Distinct Mechanisms of Human Retinal Endothelial Barrier Modulation In Vitro by Mediators of Diabetes and Uveitis

Ocular diseases such as diabetic retinopathy (DR) and uveitis are associated with injury to the blood–retinal barrier (BRB). Whereas high glucose (HG) and advanced glycation end products (AGE) contribute to DR, bacterial infections causing uveitis are triggered by endotoxins such as lipopolysaccharide (LPS). It is unclear how HG, AGE, and LPS affect human retinal endothelial cell (HREC) junctions. Moreover, tumor necrosis factor-α (TNFα) is elevated in both DR and ocular infections. In the current study, we determined the direct effects of HG, AGE, TNFα, and LPS on the expression and intracellular distribution of claudin-5, VE-cadherin, and β-catenin in HRECs and how these mediators affect Akt and P38 MAP kinase that have been implicated in ocular pathologies. In our results, whereas HG, AGE, and TNFα activated both Akt and P38 MAPK, LPS treatment suppressed Akt but increased P38 MAPK phosphorylation. Furthermore, while treatment with AGE and HG increased cell-junction protein expression in HRECs, LPS elicited a paradoxical effect. By contrast, when HG treatment increased HREC-barrier resistance, AGE and LPS stimulation compromised it, and TNFα had no effect. Together, our results demonstrated the differential effects of the mediators of diabetes and infection on HREC-barrier modulation leading to BRB injury.

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Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors

Blood ◽

10.1182/blood-2006-05-023093 ◽

2006 ◽

Vol 108 (13) ◽

pp. 4170-4177 ◽

Cited By ~ 86

Author(s):

Tony A. Navas ◽

Mani Mohindru ◽

Myka Estes ◽

Jing Ying Ma ◽

Lubomir Sokol ◽

...

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

P38 Mapk ◽

Myelodysplastic Syndromes ◽

Clinical Investigation ◽

P38 Map Kinase ◽

Hematopoietic Progenitors ◽

Molecule Inhibitor ◽

Pharmacologic Inhibition

Abstract The myelodysplastic syndromes (MDSs) are collections of heterogeneous hematologic diseases characterized by refractory cytopenias as a result of ineffective hematopoiesis. Development of effective treatments has been impeded by limited insights into any unifying pathogenic pathways. We provide evidence that the p38 MAP kinase is constitutively activated or phosphorylated in MDS bone marrows. Such activation is uniformly observed in varied morphologic subtypes of low-risk MDS and correlates with enhanced apoptosis observed in MDS hematopoietic progenitors. Most importantly, pharmacologic inhibition of p38α by a novel small molecule inhibitor, SCIO-469, decreases apoptosis in MDS CD34+ progenitors and leads to dose-dependant increases in erythroid and myeloid colony formation. Down-regulation of the dominant p38α isoform by siRNA also leads to enhancement of hematopoiesis in MDS bone marrow progenitors in vitro. These data implicate p38 MAPK in the pathobiology of ineffective hematopoiesis in lowrisk MDS and provide a strong rationale for clinical investigation of SCIO-469 in MDS.

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Activation of p38 MAP Kinase Pathway by Erythropoietin and Interleukin-3

Blood ◽

10.1182/blood.v90.3.929.929_929_934 ◽

1997 ◽

Vol 90 (3) ◽

pp. 929-934 ◽

Cited By ~ 6

Author(s):

Yuka Nagata ◽

Tetsuo Moriguchi ◽

Eisuke Nishida ◽

Kazuo Todokoro

Keyword(s):

Map Kinase ◽

Osmotic Shock ◽

Interleukin 1 ◽

P38 Map Kinase ◽

Kinase Assay ◽

Upstream Kinase ◽

In Vitro Kinase Assay ◽

Map Kinase Pathway ◽

Factor Α

Activation of p38 MAP kinase (p38) as well as JNK/SAPK has been described as being induced by a variety of environmental stresses such as osmotic shock, ultraviolet radiation, and heat shock, or the proinflammatory cytokines tumor necrosis factor-α and interleukin-1 (IL-3). We found that the hematopoietic cytokines erythropoietin (Epo) and IL-3, which regulate growth and differentiation of erythroids and hematopoietic progenitors, respectively, also activate a p38 cascade. Immunoblot analyses and in vitro kinase assay clearly showed that Epo and IL-3 rapidly and transiently phosphorylated and activated p38 in Epo– or IL-3–dependent mouse hematopoietic progenitor cells. p38 can generally be activated by the upstream kinase MKK3 or MKK6. However, in vitro kinase assays in the immunoprecipitates with anti-MKK6 antibody and anti-phosphorylated MKK3/MKK6 antibody showed that activation of neither MKK3 nor MKK6 was detected after Epo or IL-3 stimulation, while osmotic shock clearly induced activation of both MKK3/MKK6 and p38. Together with previous observations, these results suggest that both p38 and JNK cascades play an important role not only in stress and proinflammatory cytokine responses but also in hematopoietic cytokine actions.

