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• W2988505923 abstract "Severe hepatic insults can lead to acute liver failure and hepatic encephalopathy (HE). Transforming growth factor β1 (TGFβ1) has been shown to contribute to HE during acute liver failure; however, TGFβ1 must be activated to bind its receptor and generate downstream effects. One protein that can activate TGFβ1 is thrombospondin-1 (TSP-1). Therefore, the aim of this study was to assess TSP-1 during acute liver failure and HE pathogenesis. C57Bl/6 or TSP-1 knockout (TSP-1−/−) mice were injected with azoxymethane (AOM) to induce acute liver failure and HE. Liver damage, neurologic decline, and molecular analyses of TSP-1 and TGFβ1 signaling were performed. AOM-treated mice had increased TSP-1 and TGFβ1 mRNA and protein expression in the liver. TSP-1−/− mice administered AOM had reduced liver injury as assessed by histology and serum transaminase levels compared with C57Bl/6 AOM-treated mice. TSP-1−/− mice treated with AOM had reduced TGFβ1 signaling that was associated with less hepatic cell death as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and cleaved caspase 3 expression. TSP-1−/− AOM-treated mice had a reduced rate of neurologic decline, less cerebral edema, and a decrease in microglia activation in comparison with C57Bl/6 mice treated with AOM. Taken together, TSP-1 is an activator of TGFβ1 signaling during AOM-induced acute liver failure and contributes to both liver pathology and HE progression. Severe hepatic insults can lead to acute liver failure and hepatic encephalopathy (HE). Transforming growth factor β1 (TGFβ1) has been shown to contribute to HE during acute liver failure; however, TGFβ1 must be activated to bind its receptor and generate downstream effects. One protein that can activate TGFβ1 is thrombospondin-1 (TSP-1). Therefore, the aim of this study was to assess TSP-1 during acute liver failure and HE pathogenesis. C57Bl/6 or TSP-1 knockout (TSP-1−/−) mice were injected with azoxymethane (AOM) to induce acute liver failure and HE. Liver damage, neurologic decline, and molecular analyses of TSP-1 and TGFβ1 signaling were performed. AOM-treated mice had increased TSP-1 and TGFβ1 mRNA and protein expression in the liver. TSP-1−/− mice administered AOM had reduced liver injury as assessed by histology and serum transaminase levels compared with C57Bl/6 AOM-treated mice. TSP-1−/− mice treated with AOM had reduced TGFβ1 signaling that was associated with less hepatic cell death as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and cleaved caspase 3 expression. TSP-1−/− AOM-treated mice had a reduced rate of neurologic decline, less cerebral edema, and a decrease in microglia activation in comparison with C57Bl/6 mice treated with AOM. Taken together, TSP-1 is an activator of TGFβ1 signaling during AOM-induced acute liver failure and contributes to both liver pathology and HE progression. Acute liver failure results from a significant loss of liver function caused by hepatocyte necrosis in response to hepatotoxin ingestion, viral hepatitis, autoimmune disease, metabolic disease, ischemia, and various other causes. Patients with acute liver failure often present with deleterious systemic complications, including neurologic dysfunction called hepatic encephalopathy (HE).1Bernal W. Auzinger G. Dhawan A. Wendon J. Acute liver failure.Lancet. 2010; 376: 190-201Abstract Full Text Full Text PDF PubMed Scopus (747) Google Scholar HE is a serious neuropsychiatric complication that is responsible for approximately 20% to 25% of deaths resulting from acute liver failure and negatively influences health-related quality of life, clinical management strategies, liver transplant priority, and survival.1Bernal W. Auzinger G. Dhawan A. Wendon J. Acute liver failure.Lancet. 2010; 376: 190-201Abstract Full Text Full Text PDF PubMed Scopus (747) Google Scholar,2Butterworth R.F. Hepatic encephalopathy: a central neuroinflammatory disorder?.Hepatology. 2011; 53: 1372-1376Crossref PubMed Scopus (170) Google Scholar HE resulting from acute liver failure generates changes in mental status, including cognitive disruptions that can progress to hepatic coma in hours or days resulting from the development of cerebral edema and increased intracranial pressure.3Hazell A.S. Butterworth R.F. Hepatic encephalopathy: an update of pathophysiologic mechanisms.Proc Soc Exp Biol Med. 1999; 222: 99-112Crossref PubMed Scopus (246) Google Scholar,4Fridman V. Galetta S.L. Pruitt A.A. Levine J.M. MRI findings associated with acute liver failure.Neurology. 