Synergistic effect of high‐affinity binding and flow preconditioning on endothelial cell adhesion

Abstract

The current study examined whether the combined introduction of high-affinity avidin-biotin bonds and fibronectin-integrin bonds (i.e., dual ligand treatment) would further augment the adhesion of flow-preconditioned endothelial cells to model substrates via contributions to the actin cytoskeleton and the formation of focal contacts. Human umbilical vein endothelial cells (HUVEC) were grown under static conditions or exposed to a flow-preconditioning regimen for 24 h. Cell retention was determined by exposure to 75 dynes/cm(2). The combination of flow preconditioning and the dual ligand treatment yielded higher cell retention under flow compared to the cells adherent via fibronectin-integrin bonds only. This increase in adhesion strength correlated with a greater focal contact area. Elongation of the HUVEC occurred after exposure to flow preconditioning; however, orientation of dual ligand adherent cells was restricted due to the presence of the high-affinity ligand. Flow-preconditioned cells showed increased stress fiber formation compared to nonconditioned cells although the stress fibers per cell for flow-preconditioned cells were the same on both the ligand systems employed. The results indicate that enhanced adhesion strength is due to a combination of increased focal contact area, stress fiber formation, and cell alignment.

Topographic guidance of endothelial cells on silicone surfaces with micro‐ to nanogrooves: Orientation of actin filaments and focal adhesions

“…Figure 6 shows the formation of dense peripheral F-actin bundles as well as focal adhesions after 1 h. The focal adhesions were approximately 1 m wide and 1 to 5 m long, consistent with literature values. 32,33 In accordance with the orientation and localization of the majority of F-actin, particularly on 500-nm, 1-m, and 5-m grooves [ Fig. 6(C-E)], the majority of focal adhesions also aligned parallel to the channel direction and preferentially attached to the ridge edge as indicated by white arrows in Figure 6(H-J).…”

Flow and High Affinity Binding Affect the Elastic Modulus of the Nucleus, Cell Body and the Stress Fibers of Endothelial Cells

“…17 Briefly, the addition of the dual ligand and flow preconditioning increased cell retention by a factor of 5.6 as compared to the cells adherent under static conditions to the fibronectin ligand. The addition of the high-affinity ligand also increased the cell retention 3-fold as compared to fibronectin alone under static conditions.…”

“…17 Briefly, a flow system was designed to induce laminar shear stress over the endothelial monolayer adherent to either the fibronectin surface or the dual ligand surface. 17 The flow system consisted of a flow cell, a dual syringe pump (Harvard Apparatus, Holliston, MA), 5% CO 2 percolated media reservoir, and a pulse dampener (Cole Parmer Instrument Company, Chicago, IL). The flow cell consisted of two anodized aluminum plates (12 cm  7.5 cm) with steel inlet and outlet tubes.…”

“…We have previously demonstrated that high affinity avidin-biotin (Ka = 10 13 -10 15 M )1 ) binding supplements low affinity fibronectin (Fn)-integrin (Ka = 10 6 -10 9 M )1 ) binding of human umbilical vein endothelial cells (HUVEC) to an artificial surface leading to a significant increase in adhesion strength. 4, 17,18 Avidin-biotin bonds increase the focal contact area of endothelial cells, 17 possibly due to the increased numbers of bonds formed. 6 In other studies, absence of focal adhesion proteins have been directly associated with a decrease in mechanical stiffness of cells 12 whereas an increase in fibronectin density was shown to increase cytoskeletal stiffness 31 .…”

Strategies for the chemical and biological functionalization of scaffolds for cardiac tissue engineering: a review

“…The conjugation of biotin on the cell membranes of non-adhesive cells of the Ehrilich ascites carcinoma cell line produced their adhesion to avidin-coated substrata [210]. Similarly, biotinylation of endothelial cells was used to enhance the initial cell attachment to avidin-treated surfaces, in order to improve in vivo patency of vascular grafts [211][212][213][214][215]. The application of this strategy in cell culture and tissue engineering was also explored.…”