RESUMO
Urinary tract infection (UTI), one of the most common bacterial infections worldwide, is a typical example of an infection that is often polymicrobial in nature. While the overall infection course is known on a macroscale, bacterial behavior is not fully understood at the cellular level and bacterial pathophysiology during multispecies infection is not well characterized. Here, using clinically relevant bacteria, human epithelial bladder cells and human urine, we establish co-infection models combined with high resolution imaging to compare single- and multi-species bladder cell invasion events in three common uropathogens: uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae and Enterococcus faecalis. While all three species invaded the bladder cells, under flow conditions the Gram-positive E. faecalis was significantly less invasive compared to the Gram-negative UPEC and K. pneumoniae. When introduced simultaneously during an infection experiment, all three bacterial species sometimes invaded the same bladder cell, at differing frequencies suggesting complex interactions between bacterial species and bladder cells. Inside host cells, we observed encasement of E. faecalis colonies specifically by UPEC. During subsequent dispersal from the host cells, only the Gram-negative bacteria underwent infection-related filamentation (IRF). Taken together, our data suggest that bacterial multispecies invasions of single bladder cells are frequent and support earlier studies showing intraspecies cooperation on a biochemical level during UTI.
Assuntos
Enterococcus faecalis , Células Epiteliais , Klebsiella pneumoniae , Infecções Urinárias , Escherichia coli Uropatogênica , Humanos , Infecções Urinárias/microbiologia , Enterococcus faecalis/fisiologia , Células Epiteliais/microbiologia , Escherichia coli Uropatogênica/fisiologia , Klebsiella pneumoniae/fisiologia , Bexiga Urinária/microbiologia , Bexiga Urinária/citologia , Coinfecção/microbiologia , Linhagem Celular , Interações Hospedeiro-PatógenoRESUMO
Previous studies have shown that growth arrest, dedifferentiation, and loss of original function occur in cells after multiple generations of culture, which are attributed to the lack of stress stimulation. To investigate the effects of multi-modal biomimetic stress (MMBS) on the biological function of human bladder smooth muscle cells (HBSMCs), a MMBS culture system was established to simulate the stress environment suffered by the bladder, and HBSMCs were loaded with different biomimetic stress for 24 h. Then, cell growth, proliferation and functional differentiation were detected. The results showed that MMBS promoted the growth and proliferation of HBSMCs, and 80 cm H 2O pressure with 4% stretch stress were the most effective in promoting the growth and proliferation of HBSMCs and the expression level of α-smooth muscle actin and smooth muscle protein 22-α. These results suggest that the MMBS culture system will be beneficial in regulating the growth and functional differentiation of HBSMCs in the construction of tissue engineered bladder.
Assuntos
Actinas , Diferenciação Celular , Proliferação de Células , Miócitos de Músculo Liso , Estresse Mecânico , Engenharia Tecidual , Bexiga Urinária , Bexiga Urinária/citologia , Bexiga Urinária/fisiologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Humanos , Engenharia Tecidual/métodos , Actinas/metabolismo , Biomimética , Proteínas Musculares/metabolismo , Células CultivadasRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Arctostaphylos uva-ursi (L.) Spreng. (bearberry) is a well-known traditional herbal plant used as a urinary tract disinfectant. Its antiseptic and diuretic properties can be attributed to hydroquinone, obtained by hydrolysis of arbutin. AIM OF THE STUDY: This study aimed to determine the toxic profile of free hydroquinone on urinary bladder cells (T24) as a target of therapeutic action. MATERIALS AND METHODS: Quantitative and qualitative analysis of the extract and the digestive stability and bioavailability of arbutin and hydroquinone were performed by HPLC assay and simulated in vitro digestion, respectively. Cytotoxic effect, reactive oxygen species induction and proteome changes in T24 cells after hydroquinone treatment were determined using Neutral red assay, 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay and mass spectrometry, respectively. RESULTS: Through in vitro digestion, arbutin was stable, but hydroquinone increased after pepsin treatment (109.6%) and then decreased after the small intestine phase (65.38%). The recommended doses of Uva-ursi had a cytotoxic effect on T24 cells only when all hydroquinone conjugates were converted to free hydroquinone (320 and 900 µg/mL) and the toxic effect was enhanced by recovery. One cup of the therapeutic dose had a prooxidative effect after 4 h of incubation. Shorter time of cell exposure (2 h) to hydroquinone did not have any impact on reactive oxygen species induction. Proteomic analysis found 17 significantly up-regulated proteins compared to control. Hydroquinone activated proteins related to oxidative stress response, stress-adaptive signalling, heat shock response and initiation of translation. CONCLUSIONS: Despite the therapeutic properties of bearberry, up-regulated T24 cell proteins are evidence that plant compounds, although from a natural source, may exhibit negative properties.
