RESUMEN
Increasing evidences demonstrate the role of sensory innervation in bone metabolism, remodeling and repair, however neurovascular coupling in bone is rarely studied. Using microfluidic devices as an indirect co-culture model to mimic in vitro the physiological scenario of innervation, our group demonstrated that sensory neurons (SNs) were able to regulate the extracellular matrix remodeling by endothelial cells (ECs), in particular through sensory neuropeptides, i.e. calcitonin gene-related peptide (CGRP) and substance P (SP). Nonetheless, still little is known about the cell signaling pathways and mechanism of action in neurovascular coupling. Here, in order to characterize the communication between SNs and ECs at molecular level, we evaluated the effect of SNs and the neuropeptides CGRP and SP on ECs. We focused on different pathways known to play a role on endothelial functions: calcium signaling, p38 and Erk1/2; the control of signal propagation through Cx43; and endothelial functions through the production of nitric oxide (NO). The effect of SNs was evaluated on ECs Ca2+ influx, the expression of Cx43, endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production, p38, ERK1/2 as well as their phosphorylated forms. In addition, the role of CGRP and SP were either analyzed using respective antagonists in the co-culture model, or by adding directly on the ECs monocultures. We show that capsaicin-stimulated SNs induce increased Ca2+ influx in ECs. SNs stimulate the increase of NO production in ECs, probably involving a decrease in the inhibitory eNOS T495 phosphorylation site. The neuropeptide CGRP, produced by SNs, seems to be one of the mediators of this effect in ECs since NO production is decreased in the presence of CGRP antagonist in the co-culture of ECs and SNs, and increased when ECs are stimulated with synthetic CGRP. Taken together, our results suggest that SNs play an important role in the control of the endothelial cell functions through CGRP production and NO signaling pathway.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina , Células Endoteliales , Óxido Nítrico , Células Receptoras Sensoriales , Transducción de Señal , Sustancia P , Péptido Relacionado con Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/farmacología , Sustancia P/farmacología , Sustancia P/metabolismo , Transducción de Señal/fisiología , Transducción de Señal/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Animales , Óxido Nítrico/metabolismo , Técnicas de Cocultivo , Comunicación Celular/fisiología , Comunicación Celular/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Células Cultivadas , Humanos , RatasRESUMEN
Increasing evidence is implicating roles for platelets in the development and progression of ovarian cancer, a highly lethal disease that can arise from the fallopian tubes, and has no current method of early detection or prevention. Thrombosis is a major cause of mortality of ovarian cancer patients suggesting that the cancer alters platelet behavior. The objective of this study was to develop a cell culture model of the pathological interactions of human platelets and ovarian cancer cells, using normal FT epithelial cells as a healthy control, and to test effects of the anti-platelet dihomo-gamma-linolenic acid (DGLA) in the model. Both healthy and cancer cells caused platelet aggregation, however platelets only affected spheroid formation by cancer cells and had no effect on healthy cell spheroid formation. When naturally-formed spheroids of epithelial cells were exposed to platelets in transwell inserts that did not allow direct interactions of the two cell types, platelets caused increased size of the spheroids formed by cancer cells, but not healthy cells. When cancer cell spheroids formed using magnetic nanoshuttle technology were put in direct physical contact with platelets, the platelets caused spheroid condensation. In ovarian cancer cells, DGLA promoted epithelial-to-mesenchymal (EMT) transition at doses as low as 100 µM, and inhibited metabolic viability and induced apoptosis at doses ≥150 µM. DGLA doses ≤150 µM used to avoid direct DGLA effects on cancer cells, had no effect on the pathological interactions of platelets and ovarian cancer cells in our models. These results demonstrate that the pathological interactions of platelets with ovarian cancer cells can be modeled in cell culture, and that DGLA has no effect on these interactions, suggesting that targeting platelets is a rational approach for reducing cancer aggressiveness and thrombosis risk in ovarian cancer patients, however DGLA is not an appropriate candidate for this strategy.
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Ácido 8,11,14-Eicosatrienoico , Plaquetas , Células Epiteliales , Neoplasias Ováricas , Esferoides Celulares , Humanos , Plaquetas/efectos de los fármacos , Femenino , Células Epiteliales/efectos de los fármacos , Neoplasias Ováricas/patología , Ácido 8,11,14-Eicosatrienoico/farmacología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Esferoides Celulares/efectos de los fármacos , Línea Celular Tumoral , Agregación Plaquetaria/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Comunicación Celular/efectos de los fármacosRESUMEN
Biomaterial wound dressings, such as hydrogels, interact with host cells to regulate tissue repair. This study investigates how crosslinking of gelatin-based hydrogels influences immune and stromal cell behavior and wound healing in female mice. We observe that softer, lightly crosslinked hydrogels promote greater cellular infiltration and result in smaller scars compared to stiffer, heavily crosslinked hydrogels. Using single-cell RNA sequencing, we further show that heavily crosslinked hydrogels increase inflammation and lead to the formation of a distinct macrophage subpopulation exhibiting signs of oxidative activity and cell fusion. Conversely, lightly crosslinked hydrogels are more readily taken up by macrophages and integrated within the tissue. The physical properties differentially affect macrophage and fibroblast interactions, with heavily crosslinked hydrogels promoting pro-fibrotic fibroblast activity that drives macrophage fusion through RANKL signaling. These findings suggest that tuning the physical properties of hydrogels can guide cellular responses and improve healing, offering insights for designing better biomaterials for wound treatment.
