions. Both IgM and IgG1 anti-PC were inversely associated with HO, also after controlling for age and sex. When further adjusted for waist circumference, systolic blood pressure, glucose levels and smoking status, only IgG1 anti-PC remained significantly associated with HO. In multivariate models, each 1 standard deviation of increment in anti-PC IgG1 levels was inversely associated with prevalence of HO (odds ratio 0.57; CI 95% 0.33-0.98; p = 0.044). IgG2 anti-PC did not show any associations with HO. Low levels of IgM and IgG1 anti-PC are associated with higher risk of being a HO individual independent of sex and age, IgG1 anti-PC also independently of diabetes and metabolic syndrome. The anti-inflammatory properties of these antibodies may be related to inflammation in obesity and its complications.

An elevated polyclonal free light chain level reflects a strong interferon signature in patients with systemic autoimmune diseases.

High amount of polyclonal free light chains (FLC) are reported in systemic autoimmune diseases (SAD) and we took advantage of the PRECISESADS study to better characterize them. Serum FLC levels were explored in 1979 patients with SAD (RA, SLE, SjS, Scl, APS, UCTD, MCTD) and 614 healthy controls. Information regarding clinical parameters, disease activity, medications, autoantibodies (Ab) and the interferon α and/or γ scores were recorded. Among SAD patients, 28.4% had raised total FLC (from 12% in RA to 30% in SLE and APS) with a normal kappa/lambda ratio. Total FLC levels were significantly higher in SAD with inflammation, active disease in SLE and SjS, and an impaired pulmonary functional capacity in SSc, while independent from kidney impairment, infection, cancer and treatment. Total FLC concentrations were positively correlated among the 10/17 (58.8%) autoantibodies (Ab) tested with anti-RNA binding protein Ab (SSB, SSA-52/60 kDa, Sm, U1-RNP), anti-dsDNA/nucleosome Ab, rheumatoid factor and negatively correlated with complement fractions C3/C4. Finally, examination of interferon (IFN) expression as a potential driver of FLC overexpression was tested showing an elevated level of total FLC among patients with a high IFNα and IFNγ Kirou's score, a strong IFN modular score, and the detection in the sera of B-cell IFN dependent factors, such as TNF-R1/TNFRSF1A and CXCL10/IP10. In conclusion, an elevated level of FLC, in association with a strong IFN signature, defines a subgroup of SAD patients, including those without renal affectation, characterized by increased disease activity, autoreactivity, and complement reduction.

Patients with SLE have higher risk of cardiovascular events and mortality in comparison with controls with the same levels of traditional risk factors and intima-media measures, which is related to accumulated disease damage and antiphospholipid syndrome: a case-control study over 10 years.

OBJECTIVE: SLE is a strong risk factor for premature cardiovascular (CV) disease and mortality. We investigated which factors could explain poor prognosis in SLE compared with controls. METHODS: Patients with SLE and population controls without history of clinical CV events who performed carotid ultrasound examination were recruited for this study. The outcome was incident CV event and death. Event-free survival rates were compared using Kaplan-Meier curves. Relative HR (95% CI) was used to estimate risk of outcome. RESULTS: Patients (n=99, 87% female), aged 47 (13) years and with a disease duration of 12 (9) years, had mild disease at inclusion, Systemic Lupus Erythematosus Diseases Activity Index score of 3 (1-6) and Systemic Lupus International Collaborating Clinics (SLICC) Damage Index score of 0 (0-1). The controls (n=109, 91% female) were 49 (12) years old. Baseline carotid intima-media thickness (cIMT) did not differ between the groups, but plaques were more prevalent in patients (p=0.068). During 10.1 (9.8-10.2) years, 12 patients and 4 controls reached the outcome (p=0.022). Compared with the controls, the risk of the adverse outcome in patients increased threefold to fourfold taking into account age, gender, history of smoking and diabetes, family history of CV, baseline body mass index, waist circumference, C reactive protein, total cholesterol, high-density lipoprotein, low-density lipoprotein, dyslipidaemia, cIMT and presence of carotid plaque. In patients, higher SLICC score and SLE-antiphospholipid syndrome (SLE-APS) were associated with increased risk of the adverse outcome, with respective HRs of 1.66 (95% CI 1.20 to 2.28) and 9.08 (95% CI 2.71 to 30.5), as was cIMT with an HR of 1.006 (95% CI 1.002 to 1.01). The combination of SLICC and SLE-APS with cIMT significantly improved prediction of the adverse outcome (p<0.001). CONCLUSION: In patients with mild SLE of more than 10 years duration, there is a threefold to fourfold increased risk of CV events and death compared with persons who do not have SLE with similar pattern of traditional CV risk factors, cIMT and presence of carotid plaque. SLICC, SLE-APS and subclinical atherosclerosis may indicate a group at risk of worse outcome in SLE.

