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. Author manuscript; available in PMC: 2021 Dec 15.
Published in final edited form as: Toxicol Appl Pharmacol. 2020 Nov 8;409:115323. doi: 10.1016/j.taap.2020.115323

Inhibition of thrombospondin-1 reduces glutathione activity and worsens acute liver injury during acetaminophen hepatotoxicity in mice

Gabriel Frampton a,b, Priyanka Reddy a, Brandi Jefferson a, Malaika Ali a, Durreshahwar Khan a, Matthew McMillin a,b,*
PMCID: PMC8364670  NIHMSID: NIHMS1729751  PMID: 33176120

Abstract

Acetaminophen (N-Acetyl-p-Aminophenol or APAP)-induced hepatotoxicity is the most common cause of acute liver failure in the United States and Western Europe. Previous studies have shown that TGFβ1 is elevated during APAP-induced hepatotoxicity and promotes liver injury by reducing liver regeneration while inducing hepatocyte senescence. At this time, little is known about the role of proteins that activate latent TGFβ1 and their effects during APAP-induced hepatotoxicity. Thrombospondin-1 (TSP1) is a homotrimeric protein that can not only activate latent TGFβ1 but can also interact with other proteins including Nrf2 to induce antioxidant signaling. The aim of the current study was to assess the role of thrombospondin-1 (TSP1) in both TGFβ1 activation and its contribution to APAP-induced liver injury. C57Bl/6 mice or TSP1 null mice (TSP1−/−) were administered 300 mg/kg or 600 mg/kg of APAP. TGFβ1 signaling, TSP1 expression, measures of hepatic injury, Nrf2 expression, measures of oxidative/nitrosative stress and GSH metabolism were assessed. The expression of TGFβ1, TSP1 and phosphorylation of SMAD proteins increased in APAP-treated mice compared to controls. TSP1−/− mice had reduced TGFβ1 expression and phosphorylation of SMAD proteins but increased liver injury. Hepatocyte cell death was increased in TSP1−/− mice and this was associated with decreased Nrf2 activity, decreased GSH levels and increased oxidative stress in comparison to wild-type C57Bl/6 mice. Together, these data demonstrate that elimination of TSP1 protein in APAP-treated mice reduces TGFβ1 signaling but leads to increased liver injury by reducing Nrf2 expression and GSH activity, ultimately resulting in increased cell death.

Keywords: Transforming growth factor beta 1, Hepatotoxicity, Acute liver failure, Nrf2

1. Introduction

Acute liver injury resulting from acetaminophen (N-Acetyl-p-Aminophenol or APAP) hepatotoxicity is the primary cause of acute liver failure in the United States and Western Europe (Bernal et al., 2015). The disease process involves the metabolism of APAP into the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI), depleting glutathione (GSH), generating oxidative stress and ultimately leading to cell death. Acute liver injury can progress to liver failure when the rate of cell death overwhelms the regenerative capacity of the liver, leading to a loss of hepatic function and subsequent liver failure.

Recent research in the field has shown that cell signaling pathways play a significant role in regulating both cell death and cell regeneration in the context of APAP-induced liver injury. Transforming growth factor beta 1 (TGFβ1) is a protein that is involved with numerous cellular processes and has been demonstrated to have an active role in the progression of APAP-induced liver injury in pre-clinical studies. Inhibition of TGFβ1 receptor activity with GW788388 in mice has been shown to reduce hepatic necrosis, lessen liver injury and promote regeneration of hepatocytes compared to control-treated mice (McMillin et al., 2019). In addition, treatment of mice with the TGFβ receptor 1 inhibitor AZ12601011 during APAP-induced hepatotoxicity reduced both mortality and hepatic cellular senescence, giving further support for the deleterious role of TGFβ1 during this disease state (Bird et al., 2018). The activity of TGFβ1 is tightly controlled with its activation requiring interactions with other proteins or changes in physiological states. TGFβ1 is a component of a protein complex made up of latent TGFβ1 binding protein and latency associated peptide (Munger et al., 1997). Following interactions with proteins, enzymes or other factors, TGFβ1 is released from the protein complex where it can dimerize and bind its receptors, leading to phosphorylation of SMAD2 and SMAD3 proteins and ultimately influencing the transcriptional activity of numerous genes (Abdollah et al., 1997; Zhang et al., 1996). At this time, TGFβ1 has been shown to exacerbate APAP hepatotoxicity but the understanding of the factors that contribute to the activation of this protein during this disease state have not been characterized.

Thrombospondin-1 (TSP1) is a matricellular protein that has the capability to activate latent TGFβ1, resulting in a myriad of other functions including regulation of apoptosis, inflammation, extracellular matrix deposition, stem cell maturation and other cellular functions (Mirochnik et al., 2008; Lopez-Dee et al., 2011). In regards to latent TGFβ1 activation, TSP1 interacts with the LSKL peptide sequence of latency-associated peptide, leading to a conformational change in the quaternary structure of the TGFβ1 protein complex, resulting in the release of the TGFβ1 monomer (Ribeiro et al., 1999). Recently, we demonstrated that TSP1 is upregulated in the azoxymethane model of acute liver failure (Jefferson et al., 2020), demonstrating that this protein may have a role in other models of acute liver injury. Interactome studies for TSP1 have shown that outside of TGFβ1, TSP1 can interact with over 80 other proteins including cell receptors, growth factors and proteases (Resovi et al., 2014; Crawford et al., 1998). TSP1 has been shown to induce signaling via nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor involved in the antioxidant response, resulting in reduced oxidative stress and apoptosis in pancreatic β cells (Cunha et al., 2016). In hepatocyte cell cultures and mouse models investigating APAP-induced hepatotoxicity, multiple studies have determined that increased Nrf2 activity is protective and can result in reducing oxidative stress (Wang et al., 2018; Fan et al., 2018; Lv et al., 2019). It is conceivable that the hepatoprotective effects of TSP1 generated by inducing Nrf2 signaling could overcome the adverse consequences of TGFβ1 activation. Therefore, this study investigates the role of TSP1 in both the activation of TGFβ1 and its influence on oxidative stress during APAP-induced hepatotoxicity. We hypothesize that TSP1 is upregulated during APAP-induced acute liver failure and that antagonism of TSP1 will reduce TGFβ1 and Nrf2 signaling, which ultimately results in increased oxidative stress and exacerbated liver injury.

