Interleukin-17A modulates retinal inflammation by regulating microglial activation via the p38 MAPK pathway in experimental glaucoma neuropathy.

As a prototypical member of the IL-17 family, interleukin-17A (IL-17A) has received increasing attentions for its potent proinflammatory role as well as potential to be a key therapeutic target in human autoimmune inflammatory diseases; however, its roles in other pathological scenarios like neuroinflammations are not fully elucidated yet but appear essentially correlating and promising. Glaucoma is the leading cause of irreversible blindness with complicated pathogenesis still to be understood, where neuroinflammation was reported to be critically involved in its both initiation and progression. Whether IL-17A takes part in the pathogenesis of glaucoma through interfering neuroinflammation due to its potent proinflammatory effect is still unknown. In the present study, we investigated the role of IL-17A in the pathological process of glaucoma neuropathy as well as its relationship with the predominant immune inflammation mediator microglia in retina, trying to elucidate the underlying mechanisms from the view of inflammation modulation. In our study, RNA sequencing was performed for the retinas of chronic ocular hypertension (COH) and control mice. Western blot, RT-PCR, immunofluorescence, and ELISA were used to evaluate the microglial activation and proinflammatory cytokines release at conditioned levels of IL-17A, along with assessment of optic nerve integrity including retinal ganglion cells (RGCs) counting, axonal neurofilament quantification, and flash visual-evoked potential (F-VEP) examination. And the possibly involved signaling pathways were screened out to go through further validation in scenarios with conditioned IL-17A. Subsequently, IL-17A was found to be significantly upregulated in COH retina. Furthermore, suppression of IL-17A effectively diminished the loss of RGCs, improved axonal quality, and F-VEP performance in COH mice. Mechanistically, IL-17A promoted microglial activation and proinflammatory cytokines release along with enhanced phenotypic conversion of activated microglia to M2-type in early stage and to M1-type in late stage in glaucomatous retinas. Microglia elimination decreased the proinflammatory factors secretion, enhanced the RGCs survival and axonal quality mediated by IL-17A. Furthermore, IL-17A-induced the overactivation of microglia in glaucomatous condition was alleviated after blocking the p38 MAPK pathway. Taken together, IL-17A is involved in the regulation of retinal immune response and RGCs cell death in experimental glaucoma by essentially promoting retinal microglial activation via p38 MAPK signaling pathway. IL-17A dynamically regulates the phenotypic conversion of retinal microglia in experimental glaucoma partly depending on the duration of elevated intraocular pressure. Suppression of IL-17A contributes to alleviate glaucoma neuropathy and exhibits promising potential as an innovative target for therapeutic strategy in glaucoma.

Establishment of a minimally invasive distal traumatic optic neuropathy model in mice to investigate cascade reactions of retinal glial cells.

Traumatic optic neuropathy (TON) is a complication of craniocerebral, orbital and facial injuries, leading to irreversible vision loss. At present, there is no reliable, widely used animal model, although it has been confirmed that TON can cause the loss of retinal ganglion cells (RGC). However, the cascade reaction of retinal glial cells underlying TON is unclear. Therefore, the establishment of an animal model to explore the pathological mechanism of TON would be of great interest to the scientific community. In this study, we propose a novel mouse model utilizing a 3D stereotaxic apparatus combined with a 27G needle to evaluate damage to the optic nerve by micro-CT, anatomy, SD-OCT and F-VEP. Immunofluorescence, western blotting, qPCR experiments were conducted to investigate the loss of RGCs and activation or inactivation of microglia, astrocytes and Müller glial cells in the retina from the first week to the fourth week after modeling. The results showed that this minimally invasive method caused damage to the distal optic nerve and loss of RGC after optic nerve injury. Microglia cells were found to be activated from the first week to the third week; however, they were inactivated at the fourth week; astrocytes were activated at the second week of injury, while Müller glial cells were gradually inactivated following injury. In conclusion, this method can be used as a novel animal model of distal TON, that results in a series of cascade reactions of retinal glial cells, which will provide a basis for future studies aimed at exploring the mechanism of TON and the search for effective treatment methods.

miR-124-3p regulates the proliferation and differentiation of retinal progenitor cells through SEPT10.

