In vitro and in vivo pro-angiogenic effects of thymosin-ß4-derived peptides.

Thymosin-ß4 (Tß4) is a G-actin sequestering peptide involved in regeneration and remodeling of injured tissues. In this work, we have designed and synthesized three peptide sequences containing the N-terminus (TYB4-n), the central part (TYB4-i) or the C-terminus (TYB4-c) of Tß4. All fragments are overlapping on the main central binding actin site. After a structural characterization, we have evaluated in vitro and in vivo their pro-angiogenic effects. The results of this study have shown that: (i) each fragment reproduces the native conformation; (ii) Tß4-derived peptides exert both in vitro and in vivo pro-angiogenic effects; (iii) their in vitro effect seem to be related to the activation of several signaling pathways and is positively modulated by the N-terminus of Tß4.

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences.

It is well known that tumor growth is strictly dependent on neo-vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)(4)K] or the heparin-binding sequence of human vitronectin that interacts with HSPGs [HVP(351-359)]. Cell adhesion, proliferation, migration, and capillary-like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)(4)K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti-angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro-angiogenic effects induced by the Fibroblast growth factor (FGF-2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)(4)K. Our data indicate that the activity of RGD-containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti-angiogenic properties of (GRGDSP)(4)K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF-2.

Covalent surface modification of titanium oxide with different adhesive peptides: surface characterization and osteoblast-like cell adhesion.

A fundamental goal in the field of implantology is the design of innovative devices suitable for promoting implant-to-tissue integration. This result can be achieved by means of surface modifications aimed at optimizing tissue regeneration. In the framework of oral and orthopedic implantology, surface modifications concern both the optimization of titanium/titanium alloy surface roughness and the attachment of biochemical factors able to guide cellular adhesion and/or growth. This article focuses on the covalent attachment of two different adhesive peptides to rough titanium disks. The capability of biomimetic surfaces to increase osteoblast adhesion and the specificity of their biological activity due to the presence of cell adhesion signal-motif have also been investigated. In addition, surface analyses by profilometry, X-ray photoelectron spectroscopy, and time of flight-secondary ion mass spectrometry have been carried out to investigate the effects and modifications induced by grafting procedures.

Assessment of novel chemical strategies for covalent attachment of adhesive peptides to rough titanium surfaces: XPS analysis and biological evaluation.

Bioactive molecules have been proposed to promote beneficial interactions at bone-implant interfaces for enhancing integration. The main objective of this study was to develop novel methods to functionalize oxidized titanium surfaces by the covalent immobilization of bioactive peptides, through selective reaction involving single functional groups. In the first protocol, an aminoalkylsilane was covalently linked to the Ti oxide layer, followed by covalent binding of glutaric anhydride to the free NH(2) groups. The carboxylic group of glutaric anhydride was used to condense the free N-terminal group of the side-chain protected peptide sequence. Finally, the surface was treated with trifluoroacetic acid to deprotect side-chain groups. In the second protocol, the peptide was directly anchored to the Ti oxide surface via UV activation of an arylazide peptide analogue. X-ray photoelectron spectroscopy analyses confirmed that modifications induced onto surface composition were in agreement with the reactions performed. The peptide density of each biomimetic surface was determined on the basis of radiolabeling and XPS derived reaction yields. The in vitro cellular response of the biomimetic surfaces was evaluated using a primary human osteoblast cell model. Cell adhesion, proliferation, differentiation, and mineralization were examined at initial-, short-, and long-time periods. In was shown that the biomimetic surface obtained through photoprobe-marked analogue that combines an easily-performed modification provides a favorable surface for an enhanced cellular response.

Self-assembling peptides: sequence, secondary structure in solution and film formation.

Peptides of alternating charge and hydrophobic amino acids have a tendency to adopt unusually stable beta-sheet structures that can form insoluble macroscopic aggregates under physiological conditions. In this study, analogues of a well-known self-assembling peptide, characterized by the same polar/nonpolar periodicity but with different residues, were designed to study the relationship between sequence, conformation in solution and film-forming capacity in saline solution. Peptide conformation, evaluated by circular dichroism, correlated with film forming capacity observed by inverted optical microscopy after addition of saline solution and subsequent drying. We found that polar/nonpolar periodicity of several analogues is not criterion enough to induce beta-sheet and thus film formation and that conformations different from beta-sheet also allow self-assemblage. Furthermore, addition of the short adhesive sequence RGD to a known self-assembling sequence was shown to not prevent the self-assembling process. This finding might prove useful for the design of biomimetic scaffolds.

