Pentamidine blocks hepatotoxic injury in mice

ANIMAL MODELS

C57BL/6J male and female mice were maintained under 12-hour light/dark cycles with unlimited access to food and water. Mice were fed a standard chow diet (PicoLab Rodent Diet 20 #5053; LabDiet, St. Louis, MO) at all times except during alcohol diet feeding. Mice were housed in static cages with a quarter-inch corncob bedding, individual water bottles, and cotton nestlets for enrichment. During alcohol diet feeding cages also contained a paper igloo. Littermates were used as vehicle-treated controls for all experiments.

GalN/LPS liver injury was induced in 10- to 14-week old male mice by an intraperitoneal injection of 100 µg/kg of LPS (Escherichia coli 0111:B4) and 700 mg/kg of d-galactosamine (Sigma-Aldrich, St. Louis, MO) dissolved in phosphate-buffered saline (PBS), as previously performed.16 The GalN/LPS mortality studies were performed with an LPS dose of 10 µg/kg. For the pretreatment studies, an equal volume of PBS vehicle or pentamidine isethionate (VLX103; Verlyx Pharma Inc, Montreal, Canada) at a dose of 25, 40, or 50 mg/kg was injected intraperitoneally 30 minutes before GalN/LPS administration. For the posttreatment experiments, PBS or VLX103 at 40 mg/kg was injected at various times after GalN/LPS.

Additional C57BL/6J male mice were injected with GalN plus TNF as described previously.2 Mice were pretreated with PBS or VLX103 40 mg/kg intraperitoneally. At 30 minutes after this pretreatment, the mice were injected with GalN intraperitoneally, and then another 30 minutes later injected intravenously with 10 μg/kg of mouse recombinant TNF (PeproTech, Rocky Hill, NJ).

Studies of alcohol-induced injury employed a modified National Institute on Alcohol Abuse and Alcoholism (NIAAA) alcohol model.17 Female 10-week old mice were administered 5 days of liquid control diet (Bio-Serv, Flemington, NJ; #F1259SP). Mice were then randomized to receive a 5% liquid ethanol diet (Bio-Serv; #F1258SP), or pair-fed an equal caloric amount of the control diet. At this time the mice were started on a daily gavage of normal saline vehicle or VLX103 75 mg/kg dissolved in normal saline. After 10 days of control/ethanol diet feeding, a single gavage of ethanol 5 g/kg, or an equal volume and caloric intake of maltose dextrin, was administered to the mice. Mice were sacrificed 9 hours after gavage. All animal studies were approved by the Animal Care and Use Committees of the Albert Einstein College of Medicine and Emory University School of Medicine and followed the National Institutes of Health guidelines for animal care.

SERUM ASSAYS

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by commercial kits (TECO Diagnostics, Anaheim, CA).

HISTOLOGY

Hematoxylin and eosin-stained livers were evaluated for the amount of hepatic parenchyma with steatosis, apoptosis/necrosis or inflammation by a single pathologist in a blinded fashion on the following semiquantitative scale: 0, absent; 0.5, minimal; 1, mild; 1.5, mild to moderate; 2, moderate; 2.5, moderate to severe; 3, severe; and 4, markedly severe.

TERMINAL DEOXYNUCLEOTIDE TRANSFERASE–MEDIATED DEOXYURIDINE TRIPHOSPHATE NICK END-LABELING ASSAY

Terminal deoxynucleotide transferase–mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive cells in liver sections were stained with the commercial kit DeadEnd Colorimetric System (Promega, Madison, WI). Under light microscopy, the numbers of TUNEL positive cells in 10 randomly selected high-power fields (magnification × 200) were counted per liver section.

QUANTITATIVE REAL-TIME REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION

Total RNA was extracted from livers with the commercial kit RNeasy Plus (QIAGEN, Valencia, CA). Reverse transcription was performed with 1 µg of RNA in an Eppendorf Mastercycler (Hamburg, Germany) using a high-capacity complementary DNA reverse transcription kit (ABI, Foster City, CA). Annealing of primers was performed at 25 °C for 10 minutes, followed by elongation at 37 °C for 2 hours and inactivation of the enzyme at 85 °C for 5 minutes. Negative controls (no added transcriptase) were performed in parallel. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in duplicate in a 7500 Fast Real-Time PCR System (ABI). The primer sequences in Supporting Table S1 were purchased from Integrated DNA Technologies (Coralville, IA). PCR was performed using Power SYBR Green Master Mix (ABI). Taq polymerase was activated at 95 °C for 10 minutes. The cycling parameters were denaturation at 95 °C for 30 seconds and extension at 60 °C for 1 minutes (for 40 cycles). Data analysis was performed using the 2^−ΔΔCT method for relative quantification. Samples were normalized to glyceraldehyde 3-phosphate dehydrogenase.

