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Purpose The aim of this study was to estimate the 1-year costs associated with treating diabetic macular oedema (DME) patients using current intravitreal anti-vascular endothelial growth factor (anti-VEGF) biologics compared with the dexamethasone implant. Methods We conducted a descriptive cost-evaluation analysis using data from Oslo University Hospital and literature to compare three different intravitreal drugs for DME: bevacizumab, aflibercept and dexamethasone. Stratification of patients into ‘Naive’ or ‘Switch’ group was based on treatment history. We estimated the costs from healthcare and ‘extended’ healthcare perspectives. Sensitivity analysis evaluated the impact of various parameters. Results The average injections per patient per year for the Naive group (bevacizumab), Switch group (aflibercept) and dexamethasone were 9.5, 9.1 and 3.0 respectively. From a healthcare perspective, the 1-year costs for the Naive group were 15% lower (bevacizumab, €3619), and for the Switch group, 23% higher (aflibercept, €5226) compared with dexamethasone (€4252). The ‘extended’ healthcare perspective showed the cost per patient per year for bevacizumab remained nominally lower in the Naive group, while dexamethasone remained lower for the Switch group (€5116 for dexamethasone, compared to €4987 for bevacizumab and €6537 for aflibercept). Conclusions From a primary healthcare perspective, the dexamethasone as a first-line DME treatment may increase economic costs in settings where bevacizumab is used off-label. Treating resistant DMEwith dexamethasone may reduce the costs and treatment burden compared with switching to aflibercept.

PurposeTo investigate the effect of polyquaternium-1 (PQ)-preserved and benzalkonium chloride (BAK)-preserved travoprost eye drops on viability of primary human conjunctival goblet cell (GC) cultures and on secretion of mucin and cytokines. Furthermore, to evaluate the physicochemical properties of the branded travoprost eye drop Travatan® and available generics.MethodsThe effect of travoprost eye drops was evaluated on GC cultures. Cell viability was assessed through lactate dehydrogenase (LDH) and tetrazolium dye (MTT) colorimetric assays. Mucin secretion was evaluated by immunohistochemical staining. Secretion of interleukin (IL)-6 and IL-8 was measured using BD Cytometric Bead Arrays. pH, viscosity, droplet mass, osmolality and surface tension were measured for all included eye drops.ResultsIn the LDH assay, BAK travoprost caused significant GC loss after 2 hrs of incubation compared to the control. PQ travoprost caused no GC loss at any time point. Both PQ- and BAK travoprost caused secretion of mucin to the cytoplasma. No difference in IL-6 and IL-8 secretion was identified compared to controls. The pH values for the generics were lower (pH 6.0) than the pH value for Travatan (pH 6.7; p ConclusionBAK travoprost caused GC loss, indicating that PQ preservation may be preferable in treatment of glaucoma. Furthermore, physicochemical properties of branded and generic travoprost eye drops can not be assumed to be identical.

Purpose To clarify how early in the development of diabetic retinopathy (DR) can oxygen (O2 ) saturation changes be detected. Methods Retinal oximetry was performed in a cross-sectional study, involving 14- to 30-year-old individuals: 185 with type 1 diabetes (T1D) and 94 controls. The subjects were divided into four groups according to the grade of DR. One-way ANOVA and post hoc tests were used to test for differences in the mean O2 saturations between the groups. Results Fifty-eight (31 %) of the T1D patients had nonproliferative DR. There was no significant difference in O2 saturations between controls and T1D patients with no DR. Arteriolar and venular O2 saturations in T1D patients were significantly higher in moderate/severe DR than in no DR (p = 0.009 and p > 0.001), while venular O2 saturation was significantly higher in mild DR than in no DR (p = 0.013). Conclusion Increase in venular O2 saturation could not be detected before mild retinopathy had developed, and the retinal O2 saturation increase was measurable on the venular side first. Our results suggest that the increase in O2 saturation is likely a consequence of DR.

Purpose Dysfunction of the retinal pigment epithelium (RPE) causes numerous forms of retinal degeneration. RPE replacement is a modern option to save vision. We aimed to test the results of transplanting cultured RPEs on biocompatible membranes. Methods We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse on two types of membranes: commercial polyester scaffolds Transwell (Corning Inc., Kenneburg, ME, USA) with 0.4 µm pore size and prepared Poly (L-lactide-co-DL-lactide) (PDLLA) nanofibrous membranes with an average pore size of 0.4 µm. Results Five types of assays were used for the analysis: immunocytochemistry (ICC), phagocytosis assay, Western blotting, real-time qPCR (RT-qPCR) and electron microscopy. RT-qPCR demonstrated that RPEs cultured on nanofibrous membranes have higher expressions of BEST1 (bestrophin 1), RLBP1 (retinaldehyde-binding protein 1), RPE65 (retinal pigment epithelium-specific 65 kDa protein), PAX6 (transcription factor PAX6), SOX9 (transcription factor SOX9), DCT (dopachrome tautomerase) and MITF (microphthalmia-associated transcription factor). ICC of the RPEs cultured on nanofibrous membranes showed more intensive staining of markers such as BEST1, MCT1 (monocarboxylate transporter 1), Na+ /K+ ATPase, RPE65 and acetylated tubulin in comparison with commercial ones. Additionally, the absence of α-SMA proved the stability of the RPE polarization state and the absence of epithelial-to-mesenchymal transition. RPE possessed high phagocytic activity. Electron microscopy of both membranes confirmed a confluent layer of RPE cells and their genuine morphological structure, which was comparable to native RPEs. Conclusions Retinal pigment epitheliums cultured on polylactide nanofibrous membranes improved the final quality of the cell product by having better maturation and long-term survival of the RPE monolayer compared to those cultured on commercial polyester scaffolds. PDLLA-cultured RPEs are a plausible source for the replacement of non-functioning RPEs during cell therapy.

