Retinal Pigment Epithelium Function Research Articles

Progressive dysfunction of the retinal pigment epithelium and retina due to increased VEGF‐A levels

Patients with nonexudative ("dry") age-related macular degeneration (AMD) frequently also develop neovascular ("wet") AMD, suggesting a common pathomechanism. Increased vascular endothelial growth factor A (VEGF-A) has been implicated in the pathogenesis of choroidal neovascularization (CNV) in neovascular AMD, while its role in nonexudative AMD that manifests with progressive retinal pigment epithelium (RPE) and photoreceptor degeneration is not well defined. Mice with overall increased VEGF-A levels develop progressive morphological features of both forms of AMD, suggesting that an increase in VEGF-A has a direct age-dependent adverse effect on RPE and photoreceptor function independently of its CNV-promoting proangiogenic effect. Here we provide evidence for this hypothesis and show that morphological RPE abnormalities and retinal thinning in mice with increased VEGF-A levels correlate with progressive age-dependent attenuation of visual function with abnormal electroretinograms and reduced retinal rhodopsin levels. Retinoid profiling revealed a progressive reduction of 11-cis and all-trans retinal in the retinas of these mice, consistent with an impaired retinoid transport between the RPE and photoreceptors. These findings suggest that increased VEGF-A leads to an age-dependent RPE and retinal dysfunction that occurs also at sites where no CNV lesions form. The data support a central role of increased VEGF-A not only in the pathogenesis of neovascular but also of nonexudative AMD.

Spectral domain optical coherence tomography used to view and quantify choroidal vascular congestion in new subretinal fluid following encircling scleral buckling

Known complications of scleral buckling surgery for primary rhegmatogenous retinal detachment include choroidal detachment, vortex vein compression and anterior segment ischemia. Post-operative persistent subretinal fluid following scleral buckling surgery is thought to occur in as many as 55% of cases. Likely mechanisms include impaired retinal pigment epithelium (RPE) function, altered subretinal fluid composition, choroidal ischemia, incomplete drainage of subretinal fluid and altered choroidal blood flow. Indocyanine green (ICG) angiography, laser Doppler velocimetry and scanning laser Doppler flowmetry have showed altered choroidal blood flow, pulsatile ocular blood flow and choroidal venous drainage after scleral buckling.

Transport mechanisms of the retinal pigment epithelium to maintain of visual function

In the visual process the interaction between the retinal pigment epithelium (RPE) and photoreceptors involves several transport phenomena. Heat from light-absorption is eliminated by blood-flow in the choroid. Transepithelial transport eliminates water from subretinal space for close interaction between photoreceptors and RPE. A recycling transport supplies the chromophore for photoreceptors. Last but not least transmembranal K+ transport maintains excitability of photoreceptors and Ca2+ enables the regulation of RPE function.

Independent roles of methionine sulfoxide reductase A in mitochondrial ATP synthesis and as antioxidant in retinal pigment epithelial cells

The antioxidant enzyme methionine sulfoxide reductase A (MsrA) is highly expressed in the retinal pigment epithelium (RPE), a support tissue for neighboring photoreceptors. MsrA protein levels correlate with sensitivity of RPE in culture to experimental oxidative stress. To investigate whether and how MsrA affects RPE functionality regardless of oxidative stress, we tested the effects of acute silencing or overexpression of MsrA on the phagocytosis of photoreceptor outer segment fragments (POS), a demanding, daily function of the RPE that is essential for vision. Endogenous MsrA localized to mitochondria and cytosol of rat RPE in culture. RPE cells manipulated to express higher or lower levels of MsrA than control cells showed no signs of cell death but increased or decreased, respectively, POS binding as well as engulfment. These effects of altered MsrA protein concentration on phagocytosis were independent of the levels of oxidative stress. However, altering MsrA expression had no effect on phagocytosis when mitochondrial respiration was inhibited. Furthermore, ATP content directly correlated with MsrA protein levels in RPE cells that used mitochondrial oxidative phosphorylation for ATP synthesis but not in RPE cells that relied on glycolysis alone. Overexpressing MsrA was sufficient to increase specifically the activity of complex IV of the respiratory chain, whereas activity of complex II and mitochondrial content were unaffected. Thus, MsrA probably enhances ATP synthesis by increasing complex IV activity. Such contribution of MsrA to energy metabolism is independent of its function in protection from elevated oxidative stress but contributes to routine but vital photoreceptor support by RPE cells.

