title: Canonical hedgehog signalling regulates hepatic stellate cell-mediated angiogenesis in liver fibrosis.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.affiliation: Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory of Therapeutic Material of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory of Therapeutic Material of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Hao
authors.affiliation: Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory of Therapeutic Material of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, China.
authors.forename: Huanhuan
authors.initials: H
authors.lastname: Jin
authors.affiliation: Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory of Therapeutic Material of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Yao
authors.affiliation: Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory of Therapeutic Material of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, China.
authors.forename: Naqi
authors.initials: N
authors.lastname: Lian
authors.affiliation: Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory of Therapeutic Material of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, China.
authors.forename: Li
authors.initials: L
authors.lastname: Wu
authors.affiliation: Department of Pharmacy, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China.
authors.forename: Jiangjuan
authors.initials: J
authors.lastname: Shao
authors.affiliation: Department of Pathology, School of Medicine, Saint Louis University, Saint Louis, MO, USA.
authors.forename: Anping
authors.initials: A
authors.lastname: Chen
authors.affiliation: Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China.|Jiangsu Key Laboratory of Therapeutic Material of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, China.
authors.forename: Shizhong
authors.initials: S
authors.lastname: Zheng
mesh_terms: D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D002251:Carbon Tetrachloride / Q000633:toxicity; D004195:Disease Models, Animal; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D053823:Hedgehog Proteins / Q000378:metabolism*; D055166:Hepatic Stellate Cells / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008103:Liver Cirrhosis / Q000188:drug therapy / Q000503:physiopathology*; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009150:Mustard Compounds / Q000494:pharmacology; D009389:Neovascularization, Pathologic / Q000188:drug therapy / Q000473:pathology*; D010666:Phenylpropionates / Q000494:pharmacology; D011719:Pyrazines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D000072086:Smoothened Receptor / Q000378:metabolism
chemical_list: C492908:2-amino-3-(4'-N,N-bis(2-chloroethyl)amino)phenylpropionic acid N-oxide; D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D053823:Hedgehog Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047029:Lactams, Macrocyclic; D009150:Mustard Compounds; D010666:Phenylpropionates; D011719:Pyrazines; C491022:Smo protein, mouse; C498555:Smo protein, rat; D000072086:Smoothened Receptor; C112765:tanespimycin; D002251:Carbon Tetrachloride; C017953:tetramethylpyrazine