RESUMO
Tunneling nanotubes (TNTs) are elastic tubular structures that physically link cells, facilitating the intercellular transfer of organelles, chemical signals, and electrical signals. Despite TNTs serving as a multifunctional pathway for cell-cell communication, the transmission of mechanical signals through TNTs and the response of TNT-connected cells to these forces remain unexplored. In this study, external mechanical forces were applied to induce TNT bending between rat kidney (NRK) cells using micromanipulation. These forces, transmitted via TNTs, induced reduced curvature of the actin cortex and increased membrane tension at the TNT-connected sites. Additionally, TNT bending results in an elevation of intracellular calcium levels in TNT-connected cells, a response attenuated by gadolinium ions, a non-selective mechanosensitive calcium channel blocker. The degree of TNT deflection positively correlated with decreased actin cortex curvature and increased calcium levels. Furthermore, stretching TNT due to the separation of TNT-connected cells resulted in decreased actin cortex curvature and increased intracellular calcium in TNT-connected cells. The levels of these cellular responses depended on the length changes of TNTs. Moreover, TNT connections influence cell migration by regulating cell rotation, which involves the activation of mechanosensitive calcium channels. In conclusion, our study revealed the transmission of mechanical signals through TNTs and the subsequent responses of TNT-connected cells, highlighting a previously unrecognized communication function of TNTs. This research provides valuable insights into the role of TNTs in long-distance intercellular mechanical signaling.
Assuntos
Actinas , Nanotubos , Ratos , Animais , Cálcio/metabolismo , Comunicação Celular/fisiologia , Linhagem Celular , Nanotubos/químicaRESUMO
Mechanical forces generated by cells and the tension of the extracellular matrix (ECM) play a decisive role in establishment, homeostasis maintenance, and repair of tissue morphology. However, the dynamic change of cell-derived force during large-scale remodeling of soft tissue is still unknown, mainly because the current techniques of force detection usually produce a nonnegligible and interfering feedback force on the cells during measurement. Here, we developed a method to fabricate highly stretchable polymer-based microstrings on which a microtissue of fibroblasts in collagen was cultured and allowed to contract to mimic the densification of soft tissue. Taking advantage of the low-spring constant and large deflection range of the microstrings, we detected a strain-induced contraction force as low as 5.2 µN without disturbing the irreversible densification. Meanwhile, the microtissues displayed extreme sensitivity to the mechanical boundary within a narrow range of tensile stress. More importantly, results indicated that the cell-derived force did not solely increase with increased ECM stiffness as previous studies suggested. Indeed, the cell-derived force and collagen tension exchanged dramatically in dominating the microtissue strain during the densification, and the proportion of cell-derived force decreased linearly as the microtissue densified, with stiffness increasing to â¼500 Pa. Thus, this study provides insights into the biomechanical cross-talk between the cells and ECM of extremely soft tissue during large-extent densification, which may be important to guide the construction of life-like tissue by applying appropriate mechanical boundary conditions.
Assuntos
Colágeno/química , Matriz Extracelular/química , Fenômenos Biomecânicos , Desenvolvimento Embrionário , Fenômenos Mecânicos , Engenharia TecidualRESUMO
Ventilator-induced lung injury (VILI) during mechanical ventilation (MV) has been attributed to airway remodeling involving increased airway smooth muscle cells (ASMCs), but the underlying mechanism is not fully understood. Thus, we aimed to investigate whether MV-associated high stretch (>10% strain) could modulate mechanosensitive Piezo1 expression and thereby alter cell migration of ASMCs as a potential pathway to increased ASMCs in VILI. C57BL/6 mice and ASMCs were subjected to MV at high tidal volume (VT, 18 mL/kg, 3 h) and high stretch (13% strain, 0.5 Hz, 72 h), respectively. Subsequently, the mice or cells were evaluated for Piezo1 and integrin mRNA expression by immunohistochemical staining and quantitative PCR (qPCR), and cell migration and adhesion by transwell and cell adhesion assays. Cells were either treated or not with Piezo1 siRNA, Piezo1-eGFP, Piezo1 knockin, Y27632, or blebbistatin to regulate Piezo1 mRNA expression or inhibit Rho-associated kinase (ROCK) signaling prior to migration or adhesion assessment. We found that expression of Piezo1 in in situ lung tissue, mRNA expression of Piezo1 and integrin αVß1 and cell adhesion of ASMCs isolated from mice with MV were all reduced but the cell migration of primary ASMCs (pASMCs) isolated from mice with MV was greatly enhanced. Similarly, cell line mouse ASMCs (mASMCs) cultured in vitro with high stretch showed that mRNA expression of Piezo1 and integrin αVß1 and cell adhesion were all reduced but cell migration was greatly enhanced. Interestingly, such effects of MV or high stretch on ASMCs could be either induced or abolished/reversed by down/up-regulation of Piezo1 mRNA expression and inhibition of ROCK signaling. High stretch associated with MV appears to be a mechanical modulator of Piezo1 mRNA expression and can, thus, promote cell migration of ASMCs during therapeutic MV. This may be a novel mechanism of detrimental airway remodeling associated with MV, and, therefore, a potential intervention target to treat VILI.