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Fangjifuling Ameliorates Lipopolysaccharide-Induced Renal Injury via Inhibition of Inflammatory and Apoptotic Response in Mice

Cellular Physiology and Biochemistry ◽

10.1159/000493816 ◽

2018 ◽

Vol 49 (6) ◽

pp. 2124-2137 ◽

Cited By ~ 10

Author(s):

Zhonghao Su ◽

Ping Yu ◽

Lanyue Sheng ◽

Jin Ye ◽

Zhenxia Qin

Keyword(s):

Renal Injury ◽

Target Genes ◽

Bioinformatics Analysis ◽

Cell Junction ◽

Acute Glomerulonephritis ◽

Analysis Tool ◽

Renoprotective Effect ◽

Nephritic Syndrome ◽

Junction Protein

Background/Aims: Acute kidney injury (AKI) is a frequent and serious complication of sepsis; however, there is no effective treatment for it. FangJiFuling (FF) decoction is widely used to treat acute glomerulonephritis and nephritic syndrome in the clinical setting. Methods: On the basis of its anti-inflammatory properties, the renoprotective effect of FF on a mouse model of lipopolysaccharide (LPS)-induced AKI was investigated. Major compounds were identified in FF with high-performance liquid chromatography. A bioinformatics analysis tool was used to predict target genes. Quantitative real-time PCR and western blot analyses were performed to validate the targets. Furthermore, the expression of a target gene was silenced by small interfering RNA-mediated knockdown in vitro. Results: Bioinformatics analysis indicated that inflammation, apoptosis, and cell junction were closely related to the renoprotective effects of FF. Validation was confirmed by an in vivo test. A reduction of inflammatory cell infiltration and inflammatory cytokine mRNA expression (iNOS, NF-κB, MCP-1, and TNF-α) following the administration of FF (50 mg/kg) was observed in LPS-treated renal tissue. In addition, FF treatment suppressed mitochondrial-mediated apoptosis by regulating the Bax/Bcl-2 ratio in LPS-induced renal injury. Silencing Cx43, a cell-to-cell junction protein, was found to enhance the protective effect of FF against LPS-induced renal injury. Conclusion: Our study suggests that FF exhibits a renoprotective effect against LPS-induced inflammatory and apoptotic responses. In addition, Cx43 might be involved in these processes. These findings indicate the potential role of FF as a natural renoprotective product.

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p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

Blood ◽

10.1182/blood-2009-03-211706 ◽

2010 ◽

Vol 115 (9) ◽

pp. 1835-1842 ◽

Cited By ~ 67

Author(s):

Matthias Canault ◽

Daniel Duerschmied ◽

Alexander Brill ◽

Lucia Stefanini ◽

Daphne Schatzberg ◽

...

Keyword(s):

Protein Kinase ◽

P38 Mapk ◽

P38 Map Kinase ◽

Mitogen Activated Protein Kinase ◽

Platelet Recovery ◽

Mitochondrial Injury ◽

Platelet Storage ◽

Receptor Shedding

AbstractPlatelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-α and GPV. We recently demonstrated that tumor necrosis factor-α converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

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Abstract 11078: Granzyme B Deficiency Protects Against Angiotensin II-induced Cardiac Fibrosis via a Perforin-independent Mechanism

Circulation ◽

10.1161/circ.132.suppl_3.11078 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Yue Shen ◽

Fang Cheng ◽

Mehul Sharma ◽

Yulia Merkulova ◽

Sheetal A Raithatha ◽

...

