Anna G. Frostegård

Low levels of IgM antibodies to phosphorylcholine predict cardiovascular disease in 60-year old men: Effects on uptake of oxidized LDL in macrophages as a potential mechanism

OBJECTIVE We here determine the role of IgM antibodies against phosphorylcholine (anti-PC) in prediction of cardiovascular disease (CVD) and on macrophage uptake of Oxidized LDL (OxLDL). METHODS From a screening of 4232 subjects, 60-year-old (2039 men and 2193 women), 211 incident cases of CVD (myocardial infarction, ischemic stroke, or hospitalized angina pectoris) and 633 age- and sex-matched controls were identified through a 5-7 year follow-up. Serum levels of IgM anti-PC was determined by ELISA. Anti-PC was extracted from pooled human IgM and the effect of anti-PC on the uptake of OxLDL was studied by FACScan. RESULTS Relative risks (RR) with 95% confidence intervals (CI) by quartiles of anti-PC levels with quartile 4 set as the reference value (RR = 1.0) and adjusted for smoking, BMI, type II diabetes, hypercholesterolaemia, and high blood pressure yielded an excess risk for CVD only for those within the lowest quartile of anti-PC values with an RR of 1.37 (CI 0.87-2.16). However, for men stronger associations were noted with increasing multivariately adjusted RRs from quartile 4 to quartile 1. Subjects within quartile 1 (values below 29.7 U/ml) had a significantly increased RR of 1.96 (CI 1.09-3.55). Further adjustments for hsCRP gave essentially the same results. No excess risk was noted for women. Specific anti-PC could be extracted from IgM and these antibodies inhibited macrophage uptake of OxLDL. CONCLUSIONS Low IgM anti-PC could be a novel risk marker for CVD among men. One possible mechanism could be inhibition of uptake of oxLDL in macrophages.

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.

Induction of Dendritic Cell–Mediated T-Cell Activation by Modified but Not Native Low-Density Lipoprotein in Humans and Inhibition by Annexin A5 Involvement of Heat Shock Proteins

Objective—Atherosclerosis is an inflammatory disease, where activated immunocompetent cells, including dendritic cells (DCs) and T cells are abundant in plaques. Low-density lipoprotein modified either by oxidation (oxLDL) or by human group X-secreted phospholipase A2 (LDLx) and heat shock proteins (HSP), especially HSP60 and 90, have been implicated in atherosclerosis. We previously reported that Annexin A5 inhibits inflammatory effects of phospholipids, decreases vascular inflammation and improves vascular function in apolipoprotein E−/− mice. Here, we focus on the LDLx effects on human DCs and T cells. Approach and Results—Human DCs were differentiated from peripheral blood monocytes, stimulated by oxLDL or LDLx. Naive autologous T cells were cocultured with pretreated DCs. oxLDL and LDLx, in contrast to LDL, induced DC-activation and T-cell proliferation. T cells exposed to LDLx-treated DCs produced interferon-&ggr;, interleukin (IL)-17 but not IL-4 and IL-10. Annexin A5 abrogated LDLx effects on DCs and T cells and increased production of transforming growth factor-&bgr; and IL-10. Furthermore, IL-10 producing T cells suppressed primary T-cell activation via soluble IL-10, transforming growth factor-&bgr;, and cell–cell contact. Lentiviral-mediated shRNA knock-down HSP60 and 90 in DCs attenuated the effect of LDLx on DCs and subsequent T-cell proliferation. Experiments on DC and T cells derived from carotid atherosclerotic plaques gave similar results. Conclusions—Our data show that modified forms of LDL such as LDLx but not native LDL activate human T cells through DCs. HSP60 and 90 contribute to such T-cell activation. Annexin A5 promotes induction of regulatory T cells and is potentially interesting as a therapeutic agent.

IgM antibodies against malondialdehyde and phosphorylcholine are together strong protection markers for atherosclerosis in systemic lupus erythematosus: Regulation and underlying mechanisms

OBJECTIVES Phosphorylcholine (PC) and malondialdehyde (MDA) are generated during lipid peroxidation and form adducts with proteins as albumin as studied herein. Atherosclerosis and cardiovascular disease (CVD) are increased in systemic lupus erythematosus (SLE). We here investigate the role and regulation of IgM antibodies against PC (anti-PC) and MDA (anti-MDA). METHODS IgM anti-PC and anti-MDA in SLE patients (n=114) were compared with age- and sex-matched population-based controls (n=108). Common carotid intima-media thickness (IMT) and plaque occurrence were determined by B-mode ultrasound. Plaques were graded according to echogenicity (potentially vulnerability). Production of IgM anti-PC and anti-MDA by B cells was determined by ELISA and ELISPOT. The effect of anti-PC and anti-MDA on macrophage uptake of apoptotic cells and oxidative stress was studied by flow cytometry. RESULTS Above 66rd percentile together, IgM anti-PC and anti-MDA were striking protection markers for plaque prevalence and echolucency in SLE (OR: 0.08, CI: 0.01-0.46 and OR: 0.10, CI: 0.01-0.82), respectively, and risk markers for plaque prevalence when below 33rd percentile: OR: 3.79, CI: (1.10-13.00). In vitro, IgM anti-PC and anti-MDA were much higher when B cells were co-cultured with CD3 T cells. Anti-HLA-, anti-CD40 antibody or CD40 silencing abolished these effects. Uptake of apoptotic cells was increased by IgM anti-PC and anti-MDA. MDA induced increased oxidative stress, which was inhibited by IgM anti-MDA. CONCLUSIONS Unexpectedly, both IgM anti-MDA and IgM anti-PC are T-cell dependent and especially together, are strong protection markers for atherosclerosis in SLE. Underlying mechanisms include increased phagocytosis of apoptotic cells and decrease of oxidative stress.