Retinal degenerative diseases such as glaucoma, retinitis pigmentosa, and age-related macular degeneration pose serious threats to human visual health due to lack of effective therapeutic approaches. In recent years, the transplantation of retinal progenitor cells (RPCs) has shown increasing promise in the treatment of these diseases; however, the application of RPC transplantation is limited by both their poor proliferation and their differentiation capabilities. Previous studies have shown that microRNAs (miRNA) act as essential mediators in the fate determination of stem/progenitor cells. In this study, we hypothesized that miR-124-3p plays a regulatory role in the fate of RPC determination by targeting Septin10 (SEPT10) in vitro. We observed that the overexpression of miR124-3p downregulates SEPT10 expression in RPCs, leading to reduced RPC proliferation and increased differentiation, specifically towards both neurons and ganglion cells. Conversely, antisense knockdown of miR-124-3p was shown to boost SEPT10 expression, enhance RPC proliferation, and attenuate differentiation. Moreover, overexpression of SEPT10 rescued miR-124-3p-caused proliferation deficiency while weakening the enhancement of miR-124-3p-induced-RPC differentiation. Results from this study show that miR-124-3p regulates RPC proliferation and differentiation by targeting SEPT10. Furthermore, our findings enable a more comprehensive understanding into the mechanisms of proliferation and differentiation of RPC fate determination. Ultimately, this study may be useful for helping researchers and clinicians to develop more promising and effective approaches to optimize the use of RPCs in treating retinal degeneration diseases.

Betacellulin regulates the proliferation and differentiation of retinal progenitor cells in vitro.

Retinal progenitor cells (RPCs) hold great potential for the treatment of retinal degenerative diseases. However, their proliferation capacity and differentiation potential towards specific retinal neurons are limited, which limit their future clinical applications. Thus, it is important to improve the RPCs' ability to proliferate and differentiate. Currently, epidermal growth factor (EGF) is commonly used to stimulate RPC growth in vitro. In this study, we find that betacellulin (BTC), a member of the EGF family, plays important roles in the proliferation and differentiation of RPCs. Our results showed that BTC can significantly promote the proliferation of RPCs more efficiently than EGF. EGF stimulated RPC proliferation through the EGFR/ErbB2-Erk pathway, while BTC stimulated RPC proliferation more powerfully through the EGFR/ErbB2/ErbB4-Akt/Erk pathway. Meanwhile, under differentiated conditions, the BTC-pre-treated RPCs were preferentially differentiated into retinal neurons, including photoreceptors, one of the most important types of cells for retinal cell replacement therapy, compared to the EGF-pre-treated RPCs. In addition, knockdown of endogenous BTC expression can also obviously promote RPC differentiation into retinal neuronal cells. This data demonstrate that BTC plays important roles in promoting RPC proliferation and differentiation into retinal neurons. This study may provide new insights into the study of RPC proliferation and differentiation and make a step towards the application of RPCs in the treatment of retinal degenerative diseases.

Decellularized matrix of adipose-derived mesenchymal stromal cells enhanced retinal progenitor cell proliferation via the Akt/Erk pathway and neuronal differentiation.