ULTRA-WIDEFIELD AUTOFLUORESCENCE IMAGING IN THE EVALUATION OF SCLERAL BUCKLING SURGERY FOR RETINAL DETACHMENT

To investigate the use of ultra-widefield fundus autofluorescence imaging in the early postoperative evaluation of scleral buckling surgery for retinal detachment. Forty-five eyes from 44 patients with rhegmatogenous retinal detachment were included. Ultra-widefield fundus autofluorescence imaging (Optomap P200Tx) was performed from both eyes preoperatively and early (1-2 days) postoperatively. All patients were operated with 2.5-mm encircling band, 6-mm to 9-mm segmental buckle, transscleral cryopexy, and the choice of drainage and air/gas endotamponade. The mean age of the patients was 58 ± 12 years, and the ratio of macula on-off detachments was 19/26. Light cryopexy induced hyperfluorescence of the treated area (in 11% of cases). Moderate cryopexy resulted in central hypofluorescence with a hyperfluorescent halo (in 51% of cases), whereas extensive cryopexy and disruption of the retinal pigment epithelium resulted in a broad hypofluorescent area (in 36% of cases). Tightening of the indenting elements induced peripheral hyperfluorescent radial streaks in 47% of cases and distinct areas of hyperfluorescence in 58% of cases. Demarcation lines and residual subretinal fluid were observed as hyperfluorescent areas. Central autofluorescence changes were observed in 96% of macula-off surgeries, whereas only 27% of these cases showed distinct hyper- and hypo-autofluorescent streaks. Ultra-widefield fundus autofluorescence imaging is a useful adjuvant tool for evaluating early outcome and retinal pigment epithelium function after scleral buckling surgery for retinal detachment.

Loss of Otx2 in the Adult Retina Disrupts Retinal Pigment Epithelium Function, Causing Photoreceptor Degeneration

Photoreceptors are specialized neurons of the retina that receive nursing from the adjacent retinal pigment epithelium (RPE). Frequent in the elderly, photoreceptor loss can originate from primary dysfunction of either cell type. Despite intense interest in the etiology of these diseases, early molecular actors of late-onset photoreceptor degeneration remain elusive, mostly because of the lack of dedicated models. Conditional Otx2 ablation in the adult mouse retina elicits photoreceptor degeneration, providing a new model of late-onset neuronal disease. Here, we use this model to identify the earliest events after Otx2 ablation. Electroretinography and gene expression analyses suggest a nonautonomous, RPE-dependent origin for photoreceptor degeneration. This is confirmed by RPE-specific ablation of Otx2, which results in similar photoreceptor degeneration. In contrast, constitutive Otx2 expression in RPE cells prevents degeneration of photoreceptors in Otx2-ablated retinas. We use chromatin immunoprecipitation followed by massive sequencing (ChIP-seq) analysis to identify the molecular network controlled in vivo by Otx2 in RPE cells. We uncover four RPE-specific functions coordinated by Otx2 that underpin the cognate photoreceptor degeneration. Many direct Otx2 target genes are associated with human retinopathies, emphasizing the significance of the model. Importantly, we report a secondary genetic response after Otx2 ablation, which largely precedes apoptosis of photoreceptors, involving inflammation and stress genes. These findings thus provide novel general markers for clinical detection and prevention of neuronal cell death.

