title: Protein engineered variants of hepatocyte growth factor/scatter factor promote proliferation of primary human hepatocytes and in rodent liver.
abstract: OBJECTIVE
  Hepatocyte growth factor/scatter factor (HGF/SF) stimulates hepatocyte DNA synthesis and protects against apoptosis; in vivo it promotes liver regeneration and reduces fibrosis. However, its therapeutic value is limited by its complex domain structure, high cost of production, instability, and poor tissue penetration due to sequestration by heparin sulfate proteoglycans (HSPGs).
  
  
  METHODS
  Using protein engineering techniques, we created a full-length form of HGF/SF (called HP21) and a form of the small, naturally occurring HGF/SF fragment, NK1 (called 1K1), which have reduced affinity for HSPG. We characterized the stability and proliferative and anti-apoptotic effects of these variants in primary human hepatocytes and in rodents.
  
  
  RESULTS
  Analytical ultracentrifugation showed that 1K1 and NK1 were more stable than the native, full-length protein. All 4 forms of HGF/SF induced similar levels of DNA synthesis in human hepatocytes; 1K1 and NK1 required heparin, an HSPG analogue, for full agonistic activity. All the proteins reduced levels of Fas ligand-mediated apoptosis, reducing the activity of caspase-3/7 and cleavage of poly(adenosine diphosphate-ribose) polymerase. 1K1 was more active than NK1 in rodents; in healthy mice, 1K1 significantly increased hepatocyte DNA synthesis, and in mice receiving carbon tetrachloride, it reduced fibrosis. In rats, after 70% partial hepatectomy, daily administration of 1K1 for 5 days significantly increased liver mass and the bromodeoxyuridine labeling index compared with mice given NK1.
  
  
  CONCLUSIONS
  1K1, an engineered form of the small, naturally occurring HGF/SF fragment NK1, has reduced affinity for HSPG and exerts proliferative and antiapoptotic effects in cultured hepatocytes. In rodents, 1K1 has antifibrotic effects and promotes liver regeneration. The protein has better stability and is easier to produce than HGF/SF and might be developed as a therapeutic for acute and chronic liver disease.
authors.affiliation: UCL Hepatology, Royal Free Campus, University College London, London, England, UK.
authors.forename: Jacob
authors.initials: J
authors.lastname: Ross
authors.forename: Ermanno
authors.initials: E
authors.lastname: Gherardi
authors.forename: Noemi
authors.initials: N
authors.lastname: Mallorqui-Fernandez
authors.forename: Marco
authors.initials: M
authors.lastname: Bocci
authors.forename: Anna
authors.initials: A
authors.lastname: Sobkowicz
authors.forename: Myrrdin
authors.initials: M
authors.lastname: Rees
authors.forename: Arthur
authors.initials: A
authors.lastname: Rowe
authors.forename: Stephan
authors.initials: S
authors.lastname: Ellmerich
authors.forename: Isobel
authors.initials: I
authors.lastname: Massie
authors.forename: Junpei
authors.initials: J
authors.lastname: Soeda
authors.forename: Clare
authors.initials: C
authors.lastname: Selden
authors.forename: Humphrey
authors.initials: H
authors.lastname: Hodgson
mesh_terms: D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D002251:Carbon Tetrachloride; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D004261:DNA Replication; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D053222:Fas Ligand Protein / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D006498:Hepatectomy; D017228:Hepatocyte Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology / Q000601:surgery; D008103:Liver Cirrhosis / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D008115:Liver Regeneration / Q000187:drug effects*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014461:Ultracentrifugation
chemical_list: D053222:Fas Ligand Protein; C066855:HGF protein, human; D019812:Heparan Sulfate Proteoglycans; D010446:Peptide Fragments; D011994:Recombinant Proteins; D017228:Hepatocyte Growth Factor; D002251:Carbon Tetrachloride; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D053179:Caspase 7