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We propose a new organ-conditioning strategy based on mesenchymal stromal cell (MSCs)/extracellular vesicle (EVs) delivery during hypothermic perfusion. MSCs/EVs marker CD73 is present on renal proximal tubular cells, and it protects against renal ischemia-reperfusion injury by converting adenosine monophosphate into adenosine (ADO). In this study, after checking if CD73-silenced EVs (EVsi) would impact in vitro tubular-cell proliferation, we perfused kidneys of a rat model of donation after circulatory death, with Belzer solution (BS) alone, BS supplemented with MSCs, EVs, or EVsi. The ADO and ATP levels were measured in the effluents and tissues. Global renal ischemic damage score (GRS), and tubular cell proliferation index (IPT) were evaluated in the tissue. EVsi did not induce cell proliferation in vitro. Ex vivo kidneys perfused with BS or BS + EVsi showed the worst GRS and higher effluent ADO levels than the MSC- and EV-perfused kidneys. In the EV-perfused kidneys, the tissue and effluent ATP levels and IPT were the highest, but not if CD73 was silenced. Tissue ATP content was positively correlated with tissue ADO content and negatively correlated with effluent ADO level in all groups. In conclusion, kidney conditioning with EVs protects against ischemic damage by activating the CD73/ADO system.
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Vesículas Extracelulares , Células-Tronco Mesenquimais , Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Vesículas Extracelulares/metabolismo , Isquemia/metabolismo , Rim/metabolismo , Células-Tronco Mesenquimais/metabolismo , RatosRESUMO
OBJECTIVES: Critical hindlimb ischemia is a severe consequence of peripheral artery disease. Surgical treatment does not prevent skeletal muscle impairment or improve long-term patient outcomes. The present study investigates the protective/regenerative potential and the mechanism of action of adipose stem cell-derived extracellular vesicles (ASC-EVs) in a mouse model of hindlimb ischemia. Approach and Results: We demonstrated that ASC-EVs exert a protective effect on muscle damage by acting both on tissue microvessels and muscle cells. The genes involved in muscle regeneration were up-regulated in the ischemic muscles of ASC-EV-treated animals. MyoD expression has also been confirmed in satellite cells. This was followed by a reduction in muscle function impairment in vivo. ASC-EVs drive myoblast proliferation and differentiation in the in vitro ischemia/reoxygenation model. Moreover, ASC-EVs have shown an anti-apoptotic effect both in vitro and in vivo. Transcriptomic analyses have revealed that ASC-EVs carry a variety of pro-angiogenic mRNAs, while proteomic analyses have demonstrated an enrichment of NRG1 (neuregulin 1). A NRG1 blocking antibody used in vivo demonstrated that NRG1 is relevant to ASC-EV-induced muscle protection, vascular growth, and recruitment of inflammatory cells. Finally, bioinformatic analyses on 18 molecules that were commonly detected in ASC-EVs, including mRNAs and proteins, confirmed the enrichment of pathways involved in vascular growth and muscle regeneration/protection. CONCLUSIONS: This study demonstrates that ASC-EVs display pro-angiogenic and skeletal muscle protective properties that are associated with their NRG1/mRNA cargo. We, therefore, propose that ASC-EVs are a useful tool for therapeutic angiogenesis and muscle protection.