Improvement of Anselme's adhesion model for evaluating human osteoblast response to peptide-grafted titanium surfaces.

Investigations on the relationships between the properties of biomaterial surfaces and cell adhesion/proliferation processes have recently gained increasing interest. To describe the behaviour of cells adhering and proliferating over different types of (and/or differently treated) substrates, some mathematical models have been also suggested in literature; these models consider both the dependence of cell adhesion/proliferation over time, and the influence of substrate morphology in allowing (or even hampering) cell attachment. Major developments in the biochemical functionalization of the materials used for the production of endosseous devices have been achieved; the ability of the so-called "biomimetic" surfaces to promote cell adhesion, thus favoring the osseointegration process, is already well acknowledged. The aim of this study was to formulate a mathematical model for osteoblast adhesion, mediated by an adhesion peptide (sequence 351-359 mapped on the Human Vitronectin Protein) covalently grafted to a titanium-based surface. To assure a highly homogenous orientation of the peptide to cells, the "specific functionalization" strategy was properly designed. Enzymatic detachment assays allowed comparing osteoblast behaviour over three differently treated titanium substrates (i.e., oxidized, silanized, and peptide-grafted), thus determining how and how much the bioactive peptide can improve the strength of cell adhesion. The results confirmed the capacity of the peptide to increase cell adhesion and adhesion strength; moreover, the role of the peptide was described by a mathematical equation characterizing cells behaviour.

Human osteoblast-like cell adhesion on titanium substrates covalently functionalized with synthetic peptides.

Biomaterials to be used for the production of endosseous devices in dental, orthopedic and maxillo-facial applications, might be designed to support, guide and enhance osteoblast adhesion. Cell recruitment onto biomaterial surface is a fundamental step within the complex process responsible for implant osseointegration; this process involves several proteins from the extra cellular matrix (ECM), cytoskeleton and cell membrane. A new strategy to improve endosseous implant integration is based on preparing biomimetic surfaces able to present adhesive factors to cells. Osteoblast adhesion takes place by at least two different mechanisms: the most investigated one implies the interaction with RGD sequences via cell-membrane integrin receptors; a further mechanism concerns the interaction between cell-membrane heparan sulfate proteoglycans and heparin-binding sites of ECM proteins. In the present study two different biomimetic surfaces were obtained by covalently grafting two adhesive peptides on oxidized titanium substrates after silanization: an RGD-containing peptide and a peptide mapped on human vitronectin. The two sequences are known to act via different adhesive mechanisms. The amount of human osteoblasts adhered onto peptide-enriched or not enriched titanium oxidized surfaces and the strength of cell binding were estimated, thus comparing the capacity of the bioactive substrates in promoting cell adhesion.

The proprotein convertase SKI-1/S1P. In vitro analysis of Lassa virus glycoprotein-derived substrates and ex vivo validation of irreversible peptide inhibitors.

Herein we designed, synthesized, tested, and validated fluorogenic methylcoumarinamide (MCA) and chloromethylketone-peptides spanning the Lassa virus GPC cleavage site as substrates and inhibitors for the proprotein convertase SKI-1/S1P. The 7-mer MCA (YISRRLL-MCA) and 8-mer MCA (IYISRRLL-MCA) are very efficiently cleaved with respect to both the 6-mer MCA (ISRRLL-MCA) and point mutated fluorogenic analogues, except for the 7-mer mutant Y253F. The importance of the P7 phenylic residue was confirmed by digestions of two 16-mer non-fluorogenic peptidyl substrates that differ by a single point mutation (Y253A). Because NMR analysis of these 16-mer peptides did not reveal significant structural differences at recognition motif RRLL, the P7 Tyr residue is likely important in establishing key interactions within the catalytic pocket of SKI-1. Based on these data, we established through analysis of pro-ATF6 and pro-SREBP-2 cellular processing that decanoylated chloromethylketone 7-mer, 6-mer, and 4-mer peptides containing the core RRLL sequence are irreversible and potent ex vivo SKI-1 inhibitors. Although caution must be exercised in using these inhibitors in in vitro reactions, as they can also inhibit the basic amino acid-specific convertase furin, within cells and when used at concentrations < or = 100 microM these inhibitors are relatively specific for inhibition of SKI-1 processing events, as opposed to those performed by furin-like convertases.

