Role of Chondroitin Sulfate Proteoglycan 5 in Steroid-Induced Cataract.

Steroid-induced cataracts (SIC) are defined as cataracts associated with the administration of corticosteroids. Long-term glucocorticoid treatment for inflammatory diseases reportedly increases the risk of SIC, and steroids can induce cataracts by disrupting ocular growth factor balance or homeostasis. In this study, we verified the effect of chondroitin sulfate proteoglycan 5 (CSPG5) using dexamethasone (dexa)-treated human lens epithelial (HLE-B3) cells and the lens epithelium from the anterior capsule of SIC patients obtained during cataract surgery. CSPG5 expression increased in the lens epithelium of SIC patients. The downregulation of CSPG5 suppressed the dexa-induced epithelial-mesenchymal transition (EMT)-related protein expression and motility in HLE-B3 cells. The disruption of the transcription factors EZH2 and B-Myb downregulated CSPG5, dexa-induced fibronectin expression, and cell migration in HLE-B3 cells, reaffirming that CSPG5 expression regulates EMT in lens epithelial cells. Taken together, these results indicate that the steroid-induced effects on lens epithelial cells are mediated via alterations in CSPG5 expression. Therefore, our study emphasizes the potential of CSPG5 as a therapeutic target for the prevention and treatment of SIC.

Effect of Renal Ischemia Reperfusion on Brain Neuroinflammation.

Acute kidney injury (AKI) is an inflammatory sequence. It can lead to distant organ injury, including damage to the central nervous system (CNS), mediated by increased circulating cytokines and other inflammatory mediators. It can also lead to increased blood-brain barrier (BBB) permeability. However, the effect of AKI on the inflammatory response of the brain has not yet been investigated. Therefore, we observed the effect of AKI on BBB permeability, microglia and astrocyte activation, and neuronal toxicity in the brain. The striatum and ventral midbrain, known to control overall movement, secrete the neurotransmitter dopamine. The activation of microglia and astrocytes present in this area causes neuro-degenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). The activation of astrocytes and microglia in the hippocampus and cerebral cortex, which are responsible for important functions, including memory, learning, concentration, and language, can trigger nerve cell apoptosis. The activation of astrocytes and microglia at this site is also involved in the inflammatory response associated with the accumulation of beta-amyloid. In the situation of kidney ischemia reperfusion (IR)-induced AKI, activation of microglia and astrocytes were observed in the striatum, ventral midbrain, hippocampus, and cortex. However, neuronal cell death was not observed until 48 h.

MicroRNA profiles in aqueous humor between pseudoexfoliation glaucoma and normal tension glaucoma patients in a Korean population.

We aimed to obtain microRNA (miRNA) profiles of patients with pseudoexfoliation (PEX) glaucoma or normal-tension glaucoma (NTG) compared to normal controls using individual aqueous humor (AH) samples and investigate the role of miRNAs in the pathogenesis of PEX glaucoma compared to NTG in Korean. AH (80-120 µl) was collected before cataract surgery or trabeculectomy from 26 Korean subjects (eleven with PEX glaucoma, age-matched eight NTG, and seven controls). RNA sequencing was conducted for RNA samples extracted from 26 AH samples. Bioinformatics analysis was performed for targets and related pathways. A total of 334 and 291 discrete miRNAs were detected in AH samples of PEX glaucoma and NTG patients, respectively. Two significantly upregulated miRNAs (hsa-miR-30d-5p and hsa-miR-320a) and ten significantly downregulated miRNAs (hsa-miR-3156-5p, hsa-miR-4458, hsa-miR-6717-5p, hsa-miR-6728-5p, hsa-miR-6834-5p, hsa-miR-6864-5p, hsa-miR-6879-5p, hsa-miR-877-3p, hsa-miR-548e-3p, and hsa-miR-6777-5p) in PEX glaucoma patients compared to control (fold-change > 2, p < 0.05) were found. In NTG patients, ten significantly upregulated and two downregulated miRNAs compared to control were found. Only hsa-miR-6777-5p was commonly downregulated in both PEX glaucoma and NTG patients. Related pathways were proteoglycans in cancer, glioma, and TGF-beta signaling pathway in PEX glaucoma. These differentially expressed miRNAs between PEX glaucoma and NTG samples suggest the possible role of miRNA in the pathogenesis of glaucoma, further implying that pathogenic mechanisms may differ between different types of glaucoma.

