RESUMO
The major fibronectin (FN)-binding α5ß1 and αvß3 integrins exhibit cooperativity during cell adhesion, migration and mechanosensing, through mechanisms that are not yet fully resolved. Exploiting mechanically tunable nano-patterned substrates, and peptidomimetic ligands designed to selectively bind corresponding integrins, we report that focal adhesions (FAs) of endothelial cells assembled on α5ß1 integrin-selective substrates rapidly recruit αvß3 integrins, but not vice versa. Blocking of αvß3 integrin hindered FA maturation and cell spreading on α5ß1 integrin-selective substrates, indicating a mechanism dependent on extracellular ligand binding and highlighting the requirement of αvß3 integrin engagement for efficient adhesion. Recruitment of αvß3 integrins additionally occurred on hydrogel substrates of varying mechanical properties, above a threshold stiffness that supports FA formation. Mechanistic studies revealed the need for soluble factors present in serum to allow recruitment, and excluded exogenous, or endogenous, FN as the ligand responsible for αvß3 integrin accumulation to adhesion clusters. Our findings highlight a novel mechanism of integrin cooperation and a critical role for αvß3 integrins in promoting cell adhesion on α5ß1 integrin-selective substrates.
Assuntos
Adesões Focais/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , HumanosRESUMO
Platelets play a major role in hemostasis and thrombosis, by binding to the underlying extracellular matrix around injured blood vessels, via integrin receptors. In this study, we investigated the effects of adhesive ligand spacing on the stability of platelets' adhesion and the mode of their spreading on extracellular surfaces. Toward this end, we have examined the differential adhesion and spreading of human platelets onto nanogold-patterned surfaces, functionalized with the αIIbß3 integrin ligand, SN528. Combining light- and scanning electron-microscopy, we found that interaction of platelets with surfaces coated with SN528 at spacing of 30-60 nm induces the extension of filopodia through which the platelets stably attach to the nanopatterned surface and spread on it. Increasing the nanopattern-gold spacing to 80-100 nm resulted in a dramatic reduction (>95%) in the number of adhering platelets. Surprisingly, a further increase in ligand spacing to 120 nm resulted in platelet binding to the surface at substantially larger numbers, yet these platelets remained discoid and were essentially devoid of filopodia and lamellipodia. These results indicate that the stimulation of filopodia extension by adhering platelets, and the consequent spreading on these surfaces depend on different ligand densities. Thus, the extension of filopodia occurs on surfaces with a ligand spacing of 100 nm or less, while the sustainability and growth of these initial adhesions and induction of extensive platelet adhesion and spreading requires lower ligand-to-ligand spacing (≤60 nm). The mechanisms underlying this differential ligand-density sensing by platelets, as well as the unexpected retention of discoid platelets on surfaces with even larger spacing (120 nm) are discussed.
RESUMO
Adoptive cell therapies are showing very promising results in the fight against cancer. However, these therapies are expensive and technically challenging in part due to the need of a large number of specific T cells, which must be activated and expanded in vitro. Here we describe a method to activate primary human T cells using a combination of nanostructured surfaces functionalized with the stimulating anti-CD3 antibody and the peptidic sequence arginine-glycine-aspartic acid, as well as costimulatory agents (anti-CD28 antibody and a cocktail of phorbol 12-myristate 13-acetate, ionomycin, and protein transport inhibitors). Thus, we propose a method that combines nanotechnology with cell biology procedures to efficiently produce T cells in the laboratory, challenging the current state-of-the-art expansion methodologies.