Low levels of PCSK9 are associated with remission in patients with rheumatoid arthritis treated with anti-TNF-α: potential underlying mechanisms.

BACKGROUND: Proprotein convertase subtilisin kexin 9 (PCSK9) targets the LDL-receptor (LDLR) which raises LDL-levels. In addition, PCSK9 has proinflammatory immunological effects. Here, we investigate the role of PCSK9 in relation to the inflammatory activity in patients with rheumatoid arthritis (RA). METHODS: PCSK9-levels were determined at baseline by ELISA in 160 patients with RA not previously treated with biologics. The patients started anti-TNF-α (adalimumab, infliximab, or etanercept) treatment and were followed-up for 1 year. Disease activity was determined by DAS28. Effects of PCSK9 on cytokine production from macrophages of healthy individuals and synoviocytes from RA patients and inhibition by anti-PCSK9 antibodies were studied in supernatants by ELISA. RESULTS: A significantly lower level of PCSK9 at baseline, p = 0.035, was observed in patients who reached remission within 1 year, defined as DAS28 < 2.6, compared to those not in remission. At 12 months of TNF-α antagonist treatment, the mean DAS28 was reduced but was significantly greater in patients with highest quartile PCSK9 (Q4) compared to those at lowest PCSK9 (Q1) in both crude (p = 0.01) and adjusted analysis (p = 0.004). In vitro, PCSK9 induced TNF-alpha and IL-1beta in macrophages and monocyte chemoattractant protein-1 (MCP1) in synoviocytes. These effects were inhibited by anti-PCSK9 antibodies. CONCLUSIONS: Low levels of PCSK9 at baseline are associated with being DAS28-responder to anti-TNF-α treatment in RA. An underlying cause could be that PCSK9 stimulates the production of proinflammatory cytokines from macrophages and synoviocytes, effects inhibited by anti-PCSK9 antibodies. PCSK9 could thus play an immunological role in RA.

Malondialdehyde Conjugated With Albumin Induces Pro-Inflammatory Activation of T Cells Isolated From Human Atherosclerotic Plaques Both Directly and Via Dendritic Cell-Mediated Mechanism.

Human dendritic cells were differentiated from blood monocytes and treated with malondialdehyde (MDA) conjugated with human serum albumin (HSA). Autologous T cells from human plaques or blood were co-cultured with the pre-treated dendritic cells or treated directly. MDA modifications were studied by mass spectrometry. MDA-HSA induced a pro-inflammatory DC-mediated T-cell activation and also a strong direct effect on T cells, inhibited by an inhibitor of oxidative stress and antibodies against MDA. Atherogenic heat shock protein-60 was strongly induced in T cells activated by MDA-HSA. Two peptide modifications in atherosclerotic patients' HSA were similar to those present in in vitro MDA-modified HSA.

A genome-wide association study of IgM antibody against phosphorylcholine: shared genetics and phenotypic relationship to chronic lymphocytic leukemia.