2. Methods

2.1. Materials

RNeasy mini kits and real-time polymerase chain reaction (RT-PCR) primers against TSP1, TGFβ1, glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modulatory subunit (GCLM), glutathione-disulfide reductase (GSR) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were bought from either Qiagen (Germantown, MD) or Bio-Rad (Hercules, CA). The iScript cDNA kit, Laemmli buffer, running buffer and transfer buffer were purchased from Bio-Rad. Hematoxylin QS, antigen unmasking solution and VectaStain ABC kits were purchased from Vector Laboratories (Burlingame, CA). Phosphorylated SMAD2/3 (pSMAD2/3) ELISA kits were purchased from Cell Signaling Technology (Danvers, MA). TGFβ1, TSP1, CYP2E1, mouse IgG and mouse IgG1 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). The GAPDH antibody was purchased from GeneTex (Irvine, CA). The superoxide dismutase 1 (SOD1) antibody was purchased from ThermoFisher Scientific (Waltham, MA). Blocking buffer and secondary antibodies for western blotting were ordered from LI-COR Biosciences (Lincoln, NE). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, malondialdehyde (MDA) kit, pSMAD3 antibody, Nrf2 antibody, nitrotyrosine antibody and rabbit IgG antibody were purchased from Abcam (Cambridge, MA). All other chemicals and reagents used were of the highest necessary grade and were purchased from Millipore-Sigma (Burlington, MA) unless indicated otherwise.

2.2. APAP model of acute liver injury

TSP1 null mice (TSP1−/−, stock # 006141; B6.129S2-Thbs1tm1Hyn/J) or C57Bl/6 J mice (stock # 000664), used as the wild-type control, were purchased from The Jackson Laboratory (Bar Harbor, ME) and used for all in vivo experiments. All mice were fasted overnight and liver injury was induced via a single intraperitoneal injection of acetaminophen (APAP) into mice (20–25 g). The first set of mice were C57Bl/6, injected with saline as vehicle, 300 mg/kg APAP or 600 mg/kg APAP and euthanized 6 h after injection. The second group of mice were wild-type and TSP1−/− mice that were injected with vehicle or 600 mg/kg APAP and euthanized 2 h or 6 h after injection. The third group of mice were C57Bl/6 mice injected with SLLK or LSKL peptide (30 mg/kg) 1 h after saline or 600 mg/kg APAP injection. Mice were euthanized and tissue collected 6 h after APAP administration for this third group of mice. Following APAP administration, mice were placed on heating pads set to 37 °C to ensure that they did not become hypothermic. Rodent chow and hydrogel were placed on the floor of the cage to ensure easy access to food and hydration. At time of euthanasia, liver tissue and serum were collected. All animal experiments were approved by the Baylor Scott & White Health IACUC committee.

2.3. Liver histology and serum chemistry

Paraffin-embedded livers were cut into 4 μm sections and mounted onto positively-charged slides (VWR, Radnor, PA). Slides were deparaffinized and stained with Hematoxylin QS (Vector Laboratories, Burlingame, CA) followed by staining with Eosin Y (Amresco, Solon, OH) and rinsed in 95% ethanol. The slides were then dipped into 100% ethanol and subsequently through 2 xylene washes. Coverslips were mounted onto the slides using CytoSeal XYL mounting media (ThermoFisher Scientific). The slides were viewed and imaged using an Olympus BX40 microscope with a DP25 imaging system (Olympus, Center Valley, PA). Percentage area of necrosis was calculated using 20× field views, drawing areas devoid of hepatocyte nuclei and calculating area using ImageJ software (National Institutes of Health, Bethesda, MD). In each mouse, the necrosis from 5 to 10 fields were quantified and averaged together to determine necrotic area.

Liver function was assessed by measuring serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels using a Catalyst One serum chemistry analyzer from IDEXX Laboratories, Inc. (Westbrook, MA). For control groups, serum was diluted 1:2 in deionized H20 and for APAP-treated groups, serum was diluted 1:9 in deionized H20 prior to running on the analyzer.

2.4. GSH and MDA assays

Hepatic glutathione (GSH) levels were measured using a commercially available kit from Millipore-Sigma with no deviations from the instructions provided by the manufacturer. Concentrations of malondialdehyde (MDA) in liver tissue were determined using a kit from Abcam according to the manufacturer’s instructions. For both GSH and MDA, concentrations were normalized to total protein of each measured homogenate.