Assuntos
Materiais Revestidos Biocompatíveis/química , Ativação Linfocitária , Nanoestruturas/química , Linfócitos T/imunologia , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Adesão Celular , Células Cultivadas , Ouro/química , Humanos , Imunoterapia Adotiva , Ionomicina/química , Ionomicina/imunologia , Nanoestruturas/ultraestrutura , Oligopeptídeos/química , Oligopeptídeos/imunologia , Propriedades de Superfície , Linfócitos T/citologia , Acetato de Tetradecanoilforbol/química , Acetato de Tetradecanoilforbol/imunologia , Titânio/químicaRESUMO
PURPOSE: Integrins are transmembrane receptors responsible for cell-cell adhesion and cell-extracellular matrix binding and play an important role in angiogenesis and tumour metastasis. For this reason, integrins are increasingly used as targets for molecular imaging. Up to now interest has mostly been focused on the integrin subtype αvß3. However, targeting of other subtypes such as the integrin α5ß1 is also of high interest due to its central role in colonization of metastatic cells, resistance of tumour cells to chemotherapy and ionizing radiation, and tumour aggressiveness. Recently, a highly active antagonist ligand (2,2'-(7-(1-carboxy-4-((6-((3-(4-(((S)-1-carboxy-2-(2-(3-guanidinobenzamido)acetamido)ethyl)carbamoyl)-3,5-dimethylphenoxy)propyl)amino)-6-oxohexyl)amino)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, FR366) for the integrin subtype α5ß1 with high selectivity versus αvß3, has been developed and tested successfully in preliminary in vitro and in vivo experiments. Here, we present our results of an investigation of the use of (68)Ga-labelled α5ß1 ligand in PET imaging. METHODS: The free α5ß1 peptidomimetic ligand was functionalized with a spacer (6-aminohexanoic acid) and the bifunctional chelator 1-((1,3-dicarboxy)propyl)-4,7-(carboxymethyl)-1,4,7-triazacyclononane (NODAGA) to yield FR366 and labelled with (68)Ga. To confirm selective in vivo targeting of α5ß1, female BALB/c nude mice xenografted with α5ß1-expressing RKO cells in the right shoulder and α5ß1/αvß3-expressing M21 cells in the left shoulder were subjected to PET/CT scans and biodistribution experiments. Specificity of tracer uptake was proven by blocking studies. Metabolic stability of the injected tracer was measured in urine and in plasma. RESULTS: MicroPET/CT scans with radiolabelled FR366 showed a good tumour-to-normal tissue ratio with low uptake in the liver (0.32 ± 0.14 %ID/g) and good retention of (68)Ga-NODAGA-FR366 in the tumour (0.71 ± 0.20 %ID/g and 0.40 ± 0.12 %ID/g for RKO and M21 tumours, respectively, at 90 min after injection). Biodistribution experiments showed uptake in the α5ß1-expressing RKO tumour of 1.05 ± 0.23 %ID/g at 90 min after injection. Specificity of tracer uptake was demonstrated by injection of 5 mg/kg unlabelled ligand 10 min prior to tracer injection, resulting in a 67 % reduction in uptake in the RKO tumour. The tracer was found to be metabolically stable in urine and plasma 30 min after injection. CONCLUSION: Our results show that PET imaging of α5ß1 expression with the (68)Ga-labelled α5ß1-specific ligand is feasible with good image quality. Thus, FR366 is a promising new tool for investigating the role of α5ß1 in angiogenesis and the influence of this integrin subtype on cancer aggressiveness and metastatic potential.
Assuntos
Radioisótopos de Gálio/farmacocinética , Guanidinas/farmacocinética , Integrina alfa5beta1/metabolismo , Peptidomiméticos/farmacocinética , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/farmacocinética , Triazinas/farmacocinética , Animais , Linhagem Celular Tumoral , Feminino , Guanidinas/química , Guanidinas/farmacologia , Humanos , Integrina alfa5beta1/antagonistas & inibidores , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ligação Proteica , Distribuição Tecidual , Triazinas/química , Triazinas/farmacologiaRESUMO
Engineering biomaterials with integrin-binding activity is a very powerful approach to promote cell adhesion, modulate cell behavior, and induce specific biological responses at the surface level. The aim of this Review is to illustrate the evolution of surface-coating molecules in this field: from peptides and proteins with relatively low integrin-binding activity and receptor selectivity to highly active and selective peptidomimetic ligands. In particular, we will bring into focus the difficult challenge of achieving selectivity between the two closely related integrin subtypes αvß3 and α5ß1. The functionalization of surfaces with such peptidomimetics opens the way for a new generation of highly specific cell-instructive surfaces to dissect the biological role of integrin subtypes and for application in tissue engineering and regenerative medicine.
