RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Wenshen Xiaozheng Tang (WXT), a traditional Chinese medicine (TCM) decoction, is effective for treating endometriosis. However, the effect of WXT on endometrium-derived mesenchymal stem cells (eMSCs) which play a key role in the fibrogenesis of endometriosis requires further elucidation. AIMS OF THE STUDY: The aim of this study was to clarify the potential mechanism of WXT in improving fibrosis in endometriosis by investigating the regulation of WXT on differentiation and paracrine of eMSCs. MATERIALS AND METHODS: The nude mice with endometriosis were randomly divided into model group, WXT group and mifepristone group. After 21 days of treatment, the lesion volume was calculated. Fibrosis in the lesions was evaluated by Masson staining and expression of fibrotic proteins. The differentiation of eMSCs in vivo was explored using a fate-tracking experiment. To further clarify the regulation of WXT on eMSCs, primary eMSCs from the ectopic lesions of endometriosis patients were isolated and characterized. The effect of WXT on the proliferation and differentiation of ectopic eMSCs was examined. To evaluate the role of WXT on the paracrine activity of ectopic eMSCs, the conditioned medium (CM) from ectopic eMSCs pretreated with WXT was collected and applied to treat ectopic endometrial stromal cells (ESCs), after which the expression of fibrotic proteins in ectopic ESCs was assessed. In addition, transcriptome sequencing was used to investigate the regulatory mechanism of WXT on ectopic eMSCs, and western blot and ELISA were employed to determine the key mediator. RESULTS: WXT impeded the growth of ectopic lesions in nude mice with endometriosis and reduced collagen deposition and the expression of fibrotic proteins fibronectin, collagen I, α-SMA and CTGF in the endometriotic lesions. The fate-tracking experiment showed that WXT prevented human eMSCs from differentiating into myofibroblasts in the nude mice. We successfully isolated eMSCs from the lesions of patients with endometriosis and demonstrated that WXT suppressed proliferation and myofibroblast differentiation of ectopic eMSCs. Moreover, the expression of α-SMA, collagen I, fibronectin and CTGF in ectopic ESCs was significantly down-regulated by the CM of ectopic MSCs pretreated with WXT. Combining the results of RNA sequencing, western blot and ELISA, we found that WXT not only reduced thrombospondin 4 expression in ectopic eMSCs, but also decreased thrombospondin 4 secretion from ectopic eMSCs. Thrombospondin 4 concentration-dependently upregulated the expression of collagen I, fibronectin, α-SMA and CTGF in ectopic ESCs, indicating that thrombospondin 4 was a key mediator of WXT in inhibiting the fibrotic process in endometriosis. CONCLUSION: WXT improved fibrosis in endometriosis by regulating differentiation and paracrine signaling of eMSCs. Thrombospondin 4, whose release from ectopic eMSCs is inhibited by WXT, may be a potential target for the treatment of endometriosis.
Assuntos
Diferenciação Celular , Medicamentos de Ervas Chinesas , Endometriose , Endométrio , Fibrose , Células-Tronco Mesenquimais , Camundongos Nus , Comunicação Parácrina , Endometriose/tratamento farmacológico , Endometriose/patologia , Endometriose/metabolismo , Feminino , Animais , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Humanos , Diferenciação Celular/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Endométrio/patologia , Camundongos , Células Cultivadas , Adulto , Modelos Animais de DoençasRESUMO
Rotator cuff injuries present a clinical challenge for repair due to current limitations in functional regeneration of the native tendon-to-bone enthesis. A biomaterial that can regionally instruct unique tissue-specific phenotypes offers potential to promote enthesis repair. We have recently demonstrated the mechanical benefits of a stratified triphasic biomaterial made up of tendon- and bone-mimetic collagen scaffold compartments connected via a continuous hydrogel, and we now explore the potential of a biologically favorable enthesis hydrogel for this application. Here we report in vitro behavior of human mesenchymal stem cells (hMSCs) within thiolated gelatin (Gel-SH) hydrogels in response to chondrogenic stimuli as well as paracrine signals derived from MSC-seeded bone and tendon scaffold compartments. Chondrogenic differentiation media promoted upregulation of cartilage and entheseal fibrocartilage matrix markers COL2, COLX, and ACAN as well as the enthesis-associated transcription factors SCX, SOX9, and RUNX2 in hMSCs within Gel-SH. Similar effects were observed in response to TGF-ß3 and BMP-4, enthesis-associated growth factors known to play a role in entheseal development and maintenance. Conditioned media generated by hMSCs seeded in tendon- and bone-mimetic collagen scaffolds influenced patterns of gene expression regarding enthesis-relevant growth factors, matrix markers, and tendon-to-bone transcription factors for hMSCs within the material. Together, these findings demonstrate that a Gel-SH hydrogel provides a permissive environment for enthesis tissue engineering and highlights the significance of cellular crosstalk between adjacent compartments within a spatially graded biomaterial.
