Association of Tumor Cell Metabolic Subtype and Immune Response With the Clinical Course of Hepatocellular Carcinoma.

AIM: Tumor metabolism plays an important role in tumorigenesis and tumor progression. This study evaluated the potential association of tumor cell metabolism and immune cell tumor infiltration with the clinical course of hepatocellular carcinoma (HCC). METHODS: Gene-wise normalization and principal component analysis were performed to evaluate the metabolic system. A tumor microenvironment score system of tumor immune cell infiltration was constructed to evaluate its association with metabolic subtypes. Finally, we analyzed the impact of metabolism and immune cell infiltration on the clinical course of HCC. RESULTS: A total of 673 HCC patients were categorized into cholesterogenic (25.3%), glycolytic (14.6%), mixed (10.4%), and quiescent (49.8%) types based on glycolysis and cholesterol biosynthesis gene expression. The subgroups including the glycolytic genotyping expression (glycolytic and mixed types) showed a higher mortality rate. The glycolytic, cholesterogenic, and mixed types were positively correlated with M0 macrophage, resting mast cell, and naïve B-cell infiltration (P = .013, P = .019, and P = .006, respectively). In TCGA database, high CD8+ T cell and low M0 macrophage infiltration were associated with prolonged overall survival (OS, P = .0017 and P < .0001, respectively). Furthermore, in glycolytic and mixed types, patients with high M0 macrophage infiltration had a shorter OS (P = .03 and P = .013, respectively), and in quiescent type, patients with low naïve B-cell infiltration had a longer OS (P = .007). CONCLUSIONS: Tumor metabolism plays a prognostic role and correlates with immune cell infiltration in HCC. M0 macrophage and CD8+ T cell appear to be promising prognostic biomarker for HCC. Finally, M0 macrophages may represent a useful immunotherapeutic target in patients with HCC.

Roles of BATF/JUN/IRF4 complex in tacrolimus mediated immunosuppression on Tfh cells in acute rejection after liver transplantation.

Rejection injury is a serious complication after liver transplantation (LTx). Tacrolimus (Tac) is a key immunosuppressive agent in the prevention of liver rejection after transplantation. The basic leucine zipper ATF-like transcription factor (BATF)/JUN/interferon regulatory factor 4 (IRF4) complex serves critical functions in the immune response. This study aimed to explore the role of the BATF/JUN/IRF4 complex in rejection after LTx by treatment with Tac. Herein, DA and Lewis (LEW) rats were used to construct the LTx animal model. The recipient LEW rats were treated with Tac or saline, subcutaneously. Splenic mononuclear cells were treated with Tac at 1 and 10 nM after stimulation with interleukin-6 (IL-6), and the expression of factors associated with the nuclear factor of activated T cells (NFAT)-BATF/JUN/IRF4 and IL-21 were detected. The results demonstrated that Tac prolonged the allografts survival and attenuated inflammation injury, and decreased the percentage frequencies of T follicular helper (Tfh) cells in peripheral blood mononuclear cells and inhibited B-cell lymphoma 6 (Bcl-6) and IL-6 expression in Tfh cells. In addition, Tac inhibited the expression of the BATF/JUN/IRF4 complex, Bcl-6 and IL-21 NFATc1 and NFATc2 were inhibited by Tac, and interacted with the promoter of BATF and IRF4. In conclusion, the attenuation of rejection injury may be dependent on the NFAT-BATF/JUN/IRF4-IL-21 axis, and the BATF/JUN/IRF4 complex participates in the inhibition of IL-21-producing Tfh cells after treatment with Tac. These findings suggest that the BATF/JUN/IRF4 complex-IL-21 axis may be used as a potential target for attenuating rejection injury after LTx.

Administration of glycyrrhetinic acid reinforces therapeutic effects of mesenchymal stem cell-derived exosome against acute liver ischemia-reperfusion injury.

