ABSTRACT
IL-1ß is a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19, and IL-1ß blockade with anakinra and canakinumab during COVID-19 infection has entered clinical trials. Using mass cytometry of human peripheral blood mononuclear cells, we identified effector memory CD4+ T cells and CD4-CD8low/-CD161+ T cells, specifically those positive for the chemokine receptor CCR6, as the circulating immune subtypes with the greatest response to IL-1ß. This response manifested as increased phosphorylation and, thus, activation of the proinflammatory transcription factor NF-κB and was also seen in other subsets, including CD11c+ myeloid dendritic cells, classical monocytes, two subsets of natural killer cells (CD16-CD56brightCD161- and CD16-CD56dimCD161+), and lineage- (Lin-) cells expressing CD161 and CD25. IL-1ß also induced a rapid but less robust increase in the phosphorylation of the kinase p38 as compared to that of NF-κB in most of these immune cell subsets. Prolonged IL-1ß stimulation increased the phosphorylation of the transcription factor STAT3 and to a lesser extent that of STAT1 and STAT5 across various immune cell types. IL-1ß-induced production of IL-6 likely led to the activation of STAT1 and STAT3 at later time points. Interindividual heterogeneity and inhibition of STAT activation by anakinra raise the possibility that assays measuring NF-κB phosphorylation in response to IL-1ß in CCR6+ T cell subtypes could identify those patients at higher risk of cytokine storm and most likely to benefit from IL-1ß-neutralizing therapies.
Subject(s)
COVID-19/immunology , Interleukin-1beta/blood , T-Lymphocyte Subsets/immunology , COVID-19/blood , COVID-19/complications , Cytokine Release Syndrome/blood , Cytokine Release Syndrome/etiology , Cytokine Release Syndrome/immunology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Flow Cytometry , Humans , Interleukin-1beta/pharmacology , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Monocytes/classification , Monocytes/immunology , Monocytes/metabolism , NF-kappa B/blood , Pandemics , Phosphorylation , Receptors, CCR6/blood , SARS-CoV-2 , STAT Transcription Factors/blood , STAT Transcription Factors/immunology , Signal Transduction/immunology , T-Lymphocyte Subsets/metabolism , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
CXCL12, also known as stromal cell-derived factor-1, is a chemokine classified into CXC families, which exerts its function by binding to specific receptors called CXCR4 and CXCR7. Human platelets express CXCR4 and CXCR7 on the plasma membrane. It has been reported that CXCL12 potentiates to induce platelet aggregation in cooperation with agonists including collagen. However, the precise roles and mechanisms of CXCL12 in human platelet activation are not fully elucidated. In the present study, we investigated the effect of simultaneous stimulation with low doses of collagen and CXCL12 on the activation of human platelets. The simultaneous stimulation with collagen and CXCL12 induced the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble CD40 ligand (sCD40L) from human platelets in addition to their aggregation, despite the fact that the simultaneous stimulation with thrombin receptor-activating peptide (TRAP) or adenosine diphosphate (ADP), and CXCL12 had little effects on the platelet aggregation. The agonist of Glycoprotein (GP) â ¥ convulxin and CXCL12 also induced platelet aggregation synergistically. The monoclonal antibody against CXCR4 but not CXCR7 suppressed the platelet aggregation induced by simultaneous stimulation with collagen and CXCL12. The phosphorylation of p38 mitogen-activated protein kinase (MAPK), but not p44/p42 MAPK, was induced by the simultaneous stimulation. In addition, the simultaneous stimulation with collagen and CXCL12 induced the phosphorylation of HSP27 and the subsequent release of phosphorylated-HSP27 from human platelets. SB203580, a specific inhibitor of p38 MAPK, attenuated the platelet aggregation, the phosphorylation of p38 MAPK and HSP27, the PDGF-AB secretion, the sCD40L release and the phosphorylated-HSP27 release induced by the simultaneous stimulation with collagen and CXCL12. These results strongly suggest that collagen and CXCL12 in low doses synergistically act to induce PDGF-AB secretion, sCD40L release and phosphorylated-HSP27 release from activated human platelets via p38 MAPK activation.
Subject(s)
Blood Platelets/drug effects , Chemokine CXCL12/pharmacology , Collagen/pharmacology , Platelet Activation/drug effects , Blood Platelets/cytology , Blood Platelets/metabolism , CD40 Ligand/blood , Healthy Volunteers , Heat-Shock Proteins/blood , Humans , Molecular Chaperones/blood , Platelet-Derived Growth Factor/metabolism , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
BACKGROUND: The intracellular ROCK signaling pathway is an important modulator of blood pressure and of cardiovascular and renal remodeling when Rho-kinase activity is increased. Besides, in preclinical models of diabetes, ROCK activation has also a role in abnormal glucose metabolism as well as in subsequent vascular and myocardial dysfunction. In humans, there are a few data assessing ROCK activation in patients with type 2 diabetes mellitus (T2D) and no studies assessing upstream/downstream components of the ROCK pathway. We assessed here levels of ROCK activation and some of the RhoA/ROCK cascade molecules in peripheral blood mononuclear cells (PBMCs) in T2D patients under current treatment. METHODS: Cross-sectional observational study comparing 28 T2D patients under current antidiabetic treatment with 31 consecutive healthy subjects, matched by age and gender. Circulating levels of malondialdehyde, angiotensin II and inflammatory cytokines IL-6 and IL-8 were determined in all subjects. ROCK activation in PMBCs, upstream and downstream cascade proteins, and levels of the proinflammatory molecules VCAM, ICAM-1 and IL-8 were determined in their PMBCs by Western blot. RESULTS: Compared to healthy controls, ROCK activation in T2D patients measured by 2 direct ROCK targets in PBMCs was increased by 420 and 570% (p < 0001) and it correlated significantly with serum glucose levels. p38 MAPK phosphorylation (downstream from ROCK) and JAK-2 (upstream from ROCK) were significantly higher in the T2D patients by 580% and 220%, respectively. In T2D patients, significantly increased PBMC levels of the proinflammatory molecules VCAM-1, ICAM-1 and IL-8 were observed compared to control subjects (by 180%, 360% and 260%, respectively). Circulating levels of Ang II and MDA were significantly higher in T2D patients by 29 and 63%, respectively. CONCLUSIONS: T2D patients under treatment with glucose-lowering drugs, antihypertensive treatment as well as with statins have significantly increased ROCK activation in their circulating leukocytes along with higher phosphorylation of downstream cascade proteins despite pharmacologic treatment, along with increased plasma angiotensin II and MDA levels. ROCK inhibition might have an additional role in the prevention and treatment of T2D.
