The Reg3α (HIP/PAP) Lectin Suppresses Extracellular Oxidative Stress in a Murine Model of Acute Liver Failure.
OBJECTIVE
Acute liver failure (ALF) is a rapidly progressive heterogeneous illness with high mortality rate and no widely accessible cure. A promising drug candidate according to previous preclinical studies is the Reg3α (or HIP/PAP) lectin, which alleviates ALF through its free-radical scavenging activity. Here we study the therapeutic targets of Reg3α in order to gain information on the nature of the oxidative stress associated with ALF.


METHODS
Primary hepatocytes stressed with the reactive oxygen species (ROS) inducers TNFα and H2O2 were incubated with a recombinant Reg3α protein. ALF was induced in C57BL/6J mice by an anti-CD95 antibody. Livers and primary hepatocytes were harvested for deoxycholate separation of cellular and extracellular fractions, immunostaining, immunoprecipitation and malondialdehyde assays. Fibrin deposition was studied by immunofluorescence in frozen liver explants from patients with ALF.


RESULTS
Fibrin deposition occurs during experimental and clinical acute liver injuries. Reg3α bound the resulting transient fibrin network, accumulated in the inflammatory extracellular matrix (ECM), greatly reduced extracellular ROS levels, and improved cell viability. Hepatocyte treatment with ligands of death receptors, e.g. TNFα and Fas, resulted in a twofold increase of malondialdehyde (MDA) level in the deoxycholate-insoluble fractions. Reg3α treatment maintained MDA at a level similar to control cells and thereby increased hepatocyte survival by 35%. No antioxidant effect of Reg3α was noted in the deoxycholate-soluble fractions. Preventing fibrin network formation with heparin suppressed the prosurvival effect of Reg3α.


CONCLUSIONS
Reg3α is an ECM-targeted ROS scavenger that binds the fibrin scaffold resulting from hepatocyte death during ALF. ECM alteration is an important pathogenic factor of ALF and a relevant target for pharmacotherapy.
INSERM, U1193, Centre Hépatobiliaire, Villejuif, F-94800, France; Université Paris-Sud, Faculté de Médecine, Villejuif, F-94800, France.
Nicolas
N
Moniaux
INSERM, U1193, Centre Hépatobiliaire, Villejuif, F-94800, France; Université Paris-Sud, Faculté de Médecine, Villejuif, F-94800, France.
Marion
M
Darnaud
INSERM, U1193, Centre Hépatobiliaire, Villejuif, F-94800, France; Université Paris-Sud, Faculté de Médecine, Villejuif, F-94800, France.
Kévin
K
Garbin
INSERM, U1193, Centre Hépatobiliaire, Villejuif, F-94800, France; Université Paris-Sud, Faculté de Médecine, Villejuif, F-94800, France.
Alexandre
A
Dos Santos
INSERM, U1193, Centre Hépatobiliaire, Villejuif, F-94800, France; Université Paris-Sud, Faculté de Médecine, Villejuif, F-94800, France; Assistance Publique-Hôpitaux de Paris (APHP), Hôpital Universitaire Paul Brousse, Villejuif, F-94800, France.
Catherine
C
Guettier
INSERM, U1193, Centre Hépatobiliaire, Villejuif, F-94800, France; Université Paris-Sud, Faculté de Médecine, Villejuif, F-94800, France; Assistance Publique-Hôpitaux de Paris (APHP), Hôpital Universitaire Paul Brousse, Villejuif, F-94800, France.
Didier
D
Samuel
Alfact Innovation, Paris, France.
Gilles
G
Amouyal
Alfact Innovation, Paris, France.
Paul
P
Amouyal
INSERM, U1193, Centre Hépatobiliaire, Villejuif, F-94800, France; Université Paris-Sud, Faculté de Médecine, Villejuif, F-94800, France.
Christian
C
Bréchot
INSERM, U1193, Centre Hépatobiliaire, Villejuif, F-94800, France; Université Paris-Sud, Faculté de Médecine, Villejuif, F-94800, France; Assistance Publique-Hôpitaux de Paris (APHP), Hôpital Universitaire Paul Brousse, Villejuif, F-94800, France.
Jamila
J
Faivre
D000328:Adult; D000368:Aged; D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000378:metabolism*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005109:Extracellular Matrix / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism*; D005260:Female; D005337:Fibrin / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D037181:Lectins, C-Type / Q000378:metabolism*; D017114:Liver Failure, Acute / Q000378:metabolism* / Q000473:pathology*; D008297:Male; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D008954:Models, Biological; D018384:Oxidative Stress; D000073718:Pancreatitis-Associated Proteins; D019014:fas Receptor / Q000378:metabolism
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D037181:Lectins, C-Type; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D019014:fas Receptor; D005337:Fibrin