RÉSUMÉ
The interaction between the integrin and collagen is important in cell adhesion and signaling. Collagen, as the main component of extracellular matrix, is a base material for tissue engineering constructs. In tissue engineering, the collagen structure and molecule state may be altered to varying degrees in the process of processing and utilizing, thereby affecting its biological properties. In this work, the impact of changes in collagen structure and molecular state on the binding properties of collagen to integrin α2ß1 and integrin specific cell adhesion were explored. The results showed that the molecular structure of collagen is destroyed under the influence of heating, freeze-grinding and irradiation, the triple helix integrity is reduced and molecular breaking degree is increased. The binding ability of collagen to integrin α2ß1 is increased with the increase of triple helix integrity and decays exponentially with the increase of molecular breaking degree. The collagen molecular state can also influences the binding ability of collagen to cellular receptor. The collagen fibrils binding to integrin α2ß1 and HT1080 cells is stronger than to collagen monomolecule. Meanwhile, the hybrid fibril exhibits a different cellular receptor binding performance from corresponding single species collagen fibril. These findings provide ideas for the design and development of new collagen-based biomaterials and tissue engineering research.
Sujet(s)
Adhérence cellulaire , Collagène , Intégrine alpha2bêta1 , Liaison aux protéines , Intégrine alpha2bêta1/métabolisme , Intégrine alpha2bêta1/composition chimique , Humains , Collagène/composition chimique , Collagène/métabolisme , Matériaux biocompatibles/composition chimique , Matériaux biocompatibles/métabolisme , Animaux , Ingénierie tissulaire/méthodes , Lignée cellulaire tumoraleRÉSUMÉ
Collagen constructs are widely used for tissue engineering. These are frequently chemically crosslinked, using EDC, to improve their stability and tailor their physical properties. Although generally biocompatible, chemical crosslinking can modify crucial amino acid side chains, such as glutamic acid, that are involved in integrin-mediated cell adhesion. Instead UV crosslinking modifies aromatic side chains. Here we elucidate the impact that EDC, in combination with UV, exerts on the activity of integrin-binding motifs. By employing a model cell line that exclusively utilises integrin α2ß1, we found that whilst EDC crosslinking modulated cell binding, from cation-dependent to cation-independent, UV-mediated crosslinking preserved native-like cell binding, proliferation and surface colonisation. Similar results were observed using a purified recombinant I-domain from integrin α1. Conversely, binding of the I-domain from integrin α2 was sensitive to UV, particularly at low EDC concentrations. Therefore, from this in vitro study, it appears that UV can be used to augment EDC whist retaining a specific subset of integrin-binding motifs in the native collagen molecule. These findings, delineating the EDC- and UV-susceptibility of cell-binding motifs, permit controlled cell adhesion to collagen-based materials through specific integrin ligation in vitro. However, in vivo, further consideration of the potential response to UV wavelength and dose is required in the light of literature reports that UV initiated collagen scission may lead to an adverse inflammatory response. STATEMENT OF SIGNIFICANCE: Recently, there has been rapid growth in the use of extracellular matrix-derived molecules, and in particular collagen, to fabricate biomaterials that replicate the cellular micro-environment. Often chemical or physical crosslinkers are required to enhance the biophysical properties of these materials. Despite extensive use of these crosslinkers, the cell-biological consequences have not been ascertained. To address this, we have investigated the integrin-binding properties of collagen after chemically crosslinking with EDC and physically crosslinking with UV-irradiation. We have established that whilst EDC crosslinking abates all of the integrin binding sites in collagen, UV selectively inhibits interaction with integrin-α2 but not -α1. By providing a mechanistic model for this behaviour, we have, for the first time, defined a series of crosslinking parameters to systematically control the interaction of collagen-based materials with defined cellular receptors.
Sujet(s)
Matériaux biocompatibles/métabolisme , Carbodiimides/composition chimique , Collagène/métabolisme , Réactifs réticulants/composition chimique , Intégrine alpha2bêta1/métabolisme , Rayons ultraviolets , Animaux , Bovins , Adhérence cellulaire , Lignée cellulaire tumorale , Mouvement cellulaire , Prolifération cellulaire , Humains , Intégrine alpha2bêta1/composition chimique , Adhésivité plaquettaire , Liaison aux protéines , Domaines protéiquesRÉSUMÉ
Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2ß1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly â Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2ß1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2ß1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly â Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly â Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly â Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.