Subject(s)
Diabetes Mellitus, Type 2/enzymology , Leukocytes, Mononuclear/enzymology , rho-Associated Kinases/blood , Aged , Angiotensin II/blood , Antihypertensive Agents/therapeutic use , Biomarkers/blood , Case-Control Studies , Cross-Sectional Studies , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/diagnosis , Diabetes Mellitus, Type 2/drug therapy , Enzyme Activation , Female , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Hypoglycemic Agents/therapeutic use , Intercellular Adhesion Molecule-1/blood , Interleukin-8/blood , Janus Kinase 2/blood , Male , Malondialdehyde/blood , Middle Aged , Signal Transduction , Vascular Cell Adhesion Molecule-1/blood , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
The aim of this study was to investigate the roles of CD4+ T cells and transforming growth factor beta (TGFß1) in the pathological process of valvular hyperblastosis and fibrosis of patients with rheumatic heart disease (RHD). A total of 151 patients were enrolled, among whom, 78 patients were with RHD, and 73 were age and gender matched RHD negative patients. Blood samples and valve specimens were collected for analysis. Pathological changes and collagen fibers contents of valves were analyzed using HE and Masson staining. Percentage of peripheral blood CD4+ T cells was tested through flow cytometry. TGFß1 level in serum were identified by ELISA. CD4+ T cells infiltration and expression of TGFß1, p-p38, p-JNK, p-ERK in valves were detected by immunohistochemistry. The mRNA and protein levels of p38, JNK, ERK, TGFß1, I-collagen and α-SMA were detected by qRT-PCR and western blotting, respectively. The heart valve tissues of RHD patients showed higher degrees of fibrosis, calcification and lymphocytes infiltration, which were mainly CD4+ T cells. In addition, compared with control group, RHD patients had more total CD4+ T cells in peripheral blood and valve tissues. Expression of TGFß1, phosphorylation of JNK and p38, and synthesis of I-collagen in valve tissues of RHD patients were also significantly increased. Furthermore, we found a strong positive correlation between TGFß1 expression and phosphorylation of JNK and p38. CD4+ T cells, and fibrogenic cytokine TGFß1, which activate the intracellular MAPK signaling pathway may participate in the fibrosis of heart valve in RHD patients.
Subject(s)
Heart Valve Diseases/genetics , Mitral Valve Stenosis/genetics , Rheumatic Heart Disease/genetics , Transforming Growth Factor beta1/genetics , Adult , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/physiology , Extracellular Signal-Regulated MAP Kinases/blood , Extracellular Signal-Regulated MAP Kinases/genetics , Female , Fibrosis/blood , Fibrosis/genetics , Fibrosis/pathology , Gene Expression Regulation/genetics , Heart Valve Diseases/blood , Heart Valve Diseases/pathology , Humans , MAP Kinase Kinase 4/blood , MAP Kinase Kinase 4/genetics , MAP Kinase Signaling System/genetics , Male , Middle Aged , Mitral Valve Stenosis/blood , Mitral Valve Stenosis/pathology , Rheumatic Heart Disease/blood , Rheumatic Heart Disease/pathology , Transforming Growth Factor beta1/blood , p38 Mitogen-Activated Protein Kinases/blood , p38 Mitogen-Activated Protein Kinases/geneticsABSTRACT
Spinal cord injury (SCI) is associated with inflammation with concurrent oxidative stress and glial activation. The aim of this study was to evaluate whether hesperidin, a representative flavonoid in citrus fruits, ameliorates SCI-induced motor dysfunction and neuro-pathologic degeneration in rat model. Rats received hesperidin (100â¯mg/kg body weight/daily, oral administration) from 7 days prior to SCI to 7 days post SCI. Behavioral test was done on rats with SCI until 6 weeks. For the study of inflammatory molecules in SCI rats with hesperidin treatment, rats were sacrificed at day 4 post SCI, and spinal cords were collected and studied histopathologically. Behavioral tests on hind-limbs of rats with SCI revealed that treatment of hesperidin in rats with SCI significantly ameliorate the hind-limb paralysis beginning at day 21 post SCI. Hesperidin treatment in rats with SCI reduced the neuropathological changes (e.g., hemorrhage, inflammatory cell infiltration, and tissue loss) and pro-inflammatory cytokines including tumor necrotic factor-α and interleukin-1ß. In addition, oxidative stress related molecules including superoxide dismutase, catalase, nuclear factor erythroid 2-related factor-2 and heme oxygenase-1 were also increased by hesperidin treatment. Furthermore, Fe2+, bilirubin and p38 mitogen activated protein kinase, these by-product of heme catabolism in serum and spinal cord of rats with hesperidin-treatment groups were significantly increased compared with those of vehicle-treatment group. Collectively, this study implies that hesperidin accelerates recovery of locomotor function and tissue repair of damaged spinal cord, with concurrent upregulation of heme oxygenase-1 as far as rat SCI model is concerned.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Antioxidants/pharmacology , Heme Oxygenase (Decyclizing)/metabolism , Hesperidin/pharmacology , NF-E2-Related Factor 2/metabolism , Paralysis/prevention & control , Signal Transduction/drug effects , Spinal Cord Injuries/prevention & control , Animals , Bilirubin/blood , Hindlimb/drug effects , Iron/blood , Male , Rats , Spinal Cord/metabolism , Spinal Cord Injuries/complications , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
