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Background: Arterial hemodynamic parameters derived from reservoir-excess pressure analysis exhibit prognostic utility. Reservoir-excess pressure analysis may provide useful information about an influence of altered hemodynamics on target organ such as the kidneys. We determined whether the parameters derived from the reservoir-excess pressure analysis were associated with the reduction in estimated glomerular filtration rate in 542 older adults (69.4±7.9 years, 194 females) at baseline and after 3 years. Methods: Reservoir-excess pressure parameters, including reservoir pressure integral, excess pressure integral, systolic, and diastolic rate constants, were obtained by radial artery tonometry. Results: After 3 years, and in a group of 94 individuals (72.4±7.6 years, 26 females), there was an estimated glomerular filtration rate reduction of >5% per year (median reduction of 20.5% over 3 years). A multivariable logistic regression analysis revealed that higher baseline reservoir pressure integral was independently associated with a smaller reduction in estimated glomerular filtration rate after accounting for conventional cardiovascular risk factors and study centers (odds ratio: 0.660 [95% CIs, 0.494–0.883]; P =0.005). The association remained unchanged after further adjustments for potential confounders and baseline renal function (odds ratio: 0.528 [95% CIs, 0.351–0.794]; P =0.002). No other reservoir-excess pressure parameters exhibited associations with the reduction in renal function. Conclusions: This study demonstrates that baseline reservoir pressure integral was associated with the decline in renal function in older adults at 3-year follow-up, independently of conventional cardiovascular risk factors. This suggests that reservoir pressure integral may play a role in the functional decline of the kidneys.

Objective: To identify causal pathophysiological mechanisms for atherosclerosis and incident cardiovascular events using protein measurements. Approach and Results: Carotid artery atherosclerosis was assessed by ultrasound, and 86 cardiovascular-related proteins were measured using the Olink CVD-I panel in 7 Swedish prospective studies (11 754 individuals). The proteins were analyzed in relation to intima-media thickness in the common carotid artery (IMT-CCA), plaque occurrence, and incident cardiovascular events (composite end point of myocardial infarction or ischemic stroke) using a discovery/replication approach in different studies. After adjustments for traditional cardiovascular risk factors, 11 proteins remained significantly associated with IMT-CCA in the replication stage, whereas 9 proteins were replicated for plaque occurrence and 17 proteins for incident cardiovascular events. NT-proBNP (N-terminal pro-B-type natriuretic peptide) and MMP (matrix metalloproteinase)-12 were associated with both IMT-CCA and incident events, but the overlap was considerably larger between plaque occurrence and incident events, including MMP-12, TIM-1 (T-cell immunoglobulin and mucin domain 1), GDF (growth/differentiation factor)-15, IL (interleukin)-6, U-PAR (urokinase plasminogen activator surface receptor), LOX-1 (lectin-like oxidized LDL [low-density lipoprotein] receptor 1), and TRAIL-R2 (TNF [tumor necrosis factor]-related apoptosis-inducing ligand receptor 2). Only MMP-12 was associated with IMT-CCA, plaque, and incident events with a positive and concordant direction of effect. However, a 2-sample Mendelian randomization analysis suggested that increased MMP-12 may be protective against ischemic stroke ( P =5.5×10 −7 ), which is in the opposite direction of the observational analyses. Conclusions: The present meta-analysis discovered several proteins related to carotid atherosclerosis that partly differed in their association with IMT-CCA, plaque, and incident atherosclerotic disease. Mendelian randomization analysis for the top finding, MMP-12, suggests that the increased levels of MMP-12 could be a consequence of atherosclerotic burden rather than the opposite chain of events.

Objective: The aim of this study was to investigate if there is a causal relationship between circulating levels of TIM-1 (T-cell immunoglobulin and mucin domain 1) and incidence of stroke. Approach and Results: Plasma TIM-1 was analyzed in 4591 subjects (40% men; mean age, 57.5 years) attending the Malmö Diet and Cancer Study. Incidence of stroke was studied in relation to TIM-1 levels during a mean of 19.5 years follow-up. Genetic variants associated with TIM-1 (pQTLs [protein quantitative trait loci]) were examined, and a 2-sample Mendelian randomization analysis was performed to explore the role of TIM-1 in stroke using summary statistics from our pQTLs and the MEGASTROKE consortium. A total of 416 stroke events occurred during follow-up, of which 338 were ischemic strokes. After risk factor adjustment, TIM-1 was associated with increased incidence of all-cause stroke (hazards ratio for third versus first tertile, 1.44 [95% CI, 1.10–1.87]; P for trend, 0.004), and ischemic stroke (hazards ratio, 1.42 [95% CI, 1.06–1.90]; P for trend, 0.011). Nineteen independent lead SNPs, located in three genomic risk loci showed significant associations with TIM-1 ( P −8 ). A 2-sample Mendelian Randomization analysis suggested a causal effect of TIM-1 on stroke (β=0.083, P =0.0004) and ischemic stroke (β=0.102, P =7.7×10 −5 ). Conclusions: Plasma level of TIM-1 is associated with incidence of stroke. The genetic analyses suggest that this could be a causal relationship.

