RPE Cells Research Articles

Metabolite Extraction from RPE Cells and Retinas Related to Retinitis Pigmentosa.

The application of metabolomics in ophthalmology helps to identify new biomarkers and elucidate disease mechanisms in different eye diseases, as well as aiding in the development of potential treatment options. Extracting metabolites successfully is essential for potential further analysis using mass spectrometry. In this chapter, we describe how to extract metabolites from a variety of sources including (1) cells on a dish, (2) cell culture medium, and (3) tissues in vivo with and without stable isotope tracers. Samples prepared using this protocol are suitable for a range of downstream mass spectrometry analyses and are stable in solvent for weeks at -80°C.

Clinical grade of genetically corrected RPE cells for autologous cell therapy of hereditary retinal dystrophies

AbstractPurpose: The generation of genetically corrected RPE cells from patient‐derived human induced pluripotent stem cells (hiPSCs) may provide a source for autologous therapeutically‐relevant therapy for this rare retinal dystrophy as well to create the human cellular disease models. Recently we generated a faithful RPE model from patient bearing mutation in MERTK gene and genetically corrected these cells using CRISPR/Cas9 system. For further application in humans is necessary to differentiate the hiPSC towards RPE in clinical grade conditions.Methods: The RPE cells from the genetically corrected patient‐derived hiPSC (1 and 2 alleles) as well as patient's RP‐hiPSC and control healthy hiPSC were characterized using immunocytological methods for the differentiation and polarization markers, electronic microscopy, RT‐PCR, TEER measurement, ELISA test, Western Blot and phagocytic assay.Results: All generated RPE cells exhibit typical features of RPE cells We demonstrated the reestablishment of the expression of full‐length MERTK protein as well as the reversion of lost phagocyte function of hiPSC‐RPE in vitro, which represents the first example of its kind in this field.Conclusions: The generated hiPSC‐derived RPE in clinical grade conditions display the typical characteristics of mature RPE cells re‐establishing the function of phagocytosis in genetically corrected patient‐derived RPE cells in vitro. Functional in vivo studies in animal models such as RCS rats or larger animals will confirm whether this strategy can completely restore the vision and be employed as a potential treatment for patients.

The cleavage of Gasdermin E is the rate limiting step in the secretion of IL‐1β in RPE cells with non‐functional cellular clearance

AbstractPurpose: Since RPE cells has important regulatory role in retinal inflammation, we examined the secretion kinetics of proinflammatory cytokine IL‐1b.Methods: IL‐1β production and release from IL‐1a‐primed (4 ng/ml) ARPE‐19 cells was induced by inhibiting proteasomal clearance and autophagy using MG‐132 (5 μM) and Bafilomycin A1 (BafA; 50 nM), respectively. Medium samples and cell lysates were collected from the beginning of BafA exposure and continued for 24 h in 6 h intervals. IL‐1β levels were measured using the ELISA technique. The cleavage of pore forming gasdermin E was detected using the western blot method.Results: In the lysate samples, active IL‐1β levels were highest right after the BafA exposure. After 12 h, IL‐1β levels in lysates had fallen to only 54.6% of the maximal concentration. The cleavage of gasdermin E and the secretion of IL‐1β increased concurrently.Conclusions: In contrast to classical immunological cells, the secretion of IL‐1β from human RPE cells is a slow process. IL‐1β is secreted trough gasdermin E pores. Our data suggest that the secretion phase is the rate limiting step.

