Fig 1.
Pharmacological characterization of BAR501.
(A) Effect of BAR501 on GPBAR1 transactivation. BAR501 causes a concentration-dependent transactivation of GPBAR1 in HEK293 T cells transfected with a CREB responsive element. Data are mean ± SE of 3 experiments. (B) BAR501 fails to promote FXR transactivation. (C) Effect of BAR501 on mRNA expression of pro-glucagon in GLUTAg cells. Data are mean ± SE of 3 experiments. *p<0.05 versus not treated cells. (D) Effect of BAR501 on cAMP production in GLUTAg cells. Results are the mean ± SE of 3 experiments. *p<0.05 versus not treated cells (NT). F is forskolin. (E) Effect of BAR501 on vasomotor activity of nor-epinephrine (NE) on rat liver. Administering rats with 15 mg/kg/day BAR501 for 6 days attenuated the vasomotor response to NE at any concentration tested. (F) Effect of BAR501 on liver perfusion pressure caused by shear stress in rats. Data are mean ± SE of 4–6 animals per group. *p<0.05 versus shear stress alone (G) BAR501 attenuates vasomotor response to methoxamine in rats. Data are mean ± SE of 7 livers. *p < 0.05 versus methoxamine alone.
Fig 2.
Administration of BAR501 exerts a direct vasodilatory activity in the CCl4 model.
Effect of BAR501 on (A) portal pressure, (B) AST, (C) alkaline phosphatase (ALP), (D) Albumin, (E) Bilirubin in mice renedered cirrhotic by administration with CCl4. (F-H) Hematoxylin and eosin (H&E) staining. (I-M) Syrius red staining. (N) Image J quantification of Syrius red staining. Data are mean ± SE of 8–10 animals per group. *p<0.05 versus naïve mice. #p<0.05 versus CCl4.
Fig 3.
BAR501 regulates the expression of CSE and ET-1 in the CCl4 model of liver cirrhosis.
C57BL6 mice were treated for 9 weeks with CCl4 or with the combination of CCl4 plus BAR501 15 mg/kg. The relative hepatic mRNA expression of TGFβ1, COL1α1, αSMA, TNFα, IL1β, GP-BAR1 (panel A) and CBS, CSE, eNOS, iNOS, CAV-1 ET-1 (panel B) was assayed by Real-Time PCR. (C) Effect of BAR501 on CSE activity in the CCl4 model. (D) Effect of BAR501 on nitrite/nitrate in CCl4 treated mice. Results are the mean ± SE of 4–8 mice per group. *p<0.05 versus naive mice. #p<0.05 versus CCl4 alone.
Fig 4.
BAR501 correct for endtothelial dysfunction caused by methionine diet.
Gpbar1+/+ and Gpbar1-/- mice administered L-methionine for 4 weeks were randomized to receive BAR501 (20 mg/Kg daily by gavage) or vehicle (distilled water). Effect of BAR501 on (A) portal pressure, (B) ET-1 mRNA, (C) CBS mRNA, (D) CSE mRNA, (E) eNOS mRNA, (F) iNOS mRNA and (G) CAV-1. Results are the mean ± SE of 4–8 mice per group. *p<0.05 versus pbar1+/+ mice. #p<0.05 versus Gpbar1 +/+ mice fed methionine. °p<0.05 versus Gpbar1-/- mice. °°p<0.05 versus Gpbar1-/- mice fed methionine.
Fig 5.
GPBAR1 activation by BAR501 modulates the expression/activity of regulates CSE in human LSEC.
(A) Serum starved LSEC were exposed to 10 μM TLCA, OA, BA, UDCA or BAR501 for 18 hr. CSE mRNA expression was evaluated using Real-Time PCR. (B) Serum starved LSEC were exposed to 10 μM TLCA or BAR501 for 18 hr. Relative mRNA expression of CSE, CBS, eNOS and TGR5 was assayed by Real-Time PCR. Protein expression of CSE, GPBAR1 and tubulin was measured by Western blotting. (C) LSEC were transiently transfected with pCMVSPORT-hTGR5 and pGL4(CRE1)5X as described in Materials and Methods. Forty-height h post-transfection cells were stimulated 18 h with BAR501 (10 μM). (D) ChIP assay carried out in LSEC left untreated or primed with BAR501 as described in Materials and Methods. RT-PCR was performed with specific primers flanking the responsive element CRE1 on human CSE promoter. (E) CSE activity in LSEC administered 10 μM TLCA or BAR501 for 24 and 48 h. (F) Effect of BAR501 on phosphorylation of CSE on serine residues. Serine phosphorylation of CSE was assessed by immunoprecipitation of CSE followed by Western blot determination of phosphoserine and phospho-Akt1 content in SEC exposed to BAR501 (10 μM) for 0, 5, 15, 30, and 60 min. (G) Exposure of LSEC to BAR501 increases eNOS phosphorylation. All analyses were carried out in triplicate and the experiments were repeated twice. *p<0.05 versus not treated cells.
Fig 6.
GPBAR1 activation down-regulates ET-1 expression in human LSEC by displacing FOXO1 transcriptional complex from the ET-1 promoter.
(A) Serum starved LSEC were stimulated 18 h with 10 μM TLCA or BAR501. At the end of stimulation CSE mRNA expression was evaluated using Real-Time PCR method. (B) representative Western blot analysis of Akt1, phospho-Akt1, FOXO1 and phospho-FOXO1 proteins in SEC exposed to BAR501 (10 μM) for 0, 5, 15, 30, and 60 min. (C) ChIP assay carried out in SEC left untreated or primed with BAR501 as described in materials and methods. RT-PCR was performed with specific primers flanking the FOXO1 responsive element on human ET-1 promoter. Inset of Fig 6C. Representative qualitative PCR of ET-1 promoter immunoprecipitated with an anti-FOXO1 antibody on SEC left untreated or primed with BAR501 (D) Effect of PI3K inhibitor LY-294,002 (LY) on ET-1 mRNA expression in SEC coadministered with BAR501. *p< 0.05 versus not treated (NT) cells; #p<0.05 versus BAR501 treated cells.
Fig 7.
Schematic representation of the effect of BAR501 on LSEC in models of LSEC dysfunction.
CCl4 and methionine feeding alter liver sinusoidal cell function. In these settings LSEC express high levels of endothelin 1 (ET-1) along with reduces eNOS activity due to enhanced binding of eNOS with caveolin-1, and reduced expression of CSE, a H2S-generating enzyme. Activation of GPBAR1 by BAR501, increases CSE expression by CRE-mediated activity, and causes both eNOS and CSE phosphorylation by AKT-mediated mechanism. In addition, AKT-driven phosphorylation of FOXOA1, attenuates ET-1 production. However since eNOS is bound to caveolin 1 the generation of nitrite/nitrate do not increase. Thus, activation of GPBAR1 in CCl4-treated mice, leads to eNOS-independent reduction of intravascular resistance, that is mostly mediated by inhibition of ET-1 and increased release of H2S.