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1.
Nat Commun ; 11(1): 114, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31913286

RESUMO

Stem cell therapies are limited by poor cell survival and engraftment. A hurdle to the use of materials for cell delivery is the lack of understanding of material properties that govern transplanted stem cell functionality. Here, we show that synthetic hydrogels presenting integrin-specific peptides enhance the survival, persistence, and osteo-reparative functions of human bone marrow-derived mesenchymal stem cells (hMSCs) transplanted in murine bone defects. Integrin-specific hydrogels regulate hMSC adhesion, paracrine signaling, and osteoblastic differentiation in vitro. Hydrogels presenting GFOGER, a peptide targeting α2ß1 integrin, prolong hMSC survival and engraftment in a segmental bone defect and result in improved bone repair compared to other peptides. Integrin-specific hydrogels have diverse pleiotropic effects on hMSC reparative activities, modulating in vitro cytokine secretion and in vivo gene expression for effectors associated with inflammation, vascularization, and bone formation. These results demonstrate that integrin-specific hydrogels improve tissue healing by directing hMSC survival, engraftment, and reparative activities.


Assuntos
Doenças Ósseas/terapia , Integrina alfa2beta1/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Animais , Doenças Ósseas/metabolismo , Doenças Ósseas/fisiopatologia , Medula Óssea/química , Medula Óssea/metabolismo , Regeneração Óssea , Adesão Celular , Sobrevivência Celular , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Hidrogéis/química , Integrina alfa2beta1/genética , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Peptídeos/metabolismo
2.
Nanoscale ; 11(43): 20766-20776, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31651003

RESUMO

A major impediment in the clinical translation of stem cell therapy has been the inability to efficiently and reproducibly direct differentiation of a large population of stem cells. Thus, we aimed to engineer a substrate for culturing stem cells to efficiently induce cardiomyogenic lineage commitment. In this work, we present a nanopillar array on the surface of titanium that was prepared by mask-less reactive ion etching. Scanning electron and atomic force microscopy revealed that the surface was covered by vertically aligned nanopillars each of ≈1 µm with a diameter of ≈80 nm. The nanopillars supported the attachment and proliferation of human mesenchymal stem cells (hMSCs). Cardiomyogenic lineage commitment of the stem cells was more enhanced on the nanopillars than on the smooth surface. When co-cultured with neonatal rat cardiomyocytes, the cyclic pattern of calcium transport observed distinctly in cells differentiated on the arrays compared to the cells cultured on the smooth surface was the functional validation of differentiation. The use of small molecule inhibitors revealed that integrins namely, α2ß1 and αvß3, are essential for cardiomyogenesis on the nanostructured surface, which is further mediated by FAK, Erk and Akt cell signaling pathways. This study demonstrates that the nanopillar array efficiently promotes the cardiomyogenic lineage commitment of stem cells via integrin-mediated signaling and can potentially serve as a platform for the ex vivo differentiation of stem cells toward cell therapy in cardiac tissue repair and regeneration.


Assuntos
Diferenciação Celular , Nanoestruturas/química , Titânio/química , Animais , Cálcio/metabolismo , Linhagem da Célula , Proliferação de Células , Técnicas de Cocultura , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína Homeobox Nkx-2.5/metabolismo , Humanos , Integrina alfa2beta1/antagonistas & inibidores , Integrina alfa2beta1/metabolismo , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Microscopia Confocal , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Propriedades de Superfície
3.
PLoS One ; 14(8): e0216839, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31398205