Purpose To determine the structural characteristics of lens epithelial cells (LECs) found on the anterior portion of the lens capsule and their pluripotency, proliferating and migrating potential when grown ex vivo with relevance to posterior capsular opacification (PCO) after cataract surgery. Methods The explants of anterior portion of the lens capsule consisting of monolayer of LECs were obtained from uneventful cataract surgery and were cultivated under adherent conditions. The size and shape of the outgrowing cells were recorded by scanning electron microscopy (SEM), while their migration and proliferation potential were followed using light microscopy. Positivity for proliferation (Ki-67)- and pluripotency (Sox2)-specific markers were tested by immunofluorescent staining. Results The proliferation and migration of anterior portion of the lens capsule's LECs filling up the denuded and reverse side regions of the lens capsule as well as their growth on glass culture surfaces could be followed by light microscopy and SEM, while the distribution of LECs and their morphology could be analysed in detail by SEM. The expression of Ki-67 and Sox2 in LECs growing adherently on human anterior portion of the lens capsule could also be detected. Conclusions Classic light microscopy and SEM can be used to show that human anterior portion of the lens capsule harbours LECs that can proliferate and migrate, suggesting their pluripotency or putative stem cell nature. Similarly, morphological techniques can be used to study PCO and the effect different drugs or physical treatments have against PCO development.

Summary Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) provide a promising cell source for studying retinal development, for disease modelling, and retinal cell replacement therapies. Several research groups, including ours, have demonstrated that hESC-RPE structure, function, and physiology resembles that of native RPE regarding cellular fine structure and expression of many RPE signature genes and proteins. To characterize the hESC-RPE proteome in larger scale, we have compared hESC-RPE protein expression to primary human RPE using isobaric tags for relative quantitation (iTRAQ) technology. The hESC-RPE proteome reflected that of native RPE with a large number of metabolic, mitochondrial, cytoskeletal, and transport proteins expressed. No adverse signs such as increased stress, proliferation, or retinal degeneration-related changes were seen in hESC-RPE, while proteins involved in key RPE functions such as visual cycle and phagocytosis were detected. Proteomics provides valuable tools for validation of hESC-RPE, studying disease mechanisms, and discovery of new biomarkers and therapeutic targets.

Purpose To examine levels of oxidative DNA base damage and expression of selected genes and proteins related to DNA damage repair in human limbal epithelium engineered ex vivo. Methods Cells were expanded from limbal tissue on cell culture‐treated inserts in medium containing fetal bovine serum, recombinant growth factors, hormones and cholera toxin (COM) and in medium with human serum as the single growth‐promoting additive (HS). Cells were analysed after two, three and four weeks in culture for DNA strand breaks and oxidized purine bases (Comet assay using the enzyme formamidopyrimidine DNA glycosylase, Fpg) and for expression of DNA repair enzymes APE1, OGG1 and Polβ by in situ hybridization (ISH) and by immunohistochemistry (IHC). Results Levels of strand breaks were substantial while levels of net Fpg‐sensitive sites (8‐oxoguanine and ring‐opened FaPy bases) were relatively low in cells engineered in COM and in HS. Both types of medium were found to support expression of base excision repair (BER) enzymes APE1, OGG1 and Polβ at the gene level. At the protein level, expression of APE1 and OGG1 was noticeable in both conditions while expression of Polβ was low. Conclusion Our findings indicate low levels of oxidative stress and/or efficient DNA purine base damage repair in human limbal epithelium engineered in a medium with human serum as the single growth‐promoting additive as well as in traditional medium with xenobiotics.

ummary The anatomical and biological properties of stem cells in the eye have been the topic of indepth research over the last few decades. Enormous advances in the cell and tissue isolation and cultivation techniques, as well as molecular characterization of the different cell populations have been achieved, next to the major advances made in the bioscaffolds’ engineering for cell delivery and treatment of eye diseases. Advances in the cell and gene therapy have reached culmination with the human cornea being at the top and holding great promise in treating eye disorders. The eye is indeed a golden mine for stem cells. This session will include lectures from European experts in the field with high international recognition and large collaborative networks. Challenges in the clinical applications of cornea limbal stem cells will be described, along with advances in corneal endothelium engineering for future transplantation applications. In addition, examples of regulating the gene expression towards solving ocular surface diseases will be shown, as well as future directions hallmarking stem cell and cell therapy in ophthalmology.