BACKGROUND AIMS: Retinal progenitor cells (RPCs) are a promising cell therapy treatment for retinal degenerative diseases. However, problems with limited proliferation ability and differentiation preference toward glia rather than neurons restrict the clinical application of these RPCs. The extracellular matrix (ECM) has been recognized to provide an appropriate microenvironment to support stem cell adhesion and direct cell behaviors, such as self-renewal and differentiation. METHODS: In this study, decellularized matrix of adipose-derived mesenchymal stromal cells (DMA) was manufactured using a chemical agent method (0.5% ammonium hydroxide Triton + 20 mmol/L NH4OH) in combination with a biological agent method (DNase solution), and the resulting DMA were evaluated by scanning electron microscopy (SEM) and immunocytochemistry. The effect of DMA on RPC proliferation and differentiation was evaluated by quantitative polymerase chain reaction, Western blot and immunocytochemistry analysis. RESULTS: DMA was successfully fabricated, as demonstrated by SEM and immunocytochemistry. Compared with tissue culture plates, DMA may effectively enhance the proliferation of RPCs by activating Akt and Erk phosphorylation; when the two pathways were blocked, the promoting effect was reversed. Moreover, DMA promoted the differentiation of RPCs toward retinal neurons, especially rhodopsin- and recoverin-positive photoreceptors, which is the most interesting class of cells for retinal degeneration treatment. CONCLUSIONS: These results indicate that DMA has important roles in governing RPC proliferation and differentiation and may contribute to the application of RPCs in treating retinal degenerative diseases.

Progress of stem/progenitor cell-based therapy for retinal degeneration.

Retinal degeneration (RD), such as age-related macular degeneration (AMD) and retinitis pigmentosa, is one of the leading causes of blindness. Presently, no satisfactory therapeutic options are available for these diseases principally because the retina and retinal pigmented epithelium (RPE) do not regenerate, although wet AMD can be prevented from further progression by anti-vascular endothelial growth factor therapy. Nevertheless, stem/progenitor cell approaches exhibit enormous potential for RD treatment using strategies mainly aimed at the rescue and replacement of photoreceptors and RPE. The sources of stem/progenitor cells are classified into two broad categories in this review, which are (1) ocular-derived progenitor cells, such as retinal progenitor cells, and (2) non-ocular-derived stem cells, including embryonic stem cells, induced pluripotent stem cells, and mesenchymal stromal cells. Here, we discuss in detail the progress in the study of four predominant stem/progenitor cell types used in animal models of RD. A short overview of clinical trials involving the stem/progenitor cells is also presented. Currently, stem/progenitor cell therapies for RD still have some drawbacks such as inhibited proliferation and/or differentiation in vitro (with the exception of the RPE) and limited long-term survival and function of grafts in vivo. Despite these challenges, stem/progenitor cells represent the most promising strategy for RD treatment in the near future.

CX3CL1-CX3CR1 axis protects retinal ganglion cells by inhibiting microglia activation in a distal optic nerve trauma model.

BACKGROUND: The chemokine CX3CL1 has been reported to play an important role in optic nerve protection, but the underlying mechanism is still unclear. CX3CR1, the only receptor of CX3CL1, is specifically expressed on retinal microglia, whose activation plays a role in the pathological process of optic nerve injury. This study aimed to evaluate whether CX3CL1 exerts optic neuroprotection by affecting the activation of microglia by combining with CX3CR1. METHODS: A mouse model of distal optic nerve trauma (ONT) was used to evaluate the effects of the CX3CL1-CX3CR1 axis on the activation of microglia and survival or axonal regeneration of retinal ganglion cells (RGCs). The activation of microglia, loss of RGCs, and damage to visual function were detected weekly till 4 weeks after modeling. CX3CL1 was injected intravitreally immediately or delayed after injury and the status of microglia and RGCs were examined. RESULTS: Increases in microglia activation and optic nerve damage were accompanied by a reduced production of the CX3CL1-CX3CR1 axis after the distal ONT modeling. Both immediate and delayed intravitreal injection of CX3CL1 inhibited microglia activation, promoted survival of RGCs, and improved axonal regenerative capacity. Injection with CX3CL1 was no longer effective after 48 h post ONT. The CX3CL1-CX3CR1 axis promotes survival and axonal regeneration, as indicated by GAP43 protein and gene expression, of RGCs by inhibiting the microglial activation after ONT. CONCLUSIONS: The CX3CL1-CX3CR1 axis could promote survival and axonal regeneration of RGCs by inhibiting the microglial activation after optic nerve injury. The CX3CL1-CX3CR1 axis may become a potential target for the treatment of optic nerve injury. Forty-eight hours is the longest time window for effective treatment after injury. The study is expected to provide new ideas for the development of targeted drugs for the repair of optic nerve.

