Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.800
Filtrar
1.
Int J Nanomedicine ; 16: 1663-1680, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33688184

RESUMEN

Background: Intracellular tension plays a crucial role in the destruction of the blood-brain barrier (BBB) in response to lesion stimuli. Tight junction structure could be primarily affected by tension activity. In this study, we aimed to determine the effects of extracellular BBB damage on intracellular tension activity, and elucidate the mechanism underlying the effects of intracellular protein nanoparticle-related osmotic pressure on BBB permeability. Methods: The intracellular tension for tight junction proteins occludin and ZO1 was evaluated using the fluorescence resonance energy transfer (FRET)-based tension probes and cpstFRET analysis. The changes in mobility ratios of occludin were evaluated via the fluorescence recovery after photobleaching (FRAP) test. The cytoplasmic osmotic pressure (OP) was measured using Osmometer. The count rate of cytoplasmic nanoparticles was detected by Nanosight NS300. The activation of cofilin and stathmin was examined by Western blot analysis. The BBB permeability in vivo was determined via the changes of Evans Blue (EB) injected into SD rats. The tight junction formation was assessed by the measurement of transendothelial electrical resistance (TEER). Intracellular calcium or chloride ions were measured using Fluo-4 AM or MQAE dyes. Results: BBB lesions were accompanied by changes in occludin/ZO1 tension. Increases in intracellular osmotic pressure were involved in alteration of BBB permeability, possibly through the depolymerization of microfilaments or microtubules and mass production of protein nanoparticles according to the Donnan effect. Recovery of protein nanoparticle-related osmotic pressure could effectively reverse the effects of changes in occludin/ZO1 tension under BBB lesions. Outward tension of intracellular osmotic potential also caused upregulation of membrane fluidity, which promoted nonselective drug influx. Conclusion: Our results suggest a crucial mechanical mechanism underlying BBB lesions, and protein nanoparticle-related osmotic pressure could be a novel therapeutic target for BBB lesion-related brain diseases.


Asunto(s)
Barrera Hematoencefálica/metabolismo , Fluidez de la Membrana , Nanopartículas/química , Presión Osmótica , Proteínas/química , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Barrera Hematoencefálica/efectos de los fármacos , Línea Celular , Azul de Evans/metabolismo , Humanos , Masculino , Fluidez de la Membrana/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Ocludina/metabolismo , Presión Osmótica/efectos de los fármacos , Permeabilidad , Fitoquímicos/farmacología , Polimerizacion , Ratas Sprague-Dawley , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/metabolismo
2.
BMC Pulm Med ; 21(1): 58, 2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33588817

RESUMEN

BACKGROUND: Hyperoxia downregulates the tight junction (TJ) proteins of the alveolar epithelium and leads to barrier dysfunction. Previous study has showed that STE20/SPS1-related proline/alanine-rich kinase (SPAK) interferes with the intestinal barrier function in mice. The aim of the present study is to explore the association between SPAK and barrier function in the alveolar epithelium after hyperoxic exposure. METHODS: Hyperoxic acute lung injury (HALI) was induced by exposing mice to > 99% oxygen for 64 h. The mice were randomly allotted into four groups comprising two control groups and two hyperoxic groups with and without SPAK knockout. Mouse alveolar MLE-12 cells were cultured in control and hyperoxic conditions with or without SPAK knockdown. Transepithelial electric resistance and transwell monolayer permeability were measured for each group. In-cell western assay was used to screen the possible mechanism of p-SPAK being induced by hyperoxia. RESULTS: Compared with the control group, SPAK knockout mice had a lower protein level in the bronchoalveolar lavage fluid in HALI, which was correlated with a lower extent of TJ disruption according to transmission electron microscopy. Hyperoxia down-regulated claudin-18 in the alveolar epithelium, which was alleviated in SPAK knockout mice. In MLE-12 cells, hyperoxia up-regulated phosphorylated-SPAK by reactive oxygen species (ROS), which was inhibited by indomethacin. Compared with the control group, SPAK knockdown MLE-12 cells had higher transepithelial electrical resistance and lower transwell monolayer permeability after hyperoxic exposure. The expression of claudin-18 was suppressed by hyperoxia, and down-regulation of SPAK restored the expression of claudin-18. The process of SPAK suppressing the expression of claudin-18 and impairing the barrier function was mediated by p38 mitogen-activated protein kinase (MAPK). CONCLUSIONS: Hyperoxia up-regulates the SPAK-p38 MAPK signal pathway by ROS, which disrupts the TJ of the alveolar epithelium by suppressing the expression of claudin-18. The down-regulation of SPAK attenuates this process and protects the alveolar epithelium against the barrier dysfunction induced by hyperoxia.


Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Células Epiteliales Alveolares/metabolismo , Claudinas/genética , Hiperoxia/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Alveolos Pulmonares/metabolismo , Uniones Estrechas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Lesión Pulmonar Aguda/patología , Células Epiteliales Alveolares/ultraestructura , Animales , Líquido del Lavado Bronquioalveolar/química , Claudinas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hiperoxia/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Permeabilidad , Proteínas Serina-Treonina Quinasas/metabolismo , Alveolos Pulmonares/ultraestructura , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Uniones Estrechas/ultraestructura
3.
Int J Mol Sci ; 22(3)2021 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-33513818

RESUMEN

In Alzheimer's disease (AD), several studies have reported blood-brain barrier (BBB) breakdown with compromised function. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are transport proteins localized at the BBB luminal membrane and play an important role in the clearance of amyloid-ß (Aß). The purpose of this study was to investigate the effect of pharmacological inhibition of Aß efflux transporters on BBB function and Aß accumulation and related pathology. Recently, we have developed an in vitro high-throughput screening assay to screen for compounds that modulate the integrity of a cell-based BBB model, which identified elacridar as a disruptor of the monolayer integrity. Elacridar, an investigational compound known for its P-gp and BCRP inhibitory effect and widely used in cancer research. Therefore, it was used as a model compound for further evaluation in a mouse model of AD, namely TgSwDI. TgSwDI mouse is also used as a model for cerebral amyloid angiopathy (CAA). Results showed that P-gp and BCRP inhibition by elacridar disrupted the BBB integrity as measured by increased IgG extravasation and reduced expression of tight junction proteins, increased amyloid deposition due to P-gp, and BCRP downregulation and receptor for advanced glycation end products (RAGE) upregulation, increased CAA and astrogliosis. Further studies revealed the effect was mediated by activation of NF-κB pathway. In conclusion, results suggest that BBB disruption by inhibiting P-gp and BCRP exacerbates AD pathology in a mouse model of AD, and indicate that therapeutic drugs that inhibit P-gp and BCRP could increase the risk for AD.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Acridinas/farmacología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Barrera Hematoencefálica/metabolismo , Encéfalo/efectos de los fármacos , Tetrahidroisoquinolinas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Acridinas/administración & dosificación , Enfermedad de Alzheimer/patología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Barrera Hematoencefálica/patología , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Modelos Animales de Enfermedad , Inmunoglobulina G/metabolismo , Inmunohistoquímica , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Transgénicos , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Tetrahidroisoquinolinas/administración & dosificación , Uniones Estrechas/metabolismo
4.
Am J Physiol Cell Physiol ; 320(3): C448-C461, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33471620

RESUMEN

Gram-negative bacterial lipopolysaccharide (LPS) increases the susceptibility of cells to pathogenic diseases, including inflammatory diseases and septic syndrome. In our experiments, we examined whether LPS induces epithelial barrier disruption in secretory epithelia and further investigated its underlying mechanism. The activities of Ca2+-activated Cl- channels (CACC) and epithelial Na+ channels (ENaC) were monitored with a short-circuit current using an Ussing chamber. Epithelial membrane integrity was estimated via transepithelial electrical resistance and paracellular permeability assays. We found that the apical application of LPS evoked short-circuit current (Isc) through the activation of CACC and ENaC. Although LPS disrupted epithelial barrier integrity, this was restored with the inhibition of CACC and ENaC, indicating the role of CACC and ENaC in the regulation of paracellular pathways. We confirmed that LPS, CACC, or ENaC activation evoked apical membrane depolarization. The exposure to a high-K+ buffer increased paracellular permeability. LPS induced the rapid redistribution of zonula occludens-1 (ZO-1) and reduced the expression levels of ZO-1 in tight junctions through apical membrane depolarization and tyrosine phosphorylation. However, the LPS-induced epithelial barrier disruption and degradation of ZO-1 were largely recovered by blocking CACC and ENaC. Furthermore, although LPS-impaired epithelial barrier became vulnerable to secondary bacterial infections, this vulnerability was prevented by inhibiting CACC and ENaC. We concluded that LPS induces the disruption of epithelial barrier integrity through the activation of CACC and ENaC, resulting in apical membrane depolarization and the subsequent tyrosine phosphorylation of ZO-1.


Asunto(s)
Canales de Cloruro/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Lipopolisacáridos/farmacología , Canales de Sodio/metabolismo , Animales , Células Cultivadas , Masculino , Potenciales de la Membrana/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/metabolismo
5.
JCI Insight ; 6(4)2021 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-33507884

