| Capillarization of the sinusoidal endothelium includes a defenestrated sinusoidal endothelium and the presence of a subendothelial basement membrane,which is an important contributor to the hepatic cirrhosis.Liver sinusoidal endothelial cells (LSECs)possess open fenestrae that perforate the hepatic endothelial lining,but lack a basal lamina.Fenestrae,vesicles and channels together control the bulk of trans-endothelial transport between blood and tissues.Structural integrity of the fenestrated sinusoidal liver endothelium is believed to be essential for the maintenance of the normal exchange of fluids,solutes,particles and metabolites between the sinusoidal blood and hepatocytes.Although there are various causes and morphologies of hepatic cirrhosis,all forms of cirrhosis are characterized by a defenestrated sinusoidal endothelium and the presence of a subendothelial basement membrane.It has been demonstrated that the disappearance of the normal filtration barrier in cirrhotic livers results in an impaired bidirectional exchange between the sinusoidal blood and parenchymal cells.Vascular endothelial growth factor(VEGF),which plays a role in regulating vasculogenesis,induces angiogenesis and endothelial cell proliferation.In recent years,it has been proven that VEGF is relevant to the increased number of fenestrae and endothelial permeability in endothelial cells.Injection of VEGF-D plasmid into both normal and ischemic rat liver resulted in an increased number of new capillaries around hepatic sinuses.Because defenestration and basement membrane formation result in a disordered exchange between the sinusoidal blood and hepatocytes,it is necessary to restore the function of liver sinusoidal endothelial cells in order to reverse cirrhosis.VEGF provides the perfect means to achieve this,because of it promotes fenestration and permeability.On account of these ideas,we studied the effects of VEGF transfection in cirrhotic rat livers.Objective:To investigate the efficacy of EGFP/VEGF plasmid transfection and the effect of VEGF on fenestrae of LECs by primary hepatocytes and LECs being transfected by EGFP/VEGF plasmid.Cirrhotic rats were generated by thioacetamide(TAA)administration. EGFP/VEGF plasmid were injected into the portal vein to observe the super microstructural change of the liver.Gne array was carried out to clarify the mechanism of the super microstructural change.Method:Primary hepatocytes and LECs were isolated from the livers of male Wistar rats by collagenase perfusion.EGFP/VEGF transfection was confirmed by immunofluorescence microscopy and immunohistochemistry both in primary hepatocytes and in normal liver.Scanning electron microscope was used to observe the number of fenestrae in LECs.40 rats accepted 0.03%TAA administration,after 5 weeks,the concentration of TAA was changed to 0.04%.After 10 weeks the portal hypertension models were built.Determined the pressure of superior mesenteric vein and obtain cirrhosis liver example,then infused VEGF/EGFP plasmid 150μg through superior mesenteric vein.After 3 weeks,determined the pressure of superior mesenteric vein again,then sacrificed all rats,took the liver organization specimen.The expression of VEGF in protein level were identified.The change of the liver super microstructural after gene transfection was observed by electronic microscope.Taking the liver organization to go the gene chip experiment,detected the change of Angiogenesis Gene after gene transfection.Result: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 expressed immunodetectable VEGF both in vitro and in vivo.Much fenestrae in LECs was found under SEM after gene transfection.In TAA induced cirrotic rats,the transmission electronic microscope showed a reduced number of fenestrae accompanied with the increased portal vein pressure.A basement membrane appeared.Cell conjunction between hepatocytes was destroyed and particles of bilirubin overflowed into the cytoplasm of hepatocytes and LECs, even into the hepatic sinusoid.The microvilli of hepatocytes in the space of Disse and the cholangiole were ablated.After VEGF plasmid transfection,accompanied with the increased number of fenestrae the portal vein pressure decreased.Furthermore, morphology changed after transfection with the EGFP/VEGF plasmid.The fenestrae, cell conjunctions,and microvilli of hepatocytes were restored,the basement membrane disappeared and cell apoptosis decreased.Newborn capillaries formed by a single liver endothelial cell emerged Gene-array analysis revealed that the relative abundance of transcripts of VEGF family members decreased in the cirrhotic state and increased after transfection.Conclusion:Hepatic sinusoid capillarization performed an important actor in the happen of portal hypertension.The formation of basement membrane impaired the substance exchange between the sinusoidal blood and hepatic cells.Oxygen deficiency, metabolic product accumulation and cell necrosis were the basic pathologic physiology change.The VEGF plasmid transfection increased the number of fenestra and impaired basement membrane.The exchange between the sinusoidal blood and hepatic cells restored,so the liver cell regenerated and the portal vein pressure decreased.The VEGF transfection promoted endothelial cell in hepatic sinusoid growth and the basement membrane degradation through many growth factors interaction which mainly include:Vascular Endothelial Growth Factors & Receptors:Figf(VEGFD), Pgf,Vegf,Vegfb,Vegfc;Ephrin Family Members:Efna2(the Ephrin A2), Efnb2(the Ephrin B2),Ephb4(the Ephrin B4);Fibroblast Growth Factors & Receptors:Fgf1(aFGF),Fgf6 /Fgfr3;Platelet-derived Growth Factors & Receptors:The Pdgfa,Pdgfb/Pdgfrb,Transforming Growth Factors & Receptors: Tgfo1,Tgfb2,Tgfb3/Tgfbr2;Other Growth Factors:Ctgf,Egf,Igf1,Gro1. Chemokines:Nrp(neuropilin).
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