RESUMEN
Due to its high metastatic potential, malignant melanoma is one of the deadliest skin cancers. In melanoma as well as in other cancers, acidification of the tumor microenvironment (=TME, inverse pH-gradient) is a well-known driver of tumor progression and metastasis. Membrane-bound receptors, such as the proton-sensitive GPCR (pH-GPCR) GPR4, are considered as potential initiators of the signalling cascades relevant to malignant transformation. In this study, we investigated the pH-dependent migration of GPR4 wildtype/overexpressing SK-Mel-28 cells using an impedance-based electrical wounding and migration assay and classical Boyden chamber experiments. Migration of GPR4 overexpressing SK-Mel-28 cells was enhanced in a range of pH 6.5-7.5 as compared to controls in the impedance-based electrical wounding and migration assay. In Boyden chamber experiments, GPR4 overexpression only increased migration at pH 7.5 in a Matrigel-free setup, but not at pH 6.5. Results indicate that GPR4 is involved in the migration of melanoma cells, especially in the tumor periphery, and that this process is affected by pH in the TME.
Asunto(s)
Melanoma , Receptores Acoplados a Proteínas G , Neoplasias Cutáneas , Humanos , Concentración de Iones de Hidrógeno , Melanoma/patología , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Cutáneas/patología , Microambiente Tumoral , Línea Celular TumoralRESUMEN
Mitochondria exert important control over plasma membrane (PM) Orai1 channels mediating store-operated Ca2+ entry (SOCE). Although the sensing of endoplasmic reticulum (ER) Ca2+ stores by STIM proteins and coupling to Orai1 channels is well understood, how mitochondria communicate with Orai1 channels to regulate SOCE activation remains elusive. Here, we reveal that SOCE is accompanied by a rise in cytosolic Na+ that is critical in activating the mitochondrial Na+/Ca2+ exchanger (NCLX) causing enhanced mitochondrial Na+ uptake and Ca2+ efflux. Omission of extracellular Na+ prevents the cytosolic Na+ rise, inhibits NCLX activity, and impairs SOCE and Orai1 channel current. We show further that SOCE activates a mitochondrial redox transient which is dependent on NCLX and is required for preventing Orai1 inactivation through oxidation of a critical cysteine (Cys195) in the third transmembrane helix of Orai1. We show that mitochondrial targeting of catalase is sufficient to rescue redox transients, SOCE, and Orai1 currents in NCLX-deficient cells. Our findings identify a hitherto unknown NCLX-mediated pathway that coordinates Na+ and Ca2+ signals to effect mitochondrial redox control over SOCE.
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Calcio/metabolismo , Mitocondrias/metabolismo , Proteína ORAI1/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Sodio/metabolismo , Línea Celular , Humanos , Proteínas Mitocondriales , Oxidación-ReducciónRESUMEN
Solid tumors exhibit an inversed pH gradient with increased intracellular pH (pHi ) and decreased extracellular pH (pHe ). This inside-out pH gradient is generated via sodium/hydrogen antiporter 1, vacuolar-type H + ATPases, monocarboxylate transporters, (bi)carbonate (co)transporters and carboanhydrases. Our knowledge on how pHe -signals are sensed and what the respective receptors induce inside cells is scarce. Some pH-sensitive receptors (GPR4, GPR65/TDAG8, GPR68/OGR1, GPR132/G2A, possibly GPR31 and GPR151) and ion channels (acid-sensing ion channels ASICs, transient receptor potential vanilloid receptors TRPVs) transduce signals inside cells. As little is known on the expression and function of these pH sensors, we used immunostainings to study tissue samples from common and rare skin cancers. Our current and future work is directed towards investigating the impact of all the pH-sensing receptors in different skin tumors using cell culture techniques with selective knockdown/knockout (siRNA/CRISPR-Cas9). To study cell migration and proliferation, novel impedance-based wound healing assays have been developed and are used. The field of pH sensing in tumors and wounds holds great promise for the development of pH-targeting therapies, either against pH regulators or sensors to inhibit cell proliferation and migration.
