Cytotoxic effects of betaxolol on healthy corneal endothelial cells both in vitro and in vivo

AIM: To demonstrate the cytotoxic effect of betaxolol and its underlying mechanism on human corneal endothelial cells(HCE cells) in vitro and cat corneal endothelial cells(CCE cells) in vivo,providing experimental basis for safety anti-glaucoma drug usage in clinic of ophthalmology. ·METHODS: In vivo and in vitro experiments were conducted to explore whether and how betaxolol participates in corneal endothelial cell injury. The in vitro morphology,growth status,plasma membrane permeability,DNA fragmentation,and ultrastructure of HCE cells treated with 0.021875-0.28g/L betaxolol were examined by light microscope,3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide(MTT) assay,acridine orange(AO)/ethidium bromide(EB) double-fluorescent staining,DNA agarose gel electrophoresis,and transmission electron microscope(TEM). The in vivo density,morphology,and ultrastructure of CCE cells,corneal thickness,and eye pressure of cat eyes treated with 0.28g/L betaxolol were investigated by specular microscopy,applanation tonometer,alizarin red staining,scanning electron microscope(SEM),and TEM. · RESULTS: Exposure to betaxolol at doses from 0.0875g/L to 2.8g/L induced morphological and ultrastructural changes of in vitro cultured HCE cells such as cytoplasmic vacuolation,cellular shrinkage,structural disorganization,chromatin condensation,and apoptotic body appearance. Simultaneously,betaxolol elevated plasma membrane permeability and induced DNA fragmentation of these cells in a dose-dependent manner in AO/EB staining. Furthermore,betaxolol at adose of 2.8g/L also induced decrease of density of CCE cells in vivo,and non-hexagonal and shrunk apoptotic cells were also found in betaxolol-treated cat corneal endothelia. ·CONCLUSION: Betaxolol has significant cytotoxicity on HCE cells in vitro by inducing apoptosis of these cells,and induced apoptosis of CCE cells in vivo as well. The findings help provide new insight into the apoptosis-inducing effect of anti-glaucoma drugs in eye clinic.

Effects of corneal stromal cell-and bone marrow-derived endothelial progenitor cell-conditioned media on the proliferation of corneal endothelial cells

AIM： To explore the effects of conditioned media on the proliferation of corneal endothelial cells （CECa） and to compare the efficiency of different conditioned media （CM）. METHODS： Rat CECs, corneal stromal cells （CSCs）, bone marrow -derived endothelial progenitor cells （BEPCs）, and bone marrow-derived mesenchymal stem cells （BMSCs） were isolated and cultured in vitra CM was collected from CSCs, BEPCs, and BMSCSo CECs were cultivated in different culture media. Cell morphology was recorded, and gene and protein expression were analyzed.~ RESULTS： After grown in CM for 5d, CECs in each experimental group remained polygonal, in a cobblestone- like monolayer arrangement. Immunocytofluorescence revealed positive expression of Na＋/K＋-ATP, aquaporin 1 （AQP1）, and zonula occludens 1 （ZO-1）. Based on quantitative polymerase chain reaction （qPCR） analysis, Na ＋/K ＋-ATP expression in CSC-CM was notably upregulated by 1.3-fold （＋0.036） （P〈0.05, n=3）. The expression levels of ZO-1, neuron specific enolase （NSE）, Vimentin, paired homebox 6 （PAX6）, and procollagen type VII （COL8A1） were notably upregulated in each experimental group. Each CM had a positive effect on CEC proliferation, and CSC-CM had the strongest effect on proliferation.~ CONCLUSION： CSC-CM, BEPC-CM, and BMSC-CM not only stimulated the proliferation of CECs, but also maintained the characteristic differentiated phenotypes necessary for endothelial functions. CSC-CM had the most notable effect on CEC proliferation. KEYWORDS： conditioned medium; corneal endothelial cell; corneal stromal cell; bone marrow-derived endothelial progenitor cell; proliferation

Nerve Growth Factor Modulate Proliferation of Cultured Rabbit Corneal Endothelial Cells and Epithelial Cells

Summary： In order to investigate the effect of nerve growth factor （NGF） on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF.MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mI. and 500 U/mI. NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

Transforming growth factor-β2 induces morphological alteration of human corneal endothelial cells in vitro

AIM:To investigate the morphological altering effect of transforming growth factor-β2(TGF-β2) on untransfected human corneal endothelial cells(HCECs)in vitro.METHODS:After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology,cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy,immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2(9 μg/L) altered HCE cell morphology after treatment for 36 h, increased the mean optical density(P 【0.01) and the length of F-actin,reduced the mean optical density(P 【0.01) of the collagen type IV in extracellular matrix(ECM) and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72 h.·CONCLUTION: TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.