Assuntos
Arctostaphylos/química , Hidroquinonas/toxicidade , Extratos Vegetais/toxicidade , Bexiga Urinária/efeitos dos fármacos , Arbutina/química , Arbutina/isolamento & purificação , Células CACO-2 , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Humanos , Hidroquinonas/isolamento & purificação , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/química , Proteoma , Proteômica , Bexiga Urinária/citologiaRESUMO
The inflammasome-associated proteins caspase-1, caspase-4 and NLRP3 have been emphasised to be essential in the host cell response during urinary tract infection (UTI) by regulating IL-1ß release. Our aim was to investigate how the inflammasome-associated proteins regulate the cell response of bladder epithelial cells during infection with uropathogenic Escherichia coli (UPEC). Human bladder epithelial cells (5637) and CRISPR/Cas9 generated caspase-1, caspase-4 and NLRP3 knockdown cells were stimulated with the UPEC strain CFT073. Using Olink proteomics and real time RT-PCR, we showed that caspase-1, caspase-4 and NLRP3 are vital for the expression of many inflammatory genes and proteins from bladder epithelial cells. When investigating the effect of inflammasome-associated proteins on neutrophils, we found that conditioned medium from UPEC-infected caspase-4 knockdown cells significantly increased phagocytosis of CFT073 and significantly decreased ROS production from neutrophils. In contrast, conditioned medium from UPEC-infected NLRP3 knockdown cells significantly decreased the phagocytosis of CFT073 and significantly increased the ROS production from neutrophils. In conclusion, we showed that the inflammasome-associated proteins contribute to the host cell response during UPEC infection.
Assuntos
Caspase 1/fisiologia , Caspases Iniciadoras/fisiologia , Células Epiteliais/imunologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Infecções Urinárias/genética , Infecções Urinárias/imunologia , Escherichia coli Uropatogênica/imunologia , Caspases Iniciadoras/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Inflamação , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neutrófilos/metabolismo , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Bexiga Urinária/citologiaRESUMO
Single-cell RNA sequencing (scRNA-seq) data has been widely used to profile cellular heterogeneities with a high-resolution picture. Clustering analysis is a crucial step of scRNA-seq data analysis because it provides a chance to identify and uncover undiscovered cell types. Most methods for clustering scRNA-seq data use an unsupervised learning strategy. Since the clustering step is separated from the cell annotation and labeling step, it is not uncommon for a totally exotic clustering with poor biological interpretability to be generated-a result generally undesired by biologists. To solve this problem, we proposed an active learning (AL) framework for clustering scRNA-seq data. The AL model employed a learning algorithm that can actively query biologists for labels, and this manual labeling is expected to be applied to only a subset of cells. To develop an optimal active learning approach, we explored several key parameters of the AL model in the experiments with four real scRNA-seq datasets. We demonstrate that the proposed AL model outperformed state-of-the-art unsupervised clustering methods with less than 1000 labeled cells. Therefore, we conclude that AL model is a promising tool for clustering scRNA-seq data that allows us to achieve a superior performance effectively and efficiently.
