We investigated the question of whether 7-oxygenated cholesterol derivatives could affect inflammatory and/or immune responses in atherosclerosis by examining their effects on expression of IL-23 in monocytic cells. 7alpha-Hydroxycholesterol (7alphaOHChol) induced transcription of the TLR6 gene and elevated the level of cell surface TLR6 protein in THP-1 monocytic cells. Addition of an agonist of TLR6, FSL-1, to TLR6-expressing cells by treatment with 7alphaOHChol resulted in enhanced production of IL-23 and transcription of genes encoding the IL-23 subunit alpha (p19) and the IL-12 subunit beta (p40). However, treatment with 7-ketocholesterol (7K) and 7beta-hydroxycholesterol (7betaOHChol) did not affect TLR6 expression, and addition of FSL-1 to cells treated with either 7K or 7betaOHChol did not influence transcription of the genes. Pharmacological inhibition of ERK, Akt, or PI3K resulted in attenuated transcription of TLR6 induced by 7alphaOHChol as well as secretion of IL-23 enhanced by 7alphaOHChol plus FSL-1. Inhibition of p38 MAPK or JNK resulted in attenuated secretion of IL-23. These results indicate that a certain type of 7-oxygenated cholesterol like 7alphaOHChol can elicit TLR6-mediated expression of IL-23 by monocytic cells via PI3K/Akt and MAPKs pathways.
Atherosclerosis ; Cholesterol ; Interleukin-12 ; Interleukin-23* ; Macrophages ; p38 Mitogen-Activated Protein Kinases ; Toll-Like Receptor 6

We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors/PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype.
Humans ; Muscle, Smooth, Vascular* ; Myocytes, Smooth Muscle ; Pertussis Toxin ; Phenotype* ; Phosphorylation ; Protein Kinase C ; Proto-Oncogene Proteins c-raf ; Thrombin

STUDY DESIGN: A cross-sectional study. PURPOSE: To explore the impact of chronic low back pain (CLBP) on individuals' quality of life; to understand current treatment practices and level of satisfaction with treatment in patients with CLBP. OVERVIEW OF LITERATURE: Assessing subjective, patient-reported outcomes such as quality of life is essential to health care research. METHODS: Influences of the CLBP were analyzed via a questionnaire, which contained the character of CLBP, effect of pain management, Korean version Oswestry Disability Index (K-ODI) and Korean version of 12-item Short Form Health Survey (SF-12v2). RESULTS: Of 3,121 subjects who responded, 67.3% had moderate to severe pain; 43.5% presented prolonged CLBP of more than two years; and 32.4% had suffered from sleep disturbance due to pain. 22.8% of the patients were not satisfied with current pain management. The mean K-ODI score was 37.63; and it was positively correlated with the mean pain intensity (r=0.6, p<0.001). The SF-12v2 result was negatively correlated with mean pain intensity (PCS: r=-0.5, p<0.001; MCS: r=-0.4, p<0.001) and also negatively correlated with the K-ODI score (PCS: r=-0.75, p<0.001; MCS: r=-0.5, p<0.001). The conformity between patients and doctors in pain assessment was fair (kappa=0.2463). CONCLUSIONS: CLBP negatively affects quality of life. Of total 22.8% of the patients were not satisfied with current pain management. Such needs to be taken more seriously by doctors for improvement of satisfaction and quality of life in patients with CLBP.
Back Pain* ; Cross-Sectional Studies* ; Health Services Research ; Health Surveys ; Humans ; Low Back Pain ; Pain Management ; Pain Measurement ; Quality of Life* ; Surveys and Questionnaires

We investigated the question of whether cholesterol catabolite can influence expression of inflammatory cytokines via Toll-like receptors (TLR) in monocytic cells. Treatment of THP-1 monocytic cells with 27-hydroxycholesterol (27OHChol) resulted in induction of gene transcription of TLR6 and elevated level of cell surface TLR6. Addition of FSL-1, a TLR6 agonist, to 27OHChol-treated cells resulted in transcription of the IL-1alpha gene and enhanced secretion of the corresponding gene product. However, cholesterol did not affect TLR6 expression, and addition of FSL-1 to cholesterol-treated cells did not induce expression of IL-1alpha . Using pharmacological inhibitors, we investigated molecular mechanisms underlying the expression of TLR6 and IL-1alpha. Treatment with Akt inhibitor IV or U0126 resulted in significantly attenuated expression of TLR6 and IL-1alpha induced by 27OHChol and 27OHChol plus FSL-1, respectively. In addition, treatment with LY294002, SB202190, or SP600125 resulted in significantly attenuated secretion of IL-1alpha . These results indicate that 27OHChol can induce inflammation by augmentation of TLR6-mediated production of IL-1alpha in monocytic cells via multiple signaling pathways.
Cholesterol ; Cytokines ; Inflammation ; Interleukin-1 ; Interleukin-1alpha* ; Toll-Like Receptors*

