Cat Corneal Endothelial Cells Research Articles

Apoptotic effects of norfloxacin on corneal endothelial cells.

Norfloxacin, a frequently used ocular antibiotic, might have cytotoxic effect on human corneal endothelial cells (HCECs), subsequently damage the cornea and finally impair human vision. However, the possible mechanisms of cytotoxicity of norfloxacin to HCEC line are unclear. Herein, we investigated the cytotoxicity of norfloxacin and its underlying cellular and molecular mechanisms using in vitro cultured non-transfected HCECs and verified the cytotoxicity with cat corneal endothelium in vivo. In the present study, the cytotoxicity of norfloxacin in the in vitro cultured HCECs was recognized by causing abnormal morphology such as cell shrinkage and detachment from plate bottom, and decline of viability of in vitro cultured HCECs. Then, its cytotoxicity was verified by inducing reduction of cell density and morphological abnormality of in vivo cat corneal endothelial cells. Furthermore, the cytotoxicity of norfloxacin in HCECs was corroborated as apoptosis by elevation of plasma membrane permeability, S phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation in in vitro cultured HCECs and apoptosis-like swollen cells in the in vivo model. Moreover, norfloxacin induced extrinsic death receptor-mediated apoptosis pathway by activating caspase-2/-8/-3 and intrinsic mitochondrion-dependent apoptosis pathway by downregulating anti-apoptotic Bcl-2 and upregulating of pro-apoptotic Bad, which disrupted mitochondrial transmembrane potential, subsequently upregulated cytoplasmic cytochrome c and apoptosis-inducing factor and finally activated caspase-9/-3. Generally, norfloxacin induces HCE cell apoptosis via a death receptor-mediated and mitochondrion-dependent signaling pathway.

Human β-NGF gene transferred to cat corneal endothelial cells.

To transfect the cat corneal endothelial cells (CECs) with recombinant human β-nerve growth factor gene adeno-associated virus (AAV-β-NGF) and to observe the effect of the expressed β-NGF protein on the proliferation activity of cat CECs. The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco's modified Eagle's medium (DMEM) to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-β-NGF was constructed. The recombinant human AAV-β-NGF was transferred into cat CECs directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-β-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the β-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method (FCM); cell morphology was observed under inverted phase contrast microscope. The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05); there was significant difference between two control groups and recombinant CEC-AAV-β-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-β-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05). FCM result showed that the percentage of G1cells in CEC-AAV-NGF group was 76.8% while that in normal CEC control group and CEC-AAV control group was 46.6% and 49.8%. Recombinant AAV-β-NGF promotes proliferation in cat CECs by expressing bioactive β-NGF protein in high efficiency and suggests that its modulation can be used to treat vision loss secondary to corneal endothelial dysfunction.

Cytotoxicity of proparacaine to human corneal endothelial cells &lt;i&gt;in vitro &lt;/i&gt;

Proparacaine is a widely used topical anesthetic in ophthalmic optometry and surgery, and has been reported to have cytotoxic effects on rabbit corneal endothelial cells after prolonged and repeated usage. Since rabbit is an exceptive mammal whose corneal endothelial cells still maintaining proliferation abilities even in adulthood, whether proparacaine has cytotoxic effects on human corneal endothelial (HCE) cells need to be further verified. Our objectives in the present study were to investigate the cytotoxicity to HCE cells of proparacaine and its underlying mechanisms in vitro and verify the cytotoxicity using cat corneal endothelial (CCE) cells in an in vivo model of cat corneas. Cytotoxic evaluation results indicated that a dose- and time-dependent toxic response of HCE cells to proparacaine over 0.03125% was rated based on morphology and viability, and a toxic response of CCE cells to 0.5% (clinical applied dosage) proparacaine was also rated based on cell density and histology. Importantly, treatment with proparacaine resulted in significant elevation of plasma membrane permeability, cell cycle arrest at S phase, fragmentation of genomic DNA, formation of apoptotic bodies, and externalization of phosphatidylserine (PS) of HCE cells. Moreover, proparacaine demonstrated disrupting effects on mitochondrial transmembrane potential (MTP) of HCE cells and activating effects on caspase-3, -8 and -9. This study demonstrates that proparacaine has notable cytotoxicity to both HCE cells in vitro and CCE cells in vivo, and its dose- and time-dependent cytotoxicity to HCE cells is achieved by inducing apoptosis via a mitochondrion-mediated caspase-dependent pathway. These findings provide new insights into the cytotoxicity and apoptosis-inducing effect of local anesthetics which should be used with great caution in the eye clinic.

