α-Melanocyte-stimulating hormone prevents glutamate excitotoxicity in developing chicken retina via MC4R-mediated down-regulation of microRNA-194.

Glutamate excitotoxicity is a common pathology to blinding ischemic retinopathies, such as diabetic retinopathy, glaucoma, and central retinal vein or artery occlusion. The development of an effective interventional modality to glutamate excitotoxicity is hence important to preventing blindness. Herein we showed that α-melanocyte-stimulating hormone (α-MSH) time-dependently protected against glutamate-induced cell death and tissue damage in an improved embryonic chicken retinal explant culture system. α-MSH down-regulated microRNA-194 (miR-194) expression during the glutamate excitotoxicity in the retinal explants. Furthermore, pharmacological antagonists to melanocortin 4 receptor (MC4R) and lentivirus-mediated overexpression of pre-miR-194 abrogated the suppressing effects of α-MSH on glutamate-induced activities of caspase 3 or 7, the ultimate enzymes for glutamate-induced cell death. These results suggest that the protective effects of α-MSH may be due to the MC4R mediated-down-regulation of miR-194 during the glutamate-induced excitotoxicity. Finally, α-MSH attenuated cell death and recovered visual functions in glutamate-stimulated post-hatch chick retinas. These results demonstrate the previously undescribed protective effects of α-MSH against glutamate-induced excitotoxic cell death in the cone-dominated retina both in vitro and in vivo, and indicate a novel molecular mechanism linking MC4R-mediated signaling to miR-194.

Role of autophagy in photoreceptor cell survival and death.

Autophagy, a highly conserved self-degradation process that occurs under both physiological and pathological conditions, provides the raw material and energy for cell regeneration under normal circumstances. Dysregulated autophagy under diseased conditions may cause protein accumulation, organelle dysfunction, and even cell death. Recent studies have shown that autophagy regulates the structural integrity and physiological functions of retinal photoreceptor cells and contributes to the pathogenesis of retinopathies such as retinal detachment, age-related macular degeneration, retinitis pigmentosa, and Leber's congenital amaurosis. In this review, we discuss the role of autophagy in photoreceptor cell survival and death in retinal physiology and diseases, and suggest the possibility that autophagy-targeting therapy may be a new strategy for retinal diseases marked by photoreceptor cell death.

Development of retinal pigment epithelium from human parthenogenetic embryonic stem cells and microRNA signature.

PURPOSE: We investigated the potential of human parthenogenetic embryonic stem cells (hPESCs) to differentiate into RPE cells, and identified development-regulating microRNAs (miRNAs). METHODS: RPE cells were derived from hPESCs. The expression of markers and miRNA expression profiles during differentiation were studied by immunocytochemistry, real-time RT-PCR, and miRNA expression array at three time points. Human fetal RPE (hfRPE) cells also were analyzed. The target genes of candidate miRNAs then were validated. RESULTS: hPESC-derived RPE cells exhibited similar morphology and pigmentation to hfRPE cells. The expression of markers during differentiation indicated that the hPESC-derived RPE cells were immature. Most specific miRNAs had a role at some time point during the differentiation and maturation of RPE from hPESCs, except for two miRNAs (miR-204 and the miR-302 family). The miR-204 was upregulated and miR-302 was down-regulated throughout the process. Subsequently, pigmented clusters and RPE signature gene expression increased significantly in the miR-204 overexpression group and miR-302 inhibition group compared to the control groups. CTNNBIP1 and TGFBR2 were confirmed to be the target genes of miR-204 and miR-302, respectively. CONCLUSIONS: hPESCs can develop into RPE-like cells and, thus, can be additional promising sources of RPE cells in cell therapy. The miR-204, miR-302s, and their targets are involved in regulating directed differentiation during the full course, thereby contributing to the search for a new method of improving differentiation efficiency using miRNAs.

MicroRNA-204/211 alters epithelial physiology.

