Migration Of Human Lens Epithelial Cells Research Articles

Abnormal function of EPHA2/p.R957P mutant in congenital cataract.

To identify genetic defects in a Chinese family with congenital posterior polar cataracts and assess the pathogenicity. A four-generation Chinese family affected with autosomal dominant congenital cataract was recruited. Nineteen individuals took part in this study including 5 affected and 14 unaffected individuals. Sanger sequencing targeted hot-spot regions of 27 congenital cataract-causing genes for variant discovery. The pathogenicity of the variant was evaluated by the guidelines of American College of Medical Genetics and InterVar software. Confocal microscopy was applied to detect the subcellular localization of fluorescence-labeled ephrin type-A receptor 2 (EPHA2). Co-immunoprecipitation assay was implemented to estimate the interaction between EphA2 and other lens membrane proteins. The mRNA and protein expression were analyzed by reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assay, respectively. The cell migration was analyzed by wound healing assay. Zebrafish model was generated by ectopic expression of human EPHA2/p.R957P mutant to demonstrate whether the mutant could cause lens opacity in vivo. A novel missense and pathogenic variant c.2870G>C was identified in the sterile alpha motif (SAM) domain of EPHA2. Functional studies demonstrated the variant's impact: reduced EPHA2 protein expression, altered subcellular localization, and disrupted interactions with other lens membrane proteins. This mutant notably enhanced human lens epithelial cell migration, and induced a central cloudy region and roughness in zebrafish lenses with ectopic expression of human EPHA2/p.R957P mutant under differential interference contrast (DIC) optics. Novel pathogenic c.2870G>C variant of EPHA2 in a Chinese congenital cataract family contributes to disease pathogenesis.

TGF-β/Smad Signalling Activation by HTRA1 Regulates the Function of Human Lens Epithelial Cells and Its Mechanism in Posterior Subcapsular Congenital Cataract.

Congenital cataract is the leading cause of blindness among children worldwide. Patients with posterior subcapsular congenital cataract (PSC) in the central visual axis can result in worsening vision and stimulus deprivation amblyopia. However, the pathogenesis of PSC remains unclear. This study aims to explore the functional regulation and mechanism of HTRA1 in human lens epithelial cells (HLECs). HTRA1 was significantly downregulated in the lens capsules of children with PSC compared to normal controls. HTRA1 is a suppression factor of transforming growth factor-β (TGF-β) signalling pathway, which plays a key role in cataract formation. The results showed that the TGF-β/Smad signalling pathway was activated in the lens tissue of PSC. The effect of HTRA1 on cell proliferation, migration and apoptosis was measured in HLECs. In primary HLECs, the downregulation of HTRA1 can promote the proliferation and migration of HLECs by activating the TGF-β/Smad signalling pathway and can significantly upregulate the TGF-β/Smad downstream target genes FN1 and α-SMA. HTRA1 was also knocked out in the eyes of C57BL/6J mice via adeno-associated virus-mediated RNA interference. The results showed that HTRA1 knockout can significantly upregulate p-Smad2/3 and activate the TGF-β/Smad signalling pathway, resulting in abnormal proliferation and irregular arrangement of lens epithelial cells and leading to the occurrence of subcapsular cataract. To conclude, HTRA1 was significantly downregulated in children with PSC, and the downregulation of HTRA1 enhanced the proliferation and migration of HLECs by activating the TGF-β/Smad signalling pathway, which led to the occurrence of PSC.

High glucose: activating autophagy and affecting the biological behavior of human lens epithelial cells.

To clarify the effect of autophagy on human lens epithelial cells (HLECs) under high glucose conditions. HLECs were cultured with different concentrations of glucose and 3-methyladenine (3-MA); the expression of autophagy-related protein LC3B was detected by Western blotting and immunofluorescence histochemistry. The migration of HLECs was quantified by scratch wound assay and the expression of transforming growth factor-β (TGF-β) was measured by real-time polymerase chain reaction. Compared with 5 mmol/L normal glucose treatment, 40 mmol/L glucose treatment can significantly increase the generation of autophagosome in HLECs, which could be inhibited by 0.375 mmol/L 3-MA treatment. The migration of HLECs and the expression of TGF-β in HLECs induced by high glucose were significantly suppressed by 0.375 mmol/L 3-MA treatment. Autophagy promotes HLECs cell migration and increases the expression of TGF-β after exposed to high glucose, which may relate to the development of diabetic cataract.