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Adipócitos/citologia , Vesículas Extracelulares/metabolismo , Membro Posterior/irrigação sanguínea , Isquemia/patologia , Músculo Esquelético/ultraestrutura , Neuregulina-1/metabolismo , Células-Tronco/ultraestrutura , Adipócitos/metabolismo , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Vesículas Extracelulares/ultraestrutura , Isquemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Músculo Esquelético/metabolismo , Proteômica , Células-Tronco/metabolismoRESUMO
Extracellular vesicles (EVs) are membrane vesicles released virtually by all cell types. Several studies have shown that stem cell-derived EVs may mimic both in vitro and in vivo the biological effects of the cells. We recently demonstrated that non-alcoholic steatohepatitis (NASH) is inhibited by treatment with human liver stem cells (HLSCs). The aim of the present study was to evaluate whether EVs released by HLSCs influence the progression of NASH, induced by a diet deprived of methionine and choline, in immunocompromised mice. EV treatment was initiated after 2 weeks of diet with a biweekly administration of three different doses. Bio-distribution evaluated by optical imaging showed a preferential accumulation in normal and, in particular, in fibrotic liver. EV treatment significantly improved liver function and reduced signs of liver fibrosis and inflammation at both morphological and molecular levels. In particular, we observed that, out of 29 fibrosis-associated genes upregulated in NASH liver, 28 were significantly downregulated by EV treatment. In conclusion, HLSC-derived EVs display anti-fibrotic and anti-inflammatory effects in a model of chronic liver disease, leading to an improvement of liver function.
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Vesículas Extracelulares/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Fígado/citologia , Fígado/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Biomarcadores , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Inflamação/terapia , Cirrose Hepática/etiologia , Cirrose Hepática/terapia , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , TranscriptomaRESUMO
Corneal endothelial dystrophy is a relevant cause of vision loss and corneal transplantation worldwide. In the present study, we analyzed the effect of mesenchymal stem cell (MSC)-derived extracellular vesicles (MSC-EVs) in an in vitro model of corneal dystrophy, characterized by endoplasmic reticulum stress. The effects of MSC-EVs were compared with those of serum-derived EVs, reported to display a pro-angiogenic activity. MSC-EVs were able to induce a significant down-regulation of the large majority of endoplasmic reticulum stress-related genes in human corneal endothelial cells after exposure to serum deprivation and tunicamycin. In parallel, they upregulated the Akt pathway and limited caspase-3 activation and apoptosis. At variance, the effect of the serum EVs was mainly limited to Akt phosphorylation, with minimal or absent effects on endoplasmic reticulum stress modulation and apoptosis prevention. The effects of MSC-EVs were correlated to the transfer of numerous endoplasmic reticulum (ER)-stress targeting miRNAs to corneal endothelial cells. These data suggest a potential therapeutic effect of MSC-EVs for corneal endothelial endoplasmic reticulum stress, a major player in corneal endothelial dystrophy.
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Apoptose , Estresse do Retículo Endoplasmático , Células Endoteliais/patologia , Endotélio Corneano/patologia , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Separação Celular , Meios de Cultura Livres de Soro , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Fosforilação/efeitos dos fármacos , Tunicamicina/farmacologiaRESUMO
Extracellular vesicles (EVs) derived from mesenchymal stem cells isolated from both bone marrow (BMSCs) and adipose tissue (ADSCs) show potential therapeutic effects. These vesicles often show a similar beneficial effect on tissue regeneration, but in some contexts, they exert different biological properties. To date, a comparison of their molecular cargo that could explain the different biological effect is not available. Here, we demonstrated that ADSC-EVs, and not BMSC-EVs, promote wound healing on a murine model of diabetic wounds. Besides a general similarity, the bioinformatic analysis of their protein and miRNA cargo highlighted important differences between these two types of EVs. Molecules present exclusively in ADSC-EVs were highly correlated to angiogenesis, whereas those expressed in BMSC-EVs were preferentially involved in cellular proliferation. Finally, in vitro analysis confirmed that both ADSC and BMSC-EVs exploited beneficial effect on cells involved in skin wound healing such as fibroblasts, keratinocytes and endothelial cells, but through different cellular processes. Consistent with the bioinformatic analyses, BMSC-EVs were shown to mainly promote proliferation, whereas ADSC-EVs demonstrated a major effect on angiogenesis. Taken together, these results provide deeper comparative information on the cargo of ADSC-EVs and BMSC-EVs and the impact on regenerative processes essential for diabetic wound healing.