Molecular signaling from microglia impacts macroglia autophagy and neurons survival in glaucoma.

Interactions between microglia and macroglia play important roles in the neurodegeneration of the central nervous system and so is the situation between microglia and Müller cells in retina neurodegenerations like glaucoma. This study focuses on the roles of microglia-derived osteopontin (OPN) in impacting Müller cells and retinal ganglion cells (RGCs). Rat model and cell pressurization culture were used to simulate glaucoma scenarios. Animals were differently treated with anti-OPN, suppressors of OPN receptors (Itgαvß3/CD44) or microglia inhibitor minocycline, while isolated retinal Müller cells were accordingly treated with conditioned media from microglia culture pretreated with pressuring, overexpression-OPN, SiR-OPN, or minocycline. SB203580 was introduced to explore the role of p38 MAPK signaling pathway. Results revealed microglia may secret OPN to impact Müller cells' autophagy and RGCs survival via binding to Itgαvß3/CD44 receptors in glaucomatous neurodegeneration with involvement of p38 MAPK pathway. This discovery may benefit understanding neurodegenerative disorders and exploring therapeutics.

Stem/Progenitor Cells and Biodegradable Scaffolds in the Treatment of Retinal Degenerative Diseases.

BACKGROUND: Visual impairment caused by retinal degeneration is primarily attributed to the irreversible degradation of retinal neurons or the adjacent retinal pigment epithelium (RPE). No efficient clinical therapies to restore or improve visual ability are currently available. Cell therapy has been touted as a promising strategy to overcome this challenge. OBJECTIVE: This review aims to depict the effects and progresses of using stem/progenitor cells and biodegradable scaffolds in the treatment of retinal degenerative diseases, as well as discuss the challenges and opportunities of cell-based therapy for the future clinical application. RESULTS: Progenitor/stem cells may be obtained from both ocular and non-ocular tissues. The former mainly includes retinal progenitor cells (RPCs), whereas the latter comprises embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), which have been utilized in stem cell replacement therapy studies ranging from proof-of-concept animal models to clinical trials in humans. Mesenchymal stem cells (MSCs), which represent another type of stem cells, secrete anti-inflammatory cytokines and neurotrophic factors that protect and nourish retinae. Although the origins of seed cells are diverse, the cell survival rate after transplantation in vivo is limited. Therefore, cell delivery techniques that combine seed cells with polymer scaffolds are applied to improve the cell survival rate. CONCLUSION: This review summarized the different resources of stem cells and the significant progresses in the treatment of retinal degeneration combined with seed cells and scaffolds, which may pave the way for future clinical therapies.

Insulin-like growth factor-1 regulation of retinal progenitor cell proliferation and differentiation.

Strategies to improve retinal progenitor cell (RPC) capacity to yield proliferative and multipotent pools of cells that can efficiently differentiate into retinal neurons, including photoreceptors, could be vital for cell therapy in retinal degenerative diseases. In this study, we found that insulin-like growth factor-1 (IGF-1) plays a role in the regulation of proliferation and differentiation of RPCs. Our results show that IGF-1 promotes RPC proliferation via IGF-1 receptors (IGF-1Rs), stimulating increased phosphorylation in the PI3K/Akt and MAPK/Erk pathways. An inhibitor experiment revealed that IGF-1-induced RPC proliferation was inhibited when the PI3K/Akt and MAPK/Erk pathways were blocked. Furthermore, under the condition of differentiation, IGF-1-pretreated RPCs prefer to differentiate into retinal neurons, including photoreceptors, in vitro, which is crucial for visual formation and visual restoration. These results demonstrate that IGF-1 accelerates the proliferation of RPCs and IGF-1 pretreated RPCs may have shown an increased potential for retinal neuron differentiation, providing a novel strategy for regulating the proliferation and differentiation of retinal progenitors in vitro and shedding light upon the application of RPCs in retinal cell therapy.

