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Diabetic retinopathy (DR) is a major health problem, being the leading cause of vision loss in the working-age group. Novel discoveries facilitating the early diagnosis and curative treatment of DR are still greatly needed. Extracellular vesicles (EVs) are nano-sized vesicles released from different kinds of body cells including various retinal cells for intercellular communication. EVs recently attracted attention because of their non-immunogenic, biocompatible, and biodegradable characteristics. Recent studies investigated EV cargoes in DR patient samples, animal models, and in vitro models of DR. Although a thorough understanding of the trafficking of EVs in the retina still needs extensive investigations, these studies will pave the way for the discovery of novel biomarkers for early diagnosis of DR. Furthermore, EVs-based therapeutics could be of great interest in the treatment of DR. They are presented as promising delivery tools for both pharmacological and genetic therapies. Here, we highlighted the studies that discussed the role of EVs in both inflammation and angiogenesis as two main pathophysiological events occurring in DR. We also tried to portray what we know so far about the role of EVs in DR as a diagnostic, prognostic, and therapeutic tool.
Department of Oral Biology and Diagnostic Sciences, The Dental College of Georgia, Augusta University, Augusta, GA, USA
DCG Center for Excellence in Research, Scholarship, and Innovation (CERSI), Augusta University, Augusta, GA, USA
Mansoura Faculty of Medicine, Department of Medical Parasitology, Mansoura University, Mansoura, Egypt
Alaa M. Mansour
Department of Oral Biology and Diagnostic Sciences, The Dental College of Georgia, Augusta University, Augusta, GA, USA
Faculty of Dentistry, Department of Oral Biology, Mansoura University, Mansoura, Egypt
Babak Baban
Department of Oral Biology and Diagnostic Sciences, The Dental College of Georgia, Augusta University, Augusta, GA, USA
DCG Center for Excellence in Research, Scholarship, and Innovation (CERSI), Augusta University, Augusta, GA, USA
Khaled Elmasry*
Department of Oral Biology and Diagnostic Sciences, The Dental College of Georgia, Augusta University, Augusta, GA, USA
DCG Center for Excellence in Research, Scholarship, and Innovation (CERSI), Augusta University, Augusta, GA, USA
Department of Cellular Biology and Anatomy, The Medical College of Georgia, Augusta University, Augusta, GA, USA
Mansoura Faculty of Medicine, Department of Human Anatomy and Embryology, Mansoura University, Mansoura, Egypt
*Address all correspondence to: khelmasry@augusta.edu, elmasryretina@gmail.com
1. Introduction
Diabetic retinopathy (DR) is a diabetes-related complication characterized by various molecular, metabolic, functional, and structural changes in the retina. It is considered the leading cause of acquired preventable sight loss in the working-age population worldwide. It causes visual impairment that negatively impacts the individual’s quality of life [1, 2, 3, 4, 5]. DR has several risk factors such as the duration of diabetes mellitus (DM), poor glycemic control, hypertension, hyperlipidemia, obesity, smoking, alcohol intake, and pregnancy [6, 7, 8]. Moreover, genetic determinants have a role in the development and progression of DR [9].
Regular DR screening is recommended for diabetic patients to recognize high-risk DR characteristics. Therefore, it is essential to detect cases that require timely full ophthalmic examination and treatment for permanent visual loss prevention and to reduce the new cases of blindness. Additionally, screening is useful for identifying cases at the risk of cardiovascular disease, which expands their role beyond the prevention of sight-threatening disease. Screening is done using diagnostic modalities for DR which are important for the detection and classification of DR. Current diagnostic modalities include ophthalmoscopy and retinal photography, standard color fundus photography, multicolor fundus photography, fundus fluorescein angiography, optical coherence tomography, optical coherence tomography angiography, ultrawide-field imaging systems, swept-source wide-field optical coherence tomography angiography, smartphone-based imaging, and artificial intelligence systems [10].
There are several stages for DR. The reduction in visual acuity (VA), distortion of visual images, and sight loss can occur at any stage of the disease. In the early stages, the disease may remain asymptomatic, but later on, retinal examination reveals progressive retinal damage. This retinal damage appears firstly as microaneurysms, followed by the appearance of cotton wool spots, retinal hemorrhages, and intraretinal microvascular abnormalities. Eventually, this may lead to neovascularization with fragile new blood vessels, so they can be easily damaged, causing vision obstruction. Neovascularization can also detach the retina, resulting in blindness [4].
Regarding the pathophysiology of DR, oxidative stress and inflammation occupy a major role in the development and progression of DR. Sustained high-circulating glucose level activates several inflammatory pathways such as the protein kinase C pathway, the hexosamine biosynthetic pathway, and advanced glycation end-products accumulation (Figure 1). This chronic hyperglycemia increases reactive oxygen species (ROS) exacerbating retinal oxidative stress and inflammation [2, 11]. This leads to a vicious circle of ROS inducing an increase of the inflammatory factors, and then, inflammation induces ROS generation. The nuclear factor κappa-B (NF-κB) is activated also in DM. It is a transcription factor regulating many cytokines, for example, interleukin (IL)-1β, IL-8, tumor necrosis factor (TNF)-α, and several chemokines [4, 12, 13]. Moreover, oxidative stress and inflammation can cause epigenetic modifications. These epigenetic modifications are rapid and transmissible to the next generation [4, 14].