Changes in visual function associated with hydroxychloroquine retinal toxicity

AbstractIntroduction: Non‐infectious inflammatory diseases, such as systemic lupus erythematosus, rheumatoid arthritis, dermatomyositis, antiphospholipid antibody syndrome and others cause significant morbidity and are treated with immunosuppression. Hydroxychloroquine (HCQ) sulfate is used as a second line immunosuppressant and has many advantages over other agents. These include increased life expectancy, reduced risk of neoplasia, reduced risk of diabetes and hypercholesterolaemia. HCQ is safe in pregnancy. After more than 5 years of treatment, HCQ can cause a degenerative maculopathy characterized by loss of parafoveal photoreceptors and RPE cells, leading to a ring‐like visual field defect. Current detection of early retinal toxicity relies on central visual field testing, optical coherence tomography (OCT) scan and fundus autofluorescence. The reason for the parafoveal predilection for retinopathy is not understood. HCQ is concentrated in melanin‐containing cells, and there is increased melanin concentration in the macular RPE cells. A hypothesis is that HCQ concentrated in the macular RPE cells leads to toxicity, with macular pigment protecting the fovea but leaving a ring of potentially vulnerable tissue in the parafoveal region. We were interested to investigate parafoveal function in patients with mild HCQ maculopathy and report the results of detailed macular function assessment of red/green (RG) and yellow / blue (YB) colour vision and rod‐ and cone‐mediated rapid flicker sensitivity.Methods: Two patients with confirmed early hydroxychloroquine maculopathy participated in the study. RG and YB colour thresholds were measured with the Colour Assessment and Diagnosis (CAD) test. Cone and rod‐mediated rapid flicker sensitivity was also measured in both central and paracentral vision using the Flicker‐Plus test. Both patients had taken HCQ for more than 15 years and had paracentral scotomas on 10–2 field test and loss of the parafoveal outer nuclear layer and ellipsoid layer, confirming the presence of maculopathy. To test the hypothesis of parafoveal toxicity risk, 20 short wavelength (BAF) and infrared wavelength (IRAF) autofluorescence images were reviewed. The BAF images are surrogate markers of the macular pigment and the IRAF the melanin concentration. Normalized maps of the difference between BAF and IRAF were calculated to investigate the possibility of a ring of vulnerable tissue.Results: Both patients had normal RG, but abnormal YB colour thresholds. One subject had normal cone flicker thresholds, but the rod flicker thresholds were raised in all quadrants in both eyes. The second subject had significantly elevated rod thresholds and elevated cone flicker thresholds in both eyes. Of note is that the degree of retinopathy in the second patient was greater than in the first. The areas of greatest threshold elevation correlated with the areas of greatest field loss and anatomical disruption on OCT.Conclusion: These new findings show that hydroxychloroquine retinopathy affects preferentially both YB colour vision and rod‐mediated flicker sensitivity. The cone‐mediated flicker sensitivity is also affected in more advanced disease. A model for the anatomical location of the toxicity will be presented, based on the hypothesis outlined above.

Neuroprotective effects of intraocular bone marrow‐derived mononuclear cell transplants in inherited retinal degenerations

AbstractInherited retinal degenerations are diseases characterized by photoreceptor loss secondary to genetic defects. At present, only a minority are susceptible of genetic treatment and therefore are a frequent cause of blindness in infancy and adulthood.Retinitis Pigmentosa (RP) is the most frequent inherited retinal degeneration. This disease has a prevalence of around 1 in 4000 individuals and is caused by mutations in more than 300 genes. RP is characterized by progressive loss of photoreceptors, rods and then cones that may die secondarily to rod loss.The rats P23H and Royal college of Surgeon (RCS) are two well characterized animal models of RP because they bear genetic defects observed in RP patients. The P23H rat suffers an autosomal dominant mutation of the rhodopsin gene that causes protein misfolding and endoplasmic reticulum stress in rods, which die first followed by cones. The RCS rat has a mutation of MERTK gene, that impedes the normal photoreceptor outer segment phagocytosis by RPE cells thus causing simultaneous rod and cone degeneration.Therapies for inherited photoreceptor degeneration aim to prevent or delay death of the photoreceptors, or in specific circumstances try to replace them.The Bone marrow derived‐mononuclear cells (BM‐MNCs) are a mononuclear CD34+ subpopulation of bone marrow‐derived cells that can be separated from the other cellular fractions of the bone marrow aspirate using a Ficoll density gradient‐based protocol. The BM‐MNCs is a mixture of specific bone marrow cell subtypes in which is possible to find cells: Lin+, Lin−, CD34+, c‐Kit+, CD133+ or Sca‐1+ HSCs, endothelial precursor cells, and Mesenchymal Stem Cells.BM‐MNCs transplantation has shown promising results in different animal models of neuronal degeneration, including inherited photoreceptor degenerations.In our studies we have performed a syngeneic intravitreal or subretinal transplantation of BM‐MNCs in a P23H‐1 and RCS rats, and we have documented that both increase photoreceptor survival. They also improve the morphology of the photoreceptors outer segment, decrease synaptic degeneration in the outer plexiform layer and reduce glial fibrillary acidic protein (GFAP) expression in Müller cells. However, the transplanted cells did not penetrate the retina, only survived for up to 15 days, and did not modify microglial cell activation and migration or the electroretinographic responses. Their beneficial effects are maybe due to a paracrine trophic effect achieved through release of neurotrophic, anti‐angiogenic and/or immunomodulatory factors.In conclusion, the intra ocular transplantation BM‐MNCs shows photoreceptor neuroprotective actions in inherited retinal degenerations. These cells may have the potential to slow down photoreceptor loss, and therefore could be indicated for treatment of the early stages of photoreceptor degenerations.

