CD40L induces inflammation and adipogenesis in adipose cells--a potential link between metabolic and cardiovascular disease.

CD40L figures prominently in atherogenesis. Recent data demonstrate elevated levels of sCD40L in the serum of patients with the metabolic syndrome (MS). This study investigated the role of CD40L in pro-inflammatory gene expression and cellular differentiation in adipose tissue to obtain insight into mechanisms linking the MS with atherosclerosis. Human adipocytes and preadipocytes expressed CD40 but not CD40L. Stimulation with recombinant CD40L or membranes over-expressing CD40L induced a time- and dose-dependent expression of IL-6, MCP-1, IL-8, and PAI-1. Supernatants of CD40L-stimulated adipose cells activated endothelial cells, suggesting a systemic functional relevance of our findings. Neutralising antibodies against CD40L attenuated these effects substantially. Signalling studies revealed the involvement of mitogen-activated protein kinases and NFkB. Furthermore, stimulation with CD40L resulted in enhanced activation of C/EBPa and PPARg and promoted adipogenesis of preadipose cells in the presence and absence of standard adipogenic conditions. Finally, patients suffering from the metabolic syndrome with high levels of sCD40L also displayed high levels of IL-6, in line with the concept that CD40L may induce the expression of inflammatory cytokines in vivo in this population. Our data reveal potent metabolic functions of CD40L aside from its known pivotal pro-inflammatory role within plaques. Our data suggest that CD40L may mediate risk at the interface of metabolic and atherothrombotic disease.

Interleukin-18, the metabolic syndrome, and subclinical atherosclerosis: results from the Dallas Heart Study.

OBJECTIVE: Although IL-18 promotes atherogenesis in animal studies and predicts cardiovascular risk in humans, it is unknown whether elevated IL-18 levels are associated with coronary atherosclerosis in the general population. METHODS AND RESULTS: IL-18 plasma levels were determined by ELISA in 2231 subjects from the Dallas Heart Study. In univariable analysis, IL-18 levels associated with traditional cardiovascular risk factors and particularly with components of the metabolic syndrome (MS, P<0.01 for trend across the number of MS components); IL-18 also associated with coronary artery calcium (CAC) scores measured by electron beam computed tomography and aortic plaque measured by MRI (P<0.01 for each). In multivariable analyses, IL-18 remained associated with multiple components of the MS but not with CAC or aortic plaque. CONCLUSIONS: In a large population-based sample, elevated IL-18 plasma levels associated with risk factors for atherosclerosis and with the metabolic syndrome. The association between IL-18 and atherosclerosis diminished after accounting for traditional cardiovascular risk factors. These data suggest that IL-18 does not add independently to detection of atherosclerotic burden in asymptomatic individuals.

TRAF-1, -2, -3, -5, and -6 are induced in atherosclerotic plaques and differentially mediate proinflammatory functions of CD40L in endothelial cells.

OBJECTIVE: Several lines of evidence implicate CD40 ligand (CD40L, CD154) as a mediator and marker of atherosclerosis. This study investigated the involvement of tumor necrosis factor receptor-associated factors (TRAFs) in CD40 signaling in endothelial cells (ECs) and their expression in atheromata and cells involved in atherogenesis. METHODS AND RESULTS: CD40L enhanced the basal expression of TRAF-1, -2, -3, and 6, but not TRAF-5 in ECs. TRAFs associated with CD40 on ligation by CD40L. Study of ECs from TRAF-1, -2, and -5-deficient mice demonstrated functional involvement of TRAFs in proinflammatory CD40 signaling. Whereas TRAF-1 deficiency enhanced CD40L-induced IL-6 and MCP-1 expression, TRAF-2 and TRAF-5 deficiency inhibited CD40L-inducible IL-6 but not MCP-1 expression. Gene silencing in human ECs further delineated functions of TRAFs in CD40 signaling. TRAF-3 silencing in ECs showed increased CD40L-induced IL-6, MCP-1, and IL-8 expression, whereas TRAF-6 silencing increased selectively CD40L-induced MCP-1 expression. Enhanced TRAF levels in atherosclerotic lesions further supports involvement of members of this family of signaling molecules in arterial disease. CONCLUSIONS: These results implicate endothelial TRAF-1, -2, -3, -5, and -6 in CD40 signaling in atherogenesis, identifying these molecules as potential targets for selective therapeutic intervention.

