Inhibition of thrombospondin‐1 reduces transforming growth factor beta 1 signaling, hepatic fibrosis and neurological deficits during cholestasis in rats

Abstract

IntroductionPrimary sclerosing cholangitis (PSC) is a liver disease associated with biliary inflammation, fibrosis and ultimately liver cirrhosis. During decompensated liver cirrhosis, hepatic encephalopathy (HE) can develop leading to increased morbidity and mortality. One rodent model that mimics clinical aspects of PSC is extrahepatic bile duct ligation (BDL). Transforming growth factor beta 1 (TGFβ1) has been shown to induce activation of hepatic stellate cells and promote liver fibrosis. For TGFβ1 to bind and activate its receptor, it must be released from a protein complex by interactions with other proteins. One of these is a matricellular protein called thrombospondin‐1 (TSP‐1). Therefore, we hypothesize that the expression of TSP‐1 is upregulated during BDL and promotes TGFβ1 signaling, liver injury, fibrosis and HE.MethodsMale Sprague Dawley rats (150–175 g) underwent BDL surgery and were implanted with osmotic minipumps that infused a TSP‐1 inhibitory peptide (LSKL, 100 g/day for 5 weeks) or inactive peptide (SLLK, 100 g/day for 5 weeks) into the peritoneum. Rotarod performance and open‐field tests were performed bi‐weekly starting at 2 weeks post‐surgery. After 5 weeks, rats were euthanized and tissue was collected. Serum ammonia, alanine and aspartate transaminases, total bilirubin and alkaline phosphatase were measured. TGFβ1, SMAD2/3, phosphorylated SMAD2/3 and TSP‐1 expression were assessed in liver, serum and cortex. Fibrosis was determined in liver sections by Sirius Red staining, hydroxyproline assays and qPCR analyses for alpha smooth muscle actin, fibronectin 1, and matrix metalloproteinase 9. In order to assess HE, cerebral edema, microglia activation and proinflammatory cytokine expression were assessed in the cortex.ResultsTGFβ1 signaling and TSP‐1 expression were increased in the liver and serum, but not the cortex, of BDL rats. Infusion of LSKL into BDL rats reduced liver injury, as determined by improved serum chemistry, in comparison to SLLK‐infused BDL rats. Sirius red staining, hydroxyproline content and fibrosis‐associated gene expression were reduced in livers from LSKL‐infused BDL rats compared to SLLK‐infused BDL rats. Time and distance traversed on a rotarod and overall activity were greater in BDL rats infused with LSKL compared to BDL rats infused with SLLK. Ammonia concentrations in serum, cerebral edema, microglia activation and cortex cytokine expression were significantly decreased in BDL rats infused with LSKL compared to SLLK‐infused BDL rats.ConclusionsInhibition of TSP‐1 via LSKL reduced TGFβ1 signaling, liver injury, fibrosis and neurological deficits in BDL rats giving support that antagonism of TSP‐1 could be a therapeutic option for the management of cholestatic liver disease.Support or Funding InformationThis study was funded by a VA Career Development award (BX003486) from the United States Department of Veterans Affairs to Dr. Matthew McMillin. This study was also funded by NIH R01 awards (DK082435 and DK112803) and a VA Merit award (BX002638) from the United States Department of Veterans Affairs to Dr. Sharon DeMorrow.