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A trait of inflammation pathogenesis in human atherosclerosis: inflammasome driven interleukin-1 signalling in complex atherosclerotic plaques via hyperlipidemia trained innate immunity Objectives We aimed to investigate interleukin (IL)-1 generation and the regulatory role of inflammasome in human advanced atherosclerosis. Background IL-1β is key contributor to the inflammatory process associated with atherosclerosis and its complications. Recent studies suggested that IL-1β blockade reduces the burden of inflammation and recurrence of cardiovascular events. Yet, other cytokines in IL-1 family and the regulation of IL-1 generation in patients with atherosclerosis remains poorly understood. Methods and results A focused transcriptomic analysis in human atherosclerotic specimens discovered that human atherosclerotic plaques host a broad reservoir of inflammasome components, characterised by expression of canonical inflammasome gene NLRP6, NLRP12, NLRC4, NLRP3 and non-canonical inflammasome gene caspase 4 significantly elevated in the symptomatic plaques versus the asymptomatic plaques. Upregulation of NLRP3 inflammasome expression in plaque validated by immunohistochemistry staining suggested it as a distinctive characteristic of plaque vulnerability and complexity. Functional studies on atherosclerotic explants obtained from patients undergoing carotid endarterectomy revealed constitutive generation of IL-1β accompanied by secretion of comparable levels of IL-1α from the majority of the plaques, while IL-18 and IL-33 generation from some of the plaques. Stimulation of the plaques with inflammasome activators showed an inducible generation of both IL-1α and IL-1β, not IL-18 or IL-33, mediated by specific canonical and non-canonical inflammasome pathways. Analysis on the medication records of these patients indicated that plaques from patients with suboptimally controlled hyperlipidemia, imaging signs for plaque instability and inadequate statins therapy possessed higher recruitable production of IL-1β, suggesting the conventional atherogenic factor in regulation of inflammasome immunity and disease activity. Mechanistic studies on tissue and cells isolated from atheromatous plaques demonstrate that generation of mature IL-1β is via a mechanism controlled by NLRP3 and the effector caspase-1. Conclusions The study supports a profound canonical and non-canonical inflammasomes mediated plaque IL-1α/β generation, via a key mechanism by NLRP3 and caspase-1. The results provide biological insights into the clinical merit of high-intensity cholesterol lowering and anti-IL-1 signalling-directed therapies in high-risk patients with atherosclerosis. Acknowledgement/Funding KI-Mayo collaboration project, the Swedish Research Council, the Swedish Heart-Lung Foundation, European Union FP7 projects, the NIH

Aims Radiotherapy-induced cardiovascular disease is an emerging problem in a growing population of cancer survivors where traditional treatments, such as anti-platelet and lipid-lowering drugs, have limited benefits. The aim of the study was to investigate vascular inflammatory patterns in human cancer survivors, replicate the findings in an animal model, and evaluate whether interleukin-1 (IL-1) inhibition could be a potential treatment. Methods and results Irradiated human arterial biopsies were collected during microvascular autologous free tissue transfer for cancer reconstruction and compared with non-irradiated arteries from the same patient. A mouse model was used to study the effects of the IL-1 receptor antagonist, anakinra, on localized radiation-induced vascular inflammation. We observed significant induction of genes associated with inflammasome biology in whole transcriptome analysis of irradiated arteries, a finding supported by elevated protein levels in irradiated arteries of both, pro-caspase and caspase-1. mRNA levels of inflammasome associated chemokines CCL2, CCL5 together with the adhesion molecule VCAM1, were elevated in human irradiated arteries as was the number of infiltrating macrophages. A similar pattern was reproduced in Apoe−/− mouse 10 weeks after localized chest irradiation with 14 Gy. Treatment with anakinra in irradiated mice significantly reduced Ccl2 and Ccl5 mRNA levels and expression of I-Ab. Conclusion Anakinra, administered directly after radiation exposure for 2 weeks, ameliorated radiation induced sustained expression of inflammatory mediators in mice. Further studies are needed to evaluate IL-1 blockade as a treatment of radiotherapy-induced vascular disease in a clinical setting.

Objective— Cholinergic pathways of the autonomic nervous system are known to modulate inflammation. Because atherosclerosis is a chronic inflammatory condition, we tested whether cholinergic signaling operates in this disease. We have analyzed the expression of the α7 nicotinic acetylcholine receptor (α7nAChR) in human atherosclerotic plaques and studied its effects on the development of atherosclerosis in the hypercholesterolemic Ldlr –/– mouse model. Approach and Results— α7nAChR protein was detected on T cells and macrophages in surgical specimens of human atherosclerotic plaques. To study the role of α7nAChR signaling in atherosclerosis, male Ldlr –/– mice were lethally irradiated and reconstituted with bone marrow from wild-type or α7nAChR-deficient animals. Ablation of hematopoietic cell α7nAChR increased aortic atherosclerosis by 72%. This was accompanied by increased aortic interferon-γ mRNA, implying increased Th1 activity in the absence of α7nAChR signaling. Conclusions— The present study shows that signaling through hematopoietic α7nAChR inhibits atherosclerosis and suggests that it operates by modulating immune inflammation. Given the observation that α7nAChR is expressed by T cells and macrophages in human plaques, our findings support the notion that cholinergic regulation may act to inhibit disease development also in man.

Atherosclerosis is an inflammatory disease associated with the activation of innate immune TLRs and nucleotide-binding oligomerization domain-containing protein (NOD)like receptor pathways. However ...

