Yuichi Kadoya

Laminin-1 peptide-conjugated chitosan membranes as a novel approach for cell engineering

Laminin, a major component of the basement membrane, has diverse biological activities. Recently, we identified various biologically active sequences on laminin‐1 by using a large set of synthetic peptides. Chitosan, a polysaccharide, is biodegradable and has been used as a biomaterial. Here, we conjugated several biologically active laminin peptides onto chitosan membranes and measured the cell attachment activity of peptide‐conjugated chitosan membranes with various cell types. The active laminin peptide‐conjugated chitosan membranes promoted cell attachment with cell type specificity. A99 (AGTFALRGDNPQG)‐chitosan membrane promoted cell attachment with well‐organized actin stress fibers. This adhesion was inhibited by EDTA but not by heparin. AG73 (RKRLQVQLSIRT)‐chitosan membrane promoted cell attachment with filopodia formation, and this adhesion was inhibited by heparin but not by EDTA. These data suggest that the A99‐chitosan membrane interacted with an integrin cellular receptor and that the AG73‐chitosan membrane promoted proteoglycan‐mediated cell attachment, as previously reported. Furthermore, both AG73‐chitosan and A99‐chitosan membranes effectively promoted neurite outgrowth with PC12 rat pheochromocytoma cells. We conclude that conjugation on a chitosan membrane is applicable for testing quantitatively the biological activity of synthetic peptides and that these constructs have a potential ability to serve as bioadhesive materials for tissue regeneration and engineering.

Ile-Lys-Val-Ala-Val (IKVAV)-containing laminin α1 chain peptides form amyloid-like fibrils

The Ile‐Lys‐Val‐Ala‐Val (IKVAV) sequence derived from laminin‐1 promotes cell adhesion, neurite outgrowth, and tumor growth and metastasis. Here, we examined amyloid formation of an IKVAV‐containing peptide (LAM‐L: AASIKVAVSADR, mouse laminin α1 chain 2097–2108). The LAM‐L peptide was stained with Congo red and exhibited fibrils in electron microscopy with a characteristic cross‐β X‐ray diffraction pattern. Further, infrared spectra of LAM‐L suggested a β‐sheet structure. These results indicate that LAM‐L forms amyloid‐like fibrils. We also examined amyloid‐like fibril formation of LAM‐L analogs. The neurite outgrowth activity of the LAM‐L analogs was closely related to their amyloid‐like fibril formation.

Laminin α1 chain G domain peptide, RKRLQVQLSIRT, inhibits epithelial branching morphogenesis of cultured embryonic mouse submandibular gland

Active sequences from the laminin α1 and α2 chain carboxyl‐terminal globular domains (G domain) have been identified by screening overlapping synthetic peptides in a number of biological assays (Nomizu et al. [1995] J. Biol. Chem. 270:20583–20590; Nomizu et al. [1996] FEBS Lett. 396:37–42). We have tested the activity of these peptides in submandibular gland explants of embryonic day 13 mice to determine the functional sites involved in organ development. The laminin α1 chain peptide, RKRLQVQLSIRT (residues 2719–2730 and designated AG‐73), significantly inhibited epithelial branching morphogenesis. In contrast, other cell adhesive laminin α1 chain peptides including the AASIKVAVSADR and NRWHSIYITRFG failed to inhibit the branching. MG‐73, a homologue of AG‐73 from the laminin α2 chain, did not inhibit the branching. The α2 chain peptide had no effect, which may be due to the low levels of this laminin chain in day 13 mice. Laminin α2 chain‐specific monoclonal antibodies strongly reacted with the basement membranes of developed acini but only weakly stained embryonic day 13 submandibular epithelium. The expression of E‐cadherin and α6 integrin, as detected by immunofluorescence, were unchanged in both AG‐73 and control scramble peptide‐treated epithelial cells of the explants. In contrast, immunostaining of nidogen/entactin showed that explants treated with AG‐73 for 3 days had a discontinuous basement membrane. Explants treated for 3 days with control peptide showed a normal basement membrane. These results suggest that the region containing the AG‐73 sequence of the laminin α1 chain is crucial for development of submandibular gland at early embryonic stages. The discontinuous basement membrane in AG‐73‐treated explants may indicate an important role for this region in basement membrane assembly. Dev. Dyn. 1998;212:394–402.