Sujet(s)
Substitution d'acide aminé , Collagène/composition chimique , Syndrome d'Ehlers-Danlos/génétique , Intégrine alpha2bêta1/métabolisme , Peptides/métabolisme , Sites de fixation , Adhérence cellulaire , Lignée cellulaire , Collagène/métabolisme , Humains , Intégrine alpha2bêta1/composition chimique , Intégrine alpha2bêta1/génétique , Cellules souches mésenchymateuses/métabolisme , Cellules souches mésenchymateuses/physiologie , Simulation de docking moléculaire , Peptides/composition chimique , Liaison aux protéinesRÉSUMÉ
Collagen is the most abundant protein in the animal kingdom and has a unique triple-helical structure. It not only provides mechanical strength to tissues, but also performs specific biological functions as a multifaceted signaling molecule. Animal-derived collagen is therefore widely used as a biocompatible material in vitro and in vivo. In this study, we developed a novel peptide-based material that mimicked both the polymeric properties and a selected biological function of native collagen. This material was prepared by end-to-end multiple disulfide cross-linking of chemically synthesized triple-helical peptides. The peptide polymer showed a gel-forming property, and receptor-specific cell binding was observed in vitro by incorporating a peptide harboring an integrin α2ß1-binding sequence. Furthermore, cell signaling activity and biodegradability were tunable according to the polymer contents. The results demonstrated the potential of this material as a designer collagen. STATEMENT OF SIGNIFICANCE: Collagen is a useful biomaterial with the gel-forming property. It also exhibits various biological activities through the interaction of specific amino acid sequences displayed on the triple helix with functional biomacromolecules. Here we report a novel synthetic material, artificial collagen, by end-to-end cross-linking of chemically synthesized collagen-like triple-helical peptides. The material allows independent regulation of polymer properties, i.e. gel stiffness, and sequence-specific bioactivities by altering peptide compositions. This material can also be variously shaped, for example, thin films with high transparency. In addition, it has low inflamatogenic properties and tunable biodegradability in vivo.
Sujet(s)
Matériaux biomimétiques/composition chimique , Collagène/composition chimique , Réactifs réticulants/composition chimique , Disulfures/composition chimique , Hydrogels/composition chimique , Oligopeptides/composition chimique , Séquence d'acides aminés , Animaux , Sites de fixation , Adhérence cellulaire , Lignée cellulaire , Prolifération cellulaire , Module d'élasticité , Matrice extracellulaire/métabolisme , Humains , Hydrogels/métabolisme , Intégrine alpha2bêta1/composition chimique , Mâle , Souris de lignée C57BL , Liaison aux protéines , Rhéologie , Propriétés de surfaceRÉSUMÉ
The collagen binding integrin α2ß1 plays a crucial role in hemostasis, fibrosis, and cancer progression amongst others. It is specifically inhibited by rhodocetin (RC), a C-type lectin-related protein (CLRP) found in Malayan pit viper (Calloselasma rhodostoma) venom. The structure of RC alone reveals a heterotetramer arranged as an αß and γδ subunit in a cruciform shape. RC specifically binds to the collagen binding A-domain of the integrin α2 subunit, thereby blocking collagen-induced platelet aggregation. However, until now, the molecular basis for this interaction has remained unclear. Here, we present the molecular structure of the RCγδ-α2A complex solved to 3.0 Å resolution. Our findings show that RC undergoes a dramatic structural reorganization upon binding to α2ß1 integrin. Besides the release of the nonbinding RCαß tandem, the RCγ subunit interacts with loop 2 of the α2A domain as result of a dramatic conformational change. The RCδ subunit contacts the integrin α2A domain in the "closed" conformation through its helix C. Combined with epitope-mapped antibodies, conformationally locked α2A domain mutants, point mutations within the α2A loop 2, and chemical modifications of the purified toxin protein, this molecular structure of RCγδ-α2A complex explains the inhibitory mechanism and specificity of RC for α2ß1 integrin.