2009; 72: 2130-2131Crossref PubMed Scopus (17) Google Scholar Aberrant cellular signaling pathways can contribute to the pathogenesis of acute liver failure and HE. One of these pathways is transforming growth factor beta 1 (TGFβ1). Plasma concentrations of transforming growth factor β1 (TGFβ1) are increased in patients with acute liver failure.5Miwa Y. Harrison P.M. Farzaneh F. Langley P.G. Williams R. Hughes R.D. Plasma levels and hepatic mRNA expression of transforming growth factor-beta1 in patients with fulminant hepatic failure.J Hepatol. 1997; 27: 780-788Abstract Full Text PDF PubMed Scopus (42) Google Scholar In mice with acute liver failure, TGFβ1 has been shown to be up-regulated and co-localizes with markers of hepatocytes.6McMillin M. Galindo C. Pae H.Y. Frampton G. Di Patre P.L. Quinn M. Whittington E. DeMorrow S. Gli1 activation and protection against hepatic encephalopathy is suppressed by circulating transforming growth factor beta1 in mice.J Hepatol. 2014; 61: 1260-1266Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar,7McMillin M. Grant S. Frampton G. Petrescu A.D. Williams E. Jefferson B. Thomas A. Brahmaroutu A. DeMorrow S. Elevated circulating TGFbeta1 during acute liver failure activates TGFbetaR2 on cortical neurons and exacerbates neuroinflammation and hepatic encephalopathy in mice.J Neuroinflammation. 2019; 16: 69Crossref PubMed Scopus (17) Google Scholar The use of a pan-TGFβ inhibitor in mice with acute liver failure slows the progression of HE.6McMillin M. Galindo C. Pae H.Y. Frampton G. Di Patre P.L. Quinn M. Whittington E. DeMorrow S. Gli1 activation and protection against hepatic encephalopathy is suppressed by circulating transforming growth factor beta1 in mice.J Hepatol. 2014; 61: 1260-1266Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar In addition, hepatic TGFβ1 enters the brain by permeabilizing the blood-brain barrier, subsequently inducing TGFβ receptor 2–mediated signaling in neurons, leading to increased neuroinflammation in the cerebral cortex.7McMillin M. Grant S. Frampton G. Petrescu A.D. Williams E. Jefferson B. Thomas A. Brahmaroutu A. DeMorrow S. Elevated circulating TGFbeta1 during acute liver failure activates TGFbetaR2 on cortical neurons and exacerbates neuroinflammation and hepatic encephalopathy in mice.J Neuroinflammation. 2019; 16: 69Crossref PubMed Scopus (17) Google Scholar,8McMillin M.A. Frampton G.A. Seiwell A.P. Patel N.S. Jacobs A.N. DeMorrow S. TGFbeta1 exacerbates blood-brain barrier permeability in a mouse model of hepatic encephalopathy via upregulation of MMP9 and downregulation of claudin-5.Lab Invest. 2015; 95: 903-913Crossref PubMed Scopus (61) Google Scholar TGFβ1 is a member of a multifunctional cytokine family that binds and activates a heterotetramer-receptor complex made up of TGFβ receptors 1 and 2, leading to phosphorylation of SMAD2 and SMAD3 proteins, which ultimately modulate transcription of numerous genes.9Abdollah S. Macias-Silva M. Tsukazaki T. Hayashi H. Attisano L. Wrana J.L. TbetaRI phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling.J Biol Chem. 1997; 272: 27678-27685Crossref PubMed Scopus (414) Google Scholar,10Zhang Y. Feng X. We R. Derynck R. Receptor-associated Mad homologues synergize as effectors of the TGF-beta response.Nature. 1996; 383: 168-172Crossref PubMed Scopus (759) Google Scholar TGFβ1 is secreted in its inactive form associated with latency-associated peptide, and must be released from this complex to be active and bind its receptors.11Munger J.S. Harpel J.G. Gleizes P.E. Mazzieri R. Nunes I. Rifkin D.B. Latent transforming growth factor-beta: structural features and mechanisms of activation.Kidney Int. 1997; 51: 1376-1382Abstract Full Text PDF PubMed Scopus (440) Google Scholar One of the proteins responsible for the activation of TGFβ1 is thrombospondin-1 (TSP-1), which can bind the latent complex at the conserved sequence leucine–serine-lysine-leucine (LSKL) on the latency-associated peptide, resulting in the release