Once activated, the intracellular receptor NLRP3 assembles an inflammasome protein complex that facilitates the caspase-1-mediated maturation of IL-1β and IL-18. Inactive NLRP3 is guarded by a protein complex containing Hsp90. In response to stress stimuli, Hsp90 is released, and NLRP3 can be activated to promote inflammation. In this study, we blocked Hsp90 with geldanamycin and studied the fate of NLRP3 in human retinal pigment epithelial (RPE) cells. RPE cells play a central role in the development of age-related macular degeneration (AMD), a progressive eye disease causing severe vision loss in the elderly. IL-1α-primed ARPE-19 cells, human embryonal stem cell (hESC)-derived RPE cells, and primary human RPE cells were exposed to MG-132 and bafilomycin A to activate NLRP3 via the inhibition of proteasomes and autophagy, respectively. Additionally, RPE cells were treated with geldanamycin at different time points and the levels of NLRP3 and IL-1β were determined. Caspase-1 activity was measured using a commercial assay. Geldanamycin prevented the activation of the inflammasome in human RPE cells. NLRP3 released from its protective complex became degraded by autophagy or secreted from the cells. Controlled destruction of NLRP3 is a potential way to regulate the inflammation associated with chronic diseases, such as AMD.

Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.

Purpose: To investigate the mechanism by which resveratrol acts upon retinal pigment epithelial (RPE) cells and to characterize its effect upon autophagy, survival, and inflammation, with consequent implications to treatment for age-related macular degeneration (AMD). Methods: Cultured ARPE-19 cells were exposed to 10 and 50 μM resveratrol. Cell survival/death was determined by annexin-FITC/propidium iodide using flow cytometry, while autophagy was studied by detecting autophagic vacuoles formation (acridine orange and transmission electron microscopy), as well as LC3II/I ratio and p62 expression by Western blot. In addition, time-lapse confocal microscopy of a pDENDRA-LC3 expression vector was performed to detect autophagy in transfected ARPE-19 cells under the different treatment conditions. Inhibition of proteasomal and autophagy-lysosomal fusion was carried out by MG-132 and chloroquine, respectively, while induction of autophagy was achieved by rapamycin treatment. Detection of secreted cytokines by ARPE-19 cells using Human XL Cytokine Array was performed under oxidative stress (H2O2) and resveratrol treatments, respectively. Results: Resveratrol induced autophagy in ARPE-19 cells as determined by augmented presence of autophagic vacuoles, increased LC3II/I ratio and decreased p62 expression, as well as time-lapse confocal microscopy using pDENDRA-LC3 expression vector. Resveratrol acted similarly to proteasomal inhibition and downstream of mammalian target of rapamycin (mTOR), since upstream inhibition of autophagy by 3-methyladenine could not inhibit autophagy in ARPE-19 cells. Co-treatmeant by rapamycin and/or proteasome inhibition showed no additive effect upon autophagy induction. ARPE-19 cells treated by resveratrol showed lower cell death rate compared to untreated controls. Resveratrol induced a specific anti-inflammatory response in ARPE-19 cells. Conclusions: Resveratrol can induce autophagy, pro-survival, and anti-inflammatory stimuli in ARPE-19 cells, properties which could be plausible to formulate future treatment modalities for AMD.

Purpose Retinal detachment (RD) is one of the most frequently diagnosed ophthalmologic conditions requiring prompt surgical intervention. Combination of proper surgical technique and new diagnostic markers, both clinical and molecular, can help improve the diagnosis and prognosis of RD treatment. Methods 12 patients with rhegmatogenous RD (rRD) were included into the study after obtaining patient consent and Regional Ethical Approval (average age: 58.1 ± 17.4 years). OCT was performed before and after 23G vitrectomy for RD. Pure subretinal fluid (SRF) was collected during surgery and analyzed by protein array profiling on a panel of 105 inflammatory cytokines (Human XL Cytokine Array), while the effect of SRF upon human macrophages-driven phagocytosis of apoptotic retinal pigment epithelial (RPE) cells ex vivo was quantified by flow cytometry. Immunohistochemistry (IHC) of retinectomized tissue due to PVR caused by RD was performed to determine presence of markers for microglial cells (CD34), macrophages and activated microglia (CD68), regulator of the immune response to infection (NFkB), progenitor and stem cell marker (Sox2), pluripotency marker (Oct4) and intermediate filament markers (GFAP and Nestin). Results OCT of fresh RD patients contained pre-operatively hyper reflective points (HRPs) at the detached neuroretina border and proximal to the RPE layer—their size and number decreased following successful reattachment surgery. IHC of the retinectomized tissue from detached retina due to severe PVR showed presence of cell conglomerates at the detached neuroretina border which were positive for CD68, NFkB, Sox2 and GFAP, less positive for CD47 and Nestin and negative for Oct4 and CD34. The SRF contained at least 37 cytokines with higher, and 4 cytokine with lower concentration compared to that in vitreous from non-RD pathology; when used as conditional medium to human macrophages ex vivo , the SRF doubled their capacity for engulfing dying RPEs. Conclusions Fresh RD can be hallmarked by presence of HRPs at the detached neuroretina border on OCT; the HRPs decrease in size and number after successful reattachment surgery, and likely resemble the macrophage conglomerates seen by IHC. The neuroretina in RD contains progenitor/stem-like cells and signs of inflammatory reaction, while the SRF contains inflammatory cytokines and other factors which increase the ability of professional phagocytes to engulf dying RPE, or for that matter, other dying cells in the retina.