Assuntos
Asma , Camundongos , Animais , Asma/metabolismo , Respiração Artificial/efeitos adversos , Remodelação das Vias Aéreas , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/metabolismo , RNA Mensageiro/metabolismo , Proliferação de Células , Células Cultivadas , Canais Iônicos/genética , Canais Iônicos/metabolismoRESUMO
Monitoring airway impedance has significant clinical value in accurately assessing and diagnosing pulmonary function diseases at an early stage. To address the issue of large oscillator size and high power consumption in current pulmonary function devices, this study adopts a new strategy of expiration-driven oscillation. A lightweight and low-power airway impedance monitoring system with integrated sensing, control circuitry, and dynamic feedback system, providing visual feedback on the system's status, was developed. The respiratory impedance measurement experiments and statistical comparisons indicated that the system could achieve stable measurement of airway impedance at 5 Hz. The frequency spectrum curves of respiratory impedance ( R and X) showed consistent trends with those obtained from the clinical pulmonary function instrument, specifically the impulse oscillometry system (IOS). The differences between them were all less than 1.1 cm H 2O·s/L. Additionally, there was a significant statistical difference in the respiratory impedance R5 between the exercise and rest groups, which suggests that the system can measure the variability of airway resistance parameters during exercise. Therefore, the impedance monitoring system developed in this study supports subjects in performing handheld, continuous measurements of dynamic changes in airway impedance over an extended period of time. This research provides a foundation for further developing low-power, portable, and even wearable devices for dynamic monitoring of pulmonary function.
Assuntos
Resistência das Vias Respiratórias , Impedância Elétrica , Oscilometria , Testes de Função Respiratória , Humanos , Oscilometria/instrumentação , Monitorização Fisiológica/instrumentação , Monitorização Fisiológica/métodos , Testes de Função Respiratória/instrumentação , Expiração/fisiologia , Desenho de Equipamento , Exercício FísicoRESUMO
Human brain experiences vibration of certain magnitude and frequency during various physical activities such as vehicle transportation and machine operation, which may cause traumatic brain injury or other brain diseases. However, the mechanisms of brain pathogenesis due to vibration are not fully elucidated due to the lack of techniques to study brain functions while applying vibration to the brain at a specific magnitude and frequency. Here, this study reported a custom-built head-worn electromagnetic actuator that applied vibration to the brain in vivo at an accurate frequency inside a magnetic resonance imaging scanner while cerebral blood flow (CBF) was acquired. Using this technique, CBF values from 45 healthy volunteers were quantitatively measured immediately following vibration at 20, 30, 40 Hz, respectively. Results showed increasingly reduced CBF with increasing frequency at multiple regions of the brain, while the size of the regions expanded. Importantly, the vibration-induced CBF reduction regions largely fell inside the brain's default mode network (DMN), with about 58 or 46% overlap at 30 or 40 Hz, respectively. These findings demonstrate that vibration as a mechanical stimulus can change strain conditions, which may induce CBF reduction in the brain with regional differences in a frequency-dependent manner. Furthermore, the overlap between vibration-induced CBF reduction regions and DMN suggested a potential relationship between external mechanical stimuli and cognitive functions.