of TGFβ1 from the latent complex.12Ribeiro S.M. Poczatek M. Schultz-Cherry S. Villain M. Murphy-Ullrich J.E. The activation sequence of thrombospondin-1 interacts with the latency-associated peptide to regulate activation of latent transforming growth factor-beta.J Biol Chem. 1999; 274: 13586-13593Crossref PubMed Scopus (301) Google Scholar TSP-1 is a matricellular glycoprotein that was first isolated and characterized from human blood platelets.13Lawler J.W. Slayter H.S. Coligan J.E. Isolation and characterization of a high molecular weight glycoprotein from human blood platelets.J Biol Chem. 1978; 253: 8609-8616Abstract Full Text PDF PubMed Google Scholar This protein has numerous functions including regulating angiogenesis, apoptosis, inflammation, cell fate determination, extracellular matrix deposition, and other cellular functions.14Mirochnik Y. Kwiatek A. Volpert O.V. Thrombospondin and apoptosis: molecular mechanisms and use for design of complementation treatments.Curr Drug Targets. 2008; 9: 851-862Crossref PubMed Scopus (73) Google Scholar,15Lopez-Dee Z. Pidcock K. Gutierrez L.S. Thrombospondin-1: multiple paths to inflammation.Mediators Inflamm. 2011; 2011: 296069Crossref PubMed Scopus (180) Google Scholar These various functions are the result of the numerous ligands that interact with TSP-1, with interactome studies identifying 83 different targets.16Resovi A. Pinessi D. Chiorino G. Taraboletti G. Current understanding of the thrombospondin-1 interactome.Matrix Biol. 2014; 37: 83-91Crossref PubMed Scopus (171) Google Scholar Although TSP-1 has not been studied extensively during acute liver failure, TSP-1 has been shown to influence both lipid accumulation in nonalcoholic fatty liver disease and the inhibition of liver regeneration by activating latent TGFβ1.17Li Y. Turpin C.P. Wang S. Role of thrombospondin 1 in liver diseases.Hepatol Res. 2017; 47: 186-193Crossref PubMed Scopus (33) Google Scholar,18Hayashi H. Sakai K. Baba H. Sakai T. Thrombospondin-1 is a novel negative regulator of liver regeneration after partial hepatectomy through transforming growth factor-beta1 activation in mice.Hepatology. 2012; 55: 1562-1573Crossref PubMed Scopus (57) Google Scholar Currently, little data exist concerning the involvement of TSP-1 in TGFβ1 activation during acute liver failure and the development of HE. Our aim was to assess TSP-1 expression and how it influences the activation state and downstream signaling of TGFβ1 during acute liver failure and HE. All chemicals used were of the highest necessary grade and were purchased from Millipore-Sigma (Burlington, MA) unless noted otherwise. RNeasy mini kits and real-time PCR primers against TSP-1 (catalog number PPM03098F-200), TGFβ1 (catalog number PPM02991B-200), chemokine ligand 2 (catalog number PPM03151G-200), and glyceraldehyde 3-phosphate dehydrogenase (catalog number PPM02946E-200) were purchased from Qiagen (Germantown, MD). The iScript cDNA kit, Laemmli buffer, running buffer, and transfer buffer were purchased from Bio-Rad (Hercules, CA). Hematoxylin QS, antigen unmasking solution, and VectaStain ABC kits were purchased from Vector Laboratories (Burlingame, CA). TGFβ1 and TSP-1 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). The cleaved caspase 3 antibody was purchased from Cell Signaling Technology (Danvers, MA). Ionized calcium-binding adapter molecule 1 (IBA1) antibodies were purchased from Wako Chemicals USA (Richmond, VA). Blocking buffer and secondary antibodies for Western blot were ordered from Li-Cor Biosciences (Lincoln, NE). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) kits were purchased from Abcam (Cambridge, MA). Fluorescent secondary antibodies for immunofluorescence were bought from Jackson ImmunoResearch Laboratories (West Grove, PA). TSP-1 null mice (TSP-1−/−, stock 006141; B6.129S2-Thbs1tm1Hyn/J) or C57Bl/6J mice were from The Jackson Laboratory (Bar Harbor, ME) and used for all in vivo experiments. Acute liver failure and HE were caused by a single intraperitoneal injection of 100 mg/kg body weight of azoxymethane (AOM) into mice (20 to 25 g) as described previously.19Silva V.R. Secolin R. Vemuganti R. Lopes-Cendes I. Hazell A.S. Acute liver failure is associated with altered cerebral expression profiles of long non-coding RNAs.Neurosci Lett. 2017; 656: 58-64Crossref PubMed Scopus (6) Google Scholar, 20McMillin M. Frampton G. Grant S. Khan S. Diocares J. Petrescu A. Wyatt A. Kain J. Jefferson B. DeMorrow S. Bile acid-mediated sphingosine-1-phosphate receptor 2 signaling promotes neuroinflammation during hepatic encephalopathy in mice.Front Cell Neurosci. 