Inhibitory effects of glutathione 2 on excessive migration of human lens epithelial cells induced by hyperoxia

Objective To observe the influences of hyperoxia on the migration of human lens epithelial cells (HLECs) and the inhibitory effects of glutathione 2 (Grx2) on excessive migration of HLECs induced by hyperoxia. Methods The HLECs were divided into the hyperoxic model group and normoxic control group.HLECs were cultured in 80% oxygen mixture for 2 days to establish the oxidative damage model of HLECs induced by hyperoxia in vitro (hyperoxia model group), and cultured in normoxia mixture as the normoxic control group.Cell migration was observed by scratch test, cytoskeleton morphology was observed by phalloidin staining, and Grx2 mRNA level was detected by real-time quantitative PCR.HLECs in the hyperoxic model group and the normoxic control group were subdivided into empty plasmid transfection group and Grx2 plasmid transfection group, respectively.The migration behavior and cytoskeleton changes of hyperoxic model cells in different transfection groups were observed.MitoSOX staining was used to detect reactive oxygen species (ROS) content of mitochondria under flow cytometry, and JC-1 staining was used to observe the changes of mitochondrial membrane potential under laser-scanning confocal microscopy. Results In the process of cell culture, the cells grew well.Twenty-four hours and 48 hours after scratching, the scratch distance of the hyperoxic model group was smaller than that of the normoxic control group, with significant differences between them (both at P<0.05). The cell circumference of the hyperoxic model group was increased compared with that of the normoxic group, with a significant difference between them (t= -2.254, P<0.05). After 2 days culture, the relative expression level of Grx2 mRNA in the hyperoxic model group was 0.54±0.11, which was significantly lower than that in the normoxic control group (t=7.128, P<0.01). The transfection efficiency of Grx2 was higher than 90%.At 24 hours and 48 hours after scratching, the scratch distance in the Grx2 transfected hyperoxic model group was wider than that in the empty plasmid transfected hyperoxic model group, with significant differences between them (both at P<0.05). The relative expression level of Grx2 mRNA in Grx2 transfected cells was up-regulated compared with that in empty plasmid transfected cells, with a significant difference between them (P<0.01). Compared with the normoxic control group, the cell circumference of the hyperoxic model group was decreased, with a significant difference between them (P<0.05). The relative mean fluorescence intensity of mitochondrial ROS in the Grx2 transfected hyperoxic model group was lower than that in the empty plasmid transfected hyperoxic model group, with a significant difference between them (P<0.05). Under the hyperoxic condition, the red/green fluorescence intensity of Grx2 transfected group was higher than that of the empty plasmid transfected group, with a significant difference between them (P<0.01). Conclusions Grx2 may inhibit the excessive migration of HLECs under hyperoxia by regulating oxidative stress in mitochondria. Key words: Lens epithelial cells; Hyperoxia; Glutathione 2; Cell migration; Cell culture

Therapeutic potential of a dual mTORC1/2 inhibitor for the prevention of posterior capsule opacification: An in vitro study.

Mammalian target of rapamycin (mTOR) serves a central role in regulating cell growth and survival, and has been demonstrated to be involved in the pathological progression of posterior capsule opacification (PCO). In the present study, the potency of PP242, a novel dual inhibitor of mTOR complex 1/2 (mTORC1/2), in the suppression of the growth of human lens epithelial cells (HLECs) was investigated. Using a Cell Counting Kit-8 and a wound healing assay, it was demonstrated that PP242 inhibited the proliferation and migration of HLECs. In addition, western blot analysis indicated that PP242 completely inhibited mTORC1 and mTORC2 downstream signaling activities, whereas rapamycin only partially inhibited mTORC1 activity within LECs. Furthermore, PP242 treatment led to an upregulation of the expression levels of p53 and B cell lymphoma-2 (Bcl-2)-associated X and downregulation of Bcl-2. In addition, flow cytometric analysis demonstrated that PP242 induced the cell cycle arrest at the G0/G1 phase, which may have caused apoptosis and induced autophagy within the LECs. The results of the present study suggested that administration of PP242 may potentially offer a novel therapeutic approach for the prevention of PCO.

Long Non-Coding RNA H19 Regulates Human Lens Epithelial Cells Function

Background/Aims: Age-related cataract (ARC) remains the leading cause of visual impairment among the elderly population. Long non-coding RNAs (lncRNAs) have emerged as potential regulators in many ocular diseases. However, the role of lncRNAs in nuclear ARC, a subtype of ARC, requires further elucidation. Methods: LncRNA sequencing was performed to identify differentially expressed lncRNAs between the capsules of transparent and nuclear ARC lenses. Expression validation was confirmed by qRT-PCR. MTT assay, Calcein-AM and propidium iodide double staining, Rhodamine 123 and Hoechst double staining, EdU and transwell assay were used to determine the role of H19 or miR-675 in the viability, apoptosis, proliferation and migration of primary cultured human lens epithelial cells (HLECs). Bioinformatics and luciferase reporter assays were used to identify the binding target of miR-675. Results: Sixty-three lncRNAs are differentially expressed between the capsules of transparent and nuclear ARC lenses. One top abundantly expressed lncRNA, H19, is significantly up-regulated in the nuclear ARC lens capsules and positively associated with nuclear ARC grade. H19 knockdown accelerates apoptosis development and reduces the proliferation and migration of HLECs upon oxidative stress. H19 is the precursor of miR-675, and a reduction of H19 inhibits miR-675 expression. miR-675 regulates CRYAA expression by targeting the binding site within the 3’UTR. Moreover, miR-675 increases the proliferation and migration while decreasing the apoptosis of HLECs upon oxidative stress. Conclusion: H19 regulates HLECs function through miR-675-mediated CRYAA expression. This finding would provide a novel insight into the pathogenesis of nuclear ARC.

The proliferative inhibition and apoptosis promotion of Smac on human lens epithelial cells