Assuntos
Encéfalo , Vibração , Humanos , Imageamento por Ressonância Magnética , Cognição , Circulação Cerebrovascular/fisiologiaRESUMO
Inspired by the well-known phenomenon of stretch-induced airway dilation in normal lungs and the emerging stretch-responsive Piezo1 channels that can be chemically activated by specific agonists such as Yoda1, we attempted to investigate whether chemical activation of Piezo1 by Yoda1 can modulate the biomechanical behaviors of airway smooth muscle cells (ASMCs) so that it may be exploited as a novel approach for bronchodilation. Thus, we treated in vitro cultured rat ASMCs with Yoda1, and examined the cells for calcium signaling, cell stiffness, traction force, cell migration, and the mRNA expression and distribution of molecules relevant to cell biomechanics. The data show that ASMCs expressed abundant mRNA of Piezo1. ASMCs exposed to 1 µM Yoda1 exhibited a potent but transient Ca2+ signaling, and treatment with 1 µM Yoda1 for 24 h led to decreased cell stiffness and traction force, all of which were partially reversed by Piezo1 inhibitor GsMTx4 and Piezo1 knockdown, respectively. In addition, ASMCs treated with 1 µM Yoda1 for 24 h exhibited impaired horizontal but enhanced vertical cell migration, as well as significant changes in key components of cells' contractile machinery including the structure and distribution of stress fibers and alpha-smooth muscle actin (α-SMA) fibrils, the mRNA expression of molecules associated with cell biomechanics. These results provide the first evidence that chemical activation of Piezo1 by Yoda1 resulted in marked pro-relaxation alterations of biomechanical behaviors and contractile machinery of the ASMCs. These findings suggest that Piezo1-specific agonists may indeed have great potential as alternative drug agents for relaxing ASMCs.
Assuntos
Sinalização do Cálcio , Miócitos de Músculo Liso , Ratos , Animais , Células Cultivadas , Miócitos de Músculo Liso/metabolismo , RNA Mensageiro/metabolismoRESUMO
Blindness is frequently caused by corneal abnormalities, and corneal transplantation is the most effective treatment method. It is extremely important to develop high-quality artificial corneas because there are not enough donor corneas accessible for cornea transplantation. One of the most-often utilized materials is collagen, which is the primary component of natural cornea. Collagen-based corneal repair materials have good physicochemical properties and excellent biocompatibility, but how to promote the regeneration of the corneal nerve after keratoplasty is still a big challenge. In this research, in order to promote the growth of nerve cells on a collagen (Col) substrate, a novel collagen-based material was synthesized starting from the functionalization of collagen with unsaturated methacryloyl groups that three-dimensionally photopolymerize to a 3D network of chemically crosslinked collagen (ColMA), onto which taurine molecules were eventually grafted (ColMA-Tr). The physicochemical properties and biocompatibility of the Col, ColMA and ColMA-Tr films were evaluated. By analyzing the results, we found that all the three samples had good moisture retention and aq high covalent attachment of methacryloyl groups followed by their photopolymerization improved the mechanical properties of the ColMA and ColMA-Tr. Most importantly, compared with ColMA, the taurine-modified collagen-MA film significantly promoted the growth of nerve cells and corneal epithelial cells on its surface. Our preliminary results suggest that this novel ColMA-Tr film may have potential use in cornea tissue engineering in the future.
