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Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in the heart under homeostatic and pathological conditions, such as myocardial infarction (MI). However, the basic mechanisms driving cardiomyocyte‐derived EV (CM‐EV) production following stress are poorly understood. In this study, we generated human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) that express NanoLuc‐tetraspanin reporters. These modified hiPSC‐CMs allow for quantification of tetraspanin‐positive CM‐EV secretion from small numbers of cells without the need for time‐consuming EV isolation techniques. We subjected these cells to a panel of small molecules to study their effect on CM‐EV biogenesis and secretion under basal and stress‐associated conditions. We observed that EV biogenesis is context‐dependent in hiPSC‐CMs. Nutrient starvation decreases CM‐EV secretion while hypoxia increases the production of CM‐EVs in a nSmase2‐dependent manner. Moreover, the inflammatory cytokine TNF‐α increased CM‐EV secretion through a process involving NLRP3 inflammasome activation and mTOR signalling. Here, we detailed for the first time the regulatory mechanisms of EV biogenesis in hiPSC‐CMs upon MI‐associated stressors. [ABSTRACT FROM AUTHOR]

Abstract Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in the heart under homeostatic and pathological conditions, such as myocardial infarction (MI). However, the basic mechanisms driving cardiomyocyte‐derived EV (CM‐EV) production following stress are poorly understood. In this study, we generated human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) that express NanoLuc‐tetraspanin reporters. These modified hiPSC‐CMs allow for quantification of tetraspanin‐positive CM‐EV secretion from small numbers of cells without the need for time‐consuming EV isolation techniques. We subjected these cells to a panel of small molecules to study their effect on CM‐EV biogenesis and secretion under basal and stress‐associated conditions. We observed that EV biogenesis is context‐dependent in hiPSC‐CMs. Nutrient starvation decreases CM‐EV secretion while hypoxia increases the production of CM‐EVs in a nSmase2‐dependent manner. Moreover, the inflammatory cytokine TNF‐α increased CM‐EV secretion through a process involving NLRP3 inflammasome activation and mTOR signalling. Here, we detailed for the first time the regulatory mechanisms of EV biogenesis in hiPSC‐CMs upon MI‐associated stressors.

Extracellular vesicles (EVs) are involved in cell-to-cell communication. Following uptake, EV cargo molecules, including DNA, RNA, lipids, and proteins, influence gene expression and molecular signaling in recipient cells. Although various studies have identified disease-specific EV molecules, further research into their biogenesis and secretion mechanisms is needed for clinical application. Here, we investigated the role of UPF2 in regulating the biogenesis and components of EVs. Notably, UPF2 promoted the expression of CD81, a membrane protein marker of EVs, as UPF2 silencing decreased CD81 levels in EVs, both inside the cell and secreted. In contrast, the expression levels of CD63 increased, without altering the size or numbers of EVs. In addition, reducing UPF2 levels did not affect the total number of EVs but lowered production of CD81-positive EVs and reduced the efficiency of uptake by recipient cells. Collectively, our findings uncover a novel function for UPF2 in regulating the production of CD81 and changing EV properties.

Abstract Extracellular vesicles (EVs) are small, membrane-bound structures that are released from cells into the surrounding environment. These structures can be categorized as exosomes, microvesicles, or apoptotic vesicles, and they play an essential role in intercellular communication. These vesicles are attracting significant clinical interest as they offer the potential for drug delivery, disease diagnosis, and therapeutic intervention. To fully understand the regulation of intercellular communication through EVs, it is essential to investigate the underlying mechanisms. This review aims to provide a summary of the current knowledge on the intercellular communications involved in EV targeting, binding, and uptake, as well as the factors that influence these interactions. These factors include the properties of the EVs, the cellular environment, and the recipient cell. As the field of EV-related intercellular communication continues to expand and techniques improve, we can expect to uncover more information about this complex area, despite the current limitations in our knowledge.

Extracellular vesicles (EVs) are small, membrane-bound structures that are released from cells into the surrounding environment. These structures can be categorized as exosomes, microvesicles, or apoptotic vesicles, and they play an essential role in intercellular communication. These vesicles are attracting significant clinical interest as they offer the potential for drug delivery, disease diagnosis, and therapeutic intervention. To fully understand the regulation of intercellular communication through EVs, it is essential to investigate the underlying mechanisms. This review aims to provide a summary of the current knowledge on the intercellular communications involved in EV targeting, binding, and uptake, as well as the factors that influence these interactions. These factors include the properties of the EVs, the cellular environment, and the recipient cell. As the field of EV-related intercellular communication continues to expand and techniques improve, we can expect to uncover more information about this complex area, despite the current limitations in our knowledge. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are involved in cell-to-cell communication. Following uptake, EV cargo molecules, including DNA, RNA, lipids, and proteins, influence gene expression and molecular signaling in recipient cells. Although various studies have identified disease-specific EV molecules, further research into their biogenesis and secretion mechanisms is needed for clinical application. Here, we investigated the role of UPF2 in regulating the biogenesis and components of EVs. Notably, UPF2 promoted the expression of CD81, a membrane protein marker of EVs, as UPF2 silencing decreased CD81 levels in EVs, both inside the cell and secreted. In contrast, the expression levels of CD63 increased, without altering the size or numbers of EVs. In addition, reducing UPF2 levels did not affect the total number of EVs but lowered production of CD81-positive EVs and reduced the efficiency of uptake by recipient cells. Collectively, our findings uncover a novel function for UPF2 in regulating the production of CD81 and changing EV properties., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Oh, Mazan-Mamczarz, Gorospe, Noh and Kim.)