Assuntos
Diferenciação Celular , Fibrocartilagem , Gelatina , Hidrogéis , Células-Tronco Mesenquimais , Comunicação Parácrina , Humanos , Gelatina/química , Hidrogéis/química , Hidrogéis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Comunicação Parácrina/efeitos dos fármacos , Fibrocartilagem/química , Fibrocartilagem/efeitos dos fármacos , Fibrocartilagem/metabolismo , Condrogênese/efeitos dos fármacos , Manguito Rotador , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fatores de Transcrição SOX9/metabolismo , Alicerces Teciduais/química , Lesões do Manguito Rotador/terapia , Agrecanas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice BásicosRESUMO
Telmisartan, an angiotensin II type 1 receptor (AT1R) blocker, exhibits broad anti-tumor activity. However, in vitro, anti-proliferative effects are shown at doses far beyond the therapeutic plasma concentration. Considering the role of tumor microenvironment in glioma progression, glioma-astrocyte co-cultures were employed to test the anti-tumor potential of low-dose telmisartan. When a high dose was required for a direct anti-proliferative effect on glioma cell lines, a low dose significantly inhibited glioma cell proliferation and migration in the co-culture system. Under co-culture conditions, upregulated IL-6 expression in astrocytes played a critical role in glioma progression. Silencing IL-6 in astrocytes or IL-6R in glioma cells reduced proliferation and migration. Telmisartan (5 µM) inhibited astrocytic IL-6 expression, and its anti-tumor effects were reversed by silencing IL-6 or IL-6R and inhibiting signal transducer and activator of transcription 3 (STAT3) activity in glioma cells. Moreover, the telmisartan-driven IL-6 downregulation was not imitated by losartan, an AT1R blocker with little capacity of peroxisome proliferator-activated receptor-gamma (PPARγ) activation, but was eliminated by a PPARγ antagonist, indicating that the anti-glioma effects of telmisartan rely on its PPARγ agonistic activity rather than AT1R blockade. This study highlights the importance of astrocytic IL-6-mediated paracrine signaling in glioma growth and the potential of telmisartan as an adjuvant therapy for patients with glioma, especially those with hypertension.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II , Astrócitos , Proliferação de Células , Técnicas de Cocultura , Glioma , Interleucina-6 , PPAR gama , Fator de Transcrição STAT3 , Telmisartan , Telmisartan/farmacologia , Telmisartan/uso terapêutico , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Interleucina-6/metabolismo , Glioma/tratamento farmacológico , Glioma/metabolismo , Glioma/patologia , Humanos , Proliferação de Células/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Linhagem Celular Tumoral , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , PPAR gama/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Receptores de Interleucina-6/metabolismo , Losartan/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
Glucagon is critical for the maintenance of blood glucose, however nutrient regulation of pancreatic α-cells remains poorly understood. Here, we identified a role of leucine, a well-known ß-cell fuel, in the α-cell-intrinsic regulation of glucagon release. In islet perifusion assays, physiologic concentrations of leucine strongly inhibited alanine- and arginine-stimulated glucagon secretion from human and mouse islets under hypoglycemic conditions. Mechanistically, leucine dose-dependently reduced α-cell cAMP, independently of Ca2+, ATP/ADP, or fatty acid oxidation. Leucine also reduced α-cell cAMP in islets treated with somatostatin receptor 2 antagonists or diazoxide, compounds that limit paracrine signaling from ß/δ-cells. Studies in dispersed mouse islets confirmed an α-cell-intrinsic effect. The inhibitory effect of leucine on cAMP was mimicked by glucose, α-ketoisocaproate, succinate, and the glutamate dehydrogenase activator BCH and blocked by cyanide, indicating a mechanism dependent on mitochondrial metabolism. Glucose dose-dependently reduced the impact of leucine on α-cell cAMP, indicating an overlap in function; however, leucine was still effective at suppressing glucagon secretion in the presence of elevated glucose, amino acids, and the incretin GIP. Taken together, these findings show that leucine plays an intrinsic role in limiting the α-cell secretory tone across the physiologic range of glucose levels, complementing the inhibitory paracrine actions of ß/δ-cells.
