Is Secondary Vesicle Release an Underappreciated Component of Extracellular Vesicle Signalling?

Amplification is essential to biological signalling, ensuring an efficient and sensitive response to ligands. It is a well-established principle of pharmacology that a minimal amount of ligand binding can be augmented to achieve a large-scale cellular response (Esam El-Fakahany and Becky Merkey 2025). Seminal work by Rodwell and Gilman outlined the role of G-proteins in activating “signal amplifiers” that generate large quantities of second messengers (Birnbaumer 2007). Since these foundational studies, recognized with the 1994 Nobel Prize in Physiology and Medicine (The Nobel Prize in Physiology or Medicine 1994), it has become increasingly clear that a single binding event can activate multiple G-proteins or phosphorylation-triggered signalling cascades, which can amplify at each stage (Nahorski 2006). Signal amplification thus allows the body to minimize the energy spent on signal detection itself and dedicate more resources to the response (see Figure 1A). It has long been recognized that signal amplification is necessary for extracellular vesicles (EVs) to achieve their biological effects. While numerous groups, including our own, have suggested that miRNA transfer by EVs plays a role in their biological effects (Viñas et al. 2016; Gong et al. 2017; Su et al. 2020; Nail et al. 2023), a seminal study by Chevillet et al. called this mechanism of action into question, suggesting that most EVs lack sufficient copies of miRNA to achieve translational repression through direct action (Chevillet et al. 2014). One could similarly question whether EVs possess sufficient material to trigger signalling through other common mechanisms such as surface receptor activation, membrane fusion, and EV uptake (Jahnke and Staufer 2024; Mulcahy et al. 2014; Liu and Wang 2023; Hirose et al. 2022). Further complexity is the fact that much of what we know about EV signalling is from cell culture studies, where EV levels achieved at targeted cells may far exceed those seen in vivo (Hagey et al. 2023). Downstream amplification is therefore a logical explanation for how EVs may attain many of their biological effects. Consistent with this, EVs have been shown to activate many common second messenger systems, including cyclic nucleotides (i.e., cAMP, cGMP [Bhadra et al. 2022, Sayner et al. 2019]), lipid-derived second messengers (i.e., IP3, DAG [Tu et al. 2023, Xia et al. 2022]), and arachidonic acid (Barry et al. 1999, Boilard 2018). In a recent perspective in the Journal of Extracellular Vesicles, Philip Askenase, of Yale University School of Medicine, lays out a hypothesis for how EVs achieve their diverse in vivo effects (Askenase 2025). This provocative viewpoint suggests that EVs signal, at least in part, by triggering release of secondary “effector EVs” from target cells. Dr. Askenase lays out the case for this mechanism of EV-induced EV release through a series of examples. First referenced is work focusing on stimulation of immune tolerance by EVs from CD8+ suppressor T cells (Nazimek et al. 2021). These primary EVs carry miRNA-150, which is transferred to macrophages, reprogramming them to release secondary EVs. In this instance, these macrophage-derived secondary EVs suppress CD4+ Th1 effector T cells, thereby modulating the immune response. Here, the downstream actions on effector T-cells were not achieved directly, but rather through the release of secondary macrophage-derived EVs (Nazimek et al. 2021). A second example provided is with the administration of mesenchymal stromal cell (MSC) EVs in a rat model with spinal cord injury (Nakazaki et al. 2021). Referencing his own group's work, Dr. Askenase notes the demonstrated uptake of MSC EVs by M2 macrophages in the damaged spinal cord and suggests that the release of targeted macrophage-derived secondary EVs serves as the final downstream mediator of administered primary MSC EVs (Nakazaki et al. 2021); in part by altering vascular permeability through induction of endothelial tight junction proteins in the spinal cord lesions (Nakazaki et al. 2021). This proposed framework for EV signalling offers a plausible explanation for multiple unanswered questions related to EV signalling. First, this would be a novel mechanism for amplification of EV signalling, as recipient cells could release large amounts of secondary EVs in response to comparatively few primary EVs. Indeed, in the aforementioned example of suppressor T cell EV actions, there appeared to be amplification of EV-associated miRNA signalling (Nazimek et al. 2021). The primary T-cell EVs were noted to be enriched in miRNA-150, but the macrophage-released secondary EVs also acted in an miRNA-150-dependent manner. If this model holds true in other systems, then it would explain how small amounts of EV-associated miRNA can achieve meaningful translational repression in vivo. Second, as Dr. Askenase lays out in his viewpoint, this framework would also explain the relative immune privilege of EV signalling, as endogenously produced secondary EVs would be recognized as ‘self’ by the immune system. Finally, EVs have always been remarkable for their regenerative/beneficial effects in such a wide array of diseases (Toh et al. 2018; Terriaca et al. 2021; Tieu et al. 2020). The release of secondary EVs from targeted macrophages (which are implicated in a similarly wide array of diseases [Yousaf et al. 2023]) could facilitate such diversity of advantageous effects. A number of questions immediately come to mind when considering this hypothesis. First, one wonders to what extent this mechanism of action contributes to the effects of EVs?. Second, what is the mechanism by which primary EVs induce the release of secondary EVs? Does it involve ectosomal or exosomal pathways? (see Figure 1B). Third, are secondary EVs the primary determinant of EV effects or merely a contributor seen under specific circumstances? This remains to be seen, but one hopes that this viewpoint stimulates an increased focus on clarifying the role of secondary EV release in the actions of EVs. If the mechanism of secondary EV release is a major contributor to EV signalling, then one wonders if EVs are particularly potent stimuli for secondary EV release. It is possible that there are other potential signals for endogenous EV release that could be exploited therapeutically. It remains plausible that primary EVs are also contributing to the final biological action in these circumstances. However, a methodology for distinguishing between primary and secondary EVs at the target site would be essential to addressing such questions. One potential approach would be to uniquely tag proteins or RNAs within primary EVs to distinguish them from induced secondary EVs. Finally, it is unclear if there are specific cell populations that are most effective in amplifying the response to EVs through the release of secondary EVs. Dr. Askenase focuses on macrophages, particularly so-called healing M2 macrophages, and these are an attractive choice given their widespread distribution and involvement in many pathogenic processes. However, one can also envision the endothelium as another potential hub given its direct contact with circulating EVs and extensive integration across organ systems. In summary, the release of secondary EVs proposed by Dr. Askenase is a plausible explanation for how EVs achieve many of their diverse biological effects. This mechanism would explain the extensive signal amplification, immune privilege, and ability to impact multiple disease processes. We look forward to further evidence to clarify the role of secondary EV release in in vivo EV signalling. Gary Sweeney: conceptualization, writing – review and editing, writing – original draft, formal analysis. Cydney Pitre: conceptualization, writing – review and editing, writing – original draft, formal analysis. Dylan Burger: conceptualization, investigation, writing – original draft, writing – review and editing, funding acquisition, supervision, resources, formal analysis. The authors declare no conflicts of interest. Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

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