Immune responses against oxidized LDL as possible targets for prevention of atherosclerosis in systemic lupus erythematosus.

Patients suffering from systemic lupus erythematosus (SLE) are at increased risk of developing cardiovascular disease (CVD) and traditional therapies including statins provide insufficient protection. Impaired removal of apoptotic material is a common pathogenic mechanism in both SLE and atherosclerosis and is considered to be a key factor in the development of autoimmunity. Since oxidized LDL and apoptotic material bind to the same receptors, we aimed to investigate if targeting the oxidized LDL autoimmunity can affect atherosclerosis in SLE. To investigate the possible role of oxidized LDL autoimmunity in the accelerated atherosclerosis associated with SLE we used a hypercholesterolemic SLE mouse model (B6.lpr.ApoE-/- mice). Promoting LDL tolerance through mucosal immunization with an apolipoprotein B-100 peptide p45 (amino acids 661-680) and cholera toxin B-subunit fusion protein increased regulatory T cells and B cells in mesenteric lymph nodes and reduced plaque development in the aorta by 33%. Treatment with the oxidized LDL-specific antibody Orticumab reduced aortic atherosclerosis by 43%, subvalvular plaque area by 50% and the macrophage content by 31%. The present study provides support for oxLDL as a possible target for prevention of cardiovascular complications in SLE.

ILC2 transfers to apolipoprotein E deficient mice reduce the lipid content of atherosclerotic lesions.

BACKGROUND: Expansion of type 2 innate lymphoid cells (ILC2s) in hypercholesterolaemic mice protects against atherosclerosis while different ILC2 subsets have been described (natural, inflammatory) based on their suppression of tumorigenicity 2 (ST2) and killer-cell lectin like receptor G1 (KLRG1) expression. The aim of the current study is to characterize the interleukin 25 (IL25)-induced splenic ILC2 population (Lin-CD45+IL17RB+ICOS+IL7raintermediate) and address its direct role in experimental atherosclerosis by its adoptive transfer to hypercholesterolaemic apolipoprotein E deficient (apoE-/-) mice. RESULTS: Immunomagnetically enriched, FACS-sorted ILC2s from the spleens of IL-25 treated apoE-/- mice were stained for KLRG1 and ST2 directly upon cell obtainment or in vitro cell expansion for flow cytometric analysis. IL25-induced splenic ILC2s express high levels of both KLRG1 and ST2. However, both markers are downregulated upon in vitro cell expansion. In vitro expanded splenic ILC2s were intraperitoneally transferred to apoE-/- recipients on high fat diet. ApoE-/- mice that received in vitro expanded splenic ILC2s had decreased lipid content in subvalvular heart and brachiocephalic artery (BCA) plaques accompanied by increased peritoneal B1 cells, activated eosinophils and alternatively activated macrophages (AAMs) as well as anti-phosphorylcholine (PC) immunoglobulin (Ig) M in plasma. CONCLUSIONS: With the current data we designate the IL25-induced ILC2 population to decrease the lipid content of atherosclerotic lesions in apoE-/- mice and we directly link the induction of B1 cells and the atheroprotective anti-PC IgM antibodies with ILC2s.

Lack of Ability to Present Antigens on Major Histocompatibility Complex Class II Molecules Aggravates Atherosclerosis in ApoE-/- Mice.