CD40 ligand mediates inflammation independently of CD40 by interaction with Mac-1.

BACKGROUND: Strong evidence supports a role for CD40 ligand (CD40L) as marker and mediator of inflammatory diseases such as atherosclerosis. Despite extensive characterization of CD40, the classic receptor of CD40L, its role in immune defense against inflammatory diseases remains uncertain. The present study aimed to characterize the contribution of CD40 signaling to atherogenesis. METHODS AND RESULTS: Surprisingly, mice deficient in both CD40 and the low-density lipoprotein receptor did not develop smaller lesions in the aortic arch, root, and thoracoabdominal aorta compared with mice deficient only in the low-density lipoprotein receptor that consumed an atherogenic diet for 8 and 16 weeks. By flow cytometry, radioactive binding assays, and immunoprecipitation, we demonstrate that CD40L interacts with the integrin Mac-1, which results in Mac-1-dependent adhesion and migration of inflammatory cells as well as myeloperoxidase release in vitro. Furthermore, mice deficient in CD40L show significantly reduced thioglycolate-elicited invasion of inflammatory cells into the peritoneal cavity compared with mice deficient in CD40 and wild-type controls. Inhibition of Mac-1 in low-density lipoprotein receptor-deficient mice attenuates lesion development and reduces lesional macrophage accumulation. CONCLUSIONS: These observations identify the interaction of CD40L and Mac-1 as an alternative pathway for CD40L-mediated inflammation. This novel mechanism expands understanding of inflammatory signaling during atherogenesis.

Influence of pre-analytical and analytical factors on soluble CD40L measurements.

The soluble form of CD40L (CD40 ligand), a pro-atherogenic mediator, has emerged as a diagnostic and prognostic marker for cardiovascular events. However, as platelets can shed CD40L upon activation, accurate measurement has proved challenging. The present study addresses the controversy regarding the appropriate specimen and preparation for laboratory evaluation of blood sCD40L (soluble CD40L). Serum and plasma (collected in EDTA, citrate or heparin) were collected from healthy volunteers (n=20), and sCD40L was analysed by ELISA immediately or after one to three freeze-thaw cycles and at different centrifugation speeds. Urine sCD40L levels were measured in subjects with low- and high-plasma sCD40L levels. Serum sCD40L levels (5.45+/-4.55 ng/ml; P<0.001) were higher than in citrate, EDTA or heparin plasma (1.03+/-1.07, 1.43+/-1.03 or 1.80+/-1.25 ng/ml respectively), with no significant differences between plasma preparations. Increasing g values (200-13000 g), which gradually deplete plasma of platelets, yielded lower sCD40L levels. Repeated freeze-thaw cycles significantly (P<0.05) increased sCD40L concentrations in platelet-rich, but not platelet-depleted, plasma (up to 2.4-fold). Bilirubin and haemoglobin interfered positively, and triacylglycerols (triglycerides) and cholesterol quenched CD40L signalling. No sCD40L was detected in urine samples. In conclusion, serum yields higher sCD40L concentrations than plasma; accurate measurements of sCD40L require exclusion of platelets and avoiding their post-hoc activation. Samples with high concentrations of bilirubin, haemoglobin and/or triacylglycerols should be excluded, as these substances interfere with the assay.

Family history of diabetes is a major determinant of endothelial function.