Assuntos
Materiais Revestidos Biocompatíveis/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Peptidomiméticos/metabolismo , Animais , Adesão Celular , Materiais Revestidos Biocompatíveis/química , Humanos , Ligantes , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Peptidomiméticos/química , Ligação Proteica , Propriedades de SuperfícieRESUMO
BACKGROUND: Integrins are extracellular matrix receptors involved in several pathologies. Despite homologies between the RGD-binding α5ß1 and αvß3 integrins, selective small antagonists for each heterodimer have been proposed. Herein, we evaluated the effects of such small antagonists in a cellular context, the U87MG cell line, which express both integrins. The aim of the study was to determine if fibronectin-binding integrin antagonists are able to impact on cell adhesion and migration in relationships with their defined affinity and selectivity for α5ß1 and αvß3/ß5 purified integrins. METHODS: Small antagonists were either selective for α5ß1 integrin, for αvß3/ß5 integrin or non-selective. U87MG cell adhesion was evaluated on fibronectin or vitronectin. Migration assays included wound healing recovery and single cell tracking experiments. U87MG cells stably manipulated for the expression of α5 integrin subunit were used to explore the impact of α5ß1 integrin in the biological assays. RESULTS: U87MG cell adhesion on fibronectin or vitronectin was respectively dependent on α5ß1 or αvß3/ß5 integrin. Wound healing migration was dependent on both integrins. However U87MG single cell migration was highly dependent on α5ß1 integrin and was inhibited selectively by α5ß1 integrin antagonists but increased by αvß3/ß5 integrin antagonists. CONCLUSIONS: We provide a rationale for testing new integrin ligands in a cell-based assay to characterize more directly their potential inhibitory effects on integrin cellular functions. GENERAL SIGNIFICANCE: Our data highlight a single cell tracking assay as a powerful cell-based test which may help to characterize true functional integrin antagonists that block α5ß1 integrin-dependent cell migration.
Assuntos
Antineoplásicos , Glioma/tratamento farmacológico , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfaVbeta3/antagonistas & inibidores , Cadeias beta de Integrinas , Proteínas de Neoplasias/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Ensaios de Seleção de Medicamentos Antitumorais , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Humanos , Integrina alfa5beta1/biossíntese , Integrina alfa5beta1/genética , Integrina alfaVbeta3/biossíntese , Integrina alfaVbeta3/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genéticaRESUMO
To shed light on cell-adhesion-related molecular pathways, synthetic cells offer the unique advantage of a well-controlled model system with reduced molecular complexity. Herein, we show that liposomes with the reconstituted platelet integrin αIIb ß3 as the adhesion-mediating transmembrane protein are a functional minimal cell model for studying cellular adhesion mechanisms in a defined environment. The interaction of these synthetic cells with various extracellular matrix proteins was analyzed using a quartz crystal microbalance with dissipation monitoring. The data indicated that integrin was functionally incorporated into the lipid vesicles, thus enabling integrin-specific adhesion of the engineered liposomes to fibrinogen- and fibronectin-functionalized surfaces. Then, we were able to initiate the detachment of integrin liposomes from these surfaces in the presence of the peptide GRGDSP, a process that is even faster with our newly synthesized peptide mimetic SN529, which specifically inhibits the integrin αIIb ß3 .
Assuntos
Células Artificiais/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Adesão Celular , Estrutura Molecular , Técnicas de Microbalança de Cristal de QuartzoRESUMO
We present a click chemistry-based molecular toolkit for the biofunctionalization of materials to selectively control integrin-mediated cell adhesion. To this end, α5ß1-selective RGD peptidomimetics were covalently immobilized on Ti-based materials, and the capacity to promote the selective binding of α5ß1 was evaluated using a solid-phase integrin binding assay. This functionalization strategy yielded surfaces with a nine-fold increased affinity for α5ß1, in comparison to control samples, and total selectivity against the binding of the closely related integrin αvß3. Moreover, our methodology allowed the screening of several phosphonic acid containing anchoring units to find the best spacer-anchor moiety required for establishing an efficient binding to titanium and to promote selective integrin binding. The integrin subtype specificity of these biofunctionalized surfaces was further examined in vitro by inducing selective adhesion of genetically modified fibroblasts, which express exclusively the α5ß1 integrin. The versatility of our molecular toolkit was proven by shifting the cellular specificity of the materials from α5ß1- to αvß3-expressing fibroblasts by using an αvß3-selective peptidomimetic as coating molecule. The results shown here represent the first functionalization of Ti-based materials with α5ß1- or αvß3-selective peptidomimetics that allow an unprecedented control to discriminate between α5ß1- and αvß3-mediated adhesions. The role of these two integrins in different biological events is still a matter of debate and is frequently discussed in literature. Thus, such bioactive titanium surfaces will be of great relevance for the study of integrin-mediated cell adhesion and the development of new biomaterials targeting specific cell types.