RESUMO

The two main collagen receptors on platelets, GPVI and integrin α2ß1, play an important role for the recognition of exposed collagen at sites of vessel injury, which leads to platelet activation and subsequently stable thrombus formation. Both receptors are already expressed on megakaryocytes, the platelet forming cells within the bone marrow. Megakaryocytes are in permanent contact with collagen filaments in the marrow cavity and at the basal lamina of sinusoids without obvious preactivation. The role of both collagen receptors for megakaryocyte maturation and thrombopoiesis is still poorly understood. To investigate the function of both collagen receptors, we generated mice that are double deficient for Gp6 and Itga2. Flow cytometric analyses revealed that the deficiency of both receptors had no impact on platelet number and led to the expected lack in GPVI responsiveness. Integrin activation and degranulation ability was comparable to wildtype mice. By immunofluorescence microscopy, we could demonstrate that both wildtype and double-deficient megakaryocytes were overall normally distributed within the bone marrow. We found megakaryocyte count and size to be normal, the localization within the bone marrow, the degree of maturation, as well as their association to sinusoids were also unaltered. However, the contact of megakaryocytes to collagen type I filaments was decreased at sinusoids compared to wildtype mice, while the interaction to type IV collagen was unaffected. Our results imply that GPVI and α2ß1 have no influence on the localization of megakaryocytes within the bone marrow, their association to the sinusoids or their maturation. The decreased contact of megakaryocytes to collagen type I might at least partially explain the unaltered platelet phenotype in these mice, since proplatelet formation is mediated by these receptors and their interaction to collagen. It is rather likely that other compensatory signaling pathways and receptors play a role that needs to be elucidated.


Assuntos
Plaquetas/citologia , Deleção de Genes , Integrina alfa2beta1/deficiência , Integrina alfa2beta1/genética , Megacariócitos/citologia , Glicoproteínas da Membrana de Plaquetas/deficiência , Glicoproteínas da Membrana de Plaquetas/genética , Animais , Camundongos , Trombopoese/genética
4.
J Biol Chem ; 294(39): 14442-14453, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31406019

RESUMO

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 → 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 → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → 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.


Assuntos
Substituição de Aminoácidos , Colágeno/química , Síndrome de Ehlers-Danlos/genética , Integrina alfa2beta1/metabolismo , Peptídeos/metabolismo , Sítios de Ligação , Adesão Celular , Linhagem Celular , Colágeno/metabolismo , Humanos , Integrina alfa2beta1/química , Integrina alfa2beta1/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Simulação de Acoplamento Molecular , Peptídeos/química , Ligação Proteica
5.
Semin Ophthalmol ; 34(5): 365-374, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31257963

RESUMO

Purpose: In this study, we investigated the association of two polymorphisms (rs869109213 and rs2070744) in the eNOS gene and one polymorphism BglII in the α2ß1 integrin gene (ITGA2) with the risk of diabetic retinopathy (DR) in a Tunisian population. Methods: The study investigated of 110 type 2 diabetes mellitus (T2DM) and 127 DR patients. The genotypes of the eNOS 4b/4a (rs869109213) and -786T/C (rs2070744) polymorphisms and of the BglII polymorphism of ITGA2 were studied using the PCR or PCR-RFLP method. Results: The genotype distributions of the two polymorphisms in eNOS 4b4a and eNOS (-786T/C) were significantly different between T2DM and DR patients (p < .004 and p = .033, respectively). These polymorphisms were associated with the risk of DR (OR = 2.65, 95%CI [1.45-4.84], p = .002) for the eNOS 4b4a genotype and (OR = 2.43, 95%CI [1.06 - 5.56], p = .036) for the CC genotype of the eNOS gene (-786T/C). Similarly, the genotype distribution of the BglII polymorphism was significantly different between the two groups studied (p = .037). This polymorphism was associated with an increased risk of DR (OR = 4.03, 95% CI [1.17 - 7.85], p = .022) for BglII(+/+). Conclusion: The present study suggests that the polymorphisms 4b4a and -786T/C in the eNOS gene might be associated with DR. In addition, the BglII polymorphism in the ITGA2 gene was a risk factor for DR.


