Retinal Epithelial Cells In Culture Research Articles

α1-Microglobulin Binds Illuminated Flavins and Has a Protective Effect Against Sublethal Riboflavin-Induced Damage in Retinal Epithelial Cells.

Riboflavin (vitamin B2) is an important constituent of the prosthetic groups flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which are utilized as electron-carriers in energy metabolism. Excitation by UV-light leads to the generation of riboflavin radicals and reactive oxygen species (ROS), which can oxidize a wide range of biomolecules. The human protein α1-microglobulin (A1M) is a reductase and a radical scavenger, which can protect cells and matrix against oxidative damage. Here, we provide evidence of a molecular interaction between illuminated riboflavin and A1M, similar to the radical scavenging reactions previously seen between A1M and other organic radicals. Binding between riboflavin and A1M was demonstrated by gel migration shift, UV-absorbance and fluorescence spectrum analysis. The reaction between A1M and UV-light illuminated riboflavin involved covalent modification of A1M and proteolytic release of an N-terminal part of the protein. Furthermore, A1M also inhibited the ROS-induced photoreduction reaction of riboflavin, in a reaction involving the free thiol group in position C34. Finally, the results show a protective effect of A1M, analyzed by gene expression rates of stress genes, against sublethal damage in retinal epithelial cells in culture. Together, our results suggest a new role of A1M as a scavenger of riboflavin radicals and ROS produced during illumination of riboflavin.

Nrf2 activators modulate oxidative stress responses and bioenergetic profiles of human retinal epithelial cells cultured in normal or high glucose conditions

Retinal pigment epithelial cells exert an important supporting role in the eye and develop adaptive responses to oxidative stress or high glucose levels, as observed during diabetes. Endogenous antioxidant defences are mainly regulated by Nrf2, a transcription factor that is activated by naturally-derived and electrophilic compounds. Here we investigated the effect of the Nrf2 activators dimethylfumarate (DMF) and carnosol on antioxidant pathways, oxygen consumption rate and wound healing in human retinal pigment epithelial cells (ARPE-19) cultured in medium containing normal (NG, 5mM) or high (HG, 25mM) glucose levels. We also assessed wound healing using an in vivo corneal epithelial injury model. We found that Nrf2 nuclear translocation and heme oxygenase activity increased in ARPE cells treated with 10μM DMF or carnosol irrespective of glucose culture conditions. However, HG rendered retinal cells more sensitive to regulators of glutathione synthesis or inhibition and caused a decrease of both cellular and mitochondrial reactive oxygen species. Culture in HG also reduced ATP production and mitochondrial function as measured with the Seahorse XF analyzer and electron microscopy analysis revealed morphologically damaged mitochondria. Acute treatment with DMF or carnosol did not restore mitochondrial function in HG cells; conversely, the compounds reduced cellular maximal respiratory and reserve capacity, which were completely prevented by N-acetylcysteine thus suggesting the involvement of thiols in this effect. Interestingly, the scratch assay showed that wound closure was faster in cells cultured in HG than NG and was accelerated by carnosol. This effect was reversed by an inhibitor of heme oxygenase activity. Moreover, topical application of carnosol to the cornea of diabetic rats significantly accelerated wound healing. In summary, these data indicate that culture of retinal epithelial cells in HG does not affect the activation of the Nrf2/heme oxygenase axis but influences other crucial oxidative and mitochondrial-dependent cellular functions. The additional effect on wound closure suggests that results obtained in in vitro experimental settings need to be carefully evaluated in the context of the glucose concentrations used in cell culture.

Targeting epithelial-mesenchymal transition.

