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Emricasan, a pan-caspase inhibitor, improves survival and portal hypertension in a murine model of common bile-duct ligation.

ABSTRACT: Development of portal hypertension (PHT) is a central prognostic factor in patients with cirrhosis. Circulating microparticles (MPs) are released by hepatocytes in a caspase-dependent manner, are increased in circulation of patients with cirrhosis, and contribute to PHT via induction of impaired vasoconstrictor responses. Here, we tested the hypothesis that emricasan, a pan-caspase inhibitor, ameliorates PHT and reduction in release of MPs. We used a short-term and long-term protocol following common bile-duct ligation (BDL) in C57BL/6 mice (10 and 20 days, respectively). Mice were treated daily via intraperitoneal injection with 10 mg/kg/day of emricasan or placebo. Circulating MP levels were analyzed using flow cytometry and function via ex vivo angiogenesis assays. In contrast to BDL-placebo group, nearly all BDL-emricasan-treated mice survived after long-term BDL. Assessment of portal pressure showed a significant increase in BDL-placebo mice compared to sham-placebo mice. In contrast, BDL-emricasan mice had significantly lower levels of portal pressure compared to BDL-placebo mice. Although emricasan treatment resulted in a decrease in fibrosis, the changes did not reach statistical significance, suggesting that the effects on PHT are at least in part independent of the anti-fibrotic effects of the drug. Following short-term BDL, hepatocellular cell death as well as liver fibrosis had improved and circulating MPs were significantly reduced in BDL-emricasan mice compared to BDL-placebo. Circulating MPs from BDL-placebo mice induced endothelial cell activation, and this was significantly reduced in MPs from BDL-emricasan mice. Our results indicate that emricasan treatment improves survival and PHT in a murine model of long-term BDL. Emricasan is a promising agent for the treatment of PHT. KEY MESSAGE:Emricasan, a pan-caspase inhibitor, improves survival and portal hypertension induced by long-term bile-duct ligation (BDL) in mice Emricasan reduces liver damage, hepatocyte death, and fibrosis, following short-term BDL in mice, and these changes are associated with a decrease in circulating microparticle (MPs) Circulating MPs from BDL-placebo but not from BDL-emiricasan-treated mice activate endothelial cells ex vivo.

SUBMITTER: Eguchi A

PROVIDER: S-EPMC6347405 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Emricasan, a pan-caspase inhibitor, improves survival and portal hypertension in a murine model of common bile-duct ligation.

Eguchi Akiko A Koyama Yukinori Y Wree Alexander A Johnson Casey D CD Nakamura Ryota R Povero Davide D Kneiber David D Tameda Masahiko M Contreras Patricia P Spada Al A Feldstein Ariel E AE

Journal of molecular medicine (Berlin, Germany) 20180505 6

Development of portal hypertension (PHT) is a central prognostic factor in patients with cirrhosis. Circulating microparticles (MPs) are released by hepatocytes in a caspase-dependent manner, are increased in circulation of patients with cirrhosis, and contribute to PHT via induction of impaired vasoconstrictor responses. Here, we tested the hypothesis that emricasan, a pan-caspase inhibitor, ameliorates PHT and reduction in release of MPs. We used a short-term and long-term protocol following c ...[more]