Background: Stable atherosclerotic plaques are characterized by thick fibrous caps of smooth muscle cells, collagen, and macrocalcifications. Identifying factors of plaque stability is necessary to design drugs to prevent plaque rupture and symptoms. Osteomodulin, originally identified in bones, is expressed by bone synthesizing osteoblasts and involved in mineralization. In the present study, we analyzed osteomodulin expression in human carotid plaques, its link with plaque phenotype, calcification, and future cardiovascular events. Methods: Osteomodulin gene expression (OMD ; n=82) was determined by RNA sequencing and osteomodulin protein levels by immunohistochemistry (n=45) in carotid plaques obtained by endarterectomy from patients with or without cerebrovascular symptoms from the CPIP (Carotid Plaque Imaging Project) cohort, Skåne University Hospital, Sweden. Plaque components were assessed by immunohistochemistry, RNA sequencing, and multiplex analysis. Patients were followed for cardiovascular events or cardiovascular death during a median of 57 or 70 months, respectively, using national registers. Results: OMD levels were increased in plaques from asymptomatic patients compared to symptomatics. High OMD levels were associated with fewer cardiovascular events during follow-up. OMD correlated positively with smooth muscle α-actin ( ACTA2 ; r =0.73, P =10 -13 ) and collagen ( COL1A2 ; r =0.4, P =0.0002), but inversely with CD68 gene expression ( r =−0.67, P =10 -11 ), lipids ( r =−0.37, P =0.001), intraplaque hemorrhage ( r =−0.32, P =0.010), inflammatory cytokine, and matrix metalloproteinase plaque contents. OMD was positively associated with MSX2 (Msh Homeobox 2) ( r =0.32, P =0.003), a marker of preosteoblast differentiation, BMP4 (bone morphogenetic protein) ( r =0.50, P =0.000002) and BMP6 ( r =0.47, P =0.000007), plaque calcification ( r =0.35, P =0.016), and was strongly upregulated in osteogenically stimulated smooth muscle cells, which was further increased upon BMP stimulation. Osteomodulin protein was present in calcified regions. Osteomodulin protein levels were associated with plaque calcification ( r =0.41, P =0.006) and increased in macrocalcified plaques. Conclusions: These data show that osteomodulin mRNA and protein levels are associated with plaque calcification in human atherosclerosis. Furthermore, osteomodulin mRNA, but not protein levels, is associated with plaque stability.

Objective: CD4 + CD28 null T cells have been shown to be associated with recurrent coronary events and suggested as potential biomarker and therapeutic target. It is unknown whether CD4 + CD28 null T cells associate with first-time cardiovascular events. We examined CD4 + CD28 null T cells in a prospective population-based cohort and in patients with advanced atherosclerosis. Approach and Results: CD4 + CD28 null T cells were quantified in 272 individuals experiencing a first-time coronary event during up to 17 years of follow-up and 272 age- and sex-matched controls in a case-control study, nested within the population-based Malmö Diet and Cancer study. The highest tertile of CD4 + CD28 null T cells was associated with a lower incidence of first-time coronary events compared with the lowest tertile (odds ratio, 0.48 [95% CI, 0.29–0.79], P =0.004) when adjusting for Framingham risk factors. This association remained significant for events recorded after >9 years of follow-up, when most coronary events occurred, but not during the first 9 years of follow-up, despite similar odds ratio. Additionally, we analyzed CD4 + CD28 null T cells in 201 patients with advanced atherosclerosis undergoing carotid endarterectomy. The adjusted hazard ratio for cardiovascular events in patients with advanced atherosclerosis was 2.11 (95% CI, 1.10–4.05, P =0.024), comparing the highest with the lowest CD4 + CD28 null T-cell tertile. Conclusions: Our findings reveal complex associations between CD4 + CD28 null T cells and cardiovascular disease. Although we confirm the reported positive associations with an adverse prognosis in patients with already established disease, the opposite associations with first-time coronary events in the population-based cohort may limit the clinical use of CD4 + CD28 null T cells.

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 5 µm 2 versus 4.6±2.8×10 5 µm 2 ; P −/− 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.

Objective— RAGE (receptor for advanced glycation end products) and EMMPRIN (extracellular matrix metalloproteinase inducer) are immune receptors for proinflammatory mediators. These receptors can also be found in a soluble form in the circulation. Soluble RAGE (sRAGE) has shown atheroprotective properties in animal studies, possibly by acting as a decoy receptor for its ligands. Whether sEMMPRIN (soluble EMMPRIN) has similar roles is unknown. We hypothesized that sRAGE and sEMMPRIN might be associated with vascular disease progression, incident coronary events, and mortality. Approach and Results— We measured baseline sRAGE and sEMMPRIN in 4612 cardiovascular disease-free individuals from the population-based Malmö Diet and Cancer cohort. Measurements of intima-media thickness in the common carotid artery were performed at inclusion and after a median of 16.5 years. sRAGE was negatively correlated with carotid intima-media thickness progression, independently of traditional cardiovascular risk factors, kidney function, and hsCRP (high sensitive C-reactive protein). Additionally, sRAGE was associated with decreased risk for major adverse coronary events (hazard ratio=0.90 [0.82–0.97]; P =0.009) and mortality (hazard ratio=0.93 [0.88–0.99]; P =0.011) during a follow-up period of 21 years. The relationship with mortality was independent of all considered potential confounders. We found no correlations between EMMPRIN, intima-media thickness progression, or prognosis. Conclusions— Individuals with high levels of circulating sRAGE have a slower rate of carotid artery disease progression and a better prognosis. Although its predictive value was too weak to promote sRAGE as a useful clinical biomarker in the population, the findings support further research into the potential anti-inflammatory and atheroprotective properties of this soluble receptor.

Background and Purpose— ECP (eosinophil cationic protein) is a marker of eosinophil activity and degranulation, which has been linked to atherosclerosis and cardiovascular disease. We examined the relationship between ECP, carotid plaque, and incidence of stroke in a prospective population-based cohort. Methods— The subjects participated in the Malmö Diet and Cancer Study between 1991 and 1994. A total of 4706 subjects with no history of stroke were included (40% men; mean age, 57.5 years). Carotid plaque was determined by B-mode ultrasound of the right carotid artery. Incidence of stroke was followed up during a mean period of 16.5 years in relation to plasma ECP levels. Results— Subjects in the third tertile (versus first tertile) of ECP tended to have higher prevalence of carotid plaque (odds ratio: 1.18; 95% confidence interval: 1.003–1.39; P =0.044 after multivariate adjustments). A total of 258 subjects were diagnosed with ischemic stroke (IS) during follow-up. ECP was associated with increased incidence of IS after risk factor adjustment (hazard ratio, 1.57; 95% confidence interval: 1.13–2.18; for third versus first tertile; P =0.007). High ECP was associated with increased risk of IS in subjects with carotid plaque. The risk factor–adjusted hazard ratio for IS was 1.86 (95% confidence interval: 1.32–2.63) in subjects with carotid plaque and ECP in the top tertile, compared with those without plaque and ECP in the first or second tertiles. Conclusions— High ECP is associated with increased incidence of IS. The association between ECP and IS was also present in the subgroup with carotid plaque.

