PMC,a potent hydrophilic α‐tocopherol derivative,inhibits NF‐κB activation via PP2A but not IκBα‐dependent signals in vascular smooth muscle cells

The hydrophilic α‐tocopherol derivative, 2,2,5,7,8‐pentamethyl‐6‐hydroxychromane (PMC), is a promising alternative to vitamin E in clinical applications. Critical vascular inflammation leads to vascular dysfunction and vascular diseases, including atherosclerosis, hypertension and abdominal aortic aneurysms. In this study, we investigated the mechanisms of the inhibitory effects of PMC in vascular smooth muscle cells (VSMCs) exposed to pro‐inflammatory stimuli, lipopolysaccharide (LPS) combined with interferon (IFN)‐γ. Treatment of LPS/IFN‐γ‐stimulated VSMCs with PMC suppressed the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase‐9 in a concentration‐dependent manner. A reduction in LPS/IFN‐γ‐induced nuclear factor (NF)‐κB activation was also observed in PMC‐treated VSMCs. The translocation and phosphorylation of p65, protein phosphatase 2A (PP2A) inactivation and the formation of reactive oxygen species (ROS) were significantly inhibited by PMC in LPS/IFN‐γ‐activated VSMCs. However, neither IκBα degradation nor IκB kinase (IKK) or ribosomal s6 kinase‐1 phosphorylation was affected by PMC under these conditions. Both treatments with okadaic acid, a PP2A‐selective inhibitor, and transfection with PP2A siRNA markedly reversed the PMC‐mediated inhibition of iNOS expression, NF‐κB‐promoter activity and p65 phosphorylation. Immunoprecipitation analysis of the cellular extracts of LPS/IFN‐γ‐stimulated VSMCs revealed that p65 colocalizes with PP2A. In addition, p65 phosphorylation and PP2A inactivation were induced in VSMCs by treatment with H₂O₂, but neither IκBα degradation nor IKK phosphorylation was observed. These results collectively indicate that the PMC‐mediated inhibition of NF‐κB activity in LPS/IFN‐γ‐stimulated VSMCs occurs through the ROS‐PP2A‐p65 signalling cascade, an IKK‐IκBα‐independent mechanism. Therapeutic interventions using PMC may therefore be beneficial for the treatment of vascular inflammatory diseases.     

Relation between TLR4/NF‐κB signaling pathway activation by 27‐hydroxycholesterol and 4‐hydroxynonenal,and atherosclerotic plaque instability

It is now thought that atherosclerosis, although due to increased plasma lipids, is mainly the consequence of a complicated inflammatory process, with immune responses at the different stages of plaque development. Increasing evidence points to a significant role of Toll‐like receptor 4 (TLR4), a key player in innate immunity, in the pathogenesis of atherosclerosis. This study aimed to determine the effects on TLR4 activation of two reactive oxidized lipids carried by oxidized low‐density lipoproteins, the oxysterol 27‐hydroxycholesterol (27‐OH) and the aldehyde 4‐hydroxynonenal (HNE), both of which accumulate in atherosclerotic plaques and play a key role in the pathogenesis of atherosclerosis. Secondarily, it examined their potential involvement in mediating inflammation and extracellular matrix degradation, the hallmarks of high‐risk atherosclerotic unstable plaques. In human promonocytic U937 cells, both 27‐OH and HNE were found to enhance cell release of IL‐8, IL‐1β, and TNF‐α and to upregulate matrix metalloproteinase‐9 (MMP‐9) via TLR4/NF‐κB‐dependent pathway; these actions may sustain the inflammatory response and matrix degradation that lead to atherosclerotic plaque instability and to their rupture. Using specific antibodies, it was also demonstrated that these inflammatory cytokines increase MMP‐9 upregulation, thus enhancing the release of this matrix‐degrading enzyme by macrophage cells and contributing to plaque instability. These innovative results suggest that, by accumulating in atherosclerotic plaques, the two oxidized lipids may contribute to plaque instability and rupture. They appear to do so by sustaining the release of inflammatory molecules and MMP‐9 by inflammatory and immune cells, for example, macrophages, through activation of TLR4 and its NF‐κB downstream signaling.     