Sujet(s)
Venins de crotalidé/composition chimique , Intégrine alpha2bêta1/composition chimique , Venins de crotalidé/pharmacologie , Cristallographie aux rayons X , Intégrine alpha2bêta1/antagonistes et inhibiteurs , Modèles moléculaires , Liaison aux protéines , Structure tertiaire des protéinesRÉSUMÉ
Conformational activation of integrins is generally required for ligand binding and cellular signalling. However, we have previously reported that the nonactivated conformation of α2ß1 integrin can also bind to large ligands, such as human echovirus 1. In this study, we show that the interaction between the nonactivated integrin and a ligand resulted in the activation of focal adhesion kinase (FAK) in a protein kinase C dependent manner. A loss-of-function mutation, α2E336A, in the α2-integrin did not prevent the activation of FAK, nor did EDTA-mediated inactivation of the integrin. Full FAK activation was observed, since phosphorylation was not only confirmed in residue Y397, but also in residues Y576/7. Furthermore, initiation of downstream signaling by paxillin phosphorylation in residue Y118 was evident, even though this activation was transient by nature, probably due to the lack of talin involvement in FAK activation and the absence of vinculin in the adhesion complexes formed by the nonactivated integrins. Altogether these results indicate that the nonactivated integrins can induce cellular signaling, but the outcome of the signaling differs from conventional integrin signaling.
Sujet(s)
Focal adhesion protein-tyrosine kinases/métabolisme , Intégrine alpha2bêta1/métabolisme , Transduction du signal , Cellules HeLa , Humains , Intégrine alpha2bêta1/composition chimique , Conformation des protéinesRÉSUMÉ
Interactions of cells with supramolecular aggregates of the extracellular matrix (ECM) are mediated, in part, by cell surface receptors of the integrin family. These are important molecular components of cell surface-suprastructures regulating cellular activities in general. A subfamily of ß1-integrins with von Willebrand-factor A-like domains (I-domains) in their α-chains can bind to collagen molecules and, therefore, are considered as important cellular mechano-receptors. Here we show that chondrocytes strongly bind to cartilage collagens in the form of individual triple helical molecules but very weakly to fibrils formed by the same molecules. We also find that chondrocyte integrins α1ß1-, α2ß1- and α10ß1-integrins and their I-domains have the same characteristics. Nevertheless we find integrin binding to mechanically generated cartilage fibril fragments, which also comprise peripheral non-collagenous material. We conclude that cell adhesion results from binding of integrin-containing adhesion suprastructures to the non-collagenous fibril periphery but not to the collagenous fibril cores. The biological importance of the well-investigated recognition of collagen molecules by integrins is unknown. Possible scenarios may include fibrillogenesis, fibril degradation and/or phagocytosis, recruitment of cells to remodeling sites, or molecular signaling across cytoplasmic membranes. In these circumstances, collagen molecules may lack a fibrillar organization. However, other processes requiring robust biomechanical functions, such as fibril organization in tissues, cell division, adhesion, or migration, do not involve direct integrin-collagen interactions.
Sujet(s)
Chondrocytes/physiologie , Collagènes fibrillaires/composition chimique , Intégrines alpha/composition chimique , Intégrine alpha1 bêta1/composition chimique , Intégrine alpha2bêta1/composition chimique , Animaux , Cartilage articulaire/cytologie , Bovins , Adhérence cellulaire , Cellules cultivées , Embryon de poulet , Récepteurs à domaine discoïdine/physiologie , Collagènes fibrillaires/physiologie , Humains , Protéines immobilisées/composition chimique , Intégrines alpha/physiologie , Intégrine alpha1 bêta1/physiologie , Intégrine alpha2bêta1/physiologie , Liaison aux protéinesRÉSUMÉ
The replacement of one Gly in the essential repeating tripeptide sequence of the type I collagen triple helix results in the dominant hereditary bone disorder osteogenesis imperfecta. The mechanism leading to pathology likely involves misfolding and autophagy, although it has been hypothesized that some mutations interfere with known collagen interactions. Here, the effect of Gly replacements within and nearby the integrin binding GFPGER sequence was investigated using a recombinant bacterial collagen system. When a six-triplet human type I collagen sequence containing GFPGER was introduced into a bacterial collagen-like protein, this chimeric protein bound to integrin. Constructs with Gly to Ser substitutions within and nearby the inserted human sequence still formed a trypsin-resistant triple helix, suggesting a small local conformational perturbation. Gly to Ser mutations within the two Gly residues in the essential GFPGER sequence prevented integrin binding and cell attachment as predicted from molecular dynamics studies of the complex. Replacement of Gly residues C-terminal to GFPGER did not affect integrin binding. In contrast, Gly replacements N-terminal to the GFPGER sequence, up to four triplets away, decreased integrin binding and cell adhesion. This pattern suggests either an involvement of the triplets N-terminal to GFPGER in initial binding or a propagation of the perturbation of the triple helix C-terminal to a mutation site. The asymmetry in biological consequences relative to the mutation site may relate to the observed pattern of osteogenesis imperfecta mutations near the integrin binding site.
