Gunilla Nordin Fredrikson

Supplementation With Conjugated Linoleic Acid Causes Isomer-Dependent Oxidative Stress and Elevated C-Reactive Protein: A Potential Link to Fatty Acid-Induced Insulin Resistance.

Background—Conjugated linoleic acids (CLAs), a group of fatty acids shown to have beneficial effects in animals, are also used as weight loss supplements. Recently, we reported that the t 10 c 12 CLA-isomer caused insulin resistance in abdominally obese men via unknown mechanisms. The aim of the present study was to examine whether CLA has isomer-specific effects on oxidative stress or inflammatory biomarkers and to investigate the relationship between these factors and induced insulin resistance. Methods and Results—In a double-blind placebo-controlled trial, 60 men with metabolic syndrome were randomized to one of 3 groups receiving t 10 c 12 CLA, a CLA mixture, or placebo for 12 weeks. Insulin sensitivity (euglycemic clamp), serum lipids, in vivo lipid peroxidation (determined as urinary 8-iso-PGF2&agr; [F2-isoprostanes]), 15-ketodihydro PGF2&agr;, plasma vitamin E, plasma C-reactive protein, tumor necrosis factor-&agr;, and interleukin-6 were assessed before and after treatment. Supplementation with t 10 c 12 CLA markedly increased 8-iso-PGF2&agr; (578%) and C-reactive protein (110%) compared with placebo (P <0.0001 and P <0.01, respectively) and independent of changes in hyperglycemia or dyslipidemia. The increases in 8-iso-PGF2&agr;, but not in C-reactive protein, were significantly and independently related to aggravated insulin resistance. Oxidative stress was related to increased vitamin E levels, suggesting a compensatory mechanism. Conclusions—t 10 c 12 CLA supplementation increases oxidative stress and inflammatory biomarkers in obese men. The oxidative stress seems closely related to induced insulin resistance, suggesting a link between the fatty acid-induced lipid peroxidation seen in the present study and insulin resistance. These unfavorable effects of t 10 c 12 CLA might be of clinical importance with regard to cardiovascular disease, in consideration of the widespread use of dietary supplements containing this fatty acid.

Inhibition of Atherosclerosis in ApoE-Null Mice by Immunization with ApoB-100 Peptide Sequences

Objective—LDL oxidation is believed to play an important role in the development of atherosclerosis, and oxidized LDL particles have been shown to become targets for the immune system. Immunization of animals with oxidized LDL results in reduction of atherosclerosis, suggesting an atheroprotective effect of this immune response. Methods and Results—Using a polypeptide library covering the complete sequence of apoB-100, a large number of native and malondialdehyde-modified peptide sequences in apoB-100 that are recognized by antibodies in human plasma were identified. We report here that immunization with apoB-100 peptide sequences, against which high levels of IgG and IgM antibodies are present in healthy human controls, reduce atherosclerosis in apoE-null mice by about 60%. Immunizations with these peptides were also found to increase the collagen content of subvalvular lesions. Conclusions—These studies have identified peptide sequences in apoB-100 that induce immune responses, which inhibits atherosclerosis. This suggests a way of developing an immunization therapy for coronary heart disease.

Recombinant human antibodies against aldehyde-modified apolipoprotein B-100 peptide sequences inhibit atherosclerosis

Background—Accumulation and oxidation of LDL are believed to be important initiating factors in atherosclerosis. Oxidized LDL is recognized by the immune system, and animal studies have suggested that these immune responses have a protective effect against atherosclerosis. Aldehyde-modified peptide sequences in apolipoprotein B-100 (apoB-100) are major targets for these immune responses. Methods and Results—Human IgG1 antibodies against 2 malondialdehyde (MDA)-modified apoB-100 peptide sequences were produced through screening of a single-chain antibody-fragment library and subsequent cloning into a pcDNA3 vector. Three weekly doses of these antibodies were injected into male apoE−/− mice. Phosphate-buffered saline and human IgG1 antibodies against fluorescein isothiocyanate were used as controls. One of the IgG1 antibodies significantly and dose-dependently reduced the extent of atherosclerosis as well as the plaque content of oxidized LDL epitopes and macrophages. In cell culture studies, human monocytes were incubated with native LDL or oxidized LDL, in the presence of antibodies. The same antibody induced an increase in monocyte binding and uptake of oxidized LDL. Conclusions—These findings suggest that antibodies are important mediators of atheroprotective immune responses directed to oxidized LDL. Thus, passive immunization against MDA-modified apoB-100 peptide sequences may represent a novel therapeutic approach for prevention and treatment of cardiovascular disease.

