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Laminin-511, a major component of endothelial basement membrane, consists of α5, β1, and γ1 chains. The short arm region of the α5 chain is a structural feature of endothelial laminins. In this study, we identified active sequences for human umbilical vein endothelial cells (HUVECs) using recombinant proteins and synthetic peptides. The short arm of the α5 chain contains three globular domains [laminin N-terminal globular domain, laminin 4 domain a, and laminin 4 domain b (LN, L4a, and L4b)] and three rod-like elements [laminin epidermal growth factor-like domain a, b, and c (LEa, LEb, and LEc)]. The cell attachment assay using recombinant proteins showed that RGD-independent cell attachment sites were localized in the α5LN-LEa domain. Further, we synthesized 70 peptides covering the amino acid sequences of the α5LN-LEa domain. Of the 70 peptides, A5-16 (mouse laminin α5 230-243: LENGEIVVSLVNGR) potently exhibited endothelial cell attachment activity. An active sequence analysis using N-terminally and C-terminally truncated A5-16 peptides showed that the nine-amino acid sequence IVVSLVNGR was critical for the endothelial cell attachment activity. Cell adhesion to the peptides was dependent on both cations and heparan sulfate. Further, the A5-16 peptide inhibited the capillary-like tube formation of HUVECs with the cells forming small clumps with short tubes. The eight-amino acid sequence EIVVSLVN in the A5-16 peptide was critical to inhibit HUVEC tube formation. This amino acid sequence could be useful for grafts and thus modulate endothelial cell behavior for vascular surgery. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Peptide-polysaccharide matrices can mimic extracellular matrix structure and function and are useful for tissue and cell engineering. The spacer between the peptide and the polysaccharide is important for both peptide conformation and the interaction between the peptide and receptors. Here, the effect of a spacer on the biological activity of peptide-polysaccharide matrices using various lengths of spacers consisting of glycine, β-alanine, and e-aminocaproic acid has been examined. Active laminin-derived peptides, including a syndecan-binding peptide (AG73: RKRLQVQLSIRT), an integrin αvβ3-binding peptide (A99a: ALRGDN), and an integrin α6β1-binding peptide (A2G10: SYWYRIEASRTG), were used as the peptide ligands and chitosan was used as a polysaccharide matrix. The spacers did not influence the biological activity of the AG73-chitosan matrix. In contrast, the integrin-binding peptide-chitosan matrices showed spacer-dependent activity. Hydrophobic spacers enhanced the cell attachment activity of the A99a-chitosan matrix. A four-glycine spacer showed the strongest effect for the biological activity of the A2G10-chitosan matrix. These results suggested that spacer-optimization for each peptide is important for designing effective peptide-polysaccharide matrices. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 512-520, 2016.

Despite the research done on pathological angiogenesis, there is still a need for the development of new therapies against angiogenesis-related diseases. Fibulin-7 (Fbln7) is a member of the extracellular matrix fibulin protein family. The Fbln7 C-terminal fragment, Fbln7-C, binds to endothelial cells and inhibits their tube formation in culture. In this study, we screened 12 synthetic peptides, covering the fibulin-globular domain of Fbln7-C, to identify active sites for endothelial cell adhesion and in vitro antiangiogenic activity. Three peptides, fc10, fc11, and fc12, promoted Human Umbilical Vein Endothelial Cells (HUVECs) adhesion, and the morphology of HUVECs on fc10 was similar to that on Fbln7-C. EDTA and the anti-integrin β1 function-blocking antibody inhibited HUVECs adhesion to both fc10 and fc12, and heparin inhibited HUVECs adhesion to both fc11 and fc12. fc10 and fc11 inhibited HUVECs tube formation. Our results suggest that three peptides from Fbln7-C are biologically active for endothelial cell adhesion and disrupt the tube formation, suggesting a potential therapeutic use of these peptides for angiogenesis-related diseases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 184-195, 2016.

