Penehyclidine hydrochloride attenuates LPS-induced iNOS production by inhibiting p38 MAPK activation in endothelial cells

The aim of this study was to investigate the inhibitory effect of penehyclidine hydrochloride (PHC) on lipopolysaccharide (LPS)-induced nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production in human endothelial cell. Cultured endothelial cells were pretreated with PHC, followed by LPS treatment. NO activity were determined. iNOS expression and p38 mitogen-activated protein kinase (p38 MAPK) protein expression were measured by Western blot analysis. LPS treatment significantly induced p38 MAPK activation, iNOS expression, and NO production, which could be attenuated by 2 μg/ml PHC pretreatment. Furthermore, our study showed LPS-induced NO production and iNOS expression were suppressed by p38 MAPK inhibitor SB203580 pretreatment. We concluded that PHC attenuates NO production and iNOS expression by suppressing the activation of p38 MAPK pathway, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced endothelial cell injury.     

Genistein enhances TRAIL-induced apoptosis through inhibition of p38 MAPK signaling in human hepatocellular carcinoma Hep3B cells

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some carcinoma cancer cells. However, it was found that treatment with TRAIL in combination with nontoxic concentrations of genistein sensitized TRAIL-resistant human hepatocellular carcinoma Hep3B cells to TRAIL-mediated apoptosis. Combined treatment with genistein and TRAIL-induced chromatin condensation and sub-G1 phase DNA content. These indicators of apoptosis were correlated with the induction of caspase activity that resulted in the cleavage of poly(ADP-ribose) polymerase (PARP). Both cell viability and the cleavage of PARP induced by combined treatment were significantly inhibited by caspase-3, -8 and -9 inhibitors, which demonstrates the important roles of caspases in the observed cytotoxic effects. Genistein treatment also triggered the inhibition of p38-β mitogen-activated protein kinase (MAPK) activation. Pretreatment with SB203580 resulted in significantly increased sub-G1 population and loss of mitochondrial membrane potential (MMP) in TRAIL-induced apoptosis. By contrast, overexpression of p38 MAPK protected apoptosis by co-treatment with genistein and TRAIL, suggesting that the p38 MAPK act as key regulators of apoptosis in response to treatment with a combination of genistein and TRAIL in human hepatocellular carcinoma Hep3B cells.     

Sodium butyrate inhibits angiogenesis of human intestinal microvascular endothelial cells through COX-2 inhibition

We examined the effect of sodium butyrate on in vitro angiogenesis and cyclooxygenase (COX) expression using primary cultures of human intestinal microvascular endothelial cells (HIMEC). Butyrate inhibited VEGF-induced cellular proliferation, transmigration and tube formation of HIMEC. Butyrate also inhibited COX-2 expression as well as prostaglandin (PG)E2 and PGI2 production, and administration of PGI2 analog partially reversed the effect of butyrate on HIMEC angiogenesis. These results indicate that sodium butyrate inhibits HIMEC angiogenesis through down-regulation of COX-2 expression and PG production, and suggest that anti-angiogenic mechanisms may also be involved in the inhibitory effect of sodium butyrate on tumor growth.     

Heat stress prevents lipopolysaccharide-induced apoptosis in pulmonary microvascular endothelial cells by blocking calpain/p38 MAPK signalling

Pulmonary microvascular endothelial cells (PMECs) injury including apoptosis plays an important role in the pathogenesis of acute lung injury during sepsis. Our recent study has demonstrated that calpain activation contributes to apoptosis in PMECs under septic conditions. This study investigated how calpain activation mediated apoptosis and whether heat stress regulated calpain activation in lipopolysaccharides (LPS)-stimulated PMECs. In cultured mouse primary PMECs, incubation with LPS (1 μg/ml, 24 h) increased active caspase-3 fragments and DNA fragmentation, indicative of apoptosis. These effects of LPS were abrogated by pre-treatment with heat stress (43 °C for 2 h). LPS also induced calpain activation and increased phosphorylation of p38 MAPK. Inhibition of calpain and p38 MAPK prevented apoptosis induced by LPS. Furthermore, inhibition of calpain blocked p38 MAPK phosphorylation in LPS-stimulated PMECs. Notably, heat stress decreased the protein levels of calpain-1/2 and calpain activities, and blocked p38 MAPK phosphorylation in response to LPS. Additionally, forced up-regulation of calpain-1 or calpain-2 sufficiently induced p38 MAPK phosphorylation and apoptosis in PMECs, both of which were inhibited by heat stress. In conclusion, heat stress prevents LPS-induced apoptosis in PMECs. This effect of heat stress is associated with down-regulation of calpain expression and activation, and subsequent blockage of p38 MAPK activation in response to LPS. Thus, blocking calpain/p38 MAPK pathway may be a novel mechanism underlying heat stress-mediated inhibition of apoptosis in LPS-stimulated endothelial cells.     