2.5. Measurement of phospho-SMAD levels

Pathscan® pSMAD2 (Ser465/467)/pSMAD3 (Ser423/425) ELISA kits were used to determine phosphorylation of SMAD2/3 in hepatic tissue. Liver homogenates were assayed at a protein concentration of 1.0 μg/μL. These ELISA assays were performed according to the instructions from the manufacturer and values are expressed as relative phosphorylation activity of experimental group compared to control group.

2.6. mRNA analyses

Liver tissue from wild-type and TSP1−/− mice treated with vehicle or APAP was homogenized and RNA was isolated using a RNeasy Mini Kit (Qiagen, Germantown, MD) according to the manufacturer’s instructions. The concentration of RNA in each sample was measured using a ThermoFisher Scientific™ Nanodrop 2000 spectrophotometer. Synthesis of cDNA was accomplished using a Bio-Rad iScript™ cDNA Synthesis Kit. RT-PCR was performed as previously described (Frampton et al., 2012) using commercially-available primers designed against mouse TSP1, TGFβ1, GCLC, GCLM, GSR and GAPDH. SYBR green fluorescence was measured using either an AriaMX or MX3005P thermal cycler from Agilent Technologies. A ΔΔCT analysis was performed using vehicle-treated tissue as the control group (DeMorrow et al., 2008; Livak and Schmittgen, 2001).

2.7. TUNEL assay and immunohistochemistry

Immunohistochemistry and TUNEL assays were performed in liver sections (4–6 μm thick) prepared as outlined above. For immunohistochemistry, antibodies against TGFβ1, TSP1, pSMAD3, nitrotyrosine, mouse IgG, mouse IgG1 or rabbit IgG were incubated overnight at 4 °C. Subsequent secondary antibody incubation and color development using 3,3′ -diaminobenzidine (DAB) substrate was performed using kits from Vector Laboratories according to the manufacturer’s instructions. Sections were counterstained with Hematoxylin QS. The TUNEL assay was performed according to the protocol supplied from Abcam with no modifications. TUNEL and immunohistochemical stained slides were scanned by a Leica Biosystems (Buffalo Grove, IL) SCN400 digital slide scanner. Images from the digitized slides were taken using Aperio ImageScope software from Leica Biosystems. TUNEL staining was quantified by converting images to greyscale and quantifying the percentage area of positive staining using ImageJ software.

2.8. Western blotting

Liver tissue from all mouse groups was homogenized using a Miltenyi Biotec gentleMACS™ Dissociator and total protein was quantified using a ThermoFisher Pierce™ BCA Protein Assay kit. SDS-PAGE gels (10–15% v/v) were loaded with 30–40 μg of protein diluted in Laemmli buffer per each tissue sample. Specific antibodies against TGFβ1, Cyp2E1, Nrf2, SOD1, GAPDH and β-actin were used. All imaging was performed on a LI-COR Biosciences Odyssey 9120 Infrared Imaging System or a Bio-Rad Chemidoc MP Imaging System. Data are expressed as fold change in fluorescent band intensity of target antibody divided by GAPDH or β-actin, which were used as loading controls. The values of control groups were used as the baseline and relative protein expression was set to a value of 1. The fluorescence intensity quantifications were performed using ImageJ software.

2.9. Statistical analyses

All statistical analyses were performed using Graphpad Prism software (Graphpad Software, La Jolla, CA). Results were expressed as mean ± SEM. Significance was determined using the Student’s t-test when differences between two groups were analyzed, and analysis of variance when differences between three or more groups were compared. Differences were considered significant for p values less than 0.05.

3. Results

3.1. APAP administration generated acute liver injury

In order to validate that our model of APAP-induced hepatotoxicity was inducing consistent acute liver injury, C57Bl/6 mice were injected with 300 mg/kg or 600 mg/kg acetaminophen and euthanized 6 h later. Liver injury and centrilobular necrosis were observed in both 300 mg/kg and 600 mg/kg APAP-treated mice (Fig. 1A). When quantified, both APAP-treated groups had a significant increase in areas of necrosis, though a greater degree of necrosis was observed in 600 mg/kg APAP-treated mice (Fig. 1B). This was associated with a significant elevation of serum ALT (Fig. 1C) and AST (Fig. 1D) concentrations in APAP-treated mice, with the 600 mg/kg APAP-treated mice having larger elevation of serum transaminases. Total GSH levels were significantly reduced in only the 600 mg/kg APAP-treated mice, demonstrating that this group was unable to significantly replenish GSH levels 6 h following APAP-induced liver injury (Fig. 1E). However, the antioxidant capacity of the 300 mg/kg APAP-treated mice was fully restored within 6 h following APAP administration, which collectively demonstrates an APAP dose-dependent effect on GSH depletion.

Fig. 1.

Fig. 1.

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Increasing doses of APAP induce greater hepatotoxicity. (A) H&E images of liver sections from mice treated with vehicle, 300 mg/kg or 600 mg/kg APAP. Images are displayed at 200× magnification. (B) Percentage area of necrosis present in liver sections from vehicle, 300 mg/kg or 600 mg/kg APAP mouse groups. (C) Serum ALT concentration from vehicle and APAP-treated mice. (D) Levels of AST in the serum from mice administered vehicle or APAP at the indicated concentrations. (E) Hepatic GSH levels normalized to lysate protein in mice injected with vehicle, 300 mg/kg APAP or 600 mg/kg APAP. Data are presented as mean ± SEM with n = 5 for all groups. * = p < 0.05 compared to vehicle-treated mice. # = p < 0.05 compared to APAP-treated 300 mg/kg group.

