Volume 138, Issue 3, Pages 1134-1142 (March 2010) Hepatitis C Virus Infection Reduces Hepatocellular Polarity in a Vascular Endothelial Growth Factor–Dependent Manner  Christopher J. Mee, Michelle J. Farquhar, Helen J. Harris, Ke Hu, Wenda Ramma, Asif Ahmed, Patrick Maurel, Roy Bicknell, Peter Balfe, Jane A. McKeating  Gastroenterology  Volume 138, Issue 3, Pages 1134-1142 (March 2010) DOI: 10.1053/j.gastro.2009.11.047 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 VEGF regulates hepatocellular TJ integrity and polarity. (A) HepG2 polarity was quantified by fixing the cells in 3% paraformaldehyde and staining for the BC marker MRP2. Enumerating the frequency of MRP2+ BC per 100 stained nuclei using CMFDA (4′, 6′-diamidino-2-phenylindole) enabled us to determine a polarity index. TJ barrier function was measured by quantifying the frequency of BC retaining CMFDA. Representative images depict apical expressed MRP2 and BC annotated with arrows and their retention of fluorescent CMFDA. Scale bar, 10 μmol/L. (B) HepG2 cells were allowed to polarize for 3 days and treated with VEGF-A for 24 hours. Treated and untreated cells were stained for MRP2 to quantify their polarity or incubated with CMFDA to measure TJ barrier function. Polarity index and TJ integrity were determined by quantifying the number of MRP2+ BC per 100 nuclei that retained CMFDA in 5 fields of view on 3 replicate coverslips. (C) WIF-B9 cells were grown for a minimum of 11 days to develop hepatic polarity and treated with VEGF-A for 24 hours; polarity and TJ integrity were assessed as described earlier. (D) HepG2, WIF-B9, and PHHs from 2 independent donors, and liver sinusoidal endothelial cells (lsec) were plated and the extracellular media was collected from 100,000 cells over a 24-hour time period and assessed for VEGF using a human VEGF enzyme-linked immunosorbent assay. (E) HepG2 cells were allowed to polarize for 3 days and were pre-incubated in serum-free Dulbecco's modified Eagle medium for 4 hours before treating with control dimethyl sulfoxide, neutralizing anti–VEGF-A antibody VG76e (1.3 mg/mL), or receptor kinase antagonist Sorafenib (10 nmol/L) for 24 hours. Cells were fixed and their polarity index was measured. *P < .01 (t test). Gastroenterology 2010 138, 1134-1142DOI: (10.1053/j.gastro.2009.11.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 VEGF regulation of hepatoma TJ integrity and polarity is VEGFR-2–dependent. HepG2 cells were allowed to polarize for 3 days, incubated in serum-free Dulbecco's modified Eagle medium for 4 hours followed by: (A) control dimethyl sulfoxide, Sorafenib (10 nmol/L), or VEGFR-2 antagonist Ki8571 (10 nmol/L) for 24 hours, followed by treatment with VEGF-A (10 ng/mL) or IFNγ (10 ng/mL) for 1 hour; (B) VEGF-A (10 ng/mL) or IFNγ (10 ng/mL) pretreated with irrelevant IgG (5 mg/mL) or anti–VEGF 2c3 (5 mg/mL) for 1 hour, or (C) treated with mock (control), placental-induced growth factor (plgf) (50 ng/mL), VEGF-E (50 ng/mL), or VEGF-A (50 ng/mL) for 1 hour. TJ barrier function was assessed by quantifying the number of CMFDA+ BC in a minimum of 5 fields of view on 3 replicate coverslips, where *P < .01, **P < .001, ***P < .0001 (t test). Gastroenterology 2010 138, 1134-1142DOI: (10.1053/j.gastro.2009.11.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 VEGF regulates HCV entry into polarized HepG2 cells. (A) HepG2-CD81 cells at 3 days postplating were untreated or treated with VEGF-A (10 ng/mL) or VEGF-E (10 ng/mL) for 1 hour and challenged with HCVcc J6/JFH (black bars), HCVpp (white bars), or murine leukemia virus pseudotype particles (mlvpp) (gray bars). *P < .01 (t test). (B) HepG2-CD81 cells at 3 days postplating were untreated or treated with neutralizing anti–VEGF-A antibody VG76e (1.3 mg/mL) or receptor kinase antagonist Sorafenib (10 nmol/L) for 16 hours and challenged with HCVcc J6/JFH (black bars), HCVpp (white bars), or MLVpp (gray bars). Infectivity is expressed relative to control. **P < .001 (t test). Gastroenterology 2010 138, 1134-1142DOI: (10.1053/j.gastro.2009.11.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 HCV infection increases VEGF expression, which reduces hepatoma polarity. (A) HepG2-CD81, Huh-7.5, and PHHs from 2 donors were inoculated with mock or HCVcc J6/JFH, pretitrated to achieve comparable levels of infection in the different target cells. Infected HepG2-CD81, Huh-7.5, and PHHs contained 2.6 × 107, 2.9 × 107, and 1.9 × 107 HCV RNA copies/106 cells, respectively. Extracellular media was collected from 100,000 uninfected (white bars) or HCV-infected cells (black bars) over a period of 24 hours and assessed for VEGF expression using a human VEGF-A enzyme-linked immunosorbent assay. *P < .01, **P < .001, ***P < .0001 (t test). (B) HepG2-CD81 cells were infected with mock or HCV J6/JFH for 4 hours, unbound virus was removed by extensive washing, and the cells were untreated or treated with anti–VEGF-A antibody VG76e (1.3 mg/mL) or Sorafenib (10 nmol/L) for 48 hours. Cells were fixed and the frequency of NS5A+ and polarized cells in duplicate coverslips was assessed. Less than 2% of infected HepG2-CD81 cells expressed NS5A antigen at 48 hours postinfection. Relative polarity is presented as the increase in polarized cells over 48 hours for uninfected (white bars) and J6/JFH-infected (black bars) cells. **P < .001 (t test). Gastroenterology 2010 138, 1134-1142DOI: (10.1053/j.gastro.2009.11.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 VEGF modulates occludin localization and phosphorylation. (A) HepG2 cells were allowed to polarize for 3 days and were untreated (control) or treated with VEGF-A (10 ng/mL) for 1 hour, TJ integrity was assessed, and occludin (ocln) localization was ascertained by staining with an OCLN-specific antibody. VEGF-A reduced the frequency of CMFDA+ BC by 48% and representative images show a re-organization of OCLN to basolateral membranes. (B) To quantify the effect of VEGF on OCLN localization and to ascertain the effect on other TJ-associated proteins and viral co-receptor CD81, polarized HepG2 cells expressing AcGFP.CLDN1 and AcGFP.CD81 were untreated (control) or treated with VEGF-A (10 ng/mL) for 1 hour and stained with anti-OCLN or anti–ZO-1. OCLN, CLDN1, CD81, and ZO-1 localization at basolateral (black), intracellular (white), and TJ (grey) locations was quantified. (C) Polarized HepG2 cells were untreated or treated with VEGF-A (10 ng/mL) for 1 hour and OCLN immunoprecipitates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting with anti-OCLN. (D) To determine the phosphorylation status of precipitated OCLN after VEGF-A treatment, sodium dodecyl sulfate–polyacrylamide gel electrophoresis separated proteins were probed with antiphosphoserine or antiphosphotyrosine and protein loading confirmed using anti–β-actin. Gastroenterology 2010 138, 1134-1142DOI: (10.1053/j.gastro.2009.11.047) Copyright © 2010 AGA Institute Terms and Conditions