Effects of low-dose of TGF-β1 on maintaining bovine corneal stromal cell growth and retarding extra cellular matrix fibrosis in a three-dimensional culture model / 中华实验眼科杂志

Background Transforming growth factor-β1(TGF-β1) plays an important role in corneal woundhealing.The effects of TGF-β1 on the synthesis of extra cellular matrix (ECM) vary upon different concentrations.Previous studies focused on the effects of high concentration of TGF-β1 on keratocytes under the two-dimensional culture condition,and the effect of low concentration of TGF-β1 on the synthesis of ECM in keratocytes remains unclear.Objective This study was to investigate the growth of Pellet,a three-dimensional model of corneal stroma cells in vitro,and its ECM synthesis under a low concentration of TGF-β1.Methods Bovine corneal stromal cells were isolated from fresh bovine eyeballs by two-step digestion by collagenase and cultured using DMEM/F12 medium with 10％ fetal bovine serum (FBS).Pellets derived fresh bovine keratocytes with culture medium containing 0.25 ng/ml TGF-β1 +5％ FBS and 0.50 ng/ml TGF-β1 +5％ FBS were established,respectively.The morphology of Pellets was observed under the natural light at 48 hours,1 week,2 weeks and 3 weeks after culture.In 3 weeks after culture,the cell structures was observed by hematoxylin-eosin staining,and Calcein-AM/propidium (Calcein-AM/PI) staining was used to assay the cell viability.Real-time flurorescence quantitative PCR and immunofluorescence technology were applied to analyze the expressions of α-smooth muscle actin (α-SMA),fibronectin (FN),type Ⅰ collagen (Col Ⅰ) and type Ⅲ collagen (Col Ⅲ) mRNA and proteins.RT-PCR was employed to detect the expressions of lumican (LUM) mRNA and keratocan (KERA) mRNA in the cells.Results Cells in Pellet clustered throughout the culture duration.Hematoxylin-eosin staining showed the mass red-dyed collagen fibers in both 0.25 ng/ml TGF-β1 +5％ FBS group and 0.50 ng/ml TGF-β1 +5％ FBS group,and most cells possessed complete structures.The death rate of the cells was (33.60±1.65)％ in the 0.25 ng/ml TGF-β1 +5％ FBS group and (30.90±0.78) ％ in the 0.50 ng/ml TGF-β1 +5％ FBS group,showing an insignificant difference between them (t =0.144,P=0.887).The expressions of α-SMA,FN and Col Ⅲ proteins in 0.25 ng/ml TGF-β1 +5％ FBS group were lower than those in the 0.50 ng/ml TGF-β1 + 5 ％ FBS group (tα-SMA =4.622,P =0.010;tFN =2.973,P =0.040;tCol Ⅲ =7.845,P＜0.001),but the expression of Col Ⅰ in 0.25 ng/ml TGF-β1 +5％ FBS group was higher than that in 0.50 ng/ml TGF-β1+5％ FBS group (tColⅠ =4.022,P=0.016).The ratio of Col Ⅲ/Col Ⅰ in 0.25 ng/ml TGF-β1+5％ FBS group was lower than that in the 0.50 ng/ml TGF-β1 +5％ FBS group in both mRNA and protein level (tmRNA =-3.039,P =0.038;tprotein =3.215,P =0.032).The expression of LUM mRNA and KERA mRNA were detected in Pellet at different time points.The expression of LUM mRNA in 0.25 ng/ml TGF-β1 +5％ FBS group increased over time.While in 0.50 ng/ml TGF-β1 +5％ FBS group,the expression of LUM mRNA peaked at 1 week but declined at 2 weeks.The expression of KERA mRNA in two groups were all peaked at 1 week but declined at 2 weeks.Conclusions Low-dose TGF-β1 in Pellet can maintain the normal growth of keratocytes and synthesize ECM.The expression of ECM tends to the normal condition after reducing the concentration of TGF-β1,implying a scarless expression.