Assuntos
Integrina alfa5beta1/química , Integrina alfaVbeta3/química , Oligopeptídeos/química , Peptidomiméticos/química , Titânio , Animais , Materiais Biocompatíveis , Adesão Celular , Química Click , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Peptidomiméticos/farmacologia , Ligação ProteicaRESUMO
Pattern seekers: For the two angiogenic relevant integrins α5ß1 and αvß3, functionalized derivatives of the selective antagonists 1 and 2 could target and discriminate between tumor cells inâ vivo based on their different integrin patterns and also delay tumor growth inâ vivo. In addition, the first α5ß1-selective integrin antagonist that enables specific molecular imaging by positron emission tomography was developed.
Assuntos
Integrina alfa5beta1/antagonistas & inibidores , Integrina alfaVbeta3/antagonistas & inibidores , Tomografia por Emissão de Pósitrons/métodos , Moduladores da Angiogênese , Animais , Humanos , Camundongos , Peptidomiméticos , RatosRESUMO
Insufficient oral bioavailability is considered as a key limitation for the widespread development of peptides as therapeutics. While the oral bioavailability of small organic compounds is often estimated from simple rules, similar rules do not apply to peptides, and even the high oral bioavailability that is described for a small number of peptides is not well understood. Here we present two highly Caco-2 permeable template structures based on a library of 54 cyclo(-D-Ala-Ala(5)-) peptides with different N-methylation patterns. The first (all-trans) template structure possesses two ß-turns of type II along Ala(6)-D-Ala(1) and Ala(3)-Ala(4) and is only found for one peptide with two N-methyl groups at D-Ala(1) and Ala(6) [(NMe(1,6)]. The second (single-cis) template possesses a characteristic cis peptide bond preceding Ala(5), which results in type VI ß-turn geometry along Ala(4)-Ala(5). Although the second template structure is found in seven peptides carrying N-methyl groups on Ala(5), high Caco-2 permeability is only found for a subgroup of two of them [NMe(1,5) and NMe(1,2,4,5)], suggesting that N-methylation of D-Ala(1) is a prerequisite for high permeability of the second template structure. The structural similarity of the second template structure with the orally bioavailable somatostatin analog cyclo(-Pro-Phe-NMe-D-Trp-NMe-Lys-Thr-NMe-Phe-), and the striking resemblance with both ß-turns of the orally bioavailable peptide cyclosporine A, suggests that the introduction of bioactive sequences on the highly Caco-2 permeable templates may result in potent orally bioavailable drug candidates.
Assuntos
Absorção Intestinal , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Células CACO-2 , Dipeptídeos/química , Dipeptídeos/farmacocinética , Humanos , Metilação , Modelos Moleculares , Permeabilidade , Estrutura Secundária de ProteínaRESUMO
Here we present the use of surface nanopatterning of covalently immobilized BMP-2 and integrin selective ligands to determine the specificity of their interactions in regulating cell adhesion and focal adhesion assembly. Gold nanoparticle arrays carrying single BMP-2 dimers are prepared by block-copolymer micellar nanolithography and azide-functionalized integrin ligands (cyclic-RGD peptides or α5ß1 integrin peptidomimetics) are immobilized on the surrounding polyethylene glycol alkyne by click chemistry. Compared to BMP-2 added to the media, surface immobilized BMP-2 (iBMP-2) favors the spatial segregation of adhesion clusters and enhances focal adhesion (FA) size in cells adhering to α5ß1 integrin selective ligands. Moreover, iBMP-2 copresented with α5ß1 integrin ligands induces the recruitment of αvß3 integrins in FAs. When copresented with RGD, iBMP-2 induces the assembly of a higher number of FAs, which are not affected by α5ß1 integrin blocking. Our dual-functionalized platforms offer the possibility to study the crosstalk between integrins and BMP receptors, and more in general they could be used to address the spatial regulation of growth factors and adhesion receptors crosstalk on biomimetic surfaces.
