RESUMEN
The intricate interplay between biomechanical and biochemical pathways in modulating morphogenesis is an interesting research topic. How biomechanical force regulates epithelial cell tubulogenesis remains poorly understood. Here, we established a model of tubulogenesis by culturing renal proximal tubular epithelial cells on a collagen gel while manipulating contractile force. Epithelial cells were dynamically self-organized into tubule-like structures by augmentation of cell protrusions and cell-cell association. Reduction and asymmetric distribution of phosphorylated myosin light chain 2, the actomyosin contractility, in cells grown on soft matrix preceded tube connection. Notably, reducing matrix stiffness via sonication of collagen fibrils and inhibiting actomyosin contractility with blebbistatin promoted tubulogenesis, whereas inhibition of cytoskeleton polymerization suppressed it. CXC chemokine ligand 1 (CXCL1) expression was transcriptionally upregulated in cells undergoing tubulogenesis. Additionally, inhibiting actomyosin contractility facilitated CXCL1 polarization and cell protrusions preceding tube formation. Conversely, inhibiting the CXCL1-CXC receptor 1 pathway hindered cell protrusions and tubulogenesis. Mechanical property asymmetry with cell-collagen fibril interaction patterns at cell protrusions and along the tube structure supported the association of anisotropic contraction with tube formation. Furthermore, suppressing the mechanosensing machinery of integrin subunit beta 1 reduced CXCL1 expression, collagen remodeling, and impaired tubulogenesis. In summary, symmetry breaking of cell contractility on a soft collagen gel promotes CXCL1 polarization at cell protrusions which in turn facilitates cell-cell association and thus tubule connection.
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Actomiosina , Colágeno , Actomiosina/metabolismo , Matriz Extracelular/metabolismo , Morfogénesis , Células Epiteliales/metabolismoRESUMEN
BACKGROUND: Tumor vascular mimicry is an emerging issue that affects patient survival while having no treatment at the current moment. Despite several factors implicated in vascular mimicry, little is known about stromal factors that modulate tumor microenvironment and shape malignant transformation. CD248, a type-I transmembrane protein dominantly expressed in stromal cells, mediates the interaction between cells and extracellular matrix proteins. CD248 protein expression is associated with the metastatic melanoma phenotype and promotes tumor progression in the stromal cells. This study aimed to explore the cell-autonomous effects of CD248 in melanoma vascular mimicry to aid cancer therapy development. METHODS: Loss-of-function approaches in B16F10 melanoma cells were used to study the cell-autonomous effects of CD248 on cell adhesion, migration, proliferation, and vascular mimicry. A solid-phase binding assay was performed to identify the interaction between CD248 and fibronectin. Horizontal and vertical cell migration assays were performed to analyze cell migration activity, and cell-patterned network formation on Matrigel was used to evaluate vascular mimicry activity. Recombinant CD248 (rCD248) proteins were generated, and whether rCD248 interfered with melanoma CD248 functions was evaluated in vitro. An experimental lung metastasis mouse model was used to investigate the effect of rCD248 treatment in vivo. RESULTS: CD248 protein expression in melanoma cells was increased by a fibroblast-conditioned medium. Knockdown of CD248 expression significantly decreased cell adhesion to fibronectin, cell migration, and vascular mimicry in melanoma cells. The lectin domain of CD248 was directly involved in the interaction between CD248 and fibronectin. Furthermore, rCD248 proteins containing its lectin domain inhibited cell adhesion to fibronectin and slowed down cell migration and vascular mimicry. Treatment with rCD248 protein could reduce pulmonary tumor burden, accompanied by a reduction in vascular mimicry in mice with melanoma lung metastasis. CONCLUSION: CD248 expression in melanoma cells promotes malignant transformation by increasing the activity of cell adhesion, migration, and vascular mimicry, whereas rCD248 protein functions as a molecular decoy interfering with tumor-promoting effects of CD248 in melanoma cells.
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Neoplasias Pulmonares , Melanoma , Ratones , Animales , Fibronectinas , Melanoma/genética , Adhesión Celular , Neoplasias Pulmonares/genética , Lectinas/farmacología , Microambiente Tumoral , Antígenos de Neoplasias/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD/farmacologíaRESUMEN
[Figure: see text].
