Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway

AIM:To explore the effect of epidermal growth factor receptor(EGFR)inhibition by erlotinib and EGFR siRNA on epidermal growth factor(EGF)-induced activation of retinal pigment epithelium(RPE)cells.METHODS:Human RPE cell line(ARPE-19 cells)was activated by 100 ng/mL EGF.Erlotinib and EGFR siRNA were used to intervene EGF treatment.Cellular viability,proliferation,and migration were detected by methyl thiazolyl tetrazolium(MTT)assay,bromodeoxyuridine(BrdU)staining assay and wound healing assay,respectively.EGFR/protein kinase B(AKT)pathway proteins and N-cadherin,α-smooth muscle actin(α-SMA),and vimentin were tested by Western blot assay.EGFR was also determined by immunofluorescence staining.RESULTS:EGF treatment for 24h induced a significant increase of ARPE-19 cells’viability,proliferation and migration,phosphorylation of EGFR/AKT proteins,and decreased total EGFR expression.Erlotinib suppressed ARPE-19 cells’viability,proliferation and migration through down regulating total EGFR and AKT protein expressions.Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin,α-SMA,and vimentin proteins.Similarly,EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation,viability,and migration,phosphorylation of EGFR/AKT proteins,and up-regulation of N-cadherin,α-SMA,and vimentin proteins.CONCLUSION:Erlotinib and EGFR-knockdown suppress EGF-induced cell viability,proliferation,and migration via EGFR/AKT pathway in RPE cells.EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy(PVR).

Autophagy: a new mechanism for regulating VEGF and PEDF expression in retinal pigment epithelium cells

AIM: To investigate the regulation of vascular endothelial growth factors(VEGF) and pigment epithelium-derived factor(PEDF) expression by autophagy in retinal pigment epithelium(RPE) cells on exposure to hypoxia. METHODS: ARPE-19, an RPE cell line, was treated as following: the control group was kept in a normoxic incubator; the hypoxia group was incubated in a hypoxic incubator with 1% O_2/5% CO_2/94% N_2 for 24 h; the hypoxia + 3-methyladenine(3-MA) group was pretreated with 10 mmol/L 3-MA for 1 h and then in the hypoxic incubator for 24 h; and the hypoxia + chloroquine(CQ) group was pretreated with 50 μmol/L CQ for 1 h and then in the hypoxic incubator for 24 h. The morphology and ultrastructure of the cells was observed by an inverted microscope or a transmission electronic microscope(TEM). Western blot was performed to assay the expression of autophagy-associated markers, including microtubule associated protein 1 light chain 3 B(LC3 B), Beclin-1, Atg5 and p62. The concentration of VEGF and PEDF in the culture supernatant was determined by ELISA, and the ratio of VEGF/PEDF was calculated. RESULTS: There were no obvious differences in cell morphology among different groups and autolysosomes could be observed in the cytoplasm in all groups. Compared to the control cells, the LC3 B-II/I ratio and levels of Beclin-1 and Atg5 were significantly increased and p62 level was significantly decreased in the hypoxia group. With the increase of VEGF and decrease of PEDF concentration, the VEGF/PEDF ratio was significantly increased in the hypoxia group compared to the control cells. The LC3 B-II/I ratio was significantly reduced by 3-MA treatment and increased by CQ treatment. The expressions of Beclin-1 and Atg5 were significantly reduced by 3-MA or CQ treatment, while expression of p62 was increased in the 3-MA or CQ treated cells. The concentration of VEGF was significantly decreased and PEDF increased, thereby the VEGF/PEDF ratio was decreased in the hypoxia + 3-MA group and hypoxia + CQ group compared with that in the hypoxia group. CONCLUSION: Hypoxia leads to elevated autophagy in RPE cells, and expression of VEGF and PEDF might be regulated by autophagy on exposure to hypoxia to further participate in regulating the formation of retinal neovascularization.