Extracellular vesicles (EVs) are membrane-bound particles released by cells in various (patho)physiological conditions. EVs can transfer effector molecules and elicit potent responses in recipient cells, making them attractive therapeutic agents and drug delivery platforms. In contrast to their tremendous potential, only a few EV-based therapies and drug delivery have been approved for clinical use, which is largely attributed to limited therapeutic loading technologies and efficiency. As EV cargo has major influence on their functionality, understanding and translating the biology underlying the packaging and transferring of biomolecule cargos (e.g. miRNAs, pathogen antigens, small molecule drugs) into EVs is key in harnessing their therapeutic potential. In this review, through recent insights into EVs' content packaging, we discuss different mechanisms utilized by EVs during cargo packaging, and how one might therapeutically exploit this process. Apart from the well-characterized EVs like exosomes and microvesicles, we also cover the less-studied and other EV subtypes like apoptotic bodies, large oncosomes, bacterial outer membrane vesicles, and migrasomes to highlight therapeutically-diverse opportunities of EV armoury. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are membrane-bound particles released by cells in various (patho)physiological conditions. EVs can transfer effector molecules and elicit potent responses in recipient cells, making them attractive therapeutic agents and drug delivery platforms. In contrast to their tremendous potential, only a few EV-based therapies and drug delivery have been approved for clinical use, which is largely attributed to limited therapeutic loading technologies and efficiency. As EV cargo has major influence on their functionality, understanding and translating the biology underlying the packaging and transferring of biomolecule cargos (e.g. miRNAs, pathogen antigens, small molecule drugs) into EVs is key in harnessing their therapeutic potential. In this review, through recent insights into EVs’ content packaging, we discuss different mechanisms utilized by EVs during cargo packaging, and how one might therapeutically exploit this process. Apart from the well-characterized EVs like exosomes and microvesicles, we also cover the less-studied and other EV subtypes like apoptotic bodies, large oncosomes, bacterial outer membrane vesicles, and migrasomes to highlight therapeutically-diverse opportunities of EV armoury.

Extracellular vesicles (EVs), such as exosomes, microvesicles, and apoptotic bodies, are cell-derived, lipid bilayer-enclosed particles mediating intercellular communication and are therefore vital for transmitting a plethora of biological signals. The vascular endothelium substantially contributes to the circulating particulate secretome, targeting important signaling pathways that affect blood cells and regulate adaptation and plasticity of endothelial cells in a paracrine manner. Different molecular signatures and functional properties of endothelial cells reflect their heterogeneity among different vascular beds and drive current research to understand varying physiological and pathological effects of blood and lymphatic endothelial EVs. Endothelial EVs have been linked to the development and progression of various vascular diseases, thus having the potential to serve as biomarkers and clinical treatment targets. This review aims to provide a brief overview of the human vasculature, the biology of extracellular vesicles, and the current knowledge of endothelium-derived EVs, including their potential role as biomarkers in disease development. [ABSTRACT FROM AUTHOR]

Abstract Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells.

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs), such as exosomes, microvesicles, and apoptotic bodies, are cell-derived, lipid bilayer-enclosed particles mediating intercellular communication and are therefore vital for transmitting a plethora of biological signals. The vascular endothelium substantially contributes to the circulating particulate secretome, targeting important signaling pathways that affect blood cells and regulate adaptation and plasticity of endothelial cells in a paracrine manner. Different molecular signatures and functional properties of endothelial cells reflect their heterogeneity among different vascular beds and drive current research to understand varying physiological and pathological effects of blood and lymphatic endothelial EVs. Endothelial EVs have been linked to the development and progression of various vascular diseases, thus having the potential to serve as biomarkers and clinical treatment targets. This review aims to provide a brief overview of the human vasculature, the biology of extracellular vesicles, and the current knowledge of endothelium-derived EVs, including their potential role as biomarkers in disease development.

