Retinal Pigment Epithelium Apoptosis Research Articles

TIN2 modulates FOXO1 mitochondrial shuttling to enhance oxidative stress-induced apoptosis in retinal pigment epithelium under hyperglycemia.

Progressive dysfunction of the retinal pigment epithelium (RPE) and the adjacent photoreceptor cells in the outer retina plays a pivotal role in the pathogenesis of diabetic retinopathy (DR). Here, we observed a marked increase in oxidative stress-induced apoptosis in parallel with higher expression of telomeric protein TIN2 in RPE cells under hyperglycemia in vivo and in vitro. Delving deeper, we confirm that high glucose-induced elevation of mitochondria-localized TIN2 compromises mitochondrial activity and weakens the intrinsic antioxidant defense, thereby leading to the activation of mitochondria-dependent apoptotic pathways. Mechanistically, mitochondrial TIN2 promotes the phosphorylation of FOXO1 and its relocation to the mitochondria. Such translocation of transcription factor FOXO1 not only promotes its binding to the D-loop region of mitochondrial DNA-resulting in the inhibition of mitochondrial respiration-but also hampers its availability to nuclear target DNA, thereby undermining the intrinsic antioxidant defense. Moreover, TIN2 knockdown effectively mitigates oxidative-induced apoptosis in diabetic mouse RPE by preserving mitochondrial homeostasis, which concurrently prevents secondary photoreceptor damage. Our study proposes the potential of TIN2 as a promising molecular target for therapeutic interventions for diabetic retinopathy, which emphasizes the potential significance of telomeric proteins in the regulation of metabolism and mitochondrial function. Created with BioRender ( https://www.biorender.com/ ).

Kaempferol Alleviates Injury in Human Retinal Pigment Epithelial Cells via STAT1 Ubiquitination-Mediated Degradation of IRF7.

Retinal pigment epithelial (RPE) cells have a pivotal function in preserving the equilibrium of the retina and moderating the immunological interaction between the choroid and the retina. This study primarily focuses on delineating the protective effect offered by Kaempferol (Kae) against RPE cell damage. Bioinformatics analysis was performed on the GSE30719 dataset to identify hub genes associated with RPE. Subsequently, we analyzed the impact of Kae on RPE apoptosis, cell viability, and inflammatory response through cell experiments, and explored the interaction between hub genes and Kae. Based on the GSE30719 dataset, nine hub genes (ISG15, IFIT1, IFIT3, STAT1, OASL, RSAD2, IRF7, MX2, and MX1) were identified, all of which were highly expressed in the GSE30719 case group. Kae could boost the proliferative activity of RPE cells caused by lipopolysaccharide (LPS), as well as reduce apoptosis and the generation of inflammatory factors (tumor necrosis factor receptor (TNFR), interleukin-1beta (IL-1β)) and cytokines (IL-1, IL-6, IL-12). STAT1 was shown to inhibit cell proliferation, promote apoptosis, and secrete IL-1/IL-6/IL-12 in LPS-induced RPE cells. Moreover, IRF7 was found to interact with STAT1 in LPS-induced RPE cells, and STAT1 could maintain IRF7 levels through deubiquitination. In addition, we also found that the protective effect of Kae on LPS-induced RPE cell injury was mediated through STAT1/IRF7 axis. This study provided evidence that Kae protects RPE cells via regulating the STAT1/IRF7 signaling pathways, indicating its potential therapeutic relevance in the diagnosis and management of retinal disorders linked with RPE cell damage.

Diabetes and high-glucose could upregulate the expression of receptor for activated C kinase 1 in retina.