Age-related macular degeneration (AMD) is attributed to a complex interaction of genetic and environmental factors. It is characterized by degeneration involving the retinal photoreceptors, retinal pigment epithelium (RPE) and Bruch's membrane, as well as alterations in choroidal capillaries. AMD pathogenesis is strongly associated with chronic oxidative stress and inflammation that ultimately lead to protein damage, aggregation and degeneration of RPE. Specific degenerative findings for AMD are accumulation of intracellular lysosomal lipofuscin and extracellular drusens. In this review, we discuss thoroughly RPE-derived mechanisms in AMD pathology.

Purpose: The identification of cells with properties of retinal progenitor cells (RPCs) in the adult human ciliary margin (CM) prompted a number of studies of their proliferative and differentiation potential. One of the remaining challenges is to find a feasible method of isolating RPCs from the patient’s eye. In the human CM, only the iris pigment epithelium (IPE) is easily obtained by a minimally invasive procedure. In the light of recent studies questioning the existence of RPCs in the adult mammalian CM, we wanted to assess the potential of the adult human IPE as source of RPCs. Methods: The IPE were isolated from peripheral iridectomies during glaucoma surgery, and IPE and ciliary body (CB) epithelium were also isolated from post-mortem tissue. Cells were cultivated in sphere-promoting conditions or as monolayers. Whole-tissue samples, undifferentiated and differentiated cells were studied by immunocytochemistry, RT-PCR and transmission electron microscopy. Results: The adult human IPE, like the CB, expressed markers of RPCs such as Pax6, Sox2 and Nestin in vivo. Both sphere-promoting and monolayer cultures preserved this phenotype. However, both IPE ⁄ CB cultures expressed markers of differentiated epithelial cells such as Claudin, microphtalmia-associated transcription factor (MITF) and Cytokeratin-19. Ultrastructurally, IPE spheres displayed epithelial-like junctions and contained mature melanosomes. After induced differentiation, IPE-derived cells showed only partial neuronal differentiation expressing b-III-tubulin, Map-2 and Rhodopsin, whereas no mature glial markers were found. Conclusion: Proliferative cells with some properties of RPCs can be isolated from the adult human IPE by peripheral iridectomies. Yet, many cells retain properties of differentiated epithelial cells and lack central properties of somatic stem cells.

Acta Ophthalmol. 2011: 89: e30–e34 Abstract. Purpose: Failure of retinal pigment epithelial (RPE) cells and macrophages to engulf different dying cells in the retina may result in accumulation of debris and development of age-related macular degeneration (AMD). The dynamics and influence of different treatments on this clearance process can be studied in vitro using human ARPE-19 cells and macrophages as phagocytes modelling dry and wet type of AMD, respectively. Methods: Death through extracellular matrix detachment using polyHEMA-coated surfaces (anoikis) and UV irradiation (apoptosis) was induced in ARPE-19 cells. Two-coloured phagocytic assays were performed to quantify the amount of dying cells phagocytes engulfed (flow cytometry) and for visualization (fluorescent and scanning electron microscopy). The effect of phosphatidylserine inhibition with recombinant annexin-V and glucocorticoid (triamcinolone) treatment on the phagocytic process was tested. Results: The clearance of anoikic and apoptotic cells by nondying ARPE-19 cells over 8 hr of co-incubation increased over time (at 8 hr, over 53% and 35% of the phagocytes contained engulfed dying cells, respectively). The human macrophages engulfed the anoikic and apoptotic ARPE-19 cells with seven and four times lower capacity, respectively. Phosphatidylserine appearance on the dying cells did not affect, but triamcinolone treatment enhanced the phagocytosis of the dying cells by macrophages. Conclusions: ARPE-19 cells are more efficient in clearing anoikic than UV-induced apoptotic cells. Macrophages are less efficient in the clearance process than ARPE-19 cells. The present model can be used for studying both dry and wet type of AMD in vitro and for testing different pharmacological aspects affecting this disease.

