Extracellular Vesicles as Emerging Players in Glaucoma: Mechanisms, Biomarkers, and Therapeutic Targets

Abstract

In recent years, extracellular vesicles (EVs) have attracted significant scientific interest due to their widespread distribution, their potential as disease biomarkers, and their promising applications in therapy. Encapsulated by lipid bilayers these nanovesicles include small extracellular vesicles (sEV) (30–150 nm), microvesicles (100–1000 nm), and apoptotic bodies (100–5000 nm) and are essential for cellular communication, immune responses, biomolecular transport, and physiological regulation. As they reflect the condition and functionality of their originating cells, EVs play critical roles in numerous physiological processes and diseases. Therefore, EVs offer valuable opportunities for uncovering disease mechanisms, enhancing drug delivery systems, and identifying novel biomarkers. In the context of glaucoma, a leading cause of irreversible blindness, the specific roles of EVs are still largely unexplored.

This review examines the emerging role of EVs in the pathogenesis of glaucoma, with a focus on their potential as diagnostic biomarkers and therapeutic agents. Through a thorough analysis of current literature, we summarize key advancements in EV research and identify areas where further investigation is needed to fully understand their function in glaucoma.

1 -. Introduction

Glaucoma, a leading cause of irreversible blindness worldwide, is characterized by the progressive degeneration of retinal ganglion cells (RGCs), which results in the gradual deterioration of the visual field. This chronic and progressive optic neuropathy damages the optic nerve head (ONH) and retinal nerve fiber layer (RNFL), leading to the loss of peripheral vision and, in severe cases, central vision as well^1–3. Globally, more than 70 million people are diagnosed with glaucoma, a number expected to increase to 112 million by 2040^1,3. The socioeconomic impact of this condition is profound, not only due to the loss of vision but also because of the associated healthcare costs and reduced quality of life.

Primary open-angle glaucoma (POAG) is the most common type of glaucoma in adults over the age of 40 and is responsible for approximately half of all glaucoma cases⁴. Elevated intraocular pressure (IOP) is recognized as the primary risk factor for the development and progression of glaucoma, with both pharmacological and surgical interventions primarily focusing on reducing IOP. While lowering IOP typically slows disease progression in both POAG and normal tension glaucoma (NTG), treatment outcomes can vary significantly^5,6. In some patients, optic nerve damage may continue to progress even with effectively controlled IOP. This is particularly evident in NTG, where disease progression occurs despite IOP levels remaining within the normal range, highlighting the complexity of glaucoma’s underlying mechanisms^5,6.

As a multifactorial disease, the pathogenesis of glaucoma is complex and involves a variety of pathological processes, including oxidative stress, impaired microcirculation, mechanical damage from elevated intraocular pressure (IOP), chronic neuroinflammation, mitochondrial dysfunction, and extracellular matrix (ECM) remodeling, all of which contribute to irreversible RGC damage. There are also numerous sites of glaucomatous damage; the conventional outflow pathway in the anterior segment, the ONH, and RGCs^7–9. Many of the mechanisms underlying these processes continue to be poorly understood, underscoring the need for further research in this area.

The ocular drainage system, crucial for maintaining IOP, relies on the balance between AH production by the non-pigmented ciliary epithelium (NPCE) and its drainage through conventional and unconventional (uveoscleral) outflow pathways. The conventional pathway, which provides the bulk of resistance to AH outflow, is regulated by cells of the trabecular meshwork (TM), Schlemm’s canal (SC), and distal vessels. ^10,11 The primary source of outflow resistance lies within the juxtacanalicular tissue (JCT) of the TM and the inner wall basement membrane of SC, where there is a significant buildup of extracellular matrix (ECM) materials.^10,12 Acott et al recently highlighted what we currently know about the conventional outflow pathway, including the role of ECM in outflow resistance¹³.

The elevated IOP causes chronic damage at the ONH, leading to its deformation and thinning and compression of the lamina cribrosa (LC) tissue in this region. This results in vision loss due to compression of RGC axons by the thinning LC, and increased ECM material deposition. Astrocytes and microglia at the ONH have also been shown to be dysregulated in glaucoma, and likely contribute to the accumulation of excess ECM materials in this region of the eye.^14–16 For a more in-depth examination of glaucomatous damage at the ONH, please see reviews by Abe et al.¹⁷ and Hernandez and Pena¹⁸.

EVs have emerged as critical players in intercellular communication, with their role in the pathology of many diseases, including glaucoma, gaining increasing attention. They are ubiquitously present in a variety of biological fluids, including tears, aqueous and vitreous humor, and circulating blood plasma, indicating their extensive distribution and potential significance in biological processes^19–22. Discovered by Pan and Johnstone in 1983 in sheep reticulocytes, the then-called “exosomes” were initially characterized as byproducts of the cellular differentiation process, specifically associated with the disposal of transferrin receptors ²³. The composition of EVs varies according to their cell of origin and the external environment, which contributes to their diverse biological functions. These functions include facilitating intercellular communication, cellular waste management, and involvement in inflammatory and immune responses, as well as tissue repair and regeneration processes. The lipid composition of EVs not only confers stability in the extracellular environment but also provides protection against enzymatic degradation, enhancing their potential as stable carriers of molecular cargoes²⁴.

The prevalence of glaucoma and the challenges in early diagnosis and treatment, due to the eye’s complex anatomy and physiological barriers, underscore the need for innovative diagnostic and therapeutic strategies ^25,26. EVs play a role in the development of ocular diseases, with alterations in their cargo mirroring changes in disease states, thus offering potential as diagnostic biomarkers. As natural carriers, EVs exhibit superior barrier-crossing capabilities and safety compared to synthetic nano-drug carriers, making them promising candidates for drug delivery and bioactive molecule transport. This article aims to detail the role of EVs in the pathogenesis, diagnosis, and treatment of glaucoma, emphasizing their therapeutic potential and outlining critical insights into their biogenesis, composition, functions, and methodologies for isolation, purification, and engineering for clinical applications.

2-. EV Definition and Classification

EVs are now recognized as a distinct class of cell-derived particles, encapsulated by a lipid bilayer, and devoid of replicative structures such as a functional nucleus. This updated definition aligns with the latest Minimal Information for Studies of Extracellular Vesicles 2023 (MISEV2023)²⁷ guidelines, presenting a significant evolution from the MISEV2018 recommendations²⁸. The MISEV2023 broadens the scope of EV research to include both naturally occurring and artificially engineered vesicles, thereby expanding the EV research framework to include vesicles produced under various laboratory conditions. The International Society for Extracellular Vesicles (ISEV) underscores the necessity for precision in employing operational terms with ‘EV’ to enhance scientific clarity. Traditionally, EVs were classified into three subtypes based on their origin, biogenesis, size, cargo, and surface markers: exosomes (30-150 nm), microvesicles (100-1000 nm), and apoptotic bodies (500-2000 nm), each with distinct characteristics.^20–22 The MISEV2023 guidelines, however, advocate for using size descriptors like ‘small’ and ‘large’ EVs, with clearly defined criteria, acknowledging the overlapping sizes resulting from current separation techniques. Further, the guidelines urge careful use of terms related to the biogenesis of EVs, such as ‘exosomes’ for those released via multivesicular bodies (MVBs), and ‘ectosomes’ or ‘microvesicles’ for those budding from the cell surface. This is due to the challenges in distinguishing EVs based solely on their cellular origin, given the overlapping features among EV populations isolated by standard techniques. Consequently, the society advises against using the term ‘small EVs’ to specifically refer to ‘exosomes,’ since such populations may contain both small ectosomes and exosomes. Researchers are recommended to use such specific terms only when they have distinctly separated and characterized these EVs.

In this review, we will use EVs as a generic term for the entire group of vesicles secreted from cells, since most research described in the literature has utilized mixed populations of EVs, and has not characterized the isolated EVs according to these newly published guidelines.

3-. EV Biogenesis and Secretion

EVs are formed through multiple mechanisms. EVs are broadly classified into two major subtypes based on their cellular origin and biogenesis: exosomes and ectosomes, the latter are also known as microvesicles.²⁹

Exosomes are small EVs, approximately 50–150 nm in diameter, that form when the endosomal membrane buds inward, creating intraluminal vesicles (ILVs). The endosome, now termed a multivesicular endosomes (MVE), fuses with the plasma membrane to release these ILVs as exosomes into the extracellular space or converge with lysosomes for degradation.^29–31 . The formation of exosomes involves several distinct pathways.