3.2. APAP treatment increased TSP1 expression and TGFβ1 signaling

Previously, we identified a time-dependent increase of TGFβ1 in APAP-treated mice (McMillin et al., 2019). To see if there was also a dose-dependent response, we assessed TGFβ1 expression and signaling in mice treated with 300 mg/kg and 600 mg/kg of APAP. Mice treated with either 300 mg/kg or 600 mg/kg of APAP saw a significant increase of TGFβ1 mRNA expression (Fig. 2A). This resulted in a significant increase in protein, as both the 300 mg/kg and 600 mg/kg APAP-treated mice had a significant elevation of active TGFβ1 protein compared to vehicle-treated mice (Fig. 2B and C). To determine hepatic expression and cellular localization of TGFβ1, liver sections were stained, demonstrating that necrotic areas and peri-necrotic hepatocytes stained most intensely in APAP-treated mice, with IgG1-stained sections displaying faint to no staining in all groups (Fig. 2D). Staining for pSMAD3 also displayed a similar increase with the darkest staining observed in nuclei and less staining observed in the necrotic nodules (Fig. 2E). To further assess downstream TGFβ1 signaling, pSMAD2/3 levels were assessed, identifying that their expression was only significantly increased in the 600 mg/kg APAP-treated mice (Fig. 2F). This increase of pSMAD2/3 expression was due to an increase of pSMAD3 protein and not pSMAD2 protein in 600 mg/kg APAP-treated mice (Fig. S1A).

Fig. 2.

Fig. 2.

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Hepatic TGFβ1 expression and signaling are increased in APAP-treated mice. (A) TGFβ1 mRNA expression in the livers from mice treated with vehicle, 300 mg/kg APAP or 600 mg/kg APAP. (B) Representative immunoblot images for hepatic TGFβ1 protein in mice treated with vehicle or indicated doses of APAP with GAPDH used as a protein loading control. (C) Quantification of liver TGFβ1 immunoblots represented as a ratio of TGFβ1/GAPDH with vehicle normalized to 1 in vehicle and APAP-treated mice. (D) Liver sections stained for TGFβ1 or IgG1 for mice administered vehicle or indicated doses of APAP. Scale bar represents 200 μm. (E) Liver sections stained for pSMAD3 or IgG in mice administered vehicle or indicated doses of APAP. Scale bar represents 200 μm. (F) Relative levels of pSMAD2/3 in livers from vehicle or APAP-treated mice. Data are presented as mean ± SEM with n = 5 for all groups. * = p < 0.05 compared to vehicle-treated mice. # = p < 0.05 compared to APAP-treated 300 mg/kg group.

TSP1, as an activator of latent TGFβ1, was found to be significantly increased in mice treated with either 300 mg/kg or 600 mg/kg APAP, with the effect being greater in mice treated with 600 mg/kg APAP (Fig. 3A). In vehicle-treated mice, immunostaining for TSP1 was largely absent throughout the tissue, with increased expression of TSP1 observed as the dose of APAP was increased, and this observation was not observed in IgG-stained liver sections (Fig. 3B). The expression of TSP1 in livers from mice treated with APAP was localized to necrotic and peri-necrotic areas, with the cellular expression being predominately in hepatocytes (Fig. 3B).

Fig. 3.

Fig. 3.

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TSP1 expression in the liver increases during APAP-induced hepatotoxicity. (A) TSP1 mRNA expression in the livers from mice treated with vehicle, 300 mg/kg APAP or 600 mg/kg APAP. (B) Liver sections stained for TSP1 or IgG in mice administered vehicle or indicated doses of APAP. Scale bar represents 200 μm. Data are presented as mean ± SEM with n = 5 for all groups. * = p < 0.05 compared to vehicle-treated mice. # = p < 0.05 compared to APAP-treated 300 mg/kg group.

3.3. APAP-induced TGFβ1 signaling was suppressed in TSP1−/− mice

With the increased expression of TSP1 and TGFβ1 signaling observed in APAP-treated mice, TSP1−/− mice were administered 600 mg/kg APAP to determine if inhibition of TSP1 could inhibit TGFβ1 signaling during APAP-induced liver injury. TGFβ1 protein expression was significantly increased at 2 h and 6 h after APAP administration in wild-type mice (Fig. 4A and B). However, in TSP1−/− mice, TGFβ1 protein was significantly reduced at 2 h and 6 h after APAP injection compared to wild-type mice at the same time points (Fig. 4A and B). Relative levels of pSMAD2/3 were significantly increased in both groups of wild-type APAP-treated mice, though in TSP1−/− mice there was a significant decrease in pSMAD3 expression at 6 h, but not at 2 h, after APAP administration in comparison to wild-type mice (Fig. 4C). When individual pSMAD proteins were assessed, pSMAD2 was unchanged except in wild-type 300 mg/kg APAP-treated mice (Fig. S2A). However, protein expression of pSMAD3 was significantly increased in all APAP-treated mice, though there was a significant decrease in TSP1−/− mice treated with 600 mg/kg APAP compared to wild-type mice injected with the same concentration of APAP (Fig. S2B).

Fig. 4.

Fig. 4.

