Search

Objectives and Background: The expression of inflammatory genes in the heart plays an important role in organ dysfunction. However, the contribution of cardiomyocytes to this process, and in particular to the synthesis of high concentrations of nitric oxide and prostaglandins, has not been analyzed in detail. For this reason, cultured isolated cardiomyocytes were used to evaluate the response to pro-inflammatory stimuli., Methods and Results: Isolated cultured foetal, neonatal, and adult rat cardiomyocytes were stimulated with lipopolysaccharide and cytokines, and the expression of nitric oxide synthase-2 (NOS-2) and cyclooxygenase-2 (COX-2) in these cells was investigated. Only cultured foetal cardiomyocytes expressed NOS-2 and COX-2 under these conditions, whereas the neonatal counterparts required various days in culture to gain this response. Analysis of the NF-kappaB signalling pathway showed an impaired activation of IkappaB kinase in response to lipopolysaccharide and cytokines in cells maintained in culture for 1 day. These data were confirmed by DNA microarray analysis. However, other early signalling pathways, such as the p38 and Erk MAPKs, were not affected by the time in culture., Conclusions: Neonatal and adult cardiomyocytes are resistant to the expression of pro-inflammatory genes due to impairment in the activation of IkappaB kinase, a process that might contribute to preventing rapid organ dysfunction in the course of various inflammatory pathologies, such as septic shock and myocarditis.

Background: Cyclooxygenase 2 (COX-2) and matrix metalloproteinases (MMPs) have been implicated in tissue injury and fibrogenesis in animal models but little is known regarding their role in hepatitis C virus (HCV) related liver disease in humans., Aims: To characterise the intrahepatic expression pattern of COX-2 and MMPs in chronic HCV infection and determine whether HCV core and NS5A proteins could promote their expression in cultured hepatocyte derived cell lines., Patients: Thirty two anti-HCV+ and 10 anti-HCV- patients were studied., Methods: Western blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunohistochemistry were used to assess the expression pattern of COX-2 and MMPs in liver biopsy samples from all patients. COX-2 gene expression and MMP-9 protein levels were also determined by immunoblot, RT-PCR, and luciferase assays in core and NS5A transfected hepatocyte derived cells., Results: The intrahepatic expression level of COX-2, MMP-2, and MMP-9 was significantly higher in HCV+ than in HCV- patients, increasing with the fibrotic stage of liver disease. We further demonstrated that COX-2 mRNA, protein, and activity were induced in resting and activated core and NS5A transfectants. Both viral proteins induced transcriptional activity of the COX-2 gene promoter whereas core, but not NS5A, exerted an inducer effect on MMP-9 protein levels in cultured hepatocyte derived cells., Conclusions: Intrahepatic COX-2, MMP-2, and MMP-9 overexpression is associated with progressive hepatic fibrosis in chronic HCV infection, suggesting their pathogenic role in fibrogenesis. HCV core and NS5A proteins were able to upregulate COX-2 and MMP-9 gene expression in hepatocyte derived cells, providing a potential mechanism for hepatic fibrosis during chronic HCV infection.

The effect of rofecoxib, a selective cyclooxygenase-2 inhibitor, on inflammatory signaling has been investigated in elicited murine peritoneal macrophages. Macrophages treated with 10 microM rofecoxib exhibited an important inhibition in the early activation of nuclear factor kappa B (NF-kappa B) and the mitogen-activated protein kinase p38, the extracellular-regulated kinase p44, and the c-Jun N-terminal kinase. Moreover, this drug decreased the protein levels of nitric-oxide synthase-2 and cyclooxygenase-2 in lipopolysaccharide (LPS)-treated macrophages. Rofecoxib delayed and attenuated NF-kappa B activation, which impaired significantly the expression of kappa B-dependent genes. This drug and related coxibs did not affect cell viability and protected against LPS-induced apoptosis through the impairment of the inflammatory response. These data show an additional anti-inflammatory mechanism of selective cyclooxygenase-2 inhibitors through the attenuation of macrophage activation.

Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) transiently activates protein kinase C zeta (PKC zeta) and Jun N-terminal kinase (JNK) through a phosphoinositide-3-kinase (PI3-kinase)-dependent pathway. Incubation of LPS-treated cells with the cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)) promoted a sustained activation of PKC zeta and JNK and inhibited I kappa B kinase (IKK) and NF-kappa B activity. Accordingly, 15dPGJ(2) induced an imbalance between JNK and IKK activities by increasing the former signaling pathway and inhibiting the latter signaling pathway. Under these conditions, apoptosis was significantly enhanced; this response was very dependent on PKC zeta and JNK activation. The effect of 15dPGJ(2) on PKC zeta activity observed in LPS-activated macrophages was not dependent on a direct action of this prostaglandin on the enzyme but was due to the activation of a step upstream of PI3-kinase. Moreover, LPS promoted the redistribution of activated PKC zeta from the cytosol to the nucleus, a process that was enhanced by treatment of the cells with 15dPGJ(2) that favored a persistent and broader distribution of PKC zeta in the nucleus. These results indicate that 15dPGJ(2) and other cyclopentenone prostaglandins, through the sustained activation of PKC zeta, might contribute significantly to the process of resolution of inflammation by promoting apoptosis of activated macrophages.

Partial hepatectomy (PH) triggers a rapid regenerative response in the remaining tissue to reinstate the organ function and the cell numbers. Among the molecules that change in the course of regeneration is an accumulation of prostaglandin E2 in the sera of rats with PH. Analysis of the cyclooxygenase (COX) isoenzymes in the remnant liver showed the preferential expression of COX-2 in hepatocytes. Cultured regenerating hepatocytes expressed significant levels of COX-2, a process that was not observed in the sham counterparts. Maximal expression of COX-2 was detected 16 h after PH with increased levels present even at 96 h. Pharmacological inhibition of COX-2 activity with NS398 shunted the up-regulation of cell proliferation after PH, which suggests a positive interaction of prostaglandins with the progression of the cell cycle. Similar results were obtained after PH of mice lacking the COX-2 gene. The expression of COX-2 in regenerating liver was concomitant with a decrease in CCAAT-enhancer binding protein (C/EBP-a) level and an increase in the expression of C/EBP-b and C/EBP-d. These results suggest a contribution of the enhanced synthesis of prostaglandins to liver regeneration observed after PH.

Treatment of primary cultures of fetal hepatocytes with proinflammatory cytokines, lipopolysaccharide (LPS), and hepatocyte growth factor promoted the expression of cyclooxygenase-2 (COX-2) and the synthesis of high amounts of prostaglandins (PGs). Under these conditions, the active forms of the matrix metalloproteinases-2 and -9 (MMPs) were released to the extracellular medium. This process was inhibited when the synthesis of PGs was suppressed pharmacologically with COX-2 inhibitors. Addition to the cell cultures of PGE(2) promoted the release of MMPs through a mechanism that involved the expression of COX-2 and the synthesis of additional PGs. Kinetic analysis of the secretion of MMPs in response to LPS and PGE(2) showed a similar time course, with a lag period of 6 hours, which suggests that PGE(2) does not act directly on the mechanism of MMP processing and release. Inhibitors of protein kinase A, p38 MAP kinase, phosphatidylinositol-3-kinase, and nuclear factor kappaB (NF-kappaB) activation impaired the release of MMPs in response to PGE(2) challenge, indicating the involvement of multiple steps in the process. The ability of fetal hepatocytes to release MMPs in response to growth factors and inflammatory stimuli constitutes a model for the study of the extracellular matrix remodeling that accompanies most liver diseases.

Background & Aims: Expression of cyclooxygenase (COX)-2 in response to lipopolysaccharide (LPS) challenge has been analyzed in cultured fetal, neonatal, and adult hepatocytes and in hepatoma cell lines., Methods: To study the mechanisms of LPS-dependent expression of COX-2 in these cells, the activity of the COX-2 promoter and the levels of CCAAT/enhancer-binding proteins (C/EBPs) were determined., Results: COX-2 was induced in fetal hepatocytes, but this response declined rapidly after birth. This loss of inducibility of COX-2 paralleled the expression of C/EBP-alpha in neonatal hepatocytes. Transfection of fetal and adult hepatocytes with sequences corresponding to the 5'-flanking region of the rat COX-2 gene confirmed the absence of promoter activity in adult hepatocytes. Moreover, transient expression of C/EBP-alpha, but not C/EBP-delta, in the hepatoma cell line AT3F cells abolished the COX-2 promoter activity. Prolonged culture of adult hepatocytes restored the induction of COX-2 after complete disappearance of C/EBP-alpha., Conclusions: These results suggest that the presence of high levels of C/EBP-alpha is involved in the impairment of COX-2 expression in adult hepatocytes challenged with proinflammatory stimuli.