Assuntos
Córnea , Transplante de Córnea , Colágeno/química , Engenharia Tecidual/métodos , Regeneração Nervosa , Materiais Biocompatíveis/farmacologia , Materiais Biocompatíveis/químicaRESUMO
Ventilator-induced lung injury (VILI) occurs in mechanically ventilated patients of respiratory disease and is typically characterized by airway inflammation. However, recent studies increasingly indicate that a major cause of VILI may be the excessive mechanical loading such as high stretch (>10% strain) on airway smooth muscle cells (ASMCs) due to mechanical ventilation (MV). Although ASMCs are the primary mechanosensitive cells in airways and contribute to various airway inflammation diseases, it is still unclear how they respond to high stretch and what mediates such a response. Therefore, we used whole genome-wide mRNA-sequencing (mRNA-Seq), bioinformatics, and functional identification to systematically analyze the mRNA expression profiles and signaling pathway enrichment of cultured human ASMCs exposed to high stretch (13% strain), aiming to screen the susceptible signaling pathway through which cells respond to high stretch. The data revealed that in response to high stretch, 111 mRNAs with count ≥100 in ASMCs were significantly differentially expressed (defined as DE-mRNAs). These DE-mRNAs are mainly enriched in endoplasmic reticulum (ER) stress-related signaling pathways. ER stress inhibitor (TUDCA) abolished high-stretch-enhanced mRNA expression of genes associated with ER stress, downstream inflammation signaling, and major inflammatory cytokines. These results demonstrate in a data-driven approach that in ASMCs, high stretch mainly induced ER stress and activated ER stress-related signaling and downstream inflammation response. Therefore, it suggests that ER stress and related signaling pathways in ASMCs may be potential targets for timely diagnosis and intervention of MV-related pulmonary airway diseases such as VILI.
Assuntos
Pulmão , Respiração Artificial , Humanos , Pulmão/metabolismo , Estresse do Retículo Endoplasmático , Inflamação/metabolismo , Células Cultivadas , Miócitos de Músculo Liso/metabolismo , RNA Mensageiro/metabolismoRESUMO
Extracellular Matrix (ECM) assembly and remodeling are critical physiological events in vivo, and abnormal ECM assembly or remodeling is related to pathological conditions such as osteoarthritis, fibrosis, cancers, and genetic diseases. ECM assembly/remodeling driven by cells represents more physiological processes. Collagen I (COL) is very abundant in tissues, which assembly/remodeling is mediated by biochemical and mechanical factors. How cells regulate COL assembly biomechanically still remains to be well understood. Here we used fluorescent COL in the medium to study how cells assembled ECM which represents more physiological structures. The results showed that MDCK cells actively recruited COL from the medium and helped assemble the fibers, which in turn facilitated cell branching morphogenesis, both displaying highly spatial associations and mutual dependency. Inhibition of cellular contraction force by ROCK and Myosin II inhibitors attenuated but did not block the COL fiber formation, while cell motion showed high consistency with the fiber assembly. Under ROCK or Myosin II inhibition, further analysis indicated high correlation between local cell movement and COL fiber strength as quantified from different regions of the same groups. Blocking cell motion by actin cytoskeleton disruption completely inhibited the fiber formation. These suggest that cell motion coordinated COL fiber assembly from the medium, possibly through generated strain on deposited COL to facilitate the fiber growth.
Assuntos
Movimento Celular , Colágeno Tipo I/metabolismo , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Citoesqueleto/metabolismo , Cães , Células Epiteliais/citologia , Células Madin Darby de Rim Canino , Miosina Tipo II/metabolismoRESUMO
Excessive contraction of airway smooth muscle cells (ASMCs) is a hallmark feature of asthma. Intriguing, the activation of bitter taste receptor (TAS2R) in ASMCs can relax ASMCs. However, there is a lack of potent TAS2R agonists that can be used in asthma therapies since those tested agonists cannot relax ASMCs at the dose below a few hundred micromolar. Considering that sanguinarine (SA) is a bitter substance often used in small doses for the treatment of asthma in folk medicine, the present study was to determine the rapid relaxation effect of SA on ASMCs and to reveal the underlying mechanisms associated with TAS2R signaling. Here, cell stiffness, traction force, calcium signaling, cAMP levels, and the mRNA expression were evaluated by using optical magnetic twisting cytometry, traction force microscopy, Fluo-4/AM labeling, enzyme-linked immunosorbent assay (ELISA), and quantitative (q)RT-PCR, respectively. We found that 0.5 µM SA immediately decreased cell stiffness and traction force, which is comparable with the effect of 5 µM isoproterenol. In addition, 0.5 µM SA immediately increased intracellular free calcium concentration ([Ca2+]i) and decreased the mRNA expression of contractile proteins such as calponin and α-smooth muscle actin after the treatment for 24 h. Furthermore, SA-mediated decrease in cell stiffness/traction force and increase in [Ca2+]i were significantly blunted by inhibiting the TAS2Rs signaling. These findings establish the rapid relaxation effect of SA at low concentration (<1 µM) on cultured ASMCs depending on TAS2R signaling, indicating that SA might be developed as a useful bronchodilator in asthma therapy.