An amyloidogenic LAM-L peptide (AASIKVAVSADR, all-L configuration) derived from laminin promoted cell adhesion, neurite outgrowth, and angiogenesis. Here, we prepared novel matrices using double-stranded DNA and the LAM-L peptide. Double-stranded DNA promoted aggregation of amyloid-like fibrils and generated a LAM-L/DNA matrix through electrostatic interactions between the phosphate groups of DNA and the amino groups of LAM-L. This formation of peptide/DNA matrix depends on the Ile-Lys-Val-Ala-Val (IKVAV) sequence in the peptide, since LAM-RM peptide (AASVVIAKSADR), which is scrambled peptide of LAM-L, did not form a matrix with DNA. Further, LAM-D (all-D configuration of LAM-L), which forms amyloid-like fibrils and promotes similar biological activities as LAM-L, did not form amyloid-like fibrils with DNA, suggesting that DNA selectively interacts with the L-configured peptide. Moreover, the LAM-L/DNA matrices showed stronger cell attachment activity compared with LAM-L alone, suggesting the LAM-L/DNA matrices have potential for use as a novel biomaterial in tissue engineering. © 2014 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 465–472, 2014.

Laminins are a multifunctional molecule with numerous active sites that have been identified in short peptide sequences. Mixed peptide-conjugated chitosan membranes using laminin-derived active peptides have been previously demonstrated to be useful as a biomaterial for tissue engineering. In this study, two syndecan-binding peptides, AG73 (RKRLQVQLSIRT) and C16 (KAFDITYVRLKF), and three integrin-binding peptides, EF1zz (ATLQLQEGRLHFXFDLGKGR, X: Nle, binding to integrin α2β1), A99a (ALRGDN, binding to integrin αvβ3), and A2G10 (SYWYRIEASRTG, binding to integrin α6β1), were mixed in various combinations, conjugated to chitosan membranes, and evaluated for their cell attachment and spreading activities. The cell attachment and spreading activity of EF1zz, A99a, and A2G10 were enhanced by AG73. In contrast, C16 enhanced only the cell attachment and spreading activity of A99a and did not influence the activity of EF1zz and A2G10. As well as previous study, the AG73-chitosan membrane bound to only syndecan. On the other hand, the C16-chitosan membrane interacted with both syndecan and β1 integrin. These data suggest that interaction of different receptors can cause synergistic effects. Therefore, AG73 is widely applicable as a synergistic agent for mixed peptide-matrices using several types of integrin-binding peptides. Additionally, the A2G10/AG73-chitosan membrane may be useful to investigate detailed biological functions of α6β1 integrin, which is a major laminin-binding receptor. Using a combination of tissue-appropriate laminin-derived peptides, the mixed peptide-chitosan membranes may serve as functional biomaterials for tissue engineering.

Hepatocytes rapidly lose hepatic functions upon isolation from liver, perhaps due to disrupted cell/matrix interactions. The matrix macromolecule laminin-111 consists of three chains, α1, β1, and γ1; it is a major component of Matrigel, which can maintain hepatic differentiation. We previously showed that the A13 peptide (RQVFQVAYIIIKA, α1 chain 121–133) derived from mouse laminin α1 exhibits hepatocyte attachment activity and maintains hepatic differentiation. Here, we sought to identify hepatocyte adhesive sequences from the mouse laminin β1 and γ1 chains using 22 synthetic peptides that show biological activity for fibrosarcoma cells. Nine peptides showed hepatocyte attachment activity. Of these, B160 (VILQQSAADIAR, β1 chain 1607–1618), and C16 (KAFDITYVRLKF γ1 chain 139–150) exhibited the most potent activity. Hepatocytes cultured on both peptides also maintained expression of albumin, tyrosine aminotransferase, tryptophan-2,3-dioxygenase, and cytochrome P450. The morphology of hepatocytes on both peptides was a rounded shape typical for hepatic differentiation. We also characterized the nature of adhesion to the peptides. Heparin and EDTA inhibited cell attachment to both peptides, suggesting that hepatocyte attachment to the peptides was mediated by multiple receptors. The identification of active sequences regulating hepatic functions may facilitate the design of hepatocyte culture substrata that can regulate specific cellular behaviors in the context of a bioartificial liver. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.