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Inductores de la Angiogénesis/farmacología , Células Endoteliales/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Podosomas/efectos de los fármacos , Trombomodulina/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Quinasas Asociadas a rho/metabolismo , Actinas/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/enzimología , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Hiperoxia/complicaciones , Masculino , Microdominios de Membrana/enzimología , Ratones Endogámicos C57BL , Ratones Noqueados , Podosomas/enzimología , Neovascularización Retiniana/enzimología , Neovascularización Retiniana/etiología , Neovascularización Retiniana/genética , Transducción de Señal , Trombomodulina/genética , Cicatrización de Heridas , Quinasas Asociadas a rho/genéticaRESUMEN
Pathological angiogenesis (PA) contributes to various ocular diseases, including age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity, which are major causes of blindness over the world. Current treatments focus on anti-vascular endothelial growth factor (VEGF) therapy, but persistent avascular retina, recurrent intravitreal neovascularization, and general adverse effects are reported. We have previously found that recombinant thrombomodulin domain 1 (rTMD1) can suppress vascular inflammation. However, the function of rTMD1 in VEGF-induced PA remains unknown. In this study, we found that rTMD1 inhibited VEGF-induced angiogenesis in vitro. In an oxygen induced retinopathy (OIR) animal model, rTMD1 treatment significantly decreased retinal neovascularization but spared normal physiological vessel growth. Furthermore, loss of TMD1 significantly promoted PA in OIR. Meanwhile, hypoxia-inducible factor-1α, the transcription factor that upregulates VEGF, was suppressed after rTMD1 treatment. The levels of interleukin-6, and intercellular adhesion molecule-1 were also significantly suppressed. In conclusion, our results indicate that rTMD1 not only has dual effects to suppress PA and inflammation in OIR, but also can be a potential HIF-1α inhibitor for clinical use. These data bring forth the possibility of rTMD1 as a novel therapeutic agent for PA.
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Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Neovascularización Patológica/prevención & control , Neovascularización Retiniana/prevención & control , Trombomodulina/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Apoptosis , Movimiento Celular , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neovascularización Retiniana/genética , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Trombomodulina/genética , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Thrombomodulin (TM), an integral membrane protein, has long been known for its anticoagulant activity. Recent studies showed that TM displays multifaceted activities, including the involvement in cell adhesion and collective cell migration in vitro. However, whether TM contributes similarly to these biological processes in vivo remains elusive. METHODS: We adapted zebrafish, a prominent animal model for studying molecular/cellular activity, embryonic development, diseases mechanism and drug discovery, to examine how TM functions in modulating cell migration during germ layer formation, a normal and crucial physiological process involving massive cell movement in the very early stages of life. In addition, an in vivo assay was developed to examine the anti-hemostatic activity of TM in zebrafish larva. RESULTS: We found that zebrafish TM-b, a zebrafish TM-like protein, was expressed mainly in vasculatures and displayed anti-hemostatic activity. Knocking-down TM-b led to malformation of multiple organs, including vessels, heart, blood cells and neural tissues. Delayed epiboly and incoherent movement of yolk syncytial layer were also observed in early TM-b morphants. Whole mount immunostaining revealed the co-localization of TM-b with both actin and microtubules in epibolic blastomeres. Single-cell tracking revealed impeded migration of blastomeres during epiboly in TM-b-deficient embryos. CONCLUSION: Our results showed that TM-b is crucial to the collective migration of blastomeres during germ layer formation. The structural and functional compatibility and conservation between zebrafish TM-b and mammalian TM support the properness of using zebrafish as an in vivo platform for studying the biological significance and medical use of TM.