Assuntos
Algoritmos , Perfilação da Expressão Gênica/métodos , RNA-Seq/métodos , Análise de Célula Única/métodos , Aprendizado de Máquina não Supervisionado , Animais , Células Cultivadas , Análise por Conglomerados , Humanos , Rim/citologia , Rim/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Reprodutibilidade dos Testes , Bexiga Urinária/citologia , Bexiga Urinária/metabolismoRESUMO
Cell-based tissue engineering (TE) has been proposed to improve treatment outcomes in end-stage bladder disease, but TE approaches with 2D smooth muscle cell (SMC) culture have so far been unsuccessful. Here, we report the development of primary bladder-derived 3D SMC spheroids that outperform 2D SMC cultures in differentiation, maturation, and extracellular matrix (ECM) production. Bladder SMC spheroids were compared with 2D cultures using live-dead staining, qRT-PCR, immunofluorescence, and immunoblotting to investigate culture conditions, contractile phenotype, and ECM deposition. The SMC spheroids were viable for up to 14 days and differentiated rather than proliferating. Spheroids predominantly expressed the late myogenic differentiation marker MyH11, whereas 2D SMC expressed more of the general SMC differentiation marker α-SMA and less MyH11. Furthermore, the expression of bladder wall-specific ECM proteins in SMC spheroids was markedly higher. This first establishment and analysis of primary bladder SMC spheroids are particularly promising for TE because differentiated SMCs and ECM deposition are a prerequisite to building a functional bladder wall substitute. We were able to confirm that SMC spheroids are promising building blocks for studying detrusor regeneration in detail and may provide improved function and regenerative potential, contributing to taking bladder TE a significant step forward.
Assuntos
Miócitos de Músculo Liso/citologia , Esferoides Celulares/citologia , Engenharia Tecidual/métodos , Bexiga Urinária/citologia , Actinas/genética , Actinas/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Expressão Gênica , Masculino , Desenvolvimento Muscular , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Wistar , Esferoides Celulares/metabolismoRESUMO
Bladder cancer represents a highly heterogeneous disease characterized by distinct histological, molecular and clinical phenotypes, and a detailed analysis of tumor cell invasion and crosstalks within bladder tumor cells has not been determined. Here, we applied droplet-based single-cell RNA sequencing (scRNA-seq) to acquire transcriptional profiles of 36 619 single cells isolated from seven patients. Single cell transcriptional profiles matched well with the pathological basal/luminal subtypes. Notably, in T1 tumors diagnosed as luminal subtype, basal cells displayed characteristics of epithelial-mesenchymal transition (EMT) and mainly located at the tumor-stromal interface as well as micrometastases in the lamina propria. In one T3 tumor, muscle-invasive tumor showed significantly higher expression of cancer stem cell markers SOX9 and SOX2 than the primary tumor. We additionally analyzed communications between tumor cells and demonstrated its relevance to basal/luminal phenotypes. Overall, our single-cell study provides a deeper insight into the tumor cell heterogeneity associated with bladder cancer progression.
Assuntos
Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias da Bexiga Urinária/genética , Bexiga Urinária/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Músculo Liso/patologia , Invasividade Neoplásica/genética , RNA-Seq , Análise de Célula Única , Tomografia Computadorizada por Raios X , Bexiga Urinária/citologia , Bexiga Urinária/diagnóstico por imagem , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologiaRESUMO
Postmenopausal women with ovary hormone deficiency (OHD) are subject to overactive bladder (OAB) symptoms. The present study attempted to elucidate whether low-intensity extracorporeal shock wave therapy (LiESWT) alters bladder angiogenesis, decreases inflammatory response, and ameliorates bladder hyperactivity to influence bladder function in OHD-induced OAB in human clinical trial and rat model. The ovariectomized (OVX) for 12 months Sprague-Dawley rat model mimicking the physiological condition of menopause was utilized to induce OAB and assess the potential therapeutic mechanism of LiESWT (0.12 mJ/mm2, 300 pulses, and 3 pulses/second). The randomized, single-blinded clinical trial was enrolled 58 participants to investigate the therapeutic efficacy of LiESWT (0.25 mJ/mm2, 3000 pulses, 3 pulses/second) on postmenopausal women with OAB. The results revealed that 8 weeks' LiESWT inhibited interstitial fibrosis, promoted cell proliferation, enhanced angiogenesis protein expression, and elevated the protein phosphorylation of ErK1/2, P38, and Akt, leading to decreased urinary frequency, nocturia, urgency, urgency incontinence, and post-voided residual urine volume, but increased voided urine volume and the maximal flow rate of postmenopausal participants. In conclusion, LiESWT attenuated inflammatory responses, increased angiogenesis, and promoted proliferation and differentiation, thereby improved OAB symptoms, thereafter promoting social activity and the quality of life of postmenopausal participants.