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Canales Iónicos Sensibles al Ácido/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Cutáneas/química , Neoplasias Cutáneas/metabolismo , Canales Catiónicos TRPV/metabolismo , Movimiento Celular , Proliferación Celular , Humanos , Concentración de Iones de Hidrógeno , Transducción de SeñalRESUMEN
Endothelial barrier function is tightly regulated by plasma membrane receptors and is crucial for tissue fluid homeostasis; its dysfunction causes disease, including sepsis and inflammation. The ubiquitous activation of Ca2+ signaling upon phospholipase C-coupled receptor ligation leads quite naturally to the assumption that Ca2+ signaling is required for receptor-regulated endothelial barrier function. This widespread hypothesis draws analogy from smooth muscle and proposes the requirement of G protein-coupled receptor (GPCR)-generated Ca2+ signaling in activating the endothelial contractile apparatus and generating interendothelial gaps. Notwithstanding endothelia being non-excitable in nature, the hypothesis of Ca2+-induced endothelial contraction has been invoked to explain actions of GPCR agonists that either disrupt or stabilize endothelial barrier function. Here, we challenge this correlative hypothesis by showing a lack of causal link between GPCR-generated Ca2+ signaling and changes in human microvascular endothelial barrier function. We used three endogenous GPCR agonists: thrombin and histamine, which disrupt endothelial barrier function, and sphingosine-1-phosphate, which stabilizes barrier function. The qualitatively different effects of these three agonists on endothelial barrier function occur independently of Ca2+ entry through the ubiquitous store-operated Ca2+ entry channel Orai1, global Ca2+ entry across the plasma membrane, and Ca2+ release from internal stores. However, disruption of endothelial barrier function by thrombin and histamine requires the Ca2+ sensor stromal interacting molecule-1 (STIM1), whereas sphingosine-1-phosphate-mediated enhancement of endothelial barrier function occurs independently of STIM1. We conclude that although STIM1 is required for GPCR-mediated disruption of barrier function, a causal link between GPCR-induced cytoplasmic Ca2+ increases and acute changes in barrier function is missing. Thus, the cytosolic Ca2+-induced endothelial contraction is a cum hoc fallacy that should be abandoned.
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Señalización del Calcio , Células Endoteliales/metabolismo , Calcio/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Lisofosfolípidos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Trombina/genética , Trombina/metabolismoRESUMEN
Leukotriene-C4 synthase (LTC4S) generates LTC4 from arachidonic acid metabolism. LTC4 is a proinflammatory factor that acts on plasma membrane cysteinyl leukotriene receptors. Recently, however, we showed that LTC4 was also a cytosolic second messenger that activated store-independent LTC4-regulated Ca(2+) (LRC) channels encoded by Orai1/Orai3 heteromultimers in vascular smooth muscle cells (VSMCs). We showed that Orai3 and LRC currents were up-regulated in medial and neointimal VSMCs after vascular injury and that Orai3 knockdown inhibited LRC currents and neointimal hyperplasia. However, the role of LTC4S in neointima formation remains unknown. Here we show that LTC4S knockdown inhibited LRC currents in VSMCs. We performed in vivo experiments where rat left carotid arteries were injured using balloon angioplasty to cause neointimal hyperplasia. Neointima formation was associated with up-regulation of LTC4S protein expression in VSMCs. Inhibition of LTC4S expression in injured carotids by lentiviral particles encoding shRNA inhibited neointima formation and inward and outward vessel remodeling. LRC current activation did not cause nuclear factor for activated T cells (NFAT) nuclear translocation in VSMCs. Surprisingly, knockdown of either LTC4S or Orai3 yielded more robust and sustained Akt1 and Akt2 phosphorylation on Ser-473/Ser-474 upon serum stimulation. LTC4S and Orai3 knockdown inhibited VSMC migration in vitro with no effect on proliferation. Akt activity was suppressed in neointimal and medial VSMCs from injured vessels at 2 weeks postinjury but was restored when the up-regulation of either LTC4S or Orai3 was prevented by shRNA. We conclude that LTC4S and Orai3 altered Akt signaling to promote VSMC migration and neointima formation.