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Estratos Germinativos/embriología , Morfogénesis , Organogénesis , Trombomodulina/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Blastómeros/metabolismo , Embrión no Mamífero/embriología , Trombomodulina/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
RATIONALE: PhosPhatidic Acid Phosphatase type 2B (PPAP2B), an integral membrane protein known as lipid phosphate phosphatase (LPP3) that inactivates lysophosphatidic acid, was implicated in coronary artery disease (CAD) by genome-wide association studies. However, it is unclear whether genome-wide association studies-identified coronary artery disease genes, including PPAP2B, participate in mechanotransduction mechanisms by which vascular endothelia respond to local atherorelevant hemodynamics that contribute to the regional nature of atherosclerosis. OBJECTIVE: To establish the critical role of PPAP2B in endothelial responses to hemodynamics. METHODS AND RESULTS: Reduced PPAP2B was detected in vivo in mouse and swine aortic arch (AA) endothelia exposed to chronic disturbed flow, and in mouse carotid artery endothelia subjected to surgically induced acute disturbed flow. In humans, PPAP2B was reduced in the downstream part of carotid plaques where low shear stress prevails. In culture, reduced PPAP2B was measured in human aortic endothelial cells under atherosusceptible waveform mimicking flow in human carotid sinus. Flow-sensitive microRNA-92a and transcription factor KLF2 were identified as upstream inhibitor and activator of endothelial PPAP2B, respectively. PPAP2B suppression abrogated atheroprotection of unidirectional flow; inhibition of lysophosphatidic acid receptor 1 restored the flow-dependent, anti-inflammatory phenotype in PPAP2B-deficient cells. PPAP2B inhibition resulted in myosin light-chain phosphorylation and intercellular gaps, which were abolished by lysophosphatidic acid receptor 1/2 inhibition. Expression quantitative trait locus mapping demonstrated PPAP2B coronary artery disease risk allele is not linked to PPAP2B expression in various human tissues but significantly associated with reduced PPAP2B in human aortic endothelial cells. CONCLUSIONS: Atherorelevant flows dynamically modulate endothelial PPAP2B expression through miR-92a and KLF2. Mechanosensitive PPAP2B plays a critical role in promoting anti-inflammatory phenotype and maintaining vascular integrity of endothelial monolayer under atheroprotective flow.
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Aorta Torácica/enzimología , Aterosclerosis/enzimología , Células Endoteliales/enzimología , Hemodinámica , Mecanotransducción Celular , Fosfatidato Fosfatasa/metabolismo , Regiones no Traducidas 3' , Animales , Aorta Torácica/fisiopatología , Aterosclerosis/genética , Aterosclerosis/fisiopatología , Aterosclerosis/prevención & control , Sitios de Unión , Células Cultivadas , Regulación Enzimológica de la Expresión Génica , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , MicroARNs/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Fenotipo , Fosfatidato Fosfatasa/genética , Fosforilación , Interferencia de ARN , Receptores del Ácido Lisofosfatídico/metabolismo , Flujo Sanguíneo Regional , Estrés Mecánico , Porcinos , Factores de Tiempo , TransfecciónRESUMEN
AIMS/HYPOTHESIS: Chronic inflammatory processes have been increasingly shown to be involved in the pathogenesis of diabetes and diabetic nephropathy. Recently, we demonstrated that a lectin-like domain of thrombomodulin (THBD), which is known as THBD domain 1 (THBDD1) and which acts independently of protein C activation, neutralised an inflammatory response in a mouse model of sepsis. Here, therapeutic effects of gene therapy with adeno-associated virus (AAV)-carried THBDD1 (AAV-THBDD1) were tested in a mouse model of type 2 diabetic nephropathy. METHODS: To assess the therapeutic potential of THBDD1 and the mechanisms involved, we delivered AAV-THBDD1 (10(11) genome copies) into db/db mice and tested the effects of recombinant THBDD1 on conditionally immortalised podocytes. RESULTS: A single dose of AAV-THBDD1 improved albuminuria, renal interstitial inflammation and glomerular sclerosis, as well as renal function in db/db mice. These effects were closely associated with: (1) inhibited activation of the nuclear factor κB (NF-κB) pathway and the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome; (2) promotion of nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear translocation; and (3) suppression of mitochondria-derived apoptosis in the kidney of treated mice. CONCLUSIONS/INTERPRETATION: AAV-THBDD1 gene therapy resulted in improvements in a model of diabetic nephropathy by suppressing the NF-κB-NLRP3 inflammasome-mediated inflammatory process, enhancing the NRF2 antioxidant pathway and inhibiting apoptosis in the kidney.
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Antioxidantes/farmacología , Proteínas Portadoras/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/patología , Nefropatías Diabéticas/metabolismo , Terapia Genética , Inflamasomas/metabolismo , FN-kappa B/metabolismo , Trombomodulina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Portadoras/antagonistas & inhibidores , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/inmunología , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/inmunología , Terapia Genética/métodos , Inflamación/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/antagonistas & inhibidores , Proteína con Dominio Pirina 3 de la Familia NLRRESUMEN
Lewis Y Ag (LeY) is a cell-surface tetrasaccharide that participates in angiogenesis. Recently, we demonstrated that LeY is a specific ligand of the recombinant lectin-like domain of thrombomodulin (TM). However, the biologic function of interaction between LeY and TM in endothelial cells has never been investigated. Therefore, the role of LeY in tube formation and the role of the recombinant lectin-like domain of TM-TM domain 1 (rTMD1)-in antiangiogenesis were investigated. The recombinant TM ectodomain exhibited lower angiogenic activity than did the recombinant TM domains 2 and 3. rTMD1 interacted with soluble LeY and membrane-bound LeY and inhibited soluble LeY-mediated chemotaxis of endothelial cells. LeY was highly expressed on membrane ruffles and protrusions during tube formation on Matrigel. Blockade of LeY with rTMD1 or Ab against LeY inhibited endothelial tube formation in vitro. Epidermal growth factor (EGF) receptor in HUVECs was LeY modified. rTMD1 inhibited EGF receptor signaling, chemotaxis, and tube formation in vitro, and EGF-mediated angiogenesis and tumor angiogenesis in vivo. We concluded that LeY is involved in vascular endothelial tube formation and rTMD1 inhibits angiogenesis via interaction with LeY. Administration of rTMD1 or recombinant adeno-associated virus vector carrying TMD1 could be a promising antiangiogenesis strategy.
