RESUMO
This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-ß7, LAG3, TGFß/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-ß7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-ß7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.
Assuntos
Doença Enxerto-Hospedeiro , Cadeias beta de Integrinas , Neuropilina-1 , Linfócitos T Reguladores , Animais , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/metabolismo , Linfócitos T Reguladores/imunologia , Camundongos , Neuropilina-1/metabolismo , Neuropilina-1/genética , Cadeias beta de Integrinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Condicionamento Pré-Transplante/métodos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Camundongos Endogâmicos C57BL , Gastroenteropatias/imunologia , Camundongos Endogâmicos BALB C , Receptores OX40/metabolismo , Doença Aguda , Transplante de Células-Tronco Hematopoéticas , Feminino , Ligante OX40RESUMO
Extracellular vesicles (EVs) constitute a vital component of intercellular communication, exerting significant influence on metastasis formation and drug resistance mechanisms. Malignant melanoma (MM) is one of the deadliest forms of skin cancers, because of its high metastatic potential and often acquired resistance to oncotherapies. The prevalence of BRAF mutations in MM underscores the importance of BRAF-targeted therapies, such as vemurafenib and dabrafenib, alone or in combination with the MEK inhibitor, trametinib. This study aimed to elucidate the involvement of EVs in MM progression and ascertain whether EV-mediated metastasis promotion persists during single agent BRAF (vemurafenib, dabrafenib), or MEK (trametinib) and combined BRAF/MEK (dabrafenib/trametinib) inhibition.Using five pairs of syngeneic melanoma cell lines, we assessed the impact of EVs - isolated from their respective supernatants - on melanoma cell proliferation and migration. Cell viability and spheroid growth assays were employed to evaluate proliferation, while migration was analyzed through mean squared displacement (MSD) and total traveled distance (TTD) measurements derived from video microscopy and single-cell tracking.Our results indicate that while EV treatments had remarkable promoting effect on cell migration, they exerted only a modest effect on cell proliferation and spheroid growth. Notably, EVs demonstrated the ability to mitigate the inhibitory effects of BRAF inhibitors, albeit they were ineffective against a MEK inhibitor and the combination of BRAF/MEK inhibitors. In summary, our findings contribute to the understanding of the intricate role played by EVs in tumor progression, metastasis, and drug resistance in MM.
Assuntos
Movimento Celular , Vesículas Extracelulares , Melanoma , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas B-raf , Melanoma/patologia , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Humanos , Movimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Proliferação de Células/efeitos dos fármacos , Vemurafenib/farmacologia , Pirimidinonas/farmacologia , Piridonas/farmacologia , Piridonas/uso terapêutico , Imidazóis/farmacologia , Oximas/farmacologiaRESUMO
BACKGROUND: End-stage heart failure (ESHF) leads to hypoperfusion and edema formation throughout the body and is accompanied by neurohormonal and immunological alterations. Orthotopic heart transplantation (HTX) has been used as a beneficial option for ESHF. Due to the shortage of donor hearts, the ideal matching and timing of donors and recipients has become more important. PURPOSE: In this study, our aim was to explore the relationship between the clinical outcomes of HTX and the cytokine and apolipoprotein profiles of the recipient pericardial fluid obtained at heart transplantation after opening the pericardial sac. MATERIALS AND METHODS: The clinical data and the interleukin, adipokine, and lipoprotein levels in the pericardial fluid of twenty HTX recipients were investigated. Outcome variables included primer graft dysfunction (PGD), the need for post-transplantation mechanical cardiac support (MCS), International Society for Heart and Lung Transplantation grade ≥2R rejection, and mortality. Recipient risk scores were also investigated. RESULTS: Leptin levels were significantly lower in patients with PGD than in those without PGD (median: 6.36 (IQR: 5.55-6.62) versus 7.54 (IQR = 6.71-10.44); p = 0.029). Higher ApoCII levels (median: 14.91 (IQR: 11.55-21.30) versus 10.31 (IQR = 10.02-13.07); p = 0.042) and ApoCIII levels (median: 60.32 (IQR: 43.00-81.66) versus 22.84 (IQR = 15.84-33.39); p = 0.005) were found in patients (n = 5) who died in the first 5 years after HTX. In patients who exhibited rejection (n = 4) in the first month after transplantation, the levels of adiponectin (median: 74.48 (IQR: 35.51-131.70) versus 29.96 (IQR: 19.86-42.28); p = 0.039), ApoCII (median: 20.11 (IQR: 13.06-23.54) versus 10.32 (IQR: 10.02-12.84); p = 0.007), and ApoCIII (median: 70.97 (IQR: 34.72-82.22) versus 26.33 (IQR: 17.18-40.17); p = 0.029) were higher than in the nonrejection group. Moreover, the pericardial thyroxine (T4) levels (median: 3.96 (IQR: 3.49-4.46) versus 4.69 (IQR: 4.23-5.77); p = 0.022) were lower in patients with rejection than in patients who did not develop rejection. CONCLUSION: Our results indicate that apolipoproteins can facilitate the monitoring of rejection and could be a useful tool in the forecasting of early and late complications.
