Rowe IA, Galsinh SK, Wilson GK, Durant S, Lazar C, Branza-Nichita N, Bicknell R, Adams DH, Balfe P and McKeating JA.
Hepatology, 59: 375-84. 2014.
Get a PDF file of this paper
back to publications page
The liver is a large and complex organ containing multiple cell types, including sinusoidal endothelial cells (LSEC), stellate cells, Kupffer cells and biliary epithelial cells as well as hepatocytes. Hepatocytes are the major reservoir supporting HCV replication and the contribution of other cell types in the viral lifecycle remain largely unexplored. LSEC hepatocyte interactions are critical for normal liver development and function, and regulate the organ’s response to injury. One of the best- characterised paracrine signals between these endothelial and epithelial cells is vascular endothelial growth factor-A (VEGF-A) which maintains the sinusoidal endothelial fenestrated phenotype and promotes hepatocyte growth factor expression in response to injury.
We previously reported that HCV infection promotes VEGF expression (Mee et al 2010), resulting in hepatocyte depolarization and enhanced viral entry. Treating HCV infected hepatocytes with VEGF inhibitors, including sorafenib, restored their ability to polarize and limited viral infection, suggesting a therapeutic role for VEGF inhibitors in HCV infection. These reports led us to develop endothelial-hepatocyte co-cultures to study the role of LSEC in the HCV lifecycle. This study describes a new paracrine network linking these cell types that regulates HCV replication.
LSECs express bone morphogenetic protein 4 (BMP4) that promotes hepatocyte permissivity to support HCV replication. BMP4 expression is negatively regulated by VEGF-A activation of VEGF Receptor-2 (VEGFR-2) and down-stream p38 MAPK signalling. Ex vivo studies demonstrate increased BMP4 expression and reduced per endothelial cell VEGFR-2 activation in the diseased liver, highlighting new aspects of LSEC-hepatocyte cross-talk that may limit the efficacy of anti-VEGF therapies in HCV infection and suggesting therapeutic manipulation of BMP4.
Hepatitis C virus (HCV) is a major cause of global morbidity, causing chronic liver injury that can progress to cirrhosis and hepatocellular carcinoma. The liver is a large and complex organ containing multiple cell types, including hepatocytes, sinusoidal endothelial cells (LSEC), Kupffer cells and biliary epithelial cells. Hepatocytes are the major reservoir supporting HCV replication, however, the role of non-parenchymal cells in the viral lifecycle remain largely unexplored. LSEC secrete factors that promote HCV infection and transcript analysis identified bone morphogenetic protein 4 (BMP4) as a candidate endothelial expressed pro-viral molecule. Recombinant BMP4 increased HCV replication and neutralisation of BMP4 abrogated the pro-viral activity of LSEC conditioned media. Importantly, BMP4 expression was negatively regulated by vascular endothelial growth factor A (VEGF-A) via a VEGF receptor-2 (VEGFR-2) primed activation of p38 MAPK. Consistent with our in vitro observations, we demonstrate that in normal liver VEGFR-2 is activated and BMP4 expression is suppressed. In contrast, in chronic liver disease including HCV infection where there is marked endothelial cell proliferation we observed reduced per endothelial cell VEGFR-2 activation and a concomitant increase in BMP4 expression.
Conclusion: These studies identify a role for LSEC and BMP4 in HCV infection and highlight BMP4 as a new therapeutic target for treating individuals with liver disease.
Figure 1. Paracrine VEGF signalling reduces HCV infection of LSEC-hepatocyte co-cultures.
(A) CM was collected from LSEC treated with increasing concentrations of recombinant VEGF-A and screened for its effect on Huh-7.5 permissivity to support HCV JFH-1 infection. Infection was enumerated by quantifying NS5A expressing cells and the data expressed relative to mock control (black). (B) Recombinant VEGF-A (10ng/ml) was used to treat Huh-7.5 cells at the time of HCV infection. Infectivity was assessed relative to untreated control. (C) LSEC/Huh-7.5 co-cultures were established and a representative image is shown, where LSEC are labelled with CMFDA (green) cell tracker dye. LSEC/Huh-7.5 co-cultures were treated with neutralizing anti-VEGF antibody or irrelevant IgG (10μg/ml) prior to infecting with HCV.
Figure 2. BMP4 is negatively regulated by VEGF and stimulates HCV replication.
LSEC were starved of VEGF overnight and stimulated with VEGF-A (10ng/ml) as indicated for 8-hours and lysed to quantify￼BMP4 mRNA(A) or protein(B). Conditioned media (CM) from untreated (black) or VEGF-A treated LSEC were incubated with a neutralising anti-BMP4 antibody (10μg/ml) and screened for its effect on Huh-7.5 permissivity(C). BMP4 antibody clearly blocks enhancement.
Huh-7.5 were treated with recombinant BMP4 for 48-hours and the cells assessed for their ability to proliferate(D) and support HCV JFH-1 infection, enumerated either by quantifying NS5A expressing cells(E) or measuring HCV RNA(F). To assess the effects of BMP4, primary hepatocytes were treated with BMP4 (100ng/ml) for 18-hours, inoculated with HCV JFH-1 and infection assessed by measuring HCV RNA(G).
BMP4 has a clear impact on HCV infection.
Figure 3: LSEC paracrine signals regulate HCV replication.
In the normal liver BMP4 expression is suppressed through VEGFR-2/p38 MAPK signalling (A). Following HCV infection VEGF-A expression is increased. In the hepatocyte this stimulates depolarisation and increases permissivity to HCV entry. VEGFR-2 signalling in LSEC is reduced thus permitting BMP4 expression. BMP4 stimulation of hepatocytes increases HCV replication (B).