Prevention of radiotherapy-induced arterial inflammation by interleukin-1 blockade.

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.

Innate immune receptor NOD2 promotes vascular inflammation and formation of lipid-rich necrotic cores in hypercholesterolemic mice.

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, the function of most innate immune receptors in atherosclerosis remains unclear. Here, we show that NOD2 is a crucial innate immune receptor influencing vascular inflammation and atherosclerosis severity. 10-week stimulation with muramyl dipeptide (MDP), the NOD2 cognate ligand, aggravated atherosclerosis, as indicated by the augmented lesion burden, increased vascular inflammation and enlarged lipid-rich necrotic cores in Ldlr(-/-) mice. Myeloid-specific ablation of NOD2, but not its downstream kinase, receptor-interacting serine/threonine-protein kinase 2, restrained the expansion of the lipid-rich necrotic core in Ldlr(-/-) chimeric mice. In vitro stimulation of macrophages with MDP enhanced the uptake of oxidized low-density lipoprotein and impaired cholesterol efflux in concordance with upregulation of scavenger receptor A1/2 and downregulation of ATP-binding cassette transporter A1. Ex vivo stimulation of human carotid plaques with MDP led to increased activation of inflammatory signaling pathways p38 MAPK and NF-κB-mediated release of proinflammatory cytokines. Altogether, this study suggests that NOD2 contributes to the expansion of the lipid-rich necrotic core and promotes vascular inflammation in atherosclerosis.

α7 Nicotinic acetylcholine receptor is expressed in human atherosclerosis and inhibits disease in mice--brief report.

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.

The leukotriene B4 receptor (BLT) antagonist BIIL284 decreases atherosclerosis in ApoE-/- mice.

Leukotriene B4 (LTB4) induces proinflammatory signaling through BLT receptors expressed in atherosclerotic lesions. Either genetic or pharmacological targeting of the high affinity LTB4 receptor, BLT1, reduces atherosclerosis in different mouse models. The low affinity BLT2 receptor for LTB4 may transduce additional pro-atherogenic signaling, but combined BLT1 and BLT2 receptor antagonism has not previously been explored in atherosclerosis. The aim of the present study was to unravel the effects of the BLT receptor antagonist BIIL284 in apolipoprotein E deficient mice in terms of atherosclerotic lesion size and composition, as well as on arterial matrixmetalloproteinase (MMP) activity and plasma cytokines. Oral administration of BIIL284 (0.3-3mg/kg) dose-dependently decreased atherosclerotic lesion size after 12 weeks. In addition, significantly smaller aortic lesions were observed in mice treated with BIIL284 (3mg/kg) for 24 weeks. The reduced atherosclerosis was associated with less lesion smooth muscle cells, less arterial MMP activities and lower plasma levels of TNF-α and IL-6. Taken together, these results suggest a therapeutic value of BLT receptor antagonism in atherosclerosis.

NOD2-mediated innate immune signaling regulates the eicosanoids in atherosclerosis.

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 E2, 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-1ß 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 E2 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 E2 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.

Rip2 deficiency leads to increased atherosclerosis despite decreased inflammation.

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.

Identification of a danger-associated peptide from apolipoprotein B100 (ApoBDS-1) that triggers innate proatherogenic responses.

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 E2 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.

Innate immune recognition receptors and damage-associated molecular patterns in plaque inflammation.

PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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. SUMMARY: 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.

Nuclear factor {kappa}B-mediated transactivation of telomerase prevents intimal smooth muscle cell from replicative senescence during vascular repair.

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. CONCLUSIONS: 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.

Inflammasome-Driven Interleukin-1α and Interleukin-1ß Production in Atherosclerotic Plaques Relates to Hyperlipidemia and Plaque Complexity.

CANTOS (Canakinumab Antiinflammatory Thrombosis Outcome Study) confirmed interleukin (IL)-1ß as an appealing therapeutic target for human atherosclerosis and related complications. However, there are serious gaps in our understanding of IL-1 production in atherosclerosis. Herein the authors show that complex plaques, or plaques derived from patients with suboptimally controlled hyperlipidemia, or on no or low-intensity statin therapy, demonstrated higher recruitable IL-1ß production. Generation of mature IL-1ß was matched by IL-1α release, and both were attenuated by inhibition of NLR family pyrin domain containing 3 or caspase. These findings support the inflammasome as the main pathway for IL-1α/ß generation in atherosclerosis and high-intensity lipid-lowering therapies as primary and additional anti-IL-1-directed therapies as secondary interventions in high-risk patients.

