Chiara Gai

Cross Talk between Cancer and Mesenchymal Stem Cells through Extracellular Vesicles Carrying Nucleic Acids

Extracellular vesicles (EVs) are considered to be a novel complex mechanism of cell communication within the tumor microenvironment. EVs may act as vehicles for transcription factors and nucleic acids inducing epigenetic changes in recipient cells. Since tumor EVs may be present in patient biological fluids, it is important to investigate their function and molecular mechanisms of action. It has been shown that tumor cells release EVs, which are capable of regulating cell apoptosis, proliferation, invasion, and epithelial–mesenchymal transition, as well as to suppress activity of immune cells, to enhance angiogenesis, and to prepare a favorable microenvironment for metastasis. On the other hand, EVs derived from stromal cells, such as mesenchymal stem cells (MSCs), may influence the phenotype of tumor cells through reciprocal cross talk greatly influenced by the transcription factors and nucleic acids they carry. In particular, non-coding RNAs (ncRNAs), including microRNAs and long ncRNAs, have recently been identified as the main candidates for the phenotypic changes induced in the recipient cells by EVs. ncRNAs, which are important regulators of mRNA and protein expression, can function either as tumor suppressors or as oncogenes, depending on their targets. Herein, we have attempted to revise actual evidence reported in the literature on the role of EVs in tumor biology with particular regard to the cross talk of ncRNAs between cancer cells and MSCs.

Stem Cell-Derived Extracellular Vesicles and Immune-Modulation

Extra-cellular vesicles (EVs) are bilayer membrane structures enriched with proteins, nucleic acids, and other active molecules and have been implicated in many physiological and pathological processes over the past decade. Recently, evidence suggests EVs to play a more dichotomic role in the regulation of the immune system, whereby an immune response may be enhanced or supressed by EVs depending on their cell of origin and its functional state. EVs derived from antigen (Ag)-presenting cells for instance, have been involved in both innate and acquired (or adaptive) immune responses, as Ag carriers or presenters, or as vehicles for delivering active signaling molecules. On the other hand, tumor and stem cell derived EVs have been identified to exert an inhibitory effect on immune responses by carrying immuno-modulatory effectors, such as transcriptional factors, non-coding RNA (Species), and cytokines. In addition, stem cell-derived EVs have also been reported to impair dendritic cell maturation and to regulate the activation, differentiation, and proliferation of B cells. They have been shown to control natural killer cell activity and to suppress the innate immune response (IIR). Studies reporting the role of EVs on T lymphocyte modulation are controversial. Discrepancy in literature may be due to stem cell culture conditions, methods of EV purification, EV molecular content, and functional state of both parental and target cells. However, mesenchymal stem cell-derived EVs were shown to play a more suppressive role by shifting T cells from an activated to a T regulatory phenotype. In this review, we will discuss how stem cell-derived EVs may contribute toward the modulation of the immune response. Collectively, stem cell-derived EVs mainly exhibit an inhibitory effect on the immune system.

Role of Alix in miRNA packaging during extracellular vesicle biogenesis

Evidence indicates that Alix, an accessory protein of the endosomal sorting complex required for transport (ESCRT), is involved in the biogenesis of extracellular vesicles (EVs). EVs contain selected patterns of microRNAs (miRNAs or miRs); however, little is known about the mechanisms of miRNA enrichment in EVs. The aim of the present study was to evaluate whether Alix is involved in the packaging of miRNAs within EVs released by human liver stem-like cells (HLSCs). EVs released from HLSCs were enriched with miRNAs and expressed Alix and several RNA-binding proteins, including Argonaute 2 (Ago2), a member of the Argonaute family known to be involved in the transport and the processing of miRNAs. Co-immunoprecipitation experiments revealed an association between Alix and Ago2. The results from RT-qPCR indicated that in the Alix/Ago2 immunoprecipitates, miRNAs were detectable. EVs were instrumental in transferring selected miRNAs from HLSCs to human endothelial cells absent in the latter cells. Alix knockdown did not influence the number of EVs released by HLSCs, but it significantly decreased miRNA expression levels in the EVs and consequently their transfer to the endothelium. Our findings indicate that Alix binds to Ago2 and miRNAs, suggesting that it plays a key role in miRNA enrichment during EV biogenesis. These results may represent a novel function of Alix, demonstrating its involvement in the EV-mediated transfer of miRNAs.