Recent studies have shown that mesenchymal stem cell-derived exosome could attenuate ischaemia-reperfusion (I/R) injury by suppressing inflammatory response in the liver. Glycyrrhetinic acid was also shown to be capable of repressing the TLR4 signalling pathway. However, it remains to be explored as whether the combined administration of mesenchyma stem cell (MSC)-derived exosome and glycyrrhetinic acid (GA) could increase their therapeutic effects on I/R injury. Western blot was performed to evaluate the expression of proteins associated with inflammatory response in THP-1 cells and I/R rat models treated under different conditions. Flow cytometry was carried out to analyse the proportions of different subtypes of peripheral blood cells in I/R rats. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to assess the liver injury in I/R rats. Combined treatment with MSC-derived exosome and GA effectively maintained the expression of key proteins involved in inflammatory response in LPS stimulated THP-1 cells and THP-1 cells treated under hypoxia conditions. In the established of I/R rat models, GA administration reinforced the therapeutic efficiency of MSC-derived exosomes by maintaining the proportion of different subgroups of peripheral blood cells, decreasing the concentration of ALT and AST, and restoring the expression of dysregulated proteins associated with inflammation. Our results demonstrated that treatment with exosomes derived from mesenchymal stem cells (MSCs) attenuated liver I/R injury, while the pre-treatment with GA may further promote the therapeutic effect of mesenchymal stem cell-derived exosome against acute liver ischaemia-reperfusion injury.

MicroRNA-125a-3p overexpression promotes liver regeneration through targeting proline-rich acidic protein 1.

INTRODUCTION AND OBJECTIVES: Liver regeneration plays a valuable significance for hepatectomies, and is mainly attributed to hepatocyte proliferation. MicroRNA-125a-3p was reported to be highly associated with liver regeneration process. We studied the underlying mechanism of the functional role of miR-125a-3p in liver regeneration. MATERIALS AND METHODS: The miR-125a-3p mimics and inhibitor vector were constructed and transfected into primary human liver HL-7702 cells, the transfected cell viability was detected using cell counting kit-8 (CCK-8). Cell cycle distribution was analyzed by flow cytometry. With Targetscan and OUGene prediction, the potential targets of miR-125 were verified by real-time quantitative PCR (qPCR) and luciferase reporter assays in turn. The overexpression vector of proline-rich acidic protein 1 (PRAP1) was constructed and co-transfected with miR-125a-3p mimics into HL-7702 cells, detecting the changes of proliferative capacity and cell cycle distribution. Western blot and qPCR performed to analyze gene expressions. RESULTS: Overexpressed miR-125a-3p notably increased the hepatocyte viability at 48h, and decreased the number of G1 phase cells (p<0.05). However, miR-125a-3p inhibition suppressed the development of hepatocytes. PRAP1 was the target of miR-125a-3p. After co-transfection with PRAP1 vector, hepatocyte viability was decrease and the G1 phase cell number was increased (p<0.05). More importantly, overexpressed PRAP1 notably decreased the mRNA and protein levels of cyclin D1, cyclin-dependent kinase 2 (CDK2) and cell division cycle 25A (CDC25A). CONCLUSION: The elevated miR-125a-3p positively correlated with hepatocyte viability and cell cycle progression due to the modulation of PRAP1, and miR-125a-3p may contribute to improving liver regeneration.

Over-expression of MiR-122 promotes apoptosis of hepatocellular carcinoma via targeting TLR4.

INTRODUCTION AND OBJECTIVE: MiR-122 has been regarded as a tumor suppressor. Toll-like receptor 4 (TLR4) has been found to be closely related to metastasis and immune escape of hepatocellular carcinoma (HCC). In the study, we sought to investigate the effect of miR-122 on HCC and the expression of TLR4. PATIENTS OR MATERIALS AND METHODS: Real-time PCR and Western blot were performed to detect the expressions of target factors. CCK-8 and flow cytometry analysis were employed to evaluate cell viability and apoptosis, respectively. Luciferase reporter assay was used to determine whether miR-122 could directly regulate the expression of TLR4. Enzyme-linked Immuno Sorbent Assay was adopted to detect the secretion of inflammatory cytokines. RESULTS: Both down-regulation of miR-122 and up-regulation of TLR4 were found to be correlated with low overall survival rate of HCC patients. TLR4 may be a direct target gene of miR-122. Over-expression of miR-122 induced apoptosis and inhibited cell viability of HCC by down-regulating TLR4, enhanced the expression of pro-apoptotic genes and suppressed the expression of anti-apoptotic genes. MiR-122 inhibited expressions and activities of inflammatory cytokines, including vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), cyclooxygenase-2 (Cox-2) and prostaglandin E2 (PGE2) and also reduced the expression of matrix metallopeptidase 9 (MMP-9). Furthermore, activities of phosphatidylinositide 3-kinases (PI3K), Akt and nuclear factor-kappa B (NF-κB) were suppressed by miR-122. CONCLUSIONS: Down-regulation of miR-122 facilitated the immune escape of HCC by targeting TLR4, which was related to PI3K/Akt/NF-κB signaling pathways. Our study may provide a possible strategy for the treatment of HCC.

