Molecular mechanism of high glucose-induced mitochondrial DNA damage in retinal ganglion cells.

Mitochondrial DNA damage in retinal ganglion cells (RGCs) may be closely related to lesions of glaucoma. RGCs were cultured with different concentrations of glucose and grouped into 3 groups, namely normal control (NC) group, Low-Glu group, and High-Glu group. Cell viability was measured with cell counting kit-8, and cell apoptosis was measured using flow cytometry. The DNA damage was measured with comet assay, and the morphological changes of damaged mitochondria in RGCs were observed using TEM. Western blot analyzed the expression of MRE11, RAD50, and NBS1 protein. Cell viability of RGCs in Low-Glu and High-Glu groups were lower than that of NC group in 48 and 96 h. The cell apoptosis in NC group was 4.9%, the Low-Glu group was 12.2% and High-Glu group was 24.4%. The comet imaging showed that NC cells did not have tailings, but the low-Glu and high-Glu group cells had tailings, indicating that the DNA of RGCs had been damaged. TEM, mitochondrial membrane potential, ROS, mitochondrial oxygen consumption, and ATP content detection results showed that RGCs cultured with high glucose occurred mitochondrial morphology changes and dysfunction. MRE11, RAD50, and NBS1 protein expression associated with DNA damage repair pathway in High-Glu group declined compared with Low-Glu group. Mitochondrial DNA damage caused by high glucose will result in apoptosis of retinal ganglion cells in glaucoma.

Explore the molecular mechanism of angle-closure glaucoma in elderly patients induced telomere shortening of retinal ganglion cells through oxidative stress.

Senile glaucoma is a common ophthalmological disease in the elderly. It is a disease of visual papillary perfusion caused by elevated intraocular pressure, complicated by visual dysfunction. Glaucoma can cause serious damage to the normal vision of the elderly. Therefore, exploring the related molecular mechanisms of glaucoma is of great significance to the diagnosis and treatment of glaucoma. This is an exploratory study. Establish a mouse model and conduct experimental groupings. After one week of adaptive feeding, the mice were intraperitoneally injected with an anesthetic mixture: ketamine + xylazine. Then the mice were sacrificed by neck dissection, and the eyeball tissues were immediately dissected. HE staining was used to analyze the histopathological characteristics of the retina of each group of mice. MitoSOX fluorescent probe was used to analyze the content of ROS in retinal tissue. The ELISA analysis was used to detect the activation of ß-galactosidase for the aging characteristics of retinal ganglion cells in retinal tissues. Immunohistochemistry experiments were used to analyze the expression of telomerase TERT in retinal tissues. Western blot analysis was used to determine the expression of proteins POT1, TERF1, TERF2, and TINF2 in retinal tissues. The HE staining experiment showed that the damage of retinal tissue decreased from group Glaucoma to group Old, group Old to group Young. The experimental results of MitoSOX fluorescent probe show that ROS content is positively correlated with the degree of tissue damage. ELISA analysis results showed that the expression trend of ß-galactosidase was the same as the ROS content. The protein expression levels related to telomere protection (TRET, POT1, TREF1, TREF2 and TINF2) all increased from group Glaucoma to group Old, group Old to group Young. The increase in ROS content, the decrease in telomere protection-related protein expression (telomere shortening) induced by ROS, and the increase of the expression of ß-galactosidase, are all potential molecular mechanisms for the occurrence of angle-closure glaucoma in elderly patients.

Hyperglycemia induces retinal ganglion cell endoplasmic reticulum stress to the involvement of glaucoma in diabetic mice.

Glaucoma is a neurodegenerative disease leading to visual loss. Since glaucoma is associated with chronic renal diseases (RDs) their rate is higher in patients with RDs, and end-stage RDs (ESRDs) than in the general population and kidney transplant recipients. OBJECTIVE: To explore the molecular mechanism of diabetic internal environment in regulating the endoplasmic reticulum stress of the retinal ganglion cells (RGCs). METHODS: Thirty-six SPF grade type 2 diabetes models were divided into 3 groups: Diabetes mellitus (DM), DM + glaucoma and 4-phenylbutyric acid-DM (4-PBA-DM) + glaucoma group. C57BL6 mice of the same week age were taken as the negative control (NC) group. The morphology of RGCs and their axon in the 4 groups were labeled by fluorescent reactive dye Dil. The apoptosis situation of RGCs was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The protein expression values of RTN4IP1, Protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor 2A (eIF2a) and X-box-binding Protein 1 (XBP1) were determined by western blot. The relative mRNA levels of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP), Caspase12 and Bax were determined by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Glaucoma promotes the apoptosis of RGCs. The protein expression values of RTN4IP1, PERK and XBP1 in DM mouse models with glaucoma were much higher compared to only DM mouse models. Further injection of endoplasmic reticulum stress inhibitor 4-PBA decreased the expression values. The relative mRNA levels of CHOP, Cysteine aspartic acid specific protease12 (Caspase12) and BCL2-associated X protein (Bax) in DM + glaucoma were significantly higher compared to those in DM group. Further injection of endoplasmic reticulum stress inhibitor 4-PBA decreased the mRNA levels. CONCLUSION: Endoplasmic reticulum stress (ERS) is the underlying cause of glaucoma, which could promote the apoptosis of RGCs in diabetic mice.