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

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. 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. 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 a dose 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. 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.

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

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. 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. 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. The recombinant AAV-PDGF-B expresses biological active PDGF-BB protein in cat CECs, which promotes the viability and proliferation of cells.

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

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. 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™ lipofectine. The transfection efficiency of Effectene™ 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 (MTT) method. Cell morphology was observed under inverted phase contrast microscope. 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™ 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. 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 basic fibroblast growth factor on cat corneal endothelial cell proliferation.

To investigate the function of basic fibroblast growth factor (bFGF) on cat corneal endothelial cells proliferation. 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. 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. bFGF can promote proliferation of cat corneal endothelial cells. 10ng/mL is the relatively most effective dose.

Cloning, expression and functional analyses of human platelet-derived growth factor-B chain peptide for wound repair of cat corneal endothelial cells

To investigate the biological function of platelet-derived growth factor B (PDGF-B) on the survival and proliferation of cat corneal endothelial cells so as to provide bases for further studies of its role in wound repair and its clinical application. Total RNA was extracted from the placenta tissues of healthy pregnant women undergoing hysterotokotomy and PDGF cDNA was obtained with reverse transcription-polymerase chain reaction (RT-PCR). The prokaryotic expression vector pET-PDGF-B was constructed and expressed the recombinant PDGF-B in Escherichia coli (E. coli) BL21 (DE3). After purification and refolding on Ni2+-chelation affinity chromatography (NTA) column, it was used to culture cat corneal endothelial cells. Cell proliferation was tested by modified tertrazolium salt (MTT) and flow cytometer. And the morphologic change and the ultrastructure were observed under an inverted phase contrast microscope, a scanning electron microscope and a transmission electon microscope, respectively. PDGF-B chain peptide (PDGF-BB) gene was successfully inserted into the prokaryotic expression vector, pET-28a (+). The purified recombined protein pET-PDGF-B showed a single band on sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) with the molecular weight of about 27 u, which was in agreement with the deduced value. MTT and flow cytometry showed that PDGF-BB promoted the survival and proliferation of cat corneal endothelial cells. The construction of recombinant prokaryotic expression vector pET-PDGF-B and the preparation of PDGF-BB protein provide a foundation for further study of the function of PDGF-BB and producing biological PDGF-BB protein. The expressed PDGF-BB promotes the proliferation of cultured cat corneal endothelial cells.

Proliferation and regeneration effects of constructed human beta-nerve growth factor gene on transfected cat corneal endothelial cells in vitro