Background Posterior capsular opacification (PCO) is a primary complication after extracapsular cataract extraction.The mechanism of PCO is associated with proliferation, migration and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs). To explore the target treatment of PCO is very important. Objective This study was to investigate the biological effects of second mitochondria-derived activator of caspases (Smac) on the proliferation and apoptosis of LECs. Methods Human LECs line (HLE-B3) and Smac-overexpressed LECs line were cultured, and the cells were transfected using small interfering RNA (siRNA)-Smac3 plasmid with green fluorescent protein (GFP) for 24 hours.Different concentration of transforming growth factor -β2 (TGF-β2) (5, 10, 20 and 50 μg/ml) or 200 μmol/L H2O2 were added respectively into the culture medium to establish PCO model and oxidative stress model.Cell counting kit-8 (CCK-8) assay was used to compare the cell proliferative activity among PBS group, TGF-β2 group and Smac-hyperexpression+ TGF-β2 group.Flow cytometry was used to evaluate the apoptotic rate of the PBS group, H2O2 group and siRNA-Smac+ H2O2 group.The expressions of Smac, caspase-3 and proliferating cell nuclear antigen (PCNA) mRNA and their proteins in the cells were detected by real-time quantitative PCR (RT-PCR) and Western blot. Results The GFP+ cells were≥ 80% 12 hours after siRNA-Smac3 transfection, with the optimal plasmid of siRNA-Smac3.GFP+ cell rate was (72.32±2.31)% in the siRNA-Smac3 transfection group, which was significantly higher than that in the blank plasmid group([4.91±0.24]%) (t=116.342, P<0.001). The relevant expression levels of Smac was 35.21±4.11 in the Smac-hyperexpression group, and that in the blank plasmid group was 15.24±2.48, with a significant difference between them (t=215.47, P<0.05). The cell viability of 20 ng/ml TGF-β2 affected PBS group, TGF-β2 group and Smac-hyperepression+ TGF-β2 group was (98.4±1.7)%, (98.9±0.1)% and (64.2±3.1)%, and the cell viability of Smac-hyperepression+ TGF-β2 group was significantly lower in the Smac-hyperepression+ TGF-β2 group than that in the TGF-β2 group (P<0.05). The apoptotic rate in the PBS group, H2O2 group and siRNA-Smac+ H2O2 group were (2.9±1.2) %, (45.1±4.5)% and (27.5±1.8)%, and the apoptotic rate was evidently lower in the siRNA-Smac+ H2O2 group than that in the H2O2 group (P<0.05). RT-PCR results showed that the expression levels of caspase-3 mRNA in PBS group, H2O2 group and siRNA-Smac+ H2O2 group were 0.321±0.103, 0.715±0.112 and 0.479±0.209, respectively.Compared with the H2O2 group, the relative expression level of caspase-3 mRNA in siRNA-Smac+ H2O2 group was significantly decreased, the difference was statistically significant (P<0.05). The PCNA mRNA expression levels in PBS group, TGF-β2 group and Smac-hyperepression+ TGF-β2 group were 0.299±0.013, 0.645±0.102 and 0.490±0.209, respectively.Western blot results showed that the relative expression of caspase-3 protein in siRNA-Smac+ H2O2 group and H2O2 group was 0.712±0.012 and 0.973±0.051, with significant difference between the two groups (t=132.52, P<0.05). The relative expression of PCNA protein in Smac-hyperepression+ TGF-β2 group was 0.782±0.212, which was lower than 1.126±0.251 in the TGF-β2 group (P<0.05). Conclusions Smac may prevent and treat PCO by inhibiting the proliferation and promoting apoptosis of human LECs. Key words: Smac; Apoptsis; Proliferation; After-cataract; Human; Lens epithlial cells; Small interfering RNA

Role of long non-coding RNA MIAT in proliferation, apoptosis and migration of lens epithelial cells: a clinical and in vitro study.

Age-related cataract is among the most common chronic disorders of ageing and is the world's leading blinding disorder. Long non-coding RNAs play important roles in several biological processes and complicated diseases. However, the role of lncRNAs in the setting of cataract is still unknown. Here, we extracted total RNAs from the transparent and age-matched cataractous human lenses, and determined lncRNA expression profiles using microarray analysis. We found that 38 lncRNAs were differentially expressed between transparent and cataractous lenses. 17 of 20 differentially expressed lncRNAs were further verified by quantitative RT-PCRs. One top abundant lncRNA, MIAT, was specifically up-regulated both in the plasma fraction of whole blood and aqueous humor of cataract patients. MIAT knockdown could affect the proliferation, apoptosis and migration of Human lens epithelial cells (HLECs) upon oxidative stress. Posterior capsule opacification (PCO) is a common complication of cataract surgery, which is associated with abnormal production of inflammatory factors. MIAT knockdown could repress tumour necrosis factor-α-induced abnormal proliferation and migration of HLECs, suggesting a potential role of MIAT in PCO-related pathological process. Moreover, we found that MIAT acted as a ceRNA, and formed a feedback loop with Akt and miR-150-5p to regulate HLEC function. Collectively, this study provides a novel insight into the pathogenesis of age-related cataract.

Suppressing effects of MMPs inhibitor GM6001, MMP-2/9 inhibitorI, MMP-2/9 inhibitorII on migration of human lens epithelial cells

