hRPE Cells Research Articles

Infection of Human Retinal Pigment Epithelium with Chlamydia trachomatis.

PurposeLittle is known about the susceptibility of posterior segment tissues, particularly the human retinal pigment epithelium (hRPE), to Chlamydia trachomatis. The purpose of the study was to investigate the possibility of infecting the hRPE with Chlamydia trachomatis, and to examine the infectivity of different Chlamydia trachomatis clinical isolates for hRPE cells and the hRPE cell response to the infection.MethodsCultured hRPE and McCoy cells were inoculated with eight Chlamydia trachomatis (serovar E) clinical isolates at multiplicity of infection (MOI) of 2.0 or 0.3. To detect Chlamydia trachomatis, samples were stained immunohistochemically with anti-major outer membrane protein antibodies at 24h, 48h, and 72h postinoculation (PI). The changes in the expression of signaling molecules and proteins of cytoskeleton and extracellular matrix in hRPE cells were examined immunohistochemically.ResultsAll eight clinical isolates demonstrated ability to infect hRPE cells. At equal MOI of 0.3, the infectivity of Chlamydia trachomatis clinical isolates for RPE culture was found to be at least as high as that for McCoy cell culture. At 24h PI, the percentage of inclusion-containing cells varied from 1.5 ± 0.52 to 14.6 ± 3.3% in hRPE cell culture infected at MOI of 2.0 against 0.37 ± 0.34 to 8.9 ± 0.2% in McCoy cell culture infected at MOI of 0.3. Collagen type I, collagen type IV, basic fibroblast growth factor, transforming growth factor-beta and interleukin–8 expression at 48h PI were maximally increased, by 2.1-, 1.3-, 1.5-, 1.5- and 1.6-fold, respectively, in the Chlamydia trachomatis-infected compared with control hRPE cell culture specimens (P < 0.05).ConclusionsThis study, for the first time, proved the possibility of infecting hRPE cultured cells with Chlamydia trachomatis, which leads to proproliferative and proinflammatory changes in the expression of signaling molecules and extracellular matrix components.

CYP2E1 in the Human Retinal Pigment Epithelium: Expression, Activity, and Induction by Ethanol.

Cytochrome p450 2E1 (CYP2E1) is a detoxifying enzyme with particular affinity for ethanol (EtOH) expressed in several tissues. Although CYP2E1 has been identified in human RPE, nothing is known about its metabolic activity. Expression of CYP2E1 and activity after EtOH exposure have been studied in human RPE and ARPE-19 cells. Ethanol-induced CYP2E1 mRNA expression was analyzed by RT-PCR and quantitative PCR (qPCR) from human donor RPE as well as from ARPE-19 cells. Expression of CYP2E1 protein was determined by Western blot. Cytoplasmic CYP2E1 location also was demonstrated by immunocytochemistry. Cell viability was studied by the colorimetric assay XTT after EtOH treatment. Diallyl sulfide (DAS) was used to inhibit CYP2E1 activity. The microsomal CYP2E1 activity assay was determined by quantification of 4-nitrocatechol (4NC) formation through HPLC. Relevant CYP2E1 mRNA levels are present in human RPE. Ethanol augmented the formation of reactive oxygen species (ROS) in ARPE-19 cells. Expression of CYP2E1 mRNA, CYP2E1 protein activity, and ROS production were induced by ethanol in a concentration-dependent manner. Interestingly, the treatment with DAS reduced all the aforementioned increased values. The presence of CYP2E1 in both hRPE and ARPE-19 cells reinforces the protective role of the RPE and strongly suggests additional roles for CYP2E1 related to vision.

Valproic Acid Induced Human Retinal Pigment Epithelial Cell Death as Well as its Survival after Hydrogen Peroxide Damage is Mediated by P38 Kinase.

