New insight into the role of extracellular vesicles in kidney disease

Extracellular vesicles (EVs) are released to maintain cellular homeostasis as well as to mediate cell communication by spreading protective or injury signals to neighbour or remote cells. In kidney, increasing evidence support that EVs are signalling vesicles for different segments of tubules, intra‐glomerular, glomerular‐tubule and tubule‐interstitial communication. EVs released by kidney resident and infiltrating cells can be isolated from urine and were found to be promising biomarkers for kidney disease, reflecting deterioration of renal function and histological change. We have here summarized the recent progress about the functional role of EVs in kidney disease as well as challenges and future directions involved.     

Proteomic Signature of Mesenchymal Stromal Cell‐Derived Small Extracellular Vesicles

Small extracellular vesicles (EVs) are 50–200 nm vesicles secreted by most cells. They are considered as mediators of intercellular communication, and EVs from specific cell types, in particular mesenchymal stem/stromal cells (MSCs), offer powerful therapeutic potential, and can provide a novel therapeutic strategy. They appear promising and safe (as EVs are non‐self‐replicating), and eventually MSC‐derived EVs (MSC‐EVs) may be developed to standardized, off‐the‐shelf allogeneic regenerative and immunomodulatory therapeutics. Promising pre‐clinical data have been achieved using MSCs from different sources as EV‐producing cells. Similarly, a variety EV isolation and characterization methods have been applied. Interestingly, MSC‐EVs obtained from different sources and prepared with different methods show in vitro and in vivo therapeutic effects, indicating that isolated EVs share a common potential. Here, well‐characterized and controlled, publicly available proteome profiles of MSC‐EVs are compared to identify a common MSC‐EV protein signature that might be coupled to the MSC‐EVs’ common therapeutic potential. This protein signature may be helpful in developing MSC‐EV quality control platforms required to confirm the identity and test for the purity of potential therapeutic MSC‐EVs.     

Extracellular micro/nanovesicles rescue kidney from ischemia-reperfusion injury

Acute renal failure (ARF) is a clinical challenge that is highly resistant to treatment, and its high rate of mortality is alarming. Ischemia–reperfusion injury (IRI) is the most common cause of ARF. Especially IRI is implicated in kidney transplantation and can determine graft survival. Although the exact pathophysiology of renal IRI is unknown, the role of inflammatory responses has been elucidated. Because mesenchymal stromal cells (MSCs) have strong immunomodulatory properties, they are under extensive investigation as a therapeutic modality for renal IRI. Extracellular vesicles (EVs) play an integral role in cell-to-cell communication. Because the regenerative potential of the MSCs can be recapitulated by their EVs, the therapeutic appeal of MSC-derived EVs has dramatically increased in the past decade. Higher safety profile and ease of preservation without losing function are other advantages of EVs compared with their producing cells. In the current review, the preliminary results and potential of MSC-derived EVs to alleviate kidney IRI are summarized. We might be heading toward a cell-free approach to treat renal IRI.     

Urinary CD133+ Extracellular Vesicles Are Decreased in Kidney Transplanted Patients with Slow Graft Function and Vascular Damage

Extracellular vesicles (EVs) present in the urine are mainly released from cells of the nephron and can therefore provide information on kidney function. We here evaluated the presence of vesicles expressing the progenitor marker CD133 in the urine of normal subjects and of patients undergoing renal transplant. We found that EV expressing CD133 were present in the urine of normal subjects, but not of patients with end stage renal disease. The first day after transplant, urinary CD133⁺ EVs were present at low levels, to increase thereafter (at day 7). Urinary CD133⁺ EVs significantly increased in patients with slow graft function in respect to those with early graft function. In patients with a severe pre-transplant vascular damage of the graft, CD133⁺ EVs did not increase at day 7. At variance, the levels of EVs expressing the renal exosomal marker CD24 did not vary in the urine of patients with end stage renal disease or in transplanted patients in respect to controls. Sorted CD133⁺ EVs were found to express glomerular and proximal tubular markers. These data indicate that urinary CD133⁺ EVs are continuously released during the homeostatic turnover of the nephron and may provide information on its function or regenerative potential.     