Extracellular vesicle-mediated modulation of angiogenesis.

Angiogenesis is a tightly regulated process where a number of different players are involved. Recently, a role for membrane vesicles actively released from cells has been proposed. Virtually all cell types may release non-apoptotic membrane vesicles in the nano-size range containing critical components of the cell of origin. The two main categories of these vesicles include exosomes and microvesicles that differ for biogenesis but, sharing several features and mechanisms of action, have been collectively named extracellular vesicles (EV). EV are able to transfer from one cell to another bioactive lipids, proteins and nucleic acids that may induce changes in the phenotype and functions of the recipient cells. This new mechanism of cell to cell communication has been involved in modulation of the angiogenic process. Tumor cells, inflammatory cells and stem/progenitor cells were shown to release EV with angiogenic properties suggesting that they may act on vascular remodeling in different physiological and pathological conditions. In this review we discuss the evidence for the role and the mechanisms of action of EV in vascular homeostasis and in the angiogenic processes occurring in tumors, inflammation and tissue regeneration.

Extracellular Vesicles in Renal Pathophysiology

Extracellular vesicles are a heterogeneous population of microparticles released by virtually all living cells which have been recently widely investigated in different biological fields. They are typically composed of two primary types (exosomes and microvesicles) and are recently commanding increasing attention as mediators of cellular signaling. Indeed, these vesicles can affect recipient cells by carrying and delivering complex cargos of biomolecules (including proteins, lipids and nucleic acids), protected from enzymatic degradation in the environment. Their importance has been demonstrated in the pathophysiology of several organs, in particular in kidney, where different cell types secrete extracellular vesicles that mediate their communication with downstream urinary tract cells. Over the past few years, evidence has been shown that vesicles participate in kidney development and normal physiology. Moreover, EVs are widely demonstrated to be implicated in cellular signaling during renal regenerative and pathological processes. Although many EV mechanisms are still poorly understood, in particular in kidney, the discovery of their role could help to shed light on renal biological processes which are so far elusive. Lastly, extracellular vesicles secreted by renal cells gather in urine, thus becoming a great resource for disease or recovery markers and a promising non-invasive diagnostic instrument for renal disease. In the present review, we discuss the most recent findings on the role of extracellular vesicles in renal physiopathology and their potential implication in diagnosis and therapy.

Noncoding RNAs Carried by Extracellular Vesicles in Endocrine Diseases

RNA molecules are essential and fine regulators of important biological processes. Their role is well documented also in the endocrine system, both in physiological and pathological conditions. Increasing interest is arising about the function and the importance of noncoding RNAs shuttled by extracellular vesicles (EVs). In fact, EV membrane protects nucleic acids from enzyme degradation. Nowadays, the research on EVs and their cargoes, as well as their biological functions, faces the lack of standardization in EV purification. Here, the main techniques for EV isolation are discussed and compared for their advantages and vulnerabilities. Despite the possible discrepancy due to methodological variability, EVs and their RNA content are reported to be key mediators of intercellular communication in pathologies of main endocrine organs, including the pancreas, thyroid, and reproductive system. In particular, the present work describes the role of RNAs contained in EVs in pathogenesis and progression of several metabolic dysfunctions, including obesity and diabetes, and their related manifestations. Their importance in the establishment and progression of thyroid autoimmunity disorders and complicated pregnancy is also discussed. Preliminary studies highlight the attractive possibility to use RNAs contained in EVs as biomarkers suggesting their exploitation for new diagnostic approaches in endocrinology.

