Cell-binding Peptide Research Articles

Human microvascular endothelial cell growth and migration on biomimetic surfactant polymers

Successful engineering of a tissue-incorporated vascular prosthesis requires cells to proliferate and migrate on the scaffold. Here, we report on a series of “ECM-like” biomimetic surfactant polymers that exhibit quantitative control over the proliferation and migrational properties of human microvascular endothelial cells (HMVEC). The biomimetic polymers consist of a poly(vinyl amine) (PVAm) backbone with hexanal branches and varying ratios of cell binding peptide (RGD) to carbohydrate (maltose). Proliferation and migration behavior of HMVEC was investigated using polymers containing RGD: maltose ratios of 100:0, 75:25 and 50:50, and compared with fibronectin (FN) coated glass (1 μg/cm 2). A radial Teflon fence migration assay was used to examine HMVEC migration at 12 h intervals over a 48 h period. Migration was quantified using an inverted optical microscope, and HMVEC were examined by confocal microscopy for actin and focal adhesion organization/ arrangement. Over the range of RGD ligand density studied (∼0.19–0.6 peptides/nm 2), our results show HMVEC migration decreases with increasing RGD density in the polymer. HMVEC were least motile on the 100% RGD polymer (∼0.38–0.6 peptides/nm 2) with an average migration of 0.20 mm 2/h in area covered, whereas HMVEC showed the fastest migration of 0.48±0.06 mm 2/h on the 50% RGD surface (∼0.19–0.30 peptides/nm 2). In contrast, cell proliferation increased with increasing surface peptide density; proliferation on the 50% RGD surface was 1.5%±0.06/h compared with 2.2%±0.07/h on the 100% RGD surface. Our results show that surface peptide density affects cellular functions such as growth and migration, with the highest peptide density supporting the most proliferation but the slowest migration.

In vivo bone and soft tissue response to injectable, biodegradable oligo(poly(ethylene glycol) fumarate) hydrogels

This study was designed to assess in vivo bone and soft tissue behavior of novel oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels using a rabbit model. In vitro degradation of the OPF hydrogels was also investigated in order to compare with in vivo characteristics. Four groups of OPF hydrogel implants were synthesized by alternation of crosslinking density, poly(ethylene glycol) (PEG) block length of OPF, and cell-binding peptide content. The in vitro degradation rate of OPF hydrogels increased with decreasing crosslinking density of hydrogels, which was characterized by measuring weight loss and swelling ratio of hydrogels and medium pH change. Examination of histological sections of the subcutaneous and cranial implants showed that an uniform thin circumferential fibrous capsule was formed around the OPF hydrogel implants. Quantitative evaluation of the tissue response revealed that no statistical difference existed in capsule quality or thickness between implant groups, implantation sites or implantation times. At 4 weeks, there was a very limited number of inflammatory and multinuclear cells at the implant–fibrous capsule interface for all implants. However, at 12 weeks, OPF hydrogels with PEG block length of number average molecular weight 6090±90 showed extensive surface erosion and superficial fragmentation that was surrounded by a number of inflammatory cells, while OPF hydrogels with PEG block length of number average molecular weight 930±10 elicited minimal degradation. Constant fibrous capsule layers and number of inflammatory cells were observed regardless of the incorporation of cell-binding peptide and crosslinking density of OPF hydrogels with PEG block length of number average molecular weight 930±90. These results confirm that the degradation of implants can be controlled by tailoring the macromolecular structure of OPF hydrogels. Additionally, histological evaluation of implants proved that the OPF hydrogel is a promising material for biodegradable scaffolds in tissue engineering.

