Transplanted Erythropoietin-Expressing Mesenchymal Stem Cells Promote Pro-survival Gene Expression and Protect Photoreceptors From Sodium Iodate-Induced Cytotoxicity in a Retinal Degeneration Model.
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ABSTRACT: Mesenchymal stem cells (MSC) are highly regarded as a potential treatment for retinal degenerative disorders like retinitis pigmentosa and age-related macular degeneration. However, donor cell heterogeneity and inconsistent protocols for transplantation have led to varied outcomes in clinical trials. We previously showed that genetically-modifying MSCs to express erythropoietin (MSCEPO) improved its regenerative capabilities in vitro. Hence, in this study, we sought to prove its potential in vivo by transplanting MSCsEPO in a rat retinal degeneration model and analyzing its retinal transcriptome using RNA-Seq. Firstly, MSCsEPO were cultured and expanded before being intravitreally transplanted into the sodium iodate-induced model. After the procedure, electroretinography (ERG) was performed bi-weekly for 30 days. Histological analyses were performed after the ERG assessment. The retina was then harvested for RNA extraction. After mRNA-enrichment and library preparation, paired-end RNA-Seq was performed. Salmon and DESeq2 were used to process the output files. The generated dataset was then analyzed using over-representation (ORA), functional enrichment (GSEA), and pathway topology analysis tools (SPIA) to identify enrichment of key pathways in the experimental groups. The results showed that the MSCEPO-treated group had detectable ERG waves (P <0.05), which were indicative of successful phototransduction. The stem cells were also successfully detected by immunohistochemistry 30 days after intravitreal transplantation. An initial over-representation analysis revealed a snapshot of immune-related pathways in all the groups but was mainly overexpressed in the MSC group. A subsequent GSEA and SPIA analysis later revealed enrichment in a large number of biological processes including phototransduction, regeneration, and cell death (Padj <0.05). Based on these pathways, a set of pro-survival gene expressions were extracted and tabulated. This study provided an in-depth transcriptomic analysis on the MSCEPO-treated retinal degeneration model as well as a profile of pro-survival genes that can be used as candidates for further genetic enhancement studies on stem cells.
Project description:Mesenchymal stem cells (MSC) have shown promise in restoring the vision of patients in clinical trials. However, this therapeutic effect is not observed in every treated patient and is possibly due to the inefficacies of cell delivery and high cell death following transplantation. Utilizing erythropoietin can significantly enhance the regenerative properties of MSCs and hence improve retinal neuron survivability in oxidative stress. Hence, this study aimed to investigate the efficacy of conditioned medium (CM) obtained from transgenic human erythropoietin-expressing MSCs (MSC EPO ) in protecting human retinal pigment epithelial cells from sodium iodate (NaIO3)-induced cell death. Human MSC and MSC EPO were first cultured to obtain conditioned media (CM). The IC50 of NaIO3 in the ARPE-19 culture was then determined by an MTT assay. After that, the efficacy of both MSC-CM and MSC-CM EPO in ARPE-19 cell survival were compared at 24 and 48 h after NaIO3 treatment with MTT. The treatment effects on mitochondrial membrane potential was then measured by a JC-1 flow cytometric assay. The MTT results indicated a corresponding increase in cell survivability (5-58%) in the ARPE-19 cell cultures. In comparison to MSC-CM, the use of conditioned medium collected from the MSC-CM EPO further enhanced the rate of ARPE-19 survivability at 24 h (P < 0.05) and 48 h (P < 0.05) in the presence of NaIO3. Furthermore, more than 90% were found viable with the JC-1 assay after MSC-CM EPO treatment, showing a positive implication on the mitochondrial dynamics of ARPE-19. The MSC-CM EPO provided an enhanced mitigating effect against NaIO3-induced ARPE-19 cell death over that of MSC-CM alone during the early phase of the treatment, and it may act as a future therapy in treating retinal degenerative diseases.