Integrin‐linked kinase is involved in TNF‐α‐induced inducible nitric‐oxide synthase expression in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. Nitric oxide (NO) is a highly reactive nitrogen radical implicated in inflammatory responses. We investigated the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and NO production stimulated by TNF‐α in cultured myoblasts. TNF‐α stimulation caused iNOS expression and NO production in myoblasts (G7 cells). TNF‐α‐mediated iNOS expression was attenuated by integrin‐linked kinase (ILK) inhibitor (KP392) and siRNA. Pretreatment with Akt inhibitor, mammalian target of rapamycin (mTOR) inhibitor (rapamycin), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK) also inhibited the potentiating action of TNF‐α. Stimulation of cells with TNF‐α increased ILK kinase activity. TNF‐α also increased the Akt and mTOR phosphorylation. TNF‐α mediated an increase of NF‐κB‐specific DNA–protein complex formation, p65 translocation into nucleus, NF‐κB‐luciferase activity was inhibited by KP392, Akt inhibitor, and rapamycin. Our results suggest that TNF‐α increased iNOS expression and NO production in myoblasts via the ILK/Akt/mTOR and NF‐κB signaling pathway. J. Cell. Biochem. 109: 1244–1253, 2010. © 2010 Wiley‐Liss, Inc.     

Macrophage autophagy regulates mitochondria‐mediated apoptosis and inhibits necrotic core formation in vulnerable plaques

The vulnerable plaque is a key distinguishing feature of atherosclerotic lesions that can cause acute atherothrombotic vascular disease. This study was designed to explore the effect of autophagy on mitochondria‐mediated macrophage apoptosis and vulnerable plaques. Here, we generated the mouse model of vulnerable carotid plaque in ApoE^?/? mice. Application of ApoE^?/? mice with rapamycin (an autophagy inducer) inhibited necrotic core formation in vulnerable plaques by decreasing macrophage apoptosis. However, 3‐methyladenine (an autophagy inhibitor) promoted plaque vulnerability through deteriorating these indexes. To further explore the mechanism of autophagy on macrophage apoptosis, we used macrophage apoptosis model in vitro and found that 7‐ketocholesterol (7‐KC, one of the primary oxysterols in oxLDL) caused macrophage apoptosis with concomitant impairment of mitochondria, characterized by the impairment of mitochondrial ultrastructure, cytochrome c release, mitochondrial potential dissipation, mitochondrial fragmentation, excessive ROS generation and both caspase‐9 and caspase‐3 activation. Interestingly, such mitochondrial apoptotic responses were ameliorated by autophagy activator, but exacerbated by autophagy inhibitor. Finally, we found that MAPK‐NF‐κB signalling pathway was involved in autophagy modulation of 7‐KC–induced macrophage apoptosis. So, we provide strong evidence for the potential therapeutic benefit of macrophage autophagy in regulating mitochondria‐mediated apoptosis and inhibiting necrotic core formation in vulnerable plaques.     

Clinorotation upregulates inducible nitric oxide synthase by inhibiting AP‐1 activation in human umbilical vein endothelial cells

Alterations of nitric oxide contribute to post‐flight orthostatic intolerance. The aim of this study was to investigate the changes of inducible nitric oxide synthase (iNOS) and the mechanisms underlying regulation of iNOS by simulated microgravity in human umbilical vein endothelial cells (HUVECs). Clinorotation, a simulated‐model of microgravity, increased iNOS expression and promoter activity in HUVECs. The transactivations of NF‐κB and AP‐1 were suppressed by 24 h clinorotation. A key role for AP‐1, but not NF‐κB in the regulation of iNOS was shown. (1) PDTC, a NF‐κB inhibitor, had no effect on clinorotation upregulation of iNOS. (2) SP600125, a JNK‐specific inhibitor, which resulted in inhibition of AP‐1 activity, enhanced the iNOS expression and promoter activity in clinorotation. (3) Overexpression of AP‐1 remarkably attenuated the upregulation effect of clinorotation. These findings indicate that clinorotation upregulates iNOS in HUVECs by a mechanism dependent on suppression of AP‐1, but not NF‐κB. These results support a key role for AP‐1 in the signaling of postflight orthostatic intolerance. J. Cell. Biochem. 107: 357–363, 2009. © 2009 Wiley‐Liss, Inc.     