Upregulation of fetal hemoglobin expression can alleviate the severity of ßthalassaemia. This study aimed to investigate the effects of Oridonin (ORI, a diterpenoid compound) on γglobin expression in human erythroid precursor cells and the potential underlying mechanisms. Erythroid precursor cells were enriched from 12 patients with ßthalassaemia by twophase culture. The cells were then treated with different doses of ORI and the survival of erythroid precursor cells was determined. In addition, the expression levels of γglobin and potential mechanisms were analyzed by reverse transcriptionquantitative PCR, western blotting and chromatin immunoprecipitation. Treatment with 0.5 µM ORI preferably enhanced γglobin expression and exhibited little cytotoxicity. Similar to sodium butyrate (NaB, a histone deacetylase inhibitor), ORI significantly increased p38 mitogenactivated protein kinase (MAPK) activation, γglobin expression, histone H3 and H4 acetylation at the Gγ and Aγglobin promoters, and cAMPresponse element binding protein 1 (CREB1) phosphorylation. These effects were significantly mitigated by treatment with SB23580, a p38 MAPK inhibitor, in erythroid precursor cells. Therefore, ORI may effectively enhance γglobin expression by activating p38 MAPK and CREB1, leading to histone modification in γglobin gene promoters during the maturation of erythroid precursor cells. These findings suggested that ORI may be a novel and potential therapeutic agent for the treatment of ßthalassaemia.
Subject(s)
Diterpenes, Kaurane/pharmacology , Erythroid Precursor Cells/metabolism , beta-Thalassemia/metabolism , gamma-Globins/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Acetylation , Adolescent , Child , Child, Preschool , Chromatin Immunoprecipitation , Cyclic AMP Response Element-Binding Protein/blood , Cyclic AMP Response Element-Binding Protein/chemistry , Cyclic AMP Response Element-Binding Protein/metabolism , Female , Histones/blood , Histones/chemistry , Histones/metabolism , Humans , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Male , Phosphorylation , Promoter Regions, Genetic , beta-Thalassemia/genetics , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/blood , p38 Mitogen-Activated Protein Kinases/geneticsABSTRACT
Due to their significance in trophoblast differentiation and survival, we evaluated the expression of the cell signalling molecules; Extracellular signal-regulated kinase 1/2 (ERK1/2), Mitogen Activated Protein Kinase 38 (MAPK38) and p90 ribosomal protein S6 kinase (p90 RSK) in buffy coat samples. Eighty pregnant women attending a large hospital in Durban, South Africa were assigned into normotensive and pre-eclamptic groups and further stratified by their HIV status. The degree of phosphorylation of the analytes was determined using the Bio-Plex ProTM Cell Signalling Immunoassay. There was a significantly lower protein concentration of the analytes in the pre-eclamptic versus the normotensive patients, irrespective of HIV status (p < .0001). Also, there was no significant difference in expression of ERK1/2 (p = .4369), p38MAPK (p = .4720) and p90 RSK (p = .0188), according to HIV status. This study demonstrates a down-regulation of ERK1/2, p38MAPK and p90RSK prosurvival markers in pre-eclampsia. This implicates the involvement of the MAPK pathway in the pathogenesis of preeclampsia. Activation of these pathways may prove useful in increasing the body of evidence on prevention of placenta dysfunction and apoptosis. Impact statement What is already known on this subject? Preeclampsia occurring in co-morbidity with HIV is a public health problem among pregnant, black South-African women. There have been conflicting theories regarding the predisposition to the development of preeclampsia as a result of compromised immune response due to HIV infection. In normal pregnancies, the MAPK pathway plays a significant role in molecular processes involved in the cells including survival and differentiation of the placental trophoblast. ERK1/2, p38MAPK and p90RSK are members of the MAPK family, which are pro-apoptotic. Inhibition in the signalling of MAPKs has been found to result in oxidative stress, a process which contributes to the defective trophoblast invasion seen in preeclampsia. What do the results of this study add? The results from this study showed that there is no relationship between HIV infection and an increased predisposition to the development of preeclampsia. In addition, this study highlights a downregulation in the expression of ERK1/2, p38 MAPK and p90RSK in preeclampsia. What are the implications of these findings for clinical practice and/or further research? These findings demonstrate the potential of these analytes as biomarkers for the diagnosis of preeclampsia. Also, this may serve as a framework for further research in the prevention of preeclampsia by elucidating more on the pathway.
