BMX deletion mitigates neuroinflammation induced by retinal ischemia/reperfusion through modulation of the AKT/ERK/STAT3 signaling cascade.

Aims: Retinal ischemia/reperfusion (I/R) injury is implicated in the etiology of various ocular disorders. Prior research has demonstrated that bone marrow tyrosine kinase on chromosome X (BMX) contributes to the advancement of ischemic disease and inflammatory reactions. Consequently, the current investigation aims to evaluate BMX's impact on retinal I/R injury and clarify its implied mechanism of action. Main methods: This study utilized male and female systemic BMX knockout (BMX-/-) mice to conduct experiments. The utilization of Western blot assay and immunofluorescence labeling techniques was employed to investigate variations in the expression of protein and tissue localization. Histomorphological changes were observed through H&E staining and SD-OCT examination. Visual function changes were assessed through electrophysiological experiments. Furthermore, apoptosis in the retina was identified using the TUNEL assay, as well as the ELISA technique, which has been utilized to determine the inflammatory factors level. Key findings: Our investigation results revealed that the knockdown of BMX did not yield a significant effect on mouse retina. In mice, BMX knockdown mitigated the negative impact of I/R injury on retinal tissue structure and visual function. BMX knockdown effectively reduced apoptosis, suppressed inflammatory responses, and decreased inflammatory factors subsequent to I/R injury. The outcomes of the current investigation revealed that BMX knockdown partially protected the retina through downregulating phosphorylation of AKT/ERK/STAT3 pathway. Significance: Our investigation showed that BMX-/- reduces AKT, ERK, and STAT3 phosphorylation, reducing apoptosis and inflammation. Thus, this strategy protected the retina from structural and functional damage after I/R injury.

Inhibition of NF-κB ameliorates aberrant retinal glia activation and inflammatory responses in streptozotocin-induced diabetic rats.

Background: To determine the anti-inflammatory effects of IMD-0354, a specific NF-κB blocker, on glial cells in rats with streptozotocin (STZ)-induced diabetic retinopathy (DR). Methods: The following four groups of rats were used: control, control + IMD-0354, STZ, and STZ + IMD-0354. After six weeks of STZ injection, diabetic rats and nondiabetic control rats received IMD-0354 (30 mg/kg) or an equal volume of 4% dimethyl sulfoxide (DMSO) in phosphate-buffered saline intraperitoneally for six consecutive weeks. The following four groups of primary rat retinal microglia and Müller cells were used: control (5 mM), control + IMD-0354, high glucose (20 mM), and high glucose + IMD-0354. The effects of IMD-0354 on nuclear factor-κB (NF-κB) activation, oxidative stress strength, expression of inflammatory cytokines and VEGF (vascular endothelial growth factor), activation of glial cells, and apoptosis of neuron cells were evaluated by immunohistochemistry, oxidative stress assays, western blot, enzyme linked immunosorbent assay (ELISA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining respectively. Results: Nuclear translocation of NF-κB was markedly increased in diabetic rat retina and high glucose treated glial cells. Systemic administration of IMD-0354 significantly inhibited NF-κB activation in both diabetic rat retina and high glucose treated glial cells, ameliorated oxidative injury, inflammatory responses, VEGF production and glial cell activation, and protected neurons from apoptosis. Conclusions: Our findings indicated that NF-κB activation is acritical step in the abnormal reactivity of glial cells in STZ-induced diabetic rats. Inhibition effect of IMD-0354 on NF-κB activation may represent a promising therapeutic strategy for DR via a variety of mechanisms, including inflammation reduction and glial cells regulation.

The Role of Widefield Optical Coherence Tomography Angiography in the Diagnosis and Management of Acute Vogt-Koyanagi-Harada Disease.

PURPOSE: We explore the choroid vasculature changes of acute Vogt-Koyanagi-Harada (VKH) disease using widefield optical coherence tomography angiography (OCTA). METHODS: In this retrospective, observational, longitudinal study, 16 patients with acute VKH disease (32 eyes, mean age: 42.19 ±13.66 years) were measured using widefield OCTA. RESULTS: In this study, we first described the multiple dark foci in choriocapillaris and Sattler's layer in the panoramic montage of the five 12x12mm images in 30 eyes (93.8%) of acute VKH disease. OCTA follow-up in these 30 eyes demonstrated the diminished size and number of these dark foci in choriocapillaris and Sattler's layer after the initiation of treatment. CONCLUSIONS: Widefield OCTA enables noninvasive identification of characteristics of flow void at the level of superficial choroidal vessels in the acute phase and may be a novel valuable tool for diagnosis and monitoring of disease progression in VKH disease in the future.