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Complicações do Diabetes/terapia , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Úlcera/etiologia , Úlcera/terapia , Cicatrização , Tecido Adiposo/citologia , Animais , Células da Medula Óssea , Exossomos/metabolismo , Exossomos/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Citometria de Fluxo , Perfilação da Expressão Gênica , Imuno-Histoquímica , Células-Tronco Mesenquimais/citologia , CamundongosRESUMO
Cancer stem cells (CSCs) are considered as responsible for initiation, maintenance and recurrence of solid tumors, thus representing the key for tumor eradication. The antitumor activity of extracellular vesicles (EVs) derived from different stem cell sources has been investigated with conflicting results. In our study, we evaluated, both in vitro and in vivo, the effect of EVs derived from human bone marrow mesenchymal stromal cells (MSCs) and from a population of human liver stem cells (HLSCs) of mesenchymal origin on renal CSCs. In vitro, both EV sources displayed pro-apoptotic, anti-proliferative and anti-invasive effects on renal CSCs, but not on differentiated tumor cells. Pre-treatment of renal CSCs with EVs, before subcutaneous injection in SCID mice, delayed tumor onset. We subsequently investigated the in vivo effect of MSC- and HLSC-EVs systemic administration on progression of CSC-generated renal tumors. Tumor bio-distribution analysis identified intravenous treatment as best route of administration. HLSC-EVs, but not MSC-EVs, significantly impaired subcutaneous tumor growth by reducing tumor vascularization and inducing tumor cell apoptosis. Moreover, intravenous treatment with HLSC-EVs improved metastasis-free survival. In EV treated tumor explants, we observed both the transfer and the induction of miR-145 and of miR-200 family members. In transfected CSCs, the same miRNAs affected cell growth, invasion and survival. In conclusion, our results showed a specific antitumor effect of HLSC-EVs on CSC-derived renal tumors in vivo, possibly ascribed to the transfer and induction of specific antitumor miRNAs. Our study provides further evidence for a possible clinical application of stem cell-EVs in tumor treatment.
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Produtos Biológicos/administração & dosagem , Vesículas Extracelulares/metabolismo , Neoplasias Renais/terapia , Células-Tronco Mesenquimais/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Administração Intravenosa , Animais , Terapia Biológica/métodos , Fracionamento Celular , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Rim/citologia , Rim/patologia , Rim/cirurgia , Neoplasias Renais/patologia , Fígado/citologia , Camundongos , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/patologia , Nefrectomia , Cultura Primária de Células , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human liver stem-like cells (HLSC) and derived extracellular vesicles (EVs) were previously shown to exhibit anti-tumor activity. In our study, we investigated whether HLSC-derived EVs (HLSC-EVs) were able to inhibit tumor angiogenesis in vitro and in vivo, in comparison with EVs derived from mesenchymal stem cells (MSC-EVs). The results obtained indicated that HLSC-EVs, but not MSC-EVs, inhibited the angiogenic properties of tumor-derived endothelial cells (TEC) both in vitro and in vivo in a model of subcutaneous implantation in Matrigel. Treatment of TEC with HLSC-EVs led to the down-regulation of pro-angiogenic genes. Since HLSC-EVs carry a specific set of microRNAs (miRNAs) that could target these genes, we investigated their potential role by transfecting TEC with HLSC-EV specific miRNAs. We observed that four miRNAs, namely miR-15a, miR-181b, miR-320c and miR-874, significantly inhibited the angiogenic properties of TEC in vitro, and decreased the expression of some predicted target genes (ITGB3, FGF1, EPHB4 and PLAU). In parallel, TEC treated with HLSC-EVs significantly enhanced expression of miR-15a, miR-181b, miR-320c and miR-874 associated with the down-regulation of FGF1 and PLAU. In summary, HLSC-EVs possess an anti-tumorigenic effect, based on their ability to inhibit tumor angiogenesis.
