Laminin Peptide Research Articles

Surface-Functionalized Silk Fibroin Films as a Platform To Guide Neuron-like Differentiation of Human Mesenchymal Stem Cells.

Surface interactions at the biomaterial-cellular interface determine the proliferation and differentiation of stem cells. Manipulating such interactions through the surface chemistry of scaffolds renders control over directed stem cell differentiation into the cell lineage of interest. This approach is of central importance for stem cell-based tissue engineering and regenerative therapy applications. In the present study, silk fibroin films (SFFs) decorated with integrin-binding laminin peptide motifs (YIGSR and GYIGSR) were prepared and employed for in vitro adult stem cell-based neural tissue engineering applications. Functionalization of SFFs with short peptides showcased the peptide sequence and nature of functionalization-dependent differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). Intriguingly, covalently functionalized SFFs with GYIGSR hexapeptide (CL2-SFF) supported hMSC proliferation and maintenance in an undifferentiated pluripotent state and directed the differentiation of hMSCs into neuron-like cells in the presence of a biochemical cue, on-demand. The observed morphological changes were further corroborated by the up-regulation of neuronal-specific marker gene expression (MAP2, TUBB3, NEFL), confirmed through semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. The enhanced proliferation and on-demand directed differentiation of adult stem cells (hMSCs) by the use of an economically viable short recognition peptide (GYIGSR), as opposed to the integrin recognition protein laminin, establishes the potential of SFFs for neural tissue engineering and regenerative therapy applications.

Targeting cancer cells via tumor-homing peptide CREKA functional PEG nanoparticles

Targeting cell microenvironment via nano-particle based therapies holds great promise for the treatment of various diseases. One of the main challenges in targeted delivery of nanoparticles for cancer therapy is the reduced localization of delivery vehicles to the tumor site. The therapeutic efficacy of drugs can be improved by recruiting delivery vehicles towards specific region of tumorigenesis in the body. Here, we demonstrate an effective approach in creating PEG particles via water-in-water emulsion technique with a tumor-homing peptide CREKA functionalization. The CREKA conjugated hydrogel nanoparticles were found to be more effective at inducing Doxorubicin (DOX)-mediated apoptosis compared to that of particles conjugated with laminin peptide IKVAV. Fluorescence intensity analysis on confocal micrographs suggested significantly higher cellular uptake of CREKA conjugated PEG particles than internalization of nanoparticles in other groups. We observed that fibrin binding ability of PEG particles could be increased up to 94% through CREKA conjugation. Our results suggest the possibility of cancer cell targeting via CREKA-functional PEG nanoparticles.

Laminin-111 peptide C16 regulates invadopodia activity of malignant cells through β1 integrin, Src and ERK 1/2.

Laminin peptides influence tumor behavior. In this study, we addressed whether laminin peptide C16 (KAFDITYVRLKF, γ1 chain) would increase invadopodia activity of cells from squamous cell carcinoma (CAL27) and fibrosarcoma (HT1080). We found that C16 stimulates invadopodia activity over time in both cell lines. Rhodamine-conjugated C16 decorates the edge of cells, suggesting a possible binding to membrane receptors. Flow cytometry showed that C16 increases activated β1 integrin, and β1 integrin miRNA-mediated depletion diminishes C16-induced invadopodia activity in both cell lines. C16 stimulates Src and ERK 1/2 phosphorylation, and ERK 1/2 inhibition decreases peptide-induced invadopodia activity. C16 also increases cortactin phosphorylation in both cells lines. Based on our findings, we propose that C16 regulates invadopodia activity over time of squamous carcinoma and fibrosarcoma cells, probably through β1 integrin, Src and ERK 1/2 signaling pathways.