Protective effects of lipoic acid-niacin dimers against blue light-induced oxidative damage to retinal pigment epithelium cells

AIM: To evaluate the protective effects of lipoic acid-niacin(N2 L) dimers against blue light(BL)-induced oxidative damage to human retinal pigment epithelium(hRPE) cells in vitro.METHODS: h RPE cells were divided into a control group(CG), a BL group, an N2 L plus BL irradiation group, an α-lipoic acid(ALA) plus BL group, an ALA-only group, and an N2 L-only group. hRPE cellular viability was detected by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium(MTT) bromide assays, and apoptosis was evaluated by annexin-V-PE/7-AAD staining followed by flow cytometry. Ultrastructural changes in subcellular organelles were observed by transmission electron microscopy. Reactive oxygen species formation was assayed by flow cytometry. The expression levels of the apoptosis-related proteins BCL-2 associated X protein(BAX), B-cell leukmia/lymphoma 2(BCL-2), and caspase-3 were quantified by Western blot analysis.RESULTS: BL exposure with a light density of 4±0.5 mW/cm2 exceeding 6 h caused hRPE toxicity, whereas treatment with a high dose of N2 L(100 mol/L) or ALA(150 mol/L) maintained cell viability at control levels. BL exposure caused vacuole-like degeneration, mitochondrial swelling, and reduced microvillus formation;however, a high dose of N2 L or ALA maintained the ultrastructure of hRPE cells and their organelles. High doses of N2 L and ALA also protected hRPE cells from BL-induced apoptosis, which was confirmed by Western blot analysis: BCL-2 expression significantly increased, while BAX and caspase-3 expression slightly decreased compared to the CG.CONCLUSION: High-dose N2 L treatment(>100 mol/L) can reduce oxidative damage in degenerating hRPE cells exposed to BL with an efficacy similar to ALA.

In vitro reconstruction and characterization of tissue-engineered human corneal epithelium with seeder cells from an untransfected human corneal epithelial cell line

AIM: To demonstrate the morphology and structure of in vitro reconstructed tissue-engineered human corneal epithelium (TE-HCEP) with seeder cells from an untransfected HCEP cell line. METHODS: The TE-HCEPs were reconstructed in vitro with seeder cells from an untransfected HCEP cell line, and scaffold carriers of denuded amniotic membrane (dAM) in air-liquid interface culture for 3, 5, 7 and 9 days, respectively. The specimens were examined with hematoxylin-eosin (HE) staining of paraffin-section, immunocytochemical staining, scanning and transmission electron microscopy. RESULTS: During in vitro reconstruction of TE-HCEP, HCEP cells formed a 3-4, 6-7 and 8-10 layers of an HCEP-like structure on dAMs in air-liquid interface culture for 3, 5 and 7 days, respectively. But the cells deceased to 5-6 layers and the structure of straified epithelium became loose at day 9. And the cells maintained positive expression of marker proteins (keratin 3 and keratin 12), cell-junction proteins (zonula occludens-1, E-cadherin, connexin 43 and integrin beta 1) and membrane transport protein of Na+-K+ ATPase. The HCEP cells in TE-HCEP were rich in microvilli on apical surface and established numerous cell-cell and cell-dAM junctions at day 5. CONCLUSION: The morphology and structure of the reconstructed TE-HCEP were similar to those of HCEP in vivo. The HCEP cells in the reconstructed TE-HCEP maintained the properties of HCEP cells, including abilities of forming intercellular and cell-extracellular matrix junctions and abilities of performing membrane transportation. The untransfected HCEP cells and dAMs could promisingly be used in reconstruction HCEP equivalent for clinical corneal epithelium transplantation.

Gene Therapy Activates Retinal Pigment Epithelium Cell Proliferation for Age-related Macular Degeneration in a Mouse Model

Objective Age-related macular degeneration(AMD)is a degenerative retinal disease.The degeneration or death of retinal pigment epithelium(RPE)cells is implicated in the pathogenesis of AMD.This study aimed to activate the proliferation of RPE cells in vivo by using an adeno-associated virus(AAV)vector encodingβ-catenin to treat AMD in a mouse model.Methods Mice were intravitreally injected with AAV2/8-Y733F-VMD2-β-catenin for 2 or 4 weeks,andβ-catenin expression was measured using immunofluorescence staining,real-time quantitative reverse transcription polymerase chain reaction(PCR),and Western blotting.The function ofβ-catenin was determined using retinal flat mounts and laser-induced damage models.Finally,the safety of AAV2/8-Y733F-VMD2-β-catenin was evaluated by multiple intravitreal injections.Results AAV2/8-Y733F-VMD2-β-catenin induced the expression ofβ-catenin in RPE cells.It activated the proliferation of RPE cells and increased cyclin D1 expression.It was beneficial to the recovery of laser-induced damage by activating the proliferation of RPE cells.Furthermore,it could induce apoptosis of RPE cells by increasing the expression of Trp53,Bax and caspase3 while decreasing the expression of Bcl-2.Conclusion AAV2/8-Y733F-VMD2-β-catenin increasedβ-catenin expression in RPE cells,activated RPE cell proliferation,and helped mice heal from laser-induced eye injury.Furthermore,it could induce the apoptosis of RPE cells.Therefore,it may be a safe approach for AMD treatment.