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Antígenos del Grupo Sanguíneo de Lewis/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Recombinantes/farmacología , Trombomodulina/química , Animales , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/patología , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Femenino , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Células Endoteliales de la Vena Umbilical Humana/trasplante , Humanos , Lectinas/química , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica/fisiología , Unión Proteica , Estructura Terciaria de Proteína/fisiología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Trombomodulina/metabolismo , Trombomodulina/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Urokinase-type plasminogen activator (uPA) activates plasminogen (Plg) through a major pericellular proteolytic system involved in cell migration and angiogenesis; however, the Plg receptor that participates in uPA-mediated Plg activation has not yet been identified. In this study, we demonstrated that thrombomodulin (TM), a type I transmembrane glycoprotein, is a novel Plg receptor that plays a role in pericellular proteolysis and cell migration. Plg activation at the cell surface and the extent of its cell migration- and invasion-promoting effect are cellular TM expression dependent. Direct binding of Plg and the recombinant TM extracellular domain, with a KD of 0.1-0.3 µM, was determined through surface plasmon resonance analysis. Colocalization of TM, Plg, and the uPA receptor within plasma membrane lipid rafts, at the leading edge of migrating endothelial cells, was demonstrated and was also shown to overlap with areas of major pericellular proteolysis. Moreover, the roles of TM and Plg in neoangiogenesis were demonstrated in vivo through the skin wound-healing model. In conclusion, we propose that TM is a novel Plg receptor that regulates uPA/uPA receptor-mediated Plg activation and pericellular proteolysis within lipid rafts at the leading edge of migrating cells during angiogenesis.
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Neovascularización Fisiológica , Plasminógeno/metabolismo , Trombomodulina/metabolismo , Animales , Células CHO , Movimiento Celular , Cricetinae , Cricetulus , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Microdominios de Membrana/metabolismo , Ratones , Ratones Transgénicos , Factor de Crecimiento Placentario , Proteínas Gestacionales/genética , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteolisis , Piel/irrigación sanguínea , Trombomodulina/química , Trombomodulina/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Cicatrización de HeridasRESUMEN
Opioids are powerful analgesics; however, their significant systemic adverse effects and the need for frequent administration restrict their use. Nalbuphine (NA) is a κ-agonist narcotic with limited adverse effects, but needs to be frequently administrated due to its short elimination half-life. Whereas sebacoyl dinalbuphine ester (SDE) is a NA prodrug, which can effectively prolong the analgesic effect, but lacks immediate pain relief. Therefore, in this study, a rapid and sustained local delivery formulation to introduce NA and SDE directly into surgical sites was developed. An amphiphilic nanostructured lipid carrier (NLC) poloxamer 407 (P407) gel (NLC-Gel) was developed to permit concurrent delivery of hydrophobic SDE from the NLC core and hydrophilic NA from P407, offering a dual rapid and prolonged analgesic effect. Benefiting from the thermal-sensitive characteristic of P407, the formulation can be injected in liquid phase and instantly transit into gel at wound site. NLC-Gel properties, including particle size, drug release, rheology, and stability, were assessed. In vivo evaluation using a rat spinal surgery model highlighted the effect of the formulation through pain behavior test and hematology analysis. NLC-Gels demonstrated an analgesic effect comparable with that of commercial intramuscular injected SDE formulation (IM SDE), with only 15 % of the drug dosage. The inclusion of supplemental NA in the exterior gel (PA12-Gel + NA) provided rapid drug onset owing to swift NA dispersion, addressing acute pain within hours along with prolonged analgesic effects. Our findings suggest that this amphiphilic formulation significantly enhanced postoperative pain management in terms of safety and efficacy.