Manifestations of type 2 diabetes in corneal endothelial cell density,corneal thickness and intraocular pressure

We sought to evaluate central corneal thickness（CCT）,corneal endothelial cell density（ECD）and intraocular pressure（IOP）in patients with type 2 diabetes mellitus（DM）and to associate potential differences with diabetes duration and treatment modality in a prospective,randomized study.We measured ECD,CCT and IOP of125 patients with type 2 DM（mean age 57.1±11.5 years）and compared them with 90 age-matched controls.Measured parameters were analyzed for association with diabetes duration and glucose control modalities（insulin injection or oral medication）while controlling for age.In the diabetic group,the mean ECD（2511±252 cells/mm^2）,mean CCT（539.7±33.6μm）and mean IOP（18.3±2.5 mmHg）varied significantly from those the control group[ECD：2713±132 cells/mm^2（P〈0.0001）,CCT：525.0±45.3μm（P=0.003）and IOP：16.7±1.8 mmHg（P〈0.0001）].ECD was significantly reduced by about 32 cell/mm^2 for diabetics with duration of〉10 years when compared with those with duration of〈10 years（P〈0.05）.CCT was thicker and IOP was higher for diabetics with duration of〉10 years than those with duration of〈10 years（P〉0.05）.None of the measured parameters was significantly associated with diabetes duration and treatment modality（P〉0.05）.In conclusion,subjects with type 2DM exhibit significant changes in ECD,IOP and CCT,which,however,are not correlated with disease duration or if the patients receive on insulin injection or oral medications.

Small extracellular vesicles derived from cerebral endothelial cells with elevated microRNA 27a promote ischemic stroke recovery

Axonal remodeling is a critical aspect of ischemic brain repair processes and contributes to spontaneous functional recovery.Our previous in vitro study demonstrated that exosomes/small extracellular vesicles(sEVs)isolated from cerebral endothelial cells(CEC-sEVs)of ischemic brain promote axonal growth of embryonic cortical neurons and that microRNA 27a(miR-27a)is an elevated miRNA in ischemic CEC-sEVs.In the present study,we investigated whether normal CEC-sEVs engineered to enrich their levels of miR-27a(27a-sEVs)further enhance axonal growth and improve neurological outcomes after ischemic stroke when compared with treatment with non-engineered CEC-sEVs.27a-sEVs were isolated from the conditioned medium of healthy mouse CECs transfected with a lentiviral miR-27a expression vector.Small EVs isolated from CECs transfected with a scramble vector(Scra-sEVs)were used as a control.Adult male mice were subjected to permanent middle cerebral artery occlusion and then were randomly treated with 27a-sEVs or Scra-sEVs.An array of behavior assays was used to measure neurological function.Compared with treatment of ischemic stroke with Scra-sEVs,treatment with 27a-sEVs significantly augmented axons and spines in the peri-infarct zone and in the corticospinal tract of the spinal grey matter of the denervated side,and significantly improved neurological outcomes.In vitro studies demonstrated that CEC-sEVs carrying reduced miR-27a abolished 27a-sEV-augmented axonal growth.Ultrastructural analysis revealed that 27a-sEVs systemically administered preferentially localized to the pre-synaptic active zone,while quantitative reverse transcription-polymerase chain reaction and Western Blot analysis showed elevated miR-27a,and reduced axonal inhibitory proteins Semaphorin 6A and Ras Homolog Family Member A in the peri-infarct zone.Blockage of the Clathrin-dependent endocytosis pathway substantially reduced neuronal internalization of 27a-sEVs.Our data provide evidence that 27a-sEVs have a therapeutic effect on stroke recovery by promoting axonal remodeling and improving neurological outcomes.Our findings also suggest that suppression of axonal inhibitory proteins such as Semaphorin 6A may contribute to the beneficial effect of 27a-sEVs on axonal remodeling.