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Canales de Calcio/metabolismo , Glutatión Transferasa/biosíntesis , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima/metabolismo , Animales , Canales de Calcio/genética , Regulación Enzimológica de la Expresión Génica/genética , Glutatión Transferasa/genética , Hiperplasia/genética , Hiperplasia/metabolismo , Hiperplasia/patología , Masculino , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Neointima/genética , Neointima/patología , Proteína ORAI1 , Fosforilación/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/genéticaRESUMEN
Intraocular pressure (IOP) is mostly regulated by aqueous humor outflow through the human trabecular meshwork (HTM) and represents the only modifiable risk factor of glaucoma. The lack of IOP-modulating therapeutics that targets HTM underscores the need of engineering HTM for understanding the outflow physiology and glaucoma pathology in vitro. Using a 3D HTM model that allows for regulation of outflow in response to a pharmacologic steroid, a fibrotic state has been induced resembling that of glaucomatous HTM. This disease model exhibits HTM marker expression, ECM overproduction, impaired HTM cell phagocytic activity and outflow resistance, which represent characteristics found in steroid-induced glaucoma. In particular, steroid-induced ECM alterations in the glaucomatous model can be modified by a ROCK inhibitor. Altogether, this work presents a novel in vitro disease model that allows for physiological and pathological studies pertaining to regulating outflow, leading to improved understanding of steroid-induced glaucoma and accelerated discovery of new therapeutic targets. Biotechnol. Bioeng. 2016;113: 1357-1368. © 2015 Wiley Periodicals, Inc.
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Modelos Animales de Enfermedad , Glaucoma/patología , Técnicas de Cultivo de Órganos/métodos , Ingeniería de Tejidos/instrumentación , Andamios del Tejido , Malla Trabecular/patología , Animales , Células Cultivadas , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Impresión Tridimensional , Ingeniería de Tejidos/métodosRESUMEN
The past 20 years has seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists and pharmacological and toxicological compounds. Most studies on barrier function use G protein-coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance-based techniques such as electric cell-substrate impedance sensing (ECIS) resides in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications, and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research, little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency, or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling, and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine, and sphingosine-1-phosphate.
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Impedancia Eléctrica , Electrofisiología/métodos , Células Endoteliales/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Algoritmos , Células Cultivadas , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Humanos , Potenciales de la Membrana , Receptores Acoplados a Proteínas G/agonistasRESUMEN
We determined the in vivo role of stromal-interacting molecule 1 (STIM1) in the regulation of vascular function using endothelial cell (EC)- and smooth-muscle (SM)-specific knockout mice. Systolic blood pressure and glucose levels were similar in all mice (Stim1(SMC-/-), Stim1(SMC-/+), Stim1(EC-/-), Stim1(EC-/+)), but body weight was reduced in Stim1(EC-/-) and Stim1(SMC-/-) mice. The contraction of arteries in response to phenylephrine was significantly reduced in Stim1(SMC-/-) mice only. However, contraction to thromboxane and KCl was similar in all groups. The endothelium-dependent relaxation (EDR) was impaired in Stim1(EC-/+) and drastically reduced in Stim1(EC-/-) mice while the endothelium-independent vasorelaxation was similar among all groups. Acute downregulation of STIM1 in arteries reduced EDR and the contractile response to phenylephrine, while the contractile response to thromboxane was not affected. NADPH oxidase activity was increased only in Stim1(EC-/+) and Stim1(EC-/-) mice. Calcium (Ca(2+)) entry in endothelial cells stimulated with thrombin and histamine had the pharmacological features of store-operated Ca(2+) entry (SOCE) and was dependent on STIM1 expression. We conclude that STIM1 plays opposing roles in vascular smooth muscle vs. endothelial cells in the regulation of vascular reactivity.
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Canales de Calcio/metabolismo , Endotelio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Vasoconstricción , Vasodilatación , Animales , Canales de Calcio/genética , Señalización del Calcio , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/metabolismo , Arterias Mesentéricas/fisiología , Ratones , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Especificidad de Órganos , Fenilefrina/farmacología , Molécula de Interacción Estromal 1 , Tromboxanos/farmacología , Vasoconstrictores/farmacologíaRESUMEN
Store-operated Ca²âº entry (SOCE) is a receptor-regulated Ca²âº entry pathway that is both ubiquitous and evolutionarily conserved. SOCE is activated by depletion of intracellular Ca²âº stores through receptor-mediated production of inositol 1,4,5-trisphosphate (IP3). The depletion of endoplasmic reticulum (ER) Ca²âº is sensed by stromal interaction molecule 1 (STIM1). On store depletion, STIM1 aggregates and moves to areas where the ER comes close to the plasma membrane (PM; within 25 nm) to interact with Orai1 channels and activate Ca²âº entry. Ca²âº entry through store-operated Ca²âº (SOC) channels, originally thought to mediate the replenishment of Ca²âº stores, participate in active downstream signaling by coupling to the activation of enzymes and transcription factors that control a wide variety of long-term cell functions such as proliferation, growth, and migration. SOCE has also been proposed to contribute to short-term cellular responses such as muscle contractility. While there are significant STIM1/Orai1 protein levels and SOCE activity in adult skeletal muscle, the precise role of SOCE in skeletal muscle contractility is not clear. The dependence on SOCE during cardiac and smooth muscle contractility is even less certain. Here, we will hypothesize on the contribution of SOCE in muscle and its potential role in contractility and signaling.