Assuntos
Transplante de Coração , Transplante de Pulmão , Humanos , Transplante de Coração/efeitos adversos , Transplante de Coração/métodos , Doadores de Tecidos , Fatores de Risco , Apolipoproteínas , Estudos Retrospectivos , Rejeição de Enxerto/etiologiaRESUMO
The release of extracellular vesicles (EVs) is increased under cellular stress and cardiomyocyte damaging conditions. However, whether the cardiomyocyte-derived EVs eventually reach the systemic circulation and whether their number in the bloodstream reflects cardiac injury, remains unknown. Wild type C57B/6 and conditional transgenic mice expressing green fluorescent protein (GFP) by cardiomyocytes were studied in lipopolysaccharide (LPS)-induced systemic inflammatory response syndrome (SIRS). EVs were separated both from platelet-free plasma and from the conditioned medium of isolated cardiomyocytes of the left ventricular wall. Size distribution and concentration of the released particles were determined by Nanoparticle Tracking Analysis. The presence of GFP + cardiomyocyte-derived circulating EVs was monitored by flow cytometry and cardiac function was assessed by echocardiography. In LPS-treated mice, systemic inflammation and the consequent cardiomyopathy were verified by elevated plasma levels of TNFα, GDF-15, and cardiac troponin I, and by a decrease in the ejection fraction. Furthermore, we demonstrated elevated levels of circulating small- and medium-sized EVs in the LPS-injected mice. Importantly, we detected GFP+ cardiomyocyte-derived EVs in the circulation of control mice, and the number of these circulating GFP+ vesicles increased significantly upon intraperitoneal LPS administration (P = 0.029). The cardiomyocyte-derived GFP+ EVs were also positive for intravesicular troponin I (cTnI) and muscle-associated glycogen phosphorylase (PYGM). This is the first direct demonstration that cardiomyocyte-derived EVs are present in the circulation and that the increased number of cardiac-derived EVs in the blood reflects cardiac injury in LPS-induced systemic inflammation (SIRS).