Selective proteasome inhibition activates AIM2 inflammasome in human retinal pigment epithelium cells

AbstractPurpose: Dysfunctional intracellular clearance is associated with various diseases, including age‐related macular degeneration (AMD). Previously, we reported the inhibition of intracellular clearance by the non‐selective proteasome inhibitor MG‐132 resulted in oxidative stress‐mediated NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and subsequent interleukin‐1β (IL‐1β) secretion in human ARPE‐19 cells. In this study, we used the selective proteasome inhibitor epoxomicin and examined its effect on the inflammasome activation and IL‐1β production.Methods: IL‐1α‐primed human‐derived ARPE‐19 cells were exposed either to MG‐132 or epoxomicin and the autophagy inhibitor bafilomycin A (BafA) and studied using enzyme‐linked immunosorbent assay and cytotoxicity tests.Results: Our data reveal epoxomicin‐induced proteasome inhibition led to NADPH oxidase and mitochondria‐mediated oxidative stress‐related release of mitochondrial DNA to the cytosol, which then resulted in potassium efflux‐dependent absent in melanoma 2 (AIM2) inflammasome activation and subsequent release of inflammatory cytokine IL‐1β in ARPE‐19 cells.Conclusions: Our study emphasizes the role of AIM2 inflammasome in RPE cell degeneration along with the well‐known NLRP3 inflammasome and may represent a novel therapy target.

Subretinal implantation of human primary RPE cells cultured on nanofibrous membranes in minipigs

AbstractThe development of primary human retinal pigmented epithelium (hRPE) for clinical transplantation purposes on biodegradable scaffolds is indispensable. The results of the subretinal implantation of hRPE cells on nanofibrous membranes in minipigs are hereby reported.The hRPEs were collected from human cadaver donor eyes and cultivated on ultrathin nanofibrous carriers prepared via the electrospinning of poly(L‐lactide‐co‐DL‐lactide) (PDLLA). “Libechov” minipigs (12–36 months old) were used in the study, supported by preoperative tacrolimus immunosuppressive therapy. The subretinal implantation of the hRPE‐nanofibrous carrier was conducted using general anaesthesia, followed by silicone oil endotamponade. The observational period lasted up to 8 weeks, and included in vivo optical coherence tomography (OCT) of the retina, as well as post mortem immunohistochemistry.The hRPEs assumed cobblestone morphology, persistent pigmentation and measurable trans‐epithelial electrical resistance on the nanofibrous PDLLA carrier. The implanted hRPEs were positive for HNAA and STEM121 (human cell markers) and were located between the minipig's neuroretina and RPE layers at week 2 post‐implantation, which was gradually attenuated until week 8. The neuroretina over the implants showed rosette or hypertrophic reaction at week 6. The implanted cells expressed the typical RPE marker bestrophin throughout the whole observation period, and a gradual diminishing of the CRALBP expression in the area of implantation at week 8 post‐implantation was observed. The transplanted hRPEs appeared not to form a confluent layer and were less capable of keeping the inner and outer retinal segments intact. The cone photoreceptors adjacent to the implant scaffold were unchanged initially, but underwent a gradual change in structure after hRPE implantation; the retina above and below the implant appeared relatively healthy. The glial reaction of the transplanted and host retina showed Vimentin and GFAP positivity from week 1 onward. Microglial activation appeared in the retinal area of the transplant early after the surgery, which seemed to move into the transplant area over time.Altogether, the differentiated hRPEs can serve as an alternative cell source for RPE replacement in animal studies. These cells can be cultivated on nanofibrous PDLLA and implanted subretinally into minipigs using standard 23‐gauge vitrectomy and implantation injector. The hRPE‐laden scaffolds demonstrated relatively good incorporation into the host retina over an eight‐week observation period, with some indication of a gliotic scar formation, and a likely neuroinflammatory response in the transplanted area despite the use of immunosuppression.

Phloroglucinol Attenuates DNA Damage and Apoptosis Induced by Oxidative Stress in Human Retinal Pigment Epithelium ARPE-19 Cells by Blocking the Production of Mitochondrial ROS.

Phloroglucinol, a phenolic compound, is known to possess a potent antioxidant ability. However, its role in retinal cells susceptible to oxidative stress has not been well elucidated yet. Thus, the objective of this study was to evaluate whether phloroglucinol could protect against oxidative damage in cultured human retinal pigment epithelium ARPE-19 cells. For this purpose, ARPE-19 cells were stimula ted with hydrogen peroxide (H2O2) to mimic oxidative stress. Cell viability, cytotoxicity, apoptosis, reactive oxygen species (ROS) generation, mitochondrial function, DNA damage, and autophagy were then assessed. Our results revealed that phloroglucinol ameliorated cell viability, cytotoxicity, and DNA damage in H2O2-exposued ARPE-19 cells and blocked production of ROS. Phloroglucinol also counteracted H2O2-induced apoptosis by reducing Bax/Bcl-2 ratio, blocking activation of caspase-3, and inhibiting degradation of poly (ADP-ribose) polymerase. H2O2 caused mitochondrial impairment and increased expression levels of mitophagy markers such as PINK1and PARKIN known to be associated with mitochondrial ROS (mtROS) generation and cytosolic release of cytochrome c. However, these changes were significantly attenuated by phloroglucinol. Mito-TEMPO, a selective mitochondrial antioxidant, further enhanced the protective effect of phloroglucinol against dysfunctional mitochondria. Furthermore, H2O2 induced autophagy, but not when ARPE-19 cells were pretreated with phloroglucinol, meaning that autophagy by H2O2 contributed to the pro-survival mechanism and that phloroglucinol protected ARPE-19 cells from apoptosis by blocking autophagy. Taken together, these results suggest that phloroglucinol can inhibit oxidative stress-induced ARPE-19 cell damage and dysfunction by protecting DNA damage, autophagy, and subsequent apoptosis through mitigation of mtROS generation. Thus, phloroglucinol might have therapeutic potential to prevent oxidative stress-mediated damage in RPE cells.

MiR-302d inhibits TGFB-induced EMT and promotes MET in primary human RPE cells.

Transforming growth factor-beta (TGFB)-mediated epithelial-mesenchymal transition (EMT) plays a crucial role in the pathogenesis of retinal fibrosis, which is one of the leading causes of impaired vision. Current approaches to treating retinal fibrosis focus, among other things, on inhibiting the TGFB signaling pathway. Transient expression of microRNAs (miRNAs) is one way to inhibit the TGFB pathway post-transcriptionally. Our previous study identified the miRNA miR-302d as a regulator of multiple TGFB-related genes in ARPE-19 cells. To further explore its effect on primary cells, the effect of miR-302d on TGFB-induced EMT in primary human retinal pigment epithelium (hRPE) was investigated in vitro. hRPE cells were extracted from patients receiving enucleation. Transfection of hRPE cells with miR-302d was performed before or after TGFB1 stimulation. Live-cell imaging, immunocytochemistry staining, Western blot, and ELISA assays were utilized to identify the alterations of cellular morphology and EMT-related factors expressions in hRPE cells. hRPE cells underwent EMT by TGFB1 exposure. The transfection of miR-302d inhibited the transition with decreased production of mesenchymal markers and increased epithelial factors. Meanwhile, the phosphorylation of SMAD2 activated by TGFB1 was suppressed. Moreover, miR-302d expression promoted TGFB1-induced fibroblast-like hRPE cells to revert towards an epithelial stage. As confirmed by ELISA, miR-302d reduced TGFB receptor 2 (TGFBR2) and vascular endothelial growth factor A (VEGFA) levels 48 hours after transfection. The protective effect of miR-302d might be a promising approach for ameliorating retinal fibrosis and neovascularization. MiR-302d suppresses TGFB-induced EMT in hRPE cells via downregulation of TGFBR2, even reversing the process. Furthermore, miR-302d reduces the constitutive secretion of VEGFA from hRPE cells.

Protrusion of KCNJ13 Gene Knockout Retinal Pigment Epithelium Due to Oxidative Stress-Induced Cell Death.

This study was performed to elucidate the mechanisms of morphological abnormalities in a Leber congenital amaurosis 16 (LCA16) cell model using KCNJ13 knockout (KO) retinal pigment epithelial cells derived from human iPS cells (hiPSC-RPE). In KCNJ13 KO and wild-type hiPSC-RPE cells, ZO-1 immunofluorescence was performed, and confocal images were captured. The area and perimeter of each cell were measured. To detect cell death, ethidium homodimer III (EthD-III) staining and LDH assay were used. Scanning electron microscopy (SEM) was used to observe the cell surface. The expression levels of oxidative stress-related genes were examined by quantitative PCR. To explore the effects of oxidative stress, tert-butyl hydroperoxide (t-BHP) was administered to the hiPSC-RPE cells. Cell viability was tested by MTS assay, whereas oxidative damage was monitored by oxidized (GSSG) and reduced glutathione levels. The area and perimeter of KCNJ13-KO hiPSC-RPE cells were enlarged. EthD-III-positive cells were increased with more dead cells in the protruded region. The KO RPE had significantly higher LDH levels in the medium. SEM observations revealed aggregated cells having broken cell surfaces on the KO RPE sheet. The KCNJ13-deficient RPE showed increased expression levels of oxidative stress-related genes and total glutathione levels. Furthermore, t-BHP induced a significant increase in cell death and GSSG levels in the KO RPE. We suggest that in the absence of the Kir.7.1 potassium channel, human RPE cells are vulnerable to oxidative stress and ultimately die. The dying/dead cells form aggregates and protrude from the surviving KCNJ13-deficient RPE sheet.

Pigment epithelium-derived factor is an interleukin-6 antagonist in the RPE: Insight of structure-function relationships.

Retinal and choroidal inflammatory lesions increase the levels of the pro-inflammatory cytokine interleukin-6 (IL-6). Pigment epithelium-derived factor (PEDF) has anti-inflammatory properties, but it is not known if it can prevent the production of IL-6 by the retinal pigment epithelium. To investigate the anti-inflammatory effects of PEDF in the RPE, we used human ARPE-19 cells stimulated with human recombinant tumor necrosis factor-alpha (TNF-α) to induce overexpression of the IL6 gene. We found that the viability of ARPE-19 cells decreased by 22% with TNF-α at 10ng/ml, being drastically decreased at ≥50ng/ml. TNF-α at 5-100ng/ml elevated the production and secretion of IL-6 protein, as measured by ELISA. To challenge the TNF-α-mediated stimulation of IL-6, we used recombinant human PEDF protein. PEDF at 100nM recovered the TNF-α-mediated loss of cell viability and repressed IL-6 gene expression as determined by RT-PCR. PEDF at 10-100nM attenuated the IL-6 protein secretion in a dose dependent fashion (IC50 = 65nM), being abolished with 100nM PEDF. To map the region that confers the IL-6 blocking effect to the PEDF polypeptide, we used chemically synthesized peptides designed from its biologically active domains, pro-death 34-mer, and pro-survival 44-mer and 17-mer (H105A), to challenge the IL-6 overproduction. The pro-survival peptides recovered the TNF-α-mediated cell viability loss, and inhibited IL-6 secretion, while the 34-mer did not have an effect, suggesting a role for the pro-survival domain in blocking TNF-α-mediated cell death and IL-6 stimulation. Our findings position PEDF as a novel antagonistic agent of IL-6 production in RPE cells, underscoring its use for the management of retinal disease-related inflammation.

