The generation of functional retinal pigment epithelium (RPE) is of great therapeutic interest to the field of regenerative medicine and may provide possible cures for retinal degenerative diseases, including age-related macular degeneration (AMD). Although RPE cells can be produced from either embryonic stem cells or induced pluripotent stem cells, direct cell reprogramming driven by lineage-determining transcription factors provides an immediate route to their generation. By monitoring a human RPE specific Best1::GFP reporter, we report the conversion of human fibroblasts into RPE lineage using defined sets of transcription factors. We found that Best1::GFP positive cells formed colonies and exhibited morphological and molecular features of early stage RPE cells. Moreover, they were able to obtain pigmentation upon activation of Retinoic acid (RA) and Sonic Hedgehog (SHH) signaling pathways. Our study not only established an ideal platform to investigate the transcriptional network regulating the RPE cell fate determination, but also provided an alternative strategy to generate functional RPE cells that complement the use of pluripotent stem cells for disease modeling, drug screening, and cell therapy of retinal degeneration.
Animals ; Bestrophins ; Cell Differentiation ; Cell Line ; Cell Lineage ; Chloride Channels ; genetics ; metabolism ; Embryonic Stem Cells ; cytology ; metabolism ; Eye Proteins ; genetics ; metabolism ; Fibroblasts ; cytology ; metabolism ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Mice ; Pigmentation ; Retinal Pigment Epithelium ; cytology ; metabolism ; Transcription Factors ; metabolism

OBJECTIVETo investigate the in vivo inhibition of extract of Fructus lycii (FL) on the expressions of cathepsin B (Cat B) and cystatin C (Cys C) in high-fat diet and hydroquinone (HQ) induced model mice with age-related macular degeneration (AMD), and to explore the in vitro effects of lutein and zeaxanthin on hydrogen peroxide (H2O2,) induced expressions of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) on ARPE-19 cells.

METHODSFifty female 8-month-old C57BL/6 mice were recruited in this research. Ten mice fed with regular diet was taken as the age control group. The rest 40 mice were fed with high fat diet for 6 months, followed by adding HQ (0. 8%) in the drinking water for 3 consecutive months. Then the modeled mice were randomly divided into the model control group (n =10), the high (at the daily dose of 3.75 g/kg), middle (at the daily dose of 2.50 g/kg), and low dose (at the daily dose of 1.25 g/kg) FL groups, 10 in each group. The extract of FL at each dose was respectively administered to mice by gastrogavage for 3 successive months. By the end of the experiment, the mice were killed and their eyeballs were removed. The protein expressions of Cat B and Cys C were observed by immunohistochemical assay. The mRNA and protein expressions of Cat B and Cys C were detected by real-time PCR and Western blot respectively. The drug concentrations of H2O2, lutein, and zeaxanthin were screened and detected using the activity of cell proliferation. The protein expressions of MMP-2 and TIMP-2 were detected using Western blot.

RESULTSCompared with the age control group, the mRNA and protein expressions of Cat B and Cys C were significantly higher in the in vivo model control group (P <0.05, P <0.01). The mRNA expressions of Cat B and Cys C were weaker in the middle and high dose FL groups than in the model control group (P <0. 05, P <0. 01). In in vitro cells, lutein and zeaxanthin could down-regulate the protein expressions of MMP-2 and TIMP-2 in H202 induced ARPE-19 cells (P <0. 05, P <0. 01).

CONCLUSIONSExtract of FL could down-regulate the high protein expressions of Cat B and Cys C in high-fat diet and HQ induced model mice. Lutein and zeaxanthin could down-regulate the protein expressions of MMP-2 and TIMP-2 in H202 induced ARPE-19 cells.

Animals ; Cathepsin B ; metabolism ; Cystatin C ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hydrogen Peroxide ; Lutein ; pharmacology ; Macular Degeneration ; prevention & control ; Matrix Metalloproteinase 2 ; metabolism ; Mice ; Mice, Inbred C57BL ; Pigment Epithelium of Eye ; drug effects ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Xanthophylls ; pharmacology ; Zeaxanthins

BACKGROUNDRetinal pigment epithelial (RPE) cell is a monolayer of multifunctional cells between the retina and the choroid. Peroxynitrite (ONOO(-)) is known to induce toxicity on RPE cells. This study aimed to evaluate ONOO(-) induced expression of inducible nitric oxide synthase (iNOS) and complement 3 (C3) via Fas/FasL pathway in RPE cells and the values of puerarin as a therapeutic target for inhibiting the apoptosis of RPE cells.