Effect of the intermittent Pringle maneuver on liver damage after hepatectomy: a retrospective cohort study.

BACKGROUND: The Pringle maneuver (PM) interrupts the blood flow through the hepatic artery and portal vein to help control bleeding. This study analyzes the effects of the intermittent Pringle maneuver (IPM) on the surgical process and postoperative liver injury. METHODS: This study retrospectively evaluated 182 hepatocellular carcinoma patients who underwent hepatectomy. In the IPM group, hepatic blood flow was intermittently interrupted via clamping, with cycles of 10 minutes of inflow occlusion followed by 5 minutes of reperfusion that were repeated until the end of the surgery. In the non-IPM group, liver resection was performed without hepatic vascular blockage. RESULTS: For postoperative complications, the incidence rates of ascites and pleural effusion in the IPM group were significantly lower than those in the non-IPM group. The postoperative hospitalization time in the IPM group was significantly lower than that in the non-IPM group (p=0.0008). On the first day after the operation, the platelet count was significantly lower (p=0.0381) but the prothrombin time (PT) (p=0.0195) and activated partial thromboplastin time (APTT) (p=0.0071) were significantly higher in the non-IPM group than those in the IPM group. At discharge, only albumin was significantly higher in the non-IPM group than that in the IPM group (p=0.0303). Regression analysis showed that a prolonged interruption time was related to increased ALT and AST levels on the first day after surgery, but not on the seventh day or at discharge. CONCLUSION: The IPM does not cause additional liver damage during hepatectomy, and use of the IPM results in shorter hospital stays compared to surgery without using the IPM. The results of this study require further confirmation because of the retrospective design.

Tumor-Secreted Exosomal miR-222 Promotes Tumor Progression via Regulating P27 Expression and Re-Localization in Pancreatic Cancer.

BACKGROUND/AIMS: MicroRNAs (miRNAs) or exosomes have recently been shown to play vital regulatory or communication roles in cancer biology. However, the roles and mechanisms of exosomal miRNAs in pancreatic ductal adenocarcinoma (PDAC) remain unknown. We aimed to investigate the detailed roles and mechanisms of tumor-generated exosomal miRNAs in progression of PDAC. METHODS: miR-222 was identified by miRNA microarray studies in exosomes of PDAC cells, and further analyzed in plasma exosomes of PDAC patients. The regulatory mechanisms of miR-222 were explored by qRT-PCR, WB, dual-luciferase assays and immunofluorescence or confocal analysis. Other biological assays include transwell, xenograft models and so on. RESULTS: miR-222 is significantly high in tumor exosomes or highly invasive PDAC cells. miR-222 could directly regulate p27 to promote cell invasion and proliferation. miR-222 could also activate AKT by inhibiting PPP2R2A expression, thus inducing p27 phosphorylation and cytoplasmic p27 expression to promote cell survival, invasion and metastasis. Expressions of miR-222 and p27 were significantly inversely correlated, and cytoplasmic p27, instead of nuclear p27, was associated with tumor malignancy. miR-222 could be transmitted between PDAC cells via exosome communication, and the exosomal miR-222 communication is functional. Plasma exosomal miR-222 in PDAC patients was high and significantly correlated to tumor size and TNM stage, and was an independent risk factor for PDAC patient survival. CONCLUSION: Tumor-generated exosomes could promote invasion and proliferation of neighboring tumor cells via miR-222 transmission, the plasma exosomal miR-222 plays important roles and may be a useful prognostic maker in PDAC.

Combination of glycopyrronium and indacaterol inhibits carbachol-induced ERK5 signal in fibrotic processes.

