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

SUBMITTER: Bansal R

PROVIDER: S-EPMC3933682 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Targeted recombinant fusion proteins of IFNγ and mimetic IFNγ with PDGFβR bicyclic peptide inhibits liver fibrogenesis in vivo.

Bansal Ruchi R Prakash Jai J De Ruiter Marieke M Poelstra Klaas K

PloS one 20140224 2

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 inve ...[more]

PMID: 24587093

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Project description:Myosin 1c (Myo1c) is an unconventional myosin that modulates signaling pathways involved in tissue injury and repair. In this study, we observed that Myo1c expression is significantly upregulated in human chronic liver disease such as nonalcoholic steatohepatitis (NASH) and in animal models of liver fibrosis. High throughput data from the GEO-database identified similar Myo1c upregulation in mice and human liver fibrosis. Notably, transforming growth factor-β1 (TGF-β1) stimulation to hepatic stellate cells (HSCs), the liver pericyte and key cell type responsible for the deposition of extracellular matrix, upregulates Myo1c expression, whereas genetic depletion or pharmacological inhibition of Myo1c blunted TGF-β-induced fibrogenic responses, resulting in repression of α-smooth muscle actin (α-SMA) and collagen type I α 1 chain (Col1α1) mRNA. Myo1c deletion also decreased fibrogenic processes such as cell proliferation, wound healing response, and contractility when compared with vehicle-treated HSCs. Importantly, phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) and mothers against decapentaplegic homolog 3 (SMAD3) were significantly blunted upon Myo1c inhibition in GRX cells as well as Myo1c knockout (Myo1c-KO) mouse embryonic fibroblasts (MEFs) upon TGF-β stimulation. Using the genetic Myo1c-KO mice, we confirmed that Myo1c is critical for fibrogenesis, as Myo1c-KO mice were resistant to carbon tetrachloride (CCl4)-induced liver fibrosis. Histological and immunostaining analysis of liver sections showed that deposition of collagen fibers and α-SMA expression were significantly reduced in Myo1c-KO mice upon liver injury. Collectively, these results demonstrate that Myo1c mediates hepatic fibrogenesis by modulating TGF-β signaling and suggest that inhibiting this process may have clinical application in treating liver fibrosis.NEW & NOTEWORTHY The incidences of liver fibrosis are growing at a rapid pace and have become one of the leading causes of end-stage liver disease. Although TGF-β1 is known to play a prominent role in transforming cells to produce excessive extracellular matrix that lead to hepatic fibrosis, the therapies targeting TGF-β1 have achieved very limited clinical impact. This study highlights motor protein myosin-1c-mediated mechanisms that serve as novel regulators of TGF-β1 signaling and fibrosis.

Dataset Information

Targeted recombinant fusion proteins of IFN? and mimetic IFN? with PDGF?R bicyclic peptide inhibits liver fibrogenesis in vivo.

Publications

Targeted recombinant fusion proteins of IFNγ and mimetic IFNγ with PDGFβR bicyclic peptide inhibits liver fibrogenesis in vivo.

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