Purpose To compare the entry site and study the cellular content of different needle tip aspirates after transscleral intravitreal injection (IVI) on rat eyes. Methods The intravitreal injections (IVI) were performed on 20 white outbred rat eyes (10 IVI with 30-gauge subcutaneous needles (SCN), 10 with 27-gauge Pencan needle (PCN) (B. Braun)). The 1.0 cc syringes were preloaded with 0.02 cc of balanced salt solution (BSS) and connected to the needles. The penetration was performed 1 mm posterior to the limbus, followed by aspiration of 0.01 cc vitreous body. Aspirated material was evacuated onto glass slides and stained by Azure-2-Eosin. Enucleation and histological analysis of the IVI entry site was performed at magnification 100 and 400 times. Results Cellular content of the aspirated material was revealed in all cases. The aspirated cells represented conjunctival epithelial-, ciliary body non-pigmented epithelial-, sclerocyte-like cells and vitreous crystallised specimens. The amount of conjunctival epithelial cells prevailed in 27-gauge PCN IVI cases. The stained granular proteins were less significant in the case of 27-gauge PCN tips. The entry sites after 30-gauge SCN injection showed concrete cut of all tissues, while partial reassembling of the sclerocyte bindings was seen after 27-gauge PCN injections. Conclusions The use of 30-gauge SCN and 27-gauge PCN needles for transscleral IVI has resulted in trauma of all layers of the rats’ eye wall. Histological analysis of the needle tip aspirates showed less tissue damage by 27-gauge PCN; moreover, the SCN tips created complete cuts due to their sharp edges, in contrast to the PCN tips.

Purpose Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional and non-professional phagocytes can result in debris formation and development of age-related macular degeneration (AMD). We aimed to study the clearance of autophagic dying RPE cells serving as a model for dry and wet type of AMD, respectively. Methods Autophagic cell death was induced by serum deprivation and H2O2 co-treatment in ARPE-19 and primary human RPE (hRPE) cells. Annexin-V FITC/PI labelling was used to determine the cell death rate, while autophagy detection was achieved by Western blot quantification of LC3 II/LC3 I ratio. The clearance of autophagic dying cells by non-professional and professional phagocytes was quantified using flow cytometry. Results An increasing percentage of dying RPE cells was observed in a time- and concentration dependent manner upon H2O2 treatment. Paralelly, an induction of autophagy could be detected within 2hrs of H2O2 treatment. Phagocytosis studies found autophagic dying cells to be efficiently and increasingly engulfed by both professional and non-professional phagocytes over time. Conclusion The clearance of autophagic dying RPE cells can be used as a model for studying both dry and wet type of AMD in vitro, as well as for testing future pharmacological agents for treating this disease.

Purpose To test the proliferating potential and characterize the functional and molecular profile of cells growing out of human idopathic epiretinal membranes (iERMs) ex vivo. Methods iERMs were obtained from uneventful vitrectomies from cases with iERM. Preoperative and postoperative BCVA, CFT and TMV were compared. Ex vivo cultivation under adherent conditions was carried out and followed up to 6 days. Morphological and immunocytochemical characterization of the cells was carried out. The dynamics of the intracellular calcium was measured using fluorescent dye Fura-2 and imaged in response to pharmacological stimulation by acetylcholine (ACh) or mechanostimulation. Results Visual acuity improved in 9/10 patients. CFT and TMV dereased significantly 3 months after surgery. The cells from the iERMs formed sphere-like structures when cultured ex vivo. The diameter of the spheres increased by 5% at day 6 and kept an increasing tendency over a month. The cells growing out of the iERM spheres had mainly a glial- and some neuronal- like morphology. Stimulation of these cells with ACh or mechanostimulation induced intracellular calcium propagation in both cell phenotypes – in the neuronal-like cells resembling action potential from the soma to the dendrites. Immunocytochemistry confirmed presence of glial- and neuronal cell phenotype in the iERMs, as well as presence of pluripotency marker. Conclusion iERMs contain cells of neuronal- and glial- like origin which have proliferative and pluripotent potential, show functionality reflected through calcium dynamics upon ACh and mechano- stimulation, and a corresponding molecular phenotype.

Purpose We investigated the ultra-structural changes in the distribution of collagen fibrils (CF) and proteoglycans (PGs) due to hydration in the human corneal stroma. Methods Six fresh human scleral corneal rings were hydrated separately in deionized water for 2hrs and 48hrs. Corneas were divided into two parts. The first part of each cornea was fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy to assess proteoglycans. A second part of each cornea was fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer and post fixed in 1% osmium tetroxide to assess collagen fibril diameter. Results In the hydrated corneas the mean diameter of CF in the anterior stroma (23.40±2.61nm), middle stroma (22.48±3.28nm) and posterior stroma (23.58±2.46nm) was less than the CF diameter in the anterior (28.27±4.05nm), middle (28.22±3.09nm) and posterior stroma (29.73±3.72nm) of the normal cornea. Micro-fibrils within the CF in the hydrated cornea were degenerated. In the hydrated cornea, the mean area of the PGs in the anterior stroma was reduced whereas it increased in the middle stroma compared to those in the normal cornea. The density of the PGs in the hydrated cornea was reduced in the anterior, middle and posterior stroma compared to those in the normal cornea. Conclusion The effect of hydration on the uniform organisation of CF and PGs was higher in the middle and posterior stroma than in the anterior stroma. This could be due to less penetration of water in the interlacing lamellae of the anterior stroma. The reduction in the collagen fibrils diameter could be due to degeneration of microfibrils within the collagen fibrils.