To explore the mechanisms of proliferation and regeneration effects of a human nerve growth factor (beta-NGF) expression vector (pcDNA4-beta-NGF) on the transfected cat corneal endothelial cells in vitro. To provide a new method for long term cultivation of human corneal endothelial cells in vitro and to establish theoretical basis of gene therapy for corneal endothelial defects. It was a experimental study. The human pcDNA4-beta-NGF expression vector was constructed and transfected into cultured cat corneal endothelial cells by Effectene lipofectine transfection technique. The expression of the reporter gene pcDNA4-beta-LacZ expression was used to determine the transfection efficiency 48 hours after the transfection. RT-PCR and immunohistochemistry techniques were used to check the transient expression status at mRNA and protein levels in cat corneal endothelial cells. Mitotic index and methyl thiazolyl tetrazolium (MTT) value were measured and cell numbers at different stages of cell cycles were determined by flow cytometer 96 hours after transfection. An in vitro quantitative cat corneal endothelial cell traumatic model was established which was used for observing the effect of human beta-NGF expression product on the DNA synthesis of cat endothelial cells and healing process of traumatized endothelial cells. A human nerve growth factor (beta-NGF) expression vector (pcDNA4-beta-NGF)was successfully constructed and confirmed by sequence analysis. Single layered pure cat corneal endothelial cells were obtained by a modified sliced tissue culture technique and confirmed by morphological analysis, neurone specific enolase immunohistochemistry study and transmission electronic microscope. Effectene lipofectine mediated transfection efficiency of pcDNA4-beta-NGF into cat corneal endothelial cells in vitro was 11.3%. The human beta-NGF could be highly expressed in the transfected corneal endothelial cells at mRNA and protein levels. Mitotic index, MTT value and G1 stage cell numbers, as well as traumatically defected endothelial cells numbers during the healing process of human beta-NGF transfected corneal endothelial cells were statistically differed from the pre-transfected cells and control groups. Effectene lipofectine transfection technique could be effectively used for transfecting pcDNA4-beta-NGF into cat corneal endothelial cells in vitro with good efficacy and the gene could stably express to improve the proliferation and regeneration of the cat corneal endothelial cells. This method could be managed as an experimental basis to be applied in the experimental study for transfecting the human beta-NGF gene into human corneal endothelial cells. Therefore a new method for resolving the problem of impossible regeneration of corneal endothelial cells could become possible.

Using Basement Membrane of Human Amniotic Membrane as a Cell Carrier for Cultivated Cat Corneal Endothelial Cell Transplantation

Purpose: To study the feasibility of using basement membrane of human amniotic membrane (BMHAM) as a carrier for transplantation of cultivated cat corneal endothelial cells (cCCECs). Methods: BHMAM was obtained by enzymic digestion. cCCECs were seeded on the BMHAM and cultivated traditionally. The resulting continuous monolayer of cCCECs was transplanted onto the cat corneal graft stripped of the Descemet membrane with endothelium. To determine whether the transplanted cCCECs were vital and functional in vivo, the corneal grafts were examined by slit-lamp microscope every day for 6 weeks, and corneal thickness was measured by ultrasonic pachymetry. Either in vivo or in vitro, the cCCEC sheets on BMHAMs were examined morphologically by light and electron microscope, and the cell density was measured. Results: Seven to 10 days after seeding on the BMHAM, the cCCECs were confluent and formed a continuous monolayer with 3486 ± 53 cells/mm2 cell density. Like normal corneal endothelial cells, the cCCECs were almost hexagonal, squamous, and uniform in size. After transplantation, most cells were vital and functional nearly enough to maintain corneal graft thickness and transparency without rejection for at least 6 weeks. Six weeks after operation, the average thickness of the transplanted corneal grafts was only slightly greater than that before operation. Compared with that in vitro, after transplantation there was 5% to 8% reduction per week in cell density, which lasted for almost 3 weeks. After that, the average cCCEC density of corneal grafts was 2837 ± 57 cells/mm2 and quite stable maintained. Conclusions: This study demonstrated that BMHAM would be an ideal alternative for corneal Descemet membrane and a cell carrier for cCCEC transplantation.

Synergistic effect of platelet-derived growth factor BB and basic fibroblast growth factor on cat corneal endothelial cell proliferation

目的 观察血小板源性生长因子(PDGF-BB)和碱性成纤维细胞生长因子(bFGF)对猫角膜内皮细胞(CECs)增生的影响及其联合应用对细胞增生是否有协同作用.方法 猫CECs原代培养,传1代后,分别在培养液中加不同浓度的PDGF-BB和bFGF,加药后第1、3、5 d分别利用MTT法测定在490 nm处的吸光值来判断CECs的增生情况;同时应用倒置相差显微镜观察细胞形态,扫描电镜检测细胞超微结构.结果 与对照组比较,加药后第1、3、5 d,PDGF-BB和bFGF均能增加培养的猫CECs的数量,最有效质量浓度分别为100ng/ml和10 ng/ml,二者联合培养组猫CECs的增生能力更强.结论 PDGF-BB和bFGF可促进猫CECs的增生,二者具有协同作用。

