RESUMO
A number of stressors and inflammatory mediators (cytokines, proteases, oxidative stress mediators) released during inflammation or ischemia stimulate and activate cells in blood, the vessel wall or tissues. The most well-known functional and phenotypic responses of activated cells are (1) the immediate expression and/or release of stored or newly synthesized bioactive molecules, and (2) membrane blebbing followed by release of microvesicles. An ultimate response, namely the formation of extracellular traps by neutrophils (NETs), is outside the scope of this work. The main objective of this article is to provide an overview on the mechanism of plasminogen reception and activation at the surface of cell-derived microvesicles, new actors in fibrinolysis and proteolysis. The role of microvesicle-bound plasmin in pathological settings involving inflammation, atherosclerosis, angiogenesis, and tumour growth, remains to be investigated. Further studies are necessary to determine if profibrinolytic microvesicles are involved in a finely regulated equilibrium with pro-coagulant microvesicles, which ensures a balanced haemostasis, leading to the maintenance of vascular patency.
Assuntos
Micropartículas Derivadas de Células , Fibrinólise , Plasminogênio , Proteólise , Humanos , Vasos Sanguíneos/metabolismo , Fibrinolisina/metabolismo , Fibrinólise/fisiologia , Inflamação/metabolismo , Plasminogênio/metabolismo , Micropartículas Derivadas de Células/metabolismoRESUMO
In the last ten years, endothelial progenitor cells (EPCs) have gained interest as an attractive cell population in regenerative medicine for vascular applications. This population is defined as the precursor of endothelial mature cells (ECs) through a process of differentiation. To our knowledge, no single marker can be used to discriminate them from mature ECs. To effectively study their differentiation kinetics, gene expression must be assessed. Quantitative real-time PCR (RT-qPCR) is widely used to analyze gene expression. To minimize the impact of variances from RT-qPCR, a rigorous selection of reference genes must be performed prior to any experiments due to variations in experimental conditions. In this study, CD34+ mononuclear cells were extracted from human cord blood and differentiated into EPCs after seeding for a maximum period of 21 days. To choose the best combinations of reference genes, we compared the results of EPCs, CD34+ mononuclear cells, and mature endothelial cells to ensure that the differentiation kinetics did not affect the expression of our selected reference genes. The expression levels of seven genes, namely, YWHAZ, GAPDH, HPRT1, RPLP0, UBC, B2M, and TBP were thus compared. The algorithms geNorm, NormFinder, BestKeeper, and the Comparative ΔCt method were employed to assess the expression of each candidate gene. Overall results reveal that the expression stability of reference genes may differ depending on the statistical program used. YWHAZ, GAPDH, and UBC composed the optimal set of reference genes for the gene expression studies performed by RT-qPCR in our experimental conditions. This work can thus serve as a starting point for the selection of candidate reference genes to normalize the levels of gene expression in endothelial progenitor cell populations.
Assuntos
Células Progenitoras Endoteliais/metabolismo , Perfilação da Expressão Gênica , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Algoritmos , Diferenciação Celular/genética , Células Cultivadas , Sangue Fetal/metabolismo , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Humanos , Leucócitos Mononucleares/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
Annexin-A5 (AnxA5) is the smallest member of the annexins, a group of soluble proteins that bind to membranes containing negatively-charged phospholipids, principally phosphatidylserine, in a Ca(2+)-dependent manner. AnxA5 presents unique properties of binding and self-assembling on membrane surfaces, forming highly ordered two-dimensional (2D) arrays. We showed previously that AnxA5 plays a central role in the machinery of cell membrane repair of murine perivascular cells, promoting the resealing of membrane damages via the formation of 2D protein arrays at membrane disrupted sites and preventing the extension of membrane ruptures. As the placenta is one of the richest source of AnxA5 in humans, we investigated whether AnxA5 was involved in membrane repair in this organ. We addressed this question at the level of human trophoblasts, either mononucleated cytotrophoblasts or multinucleated syncytiotrophoblasts, in choriocarcinoma cells and primary trophoblasts. Using established procedure of laser irradiation and fluorescence microscopy, we observed that both human cytotrophoblasts and syncytiotrophoblasts repair efficiently a µm²-size disruption. Compared to wild-type cells, AnxA5-deficient trophoblasts exhibit severe defect of membrane repair. Through specifically binding to the disrupted site as early as a few seconds after membrane wounding, AnxA5 promotes membrane resealing of injured human trophoblasts. In addition, we observed that a large membrane area containing the disrupted site was released in the extracellular milieu. We propose mechanisms ensuring membrane resealing and subsequent lesion removal in human trophoblasts. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Assuntos
Anexina A5/metabolismo , Membrana Celular/metabolismo , Trofoblastos/metabolismo , Anexina A5/genética , Linhagem Celular Tumoral , Membrana Celular/patologia , Feminino , Humanos , Gravidez , Trofoblastos/patologiaRESUMO
In this paper, we describe a simple and powerful way to synthesize antibacterial biomaterials with applications as implants in orthopedic surgery. Such implants are obtained by covalently grafting onto the Ti90A16 V4 alloy surface with vancomycin-functionalized nanoparticles. Nanoparticles were produced by ring-opening metathesis polymerization of α-norbornenyl-ω-vancomycin poly(ethylene oxide) macromonomers. Vancomycin is an interesting candidate because of its use in the field of implant associated infection as it is a glycopeptide which acts on bacterial walls. As a consequence, vancomycin does not need to be released for it to be active. In the first part of this paper, the synthesis and the complete characterization of these materials are described. In a second part, the in vitro antibacterial behavior is analyzed and discussed.