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Fibroblastos , Hidrogeles , Macrófagos , Cicatrización de Heridas , Animales , Hidrogeles/química , Cicatrización de Heridas/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Femenino , Comunicación Celular/efectos de los fármacos , Materiales Biocompatibles/química , Ligando RANK/metabolismo , Ratones Endogámicos C57BL , Reactivos de Enlaces Cruzados/química , Gelatina/química , Inflamación/metabolismo , Inflamación/patologíaRESUMEN
Cell-cell interactions play an important role in many biological processes, and various methods have been developed for controlling the cell-cell interactions. However, the effective and rapid control of intercellular interactions remains challenging. Herein, we report a novel, rapid, and effective electrochemical strategy without destroying the basic life processes for the dynamic control of intercellular interactions via liposome fusion. In the proposed system, bioorthogonal chemical groups and hydroquinone (HQ)- and aminooxy (AO)-tethered ligands were modified on the surface of living cells on the basis of the liposome fusion, enabling dynamical intercellular assemblies. Upon application of the corresponding oxidative potential, the "off-state" HQ could be oxidized to the "on-state" quinone (Q), which subsequently reacts with AO-tethered ligands to form stable oxime linkages under physiological conditions. This reaction effectively shortens the distance between cells, promoting the formation of cell clusters. When the corresponding reverse reductive potential is applied, the oxime linkage is cleaved, resulting in the release of the cells. Furthermore, we employed HQ- and AO-tethered ligands to modify mitochondria, inducing mitochondrial aggregation. This noninvasive and label-free strategy allows for the dynamic reversible regulation of intercellular interactions, enhancing our understanding of intercellular communication networks, and has the potential for improving the antitumor therapy efficacy.
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Comunicación Celular , Hidroquinonas , Humanos , Comunicación Celular/efectos de los fármacos , Hidroquinonas/química , Hidroquinonas/farmacología , Liposomas/química , Técnicas Electroquímicas , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , LigandosRESUMEN
Titanium dioxide nanoparticles (TiO2 NPs) can reduce sperm number, but the mechanisms of defective spermatogenesis induced by TiO2 NPs have not been studied through cell-cell interactions at present. A kind of biomimetic three-dimensional blood-testis barrier microfluidic chip capable of intercellular communication was constructed with soft lithography techniques, including Sertoli cell (TM4), spermatogonia (GC-1) and vascular endothelial cell units, to study the mechanisms of TiO2 NPs-induced defective spermatogenesis. TM4 and GC-1 cells cultured in TiO2 NPs exposure and control chips were collected for transcriptomics and metabonomics analysis, and key proteins and metabolites in changed biological processes were validated. In TM4 cells, TiO2 NPs suppressed glucose metabolism, especially lactate production, which reduced energy substrate supply for spermatogenesis. TiO2 NPs also decreased the levels of key proteins and metabolites of lactate production. In GC-1 cells, TiO2 NPs disturbed chemokine signaling pathways regulating cell proliferation and interfered with glutathione metabolism. The Cxcl13, Stat3 and p-Stat3 levels and cell proliferation rate were decreased, and the GSR, GPX4 and GSH contents were increased in GC-1 cells in chips under TiO2 NPs treatment. The decrease in energy substrate supply for spermatogenesis and inhibition of spermatogonia proliferation could be the main mechanisms of defective spermatogenesis induced by TiO2 NPs.
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Barrera Hematotesticular , Células de Sertoli , Espermatogénesis , Espermatogonias , Titanio , Masculino , Titanio/toxicidad , Espermatogénesis/efectos de los fármacos , Animales , Células de Sertoli/efectos de los fármacos , Células de Sertoli/metabolismo , Barrera Hematotesticular/efectos de los fármacos , Ratones , Espermatogonias/efectos de los fármacos , Espermatogonias/metabolismo , Espermatogonias/patología , Línea Celular , Nanopartículas del Metal/toxicidad , Dispositivos Laboratorio en un Chip , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Comunicación Celular/efectos de los fármacosRESUMEN
Cellular communication mediated by messenger molecules plays an important role in the progression of cancer. Herein, pH-sensitive zeolitic imidazolate framework-8 (ZIF-8) loaded with PtCl2(OH)2(NH3)2 [i.e., Pt(IV)] bimetallic nanoplatforms were developed for prostate cancer therapy by interfering inositol-1, 4, 5-trisphosphate (IP3)-mediated cellular communication. As an important messenger in cells, the function of IP3 was found to be efficiently interfered with by the Pt(IV)-binding inositol unit. This finding effect of Pt(IV) is totally different from its traditional function as a prodrug of cis-platinum for chemotherapy. The decreased IP3 signal further downregulated the cytoplasmic Ca2+ concentration and downstream signal transduction to inhibit proliferation and invasion of tumor cells. Meanwhile, Zn2+ released from ZIF-8 under an acidic tumor microenvironment decreased adenosine triphosphate biosynthesis, which could further limit the cellular communication. Such a proposed strategy of interfering cellular communication has demonstrated its feasibility in this study, which may provide new perspectives for cancer therapy.
