Volume 142, Issue 4, Pages 897-906 (April 2012) Protein Engineered Variants of Hepatocyte Growth Factor/Scatter Factor Promote Proliferation of Primary Human Hepatocytes and in Rodent Liver  Jacob Ross, Ermanno Gherardi, Noemi Mallorqui–Fernandez, Marco Bocci, Anna Sobkowicz, Myrrdin Rees, Arthur Rowe, Stephan Ellmerich, Isobel Massie, Junpei Soeda, Clare Selden, Humphrey Hodgson  Gastroenterology  Volume 142, Issue 4, Pages 897-906 (April 2012) DOI: 10.1053/j.gastro.2011.12.006 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 (A–C) Domain structure and (D–F) sedimentation analysis of HGF/SF, HP21, NK1, and 1K1. (A) Domain structure of HGF/SF-HP21 and NK1-1K1: n, N-terminal; k1– k4, kringle; sphd, serine protease homology domain. HP21 is a single point mutant of HGF/SF (R73E). 1K1 is a double mutant of NK1 (K132E:R134E). (B and C) Electrostatic potential of kringle 1 domains of NK1 and 1K1. Atomic coordinates of NK1 and 1K1 are from Protein Data Bank (PDB) accessions 1NK1 and 3MKP, respectively. Blue areas are positively charged, and red areas are negatively charged; figure drawn with Pymol (DeLano Scientific, San Carlos, CA). (D–F) Sedimentation velocity analysis of NK1, 1K1, and HGF/SF. Results of 2 samples for each protein analyzed after dialysis against PBS (day 0) or after a further 72-hour incubation at 37°C in PBS (day 3). For HGF/SF, a further sample is shown (high salt) that, before incubation at 37°C, was adjusted to a concentration of 1.0 mol/L NaCl. Data show plots of c(s) against s*20,w and insets show s*20,w (S), frictional ratios (fr), and molecular mass (m) values for main peak components. The species with S- values between 1.22 and 1.48 in HGF/SF samples represent a small molecular weight degradation product with high A280 absorptivity, enriched due to precipitation of intact HGF/SF following dialysis against PBS. Gastroenterology 2012 142, 897-906DOI: (10.1053/j.gastro.2011.12.006) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 DNA synthesis in primary human hepatocytes in response to HGF/SF, NK1, and 1K1, with (right) and without (left) 10 μg/mL heparin. Data from ≥ 3 livers per curve, taken from a total of 5 liver preparations. Values are expressed as mean (n = 3) ± range. Statistics are from a 2-tailed t test, and asterisks indicate significance compared with basal, corresponding to the least significant data point for each concentration (*P < .05, **P < .01; ***P < .001). Results are expressed as disintegrations per minute (DPM) 3H in DNA per microgram protein. Gastroenterology 2012 142, 897-906DOI: (10.1053/j.gastro.2011.12.006) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Reduction in FasL-induced apoptosis in response to HGF/SF proteins in primary human hepatocytes. (A–C) Prevention models: caspase-3/7 activity, with HGF/SF proteins given 2 hours or 1 hour before, or at the same time as FasL insult, respectively. (D) Rescue model: HGF/SF proteins given 1 hour after FasL. (E and F) Prevention models: PARP cleavage in cells treated with HGF/SF proteins 2 hours before or at the same time as FasL insult, respectively. Data from one representative experiment, values are mean (n = 6) ± SD. Statistics are from a 2-tailed t test (*P < .05, **P < .01; ***P < .001). LU, luminescence units. Gastroenterology 2012 142, 897-906DOI: (10.1053/j.gastro.2011.12.006) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 DNA synthesis in intact, healthy mouse liver. (A) DNA synthesis in C57BL/6 mice in response to HP21 and 1K1, administered IV. Mean values (n = 3) ± range; statistics are from a 2-tailed t test (**P < .01). (B) DNA synthesis in Balb/c mice in response to 1K1, administered IP and IV. Mean values (vehicle IP, n = 2; vehicle IV, n = 2; 1K1 IP, n = 2; 1K1 IV, n = 3) ± range. (C and D) Liver sections immunostained for BrdU in Balb/c mice receiving vehicle and 1K1 (IP). Gastroenterology 2012 142, 897-906DOI: (10.1053/j.gastro.2011.12.006) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Enhanced liver regeneration in rats by engineered HGF/SF proteins 4 days after 70% hepatectomy. (A–C) DNA synthesis in liver on day 4 sections stained for BrdU. Animals treated with (A) saline vehicle or (B) 1K1 daily after surgery. (C) Percent BrdU-positive hepatocytes on day 4. (D) Liver size on day 4 (liver mass:body mass). (E) Total liver protein on day 4 (total protein:body mass). (F) Total liver DNA on day 4 (total DNA:body mass). Values are mean (n = 6) ± SD. Statistics are from 2-tailed t test (**P < .01; *P < .05). Gastroenterology 2012 142, 897-906DOI: (10.1053/j.gastro.2011.12.006) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 Preventative effects of 1K1 in CCl4-induced liver fibrosis in Balb/c mice. Mice were treated for 4 weeks with CCl4 while receiving 1K1 or saline (vehicle) alone. Sirius Red collagen-stained liver section at 4 weeks in mouse receiving (A) vehicle or (B) 1K1. (C) Quantification of red-stained fibrotic area (excluding blood vessels) in CCl4-treated mice. (D) Serum ALT level in CCl4-treated mice at 4 weeks. Values are mean (n = 8) ± SEM. Statistics are from a 2-tailed t test (*P < .05). Gastroenterology 2012 142, 897-906DOI: (10.1053/j.gastro.2011.12.006) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 Gastroenterology 2012 142, 897-906DOI: (10.1053/j.gastro.2011.12.006) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Gastroenterology 2012 142, 897-906DOI: (10.1053/j.gastro.2011.12.006) Copyright © 2012 AGA Institute Terms and Conditions