BACKGROUND: Airway fibrosis is one of the pathological features of chronic obstructive pulmonary disease (COPD), and recent studies revealed that acetylcholine plays an important role in the development of airway remodeling by stimulating proliferation and collagen synthesis of lung fibroblasts. This study was designed to examine the effects of a long-acting muscarinic receptor antagonist (LAMA) glycopyrronium and a long-acting ß2 adrenergic receptor agonist (LABA) indacaterol on acetylcholine-mediated fibrotic responses in lung fibroblasts. METHODS: After carbachol (CCh) or transforming growth factor-ß1 (TGF-ß1) exposure, the response to glycopyrronium and indacaterol was determined in vitro in fibroblasts isolated from mild-to-moderate COPD lung tissue. The ability of fibroblasts to mediate the contraction of collagen gels was assessed. The expression of α-smooth muscle actin (α-SMA) and the phosphorylation of extracellular-signal-regulated kinase 5 (ERK5) were determined by immunoblot. TGF-ß1 was quantified by ELISA and acetylcholine was quantified by liquid chromatography tandem-mass spectrometry. RESULTS: CCh stimulated fibroblast-mediated collagen gel contraction and α-SMA expression and TGF-ß1 release by fibroblasts. Blockade of autocrine TGF-ß1 attenuated CCh-mediated fibrotic responses, while TGF-ß1 did not stimulate acetylcholine release. Glycopyrronium plus indacaterol significantly attenuated CCh- and TGF-ß1-mediated fibrotic responses through inhibition of ERK5 phosphorylation. Notably, the magnitudes of CCh- and TGF-ß1-stimulated gel contraction, CCh-induced TGF-ß1 release, and ERK5 phosphorylation were greater in fibroblasts isolated from COPD subjects than in those from non-smokers. CONCLUSIONS: CCh induced TGF-ß1 self-sustaining signaling loops by potentiating ERK5 signaling and promoted myofibroblast activity. This autocrine signaling mechanism may be an attractive therapeutic target to block the fibrotic response, which was modulated by the combination of glycopyrronium and indacaterol.

Proteases in agricultural dust induce lung inflammation through PAR-1 and PAR-2 activation.

Workers exposed to aerosolized dust present in concentrated animal feeding operations (CAFOs) are susceptible to inflammatory lung diseases, such as chronic obstructive pulmonary disease. Extracts of dust collected from hog CAFOs [hog dust extract (HDE)] are potent stimulators of lung inflammatory responses in several model systems. The observation that HDE contains active proteases prompted the present study, which evaluated the role of CAFO dust proteases in lung inflammatory processes and tested whether protease-activated receptors (PARs) are involved in the signaling pathway for these events. We hypothesized that the damaging proinflammatory effect of HDE is due, in part, to the proteolytic activation of PARs, and inhibiting the proteases in HDE or disrupting PAR activation would attenuate HDE-mediated inflammatory indexes in bronchial epithelial cells (BECs), in mouse lung slices in vitro, and in a murine in vivo exposure model. Human BECs and mouse lung slice cultures stimulated with 5% HDE released significantly more of each of the cytokines measured (IL-6, IL-8, TNF-α, keratinocyte-derived chemokine/CXC chemokine ligand 1, and macrophage inflammatory protein-2/CXC chemokine ligand 2) than controls, and these effects were markedly diminished by protease inhibition. Inhibition of PARs also blunted the HDE-induced cytokine release from BECs. In addition, protease depletion inhibited HDE-induced BEC intracellular PKCα and PKCε activation. C57BL/6J mice administered 12.5% HDE intranasally, either once or daily for 3 wk, exhibited increased total cellular and neutrophil influx, bronchial alveolar fluid inflammatory cytokines, lung histopathology, and inflammatory scores compared with mice receiving protease-depleted HDE. These data suggest that proteases in dust from CAFOs are important mediators of lung inflammation, and these proteases and their receptors may provide novel targets for therapeutic intervention in CAFO dust-induced airways disease.

