LL37/FPR2 regulates neutrophil mPTP promoting the development of neutrophil extracellular traps in diabetic retinopathy.

Diabetic retinopathy (DR) is characterized by chronic, low-grade inflammation. This state may be related to the heightened production of neutrophil extracellular traps (NETs) induced by high glucose (HG). Human cathelicidin antimicrobial peptide (LL37) is an endogenous ligand of G protein-coupled chemoattractant receptor formyl peptide receptor 2 (FPR2), expressed on neutrophils and facilitating the formation and stabilization of the structure of NETs. In this study, we detected neutrophils cultured under different conditions, the retinal tissue of diabetic mice, and fibrovascular epiretinal membranes (FVM) samples of patients with proliferative diabetic retinopathy (PDR) to explore the regulating effect of LL37/FPR2 on neutrophil in the development of NETs during the process of DR. Specifically, HG or NG with LL37 upregulates the expression of FPR2 in neutrophils, induces the opening of mitochondrial permeability transition pore (mPTP), promotes the increase of reactive oxygen species and mitochondrial ROS, and then leads to the rise of NET production, which is mainly manifested by the release of DNA reticular structure and the increased expression of NETs-related markers. The PI3K/AKT signaling pathway was activated in neutrophils, and the phosphorylation level was enhanced by FPR2 agonists in vitro. In vivo, increased expression of NETs markers was detected in the retina of diabetic mice and in FVM, vitreous fluid, and serum of PDR patients. Transgenic FPR2 deletion led to decreased NETs in the retina of diabetic mice. Furthermore, in vitro, inhibition of the LL37/FPR2/mPTP axis and PI3K/AKT signaling pathway decreased NET production induced by high glucose. These results suggested that FPR2 plays an essential role in regulating the production of NETs induced by HG, thus may be considered as one of the potential therapeutic targets.

Comparison of risk factors for OBL in FS-LASIK and SMILE correction for myopia and myopia astigmatism.

BACKGROUND: To find out the incidence and risk factors of opaque bubble layer (OBL) in eyes with myopia and myopic astigmatism following femtosecond laser-assisted in situ keratomileusis (FS-LASIK) and small incision lenticule extraction (SMILE). METHODS: A total of 1076 eyes from 569 patients who had FS-LASIK or SMILE were included in the retrospective research. For each kind of surgery, eyes were separated into two groups: "OBL" groups and "no OBL" groups. In the FS-LASIK group, eyes that developed OBL were split into "hard OBL" and "soft OBL" groups. The incidence and size of OBL were analyzed after watching the surgical procedure videotaped during the operation and taking screenshots. Surgical parameters, including sphere, cylinder, keratometry, corneal thickness, flap thickness, cap thickness, lenticule thickness, and visual acuity, were compared. RESULTS: In the FS-LASIK surgery, the incidence of OBL was 63.2% (347 eyes). A thicker central corneal thickness (CCT) was the only independent risk factor affecting the OBL area (ß = 0.126, P = 0.019). One hundred and thirty of these eyes had hard OBL, and the flap thickness of these eyes was thinner than that of those with soft OBL (P = 0.027). In the SMILE group, 26.6% (140 eyes) developed OBL. A higher flat keratometry (K) and a thicker residual stromal thickness (RST) were risk factors affecting the OBL area (ß = 0.195, P = 0.024; ß = 0.281, P = 0.001). CONCLUSION: The incidence of OBL differs between the FS-LASIK surgery and the SMILE surgery. There are differences in the factors influencing OBL between the two surgeries.

Efficacy and effectiveness of anti-VEGF or steroids monotherapy versus combination treatment for macular edema secondary to retinal vein occlusion: a systematic review and meta-analysis.