2017; 11: 191Crossref PubMed Scopus (49) Google Scholar, 21Popek M. Bobula B. Sowa J. Hess G. Polowy R. Filipkowski R.K. Frontczak-Baniewicz M. Zablocka B. Albrecht J. Zielinska M. Cortical synaptic transmission and plasticity in acute liver failure are decreased by presynaptic events.Mol Neurobiol. 2018; 55: 1244-1258Crossref PubMed Scopus (8) Google Scholar, 22Chastre A. Belanger M. Nguyen B.N. Butterworth R.F. Lipopolysaccharide precipitates hepatic encephalopathy and increases blood-brain barrier permeability in mice with acute liver failure.Liver Int. 2014; 34: 353-361Crossref PubMed Scopus (46) Google Scholar After AOM administration, mice cages were placed on heating pads set to 37°C to ensure the mice remained normothermic. Hydrogel and rodent chow were placed on the floor of the cages to ensure access to food and hydration. Mice were injected subcutaneously with 5% dextrose in 250 μL saline at 12 hours and every 4 hours thereafter to ensure euglycemia and hydration. Mice were monitored every 2 hours (starting at 12 hours after AOM injection) for body temperature, weight, and neurologic score using previously published methodology.6McMillin M. Galindo C. Pae H.Y. Frampton G. Di Patre P.L. Quinn M. Whittington E. DeMorrow S. Gli1 activation and protection against hepatic encephalopathy is suppressed by circulating transforming growth factor beta1 in mice.J Hepatol. 2014; 61: 1260-1266Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar,23McMillin M. Frampton G. Tobin R. Dusio G. Smith J. Shin H. Newell-Rogers K. Grant S. DeMorrow S. TGR5 signaling reduces neuroinflammation during hepatic encephalopathy.J Neurochem. 2015; 135: 565-576Crossref PubMed Scopus (67) Google Scholar,24McMillin M. Frampton G. Thompson M. Galindo C. Standeford H. Whittington E. Alpini G. DeMorrow S. Neuronal CCL2 is upregulated during hepatic encephalopathy and contributes to microglia activation and neurological decline.J Neuroinflammation. 2014; 11: 121Crossref PubMed Scopus (49) Google Scholar After the development of neurologic dysfunction, mice were monitored with formal assessments of temperature, body weight, and neurologic score performed every hour until the mice became comatose. The neurologic score was determined by an investigator blind to the treatments (S.G. or G.F.) by assigning a score between 0 (absent) and 2 (intact) to the following parameters: the pinna reflex, corneal reflex, tail flexion, escape response, righting reflex, and ataxia. The scores of these five reflexes and ataxia were summated to provide a neurologic score between 0 and 12. Once mice lost their corneal and righting reflex, the time to coma was recorded, the mice were euthanized, and tissue was collected to be used for all analyses. Cerebral edema was assessed in all mice using the wet/dry weight method as previously described.25Baskaya M.K. Dogan A. Rao A.M. Dempsey R.J. Neuroprotective effects of citicoline on brain edema and blood-brain barrier breakdown after traumatic brain injury.J Neurosurg. 2000; 92: 448-452Crossref PubMed Scopus (96) Google Scholar This method involves weighing a micro–centrifuge tube before and after placing brain tissue inside of it to calculate the weight of the tube and the wet weight of the tissue, respectively. The microcentrifuge tube is left uncapped and put into an oven at 70°C for 2 days. The microcentrifuge tube with the tissue is weighed again to calculate the dry weight. Water content was expressed as a percentage of brain weight; calculated as follows: ((wet weight − dry weight)/wet weight) × 100%. An increase in brain water content of 1% to 2% in mice is indicative of cerebral edema and increased intracranial pressure.26Cao C. Yu X. Liao Z. Zhu N. Huo H. Wang M. Ji G. She H. Luo Z. Yue S. Hypertonic saline reduces lipopolysaccharide-induced mouse brain edema through inhibiting aquaporin 4 expression.Crit Care. 2012; 16: R186Crossref PubMed Scopus (4) Google Scholar,27Bemeur C. Vaquero