The Endosomal Sorting Complex Required for Transport (ESCRT) pathway is considered the canonical mechanism in exosome biogenesis; exosome formation also involves several ESCRT-independent pathways including the ceramide-dependent pathway and the tetraspanin-mediated pathway. The balance between these pathways varies depending on the cargo, cell type, and external signals.³¹ For instance, MHC class II proteins can be targeted to MVEs through both ESCRT-dependent and ESCRT-independent mechanisms^32,33. Similarly, the melanocyte protein PMEL is sorted by both pathways, with the luminal domain utilizing an ESCRT-independent mechanism and the transmembrane domain relying on ESCRT³⁴. These pathways often act concurrently or sequentially, resulting in subpopulations of MVEs with distinct lipid and protein compositions, which in turn leads to heterogeneous populations of ILVs and exosomes. Therefore, although the ESCRT pathway is a primary driver of exosome biogenesis, several sorting mechanisms contribute to the formation of diverse exosome subpopulations^34,35.

Regardless of the initial pathway, the MVEs merge with the cellular membrane, releasing ILVs as exosomes into the extracellular space, facilitated by RAS-related proteins, specifically RAB27A and RAB27B³¹. The intersection of exosome biogenesis with other cellular trafficking pathways introduces an additional layer of complexity, as proteins such as Rab GTPases are also involved in processes like autophagy and lysosomal degradation. This interplay underscores the multifaceted nature of intracellular vesicle trafficking and its regulation³¹.

Ectosomes, also known as microvesicles (MVs), originate from outward protrusions of the plasma membrane and are excised directly into the extracellular space^29,31. MVs generally range from 50 to 1,000 nm in diameter, but can also reach several micrometers in size. A notable example is large oncosomes, which are microvesicles produced by tumor cells and can measure between 1 and 10 μm ^29,31,36.

The biogenesis of microvesicles is less understood than that of exosomes. One proposed mechanism involves the recruitment of the ESCRT machinery, which promotes the formation of ILVs within MVBs. Both exosomes and microvesicles can form through the recruitment of ESCRT-III proteins that promote membrane curvature and subsequent vesicle fission^37,38. Specifically, the adaptor protein arrestin domain-containing protein 1 (ARRDC1) recruits ESCRT proteins TSG101 (ESCRT-I) and VPS4 (an accessory protein) to the plasma membrane, contributing to microvesicle formation ³⁹.

Vesicle budding can also occur in response to plasma membrane injury as a repair mechanism^40,41. Similar to exosome biogenesis, alterations in plasma membrane lipid asymmetry appear crucial. Enzymes like flippases, floppases, and scramblases transfer lipids between membrane leaflets, influencing membrane curvature and vesicle formation⁴². Changes in ceramide content on the outer leaflet, triggered by the activation of acid sphingomyelinase, can also induce membrane curvature and initiate microvesicle release^43,44. Shedding of vesicles has been observed from various regions of the plasma membrane, such as microvillar protrusions of intestinal epithelial cells⁴⁵, cilia⁴⁶, and from cells engineered to overexpress hyaluronan synthase⁴⁷.

4-. EV content

The molecular composition of EVs mirrors the physiological or pathological state of the originating cells, making them a potent biomarker for various diseases⁴⁸. The cargo of EVs encompasses a wide range of biological molecules. This molecular diversity includes not just proteins and nucleic acids but also lipids, enzymes, and metabolites, offering insights into the cell’s health and activity. Such molecular complexity positions EVs as key mediators of intercellular communication, with their contents having significant implications for disease progression, response to treatment, and overall understanding of pathobiology⁴⁹. Particularly, exosomes, the smallest type of EVs, encapsulate a diverse array of bioactive molecules such as proteins, nucleic acids, lipids, and sugars. The Exocarta database, which catalogs exosomal contents, lists thousands of proteins, mRNAs, miRNAs, and lipids identified across numerous studies, highlighting the variability of exosomal cargo depending on the cell’s condition⁵⁰.

Deep sequencing studies have revealed that EVs harbor a vast spectrum of RNA biotypes, including intact messenger RNA (mRNA), circular RNA (circRNA), and a variety of non-coding RNA types such as microRNA (miRNA), small nuclear RNA (snRNA), long non-coding RNA (lncRNA), vault RNA, Y-RNA, transfer RNA (tRNA), and ribosomal RNA (rRNA)^51,52. Significantly, the RNA content of EVs is stable and shielded from RNase degradation⁵³. This protection ensures that the RNAs within EVs can efficiently modify the phenotype of recipient cells following uptake, underscoring the critical role of EVs in intercellular communication and their potential in therapeutic strategies. MiRNAs play a critical role in regulating several pathways and processes implicated in glaucoma, including apoptosis, autophagy, neurogenesis, aging, extracellular matrix remodeling, oxidative stress, inflammation, and angiogenesis⁵⁴. These small non-coding RNAs are crucial for maintaining the balance of aqueous humor^55–58 modifying the trabecular meshwork (TM) ^59–62, maintaining the cells of the optic nerve head^63,64 and influencing retinal ganglion cell apoptosis^65–67. Research indicates that the expression patterns of miRNAs are distinctly altered in both human glaucoma patients and animal models, underscoring their significant impact on the disease’s pathophysiology.

EVs are not just carriers of genetic material; they are also enriched in proteins due to their biogenesis processes. Apart from cell-surface and soluble proteins present in the extracellular space, EVs also carry a variety of intracellular proteins from the secreting cell. According to the latest update of the ExoCarta database (2023)⁵⁰, EVs from various organisms have been documented to contain 41,860 proteins, 7,784 RNA entries, and 1,116 lipid species.

Furthermore, similar to nucleic acids, EV proteins are stable⁶⁸ and can directly influence target cells. Given that their protein content, along with nucleic acids like miRNA, reflects the characteristics of the originating cell, EVs offer valuable insights for diagnosing and monitoring diseases⁶⁹.

5-. EV-mediated cellular communication

EVs facilitate intercellular communication through two primary mechanisms⁷⁰: 1- surface ligand-mediated interactions, without delivery of the EV contents into the recipient cell, This process has been extensively researched in the context of immunomodulation by activating or inhibiting immune responses^71,72; and 2- cellular internalization or membrane fusion, leading to the transfer of EV contents to recipient cells. The second mechanism allows the transfer of microRNAs, messenger RNAs, and other genetic materials, thereby altering gene expression or signaling pathways in the acceptor cells. This process has been explored for therapeutic applications, leveraging EVs’ natural cargo delivery system^73–75.

Regarding the uptake of EVs by cells, similarities with viral entry mechanisms have shed light on potential pathways⁷⁶, including direct membrane fusion and various forms of endocytosis^77,78. While some studies have observed direct fusion of EVs with the plasma membrane, the majority of evidence supports endocytosis as the prevalent route for EV internalization^79,80. Endocytosis encompasses several mechanisms, such as receptor-mediated (clathrin-mediated), caveolin-mediated, and others, allowing for the specific uptake of EVs depending on the ligand-receptor interactions between the EVs and the recipient cells. Manipulating these pathways to enhance the specificity of EV uptake could advance the development of targeted EV-based therapies^77,78,81.

Once entering the cell, the mechanism by which EV cargo is released remains uncertain. A possible scenario involves the fusion of EVs with endosomal membranes, facilitating the delivery of cargo into the cytoplasm of the recipient cell³⁰. EV cargo may escape into the cytoplasm either through direct fusion with the plasma membrane or by a back-fusion process, in which EVs fuse with the limiting membrane of MVEs, releasing their contents into the cytosol before degradation. However, the exact mechanism of this back-fusion process remains largely unknown.⁸²

6-. EVs in Glaucoma

While the precise cause of POAG remains unclear, one theory proposes that the production and drainage of AH are influenced by signal transmission through autocrine, endocrine, and exocrine processes among tissues. Coca-Prados et al. initially proposed the hypothesis of intercellular communication within this system, although the mechanisms of such interactions remain to be elucidated⁸³. The detection of active proteins in the AH substantiates the concept of tissue communication, indicating a dynamic biochemical environment. These proteins include unique AH constituents and others commonly involved in cellular signaling, such as cytokines⁸⁴, kinases⁸⁵, phosphatases⁸⁶, and growth factors⁸⁷.

Empirical evidence supporting the communication between ocular drainage tissues has been further bolstered by in vitro studies. For instance, co-culturingNPCE with TMcells resulted in a marked increase in the activity of certain enzymes, including matrix metalloproteinases within TM cells ⁸⁸. However, the mechanism by which proteins and enzymes, traditionally understood to operate within cells, exert effects in the extracellularly located AH remains a subject of inquiry.