Diabetic retinopathy (DR) is a major ocular complication of diabetes mellitus, leading to visual impairment. Retinal pigment epithelium (RPE) injury is a key component of the outer blood retinal barrier, and its damage is an important indicator of DR. Receptor for activated C kinase 1 (RACK1) activates protein kinase C-ε (PKC-ε) to promote the generation of reactive oxygen species (ROS) in RPE cells, leading to apoptosis. Therefore, we hypothesize that the activation of RACK1 under hypoxic/high-glucose conditions may promote RPE cell apoptosis by modulating PKC-ε/ROS, thereby disrupting the barrier effect of the outer blood retinal barrier and contributing to the progression of DR. To investigate the role and associated underlying mechanisms of RACK1 in the development of early DR. In this study, Sprague-Dawley rats and adult RPE cell line-19 (ARPE-19) cells were used as in vivo and in vitro models, respectively, to explore the role of RACK1 in mediating PKC-ε in early DR. Furthermore, the impact of RACK1 on apoptosis and barrier function of RPE cells was also investigated in the former model. Streptozotocin-induced diabetic rats showed increased apoptosis and up-regulated expression of RACK1 and PKC-ε proteins in RPE cells following a prolonged modeling. Similarly, ARPE-19 cells exposed to high glucose and hypoxia displayed elevated mRNA and protein levels of RACK1 and PKC-ε, accompanied by an increases in ROS production, apoptosis rate, and monolayer permeability. However, silencing RACK1 significantly downregulated the expression of PKC-ε and ROS, reduced cell apoptosis and permeability, and protected barrier function. RACK1 plays a significant role in the development of early DR and might serve as a potential therapeutic target for DR by regulating RPE apoptosis and barrier function.

Therapeutic potential of photobiomodulation on hydrogen peroxide‐induced oxidative damage in retinal pigment epithelial cells

Aims/Purpose: Photobiomodulation (PBM) is a cutting‐edge therapeutic method for restoring the functioning of injured mitochondria in experimental settings. Our goal was to find out how 780 nm PBM therapy affected the apoptosis and cell survival of hydrogen peroxide‐treated retinal pigment epithelial cells.Methods: After being treated with 150 μM hydrogen peroxide for 24 h, the retinal pigment epithelial (RPE) cells were exposed to continuous wave laser radiation with the following parameters: wavelength of 780 nm, power density of 140 mW/cm2, 65 mW/cm2 and 40 mW/cm2 and duration of 10 min and 30 min. Furthermore, light therapy was given to untreated control cells to check PBM effect on healthy group. After PBM therapy in a time‐dependent manner, cell survival and apoptosis were assessed by MTT assay and Annexin V/PI staining, respectively.Results: Based on MTT findings, PBM showed no obvious effect on cell viability in H2O2‐treated RPE cells, while after 48 h post‐PBM incubation, cell viability was dramatically improved. It was significantly increased at the power density of 140 mW/cm2 for 30 min application without unacceptable effect on control cells. RPE apoptosis was strikingly decreased in post‐PBM incubation group compared to H2O2‐treated group in a time‐dependent manner, as detected by flow cytometry.Conclusions: As a result, photobiomodulation supported its potential utility in the prevention and treatment of retinal disorders associated with oxidative stress by promoting cell survival and reducing apoptosis under oxidative stress.

Phenotypic high-throughput screening identifies aryl hydrocarbon receptor agonism as common inhibitor of toxin-induced retinal pigment epithelium cell death.

The retinal pigment epithelium (RPE) is essential to maintain retinal function, and RPE cell death represents a key pathogenic stage in the progression of several blinding ocular diseases, including age-related macular degeneration (AMD). To identify pathways and compounds able to prevent RPE cell death, we developed a phenotypic screening pipeline utilizing a compound library and high-throughput screening compatible assays on the human RPE cell line, ARPE-19, in response to different disease relevant cytotoxic stimuli. We show that the metabolic by-product of the visual cycle all-trans-retinal (atRAL) induces RPE apoptosis, while the lipid peroxidation by-product 4-hydroxynonenal (4-HNE) promotes necrotic cell death. Using these distinct stimuli for screening, we identified agonists of the aryl hydrocarbon receptor (AhR) as a consensus target able to prevent both atRAL mediated apoptosis and 4-HNE-induced necrotic cell death. This works serves as a framework for future studies dedicated to screening for inhibitors of cell death, as well as support for the discussion of AhR agonism in RPE pathology.