Increased expression of TSPO-VDAC complex is correlated with NLRP3 inflammasome activation in diabetic retinopathy.

The present study aimed to investigate the level of translocator protein (TSPO) and its correlation with different inflammatory cytokines in diabetic retinopathy (DR) patients. Peripheral blood samples were obtained from 54 DR patients and 22 age-related cataract (ARC) patients. The mRNA expression of TSPO, voltage-dependent anion channel (VDAC), apoptosis-associated speck like protein with a caspase recruitment domain (ASC), NOD-like receptors pyrin domain-containing 3 (NLRP3) and caspase-1 were examined by reverse transcription-quantitative PCR. Interleukin-1ß and interleukin-18 levels were detected by enzyme-linked immunosorbent assay. The mRNA levels of TSPO, VDAC, ASC, NLRP3 and capase-1, the protein levels of IL-ß and IL-18 were all significantly higher in the DR group compared with those in the ARC group. The expression levels of those aforementioned cytokines/proteins were more significantly higher in the subgroup of active proliferative DR (PDR) compared with those in the inactive PDR group (P<0.05). Significant positive correlations between TSPO/VDAC complex and ASC, NLRP3, capase-1, IL-ß and IL-18 were found in DR patients. These outcomes suggested that TSPO/VDAC complex and NLRP3 inflammasomes may play an important role in the development and progression of DR.

Intravitreal injection of mitochondrial DNA induces cell damage and retinal dysfunction in rats.

BACKGROUND: Retinal neurodegeneration is induced by a variety of environmental insults and stresses, but the exact mechanisms are unclear. In the present study, we explored the involvement of cytosolic mitochondrial DNA (mtDNA), resulting in the cGAS-STING dependent inflammatory response and apoptosis in retinal damage in vivo. METHODS: Retinal injury was induced with white light or intravitreal injection of lipopolysaccharide (LPS). After light- or LPS-induced injury, the amount of cytosolic mtDNA in the retina was detected by PCR. The mtDNA was isolated and used to transfect retinas in vivo. WB and real-time PCR were used to evaluate the activation of cGAS-STING pathway and the levels of apoptosis-associated protein at different times after mtDNA injection. Retinal cell apoptosis rate was detected by TUNEL staining. Full-field electroretinography (ERG) was used to assess the retinal function. RESULTS: Light injury and the intravitreal injection of LPS both caused the leakage of mtDNA into the cytoplasm in retinal tissue. After the transfection of mtDNA in vivo, the levels of cGAS, STING, and IFN-ß mRNAs and the protein levels of STING, phosph-TBK1, phospho-IRF3, and IFN-ß were upregulated. mtDNA injection also induced the activation of caspase 3 and caspase 9. BAX and BAK were increased at both the mRNA and protein levels. The release of cytochrome c from the mitochondria to the cytosol was increased after mtDNA injection. The wave amplitudes on ERG decreased and retinal cell apoptosis was detected after mtDNA injection. CONCLUSIONS: Cytosolic mtDNA triggers an inflammatory response. It also promotes apoptosis and the dysfunction of the retina.

Characteristics and Visual Prognosis of Pediatric Open Globe Injury in Shanghai, China.

INTRODUCTION: The aim of the study was to describe the characteristics of open globe injury (OGI) and the relationship between the complications and visual outcomes in children with this type of injury. METHODS: This was a retrospective chart review of 1,664 children, under the age of 16 years, who were hospitalized for OGI between January 1, 2007, and December 31, 2015. Each patient's age, sex, cause and agent of injury, complications, visual acuity, and classification of ocular trauma were collected for review and analysis. RESULTS: The mean age was 5.6 ± 3.4 years. Right eyes were particularly vulnerable to injury (right eye:left eye ratio = 1.2:1). Traumatic cataract was the most common complication. The average initial and final best corrected visual acuity were logarithm of the minimum angle of resolution (logMAR) 2.04 ± 0.78 and logMAR 1.74 ± 0.88, respectively. Logistic regression analysis showed that hyphema (odds ratio [OR] = 1.850), iris prolapse (OR = 1.702), vitreous hemorrhage (OR = 9.703), retinal detachment (OR = 11.938), endophthalmia (OR = 5.377), intraocular foreign body (OR = 3.346), and initial visual acuity <0.05 (OR = 9.017) were risk factors for visual acuity <0.05 at hospital discharge. CONCLUSION: OGI was most frequent in preschool children and boys. Right eyes were more vulnerable than left eyes. Poor visual outcomes were associated with hyphema, iris prolapse, vitreous hemorrhage, retinal detachment, endophthalmia, intraocular foreign body, and an initial visual acuity <0.05.