Biofunctionalization of PEDOT films with laminin-derived peptides

Poly(3,4-ethylenedioxythiophenes) (PEDOT) have been extensively explored as materials for biomedical implants such as biosensors, tissue engineering scaffolds and microelectronic devices. Considerable effort has been made to incorporate biologically active molecules into the conducting polymer films in order to improve their long term performance at the soft tissue interface of devices, and the development of functionalized conducting polymers that can be modified with biomolecules would offer important options for device improvement. Here we report surface modification, via straightforward protocols, of carboxylic-acid-functional PEDOT copolymer films with the nonapeptide, CDPGYIGSR, derived from the basement membrane protein laminin. Evaluation of the modified surfaces via XPS and toluidine blue O assay confirmed the presence of the peptide on the surface and electrochemical analysis demonstrated unaltered properties of the peptide-modified films. The efficacy of the peptide, along with the impact of a spacer molecule, for cell adhesion and differentiation was tested in cell culture assays employing the rat pheochromocytoma (PC12) cell line. Peptide-modified films comprising the longest poly(ethylene glycol) (PEG) spacer used in this study, a PEG with ten ethylene glycol repeats, demonstrated the best attachment and neurite outgrowth compared to films with peptides alone or those with a PEG spacer comprising three ethylene glycol units. The films with PEG10-CDPGYISGR covalently modified to the surface demonstrated 11.5% neurite expression with a mean neurite length of 90μm. This peptide immobilization technique provides an effective approach to biofunctionalize conducting polymer films. Statement of SignificanceFor enhanced diagnosis and treatment, electronic devices that interface with living tissue with minimum shortcomings are critical. Towards these ends, conducting polymers have proven to be excellent materials for electrode-tissue interface for a variety of biomedical devices ranging from deep brain stimulators, cochlear implants, and microfabricated cortical electrodes. To improve the electrode-tissue interface, one strategy utilized by many researchers is incorporating relevant biological molecules within or on the conducting polymer thin films to provide a surface for cell attachment and/or provide biological cues for cell growth. The present study provides a facile means for generating PEDOT films grafted with a laminin peptide with or without a spacer molecule for enhanced cell attachment and neurite extension.

Aligned PLLA nanofibrous scaffolds coated with graphene oxide for promoting neural cell growth

The graphene oxide (GO) has attracted tremendous attention in biomedical fields. In order to combine the unique physicochemical properties of GO nanosheets with topological structure of aligned nanofibrous scaffolds for nerve regeneration, the GO nanosheets were coated onto aligned and aminolyzed poly-l-lactide (PLLA) nanofibrous scaffolds. Scanning electronic microscopy (SEM) and atomic force microscopy (AFM) revealed that the surface of aligned PLLA nanofibers after being coated with GO became rougher than those of the aligned PLLA and aminolyzed PLLA nanofibrous scaffolds. The GO nanosheets did not destroy the alignment of nanofibers. The characterizations of X-ray photoelectron spectroscopy (XPS) and water contact angle displayed that the aligned PLLA nanofibrous scaffolds were introduced with hydrophilic groups such as NH2, COOH, and OH after aminolysis and GO nanosheets coating, showing better hydrophilicity. The GO-coated and aligned PLLA nanofibrous scaffolds significantly promoted Schwann cells (SCs) proliferation with directed cytoskeleton along the nanofibers compared with the aligned PLLA and aminolyzed PLLA nanofibrous scaffolds. These scaffolds also greatly improved the proliferation of rat pheochromocytoma 12 (PC12) cells, and significantly promoted their differentiation and neurite growth along the nanofibrous alignment in the presence of nerve growth factor (NGF). This type of scaffolds with nanofibrous surface topography and GO nanosheets is expected to show better performance in nerve regeneration. Statement of SignificanceRecovery of damaged nerve functions remains a principal clinical challenge in spite of surgical intervention and entubulation. The use of aligned fibrous scaffolds provides suitable microenvironment for nerve cell attachment, proliferation and migration, enhancing the regeneration outcome of nerve tissue. Surface modification is generally required for the synthetic polymeric fibers by laminin, fibronectin and YIGSR peptides to stimulate specific cell functions and neurite outgrowth. Yet these proteins or peptides present the poor processibility, limited availability, and high cost, influencing their application in clinic. In this work, we combined GO nanosheets and topological structure of aligned nanofibrous scaffolds to direct cell migration, proliferation, and differentiation, and to induce neurite outgrowth for nerve regeneration. The GO coating improved several biomedical properties of the aligned PLLA nanofibrous scaffolds including surface roughness, hydrophilicity and promotion of cells/material interactions, which significantly promoted SCs growth and regulated cell orientation, and induced PC12 cells differentiation and neurite growth. The design of this type of structure is of both scientific and technical importance, and possesses broad interest in the fields of biomaterials, tissue engineering and regenerative medicine.