REST, regulated by RA through miR-29a and the proteasome pathway, plays a crucial role in RPC proliferation and differentiation.

One of the primary obstacles in the application of retinal progenitor cells (RPCs) to the treatment of retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), is their limited ability to proliferate and differentiate into specific retinal neurons. In this study, we revealed that repressor element-1-silencing transcription factor (REST), whose expression could be transcriptionally and post-transcriptionally mediated by retinoic acid (RA, one isomeride of a vitamin A derivative used as a differentiation-inducing agent in many disease treatments), plays a pivotal role in the regulation of proliferation and differentiation of RPCs. Our results show that direct knockdown of endogenous REST reduced RPC proliferation but accelerated RPC differentiation toward retinal neurons, which phenocopied the observed effects of RA on RPCs. Further studies disclosed that the expression level of REST could be downregulated by RA not only through upregulating microRNA (miR)-29a, which directly interacted with the 3'-untranslated region (3'-UTR) of the REST mRNA, but also through promoting REST proteasomal degradation. These results show us a novel functional protein, REST, which regulates RPC proliferation and differentiation, can be mediated by RA. Understanding the mechanisms of REST and RA in RPC fate determination enlightens a promising future for the application of REST and RA in the treatment of retinal degeneration diseases.

Effects of RPE-conditioned medium on the differentiation of hADSCs into RPE cells, and their proliferation and migration.

Age-related macular degeneration (AMD) is associated with the dysfunction and death of the retinal pigment epithelium (RPE). Recently, there has been increasing interest in stem cell-derived RPE cells for cell replacement therapies, such as those for AMD. The present study investigated whether RPE-conditioned medium (RPECM) could promote the differentiation of human adipose tissue-derived mesenchymal stromal cells (hADSCs) into RPE cells, and enhance the proliferation and migration of these cells. Reverse-transcription quantitative polymerase chain reaction analysis demonstrated that RPECM induced hADSCs to differentiate into cells expressing RPE markers, including retinoid isomerohydrolase (RPE65), cytokeratin (CK8) and Bestrophin, which were identified to be significantly upregulated by ~10-fold, 3.5-fold and 2.4-fold, respectively, compared with the control group [hADSCs cultured in ADSC-conditioned medium (ADSCCM)]. The immunocytochemistry and western blot analysis results demonstrated that the protein levels of RPE65, CK8 and Bestrophin were significantly increased in RPECM-treated hADSCs. In addition, Cell Counting Kit-8 analysis demonstrated that RPECM promoted the proliferation of induced cells. RPECM also increased the expression level of the cell proliferative marker Ki-67. Furthermore, to evaluate the migration potential, cell migration assays were performed. These assays demonstrated that following RPECM treatment hADSCs migrated more quickly compared with the control group. The results of the present study suggest that RPECM induces hADSCs to differentiate into RPE cells with higher proliferative and migratory potentials, which may aid in applications for hADSCs in RPE regenerative therapy.

miR-29a regulates the proliferation and differentiation of retinal progenitors by targeting Rbm8a.