BACKGROUND: Retinal vein occlusion (RVO) is the main cause of retinal vascular blindness. Laser photocoagulation therapy is the regarded as the standard treatment for branch retinal vein occlusion (BRVO) in the guidelines, but it is not effective for macular edema (ME) secondary to central retinal vein occlusion (CRVO). As anti-VEGF (vascular endothelial growth factor) or steroids monotherapy has been used to treat RVO, but each has its advantages and disadvantages. Our purpose was to evaluate the efficacy and safety of intraocular injection of anti-VEGF combined with steroids versus anti-VEGF or steroids monotherapy for ME secondary to RVO. METHODS: We systematically searched trials on Pubmed, Embase, Cochrane Library, Web of Science and China National Knowledge Infrastructure (CNKI) for RCTs (random clinical trials) or non-RCTs, comparing anti-VEGF or steroids monotherapy to their combination. The primary outcomes were changes in best-corrected visual acuity (BCVA), central macular thickness (CMT) and intraocular pressure (IOP). The pooled data was analyzed by random effects model. FINDINGS: A total of 10 studies selected from 366 studies were included in this meta-analysis. Our results favored anti-VEGF with steroids combination therapy in comparison with anti-VEGF {pooled SMD (standardized mean difference), 95% CI, -0.16 [-0.28, -0.04], P = 0.01} or steroids (pooled SMD, 95% CI, -0.56 [-0.73, -0.40], P < 0.00001) alone on changes of BCVA. Compared with anti-VEGF monotherapy group, the combination therapy also had a better effect {pooled MD (mean difference), 95% CI, -9.62 [-17.31, -1.93], P = 0.01)} at improvements on CMT. On the changes of IOP, assessment favored that combination therapy was associated with a better relief of IOP compared to steroids monotherapy group (pooled MD, 95% CI, -5.93 [-7.87, -3.99],P < 0.00001). What's more, the incidence of ocular hypertension was lower in the combined treatment group compared with control group treated with steroids alone (Odds Ratio, 95% CI, 0.21 [0.06, 0.77], P = 0.02). Results also showed that the combination group can prolong the average time to first anti-VEGF reinjection (MD, 95% CI, 1.74 [0.57, 2.90], P = 0.003) compared to control group treated with anti-VEGF alone. CONCLUSION: Anti-VEGF with steroids combination treatment can enable a better achievement of improving BCVA, CMT, reducing the risk of increased IOP and improving patient prognosis compared to anti-VEGF or steroids therapy alone, lengthening the average time to anti-VEGF reinjection with reducing the injections during follow-up.

TNF-α released from retinal Müller cells aggravates retinal pigment epithelium cell apoptosis by upregulating mitophagy during diabetic retinopathy.

Retinal pigment epithelium (RPE) cell damage, including mitophagy-associated cell apoptosis, accelerates the pathogenesis of diabetic retinopathy (DR), a common complication of diabetes that causes blindness. Müller cells interact with RPE cells via pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α). Herein, we investigated the role of the RPE cell epidermal growth factor receptor (EGFR)/p38 mitogen-activated protein kinase (p38)/nuclear factor kappa B (NF-κB) pathway in Müller cell-derived TNF-α-induced mitophagy-associated apoptosis during DR. Our results showed that TNF-α released from Müller cells activated the EGFR/p38/NF-κB/p62 pathway to increase mitophagy and apoptosis in RPE cells under high glucose (HG) conditions. Additionally, blockade of the TNF-α/EGFR axis alleviates blood-retina barrier breakdown in diabetic mice. Our data further illustrate the effects of the Müller cell inflammatory response on RPE cell survival, implying potential molecular targets for DR treatment.

Geniposide alleviates choroidal neovascularization by downregulating HB-EGF release from RPE cells by downregulating the miR-145-5p/NF-κB axis.

Age-related macular degeneration (AMD), mainly wet AMD, is the major reason for nonreversible vision loss worldwide. Choroidal neovascularization (CNV) is a characteristic pathological manifestation of wet AMD. Stress or injury to the retinal pigment epithelium (RPE) induces proangiogenic factors that drive CNV. An iridoid glycoside extracted from the fruit of gardenia, geniposide (GEN) plays an antiangiogenic role. In this study, GEN inhibited the transcription and expression of heparin-binding epidermal growth factor (HB-EGF), a proangiogenic factor, in hypoxic RPE cells and a mouse laser-induced CNV model. Inhibition of glucagon-like peptide-1 receptor (GLP-1R), a GEN receptor blocker, eliminated the protective effect of GEN. Additionally, GEN decreased the transcription and expression of HB-EGF in hypoxia-exposed RPE cells by downregulating the miR-145-5p/NF-κB axis. Therefore, our research provides a promising novel strategy for wet AMD therapy.

