Self-assembly of Fmoc-tetrapeptides based on the RGDS cell adhesion motif

Self-assembly in aqueous solution has been investigated for two Fmoc [Fmoc ¼ N-(fluorenyl)-9-methoxycarbonyl] tetrapeptides comprising the RGDS cell adhesion motif from fibronectin or the scrambled sequence GRDS. The hydrophobic Fmoc unit confers amphiphilicity on the molecules, and introduces aromatic stacking interactions. Circular dichroism and FTIR spectroscopy show that the self-assembly of both peptides at low concentration is dominated by interactions among Fmoc units, although Fmoc-GRDS shows b-sheet features, at lower concentration than Fmoc-RGDS. Fibre X-ray diffraction indicates b-sheet formation by both peptides at sufficiently high concentration. Strong alignment effects are revealed by linear dichroism experiments for Fmoc-GRDS. Cryo-TEM and smallangle X-ray scattering (SAXS) reveal that both samples form fibrils with a diameter of approximately 10 nm. Both Fmoc-tetrapeptides form self-supporting hydrogels at sufficiently high concentration. Dynamic shear rheometry enabled measurements of the moduli for the Fmoc-GRDS hydrogel, however syneresis was observed for the Fmoc-RGDS hydrogel which was significantly less stable to shear. Molecular dynamics computer simulations were carried out considering parallel and antiparallel b-sheet configurations of systems containing 7 and 21 molecules of Fmoc-RGDS or Fmoc-GRDS, the results being analyzed in terms of both intermolecular structural parameters and energy contributions.

Regulation of platelet biology by platelet endothelial cell adhesion molecule-1.

Platelet endothelial cell adhesion molecule-1 (PECAM-1), an immunoreceptor tyrosine-based inhibitory motif containing receptor, plays diverse and apparently contradictory roles in regulating the response of platelets to stimuli; inhibiting platelet response to immunoreceptor tyrosine-based activation motif and G protein-coupled receptor signalling following stimulation with collagen, adenosine diphosphate, and thrombin, as well as enhancing integrin outside-in signalling. These dual, and opposing, roles suggest an important and complex role for PECAM-1 in orchestrating platelet response to vascular damage. Indeed, during thrombus formation, the influence of PECAM-1 on the multiple signalling pathways combines leading to a relatively large inhibitory effect on thrombus formation.

Self-assembly and bioactivity of a polymer/peptide conjugate containing the RGD cell adhesion motif and PEG

The self-assembly and bioactivity of the peptide–polymer conjugate DGRFFF–PEG3000 containing the RGD cell adhesion motif has been examined, in aqueous solution. The conjugate is designed to be amphiphilic by incorporation of three hydrophobic phenylalanine residues as well as the RGD unit and a short poly(ethylene glycol) (PEG) chain of molar mass 3000 kg mol-1. Above a critical aggregation concentration, determined by fluorescence measurements, signals of b-sheet structure are revealed by spectroscopic measurements, as well as X-ray diffraction. At high concentration, a self-assembled fibril nanostructure is revealed by electron microscopy. The fibrils are observed despite PEG crystallization which occurs on drying. This suggests that DGRFFF has an aggregation tendency that is sufficiently strong not to be prevented by PEG crystallization. The adhesion, viability and proliferation of human corneal fibroblasts was examined for films of the conjugate on tissue culture plates (TCPs) as well as low attachment plates. On TCP, DGRFFF–PEG3000 films prepared at sufficiently low concentration are viable, and cell proliferation is observed. However, on low attachment surfaces, neither cell adhesion nor proliferation was observed, indicating that the RGD motif was not available to enhance cell adhesion. This was ascribed to the core–shell architecture of the self-assembled fibrils with a peptide core surrounded by a PEG shell which hinders access to the RGD unit.