Development of a biomaterials-based functional epithelial graft for tracheal regeneration

Event Abstract Back to Event Development of a biomaterials-based functional epithelial graft for tracheal regeneration Ratna Varma1, 2, Golnaz Karoubi2 and Thomas K. Waddell1, 2 1 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada 2 Toronto General Hospital, Latner Thoracic Surgery Research Laboratories, Canada Tracheal injury, stenosis, and malignancy demand surgical management or tracheal reconstruction [1],[2], however, the latter is an unmet clinical need as tracheal grafts and transplants fail due to the lack of a functioning epithelium and immune rejection. Tracheal immune rejection is largely associated with the epithelium, therefore we believe that the best method to optimize the use of donor tracheae is to decellularize only the epithelium and maintain the remaining tracheal structure, to replace it with a recipient-derived epithelium. We aim to develop a donor-recipient hybrid tracheal model by de-epithelializing the donor trachea and engrafting a pre-developed biomaterial-based recipient epithelial graft. The absence of a functioning, ciliated, pseudostratified epithelium is a primary concern in tracheal regeneration. Clinically applied biomaterials demonstrate limited success in allowing proper airway epithelium growth and function [3]-[5]. Promising biomaterials have not been studied systematically. Therefore, we propose to perform the first comparative study to determine the optimal biomaterial for this context. Hyaluronan-Poly-ethylene glycol (HA-PEG), Chitosan-Collagen, Collagen Vitrigel Membrane, Gelatin, Silk Fibroin, and Fibrin-PEG diacrylate, were screened to identify substrates that promote airway epithelium (cell lines: BEAS-2B, A549, CALU-3, and primary human tracheal epithelial cells: HTECs) attachment, viability and function while possessing mechanical strength. Cell viability and proliferation on the biomaterials are assessed through Live/Dead staining and Ki-67 immunocytochemistry, respectively, while cell attachment is assessed via focal adhesion kinase and vinculin immunocytochemistry. Tight junction (zonula occludins-1) formation and differentiation of HTECs into ciliated (acetylated α-tubulin) and secretory cells (mucin 5AC) in air-liquid-interface culture is examined via immunocytochemistry. Preliminary results of phase contrast microscopy revealed no attachment of BEAS-2Bs and HTECs on HA-PEG hydrogels (HA-PEG, HA-PEG with functionalized IKVAV peptide, and HA-PEG with functionalized RGD peptide) by day 7. There was limited attachment of A549s and BEAS-2Bs and no attachment of CALU-3s on Chitosan-Collagen by day 14, and successful attachment with epithelial sheet formation with all cell types on Silk Fibroin by day 14 of culture. Irregular tight junction formation on HTECs on Silk Fibroin is detected as early as day 14 of air-liquid-interface culture and organizes by day 45. While Silk Fibroin is a promising biomaterial with exceptional mechanical strength [6], it demonstrates delayed maturation of HTECs and no ciliated cell differentiation by day 45 as opposed to day 21 on the Collagen I control. Silk Fibroin can be coated with other matrix proteins to promote enhanced epithelial cell attachment and differentiation. A 72-hour extracellular matrix screen of thirty individual or matrix protein combinations demonstrates BEAS-2B and HTEC attachment to Fibronectin (50 µg/ml) + Laminin (100 µg/ml) and RGD Peptide (50 µg/ml) + Laminin (50 µg/ml), and little attachment to Collagen I (200 µg/ml or 400 µg/ml) which is often used as a standard control. With further investigation, an optimal biomaterial will be selected to develop an airway epithelium graft for human tracheal regeneration. This study will have significant clinical implications for viable tracheal transplants in future and can serve as a stepping stone for regenerating other organs comprising an epithelium. Dr. Molly Shoichet; Alexander Baker

Tailoring minimalist self-assembling peptides for localized viral vector gene delivery

