primary-culture-of-porcine-retinal-pigment-epithelial-cells

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Xiamen University

Free all--retinal (atRAL) and retinal pigment epithelium (RPE) lipofuscin are both considered to play etiological roles in Stargardt disease and age-related macular degeneration. A2E and all--retinal dimer (atRAL-dimer) are two well characterized bisretinoid constituents of RPE lipofuscin. In this study, we found that, after treatment of primary porcine RPE (pRPE) cells with atRAL, atRAL-dimer readily formed and accumulated in a concentration- and time-dependent manner, but A2E was barely detected. Cell-based assays revealed that atRAL, the precursor of atRAL-dimer, significantly altered the morphology of primary pRPE cells and decreased cell viability at a concentration of 80 μm regardless of light exposure. By contrast, atRAL-dimer was not cytotoxic and phototoxic to primary pRPE cells. Compared with atRAL and A2E, atRAL-dimer was more vulnerable to light, followed by the generation of its photocleaved products. Moreover, we observed the presence of atRAL-dimer in reaction mixtures of atRAL with porcine rod outer segments (ROS), RPE/choroid, or neural retina. Taken together, we here proposed an alternative metabolic/antidotal pathway of atRAL in the retina: atRAL that evades participation of the visual (retinoid) cycle undergoes a condensation reaction to yield atRAL-dimer in both ROS and RPE. Translocation of atRAL, all--retinylidene-phosphatidylethanolamine (NR-PE), atRAL-dimer, and photocleavage products of atRAL-dimer from ROS into RPE is accomplished by phagocytosing shed ROS on a daily basis. Without causing damage to RPE cells, light breaks up total atRAL-dimer within RPE cells to release low-molecular-weight photocleavage fragments. The latter, together with ROS-atRAL-dimer photocleavage products, may easily move across membranes and thereby be metabolically eliminated.

Conversion of all--retinal into all--retinal dimer reflects an alternative metabolic/antidotal pathway of all--retinal in the retina.

Visual (retinoid) cycle anomalies induce aberrant build-up of all-trans retinal (atRAL) in the retinal pigment epithelium (RPE), which is a cause of RPE atrophy in Stargardt disease type 1 and age-related macular degeneration. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation is implicated in the etiology of age-related macular degeneration. Here, we elucidated the relationship between NLRP3 inflammasome activation and atRAL-induced death of RPE cells.

Retinal Pigment Epithelium Cell Death Is Associated With NLRP3 Inflammasome Activation by All-trans Retinal.

The underlying mechanisms of complement activation in Stargardt disease type 1 (STGD1) and age-related macular degeneration (AMD) are not fully understood. Overaccumulation of all-trans-retinal (atRAL) has been proposed as the pathogenic factor in both diseases. By incubating retinal pigment epithelium (RPE) cells with atRAL, we showed that C5b-9 membrane attack complexes (MACs) were generated mainly through complement alternative pathway. An increase in complement factor B (CFB) expression as well as downregulation of complement regulatory proteins CD46, CD55, CD59, and CFH were observed in RPE cells after atRAL treatment. Furthermore, interleukin-1β production was provoked in both atRAL-treated RPE cells and microglia/macrophages. Coincubation of RPE cells with interleukin-1 receptor antagonist (IL1Ra) and atRAL ameliorated complement activation and downregulated CFB expression by attenuating both p38 and c-Jun N-terminal kinase (JNK) signaling pathways. Our findings demonstrate that atRAL induces an autocrine/paracrine IL-1/IL-1R signaling to promote complement alternative pathway activation in RPE cells and provide a novel perspective on the pathomechanism of macular degeneration.

IL-1/IL-1R signaling induced by all-trans-retinal contributes to complement alternative pathway activation in retinal pigment epithelium.