Brivanib, a multitargeted small-molecule tyrosine kinase inhibitor, suppresses laser-induced CNV in a mouse model of neovascular AMD.

In age-related macular degeneration (AMD), choroidal neovascularization (CNV), a major pathologic feature of neovascular AMD (nAMD), affects 10% of patients, potentially causing serious complications, including vision loss. Vascular endothelial growth factor receptor 2 (VEGFR2) and fibroblast growth factor receptor 1 (FGFR1) contribute to the pathogenesis of CNV. Brivanib is an oral selective dual receptor tyrosine kinase (RTK) inhibitor of FGFRs and VEGFRs, especially VEGFR2 and FGFR1. In this study, brivanib inhibited zebrafish embryonic angiogenesis without impairing neurodevelopment. In a mouse CNV model, brivanib intravitreal injection blocked phosphorylation of FGFR1 and VEGFR2 and reduced CNV leakage, area, and formation without causing intraocular toxicity. Moreover, brivanib oral gavage reduced CNV leakage and area. Accordingly, brivanib remained at high concentrations (above 14,000 ng/ml) in retinal/choroidal/scleral tissues following intravitreal injection. Similarly, brivanib remained at high concentrations (over 10,000 ng/ml) in retinal/choroidal/scleral tissues following oral gavage. Finally, in vitro cell experiments demonstrated that brivanib inhibited the proliferation, migration and tube formation of microvascular endothelial cells. In conclusion, our study suggested that brivanib treatment could be a novel therapeutic strategy for nAMD.

Andrographolide attenuates choroidal neovascularization by inhibiting the HIF-1α/VEGF signaling pathway.

Choroidal neovascularization (CNV), a characteristic of wet age-related macular degeneration (AMD), leads to severe vision loss amongst the elderly in the developed countries. Currently, the premier treatment for AMD is anti-VEGF therapy, which has limited efficacy, and is still controversial. Previous studies have showed that Andrographolide (Andro) had various biological effects, including anti-angiogenesis, anti-inflammation, and antioxidant. However, the effect of Andro on the formation of CNV has not been studied thus far. Here our results showed that Andro reduced the expression levels of HIF-1α and VEGF in the RF/6A cells chemical hypoxia model and the laser-induced CNV mouse model. Moreover, Andro inhibited the tube formation activity of RF/6A cells under hypoxic conditions. Furthermore, intraperitoneal injection of Andro reduced the severity of choroidal vascular leakage and the size of CNV in the laser-induced CNV mouse model, indicating that Andro attenuated the development of CNV by inhibiting the HIF-1α/VEGF signaling pathway. These results suggest that Andro could be a potential novel therapeutic agent for AMD.

YAP via interacting with STAT3 regulates VEGF-induced angiogenesis in human retinal microvascular endothelial cells.

Endothelial dysfunction is a main feature of retinal neovascular diseases which are the leading cause of blindness in developed countries. Yes-associated protein (YAP) and signal transducer and activator of transcription factor 3 (STAT3) participate in angiogenesis via vascular endothelial growth factor (VEGF) signaling. Additionally, YAP can bind STAT3 in endothelial cells. In the study, dimethyloxalylglycine (DMOG) stimulated human retinal microvascular endothelial cells (HRMECs) was used as retinal endothelial hypoxia model. The proliferation of HRMECs, as well as t-YAP, p-STAT3 (Tyr705) increased, while p-YAP (Ser127), p-YAP (Ser397) decreased following hypoxia. Meanwhile, YAP and STAT3 translocated to the nucleus. YAP knockdown inhibited the proliferation, migration and tube formation of HRMECs. YAP overexpression up-regulated phosphorylation of STAT3. The YAP overexpression-induced HRMECs proliferation, migration and tube formation were reversed by S3I-201, a selective STAT3 inhibitor. YAP interacted with STAT3 to promote STAT3 nuclear translocation. Additionally, YAP and STAT3 promoted the transcription of VEGF synergistically. Finally, inhibition of YAP alleviated retinal pathological neovascularization in mouse oxygen-induced retinopathy (OIR) model. In summary, activated YAP interacted with STAT3 to promote the activation and nuclear translocation of STAT3, hence boosted the proliferation, migration and tube formation of HRMECs via VEGF signaling following hypoxia. The data will further elucidate the mechanisms of retinal neovascular diseases.

Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo.

choroidal neovascularization (CNV), a characteristic of wet age-related macular degeneration (AMD), causes severe vision loss among elderly patients. TANK-binding kinase 1 (TBK1) is a ubiquitously expressed serine-threonine kinase and is found to induce endothelial cells proliferation, represent a novel mediator of tumor angiogenesis and exert pro-inflammatory effect. However, the role of TBK1 in choroidal neovascularization has not been investigated so far. In this study, we found that the expression of TBK1 and VEGF was up-regulated in RF/6 A cells chemical hypoxia model and laser-induced mouse CNV model. Silencing of TBK1 suppressed the proliferation and tube formation activity of RF/6 A cells. Intravitreal injection of anti-TBK1 monoclonal antibody ameliorates CNV formation. Taken together, these findings exhibit a proangiogenic role for TBK1 via upregulating the expression of VEGF, and may suggest that TBK1 inhibition offers a unique and alternative method for prevention and treatment of AMD.

iT2DMS: a Standard-Based Diabetic Disease Data Repository and its Pilot Experiment on Diabetic Retinopathy Phenotyping and Examination Results Integration.

Type 2 diabetes mellitus (T2DM) is a common chronic disease, and the fragment data collected through separated vendors makes continuous management of DM patients difficult. The lack of standard of fragment data from those diabetic patients also makes the further potential phenotyping based on the diabetic data difficult. Traditional T2DM data repository only supports data collection from T2DM patients, lack of phenotyping ability and relied on standalone database design, limiting the secondary usage of these valuable data. To solve these issues, we proposed a novel T2DM data repository framework, which was based on standards. This repository can integrate data from various sources. It would be used as a standardized record for further data transfer as well as integration. Phenotyping was conducted based on clinical guidelines with KNIME workflow. To evaluate the phenotyping performance of the proposed system, data was collected from local community by healthcare providers and was then tested using algorithms. The results indicated that the proposed system could detect DR cases with an average accuracy of about 82.8%. Furthermore, these results had the promising potential of addressing fragmented data. The proposed system has integrating and phenotyping abilities, which could be used for diabetes research in future studies.

Involvement of Fra-1 in Retinal Ganglion Cell Apoptosis in Rat Light-Induced Retina Damage Model.

Cell cycle re-entry, in which Fra-1 (transcription factor FOS-related antigen 1) plays an important role, is a key process in neuronal apoptosis. However, the expression and function of Fra-1 in retinal ganglion cell (RGC) apoptosis are unknown. To investigate whether Fra-1 was involved in RGC apoptosis, we performed a light-induced retinal damage model in adult rats. Western blot revealed that up-regulation of Fra-1 expression appeared in retina after light exposure (LE). Immunostaining indicated that increased Fra-1 was mainly expressed in RGCs in retinal ganglion cell layer (GCL) after LE. Co-localization of Fra-1 with active caspase-3 or TUNEL-positive cells in GCL after LE was also detected. In addition, Fra-1 expression increased in parallel with cyclin D1 and phosphorylated mitogen-activated protein kinase p38 (p-p38) expression in retina after LE. Furthermore, Fra-1, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of SB203580, a highly selective inhibitor of p38 MAP kinase (p38 MAPK). All these results suggested that Fra-1 may be associated with RGC apoptosis after LE regulated by p38 MAPK through cell cycle re-entry mechanism.

Tissue factor induces VEGF expression via activation of the Wnt/ß-catenin signaling pathway in ARPE-19 cells.