Assuntos
Benzofenantridinas/farmacologia , Broncodilatadores/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Isoquinolinas/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Animais , Benzofenantridinas/química , Broncodilatadores/química , Sinalização do Cálcio/fisiologia , Forma Celular/efeitos dos fármacos , Forma Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Isoquinolinas/química , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismoRESUMO
Stereospecific molecular recognition with simple and easily available proteins is of significant importance in life science and biomaterial science. Herein, we report on a chiral sensing platform, graphene quantum dots (GQDs)-functionalized bovine serum albumin (BSA), for chiral recognition of tryptophan (Trp) isomers. Amidation reaction between BSA and GQDs was directly responsible for the introduction of GQDs to BSA, resulting in significant changes in the spatial configuration of BSA and the exposure of more chiral sites at the protein surface. The BSA-GQDs-based chiral sensor exhibited good biomolecular homochirality in the recognition of Trp isomers, and the higher affinity of BSA-GQDs toward l-Trp than its isomer, d-Trp, was also revealed by density functional theory (DFT) considering the possible hydrogen bonds between the Trp isomers and the solvent-accessible residues of BSA.
Assuntos
Grafite/química , Pontos Quânticos/química , Soroalbumina Bovina/química , Triptofano/análise , Triptofano/química , Teoria da Densidade Funcional , Técnicas Eletroquímicas , Isomerismo , Solventes/química , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
By airway surface liquid, we mean a thin fluid continuum consisting of the airway lining layer and the alveolar lining layer, which not only serves as a protective barrier against foreign particles but also contributes to maintaining normal respiratory mechanics. In recent years, measurements of the rheological properties of airway surface liquid have attracted considerable clinical attention due to new advances in microrheology instruments and methods. This article reviews the clinical relevance of measurements of airway surface liquid viscoelasticity and surface tension from four main aspects: maintaining the stability of the airways and alveoli, preventing ventilator-induced lung injury, optimizing surfactant replacement therapy for respiratory syndrome distress, and characterizing the barrier properties of airway mucus to improve drug and gene delivery. Primary measuring techniques and methods suitable for determining the viscoelasticity and surface tension of airway surface liquid are then introduced with respect to principles, advantages and limitations. Cone and plate viscometers and particle tracking microrheometers are the most commonly used instruments for measuring the bulk viscosity and microviscosity of airway surface liquid, respectively, and pendant drop methods are particularly suitable for the measurement of airway surface liquid surface tension in vitro. Currently, in vivo and in situ measurements of the viscoelasticity and surface tension of the airway surface liquid in humans still presents many challenges.