One possible mechanism for this extracellular communication is the involvement of EVs. EVs mediate intercellular communication by transporting proteins, lipids, and nucleic acids between cells. EVs are secreted into the extracellular environment by a variety of cell types, such as epithelial cells, bone marrow-derived cells, and TM cells^89,90. Specifically, in ocular environments, EVs are present in tear fluid⁹¹, AH¹⁹, vitreous humor ⁹², and blood^93,94. EVs act as mediators of cells’ paracrine effects, playing a crucial role in both physiological and pathological conditions⁹⁵. This suggests that EVs could play a crucial role in the transfer of signaling molecules within the ocular drainage system, offering a novel perspective on the regulation of IOP and the pathophysiology of glaucoma (Figure 1, 2). The TM is one of the key regulators of IOP, and shows significantly increased accumulation of ECM materials throughout the tissue in patients with glaucoma. The exact mechanism that triggers ECM accumulation in the TM is unknown, however, dysfunctional EVs released from TM cells may be involved in this process.

Figure 1: Schematic Overview of Aqueous Humor Outflow and EV-Mediated Crosstalk Among Non-Pigmented Ciliary Epithelial, Trabecular Meshwork, and Schlemm’s Canal Cells.

This figure illustrates the pathway of aqueous humor flow through the trabecular meshwork (TM) structures. Aqueous humor flows from the uveal meshwork (UM), through the corneoscleral meshwork (CSM) and juxtacanalicular connective tissue (JCT), into Schlemm’s canal (SC), and finally drains into the episcleral veins. A magnified section highlights the secretion and dynamic crosstalk of EVs among non-pigmented ciliary epithelial cells (NPCE, red), TM cells (green), and Schlemm’s canal endothelial cells (orange), showcasing their roles in cellular communication and maintaining intraocular pressure.

Figure 2: Schematic Overview of the Optic Nerve Head.

This figure illustrates the optic nerve head, highlighting five key tissue regions: the sclera, lamina cribrosa, pre-laminar and post-laminar neural tissues, and the pia mater (top). For clarity, blood vessels are not depicted. A magnified section details the secretion and dynamic crosstalk of EVs among the components of the lamina cribrosa, including lamina cribrosa (LC) cells (green), astrocytes (red), and microglial cells (orange). This interaction plays a crucial role in cellular communication and the glaucomatous cupping of the lamina cribrosa.

6-1-. EVs and Extracellular Matrix Remodeling

The ECM serves as the structural scaffold of tissues and actively modulates cellular functions such as growth, migration, and differentiation. It consists of major components like collagens, laminins, fibronectin, and proteoglycans. Cellular engagement with the ECM is facilitated by adhesion molecules including integrins, cadherins, and transmembrane proteoglycans, which support the migration of cells through the ECM. Furthermore, cells can modify the ECM by either depositing new components or by breaking down existing ones using enzymes like MMPs^96–98. There are two major sites of ECM remodeling in glaucoma: the TM and ONH Pathological ECM remodeling at these locations affects the biomechanical response of the tissues to fluctuating IOP, reducing the tissues’ ability to effectively respond to elevations in IOP as part of disease pathophysiology.

While EVs are conventionally known to circulate freely in biological fluids, within tissues they inevitably interact with the ECM, which they must traverse. This movement can be either active, as seen with cancer-derived EVs, or passive, influenced by the combined properties of both EVs and the ECM⁹⁹. The ECM serves as a supportive environment, facilitating EV transport while influencing their content and release. Thus, the ECM plays a more dynamic role than merely being a medium that EVs must traverse^100,101.

TM cells are one of the key regulators of IOP and show significantly increased accumulation of ECM materials throughout the tissue in patients with glaucoma. The exact mechanism that triggers ECM accumulation in the TM is unknown; however, dysfunctional EVs released from TM cells may be involved in this process. TM cells play a crucial role in maintaining the ECM, which creates a critical microenvironment for the TM’s biomechanical function. The ECM prominently features collagen types I, III, and IV as key structural components ^102,103 Excessive accumulation of ECM, particularly collagen type I^104,105, along with reduced levels of hyaluronic acid, can impair AH outflow and lead to increased IOP¹⁰⁶. To counteract this, TM cells modulate MMPs, especially MMP-2 and MMP-9, which promote ECM turnover and maintain outflow efficiency. However, in glaucoma, this process is disrupted, leading to increased ECM accumulation in the juxtacanalicular connective tissue (JCT)region of the TM; a key factor in this is likely the imbalance between MMPs and tissue inhibitors of metalloproteinases (TIMPs)^103,107. The continuous remodeling of the ECM within the JCT is thought to be an adaptive mechanism to accommodate fluctuations in IOP, allowing variable AH flow rates through regions with differential ECM gene expression and when this is interrupted, glaucomatous disease progresses^{10,89,108,109}.

ECM remodeling in the ONH is a critical process in the development of glaucoma, marked initially by the earliest signs of damage in this region. In the human lamina cribrosa (LC), an imbalance between ECM protein formation and degradation leads to significant structural changes, which contribute directly to the damage and eventual apoptosis of RGC axons. ONH astrocytes, LC cells, and microglia are key players in this process¹⁴. The LC in the ONH is a trabeculated connective tissue composed of stacks of cribriform plates, which are densely packed with collagen fibrils and elastin fibers¹⁵. This structural composition is critical in understanding the progression of glaucoma, primarily due to its role as the primary site of RGC axonal damage¹⁶. In both human and animal models of glaucoma, ECM remodeling within the LC is marked by reductions in elastin content¹¹⁰, buckling and disconnection of elastin fibers from the ECM¹¹¹, and the loss of fiber-forming types of collagens^112,113. There is also a proliferation of type IV collagen in areas previously occupied by RGC axons before damage^114,115, along with an accumulation of chondroitin sulfate glycosaminoglycans (GAGs)¹¹⁶.

LC cells are central to ONH remodeling in glaucoma responding to mechanical strain, oxidative stress, and hypoxia. In healthy eyes exposed to mechanical strain, human LC cells in culture exhibit changes in gene expression related to ECM components, cell proliferation, growth factors, and cell surface receptors¹¹⁷. Additionally, these cells react to oxidative stress by upregulating fibrotic genes and increasing the production of collagen and alpha-smooth muscle actin (α-SMA)¹¹⁸. Under hypoxic conditions, LC cells further demonstrate heightened expression of factors such as macrophage migration inhibitory factor and discoidin domain receptor^119–121. These adaptive responses maintain a balance to allow the ocular tissues to respond to normal IOP fluctuations, but when this balance is lost, these responses are integral to the pathogenesis of glaucoma, compelling LC cells to secrete key fibrotic molecules and underscoring their critical role in the progression of glaucoma.

Previous research has demonstrated elevated levels of TGFβ in the AH and ECM of the TM as key molecular markers of glaucoma, particularly in POAG¹²². Recent findings suggest that this elevated TGFβ may trigger pro-fibrotic pathway activation in both TM and LC tissues¹²³. Such activation leads to ECM remodeling, making the TM less efficient at draining AH and rendering the LC more vulnerable to damage from elevated IOP due to ECM transformation in the LC¹²³. Moreover, MMP-2 expression and activity are increased in glaucomatous conditions^117,118,124. Thrombospondin-1 (TSP1) and MMP expression is upregulated in LC cells under mechanical stress, facilitating localized ECM degradation and allowing LC cells to migrate within the ONH^117,125. These changes lead to a stiffened ECM and diminished tissue compliance, potentially heightening the vulnerability to IOP-induced damage.

Astrocytes, like LC cells, exhibit mechanosensitive properties and respond to glaucomatous conditions in both human and animal models^126–129. Under normal conditions in the ONH, astrocytes primarily support the blood-brain barrier (BBB). However, type 1B astrocytes, which are predominant in the ONH, collaborate with LC cells in ECM production when exposed to glaucomatous stress ^113,130. These astrocytes form connections with other astrocytes and LC cells, facilitating coordinated responses to mechanical strain. When IOP increases, astrocytes undergo actin reorganization within hours, which can return to baseline levels over similar periods^131,132. Additionally, reactive astrocytes produce MMPs, which, like LC cells, may break down ECM components to enable cellular movement and ECM remodeling¹³³.

Microglia in the ONH are central to the pathophysiological transformations in the ECM associated with glaucoma. Their early activation releases damage-associated molecular patterns (DAMPs) that trigger toll-like receptor 4 (TLR4), enhancing the signaling pathways associated with fibrogenesis, particularly TGFβ2 signaling known to be upregulated in glaucomatous conditions¹⁴. This activation leads to increased ECM production, contributing to the imbalance between ECM protein formation and degradation in the LC region. This imbalance is directly linked to the damage of RGC axons and subsequent apoptosis.

A study by McDowell et al. demonstrates that microglia, through TLR4 activation, significantly influence ECM remodeling by upregulating proteins such as fibronectin and laminin, highlighting their vital role in modulating the ECM environment within the ONH during glaucoma progression¹⁴.

