Lens Epithelial Cells Of Patients Research Articles

A lipidomic study on the lens epithelial cells of patients with age related cataracts.

Age related cataracts (ARC) represent the main reason for blindness globally. The lens epithelial cells (LECs) participate not only in the metabolism of many substances in the lens but also in maintaining lens transparency. This study used lipidomics to investigate the metabolic differences in LECs of ARC patients with different severity, aiming at identifying potential metabolic biomarkers of ARC. Patients diagnosed with ARC and underwent cataract surgery at Shanghai Tongren Hospital were selected to participate in this study, which were classified as mild ARC group and severe ARC group. During their cataract surgery, anterior lens capsules(LCs) containing LECs were obtained. The lipidomics of LECs were analyzed using the liquid chromatography‑mass spectrometry (LC-MS). Potential pathways of lipids were searched for using databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) and MetaboAnalyst platform. In LEC lipids, 26 lipids have been identified as potential biomarkers between mild ARC and severe ARC, with AUC values of 0.67-0.94. The pathway analysis results revealed that the Glycerophospholipid (GPL) metabolism was significantly influenced, indicating that these metabolic markers contribute significantly to regulating this pathway. The LEC metabolic spectrum demonstrates a proficient ability to differentiate between patients with varying levels of cataracts. Herein, we have successfully identified potential metabolic biomarkers and pathways that have proven to be valuable in enhancing our understanding of ARC pathogenesis. The finding has translational value for developing new cataract treatment methods in the future.

HO-1-Mediated Autophagic Restoration Protects Lens Epithelial Cells Against Oxidative Stress and Cellular Senescence.

Oxidative stress and cellular senescence are risk factors for age-related cataract. Heme oxygenase 1 (HO-1) is a critical antioxidant enzyme and related to autophagy. Here, we investigate the crosstalk among HO-1, oxidative stress, and cellular senescence in mouse lens epithelial cells (LECs). The gene expression of HO-1, p21, LC3, and p62 was measured in human samples. The protective properties of HO-1 were examined in hydrogen peroxide (H2O2)-damaged LECs. Autophagic flux was examined by Western blot and mRFP-GFP-LC3 assay. Western blotting and lysotracker staining were used to analyze lysosomal function. Flow cytometry was used to detect intracellular reactive oxygen species and analyze cell cycle. Senescence-associated β-galactosidase assay was used to determine cellular senescence. The crosstalk between HO-1 and transcription factor EB (TFEB) was further observed in TFEB-knockdown cells. The TFEB binding site in the promoter region of Hmox1 was predicted by the Jasper website and was confirmed by chromatin immunoprecipitation assay. HO-1 gene expression decreased in LECs of patients with age-related nuclear cataract, whereas mRNA expression levels of p21, LC3, and p62 increased. Upon H2O2-induced oxidative stress, LECs showed the characteristics of autophagic flux blockade, lysosomal dysfunction, and premature senescence. Interestingly, HO-1 significantly restored the impaired autophagic flux and lysosomal function and delayed cellular senescence. TFEB gene silencing greatly reduced the HO-1-mediated autophagic restoration, leading to a failure to prevent LECs from oxidative stress and premature senescence. We demonstrated HO-1 effects on restoring autophagic flux and delaying cellular senescence under oxidative stress in LECs, which are dependent on TFEB.

The Upregulation of HLA-G1 and miRNA-34a in Lens Epithelial Cells of Diabetic Retinopathy Patients.

Retinopathy of diabetes is a chronic diabetes mellitus complication affecting retinal vessels, and some ocular complications' molecular mechanisms remain obscure. To evaluate the expression of HLA-G1, HLA-G5, miRNA-181a, and miRNA-34a in the lens epithelial cells of patients with retinopathy of diabetes. In a case-control study, 30 diabetic patients with retinopathy, 30 diabetic patients without retinopathy, and 30 cataract patients without diabetes mellitus as the control group were enrolled after a full description with details about the study methods and objectives. The expression of HLA G1, HLA G5, miRNA-181a, and miRNA-34a in lens epithelial cells was assessed by quantitative RT PCR. Moreover, the levels of HLA-G protein in aqueous humor were evaluated by the ELISA method. HLA-G1 expression was significantly upregulated in the retinopathy group (P=0.003). The aqueous humor of diabetic retinopathy patients contained significantly higher levels of HLA-G protein compared with the non-diabetic patients (P=0.001). miRNA-181a was significantly downregulated in the diabetic retinopathy group compared with the patients without diabetes (P=0.001). In addition, miRNA-34a was upregulated in the retinopathy group (P=0.009). Taken together, the present results showed that HLA-G1 and miRNA-34a can be valuable markers for diabetic retinopathy. Our data offers new perspectives for improving the control of inflammation in the lens epithelial cells by considering HLA-G and miRNA.

