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Long-term administration of aspirin (ASA, acetylsalicylic acid) in oncogenic patients has been related to a reduction in cancer risk incidence, but its precise mechanism of action is unclear. The activation of cancer-associated fibroblasts (CAFs) is a key element in tumor progression and can be triggered by cancer-derived extracellular vesicles (EVs). Targeting the communication between cancer cells and the surrounding tumor microenvironment (TME) may control cancer progression. Our aim was to investigate the effect of ASA on breast cancer cells, focusing on EV secretion and their effect on the biological properties of CAFs. As a result, ASA was shown to reduce the amount and alter the size distribution of EVs produced by MDA-MB-231 tumor cells. Fibroblasts stimulated with EVs derived from MDA-MB-231 treated with ASA (EV-ASA) showed a lower expression of alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP2) but not fibroblast activation protein (FAP) in respect to the ones stimulated with EVs from untreated breast cancer cells (EV-CTR). Furthermore, invasion assays using a three-dimensional (3D) fibroblast spheroid model showed reduced MDA-MB-231 invasion towards fibroblast spheroids pretreated with EV-ASA as compared to spheroids prepared with EV-CTR-stimulated fibroblasts. This suggests that ASA partially inhibits the ability of tumor EVs to stimulate CAFs to promote cancer invasion. In conclusion, ASA can interfere with tumor communication by reducing EV secretion by breast tumor cells as well as by interfering with their capacity to stimulate fibroblasts to become CAFs.
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Acute kidney injury (AKI) caused by ischemia followed by reperfusion (I/R) is characterized by intense anion superoxide (O2â¢-) production and oxidative damage. We investigated whether extracellular vesicles secreted by adipose tissue mesenchymal cells (EVs) administered during reperfusion can suppress the exacerbated mitochondrial O2â¢- formation after I/R. We used Wistar rats subjected to bilateral renal arterial clamping (30 min) followed by 24 h of reperfusion. The animals received EVs (I/R + EVs group) or saline (I/R group) in the kidney subcapsular space. The third group consisted of false-operated rats (SHAM). Mitochondria were isolated from proximal tubule cells and used immediately. Amplex Red™ was used to measure mitochondrial O2â¢- formation and MitoTracker™ Orange to evaluate inner mitochondrial membrane potential (Δψ). In vitro studies were carried out on human renal proximal tubular cells (HK-2) co-cultured or not with EVs under hypoxic conditions. Administration of EVs restored O2â¢- formation to SHAM levels in all mitochondrial functional conditions. The gene expression of catalase and superoxide dismutase-1 remained unmodified; transcription of heme oxygenase-1 (HO-1) was upregulated. The co-cultures of HK-2 cells with EVs revealed an intense decrease in apoptosis. We conclude that the mechanisms by which EVs favor long-term recovery of renal structures and functions after I/R rely on a decrease of mitochondrial O2â¢- formation with the aid of the upregulated antioxidant HO-1/Nuclear factor erythroid 2-related factor 2 system, thus opening new vistas for the treatment of AKI.
Assuntos
Injúria Renal Aguda , Vesículas Extracelulares , Traumatismo por Reperfusão , Injúria Renal Aguda/metabolismo , Tecido Adiposo/metabolismo , Animais , Vesículas Extracelulares/metabolismo , Isquemia/metabolismo , Rim/metabolismo , Mitocôndrias/metabolismo , Ratos , Ratos Wistar , Reperfusão , Traumatismo por Reperfusão/metabolismo , Superóxidos/metabolismoRESUMO
Induced pluripotent stem cells (iPSC) have been the focus of several studies due to their wide range of application, including in cellular therapy. The use of iPSC in regenerative medicine is limited by their tumorigenic potential. Extracellular vesicles (EV) derived from stem cells have been shown to support renal recovery after injury. However, no investigation has explored the potential of iPSC-EV in the treatment of kidney diseases. To evaluate this potential, we submitted renal tubule cells to hypoxia-reoxygenation injury, and we analyzed cell death rate and changes in functional mitochondria mass. An in vivo model of ischemia-reperfusion injury was used to evaluate morphological and functional alterations. Gene array profile was applied to investigate the mechanism involved in iPSC-EV effects. In addition, EV derived from adipose mesenchymal cells (ASC-EV) were also used to compare the potential of iPSC-EV in support of tissue recovery. The results showed that iPSC-EV were capable of reducing cell death and inflammatory response with similar efficacy than ASC-EV. Moreover, iPSC-EV protected functional mitochondria and regulated several genes associated with oxidative stress. Taken together, these results show that iPSC can be an alternative source of EV in the treatment of different aspects of kidney disease.