Purpose Purpose Stem cells in the central part of the cornea serve an important regenerative and homeostatic function. We aimed to describe the genetic fingerprint of these cells and compare that to limbal epithelial stem cells (LESC) and bone marrow-derived MSCs (bmMSCs). Methods Corneal tissue and bmMSCs were harvested from cadavers and healthy donors, respectively (according to the Guidelines of the Helsinki Declaration) and cultured ex vivo. MSC-specific cell surface markers- and genome-wide microarray analysis were performed using FACS and Affymetrix GeneChip Human Gene 1.0 ST Array (~23,000 gene transcripts). Results Genes related to stemness (228), differentiation and lineage (220), cell cycle (108) and HOX, SOCS, Notch signaling (218) were collected into functional groups and clustered hierarchically: 45 genes were found to be specific for corneal stroma (CS)-MSCs, 62 for LESCs and 9 for bmMSCs. The hierarchical clustering clearly separated the CS-MSCs from the LESCs and bmMSCs, but formed a higher cluster with the later. The top 10 genes related to the differences were VCAM1, FNDC1, MFAP5, SFRP2, IGF2, MMP, ITGA2, COLEC12, SEMA3A and MGARP. Number of molecules functioning in cellular movement (381), cellular growth and proliferation (408), development (370) and cellular development (360) were found with top biological functions in CSMSCs compared to LESCs or bmMSCs (p

To confirm that human corneal rims left over from DALK/DSEK/PK surgeries could be useful sources for ex vivo endothelial cell expansion. Human corneal rims remaining from DALK/DSEK/PK surgeries were utilized (1:1 sex ratio, age 63+20 years, endothelial cell density >2,500 cells/mm2). The time from death to use varied between 3 days and 1.5 months. Endothelial cells isolated using a two-step, peel-and-digest method, whereby the Descemet’s membrane and endothelial cells were peeled off under a dissecting microscope, followed by digestion in collagenase. The isolated cells were suspended in TrypLE prior to plating onto FNC-coated tissue culture plates. The cells were then cultured in Ham’s F12:M199 (1:1) media supplemented with, ascorbic acid, transferrin, sodium selenite and bFGF. Characterisation of the cultured cells was performed by RT-qPCR and immunofluorescence staining accordingly. The number of isolated endothelial cells was repeatedly low (< 20,000 cells). However, improved techniques allowed to reduce stromal cell contamination. It was observed that endothelial cell proliferation was improved when the culture surface area was reduced. Furthermore, typical endothelial cobble stone morphology was observed when the cell density was high. Cell morphology and growth showed notable difference related to donor age and preservation time. ZO-1, Na/K-ATPase and PITX2 were used to confirm the endothelial phenotype. Preserved human corneal rims can be utilized for ex vivo expansion of corneal endothelial cells but further optimization is needed.

Purpose The proliferation and migration of lens epithelial cells (LECs) that remain in the capsular bag after cataract surgery can lead to development of posterior capsule opacification (PCO) - a major cause of post-operative visual loss. The multipotency of anterior lens capsule (LC) LECs is still controversial. We aimed to test the proliferating and migrating potential and to characterize structurally the ex vivo cultured LECs growing out off human anterior LC. Methods The explants of LCs were obtained from uneventful cataract surgery and consisted of basal membrane and monolayer of LECs that were cultivated under adherent conditions. The size and shape of the outgrowing cells were recorded by scanning electron microscopy (SEM) and their migration and proliferation potential were followed on a daily basis throughout their continued growth using light microscope. Results The migration and proliferation of anterior LC LECs filling up the denuded region of the LC could be followed by light microscopy. These cells could also migrate to the opposite side of the capsule as shown by both, light microscopy and SEM. The migration and proliferation of LECs on glass or plastic culture surfaces could be shown by both techniques, while the distribution of the LECs and their morphology could be analyzed in details by SEM. Conclusion Classic light microscopy and SEM studies can be used to show that human anterior LC harbors LECs that can migrate and proliferate, suggesting their multipotency and putative stem cell nature. The anterior LC explants can be used to study PCO and the potential of different pharmaceutical or physical treatments against PCO development.

Purpose To characterize the cell surface marker phenotype and function of ex vivo cultured cells growing out of human epiretinal membranes (ERMs) from proliferative diabetic retinopathy (PDR). Methods All tissue collection complied with the Guidelines of the Helsinki Declaration. ERMs were obtained from vitrectomies due to intravitreal hemorrhage in PDR. Ex vivo cultivation under adherent conditions was performed in DMEM supplemented with FBS and the cell surface marker phenotype determined. Release of IL-6, IL-8 and TNFalpha was measured upon activation of the cells with TLR lingands and TNFalpha. The dynamics of the intracellular calcium was measured using fluorescent dye Fura-2 and imaged in response to mechano-stimulation. Results The cultivated ERMs formed proliferating cell monolayers when cultivated ex vivo. These cells were negative for endothelial markers (CD31, VEGFR2), partially positive for hematopoietic (CD34, CD47) and mesenchymal markers (CD90, PDGFRb, CD73) and negative for CD105. IL-6, IL-8 and TNFalpha secretion could be measured upon activation of the cells by LPS, Poly I:C and TNFalpha. Mechano-stimulation of the outgrowing cells induced intracellular calcium propagation representing their functional viability. Conclusion ERMs from PDR contain cells of hematopoietic and mesenchimal origin which have proliferative potential, release pro-inflammatory cytokines upon selective inflammatory stimulation and show functionality reflected through calcium dynamics upon mechano-stimulation.