Age-related macular degeneration (AMD) is a leading cause of legal blindness in developed countries. Several new drugs are now available to reduce the sight threatening complications of this disease, however, all are useful in only a small fraction of patients and none of them prevents disease development. An understanding of the pathogenesis of the retinal and macular degeneration is the first step in developing preventive and fully effective treatment options for this condition. Lifelong oxidative stress seems to be an etiologic factor. In this study, we used cultured human retinal pigment epithelial cells to study the mechanism of cell death and survival in cells exposed to oxidative stress. Our studies demonstrate that valproic acid (VPA), an epigenetic factor, reduces apoptosis in hRPE cells that were subjected to hydrogen peroxide-induced oxidative injury by alteration in P38 kinase activity. Since VPA has been shown to have therapeutic use in other neuronal diseases, better understanding of the mechanism of this VPA anti-apoptotic activity may enhance its development as a therapeutic agent.

Downregulating p22phox ameliorates inflammatory response in Angiotensin II-induced oxidative stress by regulating MAPK and NF-κB pathways in ARPE-19 cells.

Oxidative stress and inflammation are two interrelated biological events implicated in the pathogenesis of many diseases. Reactive oxygen species (ROS) produced under oxidative stress play a key role in pathological conditions. Inhibition of p22phox, an indispensable component of the NADPH oxidase (NOX) complex comprising the main source of ROS, plays a protective role in many ocular conditions by inhibiting the activation of NOXs and the generation of ROS. However, little is understood regarding the role of p22phox in oxidative stress-related inflammation in the eye. We used a p22phox small interfering RNA (siRNA) to transfect the retinal pigment epithelium (RPE)-derived cell line ARPE-19, and human primary RPE (hRPE) cells, then stimulated with Ang II. We observed a potent anti-inflammatory effect and studied the underlying mechanism. Downregulating p22phox resulted in decreased ROS generation, a reduction of NOXs (NOX1, 2, 4) and a decrease in inflammatory cytokine. In addition, p22phox downregulation reduced the activation of the MAPK and NF-κB signaling pathways. We conclude that inhibition of p22phox has an anti-inflammatory effect in Ang II-induced oxidative stress. Suppressing the MAPK and NF-κB pathways is involved in this protective effect. These results suggest that p22phox may provide a promising therapeutic target for oxidative stress-induced ocular inflammation

SIRT1 mediated inhibition of VEGF/VEGFR2 signaling by Resveratrol and its relevance to choroidal neovascularization

SIRT1, a NAD+ -dependent histone deacetylase, has been shown to act as a key regulator of angiogenesis. The purpose of this study was to determine the effects of resveratrol (RSV, a SIRT1 activator) on the vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathway and to establish its relevance to choroidal neovascularization (CNV), a blinding complication of age-related macular degeneration. Western blot and ELISA assay showed that RSV inhibited hypoxia-inducible factor (HIF)-1α accumulation and VEGF secretion induced by cobalt chloride (CoCl2) through SIRT1 in human retinal pigment epithelial (hRPE) cells. Furthermore, RSV down-regulated VEGFR2 phosphorylation and activation induced by VEGF in endothelial cells via SIRT1. Thus, the inhibitory effect of RSV on the HIF-1α/VEGF/VEGFR2 signaling axis is mediated, at least in part, through SIRT1. The results suggest that targeting SIRT1 could have therapeutic potential for the treatment of CNV.

Efficient delivery of NF-κB siRNA to human retinal pigment epithelial cells with hyperbranched cationic polysaccharide derivative-based nanoparticles.

A hyperbranched cationic polysaccharide derivative-mediated small interfering (si)RNA interference strategy was proposed to inhibit nuclear transcription factor-kappa B (NF-κB) activation in human retinal pigment epithelial (hRPE) cells for the gene therapy of diabetic retinopathy. Two hyperbranched cationic polysaccharide derivatives containing the same amount of cationic residues, but with different branching structures and molecular weights, including 3-(dimethylamino)-1-propylamine-conjugated glycogen (DMAPA-Glyp) and amylopectin (DMAPA-Amp) derivatives, were developed for the efficient delivery of NF-κB siRNA into hRPE cells. The DMAPA-Glyp derivative showed lower toxicity against hRPE cells. Furthermore, the DMAPA-Glyp derivative more readily condensed siRNA and then formed the nanoparticles attributed to its higher branching architecture when compared to the DMAPA-Amp derivative. Both DMAPA-Glyp/siRNA and DMAPA-Amp/siRNA nanoparticles were able to protect siRNA from degradation by nuclease in 25% fetal bovine serum. The particle sizes of the DMAPA-Glyp/siRNA nanoparticles (70–120 nm) were smaller than those of the DMAPA-Amp/siRNA nanoparticles (130–180 nm) due to the higher branching architecture and lower molecular weight of the DMAPA-Glyp derivative. In addition, the zeta potentials of the DMAPA-Glyp/siRNA nanoparticles were higher than those of the DMAPA-Glyp/siRNA nanoparticles. As a result, siRNA was much more efficiently transferred into hRPE cells using the DMAPA-Glyp/siRNA nanoparticles rather than the DMAPA-Amp/siRNA nanoparticles. This led to significantly high levels of suppression on the expression levels of NF-κB p65 messenger RNA and protein in the cells transfected with DMAPA-Glyp/siRNA nanoparticles. This work provides a potential approach to promote hyperbranched polysaccharide derivatives as nonviral siRNA vectors for the inhibition of NF-κB activation in hRPE cells.