Given the complexities of ECM dysregulation, further research is essential to unravel the mechanisms underlying glaucomatous pathophysiology. Recent studies highlight the significance of EVs in ECM remodeling, suggesting their role in intercellular communication and as carriers of molecular signals. The interplay between EVs and ECM could provide key insights into the molecular pathways driving glaucomatous neurodegeneration.

EVs first interact with the ECM when they enter the intercellular space and the interactions between EVs and ECM significantly influence their biological effects. For over 50 years, matrix EVs have been recognized for their role in the mineralization of developing hard tissues like calcified cartilage, bone, and dentin, highlighting the importance of EVs in matrix remodeling¹³⁴. Research by Huleihel et al. showed that biologic scaffold materials contain active EVs bound by matrix components, reinforcing the role of EVs as integral structural and functional components of the ECM¹⁰⁰.

EVs play an active role in ECM remodeling through surface molecular interactions and the downstream effects of their cargo^135,136. Evidence from cancer-derived and ocular EVs reveals that proteases and glycosidases on their surface contribute to matrix remodeling and degradation¹³⁶. For example, the surface enzyme heparanase, retained on EVs during their biogenesis, degrades heparan sulfate chains in the ECM, releasing associated signaling molecules; Dismuke et al identified the heparan sulfate complexes on EVs as a pathway for regulation of fibronectin within the TM ^136,137. Similarly, other cell surface enzymes like Membrane-Type 1 Matrix Metalloproteinase (MT1-MMp, MMP14,) degrade matrix proteins such as fibrillar collagens and fibronectin or activates MMP-2 , thus promoting matrix remodeling^136,138. EVs also have been identified to carry surface-bound enzymes that modify the ECM, such as lysyl oxidase (LOX), elastase, and collagenase. For instance, research on breast cancer, highlighted how LOX catalyzes the cross-linking of collagen, increasing the matrix stiffness and influencing local cancer cell behaviors by enhancing collagen cross-linking and upregulating Growth Factor Receptor-dependent Phosphoinositide 3-kinases (PI3K) signaling¹³⁹. Similar mechanisms appear to play a role in the TM of both normal and POAG eyes, where appropriate levels of cross-linking are crucial for ECM’s resistance to degradation and mechanical strength. However, excessive cross-linking can obstruct ECM remodeling, which is crucial for TM functionality. Enzymes such as LOX and LOX-like enzymes 1-4 (LOXL1-4), which cross-link lysine residues in collagens and elastin, are notably expressed in TM cells^140–143.

Hariani et al. explored the role of LOXL1 in the ONH, particularly in the context of reactive astrocytosis in primary rat ONH astrocytes (ONHA) following exposure to mechanical strain. Their finding indicated that reduced expression of LOXL1 led to decreased elastin production, which can disrupt cell-matrix interactions and impair cell signaling. This study also highlighted the role of astrocyte-derived EVs (ADEs) in neuron-glia communication. ADEs from LOXL1-deficient ONHA exhibited decreased trophic effects on neurite outgrowth, suggesting a critical role of LOXL1 in maintaining not only ECM integrity but also in modulating EV signaling essential for neuronal health in the ONH. These observations emphasize the importance of LOXL1 in the pathology of glaucoma, particularly under mechanical stress conditions, and suggest that alterations in LOXL1 and EV-mediated signaling are potential areas for further research to understand disease progression better¹⁴⁴. Despite these insights, there remains a gap in research specifically addressing the role of LOXL1 and EVs in trabecular meshwork cells in glaucoma. Given the significant impact of LOXL1 on ECM dynamics and astrocyte-mediated neuronal support in the optic nerve head, along with the established role of LOXL1 in the development of pseudoexfoliation syndrome, and pseudoexfoliation glaucoma (PEXG), it is plausible to hypothesize that similar mechanisms could influence the ECM of the trabecular meshwork in both POAG and PEXG. The potential for LOXL1-modified EVs to affect ECM remodeling and cellular signaling within the TM highlights a critical area for future research, aiming to elucidate their roles in intraocular pressure regulation and glaucoma pathophysiology.

EVs not only facilitate matrix degradation but also play a role in ECM synthesis by influencing target cells. In foreskin fibroblasts, for example, stress-relaxation triggers an ectocytic process that leads to the release of membrane-derived EVs into the collagen matrix¹⁴⁵. Research indicates that EVs significantly influence ECM formation, as their specific cargo components are key to altering collagen fiber formation and adhesion assembly in various cell types, including those in the TM. A key example is miR-29b, which is known to be involved in ECM regulation and has been shown to be a component of EVs derived from non-pigmented ciliary epithelium (NPCE) cells (see section 5–4). Its role in negatively regulating ECM synthesis genes in TM cells under oxidative stress is critical; a reduction in miR-29b levels can lead to increased ECM gene expression, potentially contributing to elevated IOP in glaucoma^61,146,147.

Additionally, TM cells, when cultured on stiffer, pathomimetic substrates, typically exhibit a robust actin cytoskeleton network, elevated α-smooth muscle actin expression (indicating a fibrotic cell type), and activated focal adhesion kinase. Conversely, on softer, homeomimetic hydrogels, these cells display significantly reduced attachment and proliferation rates^148,149. The actin cytoskeleton is crucial in mediating TM cell stiffness and their response to ECM-mediated mechanical cues through integrin-dependent adjustments¹⁵⁰. Expanding on these insights, novel glaucoma treatments designed to lower IOP have been developed using actin cytoskeleton disruptors such as latrunculin A/B, and Rho kinase inhibitors such as netarsudil^151–153. We also discovered differences in the proteomic content of EVs isolated from glaucomatous and non-glaucomatous cells demonstrating that there is an increased number of proteins found in glaucomatous TM EVs that correlate with cytoskeletal molecular pathways¹⁰. This further underscores the complex relationship between the cellular mechanical environment and critical cellular functions, including the therapeutic targeting of these interactions in glaucoma. In our recent study, EVs from TM cells revealed an abundance of ECM-related proteins, notably fibronectin, collagen, and EDIL3, which mediate ECM protein binding and internalization¹⁰. Remarkably, 72% of the top identified proteins were ECM-related, indicating their pivotal role in cell communication and ECM dynamics. Our comparative study showed a 55% decrease in fibronectin binding to EVs from glaucomatous TM (GTM) cells compared to normal TM (NTM) cells, suggesting alterations in EV function related to glaucoma ¹⁰(Figure 2). This binding capacity can be increased by 63% through mechanical stretch pre-conditioning, suggesting a pivotal regulatory mechanism in stress response within the TM, emphasizing their involvement in ECM opsonization, degradation, and homeostasis. Crucially, the findings also indicate that this mechanism may be impaired in GTM cells, possibly as a result of tissue stiffening or reduced EV opsonization and binding¹⁰. In addition, EVs from TM cells of patients with POAG are significantly smaller than those from healthy controls, a difference attributed to variations in membrane phospholipid content and the activity of phospholipid conversion enzymes¹⁵⁴. This suggests a diminished capacity for loading ECM recycling proteins^10,154. Quantitative analysis of AH in a Korean population, demonstrated significant EV differences between pseudoexfoliation glaucoma (PXFG) patients and controls ¹⁵⁵. The study revealed elevated EV counts in the PEX group, suggesting a potential role in the disease’s pathogenesis. Key EV markers, including CD63, CD81, CD9, and syntenin, were significantly higher in PEX patients, highlighting their promise as biomarkers for diagnosing and prognosticating PEX glaucoma. These initial results advocate for further research with larger sample sizes to establish EV diagnostic and prognostic utility in glaucoma, offering a new direction for understanding glaucoma at the molecular level¹⁵⁵.

In summary, the interplay between EVs and the ECM is bidirectional, with EVs actively remodeling the matrix and being influenced by the ECM’s physical properties. Their role as signaling cues within the ECM and their potential to promote tissue repair position them as promising therapeutic tools for regenerative medicine. Further research into EV biogenesis, cargo, and interaction with the ECM will expand our understanding of their role in matrix remodeling and disease progression^135–138.

6-2. Dexamethasone, Glaucoma, and the Role of Extracellular Vesicles

Dexamethasone (Dex), a type of glucocorticoid, is commonly used to reduce inflammation but can lead to increased resistance to aqueous humor outflow in the TM, subsequently raising IOP¹⁵⁶. This effect mirrors changes observed in POAG, making glucocorticoid-treated TM cells a useful in vitro model for studying this disease^157,158. However, it remains largely unclear how biomechanical alterations in the TM cells, LC cells, and the ECM influence this dysfunction¹⁵⁹. Despite uncertainties regarding the exact mechanisms, there is growing evidence that dexamethasone affects multiple signaling pathways within TM cells. A significant response of TM cells to Dex treatment includes the upregulation of myocilin, a protein implicated in glaucoma’s pathogenesis¹⁵⁸.