Effect of Sirt3 on retinal pigment epithelial cells in high glucose through Foxo3a/ PINK1-Parkin pathway mediated mitophagy

Sirt3 is closely associated with mitophagy. This study aimed to investigate the effect and potential mechanism of Sirt3 on mitophagy in retinal pigment epithelium (RPE) in a high glucose environment. The expression levels of Sirt3, Foxo3a, PINK1, Parkin and LC3B in RPE subjected to high-glucose (HG, 30 mM D-glucose) conditions were detected by RT-PCR and western blotting. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining was used to detect the level of reactive oxygen species (ROS) in RPE treated with HG. MitoTracker and LysoTracker probes were used to label mitochondria and lysosomes, respectively, to observe the occurrence of autophagy. Sirt3-dependent regulation of mitophagy through the Foxo3a/PINK1-Parkin pathway was further investigated by virus transfection-mediated Sirt3 overexpression and PINK1 silencing. The effect of Sirt3 overexpression on apoptosis was detected by flow cytometry. The Sirt3 expression was decreased, the Foxo3a/PINK1-Parkin pathway was inhibited, intracellular ROS level was increased, and mitophagy was attenuated in RPE under HG condition. Sirt3 overexpression activated the Foxo3a/PINK1-Parkin signaling pathway and mitophagy, and inhibited cell apoptosis. Silencing PINK1 inhibited the effect of Sirt3 overexpression on mitophagy. In summary, Sirt3 can activate mitophagy through the Foxo3a/PINK1-Parkin pathway and reduce HG-induced apoptosis of RPE. This study provides a new direction to understand the pathogenesis and develop a potential therapeutic target for diabetic retinopathy.

Effects of Lycium barbarum polysaccharide on the photoinduced autophagy of retinal pigment epithelium cells.

To investigate the relationship between autophagy and apoptosis in photoinduced injuries in retinal pigment epithelium (RPE) cells and how Lycium barbarum polysaccharide (LBP) contributes to the increased of RPE cells to photoinduced autophagy. In vitro cultures of human RPE strains (ARPE-19) were prepared and randomly divided into the blank control, model, low-dose LBP, middle-dose LBP, high-dose LBP, and 3-methyladenine (3MA) groups. The viability of the RPE cells and apoptosis levels in each group were tested through cell counting kit-8 (CCK8) method with a flow cytometer (Annexin V/PI double staining technique). The expression levels of LC3II, LC3I, and P62 proteins were detected with the immunofluorescence method. The expression levels of beclin1, LC3, P62, PI3K, P-mTOR, mTOR, P-Akt, and Akt proteins were tested through Western blot. LBP considerably strengthens cell viability and inhibits the apoptosis of RPE cells after photoinduction. The PI3K/Akt/mTOR signal pathway is activated because of the upregulation of the phosphorylation levels of Akt and mTOR proteins, and thus autophagy is inhibited. LBP can inhibit the excessive autophagy in RPE cells by activating the PI3K/Akt/mTOR signaling pathways and thereby protect RPE cells from photoinduced injuries.

Repressing c-Jun N-terminal kinase signaling mitigates retinal pigment epithelium degeneration in mice with failure to clear all-trans-retinal

Retinal pigment epithelium (RPE) cell apoptosis arising from all-trans-retinal (atRAL) is in close contact with the etiology of dry age-related macular degeneration (AMD) and autosomal recessive Stargardt's disease (STGD1), but its underlying mechanisms remain elusive. In this study, we reported that c-Jun N-terminal kinase (JNK) activation facilitated atRAL-induced apoptosis of RPE cells. Reactive oxygen species production and endoplasmic reticulum stress were identified as two of major upstream events responsible for activating JNK signaling in atRAL-loaded RPE cells. Inhibiting JNK signaling rescued RPE cells from apoptosis induced by atRAL through attenuating caspase-3 activation leading to poly-ADP-ribose polymerase (PARP) cleavage, and DNA damage response. Abca4−/−Rdh8−/− mice upon light exposure exhibit rapidly increased accumulation of atRAL in the retina, and display severe RPE degeneration, a primary attribute of dry AMD and STGD1. Reducing JNK signaling by intraperitoneally injected JNK–IN-8 was highly effective in preventing RPE atrophy and apoptosis in light-exposed Abca4−/−Rdh8−/− mice. These findings afford a further understanding for contribution of JNK activation by atRAL to retinal damage.

Potential Protective Role of TRPM7 and Involvement of PKC/ERK Pathway in Blue Light-Induced Apoptosis in Retinal Pigment Epithelium Cells in Vitro.

