Feasible Study On Noninvasive Assessment Liver Fibrosis Using Alpha V Beta 3 Integrin Receptor Imaging In Rats

Liver fibrosis is a reversible response of wound healing and architecture remodeling after liver cells were attacked by all kinds of insults.Assessment of hepatic fibrosis plays a key role for diagnosis and evaluation in chronic liver injury.At present liver biopsy is widely accepted as a gold standard to document liver fibrosis.However,liver biopsy procedure is limited as an invasive procedure,sampling error, potential morbidity and mortality.Hence,it is vitally important to develop a reliable non-invasive measure to dynamically detect the alteration of hepatic repair in chronic liver disease.The activation of hepatic stellate cell(HSC) is recognized as one of the critical hallmark in liver fibrosis and to decide the fate of injured liver. Recently,increasingly proofs support angiogenesis is precondition of liver tissue repairing in diverse etiologies.The accumulation of extracellular matrix(ECM) and angiogenesis is two significant pathological characteristics of advance liver fibrosis.Integrin receptor is a cell surface receptor consisting of oneα-subunit and oneβ-subunit,and at least 18 differentαsubunits and 8 differentβsubunits of integrin are currently known.Therefore, various assemblages of subunits result in more than 24 functional subtypes of integrin dimers which differ in distribution,binding ligands and signalling properties.Among them,theαVβ3 subtype is also referred to as the vitronectin receptor,which is an extensive extracellular matrix(ECM) receptor and acts as an auxiliary receptor of connective tissue growth factor(CTGF) and platelet-derived growth factor(PDGF) that mediates cell events involved in proliferation, migration,activation and survival.These actions facilitate wound healing and the remodeling response in injured tissue.Normally,the expression ofαVβ3 integrin is restricted in special cell types and only low levels are produced in adult tissue.It has been shown that expression ofαVβ3 is weakly around the portal vein in a normal liver, with the resting endothelial cells,hepatocytes,Kupffer cells and quiescent HSCs expressing even lessαVβ3 integrin.Integrin-ligand binding is well-characterized.ECM proteins can bind many integrins via the conservative motif of the arginine-glycine-aspartic acid(RGD) tripeptide,and the flank conformation of RGD determines the selectivity in different integrin subtypes.For example,vitronectin,denatured collagen,laminin, fibrinogen,osteopontin,von Willebrand factor and fibronectin interact withαVβ3 integrin through their special conformation of the RGD sequence.Based on these findings,various RGD-contained peptides have been successfully produced for experimental studies.The aim of this study was to display the expression ofαVβ3 integrin in vitro and vivo,and to explore the relationship of angiogenesis and architecture remodeling in fibrotic liver.Furthermore,intend to investigate whether ^99mTc labeled artificial Arg-Gly-Asp(RGD) containing cyclic peptides ligand can be applied to assess hepatic fibrosis by receptor imaging using SPECT.This assessing procedure is more effective and sensitive which reflect the molecular pathology mechanism of liver fibrosis.This study will provide a new perspective to explore the feasibility of developing a novel receptor-targeting strategy for non-invasive detecting liver fibrosis.Part one Isolation and culture of rat primary hepatic stellate cell and establish of activated HSC model in vitroObjective To build an efficient procedure for isolating hepatic stellate cell and establish activated HSC model.Methods Primary hepatic stellate cells(HSC) were isolated from normal Sprague-Dawley(SD) rats by infusion and combined digestion of pronase E and collagenaseâ…£ex situ.Hepatic stellate cells were purified by density centrifugation with 11%Nycodenz.Transmission electron microscope,autofluorescenes,sudanâ…¢staining,desmin andα-smooth muscle actin(α-SMA) immunofluorescence staining identified and assayed purity of HSC.Hepatic