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Analgésicos Opioides , Portadores de Fármacos , Liberación de Fármacos , Geles , Nalbufina , Dolor Postoperatorio , Poloxámero , Ratas Sprague-Dawley , Nalbufina/administración & dosificación , Dolor Postoperatorio/tratamiento farmacológico , Animales , Masculino , Poloxámero/química , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/química , Portadores de Fármacos/química , Ratas , Lípidos/química , Tamaño de la Partícula , Nanoestructuras/administración & dosificación , Nanoestructuras/química , Ésteres/químicaRESUMEN
Intervertebral disc degeneration arises from damage or degeneration of the nucleus pulposus (NP). In this study, we developed a photo-crosslinkable hydrogel incorporating FG4592 to support the growth and differentiation of bone-marrow-derived mesenchymal stem cells (BMSC). Initially, hyaluronic acid was modified with tyramine and combined with collagen to introduce riboflavin as a photo-crosslinker. This hydrogel transitioned from liquid to gel upon exposure to blue light in 3 min. The results showed that the hydrogel was biodegradable and had mechanical properties comparable to those of human NP tissues. Scanning electron microscopy after BMSC seeding in the hydrogel revealed an even distribution, and cells adhered to the collagen fibers in the hydrogel with minimal cell mortality. The effect of FG4592 on BMSC proliferation and differentiation was examined, revealing the capability of FG4592 to promote BMSC proliferation and direct differentiation resembling human NP cells. After cultivating BMSCs in the photo-crosslinked hydrogel, there was an upregulation in the expression of glycosaminoglycans, aggrecan, type II collagen, and keratin 19 proteins. Cross-species analyses of rat and human BMSCs revealed consistent results. For potential clinical applications, BMSC loaded with photo-crosslinked hydrogels can be injected into damaged intervertebral disc to facilitate NP regeneration.
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Diferenciación Celular , Proliferación Celular , Colágeno , Ácido Hialurónico , Hidrogeles , Células Madre Mesenquimatosas , Núcleo Pulposo , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Núcleo Pulposo/citología , Núcleo Pulposo/efectos de los fármacos , Núcleo Pulposo/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Humanos , Animales , Hidrogeles/química , Hidrogeles/farmacología , Colágeno/química , Ratas , Reactivos de Enlaces Cruzados/química , Ratas Sprague-Dawley , Anilidas , Ácidos FtálicosRESUMEN
BACKGROUND: In spite of new treatments, the incidence of type 2 diabetes (T2D) and its morbidities continue to rise. The key feature of T2D is resistance of adipose tissue and other organs to insulin. Approaches to overcome insulin resistance are limited due to a poor understanding of the mechanisms and inaccessibility of drugs to relevant intracellular targets. We previously showed in mice and humans that CD248, a pre/adipocyte cell surface glycoprotein, acts as an adipose tissue sensor that mediates the transition from healthy to unhealthy adipose, thus promoting insulin resistance. METHODS: Molecular mechanisms by which CD248 regulates insulin signaling were explored using in vivo insulin clamp studies and biochemical analyses of cells/tissues from CD248 knockout (KO) and wild-type (WT) mice with diet-induced insulin resistance. Findings were validated with human adipose tissue specimens. FINDINGS: Genetic deletion of CD248 in mice, overcame diet-induced insulin resistance with improvements in glucose uptake and lipolysis in white adipose tissue depots, effects paralleled by increased adipose/adipocyte GLUT4, phosphorylated AKT and GSK3ß, and reduced ATGL. The insulin resistance of the WT mice could be attributed to direct interaction of the extracellular domains of CD248 and the insulin receptor (IR), with CD248 acting to block insulin binding to the IR. This resulted in dampened insulin-mediated autophosphorylation of the IR, with reduced downstream signaling/activation of intracellular events necessary for glucose and lipid homeostasis. INTERPRETATION: Our discovery of a cell-surface CD248-IR complex that is accessible to pharmacologic intervention, opens research avenues toward development of new agents to prevent/reverse insulin resistance. FUNDING: Funded by Canadian Institutes of Health Research (CIHR), Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Foundations for Innovation (CFI), the Swedish Diabetes Foundation, Family Ernfors Foundation and Novo Nordisk Foundation.