Observation of varicella zoster virus-induced acute retinal necrosis: viral load detection and visual outcome.

OBJECTIVES: To observe the changes of viral load in aqueous humour samples and visual outcomes in varicella zoster virus (VZV)-induced acute retinal necrosis (ARN). METHODS: Observational retrospective study. Medical records and viral load measured by real-time quantitative polymerase chain reaction (qPCR) of 20 eyes with VZV-induced ARN were reviewed. RESULTS: The mean viral load at presentation was 5.7 × 107 ± 9.7 × 107 copies/mL. An initial plateau phase for viral load lasting up to 2 weeks occurred in most eyes (18 eyes, 90%). In the following logarithmic reduction phase, the mean slope of the decline in viral load was -0.103 ± 0.029 log/day, and the expected time for half reduction of the initial viral load was 3.2 ± 1.0 days. At the end of the first 8-week's antiviral treatment, the viral load was below detection threshold in all 20 eyes (100.0%). The mean logarithm of the minimum angle of resolution (logMAR) best-corrected visual acuity (BCVA) improved from 1.1 ± 0.7 (Snellen equivalent 20/250) to 0.7 ± 0.6 (Snellen equivalent 20/100) after a follow-up of 8.6 ± 2.0 months. Thirteen of the 20 eyes (65.0%) suffered retinal detachment and underwent vitrectomy. The initial viral load was the independent predictive factor of logMAR BCVA at the last follow-up (ß = 0.745, P < 0.001). CONCLUSIONS: The observation of viral load changes by qPCR was useful for better monitoring of therapeutic efficacy and deciding needed antiviral duration in VZV-induced ARN patients.

Intravitreal injection of mitochondrial DNA induces cell damage and retinal dysfunction in rats

BACKGROUND: Retinal neurodegeneration is induced by a variety of environmental insults and stresses, but the exact mechanisms are unclear. In the present study, we explored the involvement of cytosolic mitochondrial DNA (mtDNA), resulting in the cGAS-STING dependent inflammatory response and apoptosis in retinal damage in vivo. METHODS: Retinal injury was induced with white light or intravitreal injection of lipopolysaccharide (LPS). After light-or LPS-induced injury, the amount of cytosolic mtDNA in the retina was detected by PCR. The mtDNA was isolated and used to transfect retinas in vivo. WB and real-time PCR were used to evaluate the activation of cGAS-STING path-way and the levels of apoptosis-associated protein at different times after mtDNA injection. Retinal cell apoptosis rate was detected by TUNEL staining. Full-field electroretinography (ERG) was used to assess the retinal function. RESULTS: Light injury and the intravitreal injection of LPS both caused the leakage of mtDNA into the cytoplasm in retinal tissue. After the transfection of mtDNA in vivo, the levels of cGAS, STING, and IFN-ß mRNAs and the protein levels of STING, phosph-TBK1, phospho-IRF3, and IFN-ß were upregulated. mtDNA injection also induced the activation of caspase 3 and caspase 9. BAX and BAK were increased at both the mRNA and protein levels. The release of cytochrome c from the mitochondria to the cytosol was increased after mtDNA injection. The wave amplitudes on ERG decreased and retinal cell apoptosis was detected after mtDNA injection. CONCLUSIONS: Cytosolic mtDNA triggers an inflammatory response. It also promotes apoptosis and the dysfunction of the retina.

Focal choroidal excavation complicated with choroidal neovascularization in young and middle aged patients.