Assuntos
Ouro , Nanopartículas Metálicas , Adesão Celular , Integrina alfa5beta1 , Integrina alfaVbeta3 , LigantesRESUMO
BACKGROUND: Neoatherosclerosis represents an accelerated manifestation of atherosclerosis in nascent neointima after stenting, associated with adverse events. We investigated whether improved reendothelialization using RGD-coated stents results in diminished vascular permeability and reduced foam cell formation compared to standard DES in atherosclerotic rabbits. METHODS AND RESULTS: Neointimal foam cell formation was induced in rabbits (n = 7). Enhanced endothelial integrity in RGD-coated stents resulted in decreased vascular permeability relative to DES, which was further confirmed by SEM and TEM. Cell culture experiments examined the effect of everolimus on endothelial integrity. Increasing concentrations of everolimus resulted in a dose-dependent decrease of endothelial cell junctions and foam cell transformation of monocytes, confirming the relevance of endothelial integrity in preventing permeability of LDL. CONCLUSION: Incomplete endothelial integrity was confirmed as a key factor of neointimal foam cell formation following stent implantation. Pro-healing stent coatings may facilitate reendothelialization and reduce the risk of neoatherosclerosis.
Assuntos
Aterosclerose/terapia , Stents , Cicatrização , Animais , Aterosclerose/patologia , Modelos Animais de Doenças , Células Espumosas/patologia , Masculino , Coelhos , Túnica Íntima/patologiaAssuntos
Integrina alfa5beta1/antagonistas & inibidores , Integrina alfaVbeta3/antagonistas & inibidores , Animais , Adesão Celular , Linhagem Celular , Ouro/química , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Ligantes , Nanopartículas Metálicas/química , Camundongos , Peptidomiméticos , Compostos de Sulfidrila/química , Propriedades de SuperfícieRESUMO
Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC50-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins αvß3, αvß5, αvß6, αvß8, α5ß1, αIIbß3, using homogenous ELISA-like solid phase binding assay.
Assuntos
Integrinas/metabolismo , Ligantes , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Terapia Biológica , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Humanos , Imunomodulação , Concentração Inibidora 50 , Integrinas/agonistas , Ligação ProteicaRESUMO
Engineering the interface between biomaterials and tissues is important to increase implant lifetime and avoid failures and revision surgeries. Permanent devices should enhance attachment and differentiation of stem cells, responsible for injured tissue repair, and simultaneously discourage bacterial colonization; this represents a major challenge. To take first steps towards such a multifunctional surface we propose merging topographical and biochemical cues on the surface of a clinically relevant material such as titanium. In detail, our strategy combines antibacterial nanotopographical features with integrin selective synthetic ligands that can rescue the adhesive capacity of the surfaces and instruct mesenchymal stem cell (MSC) response. To this end, a smooth substrate and two different high aspect ratio topographies have been produced and coated either with an αvß3-selective peptidomimetic, an α5ß1-selective peptidomimetic, or an RGD/PHSRN peptidic molecule. Results showed that antibacterial effects of the substrates could be maintained when tested on pathogenic Pseudomonas aeruginosa. Further, functionalization increased MSC adhesion to the surfaces and the αvß3-selective peptidomimetic-coated nanotopographies promoted osteogenesis. Such a dual physicochemical approach to achieve multifunctional surfaces represents a first step in the design of novel cell-instructive biomaterial surfaces.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Integrinas/química , Antibacterianos/metabolismo , Aderência Bacteriana , Materiais Biocompatíveis/metabolismo , Diferenciação Celular , Células Cultivadas , Materiais Revestidos Biocompatíveis , Humanos , Integrinas/metabolismo , Ligantes , Células-Tronco Mesenquimais/citologia , Viabilidade Microbiana , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Propriedades de Superfície , Titânio/químicaRESUMO
