RESUMO
Telocytes (TCs) are a type of stromal cell discovered in the various organs of different animals and have many potential functions, including angiogenesis, signalling, and substance transport. However, the TCs have not been detected in the testis or epididymis of Tibetan sheep. This study investigated the position, characteristics, and distribution of TCs in the testis and epididymis of Tibetan sheep using transmission electron microscopy (TEM), toluidine blue staining, immunohistochemistry, and double immunofluorescence to elucidate their possible functions. TEM revealed that TCs were often found near basement membranes and capillaries and were characterised by large nuclei, elongated cytoplasmic protrusions, and many secretory vesicles. We also observed via toluidine staining that TCs were present near basement membrane and interstitial capillaries. Immunohistochemistry and double immunofluorescence revealed the positive expression of CD117, vimentin, platelet derived growth factor receptor α(PDGFRα), PDGFRα + CD117, and PDGFRα + vimentin in TCs. Additionally, we inferred that TCs participates in the formation of the blood-testis and blood-epididymis barriers, as well as in material transport and a stable microenvironment. This study presents the first evidence of the presence of TCs near the basement membrane and blood vessels in the testis and epididymis of Tibetan sheep. These findings provide new insights into the function of TCs in the reproductive systems of plateau animals.
Assuntos
Epididimo , Telócitos , Testículo , Animais , Masculino , Telócitos/metabolismo , Telócitos/citologia , Telócitos/ultraestrutura , Epididimo/metabolismo , Epididimo/citologia , Ovinos , Testículo/metabolismo , Testículo/citologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Microscopia Eletrônica de Transmissão , Tibet , Vimentina/metabolismo , Imuno-Histoquímica , Membrana Basal/metabolismo , Membrana Basal/ultraestruturaRESUMO
Histopathologically, oral squamous cell carcinoma (OSCC) consists of well-defined interfaces with adjacent non-cancerous epithelium. Previously, we found that SCC tissues expressed higher levels of specific proteins at this interface. Ladinin-1 (LAD1) is one of the specific molecules that has increased expressions in cancer fronts; however, its function in OSCC is unknown. Therefore, this study aimed to elucidate the function of LAD1 in human OSCC cells. LAD1 was localized on the actin arc at the distal periphery of cell clusters in the OSCC cell lines HSC-2, HSC-3, and HSC-4. When LAD1 was knocked down, cellular migration was repressed in wound scratch assays but was reversed in three-dimensional collagen gel invasion assays. Characteristic LAD1 localization along actin arcs forming the leading edge of migrating cells was diminished with loss of filopodia formation and ruffling in knockdown cells, in which the expression levels of cell motility-related genes-p21-activated kinase 1 (PAK1) and caveolin-1 (CAV1)-were upregulated and downregulated, respectively. LAD1 expression was also associated with the downregulation of vimentin and increased histological differentiation of OSCC. These results suggest that LAD1 is involved in actin dynamics during filopodia and lamellipodia formation, and in maintaining the epithelial phenotype of OSCC cells.
Assuntos
Actinas , Carcinoma de Células Escamosas , Movimento Celular , Neoplasias Bucais , Humanos , Neoplasias Bucais/patologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/genética , Linhagem Celular Tumoral , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/genética , Actinas/metabolismo , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Vimentina/metabolismo , Vimentina/genética , Caveolina 1/metabolismo , Caveolina 1/genética , Regulação Neoplásica da Expressão Gênica , Fenótipo , Células Epiteliais/metabolismo , Células Epiteliais/patologiaRESUMO
BACKGROUND: Although tumor cells undergoing epithelial-mesenchymal transition (EMT) typically exhibit spindle morphology in experimental models, such histomorphological evidence of EMT has predominantly been observed in rare primary spindle carcinomas. The characteristics and transcriptional regulators of spontaneous EMT in genetically unperturbed non-spindled carcinomas remain underexplored. METHODS: We used primary culture combined with RNA sequencing (RNA-seq), single-cell RNA-seq (scRNA-seq), and in situ RNA-seq to explore the characteristics and transcription factors (TFs) associated with potential spontaneous EMT in non-spindled breast carcinoma. RESULTS: Our primary culture