Assuntos
Diabetes Mellitus Tipo 2 , Retinopatia Diabética/genética , Variação Genética , Integrina alfa2beta1/genética , Óxido Nítrico Sintase Tipo III/genética , Adulto , Idoso , Análise de Variância , Feminino , Predisposição Genética para Doença , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Tunísia
6.
Blood Rev ; 38: 100592, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31351674

RESUMO

Much interest surrounds the receptors α2ß1 and glycoprotein VI (GPVI) whose synchronized action mediates the attachment and activation of platelets on collagen, essential for preventing blood loss but also the most thrombogenic component of the vessel wall. Subject to density variations on platelets through natural polymorphisms, the absence of α2ß1 or GPVI uniquely leads to a substantial block of hemostasis without causing major bleeding. Specific to the megakaryocyte lineage, GPVI and its signaling pathways are most promising targets for anti-thrombotic therapy. This review looks at the clinical consequences of the loss of collagen receptor function with emphasis on both the inherited and acquired loss of GPVI with brief mention of mouse models when necessary. A detailed survey of rare case reports of patients with inherited disease-causing variants of the GP6 gene is followed by an assessment of the causes and clinical consequences of acquired GPVI deficiency, a more frequent finding most often due to antibody-induced platelet GPVI shedding. Release of soluble GPVI is brought about by platelet metalloproteinases; a process induced by ligand or antibody binding to GPVI or even high shear forces. Also included is an assessment of the clinical importance of GPVI-mediated platelet interactions with fibrin and of the promise shown by the pharmacological inhibition of GPVI in a cardiovascular context. The role for GPVI in platelet function in inflammation and in the evolution and treatment of major illnesses such as rheumatoid arthritis, cancer and sepsis is also discussed.


Assuntos
Colágeno/metabolismo , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Plaquetas/metabolismo , Humanos , Integrina alfa2beta1/metabolismo , Receptores de Colágeno/metabolismo , Transdução de Sinais
7.
Acta Biomater ; 94: 361-371, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31200119

RESUMO

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.


Assuntos
Materiais Biomiméticos/química , Colágeno/química , Reagentes para Ligações Cruzadas/química , Dissulfetos/química , Hidrogéis/química , Oligopeptídeos/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Adesão Celular , Linhagem Celular , Proliferação de Células , Módulo de Elasticidade , Matriz Extracelular/metabolismo , Humanos , Hidrogéis/metabolismo , Integrina alfa2beta1/química , Masculino , Camundongos Endogâmicos C57BL , Ligação Proteica , Reologia , Propriedades de Superfície
8.
Cancer Sci ; 110(7): 2110-2118, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31120174

RESUMO

The tumor microenvironment is associated with various tumor progressions, including cancer metastasis, immunosuppression, and tumor sustained growth. Tumor-associated macrophages (TAMs) are considered an indispensable component of the tumor microenvironment, participating in the progression of tumor microenvironment remodeling and creating various compounds to regulate tumor activities. This study aims to observe enriched TAMs in tumor tissues during bladder cancer development, which markedly facilitated the proliferation of bladder cancer cells and promoted tumor growth in vivo. We determined that TAMs regulate tumor sustained growth by secreting type I collagen, which can activate the prosurvival integrin α2ß1/PI3K/AKT signaling pathway. Furthermore, traditional chemotherapeutic drugs combined with integrin α2ß1 inhibitor showed intensive anticancer effects, revealing an innovative approach in clinical bladder cancer treatment.


Assuntos
Cromonas/administração & dosagem , Colágeno/metabolismo , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Macrófagos/patologia , Morfolinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Proliferação de Células , Cromonas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Integrina alfa2beta1/genética , Integrina alfa2beta1/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Microambiente Tumoral , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Cells ; 8(4)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939867

RESUMO

A hallmark of ageing is the redistribution of body fat. Particularly, subcutaneous fat decreases paralleled by a decrease of skin collagen I are typical for age-related skin atrophy. In this paper, we hypothesize that collagen I may be a relevant molecule stimulating the differentiation of adipose-derived stem cells (ASCs) into adipocytes augmenting subcutaneous fat. In this context lipogenesis, adiponectin, and collagen I receptor expression were determined. Freshly isolated ASCs were characterized by stemness-associated surface markers by FACS analysis and then transdifferentiated into adipocytes by specific medium supplements. Lipogenesis was evaluated using Nile Red staining and documented by fluorescence microscopy or quantitatively measured by using a multiwell spectrofluorometer. Expression of adiponectin was measured by real-time RT-PCR and in cell-free supernatants by ELISA, and expression of collagen I receptors was observed by western blot analysis. It was found that supports coated with collagen I promote cell adhesion and lipogenesis of ASCs. Interestingly, a reverse correlation to adiponectin expression was observed. Moreover, we found upregulation of the collagen receptor, discoidin domain-containing receptor 2; receptors of the integrin family were absent or downregulated. These findings indicate that collagen I is able to modulate lipogenesis and adiponectin expression and therefore may contribute to metabolic dysfunctions associated with ageing.