Sujet(s)
Collagène de type I/composition chimique , Intégrine alpha2bêta1/composition chimique , Substitution d'acide aminé , Collagène de type I/génétique , Collagène de type I/métabolisme , Humains , Intégrine alpha2bêta1/génétique , Intégrine alpha2bêta1/métabolisme , Mutation faux-sens , Liaison aux protéines , Structure secondaire des protéinesRÉSUMÉ
Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2ß1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2ß1 and α1ß1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons.
Sujet(s)
Collagène de type I/composition chimique , Intégrine alpha1 bêta1/composition chimique , Intégrine alpha2bêta1/composition chimique , Peptides/composition chimique , Sites de fixation , Adhérence cellulaire , Lignée cellulaire tumorale , Diazo-méthane/pharmacologie , 1-Éthyl-3-(3-diméthylaminopropyl)carbodiimide/composition chimique , Humains , Liaison aux protéines , Succinimides/composition chimique , Structures d'échafaudage tissulaires/composition chimiqueRÉSUMÉ
Cells inside a 3D matrix (such as tissue extracellular matrix or biomaterials) sense their insoluble environment through specific binding interactions between their adhesion receptors and ligands present on the matrix surface. Despite the critical role of the insoluble matrix in cell regulation, there exist no widely-applicable methods for quantifying the chemical stimuli provided by a matrix to cells. Here, we describe a general-purpose technique for quantifying in situ the density of ligands for specific cell adhesion receptors of interest on the surface of a 3D matrix. This paper improves significantly the accuracy of the procedure introduced in a previous publication by detailed marker characterization, optimized staining, and improved data interpretation. The optimized methodology is utilized to quantify the ligands of integrins α 1 ß 1, α 2 ß 1 on two kinds of matched porous collagen scaffolds, which are shown to possess significantly different ligand density, and significantly different ability to induce peripheral nerve regeneration in vivo. Data support the hypothesis that cell adhesion regulates contractile cell phenotypes, recently shown to be inversely related to organ regeneration. The technique provides a standardized way to quantify the surface chemistry of 3D matrices, and a means for introducing matrix effects in quantitative biological models.
Sujet(s)
Matériaux biocompatibles/composition chimique , Collagène/composition chimique , Intégrine alpha1 bêta1/composition chimique , Intégrine alpha2bêta1/composition chimique , Structures d'échafaudage tissulaires/composition chimique , Animaux , Femelle , Porosité , Rats , Rats de lignée LEWRÉSUMÉ
Targeting nanoparticles to desired intracellular compartments is a major challenge. Integrin-type adhesion receptors are connected to different endocytosis routes in a receptor-specific manner. According to our previous observations, the internalization of an α2ß1-integrin-echovirus-1 complex takes place via a macropinocytosis-like mechanism, suggesting that the receptor could be used to target nanoparticles to this specific entry route. Here, silica-based nanoparticles, carrying monoclonal antibodies against the α2ß1 integrin as address labels, were synthesized. Studies with flow cytometry, atomic force microscopy and confocal microscopy showed the particles to attach to the cell surface via the α2ß1 integrin. Furthermore, quantitative analysis of nanoparticle trafficking inside the cell performed with the BioImageXD software indicated that the particles enter cells via a macropinocytosis-like process and end up in caveolin-1 positive structures. Thus, we suggest that different integrins can guide particles to distinct endocytosis routes and, subsequently, also to specific intracellular compartments. In addition, we show that with the BioImageXD software it is possible to conduct sensitive and complex analyses of the behavior of small fluorescent particles inside cells, using basic confocal microscopy images.