The expression of MMP‐1 and MMP‐9 is up‐regulated by smooth muscle cells after their cross‐talk with macrophages in high glucose conditions

Patients with diabetes mellitus have an increased risk of myocardial infarction and coronary artery disease‐related death, exhibiting highly vulnerable plaques. Many studies have highlighted the major role of macrophages (MAC) and smooth muscle cells (SMC) and the essential part of metalloproteases (MMPs) in atherosclerotic plaque vulnerability. We hypothesize that in diabetes, the interplay between MAC and SMC in high glucose conditions may modify the expression of MMPs involved in plaque vulnerability. The SMC‐MAC cross‐talk was achieved using trans‐well chambers, where human SMC were grown at the bottom and human MAC in the upper chamber in normal (NG) or high (HG) glucose concentration. After cross‐talk, the conditioned media and cells were isolated and investigated for the expression of MMPs, MCP‐1 and signalling molecules. We found that upon cross‐talk with MAC in HG, SMC exhibit: (i) augmented expression of MMP‐1 and MMP‐9; (ii) significant increase in the enzymatic activity of MMP‐9; (iii) higher levels of soluble MCP‐1 chemokine which is functionally active and involved in MMPs up‐regulation; (iv) activated PKCα signalling pathway which, together with NF‐kB are responsible for MMP‐1 and MMP‐9 up‐regulation, and (v) impaired function of collagen assembly. Taken together, our data indicate that MCP‐1 released by cell cross‐talk in diabetic conditions binds to CCR2 and triggers MMP‐1 and MMP‐9 over‐expression and activity, features that could explain the high vulnerability of atherosclerotic plaque found at diabetic patients.     

Ultrasound stimulates NF‐κB activation and iNOS expression via the Ras/Raf/MEK/ERK signaling pathway in cultured preosteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and non‐operatively clinical uses. Nitric oxide (NO) is a crucial early mediator in mechanically induced bone formation. Here we found that US‐mediated inducible nitric oxide synthase (iNOS) expression was attenuated by Ras inhibitor (manumycin A), Raf‐1 inhibitor (GW5074), MEK inhibitor (PD98059), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK). US‐induced Ras activation was inhibited by manumycin A. Raf‐1 phosphorylation at Ser³³⁸ by US was inhibited by manumycin A and GW5074. US‐induced MEK and ERK activation was inhibited by manumycin A, GW5074, and PD98059. Stimulation of preosteoblasts with US activated IκB kinase α/β (IKK α/β), IκBαphosphorylation, p65 phosphorylation at Ser²⁷⁶, p65, and p50 translocation from the cytosol to the nucleus, and κB‐luciferase activity. US‐mediated an increase of IKK α/β, IκBα, and p65 phosphorylation, κB‐luciferase activity and p65 and p50 binding to the NF‐κB element was inhibited by manumycin A, GW5074, and PD98059. Our results suggest that US increased iNOS expression in preosteoblasts via the Ras/Raf‐1/MEK/ERK/IKKαβ and NF‐κB signaling pathways. J. Cell. Physiol. 220: 196–203, 2009. © 2009 Wiley‐Liss, Inc.     

Activated α2 macroglobulin induces matrix metalloproteinase 9 expression by low‐density lipoprotein receptor‐related protein 1 through MAPK‐ERK1/2 and NF‐κB activation in macrophage‐derived cell lines