OBJECTIVES: We evaluated whether endothelial dysfunction was present in nondiabetic persons with a family history (FH) of diabetes and assessed its relationship with insulin resistance and atherosclerosis risk factors. BACKGROUND: Atherosclerosis is frequently present when type 2 diabetes (T2D) is first diagnosed. Endothelial dysfunction contributes to atherogenesis. METHODS: Oral glucose tolerance and brachial artery flow-mediated, endothelium-dependent vasodilation (EDV) were assessed in 38 nondiabetic subjects; offspring of two parents with T2D (FH+) or with no first-degree relative with diabetes (FH-). RESULTS: Although fasting glucose was higher in FH+ than FH- (5.3 +/- 0.1 mmol/l vs. 4.9 +/- 0.1 mmol/l, p < 0.03), glycemic burden assessed as 2-h or area-under-the-curve glucose after glucose load or glycosylated hemoglobin (HbA1c), and measures of insulin sensitivity or inflammation did not differ. Brachial artery flow-mediated EDV was reduced in FH+ (7.1 +/- 0.9% vs. 11.7 +/- 1.6%, p < 0.02), with no difference in nitroglycerin-induced endothelium-independent vasodilatation. In the combined cohort, only FH+ (r2 = 0.12, p < 0.02) and HbA1c (r2 = 0.14, p < 0.02) correlated with EDV. Insulin resistance, assessed by tertile of homeostasis model assessment of insulin resistance (HOMA-IR), was associated with impaired endothelium-dependent vasodilatation in FH- (p < 0.03, analysis of variance), but not in FH+, as even the most insulin-sensitive FH+ offspring had diminished endothelial function. In multiple regression analysis, including established cardiac risk factors, blood pressure and lipids, HbA1c, and HOMA-IR, FH remained a significant determinant of EDV (p = 0.04). CONCLUSIONS: Bioavailability of nitric oxide is lower in persons with a strong FH of T2D. Glycemic burden, even in the nondiabetic range, can contribute to endothelial dysfunction. Abnormalities of endothelial function may contribute to atherosclerosis before development of overt diabetes.

Metformin inhibits proinflammatory responses and nuclear factor-kappaB in human vascular wall cells.

OBJECTIVE: Metformin may benefit the macrovascular complications of diabetes independently of its conventional hypoglycemic effects. Accumulating evidence suggests that inflammatory processes participate in type 2 diabetes and its atherothrombotic manifestations. Therefore, this study examined the potential action of metformin as an inhibitor of pro-inflammatory responses in human vascular smooth muscle cells (SMCs), macrophages (Mphis), and endothelial cells (ECs). METHODS AND RESULTS: Metformin dose-dependently inhibited IL-1beta-induced release of the pro-inflammatory cytokines IL-6 and IL-8 in ECs, SMCs, and Mphis. Investigation of potential signaling pathways demonstrated that metformin diminished IL-1beta-induced activation and nuclear translocation of nuclear factor-kappa B (NF-kappaB) in SMCs. Furthermore, metformin suppressed IL-1beta-induced activation of the pro-inflammatory phosphokinases Akt, p38, and Erk, but did not affect PI3 kinase (PI3K) activity. To address the significance of the anti-inflammatory effects of a therapeutically relevant plasma concentration of metformin (20 micromol/L), we conducted experiments in ECs treated with high glucose. Pretreatment with metformin also decreased phosphorylation of Akt and protein kinase C (PKC) in ECs under these conditions. CONCLUSIONS: These data suggest that metformin can exert a direct vascular anti-inflammatory effect by inhibiting NF-kappaB through blockade of the PI3K-Akt pathway. The novel anti-inflammatory actions of metformin may explain in part the apparent clinical reduction by metformin of cardiovascular events not fully attributable to its hypoglycemic action.

Elevated release of sCD40L from platelets of diabetic patients by thrombin, glucose and advanced glycation end products.