Sujet(s)
Intégrine alpha2bêta1/composition chimique , Nanoparticules/composition chimique , Anticorps immobilisés/composition chimique , Anticorps immobilisés/métabolisme , Anticorps monoclonaux/composition chimique , Anticorps monoclonaux/immunologie , Cavéoline-1/métabolisme , Lignée cellulaire tumorale , Endocytose , Entérovirus humain B/génétique , Entérovirus humain B/métabolisme , Humains , Immunohistochimie , Intégrine alpha2bêta1/immunologie , Intégrine alpha2bêta1/métabolisme , Microscopie à force atomique , Microscopie confocale , Silice/composition chimiqueRÉSUMÉ
Secreted protein components of hookworm species include a number of representatives of the cysteine-rich/antigen 5/pathogenesis-related 1 (CAP) protein family known as Ancylostoma-secreted proteins (ASPs). Some of these have been considered as candidate antigens for the development of vaccines against hookworms. The functions of most CAP superfamily members are poorly understood, but one form, the hookworm platelet inhibitor (HPI), has been isolated as a putative antagonist of the platelet integrins αIIbß3 and α2ß1. Here, the crystal structure of HPI is described and its structural features are examined in relation to its possible function. The HPI structure is similar to those of other ASPs and shows incomplete conservation of the sequence motifs CAP1 and CAP2 that are considered to be diagnostic of CAP superfamily members. The asymmetric unit of the HPI crystal contains a dimer with an extensive interaction interface, but chromatographic measurements indicate that it is primarily monomeric in solution. In the dimeric structure, the putative active-site cleft areas from both monomers are united into a single negatively charged depression. A potential Lys-Gly-Asp disintegrin-like motif was identified in the sequence of HPI, but is not positioned at the apex of a tight turn, making it unlikely that it interacts with the integrin. Recombinant HPI produced in Escherichia coli was found not to inhibit the adhesion of human platelets to collagen or fibrinogen, despite having a native structure as shown by X-ray diffraction. This result corroborates previous analyses of recombinant HPI and suggests that it might require post-translational modification or have a different biological function.
Sujet(s)
Ancylostoma/composition chimique , Plaquettes/effets des médicaments et des substances chimiques , Adhérence cellulaire/effets des médicaments et des substances chimiques , Protéines d'helminthes/composition chimique , Motifs d'acides aminés , Ancylostoma/métabolisme , Animaux , Plaquettes/composition chimique , Domaine catalytique , Collagène/composition chimique , Collagène/métabolisme , Cristallographie aux rayons X , Escherichia coli/génétique , Escherichia coli/métabolisme , Fibrinogène/composition chimique , Fibrinogène/métabolisme , Expression des gènes , Protéines d'helminthes/génétique , Protéines d'helminthes/métabolisme , Protéines d'helminthes/pharmacologie , Humains , Liaison hydrogène , Intégrine alpha2bêta1/composition chimique , Intégrine alpha2bêta1/métabolisme , Modèles moléculaires , Données de séquences moléculaires , Complexe glycoprotéique IIb-IIIa de la membrane plaquettaire/composition chimique , Complexe glycoprotéique IIb-IIIa de la membrane plaquettaire/métabolisme , Multimérisation de protéines , Protéines recombinantes/composition chimique , Protéines recombinantes/génétique , Protéines recombinantes/métabolisme , Alignement de séquences , Similitude de séquences d'acides aminés , Électricité statiqueRÉSUMÉ
The plasma protein histidine-rich glycoprotein (HRG) affects the morphology and function of both endothelial cells (ECs) and monocytes/macrophages in cancer. Here, we examined the mechanism of action of HRG's effect on ECs. HRG suppressed adhesion, spreading and migration of ECs specifically on collagen I (COL I) whereas ECs seeded on other extracellular matrix proteins were insensitive to HRG. HRG did not bind specifically to COL I or to the α-integrin binding site on collagen, GFOGER. Furthermore, HRG's inhibition of EC adhesion was not dependent upon heparan sulfate (HS) moieties as heparitinase-treated ECs remained sensitive to HRG. C2C12 cells expressing α2 integrin, the major collagen-binding α-integrin subunit in ECs, showed increased binding of HRG compared with wild type C2C12 cells lacking the α2 subunit. Recombinant α2 I-domain protein bound HRG and to a higher extent when in active conformation. However, the α2 I-domain bound weakly to HRG compared with COL I and the purified α2ß1 ectodomain complex failed to retain HRG. We conclude that HRG binds to α2 integrin through low-affinity interactions in a HS-independent manner, thereby blocking EC-adhesion to COL I.