Assuntos
Adipócitos/citologia , Tecido Adiposo/citologia , Colágeno Tipo I/farmacologia , Células-Tronco/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adiponectina/genética , Adiponectina/metabolismo , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Integrina alfa2beta1/metabolismo , Lipogênese/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Colágeno/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos
10.
Int J Mol Sci ; 20(8)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022936

RESUMO

Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2ß1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2ß1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2ß1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2-/- mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2ß1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2ß1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke.


Assuntos
Anticorpos/uso terapêutico , Encéfalo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/prevenção & controle , Integrina alfa2beta1/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Substâncias Protetoras/uso terapêutico , Ativador de Plasminogênio Tecidual/uso terapêutico , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Colágeno/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/uso terapêutico , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/prevenção & controle
11.
Int Immunol ; 31(6): 407-412, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30783682

RESUMO

Integrin α2ß1, also known as very late antigen (VLA)-2, is a collagen-binding molecule expressed constitutively on platelets. Vatelizumab, a monoclonal antibody targeting the α2 subunit (CD49b) of VLA-2, was recently investigated for its safety and efficacy during a Phase 2 clinical study in multiple sclerosis patients, as integrin-mediated collagen binding at the site of inflammation is central to a number of downstream pro-inflammatory events. In the course of this study, we could show that VLA-2 is expressed ex vivo on platelets, platelet-T-cell aggregates, as well as a small population of highly activated memory T cells. Even though the clinical trial did not meet its primary clinical end-point (reduction in the cumulative number of new contrast-enhancing lesions on magnetic resonance imaging (MRI)), we observed enhanced frequencies of regulatory T cells (TREG) following vatelizumab treatment. Elevated TREG frequencies might be explained by the inhibition of p38 mitogen-activated protein kinase (MAPK) signaling, which is critically involved in the polarization of T helper 17 (TH17) cells and is activated by the α2 integrin cytoplasmic domain. Our findings suggest that blockade of VLA-2 might be a way to safely shift the TH17/TREG balance by inducing TREGin vivo.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Plaquetas/metabolismo , Integrina alfa2/metabolismo , Integrina alfa2beta1/metabolismo , Esclerose Múltipla/tratamento farmacológico , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Antígenos CD4/metabolismo , Colágeno/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Humanos , Memória Imunológica , Integrina alfa2/imunologia , Integrina alfa2beta1/antagonistas & inibidores , Ativação Linfocitária , Sistema de Sinalização das MAP Quinases , Transdução de Sinais
12.
J Orthop Res ; 37(3): 706-716, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30561137

RESUMO

This study was designed to investigate (i) extracellular matrix to specify adhesive substrates to human dura mater cell (hDMC); (ii) the alteration on adhesion-related molecules in hDMC; and (iii) secreted matrix metalloproteinases (MMPs) linked with extracellular matrix remodeling after exposure to inflammation. The hDMC was cultured from human dura mater tissue, and the studies were performed with hDMC after co-culturing with macrophage like THP-1 cells (Mϕ). The adhesion of co-cultured hDMC through collagen I increased 6.4-fold and through collagen IV increased 5.0-fold compared with the adhesion of naïve cells (p < 0.001). Integrin subtype α2 ß1 expression was increased 6.3-fold (p < 0.001) and α1 expression was decreased 2.0-fold (p < 0.001) in the co-cultured cells compared with the naïve cells. Co-culturing induced significant increases in MMP-1 (13.9-fold, p < 0.01), MMP-3 (7.6-fold, p < 0.01), and VEGF (VEGF: 3.8-fold, p < 0.05) expression and decreases in MMP-9 (0.1-fold, p < 0.01) compared with the sum of naïve hDMC and Mϕ values. Increased hDMC adhesion under inflammatory conditions is caused by an increased cellular affinity for collagen I and IV mediated by increased hDMC levels of integrin subtype α2 ß1 and environmental MMP-1, -3 and decreased MMP-9. Selective integrin subtype α2 ß1 inhibition assay showed 37.8% and 35.7% reduction in adhesion of co-cultured hDMC to collagen I (p < 0.001) and IV (p = 0.057), respectively. The present study provides insight into the pathological conditions related to dura mater adhesion in inflammation. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1-11, 2019.