Macrophages under certain stimuli induce matrix metalloproteinase 9 (MMP‐9) expression and protein secretion through the activation of MAPK‐ERK and NF‐κB signaling pathways. Previously, we demonstrated that activated α₂‐macroglulin (α₂M*) through the interaction with its receptor low‐density lipoprotein receptor‐related protein 1 (LRP1) induces macrophage proliferation mediated by the activation of MAPK‐ERK1/2. In the present work, we examined whether α₂M*/LRP1interaction could induce the MMP‐9 production in J774 and Raw264.7 macrophage‐derived cell lines. It was shown that α₂M* promoted MMP‐9 expression and protein secretion by LRP1 in both macrophage‐derived cell lines, which was mediated by the activation of MAPK‐ERK1/2 and NF‐κB. Both intracellular signaling pathways activated by α₂M* were effectively blocked by calphostin‐C, suggesting involvement of PKC. In addition, we demonstrate that α₂M* produced extracellular calcium influx via LRP1. However, when the intracellular calcium mobilization was inhibited by BAPTA‐AM, the α₂M*‐induced MAPK‐ER1/2 activation was fully blocked in both macrophage cell lines. Finally, using specific pharmacological inhibitors for PKC, Mek1, and NF‐κB, it was shown that the α₂M*‐induced MMP‐9 protein secretion was inhibited, indicating that the MMP production promoted by the α₂M*/LRP1 interaction required the activation of both signaling pathways. These findings may prove useful in the understanding of the macrophage LRP1 role in the vascular wall during atherogenic plaque progression. J. Cell. Biochem. 111: 607–617, 2010. © 2010 Wiley‐Liss, Inc.     

Histochemical evidence for inducible nitric oxide synthase in advanced but non-ruptured human atherosclerotic carotid arteries

In response to cytokine stimulation, the inducible isoform of the nitric oxide synthase (iNOS) produces large amounts of nitric oxide with potential consequences in the pathophysiology of atherosclerosis. Previous investigations have demonstrated the presence of iNOS in human atherosclerotic lesions. The goal of this study was to evaluate the occurrence of the expression of iNOS in ruptured versus non-ruptured human carotid atherosclerotic plaques. Using plastic-embedded sections, we performed in situ hybridization and immunohistochemistry on very advanced atherosclerotic lesions type V (non-ruptured) and type VI (ruptured) from 12 atheromatous carotid arteries from endarterectomy and six non-atherosclerotic internal mammary arteries from aorto-coronary bypass. Only one internal mammary artery expressed iNOS in the endothelium. In contrast, iNOS mRNA and protein were repeatedly expressed in advanced lesions type V in 5/7 cases, particularly in inflammatory regions. Specific cell markers identified iNOS-positive cells as macrophages and T-lymphocytes but also as smooth muscle cells and endothelial cells adjacent to these inflammatory regions. Nitration of protein tyrosines was not always associated to iNOS expression but more likely to the presence of inflammatory cells. In complicated lesions type VI, the occurrence of iNOS mRNA and protein expression diminished drastically (1/5 cases). Combined expression of iNOS mRNA and protein is frequently found in advanced but non-ruptured human atherosclerotic carotid lesions while it becomes rare after the plaque has ruptured. These findings suggest that iNOS could be an active participant in the plaque rupture event.     

A fluorescence lifetime spectroscopy study of matrix metalloproteinases‐2 and ‐9 in human atherosclerotic plaque

Matrix metalloproteinase (MMP)‐2 and ‐9 play important roles in the progression of atherosclerosis. This study aims to determine whether MMP‐2 and ‐9 content in the fibrotic caps of atherosclerotic plaque is correlated with plaque autofluorescence. A time‐resolved laser‐induced fluorescence spectroscopy (TR‐LIFS) system was used to measure the autofluorescence and assess the biochemical composition of human plaques obtained from carotid endarterectomy. Results presented here demonstrate for the first time the ability to characterize the biochemical composition as it relates to MMP‐2 and ‐9 content in the atherosclerotic plaque cap using a label‐free imaging technique implemented with a fiberoptic TR‐LIFS system. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Rosmarinic acid down‐regulates NO and PGE2 expression via MAPK pathway in rat chondrocytes