Retinal pigment epithelium (RPE) cell apoptosis arising from all-trans-retinal (atRAL) is in close contact with the etiology of dry age-related macular degeneration (AMD) and autosomal recessive Stargardt's disease (STGD1), but its underlying mechanisms remain elusive. In this study, we reported that c-Jun N-terminal kinase (JNK) activation facilitated atRAL-induced apoptosis of RPE cells. Reactive oxygen species production and endoplasmic reticulum stress were identified as two of major upstream events responsible for activating JNK signaling in atRAL-loaded RPE cells. Inhibiting JNK signaling rescued RPE cells from apoptosis induced by atRAL through attenuating caspase-3 activation leading to poly-ADP-ribose polymerase (PARP) cleavage, and DNA damage response. Abca4Rdh8 mice upon light exposure exhibit rapidly increased accumulation of atRAL in the retina, and display severe RPE degeneration, a primary attribute of dry AMD and STGD1. Reducing JNK signaling by intraperitoneally injected JNK-IN-8 was highly effective in preventing RPE atrophy and apoptosis in light-exposed Abca4Rdh8 mice. These findings afford a further understanding for contribution of JNK activation by atRAL to retinal damage.

Repressing c-Jun N-terminal kinase signaling mitigates retinal pigment epithelium degeneration in mice with failure to clear all-trans-retinal.

The breakdown of all-trans-retinal (atRAL) clearance is closely associated with photoreceptor cell death in dry age-related macular degeneration (AMD) and autosomal recessive Stargardt's disease (STGD1), but its mechanisms remain elusive. Here, we demonstrate that activation of gasdermin E (GSDME) but not gasdermin D promotes atRAL-induced photoreceptor damage by activating pyroptosis and aggravating apoptosis through a mitochondria-mediated caspase-3-dependent signaling pathway. Activation of c-Jun N-terminal kinase was identified as one of the major causes of mitochondrial membrane rupture in atRAL-loaded photoreceptor cells, resulting in the release of cytochrome c from mitochondria to the cytosol, where it stimulated caspase-3 activation required for cleavage of GSDME. Aggregation of the N-terminal fragment of GSDME in the mitochondria revealed that GSDME was likely to penetrate mitochondrial membranes in photoreceptor cells after atRAL exposure. ABC (subfamily A, member 4) and all-trans-retinol dehydrogenase 8 are two key proteins responsible for clearing atRAL in the retina. Abca4Rdh8 mice exhibit serious defects in atRAL clearance upon light exposure and serve as an acute model for dry AMD and STGD1. We found that N-terminal fragment of GSDME was distinctly localized in the photoreceptor outer nuclear layer of light-exposed Abca4Rdh8 mice. Of note, degeneration and caspase-3 activation in photoreceptors were significantly alleviated in Abca4Rdh8Gsdme mice after exposure to light. The results of this study indicate that GSDME is a common causative factor of photoreceptor pyroptosis and apoptosis arising from atRAL overload, suggesting that repressing GSDME may represent a potential treatment of photoreceptor atrophy in dry AMD and STGD1.

Gasdermin E mediates photoreceptor damage by all-trans-retinal in the mouse retina.

Excessive exposure to blue light from smartphones, computers, and other video equipment causes retinal degeneration. Cyanidin-3-glucoside (C3G) exerts protective effects on retinal cells. However, the mechanism by which C3G enhances the barrier function of retinal pigment epithelial (RPE) cells remains unclear. This study investigated the effects of C3G on blue light-irradiated A2E-containing RPE cells and explored whether or not the endoplasmic reticulum (ER) stress and downstream nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways are involved in the mechanism. Results showed that C3G (10 and 25 μM) observably increased the viability and inhibited the apoptosis of RPE cells. Furthermore, C3G enhanced the barrier function of RPE cells and upregulated the expression of tight junction proteins. Blue light irradiation triggered ER stress, but C3G significantly suppressed the PERK/eIF2α/ATF4/CHOP pathway and maintained normal ER morphology in RPE cells. C3G also activated the Nrf2 pathway to promote RPE survival, which was independent of ER stress modulating Nrf2 activity. This study suggests that C3G promotes the barrier function of RPE cells by regulating ER stress-induced apoptosis, thereby offering a new approach to preventing retinal diseases. Thus, C3G is a potential functional food ingredient to improve visual health.

Danxue He

Xiamen University

Primary Culture of Porcine Retinal Pigment Epithelial Cells