Extracellular vesicles in normal pregnancy and pregnancy‐related diseases

Extracellular vesicles (EVs) are nanosized, membranous vesicles released by almost all types of cells. Extracellular vesicles can be classified into distinct subtypes according to their sizes, origins and functions. Extracellular vesicles play important roles in intercellular communication through the transfer of a wide spectrum of bioactive molecules, contributing to the regulation of diverse physiological and pathological processes. Recently, it has been established that EVs mediate foetal‐maternal communication across gestation. Abnormal changes in EVs have been reported to be critically involved in pregnancy‐related diseases. Moreover, EVs have shown great potential to serve as biomarkers for the diagnosis of pregnancy‐related diseases. In this review, we discussed about the roles of EVs in normal pregnancy and how changes in EVs led to complicated pregnancy with an emphasis on their values in predicting and monitoring of pregnancy‐related diseases.     

Extracellular vesicle‐associated organotropic metastasis

Metastasis refers to the progressive dissemination of primary tumour cells and their colonization of other tissues and is associated with most cancer‐related mortalities. The disproportional and systematic distribution pattern of distant metastasis in different cancers has been well documented, as is termed metastatic organotropism, a process orchestrated by a combination of anatomical, pathophysiological, genetic and biochemical factors. Extracellular vesicles (EVs), nanosized cell‐derived membrane‐bound particles known to mediate intercellular communication, are now considered crucial in organ‐specific metastasis. Here, we review and summarize recent findings regarding EV‐associated organotropic metastasis as well as some of the general mechanisms by which EVs contribute to this important process in cancer and provide a future perspective on this emerging topic. We highlight studies that demonstrate a role of tumour‐derived EVs in organotropic metastasis via pre‐metastatic niche modulation. The bioactive cargo carried by EVs is of diagnostic and prognostic values, and counteracting the functions of such EVs may be a novel therapeutic strategy targeting metastasis. Further investigations are warranted to better understand the functions and mechanisms of EVs in organotropic metastasis and accelerate the relevant clinical translation.     

Recent advances in extracellular vesicle research for urological cancers: From technology to application

Urological malignancies, including prostate cancer, bladder cancer and kidney cancer, are major causes of morbidity and mortality worldwide. Because of the high incidence, diversity in biology, and especially direct interaction with urine, urological cancers are an important resource for both scientists and clinicians for novel diagnostic and therapeutic discovery. Extracellular vesicles (EVs) are lipid bilayer encapsulated particles released by cells into the extracellular space. Since EVs work as a safe way to transport important biological information through the whole body, they are now recognized as an important mechanism of cell–cell communication and have opened a new window for us to gain a better understanding of cancer biology, novel diagnostics, and therapeutic options. In recent years, numerous evolutions in EV technologies and novel biological and clinical findings continue to be reported in the research field of urological cancers. This comprehensive review aims to give an update of recent advances in EV technologies and summarize the state-of-the-art knowledge of EVs related to prostate cancer, bladder cancer and kidney cancer, particularly focusing on the potential of EV as biomarkers and their biological roles in promoting cancer and metastasis.     

Extracellular vesicles in urologic malignancies—Implementations for future cancer care

Extracellular vesicles (EVs), a heterogeneous group of vesicles differing in size and shape, cargo content and function, are membrane‐bound and nano‐sized vesicles that could be released by nearly all variations of cells. EVs have gained considerable attention in the past decades for their functions in modulating intercellular signalling and roles as potential pools for the novel diagnostic and prognostic biomarkers, as well as therapeutic targets in several cancers including urological neoplasms. In general, human and animal cells both can release distinct types of EVs, including exosomes, microvesicles, oncosomes and large oncosomes, and apoptotic bodies, while the content of EVs can be divided into proteins, lipids and nucleic acids. However, the lack of standard methods for isolation and detection platforms rein the widespread usage in clinical applications warranted furthermore investigations in the development of reliable, specific and sensitive isolation techniques. Whether and how the EVs work has become pertinent issues. With the aid of high‐throughput proteomics or genomics methods, a fully understanding of contents contained in EVs from urogenital tumours, beyond all doubt, will improve our ability to identify the complex genomic alterations in the process of cancer and, in turn, contribute to detect potential therapeutic target and then provide personalization strategy for patient.     