New cell attachment peptide sequences from conserved epitopes in the carboxy termini of fibrinogen

Fibrinogen seems to contribute significantly to cell binding and recruitment into wounds besides its major role in clot formation. We describe 19- to 21-mer cell-binding (haptotactic) peptides from the C-termini of fibrinogen β-chain (Cβ), the extended αE chain, and near the C-terminal of the γ-chain. When these peptides were covalently bound to a biologically inert matrix such as Sepharose beads (SB), they elicited beads attachment to cells, mostly of mesenchymal origin (including fibroblasts, endothelial cells, and smooth muscle cells) as well as some transformed cell lines. Based on such haptotactic activity, these peptides were termed “haptides.” By contrast, peptides homologous to fibrinogen C-termini α- and γ-chains elicited no such activity. The haptide Cβ could not block the interaction of fibroblasts with antibodies directed against integrins β 1, α v, α vβ 1, α vβ 3, and αIIβ 3. Moreover, GRGDS peptide could not inhibit enhanced cell binding to SB–Cβ, as expected from an integrin-mediated process. In soluble form the haptides were accumulated in cells with nonsaturable kinetics without any toxic or proproliferative effects in concentrations up to 80 μM. These findings suggest that the conserved haptidic sequences within fibrin(ogen) can be associated with the adhesion and migration of cells into fibrin clots and may have a significant role in normal wound healing and in various pathological conditions.

Bone regeneration of localized chronic alveolar defects utilizing cell binding peptide associated with anorganic bovine-derived bone mineral: a clinical and histological study.

Osteoinduction to treat osseous defects has been attempted by several means. Some clinical studies have demonstrated that a synthetic cell binding peptide (P-15) with anorganic bovine derived bone matrix (ABM) has the ability to enhance bone regeneration. These studies suggest that more histological data are necessary to better understand this process. We have developed a Class III chronic alveolar defect animal model to investigate space-maintaining regenerative materials. The objective of this study was to clinically and histologically evaluate the use of P-15/ABM with or without a bioabsorbable membrane (M) to regenerate localized chronic alveolar ridge defects in dogs. Six adult, male mongrel dogs were used in this study. Bilateral, Class III, alveolar defects were surgically produced following extraction of the mandibular second premolar teeth and local reduction of the alveolar ridge. After an 8-week healing interval, mucoperiosteal flaps were elevated. P-15/ABM with or without bioabsorbable membranes were implanted into contralateral defects in 10 sites. Two sites received no biomaterial (controls). Mucoperiosteal flaps were advanced over the P-15/ABM or P-15/ABM/M constructs and sutured. Pre- and postaugmentation clinical evaluation was done utilizing periodontal probes and calipers. The animals were sacrificed 12 weeks postaugmentation and block specimens processed for histologic evaluation. Clinical results showed no significant statistical augmentation on the control group (0.0 +/- 0.6 mm). In all experimental sites utilizing P-15/ABM or P-15/ABM/M, relevant ridge augmentation was observed (3.6 +/- 2.0 mm and 2.9 +/- 1.9 mm, respectively). Histologically, all experimental sites showed active bone formation with plump osteoblast and osteoid matrix deposition in the treated area. Bone ingrowth filled the area of the defects treated with P-15/ABM/M. Few P-15/ABM particles were seen in the cellular fibrous tissue surrounding the new formed bone trabeculae. P-15/ABM with or without membranes can produce a significant clinical ridge augmentation. Bone formation was histologically observed in all test areas. The association of a membrane with P-15/ABM seemed to enhance the process of bone formation.

Identification and validation of a novel cell-recognition site (KNEED) on the 8th type III domain of fibronectin

Interactions between cell-surface integrins and extracellular matrix proteins underlie a versatile recognition system providing cells with anchorage, traction for migration or matrix remodeling, as well as signals for polarity, differentiation and growth. Short peptide sequences of fibronectin (FN), most notably RGD found on a loop in the 10th type III domain, are effective in promoting cell adhesion when immobilized to a biomaterial scaffold. Additional sequences (e.g. PHSRN) have been shown to act synergistically to enhance cell adhesion and other cellular processes. Using bioinformatics, we identified a candidate cell-binding peptide sequence, KNEED, located on the loop region of the 8th domain of FN that from in vitro studies appears to participate in cell attachment and spreading. Computational analysis revealed that KNEED exhibits both high solvent accessibility and sequence conservation values across FN sequences from seven species. We demonstrate the importance of the KNEED sequence using a solution-phase competitive inhibition assay utilizing soluble peptides. Results indicate that the presence of soluble KNEED peptides inhibits the attachment and spreading of 3T3 balb/c fibroblasts on FN-coated surfaces in a concentration-dependent manner. As more sequence and crystallographic data become available, computational approaches may aid in the identification of new targets for applications where biorecognition plays a key role.