AIM: To investigate the clinical and optical coherence tomography (OCT) features of focal choroidal excavation (FCE) complicated with choroidal neovascularization (CNV) in young and middle aged patients. METHODS: We performed a retrospective review of 26 patients with FCE accompanied by CNV. All patients underwent a complete ophthalmic examination. We analyzed the clinical characteristics of patients, focusing on the spectral-domain OCT features. All patients received intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) agents. And we assessed the changes of central retinal thickness and best-corrected visual acuity (BCVA) after anti-VEGF therapy. RESULTS: The mean age of 26 patients was 35.5±7.3y (range, 21-48y). Of the 26 FCE lesions, 11 were located subfoveal, 6 were parafoveal, and 9 were extrafoveal. The mean FCE depth was 129.8±50.3 µm, and the mean width was 901.3±306.0 µm. The FCE depth was correlated positively with the width, but not correlated with age or refractive error. CNV was located within the excavation (19 eyes) or adjacent to the excavation (7 eyes). After anti-VEGF therapy, the central retinal thickness was significantly reduced and the BCVA was significantly improved. In the absorption process of subretinal fluid, we found that the fluid in the excavations needed to be absorbed at the last. A small amount of residual fluid could still be seen in a few deep excavations even after a long-term follow-up. CONCLUSION: FCE may be an important reason to cause CNV. Especially in young patients with idiopathic CNV, we should pay attention to the use of OCT to check the presence of FCE. Anti-VEGF therapy is generally effective for CNV associated with FCE.

Effect of Phosphorylated-Extracellular Regulated Kinase 1/2 Inhibitor on Retina from Light-induced Photoreceptor Degeneration.

BACKGROUND: The demonstrated role of mitogen-activated protein kinase (MAPK) in both cell apoptosis and the inflammation pathway makes it an attractive target for photoreceptor protection. The aim of this study was to investigate the protective effects of MAPK antagonists against photoreceptor degeneration and retinal inflammation in a rat model of light-induced retinal degeneration. METHODS: Sprague Dawley rats were treated with intravitreal injections of MAPK antagonists, inhibitors of p-P38, phosphorylated-extracellular regulated kinase (p-ERK) 1/2, and p-c-Jun N-terminal kinase (JNK) just before they were assigned to dark adaptation. After dark adaptation for 24 h, rats were exposed to blue light (2500 lux) in a light box for 24 h, and then returned to the normal 12-h light/12-h dark cycle. Samples were collected at different time points. MAPK expression during light exposure was examined with immunofluorescence. Photoreceptor death was detected with histopathology and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression of retinal p-ERK1/2, caspase 3, activated caspase 3, tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß was examined by Western blotting. Differences between groups were evaluated using unpaired one-way analysis of variance and least significant difference post hoc tests. RESULTS: MAPKs (P38, ERK1/2, and p-JNK) were phosphorylated and activated in the light injury groups, compared with normal group, and their expressions were mainly elevated in the outer nuclear layer (ONL). Among the selected MAPK antagonists, only the p-ERK1/2 inhibitor attenuated the loss of photoreceptors and the thinning of ONL in light injury groups. Besides, p-ERK1/2 inhibitor refrained light-induced photoreceptor apoptosis, which was presented by TUNEL positive cells. Light injury significantly increased the expression of p-ERK1/2 (1.12 ± 0.06 vs. 0.57 ± 0.08, t = 9.99, P < 0.05; 1.23 ± 0.03 vs. 0.57 ± 0.08, t = 11.90, P < 0.05; and 1.12 ± 0.12 vs. 0.57 ± 0.08, t = 9.86, P < 0.05; F = 49.55, P < 0.001), and induced caspase 3 activating (0.63 ± 0.06 vs. 0.14 ± 0.05, t = 13.67, P < 0.05; 0.74 ± 0.05 vs. 0.14 ± 0.05, t = 16.87, P < 0.05; and 0.80 ± 0.05 vs. 0.14 ± 0.05, t = 18.57, P < 0.05; F = 100.15, P < 0.001), compared with normal group. The p-ERK1/2 inhibitor significantly reduced p-ERK1/2 overexpression (0.61 ± 0.06 vs. 1.12 ± 0.06, t = -9.26, P < 0.05; 0.77 ± 0.06 vs. 1.23 ± 0.03, t = -8.29, P < 0.05; and 0.68 ± 0.03 vs. 1.12 ± 0.12, t = -7.83, P < 0.05; F = 49.55, P < 0.001) and downregulated caspase 3 activating (0.23 ± 0.04 vs. 0.63 ± 0.06, t = -11.24, P < 0.05; 0.43 ± 0.03 vs. 0.74 ± 0.05, t = -8.86, P < 0.05; and 0.58 ± 0.03 vs. 0.80 ± 0.05, t = -6.17, P < 0.05; F = 100.15, P < 0.001), compared with light injury group. No significant change in the total level of caspase 3 was seen in different groups (F = 0.56, P = 0.75). As for inflammation, light injury significantly increased the expression of TNF-α (0.42 ± 0.04 vs. 0.25 ± 0.05, t = 5.99, P < 0.05; 0.65 ± 0.03 vs. 0.25 ± 0.05, t = 14.87, P < 0.05; and 0.86 ± 0.04 vs. 0.25 ± 0.05, t = 22.58, P < 0.05; F = 160.27, P < 0.001) and IL-1ß (0.24 ± 0.01 vs. 0.19 ± 0.02, t = 2.33, P < 0.05; 0.35 ± 0.02 vs. 0.19 ± 0.02, t = 7.97, P < 0.05; and 0.48 ± 0.04 vs. 0.19 ± 0.02, t = 14.69, P < 0.05; F = 77.29, P < 0.001), compared with normal group. P-ERK1/2 inhibitor significantly decreased the overexpression of TNF-α (0.22 ± 0.02 vs. 0.42 ± 0.04, t = -7.40, P < 0.05; 0.27 ± 0.02 vs. 0.65 ± 0.03, t = -14.27, P < 0.05; and 0.33 ± 0.03 vs. 0.86 ± 0.04, t = -19.58, P < 0.05; F = 160.27, P < 0.001) and IL-1ß (0.13 ± 0.03 vs. 0.24 ± 0.01, t = -5.77, P < 0.05; 0.17 ± 0.01 vs. 0.22 ± 0.02, t = -9.18, P < 0.05; and 0.76 ± 0.05 vs. 0.48 ± 0.04, t = -13.12, P < 0.05; F = 77.29, P < 0.001), compared with light injury group. CONCLUSION: The p-ERK1/2 inhibitor might protect the retina from light-induced photoreceptor degeneration and retinal inflammation.

