The broad goal of this research is to establish the physiologic significance of mitogenic growth factors in liver repair after chronic injury. This proposal focuses on the potential roles in repair for transforming growth factors a&B (TGFalpha &beta), hepatocyte growth factor (HGF), and Kupffer cell derived tumor necrosis factor alpha (TNFalpha) which are known to regulate liver regeneration after acute injury. There are five specific aims.
Aim 1 : Determine TGFalpha&Beta, HGF, and TNFalpha gene expression in the liver during chronic, cholestatic injury and repair. The simultaneous expression of protooncogene mRNAs coding for receptors for TGFalpha and HGF, c-erbB and c-met, will be determined and protein levels will be measured. Cholestasis will be initiated in rats by vessel loop suspension of the common bile duct for increasing periods of injury; release of the loop allows biliary decompression to simulate reconstruction and initiate repair. mRNA levels will be quantified by ribonuclease protection assays and protein by Western blots.
Aim 2 : Localize the cellular sources of HGF and TGF alpha & Beta mRNA and proteins during repair after chronic injury b a combination of in situ hybridization and immunohistochemical techniques.
Aim 3 : Related changes in steady state levels of growth factor and receptor expression to in vivo hepatocellular DNA synthesis by immunohistochemistry for proliferating cell nuclear antigen. Important modulator os the regenerative response will be studied during repair after Kupffer cell blockade by the injection of gadolinium and after endotoxin restriction by the administration of Polymyxin B.
Aim 4 : Examine functional and proliferative changes to in vitro TGFalpha and HGF stimulation in hepatocytes isolated after increasing periods of cholestatic injury. Hepatocytes will be extracted and purified by centrifugal elutriation and in vitro morphology, DNA synthesis, and albumin synthesis will be measured.
Aim 5 : Establish the autocrine mechanism(s) of growth factor stimulation by TGFalpha on normal and injured hepatocytes by measuring DNA synthesis, the hepatocytes expression of c-erbB and, the levels of TGFalpha mRNA. These studies are designed to test the hypothesis that after acute injury, liver repair and regeneration share similarities in the growth factor control of hepatocyte proliferation. However, because regeneration yields a normal liver, and repair often lead to scar, quantitative differences in either growth factors and their receptors, or the hepatocyte response to growth factor stimulation must exist to slow the course, and inhibit the quality of repair. Data from this combined in vivo and in vitro approach should provide insights into the role and mechanism of action of growth factors in chronic liver injury and repair.