Previous studies using the MYOC-Y437H transgenic mouse model have shown that mutant myocilin leads to increased IOP and abnormal accumulation of ECM proteins like fibronectin, elastin, and collagen in the TM^160–168. This ECM buildup is linked to endoplasmic reticulum (ER) stress, which impairs TM function and reduces AH outflow, key factors in glaucoma development ^160–164. In a recent study by Yan et al., MYOCS341P transgenic mice exhibited significant glaucoma phenotypes, including decreased AH outflow, elevated IOP, reduced TM cell number, narrowed Schlemm’s canal, and visual impairment¹⁶⁹. Myocilin is also associated with mitochondrial functions and may contribute to apoptotic processes by affecting the mitochondrial membrane potential^170,171. For instance, the Pro370Leu mutation in myocilin induces a POAG phenotype in mice and significantly exacerbates mitochondrial dysfunction in TM cells, leading to increased reactive oxygen species (ROS) production and calcium imbalances, which contribute to cell death and could potentially accelerate the progression of primary open-angle glaucoma ¹⁷². In a recent study by Graybeal et al¹⁷³, Dex was shown to significantly impact mitochondrial function in TM cells, enhancing mitochondrial respiration and ATP production via oxidative phosphorylation (OxPhos) potentially contributing to cellular damage and the progression of glaucoma by affecting aqueous humor outflow.

Furthermore, analysis of protein expression patterns in EVs derived from human TM cells, with urine-derived EVs as control, revealed a significant presence of myocilin. This discovery was further substantiated by observing elevated levels of EV-associated myocilin upon treating TM cells with the corticosteroid dexamethasone, highlighting the EVs’ potential role in IOP regulation⁹⁰. In cancer research¹⁷⁴, EVs are shown to be involved in mitochondrial reprogramming within cells, which is crucial for supporting cancer cell survival and proliferation. This reprogramming includes alterations in mitochondrial dynamics and function, influenced by the molecular cargo delivered by EVs from the tumor microenvironment. With these studies, further research is warranted into effects of Dex on TM cells, particularly examining mitochondrial function and myocilin expression in EVs.

On the other hand, Dex has an impact on the ECM of TM cells, primarily through modifications in the protein composition of EVs. Dismuke et al. demonstrate that dexamethasone treatment significantly reduces fibronectin levels on EVs released from human TM cells. This reduction is linked to decreased annexins A2 and A6, which are crucial for fibronectin binding via heparan sulfate pathways. Additionally, they noted a reduction in the activity of dipeptidyl peptidase 4 (DPP4), on these EVs.

In the TM, EV’s fibronectin is critical for cell-matrix interactions at specialized cellular structures known as invadosomes^175,176, which TM cells utilize to remodel their surrounding matrix and ensure the proper flow of aqueous humor. These findings highlight how dexamethasone disrupts EV functions, potentially leading to ECM accumulation commonly seen in glucocorticoid-induced glaucoma.

In summary, various studies have demonstrated that dexamethasone-induced alterations in TM cells highlight the crucial role of extracellular vesicles in mediating these effects. Further investigation into the specific molecular compositions of these vesicles is essential. Understanding their mechanisms of action and identifying potential therapeutic targets are critical for advancing the management of glaucoma.

6-3. miRNAs from EVs in glaucoma

EVs are significant components of the AH, facilitating communication between ocular tissues^19,90. Beyond conditionally expressed proteins, miRNA within EVs also regulate gene expression in the TM and Schlemm’s canal, significantly impacting glaucoma pathogenesis^12,177. miRNAs, a group of non-coding RNAs, suppress the expression of their target mRNAs by cleavage, destabilization, or translation inhibition. As key post-transcriptional regulators, a single miRNA can interact with multiple mRNAs, and vice versa, significantly expanding the complexity of the regulatory network. Molecular analysis of EV cargoes reveals 584 miRNAs and 182 proteins linked to the regulation of TM metabolism^12,178.

Changes in miRNA expression profiles have been identified as markers of glaucoma in both experimental models and patients^179–181. In the study of AH samples, a spectrum of microRNAs was detected, including miR-486-5p, miR-184, miR-204, miR-181a, miR-191, miR-148a, miR-26a, miR-125a-5p, let-7a, and let-7b. Among these, miR-191 and miR-26a emerged as the predominant miRNAs in EVs extracted not only from the AH but also from the conditioned media of primary human TM cells¹⁹.

Liu et al.’s study underscores the importance of miR-182 in the pathogenesis of POAG. Their comprehensive analysis, using the large NEIGHBORHOOD dataset (3853 cases/33,480 controls with European ancestry), established a link between the rs76481776 variant in the MIR182 gene and POAG. This genetic variant is associated with elevated expression of miR-182, which was consistently observed across various ocular tissues and in the aqueous humor glaucoma patients, where miR-182 levels were found to be significantly higher compared to controls. This increased expression, particularly noted in TM-derived EVs and aqueous humor, suggests that miR-182 could influence the regulation of intraocular pressure and aqueous humor dynamics, highlighting its potential as a biomarker and therapeutic target in glaucoma management. The study’s findings provide compelling evidence that miR-182 plays a critical role in the molecular mechanisms that may contribute to the elevated intraocular pressure observed in glaucoma patients, offering a new avenue for potential therapeutic interventions ¹⁸².

In the study by Takahashi et al., the impact of EVs derived from TM cells on Schlemm’s canal endothelial (SCE) cells was explored, focusing specifically on how miRNAs transported via TM-derived EVs modulate SCE cell functions. EVs released from TGF-β2-stimulated TM cells are enriched with miR-7515, which elevates the expression of key vascular elements, including Vascular Endothelial Growth Factor A (VEGFA), Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), Platelet Endothelial Cell Adhesion Molecule (PECAM), and Tyrosine-protein kinase receptor Tie2, within Schlemm’s canal endothelial cells. This interaction suggests a pivotal role for these miRNAs in reprogramming SCE cells, potentially affecting the dynamics of aqueous outflow and IOP regulation through cellular communication mediated by EVs¹⁷⁷.

6-4. Role of EVs in Glaucomatous Optic Neuropathy

Recent studies have shown that EVs play a role in the development of neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS)¹⁸³, and Huntington’s disease (HD)^183–188. EVs also have the ability to cross the brain and blood-retina barriers ¹⁸⁹. Given these capabilities, EVs are increasingly recognized for their potential as innovative treatments for neurodegenerative disorders. Similar to other neurodegenerative disorders, glaucoma involves intricate immunoregulation by glial cells including microglia and astrocytes^190–193. In the complex pathophysiology of glaucoma, microglial cells play a pivotal role, particularly in the progression of neuroinflammation that leads to RGC loss and optic nerve damage^194,195. As the primary immune cells of the retina, microglia monitor and respond to changes in retinal health. Normally, they support retinal homeostasis by clearing debris and secreting neurotrophic factors. However, in glaucoma, sustained elevation of IOP triggers microglia to shift from a neuroprotective role to a pro-inflammatory one, exacerbating neural damage^194,196. In research utilizing the murine model of retinal microglia (BV-2 cell)¹⁹⁷, cells exposed to conditions simulating elevated IOP produced EVs that significantly altered microglial functions. The release of EVs by microglia is facilitated by adenosine triphosphate (ATP), which may elucidate the effects of elevated pressure on the augmented release of EVs. This connection is supported by observations of increased extracellular ATP when microglial cells are subjected to elevated hydrostatic pressure, as detailed by Rodrigues-Neves et al. ¹⁹⁸. This suggests that pressure-induced cellular stress could be a key driver of changes in vesicle dynamics.

The EVs isolated from microglia exposed to elevated hydrostatic pressure significantly enhanced pro-inflammatory cytokine production, including TNF and IL-1β, improved microglial mobility, increased MHC-II expression, and boosted phagocytic activity¹⁹⁷. Furthermore, these changes correlated with augmented cell death and reactive oxygen species in retinal neural cultures. Intravitreal administration of these EVs activated retinal microglia and led to RGC loss, indicating a direct influence on retinal microglia homeostasis¹⁹⁷. The significant impact of these EVs is evident from studies where the modulation of EV release or altering their molecular content significantly affected the course of glaucoma. For instance, experiments have shown that blocking the release of EVs from microglia using pharmacological inhibitors, like GW4869 which impairs the formation of EVs, can halt the progression of retinal damage induced by elevated hydrostatic pressure¹⁹⁹. This inhibition of EV release prevents the usual increase in pro-inflammatory cytokine levels and mitigates the subsequent neurodegenerative effects typically observed in glaucoma models. Moreover, recent findings have highlighted that EVs from reactive microglia, when injected into animal models, can induce a pronounced inflammatory response, exacerbating retinal cell death including that of RGCs. Conversely, EVs from microglia under normal pressure conditions do not trigger such deleterious effects, indicating that the contents of EVs are crucially altered under glaucomatous conditions¹⁹⁹ .(Figure 3)

Figure 3.