Structure and function of an early divergent form of laminin in hydra: a structurally conserved ECM component that is essential for epithelial morphogenesis.

As a major component of the extracellular matrix (ECM), laminin has been found in many vertebrate and invertebrate organisms. Its molecular structure is very similar across species lines and its biological function in the ECM has been extensively studied. In an effort to study ECM structure and function in hydra, we have cloned a partial hydra laminin alpha chain and the full-length hydra laminin beta chain using ECM-enriched cDNA libraries. Analysis of deduced amino acid sequences indicated that both polypeptides have high sequence similarity to a number of invertebrate and vertebrate laminin alpha and beta subunits. Rotary shadow analysis of isolated hydra laminin indicates it has a heterotrimeric organization that is characteristic of vertebrate laminins. A putative integrin-class protein was also identified using a cell-binding peptide sequence from the laminin beta chain as an affinity probe, indicating that integrins are possible cell surface receptors in hydra. In agreement with previous results for the hydra laminin beta chain, in situ hybridization experiments revealed that hydra laminin alpha chain mRNA is restricted to endodermal cells. As with a number of other hydra ECM components, higher levels of laminin alpha chain mRNA are localized to regions where cell migration and differentiation are actively undertaken such as the base of tentacles, the peduncle region, buds, regenerating tentacles, and at the head end during regeneration. The role of laminin in morphogenesis was studied using an antisense approach and the results indicated that translation of the laminin alpha chain is required for head regeneration.

Inhibition of Hair Follicle Growth by a Laminin-1 G-Domain Peptide, RKRLQVQLSIRT, in an Organ Culture of Isolated Vibrissa Rudiment

We established a serum-free organ culture system of isolated single vibrissa rudiments taken from embryonic day 13 mice. This system allowed us to test more than 30 laminin-derived cell adhesive peptides to determine their roles on the growth and differentiation of vibrissa hair follicles. We found that the RKRLQVQLSIRT sequence (designated AG-73), which mapped to the LG-4 module of the laminin-alpha1 chain carboxyl-terminal G domain, perturbed the growth of hair follicles in vitro. AG-73 is one of the cell-binding peptides identified from more than 600 systematically synthesized 12 amino acid peptides covering the whole amino acid sequence of the laminin-alpha1, -beta1, and -gamma1 chains, by cell adhesion assay. Other cell-adhesive laminin peptides and a control scrambled peptide, LQQRRSVLRTKI, however, failed to show any significant effects on the growth of hair follicles. The AG-73 peptide binds to syndecan-1, a transmembrane heparan-sulfate proteoglycan. Syndecan-1 was expressed in both the mesenchymal condensation and the epithelial hair peg of developing vibrissa, suggesting that AG-73 binding to the cell surface syndecan-1 perturbed the epithelial-mesenchymal interactions of developing vibrissa. The formation of hair bulbs was aberrant in the explants treated with AG-73. In addition, impaired basement membrane formation, an abnormal cytoplasmic bleb formation, and an unusual basal formation of actin bundles were noted in the AG-73-treated-hair matrix epithelium, indicating that AG-73 binding perturbs various steps of epithelial morphogenesis, including the basement membrane remodeling. We also found a region-specific loss of the laminin-alpha1 chain in the basement membrane at the distal region of the invading hair follicle epithelium, indicating that laminins play a part in hair morphogenesis.