Assuntos
Movimento Celular , Vesículas Extracelulares/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/patologia , Síndrome de Resposta Inflamatória Sistêmica/patologia , Animais , Movimento Celular/efeitos dos fármacos , Clusterina/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Glicogênio Fosforilase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Integrases/metabolismo , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Fenótipo , Síndrome de Resposta Inflamatória Sistêmica/sangue , Síndrome de Resposta Inflamatória Sistêmica/fisiopatologia , Tamoxifeno/farmacologia , Troponina I/metabolismoRESUMO
The proinflammatory cascade that is activated at the time of brain death plays a crucial role in organ procurement. Our aim of this study was to explore the relationship between the clinical outcome of orthotopic heart transplantation, as well as cytokine and apolipoprotein profiles of the pericardial fluid obtained at donation. Interleukin, adipokine and lipoprotein levels in the pericardial fluid, as well as clinical data of twenty donors after brain death, were investigated. Outcome variables included primary graft dysfunction, the need for posttransplantation mechanical cardiac support and International Society for Heart and Lung Transplantation grade ≥ 2R rejection. Hormone management and donor risk scores were also investigated. Lower levels of IL-6 were observed in primary graft dysfunction (median: 36.72 [IQR: 19.47-62.90] versus 183.67 [41.21-452.56]; p = 0.029) and in the need for mechanical cardiac support (44.12 [20.12-85.70] versus 247.13 [38.51-510.38]; p = 0.043). Rejection was associated with lower ApoAII (p = 0.021), ApoB100 (p = 0.032) and ApoM levels (p = 0.025). Lower adipsin levels were detected in those patients receiving desmopressin (p = 0.037); moreover, lower leptin levels were found in those patients receiving glucocorticoid therapy (p = 0.045), and higher T3 levels were found in those patients treated with L-thyroxine (p = 0.047) compared to those patients not receiving these hormone replacement therapies. IL-5 levels were significantly associated with UNOS-D score (p = 0.004), Heart Donor Score (HDS) and Adapted HDS (p < 0.001). The monitoring of immunological and metabolic changes in donors after brain death may help in the prediction of potential complications after heart transplantation, thus potentially optimizing donor heart allocation.
Assuntos
Transplante de Coração , Disfunção Primária do Enxerto , Obtenção de Tecidos e Órgãos , Humanos , Doadores de Tecidos , Transplante de Coração/efeitos adversos , Morte Encefálica , Interleucinas , Apolipoproteínas , Estudos Retrospectivos , Rejeição de Enxerto/etiologiaRESUMO
BACKGROUND: Cardiac cell lines and primary cells are widely used in cardiovascular research. Despite increasing number of publications using these models, comparative characterization of these cell lines has not been performed, therefore, their limitations are undetermined. We aimed to compare cardiac cell lines to primary cardiomyocytes and to mature cardiac tissues in a systematic manner. METHODS AND RESULTS: Cardiac cell lines (H9C2, AC16, HL-1) were differentiated with widely used protocols. Left ventricular tissue, neonatal primary cardiomyocytes, and human induced pluripotent stem cell-derived cardiomyocytes served as reference tissue or cells. RNA expression of cardiac markers (e.g. Tnnt2, Ryr2) was markedly lower in cell lines compared to references. Differentiation induced increase in cardiac- and decrease in embryonic markers however, the overall transcriptomic profile and annotation to relevant biological processes showed consistently less pronounced cardiac phenotype in all cell lines in comparison to the corresponding references. Immunocytochemistry confirmed low expressions of structural protein sarcomeric alpha-actinin, troponin I and caveolin-3 in cell lines. Susceptibility of cell lines to sI/R injury in terms of viability as well as mitochondrial polarization differed from the primary cells irrespective of their degree of differentiation. CONCLUSION: Expression patterns of cardiomyocyte markers and whole transcriptomic profile, as well as response to sI/R, and to hypertrophic stimuli indicate low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless their differentiation. Low resemblance of cell lines to mature adult cardiac tissue limits their potential use. Low translational value should be taken into account while choosing a particular cell line to model cardiomyocytes.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Fenótipo , TranscriptomaRESUMO
The majority of colorectal cancer (CRC) patients carry mutations in the APC gene, which lead to the unregulated activation of the Wnt pathway. Extracellular vesicles (EV) are considered potential therapeutic tools. Although CRC is a genetically heterogeneous disease, the significance of the intra-tumor heterogeneity in EV uptake of CRC cells is not yet known. By using mouse and patient-derived organoids, the currently available best model of capturing cellular heterogeneity, we found that Apc mutation induced the expression of interferon-induced transmembrane protein 1 (Ifitm1), a membrane protein that plays a major role in cellular antiviral responses. Importantly, organoids derived from IFITM1high CRC cells contained more proliferating cells and they had a markedly reduced uptake of fibroblast EVs as compared to IFITM1low/- cells. In contrast, there was no difference in the intensity of EV release between CRC subpopulations with high and low IFITM1 levels. Importantly, the difference in cell proliferation between these two subpopulations disappeared in the presence of fibroblast-derived EVs, proving the functional relevance of the enhanced EV uptake by IFITM1low CRC cells. Furthermore, inactivating IFITM1 resulted in an enhanced EV uptake, highlighting the importance of this molecule in establishing the cellular difference for EV effects. Collectively, we identified CRC cells with functional difference in their EV uptake ability that must be taken into consideration when using EVs as therapeutic tools for targeting cancer cells.