Establishment of a three-dimensional corneal stroma extracellular matrix fibrosis model induced by transforming growth factor-β1 in vitro / 中华实验眼科杂志

Background Extracellular matrix (ECM) fibrosis leads to corneal scaring during the process of cornea wound healing.Transforming growth factor-β1 (TGF-β1) is known to mediate overproduce of ECM components.Our previous study developed a three-dimensional model for corneal stromal cells culture in vitro.Objective The hypothesis of this study was to apply TGF-β1 in the three-dimensional culture system to establish a corneal stroma ECM fibrosis model.Methods Fresh bovine corneas were extracted for the culture of bovine keratocytes in constructed three-dimension culture system.The Pellets were cultured in the DMEM/F12+ 10％ fetal bovine serum (FBS) medium with 0.5 ng/ml or 1.0 ng/ml TGF-β1 or without TGF-β1,respectively.Calcein AM/(propidium iodide) PI staining was employed to assay the cell viability 2 weeks after culture.The expressions of α-smooth muscle actin (α-SMA),type Ⅰ collagen (Col Ⅰ) and Col Ⅲ mRNA and protein in the cells were detected by real-time PCR and Western blot respectively 48 hours,1 week and 2 weeks after cultured.The results were statistically analyzed.Results Cultured for 48 hours in the Pellet system,corneal stromal cells clustered and was identified alive by Calcein-AM/PI staining in 2 weeks.The relative expression levels of α-SMA,Col Ⅰ and Col m mRNA were elevated in both the 0.5 ng/ml and 1.0 ng/ml TGF-β1 supplement groups in comparison with the only DMEM/F12+10％ FBS group,with marked difference among the three groups (Fgroup =696.745,P＜0.001;Fgroup =35.166,P＜0.001;Fgroup =33.677,P＜0.001),and the expression levels increased with the lapse of culture time (Ftime =5.863,P＜0.05;Ftime =298.614,P＜0.001;Ftime =607.472,P＜0.001).The synthetic rate of Col Ⅲ mRNA was obviously faster than that of Col Ⅰ mRNA.Western blot showed that only a trace of α-SMA,Col Ⅰ and Col Ⅲ were detected 48 hours and 1 week after culture.The expression levels of α-SMA,Col Ⅰ and Col Ⅲ in Pellet system in 0.5 ng/ml TGF-β1 medium were 0.395±0.208,1.060±0.175 and 0.629±0.382,and in 1.0 ng/ml TGF-β1 medium were 0.758±0.228,1.201 ±0.187 and 0.753±0.468,respectively 2 weeks after culture,significant differences were shown among the three groups (α-SMA:F=10.691,P＜0.05;Col Ⅰ:F=14.094,P＜0.05;Col Ⅲ:F=10.995,P＜0.05).Conclusions Addition of TGF-β1 and serum enhance the assembly and fibrosis of ECM,showing the higher expressions of specific fibrotic markers in bovine keratocytes Pellet.This culture systerm can be used as a candidate three-dimensional model for corneal stroma ECM fibrosis.

Two Cases of Corneal Toxicity in Acanthamoeba Keratitis by Combined Topical Anti-Acanthamoeba Keratitis Eye Solution

PURPOSE: To present a case of corneal toxicity caused by therapeutic duplication during treatment of acanthamoeba keratitis as a complication of contact lens use. CASE SUMMARY: A 12-year-old girl with a history of wearing orthokeratology contact lenses and a 14-year-old girl with a history of wearing cosmetic contact lenses presented with ocular pain, injection, and decreased visual acuity. They were diagnosed as having acanthamoeba keratitis based on slit lamp examination, confocal microscopy and culture. After the patients were treated with polyhexamethylene biguanide (PHMB) and chlorhexidine, corneal epithelial defect and erosion occurred. Use of chlorhexidine was stopped, and PHMB was used to treat patients and recovery of the corneal epithelium with improvement in symptoms of acanthamoeba keratitis was found. CONCLUSIONS: Using PHMB and chlorhexidine together in treating acanthamoeba keratitis increases the risk of corneal toxicity. Therefore, these drugs should be avoided in combination.

Microarray Analysis for Genes Associated with Angiogenesis in Diabetic OLETF Keratocytes

The purpose of this study was to identify the differences in angiogenesis gene expression between normal and diabetic keratocytes stimulated with interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha). Primarily cultured normal and diabetic keratocytes were treated with 20 ng/mL of IL-1a and TNF-alpha for 6 hr. cDNA was hybridized to an oligonucleotide microarray. Microarray analysis was used to identify differentially expressed genes that were further evaluated by real-time polymerase chain reaction (RT-PCR). Diabetes keratocytes overexpressed vital components of angiogenesis including Agtr1, and under-expressed components related to the blood vessel maturation, including Dcn. Cytokine-treated diabetic keratocytes differentially expressed components of angiogenesis. OLETF keratocytes after treatment with IL-1alpha and TNF-alpha showed the newly expressed 15 and 14 genes, respectively. Newly and commonly under-expressed five genes followed by treatment with both IL-1alpha and TNF-alpha were also evident. RT-PCR showed results similar to the microarray results. Agtr1 and Itga1 showed an increased expression in diabetic keratocytes compared with normal corneal keratocytes, especially after TNF-alpha treatment. Il6 appeared strong expression after interleukin-1alpha treatment, but showed down expression after TNF-alpha treatment. Further studies to analyze and confirm the significance of the identified angiogenetic genes of diabetes are needed.