Evaluation of silicon dioxide-based coating enriched with bioactive peptides mapped on human vitronectin and fibronectin: in vitro and in vivo assays.

A wide range of biochemical signals promoting cell functions (adhesion, migration, proliferation, and differentiation) and thereby improving the osseointegration process are currently investigated. Unfortunately, their application for the production of bioactive implantable devices is often hampered by their insolubility; instability; and limited availability of a large amount of inexpensive, high-purity samples. An attractive alternative is the use of short peptides carrying the minimum active sequence of the natural factors. Synthetic peptides mapped on fibronectin and vitronectin have been demonstrated to enhance cell adhesion both to polystyrene and acellular bone matrix; in particular, a nonapeptide sequence from human vitronectin works via an osteoblast-specific adhesion mechanism. In this study, we incorporated these peptides into a sol-gel silica dressing applied to coat sand-blasted and acid-attacked titanium samples; measured the kinetic of peptide release; and used titanium disks, coated with a peptide-enriched film, as substrates to determine the peptide concentration that maximizes cell adhesion in vitro. We also evaluated in vivo the capacity of the vitronectin-derived peptide to improve osteogenic activity: histologic analysis revealed markedly improved osteogenic activity around peptide-enriched samples. This article also discusses the role of surface characteristics and the importance of bioactive peptides.

Effect of synthetic peptides on osteoblast adhesion.

The quality of the early cell/material interactions is responsible for the long-term functional properties of any implanted device. Accordingly, "next generation" dental/orthopedic biomaterials should be able to promote osteoblast adhesion thus improving the integration process between surgically placed implants and biological tissues. Recent studies have identified a wide range of biochemical signals that can be exploited to promote adhesion, migration, proliferation and differentiation of cells. The clinical use of natural factors to promote osteoblast adhesion is complicated because those are often insoluble and unstable macromolecules and, in addition, it is difficult to obtain them in high quantities, with good purity grade and at low cost. A valid alternative could be the use of short peptides carrying the minimum active sequence of the natural macromolecular factor. This paper describes the properties of two classes of peptides, promoting different adhesion mechanisms, to enhance rat bone marrow osteoblast adhesion both to polystyrene and to acellular bone matrix.

Conformational analysis of heparin binding peptides.

A properly engineered biomaterial for dental/orthopaedic applications must induce specific responses from the osteoblasts at the implant site. A most desirable response is an efficient adhesion, as it represents the first phase in the cell/material interaction and the quality of this phase will influence the cell's capacity to organize into a new functional tissue. The four osteoblast-adhesive peptides discussed in this paper are mapped on the 339-364 sequence (339MAPRPSLAKKQRFRHRNRKGYRSQRG364) located in the primary heparin-binding site of human vitronectin (HVP). Adsorbed on a polystyrene scaffold, these peptides display different adhesive activities towards osteoblasts. In this paper we report on the structural analysis in solution of the peptides through NMR and computational techniques. We find that the peptides with the highest adhesive activities display a hydrophobic patch opposite to the charged surface candidate to interact with heparin. These findings suggest that the peptides might adsorb on the polystyrene support in a favourable orientation for their activity. Furthermore, molecular models obtained for the four peptides in solution were used in rigid docking simulations with a heparin model. Assuming that the peptide solution conformations are not very different from the polystyrene-adsorbed structures, the simulations reveal that peptide adhesive activity is also affected by the number of ionic interactions and spacing between charged residues.

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site 3: role of site-specific mutations.

Proteolytic processing of HIV gp160 to produce gp120 and gp41 is performed by PC enzymes. This process is a prerequisite for the virus infectivity, since both gp120 and gp41 participate in the virus HIV-1 entry mechanism. The structure of the gp120/gp41 junction remains to be elucidated, and the structural features required for molecular recognition between HIV-1 gp160 and proteolytic enzymes have not been clarified. Furin is the best PC candidate for the gp160 proteolytic processing known to date. In previous studies on model peptides, we have shown the relevance of an N-terminal helix for the proper recognition of the gp160 processing site by furin. Here we analyze the effect of point mutations in peptides lacking a regular N-terminal helix. To this end, we present the structure-activity characterization of three peptide analogues of the HIV gp160 processing site that all present mutations in proline at positions P3 and/or P2', while sharing the same N-terminal sequence, containing helix-breaking D-amino acids. Conformational analysis of the peptides was carried out in solution by NMR techniques, and furin's efficiency in cleaving them was measured. Structural findings are presented and discussed in relation to the different exhibited activity.

