Exosomes in the visual system: New avenues in ocular diseases

1 Introduction

Extracellular vesicles (EVs) are diverse groups of membrane-bound nano-sized vesicles found abundantly in all body fluids such as blood, milk, cerebrospinal fluid, urine including ocular tissues, and ocular fluids like tears, aqueous, and vitreous humor (VH). Wolf first identified EVs in plasma and termed them “platelet dust” [1]. Earlier, EVs were considered waste-removing vehicles released by cells. But recent studies have established them as effectors of cellular communication in normal and pathological conditions. However, over the last decade, there has been an enormous amount of knowledge gained about EVs with specific reference to small EVs (< 200 nm) with endosomal origin called exosomes. From being considered as dust, they are now believed to be contributing to the disease progression. Evidence from research studies shows that exosomes promote tumor progression by promoting angiogenesis and tumor cell migration in metastasis [2, 3]. While the role of exosomes is important in every organ, it is of unique importance in the eye, a specialized sense organ capable of receiving visual images, which are then carried to the brain. It is not only uniquely situated in a bony orbit, connected directly with a cranial nerve to the brain and meninges, enjoys immune privilege, and has a blood-retinal barrier in the retina that regulates the nutrient flux between the circulating blood and the retina. Because of our interest in ocular pathology and ophthalmic research, and the emerging evidence of the role of exosomes, in this review, we present a brief overview of EVs, the classification of different types of EVs, and the most well-described entity “exosomes” with specific reference to ocular tissues in health and disease conditions. As clarified in the subsequent paragraphs, there is a slight ambiguity regarding small EVs vs exosomes in using terminology. Therefore, we have included the keywords “EVs”, “microvesicles (MVs)”, “small EVs” and “exosomes” for the literature search.

2 Classification of EVs

Three main subtypes of EVs are described based on their subcellular origin, release pathway, vesicle size, content (cargos: DNA, RNA, and proteins), and function [4]. The largest vesicles are apoptotic bodies having sizes varying from 1–5 μm in diameter, that are released during apoptotic processes through budding directly from the plasma membrane and then discharged into extracellular space [5]. MVs’ size ranges from 100–1000 nm. MVs are formed by direct budding of the cell’s plasma membrane. Exosomes are the smallest subpopulation among all EVs with a diameter of 30–150 nm. Exosomes are released into the extracellular space after fusion with late endosomes or multi-vesicular bodies with the plasma membrane. The characteristics of three different types of EVs are shown inTable 1.

As evident in Table 1, there is an overlap of size between exosomes and MVs and the same is reflected in various publications. Some of the authors used the size of 30–100 nm [4, 6] as the criterion of distinguishing exosomes, while others have used the size of 40–120 nm [7], 30–150 nm [8], 50–150 nm [9], and 40–200 nm [10]. One may speculate that the differences in sizes of exosomes may result from different types of methods and principles used for measuring; the hydrodynamic diameter measured by nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) is in the range of 119 and 182 nm, whereas geometric sizes of hydrated and desiccated exosomes measured by differential mobility analyzer (DMA), and scanning electron microscopy (SEM) are 37–71 nm [11]. Moreover, it became a general practice that the term “exosome” is used for all the vesicles of less than 200 nm, even when their origin is unknown/ not proved as endosomal [12]. Because of this, the International Society for Extracellular Vesicles (ISEV) framed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for uniforming nomenclature and functionally characterize different groups of EVs. As per MISEV 2014 and updated 2018 guidelines it is recommended to name the EV subtypes that refer to a) physical properties of EVs, like size (small EVs for < 100 nm and < 200 nm vesicles; large/medium for > 200 nm vesicles) or density (low, middle, high, with each range defined; b) cell surface marker/biochemical composition (CD63 + /CD81+- EVs, Annexin A5-stained EVs, etc.); or c) based on the cell of origin (podocyte EVs, hypoxic EVs, large oncosomes, apoptotic bodies) instead of terms such as “exosome” and “MV” [13]. Literature review on EVs’ role in ocular health and disease also revealed the ambiguity of using the term “exosomes”. The majority of the authors quoted small vesicles (< 200 nm) as exosomes, some of them referred to them as “small EVs” or “EVs” as such without addressing their origin. We have included all of them in the review as they fall into the definition/criteria of the size of exosomes. We also believe that the work should be quoted as such without any change from our end. Thus, the terminology used for subtypes of EVs in this review is as per source publication. Numerous methods are available for the isolation of EVs, but none of them offer substantial yields with reliable quality. Common methods used for EV isolation are density gradient centrifugation techniques, size exclusion chromatography, ultrafiltration, and microfluidics-based sorting mechanisms. Immunoaffinity methods using anti-CD63, -CD9, and -CD81 antibodies are used to isolate exosomes from mixed cell populations such as in cell cultures, tissues, and biological fluids (plasma, serum, and urine) [14, 15]. EV characterization involves the determination of vesicle size, shape, surface charge, porosity, particle concentration, stability, yield, and purity which are determined by several biophysical and molecular biology methods [16].