Oxidative Stress-Induced TRPV2 Expression Increase Is Involved in Diabetic Cataracts and Apoptosis of Lens Epithelial Cells in a High-Glucose Environment.

Cataracts are a serious complication of diabetes. In long-term hyperglycemia, intracellular Ca2+ concentration ([Ca2+]i) and reactive oxygen species (ROS) are increased. The apoptosis of lens epithelial cells plays a key role in the development of cataract. We investigated a potential role for transient receptor potential vanilloid 2 (TRPV2) in the development of diabetic cataracts. Immunohistochemical and Western blotting analyses showed that TRPV2 expression levels were significantly increased in the lens epithelial cells of patients with diabetic cataracts as compared with senile cataract, as well as in both a human lens epithelial cell line (HLEpiC) and primary rat lens epithelial cells (RLEpiCs) cultured under high-glucose conditions. The [Ca2+]i increase evoked by a TRPV2 channel agonist was significantly enhanced in both HLEpiCs and RLEpiCs cultured in high-glucose media. This enhancement was blocked by the TRPV2 nonspecific inhibitor ruthenium red and by TRPV2-specific small interfering (si)RNA transfection. Culturing HLEpiCs or RLEpiCs for seven days in high glucose significantly increased apoptosis, which was inhibited by TRPV2-specific siRNA transfection. In addition, ROS inhibitor significantly suppressed the ROS-induced increase of TRPV2-mediated Ca2+ signal and apoptosis under high-glucose conditions. These findings suggest a mechanism underlying high-glucose–induced apoptosis of lens epithelial cells, and offer a potential target for developing new therapeutic options for diabetes-related cataracts.

Immunoreactivity of ICAM-1, MMP-2, and Nesfatin-1 in lens epithelial cells of patients with diabetes mellitus with or without diabetic retinopathy

The aim of this study was to evaluate the immunoreactivity of matrix metalloproteinases-2 (MMP-2), intercellular adhesion molecule-1 (ICAM-1), and nesfatin-1 in cataract lens epithelial cells (LECs) of patients with diabetes mellitus (DM) and to investigate the relationship of these markers with DM cataract and diabetic retinopathy (DR). Ninety patients were included in the study. The patients were divided into three groups (n = 30): Group 1 (control; patients without DM or DR); Group 2 (patients with DM only), and Group 3 (patients with both DM and DR). Lens capsule samples were collected during intraoperative cataract surgery. Samples were immunohistochemically stained for MMP-2, ICAM-1, and nesfatin-1 and their immunoreactivity was evaluated. The number of immunoreactive cells was determined with a microscope at ×400 magnification. Increased MMP-2 and ICAM-1 immunoreactivity was detected in the LECs of patients with DM, and especially in patients with DR (p < 0.001, p < 0.001, respectively). Nesfatin-1 immunoreactivity was significantly lower in LECs of diabetic patients (p < 0.001). The mean of MMP-2 immunoreactive cells were 7.47 ± 8.18, 22.80 ± 15.70, and 34.80 ± 20.85 in Groups 1, 2, and 3, respectively. The mean of ICAM-1 immunoreactive cells were 17.10 ± 9.83, 38.50 ± 23.55, and 56.93 ± 20.94 in Groups 1, 2, and 3, respectively. Nesfatin-1, MMP-2, and ICAM-1 and could potentially play important roles in the pathogenesis of cataracts in patients with DM.

Expression of miR-181a and TGF-β2 in lens epithelial cells of patients with cataractous retinal detachment and its clinical significance.