Purpose:Blue light triggers apoptosis of retinal pigment epithelium (RPE) cells and causes retinal damage. The aim of this study was to elucidate the protective role of transient receptor potential melastatin 7 (TRPM7) in photodamaged RPE cells.Methods:RPE cells were isolated from Sprague-Dawley (SD) rats and exposed to varying intensities of blue light (500–5000 lux) in vitro. Cell proliferation and metabolic activity were respectively assessed by bromodeoxyuridine (BrdU) incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to analyze the TRPM7, protein kinase C (PKC), extracellular signal-regulated kinase (ERK) and Bcl2-associated x/B-cell lymphoma 2 (Bax/Bcl-2) messenger RNA (mRNA) and protein expression levels. The cells were transfected with TRPM7 small interfering RNA (siRNA) or transduced with TRPM7-overexpressing lentiviruses and cultured with or without the pigment epithelium-derived factor (PEDF).Results:Blue light inhibited the proliferation and metabolic activity of RPE cells in an intensity-dependent manner when compared to nonirradiated controls (P < 0.05). Compared to the control, photodamaged RPE cells showed decreased levels of TRPM7, PKC, ERK, and Bax, and an increase in Bcl-2 levels (P < 0.01). Forced expression of TRPM7 partially rescued the proliferative capacity of RPE cells (P < 0.01) and restored the levels of TRPM7, PKC, ERK, and Bax (P < 0.01), whereas TRPM7 knockdown had the opposite effects (P < 0.01). TRPM7 and PEDF synergistically alleviated the damaging effects of blue light.Conclusions:Blue light triggers apoptosis of RPE cells, and its deleterious effects can be partially attenuated by the synergistic action of TRPM7 and PEDF via the PKC/ERK signaling pathway.

Long Noncoding RNA MEG3 Inhibits Apoptosis of Retinal Pigment Epithelium Cells Induced by High Glucose via the miR-93/Nrf2 Axis

Diabetic retinopathy (DR) is the leading cause of visual impairment in developed nations. Though plasma microRNA-93 (miR-93) is associated with the risk of DR, the function and regulatory mechanism of miR-93 during DR remains unclear. Blood samples were collected from 12 DR patients and 12 healthy controls. Primary human retinal pigment epithelium (RPE) cells and ARPE-19 cells were cultured in 5 mmol/L or 33 mmol/L d-glucose medium. Long noncoding (lnc) RNA MEG3 and miR-93 expression was detected by real-time quantitative PCR. The effect of MEG3 and miR-93 on high glucose (HG)-induced apoptosis was detected by MTT and flow cytometry. IL-6 and tumor necrosis factor-α levels were detected by enzyme-linked immunosorbent assay. The relationships among MEG3, miR-93, and Nrf2 (also known as NFE2L2) were explored via dual-luciferase reporter assay. lncRNA MEG3 and Nrf2 were decreased and miR-93 was increased in blood samples of DR patients and HG-treated human RPE and ARPE-19 cells. Overexpression of miR-93 inhibited cell proliferation and promoted apoptosis, whereas overexpression of Nrf2 or MEG3 promoted proliferation and suppressed apoptosis and inflammation. In addition, MEG3 targeted miR-93 and down-regulated miR-93. Moreover, miR-93 directly targeted Nrf2and negatively regulated Nrf2. This study suggests that lncRNA MEG3 depresses HG-induced apoptosis and inflammation of RPE via miR-93/Nrf2 axis, providing a novel perspective on the genesis and development of DR.

S-allyl cysteine protects retinal pigment epithelium cells from hydroquinone-induced apoptosis through mitigating cellular response to oxidative stress.

Retinal pigment epithelium (RPE) degenerative death is an evident hallmark of advanced age-related macular degeneration (AMD). The present study aims to evaluate the protective effects of S-allyl L-cysteine (SAC), a bioactive component from aged garlic extracts, on the oxidative stress-related apoptosis of RPE cells and to investigate the potential underlying mechanisms. Cell Counting Kit-8 (CCK-8) assay, flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining were performed to evaluate the effects of SAC on the hydroquinone-treated human ARPE19 cells. The Reactive Oxygen Species (ROS) production was measured by virtue of flow cytometry or determined under an inverted fluorescence microscope. Furthermore, the expression of antioxidant factor Nrf2, as well as downstream antioxidant genes, including NQO1, SOD1, SOD2, and HO1 was assessed in hydroquinone stimulated ARPE19 cells, in the presence or absence of SAC pretreatment. Hydroquinone incitement contributed to a marked decrease in cell viability, but enhanced cell apoptosis, whereas SAC addition did not cause significant alterations. When cells were pre-treated with SAC, cell proliferation was dramatically enhanced whereas apoptosis was mitigated, and the ROS generation induced by hydroquinone was also significantly suppressed, indicating a prominent function of SAC in preventing ARPE19 cells from oxidant-related apoptosis. The elevated expression levels of Nrf2 and other antioxidant genes driven by hydroquinone were downregulated by SAC addition. These data suggest that SAC can effectively attenuate hydroquinone-induced oxidative damage in human RPE cells. Our work is the first to demonstrate that SAC modulates oxidative stress-induced RPE apoptosis, thereby potentially proving new insights into the treatment of AMD.

Glycyrrhizin protects against sodium iodate-induced RPE and retinal injury though activation of AKT and Nrf2/HO-1 pathway.

Glycyrrhizin is a bioactive triterpenoid saponin extracted from a traditional Chinese medicinal herb, glycyrrhiza, and has been reported to protect the organs such as liver and heart from injuries. However, there is no report about the effects of glycyrrhizin on atrophic age‐related macular degeneration (AMD). This study investigated the effects of glycyrrhizin on retinal pigment epithelium (RPE) in vitro and retina of mice in vivo treated with sodium iodate (SI). Glycyrrhizin significantly inhibited SI‐induced reactive oxygen species (ROS), and decreased apoptosis of RPE in vitro. The underlying mechanisms included increased phosphorylation of Akt, and increased expression of nuclear factor erythroid 2‐related factor2 (Nrf‐2) and HO‐1, thereby protecting RPE from SI‐induced ROS and apoptosis. Furthermore, glycyrrhizin significantly decreased the apoptosis of retinal cells in vivo, resulting in the inhibition of thinning of retina, decreasing the number of drusen and improving the function of retina. These findings suggested that glycyrrhizin may be a potential candidate for the treatment of atrophic AMD in clinical practice.

High-glucose induces retinal pigment epithelium mitochondrial pathways of apoptosis and inhibits mitophagy by regulating ROS/PINK1/Parkin signal pathway

Diabetic retinopathy (DR) seriously endangers human beings’ health, uncovering the underlying mechanism might help to cure DR. In this study, we found that the effects of glucose on retinal pigment epithelium (RPE) varies in a dose dependent manner, high-glucose (50mM) promotes reactive oxygen species (ROS) generation and cell apoptosis, inhibits cell mitophagy as well as proliferative abilities, while low-glucose (15mM) induces ROS production and cell mitophagy, but has little impacts on cell apoptosis and proliferation. Of note, the toxic effects of high-glucose (50mM) on RPE are alleviated by ROS scavengers and aggravated by autophagy inhibitor 3-methyladenine (3-MA) or mitophagy inhibitor cyclosporin A (CsA). High-glucose (50mM) induced ROS generation is merely eliminated by ROS scavengers instead of mitophagy or autophagy inhibitor. We also proved that high-glucose (50mM) inhibits cell proliferation and promotes cell apoptosis by regulating ROS mediated inhibition of mitophagy. In addition, mitophagy associated proteins PINK1 and Parkin are downregulated by high-glucose (50mM) or hydrogen peroxide treatments, which are reversed by ROS scavengers. Of note, Knock-down of PINK1 decreases phospharylated Parkin instead of total Parkin levels in RPE. Intriguingly, high-glucose’s inhibiting effects on cell mitophagy as well as proliferation and its promoting effects on cell apoptosis are reversed by either PINK1 or Parkin overexpression. Therefore, we concluded that high-glucose promotes RPE apoptosis and inhibits cell proliferation as well as mitophagy by regulating ROS mediated inactivation of ROS/PINK1/Parkin signal pathway.