Figure 1.
Schematic diagram showing different pathways involved in DR pathogenesis. These pathways induce oxidative stress and inflammation that lead to diabetic retinopathy. NADPH; Polyol pathway downregulates the nicotinamide adenine dinucleotide phosphate, AGEs; advanced glycation end products, NF-κB; nuclear factor κappa-B, IL; interleukin, TNF- α; tumor necrosis factor-α, GAPDH; glyceraldehyde 3-phosphate dehydrogenase.
The upregulation of inflammatory mediators in DM, together with growth factors such as vascular endothelial growth factor (VEGF), cause blood-retinal barrier (BRB) breakdown, vascular damage, and neuro-inflammation, as well as pathological neovascularization (NV) noticed in DR [15, 16]. VEGF is a major pathogenic factor in DR. Upregulated VEGF induces the expression of more inflammatory cytokines, inducing endothelial dysfunction and breakdown of the BRB. The sub- and intraretinal fluid accumulation causes macular thickening, known as diabetic macular edema (DME), and is considered a complication of DR [2, 17]. As DR is a neurovascular complication of diabetes, non-vascular cells, including retinal neurons and glial cells, are also affected, showing many abnormalities or accelerated apoptosis. Retinal microglia and astrocytes are two main sources of ROS causing oxidative stress in chronic neurodegeneration [4, 18, 19]. Diabetic retinal neurodegeneration is shown as a significant thinning of the inner retinal layers, including the retinal nerve fiber layer, and ganglion cell layer [20, 21, 22].
The current therapies available for DR aim to stop the inflammatory response, prevent retinal NV, and stabilize the BRB [23]. There is a wide range of therapies for DR which are always under development. The selection of the treatment modality depends on several factors. For instance, vitrectomy, where the blood and scar tissue is removed along with vitreous removal, is important in the management of certain cases in DR, for example, non-clearing vitreous hemorrhages. Laser photocoagulation of the abnormal leaky blood vessels is also used to treat pathological NV [24].
Pharmacotherapy has also been used and it produces more progress in the treatment of DR than laser or surgical therapy, but its cost is still a challenge [24]. Corticosteroids, such as dexamethasone, triamcinolone, and fluocinolone, are potent anti-inflammatory agents. They antagonize various pathologic inflammatory mediators such as TNF-α, IL-1β, and IL-6 [25]. As VEGF plays a major role in the pathogenesis of DR, it is considered a critical therapeutic target. Intravitreal injection of anti-VEGF therapy such as bevacizumab, brolucizumab, ranibizumab, and aflibercept can improve non-proliferative DR (NPDR) and prevent the onset of angiogenesis and DME [20, 26, 27, 28, 29]. Unfortunately, not all patients respond properly to anti-VEGF such as in the case of DME with subretinal fluid [2]. Another treatment modality, depending on the integration of laser therapy with pharmacotherapy, may provide a more durable and effective response [24]. It can also result in optimum clinical efficacy with reduced treatment burden for the patient. In proliferative DR (PDR), the combination treatment between anti-VEGF and pan-retinal photocoagulation may be superior to monotherapy [30].
As DR is a polygenetic disease influenced by both environmental and genetic factors, gene therapy is considered a potential alternative therapy for DR. Generally, gene therapy is proposed to introduce genetic material into the cells to compensate for defective genes or deliver therapeutic transgenes. It has several benefits, such as prolonged effect with less frequency of intravitreal injection [31, 32]. One of the methods used for gene delivery is extracellular vesicles (EVs), as it is considered an immune-friendly gene therapy-delivering approach.
Generally, EVs are lipid-bound structures released by cells into the extracellular space carrying bioactive components that interact with target cells, causing subsequent modification. They can be detected in different biological fluids. EVs represent important tools of intercellular communication that allow the exchange of various messages including proteins, lipids, and nucleic acids between adjacent and/or distant cells. These intercellular messages have critical rules in normal homeostasis and disease development [33]. Therefore, EVs are considered novel diagnostic/prognostic biomarkers, promising drug delivery vehicles, and unique tools for targeted therapy [34, 35]. EVs can effectively deliver their cargo, including drugs or nucleic acids, to targeted tissues or organs as they possess several natural advantages, such as stability in circulation, transportation capability, and natural barriers-traversing capacity [36].