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Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Músculo Esquelético/metabolismo , Músculo Liso/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Membrana Celular/metabolismo , Humanos , Proteína ORAI1 , Molécula de Interacción Estromal 1RESUMEN
We report on the first dual nanosensors for imaging of pH values and oxygen partial pressure in cells. The sensors have a unique nanostructure in that a soft core structure is rigidized with a silane reagent, while poly(ethylene glycol) chains form an outer shell. Lipophilic oxygen-sensitive probes and reference dyes are encapsulated inside the hydrophobic core, while a pH-sensitive probe is covalently attached to the poly(ethylene glycol) end-group on the shell. The core/shell structure renders the nanosensors well dispersed and highly stable in various kinds of aqueous media. Their average size is 12 nm, and they respond to both pH and oxygen in the physiological range. They do not pass cell membranes, but can be internalized into the cellular cytosol by electroporation, upon which they enable sensing and imaging of pH values and oxygen with high spatial resolution. The nanosensor strategy shown here is expected to be applicable to the development of various other kinds of multiple nanosensors for in vivo studies.
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Citosol/química , Nanotecnología/instrumentación , Nanotecnología/métodos , Oxígeno/análisis , Animales , Concentración de Iones de Hidrógeno , Riñón/citología , Modelos Moleculares , Estructura Molecular , Nanopartículas/química , Oxígeno/química , Polietilenglicoles/química , RatasRESUMEN
G protein-coupled receptors (GPCRs) are among the most heavily addressed drug targets in medicinal chemistry and pharmacology. The screening for new agonists or antagonists has been largely based on genetically engineered cells overexpressing the receptor to study binding of ligands directly or via intracellular signaling events downstream of receptor activation. These approaches are often invasive in nature, need to be conducted as endpoint assays, require isotope- or fluorophore-labeling and significant genetic manipulation. In contrast to that, non-invasive and label-free impedance measurements are capable of monitoring ligand-receptor interactions in target cells with endogenous receptor expression in real time. The cells expressing the receptor are grown on planar gold-film electrodes that are integrated into regular cell culture dishes. This article will highlight several impedance-based assay formats to characterize biomolecular interactions between ligands and their GPCRs in vitro, comprising agonist and antagonist characterization, dose-response relationships, receptor desensitization, and signal transduction profiling.
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Receptores Acoplados a Proteínas G , Transducción de Señal , Bioensayo , Impedancia Eléctrica , Ligandos , Receptores Acoplados a Proteínas G/agonistas , Transducción de Señal/fisiologíaRESUMEN
Specific intracellular manipulation of animal cells is a persistent goal in experimental cell biology. Such manipulations allow precise and targeted interference with signaling cascades, metabolic pathways, or bi-molecular interactions for subsequent tracking of functional consequences. However, most biomolecules capable of molecular recognition are membrane impermeable. The ability to introduce these molecules into the cytoplasm and then to apply appropriate readouts to monitor the corresponding cell response could prove to be an important research tool. This study describes such an experimental approach combining in situ electroporation (ISE) as a means to efficiently deliver biomolecules to the cytoplasm with an impedance-based, time-resolved analysis of cell status using electric cell-substrate impedance sensing (ECIS). In this approach, gold-film electrodes, deposited on the bottom of regular culture dishes, are used for both electroporation and monitoring. The design of the electrode layout and measurement chamber allows working with sample volumes as small as 10 µL. A miniaturized setup for combined electroporation and impedance sensing (µISE-ECIS) was applied to load different adherent cells with bioactive macromolecules including enzymes, antibodies, nucleic acids and quantum dot nanoparticles. The cell response after loading the cytoplasm with RNase A or cytochrome c (in the presence or absence of caspase inhibitors) was tracked by non-invasive impedance readings in real-time.