A comparative proteomic analysis of EVs from TM cells, showing differences in ECM proteins between glaucomatous and non-glaucomatous conditions. The EVs were separated using density gradient centrifugation, with fractions representing different EV subpopulations based on their density; small EVs are expected in fractions 5-8, however positive markers for small EVs were found in fractions 9-10 also. Panels A-C depict EVs from non-glaucomatous TM cells: (A) pooled fractions 5-10, (B) pooled fractions 5-8, and (C) pooled fractions 9-10. Panels D-F show EVs from glaucomatous TM cells: (D) pooled fractions 5-10, (E) pooled fractions 5-8, and (F) pooled fractions 9-10. The ECM protein composition varies between these subpopulations, indicating altered ECM remodeling in glaucoma. From McDonnell, Fiona S et al., “Comparison of the extracellular vesicle proteome between glaucoma and non-glaucoma trabecular meshwork cells.” Frontiers in Ophthalmology, vol. 3, 2023: 1257737.

Collectively, these studies highlight the important roles EVs play in how cells and tissues of the eye respond to glaucoma stimuli and pathogenesis.

In addition to microglia, Astrocyte-derived EVs (ADEs) are particularly notable for their regulatory and protective effects on neuronal cells, especially under stress conditions such as hypoxia and oxidative stress^200–202. Although the exact mechanisms of glaucoma progression are yet to be fully understood, it is reasonable to speculate that paracrine effects from ONH astrocytes, mediated through EVs, play a significant role. Studies have shown that astrocytes release EVs abundantly, which possess the ability to modulate neuron-glia interactions and maintain cellular homeostasis within the ONH^200,203,204. Venturini, A. et al. highlighted that EVs released from cortical astrocytic processes can selectively target neurons, enhancing their survival in vitro²⁰⁴. This suggests that ADEs are crucial not only for sustaining neuron-glia communication but also for providing trophic support to neurons under the pathophysiological conditions such as those found in glaucoma. Research also showed the varied impacts of EVs sourced from wild type neurons and neurons with a Methyl-CpG binding protein 2 (MECP2) deficiency on the proliferation of astrocytes in vitro cultures. Furthermore, there is documented evidence that neuronal EVs can regulate the expression of the astrocytic glutamate transporter 1 (GLT1), demonstrating a significant means of communication and functional regulation between neurons and astrocytes^205,206.

The diversity in astrocyte behavior, especially comparing cortical astrocytes with those in the ONH²⁰⁷, as well as differences between reactive and wild-type ONH astrocytes underscores the complexity of EV-mediated effects in glaucoma. Reactive ONH astrocytes, known for their distinct morphology and capacity to respond to biomechanical pressure, may release EVs with different cargo compared to their wild-type ONH astrocytes ^207–209. Changes in the EV profile may affect the severity of RGC axon damage and the progression of the disease. Understanding these details is key to creating targeted treatments that utilize the therapeutic benefits of astrocyte-derived EVs in treating glaucoma

In summary, EVs facilitate cellular communication in glaucomatous optic neuropathy, mirroring their role in other neurodegenerative diseases. Understanding how EVs influence the optic nerve head could unveil new therapeutic strategies for glaucoma, potentially altering its progression. As we deepen our understanding of the integral roles EVs play within the glaucomatous landscape, it becomes increasingly apparent that these vesicles could be harnessed not only for their diagnostic potential but also for therapeutic interventions. This insight transitions our focus from pathological mechanisms to exploring therapeutic applications, setting the stage for the next section on the therapeutic potential of EVs in glaucoma.

7-. Therapeutic Potential of EVs in Glaucoma:

7-1. EVs as Targeted Delivery Drug System

Recent years have seen rapid advances in the use of nanocarriers for drug delivery. However, challenges such as ensuring safety, improving delivery efficacy, and maintaining stability have hindered their widespread clinical adoption. These issues have motivated researchers to innovate and create nanocarriers that are more compatible, stable, and have extended circulation times in the body.

EVs, as naturally occurring nanocarriers of small RNAs and proteins, present a groundbreaking solution to these issues. They offer a sophisticated strategy for delivering therapeutic molecules, such as microRNAs and proteins, directly to targeted cells^210–212. Their stability under various physiological and pathological conditions²¹³, combined with key characteristics like minimal toxicity and immunogenicity, attributable to their limited tissue accumulation, establishes them as superior vehicles for drug delivery.

Furthermore, EVs’ capacity to traverse critical biological barriers, including the blood-brain and blood-retina barriers, tackles a major obstacle in systemic drug delivery, facilitating targeted treatment with minimal side effects. Additional merits of EVs include their ease of isolation and purification processes, their acellular nature, which obviates ethical concerns associated with stem cell utilization, and their stability supports straightforward storage. The small size of EVs permit not only precise dosage administration but also penetration into the ganglion cell layer from the vitreous—a feat unattainable by transplanted cellular entities.

These properties have led to the development of EV-based nanocarriers for treating a wide range of diseases, including cancer, cardiovascular disease, wound healing, ophthalmology, and neurodegenerative disorders. EVs can be used as carriers for small RNA, anticancer drugs, and anti-inflammatory agents. For instance, EVs loaded with chemotherapeutic drugs demonstrated enhanced efficacy and improved bioavailability when compared to the administration of the drugs alone^214,215.

The traditional approach for treating eye diseases, particularly those affecting the anterior segment, often relies on topical eye drops. This method suffers from drawbacks like frequent administration and low bioavailability. In response, recent advancements have been made in developing synthetic drug carriers to enhance therapeutic outcomes, although issues such as immunotoxicity and rapid clearance by the mononuclear phagocyte system remain challenges²¹⁶. Conversely, EVs, natural nanocarriers, offer promising solutions, facilitate targeted drug delivery, and enhance bioavailability. For example, milk-derived EVs have been utilized to encapsulate lutein, significantly enhancing its bioavailability for treating dry eye disease²¹⁷. This integration not only improves lutein’s water solubility and stability but also markedly increases its therapeutic efficacy, leading to substantial improvements in corneal health and tear film stability in a chronic dry eye model²¹⁷. While these approaches focus on anterior segment diseases, posterior segment conditions, such as retinal diseases, require a different strategy. Intravitreal injections are used for targeted delivery to the retina, as topical treatments do not effectively penetrate the blood-retina barrier. For retinal diseases like age-related macular degeneration (AMD), diabetic retinopathy, and retinal vein occlusion, intravitreal injections are common. However, repeated injections are often necessary, which can lead to complications and reduce patients’ quality of life. Finding more effective and longer-lasting treatments is essential for improving patient outcomes. EVs derived from cells producing adeno-associated virus type 2 have demonstrated enhanced retinal transduction efficiency²¹⁸, while those loaded with miRNA-126 have successfully reduced hyperglycemia-induced retinal inflammation²¹⁹. These advances highlight the potential of EVs for broader applications, including the treatment of RGC death in glaucoma, where they may help prevent or even reverse vision loss.

The use of EVs as drug delivery systems in glaucoma filtering surgery shows promising potential, particularly in enhancing the efficacy of antifibrotic agents²²⁰. Recent research demonstrated that EVs, specifically those derived from human embryonic kidney cells (HEK293T), can effectively encapsulate and deliver aptamer S58. This aptamer specifically targets and inhibits TGF-β2 stimulation by binding to its receptor, TβRII, which plays a critical role in fibrotic processes. This delivery method targeted fibrosis in both human conjunctival fibroblasts and a rat model of glaucoma filtration surgery, resulting in a significant reduction in cell proliferation, migration, and fibrosis compared to the administration of the naked aptamer. This approach not only prolonged the therapeutic effects of the aptamer in vivo but also demonstrated a superior antifibrotic effect. These findings suggest that EVs could be a valuable tool for improving treatment outcomes in glaucoma by providing targeted and sustained drug delivery²²⁰.

These instances highlight the versatility of EVs in delivering various biomolecules, including functional RNAs and synthetic drugs, directly to targeted sites in the eye.

7-2. EV Bioengineering

EV engineering represents a promising frontier in therapeutic molecular delivery, addressing limitations inherent in natural EVs such as suboptimal targeting and limited therapeutic payload capacity. While natural EVs offer certain benefits, they often lack specific pharmaceutical components and exhibit poor tissue and cell targeting abilities^221,222. To overcome these challenges, researchers have developed advanced strategies that include both ‘cell engineering’—the genetic modification of parent cells to incorporate desired nucleotides or proteins into EVs—and ‘EV engineering’—direct modifications after EV secretion to alter the EV membrane for improved targeting, reduced clearance, and specific therapeutic molecule loading^26,223.