Assuntos
Modelos Animais de Doenças , Tratamento por Ondas de Choque Extracorpóreas/métodos , Insuficiência Ovariana Primária/complicações , Qualidade de Vida , Regeneração , Bexiga Urinária Hiperativa/terapia , Bexiga Urinária/citologia , Adulto , Idoso , Animais , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Ratos , Ratos Sprague-Dawley , Método Simples-Cego , Bexiga Urinária Hiperativa/etiologia , Bexiga Urinária Hiperativa/patologiaRESUMO
Volume accommodation occurs via a novel mechanism involving interstitial cells in detrusor muscles. The interstitial cells in the bladder are PDGFRα+, and they restrain the excitability of smooth muscle at low levels and prevents the development of transient contractions (TCs). A common clinical manifestation of spinal cord injury (SCI)-induced bladder dysfunction is detrusor overactivity (DO). Although a myogenic origin of DO after SCI has been suggested, a mechanism for development of SCI-induced DO has not been determined. In this study we hypothesized that SCI-induced DO is related to loss of function in the regulatory mechanism provided by PDGFRα+ cells. Our results showed that transcriptional expression of Pdgfra and Kcnn3 was decreased after SCI. Proteins encoded by these genes also decreased after SCI, and a reduction in PDGFRα+ cell density was also documented. Loss of PDGFRα+ cells was due to apoptosis. TCs in ex vivo bladders during filling increased dramatically after SCI, and this was related to the loss of regulation provided by SK channels, as we observed decreased sensitivity to apamin. These findings show that damage to the mechanism restraining muscle contraction during bladder filling that is provided by PDGFRα+ cells is causative in the development of DO after SCI.
Assuntos
Contração Muscular/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/fisiologia , Traumatismos da Medula Espinal/complicações , Bexiga Urinária Hiperativa/etiologia , Bexiga Urinária/metabolismo , Bexiga Urinária/fisiopatologia , Animais , Apamina/metabolismo , Apoptose , Expressão Gênica , Camundongos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Traumatismos da Medula Espinal/genética , Bexiga Urinária/citologia , Bexiga Urinária/patologia , Bexiga Urinária Hiperativa/fisiopatologiaAssuntos
Adenocarcinoma/diagnóstico , Carcinoma de Células de Transição/diagnóstico , Neoplasias da Bexiga Urinária/diagnóstico , Bexiga Urinária/patologia , Adenocarcinoma/patologia , Idoso , Autopsia , Evolução Fatal , Humanos , Masculino , Derrame Pleural , Toracentese , Bexiga Urinária/citologiaAssuntos
Alergia e Imunologia/história , Dermatite Alérgica de Contato/imunologia , Infecções por Herpesviridae/imunologia , Memória Imunológica , Células Matadoras Naturais/imunologia , Animais , Benzenossulfonatos/administração & dosagem , Benzenossulfonatos/imunologia , Proteínas de Ligação a DNA/genética , Dinitrofluorbenzeno/administração & dosagem , Dinitrofluorbenzeno/imunologia , Modelos Animais de Doenças , Infecções por Herpesviridae/virologia , História do Século XXI , Humanos , Fígado/citologia , Fígado/imunologia , Camundongos , Camundongos Knockout , Camundongos SCID , Modelos Animais , Muromegalovirus/imunologia , Pele/citologia , Pele/efeitos dos fármacos , Pele/imunologia , Bexiga Urinária/citologia , Bexiga Urinária/imunologiaRESUMO
