NCX-1000, a NO-releasing derivative of ursodeoxycholic acid, selectively delivers NO to the liver and protects against development of portal hypertension

Abstract

Portal hypertension resulting from increased intrahepatic resistance is a common complication of chronic liver diseases and a leading cause of death in patients with liver cirrhosis, a scarring process of the liver that includes components of both increased fibrogenesis and wound contraction. A reduced production of nitric oxide (NO) resulting from an impaired enzymatic function of endothelial NO synthase and an increased contraction of hepatic stellate cells (HSCs) have been demonstrated to contribute to high intrahepatic resistance in the cirrhotic liver. 2-(Acetyloxy) benzoic acid 3-(nitrooxymethyl) phenyl ester (NCX-1000) is a chemical entity obtained by adding an NO-releasing moiety to ursodeoxycholic acid (UDCA), a compound that is selectively metabolized by hepatocytes. In this study we have examined the effect of NCX-1000 and UDCA on liver fibrosis and portal hypertension induced by i.p. injection of carbon tetrachloride in rats. Our results demonstrated that although both treatments reduced liver collagen deposition, NCX-1000, but not UDCA, prevented ascite formation and reduced intrahepatic resistance in carbon tetrachloride-treated rats as measured by assessing portal perfusion pressure. In contrast to UDCA, NCX-1000 inhibited HSC contraction and exerted a relaxing effect similar to the NO donor S-nitroso-N-acetylpenicillamine. HSCs were able to metabolize NCX-1000 and release nitrite/nitrate in cell supernatants. In aggregate these data indicate that NCX-1000, releasing NO into the liver microcirculation, may provide a novel therapy for the treatment of patients with portal hypertension

PAR1 antagonism protects against experimental liver fibrosis. Role of proteinase receptors in stellate cell activation

“…2), samples of total RNA were isolated using TRIzol reagent (Life Technologies, Milan, Italy). 28 Reverse-transcription polymerase chain reaction (RT-PCR) was performed using the following sense and antisense primers (Sigma Genosys, Milan, Italy): ␤-actin 5Ј-TAC CAC TGG CAT TGT GAT GG-3Ј and 5Ј-TTAATGTCACGCACGATT TC-3Ј; PAR 1 5Ј-TAGCTGCATCGA CCCCTTGAT T-3Ј and 5Ј-TTG-ACTCCGTTCCCATCACCTT-3Ј; PAR 2 5Ј-GAACCA-GGCTTTTCC GTTGATG-3Ј and 5Ј-ATGGCAGCAC-CGTGATGATG A-3Ј ; PAR 4 5Ј-GAACCAGGCTTT-TCCGTT GAT G-3Ј and 5Ј-ATGGACAGCACCGTG-ATGATG A-3Ј; procollagen-␣ 1 (I), 5Ј-ACAGCACGC-TTG TGGAT-3Ј and 5Ј-GTCTTCAAGCAAGAG-GACCA-3Ј; TGF-␤1 5Ј-CGGACTACTACGCCAAAG-AA-3Ј and 5Ј-TGGTTTTGTCATAGATTGCCGTT-3Ј.…”

“…Quantitative RT-PCR was performed on a BioRad I cycler (BioRad, Hercules, CA). 27,28 Values are expressed as a ratio between threshold cycles (CT). 29,30 The CT for each PAR in each sample was normalized to the CT of ␤-actin and the resulting ratio was compared with a similar ratio observed in control cells.…”

“…Contraction of HSC-T6 on collagen lattices was performed in 24-well flat-bottom tissue culture plates as described previously. 28 Cell contraction was induced by adding 10 U thrombin or 1 to 100 mol/L SF-NH 2 , SL-NH 2 , and GY-NH 2 to the HSC monolayers with or without 0.1 to 100 mol/L anti-PAR 1 . The lattice was detached by gentle circumferential dislodgment with a 200-L micropipette tip and contraction was measured by monitoring the change in lattice area over 24 hours.…”

“…HSC-T6 (4 ϫ 10 5 /mL) was incubated with 100 ng/mL of tumor necrosis factor-␣ with or without PAR 1 antagonist (50 mol/L) for 24 hours and the monocyte chemotactic protein-1 (MCP-1) released in cell supernatants assayed by a specific enzyme-linked immunosorbent assay (Endogen, Woburn, MA). 22,28 ERK-1 and ERK-2. ERK-1 and ERK-2 phosphorylation was assessed in HSCs and HSC-T6 lysates as described previously using a phosphorylated ERK-1 and ERK-2 (phospho-p44/42 MAPK [Thr 202 /Tyr 204 ]; 1:2,000 dilution) monoclonal antibody (Cell Signalling Technologies, Beverly, MA).…”

A gas, an amino acid, and an imposter: The story of nitric oxide, l-arginine, and ADMA in portal hypertension

“…In this regard, over the years it has become clear that increased portal pressure occurs secondary to increased intrahepatic resistance not only from mechanical factors, such as increased collagen deposition and development of regenerative nodules, but also through intrahepatic vasoconstriction, which occurs through signaling defects within the hepatic vascular endothelium, 1 and which is the focus of the study in this issue of HEPATOLOGY by Laleman et al 2 Indeed, a number of novel therapies for portal hypertension are now focused on increased intrahepatic vascular resistance as a target, especially through the nitric oxide (NO) pathway. 3,4 NO is a free radical gas generated by endothelial NO synthase (eNOS), whose signaling functions are a key component of endothelium-dependent vasodilation in liver through its paracrine actions on adjacent contractile cells. [5][6][7][8] Furthermore, deficient NO from hepatic endothelial cells contributes to endothelial dysfunction, which is defined as an impaired endothelium-dependent relaxation and which is present in the liver with cirrhosis.…”