PDGF-BB Carried by Endothelial Cell–Derived Extracellular Vesicles Reduces Vascular Smooth Muscle Cell Apoptosis in Diabetes

Endothelial cell–derived extracellular vesicles (CD31EVs) constitute a new entity for therapeutic/prognostic purposes. The roles of CD31EVs as mediators of vascular smooth muscle cell (VSMC) dysfunction in type 2 diabetes (T2D) are investigated herein. We demonstrated that, unlike serum-derived extracellular vesicles in individuals without diabetes, those in individuals with diabetes (D CD31EVs) boosted apoptosis resistance of VSMCs cultured in hyperglycemic condition. Biochemical analysis revealed that this effect relies on changes in the balance between antiapoptotic and proapoptotic signals: increase of bcl-2 and decrease of bak/bax. D CD31EV cargo analysis demonstrated that D CD31EVs are enriched in membrane-bound platelet-derived growth factor-BB (mbPDGF-BB). Thus, we postulated that mbPDGF-BB transfer by D CD31EVs could account for VSMC resistance to apoptosis. By depleting CD31EVs of platelet-derived growth factor-BB (PDGF-BB) or blocking the PDGF receptor β on VSMCs, we demonstrated that mbPDGF-BB contributes to D CD31EV–mediated bak/bax and bcl-2 levels. Moreover, we found that bak expression is under the control of PDGF-BB–mediated microRNA (miR)-296-5p expression. In fact, while PDGF-BB treatment recapitulated D CD31EV–mediated antiapoptotic program and VSMC resistance to apoptosis, PDGF-BB–depleted CD31EVs failed. D CD31EVs also increased VSMC migration and recruitment to neovessels by means of PDGF-BB. Finally, we found that VSMCs, from human atherosclerotic arteries of individuals with T2D, express low bak/bax and high bcl-2 and miR-296-5p levels. This study identifies the mbPDGF-BB in D CD31EVs as a relevant mediator of diabetes-associated VSMC resistance to apoptosis.

Salivary extracellular vesicle-associated miRNAs as potential biomarkers in oral squamous cell carcinoma

BackgroundSeveral studies in the past have investigated the expression of micro RNAs (miRNAs) in saliva as potential biomarkers. Since miRNAs associated with extracellular vesicles (EVs) are known to be protected from enzymatic degradation, we evaluated whether salivary EVs from patients with oral squamous cell carcinoma (OSCC) were enriched with specific subsets of miRNAs.MethodsOSCC patients and controls were matched with regards to age, gender and risk factors. Total RNA was extracted from salivary EVs and the differential expression of miRNAs was evaluated by qRT-PCR array and qRT-PCR. The discrimination power of up-regulated miRNAs as biomarkers in OSCC patients versus controls was evaluated by the Receiver Operating Characteristic (ROC) curves.ResultsA preliminary qRT-PCR array was performed on samples from 5 OSCC patients and 5 healthy controls whereby a subset of miRNAs were identified that were differentially expressed. On the basis of these results, a cohort of additional 16 patients and 6 controls were analyzed to further confirm the miRNAs that were up-regulated or selectively expressed in the previous pilot study. The following miRNAs: miR-302b-3p and miR-517b-3p were expressed only in EVs from OSCC patients and miR-512-3p and miR-412-3p were up-regulated in salivary EVs from OSCC patients compared to controls with the ROC curve showing a good discrimination power for OSCC diagnosis. The Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis suggested the possible involvement of the miRNAs identified in pathways activated in OSCC.ConclusionsIn this work, we suggest that salivary EVs isolated by a simple charge-based precipitation technique can be exploited as a non-invasive source of miRNAs for OSCC diagnosis. Moreover, we have identified a subset of miRNAs selectively enriched in EVs of OSCC patients that could be potential biomarkers.