Laminin active peptide-conjugated chitosan mambranes have been previously demonstrated as a useful biomaterial for tissue engineering. Here, three laminin active peptides, A99 (AGTFALRGDNPQG), AG73 (RKRLQVQLSIRT), and EF1zz (ATLQLQEGRLHFXFDLGKGR, X: Nle), which interact with integrin αvβ3, syndecans, and integrin α2β1, respectively, were conjugated to alginate and evaluated the biological activities. A99-alginate (3-3000 ng/mm(2)) promoted cell attachment depending on the amount of alginate. More than 300 ng/mm(2) of the A99-alginate matrices effectively promoted cell attachment, cell spreading with well-organized actin stress fibers, and neurite outgrowth. AG73- and EF1zz-alginates promoted strong cell attachment at the all amounts (3-3000 ng/mm(2)). A99-alginate (30-3000 ng/mm(2)) promoted strong neurite outgrowth but lower amounts of A99-alginate (3 ng/mm(2)) showed weak activity. In contrast, AG73-alginates (3-30 ng/mm(2)) showed strong neurite outgrowth activity but higher amounts of AG73-alginate (300-3000 ng/mm(2)) decreased the activity. These data indicate that neurite outgrowth activity of peptide-alginate matrices is peptide specific and the activity is dependent on the amount of alginate. Further, biological activities of the peptides on alginate and chitosan matrices were different, suggesting that the integrin- and syndecan-mediated cellular functions on the peptide-matrices are highly influenced by the scaffold structure including polysaccharide types and amounts. The laminin active peptide-conjugated alginate and chitosan matrices can control receptor type specific functions and are useful for tissue engineering.

Novel peptide-conjugated chitosan membranes were fabricated and used to deliver keratinocytes to dermal wounds in mice. Three active peptides of 12 or 13 amino acids each, RLVSYNGIIFFLK (A5G27), ASKAIQVFLLAG (A5G33), and AGTFALRGDNPQG (A99) were selected from a cell-adhesive peptide library of laminin, a major constituent of basement membrane. The peptides were synthesized and coupled to chitosan membranes, and the resulting peptide-chitosan membranes were tested for keratinocyte attachment. Two of the peptides that bind to cell surface heparin-like receptors (A5G27 and A5G33) were found to promote strong keratinocyte attachment, whereas the one that binds to integrin (A99) was inactive. Subsequently, A5G27- and A5G33-chitosan membranes were tested as vehicles for keratinocyte delivery in a wound model. We found that keratinocytes were delivered into the full-thickness wound with either membrane. Using the A5G33-chitosan membrane, we further evaluated the activity of the delivered keratinocytes in wound healing. Immunohistochemistry for granulation tissue markers, including tenascin and alpha-smooth muscle actin, showed that keratinocyte delivery by the present peptide-chitosan membranes in the wound bed provided a favorable condition for keratinocyte migration along the wound surface and reduced granulation tissue formation.

Peptide-conjugated polysaccharide matrices using bioactive laminin-derived peptides are useful biomaterials for tissue and cell engineering. Here, we demonstrate an easy handling preparation method for peptide-polysaccharide matrices using polyion complex with both alginate and chitosan. First, aldehyde-alginate was synthesized by oxidization of alginate using NaIO4 , and then, reacted with Cys-peptides. Next, the peptide-alginate solution was added to a chitosan-coated plate, and the peptide-polyion complex matrices (peptide-PCMs) were prepared. The peptide-PCMs using an integrin αvβ3-binding peptide (A99a: ALRGDN, mouse laminin α1 chain 1145-1150) and an integrin α2β1-binding peptide (EF1XmR: RLQLQEGRLHFXFD, X = Nle, mouse laminin α1 chain 2751-2763) showed strong cell attachment activity in a dose-dependent manner. When we examined the effect of various spacers on the biological activity of A99a-PCM, hydrophobic and long spacers enhanced the cell attachment activity. Further, the A99a-PCM with the spacers strongly promoted neurite outgrowth. The polyion complex method is an easy way to obtain insolubilized matrix and is widely applicable for various polysaccharides. The peptide-PCM is useful as a biomaterial for cell and tissue engineering.

Cell-adhesive peptides derived from extracellular matrix (ECM) proteins are potential candidates for incorporating cell-binding activities into materials for tissue engineering. We have identified a number of cell adhesive peptides from laminins, which are major components of basement membrane ECM. Our goal is the development of synthetic basement membranes using the peptides on scaffolds. We review peptide–polysaccharide complexes, which were prepared by conjugation of the peptides to chitosan and alginate, and the biological activities of the resulting matrices. The peptide–polysaccharide matrices can also be used as a biomaterial for cell transplantation. These studies suggest that the peptide–polysaccharide complexes have the potential to mimic the multifunctional basement membrane and may be useful for tissue engineering.