Background The primary pathologic mechanism of posterior capsular opacification (PCO) is proliferation, migration and epithelial-mesenchymal transformation of residuary lens epithelial cells (LECs) following cataract surgery. Matrix metalloproteinases (MMPs) play a role during the migration of LECs. Researches showed that GM6001, a broad inhibitor of MMPs, can arrest the migration of LECs, but as specific inhibitors of MMPs, the efficacy and safety of MMP-2/9 inhibitorⅠand Ⅱ on LECs migration remain unclear. Objective This study was to determine and compare the inhibitory efficacy among GM6001, MMP-2/9 inhibitorⅠand Ⅱ on human LECs and search the clinical medication to prevent PCO. Methods Human LECs were cultured and passaged in vitro, and the cells of 3-4 generation were incubated in 6-well plates. Then the cells of 70% confluent monolayer were cultured in DMEM without fetal bovine serum for 12 hours. GM6001, MMP-2/9 inhibitorⅠand Ⅱ at different concentrations (0. 25, 0. 50, 1. 00, 2. 00, 4. 00, 8. 00, 16. 00, 32. 00, 64. 00, 128. 00 μmol/L) were added into the culture medium for 24 hours separately, and regularly cultured cells served as the control group. A bare area was made by a 200 μl sterile spear on the cell layer, and the migrated distance and inhibitory rate were calculated. The second or third generation of cells were incubated in 96-well plates at a density of 5×105/ml (200 μl/well). GM6001 (128. 00 μmol/L), MMP-2/9 inhibitorⅠ(64. 00 μmol/L) and Ⅱ (32. 00 μmol/L) were added into the culture medium for 24 hours, and the cell viability was assayed by using MTT assay. Results Cultured cells grew well with irregular arrangement and presented the polygon in shape. The migrated distance was gradually reduced as the increase of concentrations of GM6001, MMP-2/9 inhibitorⅠand Ⅱ, showing significant differences among the various concentration groups (GM6001: F=248. 647, P 0. 05). Conclusions GM6001, MMP-2/9 inhibitorⅠ and Ⅱ reduce the mobility of human LECs effectively but do not affect the viability of the cells in vitro. MMP-2/9 inhibitorⅡappears to be most dominant in inhibiting migration of human LECs. Key words: Matrix metalloproteinase/inhibitor; Epithelial cells/eye; Lens/ophthalmology; Migration/drug effect; Posterior capsule opacification

Inhibition of Rho-kinase inhibitor fasudil on the growth and migration of human LECs and extracellular matrix synthesis

Background Biological behavior changes of human lens epithelial cells (LECs) and extracellular matrix (ECM) synthesis are major events in the pathogenesis of posterior capsule opacification (PCO). Rho-kinase inhibitor fasudil can inhibit the cytoskeleton remodeling, migration and fibrosis, however, the mechanism of its effect in LECs is below understood. Objective This study was to investigate the role of Rho-kinase inhibitor fasudil in proliferation, migration of human LECs and ECM synthesis in vitro. Methods Human LECs line HLEB-3 cells were regularly cultured and assigned to the control group and the drug treatment groups. Different doses of fasudil were added into the medium to make the final concentrations 10, 20, 40 and 60 μmol/L, respectively. Cell proliferation was assessed by cell counting kit-8 (CCK-8) assay in 12, 24 and 48 hours after addition of drug to calculate the growth inhibitory rate. Transwell test was employed to detect the migrating number of the cells in 12, 24, 36 and 48 hours after action of drug. The concentrations of collagen type Ⅰ (COL-Ⅰ), fibronectin (FN) in cell supernatant and α-smooth muscle actin (α-SMA) content in the cells were measured by ELISA. Results The cell growth inhibitory rates in different doses of fasudil groups were higher than that in the control group. The cell growth inhibitory rates were gradually elevated as the increases of fasudil concentrations and prolong of action time (Fconcentration=966.727, P=0.000; Ftime=280.428, P=0.000). Transwell migration assay showed that the number of migration cells across polycarbonate membrane was 29.20±1.28 in the control group after 48 hours of incubation, which was much more than 24.40±1.33, 17.00±1.10, 14.60±0.68, 6.60±1.29 in the 10, 20, 40 and 60 μmol/L fasudil groups, respectively, with a significant difference among the five groups (F=57.34, P<0.05). COL-Ⅰ and FN protein contents in supernatant were gradually declined with the increase of drug concentrations and lapse of time (COL-Ⅰ: F concentration=143.992, P=0.000; Ftime=113.745, P=0.000.FN: Fconcentration=81.761, P=0.000; Ftime=69.602, P=0.000), and the α-SMA levels in the cells were significantly reduced with the increase of drug concentrations and lapse of time (Fconcentration=78.156, P=0.000; Ftime=65.162, P=0.000). Conclusions Fasudil can inhibit the proliferation, migration and ECM synthesis of human LECs in vitro in a dose- and time-dependent manner. Key words: Rho-associated kinases/antagonists and inhibitors; Epithelial cells, lens/drug effect; Humans; Cell migration; Cell proliferation; Collagen type I; Fibronectins; Extracellular matrix; Fasudil /pharmacology