BACKGROUND: Hypercholesterolemic mice lacking factors required for activation of CD4+ T cells are characterized by reduced development of atherosclerosis. Consequently, it has been assumed that atherosclerosis involves loss of tolerance against modified self-antigens generated in response to hypercholesterolemia and that presentation of such antigens on major histocompatibility complex class II (MHCII) leads to activation of proatherogenic Th1 cells. In this study, we wanted to determine the role of antigen presentation on MHCII in atherosclerosis development. METHODS: Apolipoprotein E (ApoE-/-) mice deficient for MHCII (ApoE-/-MHCII-/-) were used to study the role of MHCII in atherosclerosis development. RESULTS: Compared with ApoE-/- mice, ApoE-/-MHCII-/- mice had reduced levels of CD4+ T cells, immunoglobulin G and M levels, and Th1 and Th2 cytokines in plasma. CD8+ T cells were increased and regulatory T cells were reduced both in spleen and in lesions of ApoE-/-MHCII-/- mice. Decreased plasma levels of inflammatory cytokines in ApoE-/-MHCII-/- mice indicated reduced systemic inflammation. Despite this, ApoE-/-MHCII-/- mice had significantly more atherosclerosis as assessed by en face Oil Red O staining of the aorta (4.7±2.9% versus 1.9±1.3%; P<0.01) and cross-sectional area of subvalvular lesions (7.7±2.2×105 µm2 versus 4.6±2.8×105 µm2; P<0.05). Cell transfer and blocking antibody studies suggested that loss of regulatory T cells is the most important cause of aggravated atherosclerosis in ApoE-/-MHCII-/- mice. CONCLUSIONS: Our observations demonstrate that antigen presentation on MHCII has important protective functions in atherosclerosis and that this is primarily the result of activation of regulatory T cells. These findings have implications for understanding the possible risks and benefits of immunosuppressive therapy in patients with cardiovascular disease.

Time-resolved small-angle neutron scattering as a probe for the dynamics of lipid exchange between human lipoproteins and naturally derived membranes.

Atherosclerosis is the main killer in the western world. Today's clinical markers include the total level of cholesterol and high-/low-density lipoproteins, which often fails to accurately predict the disease. The relationship between the lipid exchange capacity and lipoprotein structure should explain the extent by which they release or accept lipid cargo and should relate to the risk for developing atherosclerosis. Here, small-angle neutron scattering and tailored deuteration have been used to follow the molecular lipid exchange between human lipoprotein particles and cellular membrane mimics made of natural, "neutron invisible" phosphatidylcholines. We show that lipid exchange occurs via two different processes that include lipid transfer via collision and upon direct particle tethering to the membrane, and that high-density lipoprotein excels at exchanging the human-like unsaturated phosphatidylcholine. By mapping the specific lipid content and level of glycation/oxidation, the mode of action of specific lipoproteins can now be deciphered. This information can prove important for the development of improved diagnostic tools and in the treatment of atherosclerosis.

High Levels of Soluble Lectinlike Oxidized Low-Density Lipoprotein Receptor-1 Are Associated With Carotid Plaque Inflammation and Increased Risk of Ischemic Stroke.

Background When the lectinlike oxidized low-density lipoprotein (ox LDL) receptor-1 ( LOX -1), a scavenger receptor for ox LDL , binds ox LDL , processes leading to endothelial dysfunction and inflammation are promoted. We aimed to study release mechanisms of LOX -1 and how circulating levels of soluble LOX -1 ( sLOX -1) relate to plaque inflammation and future risk for ischemic stroke. Methods and Results Endothelial cells and leukocytes were used to study release of sLOX -1. Plasma levels of sLOX -1 were determined in 4703 participants in the Malmö Diet and Cancer cohort. Incidence of ischemic stroke was monitored. For 202 patients undergoing carotid endarterectomy, levels of sLOX -1 were analyzed in plasma and plaque homogenates and related to plaque inflammation factors. Endothelial cells released sLOX -1 when exposed to ox LDL . A total of 257 subjects experienced stroke during a mean follow-up of 16.5 years. Subjects in the highest tertile of sLOX -1 had a stroke hazard ratio of 1.75 (95% CI, 1.28-2.39) compared with those in the lowest tertile after adjusting for age and sex. The patients undergoing carotid endarterectomy had a significant association between plasma sLOX -1 and the plaque content of sLOX -1 ( r=0.209, P=0.004). Plaques with high levels of sLOX -1 had more ox LDL , proinflammatory cytokines, and matrix metalloproteinases. Conclusions Our findings demonstrate that ox LDL induces the release of sLOX -1 from endothelial cells and that circulating levels of sLOX -1 correlate with carotid plaque inflammation and risk for ischemic stroke. These observations provide clinical support to experimental studies implicating LOX -1 in atherosclerosis and its possible role as target for cardiovascular intervention.