Molecular and functional characterization of circulating extracellular vesicles from diabetic patients with and without retinopathy and healthy subjects

&NA; Diabetic retinopathy is a sight‐threatening complication of diabetes, characterized by loss of retinal pericytes and abnormal angiogenesis. We previously demonstrated that extracellular vesicles (EVs) derived from mesenchymal stem cells cultured in diabetic‐like conditions are able to enter the pericytes, causing their detachment and migration, and stimulating angiogenesis in vitro. The purpose of this work was the molecular and functional characterization of EVs derived from diabetic subjects with or without diabetic retinopathy, compared with healthy controls. Characterization of EVs extracted from serum/plasma of diabetic patients with or without retinopathy, and healthy controls, was performed by FACS and microarray analysis of microRNA (miRNA) content. Relevant miRNA expression was validated through qRT‐PCR. EV influence on pericyte detachment, angiogenesis and permeability of the blood‐retinal barrier was also investigated. Diabetic subjects had a 2.5 fold higher EV concentration than controls, while expression of surface molecules was unchanged. Microarray analysis revealed 11 differentially expressed miRNAs. Three of them (miR‐150‐5p, miR‐21‐3p and miR‐30b‐5p) were confirmed by qRT‐PCR. Plasma EVs from subjects with diabetic retinopathy induced pericyte detachment and pericyte/endothelial cell migration, increased the permeability of pericyte/endothelial cell bilayers and the formation of vessel‐like structures, when compared with EVs from controls. In conclusion, circulating EVs show differences between diabetic patients and healthy subjects. EVs extracted from plasma of diabetic retinopathy patients are able to induce features of retinopathy in in vitro models of retinal microvasculature. Our data suggest a role for miR‐150‐5p, miR‐21‐3p and miR‐30b‐5p as potential biomarkers of the onset of diabetic retinopathy. HighlightsCirculating extracellular vesicles are different in diabetic and healthy subjects.Vesicles from diabetic retinopathy patients induce features of retinopathy in vitro.MiRNAs shuttled by extracellular vesicles are involved.miR‐150‐5p, miR‐21‐3p, miR‐30b‐5p are potential biomarkers of diabetic retinopathy.

Protective Role of Stem Cell Derived Extracellular Vesicles in an In VitroModel of Hyperglycemia-Induced Endothelial Injury

Background: Adipose and bone marrow derived mesenchymal stem cells are two populations of multipotent adult stem cells with immunosuppressive, anti-inflammatory, and regenerative properties. It has been previously described that extracellular vesicles (EVs) derived from stem cells possess pro-regenerative and pro-angiogenic abilities. Hyperglycemia is a pathological condition affecting diabetic patients. Long term effects of hyperglycemia are endothelial dysfunction and vascular lesions leading to diabetic microangiopathy. The aim of the present study was to evaluate whether stem cell-derived EVs may inhibit endothelial cells dysfunction induced by hyperglycemia to mimic human microangiopathy. Methods: We set up an in vitro hyperglycemic model by culturing human microvascular endothelial cells in hyperglycemic constant or intermittent conditions for 7 days, in order to mimic a chronic damage. At day 5, endothelial cells were incubated with adipose and mesenchymal stem cell-derived EVs or vehicle alone for 48 hr. At day 7, we evaluated apoptosis, oxidative stress, and capillary-like formation ability on Matrigel. Results: Intermittent and constant high glucose models significantly decreased endothelial cell proliferation, increased number of apoptotic cells, promoted oxidation of intercellular proteins, and reduced capillary-like structure formation. Treatment with both kinds of EVs significantly restored proliferation, inhibited apoptosis and oxidation, and restored capillary-like formation. Conclusions: The results of the present study demonstrate that adipose and bone marrow mesenchymal stem cell-derived EVs may inhibit the endothelial dysfunction induced by high glucose concentration, which mimic diabetic microvascular injury.