Coordination of the specific functions of α5ß1 and αvß3 integrins is crucial for the precise regulation of cell adhesion, spreading and migration, yet the contribution of differential integrin-specific crosstalk to these processes remains unclear. To determine the specific functions of αvß3 and α5ß1 integrins, we used nanoarrays of gold particles presenting immobilized, integrin-selective peptidomimetic ligands. Integrin binding to the peptidomimetics is highly selective, and cells can spread on both ligands. However, spreading is faster and the projected cell area is greater on α5ß1 ligand; both depend on ligand spacing. Quantitative analysis of adhesion plaques shows that focal adhesion size is increased in cells adhering to αvß3 ligand at 30 and 60 nm spacings. Analysis of αvß3 and α5ß1 integrin clusters indicates that fibrillar adhesions are more prominent in cells adhering to α5ß1 ligand, while clusters are mostly localized at the cell margins in cells adhering to αvß3 ligand. αvß3 integrin clusters are more pronounced on αvß3 ligand, though they can also be detected in cells adhering to α5ß1 ligand. Furthermore, α5ß1 integrin clusters are present in cells adhering to α5ß1 ligand, and often colocalize with αvß3 clusters. Taken together, these findings indicate that the activation of αvß3 integrin by ligand binding is dispensable for initial adhesion and spreading, but essential to formation of stable focal adhesions.
Assuntos
Movimento Celular , Adesões Focais/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Humanos , LigantesRESUMO
The interplay between specific integrin-mediated matrix adhesion and directional persistence in cell migration is not well understood. Here, we characterized fibroblast adhesion and migration on the extracellular matrix glycoproteins fibronectin and vitronectin, focusing on the role of α5ß1 and αvß3 integrins. Fibroblasts manifested high directional persistence in migration on fibronectin-, but not vitronectin-coated substrates, in a ligand density-dependent manner. Fibronectin stimulated α5ß1-dependent organization of the actin cytoskeleton into oriented, ventral stress fibers, and assembly of dynamic, polarized protrusions, characterized as regions free of stress fibers and rich in nascent adhesions at their edge. Such protrusions correlated with persistent, local leading edge advancement, but were not sufficient, nor necessary for directional migration over longer times. Selective blocking of αvß3 or α5ß1 integrins using small molecule integrin antagonists reduced directional persistence on fibronectin, indicating integrin cooperativity in maintaining directionality. On the other hand, patterned substrates, designed to selectively engage either integrin, or their combination, were not sufficient to establish directional migration. Overall, our study demonstrates adhesive coating-dependent regulation of directional persistence in fibroblast migration and challenges the generality of the previously suggested role of ß1 and ß3 integrins in directional migration.
Assuntos
Fibroblastos/citologia , Fibronectinas/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Animais , Adesão Celular , Linhagem Celular , Movimento Celular , Análise de Fourier , Humanos , Microscopia de Força Atômica , Ratos , Bibliotecas de Moléculas PequenasRESUMO
UNLABELLED: Despite in vivo mapping of integrin αvß3 expression being thoroughly investigated in recent years, its clinical value is still not well defined. For imaging of angiogenesis, the integrin subtype α5ß1 appears to be a promising target, for which purpose we designed the PET radiopharmaceutical (68)Ga-aquibeprin. METHODS: (68)Ga-aquibeprin was obtained by click-chemistry (CuAAC) trimerization of a α5ß1 integrin-binding pseudopeptide on the triazacyclononane-triphosphinate (TRAP) chelator, followed by automated (68)Ga labeling. Integrin α5ß1 and αvß3 affinities were determined in enzyme linked immune sorbent assay on immobilized integrins, using fibronectin and vitronectin, respectively, as competitors. M21 (human melanoma)-bearing severe combined immunodeficient mice were used for biodistribution, PET imaging, and determination of in vivo metabolization. The expression of α5 and ß3 subunits was determined by immunohistochemistry on paraffin sections of M21 tumors. RESULTS: (68)Ga-aquibeprin shows high selectivity for integrin α5ß1 (50% inhibition concentration [IC50] = 0.088 nM) over αvß3 (IC50 = 620 nM) and a pronounced hydrophilicity (log D = -4.2). Severe combined immunodeficient mice xenografted with M21 human melanoma