Engineering a Blood-Retinal Barrier With Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium: Transcriptome and Functional Analysis

Retinal degenerations are a major cause of impaired vision in the elderly. Degenerations originate in either photoreceptors or the retinal pigment epithelium (RPE). RPE forms the outer blood-retinal barrier and functions intimately with photoreceptors. Animal models and cultures of RPE are commonly used to screen potential pharmaceuticals or explore RPE replacement therapy, but human RPE differs from that of other species. Human RPE forms a barrier using tight junctions composed of a unique set of claudins, proteins that determine the permeability and selectivity of tight junctions. Human adult RPE fails to replicate these properties in vitro. To develop a culture model for drug development and tissue-engineering human retina, RPE were derived from human embryonic stem cells (hESCs). Barrier properties of RPE derived from the H1 and H9 hESC lines were compared with a well-regarded model of RPE function, human fetal RPE isolated from 16-week-gestation fetuses (hfRPE). A serum-free medium (SFM-1) that enhanced the redifferentiation of hfRPE in culture also furthered the maturation of hESC-derived RPE. In SFM-1, the composition, selectivity, and permeability of tight junctions were similar to those of hfRPE. Comparison of the transcriptomes by RNA sequencing and quantitative reverse transcription-polymerase chain reaction revealed a high correlation between the hESCs and hfRPE, but there were notable differences in the expression of adhesion junction and membrane transport genes. These data indicated that hESC-derived RPE is highly differentiated but may be less mature than RPE isolated from 16-week fetuses. The study identified a panel of genes to monitor the maturation of RPE.

The p38 MAPK NF-κB pathway, not the ERK pathway, is involved in exogenous HIV-1 Tat-induced apoptotic cell death in retinal pigment epithelial cells

The mechanism through which human immunodeficiency virus (HIV) infection causes retinal disease and the loss of vision in AIDS patients remains unknown. The HIV-1 transactivator protein Tat (HIV-1 Tat) plays a pivotal role in the pathogenesis of HIV-1 infection and is often described as pleiotropic at different concentrations. In a previous study, we demonstrated that the HIV-1 Tat protein can disrupt the barrier function of retinal pigment epithelium (RPE) at 100nM without affecting cell viability. The present study was undertaken to determine if HIV-1 Tat can induce RPE cell death at different concentrations and to determine the mechanism of any observed cell death. The results demonstrated that two RPE cell lines (ARPE-19 and D407) treated with Tat at concentrations of 200nM and above exhibited reduced growth and apoptosis in a dose- and time-dependent manner. The disruption of mitochondrial outer membrane permeabilisation, the activation of caspase-3/7 and 9, the downregulation of Bcl-2, the upregulation of Bax, and the activation of p38 MAPK, ERK and NF-κB were all observed in HIV-1 Tat-treated RPE cells. When exposed to an inhibitor or transfected with siRNA of p38 MAPK and NF-κB, the level of cell death and deregulation of the expression of the apoptotic protein were attenuated. These effects were not observed with an ERK inhibitor or siRNA. Based on these results, we suggest that the induction of apoptosis by HIV-1 Tat in RPE cells may be mediated by p38 MAPK and NF-κB activation and involve the mitochondrial pathway. Therefore, these pathways may provide clues leading to novel therapeutic approaches for the retinopathy induced by HIV infection.

Ultraviolet (UV) and Hydrogen Peroxide Activate Ceramide-ER Stress-AMPK Signaling Axis to Promote Retinal Pigment Epithelium (RPE) Cell Apoptosis

Ultraviolet (UV) radiation and reactive oxygen species (ROS) impair the physiological functions of retinal pigment epithelium (RPE) cells by inducing cell apoptosis, which is the main cause of age-related macular degeneration (AMD). The mechanism by which UV/ROS induces RPE cell death is not fully addressed. Here, we observed the activation of a ceramide-endoplasmic reticulum (ER) stress-AMP activated protein kinase (AMPK) signaling axis in UV and hydrogen peroxide (H2O2)-treated RPE cells. UV and H2O2 induced an early ceramide production, profound ER stress and AMPK activation. Pharmacological inhibitors against ER stress (salubrinal), ceramide production (fumonisin B1) and AMPK activation (compound C) suppressed UV- and H2O2-induced RPE cell apoptosis. Conversely, cell permeable short-chain C6 ceramide and AMPK activator AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide) mimicked UV and H2O2’s effects and promoted RPE cell apoptosis. Together, these results suggest that UV/H2O2 activates the ceramide-ER stress-AMPK signaling axis to promote RPE cell apoptosis.