Project description:Oxidative stress, mitochondrial impairment, and pathological amyloid beta (Aβ) deposition are involved in the pathogenesis of dry age-related macular degeneration (AMD). The natural flavonoid (-)-epicatechin (EC) is known to be an antioxidant and neuroprotective compound. Whether EC plays a therapeutic role in AMD is unknown. In this work, we aimed to assess the efficacy and molecular mechanisms of EC against sodium iodate (NaIO3)-induced retinal degeneration in C57BL/6 mice via bioinformatic, morphological, and functional methods. We demonstrated that EC had no toxic effects on the retina and could ameliorate retinal deformation and thinning. EC treatment prevented outer retinal degeneration, reduced drusen-like deposits, increased b-wave amplitude in electroretinography, blocked retinal gliosis, and increased the number and quality of mitochondria. Importantly, EC increased the protein expression of OPA1 and decreased the expression of PINK1, indicating the role of EC in mitochondrial fusion that impaired by NaIO3. Moreover, EC downregulated APP and TMEM97 levels, upregulated PGRMC1 levels, and reduced subretinal Aβ accumulation. This study illustrated that EC, which may become a promising therapeutic strategy for AMD, prevented NaIO3-induced retinal degeneration, and this improvement may be associated with the mitochondrial quality control and the TMEM97/PGRMC1/Aβ signaling pathway.
Project description:Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD and αB crystallin expression is increased in RPE and associated drusen in AMD. The purpose of this study was to investigate the role of αB crystallin in sodium iodate (NaIO3)-induced retinal degeneration, a model of AMD in which the primary site of pathology is the RPE. Dose dependent effects of intravenous NaIO3 (20-70 mg/kg) on development of retinal degeneration (fundus photography) and RPE and retinal neuronal loss (histology) were determined in wild type and αB crystallin knockout mice. Absence of αB crystallin augmented retinal degeneration in low dose (20 mg/kg) NaIO3-treated mice and increased retinal cell apoptosis which was mainly localized to the RPE layer. Generation of reactive oxygen species (ROS) was observed with NaIO3 in mouse and human RPE which increased further after αB crystallin knockout or siRNA knockdown, respectively. NaIO3 upregulated AKT phosphorylation and peroxisome proliferator-activator receptor-γ (PPARγ) which was suppressed after αB crystallin siRNA knockdown. Further, PPARγ ligand inhibited NaIO3-induced ROS generation. Our data suggest that αB crystallin plays a critical role in protection of NaIO3-induced oxidative stress and retinal degeneration in part through upregulation of AKT phosphorylation and PPARγ expression.
Project description:Age-related macular degeneration (AMD) is a global health challenge. AMD causes visual impairment and blindness, particularly in older individuals. This multifaceted disease progresses through various stages, from asymptomatic dry to advanced wet AMD, driven by various factors including inflammation and oxidative stress. Current treatments are effective mainly for wet AMD; the therapeutic options for dry AMD are limited. Photobiomodulation (PBM) using low-energy light in the red-to-near-infrared range is a promising treatment for retinal diseases. This study investigated the effects of multi-wavelength PBM (680, 780, and 830 nm) on sodium iodate-induced oxidatively damaged retinal tissue. In an in vivo rat model of AMD induced by sodium iodate, multi-wavelength PBM effectively protected the retinal layers, reduced retinal apoptosis, and prevented rod bipolar cell depletion. Furthermore, PBM inhibited photoreceptor degeneration and reduced retinal pigment epithelium toxicity. These results suggest that multi-wavelength PBM may be a useful therapeutic strategy for AMD, mitigating oxidative stress, preserving retinal integrity, and preventing apoptosis.