Assuntos
Adesão Celular , Dura-Máter/citologia , Matriz Extracelular/fisiologia , Inflamação/fisiopatologia , Metaloproteinases da Matriz/metabolismo , Adulto , Idoso , Técnicas de Cocultura , Colágeno Tipo I/fisiologia , Colágeno Tipo IV/fisiologia , Dura-Máter/enzimologia , Dura-Máter/fisiopatologia , Feminino , Humanos , Integrina alfa2beta1/fisiologia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Células THP-1
13.
Front Immunol ; 9: 2269, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30374344

RESUMO

ß1 integrins are critical for T cell migration, survival and costimulation. The integrin α2ß1, which is a receptor for collagen, also named VLA-2, is a major costimulatory pathway of effector T cells and has been implicated in arthritis pathogenesis. Herein, we have examined its ability to promote methotrexate (MTX) resistance by enhancing effector T cells survival. Our results show that attachment of anti-CD3-activated human polarized Th17 cells to collagen but not to fibronectin or laminin led to a significant reduction of MTX-induced apoptosis. The anti-CD3+collagen-rescued cells still produce significant amounts of IL-17 and IFNγ upon their reactivation indicating that their inflammatory nature is preserved. Mechanistically, we found that the prosurvival role of anti-CD3+collagen involves activation of the MTX transporter ABCC1 (ATP Binding Cassette subfamily C Member 1). Finally, the protective effect of collagen/α2ß1 integrin on MTX-induced apoptosis also occurs in memory CD4+ T cells isolated from rheumatoid arthritis (RA) patients suggesting its clinical relevance. Together these results show that α2ß1 integrin promotes MTX resistance of effector T cells, and suggest that it could contribute to the development of MTX resistance that is seen in RA.


Assuntos
Apoptose/efeitos dos fármacos , Integrina alfa2beta1/imunologia , Metotrexato/farmacologia , Células Th17/efeitos dos fármacos , Apoptose/imunologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Células Cultivadas , Colágeno/farmacologia , Fibronectinas/farmacologia , Humanos , Integrina alfa2beta1/metabolismo , Laminina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Substâncias Protetoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Células Th17/imunologia
14.
Biochemistry (Mosc) ; 83(6): 738-745, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30195330

RESUMO

Blocking the expression of integrin α2ß1, which was accomplished by transduction of α2-specific shRNA, resulted in significant inhibition of proliferation and clonal activity in human MCF-7 breast carcinoma and SK-Mel-147 melanoma cells. Along with these changes, deprivation of α2ß1 caused a sharp decrease in melanoma cell invasion in vitro. Analysis of integrin-mediating signal pathways that control cell behavior revealed a significant increase in activity of Akt protein kinase in response to depletion of α2ß1. The increase in Akt activity that accompanies a suppressive effect on cell invasion contradicts well-known Akt function aimed at stimulation of tumor progression. This contradiction could be explained by the "reversed" (noncanonical) role played by Akt in some cells that consists in suppression rather than promotion of invasive phenotype. To test this suggestion, the effects of Akt inhibitors on invasive activity of SK-Mel-147 cells were investigated. If the above suggestion is true, then inhibition of Akt in cells depleted of α2ß1 should result in the restoration of their invasive activity. It appeared that treatment with LY294002, which inhibits all Akt isoforms (Akt1, Akt2, Akt3), not only failed to restore the invasive phenotype of melanoma cells but further attenuated their invasive activity. However, treatment of the cells with an Akt1-specific inhibitor significantly increased their invasion. Thus, the stimulating effect of α2ß1 integrin on invasion of melanoma cells is realized through a mechanism based on inhibition of one of the Akt isoforms, which in these cells exhibits a noncanonical function consisting in suppression of invasion.