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Antineoplásicos , Neoplasias de la Próstata , Humanos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Masculino , Antineoplásicos/farmacología , Antineoplásicos/química , Comunicación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Estructuras Metalorgánicas/química , Estructuras Metalorgánicas/farmacología , Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/química , Concentración de Iones de Hidrógeno , Imidazoles/química , Imidazoles/farmacología , Zinc/química , Zinc/farmacologíaRESUMEN
Atherosclerosis represents the major cause of mortality worldwide and triggers higher risk of acute cardiovascular events. Pericytes-endothelial cells (ECs) communication is orchestrated by ligand-receptor interaction generating a microenvironment which results in intraplaque neovascularization, that is closely associated with atherosclerotic plaque instability. Notoginsenoside R1 (R1) exhibits anti-atherosclerotic bioactivity, but its effect on angiogenesis in atherosclerotic plaque remains elusive. The aim of our study is to explore the therapeutic effect of R1 on vulnerable plaque and investigate its potential mechanism against intraplaque neovascularization. The impacts of R1 on plaque stability and intraplaque neovascularization were assessed in ApoE-/- mice induced by high-fat diet. Pericytes-ECs direct or non-direct contact co-cultured with VEGF-A stimulation were used as the in vitro angiogenesis models. Overexpressing Ang1 in pericytes was performed to investigate the underlying mechanism. In vivo experiments, R1 treatment reversed atherosclerotic plaque vulnerability and decreased the presence of neovessels in ApoE-/- mice. Additionally, R1 reduced the expression of Ang1 in pericytes. In vitro experiments demonstrated that R1 suppressed pro-angiogenic behavior of ECs induced by pericytes cultured with VEGF-A. Mechanistic studies revealed that the anti-angiogenic effect of R1 was dependent on the inhibition of Ang1 and Tie2 expression, as the effects were partially reversed after Ang1 overexpressing in pericytes. Our study demonstrated that R1 treatment inhibited intraplaque neovascularization by governing pericyte-EC association via suppressing Ang1-Tie2/PI3K-AKT paracrine signaling pathway. R1 represents a novel therapeutic strategy for atherosclerotic vulnerable plaques in clinical application.
Asunto(s)
Angiopoyetina 1 , Aterosclerosis , Células Endoteliales , Ginsenósidos , Neovascularización Patológica , Pericitos , Placa Aterosclerótica , Receptor TIE-2 , Animales , Ginsenósidos/farmacología , Ratones , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Aterosclerosis/tratamiento farmacológico , Placa Aterosclerótica/tratamiento farmacológico , Receptor TIE-2/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Angiopoyetina 1/metabolismo , Ratones Endogámicos C57BL , Masculino , Comunicación Celular/efectos de los fármacos , Humanos , Transducción de Señal/efectos de los fármacos , Apolipoproteínas E , Dieta Alta en Grasa , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Binge drinking in obese patients positively correlates with accelerated liver damage and liver-related death. However, the underlying mechanism and the effect of alcohol use on the progression of metabolic-dysfunction-associated steatotic liver disease (MASLD) remain unexplored. Here, we show that short-term feeding of a metabolic-dysfunction-associated steatohepatitis (MASH) diet plus daily acute alcohol binges for three days induce liver injury and activation of the NLRP3 inflammasome. We identify that a MASH diet plus acute alcohol binges promote liver inflammation via increased infiltration of monocyte-derived macrophages, neutrophil recruitment, and NET release in the liver. Our results suggest that both monocyte-derived macrophages and neutrophils are activated via NLRP3, while the administration of MCC950, an NLRP3 inhibitor, dampens these effects.In this study, we reveal important intercellular communication between hepatocytes and neutrophils. We discover that the MASH diet plus alcohol induces IL-1ß via NLRP3 activation and that IL-1ß acts on hepatocytes and promotes the production of CXCL1 and LCN2. In turn, the increase in these neutrophils recruits chemokines and causes further infiltration and activation of neutrophils in the liver. In vivo administration of the NLRP3 inhibitor, MCC950, improves the early phase of MetALD by preventing liver damage, steatosis, inflammation, and immune cells recruitment.