Role of IRAK4 and IRF3 in the control of intracellular infection with Chlamydia pneumoniae.

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.

Augmented Th17 differentiation in Trim21 deficiency promotes a stable phenotype of atherosclerotic plaques with high collagen content.

Aims: Patients 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 results: Trim21-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. Conclusion: In 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.

Involvement of the antimicrobial peptide LL-37 in human atherosclerosis.

OBJECTIVE: Antimicrobial peptides are effector molecules of the innate immune system. To understand the function of vascular innate immunity in atherosclerosis, we investigated the role of LL-37, a cathelicidin antimicrobial peptide, in the disease process. METHODS AND RESULTS: Using real-time polymerase chain reaction, we found a 6-fold increase in human cationic antimicrobial protein 18/LL-37 transcript in human atherosclerotic lesions compared with normal arteries. Immunohistochemical analysis of atherosclerotic plaques showed that LL-37 was expressed mainly by macrophages and some endothelial cells. Western blot demonstrated existence of active LL-37 peptide and abundant proprotein in atheroma specimens. To understand the functional implication of LL-37 production in atherosclerosis, the transcription profile was assessed in endothelial cells treated with LL-37. Our data show that LL-37 induces expression of the adhesion molecule intercellular adhesion molecule-1 and the chemokine monocyte chemoattractant protein 1 in endothelial cells. Intriguingly, Chlamydia pneumoniae withstood the antimicrobial activity of LL-37 in vitro, although inflammatory response was induced on infection. CONCLUSIONS: LL-37 is produced in atherosclerotic lesions, where it may function as an immune modulator by activating adhesion molecule and chemokine expression, thus enhancing innate immunity in atherosclerosis.

Countervailing effects of rapamycin (sirolimus) on nuclear factor-kappa B activities in neointimal and medial smooth muscle cells.

OBJECTIVE: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

IKKbeta-dependent NF-kappaB pathway controls vascular inflammation and intimal hyperplasia.

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.

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.

Neointimal smooth muscle cells display a proinflammatory phenotype resulting in increased leukocyte recruitment mediated by P-selectin and chemokines.

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.

Expression of toll-like receptors in human atherosclerotic lesions: a possible pathway for plaque activation.

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-kappaB. 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.

HMG-CoA reductase inhibitors induce apoptosis in neointima-derived vascular smooth muscle cells.

In the context of atherogenesis and restenosis, vascular smooth muscle cell (SMC) proliferation and apoptosis play a crucial role. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) have been shown to inhibit the migration and proliferation of SMC, and to induce apoptosis in different cell types including SMC. However, it is not known whether these agents induce apoptosis in neointimal SMC. We investigated the effects of statin treatment on neointimal SMC as compared to medial cells by using trypan blue counting, MTT test, Annexin V staining, cell cycle analysis and a co-culture model. The incubation of neointimal or medial SMC with lovastatin reduced the MTT activity as well as the total cell number, and increased the amount of trypan blue positive cells, indicative of cell death. We tested by staining with Annexin V/propidium iodide, specific antibodies to active caspase-3, TUNEL reaction, and by the appearance of a sub-G1 peak, whether the observed increase in cell death was due to apoptosis. After treatment with lovastatin, programmed cell death was slightly increased in medial SMC, while neointimal cells showed a pronounced rate of apoptosis. In an attempt to mimic early phases of restenosis in vitro by seeding low density neointimal cells onto high density medial cells, we found that statin treatment induced cell death preferentially in the neointimal SMC. Our results suggest that statins enhance the rate of apoptosis in neointimal SMC, which may be an interesting feature to reduce restenosis after successful angioplasty.

Activation of macrophage nuclear factor-kappa B and induction of inducible nitric oxide synthase by LPS.

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-kappaB (NF-kappaB) 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-kappaB. RESULTS: We found that LPS stimulated the rat alveolar macrophages to produce NO in a dose (>or=10 ng/ml) and time dependent manner (p < 0.05). This effect was further enhanced by IFN-gamma (>or=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-kappaB 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.