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Activation of TGFβ1 reduced in TSP1−/− mice. (A) Immunoblot images for TGFβ1 protein in the livers of wild-type and TSP1−/− mice treated with vehicle or 600 mg/kg APAP. β-actin is used as a protein input control. (B) Quantification of hepatic TGFβ1 immunoblot images with data expressed as TGFβ1/β-actin ratio with wild-type vehicle set to 1. (C) Relative protein expression of pSMAD2/3 as determined by ELISA in livers of wild-type and TSP1−/− mice administered vehicle or APAP. Data are presented as mean ± SEM with n = 5 for all groups. * = p < 0.05 compared to wildtype vehicle-treated mice. # = p < 0.05 compared to time-matched APAP-treated wild-type group.

3.4. TSP1−/− mice had elevated serum transaminases during APAP-induced liver injury

In APAP-treated mice, we observed no significant changes in necrosis between wild-type and TSP1−/− mice, though there was a greater increase of steatosis and vascular injury in TSP1−/− mice 6 h after APAP administration in comparison to to wild-type time-matched mice (Fig. 5A and B). Interestingly, in vehicle-injected mice, we observed a significant increase of necrosis in TSP1−/− compared to wild-type controls, indicating that cellular stress is occurring in this strain of mice under normal conditions (Fig. 5B). Serum ALT levels were significantly increased at 2 h and 6 h after APAP administration in wild-type and TSP1−/− mice, with TSP1−/− mice concentrations being significantly higher 6 h after APAP (Fig. 5C). Similar observations were found with AST concentrations in the serum, with a significant increase in all APAP-treated mice, and TSP1−/− mice had a significant increase from wild-type mice at 6 h after APAP injection (Fig. 5D). In order to see if the effects observed were due to changes in the metabolism of APAP, Cyp2E1 protein expression was assessed by immunoblots in all groups (Fig. 5E). No significant changes in hepatic Cyp2E1 expression were observed in any of the wild-type or TSP1−/− mice groups (Fig. 5F).

Fig. 5.

Fig. 5.

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TSP1−/− mice have worse APAP-induced liver injury. (A) Photomicrographs of H&E staining in livers from wild-type and TSP1−/− mice injected with vehicle or 600 mg/kg APAP. Images are shown as 200× magnification. (B) Area of hepatic necrosis as determined from quantitative analysis of H&E stained liver section from wild-type and TSP1−/− mice administered vehicle or 600 mg/kg APAP. (C) Serum ALT concentrations in wild-type of TSP1−/− mice treated with either vehicle or 600 mg/kg APAP. (D) Levels of AST in the serum of wild-type and TSP1−/− mice administered vehicle or APAP. (E) Representative Cyp2E1 immunoblot image from wild-type and TSP1−/− mice injected with vehicle or APAP with β-actin used as a protein input control. (F) Quantification of Cyp2E1 immunoblot images in wild-type and TSP1−/− mice treated with vehicle or APAP with data expressed as Cyp2E1/β-actin with wild-type vehicle set to the value of 1. Data are presented as mean ± SEM with n = 5 for all groups. * = p < 0.05 compared to wild-type vehicle-treated mice. # = p < 0.05 compared to time-matched APAP-treated wild-type group.

3.5. Mice injected with LSKL had no change in APAP-induced acute liver injury

In order to determine if the observations observed in TSP1−/− mice regarding APAP-induced liver injury was dependent upon activation of TGFβ1 alone, wild-type mice were injected with the peptide LSKL, or the inactive peptide SLLK, to inhibit the ability of TSP1 to activate latent TGFβ1. LKSL-injected APAP-treated mice had roughly the same degree of liver injury as SLLK-injected APAP-treated mice (Fig. S3A). In support of these histological assessments, the relative levels of ALT were equivalent between SLLK and LSKL-injected mice, giving support that inhibition of TSP1-dependent activation of TGFβ1 did not significantly influence liver injury (Fig. S3B). Therefore, these data support a second mechanism of action of TSP1 outside of TGFβ1 signaling to influence liver injury.

3.6. TSP1−/− mice have increased cell death and oxidative stress

With TSP1−/− mice demonstrating increased liver injury, mechanisms of cell death and oxidative stress were examined. Increased TUNEL staining was not observed in vehicle or 2 h after APAP treatment when comparing wild-type and TSP1−/− mice (Fig. 6A). However, 6 h after APAP injection, increased TUNEL staining was observed in both mouse strains (Fig. 6A). In TSP1−/− mice, the increase was significantly larger than in wild-type mice at 6 h following APAP administration, demonstrating that TSP1 can reduce cell death during this model of liver injury (Fig. 6B).

Fig. 6.

Fig. 6.

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APAP-induced liver injury is exacerbated in TSP1−/− mice. (A) Representative photomicrographs of TUNEL-stained liver section from wild-type and TSP1−/− mice injected with vehicle or 600 mg/kg APAP. Images are shown at 200× magnification. (B) Quantification of TUNEL-stained liver sections expressed as % area per field in livers from wild-type or TSP1−/− mice administered vehicle or APAP.