Subject(s)
HIV Infections/complications , MAP Kinase Signaling System/physiology , Pre-Eclampsia/virology , Ribosomal Protein S6 Kinases, 90-kDa/physiology , p38 Mitogen-Activated Protein Kinases/physiology , Adult , Female , HIV Seropositivity , Humans , Phosphorylation , Pre-Eclampsia/physiopathology , Pregnancy , Retrospective Studies , Ribosomal Protein S6 Kinases, 90-kDa/blood , Signal Transduction , South Africa , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
INTRODUCTION: p38 MAPK signaling molecules plays a dual role in cancer, both progression and suppression. Elevated expression of p38α was reported in lung cancer tissue in rat model. Our objective was to explore the concentration of all 4 isoforms of p38MAPK in serum of Non Small Cell Lung Cancer (NSCLC). MATERIAL AND METHODS: The blood samples were collected from 77 NSCLC patients, 52 ethically matched healthy controls and 18 follow up patients were collected as some patients expired and some discontinued the treatment. The concentration of all isoforms of p38 (p38α, p38ß, p38γ, and p38δ) were evaluated by Surface Plasmon Resonance (SPR) technology. RESULT: The levels of all isoforms of serum p38 were significantly elevated at pre-therapy compare to control. Only p38α expression was significantly associated with tumor stage and its expression reduced after treatment which is then validated by western blot. However, no changes were observed in other isoforms after therapy. CONCLUSION: Our study revealed that, p38α is more efficient among all the isoform to predict the disease accurately and it can be concluded that p38 MAPK may be used as diagnostic as well as prognostic marker of NSCLC disease.
Subject(s)
Biomarkers, Tumor/blood , Carcinoma, Non-Small-Cell Lung/blood , Lung Neoplasms/blood , p38 Mitogen-Activated Protein Kinases/blood , Adult , Aged , Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/mortality , Female , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/mortality , Male , Middle Aged , PrognosisABSTRACT
BACKGROUND: Inflammation helps regulate normal growth and tissue repair. Although bone morphogenetic proteins (BMPs) and inflammation are known contributors to abnormal bone formation, how these pathways interact in ossification remains unclear. METHODS: We examined this potential link in patients with fibrodysplasia ossificans progressiva (FOP), a genetic condition of progressive heterotopic ossification caused by activating mutations in the Activin A type I receptor (ACVR1/ALK2). FOP patients show exquisite sensitivity to trauma, suggesting that BMP pathway activation may alter immune responses. We studied primary blood, monocyte, and macrophage samples from control and FOP subjects using multiplex cytokine, gene expression, and protein analyses; examined CD14+ primary monocyte and macrophage responses to TLR ligands; and assayed BMP, TGF-ß activated kinase 1 (TAK1), and NF-κB pathways. RESULTS: FOP subjects at baseline without clinically evident heterotopic ossification showed increased serum IL-3, IL-7, IL-8, and IL-10. CD14+ primary monocytes treated with the TLR4 activator LPS showed increased CCL5, CCR7, and CXCL10; abnormal cytokine/chemokine secretion; and prolonged activation of the NF-κB pathway. FOP macrophages derived from primary monocytes also showed abnormal cytokine/chemokine secretion, increased TGF-ß production, and p38MAPK activation. Surprisingly, SMAD phosphorylation was not significantly changed in the FOP monocytes/macrophages. CONCLUSIONS: Abnormal ACVR1 activity causes a proinflammatory state via increased NF-κB and p38MAPK activity. Similar changes may contribute to other types of heterotopic ossification, such as in scleroderma and dermatomyositis; after trauma; or with recombinant BMP-induced bone fusion. Our findings suggest that chronic antiinflammatory treatment may be useful for heterotopic ossification.
Subject(s)
Activin Receptors, Type I/blood , Inflammation/complications , Myositis Ossificans/complications , NF-kappa B/blood , Ossification, Heterotopic/etiology , Chemokines/blood , Cytokines/blood , Humans , Inflammation/blood , Macrophages/metabolism , Monocytes/metabolism , Myositis Ossificans/blood , Myositis Ossificans/immunology , Ossification, Heterotopic/blood , Ossification, Heterotopic/immunology , Signal Transduction , Transforming Growth Factor beta/blood , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
Nuclear factor erythroid-related factor 2 (Nrf2) has been implicated in several detoxifying and antioxidant defense processes. Nrf2-mediated heme oxygenase-1 (HO-1) expression was demonstrated to play a key role against oxidative stress. Gastrodin (GSTD) is a well-known active compound isolated from the roots of Rhizoma gastrodiae, a plant used in ancient Chinese traditional medicine. The aim of this work was to investigate whether GSTD could alleviate H2O2-induced oxidative stress in mouse liver sinusoidal endothelial cells (LSECs). In LSECs exposed to 1 mM H2O2, treatment with GSTD (1, 10, or 50 µM) resulted in higher cell viability than the untreated control. Treated cells maintained a higher Bcl2/Bax ratio and suppressed caspase-9 expression compared with untreated cells, reducing cell apoptosis. GSTD was protective for H2O2-induced oxidative injury by reducing the generation of intracellular reactive oxygen species and malondialdehyde. HO-1 and Nrf2 expressions were synergistically upregulated by GSTD. Inhibition of HO-1 by 10 µM zinc protoporphyrin resulted in less protective effects on cell viability and malondialdehyde reduction by GSTD treatment in H2O2-exposed LSECs. Additionally, phosphorylated p38 in LSECs exposed to H2O2 was elevated by GSTD. Inhibition of p38 phosphorylation by SB203580 did not induce Nrf2 and HO-1 expression after 1 or 10 µM GSTD treatment and the protective effect on cell viability and malondialdehyde reduction in H2O2-exposed LSECs was reduced. The data conclusively demonstrated that GSTD-induced HO-1 and Nrf2 expression is involved in protection of LSECs from H2O2-induced oxidative injury, which may be regulated by p38 phosphorylation.