Assuntos
Bronquite/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Surfactantes Pulmonares/administração & dosagem , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia , Animais , Bronquite/tratamento farmacológico , Doença Crônica , Humanos , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Mecânica Respiratória , Reologia , Sensibilidade e Especificidade , Tensão Superficial/efeitos dos fármacos , Lesão Pulmonar Induzida por Ventilação Mecânica/tratamento farmacológico , ViscosidadeRESUMO
NEW FINDINGS: What is the central question of this study? The aim of this study was to evaluate artesunate for its use as a bronchodilator in asthma treatment. What is the main finding and its importance? We found that artesunate reduces airway resistance in both normal and ovalbumin-treated Balb/c mice in vivo. Artesunate reduces traction force while inducing Ca2+ influx into cultured airway smooth muscle cells in vitro, most probably via the bitter taste receptor. These findings provide important evidence at both animal and cellular levels that artesunate might potentially be used as a bronchodilator for treating obstructive airway diseases, such as asthma. ABSTRACT: Following the surprising discovery that bitter taste receptors (TAS2Rs) expressed in the lung and can be stimulated to relax airway smooth muscle cells (ASMCs), there is great interest in searching for a bitter taste receptor agonist as a new bronchodilator for asthma therapy. Among the great many other natural bitter substances, artesunate is of special interest to be evaluated for this purpose because of its pharmacological value as a derivative from the well-known anti-malarial, artemisinin. Therefore, in this study we treated either normal or ovalbumin (OVA)-induced asthmatic Balb/c mice in vivo with artesunate (30, 60 or 120 µg) via aerosol inhalation. Subsequently, we measured the airway resistance of the mice in the presence or absence of artesunate. In addition, we treated either mouse or human ASMCs cultured in vitro with artesunate (0.25-2.0 mM) and then measured the traction force and [Ca2+ ]i flux of the cells in the presence or absence of artesunate. The results demonstrate that artesunate attenuated airway resistance in a dose-dependent manner in both the normal and the OVA-treated mice, but more potently in the latter. The in vivo efficacy of artesunate at 120 µg was comparable to that of the conventional bronchodilator, salbutamol, at 3 µg in terms of the reduction in airway resistance. Artesunate also reduced traction force and induced an increase in [Ca2+ ]i in the cultured ASMCs, which was mediated, at least in part, by TAS2R signalling in the human ASMCs. These results together suggest that artesunate might potentially be a cheap and safe bronchodilator to complement the current therapy of asthma.
Assuntos
Resistência das Vias Respiratórias/efeitos dos fármacos , Artesunato/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Paladar/efeitos dos fármacos , Animais , Artemisininas/farmacologia , Asma/tratamento farmacológico , Asma/metabolismo , Broncoconstrição/efeitos dos fármacos , Broncodilatadores/farmacologia , Cálcio/metabolismo , Feminino , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Conductive materials are valuable supports and widely applied in electrochemical analysis. In the current article, a mesoporous organosilica sphere (S,S)-CPMO-3 with the chiral group and electroactive units is presented. The organosilicon framework is composed of free ions belonging to the ionic liquid and the chiral group arising from (1S,2S)-1,2-diaminocyclohexane. Next, a bare electrode was modified by the as-synthesized composite material to construct the new electrochemical sensor (S,S)-CPMO-3-GCE. It was observed that (S,S)-CPMO-3-GCE exhibited favourable enantioselective recognition in the response of the peak current (Ip) and the peak potential (Ep) to the different configurations of amino acids. Taking tryptophan as an example, the value of IL/ID is 13.84 and the peak-to-peak potential approaches 48 mV. In addition, cysteine and tyrosine were successfully distinguished by the sensor. In summary, this study not only introduces a route for the synthesis of a conductive material, but also opens up its further potential in enantioselective recognition.
Assuntos
Aminoácidos/análise , Técnicas Eletroquímicas/métodos , Dióxido de Silício/química , Aminoácidos/química , Cicloexilaminas/síntese química , Cicloexilaminas/química , Cisteína/química , Líquidos Iônicos/síntese química , Líquidos Iônicos/química , Porosidade , Estereoisomerismo , Triptofano/química , Tirosina/químicaRESUMO
BACKGROUND: Allergic inflammation has long been implicated in asthmatic hyperresponsiveness of airway smooth muscle (ASM), but its underlying mechanism remains incompletely understood. Serving as G protein-coupled receptor agonists, several inflammatory mediators can induce membrane depolarization, contract ASM, and augment cholinergic contractile response. We hypothesized that the signal cascade integrating on membrane depolarization by the mediators might involve asthmatic hyperresponsiveness. OBJECTIVE: We sought to investigate the signaling transduction of inflammatory mediators in ASM contraction and assess its contribution in the genesis of hyperresponsiveness. METHODS: We assessed the capacity of inflammatory mediators to induce depolarization currents by electrophysiological analysis. We analyzed the phenotypes of transmembrane protein 16A (TMEM16A) knockout mice, applied pharmacological reagents, and measured the Ca2+ signal during ASM contraction. To study the role of the depolarization signaling in asthmatic hyperresponsiveness, we measured the synergistic contraction by methacholine and inflammatory mediators both ex vivo and in an ovalbumin-induced mouse model. RESULTS: Inflammatory mediators, such as 5-hydroxytryptamin, histamine, U46619, and leukotriene D4, are capable of inducing Ca2+-activated Cl- currents in ASM cells, and these currents are mediated by TMEM16A. A combination of multiple analysis revealed that a G protein-coupled receptor-TMEM16A-voltage-dependent Ca2+ channel signaling axis was required for ASM contraction induced by inflammatory mediators. Block of TMEM16A activity may significantly inhibit the synergistic contraction of acetylcholine and the mediators and hence reduces hypersensitivity. CONCLUSIONS: A G protein-coupled receptor-TMEM16A-voltage-dependent Ca2+ channel axis contributes to inflammatory mediator-induced ASM contraction and synergistically activated TMEM16A by allergic inflammatory mediators with cholinergic stimuli.