As cell death can result from increased oxidative stress, signaling pathways involved with this process were investigated. Nrf2 is a transcription factor that plays a central role in mitigating oxidative stress. The protein levels of Nrf2 were relatively static in wild-type mice groups (Fig. 7A and B). However, in TSP1−/− mice at 6 h after APAP injection, there was a significant decrease of Nrf2 protein levels (Fig. 7A and B). SOD1 is a protein that is transcribed by Nrf2 and has antioxidant activity (Akino et al., 2018). Similar to Nrf2, levels were static except in 6 h APAP-treated TSP1−/− mice, where levels were significantly decreased compared to wild-type vehicle-treated mice (Fig. 7C and D). Associated with this, there was increased immunostaining for nitrotyrosine in all APAP-treated groups with this staining increasing over time following APAP administration (Fig. 7E). In addition, TSP1−/− mice at 6 h following APAP injection had the most intense staining for nitrotyrosine (Fig. 7E). While lipid peroxidation is generally not a primary mechanism of APAP-induced liver injury, we observed an increase of MDA levels in both wild-type and TSP1−/− mice 6 h after APAP-administration, with TSP1−/− mice having a significant increase over wild-type at 6 h post-APAP injection (Fig. S4A). Collectively, these data demonstrate that deletion of TSP1 in mice leads to increased cell death and oxidative stress in the context of APAP-induced liver injury.

Fig. 7.

Fig. 7.

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Nrf2 and downstream transcriptional targets are suppressed in TSP1−/− mice. (A) Nrf2 immunoblots images in homogenized whole liver lysates from wild-type or TSP1−/− mice treated with vehicle or 600 mg/kg APAP. β-actin is used as a protein input control. (B) Quantification of Nrf2 immunoblot images in wild-type and TSP1−/− mice administered vehicle or 600 mg/kg APAP with data expressed as Nrf2/β-actin with the wild-type vehicle group set to the value of 1. (C) Representative SOD1 western blot images in whole liver homogenates from vehicle- or 600 mg/kg APAP-treated wild-type or TSP1−/− mice with β-actin used to normalize protein expression. (D) SOD1 immunoblot quantification from western blot images in vehicle- or 600 mg/kg APAP-treated wild-type or TSP1−/− mice. Data are presented as SOD1/β-actin with the vehicle-treated wild-type group set to the value of 1. (E) Immunohistochemistry images for nitrotyrosine in liver sections from wild-type and TSP1−/− mice administered vehicle or 600 mg/kg APAP. Photomicrographs are presented at 200× magnification. Data are presented as mean ± SEM with n = 5 for all groups. * = p < 0.05 compared to wild-type vehicle-treated mice. # = p < 0.05 compared to time-matched APAP-treated wild-type group.

3.7. Impaired GSH activity in TSP1−/− mice

During APAP-induced liver injury, GSH acts as a detoxifying agent for NAPQI and therefore, modulation of GSH levels or metabolism can directly influence oxidative stress and hepatotoxicity in this disease state. Levels of GSH were fully depleted at 2 h following APAP administration, with wild-type mice displaying a small recovery of GSH 6 h following APAP administration, while TSP1−/− mice had significantly less GSH at this time point (Fig. 8A). GSH synthesis is controlled by numerous enzymes including GCLC, GCLM and GSR (Bachhawat and Yadav, 2018). The expression of GCLC was significantly increased in wild-type mice 6 h after APAP administration, though TSP1−/− mice at this same time point had significantly less GCLC mRNA expression (Fig. 8B). GCLM levels were fairly static in all groups, though at 6 h after APAP, TSP1−/− mice had significantly lower levels compared to wild-type mice (Fig. 8C). GSR levels were suppressed in TSP1−/− mice for all groups including vehicle, with wild-type mice showing a significant increase compared to vehicle at 6 h after APAP administration (Fig. 8D).

Fig. 8.

Fig. 8.

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TSP1−/− mice have reduced hepatic GSH levels after APAP administration. (A) GSH concentrations in liver homogenates normalized to input protein from wild-type and TSP1−/− mice injected with vehicle or 600 mg/kg APAP. (B) Hepatic GCLC mRNA fold change in wild-type and TSP1−/− mice administered vehicle or APAP. (C) GCLM mRNA expression in livers from wild-type and TSP1−/− mice treated with vehicle or APAP. (D) Hepatic GSR mRNA fold change in wild-type and TSP1−/− mice injected with vehicle or APAP. Data are presented as mean ± SEM with n = 5 for all groups. * = p < 0.05 compared to wild-type vehicle-treated mice. # = p < 0.05 compared to time-matched APAP-treated wild-type group.

4. Discussion

The significant findings from this study relate to the role of TSP1 in the pathology associated with APAP-induced liver injury. Increased concentrations of APAP were found to lead to increased TSP1 expression and TGFβ1 signaling. This increase of active TGFβ1 protein was found to rely heavily on TSP1 as TSP1−/− mice were observed to have a significant reduction in active TGFβ1 protein expression. That being said, the use of LSKL peptide treatment demonstrated that TGFβ1 activation by TSP1 in this model plays little role in overall liver injury. However, TSP1−/− mice had worse APAP-induced liver injury, increased cell death, reduced Nrf2 expression and elevated oxidative stress and this was not due to a change in Cyp2E1 expression. TSP1−/− mice did have a significant reduction in GSH levels and suppressed expression of genes involved in its synthesis. Therefore, these data support that while TSP1 inhibition can reduce TGFβ1 activation during APAP-induced liver injury, it leads to a suppression of GSH levels which worsens hepatic damage.