Production of extracellular vesicles (EVs) involved in intercellular communication is a common capacity of most cell types. Upon encountering opsonized microorganisms, neutrophilic granulocytes release EVs that compromise bacterial growth. We carried out a systematic investigation of the involvement of potential opsonin receptors in EV-generation from human and murine neutrophils. Applying flow cytometric, proteomic and functional analysis as well as using genetically modified mice, we demonstrate that formation of antibacterial EVs depends upon stimulation of the multifunctional Mac-1 integrin complex, also called as complement receptor 3 (CR3), whereas activation of immunoglobulin binding Fc receptors or pattern recognition receptors alone or in combination is ineffective. Mac-1/CR3 stimulation and downstream tyrosine kinase signalling affect both the numbers, the cargo content and the antibacterial capacity of the produced vesicles. In contrast, Mac-1/CR3 signalling is not required for spontaneous EV formation, clearly indicating the existence of separate molecular pathways in EV biogenesis. We propose that EVs are “tailor-made” with different composition and functional properties depending on the environmental circumstances.

Production of extracellular vesicles (EVs) involved in intercellular communication is a common capacity of most cell types. Upon encountering opsonized microorganisms, neutrophilic granulocytes release EVs that compromise bacterial growth. We carried out a systematic investigation of the involvement of potential opsonin receptors in EV-generation from human and murine neutrophils. Applying flow cytometric, proteomic and functional analysis as well as using genetically modified mice, we demonstrate that formation of antibacterial EVs depends upon stimulation of the multifunctional Mac-1 integrin complex, also called as complement receptor 3 (CR3), whereas activation of immunoglobulin binding Fc receptors or pattern recognition receptors alone or in combination is ineffective. Mac-1/CR3 stimulation and downstream tyrosine kinase signalling affect both the numbers, the cargo content and the antibacterial capacity of the produced vesicles. In contrast, Mac-1/CR3 signalling is not required for spontaneous EV formation, clearly indicating the existence of separate molecular pathways in EV biogenesis. We propose that EVs are "tailor-made" with different composition and functional properties depending on the environmental circumstances. [ABSTRACT FROM AUTHOR]

We sought to characterize the lipid profile of skeletal muscle cell-derived Extracellular Vesicles (EVs) to determine if a hypertrophic stimulus would affect the lipid composition of C2C12 myotube-derived EVs. Analyses included C2C12 murine myoblasts differentiated into myotubes and treated with Insulin-Like Growth Factor 1 (IGF-1) for 24 h to induce hypertrophic growth. EVs were isolated from cell culture media, quantified using Nanoparticle Tracking Analysis (NTA) and analyzed using Transmission Electron Microscopy (TEM). EVs were homogenized and lipids extracted for quantification by Mass Spectrometry followed by downstream lipid class enrichment and lipid chain analysis. IGF-1 treatment elicited an increase in CD63 and CD81 levels (39% and 21%) compared to the controls (16%), respectively. Analysis revealed that skeletal muscle-derived EVs are enriched in bioactive lipids that are likely selectively incorporated into EVs during hypertrophic growth. IGF-1 treatment of myotubes had a significant impact on the levels of diacylglycerol (DG) and ceramide (Cer) in secreted EVs. Specifically, the proportion of unsaturated DG was two- to three-fold higher in EVs derived from IGF-treated cells, as compared to those from control cells. The levels of saturated DG were unaffected. Selective increases were similarly seen in C16- and C24-Cer but not in other species. Levels of free sphingoid bases tended to decrease, while those of sphingosine-1-phosphate was unaffected. Our results suggest that the lipid composition and biogenesis of skeletal muscle-derived EVs, are specific and highly selective during hypertrophic growth.

Epidermal growth factor receptor (EGFR) takes centre stage in carcinogenesis throughout its entire cellular trafficking odyssey. When loaded in extracellular vesicles (EVs), EGFR is one of the key proteins involved in the transfer of information between parental cancer and bystander cells in the tumour microenvironment. To hijack EVs, EGFR needs to play multiple signalling roles in the life cycle of EVs. The receptor is involved in the biogenesis of specific EV subpopulations, it signals as an active cargo, and it can influence the uptake of EVs by recipient cells. EGFR regulates its own inclusion in EVs through feedback loops during disease progression and in response to challenges such as hypoxia, epithelial-to-mesenchymal transition and drugs. Here, we highlight how the spatiotemporal rules that regulate EGFR intracellular function intersect with and influence different EV biogenesis pathways and discuss key regulatory features and interactions of this interplay. We also elaborate on outstanding questions relating to EGFR-driven EV biogenesis and available methods to explore them. This mechanistic understanding will be key to unravelling the functional consequences of direct anti-EGFR targeted and indirect EGFR-impacting cancer therapies on the secretion of pro-tumoural EVs and on their effects on drug resistance and microenvironment subversion.