UVB radiation induces human lens epithelial cell migration via NADPH oxidase-mediated generation of reactive oxygen species and up-regulation of matrix metalloproteinases

Ultraviolet (UV) radiation is one of the important cataract risk factors. The migration of human lens epithelial cells (HLECs) plays a crucial role in the remodeling of lens capsule and cataract formation. The purpose of the present study was to investigate the molecular mechanisms of UV-induced lens cell migration. We found that UVB radiation induces cell migration in cultured human lens epithelial cells. Further, we observed that UVB radiation induces NADPH oxidase activity and ROS generation which are inhibited by NADPH oxidase inhibitor diphenylene iodonium or DPI and antioxidant epigallocatechin gallate (EGCG). In addition, DPI and EGCG also block UVB irradiation-induced MMP-2, and -9 activity and expression and nuclear translocation of NF-kappaB. Collectively, our data suggest that NADPH oxidase may be a major source for the UVB-induced ROS generation, and it plays an essential role in the activation of NF-kappaB, which is involved in the activities of MMP-2 and MMP-9 and cell migration induced by UVB in HELCs. Understanding the cell signaling pathways may constitute potential therapeutic targets in for UVB-induced cataract.

Study on inhibitory effects of matrix metalloproteinase inhibitor on migration of cultured human lens epithelial cells

To determine whether matrix metalloproteinases inhibitor can prevent human lens epithelial cell migration in lens capsule bag model from the donor eyes. This study was an experimental investigation. Sham cataract surgery were performed in 16 human donor eyes. The donor lens capsule bags were cultured and treated with a broad MMP inhibitor (GM6001) at different concentrations (1, 10 and 100 micromol/L) and without GM6001 (negative control). The distance of lens epithelial cells migration on the posterior capsule was measured in all capsule bags under microscope and total MMP-2 and MMP-9 protein production was determined by ELISA. The cell viability in all groups was determined using methylthiazol tetrazolium (MTT) test. Lens epithelial cells at the equator began to migrate by day 4. Epithelial cells migration onto the posterior capsule was significantly reduced by GM6001 in a dose dependent manner as compared with the controls (F = 53.79, P < 0.01). Seventy percent reduction (P < 0.01) and 98% reduction (P <0. 01) of migration were observed in 10 micromol/L and 100 micromol/L GM6001 treated cultures, respectively, as compared with that in control on day 20. MMP levels in capsular bag were reduced by GM6001, the greatest inhibitory effect was found at the highest concentrations of GM6001 (F = 86.59,72.96; P < 0.01). By day 20, the reduction in total MMP-2 level (P < 0.01) and MMP-9 level (P < 0.01) in 10 micromol/L GM6001 treated cultures was 70% as compared with that in control. Ninety percent reduction in MMP-2 level (P < 0.01) and 87% reduction in MMP-9 level (P < 0.01) were found in 100 micromol/L GM6001 treated cultures. Lens epithelial cells remained viable and proliferated in the presence of all concentrations of GM6001. No significant difference of cell viability between the controls and GM6001 treated cultures (1,10, and 100 micromol/L) was found by the MTT assay (F = 0.62, P > 0.05). MMP inhibitor inhibits the lens epithelial cell migration onto the posterior capsule and does not affect the cell viability in cultured lens capsule bags in vitro. Therefore, MMP inhibition may have a role in the treatment of posterior capsule opacification.