The role of extracellular vesicles in podocyte autophagy in kidney disease

Podocytes are the key cells involved in protein filtration in the glomerulus. Once proteins appear in the urine when podocytes fail, patients will end with renal failure due to the progression of glomerular damage if no proper treatment is applied. The injury and loss of podocytes can be attributed to diverse factors, such as genetic, immunologic, toxic, or metabolic disorders. Recently, autophagy has emerged as a key mechanism to eliminate the unwanted cytoplasmic materials and to prolong the lifespan of podocytes by alleviating cell damage and stress. Typically, the fundamental function of extracellular vesicles (EVs) is to mediate the intercellular communication. Recent studies have suggested that, EVs, especially exosomes, play a certain role in information transfer by communicating proteins, mRNAs, and microRNAs with recipient cells. Under physiological and pathological conditions, EVs assist in the bioinformation interchange between kidneys and other organs. It is suggested that EVs are related to the pathogenesis of acute kidney injury and chronic kidney disease, including glomerular disease, diabetic nephropathy, renal fibrosis and end-stage renal disease. However, the role of EVs in podocyte autophagy remains unclear so far. Here, this study integrated the existing information about the relevancy, diagnostic value and therapeutic potential of EVs in a variety of podocytes-related diseases. The accumulating evidence highlighted that autophagy played a critical role in the homeostasis of podocytes in glomerular disease.     

Exploring the utility of extracellular vesicles in ameliorating viral infection-associated inflammation,cytokine storm and tissue damage

Extracellular vesicles (EVs) have emerged as potential mediators of intercellular communication. EVs are nano-sized, lipid membrane–bound vesicles that contains biological information in the form of proteins, metabolites and/or nucleic acids. EVs are key regulators of tissue repair mechanisms, such as in the context of lung injuries. Recent studies suggest that EVs have the ability to repair COVID19-associated acute lung damage. EVs hold great promise for therapeutic treatments, particularly in treating a potentially fatal autoimmune response and attenuate inflammation. They are known to boost lung immunity and are involved in the pathogenesis of various lung diseases, including viral infection. EV-based immunization technology has been proven to elicit robust immune responses in many models of infectious disease, including COVID-19. The field of EV research has tremendous potential in advancing our understanding about viral infection pathogenesis, and can be translated into anti-viral therapeutic strategies.     

Mitochondrial DNA in extracellular vesicles declines with age

The mitochondrial free radical theory of aging suggests that accumulating oxidative damage to mitochondria and mitochondrial DNA (mtDNA) plays a central role in aging. Circulating cell‐free mtDNA (ccf‐mtDNA) isolated from blood may be a biomarker of disease. Extracellular vesicles (EVs) are small (30–400 nm), lipid‐bound vesicles capable of shuttling proteins, nucleic acids, and lipids as part of intercellular communication systems. Here, we report that a portion of ccf‐mtDNA in plasma is encapsulated in EVs. To address whether EV mtDNA levels change with human age, we analyzed mtDNA in EVs from individuals aged 30–64 years cross‐sectionally and longitudinally. EV mtDNA levels decreased with age. Furthermore, the maximal mitochondrial respiration of cultured cells was differentially affected by EVs from old and young donors. Our results suggest that plasma mtDNA is present in EVs, that the level of EV‐derived mtDNA is associated with age, and that EVs affect mitochondrial energetics in an EV age‐dependent manner.     

Cardioprotective role of extracellular vesicles: A highlight on exosome beneficial effects in cardiovascular diseases

Extracellular vesicles (EVs) are nano-sized vesicles, released from many cell types including cardiac cells, have recently emerged as intercellular communication tools in cell dynamics. EVs are an important mediator of signaling within cells that influencing the functional behavior of the target cells. In heart complex, cardiac cells can easily use EVs to transport bioactive molecules such as proteins, lipids, and RNAs to the regulation of neighboring cell function. Cross-talk between intracardiac cells plays pivotal roles in the heart homeostasis and in adaptive responses of the heart to stress. EVs were released by cardiomyocytes under baseline conditions, but stress condition such as hypoxia intensifies secretome capacity. EVs secreted by cardiac progenitor cells and cardiosphere-derived cells could be pinpointed as important mediators of cardioprotection and cardiogenesis. Furthermore, EVs from many different types of stem cells could potentially exert a therapeutic effect on the damaged heart. Recent evidence shows that cardiac-derived EVs are rich in microRNAs, suggesting a key role in the controlling of cellular processes. EVs harboring exosomes may be clinically useful in cell-free therapy approaches and potentially act as prognosis and diagnosis biomarkers of cardiovascular diseases.     