revealed carcinoma cells expressing diverse epithelial-mesenchymal traits, consistent with epithelial-mesenchymal plasticity. Importantly, carcinoma cells undergoing spontaneous EMT did not necessarily exhibit spindle morphology, even when undergoing complete EMT. EMT was a favored process, whereas mesenchymal-epithelial transition appeared to be crucial for secondary tumor growth. Through scRNA-seq, we identified TFs that were sequentially and significantly upregulated as carcinoma cells progressed through the EMT process, which correlated with increasing VIM expression. Once upregulated, the TFs remained active throughout the EMT process. ZEB1 was a key initiator and sustainer of EMT, as indicated by its earliest significant upregulation in the EMT process, its exact correlation with VIM expression, and the reversal of EMT and downregulation of EMT-upregulated TFs upon ZEB1 knockdown. The correlation between ZEB1 and vimentin expression in triple-negative breast cancer and metaplastic breast carcinoma tumor cohorts further highlighted its role. The immediate upregulation of ZEB2 following that of ZEB1, along with the observation that the knockdown of ZEB1 or ZEB2 downregulates both ZEB1 and ZEB2 concomitant with the reversal of EMT, suggests their functional cooperation in EMT. This finding, together with that of a lack of correlation of SNAI1, SNAI2, and TWIST1 expression with the mesenchymal phenotype, indicated EMT-TFs have a context-dependent role in EMT. Upregulation of EMT-related gene signatures during EMT correlated with poor patient outcomes, highlighting the biological importance of the model. Elevated EMT gene signatures and increased ZEB1 and ZEB2 expression in vimentin-positive compared to vimentin-negative carcinoma cells within the corresponding primary tumor tissue confirmed ZEB1 and ZEB2 as intrinsic, instead of microenvironmentally-induced, EMT regulators, and vimentin as an in vivo indicator of EMT. CONCLUSIONS: Our findings provide insights into the characteristics and transcriptional regulators of spontaneous EMT in primary non-spindled carcinoma.
Assuntos
Neoplasias da Mama , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição , Transição Epitelial-Mesenquimal/genética , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vimentina/metabolismo , Vimentina/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Linhagem Celular Tumoral , Animais , Camundongos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismoRESUMO
This study aims to investigate the mechanism of Xueshuantong Injection(XST) on pulmonary fibrosis induced by bleomycin(BLM) in rats based on the coagulation cascade pathway. Sixty SD rats were randomly divided into sham surgery group,model group, pirfenidone(PFD, 50 mg·kg~(-1)) group, and 27, 54, and 81 mg·kg~(-1) XST groups. The rat model of pulmonary fibrosis was established by intratracheal injection of BLM(5 mg·kg~(-1)). After 24 hours, the administration groups were given corresponding drugs, while the sham surgery group and model group were given equal volumes of saline. On the 28th day, samples were collected,and the imaging and collagen fiber changes in the lungs of rats were observed. Immunofluorescence(IF) method was used to detect the expression level of alpha-smooth muscle actin(α-SMA), collagen â (Col-â ), E-cadherin(E-cad), and vimentin(Vim). Western blot was used to determine the protein expression of α-SMA, Col-â , Vim, and E-cad. Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of prothrombin fragment(F1 + 2), thrombin-antithrombin complex(TAT), soluble fibrin monomer complex(SFMC), and rat fibrinogen degradation products(FDP) in rat lung tissue. Finally, the mRNA and protein levels of protease activated receptor 1(PAR-1) were detected by RT-qPCR, western blot, and IF. Compared with the model group, the scanning of the lungs of rats receiving XST treatment also exhibited patchy and non-homogeneous shadows, but these shadows were less dense than those in the model group. At the same time, there was a significant decrease in Col-â fibers in the lungs of rats, and XST could inhibit epithelial-mesenchymal transition(EMT) and downregulate α-SMA and Col-â protein expression. In the aspect of the coagulation system, administration of 81 mg·kg~(-1) XST significantly reduced the levels of SFMC and FDP. Meanwhile, 81 mg·kg~(-1) XST significantly downregulated the mRNA and protein levels of PAR-1. XST has an anti-pulmonary fibrosis effect in rats, and its mechanism may be related to the downregulation of PAR-1 to rebalance the coagulation cascade pathway.