SummaryLow-density lipoproteins (LDL), occurring in vivo in both their native and oxidative form, modulate platelet function and thereby contribute to atherothrombosis. We recently identified and demonstrated that ‘ApoB100 danger-associated signal 1’ (ApoBDS-1), a native peptide derived from Apolipoprotein B-100 (ApoB100) of LDL, induces inflammatory responses in innate immune cells. Platelets are critically involved in the development as well as in the lethal consequences of atherothrombotic diseases, but whether ApoBDS-1 has also an impact on platelet function is unknown. In this study we examined the effect of ApoBDS-1 on human platelet function and platelet-leukocyte interactions in vitro. Stimulation with ApoBDS-1 induced platelet activation, degranulation, adhesion and release of proinflammatory cytokines. ApoBDS-1-stimulated platelets triggered innate immune responses by augmenting leukocyte activation, adhesion and transmigration to/through activated HUVEC monolayers, under flow conditions. These platelet-activating effects were sequence-specific, and stimulation of platelets with ApoBDS-1 activated intracellular signalling pathways, including Ca2+, PI3K/Akt, PLC, and p38– and ERKMAPK. Moreover, our data indicates that ApoBDS-1-induced platelet activation is partially dependent of positive feedback from ADP on P2Y1 and P2Y12, and TxA2. In conclusion, we demonstrate that ApoBDS-1 is an effective platelet agonist, boosting platelet-leukocyte’s proinflammatory responses, and potentially contributing to the multifaceted inflammatory-promoting effects of LDL in the pathogenesis of atherothrombosis.

AimsPatients with hyperlipidemia are at risk of atherosclerosis, but not all develop cardiovascular disease, highlighting the importance of other risk factors such as inflammation. Both the innate and adaptive arms of the immune system have been suggested in the initiation and propagation of plaque formation. Tri-partite motif (TRIM) 21 is a regulator of tissue inflammation and pro-inflammatory cytokine production, and has been implicated in chronic inflammatory disease. Here, we investigate a potential role for TRIM21 in coronary artery disease.Methods and resultsTrim21-deficient or wild-type bone marrow was transplanted into Ldlr-/- mice fed a hypercholesterolemic diet. The Trim21-/-->Ldlr-/- mice developed larger atherosclerotic plaques, with significantly higher collagen content compared to mice transplanted with wild-type cells. High collagen content of the atheroma is stabilizing, and has recently been linked to IL-17. Interestingly, Trim21-/-->Ldlr-/- mice had elevated CD4 and IL-17 mRNA expression in plaques, and increased numbers of activated CD4+ T cells in the periphery. An increased differentiation of naïve T cells lacking Trim21 into Th17 cells was confirmed in vitro, with transcriptomic analysis revealing upregulation of genes of a non-pathogenic Th17 phenotype. Also, decreased expression of matrix metalloproteinases (MMPs) was noted in aortic plaques. Analysis of human carotid plaques confirmed that TRIM21 expression negatively correlates with the expression of key Th17 genes and collagen, but positively to MMPs also in patients, linking our findings to a clinical setting.ConclusionIn this study, we demonstrate that TRIM21 influences atherosclerosis via regulation of Th17 responses, with TRIM21 deficiency promoting IL-17 expression and a more fibrous, stable, phenotype of the plaques.

Clinical studies show an increase risk for cardiovascular disease (CVD) at the site of irradiation years after exposure. Radiotherapy together with other cancer therapies contributes to a growing population of cancer survivors in risk of developing radiotherapy induced vascular disease. However, traditional treatments for CVD such as thrombolytic inhibitors and lipid lowering drugs (statins) seem to have no effect on radiation induced vascular disease in mouse models. We have previously demonstrated a sustained chronic inflammation by NF-kB activation in irradiated human arteries. To further investigate radiation induced vascular disease and potential therapies against innate inflammation, we established a mouse model of local irradiation in an atherosclerotic prone ApoE-/- background. ApoE-/- mice fed chow diet were exposed to a single dose of 0 Gy (sham/controls) or 14 Gy at the upper chest and neck area and harvested 10 weeks later. Plasma, thoracic aorta, aortic arch, heart were retrieved and taken for plasma, blood count, en face, mRNA, immunohistochemistry and immunofluorescence analysis. Irradiated mice presented higher total plasma cholesterol levels than controls. En face analysis of aortic arches showed a decreased percentage of lesion size in irradiated arteries compared to controls. However, an increase of MHC class II (IA-b) staining in irradiated aortic roots suggested an induction of inflammation in the context of radiotherapy. These data were supported by the quantification of monocyte chemotactic protein 1 mRNA expression in the thoracic aorta showing a significant increase in irradiated animals compared to unirradiated (p≤0,001). The current data demonstrated that radiotherapy induces a macrophage-rich and inflamed plaque that may explain the increased risk of developing severe clinical events i.e. myocardial infarction or stroke. Further analyses of this partial irradiation mouse model are on-going to assess the potential therapeutic targets involved in innate immunity and NF-kB activation. Results of currently performed therapeutic experiments will be further presented.

Rationale: The innate immune system and in particular the pattern-recognition receptors Toll-like receptors have recently been linked to atherosclerosis. Consequently, inhibition of various signaling molecules downstream of the Toll-like receptors has been tested as a strategy to prevent progression of atherosclerosis. Receptor-interacting protein 2 (Rip2) is a serine/threonine kinase that is involved in multiple nuclear factor-κB (NFκB) activation pathways, including Toll-like receptors, and is therefore an interesting potential target for pharmaceutical intervention. Objective: We hypothesized that inhibition of Rip2 would protect against development of atherosclerosis. Methods and Results: Surprisingly, and contrary to our hypothesis, we found that mice transplanted with Rip2 −/− bone marrow displayed markedly increased atherosclerotic lesions despite impaired local and systemic inflammation. Moreover, lipid uptake was increased whereas immune signaling was reduced in Rip2 −/− macrophages. Further analysis in Rip2 −/− macrophages showed that the lipid accumulation was scavenger-receptor independent and mediated by Toll-like receptor 4 (TLR4)–dependent lipid uptake. Conclusions: Our data show that lipid accumulation and inflammation are dissociated in the vessel wall in mice with Rip2 −/− macrophages. These results for the first time identify Rip2 as a key regulator of cellular lipid metabolism and cardiovascular disease.

To highlight critical advances achieved over the last year in the study of endogenous proatherogenic danger signals and corresponding molecular mechanism of innate immune signalling in atherosclerosis.The identity and signalling mechanisms of LDL-derived inflammatory components are central in understanding the pathogenic role of modified LDL in the development of atherosclerosis. Studies in the preceding years have revealed LDL-derived phospholipids and cholesterol crystals as endogenous danger signals. These danger signals trigger Toll-like receptors and nucleotide-binding oligomerization domain-like receptors inflammasome respectively, thereby instigating inflammatory responses and promoting disease progression.Recent understandings of the causal role of LDL in atherosclerosis provide a new perspective on modified LDL-derived danger signals. These insights suggest dysregulated Toll-like receptor and nucleotide-binding oligomerization domain inflammasome signalling as an important mechanism underlying atherogenesis.