Resveratrol prevents hypoxia-induced retinal ganglion cell death related with ErbB2.

AIM: To confirm the changes in proteins related with hypoxia-induced retinal cell death and to assess the effects of resveratrol (Res). METHODS: The therapeutic effect of Res was verified using an ischemic/reperfusion (I/R) model in vivo and a hypoxia modelin retinal ganglion cells (RGCs) in vitro. Death of RGCs were confirmed by TUNEL assay. Protein expression was confirmed by Western blotting and immunohistochemistry. In addition, flow cytometric analysis was used to confirm the response in the cell unit to obtain more accurate data. RESULTS: ErbB2 expression and apoptosis in the ganglion cell layer (GCL) increased after I/R injury. Treatment of Res rescued I/R-induced ganglion cell death, downregulated apoptosis and ErbB2 protein expression in the retina. In subsequent in vitro models, Res affects apoptosis by regulating the phosphorylation and expression of mouse double minute 2 homolog (MDM2), along with those of ErbB2. These results suggest that Res reverses GCL-specific apoptosis via downregulation of ErbB2 in ischemic injury. CONCLUSION: In light of Res favorable properties, it should be evaluated in the treatment of RGC death and related retinal disease characterized by ErbB2 and MDM2 expression. Therefore, Res is appropriate therapeutic agent for treating ischemic injury-related eye diseases by targeting the expression of ErbB2 and MDM2.

Profiles of microRNA in aqueous humor of normal tension glaucoma patients using RNA sequencing.

We aimed to identify and compare microRNAs (miRNAs) from individual aqueous humor samples between normal-tension glaucoma (NTG) patients and normal controls. Aqueous humor (80 to 120 µl) was collected before cataract surgery. Six stable NTG patients and seven age-matched controls were included in the final analysis. RNA sequencing was conducted for RNA samples extracted from the 13 aqueous humor samples, and bioinformatics analysis was employed for the miRNA targets and related pathways. Two hundred and twenty-eight discrete miRNAs were detected in the aqueous humor and consistently expressed in all samples. Eight significantly upregulated miRNAs were found in the NTG patients compared to the controls (fold-change > 2, p < 0.05). They were hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa-miR-375, and hsa-miR-143-3p. These miRNAs were predicted to be associated with the biological processes of apoptosis, autophagy, neurogenesis, and aging in the gene ontology categories. The related Kyoto encyclopedia of genes and genomes pathways were extracellular matrix-receptor interaction, mucin-type O-glycan biosynthesis, biotin metabolism, and signaling pathways regulating the pluripotency of stem cells. The differentially expressed miRNA in the NTG samples compared to the controls suggest the possible roles of miRNA in the pathogenesis of NTG. The underlying miRNA-associated pathways further imply novel targets for the pathogenesis of NTG.

The Role of Nuclear Factor of Activated T Cells 5 in Hyperosmotic Stress-Exposed Human Lens Epithelial Cells.

We investigated the role of nuclear factor of activated T cells 5 (NFAT5) under hyperosmotic conditions in human lens epithelial cells (HLECs). Hyperosmotic stress decreased the viability of human lens epithelial B-3 cells and significantly increased NFAT5 expression. Hyperosmotic stress-induced cell death occurred to a greater extent in NFAT5-knockout (KO) cells than in NFAT5 wild-type (NFAT5 WT) cells. Bcl-2 and Bcl-xl expression was down-regulated in NFAT5 WT cells and NFAT5 KO cells under hyperosmotic stress. Pre-treatment with a necroptosis inhibitor (necrostatin-1) significantly blocked hyperosmotic stress-induced death of NFAT5 KO cells, but not of NFAT5 WT cells. The phosphorylation levels of receptor-interacting protein kinase 1 (RIP1) and RIP3, which indicate the occurrence of necroptosis, were up-regulated in NFAT5 KO cells, suggesting that death of these cells is predominantly related to the necroptosis pathway. This finding is the first to report that necroptosis occurs when lens epithelial cells are exposed to hyperosmolar conditions, and that NFAT5 is involved in this process.