Assuntos
Anoctamina-1/metabolismo , Asma/metabolismo , Hiper-Reatividade Brônquica/metabolismo , Canais de Cálcio/metabolismo , Contração Muscular , Músculo Liso/fisiopatologia , Transdução de Sinais , Animais , Asma/fisiopatologia , Biomarcadores/metabolismo , Hiper-Reatividade Brônquica/fisiopatologia , Fenômenos Eletrofisiológicos , Feminino , Cobaias , Masculino , Camundongos , Camundongos Knockout , FenótipoRESUMO
Polypyrrole (PPy) was synthesized by a galvanostatic method using (1 S)-(+)-10-camphorsulfonic acid ((+)-CSA) as the dopant, and the produced PPy was further overoxidized in a solution of (+)-CSA. A chiral microenvironment was successfully formed in the overoxidized PPy (OPPy) as a result of the synergistic effects of overoxidation and (+)-CSA, resulting in a twisted helical architecture of the OPPy chains. The formation of optically active OPPy was confirmed from aspects of its morphology (SEM and AFM) and circular-dichroism (CD) spectra. Finally, an electrochemical chirality sensor was fabricated on the basis of the resultant OPPy, which exhibited excellent biomolecular homochirality in the discrimination of tryptophan (Trp) enantiomers.
RESUMO
NEW FINDINGS: What is the central question of this study? What is the effect of catenin alpha-like 1 (CTNNAL1), an asthma-related epithelial adhesion molecule that plays a vital role in airway epithelial wound repair, on airway epithelial-mesenchymal transition? What is the main finding and its importance? CTNNAL1 inhibits ozone-induced airway epithelial-mesenchymal transition features, mediated by repressing the expression of Twist1 mRNA and reducing TGF-ß1 levels. These findings contribute to our understanding of the pathology of airway EMT and may indicate a possible therapeutic target for airway remodelling in bronchial asthma. ABSTRACT: Epithelial-mesenchymal transition (EMT), a crucial event occurring during epithelial and mesenchymal repair, was reported to be a possible mechanism for airway remodelling. Our previous work showed that the expression of catenin alpha-like 1 (CTNNAL1) was down-regulated in the bronchial epithelial cells of asthmatic models and played a vital role in airway epithelial wound repair. The aim of this study was to investigate the effect of CTNNAL1 on airway EMT. Overexpression or silencing of CTNNAL1 in human bronchial epithelial cells was induced by stable transfection. CTNNAL1 was silenced in primary mouse airway epithelial cells with an effective siRNA vector. Cells were stressed by ozone for 4 days at 30 min day-1 to induce EMT. EMT features, changes in the function of co-cultured lung fibroblasts, changes in the expression of the transcriptional repressors Snail/Slug and Twist1/Twist2 and changes in the secretion of transforming growth factor ß1 (TGF-ß1) were assayed in different cell lines with or without ozone exposure. Both ozone exposure and silencing of CTNNAL1 induced EMT features in airway epithelial cells. Functional changes in lung fibroblasts increased after co-culture with (ozone-stressed) CTNNAL1-silenced cells. Snail and Twist1 expression increased, and the level of TGF-ß1 was enhanced. Conversely, CTNNAL1 overexpression reversed EMT features, repressed mRNA levels of Twist1 and reduced the secretion of TGF-ß1, both alone and in combination with ozone exposure. Our results indicate that ozone exposure induces airway EMT and that CTNNAL1 inhibits ozone-induced airway EMT. CTNNAL1 may play a role in airway EMT by repressing the expression of Twist1 mRNA and reducing the level of TGF-ß1.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Ozônio/administração & dosagem , Animais , Linhagem Celular , Proliferação de Células , Proteínas do Citoesqueleto/genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Proteína 2 Relacionada a Twist/metabolismo , alfa CateninaRESUMO