Regulation of Novel Calcium‐Sensitive Dicarboxylate Transport by the Calcium‐Sensing Receptor

Citrate inhibits calcium nephrolithiasis by complexing calcium (Ca) in a soluble form. Lowering apical extracellular Ca from 1.2mM (normal levels, Nl) to <60μM (low) results in an increase in Cit and succinate (Suc) transport in OK cells. Activation of the calcium-sensing receptor (CaSR) with spermine inhibits transport in both low and Nl Ca. But activation of PKC with PMA inhibited transport in low Ca only, implying that Ca-sensitive dicarboxylate transport is regulated by the CaSR through the Gq/PKC pathway. The purpose here is to determine the role of the Gi pathway. 14C-Suc uptakes were performed in OK cells and HRPE cells transfected with NaDC1 (CUBS). CaSRs inhibit adenylate cyclase (AC) through Gi. To mimic Gi, cells were pre-incubated with an AC inhibitor, MDL-12,330A (50μM for 30min) followed by uptake. The addition of MDL inhibited uptake in both Nl (0.09±0.01 to 0.05±0.01, p<0.01) and low Ca (0.13±0.01 to 0.08±0.01, p<0.01). In CUBS uptake was also inhibited in Nl (0.6±0.02 to 0.3±0.03, p<0.01) and low Ca (0.5±0.04 to 0.2±0.03, p<0.01). These results are similar to our studies of CaSR activation by spermine. If MDL inhibits transport via AC inhibition, 8-Br-cAMP (8-Br) should reverse MDL action. In OK cells, MDL eliminated the Ca-sensitivity; i.e. Nl (0.05±0.004) vs low Ca (0.05±0.002). In low Ca, 8-Br negated the inhibitory effects of MDL thus significantly increasing uptake (0.049±0.002 to 0.063±0.002, p<0.05). Thus stimulation of the CaSR through both Gq and Gi indicates CaSR involvement in the regulation of Ca-sensitive citrate transport. NIH/NIDDK

Role of NaDC1 in Control of Urinary Excretion of α‐Ketoglutarate

Recent studies have suggested that urinary α‐Ketoglutarate (α‐KG) increases with alkali loads and decreases with acid loads. Also luminal α‐KG stimulates distal bicarbonate secretion via the Oxgr1 (GPR99) receptor.To investigate the role of sodium dependent dicarboxylate transporter, NaDC1, in regulation of α‐KG excretion, we studied NaDC1 knockout (KO) and Heterozygous (Het) mice on normal diet and with 72 hrs acid loading. Plasma and urine samples were analyzed by LC/Mass Spec.Also 14C‐α‐KG uptake was studied using HRPE cells overexpressing hNaDC1 (CUBS) and the proximal tubule (PT) OK cell line. OK cells express a novel calcium (Ca)‐sensitive dicarboxylate transport system.In mice, fractional excretion (FE) of α‐KG was dramatically increased in the KO and Het mice (WT 0.38+/‐0.05, Het 2.8+/‐1.3, KO 4.7+/‐0.9; p&lt;0.05). Acidosis lowered FE significantly in all mice.In CUBS α‐KG uptake decreased when competed with the dicarboxylate succinate (Suc 2 mM) in both normal and &lt;60 µM Ca buffers (0.64±0.11 to 0.25±0.10 and 0.63±0.15 to 0.29±0.11, p&lt;0.01).In OK cells, α‐KG uptake increased when extracellular Ca was lowered to &lt;60 µM (1.7±0.49 vs 2.5±0.6, p&lt;0.01). Transport decreased when competed with Suc in both Ca buffers (1.3±0.39 vs 1.6±0.34, p&lt;0.01).NaDC1 transports α‐KG and is the predominant, but not exclusive, mechanism of control of distal α‐KG delivery. Also α‐KG is transported via a Ca‐sensitive transporter in OK cells. Furthermore based on the FEs &gt; 1.0, α‐KG is secreted into the PT simultaneously with reabsorption.Thus NaDC1 in the PT regulates acid‐base transport in the distal tubule via the control of α‐KG transport. NaDC1 represents a novel mechanism of PT–distal tubule communication in the kidney.NIH/NIDDK