Assuntos
Antibacterianos/química , Materiais Revestidos Biocompatíveis/síntese química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Nanopartículas/química , Próteses e Implantes/microbiologia , Vancomicina/química , Antibacterianos/farmacologia , Materiais Revestidos Biocompatíveis/química , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Polietilenoglicóis/química , Propriedades de Superfície , Titânio/química , Vancomicina/farmacologiaRESUMO
BACKGROUND: Plasminogen activation is a ubiquitous source of fibrinolytic and proteolytic activity. Besides its role in prevention of thrombosis, plasminogen is involved in inflammatory reactions in the central nervous system. Plasminogen has been detected in the cerebrospinal fluid (CSF) of patients with inflammatory diseases; however, its origin remains controversial, as the blood-CSF barrier may restrict its diffusion from blood. METHODS: We investigated the origin of plasminogen in CSF using Alexa Fluor 488-labelled rat plasminogen injected into rats with systemic inflammation and blood-CSF barrier dysfunction provoked by lipopolysaccharide (LPS). Near-infrared fluorescence imaging and immunohistochemistry fluorescence microscopy were used to identify plasminogen in brain structures, its concentration and functionality were determined by Western blotting and a chromogenic substrate assay, respectively. In parallel, plasminogen was investigated in CSF from patients with Guillain-Barré syndrome (n = 15), multiple sclerosis (n = 19) and noninflammatory neurological diseases (n = 8). RESULTS: Endogenous rat plasminogen was detected in higher amounts in the CSF and urine of LPS-treated animals as compared to controls. In LPS-primed rats, circulating Alexa Fluor 488-labelled rat plasminogen was abundantly localized in the choroid plexus, CSF and urine. Plasminogen in human CSF was higher in Guillain-Barré syndrome (median = 1.28 ng/µl (interquartile range (IQR) = 0.66 to 1.59)) as compared to multiple sclerosis (median = 0.3 ng/µl (IQR = 0.16 to 0.61)) and to noninflammatory neurological diseases (median = 0.27 ng/µl (IQR = 0.18 to 0.35)). CONCLUSIONS: Our findings demonstrate that plasminogen is transported from circulating blood into the CSF of rats via the choroid plexus during inflammation. Our data suggest that a similar mechanism may explain the high CSF concentrations of plasminogen detected in patients with inflammation-derived CSF barrier impairment.
Assuntos
Barreira Hematoencefálica/fisiologia , Inflamação/sangue , Inflamação/líquido cefalorraquidiano , Plasminogênio/líquido cefalorraquidiano , Animais , Western Blotting , Humanos , Masculino , Microscopia de Fluorescência , Ratos , Ratos WistarRESUMO
Exposure to ionizing radiation can induce genetic aberrations via unrepaired DNA strand breaks. To investigate quantitatively the dose-effect relationship at the molecular level, we irradiated dry pBR322 plasmid DNA with 3 MeV protons and assessed fragmentation yields at different radiation doses using long-read sequencing from Oxford Nanopore Technologies. This technology applied to a reference DNA model revealed dose-dependent fragmentation, as evidenced by read length distributions, showing no discernible radiation sensitivity in specific genetic sequences. In addition, we propose a method for directly measuring the single-strand break (SSB) yield. Furthermore, through a comparative study with a collection of previous works on dry DNA irradiation, we show that the irradiation protocol leads to biases in the definition of ionizing sources. We support this scenario by discussing the size distributions of nanopore sequencing reads in the light of Geant4 and Geant4-DNA simulation toolkit predictions. We show that integrating long-read sequencing technologies with advanced Monte Carlo simulations paves a promising path toward advancing our comprehension and prediction of radiation-induced DNA fragmentation.