Assuntos
Bleomicina , Medicamentos de Ervas Chinesas , Fibrose Pulmonar , Ratos Sprague-Dawley , Animais , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/genética , Bleomicina/efeitos adversos , Ratos , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/farmacologia , Masculino , Coagulação Sanguínea/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Humanos , Actinas/metabolismo , Actinas/genética , Caderinas/genética , Caderinas/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Vimentina/metabolismo , Vimentina/genética , InjeçõesRESUMO
BACKGROUND: Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), can undergo erythroid differentiation, offering a potentially invaluable resource for generating large quantities of erythroid cells. However, the majority of erythrocytes derived from hPSCs fail to enucleate compared with those derived from cord blood progenitors, with an unknown molecular basis for this difference. The expression of vimentin (VIM) is retained in erythroid cells differentiated from hPSCs but is absent in mature erythrocytes. Further exploration is required to ascertain whether VIM plays a critical role in enucleation and to elucidate the underlying mechanisms. METHODS: In this study, we established a hESC line with reversible vimentin degradation (dTAG-VIM-H9) using the proteolysis-targeting chimera (PROTAC) platform. Various time-course studies, including erythropoiesis from CD34+ human umbilical cord blood and three-dimensional (3D) organoid culture from hESCs, morphological analysis, quantitative real-time PCR (qRT-PCR), western blotting, flow cytometry, karyotyping, cytospin, Benzidine-Giemsa staining, immunofluorescence assay, and high-speed cell imaging analysis, were conducted to examine and compare the characteristics of hESCs and those with vimentin degradation, as well as their differentiated erythroid cells. RESULTS: Vimentin expression diminished during normal erythropoiesis in CD34+ cord blood cells, whereas it persisted in erythroid cells differentiated from hESC. Depletion of vimentin using the degradation tag (dTAG) system promotes erythroid enucleation in dTAG-VIM-H9 cells. Nuclear polarization of erythroblasts is elevated by elimination of vimentin. CONCLUSIONS: VIM disappear during the normal maturation of erythroid cells, whereas they are retained in erythroid cells differentiated from hPSCs. We found that retention of vimentin during erythropoiesis impairs erythroid enucleation from hPSCs. Using the PROTAC platform, we validated that vimentin degradation by dTAG accelerates the enucleation rate in dTAG-VIM-H9 cells by enhancing nuclear polarization.
Assuntos
Diferenciação Celular , Células Eritroides , Proteólise , Vimentina , Vimentina/metabolismo , Vimentina/genética , Humanos , Diferenciação Celular/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Células Eritroides/metabolismo , Células Eritroides/citologia , Células Eritroides/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Linhagem CelularRESUMO
BACKGROUND/AIM: Elevated blood fibronectin (FN) levels have been observed in various cancers; however, their significance remains controversial. Herein, we measured the levels of sialyl-fibronectin (S-FN), a type of FN secreted by tumor cells, and investigated whether blood S-FN secretion is associated with recurrent metastasis and epithelial-mesenchymal transition (EMT). PATIENTS AND METHODS: An ELISA system recognizing S-FN was constructed, and the amount of S-FN in blood samples from 63 patients with thyroid carcinoma was measured. The relationship between S-FN secretion and clinical prognosis was also examined. Vimentin immunostaining was performed to identify the mesenchymal status of the cells during EMT. RESULTS: After 12 years of observation, 17/63 patients had recurrent metastases, including nine cases of lymph node recurrence (LNR) and eight cases of remote metastasis (RM). LNR occurred in 7/39 (17.9%) of S-FN-negative cases, where 4/7 (57.1%) had two or more repeat recurrences. In S-FN-positive cases, LNR was observed in 2/24 cases (8.3%), and no repeat recurrence was observed. For RM, 6/39 (15.4%) patients were S-FN-negative, of which 5/6 (83.3%) had progressive disease even during treatment at metastasis. Of the S-FN-positive cases, RM was observed in 2/24 (8.3%) patients; progressive disease was observed in 1/2 (50.0%) patients. In 9/11 S-FN-negative recurrent metastasis cases (81.8%) and 2/4 S-FN-positive cases (50.0%), many vimentin-positive, FN-secreting cells were found in the interstitial tissue around the tumor. CONCLUSION: S-FN-negative thyroid cancer has a poor prognosis because of the progression of EMT associated with increased paracrine FN levels in the stroma.
Assuntos
Transição Epitelial-Mesenquimal , Fibronectinas , Neoplasias da Glândula Tireoide , Humanos , Fibronectinas/metabolismo , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Feminino , Masculino , Prognóstico , Pessoa de Meia-Idade , Idoso , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/metabolismo , Adulto , Progressão da Doença , Metástase Linfática , Vimentina/metabolismo , Biomarcadores Tumorais/metabolismoRESUMO
Purpose: To investigate the inhibitory effect of antimicrobial peptide merecidin on triple-negative breast cancer (TNBC) and the mechanism of inhibiting epithelial-mesenchymal transformation (EMT) by regulating miR-30d-5p/vimentin. Methods: TNBC cell lines (MDA-MB-231, MDA-MB-468) were treated with merecidin to assess proliferation, migration, invasion ability, and EMT. Confocal laser localization was used to examine the role of merecidin and TNBC cells. The relationship between merecidin and miR-30d-5p was determined through RT-qPCR and dual-luciferase reporter gene, and the relationship between merecidin and vimentin was verified through pulling down the experiment. The effects of miR-30d-5p on the migration and invasion ability of TNBC cells were confirmed through scratch and transwell experiments. Vimentin levels, tumor volume, shape, size, and weight were observed in the MDA-MB-231 subcutaneous tumor model in nude mice. Results: merecidin inhibited the proliferation, migration, invasion, and EMT of TNBC cells. merecidin was primarily located in the cytoplasm of TNBC cells, and the expression of miR-30d-5p was low in TNBC cells. merecidin significantly up-regulated the expression of miR-30d-5p. miR-30d-5p negatively regulated vimentin. merecidin could bind to vimentin in vitro. miR-30d-5p inhibited the migration and invasion ability of TNBC cells, while vimentin promoted their migration and invasion ability. Down-regulation of miR-30d-5p or overexpression of vimentin partially counteracted the inhibitory effects of merecidin on TNBC cell migration, invasion ability, and EMT. In nude mouse tumor models, merecidin significantly suppressed tumor growth. Conclusion: Merecidin effectively blocks the EMT process and inhibits the migration and invasion of TNBC cells by regulating miR-30d-5p/vimentin.
