Thrombospondin‐1 Contributes to Hepatic Pathology and Systemic Complications in the Acetaminophen and Azoxymethane Mouse Models of Acute Liver Failure

Acute liver failure results from a significant loss of liver function and has few effective treatments and poor clinical outcomes. Numerous systemic complications can occur following acute liver failure though the development of neurological complications, a disease state called hepatic encephalopathy, greatly increases mortality. Previously we have demonstrated that transforming growth factor beta 1 (TGFβ1) is upregulated during acute liver failure and promotes disease progression. TGFβ1 requires interactions with other proteins, such as thrombospondin‐1 (TSP‐1), to become activated and be capable of binding its receptors. Therefore, we hypothesize that TSP‐1 contributes to acute liver failure disease progression through its activation of TGFβ1. Methods Male C57Bl/6 mice or TSP‐1 knockout mice were injected with acetaminophen (APAP; 300 and 600 mg/kg) to generate acute liver injury or azoxymethane, (AOM; 100 mg/kg) to induce acute liver failure and hepatic encephalopathy. APAP‐treated mice had serum and liver collected at 6 hours and 24 hours post injection while AOM‐treated mice had serum, liver and brain collected once at coma. Liver pathology was assessed in both models by H&E staining, cytokine ELISAs and reactive oxygen species (ROS), reactive nitrogen species (RNS), nitrotyrosine and transaminase assessments. Hepatic glutathione concentrations were assessed in APAP‐treated mice using a fluorometic assay. TGFβ1 and TSP‐1 expression was assessed by immunoblotting, immunohistochemistry, ELISA and/or real‐time PCR in both models. In AOM‐treated mice, cognitive impairment was monitored by assessment of reflex responses and ataxia and cerebral edema, microglia activation and neuroinflammation via cytokine ELISAs. Results Mice injected with AOM or APAP had elevated hepatic TGFβ1 and TSP‐1 levels, which colocalized primarily to hepatocytes. AOM and APAP‐treated mice with genetic knockout of TSP‐1 had reduced hepatocyte necrosis, injury and oxidative stress compared to wildtype controls as assessed by H&E staining, ROS, RNS and serum transaminase concentrations. Levels of hepatic and circulating proinflammatory cytokines were also reduced in both models in TSP‐1 knockout mice compared to wildtype controls. APAP‐treated mice had reduced glutathione concentrations that were increased in TSP‐1 knockout mice. AOM‐treated TSP‐1 knockout mice also had delayed development of hepatic encephalopathy as indicated by a delay in the time to coma, a reduction in cerebral edema, attenuated microglia activation, and reduced neuroinflammation compared to wildtype controls. Conclusions Strategies employed to reduce TSP‐1 signaling in APAP‐treated mice reduced liver damage and pathology while AOM‐treated mice had reduced liver damage, neuroinflammation and improved neurological outcomes. Therefore, targeting TSP‐1 signaling may be a novel therapeutic target for the management of acute liver failure and/or hepatic encephalopathy. Support or Funding Information The study was funded by a VA Career Development Award‐2 (BX003486‐01) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service (BLR&D) to Dr. McMillin, an NIH R01 award (DK082435) to Dr. DeMorrow and a VA Merit award (BX002638‐01) from the United States Department of Veterans Affairs BLR&D to Dr. DeMorrow. 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 views are those of the authors and do not necessarily represent the views of the Department of Veterans Affairs.