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Vesículas Extracelulares , Hepatócitos , Neovascularização Patológica , Células-Tronco , Animais , Humanos , Fígado/citologia , Camundongos , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) possess pro-regenerative potential in different animal models with renal injury. EVs contain different molecules, including proteins, lipids and nucleic acids. Among the shuttled molecules, miRNAs have a relevant role in the pro-regenerative effects of EVs and are a promising target for therapeutic interventions. The aim of this study was to increase the content of specific miRNAs in EVs that are known to be involved in the pro-regenerative effect of EVs, and to assess the capacity of modified EVs to contribute to renal regeneration in in vivo models with acute kidney injuries. To this purpose, MSCs were transiently transfected with specific miRNA mimics by electroporation. Molecular analyses showed that, after transfection, MSCs and derived EVs were efficiently enriched in the selected miRNAs. In vitro and in vivo experiments indicated that EVs engineered with miRNAs maintained their pro-regenerative effects. Of relevance, engineered EVs were more effective than EVs derived from naïve MSCs when used at suboptimal doses. This suggests the potential use of a low amount of EVs (82.5 × 106) to obtain the renal regenerative effect.
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Injúria Renal Aguda/terapia , Vesículas Extracelulares/transplante , Transplante de Células-Tronco Mesenquimais/métodos , MicroRNAs/genética , Terapêutica com RNAi/métodos , Regeneração , Animais , Células Cultivadas , Vesículas Extracelulares/genética , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos SCID , MicroRNAs/metabolismoRESUMO
BACKGROUND: Tumor immune-escape has been related to the ability of cancer cells to inhibit T cell activation and dendritic cell (DC) differentiation. We previously identified a tumor initiating population, expressing the mesenchymal marker CD105, which fulfills the criteria for definition as cancer stem cells (CD105(+) CSCs) able to release extracellular vesicles (EVs) that favor tumor progression and metastases. The aim of the present study was to compare the ability of renal CSCs and derived EVs to modulate the behavior of monocyte-derived DCs with a non-tumor initiating renal cancer cell population (CD105(-) TCs) and their EVs. METHODS: Maturation of monocyte-derived DCs was studied in presence of CD105(+) CSCs and CD105(-) TCs and their derived EVs. DC differentiation experiments were evaluated by cytofluorimetric analysis. T cell proliferation and ELISA assays were performed. Monocytes and T cells were purified from peripheral blood mononuclear cells obtained from healthy donors. RESULTS: The results obtained demonstrate that both CD105(+) CSCs and CD105(-) TCs impaired the differentiation process of DCs from monocytes. However, the immune-modulatory effect of CD105(+) CSCs was significantly greater than that of CD105(-) TCs. EVs derived from CD105(+) CSCs and in less extent, those derived from CD105(-) TCs retained the ability to impair monocyte maturation and T cell activation. The mechanism has been mainly related to the expression of HLA-G by tumor cells and to its release in a form associated to EVs. HLA-G blockade significantly reduced the inhibitory effect of EVs on DC differentiation. CONCLUSIONS: In conclusion, the results of the present study indicate that renal cancer cells and in particular CSCs and derived EVs impair maturation of DCs and T cell immune response by a mechanism involving HLA-G.
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Carcinoma de Células Renais/imunologia , Diferenciação Celular , Células Dendríticas/fisiologia , Vesículas Extracelulares/fisiologia , Antígenos HLA-G/fisiologia , Neoplasias Renais/imunologia , Monócitos/fisiologia , Adulto , Carcinoma de Células Renais/fisiopatologia , Técnicas de Cocultura , Células Dendríticas/citologia , Vesículas Extracelulares/imunologia , Feminino , Antígenos HLA-G/imunologia , Humanos , Neoplasias Renais/fisiopatologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/fisiologia , Linfócitos T/imunologiaRESUMO
Quantitative measurement of marker expression in diseased cells is still a topic of considerable interest and different methodologies are currently under intense scrutiny. This work aims at developing an inâ vitro diagnostic method based on the release of paramagnetic species from relaxometrically "silent" liposomes operated by the action of a phospholipase A2 (PLA2) previously targeted to the epitope of interest. The released paramagnetic species causes an increase of the longitudinal water proton relaxation rate proportional to the number of PLA2 bound to the cell outer surface. The sensitivity of the herein proposed method, named R-ELISA, was attempted in the detection of folate receptor expression on human ovarian cancer cells by functionalizing PLA2 with folic acid. Receptor/cell number of 8.3×10(5) has been measured on IGROV-1 cells. The R-ELISA assay can detect nanomolar cell suspension receptor concentrations and has been validated by well-established spectrofluorimetric procedures.