Inflammation underpins the development of atherosclerosis. Initiation and progression of vascular inflammation involves a complex cellular network, with macrophages as major contributors. Activated macrophages produce proinflammatory mediators, bridge innate and adaptive immunity, regulate lipid retention, and participate directly in vascular repair and remodeling. Recent efforts to elucidate molecular mechanisms involved in the regulation of vascular inflammation in atherosclerosis have implicated several families of innate immune recognition receptors in inflammatory activation during the course of this disease. This article reviews our current understanding of innate immune recognition receptors, signaling pathways, and putative ligands implicated in activation of macrophages in the disease. In its final section, we propose a model for the role of macrophages in bridging inflammation and atherosclerosis from the perspective of innate immune recognition and activation.

The role of nuclear factor kappa-B (NF-kappaB) p105 for vascular inflammatory gene expression and neointima formation after arterial injury was studied. Mice carotid arteries were injured by ligation. Vascular NF-kappaB activation was monitored using a NF-kappaB luciferase reporter mouse. Mice with gene deletion of the NF-kappaB p105 subunit (p50 precursor) and the corresponding wild types were assessed for vascular gene expression and neointimal hyperplasia. NF-kappaB was activated in the injured vessel wall in wild type mice, and this was accompanied by increased expression of the proinflammatory genes tumor necrosis factor alpha, interleukin 1 beta, and inducible nitric oxide synthase. In contrast, NF-kappaB p105 knockout mice had reduced expression of the inflammatory genes and enhanced neointima formation four weeks after ligation. Basic fibroblast growth factor (bFGF) gene expression increased after arterial ligation. A higher percentage of bFGF positive cells were found in lesions from NF-kappaB p105 knock out mice. These data indicate that the p105 subunit of NF-kappaB plays an essential role in vascular healing, and defects in NF-kappaB p105 promote neointima hyperplasia.

Leukotriene B 4 (LTB 4 ), a potent leukocyte chemoattractant derived from the 5-lipoxygenase metabolism of arachidonic acid, exerts its action by means of specific cell surface receptors, denoted BLT 1 and BLT 2 . In this study, BLT 1 receptor proteins were detected in human carotid artery atherosclerotic plaques, colocalizing with markers for macrophages, endothelial cells, and vascular smooth muscle cells (SMC). Challenge of human coronary artery SMC with either LTB 4 or U75302, a partial agonist that is selective for the BLT 1 receptor, induced an ≈4-fold increase of whole-cell currents by using the patch-clamp technique, indicating that these cells express functional BLT 1 receptors. LTB 4 induced migration and proliferation of SMC in vitro , and treatment with the BLT receptor antagonist BIIL 284 (10 mg/kg, once daily) for 14 days after carotid artery balloon injury in vivo inhibited intimal hyperplasia in rats. In the latter model, SMC derived from the intima exhibited increased levels of BLT 1 receptor mRNA compared with medial SMC. BLT receptor up-regulation in the intima in vivo , as well as that induced by IL-1β in vitro , were prevented by transfection with a dominant-negative form of Iκ kinase β carried by adenovirus, indicating that BLT 1 receptor expression depends on NF-κΒ. These results show that LTB 4 activates functional BLT 1 receptors on vascular SMC, inducing chemotaxis and proliferation, and that BLT 1 receptors were up-regulated through an Iκ kinase β/NF-κB-dependent pathway. Inhibition of LTB 4 /BLT 1 signaling during the response to vascular injury reduced intimal hyperplasia, suggesting this pathway as a possible target for therapy.

Aim Toll-like receptor 4 (TLR4) is a receptor for bacterial lipopolysaccharide (LPS) and heat shock protein essential for innate immunity. Recent studies imply that TLR4 polymorphisms might affect atherogenesis. In this study we investigated the impact of LPS-hypo-responsive TLR4 variants on the risk of myocardial infarction (MI). Methods and results Using TaqMan PCR technology, we determined the prevalence of the Asp299Gly and Thr399Ile polymorphisms in the TLR4 gene, and their association with MI in a study of 1213 survivors of a first MI and 1561 controls from the Stockholm region. The frequency was 0.096 for carriers of both 299Gly and 399Ile, and 0.006 for carriers of 399Ile alone. Carriers of both 299Gly and 399Ile were more frequent among the male cases than the male controls (10.7% vs 7.9%, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(p=0.004\) \end{document}). Compared with wild-type carriers, men with the 299Gly and the 399Ile TLR4 genotype had an increased risk of MI (OR [95% CI]: 1.4 [1.0;1.9]) whereas no association was observed for women. Furthermore a synergistic interaction was found between the TLR4 polymorphism and smoking in men. Conclusion The association found between TLR4 genotype and risk of MI suggests that TLR4 genetic variants could potentially affect the susceptibility to MI and that TLR4-mediated innate immunity is implicated in the pathogenesis of MI.

Leukocyte recruitment is crucial for the response to vascular injury in spontaneous and accelerated atherosclerosis. Whereas the mechanisms of leukocyte adhesion to endothelium or matrix-bound platelets have been characterized, less is known about the proadhesive role of smooth muscle cells (SMCs) exposed after endothelial denudation. In laminar flow assays, neointimal rat SMCs (niSMCs) supported a 2.5-fold higher arrest of monocytes and "memory" T lymphocytes than medial SMCs, which was dependent on both P-selectin and VLA-4, as demonstrated by blocking antibodies. The increase in monocyte arrest on niSMCs was triggered by the CXC chemokine GRO-alpha and fractalkine, whereas "memory" T cell arrest was mediated by stromal cell-derived factor (SDF)-1alpha. This functional phenotype was paralleled by a constitutively increased mRNA and surface expression of P-selectin and of relevant chemokines in niSMCs, as assessed by real-time PCR and flow cytometry. The increased expression of P-selectin in niSMCs versus medial SMCs was associated with enhanced NF-kappaB activity, as revealed by immunofluorescence staining for nuclear p65 in vitro. Inhibition of NF-kappaB by adenoviral IkappaBalpha in niSMCs resulted in a marked reduction of increased leukocyte arrest in flow. Furthermore, P-selectin expression by niSMCs in vivo was confirmed in a hypercholesterolemic mouse model of vascular injury by double immunofluorescence and by RT-PCR after laser microdissection. In conclusion, we have identified a NF-kappaB-mediated proinflammatory phenotype of niSMCs that is characterized by increased P-selectin and chemokine expression and thereby effectively supports leukocyte recruitment.