Project description:Given the high genetic heterogeneity of inherited retinal degenerations (IRDs), a wide applicable treatment would be desirable to halt/slow progressive photoreceptor (PR) cell loss in a mutation-independent manner. In addition to its erythropoietic activity, erythropoietin (EPO) presents neurotrophic characteristics. We have previously shown that adeno-associated viral (AAV) vector-mediated systemic EPO delivery protects from PR degeneration. However, this is associated with an undesired hematocrit increase that could contribute to PR protection. Non-erythropoietic EPO derivatives (EPO-D) are available which allow us to dissect erythropoiesis's role in PR preservation and may be more versatile and safe than EPO as anti-apoptotic agents. We delivered in animal models of light-induced or genetic retinal degeneration either intramuscularly or subretinally AAV vectors encoding EPO or one of the three selected EPO-D: the mutant S100E, the helix A- and B-derived EPO-mimetic peptides. We observed that (i) systemic expression of S100E induces a significantly lower hematocrit increase than EPO and provides similar protection from PR degeneration, and (ii) intraocular expression of EPO-D protects PR from degeneration in the absence of significant hematocrit increase. On the basis of this, we conclude that erythropoiesis is not required for EPO-mediated PR protection. However, the lower efficacy observed when EPO or S100E is expressed intraocularly rather than systemically suggests that hormone systemic effects contribute to PR protection. Unlike S100E, EPO-mimetic peptides preserve PR only when given locally, suggesting that different EPO-D have a different potency or mode of action. In conclusion, our data show that subretinal delivery of AAV vectors encoding EPO-D protects from light-induced and genetic PR degeneration.
Project description:Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO3)-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology. RNA sequencing (RNA-seq), qRT-PCR, Western blot, immunohistochemistry of the retinas and multiplex ELISA of the mouse serum were applied to find the pathways involved in the degeneration. NaIO3 caused patchy RPE loss and thinning of the photoreceptor layer. This was accompanied by the increased retinal expression of complement components c1s, c3, c4, cfb and cfh. C1s, C3, CFH and CFB were complement proteins, with enhanced deposition at day 3. C4 was upregulated in retinal degeneration at day 10. Consistently, the transcript levels of proinflammatory ccl-2, -3, -5, il-1β, il-33 and tgf-β were increased in the retinas of NaIO3 mice, but vegf-a mRNA was reduced. Macrophages, microglia and gliotic Müller cells could be a cellular source for local retinal inflammatory changes in the NaIO3 retina. Systemic complement and cytokines/chemokines remained unaltered in this model of NaIO3-dependent retinal degeneration. In conclusion, systemically administered NaIO3 promotes degenerative and inflammatory processes in the retina, which can mimic the hallmarks of geographic atrophy.
Project description:We developed and characterized a canine model of outer retinal degeneration induced by sodium iodate (SI) intravitreal injection after vitrectomy. In the preliminary study, we repeatedly injected SI intravitreally into the eyes of three canines to develop outer retinal degeneration two weeks after vitrectomy. Based on the preliminary study, a single dose of either 1.2 mg or 1.0 mg SI/0.05 mL was also injected (1.2 mg in n = 5 canines, 1.0 mg in n = 2 canines). Spectral domain-optical coherence tomography (OCT), electroretinography (ERG), and histological examinations were performed at baseline and following intravitreal injection. In the preliminary study, after a 0.5-mg SI injection and a 1.0-mg SI injection and after two 0.8-mg SI injections, retinal degeneration with retinal thinning was observed on OCT imaging. In the second study, after a single 1.0- or 1.2-mg SI injection, outer retinal degeneration was induced. All eyes showed diffuse outer retinal degeneration on OCT and a loss of both cone and rod responses in ERG. Histological examination also showed the loss of outer retinal layer. Intravitreally injected SI (1.0-1.2 mg) in a vitrectomized canine model induced outer retinal degeneration effectively, and could be evaluated through in vivo ophthalmic examination.