J. Desjardins P. Butterworth R.F. N-acetylcysteine attenuates cerebral complications of non-acetaminophen-induced acute liver failure in mice: antioxidant and anti-inflammatory mechanisms.Metab Brain Dis. 2010; 25: 241-249Crossref PubMed Scopus (60) Google Scholar Paraffin-embedded livers were cut into 4-μm sections and mounted onto positively charged slides (VWR, Radnor, PA). Slides were deparaffinized with xylene and rehydrated with ethanol at decreasing concentrations. The liver tissue then was stained with Hematoxylin QS (Vector Laboratories) and Eosin Y (Amresco, Solon, OH) and rinsed with 95% ethanol. The slides were submerged into 100% ethanol and then through two xylene washes. Coverslips were mounted onto the slides using CytoSeal XYL mounting media (ThermoFisher Scientific, Waltham, MA). The slides were imaged using an Olympus BX40 microscope with a DP25 imaging system (Olympus, Center Valley, PA). Liver function was assessed by measuring serum alanine aminotransferase levels using a Catalyst One serum chemistry analyzer from IDEXX Laboratories, Inc. (Westbrook, MA). Liver and cortex tissue from vehicle, AOM, and TSP-1−/− mice were homogenized and RNA was isolated using an RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. The concentration of RNA in each sample was measured using a ThermoFisher Scientific Nanodrop 2000 spectrophotometer. cDNA was synthesized using a Bio-Rad iScript cDNA Synthesis Kit and real-time PCR was performed as described previously28Frampton G. Invernizzi P. Bernuzzi F. Pae H.Y. Quinn M. Horvat D. Galindo C. Huang L. McMillin M. Cooper B. Rimassa L. DeMorrow S. Interleukin-6-driven progranulin expression increases cholangiocarcinoma growth by an Akt-dependent mechanism.Gut. 2012; 61: 268-277Crossref PubMed Scopus (90) Google Scholar using commercially available primers designed against mouse TSP-1, TGFβ1, Ccl2, and GAPDH. SYBR green fluorescence was measured using a MX3005P thermal cycler from Agilent Technologies (Santa Clara, CA). A ΔΔCt analysis was performed using vehicle-treated tissue as the control group.29DeMorrow S. Francis H. Gaudio E. Venter J. Franchitto A. Kopriva S. Onori P. Mancinelli R. Frampton G. Coufal M. Mitchell B. Vaculin B. Alpini G. The endocannabinoid anandamide inhibits cholangiocarcinoma growth via activation of the noncanonical Wnt signaling pathway.Am J Physiol Gastrointest Liver Physiol. 2008; 295: G1150-G1158Crossref PubMed Scopus (67) Google Scholar,30Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123424) Google Scholar TUNEL assays and immunohistochemistry were performed in liver sections (4- to 6-μm thick) prepared as outlined in Liver Histology and Serum Chemistry. The TUNEL assays were performed according to the manufacturer's protocols with no modifications. For immunohistochemistry, antibodies against TSP-1, TGFβ1, phosphorylated mothers against decapentaplegic homolog 3 (pSMAD3), and cleaved caspase 3 were incubated overnight at 4°C. Subsequent incubation with secondary antibody and color development using 3,3′-diaminobenzidine substrate was performed using Vector Laboratories kits according to the manufacturer's instructions. Sections were counterstained with Hematoxylin QS. TUNEL and immunohistochemical stained tissue were scanned by a Leica SCN400 digital slide scanner (Leica Microsystems, Buffalo Grove, IL). The percentage area staining positive for pSMAD3 or TUNEL staining was quantified by converting images to grayscale and quantifying positive staining area using ImageJ software version 1.52a (NIH, Bethesda, MD; https://imagej.nih.gov/ij). Liver and cortex tissue from each mouse group were homogenized using a Miltenyi Biotec (Auburn, CA) gentleMACS Dissociator and total protein concentrations were measured using a ThermoFisher BCA Protein Assay kit. SDS-PAGE gels (10% to 15% v/v) were loaded with 30 to 40 μg protein diluted in Laemmli buffer per tissue sample. TSP-1, TGFβ1, cytochrome p450 2E1, cleaved caspase 3, and β-actin antibodies were used. Imaging was performed on a Li-Cor Odyssey 9120 Infrared Imaging System. Data are expressed as fold change in fluorescent band intensity of the target antibody compared with β-actin, which was used as a loading control. The control group values were used as a baseline and set to a relative protein