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.

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 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.