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Interleucina-1beta , Hígado , Proteína con Dominio Pirina 3 de la Familia NLR , Infiltración Neutrófila , Neutrófilos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Hígado/patología , Hígado/metabolismo , Hígado/efectos de los fármacos , Interleucina-1beta/metabolismo , Infiltración Neutrófila/efectos de los fármacos , Masculino , Neutrófilos/metabolismo , Neutrófilos/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones , Inflamasomas/metabolismo , Consumo Excesivo de Bebidas Alcohólicas/patología , Consumo Excesivo de Bebidas Alcohólicas/complicaciones , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Comunicación Celular/efectos de los fármacos , Sulfonas/farmacología , Sulfonamidas/farmacología , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Furanos/farmacología , Humanos , Indenos/farmacología , Dieta , Transducción de Señal/efectos de los fármacos , Trampas Extracelulares/metabolismo , Trampas Extracelulares/efectos de los fármacos , Hígado Graso/patología , Hígado Graso/metabolismo , Sulfóxidos/farmacologíaRESUMEN
Cell-cell interactions, which allow cells to communicate with each other through molecules in their microenvironment, are critical for the growth, health, and functions of cells. Previous studies show that drug-resistant cells can interact with drug-sensitive cells to elevate their drug resistance level, which is partially responsible for cancer recurrence. Studying protein targets and pathways involved in cell-cell communication provides essential information for fundamental cell biology studies and therapeutics of human diseases. In the current studies, we performed direct coculture and indirect coculture of drug-resistant and drug-sensitive cell lines, aiming to investigate intracellular proteins responsible for cell communication. Comparative studies were carried out using monoculture cells. Shotgun bottom-up proteomics results indicate that the P53 signaling pathway has a strong association with drug resistance mechanisms, and multiple TP53-related proteins were upregulated in both direct and indirect coculture systems. In addition, cell-cell communication pathways, including the phagosome and the HIF-signaling pathway, contribute to both direct and indirect coculture systems. Consequently, AK3 and H3-3A proteins were identified as potential targets for cell-cell interactions that are relevant to drug resistance mechanisms. We propose that the P53 signaling pathway, in which mitochondrial proteins play an important role, is responsible for inducing drug resistance through communication between drug-resistant and drug-sensitive cancer cells.
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Comunicación Celular , Técnicas de Cocultivo , Resistencia a Antineoplásicos , Proteómica , Transducción de Señal , Proteína p53 Supresora de Tumor , Humanos , Proteómica/métodos , Comunicación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias/metabolismo , Neoplasias/tratamiento farmacológicoRESUMEN
BACKGROUND: Astrocytes are the most abundant cell type of the central nervous system and are fundamentally involved in homeostasis, neuroprotection, and synaptic plasticity. This regulatory function of astrocytes on their neighboring cells in the healthy brain is subject of current research. In the ischemic brain we assume disease specific differences in astrocytic acting. The renin-angiotensin-aldosterone system regulates arterial blood pressure through endothelial cells and perivascular musculature. Moreover, astrocytes express angiotensin II type 1 and 2 receptors. However, their role in astrocytic function has not yet been fully elucidated. We hypothesized that the angiotensin II receptors impact astrocyte function as revealed in an in vitro system mimicking cerebral ischemia. Astrocytes derived from neonatal wistar rats were exposed to telmisartan (angiotensin II type 1 receptor-blocker) or PD123319 (angiotensin II type 2 receptor-blocker) under normal conditions (control) or deprivation from oxygen and glucose. Conditioned medium (CM) of astrocytes was harvested to elucidate astrocyte-mediated indirect effects on microglia and cortical neurons. RESULT: The blockade of angiotensin II type 1 receptor by telmisartan increased the survival of astrocytes during ischemic conditions in vitro without affecting their proliferation rate or disturbing their expression of S100A10, a marker of activation. The inhibition of the angiotensin II type 2 receptor pathway by PD123319 resulted in both increased expression of S100A10 and proliferation rate. The CM of telmisartan-treated astrocytes reduced the expression of pro-inflammatory mediators with simultaneous increase of anti-inflammatory markers in microglia. Increased neuronal activity was observed after treatment of neurons with CM of telmisartan- as well as PD123319-stimulated astrocytes. CONCLUSION: Data show that angiotensin II receptors have functional relevance for astrocytes that differs in healthy and ischemic conditions and effects surrounding microglia and neuronal activity via secretory signals. Above that, this work emphasizes the strong interference of the different cells in the CNS and that targeting astrocytes might serve as a therapeutic strategy to influence the acting of glia-neuronal network in de- and regenerative context.