Identification of Immune Responses Against Aldehyde-Modified Peptide Sequences in ApoB Associated With Cardiovascular Disease

Atherosclerosis develops as a result of a chronic arterial inflammation and intimal fibrosis. The disease represents in many respects a vascular repair process activated in response to injury caused by toxic breakdown products of aggregated and oxidized lipoproteins. The initial response of the artery involves expression of adhesion molecules and recruitment of leukocytes. Degenerated lipoproteins are removed from the extracellular space by macrophages. If lipoproteins continue to accumulate, the inflammatory process becomes chronic and cytokines stimulate smooth muscle to migrate into the intima. These cells proliferate and form an atherosclerotic plaque. Plaque cell death and inflammation in response to oxidized lipids and other toxic factors may cause plaques to rupture.Objective—Atherosclerosis is associated with an immune response against oxidized LDL, which modulates the progression of the disease process. Methods and Results—Using a library of polypeptides covering the complete sequence of apoB-100, the only major protein of LDL, we have identified over 100 different human antibodies reacting against aldehyde-modified apoB-100 sequences. IgM antibody titer levels decreased with age and were associated with the intima-media thickness of the carotid artery in subjects younger than 60 years. There were also inverse associations between IgM levels and oxidized LDL in plasma. In prospective clinical studies, antibody levels against several aldehyde-modified apoB-100 sites were associated with cardiovascular disease in this age group. Whether this immune response is adaptive (protective) or maladaptive (causal) in atherosclerosis requires further investigation. Conclusions—We have characterized a large number of epitopes within the apoB-100 component of oxidized LDL that provoke an immune response in humans. These findings will make it possible to study the role of immune responses against specific sites in oxidized LDL and to determine whether these immune responses influence the risk for future cardiac events.

Intranasal Immunization With an Apolipoprotein B-100 Fusion Protein Induces Antigen-Specific Regulatory T Cells and Reduces Atherosclerosis

Objective—Atherosclerosis is an inflammatory disease. Autoimmune responses to low-density lipoproteins (LDL) contribute to its progression, whereas immunization with LDL may induce atheroprotective or proatherogenic responses. The objective of this study was to develop an atheroprotective vaccine by targeting a peptide of the LDL protein constituent apolipoprotein B-100 (apoB-100) to the nasal mucosa to induce a protective mucosal immune response. Methods and Results—A peptide comprising amino acids 3136 to 3155 of apoB-100 (p210) was fused to the B subunit of cholera toxin (CTB), which binds to a ganglioside on mucosal epithelia. The effect of nasal administration of the p210-CTB fusion protein on atherogenesis was compared with that of an ovalbumin peptide fused to CTB and with untreated controls. Immunization with p210-CTB for 12 weeks caused a 35% reduction in aortic lesion size in Apoe−/− mice. This effect was accompanied by induction of regulatory T cells that markedly suppressed effector T cells rechallenged with apoB-100 and increased numbers of interleukin (IL)-10+ CD4+ T cells. Furthermore, a peptide-specific antibody response was observed. Atheroprotection was also documented in apoe−/− mice lacking functional transforming growth factor-&bgr; receptors on T cells. Conclusion—Nasal administration of an apoB-100 peptide fused to CTB attenuates atherosclerosis and induces regulatory Tr1 cells that inhibit T effector responses to apoB-100.

Elevated CD14++CD16- Monocytes Predict Cardiovascular Events

Background— Although monocytes in peripheral blood are no longer considered to be a homogeneous population, associations between distinct monocyte subsets and cardiovascular disease have not been highlighted in large epidemiological studies. Methods and Results— The study included 700 randomly selected subjects from the cardiovascular arm of the Malmö Diet and Cancer study. Among these, 123 subjects experienced ischemic cardiovascular events during the follow-up until December 2008. Mononuclear leukocytes frozen at the baseline investigation in 1991 to 1994 were thawed and analyzed with flow cytometry to enumerate monocyte subsets, based on CD14 and CD16 expression. The percentage and number of classical CD14++CD16− monocytes were increased in the cardiovascular-event group compared with the event-free subjects (median, 69% [interquartile range, 62% to 76%] versus 67% [59% to 72%], P=0.017; 344 [251 to 419] cells/&mgr;L versus 297 [212 to 384] cells/&mgr;L, P=0.003). The hazard ratio was 1.66 for suffering a cardiovascular event in the highest tertile of the number of CD14++CD16− monocytes compared with the lowest tertile, even after adjustment for common risk factors (HR, 1.66; 95% CI: 1.02 to 2.72). CD14++CD16− monocytes did not, however, associate with the extent of atherosclerosis at baseline. In contrast, the percentage of monocytes expressing CD16 was negatively associated to the extent of carotid atherosclerosis measured as intima-media thickness at baseline. The chemokine receptors CCR2, CX3CR1, and CCR5 were not differentially expressed between cases and controls on any of the monocyte subsets, but CCR5 expression on CD14+CD16++ monocytes was negatively associated to carotid intima-media thickness. Conclusions— This study shows that classical CD14++CD16− monocytes can predict future cardiovascular risk independently of other risk factors in a randomly selected population.

