A fluorophore-tagged RGD peptide to control endothelial cell adhesion to micropatterned surfaces

Abstract

The long-term patency rates of vascular grafts and stents are limited by the lack of surface endothelialisation of the implanted materials. We have previously reported that GRGDS and WQPPRARI peptide micropatterns increase the endothelialisation of prosthetic materials in vitro. To investigate the mechanisms by which the peptide micropatterns affect endothelial cell adhesion and proliferation, a TAMRA fluorophore-tagged RGD peptide was designed. Live cell imaging revealed that the micropatterned surfaces led to directional cell spreading dependent on the location of the RGD-TAMRA spots. Focal adhesions formed within 3 h on the micropatterned surfaces near RGD-TAMRA spot edges, as expected for cell regions experiencing high tension. Similar levels of focal adhesion kinase phosphorylation were observed after 3 h on the micropatterned surfaces and on surfaces treated with RGD-TAMRA alone, suggesting that partial RGD surface coverage is sufficient to elicit integrin signaling. Lastly, endothelial cell expansion was achieved in serum-free conditions on gelatin-coated, RGD-TAMRA treated or micropatterned surfaces. These results show that these peptide micropatterns mainly impacted cell adhesion kinetics rather than cell proliferation. This insight will be useful for the optimization of micropatterning strategies to improve vascular biomaterials.

The spatial patterning of RGD and BMP‐2 mimetic peptides at the subcellular scale modulates human mesenchymal stem cells osteogenesis

“…Finally, the substrates were rinsed three times for 15 min in DMSO, thoroughly washed with deionized water, and stored for at most 1 month in PBS until use. 37 Unless otherwise indicated, all reactions needed for surface modification were carried out with 50 rpm agitation, at room temperature, away from light, and cleaning steps were performed in ultrasonic bath. Patterns of RGD-TAMRA and BMP-2-FITC peptides developed using the underlying protocol are shaped as triangles, squares or rectangles and exhibit a constant overall area at subcellular scale.…”

Multifunctional biomaterial coatings: synthetic challenges and biological activity

“…A similar assembly consists of a linear RGD peptide, which was modified with a fluorescent dye and immobilized by silanization on glass (Table 1, line b). With this tag it was possible to calculate bound peptide, to visualize generated micropatterns and to investigate their potential co-localization with focal contacts by fluorescence microscopy (Hoesli et al, 2014). Fluorescence resonance energy transfer (FRET) between two fluorescent dyes is used to evaluate distances between a donor and an acceptor chromophore and thereby investigate interactions of biomolecules.…”

Micro-structured peptide surfaces for the detection of high-affinity peptide–receptor interactions in living cells

“…So far, micro-structured peptide surfaces have been described for the spatially resolved adhesion and growth of cells, as well as for the improvement of vascular biomaterials. Micro-contact printing (Csucs et al, 2003), inkjetprinting (Boivin et al, 2013) and spraying (Hoesli et al, 2014) have been used as micro-structuring methods. Here we employed photolithography for micro-structuring peptides on glass surfaces and used these surfaces for the validation of high-affinity peptidereceptor interactions in living cells.…”