In blood vessels, substrate stiffness of the endothelium varies between different body locations and increases during the progression of multiple sclerosis. As a crucial step of the immune response, lymphocyte function associated antigen-1 (LFA-1)/ intercellular adhesion molecule-1 (ICAM-1) interaction occurs in various tissues and plays a pivotal role in atherosclerosis. However, the contribution of the physical property of endothelium substrate, such as the stiffness, to LFA-1/ICAM-1 interaction and immune-related diseases progression remains largely unknown. In this study, we investigated the influence of substrate stiffness on the adhesion force of LFA-1/ICAM-1 bond and ICAM-1 expression on the endothelial cell apical surface with an improved in vitro model. A silica microsphere-functionalized atomic force microscopy (AFM) tip was linked to LFA-1 via a polyethylene glycol (PEG) chain, and then approached toward human aortic endothelial cells (HAECs) on polyacrylamide gels of different stiffnesses. The results showed that the adhesion force was elevated on stiff substrates, while the expression of ICAM-1 on the HAECs surface was not influenced by substrate stiffness. A low-dose blebbistatin treatment (5μmol/L) reduced the adhesion force on both substrates while a high dose blebbistatin treatment (50μmol/L)) eliminated the adhesion between LFA-1 and ICAM-1, indicating that endothelium substrate stiffness directs the LFA-1/ICAM-1 interaction in a myosin II-dependent manner. These results help to describe the relationship between substrate stiffness and myosin II-dependent LFA-1/ICAM-1 interaction, and may increase the understanding of the pathogenesis and treatment of immune-related diseases.
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