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Biomarcadores Tumorais/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Neoplasias Ovarianas/química , Neoplasias Ovarianas/metabolismo , Fosfolipases A2/metabolismo , Feminino , HumanosRESUMO
Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro. Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system, guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study, we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation. Methods: We evaluated the expression of hepatic markers using qRT-PCR, immunofluorescence, and Western blot analysis. Additionally, we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality. Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes, including CYP450, urea cycle enzymes, and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes, evident as early as 1 day post-differentiation. Interestingly, HLSCs transiently upregulated stem cell markers during differentiation, followed by downregulation after 7 days. Furthermore, differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24 h, with accumulation over time. Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system, its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications.
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Chronic hepatitis (CH) of dysmetabolic or viral etiology has been associated with poor prognosis in patients who experienced the severe acute respiratory coronavirus virus-2 (SARS-Cov-2) infection. We aimed to explore the impact of SARS-Cov-2 infection on disease severity in a group of patients with CH. Forty-two patients with CH of different etiology were enrolled (median age, 56 years; male gender, 59%). ACE2 and TMPRSS2 were measured in plasma samples of all patients by ELISA and in the liver tissue of a subgroup of 15 patients by Western blot. Overall, 13 patients (31%) experienced SARS-Cov-2 infection: 2/15 (15%) had CHB, 5/12 (39%) had CHC, and 6/15 (46%) had non-alcoholic fatty liver disease (NAFLD). Compared to viral CH patients, NAFLD subjects showed higher circulating ACE2 levels (p = 0.0019). Similarly, hepatic expression of ACE2 was higher in subjects who underwent SARS-Cov-2 infection compared to the counterpart, (3.24 ± 1.49 vs. 1.49 ± 1.32, p = 0.032). Conversely, hepatic TMPRSS2 was significantly lower in patients who experienced symptomatic COVID-19 disease compared to asymptomatic patients (p = 0.0038). Further studies are necessary to understand the impact of COVID-19 in patients with pre-existing liver diseases.
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COVID-19 , Hepatopatia Gordurosa não Alcoólica , Humanos , Masculino , Pessoa de Meia-Idade , Enzima de Conversão de Angiotensina 2 , Hepatite Crônica , Peptidil Dipeptidase A/metabolismo , SARS-CoV-2 , FemininoRESUMO
Liver fibrosis occurs in response to chronic liver injury and is characterized by an excessive deposition of extracellular matrix. Activated hepatic stellate cells are primarily responsible for this process. A possible strategy to counteract the development of hepatic fibrosis could be the reversion of the activated phenotype of hepatic stellate cells. Extracellular vesicles (EVs) are nanosized membrane vesicles involved in intercellular communication. Our previous studies have demonstrated that EVs derived from human liver stem cells (HLSCs), a multipotent population of adult stem cells of the liver with mesenchymal-like phenotype, exert in vivo anti-fibrotic activity in the liver. However, the mechanism of action of these EVs remains to be determined. We set up an in vitro model of hepatic fibrosis using a human hepatic stellate cell line (LX-2) activated by transforming growth factor-beta 1 (TGF-ß1). Then, we investigated the effect of EVs obtained from HLSCs and from human bone marrow-derived mesenchymal stromal cells (MSCs) on activated LX-2. The incubation of activated LX-2 with HLSC-EVs reduced the expression level of alpha-smooth muscle actin (α-SMA). Conversely, MSC-derived EVs induced an increase in the expression of pro-fibrotic markers in activated LX-2. The analysis of the RNA cargo of HLSC-EVs revealed the presence of several miRNAs involved in the regulation of fibrosis and inflammation. Predictive target analysis indicated that several microRNAs (miRNAs) contained into HLSC-EVs could possibly target pro-fibrotic transcripts. In particular, we demonstrated that HLSC-EVs shuttled miR-146a-5p and that treatment with HLSC-EVs increased miR-146a-5p expression in LX-2. In conclusion, this study demonstrates that HLSC-EVs can attenuate the activated phenotype of hepatic stellate cells and that their biological effect may be mediated by the delivery of anti-fibrotic miRNAs, such as miR-146a-5p.