expression value of 1. Band intensity quantifications were performed using ImageJ software. Free-floating immunofluorescence staining was performed on 30-μm thick brain sections. Brain sections were blocked with 5% goat serum and then were incubated with IBA1 antibodies to detect morphology and relative staining of microglia. Cy3 fluorescent secondary antibodies were used to visualize immunoreactivity. Brain sections subsequently were moved to positively charged slides and coverslips were mounted using ProLong Gold Antifade Reagent (ThermoFisher) containing DAPI. Brain sections were imaged using a Leica TCS SP5-X inverted confocal microscope (Leica Microsystems). The field fluorescence area of IBA1 was calculated by converting images to grayscale, inverting their color, and quantifying field staining with ImageJ software. Statistical analyses were performed using GraphPad Prism 8 version 8.2.1 (GraphPad Software, La Jolla, CA). Results were expressed as means ± SEM. Significance was determined using the t-test when differences between two groups were assessed, and analysis of variance when differences between three or more groups were compared. A two-way analysis of variance was performed for the neurologic score analyses followed by a Bonferroni multiple comparison post hoc test. Differences were considered significant for P values < 0.05. Mice were administered AOM to induce acute liver failure and HE. After AOM treatment, there was a significant increase of hepatic TSP-1 mRNA expression compared with vehicle-treated mice (Figure 1A). In liver sections from AOM-treated mice, staining for TSP-1 was increased compared with vehicle-treated mice, with distinct hepatocyte populations near areas of necrosis expressing high TSP-1 levels (Figure 1B). TSP-1 protein expression in the liver was increased significantly in AOM-treated mice, with levels nearly undetectable in vehicle-treated mice (Figure 1, C and D). Collectively, these data support an increase of hepatic TSP-1 expression in response to AOM-induced acute liver injury. Because TSP-1 can increase TGFβ1 activity and subsequent signaling, the expression of TGFβ1 was assessed in vehicle and AOM-treated mice. AOM-induced acute liver failure generated an increase of TGFβ1 mRNA expression in the liver compared with vehicle-treated mice (Figure 2A). In liver sections from AOM-treated mice, there was increased staining for TGFβ1 compared with sections from vehicle-treated mice (Figure 2B). In support of these data, TGFβ1 protein expression was increased significantly in liver homogenates from AOM-treated mice compared with vehicle-treated mice (Figure 2, C and D). SMAD3 phosphorylation, as a measure of downstream TGFβ1 activity, was increased significantly in the livers of AOM-treated mice, however, staining was not observed in vehicle-treated livers (Figure 2, E and F). Because of the correlation between TSP-1 expression and increased TGFβ1 expression and activity in the livers of AOM-treated mice, the effects of genetic ablation of TSP-1 were examined during AOM-induced HE. Vehicle-treated wild-type (WT) or TSP-1−/− mice administered AOM had low TSP-1 expression in the liver, whereas hepatic TSP-1 expression was increased significantly in WT AOM-treated mice (Figure 3, A and B ). To validate that TSP-1 exacerbated AOM-induced hepatotoxicity, liver injury and function were assessed. In WT AOM-treated mice there was extensive necrosis, microvesicular steatosis, and hemorrhage present, which were reduced in TSP-1−/− mice injected with AOM (Figure 3C). These histologic findings are mirrored by alanine aminotransferase levels, which show a large increase in WT AOM-treated mice that was reduced significantly in TSP-1−/− mice administered AOM (Figure 3D). Because it is possible that the hepatoprotective effects of TSP-1−/− compared with WT mice were the result of differences in AOM metabolism, cytochrome p450 2E1 expression was assessed. Cytochrome p450 2E1 was reduced significantly in both WT AOM-treated and TSP-1−/− AOM-treated mice, with no significant differences between groups (Figure 3, E and F). With TGFβ1 being known to contribute to AOM pathogenesis, the hypothesis that TSP-1 activates TGFβ1 during AOM-induced liver injury was evaluated. TGFβ1 mRNA expression was increased significantly