Interleukin-25 reduces Th17 cells and inflammatory responses in human peripheral blood mononuclear cells.

BACKGROUND: The absence of interleukin-25 (IL-25) favors the induction of Th1 and Th17 immune responses in mice. Th1 immune responses have been associated with the pathology of atherosclerosis, a lipid and inflammation driven disease of the arterial wall. PURPOSE OF RESEARCH: To evaluate the effect of IL-25 on human peripheral blood mononuclear cells (hPBMCs) in the presence and absence of oxidized low density lipoprotein (oxLDL), a key player in atherosclerosis development. PRINCIPAL RESULTS: Human PBMCs were incubated with recombinant human IL-25 (rhIL-25) in the presence and absence of oxLDL and analyzed with flow cytometry while cytokine secretion was measured in cell culture supernatants. The IL-25 receptor, IL-17RB, was mostly expressed on T cells. Incubation of hPBMCs with IL-25 reduced the frequency of Th17 cells. Furthermore, IL-25 inhibited the release of the Th17-inducing cytokine IL-6 from dendritic cells isolated from hPBMCs indicating that the IL-25 mediated Th17 suppression may be indirect. Moreover, IL-25 reduced the secretion of the proinflammatory cytokine IFNγ from hPBMCs. OxLDL decreased IFNγ release from hPBMCs regardless of the presence or absence of IL-25. CONCLUSIONS: IL-25 reduces Th1 and Th17 immune responses in hPBMCs raising the interesting possibility that IL-25 could have a protective role in human atherosclerosis.

Interleukin-25 (IL-25) has a protective role in atherosclerosis development in the aortic arch in mice.

Atherosclerosis is a chronic inflammatory disease characterized by the entrapment of apolipoprotein B-containing lipoproteins in the arterial intima, leading to local inflammation. T helper (Th) cell 1-mediated immune responses have been associated with atherosclerosis, and the cytokine interleukin-25 (IL-25 or IL-17E) has been reported to potentially regulate Th1 cell- and Th17 cell-related immune responses. In this study, we evaluated the effects of complete IL-25 deficiency or of a temporal IL-25 blockade on atherosclerosis development in apolipoprotein E-deficient (Apoe-/-) mice. Mice deficient in both apolipoprotein E and IL-25 (Apoe-/-/IL-25-/-) had more Th1 cells in the spleen, along with elevated plasma levels of IL-17 and an increased release of splenic interferon-γ (INF-γ). In support of this observation, a 4-week-long treatment of young Apoe-/- mice (at 10-14 weeks of age) with an IL-25-blocking antibody increased the release of Th1/Th17-associated cytokines in the spleen. In both mouse models, these findings were associated with increased atherosclerotic plaque formation in the aortic arch. We conclude that complete IL-25 deficiency and a temporal IL-25 blockade during early plaque development aggravate atherosclerosis development in the aortic arch of Apoe-/- mice, accompanied by an increase in Th1/Th17-mediated immune responses. Our finding that endogenous IL-25 has an atheroprotective role in the murine aortic arch has potential implications for atherosclerosis development and management in humans.

Cardiovascular disease in systemic lupus erythematosus is associated with increased levels of biomarkers reflecting receptor-activated apoptosis.