CRISPR-mediated SOX9 knockout inhibits GFAP expression in retinal glial (Müller) cells.

Müller cells, as the predominant glial element in the sensory retina, play a crucial role in healthy and diseased retina. Overactivation of Müller cells in response to damage is detrimental to the retina tissue. Current research shows that inhibiting glial fibrillary acidic protein (GFAP), a sensitive indicator of Müller cell activation, attenuated glial reactions and promoted neuroprotection. Recent evidence suggests that the transcript factor SOX9 (sex-determining region Y box 9), part of the SOX family, regulates GFAP expression of astrocytes in the central nervous system. However, in retina Müller cells, it is still unknown whether GFAP can be downregulated by reduced SOX9 function. The present results show that clustered regularly interspaced short palindromic repeats/Cas9-mediated SOX9 knockout not only inhibited GFAP expression in rat Müller cells but also attenuated cell migration ability. These results suggest that inhibition of SOX9 activity may be a novel therapeutic strategy for reduction of glial cell activity.

Microglia enhanced the angiogenesis, migration and proliferation of co-cultured RMECs.

BACKGROUND: Attention is increasingly being given to microglia-related inflammation in neovascular diseases, such as diabetic retinopathy and age-related macular disease. Evidence shows that activated microglia contribute to disruption of the blood-retinal barrier, however, the mechanism is unclear. In this study, we aimed to clarify whether and how microglia affect the function of retinal microvascular endothelial cells (RMECs). METHODS: We activated microglia by Lipopolysaccharides (LPS) stimulation. After co-culturing static or activated microglia with RMECs using the Transwell system, we evaluated the function of RMECs. Vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) levels in the supernatant from the lower chamber were evaluated by ELISA. Angiogenesis, migration, and proliferation of RMECs were assessed by tube formation, wound healing, and WST-1 assays. The expression levels of tight junction proteins (ZO-1 and occludin) and endothelial markers (CD31 and CD34) were examined by Western blot analysis. RESULTS: We successfully established an LPS-activated microglia model and co-culture system of static or activated microglia with RMECs. In the co-culture system, we showed that microglia, especially activated microglia stimulated VEGF-A and PDGF-BB expression, enhanced angiogenesis, migration, proliferation, and permeability, and altered the phenotype of co-cultured RMECs. CONCLUSIONS: Microglia, especially activated microglia, play important roles in angiogenesis and maintenance of vascular function hemostasis in the retinal microvasculature. The mechanism needs further investigation and clarification.