Fallopia Japonica and Prunella vulgaris inhibit myopia progression by suppressing AKT and NFκB mediated inflammatory reactions

BackgroundThe increased global incidence of myopia requires the establishment of therapeutic approaches. This study aimed to investigate the effect of Fallopia Japonica (FJ) and Prunella vulgaris (PV) extract on myopia caused by monocular form deprivation (MFD).MethodsWe used human retinal pigment epithelial cell to study the molecular mechanisms on how FJ extract (FJE) and PV extract (PVE) lowering the inflammation of the eye. The effect of FJE and PVE in MFD induced hamster model and explore the role of inflammation cytokines in myopia.ResultsFJE + PVE reduced IL-6, IL-8, and TNF-α expression in RPE cells. Furthermore, FJE and PVE inhibited inflammation by attenuating the phosphorylation of protein kinase B (AKT), and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) pathway. In addition, we report two resveratrol + ursolic acid compounds from FJ and PV and their inhibitory activities against IL-6, IL-8, and TNF-α expression levels in RPE cells treated with IL-6 and TNF-α. FJE, PVE, and FJE + PVE were applied to MFD hamsters and their axial length was measured after 21 days. The axial length showed statistically significant differences between phosphate-buffered saline- and FJE-, PVE-, and FJE + PVE-treated MFD eyes. FJE + PVE suppressed expressions of IL-6, IL-8, and TNF-α. They also inhibited myopia-related transforming growth factor-beta (TGF)-β1, matrix metalloproteinase (MMP)-2, and NF-κB expression while increasing type I collagen expression.ConclusionsOverall, these results suggest that FJE + PVE may have a therapeutic effect on myopia and be used as a potential treatment option.

Multifunctional Interleukin-24 Resolves Neuroretina Autoimmunity via Diverse Mechanisms.

IL-24 is a multifunctional cytokine that regulates both immune cells and epithelial cells. Although its elevation is associated with a number of autoimmune diseases, its tolerogenic properties against autoreactive T cells have recently been revealed in an animal model of central nervous system (CNS) autoimmunity by inhibiting the pathogenic Th17 response. To explore the potential of IL-24 as a therapeutic agent in CNS autoimmunity, we induced experimental autoimmune uveitis (EAU) in wildtype mice and intravitreally injected IL-24 into the inflamed eye after disease onset. We found that the progression of ocular inflammation was significantly inhibited in the IL-24-treated eye when compared to the control eye. More importantly, IL-24 treatment suppressed cytokine production from ocular-infiltrating, pathogenic Th1 and Th17 cells. In vitro experiments confirmed that IL-24 suppressed both Th1 and Th17 differentiation by regulating their master transcription factors T-bet and RORγt, respectively. In addition, we found that intravitreal injection of IL-24 suppressed the production of proinflammatory cytokines and chemokines from the retinas of the EAU-inflamed eyes. This observation appears to be applicable in humans, as IL-24 similarly inhibits human retinal pigment epithelium cells ARPE-19. In conclusion, we report here that IL-24, as a multifunctional cytokine, is capable of resolving ocular inflammation in EAU mice by targeting both uveitogenic T cells and RPE cells. This study sheds new light on IL-24 as a potential therapeutic candidate for autoimmune uveitis.

RO4929097, a Selective γ-Secretase Inhibitor, Inhibits Subretinal Fibrosis Via Suppressing Notch and ERK1/2 Signaling in Laser-Induced Mouse Model.

PurposeThis study aimed to explore whether RO4929097 (RO), a specific γ-secretase inhibitor, could inhibit the subretinal fibrosis in laser-induced mouse model and the relevant molecular mechanisms.MethodsMale C57BL/6J mice were used to produce choroidal neovascularization (CNV) and subretinal fibrosis by laser photocoagulation, and RO was administered intravitreally 1 day after laser induction. The sizes of CNV and subretinal fibrosis were measured and quantified in both 2D and 3D constructions. The ARPE-19 cell line and primary human RPE (phRPE) cells were treated with TGFβ1, in combination with or without RO, to examine Notch related molecules, epithelial mesenchymal transition (EMT), cell viability, migration, and contractile function, as well as the crosstalk between Notch and other EMT relevant signaling pathways.ResultsIntravitreal injection of RO reduced the sizes of both CNV and subretinal fibrosis in laser-induced young and old mice at day 7 and day 14 after laser induction. Moreover, EMT and Notch activation in RPE-choroid complexes from laser-induced mice were significantly attenuated by RO. In vitro, TGFβ1 activated Notch signaling and induced EMT in ARPE-19 cells, accompanied by enhanced EMT-related function, which were inhibited by RO. The inhibition of RO on EMT was further confirmed in TGFβ1-treated phRPE cells. Blockage of Notch signaling by RO could inhibit ERK1/2 signaling; whereas ERK1/2 inhibition had no effect on Notch. The action of RO was independent of Smad2/3 or p38, and co-inhibition of Notch and Smad2/3 showed synergistic effect on EMT inhibition.ConclusionsRO exerts its antifibrotic effect by directly inhibiting Notch signaling and indirectly suppressing ERK1/2 signaling. Targeting Notch signaling might provide a therapeutic strategy in prevention and treatment of subretinal fibrosis in neovascular age-related macular degeneration (nAMD).