According to the pathways involved in their biogenesis and release, size, contents, and functions, EVs are classified into exosomes, microvesicles (MVs), and apoptotic bodies [37]. Exosomes are generated via the inward budding of the limiting membrane of early endosomes during maturation into multivesicular bodies (MVBs). They vary in size from 50 to 150 nm [38]. Their formation and release are regulated by endosomal sorting complexes required for transport (ESCRT) [39]. Nonetheless, ESCRT-independent pathways have also been identified, where sphingomyelinase converts sphingomyelin into ceramides, which allows the sorting of the cargoes in the exosomes and enhances the formation of curvatures in the membrane [40]. Exosomes were shown to have higher levels of transmembrane proteins and glycoproteins compared to whole-cell lysate [41]. Based on the cholesterol contents, MVBs are degraded by lysosomes, or they merge with the plasma membrane and liberate their exosome contents into the extracellular space [42, 43]. Exosomes interact with the target cells by micropinocytosis, endocytosis, or they fuse with the cell membrane and deliver their contents into the cell [44]. Figure 2 describes the biogenesis and uptake of exosomes.
Figure 2.
A schematic diagram illustrating different steps of EVs biogenesis and release from donor cells. EVs uptake by the recipient cells can occur via endocytosis, pinocytosis, or membrane fusion.
Microvesicles are formed by external budding and then scission from the plasma membrane of both healthy and damaged cells. Their size ranges between 50 and 500 nm, and up to 10 μm in cases of cancer-derived EVs, known as oncosomes. Cytoskeletal components, molecular motors, and fusion machinery were shown to contribute to MVs formation [45]. In comparison to exosomes, MVs tend to contain higher levels of proteins with post-translational modifications [41]. Different mechanisms are adopted to mediate MVs-cellular communications such as receptor-ligand interaction, fusion of MVs membrane with that of the recipient cell, endocytic-mediated internalization of MVs contents, and membrane protease cleavage resulting in the release of MVs soluble proteins, which interact with specific receptors on the target cells [46].
Regarding apoptotic bodies, they are typically larger, ranging from 50 to 5000 nm. They are generated during the process of programmed cell death (apoptosis) as blebbing from the plasma membrane of cells [47]. They enclose intact organelles, chromatin, and proteins [48]. In contrast to exosomes and MVs, the proteomic analysis of apoptotic bodies is similar to that of cell lysate [49]. The features of different subtypes of EVs were summarized by Hu et al. [50].
The basics of uptake and intercellular trafficking of different subtypes of EVs are thought to be shared despite different sizes and components [36]. Inside the recipient cell, endocytosed EVs either follow the degradation pathway or release their contents into the cytoplasm [35]. Van der Pol et al. [51] and Lv et al. [52] have reviewed the functions of exosomes and MVs and highlighted their crucial roles in cell-to-cell communication and signaling, particularly in the immune response, tumorigenesis, thrombus formation, and waste removal. Figure 3 portrays retinal intercellular communication via EVs [51, 52].
Figure 3.
Different retinal cells use EVs as a tool for intercellular communication. This diagram illustrates different sources of color-coded EVs from various retinal cells.
In this book chapter, we briefly discuss the EVs’ isolation. We emphasize EVs originating from various sources, either intraocular or extraocular sources, and explain how they contribute to inflammation and angiogenesis, as two major arms of DR pathophysiology. Additionally, we highlight the EVs as promising diagnostic biomarkers and therapeutic tools in the field of DR. Finally, we list the related challenges and future recommendations.
As most isolation methods depend on the distinction of EVs according to the size, and due to size overlap between exosomes and MVs, it is hard to obtain a pure population of EVs subtypes [53]. Commonly used isolation methods include immunoprecipitation, polymer-based precipitation, ultrafiltration, ultracentrifugation, density gradient, microfluidics, and size exclusion chromatography. An interesting study by Helwa et al. [54] compared ultracentrifugation versus commercially available kits for exosome isolation and showed that even with limited volumes of biological samples, commercially available isolation kits were feasible alternatives to the ultracentrifugation technique. The principle of the techniques, advantages, disadvantages, yield, and purity are neatly tabled by Massoumi et al. [55]. Regarding identification and characterization, tetraspanins, such as CD9, CD63, and CD81, are generally used as markers for exosomes. CD40, selectins, integrins, and annexin 1 are used for MVs characterization. As far as apoptotic bodies, DNA, histone, and phosphatidylserine are commonly used [55, 56, 57]. Electron microscopy (EM) studies including transmission EM, scanning EM, or immune-gold labeling using different EVs markers are generally used as an additional confirmatory step for EVs characterization [58, 59]. Nanoparticle tracking analysis is commonly used to report the concentration and the size range of EVs in isolated samples [54].