The adaptability of EVs has been demonstrated across various medical applications through modifications of their surface ligands, cargos, and the incorporation of specific genetic materials²²⁴. Examples of this include EVs engineered to carry integrin-binding RGD peptides for targeted drug delivery to Glioblastoma multiforme (GBM), a notably aggressive brain tumor²²⁵. These RGD-EVs, produced from human embryonic kidney 293FT (HEK 293FT) cells, show selective uptake and increased accumulation in GBM cells overexpressing integrins. Their use significantly improves the delivery and effectiveness of doxorubicin and siRNA targeting the GAPDH gene²²⁵. Similarly, EVs modified to carry the surface protein Lamp2b have shown potential in delivering therapeutic agents across the blood-brain barrier to reduce neuroinflammation in Alzheimer’s disease²²⁶. Additionally, EVs engineered to express the human epidermal growth factor receptor 2 (HER2) have been effective in reducing tumor growth in HER2-positive breast cancer models²²⁴.

In another innovative approach, EVs have been engineered to carry superparamagnetic iron oxide nanoparticles and chemotherapeutic drugs for targeted tumor therapy under an external magnetic field, significantly reducing tumor growth and extending survival in animal models²²⁷. The use of biocompatible scaffolds for EV delivery further facilitates targeted and sustained release, enhancing therapeutic outcomes²²⁷.

In a study by Haney et al., EVs from monocytes and macrophages were developed as drug delivery vehicles for treating Parkinson’s Disease (PD). They loaded catalase into EVs using methods such as saponin permeabilization and sonication, achieving high efficiency and protection against degradation. These catalase-loaded EVs, were effectively taken up by neuronal cells and reached the brains of PD model mice through intranasal administration, showing significant neuroprotective effects in PD models²²⁸.

Specifically, in the context of ocular diseases, engineered EVs show great promise in enhancing treatment efficacy by enabling precise delivery of therapeutic agents directly to pathological sites. For instance, EVs derived from NPCE cells carrying SMAD7 siRNA have led to significant knockdowns and subsequent biochemical changes in trabecular meshwork cells²²⁹. This modification leads to an increase in β-catenin and tumor growth factor β2 levels in TM cells following in vivo incubation²²⁹. Additionally, by attaching Arg-Gly-Asp (RGD) peptides, which target integrins overexpressed in choroidal neovascularization (CNV) tissues, EVs from Müller glial cells or the retina can preferentially accumulate in CNV areas after intravitreal injection, enhancing therapeutic outcomes²³⁰. In glaucoma treatment, modifying the surface of mesenchymal stem cell-derived EVs could improve targeting specificity towards cells such as microglia and retinal ganglion cells. Ongoing investigation and validation are necessary to fully understand and optimize the use of EV-based strategies for these therapies.

In addition to directly engineered EVs, emerging preclinical studies highlight how certain drugs can indirectly influence EV properties, enhancing their therapeutic potential. The protein kinase inhibitor sunitinib has been recognized for its capacity to target neuroprotective pathways ²³¹, thereby enhancing the survival of RGCs in glaucoma models. In a study investigating the effects of tyrosine kinase inhibitors (TKIs) on human renal cell carcinoma (RCC) cells, a dose-dependent increase in the secretion of both large and small EVs was noted. Quantitative proteomic analysis revealed a marked enrichment of metabolic proteins, notably GLUT1, in sEVs from Sunitinib-treated cells, indicating a TKI-induced shift in the metabolic profile of vesicular cargo. These sEVs demonstrated enhanced glucose uptake and glycolysis in comparison to control sEVs. Further, RCC cells overexpressing GLUT1 produced sEVs with augmented GLUT1 levels and, consequently, increased metabolic activity. This evidence suggests that TKIs can influence the metabolic content and functionality of sEVs derived from RCC cells. Nonetheless, analogous investigations in ocular systems remain to be conducted, presenting a promising avenue for future research²³¹.

Similarly, Netarsudil impacts the trabecular meshwork cells’ actin cytoskeleton, affecting the generation and uptake of actin-rich EVs. The drug stimulates the phagocytic uptake of these EVs by trabecular meshwork cells ²³², an activity that could be critical for therapies aimed at lowering intraocular pressure in glaucoma. Through mechanisms such as exocytosis of intracellular vesicles and the cleavage of filopodial tips—which allows for the detachment and retraction of filopodia—Netarsudil facilitates the generation of EVs. These findings underscore that while some therapies may not be based directly on EVs, they can significantly modify the functionality and deployment of EVs within biological systems²³².

7-3. Stem Cell (MSC)-derived EVs

Mesenchymal Stem Cell (MSC)-derived EVs are currently the most extensively studied therapeutic EVs across various medical fields, including ophthalmology. MSCs are self-renewing, multipotent progenitor cells of mesodermal origin, notable for their extensive presence in various tissues and ease of in vitro expansion^233–235. MSCs derived from bone marrow, adipose tissue, and umbilical cords have demonstrated protective effects across a range of ocular injuries, including those affecting the cornea^236,237 , lacrimal gland^{238 239}, retina^240–243, and photoreceptors. The therapeutic strategy of retinal cell replacement using stem cells is an emerging frontier for neuroprotection and regeneration in ophthalmology ^211,244. Recent evidence from the intravitreal transplantation of umbilical cord-derived MSCs (UC-MSCs) has shown a decrease in retinal ganglion cell (RGC) loss and mitigation of retinal damage in chronic ocular hypertension models,²⁴⁵ suggesting a viable approach for treating optic neuropathies such as glaucoma. Nonetheless, challenges such as low cell integration rates and potential for aberrant growth currently limit MSCs’ clinical application efficacy²⁴⁶.

The use of MSC-derived EVs presents a viable method to bypass the limitations and complications associated with direct stem cell therapies in ocular applications²⁴⁷. MSC-derived EVs demonstrate exceptional ability to traverse the retinal barrier, highlighting their potential as therapeutic vectors in ophthalmology. Intravitreal injection of MSC-derived EVs exhibit pronounced retinal tropism, allowing for their widespread distribution within the retinal layers. Specifically, experimental data indicate that upon administration into the mouse vitreous cavity, these EVs can effectively permeate the inner nuclear layer and outer plexiform layer, with notable, albeit reduced, penetration into the outer nuclear layer^218,242,248. The capacity for rapid internalization by retinal and microglial cells serves to decrease EV clearance rates, consequently extending the duration of their therapeutic effects²⁴². In rat models, EVs introduced into the vitreous body have been detected up to four weeks post-injection, indicating a prolonged presence and sustained interaction with the vitreous humor constituents. This extended detectability and binding efficiency underline the promising utility of MSC-derived EVs in delivering targeted therapies across the retina, offering a novel approach to modulate retinal cellular activities and potentially address a range of ophthalmological conditions²⁴².

Further, intravitreal injections of bone marrow MSC-EVs have been successful in increasing retinal ganglion cell (RGC) survival and promoting axon regeneration in various models of optic nerve damage. This includes the optic nerve crush model, the DBA/2J genetically modified mouse model of glaucoma²⁴⁹, and rat models of glaucoma induced by microbead injection into the anterior chamber or by laser photocoagulation of circumferential limbal vessels^240,250.

The neuroprotective potential of bone marrow stem cell (BMSC)-derived EVs in glaucoma treatment emerges through their molecular cargo, particularly miRNAs like MIR-106A-5P and MIR-486-5P. These miRNAs target key pathways that promote l RGC survival and reduce oxidative stress, laying the foundation for their therapeutic efficacy. Moreover, the modification of mRNA cargo, through strategies such as TNFα priming, can further enhance this protective effect by increasing the levels of neuroprotective factors such as pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor A (VEGF-A). This enhancement underscores the dynamic nature of EVs’ therapeutic properties and the critical role of their cargo²⁵¹.The distinction in effectiveness between fibroblast-derived EVs and those from primed BMSCs highlights the importance of the source and preconditioning of cells in determining the EVs’ therapeutic impact. Specifically, in a chronic glaucoma mouse model, EVs from tumor necrosis factor alpha (TNFα)-primed BMSCs provided superior protection for RGCs compared to those from unprimed cells, emphasizing the potential of targeted preconditioning to augment the therapeutic outcomes²⁵¹.

However, despite these promising insights, the neuroprotective effects conferred by BMSC-derived EVs have shown to be transient, waning after six months. This transient nature signals a critical need for periodic administrations to maintain therapeutic benefits, presenting a challenge for long-term treatment strategies²⁴⁹. Additionally, this underscores the necessity for a deeper understanding of the molecular mechanisms underpinning these effects. Ongoing research is essential to elucidate these pathways and validate the long-term efficacy and safety of EV-based therapies in the management of glaucoma, paving the way for novel interventions that could offer sustained neuroprotection and ultimately alter the course of glaucoma treatment.