Project description:BackgroundSodium iodate (SI) is a chemical widely applied to induce retina degeneration in animal models. SI treatment caused formation of rosettes/folds in the outer nuclear layer (ONL) of the rat retina, but it was previously unclear whether SI also forms rosettes in mice. In addition, SI induced retina degeneration was never addressed in non-separate sclerochoroid/retina pigment epithelium/retina whole mount. Here we displayed features of retina degeneration including rosette formation in mice and developed a morphological analytic assessment using sclerochoroid/retina pigment epithelium/retina whole mounts.MethodsSI was intraperitoneally injected in Sprague-Dawley (SD) rats and C57BL/6J mice using a single dose (50 mg/kg) or with a dose range (10 to 50 mg/kg) in BALB/C mice. Rat retinas were investigated up to 2-week post-injection by histology and whole mounts, and mouse retinas were investigated up to 3-week post-injection by histology, fluorescent staining of sections and/or sclerochoroid/retina pigment epithelium/retina whole mounts for the morphological evaluations of the SI-induced retina damage.ResultsSI-induced retina damage caused photoreceptor (PR) degeneration and rosettes/folds formation, as well as retina pigment epithelium degeneration and inward migration. It displayed mixed nuclei from choroid to PRs, due to layer disorganization, as shown by single horizontal images in the sclerochoroid/retina pigment epithelium/retina whole mounts. Measurement of the PR rosette area induced by SI provided a quantitative, morphological evaluation of retina degeneration.ConclusionsThe method of non-separate sclerochoroid/retina pigment epithelium/retina whole staining and mount allows us to observe the integral horizontal view of damage from sclera to PR layers, which cannot be addressed by using sectioned and separate whole mount methods. This method is applicable for morphological evaluation of retina damage, especially in the subretinal layer.
Project description:Excessive reactive oxygen species (ROS) contribute to damage of retinal cells and the development of retinal diseases including age-related macular degeneration (AMD). ROS result in increased metabolites of lipoxygenases (LOXs), which react with ROS to induce lipid peroxidation and may lead to ferroptosis. In this study, the effect of 5-LOX inhibition on alleviating ROS-induced cell death was evaluated using sodium iodate (NaIO3) in the retinal pigment epithelium (RPE) cell line ARPE-19 and a mouse model investigating oxidative stress in AMD. We demonstrated that NaIO3 induced cell death in the RPE cells through mechanisms including ferroptosis. Inhibition of 5-LOX with specific inhibitor, Zileuton, or siRNA knockdown of ALXO5 mitigated NaIO3-induced lipid peroxidation, mitochondrial damage, DNA impairment, and cell death in ARPE-19 cells. Additionally, in the mouse model, pretreatment with Zileuton reduced the NaIO3-induced lipid peroxidation of RPE cells, cell death in the photoreceptor layer of the retina, inflammatory responses, and degeneration of both the neuroretina and RPE monolayer cells. Our results suggest that 5-LOX plays a crucial role in ROS-induced cell death in the RPE and that regulating 5-LOX activity could be a useful approach to control ROS and ferroptosis-induced damage, which promote degeneration in retinal diseases.
Project description:Green tea extracts exhibit anti-oxidative and anti-inflammatory actions in different disease conditions. We hypothesized that green tea extract and its catechin constituents ameliorate sodium iodate-induced retinal degeneration in rats by counteracting oxidative stress. In this study, adult Sprague-Dawley rats were intravenously injected with a single dose of sodium iodate. Green tea extract (GTE; Theaphenon-E) or combinations of its catechin constituents, including (-)-epigallocatechin gallate (EGCG), were administered intra-gastrically before injection. Live imaging analysis using confocal scanning laser ophthalmoscopy and spectral-domain optical coherence tomography showed a progressive increase of degenerating profile across the retinal surface and decrease in thickness of outer nuclear layer (ONL) at Day-14 of post-injection. These lesions were significantly ameliorated by Theaphenon-E and catechin combinations with EGCG. Catechins with exclusion of EGCG did not show obvious protective effect. Histological analyses confirmed that Theaphenon-E and catechins containing EGCG protect the retina by reducing ONL disruption. Retinal protective effects were associated with reduced expression of superoxide dismutase, glutathione peroxidase and caspase-3, and suppression of 8-iso-Prostaglandin F2α generation in the retina. In summary, GTE and its catechin constituents are potent anti-oxidants that offer neuroprotection to the outer retinal degeneration after sodium iodate insult, among which EGCG is the most active constituent.