Wogonin induces retinal neuron-like differentiation of bone marrow stem cells by inhibiting Notch-1 signaling.

Age-related macular degeneration and retinitis pigmentosa are major causes of irreversible vision loss in the elderly and, despite sustained efforts, current treatments are largely ineffective. Wogonin is a bioactive plant flavonoid possessing a range of beneficial properties, including neuroprotective effects. We investigated the ability of wogonin to promote retinal neuron-like differentiation of bone marrow stem cells (BMSCs) and assessed the involvement of Notch-1 signaling in this process. Cultured mouse BMSCs were left untreated or exposed to neurotrophic factors in the presence or absence of wogonin, and western blotting, RT-PCR and immunofluorescence were used to identify changes in molecular markers of stemness and neuroretinal differentiation. Proteins in the Notch-1 signaling pathway, a main negative regulator of neurogenesis, were also examined by western blotting. We found that expression of stem cell markers was reduced, while markers of mature retinal neurons, bipolar cells and photoreceptors were increased in wogonin-treated BMSCs. Wogonin also dose-dependently decreased expression of Notch-1 signaling proteins. Moreover, blockade of Notch-1 both mimicked and enhanced the effect of wogonin to facilitate BMSC differentiation into retinal neuron-like cells. Wogonin thus appears to promote retinal neuron-like differentiation of BMSCs by antagonizing the inhibitory actions of Notch-1 signaling on neurogenesis and may be useful in the treatment of retinal degenerative diseases.

Glucocorticoid-induced leucine zipper overexpression inhibits lipopolysaccharide-induced retinal inflammation in rats.

Glucocorticoid-induced leucine zipper (GILZ) mediates several effects of glucocorticoids and has important anti-inflammatory properties. Here, we explored the role of GILZ in inhibiting retinal inflammation. Endotoxin-induced uveitis (EIU) was established in rats by intravitreal injection of lipopolysaccharide (LPS). GILZ levels decreased in the EIU retina after LPS injection. Retinal GILZ was downregulated by recombinant lentivirus-delivered short-hairpin RNA targeting GILZ (shRNA-GILZ-rLV) and upregulated by recombinant lentivirus-mediated GILZ overexpression (Oe-GILZ-rLV). GILZ silencing attenuated the anti-inflammatory effects of intravitreal injection of triamcinolone acetonide (TA) in the EIU retina, as demonstrated by increased retinal interleukin (IL)-1ß, monocyte chemoattractant protein (MCP)-1and intercellular cell adhesion molecule-1 expression at 18 h after TA injection. A Bio-Plex cytokine assay and western blotting demonstrated that GILZ overexpression inhibited the effects of LPS, downregulating retinal IL-1ß, MCP-1, MIP-1α, and IL-17 and inhibiting LPS-induced activation of the retinal toll-like receptor 4-myeloid differentiation factor 88 signaling pathway. At 48 and 72 h after LPS injection, the clinical score of inflammation was significantly lower in Oe-GILZ-rLV-transfected eyes than in blank-rLV-transfected eyes. Histological examination showed a 67.85% reduction of infiltrating inflammatory cells in the anterior chamber and a 58.97% reduction in vitreous cavity of Oe-GILZ-rLV transfected eyes at 48 h after LPS injection. Taken together, our results suggest that GILZ is a novel therapeutic target for the treatment of retinal inflammatory diseases.

Secretion of Down Syndrome Critical Region 1 Isoform 4 in Ischemic Retinal Ganglion Cells Displays Anti-Angiogenic Properties Via NFATc1-Dependent Pathway.