Inflammation and immune dysfunction result from chronic hyperglycemia and hyperlipidemia, are believed to be key mechanisms in the development of DR. Increased levels of inflammatory cytokines and angiogenic factors were noticed in plasma EVs from diabetic patients, which find their way to the retina because of increased permeability of BRB and are known to contribute to the pathophysiology of DR [60]. Our previous work highlighted the pathway analysis of retinal pigment epithelium (RPE)-derived exosomal microRNAs (miRNAs or miRs) that showed their involvement in different cellular pathways related to the development of DR such as oxidative stress, endoplasmic reticulum (ER) stress, and VEGF signaling [61]. Our ongoing research noticed a differential exosomal miRNA expression profile of retinal endothelial cells treated with proinflammatory lipid mediators. Inflammatory mediators such as TNF-α, thrombin, and C-reactive protein (CRP) were found to enhance endothelial cells (ECs) to form MVs in vitro. These MVs promote inflammation by triggering target cells to produce ROS, leading to oxidative stress [62, 63, 64]. High glucose causes increased NADPH oxidase activity in endothelium-derived microparticles, contributing to disturbed retinal barrier functions [65]. EVs derived from RPE under oxidative stress promoted apoptosis and inflammation through Apaf1/caspase-9 axis [66]. Additionally, Tokarz et al. [67] found a correlation between chemokine receptor (CCR)-5 positive MVs with NPDR progression.
Platelet-derived MVs from patients with DR enhanced the interaction between monocytes and endothelial cells [68]. Also, in diabetic patients, high levels of CXCL-10 in platelet-derived EVs stimulated the Toll-like receptor (TLR)-4 pathway, triggered ROS production, inhibited superoxide dismutase (SOD) activity, and lessened tight junction proteins, with the net result of increased BRB permeability [69]. Additionally, exosomes derived from platelet-rich plasma from diabetic rats enhanced the proliferation and fibrogenic activity of human retinal Müller cells (hMCs) through PI3K/AKT signaling [70].
Microglia are the principal immune cells in the retina which respond to paracrine signals carried by EVs. DM promotes a chronic inflammatory status in the body, which causes polarization of the microglia to the proinflammatory M1 phenotype [71]. Furthermore, M1-polarized microglia accumulate around the capillaries and release EVs, which heighten neuro-inflammation and increase vascular permeability [72, 73]. Beltramo et al. [74] reported a significant increase in inflammatory markers, including chemokine ligand (CCL)-2, matrix metalloproteinase (MMP)-2, and MMP-9, in M1-polarized microglia-derived EVs. A significant increase in the miRNAs related to inflammation and angiogenesis (miR-21 and miR-155) was also marked. Interestingly, exposure of human retinal pericytes (HRPs) and Human retinal endothelial cells (HRECs) to EVs derived from M1-polarized microglia caused a significant increase in their proliferation, apoptosis, migration, and ROS production. In addition, HRPs displayed significant production of IL-1β, IL-6, MMP-9, CCL-2, and VEGF, while HRECs exhibited significant release of TNF-α, angiopoietin (Ang)-2, VEGF, and platelet-derived growth factor (PDGF)-B, when exposed to M1-polarized microglia-derived EVs. Of note, Müller glia cells-derived exosomes were found to aggravate vascular dysfunction under high glucose through exosomal miR-9-3p, which is transferred to retinal endothelial cells and binds to the sphingosine-1-phosphate receptor S1P1 coding sequence, which subsequently activates VEGFR2 phosphorylation [75].
The complement system is a key mechanism deployed by the innate and adaptive immune responses and causes damage to opsonized pathogens and cells via deposition of the membrane attack complex (MAC) on the plasma membrane, resulting in osmotic disturbance and lysis of the target cell [76, 77]. The expression of MAC was marked in the chorio-capillaries and around larger choroidal vessels in patients with DR [78]. This complement-mediated damage is attributed to continuous complement activation, in addition to the lack of complement regulatory proteins on the retinal endothelium [79]. Interestingly, Huang et al. [80, 81] reported robust complement activation in the plasma of diabetic rats by immunoglobulin (Ig) G-enriched EVs. Further, these IgG-laden EVs caused MAC deposition and lysis of HRECs in vitro, which was abrogated by the depletion of EVs from the plasma.
The involvement of EVs in the pathogenesis of DR, particularly in establishing microvascular dysfunction and angiogenesis, has been investigated in numerous studies. Although RPE-derived EVs are believed to induce protective effects in physiological states and during the early stages of DR, EVs originated from stressed ARPE-19 cells enhanced angiogenesis, through upregulation of VEGF receptors on HUVEC cells [82]. A longitudinal cohort study conducted by Wu et al. [83] reported the significant link between EVs levels of VEGF-A, the diabetes situation, and parameters of insulin resistance, in diabetic patients when compared to euglycemic controls. Moreover, when endothelial cells were treated with EVs from diabetic patients, the formation of lamellipodia increased and led to enhanced migration of endothelial cells.
Plasma-derived EVs from patients with DR were capable of inducing retinopathy indices in retinal microvascular in vitro models in the form of detachment and migration of pericytes, increased permeability, and new vessel-like structure formation [84]. TNF-α-induced protein 8 (TNFAIP8) was demonstrated in the small EVs (sEVs) isolated from DR patients’ plasma and was linked to increased viability, proliferation, migration, and tube formation of HRECs [85].
