The Effects Of Substrate Rigidity And Surface Topography On Epithelial-Mesenchymal Transitions In Alveolar Epithelial Cells And Its Mechanisms

Introduction: Pulmonary fibrosis is a fatal progressive lung disease.It is characterized by the proliferation of fibroblasts and extracellular matrix protein deposition,and thus destructs lung tissue structure,which result in the loss of the lung function.In recent years,the morbidity and mortality of pulmonary fibrosis have increased,which has been a serious threat to public health.So far,the cause of pulmonary fibrosis is still unknown,there was also lack of efficient therapeutic methods.Therefore,it's very important and urgent to identify new therapeutic strategies for effective treatment.An epithelial to mesenchymal trasition(EMT)is a fundmental phenomena in vivo,which allows a polarized epithelial cell loss intercellular tight junctions,to abtain a mesenchymal cell phenotype,including migratory capacity and invasiveness.The change of extracellular matrix mechanical micro-environment(rigidity and topography)is an important characteristic of pulmonary fibrosis.Although researchers have shown that the epithelial to mesenchymal transition plays an important role in the pulmonary fibrosis,the regulation of mechanical micro-environment to EMT and its mechanism are still unclear.Objectives: This study was designed to investigate the relationship between mechanical micro-environment and EMT,and to explore the role of PI3K/Akt signaling pathway in mechanical micro-environment regulation of EMT in human A549 cells.Based on the studies above,the regulation mechanism of mechanical microenvironment to EMT progress will be explored.Methods: Polydimethylsiloxane(PDMS)was selected as the substrate material to culture human alveolar epithelial cells A549 in the study.Three kinds of substrate molds were constructed by PDMS with different stiffness or micro-topography.The morphological changes of A459 cells were observed by SEM,the EMT-related proteins expression of E-cadherin,N-cadherin and Vimentin were detected by immunofluoresence staining.Then,the cells were treated with PI3 K inhibitor LY294002,the expression of p-Akt,E-cadherin and Vimentin were detected by immunofluoresence staining.Results:1)PDMS substrates with different stiffness have no effect on EMT.We made different stiffness of substrates(10:1,30:1 and 50:1)to culture A549 cells.After 3 days,the proteins expression of E-cadherin and Vimentin were examined and have no significant differences.2)Different topographic substrates can affect EMT in A549 cells.PDMS micropillar arrayed topographic substrates were fabricated,with dimensions of 4um and 10 um in pillar diameter,2um,4um and 7um in spacing and 4um in height,to culture A549 cells.Compared with control group,the protein expression of Vimentin increased and E-cadherin decreased with increasing diameter or decreasing spacing.Furthermore,the variation of micropillar spacing effect EMT process in A549 cell more obviously.3)Stiffness and topography of substrates interactions co-regulate EMT.We fabricate two kinds of stiffness of substrates(10:1 and 30:1)with different topography.We detected EMT-associated proteins by immunofluoresence staining,and found that A549 cells decreased expression of E-cadherin,Vimentin expression was significantly enhanced with decreasing spacing of micropillars on hard substrate.However,the proteins expression of E-cadherin decreased and Vimentin increased with increasing spacing on soft substrate.The results further proved that the mechanical microenvironment can regulate EMT process in human alveolar epithelial cells.4)The effect of stiffness and topographic substrates on PI3K/Akt signaling pathway.We found that the proteins expression of E-cadherin increased,p-Akt and Vimentin both decreased after PI3 K inhibitor LY294002 treated by immunofluoresence staining.Conclusion:1)PDMS substrates with different stiffness have no effect on EMT.2)Different topographic substrates can affect EMT in A549 cells.Compared with planar,EMT process can be induced by increasing dimension or decreasing spacing of micropillars.Notably,the variation of spacing plays an even more important role in EMT.3)Stiffness and topography of substrates interactions co-regulate EMT.The proteins expression of E-cadherin decreased and Vimentin increased with decreasing spacing on hard substrate.While the similar phenomenon was found by increasing spacing of micropillars on soft substrate.4)PI3K/Akt signaling pathway was involved in the EMT process of human alveolar epithelial cells.