Role of glycolysis in retinal vascular endothelium, glia, pigment epithelium, and photoreceptor cells and as therapeutic targets for related retinal diseases

Glycolysis produces large amounts of adenosine triphosphate(ATP)in a short time.The retinal vascular endothelium feeds itself primarily through aerobic glycolysis with less ATP.But when it generates new vessels,aerobic glycolysis provides rapid and abundant ATP support for angiogenesis,and thus inhibition of glycolysis in endothelial cells can be a target for the treatment of neovascularization.Aerobic glycolysis has a protective effect on Müller cells,and it can provide with a target for visual protection and maintenance of the blood-retinal barrier.Under physiological conditions,the mitochondria of RPE can use lactic acid produced by photoreceptor cells as an energy source to provide ATP for survival.In pathological conditions,because RPE cells avoid their oxidative damage by increasing glycolysis,a large number of glycolysis products accumulate,which in turn has a toxic effect on photoreceptor cells.This shows that stabilizing the function of RPE mitochondria may become a target for the treatment of diseases such as retinal degeneration.The decrease of aerobic glycolysis leads to the decline of photoreceptor cell function and impaired vision;therefore,aerobic glycolysis of stable photoreceptor cells provides a reliable target for delaying vision loss.It is of great significance to study the role of glycolysis in various retinal cells for the targeted treatment of ocular fundus diseases.

Chordin-like 2 influences the differentiation fate of retinal pigment epithelium cells by dynamically regulating BMP pathway

AIM:To explore the functions of Chordin-like 2,which is encoded by CHRDL2,in the process of retinal pigmented epithelium(RPE)differentiation and damage repair.METHODS:The fetal RPE cells(f RPE)was obtained from aborted fetus which obeyed medical ethics.Real-time quantitative polymerase chain reaction was used to measure expression quantity of CHRDL2 and other functional genes expression.Knocking down and overexpression was used to analyze the functions about Chordin-like 2.Enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of bone morphogenetic proteins 4(BMP4).Flow cytometry was used to analyze cell cycle.Cell morphology was observed by phase contrast microscope(PCM).RESULTS:In normal RPE cells,CHRDL2 was firstly upregulated and followed a downregulation but eventually,it was expressed higher than the cells which undergone epithelial-mesenchymal transition(EMT).After knocking down CHRDL2,the secretion of BMP4 was decreased,RPErelated genes(OTX2,MITF,RPE65)were downregulated while EMT-related genes(SNAI1,VIM)were upregulated.However,the expression of these related genes after overexpression of CHRDL2 had contrary results.Chordin-like 2 also regulated the cell cycle by regulating BMP pathway.When CHRDL2 was knocked down,more f RPE cells stayed in S phase of cell cycle,while adding BMP4 reduced the proportion of the cells in S phase.However,overexpression of CHRDL2 increased more BMP4 secretion,this effect decreased the number of cells in S phase,but exogenous BMP inhibitor also could change this effect.At last,in the process of RPE cells differentiation,adding BMP4 at early stage could intervene normal RPE differentiation.Compared with BMP4,inhibiting BMP pathway had no significant negative effect at early stage,but suppressed differentiation at late stage.CONCLUSION:BMP pathway can be activated in a correct temporal order,otherwise,the cells have incorrect differentiation orientation.And Chordin-like 2 plays a role in dynamic regulation of BMP pathway and it also regulates the differentiation of RPE cells.Therefore,this research enlightens a new direction to inhibit EMT and promote cell redifferentiation after injury.