Phosphorylcholine (PC) is an epitope on oxidized low-density lipoprotein (oxLDL), apoptotic cells and several pathogens like Streptococcus pneumoniae. Immunoglobulin M against PC (IgM anti-PC) has the ability to inhibit uptake of oxLDL by macrophages and increase clearance of apoptotic cells. From our genome-wide association studies (GWASs) in four European-ancestry cohorts, six single nucleotide polymorphisms (SNPs) in 11q24.1 were discovered (in 3002 individuals) and replicated (in 646 individuals) to be associated with serum level of IgM anti-PC (the leading SNP rs35923643-G, combined ß = 0.19, 95% confidence interval 0.13-0.24, P = 4.3 × 10-11). The haplotype tagged by rs35923643-G (or its proxy SNP rs735665-A) is also known as the top risk allele for chronic lymphocytic leukemia (CLL), and a main increasing allele for general IgM. By using summary GWAS results of IgM anti-PC and CLL in the polygenic risk score (PRS) analysis, PRS on the basis of IgM anti-PC risk alleles positively associated with CLL risk (explained 0.6% of CLL variance, P = 1.2 × 10-15). Functional prediction suggested that rs35923643-G might impede the binding of Runt-related transcription factor 3, a tumor suppressor playing a central role in the immune regulation of cancers. Contrary to the expectations from the shared genetics between IgM anti-PC and CLL, an inverse relationship at the phenotypic level was found in a nested case-control study (30 CLL cases with 90 age- and sex-matched controls), potentially reflecting reverse causation. The suggested function of the top variant as well as the phenotypic association between IgM anti-PC and CLL risk needs replication and motivates further studies.

IgM antibodies against phosphorylcholine promote polarization of T regulatory cells from patients with atherosclerotic plaques, systemic lupus erythematosus and healthy donors.

BACKGROUND AND AIMS: IgM antibodies against phosphorylcholine (anti-PC) are negatively associated with atherosclerosis, cardiovascular disease (CVD) and systemic lupus erythematosus (SLE), where the risk of CVD and atherosclerosis is high. We here study the effects of IgM anti-PC immune regulation. METHODS: Mononuclear leukocytes were isolated from peripheral blood (PBMC) obtained from healthy blood donors, six SLE patients with age- and sex-matched controls, and symptom-giving human atherosclerotic plaques. The proportion of Th17 (CD4+CCR6+) and Treg (CD4+CD25+CD127dim/-) cells was determined by flow cytometry in CD4+T cells after 6 days of culture with Th17 or Treg-polarizing cytokines, with PMA and Ionomycin stimulation. IgM anti-PC were extracted from total IgM, with flow-through IgM as controls. Dendritic cells (DC) were differentiated from PBMC. Antibody peptide/protein characterization was done by a proteomics de novo sequencing approach. RESULTS: IgM anti-PC increased significantly the proportion of Tregs from healthy donors, SLE patients and atherosclerotic plaque cells while control antibodies did not. T cells from SLE patients had a significantly lower proportion of Tregs and a higher proportion of Th17 cells as compared to matched controls. IgM anti-PC, but not control antibodies, significantly reduced the production of IL-17 and TNF-α in cell cultures from SLE patients and atherosclerotic plaque cells. IgM anti-PC interacted with CD40 and kept DCs in an immature stage, potentially being tolerogenic. We observed differences in the IgM peptide expression levels in anti-PC compared to control antibodies. CONCLUSIONS: IgM anti-PC promote polarization of Tregs, which could represent a novel protective mechanism in atherosclerosis and autoimmune conditions as SLE.

IgM antibodies to oxidized phosphatidylserine as protection markers in cardiovascular disease among 60-year olds.

OBJECTIVE: Phosphatidylserine is exposed on apoptotic cells and is prone to oxidation (OxPS). Here we analyze the association of IgM antibodies against OxPS (anti-OxPS) with the risk of cardiovascular disease (CVD). METHODS: Among sixty-year olds from Stockholm County in Sweden, previously screened for cardiovascular risk factors (2039 men, 2193 women), there were 210 incident CVD-cases identified during a 5-year follow-up. Using a nested case-control design, 622 age- and sex-matched controls were selected. Odds ratios (OR) with 95% intervals (CI) were calculated by conditional logistic regression. IgM anti-OxPS was measured by ELISA. Phagocytosis of apoptotic Jurkat-cells by macrophages was studied by flow cytometry. RESULTS: Anti-OxPS levels were lower among cases (median (interquartile range): 80.7 (60.9-101.0 vs. 84.6 (65.8-109.6); p = 0.047); among men (76.6 (55.8-99.2) vs. 82.0 (63.1-105.1); p = 0.022) and among women 89.6 (72.3-110.1) vs. 89.8 (69.9-114.4); p = 0.79). After adjustment for smoking, BMI, diabetes mellitus type II, hypercholesterolaemia and hypertension, and dividing into quartiles, using the highest quartile (quartile 4) as reference, quartile 3 was associated with a OR of 1.74 (CI 1.08-2.81). Quartiles 2 and 1 had similar associations, the later reaching statistical significance. Among men associations were stronger whereas no significant associations were observed in women. The OR of MI/angina comparing quartile 3 with quartile 4 was 2.31 (CI 1.30-4.11). The OR for quartile 2 and 1, respectively, were similar as for quartile 3. Total IgM increased uptake of apoptotic cells, which was reversed if incubated with OxPS. CONCLUSIONS: IgM anti-OxPS is a novel potential protection marker for CVD, in particular in men. Increased phagocytosis of dying/dead cells could be one potential underlying mechanism.