Sujet(s)
Héparitine sulfate/composition chimique , Intégrine alpha2bêta1/composition chimique , Sous-unités de protéines/composition chimique , Protéines/composition chimique , Animaux , Adhérence cellulaire , Lignée cellulaire , Chimiotaxie/effets des médicaments et des substances chimiques , Femelle , Expression des gènes , Héparitine sulfate/métabolisme , Cellules endothéliales de la veine ombilicale humaine , Humains , Intégrine alpha2bêta1/génétique , Intégrine alpha2bêta1/métabolisme , Souris , Souris de lignée C57BL , Myoblastes/cytologie , Myoblastes/effets des médicaments et des substances chimiques , Myoblastes/métabolisme , Liaison aux protéines , Structure tertiaire des protéines , Sous-unités de protéines/génétique , Sous-unités de protéines/métabolisme , Protéines/génétique , Protéines/métabolisme , Protéines recombinantes/composition chimique , Protéines recombinantes/génétique , Protéines recombinantes/métabolisme , Facteur de croissance endothéliale vasculaire de type A/pharmacologieRÉSUMÉ
The ability to early detect and assess the treatment response of recurrent and/or disseminated metastatic glioblastoma is critical for the effective management of this group of patients. Accumulating experimental evidence indicates that integrin α2ß1 might be a prognostic biomarker for advanced phenotype of cancers. In this study, a novel (68)Ga-labeled integrin α2ß1-targeted PET tracer (68)Ga-NOTA-PEG4-cyclo (GDGEAyK) ((68)Ga-A2B1) was designed and evaluated for the potential prognostic imaging of glioblastoma tumor in preclinical model. To prospectively verify the prognostic value of integrin α2ß1, the in vitro Western blot and flow cytometry studies were performed to validate the integrin expression level of human glioblastoma (U87MG) cells. Extremely high expression level of integrin α2ß1 justifies its role as a potential targeting marker. Thus, (68)Ga-A2B1 positron emission tomography was performed in subcutaneous U87MG tumor bearing athymic mice at 15 min postinjection after injection of 7-8MBq tracers. The receptor targeting specificity was confirmed in a competition blocking experiment. The tumor uptake of (68)Ga-A2B1 in the control and blockage groups was 1.57 ± 0.13 %ID/g (n = 3) and 0.96 ± 0.23 %ID/g** (n = 3), respectively. However, because of the quick renal washout rate and labile nature of peptide tracers in circulation conditions, the focus ultrasound (FUS) mediated delivery method was adopted to enhance tumor uptake and retention of tracers. To test the FUS delivery efficacy in vivo, three experimental arms were designed as follows: tumor bearing mice were administrated with (68)Ga-A2B1 only or microbubbles (MBs) with FUS treatment ((68)Ga-A2B1 + FUS + MBs) or embedded (68)Ga-A2B1-microbubbles ((68)Ga-A2B1-MBs + FUS) followed with FUS sonication. The average radioactivity accumulation within a tumor was quantified from the multiple region of interest volumes using the %ID/g value and was analyzed in accordance with the ex vivo autoradiographic and pathologic data. The significant tumor uptake in (68)Ga-A2B1 + FUS +MBs group (n = 6) and (68)Ga-A2B1-MBs + FUS group (n = 4) following FUS treatment were calculated as 2.25 ± 0.50 %ID/g* and 2.6 ± 0.49 %ID/g**, comparing with (68)Ga-A2B1 only group 1.48 ± 0.42 %ID/g (n = 10). These results suggest that there is significant difference in (68)Ga-A2B1 tumor uptake by FUS treatment either with or without tracer integration with microbubbles, which demonstrate a promising delivery strategy and critical multimodal setting for phenotyping imaging of aggressive glioma tumor. In conclusion, (68)Ga labeled (68)Ga-A2B1 allows noninvasive imaging of tumor-associated α2ß1 expression and can be embedded in MB lipid shell for enhanced delivery and controlled release by sonoporation.
