Advanced glycation end products induce production of reactive oxygen species via the activation of NADPH oxidase in murine hepatic stellate cells.
OBJECTIVE
Advanced glycation end products are known to play an important role in the metabolic syndrome and were recently suggested to contribute to liver fibrosis development. However, little is known about the effect of advanced glycation end products on hepatic stellate cells, the major contributors to liver fibrosis development. We therefore studied the effect of advanced glycation end products on reactive oxygen species generation, a main feature for the activation hepatic stellate cells.


METHODS
Three different types of advanced glycation end products were generated by BSA incubation with different substrates. The presence of advanced glycation end product receptors was examined by RTq-PCR, immunofluorescence and western blotting. Reactive oxygen species production was measured using DCFH-DA.


RESULTS
Hepatic stellate cells express five advanced glycation end product receptors: Galectin-3, CD36, SR-AI, SR-BI and RAGE. All receptors, except SR-BI, showed up-regulation during HSC activation. All three advanced glycation end product types induced reactive oxygen species generation. DPI and NSC, a NADPH oxidase and a Rac1 inhibitor respectively, inhibited reactive oxygen species production. Rottlerin, a molecule often used as a PKCdelta inhibitor, also abrogated reactive oxygen species production. SiRNA mediated knockdown of p47(phox), Rac1 and PKCdelta decreased reactive oxygen species production induced by advanced glycation end products, establishing a role for these proteins in reactive oxygen species induction.


CONCLUSIONS
The demonstration of advanced glycation end product-induced reactive oxygen species generation in hepatic stellate cells unveils a potential new route through which advanced glycation end products induce liver fibrosis in the metabolic syndrome.
Department of Cell Biology, Vrije Universiteit, Laarbeeklaan 103, Brussels, Belgium.
Eduardo L M
EL
Guimarães
Christophe
C
Empsen
Albert
A
Geerts
Leo A
LA
van Grunsven
D000818:Animals; D002478:Cells, Cultured; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D055166:Hepatic Stellate Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D051379:Mice; D023421:Models, Animal; D019255:NADPH Oxidases / Q000378:metabolism*; D009479:Neuropeptides / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020830:rac1 GTP-Binding Protein
D017127:Glycation End Products, Advanced; D009479:Neuropeptides; D034741:RNA, Small Interfering; C484602:Rac1 protein, mouse; D017382:Reactive Oxygen Species; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D051745:Protein Kinase C-delta; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein