Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases： present and future

As a constituent of blood-retinal barrier and retinal outer segment（ROS） scavenger, retinal pigmented epithelium（RPE） is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE（h RPESC-RPE） has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE（h ESC-RPE） can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE（i PSC-RPE） is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received i PSCRPE transplant, which is a hallmark of i PSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE（SC-RPE） especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.

AB044.Phototoxic stress induced in retinal pigmented epithelium cells by the synergy between polycyclic aromatic hydrocarbons and blue light

Background:Lesion to the retinal pigment epithelium(RPE)is a crucial event in age-related macular degeneration(AMD)development.Although the pathogenesis of this complex disease is poorly understood,sunlight exposure and smoking are major environmental risk factors associated with AMD.High-energy visible blue light(HEV;400-500 nm)is the most energetic and potentially harmful solar wavelengths reaching adults retina.On the other hand,RPE cells can be exposed to a large range of pollutants from cigarette smoke,with polycyclic aromatic hydrocarbons(PAH)being among the most toxic.Some PAH from cigarette smoke can absorb HEV light.This led us hypothesize that in RPE cells,the combination of PAH and HEV could synergize to exacerbate the stress caused by either factor alone.We thus investigate the combined effect of PAH and HEV light in RPE cells.Methods:Confluent RPE immortalized cells(ARPE19)were exposed to nanomolar concentrations of benzo[a]pyrene(BaP)or indeno[1,2,3-cd]pyrene(IcdP).While IcdP efficiently absorbs HEV wavelengths,BaP,the most studied PAH,does not significantly absorb HEV light and was used as a control.BaP or IcdP contaminated ARPE19 were then irradiated with increasing sub-lethal doses of HEV light(150-500 J/cm2)using a setup that mimics the light spectrum normally reaching the retina.Cytotoxicity,apoptosis and reactive oxygen species(ROS)generation were assessed in each condition.Results:In presence of low concentrations of IcdP,sub-lethal amounts of HEV light trigger,in a dose-dependent way,up to 70%of apoptotic cell death.Co-exposure to IcdP and HEV also leads to a synergistic ROS generation in ARPE19 cells,thus inducing oxidative stress.None of these effects were observed with BaP.Efficient inhibition of ROS production by specific antioxidants only decreases death by 20%in cells simultaneously exposed to both IcdP and HEV light.Conclusions:Low concentrations of IcdP synergize with HEV light to induce phototoxicity in ARPE19 cells.An increased oxidative stress results from the interaction between both agents and partially explains the enhanced HEV phototoxicity in IcdP contaminated ARPE19 cells.This suggests that another major mechanism is involved in the synergetic toxicity.For smokers,this synergy between HEV and PAH may accelerate RPE cells loss and contribute to their greater risk of developing AMD.

Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway

AIM:To explore the effect of epidermal growth factor receptor(EGFR)inhibition by erlotinib and EGFR siRNA on epidermal growth factor(EGF)-induced activation of retinal pigment epithelium(RPE)cells.METHODS:Human RPE cell line(ARPE-19 cells)was activated by 100 ng/mL EGF.Erlotinib and EGFR siRNA were used to intervene EGF treatment.Cellular viability,proliferation,and migration were detected by methyl thiazolyl tetrazolium(MTT)assay,bromodeoxyuridine(BrdU)staining assay and wound healing assay,respectively.EGFR/protein kinase B(AKT)pathway proteins and N-cadherin,α-smooth muscle actin(α-SMA),and vimentin were tested by Western blot assay.EGFR was also determined by immunofluorescence staining.RESULTS:EGF treatment for 24h induced a significant increase of ARPE-19 cells’viability,proliferation and migration,phosphorylation of EGFR/AKT proteins,and decreased total EGFR expression.Erlotinib suppressed ARPE-19 cells’viability,proliferation and migration through down regulating total EGFR and AKT protein expressions.Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin,α-SMA,and vimentin proteins.Similarly,EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation,viability,and migration,phosphorylation of EGFR/AKT proteins,and up-regulation of N-cadherin,α-SMA,and vimentin proteins.CONCLUSION:Erlotinib and EGFR-knockdown suppress EGF-induced cell viability,proliferation,and migration via EGFR/AKT pathway in RPE cells.EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy(PVR).

RNA-sequencing expression profile and functional analysis of retinal pigment epithelium in atrophic age-related macular degeneration

The retinal pigment epithelium(RPE)is fundamental to sustaining retinal homeostasis.RPE abnormality leads to visual defects and blindness,including age-related macular degeneration(AMD).Although breakthroughs have been made in the treatment of neovascular AMD,effective intervention for atrophic AMD is largely absent.The adequate knowledge of RPE pathology is hindered by a lack of the patients'RPE datasets,especially at the single-cell resolution.In the current study,we delved into a large-scale single-cell resource of AMD donors,in which RPE cells were occupied in a substantial proportion.Bulk RNA-seq datasets of atrophic AMD were integrated to extract molecular characteristics of RPE in the pathogenesis of atrophic AMD.Both in vivo and in vitro models revealed that carboxypeptidase X,M14 family member 2(CPXM2),was specifically expressed in the RPE cells of atrophic AMD,which might be induced by oxidative stress and involved in the epithelial-mesenchymal transition of RPE cells.Additionally,silencing of CPXM2 inhibited the mesenchymal phenotype of RPE cells in an oxidative stress cell model.Thus,our results demonstrated that CPXM2 played a crucial role in regulating atrophic AMD and might serve as a potential therapeutic target for atrophic AMD.

Effect of acetyl L-carnitine on human retinal pigment epithelium-19 cells in hypoxic conditions

AIM:To investigate the effect of acetyl-L-carnitine(ALCAR)on cell viability,morphological integrity,and vascular endothelial growth factor(VEGF)expression in human retinal pigment epithelium(ARPE-19)cells using a hypoxic model.METHODS:In the first set of experiments,the optimal CoCl_(2) dose was determined by exposing ARPE-19 cell cultures to different concentrations.To evaluate the effect of ALCAR on cell viability,five groups of ARPE-19 cell culture were established that included a control group,a sham group(200μM CoCl_(2)),and groups that received 1,10 and 100 mM doses of ALCAR combined with 200μM CoCl_(2),respectively.The cell viability was measured by MTT assay.The morphological characteristics of cells were observed by an inverted phase contrast microscope.The levels of VEGF and HIF-1α secretion by ARPE-19 cells were detected by enzyme linked immunosorbent assay(ELISA)assay.RESULTS:ARPE-19 cells were exposed to different doses of CoCl_(2) in order to create a hypoxia model.Nevertheless,when exposed to a concentration of 200μM CoCl_(2),a notable decrease in viability to 83% was noted.ALCAR was found to increase the cell viability at 1 mM and 10 mM concentrations,while the highest concentration(100 mM)did not have an added effect.The cell viability was found to be significantly higher in the groups treated with a concentration of 1 mM and 10 mM ALCAR compared to the Sham group(P=0.041,P=0.019,respectively).The cell viability and morphology remained unaffected by the greatest dose of ALCAR(100 mM).The administration of 10 mM ALCAR demonstrated a statistically significant reduction in the levels of VEGF and HIF-1α compared with the Sham group(P=0.013,P=0.033,respectively).CONCLUSION:The findings from the current study indicate that ALCAR could represent a viable therapeutic option with the potential to open up novel treatment pathways for retinal diseases,particular relevance for age-related macular degeneration(AMD).However,to fully elucidate ALCAR’s application potential in retinal diseases,additional investigation is necessary to clearly define the exact mechanisms involved.

Oxidative stress in retinal pigment epithelium degeneration:from pathogenesis to therapeutic targets in dry age-related macular degeneration

Age-related macular degeneration is a primary cause of blindness in the older adult population. Past decades of research in the pathophysiology of the disease have resulted in breakthroughs in the form of anti-vascular endothelial growth factor therapies against neovascular age-related macular degeneration;however, effective treatment is not yet available for geographical atrophy in dry agerelated macular degeneration or for preventing the progression from early or mid to the late stage of age-related macular degeneration. Both clinical and experimental investigations involving human agerelated macular degeneration retinas and animal models point towards the atrophic alterations in retinal pigment epithelium as a key feature in age-related macular degeneration progression. Retinal pigment epithelium cells are primarily responsible for cellular-structural maintenance and nutrition supply to keep photoreceptors healthy and functional. The retinal pigment epithelium constantly endures a highly oxidative environment that is balanced with a cascade of antioxidant enzyme systems regulated by nuclear factor erythroid-2-related factor 2 as a main redox sensing transcription factor. Aging and accumulated oxidative stress triggers retinal pigment epithelium dysfunction and eventually death. Exposure to both environmental and genetic factors aggravates oxidative stress damage in aging retinal pigment epithelium and accelerates retinal pigment epithelium degeneration in age-related macular degeneration pathophysiology. The present review summarizes the role of oxidative stress in retinal pigment epithelium degeneration, with potential impacts from both genetic and environmental factors in age-related macular degeneration development and progression. Potential strategies to counter retinal pigment epithelium damage and protect the retinal pigment epithelium through enhancing its antioxidant capacity are also discussed, focusing on existing antioxidant nutritional supplementation, and exploring nuclear factor erythroid-2-related factor 2 and its regulators including REV-ERBα as therapeutic targets to protect against age-related macular degeneration development and progression.

Combined hamartoma of the retina and retinal pigment epithelium:A case report

BACKGROUND Combined hamartoma of the retina and retinal pigment epithelium(CHRRPE)is a rare congenital benign tumor which is commonly monocular.Typical CHRRPE comprises slightly raised lesions at the posterior pole,with proliferation membrane often leading to vascular distortion.In severe cases,macular edema,macular hole,retinal detachment or vitreous hemorrhage may occur.Patients with atypical clinical manifestations are prone to misdiagnosis by inexperienced ophthalmologists.CASE SUMMARY A 33-year-old man reported onset of right eye blurred vision for one week prior.Anterior segment and intraocular pressure were normal in both eyes.Left eye fundus photography was normal.Right eye ophthalmoscopy showed vitreous hemorrhage and off-white raised retinal lesions below the optic disc.Proliferative membranes on the lesion surfaces resulted in superficial retinal detachment and tortuosity and occlusion of peripheral blood vessels.A horseshoe-like tear in the temporal periphery was surrounded by retinal detachment.Optical coherence tomography revealed retinal thickening at the focal site with structural disturbance indicated by high reflectance.Right eye ultrasound showed retinal thickening at the lesion,stretching and uplifting of the proliferative membrane,with moderately patchy echo at the optic disc edge.Cytokines and antibodies were detected in vitreous fluids during the operation to rule out other diseases.Fundus fluorescein angiography(FFA)at postoperative follow-up led to final diagnosis of CHRRPE.CONCLUSION FFA is helpful in diagnosing retinal and retinal pigment epithelial combined hamartoma.In addition,other cytokine and etiological tests facilitate further differential diagnosis to rule out other suspected diseases.

Gene Therapy Activates Retinal Pigment Epithelium Cell Proliferation for Age-related Macular Degeneration in a Mouse Model

Objective Age-related macular degeneration(AMD)is a degenerative retinal disease.The degeneration or death of retinal pigment epithelium(RPE)cells is implicated in the pathogenesis of AMD.This study aimed to activate the proliferation of RPE cells in vivo by using an adeno-associated virus(AAV)vector encodingβ-catenin to treat AMD in a mouse model.Methods Mice were intravitreally injected with AAV2/8-Y733F-VMD2-β-catenin for 2 or 4 weeks,andβ-catenin expression was measured using immunofluorescence staining,real-time quantitative reverse transcription polymerase chain reaction(PCR),and Western blotting.The function ofβ-catenin was determined using retinal flat mounts and laser-induced damage models.Finally,the safety of AAV2/8-Y733F-VMD2-β-catenin was evaluated by multiple intravitreal injections.Results AAV2/8-Y733F-VMD2-β-catenin induced the expression ofβ-catenin in RPE cells.It activated the proliferation of RPE cells and increased cyclin D1 expression.It was beneficial to the recovery of laser-induced damage by activating the proliferation of RPE cells.Furthermore,it could induce apoptosis of RPE cells by increasing the expression of Trp53,Bax and caspase3 while decreasing the expression of Bcl-2.Conclusion AAV2/8-Y733F-VMD2-β-catenin increasedβ-catenin expression in RPE cells,activated RPE cell proliferation,and helped mice heal from laser-induced eye injury.Furthermore,it could induce the apoptosis of RPE cells.Therefore,it may be a safe approach for AMD treatment.

Therapeutic effect of microencapsulated porcine retinal pigmented epithelial cells transplantation on rat model of Parkinson's disease

Object To investigate the therapeutic effect of microencapsulated porcine retinal pigmented epithelial cells （RPE-M） transplantation on rat model of Parkinson＇s disease （PD）. Methods Primary porcine RPE cells were harvested by enzyme digestion and expanded in culture medium. Determine the levels ofdopamine （DA） and homovanillic acid （HVA） by high performance liquid chromatography electrochemical （HPLC） assay, and the levels of brain-derived neurotrophic factor （BDNF） and glial-derived neurotrophic factor （GDNF） were detected by ELISA. Alginate-polylysine-alginate （APA） microencapsulated cells were produced by using a high voltage electrostatic system. PD rat model was established by unilateral injection of 6-hydroxydopamine （6-OHDA） into the medial forebrain bundle （MFB）. After that, the RPE-M was transplanted into the corpus striatum of PD rat, and then the rotation test scores were recorded and biochemical changes of the corpus striatum were tested. Results The levels of DA, HVA, BDNF and GDNF secreted by RPE were stable in the RPE culture supernatant and were not changed by the microencapsulation. Eighty-three percent rats developed PD by unilateral lesion of 6-OHDA in the MFB. The RPE-M transplantation had therapeutic effect on 33% PD rats. Conclusion Porcine RPE cells grow actively in vitro and could secrete DA, HVA, BDNF, and GDNF constantly, which does not be affected by the passage culture and the APA miroencapsulation. RPE-M transplantation of may be a curative therapy for PD.

Chordin-like 2 influences the differentiation fate of retinal pigment epithelium cells by dynamically regulating BMP pathway

AIM:To explore the functions of Chordin-like 2,which is encoded by CHRDL2,in the process of retinal pigmented epithelium(RPE)differentiation and damage repair.METHODS:The fetal RPE cells(f RPE)was obtained from aborted fetus which obeyed medical ethics.Real-time quantitative polymerase chain reaction was used to measure expression quantity of CHRDL2 and other functional genes expression.Knocking down and overexpression was used to analyze the functions about Chordin-like 2.Enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of bone morphogenetic proteins 4(BMP4).Flow cytometry was used to analyze cell cycle.Cell morphology was observed by phase contrast microscope(PCM).RESULTS:In normal RPE cells,CHRDL2 was firstly upregulated and followed a downregulation but eventually,it was expressed higher than the cells which undergone epithelial-mesenchymal transition(EMT).After knocking down CHRDL2,the secretion of BMP4 was decreased,RPErelated genes(OTX2,MITF,RPE65)were downregulated while EMT-related genes(SNAI1,VIM)were upregulated.However,the expression of these related genes after overexpression of CHRDL2 had contrary results.Chordin-like 2 also regulated the cell cycle by regulating BMP pathway.When CHRDL2 was knocked down,more f RPE cells stayed in S phase of cell cycle,while adding BMP4 reduced the proportion of the cells in S phase.However,overexpression of CHRDL2 increased more BMP4 secretion,this effect decreased the number of cells in S phase,but exogenous BMP inhibitor also could change this effect.At last,in the process of RPE cells differentiation,adding BMP4 at early stage could intervene normal RPE differentiation.Compared with BMP4,inhibiting BMP pathway had no significant negative effect at early stage,but suppressed differentiation at late stage.CONCLUSION:BMP pathway can be activated in a correct temporal order,otherwise,the cells have incorrect differentiation orientation.And Chordin-like 2 plays a role in dynamic regulation of BMP pathway and it also regulates the differentiation of RPE cells.Therefore,this research enlightens a new direction to inhibit EMT and promote cell redifferentiation after injury.

Biomaterial engineering strategies for modeling the Bruch's membrane in age-related macular degeneration

Age-related macular degeneration,a multifactorial inflammatory degenerative retinal disease,ranks as the leading cause of blindness in the elderly.Strikingly,there is a scarcity of curative therapies,especially for the atrophic advanced form of age-related macular degeneration,likely due to the lack of models able to fully recapitulate the native structure of the outer blood retinal barrier,the prime to rget tissue of age-related macular degeneration.Standard in vitro systems rely on 2D monocultures unable to adequately reproduce the structure and function of the outer blood retinal barrier,integrated by the dynamic interaction of the retinal pigment epithelium,the Bruch's membrane,and the underlying choriocapillaris.The Bruch's membrane provides structu ral and mechanical support and regulates the molecular trafficking in the outer blood retinal barrier,and therefo re adequate Bruch's membrane-mimics are key for the development of physiologically relevant models of the outer blood retinal barrie r.In the last years,advances in the field of biomaterial engineering have provided novel approaches to mimic the Bruch's membrane from a variety of materials.This review provides a discussion of the integrated properties and function of outer blood retinal barrier components in healt hy and age-related macular degeneration status to understand the requirements to adequately fabricate Bruch's membrane biomimetic systems.Then,we discuss novel materials and techniques to fabricate Bruch's membrane-like scaffolds for age-related macular degeneration in vitro modeling,discussing their advantages and challenges with a special focus on the potential of Bruch's membrane-like mimics based on decellularized tissue.

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

BACKGROUND 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.AIM To investigate the role and associated underlying mechanisms of RACK1 in the development of early DR.METHODS 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-εin early DR.Furthermore,the impact of RACK1 on apoptosis and barrier function of RPE cells was also investigated in the former model.RESULTS Streptozotocin-induced diabetic rats showed increased apoptosis and upregulated expression of RACK1 and PKC-εproteins 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-εand ROS,reduced cell apoptosis and permeability,and protected barrier function.CONCLUSION 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.

Potential therapeutic effects of pigment epithelium-derived factor for treatment of diabetic retinopathy

Diabetic retinopathy (DR), a major micro-vascular complication of diabetes, has emerged as a leading cause of visual impairment and blindness among working adults in the worldwide. The pathobiology of DR involves multiple molecular pathways and is characterized chronic neurovascular degeneration. Current approaches to prevent or to treat DR are still far from satisfactory. Therefore, it is important to develop new therapeutic strategies for the prevention and treatment to DR. Pigment epithelium-derived factor (PEDF), a 50-kDa secreted glycoprotein, has been described as a multi-functional protein. Some emerging evidences indicate that PEDF are able to target multiple pathways exerting neurotropic, neuroprotective, anti-angiogenic, antivasopermeability, anti-inflammation, anti-thrombogenic and anti-oxidative effects in DR. In this review, we addressed the functions of PEDF in different pathways, which could lead to potential therapeutics on the treatment to DR.

Long-term outcomes of drusenoid pigment epithelium detachment in intermediate AMD treated with 577 nm subthreshold micropulse laser: a preliminary clinical study

AIM: To evaluate the long-term anatomical and visual outcomes of drusenoid pigment epithelial detachment(D-PED) in intermediate age-related macular degeneration(AMD) eyes treated with 577 nm yellow subthreshold micropulse laser(SML).METHODS: In this retrospective study, 21 eyes of 16 patients with D-PED in intermediate AMD were consecutively included and assessed.All the eyes were treated with 577 nm SML in several sessions according to D-PED growth status.The logarithm of the minimum angle of resolution(logMAR) best-corrected visual acuity(BCVA) were assessed at the initial visit and after treatment.Spectral-domain optical coherence tomography(SD-OCT) was performed to evaluate the D-PED lifecycle by volumetric calculations.Regression analysis was used to determine the breakpoint, growth, and collapse rate of the D-PED lesions.The progression to advanced AMD was also documented.RESULTS: All the eyes were treated with SML for 2.9±1.0 sessions.The mean follow-up period was 25.3±12.6 mo.The BCVA was stable from the baseline to final visit.All the eyes were categorized into two groups according to the anatomical changes of the D-PED lesion: the collapse group(n=6, 28.6%) and non-collapse group(n=15, 71.4%).The change in logMAR BCVA did not differ significantly between the collapse group 0.00(-0.31, 0.85) and non-collapse group 0.00(0.00, 0.00;P=1).Regression analysis showed that the growth rate was significantly higher in the collapse group(0.090±0.095 mm;/mo) than in the non-collapse group(0.025±0.035 mm;/mo;P<0.001).One eye(4.8%) developed macular neovascularization at 11 mo after SML treatment in the non-collapse group.Three eyes(14.3%) developed geographic atrophy(GA) in the collapse group.CONCLUSION: Compared to the natural course of D-PED reported by previous studies, our results preliminarily show that SML can alleviate visual loss and possibility of progression to advanced AMD in eyes with D-PED in intermediate AMD.A controlled clinical trial needs to further verify the benefit of the intervention.

Melanin change of retinal pigment epithelium and choroid in the convalescent stage of Vogt-Koyanagi-Harada disease

AIM:To observe the melanin change of the retinal pigment epithelium(RPE)and choroid in the convalescent stage of Vogt-Koyanagi-Harada(VKH).METHODS:A retrospective study was performed on 40 eyes of 20 patients in the convalescent stage of VKH.Fundus photography(FP),multi-spectral imaging(MSI),and optical coherence tomography(OCT)were performed.RESULTS:In the VKH convalescent stage,focal RPE melanin accumulation(FRMA)was detected in 34 eyes(85%)on MSI and in 7 eyes(17.5%)on FP.FRMA was limited to the previous retinal detachment area in all 28 eyes(FRMA was detected in 34 eyes on MSI,which were enrolled,and 6 eyes lacked data in the acute stage).Sunset-glow fundus was detected in 20 eyes(50%)on FP.The mean density of FRMA in a 1-mm-diameter circular area of the fovea was 0.04±0.07 on MSI,which was significantly correlated with sunset-glow fundus(ρ=0.467,P=0.02).CONCLUSION:In the VKH convalescent stage,FRMA is derived from the RPE melanin change,and sunset-glow fundus is derived from the choroid melanin change.A higher density of FRMA in the fovea and sunset-glow fundus represents more serious depigmentation of melanin.

Age-related maculopathy susceptibility 2 participates in the phagocytosis functions of the retinal pigment epithelium

AIM: Age-related macular degeneration (AMD) is a multifactorial disease and a prevalent cause of visual impairment in developed countries. Many studies suggest that age-related maculopathy susceptibility 2 (ARMS2) is a second major susceptibility gene for AMD. At present, there is no functional information on this gene. Therefore, the purpose of the present study was to detect the expression of ARMS2 in retinal pigment epithelium (RPE) cells and to investigate the effect of ARMS2 on the phagocytosis function of RPE cells. METHODS: Immunofluorescence and reverse transcriptase PCR were used to demonstrate the presence and location of ARMS2 in ARPE-19 (human retinal pigment epithelial cell line, ATCC, catalog No.CRL-2302) cells. siRNA was used to knock down ARMS2 mRNA, and the effects of the knockdown on the phagocytosis function of the ARPE-19 cells were evaluated via Fluorescence Activated Cell Sorting (FACS). RESULTS: ARMS2 was present in ARPE-19 cells, localized in the cytosol of the perinuclear region. The expression of ARMS2 mRNA (messenger RNA) in ARPE-19 cells transfected with ARMS2-siRNA (small interfering RNA, 0.73+/- 0.08) was decreased compared with normal cells (1.00+/- 0.00) or with cells transfected with scrambled siRNA (0.95+/- 0.13) (P<0.05). After incubation of RPE cells with a latex beads medium for 12, 18, or 24 hours, the fluorescence intensities were 38.04 +/- 1.02, 68.92 +/- 0.92, and 78.00 +/- 0.12 in the ARMS2-siRNA-transfected groups, respectively, and 77.98 +/- 5.43, 94.87 +/- 0.60, and 98.30 +/- 0.11 in the scrambled siRNA-transfected groups, respectively. The fluorescent intensities of the same time points in the two groups were compared using Student's t-test, and the p values were all less than 0.001 at the three different time points. CONCLUSION: There is endogenous expression of ARMS2 in ARPE-19 cells. ARMS2 plays a role in the phagocytosis function of RPE cells, and this role may be one of the mechanisms that participates in the development of AMD.

The thickness and volume of the choroid,outer retinal layers and retinal pigment epithelium layer changes in patients with diabetic retinopathy

AIM: To evaluate the thickness and volume changes of the choroidal, outer retinal layers(ORL) and retinal pigment epithelium(RPE) in patients with diabetic retinopathy(DR) using optical coherence tomography(OCT) and correlate them with visual acuity.METHODS: We carried out a retrospective observational case series. Consecutive DR patients were recruited for color fundus photography and OCT assessment. The RPE, ORL and choroidal thickness were measured. The correlation with the best-corrected visual acuity(BCVA) was also investigated.RESULTS: The study included 128 eyes, comprising 45 eyes of 25 diabetic macular edema(DME) patients, 34 eyes of 20 DR without DME(non-DME) patients, and 49 eyes of 25 age-matched normal individuals. The choroidal thickness in DR patients were decreased statistically significantly compared with the control group(P<0.05). The mean macular ORL thickness in DME(73.02±15.34 μm) and non-DME groups(76.35±7.32 μm) were decreased statistically significantly compared with the control group(80.20±5.85 μm; P=0.006, P=0.013, respectively). In both the non-DME and DME groups, the RPE thickness were decreased compared with the control group(P<0.05), except in the macular and central ring. The BCVA were significant interactions with the total inner retinal volume and macular RPE thickness in the DME group(r=0.115, P<0.001, r=-0.013, P=0.017, respectively).CONCLUSION: The choroid, ORL and RPE thickness are significantly decreased in DR patients compared with controls in different segments.

Amyloid beta deposition related retinal pigment epithelium cell impairment and subretinal microglia activation in aged APPswePS1 transgenic mice

AIM：To identify the pathological role of amyloid beta（Aβ） deposition in retinal degeneration,and explore Aβ deposition on the retinal pigment epithelium cells（RPE） layer and the associated structural and functional changes in Alzheimer＇s disease transgenic mice.METHODS：RPE changes in the eyes of APPswe/PS1 transgenic and none transgenic（NTG） mice over 20 months old were examined.Histological changes were investigated via hematoxylin and eosin（H＆E） staining and transmission electron microscopy（TEM） examination,whereas the expression of amyloid precursor protein（APP）,Aβ,Zonula occludens-1（ZO-1） and Ionized calcium binding adaptor molecule-1（IBA-1） were investigated using immunohistochemistry and immunofluorescence techniques.All of the obtained results were quantitatively and statistically analyzed.RESULTS：In aged transgenic mice,an APP-positive immunoreaction and Aβ deposition were detected on the RPE layer but were undetectable in NTG mice.The RPE demonstrated some vacuole changes,shortened basal infoldings and basal deposition in histopathological examination and TEM tests,wherein irregular shapes were indicated by ZO-1 disorganization through fluorescence.Furthermore,IBA-1 positive cells were observed to have accumulated and infiltrated into the RPE layer and localized beneath the RPE/Bruch＇s membrane（Br M） complex,which was accompanied by an increase in BrM thickness in aged transgenic mice in comparison to NTG mice.The IBA-1 positive cells were found to be co-stained with Aβ deposition on the RPE flat mounts.CONCLUSION：The observed Aβ deposition in the RPE layer may cause RPE dysfunction,which is associated with microglia cells infiltration into the retina of aged transgenic mice,suggesting that Aβ deposition probably plays a significant role in RPE-related degenerative disease.

Role of glycolysis in retinal vascular endothelium, glia, pigment epithelium, and photoreceptor cells and as therapeutic targets for related retinal diseases

Glycolysis produces large amounts of adenosine triphosphate(ATP)in a short time.The retinal vascular endothelium feeds itself primarily through aerobic glycolysis with less ATP.But when it generates new vessels,aerobic glycolysis provides rapid and abundant ATP support for angiogenesis,and thus inhibition of glycolysis in endothelial cells can be a target for the treatment of neovascularization.Aerobic glycolysis has a protective effect on Müller cells,and it can provide with a target for visual protection and maintenance of the blood-retinal barrier.Under physiological conditions,the mitochondria of RPE can use lactic acid produced by photoreceptor cells as an energy source to provide ATP for survival.In pathological conditions,because RPE cells avoid their oxidative damage by increasing glycolysis,a large number of glycolysis products accumulate,which in turn has a toxic effect on photoreceptor cells.This shows that stabilizing the function of RPE mitochondria may become a target for the treatment of diseases such as retinal degeneration.The decrease of aerobic glycolysis leads to the decline of photoreceptor cell function and impaired vision;therefore,aerobic glycolysis of stable photoreceptor cells provides a reliable target for delaying vision loss.It is of great significance to study the role of glycolysis in various retinal cells for the targeted treatment of ocular fundus diseases.

Inhibition of cell proliferation,migration and apoptosis in blue-light illuminated human retinal pigment epithelium cells by down-regulation of HtrA1

AIM：To investigate the effect of Htr A1 on the proliferation,migration and apoptosis of human retinal pigment epithelium（RPE） cells in the light injured model,as well as the expression of the apoptosis related molecules.METHODS：The human RPE cell line ARPE-19 was exposed to blue light to establish the light injured model.The cells were transfected with Htr A1 si RNA to knockdown Htr A1 expression.Subsequent expression of Htr A1 was determined by real-time polymerase chain reaction（RTPCR） and Western blot,respectively.Changes in cell proliferation,migration and apoptosis were assessed by cell counting kit-8（CCK-8）,Transwell assay and flow cytometry respectively,as well as changes in the m RNA and protein levels of Bax,Caspase-3 and Bcl-2 expression.RESULTS：Htr A1 was highly expressed in ARPE-19 cells after blue light irradiation.Knockdown of Htr A1 expression inhibited the proliferation,migration and apoptosis of the blue-light-irradiated ARPE-19 cells（P〈0.05）.Bax and Caspase-3 expression were significantly reduced both at m RNA and protein levels（P〈0.05） after si RNA treatment.Bcl-2 expression significantly increased in blue-lightirradiated ARPE-19 cells after si RNA interference（P〈0.05）.CONCLUSION：Silence of Htr A1 may inhibit the proliferation,migration and apoptosis of ARPE-19 cells in light injured model.Moreover,Htr A1 suppression in blue-lightirradiated ARPE-19 cells may ameliorate cell apoptosis through down-regulation of Bax and Caspase-3,and upregulation of Bcl-2 expression.