Bioactive films produced from self-assembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions

The development of versatile bioactive surfaces able to emulate in vivo conditions is of enormous importance to the future of cell and tissue therapy. Tuning cell behaviour on two-dimensional surfaces so that the cells perform as if they were in a natural three-dimensional tissue represents a significant challenge, but one that must be met if the early promise of cell and tissue therapy is to be fully realised. Due to the inherent complexities involved in the manufacture of biomimetic three-dimensional substrates, the scaling up of engineered tissue-based therapies may be simpler if based upon proven two-dimensional culture systems. In this work, we developed new coating materials composed of the self-assembling peptide amphiphiles (PAs) C16G3RGD (RGD) and C16G3RGDS (RGDS) shown to control cell adhesion and tissue architecture while avoiding the use of serum. When mixed with the C16ETTES diluent PA at 13 : 87 (mol mol-1) ratio at 1.25 times 10-3 M, the bioactive {PAs} were shown to support optimal adhesion, maximal proliferation, and prolonged viability of human corneal stromal fibroblasts ({hCSFs)}, while improving the cell phenotype. These {PAs} also provided stable adhesive coatings on highly-hydrophobic surfaces composed of striated polytetrafluoroethylene ({PTFE)}, significantly enhancing proliferation of aligned cells and increasing the complexity of the produced tissue. The thickness and structure of this highly-organised tissue were similar to those observed in vivo, comprising aligned newly-deposited extracellular matrix. As such, the developed coatings can constitute a versatile biomaterial for applications in cell biology, tissue engineering, and regenerative medicine requiring serum-free conditions.

Alanine-rich amphiphilic peptide containing the RGD cell adhesion motif: a coating material for human fibroblast attachment and culture

We studied the self-assembly of peptide A6RGD (A: alanine, R: arginine, G: glycine, D: aspartic acid) in water, and the use of A6RGD substrates as coatings to promote the attachment of human cornea stromal fibroblasts (hCSFs). The self-assembled motif of A6RGD was shown to depend on the peptide concentration in water, where both vesicle and fibril formation were observed. Oligomers were detected for 0.7 wt% A6RGD, which evolved into short peptide fibres at 1.0 wt% A6RGD, while a co-existence of vesicles and long peptide fibres was revealed for 2–15 wt% A6RGD. A6RGD vesicle walls were shown to have a multilayer structure built out of highly interdigitated A6 units, while A6RGD fibres were based on β-sheet assemblies. Changes in the self-assembly motif with concentration were reflected in the cell culture assay results. Films dried from 0.1–1.0 wt% A6RGD solutions allowed hCSFs to attach and significantly enhanced cell proliferation relative to the control. In contrast, films dried from 2.5 wt% A6RGD solutions were toxic to hCSFs.

Comparative effect of dairy fatty acids on cell adhesion molecules, nitric oxide and relative gene expression in healthy and diabetic human aortic endothelial cells

Dairy intake, despite its high saturated fatty acid (SFA) content, is associated with a lower risk of cardiovascular disease and diabetes. This in vitro study determined the effect of individual fatty acids (FA) found in dairy, and FA mixtures representative of a high SFA and a low SFA dairy lipid on markers of endothelial function in healthy and type II diabetic aortic endothelial cells.

Serum protein layers on parylene-C and silicon oxide: effect on cell adhesion

Among the range of materials used in bioengineering, parylene-C has been used in combination with silicon oxide and in presence of the serum proteins, in cell patterning. However, the structural properties of adsorbed serum proteins on these substrates still remain elusive. In this study, we use an optical biosensing technique to decipher the properties of fibronectin (Fn) and serum albumin adsorbed on parylene-C and silicon oxide substrates. Our results show the formation of layers with distinct structural and adhesive properties. Thin, dense layers are formed on parylene-C, whereas thicker, more diffuse layers are formed on silicon oxide. These results suggest that Fn acquires a compact structure on parylene-C and a more extended structure on silicon oxide. Nonetheless, parylene-C and silicon oxide substrates coated with Fn host cell populations that exhibit focal adhesion complexes and good cell attachment. Albumin adopts a deformed structure on parylene-C and a globular structure on silicon oxide, and does not support significant cell attachment on either surface. Interestingly, the co-incubation of Fn and albumin at the ratio found in serum, results in the preferential adsorption of albumin on parylene-C and Fn on silicon oxide. This finding is supported by the exclusive formation of focal adhesion complexes in differentiated mouse embryonic stem cells (CGR8), cultured on Fn/albumin coated silicon oxide, but not on parylene-C. The detailed information provided in this study on the distinct properties of layers of serum proteins on substrates such as parylene-C and silicon oxide is highly significant in developing methods for cell patterning.