Expression and clinical significance of miR-181a and TGF-β2 in lens epithelial cells of patients with cataract-induced retinal detachment were investigated. Forty patients with rhegmatogenous retinal detachment combined with age-related cataract (cast-off group) and another 40 patients with simple age-related cataract (non-exfoliated group) in Tongren Hospital were enrolled between January 2017 and June 2018. Lens epithelial cells were collected, and expression levels of miR-181a and TGF-β2 mRNA in lens epithelial cells were measured by RT-qPCR. Expression of protein was detected by western blot analysis. miR-181a overexpression vector (miR-181a-mimic group) was constructed and transfected into lens epithelial cells isolated from patients with rhegmatogenous retinal detachment combined with age-related cataract. Empty vector (miR-control group) transfection was performed. Untransfected lens epithelial cells were the control group. Independent sample t-test was performed for comparison between groups. Correlation between miR-181a and TGF-β2 protein expression levels was analyzed by Pearson's correlation analysis. Relative expression level of miR-181a in the non-exfoliated group was significantly higher than that in the cast-off group (P<0.05). Relative expression levels of TGF-β2 mRNA and TGF-β2 protein in the cast-off group were significantly higher than those in the non-exfoliated group (P<0.05). Relative expression level of E-cadherin in the miR-control group was significantly lower than that in the miR-181a-mimic group (P<0.05). Relative expression level of vimentin in the miR-control group was significantly higher than that in the miR-181a-mimic group (P<0.05). Relative expression level of TGF-β2 protein in the miR-control group was significantly higher than that in the miR-181a-mimic group (P<0.05). The expression level of miR-181a was negatively correlated with the expression level of TGF-β2 protein (r= −0.875, P<0.001). miR-181a expression is decreased, while TGF-β2 expression is increased in lens epithelial cells of patients with rhegmatogenous retinal detachment combined with age-related cataract. Over-expression of miR-181a may inhibit epithelial cell epithelial-inhibition by inhibiting TGF-β2 expression. Our findings may provide guidance for future postoperative prevention and treatment of rhegmatogenous retinal detachment in cataract patients.

Expression of thioredoxin-2 in human lens epithelial cells with oxidative damage and its significance

To explore whether thioredoin-2 (Trx-2) is involved in the development of cataract and to study the effect of Trx-2 on hydrogen peroxide (H2O2)-induced injury in human lens epithelial cells. Methods: A total of 10 volunteers (removing the lens due totraumatism) and 30 patients received phacoemulsification (age more than 60 years) were selected. The expression of Trx-2 protein in lens epithelial cells from cataract patients and volunteers were detected by the immunohistochemical streptavidin-peroxidase (SP) method. SRA01/04 cells were cultured and were divided into six groups according to different treatment: a control group, H2O2-treated groups at 20, 50 or 100 μmol/L, a negative control group (transfected with pCMV6 plasmid plus 100 μmol/L H2O2), and a Trx-2 overexpression group (transfected with pCMV6-Trx-2 plasmid plus 100 μmol/L H2O2). Methyl thiazolyltetrazolium (MTT) assay and flow cytometry was performed to measure the cell viability and apoptosis for SRA01/04 cells, respectively. The activities of superoxide dismutase (SOD) and catalase (CAT), the content of glutathione (GSH) and malondialdehyde (MDA) in human lens epithelial cells were measured via chemical chromatometry. Western blot was used to measure the protein levels of Trx-2, B-cell lymphoma 2 protein (Bcl-2), Bcl-2 associated X protein (Bax) and caspase-3. Results: Compared with the volunteers, the expression of Trx-2 was significantly decreased in lens epithelial cells in patients with cataract (P<0.05). Compared with the control group, the expression of Trx-2 protein in the 20, 50 or 100 μmol/L H2O2 groups was decreased (all P<0.05). Compared with the control group, the cell survival rates were decreased in the 100 μmol/L H2O2 group and the negative control group (both P<0.05), along with enhanced apoptotic rates, inhibited cellular SOD activities and CAT activities, reduced GSH contents, augmented MDA contents, down-regulated Trx-2 and Bcl-2 expression and up-regulated Bax and caspase-3 expression (all P<0.05). Compared with the negative control group, the cell survival rate was increased in the Trx-2 overexpression group (P<0.05), along with suppressed apoptosis, increased SOD activities and CAT activities, elevated GSH contents, decreased MDA content, up-regulated Trx-2 and Bcl-2 expression and down-regulated Bax and caspase-3 expression (P<0.05). Conclusion: Trx-2 might be involved in the apoptosis of lens epithelial cells in patients with cataract. The overexpression of Trx-2 obviously attenuated H2O2-induced injury of human lens epithelial cells, which might be associated with the inhibition of H2O2-mediated oxidative stress.