A novel xeno-free culture system for human retinal pigment epithelium cells

AIM: To find out an animal-free, xeno-free culture method for human fetal retinal pigment epithelium(fRPE) cells aiming for cell-replacement therapy. METHODS: Human AB serum, knock-out serum replacement(KSR) and B27 were supplemented as a substitute of fetal bovine serum(FBS) in culture medium of human fRPE cells. Cell morphology was examined by light microscope and transmission electron microscope. Proliferation ability was detected by cell cycle analysis and examination of KI67 expression. Apoptosis was analyzed using FACS. The expression ofRPE-specific markers was demonstrated by quantitative real-time polymerase chain reaction(qPCR), Western blot(WB) and immunocytochemistry. Paracrine function was determined using enzyme-linked immunosorbent assay method.RESULTS: Our results indicated that the optimum concentration of KSR was 15%, the optimum concentration of B27 was 2%, and the optimum concentration of human AB serum was 10%. fRPE cells cultured in 15% KSR and 2% B27 media showed repressed propagation and differentiation ability, and gradually lost epithelial morphology and RPE function. While fRPE cells cultured in 10% human AB serum media showed a typical cobblestone morphology with pigmentation, elevated proliferation ability, satisfying paracrine function and expressed RPE-specific markers. CONCLUSION: Our study indicates that culturing fRPE cells in 10% human AB serum-supplemented medium is more favorable compared with KSR, B27 and traditional FBS-supplemented mediums when fRPE cells are to be applied in cell-based therapy.

Pigment Epithelium-derived Factor in Cataractous Aqueous Humor and Lens Epithelial Cells

Purpose: To study the characteristics of PEDF in cataractous aqueous humor and its expression in human lens epithelium. Methods: The PEDF concentration in the aqueous humor was measured by enzyme -linked immunosorbent assay in senile (130cases) and congenital (18cases) cataract patients who underwent cataract phacoemulsification extraction surgery. Anterior lens capsular specimens were obtained from these patients to count lens epithelial cells (LEC) density. The Lens Opacities Classification System Ⅲ was used to classify the senile cataracts as cortical, nuclear, posterior subcapsular and mixed types of opacity, and quantitative analysis of the nuclear opacities was performed by Pentacam Scheimpflug imaging system. Anterior lens capsular specimens from another senile (10cases) and congenital (10cases) cataract were collected for immunofluorescence with polyclonal antibodies specific to human pigment epithelium -derived factor (PEDF). Results:The mean aqueous level of PEDF was(178. 9±87. 5)ng/ml, and there was negative linear correlation of PEDF level and age (r=0. 811, P<0. 001). In senile cases, the aqueous PEDF concentration decreased with increasing nuclear opacities (r=0. 447, P < 0.01) , and the mean PEDF level in nuclear cataract was significantly lower than that in posterior subcapsular opacity (P < 0.01) . PEDF immunostaining was detected in LEC of all capsular specimens. Conclusion : The PEDF level in human aqueous humor is related to age, types of cataracts and lens opacity. PEDF also express in human LEC. The study results suggest PEDF may regulate and/or protect LEC by paracrine and autocrine, and lack of PEDF may play a role in cataractogenesis.

Down regulation of UCP2 expression in retinal pigment epithelium cells under oxidative stress: an in vitro study

AIM: To evaluate the expression of uncoupling protein 2(UCP2) in a retinal pigment epithelium cell line(ARPE-19), under oxidative stress(OS).METHODS: ARPE-19 cells were divided into groups treated with various concentrations of hydrogen peroxide(H2 O2;0, 150, 300, 500, 700, and 900 μmol/L) for 24 h, to induce oxidative damage and cell viability was assessed by MTT assay. UCP2 mRNA expression in cells treated with H2 O2 was investigated by reverse transcription-polymerase chain reaction(RT-PCR). UCP2 protein expression was assessed by Western blotting and ROS levels analyzed by flow cytometry(FCM). Further, UCP2-siRNA treated cultures were exposed to H2 O2(0, 75, 150, and 300 μmol/L) for 2 h and cell viability determined by MTT assay.RESULTS: Cells treated with higher concentrations of H2 O2 appeared shrunken;their adhesion to adjacent cells was disrupted, and the number of dead cells increased. The results of cell viability assays demonstrated that the numbers of cells were decreased in a dose-dependent manner following treatment with H2 O2. Compared with untreated controls, cell viability was significantly reduced after treatment with >300 μmol/L H2 O2(P<0.05). Cell metabolic activity was decreased with increased concentrations of H2 O2 as detected by MTT assay. Levels of OS were further decreased in cells treated with UCP2-siRNA compared with those treated with H2 O2 alone(P<0.05). The results of RT-PCR and Western blotting demonstrated that UCP2 expression was reduced in H2 O2-treated groups compared with controls(P<0.05). FCM analysis showed that cell reactive oxygen species(ROS) levels were increased in H2 O2-treated groups and further upregulated by UCP2-si RNA treatment(P<0.05).CONCLUSION: Expression levels of UCP2 are decreased in ARPE-19 cells treated with H2 O2. ROS levels are further increased in cells treated with UCP2-siRNA relative to those treated with H2 O2 alone. UCP2 may have a protective role in ARPE-19 cells during oxidative injury.