RESUMEN

An intact lung epithelial barrier is essential for lung homeostasis. The Na+, K+-ATPase (NKA), primarily serving as an ion transporter, also regulates epithelial barrier function via modulation of tight junctions. However, the underlying mechanism is not well understood. Here, we show that overexpression of the NKA ß1 subunit upregulates the expression of tight junction proteins, leading to increased alveolar epithelial barrier function by an ion transport-independent mechanism. Using IP and mass spectrometry, we identified a number of unknown protein interactions of the ß1 subunit, including a top candidate, myotonic dystrophy kinase-related cdc42-binding kinase α (MRCKα), which is a protein kinase known to regulate peripheral actin formation. Using a doxycycline-inducible gene expression system, we demonstrated that MRCKα and its downstream activation of myosin light chain is required for the regulation of alveolar barrier function by the NKA ß1 subunit. Importantly, MRCKα is expressed in both human airways and alveoli and has reduced expression in patients with acute respiratory distress syndrome (ARDS), a lung illness that can be caused by multiple direct and indirect insults, including the infection of influenza virus and SARS-CoV-2. Our results have elucidated a potentially novel mechanism by which NKA regulates epithelial tight junctions and have identified potential drug targets for treating ARDS and other pulmonary diseases that are caused by barrier dysfunction.


Asunto(s)
Proteína Quinasa de Distrofia Miotónica/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Uniones Estrechas/metabolismo , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Animales , Células HEK293 , Humanos , Proteína Quinasa de Distrofia Miotónica/genética , Cultivo Primario de Células , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , /virología , ATPasa Intercambiadora de Sodio-Potasio/genética
6.
Metabolism ; 114: 154409, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33096076

RESUMEN

BACKGROUND AND OBJECTIVES: The gut-liver axis plays an important role in the pathogenesis of nonalcoholic steatohepatitis (NASH), and increased intestinal permeability causes transfer of endotoxin to the liver, which activates the immune response, ultimately leading to hepatic inflammation. Nuclear receptor Rev-erbα is a critical regulator of circadian rhythm, cellular metabolism, and inflammatory responses. However, the role and mechanism of Rev-erbα in gut barrier function and NASH remain unclear. In the present study, we investigated the involvement of Rev-erbα in the regulation of intestinal permeability and the treatment of NASH. METHODS AND RESULTS: The expression of tight junction-related genes and Rev-erbs decreased in the jejunum, ileum and colon of mice with high cholesterol, high fat diet (CL)-induced NASH. Chromatin immunoprecipitation analysis indicated that REV-ERBα directly bound to the promoters of tight junction genes to regulate intestinal permeability. Pharmacological activation of REV-ERBα by SR9009 protected against lipopolysaccharide-induced increased intestinal permeability both in vitro and in vivo, and these effects were associated with the activation of autophagy and decreased apoptotic signaling of epithelial cells. In addition, the chronopharmacological effects of SR9009 were more potent at Zeitgeber time 0 (ZT0) than at ZT12, which was contrary to the rhythm of Rev-erbs in the gastrointestinal tract. The administration of SR9009 attenuated hepatic lipid accumulation, insulin resistance, inflammation, and fibrosis in mice with CL diet-induced NASH, which might be partly attributed to the enhancement of intestinal barrier function. CONCLUSION: Chronopharmacological activation of REV-ERBα might be a potential strategy to treat intestinal barrier dysfunction-related disorders and NASH.


Asunto(s)
Mucosa Intestinal/efectos de los fármacos , Intestinos/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Pirrolidinas/uso terapéutico , Receptores Citoplasmáticos y Nucleares/agonistas , Proteínas Represoras/agonistas , Tiofenos/uso terapéutico , Uniones Estrechas/efectos de los fármacos , Animales , Autofagia/efectos de los fármacos , Glucemia , Células CACO-2 , Colesterol/sangre , Humanos , Insulina/sangre , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Masculino , Ratones , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Permeabilidad/efectos de los fármacos , Pirrolidinas/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Tiofenos/farmacología , Uniones Estrechas/metabolismo , Triglicéridos/sangre
7.
Methods Mol Biol ; 2217: 301-311, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33215388

RESUMEN

In endothelial cells (ECs), the onset of apicobasal polarity is primarily regulated by the interaction of integrins with the surrounding extracellular matrix (ECM). ECs secrete and polymerize fibronectin (FN), a unique, permissive substrate that allows for vascular morphogenesis and lumen formation. We previously identified a signaling pathway that, under the control of the adhesion site adaptor protein PPFIA1, integrates the polarized secretion of freshly synthesized FN with the recycling of conformationally active α5ß1 integrin, the main FN receptor in ECs. To characterize the functional role of PPFIA1-dependent signaling in ECs, we set up a Transwell-based assay to quantify the polarized secretion of ECM proteins. To this aim, we allowed ECs to form a confluent monolayer on the Transwell membrane and checked its integrity by measuring transendothelial electric resistance and controlling the stability of tight junctions over time by fluorescent confocal microscope analysis. Finally, we quantified apical and basolateral FN secretion in control and PPFIA1-silenced EC culture medium by western blot analysis coupled to spike-in normalization.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/genética , Integrina alfa5beta1/genética , Uniones Estrechas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transporte Biológico , Polaridad Celular , Cámaras de Difusión de Cultivos , Células Endoteliales/ultraestructura , Matriz Extracelular/ultraestructura , Fibronectinas/metabolismo , Regulación de la Expresión Génica , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Humanos , Integrina alfa5beta1/metabolismo , Microscopía Fluorescente/métodos , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Uniones Estrechas/ultraestructura
8.
Biochim Biophys Acta Mol Basis Dis ; 1867(1): 165994, 2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-33184034