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.

Background — Innate immune reactions against bacteria and viruses have been implicated in the pathogenesis of atherosclerosis. To explore the molecular mechanism by which microbe recognition occurs in the artery wall, we characterized the expression of toll-like receptors (TLRs), a family of pathogen pattern recognition receptors, in atherosclerotic lesions. Methods and Results — Semiquantitative polymerase chain reaction and immunohistochemical analysis demonstrated that of 9 TLRs, the expression of TLR1, TLR2, and TLR4 was markedly enhanced in human atherosclerotic plaques. A considerable proportion of TLR-expressing cells were also activated, as shown by the nuclear translocation of nuclear factor-κB. Conclusion — Our findings illustrate a repertoire of TLRs associated with inflammatory activation in human atherosclerotic lesions, and they encourage further exploration of innate immunity in the pathogenesis of atherosclerosis.

Objective: The aim was to examine the expression of the alpha 7 nicotinic acetylcholine receptor (α7R) in human atherosclerotic plaques and to investigate α7R effects on progression of atherosclerosis in hypercholesterolemic Ldlr-/- mice. Methods: Human carotid plaques and aortic lesions of hypercholesterolemic mice were stained for α7R, using immunohistochemistry. To study the role of cholinergic signaling in atherosclerosis, male Ldlr-/- mice were lethally irradiated and reconstituted with bone marrow from wildtype (WT) or α7R deficient animals. Results: α7R was detected on T cells and macrophages in human carotid plaques. Ablation of hematopoietic cell α7R in Ldlr-/- mice increased aortic atherosclerosis by 38%. This was accompanied by increased aortic interferon-γ mRNA, implying increased Th1 activity in the absence of α7R signaling. Conclusion: α7R is expressed by T cells and macrophages in human plaques. Lack of α7R in bone marrow dramatically accelerates atherosclerosis in LDLr-/- mice. The present...

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.

Septic shock, a major cause of death, is characterized by a pathophysiologic increased production of nitric oxide (NO), which leads to vasodilation and myocardial toxicity. Septic Escherichia coli frequently express proteinaceous curli fibers. In this study, curliated E. coli induced high levels of NO by directly inducing type 2 nitric oxide synthase (NOS2) both in vitro and in vivo. More severe hypotension and higher plasma nitrite/nitrate levels were seen in wild type mice systemically infected with curliated E. coli than in animals infected with E. coli mutants that lacked curli proteins. Blood pressure remained stable in NOS2-deficient mice with curliated bacteria. Increased heart rates, transient hypothermia, and loss of gross activity were seen in all mice, regardless of curli expression. Study results suggest that expression of curli fibers by E. coli activates the NO/NOS2 arm of the innate immune system, which leads to a significant fall in blood pressure.

Abstract —We have recently shown that all- trans retinoic acid (tRA) modulates arterial smooth muscle cell (SMC) morphologic features and biochemical composition in vitro. It has been proposed that different SMC phenotypes coexist in arteries, which may be retrieved in culture: hence, a differential action of tRA on distinct SMC subsets is conceivable. We have examined the effect of tRA on SMC proliferation, migration, plasminogen activator activity, and α-smooth muscle actin expression in 2 phenotypically different rat SMC populations, cultured respectively from the normal aortic media and from the intimal thickening (IT) after endothelial injury. tRA inhibited proliferation and increased migration and tissue-type plasminogen activator activity in both SMC populations, but decreased α-smooth muscle actin only in SMC cultured from the IT. The action of tRA is mediated by 2 families of nuclear receptors, RAR and RXR, each containing 3 isoforms, α, β, and γ. RAR and RAR-α agonists, but not RXR agonists, inhibited SMC proliferation in both cell populations and α-smooth muscle actin expression only in IT SMC. When administered intraperitoneally to balloon-injured rats, tRA and RAR-α agonists reduced the intimal hyperplasia in the carotid artery. Our results show that tRA and synthetic retinoids can affect the proliferation, migration, and differentiation of SMC in vitro. Furthermore, retinoids are able to reduce the IT induced by endothelial injury in vivo.

Abstract —The formation of a neointima represents an important repair mechanism in response to vascular injury. It is associated with the expression of a specific set of genes by the intimal smooth muscle cells. Recently, expression of the inducible isoform of NO synthase (iNOS) has been identified in injured arteries during neointimal formation, suggesting that intimal SMCs have a unique mechanism for regulating NO production. Therefore, we have analyzed the expression of iNOS in intimal SMCs. Although first expressed in the media within 1 day after injury, iNOS was confined to neointimal smooth muscle cells at 1 to 2 weeks after injury. Isolated intimal SMCs were found to consistently reexpress iNOS in reaction to proinflammatory mediators. This was associated with a 5- to 8-fold higher output of NO in comparison with SMCs derived from the media of uninjured arteries. Western blot and Northern blot analyses likewise revealed that the high production of NO by intimal SMCs was due to overexpression of iNOS. Moreover, the same stimuli induced a higher transcriptional activity in intimal than in medial SMCs, as detected by transfection of a reporter gene under the iNOS promoter. Induction of iNOS led to a reduced proliferation in both medial and intimal SMCs. This inhibitory effect was, however, less pronounced in intimal than in medial SMCs. Similarly, intimal cells were less sensitive to NO-induced inhibition of mitochondrial respiration. When SMC clones were analyzed, there was no correlation between iNOS expression and growth pattern, suggesting that iNOS expression is independent of the morphological phenotype of SMCs. Together, our data show that the intimal SMC is the main iNOS-expressing cell type in the injured artery, that it responds more vividly to iNOS-inducing cytokines because of a more efficient activation of the iNOS promoter, and that it is more resistant to the actions of NO compared with medial SMCs. Intimal production of NO via the inducible pathway may be important for the restoration of vascular homeostasis after injury.