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Bloqueadores del Receptor Tipo 1 de Angiotensina II , Bloqueadores del Receptor Tipo 2 de Angiotensina II , Astrocitos , Accidente Cerebrovascular Isquémico , Microglía , Neuronas , Ratas Wistar , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Telmisartán , Animales , Ratas , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Bloqueadores del Receptor Tipo 2 de Angiotensina II/farmacología , Animales Recién Nacidos , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Bencimidazoles/farmacología , Comunicación Celular/fisiología , Comunicación Celular/efectos de los fármacos , Células Cultivadas , Imidazoles/farmacología , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/patología , Microglía/metabolismo , Microglía/efectos de los fármacos , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Piridinas/farmacología , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Telmisartán/farmacologíaRESUMEN
Microfluidic cell encapsulation has provided a platform for studying the behavior of individual cells and has become a turning point in single-cell analysis during the last decade. The engineered microenvironment, along with protecting the immune response, has led to increasingly presenting the results of practical and pre-clinical studies with the goals of disease treatment, tissue engineering, intelligent control of stem cell differentiation, and regenerative medicine. However, the significance of cell-substrate interaction versus cell-cell communications in the microgel is still unclear. In this study, monodisperse alginate microgels were generated using a flow-focusing microfluidic device to determine how the cell microenvironment can control human bone marrow-derived mesenchymal stem cells (hBMSCs) viability, proliferation, and biomechanical features in single-cell droplets versus multi-cell droplets. Collected results show insufficient cell proliferation (234 % and 329 %) in both single- and multi-cell alginate microgels. Alginate hydrogels supplemented with poly-l-lysine (PLL) showed a better proliferation rate (514 % and 780 %) in a comparison of free alginate hydrogels. Cell stiffness data illustrate that hBMSCs cultured in alginate hydrogels have higher membrane flexibility and migration potency (Young's modulus equal to 1.06 kPa), whereas PLL introduces more binding sites for cell attachment and causes lower flexibility and migration potency (Young's modulus equal to 1.83 kPa). Considering that cell adhesion is the most important parameter in tissue engineering, in which cells do not run away from a 3D substrate, PLL enhances cell stiffness and guarantees cell attachments. In conclusion, cell attachment to PLL-mediated alginate hydrogels is crucial for cell viability and proliferation. It suggests that cell-cell signaling is good enough for stem cell viability, but cell-PLL attachment alongside cell-cell signaling is crucial for stem cell proliferation and self-renewal.
Asunto(s)
Alginatos , Adhesión Celular , Proliferación Celular , Células Madre Mesenquimatosas , Microgeles , Polilisina , Alginatos/química , Alginatos/farmacología , Polilisina/química , Polilisina/farmacología , Humanos , Adhesión Celular/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Proliferación Celular/efectos de los fármacos , Microgeles/química , Microfluídica/métodos , Comunicación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Hidrogeles/química , Hidrogeles/farmacología , Encapsulación Celular/métodos , Análisis de la Célula Individual , Autorrenovación de las Células/efectos de los fármacos , Diferenciación Celular/efectos de los fármacosRESUMEN
Lupus nephritis (LN) occurs in 50% of cases of systemic lupus erythematosus (SLE) and is one of the most serious complications that can occur during lupus progression. Mesangial cells (MCs) are intrinsic cells in the kidney that can regulate capillary blood flow, phagocytose apoptotic cells, and secrete vasoactive substances and growth factors. Previous studies have shown that various types of inflammatory cells can activate MCs for hyperproliferation, leading to disruption of the filtration barrier and impairment of renal function in LN. Here, we characterized the heterogeneity of kidney cells of LN mice by single-nucleus RNA sequencing (snRNA-seq) and revealed the interaction between macrophages and MCs through the CXC motif chemokine ligand 12 (CXCL12)/dipeptidyl peptidase 4 (DPP4) axis. In culture, macrophages modulated the proliferation and migration of MCs through this ligand-receptor interaction. In LN mice, treatment with linagliptin, a DPP4 inhibitor, effectively inhibited MC proliferation and reduced urinary protein levels. Together, our findings indicated that targeting the CXCL12/DPP4 axis with linagliptin treatment may serve as a novel strategy for the treatment of LN via the CXCL12/DPP4 axis.