Effect of Amoebicidal Agents on the Human Corneal Keratocytes in Vitro

PURPOSE: To evaluate the effect of polyhexamethylene biguanide (PHMB) and chlorhexidine on Acanthamoeba cysts and cultured human keratocytes. METHODS: Each well of two-fold diluted PHMB and chlorhexidine were treated on the Acanthamoeba cyst suspension of 5 x 10(4) cysts/ml for 8, 24, and 48 hours to measure the minimal cysticidal concentration (MCC) of each disinfectant and was exposed to the human corneal keratocytes of 5 x 10(4) cells/ml for same hours to measure the survival rate of keratocytes. Inverted phase-contrast micrograph and electron microscopy for observing the morphologic changes were evaluated. RESULTS: MCC of PHMB was 9.42 microgram/ml, 5.62 microgram/ml, and 2.37 microgram/ml, and chlorhexidine was 24.32 microgram/ml, 10.02 microgram/ml, and 7.02 microgram/ml respecitvely in 8, 24, and 48 hours. The survival rate of keratocytes at MCC was 91.7%, 64.6%, and 49.7% in PHMB of which significant decreases were found at 24 and 48 hours, and 95.7%, 90.6%, and 78.1% in chlorhexidine of which significant decrease was only found at 48 hours. The higher the concentration of disinfectants, cysts and keratocytes demonstrated more damaged appearance. CONCLUSIONS: The amoebicidal efficacy of PHMB and chlorhexidine was similar. However, in consideration of toxic effect on keratocytes by disinfectants, chlorhexidine is suggested to be more clinically useful than PHMB.

Effect of Tranilast on the Proliferation of Human Corneal Keratocytes in Vitro

PURPOSE: To evaluate the inhibitory effect of tranilast on proliferation of cultured human keratocytes, and to investigate the apoptotic response and the cellular morphologic changes associated with tranilast in vitro. METHODS: Human corneal keratocytes were exposed to tranilast at a concentration of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg/ml for a period of 4, 24, and 48 hours. Evaluations were conducted with MTT-based-calorimetric assay for measuring the metabolic activity, flow cytometric analysis and fluorescent micrograph for assessing the apoptotic response, and inverted phase-contrast micrograph and electron microscopy for observing the morphologic changes. RESULTS: The inhibitory effect of human keratocyte proliferation was found to have a dose and time dependent pattern (p<0.05). In flow cytometry, the maximal apoptotic response developed at 0.8 mg/ml concentration after 4 and 24 hours of exposure time, and apoptotic cells were demonstrated in fluorescent micrograph. At higher concentration of Tratnilast, human corneal keratocytes were more swollen rather than having a spindle shape and being detached from the bottom of the dish. The damaged keratocytes had degenerative and apoptotic changes like the formation of phagolysosomal granule, marginal condensation in the nucleus, and bleb formation of the nuclear membrane. CONCLUSIONS: The apoptotic response of tranilast is concerned with the inhibitory effect of human corneal keratocyte proliferation. Therefore, tranilast shows promise in clinical use for the inhibition of postoperative excimer laser induced corneal opacity or haze with fewer side effects.

Effect of Dexamethasone and Diclofenac on the Proliferation of Human Corneal Keratocytes