The epithelial–mesenchymal transition (EMT) is a process in which epithelial cells dedifferentiate to become mesenchymal cells [1–3]. The process requires them to lose their cell polarity, to relinquish their cell–cell adhesion, and to gain migratory and invasive properties. The mesenchymal cells are multipotent stromal cells (mesenchymal stem cells) that can differentiate into various cell types. EMT is essential for the developing embryo to enable mesoderm and neural tube formation. EMT is also necessary for wound healing and other physiological processes. On the other hand, EMT can also be an initiator for metastasis, cancer progression, organ fibrosis, and other pathological conditions. These are disorderly processes. The induction of EMT is complex (Fig. 1) [4]. A fundamental event appears to be the loss of E-cadherin. Transcription factors that can repress E-cadherin directly or indirectly are important to this process. SNAI1/Snail 1, SNAI2/Snail 2 (also known as Slug), ZEB1, ZEB2, E47, and Kruppel-like factor-8 (KLF8) can repress E-cadherin transcription by binding to its promoter. Indirect repressors, such as Twist, Goosecoid, TCF4 (sometimes referred to as immunoglobulin transcription factor 2), homeobox protein SIX1, and FOXC2 (fork-head box protein C2) are able to repress E-cadherin indirectly [5]. Other junction proteins, such as claudins and desmosomes, are also suppressed, facilitating EMT. Various transcription factors such as grainyhead-like protein 2 homologue (GRHL2) and ETS-related transcription factors ELF3 and ELF5 are downregulated during EMT [6, 7]. The induction of EMT is dependent on numerous signaling pathways including TGF-beta, FGF, EGF, HGF, Wnt/beta-catenin, Notch, and hypoxia-induced mechanisms. Ras-MAPK signaling activates Snail and Slug during EMT induction. Slug is involved in disrupting desmosomes, inducing cell spreading, and separating the cell–cell borders [8]. However, EMT involves a second phase that cannot be triggered by Slug. This phase includes the induction of cell motility, repression of the cytokeratin expression, and activation of vimentin expression [9]. Phosphatidylinositol 3′ kinase (PI3K)/AKT-axis activation is emerging as pivotal to EMT. Similarly, hedgehog, nuclear factorkappaB (NF-κB), and activating transcription factor-2 have been implicated [10]. Of course, the EMT processes are specific, so that while Wnt signaling pathways regulate EMT in embryonic development, cardiac valve formation, and cancer, other pathways are important towound healing and fibrosis [11]. EMT is central to two recent J Mol Med contributions. Kimura et al. report on the attenuation of EMT in retinal pigment epithelial cells and subretinal fibrosis by a retinoic-acid receptor-gamma (RAR-γ) agonist [12]. The retinal pigment epithelium belongs the neural retina and is indispensable for vision. In humans, proliferation and transformation (EMT switching) of retinal pigment epithelial cells after injury causes retinal disorders and loss of vision [13]. On the other hand, EMT could develop into a therapeutic target [14]. Subretinal fibrosis contributes to the loss of vision associated with age-related macular degeneration (AMD), a devastating and common disease in older people. The authors began with retinal epithelial cells in culture. When subjected to TGF-β2, the cells expressed mesenchymal markers including α-smooth muscle actin and fibronectin. The cells also released interleukin-6. Paxillin was phosphorylated; MAPK constituents such as ERK, p38, and JNK were activated, as were Smad2 and AKT. A RAR-γ agonist, R667, attenuated the EMT responses. Furthermore, the cells treated * Friedrich C. Luft luft@charite.de

Culture of retinal pigment epithelial cells of rat

目的 探索体外分离、培养大鼠视网膜色素上皮(RPE)细胞的方法,建立其体外培养模式.方法 采用两步酶分层消化法分离获取大鼠RPE细胞,F12培养液培养;生长曲线、细胞分裂时间等指标观察细胞生存活力及增生状态;光镜、免疫组织化学法形态学鉴别及细胞鉴定,电镜观察超微结构.结果 收获的大鼠RPE细胞纯度高,量多,体外生长旺盛,达到融合的时间较短.原代细胞呈多角形,含大量色素;传代细胞呈梭形.电镜显示胞浆内含黑色素颗粒及各种细胞器.结论 两步酶分层消化法是分离培养大鼠RPE细胞的理想方法,培养的细胞可用于RPE的体外实验研究。