Assuntos
AMP Cíclico , Células Secretoras de Glucagon , Glucagon , Leucina , Comunicação Parácrina , Animais , Glucagon/metabolismo , AMP Cíclico/metabolismo , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/efeitos dos fármacos , Camundongos , Humanos , Leucina/farmacologia , Comunicação Parácrina/efeitos dos fármacos , Glucose/metabolismo , Cetoácidos/farmacologia , Masculino , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismoRESUMO
BACKGROUND: Hypertrophic scarring results from myofibroblast differentiation and persistence during wound healing. Currently no effective treatment for hypertrophic scarring exists however, autologous fat grafting has been shown to improve scar elasticity, appearance, and function. The aim of this study was to understand how paracrine factors from adipose tissues and adipose-derived stromal cells (ADSC) affect fibroblast to myofibroblast differentiation. METHODS: The transforming growth factor-ß1 (TGF-ß1) induced model of myofibroblast differentiation was used to test the effect of conditioned media from adipose tissue, ADSC or lipid on the proportion of fibroblasts and myofibroblasts. RESULTS: Adipose tissue conditioned media inhibited the differentiation of fibroblasts to myofibroblasts but this inhibition was not observed following treatment with ADSC or lipid conditioned media. Hepatocyte growth factor (HGF) was readily detected in the conditioned medium from adipose tissue but not ADSC. Cells treated with HGF, or fortinib to block HGF, demonstrated that HGF was not responsible for the inhibition of myofibroblast differentiation. Conditioned media from adipose tissue was shown to reduce the proportion of myofibroblasts when added to fibroblasts previously treated with TGF-ß1, however, conditioned media treatment was unable to significantly reduce the proportion of myofibroblasts in cell populations isolated from scar tissue. CONCLUSIONS: Cultured ADSC or adipocytes have been the focus of most studies, however, this work highlights the importance of considering whole adipose tissue to further our understanding of fat grafting. This study supports the use of autologous fat grafts for scar treatment and highlights the need for further investigation to determine the mechanism.
Assuntos
Tecido Adiposo , Diferenciação Celular , Fator de Crescimento de Hepatócito , Miofibroblastos , Fator de Crescimento Transformador beta1 , Miofibroblastos/metabolismo , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/citologia , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Humanos , Fator de Crescimento de Hepatócito/farmacologia , Fator de Crescimento de Hepatócito/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Fenótipo , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/citologia , Adipócitos/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacosRESUMO
BACKGROUND: Glioblastoma multiforme (GBM) is a highly invasive brain tumour, characterized by its ability to secrete factors promoting its virulence. Brain endothelial cells (BECs) in the GBM environment are physiologically modulated. The present study investigated the modulatory effects of normoxically and hypoxically induced glioblastoma U-87 cell secretions on BECs. METHODS: Conditioned media (CM) were derived by cultivating U-87 cells under hypoxic incubation (5% O2) and normoxic incubation (21% O2). Treated bEnd.3 cells were evaluated for mitochondrial dehydrogenase activity, mitochondrial membrane potential (ΔΨm), ATP production, transendothelial electrical resistance (TEER), and endothelial tight-junction (ETJ) gene expression over 96 h. RESULTS: The coculture of bEnd.3 cells with U-87 cells, or exposure to either hypoxic or normoxic U-87CM, was associated with low cellular viability. The ΔΨm in bEnd.3 cells was hyperpolarized after hypoxic U-87CM treatment (p < 0.0001). However, normoxic U-87CM did not affect the state of ΔΨm. BEC ATP levels were reduced after being cocultured with U-87 cells, or with hypoxic and normoxic CM (p < 0.05). Suppressed mitochondrial activity in bEnd.3 cells was associated with increased transendothelial permeability, while bEnd.3 cells significantly increased the gene expression levels of ETJs (p < 0.05) when treated with U-87CM. CONCLUSIONS: Hypoxic and normoxic glioblastoma paracrine factors differentially suppressed mitochondrial activity in BECs, increasing the BECs' barrier permeability.
Assuntos
Neoplasias Encefálicas/patologia , Encéfalo/patologia , Células Endoteliais/patologia , Glioblastoma/patologia , Comunicação Parácrina , Hipóxia Tumoral , Trifosfato de Adenosina/metabolismo , Animais , Neoplasias Encefálicas/genética , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Claudina-5/genética , Claudina-5/metabolismo , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ocludina/genética , Ocludina/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Comunicação Parácrina/genética , Hipóxia Tumoral/efeitos dos fármacos , Hipóxia Tumoral/genéticaRESUMO
BACKGROUND AND AIMS: Patients with pancreatic ductal adenocarcinoma (PDA) have not yet benefitted from the revolution in cancer immunotherapy due in large part to a dominantly immunosuppressive tumor microenvironment. MEK inhibition combined with autophagy inhibition leads to transient tumor responses in some patients with PDA. We examined the functional effects of combined MEK and autophagy inhibition on the PDA immune microenvironment and the synergy of combined inhibition of MEK and autophagy with CD40 agonism (aCD40) against PDA using immunocompetent model systems. METHODS: We implanted immunologically "cold" murine PDA cells orthotopically in wide type C57BL/6J mice. We administered combinations of inhibitors of MEK1/2, inhibitors of autophagy, and aCD40 and measured anticancer efficacy and immune sequelae using mass cytometry and multiplexed immunofluorescence imaging analysis to characterize the tumor microenvironment. We also used human and mouse PDA cell lines and human macrophages in vitro to perform functional assays to elucidate the cellular effects induced by the treatments. RESULTS: We find that coinhibition of MEK (using cobimetinib) and autophagy (using mefloquine), but not either treatment alone, activates the STING/type I interferon pathway in tumor cells that in turn activates paracrine tumor associated macrophages toward an immunogenic M1-like phenotype. This switch is further augmented by aCD40. Triple therapy (cobimetinib + mefloquine + aCD40) achieved cytotoxic T-cell activation in an immunologically "cold" mouse PDA model, leading to enhanced antitumor immunity. CONCLUSIONS: MEK and autophagy coinhibition coupled with aCD40 invokes immune repolarization and is an attractive therapeutic approach for PDA immunotherapy development.