Assuntos
Fragmentação do DNA , Método de Monte Carlo , Plasmídeos , Plasmídeos/genética , Fragmentação do DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Análise de Sequência de DNA/métodos , Quebras de DNA de Cadeia Simples/efeitos da radiação , DNA/genéticaRESUMO
This study aimed to explore the potential of metal oxides such as Titanate Scrolled Nanosheets (TNs) in improving the radiosensitivity of sarcoma cell lines. Enhancing the response of cancer cells to radiation therapy is crucial, and one promising approach involves utilizing metal oxide nanoparticles. We focused on the impact of exposing two human sarcoma cell lines to both TNs and ionizing radiation (IR). Our research was prompted by previous in vitro toxicity assessments, revealing a correlation between TNs' toxicity and alterations in intracellular calcium homeostasis. A hydrothermal process using titanium dioxide powder in an alkaline solution produced the TNs. Our study quantified the intracellular content of TNs and analyzed their impact on radiation-induced responses. This assessment encompassed PIXE analysis, cell proliferation, and transcriptomic analysis. We observed that sarcoma cells internalized TNs, causing alterations in intracellular calcium homeostasis. We also found that irradiation influence intracellular calcium levels. Transcriptomic analysis revealed marked disparities in the gene expression patterns between the two sarcoma cell lines, suggesting a potential cell-line-dependent nano-sensitization to IR. These results significantly advance our comprehension of the interplay between TNs, IR, and cancer cells, promising potential enhancement of radiation therapy efficiency.
Assuntos
Nanopartículas Metálicas , Sarcoma , Humanos , Cálcio , Óxidos , Perfilação da Expressão Gênica , Sarcoma/genética , Tolerância a RadiaçãoRESUMO
We describe a methodology to manipulate Caenorhabditis elegans (C. elegans) and irradiate the stem progenitor gonad region using three MeV protons at a specific developmental stage (L1). The consequences of the targeted irradiation were first investigated by considering the organogenesis of the vulva and gonad, two well-defined and characterized developmental systems in C. elegans. In addition, we adapted high-throughput analysis protocols, using cell-sorting assays (COPAS) and whole transcriptome analysis, to the limited number of worms (>300) imposed by the selective irradiation approach. Here, the presented status report validated protocols to (i) deliver a controlled dose in specific regions of the worms; (ii) immobilize synchronized worm populations (>300); (iii) specifically target dedicated cells; (iv) study the radiation-induced developmental alterations and gene induction involved in cellular stress (heat shock protein) and cuticle injury responses that were found.
RESUMO
Fibrinolysis and pericellular proteolysis depend on molecular coassembly of plasminogen and its activator on cell, fibrin, or matrix surfaces. We report here the existence of a fibrinolytic cross-talk mechanism bypassing the requirement for their molecular coassembly on the same surface. First, we demonstrate that, despite impaired binding of Glu-plasminogen to the cell membrane by epsilon-aminocaproic acid (epsilon-ACA) or by a lysine-binding site-specific mAb, plasmin is unexpectedly formed by cell-associated urokinase (uPA). Second, we show that Glu-plasminogen bound to carboxy-terminal lysine residues in platelets, fibrin, or extracellular matrix components (fibronectin, laminin) is transformed into plasmin by uPA expressed on monocytes or endothelial cell-derived microparticles but not by tissue-type plasminogen activator (tPA) expressed on neurons. A 2-fold increase in plasmin formation was observed over activation on the same surface. Altogether, these data indicate that cellular uPA but not tPA expressed by distinct cells is specifically involved in the recognition of conformational changes and activation of Glu-plasminogen bound to other biologic surfaces via a lysine-dependent mechanism. This uPA-driven cross-talk mechanism generates plasmin in situ with a high efficiency, thus highlighting its potential physiologic relevance in fibrinolysis and matrix proteolysis induced by inflammatory cells or cell-derived microparticles.