Effects of Collagen IV on Neuroblastoma Cell Matrix-Related Functions

Integrin-mediated interactions with collagen IV and its domains were examined in a human neuroblastoma cell line (SK-N-SH). By adhesion assays we demonstrated that neuroblastoma cells bound to solid-phase intact collagen IV and synthetic cell-binding peptide HEP-III, derived from the collagenous part of the molecule, but not to the main noncollagenous NC1 domain or to the synthetic cell-binding peptide HEP-I, derived from this domain. Monoclonal antibodies against β1, α3, and αvβ3 integrins resulted in inhibition of cell binding to collagenous substrates by 95, 30, and 35%, respectively. By flow cytometry and immunoblotting it was shown that culture of SK-N-SH cells on collagen IV resulted in alteration in the expression of major neuroblastoma cell integrins. Binding to collagen IV induced the expression and activation of matrix metalloproteinases A and B (MMP-2, MMP-9), with a concomitant increase at the protein level of tissue-specific inhibitors of metalloproteinases (TIMP-1, TIMP-2). Finally, the expression of MMP-2 was significantly up-regulated by anti-α3β1 antibodies, whereas ligation of anti-αvβ3 antibodies resulted in a modest down-regulation of MMP-2. Our results indicate that the presence of collagen IV modulates the expression of integrins, which are used for binding to this glycoprotein, and MMP-2 secreted by SK-N-SH cells.

Fibronectin facilitates Mycobacterium tuberculosis attachment to murine alveolar macrophages.

Mycobacterium tuberculosis remains a major cause of pulmonary infection worldwide. Attachment of M. tuberculosis organisms to alveolar macrophages (AMs) represents the earliest phase of primary infection in pulmonary tuberculosis. In this study fibronectin (Fn), an adhesive protein, is shown to bind M. tuberculosis organisms and facilitates attachment of M. tuberculosis to murine AMs. A monoclonal antibody (MAb) specific to the heparin binding domain (HBD) of Fn decreases (125)I-Fn binding to M. tuberculosis; whereas MAbs specific to either the cell binding domain (CBD) or the gelatin binding domain (GBD) have no effect on Fn binding to M. tuberculosis. In the presence of exogenous Fn (10 microg/ml) M. tuberculosis attachment to AMs increased significantly from control levels (means +/- standard errors of the means) of 11.5% +/- 1.1% to 44.2% +/- 4.2% (P < 0.05). Fn-enhanced attachment was significantly decreased from 44.2% +/- 4.2% to 10.8% +/- 1.2% (P < 0.05) in the presence of anti-Fn polyclonal antibodies. The attachment is also inhibited in the presence of MAbs specific for the HBD and CBD, whereas MAbs specific to GBD did not affect the attachment. Further, an Fn cell binding peptide, Arg-Gly-Asp-Ser (RGDS), decreased the attachment from 44.2% +/- 4.2% to 15.3% +/- 1.2% (P < 0.05), whereas addition of a control peptide, Arg-Gly-Glu-Ser (RGES) did not affect the attachment (40.5% +/- 1.8%). These results suggest that Fn-mediated attachment of M. tuberculosis can occur through the binding of Fn to the AM via the CBD and to M. tuberculosis organisms via the HBD.

Clinical effects of combination anorganic bovine-derived hydroxyapatite matrix(ABM)/cell binding peptide (P-15) in periodontal intrabony defects

Clinical effects of combination anorganic bovine-derived hydroxyapatite matrix(ABM)/cell binding peptide (P-15) in periodontal intrabony defects

Neuroblastoma tumor cell-binding peptides identified through random peptide phage display