Assuntos
Antígenos de Diferenciação/genética , Neoplasias Colorretais/genética , Vesículas Extracelulares/genética , Animais , Transporte Biológico/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Organoides/fisiologia , Via de Sinalização Wnt/genéticaRESUMO
Extracellular vesicles (EV) are considered as a promising diagnostic tool for pancreatic ductal adenocarcinoma (PDAC), a disease with a poor 5-year survival that has not improved in the past years. PDAC patient-derived 3D organoids maintain the intratumoral cellular heterogeneity, characteristic for the tumor in vivo.Thus, they represent an ideal in vitro model system to study human cancers. Here we show that the miRNA cargo of EVs from PDAC organoids largely differs among patients. However, we detected a common set of EV miRNAs that were present in matched organoids and blood plasma samples of individual patients. Importantly, the levels of EV miR-21 and miR-195 were higher in PDAC blood EV preparations than in healthy controls, albeit we found no difference compared to chronic pancreatitis (CP) samples. In addition, here we report that the accumulation of collagen I, a characteristic change in the extracellular matrix (ECM) in both CP and PDAC, largely increases EV release from pancreatic ductal organoids. This provides a possible explanation why both CP and PDAC patient-derived plasma samples have an elevated amount of CD63 + EVs. Collectively, we show that PDAC patient-derived organoids represent a highly relevant model to analyze the cargo of tumor cell-derived EVs. Furthermore, we provide evidence that not only driver mutations, but also changes in the ECM may critically modify EV release from pancreatic ductal cells.
Assuntos
Carcinoma Ductal Pancreático/patologia , Vesículas Extracelulares/genética , MicroRNAs/metabolismo , Organoides/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo I/farmacologia , Citocinas/farmacologia , Matriz Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/sangue , Organoides/citologia , Organoides/efeitos dos fármacos , Ductos Pancreáticos/citologia , Ductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Pancreatite/genética , Pancreatite/metabolismo , Pancreatite/patologiaRESUMO
Liver plays a central role in elimination of circulating extracellular vesicles (EVs), and it also significantly contributes to EV release. However, the involvement of the different liver cell populations remains unknown. Here, we investigated EV uptake and release both in normolipemia and hyperlipidemia. C57BL/6 mice were kept on high fat diet for 20-30 weeks before circulating EV profiles were determined. In addition, control mice were intravenously injected with 99mTc-HYNIC-Duramycin labeled EVs, and an hour later, biodistribution was analyzed by SPECT/CT. In vitro, isolated liver cell types were tested for EV release and uptake with/without prior fatty acid treatment. We detected an elevated circulating EV number after the high fat diet. To clarify the differential involvement of liver cell types, we carried out in vitro experiments. We found an increased release of EVs by primary hepatocytes at concentrations of fatty acids comparable to what is characteristic for hyperlipidemia. When investigating EV biodistribution with 99mTc-labeled EVs, we detected EV accumulation primarily in the liver upon intravenous injection of mice with medium (326.3 ± 19.8 nm) and small EVs (130.5 ± 5.8 nm). In vitro, we found that medium and small EVs were preferentially taken up by Kupffer cells, and liver sinusoidal endothelial cells, respectively. Finally, we demonstrated that in hyperlipidemia, there was a decreased EV uptake both by Kupffer cells and liver sinusoidal endothelial cells. Our data suggest that hyperlipidema increases the release and reduces the uptake of EVs by liver cells. We also provide evidence for a size-dependent differential EV uptake by the different cell types of the liver. The EV radiolabeling protocol using 99mTc-Duramycin may provide a fast and simple labeling approach for SPECT/CT imaging of EVs biodistribution.
