Xiao Y, Truskey G A
Center for Biochemical Engineering, Duke University, Durham, North Carolina 27708, USA.
Biophys J. 1996 Nov;71(5):2869-84. doi: 10.1016/S0006-3495(96)79484-5.
The objective of this study was to determine the effect of receptor-ligand affinity on the strength of endothelial cell adhesion. Linear and cyclic forms of the fibronectin (Fn) cell-binding domain peptide Arg-Gly-Asp (RGD) were covalently immobilized to glass, and Fn was adsorbed onto glass slides. Bovine aortic endothelial cells attached to the surfaces for 15 min. The critical wall shear stress at which 50% of the cells detached increased nonlinearly with ligand density and was greater with immobilized cyclic RGD than with immobilized linear RGD or adsorbed Fn. To directly compare results for the different ligand densities, the receptor-ligand dissociation constant and force per bond were estimated from data for the critical shear stress and contact area. Total internal reflection fluorescence microscopy was used to measure the contact area as a function of separation distance. Contact area increased with increasing ligand density. Contact areas were similar for the immobilized peptides but were greater on surfaces with adsorbed Fn. The dissociation constant was determined by nonlinear regression of the net force on the cells to models that assumed that bonds were either uniformly stressed or that only bonds on the periphery of the contact region were stressed (peeling model). Both models provided equally good fits for cells attached to immobilized peptides whereas the peeling model produced a better fit of data for cells attached to adsorbed Fn. Cyclic RGD and linear RGD both bind to the integrin alpha v beta 3, but immobilized cyclic RGD exhibited a greater affinity than did linear RGD. Receptor affinities of Fn adsorbed to glycophase glass and Fn adsorbed to glass were similar. The number of bonds was calculated assuming binding equilibrium. The peeling model produced good linear fits between bond force and number of bonds. Results of this study indicate that 1) bovine aortic endothelial cells are more adherent on immobilized cyclic RGD peptide than linear RGD or adsorbed Fn, 2) increased adhesion is due to a greater affinity between cyclic RGD and its receptor, and 3) the affinity of RGD peptides and adsorbed Fn for their receptors is increased after immobilization.
本研究的目的是确定受体 - 配体亲和力对内皮细胞黏附强度的影响。将纤连蛋白（Fn）细胞结合域肽Arg - Gly - Asp（RGD）的线性和环状形式共价固定在玻璃上，并将Fn吸附到载玻片上。牛主动脉内皮细胞在这些表面上附着15分钟。50%的细胞脱离时的临界壁面剪应力随配体密度呈非线性增加，并且固定化环状RGD的临界壁面剪应力大于固定化线性RGD或吸附的Fn的临界壁面剪应力。为了直接比较不同配体密度的结果，根据临界剪应力和接触面积的数据估算受体 - 配体解离常数和每个键的力。使用全内反射荧光显微镜测量接触面积作为分离距离的函数。接触面积随配体密度增加而增加。固定化肽的接触面积相似，但吸附有Fn的表面上的接触面积更大。解离常数通过将细胞上的净力进行非线性回归到假设键要么均匀受力要么仅接触区域周边的键受力的模型（剥离模型）来确定。对于附着在固定化肽上的细胞，这两种模型拟合效果同样良好，而对于附着在吸附的Fn上的细胞，剥离模型对数据的拟合更好。环状RGD和线性RGD都与整合素αvβ3结合，但固定化环状RGD表现出比线性RGD更高的亲和力。吸附到糖相玻璃上的Fn和吸附到玻璃上的Fn的受体亲和力相似。假设结合平衡计算键的数量。剥离模型在键力和键的数量之间产生了良好的线性拟合。本研究结果表明：1）牛主动脉内皮细胞在固定化环状RGD肽上比在线性RGD或吸附的Fn上更具黏附性；2）黏附增加是由于环状RGD与其受体之间具有更高的亲和力；3）固定化后，RGD肽和吸附的Fn对其受体的亲和力增加。