PMID: 29728708

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Project description:Male Sprague-Dawley rats weighing 250-300 g were purchased from Japan SLC Co. (Shizuoka, Japan). The animals were housed under a daily controlled 12-h light and 12-h dark cycle at 23 °C with free access to rat chow (Japan SLC Co.) and water for 1 week prior to the experiments. Rats were laparotomized under ether anesthesia in each experiment, and the common bile duct of each rat was ligated between the liver hilus and small intestine. Control animals underwent a sham operation with exposure but without ligation of the common bile duct. After the CBDL or sham operation, animals were allowed access to food and water ad libitum. Groups of three rats were sacrificed under ether anesthesia at 1, 3 and 12 hr after the CBDL or sham operation. At the time of sacrifice, the right lateral liver lobe was removed and flash-frozen in liquid nitrogen and stored at -80 °C. Total RNA isolated from frozen livers using TRIzol reagent (Invitrogen Co., Carlsbad, CA) was mixed to minimize variation among animals. Poly(A) RNA was purified using Oligotex-dT30 (Takara Shuzo Co., Ltd., Kyoto, Japan) in accordance with manufacturer’s instructions. Fluorescence-labeled probes were prepared by reverse transcription using a superscript II reverse transcriptase (Invitrogen Co.) and cyanine-3- and cyanine-5-dUTP (Perkin-Elmer Inc., Wellesley, MA). Poly(A) RNAs derived from rats that has undergone CBDL and from those that has undergone a sham operation were labeled with cyanine-3 and cyanine-5, respectively, and vise varsa. Fluorescence-labeled probes were purified using a MinElute PCR Products Purification Kit (Quiagen GmbH, Hilden, Germany) in accordance with manufacturer’s instructions. Each purified probe was suspended in hybridization buffer containing 1.6 mg/mL poly(A) (Roche Diagnostics, Basel, Switzerland) and yeast tRNA (Roche Diagnostics), 0.67 mg/mL herring sperm, 16% 20 x SSC and 0.3% SDS and applied to a cDNA microarray containing 1,800 rat genes on a slide glass (Asahi Technoglass Co., Tokyo, Japan). Three house-keeping genes (GAPDH, HPRT and b-actin) and rat unrelated traits (Lambda DNA) were also spotted on the slide as internal positive and negative controls, respectively. Hybridization was carried out twice to eliminate any dye bias. In one experiment, duplicate slides were hybridized with probes derived from CBDL and control rats that had been labeld with cyanine-3 and cyanine-5, respectively. In a replicate experiment, other duplicate slides were hybridized with probes derived from CBDL rats and control rats that had been labeled with cyanine-5 and cyanine-3, respectively (color swap). Then, the slides were cover-slipped and incubated in a sealed chamber (Asahi Technoglass Co.) for 16 hrs under a humidified (65 °C) condition. After being washed in low-stringency buffer (2 x SSC and 0.1% SDS), high-stringency buffer (0.2 x SSC and 0.1% SDS) and 0.2 x SSC and then rinsing with 99.5% ethanol, the slide was dried by centrifugation at low speed and used for scanning. Fluorescence was scanned by using a ScanArray 4000 (Packard BioChip Technologies, Billerica, MA) and quantified by using QuantArray Software (Packard BioChip Technologies, Billerica, MA). Keywords: time-course

Project description:BackgroundCirculating cirrhotic endothelial progenitor cells (EPC) interact with both liver sinusoidal endothelial cells (LSEC) and hepatic stellate cells (HSC) and promote angiogenesis in vitro. This study evaluated the effect of cirrhotic and control EPCs on hepatic angiogenesis, microcirculation, and fibrosis in vivo in rat models of cirrhosis.MethodologyAnimal models of cirrhosis were prepared by bile duct ligation (BDL). Circulating EPCs isolated from healthy human and cirrhotic blood were characterized by flow cytometry, cultured and administered through the tail vein in BDL rats after 2 weeks of ligation. The cells were given thrice a week for 2 weeks. The untreated group of BDL rats received only saline. Fibrosis was evaluated by Masson's trichrome staining. Dedifferentiated LSECs were identified by the expression of CD31, and activated HSCs were marked as alpha-SMA-positive cells and were studied by immunohistochemistry and western blotting in saline-, healthy EPC-, and cirrhotic EPC-treated rats. In vivo, hepatic and systemic hemodynamic parameters were evaluated. Liver functions were evaluated.ResultsIn comparison to controls, BDL rats revealed an increase of fibrosis and angiogenesis. Among the treated rats, cirrhotic EPC-treated rats had increased fibrosis grade as compared to healthy EPC-treated and saline-treated rats. There was an increase of both fibrosis and angiogenesis markers, alpha-SMA and CD31 in cirrhotic EPC-treated rats as compared to healthy EPC-treated and saline-treated rats in immunohistochemistry and western blot studies. Cirrhotic EPC-treated BDL rats had high portal pressure and portal blood flow with significantly elevated hepatic vascular resistance in comparison with healthy EPC- and saline-treated BDL animals, without significant differences in mean arterial pressure. Cirrhotic EPC-treated BDL rats also showed a substantial increase in the hepatic expression of angiogenic receptors, VEGFR2 and CXCR4 in comparison with saline-treated rats.ConclusionThe study suggests that transplantation of cirrhotic EPCs enhances LSEC differentiation and angiogenesis, activates HSCs and worsens fibrosis, thus resulting in hepatic hemodynamic derangements in BDL-induced cirrhosis.