Phosphorylation of STAT3 and ERBB2 mediates hypoxia­induced VEGF release in ARPE­19 cells.

Neovascularization in the retina can cause loss of vision. Vascular endothelial growth factor (VEGF) serves an important role in the pathogenesis of retinal vascular diseases. Hypoxia is a notable cause of VEGF release and both STAT3 and ERBB2 are known to be associated with VEGF. In addition, STAT3 and ERBB2 interact with each other. In the present study, it was hypothesized that signal transducer and activator of transcription 3 (STAT3) and erbB­2 receptor tyrosine kinase 2 (ERBB2) may be involved in the regulation of hypoxia­induced VEGF in the retina. Cells of the retinal pigment epithelium (RPE) are an important source of VEGF. Therefore, the RPE­derived human cell line ARPE­19 was exposed to hypoxia. Hypoxia­induced phosphorylation of STAT3 and ERBB2 in ARPE­19 cells was decreased by AG490, an inhibitor of Janus kinase 2, as were hypoxia­induced VEGF release and tube formation in human umbilical vein endothelial cells. Thus, phosphorylation of ERBB2 and STAT3 regulates hypoxia­induced VEGF release in ARPE­19 cells. The results of the present study suggested that inhibition of ERBB2 and STAT3­mediated pathways under hypoxia may represent a new strategy for treating retinal vascular disease.

Resveratrol Ameliorates Retinal Ischemia/Reperfusion Injury in C57BL/6J Mice via Downregulation of Caspase-3.

PURPOSE: Ischemia/reperfusion (I/R) injury induces apoptosis in retinal ganglion cells (RGCs). Resveratrol (Res) is a potent natural antioxidant with beneficial effects in many ocular diseases, such as age-related macular degeneration, diabetic retinopathy, and glaucoma. Because caspase-3 expression is highly correlated with activation of the apoptotic pathway, the present study aimed to determine whether Res regulates the expression of caspase-3 using an I/R retinal injury mouse model. METHODS: Male C57BL/6J mice were injected with Res for 2 consecutive days before I/R retinal injury. I/R retinal injury was induced by increasing the intraocular pressure for 1 h. Res was then injected for 3 consecutive days. Changes in retinal morphology were monitored for 3 days after injury by histochemistry using hematoxylin and eosin staining. mRNAs and proteins were extracted 2 days after injury. The expression levels of caspase-8 and caspase-3 mRNA and protein were determined using reverse-transcriptase polymerase chain reaction (RT-PCR) and western blot analyses. RESULTS: I/R injury induced declines in retinal thickness and number of RGCs during 5 days after injury. Caspase-8 and caspase-3 mRNA and protein activation increased. Res treatment reduced the significant loss of retinal morphology and downregulated the expression of mRNA and activation of caspase-8 and caspase-3 protein. CONCLUSIONS: The observed changes in retinal morphology suggest that I/R injury promotes retinal degeneration. Increased expression of caspase-8 and caspase-3 mRNA indicates apoptosis activation. Res, however, suppresses apoptosis via downregulation of caspase-8 and caspase-3 expression.

Tristetraprolin regulates the decay of the hypoxia-induced vascular endothelial growth factor mRNA in ARPE-19 cells.