Interactions between lipid-free apolipoprotein-AI and a lipopeptide incorporating the RGDS cell adhesion motif

The interaction of a designed bioactive lipopeptide C16-GGGRGDS, comprising a hexadecyl lipid chain attached to a functional heptapeptide, with the lipid-free apoliprotein, Apo-AI, is examined. This apolipoprotein is a major component of high density lipoprotein and it is involved in lipid metabolism and may serve as a biomarker for cardiovascular disease and Alzheimers’ disease. We find via isothermal titration calorimetry that binding between the lipopeptide and Apo-AI occurs up to a saturation condition, just above equimolar for a 10.7 μM concentration of Apo-AI. A similar value is obtained from circular dichroism spectroscopy, which probes the reduction in α-helical secondary structure of Apo-AI upon addition of C16-GGGRGDS. Electron microscopy images show a persistence of fibrillar structures due to self-assembly of C16-GGGRGDS in mixtures with Apo-AI above the saturation binding condition. A small fraction of spheroidal or possibly “nanodisc” structures was observed. Small-angle X-ray scattering (SAXS) data for Apo-AI can be fitted using a published crystal structure of the Apo-AI dimer. The SAXS data for the lipopeptide/ Apo-AI mixtures above the saturation binding conditions can be fitted to the contribution from fibrillar structures coexisting with flat discs corresponding to Apo-AI/lipopeptide aggregates.

Self-assembly of a dual functional bioactive peptide amphiphile incorporating both matrix metalloprotease substrate and cell adhesion motifs

We describe a bioactive lipopeptide that combines the capacity to promote the adhesion and subsequent self-detachment of live cells, using template-cell-environment feedback interactions. This self-assembling peptide amphiphile comprises a diene-containing hexadecyl lipid chain (C16e) linked to a matrix metalloprotease-cleavable sequence, Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, and contiguous with a cell-attachment and signalling motif, Arg-Gly-Asp-Ser. Biophysical characterisation revealed that the PA self-assembles into 3 nm diameter spherical micelles above a critical aggregation concentration (cac). In addition, when used in solution at 5–150 nM (well below the cac), the PA is capable of forming film coatings that provide a stable surface for human corneal fibroblasts to attach and grow. Furthermore, these coatings were demonstrated to be sensitive to metalloproteases expressed endogenously by the attached cells, and consequently to elicit the controlled detachment of cells without compromising their viability. As such, this material constitutes a novel class of multi-functional coating for both fundamental and clinical applications in tissue engineering.

In vitro study on the cell adhesion ability of immobilized lactobacilli on natural supports

The aim of the present study was to investigate the effect of probiotic immobilization onto wheat grains, both wet and freeze dried, on the adhesion properties of the probiotic cells and make comparisons with wet and freeze dried free cells. Lactobacillus casei ATCC 393 and Lactobacillus plantarum NCIMB 8826 were used as model probiotic strains. The results showed satisfactory adhesion ability of free cells to a monolayer of Caco-2 cells (> 1000 CFU/100 Caco-2 cells for wet cells). Cell immobilization resulted in a significant decrease in adhesion, for both wet and freeze dried formulations, most likely because immobilized cells did not have direct access to the Caco-2 cells, but it still remained in adequate levels (> 100 CFU/100 Caco-2 cells for wet cells). No clear correlation could be observed between cell adhesion and the hydrophobicity of the bacterial cells, measured by the hexadecane adhesion assay. Most notably, immobilization enhanced the monolayer integrity of Caco-2 cells, demonstrated by a more than 2-fold increase in transepithelial electrical resistance (TEER) compared to free cells. SEM micrographs ascertained the adhesion of both immobilized and free cells to the brush border microvilli. Finally, the impact of the food matrix on the adhesion properties of probiotic bacteria and on the design of novel functional products is discussed.

