PBC is a cholangiopathy characterized by initial biliary proliferation followed by progressive ductopenia and cirrhosis. The dominant negative transforming growth factor-β receptor II (dnTGFβRII) murine model presents with features of human PBC. Knockdown of the SCT/SR axis (that stimulates bicarbonate secretion via CFTR and AE2) ablates biliary growth and bicarbonate secretion, and increases apoptosis during cholestasis. Aim: to determine the mechanisms that regulate biliary proliferation, apoptosis and fibrosis in dnTGFβRII mice and human PBC samples. Methods: Serum, total liver and cholangiocytes were obtained from male and female wild-type (WT) and dnTGFβRII mice at 12 and 34 wk of age. Biliary proliferation was evaluated by qPCR for PCNA in cholangiocytes. Intrahepatic bile duct mass (IBDM) was measured. Apoptosis was evaluated by qPCR for Bax and cleaved caspase 3 in cholangiocytes, and TUNEL analysis in liver sections. Fibrosis was measured by qPCR for fibronectin, collagen type1a, and α-smooth muscle actin in liver samples and Sirius Red staining in liver samples. SCT/SR/CFTR/AE2 axis expression was assessed by qPCR in cholangiocytes. Secretin was measured in serum and cholangiocyte supernatants by EIA. In total liver from controls (from healthy part of the liver in a patient with hepatic metastasis from colon cancer) and PBC patients (stage I-III, female) the expression of SCT/SR/CFTR/AE2 and fibrotic genes was assessed by qPCR. Secretin was measured in serum and bile from control and PBC patients. Results: In dnTGFβRII mice there was increased duct proliferation and IBDM compared to 12 wk old WT mice, but this was reduced in 34 wk old dnTGFβRII mice compared to WT. Collagen deposition and fibrotic gene expression increased in dnTGFβRII compared to WT mice in both 12 and 34 wk old mice. dnTGFβRII mice had increased liver damage and biliary expression of SCT/SR/ CFTR/AE2 and decreased apoptosis in 12 wk old dnTGFβRII mice compared to WT; however, 34 wk old dnTGFβRII mice had increased liver damage and apoptosis but decreased biliary expression of SCT/SR/CFTR/AE2 compared to WT. SCT levels in biliary supernatants was decreased, but increased in serum in dnTGFβRII mice compared to WT, suggesting that SCT comes from other sources such as S cells. There was increased fibrosis and SCT/SR/CFTR/AE2 in early stage PBC samples, but decreased expression of SCT/SR/CFTR/AE2 in advanced PBC samples compared to controls. SCT levels increased in serum but decreased in bile in PBC compared to control. Conclusion: Loss of biliary bicarbonate secretion may contribute to disease progression, which may be partly counteracted by peripheral sources of SCT.
Down-regulation of the secretin/SR (SCT/SR) axis is coupled with functional biliary damage in dnTGF beta RII mice and human primary biliary cirrhosis (PBC)
FRANCHITTO, Antonio;
2015-01-01
Abstract
PBC is a cholangiopathy characterized by initial biliary proliferation followed by progressive ductopenia and cirrhosis. The dominant negative transforming growth factor-β receptor II (dnTGFβRII) murine model presents with features of human PBC. Knockdown of the SCT/SR axis (that stimulates bicarbonate secretion via CFTR and AE2) ablates biliary growth and bicarbonate secretion, and increases apoptosis during cholestasis. Aim: to determine the mechanisms that regulate biliary proliferation, apoptosis and fibrosis in dnTGFβRII mice and human PBC samples. Methods: Serum, total liver and cholangiocytes were obtained from male and female wild-type (WT) and dnTGFβRII mice at 12 and 34 wk of age. Biliary proliferation was evaluated by qPCR for PCNA in cholangiocytes. Intrahepatic bile duct mass (IBDM) was measured. Apoptosis was evaluated by qPCR for Bax and cleaved caspase 3 in cholangiocytes, and TUNEL analysis in liver sections. Fibrosis was measured by qPCR for fibronectin, collagen type1a, and α-smooth muscle actin in liver samples and Sirius Red staining in liver samples. SCT/SR/CFTR/AE2 axis expression was assessed by qPCR in cholangiocytes. Secretin was measured in serum and cholangiocyte supernatants by EIA. In total liver from controls (from healthy part of the liver in a patient with hepatic metastasis from colon cancer) and PBC patients (stage I-III, female) the expression of SCT/SR/CFTR/AE2 and fibrotic genes was assessed by qPCR. Secretin was measured in serum and bile from control and PBC patients. Results: In dnTGFβRII mice there was increased duct proliferation and IBDM compared to 12 wk old WT mice, but this was reduced in 34 wk old dnTGFβRII mice compared to WT. Collagen deposition and fibrotic gene expression increased in dnTGFβRII compared to WT mice in both 12 and 34 wk old mice. dnTGFβRII mice had increased liver damage and biliary expression of SCT/SR/ CFTR/AE2 and decreased apoptosis in 12 wk old dnTGFβRII mice compared to WT; however, 34 wk old dnTGFβRII mice had increased liver damage and apoptosis but decreased biliary expression of SCT/SR/CFTR/AE2 compared to WT. SCT levels in biliary supernatants was decreased, but increased in serum in dnTGFβRII mice compared to WT, suggesting that SCT comes from other sources such as S cells. There was increased fibrosis and SCT/SR/CFTR/AE2 in early stage PBC samples, but decreased expression of SCT/SR/CFTR/AE2 in advanced PBC samples compared to controls. SCT levels increased in serum but decreased in bile in PBC compared to control. Conclusion: Loss of biliary bicarbonate secretion may contribute to disease progression, which may be partly counteracted by peripheral sources of SCT.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
