Feng Zhang1,2,3, Meng Hao1,2,3, Huanhuan Jin1,2,3, Zhen Yao1,2,3, Naqi Lian1,2,3, Li Wu1,2,3, Jiangjuan Shao4, Anping Chen5, Shizhong Zheng1,2,3. 1. Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China. 2. Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China. 3. Jiangsu Key Laboratory of Therapeutic Material of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, China. 4. Department of Pharmacy, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China. 5. Department of Pathology, School of Medicine, Saint Louis University, Saint Louis, MO, USA.
Abstract
BACKGROUND AND PURPOSE: Hepatic stellate cells (HSCs) are liver-specific pericytes regulating angiogenesis during liver fibrosis. We aimed to elucidate the mechanisms by which hedgehog signalling regulated HSC angiogenic properties and to validate the therapeutic implications. EXPERIMENTAL APPROACH: Rats and mice were treated with carbon tetrachloride for in vivo evaluation of hepatic angiogenesis and fibrotic injury. Diversified molecular approaches including real-time PCR, Western blot, luciferase reporter assay, chromatin immunoprecipitation, electrophoretic mobility shift assay and co-immunoprecipitation were used to investigate the underlying mechanisms in vitro. KEY RESULTS: Angiogenesis was concomitant with up-regulation of Smoothened (SMO) and hypoxia inducible factor-1α (HIF-1α) in rat fibrotic liver. The SMO inhibitor cyclopamine and Gli1 inhibitor GANT-58 reduced expression of VEGF and angiopoietin 1 in HSCs and suppressed HSC tubulogenesis capacity. HIF-1α inhibitor PX-478 suppressed HSC angiogenic behaviour, and inhibition of hedgehog decreased HIF-1α expression. Furthermore, heat shock protein 90 (HSP90) was characterized as a direct target gene of canonical hedgehog signalling in HSCs. HSP90 inhibitor 17-AAG reduced HSP90 binding to HIF-1α, down-regulated HIF-1α protein abundance and decreased HIF-1α binding to DNA. 17-AAG also abolished 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) (a SMO agonist)-enhanced HSC angiogenic properties. Finally, the natural compound ligustrazine was found to inhibit canonical hedgehog signalling leading to suppressed angiogenic properties of HSCs in vitro and ameliorated liver fibrosis and sinusoidal angiogenesis in mice. CONCLUSION AND IMPLICATIONS: We have provided evidence that the canonical hedgehog pathway controlled HSC-mediated liver angiogenesis. Selective inhibition of HSC hedgehog signalling could be a promising therapeutic approach for hepatic fibrosis.
BACKGROUND AND PURPOSE: Hepatic stellate cells (HSCs) are liver-specific pericytes regulating angiogenesis during liver fibrosis. We aimed to elucidate the mechanisms by which hedgehog signalling regulated HSC angiogenic properties and to validate the therapeutic implications. EXPERIMENTAL APPROACH: Rats and mice were treated with carbon tetrachloride for in vivo evaluation of hepatic angiogenesis and fibrotic injury. Diversified molecular approaches including real-time PCR, Western blot, luciferase reporter assay, chromatin immunoprecipitation, electrophoretic mobility shift assay and co-immunoprecipitation were used to investigate the underlying mechanisms in vitro. KEY RESULTS: Angiogenesis was concomitant with up-regulation of Smoothened (SMO) and hypoxia inducible factor-1α (HIF-1α) in rat fibrotic liver. The SMO inhibitor cyclopamine and Gli1 inhibitor GANT-58 reduced expression of VEGF and angiopoietin 1 in HSCs and suppressed HSC tubulogenesis capacity. HIF-1α inhibitor PX-478 suppressed HSC angiogenic behaviour, and inhibition of hedgehog decreased HIF-1α expression. Furthermore, heat shock protein 90 (HSP90) was characterized as a direct target gene of canonical hedgehog signalling in HSCs. HSP90 inhibitor 17-AAG reduced HSP90 binding to HIF-1α, down-regulated HIF-1α protein abundance and decreased HIF-1α binding to DNA. 17-AAG also abolished 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) (a SMO agonist)-enhanced HSC angiogenic properties. Finally, the natural compound ligustrazine was found to inhibit canonical hedgehog signalling leading to suppressed angiogenic properties of HSCs in vitro and ameliorated liver fibrosis and sinusoidal angiogenesis in mice. CONCLUSION AND IMPLICATIONS: We have provided evidence that the canonical hedgehog pathway controlled HSC-mediated liver angiogenesis. Selective inhibition of HSC hedgehog signalling could be a promising therapeutic approach for hepatic fibrosis.
Authors: Michael J Curtis; Richard A Bond; Domenico Spina; Amrita Ahluwalia; Stephen P A Alexander; Mark A Giembycz; Annette Gilchrist; Daniel Hoyer; Paul A Insel; Angelo A Izzo; Andrew J Lawrence; David J MacEwan; Lawrence D F Moon; Sue Wonnacott; Arthur H Weston; John C McGrath Journal: Br J Pharmacol Date: 2015-07 Impact factor: 8.739
Authors: Sònia Tugues; Guillermo Fernandez-Varo; Javier Muñoz-Luque; Josefa Ros; Vicente Arroyo; Juan Rodés; Scott L Friedman; Peter Carmeliet; Wladimiro Jiménez; Manuel Morales-Ruiz Journal: Hepatology Date: 2007-12 Impact factor: 17.425