Surface treatments and roughness properties of Ti-based biomaterials.

Nowadays, the use of implanted devices is a well-acknowledged practice in the field of orthopaedic and dental surgery. Scientific research and clinical experience suggest that the successful exploitation of these devices mainly depends on osseointegration, considered as both anatomical congruency and load-bearing capacity. Indeed, the osseointegration process is influenced by a wide range of factors: anatomical location, implant size and design, surgical procedure, loading effects, biological fluids, age and sex, and, in particular, surface characteristics. For this reason, several attempts have been aimed at modifying implant surface composition and morphology to optimise implant-to-bone contact and improve integration. Preliminary interactions between implanted materials and biological environment are deemed to be governed by the surface properties; they control the amount and quality of cell adhesion on the surface and, consequently, cell/tissue growth. Thus, surface properties govern new bone tissue formation and implant osseointegration. This paper reviews the state of art in the field of physical, chemical and biochemical treatments commonly used on Ti-based biomaterials for the production of biomedical devices. In particular, roughness characteristics due to physical and chemical techniques are investigated; the development of biologically active surfaces by means of biochemical functionalisation is also considered.

Synthetic peptides for AIDS research.

In spite of the relevance of the results obtained through the clinical application of chemotherapeutic agents (reverse transcriptase and proteinase inhibitors) that are able to prolong the life span of affected people, acquired immunodeficiency syndrome (AIDS) remains a serious and lethal disease. AIDS is caused by a type 1 human immunodeficiency virus (HIV-1) and formation of a complex among the gp120, CD4 and CCR5/CXCR4 surface proteins represents a key-step in the infection. The use of synthetic peptides reproducing reduced sequences of these proteins has contributed to increase the knowledge of the mechanism that determines the penetration of the HIV viruses into the target-cells. In addition, short peptides with minimum structural requirements for anti-HIV activity hold greater potential as lead compounds for rational drug design than macromolecular proteins. In this context, our studies concern: the role of gp120 V3 loop in CD4 binding, the importance of the N-terminal sequence of HIV CCR5 coreceptor, the potential inhibitory properties of sequences patterned on CXCR4 natural ligand (SDF-1) and the role of secondary structure in determining gp160 enzymatic processing into gp120.

Anti-HIV activity and conformational studies of peptides derived from the C-terminal sequence of SDF-1.

The entry of the human immunodeficiency virus type 1 (HIV-1) into target cells requires the interaction of viral envelope glycoprotein, gp120, with the human CD4 glycoprotein and a chemokine receptor, usually CCR5 or CXCR4. The natural ligand for CXCR4 is the chemokine SDF-1 that inhibits entry and replication of X4 HIV-1 strains. SDF-1 is produced in two forms, SDF-1alpha (68 residues) and SDF-1beta (72 residues); the difference between them lies in the additional four C-terminal amino acids in the SDF-1beta sequence. Despite the relevance of the N-terminal site in determining the SDF anti HIV-1 activity, SDF-1beta has a stronger activity than SDF-1alpha. Here we demonstrate that a synthetic peptide mapped on the C-terminus of SDF-1beta presents inhibitory activity, whereas an analogue reproducing the C-terminal trait of SDF-1alpha does not show any activity. The opposite biological effect of the two peptides correlates with the type of interaction they each have with heparin and chondroitin sulfate.

Contact profilometry and correspondence analysis to correlate surface properties and cell adhesion in vitro of uncoated and coated Ti and Ti6Al4V disks.

