In the two years of the granting period we have completed the localization by light and electron microscopy of four components of the hepatic extracellular matrix in normal, fibrotic and cirrhotic liver. Collagen deposition in our system is a late event in the cirrhotic process, the earliest detectable change, is increased synthesis of fibronectin by the hepatocyte with subsequent deposition of this fibronectin in the space of Disse. Our working hypothesis is that not only does fibronectin play a critical role in the fibrotic process, but that this role may be mediated by post-translational modifications in the fibronectin secreted by the injured hepatocyte. We are proposing to complete the morphological studies initiated two years ago, obtaining precise quantitation (with computer-aided image analysis) of the changes in the hepatic extracellular matrix in the normal, fibrotic and cirrhotic liver. New studies include: The reversal of the cirrhosis induced by CCl4 and characterization and quantitation of the catabolism of this extracellular matrix during the reversal of the cirrhosis. These studies will use light and electron immunohistochemistry combined with image analysis. To characterize and quantitate the matrix produced by primary cultures of hepatocytes obtained from normal, fibrotic and cirrhotic livers using immunohistochemistry, quantitative rocket immunoelectrophoresis and electroimmunobloting. We will use in vitro sublethal hepatocyte injury with CCl4 and compare the matrix secreted in vitro by these injured hepatocytes with that secreted by normal hepatocytes. The last part of the proposal will initiate studies designed to characterize any possible changes in the fibronectin secreted by hepatocytes as a response to injury. Plasma and tissue fibronectin from rats exposed to CCl4 will be compared with plasma and tissue fibronectin from normal rats. The fibronectins will be characterized by PAGE, IEF, electroimmunoblotting and crossed immunoaffinity electrophoresis with ConA in the first dimension. Using this combination of techniques it should be possible to detect any post-translational modifications in the fibronectin secreted by rat hepatocytes in response to chronic injury.