Assuntos
Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , MicroRNAs , Neoplasias de Mama Triplo Negativas , Vimentina , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , MicroRNAs/genética , Animais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Vimentina/metabolismo , Camundongos , Feminino , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Modelos Animais de Doenças , Metástase Neoplásica , Peptídeos Catiônicos Antimicrobianos/farmacologiaRESUMO
Fibronectin (FN) is an essential component of the extracellular matrix (ECM) that protects the integrity of the microvascular endothelial barrier (MEB). However, Treponema pallidum subsp. pallidum (Tp) breaches this barrier through elusive mechanisms and rapidly disseminates throughout the host. We aimed to understand the impact of Tp on the surrounding FN matrix of MEB and the underlying mechanisms of this effect. In this study, immunofluorescence assays (IF) were conducted to assess the integrity of the FN matrix surrounding human microvascular endothelial cell-1 (HMEC-1) with/without Tp co-culture, revealing that only live Tp exhibited the capability to mediate FN matrix disaggregation in HMEC-1. Western blotting and IF were employed to determine the protein levels associated with the FN matrix during Tp infection, which showed the unaltered protein levels of total FN and its receptor integrin α5ß1, along with reduced insoluble FN and increased soluble FN. Simultaneously, the integrin α5ß1-binding protein-intracellular vimentin maintained a stable total protein level while exhibiting an increase in the soluble form, specifically mediated by the phosphorylation of its 39th residue (pSer39-vimentin). Besides, this process of vimentin phosphorylation, which could be hindered by a serine-to-alanine mutation or inhibition of phosphorylated-AKT1 (pAKT1), promoted intracellular vimentin rearrangement and FN matrix disaggregation. Moreover, within the introduction of additional cellular FN rather than other Tp-adhered ECM protein, in vitro endothelial barrier traversal experiment and in vivo syphilitic infectivity test demonstrated that viable Tp was effectively prevented from penetrating the in vitro MEB or disseminating in Tp-challenged rabbits. This investigation revealed the active pAKT1/pSer39-vimentin signal triggered by live Tp to expedite the disaggregation of the FN matrix and highlighted the importance of FN matrix stability in syphilis, thereby providing a novel perspective on ECM disruption mechanisms that facilitate Tp dissemination across the MEB.
Assuntos
Células Endoteliais , Fibronectinas , Treponema pallidum , Vimentina , Fibronectinas/metabolismo , Humanos , Vimentina/metabolismo , Treponema pallidum/metabolismo , Animais , Fosforilação , Células Endoteliais/metabolismo , Células Endoteliais/microbiologia , Matriz Extracelular/metabolismo , Sífilis/metabolismo , Sífilis/microbiologia , Coelhos , Endotélio Vascular/metabolismo , Endotélio Vascular/microbiologiaRESUMO
OBJECTIVE: Sox2 plays crucial roles in tissues homeostasis and regeneration. However, there are lack of a comprehensive examination of Sox2 expression and its functional role in submandibular gland regeneration. Therefore, we aimed to elucidate the impact of Sox2 on submandibular gland regeneration. MATERIALS AND METHODS: A Sprague-Dawley rat submandibular gland duct ligation/de-ligation regeneration model was conducted in this study. Sox2-shRNA vectors were retro-ductally administered into the submandibular gland to establish a stable Sox2 knockdown model. Conventional histopathological and molecular biological methods were used to investigate phenotypic changes. RESULTS: The submandibular gland normalized completely 28 days after ligature removal (following 7 days of duct ligation). AQP5 expression gradually increased after ligation removal until returning to normal levels. In submandibular gland regeneration, Sox2 re-expressed and co-expressed with AQP5+ acinar cells, and Sox2 expression peaked on day 14, recovered to normal on day 28, reproducing the developmental pattern. Sox2 knockdown hindered gland regeneration and induced irreversible fibrosis. The AQP5 expression was significantly lower than the contemporaneous solely ligated group, while the blue collagen deposition and the Vimentin expression increased prominently. The expression of CD68, IL-1ß, TNF-α and IL-17A increased significantly, and epithelial cells in the Sox2 knockdown group expressed higher levels of IL-17A. CONCLUSIONS: These findings highlight Sox2 as a crucial regulator of the acinar cell lineage. Sox2+ progenitor cells are pivotal for acinar cell maintenance, which is indispensable for submandibular gland regeneration. Collectively, our findings may help develop targeted interventions for enhancing tissue repair and preventing irreversible fibrosis in salivary gland disorders.