Effect of recombinant human platelet-derived growth factor B on cat corneal endothelial cell viability mediated by adeno-associated virus

AIM: To transduce recombinant human platelet-derived growth factor B (PDGF-B) gene adeno-associated virus(AAV) to in vitro cultured cat corneal endothelial cell (CEC) and observe the effect of the expressed PDGF-BB protein on the viability of cat CEC. METHODS: Cat cornea endothelium was torn under microscope and rapidly cultivated in DMEM to form single layer CEC and the passage 2 endothelial cells were used in this study. The recombinant human PDGF-B gene AAV was constructed and transduced into cat CEC directly. Three groups were as following: blank control group, AAV control group and recombinant AAV group. At 24 hours, 48 hours, and 5 days after transduction, total RNA was extracted from the CEC by Trizol and the expression of PDGF-B gene was detected by fluorescence quantitative polymerase chain reaction. Viability of the transduced CEC was detected at 48 hours after transduction by MTT assay. Cell morphology was observed under inverted phase contrast microscope. RESULTS: With the torn endothelium culture technique, we rapidly got single layer cat CEC. At 24 hours, 48 hours and 5 days after transduction, fluorescence quantitative polymerase chain reaction showed there was no significant difference of the expressed PDGF-B gene mRNA between blank control group and AAV control group (P>0.05). In contrast, there were significant differences between two control groups and recombinant AAV group (P<0.05). MTT assay showed that in recombinant AAV group, the expressed PDGF-BB protein could promote the viability of cat CEC. Morphology observation showed at 48 hours after transduction, cells in CEC-AAV-PDGF-B group proliferated into bigger scales in regular triangle to hexagon shape with distinct boundary, while the number of cells was significantly less in the two control groups. CONCLUSION: The recombinant AAV-PDGF-B expresses biological active PDGF-BB protein in cat CEC, which promotes the viability and proliferation of cells.

Corneal endothelial cell density and morphology and central corneal thickness in Guangxi Maonan and Han adolescent students of China

AIM: To investigate the corneal endothelial cell density and morphology and central corneal thickness in the Guangxi Maonan and Han adolescent students of China.METHODS: Noncontact specular microscope(Topcon SP3000 P, Tokyo, Japan) was performed in 133 adolescent students of Maonan nationality(M:F 54:79)and 105 adolescent students of Han nationality(M:F 50:55),5 to 20 y of age, who were randomly selected from 3schools in Huanjiang Maonan Autonomous County of Guangxi Zhuang Autonomous Region of China.Parameters studied included endothelial cell density,mean cell area, coefficient of variation in cell size,percentage hexagonality and central corneal thickness. RESULTS: Endothelial cell density, mean cell area,coefficient of variation in cell size, percentage hexagonality and central corneal thickness in the study population were(2969.50 ±253.93) cells/mm2,(339.23 ±29.44) μm2,(29.96 ±4.07) %,(64.58 ±9.41) % and(523.71 ±32.82) μm in Maonan and(2998.26 ±262.65) cells/mm2,(336.11±30.07) μm2,(29.89±5.03) %,(64.91±11.64) % and(524.39 ±33.15) μm in Han, respectively. No significant differences were observed in endothelial cell density,mean cell area, coefficient of variation in cell size,percentage hexagonality and central corneal thickness between Maonan and Han(P =0.615, 0.659, 0.528, 0.551,0.999). In Maonan and Han, we found age was negatively correlated with endothelial cell density and percentagehexagonality and positively correlated with mean cell area and coefficient of variation in cell size. Negative correlation was also found between central corneal thickness and age in Han, whereas no correlation was found in Maonan. CONCLUSION: There were no differences between Maonan and Han in corneal endothelial cell density and morphology and central corneal thickness. In these two nationalities, there were statistically significant decrease in endothelial cell density and percentage hexagonality with increasing age and statistically significant increase in cell area and coefficient of variation in cell size with increasing age. Central corneal thinned with increasing age in Han, whereas difference did not attain statistical significance in Maonan.