This study aimed to evaluate the effect of sanguinarine on biomechanical properties of rat airway smooth muscle cells (rASMCs) including stiffness, traction force and cytoskeletal stress fiber organization. To do so, rASMCs cultured in vitro were treated with sanguinarine solution at different concentrations (0.005~5 µmol/L) for 12 h, 24 h, 36 h, and 48 h, respectively. Subsequently, the cells were tested for their viability, stiffness, traction force, migration and microfilament distribution by using methylthiazolyldiphenyl-tetrazolium bromide assay, optical magnetic twisting cytometry, Fourier transform traction microscopy, scratch wound healing method, and immunofluorescence microscopy, respectively. The results showed that at concentration below 0.5 µmol/L sanguinarine had no effect on cell viability, but caused dose and time dependent effect on cell biomechanics. Specifically, rASMCs treated with sanguinarine at 0.05 µmol/L and 0.5 µmol/L for 12 and 24 h exhibited significant reduction in stiffness, traction force and migration speed, together with disorganization of the cytoskeletal stress fibers. Considering the essential role of airway smooth muscle cells (ASMCs) biomechanics in the airway hyperresponsiveness (AHR) of asthma, these findings suggest that sanguinarine may ameliorate AHR via alteration of ASMCs biomechanical properties, thus providing a novel approach for asthma drug development.
RESUMO
Construction of convenient systems for isomer discrimination is of great importance for medical and life sciences. Here, we report a simple and effective chiral sensing device based on a highly ordered self-assembly framework. Cu2+-modified ß-cyclodextrin (Cu-ß-CD) was self-assembled to the ammonia-ethanol cotreated chitosan (ae-CS), and the highly ordered framework was gradually formed during the "re-growth" process of the shrinked ae-CS films. Tryptophan (Trp) isomers were well discriminated with the highly ordered framework by electrochemical approach. This study is the first example showing how an ordered structure influences chiral recognition.
RESUMO
A disintegrin and metalloproteinase 33 (ADAM33) has been identified as a susceptibility gene for asthma, but details of the causality are not fully understood. We hypothesize that soluble ADAM33 (sADAM33) overexpression can alter the mechanical behaviors of airway smooth muscle cells (ASMCs) via regulation of the cell's contractile phenotype, and thus contributes to airway hyperresponsiveness (AHR) in asthma. To test this hypothesis, we either overexpressed or knocked down the sADAM33 in rat ASMCs by transfecting the cells with sADAM33 coding sequence or a small interfering RNA (siRNA) that specifically targets the ADAM33 disintegrin domain, and subsequently assessed the cells for stiffness, contractility and traction force, together with the expression level of contractile and proliferative phenotype markers. We also investigated whether these changes were dependent on Rho/ROCK pathway by culturing the ASMCs either in the absence or presence of ROCK inhibitor (H1152). The results showed that the ASMCs with sADAM33 overexpression were stiffer and more contractile, generated greater traction force, exhibited increased expression levels of contractile phenotype markers and markedly enhanced Rho activation. Furthermore these changes were largely attenuated when the cells were cultured in the presence of H-1152. However, the knock-down of ADAM33 seemed insufficient to influence majority of the mechanical behaviors of the ASMCs. Taken together, we demonstrated that sADAM33 overexpression altered the mechanical behaviors of ASMCs in vitro, which was most likely by promoting a hypercontractile phenotype transition of ASMCs through Rho/ROCK pathway. This revelation may establish the previously missing link between ADAM33 expression and AHR, and also provide useful insight for targeting sADAM33 in asthma prevention and therapy.