Rosmarinic acid (RosA) is a water‐soluble polyphenol, which can be isolated from many herbs such as orthosiphon diffuses and rosmarinus officinalis. Previous studies have shown that RosA possesses various biological properties. In this study, we investigate the anti‐osteoarthritic effects of RosA in rat articular chondrocytes. Chondrocytes were pre‐treated with RosA, followed by the stimulation of IL‐1β. Real‐time PCR and Western blot were performed to detect the expression of matrix metalloproteinase (MMP)‐1, MMP‐3 and MMP‐13. Nitric oxide and PGE₂ production were measured by Griess reagent and enzyme‐linked immunosorbent assay (ELISA). The expression of mitogen‐activated protein kinase (MAPK) and nuclear factor‐κB (NF‐κB) was also investigated by Western blot analysis. We found that RosA down‐regulated the MMPs expression as well as nitric oxide and PGE₂ production in IL‐1β‐induced chondrocytes. In addition, RosA inhibited p38 and JNK phosphorylation as well as p65 translocation. The results suggest that RosA may be considered a possible agent in the treatment of OA.     

p75 Neurotrophin receptor mediates neurotrophin activation of NF‐kappa B and induction of iNOS expression in P19 neurons

P19 embryonic carcinoma cells can be differentiated into neurons that form synaptic connections and that produce a variety of neurotransmitters. Results of RT‐PCR indicate that P19 neurons express several neurotrophin receptors (p75^NTR, trkB, and trkC, but not trkA) but they do not express any of the four neurotrophins. Consistent with the presence of trkB but not trkA, BDNF causes rapid phosphorylation of MAP kinases ERK1 and ERK2, but NGF does not. Neurotrophins induce translocation of NF‐κB into the nucleus. All four neurotrophins induce activation of NF‐κB in a biphasic manner. This effect is apparently mediated by p75^NTR, because an inhibitor of trk receptors, K252a, does not inhibit activation of NF‐κB. Instead, K252a itself promotes activation of NF‐κB and this effect is additive with the effect of neurotrophins. Inhibition of reactive oxygen intermediates with PDTC completely abolishes basal activity of NF‐κB and strongly inhibits activation of NF‐κB by neurotrophins, indicating an important role of reactive oxygen intermediates in the pathway by which neurotrophins activate NF‐κB. NF‐κB is known to promote expression of the iNOS gene. We found that all four neurotrophins increased iNOS mRNA levels, resulting in increased accumulation of iNOS protein. In contrast, none of the neurotrophins stimulated nNOS mRNA or protein synthesis. PDTC abolishes constitutive and neurotrophin‐induced expression of iNOS mRNA and protein and abolishes constitutive expression of nNOS mRNA, suggesting that reactive oxygen intermediates promote expression of nNOS. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 191–203, 2003     

Effect of arachidonic acid on hypoxia‐induced IL‐6 production in mouse ES cells: Involvement of MAPKs,NF‐κB,and HIF‐1α

This study examined the role of arachidonic acid (AA) in hypoxia‐induced production of interleukin (IL)‐6 and its related signaling pathways in mouse embryonic stem (ES) cells. Hypoxia with AA induced IL‐6 production, which was mediated by reactive oxygen species (ROS). In addition, hypoxia increased the levels of p38 mitogen‐activated protein kinases (MAPKs) and stress‐activated protein kinase/c‐jun NH₂‐terminal kinase (SAPK/JNK) phosphorylation, which were blocked by antioxidant (vitamin C). Inhibition of p38 MAPK and SAPK/JNK blocked hypoxia‐ or hypoxia with AA‐induced nuclear factor‐kappa B (NF‐κB) activation. Furthermore, hypoxia‐induced increase in hypoxia‐inducible factor‐1α (HIF‐1α) expression was regulated by NF‐κB activation. Consequently, the increased HIF‐1α expression induced activation of matrix metalloproteinase (MMP)‐2 and MMP‐9. The expression of each signaling molecule stimulated an increase in IL‐6 production that was greater in hypoxic conditions with AA than with hypoxia alone. Finally, inhibition of IL‐6 production using IL‐6 antibody or soluble IL‐6 receptor attenuated the hypoxia‐induced increases in DNA synthesis of mouse ES cells. In conclusion, AA potentiates hypoxia‐induced IL‐6 production through the MAPKs, NF‐κB, and HIF‐1α pathways in mouse ES cells. J. Cell. Physiol. 222: 574–585, 2010. © 2009 Wiley‐Liss, Inc.     