Effect of basic fibroblast growth factor on cat corneal endothelial cell proliferation

AIM: To investigate the function of basic fibroblast growth factor (bFGF) on cat corneal endothelial cells proliferation. METHODS: Cat corneal endothelial cells were primarily cultured, stimulated with bFGF for different period, the proliferation of cells was assayed by modified tertrozalium salt (MTT) method, and the morphologic changes were observed with inverted phase contrast microscope and transmission electron microscope. RESULTS: At 1, 3 and 5 days after bFGF was added to cat corneal endothelial cells, the result of MTT in 490nm showed significant difference than that in control group, and the difference was most significant in 10ng/mL group. CONCLUSION: bFGF can promote proliferation of cat corneal endothelial cells. 10ng/mL is the relatively most effective dose.

The study of human PDGF-B gene transferred to cat corneal endothelial cells

AIM: To demonstrate that human platelet-derived growth factor-B (PDGF-B) cDNA could be Expressed in primary cultured cat corneal endothelia cells by using gene transfer techniques; to explore a useful tool for the further studies of the molecular mechanisms of corneal endothelium failure and provide a potential effective genetic therapy for the blind patients. ' METHODS: Human PDGF-B cDNA was isolated from human placent by RT-PCR and inserted into pcDNA(4) vector to construct recombinant eukaryotic expression plasmid pcDNA(4)-PDGF-B. The full length was confirmed by the DNA sequencing analysis. By tearing endothelium technique we obtained pure single layer of cat corneal endothelial cells. The pcDNA(4)-PDGF-B eukaryotic Expression vector was transferred into cat corneal endothelial cells by Effectene (TM) lipofectine. The transfection efficiency of Effectene (TM) lipofectine in pcDNA(4)-B was detected with pcDNA(4)-GFP. 5 days later, RT-PCR was used to check the PDGF-B expression. Cell viability was tested by modified tertrozalium salt (MU) method. Cell morphology was observed under inverted phase contrast microscope. RESULTS: The human PDGF-B cDNA was isolated successfully from healthy parturien placent tissue and the sequence was confirmed by computer automatic sequence and PCR analysis. Pure single layer cat corneal endothelial cells were successfully cultured by tearing endothelium technique. Effectene (TM) lipofectine transfection technique could be effectively used to transfer pcDNA(4)-PDGF-B into cat corneal endothelial cells in vitro, the transfection efficiency was 30%. RT-PCR result showed that human PDGF-B gene was highly expressed in transfected cat corneal endothelial cells. The expressed PDGF-BB protein promoted the viability of cat corneal endothelial cells. CONCLUSION: Human platelet-derived growth factor-B (PDGF-B) cDNA could be highly Expressed in cultured cat corneal endothelial cells by gene transfection techniques. Expressed PDGF-BB protein significantly promoted the viability of cat corneal endothelial cells, thus provided a potential effective method for corneal endothelium blindness genetic therapy.

Effect of p27Kip1 Inhibition on Proliferation of Bovine Corneal Endothelial Cells by RNA Interference

Three plasmids （pGenesil-P1, pGenesil-P2, pGenesil-P3） with different p27Kip1-shRNA sequences were designed and synthesized. Their effects on the proliferation of bovine corneal endothelial cells （bCEC） were investigated. Plasmid expressing irrelevant shRNA with a random combination was used as negative control （pGenesil-HK）. The recombination of four plamids was confirmed by restrictive enzyme digestion and sequence analysis. The expression of mRNA and protein of p27Kip1 was detected by RT-PCR and Western blotting after stable transfection. The expressions of p27Kip1 mRNA and p27Kip1 protein of pGenesil-P1 group, pGenesil-P2 group and pGenesil-P3 group were all lower than those in the pGenesil-HK group and the blank group （non-transfected group）, pGenesil-P3 had the strongest inhibitory effect and was selected for the next steps. The proliferation rates of the pGenesil-P3 group, the pGenesil-HK group and the blank group were assessed by MTT. The influence of shRNA-p27Kip1 on bCEC cell cycle was detected by flow cytometry （FCM）. Compared with the control groups, the proliferation rate of the pGenesil-P3 group was increased significantly, and the ratio of S-phase also increased. It is concluded that shRNA-p27Kip1 could down-regulate the expression of p27Kip1 effectively and increase the proliferation of bCEC. RNA interference （RNAi） may be an effective means to promote the proliferation of CEC.