Assuntos
Proliferação de Células , Integrina alfa2beta1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Integrina alfa2beta1/antagonistas & inibidores , Integrina alfa2beta1/genética , Células MCF-7 , Metaloproteinase 2 da Matriz/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Morfolinas/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
15.
PLoS One ; 13(6): e0198332, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29912899

RESUMO

BACKGROUND: Nasopharyngeal carcinoma is a rare form of cancer across the world except in certain areas such as Southern China, Hong Kong and Malaysia. NPC is considered a relatively radiosensitive tumor and patients diagnosed at early stages tend to survive longer compared to those with advanced disease. Given that early symptoms of NPC are non-specific and that the nasopharynx is relatively inaccessible, less invasive screening methods such as biomarker screening might be the key to improve NPC survival and management. A number of genes with their respective polymorphisms have been shown in past studies to be associated with survival of various cancers. hOGG1 and XPD genes encode for a DNA glycosylase and a DNA helicase respectively; both are proteins that are involved in DNA repair. ITGA2 is the alpha subunit of the transmembrane receptor integrin and is mainly responsible for cell-cell and cell-extracellular matrix interaction. TNF-α is a cytokine that is released by immune cells during inflammation. METHODS: Restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) was used to genotype all the aforementioned gene polymorphisms. Kaplan-Meier survival function, log-rank test and Cox regression were used to investigate the effect of gene polymorphisms on the all-cause survival of NPC. RESULTS: NPC cases carrying T/T genotype of ITGA2 C807T have poorer all-cause survival compared to those with C/C genotypes, with an adjusted HR of 2.06 (95% CI = 1.14-3.72) in individual model. The 5-year survival rate of C/C carriers was 55% compared to those with C/T and T/T where the survival rates were 50% and 43%, respectively. CONCLUSION: The finding from the present study showed that ITGA2 C807T polymorphism could be potentially useful as a prognostic biomarker for NPC. However, the prognostic value of ITGA2 C807T polymorphism has to be validated by well-designed further studies with larger patient numbers.


Assuntos
DNA Glicosilases/genética , Integrina alfa2beta1/genética , Carcinoma Nasofaríngeo/mortalidade , Neoplasias Nasofaríngeas/mortalidade , Fator de Necrose Tumoral alfa/genética , Proteína Grupo D do Xeroderma Pigmentoso/genética , Adulto , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Técnicas de Genotipagem , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Estadiamento de Neoplasias , Polimorfismo de Nucleotídeo Único , Análise de Sobrevida
16.
Biochim Biophys Acta Rev Cancer ; 1869(2): 321-332, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29673969

RESUMO

We propose a new cadherin family classification comprising epithelial cadherins (cadherin 17 [CDH17], cadherin 16, VE-cadherin, cadherin 6 and cadherin 20) containing RGD motifs within their sequences. Expression of some RGD cadherins is associated with aggressive forms of cancer during the late stages of metastasis, and CDH17 and VE-cadherin have emerged as critical actors in cancer metastasis. After binding to α2ß1 integrin, these cadherins promote integrin ß1 activation, and thereby cell adhesion, invasion and proliferation, in liver and lung metastasis. Activation of α2ß1 integrin provokes an affinity increase for type IV collagen, a major component of the basement membrane and a critical partner for cell anchoring in liver and other metastatic organs. Activation of α2ß1 integrin by RGD motifs breaks an old paradigm of integrin classification and supports an important role of this integrin in cancer metastasis. Recently, synthetic peptides containing the RGD motif of CDH17 elicited highly specific and selective antibodies that block the ability of CDH17 RGD to activate α2ß1 integrin. These monoclonal antibodies inhibit metastatic colonization in orthotopic mouse models of liver and lung metastasis for colorectal cancer and melanoma, respectively. Hopefully, blocking the cadherin RGD ligand capacity will give us control over the integrin activity in solid tumors metastasis, paving the way for development of new agents of cancer treatment.