The pro-inflammatory CD40/CD40L dyad participates in atherogenesis. Plasma levels of the soluble ligand (sCD40L) predict cardiovascular events and are elevated in diabetic patients. This study compared CD40/CD40L surface expression on platelets and T lymphocytes of diabetic and control subjects, and tested whether glucose and advanced glycation end products (AGEs) stimulate sCD40L release. Constitutive and inducible surface expression of CD40/CD40L on platelets or T lymphocytes did not differ between diabetic patients (n = 9) and controls (n = 13). Platelets from diabetic patients contained higher intracellular CD40L than controls (p < 0.05) and thrombin stimulated greater platelet sCD40L release in diabetic patients (15.11 +/- 16.77 ng/ml) compared to controls (3.64 +/- 2.03 ng/ml; p < 0.05). Glucose and AGEs induced platelet sCD40L release and CD40L expression in mouse megakaryocytes. This study demonstrates elevated CD40L content and inducible release from platelets of diabetic patients, and identifies glucose and AGEs as potential triggers of expression and release accounting for the elevated sCD40L plasma levels in these patients.

Associations between soluble CD40 ligand, atherosclerosis risk factors, and subclinical atherosclerosis: results from the Dallas Heart Study.

OBJECTIVE: The purpose of this study was to evaluate the associations between plasma levels of soluble CD40 ligand (sCD40L), atherosclerosis risk factors, and evidence of subclinical atherosclerosis. METHODS AND RESULTS: Plasma levels of sCD40L were measured in 2811 subjects from the Dallas Heart Study, a multiethnic population-based cross-sectional study. Electron Beam Computed Tomography measurements of coronary artery calcium (CAC) and MRI measurements of aortic plaque were performed in 2198 and 1965 subjects, respectively. No association was observed between quartiles of sCD40L and age, sex, race, body mass index, diabetes, smoking, creatinine clearance, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, or C-reactive protein. In contrast, weak but statistically significant associations were observed between sCD40L and total cholesterol and triglycerides. The prevalence of detectable CAC (CAC score > or =10) and aortic plaque did not differ across sCD40L quartiles, and individuals with CAC scores <10, > or =10 to 100, >100 to 400, and >400 had similar sCD40L levels. CONCLUSIONS: In a large and representative multiethnic population-based sample, sCD40L was not associated with most atherosclerotic risk factors or with subclinical atherosclerosis. These findings suggest that sCD40L will not be useful as a tool to screen for the presence of subclinical atherosclerosis in the population. Further evaluation of this biomarker should focus on settings in which platelet activation is common, such as following acute coronary syndromes or coronary revascularization procedures.

Atherogenesis in mice does not require CD40 ligand from bone marrow-derived cells.

OBJECTIVE: Recent research suggests a central role for CD40 ligand (CD40L) in atherogenesis. However, the relevant cellular source of this proinflammatory cytokine remains unknown. To test the hypothesis that CD40L expressed on hematopoietic cell types (eg, macrophages, lymphocytes, platelets) is crucial to atherogenesis, we performed bone marrow reconstitution experiments using low-density receptor-deficient (ldlr-/-) and ldlr-/-/cd40l-/- compound-mutant mice. METHODS AND RESULTS: As expected, systemic lack of CD40L in hypercholesterolemic ldlr-/- mice significantly reduced the development of atherosclerotic lesions in the aortic arch, aortic root, and abdominal aorta compared with ldlr-/- mice. Furthermore, atheromata in ldlr-/-/cd40l-/- mice showed reduced accumulation of macrophages and lipids and increased content in smooth muscle cells and collagen compared with ldlr-/- mice. Surprisingly, reconstitution of irradiated ldlr-/- mice with ldlr-/-/cd40l-/- bone marrow did not affect the size or composition of atherosclerotic lesions in the root or arch of hypercholesterolemic ldlr-/- mice. Moreover, lipid deposition in the abdominal aorta diminished only marginally compared with mouse aortas reconstituted with ldlr-/- bone marrow. CONCLUSIONS: These experiments demonstrate that CD40L modulates atherogenesis, at least in mice, primarily by its expression on nonhematopoietic cell types rather than monocytes, T lymphocytes, or platelets, a surprising finding with important pathophysiologic and therapeutic implications.