Prostaglandin E2 switches from a stimulator to an inhibitor of cell migration after epithelial-to-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is critical for embryonic development, and this process is recapitulated in adults during wound healing, tissue regeneration, fibrosis and cancer progression. Cell migration is believed to play a key role in both normal wound repair and in abnormal tissue remodeling. Prostaglandin E2 (PGE2) inhibits fibroblast chemotaxis, but stimulates chemotaxis in airway epithelial cells. The current study was designed to explore the role of PGE2 and its four receptors on airway epithelial cell migration following EMT using both the Boyden blindwell chamber chemotaxis assay and the wound closure assay. EMT in human bronchial epithelial cells (HBECs) was induced by TGF-ß1 and a mixture of cytokines (IL-1ß, TNF-α, and IFN-γ). PGE2 and selective agonists for all four EP receptors stimulated chemotaxis and wound closure in HBECs. Following EMT, the EP1 and EP3 agonists were without effect, while the EP2 and EP4 agonists inhibited chemotaxis as did PGE2. The effects of the EP2 and EP4 receptors on HBEC and EMT cell migration were further confirmed by blocking the expected signaling pathways. Taken together, these results demonstrate that PGE2 switches from a stimulator to an inhibitor of cell migration following EMT of airway epithelial cells and that this inhibition is mediated by an altered effect of EP2 and EP4 signaling and an apparent loss of the stimulatory effects of EP1 and EP3. Change in the PGE2 modulation of chemotaxis may play a role in repair following injury.

Vitamin E intake and pancreatic cancer risk: a meta-analysis of observational studies.

BACKGROUND: Some epidemiological studies have suggested that vitamin E intake reduces the risk of pancreatic cancer; however, this conclusion has not been supported by all the published studies. We conducted a meta-analysis to assess the relationship between vitamin E intake and the risk of pancreatic cancer by combining the results from published articles. MATERIAL/METHODS: We searched the published studies that reported the relationship between vitamin E intake and pancreatic cancer risk using the PubMed, Web of Science, and Embase databases through December 31st, 2014. Based on a fixed-effects or random-effects model, the RR and 95% CI were used to assess the combined risk. RESULTS: In total, 10 observational studies (6 case-control studies and 4 cohort studies) were included. The overall RR (95% CI) of pancreatic cancer for the highest vs. the lowest level of vitamin E intake was 0.81 (0.73, 0.89). We found little evidence of heterogeneity (I2=19.8%, P=0.255). In the subgroup analyses, we found an inverse association between vitamin E intake and pancreatic cancer risk both in the case-control and cohort studies. Additionally, this inverse association was not modified by different populations. CONCLUSIONS: In our meta-analysis, there was an inverse association between vitamin E intake and the risk of pancreatic cancer. A high level of vitamin E might be a protective factor for populations at risk for pancreatic cancer.

Vitamin D modulates prostaglandin E2 synthesis and degradation in human lung fibroblasts.

Vitamin D insufficiency has been increasingly recognized in the general population worldwide and has been associated with several lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), and respiratory tract infections. Fibroblasts play a critical role in tissue repair and remodeling, which is a key feature of COPD and asthma. Fibroblasts modulate tissue repair by producing and modifying extracellular matrix components and by releasing mediators that act as autocrine or paracrine modulators of tissue remodeling. The current study was designed to investigate if vitamin D alters fibroblast release of key autocrine/paracrine repair factors. First, we demonstrated that human fetal lung (HFL)-1 cells express the vitamin D receptor (VDR) and that vitamin D, 25-hydroxyvitamin D [25(OH)D], or 1,25-dihydroxyvitamin D [1,25(OH)2D] induce VDR nuclear translocation and increase VDR-DNA binding activity. We next demonstrated that vitamin D, 25(OH)D, and 1,25(OH)2D significantly reduced prostaglandin (PG)E2 production by human lung fibroblasts (HFL-1) but had no effect on transforming growth factor ß1, vascular endothelial growth factor, or fibronectin production. Vitamin D, 25(OH)D, and 1,25(OH)2D significantly inhibited IL-1ß-induced microsomal PGE synthase (mPGES)-1 expression; in contrast, all three forms of vitamin D stimulated 15-hydroxy PG dehydrogenase, an enzyme that degrades PGE2. Cyclooxygenase-1 and -2 and the other two PGE2 synthases (mPGES-2 and cytosolic PGE synthase) were not altered by vitamin D, 25(OH)D, or 1,25(OH)2D. Finally, the effect of PGE2 inhibition by 25(OH)D was observed in adult lung fibroblasts. These findings suggest that vitamin D can regulate PGE2 synthesis and degradation and by this mechanism can modulate fibroblast-mediated tissue repair function.

