Vascular endothelial growth factor attenuates hepatic sinusoidal capillarization in thioacetamide-induced cirrhotic rats

AIM: To investigate the effect of vascular endothelial growth factor (VEGF) transfection on hepatic sinusoidal capillarization. METHODS: Enhanced green fluorescent protein (EGFP)/ VEGF transfection was confirmed by immunofluorescence microscopy and immunohistoche-mistry both in primary hepatocytes and in normal liver. Cirrhotic rats were generated by thioacetamide (TAA) administration and then divided into a treatment group, which received injections of 400 μg of plasmid DNA encoding an EGFP- VEGF fusion protein, and a blank group, which received an equal amount of normal saline through the portal vein. The portal vein pressure was measured in the normal and cirrhotic state, in treated and blank groups. The average number of fenestrae per hepatic sinusoid was determined using transmission electron microscopy (TEM), while the relative abundance of VEGF transcripts was examined by Gene array. RESULTS: Green fluorescent protein was observed in the cytoplasms of liver cells under immunofluorescence microscopy 24 h after transfection with EGFP/VEGF plasmid in vitro. Staining with polyclonal antibodies against VEGF illustrated that hepatocytes expressedimmunodetectable VEGF both in vitro and in vitro. There were significant differences in the number of fenestrae and portal vein pressures between normal and cirrhotic rats (7.40 ± 1.71 vs 2.30 ± 1.16 and 9.32 ± 0.85 cmH2O vs 17.92 ± 0.90 cmH2O, P < 0.01), between cirrhotic and treated rats (2.30 ± 1.16 cmH2O vs 4.60 ± 1.65 and 17.92 ± 0.90 cmH2O vs 15.52 ± 0.93 cmH2O, P < 0.05) and between the treatment group and the blank group (4.60 ± 1.65 cmH2O vs 2.10 ± 1.10 cmH2O and 15.52 ± 0.93 cmH2O vs 17.26 ± 1.80 cmH2O, P < 0.05). Gene- array analysis revealed that the relative abundance of transcripts of VEGF family members decreased in the cirrhotic state and increased after transfection. CONCLUSION: Injection of a plasmid encoding VEGF through the portal vein is an effective method to induce the formation of fenestrae and decrease portal vein pressure in cirrhotic rats. Therefore, it may be a good choice for treating hepatic cirrhosis and portal hypertension.

Reconstruction of liver organoid using a bioreactor

AIM： To develop the effective technology for reconstruction of a liver organ in vitro using a bio-artificial liver. METHODS： We previously reported that a radial-flow bioreactor （RFB） could provide a three-dimensional highdensity culture system. We presently reconstructed the liver organoid using a functional human hepatocellular carcinoma cell line （FLC-5） as hepatocytes together with mouse immortalized sinusoidal endothelial cell （SEC） line M1 and mouse immortalized hepatic stellate cell （HSC） line A7 as non parenchymal cells in the RFB. Two × 10^7 FLC-5 cells were incubated in the RFB. After 5 d, 2 × 10^7 A7 cells were added in a similar manner followed by another addition of 10^7 M1 cells 5 d later. After three days of perfusion, some cellulose beads with the adherent cells were harvested. The last incubation period included perfusion with 200 nmol/L swinholide A for 2 h and then the remaining cellulose beads along with adherent cells were harvested from the RFB. The cell morphology was observed by transmission electron microscopy （TEM） and scanning electron microscopy （SEM）. To assess hepatocyte function, we compared mRNA expression for urea cycle enzymes as well as albumin synthesis by FLC-5 in monolayer cultures compared to those of single-type cultures and cocultures in the RFB. RESULTS： By transmission electron microscopy, FLC-5, M1, and A7 were arranged in relation to the perfusion side in a liver-like organization. Structures resembling bile canaliculi were seen between FCL-5 cells. Scanning electron microscopy demonstrated fenestrae on SEC surfaces. The number of vesiculo-vacuolar organelles （WO） and fenestrae increased when we introduced the actin-binding agent swinholide-A in the RFB for 2h. With respect to liver function, urea was found in the medium, and expression of mRNAs encoding arginosuccinate synthetase and arginase increased when the three cell types were cocultured in the RFB. However, albumin synthesis decreased. CONCLUSION： Co-culture in the RFB system can dramatically change the structure and function of all cell types, including the functional characteristics of hepatocytes. Our system proves effective for reconstruction of a liver organoid using a bio-artificial liver.

Hydrodynamics based transfection in normal and fibrotic rats

AIM： Hydrodynamics based transfection （HBT）, the injection of a large volume of naked plasmid DNA in a short time is a relatively simple, efficient and safe method for in vivo transfection of liver cells. Though used for quite some time, the mechanism of gene transfection has not yet been elucidated. METHODS： A lucJferase encoding plasmid was injected using the hydrodynamics based procedure into normal and thioacetamide-induced fibrotic Sprague Dawley rats. Scanning and transmission electron microscopy images were taken. The consequence of a dual injection of Ringer solution and luciferase pDNA was followed. Halofuginone, an anti collagen type I inhibitor was used to reduce ECM load in fibrotic rats prior to the hydrodynamic injection. RESULTS： Large endothelial gaps formed as soon as 10＇ following hydrodynamic injection; these gradually returned to normal 10 d post injection. Hydrodynamic administration of Ringer 10 or 30 m prior to moderate injection of plasmid did not result in efficient transfection suggesting that endothelial gaps by themselves are not sufficient for gene expression. Gene transfection following hydrodynamic injection in thioacetamide induced fibrotic rats was diminished coinciding with the level of fibrosis. Halofuginone, a specific collagen type I inhibitor, alleviated this effect. CONCLUSION： The hydrodynamic pressure formed following HBT results in the formation of large endothelial gaps. These gaps, though important in the transfer of DNA molecules from the blood to the space of Disse are not enough to provide the appropriate conditions for hepatocyte transfection. Hydrodynamics based injection is applicable in fibrotic rats provided that ECM load is reduced.