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Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Humanos , Ratones , Animales , Insulina/metabolismo , Resistencia a la Insulina/genética , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Ratones Noqueados , Canadá , Tejido Adiposo/metabolismo , Obesidad/metabolismo , Antígenos de Neoplasias/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismoRESUMEN
OBJECTIVE: To investigate whether recombinant thrombomodulin containing all the extracellular domains (rTMD123) has therapeutic potential against aneurysm development. SUMMARY BACKGROUND DATA: The pathogenesis of abdominal aortic aneurysm (AAA) is characterized by chronic inflammation and proteolytic degradation of extracellular matrix. Thrombomodulin, a transmembrane glycoprotein, exerts anti-inflammatory activities such as inhibition of cytokine production and sequestration of proinflammatory high-mobility group box 1 (HMGB1) to prevent it from engaging the receptor for advanced glycation end product (RAGE) that may sustain inflammation and tissue damage. METHODS: The in vivo effects of treatment and posttreatment with rTMD123 on aortic dilatation were measured using the CaCl2-induced AAA model in mice. RESULTS: Characterization of the CaCl2-induced model revealed that HMGB1 and RAGE, both localized mainly to macrophages, were persistently upregulated during a 28-day period of AAA development. In vitro, rTMD123-HMGB1 interaction prevented HMGB1 binding to macrophages, thereby prohibiting activation of HMGB1-RAGE signaling in macrophages. In vivo, short-term treatment with rTMD123 upon AAA induction suppressed the levels of proinflammatory cytokines, HMGB1, and RAGE in the aortic tissue; reduced the infiltrating macrophage number; and finally attenuated matrix metalloproteinase production, extracellular matrix destruction, and AAA formation without disturbing vascular calcification. Consistently, posttreatment with rTMD123 seven days after AAA induction alleviated vascular inflammation and retarded AAA progression. CONCLUSIONS: These data suggest that rTMD123 confers protection against AAA development. The mechanism of action may be associated with reduction of proinflammatory mediators, blockade of macrophage recruitment, and suppression of HMGB1-RAGE signaling involved in aneurysm formation and downstream macrophage activation.
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Aneurisma de la Aorta Abdominal/prevención & control , Trombomodulina/uso terapéutico , Animales , Aneurisma de la Aorta Abdominal/inducido químicamente , Cloruro de Calcio/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/fisiología , Proteínas Recombinantes/uso terapéuticoRESUMEN
Adhesive interactions between cells are needed to maintain tissue architecture during development, tissue renewal and wound healing. Thrombomodulin (TM) is an integral membrane protein that participates in cell-cell adhesion through its extracellular lectin-like domain. However, the molecular basis of TM-mediated cell-cell adhesion is poorly understood. Here, we demonstrate that TM is linked to the actin cytoskeleton via ezrin. In vitro binding assays showed that the TM cytoplasmic domain bound directly to the N-terminal domain of ezrin. Mutational analysis of the TM cytoplasmic domain identified (522)RKK(524) as important ezrin-binding residues. In epidermal epithelial A431 cells, TM colocalized with ezrin and actin filaments at cell-cell contacts. Knockdown of endogenous TM expression by RNA interference induced morphological changes and accelerated cell migration in A431 cells. Moreover, epidermal growth factor, upstream of ezrin activation, stimulated the interaction between ezrin and TM. In skin wound healing of mice, TM and ezrin were highly expressed in neoepidermis, implying that both proteins are key molecules in reepithelialization that requires collective cell migration of epithelial cells. Finally, exogenous expression of TM in TM-deficient melanoma A2058 cells promoted collective cell migration. In summary, TM, which associates with ezrin and actin filaments, maintains epithelial morphology and promotes collective cell migration.
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Movimiento Celular , Proteínas del Citoesqueleto/metabolismo , Trombomodulina/fisiología , Actinas/metabolismo , Animales , Sitios de Unión , Adhesión Celular , Línea Celular , Células Epidérmicas , Factor de Crecimiento Epidérmico , Humanos , Ratones , Estructura Terciaria de Proteína , Trombomodulina/genética , Cicatrización de Heridas/fisiologíaRESUMEN
Despite several extraordinary improvements in cancer immunotherapy, its therapeutic effectiveness against many distinct cancer types remains mostly limited and requires further study. Different microfluidic-based cancer immunotherapy-on-a-chip (ITOC) systems have been developed to help researchers replicate the tumor microenvironment and immune system. Numerous microfluidic platforms can potentially be used to perform various on-chip activities related to early clinical cancer immunotherapy processes, such as improving immune checkpoint blockade therapy, studying immune cell dynamics, evaluating cytotoxicity, and creating vaccines or organoid models from patient samples. In this review, we summarize the most recent advancements in the development of various microfluidic-based ITOC devices for cancer treatment niches and present future perspectives on microfluidic devices for immunotherapy research.