BACKGROUND AND AIMS: There is convincing evidence that adaptive immune responses affect the development of atherosclerosis and thrombosis and several autoimmune diseases are associated with increased cardiovascular risk. However, our understanding of the underlying mechanisms remains limited. We investigated how biomarkers reflecting four aspects of autoimmunity: apoptosis, inflammation, tissue degradation and repair, associate with cardiovascular disease (CVD) in subjects with systemic lupus erythematosus (SLE). METHODS: We investigated 484 well-characterized SLE patients, 69 of whom had CVD (coronary artery disease, cerebrovascular disease or peripheral artery disease), and 253 controls. Occurrence of carotid plaques was investigated with ultrasound. Plasma levels of biomarkers reflecting apoptosis (Fas, TNF receptor 1, TRAIL receptor 2), inflammation (IL-6, IL-8, monocyte chemotactic protein-1), tissue degradation (matrix metalloproteinase (MMP)-1, MMP-3, MMP-7), and tissue repair (platelet-derived growth factor, epidermal growth factor and stem cell factor) were analyzed by Proximity Extension Assay. RESULTS: Subjects with SLE had markedly elevated plasma levels of biomarkers reflecting apoptosis, inflammation and tissue degradation as compared to controls. SLE patients with CVD had higher levels of Fas, TNF receptor 1, TRAIL receptor 2, MMP-1 and -7 than those without CVD. The same associations were found for the presence of a carotid plaque. When controlling for the factors included in the Framingham risk score, all biomarkers except MMP-1 remained associated with the presence of a carotid plaque, while only TRAIL receptor 2 levels remained significantly associated with CVD. CONCLUSIONS: Our findings argue that the cardiovascular risk in SLE is associated with increased cell death by apoptosis and tissue degradation.

FADD, Caspase-3, and Caspase-8 and Incidence of Coronary Events.

OBJECTIVE: To investigate the relationship between 3 markers of apoptosis, that is, FADD (Fas-associated death domain-containing protein), caspase-3, and caspase-8, and incidence of coronary events (CEs) in a population-based cohort study. APPROACH AND RESULTS: In vitro experiments were performed to assess the response of the apoptotic biomarkers after Fas stimulation of peripheral blood mononuclear cells. The experiments showed significantly increased releases of FADD, caspase-3, and caspase-8 after Fas stimulation. The relationship between FADD, caspase-3, and caspase-8, respectively, and incidence of CEs was studied in 4284 subjects from the population-based Malmö Diet and Cancer Study. Cox' proportional hazards regression was used to examine the association between the apoptotic biomarkers and incidence of CE over a mean follow-up of 19 years. A total of 381 individuals had CE during the follow-up. High FADD at baseline was significantly associated with incident CE. In the highest compared with the lowest quartile of FADD, the risk factor adjusted hazards ratio for CE was 1.82 (95% confidence interval, 1.35-2.46; P for trend <0.001). A significant association was also found between caspase-8 and CE; the hazards ratio (Q4 versus Q1) was 1.90 (95% confidence interval, 1.39-2.60; P for trend <0.001) after adjustment for risk factors. No association was found between caspase-3 and CEs. CONCLUSIONS: High levels of FADD and caspase-8, but not caspase-3, were associated with increased incidence of CE in subjects from the general population. The in vitro experiments support the view that these biomarkers could reflect activation of the extrinsic apoptotic pathway.

oxLDL antibody inhibits MCP-1 release in monocytes/macrophages by regulating Ca2+ /K+ channel flow.

oxLDL peptide vaccine and its antibody adoptive transferring have shown a significantly preventive or therapeutic effect in atherosclerotic animal model. The molecular mechanism behind this is obscure. Here, we report that oxLDL induces MCP-1 release in monocytes/macrophages through their TLR-4 (Toll-like receptor 4) and ERK MAPK pathway and is calcium/potassium channel-dependent. Using blocking antibodies against CD36, TLR-4, SR-AI and LOX-1, only TLR-4 antibody was found to have an inhibitory effect and ERK MAPK-specific inhibitor (PD98059) was found to have a dramatic inhibitory effect compared to inhibitors of other MAPK group members (p38 and JNK MAPKs) on oxLDL-induced MCP-1 release. The release of cytokines and chemokines needs influx of extracellular calcium and imbalance of efflux of potassium. Nifedipine, a voltage-dependent calcium channel (VDCC) inhibitor, and glyburide, an ATP-regulated potassium channel (K+ATP ) inhibitor, inhibit oxLDL-induced MCP-1 release. Potassium efflux and influx counterbalance maintains the negative potential of macrophages to open calcium channels, and our results suggest that oxLDL actually induces the closing of potassium influx channel - inward rectifier channel (Kir ) and ensuing the opening of calcium channel. ERK MAPK inhibitor PD98059 inhibits oxLDL-induced Ca2+ /Kir channel alterations. The interfering of oxLDL-induced MCP-1 release by its monoclonal antibody is through its FcγRIIB (CD32). Using blocking antibodies against FcγRI (CD64), FcγRIIB (CD32) and FcγRIII (CD16), only CD32 blocking antibody was found to reverse the inhibitory effect of oxLDL antibody on oxLDL-induced MCP-1 release. Interestingly, oxLDL antibody specifically inhibits oxLDL-induced ERK MAPK activation and ensuing Ca2+ /Kir channel alterations, and MCP-1 release. Thus, we found a molecular mechanism of oxLDL antibody on inhibition of oxLDL-induced ERK MAPK pathway and consequent MCP-1 release.