PURPOSE: The purpose of the present study was to investigate the potential signal mechanism of tissue factor (TF) in the regulation of the expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (ARPE-19) cells. METHODS: An in vitro RPE cell chemical hypoxia model was established by adding cobalt chloride (CoCl2) in the culture medium. The irritative concentration of CoCl2 was determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit. VEGF production in ARPE-19 cells was measured with enzyme-linked immunosorbent assay (ELISA) and western blotting. The Wnt signaling pathway-associated molecules, including phospho-glycogen synthase kinase 3ß (p-GSK3ß), GSK3ß, p-ß-catenin and ß-catenin, were detected with western blotting. pEGFP-N3-hTF was constructed and verified with digestion of the restriction enzyme and sequencing analysis. Human TF overexpression and silencing plasmids were transfected into the ARPE-19 cells to clarify the causal relationship between TF and VEGF expression. The Transwell coculture system of ARPE-19 cells and RF/6A rhesus macaque choroid-retinal endothelial cells was performed to evaluate cell invasion and tube formation ability. RESULTS: Our anoxic model of ARPE-19 cells showed that TF expression was upregulated in accordance with variations in hypoxia-inducible factor 1-alpha (HIF-1α) and VEGF levels. Silencing and overexpression of TF decreased and increased VEGF expression, respectively. The Wnt/ß-catenin signaling pathway played an important role in this effect. Results from the ARPE-19 cell and RF/6A cell coculture system showed that the enhancement of TF expression in the ARPE-19 cells led to significantly faster invasion and stronger tube-forming ability of the RF/6A cells, while siRNA-mediated TF silencing caused the opposite effects. Pharmacological disruption of Wnt signaling IWR-1-endo inhibited the effects compared to the TF-overexpressing group, indicating the importance of the Wnt/ß-catenin signaling pathway in the process of TF-induced VEGF expression and angiogenesis. CONCLUSIONS: Involvement of the activation of the Wnt/ß-catenin signaling pathway is an important mediator for TF-induced VEGF production during the process of angiogenesis. Thus, our findings may ascertain the molecular regulation of TF in neovessel formation and show significant therapeutic implications.

PKR promotes choroidal neovascularization via upregulating the PI3K/Akt signaling pathway in VEGF expression.

PURPOSE: The aim of this study was to investigate the functions of dsRNA-activated protein kinase (PKR) in choroidal neovascularization (CNV) and related signaling pathways in the production of vascular endothelial growth factor (VEGF). METHODS: A chemical hypoxia model of in vitro RF/6A cells, a rhesus choroid-retinal endothelial cell line, was established by adding cobalt chloride (CoCl2) to the culture medium. PKR, phosphophosphatidylinositol 3-kinase (p-PI3K), phosphoprotein kinase B (p-Akt), and VEGF protein levels in RF/6A cells were detected with western blotting. PKR siRNA and the PI3K inhibitor LY294002 were used to evaluate the roles of the PKR and PI3K signaling pathways in VEGF expression with western blotting. In an ARPE-19 (RPE cell line) and RF/6A cell coculture system, proliferation, migration, and tube formation of RF/6A cells under hypoxic conditions were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Transwell, and Matrigel Transwell assays, respectively. In vivo CNV lesions were induced in C57BL/6J mice using laser photocoagulation. The mice were euthanized in a timely manner, and the eyecups were dissected from enucleated eyes. PKR, p-PI3K, p-Akt, and VEGF protein levels in tissues were detected with western blotting. To evaluate the leakage area, fundus fluorescein angiography and choroidal flat mount were performed on day 7 after intravitreal injection of an anti-PKR monoclonal antibody. RESULTS: The in vitro RF/6A cell chemical hypoxia model showed that PKR expression was upregulated in parallel with p-PI3K, p-Akt, and VEGF expression, peaking at 12 h. PKR siRNA downregulated PKR, p-PI3K, p-Akt, and VEGF expression. In addition, the PI3K inhibitor LY294002 greatly decreased the p-PI3K, p-Akt, and VEGF protein levels, but PKR expression was unaffected, indicating that Akt was a downstream molecule of PKR that upregulated VEGF expression. In the ARPE-19 (RPE cell line) and RF/6A cell coculture system, PKR siRNA reduced the migration and tube formation of the RF/6A cells. In vivo, PKR, p-PI3K, p-Akt, and VEGF expression increased and peaked at 7 days in the mouse CNV model induced by laser photocoagulation. Furthermore, on the RPE and choroid cryosections, PKR colocalized with CD31, suggesting that PKR was expressed by the vascular endothelium. The intravitreal injection of an anti-PKR monoclonal antibody decreased the progression and leakage area of CNV in mice. CONCLUSIONS: PKR promotes CNV formation via the PI3K/Akt signaling pathway in VEGF expression. Additionally, the anti-PKR monoclonal antibody significantly decreased CNV in a mouse model, showing the antibody may have therapeutic potential in human CNV.