Matrix metalloproteinase-9 activates TGF-ß and stimulates fibroblast contraction of collagen gels.

Matrix metalloproteinase-9 (MMP-9) is a matrix-degrading enzyme implicated in many biological processes, including inflammation. It is produced by many cells, including fibroblasts. When cultured in three-dimensional (3D) collagen gels, fibroblasts contract the surrounding matrix, a function that is thought to model the contraction that characterizes both normal wound repair and fibrosis. The current study was designed to evaluate the role of endogenously produced MMP-9 in fibroblast contraction of 3D collagen gels. Fibroblasts from mice lacking expression of MMP-9 and human lung fibroblasts (HFL-1) transfected with MMP-9 small-interfering RNA (siRNA) were used. Fibroblasts were cast into type I collagen gels and floated in culture medium with or without transforming growth factor (TGF)-ß1 for 5 days. Gel size was determined daily using an image analysis system. Gels made from MMP-9 siRNA-treated human fibroblasts contracted less than control fibroblasts, as did fibroblasts incubated with a nonspecific MMP inhibitor. Similarly, fibroblasts cultured from MMP-9-deficient mice contracted gels less than did fibroblasts from control mice. Transfection of the MMP-9-deficient murine fibroblasts with a vector expressing murine MMP-9 restored contractile activity to MMP-9-deficient fibroblasts. Inhibition of MMP-9 reduced active TGF-ß1 and reduced several TGF-ß1-driven responses, including activity of a Smad3 reporter gene and production of fibronectin. Because TGF-ß1 also drives fibroblast gel contraction, this suggests the mechanism for MMP-9 regulation of contraction is through the generation of active TGF-ß1. This study provides direct evidence that endogenously produced MMP-9 has a role in regulation of tissue contraction of 3D collagen gels mediated by fibroblasts.

Fibroblasts that resist cigarette smoke-induced senescence acquire profibrotic phenotypes.

This study assessed the effect of extended exposure to cigarette smoke extract (CSE) on tissue repair functions in lung fibroblasts. Human fetal (HFL-1) and adult lung fibroblasts were exposed to CSE for 14 days. Senescence-associated ß-galactosidase (SA ß-gal) expression, cell proliferation, and tissue repair functions including chemotaxis and gel contraction were assessed. HFL-1 proliferation was inhibited by CSE and nearly half of the CSE-exposed cells were SA ß-gal positive after 14 days exposure, whereas 33% of adult lung fibroblasts were SA ß-gal positive in response to 10% CSE exposure. The SA ß-gal-positive cells did not proliferate as indicated by bromodeoxyuridine incorporation. In contrast, cells negative for SA ß-gal after CSE exposure proliferated faster than cells never exposed to CSE. These nonsenescent cells migrated more and contracted collagen gels more than control cells. CSE exposure stimulated TGF-ß1 production, and both inhibition of TGF-ß receptor kinase and TGF-ß1 siRNA blocked CSE modulation of fibroblast function. Extended exposure to CSE might induce two different fibroblast phenotypes, a senescent and a profibrotic phenotype. The fibroblasts that resist CSE-induced cellular senescence may contribute to the pathogenesis of idiopathic pulmonary fibrosis and could contribute to fibrotic lesions in chronic obstructive pulmonary disease acting through a TGF-ß1-mediated pathway. In contrast, the senescent cells may contribute to the pathogenesis of emphysema.