Assuntos
Autofagia/imunologia , Azetidinas/farmacologia , Antígenos CD40/agonistas , Carcinoma Ductal Pancreático/imunologia , Mefloquina/farmacologia , Neoplasias Pancreáticas/imunologia , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologia , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Hidroxicloroquina/farmacologia , Imunoterapia , Interferon Tipo I/efeitos dos fármacos , Interferon Tipo I/imunologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Macrófagos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/imunologia , Camundongos , Comunicação Parácrina/efeitos dos fármacos , Comunicação Parácrina/imunologia , Evasão Tumoral , Microambiente Tumoral/efeitos dos fármacos , Macrófagos Associados a Tumor/efeitos dos fármacosRESUMO
Doxorubicin (Dox), an important anthracycline, is a potent anticancer agent that is used for treating solid tumors and hematologic malignancies. However, its clinical use is hampered by cardiac cardiotoxicity. This study aimed to investigate the cardioprotective potential of miR-199a-3p. Continuous Dox treatment not only markedly induced cardiomyocyte senescence but also resulted in a growing number of senescence-associated secretory phenotype (SASP) cardiomyocytes, frequently leading to heart senescence. This study showed that miR-199a-3p was downregulated in cardiomyocytes when exposed to Dox. The cardiac-specific overexpression of miR-199a-3p promoted cell cycle re-entry and cell proliferation, resulting in relief from cardiac senescence. Also, the elevation of miR-199a-3p inhibited the generation of SASP, thus, hampering the spread of senescence. In cardiomyocytes, the modulation of miR-199a-3p changed the levels of senescence-related protein GATA4. The ectopic expression of GATA4 blunted the anti-senescence effect of miR-199a-3p. Together, the data supported a role for miR-199a-3p during Dox cardiotoxicity. The elevation of miR-199a-3p might provide a dual therapeutic advantage in Dox cardiotoxicity therapy by simultaneously preventing cardiac senescence and reducing the spread of senescence.
Assuntos
Envelhecimento/efeitos dos fármacos , Antibióticos Antineoplásicos/toxicidade , Cardiotoxicidade/tratamento farmacológico , Doxorrubicina/toxicidade , MicroRNAs/uso terapêutico , Comunicação Parácrina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Cardiotoxicidade/etiologia , Proliferação de Células/efeitos dos fármacos , Imunofluorescência , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The limited ability of mammalian adult cardiomyocytes to proliferate following an injury to the heart, such as myocardial infarction, is a major factor that results in adverse fibrotic and myocardial remodeling that ultimately leads to heart failure. The continued high degree of heart failure-associated morbidity and lethality requires the special attention of researchers worldwide to develop efficient therapeutics for cardiac repair. Recently, various strategies and approaches have been developed and tested to extrinsically induce regeneration and restoration of the myocardium after cardiac injury have yielded encouraging results. Nevertheless, these interventions still lack adequate success to be used for clinical interventions. This review highlights and discusses both cell-based and cell-free therapeutic approaches as well as current advancements, major limitations, and future perspectives towards developing an efficient therapeutic method for cardiac repair.
Assuntos
Infarto do Miocárdio/patologia , Comunicação Parácrina , Animais , Terapia Baseada em Transplante de Células e Tecidos , Exossomos/metabolismo , Exossomos/transplante , Humanos , Infarto do Miocárdio/terapia , Comunicação Parácrina/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
The pyrazolyl-urea Gege3 molecule has shown interesting antiangiogenic effects in the tumor contest. Here, we have studied the role of this compound as interfering with endothelial cells activation in response to the paracrine effects of annexin A1 (ANXA1), known to be involved in promoting tumor progression. ANXA1 has been analyzed in the extracellular environment once secreted through microvesicles (EVs) by pancreatic cancer (PC) cells. Particularly, Gege3 has been able to notably prevent the effects of Ac2-26, the ANXA1 mimetic peptide, and of PC-derived EVs on endothelial cells motility, angiogenesis, and calcium release. Furthermore, this compound also inhibited the translocation of ANXA1 to the plasma membrane, otherwise induced by the same ANXA1-dependent extracellular stimuli. Moreover, these effects have been mediated by the indirect inhibition of protein kinase Cα (PKCα), which generally promotes the phosphorylation of ANXA1 on serine 27. Indeed, by the subtraction of intracellular calcium levels, the pathway triggered by PKCα underwent a strong inhibition leading to the following impediment to the ANXA1 localization at the plasma membrane, as revealed by confocal and cytofluorimetry analysis. Thus, Gege3 appeared an attractive molecule able to prevent the paracrine effects of PC cells deriving ANXA1 in the tumor microenvironment.