Viral vector gene delivery is a promising technique for the therapeutic administration of proteins to damaged tissue for the improvement of regeneration outcomes in various disease settings including brain and spinal cord injury, as well as autoimmune diseases. Though promising results have been demonstrated, limitations of viral vectors, including spread of the virus to distant sites, neutralization by the host immune system, and low transduction efficiencies have stimulated the investigation of biomaterials as gene delivery vehicles for improved protein expression at an injury site. Here, we show how Nfluorenylmethyloxycarbonyl (Fmoc) self-assembling peptide (SAP) hydrogels, designed for tissue-specific central nervous system (CNS) applications via incorporation of the laminin peptide sequence isoleucine–lysine–valine–alanine–valine (IKVAV), are effective as biocompatible, localized viral vector gene delivery vehicles in vivo. Through the addition of a C-terminal lysine (K) residue, we show that increased electrostatic interactions, provided by the additional amine side chain, allow effective immobilization of lentiviral vector particles, thereby limiting their activity exclusively to the site of injection and enabling focal gene delivery in vivo in a tissue-specific manner. When the C-terminal lysine was absent, no difference was observed between the number of transfected cells, the volume of tissue transfected, or the transfection efficiency with and without the Fmoc-SAP. Importantly, immobilization of the virus only affected transfection cell number and volume, with no impact observed on transfection efficiency. This hydrogel allows the sustained and targeted delivery of growth factors post injury. We have established Fmoc-SAPs as a versatile platform for enhanced biomaterial design for a range of tissue engineering applications.

Peptide modified polymer poly (glycerol- dodecanedioate co-fumarate) for efficient control of motor neuron differentiation

Neural tissue engineering is one of the most promising approaches for healing nerve damage, which bypasses the limits of contemporary conventional treatments. In a previous study, we developed a fibrous scaffold via electrospinning poly (glycerol dodecanedioate) (PGD) and gelatin that mimics the structure of a native extracellular matrix (ECM) for soft tissue engineering application. In this study, fumaric acid (FA) was incorporated into the PGD synthesis process, which produced a PGD derivative referred to as poly (glycerol dodecanedioate co-fumarate) (PGDF). This introduced a new functional group, a double bond, into the polymer thus providing new modification possibilities. Arg-Gly-Asp-Cys (RGDC) and laminin peptides were chosen as biomolecules to modify the fiber and facilitate cell attachment and differentiation efficiency. The release of FA into the medium was quantified to investigate the bioreactivity of the derived scaffolds. In combination with UV crosslinking, the developed PGDF fiber mats were able to withstand degradation processes for up to 2 months, which ensures that neural tissue engineering applications are viable. Cell viability and motor neuron differentiation efficiency were demonstrated to be significantly improved with the addition of FA, RGDC and laminin peptides.

Screening of integrin-binding peptides in a laminin peptide library derived from the mouse laminin β chain short arm regions

Laminins, major components of basement membrane, consist of three different subunits, α, β, and γ chains, and so far, five α, three β, and three γ chains have been identified. We have constructed synthetic peptide libraries derived from the laminin sequences and identified various cell-adhesive peptides. Ten active peptides from the laminin α chain sequences (α1–α5) were found to promote integrin-mediated cell adhesion. Previously, we found fourteen cell-adhesive peptides from the β1 chain sequence but their receptors have not been analyzed. Here, we expanded the synthetic peptide library to add peptides from the short arm regions of the laminin β2 and β3 chains and screened for integrin-binding peptides. Twenty-seven peptides promoted human dermal fibroblast (HDF) attachment in a peptide-coated plate assay. The morphological appearance of HDFs on the peptide-coated plates differed depending on the peptides. B34 (REKYYYAVYDMV, mouse laminin β1 chain, 255–266), B67 (IPYSMEYEILIRY, mouse laminin β1 chain, 604–616), B2–105 (APNFWNFTSGRG, mouse laminin β2 chain, 1081–1092), and B3–19 (GHLTGGKVQLNL, mouse laminin β3 chain, 182–193) promoted HDF spreading and HDF attachment was inhibited by EDTA, suggesting that the peptides interact with integrins. Immunostaining analyses revealed that B67 induced well-organized actin stress fibers and focal contacts containing vinculin, however, B34, B2–105, and B3–19 did not exhibit stress fiber formation or focal contacts. The inhibition assay using anti-integrin antibodies indicated that B67 interacts with α3, α6, and β1 integrins, and B34 and B3–19 interact with β1 integrin. Based on adhesion analysis of peptides modified with an alanine scan and on switching analysis with the homologous inactive sequence B2–64 (LPRAMDYDLLLRW, mouse laminin β2 chain, 618–630), the Glu8 residue in the B67 peptide was critical for HDF adhesion. These findings are useful for identifying an integrin binding motif. The B67 peptide has potential for use as a molecular probe for integrins.

Short laminin peptide for improved neural stem cell growth.