Can Carrier-Mediated Delivery System Promote the Development of Antisense Imaging?

We aimed to explore the feasibility of transfection methods for antisense imaging. Antisense oligonucleotides (ASON) targeted to the mRNA of hTERT gene were synthesized and labeled with Technetium-99m and fluorescein isothiocyanate (FITC), respectively. Then, ASON was combined with transfection reagent Lipofectamine 2000 and Xfect(TM), named Lipo-ASON and Xfect-ASON, respectively. After transfection, the labeled ASON was characterized in hNPCs-G3 and hRPE cells. Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were performed to assay the hTERT mRNA and protein levels after hNPCs-G3 cells were incubated with Lipo-ASON, Xfect-ASON, and naked ASON. In addition, Lipo-ASON, Xfect-ASON, and naked ASON were injected into tumor-bearing mice, and the biodistribution in vivo was performed. The presence of two transfection reagents significantly increased intracellular uptake of radiolabeled ASON in both cell lines compared with naked ASON (p < 0.05). However, there was no significant difference in cellular uptake rates of Lipo-ASON and Xfect-ASON between hNPCs-G3 and hRPE cells. In comparison with naked ASON, the fluorescence intensity was strongly enhanced after binding to transfection reagents. Furthermore, the levels of hTERT mRNA and protein were significantly reduced in cells treated with Lipo-ASON and Xfect-ASON (p < 0.05), but naked ASON had no significant effect on hTERT expression level. The biodistribution study indicated that tumor radioactivity uptake of radiolabeled ASON for naked ASON, Lipo-ASON, and Xfect-ASON group was low and shown no significant difference in vivo. Lipofectamine transfection and Xfect(TM) transfection were not effective delivery methods of ASON for antisense imaging.

Behavior of a spontaneously arising human retinal pigment epithelial cell line cultivated on thin alginate film

Purpose:A cell line spontaneously derived from human retinal pigment epithelium (hRPE) was cultured on alginate film gelatinized with different concentrations of neurobasal cell culture medium (NCCM) to assess its growth and morphological behavior on this naturally occurring polysaccharide.Methods:Neonatal human globes were used to isolate hRPE cells. They were cultured in Dulbecco's modified Eagle’s-medium-and-Ham’s-F12-medium-(DMEM/F12) supplemented with 10% fetal bovine serum (FBS). Cultures were continuously studied using phase contrast microscopy. After the nineth passage, cells were characterized through immunocytochemical analysis for Oct4, Chx10, and Pax6 and Ki67 markers. In each well of a 6-well microplate, 1 and 2% weight/volume (w/v) alginate in deionized water was added and gelatinized using 1× and 10× NCCM. hRPE cells were cultured at a density of 2 × 105 cells/well in alginate-coated microplates. After 5 days, hRPE colonies were harvested and re-plated on polystyrene substrates. Morphology and growth of hRPE cultures were determined during the next 2 weeks.Results:The first few passages of the cultures were purely hRPE cells that revealed typical morphological features of the pigmented epithelium. They made spaces, devoid of cells, between hRPE cell monolayer and fill in the unoccupied spaces. They grew faster than native RPE cells and rapidly overgrew. Immunocytochemical test revealed that the founded cells expressed Chx10, Pax6, Ki67 and Oct4. The hRPE cells survived unlimitedly on alginate film and formed giant adjoining colonies. After re-plating, hRPE colonies adhered quickly on polystyrene and displayed native hRPE morphological features.Conclusion:Alginate film can support the survival and growth of hRPE cells and induce the cells to re-organize in tissue-like structures.