Expression Patterns of Cell Adhesion Molecules in Mice`s Lung After Administration of Meso-2,3-Dimercaptosuccinic Acid-Coated Maghemite Nanoparticles

We studied the expression pattern of cell adhesion molecules associated to transendothelial migration of leukocytes in different lung`s vascular compartments after administration of a magnetic fluid sample containing maghemite nanoparticles surface-coated with meso-2,3-dimercaptosuccinic acid. The analyses were conducted in mice 4 and 12 h after endovenous administration of the magnetic fluid in control mice. Firstly, the migratory activity of leukocytes after magnetic fluid surface-coated with meso-2,3-dimercaptosuccinic acid administration was confirmed using broncho-alveolar lavage and light microscopy. Then, the expression of cell adhesion molecules in the lung`s vascular compartments was investigated by immunofluorescence microscopy of frozen sections, using antibodies against L-selectin, P-selectin, E-selectin, macrophage antigen-1, and leukocyte function associated antigen-1. L- and P-selectin showed similar pattern of expression in the pulmonary vasculature in animals treated with magnetic fluid and in the control group. In contrast, macrophage antigen-1 and leukocyte function associated antigen-1 were found in capillary only in animals treated with magnetic fluid surface-coated with meso-2,3-dimercaptosuccinic acid administration. In addition, after magnetic fluid administration E-selectin was found in post-capillary sites. Our findings demonstrated that magnetic fluid surface-coated with meso-2,3-dimercaptosuccinic acid administration exhibits modulation effects on expression patterns of E-selectin, macrophage antigen-1, and leukocyte function associated antigen-1 in the lung`s vascular compartments. These findings are very important in a strategy to reduce the potential toxicity of magnetic fluid surface-coated with meso-2,3-dimercaptosuccinic acid administration for medical applications.

Mucosal vascular addressin cell adhesion molecule-1 is expressed outside the endothelial lineage on fibroblasts and melanoma cells

Mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) is predominantly expressed on high endothelial venules in inflamed tissues where it assists with leucocyte extravasation. Here we report that MAdCAM-1 has the potential to be more widely expressed outside the endothelial cell lineage than previously appreciated. Thus, MAdCAM-1 RNA transcripts and cell-surface protein were expressed by NIH 3T3 fibroblasts following activation with tumour necrosis factor-alpha (TNF-alpha), and by freshly isolated and cultured primary mouse splenic and tail fibroblasts in the absence of TNF-alpha stimulation. They were constitutively expressed by B16F10 melanoma cells, and expression was enhanced by cell activation with TNF-alpha. Mucosal vascular addressin cell adhesion molecule-1 was expressed on the apical surface of isolated cells, but became predominantly localized to cell junctions in confluent cell monolayers, suggesting it may play a role in the homotypic aggregation of cells. Tumour necrosis factor-alpha enhanced the expression of a firefly luciferase reporter directed by the MAdCAM-1 promoter in NIH 3T3 and B16F10 cells. A DNA fragment extending from nt -1727 to -673 was sufficient to confer cell-type selective expression. Mucosal vascular addressin cell adhesion molecule-1 expressed by NIH 3T3 cells was biologically active, as it supported the adhesion of TK-1 T cells in an alpha4beta7-dependent fashion. The expression of MAdCAM-1 by fibroblasts, and melanomas suggests MAdCAM-1 may play a role in regulating host responses in the periphery, leucocyte transmigration across nonendothelial boundaries, or the homotypic interactions of some malignant melanomas.

Prevention of a chronic progressive form of experimental autoimmune encephalomyelitis by an antibody against mucosal addressin cell adhesion molecule-1, given early in the course of disease progression.

