Andrea Mencarelli

The third gas: H2S regulates perfusion pressure in both the isolated and perfused normal rat liver and in cirrhosis

The regulation of sinusoidal resistance is dependent on the contraction of hepatic stellate cells (HSC) around sinusoidal endothelial cell (SEC) through paracrine cross‐talk of vasoconstrictor and vasodilator agents. Hydrogen sulfide (H2S), a recently discovered gas neurotransmitter, is a putative vasodilator whose role in hepatic vascular regulation and portal hypertension is unexplored. Four‐week bile duct–ligated (BDL) rats with cirrhosis and control rats were treated daily with NaHS (56 μmol/kg) for 5 days. Isolated livers were perfused first with NaHS for 20 minutes and then with norepinephrine (NE) and the intrahepatic resistance studied. In normal rats and animals with cirrhosis, administration of NE resulted in a dose‐dependent increase of portal pressure. This effect was attenuated by H2S treatment (P < .05). The H2S‐induced relaxation of hepatic microcirculation was attenuated by glibenclamide, an adenosine triphosphate (ATP)‐sensitive K+ channel inhibitor. L‐Cysteine, a substrate of cystathionine‐gamma‐lyase (CSE), decreased vasoconstriction in normal rat livers (P < .05) but failed to do so in livers with cirrhosis. BDL resulted in a downregulation of CSE mRNA/protein levels and activity (P < .05). Our in vitro data demonstrate that CSE is expressed in hepatocytes, HSCs, but not in sinusoidal endothelial cells (SEC). HSC activation downregulates CSE mRNA expression, resulting in a defective production of H2S and abrogation of relaxation induced by L‐cysteine. In conclusion, CSE‐derived H2S is involved in the maintenance of portal venous pressure. The reduction of CSE expression in the liver with cirrhosis contributes to the development of increased intrahepatic resistance and portal hypertension. (HEPATOLOGY 2005.)

The Bile Acid Receptor FXR Is a Modulator of Intestinal Innate Immunity

The farnesoid X receptor (FXR) is a bile acid-regulated nuclear receptor expressed in enterohepatic tissues. In this study we investigated whether FXR is expressed by cells of innate immunity and regulates inflammation in animal models of colitis. Acute (7 days) and chronic (8 wk) colitis were induced in wild-type and FXR−/− mice by intrarectal administration of trinitrobenzensulfonic acid or by 7-day administration of 5% dextran sulfate in drinking water. The results of this experiment demonstrate that FXR is expressed by and exerts counterregulatory effects on cells of innate immunity. Exposure of LPS-activated macrophages to 6-ethyl chenodeoxycholic acid (6E-CDCA; INT-747) a synthetic FXR ligand, results in a reciprocal regulation of NF-κB dependent-genes (TNF-α, IL-1β, IL-6, COX-1, COX-2, and iNOS) and induction of SHP, a FXR-regulated gene. FXR activation stabilizes the nuclear corepressor NCoR on the NF-κB responsive element on the IL-1β promoter. Colon inflammation in Crohn’s disease patients and in rodent models of colitis is associated with a reduced expression of FXR mRNA. Using two rodent models of colon inflammation, we show that progression of these immune-mediated disorders is exacerbated in FXR−/− mice (p < 0.01). In vivo treatment with INT-747 attenuates organ injury and immune cell activation. FXR activation increased the colon expression of I-BABP, FXR, and SHP while reducing IL-1β, IL-2, IL-6, TNF-α, and IFN-γ mRNA expression and attenuating disease severity. In aggregate, these findings provide evidence that FXR is an essential component of a network of nuclear receptors that regulate intestinal innate immunity and homeostasis.

Genomic variability and alternative splicing generate multiple PML/RAR alpha transcripts that encode aberrant PML proteins and PML/RAR alpha isoforms in acute promyelocytic leukaemia.

The acute promyelocytic leukaemia (APL) 15;17 translocation generates a PML/RAR alpha chimeric gene which is transcribed as a fusion PML/RAR alpha mRNA. Molecular studies on a large series of APLs revealed great heterogeneity of the PML/RAR alpha transcripts due to: (i) variable breaking of chromosome 15 within three PML breakpoint cluster regions (bcr1, bcr2 and bcr3), (ii) alternative splicings of the PML portion and (iii) alternative usage of two RAR alpha polyadenylation sites. Nucleotide sequence analysis predicted two types of proteins: multiple PML/RAR alpha and aberrant PML. The PML/RAR alpha proteins varied among bcr1, 2 and 3 APL cases and within single cases. The fusion proteins contained variable portions of the PML N terminus joined to the B‐F RAR alpha domains; the only PML region retained was the putative DNA binding domain. The aberrant PML proteins lacked the C terminus, which had been replaced by from two to ten amino acid residues from the RAR alpha sequence. Multiple PML/RAR alpha isoforms and aberrant PML proteins were found to coexist in all APLs. These findings indicate that two potential oncogenic proteins are generated by the t(15;17) and suggest that the PML activation pathway is altered in APLs.

FXR activation reverses insulin resistance and lipid abnormalities and protects against liver steatosis in Zucker (fa/fa) obese rats.