Triamcinolone regulated apopto-phagocytic gene expression patterns in the clearance of dying retinal pigment epithelial cells. A key role of Mertk in the enhanced phagocytosis

BackgroundThe apopto-phagocytic gene expression patterns during clearance of dying cells in the retina and the effect of triamcinolone (TC) upon these processes have relevance to development of age-related macular degeneration (AMD). MethodsARPE-19 cells and primary human retinal pigment epithelium (hRPE) were induced to undergo cell death by anoikis and the clearance of these cells by living hRPE/ARPE-19 or human monocyte-derived macrophages (HMDMs) in the presence or absence of TC was quantified by flow cytometry. TaqMan low-density gene expression array determining known markers of phagocytosis and loss-of-function studies on selected apopto-phagocytic genes was carried out in HMDM engulfing anoikic cells. ResultsThe glucocorticoid TC had a profound phagocytosis-enhancing effect on HMDM engulfing anoikic ARPE-19 or hRPE cells, causing a selective upregulation of the Mer tyrosine kinase (MERTK) receptor, while decreasing the expression of the AXL receptor tyrosine kinase and thrombospondin-1 (THSB-1). The key role of the MERTK could be demonstrated in HMDM engulfing dying cells using gene silencing as well as blocking antibodies. Similar pathways were found upregulated in living ARPE-19 engulfing anoikic ARPE-19 cells. Gas6 treatment enhanced phagocytosis in TC-treated HMDMs. ConclusionsSpecific agonists of the Mertk receptor may have a potential role as phagocytosis enhancers in the retina and serve as future targets for AMD therapy. General significanceThe use of Gas6 as enhancer of retinal phagocytosis via the MerTK receptor, alone or in combination with other specific ligands of the tyrosine kinase receptors' family may have a potential role in AMD therapy.

Human retinal pigment epithelial cells dying through autophagy are engulfed by professional and non‐professional phagocytes

AbstractPurpose Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional and non‐professional phagocytes can result in debris formation and development of age‐related macular degeneration (AMD). We aimed to study the clearance of autophagic dying RPE cells serving as a model for dry and wet type of AMD, respectively.Methods Autophagic cell death was induced by serum deprivation and H2O2 co‐treatment in ARPE‐19 and primary human RPE (hRPE) cells. Annexin‐V FITC/PI labelling was used to determine the cell death rate, while autophagy detection was achieved by Western blot quantification of LC3 II/LC3 I ratio. The clearance of autophagic dying cells by non‐professional and professional phagocytes was quantified using flow cytometry.Results An increasing percentage of dying RPE cells was observed in a time‐ and concentration dependent manner upon H2O2 treatment. Paralelly, an induction of autophagy could be detected within 2hrs of H2O2 treatment. Phagocytosis studies found autophagic dying cells to be efficiently and increasingly engulfed by both professional and non‐professional phagocytes over time.Conclusion The clearance of autophagic dying RPE cells can be used as a model for studying both dry and wet type of AMD in vitro, as well as for testing future pharmacological agents for treating this disease.

Role of p42/p44MAPK signal transduction pathway in expression of VEGF induced by elevated glucose concentration in cultured hRPE cells

Role of p42/p44MAPK signal transduction pathway in expression of VEGF induced by elevated glucose concentration in cultured hRPE cells

Protective effect of complement receptor 1 on barrier of cultured human retinal epithelial cells under complement-activated oxidative stress