A role for α4 and β7 integrins in mediating leucocyte entry into the central nervous system in the multiple sclerosis (MS)-like disease experimental autoimmune encephalomyelitis (EAE) has been demonstrated. However, the individual contributions of their respective ligands mucosal addressin cell adhesion molecule-1 (MAdCAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-cadherin expressed on the blood-brain barrier has not been determined. In the present paper, it is shown that an antibody directed against MAdCAM-1, the preferential ligand for α4β7, effectively prevented the development of a progressive, non-remitting, form of EAE, actively induced by injection of myelin oligodendrocyte glycoprotein peptide (MOG(_35-55)) autoantigen. Combinational treatment with both anti-MAdCAM-1, VCAM-1, and intercellular adhesion molecule-1 (ICAM-1) (ligand for integrin lymphocyte function-associated antigen (LFA)-1) mAbs led to more rapid remission than that obtained with anti-MAdCAM-1 antibody alone. However, neither MAdCAM-1 monotherapy, nor combinational antibody blockade was preventative when administered late in the course of disease progression. In conclusion, MAdCAM-1 plays a major contributory role in the progression of chronic EAE and is a potential therapeutic target for the treatment of MS. Critically, antivascular addressin therapy must be given eaA role for alpha4 and beta7 integrins in mediating leucocyte entry into the central nervous system in the multiple sclerosis (MS)-like disease experimental autoimmune encephalomyelitis (EAE) has been demonstrated. However, the individual contributions of their respective ligands mucosal addressin cell adhesion molecule-1 (MAdCAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-cadherin expressed on the blood-brain barrier has not been determined. In the present paper, it is shown that an antibody directed against MAdCAM-1, the preferential ligand for alpha4beta7, effectively prevented the development of a progressive, non-remitting, form of EAE, actively induced by injection of myelin oligodendrocyte glycoprotein peptide (MOG(35-55)) autoantigen. Combinational treatment with both anti-MAdCAM-1, VCAM-1, and intercellular adhesion molecule-1 (ICAM-1) (ligand for integrin lymphocyte function-associated antigen (LFA)-1) mAbs led to more rapid remission than that obtained with anti-MAdCAM-1 antibody alone. However, neither MAdCAM-1 monotherapy, nor combinational antibody blockade was preventative when administered late in the course of disease progression. In conclusion, MAdCAM-1 plays a major contributory role in the progression of chronic EAE and is a potential therapeutic target for the treatment of MS. Critically, antivascular addressin therapy must be given early in the course of disease prior to the establishment of irreversible damage if it is to be effective, as a single treatment modality.

Target-specific delivery of doxorubicin to retinoblastoma using epithelial cell adhesion molecule aptamer

PURPOSE: To study target-specific delivery of doxorubicin (Dox) using an RNA aptamer against epithelial cell adhesion molecule (EpCAM) in retinoblastoma (RB) cells. METHODS: The binding affinity of the EpCAM aptamer to RB primary tumor cells, Y79 and WERI-Rb1 cells, and Müller glial cell lines were evaluated with flow cytometry. Formation of physical conjugates of aptamer and Dox was monitored with spectrofluorimetry. Cellular uptake of aptamer-Dox conjugates was monitored through fluorescent microscopy. Drug efficacy was monitored with cell proliferation assay. RESULTS: The EpCAM aptamer (EpDT3) but not the scrambled aptamer (Scr-EpDT3) bound to RB tumor cells, the Y79 and WERI-Rb1 cells. However, the EpCAM aptamer and the scrambled aptamer did not bind to the noncancerous Müller glial cells. The chimeric EpCAM aptamer Dox conjugate (EpDT3-Dox) and the scrambled aptamer Dox conjugate (Scr-EpDT3-Dox) were synthesized and tested on the Y79, WERI-Rb1, and Müller glial cells. The targeted uptake of the EpDT3-Dox aptamer caused cytotoxicity in the Y79 and WERI-Rb1 cells but not in the Müller glial cells. There was no significant binding or consequent cytotoxicity by the Scr-EpDT3-Dox in either cell line. The EpCAM aptamer alone did not cause cytotoxicity in either cell line. CONCLUSIONS: The results show that the EpCAM aptamer-Dox conjugate can selectively deliver the drug to the RB cells there by inhibiting cellular proliferation and not to the noncancerous Müller glial cells. As EpCAM is a cancer stem cell marker, this aptamer-based targeted drug delivery will prevent the undesired effects of non-specific drug activity and will kill cancer stem cells precisely in RB.