MicroRNA (miRNA) expression in fetal human retinal pigment epithelium (hfRPE), retina, and choroid were pairwise compared to determine those miRNAs that are enriched by 10-fold or more in each tissue compared with both of its neighbors. miRs-184, 187, 200a/200b, 204/211, and 221/222 are enriched in hfRPE by 10- to 754-fold compared with neuroretina or choroid (P<0.05). Five of these miRNAs are enriched in RPE compared with 20 tissues throughout the body and are 10- to 20,000-fold more highly expressed (P<0.005). miR-204 and 211 are the most highly expressed in the RPE. In addition, expression of miR-204/211 is significantly lower in the NCI60 tumor cell line panel compared with that in 13 normal tissues, suggesting the progressive disruption of epithelial barriers and increased proliferation. We demonstrated that TGF-beta receptor 2 (TGF-betaR2) and SNAIL2 are direct targets of miR-204 and that a reduction in miR-204 expression leads to reduced expression of claudins 10, 16, and 19 (message/protein) consistent with our observation that anti-miR-204/211 decreased transepithelial resistance by 80% and reduced cell membrane voltage and conductance. The anti-miR-204-induced decrease in Kir7.1 protein levels suggests a signaling pathway that connects TGF-betaR2 and maintenance of potassium homeostasis. Overall, these data indicate a critical role for miR-204/211 in maintaining epithelial barrier function and cell physiology.

PDGF-C and -D induced proliferation/migration of human RPE is abolished by inflammatory cytokines.

PURPOSE: The role of growth factors and inflammation in regulating retinal pigment epithelial (RPE) function is complex and still poorly understood. The present study investigated human RPE cell proliferation and migration mediated by platelet-derived growth factor (PDGF) and inflammatory cytokines. METHODS: Human fetal RPE (hfRPE) cells were obtained as previously described. Gene expressions of PDGF isoforms and their receptors were detected using real-time PCR. Protein expression, activity, and localization of PDGFR-alpha and -beta were analyzed by Western blot and immunohistochemistry. BrdU incorporation and wound healing assays were used to test the effects of different PDGF isoforms and inflammatory cytokines on hfRPE proliferation and migration. Annexin-V and phalloidin staining were used to detect apoptosis and the actin cytoskeleton, respectively. RESULTS: PDGF-C and PDGF-D proteins are expressed in native human adult RPE, and mRNA levels are up to 100-fold higher than PDGF-A and -B. PDGFR-alpha and -beta proteins are expressed in native adult RPE and hfRPE (mainly localized to the apical membrane). In hfRPE, these receptors can be activated by PDGF-CC and -DD. PDGF-CC, -DD, and -BB significantly increased hfRPE proliferation, whereas PDGF-DD, -BB, and -AB significantly increased cell migration. An inflammatory cytokine mixture (TNF-alpha/IL-1beta/IFN-gamma) completely inhibited the stimulatory effect of PDGF-BB, -CC, and -DD; in contrast, this mixture stimulated the proliferation of choroidal cells. This inflammatory cytokine mixture also induced apoptosis, significant disruption of actin filaments and zonula occludens (ZO-1), and a decrease in transepithelial resistance. CONCLUSIONS: These results suggest that proinflammatory cytokines in vivo can inhibit the proliferative effect of PDGF on human RPE and, at the same time, stimulate the proliferation of choroidal cells. They also suggest an important role of proinflammatory cytokines in overcoming local proliferative/wound-healing responses, thereby controlling the development of disease processes at the retina/RPE/choroid interface.

Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.

PURPOSE: Provide a reproducible method for culturing confluent monolayers of hfRPE cells that exhibit morphology, physiology, polarity, and protein expression patterns similar to native tissue. METHODS: Human fetal eyes were dissected on arrival, and RPE cell sheets were mechanically separated from the choroid and cultured in a specifically designed medium comprised entirely of commercially available components. Physiology experiments were performed with previously described techniques. Standard techniques were used for immunohistochemistry, electron microscopy, and cytokine measurement by ELISA. RESULTS: Confluent monolayers of RPE cell cultures exhibited epithelial morphology and heavy pigmentation, and electron microscopy showed extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. The mean transepithelial potential (TEP) was 2.6 +/- 0.8 mV, apical positive, and the mean transepithelial resistance (R(T)) was 501 +/- 138 Omega . cm(2) (mean +/- SD; n = 35). Addition of 100 microM adenosine triphosphate (ATP) to the apical bath increased net fluid absorption from 13.6 +/- 2.6 to 18.8 +/- 4.6 microL . cm(-2) per hour (mean +/- SD; n = 4). In other experiments, VEGF was mainly secreted into the basal bath (n = 10), whereas PEDF was mainly secreted into the apical bath (n = 10). CONCLUSIONS: A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied native human fetal and bovine RPE-choroid explants.