were found suitable for in vivo evaluation, as M21 immunohistochemistry showed not only an endothelial and strong cytoplasmatic expression of the ß3 integrin subunit but also an intense expression of the α5 integrin subunit particularly in the endothelial cells of intratumoral small vessels. Ex vivo biodistribution (90 min after injection) showed high uptake in M21 tumor (2.42 ± 0.21 percentage injected dose per gram), fast renal excretion, and low background; tumor-to-blood and tumor-to-muscle ratios were 10.6 ± 2.5 and 20.9 ± 2.4, respectively. (68)Ga-aquibeprin is stable in vivo; no metabolites were detected in mouse urine, blood serum, kidney, and liver homogenates 30 min after injection. PET imaging was performed for (68)Ga-aquibeprin and the previously described, structurally related c(RGDfK) trimer (68)Ga-avebetrin, which shows an inverse selectivity for integrin αvß3 (IC50 = 0.22 nM) over α5ß1 (IC50 = 39 nM). In vivo target specificity was proven by cross-competition studies; tumor uptake of either tracer was not affected by the coadministration of 40 nmol (â¼5 mg/kg) of the respective other compound. CONCLUSION: (68)Ga-aquibeprin and (68)Ga-avebetrin are recommendable for complementary mapping of integrins α5ß1 and αvß3 by PET, allowing for future studies on the role of these integrins in angiogenesis, tumor progression, metastasis, and myocardial infarct healing.
Assuntos
Complexos de Coordenação , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Animais , Ligação Competitiva , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Integrina alfa5beta1/biossíntese , Integrina alfaVbeta3/biossíntese , Camundongos , Músculos/diagnóstico por imagem , Transplante de Neoplasias , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismo , Inclusão em Parafina , Especificidade por Substrato , Distribuição TecidualRESUMO
Interaction between the surface of implants and biological tissues is a key aspect of biomaterials research. Apart from fulfilling the non-toxicity and structural requirements, synthetic materials are asked to direct cell response, offering engineered cues that provide specific instructions to cells. This work explores the functionalization of titanium with integrin-binding peptidomimetics as a novel and powerful strategy to improve the adhesion, proliferation and differentiation of osteoblast-like cells to implant materials. Such biomimetic strategy aims at targeting integrins αvß3 and α5ß1, which are highly expressed on osteoblasts and are essential for many fundamental functions in bone tissue development. The successful grafting of the bioactive molecules on titanium is proven by contact angle measurements, X-ray photoelectron spectroscopy and fluorescent labeling. Early attachment and spreading of cells are statistically enhanced by both peptidomimetics compared to unmodified titanium, reaching values of cell adhesion comparable to those obtained with full-length extracellular matrix proteins. Moreover, an increase in alkaline phosphatase activity, and statistically higher cell proliferation and mineralization are observed on surfaces coated with the peptidomimetics. This study shows an unprecedented biological activity for low-molecular-weight ligands on titanium, and gives striking evidence of the potential of these molecules to foster bone regeneration on implant materials.
Assuntos
Materiais Revestidos Biocompatíveis/farmacologia , Integrina alfaVbeta3/metabolismo , Osteoblastos/efeitos dos fármacos , Peptidomiméticos/farmacologia , Receptores de Vitronectina/metabolismo , Titânio/farmacologia , Fosfatase Alcalina/metabolismo , Biomarcadores/metabolismo , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/química , Implantes Dentários , Proteínas da Matriz Extracelular/química , Expressão Gênica , Humanos , Integrina alfaVbeta3/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Ligação Proteica , Receptores de Vitronectina/genética , Propriedades de Superfície , Titânio/químicaRESUMO
Orthogonally functionalized binary micropatterned substrates are produced using a novel protocol. The use of adequate peptido-mimetics enables an unprecedented segregation of purified αvß3 and α5ß1 integrins in adjacent microislands and evidences the preference of U2OS cells to colocalize such receptors. Moreover, this tendency can be altered by varying the geometry and composition of the micropatterns.