Human neural stem/progenitor cells (hNSCs) are very difficult to culture and require human or animal source extracellular matrix molecules, such as laminin or collagen type IV, to support attachment and to regulate their survival and proliferation. These extracellular matrix molecules are difficult to purify from human or animal tissues, have high batch-to-batch variability, and may cause an immune response if used in clinical applications. Although several laminin- and collagen IV-derived peptides are commercially available, they do not support long-term hNSC attachment and growth. To solve this problem, we developed a novel peptide sequence with only 12 amino acids based on the Ile-Lys-Val-Ala-Val, or IKVAV, sequence: Ac-Cys-Cys-Arg-Arg-Ile-Lys-Val-Ala-Val-Trp-Leu-Cys. This short peptide sequence, similar to tissue-derived full laminin molecules, supported hNSCs to attach and proliferate to confluence for continuous passage and subculture. This short peptide also directed hNSCs to differentiate into neurons. When conjugated to poly(ethylene glycol) hydrogels, this short peptide benefited hNSC attachment and proliferation on the surface of hydrogels and promoted cell migration inside the hydrogels with maximum enhancement at a peptide density of 10 μM. This novel short peptide shows great promise in artificial niche development for supporting hNSC culture in vitro and in vivo and for promoting hNSC transplantation in future clinical therapy.

Comparative adherence of Candida albicans and Candida dubliniensis to human buccal epithelial cells and extracellular matrix proteins

Candida albicans and Candidadubliniensis are very closely related pathogenic yeast species. Despite their close relationship, C.albicans is a far more successful colonizer and pathogen of humans. The purpose of this study was to determine if the disparity in the virulence of the two species is attributed to differences in their ability to adhere to human buccal epithelial cells (BECs) and/or extracellular matrix proteins. When grown overnight at 30°C in yeast extract peptone dextrose, genotype 1 C.dubliniensis isolates were found to be significantly more adherent to human BECs than C.albicans or C.dubliniensis genotypes 2-4 (P<0.001). However, when the yeast cells were grown at 37°C, no significant difference between the adhesion of C.dubliniensis genotype 1 and C.albicans to human BECs was observed, and C.dubliniensis genotype 1 and C.albicans adhered to BECs in significantly greater numbers than the other C.dubliniensis genotypes (P<0.001). Using surface plasmon resonance analysis, C.dubliniensis isolates were found to adhere in significantly greater numbers than C.albicans to type I and IV collagen, fibronectin, laminin, vitronectin, and proline-rich peptides. These data suggest that C.albicans is not more adherent to epithelial cells or matrix proteins than C.dubliniensis and therefore other factors must contribute to the greater levels of virulence exhibited by C.albicans.

Recombinant silk fibroin incorporated cell-adhesive sequences produced by transgenic silkworm as a possible candidate for use in vascular graft.

Interest in vascular grafts has recently grown because more patients are undergoing procedures that involve these grafts. However, smaller grafts with diameters <6 mm made from conventional biomaterials are associated with a high incidence of thrombosis, and therefore the development of improved materials suitable for small vascular grafts is highly desirable. In this paper, four kinds of recombinant Bombyx mori silk fibroins were prepared using transgenic techniques for use as silk vascular graft with a diameter of <6 mm. The peptide sequence TS(CDPGYIGSRAS)8 derived from the laminin B1 chain or the combination of two kinds of sequences, TS(CDPGYIGSRAS)8 and (TGRGDSPAS)8 derived from fibronectin, was incorporated into the light (L)-chain or the heavy (H)-chain of the silk fibroin. The fractions of the incorporated peptide sequences range from 0.8% to 7.2% by weight in the recombinant silk fibroins. This incorporation causes a very small increase in the random coil fraction of silk fibroin and a decrease in the tensile strength. Compared with native silk fibroin, the adhesive activities of mouse endothelial and smooth muscle cells increase significantly with the recombinant silk fibroin films incorporating only the TS(CDPGYIGSRAS)8 sequence independent of the L- or H-chains. A similar tendency was observed for the high migration activities of the endothelial cells in vitro and also the longer migration distance of the endothelial cell from the anastomotic part of rat abdominal aorta in vivo when this recombinant silk fibroin was used as a coating material for the silk graft. In view of the results, the recombinant silk fibroin incorporating the laminin peptide sequence can be potentially used as a vascular graft material.

Cell attachment and spreading activity of mixed laminin peptide–chitosan membranes

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.