CYTOKINE ASSAY

Cytokine levels were measured in serum samples with a multiplex kit (MCYTOMAG-70K; EMD Millipore, Billerica, MA) and Luminex xMAP Technology.

MACROPHAGE ISOLATION AND TREATMENTS

Bone marrow cells were isolated from untreated C57BL/6J mice and differentiated into bone marrow–derived macrophages (BMDMs) as described previously.18 BMDMs were pretreated with VLX103 at various concentrations for 1 hour, administered LPS (10 ng/mL) and IFNγ (100 ng/mL) (R&D Systems, Minneapolis, MN), and harvested at 12 hours for qRT-PCR.

Hepatic macrophages were isolated by Nycodenz gradient from untreated C57BL/6J mice and cultured as described previously.19 Following overnight culture, the cells were pretreated with 10 μM VLX103 and 1 hour later treated with LPS and IFNγ for harvest at 6 hours for qRT-PCR analysis.

PROTEIN ISOLATION AND WESTERN BLOTTING

Total protein and mitochondrial and cytosolic protein fractions were isolated from mouse liver and western blotting was performed as described previously.20, 21 Membranes were probed with the following antibodies: caspase 3 (#9665; Cell Signaling, Beverly, MA), caspase 7 (#9492; Cell Signaling), poly (ADP-ribose) polymerase (PARP) (#9542; Cell Signaling), tubulin (#2148; Cell Signaling), cytochrome c (#556433; BD Biosciences, San Jose, CA), cytochrome oxidase (#14744; Abcam, Cambridge, MA), caspase 8 (#AF1650; R&D Systems), p50 nuclear factor kappa B (NF-κB) (#SC-114; Santa Cruz Biotechnology, Dallas, TX), p65 NF-κB (#SC-109; Santa Cruz Biotechnology), lamin A/C (#2032; Cell Signaling), cIAP1 (#ALX-803-335-C100; Enzo Life Sciences, Farmingdale, NY), cIAP2 (#SC-7944; Santa Cruz Biotechnology), superoxide dismutase 2 (SOD2) (#ADI-SOD-111-D; Enzo Life Sciences), Bcl-X_L (#2764; Cell Signaling), c-Jun N-terminal kinase (JNK) (#9252; Cell Signaling), phosphorylated JNK (#4671; Cell Signaling), and Bid (a gift from Xiao-Ming Yin, University of Indiana).

CASPASE 8 ACTIVITY

Caspase 8 activity was detected using a commercial fluorometric kit (Biovision, Milpitis, CA). Reactions were performed on 200 μg protein at 37 °C for 2 hours, and the samples were read in a fluorometer with 400-nm excitation and 505-nm emission filters.

TRIGLYCERIDE CONTENT

Hepatic triglyceride content was determined using a kit according to the manufacturer's instructions (Sigma) and values normalized to wet liver weight.

STATISTICAL ANALYSIS

Numerical results are reported as the mean ± standard error and were derived from at least three independent experiments unless indicated otherwise. An unpaired Student t test was used to assess the significance between control and treated groups. Survival rates were compared by the Tukey multiple comparison test. Statistical significance was defined as P < 0.05.

VLX103 PREVENTS LIVER INJURY FROM GalN/LPS

To examine the possible efficacy of VLX103 treatment in hepatotoxic liver injury, we determined whether pretreatment altered acute liver injury from GalN/LPS. C57BL/6J mice were injected with PBS vehicle or 25 or 40 mg/kg of VLX103 30 minutes prior to GalN/LPS. In C57BL/6J mice, GalN/LPS-induced liver injury was detectable at 4 hours and became severe by 6 hours, at which time lethality ensued. VLX103 pretreatment remarkably improved gross liver appearance after GalN/LPS with an absence of the typical signs of hemorrhage (Fig. 1A). Liver injury as determined by serum ALT levels was markedly decreased by both VLX103 concentrations (Fig. 1B). Blinded, histological grading confirmed that VLX103 significantly reduced liver injury at 6 hours. (Fig. 1C,D). Hepatocyte death was decreased as determined by significantly reduced numbers of TUNEL-positive cells in VLX103-treated livers (Fig. 2A,B). VLX103 pretreatment therefore dramatically reduced liver injury from GalN/LPS.

