Christopher J Mee, Michelle J Farquhar, Helen J Harris, Wenda Ramma, Asif Ahmed, Patrick Maurel, Roy Bicknell, Peter Balfe and Jane A McKeating.
Gastroenterology 138:1134-42. 2010
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Vascular endothelial growth factor (VEGF) was originally discovered because of its effect(s) on endothelial cell permeability. Its critical role in pathological angiogenesis has lead to the development and clinical testing of VEGF inhibitors to limit tumour growth. However, recent research suggests a diversity of roles for VEGF in maintaining normal adult tissue. We found a role for VEGF in regulating hepatocyte tight junction integrity, polarity and permissivity to HCV infection. Neutralization of endogenous VEGF expressed by the hepatoma cell line HepG2, promotes polarization and significantly inhibits HCV entry, confirming that an autocrine pathway is in operation. HCV infection increases primary hepatocyte and hepatoma VEGF expression, which reduces their polarity. Importantly, VEGF antagonists restore the ability of infected hepatoma cells to polarize.
Our data support a model where HCV upregulation of VEGF expression induces a localized disruption of hepatocellular tight junctions that promotes viral transmission in the liver, providing a potential therapeutic opportunity for the use of VEGF antagonists to treat chronic hepatitis C infection.
Background and aims: Hepatitis C virus (HCV) infection leads to progressive liver disease, frequently culminating in fibrosis and hepatocellular carcinoma. The mechanisms underlying liver injury in chronic hepatitis C are poorly understood. This study evaluated the role of vascular endothelial growth factor (VEGF) in hepatocyte polarity and HCV infection.
Methods: We utilized polarized hepatoma cell lines and the recently described infectious HCV JFH-1 cell culture system to study the role of VEGF in regulating hepatoma permeability and HCV infection.
Results: VEGF negatively regulates hepatocellular tight junction (TJ) integrity and cell polarity by a novel VEGF receptor 2 dependent pathway. VEGF reduced hepatoma TJ integrity, induced a re-organization of occludin and promoted HCV entry. Conversely, inhibition of hepatoma expressed VEGF with the receptor kinase inhibitor Sorafenib or with neutralizing anti-VEGF antibodies promoted polarization and inhibited HCV entry, demonstrating an autocrine pathway. HCV infection of primary hepatocytes or hepatoma cell lines promoted VEGF expression and reduced their polarity. Importantly, treatment of HCV infected cells with VEGF inhibitors restored their ability to polarize, demonstrating a VEGF-dependent pathway.
Conclusion: Hepatic polarity is critical to normal liver physiology. HCV infection promotes VEGF expression that depolarizes hepatoma cells, promoting viral transmission and lymphocyte migration into the parenchyma that may promote hepatocyte injury.
Figure 1: VEGF regulates hepatocellular TJ integrity and polarity.
A. HepG2 cells were allowed to polarize for 3 days before treating with VEGF-A for 24h. Cells were fixed in 3% paraformaldehyde and stained for the BC-expressed marker MRP2 to quantify polarity or incubated with CMFDA to measure TJ barrier function. The polarity index was assessed by quantifying the number of MRP2-positive BC per 100 nuclei for five fields of view on three replicate coverslips. TJ barrier function was measured by quantifying the number of BC retaining CMFDA compared to total BC in a minimum of five fields of view on three replicate coverslips.
B. WIF-B9 cells were grown for a minimum of 11 days to develop „hepatic‟ polarity and treated with VEGF-A for 24h, polarity and TJ integrity assessed as before.
C. HepG2, WIF-B9, primary human hepatocytes (PHH) from two independent donors and LSEC were plated and the extracellular media collected from 100,000 cells over a 24h time period and assessed for VEGF using a human VEGF ELISA.
D. Polarized HepG2 cells were incubated in serum free DMEM for 4h before treatment with control DMSO, neutralizing anti-VEGF-A antibody VG76e (1.3 mg/ml) or receptor kinase antagonist Sorafenib (10nM) for 24h. Cells were fixed and their polarity index measured. * P <0.01 (t test).
Figure 2: VEGF regulates HCV entry into polarized HepG2 cells.
A. HepG2-CD81 cells at 3 days post-plating were untreated or treated with VEGF-A (10ng/ml) or VEGF-E (10ng/ml) for 1h and challenged with HCVcc J6/JFH (black bars); HCVpp (white bars) or MLVpp (gray bars). * P <0.01 (t test).
B. HepG2-CD81 cells at 3 days post-plating were untreated or treated with neutralizing anti-VEGF-A antibody VG76e (1.3mg/ml) or receptor kinase antagonist Sorafenib (10nM) for 16h and challenged with HCVcc J6/JFH (black bars); HCVpp (white bars) or MLVpp (gray bars). Infectivity is expressed relative to control. ** P <0.001 (t test).
Figure 3: HCV infection increases VEGF expression that reduces hepatoma polarity.
A. HepG2-CD81, Huh-7.5 and primary human hepatoctytes (PHH) from two donors were inoculated with mock or HCVcc J6/JFH inocula, pre-titrated to achieve comparable levels of infection in the different target cells. Infected HepG2-CD81, Huh-7.5 and PHH cells contained 2.6x107, 2.9x107 and 1.9x107 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 24h and assessed for VEGF expression using a human VEGF-A ELISA. * P < 0.01, ** P < 0.001, *** P < 0.0001 (t test).
B. HepG2-CD81 cells were infected with mock or HCV J6/JFH for 4h, unbound virus removed by extensive washing and the cells untreated or treated with anti-VEGF-A antibody VG76e (1.3mg/ml) or Sorafenib (10nM) for 48h. Cells were fixed and the frequency of NS5A+ and polarized cells in duplicate coverslips assessed. Less than 2% of infected HepG2-CD81 cells expressed NS5A antigen at 48h post infection. Relative polarity is presented as the increase in polarized cells over 48h for uninfected (white bars) and J6/JFH infected (black bars) cells. **P <0.001 (t test).