Project description:Background: In cirrhosis, the nitric oxide-soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway is impaired, which contributes to increased intrahepatic vascular resistance (IHVR) and fibrogenesis. We investigated if sGC stimulation (riociguat (RIO)), sGC activation (cinaciguat (CINA)) or phosphodiesterase (PDE)-5 inhibition (tadalafil (TADA)) improves portal hypertension (PHT) and liver fibrosis.Methods: Fifty male Sprague-Dawley rats underwent bile-duct ligation (BDL) or sham operation. RIO (0.5?mg/kg), CINA (1?mg/kg), TADA (1.5?mg/kg) or vehicle (VEH) was administered from weeks 2 to 4 after BDL. At week 4, invasive haemodynamic measurements were performed, and liver fibrosis was assessed by histology (chromotrope-aniline blue (CAB), Picro-Sirius red (PSR)) and hepatic hydroxyproline content.Results: Cirrhotic bile duct-ligated rats presented with PHT (13.1?±?1.0?mmHg) and increased IHVR (4.9?±?0.5?mmHg?min/mL). Both RIO (10.0?±?0.7?mmHg, p?=?0.021) and TADA (10.3?±?0.9?mmHg, p?=?0.050) decreased portal pressure by reducing IHVR (RIO: -41%, p?=?0.005; TADA: -21%, p?=?0.199) while not impacting heart rate, mean arterial pressure and portosystemic shunting. Hepatic cGMP levels increased upon RIO (+239%, p?=?0.006) and TADA (+32%, p?=?0.073) therapy. In contrast, CINA dosed at 1?mg/kg caused weight loss, arterial hypotension and hyperlactataemia in bile duct-ligated rats. Liver fibrosis area was significantly decreased by RIO (CAB: -48%, p?=?0.011; PSR: -27%, p?=?0.121) and TADA (CAB: -21%, p?=?0.342; PSR: -52%, p?=?0.013) compared to VEH-treated bile duct-ligated rats. Hepatic hydroxyproline content was reduced by RIO (from 503?±?20 to 350?±?30?µg/g, p?=?0.003) and TADA (282?±?50?µg/g, p?=?0.003), in line with a reduction of the hepatic stellate cell activation markers smooth-muscle actin and phosphorylated moesin. Liver transaminases decreased under RIO (AST: -36%; ALT: -32%) and TADA (AST: -24%; ALT: -27%) treatment. Hepatic interleukin 6 gene expression was reduced in the RIO group (-56%, p?=?0.053).Conclusion: In a rodent model of biliary cirrhosis, the sGC stimulator RIO and the PDE-5 inhibitor TADA improved PHT. The decrease of sinusoidal vascular resistance was paralleled by a reduction in liver fibrosis and hepatic inflammation, while systemic haemodynamics were not affected.

Dataset Information

Emricasan, a pan-caspase inhibitor, improves survival and portal hypertension in a murine model of common bile-duct ligation.

Publications

Emricasan, a pan-caspase inhibitor, improves survival and portal hypertension in a murine model of common bile-duct ligation.

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