VLX103 DECREASES GalN/LPS-INDUCED MORTALITY

GalN/LPS triggers fulminant hepatic failure and death. To determine whether VLX103 merely delays liver injury or has a sustained protective effect that prevents mortality, survival was examined. PBS-pretreated mice all died within 12 hours of GalN/LPS, whereas VLX103-treated mice had a dose-dependent improvement in survival (Fig. 2C). Mice pretreated with 25 mg/kg had a prolonged survival time but succumbed to death within 18 hours. Mice receiving the 40 or 50 mg/kg doses had a significantly improved survival, with 50% surviving with the highest dose (Fig. 2C). Subsequent GalN/LPS studies were largely performed with the 40 mg/kg dose, which significantly blocked both liver injury and improved survival.

VLX103 REDUCES TNF INDUCTION

GalN/LPS liver injury is mediated by hepatocyte sensitization to cytotoxicity from LPS-induced macrophage cytokines, in particular TNF.14, 22 To determine whether VLX103 was protective through TNF down-regulation, hepatic cytokine mRNA induction was assayed by qRT-PCR. Tnf mRNA levels after GalN/LPS treatment were decreased significantly by 47% at 1 hour, but unchanged at 2 and 4 hours in VLX103-treated mice (Fig. 3A). VLX103 had no significant effect on Ifnγ, Il-1β, or Il-6 mRNA levels (Fig. 3A). Consistent with the mRNA findings, serum TNF levels were decreased significantly by 54% at 1 hour but unaffected at later times, and other cytokines were unchanged by VLX103 (Fig. 3B). Decreased liver injury from VLX103 was therefore associated with an early decrease in TNF but not other proinflammatory cytokines.

VLX103 DECREASES HEPATIC MACROPHAGE ACTIVATION AND TNF-INDUCED LIVER INJURY

The finding of decreased TNF production, together with reports that pentamidine is anti-inflammatory in alveolar macrophages,9, 10 suggested that VLX103 may directly suppress injurious proinflammatory hepatic macrophage activation. Cultured BMDMs were pretreated with increasing VLX103 concentrations and stimulated with LPS/IFNγ for polarization into a proinflammatory M1 phenotype.18 VLX103 failed to inhibit induction of the cytokine genes Tnfα, Il-1β, and Il-6 or M1-marker gene Nos2 (Fig. 3C). In hepatic macrophages, however, the drug did inhibit proinflammatory activation as indicated by significant reductions in these same genes (Fig. 3D).

To determine whether TNF suppression is a mechanism of VLX103 hepatoprotection, drug effect was examined in GalN-sensitized mice cotreated with TNF. VLX103 pretreatment partially but significantly reduced liver injury from GalN/TNF as determined by decreased ALT levels and TUNEL staining (Fig. 4A,B). There was a trend to improved histology that did not reach statistical significance (Fig. 4C). Although significant, the reduction in liver injury was markedly less than in the GalN/LPS model (Figs. 1 and 2). Together these in vitro and in vivo findings demonstrate that a partial mechanism of VLX103's hepatoprotective effect is through inhibition of macrophage generation of TNF.

VLX103 BLOCKS ACTIVATION OF THE MITOCHONDRIAL DEATH PATHWAY

Hepatic toxicity from GalN/LPS occurs through the classical TNF death cascade of caspase 8-mediated Bid cleavage into tBid, which translocates to mitochondria to trigger cytochrome c release, effector caspase cleavage, and apoptosis.7 Decreased TUNEL positivity suggested that VLX103 blocked TNF-induced apoptosis at some point along this pathway. Supportive of this idea was that VLX103 induced a dose-dependent decrease in the active, cleaved forms of effector caspases 3 and 7 at 6 hours after GalN/LPS (Fig. 5A). Decreased caspase activity was confirmed by parallel findings of a dose-dependent reduction in cleavage of the caspase substrate PARP in VLX103-treated livers (Fig. 5A).