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Extracellular vesicles are considered a novel therapeutic tool, due to their ability to transfer their cargoes to target cells. Different strategies to directly load extracellular vesicles with RNA species have been proposed. Electroporation has been used for the loading of non-active vesicles; however, the engineering of vesicles already carrying a therapeutically active cargo is still under investigation. Here, we set up a coincubation method to increase the anti-tumor effect of extracellular vesicles isolated from human liver stem cells (HLSC-EVs). Using the coincubation protocol, vesicles were loaded with the anti-tumor miRNA-145, and their effect was evaluated on renal cancer stem cell invasion. Loaded HLSC-EVs maintained their integrity and miR transfer ability. Loaded miR-145, but not miR-145 alone, was protected by RNAse digestion, possibly due to its binding to RNA-binding proteins on HLSC-EV surface, such as Annexin A2. Moreover, miR-145 coincubated HLSC-EVs were more effective in inhibiting the invasive properties of cancer stem cells, in comparison to naïve vesicles. The protocol reported here exploits a well described property of extracellular vesicles to bind nucleic acids on their surface and protect them from degradation, in order to obtain an effective miRNA loading, thus increasing the activity of therapeutically active naïve extracellular vesicles.
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Human liver stem cells (HLSCs) were described for the first time in 2006 as a new stem cell population derived from healthy human livers. Like mesenchymal stromal cells, HLSCs exhibit multipotent and immunomodulatory properties. HLSCs can differentiate into several lineages under defined in vitro conditions, such as mature hepatocytes, osteocytes, endothelial cells, and islet-like cell organoids. Over the years, HLSCs have been shown to contribute to tissue repair and regeneration in different in vivo models, leading to more than five granted patents and over 15 peer reviewed scientific articles elucidating their potential therapeutic role in various experimental pathologies. In addition, HLSCs have recently completed a Phase 1 study evaluating their safety post intrahepatic injection in infants with inherited neonatal onset hyperammonemia. Even though a lot of progress has been made in understanding HLSCs over the past years, some important questions regarding the mechanisms of action remain to be elucidated. Among the mechanisms of interaction of HLSCs with their environment, a paracrine interface has emerged involving extracellular vesicles (EVs) as vehicles for transferring active biological materials. In our group, the EVs derived from HLSCs have been studied in vitro as well as in vivo. Our attention has mainly been focused on understanding the in vivo ability of HLSC-derived EVs as modulators of tissue regeneration, inflammation, fibrosis, and tumor growth. This review article aims to discuss in detail the role of HLSCs and HLSC-EVs in these processes and their possible future therapeutic applications.
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Extracellular vesicles (EVs) have emerged in the last decades as a cell-to-cell communication mechanism. One of their mechanism of action is the direct delivery of their cargo, composed of bioactive molecules to target cells. Different methods (direct electroporation, cell transfection, chemical transfection) were developed to vehicle therapeutic molecules through EVs. However, most of these techniques presented some limitations such as EV disruption and aggregation. In the present study, we demonstrated that a direct temperature-controlled co-incubation of EVs with defined miRNAs is a stable method to deliver information to target cells without affecting EV constitutive content. We chose serum as an easy and abundant source of EVs applicable to autologous treatment after EV modification. Exogenous cel-miR-39 loaded on serum EVs (SEVs) was taken up by human endothelial cells, demonstrating an adequate miRNA loading efficacy based on the co-incubation method. Moreover, SEVs co-incubation with the angiomiRNA-126 (miR-126) enhanced their angiogenic properties in vitro and in vivo by increasing the capacity to induce capillary-like structure formation of human endothelial cells. MiR-126 loaded EVs were also shown to stimulate mouse endothelial cells to invade Matrigel plugs and create more vessels with respect to the EV naive counterpart. When SEVs were loaded with miR-19b, an anti-angiogenic miRNA, they were able to reduce Vascular endothelial growth factors (VEGF) pro-angiogenic capacity, supporting the selective biological effect mediated by the carried miRNA. Lastly, we identified Annexin A2 (ANXA2) as one of the molecules involved in the exogenous RNA binding to serum EV surface, favoring miRNA delivery to target endothelial cells for potential therapeutic application.