Enhanced expression of CD44 variants in human atheroma and abdominal aortic aneurysm: possible role for a feedback loop in endothelial cells.

CD44, a polymorphic hyaluronate receptor, may participate in chronic inflammation. We hypothesized that CD44 variants contribute to the development of arterial diseases. CD44 levels vary in normal and diseased arterial tissues in the following order: unaffected arteries < fibrous plaques < or = abdominal aortic aneurysm < atheromatous plaques; and correlate with macrophage content. Furthermore, plaque microvessels express CD44, and anti-CD44v3 or anti-CD44v6 treatment reduces endothelial cell proliferation but not apoptosis in vitro, suggesting functionality of these receptors. Endothelial cells express CD44H and CD44v6 after exposure to interleukin-1beta and tumor necrosis factor-alpha. Macrophages, a major source of abundant CD44 in vitro, express not only CD44H but also variants CD44v4/5, CD44v6, and CD44v7/8, isoforms distinctively regulated by proinflammatory cytokines. Several proinflammatory cytokines induce shedding of CD44 from the surface of macrophages and endothelial cells. Soluble CD44 stimulates the expression and release of interleukin-1beta from endothelial cells, suggesting a positive feedback loop of this cytokine. By demonstrating augmented expression of CD44 and variants within human atheroma and in abdominal aortic aneurysm as well as the vascular cell release of sCD44, a process regulated by proinflammatory cytokines, this study provides new insights on the functions of CD44 in arterial diseases.

Inflammation, immunity, and HMG-CoA reductase inhibitors: statins as antiinflammatory agents?

According to traditional thinking, atherosclerosis results from passive lipid deposition in the vascular wall. Thus, therapies predominantly targeted lipid metabolism. The contemporary view of atherosclerosis, however, has broadened to include an active and complex role for inflammation, orchestrated in part by mediators of the immune system. This recognition prompted the question of whether antiinflammatory interventions might provide a novel avenue for the treatment of atherosclerosis. Uncertainties about the type of antiinflammatory regimen and appropriate patient selection currently hamper clinical investigation. Yet cardiovascular scientists have begun to address these questions at the bench, in experimental models, and indirectly in humans. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A HMG-CoA reductase (statins) have emerged as promising tools with dual functions. Originally designed to target elevated lipids, the "traditional" cause of atherosclerosis, statins might also confer cardiovascular benefit by directly or indirectly modulating the inflammatory component of this prevalent disease. Yet controversy persists regarding the (clinical) relevance of these potential non-LDL-lowering "pleiotropic" functions of statins. This overview addresses the controversy by reviewing in vitro and in vivo evidence regarding statins as antiinflammatory agents.

Diseased vein grafts express elevated inflammatory cytokine levels compared with atherosclerotic coronary arteries.

BACKGROUND: The pathologic modifications characterizing vein graft disease resemble those observed in native arteriosclerosis, but in accelerated form. Although both disorders are considered to be inflammatory diseases, it remains to be determined whether diseased vein grafts and atherosclerotic coronary arteries differentially express inflammatory mediators. Therefore, we examined whether differences in the expression of proinflammatory cytokines by these two distinct vascular pathologies favor the accelerated inflammation within diseased vein grafts. METHODS: The messengerRNA expression of various cytokines (interleukin-1 beta [IL-1 beta], IL-6, IL-8, tumor necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma]) was quantified using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in tissue samples of native saphenous veins (NSV, n = 5), diseased coronary arteries (CAD, n = 25), and diseased vein grafts (VG, n = 13). RESULTS: Native saphenous veins did not contain any detectable transcripts except for IFN-gamma. As expected, CAD was characterized by the expression of IL-1 beta, IL-6, IL-8, IFN-gamma, and TNF-alpha mRNA. Interestingly VG also expressed these mediators, but at markedly higher levels. Quantification by RT-PCR revealed that, compared with specimens from the CAD group, VG specimens contained 5.8 +/- 1.2 times, 286 +/- 22 times, and 29 +/- 7.3 times as many transcripts for the cytokines IL-1 beta, IL-6 and TNF-alpha, respectively, as well as 25 +/- 8.3 times more transcripts for the chemokine IL-8. In contrast, the expression of IFN-gamma transcripts did not differ among the groups. CONCLUSIONS: The elevated expression of proinflammatory cytokine transcripts supports the hypothesis that diseased vein grafts, compared with atherosclerotic coronary arteries, are characterized by enhanced inflammatory activity that might accelerate atherosclerotic modifications. This may implicate new therapeutic strategies for the prevention of vein graft disease.