Assuntos
Anexina A1/metabolismo , Regulação para Baixo , Vesículas Extracelulares/metabolismo , Neoplasias Pancreáticas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ureia/química , Anexina A1/farmacologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Vesículas Extracelulares/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Comunicação Parácrina/efeitos dos fármacos , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Microambiente Tumoral/efeitos dos fármacosRESUMO
Cancer cells experience endoplasmic reticulum (ER) stress due to activated oncogenes and conditions of nutrient deprivation and hypoxia. The ensuing unfolded protein response (UPR) is executed by ATF6, IRE1 and PERK pathways. Adaptation to mild ER stress promotes tumor cell survival and aggressiveness. Unmitigated ER stress, however, will result in cell death and is a potential avenue for cancer therapies. Because of this yin-yang nature of ER stress, it is imperative that we fully understand the mechanisms and dynamics of the UPR and its contribution to the complexity of tumor biology. The PERK pathway inhibits global protein synthesis while allowing translation of specific mRNAs, such as the ATF4 transcription factor. Using thapsigargin and tunicamycin to induce acute ER stress, we identified the transcription factor C/EBPδ (CEBPD) as a mediator of PERK signaling to secretion of tumor promoting chemokines. In melanoma and breast cancer cell lines, PERK mediated early induction of C/EBPδ through ATF4-independent pathways that involved at least in part Janus kinases and the STAT3 transcription factor. Transcriptional profiling revealed that C/EBPδ contributed to 20% of thapsigargin response genes including chaperones, components of ER-associated degradation, and apoptosis inhibitors. In addition, C/EBPδ supported the expression of the chemokines CXCL8 (IL-8) and CCL20, which are known for their tumor promoting and immunosuppressive properties. With a paradigm of short-term exposure to thapsigargin, which was sufficient to trigger prolonged activation of the UPR in cancer cells, we found that conditioned media from such cells induced cytokine expression in myeloid cells. In addition, activation of the CXCL8 receptor CXCR1 during thapsigargin exposure supported subsequent sphere formation by cancer cells. Taken together, these investigations elucidated a novel mechanism of ER stress-induced transmissible signals in tumor cells that may be particularly relevant in the context of pharmacological interventions.
Assuntos
Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Quimiocina CCL20/metabolismo , Estresse do Retículo Endoplasmático , Imunomodulação , Interleucina-8/metabolismo , Transdução de Sinais , eIF-2 Quinase/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/genética , Linhagem Celular Tumoral , Quimiocina CCL20/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunomodulação/efeitos dos fármacos , Interleucina-8/genética , Janus Quinases/metabolismo , Modelos Biológicos , Comunicação Parácrina/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tapsigargina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genéticaRESUMO
Increased expression and activity of the Ca2+ channel transient receptor potential channel 6 (TRPC6) is associated with focal segmental glomerulosclerosis, but therapeutic strategies to target TRPC6 are currently lacking. Nitric oxide (NO) is crucial for normal glomerular function and plays a protective role in preventing glomerular diseases. We investigated if NO prevents podocyte injury by inhibiting injurious TRPC6-mediated signaling in a soluble guanylate cyclase (sGC)-dependent manner and studied the therapeutic potential of the sGC stimulator Riociguat. Experiments were performed using human glomerular endothelial cells and podocytes. Podocyte injury was induced by Adriamycin incubation for 24 h, with or without the NO-donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP), the sGC stimulator Riociguat or the TRPC6 inhibitor Larixyl Acetate (LA). NO and Riociguat stimulated cGMP synthesis in podocytes, decreased Adriamycin-induced TRPC6 expression, inhibited the Adriamycin-induced TRPC6-mediated Ca2+ influx and reduced podocyte injury. The protective effects of Riociguat and NO were blocked when sGC activity was inhibited with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or when TRPC6 activity was inhibited by LA. Our data demonstrate a glomerular (e)NOS-NO-sGC-cGMP-TRPC6 pathway that prevents podocyte injury, which can be translated to future clinical use by, e.g., repurposing the market-approved drug Riociguat.