The male urogenital system is composed of the reproductive system and the urinary tract; they have an interconnected embryonic development and share one of their anatomical components, the urethra. This system has a highly complex physiology deeply interconnected with the circulatory and nervous systems, as well as being capable of adapting to environmental variations; it also undergoes changes with aging and, in the case of the reproductive system, with seasonality. The stroma is an essential component in this physiological plasticity and its complexity has increased with the description in the last decade of a new cell type, the telocyte. Several studies have demonstrated the presence of telocytes in the organs of the male urogenital system and other systems; however, their exact function is not yet known. The present review addresses current knowledge about telocytes in the urogenital system in terms of their locations, interrelationships, possible functions and pathological implications. It has been found that telocytes in the urogenital system possibly have a leading role in stromal tissue organization/maintenance, in addition to participation in stem cell niches and an association with the immune system, as well as specific functions in the urogenital system, lipid synthesis in the testes, erythropoiesis in the kidneys and the micturition reflex in the bladder. There is also evidence that telocytes are involved in pathologies in the kidneys, urethra, bladder, prostate, and testes.
Assuntos
Telócitos/patologia , Telócitos/fisiologia , Sistema Urogenital/patologia , Sistema Urogenital/fisiologia , Animais , Doenças dos Genitais Masculinos/patologia , Doenças dos Genitais Masculinos/fisiopatologia , Humanos , Metabolismo dos Lipídeos/fisiologia , Masculino , Próstata/citologia , Próstata/patologia , Próstata/fisiologia , Células-Tronco/patologia , Células-Tronco/fisiologia , Testículo/citologia , Testículo/patologia , Testículo/fisiologia , Bexiga Urinária/citologia , Bexiga Urinária/patologia , Bexiga Urinária/fisiologia , Sistema Urogenital/citologiaRESUMO
BACKGROUND: Extracellular matrix (ECM) bioscaffolds produced by decellularization of source tissue have been effectively used for numerous clinical applications. However, decellularized tracheal constructs have been unsuccessful due to the immediate requirement of a functional airway epithelium on surgical implantation. ECM can be solubilized to form hydrogels that have been shown to support growth of many different cell types. The purpose of the present study is to compare the ability of airway epithelial cells to attach, form a confluent monolayer, and differentiate on homologous (trachea) and heterologous (urinary bladder) ECM substrates for potential application in full tracheal replacement. MATERIALS AND METHODS: Porcine tracheas and urinary bladders were decellularized. Human bronchial epithelial cells (HBECs) were cultured under differentiation conditions on acellular tracheal ECM and urinary bladder matrix (UBM) bioscaffolds and hydrogels and were assessed by histology and immunolabeling for markers of ciliation, goblet cell formation, and basement membrane deposition. RESULTS: Both trachea and urinary bladder tissues were successfully decellularized. HBEC formed a confluent layer on both trachea and UBM scaffolds and on hydrogels created from these bioscaffolds. Cells grown on tracheal and UBM hydrogels, but not on bioscaffolds, showed positive-acetylated tubulin staining and the presence of mucus-producing goblet cells. Collagen IV immunolabeling showed basement membrane deposition by these cells on the surface of the hydrogels. CONCLUSIONS: ECM hydrogels supported growth and differentiation of HBEC better than decellularized ECM bioscaffolds and show potential utility as substrates for promotion of a mature respiratory epithelium for regenerative medicine applications in the trachea.