During development, tight regulation of the expansion of retinal progenitor cells (RPCs) and their differentiation into neuronal and glial cells is important for retinal formation and function. Our study demonstrated that microRNA (miR)-29a modulated the proliferation and differentiation of RPCs by suppressing RBM8A (one of the factors in the exon junction complex). Particularly, overexpression of miR-29a reduced RPC proliferation but accelerated RPC differentiation. By contrast, reduction of endogenous miR-29a elicited the opposite effects. Overexpression of miR-29a repressed the translation of Rbm8a, thus negatively regulating RPC proliferation and promoting the neuronal and glial differentiation of RPCs, and knockdown of endogenous Rbm8a phenocopied the observed effects of miR-29a overexpression. Furthermore, a luciferase reporter assay showed that miR-29a directly interacted with the Rbm8a mRNA 3'UTR, which indicated that Rbm8a is the direct target of miR-29a. To further verify the result, co-overexpression of the Rbm8a 3' UTR-wt (plasmids into which the Rbm8a 3' UTR sequence had been introduced) and miR-29a in RPCs rescued the phenotype associated with miR-29a overexpression, reversing the promotion of differentiation and inhibition of proliferation. These results show a novel mechanism by which miR-29a regulates the proliferation and differentiation of RPCs through Rbm8a.

Poly(1,3-propylene sebacate) and Poly(sebacoyl diglyceride): A Pair of Potential Polymers for the Proliferation and Differentiation of Retinal Progenitor Cells.

Using suitable polymers as a carrier for growing and delivering retinal progenitor cells (RPCs) is a promising therapeutic strategy in retinal cell-replacement therapy. Herein recently developed polymer, poly(sebacoyl diglyceride) (PSeD), is selected and its nonhydroxylized counterpart poly(1,3-propylene sebacate) (PPS) is designed to evaluate their potentials for RPC growth and future RPC application. The structures and mechanical properties of the polymers are characterized. The cytocompatibility and effects of these polymers on RPC proliferation, differentiation, and migration are systematically investigated in vitro. Our data show that PPS and PSeD display excellent cytocompatibility with low expression of inflammation and apoptosis factors, which benefit RPC growth. In proliferation assays reveal that RPCs expands well on the polymers, but PPS performs the best for RPC expansion, indicating that PPS can remarkably promote RPC proliferation. In differentiation conditions, RPCs grown on PSeD are more likely to differentiate toward retinal neurons, including photoreceptors, the most interesting type of cells for retinal cell-replacement therapy. Additionally, our results demonstrate that RPCs grown on PSeD display an outstanding ability to migrate. In conclusion, PPS can markedly promote RPC proliferation, whereas PSeD can enhance RPC differentiation toward retinal neurons, suggesting that PSeD and PPS have potential applications in future retinal cell-replacement therapies.

Electrospun SF/PLCL nanofibrous membrane: a potential scaffold for retinal progenitor cell proliferation and differentiation.

Biocompatible polymer scaffolds are promising as potential carriers for the delivery of retinal progenitor cells (RPCs) in cell replacement therapy for the repair of damaged or diseased retinas. The primary goal of the present study was to investigate the effects of blended electrospun nanofibrous membranes of silk fibroin (SF) and poly(L-lactic acid-co-ε-caprolactone) (PLCL), a novel scaffold, on the biological behaviour of RPCs in vitro. To assess the cell-scaffold interaction, RPCs were cultured on SF/PLCL scaffolds for indicated durations. Our data revealed that all the SF/PLCL scaffolds were thoroughly cytocompatible, and the SF:PLCL (1:1) scaffolds yielded the best RPC growth. The in vitro proliferation assays showed that RPCs proliferated more quickly on the SF:PLCL (1:1) than on the other scaffolds and the control. Quantitative polymerase chain reaction (qPCR) and immunocytochemistry analyses demonstrated that RPCs grown on the SF:PLCL (1:1) scaffolds preferentially differentiated toward retinal neurons, including, most interestingly, photoreceptors. In summary, we demonstrated that the SF:PLCL (1:1) scaffolds can not only markedly promote RPC proliferation with cytocompatibility for RPC growth but also robustly enhance RPCs' differentiation toward specific retinal neurons of interest in vitro, suggesting that SF:PLCL (1:1) scaffolds may have potential applications in retinal cell replacement therapy in the future.