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 P for trend 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.

Objective— Lipids are central to the development of atherosclerotic plaques. Specifically, which lipids are culprits remains controversial, and promising targets have failed in clinical studies. Sphingolipids are bioactive lipids present in atherosclerotic plaques, and they have been suggested to have both proatherogenic and antiatherogenic. However, the biological effects of these lipids remain unknown in the human atherosclerotic plaque. The aim of this study was to assess plaque levels of sphingolipids and investigate their potential association with and contribution to plaque vulnerability. Approach and Results— Glucosylceramide, lactosylceramide, ceramide, dihydroceramide, sphingomyelin, and sphingosine-1-phosphate were analyzed in homogenates from 200 human carotid plaques using mass spectrometry. Inflammatory activity was determined by analyzing plaque levels of cytokines and plaque histology. Caspase-3 was analyzed by ELISA technique. Expression of regulatory enzymes was analyzed with RNA sequencing. Human coronary artery smooth muscle cells were used to analyze the potential role of the 6 sphingolipids as inducers of plaque inflammation and cellular apoptosis in vitro. All sphingolipids were increased in plaques associated with symptoms and correlated with inflammatory cytokines. All sphingolipids, except sphingosine-1-phosphate, also correlated with histological markers of plaque instability. Lactosylceramide, ceramide, sphingomyelin, and sphingosine-1-phosphate correlated with caspase-3 activity. In vitro experiments revealed that glucosylceramide, lactosylceramide, and ceramide induced cellular apoptosis. All analyzed sphingolipids induced an inflammatory response in human coronary artery smooth muscle cells. Conclusions— This study shows for the first time that sphingolipids and particularly glucosylceramide are associated with and are possible inducers of plaque inflammation and instability, pointing to sphingolipid metabolic pathways as possible novel therapeutic targets.

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.

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.

Background: The innate immune response plays an important role in cardiac repair following an acute myocardial infarction. The pro-inflammatory alarmin S100A8/A9 is released in high amounts locally and systemically during acute coronary events. Here, we studied the effects of S100A9 blockade on the innate immune responses involved in post-ischemic myocardial inflammation and repair. Methods: We induced MI by permanent left coronary artery ligation in C57BL/6 mice, which were subsequently treated with the specific S100A8/A9 blocker ABR-238901 (30mg/kg) or with buffer for 21 days. Left ventricular function was assessed by echocardiography. Immune cell populations in the myocardium, blood, bone marrow and spleen were analysed by flow cytometry. Results: The treatment induced progressive deterioration of the left ventricular systolic function and increased left ventricular volumes in ABR-238901-treated mice compared with controls, suggesting defective repair and negative myocardial remodelling. After 21 days of treatment, infarction size was significantly higher in mice receiving S100A8/A9 blockade (16.1±5.4% vs. 9.9±3.4%, P=0.03). S100A9 blockade inhibited proliferation of hematopoietic stem cells in the bone marrow, and reduced neutrophil and monocyte trafficking from bone marrow and spleen to the circulation and myocardium. Neutrophil and monocyte counts increased in the bone marrow and spleen of mice receiving S100A8/A9 blockade, possibly due to impaired egression. The presence of reparatory CD11b + F4/80 + Ly6C low macrophages expressing the efferocytosis receptor MerTK was potently decreased in the myocardium by day 7 post-MI compared to controls (36 820 ± 2 538 vs. 72 371 ± 4 482 cells/heart, P=0.0001). The transcription factor Nur77 mediates phenotype switching from inflammatory Ly6C hi monocytes to reparatory Ly6C lo macrophages in MI. ABR-238901 lowered the expression of Nur77 in Ly6C hi/int monocytes in-vivo , total bone marrow derived monocytes in-vitro , and the activity of Nur77 in macrophages in-vitro . Conclusions: S100A8/A9 plays an important role in leukocyte trafficking after MI. Long-term S100A8/A9 blockade impairs efferocytosis and myocardial repair, leading to systolic dysfunction and detrimental remodelling.

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.

Objective— Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins and play important roles in development and tissue repair. They have also been shown to have both protective and pathogenic effects in atherosclerosis, and experimental studies have suggested that MMP-12 contributes to plaque growth and destabilization. The objective of this study was to investigate the associations between circulating MMPs, atherosclerosis burden, and incidence of cardiovascular disease with a particular focus on type 2 diabetes mellitus. Approach and Results— Plasma levels of MMP-1, -3, -7, -10, and -12 were analyzed by the Proximity Extension Assay technology in 1500 subjects participating in the SUMMIT (surrogate markers for micro- and macrovascular hard end points for innovative diabetes tools) study, 384 incident coronary cases, and 409 matched controls in the Malmö Diet and Cancer study and in 205 carotid endarterectomy patients. Plasma MMP-7 and -12 were higher in subjects with type 2 diabetes mellitus, increased with age and impaired renal function, and was independently associated with prevalent cardiovascular disease, atherosclerotic burden (as assessed by carotid intima-media thickness and ankle-brachial pressure index), arterial stiffness, and plaque inflammation. Baseline MMP-7 and -12 levels were increased in Malmö Diet and Cancer subjects who had a coronary event during follow-up. Conclusions— The plasma level of MMP-7 and -12 are elevated in type 2 diabetes mellitus, associated with more severe atherosclerosis and an increased incidence of coronary events. These observations provide clinical support to previous experimental studies, demonstrating a role for these MMPs in plaque development, and suggest that they are potential biomarkers of atherosclerosis burden and cardiovascular disease risk.