During normal conditions, TSP1 is found to be expressed primarily in platelets and endothelial cells, with concentrations in the blood ranging from 20 ng/ml to 40 ng/ml in healthy patients (Starlinger et al., 2011; Baenziger et al., 1972; Mosher et al., 1982). These relatively static values and sites of expression can change, as increases of TSP1 expression have been shown during numerous disease states including pancreatic injury, cardiac dysfunction, glioblastoma multiforme, rheumatoid arthritis and others (Neuschwander-Tetri et al., 2006; Kim et al., 2017; Daubon et al., 2019; Suzuki et al., 2015). In the liver, TSP1 expression is minimal in normal states. However, during hepatic fibrosis, increased TSP1 protein expression is observed in hepatocytes and in sinusoidal endothelial cells (El-Youssef et al., 1999). During acute liver injury, not much is known about TSP1 and its cellular expression. Recently, we found that mice with acute liver failure due to azoxymethane-induced liver injury have a large increase of TSP1 expression and this was observed primarily in hepatocytes (Jefferson et al., 2020). In the current study, we found similar results with TSP1 being upregulated during APAP-induced hepatotoxicity and that this increase is localized primarily in hepatocytes. In addition, we observed a dose-dependent response, with higher concentrations of APAP leading to a larger increase of TSP1 expression. These data support that TSP1 is upregulated in the context of APAP-induced liver injury, similar to what is observed during models of liver disease and other disorders.

In the context of the current study, TSP1 activity was investigated for its role in TGFβ1 activation. TSP1 has N- and C-terminal globular domains, a region that shows sequence similarity to procollagen and three repeated sequence motifs (Chen et al., 2000). Of these motifs, type 1 has been shown to facilitate the activation of TGFβ1 (Chen et al., 2000). That being said, the activation of latent TGFβ1 in vitro has been shown to be a result of various stimuli including a change in pH to below 2 or above 8, increasing the temperature of cells to over 100 °C, urea treatment and application of strong detergents like sodium dodecyl sulfate (Khalil, 1999). More relevant activators of latent TGFβ1 in vivo include changes in expression or activity of TSP1, matrix metalloproteinase 9, pregnancy-specific beta 1-glycoproteins, αvβ6 integrin and others (Warren et al., 2018; Munger et al., 1999; Yu and Stamenkovic, 2000; Schultz-Cherry and Murphy-Ullrich, 1993). In the context of APAP-induced liver injury, it is evident that activation of TGFβ1 is a consequence of the increase of TSP1 expression and activity. In TSP1−/− mice, the substantial increase of active TGFβ1 protein was significantly reduced. That being said, pSMAD2/3 activity was significantly suppressed at 6 h post-APAP in TSP1−/− mice, but not at 2 h when compared to wild-type mice administered APAP. When investigated individually, the change of pSMAD2/3 protein was mostly a result of changes in pSMAD3 and not pSMAD2. Studies outside the liver have shown that there may be differential effects of pSMAD2 and pSMAD3 in transducing TGFβ1 signaling, but the potential effects of this during acute liver failure is currently unknown (Liu et al., 2016). With the disparity in changes of pSMAD2 and pSMAD3, it appears that post-translational mechanisms could influence their expression in TSP1−/− mice administered APAP. One possible mechanism is that decreased SMAD7 activity results in reduced degradation of TGFβ receptors via ubiquitylation and degradation (Ebisawa et al., 2001; Kavsak et al., 2000). Another potential mechanism is a decrease in phosphatase activity, such as protein phosphatase 1, that would increase phosphorylation of SMAD proteins independently of TGFβ1-dependent signaling (Shi et al., 2004). However, with the data presented demonstrating that LSKL treatment led to no significant change in liver injury in APAP-treated mice compared to SLLK-injected controls, TGFβ1 activation by TSP1 during APAP-induced liver injury plays a minimal role in liver injury and therefore, other aspects of TSP1 signaling need to be investigated.

TSP1 has a large interactome, with Resovi et al. reporting 83 proteins that interact specifically with TSP1 (Resovi et al., 2014). Of these, most are extracellular matrix proteins, proteases, growth factors or receptors. Due to the influences of these various factors, TSP1 can influence disease states in different manners depending upon the involvement of specific proteins that contribute to this disease state. For example, during pulmonary injury due to acute infection with P. aeruginosa, TSP1−/− mice show reduced survival, increased weight loss, impaired immune function and increased inflammatory injury compared to wild-type mice (Qu et al., 2018). However, during pulmonary hypertension induced by hypoxia or Schistosoma, TSP1 inhibition by LSKL peptide was found to improve outcomes compared to mice administered an inactive control peptide (Kumar et al., 2017). These diverging consequences of TSP1 have been observed during acute liver injury as well. In the azoxymethane model, inhibition of TSP1 signaling improved hepatic outcomes and measures of hepatic encephalopathy, while in the current study inhibition of TSP1 led to increased liver injury (Jefferson et al., 2020). One possibility as to why these differing effects may be observed during acute liver injury is due to the experimental model being used as 600 mg/kg APAP-treated mice have a rapid metabolism of APAP, NAPQI accumulation and generation of oxidative stress. On the other hand, in the azoxymethane model, there are no signs of overt liver injury or elevation of ALT for at least 4 h after injection and therefore there is a slower rate of liver injury, demonstrating less demand on the antioxidant capacity of the liver (Grant et al., 2018; Matkowskyj et al., 1999). With the increased oxidative stress observed in APAP-treated mice, TSP1 can induce Nrf2 signaling, potentially reducing oxidative injury. In the current study, TSP1−/− mice had reduced Nrf2 and SOD1 protein expression, as well as greatly increased levels of MDA compared to wild-type mice, demonstrating an impairment of the antioxidant response by the liver. While this likely is the primary contributor to increased liver injury observed in this study, another possibility is that antagonism of TSP1 could disrupt platelet adhesion (Seif et al., 2018). Therefore, TSP1−/− mice administered APAP may have increased vascular injury, though this is not supported in the current study as necrosis values were not significantly changed when compared to wild-type mice administred APAP. Together, the data presented support that TSP1 is a necessary component of the injury response by inducing Nrf2 signaling and reducing oxidative stress and injury in APAP-treated mice.