METHODSRPE cells were obtained from eyes of C57BL/6 mice. RPE cells were divided into control, ONOO(-) and puerarin groups. Control group was treated with saline, ONOO(-) group was treated with ONOO(-), and puerarin group was treated with puerarin after added with ONOO(-). All changes were observered at 6, 12 and 24 hours after treatment. Western blotting analysis was used to determine the expression of nitrotyrosine (NT, the foot print of ONOO(-)) and C3; flow cytometry was used to determine the apoptosis of RPE cells. Immunohistochemistry and Western blotting were used to determine Fas/FasL signal transduction. Gene array analysis, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to determine the expression of iNOS mRNA and iNOS protein in RPE cells.

RESULTSThere were minor expression of NT, C3, Fas/FasL and iNOS mRNA in control group, and strong expression of NT and C3 in ONOO(-) group, while in puerarin group weak expressions of NT and C3 were detected as time passed by (P < 0.001). Apoptosis of RPE cells occured and reached a higher level at 6 and 24 hours after addition of ONOO(-) respectively in ONOO(-) group, but delayed apoptosis in puerarin group (P < 0.05). Compared to control group, the expression of Fas/FasL was up-regulated in ONOO(-) group, but was down-regulated in puerarin group (P < 0.001). Similarly, the expressions of iNOS mRNA and iNOS protein in ONOO(-)group were up-regulated in ONOO(-) group, but down-regulated in puerarin group (P < 0.001).

CONCLUSIONSONOO(-) expresseion in RPE cells may constitute the new way of oxidant stress. Fas/FasL signal transduction pathway and C3 may affect and reinforce apoptosis mediated by ONOO(-). Puerarin could reverse ONOO(-) damage on RPE cells. The antagonizing mechanism of puerarin may be related to its inhibitory to the expression of iNOS mRNA, and therefore decrease ONOO(-) formation as well as directly antagonize the effect of ONOO(-). Furthermore, puerarin may be an useful therapeutic agent against apoptosis of RPE cells.

Animals ; Blotting, Western ; Cells, Cultured ; Complement C3 ; genetics ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; Fas Ligand Protein ; genetics ; metabolism ; Flow Cytometry ; Immunohistochemistry ; Isoflavones ; pharmacology ; Mice ; Mice, Inbred C57BL ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Peroxynitrous Acid ; pharmacology ; Pigment Epithelium of Eye ; cytology ; Reverse Transcriptase Polymerase Chain Reaction ; fas Receptor ; genetics ; metabolism

BACKGROUNDThe cell layer of the ciliary epithelium is responsible for aqueous humor secretion and maintenance. Ion channels play an important role in these processes. The main aim of this study was to determine whether the well-characterized members of the Kv1 family (Kv1.3) contribute to the Kv currents in ciliary epithelium.

METHODSNew Zealand White rabbits were maintained in a 12 hours light/dark cycle. Ciliary epithelium samples were isolated from the rabbits. We used Western blotting and immunocytochemistry to identify the expression and location of a voltage-gated potassium channel Kv1.3 in ciliary body epithelium. Membrane potential change after adding of Kv1.3 inhibitor margatoxin (MgTX) was observed with a fluorescence method.

RESULTSWestern blotting and immunocytochemical studies showed that the Kv1.3 protein expressed in pigment ciliary epithelium and nonpigment ciliary epithelium, however it seemed to express more in the apical membrane of the nonpigmented epithelial cells. One nmol/L margatoxin, a specific inhibitor of Kv1.3 channels caused depolarization of the cultured nonpigmented epithelium (NPE) membrane potential. The cytosolic calcium increased after NPE cell depolarization, this increase of cytosolic calcium was partially blocked by 12.5 micromol/L dantrolene and 10 micromol/L nifedipine. These observations suggest that Kv1.3 channels modulate ciliary epithelium potential and effect calcium dependent mechanisms.

CONCLUSIONKv1.3 channels contribute to K+ efflux at the membrane of rabbit ciliary epithelium.

