Thrombospondin‐1 Promotes Hepatic Injury and Extrahepatic Complications during Acute Liver Failure in Mice

Acute liver failure is a loss of liver function as a result of significant hepatic dysfunction and is associated with poor clinical outcomes. Patients with acute liver failure often present with neurological complications, called hepatic encephalopathy, which further increases mortality associated with this disease state. Transforming growth factor beta 1 (TGFβ1) becomes upregulated following liver damage and we have shown that this protein promotes acute liver failure pathogenesis. However, TGFβ1 can only exert an effect following proteolytic activation of latent TGFβ1, which can occur via thrombospondin‐1 (TSP‐1). Therefore, we hypothesize that circulating hepatic‐derived TGFβ1 is activated by TSP‐1, which exacerbates the hepatic and extrahepatic complications associated with acute liver failure. Methods Male C57Bl/6 mice were injected with azoxymethane, (AOM; 100 mg/kg) to induce acute liver failure and hepatic encephalopathy. In parallel, male C57Bl/6 mice were injected with acetaminophen (APAP; 300 mg/kg and 600 mg/kg) as a second model of liver failure. TSP‐1 knockout mice and wild‐type controls were injected with AOM to assess TSP‐1 dependent effects in this model. In AOM‐treated mice, cognitive impairment was monitored by assessment of reflex responses and ataxia. At coma in AOM‐treated mice or 24 hours post injection in APAP‐treated mice, liver, brain and serum were collected. Liver histology was assessed by H&E staining, hepatic apoptosis was assessed through cleaved caspase 3 immunohistochemistry, and hepatic inflammation was determined via IL‐1β mRNA expression. TGFβ1 and TSP‐1 expression was assessed by immunoblotting, immunohistochemistry and/or real‐time PCR. Cerebral edema and microglia activation were assessed in AOM‐treated mice. Results Mice injected with AOM or APAP had elevated hepatic and circulating TGFβ1 levels, which colocalized primarily to hepatocytes. AOM and APAP‐treated mice also had elevations of hepatic TSP‐1 levels that again localized to hepatocytes. AOM‐treated mice with genetic knockout of TSP‐1 had reduced hepatocyte apoptosis and hepatic injury compared to wildtype controls. 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 and an attenuation of microglia activation compared to wildtype controls. Conclusions TGFβ1 and TSP‐1 were elevated in the livers of AOM and APAP‐treated mice. Strategies employed to reduce TSP‐1 signaling in AOM‐treated mice reduced liver damage and neuroinflammation associated with acute liver failure and led to improved outcomes. Therefore, targeting TSP‐1 signaling may be a novel therapeutic target for the management of acute liver failure and hepatic encephalopathy. Support or Funding Information This study was funded by an NIH R01 award (DK082435) and a VA Merit Award (BX002638). 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 contents do not represent the views of the U.S. Department of Veterans Affairs of the United States Government.