Development-related mitochondrial properties of retinal pigment epithelium cells derived from hEROs

AIM:To explore the temporal mitochondrial characteristics of retinal pigment epithelium(RPE)cells obtained from human embryonic stem cells(hESC)-derived retinal organoids(hEROs-RPE),to verify the optimal period for using hEROs-RPE as donor cells from the aspect of mitochondria and to optimize RPE cell-based therapeutic strategies for age-related macular degeneration(AMD).METHODS:RPE cells were obtained from hEROs and from spontaneous differentiation(SD-RPE).The mitochondrial characteristics were analyzed every 20 d from day 60 to 160.Mitochondrial quantity was measured by MitoTracker Green staining.Transmission electron microscopy(TEM)was adopted to assess the morphological features of the mitochondria,including their distribution,length,and cristae.Mitochondrial membrane potentials(MMPs)were determined by JC-1 staining and evaluated by flow cytometry,reactive oxygen species(ROS)levels were evaluated by flow cytometry,and adenosine triphosphate(ATP)levels were measured by a luminometer.Differences between two groups were analyzed by the independentsamples t-test,and comparisons among multiple groups were made using one-way ANOVA or Kruskal-Wallis H test when equal variance was not assumed.RESULTS:hEROs-RPE and SD-RPE cells from day 60 to 160 were successfully differentiated from hESCs and expressed RPE markers(Pax6,MITF,Bestrophin-1,RPE65,Cralbp).RPE features,including a cobblestonelike morphology with tight junctions(ZO-1),pigments and microvilli,were also observed in both hEROs-RPE and SDRPE cells.The mitochondrial quantities of hEROs-RPE and SD-RPE cells both peaked at day 80.However,the cristae of hEROs-RPE mitochondria were less mature and abundant than those of SD-RPE mitochondria at day 80,with hEROsRPE mitochondria becoming mature at day 100.Both hEROs-RPE and SD-RPE cells showed low ROS levels from day 100 to 140 and maintained a normal MMP during this period.However,hEROs-RPE mitochondria maintained a longer time to produce high levels of ATP(from day 120 to 140)than SD-RPE cells(only day 120).CONCLUSION:hEROs-RPE mitochondria develop more slowly and maintain a longer time to supply high-level energy than SD-RPE mitochondria.From the mitochondrial perspective,hEROs-RPE cells from day 100 to 140 are an optimal cell source for treating AMD.

LRG1 promotes epithelial-mesenchymal transition of retinal pigment epithelium cells by activating NOX4