stellate cells were activated by cultured on uncoated plastic tissue culture dish and cultured in a higher glucose Dulbecco's modified eagles medium(DMEM) supplemented with 10%fetal calf serum under 37℃contained 5%CO2 95%air incubater.Results The harvest rate of hepatic stellate cells is about 3.5±0.8×10⁷ per rat,and the viability is more than 90%.Hepatic stellate cells can be activated by cultured more than 7 days.Conclusions This reformed method is more efficient to isolate hepatic stellate cell and by culture the hepatic stellate cells can be activated.Part two Expression ofαVβ3 integrin receptor during activation of hepatic stellate cellsObjective To explore the expression ofαVβ3 integrin receptor in hepatic stellate cell(HSC),and to demonstrate whetherαVβ3 integrin is a phenotypical receptor for activated hepatic stellate cell.Methods HSCs were isolated from Sprague-Dawley rats.HSCs were activated by prolonged cultured,and identified by autofluorescenes and immunocytochemical staining respectively.Immunofluorescence staining of integrinβ3 and integrinαV combinedβ3,observed both of quiescent HSC and activated HSC.The cells were harvested and real-time PCR quantified mRNA ofαV,β3,α-SMA and Western Blot for protein.Results HSCs were isolated successfully,and the purity was＞90%.HSC phenotype changed following culture.Integrinβ3 subunits exclusively expressed on activated HSC,and co-located with integrinαv subunits on the membrane of activated HSCs.The expression ofβ3 andα-SMA were low at the first day of isolation,and significant up-regulated following activation.Compared lday with 14day theβ3 mRNA enhanced 18 fold and 5.2 fold on protein.ConclusionsαVβ3 integrin is a remarkable phenotypical receptor of activated HSC.It provides a promising strategy to target the activated HSC. Part three Binding detection ofαvβ3 integrin receptor and cyclic RGD ligand in hepatic stellate cellsObjective To detect the capability ofαvβ3 integrin receptor binding with cyclic RGD ligand in hepatic stellate cells.Methods Using solid-phase synthesis to create theαVβ3 integrin ligands of cyclic arginine-glycine-aspartate-D-phenylalanine-lysine and fluorescently labeled them with 5' -carboxyfluorescein(FAM) as probes (FAM-cRGD) to targetαVβ3 integrin.HSCs were incubated with the probes at a concentration of 10μM,and fluorescence microscopy was used to observe the internalization and distribution of probes.Flow cytometry recorded the fluorescence intensity,concentration and time effect.The equilibrium dissociationconstant(Kd) and maximal binding capacity(Bmax) for HSCs were calculated by radioligand binding assay(RBA).Results:Fluorescent traces demonstrated that qHSCs did not internalize the probes but aHSC took up the probes into the cytoplasm.The aHSCs incubated with 10μM probes for 45 minutes increased their fluorescence intensity by 6.6-fold as compared to background.Furthermore,the intensity was enhanced in a concentration-and time-dependent manner,and it was inhibited 40%after co-incubation with 100μM unlabeled cRGD.RBA analyzed shown the Kd was 4.81×10^-9 mol/L and Bmax was 2.112×10^-10mol/L.Conclusions During activation of HSC,anαVβ3 integrin receptor mediated pathway is utilized by aHSCs to bind and internalize ligands of FAM-cRGD.These results support a promising strategy developing therapeutic or diagnostic agents to aim at aHSC.Part four Relationship betweenαVβ3 integrin expression and angiogenesis in progressive liver fibrosis in ratsObjective To investigate the expression ofαVβ3 integrin and platelet endothelial cell adhesion molecule-1 in progressive liver fibrosis of rats. Methods Rats two liver fibrosis models were induced by peritoneal injected 10%thioacetamine 175mg.kg^-1 twice a week(TAA)and common bile duct ligation(BDL) respectively.Treated rats were sacrificed on the third week,ninth week in TAAmodel and on the first week,four week in BDL model respectively.Sirius red stained liver tissue and computer quantified the percent of fibrotic area.Immunohistochemical staining analyzed the expression ofαVβ3 integrin and platelet-endothelial