Tartrate-Resistant Acid Phosphatase Deficiency in the Predisposition to Systemic Lupus Erythematosus.

OBJECTIVE: Mutations in the ACP5 gene, which encodes tartrate-resistant acid phosphatase (TRAP), cause the immuno-osseous disorder spondyloenchondrodysplasia, which includes as disease features systemic lupus erythematosus (SLE) and a type I interferon (IFN) signature. Our aims were to identify TRAP substrates, determine the consequences of TRAP deficiency in immune cells, and assess whether ACP5 mutations are enriched in sporadic cases of SLE. METHODS: Interaction between TRAP and its binding partners was tested by a yeast 2-hybrid screening, confocal microscopy, and immunoprecipitation/Western blotting. TRAP knockdown was performed using small interfering RNA. Phosphorylation of osteopontin (OPN) was analyzed by mass spectrometry. Nucleotide sequence analysis of ACP5 was performed by Sanger sequencing or next-generation sequencing. RESULTS: TRAP and OPN colocalized and interacted in human macrophages and plasmacytoid dendritic cells (PDCs). TRAP dephosphorylated 3 serine residues on specific OPN peptides. TRAP knockdown resulted in increased OPN phosphorylation and increased nuclear translocation of IRF7 and P65, with resultant heightened expression of IFN-stimulated genes and IL6 and TNF following Toll-like receptor 9 stimulation. An excess of heterozygous ACP5 missense variants was observed in SLE compared to controls (P = 0.04), and transfection experiments revealed a significant reduction in TRAP activity in a number of variants. CONCLUSION: Our findings indicate that TRAP and OPN colocalize and that OPN is a substrate for TRAP in human immune cells. TRAP deficiency in PDCs leads to increased IFNα production, providing at least a partial explanation for how ACP5 mutations cause lupus in the context of spondyloenchondrodysplasia. Detection of ACP5 missense variants in a lupus cohort suggests that impaired TRAP functioning may increase susceptibility to sporadic lupus.

Human IgM Antibodies to Malondialdehyde Conjugated With Albumin Are Negatively Associated With Cardiovascular Disease Among 60-Year-Olds.

BACKGROUND: Malondialdehyde (MDA) is generated during lipid peroxidation as in oxidized low-density lipoprotein, but antibodies against oxidized low-density lipoprotein show variable results in clinical studies. We therefore studied the risk of cardiovascular disease (CVD) associated with IgM antibodies against MDA conjugated with human albumin (anti-MDA). METHODS AND RESULTS: In a 5- to 7-year follow-up of 60-year-old men and women from Stockholm County previously screened for cardiovascular risk factors (2039 men, 2193 women), 209 incident CVD cases (defined as new events of coronary heart disease, fatal and nonfatal myocardial infarction, ischemic stroke, and hospitalization for angina pectoris) and 620 age- and sex-matched controls were tested for IgM anti-MDA by ELISA. Antibody peptide/protein characterization was done using a proteomics de novo sequencing approach. After adjustment for smoking, body-mass index, type 2 diabetes mellitus, hyperlipidemia, and hypertension, an increased CVD risk was observed in the low IgM anti-MDA percentiles (below 10th and 25th) (odds ratio and 95% CI: 2.0; 1.19-3.36 and 1.67; 1.16-2.41, respectively). Anti-MDA above the 66th percentile was associated with a decreased CVD risk (odds ratio 0.68; CI: 0.48-0.98). After stratification by sex, associations were only present among men. IgM anti-MDA levels were lower among cases (median [interquartile range]: 141.0 [112.7-164.3] versus 147.4 [123.5-169.6]; P=0.0177), even more so among men (130.6 [107.7-155.3] versus 143.0 [120.1-165.2]; P=0.001). The IgM anti-MDA variable region profiles are distinctly different and also more homologous in their content (correlates strongly with fewer peptides) than control antibodies (not binding MDA). CONCLUSIONS: IgM anti-MDA is a protection marker for CVD. This finding could have diagnostic and therapeutic implications.