Exosomes originating from late endosomes along with their cargo are considered of great importance due to their key role in cell-cell communication, neovascularization, stem cell differentiation, in maintaining cellular integrity and homeostasis in normal and diseased conditions such as cancers, neurodegenerative diseases, cardiovascular illnesses, dry eye syndrome, age-related macular degeneration (AMD) and glaucoma [17]. Exosomes were first described by two different research groups led by Harding and Pan in 1983 and found that transferrin receptors moved from the plasma membrane to the reticulocytes in a ∼50 nm-sized vesicle and then released the receptors from the maturing reticulocytes which then repacked with ∼50 nm-sized vesicles inside them. Afterward, they are released from maturing blood reticulocytes into the extracellular space without undergoing its usual process, the lysosomal destruction [18, 19]. Rose Johnstone named these vesicles as exosomes in 1987 [20]. Since then, exosomes derived from different types of cultured cells such as reticulocytes, immune cells, neurons, fibroblasts, primary tumors, and also from above-stated disease conditions have been isolated and characterized [21–23]. In addition to this, exosomes have also been purified from several body fluids such as saliva, blood, urine, breast milk, cerebrospinal fluid, bronchoalveolar fluid, epididymal fluid, amniotic fluid, and in aqueous humor (AH), suggesting their key role in normal physiological processes [24–28].

It is interesting to note that also vesicles derived from different kinds of stem cells (pluripotent, embryonic, mesenchymal, and cardiac progenitor cells) are being evaluated, and given the nature of these vesicles, they are emerging as new therapeutic aids in the treatment of cardiovascular, neurodegenerative, and inflammatory ocular diseases and are being studied in various disease models [29–31]. For instance, exosomes derived from mesenchymal stem cells (MSCs) can improve cognition by repairing oxidative damage in neurons and astrocytes in cognitively impaired diabetic animals [32]. Another study reported that exosomes secreted from MSCs delivered into a rodent model of optic nerve crush can protect retinal ganglion cells (RGC) from death and preserve its function by exerting their neuroprotective and neurogenic effects [33]. The clinical significance of these vesicles is due to the following properties: a) significant difference in vesicle content during normal and disease conditions b) non-cytotoxicity, c) low immunogenicity, d) easy to determine their cellular origin as numerous cells express specific cell surface molecules, e) non-invasive specimen collection because of their presence in biofluids (saliva, tears, blood, and urine, etc), f) stable and can be stored for longer periods for analysis, g) high cargo loading capacity [34–36]. Based on these properties, exosomes are being studied for their role in corneal wound healing and regeneration, immune modulation, potential biomarkers, and drug delivery vehicles. The therapeutic applications of exosomes are depicted in Fig. 1.

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Fig. 1

Therapeutic Applications of Exosomes.

