title: Targeted recombinant fusion proteins of IFNγ and mimetic IFNγ with PDGFβR bicyclic peptide inhibits liver fibrogenesis in vivo.
abstract: Hepatic stellate cells (HSCs), following transdifferentiation to myofibroblasts plays a key role in liver fibrosis. Therefore, attempts to attenuate this myofibroblastic phenotype would be a promising therapeutic approach. Interferon gamma (IFNγ) is a potent anti-fibrotic cytokine, but its pleiotropic receptor expression leading to severe adverse effects has limited its clinical application. Since, activated HSC express high-level of platelet derived growth factor beta receptor (PDGFβR), we investigated the potential of PDGFβR-specific targeting of IFNγ and its signaling peptide that lacks IFNγR binding site (mimetic IFNγ or mimIFNγ) in liver fibrosis. We prepared DNA constructs expressing IFNγ, mimIFNγ or BiPPB (PDGFβR-specific bicyclic peptide)-IFNγ, BiPPB-mimIFNγ fusion proteins. Both chimeric proteins alongwith IFNγ and mimIFNγ were produced in E.coli. The expressed proteins were purified and analyzed for PDGFβR-specific binding and in vitro effects. Subsequently, these recombinant proteins were investigated for the liver uptake (pSTAT1α signaling pathway), for anti-fibrotic effects and adverse effects (platelet counts) in CCl4-induced liver fibrogenesis in mice. The purified HSC-targeted IFNγ and mimIFNγ fusion proteins showed PDGFβR-specific binding and significantly reduced TGFβ-induced collagen-I expression in human HSC (LX2 cells), while mouse IFNγ and mimIFNγ did not show any effect. Conversely, mouse IFNγ and BiPPB-IFNγ induced activation and dose-dependent nitric oxide release in mouse macrophages (express IFNγR while lack PDGFβR), which was not observed with mimIFNγ and BiPPB-mimIFNγ, due to the lack of IFNγR binding sites. In vivo, targeted BiPPB-IFNγ and BiPPB-mimIFNγ significantly activated intrahepatic IFNγ-signaling pathway compared to IFNγ and mimIFNγ suggesting increased liver accumulation. Furthermore, the targeted fusion proteins ameliorated liver fibrogenesis in mice by significantly reducing collagen and α-SMA expression and potentiating collagen degradation. IFNγ also induced reduction in fibrogenesis but showed significant decrease in platelet counts, which was restored with targeted proteins. These results suggest that these rationally designed proteins can be further developed as novel anti-fibrotic therapeutics.
authors.affiliation: Department of Controlled Drug Delivery (Targeted Therapeutics), MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands ; Department of Pharmacokinetics, Toxicology and Targeting, University of Groningen, Groningen, The Netherlands.
authors.forename: Ruchi
authors.initials: R
authors.lastname: Bansal
authors.affiliation: Department of Controlled Drug Delivery (Targeted Therapeutics), MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands ; Department of Pharmacokinetics, Toxicology and Targeting, University of Groningen, Groningen, The Netherlands.
authors.forename: Jai
authors.initials: J
authors.lastname: Prakash
authors.affiliation: Department of Pharmacokinetics, Toxicology and Targeting, University of Groningen, Groningen, The Netherlands.
authors.forename: Marieke
authors.initials: M
authors.lastname: De Ruiter
authors.affiliation: Department of Pharmacokinetics, Toxicology and Targeting, University of Groningen, Groningen, The Netherlands.
authors.forename: Klaas
authors.initials: K
authors.lastname: Poelstra
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002251:Carbon Tetrachloride / Q000633:toxicity; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D005455:Fluorescent Antibody Technique; D055166:Hepatic Stellate Cells / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D050832:Interferon-Stimulated Gene Factor 3 / Q000378:metabolism; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism*; D008103:Liver Cirrhosis / Q000139:chemically induced / Q000517:prevention & control*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D010976:Platelet Count; D060888:Real-Time Polymerase Chain Reaction; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000009:adverse effects / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D017931:DNA Primers; D050832:Interferon-Stimulated Gene Factor 3; D011993:Recombinant Fusion Proteins; C079868:gamma interferon activation factor; D009569:Nitric Oxide; D007371:Interferon-gamma; D002251:Carbon Tetrachloride; D020797:Receptor, Platelet-Derived Growth Factor beta