The farnesoid X receptor (FXR) is a bile acid activated nuclear receptor. Zucker (fa/fa) rats, harboring a loss of function mutation of the leptin receptor, develop diabetes, insulin resistance, obesity, and liver steatosis. In this study, we investigated the effect of FXR activation by 6-ethyl-chenodeoxycholic acid, (6E-CDCA, 10 mg/kg) on insulin resistance and liver and muscle lipid metabolism in fa/fa rats and compared its activity with rosiglitazone (10 mg/kg) alone or in combination with 6E-CDCA (5 mg/kg each). In comparison to lean (fa/+), fa/fa rats on a normal diet developed insulin resistance and liver steatosis. FXR activation protected against body weight gain and liver and muscle fat deposition and reversed insulin resistance as assessed by insulin responsive substrate-1 phosphorylation on serine 312 in liver and muscles. Activation of FXR reduced liver expression of genes involved in fatty acid synthesis, lipogenesis, and gluconeogenesis. In the muscles, FXR treatment reduced free fatty acid synthesis. Rosiglitazone reduced blood insulin, glucose, triglyceride, free fatty acid, and cholesterol plasma levels but promoted body weight gain (20%) and liver fat deposition. FXR activation reduced high density lipoprotein plasma levels. In summary, FXR administration reversed insulin resistance and correct lipid metabolism abnormalities in an obesity animal model.

Galectin-1 suppresses experimental colitis in mice

BACKGROUND & AIMS Uncontrolled T-cell activation plays a critical role in the pathogenesis of inflammatory bowel diseases. Therefore, pharmacologic strategies directed to restore the normal responsiveness of the immune system by deleting inappropriately activated T cells could be efficacious in the treatment of these pathologic conditions. Galectin-1 is an endogenous lectin expressed in lymphoid organs that plays a role in the maintenance of central and peripheral tolerance. The aim of the present study was to evaluate the therapeutic effects of galectin-1 on T-helper cell type 1-mediated experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. METHODS Cells and tissues from mice with TNBS colitis receiving treatment with several doses of human recombinant galectin-1 (hrGAL-1) were analyzed for morphology, cytokine production, and apoptosis. RESULTS Prophylactic and therapeutic administration of rhGAL-1 resulted in a striking improvement in the clinical and histopathologic aspects of the disease. hrGAL-1 reduced the number of hapten-activated spleen T cells, decreased inflammatory cytokine production, and profoundly reduced the ability of lamina propria T cells to produce IFN gamma in vitro. Moreover, hrGAL-1 led to the appearance of apoptotic mononuclear cells in colon tissue when administered in vivo and induced selective apoptosis of TNBS-activated lamina propria T cells in vitro. CONCLUSION Collectively, these data show that hrGAL-1 exerts protective and immunomodulatory activity in TNBS-induced colitis and it might be effective in the treatment of inflammatory bowel diseases.

Bile-acid-activated receptors: targeting TGR5 and farnesoid-X-receptor in lipid and glucose disorders.

Bile acids are a family of steroid molecules generated in the liver by cholesterol oxidation. In addition to their role in nutrient absorption, bile acids are signaling molecules that exert genomic and non-genomic effects by activating TGR5 (M-BAR, GP-BAR1 or BG37) a G-protein-coupled receptor, and farnesoid X receptor (FXR), a member of the nuclear hormone receptor superfamily. Ligands for these receptors might be beneficial in treating disorders of lipid and glucose homeostasis. TGR5 ligands decrease blood glucose levels and increase energy expenditure by promoting intracellular thyroid hormone activation in thermogenically competent tissues. FXR agonists repress the synthesis of endogenous bile acids and reduce triglyceride, cholesterol and glucose plasma levels and are currently being tested in nonalcoholic steatohepatitis. FXR modulators are being developed to target selective gene clusters and avoid the negative impact of FXR on HDL biosynthesis. The development of dual FXR and TGR5 ligands could provide new opportunities for the treatment of lipid and glucose disorders.

Proteinase-activated receptor 2 is an anti-inflammatory signal for colonic lamina propria lymphocytes in a mouse model of colitis.

The proteinase-activated receptor 2 (PAR-2) is a member of a family of G protein-coupled receptors for proteases. Proteases cleave PARs within the extracellular N-terminal domains to expose tethered ligands that bind to and activate the cleaved receptors. PAR-2 is highly expressed in colon in epithelial and neuronal elements. In this study we show that PAR-2 activation prevents the development and induces healing of T helper cell type 1-mediated experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. A role for PAR-2 in the protection against colon inflammation was explored by the use of SLIGRL-NH2, a synthetic peptide that corresponds to the mouse tethered ligand exposed after PAR-2 cleavage. TNBS-induced colitis was dose-dependently reduced by the administration of SLIGRL-NH2, whereas the scramble control peptide, LSIGRL-NH2, was uneffective. This beneficial effect was reflected by increased survival rates, improvement of macroscopic and histologic scores, decrease in mucosal content of T helper cell type 1 cytokines, protein, and mRNA, and a diminished myeloperoxidase activity. SLIGRL-NH2, but not the scramble peptide, directly inhibited IFN-γ secretion and CD44 expression on lamina propria T lymphocytes. Protection exerted by PAR-2 in TNBS-treated mice was reverted by injecting mice with a truncated form of calcitonin gene-related peptide and by sensory neurons ablation with the neurotoxin capsaicin. Collectively, these studies show that PAR-2 is an anti-inflammatory receptor in the colon and suggest that PAR-2 ligands might be effective in the treatment of inflammatory bowel diseases.