Objective— Rupture of atherosclerotic plaques is the major cause of acute coronary events (CEs). Plaque destabilization is the consequence of an imbalance between inflammatory-driven degradation of fibrous tissue and smooth muscle cell–dependent tissue repair. Proinflammatory factors have been documented extensively as biomarkers of cardiovascular risk but factors that contribute to stabilization of atherosclerotic plaques have received less attention. The present study aimed to investigate whether plasma levels of the smooth muscle cell growth factor epidermal growth factor (EGF), heparin-binding-EGF (HB-EGF), and platelet-derived growth factor correlate with plaque phenotype and incidence of CEs. Approach and Results— HB-EGF, EGF and platelet-derived growth factor were measured in plasma from 202 patients undergoing carotid endarterectomy and in 384 incident CE cases and 409 matched controls recruited from the Malmö Diet and Cancer cohort. Significant positive associations were found between the plasma levels of all 3 growth factors and the collagen and elastin contents of the removed plaques. CE cases in the Malmö Diet and Cancer cohort had lower levels of HB-EGF in plasma, whereas no significant differences were found for EGF and platelet-derived growth factor. After adjusting for cardiovascular risk factors in a Cox proportional hazard model, the hazard ratio for the highest HB-EGF tertile was 0.61 (95% confidence interval, 0.47–0.82; P Conclusions— The associations between high levels of smooth muscle cell growth factors in plasma and a more fibrous plaque phenotype as well as the association between low levels of HB-EGF and incident CEs point to a potential clinically important role for factors that contribute to plaque stabilization by stimulating smooth muscle cells.

Objective: Myocardial infarction (MI) is an ischemic injury of the heart leading to excessive local inflammation. The alarmin complex S100A8/9 is potently upregulated locally and systemically after MI and promotes the influx of neutrophils and monocytes into the myocardium. We studied whether S100A8/A9 inhibition with the blocking compound ABR-238901 might reduce inflammation and improve myocardial function after MI. Methods: MI was induced by left coronary artery ligation in 8-12 weeks old mice. Echocardiography was used to assess left ventricular function. Results: Two days after MI, mice treated with ABR-238901 i.p. showed improved ejection fraction in comparison with buffer-treated animals, and had significantly decreased numbers of neutrophils and monocytes in the blood. More importantly, the presence of neutrophils and monocytes in the myocardium was reduced, leading to lower numbers of infiltrating macrophages. After 7 days of continuous treatment, cardiac function and the total counts of neutrophils and monocytes in blood and heart became similar in the two groups. However, the numbers of reparatory F4/80 + MerTK hi Ly6C low macrophages were lower in the hearts of ABR-238901 treated mice after 7 days. In contrast, we found that a balanced treatment strategy using ABR-238901 for 3 days followed by 4 days buffer increased the numbers and the proliferative capacity of this macrophage subset, promoting cardiac repair. Conclusion: We report that 3-day blockage of S100A8/9 has beneficial effects on cardiac function in the immediate period after an MI and enhances the presence of reparatory macrophages in the myocardium.

Objective— Diabetes mellitus (DM) type II is increasing rapidly worldwide. Patients with DM II have a greater atherosclerotic burden and higher risk of developing cardiovascular complications. Inflammation has been proposed as the main cause for the high risk of atherosclerotic disease in DM II. In this study, we compared markers of inflammation and fibrous repair in plaques from subjects with and without DM II. Approach and Results— Carotid endarterectomy specimens were obtained from 63 patients with and 131 without DM. Plaque structure, connective tissue proteins, inflammatory cells, and markers were analyzed by immunohistochemistry, ELISA, Mesoscale, and Luminex technology. Carotid plaques from diabetics had lower levels of extracellular matrix proteins, elastin, and collagen, which are critical for plaque stability. Plaques from diabetics had reduced levels of platelet-derived growth factor and matrix metalloproteinase-2, both important for tissue repair. No differences were observed in inflammatory markers in plaques from diabetic and nondiabetic patients. Conclusion— This study suggests that atherosclerotic plaques in subjects with DM II are more prone to rupture because of impaired repair responses rather than to increased vascular inflammation. Although this study did not have a mechanistic design, our findings suggest that targeting impaired repair responses in carotid plaques may help to increase our understanding of atherosclerotic plaque development and vulnerability in patients with DM II.

Objective:Altered central haemodynamics assessed by reservoir pressure analysis (RPA) could have a detrimental effect on target organs like the kidneys and heart. In this cross-sectional study, we aimed to determine whether RPA parameters would be associated with biomarkers of renal and cardiac dama

Objective— Accumulating evidence shows that immune cells play an important role in atherosclerosis. Most attention has focused on the role of different T cell subsets, whereas the possible involvement of B cells has been less studied. In this study, we assessed the association of 2 different B cell subsets, CD19 + CD40 + and CD19 + CD86 + B cells, with risk for development of acute cardiovascular events. Approach and Results— The prospective study included 700 subjects randomly selected from the cardiovascular cohort of the Malmö Diet and Cancer study. Mononuclear leukocytes, stored at –140 ○ C at the baseline investigation in 1991–1994, were thawed and B cell subsets analyzed by flow cytometry. Cytokine release from CD3/CD28-stimulated mononuclear leukocytes was measured with multiplex ELISA. Baseline carotid intima-media thickness and stenosis were assessed by ultrasonography, and clinical events were monitored through validated national registers during a median/mean follow-up time of 15 years. The subjects in the highest tertile of CD19 + CD40 + B cells had a significantly lower risk of incident stroke after adjustment for other risk factors. In contrast, CD19 + CD86 + B cells were associated with higher risk for development of a stroke event and increased release of proinflammatory cytokines from mononuclear leukocytes. Conclusions— These observations provide evidence for an involvement of B cells in the incidence of stroke and suggest that both pathogenic and protective B cell subsets exist.