CRB2 mutation causes autosomal recessive retinitis pigmentosa

Retinitis pigmentosa (RP), the most common form of inherited retinal dystrophies, exhibits significant genetic heterogeneity. The crumbs homolog 2 (CRB2) protein, together with CRB1 and CRB3, belongs to the Crumbs family. Given that CRB1 mutations account for 4% of RP cases, the role of CRB2 mutations in RP etiology has long been hypothesized but never confirmed. Herein, we report the identification of CRB2 as a novel RP causative gene in a Chinese consanguineous family and have analyzed its pathogenic effects. Comprehensive ophthalmic and systemic evaluations confirmed the clinical diagnosis of the two patients in this family as RP. WES revealed a homozygous missense mutation, CRB2 p.R1249G, to segregate the RP phenotype, which was highly conserved among multiple species. In vitro cellular study revealed that this mutation not only interrupted the stability of the transcribed CRB2 mRNA and the encoded CRB2 protein, but also interfered with the wild type CRB2 mRNA/protein and decreased their expression. This mutation was also shown to trigger epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells, thus impairing regular RPE phagocytosis and induce RPE degeneration and apoptosis. Thus, we conclude that CRB2 p.R1249G mutation causes RP via accelerating EMT, dysfunction and loss of RPE cells, and establish CRB2 as a novel Crumbs family member associated with non-syndromic RP. We provide important hints for understanding of CRB2 defects and retinopathy, and for the involvement of EMT of RPE cells in RP pathogenesis.

Dexmedetomidine protects high-glucose induced apoptosis in human retinal pigment epithelial cells through inhibition on p75(NTR)

BackgroundIn both human patients and animals, diabetic condition of high blood glucose induced significant apoptotic responses in retinal pigment epithelial (RPE) cells. In this work, we used an in vitro culture model of human ARPE-19 cells to evaluate whether dexmedetomidine (DEX) may protect high d-glucose (DG)-induced diabetic apoptosis. MethodsARPE-19 cells were incubated with DG in vitro to induce apoptosis. Cells were also pre-incubated with different concentrations of DEX prior to DG treatment. The apoptotic injury by DG, and possible protection by DEX were evaluated using a TUNEL assay. Western blot assay was used to evaluate DEX-associated signaling pathway proteins, including Casp-3, precursor of the nerve growth factor (proNGF) and p75 neurotrophin receptor (p75(NTR)). Moreover, p75(NTR) was overexpressed in ARPE-19 cells, to assess its mechanistic role in DEX-mediated protection on DG-induced apoptosis. ResultsIn ARPE-19 culture, DG induced significant apoptosis, which was protected by pre-incubation of DEX, in a concentration-dependent manner. DG-induce apoptosis was associated with protein upregulation of Casp-3, proNGF and p75(NTR). Among them, Casp-3 and p75(NTR) were inversely reduced by DEX pre-incubation, but not proNGF. In ARPE-19 cells, p75(NTR) overexpression was shown to reverse the protective effect of DEX on DG-induced apoptosis. ConclusionDEX was proven to have protective effect on DG-induced RPE apoptosis, possible through inhibition on p75(NTR) and its associated signaling pathways.

Intra-vitreal αB crystallin fused to elastin-like polypeptide provides neuroprotection in a mouse model of age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of severe and irreversible central vision loss, and the primary site of AMD pathology is the retinal pigment epithelium (RPE). Geographic atrophy (GA) is an advanced form of AMD characterized by extensive RPE cell loss, subsequent degeneration of photoreceptors, and thinning of retina. This report describes the protective potential of a peptide derived from the αB crystallin protein using a sodium iodate (NaIO3) induced mouse model of GA. Systemic NaIO3 challenge causes degeneration of the RPE and neighboring photoreceptors, which have similarities to retinas of GA patients. αB crystallin is an abundant ocular protein that maintains ocular clarity and retinal homeostasis, and a small peptide from this protein (mini cry) displays neuroprotective properties. To retain this peptide for longer in the vitreous, mini cry was fused to an elastin-like polypeptide (ELP). A single intra-vitreal treatment by this crySI fusion significantly inhibits retinal degeneration in comparison to free mini cry. While mini cry is cleared from the eye with a mean residence time of 0.4 days, crySI is retained with a mean residence time of 3.0 days; furthermore, fundus photography reveals evidence of retention at two weeks. Unlike the free mini cry, crySI protects the RPE against NaIO3 challenge for at least two weeks after administration. CrySI also inhibits RPE apoptosis and caspase-3 activation and protects the retina from cell death up to 1-month post NaIO3 challenge. These results show that intra-ocular ELP-linked peptides such as crySI hold promise as protective agents to prevent RPE atrophy and progressive retinal degeneration in AMD.