Assuntos
Fibrinolisina/metabolismo , Fibrinólise/fisiologia , Ácido Aminocaproico/farmacologia , Animais , Antifibrinolíticos/farmacologia , Comunicação Celular/fisiologia , Células Cultivadas , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibrinólise/efeitos dos fármacos , Humanos , Camundongos , Plasminogênio/metabolismo , Ativadores de Plasminogênio/metabolismo , Processamento de Proteína Pós-Traducional , Receptor Cross-Talk/efeitos dos fármacos , Receptor Cross-Talk/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
BACKGROUND: We recently assigned a new fibrinolytic function to cell-derived microparticles in vitro. In this study we explored the relevance of this novel property of microparticles to the in vivo situation. DESIGN AND METHODS: Circulating microparticles were isolated from the plasma of patients with thrombotic thrombocytopenic purpura or cardiovascular disease and from healthy subjects. Microparticles were also obtained from purified human blood cell subpopulations. The plasminogen activators on microparticles were identified by flow cytometry and enzyme-linked immunosorbent assays; their capacity to generate plasmin was quantified with a chromogenic assay and their fibrinolytic activity was determined by zymography. RESULTS: Circulating microparticles isolated from patients generate a range of plasmin activity at their surface. This property was related to a variable content of urokinase-type plasminogen activator and/or tissue plasminogen activator. Using distinct microparticle subpopulations, we demonstrated that plasmin is generated on endothelial and leukocyte microparticles, but not on microparticles of platelet or erythrocyte origin. Leukocyte-derived microparticles bear urokinase-type plasminogen activator and its receptor whereas endothelial microparticles carry tissue plasminogen activator and tissue plasminogen activator/inhibitor complexes. CONCLUSIONS: Endothelial and leukocyte microparticles, bearing respectively tissue plasminogen activator or urokinase-type plasminogen activator, support a part of the fibrinolytic activity in the circulation which is modulated in pathological settings. Awareness of this blood-borne fibrinolytic activity conveyed by microparticles provides a more comprehensive view of the role of microparticles in the hemostatic equilibrium.
Assuntos
Doenças Cardiovasculares/sangue , Micropartículas Derivadas de Células/metabolismo , Endotélio Vascular/patologia , Fibrinólise/fisiologia , Leucócitos/patologia , Púrpura Trombocitopênica Trombótica/sangue , Doenças Cardiovasculares/patologia , Estudos de Casos e Controles , Células Cultivadas , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Fibrinolisina/metabolismo , Citometria de Fluxo , Humanos , Leucócitos/metabolismo , Púrpura Trombocitopênica Trombótica/patologia , Artéria Renal/citologia , Artéria Renal/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Cell activation by stressors is characterized by a sequence of detectable phenotypic cell changes. A given stimulus, depending on its strength, induces modifications in the activity of membrane phospholipid transporters and calpains, which lead to phosphatidylserine exposure, membrane blebbing and the release of microparticles (nanoscale membrane vesicles). This vesiculation could be considered as a warning signal that may be followed, if the stimulus is maintained, by cell detachment-induced apoptosis. In the present study, plasminogen incubated with adherent cells is converted into plasmin by constitutively expressed tPA (tissue-type plasminogen activator) or uPA (urokinase-type plasminogen activator). Plasmin formed on the cell membrane then induces a unique response characterized by membrane blebbing and vesiculation. Hitherto unknown for plasmin, these membrane changes are similar to those induced by thrombin on platelets. If plasmin formation persists, matrix proteins are then degraded, cells lose their attachments and enter the apoptotic process, characterized by DNA fragmentation and specific ultrastructural features. Since other proteolytic or inflammatory stimuli may evoke similar responses in different types of adherent cells, the proposed experimental procedure can be used to distinguish activated adherent cells from cells entering the apoptotic process. Such a distinction is crucial for evaluating the effects of mediators, inhibitors and potential therapeutic agents.
Assuntos
Plaquetas/fisiologia , Adesão Celular/fisiologia , Fibrinolisina/fisiologia , Animais , Apoptose , Plaquetas/citologia , Western Blotting , Células CHO , Sobrevivência Celular/fisiologia , Cricetinae , Cricetulus , Fibrinolisina/biossíntese , Fibrinolisina/química , Humanos , Marcação In Situ das Extremidades Cortadas , Cinética , Microscopia Eletrônica , Plasminogênio/química , Plasminogênio/genética , Plasminogênio/isolamento & purificação , Ativador de Plasminogênio Tecidual/fisiologiaRESUMO
Accurate sizing of nanoparticles in biological media is important for drug delivery and biomedical imaging applications since size directly influences the nanoparticle processing and nanotoxicity in vivo. Using fluorescence single particle tracking we have succeeded for the first time in following the aggregation of drug delivery nanoparticles in real time in undiluted whole blood. We demonstrate that, by using a suitable surface functionalization, nanoparticle aggregation in the blood circulation is prevented to a large extent.