The aim of the present study was to investigate the effects of tristetraprolin (TTP) on the vascular endothelial growth factor (VEGF) mRNA and protein expression levels in retinal pigment epithelial cells under hypoxic conditions, and to consider the possibility of using TTP as a novel treatment tool for neovascular age­related macular degeneration (AMD). Overexpression of TTP reduced the expression and secretion levels of VEGF in ARPE­19 cells under hypoxic conditions. TTP destabilized the VEGF mRNA by binding to adenosine and uridine­rich elements regions in its 3'­untranslated region. Furthermore, conditioned medium (CM) from TTP­overexpressing ARPE­19 cells suppressed the tube formation in human umbilical vein endothelial cells compared with hypoxic CM. These findings indicate that regulation of TTP expression may be a promising therapeutic tool for neovascular AMD, however, further research is required.

Resveratrol Induces Glioma Cell Apoptosis through Activation of Tristetraprolin.

Tristetraprolin (TTP) is an AU-rich elements (AREs)-binding protein, which regulates the decay of AREs-containing mRNAs such as proto-oncogenes, anti-apoptotic genes and immune regulatory genes. Despite the low expression of TTP in various human cancers, the mechanism involving suppressed expression of TTP is not fully understood. Here, we demonstrate that Resveratrol (3,5,4'-trihydroxystilbene, Res), a naturally occurring compound, induces glioma cell apoptosis through activation of tristetraprolin (TTP). Res increased TTP expression in U87MG human glioma cells. Res-induced TTP destabilized the urokinase plasminogen activator and urokinase plasminogen activator receptor mRNAs by binding to the ARE regions containing the 3' untranslated regions of their mRNAs. Furthermore, TTP induced by Res suppressed cell growth and induced apoptosis in the human glioma cells. Because of its regulation of TTP expression, these findings suggest that the bioactive dietary compound Res can be used as a novel anti-cancer agent for the treatment of human malignant gliomas.

Resveratrol suppresses vascular endothelial growth factor secretion via inhibition of CXC-chemokine receptor 4 expression in ARPE-19 cells.

The present study characterizes the effects of resveratrol (Res) on vascular endothelial growth factor (VEGF) secretion in retinal pigment epithelial (RPE) cells. ARPE-19 cells were treated with CoCl2, a hypoxia mimetic agent. CoCl2 treatment increased protein levels of hypoxia inducible factor-1α (HIF-1α) and CXC-chemokine receptor 4 (CXCR4), and secretion of VEGF. To confirm the effects of Res on VEGF secretion, the human umbilical vein endothelial cell tube formation assay was performed with conditioned medium from Res-treated ARPE-19 cells. The well-known antioxidant Res effectively blocked these effects and reduced phosphorylation of nuclear factor (NF)-κB, an upstream activator of CXCR4. Furthermore, Res also suppressed VEGF secretion induced by SDF-1, a ligand of CXCR4. Conditioned medium from Res-treated ARPE-19 cells clearly suppressed tube formation compared with hypoxia-treated conditioned medium. The results demonstrated that Res inhibited the hypoxia mimetic CoCl2-induced expression of VEGF in ARPE-19 cells. Res suppressed CXCR4 expression through decreased phosphorylation of NF-κB, resulting in downregulation of VEGF secretion.

Tristetraprolin inhibits the growth of human glioma cells through downregulation of urokinase plasminogen activator/urokinase plasminogen activator receptor mRNAs.

Urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) play a major role in the infiltrative growth of glioblastoma. Downregulatoion of the uPA and uPAR has been reported to inhibit the growth glioblastoma. Here, we demonstrate that tristetraprolin (TTP) inhibits the growth of U87MG human glioma cells through downregulation of uPA and uPAR. Our results show that expression level of TTP is inversely correlated with those of uPA and uPAR in human glioma cells and tissues. TTP binds to the AU-rich elements within the 3' untranslated regions of uPA and uPAR and overexpression of TTP decreased the expression of uPA and uPAR through enhancing the degradation of their mRNAs. In addition, overexpression of TTP inhibited the growth and invasion of U87MG cells. Our findings implicate that TTP can be used as a promising therapeutic target to treat human glioma.