The effect of polypyrimidine tract binding protein-associated splicing factor on hydrogen peroxide induced apoptosis of retinal pigment epithelial

Objective To observe the effect of polypyramidine tract binding protein-associated splicing factor (PSF) on hydrogen peroxide (H2O2) induced apoptosis of retinal pigment epithelial (RPE) cells in vitro. Methods RPE cells were cultured and divided into a normal group, normal+H2O2 group, Vec+H2O2group, PSF+H2O2 group according to the experimental design. Overexpression of PSF in RPE cells were achieved by pEGFP-PSF plasmid transient transfection into RPE cells, then RPE cells were exposed to H2O2. The morphological changes were observed by hematoxylin-eosin (HE) staining and Live/Dead staining while the survival rate of cells was detected by MTT assay. The effect of PSF on H2O2-induced RPE apoptosis was analyzed by Cell Death Detection ELISA kit. Meanwhile, intracellular reactive oxygen species (ROS) level was detected by using DCFH-DA method. Results Overexpression of PSF could effectively alleviate the morphological changes induced by H2O2 stimulation shown by HE staining, and effectively reduce dead cells number shown by Live/Dead staining. After H2O2 stimulation, the survival rate, apoptosis rate and ROS production level in PSF overexpression group were 0.68±0.12, 0.44±0.08 and 18 616±3 382.54 respectively, showing significant difference in comparison with the vector plasmid group and normal group (P<0.05). Conclusion PSF overexpression plays a protective role in H2O2-induced apoptosis by inhibiting the production of ROS in RPE cells. Key words: Retinal pigment epithelium; Polypyrimidine tract-binding protein; Apoptosis; Hydrogen peroxide

Over-expression of CNTF in bone marrow mesenchymal stem cells protects RPE cells from short-wavelength, blue-light injury.

Increasing evidence has demonstrated that excessive blue-light (BL) with high photochemical energy and phototoxicity could induce apoptosis in retinal pigment epithelium (RPE) cells. RPE apoptosis leads to retina damage and further aggravate age-related macular degeneration (ARMD). Because of their neuroprotective, plasticity, and immunomodulatory ability, bone marrow mesenchymal stem cells (BMSCs) are recognized for retinal neuroprotection. RPE cells possess ciliary neurotrophic factor (CNTF) receptor complexes and can respond to CNTF; hence, we investigated the effects of BMSCs over-expressing CNTF on BL-injured RPE cells. BL-injured RPE cells were co-cultured with CNTF-BMSCs and GFP-BMSCs for 24 and 48h. Superoxide dismutase and malondialdehyde assays were conducted to examine the effects of CNTF-BMSCs on the oxidative stress of RPE cells. VEGF protein secretion by RPE was determined by ELISA, and western blotting analysis was used to determine apoptotic protein expression and autophagic flux. Immunofluorescence was used to demonstrate the relationship between autophagy and apoptosis. We found that CNTF-BMSCs enhanced antioxidant capacity, decreased VEGF secretion, promoted autophagic flux, and inhibited apoptosis in BL-injured RPE cells, compared to GFP-BMSCs. Our findings suggest that CNTF over-expression enhances the protective effects of BMSCs on RPE cells, thus indicating subretinal-transplantation of CNTF-BMSCs may be a promising therapy for BL-injured retina.

MiR-25 Mediates Retinal Degeneration Via Inhibiting ITGAV and PEDF in Rat.

Age-related macular degeneration (AMD) is the main cause of irreversible blindness in the elderly. Oxidative stress in retinal pigment epithelium (RPE) is deemed to play a pivotal role in the pathogenesis of AMD. miR-25 functions as an essential modulator in response to oxidative-stress in several cell types, but its function in RPE cells is poorly understood. To explore the roles of miR-25 in RPE cells and in the development of AMD. A rat model of retinal degeneration was induced by sodium iodate (SI). Subretinal injection of antagomiR-25 was performed for the intervention while the scramble as control. Visual responses were recorded with Electroretinogram (ERG). TUNEL assay was performed to detect apoptosis. Phagosome quantification in vivo was performed to evaluate RPE cell function. Oxygen-glucose deprivation treatment was performed to mimic in vitro oxidative stress. Gene expression at mRNA level and protein level were performed by quantitative polymerase chain reaction (qPCR) and Western Blot, respectively. The pigment epithelium derived factor (PEDF) level in the cultured medium was measured by Enzyme-linked immunosorbent assay (ELISA). The interaction between miR-25 and integrin αV (IGTAV) / PEDF 3'UTR was examined by dual luciferase assay. Chromatin immunoprecipitation (ChIP) assay was performed to examine its transcriptional regulation of miR-25. Oxidative stress up-regulated miR-25 in RPE cells in very early stage, accompanied by decreased phagocytosis and reduced growth factor secretion in those cells. Such changes preceded RPE cell apoptosis and visual impairment in the SItreated rats. Furthermore, antagomiR-25 intervention effectively rescued RPE cells from degeneration in such model. The increased miR-25 was confirmed to mediate RPE degeneration through direct targeting IGTAV and PEDF. On the other hand, upstream, miR-25 was found to be up-regulated by STAT3 signaling under oxidative stress in both in vivo and in vitro models. Our findings demonstrate that, in SI-treated rats, oxidative stress activates STAT3 signaling which up-regulates miR-25 expression, in a very early stage. The increased miR-25 then inhibits ITGAV and PEDF expressions, resulting in RPE phagocytosis dysfunction and then RPE apoptosis and visual impairment as observed in patients with AMD. These findings lead us to a better understanding of AMD pathogenesis, and suggest that miR-25 could be a potential therapeutic target for oxidative stress related RPE diseases, like AMD.