Assuntos
Modelos Animais de Doenças , Vesículas Extracelulares/metabolismo , Hepatócitos/metabolismo , Hiperlipidemias/fisiopatologia , Fígado/metabolismo , Animais , Dieta Hiperlipídica , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Extracellular vesicles (EV) are released by virtually all cells and they transport biologically important molecules from the release site to target cells. Colorectal cancer (CRC) is a leading cause of cancer-related death cases, thus, it represents a major health issue. Although the EV cargo may reflect the molecular composition of the releasing cells and thus, EVs may hold a great promise for tumor diagnostics, the impact of intratumoral heterogeneity on the intensity of EV release is still largely unknown. By using CRC patient-derived organoids that maintain the cellular and molecular heterogeneity of the original epithelial tumor tissue, we proved that CD44high cells produce more organoids with a higher proliferation intensity, as compared to CD44low cells. Interestingly, we detected an increased EV release by CD44high CRC cells. In addition, we found that the miRNA cargos of CD44high and CD44low cell derived EVs largely overlapped and only four miRNAs were specific for one of the above subpopulations. We observed that EVs released by CD44high cells induced the proliferation and activation of colon fibroblasts more strongly than CD44low cells. However, this effect was due to the higher EV number rather than to the miRNA cargo of EVs. Collectively, we identified CRC subpopulations with different EV releasing capabilities and we proved that CRC cell-released EVs have a miRNA-independent effect on fibroblast proliferation and activation.
Assuntos
Neoplasias Colorretais , Vesículas Extracelulares , MicroRNAs , Comunicação Celular , Neoplasias Colorretais/patologia , Vesículas Extracelulares/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Organoides/metabolismoRESUMO
The purpose of the study was to carry out an immunophenotypical characterization with a special focus on natural killer cells of junior swimmers from the Hungarian National Swim Team before and after an intensive acute exercise. Nineteen swimmers, ten females and nine males, completed the exercise protocol. Sixteen swimmers experienced delayed-onset muscle soreness. Most of our findings substantiated earlier results, such as the increase in the percentage of the CD3-/CD56+ natural killer cells and the CD3-/CD56dim+ NK cells, and the decrease in the percentage of CD3+ T cells among lymphocytes after the exercise protocol. The drop of natural killer cell activity back to the pre-exercise level was in line with earlier findings. Interestingly, the percentage of CD3+/CD56+ NKT-like cells did not change significantly in those three swimmers who did not report delayed-onset muscle soreness. On the contrary, the percentage of CD3+/CD56+ NKT-like cells among lymphocytes increased in fourteen and decreased in two swimmers reporting delayed-onset muscle soreness. This study for the first time demonstrated a link between the delayed-onset muscle soreness and the imbalanced control of CD3+/CD56+ NKT-like cells among lymphocytes. However, validation of this association in a larger sample size study will be necessary.
Assuntos
Células T Matadoras Naturais , Complexo CD3/metabolismo , Antígeno CD56/metabolismo , Exercício Físico , Feminino , Humanos , Masculino , Mialgia/etiologia , Mialgia/metabolismo , Células T Matadoras Naturais/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismoRESUMO
Extracellular vesicles (EV) are membrane-surrounded vesicles that represent a novel way of intercellular communication by carrying biologically important molecules in a concentrated and protected form. The intestinal epithelium is continuously renewed by a small proliferating intestinal stem cell (ISC) population, residing at the bottom of the intestinal crypts in a specific microenvironment, the stem cell niche. By using 3D mouse and human intestinal organoids, we show that intestinal fibroblast-derived EVs are involved in forming the ISC niche by transmitting Wnt and epidermal growth factor (EGF) activity. With a mouse model that expresses EGFP in the Lgr5+ ISCs, we prove that loss in ISC number in the absence of EGF is prevented by fibroblast-derived EVs. Furthermore, we demonstrate that intestinal fibroblast-derived EVs carry EGF family members, such as amphiregulin. Mechanistically, blocking EV-bound amphiregulin inhibited the EV-induced survival of organoids. In contrast, EVs have no role in transporting R-Spondin, a critical niche factor amplifying Wnt signaling. Collectively, we prove the important role of fibroblast-derived EVs as a novel transmission mechanism of factors in the normal ISC niche.