Recent reports suggested that the delivery of exosomes into the eye can produce promising therapeutic applications in several models of retinal disease, which includes preventing retinal injury and restoration of RGC that are lost during optic neuropathy and glaucoma [37, 38]. It has been shown that the corneal wound healing and neovascularization properties of epithelial cell-derived exosomes are mediated via intercellular communication between corneal epithelial cells, corneal keratocytes, and vascular endothelial cells. As a result, exosomes can be considered as ideal targets for possible therapeutic interventions [39]. Samaeekia et al. reported that exosomes isolated from corneal MSCs could elicit corneal epithelial wound healing and may serve as a therapeutic approach for ocular surface injuries [40]. In a recent study, Tao et al. isolated EVs having a size within the range of exosomes from human placenta-derived MSCs (hP-MSCs) and showed their corneal wound healing properties in a mouse alkali-burn corneal injury model. hP-MSCs effectively enhanced wound healing by inhibiting inflammation and angiogenesis [41]. Exosomes can also be genetically modified so that they can easily pass through the biological barriers, the blood-brain barrier, penetrate tissues, help in cell-specific targeting through their cargo, and efficiently carry synthetic drugs and thus are considered natural nanocarriers [42, 43]. Some of the clinically tested drugs or entities that are delivered through exosomes are curcumin, doxorubicin, miR-122, etc. for the treatment of various cancers, ulcers and type I diabetes mellitus [42, 44].

The eye is composed of three layers- the outer protective sclera with cornea and conjunctiva, middle vascular uveal tissues (iris, ciliary body, and choroid), and the inner retinal layer that receives the visual signals. Some of the unique features of these ocular tissues are transparency and avascularity of cornea, transparent media and lens, and neurosensory retina that carries the images to the brain for interpretation. Added to this are an immune privilege, blood-retinal barriers, and direct visualization of retinal blood vessels which are believed to be the window to the body. This unique feature makes the eye susceptible to a wide variety of diseases and each part of the eye can give important clues to the diagnosis of systemic diseases, eg. diabetes, hypertension, jaundice, anemia. Hence, it is apparent that the exosome research in the eye is an exciting field, as exosomes have a potential as drug delivery vehicles, biomarkers, and therapeutic agents. Several research groups have identified exosomes in different eye structures and ascertained their role in normal physiology and diseased conditions.

Exosomes have been reported in various ocular tissues such as cornea (corneal epithelial cells), ciliary body (CB), lacrimal gland (LG), retina (retinal pigment epithelial cells (RPE), trabecular meshwork (TM), and ocular fluids like AH, VH, and also in tears [45, 46]. The presence of exosomes in different ocular tissues/fluids is depicted in Fig. 2. Exosomes were identified in vitreous samples of normal humans (vitrectomized), cadavers, and mouse eyes. It is hypothesized that the interaction between the vitreous, and retina is mediated through exosomes present in the vitreous [47].

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Fig. 2

The presence of exosomes in different ocular tissues: Exosomes are present in the ocular tissues such as cornea (corneal epithelial cells), ciliary body, lacrimal gland, retina (retinal pigment epithelial cells), trabecular meshwork, and ocular fluids like aqueous humor, vitreous humor, and tears.

4.2 Exosomes in inflammatory and autoimmune diseases of the eye

The eye is considered an immune-privileged (IP) site that prevents immune-mediated inflammation within the intraocular compartments, and this phenomenon is believed to be essential for the protection of vision. With the breach in immune privilege status several inflammatory and immune-mediated vision-threatening diseases of the eye, such as glaucoma and uveitis, and several corneal infections occur [54, 66]. In these disease conditions, as a consequence of responses from the immune cells, expression of pro-inflammatory cytokines and chemokines cause local inflammatory responses resulting in ocular tissue damage that can be repaired by exosomes derived from MSCs, and could serve as a promising cell-free therapy [54, 67].