Down syndrome candidate region 1 (DSCR1) has two differentially regulated isoforms (DSCR1-1 and DSCR1-4) and is reported to play a role in a number of physiological processes, such as the inhibition of cardiac hypertrophy, attenuation of angiogenesis and carcinogenesis, and protection against neuronal death. However, the function of DSCR1 in the retina is still not clear. Therefore, we analyzed the expression and location of DSCR1 in the retina of neonatal mice with oxygen-induced retinopathy (OIR), and studied its effects on angiogenesis. The neonatal C57BL/6J mice were exposed to 75 % O2 for 5 days from postnatal day 7 (P7) to P12. At P12, the mice were returned to 21 % O2 at room air. The primary retinal ganglion cells (RGCs) were exposed to hypoxia (93 % N2, 5 % CO2, and 2 % O2) at 37 °C for 36 h. And then the mouse retinal microvascular endothelial cells (mRMECs) were treated with 25 ng/mL vascular endothelial growth factor (VEGF) or culture medium conditioned by hypoxic RGCs alone, hypoxic RGCs treated with DSCR1-4-siRNA (siDSCR1-4) or hypoxic RGCs treated with siDSCR1-4 and 200 ng/mL cyclosporin A (CsA), and then primed with VEGF (25 ng/mL). The expression of DSCR1-4 increased strongly at P16 after OIR. There was no change in messenger RNA (mRNA) expression of DSCR1-1 at P16 after OIR. The increased DSCR1 was mainly located in the RGCs of avascular retina. In addition, DSCR1-4 expression was increased in primary RGCs after hypoxia exposure. There was no change in mRNA expression of DSCR1-1 in primary RGCs after hypoxia exposure. Moreover, DSCR1-4 produced by hypoxic RGCs showed anti-angiogenic properties, with decreased cell proliferation, migration, tube formation, and inflammatory cytokines production. These properties were due to inhibited nuclear factor of activated T cell (NFATc) 1 dephosphorylation and translocation into nuclear in VEGF-treated mRMECs. Using siRNA-mediated knockdown of DSCR1-4 and NFATc1 inhibitor (Cs A) further demonstrated the inhibitory effect of DSCR1-4 on angiogenic properties in VEGF-induced mRMECs, and this effect was NFATc1-dependent. This report describes a novel effect of DSCR1-4 in the aspect of anti-angiogenesis, suggesting potential therapeutic strategies for proliferative retinopathies.

Altered expression of microRNAs in the neuronal differentiation of human Wharton's Jelly mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have the capacity to generate multiple tissues of mesodermal origin, and also have the potential to trans-differentiate into neurons. We isolated MSCs from the Wharton's jelly of the human umbilical cord (WJ-MSCs), and efficiently induced WJ-MSCs into neuron-like cells using a modified method. After neuronal induction for 12 days, most of WJ-MSCs expressed mature neuronal marker MAP2 (83 ± 7%), and meanwhile some adopted neuronal morphology. WJ-MSCs also expressed Nestin (34 ± 6%), NSE (30 ± 5%), and GFAP (12 ± 3%). Moreover, we used miRNA microarray to analyze the differentially expressed miRNAs in neuronal differentiation of WJ-MSCs. Microarray analysis revealed discrepant miRNA profiles in the uninduced WJ-MSCs and WJ-MSCs derived neurons. Six miRNAs were chosen for further qRT-PCR validation. Among these 6 miRNAs, four miRNAs (miR-1290, miR-26b, miR-194, and miR-124a) were up-regulated and 2 miRNAs (miR-4521 and miR-543) were down-regulated in the WJ-MSCs derived neurons. In conclusion, WJ-MSCs could be efficiently induced into neuron-like cells. More importantly, our findings suggested that miRNAs might play important roles in the neuronal differentiation of WJ-MSCs.

Changes of TGF-ß2, MMP-2, and TIMP-2 levels in the vitreous of patients with high myopia.

PURPOSE: To investigate the concentrations of transforming growth factor (TGF)-ß2, matrix metalloproteinase (MMP)-2, and tissue inhibitor of metalloproteinase (TIMP)-2 in the vitreous of patients with high myopia. METHODS: Twenty-six patients with high myopia (HM) who received vitrectomy for macular retinoschisis or macular hole were enrolled in this prospective study. Twenty-six patients with idiopathic macular hole or macular epiretinal membrane were chosen as a control group. Vitreous samples were obtained during the vitrectomy surgery. The levels of TGF-ß2、MMP-2、TIMP-2 in the vitreous samples were measured by enzyme-linked immunosorbent assay. The MMP activity was determined by a fluorometric assay. RESULTS: There was no significant difference in the vitreous level of TGF-ß2 between HM (1.64 ± 0.38 ng/ml) and the control group (1.56 ± 0.32 ng/ml, p = 0.56). The vitreous levels of MMP-2 in HM (32.40 ± 14.90 ng/ml) were significantly higher than in the control group (21.42 ± 6.74 ng/ml, p < 0.01). The ratio of MMP-2/TIMP-2 was significantly elevated in the vitreous samples from HM (0.61 ± 0.19), compared to the control group (0.48 ± 0.11, p < 0.05). The MMP activity was also significantly elevated in the vitreous samples from HM (4,030.8 ± 1,257.3 FIU), compared to the control group (3,245.8 ± 835.6 FIU, p < 0.05). CONCLUSIONS: The elevated MMP/TIMP ratio and MMP activity may play a role in the pathogenesis of human high myopia. Large prospective studies are needed to further investigate the effect of MMPs in the pathogenesis of human high myopia.

The expression changes of myelin and lymphocyte protein (MAL) following optic nerve crush in adult rats retinal ganglion cells.

Myelin and lymphocyte protein (MAL), a component of compact myelin, is highly expressed in oligodendrocytes and Schwann cells. It has been reported that MAL may play a vital role in the process of neuronal apoptosis following acute spinal cord injury. However, acquaintance regarding its distribution and possible function in the retina is limited. Therefore, in a rodent model of optic nerve crush (ONC), the dynamic changes of MAL in retina was detected. The expression of MAL was mainly located in the retinal ganglion cells (RGCs) and was increased strongly after ONC. The peak of MAL expression appeared on the third day. In addition, there was a concomitant upregulation of active-caspase-3, which also co-localized with MAL in RGCs. Moreover, co-localization of MAL with terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (TUNEL) was detected in RGCs after ONC. Collectively, all these results suggested that the upregulation of MAL might play an important role in the pathophysiology of RGCs after ONC.

Ras homolog enriched in the brain is linked to retinal ganglion cell apoptosis after light injury in rats.

Ras homolog enriched in the brain (Rheb) is a small GTPase of the Ras family. It has been confirmed that Rheb activation not only regulates cell growth and migration but also induces neuron apoptosis after toxic stimuli. However, the function of Rheb in the retina is still not fully understood. To find out whether Rheb was involved in retinal neuron death, the expression profile of Rheb in light-damaged retinal ganglion cells (RGCs) of adult rats was investigated. Western blotting showed the expression of Rheb was significantly upregulated in the injured retina. Rheb was mainly detected in apoptotic RGCs by using double immunofluorescent staining. Active caspase-3 was upregulated and co-labeled with Rheb. Meanwhile, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that Rheb-positive RGCs underwent apoptosis after light exposure, which suggested that Rheb might be relevant to RGC apoptosis following phototoxicity. Furthermore, Western blotting and immunofluorescence showed that the expression profiles of CyclinD1 and cyclin-dependent kinase 4 (CDK4) were parallel with that of Rheb in a time-space dependent manner. Based on this study, it is speculated that Rheb might play an important role in physiological and pathological process in light-induced retina damage, which might provide a potential therapeutic avenue of retinal degeneration.

Upregulation of CREM-1 relates to retinal ganglion cells apoptosis after light-induced damage in vivo.

Previous studies have shown activation of cyclic AMP response element-binding protein (CREB) family is involved in the retinal ganglion cells (RGCs) protection. However, the function of cyclic AMP response element modulator-1 (CREM-1), one member of the CREB family, is still with limited acquaintance. To investigate whether CREM-1 is involved in RGCs death, we performed a light-induced retinal damage model in adult rats. Upregulation of CREM-1 was observed in retina after light-induced damage by performing western blot. Immunofluorescent labeling indicated that upregulated CREM-1 was localized mainly in the RGCs. We also investigated co-localization of CREM-1 with active-caspase-3 and TUNEL (apoptotic markers) in the retina after light-induced damage. In addition, the expression patterns of B cell lymphoma/leukemia-2 and Bcl-2 associated X protein were parallel with that of CREM-1. Collectively, we hypothesized upregulation of CREM-1 in the retina was associated with RGCs death after light-induced damage.