Assuntos
Caderinas/metabolismo , Movimento Celular , Integrina alfa2beta1/metabolismo , Neoplasias/metabolismo , Oligopeptídeos/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Sítios de Ligação , Caderinas/antagonistas & inibidores , Caderinas/imunologia , Adesão Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Humanos , Integrina alfa2beta1/antagonistas & inibidores , Integrina alfa2beta1/imunologia , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ligação Proteica , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/imunologia , Receptores de Peptídeos/antagonistas & inibidores , Receptores de Peptídeos/imunologia , Transdução de Sinais
17.
Thromb Haemost ; 118(6): 1009-1020, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29695020

RESUMO

Neonatal platelets are hypo-reactive to the tyrosine kinase-linked receptor agonist collagen. Here, we have investigated whether the hypo-responsiveness is related to altered levels of glycoprotein VI (GPVI) and integrin α2ß1, or to defects in downstream signalling events by comparison to platelet activation by C-type lectin-like receptor 2 (CLEC-2). GPVI and CLEC-2 activate a Src- and Syk-dependent signalling pathway upstream of phospholipase C (PLC) γ2. Phosphorylation of a conserved YxxL sequence known as a (hemi) immunotyrosine-based-activation-motif (ITAM) in both receptors is critical for Syk activation. Platelets from human pre-term and full-term neonates display mildly reduced expression of GPVI and CLEC-2, as well as integrin αIIbß3, accounted for at the transcriptional level. They are also hypo-responsive to the two ITAM receptors, as shown by measurement of integrin αIIbß3 activation, P-selectin expression and Syk and PLCγ2 phosphorylation. Mouse platelets are also hypo-responsive to GPVI and CLEC-2 from late gestation to 2 weeks of age, as determined by measurement of integrin αIIbß3 activation. In contrast, the response to G protein-coupled receptor agonists was only mildly reduced and in some cases not altered in neonatal platelets of both species. A reduction in response to GPVI and CLEC-2, but not protease-activated receptor 4 (PAR-4) peptide, was also observed in adult mouse platelets following immune thrombocytopenia, whereas receptor expression was not impaired. Our results demonstrate developmental differences in platelet responsiveness to GPVI and CLEC-2, and also following immune platelet depletion leading to reduced Syk activation. The rapid generation of platelets during development or following platelet depletion is achieved at the expense of signalling by ITAM-coupled receptors.


Assuntos
Plaquetas/fisiologia , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Nascimento Prematuro/metabolismo , Púrpura Trombocitopênica Idiopática/metabolismo , Quinase Syk/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Recém-Nascido , Integrina alfa2beta1/metabolismo , Camundongos , Selectina-P/metabolismo , Fosfolipase C gama/metabolismo , Ativação Plaquetária , Gravidez , Nascimento Prematuro/patologia , Púrpura Trombocitopênica Idiopática/patologia , Receptores de Trombina/metabolismo , Transdução de Sinais
18.
Biomaterials ; 167: 107-120, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29567387

RESUMO

Collagen, a strong platelet activator, is recognized by integrin α2ß1 and GPVI. It induces aggregation, if added to suspended platelets, or platelet adhesion if immobilized to a surface. The recombinant non-prolylhydroxylated mini-collagen FC3 triple helix containing one α2ß1 integrin binding site is a tool to specifically study how α2ß1 integrin activates platelet. Whereas soluble FC3 monomers antagonistically block collagen-induced platelet activation, immobilization of several FC3 molecules to an interface or to colloidal nanobeads determines the agonistic action of FC3. Nanopatterning of FC3 reveals that intermolecular distances below 64 nm between α2ß1 integrin binding sites trigger signaling through dot-like clusters of α2ß1 integrin, which are visible in high resolution microscopy with dSTORM. Upon signaling, these integrin clusters increase in numbers per platelet, but retain their individual size. Immobilization of several FC3 to 100 nm-sized nanobeads identifies α2ß1 integrin-triggered signaling in platelets to occur at a twentyfold slower rate than collagen, which activates platelet in a fast integrative signaling via different platelet receptors. As compared to collagen stimulation, FC3-nanobead-triggered signaling cause a significant stronger activation of the protein kinase BTK, a weak and dispensable activation of PDK1, as well as a distinct phosphorylation pattern of PDB/Akt.