Objective— Experimental studies in mice have attributed T-helper (Th) 1 and Th2 cells important roles in atherosclerosis, but the clinical importance of these cells in cardiovascular disease (CVD) remains to be clarified. Here, we investigated associations between Th1 and Th2 cells, carotid intima-media thickness, and cardiovascular risk. Methods and Results— Blood drawn at baseline and incident cardiovascular events during 15-year follow-up were assessed in 700 participants. Baseline Th1 (CD3 + CD4 + interferon-γ + ) and Th2 (CD3 + CD4 + interleukin-4 + ) cells were analyzed by flow cytometry, and cytokine-release from activated mononuclear leukocytes was measured by multiplex technology. High numbers of Th2 cells were independently associated with decreased mean common carotid intima-media thickness. High numbers of Th2 cells were also independently associated with a reduced risk of acute myocardial infarction in women (hazard ratio, 0.19; 95% confidence interval, 0.06–0.56; P =0.002 for the highest versus the lowest tertile of Th2 cells). Moreover, release of the Th2 cytokine interleukin-4 from activated mononuclear leukocytes was independently associated with a reduced risk of CVD. No independent associations between Th1 cells and carotid intima-media thickness or CVD risk were found. Conclusion— Our observations provide the first clinical evidence for a protective role of Th2 immunity in CVD. They also suggest this protection is more prominent in women than in men. In spite of convincing evidence from experimental studies, we found no support for a role of Th1 immunity in CVD.

Objective— The aim of this study was to analyze how an altered collagen structure affects development of atherosclerotic plaques. Methods and Results— Fibromodulin-null mice develop an abnormal collagen fibril structure. In apolipoprotein E (ApoE)-null and ApoE/fibromodulin-null mice, a shear stress-modifying carotid artery cast induced formation of atherosclerotic plaques of different phenotypes; inflammatory in low-shear stress regions and fibrous in oscillatory shear stress regions. Electron microscopy showed that collagen fibrils were thicker and more heterogeneous in oscillatory shear stress lesions from ApoE/fibromodulin-null mice. Low-shear stress lesions were smaller in ApoE/fibromodulin-null mice and contained less lipids. Total plaque burden in aortas stained en face with Oil Red O, as well as lipid accumulation in aortic root lesions, was also decreased in ApoE/fibromodulin-null mice. In addition, lipid accumulation in RAW264.7 macrophages cultured on fibromodulin-deficient extracellular matrix was decreased, whereas levels of interleukin-6 and -10 were increased. Our results show that an abnormal plaque collagen fibril structure can influence atherosclerotic plaque development. Conclusion— The present findings suggest a more complex role for collagen in plaque stability than previously anticipated, in that it may promote lipid-accumulation and inflammation at the same time as it provides mechanical stability.

Objectives— To determine whether the level of lysophosphatidylcholine (lysoPC) generated by lipoprotein-associated phospholipase A2 (Lp-PLA2) is associated with severity of inflammation in human atherosclerotic plaques. Elevated plasma Lp-PLA2 is associated with increased cardiovascular risk. Lp-PLA2 inhibition reduces atherosclerosis. Lp-PLA2 hydrolyzes low-density lipoprotein–oxidized phospholipids generating lysoPCs. According to in vitro studies, lysoPCs are proinflammatory but the association between their generation and plaque inflammation remains unknown. Methods and Results— Inflammatory activity in carotid plaques (162 patients) was determined immunohistochemically and by analyzing cytokines in homogenates (multiplex immunoassay). LysoPCs were quantified using mass spectrometry and Lp-PLA2 and the lysoPC metabolite lysophosphatidic acid (LPA) by ELISA. There was a strong correlation among lysoPC 16:0, 18:0, 18:1, LPA, and Lp-PLA2 in plaques. LysoPC 16:0, 18:0, 18:1, LPA, and Lp-PLA2 correlated with interleukin-1β, interleukin-6, monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, regulated on activation normal T-cell expressed and secreted, and tumor necrosis factor-α in plaques. High lysoPC and Lp-PLA2 correlated with increased plaque macrophages and lipids and with low content of smooth muscle cells, whereas LPA only correlated with plaque macrophages. Lp-PLA2, lysoPC 16:0, 18:0, and 18:1, but not LPA were higher in symptomatic than in asymptomatic plaques. Conclusions— The associations among Lp-PLA2, lysoPCs, LPA, and proinflammatory cytokines in human plaques suggest that lysoPCs play a key role in plaque inflammation and vulnerability. Our findings support Lp-PLA2 inhibition as a possible strategy for the prevention of cardiovascular disease.

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/μL versus 297 [212 to 384] cells/μL, 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.

Objective— Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca 2+ /calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications. Methods and Results— An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. The calcineurin inhibitor cyclosporin A or the novel NFAT blocker A-285222 prevented glucose-induced OPN expression. Furthermore, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A-285222 for 4 weeks or prevented in arteries from NFATc3 −/− mice. Conclusions— These results identify a glucose-sensitive transcription pathway in vivo, revealing a novel molecular mechanism that may underlie vascular complications of diabetes.

Background and Purpose— Autoantibodies against antigens in oxidized low-density lipoprotein are common in people; experimental studies suggest that these immune responses have a functional role in the disease process. The aim of this study was to evaluate the relationship between the immune response against one defined oxidized low-density lipoprotein antigen, the aldehyde-modified peptide corresponding amino acids 3136 and 3155 (MDA-p210) in apolipoprotein (apo) B-100, and progression of carotid intima media thickness (IMT). Methods— IgM and IgG against MDA-p210 were determined by enzyme-linked immunosorbent assay at baseline and after 12 months of treatment with placebo, metoprolol, fluvastatin, or metoprolol/fluvastatin in 751 individuals participating in the BCAPS. Carotid IMT was assessed by ultrasonography at baseline and after 18 and 36 months of treatment. Results— Antibody levels did not change in response to treatment, but high baseline MDA-p210 IgM levels were associated with a more rapid progression of carotid disease both at 18 ( r =0.09, P r =0.12, P P Conclusions— IgM against the aldehyde-modified peptide corresponding amino acids 3136 and 3155 in apo B-100 is common in subjects with asymptomatic carotid disease, and high levels are associated with a more rapid progression of carotid IMT. The observation that the effect of fluvastatin was restricted to subjects with high MDA-p210 IgM levels may reflect the increased rate of disease progression in this group.

