Ex vivo adherent cultures for ocular disease modeling, characterisation and inflammation studies

Development of ex vivo models to study pathogenesis, inflammation and treatment modalities for different eye disorders such as pterygium, cataract and fibrovascular epiretinal membranes (fvERMs) from proliferative diabetic retinopathy (PDR) is much needed. A novel, simple, and reproducible method for cultivating such pathological tissues obtained from human eyes during surgery was hereby developed using viscoelastic material as a tissue adherent to facilitate cell attachment. Pterygium obtained from surgery was cultivated until reaching 3D stratification over long periods of cultivation ex vivo (3 months). Surface profiling of the multi-layered outgrowing cells from the pterygium was performed to test for presence of hematopoietic- and mesenchymal- stem cell markers, as well as determine the origin of the outgrowing cells. Examination of these cells by immunohistochemistry using pluripotency, oxidative stress, stemness, migration and proliferation, epithelial and secretory markers was also performed, while the effect of anti-proliferative agent Mitomycin C upon secretion of pro-inflammatory cytokines IL-6 and IL-8 was assessed. The 3D outgrowing cells from the pterygium 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 weakly expressed. 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. The 3D outgrowing cells secreted low levels of IL-6 and IL-8 ex vivo, which were inhibited upon Mitomycin C treatment. The 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. Furthermore, anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and fvERMs from human eyes undergoing vitrectomy for PDR were used. The aLC-LECs and the fvERM cells were expanded ex vivo in a 2D-monolayer culture. The aLC-LECs underwent calcium imaging upon challenge by mechanical and acetylcholine (ACh) stimulation, while the fvERM outgrowing cells underwent inflammatory studies. The aLC-LECs stimulated by mechanical and ACh treatment showed active calcium signaling and presence of ACh receptors in these cells. The fvERMs could be activated to undergo an inflammatory response upon TNFα treatment, which can surve as an ex vivo study model for studying PDR and inflammation in vivo –like conditions

Cultivation and characterization of pterygium as an ex vivo study model for disease and therapy

PURPOSE: 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-1alpha) and proliferation- (Ki-67) markers. Moderate and low expression of the pluripotency markers (Vimentin and DeltaNp63) 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

AEP319 – Angiotensin-converting enzyme gene (I/D) polymorphism in association with the diabetic nephropathy

Polymorphisms in the Angiotensin-converting enzyme (ACE) gene have been associated with development of Diabetic nephropathy (DN), a major microvascular complication of the Type 2 Diabetes mellitus (T2DM). Since the genetic predisposition plays an important role in development of DN in patients with T2DM, genetic testing might largely contribute to better assessment of the risk of DN in such patients. The aim of this study is to investigate the association of the ACE gene’s I/D polymorphism with DN in T2DM patient

Clinical and molecular markers in retinal detachment: From hyperreflective points to stem cells and inflammation

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A simple method for establishing adherent ex vivo explant cultures from human eye pathologies for use in subsequent calcium imaging and inflammatory studies

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 TNFalpha treatment

Resveratrol as Inducer of Autophagy, Pro-Survival, and Anti-Inflammatory Stimuli in Cultured Human RPE Cells.

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).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.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.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

Biomacromolecular Profile in Human Primary Retinal Pigment Epithelial Cells—A Study of Oxidative Stress and Autophagy by Synchrotron-Based FTIR Microspectroscopy

Synchrotron radiation-based Fourier Transform Infrared (SR-FTIR) microspectroscopy is a non-destructive and chemically sensitive technique for the rapid detection of changes in the different components of the cell’s biomacromolecular profile. Reactive oxygen species and oxidative stress may cause damage to the DNA, RNA, and proteins in the retinal pigment epithelium (RPE), which can further lead to age-related macular degeneration (AMD) and visual loss in the elderly. In this study, human primary RPEs (hRPEs) were used to study AMD pathogenesis by using an established in vitro cellular model of the disease. Autophagy—a mechanism of intracellular degradation, which is altered during AMD, was studied in the hRPEs by using the autophagy inducer rapamycin and treated with the autophagy inhibitor bafilomycin A1. In addition, oxidative stress was induced by the hydrogen peroxide (H2O2) treatment of hRPEs. By using SR-FTIR microspectroscopy and multivariate analyses, the changes in the phosphate groups of nucleic acids, Amide I and II of the proteins, the carbonyl groups, and the lipid status in the hRPEs showed a significantly different pattern under oxidative stress/autophagy induction and inhibition. This biomolecular fingerprint can be evaluated in future drug discovery studies affecting autophagy and oxidative stress in AMD