Enhanced activity of a hydrogen sulphide-releasing derivative of mesalamine (ATB-429) in a mouse model of colitis.

Mesalamine is the first‐line therapy for colitis, but it lacks potency and is only effective for mild‐to‐moderate forms of this disease. Hydrogen sulphide has been shown to be a potent, endogenous anti‐inflammatory substance, modulating leukocyte‐endothelial adhesion and leukocyte migration. The purpose of this study was to determine if an H2S‐releasing derivative of mesalamine (ATB‐429) would exhibit increased potency and effectiveness in a mouse model of colitis.

5-Amino-2-hydroxybenzoic Acid 4-(5-Thioxo-5H-[1,2]dithiol-3yl)-phenyl Ester (ATB-429), a Hydrogen Sulfide-Releasing Derivative of Mesalamine, Exerts Antinociceptive Effects in a Model of Postinflammatory Hypersensitivity

H2S functions as a neuromodulator and exerts anti-inflammatory activities. Recent data indicate that irritable bowel syndrome (IBS) is linked to inflammation of the gastrointestinal tract. In this study, we have investigated the role of a novel H2S-releasing derivative of mesalamine (5-amino-2-hydroxybenzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3yl)-phenyl ester, ATB-429) in modulating nociception to colorectal distension (CRD), a model that mimics some features of IBS, in healthy and postcolitic rats. Four graded (0.4-1.6 ml of water) CRDs were produced in conscious rats, and colorectal sensitivity and pain were assessed by measuring the abdominal withdrawal response and spinal c-Fos expression. In healthy rats, ATB-429 dose dependently (25, 50, or 100 mg/kg) attenuated CRD-induced hypersensitivity and significantly inhibited CRD-induced overexpression of spinal c-FOS mRNA, whereas mesalamine had no effect. ATB-429-induced antinociception was reversed by glibenclamide, a ATP-sensitive K+ (KATP) channel inhibitor. The antinociceptive effect of ATB-429 was maintained in a rodent model of postinflammatory hypersensitivity (4 weeks after colitis induction). At a dose of 100 mg/kg, ATB-429 reversed the allodynic response caused by CRD in postcolitic rats. Colonic cyclooxygenase-2 and interkeukin-1β mRNA and spinal c-FOS mRNA expression were significantly down-regulated by ATB-429, but not by mesalamine. ATB-429, but not mesalamine, increased blood concentrations of H2S in both healthy and postcolitic rats. Taken together, these data suggest that ATB-429 inhibits hypersensitivity induced by CRD in both healthy and postcolitic, allodynic rats by a KATP channel-mediated mechanism. This study provides evidence that H2S-releasing drugs might have beneficial effects in the treatment of painful intestinal disorders.

The bile acid receptor GPBAR-1 (TGR5) modulates integrity of intestinal barrier and immune response to experimental colitis

Background GP-BAR1, a member G protein coupled receptor superfamily, is a cell surface bile acid-activated receptor highly expressed in the ileum and colon. In monocytes, ligation of GP-BAR1 by secondary bile acids results in a cAMP-dependent attenuation of cytokine generation. Aims To investigate the role GP-BAR1 in regulating intestinal homeostasis and inflammation-driven immune dysfunction in rodent models of colitis. Methods Colitis was induced in wild type and GP-BAR1−/− mice by DSS and TNBS administration. Potential GP-BAR1 agonists were identified by in silico screening and computational docking studies. Results GP-BAR1−/− mice develop an abnormal morphology of colonic mucous cells and an altered molecular architecture of epithelial tight junctions with increased expression and abnormal subcellular distribution of zonulin 1 resulting in increased intestinal permeability and susceptibility to develop severe colitis in response to DSS at early stage of life. By in silico screening and docking studies we identified ciprofloxacin as a GP-BAR1 ligand. In monocytes, ciprofloxacin increases cAMP concentrations and attenuates TNFα release induced by TLR4 ligation in a GP-BAR1 dependent manner. Treating mice rendered colitic by TNBS with ciprofloxacin and oleanolic acid, a well characterized GP-BAR1 ligand, abrogates signs and symptoms of colitis. Colonic expression of GP-BAR1 mRNA increases in rodent models of colitis and tissues from Crohns disease patients. Flow cytometry analysis demonstrates that ≈90% of CD14+ cells isolated from the lamina propria of TNBS-treated mice stained positively for GP-BAR1. Conclusions GP-BAR1 regulates intestinal barrier structure. Its expression increases in rodent models of colitis and Crohns disease. Ciprofloxacin is a GP-BAR1 ligand.