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Proliferación Celular , Quimiocina CXCL12 , Dipeptidil Peptidasa 4 , Nefritis Lúpica , Macrófagos , Células Mesangiales , Nefritis Lúpica/patología , Nefritis Lúpica/metabolismo , Animales , Dipeptidil Peptidasa 4/metabolismo , Quimiocina CXCL12/metabolismo , Células Mesangiales/metabolismo , Células Mesangiales/patología , Células Mesangiales/efectos de los fármacos , Ratones , Macrófagos/metabolismo , Proliferación Celular/efectos de los fármacos , Humanos , Femenino , Movimiento Celular/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Linagliptina/farmacología , Transducción de Señal , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Ratones Endogámicos C57BLRESUMEN
Male fertility depends on normal pubertal development. Di-(2-ethylhexyl) phthalate (DEHP) is a potent antiandrogen chemical, and exposure to DEHP during peripuberty can damage the developing male reproductive system, especially the testis. However, the specific cellular targets and differentiation processes affected by DEHP, which lead to testicular toxicity, remain poorly defined. Herein, we presented the first single-cell transcriptomic profile of the pubertal mouse testis following DEHP exposure. To carry out the experiment, 2 groups (n = 8 each) of 3-week-old male mice were orally administered 0.5% carboxymethylcellulose sodium salt or 100 mg/kg body weight DEHP daily from postnatal day 21-48, respectively. Using single-cell RNA sequencing, a total of 31 distinct cell populations were identified, notably, Sertoli and Leydig cells emerged as important targets of DEHP. DEHP exposure significantly decreased the proportions of Sertoli cell clusters expressing mature Sertoli markers (Sox9 and Ar), and selectively reduced the expression of testosterone synthesis genes in fetal Leydig cells. Through cell-cell interaction analyses, we observed changed numbers of interactions in Sertoli cells 1 (SCs1), Leydig cells 1 (LCs1), and interstitial macrophages, and we also identified cell-specific ligand gene expressions in these clusters, such as Inha, Fyn, Vcam1, and Apoe. Complementary in vitro assays confirmed that DEHP directly reduced the expression of genes related to Sertoli cell adhesion and intercellular communication. In conclusion, peripubertal DEHP exposure reduced the number of mature Sertoli cells and may disrupt testicular steroidogenesis by affecting the testosterone synthesis genes in fetal Leydig cells rather than adult Leydig cells.
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Dietilhexil Ftalato , Células Intersticiales del Testículo , Células de Sertoli , Testículo , Animales , Masculino , Dietilhexil Ftalato/toxicidad , Células de Sertoli/efectos de los fármacos , Células de Sertoli/metabolismo , Células de Sertoli/patología , Testículo/efectos de los fármacos , Testículo/metabolismo , Testículo/patología , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/patología , Ratones , Análisis de la Célula Individual , Maduración Sexual/efectos de los fármacos , Testosterona/sangre , Transcriptoma/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Ratones Endogámicos C57BLRESUMEN
Tert-butyl hydroperoxide (t-BuOOH) is an organic hydroperoxide widely used as a model compound to induce oxidative stress. It leads to a plethora of cellular damage, including lipid peroxidation, DNA double-strand breaks (DNA DSBs), and breakdown of the mitochondrial membrane potential (MMP). We could show in several cell lines that t-BuOOH induces ferroptosis, triggered by iron-dependent lipid peroxidation. We have further revealed that not only t-BuOOH-mediated ferroptosis, but also DNA DSBs and loss of MMP are prevented by cell-cell contacts. The underlying mechanisms are not known. Here, we show in murine fibroblasts and a human colon carcinoma cell line that t-BuOOH (50 or 100 µM, resp.) causes an increase in intracellular Ca2+, and that this increase is key to lipid peroxidation and ferroptosis, DNA DSB formation and dissipation of the MMP. We further demonstrate that cell-cell contacts prevent t-BuOOH-mediated raise in intracellular Ca2+. Hence, we provide novel insights into the mechanism of t-BuOOH-triggered cellular damage including ferroptosis and propose a model in which cell-cell contacts control intracellular Ca2+ levels to prevent lipid peroxidation, DNA DSB-formation and loss of MMP. Since Ca2+ is a central player of toxicity in response to oxidative stress and is involved in various cell death pathways, our observations suggest a broad protective function of cell-cell contacts against a variety of exogenous toxicants.
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Calcio , Roturas del ADN de Doble Cadena , Ferroptosis , Peroxidación de Lípido , Potencial de la Membrana Mitocondrial , terc-Butilhidroperóxido , Ferroptosis/efectos de los fármacos , Calcio/metabolismo , Humanos , terc-Butilhidroperóxido/toxicidad , Animales , Peroxidación de Lípido/efectos de los fármacos , Ratones , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Línea Celular TumoralRESUMEN
In tissue engineering, crosslinking with carbodiimides such as EDC is omnipresent to improve the mechanical properties of biomaterials. However, in collagen biomaterials, EDC reacts with glutamate or aspartate residues, inactivating the binding sites for cellular receptors and rendering collagen inert to many cell types. In this work, we have developed a crosslinking method that ameliorates the rigidity, stability, and degradation rate of collagen biomaterials, whilst retaining key interactions between cells and the native collagen sequence. Our approach relies on the UV-triggered reaction of diazirine groups grafted on lysines, leaving critical amino acid residues intact. Notably, GxxGER recognition motifs for collagen-binding integrins, ablated by EDC crosslinking, were left unreacted, enabling cell attachment, spreading, and colonization on films and porous scaffolds. In addition, our procedure conserves the architecture of biomaterials, improves their resistance to collagenase and cellular contraction, and yields material stiffness akin to that obtained with EDC. Importantly, diazirine-crosslinked collagen can host mesenchymal stem cells, highlighting its strong potential as a substrate for tissue repair. We have therefore established a new crosslinking strategy to modulate the mechanical features of collagen porous scaffolds without altering its biological properties, thereby offering an advantageous alternative to carbodiimide treatment. STATEMENT OF SIGNIFICANCE: This article describes an approach to improve the mechanical properties of collagen porous scaffolds, without impacting collagen's natural interactions with cells. This is significant because collagen crosslinking is overwhelmingly performed using carbodiimides, which results in a critical loss of cellular affinity. By contrast, our method leaves key cellular binding sites in the collagen sequence intact, enabling cell-biomaterial interactions. It relies on the fast, UV-triggered reaction of diazirine with collagen, and does not produce toxic by-products. It also supports the culture of mesenchymal stem cells, a pivotal cell type in a wide range of tissue repair applications. Overall, our approach offers an attractive option for the crosslinking of collagen, a prominent material in the growing field of tissue engineering.