Subject(s)
Benzyl Alcohols/pharmacology , Endothelial Cells/drug effects , Glucosides/pharmacology , Heme Oxygenase-1/metabolism , Hydrogen Peroxide/pharmacology , NF-E2-Related Factor 2/metabolism , Oxidative Stress/drug effects , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Apoptosis/drug effects , Cell Survival/drug effects , Liver/cytology , Liver/drug effects , Malondialdehyde/metabolism , Mice , Models, Theoretical , Oxidative Stress/physiology , Phosphorylation/drug effects , Reactive Oxygen Species/metabolism , Up-Regulation/drug effects , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
Hyperhomocystinemia (HHcy) is known as an independent risk factor for cardiovascular disease. Our previous study showed that ginsenoside Rb1, the major active constituent of ginseng, prevents homocysteine (Hcy)-induced endothelial damage. However, the role of ginsenoside Rb1 in Hcy-induced dysfunction in endothelial progenitor cells (EPCs) remains unknown. In the study, we found that ginsenoside Rb1 reversed the Hcy-induced impairment of adhesive and migratory ability in EPCs which were significantly abolished by CXCR4 antagonist AMD3100 and VEGFR2 inhibitor SU5416. Ginsenoside Rb1 significantly reversed Hcy-induced SDF-1 reduction in the supernatant and in the serum. Ginsenoside Rb1 reversed downregulation of SDF-1 and VEGFR2 protein expression, inhibition of p38MAPK phosphorylation induced by Hcy. Re-endothelialization in balloon-injured carotid arteries significantly increased with EPCs transplant, and was even better with Rb1 treatment. This effect was significantly abolished by AMD3100. AMD3100 also decreased the number of CM-DiI labeled EPCs in injured arteries. Here we show for the first time that Rb1 prevents Hcy-induced EPC dysfunction via VEGF/p38MAPK and SDF-1/CXCR4 activation. These findings demonstrate a novel mechanism of the action of Rb1 that may have value in prevention of HHcy associated cardiovascular disease.
Subject(s)
Endothelial Progenitor Cells/drug effects , Endothelial Progenitor Cells/metabolism , Ginsenosides/pharmacology , Homocysteine/pharmacology , Vascular Endothelial Growth Factor A/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Benzylamines , Cell Movement/drug effects , Cell Survival/drug effects , Chemokine CXCL12/blood , Chemokine CXCL12/metabolism , Cyclams , Heterocyclic Compounds/pharmacology , Indoles/pharmacology , Male , Phosphorylation/drug effects , Pyrroles/pharmacology , Rats , Rats, Sprague-Dawley , Vascular Endothelial Growth Factor A/blood , Vascular Endothelial Growth Factor Receptor-2/blood , Vascular Endothelial Growth Factor Receptor-2/metabolism , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
Fibrocytes, ahematopoietic stem cell source of fibroblasts/myofibroblasts, were previously implicated to infiltrate into the intestinal and enhance inflammation.The aims of the present study were to elucidate the role of fibrocytes in necrotizing enterocolitis (NEC) pathogenesis and to explore the mechanisms by which fibrocytes contributed to the inflammatory responses.We investigated circulating and intestinal local fibrocytes from 32 patients with NEC, 8 patients with noninflammatory conditions of the gastrointestinal tract and 12 normal subjects.Significantly higher numbers of circulating fibrocytes were found in the peripheral blood from NEC patients than the controls (Pâ<â.01). Numerous fibrocytes were found infiltrating the NEC intestinal mucous membranes. The percentage of fibrocytes to total leukocytes in the NEC inflammatory lesions was significantly increased compared with the percentage in the noninflammatory gastrointestinal tract. The fibrocyte attractant chemokine C-X-C motif chemokine ligand 12 (CXCL12) was significantly increased in the plasma and was detectable in 80% of the peritoneal lavage fluid from NEC patients but not the controls. Furthermore, chemokine expression was increased in fibrocytes infiltrating and trafficking to leukocyte sites. In culture, lipopolysaccharide (LPS) induced a significant increase in the expression of the Toll-like receptor (TLR4) signal, with the upregulation of p38 in both the isolated fibrocytes and macrophages. Similarly, interleukin (IL)-1ß induced increased the upregulation of the IL-6, tumor necrosis factor (TNF)-α, and intercellular cell adhesion molecule-1 mRNAs but downregulated ColI in fibrocytes isolated from NEC patients compared with the controls.These findings indicate that circulating fibrocytes are increased in NEC patients and may be recruited to the inflammatory intestinal track, most likely through the CXCR4/CXCL12 axis. These cells may contribute to intestinal inflammation through TLR4 signaling by producing the TNF-α and IL-6 cytokines.
Subject(s)
Enterocolitis, Necrotizing/blood , Enterocolitis, Necrotizing/immunology , Leukocytes/immunology , Biomarkers/blood , Cell Count , Cell Movement/physiology , Cells, Cultured , Chemokine CXCL12/blood , Enterocolitis, Necrotizing/pathology , Enterocolitis, Necrotizing/surgery , Humans , Infant, Newborn , Intercellular Adhesion Molecule-1/blood , Interleukin-1beta/blood , Interleukin-6/blood , Leukocytes/pathology , Lipopolysaccharides , RNA, Messenger/blood , Toll-Like Receptor 4/blood , Tumor Necrosis Factor-alpha/blood , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
The effect of lipopolysaccharide (LPS) on platelet aggregation is still controversial. We performed in vitro and ex vivo studies in controls and in patients with community-acquired pneumonia (CAP) to assess the effect of LPS on platelet activation (PA). LPS (15-100 pg/ml) significantly increased PA only if combined with sub-threshold concentrations (STC) of collagen or ADP; this effect was associated with increased platelet H2O2 production, Nox2 activation, PLA2 phosphorylation, thromboxane (Tx)A2 and 8-iso-PGF2α-III, and was inhibited by aspirin, TxA2 receptor antagonist or by Toll-like receptor 4 blocking peptide (TLR4bp). Analysis of up-stream signalling potentially responsible for Nox2 and PLA2 activation demonstrated that LPS-mediated PA was associated with phosphorylation of AKT, p38 and p47phox translocation. In 10 consecutive CAP patients serum endotoxins were significantly higher compared to 10 controls (145 [115-187] vs 18 [6-21] pg/ml; p<0.01). Ex vivo study showed that agonist-stimulated platelets were associated with enhanced PA (p<0.01), Toll-like receptor 4 (TLR4) expression (p<0.05), TxA2 (p<0.01) and 8-iso-PGF2α-III (p<0.01) production in CAP patients compared to controls. The study provides evidence that LPS amplifies the platelet response to common agonists via TLR4-mediated eicosanoid production and suggests LPS as a potential trigger for PA in CAP.