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ADN/análisis , Electroporación/métodos , Nanopartículas/química , Proteínas/análisisRESUMEN
The proton-sensing GPCRs (pH-GPCRs) GPR4 (GPR19), TDAG8 (GPR65, T-cell death associated gene 8), OGR1 (GPR68, ovarian cancer GPCR1), and G2A (GPR132, G2 accumulation protein) are involved in sensing and transducing changes in extracellular pH (pHe). Extracellular acidification is a central hallmark of solid cancer. pH-GPCR function has been associated with cancer cell proliferation, adhesion, migration and metastasis, as well as with modulation of the immune system. Little is known about the expression levels and role of pH-GPCRs in skin cancer. To better understand the functions of pH-GPCRs in skin cancer in vivo, we examined the expression-profiles of GPR4, TDAG8, OGR1 and G2A in four common skin tumors, i.e. squamous cell carcinoma (SCC), malignant melanoma (MM), compound nevus cell nevi (NCN), basal cell carcinoma (BCC). We performed immunohistochemistry and immunofluorescence staining on paraffin-embedded tissue samples acquired from patients suffering from SCC, MM, NCN or BCC. We show the expression of pH-GPCRs in four common skin cancers. Different expression patterns in the investigated skin cancer types indicate that the different pH-GPCRs may have distinct functions in tumor progression and serve as novel therapeutic targets.
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Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Cutáneas/metabolismo , Carcinoma Basocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Humanos , Melanoma/metabolismo , Neoplasias de Células Escamosas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nevo/metabolismo , Receptores de Neurotransmisores/metabolismo , Neoplasias Cutáneas/patología , Análisis de Matrices Tisulares , Melanoma Cutáneo MalignoRESUMEN
Mats of cytocompatible polymer fibers are needed as scaffolds in tissue engineering or as wound healing supports. Most recently, they have emerged as matrix-material to allow for in situ chemo- and biosensing inside intact tissue fragments or surrogates. Electrospinning of such fibers from polymer solutions provides extended options to control the structural and functional properties of the resulting fiber mats. We have prepared electrospun polymeric fiber mats from poly(lactic acid) (PLA), polystyrene (PS), and poly(vinyl pyrrolidone) (PVP) with two different fiber densities. Mats and individual fibers were characterized with respect to their dimensions, morphology, and their compatibility with human keratinocytes (HaCaT) selected as a biological model. Microscopic inspection revealed that HaCaT cells were viable on mats from all three polymers with only a negligible fraction of dead cells, similar to planar control surfaces. Growth in the presence of the fiber mats did not alter cellular metabolism (ATP, redox state) and did not induce significant production of cytokines (interleukin-6 (IL-6); monocyte chemoattractant protein-1 (MCP-1)). However, we did observe that fiber density changed the overall topography of the resulting mats and led to differences in the establishment of continuous cell sheets. In conclusion, the findings support the suitability of electrospun polymeric fiber mats made from PLA, PS, or PVP as potential biocompatible matrices for future two-dimensional (2D) or three-dimensional (3D) sensing of vital parameters from tissue in health and disease.
RESUMEN
Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample's impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).
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Bioensayo/métodos , Impedancia Eléctrica , Glioma/metabolismo , Histamina/metabolismo , Receptores Histamínicos/química , Receptores Histamínicos/metabolismo , Transducción de Señal , Glioma/patología , Humanos , Ligandos , Células Tumorales CultivadasRESUMEN
A new sacrificial molding process using a single mask has been developed to fabricate ultrathin 2-dimensional membranes from several biocompatible polymeric materials. The fabrication process is similar to a sacrificial microelectromechanical systems (MEMS) process flow, where a mold is created from a material that can be coated with a biodegradable polymer and subsequently etched away, leaving behind a very thin polymer membrane. In this work, two different sacrificial mold materials, silicon dioxide (SiO2 ) and Liftoff Resist (LOR) were used. Three different biodegradable materials; polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and polyglycidyl methacrylate (PGMA), were chosen as model polymers. We demonstrate that this process is capable of fabricating 200-500 nm thin, through-hole polymer membranes with various geometries, pore-sizes and spatial features approaching 2.5 µm using a mold fabricated via a single contact photolithography exposure. In addition, the membranes can be mounted to support rings made from either SU8 or PCL for easy handling after release. Cell culture compatibility of the fabricated membranes was evaluated with human dermal microvascular endothelial cells (HDMECs) seeded onto the ultrathin porous membranes, where the cells grew and formed confluent layers with well-established cell-cell contacts. Furthermore, human trabecular meshwork cells (HTMCs) cultured on these scaffolds showed similar proliferation as on flat PCL substrates, further validating its compatibility. All together, these results demonstrated the feasibility of our sacrificial fabrication process to produce biocompatible, ultra-thin membranes with defined microstructures (i.e., pores) with the potential to be used as substrates for tissue engineering applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1192-1201, 2016.