Soluble CD40L: risk prediction after acute coronary syndromes.

BACKGROUND: Elevated plasma concentrations of soluble CD40 ligand (sCD40L) indicate increased risk for future cardiovascular events in apparently healthy women. This study tested the hypothesis that plasma sCD40L, alone or in combination with troponin (cTnI) or C-reactive protein (CRP), may identify patients with acute coronary syndromes at heightened risk for recurrent cardiac events. METHODS AND RESULTS: In a nested case-control study (cases, n=195; controls, n=195) within the OPUS-TIMI16 trial, patients with the prespecified study end points death, myocardial infarction (MI), or congestive heart failure (CHF) within 10 months had significantly higher median (25th, 75th percentiles) sCD40L plasma levels than did controls (0.78 [0.34, 1.73] ng/mL versus 0.52 [0.16, 1.42] ng/mL, P<0.002). After adjustment for other risk predictors and levels of cTnI and CRP, sCD40L levels above median were associated with higher risk for death, MI, and the composite death/MI or death/MI/CHF (adjusted hazard ratios, 1.9 [P<0.05], 1.9 [P<0.001], 1.9 [P<0.001], and 1.8 [P<0.01], respectively). Interestingly, patients with elevated plasma levels of sCD40L and cTnI showed a markedly increased risk of death, MI, or death/MI/CHF compared with patients with the lowest levels of both markers (adjusted hazard ratios, 12.1, 7.2, and 4.3, respectively; all P<0.01). CONCLUSIONS: Elevated plasma levels of sCD40L identify patients with acute coronary syndromes at heightened risk of death and recurrent MI independent of other predictive variables, including cTnI and CRP. Notably, combined assessment of sCD40L with cTnI complements prognostic information for death and MI.

Elevated plasma levels of the atherogenic mediator soluble CD40 ligand in diabetic patients: a novel target of thiazolidinediones.

BACKGROUND: Considerable evidence implicates the proinflammatory cytokine CD40 ligand (CD40L) in atherosclerosis and accumulating data link type 1 and 2 diabetes, conditions associated with accelerated atherosclerosis, to inflammation. This study therefore evaluated the hypothesis that diabetic patients have elevated plasma levels of soluble CD40L (sCD40L) and that treatment with the insulin-sensitizing thiazolidinediones lowers this index of inflammation. METHODS AND RESULTS: Subjects with type 1 (n=49) or type 2 diabetes (n=48) had higher (P<0.001) sCD40L plasma levels (6.56+/-3.27 and 6.67+/-2.90 ng/mL, respectively) compared with age-matched control groups (1.40+/-2.21 and 1.32+/-2.68 ng/mL, respectively). Multiple regression analysis demonstrated a significant (P<0.001) association between plasma sCD40L and type 1 as well as type 2 diabetes, independent of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, blood pressure, body mass index, gender, C-reactive protein, and soluble intracellular adhesion molecule-1. Furthermore, in a pilot study, administration of troglitazone (12 weeks, 600 mg/day), but not placebo, to type 2 diabetics (n=68) significantly (P<0.001) diminished sCD40L plasma levels by 29%. The thiazolidinedione lowered plasma sCD40L in type 2 diabetic patients with long-standing disease (>3 years) with or without macrovascular complications (-34% and -29%, respectively) as well as in type 2 diabetic patients with more recent (<3 years) onset of the disease (-27%; all P<0.05). CONCLUSIONS: This study provides new evidence that individuals with type 1 or 2 diabetes have a proinflammatory state as indicated by elevated levels of plasma sCD40L. Troglitazone treatment of type 2 diabetic patients diminishes sCD40L levels, suggesting a novel antiinflammatory mechanism for limiting diabetes-associated arterial disease.