Objective To observe the effect of complement receptor 1 (CR1) on barrier of cultured human retinal epithelial cells (hRPE) under complement-activated oxidative stress.Methods The third to fifth passage of hRPE cultured on Transwell insert were used to establish a stable hRPE monolayer barrier.The hRPE monolayer barrier was exposed to 500 μmol/L ten-butyl hydroperoxide and 10％ normal human serum to establish the hRPE monolayer barrier model of complement-activated oxidative stress in vitro.hRPE monolayer barriers under complement-activated oxidative stress were divided into two groups including model group and CR1 treatment (1 μg/ml) group.Model group and CR1 treatment group were treated with 1 μl phosphate buffer solution (PBS) or CR1 for 4 hours.Normal hRPE monolayer barrier were used as control in transepithelial resistance (TER) measurement experiment.TER was measured to evaluate the barrier function of hRPE.The hRPE-secreted vascular endothelial growth factor (VEGF) and chemokine (C-C Motif) Ligand 2 (CCL2),together with complement bioactive fragments (C3a,C5a) and membrane-attack complex (MAC) in the supernatant were detected by enzyme-linked immune sorbent assay.Results Stable hRPE monolayer barrier was established 3 weeks after hRPE seeded on Transwell insert.Complement-activated oxidative stress resulted in a sharp decrease of TER to 54.51％ compared with normal hRPE barrier.CR1 treatment could significantly improve TER of barrier under complement-activated oxidative stress to 63.48％ compared with normal hRPE barrier(t =21.60,P＜0.05).Compared with model group,CR1 treatment could significantly decrease the concentration of VEGF and CCL2 by 11.48％ and 23.47 ％ secreted by hRPE under complement-activated oxidative stress (t =3.26,2.43; P＜0.05).Compared with model group,CR1 treatment could also decreased the concentration of C3a,C5a and MAC by 24.00 ％,27.87 ％,22.44 ％.The difference were statistically significant (t =9.86,2.63,6.94 ; P＜0.05).Conclusions CR1 could protect the barrier function of hRPE cells against complement-activated oxidative stress.The underlying mechanism may involve inhibiting complement activation and downregulating the expression of VEGF and CCL2. Key words: Retinal pigment epithelium; Receptors, complement 3b/antagonists & inhibitors; Complement activation; Animal experimentation

Effect of hypoxia and PDGF-BB siRNA transfected on the production of PDGF-BB and proliferation in cultured human RPE cells

Background Proliferative vitreoretinopathy (PVR):no blood vessels,fibrous membrane cell proliferation occurs retinal surface.The development of specific mechanisms has not been completely clarified,but the retinal pigment epithelium(RPE) and platelet-derived growth factor(PDGF) for the past few years are the research hotspot.Objective To explore the effects of hypoxia on the production of PDGF-BB and proliferation in cultured human RPE (hRPE) cells.Methods In the experimental group,10,15,20,30,40 μmol/L CoCl2 were used to mimic hypoxia environment of hRPE cells,the control group did not add CoCl2 on cultured hRPE cells.By using reverse transcriptive PCR (RT-PCR) and enzyme linked immunosorbent assay(ELISA),the expression of PDGF-BB mRNA and protein was detected,and detected proliferation of hRPE cells by MTT.The cells were divided into 6 groups,different siRNAs were transfected into PDGF-BB siRNA1 group,PDGF-BB siRNA2 group,PDGF-BB siRNA3 group (because PDGF-BB has three different siRNAs,one of which is a valid siRNA),β-actin siRNA group,unrelated sequence siRNA group,only Lip2000 was plused in the blank control group.After transfection of 4-6 hours,except for blank control group,the other five groups were added to PDGF-BB mRNA and protein and its effect on the proliferation of hRPE cells obviously CoCl2 concentrations (15 μmol/L) simulated hypoxia environment of 24 hours,the detection of PDGF-BB mRNA and protein and hRPE cell proliferation rate.Results Neither PDGF-BB mRNA or protein was found in the blank control group.The production of PDGF-BB mRNA and protein in hRPE cells cultured by different concentrations were different,with significant differences among them (F=43.737,P＜0.01;F=54.612,P＜0.05),and the expressions of PDGF-BB mRNA and protein of 15 μmol/L CoCl2 group were more than other groups,with significant differences between the two groups (all at P＜0.05).MTT showed that PDGF-BB protein and hRPE cell proliferation rate in different concentions of CoCl2 groups were different,with significant differences among them(F=95.379,P＜0.01 ; F =63.375,P＜0.05),the expression of PDGF-BB protein was more and hRPE cell proliferation rate was higher in 15 μmol/L CoCl2group than other groups,with significant differences between the two groups (all at P＜0.05),postive correlation was found between PDGF-BB protein and hRPE cell proliferation rate (r =0.994,P＜0.05).The production of PDGF-BB mRNA and protein in the different siRNA transfected groups were different,with significant differences among them (F =156.330,125.650,both at P＜0.01),and the expressions of PDGF-BB mRNA and protein of PDGF-BB siRNA2 group were more than other groups,with significant differences between them(all at P＜0.05).MTT showed that PDGF-BB protein and hRPE cell proliferation rate in different siRNA transfectedgroups were different,with significant differences among them (F =73.131,98.564,both at P＜ 0.01).The expression of PDGF-BB protein was less and hRPE cell proliferation rate was lower in PDGF-BB siRNA2 group than other groups,with significant differences between them (all at P ＜ 0.05),postive correlation was found between PDGF-BB protein and hRPE cell proliferation rate (r =0.996,P＜0.05).Conclusions Proper hypoxia can induce the expression of PDGF-BB,PDGF-BB expression can significantly promote the proliferation of hRPE cells.In the transfection targeting PDGF-BB siRNA,PDGF-BB expression is suppressed,can effectively reduce the value of hRPE cell hyperplasia. Key words: Platelet-derived growth factor-BB protein; Human retinal pigment epithelium; CoCl2; Proliferation ; siRNA