RESUMEN

The hepatic barrier is indispensable for the physiological functions of the liver and is impaired under various pathological conditions. Tight junctions reportedly play a central role in hepatic barrier regulation; however, there is limited direct evidence supporting this observation, with few in vivo models or confirmations of the implicated molecular mechanisms presented to date. We inactivated the tight junction component gene, Tjp2/ZO-2, and the related molecule, Tjp1/ZO-1, in mouse livers. In humans, TJP2/ZO-2 mutations have been implicated in the development of human progressive familial intrahepatic cholestasis 4 (PFIC4). The mice deficient in either ZO-1 or ZO-2 in the liver did not exhibit major abnormalities. However, the ablation of both molecules impaired the molecular architecture as well as the structure and function of hepatocyte tight junctions, which disrupted the hepatic barrier and was lethal to the mice by 6 weeks of age. In mutant mice, bile canaliculus formation and cellular polarity were compromised; also, transporter expression and localization were deregulated. Moreover, typical hepatic zonation and bile duct formation were inhibited, and sinusoidal vessels were disorganized. These findings clarify the role of tight junctions and polarity in the hepatic barrier as well as the effect that their disruption has on liver tissue. The observations also suggest that liver-specific ZO-1-/- and ZO-2-/- mice could be used as models for PFIC4, and this will provide new insights into liver pathophysiology and clinical applications.


Asunto(s)
Conductos Biliares/metabolismo , Colestasis Intrahepática/metabolismo , Hígado/metabolismo , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-2/metabolismo , Animales , Conductos Biliares/patología , Colestasis Intrahepática/genética , Colestasis Intrahepática/patología , Humanos , Hígado/patología , Ratones , Ratones Noqueados , Uniones Estrechas/genética , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-2/genética
9.
ACS Appl Mater Interfaces ; 13(1): 1943-1955, 2021 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-33373205

RESUMEN

In an in vitro nanotoxicity system, cell-nanoparticle (NP) interaction leads to the surface adsorption, uptake, and changes into nuclei/cell phenotype and chemistry, as an indicator of oxidative stress, genotoxicity, and carcinogenicity. Different types of nanomaterials and their chemical composition or "corona" have been widely studied in context with nanotoxicology. However, rare reports are available, which delineate the details of the cell shape index (CSI) and nuclear area factors (NAFs) as a descriptor of the type of nanomaterials. In this paper, we propose a machine-learning-based graph modeling and correlation-establishing approach using tight junction protein ZO-1-mediated alteration in the cell/nuclei phenotype to quantify and propose it as indices of cell-NP interactions. We believe that the phenotypic variation (CSI and NAF) in the epithelial cell is governed by the physicochemical descriptors (e.g., shape, size, zeta potential, concentration, diffusion coefficients, polydispersity, and so on) of the different classes of nanomaterials, which critically determines the intracellular uptake or cell membrane interactions when exposed to the epithelial cells at sub-lethal concentrations. The intrinsic and extrinsic physicochemical properties of the representative nanomaterials (NMs) were measured using optical (dynamic light scattering, NP tracking analysis) methods to create a set of nanodescriptors contributing to cell-NM interactions via phenotype adjustments. We used correlation function as a machine-learning algorithm to successfully predict cell and nuclei shapes and polarity functions as phenotypic markers for five different classes of nanomaterials studied herein this report. The CSI and NAF as nanodescriptors can be used as intuitive cell phenotypic parameters to define the safety of nanomaterials extensively used in consumer products and nanomedicine.


Asunto(s)
Forma del Núcleo Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Aprendizaje Automático , Nanopartículas del Metal/toxicidad , Citoesqueleto de Actina/metabolismo , Animales , Carbono/química , Proliferación Celular/efectos de los fármacos , Dendrímeros/química , Perros , Células Epiteliales/citología , Oro/química , Células de Riñón Canino Madin Darby , Nanopartículas del Metal/química , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/metabolismo
10.
Microvasc Res ; 133: 104103, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33181170

RESUMEN

Diabetic retinopathy (DR) is a disease that causes blindness due to vascular leakage or abnormal angiogenesis. Hepatocyte growth factor (HGF) is increased in the serum or vitreous fluid in proliferative diabetic retinopathy (PDR) patients, although the effect of HGF on the blood vessels remains unclear. This study focused on the effect of HGF on pericyte (PC) survival and endothelial cell (EC) permeability. It was demonstrated that HGF was increased in the diabetic mouse retina. However, HGF prevented PC apoptosis caused by TNF-α, which increased in the diabetic retinas both in vitro and in vivo. In addition, HGF was involved in PC survival by increasing the Akt signaling pathway. Moreover, HGF strengthened the EC tight junction in co-cultures of PCs and ECs by promoting PC survival, thereby reducing EC permeability. These results suggest that HGF may play a role in the prevention of increased vascular leakage by inhibiting the PC loss that occurs in DR to some extent. However, careful HGF reduction in DR might avoid an increase in PC loss.