Assuntos
Brônquios/citologia , Células Epiteliais/fisiologia , Alicerces Teciduais , Traqueia/transplante , Bexiga Urinária/citologia , Adulto , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Proliferação de Células , Matriz Extracelular , Feminino , Humanos , Hidrogéis , Masculino , Projetos Piloto , Cultura Primária de Células , Suínos , Engenharia Tecidual/métodos , Traqueia/citologia , Transplante Heterólogo , Transplante Homólogo , Adulto JovemRESUMO
BACKGROUND: Microscopic analysis of urine sediment is probably the most commonly used diagnostic procedure in nephrology. The urinary cells, however, have not yet undergone careful unbiased characterization. METHODS: Single-cell transcriptomic analysis was performed on 17 urine samples obtained from five subjects at two different occasions, using both spot and 24-hour urine collection. A pooled urine sample from multiple healthy individuals served as a reference control. In total 23,082 cells were analyzed. Urinary cells were compared with human kidney and human bladder datasets to understand similarities and differences among the observed cell types. RESULTS: Almost all kidney cell types can be identified in urine, such as podocyte, proximal tubule, loop of Henle, and collecting duct, in addition to macrophages, lymphocytes, and bladder cells. The urinary cell-type composition was subject specific and reasonably stable using different collection methods and over time. Urinary cells clustered with kidney and bladder cells, such as urinary podocytes with kidney podocytes, and principal cells of the kidney and urine, indicating their similarities in gene expression. CONCLUSIONS: A reference dataset for cells in human urine was generated. Single-cell transcriptomics enables detection and quantification of almost all types of cells in the kidney and urinary tract.
Assuntos
Rim/citologia , Idoso , Código de Barras de DNA Taxonômico , Feminino , Biblioteca Gênica , Humanos , Rim/metabolismo , Nefropatias/genética , Nefropatias/patologia , Nefropatias/urina , Masculino , Pessoa de Meia-Idade , Podócitos/citologia , Podócitos/metabolismo , RNA-Seq , Análise de Célula Única/métodos , Análise de Célula Única/estatística & dados numéricos , Transcriptoma , Bexiga Urinária/citologia , Bexiga Urinária/metabolismo , Urina/citologiaRESUMO
OBJECTIVES: Much of the information to date in terms of subtypes and function of bladder urothelial cells were derived from anatomical location or by the expression of a small number of marker genes. To have a comprehensive map of the cellular anatomy of bladder urothelial cells, we performed single-cell RNA sequencing to thoroughly characterize mouse bladder urothelium. MATERIALS AND METHODS: A total of 18,917 single cells from mouse bladder urothelium were analysed by unbiased single-cell RNA sequencing. The expression of the novel cell marker was confirmed by immunofluorescence using urinary tract infection models. RESULTS: Unsupervised clustering analysis identified 8 transcriptionally distinct cell subpopulations from mouse bladder urothelial cells. We discovered a novel type of bladder urothelial cells marked by Plxna4 that may be involved with host response and wound healing. We also found a group of basal-like cells labelled by ASPM that could be the progenitor cells of adult bladder urothelium. ASPM+ urothelial cells are significantly increased after injury by UPEC. In addition, specific transcription factors were found to be associated with urothelial cell differentiation. At the last, a number of interstitial cystitis/bladder pain syndrome-regulating genes were found differentially expressed among different urothelial cell subpopulations. CONCLUSIONS: Our study provides a comprehensive characterization of bladder urothelial cells, which is fundamental to understanding the biology of bladder urothelium and associated bladder disease.