Loss of Hes1 in embryonic stem cells caused developmental disorders in retinal pigment epithelium morphogenesis and specification

Hairy and enhancer of split homolog-1 (Hes1) is a member of an extensive family of basic helix-loop-helix (bHLH) proteins and plays a crucial role in neurogenesis, myogenesis, hematopoiesis, and sex determination. It has been reported that Hes1 is essential for precursors maintenance, optic cup-stalk boundary maintenance, and morphogenesis of the retina. However, it still reminds questions about the role and mechanism of Hes1 in the development of retinal pigment epithelial cells. In our study, We generated Hes1−/− human embrsyonic stem cells, and attempted to induce them into retinal pigment epithelial cells by our previous protocol, found that the cells induced by Hes1−/− hESCs hardly expressed RPE-related genes, and rarely appeared RPE cell morphology. Additionally, Hes1 may affect the development of RPE cells via Wnt4 pathway by analyzing the RNA-seq data of differently expressed genes between normal RPE cells development and Hes1−/− RPE cells development. Overall, depletion of Hes1 may result in the failure of Wnt4 signal activation, and contributed to the developmental disorder in retinal pigment epithelium morphogenesis and specification.

Scribble basal polarity acquisition in RPE cells and its mislocalization in a pathological AMD-like model.

Apicobasal polarity is a hallmark of retinal pigment epithelium cells and is required to perform their functions; however, the precise roles of the different proteins that execute polarity are still poorly understood. Here, we have studied the expression and location of Scribble, the core member of the polarity basal protein complex in epithelial-derived cells, in human and mouse RPE cells in both control and pathological conditions. We found that Scribble specifically localizes at the basolateral membrane of mouse and human RPE cells. In addition, we observed an increase in the expression of Scribble during human RPE development in culture, while it acquires a well-defined basolateral pattern as this process is completed. Finally, the expression and location of Scribble were analyzed in human RPE cells in experimental conditions that mimic the toxic environment suffered by these cells during AMD development and found an increase in Scribble expression in cells that develop a pathological phenotype, suggesting that the protein could be altered in cells under stress conditions, as occurs in AMD. Together, our results demonstrate, for the first time, that Scribble is expressed in both human and mouse RPE and is localized at the basolateral membrane in mature cells. Furthermore, Scribble shows impaired expression and location in RPE cells in pathological conditions, suggesting a possible role for this protein in the development of pathologies, such as AMD.

The transcriptome profile of RPE cells by the fullerenol against hydrogen peroxide stress.

Age-related macular degeneration (AMD) causes central vision impairment with increased incidence. In the pathogenesis of AMD, reactive oxygen species (ROS) are associated with RPE cell apoptosis. H2O2 is an oxidative toxicant and is used to establish the AMD in vitro model. However, the mechanisms of ROS in H2O2-induced AMD are still unclear. Fullerenol, a promising antioxidant of nanomaterials, protects RPE cells from ROS attack. In addition to working as a scavenger, little is known about the antioxidant mechanism of fullerenol in RPE cells. In this study, transcriptome sequencing was performed to examine the global changes in mRNA transcripts induced by H2O2 in human ARPE-19 cells. Moreover, we comprehensively investigated the protective effects of fullerenol against H2O2-induced oxidative injury by RNA sequencing. Gene Ontology enrichment analysis showed that those pathways related to the release of positive regulation of DNA-templated transcription and negative regulation of apoptotic process were affected. Finally, we found that 12 hub genes were related to the oxidative-protection function of fullerenol. In summary, H2O2 affected these hub genes and signaling pathways to regulate the senescence of RPE cells. Moreover, fullerenol is a potent nanomaterial that protects the RPE and would be a promising approach for AMD prevention.

Inhibition of BRD4 Promotes Pexophagy by Increasing ROS and ATM Activation.