Objective—Inflammation plays an important role in atherosclerosis. One of the most potent pro-inflammatory cytokines is tumor necrosis factor-α (TNF-α), a cytokine identified to have a pathogenic role in chronic inflammatory diseases such as rheumatoid arthritis (RA). The aim of the study was to evaluate the importance of TNF-α in atherogenesis.Methods and Results—Mice deficient in both apolipoprotein E (apoE) and TNF-α were compared regarding their atherosclerotic burden. Mice were fed a Western-style diet (WD) or normal chow. Mice deficient in both apoE and TNF-α exhibited a 50% (P=0.035) reduction of relative lesion size after 10 weeks of WD. Bone marrow transplantation ofapoEomice withapoEotnf-αobone marrow resulted in a 83% (P=0.021) reduction after 25 weeks on WD. InapoEknockout mice treated with recombinant soluble TNF receptor I releasing pellets, there was a reduction in relative lesion size after 25 weeks of 75% (P=0.018).Conclusions—These findings demonstrate that TNF-α is actively involved in the progression of atherosclerosis. Accordingly, TNF-α represents a possible target for prevention of atherosclerosis. This may be of particular importance in rheumatoid arthritis because these patients have an increased risk for cardiovascular disease.

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.

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.

Background and Purpose— Many processes involved in the pathogenesis of atherosclerosis result in modifications of the extracellular matrix. These changes not only determine the mechanical stability of atherosclerotic lesions but can directly or indirectly influence further development of the lesions. The purpose of the present study was to compare the matrix composition of human carotid plaques from symptomatic patients with those obtained from patients without symptoms. Furthermore, matrix changes related to age were studied. Methods— Thirty atherosclerotic carotid plaques were removed by endarterectomy from 27 patients and divided into 2 groups on the basis of the presence of ipsilateral symptoms. The plaques were homogenized, and the total levels of the major components of the extracellular matrix were determined. Results— Plaques associated with symptoms were characterized by increased levels of elastin (1.58±0.46 versus 1.24±0.40 mg/g wet wt; P =0.03) and decreased levels of hydroxyapatite (45.1±46.3 versus 131.4±111.7 mg/g wet wt; P =0.02) compared with asymptomatic plaques. The increase in elastin in plaques from symptomatic patients was due to elevated levels of an intermediate-size fraction, as determined by liquid chromatography. Collagen and sulfated glycosaminoglycans were present in equal amounts in both groups. Elastin content in carotid plaques decreased with age. Conclusions— Carotid plaques from symptomatic patients have lower levels of hydroxyapatite than those from asymptomatic patients. The present study also raises the possibility that non–cross-linked forms of elastin, increased in plaques associated with symptoms, could be a marker of plaque vulnerability and/or directly induce harmful cellular activities or increase lipoprotein retention in the vascular wall.

We investigated the effect of B-cell reconstitution in immune-deficient Rag-1 knockout (KO) mice subjected to arterial injury. After 21 days, injury induced a 4- to 5-fold increase in neointimal formation in Rag-1 KO mice fed normal chow compared with wild-type (WT) mice (0.020±0.0160 [n=8] versus 0.0049±0.0022 [n=8] mm2, respectively;P2, respectively;P2, respectively;P2, respectively;P

Abstract — Intimal proliferation of smooth muscle cells (SMCs) is a key event in the vascular response to injury, including the early stages of atherosclerosis and restenosis after angioplasty. Tumor necrosis factor-α (TNF-α) has been reported to stimulate growth of cultured human SMCs, but activation of TNF receptors is also known to induce cell death by apoptosis. We report here that SMCs isolated from the neointima of injured rat aortas are characterized by increased expression of TNF-α in response to interleukin-1β and γ-interferon compared with medial SMCs. Basal and serum-stimulated DNA synthesis was higher in intimal than in medial SMCs. In contrast to previous findings on human SMCs, exposure to interleukin-1β/γ-interferon or TNF-α did not affect the growth of rat medial SMCs, inhibited DNA synthesis, and decreased cell numbers in cultures of intimal SMCs. Incubation of intimal SMCs with these cytokines also resulted in induction of terminal dUTP nick end-labeling positivity and caspase-3 expression, suggesting cell death by apoptosis, whereas medial cells were markedly less sensitive in this respect. Cytokine-induced apoptosis in intimal cells was effectively inhibited by treatment with antibodies against TNF receptors. These findings suggest that endogenous activation of TNF receptors may represent a way to limit accumulation of SMCs in injured arteries. This mechanism may also be important in SMC death in advanced atherosclerotic plaques.

“ An invasion of armies can be resisted but not an idea whose time has come.” —Victor Hugo. Vaso-occlusive disease resulting from atherosclerosis and thrombosis is the leading cause of death and morbidity in the United States and other industrialized nations. Although the precise cause of atherosclerosis is unclear, an emerging paradigm suggests that atherosclerosis involves multiple pathways in which lipoprotein entry and retention, injury to the vessel wall from diverse stimuli, and an associated long-term inflammatory and immune response seem to play a key role.1–4 Dyslipidemia characterized predominantly by elevated levels of one or more circulating non-HDL cholesterol lipoproteins [LDL, VLDL, lipoprotein(a), triglycerides] and/or reduced HDL cholesterol is one of the key risk factors for atherosclerosis and cardiovascular disease.5–16 Over the past several years, a number of large, prospective, randomized, controlled clinical trials have demonstrated both angiographic and clinical benefits of lipid-lowering therapy, with a significant reduction in fatal and nonfatal cardiovascular events.17–23 These studies have primarily targeted LDL cholesterol through pharmacotherapy (mostly statins), with or without dietary counseling, lifestyle modification, or surgery (intestinal bypass in Program on the Surgical Control of the Hyperlipidemias [POSCH] trial). Overall, a significant and clinically worthwhile relative risk reduction ranging from 20% to 40% in major cardiovascular events has been achieved with these strategies, without significant adverse effects or increased noncardiovascular mortality. These remarkable results prompted Brown and Goldstein24 to predict that heart attacks will be gone with the century. This clearly reflects an overoptimistic point of view, because 60% to 70% of adverse cardiovascular events continue to occur despite LDL-lowering therapy. Potential reasons why cardiovascular events may continue to occur despite low LDL levels or despite LDL-lowering therapy include the following: (1) we may not be lowering LDL cholesterol to optimal levels, because optimal levels are not clearly …

Background —The clinical benefits of lipid lowering with statins are attributed to changes in plaque composition leading to lesion stability, but supporting clinical data from human studies are lacking. Therefore, we investigated the effect of 3 months of pravastatin treatment on composition of human carotid plaques removed during carotid endarterectomy. Methods and Results —Consecutive patients with symptomatic carotid artery stenosis received 40 mg/d pravastatin (n=11) or no lipid-lowering therapy (n=13; control subjects) for 3 months before scheduled carotid endarterectomy. Carotid plaque composition was assessed with special stains and immunocytochemistry with quantitative image analysis. Plaques from the pravastatin group had less lipid by oil red O staining (8.2±8.4% versus 23.9±21.1% of the plaque area, P P P P P P P P Conclusions —Pravastatin decreased lipids, lipid oxidation, inflammation, MMP-2, and cell death and increased TIMP-1 and collagen content in human carotid plaques, confirming its plaque-stabilizing effect in humans.