Recently isolated trophoblasts express nitric oxide synthase 2 (NOS-2) and cyclooxygenase 2 (COX-2), decreasing the levels of the corresponding mRNAs when the cells were maintained in culture. The sustained expression of COX-2 and NOS-2 in trophoblasts was dependent on the activation of nuclear factor kappaB (NF-kappaB) since proteasome inhibitors and antioxidants that abrogated NF-kappaB activity suppressed the induction of both genes. The time-dependent fall of the mRNA levels of NOS-2 and COX-2 paralleled the inhibition of NF-kappaB, determined by electrophoretic mobility shift assays, and the increase of the IkappaBalpha and IkappaBbeta inhibitory proteins. Isolated trophoblasts synthesized reactive oxygen intermediates (ROI), a process impaired after culturing the cells, and that might be involved in the NF-kappaB activation process. Moreover, treatment of recently isolated cells with ROI scavengers suppressed the expression of COX-2 and NOS-2. Challenge of trophoblasts with interleukin-1beta up-regulated the expression of both proteins, an effect that was potentiated by lipopolysaccharide. These results indicate that the physiological expression of NOS-2 and COX-2 in trophoblasts involves a sustained activation of NF-kappaB which inhibition abrogates the inducibility of both genes.

Primary cultures of fetal hepatocytes expressed cyclooxygenase-2 (COX-2) upon stimulation with bacterial lipopolysaccharide (LPS) or peroxisomal proliferators. This enzyme was active and a good correlation between the mRNA levels, the amount of protein, and the synthesis of prostaglandin E2 was observed. However, when cells were incubated in the presence of indomethacin or the COX-2-specific inhibitor NS398, the amount of COX-2 protein increased 5-fold after activation with LPS and 2-fold after treatment with clofibrate. This up-regulation of COX-2 was not observed at the mRNA level. The mechanism of protein accumulation might involve either a direct stabilization of the enzyme by the inhibitors or the absence of prostaglandins involved in the regulation of its turnover. Among the prostaglandins assayed, only 15-deoxy-Prostaglandin J2 exerted a statistically significant decrease in the COX-2 levels in cells stimulated with LPS or LPS plus NS398. The accumulation of COX-2 in the presence of inhibitors was also observed in peritoneal macrophages treated under identical conditions. These results indicate that COX-2 protein accumulates after enzyme inhibition, and because removal of the inhibitors restored the enzyme activity, suppression of treatment with reversible COX-2 inhibitors may cause a transient overproduction of prostaglandins.

Cyclooxygenase-2 (COX-2) is involved in the biosynthesis of prostanoids in the course of inflammatory reactions. This isoenzyme is regulated at the transcription level and many cells express COX-2 upon challenge with lipopolysaccharide (LPS) or pro-inflammatory cytokines. Since hepatocytes respond to LPS and pro-inflammatory stimuli, we investigated the expression of COX-2 in foetal and adult hepatocytes upon challenge with these substances. COX-2 was expressed in foetal hepatocytes incubated with LPS, tumour necrosis factor-alpha and interleukin-1beta. This response rapidly decreased after birth and was absent in hepatocytes from animals aged 2 days or more and treated under identical conditions. The expression of COX-2 was determined at the mRNA, protein and enzyme activity levels using Northern and Western blot, and following the synthesis of prostaglandin E2, respectively. The use of NS 398, a specific pharmacological inhibitor of COX-2, confirmed the expression of this isoenzyme in activated foetal hepatocytes. Synergism in COX-2 expression was observed between LPS, tumour necrosis factor-alpha and interleukin-1beta. Interleukin-6 and permeant analogues of cyclic AMP failed to induce COX-2 or to synergize with LPS. Also, transforming growth factor-beta inhibited the LPS- and pro-inflammatory cytokines-dependent expression of COX-2. These results indicate that foetal hepatocytes are competent to express COX-2 upon challenge with pro-inflammatory stimuli, a process lost completely in hepatocytes isolated from animals aged 2 days.