Oxidized Low-Density Lipoprotein (OxLDL)-Treated Dendritic Cells Promote Activation of T Cells in Human Atherosclerotic Plaque and Blood, Which Is Repressed by Statins: microRNA let-7c Is Integral to the Effect.

BACKGROUND: Activated T cells and dendritic cells (DCs) are colocalized in atherosclerotic plaques in association with plaque rupture. Oxidized low-density lipoprotein (oxLDL) promotes immune activation and inflammation. We studied the effects of statins (atorvastatin and simvastatin) on human DC maturation and T-cell activation. METHODS AND RESULTS: Human peripheral blood monocytes were differentiated to DCs and stimulated with oxLDL. T cells were isolated from carotid endarterectomy specimens from patients undergoing carotid endarterectomy or from healthy individuals. Naïve T cells were cocultured with pretreated DCs. The effects of statin were studied. OxLDL induced DC maturation and T-cell activation. OxLDL induced atherogenic heat shock proteins (HSP) 60 and 90 and decreased potentially atheroprotective heat shock protein 27, effects restored by atorvastatin. T cells exposed to oxLDL-treated DCs produced interferon-γ and interleukin (IL)-17. Atorvastatin and simvastatin suppressed the DC maturation showing lower expression of CD80, CD83, and CD86, and limited their production of tumor necrosis factor-α, IL-1ß and IL-6, and increased transforming growth factor-ß and IL-10 secretion. Statin-treated DCs inhibited Th1 and/or Th17 polarization by downregulation of transcriptional factors T-bet and RORγt expression, and induced T regulatory cells with IL-10 production. OxLDL-induced miRNA let7c and phosphorylation of Akt and ERK were repressed by statins. Let-7c had a pivotal role in mediating effect of oxLDL. Experiments on T cells derived from carotid atherosclerotic plaques or healthy individuals showed similar results. CONCLUSIONS: Statins repress human DC maturation induced by oxLDL, limit T-cell activation, and repress an atherogenic heat shock protein profile and promote induction of T regulatory cells. MicroRNA let-7c is integral to the effects.

Coenzyme Q10 and oxidative stress, the association with peripheral sensory neuropathy and cardiovascular disease in type 2 diabetes mellitus.

OBJECTIVE: Our study aimed to explore associations between metabolic control, oxidative stress and coenzyme Q10 (CoQ10) in relation to diabetes complications in a representative population of type 2 diabetes. RESEARCH DESIGN AND METHODS: A geographic cohort of 156 subjects was recruited. Serum concentrations of CoQ10 and vitamin E were measured by HPLC. ROS was determined by free oxygen radicals testing (FORT). Glutaredoxin (Grx) activity, oxidized LDL cholesterol (oxLDLc), high sensitive CRP (hsCRP), HbA1c, urine albumin, serum creatinine, serum cystatin C, and plasma lipids were assayed with routine laboratory protocols. RESULTS: Serum CoQ10 was higher than in nondiabetics. HbA1c, fP-glucose, hyperlipidemia, inflammation (hsCRP), and increased BMI were associated with signs of oxidative stress as increased levels of FORT, Grx activity and/or increased levels of oxLDLc Oxidative stress was found to be strongly correlated with prevalence of cardiovascular disease (CVD) and peripheral sensory neuropathy (PSN). In both gender groups there were positive correlations between CoQ10 and oxLDLc, and between BMI and the ratio CoQ10/chol. Grx activity was inversely correlated to oxLDLc and CoQ10. Women with CVD and PSN had higher waist index, oxLDLc, and FORT levels compared to men but lower CoQ10 levels. Men had worse kidney function and lower vitamin E. Multiple regression analysis showed increased levels of CoQ10 to be significantly correlated with increased levels of cholesterol, triglycerides, vitamin E, fB-glucose and BMI. CONCLUSIONS: Hyperlipidemia, hyperglycemia and inflammation were associated with oxidative stress, which was correlated to the prevalence of diabetes complications. CoQ10 was increased in response to oxidative stress. There were gender differences in the risk factors associated with diabetes complications.