Overexpression of HTRA1 increases the proliferation and migration of retinal pigment epithelium cells.

BACKGROUND: Age-related macular degeneration (AMD) mainly affects the central region of retina and has many late-stage manifestations. OBJECTIVES: Age-related macular degeneration is a leading cause of irreversible blindness in older people. The main feature of AMD is retinal pigment epithelium (RPE) degeneration. In this study, we aimed to explore the influence of HTRA1 expression on the proliferation and migration of RPE cells. MATERIAL AND METHODS: Human ARPE-19 cells were transfected with an HTRA1 overexpression lentivirus or HTRA1 siRNA to silence HtrA1 expression. Quantitave reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting were used to verify the relative level of HTRA1 mRNA and expression of HTRA1 protein of transfected human ARPE-19 cells. The MTT clone formation and transwell assays were used to confirm the effect of HTRA1 expression on the proliferation, colony forming ability and migration of ARPE-19 cells. RESULTS: The proliferation capacity (shown as optical density value) of ARPE-19 cells in the HTRA1-overexpressing group at culture times of 24 h and 48 h were 0.595 ±0.032 and 0.867 ±0.037 respectively, which were much higher than in the mock group. However, the proliferative capacity of cells in the HTRA1-silenced group decreased with increasing time of culture, compared with the mock group. The number of cloned and migrating cells in the HTRA1-overexpressing group were much higher than in the mock group, whereas the numbers in the HTRA1-silenced group were significantly lower. CONCLUSIONS: Overexpression of HTRA1 promotes proliferation and migration of RPE cells, which can help maintain the function of sensory neurons in the retina. Therefore, HTRA1 may be a suitable target for AMD treatments.

Role of mammalian target of rapamycin in regulating HIF-1α and vascular endothelial growth factor signals in glaucoma.

Hypoxia inducible factor subtype 1α (HIF-1α) in retinal tissues is involved in the development of glaucoma. This study examined the role played by mammalian target of rapamycin (mTOR) in regulating expression of HIF-1α and its downstream pathway, vascular endothelial growth factor (VEGF). Glaucoma was induced by chronic elevation of intraocular pressure using laser burns in rats. ELISA and western blot analysis were employed to determine the levels of HIF-1α, VEGF and mTOR in retinal tissues of eyes with high intraocular pressure. In results, HIF-1α, VEGF and VEGF receptor subtype 2 were increased in laser eyes. The p-mTOR, mTOR-mediated phosphorylation of 4E-binding protein 4, p70 ribosomal S6 protein kinase 1 were also amplified in retina of laser eyes. Blocking mTOR using rapamycin attenuated HIF-1α-VEGF pathways, accompanied with downregulation of apoptotic Caspase-3. Our data revealed potential signalling pathways engaged in the development of glaucoma, including the activation of mTOR and HIF-1α-VEGF mechanism.

Madecassoside protects retinal pigment epithelial cells against hydrogen peroxide-induced oxidative stress and apoptosis through the activation of Nrf2/HO-1 pathway.

Age-related macular degeneration (AMD) is a progressive and degenerative ocular disease associated with oxidative stress. Madecassoside (MADE) is a major bioactive triterpenoid saponin that possesses antioxidative activity. However, the role of MADE in AMD has never been investigated. In the current study, we aimed to evaluate the protective effect of MADE on retinal pigment epithelium (RPE) cells under oxidative stress condition. We used hydrogen peroxide (H2O2) to induce oxidative damage in human RPE cells (ARPE-19 cells). Our results showed that H2O2-caused significant decrease in cell viability and increase in lactate dehydrogenase (LDH) release were dose-dependently attenuated by MADE. MADE treatment also attenuated H2O2-induced reactive oxygen species (ROS) and malondialdehyde (MDA) production in RPE cells. The reduced glutathione (GSH) level and superoxide dismutase (SOD) activity in H2O2-induced ARPE-19 cells were elevated after MADE treatment. MADE also suppressed caspase-3 activity and bax expression, as well as increased bcl-2 expression. Furthermore, H2O2-induced increase in expression levels of HO-1 and nuclear Nrf2 were enhanced by MADE treatment. Finally, knockdown of Nrf2 reversed the protective effects of MADE on H2O2-induced ARPE-19 cells. In conclusion, these findings demonstrated that MADE protected ARPE-19 cells from H2O2-induced oxidative stress and apoptosis by inducing the activation of Nrf2/HO-1 signaling pathway.

DKK1 inhibits proliferation and migration in human retinal pigment epithelial cells via the Wnt/ß-catenin signaling pathway.