Random peptide phage display libraries have been employed widely to identify protein–protein interactions, using as targets either purified proteins, intact cells, or organs. To isolate peptides that bind to human neuroblastoma cells, we have used a phage display approach with the neuroblastoma cell line WAC 2 as the target. In particular, two bacteriophages, t147 and t160, displaying peptides p147 and p160, respectively, were isolated by repeated display cycles. Binding of t147 and t160 to WAC 2 cells was abrogated by pretreatment with the peptides p147 and p160, respectively, which strongly support that cellular binding of both phages is dictated by their displayed peptides. Immunofluorescence analysis by confocal light microscopy revealed that the major proportion of t147 remains on the surface of WAC 2 cells and that only a fraction is taken up into the cells. In contrast, the vast majority of t160 is internalized. K + depletion reduced the number of the phages internalized by the cells to approximately 20% for t160 and to 10% for t147, indicating that the phage internalization was through receptor-mediated endocytosis. Phage t147 appears to bind to a range of tumor cell lines, including neuroblastoma, breast cancer, glioblastoma and C-cell carcinoma, but less so to non-tumor lines, such as erythrocytes, lymphocytes, monocytes and epithelial cells. Phage t160 bound to a range of neuroblastoma cell lines and a breast cancer cell line, but not to other tested cell lines. While neither of the displayed peptides conferred a narrow tissue specific binding ability, they do provide a basis for targeted drug delivery in selected experimental or natural tumor systems.

Infection by Trypanosoma cruzi: IDENTIFICATION OF A PARASITE LIGAND AND ITS HOST CELL RECEPTOR

The infective trypomastigote stage of Trypanosoma cruzi expresses a set of surface glycoproteins that are known collectively as Tc85 and belong to the gp85/trans-sialidase supergene family. A member of this family, Tc85-11, with adhesive properties to laminin and cell surfaces was recently cloned. In this report, the Tc85-11 domain for cell binding and its corresponding receptor on epithelial cell LLC-MK(2) are described. Using synthetic peptides corresponding to the Tc85-11 carboxyl-terminal segment, we show that the mammalian cell-binding domain colocalizes to the most conserved motif of the trypanosome gp85/trans-sialidase supergene family (VTVXNVFLYNR). Even though Tc85-11 binds to laminin, the 19-residue cell-binding peptide (peptide J) does not contain the laminin-binding site, because it does not bind to laminin or inhibit cell binding to this glycoprotein. The host cell receptor for the peptide was characterized as cytokeratin 18. Addition of anti-cytokeratin antibodies to the culture medium significantly inhibited the infection of epithelial cells by T. cruzi. Tc85-11 is a multiadhesive glycoprotein, encoding at least two different binding sites, one for laminin and one for cytokeratin 18, that allow the parasite to overcome the barriers imposed by cell membranes, extracellular matrices, and basal laminae to reach the definitive host cell. This is the first description of a direct interaction between cytokeratin and a protozoan parasite.

Grafton demineralized bone matrix: performance consistency, utility, and value.

Tissue EngineeringVol. 6, No. 4 Portland Bone SymposiumGrafton® Demineralized Bone Matrix: Performance Consistency, Utility, and ValueJames L. RussellJames L. RussellSearch for more papers by this authorPublished Online:9 Jul 2004https://doi.org/10.1089/107632700418137AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byA triple minimally invasive surgery combination for subacute osteoporotic lower lumbar vertebral collapse with neurological compromise: a potential alternative to the vertebral corpectomy/expandable cage strategyNeurosurgical Focus, Vol. 54, No. 1Applications of xenografts in periodontal regeneration15 February 2022 | IP International Journal of Periodontology and Implantology, Vol. 6, No. 4Minor Review: An Overview of a Synthetic Nanophase Bone Substitute29 August 2018 | Materials, Vol. 11, No. 96.2 Bone Tissue Engineering: Growth Factors and Cytokines ☆Bone Tissue EngineeringCore decompression combined with implantation of a demineralised bone matrix for non-traumatic osteonecrosis of the femoral head8 May 2012 | Archives of Orthopaedic and Trauma Surgery, Vol. 132, No. 8Bone Tissue Engineering: Growth Factors and CytokinesStructural Augmentation with Biomaterial-Loaded Allograft Threaded Cage for the Treatment of Femoral Head OsteonecrosisThe Journal of Arthroplasty, Vol. 25, No. 8Biomaterial-loaded allograft threaded cage for the treatment of femoral head osteonecrosis in a goat model1 January 2008 | Biotechnology and Bioengineering, Vol. 100, No. 3Tissue engineering of osteochondral constructs in vitro using bioreactorsInjury, Vol. 39, No. 1Effects of a Putty-Form Hydroxyapatite Matrix Combined With the Synthetic Cell-Binding Peptide P-15 on Alveolar Ridge PreservationJournal of Periodontology, Vol. 79, No. 2Intervariability and Intravariability of Bone Morphogenetic Proteins in Commercially Available Demineralized Bone Matrix ProductsSpine, Vol. 31, No. 12Repair of mandible defect with tissue engineering bone in rabbitsANZ Journal of Surgery, Vol. 75, No. 11Scaffolding in Periodontal Engineering1 January 2010Tissue-Engineering von Röhrenknochen mit einer vaskularisierten Matrix in einem BioreaktorDer Orthopäde, Vol. 33, No. 12Donor age and gender effects on osteoinductivity of demineralized bone matrix1 January 2004 | Journal of Biomedical Materials Research, Vol. 70B, No. 1Tissue engineering of bone10 July 2009 | Minimally Invasive Therapy & Allied Technologies, Vol. 11, No. 3 Volume 6Issue 4Aug 2000 To cite this article:James L. Russell.Grafton® Demineralized Bone Matrix: Performance Consistency, Utility, and Value.Tissue Engineering.Aug 2000.435-440.http://doi.org/10.1089/107632700418137Published in Volume: 6 Issue 4: July 9, 2004PDF download