Asunto(s)
Apoptosis/efectos de los fármacos , Retinopatía Diabética/tratamiento farmacológico , Células Endoteliales/efectos de los fármacos , Factor de Crecimiento de Hepatocito/farmacología , Pericitos/efectos de los fármacos , Vasos Retinianos/efectos de los fármacos , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Pericitos/metabolismo , Pericitos/patología , Permeabilidad , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/patología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Uniones Estrechas/patología
11.
Biomed Pharmacother ; 133: 111026, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33378942

RESUMEN

The main pathophysiological mechanism of acute respiratory distress syndrome (ARDS) invovles the increase in alveolar barrier permeability that is primarily caused by epithelial glycocalyx and tight junction (TJ) protein destruction. This study was performed to explore the effects of the alveolar epithelial glycocalyx on the epithelial barrier, specifically on TJ proteins, in ARDS. We used C57BL/6 mice and human lung epithelial cell models of lipopolysaccharide (LPS)-induced ARDS. Changes in alveolar permeability were evaluated via pulmonary histopathology analysis and by measuring the wet/dry weight ratio of the lungs. Degradation of heparan sulfate (HS), an important component of the epithelial glycocalyx, and alterations in levels of the epithelial TJ proteins (occludin, zonula occludens 1, and claudin 4) were assessed via ELISA, immunofluorescence analysis, and western blotting analysis. Real-time quantitative polymerase chain reaction was used to detect the mRNA of the TJ protein. Changes in glycocalyx and TJ ultrastructures in alveolar epithelial cells were evaluated through electron microscopy. In vivo and in vitro, LPS increased the alveolar permeability and led to HS degradation and TJ damage. After LPS stimulation, the expression of the HS-degrading enzyme heparanase (HPA) in the alveolar epithelial cells was increased. The HPA inhibitor N-desulfated/re-N-acetylated heparin alleviated LPS-induced HS degradation and reduced TJ damage. In vitro, recombinant HPA reduced the expression of the TJ protein zonula occludens-1 (ZO-1) and inhibited its mRNA expression in the alveolar epithelial cells. Taken together, our results demonstrate that shedding of the alveolar epithelial glycocalyx aggravates the epithelial barrier and damages epithelial TJ proteins in ARDS, with the underlying mechanism involving the effect of HPA on ZO-1.


Asunto(s)
Células Epiteliales Alveolares/patología , Barrera Alveolocapilar/patología , Glicocálix/patología , Uniones Estrechas/patología , Células A549 , Células Epiteliales Alveolares/metabolismo , Animales , Barrera Alveolocapilar/metabolismo , Líquido del Lavado Bronquioalveolar/química , Modelos Animales de Enfermedad , Glicocálix/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Permeabilidad , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismo
12.
Elife ; 92020 12 30.
Artículo en Inglés | MEDLINE | ID: mdl-33377869

RESUMEN

Loss of ESCRT function in Drosophila imaginal discs is known to cause neoplastic overgrowth fueled by mis-regulation of signaling pathways. Its impact on junctional integrity, however, remains obscure. To dissect the events leading to neoplasia, we used transmission electron microscopy (TEM) on wing imaginal discs temporally depleted of the ESCRT-III core component Shrub. We find a specific requirement for Shrub in maintaining septate junction (SJ) integrity by transporting the claudin Megatrachea (Mega) to the SJ. In absence of Shrub function, Mega is lost from the SJ and becomes trapped on endosomes coated with the endosomal retrieval machinery retromer. We show that ESCRT function is required for apical localization and mobility of retromer positive carrier vesicles, which mediate the biosynthetic delivery of Mega to the SJ. Accordingly, loss of retromer function impairs the anterograde transport of several SJ core components, revealing a novel physiological role for this ancient endosomal agent.


Asunto(s)
Polaridad Celular/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Uniones Intercelulares/metabolismo , Transporte de Proteínas/fisiología , Uniones Estrechas/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Proteínas de la Membrana/metabolismo
13.
J Stroke Cerebrovasc Dis ; 29(9): 105071, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32807473