Healthy cells constitutively release lipid bilayered vesicles of different sizes and recognizing different biogenesis, collectively referred to as extracellular vesicles (EVs). EVs can be distinguished in exosomes and microvesicles. Biological and biomedical research on EVs is an emerging field that is rapidly growing. Many EV features including biogenesis, cell uptake, and functions still require unambiguous elucidation. Nevertheless, it has been well established that EVs are involved in communication among cells, tissues, and organs under both healthy and disease conditions by virtue of their ability to deliver macromolecules to target cells. Here, we summarize most recent findings regarding biogenesis, structure, and functions of both exosomes and microvesicles. In addition, the use of EVs as delivery tools to induce CD8 + T cell immunity is addressed compared to current designs exploiting enveloped viral vectors and virus-like particles. Finally, we describe a both safe and original approach conceived for the induction of strong CTL immunity against antigens uploaded in EVs constitutively released by muscle cells., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

We sought to characterize the lipid profile of skeletal muscle cell-derived Extracellular Vesicles (EVs) to determine if a hypertrophic stimulus would affect the lipid composition of C2C12 myotube-derived EVs. Analyses included C2C12 murine myoblasts differentiated into myotubes and treated with Insulin-Like Growth Factor 1 (IGF-1) for 24 h to induce hypertrophic growth. EVs were isolated from cell culture media, quantified using Nanoparticle Tracking Analysis (NTA) and analyzed using Transmission Electron Microscopy (TEM). EVs were homogenized and lipids extracted for quantification by Mass Spectrometry followed by downstream lipid class enrichment and lipid chain analysis. IGF-1 treatment elicited an increase in CD63 and CD81 levels (39% and 21%) compared to the controls (16%), respectively. Analysis revealed that skeletal muscle-derived EVs are enriched in bioactive lipids that are likely selectively incorporated into EVs during hypertrophic growth. IGF-1 treatment of myotubes had a significant impact on the levels of diacylglycerol (DG) and ceramide (Cer) in secreted EVs. Specifically, the proportion of unsaturated DG was two- to three-fold higher in EVs derived from IGF-treated cells, as compared to those from control cells. The levels of saturated DG were unaffected. Selective increases were similarly seen in C16- and C24-Cer but not in other species. Levels of free sphingoid bases tended to decrease, while those of sphingosine-1-phosphate was unaffected. Our results suggest that the lipid composition and biogenesis of skeletal muscle-derived EVs, are specific and highly selective during hypertrophic growth. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (EGFR) takes centre stage in carcinogenesis throughout its entire cellular trafficking odyssey. When loaded in extracellular vesicles (EVs), EGFR is one of the key proteins involved in the transfer of information between parental cancer and bystander cells in the tumour microenvironment. To hijack EVs, EGFR needs to play multiple signalling roles in the life cycle of EVs. The receptor is involved in the biogenesis of specific EV subpopulations, it signals as an active cargo, and it can influence the uptake of EVs by recipient cells. EGFR regulates its own inclusion in EVs through feedback loops during disease progression and in response to challenges such as hypoxia, epithelial-to-mesenchymal transition and drugs. Here, we highlight how the spatiotemporal rules that regulate EGFR intracellular function intersect with and influence different EV biogenesis pathways and discuss key regulatory features and interactions of this interplay. We also elaborate on outstanding questions relating to EGFR-driven EV biogenesis and available methods to explore them. This mechanistic understanding will be key to unravelling the functional consequences of direct anti-EGFR targeted and indirect EGFR-impacting cancer therapies on the secretion of pro-tumoural EVs and on their effects on drug resistance and microenvironment subversion. [ABSTRACT FROM AUTHOR]

Production of extracellular vesicles (EVs) involved in intercellular communication is a common capacity of most cell types. Upon encountering opsonized microorganisms, neutrophilic granulocytes release EVs that compromise bacterial growth. We carried out a systematic investigation of the involvement of potential opsonin receptors in EV-generation from human and murine neutrophils. Applying flow cytometric, proteomic and functional analysis as well as using genetically modified mice, we demonstrate that formation of antibacterial EVs depends upon stimulation of the multifunctional Mac-1 integrin complex, also called as complement receptor 3 (CR3), whereas activation of immunoglobulin binding Fc receptors or pattern recognition receptors alone or in combination is ineffective. Mac-1/CR3 stimulation and downstream tyrosine kinase signalling affect both the numbers, the cargo content and the antibacterial capacity of the produced vesicles. In contrast, Mac-1/CR3 signalling is not required for spontaneous EV formation, clearly indicating the existence of separate molecular pathways in EV biogenesis. We propose that EVs are "tailor-made" with different composition and functional properties depending on the environmental circumstances., (© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)