A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh) stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and proliferative diabetic retinopathy (PDR) fibrovascular epiretinal membranes (fvERMs) from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh) stimulation and indicate presence of ACh receptors in these cells. Furthermore, an ex vivo study model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment.

A chronic low-level inflammation contributes to the pathogenesis of age-related macular degeneration (AMD), the most common cause of blindness in the elderly in Western countries. The loss of central vision results from attenuated maintenance of photoreceptors due to the degeneration of retinal pigment epithelium (RPE) cells beneath the photoreceptor layer. It has been proposed that pathologic inflammation initiated in RPE cells could be regulated by the activation of type 2 cannabinoid receptors (CB2). Here, we have analysed the effect of CB2 activation on cellular survival and inflammation in human RPE cells. RPE cells were treated with the selective CB2 agonist JWH-133 in the presence or absence of the oxidative stressor 4-hydroxynonenal. Thereafter, cellular viability as well as the release of pro-inflammatory cytokines and potential underlying signalling pathways were analysed. Our results show that JWH-133 led to increased intracellular Ca2+ levels, suggesting that RPE cells are capable of responding to a CB2 agonist. JWH-133 could not prevent oxidative stress-induced cell death. Instead, 10 µM JWH-133 increased cell death and the release of proinflammatory cytokines in an ERK1/2-dependent manner. In contrast to previous findings, CB2 activation increased, rather than reduced inflammation in RPE cells.

A B S T R A C TPurpose: Development of ex vivo model to study pathogenesis, inflammation and treatment modalities for pterygium. Methods: Pterygium obtained from surgery was cultivated (3 months). Gravitational attachment method using viscoelastic facilitated adherence of graft and outgrowing cells. Medium contained serum as the only growth supplement with no use of scaffolds. Surface profiling of the multi-layered cells for hematopoietic-and mesenchymal stem cell markers was performed. Examination of cells by immunohistochemistry using pluripotency, oxidative stress, stemness, migration and proliferation, epithelial and secretory markers was performed. The effect of anti-proliferative agent Mitomycin C upon secretion of pro-inflammatory cytokines IL-6 and IL-8 was assessed. Results: Cells showed high expression of migration-(CXCR4), secretory-(MUC1, MUC4) and oxidative damage-(8-OHdG) markers, and low expression of hypoxia-(HIF-1α) and proliferation-(Ki-67) markers. Moderate and low expression of the pluripotency markers (Vimentin and ΔNp63) was present, respectively, while the putative markers of stemness (Sox2, Oct4, ABCG-2) and epithelial cell markers-(CK19, CK8-18) were weak. The surface marker profile of the outgrowing cells revealed high expression of the hematopoietic marker CD47, mesenchymal markers CD90 and CD73, minor or less positivity for the hematopoietic marker CD34, mesenchymal marker CD105, progenitor marker CD117 and attachment protein markers while low levels of IL-6 and IL-8 secretion ex vivo, were inhibited upon Mitomycin C treatment. Conclusion: Ex vivo tissue engineered pterygium consists of a mixture of cells of different lineage origin, suitable for use as a disease model for studying pathogenesis ex vivo, while opening possibilities for new treatment and prevention modalities.

Injury of the cornea is a complex biological process. Regeneration of the corneal stroma can be facilitated by the presence of mesenchymal stromal cells (MSCs) and application of tissue equivalents. A new tissue‐engineering strategy for corneal stroma regeneration is presented using cellularized 3D bioprinted hydrogel constructs implanted into organ cultured porcine corneas using femtosecond laser‐assisted intrastromal keratoplasty. The ex vivo cultured, MSC‐loaded 3D bioprinted structures remain intact, support cell survival, and contain de novo synthesized extracellular matrix components and migrating cells throughout the observation period. At day 14 postimplantation, the cellularized tissue equivalents contain few or no cells, as demonstrated by optical coherence tomography imaging and immunofluorescent staining. This study successfully combines a laboratory‐based method with modern, patient‐care practice to produce a cell‐laden tissue equivalent for corneal implantation. Optimal bioink composition and cellularization of tissue equivalents are essential in fine‐tuning a method to promote the current technique as a future treatment modality.