Assuntos
Tirosina Quinase da Agamaglobulinemia/imunologia , Plaquetas/citologia , Colágeno/imunologia , Integrina alfa2beta1/imunologia , Ativação Plaquetária , Sítios de Ligação , Plaquetas/imunologia , Colágeno/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Glicoproteínas da Membrana de Plaquetas/imunologia , Transdução de Sinais
19.
Exp Cell Res ; 362(2): 498-503, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29253536

RESUMO

Peritoneal metastasis is a major cause of recurrence of gastric cancer and integrins are key molecules involved in gastric cancer cells attachment to the peritoneum. The peptide hormone, gastrin, initially identified for its role in gastric acid secretion is also a growth factor for gastric mucosa. Gastrin has also been shown to contribute to gastric cancers progression. Here, we provide the first evidence that gastrin increases the adhesion of gastric cancer cells. Gastrin treatment induces the expression of α2 integrin subunit through a mechanism that involves the ERK pathway. We also observed in response to gastrin an increase in the amount of α2 integrin associated with ß1subunit. In addition, gastrin-stimulated cell adhesion was blocked with an anti-α2ß1 integrin neutralizing antibody. We also show that gastrin activates the integrin pathway via the phosphorylation of ß1 integrin by a Src family kinase. This mechanism may contribute to the enhancement of cell adhesion observed in response to gastrin since we found an inhibition of gastrin-mediated cell adhesion when cells were treated with a Src inhibitor. By regulating one of the key step of the metastatic process gastrin might contribute to increase the aggressive behaviour of human gastric tumours.


Assuntos
Gastrinas/farmacologia , Integrina alfa2beta1/genética , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Gástricas/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metástase Neoplásica , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/secundário , Peritônio/patologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
20.
J Biol Regul Homeost Agents ; 31(4): 911-921, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29254293

RESUMO

Myocardial infarction is the leading cause of morbidity and mortality in developed countries. It causes a left ventricular dysfunction, mainly due to the loss of functional tissue, resulting in heart failure. New therapies are being developed, using a tissue engineering approach, with the ultimate goal of restoring cardiac function by regenerating and repairing the damaged myocardium. In the present study we investigated the behaviour of a specific population of c-kit positive human cardiac stem cells, called Multipotent Adult Stem Cells (MASCs), grown within three-dimensional collagen scaffolds (3D), to establish whether they could be used in post-infarction cardiac regeneration. We also evaluated the expression levels of the Granulocyte Macrophage-Colony Stimulating Factor Receptor (GM-CSFR) and endoglin, a component of the Transforming Growth Factor beta (TGF-ß) receptor complex. Finally, we also evaluated the expression of the α2ß1integrin. MASCs cultured within 3D collagen matrices are able to proliferate and migrate even in the absence of chemotactic agents and express high levels of factors involved in cell proliferation and migration, such as GM-CSFRα chain and integrins. They therefore represent a promising approach to tissue engineering aimed to restore cardiac function. Our results also suggest a role of GM-CSF in cell proliferation, while TGF-ß does not seem to be relevant.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Multipotentes/citologia , Engenharia Tecidual/métodos , Tecidos Suporte , Células-Tronco Adultas/metabolismo , Técnicas de Cultura de Células , Movimento Celular , Proliferação de Células , Separação Celular , Colágeno/química , Endoglina/genética , Endoglina/metabolismo , Expressão Gênica , Humanos , Integrina alfa2beta1/genética , Integrina alfa2beta1/metabolismo , Células-Tronco Multipotentes/metabolismo , Infarto do Miocárdio , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
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