RESUMEN
Retinal neovascularization (RNV) and cell apoptosis observed in retinopathy are the most common cause of vision loss worldwide. Increasing vascular endothelial growth factor (VEGF), which was driven by hypoxia or inflammation, would result in RNV. This study investigated the anti-inflammatory and anti-apoptotic xanthine-based derivative KMUP-1 on hypoxia-induced conditions in vitro and in vivo. In the oxygen-induced retinopathy animal model, KMUP-1 mitigated vaso-obliteration and neovascularization. In the cell model of hypoxic endothelium cultured at 1% O2, KMUP-1 inhibited endothelial migration and tube formation and had no cytotoxic effect on cell growth. Upregulation of pro-angiogenic factors, HIF-1α and VEGF, and pro-inflammatory cytokines, IL-1ß and TNF-α, expression in the retinal-derived endothelial cells, RF/6 A cells, upon hypoxia stimulation, was suppressed by KMUP-1 treatment. RF/6 A cells treated with KMUP-1 showed a reduction of PI3K/Akt, ERK, and RhoA/ROCKs signaling pathways and induction of protective pathways such as eNOS and soluble guanylyl cyclase at 1% O2. Furthermore, KMUP-1 decreased the expression of VEGF, ICAM-1, TNF-α, and IL-1ß and increased the BCL-2/BAX ratio in the oxygen-induced retinopathy mouse retina samples. In conclusion, the results of this study suggest that KMUP-1 has potential therapeutic value in retinopathy due to its triple effects on anti-angiogenesis, anti-inflammation, and anti-apoptosis in hypoxic endothelium.
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Enfermedades de la Retina , Neovascularización Retiniana , Animales , Ratones , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Endoteliales , Factor de Necrosis Tumoral alfa/farmacología , Fosfatidilinositol 3-Quinasas , Enfermedades de la Retina/tratamiento farmacológico , Neovascularización Retiniana/tratamiento farmacológico , Xantinas/farmacología , Oxígeno/farmacología , Hipoxia/tratamiento farmacológico , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Subunidad alfa del Factor 1 Inducible por HipoxiaRESUMEN
During menopause, the sharp decline in estrogen levels leads to an increased risk of cardiovascular disease in women. The inflammatory response and oxidative stress are reportedly involved in the development of cardiovascular disorders postmenopause. In this study, we evaluated the cardioprotective effects of puerarin, a phytoestrogen derived from the root of Pueraria lobate, and investigated its underlying molecular mechanisms. Puerarin alleviated cytotoxicity and the production of reactive oxygen species (ROS) in lipopolysaccharide (LPS)- and hydrogen peroxide-stimulated H9c2 cardiomyoblasts. Puerarin scavenges free radicals and reduces apoptosis, thereby suppressing NADPH oxidase-1 and Bax activation to attenuate the production of ROS and restore Bcl-2 expression. Additionally, puerarin inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2, and nitric oxide production and decreased the hypertrophic phenotype under LPS stimulation. Treatment with puerarin reduced the levels of malondialdehyde and restored glutathione levels when facing oxidative stress. Mechanistically, puerarin inhibited both the LPS-induced Toll-like receptor 4/NF-[Formula: see text]B and mitogen-activated protein kinase signaling pathways. Furthermore, it reversed both the LPS-mediated downregulation of Akt activation and heme oxygenase-1 (HO-1) expression. The cardioprotective effects of puerarin were abolished by inhibitors of Akt and HO-1 and the estrogen receptor antagonist fulvestrant (ICI). This indicated that the estrogen receptor mediated by these two molecules plays important roles in conferring the anti-inflammatory and anti-oxidative functions of puerarin. These results demonstrate the therapeutic potential of puerarin for treating heart disease in postmenopausal women through Akt and HO-1 activation.