Intercellular Ca2+ wave propagation in human retinal pigment epithelium cells induced by mechanical stimulation

Ca2+ signaling is vitally important in cellular physiological processes and various drugs also affect Ca2+ signaling. Thus, knowledge of Ca2+ dynamics is important toward understanding cell biology, as well as the development of drug-testing assays. ARPE-19 cells are widely used for modeling human retinal pigment epithelium functions and drug-testing, but intercellular communication has not been assessed in these cells. In this study, we investigated intercellular Ca2+ communication induced by mechanical stimulation in ARPE-19 cells. An intercellular Ca2+ wave was induced in ARPE-19 monolayer by point mechanical stimulation of a single cell. Dynamic changes of intracellular Ca2+ concentration ([Ca2+]i) in the monolayer were tracked with fluorescence microscopy imaging using Ca2+-sensitive fluorescent dye fura-2 in presence and absence of extracellular Ca2+, after depletion of intracellular Ca2+ stores with thapsigargin, and after application of gap junction blocker α-glycyrrhetinic acid and P2-receptor blocker suramin. Normalized fluorescence values, reflecting amplitude of [Ca2+]i increase, and percentage of responsive cells were calculated to quantitatively characterize Ca2+ wave propagation. Mechanical stimulation of a single cell within a confluent monolayer of ARPE-19 cells initiated an increase in [Ca2+]i, which propagated to neighboring cells in a wave-like manner. Ca2+ wave propagated to up to 14 cell tiers in control conditions. The absence of extracellular Ca2+ reduced [Ca2+]i increase in the cells close to the site of mechanical stimulation, whereas the depletion of intracellular Ca2+ stores with thapsigargin blocked the wave spreading to distant cells. The gap junction blocker α-glycyrrhetinic acid reduced [Ca2+]i increase in the cell tiers close to the site of mechanical stimulation, indicating involvement of gap junctions in Ca2+ wave propagation. The P2-receptor blocker suramin reduced the percentage of responsive cells participating in Ca2+ wave spreading beyond the fourth cell tier, showing the necessity of P2-receptors for Ca2+ wave propagation. In disconnected, i.e., subconfluent, ARPE-19 cell clusters Ca2+ wave spreading was considerably less efficient compared to that in confluent ARPE-19 monolayer at the same distances. ARPE-19 cells showed repeatable and robust Ca2+ dynamics after mechanical stimulus. The ARPE-19 cells exhibited two different mechanisms of Ca2+ wave propagation dependent on the cell location: in the cells close to the site of mechanical stimulation the Ca2+ wave propagated mainly through gap junctions and required Ca2+ from both intracellular Ca2+ stores and extracellular media, while farther away the propagation was more dependent on the purinergic receptors and did not require extracellular Ca2+. The proposed method could provide a tool to assess the drug-induced changes in intercellular communication in in vitro assays in human retinal pigment epithelial cells.

Human Bone Marrow Stromal Cells can Differentiate to a Retinal Pigment Epithelial Phenotype when Co-Cultured with Pig Retinal Pigment Epithelium using a Transwell System

Background: There is an increasing interest in generating retinal pigment epithelial (RPE) cells from stem cells for therapy against degenerative eye diseases. Human bone marrow stromal cells (hBMSCs) can be induced to express retinal neuron-specific markers when co-cultured with retinal neurons, however, whether hBMSCs can differentiate into RPE-like cells in a co-culture system has not been clarified. Methods: The induction of hBMSCs into RPE-like cells was performed by combining hBMSCs and pig RPE cells in a transwell system. The biomarkers of hBMSCs-derived RPE cells were determined by quantitative RT-PCR and immunofluorescence. The function of induced cells was assayed by ELISA for secretion of neurotrophic factors. Results: Intracellular pigment granules and many RPE markers existed in hBMSCs-derived RPE cells after co-culturing with pig RPE cells for 14 days. Typical RPE functions, such as phagocytosis of photoreceptor outer segments and secretion of the trophic factors, brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF), were observed in these induced cells. Conclusion: hBMSCs can be induced toward functional RPE cells simply by transwell-based co-culture with RPE cells.