Mesenchymal stem cell–derived extracellular vesicles: a new impetus of promoting angiogenesis in tissue regeneration

Over the past few decades, extracellular vesicles (EVs) have emerged as crucial mediators of intercellular communication. EVs encapsulate and convey information to surrounding cells or distant cells, where they mediate cellular biological responses. Among their multifaceted roles in the modulation of biological responses, the involvement of EVs in vascular development, growth and maturation has been widely documented and their potential therapeutic application in regenerative medicine or in the treatment of angiogenesis-related diseases is drawing increasing interest. In this review, we have summarized the details about the current knowledge on biogenesis of EVs and conventional isolation methods. Evidence supporting the use of EVs derived from mesenchymal stromal cells (MSCs) to enhance angiogenesis in the development of insufficient angiogenesis, such as chronic wounds, stroke and myocardial infarction, will also be discussed critically. Finally, the main challenges and prerequisites for their therapeutic applications will be evaluated.     

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles: A novel therapeutic paradigm

Mesenchymal stem cells (MSCs) have been revealed to hold great potential for the development of new treatment approaches for various diseases. However, the clinical use of these cells is limited due to their tumorigenic effects. The therapeutic benefits of MSCs are largely dependent on paracrine factors including extracellular vesicles (EVs). EVs are nano-sized bilayer membrane structures containing lipids, microRNAs and proteins which play key roles in cell-to-cell communications. Because of their lower immunogenicity, tumorigenicity, and easier management, EVs have emerged as a new promising alternative to whole-cell therapy. Therefore, this paper reviews current preclinical studies on the use of EVs derived from human umbilical cord MSCs (hucMSCs) as a therapeutic approach in treatment of several diseases including neurological, cardiovascular, liver, kidney, and bone diseases as well as the cutaneous wound, inflammatory bowel disease, cancers, infertility, and other disorders.     

Placenta Stem/Stromal Cell–Derived Extracellular Vesicles for Potential Use in Lung Repair

Many acute and chronic lung injuries are incurable and rank as the fourth leading cause of death globally. While stem cell treatment for lung injuries is a promising approach, there is growing evidence that the therapeutic efficacy of stem cells originates from secreted extracellular vesicles (EVs). Consequently, EVs are emerging as next‐generation therapeutics. While EVs are extensively researched for diagnostic applications, their therapeutic potential to promote tissue repair is not fully elucidated. By housing and delivering tissue‐repairing cargo, EVs refine the cellular microenvironment, modulate inflammation, and ultimately repair injury. Here, the potential use of EVs derived from two placental mesenchymal stem/stromal cell (MSC) lines is presented; a chorionic MSC line (CMSC29) and a decidual MSC cell line (DMSC23) for applications in lung diseases. Functional analyses using in vitro models of injury demonstrate that these EVs have a role in ameliorating injuries caused to lung cells. It is also shown that EVs promote repair of lung epithelial cells. This study is fundamental to advancing the field of EVs and to unlock the full potential of EVs in regenerative medicine.     

Anti‐human CD9 antibody Fab fragment impairs the internalization of extracellular vesicles and the nuclear transfer of their cargo proteins

The intercellular communication mediated by extracellular vesicles (EVs) has gained international interest during the last decade. Interfering with the mechanisms regulating this cellular process might find application particularly in oncology where cancer cell‐derived EVs play a role in tumour microenvironment transformation. Although several mechanisms were ascribed to explain the internalization of EVs, little is our knowledge about the fate of their cargos, which are crucial to mediate their function. We recently demonstrated a new intracellular pathway in which a fraction of endocytosed EV‐associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into the nucleoplasmic reticulum. Silencing tetraspanin CD9 both in EVs and recipient cells strongly decreased the endocytosis of EVs and abolished the nuclear transfer of their cargos. Here, we investigated whether monovalent Fab fragments derived from 5H9 anti‐CD9 monoclonal antibody (referred hereafter as CD9 Fab) interfered with these cellular processes. To monitor the intracellular transport of proteins, we used fluorescent EVs containing CD9‐green fluorescent protein fusion protein and various melanoma cell lines and bone marrow‐derived mesenchymal stromal cells as recipient cells. Interestingly, CD9 Fab considerably reduced EV uptake and the nuclear transfer of their proteins in all examined cells. In contrast, the divalent CD9 antibody stimulated both events. By impeding intercellular communication in the tumour microenvironment, CD9 Fab‐mediated inhibition of EV uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti‐melanoma therapeutic strategies.     