Assuntos
Aquaporina 5 , Ratos Sprague-Dawley , Regeneração , Fatores de Transcrição SOXB1 , Células-Tronco , Glândula Submandibular , Animais , Glândula Submandibular/metabolismo , Ratos , Regeneração/fisiologia , Aquaporina 5/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/metabolismo , Masculino , Ligadura , Células Acinares/metabolismo , Vimentina/metabolismo , RNA Interferente Pequeno , Molécula CD68RESUMO
Several cells and molecules in the tumor microenvironment have been introduced as effective factors in the prognosis and progression of colorectal cancer. As a key element of the intermediate filament family, vimentin is expressed by mesenchymal cells in a ubiquitous manner and contributes significantly to cellular integrity and stress resistance in colorectal cancer. Recent studies have shown that alterations in the expression patterns of intermediate filaments are significantly related to cancer progression, especially in phenotypes associated with cellular migration and invasion. In addition to its multiple biological roles, vimentin also has a substantial function in mediating the epithelial-mesenchymal transition. Therefore, evaluating vimentin as an effective factor involved in the prognosis of colorectal cancer and targeting it as a novel approach to cancer therapy have become one of the main goals of many researchers worldwide. In this article, we will review the various biological functions of vimentin, as well as its relationship with colorectal cancer with the aim of providing novel insights into its clinical importance in the prognosis and treatment of colorectal cancer.
Assuntos
Neoplasias Colorretais , Transição Epitelial-Mesenquimal , Vimentina , Humanos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/terapia , Neoplasias Colorretais/genética , Neoplasias Colorretais/tratamento farmacológico , Vimentina/metabolismo , Prognóstico , Microambiente Tumoral , Biomarcadores Tumorais/metabolismo , Movimento Celular , Regulação Neoplásica da Expressão GênicaRESUMO
We report on the synthesis of two fluorescent probes which can be activated by ß-Galactosidase (ß-Gal) enzymes and/or light. The probes contained 2-nitro-4-oxybenzyl and 3-nitro-4-oxybenzyl fragments, with ß-Gal residues linked to C-4. We performed the enzymatic and photoactivation of the probes in a cuvette and compared them, prior to the labeling of Vimentin-Halo fusion protein in live cells with overexpressed ß-galactosidase. The dye fluorescence afforded the observation of enzyme activity by means of confocal and super-resolution optical microscopy based on stimulated emission depletion (STED). The tracing of enzymatic activity with the retention of activated fluorescent products inside cells was combined with super-resolution imaging as a tool for use in biomedicine and life science.
Assuntos
Corantes Fluorescentes , beta-Galactosidase , beta-Galactosidase/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Humanos , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Microscopia Confocal , Vimentina/metabolismoRESUMO
The intermediate filament vimentin is present in immune cells and is implicated in proinflammatory immune responses. Whether and how it supports antimicrobial activities of neutrophils are not well established. Here, we developed an immortalized neutrophil model to examine the requirement of vimentin. We demonstrate that vimentin restricts the production of proinflammatory cytokines and reactive oxygen species (ROS), but enhances phagocytosis and swarming. We observe that vimentin is dispensable for neutrophil extracellular trap (NET) formation, degranulation, and inflammasome activation. Moreover, gene expression analysis demonstrated that the presence of vimentin was associated with changes in expression of multiple genes required for mitochondrial function and ROS overproduction. Treatment of wild-type cells with rotenone, an inhibitor for complex I of the electron transport chain, increases the ROS levels. Likewise, treatment with mitoTEMPO, a SOD mimetic, rescues the ROS production in cells lacking vimentin. Together, these data show vimentin regulates neutrophil antimicrobial functions and alters ROS levels through regulation of mitochondrial activity.