Treatment of rats at 21 days' gestation with lipopolysaccharide (LPS) induced type II NO synthase (iNOS) expression in maternal liver but completely failed to elicit this response in the corresponding fetal tissue. The impaired response of fetal liver cannot be attributed to alterations in the sensitivity of this organ to LPS or to proinflammatory cytokines involved in iNOS expression (tumor necrosis factor alpha [TNF-alpha], interferon gamma [IFN-gamma]), because cultured fetal and adult hepatocytes similarly responded to these factors. Measurement of TNF-alpha and IFN-gamma levels in maternal and fetal serum showed a restricted permeability of placenta to these cytokines, results that were in agreement with the absence of iNOS expression in fetal liver. Moreover, analysis of nuclear factor kappaB, which is required for iNOS expression, showed an activation in nuclear extracts from livers of mothers under septic shock conditions but not in the corresponding fetal livers. However, cultured fetal hepatocytes prepared from mothers in septic shock displayed an anomalous pattern of iNOS expression, including a decreased response to LPS as well as a shift in sensitivity to TNF-alpha. These hepatocytes from LPS-treated mothers showed significant expression of iNOS in the absence of added stimuli. These results suggest that there is a priming of the fetal hepatocytes from mothers with septic shock. The characterization of the factors causing this priming process might provide additional clues understanding the control of iNOS expression in liver.

The presence of the calcium-independent cytokine-inducible nitric oxide synthase (iNOS) has been investigated in rat placenta from day 19 of gestation till delivery. iNOS has been detected at the mRNA, enzyme activity and protein levels in complete placenta. Immunocytochemical detection of iNOS was heterogeneously distributed in control placenta. Intraperitoneal injection of pregnant rats at 21 days of gestation with lipopolysaccharide (LPS) increased the iNOS immunoreactivity in the decidua basalis of the placenta, and, when the mRNA levels and enzyme activity were measured in total tissue, a moderate increase (approx. 160%) was observed. A constitutive nuclear factor kappaB activity was observed in placenta from both control and LPS-treated animals. These results indicate constitutive expression of iNOS in rat placenta.

The pattern of expression of liver-specific and extrahepatic S-adenosylmethionine (SAM) synthetase in developing rat liver was established by determining steady-state levels of the respective messenger RNAs (mRNAs) and protein content. Levels of liver-specific SAM synthetase mRNA increased progressively from day 20 of gestation, increased 10-fold immediately after birth, and reached a peak at 10 days of age, decreasing slightly by adulthood. Conversely, mRNA levels of extrahepatic isoenzyme decreased toward birth, increased threefold in the newborn, and decreased further in the postnatal life, reaching a minimum in the adult. Similar expression profiles were observed in isolated hepatocytes, indicating that both mRNAs are differentially regulated in the same cell type. Western blot analysis showed that levels of immunoreactive liver-specific isoenzyme followed a trend similar to the mRNA, indicating that developmental regulation of this enzyme is mediated at the mRNA level. Developmental patterns of expression of albumin and alpha-fetoprotein (AFP) mRNAs were closely related to those for liver-specific and extrahepatic isoenzymes, respectively. Therefore, it is suggested that liver-specific SAM synthetase may be a marker for hepatocyte differentiation. Incubation of primary cultures of hepatocytes from 21-day-old fetuses with permeant cyclic adenosine monophosphate (cAMP) analogues elicited an up-regulation of the mRNA for the liver-specific isoenzyme with a concomitant down-regulation of the extrahepatic message, suggesting a physiological role for the increased postnatal glucagonemia in the control of this isoenzyme switching. In contrast with the isoenzyme expression profiles, the levels of SAM, the product of SAM synthetase reaction, were determined to be greater during gestation than in immediate postnatal periods. These results indicate that synthesis and utilization of SAM may be regulated differentially in fetal and adult hepatocytes.

Fetal rat liver expresses a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/Fru-2,6-Pase2) form that differs from the adult liver enzyme in the inhibition by phosphorylation by the adenosine 3',5'-cyclic monophosphate-dependent protein kinase and in the recognition by an antibody specific for the NH2-terminal domain of the adult liver enzyme. Northern blot analysis shows that fetal hepatocytes contain a species of mRNA that is 2.2 kb in size and that exhibits the maximal levels after delivery. PFK-2/Fru-2,6-Pase2 mRNA analysis using a sensitive ribonuclease protection assay reveals the presence of nearly similar amounts of adult liver-specific and skeletal muscle-specific mRNA in fetal liver and hepatocytes during the last days of gestation, as well as a 233-bp protected fragment present in fetal liver. These results were confirmed by polymerase chain reaction using specific oligonucleotide pairs. Primer extension of fetal liver cDNA suggests the presence of two initiation sites of transcription. Analysis of the adult liver PFK-2/Fru-2,6-Pase2 protein during the perinatal transition using a specific antibody shows a marked accumulation of this form immediately after birth.