Keyword(s):

Angiotensin Ii ◽

Cardiac Fibrosis ◽

Granzyme B ◽

Cell Junction ◽

Pcr Analysis ◽

Ang Ii ◽

Junction Protein ◽

Independent Process

Introduction: Granzyme B (GzmB) is a serine protease involved in immune cell-mediated apoptosis that is enabled through a mechanism involving the pore-forming protein, perforin that facilitates internalization. However, recent evidence suggests that GzmB contributes to matrix remodeling and fibrosis through an extracellular, perforin-independent process. Hypothesis: GzmB contributes to cardiac fibrosis through a perforin-independent pathway involving extracellular proteolysis. Methods: Using a murine model of Angiotensin II (Ang II)-induced cardiac fibrosis, wild-type, GzmB deficient and Perforin deficient mice were treated with Ang II for 4 weeks, and were examined for the presence of cardiac fibrosis. Echocardiography was performed in these mice to examine the cardiac function. The level of Inflammation and inflammatory cells infiltration were examined by immunohistochemistry and RT-PCR analysis. The in vitro endothelial barrier function was measured by electric cell-substrate impedance sensing. Results: GzmB was highly up-regulated in both murine and human cardiac fibrosis. Genetic deficiency of GzmB markedly reduced Ang II-induced cardiac dysfunction, hypertrophy and fibrosis, independently of perforin. GzmB deficiency also decreases microhemorrhage, inflammation, and fibroblast accumulation in vivo. In vitro studies identified VE-cadherin as a GzmB substrate. VE-cadherin is a key endothelial cell-cell junction protein. GzmB-mediated VE-cadherin cleavage resulted in increased endothelial permeability, and increased transcellular conductance. These results were also observed in vivo. Conclusions: GzmB contributes to the onset and progression of cardiac fibrosis through a perforin-independent process involving the cleavage of VE-cadherin.

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Activation of TAO2 Kinase by Arsenic Trioxide in Leukemic Cells

Blood ◽

10.1182/blood.v112.11.5324.5324 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5324-5324

Author(s):

Jennifer L. McNeer ◽

Blazej Dolniak ◽

Barbara Kroczynska ◽

Antonella Sassano ◽

Leonidas Platanias

Keyword(s):

Arsenic Trioxide ◽

P38 Mapk ◽

Mapk Pathway ◽

Leukemic Cell ◽

Buthionine Sulfoximine ◽

P38 Map Kinase ◽

Leukemic Cells ◽

Kinase Assay ◽

P38 Mapk Pathway

Abstract Arsenic Trioxide (As2O3) has major efficacy in the treatment of acute promyelocytic leukemia (APL), but its use in other malignancies is limited by the need for high intracellular concentrations to induce apoptosis. Prior work in our laboratory has demonstrated that the p38 MAP kinase (MAPK) pathway is activated following treatment of cells with As2O3 and exhibits negative regulatory effects on As2O3-induced apoptosis and growth suppression. In the current study, we sought to identify upstream effector mechanisms by which the p38 pathway is activated by As2O3 in leukemic cells. We found that the MAPK kinase kinase TAO2 (thousand and one amino acid protein kinase 2) is phosphorylated on Ser181 after treatment of NB4, NB4.306, and U937 cells with arsenic. Such phosphorylation was rapid, occurring as early as after 5 minutes of As2O3 treatment. In addition, our data indicate that such phosphorylation occurs downstream of As2O3-induced redox reactions, as demonstrated by increased phosphorylation in cells pretreated with the oxidizing agent buthionine sulfoximine (BSO) and decreased phosphorylation following pretreatment with the reducing agent dithiothreitol (DTT). Arsenic treatment of the cells also resulted in activation of the kinase domain of TAO2, as evidenced in in vitro kinase assay studies using ATF2 as an exogenous substrate. siRNA-mediated TAO2 knockdown resulted in inhibition of As2O3-induced p38 phosphorylation, suggesting that this kinase acts as an upstream effector of the arsenic-activated p38 MAPK pathway. Moreover, in studies to determine the functional relevance of TAO2 in the induction of As2O3-dependent antileukemic responses we found that siRNA-mediated TAO2 knockdown enhanced the suppressive effects of As2O3 on KT1-derived leukemic progenitor (CFU-L) growth in clonogenic assays in methylcellulose. Altogether, our data demonstrate that TAO2 is activated during arsenic treatment of leukemic cells lines and acts as an upstream activator of the p38 MAPK pathway. Such activation appears to occur in a negative feedback regulatory manner to compensate for the suppressive effects of As2O3 on leukemic cell growth. Importantly, these findings raise the possibility that targeting TAO2 may provide a novel approach to enhance the generation of the antileukemic properties of As2O3.