Purpose The clearance of dying cells in the retina has known relevance to development of age-related macular degeneration (AMD). We aimed to investigate the gene expression patterns present during such a clearance process and the effect of triamcinolone (TC) treatment on them. Methods Primary human retinal pigment epithelium (hRPE) and ARPE-19 cells were induced to undergo cell death by loss of extracellular matrix attachment (anoikis). Phagocytic clearance assays of the engulfment of anoikic cells by human monocyte-derived macrophages (HMDMs) were performed in the presence or absence of TC. Phagocytosis was quantified using a standard double-staining method on flow cytometry. TaqMan low-density array determining the gene expression of known markers of phagocytosis was carried out in HMDMs engulfing anoikic cells. Loss-of-function studies using siRNA were also performed on selected apopto-phagocytic genes. Results The glucocorticoid TC had a profound phagocytosis-enhancing effect on HMDMs engulfing anoikic hRPE or ARPE-19 cells, causing a selective upregulation of the Mer tyrosine kinase (MERTK) receptor, while decreasing the expression of the AXL receptor tyrosine kinase and THSB-1 (phagocytosis bridging molecule). Similar expression patterns were also observed in the anoikic cells. The key role of the MERTK could be demonstrated in HMDMs engulfing the dying cells using gene silencing as well as blocking antibodies. Conclusion Specific agonists of the tyrosine kinase receptors may have a potential role as phagocytosis enhancers in the retina and serve as future targets for AMD therapy.

Purpose Mesenchymal stem cells (MSC) are the stromal cells of bone marrow, but they can be also found in other tissues including the cornea. Our goal was to isolate and cultivate human corneal stroma MSC-like cells (CSMSCs) and study their role in immunity and wound healing. Methods Corneal buttons were harvested from cadavers (according to the Guidelines of the Helsinki Declaration). The isolated stromal cells were cultured ex vivo in human serum containing medium. Fluorescent microscopy, FACS and gene array analysis, as well as standardized in vitro differentiation assays were performed to investigate the stemness and phenotype of the CSMSCs. To investigate the immunosuppressive function of these cells, mitogen activated lymphocyte reaction and activation by pro-inflammatory cytokines were used. Results According to the definition of the ISCT, the most important MSC markers (CD73, CD90 and CD105) were highly expressed on the surface of the CSMSCs with the absence of endothelial or hematopoietic cell markers. The CSMSCs were able to differentiate into fat, bone, cartilage tissues and close wounds within 24 hrs in vitro. They could suppress the proliferation of activated peripheral blood lymphocytes and secrete suppressive cytokines upon pro-inflammatory activation. Conclusion We demonstrate a method for isolating and cultivating MSC-like cells from human corneal stroma. The ex vivo data suggest that these cells may have a role in wound healing and immunological processes in the eye that can possibly be used in future treatments of ocular diseases and corneal stroma injuries.

Purpose Besides bone marrow, mesenchymal stem cells (MSCs) can be separated from various tissues including the cornea. We investigated whether human corneal stroma-derived cells resemble MSCs and if they can be grown in animal-materials free medium containing human serum as the only supplement. Methods Human cornea stromal cells were isolated from cadavers after removal of the epithelial and endothelial layers (approved by the Hungarian Regional Ethical Committee). The cells were grown in DMEM containing human serum as the only supplement. Immunophenotyping with MSC markers, integrins/cell-adhesion-, endothelial- and hematopoietic markers was carried out by FACS analysis. Standard manufacturer protocols were used for differentiating the cells into fat, cartilage or bone. Results Cells isolated from human corneal stroma grew as monolayers in vitro and could be maintained in culture for more than 10 passages (n=6). They expressed the most important markers for MSCs (CD73, CD90, CD105, CD44, CD147, PDGFRb) and were negative for the hematopoietic markers CD34, CD45, HLA-DR, CD69 and CD133. High per cent of the cells expressed the pluripotency markers CD117, C-kit and CD47, but not the endothelial cell markers CD31, CD105/V-CAM, VEGFR2. One of the hallmarks of human MSCs being capable to differentiate towards adipocytes, chondrocytes and osteocytes could also be demonstrated. Conclusion Our results indicate the presence of MSC-like cells in the human corneal stroma, which can be grown in human serum-containing medium. This opens the door for studying human keratopathies, as well as corneal tissue engineering and cell-based therapies.