Involvement of p38 MAPK pathway in GLP-1-induced inhibition of apoptosis in human umbilical vein endothelial cells

The aim of the present study was to investigate the effect of glucagon-like peptide-1 (GLP-1) on palmitate-induced apoptosis of human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. HUVECs were cultured in vitro, and then treated by palmitate to induce apoptosis. Meanwhile, GLP-1 was added to explore its effect. After 24 h of the treatments, Caspase-3 activity and DNA fragmentation were measured using ELISA kits. Phospho-p38 mitogen-activated protein kinase (p-p38 MAPK) expression was detected by Western blot. The results showed that incubating HUVECs with 0.125 mmol/L GLP-1 increased Caspase-3 activity and DNA fragmentation. GLP-1 significantly inhibited palmitate-induced increases of Caspase-3 activity and DNA fragmentation in a concentration-dependent manner. Moreover, GLP-1 inhibited the up-regulation of p-p38 MAPK expression induced by palmitate in HUVECs. These results suggest GLP-1 protects HUVECs against lipo-apoptosis, and this effect may be mediated through inhibiting p38 MAPK pathway.     

Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells

Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22^phox expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-β no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22^phox. The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.     

Inhibition by eicosapentaenoic acid of IL-1beta-induced PGHS-2 expression in human microvascular endothelial cells: involvement of lipoxygenase-derived metabolites and p38 MAPK pathway

Prostaglandin H synthase 2 (PGHS-2), a highly inducible isoenzyme, is responsible for overproduction of the prostaglandins (PGs) in inflammatory sites.We established that among fish oil polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), greatly decreased interleukin-1beta (IL-1beta)-induced PGHS-2 expression in human pulmonary microvascular endothelial cells (HPMECs). Lipoxygenase products 12 (S)-hydroperoxyeicosapentaenoic acid ((S)-HpEPE), 15 (S)-HpEPE and leukotriene (LT) D5 reproduced similar inhibitory effect, suggesting that they may be the intermediate metabolites responsible for PGHS-2 down-regulation by EPA. Accordingly, the EPA effect is prevented by nordihydroguaiaretic acid (NDGA) and by REV 5901, nonspecific and specific 5-lipoxygenase inhibitors, respectively. Besides, inhibition of cyclooxygenase activity by ibuprofen, indomethacin or aspirin was not able to prevent this effect. Moreover, cyclooxygenase metabolites of EPA (PGs D3, E3 and I3) markedly potentiate IL-1beta-induced PGHS-2 expression, probably by increasing intracellular cAMP levels. Peroxisome proliferator-activated receptors (PPARs) are known to be activated by fatty acids (FAs) such as EPA. We found here that HPMECs express only weak amounts of PPARalpha and PPARgamma whose activation by synthetic agonists, Wy-14,643 and ciglitazone, does not cause any inhibition of IL-1beta-induced PGHS-2 expression. This finding ruled out the involvement of PPARs in the EPA inhibitory effect. In addition, we established that EPA, which failed to inhibit nuclear factor-kappaB (NF-kappaB) activation, suppressed p38 mitogen-activated protein kinase (MAPK) phosphorylation in stimulated HPMECs.Our data demonstrate that EPA, unlike DHA, down-regulates PGHS-2 expression in HPMECs probably through its 5-lipoxygenase-dependent metabolites and advocates a beneficial role for this FA in limiting inflammatory response.     

Probiotic-derived polyphosphate enhances the epithelial barrier function and maintains intestinal homeostasis through integrin-p38 MAPK pathway