Sujet(s)
Tumeurs du cerveau/anatomopathologie , Encéphale/imagerie diagnostique , Gliome/imagerie diagnostique , Intégrine alpha2bêta1/composition chimique , Radiopharmaceutiques , Animaux , Fixation compétitive , Tumeurs du cerveau/imagerie diagnostique , Lignée cellulaire tumorale , Chromatographie en phase liquide à haute performance , Évolution de la maladie , Électroencéphalographie , Cellules HEK293 , Humains , Mâle , Souris , Transplantation tumorale , Peptides/composition chimique , Phénotype , Tomographie par émission de positons , Distribution tissulaire , ÉchographieRÉSUMÉ
Multifunctional nanoprobes open exciting possibilities for accurate diagnosis and therapy. In this research, we developed a (64)Cu-labeled GdVO4:4%Eu two-dimension (2D) tetragonal ultrathin nanosheets (NSs) that simultaneously possess radioactivity, fluorescence, and paramagnetic properties for multimodal imaging. The carboxyl-functionalized Eu(3+)-doped GdVO4 NSs were synthesized by a facile solvothermal reaction, followed by ligand exchange with polyacrylic acid (PAA). With ultrathin thickness of â¼5 nm and width of â¼150 nm, the carboxyl-functionalized NSs were further modified by DOTA chelator for (64)Cu labeling and Asp-Gly-Glu-Ala (DGEA) peptide for integrin α2ß1 targeting. After initial evaluation of the cytotoxicity and targeting capability with PC-3 cells, the obtained multifunctional nanoprobes ((64)Cu-DOTA-GdVO4:4%Eu-DGEA) were further explored for targeted positron emission tomography (PET) and T1-weighted magnetic resonance imaging (MRI) of PC-3 tumor (prostate cancer, high integrin α2ß1 expression) in vivo. Based on the strong fluorescence of the NSs, the particle distribution in mouse tissues was also determined by fluorescent microscopy. In summary, GdVO4:4%Eu NS is a potential multimodal multiscale nanoprobe that could not only be used for in vivo imaging, but also be tracked in cellular scale and ex vivo due to its fluorescent property.
Sujet(s)
Europium , Gadolinium , Intégrine alpha2bêta1/composition chimique , Intégrine alpha2bêta1/métabolisme , Imagerie par résonance magnétique , Imagerie multimodale , Nanoparticules , Tomographie par émission de positons , Vanadates , Animaux , Mort cellulaire , Lignée cellulaire tumorale , Survie cellulaire , Composés hétéromonocycliques/synthèse chimique , Composés hétéromonocycliques/composition chimique , Humains , Mâle , Souris nude , Nanoparticules/ultrastructure , Acide oléique/synthèse chimique , Acide oléique/composition chimique , Oligopeptides/synthèse chimique , Oligopeptides/composition chimique , Spectrométrie de fluorescence , Spectroscopie infrarouge à transformée de Fourier , Distribution tissulaire , Diffraction des rayons X , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
The α2ß1 integrin, also known as VLA-2, GPIa-IIa, CD49b, was first identified as an extracellular matrix receptor for collagens and/or laminins [55, 56]. It is now recognized that the α2ß1 integrin serves as a receptor for many matrix and nonmatrix molecules [35, 79, 128]. Extensive analyses have clearly elucidated the α2 I domain structural motifs required for ligand binding, and also defined distinct conformations that lead to inactive, partially active or highly active ligand binding [3, 37, 66, 123, 136, 137, 140]. The mechanisms by which the α2ß1 integrin plays a critical role in platelet function and homeostasis have been carefully defined via in vitro and in vivo experiments [76, 104, 117, 125]. Genetic and epidemiologic studies have confirmed human physiology and disease states mediated by this receptor in immunity, cancer, and development [6, 20, 21, 32, 43, 90]. The role of the α2ß1 integrin in these multiple complex biologic processes will be discussed in the chapter.
Sujet(s)
Intégrine alpha2bêta1/physiologie , Animaux , Hémostase , Humains , Immunité innée , Intégrine alpha2bêta1/composition chimique , Néovascularisation physiologique , Structure tertiaire des protéines , Transduction du signal , Cicatrisation de plaieRÉSUMÉ
Platelet adhesion on a collagen surface through integrin α2ß1 has been proven to be significant for the formation of arterial thrombus. However, the molecular determinants mediating the integrin-collagen complex remain unclear. In the present study, the dynamics of integrin-collagen binding and molecular interactions were investigated using molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) analysis. Hydrophobic interaction is identified as the major driving force for the formation of the integrin-collagen complex. On the basis of the MD simulation and MM-PBSA results, an affinity binding model (ABM) of integrin for collagen is constructed; it is composed of five residues, including Y157, N154, S155, R288, and L220. The ABM has been proven to capture the major binding motif contributing 84.8% of the total binding free energy. On the basis of the ABM, we expect to establish a biomimetic design strategy of platelet adhesion inhibitors, which would be beneficial for the development of potent peptide-based drugs for thrombotic diseases.