4.2.1 Glaucoma

Glaucoma is a diverse group of disorders characterized by optic neuropathy leading to loss of vision and caused by elevated intraocular pressure (IOP). IOP is based on the equilibrium between inflow and outflow of AH through the TM. The TM inducible glucocorticoid response protein (MYOC) also called myocilin is highly expressed in the TM and plays a key role in the regulation of IOP. Abnormal expression of MYOC leads to TM dysfunction resulting in increased IOP. The mechanism of AH homeostasis and release of abnormal EVs with myocilin proteins is illustrated in Fig. 3. Numerous studies have suggested that the soluble components such as hormones, growth factors, neuropeptides and exosomes present in AH play a crucial role in the crosstalk between inflow and outflow tissues [68, 69]. In 2005, Hardy et al. proved that the release of MYOC from TM cells into its conditioned medium is possible via exosome-like vesicles. They explicated the mechanism by which tissue-specific localization and pathology of MYOC is achieved, and proposed that in most cells MYOC is an intracellular protein, but is released with exosomes by the TM [70]. A similar kind of study was carried out by the Perkumas group. The authors demonstrated that MYOC-associated exosomes are present in different human ocular samples such as effluent from the perfused human anterior segments and aqueous humor from live and cadaveric human eyes. They are also present in a conditioned medium from cultured human TM cells. This study revealed that exosomes may assist the transport of MYOC into the extracellular space of human ocular cells [45]. In another study, Lerner et al. demonstrated that non-pigmented ciliary epithelium (NPCE) derived exosomes can affect the canonical Wnt signaling pathway in TM cells through down regulation of expression of phospho-GSK3 and β-catenin expression [71]. These results are consistent with previous reports that canonical Wnt signaling is involved in IOP regulation and AH homeostasis [72]. Thus, NPCE-derived exosomes affecting Wnt signaling pathway molecules could be novel targets for glaucoma treatment. In addition, Dismuke et al. hypothesized that exosomes are part of AH and they primarily originate from NPCE cells. Furthermore, the authors revealed that the communication between inflow and outflow tissues homeostasis is mediated via exosomes released from AH and their cargo molecules chiefly via miRNAs. AH exosomal miRNAs such as miR-204 and miR-148a are shown to be involved in inflow and outflow facility regulation to maintain proper IOP homeostasis [68]. It is possible that in near future the exosome profile and their cargo in AH could be used as prognostic and diagnostic markers for ocular diseases including glaucoma [73, 74].

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Fig. 3

Schematic representation of the role of exosomes in aqueous humor (AH) homeostasis under normal and disease conditions. Red arrows show the movement of exosomes from the ciliary body (CB -site of AH production) to trabecular meshwork (TM). The green arrow show the movement of exosomes from TM to CB. Based on the available literature, we speculate that exosomes control AH outflow and inflow regulation, maintain AH equilibrium and normal intraocular pressure (IOP). 1. Glaucoma: Myocilin-derived EVs (red circles) released from CB (yellow arrow) diffuse into the AH causing disrupted AH homeostasis blocking TM resulting in elevated IOP leads to impaired vision. 2. Age-related macular degeneration (AMD): In response to oxidative stress and autophagy retinal pigment epithelial (RPE) cells, the outer layer of retina cells release higher concentration of exosomes having elevated expression of VEGFR2 into the anterior chamber (pink arrow) induces new blood vessels formation and drusen leads to AMD condition. 3. Retinoblastoma (RB): Exosomes released from RB tumors enter into the systemic circulation. 4. Uveal melanoma (UM): Exosomes released from primary UM involve tumor invasion, migration, and formation of metastatic niche in liver, the most common metastatic site for UM. Exosomes and their molecular content released into the systemic circulation during the above disease conditions serve as disease signature markers and possess diverse diagnostic and prognostic potential.