Development of RGD peptides grafted onto silica surfaces: XPS characterization and human endothelial cell interactions.

The attachment of human umbilical vein endothelial cells (HUVECs) on substrates that had been covalently grafted with the cell adhesion peptides Arg-Gly-Asp (RGD) was investigated. This approach was used to provide substrates that are adhesive to cells even in the absence of serum proteins and to cells that have had no prior treatment of the surface with proteins that promote cell adhesion. We wanted to improve control of cellular interactions with cell-adhesive materials by providing fixedly bound adhesion ligands. Silica was examined as a model surface. The peptides were grafted using three different steps: grafting of aminosilane molecules; reaction with a maleimide molecule; and immobilization of cell-binding peptides containing the RGD sequence. The RGD-grafted surface was characterized by X-ray photoelectron spectroscopy (XPS) and contact-angle measurements.

The Carboxy-Terminal Cell-Binding Domain of Thrombospondin Is Essential for Sickle Red Blood Cell Adhesion

Sickle red blood cells (SS-RBCs) have enhanced adhesion to the plasma and subendothelial matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. TSP has at least four domains that mediate cell adhesion. The goal of this study was to map the site(s) on TSP that binds SS-RBCs. Purified TSP proteolytic fragments containing either the N-terminal heparin-binding domain, or the type 1, 2, or 3 repeats, failed to sustain SS-RBC adhesion (<10% adhesion). However, a 140-kD thermolysin TSP fragment, containing the carboxy-terminal cell-binding domain in addition to the type 1, 2, and 3 repeats fully supported the adhesion of SS-RBCs (126% +/- 25% adhesion). Two cell-binding domain adhesive peptides, 4N1K (KRFYVVMWKK) and 7N3 (FIRVVMYEGKK), failed to either inhibit or support SS-RBC adhesion to TSP. In addition, monoclonal antibody C6. 7, which blocks platelet and melanoma cell adhesion to the cell-binding domain, did not inhibit SS-RBC adhesion to TSP. These data suggest that a novel adhesive site within the cell binding domain of TSP promotes the adhesion of sickle RBCs to TSP. Furthermore, soluble TSP did not bind SS-RBCs as detected by flow cytometry, nor inhibit SS-RBC adhesion to immobilized TSP under conditions of flow, indicating that the adhesive site on TSP that recognizes SS-RBCs is exposed only after TSP binds to a matrix. We conclude that the intact carboxy-terminal cell-binding domain of TSP is essential for the adhesion of sickle RBCs under flow conditions. This study also provides evidence for a unique adhesive site within the cell-binding domain that is exposed after TSP binds to a matrix.

Design of biomimetic habitats for tissue engineering with P-15, a synthetic peptide analogue of collagen.