Reprogramming of COPD lung fibroblasts through formation of induced pluripotent stem cells.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) eliminates many epigenetic modifications that characterize differentiated cells. In this study, we tested whether functional differences between chronic obstructive pulmonary disease (COPD) and non-COPD fibroblasts could be reduced utilizing this approach. Primary fibroblasts from non-COPD and COPD patients were reprogrammed to iPSCs. Reprogrammed iPSCs were positive for oct3/4, nanog, and sox2, formed embryoid bodies in vitro, and induced teratomas in nonobese diabetic/severe combined immunodeficient mice. Reprogrammed iPSCs were then differentiated into fibroblasts (non-COPD-i and COPD-i) and were assessed either functionally by chemotaxis and gel contraction or for gene expression by microarrays and compared with their corresponding primary fibroblasts. Primary COPD fibroblasts contracted three-dimensional collagen gels and migrated toward fibronectin less robustly than non-COPD fibroblasts. In contrast, redifferentiated fibroblasts from iPSCs derived from the non-COPD and COPD fibroblasts were similar in response in both functional assays. Microarray analysis identified 1,881 genes that were differentially expressed between primary COPD and non-COPD fibroblasts, with 605 genes differing by more than twofold. After redifferentiation, 112 genes were differentially expressed between COPD-i and non-COPD-i with only three genes by more than twofold. Similar findings were observed with microRNA (miRNA) expression: 56 miRNAs were differentially expressed between non-COPD and COPD primary cells; after redifferentiation, only 3 miRNAs were differentially expressed between non-COPD-i and COPD-i fibroblasts. Interestingly, of the 605 genes that were differentially expressed between COPD and non-COPD fibroblasts, 293 genes were changed toward control after redifferentiation. In conclusion, functional and epigenetic alterations of COPD fibroblasts can be reprogrammed through formation of iPSCs.

Oncostatin M modulates fibroblast function via signal transducers and activators of transcription proteins-3.

Oncostatin M (OSM), an inflammatory cytokine of the interleukin-6 (IL-6) superfamily, plays a key role in various biological processes such as modulation of extracellular matrix (ECM), cell proliferation, cell survival, and induction of inflammation. It has been reported that OSM was increased in asthma and pulmonary fibrosis, and thus OSM may play a role in airway remodeling and the development of lung parenchymal fibrosis. Recruitment of lung fibroblasts to the sites of airway injury and subsequent differentiation into myofibroblasts is believed to contribute to excess ECM deposition. In the current study, we assessed the ability of OSM to modulate fibroblast collagen gel contraction, migration toward fibronectin, and expression of α-smooth muscle actin (α-SMA). We demonstrated that OSM augments gel contraction, chemotaxis, and α-SMA expression. OSM-augmented fibroblast chemotaxis was mediated by the signal transducer and activator of transcription (STAT3) and p38 mitogen-activated protein kinase, while augmentation on gel contraction and α-SMA expression was mediated by STAT3. Neither transforming growth factor-ß1 nor PGE2 was involved in mediating OSM effect on the cells. The Th2 cytokines IL-4 and IL-13, which also are believed to play an important role in promoting lung fibrosis and airway remodeling, act through STAT3, and we demonstrated the potential for additive effects of OSM with IL-4 and IL-13. The present study supports the concept that OSM may contribute to tissue remodeling, which may be additive with IL-4 or IL-13. Blockade of OSM or OSM-mediated STAT3 signaling could be a therapeutic target to regulate lung fibrotic mechanisms.

Phosphodiesterase-4 inhibition augments human lung fibroblast vascular endothelial growth factor production induced by prostaglandin E2.

Lung fibroblasts are believed to be a major source of vascular endothelial growth factor (VEGF), which supports the survival of lung endothelial cells and modulates the maintenance of the pulmonary microvasculature. VEGF has been related to the pathogenesis of lung diseases, including chronic obstructive pulmonary disease (COPD). Prostaglandin E2 (PGE2) stimulates VEGF production from lung fibroblasts via the E-prostanoid (EP)-2 receptor. The EP2 signaling pathway uses cyclic adenosine monophosphate (cAMP) as a second messenger, and cAMP is degraded by phosphodiesterases (PDEs). This study investigates whether phosphodiesterase inhibition modulates the human lung fibroblast VEGF production induced by PGE2. Human fetal lung fibroblasts were cultured with PGE2 and PDE inhibitors. The PDE4 inhibitors roflumilast, roflumilast N-oxide, and rolipram with PGE2 increased VEGF release, as quantified in supernatant media by ELISA. In contrast, PDE3, PDE5, and PDE7 inhibitors did not affect VEGF release. Roflumilast increased VEGF release with either an EP2 or an EP4 agonist. Roflumilast augmented the cytosolic cAMP levels induced by PGE2 and VEGF release with other agents that use the cAMP signaling pathway. Roflumilast-augmented VEGF release was completely inhibited by a protein kinase A (PKA) inhibitor. Roflumilast with PGE2 increased VEGF mRNA levels, and the blockade of mRNA synthesis inhibited the augmented VEGF release. The stimulatory effect of roflumilast on VEGF release was replicated using primary healthy and COPD lung fibroblasts. These findings demonstrate that PDE4 inhibition can modulate human lung fibroblast VEGF release by PGE2 acting through the EP2 and EP4 receptor-cAMP/PKA signaling pathway. Through this action, PDE4 inhibitors such as roflumilast could contribute to the survival of lung endothelial cells.