Toll-like receptor (TLR) 4 is the first identified mamalian homolog of the Drosophila Toll protein.1 TLR4 recognizes bacterial lipopolysaccharide (LPS), and also senses endogenous ligands including hyaluronic acid, oxidized low-density lipoprotein, and heat-shock proteins. Engagement of TLR4 with its ligand triggers a cascade of cellular signals through the intracellular signal transduction domain, known as Toll/interleukin (IL)-1 receptor (TIR), leading to the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signal transduction pathways, consequently inducing expression of inflammatory genes.2 Although the TLR family of molecules normally contributes to host defense, excess TLR signaling has been implicated in many inflammatory diseases. That TLR4 is implicated in cardiovascular diseases was first suggested by observations of increased expression of TLR4 in failing hearts3 and in atherosclerotic lesions.4,5 The functional importance of TLR4 has subsequently been demonstrated by the attenuated vascular inflammation and reduced lesion size in TLR4-deficient atherosclerosis prone mice,6 and by the association of hyporesponsive TLR4 variants with disease development.7,8 Furthermore, excessive activation of TLR4 by LPS induces endotoxin shock, a serious systemic disorder with a high mortality rate. Therefore TLR signaling must be tightly controlled to avoid improper activation or suppression. Yet regulation of TLR4 expression in cardiovascular disease remains elusive. In the current issue of Arteriosclerosis, Thrombosis, and Vascular Biology , two sister papers by Feng-Yen Lin and colleagues9,10 lend new mechanistic insights that oxidative stress induces posttranscriptional stabilization of TLR4 mRNA in vascular cells. See pages 2622 and 2630 Posttranscriptional regulation of mRNA stability is a critical determinant in gene expression, in particular for genes encoding cytokines, transcription factors, growth factors and oncoproteins that …

NO generated by endothelial cells is vasoprotective by antagonizing platelet adhesion and smooth muscle contraction. Since vascular smooth muscle cells (VSMCs) produce NO in response to cytokine stimulation and after arterial injury, we speculated that NO produced by VSMCs could compensate for the loss of endothelium. Using balloon catheter denudation of the rat carotid artery as a model for arterial injury and restenosis, we have evaluated the time course of expression of inducible NO synthase (iNOS) by in situ hybridization and immunohistochemistry and studied the effect of iNOS on platelet adhesion and blood flow of the injured vessel. iNOS mRNA and protein were expressed in the innermost layer of the media from day 1 and were subsequently detected in the neointima, whereas no expression was detectable in the uninjured artery. Systemic administration of N ω -nitro- l -arginine methyl ester (L-NAME) resulted in a twofold to threefold increase in the adhesion of 111 In-labeled platelets to the injured vessel wall. Platelet adhesion was also enhanced threefold by local delivery of L-NAME from a gel surrounding the injured vessel, whereas the stereoisomer, D-NAME, had no effect. Finally, inhibition of NO synthase led to a 24% reduction of the blood flow in the injured carotid artery. These results demonstrate that arterial injury triggers the expression of iNOS in the lesion and that NO produced by iNOS inhibits platelet adhesion and restores blood flow. This could explain the disappearance of platelet thrombi from deendothelialized arterial surfaces within a few days after injury and indicates the importance of NO generated by iNOS for the maintenance of vascular tone. Thus, expression of iNOS in lesions may represent a protective mechanism that compensates for the loss of endothelium.

Objective— The activity of eicosanoid pathways is critical to the inflammatory and immune responses that are associated with the progression of atherosclerosis. Yet, the signals that regulate these pathways are poorly understood. Here, we address whether the innate immune signals of nucleotide-binding oligomerization domain–containing protein (NOD) 2 affect eicosanoids metabolism in atherosclerosis. Approach and Results— Analysis of human carotid plaques revealed that NOD2 was abundantly expressed at both mRNA and protein levels by endothelial cells and macrophages. Stimulation of NOD2 in ex vivo–cultured carotid plaques by muramyl dipeptide, an extrinsic ligand of NOD2, led to release of prostaglandin E 2 , upregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, and to downregulation of cyclooxygenase-1. NOD2 was coexpressed with cyclooxygenase-2 in lesional macrophages. NOD2-induced cyclooxygenase-2 expression in macrophages was dependent on p38 mitogen-activated protein kinase activation and was mediated by interleukin-1β and tumor necrosis factor-α. Selective lipidomic analysis of the eicosanoids released by the carotid plaques characterized the metabolites of 12-, 5-, and 15-lipoxygenase as the predominant eicosanoids that were produced by the atherosclerotic lesion in the absence of additional stimuli. Unlike the prostaglandin E 2 pathway, metabolic activity of the lipoxygenase pathways was not altered on the short-term activation of NOD2 in carotid plaques. Conclusions— These results suggest that atherosclerosis may involve enhanced NOD2-mediated innate immunity. Activation of NOD2 preferentially upregulates the prostaglandin E 2 pathway. Nevertheless, lipoxygenase pathways, such as 12-lipoxygenase, predominate the basal synthesis and metabolism of eicosanoids in atherosclerotic plaques. These findings provide new insights into the regulation of eicosanoids in atherosclerosis.