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Role of p38 MAP kinase in diperoxovanadate-induced phospholipase D activation in endothelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.281.2.l435 ◽

2001 ◽

Vol 281 (2) ◽

pp. L435-L449 ◽

Cited By ~ 23

Author(s):

Viswanathan Natarajan ◽

William M. Scribner ◽

Andrew J. Morris ◽

Shukla Roy ◽

Suryanarayana Vepa ◽

...

Keyword(s):

Endothelial Cells ◽

P38 Mapk ◽

Phospholipase D ◽

Tyrosine Phosphatase ◽

P38 Map Kinase ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Mapk Activity

We previously demonstrated that diperoxovanadate (DPV), a synthetic peroxovanadium compound and cell-permeable oxidant that acts as a protein tyrosine phosphatase inhibitor and insulinomimetic, increased phospholipase D (PLD) activation in endothelial cells (ECs). In this report, the regulation of DPV-induced PLD activation by mitogen-activated protein kinases (MAPKs) was investigated. DPV activated extracellular signal-regulated kinase, c-Jun NH2-terminal kinase (JNK), and p38 MAPK in a dose- and time-dependent fashion. Treatment of ECs with p38 MAPK inhibitors SB-203580 and SB-202190 or transient transfection with a p38 dominant negative mutant mitigated the PLD activation by DPV but not by phorbol ester. SB-202190 blocked DPV-mediated p38 MAPK activity as determined by activated transcription factor-2 phosphorylation. Immunoprecipitation of PLD from EC lysates with PLD1 and PLD2 antibodies revealed both PLD isoforms associated with p38 MAPK. Similarly, PLD1 and PLD2 were detected in p38 immunoprecipitates from control and DPV-challenged ECs. Binding assays demonstrated interaction of glutathione S-transferase-p38 fusion protein with PLD1 and PLD2. Both PLD1 and PLD2 were phosphorylated by p38 MAPK in vitro, and DPV increased phosphorylation of PLD1 and PLD2 in vivo. However, phosphorylation of PLD by p38 failed to affect PLD activity in vitro. These results provide evidence for p38 MAPK-mediated regulation of PLD in ECs.

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Lipopolysaccharide Induces Upregulation of Neutral Endopeptidase 24.11 on Human Neutrophils: Involvement of the CD14 Receptor

Clinical Science ◽

10.1042/cs0890083 ◽

1995 ◽

Vol 89 (1) ◽

pp. 83-89 ◽

Cited By ~ 3

Author(s):

Christine Fagny ◽

Arnaud Marchant ◽

Eric De Prez ◽

Michel Goldman ◽

Monique Deschodt-Lanckman

Keyword(s):

Monoclonal Antibody ◽

Whole Blood ◽

Bacterial Infections ◽

Direct Interaction ◽

Neutral Endopeptidase ◽

Human Neutrophils ◽

Membrane Expression ◽

Endopeptidase 24.11 ◽

Factor Α

1. As lipopolysaccharide is a major stimulator of neutrophil responses during Gram-negative bacterial infections, we studied its effect on the membrane expression of neutral endopeptidase 24.11/CD10 on neutrophils in a model of endotoxaemia in vitro. Lipopolysaccharide added to human whole-blood induced a marked and sustained CD10/neutral endopeptidase upregulation that was already detectable at 0.1 ng/ml and was maximal at a lipopolysaccharide concentration of 10 ng/ml. 2. We observed that neither tumour necrosis factor-α nor any newly synthesized protein was involved in the upregulation observed after 1 h incubation with 10 ng/ml lipopolysaccharide. 3. We further studied whether the lipopolysaccharide-induced CD10/neutral endopeptidase upregulation was mediated by lipopolysaccharide binding to the neutrophil CD14 receptor. Incubation of whole blood with an anti-CD14 monoclonal antibody before the addition of 0.1 ng/ml or 0.5 ng/ml lipopolysaccharide resulted in complete inhibition of CD10/neutral endopeptidase upregulation. In contrast, at a lipopolysaccharide concentration of 10 ng/ml, the anti-CD14 monoclonal antibody had an incomplete blocking effect. 4. The differential requirement for the CD14 receptor, depending on the lipopolysaccharide dose, was confirmed by the study of a patient suffering from paroxysmal nocturnal haemoglobinuria (in whom a complete defect in neutrophil CD14 expression was previously documented). 5. We finally confirmed these results using purified neutrophils, demonstrating that lipopolysaccharide-induced CD10/neutral endopeptidase upregulation depends on direct interaction with neutrophil CD14.