Abstract —Activation of vascular inflammation in response to hyperlipidemia is believed to play an important role during the early stages of atherogenesis. We demonstrate here that exposure of cultured, rat aortic smooth muscle cells to low density lipoprotein (LDL) stimulated tumor necrosis factor-α (TNF-α) mRNA and protein expression. Oxidative modification of LDL resulted in a reduction of this stimulatory effect. To analyze whether a similar response also occurs in vivo, we used a recently developed model in which the effects of a rapid accumulation of human LDL in rat arteries can be studied. As previously reported, epitopes specific for human apolipoprotein B began to accumulate in the aorta within 2 to 6 hours after injection of 6 mg of human LDL. This was followed by expression of oxidized LDL–specific epitopes after 12 hours. There was no vascular expression of TNF-α at baseline or in phosphate-buffered saline–injected control rats. However, 24 hours after injection of native LDL, there was a marked induction of TNF-α mRNA and immunoreactivity in the aorta and other large arteries, whereas injection of oxidized LDL was without effect in this respect. Preincubation of LDL with the antioxidant probucol before injection markedly decreased the expression of TNF-α immunoreactivity. The present findings support the notion that LDL may activate arterial expression of TNF-α and suggest 1 possible mechanism for the inflammatory response in the early stages of atherosclerosis. The role of LDL oxidation in this process remains to be fully elucidated.

Abstract —Human obesity is associated with an increased tumor necrosis factor-α (TNF-α) mRNA expression in adipose tissue. TNF-α decreases insulin-dependent glucose uptake by inhibiting autophosphorylation of the insulin receptor, suggesting that TNF-α may play a role in insulin resistance. In this study, we analyzed plasma levels of TNF-α in 40 70-year-old men with newly detected non–insulin-dependent diabetes mellitus and in 20 age-matched controls. Twenty of the patients had a moderate level of insulin resistance and 20 were severely insulin resistant. The plasma levels of TNF-α were higher in patients (4.00±1.53 pg/mL in moderately insulin resistant and 4.91±1.43 pg/mL in severely insulin resistant subjects) than in controls (3.27±0.79 pg/mL, P

Abstract —Oxidation of LDL is associated with degradation of phosphatidylcholine into platelet-activating factor (PAF)–like phospholipids and lysophosphatidylcholine (LPC). Exposure of cultured human smooth muscle cells to PAF and LPC in a concentration of 25 μmol/L was found to result in complete cell death, as assessed by the MTT cytotoxicity assay and cell counting. Addition of 50 μg/mL apolipoprotein A-I– and apolipoprotein A-I Milano –containing phospholipid particles completely inhibited this cytotoxicity. Phospholipid complexes alone were almost as effective, whereas free apolipoprotein A-I Milano and albumin were without effect, suggesting that the effect was phospholipid dependent. Experiments using [ 14 C]LPC demonstrated that apolipoprotein A-I– and apolipoprotein A-I Milano –containing phospholipid particles effectively bind LPC. The results show that HDL-like phospholipid particles effectively inhibit the toxic effect of phospholipids and other lipid-soluble factors. The ability of HDL to inhibit the proinflammatory and toxic effects of phospholipids generated during oxidation of LDL may be responsible for part of the antiatherogenic properties of HDL.

Aims S100A8/A9 is an inflammatory mediator secreted by activated neutrophils and monocytes/macrophages. Serum S100A8/A9 levels were previously found to correlate with the severity of coronary artery disease in diabetic patients and to predict cardiovascular (CV) events in healthy postmenopausal women. Our aim was to investigate whether S100A8/A9 correlates with carotid artery disease and whether it can predict CV risk in healthy individuals. Methods We measured baseline levels of S100A8/A9 in 664 individuals with no previous history of CV disease, randomly selected from the Malmö Diet and Cancer cohort. We examined the correlation between S100A8/A9 and traditional CV risk factors, circulating cell populations, plasma cytokines, carotid intima media thickness (IMT) and area (IM area), and the incidence of CV events during a follow-up period of 16.2 years. Results We detected significant positive correlations between baseline S100A8/A9 and the number of circulating neutrophils, IFNγ, TNFα, IL-1β, smoking, BMI, systolic blood pressure and an inverse correlation with HDL. In a multivariate linear regression model adjusted for age and sex, S100A8/A9 and the neutrophil counts were correlated with IMT and IM area in the common carotid artery. The associations with IM area remained significant after further adjustment for CV risk factors. In a Cox regression model corrected for age and sex, S100A8/A9 and circulating neutrophils predicted the incidence of coronary events (fatal or non-fatal myocardial infarction) [HR (95%CI) per SD: 1.28 (1.03-1.59), P =0.026 and 1.26 (1.04-1.53), P =0.020, respectively] and CV death [1.34 (1.06-1.71), P =0.015 and 1.59 (1.33-1.90), P Conclusions S100A8/A9 correlates strongly with circulating neutrophil numbers, but not with monocytes or monocyte sub-populations. Baseline S100A8/A9 concentrations and circulating neutrophils are significantly associated with carotid artery disease and predict coronary events and CV death in apparently healthy individuals. Our study provides further evidence for the involvement of activated neutrophils in atherogenesis and acute coronary events.