Bone mineral density and carotid atherosclerosis in systemic lupus erythematosus: a controlled cross-sectional study.

INTRODUCTION: As osteoporosis is reported to be associated with atherosclerosis in the general population we examined the relationship between bone mass and carotid measurements in patients with systemic lupus erythematosus (SLE) and controls, and possible links between them in SLE. METHODS: In a cross-sectional study, 111 SLE-patient were compared with 111 age- and sex-matched controls, mean age 48.7(12.9) years, 89% were women, of which 51% postmenopausal. Carotid intima media thickness (cIMT), carotid plaque occurrence and echogenicity were determined by B-mode ultrasound and bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA). RESULTS: BMD and cIMT were inversely associated both in patients and controls. Patients, but not controls, with carotid plaque had higher cIMT at low BMD than at normal BMD, p = 0.010. Logistic regression indicated more than doubled odds ratio (OR) of carotid plaque in patients, particularly in post-menopausal women, than in controls in relation to all BMD measurements. For low BMD at hip, significant increased OR for echolucent plaque was shown for patients compared with controls. In patients, significant impact of age, body mass index, smoking, systolic blood pressure, blood lipids, diabetes mellitus, impaired renal function, low levels of complement C3 and C4, history of nephritis, SLE-damage index and ever use of antimalarial was found for association between BMD and higher cIMT and carotid plaque. In multivariate regression, low C4 was independent contributor to association between total BMD and upper cIMT tertile, accounted for OR (95% confidence interval) of 3.2 (1.03-10.01), and also for association with bilateral carotid plaque, OR of 4.8 (1.03-22.66). The contribution of low C4 for the association between BMD and carotid atherosclerosis was enhanced within the second and third tertiles of total BMD. CONCLUSION: This study is the first to demonstrate inverse association between BMD and carotid measurements in both SLE-patients and controls. Our results suggest that SLE-patients may suffer higher burden of (sub)clinical atherosclerotic disease, especially presence of both echolucent and echogenic plaque, than controls with the same bone mineral status. Low complement C4 seems to play an important role in earlier development of carotid atherosclerosis already within (sub)normal ranges of total BMD in patients.

Prediction and management of cardiovascular outcomes in systemic lupus erythematosus.

Atherosclerosis is the major cause of cardiovascular disease (CVD), which represents the major cause of death. During recent years, it has become clear that atherosclerosis is a chronic inflammatory condition where immunity could play an important role. Usually, it is when atherosclerotic plaques rupture that CVD follows, but some cases of CVD can occur without apparent atherosclerosis. In systemic lupus erythematosus, the risk of CVD is very high and the prevalence of atherosclerotic plaques, including vulnerable ones, is increased. A combination of traditional and non-traditional risk factors is implicated for the prediction of CVD in systemic lupus erythematosus. Traditional risk factors include hypertension, dyslipidemia, smoking and diabetes, though the exact importance of each of these in systemic lupus erythematosus is not clear. Anti-phospholipid antibodies, systemic inflammation and low levels of natural antibodies such as those against phosphorylcholine (anti-PC) are examples of non-traditional risk factors. Control of disease activity and disease manifestations and of established risk factors is important.

Oxidized but not native cardiolipin has pro-inflammatory effects, which are inhibited by Annexin A5.