Subject(s)
Blood Platelets/drug effects , Community-Acquired Infections/blood , Dinoprost/analogs & derivatives , Lipopolysaccharides/pharmacology , Platelet Aggregation/drug effects , Pneumonia, Bacterial/blood , Thromboxane A2/blood , Adult , Aged , Blood Platelets/metabolism , Blood Platelets/microbiology , Calcium Signaling/drug effects , Case-Control Studies , Community-Acquired Infections/diagnosis , Community-Acquired Infections/microbiology , Dinoprost/blood , Dose-Response Relationship, Drug , Enzyme Activation , Female , Group IV Phospholipases A2/blood , Humans , Hydrogen Peroxide/blood , Male , Middle Aged , NADPH Oxidase 2/blood , NADPH Oxidases/blood , Oxidative Stress/drug effects , Phosphorylation , Pneumonia, Bacterial/diagnosis , Pneumonia, Bacterial/microbiology , Proto-Oncogene Proteins c-akt/blood , Time Factors , Toll-Like Receptor 4/blood , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
BACKGROUND: The present study was intended to explore whether three proteins within MAPK signaling pathway (i.e. p38MAPK-1, HIF-1 and HO-1) were correlated with peri-menopausal women's coronary lesion features and prognosis. METHODS: Altogether 1449 peri-menopausal women were divided into non-coronary artery disease (CAD) group (n = 860) and CAD group (n = 589), including 167 pre-menopausal CAD populations and 422 post-menopausal CAD populations. General information about CAD risk parameters were gathered, including age, family history of CAD or hypertension or diabetes mellitus, bilirubin, cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) and so on. Coronary angiography results were judged, and CAD score was calculated with application of Genisin scoring method. Besides, detection of MAPK-1 levels was implemented with Strept Avidin-Biotin Complex (SABC) method, while HIF-1 and HO-1 expressions in the serum were determined utilizing ELISA detection kit. Correlations among protein expressions, characteristics of coronary lesions and prognosis of CAD populations were finally evaluated. RESULTS: Hypertension, hyperlipoidemia, diabetes and smoking history were more prevalent among postmenopausal CAD women than premenopausal CAD women (P < 0.05). Furthermore, postmenopausal women seemed to be significantly associated with multiple (i.e. double and triple) vessel lesions and severe lesion types (type B and C), when compared with premenopausal CAD group (P < 0.05). Similarly, remarkably elevated expressions of p38MAPK-1, HIF-1 and HO-1 were found within postmenopausal CAD populations in comparison to premenopausal ones (P < 0.05). The internal CysC, hs-CRP, TG and LDL-C concentrations all accorded with the following tendency: postmenopausal CAD women > premenopausal CAD women > non-CAD women. Moreover, p38MAPK-1, HIF-1 and HO-1 expressions were up-regulated with increasing number of vessel lesions and severity of coronary lesions among peri-menopausal women. Besides, among both pre-menopausal and post-menopausal CAD groups, positive correlations could be observed between MAPK-1 and TG (r s = 0.271; r s = 0.476), between HIF-1α and LDL-C (r s = 0.077; r s = 0.470), as well as between HO-1 and CysC (r s = 0.492; r s = 0.190) or hs-CRP (r s = 0.569; r s = 0.542) (all P < 0.05). MAPK-1, HIF-1α and HO-1 were also, respectively, positively correlated with CysC (r s = 0.415), hs-CRP (r s = 0.137), and TG (r s = 0.142), regarding post-menopausal CAD women (all P < 0.05). Finally, only SBP and TG were regarded as independent risk factors for CAD prognosis (i.e. high Genisin score) among premenopausal women (OR = 1.02, 95%CI: 1.01-1.18, P = 0.043; OR = 1.82, 95%CI: 1.01-3.33, P = 0.047). CONCLUSIONS: Expressions of p38MAPK-1, HIF-1 and HO-1 could serve as predictive roles for coronary lesions among peri-menopausal women.
Subject(s)
Coronary Artery Disease/blood , Heme Oxygenase-1/blood , Hypoxia-Inducible Factor 1, alpha Subunit/blood , p38 Mitogen-Activated Protein Kinases/blood , Adult , Aged , Coronary Angiography , Coronary Artery Disease/diagnostic imaging , Coronary Artery Disease/pathology , Female , Gene Expression Regulation , Humans , Middle Aged , Postmenopause/blood , Premenopause/blood , Prognosis , Risk Factors , Signal TransductionABSTRACT
Personalised health surveillance is infrequent or absent in occupational and environmental medicine. The shortage of functional tests in relevant cells and tissues greatly limits our understanding of environmental exposures and associated disease risk. We evaluated single cell signalling in peripheral blood mononuclear cells from 301 individuals in a cross sectional health survey 18 months after a chemical explosion of sulphorous coker gasoline. The accident created a malodourous environment leading to long-term health complaints. Multiple regression analysis revealed T-cell specific elevated phosphorylation of the stress kinase p-p38 (T180/Y182) among tobacco smokers and monocyte-specific elevated phosphorylation in employees at the explosion site. Other studies of the accident reported reduced tear film stability, and more airway obstruction and subjective health complaints among the employees at the accident site. Elevated monocyte p-p38 in the employee group was independent of such health effects, and could therefore be dependent on the sulphuric malodorous environment. The present study proposes signalling status in leukocytes as a scalable biomarker providing information about environmental exposures.