Probiotics exhibit beneficial effects on human health, particularly in the maintenance of intestinal homeostasis in a complex manner notwithstanding the diversity of an intestinal flora between individuals. Thus, it is highly probable that some common molecules secreted by probiotic and/or commensal bacteria contribute to the maintenance of intestinal homeostasis and protect the intestinal epithelium from injurious stimuli. To address this question, we aimed to isolate the cytoprotective compound from a lactobacillus strain, Lactobacillus brevis SBC8803 which possess the ability to induce cytoprotective heat shock proteins in mouse small intestine. L. brevis was incubated in MRS broth and the supernatant was passed through with a 0.2-μm filter. Caco2/bbe cells were treated with the culture supernatant, and HSP27 expression was evaluated by Western blotting. HSP27-inducible components were separated by ammonium sulfate precipitation, DEAE anion exchange chromatography, gel filtration, and HPLC. Finally, we identified that the HSP27-inducible fraction was polyphosphate (poly P), a simple repeated structure of phosphates, which is a common product of lactobacilli and other bacteria associated with intestinal microflora without any definitive physiological functions. Then, poly P was synthesized by poly P-synthesizing enzyme polyphosphate kinase. The synthesized poly P significantly induced HSP27 from Caco2/BBE cells. In addition, Poly P suppressed the oxidant-induced intestinal permeability in the mouse small intestine and pharmacological inhibitors of p38 MAPK and integrins counteract its protective effect. Daily intrarectal administration of poly P (10 μg) improved the inflammation grade and survival rate in 4% sodium dextran sulfate-administered mice. This study, for the first time, demonstrated that poly P is the molecule responsible for maintaining intestinal barrier actions which are mediated through the intestinal integrin β1-p38 MAPK.     

Sphingosine kinase inhibitor suppresses IL-18-induced interferon-gamma production through inhibition of p38 MAPK activation in human NK cells

Natural killer (NK) cells play an important role in the innate immune response. Interleukin-18 (IL-18) is a well-known interferon-gamma (IFN-γ inducing factor, which stimulates immune response in NK and T cells. Sphingosine kinase (SPHK) catalyzes the formation of sphingosine 1-phosphate (S1P), which acts as a second messenger to function as an anti-apoptotic factor and proliferation stimulator of immune cells. In this study, to elucidate whether SPHK is involved in IL-18-induced IFN-γ production, we measured IL-18-induced IFN-γ production after pre-treatment with SPHK inhibitor (SKI) in NK-92MI cells. We found that IL-18-induced IFN-γ expression was blocked by SKI pre-treatment in both mRNA and protein levels. In addition, the increased IFN-γ production by stimulation with IL-18 is mediated through both SPHK and p38 MAPK. To determine the upstream signals of SKI and p38 MAPK in IL-18-induced IFN-γ production, phosphorylation levels of p38 MAPK was measured after SKI pre-treatment. As a result, inhibition of SPHK by SKI blocked phosphorylation of p38 MAPK, showing that SPHK activation by IL-18 is an upstream signal of p38 MAPK activation. Inhibition of SPHK by SKI also inhibited IL-18-induced IFN-γ production in human primary NK cells. In conclusion, SPHK activation is an essential factor for IL-18-induced IFN-γ production via p38 MAPK.     

Attenuating regulatory T cell induction by TLR agonists through inhibition of p38 MAPK signaling in dendritic cells enhances their efficacy as vaccine adjuvants and cancer immunotherapeutics

TLR ligands are potent adjuvants and promote Th1 responses to coadministered Ags by inducing innate IL-12 production. We found that TLR ligands also promote the induction of IL-10-secreting regulatory T (Treg) cells through p38 MAPK-induced IL-10 production by dendritic cells (DC). Inhibition of p38 suppressed TLR-induced IL-10 and PGE(2) and enhanced IL-12 production in DC. Incubation of Ag-pulsed CpG-stimulated DC with a p38 inhibitor suppressed their ability to generate Treg cells, while enhancing induction of Th1 cells. In addition, inhibition of p38 enhanced the antitumor therapeutic efficacy of DC pulsed with Ag and CpG and this was associated with an enhanced frequency of IFN-gamma-secreting T cells and a reduction of Foxp3(+) Treg cells infiltrating the tumors. Furthermore, addition of a p38 inhibitor to a pertussis vaccine formulated with CpG enhanced its protective efficacy in a murine respiratory challenge model. These data demonstrate that the adjuvant activity of TLR agonists is compromised by coinduction of Treg cells, but this can be overcome by inhibiting p38 signaling in DC. Our findings suggest that p38 is an important therapeutic target and provides a mechanism to enhance the efficacy of TLR agonists as vaccine adjuvants and cancer immunotherapeutics.     

p38 MAPK mediates gamma-irradiation-induced endothelial cell apoptosis, and vascular endothelial growth factor protects endothelial cells through the phosphoinositide 3-kinase-Akt-Bcl-2 pathway