Immobilization of Cell-Adhesive Laminin Peptides in Degradable PEGDA Hydrogels Influences Endothelial Cell Tubulogenesis

Attachment, spreading, and organization of endothelial cells into tubule networks are mediated by interactions between cells in the extracellular microenvironment. Laminins are key extracellular matrix components and regulators of cell adhesion, migration, and proliferation. In this study, laminin-derived peptides were conjugated to poly(ethylene glycol) (PEG) monoacrylate and covalently incorporated into degradable PEG diacrylate (PEGDA) hydrogels to investigate the influence of these peptides on endothelial cellular adhesion and function in organizing into tubule networks. Degradable PEGDA hydrogels were synthesized by incorporating a matrix metalloproteinase (MMP)–sensitive peptide, GGGPQGIWGQGK (abbreviated PQ), into the polymer backbone. The secretion of MMP-2 and MMP-9 by endothelial cells promotes polymer degradation and consequently cell migration. We demonstrate the formation of extensive networks of tubule-like structures by encapsulated human umbilical vein endothelial cells in hydrogels with immobilized synthetic peptides. The resulting structures were stabilized by pericyte precursor cells (10T1/2s) in vitro. During tubule formation and stabilization, extracellular matrix proteins such as collagen IV and laminin were deposited. Tubules formed in the matrix of metalloproteinase sensitive hydrogels were visualized from 7 days to 4 weeks in response to different combination of peptides. Moreover, hydrogels functionalized with laminin peptides and transplanted in a mouse cornea supported the ingrowth and attachment of endothelial cells to the hydrogel during angiogenesis. Results of this study illustrate the use of laminin-derived peptides as potential candidates for modification of biomaterials to support angiogenesis.

Prostate specific membrane antigen produces pro-angiogenic laminin peptides downstream of matrix metalloprotease-2

Prostate specific membrane antigen (PSMA) is a pro-angiogenic cell-surface protease that we previously demonstrated regulates blood vessel formation in a laminin and integrin β1-dependent manner. Here, we examine the principal mechanism of PSMA activation of integrin β1. We show that digesting laminin sequentially with recombinant matrix metalloprotease-2 (MMP-2) and PSMA generates small peptides that enhance endothelial cell adhesion and migration in vitro. We also provide evidence that these laminin peptides activate adhesion via integrin α6β1 and focal adhesion kinase. Using an in vivo Matrigel implant assay, we show that these MMP/PSMA-derived laminin peptides also increase angiogenesis in vivo. Together, our results reveal a novel mechanism of PSMA activation of angiogenesis by processing laminin downstream of MMP-2.

Amyloid Precursor Protein Is an Autonomous Growth Cone Adhesion Molecule Engaged in Contact Guidance

Amyloid precursor protein (APP), a transmembrane glycoprotein, is well known for its involvement in the pathogenesis of Alzheimer disease of the aging brain, but its normal function is unclear. APP is a prominent component of the adult as well as the developing brain. It is enriched in axonal growth cones (GCs) and has been implicated in cell adhesion and motility. We tested the hypothesis that APP is an extracellular matrix adhesion molecule in experiments that isolated the function of APP from that of well-established adhesion molecules. To this end we plated wild-type, APP-, or β1-integrin (Itgb1)- misexpressing mouse hippocampal neurons on matrices of either laminin, recombinant L1, or synthetic peptides binding specifically to Itgb1 s or APP. We measured GC adhesion, initial axonal outgrowth, and substrate preference on alternating matrix stripes and made the following observations: Substrates of APP-binding peptide alone sustain neurite outgrowth; APP dosage controls GC adhesion to laminin and APP-binding peptide as well as axonal outgrowth in Itgb1− independent manner; and APP directs GCs in contact guidance assays. It follows that APP is an independently operating cell adhesion molecule that affects the GC's phenotype on APP-binding matrices including laminin, and that it is likely to affect axon pathfinding in vivo.

Pure Graphene Oxide Doped Conducting Polymer Nanocomposite for Bio-interfacing.