Bisretinoid-mediated Complement Activation on Retinal Pigment Epithelial Cells Is Dependent on Complement Factor H Haplotype

Age-related macular degeneration (AMD) is a common central blinding disease of the elderly. Homozygosity for a sequence variant causing Y402H and I62V substitutions in the gene for complement factor H (CFH) is strongly associated with risk of AMD. CFH, secreted by many cell types, including those of the retinal pigment epithelium (RPE), is a regulatory protein that inhibits complement activation. Recessive Stargardt maculopathy is another central blinding disease caused by mutations in the gene for ABCA4, a transporter in photoreceptor outer segments (OS) that clears retinaldehyde and prevents formation of toxic bisretinoids. Photoreceptors daily shed their distal OS, which are phagocytosed by the RPE cells. Here, we investigated the relationship between the CFH haplotype of human RPE (hRPE) cells, exposure to OS containing bisretinoids, and complement activation. We show that hRPE cells of the AMD-predisposing CFH haplotype (HH402/VV62) are attacked by complement following exposure to bisretinoid-containing Abca4(-/-) OS. This activation was dependent on factor B, indicating involvement of the alternative pathway. In contrast, hRPE cells of the AMD-protective CFH haplotype (YY402/II62) showed no complement activation following exposure to either Abca4(-/-) or wild-type OS. The AMD-protective YY402/II62 hRPE cells were more resistant to the membrane attack complex, whereas HH402/VV62 hRPE cells showed significant membrane attack complex deposition following ingestion of Abca4(-/-) OS. These results suggest that bisretinoid accumulation in hRPE cells stimulates activation and dysregulation of complement. Cells with an intact complement negative regulatory system are protected from complement attack, whereas cells with reduced CFH synthesis because of the Y402H and I62V substitutions are vulnerable to disease.

Migration of Retinal Pigment Epithelium Cells Is Regulated by Protein Kinase Cα In Vitro