Nystatin, a polyene antifungal antibiotic, is a cholesterol sequestering agent. The antifungal agent alters composition of the plasma membrane of eukaryotic cells, whereas its effects on cells are poorly investigated. In the current study, we investigated the question of whether nystatin was able to induce expression of macrophage inflammatory protein-1 (MIP-1). THP-1 cells rarely express MIP-1alpha and MIP-1beta, however, upon exposure to nystatin, significantly elevated expression of MIP-1alpha and MIP-1beta was observed in a dose-dependent fashion at the messenger and protein levels. Cellular factors activated by nystatin as well as involved in nystatin-induced expression of MIP-1 proteins were identified in order to understand the molecular mechanisms of action of the anti-fungal agent. Treatment with nystatin resulted in enhanced phosphorylation of Akt, ERK, p38 MAPK, and JNK. Abrogation or significant attenuation of nystatin-induced expression of MIP-1alpha and MIP-1beta was observed by treatment with Akt inhibitor IV, LY294002, and SP6001250. Inhibition of ERK or p38MAPK using U0126 and SB202190 did not lead to attenuation of MIP-1 expression. In addition, inhibitors of protein kinase C, such as GF109203X and Ro-318220, also attenuated expression of MIP-1. These results indicate that nystatin is able to activate multiple cellular kinases and, among them, Akt and JNK play primary roles in nystatin-induced expression of MIP-1 proteins.
Cell Membrane ; Chemokine CCL3 ; Chemokine CCL4 ; Cholesterol ; Eukaryotic Cells ; Macrophage Inflammatory Proteins* ; Macrophages* ; Nystatin* ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Phosphotransferases ; Protein Kinase C

TLR6 forms a heterodimer with TLR2 and TLR4. While proinflammatory roles of TLR2 and TLR4 are well documented, the role of TLR6 in inflammation is poorly understood. In order to understand mechanisms of action of TLR6 in inflammatory responses, we investigated the effects of FSL-1, the TLR6 ligand, on expression of chemokine CCL2 and cytokine IL-1beta and determined cellular factors involved in FSL-1-mediated expression of CCL2 and IL-1beta in mononuclear cells. Exposure of human monocytic leukemia THP-1 cells to FSL-1 resulted not only in enhanced secretion of CCL2 and IL-1beta, but also profound induction of their gene transcripts. Expression of CCL2 was abrogated by treatment with OxPAPC, a TLR-2/4 inhibitor, while treatment with OxPAPC resulted in partially inhibited expression of IL-1beta. Treatment with FSL-1 resulted in enhanced phosphorylation of Akt and mitogen-activated protein kinases and activation of protein kinase C. Treatment with pharmacological inhibitors, including SB202190, SP6001250, U0126, Akt inhibitor IV, LY294002, GF109203X, and RO318220 resulted in significantly attenuated FSL-1-mediated upregulation of CCL2 and IL-1beta. Our results indicate that activation of TLR6 will trigger inflammatory responses by upregulating expression of CCL2 and IL-1beta via TLR-2/4, protein kinase C, PI3K-Akt, and mitogen-activated protein kinases.
Butadienes ; Chemokine CCL2 ; Chromones ; Humans ; Imidazoles ; Indoles ; Inflammation ; Leukemia ; Macrophages ; Maleimides ; Mitogen-Activated Protein Kinases ; Morpholines ; Nitriles ; Phosphatidylcholines ; Phosphorylation ; Protein Kinase C ; Pyridines ; Toll-Like Receptor 6 ; Toll-Like Receptors ; Up-Regulation

To understand the roles of purinergic receptors and cellular molecules below the receptors in the vascular inflammatory response, we determined if extracellular nucleotides up-regulated chemokine expression in vascular smooth muscle cells (VSMCs). Human aortic smooth muscle cells (AoSMCs) abundantly express P2Y1, P2Y6, and P2Y11 receptors, which all respond to extracellular nucleotides. Exposure of human AoSMCs to NAD+ , an agonist of the human P2Y11 receptor, and NADP+ as well as ATP, an agonist for P2Y1 and P2Y11 receptors, caused increase in chemokine (C-C motif) ligand 2 gene (CCL2) transcript and CCL2 release; however, UPT did not affect CCL2 expression. CCL2 release by NAD+ and NADP+ was inhibited by a concentration dependent manner by suramin, an antagonist of P2-purinergic receptors. NAD+ and NADP+ activated protein kinase C and enhanced phosphorylation of mitogen-activated protein kinases and Akt. NAD(+)- and NADP(+)-mediated CCL2 release was significantly attenuated by SP6001250, U0126, LY294002, Akt inhibitor IV, RO318220, GF109203X, and diphenyleneiodium chloride. These results indicate that extracellular nucleotides can promote the proinflammatory VSMC phenotype by up-regulating CCL2 expression, and that multiple cellular elements, including phosphatidylinositol 3-kinase, Akt, protein kinase C, and mitogen-activated protein kinases, are involved in that process.
Adenosine Triphosphate ; Butadienes ; Chromones ; Humans ; Indoles ; Maleimides ; Mitogen-Activated Protein Kinases ; Morpholines ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle ; Nitriles ; Nucleotides ; Onium Compounds ; Phenotype ; Phosphatidylinositol 3-Kinase ; Phosphorylation ; Protein Kinase C ; Receptors, Purinergic ; Suramin