Circulating EVs were reported to increase in diabetic patients and to induce phenotypic changes similar to those of DR in different study models owing to their bioactive cargoes, especially miRNAs [86]. Free miRNAs in the plasma are liable to degradation by enzymes; however, EVs’ lipid membrane protects them [74]. Plasma-derived EVs from patients with DR did not display differences in the surface markers when compared to diabetic patients with no DR or healthy controls; however, microarray analysis revealed significant changes in several miRNAs [84]. One of the important miRNAs reported to promote angiogenesis is miR-30b, which was found to be prominently enriched in plasma-derived EVs and was the most differentially expressed miRNA in the retinal tissues of mice with PDR, compared to control mice. miR-30b exerts its pro-angiogenic effect on retinal microvascular endothelial cells (RMECs) through repression of sirtuin (SIRT)-1; the latter was stated to preserve vascular integrity of the retina and protect against the development of DR in the early phases [87, 88]. Besides, upregulation of miR-30a-5p was recognized in the vitreous specimens of patients diagnosed with PDR [89]. It promotes angiogenesis by enhancing endothelial cells’ motility and tube formation [90]. miR-21 was also found to enhance angiogenesis through repression of peroxisome proliferator-activated receptor-α (PPARα) [91]. In addition, the expression of miR-30b-5p, miR-21-3p, and hypoxia-inducible factor (HIF)-1α increased, and that of miR-150-5p decreased in EVs derived from DR patients, compared to healthy controls and diabetic patients with no DR [92]. Noteworthy, miR-150 was stated to inhibit the migration of endothelial cells (ECs) through inhibition of numerous angiogenic factors [93].
Pericytes regulate the blood capillaries tone, control the interchange of molecules between the blood and the neuroretina, and limit the proliferation and motility of ECs; thus, their loss leads to the development of angiogenesis, which is a key mechanism in the early stages of DR [94]. EVs control the interplay between pericytes and ECs in DR, where circular RNA (circRNA) cPWWP2A is shuttled from pericytes to ECs by EVs, inducing retinal vascular malformations through enhanced expression of Ang-1, occludin, and SIRT-1 [95]; however, a protective role of Ang-1 in DR has been described [96]. EVs derived from microglia and photoreceptors were reported to increase in the vitreous of PDR patients and were found to promote ECs proliferation and angiogenesis [97]. Maisto et al. [98, 99] demonstrated a decrease in the anti-angiogenic miRNAs (miR-20a-3p, miR-20a-5p, miR-20b, and miR-106a-5p), together with increased expression of VEGF in retinal photoreceptors/ARPE-19 cells and their derived EVs, when challenged with high glucose.
The role of MVs in the development of DR was summarized by Zhang et al. [46], where the diabetic milieu causes MVs of platelet origin to activate MMPs, which cause mitochondrial damage and release of cytochrome c, which in turn enhances apoptosis of capillary endothelial cells and consequently, capillary hypoxia, which leads to NV over time. MVs of monocyte origin were claimed to cause damage to RPE, compromising the barrier function and increasing the permeability. Noteworthy, monocyte-derived MVs were the most prominent during the transition of DR from the non-proliferative to the proliferative stage, where abnormal leaky blood vessels are formed [100].
Significantly, mesenchymal stem cells (MSCs) were established to contribute to the homeostasis and the pathology of different tissues, including the retina, through the paracrine effect of EVs [101]. Beltramo et al. [102] reported that HRPs display enhanced detachment when exposed to EVs isolated from MSCs, which were grown in high glucose and/or hypoxia to mimic diabetic conditions, regardless of the origin of MSCs. The detached cells were viable and were able to re-attach again, suggesting that these cells tend to localize in the walls of the newly formed vessels for stabilization. They also described increased permeability of the blood barrier, and enhanced formation of vessel-like structures in HRP/EC co-cultures on Matrigels when treated with the same EVs. Moreover, they attributed the effect of MSCs-derived EVs on HRPs and ECs to the active form of MMP-2, which causes degradation of the proteins in the extracellular matrix, facilitating pericyte loss and angiogenesis. Interestingly, pancreatic β cells were proved to release EVs rich in miR-15a, which spread to distant sites and promote DR through induction of oxidative stress and apoptosis in Müller cells [103].
It is worth mentioning that procoagulant MVs in patients with DR correlate with the duration of diabetes [104]. Phosphatidyl serine (PS)-positive MVs are linked to enhanced coagulant activity in patients with PDR and NPDR [105]. Additionally, endothelial-derived MVs are considered a risk factor for the development of thrombo-embolic DR [106]. The roles of EVs in the initiation and progression of DR are summarized in Table 1.
Source of EVs
Method of isolation and/or purification
Role of EVs in DR
Refs.