AIM:To investigate the effect of leucine-rich-alpha-2-glycoprotein 1(LRG1)on epithelial-mesenchymal transition(EMT)in retinal pigment epithelium(RPE)cells,and to explore the role of NADPH oxidase 4(NOX4).METHODS:RPE cells(ARPE-19 cell line)were treated with transforming growth factor-β1(TGF-β1)to induce EMT.Changes of the m RNA and protein expression levels of LRG1 were tested in the TGF-β1 treated cells.The recombinant human LRG1 protein(r LRG1)and si RNA of LRG1 were used to establish accumulation of exogenous LRG1 model and the down-regulation of LRG1 model in ARPE-19 cells respectively,and to detect EMT-related markers including fibronectin,α-smooth muscle actin(α-SMA)and zonula occludens-1(ZO-1).The m RNA and protein expression level of NOX4 were measured according to the above treatments.VAS2870 was used as a NOX4 inhibitor in r LRG1-treated cells.EMT-related markers were detected to verify the effect of NOX4 in the process of EMT.RESULTS:TGF-β1 promoted the expression of LRG1 at both the m RNA and protein levels during the process of EMT which showed the up-regulation of fibronectin andα-SMA,as well as the down-regulation of ZO-1.Furthermore,the r LRG1 promoted EMT of ARPE-19 cells,which manifested high levels of fibronectin andα-SMA and low level of ZO-1,whereas knockdown of LRG1 prevented EMT by decreasing the expressions of fibronectin andα-SMA and increasing the expression of ZO-1 in ARPE-19 cells.Besides,the r LRG1 activated and LRG1 si RNA suppressed NOX4 expression.EMT was inhibited when VAS2870 was used in the r LRG1-treated cells.CONCLUSION:These results for the first time demonstrate that LRG1 promotes EMT of RPE cells by activating NOX4,which may provide a novel direction to explore the mechanisms of subretinal fibrosis.

Pigment epithelium-derived factor protects retinal ganglion cells from hypoxia-induced apoptosis by preventing mitochondrial dysfunction

AIM： To investigate the potential of pigment epitheliumderived factor（PEDF） to protect the immortalized rat retinal ganglion cells-5（RGC-5） exposed to Co Cl2-induced chemical hypoxia. METHODS： After being differentiated with staurosporine（SS）, RGC-5 cells were cultured in four conditions： control group cells cultured in Dulbecco ＇s modified eagle medium（DMEM） supplemented with 10% fetal bovine serum, 100 μmol/m L streptomycin and penicillin（named as normal conditions）; hypoxia group cells cultured in DMEM containing 300 μmol/m L Co Cl2; cells in the group protected by PEDF were first pretreated with 100 ng/m L PEDF for 2h and then cultured in the same condition as hypoxia group cells; and PEDF group cells that were cultured in the presence of 100 ng/m L PEDF under normal conditions. The cell viability was assessed by MTT assay, the percentage of apoptotic cells was quantified using Annexin V-FITC apoptosis kit, and intra-cellar reactive oxygen species（ROS） was measured by dichloro-dihydro-fluorescein diacetate（DCFH-DA） probe. The mitochondria-mediated apoptosis was also examined to further study the underlying mechanism of the protective effect of PEDF. The opening of mitochondrial permeability transition pores（m PTPs） and membrane potential（Δψm） were tested as cellular adenosine triphosphate（ATP） level and glutathione（GSH）. Also, the expression and distribution of Cyt C and apoptosis inducing factor（AIF） were observed.RESULTS： SS induced differentiation of RGC-5 cells resulting in elongation of their neurites and establishing contacts between outgrowths. Exposure to 300 μmol/m L Co Cl2 triggered death of 30% of the total cells in cultures within 24 h. At the same time, pretreatment with 100 ng/m L PEDF significantly suppressed the cell death induced by hypoxia（P〈0.05）. The apoptosis induced by treatment of Co Cl2 was that induced cell death accompanied with increasing intracellar ROS and decreasing GSH and ATP level. PEDF pretreatment suppressed these effects（P〈0.05）. Additionally, PEDF treatment inhibited the opening of m PTPs and suppressed decreasing of Δψm in RGC-5 cells, resulting in blocking of the mitochondrial apoptotic pathway.CONCLUSION： Pretreatment of RGC-5 cells with 100 ng/m L PEDF significantly decreases the extent of apoptosis. PEDF inhibits the opening of m PTPs and suppresses decreasing of Δψm. Moreover, PEDF also reduces ROS production and inhibits cellular ATP level＇s reduction. Cyt C and AIF activation in PEDF-pretreated cultures are also reduced. These results demonstrate the potential for PEDF to protect RGCs against hypoxic damage in vitro by preventing mitochondrial dysfunction.

Adeno-associated Virus Mediated LacZ Gene Transfect to Cultured Human Iris Pigment Epithelium Cells

Purpose: To study the feasibility of adeno-associated virus mediated gene transfection tocultured human iris pigment epithelium (IPE) cells in vitro.Methods: Recombinant replication deficient adeno-associated viruses (AAV) expressingLacZ gene were produced without helper virus. The LacZ gene was transduced into culturedhuman IPE cells.Results: Cultured human IPE cells stained positively anticytokeratin, The titer ofrAAV-LacZ was 2.1 × 108 virus particles/ml, 42% cultured human IPE cells expressedβ-galactosidase 7 days after transfection and 67% after 14 days.Conclusions: Recombined AAV produced without helper virus can transfer a foreign geneinto human IPE cells with high efficiency in vitro.