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Even with high-dose post-transplant cyclophosphamide (PT-Cy) which was initially introduced for graft-versus-host disease (GvHD) prevention in the setting of HLA-haploidentical transplantation, both acute and chronic GvHDs remain a major clinical challenge. Despite improvements in the understanding of the pathogenesis of both acute and chronic GvHDs, reliable biomarkers that predict their onset have yet to be identified. We recently studied the potential correlation between extracellular vesicles (EVs) and the onset of acute (a)GvHD in transplant recipients from related and unrelated donors. In the present study, we further investigated the role of the expression profile of membrane proteins and their microRNA (miRNA) cargo (miRNA100, miRNA155, and miRNA194) in predicting the onset of aGvHD in haploidentical transplant recipients with PT-Cy. Thirty-two consecutive patients were included. We evaluated the expression profile of EVs, by flow cytometry, and their miRNA cargo, by real-time PCR, at baseline, prior, and at different time points following transplant. Using logistic regression and Cox proportional hazard models, a significant association between expression profiles of antigens such as CD146, CD31, CD140a, CD120a, CD26, CD144, and CD30 on EVs, and their miRNA cargo with the onset of aGvHD was observed. Moreover, we also investigated a potential correlation between EV expression profile and cargo with plasma biomarkers (e.g., ST2, sTNFR1, and REG3a) that had been associated with aGVHD previously. This analysis showed that the combination of CD146, sTNFR1, and miR100 or miR194 strongly correlated with the onset of aGvHD (AUROC >0.975). A large prospective multicenter study is currently in progress to validate our findings.
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Biomarcadores , Ciclofosfamida/uso terapêutico , Vesículas Extracelulares/metabolismo , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/metabolismo , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Adulto , Idoso , Ciclofosfamida/administração & dosagem , Feminino , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Cuidados Pós-Operatórios , Prognóstico , Modelos de Riscos Proporcionais , Curva ROC , Transplante Haploidêntico , Resultado do Tratamento , Adulto JovemRESUMO
Extracellular vesicles (EVs) play an important role in the cellular crosstalk by transferring bioactive molecules through biological barriers from a cell to another, thus influencing recipient cell functions and phenotype. Therefore, EVs are increasingly being explored as biomarkers of disease progression or response to therapy and as potential therapeutic agents in different contexts including in hematological malignancies. Recently, an EV role has emerged in allogeneic hematopoietic cell transplantation (allo-HCT) as well. Allogeneic hematopoietic cell transplantation often represents the only curative option in several hematological disorders, but it is associated with potentially life-threatening complications that can have a significant impact on clinical outcomes. The most common complications have been well-established and include graft-versus-host disease and infections. Furthermore, relapse remains an important cause of treatment failure. The aim of this review is to summarize the current knowledge, the potential applications, and clinical relevance of EVs in allo-HCT. Herein, we will mainly focus on the immune-modulating properties of EVs, in particular those derived from mesenchymal stromal cells, as potential therapeutic strategy to improve allo-HCT outcome. Moreover, we will briefly describe the main findings on EVs as biomarkers to monitor graft-versus-host disease onset and tumor relapse.