Assuntos
Mitocôndrias , Neutrófilos , Espécies Reativas de Oxigênio , Vimentina , Espécies Reativas de Oxigênio/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Vimentina/metabolismo , Mitocôndrias/metabolismo , Animais , Camundongos , Inflamação/imunologia , Inflamação/metabolismo , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Fagocitose , Inflamassomos/metabolismo , Inflamassomos/imunologia , Citocinas/metabolismo , Humanos , Rotenona/farmacologiaAssuntos
Neoplasias do Endométrio , Músculo Liso , Sarcoma do Estroma Endometrial , Humanos , Feminino , Neoplasias do Endométrio/patologia , Sarcoma do Estroma Endometrial/patologia , Sarcoma do Estroma Endometrial/diagnóstico , Diagnóstico Diferencial , Músculo Liso/patologia , Diferenciação Celular , Vimentina/metabolismo , Adulto , Leiomioma/patologia , Tumores do Estroma Gonadal e dos Cordões Sexuais/patologia , Tumores do Estroma Gonadal e dos Cordões Sexuais/diagnóstico , Tumores do Estroma Endometrial/patologia , Tumores do Estroma Endometrial/diagnóstico , Actinas/metabolismoRESUMO
Background: Telocytes are interstitial stromal cells identified in various human organs, including the kidney. Their presence and role in human diabetic kidney disease remain unknown. Methods: To identify and localize telocytes in glomerular and tubule-interstitial compartments, both normal and diabetic human renal tissues were examined using immunohistochemistry, immunofluorescence, and transmission electron microscopy. Results: Renal telocytes are elongated interstitial cells with long, thin telopodes, showing alternating thin and thick segments. They expressed CD34, Nestin, α-SMA, and Vimentin markers. Occasionally, c-Kit expression was observed in some rounded and spindle cells, while no positivity was detected for PDGFR-ß and NG2. Telocytes were identified around Bowman's capsule, tubules, and peritubular capillaries in both normal and diabetic conditions. In diabetic renal samples, there was a significant increase in α-SMA expressing telocytes, leading to periglomerular fibrosis. These telocytes also exhibited a synthetic phenotype with proteoglycan deposition in the extracellular matrix and, in some cases, showed pre-adipocytic differentiation. Conclusions: Telocytes were identified in normal and diabetic human kidneys. These cells form an elastic mechanical scaffold in the interstitium and are present in all renal cortical compartments. In diabetic samples, their increased α-SMA expression and synthetic phenotype suggest their potential role in the pathogenesis of diabetic nephropathy.
Assuntos
Antígenos CD34 , Nefropatias Diabéticas , Telócitos , Vimentina , Humanos , Telócitos/metabolismo , Telócitos/patologia , Telócitos/ultraestrutura , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/metabolismo , Antígenos CD34/metabolismo , Vimentina/metabolismo , Rim/metabolismo , Rim/patologia , Imuno-Histoquímica , Actinas/metabolismo , Nestina/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Masculino , Pessoa de Meia-Idade , Feminino , Microscopia Eletrônica de Transmissão , IdosoRESUMO
In goldfish, spinal cord injury triggers the formation of a fibrous scar at the injury site. Regenerating axons are able to penetrate the scar tissue, resulting in the recovery of motor function. Previous findings suggested that regenerating axons enter the scar through tubular structures surrounded by glial elements with laminin-positive basement membranes and that glial processes expressing glial fibrillary acidic protein (GFAP) are associated with axonal regeneration. How glia contribute to promoting axonal regeneration, however, is unknown. Here, we revealed that glial processes expressing vimentin or brain lipid-binding protein (BLBP) also enter the fibrous scar after spinal cord injury in goldfish. Vimentin-positive glial processes were more numerous than GFAP- or BLBP-positive glial processes in the scar tissue. Regenerating axons in the scar tissue were more closely associated with vimentin-positive glial processes than GFAP-positive glial processes. Vimentin-positive glial processes co-expressed matrix metalloproteinase (MMP)-14. Our findings suggest that vimentin-positive glial processes closely associate with regenerating axons through tubular structures entering the scar after spinal cord injury in goldfish. In intact spinal cord, ependymo-radial glial cell bodies express BLBP and their radial processes express vimentin, suggesting that vimentin-positive glial processes derive from migrating ependymo-radial glial cells. MMP-14 expressed in vimentin-positive glial cells and their processes might provide a beneficial environment for axonal regeneration.