Assuntos
Vesículas Extracelulares/metabolismo , Mucosa Intestinal/fisiopatologia , Intestinos/fisiopatologia , Nicho de Células-Tronco/genética , Idoso , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Extracellular vesicles (EVs) are membrane-surrounded structures that transmit biologically important molecules from the releasing to target cells, thus providing a novel intercellular communication mechanism. Since EVs carry their cargo in a protected form and their secretion is generally increased in tumorigenesis, EVs hold a great potential for early cancer diagnosis. By 3D culturing, we provide evidence that colorectal cancer (CRC) patient-derived organoids, representing a state-of-the-art established and essential approach for studying human CRC, is a suitable model for EV analysis. When testing the effects of major factors promoting CRC progression on EV release in the organoid model, we observed that Apc mutation, leading to uncontrolled Wnt activation and thus to tumorigenesis in the vast majority in CRC patients, critically induces EV release by activating the Wnt pathway. Furthermore, the extracellular matrix component collagen, known to accumulate in tumorigenesis, enhances EV secretion as well. Importantly, we show that fibroblast-derived EVs induce colony formation of CRC organoid cells under hypoxia. In contrast, there was no major effect of tumor cell-derived EVs on the activation of fibroblasts. Collectively, our results with CRC and Apc-mutant adenoma organoids identify Apc mutation and collagen deposition as critical factors for increasing EV release from tumors. Furthermore, we provide evidence that stromal fibroblast-derived EVs contribute to tumorigenesis under unfavorable conditions in CRC.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/patologia , Vesículas Extracelulares/patologia , Intestinos/patologia , Organoides/patologia , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Progressão da Doença , Vesículas Extracelulares/genética , Humanos , Camundongos Endogâmicos C57BL , Mutação , Organoides/metabolismo , Células Tumorais Cultivadas , Via de Sinalização WntRESUMO
Mast cells are multifunctional master cells implicated in both innate and adaptive immune responses. Their role has been best characterized in allergy and anaphylaxis; however, emerging evidences support their contribution to a wide variety of human diseases. Mast cells, being capable of both degranulation and subsequent recovery, have recently attracted substantial attention as also being rich sources of secreted extracellular vesicles (including exosomes and microvesicles). Along with secreted de novo synthesized soluble molecules and secreted preformed granules, the membrane-enclosed extracellular vesicles represent a previously unexplored part of the mast cell secretome. In this review article we summarize available data regarding the different soluble molecules and membrane-enclosed structures secreted by mast cells. Furthermore, we provide an overview of the release mechanisms including degranulation, piecemeal degranulation, transgranulation, and secretion of different types of extracellular vesicles. Finally, we aim to give a summary of the known biological functions associated with the different mast cell-derived secretion products. The increasingly recognized complexity of mast cell secretome may provide important novel clues to processes by which mast cells contribute to the development of different pathologies and are capable of orchestrating immune responses both in health and disease.