RESUMEN

BACKGROUND: Chinese medicine Tongxinluo capsule (TXL) has been extensively used to treat ischemic stroke in China, and one of its mechanisms is to protect against blood brain barrier (BBB) disruption after stroke. However, the underlying protective mechanisms are not fully illuminated. It is reported that the low-density lipoprotein receptor-related protein 1 (LRP-1) is involved in BBB disruption after brain ischemia. In this study, we explored whether TXL could downregulate LRP-1 expression and subsequently protect against BBB disruption after stroke using permanent middle cerebral artery occlusion (pMCAO) in mice. METHODS: The animal model of ischemic stroke was induced by pMCAO in male adult C57BL/6J mice. The mice were orally administered TXL (3.0 g/kg) at 1, 3 and 21 h after pMCAO. Meanwhile, the LRP-1 antagonist receptor associated protein (RAP) was intracerebroventricularly injected at 1 and 21 h after stroke. We measured the following parameters at 6 and 24 h: LRP-1 protein level, BBB leakage, and the expression of tight junction (TJ) proteins including occludin, claudin-5 and zonula occludens-1 (ZO-1). RESULTS: Our results showed that TXL downregulated LRP-1 level, upregulated these TJ proteins level, and reduced BBB leakage in peri-infarct regions after pMCAO. Further study found that the inhibitor RAP played the same role as did TXL in upregulating these TJ proteins level and reducing BBB leakage after stroke. CONCLUSION: Our study demonstrates that TXL protects against BBB disruption after stroke via inhibiting the LRP-1 pathway.


Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Medicamentos Herbarios Chinos/administración & dosificación , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Administración Oral , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/fisiopatología , Cápsulas , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Masculino , Ratones Endogámicos C57BL , Transducción de Señal , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Uniones Estrechas/patología
14.
Am J Physiol Heart Circ Physiol ; 319(4): H730-H743, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-32795184

RESUMEN

Insulin-like growth factor-1 (IGF-1) decreases atherosclerosis in apolipoprotein E (Apoe)-deficient mice when administered systemically. However, mechanisms for its atheroprotective effect are not fully understood. We generated endothelium-specific IGF-1 receptor (IGF1R)-deficient mice on an Apoe-deficient background to assess effects of IGF-1 on the endothelium in the context of hyperlipidemia-induced atherosclerosis. Endothelial deficiency of IGF1R promoted atherosclerotic burden, when animals were fed on a high-fat diet for 12 wk or normal chow for 12 mo. Under the normal chow feeding condition, the vascular relaxation response to acetylcholine was increased in the endothelial IGF1R-deficient aorta; however, feeding of a high-fat diet substantially attenuated the relaxation response, and there was no difference between endothelial IGF1R-deficient and control mice. The endothelium and its intercellular junctions provide a barrier function to the vasculature. In human aortic endothelial cells, IGF-1 upregulated occludin, claudin 5, VE-cadherin, JAM-A, and CD31 expression levels, and vice versa, specific IGF1R inhibitor, picropodophyllin, an IGF1R-neutralizing antibody (αIR3), or siRNA to IGF1R abolished the IGF-1 effects on junction and adherens proteins, suggesting that IGF-1 promoted endothelial barrier function. Moreover, endothelial transwell permeability assays indicated that inhibition of IGF-1 signaling elevated solute permeability through the monolayer of human aortic endothelial cells. In summary, endothelial IGF1R deficiency increases atherosclerosis, and IGF-1 positively regulates tight junction protein and adherens junction protein levels and endothelial barrier function. Our findings suggest that the elevation of the endothelial junction protein level is, at least in part, the mechanism for antiatherogenic effects of IGF-1.NEW & NOTEWORTHY Endothelial insulin-like growth factor-1 (IGF-1) receptor deficiency significantly elevated atherosclerotic burden in apolipoprotein E-deficient mice, mediated at least in part by downregulation of intercellular junction proteins and, thus, elevated endothelial permeability. This study revealed a novel role for IGF-1 in supporting endothelial barrier function. These findings suggest that IGF-1's ability to promote endothelial barrier function may offer a novel therapeutic strategy for vascular diseases such as atherosclerosis.


Asunto(s)
Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Permeabilidad Capilar , Células Endoteliales/metabolismo , Receptor IGF Tipo 1/deficiencia , Animales , Antígenos CD/metabolismo , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/patología , Cadherinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/patología , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Placa Aterosclerótica , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Células THP-1 , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(33): 19854-19865, 2020 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-32759214

RESUMEN

The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-ß pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.


Asunto(s)
Células Endoteliales/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Barrera Hematorretinal/efectos de los fármacos , Barrera Hematorretinal/metabolismo , Diferenciación Celular , Línea Celular , Proliferación Celular/efectos de los fármacos , Claudina-5/genética , Claudina-5/metabolismo , Evaluación Preclínica de Medicamentos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Edición Génica , Genoma , Humanos , Ratones , Ratones Noqueados , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Uniones Estrechas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo
16.
J Anim Sci ; 98(7)2020 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-32607561