Sujet(s)
Biomimétique/méthodes , Protéines du sang/composition chimique , Collagène/composition chimique , Intégrine alpha2bêta1/composition chimique , Interactions hydrophobes et hydrophiles , Simulation de dynamique moléculaire , Liaison aux protéinesRÉSUMÉ
Platelet adhesion on collagen mediated by integrin α2ß1 has been proven important in arterial thrombus formation, leading to an exigent demand on development of potent inhibitors for the integrin α2ß1-collagen binding. In the present study, a biomimetic design strategy of platelet adhesion inhibitors was established, based on the affinity binding model of integrin proposed in part I. First, a heptapeptide library containing 8000 candidates was designed to functionally mimic the binding motif of integrin α2ß1. Then, each heptapeptide in the library was docked onto a collagen molecule for the assessment of its affinity, followed by a screening based on its structure similarity to the original structure in the affinity binding model. Eight candidates were then selected for further screening by molecular dynamics (MD) simulations. Thereafter, three candidates chosen from MD simulations were separately added into the physiological saline containing separated integrin and collagen, to check their abilities for blocking the integrin-collagen interaction using MD simulations. Of these three candidates, significant inhibition was observed in the presence of LWWNSYY. Finally, the binding affinity of LWWNSYY for collagen was demonstrated by isothermal titration calorimetry. Moreover, significant inhibition of platelet adhesion in the presence of LWWNSYY has been experimentally validated. This work has thus developed an effective strategy for the biomimetic design of peptide-based platelet adhesion inhibitors.
Sujet(s)
Biomimétique/méthodes , Protéines du sang/composition chimique , Collagène/composition chimique , Intégrine alpha2bêta1/composition chimique , Interactions hydrophobes et hydrophiles , Simulation de dynamique moléculaire , Liaison aux protéinesRÉSUMÉ
Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2ß1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential.
Sujet(s)
Protéine morphogénétique osseuse de type 2/génétique , Régénération osseuse/effets des médicaments et des substances chimiques , Techniques de transfert de gènes , Hydrogels/pharmacologie , Intégrine alpha2bêta1/composition chimique , Animaux , Protéine morphogénétique osseuse de type 2/métabolisme , Régénération osseuse/physiologie , Os et tissu osseux/cytologie , Os et tissu osseux/effets des médicaments et des substances chimiques , Différenciation cellulaire/effets des médicaments et des substances chimiques , Collagène/composition chimique , Collagène/pharmacologie , Humains , Hydrogels/composition chimique , Mâle , Cellules souches mésenchymateuses , Souris , Ostéogenèse/effets des médicaments et des substances chimiques , Peptides/composition chimique , Peptides/pharmacologie , Polyéthylène glycols/composition chimique , Polyéthylène glycols/pharmacologie , Protéines recombinantes/génétique , Protéines recombinantes/métabolisme , Ingénierie tissulaire , Cicatrisation de plaie/effets des médicaments et des substances chimiquesRÉSUMÉ
BACKGROUND: Antibodies against human platelet antigens (HPA) are a cause of thrombocytopenia. Detection of rare anti-HPA antibodies using platelet preparations is difficult and would be improved by an alternative method that does not require platelets. In the present study, we describe the establishment of cell lines that stably express specific HPA associated with integrin α2ß1 and the application of these cell lines for detecting anti-HPA-5a and anti-HPA-5b antibodies. MATERIALS AND METHODS: Complementary DNA of the integrin α2 variants HPA-5b, -13b and -18b were individually transfected into K562 cells using retroviral vectors. Expression of integrin α2 was confirmed by flow cytometric analysis, immunoprecipitation and western blotting analysis. To verify whether the cell line panel was suitable for clinical diagnosis, we analysed its properties using monoclonal antibody-specific immobilisation of platelet antigens (MAIPA) and well-characterised serum samples. RESULTS: Exogenous integrin α2 expression was observed in the transfected cells for over 6 months. The cell line panel specifically detected previously characterised anti-HPA-5a and anti-HPA-5b antisera. No reactivity was observed with control sera, including normal sera and HLA antisera. DISCUSSION: We successfully established a cell line panel to facilitate the sensitive and reliable detection of anti-HPA-5a and anti-HPA-5b antibodies.