Purpose Proliferative vitreoretinopathy (PVR) is an important cause of retinal diseases such as retinal detachment (RD). In lower vertebrates, retinal damage is known to activate neuroepithelial stem cells (NSCs) in the ciliary margin in an attempt to regenerate the neuroretina. Cells expressing some markers of NSCs are also present in the retina and the ciliary body epithelium (CB) of the adult human eye. We hypothesized that if NSCs exist in the adult human eye, they should be activated by PVR formation. Methods Cells isolated from vitreous samples (n=25) obtained during vitrectomies for RD were directly fixed or cultured in a stem cell-promoting medium, and compared to cells isolated from post mortem CB and peripheral retina (PR) using sphere-forming assay, immunohistochemistry, molecular biology and electron microscopy. Markers of NSCs were also studied in whole retinal control/PVR sections obtained from enucleations. Results Spheres formed in 7/10 vitreous samples from patients with PVR compared to 2/15 samples from patients with no known PVR. These spheres stained for markers of NSCs both in vivo and after repetitive passages. Their mRNA and immunohistochemical profile resembled sphere-forming cells from the PR with only a few characteristics of CB cells. In situ characterization of the CB revealed that although there were higher numbers of dividing cells in PVR eyes than in controls, we did not detect markers of NSCs. Interestingly, markers of NSCs were evident around PR cysts with some evidence of activation following PVR formation. Conclusion A population of NSC-like cells are found in the vitreous of patients with PVR. These cells seem to be originated from the retina itself and not the CB.

Purpose To investigate the effect of the polyphenol resveratrol (3,5,4'-trihydroxy-trans-stilbene), rapamycin (RAP) and proteasome inhibitor MG-132 on cell death and autophagy in human retinal pigment epithelium-derived ARPE-19 cells. Methods ARPE-19 cells were exposed to different treatment regimens: 10-50microM resveratrol, 50-100 nM RAP, 50 microM chloroquine (CQ) and 50-100 nM MG-132 over 48 hours. The levels of LC3-II, mammalian target of rapamycin (mTOR), Hsp70, p62 were determined by Western blot analysis; autophagic vacuoles (AVs) formation was detected by acridine orange, pDendra2-hLC3 expression and transmission electron microscopy; cell death was quantified using annexin-V-FITC/propidium iodide (PI) labeling on flow cytometry. Results Exposure to RAP and MG-132 caused a time- and concentration dependent induction of autophagy that could be inhibited by 3-methyladenine (3-MA), while the induction of an active autophagic flux could be verified with CQ treatment, a blocker of the autophagosome-lysosome fusion. Similarly, resveratrol alone could induce autophagy in ARPE-19 cells, serving as a pro-survival signal in ARPE-19 cells. Inhibition with 3-MA increased the death rate of resveratrol treated ARPE-19 cell, further proving the autophagy-related protective role of resveratrol. Conclusion Resveratrol at lower concentrations, RAP and MG-132 can provide a pro-survival stimulus to ARPE-19 cells by inducing autophagy. This property can possibly be used for prolonging the lifespan of retinal pigment epithelium in diseases such as age-related macular degeneration.

Purpose The pathogenesis of age-related macular degeneration involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin-proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Recently, p62/sequestosome 1 (p62) has been shown to be a key player linking the proteasomal and lysosomal clearance systems. In the present study, the effects of AICAR (AICA ribonucleotide, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) and MG-132 (proteasome inhibitor) on autophagy regulation in ARPE-19 cells were evaluated. Methods The AMP activated protein kinase (AMPK), p62 and ubiquitin protein levels were analyzed by western blotting. pDendra2-hLC3 construct was used to detect macroautophagy in confocal microscopy analysis. Transmission electron microscopy was used to detect protein aggregates and autophagosomes. Cellular permeability was measured by analyzing lactate dehydrogenase levels in culture medium. Results MG-132 (5 microM) triggered the accumulation of perinuclear aggregates that strongly colocalized with p62 and ubiquitin. AICAR (2mM) induced autophagy clearance of p62 and ubiquitin positive protein aggregates without increasing cellular permeability. Cellular energy status regulator AMPK or p-AMPK levels were not significantly changed in response to AICAR treatment. Conclusion Our findings open new avenues for understanding the mechanisms of proteolytic processes and indicate that AICAR could be useful in the acceleration of protein clearance in RPE cells.

Failure of retinal pigment epithelial (RPE) cells and macrophages to engulf different dying cells in the retina may result in accumulation of debris and development of age-related macular degeneration (AMD). The dynamics and influence of different treatments on this clearance process can be studied in vitro using human ARPE-19 cells and macrophages as phagocytes modelling dry and wet type of AMD, respectively.Death through extracellular matrix detachment using polyHEMA-coated surfaces (anoikis) and UV irradiation (apoptosis) was induced in ARPE-19 cells. Two-coloured phagocytic assays were performed to quantify the amount of dying cells phagocytes engulfed (flow cytometry) and for visualization (fluorescent and scanning electron microscopy). The effect of phosphatidylserine inhibition with recombinant annexin-V and glucocorticoid (triamcinolone) treatment on the phagocytic process was tested.The clearance of anoikic and apoptotic cells by nondying ARPE-19 cells over 8 hr of co-incubation increased over time (at 8 hr, over 53% and 35% of the phagocytes contained engulfed dying cells, respectively). The human macrophages engulfed the anoikic and apoptotic ARPE-19 cells with seven and four times lower capacity, respectively. Phosphatidylserine appearance on the dying cells did not affect, but triamcinolone treatment enhanced the phagocytosis of the dying cells by macrophages.ARPE-19 cells are more efficient in clearing anoikic than UV-induced apoptotic cells. Macrophages are less efficient in the clearance process than ARPE-19 cells. The present model can be used for studying both dry and wet type of AMD in vitro and for testing different pharmacological aspects affecting this disease.