Significant role of laminin-1 in branching morphogenesis of mouse salivary epithelium cultured in basement membrane matrix.

Mouse submandibular epithelium shows branching morphogenesis in mesenchyme‐free conditions when covered with a basement membrane matrix (Matrigel) in medium supplemented with epidermal growth factor. In the present study, the role of laminin‐1 (LN1), a major glycoprotein of Matrigel, in this culture system was defined. When the epithelium was cultured in a LN1‐nidogen gel, the epithelium showed much branching, comparable to that observed with Matrigel. By electron microscopy, only a felt‐like matrix was formed on the epithelial surface in the LN1‐nidogen gel cultures, while an organized basal lamina structure was formed on the epithelial surface in direct or transfilter recombination cultures with mesenchyme. Next, the epithelium covered with Matrigel was cultured in medium containing either biologically active peptides from LN1, IKVAV‐including peptide (2097–2108), AG10 (2183–2194), AG32 (2370–2381) or AG73 (2719–2730) from the α1 chain, or YIGSR‐including peptide (926–933) from the β1 chain. Only AG73 (RKRLQVQLSIRT from the α1 chain carboxyl‐terminal globular domain) inhibited the epithelial branching in Matrigel. These results suggest that LN1‐nidogen can support the branching morphogenesis of submandibular epithelium even if LN1‐nidogen is not assembled into an intact basal lamina, and that the AG73 sequence is an important site on LN1, which interacts with submandibular epithelial cells.

Role for laminin-α5 chain LG4 module in epithelial branching morphogenesis

Laminin-alpha5 chain was localized in all epithelial basement membranes (BMs) of mouse submandibular gland (SMG) from the onset of branching morphogenesis and became restricted to BMs of epithelial ducts in the adult. To investigate whether the laminin-alpha5 chain plays a role in branching morphogenesis, a set of cell-adhesive peptides from the C-terminal globular domains (LG1-5) was tested for their effects in SMG organ cultures. One peptide, LVLFLNHGH (A5G77f), which represents a sequence located in the connecting loop between strands E and F of LG4. perturbed branching morphogenesis and resulted in irregularities in the contours of epithelial structures, with formation of deep clefts. The data suggest a role for the laminin-alpha5 LG4 module in the development of the duct system, rather than in the bifurcation of epithelial clusters. The epithelial BM of A5G77f-peptide-treated explants was continuous, which was in contrast to our previous finding of impaired epithelial BM assembly in explants treated with the larninin-alpha1 LG4 module peptide, or with a monoclonal antibody against this domain. A5G77f also perturbed in vitro development of lung and kidney. These results suggest a crucial role for the LG4 module of larninin-alpha5 in epithelial morphogenesis that is distinct from that of the laminin-alpha1 LG4

Intracellular accumulation of basement membrane components during morphogenesis of rat submandibular gland.

The distribution of two basement membrane (BM) components, laminin (LN) and type IV collagen (COLL IV), during acino-tubular morphogenesis of rat submandibular gland was examined immunohistochemically to determine the role of BM in the development of acino-tubular structures. On day 14 of gestation, LN could be found only in the BM separating an undifferentiated cell cluster of gland epithelium from surrounding mesenchyme. However, during a short period through days 15 to 17, LN was detected not only in the BM but also in intracellular vesicles of the cells of the terminal cluster. Immunoelectron microscopy showed the intracellular immunoreactive sites to be rough endoplasmic reticulum, indicating that active LN synthesis occurs in the cells of the terminal cluster. Intracellular immunostaining of LN disappeared completely on day 19 with the development of simple epithelium from the cell cluster, even though BM remained reactive. COLL IV also was accumulated in the intracellular vesicles of terminal cluster cells on day 16 of gestation but not on day 19. These results indicate that synthesis of certain BM components is transiently stimulated in gland epithelium before the formation of simple epithelial structure, and that these components are significantly involved in morphogenesis of the submandibular gland.

