Retinal ganglion cell neuroprotection by growth factors and exosomes: Lessons from mesenchymal stem cells

Retinal ganglions cells (RGCs) are responsible for propagating electrochemical information from the eye to the brain along their axons which make up the optic nerve. The loss of RGCs is characteristic in several conditions such as glaucoma and traumatic optic neuropathy and leads to visual loss and blindness. While no therapy exists to directly treat RGCs, the use of bone marrow-derived mesenchymal stem cells (BMSCs) has shown promise in eliciting significant RGC neuroprotection

The role of miRNA in retinal ganglion cell health and disease

miRNA are short non-coding RNA responsible for the knockdown of proteins through their targeting and silencing of complimentary mRNA sequences. The miRNA landscape of a cell thus affects the levels of its proteins and has significant consequences to its health. Deviations in this miRNA landscape have been implicated in a variety of neurodegenerative diseases and have also garnered interest as targets for treatment. Retinal ganglion cells are the sole projection neuron of the retina with their axons making up the optic nerve. They are a focus of study not only for their importance in vision and the myriad of blinding diseases characterized by their dysfunction and loss, but also as a model of other central nervous system diseases such as spinal cord injury and traumatic brain injury. This review summarizes current knowledge on the role of miRNA in retinal ganglion cell function, highlighting how perturbations can result in disease, and how modulating their abundance may provide a novel avenue of therapeutic research

Extracellular vesicle therapy for retinal diseases

Extracellular vesicles (EV), which include exosomes and microvesicles, are secreted from virtually every cell. EV contain mRNA, miRNA, lipids and proteins and can deliver this expansive cargo into nearby cells as well as over long distances via the blood stream. Great interest has been given to them for their role in cell to cell communication, disease progression, or as biomarkers, and more recent studies have interrogated their potential as a therapeutic that may replace paracrine-acting cell therapies. The retina is a conveniently accessible component of the central nervous system and the proposed paradigm for the testing of many cell therapies. Recently, several studies have been published demonstrating that the delivery of EV/exosomes into the eye can elicit significant therapeutic effects in several models of retinal disease. We summarize results from currently available studies, demonstrating their efficacy in multiple eye disease models as well as highlighting where future research efforts should be directed

Bone marrow-derived mesenchymal stem cells-derived exosomes promote survival of retinal ganglion cells through miRNA-dependent mechanisms

The loss of retinal ganglion cells (RGC) and their axons is one of the leading causes of blindness and includes traumatic (optic neuropathy) and degenerative (glaucoma) eye diseases. Although no clinical therapies are in use, mesenchymal stem cells (MSC) have demonstrated significant neuroprotective and axogenic effects on RGC in both of the aforementioned models. Recent evidence has shown that MSC secrete exosomes, membrane enclosed vesicles (30–100 nm) containing proteins, mRNA and miRNA which can be delivered to nearby cells. The present study aimed to isolate exosomes from bone marrow‐derived MSC (BMSC) and test them in a rat optic nerve crush (ONC) model. Treatment of primary retinal cultures with BMSC‐exosomes demonstrated significant neuroprotective and neuritogenic effects. Twenty‐one days after ONC and weekly intravitreal exosome injections; optical coherence tomography, electroretinography, and immunohistochemistry was performed. BMSC‐derived exosomes promoted statistically significant survival of RGC and regeneration of their axons while partially preventing RGC axonal loss and RGC dysfunction. Exosomes successfully delivered their cargo into inner retinal layers and the effects were reliant on miRNA, demonstrated by the diminished therapeutic effects of exosomes derived from BMSC after knockdown of Argonaute‐2, a key miRNA effector molecule. This study supports the use of BMSC‐derived exosomes as a cell‐free therapy for traumatic and degenerative ocular disease

Mesenchymal stem cell-derived small extracellular vescicles promote neuroprotection in a genetic DBA/2J mouse model of glaucoma