Purpose To develop simple, reproducible, animal-materials free method for cultivating limbal stem-cells and differentiating them into corneal epithelium on different human biologic materials for clinical transplantation. Methods The limbal tissues (2x2mm) were harvested from cadavers not more than 8 hours after death and proliferated in vitro on cell culture tissue plates, human amniotic membranes (HAM) or human lens capsules in medium containing human AB serum. Cell viability was tested using the MTT assay and annexin-FITC/Propidium Iodide positivity methods. Molecular gene and immunofluorescent marker studies for stemness, proliferation and differentiation were used for the analysis. Results Over a period of one year, 50 limbal tissue explants were cultivated. Emergence of cells at one edge of the explants occurred within 24 hours from culturing and formed monolayer within 14 days. Although the speed of cell growth varied among donors and types of media for growth, inadequate growth at two weeks was never recorded. The viability of the cells at 7 and 14 days of cultivation was higher than 96% except in case of HAM use where viability was below 80%. The growing cells were characterized for their positivity for stemness (P63, ABCG2), proliferation (ki67) and epithelial cell markers CK 3, 8, 12, 14, 18 and 19. Conclusion We demonstrate a simple, animal-materials free technique for generating corneal epithelium from cadavers or alternatively from autologous donors for viable cell growth on different biologic materials for transplantation. The growth of corneal epithelium on lens capsules proved to be superior compared to the other cultivation techiques.

Purpose To report a detailed spectroscopic analysis of explanted hydrophilic acrylic intraocular lenses (IOLs) that were removed because of postoperative opacification of the lens optic. Methods Thirteen Hydroview H60M (Bausch & Lomb Surgical) IOLs were explanted from 13 different patients on average 56 months after phacoemulsification and IOL implantation. All patients had decreased visual acuity because of a fine granularity of the optical surface of the IOLs. The surface was investigated by gross, microscopic, histochemical and scanning electron microscopic analysis, and the elemental composition of the opacified IOLs was determined by X-ray fluorescence spectroscopy (XRF). The spectrograms were compared to three different originally packed and never-implanted hydrophilic acrylic IOLs. Results Light and scanning electron microscopy of the optical surface of explanted IOLs revealed multiple fine granular deposits varying in size and shape that were positive for alizarin red. XRF confirmed that the explanted IOLs contained not only the previously reported calcium and phosphorous (calcium apatite), chlorine, silicone, sodium, aluminum and magnesium but also iron, sulfur, potassium as well as lesser amounts of iodine, zinc, strontium and yttrium. Conclusion This is the first spectroscopic analysis determining the content of more than 10 elements of explanted and originally packed never-implanted hydrophilic acrylic IOLs. The possible origin of the different elements obtained from the spectrograms and their implications are discussed.

Purpose The retina is the place with the highest daily phagocytic turnover in the whole human body. Besides photoreceptor outer segments, retinal pigment epithelial (RPE) cells can engulf other dying cells as well (epithelial, neural, etc.). Failure to do so may result in accumulation of debris that could lead to development of age-related macula degeneration (AMD). The in vitro dynamics of this clearance process can be modelled using human ARPE-19 cells and macrophages. Methods Different death patterns were induced in vitro in ARPE-19 cells: death through detachment from the extracellular matrix on polyHEMA coated surfaces known as anoikis, UV induced apoptosis and Argon-laser induced necrosis. Two-colored phagocytic assays were carried out where different phagocytes (living ARPE-19 cells or human macrophages) engulfed different dying cells. Flow cytometry (FACS Calibur), fluorescent and time-lapse microscopy (the later not shown) were used to quantify and visualize the phagocytic process. Results The clearance of the anoikic ARPE-19 cells by the living ARPE-19 cells (serving as a model for dry AMD) over 8 hours of co-incubation proved efficient and increasing over time (at 8 hours, over 50% of the phagocytes contained engulfed anoikic ARPE-19 cells inside). The human macrophages could also engulf the anoikic ARPE-19 cells (serving as a model for wet AMD), although less efficiently at five times lower rate over the same period. Conclusion The clearance of the different dying ARPE-19 cells can serve as a good in vitro model for studying AMD, both dry and wet type, as well as for testing different immunological and pharmacological aspects affecting this process.