Galectin-3 Regulates Tissue-engineered Seed Cell Function Via The FAK/paxillin Signaling Pathway

Objective:To investigate the effect of Galectin-3 on seed cell function during tissue-engineered valve recellularization andtoverify whether it is related tothe FAK/Paxillin signaling pathway.Methods:(1)Preparation of decellularized valve scaffolds: Trypsin+Triton-100 digestion of the valves was used to prepare decellularized valve scaffolds.(2)Comparison of the effect of Galectin-3 on the adhesion rate of two seed cells: human umbilical vein endothelial cells and human adipose mesenchymal stem cells were used as seed cells,and Galectin-3 was added to the culture medium and pre-surfaced on the surface of the culture dish in two ways.Free gal-3group: Each of the two seed cells was divided into 6 groups,6 replicate wells of each concentration were inoculated in 96-well plates,and each group added gal-3 at the indicated concentrations(0.0u M,0.5u M,1.0u M,1.5u M,2.0u M,2.5u M).pre-prepared group: 96-well plates were closed at room temperature for 1 hour by adding 0.1% BSA,and 6 concentrations of each cell were used for both types of cells.For each concentration,6 replicate wells were prepopulated with gal-3 for 1 h at room temperature and inoculated with HUVEC and AMSC,respectively.the above cells were inoculated after 16 h of starvation in serum-free medium.After inoculation of the above cells,the cells were incubated for 2 h.Then the cells were stained with crystalline violet,lysed by SDS and the absorbance value at 540 nm was measured by enzyme marker.(3)Effect of Galectin-3 on cell microstructure: AMSC and HUVEC were inoculated by serum-free starvation in confocal culture dishes closed by BSA(control group),closed by 10 u Mgal-3 pre-spread(experimental group),and incubated for 0.5h,1 h,and 1.5 h in the three experimental groups,respectively,and treated for 1.5 h in the control group.After treatment,vinculin,F-actin,and nuclei were labeled to observe cell morphology and data were measured with image J,graphs were made with Graphpad Prism,and analyzed.(4)Effect of Galectin-3 on FAK/Paxillin signaling pathway: Each of the two seeded cells was divided into control group for BSAclosure and experimental group for gal-3pre-paving after closure,extracting total cell protein,quantifying BCA,performing immunoblotting experiments to detect the expression of FAK,p-FAK,Paxillin,p-Paxillin,and performing grayscale value analysis.(5)Effect of Galectin-3 on cell function through FAK/Paxillin signaling pathway: The optimal inhibitory concentration of FAK inhibitor PF-573228 for HUVEC was detected,and HUVEC as seed cells were divided into 3 groups,BSAclosed group,gal-3 prepopulated group and inhibitor group to detect the adhesion ability of cells.The change of adhesion spot position was observed in HUVEC closed group,prepopulated group and inhibitor group,and the datawere measured by image J and plotted by Graphpad Prism.The same grouping treatment method was used to detect FAK,p-FAK,Paxillin,and p-Paxillin expressions,and grayscale analysis was performed.The same grouping methods were performed for EDU assay and scratch assay to detect their ability to proliferate and migrate.The same grouping method was used to perform decellularized valve scaffoldrecellularization assay with HEand MASSON staining.Results:The nuclei were not seen under HE staining and MASSON staining for successful preparation of valve decellularized scaffolds.(2)Galectin3 pre-layered in culture dishes increased cell-matrix adhesion at an optimal promoting concentration of 10 u M,but direct addition of Galectin-3 to the culture medium inhibited cell-matrix adhesion function.(3)AMSC at 0.5 h had more regular cell morphology,smaller cell circumference,greater roundness and better solidity than HUVEC(these kinds of data can reflect the stability of cell adhesion to some extent).And with time,the trend started to change,with HUVEC showing better morphology and starting to show adhesion spots,while AMSC substructure was not obvious.(4)The phosphorylation of FAKand Paxillin in the experimental groups inboth seed cells was positively correlated with their adhesion efficacy,and the phosphorylation of the above proteins was enhanced in the treated cells with enhanced adhesion,and the adhesion efficacy of HUVEC was stronger than that of AMSC with higher expression of p-FAK and p-Paxillin in the comparison between groups.(5)10μMPF573228 had significantly inhibited the phosphorylation of FAK,and the HUVECgal-3 prepopulated group showed enhanced cell adhesion,higher expression of p-FAK and p-Paxillin,and enhanced cell migration and proliferation ability compared with the control group.While 10μMPF-573228 treatment weakened cell adhesion,lower expression of p-FAK and p-Paxillin,and weakened cell migration ability and proliferation ability than gal-3 with pavement lease.(6)The adhesion spots were increased in the Gal-3 pre-paved group compared with the control group and were located inside the cells after the addition of inhibitors.(7)In HE and MASSON staining of recellularized valves,the cells in the Gal-3 prepopulated group were denser and appeared to be multilayered than those in the control group,and the recellularization in the inhibitor group became worse andcould not forma complete cell layer.Conclusion:1.Galectin-3 inhibits cell adhesion in the free state and promotes cell adhesion in the prepopulated state,and the adhesion function of HUVEC is superior to that of AMSC under the same treatment conditions.2.Galectin-3 promotes cell adhesion,migration,and proliferation functions through the FAK/Paxillin signaling pathway,and can alter the localization of adhesion spots and enhance recellularization of HUVEC on decellularized flap scaffolds.