Effect of Chondroitin Sulfate on the Endothelium in Corneal Storage

The scope of this study includes investigations on uptake of dextran and chondroitin sulfate in human donor corneas, and effects of chondroitin sulfate on adhesion and growth of rabbit and cat corneal endothelial cells. Fibronectin and dextran sulfate served as controls. Nuclear magnetic resonance spectroscopy revealed no detectable osmotic agents in the corneas stored in either Dexsol or Optisol at 4 degrees C for 6 days. The study showed that chondroitin sulfate enhanced cell adhesion marginally at 2.5%, but >40% at 0.5%, comparable to that of 10 mu g/ml fibronectin, whereas 2% dextran sulfate abolished approximately 70% of cell adhesion capability. In cultures with fibronectin present, the duration for cells to reach confluence was extended from 1.75 to 3 days. Chondroitin sulfate (2%) elicited no apparent cytotoxic effect, with cells becoming polygonal and reaching confluence in approximately 9 days. Cell growth was retarded by 2% dextran sulfate, with signs of senescence on day 3 and clear evidence of cell degeneration on day 9. The cells did not survive without serum in cultures, especially at 36 degrees C. These findings suggest that corneal deterioration probably is linked primarily to serum-free storage conditions, not uptake of osmotic agents in the cornea. The highly negatively charged sulfate group, however, may have rendered chondroitin sulfate less effective as an additive.

Changes in corneal endothelial morphology in cats as a function of age.

A cross-sectional study of changes in cat corneal endothelial cell morphology with age was conducted. The central corneal endothelium of 12 kittens and 70 adult cats was photographed using specular microscopy. Endothelial cell density (ECD), coefficient of variation of cell size (used as an index of polymegethism), and cellular shape factor (perimeter 2/area) were determined for each animal and analysed as a function of age. We found a rapid non-linear decrease in ECD and polymegethism in the first nine months of post-natal life. Subsequently there was a slight central cell loss of 11 cells/mm2 or 0.37% per year during adult life which was not statistically significant. However, polymegethism increased significantly with age during adult life. The shape factor for endothelial cells was 13.61 throughout adult life, indicating that the cat corneal endothelium consists predominantly of six-sided cells. Possible explanations for the finding of no significant decrease in cell density with age could include the higher peripheral ECD in the cat which may compensate for central loss, the short life expectancy of the cat and the large individual variation in corneal diameter.

Regeneration of cat corneal endothelium induced in vivo by fibroblast growth factor

The corneal endothelium of man, primates and carnivores has limited regenerative ability. Fibroblast growth factor (FGF) stimulates the proliferation of various mesoderm-derived cells, including corneal endothelial cells, in vitro. In the present work the effect of FGF on the regeneration of injured cat endothelium in vivo was studied. Scraping of cat corneal endothelium off Descemet's membrane was carried out by a specially designed instrument. FGF from crude preparations (25 micrograms in 50 microliter saline) or affinity purified FGF (0.5 micrograms in 25 microliter saline) were injected immediately after the scraping of the endothelium, and in both cases significant stimulation of endothelium regeneration was observed. During the first 2 weeks after scraping, the endothelial cell density (ECD) in FGF-treated eyes was higher by a factor of 1.7-2.3 than that in the control eyes. This marked difference decreased with time, but even after 12 weeks the ECD in FGF-treated eyes was higher by a factor of 1.15-1.30 than in the control eyes. Furthermore, FGF improved the polygonal shape of the cells and decreased the corneal thickness. These results clearly demonstrate the efficacy of FGF in inducing the proliferation of cat corneal endothelial cells in vivo and indicate its potential application in clinical practice.