Immunohistochemical analysis of microsomal glutathione S-transferase 1 and clusterin expression in lens epithelial cells of patients with pseudoexfoliation syndrome.

Pseudoexfoliation syndrome (PEX) is an age-associated, sight disorder affecting elastic fibers in the eye and visceral organs but its exact etiology remains unknown. The purpose of the current study was to determine the morphology and ultrastructure of lens epithelial cells (LECs), and to use immunohistochemistry to examine localization of microsomal glutathione S-transferase 1 (MGST1) and clusterin. Anterior lens capsules were obtained from 24 patients (13 PEX and 11 controls) who underwent phacoemulsification. Immunohistochemistry was performed, using antibodies against MGST1 and clusterin, to determine their expression. The morphology and ultrastructure of LECs were evaluated by light and transmission electron microscopy, respectively. The PEX LECs were characterized by significantly lower MGST1 (P=0.0001) and clusterin expression (P=0.0005) compared with the control group patients. PEX LECs were also observed to have significantly increased thickness compared with the control group patients (P=0.0002). The current findings suggest that low MGST1 and clusterin expression levels may be an early clinical indicator of PEX, and that oxidative stress may serve an important role, but that the specific etiology of this disease has yet to be revealed.

Pathological study on effects of preservative-free 1% lidocaine on lens epithelial cells of patients with age-related cataract

To assess whether preservative-free 1% lidocaine is capable of destroying the LECs in age-related cataract (ARC) in order to provide scientific basis for pursuing safe and effective drugs to eliminate LECs in cataract surgery. Lens anterior capsule (LAC) specimens were collected from 75 patients (82 eyes) with age-related cataract (ARC), including forty males (44 eyes) and thirty-five females (38 eyes). The age range was 41 - 85 years, the mean age was 67.97 years. There were 34 cortical cataracts, 22 nuclear cataracts and 26 subcapsular cataracts. Capsule specimens were divided into 4 groups: balanced salt solution (BSS) group I and group II (exposed to BSS for 1 minute), lidocaine group (exposed to preservative-free 1% lidocaine for 1 minute) and the control group. Specimens were stained with trypan blue and alizarin red. Photomicrographs of each capsule were taken to observe the viability of LECs and to count the number of necrosis LECs. The pathologic changes of LECs were evaluated by histological methods (11 LAC, 22 pieces) as well as transmission and scanning electron microscopes (5 LAC, 10 pieces). In the control and BSS group I (23 LAC), one half of each capsule specimen was used for the control group and the other half was used for BSS group I. In lidocaine group and BSS group II (43 LAC), one half of each capsule specimen was used for lidocaine group and the other half was used for BSS group II. The rate of necrosis LECs of the anterior capsules in the control group and BSS group I was (56.19 +/- 2.71)% and (57.23 +/- 1.98)%, respectively. The rate of necrosis LECs of the capsules in lidocaine group and BSS group II was (99.86 +/- 8.22)% and (57.64 +/- 7.00)%, respectively. Matching t-test showed that the rate of necrosis LECs in lidocaine group was greater than that in the BSS group II (t = 27.6781, P = 0.0000). There was no significant difference in the number of necrosis LECs between the control group and BSS group I (t = 2.0693, P = 0.0505). There was also no significant difference between males and females; between different cataract, types and between varying age groups (P > 0.05). After irrigated with lidocaine, LECs showed vacuoles and detached from the capsule, and many cavities appeared between the LECs and the capsule. The capsules of BSS and control group showed a normal layer of LECs attached to the capsule. Under transmission and scanning electron microscopes, in lidocaine group, the junction between the LECs and between the cells and the capsule were destroyed; many cells detached from the capsule and the rest arranged loosely. Some LECs dented and many vacuoles emerged, resulting in destruction of the cellular structures. Preservative-free 1% lidocaine may loose the junction between LECs and between the cells and the capsule, and also destroy cellular structures, resulting in degeneration and necrosis of the LECs.

