Publikationsserver der RWTH Aachen University
Therapeutic application of dominant-negative PDGF beta-receptor in liver fibrosis
Abstract
dc:descriptionUpon liver injury hepatic stellate cells (HSC) undergo phenotype transformation with acquisition of myofibroblast-like features characterized by increased cell proliferation, motility, contractility and extra cellular matrix production. HSC activation is regulated by several cytokines and growth factors including platelet derived growth factor B-chain (PDGF-BB) which exerts cellular effects by binding to PDGF receptors (PDGFR) and further inducing receptor dimerization and tyrosine-autophosphorylation, thus initiating various signaling pathways, amongst which MAPK and PI3K as principals. These PDGF signaling pathways may provide a potential therapeutic target to modulate the fibrotic response in liver. We therefore generated a dominant-negative soluble PDGFR consisting of PDGFRBeta extracellular domains connected to the IgG-Fc part of human immoglobulin, and used adenovirus type 5 mediated gene transfer into Cos-7 cells and HSC. This Ad5-CMV-sPDGFRBeta construct produced a secreted chimeric protein with two cystein residues at the hinge region of the IgG-Fc part, culminating in predimerized soluble receptors. This PDGFRBeta secreted efficiently into the conditioned media of both Cos-7 cells and HSC and binds PDGF-BB. The secreted protein from Cos-7 cells was purified and applied to test its inhibitory effects on PDGF signaling in HSC. The soluble PDGFRBeta did inhibit autophosphorilation of endogenous membrane-bound receptors upon PDGF stimulation and completely blocked p44 and p42 (ERK1 and ERK2) phosphorylation. PDGF activation of ERK triggers proliferative responses in HSC, further underlining that our sPDGFRBeta inhibits HSC proliferation and DNA synthesis along with inhibition of PDGF-induced PI3K/Akt activation, thus leading to reduced collagen type I(Alphal) gene expression. Adenovirus-mediated sPDGFRBeta gene transfer in HSC in vitro proved highly effective. Infected HSC produced the soluble receptor to scavenge available PDGF-BB and inhibit HSC proliferation. Ad5-CMV-sPDGFRBeta infected HSC did inhibit also autocrine looping on PDGF mRNA production resulting in decreased expression of thrombospondin-1, a latent TGF-Beta activator, leading to decreased fibrogenesis as proven by reduced levels of collagen type I(Alphal) mRNA compared to the control cultures.We then used a bile duct ligation (BDL) rat model to examine the effects of sPDGFRBeta on hepatic fibrogenesis. It is known that expression of PDGFRBeta-subunit does increase during experimental liver injury. In our model both approaches, adenovirus mediated sPDGFRBeta gene transfer and direct application of sPDGFRBeta as purified protein, significantly attenuated ongoing fibrosis in BDL rats as evidenced by reduced expression of Alpha-SMA and collagen type I(AlphaI). In conclusion, our experiments did demonstrate the potent antifibrogenetic effects of sPDGFRBeta, a PDGF antagonist, in experimentally induced liver fibrosis.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2004
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Borkham-Kamphorst, Erawan
- Contributors dc:contributor
-
- Gressner, Axel M.
Subjects
dc:subject × 6Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- eng
Identifiers
dc:identifier.*- OAI identifier oai:identifier
- oai:publications.rwth-aachen.de:62084