Soluble CD40 ligand levels indicate lipid accumulation in carotid atheroma: an in vivo study with high-resolution MRI.

OBJECTIVE: The CD40/CD40 ligand pathway mediates inflammatory processes important in atherogenesis and the formation of the intraplaque lipid pool. We tested the hypothesis that plasma levels of soluble CD40 ligand are elevated in patients with evidence of a lipid pool on high-resolution magnetic resonance imaging (MRI) of carotid stenoses. METHODS AND RESULTS: We recruited 49 patients with evidence of carotid atherosclerosis on ultrasonography; 3 patients could not undergo carotid MRI because of claustrophobia. The remaining 46 patients underwent high-resolution MRI of the carotid arteries. Two radiologists blinded to all other data determined the presence or absence of an intraplaque lipid pool based on the loss of signal between the 20-ms echo time (TE20) and the fat-suppressed TE55 fast spin-echo images. Plasma levels of soluble CD40 ligand were determined by ELISA. Baseline levels of soluble CD40 ligand were higher among patients with evidence of intraplaque lipid (n=14) than among those without it (n=32; median, 2.54 ng/mL; interquartile range [IQR], 1.85 to 3.52 vs median, 1.58 ng/mL; IQR, 1.21 to 2.39; P=0.02). In contrast, soluble CD40 ligand levels were not correlated with percent diameter stenosis (r=-0.19; P=0.21). The relative risk for intraplaque lipid associated with soluble CD40 ligand levels above the median was 6.0 (95% confidence interval, 1.15 to 31.23; P=0.03). The magnitude of this predictive effect did not substantially change after adjustment for traditional cardiovascular risk factors (relative risk, 5.12; 95% confidence interval, 0.78 to 33.73; P=0.09). CONCLUSIONS: Plasma levels of soluble CD40 ligand may predict patients with features of high-risk atherosclerotic lesions. These data provide novel insight into the mechanism through which elevated levels of soluble CD40 ligand may reflect cardiovascular risk in humans and illustrate the potential value of interfacing high-resolution MRI with studies of vascular inflammation.

Oxidized low-density lipoprotein augments and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors limit CD40 and CD40L expression in human vascular cells.

BACKGROUND: Although CD40 signaling participates in atherosclerosis, links between lipid risk factors and this inflammatory pathway remain obscure. Cardiovascular risk reduction by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may involve actions beyond lipid lowering, including reduced inflammation. Therefore, this study analyzed whether oxidized low-density lipoprotein (oxLDL) induces CD40/CD40L expression on cells implicated in atherogenesis and whether statins affect their expression in vitro as well as the expression of soluble CD40L (sCD40L) in vivo. METHODS AND RESULTS: Treatment of human vascular endothelial and smooth muscle cells and mononuclear phagocytes with oxLDL augmented the basal expression of CD40 and CD40L mRNA and protein. In contrast, cerivastatin, atorvastatin, or simvastatin concentration-dependently diminished the constitutive as well as oxLDL- or cytokine-induced expression of the receptor/ligand dyad, an effect reversed by mevalonate. Patients treated with statins had diminished sCD40L plasma levels compared with untreated control patients (8.3+/-3.1 ng/mL [n=11] versus 13.1+/-2.5 ng/mL [n=16], P<0.05), supporting the clinical relevance of the in vitro observations. Platelet-enriched plasma of mice deficient in CD40L showed markedly delayed fibrin clot formation, suggesting a role for the ligand in blood coagulation and supporting the hypothesis that statin-mediated reduction in CD40/CD40L expression might limit thrombosis. CONCLUSIONS: OxLDL may promote expression of CD40 and CD40L in human atheroma. Statins may limit the expression of the CD40 receptor/ligand dyad in two ways, directly as well as through diminished lipoprotein levels. Thus, reduced CD40 signaling may account for some of the statins' antiinflammatory action.

Cytokines in the pathogenesis of atherosclerosis.

Research during the last two decades established atheromatous lesions as active sites of inflammation and immune responses, contrasting to the traditional view of atherosclerosis as an acellular lesion composed of lipid deposits. In particular, cytokines appear to orchestrate the chronic development of atherosclerosis, eventually leading to the formation of complex atherosclerotic plaques, which can trigger acute thromboembolic complications, such as myocardial infarction or stroke. Indeed the rupture-prone plaque, characterized by a thin fibrous cap overlaying a voluminous lipid core, exhibits accumulation of various pro-inflammatory cytokines. These cytokines may control the clinical consequences of plaques by mediating infiltration and accumulation of immunocompetent cells, directing the turnover of fibrillar collagens (governing the fragility of the fibrous cap), or enhancing foam cell formation and thrombogenicity of the lipid core, among other processes outlined in this review. Thus, understanding the role of cytokines in the pathophysiology of the atherosclerotic plaque might provide a promising therapeutic avenue for this prevalent human disease. This review will focus on members of the interleukin, tumor necrosis factor, and interferon families of cytokines in modulating key processes of atherogenesis.

Innate and adaptive immunity in the pathogenesis of atherosclerosis.

This review considers critically the evidence for the involvement of mediators of innate and acquired immunity in various stages of atherosclerosis. Rapidly mobilized arms of innate immunity, including phagocytic leukocytes, complement, and proinflammatory cytokines, contribute to atherogenesis. In addition, adaptive immunity, with its T cells, antibodies, and immunoregulatory cytokines, powerfully modulates disease activity and progression. Atherogenesis involves cross talk between and shared pathways involved in adaptive and innate immunity. Immune processes can influence the balance between cell proliferation and death, between synthetic and degradative processes, and between pro- and antithrombotic processes. Various established and emerging risk factors for atherosclerosis modulate aspects of immune responses, including lipoproteins and their modified products, vasoactive peptides, and infectious agents. As we fill in the molecular details, new potential targets for therapies will doubtless emerge.

T(H)2 predominant immune responses prevail in human abdominal aortic aneurysm.

T lymphocytes localize within lesions of two diametrically opposed expressions of atherosclerosis: stenosis-producing plaques and ectasia-producing abdominal aortic aneurysm (AAA). T(H)1 immune responses appear to predominate in human stenotic lesions. However, little information exists regarding the nature of the T-cell infiltrate in AAAs. We demonstrate here that AAAs predominantly express T(H)2-associated cytokines and correspondingly lack mediators associated with the T(H)1 response as determined by Western blot and immunohistochemical analysis. In particular, aneurysmal tissue expressed interleukin (IL)-4, IL-5, and IL-10, cytokines not or only faintly detected in nondiseased tissue or stenotic atheroma. In contrast, AAAs contained low levels of the T(H)1 characteristic cytokines IL-2 and IL-15, which are amply expressed in stenotic lesions. Notably, stenotic lesions, but not AAAs, contained mature forms of the interferon-gamma-inducing cytokines IL-12 and IL-18 as well as the IL-18-processing enzyme caspase-1. Moreover, aneurysmal tissue lacked the receptor for interferon-gamma, although both types of lesions contained this T(H)1-promoting cytokine. These findings suggest that the functional repertoire of T cells differs in stenotic and aneurysmal lesions, and provide a novel framework for understanding the mechanisms of these diametrically opposite expressions of atherosclerosis.