UC-MSCs represent an alternative MSC source, offering advantages over BMSCs-due to their noninvasive, effortless collection and superior growth and proliferation capabilities²⁵². The neuroprotective efficacy of EVs from UC-MSCs was explored in the context of glaucoma. To this end, rat models of chronic ocular hypertension were established by injecting conjunctival fibroblasts into the anterior chamber, simulating optic nerve damage comparable to that observed in glaucoma. A week post-injury, UC-MSC-derived EVs were administered into the vitreous cavity. These EVs significantly improved retinal damage, increased retinal ganglion cell numbers, and suppressed caspase-3 activation, indicating a reduction in apoptotic cell death. These outcomes highlight the potential of UC-MSC-derived EVs in alleviating optic nerve damage associated with chronic ocular hypertension, primarily through the inhibition of apoptosis²⁵³.

Additionally, BM-MSC-derived EVs influence TM functionality, crucial for managing IOP. Research by Li et al. demonstrated that treating TM cells damaged by hydrogen peroxide with BM-MSC EVs not only enhances cell survival but also modulates the expression of 23 miRNAs. This treatment improved AH drainage and reduced elevated IOP, showcasing the therapeutic potential of MSC-derived EVs in comprehensive glaucoma management²⁵⁴.

7-4. EV-mediated crosstalk between NPCE and TM cells

The TM is the most sensitive tissue to oxidative stress in the anterior segment of the eye ²⁵⁵. In glaucoma-affected TM, significant increases in markers such as 8-hydroxy-2’-deoxyguanosine (8-OH-dG), HSP72²⁵⁶, and glutamine synthetase²⁵⁷ were found, indicating DNA oxidative stress damage, stress, and excitotoxicity-related protein expression, respectively²⁵⁸. Chronic glaucoma features mitochondrial dysfunctions and a weakened antioxidant defense, resulting in an excessive buildup of reactive oxygen species in the AH and ocular tissues²⁵⁹. Oxidative stress factors like malondialdehyde are notably higher in the AH and blood of glaucoma patients, particularly those with POAG, compared to healthy individuals²⁶⁰. This persistent oxidative stress prompts inflammatory responses and apoptotic pathways that harm TM and lead to aqueous drainage system dysfunction²⁶¹.

Interestingly, recent studies have shown that EVs from the NPCE can mitigate oxidative stress on the TM by modulating its metabolic activity and rejuvenating the ECM in glaucoma^10,11,262. These EVs, under oxidative stress conditions, activate catalase and superoxide dismutase in TM cells increasing the activity of these enzymes by 20% (catalase) and 50% (superoxide dismutase) when compared to untreated TM cells²⁶². TM cells exposed to EVs isolated from oxidatively stressed NPCE cells show reduced oxidative stress, credited to the activation of key antioxidant genes. It is thought that the changes in NPCE cells due to oxidative stress are applied to EVs released from these cells, the EVs then carry the “alert” to TM cells protecting them from oxidative stress. This protective response is absent with EVs from non-stressed NPCE, highlighting their potential to enhance target cell resistance in stressful environments²⁶². Moreover, chronic TM stress disrupts ECM regulation, leading to collagen deposition and aqueous drainage blockage, elevating IOP in glaucoma²⁵⁸. The mechanism of increased antioxidant activity has yet to be elucidated.

One study focused on extracting EVs from NPCE cell lines and examined their effects on the expression of Wnt proteins in TM cells. Proteomic analysis of NPCE EV content identified 584 miRNAs and 182 proteins that are implicated in regulating various TM cell processes, including the wNT/β-catenin signaling pathway, cell adhesion, and ECM deposition. This research aligned its miRNA findings with existing aqueous humor miRNA profiles, identifying 77 matching miRNA sequences. Notably, the predominant miRNAs found in the NPCE-derived EVs were miR-21, miR-638, let-7a, miR-100, and miR-16, which have been implicated in the regulation of Wnt signaling in the trabecular meshwork. Furthermore, miRNAs such as miR-29, miR-17, and miR-21 were associated with collagen production modulation, suggesting the capacity of NPCE-derived EVs to influence both the Wnt pathway and collagen production in trabecular meshwork cells, potentially impacting intraocular pressure regulation mechanisms ¹².

The Wnt/β-catenin signaling pathway, often activated in response to oxidative damage, plays a crucial role in a range of physiological and pathological processes, including ECM remodeling, fibrogenesis, cell proliferation, and apoptosis^263–265. Inhibition of this pathway, as evidenced by the overactivity of glycogen synthase kinase-3β (GSK-3β), is a characteristic feature in glaucoma and is associated with elevated IOP²⁶⁶. NPCE-derived EVs have been found to intricately modulate this signaling cascade, a process that is both dose- and time-dependent. Lerner et al. conducted a study to explore how EV from NPCE cells influence Wnt protein expression in human primary TM cells and the underlying mechanisms of this EV-mediated regulation¹². Their findings revealed that EVs from both primary NPCE cells and NPCE cell lines reduce phosphorylation of Glycogen Synthase Kinase 3 beta (GSK3β) and lower cytosolic β-catenin levels in TM cells. At a molecular level, treatment with NPCE EVs led to a decreased expression of the AKT protein, a positive regulator of GSK3β, and an increase in the Protein Phosphatase 2A (PP2A)protein, a negative regulator of GSK3β¹².

Furthermore, TM cells exhibit a bimodal response to NPCE EVs, with differing levels of β-catenin and its nuclear receptor (lymphoid enhancer factor/LEF-1) depending on the EV concentration, suggesting a complex regulation of TM metabolism¹¹.

In summary, the continuous release of EVs by the NPCE and TM is crucial for maintaining their homeostasis, responding to oxidative stress, managing apoptosis, and modulating the ECM. In the context of glaucoma, disrupted EV function and elevated oxidative stress form a harmful feedback loop that obstructs AH outflow, leading to increased IOP.

8-. Challenges and future direction

Utilizing EVs as drug delivery vehicles presents several advantages over nano-based or liposome-based therapies, notably their ability to evade phagocytosis or degradation by macrophages and to circulate for extended periods²²¹. Nonetheless, the translation of EV-based therapies into clinical settings is impeded by numerous limitations and challenges. A principal issue hindering the exploration of EVs in clinical applications is the shortage of pure EVs obtainable from the body. Given the low abundance of EVs, their purification poses a significant challenge^267,268. The diverse methods for isolating EVs from cell culture supernatants or various biological fluids—such as milk, urine, plasma, amniotic fluid, saliva, and cerebrospinal fluid—each offer distinct advantages and drawbacks. It is imperative to identify the most suitable source from which to derive highly enriched, well-characterized EVs of the highest quality^{267,269–271}.

Characterization of EVs through currently available techniques, including electron microscopy, FACS, and Western blot analyses, is limited and therefore cannot independently verify the biophysical and biochemical properties of EVs ^268,271There exists a necessity for definitive and reproducible techniques for the clinical characterization of EVs, facilitating their use as distinctive biomarkers for various pathological conditions^268,271.

In the realm of ophthalmology, the use of EVs for intraocular injection warrants stringent standards due to the eye’s immune-privileged status, despite their demonstrated safety in animal models^272,273. A critical challenge for EV-based therapies in glaucoma—a chronic and progressive condition—lies in their relatively short duration of action, necessitating frequent injections to maintain therapeutic effects. This requirement raises concerns about the practicality of such treatments, given glaucoma’s demand for long-term management to control IOP and protect the retina. Animal studies suggest monthly or even weekly injections are needed for sustained benefits, yet the pharmacokinetics and efficacy of these interventions, particularly in larger, non-human primate eyes, remain inadequately understood²⁴⁹. One potential strategy to extend the retention time of intravitreally injected EVs involves embedding the EVs within hydrogel implants. These implants, acting as reservoirs within the vitreous humor, could facilitate sustained EV release. This approach may significantly widen the therapeutic window, allowing for enhanced treatment efficacy from a single injection into the eye²⁷⁴. However, the optimal interval between treatments in human patients is yet to be determined.

The discrepancy between animal models and human glaucoma, including structural differences in eyes and the inability of acute models to fully replicate the chronic pathogenesis of the disease, further complicates the translation of EV-based therapies to clinical practice. It remains to be seen whether such treatments can offer advantages over traditional methods, like eye drops or surgical interventions, warranting additional research into regimens that could reduce the frequency of injections. Exploring alternative delivery methods, such as sustained-release systems or suprachoroidal and subretinal injections, might improve patient compliance by lessening the need for frequent interventions. Nevertheless, the increasing exploration of EVs in clinical trials across different fields suggests a hopeful outlook for their eventual integration into glaucoma therapy, pending further research to address these challenges²⁷⁵.