Negative regulation of NF‐κB action by Set9‐mediated lysine methylation of the RelA subunit

Proper regulation of NF‐κB activity is critical to maintain and balance the inflammatory response. Inactivation of the NF‐κB complex relies in part on the proteasome‐mediated degradation of promoter‐bound NF‐κB, but the detailed molecular mechanism initiating this process remains elusive. Here, we show that the methylation of the RelA subunit of NF‐κB has an important function in this process. Lysine methyltransferase Set9 physically associates with RelA in vitro and in vivo in response to TNF‐α stimulation. Mutational and mass spectrometric analyses reveal that RelA is monomethylated by Set9 at lysine residues 314 and 315 in vitro and in vivo. Methylation of RelA inhibits NF‐κB action by inducing the proteasome‐mediated degradation of promoter‐associated RelA. Depletion of Set9 by siRNA or mutation of the RelA methylation sites prolongs DNA binding of NF‐κB and enhances TNF‐α‐induced expression of NF‐κB target genes. Together, these findings unveil a novel mechanism by which methylation of RelA dictates the turnover of NF‐κB and controls the NF‐κB‐mediated inflammatory response.     

Effects of simvastatin and rosuvastatin on RAS protein,matrix metalloproteinases and NF‐κB in lung cancer and in normal pulmonary tissues

Objectives

In this study, we have evaluated effects of 24‐hour treatments with simvastatin or rosuvastatin on RAS protein, NF‐κB and MMP expression in LC tissues obtained from 12 patients undergoing thoracic surgery.

Materials and methods

Normal and lung tumour tissues obtained from each sample were exposed to simvastatin (2.5–30 μm ) or rosuvastatin (1.25–30 μm ) and western blot analysis was then performed.

Results

We documented increased expression of proteins, MMP‐2, MMP‐9 and NF‐κB‐p65 in LC tissues, with respect to normal tissues (P < 0.01). In the malignant tissues, simvastatin and rosuvastatin significantly (P < 0.01) and dose‐dependently reduced RAS protein, MMP‐2/9 and NF‐κB‐p65 expression.

Conclusions

In conclusion, our results suggest that simvastatin and rosuvastatin could play a role in LC treatment by modulation of RAS protein, MMP‐2/9 and NF‐κB‐p65.

     

Simvastatin abolishes nitric oxide‐ and reactive oxygen species‐induced cyclooxygenase‐2 expression by blocking the nuclear factor κB pathway in rabbit articular chondrocytes

Nitric oxide (NO) and reactive oxygen species (ROS) have been shown to be linked with numerous diseases, including osteoarthritis (OA). Our study aimed to examine the effect of simvastatin on NO‐ or ROS‐induced cyclooxygenase‐2 (COX‐2) expression in OA. Simvastatin has attracted considerable attention since the discovery of its pharmacological effects on different pathogenic processes, including inflammation. Here, we report that simvastatin treatment blocked sodium nitroprusside (SNP)‐ and interleukin 1 beta (IL‐1β)‐induced COX‐2 production. In addition, simvastatin attenuated SNP‐induced NO production and IL‐1β‐induced ROS generation. Treatment with simvastatin prevented SNP‐ and IL‐1β‐induced nuclear factor kappa B (NF‐κB) activity. Inhibiting NO production and ROS generation using N‐acetylcysteine (NAC) and NG‐monomethyl‐ l ‐arginine ( l ‐NMMA), respectively, accelerated the influence of simvastatin on NF‐κB activity. In addition, NAC blocked SNP and simvastatin‐mediated COX‐2 production and NF‐κB activity but did not alter IL‐1β and simvastatin‐mediated COX‐2 expression. l ‐NMMA treatment also abolished IL‐1β‐mediated COX‐2 expression and NF‐κB activation, whereas SNP and simvastatin‐mediated COX‐2 expression were not altered compared with the levels in the SNP and simvastatin‐treated cells. Our findings suggested that simvastatin blocks COX‐2 expression by inhibiting SNP‐induced NO production and IL‐1β‐induced ROS generation by blocking the NF‐κB pathway.