Assuntos
Degranulação Celular/imunologia , Grânulos Citoplasmáticos/imunologia , Vesículas Extracelulares/imunologia , Hipersensibilidade/metabolismo , Linfócitos/imunologia , Mastócitos/metabolismo , Cálcio/imunologia , Cálcio/metabolismo , Comunicação Celular , Citocinas/genética , Citocinas/imunologia , Grânulos Citoplasmáticos/patologia , Células Endoteliais/imunologia , Células Endoteliais/patologia , Vesículas Extracelulares/patologia , Regulação da Expressão Gênica , Humanos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Imunidade Inata , Imunoglobulina E/genética , Imunoglobulina E/metabolismo , Linfócitos/patologia , Mastócitos/patologia , Receptores de IgE/genética , Receptores de IgE/imunologia , Transdução de SinaisRESUMO
This study sought to identify novel CD8+ T cell homing markers by studying acute graft versus host disease (aGvHD), typically involving increased T cell homing to the skin and gut. FACS-sorted skin-homing (CD8ß+ /CLA+ ), gut-homing (CD8ß+ /integrinß7+ ), and reference (CD8ß+ /CLA- /integrinß7- ) T cells were compared in patients affected by cutaneous and/or gastrointestinal aGVHD. Microarray analysis, qPCR, and flow cytometry revealed increased expression of peptidase inhibitor 16 (PI16) in skin-homing CD8+ T cells. Robust association of PI16 with skin homing was confirmed in all types of aGvHD and in healthy controls, too. PI16 was not observed on CLA+ leukocytes other than T cells. Induction of PI16 expression on skin-homing T cells occurred independently of vitamin D3. Among skin-homing T cells, PI16 expression was most pronounced in memory-like CD45RO+ /CD127+ /CD25+ /CD69- /granzyme B- cells. PI16 was confined to the plasma membrane, was GPI-anchored, and was lost upon restimulation of memory CD8+ T cells. Loss of PI16 occurred by downregulation of PI16 transcription, and not by Phospholipase C (PLC)- or Angiotensin-converting enzyme (ACE)-mediated shedding, or by protein recycling. Inhibitor screening and pull-down experiments confirmed that PI16 inhibits cathepsin K, but may not bind to other skin proteases. These data link PI16 to skin-homing CD8+ T cells, and raise the possibility that PI16 may regulate cutaneous cathepsin K.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Pele/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Catepsina K/antagonistas & inibidores , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Citometria de Fluxo , Humanos , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Receptores de Retorno de Linfócitos/metabolismoRESUMO
Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Coleta de Amostras Sanguíneas/normas , Vesículas Extracelulares/metabolismo , Biomarcadores/sangue , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/diagnóstico , Exossomos/metabolismo , Citometria de Fluxo/métodos , HumanosRESUMO
Regulatory T cells (Treg) are mandatory elements in the maintenance of human pregnancy, but their de novo differentiation has not been completely exposed. HSPE1 chaperone expressing trophoblast cells may have a role in it. Trophoblast-derived extracellular vesicles (EVs), either at the feto-maternal interface or in circulation, target CD4+ T cells. We hypothesized that HSPE1-associated trophoblastic cell line (BeWo)-derived EVs are active mediators of Treg cell differentiation. We proved at first that recombinant HSPE1 promote human Treg cell differentiation in vitro. Developing a CRISPR-Cas9 based HSPE1 knockout BeWo cell line we could also demonstrate, that EV-associated HSPE1 induces Treg development. Next-generation sequencing of miRNA cargo of BeWo-EVs characterized the regulatory processes of Treg polarization. By the use of single-cell transcriptomics analysis, seven Treg cell subtypes were distinguished and we demonstrated for the first time that the expression level of HSPE1 was Treg subtype dependent, and CAPG expression is characteristic to memory phenotype of T cells. Our data indicate that HSPE1 and CAPG may be used as markers for identification of Treg subtypes. Our results suggest, that trophoblastic-derived iEVs-associated HSPE1 and miRNA cargo have an important role in Treg cell expansion in vitro and HSPE1 is a useful marker of Treg subtype characterization.
Assuntos
Comunicação Celular , Diferenciação Celular , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Trofoblastos/metabolismo , Proliferação de Células , Vesículas Extracelulares/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Proteoma , Proteômica/métodos , Linfócitos T Reguladores/imunologia , TranscriptomaRESUMO
PURPOSE OF REVIEW: Persuasive statistics support the clinical observation that because of cardiovascular comorbidities patients with inflammatory joint disease die significantly earlier despite anti-inflammatory therapy. RECENT FINDINGS: The reason for this earlier death is multifactorial and involves a combination of a complex genetic background, environmental influences, classical cardiovascular risk factors and the impact of anti-inflammatory therapy. We will describe the importance of several new mechanisms, especially the diverse intercellular communication routes including extracellular vesicles and microRNAs that support the development of cardiovascular comorbidities. SUMMARY: The aim of this review is to give an updated overview about the known risk factors in the development of cardiovascular comorbidities with the latest insights about their mechanism of action. Furthermore, the impact of newly identified risk factors and significance will be discussed.