Association between renin and atherosclerotic burden in subjects with and without type 2 diabetes.

BACKGROUND: Activation of the renin-angiotensin-aldosterone-system (RAAS) has been proposed to contribute to development of vascular complications in type 2 diabetes (T2D). The aim of the present study was to determine if plasma renin levels are associated with the severity of vascular changes in subjects with and without T2D. METHODS: Renin was analyzed by the Proximity Extension Assay in subjects with (n = 985) and without (n = 515) T2D participating in the SUMMIT (SUrrogate markers for Micro- and Macro-vascular hard endpoints for Innovative diabetes Tools) study and in 205 carotid endarterectomy patients. Vascular changes were assessed by determining ankle-brachial pressure index (ABPI), carotid intima-media thickness (IMT), carotid plaque area, pulse wave velocity (PWV) and the reactivity hyperemia index (RHI). RESULTS: Plasma renin was elevated in subjects with T2D and demonstrated risk factor-independent association with prevalent cardiovascular disease both in subjects with and without T2D. Renin levels increased with age, body mass index, HbA1c and correlated inversely with HDL. Subjects with T2D had more severe carotid disease, increased arterial stiffness, and impaired endothelial function. Risk factor-independent associations between renin and APBI, bulb IMT, carotid plaque area were observed in both T2D and non-T2D subjects. These associations were independent of treatment with RAAS inhibitors. Only weak associations existed between plasma renin and the expression of pro-inflammatory and fibrous components in plaques from 205 endarterectomy patients. CONCLUSIONS: Our findings provide clinical evidence for associations between systemic RAAS activation and atherosclerotic burden and suggest that this association is of particular importance in T2D.

Associations Between Macrophage Colony-Stimulating Factor and Monocyte Chemotactic Protein 1 in Plasma and First-Time Coronary Events: A Nested Case-Control Study.

BACKGROUND: Myeloid cells play a central role in atherosclerosis. We investigated the associations between the plasma levels of growth factors and chemokines that regulate myeloid cell homeostasis and function and the risk of first-time acute coronary events in middle-aged persons. METHODS AND RESULTS: We measured baseline plasma levels of macrophage colony-stimulating factor; monocyte chemotactic protein 1; C-C motif chemokine ligands 3, 4, and 20; C-X-C motif chemokine ligands 1, 6, and 16; and C-X3-C motif chemokine ligand 1 in 292 participants who had a coronary event during follow-up and 366 controls matched for age, sex, and time of inclusion who remained event free. Study participants were recruited from the Malmö Diet and Cancer Study population cohort and had no previous history of coronary artery disease. We found a strong independent negative association between macrophage colony-stimulating factor and incident coronary events in a forward stepwise Cox proportional hazards model including all biomarkers alongside the classic Framingham risk factors (age, sex, smoking, total cholesterol, high-density lipoprotein cholesterol, systolic blood pressure), diabetes mellitus, and medication. Conversely, monocyte chemotactic protein 1 had the strongest independent positive association with the outcome. The addition of macrophage colony-stimulating factor and monocyte chemotactic protein 1 significantly improved the predictive ability of a model including traditional risk factors alone (C statistic 0.81 [95% CI 0.78-0.84] versus 0.67 [95% CI 0.63-0.71]; net reclassification index 0.52 [0.42-0.62]; P<0.001). The combined model led to a 54% net downclassification of participants who did not have a coronary event during follow-up and was particularly effective in the intermediate-risk group. CONCLUSIONS: High levels of macrophage colony-stimulating factor and low levels of monocyte chemotactic protein 1 in plasma characterize middle-aged persons at low risk to develop clinically manifested coronary artery disease.