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Injúria Renal Aguda/fisiopatologia , Vesículas Extracelulares/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Humanos , Masculino , Ratos , Ratos Wistar , Espécies Reativas de OxigênioRESUMO
Hypertension is a long-term condition that can increase organ susceptibility to insults and lead to severe complications such as chronic kidney disease (CKD). Extracellular vesicles (EVs) are cell-derived membrane structures that participate in cell-cell communication by exporting encapsulated molecules to target cells, regulating physiological and pathological processes. We here demonstrate that multiple administration of EVs from adipose-derived mesenchymal stromal cells (ASC-EVs) in deoxycorticosterone acetate (DOCA)-salt hypertensive model can protect renal tissue by maintaining its filtration capacity. Indeed, ASC-EVs downregulated the pro-inflammatory molecules monocyte chemoattracting protein-1 (MCP-1) and plasminogen activating inhibitor-1 (PAI1) and reduced recruitment of macrophages in the kidney. Moreover, ASC-EVs prevented cardiac tissue fibrosis and maintained blood pressure within normal levels, thus demonstrating their multiple favorable effects in different organs. By applying microRNA (miRNA) microarray profile of the kidney of DOCA-salt rats, we identified a selective miRNA signature associated with epithelial-mesenchymal transition (EMT). One of the key pathways found was the axis miR-200-TGF-ß, that was significantly altered by EV administration, thereby affecting the EMT signaling and preventing renal inflammatory response and fibrosis development. Our results indicate that EVs can be a potent therapeutic tool for the treatment of hypertension-induced CKD in cardio-renal syndrome.
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Omics approaches have significantly impacted knowledge about molecular signaling pathways driving cell function. Induced pluripotent stem cells (iPSC) have revolutionized the field of biological sciences and proteomics and, in particular, has been instrumental in identifying key elements operating during the maintenance of the pluripotent state and the differentiation process to the diverse cell types that form organisms. This review covers the evolution of conceptual and methodological strategies in proteomics; briefly describes the generation of iPSC from a historical perspective, the state-of-the-art of iPSC-based proteomics; and compares data on the proteome and transcriptome of iPSC to that of embryonic stem cells (ESC). Finally, proteomics of healthy and diseased cells and organoids differentiated from iPSC are analyzed.
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Células-Tronco Pluripotentes Induzidas , Proteoma/metabolismo , Proteômica/métodos , Transcriptoma , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
BACKGROUND/AIMS: The therapeutic potential of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) in kidney injury has been largely reported. However, new approaches are necessary to optimize the efficacy in the treatment of renal diseases. MSCs physiologically are under a low O2 partial pressure (pO2), and culturing adipose-derived MSCs (ADMSCs) in hypoxia alters their secretory paracrine properties. The aim of this study was to evaluate whether hypoxia preconditioning of ADMSCs alters the properties of secreted EVs to improve renal recovery after ischemia-reperfusion injury (IRI). METHODS: The supernatants of ADMSCs cultivated under 21% pO2 (control) or 1% pO2 (hypoxia) were ultracentrifuged for EVs isolation that were posteriorly characterized by flow cytometry and electron microscopy. The uptake and effects of these EVs were analyzed by using in vitro and in vivo models. HK-2 renal tubule cell line was submitted do ATP depletion injury model. Proteomic analyses of these cells treated with EVs after injury were performed by nano-UPLC tandem nano-ESI-HDMSE method. For in vivo analyses, male Wistar rats were submitted to 45 min bilateral ischemia, followed by renal intracapsular administration of ADMSC-EVs within a 72 h reperfusion period. Histological, immunohistochemical and qRT-PCR analysis of these kidneys were performed to evaluate cell death, inflammation and oxidative stress. Kidney function was evaluated by measuring the blood levels of creatinine and urea. RESULTS: The results demonstrate that hypoxia increases the ADMSCs capacity to secrete EVs that trigger different energy supply, antiapoptotic, immunomodulatory, angiogenic and anti-oxidative stress responses in renal tissue compared with EVs secreted in normoxia. Proteomic analyses of renal tubule cells treated with EVs from ADMSCs in normoxia and hypoxia give a specific signature of modulated proteins for each type of EVs, indicating regulation of distinct biological processes. CONCLUSION: In summary, hypoxia potentially offers an interesting strategy to enhance the properties of EVs in the treatment of acute kidney disease.