Animals ; Blotting, Western ; Calcium ; metabolism ; Ciliary Body ; cytology ; metabolism ; physiology ; Immunohistochemistry ; In Vitro Techniques ; Kv1.3 Potassium Channel ; metabolism ; physiology ; Membrane Potentials ; physiology ; Pigment Epithelium of Eye ; cytology ; metabolism ; physiology ; Rabbits

PURPOSE: Hepatocyte growth factor (HGF) and its receptor (HGFR/c-Met) regulate motility, mitogenesis, and morphogenesis in a cell type-dependent fashion. We report the role of HGF and c-Met on stress-induced ARPE-19 human retinal pigment epithelial (RPE) cells in this study. METHODS: The cells were cultured either with or without serum. Southern and Western blot analyses were done to determine the expression patterns of HGF/c-Met in serum-starved ARPE-19 cells. The cell proliferation pattern in serum-starved condition was analyzed using MTS assay. Inhibition level of cell proliferation was analyzed using a neutralizing monoclonal antibody against c-Met (2 microgram/ml). RESULTS: Abnormal cell proliferation and scattering of ARPE-19 cells was observed under serum starvation. HGF/c-Met were expressed in serum-starved ARPE-19 cells. ARPE-19 cell proliferation was also enhanced with recombinant HGF treatment. Neutralization against c-Met inhibited the proliferation of serum-deprived ARPE-19 by 64.5% (n=9, S.D. 5.5%). Serum starvation appears to induce epithelial-mesenchymal transition of ARPE-19 cells, resulting in scatter, and the expression of alpha-smooth muscle actin (alpha-SMA), a marker for fibrosis. CONCLUSIONS: In conclusion, c-Met induced under non-physiologic conditions has significant effects on the activation of RPE cells.
Blotting, Southern ; Blotting, Western ; Cell Movement/physiology ; Cell Proliferation ; Cells, Cultured ; Culture Media, Serum-Free ; *Gene Expression ; Hepatocyte Growth Factor/biosynthesis/*genetics ; Humans ; Mitosis/physiology ; Pigment Epithelium of Eye/cytology/*metabolism ; Polymerase Chain Reaction ; Proto-Oncogene Proteins c-met/biosynthesis/*genetics ; RNA/*genetics

Small hairpin RNA (shRNA) was used to silence the HIF1alpha gene in human retinal pigment epithelial cells (RPE) under hypoxia in order to observe the effect of gene silencing on the expression of matrix metalloproteinase tissue inhibitor 1 (TIMP1). By using chemical hypoxic inducer CoCl2, to mimic RPE hypoxic environment, shRNA against the targeting region of HIF1alpha mRNA sequence was synthesized by a method of in vitro transcription, and the HIF1alpha was interfered in RPE cultured under hypoxia (induced by 150 micromol/L CoCl2). RT-PCR was employed to detect the expression of HIF1alpha and TIMP1. The expression levels of HIF1alpha and TIMP1 were measured by using Western blotting. The results showed that after the RPE were transfected with specific shRNA against HIF1alpha mRNA, RT-PCR revealed that under hypoxia, the efficacy of HIF1alpha gene silencing in RPE was 83.4%. Western blotting revealed that the expression levels of HIF1alpha protein was dramatically dropped. In addition. RT-PCR results demonstrated that the expression of TIMP1 mRNA was decreased by 28.9%, and the expression levels of TIMP1 protein were also significantly reduced by Western blotting. It was suggested that shRNA targeted against HIF1alpha mRNA could effectively silence the HIF1alpha gene, subsequently effectively inhibit the hypoxia-induced up-regulation of TIMP1.
Cell Hypoxia ; Cells, Cultured ; Gene Silencing ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics ; Pigment Epithelium of Eye/cytology ; Pigment Epithelium of Eye/*metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; RNA, Small Interfering/*pharmacology ; Retina/cytology ; Retina/*metabolism ; Tissue Inhibitor of Metalloproteinase-1/*biosynthesis ; Tissue Inhibitor of Metalloproteinase-1/genetics

BACKGROUNDRetinal pigment epithelial (RPE) cells play an important role in the occurrence of choroidal neovascularization (CNV). Vascular endothelial growth factor (VEGF) as a positive regulatory growth factor is produced by the RPE in an autocrine or paracrine manner, promoting CNV development. Duplexes of 21 nt RNAs, known as short interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference when introduced into mammalian cells. We searched for an efficient siRNA to interfere with VEGF expression in RPE cells and shed light on the treatment of CNV.

METHODSHuman primary RPE (hRPE) cells were cultured and identified. Three pairs of siRNAs were designed according to the sequence of VEGF 1-5 extrons and synthesized by T7 RNA polymerase transcription in vitro. To evaluate the inhibitory activity of T7-siRNAs, hRPE cells were transfected via siPORT Amine. The interfering effect of T7-siRNAs in hRPE cells was examined by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence.