Although autophagy regulates the quality and quantity of cellular compartments, the regulatory mechanisms underlying peroxisomal autophagy (pexophagy) remain largely unknown. In this study, we identified several BRD4 inhibitors, including molibresib, a novel pexophagy inducer, via chemical library screening. Treatment with molibresib promotes loss of peroxisomes selectively, but not mitochondria, ER, or Golgi apparatus in HeLa cells. Consistently, depletion of BRD4 expression also induced pexophagy in RPE cells. In addition, the inhibition of BRD4 by molibresib increased autophagic degradation of peroxisome ATG7-dependency. We further found that molibresib produced reactive oxygen species (ROS), which potentiates ATM activation. Inhibition of ROS or ATM suppressed the loss of peroxisomes in molibresib-treated cells. Taken together, our data suggest that inhibition of BRD4 promotes pexophagy by increasing ROS and ATM activation.

HTRA1 Regulates Subclinical Inflammation and Activates Proangiogenic Response in the Retina and Choroid.

High-temperature requirement A1 (HtrA1) has been identified as a disease-susceptibility gene for age-related macular degeneration (AMD) including polypoidal choroidal neovasculopathy (PCV). We characterized the underlying phenotypic changes of transgenic (Tg) mice expressing ubiquitous CAG promoter (CAG-HtrA1 Tg). In vivo imaging modalities and histopathology were performed to investigate the possible neovascularization, drusen formation, and infiltration of macrophages. Subretinal white material deposition and scattered white-yellowish retinal foci were detected on CFP [(Tg—33% (20/60) and wild-type (WT)—7% (1/15), p < 0.05]. In 40% (4/10) of the CAG-HtrA1 Tg retina, ICGA showed punctate hyperfluorescent spots. There was no leakage on FFA and OCTA failed to confirm vascular flow signals from the subretinal materials. Increased macrophages and RPE cell migrations were noted from histopathological sections. Monocyte subpopulations were increased in peripheral blood in the CAG-HtrA1 Tg mice (p < 0.05). Laser induced CNV in the CAG-HtrA1 Tg mice and showed increased leakage from CNV compared to WT mice (p < 0.05). Finally, choroidal explants of the old CAG-HtrA1 Tg mice demonstrated an increased area of sprouting (p < 0.05). Signs of subclinical inflammation was observed in CAG-HtrA1 Tg mice. Such subclinical inflammation may have resulted in increased RPE cell activation and angiogenic potential.

Determining the optimal stage for cryopreservation of human embryonic stem cell-derived retinal pigment epithelial cells

BackgroundHuman embryonic stem cell-derived retinal pigment epithelial cells (hESC-derived RPE) are a promising source for cell-replacement therapy to treat retinal degenerative diseases, but research on RPE cryopreservation is limited. This study aimed to determine the best phase for RPE cryopreservation to preserve the post-thaw function and uncover the mechanism underlying RPE freezing tolerance.MethodshESC-derived RPE cells were cryopreserved at various time points after seeding. After thawing, the survival and attachment rates, RPE marker gene expression, apical-basal polarity, PEDF secretion, transepithelial resistance, and phagocytotic ability of post-thaw RPE cells were evaluated. RNA sequencing was performed on RPE cells at three-time points, differentially expressed genes were identified, and gene ontology, Kyoto encyclopedia of genes and genomes, and protein–protein interaction analyses were used to investigate the key pathways or molecules associated with RPE cell freezing tolerance.ResultsRPE frozen at passage 2 day 5 (P2D5) had the highest cell viability and attachment after thawing. They also retained properly localized expression of RPE marker genes and biological functions such as PEDF secretion, high transepithelial resistance, and phagocytic ability. The RNA-sequencing analysis revealed that RPE cells at P2D5 expressed high levels of cell cycle/DNA replication and ECM binding associated genes, as well as THBS1, which may serve as a possible hub gene involved in freezing tolerance. We also confirmed that the RPE cells at P2D5 were in the exponential stage with active DNA replication.ConclusionsWe propose that freezing hESC-derived RPE cells during their exponential phase results in the best post-thawing outcome in terms of cell viability and preservation of RPE cell properties and functions. The high expression levels of the cell cycle and ECM binding associated genes, particularly THBS1, may contribute to better cell recovery at this stage.