Advanced materials that are highly biocompatible and easily modifiable with biomolecules are of great importance for bio-interfacing and the development of biodevices. Here, a biocompatible conducting polymer based nanocomposite was electrochemically synthesized through the electropolymerization of poly(3, 4-ethylene dioxythiophene) (PEDOT) in the presence of graphene oxide (GO) as the only dopant. GO contains many negatively charged carboxyl functional groups and is highly dispersible in aqueous solutions, enabling its facile incorporation and even distribution throughout the conducting polymer. PEDOT/GO films exhibited minimal cytotoxicity after 24 h and supported neuron growth with significantly longer neurites than a control PEDOT/PSS film, indicating that the PEDOT/GO film provides a positive growth signal to developing neurons. While some of the negatively charged functional carboxyl groups of GO "dope" the PEDOT, others are exposed freely on the surface of the nanocomposite allowing easy functionalization of the PEDOT/GO composite with biomolecules. Functional laminin peptide, RNIAEIIKDI (p20), was covalently bound to the surface of the PEDOT/GO film and maintained its bioactivity, as evidenced by an increased neurite outgrowth from neurons cultured on the functionalized composite surface. The ease of biomolecule functionalization of the PEDOT/GO nanocomposite, along with its low electrochemical impedance, minimal toxicity and permissiveness to neuron growth, underlines its potential as a material for widespread biosensing, neural interfacing and tissue engineering applications.

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929–933 sequence of the β1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67kDa laminin receptor.In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

Troponin T and Plasma Collagen Peptides in Heart Failure

The extracellular matrix of the heart is made up of a number of structural proteins including fibrillar collagen, smaller amounts of elastin, laminin, fibronectin, and signaling peptides. The complex collagen 3-dimensional weave, mainly consisting of type I collagen, interconnects individual myocytes through a collagen–integrin–cytoskeletal–myofibril arrangement.1 This network supports cardiac myocytes during contraction and relaxation and also provides a mechanism for translating individual myocyte shortening and force generation into ventricular contraction. It is also responsible for much of the ventricle’s passive diastolic stiffness.2 In both human and animal studies, progressive left ventricular remodeling and dysfunction are associated with significant changes in the extracellular matrix.3–5 Article see p 406 The structural hallmark of prolonged pressure-overload hypertrophy is increased collagen accumulation between individual myocytes and myocyte fascicles (Figure 1).6,7 Thus, the highly organized architecture of the extracellular matrix undergoes significant alterations in collagen structure, composition, and geometry caused by increased collagen synthesis, postsynthetic processing, posttranslational modification, and decreased degradation and turnover. This “reactive” collagen deposition is characterized by both perivascular and interstitial fibrosis.2,8,9 The changes in collagen homeostasis that occur during the development of chronic pressure-overload hypertrophy are directly associated with increased myocardial diastolic stiffness properties, which in turn cause abnormal diastolic filling.7,10,11 Indeed, clinical evidence suggests that progressive extracellular matrix accumulation and diastolic dysfunction are important underlying pathophysiological mechanisms for heart failure in patients with pressure-overload hypertrophy.12,13 Figure 1. Scanning electron micrographs taken from normal nonhuman primate left ventricular myocardium following the induction of pressure-overload hypertrophy (POH). These microscopic studies demonstrated thickening of the collagen weave and overall increased relative content between myocytes with POH. Reproduced with permission.6 In volume-overload hypertrophy because of the persistently elevated preload, a much different pattern of extracellular …

Laminin active peptide/agarose matrices as multifunctional biomaterials for tissue engineering

Cell adhesive peptides derived from extracellular matrix components are potential candidates to afford bio-adhesiveness to cell culture scaffolds for tissue engineering. Previously, we covalently conjugated bioactive laminin peptides to polysaccharides, such as chitosan and alginate, and demonstrated their advantages as biomaterials. Here, we prepared functional polysaccharide matrices by mixing laminin active peptides and agarose gel. Several laminin peptide/agarose matrices showed cell attachment activity. In particular, peptide AG73 (RKRLQVQLSIRT)/agarose matrices promoted strong cell attachment and the cell behavior depended on the stiffness of agarose matrices. Fibroblasts formed spheroid structures on the soft AG73/agarose matrices while the cells formed a monolayer with elongated morphologies on the stiff matrices. On the stiff AG73/agarose matrices, neuronal cells extended neuritic processes and endothelial cells formed capillary-like networks. In addition, salivary gland cells formed acini-like structures on the soft matrices. These results suggest that the peptide/agarose matrices are useful for both two- and three-dimensional cell culture systems as a multifunctional biomaterial for tissue engineering.