Objective Atherosclerosis is an inflammatory condition, and rupture of atherosclerotic plaques is a major cause of cardiovascular disease (CVD). Lysophosphatidylcholine (LPC) is generated in low-density lipoprotein (LDL) during oxidation and/or enzymatic modification and has been implicated in atherosclerosis. Annexin A5 (ANXA5) is an antithrombotic and atheroprotective plasma protein. Here, we demonstrate novel pro-inflammatory and atherogenic properties of LPC, and inhibitory effects of ANXA5. Methods Endothelial cells and macrophages (differentiated from, THP-1 a monocytic cell line) were co-cultured. Expression of MMP-9 and OxLDL uptake by macrophages were studied by flow cytometry. The effect of LPC on leukotriene B4 (LTB4) synthesis in macrophages was studied by enzyme immunoassay (EIA). Chemotactic properties of LPC were investigated using a mouse intra-peritoneal recruitment model. Results Co-culture of macrophages and endothelial cells enhanced MMP-9 expression in both cell types. This effect was increased by LPC and diminished by ANXA5. Likewise, LPC induced LTB4 production by macrophages, whereas native LDL or phosphatidylcholine (PTC) had no effect. ANXA5 inhibited uptake of OxLDL in macrophages. LPC induced cell infiltration in vivo, as determined by increased cell count in mouse peritoneal exudates, and this effect was inhibited by ANXA5. Conclusions ANXA5 could potentially play an important protective role in both atherogenesis and atherosclerotic plaque rupture by reducing pro-inflammatory effects of OxLDL and LPC as well as inhibiting OxLDL binding and uptake by macrophages. The possibility that ANXA5 could be developed into a novel therapy against CVD deserves further study.

Background— Subendothelial deposited low-density lipoprotein particles are a known inflammatory factor in atherosclerosis. However, the causal components derived from low-density lipoprotein are still poorly defined. Apolipoprotein B100 (ApoB100) is the unexchangeable protein component of low-density lipoprotein, and the progression of atherosclerosis is associated with immune responses to ApoB100-derived peptides. In this study, we analyzed the proinflammatory activity of ApoB100 peptides in atherosclerosis. Methods and Results— By screening a peptide library of ApoB100, we identified a distinct native peptide referred to as ApoB100 danger-associated signal 1 (ApoBDS-1), which shows sequence-specific bioactivity in stimulation of interleukin-8, CCL2, and interleukin-6. ApoBDS-1 activates mitogen-activated protein kinase and calcium signaling, thereby effecting the expression of interleukin-8 in innate immune cells. Ex vivo stimulation of carotid plaques with ApoBDS-1 enhances interleukin-8 and prostaglandin E 2 release. Furthermore, we demonstrated that ApoBDS-1–positive peptide fragments are present in atherosclerotic lesions using immunoassays and that low-molecular-weight fractions isolated from plaque show ApoBDS-1 activity inducing interleukin-8 production. Conclusions— Our data show that ApoBDS-1 is a previously unrecognized peptide with robust proinflammatory activity, contributing to the disease-promoting effects of low-density lipoprotein in the pathogenesis of atherosclerosis.

Objective— To gain insights into mechanisms by which intimal hyperplasia interferes with the repair process by investigating expression and function of the catalytic telomerase reverse transcriptase (TERT) subunit after vascular injury. Methods and Results— Functional telomerase is essential to the replicative longevity of vascular cells. We found that TERT was de novo activated in the intima of injured arteries, involving activation of the nuclear factor κB pathway. Stimulation of the isolated intimal smooth muscle cell (SMC) by basic fibroblast growth factor or tumor necrosis factor α resulted in increased TERT activity. This depends on the activation of c-Myc signaling because mutation of the E-box in the promoter or overexpression of mitotic arrest deficient 1 (MAD1), a c-Myc competitor, abrogated the transcriptional activity. Inhibition of nuclear factor κB in both intimal SMCs and the injured artery attenuated TERT transcriptional activity through reduction of c-Myc expression. Pharmacological blockade of TERT led to SMC senescence. Finally, depletion of telomerase function in mice resulted in severe intimal SMC senescence after vascular injury. Conclusion— These results support a model in which vascular injury induces de novo expression of TERT in intimal SMCs via activation of nuclear factor κB and upregulation of c-Myc. The resumed TERT activity is critical for intimal hyperplasia.

TLR signal transduction involves a MyD88-mediated pathway, which leads to recruitment of the IL-1 receptor (IL-1R)-associated kinase 4 (IRAK4) and Toll/IL-1R translation initiation region domain-containing adaptor-inducing IFN-beta-mediated pathway, resulting in the activation of IFN regulatory factor (IRF)3. Both pathways can lead to expression of IFN-beta. TLR-dependent and -independent signals converge in the TNF receptor-associated factor 6 (TRAF6) adaptor, which mediates the activation of NF-kappaBeta. Infection of murine bone marrow-derived macrophages (BMM) with Chlamydia pneumoniae induces IFN-alpha/beta- and NF-kappaBeta-dependent expression of IFN-gamma, which in turn, will control bacterial growth. The role of IRAK4 and IRF3 in the regulation of IFN-alpha/beta expression and NF-kappaBeta activation was studied in C. pneumoniae-infected BMM. We found that levels of IFN-alpha, IFN-beta, and IFN-gamma mRNA were reduced in infected IRAK4(-/-) BMM compared with wild-type (WT) controls. BMM also showed an IRAK4-dependent growth control of C. pneumoniae. No increased IRF3 activation was detected in C. pneumoniae-infected BMM. Similar numbers of intracellular bacteria, IFN-alpha, and IFN-gamma mRNA titers were observed in C. pneumoniae-infected IRF3(-/-) BMM. On the contrary, IFN-beta(-/-) BMM showed lower IFN-alpha and IFN-gamma mRNA levels and higher bacterial titers compared with WT controls. C. pneumoniae infection-induced activation of NF-kappaBeta and expression of proinflammatory cytokines were shown to be TRAF6-dependent but did not require IRAK4 or IRF3. Thus, our data indicate that IRAK4, but not IRF3, controls C. pneumoniae-induced IFN-alpha and IFN-gamma secretion and bacterial growth. IRAK4 and IRF3 are redundant for infection-induced NF-kappaB activation, which is regulated by TRAF6.