A fundamental goal in the field of implantology is the design of specific devices able to induce a controlled and rapid "osseointegration". This result has been achieved by means of surface modifications aimed at optimizing implant-to-bone contact; furthermore, bone cell adhesion on implant surface has been directly improved by the application of biomolecules that stimulate new tissue formation, thus controlling interactions between biological environment and implanted materials. Actually, methods for biochemical factor delivery at the interface between implant surface and biological tissues are under investigation; a reliable technique is represented by the inclusion of biologically active molecules into biocompatible and biodegradable materials used for coating implant surface. This paper focuses the application of three polymeric materials already acknowledged in the clinical practice, i.e. poly-L-lactic acid (PLLA), poly-DL-lactic acid (PDLA), and sodium alginate hydrogel. They have been used to coat Ti (Ti2) and Ti6Al4V (Ti5) disks; their characteristics have been determined and their performances compared, with specific regard to the ability in allowing osteoblast adhesion in vitro. Moreover, profilometry data analysis permitted to identify a specific roughness parameter (peak density) which mainly controls the amount of osteoblast adhesion.

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site, 2: relevance of an N-terminal helix.

Proteolytic activation of the HIV-1 envelope glycoprotein gp160 is selectively performed by the proprotein convertase furin at the C-terminus of the sequence R508-E-K-R511 (site 1), in spite of the presence of another consensus sequence, Lys500-Ala-Lys-Arg503 (site 2). On the basis of the solution structural analysis of the synthetic peptide p498, spanning the gp160 sequence Pro498-Gly516, we previously suggested a possible role of an N-terminal helix in regulating the exposure and accessibility of the gp160 physiological cleavage site, enclosed in a loop. Here we report on the activity and conformation of the 23-residue peptide h-REKR, designed to exhibit a large N-terminal helix, followed by the gp160 native sequence, Arg508-Gly516. h-REKR is digested by furin with high efficiency, comparable to the full native p498. Circular dichroism analyses, in mixtures from pure water to 98 % trifluoroethanol, outline a significant content of helical structure in the peptide conformation. The molecular model obtained from NMR data collected in trifluoroethanol/water, by means of DYANA and AMBER simulations, indeed has helical structure on a large N-terminal segment. Such a long helix does not seem to affect the loop conformation of the C-terminal site 1-containing sequence, which exhibits the same proton chemical shifts already observed for the full native p498.

Is the V3 loop involved in HIV binding to CD4?

The entry of the human immunodeficiency virus into cells requires the interaction of the viral envelope glycoprotein gp120 with CD4 and a chemokine receptor. The gp120 binding site has been previously mapped to the Ig-CDR2-like region of CD4 first domain. A second area of this domain (Ig-CDR3-like region) is involved in gp120-CD4 interactions, but its gp120 counterpart remained so far unknown. Using a photoaffinity labeling experiment, we demonstrate that a peptide, mapping the (307-330)m region of HIV-MN-gp120 V3 loop, binds a sequence including a part of the Ig-CDR3-like region. These results may contribute to explain the complex mechanism of human immunodeficiency virus penetration, helping the development of new therapeutic agents.

Plasminogen activator inhibitor-2: a molecular biomarker for head and neck cancer progression.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy in which the early diagnosis of premalignant lesions is known to directly correlate with increased survival. However, only a portion of biopsies showing dysplasia will progress to cancer, and there are no currently accepted criteria for predicting which lesions will progress. Therefore, diagnostic protocols that can identify the lesions that are likely to become HNSCC are required. RNA was isolated from normal keratinocytes, the immortalized but nontumorigenic HaCat cell line, and the tumor cell lines SCC-4, SCC-9, SCC-25, and OSCC-3. The RNA was then labeled and used to probe nylon microarray filters that contained a total of 9184 genes (5295 named and 3889 Expression Sequence Tags). Genes whose expression demonstrated a 3-fold or greater change were considered significant. Comparison of expression profiles from normal, HaCat, and four tumor cell lines revealed changes in gene expression in a total of 508 genes. Of these, 16 genes showed a consistent loss of expression when comparing normal to immortalized keratinocytes. In addition, 10 genes demonstrated a consistent loss of expression in the tumor cell lines only. In this latter group of genes, plasminogen activator inhibitor-2 (PAI-2), a gene whose expression has been linked to cell invasion, was additionally investigated. Altered expression of PAI-2 in the different cultured cells was validated via real-time quantitative-PCR. In addition, immunohistochemical evaluation of biopsy samples revealed a high expression of PAI-2 in both normal and dysplastic epithelium with a marked decrease of expression in areas of the biopsies containing HNSCC. These data demonstrate that genomic profiling can then be used to identify potential genotypic/phenotypic biomarkers that may predict which dysplastic lesions are most likely to progress to HNSCC.