Purpose: To determine if bone marrow-derived stem cell (BMSC) small extracellular vesicles (sEV) promote retinal ganglion cell (RGC) neuroprotection in the genetic DBA/2J mouse model of glaucoma for 12 months. Methods: BMSC sEV and control fibroblast-derived sEV were intravitreally injected into 3-month-old DBA/2J mice once a month for 9 months. IOP and positive scotopic threshold responses were measured from 3 months: IOP was measured monthly and positive scotopic threshold responses were measured every 3 months. RGC neuroprotection was determined in wholemounts stained with RNA binding protein with multiple splicing (RBPMS), whereas axonal damage was assessed using paraphenylenediamine staining. Results: As expected, DBA/2J mice developed chronic ocular hypertension beginning at 6 months. The delivery of BMSC sEV, but not fibroblast sEV, provided significant neuroprotective effects for RBPMS+ RGC while significantly reducing the number of degenerating axons seen in the optic nerve. BMSC sEV significantly preserved RGC function in 6-month-old mice, but provided no benefit at 9 and 12 months. Conclusions: BMSC sEV are an effective neuroprotective treatment in a chronic model of ocular hypertension for 1 year, preserving RGC numbers and protecting against axonal degeneration

Mesenchymal stem cell-derived small extracellular vesicles promote neuroprotection in rodent models of glaucoma

Purpose: To investigate the benefit of bone marrow mesenchymal stem cell (BMSC)-derived small extracellular vesicles (sEV) as an intravitreal (ivit) therapy in two rat models of glaucoma and to determine and identify candidate miRNA involved in the mechanism. Methods: sEV were isolated from human BMSC and fibroblasts and ivit injected into adult rats after induction of elevated IOP. IOP was elevated using either intracameral injection of microbeads or laser photocoagulation of circumferential limbal vessels and the trabecular meshwork. Retinal nerve fiber layer (RNFL) thickness was measured using optical coherence tomography, positive scotopic threshold response (pSTR) recorded using ERG, and RNA binding protein with multiple splicing (RBPMS+) retinal ganglion cell (RGC) counted using retinal wholemounts. sEV miRNA were sequenced using RNAseq. Results: sEV isolated from BMSC promoted significant neuroprotection of RGC while preventing RNFL degenerative thinning and loss of pSTR. sEV proved therapeutically efficacious when ivit injected every week or every month, but ineffective with longer delays between treatments. Knockdown of Argonaute2 (AGO2), a protein critical for miRNA function and packing into sEV prior to sEV isolation, significantly attenuated the above effects. Addition of BMSC sEV (but not fibroblast sEV) reduced death of cultured purified RGC. RNAseq identified 43 miRNA upregulated in BMSC sEV in comparison to fibroblast sEV, which yielded no neuroprotective effects. Conclusions: Injection of BMSC-derived sEV into the vitreous provided significant therapeutic benefit to glaucomatous eyes. The neuroprotective effect of sEV, at least partially, may be explained by direct action on RGC through miRNA-dependent mechanisms

TNFα-mediated priming of mesenchymal stem cells enhances their neuroprotective effect on retinal ganglion cells

Purpose: To determine whether priming of bone marrow mesenchymal stem cells (MSCs) by signals from injured retina, particularly tumor necrosis factor α (TNFα), increase their exosomes’ neuroprotective efficacy on retinal ganglion cells (RGCs). Methods: MSCs were primed with retinal cell culture conditioned medium, with or without the TNFα blocker etanercept or TNFα prior to isolation of exosomes. MSC conditioned medium or exosomes were added to rat retinal cultures or human stem cell–derived retinal ganglion cell (hRGC) cultures, and RGC neuroprotective effects were quantified. Luminex assays were used to compare primed versus unprimed exosomes. Results: MSC conditioned medium and exosomes exerted a significant neuroprotective effect on injured rat and hRGC. This effect was significantly increased after MSCs were primed with retinal conditioned medium or TNFα. Blocking of TNFα signaling with etanercept prevented priming-induced RGC neuroprotective efficacy. Priming increased PEDF and VEGF-AA exosomal abundance. Conclusions: MSC exosomes promote RGC survival not just in rodent retinal cultures but also with hRGC. Their efficacy can be further enhanced through TNFα priming with the mechanism of action potentially mediated, at least in part, through increased levels of PEDF and VEGF-AA

miRNA Changes in retinal ganglion cells after optic nerve crush and glaucomatous damage