Retinal pigment epithelium (RPE) cell migration and proliferation are considered key elements in proliferative vitreoretinopathy (PVR). Downregulation of protein kinase Cα (PKCα) can inhibit RPE cell proliferation. Here, we sought to analyze whether PKCα affects the migration of RPE cells. Human RPE (hRPE) cells were cultured, confirmed by immunofluorescence staining, and divided into four groups: control, thymeleatoxin, non-small interfering RNA (siRNA), and siRNA-PKCα. Thymeleatoxin was used to activate PKCα, and siRNA-PKCα was used to knock it down. Expression of PKCα was confirmed by quantitative RT-PCR (qRT-PCR). Cell migration ability was analyzed by wound healing assay and transwell chamber assay. Expression of zonula occludens (ZO)-1 and occludin was determined by immunofluorescence. Pure populations of hRPE cell cultures were observed using light and fluorescence microscopy. The mRNA levels of PKCα were not significantly increased by thymeleatoxin, but were reduced by siRNA-PKCα as determined by qRT-PCR assay. The wound healed faster in the thymeleatoxin group than in the control group at time points 12, 15, and 20 hours. The wound healed more slowly in the siRNA-PKCα group than in the non-siRNA group at the three time points. A similar tendency among the four groups was consistently observed in regard to cell numbers counted in the transwell chamber assay. The expression of ZO-1 was highest in the siRNA-PKCα group, similar in the control and non-siRNA groups, and lowest in the thymeleatoxin group. After migration, the fluorescence intensity of ZO-1 was reduced to similarly weak levels among the four groups. Retinal pigment epithelium cell migration is enhanced by a PKCα agonist and suppressed by a PKCα antagonist. The results suggest that a PKCα-mediated signal transduction pathway plays a crucial role in hRPE cell migration and may be a potential therapeutic target against hRPE cell migration and PVR disease.

Pro-inflammatory and anti-angiogenic effects of bisphosphonates on human cultured retinal pigment epithelial cells

AimBisphosphonates have been shown to induce ocular inflammatory diseases such as uveitis and scleritis, while being protective against angiogenic diseases like neovascular age-related macular degeneration (AMD). Therefore, we studied the...

Bioactive substrates for human retinal pigment epithelial cell growth from elastin-like recombinamers

The aim of this study was to investigate the use of bioactive RGD-containing elastin-like recombinamers (ELR-RGDs) as a substrate that can maintain human retinal pigment epithelial cell (hRPE) phenotype and growth pattern. Results obtained are compared with previously published behavior of ARPE19 cells. The extension of these results to hRPE is required because ARPE19 cells cannot be used clinically to treat age-related macular degeneration. hRPE cells were isolated, cultured, seeded, and grown on surface of glass, treated polystyrene (TCP), and solvent-cast ELR-RGD and ELR-IK film with no specific sequence. Cells were analyzed to study cell adhesion, proliferation, morphology, and RPE65 protein expression by staining with diamidino-2-phenylindole, Rhodamine-Phalloidin, and anti-RPE65 antibody at 12, 24, 72, 120, 168, and 360 h. hRPE cells always grew better on ELR-RGD than on glass and ELR-IK but not on TCP. The kinetic hRPE growth curves confirmed that growth differences started to appear at 24 h for these surfaces in ascending order of cell growths, namely glass, ELR-IK, ELR-RGD, and TCP. There was a clear difference at 360 h. ELR-RGD maintained hRPE cells stable morphology and RPE65 protein expression. ELR-RGD seems to be a good substrate for growing hRPE cells with stable morphology and RPE65 protein expression. As such, this work confirms our hypothesis regarding ELR-RGD substrates viability, which can be used as a Bruch's membrane prosthesis for further studies in animals. However, these results must subsequently be extrapolated to use of hRPE cells in animals to evaluate them as a transplantation vehicle in human.

Flow cytometry assessment of the purity of human retinal pigment epithelial primary cell cultures

Culturing of human retinal pigment epithelial cells (hRPE) is the initial step in cell therapy of some retinal diseases. To transfer these cells into clinical use, it is necessary to guarantee that they are well differentiated and contamination free. Fluorescence microscopy is the easiest method to do this, but it is associated with operator subjectivity, and the results are highly variable. The aim of this study was to demonstrate the practicality of implementing flow cytometry (FC) analysis to determine the purity of human RPE primary cell cultures. An ARPE19 cell line, human skin fibroblasts, hRPE, and human corneal epithelial cells were analysed by FC to determine the percentage of the hRPE population expressing RPE65 and epithelial and fibroblast proteins. The cell viability and DNA content also were determined. FC analysis showed that the hRPE cells were healthy, stable, and expressed RPE65 protein in the study working conditions. The density of RPE65 protein expression decreased during passages 2 to 10, which was confirmed using a Western blot technique. However, the hRPE cells did not express the 112-kDa epithelial and fibroblast proteins in the current working conditions. These findings suggested that FC facilitates a detailed analysis of human RPE primary cell cultures, a necessary step in developing new cell therapies for retinal diseases.