Mediated effects of endoplasmic reticulum stress on light-induced apoptosis and inflammation of human retinal pigment epithelial cell

Background The light damage model of retinal pigment epithelium (RPE) cells is a research direction of retinal degeneration diseases, and RPE cell apoptosis induced by light damage and inflammation is an important pathologic basis of light-induced RPE cell damage.However, whether endoplasmic reticulum stress (ERS) paticipates in light-induced RPE cell damage is rarely reported. Objective This study was to explore the effects of ERS on light-induced RPE cell damage. Methods Human RPE cell line (ARPE-19) was cultured, and light damage models were created by irradiating the cells for 3-, 6-, 12- and 24-hours with white fluorescent lamp with the intensity of (2 000±500)lx for the selection of optimal irradiating time, and the cells in the normal control group were cultured in the dark environment.The cells were divided into normal control group, light exposure group and 4-phenylbutyric acid (4-PBA) pretreated+ light exposure group.The cells from 4-PBA pretreated+ light exposure group were cultued firstly with 4-PBA for 30 minutes and followed by light exposure for 12 hours.The apoptisis rate of the cells and intracellular reactive oxygen species (ROS) content were detected by flow cytometry; the concentrations of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell supernatant were assyed by ELISA.The relative expressing levels of activating transcription factor 6 (ATF-6), C/enhancer binding protein homologous protein (CHOP) and caspase-12 mRNA and protein in the cells were detected by real-time quantitative PCR and Western blot, respectively. Results The cultured cells showed a long spindle shape, the border was not clear, the cytoplasm was degranulation, and the cell fragments increased.Flow cytometry showed that compared with the normal control group, the ROS content in the cells and the apoptosis rate were evidently increased with the lapse of light exposure time (F=763.00, 119.30, both at P<0.01). ELISA results showed that the concentrations of IL-1β and TNF-α in the cell supernatant were significantly higher in the light exposure 6-hour group than those in the normal control group with the peak value in the light exposure 12-hour group.Compared with the normal control group, the relative expression levels of ATF-6, CHOP and caspase-12 mRNA and protein in the cells were elevated in the light exposure group and peaked in the light exposure 12-hour group.In addition, the relative expression levels of ATF-6 mRNA, CHOP mRNA and caspase-12 mRNA in the cells were significantly reduced in 4-PBA pretreated+ light exposure group compared with the light exposure group (F=281.69, 473.88, 308.45, all at P<0.01), and their proteins were also significantly reduced (F=47.86, 57.93, 106.59, all at P<0.01). The apoptosis rate, concentrations of IL-1β and TNF-α in the cell supernatant were significantly reduced in 4-PBA pretreated+ light exposure group compared with the light exposure group (F=88.64, 245.47, 101.01, all at P<0.01). Conclusions The light exposure at (2 000±500)lx induces intracellular ROS accumulation and activates the ERS response, which results in apoptosis and inflammatory process of human RPE cells.4-PBA, a inhibitor of ERS, can suppress light-induced ERS response and therefore reduces the apoptosis rate and inhibits inflammatory process. Key words: Oxidative stress; Light/adverse effects; Endoplasmic reticulum/pathology; Pigment epithelium of eye/pathology; Cell line; Human; Apotosis; Inflammation