In tissues, collagen forms the scaffold for cell attachment and migration, and it modulates cell differentiation and morphogenesis by mediating the flux of chemical and mechanical stimuli. We are constructing biomimetic environments by immobilizing a collagen-derived high-affinity cell-binding peptide P-15 in three-dimensional (3-D) templates. The cell-binding peptide can be expected to transduce mechanical forces. In their physiological environment, periodontal ligament fibroblasts (PDLF) are subject to significant mechanical forces. We have examined the behavior of human PDLF in culture on particulate bovine anorganic bone mineral (ABM) coated with P-15 (ABM-P-15). Greater numbers of cells associated with ABM-P-15 compared to ABM alone. Higher levels of incorporation of radiolabeled precursors in DNA and protein were consistent with the presence of larger numbers of cells on ABM-P-15 compared to ABM cultures. Scanning electron microscopic examination showed that cultures on ABM-P-15 generated highly oriented 3-D colonies of elongated cells and formed copious amounts of fibrous as well as membranous matrix reminiscent of ligamentous structures. PDLF cultured on ABM formed sparse monolayers with little order and a meager matrix. Alizarin Red stained the matrix of particle associated cells and inter-particle cellular bridges in P-15-associated cultures, indicating mineralization. 3-D colony formation and ordering of cells along with increased mineralization suggests that the coupling of cells to the ABM matrix through P-15 may provide a biomimetic environment permissive for cell differentiation and morphogenesis. Our studies suggest that ABM-P-15 templates may be effective as endosseous grafts, and, when seeded with PDLF, these matrices may serve as tissue engineered substitutes for autologous bone grafts.

The Role in Cell Binding of a β1-bend within the Triple Helical Region in Collagen αl(I) Chain: Structural and Biological Evidence for Conformational Tautomerism on Fiber Surface

Summary In its physiological solid state, type I collagen serves as a host for many types of cells. Only the molecules on fiber surface are available for interaction. In this interfacial environment, the conformation of a cell binding domain can be expected to fluctuate between the collagen fold and a distinctive non-collagen molecular marker for recognition and allosteric binding. If the cell binding domain can be localized in contiguous residues within the exposed half of a turn of the triple helix (approximately 15 residues), the need for extensive structural modification and unraveling of the triple helix is avoided. We examined the conformational preferences and biological activity of a synthetic 15- residue peptide (P-15), analogous to the sequence 766GTPGPQGIAGQRGVV780 in the al (I) chain. Theoretical studies showed a high potential for a stable β-bend for the central GIAG sequence. The flanking sequences showed facile transition to extended conformations. Circular dichroism of the synthetic peptide in anisotropic solvents confirmed the presence of β-strand and β-bend structures. P-15 inhibited fibroblast binding to collagen in a concentration dependent manner, with near maximal inhibition occurring at a concentration of 7.2×10−6 M. The temporal pattern of cell attachment was altered markedly in the presence of P-15. No inhibition was seen with a peptide P-15 (AI), an analogue of P-15 with the central IA residues reversed to AI or with collagen-related peptides (Pro-Pro-Gly)10, (Pro-Pro-Gly)10, and polyproline, and with several unrelated peptides. Our studies suggest a molecular mechanism for cell binding to collagen fibers based on a conformational transition in collagen molecules on the fiber surface. Since the energy barrier between the collagen fold and β-strand conformation is low, a local conformational change may be possible in molecules on the fiber surface because of their location in an anisotropic environment. Our observations also suggest that the sequence incorporated in P-15 may be a specific ligand for cells. Unlike other reported cell binding peptides, the residues involved in this interaction are non-polar.

Binding site on human C-reactive protein (CRP) recognized by the leukocyte CRP-receptor.