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Plásticos Biodegradables/química , Células Endoteliales/metabolismo , Membranas Artificiales , Dióxido de Silicio/química , Células Endoteliales/citología , Humanos , Ácido Láctico/química , Metilmetacrilatos/química , Poliésteres/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
Calcium (Ca(2+)) is a ubiquitous second messenger that regulates a plethora of physiological functions. Deregulation of calcium homeostasis has been reported in a wide variety of pathological conditions including cardiovascular disorders, cancer and neurodegenerative diseases. One of the most ubiquitous pathways involved in regulated Ca(2+) influx into cells is the store-operated Ca(2+) entry (SOCE) pathway. In 2006, Orai1 was identified as the channel protein that mediates SOCE in immune cells. Orai1 has two mammalian homologs, Orai2 and Orai3. Although Orai1 has been the most widely studied Orai isoform, Orai3 has recently received significant attention. Under native conditions, Orai3 was demonstrated to be an important component of store-independent arachidonate-regulated Ca(2+) (ARC) entry in HEK293 cells, and more recently of a store-independent leukotrieneC4-regulated Ca(2+) (LRC) entry pathway in vascular smooth muscle cells. Recent studies have shown upregulation of Orai3 in estrogen receptor-expressing breast cancers and a critical role for Orai3 in breast cancer development in immune-compromised mice. Orai3 upregulation was also shown to contribute to vascular smooth muscle remodeling and neointimal hyperplasia caused by vascular injury. Furthermore, Orai3 has been shown to contribute to proliferation of effector T-lymphocytes under oxidative stress. In this review, we will discuss the role of Orai3 in reported pathophysiological conditions and will contribute ideas on the potential role of Orai3 in native Ca(2+) signaling pathways and human disease.
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Canales de Calcio/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Calcio/metabolismo , Señalización del Calcio , Humanos , Leucemia/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Estrés OxidativoRESUMEN
We report on the preparation of ultra-small fluorescent nanosensors for oxygen via a one-pot approach. The nanoparticles have a hydrophobic core capable of firmly hosting hydrophobic luminescent oxygen probes. Their surface is composed of a dense and long-chain poly(ethylene glycol) shell, which renders them cell-membrane impermeable but yet highly sensitive to oxygen, and also highly stable in aqueous solutions and cell culture media. These features make them potentially suitable for sensing oxygen in extracellular fluids such as blood, interstitial and brain fluid, in (micro) bioreactors and micro- or nanoscale fluidic devices. Four kinds of nanosensors are presented, whose excitation spectra cover a wide spectral range (395-630 nm), thus matching many common laser lines, and with emission maxima ranging from 565 to 800 nm, thereby minimizing interference from background luminescence of biomatter. The unquenched lifetimes are on the order of 5.8-234 µs, which-in turn-enables lifetime imaging and additional background separation via time-gated methods.
RESUMEN
In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250 µm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature. The gold-film electrodes were also used to deliver well-defined AC voltage pulses of several volts amplitude and several hundred milliseconds duration to the adherent cells in order to achieve reversible membrane electroporation (in situ electroporation=ISE). Electroporation-assisted introduction of membrane impermeable molecules into the cytoplasm was studied by using FITC-labeled dextran molecules of different molecular weights. Probes as big as 2MDa were successfully loaded into the cells residing on the electrode surface. Time-resolved impedance measurements before and immediately after the electroporation pulse revealed the kinetics of membrane resealing as well as subsequent changes in cell morphology. Cells recovered from the electroporation pulse within less than 90 min. When membrane-impermeable, bioactive compounds like N(3)(-) or bleomycin were introduced into the cells by in situ electroporation, concomitant ECIS readings sensitively reported on the associated response of the cells to these toxins as a function of time (ISE-ECIS).