Neutrophil gelatinase-associated lipocalin (NGAL) has recently emerged as an important modulator of cell homeostasis. Elevated plasma NGAL levels, possibly because of activation of blood leukocytes, are associated with atherosclerosis. However, little is known about induction of NGAL expression in blood vessels. Using a rat carotid artery injury model, we found that NGAL was highly induced in the intima after angioplasty but was attenuated by adenovirus-mediated expression of a dominant-negative mutant of inhibitor of nuclear factor (NF)-kappaB kinase beta (dnIKKbeta). Expression of NGAL mRNA and protein was also up-regulated in an NF-kappaB-dependent manner in rat and human vascular smooth muscle cells (SMCs) in response to interleukin-1beta stimulation. Rat SMC-produced NGAL was present as mono- and homomeric forms in the cytosol and in a complex containing matrix metalloproteinase-9 (MMP-9) after secretion. In agreement with levels of NGAL, proteolytic activity of MMP-9 was markedly high in the intima of injured vessels and in the culture supernatant of activated intimal SMCs but was reduced in the vessels transduced with dnIKKbeta. The present study reveals a previously unrecognized vascular response to an-gioplastic injury, characterized by NF-kappaB-dependent expression of NGAL in vascular SMCs. Further-more, SMC-produced NGAL interacts with MMP-9, a mechanism by which NGAL may modulate MMP-9 proteolytic activity in the vascular repair process.

Nuclear factor-kappaB (NF-kappaB)-mediated vascular inflammation is a prominent characteristic of atherogenesis and restenosis. We noted that angioplastic injury to carotid artery elicited two phases of NF-kappaB activation characterized by an early activation in the arterial media and a late activation coupled with high levels of inhibitor of IkappaB kinase (IKK) activity in intima. These findings prompted us to elucidate the role for the different phases of NF-kappaB activation and IKK in the progress of vascular repair. Our results show that blockade of the early NF-kappaB activation by perivascular administration of pyrrolidine dithiocarbamate transiently attenuates the expression of proinflammatory genes in the injured vessels but does not affect intimal formation. Interruption of IKKbeta by overexpressing a dominant-negative IKKbeta in the injured artery effectively inhibited the late phase of NF-kappaB activation, resulting in down-regulation of inducible nitric oxide synthase, tumor necrosis factor alpha, and monocyte chemoattractant protein-1 expression in conjunction with a 36% reduction in intima size, albeit with a lack of inhibitory effect on the early NF-kappaB activation. Collectively, these findings show that the IKKbeta-mediated late-phase NF-kappaB activation contributes to intimal hyperplasia and the accompanied vascular inflammatory responses.

Local application of rapamycin (sirolimus) by drug-eluting stents prevents lumen obliteration after angioplasty by inhibition of neointimal hyperplasia. The effects of rapamycin on neointimal smooth muscle cells (niSMC) which are responsible for the occurrence of restenosis have not been investigated so far.Rat niSMC and medial SMC (mSMC) were obtained from balloon catheter-injured arteries. The niSMC exhibited higher basal NF-kappaB activity and TNF-alpha mRNA levels. Nuclear protein binding to NF-kappaB-DNA was attenuated in niSMC by incubation with rapamycin (0.1 and 1 microg/ml) for 24 and 48 h. In contrast in mSMC, 0.1 microg/ml rapamycin had no effect and at 1 microg/ml even increased nuclear protein binding to NF-kappaB-DNA. After 12 h incubation, rapamycin (0.001-10 microg/ml) induced IkappaB-alpha protein in niSMC, whereas in mSMC it stimulated IkappaB-alpha at much lower levels. Prolonged rapamycin treatment (1 microg/ml for 72 h) had no effect on TNF-alpha mRNA level and NF-kappaB activity in niSMC, whereas it led to their increase in mSMC. Vascular endothelial growth factor (VEGF) secretion was higher in mSMC than in niSMC; rapamycin decreased VEGF levels in both cell types. Ultrastructural analysis suggested that rapamycin caused early signs of degeneration in niSMC, but enhanced protein synthesis in mSMC.This study shows that rapamycin influences the inflammatory phenotypes of SMC in opposite directions: it reduces the high basal NF-kappaB activity in niSMC and enhances NF-kappaB activity and TNF-alpha expression in mSMC. In addition, rapamycin inhibits VEGF production regardless of the phenotype of SMC. These findings shed light on molecular mechanisms and structural changes underlying therapeutic applications of rapamycin in prevention of restenosis, inhibition of chronic transplant arteriosclerosis and reduction of secondary malignoma formation due to immunosuppression.

Background Chronic lung disease (CLD) of prematurity is a major problem of neonatal care. Bacterial infection and inflammatory response have been thought to play an important role in the development of CLD and steroids have been given, with some benefit, to neonates with this disease. In the present study, we assessed the ability of lipopolysaccharide (LPS) to stimulate rat alveolar macrophages to produce nitric oxide (NO), express inducible nitric oxide synthase (iNOS) and activate nuclear factor-κB (NF-κB) in vitro. In addition, we investigated the impact of dexamethasone and budesonide on these processes. Methods Griess reaction was used to measure the nitrite level. Western blot and a semi-quantitative RT-PCR were performed to detect iNOS expression. Electrophoretic mobility shift assay (EMSA) was performed to analyze the activation of NF-κB. Results We found that LPS stimulated the rat alveolar macrophages to produce NO in a dose (≥10 ng/ml) and time dependent manner (p < 0.05). This effect was further enhanced by IFN-γ (≥10 IU/ml, p < 0.05), but was attenuated by budesonide (10-4–10-10 M) and dexamethasone (10-4–10-6 M) (p < 0.05). The mRNA and protein levels of iNOS were also induced in response to LPS and attenuated by steroids. LPS triggered NF-κB activation, a mechanism responsible for the iNOS expression. Conclusion Our findings imply that Gram-negative bacterial infection and the inflammatory responses are important factors in the development of CLD. The down-regulatory effect of steroids on iNOS expression and NO production might explain the beneficial effect of steroids in neonates with CLD.

Nuclear factor kappa-B (NFkappaB), a redox-sensitive transcription factor regulating a battery of inflammatory genes, has been indicated to play a role in the development of numerous pathological states. Activation of NFkappaB induces gene programs leading to transcription of factors that promote inflammation, such as leukocyte adhesion molecules, cytokines, and chemokines, although some few substances with possible anti-inflammatory effects are also NFkappaB regulated. The present article reviews basic regulation of NFkappaB and its activation, cell biological effects of NFkappaB activation and the role of NFkappaB in apoptosis. Evidence involving NFkappaB as a key factor in the pathophysiology of ischemia-reperfusion injury and heart failure is discussed. Although activation of NFkappaB induces pro-inflammatory genes, it has lately been indicated that the transcription factor is involved in the signaling of endogenous myocardial protection evoked by ischemic preconditioning. A possible role of NFkappaB in the development of atherosclerosis and unstable coronary syndromes is discussed. Nuclear factor kappa-B may be a new therapeutic target for myocardial protection.