Extraocular cell-derived EVs
Plasma
Isolated by ultracentrifugation and purified by OptiPrep Density Gradient
Circulating IgG-laden exosomes are responsible for the activation of the complement cascade that subsequently upregulates proinflammatory cytokines and chemokines and causes MAC deposition and lysis of HRECs in vitro.
Isolated using total exosome isolation reagent for cell culture media
Plasma-EVs transferred miR-30b to retinal microvascular endothelial cells (RMECs), where miR-30b increases VEGF expression by downregulating SIRT1. This enhances angiogenesis and proliferative DR.
Hyperglycemia elevates the level of circulating PRP-Exos which activates the TLR4 pathway through the release of CXCL10. This led to endothelial injury and dysfunction in early DR.
Platelet rich plasma derived exosomes in hyperglycemia enhance both the proliferation and fibrogenic activity of Müller cells through PI3K/AKT signaling.
Isolated using total exosome isolation reagent for cell culture media
Exposing Müller cells to exosomes derived from pancreatic β-cells under high-glucose conditions caused miR-15a overexpression in Müller cells and increased the oxidative stress and apoptotic cell death.
Apoptosis and autophagy levels in RACs were positively correlated with the oxidative stress level, and the exosomes generated from RACs under oxidative stress condition. These exosomes can promote endothelial cell proliferation, migration, and tube formation.
High glucose increased VEGF levels and decreased anti-angiogenic miR-20a-3p, miR-20a-5p, miR-106a-5p, and miR-20b expression in retinal photoreceptor-derived EVs due to ROS-induced NF-κB signaling.
Müller glia-derived exosomes aggravate vascular dysfunction under high glucose, through exosomal miRNA-9-3p which is transferred to retinal endothelial cells and binds to the sphingosine-1-phosphate receptor S1P1 coding sequence, with subsequent activation of VEGFR2 phosphorylation.
Pericytes transferred cPWWP2A, miR-579, and angiopoietin 1/occludin/SIRT1 to endothelial cells by exosomes. These transferred materials induced retinal vascular dysfunction.
The clinical diagnosis of DR is challenging since the disease is asymptomatic during the early stages. Development of symptoms in the form of blurred vision may occur with more progressive pathology [86]. Given that EVs reflect the molecular profile of the parent cells and the important role played by EVs, particularly the miRNAs shuttled by EVs in the pathophysiology of DR [107], it appeared valuable to use them as diagnostic biomarkers. Several studies aimed at the detection of EVs-miRNAs exclusive to DR.
As mentioned before, cPWWP2A circRNA is shuttled by EVs derived from pericytes in DR, and it is thought to have a protective role in refining pericytes-endothelial cells communication and amelioration of vascular pathology. This circRNA can be used as a diagnostic biomarker [95]. Likewise, EVs positive for miR-15a [103], IgG [108], or TNFAIP8 [85], all can be used for the diagnosis of DR. EVs with increased expression of miR-30b-5p, miR-21-3p, miR-26b-5p, or decreased expression of miR-150-5p were identified in the sera of DR patients [92, 109]. Additionally, EVs carrying CCR-5 can be used to detect NPDR progression [67]. Remarkably, Mighty et al. [110] confirmed the expression of junction plakoglobin protein (JUP) in the EVs isolated from retinal tissue explants and urine of DR patients but not in healthy controls. This was the first study to provide a novel non-invasive diagnosis for DR through urinary specimens. Table 2 shows examples of EV-derived cargoes that could be used as biomarkers for DR.
EVs marker
Origin
Role
Reference
cPWWP2A
Pericytes
Refining pericytes-endothelial cells communication and amelioration of vascular pathology
Examples of EVs cargoes that could serve as potential biomarkers for DR.
Interestingly, some studies have proved the effectiveness of certain miRNAs as diagnostic markers; however, these studies did not consider their expression in EVs. For instance, miR-181c, miR-21, and miR-1179 were reported to increase significantly in the sera of PDR patients when compared to NPDR patients [111]. Detection of such miRNAs in serum EVs may provide higher diagnostic values. Similarly, Friedrich et al. [112] found higher levels of six miRNAs in the vitreous of PDR patients when compared to non-diabetic patients namely, hsa-miR-23b-3p, hsa-miR-20a-5p, hsa-miR-142-3p, hsa-miR-185-5p, hsa-miR-362-5p, and has-miR-326. The first one, hsa-miR-23b-3p, was found to be downregulated in anti-VEGF-treated patients in comparison to non-treated PDR patients. On the other hand, miR-126 decreased in DR patients and could serve as a diagnostic biomarker for PDR [113]. We recommend conducting future studies to verify the expression of these miRNAs in circulating EVs, which might spot the time of alteration in their levels and allow their use as diagnostic and prognostic markers through non-invasive sampling procedures.
