Septicemia with resultant exposure to gram-negative bacterial lipopolysaccharide (LPS) is a major clinical problem associated with high mortality and a myriad of pathophysiological alterations, including liver failure. In vivo, LPS triggers a cascade of events involving cellular and soluble mediators of inflammation that culminates in liver dysfunction and loss of integrity of hepatic parenchymal cells. Kupffer cells, neutrophils and platelets are among the cellular mediators critical to the development of LPS-induced liver injury. The investigator's recent studies in rats indicate that thrombin activated during LPS exposure in vivo is essential to the pathogenesis. Moreover, its critical role is independent of its ability to form obstructive fibrin clots in the vasculature, which raises interest in the possibility that its important action occurs through receptor-mediated interaction with cells. Thrombin appears to act late in the cascade of inflammatory events that leads to hepatic dysfunction, that is, close in time to the genesis of parenchymal cell injury. Knowledge of such """"""""distal events"""""""" is likely to be useful in developing effective clinical intervention. The overall goal of this proposal is to understand the mechanisms by which thrombin participates in liver injury in the rat.
In Aim 1, studies in vivo and in isolated livers will be performed to define the temporal relationship between thrombin's critical action(s) and the onset of overt liver injury. Experiments in Aim 2 will test the hypothesis that thrombin acts by increasing the sensitivity of hepatic parenchymal cells (HCs) to injury during LPS exposure. Studies will be performed using isolated, perfused livers or HCs in culture to determine if thrombin exposure increases susceptibility of HCs to potentially injurious insults. Mechanisms by which thrombin acts on HCs will be addressed. The hypothesis that thrombin participates in hepatic injury by stimulating critical inflammatory cells to release toxic products will be addressed in Aim 3. This will be accomplished in part by evaluating thrombin's potential to cause injury to perfused livers from rats which had been depleted of neutrophils, platelets or Kupffer cells and then treated with LPS. Experiments employing cocultures of HCs with neutrophils, platelets or Kupffer cells will determine if thrombin causes the receptor-mediated release of a cytotoxic factor from a critical inflammatory cell and/or acts by proteolytically bioactivating a soluble inflammatory mediator (Aim 4). Results from these studies will reveal critical interactions between thrombin and cells and mechanisms by which they result in liver injury during LPS exposure. The long-term goal of these studies is to develop an understanding of inflammatory responses that will result in interventions that are effective in reducing mortality in people with organ failure from gram-negative bacterial sepsis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK050728-05
Application #
6177491
Study Section
Special Emphasis Panel (ZRG4-ALTX-4)
Program Officer
Serrano, Jose
Project Start
1996-08-01
Project End
2001-07-31
Budget Start
2000-08-01
Budget End
2001-07-31
Support Year
5
Fiscal Year
2000
Total Cost
$223,916
Indirect Cost
Name
Michigan State University
Department
Pharmacology
Type
Schools of Veterinary Medicine
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Copple, Bryan L; Rondelli, Catherine M; Maddox, Jane F et al. (2004) Modes of cell death in rat liver after monocrotaline exposure. Toxicol Sci 77:172-82
Copple, Bryan L; Moulin, Frederic; Hanumegowda, Umesh M et al. (2003) Thrombin and protease-activated receptor-1 agonists promote lipopolysaccharide-induced hepatocellular injury in perfused livers. J Pharmacol Exp Ther 305:417-25
Copple, Bryan L; Woolley, Brook; Banes, Amy et al. (2002) Anticoagulants prevent monocrotaline-induced hepatic parenchymal cell injury but not endothelial cell injury in the rat. Toxicol Appl Pharmacol 180:186-96
Moulin, F; Copple, B L; Ganey, P E et al. (2001) Hepatic and extrahepatic factors critical for liver injury during lipopolysaccharide exposure. Am J Physiol Gastrointest Liver Physiol 281:G1423-31