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Injúria Renal Aguda/terapia , Vesículas Extracelulares/transplante , Células-Tronco Mesenquimais/metabolismo , Traumatismo por Reperfusão/terapia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Tecido Adiposo/citologia , Animais , Hipóxia Celular , Linhagem Celular , Células Cultivadas , Vesículas Extracelulares/metabolismo , Humanos , Rim/metabolismo , Rim/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Masculino , Células-Tronco Mesenquimais/citologia , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
The tumor microenvironment comprises a heterogeneous population of tumorigenic and non-tumorigenic cells. Cancer stem cells (CSCs) and mesenchymal stem cells (MSCs) are components of this microenvironment and have been described as key regulators of different aspects of tumor physiology. They act differently on the tumor: CSCs are described as tumor initiators and are associated with tumor growth, drug resistance and metastasis; MSCs can integrate the tumor microenvironment after recruitment and interact with cancer cells to promote tumor modifications. Extracellular vesicles (EVs) have emerged as an important mechanism of cell communication under the physiological and pathological conditions. In cancer, secretion of EVs seems to be one of the main mechanisms by which stem cells interact with other tumor and non-tumor cells. The transfer of bioactive molecules (lipids, proteins and RNAs) compartmentalized into EVs triggers different responses in the target cells, regulating several processes in the tumor as angiogenesis, tumor invasiveness and immune escape. This review focuses on the role of CSCs and MSCs in modulating the tumor microenvironment through secretion of EVs, addressing different aspects of the multidirectional interactions among stem cells, tumor and tumor-associated cells.
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Several studies have suggested that extracellular vesicles (EVs) released from mesenchymal stem cells (MSCs) may mediate MSC paracrine action on kidney regeneration. This activity has been, at least in part, ascribed to the transfer of proteins/transcription factors and different RNA species. Information on the RNA/protein content of different MSC EV subpopulations and the correlation with their biological activity is currently incomplete. The aim of this study was to evaluate the molecular composition and the functional properties on renal target cells of MSC EV sub-populations separated by gradient floatation. The results demonstrated heterogeneity in quantity and composition of MSC EVs. Two peaks of diameter were observed (90-110 and 170-190 nm). The distribution of exosomal markers and miRNAs evaluated in the twelve gradient fractions showed an enrichment in fractions with a flotation density of 1.08-1.14 g/mL. Based on this observation, we evaluated the biological activity on renal cell proliferation and apoptosis resistance of low (CF1), medium (CF2) and high (CF3) floatation density fractions. EVs derived from all fractions, were internalized by renal cells, CF1 and CF2 but not CF3 fraction stimulated significant cell proliferation. CF2 also inhibited apoptosis on renal tubular cells submitted to ischemia-reperfusion injury. Comparative miRNomic and proteomic profiles reveal a cluster of miRNAs and proteins common to all three fractions and an enrichment of selected molecules related to renal regeneration in CF2 fraction. In conclusion, the CF2 fraction enriched in exosomal markers was the most active on renal tubular cell proliferation and protection from apoptosis.
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Micropartículas Derivadas de Células/metabolismo , Células Epiteliais/metabolismo , Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Apoptose , Western Blotting , Linhagem Celular , Proliferação de Células , Separação Celular/métodos , Micropartículas Derivadas de Células/ultraestrutura , Células Cultivadas , Centrifugação com Gradiente de Concentração/métodos , Células Epiteliais/citologia , Exossomos/ultraestrutura , Túbulos Renais/citologia , Células-Tronco Mesenquimais/citologia , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Microscopia Eletrônica , Proteoma/metabolismo , Proteômica/métodos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The mechanisms of cell-cell communications are now under intense study by proteomic approaches. Proteomics has unraveled changes in protein profiling as the result of cell interactions mediated by ligand/receptor, hormones, soluble factors, and the content of extracellular vesicles. Besides being a brief overview of the main and profitable methodologies now available (evaluating theory behind the methods, their usefulness, and pitfalls), this review focuses on-from a proteome perspective-some signaling pathways and post-translational modifications (PTMs), which are essential for understanding ischemic lesions and their recovery in two vital organs in mammals, the heart, and the kidney. Knowledge of misdirection of the proteome during tissue recovery, such as represented by the convergence between fibrosis and cancer, emerges as an important tool in prognosis. Proteomics of cell-cell interaction is also especially useful for understanding how stem cells interact in injured tissues, anticipating clues for rational therapeutic interventions. In the effervescent field of induced pluripotency and cell reprogramming, proteomic studies have shown what proteins from specialized cells contribute to the recovery of infarcted tissues. Overall, we conclude that proteomics is at the forefront in helping us to understand the mechanisms that underpin prevalent pathological processes.