Expression of Epidermal Growth Factor Receptor in Fetal Mouse Submandibular Gland Detected by a Biotinyltyramide-based Catalyzed Signal Amplification Method

Branching morphogenesis of the fetal mouse submandibular gland (SMG) can be modulated in vitro by stimulation or inhibition of the epidermal growth factor receptor (EGFR). Because the mRNAs for EGF and EGFR are detectable in RNA of SMG rudiments isolated directly from fetuses, the EGF system probably operates physiologically as a regulator of SMG morphogenesis. However, neither EGFR protein nor its precise cellular localization has been characterized in the fetal SMG. Here we show EGFR protein in fetal mouse SMG by immunoprecipitation, affinity labeling, ligandinduced autophosphorylation, and immunohistochemistry. SMGs from E16 fetuses (day of vaginal plug = EO) were labeled with [35S]-cysteine/methionine and homogenized. After addition of specific antibody to EGFR, the immunoprecipitate was isolated, resolved by polyacrylamide gel electrophoresis, and detected by autoradiography. A single band of 170 kD was detected, corresponding to the EGFR protein. Affinity labeling with [125I]-EGF of the membrane fraction of E18 SMG also revealed a prominent band at 170 kD, showing that this EGFR protein can bind specifically to its ligand. Incubation of SMG membranes from E18 fetuses with EGF in the presence of [γ-32P]-ATP, followed by immunoprecipitation with anti-phosphotyrosine antibody also showed a single band at 170 kD, demonstrating autophosphorylation of the EGFR in response to binding of its ligand. Immunohistochemical localization of the cellular sites of EGFR in the fetal SMG required use of a catalyzed signal amplification procedure, with biotinyltyramide as the amplifying agent. EGFR was localized predominantly, if not exclusively, in cell membranes of epithelial cells of the rudiment, whereas staining of mesenchymal cells was equivocal. Staining was strongest on duct cells, and weak on cells of the end-pieces. These findings clearly show that a functional EGFR protein is expressed in fetal SMG chiefly, if not exclusively, on epithelial cells.

A collagen-mimetic triple helical supramolecule that evokes integrin-dependent cell responses.

Collagen is an abundantly distributed extracellular matrix protein in mammalian bodies that maintains structural integrity of the organs and tissues. Besides its function as a structural protein, collagen has various biological functions which regulate cell adhesion, migration and differentiation. In order to develop totally synthetic collagen-surrogates, we recently reported a basic concept for preparing collagen-like triple helical supramolecules based on the self-assembly of staggered trimeric peptides with self-complementary shapes. In this paper, we add one of the specific cellular functions of the native collagen to the collagen-mimetic supramolecule. We synthesized a self-assembling peptide unit containing the integrin-binding sequence Gly-Phe-Hyp-Gly-Glu-Arg. The supramolecule carrying the sequence exhibited significant binding activity to human dermal fibroblasts. The supramolecular structure was found to be essential for function in in vitro cell culture. Cell adhesion was shown to be comparable to that of native collagen, and was further demonstrated to be mediated solely by integrin alpha 2 beta 1. Well-grown focal contacts and stress fibers were observed in cells spread on the supramolecular collagen-mimetic. The results demonstrate the potential of peptide-based artificial collagen as a biomaterial for regulating specific cellular function and fate.

Mixed peptide–chitosan membranes to mimic the biological activities of a multifunctional laminin α1 chain LG4 module

Laminin alpha1 chain LG4 module is multifunctional and interacts with syndecans and integrin alpha2beta1 via AG73 (RKRLQVQLSIRT) and EF-I (DYATLQLQEGRLHFMFDLG) sites, respectively. Here, we conjugated the AG73 and EF1zz (ATLQLQEGRLHFXFDLGKGR, X: Nle) peptides on a chitosan membrane in various ratios to develop an LG4 mimic biomaterial. The AG73-chitosan membrane promoted strong cell attachment with membrane ruffling and the EF1zz-chitosan membrane promoted integrin-mediated cell adhesion with well-organized actin stress fibers. When AG73 and EF1zz were conjugated on a chitosan membrane with 1:9 molar ratio, the mixed peptide-chitosan membrane promoted the strong cell attachment and neurite outgrowth similar to that on the recombinant LG4 protein. Well-organized actin stress fibers and vinculin accumulated focal contacts were observed in the cells attached on the AG73:EF1zz (molar ratio=1:9)-chitosan membrane. These results suggest that the mixed peptide-chitosan membrane interacts with both syndecans and integrin alpha2beta1 and mimics the cell adhesion of a multifunctional LG4 protein. The mixed peptide-chitosan approach has potential as a multifunctional biomaterial for cell and tissue engineering.

A novel cell‐adhesive scaffold material for delivering keratinocytes reduces granulation tissue in dermal wounds

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 α‐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.