6. Are EVs a promising new therapeutic tool for DR?
Impairment and loss of vision are unavoidable if DR is left untreated. During the initial stages, proper control of blood glucose levels may prevent the deterioration of the disease. Treatment of hypertension and hyperlipidemia is also important; however, medical intervention is necessary in the late stages. Anti-VEGF, corticosteroids, lipid-lowering drugs, argon laser photocoagulation, and vitrectomy are commonly used, as reviewed by Martins et al. [86]. Immunotherapy such as anti-TNF-α and anti-IL-1β are being tested [114, 115]. Although current interventions may help prevent or delay the sequelae of the disease, lack of curative treatment, adverse reactions, and ineffectiveness of these regimens in some patients are still considerable challenges [116]. Therefore, it is important to seek novel and more tolerable therapies, such as EVs, which can deliver many factors including miRNAs and medications to the retina, in addition to enhancing the efficacy of certain drugs (Figure 4) [117].
Figure 4.
EVs derived from intra- or extra-ocular sources can be used as a therapy for DR directly, as a drug delivery nanocarrier, or engineered EVs with elevated levels of targeted miRNAs.
Intriguingly, intraocular injection of exosomes derived from rabbit adipose tissue MSCs in diabetic rabbits resulted in well-organized retinal layers. The link between increased miR-222 in MSCs-derived exosomes and the regenerative events in the retina was highlighted [118]. In addition, Zhang and colleagues [119], used umbilical cord MSCs-derived exosomes as a vehicle for miR-126. Intravitreal injection of these exosomes in diabetic rats suppressed retinal inflammation. The authors confirmed the role of miR-126-over expression in MSCs-derived exosomes in the reduction of high-mobility group box (HMGB)-1 and inflammasome, which resulted in amelioration of retinal inflammation. Gu et al. [120] reported that MSCs-derived EVs, which shuttle miR-192, ameliorated retinal inflammation and angiogenesis. Reddy et al. [121] reported that intravitreal injection of MSCs-derived sEVs loaded with anti-VEGF caused prolonged inhibition of VEGF and leukostasis and decreased exudates in diabetic rats for more than 2 months following injection, supporting the value of sEVs as drug delivery system. Furthermore, the anti-oxidant and anti-apoptotic effect of MSCs-derived EVs was reported in an animal model of DR. The in vitro experiments attributed this therapeutic effect to neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4), which regulates PTEN/AKT/NRF2 pathway [122]. On the other hand, EVs derived from MSCs, which were grown in high glucose, were claimed to accelerate vascular dysfunction [102]; therefore, the use of MSCs-derived EVs in the treatment of DR needs more extensive studies.
Interestingly, exosomes derived from retinal astroglial cells (RACs), but not from RPE, inhibited vessel leakage and choroidal neovascularization (CNV) in a laser-induced CNV model. These anti-angiogenic properties may be useful when applied in DR models [123]. RPE-derived exosomes were found to inhibit endothelial-to-mesenchymal transition (EMT) and retinal fibrosis due to their miR-202-5p content, which inhibits transforming growth factor/Smad signaling [124]. Of note, our study showed that RPE-treated with homocysteine, which is reported as a risk factor for retinopathies, released exosomes with micro-RNA cargo close to the miRs profile noticed in the diabetic mouse retina, such as miR-17, let-7, and miR-122 [61]. Recently, Huang et al. [125] analyzed sEVs derived from human embryonic stem cell-derived retinal organoids (hERO-RPCs) and found them to delay the degeneration of photoreceptors, inhibit gliosis of Müller glia cells, and support retinal functions in RCS rats. They attributed these effects to miRNA-mediated downregulation of nuclear factor I transcription factors B (NFIB).
Lymphocyte-derived microparticles (LMPs) were found to contain high levels of miR-181a. This miRNA interferes with VEGF signaling in HREC; therefore, it could be used for the treatment of DR [126]. Besides, control of complement activation was achievable through the exclusion of IgG-laden plasma EVs, which led to the protection of the ECs from cytolytic damage [80]. A recent study by Lamb et al. [127] investigated the Müller glia cells-derived EVs and highlighted their neuroprotective functions through enrichment with miRNAs known to support the stem cells, such as miR-21 and miR-16, and miRNAs which keep the retinal homeostasis; such as miR-125b, miR-9, and let-7 family. Mathew et al. [128] treated retinal cells suffering from ischemic insult with functionally engineered EVs overexpressing miR-424 (FEE424), and found them to achieve anti-inflammatory and neuroprotective functions. They also used FEE424 in an animal model of retinal ischemia and reported that retinal functions were restored.
