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 (IFN gamma) 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 (PDGF beta R), we investigated the potential of PDGFbR-specific targeting of IFN gamma and its signaling peptide that lacks IFN gamma R binding site (mimetic IFN gamma or mimIFNc) in liver fibrosis. We prepared DNA constructs expressing IFN gamma, mimIFN gamma or BiPPB (PDGF beta R-specific bicyclic peptide)-IFN gamma, BiPPB-mimIFN gamma fusion proteins. Both chimeric proteins alongwith IFN gamma and mimIFN gamma were produced in E. coli. The expressed proteins were purified and analyzed for PDGF beta R-specific binding and in vitro effects. Subsequently, these recombinant proteins were investigated for the liver uptake (pSTAT1 alpha signaling pathway), for antifibrotic effects and adverse effects (platelet counts) in CCl4-induced liver fibrogenesis in mice. The purified HSC-targeted IFN gamma and mimIFN gamma fusion proteins showed PDGF beta R-specific binding and significantly reduced TGF beta-induced collagen-I expression in human HSC (LX2 cells), while mouse IFN gamma and mimIFN gamma did not show any effect. Conversely, mouse IFN gamma and BiPPB-IFN gamma induced activation and dose-dependent nitric oxide release in mouse macrophages (express IFN gamma R while lack PDGF beta R), which was not observed with mimIFNc and BiPPB-mimIFN gamma, due to the lack of IFN gamma R binding sites. In vivo, targeted BiPPB-IFN gamma and BiPPB-mimIFN gamma significantly activated intrahepatic IFNc-signaling pathway compared to IFN gamma and mimIFN gamma suggesting increased liver accumulation. Furthermore, the targeted fusion proteins ameliorated liver fibrogenesis in mice by significantly reducing collagen and alpha-SMA expression and potentiating collagen degradation. IFN gamma 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.
- HEPATIC STELLATE CELLS
- GROWTH-FACTOR RECEPTOR