Assuntos
Biomarcadores/metabolismo , Transcriptoma , Urotélio/metabolismo , Animais , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Diferenciação Celular , Linhagem da Célula , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Bexiga Urinária/citologia , Infecções Urinárias/metabolismo , Infecções Urinárias/patologia , Urotélio/citologiaRESUMO
Arsenic is a natural chemical element that is strongly associated with bladder cancer. Understanding the underlying mechanisms behind the association between arsenic and bladder cancer as well as identifying effective preventive interventions will help reduce the incidence and mortality of this disease. The epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties play key roles in cancer development and progression. Here, we reported that chronic exposure to arsenic resulted in EMT and increased levels of the CSC marker CD44 in human uroepithelial cells. Furthermore, IL-8 promoted a mesenchymal phenotype and upregulated CD44 by activating the ERK, AKT and STAT3 signaling. Phosphorylation of the human epidermal growth factor receptor 2 (HER2) was key for arsenic-induced IL-8 overexpression and depended on the simultaneous activation of the MAPK, JNK, PI3K/AKT and GSK3ß signaling pathways. We also found that genistein inhibited arsenic-induced HER2 phosphorylation and downregulated its downstream signaling pathways, thereby inhibiting progression of EMT, and reducing CD44 expression levels. These results demonstrate that the HER2/IL-8 axis is related to the acquisition of an EMT phenotype and CSCs in arsenic-treated cells. The inhibitory effects of genistein on EMT and CSCs provide a new perspective for the intervention and potential chemotherapy against arsenic-induced bladder cancer.
Assuntos
Arsênio/toxicidade , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Interleucina-8/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptor ErbB-2/genética , Bexiga Urinária/metabolismo , Arsênio/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/imunologia , Humanos , Receptores de Hialuronatos/genética , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bexiga Urinária/citologiaRESUMO
Basal and luminal subtypes of invasive bladder tumors have significant prognostic and predictive impacts for patients. However, it remains unclear whether tumor subtype commitment occurs in noninvasive urothelial lesions or in carcinoma in situ (CIS) and which gene pathways are important for bladder tumor progression. To understand the timing of this commitment, we used gene expression and protein analysis to create a global overview of 36 separate tissues excised from a whole bladder encompassing urothelium, noninvasive urothelial lesions, CIS, and invasive carcinomas. Additionally investigated were matched CIS, noninvasive urothelial lesions, and muscle-invasive bladder cancers (MIBC) from 22 patients. The final stage of subtype commitment to either a luminal or basal MIBC occurred at the CIS transition. For all tissues combined, hierarchical clustering of subtype gene expression revealed three subtypes: "luminal," "basal," and a "luminal p53-/extracellular matrix (ECM)-like" phenotype of ECM-related genes enriched in tumor-associated urothelium, noninvasive urothelial lesions, and CIS, but rarely invasive, carcinomas. A separate cohort of normal urothelium from noncancer patients showed significantly lower expression of ECM-related genes compared with tumor-associated urothelium, noninvasive urothelial lesions, and CIS. A PanCancer Progression Panel of 681 genes unveiled pathways specific for the luminal p53-/ECM-like cluster, for example, ECM remodeling, angiogenesis, epithelial-to-mesenchymal transition, cellular discohesion, cell motility involved in tumor progression, and cell proliferation and oncogenic ERBB2/ERBB3 signaling for invasive carcinomas. In conclusion, this study provides insights into bladder cancer subtype commitment and associated signaling pathways, which could help predict therapy response and enhance our understanding of therapy resistance. SIGNIFICANCE: This study demonstrates that CIS is the stage of commitment for determining MIBC tumor subtype, which is relevant for patient prognosis and therapy response.
Assuntos
Carcinoma in Situ/diagnóstico , Carcinoma de Células de Transição/diagnóstico , Matriz Extracelular/patologia , Neoplasias da Bexiga Urinária/diagnóstico , Bexiga Urinária/patologia , Urotélio/patologia , Administração Intravesical , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Vacina BCG/farmacologia , Vacina BCG/uso terapêutico , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Carcinoma in Situ/genética , Carcinoma in Situ/patologia , Carcinoma in Situ/terapia , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/patologia , Carcinoma de Células de Transição/terapia , Quimioterapia Adjuvante/métodos , Cistectomia , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Liso/patologia , Terapia Neoadjuvante/métodos , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Estadiamento de Neoplasias , Prognóstico , RNA-Seq , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Bexiga Urinária/citologia , Bexiga Urinária/cirurgia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/terapia , Urotélio/citologia , Sequenciamento Completo do GenomaRESUMO
Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments1,2. Here we create multilayer bladder 'assembloids' by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1-BMP-hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity.
