Vascular Endothelial Cell Derived-matrix Promotes The Metabolic Functional Maturation Of Hepatocyte Via Integrin-Src Signalling

Hepatocyte phenotype and function are depended on the the regulation of extracellular matrix(ECM)microenvironment.The endothelial cell-derived matrix(EC-matrix)and its signaling molecules are important parts of liver microenvironment.Recently,it has been reported that EC-matrix are involved in endothelial cell differentiation and vascular remodeling.However,whether EC-matrix could regulate hepatocyte differentiation and function maturation is still unknown.Here,we prepared the ECM secreted by primary human umbilical vein endothelial cells(HUVECs)and detected the properties of it.Futhermore,we explored the role of EC-matrix on metabolic functional maturation of human adipose stem cells-derived hepatocyte-like cells(h ASC-HLCs)and the mechanism.The main results are as follows1.Properties of the EC-matrix The major components of the ECM,such as fibronectin(FN),collagen I,collagen IV,and laminin(LN),were identified in cultured HUVECs.Immunofluorescence staining showed that FN,collagen I,and collagen IV,were located both in and under the cells.FN appeared as abundant fibrils distributed around the cells.In contrast,LN was located in the cells.SEM revealed that the suprastructure of the EC-matrix was composed of fibrillar network which built on alayer of dense substance,while the the surfaces of collagen I-coated substrate has no such structure.2.EC-matrix promotes the metabolic functional maturation of h ASC-HLCsTo assess the ability of EC-matrix to influence h ASC-HLCs functional maturation,the cells were seeded onto the EC-matrix or the collagen I-coated substrate for different time points.Real time RT-PCR results showed that the expression levels of phase I drug metabolism enzymes,including CYP1A2,CYP2B6,CYP2C9,CYP2E1,and CYP3A4;phase II drug metabolism enzymes,including N-acetyltransferase 2(NAT2);and drug transporters,including ATP-binding cassette,sub-family C(CFTR/MRP),member 2(ABCC2),solute carrier family 22(organic cation/carnitine transporter),member 5(SLC22A5),and dipeptidyl peptidase 4(DPP4)in h ASC-HLCs cultured on the EC-matrix were significantly higher than in the cells cultured on the collagen I-coated substrate.Immunofluorescence staining confirmed that the protein level of DPP4,the specific transporter localized on the apical membrane of hepatocyte,in the h ASC-HLCs cultured on the EC-matrix was significantly higher than that in the cells cultured on the collagen I-coated substrate.The metabolic activities of CYP2C9 and CYP3A4 enzyme and the expression of FOXA2,HNF4? and PXR in the h ASC-HLCs cultured on the EC-matrix were also higher than that in the cells cultured on the collagen I-coated substrate.3.Selective silencing of FN attenuates the effect of the EC-matrixTo investigate the potential components in the EC-matrix contributing to the functional maturation of h ASC-HLCs,the FN were selectively silenced by delivering FN-specific si RNA to HUVECs.The FN deficient in EC-matrix had significantly decreased the expression levels of phase I drug metabolism enzymes(CYP1A2,CYP2B6,CYP2C9,CYP2E1,and CYP3A4),drug transporters(ABCC2 and DPP4)and transcription factors(FOXA2,HNF4?,and PXR)in h ASC-HLCs.Immunofluorescence staining confirmed the protein level of DPP4 in the h ASC-HLCs cultured on the EC-matrix-FN si RNA was significantly lower than the cells cultured on the EC-matrix-control si RNA.The metabolic activities of CYP2C9 and CYP3A4 upon rifampicin induction in the h ASC-HLCs cultured on the EC-matrix-FN si RNA were also significantly decreased than the cells cultured on the EC-matrix-control si RNA.The protein levels of FOXA2,HNF4?,and PXR in the h ASC-HLCs cultured on the EC-matrix-FN si RNA were significantly lower than those in the cells cultured on the EC-matrix-control si RNA.4.Integrin signalling activation modulates the effects of the EC-matrix on h ASC-HLCsReal time RT-PCR results showed that the expression of the ?5 integrin was significantly increased in the h ASC-HLCs cultured on the EC-matrix.Immunofluorescence staining and quantitative analyses showed that the protein level of activated ?5?1 integrin in h ASC-HLCs cultured on the EC-matrix was also significantly higher than that in the cells cultured on the collagen I-coated substrate.The expression of ?5,?1,and ?5?1 integrin in the h ASC-HLCs cultured on the different substrates was further examined by flow cytometric analysis.The percentages of the ?5 integrin and the ?5?1 integrin in the h ASC-HLCs were increased,whereas the levels of subunit ?1 integrin present in the h ASC-HLCs did not vary between the two groups.5.EC-matrix on the metabolic functional maturation of h ASC-HLCs depended on an ?5?1 integrin-mediated signaling pathway.The results showed that the h ASC-HLCs deficient in ?5 integrin expression had significantly decreased m RNA levels of phase I drug metabolism enzymes(CYP2B6,CYP2C9,and CYP3A4),phase II drug metabolism enzyme(NAT2),drug transporters(ABCC2,SLC22A5,and DPP4)and transcription factors(FOXA2,HNF4?,and PXR).Immunofluorescence staining confirmed that the protein level of DPP4 in the h ASC-HLCs-integrin ?5 si RNA was significantly lower than that in the h ASC-HLCs-control si RNA.The metabolic activities of CYP2C9 and CYP3A4 in the h ASC-HLCs-integrin ?5 si RNA were also decreased.The protein levels of FOXA2,HNF4?,and PXR in the h ASC-HLCs-integrin ?5 si RNA significantly decreased compared to the h ASC-HLCs-control si RNA.6.EC-matrix promotes the functional maturation of h ASC-HLCs via Src phosphorylationThe results showed that the activities of CYP2C9 and CYP3A4 were attenuated by the pharmacological inhibition of SFK with PP2.The activity of CYP3A4 was attenuated by pharmacological inhibition of FAK with PF228 and ILK with Cpd22.However,neither PF228 nor Cpd22 treatment prevented the activities of CYP2C9.The Src deficient in h ASC-HLCs significantly decreased the m RNA and the activities of CYP2C9 and CYP3A4.The results of Western blot confirmed that protein levels of FOXA2,HNF4?,and PXR in the h ASC-HLCs-Src si RNA significantly decreased compared to the h ASC-HLCs-control si RNA.Collectively,these results indicate that the EC-matrix promotes the expression of ?5 integrin in h ASC-HLCs;?5?1 integrin specific interacts with FN in the EC-matrix,and activated the Src phosphorylation in the cells,then up-regulated the expression of FOXA2,HNF4?,and PXR,which promote the expression and activities of CYP2C9 and CYP3A4,that result in the metabolic functional maturation of h ASC-HLCs.