Assuntos
Análise Química do Sangue/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Nanoestruturas/análise , Nanoestruturas/ultraestrutura , Tamanho da Partícula , HumanosRESUMO
Recombinant tissue-type plasminogen activator (tPA) is the fibrinolytic drug of choice to treat stroke patients. However, a growing body of evidence indicates that besides its beneficial thrombolytic role, tPA can also have a deleterious effect on the ischaemic brain. Although ageing influences stroke incidence, complications and outcome, age-dependent relationships between endogenous tPA and stroke injuries have not been investigated yet. Here, we report that ageing is associated with a selective lowering of brain tPA expression in the murine brain. Moreover, our results show that albumin D site-binding protein (DBP) as a key age-associated regulator of the neuronal transcription of tPA. Additionally, inhibition of DBP-mediated tPA expression confers in vitro neuroprotection. Accordingly, reduced levels of tPA in old mice are associated with smaller excitotoxic/ischaemic injuries and protection of the permeability of the neurovascular unit during cerebral ischaemia. Likewise, we provide neuroradiological evidence indicating the existence of an inverse relationship between age and the volume of the ischaemic lesion in patients with acute ischaemic stroke. Together, these results indicate that the relationship among DBP, tPA and ageing play an important role in the outcome of cerebral ischaemia.
Assuntos
Envelhecimento/metabolismo , Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Proteínas de Ligação a DNA/fisiologia , Ativador de Plasminogênio Tecidual/metabolismo , Fatores de Transcrição/fisiologia , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Animais , Isquemia Encefálica/patologia , Morte Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes/métodos , Humanos , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , N-Metilaspartato/toxicidade , Neurônios/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Transcrição/genéticaRESUMO
Microparticles (MPs) are membrane fragments shed by cells activated by a variety of stimuli including serine proteases, inflammatory cytokines, growth factors, and stress inducers. MPs originating from platelets, leukocytes, endothelial cells, and erythrocytes are found in circulating blood at relative concentrations determined by the pathophysiological context. The procoagulant activity of MPs is their most characterized property as a determinant of thrombosis in various vascular and systemic diseases including myocardial infarction and diabetes. An increase in circulating MPs has also been associated with ischemic cerebrovascular accidents, transient ischemic attacks, multiple sclerosis, and cerebral malaria. Recent data indicate that besides their procoagulant components and identity antigens, MPs bear a number of bioactive effectors that can be disseminated, exchanged, and transferred via MPs cell interactions. Furthermore, as activated parenchymal cells may also shed MPs carrying identity antigens and biomolecules, MPs are now emerging as new messengers/biomarkers from a specific tissue undergoing activation or damage. Thus, detection of MPs of neurovascular origin in biological fluids such as CSF or tears, and even in circulating blood in case of blood-brain barrier leakage, would not only improve our comprehension of neurovascular pathophysiology, but may also constitute a powerful tool as a biomarker in disease prediction, diagnosis, prognosis, and follow-up.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Hemostasia/fisiologia , Humanos , Mediadores da Inflamação/sangue , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Neovascularização Patológica/sangue , Neovascularização Patológica/patologiaRESUMO
Advances in microscopy with new visualization possibilities often bring dramatic progress to our understanding of the intriguing cellular machinery. Picosecond optoacoustic micro-spectroscopy is an optical technique based on ultrafast pump-probe generation and detection of hypersound on time durations of picoseconds and length scales of nanometers. It is experiencing a renaissance as a versatile imaging tool for cell biology research after a plethora of applications in solid-state physics. In this emerging context, this work reports on a dual-probe architecture to carry out real-time parallel detection of the hypersound propagation inside a cell that is cultured on a metallic substrate, and of the hypersound reflection at the metal/cell adhesion interface. Using this optoacoustic modality, several biophysical properties of the cell can be measured in a noncontact and label-free manner. Its abilities are demonstrated with the multiple imaging of a mitotic macrophage-like cell in a single run experiment.