The primary regulator of injury during APAP hepatotoxicity is GSH, as its depletion leads to the accumulation of NAPQI. In this study, hepatic GSH levels were essentially depleted 2 h after 600 mg/kg of APAP administration in both wild-type and TSP1−/− mice. However, at 6 h after APAP administration, wild-type mice began to have a recovery of hepatic GSH, though this did not occur in TSP1−/− mice. In mammals, GSH synthesis requires the activity of two ATP-dependent glutamate-cysteine ligases, GCLC and GCLM, as well as GSR (Bachhawat and Yadav, 2018). The findings from this study show that the mRNA expression of GCLC, GCLM and GSR were significantly suppressed 6 h after APAP administration in TSP1−/− mice, likely contributing to a reduction in GSH concentrations. This may be associated with TSP1−/− mice as GSR mRNA was significantly reduced in vehicle-treated mice, but GCLC mRNA, GCLM mRNA and GSH concentrations were not. Another potential influence is that GCLC and GCLM are transcribed in response to Nrf2. Therefore, the reduced Nrf2 expression in TSP1−/− mice after APAP administration likely leads to reduced expression of these ligases and subsequently less GSH (Jin et al., 2019). The impaired recovery of GSH in TSP1−/− mice was also likely not a result of dysregulation of metabolism of APAP, as Cyp2E1 is one of the primary cytochrome p 450 enzymes responsible for metabolism of APAP and protein levels of Cyp2E1 were static across all groups (Raucy et al., 1989). Based upon these findings, it is most likely that the increased hepatic damage found in TSP1−/− mice administered APAP is due to a suppression of GSH synthesis and subsequent activity and this leads to increased oxidative stress and liver injury.

During APAP-induced liver injury, necrosis is the primary histological observation that can help determine the extent of liver injury. In the current study, there is a dose-dependent increase of necrosis as the concentration of APAP was increased from 300 mg/kg to 600 mg/kg in mice, which is expected. However, at the 600 mg/kg dose in wild-type and TSP1−/−, there was also no change observed in the necrotic area of the liver. That being said, there was increased TUNEL staining, serum tranasminases and oxidative/nitrosative stress giving support that the liver was impaired to a greater degree with increased DNA damage occurring in TSP1−/− mice. With the increase of TUNEL staining, it is possible that more apoptosis was leading to DNA fragmentation. We assessed a variety of different measures of apoptosis and did not see any significant changes between wild-type and TSP1−/− mice (data not shown). Another possibility for the increase of TUNEL staining is that DNA repair mechanisms are impaired. This has been investigated in retinoblastoma cells where TSP1 is silenced and reintroducing TSP1 can increase the expression of γ-H2AX, which is a biomarker for DNA double-strand breaks (Kuo and Yang, 2008; Chen et al., 2016). Therefore, our results with TSP1−/− are likely not explained through DNA repair mechanisms. Investigating the relationship between TSP1 and cell death in the context of liver injury is currently ongoing.

In conclusion, the results from the present study demonstrate that during APAP-induced liver injury, there is increased expression and signaling for TGFβ1 and TSP1 directly contributes to this increase in signaling. However, inhibition of this process with LSKL peptide injection led to little effect on the liver, giving support that TSP1 influences other aspects of APAP-induced liver injury. Global inhibition of TSP1 signaling, via the use of TSP1−/− mice, demonstrated that liver injury, measures of oxidative stress and cell death were increased, and these effects were associated with a decrease in Nrf2 expression and GSH activity. Therefore, TSP1 confers protection from APAP-induced liver injury by increasing the antioxidant capabilities of the liver, reducing cell death and improving liver function.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.taap.2020.115323.

Supplementary Material

Supplemental Figure 1
Supplemental Figure 4
Supplemental Figure 2
Supplemental Figure 3
Supplement Text

Acknowledgements

This material is the result of work supported with resources and the use of facilities at the Central Texas Veterans Health Care System, Temple, Texas. The content is the responsibility of the author(s) alone and does not necessarily reflect the views or policies of the Department of Veterans Affairs or the United States Government.

Funding

This work was supported by the Department of Veterans Affairs Biomedical Laboratory Research & Development Service [grant number BX003486].

Abbreviations:

ALT

alanine aminotransferase

APAP

N-Acetyl-p-Aminophenol

AST

aspartate aminotransferase

cDNA

complementary deoxyribonucleic acid

DAPI

4′6-diamidino-2-phenylindole

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

GCLC

glutamate-cysteine ligase catalytic subunit

GCLM

glutamate-cysteine ligase modulatory subunit

GSH

glutathione

GSR

glutathione-disulfide reductase

HE

hepatic encephalopathy

MDA

malondialdehyde

mRNA

messenger ribonucleic acid

NAPQI

N-acetyl-p-benzoquinone imine

Nrf2

nuclear factor erythroid 2-related factor 2

RT-PCR

real-time PCR

SEM

standard error of the mean

SOD1

superoxide dismutase 1

TGFβ1

transforming growth factor beta 1

TSP1

thrombospondin 1

TUNEL

terminal deoxynucleotidyl transferase dUTP nick end labeling

WT

wild-type

Footnotes

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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