Assuntos
Organoides/patologia , Organoides/fisiologia , Regeneração , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/fisiopatologia , Bexiga Urinária/patologia , Bexiga Urinária/fisiologia , Adulto , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Feminino , Ouriços/metabolismo , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Organoides/fisiopatologia , Análise de Célula Única , Células-Tronco/citologia , Células-Tronco/patologia , Células-Tronco/fisiologia , Transcriptoma , Bexiga Urinária/citologia , Infecções Urinárias/metabolismo , Infecções Urinárias/patologiaRESUMO
Henry Miller stated that "to relieve a full bladder is one of the great human joys". Urination is critically important in health and ailments of the lower urinary tract cause high pathological burden. Although there have been advances in understanding the central circuitry in the brain that facilitates urination1-3, there is a lack of in-depth mechanistic insight into the process. In addition to central control, micturition reflexes that govern urination are all initiated by peripheral mechanical stimuli such as bladder stretch and urethral flow4. The mechanotransduction molecules and cell types that function as the primary stretch and pressure detectors in the urinary tract mostly remain unknown. Here we identify expression of the mechanosensitive ion channel PIEZO2 in lower urinary tract tissues, where it is required for low-threshold bladder-stretch sensing and urethral micturition reflexes. We show that PIEZO2 acts as a sensor in both the bladder urothelium and innervating sensory neurons. Humans and mice lacking functional PIEZO2 have impaired bladder control, and humans lacking functional PIEZO2 report deficient bladder-filling sensation. This study identifies PIEZO2 as a key mechanosensor in urinary function. These findings set the foundation for future work to identify the interactions between urothelial cells and sensory neurons that control urination.
Assuntos
Canais Iônicos/metabolismo , Mecanotransdução Celular/fisiologia , Células Receptoras Sensoriais/metabolismo , Bexiga Urinária/inervação , Bexiga Urinária/fisiologia , Micção/fisiologia , Urotélio/citologia , Animais , Feminino , Humanos , Canais Iônicos/deficiência , Camundongos , Pressão , Reflexo/fisiologia , Bexiga Urinária/citologia , Bexiga Urinária/fisiopatologia , Sistema Urinário/inervação , Sistema Urinário/metabolismo , Urotélio/metabolismoRESUMO
Aster tataricus L.f. is a traditional Eastern Asian herbal medicine used for the relief of uroschesis-related illnesses and has been demonstrated clinically to exert satisfied effects. However, the mechanism of its therapeutic action remains unclear. The present study aimed to evaluate the protective mechanism of Aster tataricus extract (ATE) on CYP or LPS + ATP-induced interstitial cystitis (IC), we successfully constructed the induced IC Sprague-Dawley (SD) rat model and IC human urothelium cell (SV-HUC-1) model. The main compounds of ATE were determined by LC-MS. After intervention, the changes on the bladder wall morphology and inflammation were observed in each group. SV-HUC1 cell viability was measured by MTT and double stained with Hoechst 33342 and propidium iodide (PI). The expression levels of NLRP3, Pro-caspase-1, Caspsae-1 p20, GSDMD, GSDMD-N and Cleave-IL-1ß in vivo and in vitro in different groups were detected by Western blotting. ATE significantly alleviated oedema and haemorrhage and reduced the inflammation index and histopathological score in SD rat bladder. The results of cell revealed that ATE could improve cell viability and decrease pyroptosis ratio. The expression of NLRP3 and other pyroptosis-related protein was remarkably decreased by ATE both in vivo and in vitro. ATE may be used as an inhibitor of NLRP3 in treating IC. The discovery of NLRP3/Caspase-1/GSDMD-N as a new protective pathway provides a new direction for protecting cell against IC.