Meconium aspiration causes intensive inflammatory reactions in the lungs, and may lead to neonatal respiratory disorder. Infiltrated inflammatory cells, particularly macrophages, play an important role in such an inflammation. A rat alveolar macrophage cell line (ATCC8383) was exposed to meconium alone or in combination with dexamethasone, budesonide, or interferon-gamma. Nitric oxide (NO) accumulation in the supernatant of the cell culture was detected by Griess reaction, and mRNA of inducible NO synthase (iNOS) expression was detected by reverse transcriptase-PCR. Nuclear factor-kappa B was analyzed by electrophoretic mobility shift assay, and iNOS location and nuclear factor-kappa B transactivation were determined by immunostaining. Our results showed that meconium was capable of inducing production of NO and expression of iNOS in alveolar macrophages in a dose- (1-25 mg/mL, p < 0.05) and time- (4-48 h, p < 0.05) dependent manner. This capability of meconium could be further enhanced in the presence of interferon-gamma (100 IU/mL, p < 0.05). Budesonide (10(-4)-10(-10) M) or dexamethasone (10(-4)-10(-6) M) effectively inhibited the meconium-induced NO production (p < 0.05). Using the protein synthesis inhibitor cycloheximide, we demonstrated that meconium directly induced iNOS in macrophages. Furthermore, meconium also triggered nuclear factor-kappa B activation, a mechanism possibly responsible for the iNOS expression. Our findings suggest that meconium is a potent inflammatory stimulus, resulting in iNOS expression, leading to overproduction of NO from the macrophages, which may be of pathogenic importance in meconium aspiration syndrome. In vitro steroids down-regulated the iNOS expression, thus suggesting a potential to down-regulate NO-mediated inflammation in neonates with meconium aspiration syndrome.

Abstract —Vascular smooth muscle cells (SMCs) are important targets for endothelium-derived nitric oxide (NO), but this production is attenuated in injured and diseased arteries and during aging. However, SMCs can produce NO themselves by expressing an inducible form of NO synthase (iNOS) under inflammatory conditions and in the repair process after arterial injury. We examined iNOS expression in SMCs derived from the aortic media of newborn, young adult, and old rats. Our results show that SMCs from newborn rats cannot produce significant amounts of NO on stimulation with interferon-γ plus lipopolysaccharide or interleukin-1β. In contrast, SMCs from old rats exhibit markedly enhanced iNOS activity. The difference in iNOS activity between the newborn and the old SMCs was closely correlated with levels of iNOS protein, mRNA, and gene promoter activity. Similarly, intercellular adhesion molecule-1 (ICAM-1) was also expressed more abundantly in the old than in the newborn SMCs in response to cytokines. Both iNOS and ICAM-1 are transcriptionally regulated by nuclear factor κB (NF-κB). Our data demonstrate an intense transactivation of NF-κB in old SMCs on tumor necrosis factor-α stimulation but only a weak one in newborn SMCs. The difference in the NF-κB activation could be explained by a much faster and more extensive IκBα degradation in old than in newborn SMCs. These data indicate that the capability to respond to proinflammatory stimuli by activating NF-κB differs between SMCs at different stages of development. This results in differential capability to express NF-κB–dependent genes such as iNOS and ICAM-1, which could have implications for host defense and the pathogenesis of vascular diseases.

Background: Although the association between inflammation and atherosclerosis is well established, the biologic events that trigger the local inflammatory response within plaque are not fully understood. Cytotoxic free radicals and infectious agents, both of which are associated with an inflammatory response, have previously been implicated in the initiation and progression of atherosclerosis. In this study, we analyzed carotid plaque for evidence of oxidative vascular injury by determining the presence and distribution of inducible nitric oxide synthase (iNOS) expression and nitrotyrosine formation and for evidence of infection with cytomegalovirus. Methods: Carotid plaque from 51 patients who underwent endarterectomy for either primary (n = 37) or recurrent (n = 14) stenosis were examined histologically (hematoxylin-eosin staining and Masson's trichrome staining) and with immunohistochemistry with specific antibodies to α–smooth muscle actin, macrophages (CD68), T-lymphocytes (CD3), and T-cell activation (human leukocyte antigen–DR). Twenty-eight specimens from patients with primary (n = 15) and recurrent (n = 13) stenosis were examined for the presence of iNOS and nitrotyrosine with immunohistochemistry and in situ hybridization (iNOS). Twenty-three additional specimens (22 primary, and 1 recurrent) were analyzed with antibodies to p53, cytomegalovirus, and the polymerase chain reaction (cytomegalovirus, n=8). Results: Primary atherosclerotic lesions were either complex heterogenous cellular plaques (n = 29) or relatively acellular fibrous plaques (n = 8). Ten of 14 recurrent plaques were either complex or fibrous lesions, and the remaining four were typical of myointimal thickening. CD68-positive staining cells were detected in all specimens regardless of their structural morphology. CD3-positive cells were interspersed between macrophages in all heterogeneous cellular plaques and only infrequently noted in fibrous plaques. iNOS and nitrotyrosine immunoreactivity were detected in macrophages and smooth muscle cells in all complex and fibrous plaques and in two of four myointimal plaques. The presence of iNOS and nitrotyrosine in plaque correlated with the existence of symptoms in 80% of primary and 62% of recurrent lesions. Cytomegalovirus was detected in only two of 23 carotid specimens (9%). Conclusion: The association between ischemic cerebrovascular symptoms and iNOS and nitrotyrosine immunoreactivity in complex primary and recurrent carotid plaque and the infrequent occurrence of cytomegalovirus in primary carotid lesions suggests that ongoing free radical oxidative damage rather than viral infection may contribute to plaque instability in patients with complex and fibrous carotid plaques. (J Vasc Surg 1999;30:36-50.)