Effect And Mechanism Of VEGF On Liver Injury And Regeneration At Early Stage After Transplantation

Background:Liver transplantation is the only effective therapy for end-stage liver diseases, and has been carried out successfully all over the world. However, cold preservation /reperfusion injury (CP/RI) remains one of the most important factors in determining the effect of liver transplantation. CP/RI is closely related to primary nonfunction (PNF) and prolonged function recovery of the graft.Sinusoidal endothelial cell (SEC) is believed to be the primary target of CP/RI because SEC is more sensitive to this kind of injury than hepatocyte.In the past 20 years, people have neglected the CP/RI to the non-parenchymal cell, especially to the SEC. Recent research results suggest that CP/RI mainly causes the injury to the SEC, and followed by the injury to the hepatocyte. Presently, the relationship among the extent of injury to SEC, the function of graft and the survival rate of recipients is still unclear. Additionally, the precise process and mechanism of injury to SEC remain largely unknown. The powerful regenerative capacity of the liver is well accepted and is triggered by hepatic injuries or hepatectomy. During the recovery process of liver transplantation, liver regeneration also happens. SEC appears to play an important role in the process of liver regeneration. However, the precise process and regulatory mechanism of liver regeneration after transplantation is very controversial.In addition to this, the relationship between injury and regeneration of SEC after liver transplantation has been poorly understood.VEGF is an endothelial cell specific growth factor that can prevent apoptosis and can promote proliferation of cultured SEC in vitro. This characteristic of VEGF may be an ideal choice for us to improve the effect of liver transplantation.Our experiments are designed to study the influence and regulatory mechanisms of exogenous VEGF on SEC after liver transplantation in rats.Part I Study on the injury and regeneration of hepatocyte and SEC at the early stage after liver transplantation in rats.Materials and Methods:Orthotopic liver transplantation(OLT) was performed in male SD rats using the cuff technique as described by Kamada with modifications, the survival rates of rats within 168h in different groups (different in duration of cold preservation ) were observed.Rats were divided into three groups randomly: Group A (Sham ), group B (UWlh ), group C (UW12h ). Eight time points were predetermined as lh, 6h, 12h, 24h, 48h, 72h, 96h, 168h postoperation. Six animals were used per time-point. Rats were sacrificed at each time-point and the liver specimens and blood samples were collected. The hepatocyte damage was evaluated by serum ALT, AST ,LDH, TBA levels, and SEC function was evaluated by HA levels. Morphometry was observed under light microscope and TEM. Additionally, the incidence of apoptosis in hepatocytes and SECs were measured separately by Tunel method. Expression of Bcl-2, Cleaved Caspase-3, PCNA, VEGF, flt-1, flk-1 etc in the liver were assessed by immunohistochemistry. To compare the proliferation of hepatocyte with proliferation of SEC, expression of BrdU in each type of cells was also detected by immuno double staining technique. Finally, the mRNA expression of VEGF was detected by a single step RT-PCR method. The data were analyzed with SPSS 10.0. Multiple-groups were compared using MANOVA followed by pairwise comparison with Tukey's post-hoc analysis. P<0.05 was considered statistically significant. Animal survival was evaluated using the Kaplan-Meier method.Results:1. Survival analysis showed that the survival rate of rats in UW12h group within 168h was 50%. The rats underwent 12 hours' cold preservation were an ideal model for studing injury and regeneration after transplantation.2. Biochemical data showed that the level of serum ALT, AST, LDH, TBA and HA was significantly higher in UW12h group than that in UWlh group.3. The histological changes were more severe in UW12h group than that in UWlh group.4. The AI was significantly higher in UW12h group than that in UWlh group butreached to peak at 6-hour time-point after operation in both groups. Additionally, the AI of SEC was significantly higher than that of hepatocyte.5. Comparing with UWIh group, the expression of Bcl-2 in UW12h group significantly reduced. Expression of Cleaved Caspase-3 was increased in UW12h group than in UWIh group, but no statistically significant difference was found between these two groups.6. In UW12h group, the AI of SEC was negatively related to the expression of Bcl-2, but positively related to the expression of Cleaved Caspase-3.7. The double staining results indicated that the BrdU LI in UW12h group was significantly higher compared with that in UWIh group, and reached the peak at 48-h after operation in hepatocyte in both groups. But in SEC, BrdU LI reached its peak at 72-h after operation in UWIh group, and at 96-h after operation in UW12h group.8. The expression of VEGF delayed in UW12h group comparing with that in UWIh group and the VEGF positive cells were hepatocytes. RT-PCR results indicated that in UWIh group, the expression of VEGF mRNA reached to peak at 72-h after operation.9. In UWIh group, the expression of flt-1 reached peak at 96-h after operation, and the expression of flt-1 was observed along the sinusoids. In UW12h group, the expression of fit-1 is relatively reduced comparing with UWIh group. Besides, In UWIh group, the expression of flk-1 reached peak at 1-h after operation, and the positive staining was observed in Kupffer cells and SEC, but in UW12h group, the peak of expression was at 96-h after operation.Conclusions:1. The graft subjected to 12 hours of cold preservation in liver transplantation is a stable model for the research of injury and regeneration in SD rats.2. The death ways in hepatocyte and SEC after liver transplantation are different. Apoptosis is mainly found in SEC and necrosis is found in hepatocyte. And the necrosis occurs preceded by apoptosis.3. The functional damages and pathological changes are more serious in SEC with the increase of AI, which is related to survival rate of liver transplantations.4. Cold preservation reperfusion injury may induce the apoptosis of SEC by down-regulating the expression of Bcl-2 and up-regulating the expression of Cleaved Caspase-3.5. The regeneration of hepatocyte is followed by the regeneration of SEC.6. In UW12h group, the regeneration of SEC is more obvious but delayed compared with that in UWlh group.7. In UW12h group, the expression of VEGF as well as flt-1 decreased in UWlh group, but the expression of flk-1 increased.Part II Influence of VEGF on injury and regeneration ofhepatocyte and SEC after cold preserved livertransplantation in rats.Materials and Methods:Rats were divided into two groups randomly: VEGF(-) group (treatment with PBS ), VEGF(+) group (treatment with VEGF).Eight time points were predetermined as lh, 6h, 12h, 24h, 48h, 72h, 96h, 168h, postoperation.Four animals were used per time-point to collect the liver specimens and blood samples.The others are the same as those in Part I .Results:1. The survival rate in VEGF (+) treatment group was 80%,it is higher than that of VEGF(-) group.2. Treatment with VEGF, the serum level of ALT , AST, TBA and HA significantly dropped.3. Morphology of liver showed normal morphology and ultrastructure after treatment with VEGF.4. Tunel test indicated that VEGF reduced the AI, up-regulated the expression of Bcl-2 and down-regulated the expression of Cleaved Caspase-3.5. VEGF treatment increases BrdU integrated into cells.6. Immunohistochemistry showed that the expression of flt-1 was increased comparing with control, and reached to peak at 6-h time -point after operation.Conclusions:1. Treatment with VEGF can improve the survival rate of rats.2. VEGF promotes SEC regeneration in early stage, and the regenerative degree was notcorrelated with the severity of injury.3. VEGF promotes hyperplasia of Collagen, increases BcI-2 expression, and decreases Cleaved Caspase-3 expression,which may be involved in the anti-apoptosis mechanism .4. VEGF may promote the proliferation of SEC by up-regulating the expression of fit-1.