C-reactive protein (CRP), the prototypical inflammatory acute phase reactant in humans, interacts with monocytes and neutrophils via a specific receptor. To map the site on CRP recognized by the CRP receptor (CRP-R), synthetic peptides corresponding to the surface region on each of the five identical subunits were tested as competitors vs. [125I]-CRP for cell binding. A peptide of residues 27-38 (TKPLKAFTVCLH) efficiently inhibited CRP binding when compared to other nonoverlapping peptides. This peptide was termed the cell-binding peptide (CB-Pep). The F(ab')2 of an IgG Ab to the CB-Pep specifically inhibited CRP binding upon reacting with the ligand. Competitive binding studies with synthetic peptides truncated from either the NH2- or COOH-terminus of the CB-Pep revealed that the minimum length recognized by the CRP-R consisted of residues 31-36: KAFTVC. Conservative substitutions of residues within the CB-Pep indicated that the four residues AFTV were critical for CRP-R binding. The CB-Pep also inhibited induced superoxide generation by HL-60 granulocytes. The minimum length required for the inhibition was also KAFTVC; however, only Phe-33 and Leu-37 were critical residues in this assay. Anti-CB-Pep IgG Ab reacted more extensively with heat-modified CRP, suggesting that an altered conformation of CRP is preferentially recognized by the CRP-R. The results suggest that this contiguous sequence on a beta-strand on one face of each of five subunits of the CRP pentamer serves as a unique recognition motif for inflammatory leukocytes.

Immobilization of the cell-adhesive peptide Arg-Gly-Asp-Cys (RGDC) on titanium surfaces by covalent chemical attachment.

Surface modification of acid-pretreated titanium with 3-aminopropyltriethoxylsilane (APTES) in dry toluene resulted in covalently bonded siloxane films with surface coverage that was relatively controllable by regulating the reaction conditions. A hetero-bifunctional cross-linker, N-succinimidyl-3-maleimidopropionate (SMP), reacted with the terminal amino groups, forming the exposed maleimide groups. Finally, a model cell-binding peptide, Arg-Gly-Asp-Cys (RGDC), was immobilized on the surface through covalent addition of the cysteine thiol groups to the maleimide groups. X-ray photoelectron spectroscopy, radiolabelling techniques, and ellipsometry were used to quantify and characterize the modified surfaces.

Effect of receptor-ligand affinity on the strength of endothelial cell adhesion

The objective of this study was to determine the effect of receptor-ligand affinity on the strength of endothelial cell adhesion. Linear and cyclic forms of the fibronectin (Fn) cell-binding domain peptide Arg-Gly-Asp (RGD) were covalently immobilized to glass, and Fn was adsorbed onto glass slides. Bovine aortic endothelial cells attached to the surfaces for 15 min. The critical wall shear stress at which 50% of the cells detached increased nonlinearly with ligand density and was greater with immobilized cyclic RGD than with immobilized linear RGD or adsorbed Fn. To directly compare results for the different ligand densities, the receptor-ligand dissociation constant and force per bond were estimated from data for the critical shear stress and contact area. Total internal reflection fluorescence microscopy was used to measure the contact area as a function of separation distance. Contact area increased with increasing ligand density. Contact areas were similar for the immobilized peptides but were greater on surfaces with adsorbed Fn. The dissociation constant was determined by nonlinear regression of the net force on the cells to models that assumed that bonds were either uniformly stressed or that only bonds on the periphery of the contact region were stressed (peeling model). Both models provided equally good fits for cells attached to immobilized peptides whereas the peeling model produced a better fit of data for cells attached to adsorbed Fn. Cyclic RGD and linear RGD both bind to the integrin alpha v beta 3, but immobilized cyclic RGD exhibited a greater affinity than did linear RGD. Receptor affinities of Fn adsorbed to glycophase glass and Fn adsorbed to glass were similar. The number of bonds was calculated assuming binding equilibrium. The peeling model produced good linear fits between bond force and number of bonds. Results of this study indicate that 1) bovine aortic endothelial cells are more adherent on immobilized cyclic RGD peptide than linear RGD or adsorbed Fn, 2) increased adhesion is due to a greater affinity between cyclic RGD and its receptor, and 3) the affinity of RGD peptides and adsorbed Fn for their receptors is increased after immobilization.