The global objective is to determine the mechanism of cold preservation injury to hepatic sinusoidal endothelial cells and thereby develop more efficient liver preservation solutions. Currently, preservation injury results in immediate or early (1 year) failure of about 10% of liver allografts, and prevents the initiation of programs to split one liver for use in two adults. The critical injury in liver preservation is to sinusoidal endothelial cells (SEC). When the liver is cold preserved, SEC partly detach from the underlying connective tissue matrix, assume a rounded shape, and become activated. In this state SEC present an adhesive surface to leukocyte and platelets and also promote coagulation, so that on reperfusion the graft is injured or destroyed. Despite an extensive literature in this area virtually nothing is known of injury on reperfusion. Our preliminary studies suggest cold induces elevations in intracellular calcium which leads to disassembly of actin. Actin disassembly is the central event in cold preservation injury. Actin disassembly in turn induces secretion of matrix metalloproteinases (MMPs) by SEC and conversion of adhesion molecules to an activated state.
The specific aims are: 1. To define the mechanisms of cold induced actin disassembly in sinusoidal endothelial cells and find methods to inhibit them. 2. To determine the mechanisms by which actin disassembly activates the endothelial cell surface. 3. To produce improved preservation solutions. Preliminary findings are most consistent with the following hypotheses. Cold induces a number of events which favor actin disassembly. Chief among these is elevation of [Ca++]i which probably occurs due to inactivation of Ca++ ATPase by cold, allowing unrestricted lead of calcium form stores into cytoplasm. Elevated [Ca++]i initiates actin disassembly by increased activity of the actin capping and severing protein gelsolin, or through increased activity of the calcium dependent enzyme calpain. Once actin disassembly occurs there is rapid activation of preformed cell surface MMPs and of cell surface proteins responsible for platelet and neutrophil adhesion. MMP activity seems to be needed for activation of these adhesion molecules. These hypotheses will be tested in isolated SEC from normal rats and mice and in SEC from gelsolin and MMP9 deficient mice. A potentially beneficial improvements in preservation solutions are identified they will be tested in whole isolated livers and by transplantation in the rat.
| Topp, Stefan A; Upadhya, Gundumi A; Strasberg, Steven M (2004) Cold preservation of isolated sinusoidal endothelial cells in MMP 9 knockout mice: effect on morphology and platelet adhesion. Liver Transpl 10:1041-8 |
| Topp, Stefan A; Upadhya, Gundumi A; Strasberg, Steven M (2003) Leukocyte adhesion to cold-preserved rat endothelial cells: role of actin disassembly and ICAM-1. Liver Transpl 9:1286-94 |
| Upadhya, G Aravinda; Topp, Stefan A; Hotchkiss, Richard S et al. (2003) Effect of cold preservation on intracellular calcium concentration and calpain activity in rat sinusoidal endothelial cells. Hepatology 37:313-23 |
| Upadhya, G Aravinda; Strasberg, Steven M (2002) Platelet adherence to isolated rat hepatic sinusoidal endothelial cells after cold preservation. Transplantation 73:1764-70 |