Histology and immunochemistry evaluation of autologous translocation of retinal pigment epithelium‐choroid graft in porcine eyes

To evaluate structure and cellular functionality of retinal pigment epithelium (RPE)-choroid grafts after autologous translocation in porcine eyes. Retinal pigment epithelium-choroid grafts were obtained from the nasal midperiphery donor site and translocated to the central area in 12 pigs (12 eyes). Grafts were placed under the central retina through a retinotomy. Ophthalmoscopic and pathological evaluations were performed immediately (n = 1) and at 15 (n = 3) and 30 (n = 3) days after surgery. Untranslocated nasal RPE-choroid grafts were obtained at time of surgery and used as controls. Specimens were evaluated by standard histology and by immunochemical studies of RPE65, CRALBP and GFAP. Five animals were lost to follow-up owing to surgery or anaesthesia complications. Ophthalmoscopic examination revealed that the grafts remained in place at all time-points studied. Fifteen and thirty days postsurgery, some areas of the transplanted RPE maintained a monolayered structure. Retinal pigment epithelium cells were firmly attached to Bruch's membrane and predominantly preserved polarity and pigment distribution. However, RPE65, CRALBP and GFAP patterns of expression and distribution were diminished and modified during follow-up. Ophthalmoscopic retinal detachment and proliferative vitreoretinopathy (PVR), confirmed by microscopic evaluation, complicated all cases at 30 days of follow-up. Autologous RPE-choroid grafts survived up to 30 days in porcine eyes. Histological and immunochemical evaluation revealed preserved transplanted RPE cells morphology accompanied by alterations in the immunoreactivity expression of functional proteins, and development of significant PVR. The data presented in this manuscript provide insights into the fate, viability and cellular functionality of the transplanted RPE-choroid graft, serving as foundation for further knowledge and improvement of this technique.

Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses’ vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies.

C-Type Natriuretic Peptide Protects the Retinal Pigment Epithelium against Advanced Glycation End Product–Induced Barrier Dysfunction

In diabetic retinopathy, vision loss is usually secondary to macular edema, which is thought to depend on the functional integrity of the blood-retina barrier. The levels of advanced glycation end products in the vitreous correlate with the progression of diabetic retinopathy. Natriuretic peptides (NP) are expressed in the eye and their receptors are present in the retinal pigment epithelium (RPE). Here, we investigated the effect of glycated-albumin (Glyc-alb), an advanced glycation end product model, on RPE-barrier function and the ability of NP to suppress this response. Transepithelial electrical resistance (TEER) measurements were used to assess the barrier function of ARPE-19 and human fetal RPE (hfRPE) monolayers. The monolayers were treated with 0.1-100 μg/ml Glyc-alb in the absence or presence of 1 pM to 100 nM apical atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), or C-type natriuretic peptide (CNP). Glyc-alb induced a significant reduction in TEER within 2 hours. This response was concentration-dependent (EC(50)= 2.3 μg/ml) with a maximal reduction of 40 ± 2% for ARPE-19 and 27 ± 7% for hfRPE at 100 μg/ml 6 hours post-treatment. One hour pretreatment with ANP, BNP, or CNP blocked the reduction in TEER induced by Glyc-alb (100 μg/ml). The suppression of the Glyc-alb response by NP was dependent on the generation of cyclic guanosine monophosphate and exhibited a rank order of agonist potency consistent with the activation of natriuretic-peptide-receptor-2 (NPR2) subtype (CNP >> BNP ≥ ANP). Our data demonstrate that Glyc-alb is effective in reducing RPE-barrier function, and this response is suppressed by NP. Moreover, these studies support the idea that NPR2 agonists can be potential candidates for treating retinal edema in diabetic patients.