RESUMEN

The study was conducted to determine the effects of high levels of phytase on growth performance, nutrient digestibility, phytate breakdown, and expression of mucosal tight junction and nutrient transporter genes in weanling pigs. A total of 128 barrows were penned in groups of four and used in a randomized completely block design and assigned to four treatments for a 28-d study. A two-phase feeding was implemented (phase 1: day 1 to 14; phase 2: day 15 to 28). The diets differed in dietary calcium (Ca) and phosphorus (P) levels (positive control [PC]: 8.1 to 7.1 g/kg Ca and 6.5 to 6.8 g/kg P; negative control [NC]: 6.6 to 5.5 g/kg Ca and 5.6 to 5.3 g/kg P) from phase 1 to phase 2, respectively. NC diets were supplemented with phytase at 0 (NC), 1,500 (NC + 1,500), or 3,000 (NC + 3,000) phytase units (FTU)/kg. Blood was collected after fasting (day 27) or feeding (day 28) for the measurement of plasma inositol concentrations. On day 28, two pigs per pen were euthanized. Duodenal-jejunal and ileal digesta samples and feces were collected to determine inositol phosphates (InsP3-6) concentrations. Phytase supplementation increased the body weight on days 14 and 28 (P < 0.05). Average daily gain and feed efficiency compared with NC were increased by phytase with the majority of its effect in phase 1 (P < 0.05). The apparent ileal digestibility and apparent total tract digestibility of P were increased in piglets fed phytase-supplemented diets (P < 0.01) compared with NC piglets. Disappearance of InsP6 and total InsP3-6 up to the duodenum-jejunum, ileum, and in feces was increased by both phytase application rates (P < 0.01). Plasma concentrations of myo-inositol were higher (P < 0.001) in the phytase-supplemented diets than PC and NC diets, irrespective of whether pigs were fed or fasted. Expression of claudin 3 was higher in pigs fed both phytase-supplemented diets in the duodenum and jejunum compared with PC and NC. Mucin 2 expression was lower in the ileum of NC + 3,000 fed piglets compared with PC (P < 0.05), whereas expression of GLUT2 (solute carrier family 2-facilitated glucose transporter member 2) was increased (P < 0.05) by the NC + 3,000 treatment in all sections. In summary, high phytase supplementation increased the growth performance of nursery pigs. The increased expression of GLUT2 by phytase may indicate an upregulation of glucose absorption from the intestine by phytase.


Asunto(s)
6-Fitasa/farmacología , Digestión/efectos de los fármacos , Porcinos/fisiología , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/genética , Alimentación Animal/análisis , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Calcio en la Dieta/metabolismo , Dieta/veterinaria , Suplementos Dietéticos , Digestión/fisiología , Heces , Tracto Gastrointestinal/metabolismo , Íleon/metabolismo , Inositol/administración & dosificación , Masculino , Nutrientes , Fósforo/metabolismo , Fósforo Dietético/metabolismo , Ácido Fítico/metabolismo , Proteínas de Uniones Estrechas/genética , Uniones Estrechas/metabolismo
17.
Anim Sci J ; 91(1): e13429, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32696533

RESUMEN

To evaluate the effects of crude protein (CP) and lactose (LAC) for weaned piglets on performance, intestinal morphology, and expression of genes related to intestinal integrity and immune system, 144 piglets with initial weight 7.17 ± 0.97 kg were allotted in a randomized design, in a 2 × 3 factorial arrangement (20.0% and 24.0% CP and 8.0%, 12.0%, and 16.0% LAC) with eight replicates. Piglets fed 20.0% CP had greater weight gain and feed intake. Including 12.0% LAC in the 20.0% CP diet provided higher villous height in the duodenum than 8.0% LAC, and 12.0% or 16.0% LAC in the 24.0% CP diet resulted in higher villous height in the jejunum and ileum, and higher villi/crypt ratio in the ileum than 8.0% LAC. No effects of CP and LAC on interleukin-1ß and tumor necrosis factor-α mRNA were observed. The 16.0% LAC diet provided higher gene expression of transforming-ß1 growth factor. Feeding 20.0% CP resulted in better performance than 24.0% CP. The 12.0% LAC diet promoted greater genetic expression of occludin and zonula occludens. Including 12.0% LAC in the diet may improve intestinal epithelial morphology and integrity, and these improvements are more evident when piglets are fed diets with 24.0% CP.


Asunto(s)
Alimentación Animal/análisis , Fenómenos Fisiológicos Nutricionales de los Animales , Dieta/veterinaria , Proteínas en la Dieta/análisis , Expresión Génica , Mucosa Intestinal/anatomía & histología , Mucosa Intestinal/inmunología , Lactosa/análisis , Porcinos/crecimiento & desarrollo , Porcinos/genética , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Ingestión de Alimentos , Femenino , Masculino , Ocludina/genética , Ocludina/metabolismo , Porcinos/anatomía & histología , Porcinos/inmunología , Uniones Estrechas/genética , Uniones Estrechas/metabolismo , Aumento de Peso
18.
Science ; 369(6505): 787-793, 2020 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-32675289

RESUMEN

Although Huntington's disease is a late-manifesting neurodegenerative disorder, both mouse studies and neuroimaging studies of presymptomatic mutation carriers suggest that Huntington's disease might affect neurodevelopment. To determine whether this is actually the case, we examined tissue from human fetuses (13 weeks gestation) that carried the Huntington's disease mutation. These tissues showed clear abnormalities in the developing cortex, including mislocalization of mutant huntingtin and junctional complex proteins, defects in neuroprogenitor cell polarity and differentiation, abnormal ciliogenesis, and changes in mitosis and cell cycle progression. We observed the same phenomena in Huntington's disease mouse embryos, where we linked these abnormalities to defects in interkinetic nuclear migration of progenitor cells. Huntington's disease thus has a neurodevelopmental component and is not solely a degenerative disease.