PURPOSE: To evaluate the inhibitory effect of dexamethasone and diclofenac on the proliferation of cultured human keratocytes, and to investigate the apoptotic response and the cellular morphologic changes associated with dexamethasone and diclofenac in vitro. METHODS: Human corneal keratocytes were exposed to 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mM concentration of dexamethasone and diclofenac for 4, 24, and 48 hours. MTT based calorimetric assay, flow cytometric analysis, fluorescent micrograph, inverted phase-contrast micrograph, and electron microscopy were used to evaluate the results. RESULTS: The inhibitory effect of human keratocyte proliferation increased at higher concentrations and longer exposure times of dexamethasone and diclofenac (p<0.05). In flow cytometry, the maximal apoptotic response developed at 0.4 mM concentration of dexamethasone and 1.6 mM concentration of diclofenac after 4 hours. Apoptotic cells were demonstrated in fluorescent micrograph. Dexamethasone-treated cases showed a more damaged appearance, more swollen rather than spindle shaped, with greater detachment from the bottom of the dish and the chromatin of the nuclear remnant condensed along the nuclear periphery with cytoplasmic vesication and cytoplasmic blebs formation, and partial disruption of the nuclear membrane compared with diclofenac. CONCLUSIONS: The apoptotic response of dexamethasone and diclofenac is associated with the inhibitory effect of human corneal keratocyte proliferation. For inhibition of cellular proliferation of human corneal keratocytes, dexamethasone may be more effective at lower concentration and shorter exposure time than diclofenac.

Inhibitory Effect of Mitomycin C (MMC) on the Proliferation of Human Corneal Keratocyte

PURPOSE: To evaluate the anti-proliferative effect of mitomycin C (MMC) on human corneal keratocyte, and to investigate the cellular morphology of keratocyte according to the concentration and exposure time in vitro. METHODS: Human corneal keratocytes using endothelium-free explant method were exposed to 0.005%, 0.01%, and 0.05% concentration of MMC for 3, 5, and 10 minutes. MTT based colorimetric assay was performed to assess the inhibition of cellular proliferation, and cellular morphology was evaluated by inverted phase-contrast light microscope and electron microscope. RESULTS: Use of higher concentration MMC and prolongation of exposure time resulted in greater inhibitory effect on cellular proliferation. When exposed to 0.005% MMC for 3, 5 and 10 minutes, the survival rate of keratocyte was 100%, 95.7% and 74.0% respectively. At 0.01% MMC, the survival rate was 98.6%, 92.9%, and 66.9%. At 0.05% MMC, it was 74.0%, 73.4%, and 38.8%. Exposure to the highest concentration (0.05%) among the 3 preparations for 3 or 5 minutes showed significant inhibition of keratocyte proliferation (p<0.05), and when exposed for 10 minutes, all 3 preparations showed significant inhibition of keratocyte proliferation (p<0.05). Inverted phase-contrast light microscopy showed that human corneal keratocytes lost their adherence to the bottom of the dish and assumed round and swollen shape rather than spindle shape when exposed to higher concentration of MMC for a prolonged time. The damaged keratocytes showed the degenerative changes like cellular membrane disruption, disappearance of microvilli, enlargement of rough surfaced endoplasmic reticulum and mitochondria, and vacuole formation by electronic microscope. CONCLUSIONS: When MMC is applied to inhibit the proliferation of keratocytes involved in corneal wound healing, it seems to be a valuable application at least 0.05% concentration for 3 minutes. Further studies should be followed for the biological effect of MMC including drug toxicity associated with human corneal tissue in vivo.

Effect of Anti-inflammatory Mediator on the Proliferation of Human Corneal Keratocyte

PURPOSE: The purpose of this study was to evaluate the effect of anti-inflammatory mediators like dexamethasone, nordihyroguaratic acid (NDGA), and diclofenac sodium on proliferation of human corneal keratocytes, and to investigate the cellular morphology of keratocyte. METHODS: Human corneal keratocytes were exposed to 0.05, 0.1, 0.3, 0.8, and 1.0 mM concentration of each drug for period of 24, 48, and 72 hours. MTT based colorimetric assay was performed to assess the metabolic activity and inhibition of cellular proliferation. Cellular morphology was evaluated by inverted phase contrast micrograph and electron microscopy. RESULTS: The higher the concentration of inoculated each drugs was, the more the inhibitory effect of human keratocyte proliferation was found (P<0.05). NDGA, over 0.3 mM and diclofenac, more than 0.1 mM had significant more inhibitory effect on keratocyte proliferation compared with dexamethasone within 48 hours of exposure to each drug. With the concentration and exposure time of each drug, human corneal keratocytes were visible more rounded and swollen rather than spindle shape, and detached from the bottom of the dish. The damaged keratocytes had degenerative changes like cellular membrane disruption, microvilli disappearance, enlarged rough surfaced endoplasmic reticulum and mitochondria, vacuole formation and nuclear membrane damage by TEM. CONCLUSIONS: On basis of this study, the anti-inflammatory mediators such as NDGA and diclofenac sodium have less side effects and stronger inhibitory effects of human keratocyte proliferation than dexamethasone.