Asunto(s)
Materiales Biocompatibles , Colágeno , Reactivos de Enlaces Cruzados , Diazometano , Células Madre Mesenquimatosas , Diazometano/química , Reactivos de Enlaces Cruzados/química , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Colágeno/química , Animales , Andamios del Tejido/química , Comunicación Celular/efectos de los fármacos , Humanos , Ensayo de Materiales , Adhesión Celular/efectos de los fármacos , PorosidadRESUMEN
Cytokines mediate cell-cell communication in the immune system and represent important therapeutic targets1-3. A myriad of studies have highlighted their central role in immune function4-13, yet we lack a global view of the cellular responses of each immune cell type to each cytokine. To address this gap, we created the Immune Dictionary, a compendium of single-cell transcriptomic profiles of more than 17 immune cell types in response to each of 86 cytokines (>1,400 cytokine-cell type combinations) in mouse lymph nodes in vivo. A cytokine-centric view of the dictionary revealed that most cytokines induce highly cell-type-specific responses. For example, the inflammatory cytokine interleukin-1ß induces distinct gene programmes in almost every cell type. A cell-type-centric view of the dictionary identified more than 66 cytokine-driven cellular polarization states across immune cell types, including previously uncharacterized states such as an interleukin-18-induced polyfunctional natural killer cell state. Based on this dictionary, we developed companion software, Immune Response Enrichment Analysis, for assessing cytokine activities and immune cell polarization from gene expression data, and applied it to reveal cytokine networks in tumours following immune checkpoint blockade therapy. Our dictionary generates new hypotheses for cytokine functions, illuminates pleiotropic effects of cytokines, expands our knowledge of activation states of each immune cell type, and provides a framework to deduce the roles of specific cytokines and cell-cell communication networks in any immune response.
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Citocinas , Inmunidad , Análisis de la Célula Individual , Animales , Ratones , Comunicación Celular/efectos de los fármacos , Citocinas/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunidad/efectos de los fármacos , Interleucina-18/inmunología , Interleucina-1beta/inmunología , Células Asesinas Naturales/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Transducción de Señal/efectos de los fármacos , Programas InformáticosRESUMEN
2-carba-cyclic phosphatidic acid (2ccPA) suppresses microglial and astrocyte inflammation for neuronal survival following traumatic brain injury. However, it remains unknown how 2ccPA regulates microglial activation. In this study, to elucidate the 2ccPA behavior in glial communication, we collected the astrocyte conditioned media (ACM) from primary astrocyte cultures that were treated by lipopolysaccharide (LPS) and 2ccPA and analyzed the alteration of microglial inflammation caused by the ACM treatment. The addition of the ACM derived from LPS- and 2ccPA-double treated astrocytes to microglia decreased the CD86+ pro-inflammatory M1 microglia, which were upregulated with the ACM collected from astrocytes treated by LPS without 2ccPA, while the direct addition of LPS and 2ccPA to microglia failed to decrease the CD86+ microglia to the basal level. We confirmed that the ACM from LPS- and 2ccPA-treated astrocytes increased the ratio of CD206+ anti-inflammatory M2 microglia to total microglia, whereas direct treatment of microglia with LPS and 2ccPA had no effect on the CD206+ microglia ratio, demonstrating the importance of astrocyte intervention in microglial polarization. In addition, we examined whether astrocytes modulate the 2ccPA-regulated proinflammatory cytokine production derived from microglia. The addition of the ACM from LPS- and 2ccPA-treated astrocytes to microglia remarkably canceled the LPS-induced upregulation of IL-1ß, IL-6, and TNF-α secreted from microglia, while the direct addition of LPS and 2ccPA to microglia showed no affect. Therefore, our results indicate that astrocytes mediate the 2ccPA function to shift microglia towards the M2 phenotype by interfering with the polarization of M1 microglia and to suppress cytokine production.
