Aims/hypothesis Glucagon-like peptide-1 (GLP-1), a member of the proglucagon-derived peptide family, was seen to exert favourable actions on cardiovascular function in preclinical and clinical studies. The mechanisms through which GLP-1 modulates cardiovascular function are complex and incompletely understood. We thus investigated whether the GLP-1 analogue, liraglutide, which is an acylated GLP-1, has protective effects on vascular endothelial cells. Methods Nitrite and nitrate were measured in medium with an automated nitric oxide detector. Endothelial nitric oxide synthase (eNOS) activation was assessed by evaluating the phosphorylation status of the enzyme and evaluating eNOS activity by citrulline synthesis. Nuclear factor κB (NF-κB) activation was assessed by reporter gene assay. Results Liraglutide dose-dependently increased nitric oxide production in HUVECs. It also caused eNOS phosphorylation, potentiated eNOS activity and restored the cytokineinduced downregulation of eNOS (also known as NOS3) mRNA levels, which is dependent on NF-κB activation. We therefore examined the effect of liraglutide on TNFα-induced NF-κB activation and NF-κB-dependent expression of proinflammatory genes. Liraglutide dose-dependently inhibited NF-κB activation and TNFα-induced IκB degradation. It also reduced TNFα-induced MCP-1 (also known as CCL2), VCAM1, ICAM1 and E-selectin mRNA expression. Liraglutide-induced enhancement of nitric oxide production and suppression of NF-κB activation were attenuated by the AMP-activated protein kinase (AMPK) inhibitor compound C or AMPK (also known as PRKAA1) small interfering RNA. Indeed, liraglutide induced phosphorylation of AMPK, which occurs through a signalling pathway independent of cyclic AMP. Conclusions/interpretation Liraglutide exerts an antiinflammatory effect on vascular endothelial cells by increasing nitric oxide production and suppressing NF-κB activation, partly at least through AMPK activation. These effects may explain some of the observed vasoprotective properties of liraglutide, as well as its beneficial effects on the cardiovascular system.

No abstract

Cochlear fibrocytes play important roles in normal hearing as well as in several types of sensorineural hearing loss attributable to inner ear homeostasis disorders. Recently, we developed a novel rat model of acute sensorineural hearing loss attributable to fibrocyte dysfunction induced by a mitochondrial toxin. In this model, we demonstrate active regeneration of the cochlear fibrocytes after severe focal apoptosis without any changes in the organ of Corti. To rescue the residual hearing loss, we transplanted mesenchymal stem cells into the lateral semicircular canal; a number of these stem cells were then detected in the injured area in the lateral wall. Rats with transplanted mesenchymal stem cells in the lateral wall demonstrated a significantly higher hearing recovery ratio than controls. The mesenchymal stem cells in the lateral wall also showed connexin 26 and connexin 30 immunostaining reminiscent of gap junctions between neighboring cells. These results indicate that reorganization of the cochlear fibrocytes leads to hearing recovery after acute sensorineural hearing loss in this model and suggest that mesenchymal stem cell transplantation into the inner ear may be a promising therapy for patients with sensorineural hearing loss attributable to degeneration of cochlear fibrocytes. Mammalian cochlear fibrocytes of the mesenchymal nonsensory regions play important roles in the cochlear physiology of hearing, including the transport of potassium ions to generate an endocochlear potential in the endolymph that is essential for the transduction of sound by hair cells.1-3 It has been postulated that a potassium recycling pathway toward the stria vascularis via fibrocytes in the cochlear lateral wall is critical for proper hearing, although the exact mechanism has not been definitively determined.2 One candidate model for this ion transport system consists of an extracellular flow of potassium ions through the scala tympani and scala vestibuli and a transcellular flow through the organ of Corti, supporting cells, and cells of the lateral wall.4,5 The fibrocytes within the cochlear lateral wall are divided into type I to V based on their structural features, immunostaining patterns, and general location.5 Type II, type IV, and type V fibrocytes resorb potassium ions from the surrounding perilymph and from outer sulcus cells via the Na,KATPase. The potassium ions are then transported to type I fibrocytes, strial basal cells, and intermediate cells through gap junctions and are secreted into the intrastrial space through potassium channels. The secreted potassium ions are incorporated into marginal cells by the Na,K-ATPase and the Na-K-Cl co-transporter, and are finally secreted into the endolymph through potassium channels.Degeneration and alteration of the cochlear fibrocytes have been reported to cause hearing loss without any other changes in the cochlea in the Pit-Oct-Unc (POU)-domain transcription factor Brain-4 (Brn-4)-deficient mouse 6 and the otospiralin-deficient mouse.3 Brn-4 is the gene responsible f...

Previous studies have demonstrated that patients with halothane-induced hepatitis have serum antibodies that are directed against novel liver microsomal neoantigens and have suggested that these neoantigens may play an immunopathological role in development of the patients' liver damage. These investigations have further revealed that the antibodies are directed against distinct polypeptide fractions (100 kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) that have been covalently modified by the reactive trifluoroacetyl halide metabolite of halothane. In this paper, the trifluoroacetylated (TFA) 59-kDa neoantigen (59-kDa-TFA) recognized by the patients' antibodies was isolated from liver microsomes of halothane-treated rats by chromatography on an immunoaffinity column of anti-TFA IgG. Antibodies were raised against the 59-kDa-TFA protein and were used to purify the native protein from liver microsomes ofuntreated rats. Based upon its apparent monomeric molecular mass, NH2-terminal amino acid sequence, catalytic activity, and other physical properties, the protein has been identified as a previously characterized microsomal carboxylesterase (EC 3.1.1.1). A similar strategy may be used to purify and characterize neoantigens associated with other drug toxicities that are believed to have an immunopathological basis.It has been estimated that between 3% and 25% of all drug toxicities, which can include anaphylaxis, serum sickness, asthma, urticaria, dermatitis, fever, hemolytic anemia, thrombocytopenia, granulocytopenia, hepatitis, nephritis, vasculitis, pneumonitis, and lupus-erythematosus-like syndrome, are due to hypersensitivity (allergic) reactions (1). Although most of these drug-induced hypersensitivities have been presumed to be mediated by immunogens formed by the covalent interaction of a reactive drug metabolite with tissue carrier macromolecules (2-6), it is only in the case ofhepatitis caused by the inhalation of anesthetic halothane that this mechanism has been supported substantially by experimental evidence.Previous studies have demonstrated that the majority of halothane hepatitis patients have unique serum antibodies that react with novel neoantigens in livers of animals (1,7,8) and humans (9) treated with halothane and have suggested that these neoantigens may play an immunopathological role in development of the patients' liver damage. Characterization of these neoantigens by immunoblotting with haptenspecific anti-trifluoroacetyl (TFA) antibodies and sera from several halothane hepatitis patients has revealed that they correspond to distinct liver microsomal protein fractions (100 kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) (10, 11) that have been covalently modified by the reactive TFA halide metabolite of halothane (11).To investigate the role of the halothane-induced neoantigens in the pathogenesis of halothane hepatitis, a general approach for their purification and characterization has been developed and utilized to identify one of them. MATERIALS AND METHODSPurification of 59-kDa-TFA Protein from Hal...

1. A high throughput screening (HTS) method for the evaluation of the seven major human hepatic CYP isoform activities was developed on a 96-well format, with automation. The method utilized pooled human liver microsomes and seven probe substrates, generic conditions for incubation, reaction termination and metabolite extraction with solid phase extraction (SPE) plates. Metabolites from the seven reactions were pooled and quantified using a generic liquid chromatography and tandem mass spectrometry (LCMS/MS) method. 2. The HTS method was validated based on Km values obtained, which were in agreement with literature data. 3. The isoform inhibition profiles of ketoconazole, quinidine, sulfaphenazole, tranylcypromine, alpha-naphthoflavone, and 4-methylpyrazole against CYPs 3A4, 2D6, 2C9, 2A6 land 2C19), 1A2 and 2E1, respectively, were obtained by this HTS method. Graphically obtained IC50 values are in agreement with literature reported values. 4. The HTS method represents a significant efficiency and selectivity improvement over traditional methods, and can be used for CYP inhibition assay and can be extended for liver activity profiling.

Mesenchymal stem cells (MSCs) possess the capacity for site-specific differentiation of cell types in response to cues provided by different organs. This phenomenon suggests that MSCs participate in cutaneous wound regeneration. However, there are no prior reports on the influence of the local application of MSCs on cutaneous wound regeneration. To examine the effects of MSCs on wound regeneration, we cultured bone marrow cells of the femur of rats and treated the plastic adherent cells with a differentiation medium to induce differentiation. After treatment, we found that the bone marrow-derived plastic adherent cells possessed myogenesis, chondrogenesis, and adipogenesis capabilities, indicating that these cells are MSCs. The bone marrow-derived plastic adherent cells were injected intradermally into the skin of rats, and linear full-thickness incisional wounds were made immediately through the injected area. At 14 days after operation, wounds transplanted with bone marrow-derived plastic adherent cells had healed with very fine scars. Collagen architecture was thick and appeared to be similar to normal dermis. Histomorphologic scale analysis demonstrated significant differences between the control and the wounds transplanted with bone marrowderived plastic adherent cells. These results indicate that transplanted MSCs can respond quite normally to wound healing and regenerate dermal structure.

In the light of these findings, we suggest that cilostazol might attenuate the cytokine-induced expression of adhesion molecule genes by inhibiting NF-kappaB following AMPK activation.

Recently, a practical method of speckle reduction in laser rear projection displays that uses an optical system with a small moving diffuser has attracted much attention. In this paper, a model of the speckle generation and reduction mechanism in the system is presented. We investigated the speckle, focusing on the physical aspects of its generation, rather than treating it statistically. We found that the granularity of the speckle patterns generated by the small diffuser corresponded to the size of the coherent regions on the projection screen. This determined the efficiency of the speckle reduction when the small diffuser was rotated.