The uptake of oxidatively modified low density lipoprotein (Ox-LDL) by intimal macrophages is believed to play a key role in the development of atherosclerosis. The present study demonstrates that Ox-LDL in low concentrations activates monocyte/macrophage release of factors that stimulate smooth muscle cell growth, whereas higher concentrations are inhibitory. Exposure of monocytes/macrophages to 8 μg/mL Ox-LDL increased expression of tumor necrosis factor-α (TNF-α) mRNA but had no effect on interleukin-1β, platelet-derived growth factor B and heparin-binding epidermal growth factor–like mitogen mRNA levels. Ox-LDL also stimulated monocyte/macrophage release of TNF-α in a dose-dependent manner, with maximal effect at an LDL concentration of 8 μg/mL. Addition of TNF-α–blocking antibodies to conditioned medium from monocytes/macrophages already exposed to Ox-LDL reduced mitogenic activity by 44.7±8.4% ( P

Although the existence of an immune response against modified lipoproteins in atherosclerosis has been observed in experimental animals as well as in humans, the precise pathophysiological relevance of these findings remains unclear. In this study we determined the effect of an immunization with homologous LDL and copper-oxidized LDL on the formation of atherosclerotic plaque in hypercholesterolemic rabbits. Immunizations were performed at the start of a cholesterol-rich diet and 3 weeks later. After 16 weeks, antibodies against oxidized LDL had developed in rabbits given hypercholesterolemic diet alone, but the titers were increased by twofold in rabbits immunized with oxidized LDL as well as in rabbits immunized with LDL, suggesting that the LDL had also become oxidized during the preparation and/or immunization procedure. Immunization with LDL and oxidized LDL reduced atherosclerotic lesions in the proximal aorta by 74% ( P P =NS), respectively. The cellular composition of the lesions was not affected by the immunizations. These results support the hypothesis that an immune response against modified LDL has a protective effect against the development of early atherosclerotic lesions.

We studied the effects of coil orientation, stimulus intensity, and shape of the induced current pulse on the amplitudes of motor evoked potentials in the left abductor pollicis brevis of 10 normal adults who had transcranial magnetic stimulation. The optimal direction of currents induced in the brain is approximately perpendicular to the central sulcus, flowing diagonally from back to front. The most effective coil orientation depends on the shape of the induced current pulse and, when the first and second phases of the pulse are of similar size, also on the intensity of stimulation. Optimal mapping of the human motor cortex with magnetic stimulation requires knowledge of the influences of all these factors.

The immune system represents a promising novel target for prevention of atherosclerosis. Several pilot vaccines that reduce atherosclerosis in experimental animals have been developed. The aluminum hydroxide adjuvant Alum has been shown to have antiatherogenic properties in itself, suggesting that it may be a suitable adjuvant in possible future atherosclerosis vaccines. To characterize the immune pathways mediating this protection, we treated wild-type C57BL/6 andApoe−/−mice with Alum or PBS. Analyses of splenocytes isolated from 12-week-old mice demonstrated that Alum increased the presence of CD4+CD25+FoxP3+regulatory T cells and downregulated the expression of T cell activation markers CD28 and ICOS inApoe−/−mice but not in C57BL/6 wild-type mice. A similar immunosuppressive phenotype was found also in 25-week-oldApoe−/−mice and was associated with reduced atherosclerosis. Alum precipitates recovered from the injection site ofApoe−/−mice contained antigens derived from oxidized LDL. These findings demonstrate that treatment ofApoe−/−mice with Alum results in an increase of regulatory T cells and suggest that these are activated by tolerogenic antigen-presenting cells presenting oxidized LDL antigens. Our findings provide improved mechanistic understanding of the atheroprotective properties of aluminum hydroxide adjuvants but also point to the importance of determining if hypercholesterolemia may compromise the efficacy of Alum-containing vaccines used clinically today.

C-reactive protein (CRP) has emerged as an interesting novel and potentially clinically useful marker for increased cardiovascular risk.1,2 This is an attractive concept because atherosclerosis is a disease characterized by chronic arterial inflammation3,4 and suggests the possibility that subclinical states of atherosclerosis can be identified by an increase in circulating markers of inflammation before acute events occur. Based on data obtained primarily from in vitro studies it has also been proposed that CRP in itself is actively contributing to disease progression and that it should be considered as true risk factor and consequently as a target for intervention. However, in the present issue of Arteriosclerosis, Thrombosis, and Vascular Biology , two independent reports demonstrate that transgenic overexpression of CRP does not affect the development of atherosclerosis in mice, suggesting that this is not the case. See pages 1635 and 1641 The association between moderately elevated CRP levels and an increased risk for development of cardiovascular disease is well established. Although the increase in risk may have been overestimated in initial studies, recent meta-analysis comprising >7000 patients with coronary events shows that subjects with CRP in the upper tertile have a 50% increased risk for development of acute cardiovascular events.2 There are several possible explanations for this association (Figure). (1) The inflammatory process in arterial tissue affected by atherosclerosis results in release of cytokines into the circulation that subsequently activates expression of CRP in the liver. Plasma CRP levels would then reflect the severity of atherosclerosis and, as a consequence, the risk for development of clinical events. If CRP in addition …

The H-reflex recovery curve of the lower limb is considered a useful test for the diagnosis of spasticity, and recently the reciprocal inhibition of the H-reflex has proven to be abnormal in patients affected with spasticity. We studied the H-reflex recovery curve and the reciprocal inhibition of the H-reflex in the upper limb of a group of 33 patients with different degrees of spasticity secondary to stroke. Results were compared with those of 25 controls. The aim of this study was to investigate if the two tests showed any direct correlation with the degree of spasticity and, furthermore, with other clinical measures that are present in patients with spasticity as part of an upper motoneuron syndrome (i.e., changes in muscle tone, reflexes, force, etc.). The results showed an abnormality of both tests in most patients (decrease of the three phases of inhibition in the reciprocal inhibition test and increase of the late facilitation part of the H-reflex recovery curve), and these abnormalities seem mostly to be related to muscle tone, most important being the degree of correlation between tone and changes in abnormality of the H-reflex recovery curve (P < 0.03).