OBJECTIVE: Cardiolipin (CL) is a phospholipid with an unusual dimeric structure containing four double-bonds and is easily oxidized. CL is present in mitochondria. Here we explored potential pro-inflammatory properties implicated in cardiovascular disease (CVD): activation of endothelial cells, 5-lipoxygenase (5-LOX) and leukotriene B4 (LTB4), by oxidized CL (oxCL) and inhibitory effects of Annexin A5, an antithrombotic and antiinflammatory plasma protein. METHODS: In monocytes/macrophages and neutrophils, calcium mobilization was monitored spectrophotometrically with Fura-2 and synthesis of LTB4 was analyzed by EIA. Expression of adhesion molecules on endothelial cells was studied by FACScan. Binding of Annexin A5 were analyzed by ELISA. The mRNA expression of 5-LOX and cyclooxygenase-2 was assessed by Real-Time PCR. RESULTS: We demonstrate that oxCL but not its non-oxidized counterpart CL induces biosynthesis of LTB4 and increases intracellular concentrations of calcium in monocytes/macrophages and neutrophils. oxCL rather than CL selectively elevates gene expression of 5-LOX but not COX-2 in human macrophages. Furthermore, oxCL but not CL raises levels of adhesion molecules ICAM-1 and VCAM-1 in endothelial cells. Annexin A5 can bind oxCL to abolish all these oxCL-induced effects. CONCLUSIONS: oxCL may promote inflammation and related diseases especially in conditions involving unresolved apoptosis and necrosis, such as atherosclerosis, where free oxCL is likely to be released from liberated mitochondria. Increased intracellular calcium could activate 5-LOX to produce Leukotriene B4 (LTB4). Annexin A5 inhibits the pro-inflammatory effects of oxCL and its potential therapeutic use when oxCL is implicated in inflammation could be of interest.

Naturally occurring human phosphorylcholine antibodies are predominantly products of affinity-matured B cells in the adult.

Phosphorylcholine (PC) is a classic T-independent Ag that is exposed on apoptotic cells, oxidized phospholipids, and bacterial polysaccharides. Experimental as well as epidemiological studies have over the past decade implicated Abs against PC (anti-PC) as anti-inflammatory and a strong protective factor in cardiovascular disease. Although clinically important, little is known about the development of anti-PC in humans. This study was conceived to dissect the human anti-PC repertoire and generate human mAbs. We designed a PC-specific probe to identify, isolate, and characterize PC-reactive B cells from 10 healthy individuals. The donors had all mounted somatically mutated Abs toward PC using a broad variety of Ig genes. PC-reactive B cells were primarily found in the IgM(+) memory subset, although significant numbers also were detected among naive, IgG(+), and CD27(+)CD43(+) B cells. Abs from these subsets were clonally related, suggesting a common origin. mAbs derived from the same donors exhibited equivalent or higher affinity for PC than the well-characterized murine T-15 clone. These results provide novel insights into the cellular and molecular ontogeny of atheroprotective PC Abs, thereby offering new opportunities for Ab-based therapeutic interventions.

Immune mechanisms in atherosclerosis, especially in diabetes type 2.

Atherosclerosis and ensuing cardiovascular disease (CVD) are major complications of diabetes type 2. Atherosclerosis is a chronic inflammatory condition involving immunocompetent cells of different types present in the lesions. Even though inflammation and immune activation may be more pronounced in atherosclerosis in diabetes type 2, there does not appear to be any major differences between diabetics and non-diabetics. Similar factors are thus implicated in atherosclerosis-associated immune activation in both groups. The cause of immune activation is not known and different mutually non-exclusive possibilities exist. Oxidized and/or enzymatically modified forms of low-density lipoprotein (OxLDL) and dead cells are present in atherosclerotic plaques. OxLDL could play a role, being pro-inflammatory and immunostimulatory as it activates T-cells and is cytotoxic at higher concentrations. Inflammatory phospholipids in OxLDL are implicated, with phosphorylcholine (PC) as one of the exposed antigens. Antibodies against PC (anti-PC) are anti-atherogenic in mouse studies, and anti-PC is negatively associated with development of atherosclerosis and CVD in humans. Bacteria and virus have been discussed as potential causes of immune activation, but it has been difficult to find direct evidence supporting this hypothesis, and antibiotic trials in humans have been negative or inconclusive. Heat shock proteins (HSP) could be one major target for atherogenic immune reactions. More direct causes of plaque rupture include cytokines such as interleukin 1ß (IL-1ß), tumor necrosis factor (TNF), and also lipid mediators as leukotrienes. In addition, in diabetes, hyperglycemia and oxidative stress appear to accelerate the development of atherosclerosis, one mechanism could be via promotion of immune reactions. To prove that immune reactions are causative of atherosclerosis and CVD, further studies with immune-modulatory treatments are needed.