Assuntos
Microscopia/métodos , Técnicas Fotoacústicas/métodos , Análise de Célula Única , Linhagem Celular , Humanos , Razão Sinal-Ruído , Fatores de TempoRESUMO
Cell morphological analysis has long been used in cell biology and physiology for abnormality identification, early cancer detection, and dynamic change analysis under specific environmental stresses. This work reports on the remote mapping of cell 3D morphology with an in-plane resolution limited by optics and an out-of-plane accuracy down to a tenth of the optical wavelength. For this, GHz coherent acoustic phonons and their resonance harmonics were tracked by means of an ultrafast opto-acoustic technique. After illustrating the measurement accuracy with cell-mimetic polymer films we map the 3D morphology of an entire osteosarcoma cell. The resulting image complies with the image obtained by standard atomic force microscopy, and both reveal very close roughness mean values. In addition, while scanning macrophages and monocytes, we demonstrate an enhanced contrast of thickness mapping by taking advantage of the detection of high-frequency resonance harmonics. Illustrations are given with the remote quantitative imaging of the nucleus thickness gradient of migrating monocyte cells.
Assuntos
Forma Celular , Imageamento Tridimensional , Fônons , Análise de Célula Única , Acústica , Linhagem Celular Tumoral , Humanos , Macrófagos/patologia , Monócitos/patologia , Óptica e Fotônica , Osteossarcoma/diagnóstico por imagem , Osteossarcoma/patologia , Polimetil Metacrilato/químicaAssuntos
Membrana Celular/fisiologia , Vesículas Citoplasmáticas/fisiologia , Fibrinólise/fisiologia , Plasminogênio/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Trombina/antagonistas & inibidores , Trombina/metabolismo , Trombose/prevenção & controle , Animais , Vesículas Citoplasmáticas/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Ativadores de Plasminogênio/biossíntese , Ativadores de Plasminogênio/genética , Receptor Cross-Talk/fisiologia , Trombose/metabolismo , Trombose/patologiaRESUMO
Laser-generated GHz-ultrasonic-based technologies have shown the ability to image single cell adhesion and stiffness simultaneously. Using this new modality, we here demonstrate quantitative indicators to investigate contact mechanics and adhesion processes of the cell. We cultured human cells on a rigid substrate, and we used an inverted pulsed opto-acoustic microscope to generate acoustic pulses containing frequencies up to 100 GHz in the substrate. We map the reflection of the acoustic pulses at the cell-substrate interface to obtain images of the acoustic impedance of the cell, Zc, as well as of the stiffness of the interface, K, with 1 µm lateral resolution. Our results show that the standard deviation ΔZc reveals differences between different cell types arising from the multiplicity of local conformations within the nucleus. From the distribution of K-values within the nuclear region, we extract a mean interfacial stiffness, Km, that quantifies the average contact force in areas of the cell displaying weak bonding. By analogy with classical contact mechanics, we also define the ratio of the real to nominal contact areas, Sr/St. We show that Km can be interpreted as a quantitative indicator of passive contact at metal-cell interfaces, while Sr/St is sensitive to active adhesive processes in the nuclear region. The ability to separate the contributions of passive and active adhesion processes should allow gaining insight into cell-substrate interactions, with important applications in tissue engineering.
Assuntos
Adesão Celular , Microscopia Acústica , Humanos , Engenharia Tecidual , UltrassomRESUMO
Within the native microenvironment, extracellular matrix (ECM) components are thought to display a complex and heterogeneous distribution, spanning several length scales. Herein, the objective is to mimic, in vitro, the hierarchical organization of proteins and growth factors as well as their crosstalk. Photolithography technique was used to adjacently pattern geometrically defined regions of RGD and BMP-2 mimetic peptides onto glass substrates. These ECM-derived ligands are known to jointly regulate mesenchymal stem cells (MSCs) osteogenic differentiation. By manipulating the spatial distribution of dually grafted peptides, the extent of human MSCs osteogenic differentiation was significantly affected, depending on the shape of peptide micropatterns. Our data highlight the existence of a strong interplay between geometric cues and biochemical signals. Such in vitro systems provide a valuable tool to investigate mechanisms by which multiple ECM cues overlap to regulate stem cell fate, thereby contributing to the design of bioinspired biomaterials for bone tissue engineering applications.
RESUMO
Intravascular devices for continuous glucose monitoring are promising tools for the follow up and treatment of diabetic patients. Limiting the inflammatory response to the implanted devices in order to achieve better biocompatibility is a critical challenge. Herein we report on the production and the characterization of gold surfaces covalently derivatized with the peptide α-alpha-melanocyte stimulating hormone (α-MSH), with a quantifiable surface density. In vitro study demonstrated that the tethered α-MSH is able to decrease the expression of an inflammatory cytokine produced by endothelial cells.