The Effect Of CAF On The Migration And Angiogenesis Of Endothelial Cells

Objective:Among all the stromal cells,cancer-associated fibroblasts(CAFs)are the most abundant and heterogeneous mesenchymal fibroblast population,which is closely related to cancer.It not only remodels the ECM,but also regulate the homeostasis of microenvironment or angiogenesis.However,endothelial cells,located between interstitial and tumors,are playing a key role in cell signal transduction and presenting the epitopes of vascular tissues to the immune system.Current studies are mainly focus on the solid tumors,little pay attention to the information exchange among different stromal cells.To reveal the mechanism of signal transduction between CAF and human vascular endothelial cells(HUVEC),which will provide a new idea for inhibiting the tumor metastasis.Methods:First,we constructed a subcutaneous xenograft model by inoculating with Lewis lung cancer cells(LLC),and then isolated CAF or normal fibroblast(NF)with enzymes.After that they were verify by Flow cytometry,Western Blotting and Immunofluorescence according to its markers.The gel shrinkage and Western Blotting assays were performed to examine the effect of CAF on ECM remodeling and fibrosis.To detect the effect on migration and proliferation of LLC after co-culture with CAF,which compare with the adding of HUVEC to co-culture in vitro.The optimal collecting time for conditioned medium(CM)was confirmed by using CCK8 and Clone formation assays.Using the Transwell,Cell scratch and 2D angiogenesis assays to examine the CAF on the effect of enrichment and angiogenesis in HUVEC.In addition,to detect the expression of vascular-related factors in CAF and integrin,stromal membrane and endoplasmic reticulum stress-related proteins in co-cultured HUVEC by Western Blotting and Real-Time PCR(RT-PCR)assays.Moreover,we further to investigate the effect of CAF on the activation of AKT/MAPK signaling pathways in endothelial cells,and then the siVEGFA and siIntegrin β1 were separately transfected into CAF and HUVEC to explore the relationship between VEGFA and integrin β1 and its effect on the activation of AKT signaling pathway.Finally,adding the exogenous factors or inhibitors to co-culture with CAF and HUVEC,and detected the apoptosis and invasion of HUVEC by Annexin V-FITC/PI double staining and invasion assays.Result:1.We have isolated CAF and NF successfully.The purified CAF and NF were detected by Flow cytometry,and the a-SMA+cells were 98.3%and 85.7%among the cells,respectively.Immunofluorescence assay demonstrated that CAF and NF are expressing a-smooth muscle actin(a-SMA)and locates on the cell membrane.We further verified the expression of a-SMA and Vimentin,fibroblast markers,and found that they were significantly increased in CAF compare to NF and its P values were 0.0210 and 0.0328,respectively.2.Western Blotting and Gel shrinkage assays illustrated that CAF significantly promote the contraction of ECM,up to 60%,which depended on its density.The expression of matrix metalloprotein-2(MMP2),MMP9 and N-cadherin was increased,while tissue inhibitor of metalloproteinase-3(TIMP3)is reduced in CAF.Moreover,the ratio of MMP2/TIMP3 and MMP9/TIMP3 were significantly increased.These results revealed that CAF could promote the remodeling of ECM and form a fibrotic microenvironment.3.Transwell,CCK-8,Cell cycle and Cell scratch assays demonstrated that CAF not only significantly enhanced the wound healing and migration of LLC,but also improved its cell viability.The inductive effects on migration and proliferation of LLC is further augmented by inclusion of HUVEC to co-culture with CAF,its P values was 0.0328 and 0.0595,respectively.4.CAF co-culture with HUVEC could significantly accelerated the Wound healing and promote the enrichment of HUVEC.In addition,CAF and LLC co-cultured NF could significantly promoted the angiogenesis,its P value was 0.0026 and 0.0111,respectively.5.The expression of integrin β1,platelet derived growth factor β(PDGFβ)and N-cadherin was significantly up-regulated in HUVEC after co-culture with CAF,its P values was 0.0022,0.0068 and 0.0103,respectively.However,it also altered the expression of TIMP3,Laminin and endoplasmic reticulum stress related proteins in HUVEC.6.Western Blotting results revealed that AKT/MAPK signaling pathways were activated in HUVEC when co-culture with CAF.Although the expression of total AKT and MAPK proteins were no change,the phosphorylated of AKT and MAPK were significantly up-regulated,and its P values were 0.0419 and 0.0021,respectively.The siVEGFA and siIntegrin β1 was separately transfected in CAF and HUVEC,we found that VEGFA interacts with integrin β1 and decreases the expression of p-AKT and TIMP3 in HUVEC.7.Compared with the control,CAF significantly inhibited the apoptosis of HUVEC,and its P value was 0.0082.In addition,it also promotes the invasion of HUVEC.The adding of bFGF or TIMP3 inhibitor into co-culture system could significantly enhanced the invasion of HUVEC,but TIMP3 inhibitor had little effect on the apoptosis of HUVEC.Conclusion:Compared with LLC co-culture with CAF,we discovered the migration and proliferation of LLC was augmented after LLC co-culture with CAF and HUVEC.CAF significantly regulated the migration,proliferation,apoptosis and angiogenesis of HUVEC,and LLC co-cultured NF also have some functions similar to CAF.CAF not only reconstitute the ECM,but also abnormally secreted VEGFA,MMP2,MMP9,TIMP3 to promote the angiogenesis or form a fibrotic microenvironment.In addition,when CAF co-culture with HUVEC,it activates the AKT/MAPK signaling pathways and causes PDGFβ,TIMP3,integrinβ1 and other proteins abnormal expression in HUVEC.We further found that VEGFA interacts with integrin β1 and then regulates the activation of the AKT signaling pathways and simultaneously down-regulate the expression of TIMP3 in HUVEC.Therefore,this finding provides a new idea for clinical treatment or drug development for lung cancer.