Limbal stem cells: Central concepts of corneal epithelial homeostasis

A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent stud-ies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal

Human papillomavirus 16 E6 is associated with the nuclear matrix of esophageal carcinoma cells

AIM: To explore the etiologic role of HPV infection in esophageal carcinoma, and the association of HPV-16 E6with the nuclear metrix of carcinoma cells.METHODS: Two esophageal carcinoma cell lines, EC/CUHK1 and EC/CUHK2, were tested for HPV-16 E6subgenetic fragment by polymerase chain reaction amplification of virus DNA associated nuclear matrix. RT-PCR and immunocytochemistry were also used to visualizethe expression of E6 subgene in the cells.RESULTS: The HPV-16 E6 subgenetic fragment wes found to be present in nuclear metrix-associeted DNA, E6oncoprotein localized in the nucleus where it is tightly associated with nuclear matrix after sequential extraction in EC/CUHK2 cells. It was not detected, however, in EC/CUHK1 cells.CONCLUSION: The interaction between HPV-16 E6 and nuclear matrix may contribute to the virus induced carcinogenesis in esophageal carcinoma.

Notch pathway inhibitor DAPT enhances Atoh1 activity to generate new hair cells in situ in rat cochleae

Atoh1 overexpression in cochlear epithelium induces new hair cell formation. Use of adenovirus-mediated Atoh1 overexpression has mainly focused on the rat lesser epithelial ridge and induces ectopic hair cell regeneration. The sensory region of rat cochlea is difficult to transfect, thus new hair cells are rarely produced in situ in rat cochlear explants. After culturing rat cochleae in medium containing 10% fetal bovine serum, adenovirus successfully infected the sensory region as the width of the supporting cell area was significantly increased. Adenovirus encoding Atoh1 infected the sensory region and induced hair cell formation in situ. Combined application of the Notch inhibitor DAPT and Atoh1 increased the Atoh1 expression level and decreased hes1 and hes5 levels, further promoting hair cell generation. Our results demonstrate that DAPT enhances Atoh1 activity to promote hair cell regeneration in rat cochlear sensory epithelium in vitro.

Niche regulation of corneal epithelial stem cells at the limbus

Among all adult somatic stem cells, those of the corneal epithelium are unique in their exclusive location in a defined limbal structure termed Palisades of Vogt. As a result, surgical engraftment of limbal epithelial stem cells with or without ex vivo expansion has long been practiced to restore sights in patients inflicted with limbal stem cell deficiency. Neverthe- less, compared to other stem cell examples, relatively little is known about the limbal niche, which is believed to play a pivotal role in regulating self-renewal and fate decision oflimbal epithelial stem cells. This review summarizes relevant literature and formulates several key questions to guide future research into better understanding of the pathogenesis of limbal stem cell deficiency and further improvement of the tissue engineering of the corneal epithelium by focusing on the limbal niche.

Recurrent hepatitis C virus after transplant and the importance of plasma cells on biopsy

Hepatitis C virus(HCV) is the leading indication for liver transplantation in the United States.It recurs universally after transplant but the rate of fibrosis and the development of graft failure is variable.Different donor and recipient features have been demonstrated to impact fibrosis.Plasma cell hepatitis,a histologic finding,is one feature associated with poor graft and patient outcomes.The pathogenic mechanism resulting in plasma cell hepatitis is poorly understood,with evidence suggesting a role for both the HCV and the immune system.A recent publication described plasma cell hepatitis in a larger context of immune medicated graft dysfunction in transplant recipients receiving interferon based therapy.This manuscript will highlight the topic of plasma cell hepatitis and provide commentary on the lack of recognition,the data regarding pathophysiologic mechanisms and the potential management options.

Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models

AIM: To evaluate the biological functions of tissue-engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models. METHODS: TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy. RESULTS: After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575 mu m in thickness during the monitoring period. A 45 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. CONCLUSION: The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.