In vitro electroretinogram for the study of the functionality of differentiated retinal pigment epithelium cells

The purpose of this study is to develop and test a method to reveal if the retinal pigment epithelium (RPE) cells differentiated from human embryonic stem cells (hESC) support the functions of photoreceptors. hESC-derived RPE (hESC-RPE) cells offer a potent cell source for cell replacement therapy that may be used to prevent certain eye diseases. Methods to assure the functionality of the RPE cells are well warranted. Electroretinograms (ERG) measure the electrophysiological response of the retina to light stimuli. A setup was developed that enables the measurement of ERG in vitro from mice retinas cultured together with hESC-RPE cells. The co-culture of RPE and retinas seems to be a viable tool to assess the functionality of RPE in vitro. However, owing to limited sample size results were somewhat mixed, and thus it was not possible to prove that hESC-RPE cells enhance the ERG response of a mouse retina in vitro. The long-term culturing of the retinas needs to be refined to acquire more conclusive evidence of the supporting role of the RPE and to explore the full potential of the co-culture and ERG methods in assessing RPE functionality.

Generation of hESC-derived retinal pigment epithelium on biopolymer coated polyimide membranes

The in vitro generation of a functional retinal pigment epithelium (RPE) for therapeutic applications requires a limitless source of RPE cells and a supporting scaffold, which improves cell survival and promotes the acquisition of the RPE phenotype. We successfully differentiated human embryonic stem cells (hESCs) toward RPE on a transplantable, biopolymer coated polyimide (PI) membrane. We studied various membrane coatings of which several lead to the generation of a tight and highly polarized epithelium having typical characteristics and functions of human RPE. The cells established a distinctive hexagonal, cobblestone morphology with strong pigmentation, expressed RPE specific genes and proteins, and phagocytosed photoreceptor outer segments (POS) after co-culture with rat retinal explants. The barrier function of hESC-derived RPE (hESC-RPE) monolayers was confirmed by transepithelial electrical resistance and permeability measurements. In conclusion, we show that the PI biomembrane is a suitable scaffold for hESC-RPE tissue engineering.

Assessment of the retinal pigment epithelial functions‐ modelling approach

AbstractPurpose Our purpose is to construct mathematical models of functions of retinal pigment epithelium (RPE). Here we show the development and results of two models: 1) a compartmental model of Ca2+ dynamics 2) finite element model of epithelial transport and trans‐epithelial resistance.Methods A model of Ca2+ dynamics of APRE‐19 cells based on an experimental data and literature was constructed. Ca+2 dynamics of APRE‐19 cells were recorded using fluorescent microscope after mechanical stimulation. Various Ca+2 functional conditions were tested. The computational model constructed (Matlab SimBiology ) comprises over 40 cell function parameters and twelve variables ranging from stretch‐sensitive ion channels to ryanodine receptor dynamics and sarco/endoplasmic reticulum ATPases. Further, a model of RPE epithelia barrier was developed based on finite element modelling (FEM) of the epithelia and trans‐epithelial resistance simulating the spatial distribution of epithelial properties originating of the tight junction distribution on epithelia.Results The Ca+2 kinetics model of RPE was able to reproduce the Ca+2 dynamics of APRE cells with high accuracy with above 0.97 cross‐correlation. Further, including stretch‐sensitive ion channels explained the Ca+2 dynamics of the cells close to the mechanically stimulated cell. The epithelial model showed that epithelial inhomogeneity may play a crucial role in epithelial tightness and even very small cellular changes produce large variation on the measures of the epithelial properties.Conclusion The models constructed provide new insight of the functions of the RPE with applications from drug transport studies to assessment of the functionality of stem cell derived RPE.

How to remove subretinal fluid?