Recent animal studies have demonstrated the possible therapeutic applications of exosomes against glaucoma-related vision loss. Mead et al. compared the neuroprotective effect of BMC derived and fibroblast-derived small EVs having size within the range of exosomes (100–120 nm) among rats with ocular hypertension induced either by intracameral injection of microbeads or by laser photocoagulation of the TM and limbal vessels. The results show that injection of BMSC-derived small EVs into the vitreous provided significant therapeutic benefit to glaucomatous eyes possibly via their direct effect on RGC survival through miRNA-dependent mechanisms [75]. Similarly, the neuroprotective effects of BMSC small EVs were also reported in a chronic model of ocular hypertension [76]. Later, the same group demonstrated that BMSC small EVs can effectively elevate RGC survival in both rat retinal cultures and in human stem cell-derived RGC cultures. Their therapeutic efficacy can be elevated via TNFα priming possibly mediated through the increased levels of PEDF and VEGF-AA [77].

4.2.2 Autoimmune uveitis (AU)

Inflammation of the uvea and surrounding tissues around it leads to AU. It is caused by either an abrupt immune response mediated by pathogenic Th17 cells or by the involvement of the innate inflammatory molecules in response to external stimulus [78, 79]. RPE cells are shown to have immunosuppressive properties and these cells are getting damaged in uveitis conditions. Previous studies have suggested that EVs secreted from RPE cells suppress the proliferation of T lymphocytes harvested from peripheral blood of noninfectious uveitis patients [80]. Exosomes from MSCs derived from different sources such as human bone marrow and human umbilical cord inhibit the experimental autoimmune uveoretinitis (EAU) by different anti-inflammatory effects, either by inhibiting T cell proliferation or by inhibiting inflammatory cell migration or by suppressing dendritic cell-driven Th17 responses [54, 82]. The above results claim the therapeutic potential of exosomes against AU and future studies are warranted to explore their possible mechanism of action. Apart from in vitro studies and pre-clinical studies, a single clinical trial (NCT00331331) related to uveitis, vasculitis, and ocular inflammatory diseases was initiated in 2007 on patients aged above 18 years (n = 147) in order to evaluate the vitreous proteome and inflammatory mediators such as cytokines/lymphokines, biomarkers, proteome and exosomes present in the vitreous and anterior chamber. Vitreous specimens and corresponding blood samples were collected to compare inflammatory products present in both specimens. The study results of the same are awaited as per the latest update in April 2022 [83].

5 Exosomes in ocular cancers

Emerging evidence from recent reports states that exosomes play a decisive role in the progression of ocular cancers. Tumor-derived exosomes, fibroblasts, and cancer-associated fibroblasts (CAFs) within the TM influence various cancer signaling pathways such as those linked to EMT, angiogenesis, and metastasis [100–102]. The presence of exosomes and their possible applications in diagnosis and prognosis in ocular cancers, such as uveal melanoma (UM) and retinoblastoma (RB), are well documented.

6 Summary

Exosomes are the smallest vesicles having multiple roles in intercellular communication, immune response, and tumor-associated pathways. They have a dual role as tumor progression mediators and tumor antagonist factors. Over the years, numerous exosome isolation and characterization methods have developed, which poses a challenge in comparing the data and drawing parallels. Exosome samples are often contaminated with other EVs as separation techniques rely on vesicle size and cell surface marker expression. MVs and exosomes share common cell surface markers and have overlapping cell sizes, which leads to ambiguity in the literature regarding nomenclature. Hence there is a dire need to harmonize the tools for isolation and characterization of EVs and their naming. It is even more relevant to the ocular tissue as the available tissue samples from the eye are very small and rare. Hence samples obtained by noninvasive or minimally invasive methods to diagnose, prognosticate or provide targeted therapy would be of immense value. Evaluation of exosomal cargo and their biology will aid in understanding the pathogenesis of ocular diseases such as AMD, glaucoma to restore vision by improving antiangiogenic therapies. Similarly, in ocular tumors, understanding the interactions between tumor-derived exosomes with cells of the tumor microenvironment and cells of metastatic sites would aid in unraveling the molecular mechanisms responsible for tumor development, angiogenesis, metastasis, and invasion. The cumulative knowledge obtained from the exosomal cargo would pave the way for the identification of new biomarkers and also in the development of novel therapeutics that exclusively target tumor-associated pathways.