Assuntos
Guanilato Ciclase/genética , Óxido Nítrico/genética , Podócitos/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Canal de Cátion TRPC6/genética , Animais , Sinalização do Cálcio/efeitos dos fármacos , GMP Cíclico/genética , Reposicionamento de Medicamentos , Células Endoteliais/efeitos dos fármacos , Humanos , Nefropatias/tratamento farmacológico , Nefropatias/genética , Nefropatias/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Camundongos , Comunicação Parácrina/efeitos dos fármacos , Podócitos/patologiaRESUMO
COPD is characterized by irreversible lung tissue damage. We hypothesized that lung-derived mesenchymal stromal cells (LMSCs) reduce alveolar epithelial damage via paracrine processes, and may thus be suitable for cell-based strategies in COPD. We aimed to assess whether COPD-derived LMSCs display abnormalities. LMSCs were isolated from lung tissue of severe COPD patients and non-COPD controls. Effects of LMSC conditioned-medium (CM) on H2O2-induced, electric field- and scratch-injury were studied in A549 and NCI-H441 epithelial cells. In organoid models, LMSCs were co-cultured with NCI-H441 or primary lung cells. Organoid number, size and expression of alveolar type II markers were assessed. Pre-treatment with LMSC-CM significantly attenuated oxidative stress-induced necrosis and accelerated wound repair in A549. Co-culture with LMSCs supported organoid formation in NCI-H441 and primary epithelial cells, resulting in significantly larger organoids with lower type II-marker positivity in the presence of COPD-derived versus control LMSCs. Similar abnormalities developed in organoids from COPD compared to control-derived lung cells, with significantly larger organoids. Collectively, this indicates that LMSCs' secretome attenuates alveolar epithelial injury and supports epithelial repair. Additionally, LMSCs promote generation of alveolar organoids, with abnormalities in the supportive effects of COPD-derived LMCS, reflective of impaired regenerative responses of COPD distal lung cells.
Assuntos
Células Epiteliais Alveolares/patologia , Células-Tronco Mesenquimais/patologia , Comunicação Parácrina , Idoso , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Organoides/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regeneração/efeitos dos fármacos , Esferoides Celulares/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Successful implantation requires the coordinated migration and invasion of trophoblast cells from out of the blastocyst and into the endometrium. This process relies on signals produced by cells in the maternal endometrium. However, the relative contribution of stroma cells remains unclear. The study of human implantation has major technical limitations, therefore the need of in vitro models to elucidate the molecular mechanisms. Using a recently described 3D in vitro models we evaluated the interaction between trophoblasts and human endometrial stroma cells (hESC), we assessed the process of trophoblast migration and invasion in the presence of stroma derived factors. We demonstrate that hESC promotes trophoblast invasion through the generation of an inflammatory environment modulated by TNF-α. We also show the role of stromal derived IL-17 as a promoter of trophoblast migration through the induction of essential genes that confer invasive capacity to cells of the trophectoderm. In conclusion, we describe the characterization of a cellular inflammatory network that may be important for blastocyst implantation. Our findings provide a new insight into the complexity of the implantation process and reveal the importance of inflammation for embryo implantation.
Assuntos
Movimento Celular , Implantação do Embrião , Endométrio/efeitos dos fármacos , Interleucina-17/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Trofoblastos/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adesão Celular , Diferenciação Celular , Linhagem Celular , Endométrio/imunologia , Endométrio/metabolismo , Feminino , Humanos , Interleucina-17/genética , Receptores Tipo I de Fatores de Necrose Tumoral/agonistas , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Via Secretória , Transdução de Sinais , Células Estromais/imunologia , Células Estromais/metabolismo , Trofoblastos/imunologiaRESUMO
Multicellular spheroids show three-dimensional (3D) organization with extensive cell-cell and cell-extracellular matrix interactions. Owing to their native tissue-mimicking characteristics, mesenchymal stem cell (MSC) spheroids are considered promising as implantable therapeutics for stem cell therapy. Herein, we aim to further enhance their therapeutic potential by tuning the cultivation parameters and thus the inherent niche of 3D MSC spheroids. Significantly increased expression of multiple pro-regenerative paracrine signaling molecules and immunomodulatory factors by MSCs was observed after optimizing the conditions for spheroid culture. Moreover, these alterations in cellular behaviors may be associated with not only the hypoxic niche developed in the spheroid core but also with the metabolic reconfiguration of MSCs. The present study provides efficient methods for manipulating the therapeutic capacity of 3D MSC spheroids, thus laying solid foundations for future development and clinical application of spheroid-based MSC therapy for regenerative medicine.
Assuntos
Imunomodulação , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Nicho de Células-Tronco , Autofagia/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Hidrogéis/farmacologia , Imunomodulação/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Nicho de Células-Tronco/efeitos dos fármacosRESUMO
Dimeric translationally controlled tumor protein (dTCTP), also known as histamine-releasing factor, amplifies allergic responses and its production has been shown to increase in inflammatory diseases such as allergic asthma. Despite the critical role of dTCTP in allergic inflammation, little is known about its production pathways, associated cellular networks, and underlying molecular mechanisms. In this study, we explored the dTCTP-mediated inflammatory networks and molecular mechanisms of dTCTP associated with lipopolysaccharides (LPS)-induced severe asthma. LPS stimulation increased dTCTP production by mast cells and dTCTP secretion during degranulation, and extracellular dTCTP subsequently increased the production of pro-inflammatory molecules, including IL-8, by airway epithelial cells without affecting mast cell activation. Furthermore, dimeric TCTP-binding peptide 2 (dTBP2), a dTCTP inhibitor peptide, selectively blocked the dTCTP-mediated signaling network from mast cells to epithelial cells and decreased IL-8 production through IkB induction and nuclear p65 export in airway epithelial cells. More importantly, dTBP2 efficiently attenuated LPS-induced severe airway inflammation in vivo, resulting in decreased immune cell infiltration and IL-17 production and attenuated dTCTP secretion. These results suggest that dTCTP produced by mast cells exacerbates airway inflammation through activation of airway epithelial cells in a paracrine signaling manner, and that dTBP2 is beneficial in the treatment of severe airway inflammation by blocking the dTCTP-mediated inflammatory cellular network.
Assuntos
Antiasmáticos/farmacologia , Anti-Inflamatórios/farmacologia , Asma/prevenção & controle , Células Epiteliais/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Pulmão/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Peptídeos/farmacologia , Pneumonia/prevenção & controle , Proteína Tumoral 1 Controlada por Tradução/metabolismo , Animais , Asma/induzido quimicamente , Asma/imunologia , Asma/metabolismo , Técnicas de Cocultura , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Lipopolissacarídeos , Pulmão/imunologia , Pulmão/metabolismo , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos Endogâmicos C57BL , Ovalbumina , Comunicação Parácrina/efeitos dos fármacos , Pneumonia/induzido quimicamente , Pneumonia/imunologia , Pneumonia/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
Active cancer-associated fibroblasts (CAFs) are major components of the tumor microenvironment, which promote carcinogenesis and modulate response to therapy. Therefore, targeting these cells or reducing their paracrine pro-carcinogenic effects could be of great therapeutic value. To this end, we sought to investigate the effect of eugenol, a natural phenolic molecule, on active breast CAFs. We have shown that decitabine (5-Aza-2'-deoxycytidine, DAC) and eugenol inhibit the expression of the DNA methyltransferase genes DNMT1 and DNMT3A at both the protein and mRNA levels in breast CAF cells. While the effect of eugenol was persistent, DAC had only a transient inhibitory effect on the mRNA level of both DNMT genes. Furthermore, eugenol and DAC suppressed the invasive/migratory and proliferative potential of CAF cells as well as their paracrine pro-carcinogenic effects both in vitro and in humanized orthotopic tumor xenografts. Interestingly, these inhibitory effects of decitabine and eugenol were mediated through E2F1 downregulation. Indeed, ectopic expression of E2F1 upregulated both genes and attenuated the effects of eugenol. Additionally, we provide clear evidence that eugenol, like DAC, strongly modulates the methylation pattern in active CAF cells, through methylating several oncogenes and demethylating various important tumor suppressor genes, which affected their mRNA expression levels. Importantly, the E2F1 promoter was also hypermethylated and the gene downregulated in response to eugenol. Together, these findings show that the active features of breast CAF cells can be normalized through eugenol-dependent targeting of DNMT1/DNMT3A and the consequent modulation in gene methylation.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Fibroblastos Associados a Câncer/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA Metiltransferase 3A/genética , Decitabina/administração & dosagem , Fator de Transcrição E2F1/genética , Eugenol/administração & dosagem , Animais , Neoplasias da Mama/genética , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/efeitos dos fármacos , DNA Metiltransferase 3A/metabolismo , Decitabina/farmacologia , Regulação para Baixo , Sinergismo Farmacológico , Fator de Transcrição E2F1/metabolismo , Eugenol/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Comunicação Parácrina/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ovarian cancer is the leading cause of gynecological malignancy-related deaths, due to its widespread intraperitoneal metastases and acquired chemoresistance. Mesothelial cells are an important cellular component of the ovarian cancer microenvironment that promote metastasis. However, their role in chemoresistance is unclear. Here, we investigated whether cancer-associated mesothelial cells promote ovarian cancer chemoresistance and stemness in vitro and in vivo. We found that osteopontin is a key secreted factor that drives mesothelial-mediated ovarian cancer chemoresistance and stemness. Osteopontin is a secreted glycoprotein that is clinically associated with poor prognosis and chemoresistance in ovarian cancer. Mechanistically, ovarian cancer cells induced osteopontin expression and secretion by mesothelial cells through TGF-ß signaling. Osteopontin facilitated ovarian cancer cell chemoresistance via the activation of the CD44 receptor, PI3K/AKT signaling, and ABC drug efflux transporter activity. Importantly, therapeutic inhibition of osteopontin markedly improved the efficacy of cisplatin in both human and mouse ovarian tumor xenografts. Collectively, our results highlight mesothelial cells as a key driver of ovarian cancer chemoresistance and suggest that therapeutic targeting of osteopontin may be an effective strategy for enhancing platinum sensitivity in ovarian cancer.