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Antiinflamatorios , Astrocitos , Comunicación Celular , Polaridad Celular , Inflamación , Microglía , Humanos , Antiinflamatorios/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/patología , Células Cultivadas , Inflamación/metabolismo , Inflamación/patología , Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Microglía/patología , Fenotipo , Factor de Necrosis Tumoral alfa , Comunicación Celular/efectos de los fármacosRESUMEN
Long-term memory formation relies on synaptic plasticity, neuronal activity-dependent gene transcription, and epigenetic modifications. Multiple studies have shown that HDAC inhibitor (HDACi) treatments can enhance individual aspects of these processes and thereby act as putative cognitive enhancers. However, their mode of action is not fully understood. In particular, it is unclear how systemic application of HDACis, which are devoid of substrate specificity, can target pathways that promote memory formation. In this study, we explore the electrophysiological, transcriptional, and epigenetic responses that are induced by CI-994, a class I HDACi, combined with contextual fear conditioning (CFC) in mice. We show that CI-994mediated improvement of memory formation is accompanied by enhanced long-term potentiation in the hippocampus, a brain region recruited by CFC, but not in the striatum, a brain region not primarily implicated in fear learning. Furthermore, using a combination of bulk and single-cell RNA-sequencing, we find that, when paired with CFC, HDACi treatment engages synaptic plasticity-promoting gene expression more strongly in the hippocampus, specifically in the dentate gyrus (DG). Finally, using chromatin immunoprecipitation-sequencing (ChIP-seq) of DG neurons, we show that the combined action of HDACi application and conditioning is required to elicit enhancer histone acetylation in pathways that underlie improved memory performance. Together, these results indicate that systemic HDACi administration amplifies brain region-specific processes that are naturally induced by learning.
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Benzamidas , Giro Dentado , Inhibidores de Histona Desacetilasas , Memoria a Largo Plazo , Fenilendiaminas , Animales , Benzamidas/farmacología , Comunicación Celular/efectos de los fármacos , Giro Dentado/citología , Giro Dentado/efectos de los fármacos , Giro Dentado/fisiología , Inhibidores de Histona Desacetilasas/farmacología , Memoria a Largo Plazo/efectos de los fármacos , Ratones , Plasticidad Neuronal , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fenilendiaminas/farmacología , RNA-Seq , Análisis de la Célula IndividualRESUMEN
The polarized flow of information through neural circuits depends on the orderly arrangement of neurons, their processes, and their synapses. This polarity emerges sequentially in development, starting with the directed migration of neuronal precursors, which subsequently elaborate neurites that form synapses in specific locations. In other organs, Fat cadherins sense the position and then polarize individual cells by inducing localized changes in the cytoskeleton that are coordinated across the tissue. Here, we show that the Fat-related protein Fat3 plays an analogous role during the assembly of polarized circuits in the murine retina. We find that the Fat3 intracellular domain (ICD) binds to cytoskeletal regulators and synaptic proteins, with discrete motifs required for amacrine cell migration and neurite retraction. Moreover, upon ICD deletion, extra neurites form but do not make ectopic synapses, suggesting that Fat3 independently regulates synapse localization. Thus, Fat3 serves as a molecular node to coordinate asymmetric cell behaviors across development.
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Cadherinas/metabolismo , Comunicación Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Factor de Crecimiento Epidérmico/metabolismo , Células Amacrinas/metabolismo , Secuencia de Aminoácidos/efectos de los fármacos , Animales , Humanos , Ratones Transgénicos , Neuritas/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Sinapsis/efectos de los fármacosRESUMEN
Connexin 43 (Cx43) is the most important protein in the gap junction channel between cardiomyocytes. Abnormalities of Cx43 change the conduction velocity and direction of cardiomyocytes, leading to reentry and conduction block of the myocardium, thereby causing arrhythmia. It has been shown that IL-1ß reduces the expression of Cx43 in astrocytes and cardiomyocytes in vitro. However, whether caspase-1 and IL-1ß affect connexin 43 after myocardial infarction (MI) is uncertain. In this study we investigated the effects of VX765, a caspase-1 inhibitor, on the expression of Cx43 and cell-to-cell communication after MI. Rats were treated with VX765 (16 mg/kg, i.v.) 1 h before the left anterior descending artery (LAD) ligation, and then once daily for 7 days. The ischemic heart was collected for histochemical analysis and Western blot analysis. We showed that VX765 treatment significantly decreased the infarct area, and alleviated cardiac dysfunction and remodeling by suppressing the NLRP3 inflammasome/caspase-1/IL-1ß expression in the heart after MI. In addition, VX765 treatment markedly raised Cx43 levels in the heart after MI. In vitro experiments were conducted in rat cardiac myocytes (RCMs) stimulated with the supernatant from LPS/ATP-treated rat cardiac fibroblasts (RCFs). Pretreatment of the RCFs with VX765 (25 µM) reversed the downregulation of Cx43 expression in RCMs and significantly improved intercellular communication detected using a scrape-loading/dye transfer assay. We revealed that VX765 suppressed the activation of p38 MAPK signaling in the heart tissue after MI as well as in RCMs stimulated with the supernatant from LPS/ATP-treated RCFs. Taken together, these data show that the caspase-1 inhibitor VX765 upregulates Cx43 expression and improves cell-to-cell communication in rat heart after MI via suppressing the IL-1ß/p38 MAPK pathway.