Other miRNAs were confirmed to have a protective effect on the retinal tissue. For instance, Shaker et al. [129] found that miR-20b and miR-17-3p decrease significantly in the sera of both PDR and NPDR patients, in comparison to healthy controls. miR-20b and miR-17-3p are believed to regulate the expression of HIF-1α and VEGF-A genes. Another study reported downregulation of four miRNAs in the sera of DR patients namely, miR-338-3p, miR-4448, miR-9-5p, and miR-485-5p [130]. These miRNAs regulate inflammation, oxidative stress, and angiogenesis through the regulation of dozens of genes. miR-200b expression in different DR animal models revealed controversial results; nevertheless, McArthur et al. [131] reported its decrease in the retinas and endothelial cells of diabetic rats and linked this decrease to enhanced VEGF expression. Blum et al. [132] confirmed the link between decreased levels of miRNA-423 and increased levels of VEGF in the sera of DR patients. Also, Qin et al. [113] highlighted miR-126 lower levels in DR patients. While EVs were not included in these studies, it is possible to establish a drug delivery system using those EVs to deliver such deficient miRNAs.
In contrast, some studies reported higher values of certain miRNAs in DR patients without the inclusion of EVs in these studies. For example, miR-181c, miR-21, and miR-1179 were proven to increase the sera of DR patients and were known to play important roles in ischemia and angiogenesis [111]. Using EVs as vehicles to deliver such deficient miRNAs may be a valuable drug delivery tool in DR. Sun et al. [133] developed engineered MSCs-EVs with increased levels of miR-5068 and miR-10228 as therapeutic molecules. They used both MSCs-EVs and engineered ones to treat two diabetic animal models, including db/db mice and STZ-induced diabetic rats. The presence of miR-5068 and miR-10228 in both EVs targeted the HIF-1α/EZH2/PGC-1α pathway. However, the engineered MSCs-EVs showed more enhanced retinal repair efficiency than MSCs-EVs.
The therapeutic potential of EVs requires the construction of clinical-grade EVs, where enormous quantities, in addition to preserved phenotypes, are necessary. As different cell culture procedures aim at increasing the harvest, such as using different stimuli to expand the production or using 3D culture technology, the phenotype of the cultured cells and, consequently, their EVs may change [134]. Passage of the cultured cells, media composition, and oxygen levels could affect the cells/EVs number and quality [135]. The use of the third to fifth passage was recommended to preserve consistency in MSCs [136]. Regarding media composition, while fetal bovine serum was found to influence the behavior of cells, serum-free media changed the protein content of EVs [137]. Platelet lysate with an EV-depleted medium was reported to preserve the phenotype of MSCs and the RNA profile of their EVs [138].
Another challenge is the isolation of EVs. Although several techniques are available, there is no standardized technique for isolation of clinical-grade EVs. Low yield in ultracentrifugation, low purity, or chemical contamination in the other techniques interfere with their use in such settings [50]. Ultrafiltration followed by size exclusion chromatography was confirmed to obtain a higher yield of EVs and preserve their phenotype [139, 140]. Cryopreservation using glycerol and dimethyl sulfoxide (DMSO) was found to disintegrate EVs [141]. Using phosphate-buffered saline (PBS) was reported to form calcium nanoparticles, which intervene with quantification of EVs [142]. Concerning the temperature of storage, different studies reported different results, such as −20 or −80° C [141, 143]. To extend the shelf life and decrease the cost, lyophilization was proposed, with a storage temperature of 4°C [144].
Directing EVs to specific cells is another challenge. While the factors that control the targeting of EVs to certain cells are not fully described, surface molecules, the physiological status of the target cells, and the route of administration play important roles in the process [50, 118]. Using certain labels to track EVs was confirmed to enhance their targeting of certain cells [145, 146]. Improving the therapeutic properties of EVs to reach the target cells is required to decrease the dose or the frequency of administration. Priming, loading, engineering, artificial EVs, and bio nanotechnology were developed for that purpose [147].
Finally, the safety of EV-based therapy is crucial. Although cell therapy may induce tumorigenesis, EVs have been shown to be well tolerated in several animal models. Immunogenicity and toxicity monitoring in humans must be extensively studied [53].
DR is a neurovascular disease where different retinal cells may be involved in pathophysiological changes occurring throughout the disease course. Retinal endothelial cells exposed to high glucose levels during diabetes may send early warning messages to other retinal cells, initiating various signaling pathways via endothelial-derived EVs. Retinal Müller glia cells, as important master regulators of retinal homeostasis, may talk to other retinal cells via EVs, trying to fight against the high-glucose-induced retinal injury. Other retinal cells may use this salient method of communication to synergize their actions during the disease. Understanding these EVs-carried messages may help in understanding a more detailed picture of the process of DR development. That is why EVs are promising new tools that could carry novel biomarkers for early diagnosis and prognosis of DR. EVs have been used recently as an encouraging drug delivery method in cases of DR. The field of EVs is an emerging area of research that still needs more extensive studies to portray a thorough understanding of EVs’ retinal trafficking, various cargoes, isolation purity, proper characterization, and therapeutic safety.
The authors have no conflict of interest to declare.
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Written By
Samar Habib, Alaa M. Mansour, Babak Baban and Khaled Elmasry
Submitted: 17 February 2024Reviewed: 23 April 2024Published: 24 June 2024
Open access peer-reviewed chapter - ONLINE FIRST
