The Role Of Tmod1 In The Biomechanical Changes Of Macrophages Induced By Matrix Stiffness

The impairment of macrophage deformation and migration abilities is a key event in the occurrence of atherosclerosis.An increase in the stiffness of the vascular matrix is one of the important factors that cause the mechanical properties of macrophages to change.The assembly of focal adhesion and cytoskeleton determines the mechanical properties and migration of cells.Tropomodulin1(Tmod1)is a capping protein of actin microfilament(F-actin),which regulates the length and structure of F-actin.Objective:The purpose of this study was to investigate the role of Tmod1 in regulating the changes of mechanical properties in macrophages induced by matrix stiffness.Methods:We plated mouse macrophages Raw264.7 cells on polyacrylamide gels with stiffness of 2 and 20 k Pa,and cultured for 24,48,and 72 hours.Tmod1 expression was detected using real-time quantitative PCR and Western blotting.Tmod1 was overexpressed in Raw264.7 cells by adenovirus and the cells were seeded on matrices with different stiffness.The F-actin content in the cells was measured by flow cytometry.The focal adhesions were observed with a laser confocal microscope.Quantity and Differential interference phase contrast microscopy was used to detect the cell adhesion and spreading area on different matrices.Results:The results showed that compared with 2 k Pa matrigel,the protein expression level of Tmod1 in macrophages cultured on 20 k Pa matrigel increased by 1.8 ± 0.5 times(p <0.05).After overexpression of Tmod1,the number of macrophage adhesion on 2 k Pa matrigel increased by 2.00 ± 0.30 times,while the number of macrophage adhesion on 20 k Pa matrigel increased by 4.00 ± 0.78 times.Tmod1 overexpression significantly increased the stretched area of macrophages cultured on 20 k Pa Matrigel(p <0.05).The overexpression of Tmod1 significantly increased the number of focal spots and F-actin content in cells cultured on 20 k Pa matrigel(p <0.05).At the same time,after knocking down Tmod1,the number of macrophage adhesion on 2 k Pa matrigel decreased by 2.10 ± 0.67 times,and the number of adhesion on 20 k Pa matrigel decreased by 2.2 ± 0.52 times.Tmod1 knockdown caused a significant reduction in the stretched area of macrophages cultured on 20 k Pa Matrigel(p <0.05).The knockdown of Tmod1 significantly reduced the number of focal spots and the F-actin content in macrophages cultured on 20 k Pa matrigel(p <0.05).Conclusions:The results indicate that Tmod1 expression is increased in macrophages cultured on matrix with high stiffness,and Tmod1 may participate in the regulation of macrophage mechanical properties by regulating cytoskeleton and focal plaques.