Examination of the upstream mitochondrial pathway that triggers effector caspase cleavage revealed that VLX103 prevented its activation. Cytosolic tBid cleavage was blocked by VLX103 (Fig. 5B), and tBid was virtually absent in mitochondria from VLX103-treated livers in contrast to the high levels in PBS-treated mice (Fig. 5C). As a result, the decrease in mitochondrial levels of cytochrome c (Fig. 5C) and its release into the cytoplasm (Fig. 5B) were blocked in VLX103-treated mice. VLX103 therefore decreased apoptosis from GalN/LPS by mitigating mitochondrial death pathway and effector caspase activation.

VLX103 INHIBITS CASPASE 8 ACTIVATION

The marked decrease in mitochondrial death pathway activation suggested that VLX103 blocked hepatocyte death from GalN/LPS through inhibition of upstream caspase 8. Increased hepatic caspase 8 activity was detectable in PBS-treated mice at 2-6 hours after GalN/LPS treatment, but activity was decreased significantly at 4 and 6 hours by VLX103 (Fig. 6A). Immunoblots confirmed that cleavage of caspase 8 to its active p18 form was decreased by VLX103 (Fig. 6B). VLX103 therefore blocked caspase 8 activation, preventing Bid cleavage and mitochondrial death pathway activation that induce liver injury from GalN/LPS.

TNF induction of hepatocyte caspase 8 activation is down-regulated by NF-κB, which curtails JNK phosphorylation.21, 23 Examination of VLX103's effect on NF-κB signaling revealed that hepatic NF-κB activation occurred in both vehicle- and VLX103-treated mice as indicated by nuclear translocation of the phosphorylated p50 and p65 NF-κB subunits (Fig. 6C). Levels of proteins that mediate hepatocyte resistance to TNF-dependent apoptosis such as cIAP1, cIAP2, SOD2, and Bcl-X_L7 were unaffected by VLX103 (Fig. 6D). Despite the absence of an effect on NF-κB activation, VLX103 did markedly reduce levels of JNK activation, as indicated by decreased levels of phosphorylated JNK (Fig. 6E).

VLX103 TREATMENT IS HEPATOPROTECTIVE POST-GALN/LPS ADMINISTRATION

For human liver disease, a therapeutic agent must be effective when administered after the onset of injury. We therefore determined whether VLX103 was hepatoprotective when administered after the initiation of GalN/LPS-induced injury. Although the greatest reduction in hepatic injury was afforded by VLX103 pretreatment, administration of this agent even at 2 and 3 hours after GalN/LPS significantly decreased liver injury. Serum ALT levels were reduced 75% by VLX103 given 2-3 hours after GalN/LPS (Fig. 7A). TUNEL staining revealed a diminished but still significant decrease in hepatocyte death with VLX103 posttreatment (Fig. 7B). VLX103 treatment 3 hours post-GalN/LPS was highly effective in reducing cytosolic (Fig. 7C) and mitochondrial levels of tBid, as well as mitochondrial cytochrome c loss (Fig. 7D) and release into the cytosol (Fig. 7C). As a result, VLX103 posttreatment was highly effective in decreasing caspase 3 and 7 and PARP cleavage (Fig. 7E). These findings indicate that VLX103 is therapeutic when administered significantly after GalN/LPS, even immediately before the onset of liver injury.

VLX103 PROTECTS AGAINST ALCOHOLIC LIVER INJURY

The therapeutic potential of oral VLX103 was examined in a more chronic and nonlethal modified NIAAA model of alcoholic liver disease.17 Mice received a daily gavage of vehicle or VLX103 (75 mg/kg) during 10 days of ethanol feeding, and then a binge dose of alcohol by gavage for analysis 9 hours later. VLX103 decreased hepatic triglyceride content by 34% (Fig. 8A). In vehicle-treated mice, serum ALT and AST levels increased 3.3- and 2.5-fold, respectively, but levels were significantly decreased 50% by VLX103 (Fig. 8B,C). Histologically, there was a nonsignificant trend to decreased steatosis, but differences were not detected in injury or inflammation, as the levels of both histological features were low with alcohol feeding (Fig. 8D). A reduction in liver injury was confirmed by findings of a significant decrease in TUNEL-positive cells with VLX103 (Fig. 8E) in association with decreased proinflammatory cytokine gene expression (Fig. 8F). Oral VLX103 was therefore effective in decreasing hepatotoxic liver injury from alcohol.