Retinal pigment epithelial (RPE) cells play important roles in diabetic retinopathy (DR). Dickkopf 1 (DKK1) has been reported to be important in the regulation of cell proliferation and migration. However, there are few previous studies regarding DKK1 in RPE cells. Therefore, in the present study, we investigated the effect of DKK1 on the proliferation and migration of human RPE cells, and the signaling mechanisms underlying these effects. The results showed that the overexpression of DKK1 significantly inhibited the proliferation and migration of ARPE-19 cells. In addition, overexpression of DKK1 markedly inhibited the expression of ß-catenin and cyclin D1 in ARPE-19 cells. Collectively, the present findings suggest that the overexpression of DKK1 inhibited the proliferation and migration of RPE cells by suppressing the Wnt/ß-catenin signaling pathway. Therefore, DKK1 are able to augment the growth of human RPE, and further studies are warranted to investigate the effects of DKK1 effects on DR.

Oncogenic role of microRNA­20a in human uveal melanoma.

As a member of the microRNA (miR)-17-92 cluster, miR­20a has been indicated to be involved in the regulation of the proliferation and invasion of various cancer cells. Previous studies have observed elevated plasma levels of miR­20a in patients with uveal melanoma (UM), compared with normal controls. In the present study, the potential function of miR­20a in UM was investigated. Reverse transcription­quantitative polymerase chain reaction analysis was performed to detect the expression levels of miR­20a in UM cells and tissues. The functions of miR­20a on cell proliferation, migration and invasion were determined in vitro using 3­(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell assays, respectively. The expression levels of miR­20a were significantly increased in the UM cells and tissues (P<0.05). Subsequently, miR­20a mimics were transfected into UM cells, which led to increases in cell growth, migration and invasion activities. By contrast, miR­20a inhibition markedly suppressed the viability and motility of UM cells in vitro. These data provided convincing evidence that miR­20a may function as an oncogenic miRNA, and may be involved in promoting cell growth and motility in the molecular etiology of UM, suggesting its potential as a candidate therapeutic target for the treatment of patients with UM.

Epithelial cell adhesion molecule-1 (ECAM1) is required in the maintenance of corneal epithelial barrier integrity.

Corneal epithelial barrier integrity is critical in the maintenance of the corneal homeostasis. The corneal barrier dysfunction may be associated with the pathogenesis of a number of eye diseases. In this study, we assessed the expression of epithelial cell adhesion molecule-1 (ECAM1) in human corneal epithelial cells (HCE). The epithelial barrier function of the corneal epithelial monolayer was determined in Transwells. We found that the HCE cells expressed ECAM1. Knockdown of ECAM1 markedly compromised the HCE monolayer barrier function. A complex of ECAM1, claudin1, and occludin was detected in the HCE monolayers, which was not detected in the ECAM1-null HCE monolayers. Exposure to the proinflammatory cytokine, interleukin-13, inhibited the expression of ECAM1 in HCE cells and compromised the barrier function, which was prevented in the HCE monolayer with the ECAM1 overexpression. In conclusion, ECAM1 is required in the formation of the tight junction complex and maintaining the corneal epithelial barrier function.

Role of intravitreal inflammatory cytokines and angiogenic factors in proliferative diabetic retinopathy.

OBJECTIVE: Inflammatory reaction has been shown to involve the progress of type 2 (non-insulin-dependent) diabetes. We, therefore, examined the effects of inflammatory cytokines and angiogenic factors in the pathogenesis of proliferative diabetic retinopathy (PDR) in type 2 diabetes. PATIENTS AND METHODS: Vitreous fluid samples were obtained by vitrectomy from 62 eyes of PDR patients with type 2 diabetes and from 20 eyes of age-matched non-diabetic patients. The concentrations of interleukin 1 beta (IL1B), IL6, IL8, IL10, chemokine (C-C motif) ligand 2 (CCL2), endothelin 1 (EDN1), vascular endothelial growth factor (VEGF), and tumor necrosis factor (TNF) in the vitreous samples were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The concentrations of LI1B, IL6, IL8, CCL2, EDN1, VEGF, and TNF in the vitreous samples were considerably higher in PDR patients in comparison with the controls. However, the level of IL10 in PDR patients was similar to that obtained in the controls. Analysis of the correlations of the studied factors revealed the correlation of VEGF and IL6, VEGF and EDN1, IL8 and CCL2, and EDN1 and TNF in PDR patients. In addition, a significant positive correlation was observed between vitreous TNF as well as EDN1 and serum HbA(1)c levels in PDR patients. CONCLUSIONS: The inflammatory cytokines and angiogenic factors IL1B, IL6, IL8, CCL2, EDN1, VEGF, and TNF are increased in the vitreous of PDR patients without an increase in IL-10. These results add support to the role of inflammatory cytokines and angiogenic factors in the genesis of PDR. Understanding the implication of these cytokines may provide diagnostic tools and therapeutic targets for treatment and prevention of PDR.