Low levels of antibodies against oxidized but not nonoxidized cardiolipin and phosphatidylserine are associated with atherosclerotic plaques in systemic lupus erythematosus.

Objective. We have reported that the prevalence of atherosclerotic plaques and their echolucency was increased in systemic lupus erythematosus (SLE). We here study antibodies against oxidized cardiolipin (anti-OxCL) and phosphatidylserine (anti-OxPS) in SLE and in relation to atherosclerosis measures. Methods. Patients with SLE (n = 114) were compared with age- and sex-matched population-based controls (n = 122). Common carotid intima-media thickness and plaque occurrence were determined by B-mode ultrasonography. Plaques were graded according to echogenicity as 1–4, with 1 being echolucent. Antibodies were determined by ELISA. Results. In the SLE group, the prevalence of low IgM anti-OxPS and low total IgM levels (below 33rd percentile) was increased compared to controls (p = 0.045 and p = 0.0079, respectively). Among SLE patients with atherosclerotic plaques, the prevalence of low IgM anti-OxPS (p = 0.0019) and anti-OxCL (p = 0.031) was increased. Only IgM anti-OxPS remained significant (p = 0.019) after adjusting for other significant factors. Echolucent plaques (total, or left side) were more prevalent among SLE patients with low IgM anti-OxCL and anti-OxPS when controlled for other significant factors (p < 0.05). Low total IgM was independently associated with echolucent plaque on left side (p < 0.05), but not other atherosclerosis measures. IgM anticardiolipin antibodies (aCL) and antiphosphatidylserine antibodies (anti-PS) were higher among SLE patients with cardiopulmonary disease, including arterial, valvular, and venous disease (p < 0.05). There were no associations between antibodies and other disease manifestations or activity. Both anti-OxCL and anti-OxPS, in contrast to aCL, and anti-PS, were cofactor−β2-glycoprotein I (β2-GPI)-independent. Conclusion. The prevalence of low levels of IgM anti-OxCL and anti-OxPS (both cofactor-β2-GPI-independent) is associated with the presence of plaques and echolucent plaques in SLE.

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.

Antibodies against native and oxidized cardiolipin and phosphatidylserine and phosphorylcholine in atherosclerosis development.

Background Antibodies against cardiolipin and phosphatidylserine (anti-CL and anti-PS) are associated with thrombosis. In contrast, we determined that IgM antibodies against oxidized CL and PS (OxCL and OxPS) and phosphorylcholine (anti-PC) could be protection markers for cardiovascular disease (CVD). Methods 226 individuals with established hypertension (diastolic pressure>95 mmHg) from the European Lacidipine Study on Atherosclerosis. Antibodies were tested by ELISA. As a surrogate measure of atherosclerosis, the mean of the maximum intima-media thicknesses (IMT) in the far walls of common carotids and bifurcations was determined by ultrasonography at the time of inclusion and 4 years following inclusion. Results Increases in IMT measures at follow-up were significantly less common in subjects which at baseline had high IgM anti-OxPS and anti-PC at above 75th percentile: OR 0,45, CI (0,23–0,86) and OR 0.37, CI (0,19–0,71), p = 0.0137 respectively and above 90th percentile: OR 0.32, CI (0,12–0,84) and OR 0.39, CI (0,15–1.00), p = 0.050 and OR 0,22, CI (0,08–0,59) p = 0,0029. IgM anti-OxCL was negatively associated with IMT increases (OR, 0.32, CI (0,12–0,84), p = 0231). There were no associations for IgM anti-PS or anti-CL. Anti-PC, as determined herein by a commercial ELISA, was strongly associated with data from our previously published in house ELISA (R = 0,87; p<0,0001).) Anti-PC was also a risk marker at low levels (below 25th percentile; OR = 2,37 (1,16–4,82), p = 0,0177). Conclusions High levels of IgM anti-OxPS and anti-OxCL, but not traditional anti-phospholipid antibodies (anti-PS and anti-CL), are associated with protection against atherosclerosis development. In addition, low IgM anti-PC was a risk marker but high a protection marker.

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.

IgM-Antibodies against Phosphorylcholine in Mothers and Normal or Low Birth Weight Term Newborn Infants

Objective To determine levels of athero-protective IgM antibodies against phosphorylcholine in mothers and term-born normal or low birth weight infants. Approach Twenty three mother-infant pairs were studied, of whom 16 infants were within the normal weight range for gestational age (NGA; 3652[504] g) and 7 were small for gestational age (SGA; birth weight: 2715[255] g), the latter <2SD below the Swedish reference data mean for normal fetal growth. All infants were born at term (mean±SD 40.5±1.1 weeks). Serum was available from 6 mothers with SGA and 14 with NGA infants. Participating mothers were aged 34.0±3.9 years (no difference between groups). Fourteen neonates were boys and seven were girls. Levels of anti-PC IgM were determined by ELISA. Results Neonatal IgM anti-PC levels were low (undetectable in 8 infants out of which 3 were SGA) with a median of 76[range 0–2.51] U/ml. Maternal IgM anti-PC levels were significantly higher (median 7198[range: 25.32–656.0]) U/ml) and the proportion of mothers in highest quartile (>75th percentile) was larger in mothers of NGA-infants (43%) vs. those of SGA-infants (0%, p = 0.032). Conclusions IgM anti-PC levels are low at birth, which suggests that these antibodies do not play a “housekeeping” role in immune function during fetal life/development, but arise predominately on exposure to external antigens after birth. Furthermore, low maternal IgM anti-PC levels may play a role in placental insufficiency, contributing to poor fetal growth and a small-for-date baby. This preliminary observation may have implications for the future risk of atherosclerosis/cardiovascular disease development in pregnant women and their offspring.

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.