in WT AOM-treated mice, but was reduced significantly in TSP-1−/− mice treated with AOM (Figure 4A). TGFβ1 staining in liver sections was observed throughout WT AOM-treated mice whereas TSP-1−/− mice treated with AOM showed reduced staining, to levels near WT vehicle-treated mice (Figure 4B). These staining results were validated by immunoblot data, showing a significant increase of active TGFβ1 in WT AOM-treated mice, which was reduced significantly in TSP-1−/− mice injected with AOM (Figure 4, C and D). Staining for pSMAD3 was widespread in liver sections from WT AOM-treated mice and the area of staining was reduced significantly in TSP-1−/− AOM-treated mice (Figure 4, E and F). TGFβ1 can be an inducer of apoptosis, which could drive AOM-induced liver injury. TUNEL staining was increased significantly in WT AOM-treated mice in areas neighboring necrotic tissue, although there was a significant reduction in TUNEL-positive cells in TSP-1−/− AOM-treated mice (Figure 5, A and B ). In addition, cleaved caspase 3 staining was increased in WT AOM-treated mice and significantly reduced in TSP-1−/− AOM-treated mice, although a small degree of staining still was observed (Figure 5C). Immunoblots for cleaved caspase 3 show similar results, with a significant increase of cleaved caspase 3 expression in WT AOM-treated mice, which was reduced significantly in TSP-1−/− AOM-treated mice (Figure 5, D and E). The rate of neurologic decline was measured in the WT and TSP-1−/− mice after the injection of AOM. The mice were assigned a neurologic score ranging between 0 and 12, with 12 indicating full neurologic capabilities and 0 indicating an absence of reflexes and the onset of hepatic coma. TSP-1−/− knockout mice injected with AOM had a delayed rate of neurologic decline compared with WT AOM-treated mice (Figure 6A). TSP-1−/− AOM-treated mice had a significantly increased latency to reach coma in comparison with the AOM-injected WT mice (Figure 6B). Cerebral edema was increased significantly in WT AOM-treated mice and reduced significantly in TSP-1−/− AOM-treated mice, to levels similar to WT vehicle-treated mice (Figure 6C). Microglia in the brain are activated during HE and, as a result, experiments were conducted to assess microglial activation after AOM injection. Immunofluorescence was conducted using the microglia marker IBA1, which showed that WT AOM-treated mice have increased field staining for IBA1 that is reduced in TSP-1−/− AOM-treated mice (Figure 7, A and B ). In addition, the morphology of microglia in WT AOM-treated mice show more of an amoeboid-type shape with shorter processes, an indication of activation (Figure 7A). This change in morphology was not observed in the TSP-1−/− AOM-treated mice, which have microglia similar to WT vehicle-treated mice (Figure 7A). Microglia activation during HE can be a result of increased chemokine ligand 2 expression,31Dhanda S. Gupta S. Halder A. Sunkaria A. Sandhir R. Systemic inflammation without gliosis mediates cognitive deficits through impaired BDNF expression in bile duct ligation model of hepatic encephalopathy.Brain Behav Immun. 2018; 70: 214-232Crossref PubMed Scopus (21) Google Scholar,32Zhang L. Tan J. Jiang X. Qian W. Yang T. Sun X. Chen Z. Zhu Q. Neuron-derived CCL2 contributes to microglia activation and neurological decline in hepatic encephalopathy.Biol Res. 2017; 50: 26Crossref PubMed Scopus (29) Google Scholar and Ccl2 mRNA levels were found to be up-regulated significantly in WT AOM-treated mice, with a significant decrease in TSP-1−/− AOM-treated mice (Figure 7C). The data presented in the current study support that AOM-induced acute liver injury leads to increased TSP-1 expression in the liver and increased pSMAD3 signaling as a result of increased levels of activated TGFβ1 in the liver. During AOM-induced acute liver injury, TSP-1 expression was increa" @default.
• W2988505923 created "2019-11-22" @default.
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• W2988505923 date "2020-02-01" @default.
• W2988505923 modified "2023-09-24" @default.
• W2988505923 title "Thrombospondin-1 Exacerbates Acute Liver Failure and Hepatic Encephalopathy Pathology in Mice by Activating Transforming Growth Factor β1" @default.
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