Assuntos
Axônios , Carpa Dourada , Regeneração Nervosa , Neuroglia , Traumatismos da Medula Espinal , Vimentina , Animais , Carpa Dourada/metabolismo , Vimentina/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Neuroglia/metabolismo , Axônios/metabolismo , Regeneração Nervosa/fisiologia , Metaloproteinase 14 da Matriz/metabolismo , Cicatriz/metabolismo , Cicatriz/patologia , Proteínas de Ligação a Ácido Graxo/metabolismoRESUMO
Metastasis is responsible for the majority of cancer-related fatalities. We previously identified specific cancer cell populations responsible for metastatic events which are cytokeratin-14 (CK14) and E-cadherin positive in luminal tumors, and E-cadherin and vimentin positive in triple-negative tumors. Since cancer cells evolve within a complex ecosystem comprised of immune cells and stromal cells, we sought to decipher the spatial interactions of these aggressive cancer cell populations within the tumor microenvironment (TME). We used imaging mass cytometry to detect 36 proteins in tumor microarrays containing paired primary and metastatic lesions from luminal or triple-negative breast cancers (TNBC), resulting in a dataset of 1,477,337 annotated cells. Focusing on metastasis-initiating cell populations, we observed close proximity to specific fibroblast and macrophage subtypes, a relationship maintained between primary and metastatic tumors. Notably, high CK14 in luminal cancer cells and high vimentin in TNBC cells correlated with close proximity to specific macrophage subtypes (CD163intCD206intPDL1intHLA-DR+ or PDL1highARG1high). Our in-depth spatial analysis demonstrates that metastasis-initiating cancer cells consistently colocalizes with distinct cell populations within the TME, suggesting a role for these cell-cell interactions in promoting metastasis.
Assuntos
Macrófagos , Neoplasias de Mama Triplo Negativas , Microambiente Tumoral , Humanos , Feminino , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Macrófagos/patologia , Macrófagos/metabolismo , Macrófagos/imunologia , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/metabolismo , Metástase Neoplásica , Linhagem Celular Tumoral , Vimentina/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismoRESUMO
The fatal gouty disease caused by goose astrovirus genotype 2 (GAstV-2) still seriously endangers the goose industry in China, causing great economic losses. However, research on its infection mechanism has progressed relatively slowly. VP70 is the structural protein of GAstV-2 and is closely related to virus invasion and replication. To better understand the role of VP70 during GAstV-2 infection, we used immunoprecipitation and mass spectrometry to identify host proteins that interact with VP70. Here, we report that cellular vimentin (VIM) is a host binding partner of VP70. Site-directed mutagenesis showed that amino acid residues 399 to 413 of VP70 interacted with VIM. Using reverse genetics, we found that VP70 mutation disrupts the interaction of VP70 with VIM, which is essential for viral replication. Overexpression of VIM significantly promoted GAstV-2 replication, while knockdown of VIM significantly inhibited GAstV-2 replication. Laser confocal microscopy showed that VP70 protein expression induced the rearrangement of VIM, gradually aggregating from the original uniform grid to the side of the nucleus, and aggregated the originally dispersed GAstV-2 RNA in VIM. This rearrangement was associated with increased VIM phosphorylation caused by GAstV-2. Meanwhile, blocking VIM rearrangement with acrylamide substantially inhibited viral replication. These results indicate that VIM interacts with VP70 and positively regulates GAstV-2 replication, and VIM-VP70 interaction and an intact VIM network are needed for GAstV-2 replication. This study provides a theoretical basis and novel perspective for the further characterization of the pathogenic mechanism of GAstV-2-induced gouty disease in goslings.
Assuntos
Avastrovirus , Gansos , Doenças das Aves Domésticas , Vimentina , Replicação Viral , Animais , Gansos/virologia , Doenças das Aves Domésticas/virologia , Vimentina/metabolismo , Vimentina/genética , Avastrovirus/genética , Avastrovirus/fisiologia , Avastrovirus/metabolismo , Infecções por Astroviridae/veterinária , Infecções por Astroviridae/virologia , Genótipo , Proteínas Aviárias/metabolismo , Proteínas Aviárias/genéticaRESUMO
There is increasing evidence that the secretory/excretory antigens of the larval stage of Echinococcus granulosus can induce both anticancer and oncogenic effects between parasite-derived metabolites and various cancer cells. The dual role of miR-145 as either a tumor suppressor or oncogene has already been reported in cancer. However, the mechanism by which miR-145 induces apoptosis in lung cancer cells treated with hydatid cyst fluid (HCF) remains unclear. The fertile HCF was obtained from sheep, purified and lyophilized. H1299 human lung cancer cells were then cultured into two groups: HCF-treated H1299 lung cancer cells and untreated H1299 cancer cells as control cells. Cell viability was assessed using MTT assay to evaluate the effects of HCF on the H1299 cells. Caspase-3 activity was assessed by fluorometric assay. In addition, mRNA expression levels of VGEF, vimentin, caspase-3, miRNA-145, Bax and Bcl-2 genes were quantified by real-time PCR. A scratch test was also performed to assess the effects of HCF on cell migration. The MTT assay revealed that the growth of H1299 cells increased when treated with 60 µg/mL of fertile HCF for 24 h. The fold change of caspase-3, miRNA-145, Bax/Bcl-2 ratio and caspase-3 activity was lower in HCF-treated H1299 cells compared to the control cell. The fold change in VGEF and vimentin gene expression was higher in the HCF-treated H1299 cells than in the control cell. The scratch test results showed that H1299 cell mobility increased 24 and 48 h after exposure to HCF. Our results suggest that the downregulation of miR-145 in HCF-treated H1299 cells may play a role as a possible oncogenic regulator of lung cancer growth. To confirm this assumption, further studies are required to evaluate the microRNA profile and effective oncogenes in vivo.