Matrix metalloproteinase-9 activity in human lens epithelial cells of cortical, posterior subcapsular, and nuclear cataracts

To evaluate the level of matrix metalloproteinase-9 (MMP-9) activity in lens epithelial cells (LECs) derived from different types of cataract in patients having phacoemulsification. Iladevi Cataract & IOL Research Centre, Memnagar, Ahmedabad, India. This observational study of 275 patients having phacoemulsification was performed to evaluate the level of MMP-9 activity in LECs. All anterior lens capsules harboring the LECs and derived from the surgical curvilinear capsulorhexis were obtained during phacoemulsification. The anterior lens capsule samples were processed to analyze MMP-9 activity using a succinylated gelatin assay. The samples were grouped based on age and on pure cataract type. The level of MMP-9 activity in LECs was highest in eyes with cortical cataract. A significant difference in the level of MMP-9 activity was found in different types of cataract (P<.001). The highest level of MMP-9 activity was in patients older than 60 years. The MMP-9 activity increased gradually with age irrespective of cataract type (P<.001). A significant difference was observed in the level of MMP-9 activity between different types of cataract. The activity of MMP-9 was highest in eyes with cortical cataract. The level of MMP-9 activity increased with age in the LECs of patients with age-related cataract.

Activity of superoxide dismutase isoenzymes in lens epithelial cells derived from different types of age-related cataract

To evaluate the level of superoxide dismutase (SOD) and SOD isoenzyme activity in lens epithelial cells (LECs) derived from different types of cataract in patients having phacoemulsification. Iladevi Cataract & IOL Research Centre, Memnagar, Ahmedabad, India. This observational study of 109 patients having phacoemulsification was performed to evaluate the level of activity in LECs of total superoxide dismutase (TSOD) and 2 superoxide dismutase isoenzymes: copper- (Cu) and zinc (Zn)-dependent SOD (Cu/Zn-SOD) and manganese (Mn)-dependent SOD (Mn-SOD). The curvilinear capsulorhexis (lens capsule) harboring LECs was obtained during phacoemulsification. The anterior lens capsule samples were processed for assaying SOD activity using the nitro blue tetrazolium reduction assay. The samples were grouped based on age and on pure cataract types. The highest level of TSOD, Cu/Zn-SOD, and Mn-SOD activity was in patients 50 years or younger. The activity declined gradually with age (P<.001). The level of TSOD activity increased in cortical cataract. The level of Cu/Zn-SOD and Mn-SOD isoenzyme activity in LECs was higher in cortical cataracts. A significant difference in the level of TSOD and Cu/Zn-SOD activity was found with different types of cataract (P<.001). The level of TSOD, Cu/Zn-SOD, and Mn SOD isoenzyme activity decreased with age in LECs of patients with age-related cataract. A significant difference in the level of TSOD and Cu/Zn-SOD isoenzyme activity between different types of cataract was observed. The activity of all 3 SOD isoenzymes was highest in cortical cataracts.

Evidence for oxidative stress in lens epithelial cells in pseudoexfoliation syndrome

To investigate the oxidative status in lens epithelial cells of patients with pseudoexfoliation (PEX) syndrome. Lens capsule samples obtained during cataract surgery of patients with PEX syndrome and normal age-matched control subjects were examined for changes in the levels of glutathione (GSH), glutathione disulphide (GSSG), protein concentration, and lipid peroxidation. Concentrations of GSH, GSSG, lipid peroxidation, and protein concentration were determined by specific fluorescent assays. This study shows a 2.2- and 2.0-fold decrease in GSH and GSSG levels, respectively, in PEX lens epithelial lens compared with non-PEX lens epithelial cells, as well as a 2.5-fold increase in lipid peroxidation product malondialdehyde (MDA) levels. The increased MDA and decreased GSH levels indicate high oxidative stress. On the other hand, GSSG usually increases in cases of high-oxidative stress, but this is not always the case, as it may not always accumulate in cells. Our findings suggest a role for oxidative stress in the pathogenesis and the progression of PEX syndrome.

