HCV Tumor Promoting Effect Is Dependent on Host Genetic Background

BACKGROUND: The hepatitis C virus (HCV) is one of the major risk factors for the development of hepatocellular carcinoma (HCC). Nevertheless, transgenic mice which express the whole HCV polyprotein (HCV-Tg) do not develop HCC. Whereas chronic HCV infection causes inflammation in patients, in HCV-Tg mice, the host immune reaction against viral proteins is lacking. We aimed to test the role of HCV proteins in HCC development on the background of chronic inflammation in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We crossed HCV-Tg mice that do not develop HCC with the Mdr2-knockout (Mdr2-KO) mice which develop inflammation-associated HCC, to generate Mdr2-KO/HCV-Tg mice. We studied the effect of the HCV transgene on tumor incidence, hepatocyte mitosis and apoptosis, and investigated the potential contributing factors for the generated phenotype by gene expression and protein analyses. The Mdr2-KO/HCV-Tg females from the N2 generation of this breeding (having 75% of the FVB/N genome and 25% of the C57BL/6 genome) produced significantly larger tumors in comparison with Mdr2-KO mice. In parallel, the Mdr2-KO/HCV-Tg females had an enhanced inflammatory gene expression signature. However, in the N7 generation (having 99.2% of the FVB/N genome and 0.8% of the C57BL/6 genome) there was no difference in tumor development between Mdr2-KO/HCV-Tg and Mdr2-KO animals of both sexes. The HCV transgene was similarly expressed in the livers of Mdr2-KO/HCV-Tg females of both generations, as revealed by detection of the HCV transcript and the core protein. CONCLUSION: These findings suggest that the HCV transgene accelerated inflammation-associated hepatocarcinogenesis in a host genetic background-dependent manner

A Transgenic Model for Conditional Induction and Rescue of Portal Hypertension Reveals a Role of VEGF-Mediated Regulation of Sinusoidal Fenestrations

Portal hypertension (PH) is a common complication and a leading cause of death in patients with chronic liver diseases. PH is underlined by structural and functional derangement of liver sinusoid vessels and its fenestrated endothelium. Because in most clinical settings PH is accompanied by parenchymal injury, it has been difficult to determine the precise role of microvascular perturbations in causing PH. Reasoning that Vascular Endothelial Growth Factor (VEGF) is required to maintain functional integrity of the hepatic microcirculation, we developed a transgenic mouse system for a liver-specific-, reversible VEGF inhibition. The system is based on conditional induction and de-induction of a VEGF decoy receptor that sequesters VEGF and preclude signaling. VEGF blockade results in sinusoidal endothelial cells (SECs) fenestrations closure and in accumulation and transformation of the normally quiescent hepatic stellate cells, i.e. provoking the two processes underlying sinusoidal capillarization. Importantly, sinusoidal capillarization was sufficient to cause PH and its typical sequela, ascites, splenomegaly and venous collateralization without inflicting parenchymal damage or fibrosis. Remarkably, these dramatic phenotypes were fully reversed within few days from lifting-off VEGF blockade and resultant re-opening of SECs' fenestrations. This study not only uncovered an indispensible role for VEGF in maintaining structure and function of mature SECs, but also highlights the vasculo-centric nature of PH pathogenesis. Unprecedented ability to rescue PH and its secondary manifestations via manipulating a single vascular factor may also be harnessed for examining the potential utility of de-capillarization treatment modalities

Liver phenotype is reversible upon relieving sVEGF-R1.

<p>One week after sVEGF-R1 shut off (on→off) (a) Quantification of the percent of fenestrations' surface area in the sinusoids. ‘on’−8.5%, ‘on→off’−34.4% (b)-quantification of area covered by HSCs ‘on’−43.7%, ‘on→off’ (‘on’ for one month then ‘off’ for one week)−14.4% (c) Goldner staining highlighting collagen fibers in green showing reduced extracellular-matrix deposition perisinusoidally compared to ‘on’ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021478#pone-0021478-g003" target="_blank">fig 3E</a>).</p

Complications of portal hypertension resulting from sVEGF-R1 expression in the liver.

<p>(a) Anesthetized mice showing abdominal distension. ‘on’-sVEGF-R1 expression for one month. (b) Abdominal ultrasonography documenting ascites after one month of sVEGF-R1 expression in the liver. L-liver lined with pink line, ascites (hypoechogenic) is marked by a yellow asterisk and a yellow line. (c) Representative spleens taken from control (‘off’) and after one month of sVEGF-R1 expression (‘on’) in the liver. Average spleen weight is 100 mg and 193 mg in control and transgenic mice, respectively.</p

Sinusoidal capillarization in sVEGF-R1 expressing livers without parenchymal damage.

<p>(a,b) Immunohistochemical staining for vWF (Von-Willebrand Factor) on liver sections (black arrows-sinusoids, white arrows-larger blood vessels). (c) H&E staining of liver sections showing normal appearance.</p

sVEGF-R1 expression in the adult liver causes activation of HSCs.

<p>(a) Scanning electron microscopy of sinusoids in ‘off’ (a control littermate) vs. switch ‘on’ liver for one month showing prominent HSCs surrounding sinusoids (arrows) (b) Scanning electron microscopy showing transformation of HSCs from lipid droplets (L) containing cells (‘off’) to myofibroblasts like cells (arrows). (c) Quantification of the surface area of HSCs. ‘off’-5.8%, ‘on’ (sVEGF-R1 expression for one month)-43.7%. (d) Western blot analysis with anti α-smooth muscle actin antibody performed on liver extracts (e) Goldner staining for collagen fibers (green) indicating perisinusoidal accumulation of extra-cellular matrix.</p