The purpose of this study was to characterize the miRNA profile of purified retinal ganglion cells (RGC) from healthy and diseased rat retina. Diseased retina includes those after a traumatic optic nerve crush (ONC), and after ocular hypertension/glaucoma. Rats were separated into four groups: healthy/intact, 7 days after laser-induced ocular hypertension, 2 days after traumatic ONC, and 7 days after ONC. RGC were purified from rat retina using microbeads conjugated to CD90.1/Thy1. RNA were sequenced using Next Generation Sequencing. Over 100 miRNA were identified that were significantly different in diseased retina compared to healthy retina. Considerable differences were seen in the miRNA expression of RGC 7 days after ONC, whereas after 2 days, few changes were seen. The miRNA profiles of RGC 7 days after ONC and 7 days after ocular hypertension were similar, but discrete miRNA differences were still seen. Candidate mRNA showing different levels of expression after retinal injury were manipulated in RGC cultures using mimics/AntagomiRs. Of the five candidate miRNA identified and subsequently tested for therapeutic efficacy, miR-194 inhibitor and miR-664-2 inhibitor elicited significant RGC neuroprotection, whereas miR-181a mimic and miR-181d-5p mimic elicited significant RGC neuritogenesis

Differential Prox-1 and CD 31 expression in mucousae, cutaneous and soft tissue vascular lesions and tumors

The study of lymphatic vessels and lymphatic tumors has been hampered with difficulty due to the overlapping morphological features between blood and lymphatic endothelial cells, as well as to the lack of specific lymphatic endothelial markers. Over the last few years, lymphatic vessels and lymphangiogenesis have received great attention owing to their putative implications in terms of metastatic dissemination and the promise of targets for lymphangiogenic therapy. Prox-1 is a nuclear transcription factor that plays a major role during embryonic lymphangiogenesis and is deemed to be a useful marker for differentiating lymphatic endothelial cells from the other blood vessels endothelial cells. Here, we describe a double-immunostaining strategy for formalin-fixed, paraffinembedded tissues that aims at evaluating the distribution of Prox-1 and CD 31 – a cytoplasmic pan-endothelial marker -in a series of 28 mucousae, cutaneous and soft tissue vascular lesions and tumors, including hemangiomas, lymphangiomas, lymphangiectasia, and Kaposi’s sarcomas. Our results showed that in non-lesional mucousae and skin, Prox-1 decorated exclusively the nuclei of endothelial cells in lymphatic vessels. Prox-1 stained almost all the benign lymphatic vascular lesions/tumors (91%) and was absent or only focally positive in 75% of blood vascular tumors. CD 31 stained endothelial cells of blood vessels of superficial and deep dermal plexuses, lymphatics, and all blood vascular lesions/tumors. Kaposi’s sarcomas were all positive for both CD 31 and Prox-1 markers. In conclusion, although Prox-1 expression in vascular lesions/tumors was not entirely restricted to tumors with known lymphatic differentiation, CD 31/Prox-1 double-immunolabeling can be used as an adjunct marker to identify lymphatic vessels in routinely processed formalin-fixed, paraffin-embedded samples

Overexpression of optineurin E50K disrupts Rab8 interaction and leads to a progressive retinal degeneration in mice

Glaucoma is one of the leading causes of bilateral blindness affecting nearly 8 million people worldwide. Glaucoma is characterized by a progressive loss of retinal ganglion cells (RGCs) and is often associated with elevated intraocular pressure (IOP). However, patients with normal tension glaucoma (NTG), a subtype of primary open-angle glaucoma (POAG), develop the disease without IOP elevation. The molecular pathways leading to the pathology of NTG and POAG are still unclear. Here, we describe the phenotypic characteristics of transgenic mice overexpressing wild-type (Wt) or mutated optineurin (Optn). Mutations E50K, H486R and Optn with a deletion of the first (amino acids 153–174) or second (amino acids 426–461) leucine zipper were used for overexpression. After 16 months, histological abnormalities were exclusively observed in the retina of E50K mutant mice with loss of RGCs and connecting synapses in the peripheral retina leading to a thinning of the nerve fiber layer at the optic nerve head at normal IOP. E50K mice also showed massive apoptosis and degeneration of entire retina, leading to approximately a 28% reduction of the retina thickness. At the molecular level, introduction of the E50K mutation disrupts the interaction between Optn and Rab8 GTPase, a protein involved in the regulation of vesicle transport from Golgi to plasma membrane. Wt Optn and an active GTP-bound form of Rab8 complex were localized at the Golgi complex. These data suggest that alternation of the Optn sequence can initiate significant retinal degeneration in mice