cell adhesion molecule-1(CD31).Immunofluorescence staining and confocal microscopy observed the co-location of bothαVβ3 integrin andα-SMA in liver tissue.The expression ofαVβ3 integrin and CD31 were quantitatively analyzed by real-time PCR and Western blot.Results The expression ofαVβ3 integrin significantly increased in both models,and parallel with the fibrotic area.The co-location showed corresponded to bothαVβ3 andα-SMA.The expression of CD31 was extended and new vascularization located in the area of portal vein and fibrostic septum in two models.Quantitative analysis suggestedαVβ3 and CD31 mRNA were up-regulated in both TAA and BDL groups(F=28.66,P＜0.01,F=19.62, P＜0.01 and F=32.60 P＜0.01,F=42.36 P＜0.01,respectively) as well on protein that correlated with fibrotic level.ConclusionsαVβ3 integrin and CD31 were upregulated in liver fibrotic tissue.The overexpression involved in activation of hepatic stellate cells and angiogenesis that correspond with fibrotic remodeling.Part five Tissue autoradiography ofαVβ3 integrin and distribution of ¹²⁵I-cRGD in liver fibrosis ratsObjective To determine the autoradiography of fibrotic tissue and the distribution of ¹²⁵I-cRGD in rats.Methods Rats liver fibrosis were induced by injected thioacetamide(TAA). Frosted section of liver tissue incubated with ¹²⁵I-cRGD and cRGD respectively.Ligand binding in tissues was observed by autoradiography. Injected ¹²⁵I-cRGD and cRGD by tail vein,rats were sacrificed and the liver, spleen,kidney,heart,lung,muscle and brain weighed.Radioactivity percent of the injected dose in per gram(%ID/g) tissue in different organs were counted after 45 minutes.Results The gray level of autoradiography is significantly difference between normal liver tissue and fibrotic tissue(P＜0.05).Compared with the sections of without block the gray level is reduced significantly in block sections(P＜0.05).Biodistribution results revealed that ¹²⁵I-cRGD in kidney is the highest in rats,next liver,and the brain is the lowest. Compared with control group,the block group and fibrosis group uptake of%ID/g of liver tissue is 0.11±0.03,0.12±0.03 and 0.17±0.02 respectively,and the difference is significantly(F=5.175,P=0.049). In other organs the difference of uptake is insignificantly.Conclusions The ligand binding of ¹²⁵I-cRGD and tissue distribution is higher in fibrotic liver tissue than control rats.Part six Noninvasive assessment of liver fibrosis usingαVβ3 integrin receptor SPECT imaging in ratsObjective To evaluate liver fibrosis by ^99mTc-DTPA-cRGD as radiotracer in the receptor imaging ofαVβ3 integrin using single photon emission computed tomography(SPECT).Methods Rats liver fibrosis were induced by injected thioacetamide (TAA).Using solid-phase synthesis creates theαVβ3 integrin ligands of cyclic arginine-glycine-aspartate-D-phenylalanine-lysine and coupled to the chelator of diethyleletriamepentaacetic acid(DTPA-cRGD).Direct labeled ^99mTc04- with DTPA-cRGD by stannous chloride reduction and Paper chromatography was performed to assay the radiochemical purity of ^99mTc-DTPA-cRGD.On the three weeks and nine weeks of TAA treated the rats were injected 6μCi ^99mTc-DTPA-cRGD by tail vein,and detect the radioactivity using SPECT scaning,after 45 minutes computed the average rate of count per minute(CPM) at the region of interest(ROI) in the target organs and compared with control.Results The radiochemicalpurity is 95%,and ^99mTc-DTPA-cRGD is stable over 4 h at room temperature with radiolabeling rate more than 90%.The average CPM rate of liver/kidney at ROI in control rats,TAA treated 3 weeks rats and TAA treated 9 weeks rats is 1.73±0.35,2.7±0.44 and 3.73±0.65 respectively,and the difference is significant(F=13.1,P=0.006). Compared with B ultrasonography and MRI the SPECT can distinguish the difference imaging of early stage fibrosis.Conclusions ^99mTc-DTPA-cRGD as a radiotracer in the receptor imaging ofαVβ3 integrin by SPECT can display early liver fibrosis which possess many merits such as high sensitivity and specificity,and relative high target/non-target ratio.