Therapeutic radiation is widely used in cancer treatments. The success of radiation therapy depends not only on the radiosensitivity of tumor cells but also on the radiosensitivity of endothelial cells lining the tumor vasculature. Vascular endothelial growth factor (VEGF) plays a critical role in protecting endothelial cells against a number of antitumor agents including ionizing radiation. Strategies designed to overcome the survival advantage afforded to endothelial cells by VEGF might aid in enhancing the efficacy of radiation therapy. In this report we examined the signaling cascade(s) involved in VEGF-mediated protection of endothelial cells against gamma-irradiation. gamma-Irradiation-induced apoptosis of human dermal microvascular endothelial cells (HDMECs) was predominantly mediated through the p38 MAPK pathway as an inhibitor of p38 MAPK (PD169316), and dominant negative mutants of p38 MAPK could significantly enhance HDMEC survival against gamma-irradiation. Inhibition of the PI3K and MAPK pathways markedly up-regulated gamma-irradiation-mediated p38 MAPK activation resulting in enhanced HDMEC apoptosis. In contrast, VEGF-treated HDMECs were protected from gamma-irradiation-induced apoptosis predominantly through the PI3K/Akt pathway. Bcl-2 expression was markedly elevated in VEGF-treated HDMECs, and it was significantly inhibited by the PI3K inhibitor LY294002. HDMECs exposed to irradiation showed a significant decrease in Bcl-2 expression. In contrast, VEGF-stimulated HDMECs, when irradiated, maintained higher levels of Bcl-2 expression. Taken together our results suggest that gamma-irradiation induces endothelial cell apoptosis predominantly via the activation of p38 MAPK, and VEGF protects endothelial cells against gamma-irradiation predominantly via the PI3K-Akt-Bcl-2 signaling pathway.     

Activation of PAR4 induces a distinct actin fiber formation via p38 MAPK in human lung endothelial cells.

Protease-activated receptors (PARs) are multifunctional G protein-coupled receptors. Among the four existing PARs, PAR4 is preferentially expressed in the human lung tissue. However, the function of PAR4 has not been defined in the lung endothelial cells. Because PAR1-mediated cellular effects are deeply related to the morphological changes, we focused on the actin fiber and p38 mitogen-activated protein kinase (MAPK) signaling involved in actin polymerization to elucidate the role of PAR4. RT-PCR and Western blot analyses identified PAR4 expression in human pulmonary artery endothelial cells and in human microvascular endothelial cells from lung. We then examined the changes in actin fibers in endothelial cells treated with PAR4-activating peptide. PAR1-activating peptide was used for comparison. Activation of PAR4 and PAR1 by their corresponding peptides induced actin fiber formation; however, the actin filaments were broadly bundled in PAR4 as compared with the ringlike actin filaments in PAR1 activation. Correspondingly, the magnitude of p38 MAPK phosphorylation was different between cells treated with PAR4 and PAR1, with PAR4-activating peptide showing a significantly higher sensitivity to p38 MAPK inhibitor, SB203580. Taken together, these results demonstrate that activation of PAR4 results in the formation of actin fiber distinct from that by PAR1 activation, suggesting PAR4 may play specific roles in the lung endothelial cells.     

The antiapoptotic effect of heme oxygenase-1 in endothelial cells involves the degradation of p38 alpha MAPK isoform

Heme oxygenase-1 (HO-1) protects endothelial cells (EC) from undergoing apoptosis. This effect is mimicked by CO, generated via the catabolism of heme by HO-1. The antiapoptotic effect of CO in EC was abrogated when activation of the p38alpha and p38beta MAPKs was inhibited by the pyridinyl imidazole SB202190. Using small interfering RNA, p38beta was found to be cytoprotective in EC, whereas p38alpha was not. When overexpressed in EC, HO-1 targeted specifically the p38alpha but not the p38beta MAPK isoform for degradation by the 26S proteasome, an effect reversed by the 26S proteasome inhibitors MG-132 or lactacystin. Inhibition of p38alpha expression was also observed when HO-1 was induced physiologically by iron protoporphyrin IX (hemin). Inhibition of p38alpha no longer occurred when HO activity was inhibited by tin protoporphyrin IX, suggesting that p38alpha degradation was mediated by an end product of heme catabolism. Exogenous CO inhibited p38alpha expression in EC, suggesting that CO is the end product that mediates this effect. The antiapoptotic effect of HO-1 was impaired when p38alpha expression was restored ectopically or when its degradation by the 26S proteasome was inhibited by MG-132. Furthermore, the antiapoptotic effect of HO-1 was lost when p38beta expression was targeted by a specific p38beta small interfering RNA. In conclusion, the antiapoptotic effect of HO-1 in EC is dependent on the degradation of p38alpha by the 26S proteasome and on the expression of p38beta.     