Mechanism of induction of fibroblast to corneal endothelial cell

Objective:To explore mechanism of nduction of fibroblast to corneal endothelial cell.Methods:Rabbit conjunctiva fibroblasts were used as feeder cells,rabbit oral mucosa epithelial cells were used as seed cells,and human denuded amniotic membrane was used as carrier to establish tissue engineering corneal endothelium.The transformation effect was observed.Results:As concentration of mitomycin C increased,cell survival rale gradually decreased,cell proliferation was obviously inhibited when concentration ≥25μg/mL;5 days alter being treated by 5μg/mL.mitomycin C,cell body was enlarged and extended without cell fusion,however after being treated by 0.5 μg/mL mitomycin C,cell body was significantly proliferated and gradually fused:alter 3 weeks of culture,stratified epithelium appeared on rabbit oral mucosa epithelial cells,differentiation layers were 4-5 and were well differentiated,the morphology was similar to corneal endothelial cells;Under electron microscope,surface layer of cells were polygonal,tightly connected to another with microvilli on the bonier,there was hemidesmosome between basal cells and human denuded amniotic membrane.Conclusions:Fibroblast cells have the potential of multi-directional differentiation,effective induction can promote emergence of intercellular desmosomes between seed cells and emergence ol epithelial surface microvilli,and differentiate to the corneal endothelial cell.However,clinical application still needs more research and safetv evaluation.

A study of chitosan blend film as a carrier of corneal endothelial cells

In order to evaluate the cytocompatibility and biocompatibility of a new kind of chitosan blend film as a carrier of corneal endothelial cell, rabbit corneal endothelial cells cultured in vitro were breeded onto the film. After a cell monolayer formed, the scanning electron micrography was performed. After inplanted into anterior chamber, slit lamp observation, thickness metering, specular microscopy and HE staining were performed at random time after operation to evaluate the biocompatibility. Inflmmnation in anterior, thickness of cornea, cell density, hexagonality and cell size of the surgical cornea were taken as the indexes of biocompatibility. The cultured cells exhibited a confluent monolayer 10 days after incubation, which proved the satisfactory cytocompatibility of this film. Biocompatibility assay results suggested the implantation feasibility of the film as a carder of corneal endothelial cells.

Gamma-glutamyl transferase 5 overexpression in cerebrovascular endothelial cells improves brain pathology,cognition,and behavior in APP/PS1 mice

In patients with Alzheimer’s disease,gamma-glutamyl transferase 5(GGT5)expression has been observed to be downregulated in cerebrovascular endothelial cells.However,the functional role of GGT5 in the development of Alzheimer’s disease remains unclear.This study aimed to explore the effect of GGT5 on cognitive function and brain pathology in an APP/PS1 mouse model of Alzheimer’s disease,as well as the underlying mechanism.We observed a significant reduction in GGT5 expression in two in vitro models of Alzheimer’s disease(Aβ_(1-42)-treated hCMEC/D3 and bEnd.3 cells),as well as in the APP/PS1 mouse model.Additionally,injection of APP/PS1 mice with an adeno-associated virus encoding GGT5 enhanced hippocampal synaptic plasticity and mitigated cognitive deficits.Interestingly,increasing GGT5 expression in cerebrovascular endothelial cells reduced levels of both soluble and insoluble amyloid-βin the brains of APP/PS1 mice.This effect may be attributable to inhibition of the expression ofβ-site APP cleaving enzyme 1,which is mediated by nuclear factor-kappa B.Our findings demonstrate that GGT5 expression in cerebrovascular endothelial cells is inversely associated with Alzheimer’s disease pathogenesis,and that GGT5 upregulation mitigates cognitive deficits in APP/PS1 mice.These findings suggest that GGT5 expression in cerebrovascular endothelial cells is a potential therapeutic target and biomarker for Alzheimer’s disease.

Multifaceted roles of lymphatic and blood endothelial cells in the tumor microenvironment of hepatocellular carcinoma:A comprehensive review

The tumor microenvironment is a complex network of cells,extracellular matrix,and signaling molecules that plays a critical role in tumor progression and metastasis.Lymphatic and blood vessels are major routes for solid tumor metastasis and essential parts of tumor drainage conduits.However,recent studies have shown that lymphatic endothelial cells(LECs)and blood endothelial cells(BECs)also play multifaceted roles in the tumor microenvironment beyond their structural functions,particularly in hepatocellular carcinoma(HCC).This comprehensive review summarizes the diverse roles played by LECs and BECs in HCC,including their involvement in angiogenesis,immune modulation,lymphangiogenesis,and metastasis.By providing a detailed account of the complex interplay between LECs,BECs,and tumor cells,this review aims to shed light on future research directions regarding the immune regulatory function of LECs and potential therapeutic targets for HCC.

Effects of Intraocular Lens Implantation without Viscoelastic Agents on Corneal Endothelial Cells

Objective: To determine the protective effects of intraocular lens implantation without viscoelastic agents on corneal endothelial cells. Methods: Patients with age-related cataract were randomly divided into two groups: Group A (24 patients, 30 eyes) underwent phacoemulsification and intraocular lens implantation without viscoelastic agents, and group B (21 patients, 30 eyes) underwent phacoemulsification and intraocular lens implantation with viscoelastic agents. The corneal endothelial cell counts, percentages of hexagonal cells, and central corneal thicknesses were evaluated at 1 week, 1 month, and 6 months after surgery. Results: There was no significant difference in preoperative basic characteristics between the two groups (p > 0.05). The postoperative corneal endothelial cell count and percentage of hexagonal cells in both groups decreased compared with preoperative values at 1 week, 1 month, and 6 months after surgery, and the decrease of group A was significantly lower than that of group B at all time points (p p > 0.05). Conclusions: Compared with regular intraocular lens implantation, intraocular lens implantation without viscoelastic agents is less damaging to corneal endothelial cells, resulting in greater corneal safety.

Age-related changes of corneal endothelial cell in healthy Chinese tree shrew measured by non-contact specular microscope

AIM：To determine the impact of age on the morphology of endothelial cells and central corneal thickness（CCT） in Chinese tree shrew.METHODS：One-hundred and twenty eyes of 60 healthy Chinese tree shrews were studied.Based on age,the tree shrews were divided into four groups.After general anesthesia,the images of endothelium were acquired using non-contact specular microscope Topcon 3000 P.Eight parameters of corneal endothelial cells were measured by built-in software,including CCT,endothelial cell density（ECD）,percent hexagonality（HG%）,coeffi cient of variability（CV）,size of minimal cell（Smin）,size of maximal cell（Smax）,average cells size（Savg） and size standard deviation（Ssd）.Data were analyzed using STATA software.The differences of eight parameters among groups and correlations with age were analyzed.RESULTS：In all studied animals,the average CCT was 2 4 9.6 ± 2 .29 μ m（202-301 μm）,ECD was 308 0.72 ± 460.76 cells/mm^2（1239.6-4047.6 cells/mm^2） and CV was 29.10±7.60（13.6-54.6）.CV was significantly different among different groups（P〈0.001）.Strong correlation with age was found in ECD,Smax,Savg,Ssd and CV.CONCLUSION：Cornea of Chinese tree shrews had half CCT of human cornea and similar ECD,CV and size of corneal endothelial cells.Young adult tree shrews had higher ECD,HG% and low CV.ECD,Smax,Savg,Ssd and CV correlated with age signifi cantly.

Mechanism of 5-Fluorouracil-Induced Apoptosis on Cultured Corneal Endothelial Cells

Repeated subconjunctival injections with 5-fluorouacil (5-FU) after trabeculectomy are widely used in glaucoma patients for the inhibition of excess scar formation in wound site. The aim of this study was to evaluate the toxic effects of 5-FU and mechanisms of drug-induced apoptosis in cultured porcine corneal endothelial cells. Cellular damage and the caspase pathway were estimated with a MTT assay. The apoptotic characteristics were detected with flow cytometry, a TUNEL test and Western blotting. The results indicated that 5-FU was toxic to corneal endothelial cells in a time- and dose-dependent manner. Pretreatment with a general caspase inhibitor, Z-VAD-FMK, a caspase-8 inhibitor, Z-IETD-FMK, and a caspase-9 inhibitor, Z-LEHD-FMK, reversed 5-FU-induced cellular damage. Following exposure to 5-FU, a flow cytometric assay with MitoLight dye demon-strated the loss of mitochondrial membrane potential. A positive TUNEL test revealed that cellular DNA apoptosis occurred following exposure to 0.05, 0.1, and 0.5 mg/ml 5-FU for 15 h. Annexin V-FITC and negative propidium iodide (PI) staining indicated that the cell membrane underwent apoptosis upon exposure to 0.1 and 0.5 mg/ml 5-FU for 15 h. The Western blot assay demonstrated up-regulation of the Bax, p53 and p21 proteins induced by 5-FU. Taken together, these data reveal that 5-FU-induced cellular apoptosis in corneal endothelial cells may be mediated through caspase-8, caspase-9 and mitochondrial regulated pathways, as well as by up-regulation of Bax-, p53-, and p21-dependent signal transduction pathways.