RESULTSThree pairs of T7-siRNAs synthesized by in vitro transcription with T7 RNA polymerase suppressed VEGF gene expression with efficiency from 65% to 90%. T7-siRNA (B), targeted region at 207 nt to 228 nt and double stranded for 21 nt with 2 nt UU 3' overhangs, was the most effective sequence tested for inhibition of VEGF expression in hRPE cells. Compared with nontransfected cells, the mean fluorescence in hRPE cells transfected with T7-sRNAs was significantly less (P < 0.01). siRNA with a single-base mismatch and ssRNA(+) did not show suppressing effect. Furthermore, it was found that siRNAs had a dose dependent inhibitory effect (5 to 10 pmol).

CONCLUSIONT7-siRNA can effectively and specifically suppress VEGF expression in hRPE cells and may be a new way to treat CNV.

Base Sequence ; Cells, Cultured ; Choroidal Neovascularization ; therapy ; DNA-Directed RNA Polymerases ; metabolism ; Humans ; Molecular Sequence Data ; Pigment Epithelium of Eye ; cytology ; metabolism ; RNA Interference ; RNA, Small Interfering ; biosynthesis ; pharmacology ; Transcription, Genetic ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; genetics ; Viral Proteins ; metabolism

Blotting, Western ; Cells, Cultured ; Humans ; Pigment Epithelium of Eye ; metabolism ; Plasmids ; RNA Interference ; RNA, Small Interfering ; pharmacology ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; genetics

PURPOSE: To study the effect of systemic administration of phenyl-N-tert-butylnitrone (PBN) on the degeneration of photoreceptor cells in rd mice. METHODS: PBN was injected intraperitoneally into FVB/rd mice on postnatal days (P) 5 to 14 (group A), and P10 to 18 (group B). At days P14, 16, 18, 20 and 27, morphological changes and apoptosis were analyzed by staining with hematoxylin and eosin or DAPI. The effect of PBN on apoptosis was analyzed in retinal pigment epithelial (RPE) cells by the measurement of caspase-3 activity. RESULTS: In control and group B mice, the outer nuclear layer (ONL) of the retina was composed of 8-10 rows at P12, and rapidly decreased to one row at P18. In group A mice, the ONL was preserved with 5-7 rows at P18, and decreased to one row at P22. PBN inhibited caspase-3 activity in cultured RPE cells. CONCLUSIONS: PBN delayed, but did not block, the degeneration of photoreceptor cells in rd mice. PBN may exert its inhibitory effect during the early phase of photoreceptor cell degeneration.
Retinal Degeneration/*drug therapy/metabolism/pathology ; Pigment Epithelium of Eye/drug effects/metabolism/pathology ; Photoreceptors, Vertebrate/drug effects/metabolism/*pathology ; Nitrogen Oxides/*administration & dosage/pharmacokinetics/therapeutic use ; Neuroprotective Agents/*administration & dosage/pharmacokinetics/therapeutic use ; Mice ; Male ; Injections, Intraperitoneal ; Free Radical Scavengers/*administration & dosage/pharmacokinetics/therapeutic use ; Follow-Up Studies ; Female ; Enzyme Precursors/metabolism ; Disease Models, Animal ; Cells, Cultured ; Caspases/metabolism ; Caspase 3 ; Apoptosis/drug effects ; Animals

OBJECTIVE: To demonstrate the mechanism of positive effects of the sequential air-fluid exchange on the use of complicated vitroretinal microsurgery. METHODS: Seventy-one patients who underwent vitreoretinal microsurgery were performed 2 or 3 times sequential air-fluid exchange. The regenerated fliud in vitreous cavity at various periods was collected to act on cultured human retinal pigment epithelial (RPE) cells,and then the secretion of bFGF and the expressions of bcl-2 and ki-67 by RPE cells were observed. RESULTS: The expressions of bcl-2 and ki-67 were up-regulated and the secretion of bFGF significantly increased after RPE cells was acted with the regenerated fluid in the vitreous cavity. CONCLUSION The sequential air-fluid exchange can mechanically reduce intraocular growth factors after the vitreoretinal microsurgery, indirectly restrain the proliferation of RPE cell, and improve the successful rate of vitreoretinal microsurgery.
Cell Division ; Cells, Cultured ; Fibroblast Growth Factor 2 ; metabolism ; Humans ; Ki-67 Antigen ; biosynthesis ; genetics ; Microsurgery ; Pigment Epithelium of Eye ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Regeneration ; Retina ; surgery ; Retinal Detachment ; prevention & control ; Secondary Prevention ; Vitrectomy ; Vitreoretinopathy, Proliferative ; surgery