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Hemo-Oxigenasa 1 , Proteínas Proto-Oncogénicas c-akt , Femenino , Animales , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Posmenopausia , Lipopolisacáridos , Antiinflamatorios/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismoRESUMEN
Rationale: CD93, a C-type lectin-like transmembrane glycoprotein, can be shed in a soluble form (sCD93) upon inflammatory stimuli. sCD93 effectively enhances apoptotic cell clearance and has been proposed as an inflammatory disease biomarker. The function of sCD93 involved directly in inflammation remains to be determined. Herein, we attempted to examine the hypothesis that sCD93 might sequester proinflammatory high-mobility group box 1 protein (HMGB1), exerting anti-inflammatory properties. Methods: Different forms of soluble recombinant human CD93 (rCD93) were prepared by a mammalian protein expression system. rCD93-HMGB1 interaction was assessed using co-immunoprecipitation and solid-phase binding assays. Effects of soluble rCD93 were evaluated in HMGB1-induced macrophage and vascular smooth muscle cells (VSMC) activation and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, CaCl2-induced and angiotensin II-infused abdominal aortic aneurysm (AAA) formation and ovariectomized-induced osteoporosis in mice. Results: Protein binding studies revealed that soluble rCD93, via the lectin-like domain (D1), can bind to HMGB1 and intercept HMGB1-receptor interaction. Soluble rCD93 containing D1 inhibited HMGB1-induced proinflammatory cytokine production and intracellular mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB activation in macrophages and VSMCs, thereby attenuating CaCl2-induced and angiotensin II-infused AAA models. During osteoclastogenesis, RANKL stimulated HMGB1 secretion that promoted RANKL-induced osteoclastogenesis in return. Soluble rCD93 containing D1 impeded RANKL-induced osteoclastogenic marker gene expression and intracellular MAPK/NF-κB signaling, thereby mitigating ovariectomized-induced osteoporosis. Conclusion: These findings demonstrate the therapeutic potential of soluble recombinant CD93 containing D1 in inflammatory diseases. Our study highlights a novel anti-inflammatory mechanism, i.e., sequestration of HMGB1, through which sCD93 prevents HMGB1-receptor interaction on effector cells and alleviates inflammation.
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Proteína HMGB1 , Humanos , Animales , Ratones , Proteína HMGB1/metabolismo , Lectinas , Angiotensina II , Cloruro de Calcio , Inflamación , Mamíferos/metabolismoRESUMEN
Thrombomodulin (TM) is a cell surface glycoprotein which is widely expressed in a variety of cell types. It is a cofactor for thrombin binding that mediates protein C activation and inhibits thrombin activity. In addition to its anticoagulant activity, recent evidence has revealed that TM, especially its lectin-like domain, has potent anti-inflammatory function through a variety of molecular mechanisms. The lectin-like domain of TM plays an important role in suppressing inflammation independent of the TM anticoagulant activity. This article makes an extensive review of the role of TM in inflammation. The molecular targets of TM lectin-like domain have also been elucidated. Recombinant TM protein, especially the TM lectin-like domain may play a promising role in the management of sepsis, glomerulonephritis and arthritis. These data demonstrated the potential therapeutic role of TM in the treatment of inflammatory diseases.
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Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Estructura Terciaria de Proteína/genética , Trombina/metabolismo , Trombomodulina/metabolismo , Trombomodulina/uso terapéutico , Artritis/tratamiento farmacológico , Coagulación Sanguínea , Glomerulonefritis/tratamiento farmacológico , Proteína HMGB1/farmacología , Humanos , Inflamación/inducido químicamente , Proteína C/metabolismo , Sepsis/tratamiento farmacológico , Trombomodulina/genéticaRESUMEN
Single crystalline Sn doped In(2)O(3) (ITO) NWs (nanowires) were synthesized via an Au-catalyzed VLS (vapor-liquid-solid) method at 600 °C. The different sizes (~20, ~40, ~80 nm) of the Au NPs (nanoparticles) provided the controllable diameters for ITO NWs during growth. Phase and microstructures confirmed by high-resolution transmission electron microscope images (HRTEM) and X-ray diffraction (XRD) spectra indicated that the phase of In(2)O(3) NWs had a growth direction of [100]. X-ray photoelectron spectroscopy (XPS) was employed to obtain the chemical compositions of the ITO NWs as well as the ratio of Sn/In and oxygen concentrations. The findings indicated that low resistivity was found for ITO NWs with smaller diameters due to higher concentrations of oxygen vacancies and less incorporation of Sn atoms inside the NWs. The resistivity of NWs increases with increasing diameter due to more Sn atoms being incorporated into the NW and their reduction of the amount of oxygen vacancies. Low resistivity NWs could be achieved again due to excess Sn atoms doped into the large diameter NWs. Therefore, by optimizing the well-controlled growth of the NW diameter and interface states, we are able to tune the electrical properties of Sn-doped ITO NWs.