Comparison of the Effects of Different Coating Matrices on Cell Binding and Nitric Oxide-mediated Protein S-nitrosylation in Endothelial Cells under Shear Flow

Abstract

Objective: Shear flow is a mechanical signal regulating the function of Endothelial Cells (ECs). The present study aimed to investigate the effects of different matrices on cell binding, Nitric Oxide (NO) production, protein S-nitrosylation, expression of adhesion proteins, ROS generation, and cell viability in ECs under shear flow. Methods: The ECs growing on glass slides separately coated with poly-L-lysine (p-Lys), collagen (Colla), fibronectin (Fibro), and a combined matrix (Colla+Fibro) were exposed to shear flow (25 dyne/cm2) for 0, 1, 4, 8 h. The number of ECs remaining attached on the glass slide was calculated. The expressions of endothelial Nitric Oxide Synthase (eNOS), peNOSS1177, VE-cadherin, FAK, and S-nitrosylated proteins were investigated by western blotting. The production of Nitric Oxide (NO) was measured by a specific reagent. Finally, the levels of ROS and cell viability were monitored. Results: Under a constant shear flow for 1 h, the physiological responses of ECs were similar between these four matrices. When shear flow was extended to 4 and 8 h, higher cell binding, elevated NO production, increased S-nitrosylated proteins, enhanced expressions of FAK and VE-cadherin, mildly accumulated ROS, and cell death were observed in the matrix of Fibro and Colla+Fibro. Conclusion: We have concluded fibronectin to be the optimal matrix facilitating NO-mediated Snitrosylation that might be essential for superior binding efficiency, thereby preventing the stripping of ECs under shear flow. The results can be broadly applied to diverse biomechanical studies.