Assuntos
Osteopontina/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Epitélio/patologia , Feminino , Humanos , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Osteopontina/antagonistas & inibidores , Neoplasias Ovarianas/patologia , Comunicação Parácrina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RATIONALE: In recent decades, the great potential of human epicardium-derived cells (EPDCs) as an endogenous cell source for cardiac regeneration has been recognized. The limited availability and low proliferation capacity of primary human EPDCs and phenotypic differences between EPDCs obtained from different individuals hampers their reproducible use for experimental studies. AIM: To generate and characterize inducible proliferative adult human EPDCs for use in fundamental and applied research. METHODS AND RESULTS: Inducible proliferation of human EPDCs was achieved by doxycycline-controlled expression of simian virus 40 large T antigen (LT) with a repressor-based lentiviral Tet-On system. In the presence of doxycycline, these inducible EPDCs (iEPDCs) displayed high and long-term proliferation capacity. After doxycycline removal, LT expression ceased and the iEPDCs regained their cuboidal epithelial morphology. Similar to primary EPDCs, iEPDCs underwent an epithelial-to-mesenchymal transition (EMT) after stimulation with transforming growth factor ß3. This was confirmed by reverse transcription-quantitative polymerase chain reaction analysis of epithelial and mesenchymal marker gene expression and (immuno) cytochemical staining. Collagen gel-based cell invasion assays demonstrated that mesenchymal iEPDCs, like primary EPDCs, possess increased invasion and migration capacities as compared to their epithelial counterparts. Mesenchymal iEPDCs co-cultured with sympathetic ganglia stimulated neurite outgrowth similarly to primary EPDCs. CONCLUSION: Using an inducible LT expression system, inducible proliferative adult human EPDCs were generated displaying high proliferative capacity in the presence of doxycycline. These iEPDCs maintain essential epicardial characteristics with respect to morphology, EMT ability, and paracrine signaling following doxycycline removal. This renders iEPDCs a highly useful new in vitro model for studying human epicardial properties.
Assuntos
Pericárdio/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Doxiciclina/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Gânglios Simpáticos/citologia , Gânglios Simpáticos/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Modelos Biológicos , Neuritos/fisiologia , Comunicação Parácrina/efeitos dos fármacos , Pericárdio/citologia , Fator de Crescimento Transformador beta3/farmacologiaRESUMO
Glomerulonephritis are renal inflammatory processes characterized by increased permeability of the Glomerular Filtration Barrier (GFB) with consequent hematuria and proteinuria. Glomerular endothelial cells (GEC) and podocytes are part of the GFB and contribute to the maintenance of its structural and functional integrity through the release of paracrine mediators. Activation of the complement cascade and pro-inflammatory cytokines (CK) such as Tumor Necrosis Factor α (TNF-α) and Interleukin-6 (IL-6) can alter GFB function, causing acute glomerular injury and progression toward chronic kidney disease. Endothelial Progenitor Cells (EPC) are bone-marrow-derived hematopoietic stem cells circulating in peripheral blood and able to induce angiogenesis and to repair injured endothelium by releasing paracrine mediators including Extracellular Vesicles (EVs), microparticles involved in intercellular communication by transferring proteins, lipids, and genetic material (mRNA, microRNA, lncRNA) to target cells. We have previously demonstrated that EPC-derived EVs activate an angiogenic program in quiescent endothelial cells and renoprotection in different experimental models. The aim of the present study was to evaluate in vitro the protective effect of EPC-derived EVs on GECs and podocytes cultured in detrimental conditions with CKs (TNF-α/IL-6) and the complement protein C5a. EVs were internalized in both GECs and podocytes mainly through a L-selectin-based mechanism. In GECs, EVs enhanced the formation of capillary-like structures and cell migration by modulating gene expression and inducing the release of growth factors such as VEGF-A and HGF. In the presence of CKs, and C5a, EPC-derived EVs protected GECs from apoptosis by decreasing oxidative stress and prevented leukocyte adhesion by inhibiting the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin). On podocytes, EVs inhibited apoptosis and prevented nephrin shedding induced by CKs and C5a. In a co-culture model of GECs/podocytes that mimicked GFB, EPC-derived EVs protected cell function and permeselectivity from inflammatory-mediated damage. Moreover, RNase pre-treatment of EVs abrogated their protective effects, suggesting the crucial role of RNA transfer from EVs to damaged glomerular cells. In conclusion, EPC-derived EVs preserved GFB integrity from complement- and cytokine-induced damage, suggesting their potential role as therapeutic agents for drug-resistant glomerulonephritis.