Endarterectomy patients with elevated levels of circulating IL-16 have fewer cardiovascular events during follow-up.

BACKGROUND AND PURPOSE: Increased interleukin 16 (IL-16) levels in carotid plaques have been associated with reduced incidence of cardiovascular (CV) events during follow-up in patients who underwent carotid endarterectomy (CEA). In the present study we aimed to determine whether high circulating levels of IL-16 also are associated with a decreased risk of CV events after CEA. METHODS: Patients, who had their carotid plaques surgically removed (n=473), were followed for a mean follow-up time of 3.1years. Plasma levels of IL-16 the day before surgery were analyzed by proximity extension assay (PEA) and associated with the occurrence of CV events during follow-up (n=98). RESULTS: High levels of circulating IL-16 were independently associated with a decreased risk of CV events when comparing the highest versus the lowest IL-16 tertile (hazard ratio [HR] 0.47; 95% CI 0.27-0.81; P=0.007), as well as with CV deaths (HR 0.25; 95% CI 0.09-0.70; P=0.008). CONCLUSION: These present findings indicate an association between IL-16 and less clinical complications of atherosclerosis in a population with known advanced carotid disease.

Apolipoprotein B-100 Antibody Interaction With Atherosclerotic Plaque Inflammation and Repair Processes.

BACKGROUND AND PURPOSE: Treatment with IgG against the malondialdehyde (MDA)-modified apolipoprotein B-100 epitope p45 reduces atherosclerosis in experimental models. This study investigated the association between p45 IgG autoantibodies and plaque inflammation in subjects with advanced cardiovascular disease. METHODS: Native and MDA-p45 IgG levels were analyzed by ELISA in 349 carotid endarterectomy patients. In a subcohort of 195 subjects, endarterectomy samples were analyzed by immunohistochemistry and ELISA to determine plaque constituents and inflammation. Peripheral blood mononuclear cells were isolated from healthy donors. RESULTS: Patients with preoperative events of neurological ischemia had lower levels of native p45 IgG. Low levels of MDA-p45 IgG were associated with increased risk of postoperative cardiovascular death during a mean follow-up of 54 months. High plasma levels of native p45 IgG were associated with increased plaque content of collagen and smooth muscle cell growth factors, as well as with lower levels of proinflammatory cytokines. Exposure of peripheral blood mononuclear cells from healthy donors to recombinant MDA-p45 IgG in presence of oxidized low-density lipoprotein reduced the expression of tumor necrosis factor-α and stimulated release of smooth muscle cell growth factors. CONCLUSIONS: This study confirms previous experimental findings of anti-inflammatory properties of apolipoprotein B-100 p45 antibodies and provides the first clinical evidence of associations between p45 IgG autoantibody levels and atherosclerotic plaque inflammation, plaque repair as well as prevalent and incident cardiovascular events in carotid endarterectomy patients. These findings suggest the possibility that treatment with anti-p45 antibodies may have beneficial effects in advanced cardiovascular disease.

Low Levels of Apolipoprotein B-100 Autoantibodies Are Associated With Increased Risk of Coronary Events.