Annexin A5 inhibits atherogenic and pro-inflammatory effects of lysophosphatidylcholine.

OBJECTIVE: Atherosclerosis is an inflammatory condition, and rupture of atherosclerotic plaques is a major cause of cardiovascular disease (CVD). Lysophosphatidylcholine (LPC) is generated in low-density lipoprotein (LDL) during oxidation and/or enzymatic modification and has been implicated in atherosclerosis. Annexin A5 (ANXA5) is an antithrombotic and atheroprotective plasma protein. Here, we demonstrate novel pro-inflammatory and atherogenic properties of LPC, and inhibitory effects of ANXA5. METHODS: Endothelial cells and macrophages (differentiated from, THP-1 a monocytic cell line) were co-cultured. Expression of MMP-9 and OxLDL uptake by macrophages were studied by flow cytometry. The effect of LPC on leukotriene B4 (LTB4) synthesis in macrophages was studied by enzyme immunoassay (EIA). Chemotactic properties of LPC were investigated using a mouse intra-peritoneal recruitment model. RESULTS: Co-culture of macrophages and endothelial cells enhanced MMP-9 expression in both cell types. This effect was increased by LPC and diminished by ANXA5. Likewise, LPC induced LTB4 production by macrophages, whereas native LDL or phosphatidylcholine (PTC) had no effect. ANXA5 inhibited uptake of OxLDL in macrophages. LPC induced cell infiltration in vivo, as determined by increased cell count in mouse peritoneal exudates, and this effect was inhibited by ANXA5. CONCLUSIONS: ANXA5 could potentially play an important protective role in both atherogenesis and atherosclerotic plaque rupture by reducing pro-inflammatory effects of OxLDL and LPC as well as inhibiting OxLDL binding and uptake by macrophages. The possibility that ANXA5 could be developed into a novel therapy against CVD deserves further study.

Low levels of IgM antibodies to oxidized cardiolipin increase and high levels decrease risk of cardiovascular disease among 60-year olds: a prospective study.

BACKGROUND: Antibodies against cardiolipin (aCL) are associated with increased risk of cardiovascular disease (CVD). We here determine the role of antibodies against oxidized CL (aOxCL). METHODS: One third of sixty-year olds from the Stockholm County were screened (2039 men, 2193 women), where 211 incident CVD-cases and 633 age- and sex-matched controls were identified (5-7 year follow-up). Antibodies were determined by ELISA and uptake of oxLDL in macrophages by FACScan. RESULTS: IgM aOxCL was lower among CVD cases than controls (p=0.024). aOxCL-levels were divided in quartiles with the highest quartile set as the reference group. After adjustment for smoking, BMI, type II diabetes, hypercholesterolaemia and hypertension, an increased risk was determined in the lowest quartile of IgM aOxCL (OR: 1.80, CI: 1.12-2.91, p=0.0159); OR for men in the lowest quartile was 2.46 (CI 1.34-4.53, p=0.0037) for CVD and for stroke: 12.28 (CI: 1.48-101.77, p=0.02). IgG aOxCL levels did not differ between quartiles in CVD-risk. High levels of IgM aOxCL (reaching significance above 86th) and IgG aOxCL (above 95th percentile) were associated with decreased risk of CVD (OR: 0.485, CI: 0.283-0.829; p=0.0082 and OR: 0.23, CI: 0.07-0.69; p=0.0091). aCL were not associated with CVD. oxCL but not CL competed out uptake of OxLDL in macrophages, and aOxLDL recognized oxCL but not CL. In contrast to aCL, aOxCL was not dependent on co-factor Beta2-glycoprotein-I. CONCLUSIONS: aOxCL is a novel risk/protection marker for CVD, with therapeutic implications. OxCL competes with oxLDL for uptake in macrophages and the possibility that aOxCL inhibits such uptake by interfering with same or similar epitopes in oxCL and oxLDL should be further studied.