Three-dimensional Migration of Neurites Is Mediated by Adhesion Site Density and Affinity

Three-dimensional neurite outgrowth rates within fibrin matrices that contained variable amounts of RGD peptides were shown to depend on adhesion site density and affinity. Bi-domain peptides with a factor XIIIa substrate in one domain and a RGD sequence in the other domain were covalently incorporated into fibrin gels during coagulation through the action of the transglutaminase factor XIIIa, and the RGD-dependent effect on neurite outgrowth was quantified, employing chick dorsal root ganglia cultured two- and three-dimensionally within the modified fibrin. Two separate bi-domain peptides were synthesized, one with a lower binding affinity linear RGD domain and another with a higher binding affinity cyclic RGD domain. Both peptides were cross-linked into fibrin gels at concentrations up to 8.2 mol of peptide/mol of fibrinogen, and their effect on neurite outgrowth was measured. Both two- and three-dimensional neurite outgrowth demonstrated a bi-phasic dependence on RGD concentration for both the linear and cyclic peptide, with intermediate adhesion site densities yielding maximal neurite extension and higher densities inhibiting outgrowth. The adhesion site density that yielded maximal outgrowth depended strongly on adhesion site affinity in both two and three dimensions, with lower densities of the higher affinity ligand being required (0.8-1.7 mol/mol for the linear peptide versus 0.2 mol/mol for the cyclic peptide yielding maximum neurite outgrowth rates in three-dimensional cultures)

Cross-Linking Exogenous Bifunctional Peptides into Fibrin Gels with Factor XIIIa

Bi-domain peptides with a factor XIIIa substrate in one domain and a bioactive peptide in another domain were covalently incorporated into fibrin gels during coagulation through the action of the transglutaminase factor XIIIa. The cross-linking characteristics were determined for two bi-domain peptides with factor XIIIa substrates based on fibrinogen, dYRGDTIGEGQQHHLGG-NH_2, and dLRGDGAKDV-NH_2, as well as one bi-domain peptide with a substrate sequence based on α_2-plasmin inhibitor, dLNQEQVSPLRGD-NH_2, and another with a nonbiological, oligolysine substrate, dLRGDKKKKG-NH_2 (substrate domains in italic). Each of these peptides was able to cross-link into the fibrin gels during coagulation, with the peptide containing the factor XIIIa substrate based on α2-plasmin inhibitor being incorporated at levels in excess of 8 mol/mol fibrinogen. The structural characteristics of these peptide-modified gels proved to be the same as those for a native fibrin gel. The bioactivity of the incorporated active factors was tested in a neuronal culture model with day 8 chicken dorsal root ganglia using two bioactive sequences, RGD and DGEA, and one inactive control sequence, RDG. Each of these peptides influenced the extension of neurites from the ganglia as expected, indicating that the incorporated factors retained their activity. With the use of soluble competitive inhibitors, it was shown that this effect was due to the covalently incorporated peptides. Through exploiting the role of factor XIIIa in coagulation, we have developed a method by which to impart the character of nonfibrin proteins, such as extracellular matrix proteins, to fibrin, a biological material with many potential therapeutic and academic applications

Cross-Linking Exogenous Bifunctional Peptides into Fibrin Gels with Factor XIIIa

Bi-domain peptides with a factor XIIIa substrate in one domain and a bioactive peptide in another domain were covalently incorporated into fibrin gels during coagulation through the action of the transglutaminase factor XIIIa. The cross-linking characteristics were determined for two bi-domain peptides with factor XIIIa substrates based on fibrinogen, dYRGDTIGEGQQHHLGG-NH_2, and dLRGDGAKDV-NH_2, as well as one bi-domain peptide with a substrate sequence based on α_2-plasmin inhibitor, dLNQEQVSPLRGD-NH_2, and another with a nonbiological, oligolysine substrate, dLRGDKKKKG-NH_2 (substrate domains in italic). Each of these peptides was able to cross-link into the fibrin gels during coagulation, with the peptide containing the factor XIIIa substrate based on α2-plasmin inhibitor being incorporated at levels in excess of 8 mol/mol fibrinogen. The structural characteristics of these peptide-modified gels proved to be the same as those for a native fibrin gel. The bioactivity of the incorporated active factors was tested in a neuronal culture model with day 8 chicken dorsal root ganglia using two bioactive sequences, RGD and DGEA, and one inactive control sequence, RDG. Each of these peptides influenced the extension of neurites from the ganglia as expected, indicating that the incorporated factors retained their activity. With the use of soluble competitive inhibitors, it was shown that this effect was due to the covalently incorporated peptides. Through exploiting the role of factor XIIIa in coagulation, we have developed a method by which to impart the character of nonfibrin proteins, such as extracellular matrix proteins, to fibrin, a biological material with many potential therapeutic and academic applications

Enzymatic incorporation of bioactive peptides into fibrin matrices enhances neurite extension

Fibrin plays an important role in wound healing and regeneration, and enjoys widespread use in surgery and tissue engineering. The enzymatic activity of Factor XIIIa was employed to covalently incorporate exogenous bioactive peptides within fibrin during coagulation. Fibrin gels were formed with incorporated peptides from laminin and N-cadherin alone and in combination at concentrations up to 8.2 mol peptide per mole of fibrinogen. Neurite extension in vitro was enhanced when gels were augmented with exogenous peptide, with the maximal improvement reaching 75%. When this particular fibrin derivative was evaluated in rats in the repair of the severed dorsal root within polymeric tubes, the number of regenerated axons was enhanced by 85% relative to animals treated with tubes filled with unmodified fibrin. These results demonstrate that it is possible to enhance the biological activity of fibrin by enzymatically incorporating exogenous oligopeptide domains of morphoregulatory proteins

Fibronectin modulates macrophage adhesion and FBGC formation: The role of RGD, PHSRN, and PRRARV domains

To probe the role of human plasma fibronectin in modulating human blood-derived macrophage adhesion and fusion to form multinucleated foreign-body giant cells (FBGC), a series of biomimetic oligopeptides based on the functional structure of fibronectin was designed and synthesized. Peptides incorporated the RGD and PHSRN integrin-binding sequences from FIII-10 and FIII-9 modules, respectively, and the PRRARV sequence from the C-terminal heparin-binding domain, either alone or in combination. Peptides were immobilized onto a polyethyleneglycol-based polymer substrate. The following conclusions were reached. Fibronectin modulated macrophage adhesion and the extent (i.e., size) of FBGC formation on control surfaces in the presence of serum proteins. Macrophages adhered to all substrates with relatively subtle differences between adhesion mediated by RGD, PHSRN, PRRARV, or combinations thereof. β1-integrin subunit was essential in macrophage adhesion to peptide-grafted networks in a receptor-peptide specific manner, whereas β3-integrin subunit was less important. Macrophage adhesion to PRRARV was mediated primarily by the direct interaction with integrins. RGD or PHSRN alone did not provide an adequate substrate for macrophage fusion to form FBGCs. However, the PHSRN synergistic site and the RGD site in a single oligopeptide provided a substrate for FBGC formation that was statistically comparable to that on the positive control material in the presence of serum proteins. This response was highly dependent upon the relative orientation between RGD and PHSRN. PRRARV did not support FBGC formation. These results demonstrate the importance of fibronectin and, specifically, the synergy between RGD and PHSRN domains, in supporting macrophage fusion to form FBGCs. © 2001 John Wiley & Sons, Inc.Link_to_subscribed_fulltex