Subject(s)
Environmental Exposure/analysis , Gasoline/toxicity , Occupational Exposure/analysis , p38 Mitogen-Activated Protein Kinases/blood , Adolescent , Adult , Aged , Environmental Biomarkers/genetics , Female , Humans , Leukocytes, Mononuclear/drug effects , Male , Middle Aged , Norway , Phosphorylation/genetics , Single-Cell Analysis , T-Lymphocytes/drug effects , Young Adult , p38 Mitogen-Activated Protein Kinases/geneticsABSTRACT
BACKGROUND: Coronary artery bypass grafting (CABG) with conventional cardiopulmonary bypass (CPB) induces systemic inflammation. Miniaturized CPB may attenuate systemic inflammatory activation. The intracellular signaling pathways promoting inflammation in cardiac operations and the relative effects of CPB on these processes are uncertain. In this study, induction of reactive oxygen species (ROS) and activation of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK) within leukocytes, and leukocyte accumulation in cantharidin-induced blisters was compared in patients exposed to miniaturized CPB (mCPB) and those who underwent conventional CPB (cCPB). METHODS: Patients undergoing CABG were randomized to receive either cCPB (n = 13) or mCPB (n = 13). Blood samples were collected preoperatively and 5 times after initiating CPB (up to 5 hours) and analyzed by flow cytometry for intracellular markers of activation (ROS, p38-MAPK, and NF-κB phosphorylation). RESULTS: ROS in lymphocytes were elevated in cCPB compared with mCPB (p < 0.01), whereas ROS in granulocytes and monocytes were similar between groups. After initiation of CPB, p38-MAPK was higher in patients receiving cCPB compared with those receiving mCPB (p < 0.05). NF-κB phosphorylation in leukocyte subsets was similar in patients exposed to cCPB and those exposed to mCPB. Leukocyte accumulation in cantharidin-induced blisters, white cell counts, and serum C-reactive protein (CRP) was enhanced in response to cardiac operations, but no differences were observed between mCPB and cCPB groups. Postoperative serum creatinine levels were reduced in the mCPB group compared with the cCPB group (p < 0.05). CONCLUSIONS: Both p38-MAPK activation and ROS were attenuated with the use of mCPB compared with cCPB, providing a potential mechanism for reduced inflammation in association with CPB miniaturization.
Subject(s)
Cardiopulmonary Bypass/methods , Coronary Artery Bypass/methods , Inflammation Mediators/blood , Miniaturization/methods , NF-kappa B/blood , p38 Mitogen-Activated Protein Kinases/blood , Aged , C-Reactive Protein/metabolism , Cardiopulmonary Bypass/adverse effects , Cell Movement/physiology , Confidence Intervals , Coronary Artery Bypass/adverse effects , Coronary Stenosis/blood , Coronary Stenosis/surgery , Female , Follow-Up Studies , Humans , Inflammation/blood , Inflammation/prevention & control , Leukocytes/metabolism , Male , Middle Aged , Operative Time , Postoperative Complications/blood , Postoperative Complications/epidemiology , Postoperative Complications/physiopathology , Reactive Oxygen Species/blood , Risk Assessment , Treatment OutcomeABSTRACT
p38 mitogen-activated protein kinase (MAPK) is a pivotal enzyme in the biosynthesis of pro-inflammatory cytokines like IL-1 and TNF. Therefore, the success of anti-cytokine therapy for treatment of inflammatory processes qualified p38-MAPK as a solid target in drug research concerning chronic inflammatory diseases including infectious vascular, neurobiological, and autoimmune disorders. However, the discovery of new kinase inhibitors is limited by the need for a high biological activity combined with restricted activity to the target enzyme or pathway interaction. As a consequence, no p38 MAPK inhibitor has been introduced to the market so far, although several p38 inhibitors have proceeded into clinical trials. The development of novel inhibitor types and optimization of already known structural classes of MAPK inhibitors require appropriate testing systems reaching across these crucial parameters. As a new approach, we describe the sequential arrangement of three testing systems custom-tailored to the requirements of drug discovery programs with focus on p38 inhibition. Integrated analysis of the obtained results enables a concerted step-by-step selection of tested molecules in order to screen a compound library for the most suitable inhibitor. First, evaluation of the inhibitor's activity on the isolated p38 MAPK enzyme via an ELISA assay gives a first idea about the inhibitory potency of the molecule. Moreover, structure-activity relationships can be elucidated when comparing molecules within inhibitor series. Second, screening in living cells via a p38 substrate-specific MK2-EGFP translocation assay supplies further information about efficacy, but provides also a first notion concerning selectivity and toxicity. Third, efficacy is evaluated more specifically in vivo in LPS-stimulated human whole blood with regard to in vivo parameters, e.g., pharmacokinetic characteristics like plasma protein binding and cellular permeability. These three testing systems complement one another synergistically by providing a high overlap and predictability. Clear advantages of all presented systems are their realizability in an academic environment as well as their applicability for high-throughput screenings on a larger scale.