Differential roles of JNK, ERK1/2, and p38 mitogen-activated protein kinases on endothelial cell tissue repair functions in response to tumor necrosis factor-α.

Tumor necrosis factor (TNF)-α can alter tissue repair functions in a variety of cells including endothelial cells. However, the mechanism by which TNF-α mediates these functional changes has not fully been studied. We investigated the role of mitogen-activated protein kinases (MAPKs) on mediating the regulatory effect of TNF-α on the tissue repair functions of human pulmonary artery endothelial cells (HPAECs). TNF-α protected HPAECs from undergoing apoptosis induced by serum and growth factor deprivation, augmented collagen gel contraction, and stimulated wound closure. TNF-α activated c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38. Inhibitors of JNK (SP600125, 5 µM) or ERK1/2 (PD98059, 5 µM) significantly inhibited TNF-α-stimulated cell survival, contraction of collagen gels, and wound closure. In contrast, the p38 inhibitor SB203580 (5 µM) further amplified all of the TNF-α effects on HPAECs. TNF-α specifically activated p38α but not other p38 isoforms and suppression of p38α by an siRNA resulted in further amplification of the TNF-α effect. These results suggest that TNF-α stimulates tissue repair functions of HPAECs, and this may be mediated, at least in part, positively via JNK and ERK1/2, and negatively through p38α. MAPKs may play a role in endothelial cell-mediated tissue repair, especially in an inflammatory milieu where TNF-α is present.

PGE 2 desensitizes ß -agonist effect on human lung fibroblast-mediated collagen gel contraction through upregulating PDE4.

In the current study, we investigated the effect of a long-acting ß -agonist (salmeterol) and a phosphodiesterase 4 (PDE4) inhibitor (cilomilast) on human lung fibroblast-mediated collagen gel contraction. Higher concentrations of salmeterol (10(-7) and 10(-6) M) inhibited fibroblast-mediated collagen gel contraction. No effect was observed with cilomilast alone (up to 10(-5) M). In the presence of 10(-8) M salmeterol, however, cilomilast could significantly inhibit fibroblast-mediated collagen gel contraction in a concentration-dependent manner (10(-7) ~10(-5) M). Blockade of endogenous PGE2 by indomethacin further potentiated the inhibitory effect of salmeterol on fibroblast-mediated collagen gel contraction, but it did not affect cilomilast's effect. Pretreatment with PGE2 abolished the inhibitory effect of salmeterol, but it potentiated the inhibitory effect of cilomilast on fibroblast-mediated collagen gel contraction. Finally, indomethacin slightly inhibited PDE4C expression, while PGE2 stimulated the expression of PDE4A and -4C in human lung fibroblasts. These findings suggest that long-acting ß -agonist and PDE4 inhibitor have a synergistic effect in regulating fibroblast tissue repair functions and that PGE2 can modulate the effect of ß -agonist and PDE4 inhibitor at least in part through the mechanism of regulating PDE4 expression.

Research on permanent magnet synchronous motor algorithm based on linear nonlinear switching self-disturbance rejection control.

This paper presents a linear-nonlinear switching control strategy, called Switching Active Disturbance Rejection Control (SADRC), to enhance the disturbance rejection capability of the speed controller in a servo system. SADRC combines the advantages of Linear Active Disturbance Rejection Control (LADRC) and Nonlinear Active Disturbance Rejection Control (NLADRC), and introduces a parameter to switch between nonlinear and linear control, thereby improving the robustness of the servo system. Firstly, the mathematical model of the motor is analyzed as the starting point of the paper. Then, the basic principles of Active Disturbance Rejection Control (ADRC) are analyzed, and improvements are made to address its limitations, resulting in the design of SADRC. The parameters introduced in SADRC are analyzed to determine their appropriate ranges. Finally, the performance of SADRC is validated by comparing the rotational effects of Permanent Magnet Synchronous Motor (PMSM).