Flavonoids inhibit high glucose-induced up-regulation of ICAM-1 via the p38 MAPK pathway in human vein endothelial cells

Recently, several flavonoids have been shown to have cardioprotective, cancer preventive, or anti-inflammatory properties. However, the specific mechanisms underlying their protective effects remain unclear. We aimed to investigate the different effects of three representative flavonoids—hesperidin, naringin, and resveratrol—on intracellular adhesion molecule-1 (ICAM-1) induction in human umbilical vein endothelial cells (HUVECs) by using high-glucose (HG) concentrations and the possible underlying molecular mechanisms. In HG-induced HUVEC cultures, the effects of three different flavonoids on ICAM-1 production and p38 phosphorylation were examined in the presence or absence of inhibitors targeting the mitogen-activated protein kinase (MAPK) signal transduction pathway. HG stimulation of HUVECs increased the levels of the adhesion molecules ICAM-1 and endothelial selectin (E-selectin). Pretreatment with all the three flavonoids drastically inhibited ICAM-1 expression in a time-dependent manner, but did not alter VCAM-1 and E-selectin expressions. Moreover, we investigated the effects of flavonoids on the MAPK signal transduction pathway, because MAPK families are associated with vascular inflammation under stress. These flavonoids did not block HG-induced phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but completely inhibited the HG-induced phosphorylation of p38 MAPK. SB202190, an inhibitor of p38 MAPK, also inhibited the HG-induced enrichment of ICAM-1. This study demonstrated that hesperidin, naringin, and resveratrol reduced the HG-induced ICAM-1 expression via the p38 MAPK signaling pathway, contributing to the inhibition of monocyte adhesion to endothelial cells.     

Phosphatidylinositol 3-kinase controls human intestinal epithelial cell differentiation by promoting adherens junction assembly and p38 MAPK activation.

The signaling pathways mediating human intestinal epithelial cell differentiation remain largely undefined. Phosphatidylinositol 3-kinase (PI3K) is an important modulator of extracellular signals, including those elicited by E-cadherin-mediated cell-cell adhesion, which plays an important role in maintenance of the structural and functional integrity of epithelia. In this study, we analyzed the involvement of PI3K in the differentiation of human intestinal epithelial cells. We showed that inhibition of PI3K signaling in Caco-2/15 cells repressed sucrase-isomaltase and villin protein expression. Morphological differentiation of enterocyte-like features in Caco-2/15 cells such as epithelial cell polarity and brush-border formation were strongly attenuated by PI3K inhibition. Immunofluorescence and immunoprecipitation experiments revealed that PI3K was recruited to and activated by E-cadherin-mediated cell-cell contacts in confluent Caco-2/15 cells, and this activation appears to be essential for the integrity of adherens junctions and association with the cytoskeleton. We provide evidence that the assembly of calcium-dependent adherens junctions led to a rapid and remarkable increase in the state of activation of Akt and p38 MAPK pathways and that this increase was blocked in the presence of anti-E-cadherin antibodies and PI3K inhibitor. Therefore, our results indicate that PI3K promotes assembly of adherens junctions, which, in turn, control p38 MAPK activation and enterocyte differentiation.     

Inhibition of p38 MAPK reduces tumor conditioned medium-induced angiogenesis in co-cultured human umbilical vein endothelial cells and fibroblasts

Tumor conditioned medium (CM) has been widely used to stimulate endothelial cells to form capillary-like structures in in vitro angiogenesis models. We report herein the effect of HT1080 and A549 CM after they were mixed with microvascular endothelial cells medium-2 (EGM-2) on angiogenesis in human umbilical vein endothelial cells (HUVECs). Both HT1080 and A549 CM decreased HUVEC proliferation, to different extents. While A549 CM significantly increased capillary-like structure formation in a co-culture system, no effect of HT1080 was apparent. Inhibition of p38 mitogen-activated protein kinase (MAPK) blocked both basal and A549 CM induced capillary-like structure formation, but inhibition of extracellular signal-regulated kinases (ERK) and that of c-Jun N-terminal protein kinases (JNK) MAPK had no such effect. Activation of ERK MAPK was inhibited by both CMs, whereas p38 MAPK was inactivated by HT1080 and activated by A549 CM and a control. Neither CM had an effect on JNK MAPK. The results suggest that p38 MAPK played a critical role in capillary-like structure formation in the co-culture, partly via promotion of apoptosis in HUVECs.