Subject(s)
High-Throughput Screening Assays/methods , Protein Kinase Inhibitors/isolation & purification , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Blood Proteins/metabolism , Drug Discovery/methods , Enzyme-Linked Immunosorbent Assay/methods , Genes, Reporter , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Humans , Indicators and Reagents , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lipopolysaccharides/pharmacology , Protein Binding , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Transport , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/metabolism , Structure-Activity Relationship , Substrate Specificity , p38 Mitogen-Activated Protein Kinases/blood , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Alterations in central serotonin (5-hydroxytryptamine, 5-HT) neurotransmission and peripheral immune activation have been linked to multiple neuropsychiatric disorders, including depression, schizophrenia and autism. The antidepressant-sensitive 5-HT transporter (SERT, SLC6A4), a critical determinant of synaptic 5-HT inactivation, can be regulated by pro-inflammatory cytokine signaling. Systemic innate immune system activation via intraperitoneal lipopolysaccharide (LPS) injection rapidly elevates brain SERT activity and 5-HT clearance. Moreover, the pro-inflammatory cytokine interleukin (IL)-1ß rapidly stimulates SERT activity in raphe nerve terminal preparations ex vivo, effects that are attenuated by pharmacological p38 MAPK inhibition. To establish a role of serotonergic p38α MAPK signaling in LPS/IL-1ß-induced SERT regulation and attendant behavioral responses, we pursued studies in mice that afford conditional elimination of p38α MAPK in 5-HT neurons (p38α(5HT-)). We found p38α(5HT-) and control (p38α(5HT+)) littermates to be indistinguishable in viability and growth and to express equivalent levels of SERT protein and synaptosomal 5-HT transport activity. Consistent with pharmacological studies, however, IL-1ß fails to increase SERT activity in midbrain synaptosomes prepared from p38α(5HT-) animals. Moreover, although LPS elevated plasma corticosterone and central/peripheral pro-inflammatory cytokines in p38α(5HT-) animals, elevations in midbrain SERT activity were absent nor were changes in depressive and anxiety-like behaviors observed. Our studies support an obligate role of p38α MAPK signaling in 5-HT neurons for the translation of immune activation to SERT regulation and 5-HT-modulated behaviors.
Subject(s)
Immune System/physiology , Mesencephalon/physiology , Serotonin/metabolism , p38 Mitogen-Activated Protein Kinases/immunology , Animals , Behavior, Animal/physiology , Female , Lipopolysaccharides/administration & dosage , Male , Mesencephalon/immunology , Mesencephalon/metabolism , Mice , Mice, Inbred C57BL , Serotonin/blood , Serotonin/immunology , Synaptic Transmission/immunology , Synaptic Transmission/physiology , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
The relationship of inflammation and the expression of full-length receptor for advanced glycation end products (flRAGE) on monocytes, plasma levels of RAGE ligand high mobility group box protein 1 (HMGB1), soluble RAGE (sRAGE) and endogenous secretory RAGE (esRAGE) was assessed to elucidate the effect of HMGB1/DNA/RAGE-mediated innate inflammatory responses in patients with lupus nephritis. Cell surface expression of flRAGE was elevated on the monocytes of lupus patients, correlated with plasma HMGB1 levels. Plasma sRAGE level negatively correlated with systemic lupus erythematosus (SLE) disease activity index. Plasma esRAGE level was significantly lower in SLE patients with flare while esRAGE/sRAGE ratio negatively correlated with complement C3 level. HMGB1 alone could moderately induce ex vivo IL-6 production from monocytes, resulting in activation of intracellular p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase and nuclear factor (NF)-κB. Moreover, toll-like receptor-9 ligand together with HMGB1 exhibited a synergistic effect on IL-6 and IL-12p70 secretions and the phosphorylation of p38 MAPK and NF-κB. Therefore, over-expression of flRAGE in lupus may lead to the amplification of RAGE ligands-mediated inflammatory responses through the activation of p38 MAPK and NF-κB. Plasma sRAGE may serve as a potential biomarker for disease activity and a future therapeutic target in SLE.
Subject(s)
Lupus Nephritis/blood , Receptor for Advanced Glycation End Products/blood , Adult , Biomarkers/blood , Case-Control Studies , Cross-Sectional Studies , Cytokines/blood , Female , Flow Cytometry/methods , Glomerulonephritis/blood , Glomerulonephritis/pathology , Glycation End Products, Advanced/blood , HMGB1 Protein/blood , Humans , Inflammation/blood , Interleukin-6/blood , Lupus Erythematosus, Systemic/blood , Middle Aged , Monocytes/metabolism , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
BACKGROUND: Hyperketonemia is a pathological condition observed in patients with type 1 diabetes and ketosis-prone diabetes (KPD), which results in increased blood levels of acetoacetate (AA) and ß-hydroxybutyrate (BHB). Frequent episodes of hyperketonemia are associated with a higher incidence of vascular disease. We examined the hypothesis that hyperketonemia activates the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways that regulate intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured with AA (0-8 mM) or BHB (0-10 mM) for 0-24 hr. Western blotting was used to determine NF-κB activation in whole-cell lysates. ICAM-1 expression was measured using flow cytometry. RESULTS: RESULTS show a 2.4-fold increase in NF-κB activation in cells treated with 8 mM AA compared to the control. BHB had little or no effect on NF-κB activation. Pretreatment with a reactive oxygen species (ROS) inhibitor [N-acetyl-l-cysteine (NAC)] reduced NF-κB to near-control levels. The expression of AA-induced ICAM-1 was significantly reduced when cells were pretreated with either NAC or p38 MAPK inhibitor. CONCLUSIONS: These results suggest that NF-κB and p38 MAPK mediate upregulation of ICAM-1 expression in endothelial cells exposed to elevated levels of AA, which may contribute to the development of vascular disease in diabetes.