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Crosstalk of cardiomyocytes and fibroblasts in co-cultures

Open Biology ◽

10.1098/rsob.150038 ◽

2015 ◽

Vol 5 (6) ◽

pp. 150038 ◽

Cited By ~ 34

Author(s):

J. Rother ◽

C. Richter ◽

L. Turco ◽

F. Knoch ◽

I. Mey ◽

...

Keyword(s):

Cardiac Fibrosis ◽

Cardiac Tissue ◽

Contractile Function ◽

Beat Frequency ◽

Cell Junction ◽

Impedance Sensing ◽

Barrier Resistance ◽

Spreading Rates ◽

The Impact

Electromechanical function of cardiac muscle depends critically on the crosstalk of myocytes with non-myocytes. Upon cardiac fibrosis, fibroblasts translocate into infarcted necrotic tissue and alter their communication capabilities. In the present in vitro study, we determined a multiple parameter space relevant for fibrotic cardiac tissue development comprising the following essential processes: (i) adhesion to substrates with varying elasticity, (ii) dynamics of contractile function, and (iii) electromechanical connectivity. By combining electric cell-substrate impedance sensing (ECIS) with conventional optical microscopy, we could measure the impact of fibroblast–cardiomyocyte ratio on the aforementioned parameters in a non-invasive fashion. Adhesion to electrodes was quantified via spreading rates derived from impedance changes, period analysis allowed us to measure contraction dynamics and modulations of the barrier resistance served as a measure of connectivity. In summary, we claim that: (i) a preferred window for substrate elasticity around 7 kPa for low fibroblast content exists, which is shifted to stiffer substrates with increasing fibroblast fractions. (ii) Beat frequency decreases nonlinearly with increasing fraction of fibroblasts, while (iii) the intercellular resistance increases with a maximal functional connectivity at 75% fibroblasts. For the first time, cardiac cell–cell junction density-dependent connectivity in co-cultures of cardiomyocytes and fibroblasts was quantified using ECIS.

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Tumor Evasion of the Immune System: Inhibiting p38 MAP Kinase Signaling Restores the Function of Dendritic Cells.

Blood ◽

10.1182/blood.v106.11.2512.2512 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2512-2512

Author(s):

Qing Yi ◽

Siqing Wang ◽

Jing Yang ◽

Jianfei Wang ◽

Michele Wezeman ◽

...

Keyword(s):

Dendritic Cells ◽

P38 Mapk ◽

Neutralizing Antibodies ◽

Molecular Mechanisms ◽

P38 Map Kinase ◽

Mitogen Activated Protein Kinase ◽

Myeloma Cells ◽

And Function

Abstract Dendritic cells (DCs) from cancer patients are functionally defective, however, molecular mechanisms underlying are still poorly understood. In this study, we used the murine 5TGM1 myeloma model to examine the effect and mechanism of tumor-derived factors on the differentiation and function of DCs. Myeloma cells (5TGM1) or tumor culture conditioning medium (TCCM) were shown to inhibit differentiation and function of BM-derived DCs (BMDCs), evidenced by the downregulated expression of DC-related surface molecules, decreased IL-12 but increased IL-10 secretion, and compromised capacity of the cells to activate allospecific T cells in vitro. Similar results were obtained with other murine myeloma cells MOPC-315 and MPC-11. Moreover, TCCM-treated BMDCs were inferior to normal BMDCs at priming tumor-specific humoral and cellular immune responses in vivo (in the 5TGM1 mouse model). Neutralizing antibodies against IL-6, IL-10, and TGF-β partially abrogated the effects. TCCM treatment activated p38 mitogen-activated protein kinase (MAPK) and JNK but inhibited extracellular signal-related kinase (ERK). Inhibiting p38 MAPK by three different specific inhibitors was found to restore the phenotype, cytokine secretion, and function of TCCM-treated BMDCs. Vaccinating mice with BMDCs obtained from cultures in which both TCCM and p38 inhibitor were added was as efficacious as normal BMDCs at inducing tumor-specific antibody, type-1 (IFN-γ) T-cell, and CTL responses. Thus, our results suggest that tumor-induced p38 MAPK activation and ERK inhibition in DCs may be a new mechanism for tumor evasion, and regulating these signaling pathways in vivo or during DC differentiation may provide new strategies for generating potent DC vaccines for immunotherapy of multiple myeloma and other tumors.

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