Assuntos
Vesículas Extracelulares/fisiologia , Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Imunidade Adaptativa , Aloenxertos , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/fisiologia , Células Dendríticas/imunologia , Portadores de Fármacos , Endossomos/imunologia , Exossomos/fisiologia , Vesículas Extracelulares/imunologia , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/fisiopatologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Reconstituição Imune , Imunidade Inata , Células Matadoras Naturais/imunologia , Células-Tronco Mesenquimais/imunologia , MicroRNAs/genética , RecidivaRESUMO
Serum-derived extracellular vesicles (sEV) from healthy donors display in-vivo pro-angiogenic properties. To identify patients that may benefit from autologous sEV administration for pro-angiogenic purposes, sEV angiogenic capability has been evaluated in type 2 diabetic (T2DM) subjects (D), in obese individuals with (OD) and without (O) T2DM, and in subjects with ischemic disease (IC) (9 patients/group). sEV display different angiogenic properties in such cluster of individuals. miRNomic profile and TGFß content in sEV were evaluated. We found that miR-130a and TGFß content correlates with sEV in-vitro and in-vivo angiogenic properties, particularly in T2DM patients. Ingenuity Pathway Analysis (IPA) identified a number of genes as among the most significant miR-130a interactors. Gain-of-function experiments recognized homeoboxA5 (HOXA5) as a miR-130a specific target. Finally, ROC curve analyses revealed that sEV ineffectiveness could be predicted (Likelihood Ratio+ (LH+) = 3.3 IC 95% from 2.6 to 3.9) by comparing miR-130a and TGFß content 'in Series'. We demonstrate that sEV from high cardiovascular risk patients have different angiogenic properties and that miR-130a and TGFß sEV content predicts 'true ineffective sEVs'. These results provide the rationale for the use of these assays to identify patients that may benefit from autologous sEV administration to boost the angiogenetic process.
Assuntos
Doenças Cardiovasculares/sangue , Diabetes Mellitus Tipo 2/sangue , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Fator de Crescimento Transformador beta/metabolismo , Regiões 3' não Traduzidas , Adulto , Idoso , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Limitations in the current therapeutic strategies for the prevention of progression of chronic kidney disease (CKD) to end stage renal disease has been a drawback to improving patient recovery. It is therefore imperative that a solution is found to alleviate this problem and improve the health and well-being of patients overall. Aristolochic acid (AA) induced nephropathy, a type of nephrotoxic CKD is characterised by cortical tubular injury, inflammation, leading to interstitial fibrosis. Extracellular vesicles derived from human bone marrow mesenchymal stem cells (MSC-EVs) display therapeutic properties in various disease models including kidney injury. In the current study, we intended to investigate the ability of MSC-EVs on ameliorating tubular injury and interstitial fibrosis in a mouse model of aristolochic acid nephropathy (AAN). The chronic model of AAN is comprised of an intraperitoneal injection of AA in NSG mice, followed by a three-day incubation period and then inoculation of MSC-EVs intravenously. This routine was performed on a weekly basis for four consecutive weeks, accompanied by the monitoring of body weight of all mice. Blood and tissue samples were collected post sacrifice. All animals administered with AA developed kidney injury and renal fibrosis. A gradual loss of body weight was observed, together with a deterioration in kidney function. Although no significant recovery was observed in weight loss following treatment with MSC-EVs, a significant reduction in: blood creatinine and blood urea nitrogen (BUN), tubular necrosis, and interstitial fibrosis was observed. In addition, infiltration of CD45 positive immune cells, fibroblasts, and pericytes which were elevated in the interstitium post AA induced injury, were also significantly reduced by MSC-EVs. Kidneys were also subjected to molecular analyses to evaluate the regulation of pro-fibrotic genes. MSC-EVs significantly reduced AA induction of the pro-fibrotic genes α-Sma, Tgfb1 and Col1a1. A downregulation in pro-fibrotic genes was also observed in fibroblasts activated by AA injured mTECs in vitro. Furthermore, meta-analyses of miRNAs downregulated by MSC-EVs, such as miR21, revealed the regulation of multiple pathways involved in kidney injury including fibrosis, inflammation, and apoptosis. These results therefore suggest that MSC-EVs could play a regenerative and anti-fibrotic role in AAN through the transfer of biologically active cargo that regulates the disease both at a protein and genetic level.