Atheroprotective immunization with MDA-modified apo B-100 peptide sequences is associated with activation of Th2 specific antibody expression.

Objective: The objective of this study was to evaluate if immunization with MDA-modified human apo B-100 fragments is associated with a shift in the Th1/Th2 balance. Methods and Results: Apo E deficient mice were immunized with one of the peptides (P45; amino acids 688–707, P74; amino acids 1123–1142 or P240; amino acids 3613–3632) at 6, 9 and 11 weeks of age and compared to controls given carrier alone. Immunization with P45 and P74 reduced atherosclerosis in the aorta of 25-week-old mice by 48% (p=0.02) and 31% (p=0.06) and macrophage content in atherosclerotic plaques by 33% (p=0.02) and 39% (p=0.02), respectively. The levels of Th2-specific IgG1 against each peptide increased more than 50-fold in response to immunization, whereas the levels of specific IgM and Th1-associated IgG2a were only marginally affected. However, there was an increase in the plaque expression of both Th1 and Th2 cytokines as assessed by real time PCR. Immunization with P240, a non-homologous peptide used as control, induced a 10-fold increase of specific IgG1 but did not influence atherosclerosis or plaque content. Conclusions: Immunization with MDA apo B-100 fragments induce a shift from Th1 to a Th2 specific oxidized LDL antibody expression, but without a concomitant downregulation of plaque IFN-γ expression.

Regulatory T-Cell Response to Apolipoprotein B100–Derived Peptides Reduces the Development and Progression of Atherosclerosis in Mice

Objective—The immunoinflammatory response plays a critical role in the development and progression of atherosclerosis. Recent studies suggested an important role for regulatory T (Treg) cells in the inhibition of disease-related vascular inflammation. We hypothesized that induction of a specific Treg cell response to atherosclerosis-relevant antigens would be an attractive strategy to limit the development and progression of atherosclerosis through the promotion of immune tolerance. Methods and Results—Young or old Apoe−/− mice were subcutaneously infused for 2 weeks with either a control ovalbumin (OVA) peptide or with apolipoprotein B100 (ApoB100)–derived peptides without adjuvant. Atherosclerosis development, progression and immunologic status were assessed at 8 weeks after the end of the infusion. Treatment with ApoB100 peptides led to significant reduction of lesion development in young Apoe−/− mice (P=0.001 versus OVA group) and abrogated atherosclerosis progression in old Apoe−/− mice with already established lesions (0% progression in ApoB100 versus 17% in OVA group, P<0.005). Limitation of plaque progression was associated with reduced vascular inflammation and increased collagen content, indicative of plaque stabilization. Infusion of ApoB100 peptides did not alter antibody production but promoted a specific Treg cell response, which was associated with a reduction of both T helper type 1–related and T helper type 2–related cytokines. Interestingly, depletion of CD4+CD25+ Treg cells abrogated ApoB100 peptides-dependent immune modulation and atheroprotection. Conclusion—Subcutaneous infusion of adjuvant-free ApoB100-derived peptides to Apoe−/− mice reduces atherosclerosis through the induction of a specific Treg cell response.

Treatment with apo B peptide vaccines inhibits atherosclerosis in human apo B-100 transgenic mice without inducing an increase in peptide-specific antibodies

Objectives.  Autoantibodies to apolipoprotein (apo) B‐100 peptides are present in human plasma and have been shown to be associated with decreased cardiovascular risk. The present study aimed to determine if apo B‐100 peptide vaccines are atheroprotective in mice expressing human apo B‐100 and if the effectiveness of the vaccines is influenced by the level of pre‐existing peptide‐specific autoantibodies.

Evidence for a role of regulatory T cells in mediating the atheroprotective effect of apolipoprotein B peptide vaccine

Abstract.  Wigren M, Kolbus D, Dunér P, Ljungcrantz I, Söderberg I, Björkbacka H, Fredrikson GN, Nilsson J. (Malmö University Hospital, Lund University; Malmö University, Malmo; Sweden) Evidence for a role of regulatory T cells in mediating the atheroprotective effect of apolipoprotein B peptide vaccine. J Intern Med 2011; 269: 546–556.