Study of roles of cytoskeletons in morphological responses of vascular endothelial cells to mechanical stimuli

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1) Evaluation of mechanical property of stress fibersMechanical properties of stress fibers extracted from smooth muscle cells (SMCs) and endothelial cells (ECs) were measured with a microneedle-based tensile test system developed in this study. Strain-induced stiffening was observed in the force-strain curve. We also found that the extracted SFs shortened to approximately 80% of the original length after they were dislodged from the substrate, suggesting that SFs had preexisting strain in the cytoplasm. The force required for stretching the single SFs from the zero-stress length back to the original length was approximately 10 nN for SMCs and 4.1 nN for ECs.2) Measurement of traction forces of cells using microfabricated micropost arraysA mold of silicon substrates with arrays of micro-needle-like posts (3μm in diameter, 10μm in height, and 8μm intervals) was fabricated to estimate cellular traction forces using microfabrication techniques. SMCs spreading on the substrates produced deflection of the posts and was associated with organization of stress fibers of actin filaments. Traction forces varied considerably among cells, showing an average of approximately 12 nN.3) Cyclic stretch test with microsubstratesWe applied cyclic stretch to ECs using a microstructured substrate with arrays of micropost, on which cells were selectively stretched between FAs but FA-substrate contact area were hardly stretched. After exposure ECs to cyclic stretch for 3h, cells on both a flat and the micropost substrates aligned perpendicular to the direction of stretch. Stress fibers oriented about 60° to the stretch direction for the flat substrate at 3h, while stress fibers on the micropost substrate oriented approximately 90° to the stretch direction after 6h stretching. These results suggest that strain in FA-substrate contact area may have impact on rates of reorienation of stress fibers in ECs in response to cyclic stretch.