AbstractPurpose Rhegmatogenous retinal detachments (RRD) can be repaired surgically using non‐drainage techniques. However,removal of subretinal fluid (SRF) remains an important surgical step in retinal detachments treated by scleral buckling, particularly in cases of bullous detachments, inferior breaks, proliferative vitreoretinopathy, high myopia, chronic detachments, cases with poor retinal pigment epithelium function and eyes intolerant to sustained intraocular pressure rise.Methods Conventional drainage techniques include passive needle drainage of subretinal fluid through a 3 to 4 mm radial sclerotomy within the bed of the buckle and closure with a preplaced suture. Many modified SRF drainage techniques have been described. Internal subretinal fluid drainage with simultaneous automated air‐fluid infusion is an important part of modern vitreoretinal surgery.Results Needle drainage, where the needle shaft is observed using an indirect ophthalmoscope, seemed to be associated with a higher success rate than conventional drainage. The most common complications of subretinal fluid drainage include subretinal hemorrhage, retinal incarceration, and retinal perforation. Using perfluorocarbon liquid or infusion of low‐molecular weight silicone oil as a peroperative tool, retinal flattening posterior to equator is an important step of the vitreoretinal approach and internal drainage for the treatment of RRD. Subretinal fluid is drained through a preexisting retinal break or an intentionally created drainage retinotomy using an extrusion cannula. It allows the immediate and complete reattachment of the retina and enables laser endocoagulation of the breaks. If the original retinal break can be treated by laser photocoagulation or cryopexy, the subretinal fluid is not necessarily removed completely.

Identification of miRNA Signatures during the Differentiation of hESCs into Retinal Pigment Epithelial Cells

Retinal pigment epithelium (RPE) cells can be obtained through in vitro differentiation of both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We have previously identified 87 signature genes relevant to RPE cell differentiation and function through transcriptome analysis of both human ESC- and iPSC-derived RPE as well as normal fetal RPE. Here, we profile miRNA expression through small RNA-seq in human ESCs and their RPE derivatives. Much like conclusions drawn from our previous transcriptome analysis, we find that the overall miRNA landscape in RPE is distinct from ESCs and other differentiated somatic tissues. We also profile miRNA expression during intermediate stages of RPE differentiation and identified unique subsets of miRNAs that are gradually up- or down-regulated, suggesting that dynamic regulation of these miRNAs is associated with the RPE differentiation process. Indeed, the down-regulation of a subset of miRNAs during RPE differentiation is associated with up-regulation of RPE-specific genes, such as RPE65, which is exclusively expressed in RPE. We conclude that miRNA signatures can be used to classify different degrees of in vitro differentiation of RPE from human pluripotent stem cells. We suggest that RPE-specific miRNAs likely contribute to the functional maturation of RPE in vitro, similar to the regulation of RPE-specific mRNA expression.

Microphthalmia-associated Transcription Factor (MITF) Promotes Differentiation of Human Retinal Pigment Epithelium (RPE) by Regulating microRNAs-204/211 Expression

The retinal pigment epithelium (RPE) plays a fundamental role in maintaining visual function and dedifferentiation of RPE contributes to the pathophysiology of several ocular diseases. To identify microRNAs (miRNAs) that may be involved in RPE differentiation, we compared the miRNA expression profiles of differentiated primary human fetal RPE (hfRPE) cells to dedifferentiated hfRPE cells. We found that miR-204/211, the two most highly expressed miRNAs in the RPE, were significantly down-regulated in dedifferentiated hfRPE cells. Importantly, transfection of pre-miR-204/211 into hfRPE cells promoted differentiation whereas adding miR-204/211 inhibitors led to their dedifferentiation. Microphthalmia-associated transcription factor (MITF) is a key regulator of RPE differentiation that was also down-regulated in dedifferentiated hfRPE cells. MITF knockdown decreased miR-204/211 expression and caused hfRPE dedifferentiation. Significantly, co-transfection of MITF siRNA with pre-miR-204/211 rescued RPE phenotype. Collectively, our data show that miR-204/211 promote RPE differentiation, suggesting that miR-204/211-based therapeutics may be effective treatments for diseases that involve RPE dedifferentiation such as proliferative vitreoretinopathy.