OBJECTIVE: Previous smaller studies have indicated inverse associations between autoantibodies to oxidized low-density lipoprotein epitopes, and cardiovascular disease. The present study investigated associations between autoantibodies against the apolipoprotein B-100 peptides p45 and p210, respectively, and risk of incident cardiovascular disease in a large population-based cohort. APPROACH AND RESULTS: Apolipoprotein B-100 autoantibodies were analyzed by ELISA in a prospective study, including 5393 individuals (aged 46-68 years) belonging to the cardiovascular arm of the Malmö Diet and Cancer study with a follow-up time of >15 years. Subjects that suffered an acute coronary event during follow-up (n=382) had lower levels at baseline of IgM autoantibodies recognizing the native and malondialdehyde-modified apolipoprotein B-100 peptides p45 and p210 and also lower IgG levels recognizing native p210, whereas no association was found with risk for stroke (n=317). Subjects in the highest compared with lowest tertile of IgM-p45MDA (hazard ratio [95% confidence interval]: 0.72 [0.55, 0.94]; P=0.017) and IgG-p210native (hazard ratio [95% confidence interval]: 0.73 [0.56, 0.97]; P=0.029) had lower risk for incident coronary events after adjustment for cardiovascular risk factors in Cox proportional hazard regression models. Moreover, subjects with high levels of IgG-p210native were less likely to have carotid plaques as assessed by ultrasonography at baseline (odds ratio=0.81, 95% confidence interval 0.70-0.95, P=0.008 after adjustment for risk factors). CONCLUSIONS: This large prospective study demonstrates that subjects with high levels of apolipoprotein B-100 autoantibodies have a lower risk of coronary events supporting a protective role of these autoantibodies in cardiovascular disease.

High levels of cathepsin D and cystatin B are associated with increased risk of coronary events.

OBJECTIVE: The majority of acute coronary syndromes are caused by plaque ruptures. Proteases secreted by macrophages play an important role in plaque ruptures by degrading extracellular matrix proteins in the fibrous cap. Matrix metalloproteinases have been shown to be markers for cardiovascular disease whereas the members of the cathepsin protease family are less studied. METHODS: Cathepsin D, cathepsin L and cystatin B were measured in plasma at baseline from 384 individuals who developed coronary events (CEs), and from 409 age-matched and sex-matched controls from the Malmö Diet and Cancer cardiovascular cohort. RESULTS: Cathepsin D (180 (142-238) vs 163 (128-210), p<0.001), cathepsin L (55 (44-73) vs 52 (43-67), p<0.05) and cystatin B levels (45 (36-57) vs 42 (33-52), p<0.001) were significantly increased in CE cases compared to controls. In addition, increased cathepsin D (220 (165-313) vs 167 (133-211), p<0.001), cathepsin L (61 (46-80) vs 53 (43-68), p<0.05) and cystatin B (46 (38-58) vs 43 (34-54), p<0.05) were associated with prevalent diabetes. Furthermore, cathepsin D and cystatin B were increased in smokers. The HRs for incident CE comparing the highest to the lowest tertile(s) of cathepsin D and cystatin B were 1.34 (95% CI 1.02 to 1.75) and 1.26 (95% CI 1.01 to 1.57), respectively, after adjusting for age, sex, low-density lipoprotein/high-density lipoprotein ratio, triglycerides, body mass index, hypertension and glucose, but these associations did not remain significant after further addition of smoking to the model. In addition, cathepsin D was increased in incident CE cases among smokers after adjusting for cardiovascular risk factors. CONCLUSIONS: The associations of cathepsin D and cystatin B with future CE provide clinical support for a role of these factors in cardiovascular disease, which for cathepsin D may be of particular importance for smokers.

High Plasma Levels of Galectin-3 Are Associated with Increased Risk for Stroke after Carotid Endarterectomy.

BACKGROUND: Galectin-3 (Gal-3) has been suggested to have both pro- and anti-atherogenic properties. High plasma Gal-3 levels are associated with increased risk for cardiovascular (CV) death. However, it has so far not been investigated if plasma Gal-3 levels can predict the risk for future stroke in patients suffering from carotid atherosclerosis. The aim of this study was to investigate whether Gal-3 could be used as a marker to predict postoperative cerebrovascular ischemic events among patients who underwent carotid endarterectomy (CEA). METHODS: Plasma samples were obtained from 558 CEA patients and Gal-3 levels were analyzed by the proximity extension assay technique. The Swedish national in-patient health register was used to identify postoperative cerebrovascular events during the follow-up period (42.6 ± 26.2 months). RESULTS: Plasma Gal-3 was increased in patients treated for a symptomatic carotid stenosis (p = 0.013). Patients with Gal-3 levels above the median value had an increased incidence of stroke as shown by Kaplan-Meier curves of event-free survival (p = 0.007). Gal-3 was a predictor of postoperative stroke among women (hazard ratio 15.1, 95% CI 1.3-172.2; p = 0.028) even after correction for traditional CV risk factors. CONCLUSIONS: This study is the first to show that increased plasma levels of Gal-3 can help in predicting the occurrence of postoperative strokes among female subjects who undergo CEA, independently of traditional risk factors for cerebrovascular disease. This finding suggests that Gal-3 could be used as a marker to identify patients in need of intensified postoperative medical care.