Assuntos
Apoptose , Caspase 3 , Echinococcus granulosus , Neoplasias Pulmonares , MicroRNAs , Animais , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Echinococcus granulosus/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/parasitologia , Ovinos , Caspase 3/metabolismo , Caspase 3/genética , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Vimentina/metabolismo , Vimentina/genética , Equinococose/parasitologia , Líquido Cístico/química , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , RNA Mensageiro/metabolismoRESUMO
Photodynamic therapy (PDT) has significant advantages in the treatment of malignant lung tumors. The research on the mechanism of PDT mediated by hematoporphyrin derivatives (HPD) and its cytotoxic effects on lung cancer cells has primarily focused on lung adenocarcinoma cells. However, the impact of HPD-PDT on lung squamous cell carcinoma has not been thoroughly studied. This study aimed to investigate the effects of 630 nm laser on apoptosis, metastasis, invasion, and epithelial-mesenchymal transition (EMT) in human lung squamous cell carcinoma H520 cells mediated by HPD. H520 cells were divided into four groups: control group, photosensitizer group, irradiation group, and HPD-PDT group. Cell proliferation was assessed using CCK8 assay; cell apoptosis was detected by Hoechst 33258 staining and flow cytometry; cell migration and invasion abilities were evaluated using wound-healing and invasion assays; and protein and mRNA expressions were analyzed by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) respectively. Results showed that HPD-PDT significantly inhibited cell proliferation, promoted apoptosis (P < 0.05), suppressed cell migration and invasion (P < 0.05), decreased Bcl-2 mRNA expression, and increased Bax and Caspase-9 mRNA expression(P < 0.05). Western blotting analysis indicated increased expression of Bax, Caspase-9, and E-cadherin, and decreased expression of Bcl-2, N-cadherin, and Vimentin (P < 0.05). In conclusion, 630 nm laser mediated by HPD promoted cell apoptosis via upregulation of Bax and caspase-9, and downregulation of Bcl-2, and inhibited cell migration and invasion by regulating EMT in H520 cells.
Assuntos
Apoptose , Carcinoma de Células Escamosas , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares , Invasividade Neoplásica , Fotoquimioterapia , Humanos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos da radiação , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/tratamento farmacológico , Linhagem Celular Tumoral , Fotoquimioterapia/métodos , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/terapia , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Derivado da Hematoporfirina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Caderinas/metabolismo , Vimentina/metabolismo , Caspase 9/metabolismo , Caspase 9/genéticaRESUMO
PURPOSE: The abnormal growth factors-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells was known as a vital pathogenesis of proliferative vitreoretinopathy (PVR). This study aims to explore how survivin inhibition affects EMT induced by epidermal growth factor (EGF) in RPE cells. METHODS: Human primary RPE cells were identified in vitro. EMT in RPE cells was induced by EGF. Inhibition of survivin in RPE cells was accomplished through the use of a survivin inhibitor (YM155) and survivin siRNA. The viability, proliferation and migration of RPE cells was detected by methylthiazol tetrazolium assay, bromodeoxyuridine labeling assay, and wound healing assay, respectively. The EGF receptor /mitogen-activated protein kinase (EGFR/MAPK) proteins and EMT-related proteins were measured by western blot and immunofluorescence assay. RESULTS: EGF induced significant EMT in RPE cells, activated the phosphorylation of EGFR/MAPK signaling proteins, and caused changes to EMT-related proteins. YM155 suppressed RPE cells' viability, proliferation, and migration; induced the phosphorylation of EGFR, JNK, and P38MAPK; and down regulated EGFR and phosphorylated ERK. YM155 also increased expression of E-cadherin and ZO-1 proteins and reduced expression of N-cadherin, Vimentin, and α-SMA proteins. The EGF-induced increase of RPE cell proliferation and migration was constrained by survivin inhibition. Moreover, survivin inhibition in RPE cells suppressed the EGF-caused phosphorylation of EGFR/MAPK proteins and attenuated the EGF-induced reduction of E-cadherin and ZO-1 proteins and increase of N-cadherin, Vimentin, and α-SMA proteins. CONCLUSIONS: Survivin inhibition attenuates EGF-induced EMT of RPE cells by affecting the EGFR/MAPK signaling pathway. Survivin might be a promising target for preventing PVR.