Fungal Extracellular Vesicles with a Focus on Proteomic Analysis

Extracellular vesicles (EVs) perform crucial functions in cell–cell communication. The packaging of biomolecules into membrane‐enveloped vesicles prior to release into the extracellular environment provides a mechanism for coordinated delivery of multiple signals at high concentrations that is not achievable by classical secretion alone. Most of the understanding of the biosynthesis, composition, and function of EVs comes from mammalian systems. Investigation of fungal EVs, particularly those released by pathogenic yeast species, has revealed diverse cargo including proteins, lipids, nucleic acids, carbohydrates, and small molecules. Fungal EVs are proposed to function in a variety of biological processes including virulence and cell wall homeostasis with a focus on host–pathogen interactions. EVs also carry signals between fungal cells allowing for a coordinated attack on a host during infection. Research on fungal EVs in still in its infancy. Here a review of the literature thus far with a focus on proteomic analysis is provided with respect to techniques, results, and prospects.     

Proteomic Analysis of Extracellular Vesicles for Cancer Diagnostics

Extracellular vesicles (EVs) including exosomes and microvesicles are lipid bilayer‐encapsulated nanoparticles released by cells, ranging from 40 nm to several microns in diameter. Biological cargoes including proteins, RNAs, and DNAs can be ferried by EVs to neighboring and distant cells via biofluids, serving as a means of cell‐to‐cell communication under normal and pathological conditions, especially cancers. On the other hand, EVs have been investigated as a novel “information capsule” for early disease detection and monitoring via liquid biopsy. This review summarizes current advancements in EV subtype characterization, cancer EV capture, proteomic analysis technologies, as well as possible EV‐based multiomics for cancer diagnostics.     

Ischaemia alters the effects of cardiomyocyte‐derived extracellular vesicles on macrophage activation

Myocardial ischaemia is associated with an exacerbated inflammatory response, as well as with a deregulation of intercellular communication systems. Macrophages have been implicated in the maintenance of heart homeostasis and in the progression and resolution of the ischaemic injury. Nevertheless, the mechanisms underlying the crosstalk between cardiomyocytes and macrophages remain largely underexplored. Extracellular vesicles (EVs) have emerged as key players of cell‐cell communication in cardiac health and disease. Hence, the main objective of this study was to characterize the impact of cardiomyocyte‐derived EVs upon macrophage activation. Results obtained demonstrate that EVs released by H9c2 cells induced a pro‐inflammatory profile in macrophages, via p38MAPK activation and increased expression of iNOS, IL‐1β and IL‐6, being these effects less pronounced with ischaemic EVs. EVs derived from neonatal cardiomyocytes, maintained either in control or ischaemia, induced a similar pattern of p38MAPK activation, expression of iNOS, IL‐1β, IL‐6, IL‐10 and TNFα. Importantly, adhesion of macrophages to fibronectin was enhanced by EVs released by cardiomyocytes under ischaemia, whereas phagocytic capacity and adhesion to cardiomyocytes were higher in macrophages incubated with control EVs. Additionally, serum‐circulating EVs isolated from human controls or acute myocardial infarction patients induce macrophage activation. According to our model, in basal conditions, cardiomyocyte‐derived EVs maintain a macrophage profile that ensure heart homeostasis, whereas during ischaemia, this crosstalk is affected, likely impacting healing and post‐infarction remodelling.     

The role of extracellular vesicles in iron homeostasis and ferroptosis: Focus on musculoskeletal diseases

Iron homeostasis is crucial for maintaining proper cellular function, and its disruption is considered one of the pathogenic mechanisms underlying musculoskeletal diseases. Under conditions of oxidative stress, the accumulation of cellular iron overload and lipid peroxidation can lead to ferroptosis. Extracellular vesicles (EVs), serving as mediators in the cell-to-cell communication, play an important role in regulating the outcome of cell ferroptosis. Growing evidence has proven that EV biogenesis and secretion are tightly associated with cellular iron export. Furthermore, different sources of EVs deliver diverse cargoes to bring about phenotypic changes in the recipient cells, either activating or inhibiting ferroptosis. Thus, delivering therapies targeting ferroptosis through EVs may hold significant potential for treating musculoskeletal diseases. This review aims to summarize current knowledge on the role of EVs in iron homeostasis and ferroptosis, as well as their therapeutic applications in musculoskeletal diseases, and thereby provide valuable insights for both research and clinical practice.