Human Carotid Plaques With High Levels of Interleukin-16 Are Associated With Reduced Risk for Cardiovascular Events.

BACKGROUND AND PURPOSE: Interleukin-16 (IL-16) functions as a regulator of T-cell growth and acts as an inducer of cell migration. The aim of this study was to determine whether IL-16 measured in human carotid plaques was associated with symptoms (eg, stroke, transient ischemic attack, or amaurosis fugax), markers of plaque stability, and postoperative cardiovascular events. METHODS: Plaques obtained from patients who had ≥1 cerebrovascular ischemic events within 1 month before endarterectomy (n=111) were compared with plaques from patients without symptoms (n=95). Neutral lipids, smooth muscle cell, and macrophage contents were evaluated histologically, and collagen, elastin, and caspase-3 activity were measured biochemically. IL-16, matrix metalloproteinases, and tissue inhibitors of metalloproteinases were measured in plaque homogenates using a multiplex immunoassay. IL-16, CD3, CD4, and FoxP3 mRNA expressions in carotid plaques were analyzed with quantitative real-time polymerase chain reaction. RESULTS: Carotid plaques from asymptomatic patients had higher levels of IL-16 mRNA. High plaque IL-16 protein levels (above median) were associated with reduced incidence of postoperative cardiovascular events during a mean follow-up of 21 months (hazard ratio, 0.47; 95% confidence interval, 0.22-0.99; P=0.047). IL-16 levels correlated with the plaque-stabilizing components: elastin, collagen, matrix metalloproteinase-2, tissue inhibitors of metalloproteinase-1, tissue inhibitors of metalloproteinase-2 and FoxP3 mRNA. CONCLUSIONS: This study shows that high levels of IL-16 are associated with asymptomatic carotid plaques, expression of factors contributing to plaque stability, and decreased risk of new cardiovascular events during a 2-year period after surgery, suggesting that IL-16 might have a protective role in human atherosclerotic disease.

IL-22 affects smooth muscle cell phenotype and plaque formation in apolipoprotein E knockout mice.

OBJECTIVE: IL-22 is a recently discovered cytokine that belongs to the family of IL-10 related cytokines. It is produced by activated T-cells and innate lymphoid cells and has been suggested to be involved in tissue repair. As both inflammation and repair play important roles in atherosclerosis we investigated if IL-22 deficiency influences the disease process in Apoe(-/-) mice. METHODS: We generated IL-22(-/-)Apoe(-/-) mice and fed them high-fat-diet for 14 weeks to characterize atherosclerosis development. RESULTS: IL-22(-/-)Apoe(-/-) mice exhibited reduced plaque size both in the aorta (p = 0.0036) and the aortic root compared (p = 0.0012) with Apoe(-/-) controls. Moreover, plaque collagen was reduced in IL-22(-/-)Apoe(-/-) mice (p = 0.02) and this was associated with an increased expression of smooth muscle cell (SMC)-α-actin (p = 0.04) and caldesmon (p = 0.016) in the underlying media. Carotid arteries from IL-22(-/-)Apoe(-/-) mice displayed increased expression of genes associated with a contractile SMC phenotype e.g. α-actin (p = 0.004) and caldesmon (p = 0.03). Arterial SMCs were shown to express the IL-22 receptor and in vitro exposure to IL-22 resulted in a down-regulation of alpha actin and caldesmon gene expression in these cells. CONCLUSION: Our observations demonstrate that IL-22 is involved in plaque formation and suggest that IL-22 released by immune cells is involved in activation of vascular repair by stimulating medial SMC dedifferentiation into a synthetic phenotype. This response contributes to plaque growth by enabling SMC migration into the intima but may also help to stabilize the plaque.