Assuntos
Doenças Autoimunes/epidemiologia , Doenças Cardiovasculares/epidemiologia , Artropatias/epidemiologia , Doenças Autoimunes/fisiopatologia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/mortalidade , Doenças Cardiovasculares/fisiopatologia , Comunicação Celular/fisiologia , Comorbidade , Vesículas Extracelulares/fisiologia , Humanos , Inflamação/fisiopatologia , Artropatias/fisiopatologia , MicroRNAs/fisiologia , Fatores de RiscoRESUMO
AIMS: The prognosis of patients with pancreatic cancer has remained virtually unchanged with a high mortality rate compared to other types of cancers. An earlier detection would provide a time window of opportunity for treatment and prevention of deaths. In the present study we investigated extracellular vesicle (EV)-associated potential biomarkers for pancreatic cancer by directly assessing EV size-based subpopulations in pancreatic juice samples of patients with chronic pancreatitis or pancreatic cancer. In addition, we also studied blood plasma and pancreatic cancer cell line-derived EVs. METHODS: Comparative proteomic analysis was performed of 102â¯EV preparations from human pancreatic juices, blood, and pancreatic cancer cell lines Capan-1 and MIA PaCa-2. EV preparations were also characterized by electron microscopy, tunable resistive pulse sensing, and flow cytometry. RESULTS: Here we describe the presence of EVs in human pancreatic juice samples. Pancreatic juice EV-associated proteins that we identified as possible candidate markers for pancreatic cancer included mucins, such as MUC1, MUC4, MUC5AC, MUC6 and MUC16, CFTR, and MDR1 proteins. These candidate biomarkers could also be detected by flow cytometry in EVs found in pancreatic juice and those secreted by pancreatic cancer cell lines. CONCLUSIONS: Together our data show that detection and characterization of EVs directly in pancreatic juice is feasible and may prove to be a valuable source of potential biomarkers of pancreatic cancer.
Assuntos
Adenocarcinoma/diagnóstico , Biomarcadores Tumorais/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Vesículas Extracelulares/química , Mucinas/genética , Neoplasias Pancreáticas/diagnóstico , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diagnóstico Diferencial , Vesículas Extracelulares/metabolismo , Expressão Gênica , Humanos , Mucinas/metabolismo , Pâncreas , Suco Pancreático/química , Suco Pancreático/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite Crônica/diagnóstico , Pancreatite Crônica/genética , Pancreatite Crônica/metabolismo , Pancreatite Crônica/patologia , Prognóstico , Proteoma/genética , Proteoma/metabolismo , ProteômicaRESUMO
OBJECTIVE: Extracellular vesicles (EVs) are subcellular signalosomes. Although characteristic EV production is associated with numerous physiological and pathological conditions, the effect of blood-derived EVs on bone homeostasis is unknown. Herein we evaluated the role of circulating EVs on human osteoclastogenesis. METHODS: Blood samples from healthy volunteers, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients were collected. Size-based EV sub-fractions were isolated by gravity-driven filtration and differential centrifugation. To investigate the properties of EV samples, resistive pulse sensing technique, transmission electron microscopy, flow cytometry and western blot were performed. CD14+ monocytes were separated from PBMCs, and stimulated with recombinant human M-CSF, RANKL and blood-derived EV sub-fractions. After 7 days, the cells were fixed and stained for tartrate-resistant acid phosphatase and counted. RESULTS: EVs isolated by size-based sub-fractions were characterized as either microvesicles or exosomes (EXO). Healthy (n = 11) and RA-derived (n = 12) EXOs profoundly inhibited osteoclast differentiation (70%, p < 0.01; 65%, p < 0.01, respectively). In contrast, PsA-derived (n = 10) EXOs had a stimulatory effect (75%, p < 0.05). In cross-treatment experiments where EXOs and CD14+ cells were interchanged between the three groups, only healthy (n = 5) and RA (n = 5)-derived EXOs inhibited (p < 0.01, respectively) the generation of osteoclasts in all groups, whereas PsA (n = 7)-derived EXOs were unable to mediate this effect. CONCLUSIONS: Our data suggest that blood-derived EXOs are novel regulators of the human osteoclastogenesis and may offer discrete effector function in distinct inflammatory arthropathies.