Accumulation and Aberrant Modifications of α-Crystallins in Anterior Polar Cataracts

Crystallins are the major proteins found in the lens, and the localization of specific crystallins is well known. Overexpression and accumulation of alphaB-crystallin has been observed in response to stress conditions or in certain diseases, such as brain tumors and neurodegenerative diseases. The purpose of this study was to examine whether alpha-crystallins are modified during pathological myofibroblastic changes in lens epithelial cells. Lens epithelial cells attached to the anterior capsules of patients with nuclear or anterior polar cataracts were analyzed quantitatively for alpha-crystallin proteins and mRNAs using Western blot and RT-PCR analysis., respectively. The degree of modification of alpha-crystallins was determined by 2-dimensional gel electrophoresis followed by Western blotting. Higher molecular weight protein bands that were immunoreactive to anti-alphaA- and anti-alphaB-crystallin antibodies around 45 kDa accumulated more in the anterior polar cataract samples than in those with the nuclear type of cataracts. Also monomeric alphaB-crystallins accumulated more in lens epithelial cells of patients with anterior polar cataracts. By comparison, no significant changes were found in the levels of the mRNAs encoding alphaA- and alphaB-crystallins in the different types of cataracts. Both alphaA- and alphaB-crystallin proteins seemed to undergo more extensive modification in anterior polar cataracts. Conclusion. In addition to fibrotic changes, which accompany increased levels of extracellular matrix molecules, accumulation and abnormal modification of alpha-crystallins might be implicated in the pathogenic mechanism of this type of cataract.

Sex hormones and their receptors in patients with age-related cataract

Purpose To determine the sex hormone levels in the serum and aqueous humor in patients with age-related cataract and the corresponding hormone receptors in cataract lens epithelial cells (LECs). Setting Research Laboratory, International Intraocular Implant Training Center, Tianjin Medical University, Tianjin, China. Methods Serum and aqueous humor were drawn from patients with cataract and a control group. Enzyme-linked immunosorbent assay was used to determine the levels of estradiol, progesterone, and testosterone in the serum and the aqueous. The anterior lens capsules with attached LECs were obtained during cataract surgery, and the expressions of estrogen receptor, progesterone receptor, or androgen receptor were examined by immunohistochemistry. Results The testosterone level in the serum was significantly higher in the control group than in the cataract group. In the cataract group, there was no difference between sexes in the serum levels of estradiol or progesterone; however, the testosterone level in men was significantly higher than in women. The aqueous level of each hormone was lower than in serum; however, there was no difference between sexes and no association with corresponding serum levels. No estrogen, progesterone, or androgen receptor was detected in the LECs of patients with age-related cataract. Conclusions There was no statistical difference between men and women or between the cataract and control groups in the levels of estradiol or progesterone in serum. There was no between-sex difference in the aqueous levels. It appears that sex hormone levels can be regarded only as a risk factor for cataractogenesis, not as a key factor.

Expression of Stress-Response Protein 60 in Lens Epithelial Cells in Atopic Cataract

Purpose: To clarify the pathogenesis of atopic cataract, especially to determine if there is any relationship between autoimmunity and atopic cataract. Methods: We investigated the lens epithelia obtained at surgery from 12 patients (12 eyes) with atopic cataract; from 8 patients (8 eyes) with nonatopic cataract (5 with senile cataract, 2 with juvenile cataract, and one with secondary cataract due to anterior uveitis); and from 4 autopsy eyes as controls. Results: Histopathological findings in the lens epithelial cells from atopic and nonatopic cataract patients were essentially the same: atrophy of the cells, presence of the superimposed cells, migration of cells into the lens cortex, cytoplasmic vacuolation, and loss of cells. In an immunohistochemical study, the expression of stress-response protein 60 (srp 60), srp 27, and srp 72 was examined in the lens epithelial cells. In atopic cataract specimens, 71%–87% of the lens epithelial cells were stained with the antibody against srp 60, but the cells in nonatopic cataract and control specimens were not stained. Conclusions: Srp 27 and srp 72 were not expressed in any observed epithelial cells. The expression of srp 60 may reflect a protective mechanism of the epithelial cells against injury triggered by immunorelated agents. These findings suggest that the pathogenesis of degeneration of the lens epithelial cells in patients with atopic cataract may be related to autoimmunity.