Efficacy of ICL combined with Trans-PRK on corneal endothelial cell density in patients with high myopia

Objective:To explore the efficacy of implantable collamer lense (ICL) combined with laser keratectomy via corneal epithelium Trans-PRK in the treatment of patients with high myopia and its effect on corneal endothelial cell density.Methods: A prospective analysis was performed on patients with ultra-high myopia admitted to the department of ophthalmology of our hospital from March 2014 to March 2017. Six months after ICL implantation, the patients still had myopia and received Trans PRK treatment again. The number of cases was 30 and 52 eyes. Patients were followed up postoperatively. Uncorrected visual acuity (UCVA), best corrected visual acuity (BCVA), dioptre (D), intraocular pressure, ICL arch height, anterior chamber volume, anterior chamber depth, and changes in corneal endothelial cell counts were observed preoperatively, 6 months after ICL implantation and 6 months after Trans PRK so as to assess the effectiveness and safety of the surgery.Results: UCVA and BCVA of the eyes were significantly higher 6 months after ICL implantation and 6 months after Trans-PRK than those before the surgery, and UCVA of the eyes 6 months after Trans-PRK was significantly higher than that 6 months after ICL implantation. There was no increase in intraocular pressure in all patients after surgery. There was no significant difference in intraocular pressure preoperatively, 6 months after ICL implantation and 6 months after Trans PRK. The ICL arch height of the eyes 6 months after Trans-PRK was slightly lower than that 6 months after ICL implantation, but the difference was not statistically significant. Both anterior chamber volume and anterior chamber depth were significantly lower 6 months after ICL implantation and 6 months after Trans PRK than whose before the surgery, and anterior chamber depth 6 months after Trans PRK was significantly lower than that 6 months after ICL implantation. The number of corneal endothelial cells 6 months after ICL implantation was slightly less than that before surgery, but it was not statistically significant. The number of corneal endothelial cells 6 months after Trans PRK was higher than that 6 months after ICL implantation and close to the preoperative level.Conclusion: Trans PRK can effectively improve the eye diopter of patients with high myopia and residual myopia after ICL implantation. It has little effect on the density of corneal endothelial cells, can significantly improve visual acuity, and achieve the expected operation effect with safe operation, which is worthy of clinical promotion.

Inhibition of viability of human retinal microvascular endothelial cells by vialinin A under high glucose condition

AIM:To investigate the effects of vialinin A on viability of human retinal endothelial cells(HRECs)under high glucose condition and its potential mechanism.METHODS:The HRECs were divided into four groups:normal glucose control group(NG,5 mmol/L D-glucose),high glucose group(HG,30 mmol/L D-glucose),HG+1μmol/L vialinin A group,and HG+5μmol/L vialinin A group.The cell viabilities were measured with cell counting kit-8(CCK-8)assay for proliferation,with scratch assay for migration,and tube formation,for evaluation of the impact of vialinin A on cellular behaviour.Real-time PCR and Western blotting were used to determine the expression level of vascular endothelial growth factor(VEGF).RESULTS:The proliferative capacity and migration of HRECs was reduced by 5μmol/L vialinin A in high glucose environment(both P<0.05).Vialinin A also inhibited highglucose-induced tube formation of HRECs.The expression level of VEGF and PI3K in HRECs was also significantly decreased by vialinin A(P<0.05).CONCLUSION:Vialinin A inhibits the cell viability of HRECs.It may serve as a potential target for anti-angiogenic therapy.