Angiotensin II (AngII) is a crucial hormone that affects vasoconstriction and exerts hypertrophic effects on vascular smooth muscle cells. Here, we showed that phosphatidylinositol 3-kinase-dependent calcium mobilization plays pivotal roles in AngII-induced vascular constriction. Stimulation of rat aortic vascular smooth muscle cell (RASMC)-embedded collagen gel with AngII rapidly induced contraction. AngII-induced collagen gel contraction was blocked by pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) whereas ERK inhibitor (PD98059) was not effective. AngII-induced collagen gel contraction was significantly blocked by extracellular calcium depletion by EGTA or by nifedipine which is an L-type calcium channel blocker. In addition, AngII-induced calcium mobilization was also blocked by nifedipine and EGTA, whereas intracellular calcium store-depletion by thapsigargin was not effective. Finally, pretreatment of rat aortic ring with LY294002 and nifedipine significantly reduced AngII-induced constriction. Given these results, we suggest that PI3K-dependent activation of L-type calcium channels might be involved in AngII-induced vascular constriction.
1-Phosphatidylinositol 3-Kinase/*metabolism/pharmacology ; Angiotensin II/metabolism/*pharmacology ; Animals ; Aorta, Thoracic/*drug effects/physiology ; Calcium Channels, L-Type/drug effects/*metabolism ; Muscle Contraction/drug effects ; Muscle, Smooth, Vascular/drug effects/enzymology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction/*drug effects ; Specific Pathogen-Free Organisms ; Vasoconstriction/*drug effects

Heat shock proteins (HSPs) serve as molecular chaperones and play a role in cell protection from damage in response to stress stimuli. The aim of this article is to investigate whether HSP22 affects IL-8 expression in vascular smooth muscle cells (VSMCs), and which cellular factors are involved in the HSP-mediated IL-8 induction in that cell type in terms of mitogen activated protein kinase (MAPK) and transcription element. Exposure of aortic smooth muscle cells (AoSMCs) to HSP22 not only enhanced IL-8 release but also induced IL-8 transcript via promoter activation. HSP22 activated ERK and p38 MAPK in AoSMCs. HSP22-induced IL-8 release was inhibited by U0126, but not by SB202190. A mutation in the IL-8 promoter region at the binding site of NF-kappa B, but not AP-1 or C/EBP, impaired promoter activation in response to HSP22. Delivery of I kappa B, but not dominant negative c-Jun, lowered HSP22-induced IL-8 release from AoSMCs. These results suggest that HSP22 induces IL-8 in VSMCs via ERK1/2, and that transcription factor NF-kB may be required for the HSP22-induced IL-8 up-regulation.
Binding Sites ; Butadienes ; Cytoprotection ; Heat-Shock Proteins ; Hot Temperature ; I-kappa B Proteins ; Imidazoles ; Interleukin-8 ; Molecular Chaperones ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle ; NF-kappa B ; Nitriles ; p38 Mitogen-Activated Protein Kinases ; Promoter Regions, Genetic ; Protein Kinases ; Proteins ; Pyridines ; Shock ; Transcription Factor AP-1

In the present study, we aimed to identify the synergistic effects of concurrent treatment of low concentrations of cilostazol and probucol to inhibit the oxidative stress with suppression of inflammatory markers in the cultured human coronary artery endothelial cells (HCAECs). Combination of cilostazol (0.3~3micrometer) with probucol (0.03~0.3micrometer) significantly suppressed TNF-alpha-stimulated NAD(P)H-dependent superoxide, lipopolysaccharide (LPS)-induced intracellular reactive oxygen species (ROS) production and TNF-alpha release in comparison with probucol or cilostazol alone. The combination of cilostazol (0.3~3micrometer) with probucol (0.1~0.3micrometer) inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) more significantly than did the monotherapy with either probucol or cilostazol. In line with these results, combination therapy significantly suppressed monocyte adhesion to endothelial cells. Taken together, it is suggested that the synergistic effectiveness of the combination therapy with cilostazol and probucol may provide a beneficial therapeutic window in preventing atherosclerosis and protecting from cerebral ischemic injury.
Atherosclerosis ; Chemokine CCL2 ; Coronary Vessels ; Endothelial Cells ; Humans ; Monocytes ; Oxidative Stress ; Probucol ; Reactive Oxygen Species ; Superoxides ; Tetrazoles ; Tumor Necrosis Factor-alpha ; Vascular Cell Adhesion Molecule-1