Asunto(s)
Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Sistema Nervioso/embriología , Animales , Ciclo Celular , Endosomas/metabolismo , Feto , Humanos , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Ratones , Ratones Mutantes , Mitosis , Mutación , Células Neuroepiteliales/metabolismo , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/metabolismo
19.
Toxicol Lett ; 333: 33-41, 2020 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-32687961

RESUMEN

Recent studies have revealed that increased reactive oxidative stress (ROS) induced by particulate matter (PM) affects tight junction (TJ) functions; however, the molecular mechanisms underlying this effect have not been evaluated fully. Cultured human epithelial cells obtained from inferior turbinate tissues were exposed to an urban PM (UPM) standard reference material (SRM 1648a). Intracellular ROS level and expression of proinflammatory cytokines and TJ proteins were examined. Expression level of phosphorylated (p)-Akt, p38, p65 were compared between exposed and unexposed cells. Cells were pretreated with the ROS scavenger N-acetylcysteine (NAC) or Akt inhibitor MK-2206 before exposure to determine whether the changes in cellular ROS and TJ protein expression could be reversed. Exposure to UPM significantly increased ROS levels and inflammatory cytokine expression levels, and decreased expression of TJ proteins zonula occludins (ZO)-1, occludin, claudin-1, and E-cadherin. UPM exposure increased p-Akt, p-p38, and p65 expression levels, and NAC pretreatment reversed these effects. Akt inhibition decreased UPM-induced ROS formation and p38 and p65 protein phosphorylation, and restored the decreased ZO-1 and E-cadherin expression. Akt inhibition and ROS scavenging may provide targets for maintaining epithelial integrity by restoring decreased TJ protein expression during exposure to UPM.


Asunto(s)
Contaminantes Atmosféricos/toxicidad , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Mucosa Nasal/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Material Particulado/toxicidad , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Acetilcisteína/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Células Epiteliales/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mucosa Nasal/metabolismo , Estrés Oxidativo/genética , Transducción de Señal , Proteínas de Uniones Estrechas/genética , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Cornetes Nasales/efectos de los fármacos , Cornetes Nasales/metabolismo , Urbanización
20.
Toxicol Lett ; 331: 242-253, 2020 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-32579994

RESUMEN

Dysregulated bile acid (BA) homeostasis is an extremely significant pathological phenomenon of intrahepatic cholestasis, and the accumulated BA could further trigger hepatocyte injury. Here, we showed that the expression of sphingosine-1-phosphate receptor 1 (S1PR1) was down-regulated by α-naphthylisothiocyanate (ANIT) in vivo and in vitro. The up-regulated S1PR1 induced by SEW2871 (a specific agonist of S1PR1) could improve ANIT-induced deficiency of hepatocyte tight junctions (TJs), cholestatic liver injury and the disrupted BA homeostasis in mice. BA metabolic profiles showed that SEW2871 not only reversed the disruption of plasma BA homeostasis, but also alleviated BA accumulation in the liver of ANIT-treated mice. Further quantitative analysis of 19 BAs showed that ANIT increased almost all BAs in mice plasma and liver, all of which were restored by SEW2871. Our data demonstrated that the top performing BAs were taurine conjugated bile acids (T-), especially taurocholic acid (TCA). Molecular mechanism studies indicated that BA transporters, synthetase, and BAs nuclear receptors (NRs) might be the important factors that maintained BA homeostasis by SEW2871 in ANIT-induced cholestasis. In conclusion, these results demonstrated that S1PR1 selective agonists might be the novel and potential effective agents for the treatment of intrahepatic cholestasis by recovering dysregulated BA homeostasis.


Asunto(s)
1-Naftilisotiocianato/toxicidad , Ácidos y Sales Biliares/sangre , Colestasis/prevención & control , Hígado/efectos de los fármacos , Oxadiazoles/farmacología , Receptores de Esfingosina-1-Fosfato/agonistas , Tiofenos/farmacología , Animales , Ácidos y Sales Biliares/metabolismo , Colestasis/inducido químicamente , Colestasis/metabolismo , Regulación hacia Abajo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Homeostasis , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Receptores de Esfingosina-1-Fosfato/genética , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Uniones Estrechas/patología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...