Inhibition of RACK1 ameliorates choroidal neovascularization formation in vitro and in vivo.

Choroidal neovascularization (CNV) occurs as a result of age-related macular degeneration (AMD) and causes severe vision loss among elderly patients. The receptor for activated C-kinase 1 (RACK1) serves as a scaffold protein which is recently found to promote angiogenesis. However, the impact of RACK1 on the vascular endothelial growth factor (VEGF) expression in endothelial cells and subsequent choroidal angiogenesis formation remains to be elucidated. In this study, we found that RACK1 and VEGF expression increased, and reached the peak at 7d in mouse CNV model by laser application. Furthermore, on RPE/choroid cryosections, RACK1 co-localized with CD31, suggesting that RACK1 was expressed in endothelial cells. In vitro, RF/6A cell hypoxia model showed that RACK1 expression was up-regulated in parallel with hypoxia-induced factor 1 (HIF-1α) and VEGF expression, reaching the peak at 6h. Silencing of RACK1 suppressed the invasion and tube formation activity of RF/6A cells in ARPE-19 and RF/6A co-culture system, possibly through VEGF signal pathway. Overexpression of RACK1 showed the opposite effect. Intravitreal injection of anti-RACK1 monoclonal antibody predominantly decreased RACK1 and VEGF expression in mouse laser-induced CNV model. Meanwhile, anti-RACK1 monoclonal antibody intravitreal injection also decreased incidence of CNV and leakage area. These data indicated that RACK1 promoted CNV formation via VEGF pathway. Additionally, anti-RACK1 monoclonal antibody significantly decreased CNV in mouse model and may have therapeutic potential in human CNV.

Expression of RBMX in the light-induced damage of rat retina in vivo.

RNA-binding motif protein, X-linked (RBMX) is a 43 kDa nuclear protein in the RBM family and functions on alternative splicing of RNA. The gene encoding RBMX is located on chromosome Xq26. To investigate whether RBMX is involved in retinal neuron apoptosis, we performed a light-induced retinal damage model in adult rats. Western blotting analysis showed RBMX gradually increased, reached a peak at 12 h and then declined during the following days. The association of RBMX in retinal ganglion cells (RGCs) with light exposure was found by immunofluorescence staining. The injury-induced expression of RBMX was detected in active caspase-3 and TUNEL positive cells. We also examined the expression profiles of active caspase-3, bcl-2 and Bax, whose changes were correlated with the expression profiles of RBMX. To summarize, we uncovered the dynamic changes of RBMX in the light-induced retinal damage model for the first time. RBMX might play a significant role in the degenerative process of RGCs after light-induced damage in the retina.

Up-Regulation of PKM2 Relates to Retinal Ganglion Cell Apoptosis After Light-Induced Retinal Damage in Adult Rats.

Pyruvate kinase isozyme type M2 (PKM2), a key glycolytic enzyme, which is involved in ATP generation and pyruvate production, participates in tumor metabolism, growth, and other multiple cellular processes. However, one attractive biological function of PKM2 is that it translocates to the nucleus and induces cell apoptosis. Recently, increased PKM2 has been found in age-related macular degeneration (AMD), but little is known regarding its function in the AMD pathophysiology. To investigate whether PKM2 participated in retinal degeneration, we performed a light-induced retinal damage model in adult rats. Western blot and immunohistochemistry analysis showed a significant up-regulation of PKM2 in retinal ganglion cells (RGCs) layer (GCL) after light exposure. Immunofluorescent labeling indicated that PKM2 located mainly in RGCs. Co-localization of PKM2 and active caspase-3 as well as TUNEL in RGCs suggested that PKM2 might participate in RGC apoptosis. In addition, the expression patterns of cyclin D1 and phosphorylated extracellular signal-regulated kinase (p-ERK) were parallel with that of PKM2. Furthermore, PKM2, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of U0126, a highly selective inhibitor of MAPK/ERK kinase. Collectively, we hypothesized that PKM2 might participate in RGC apoptosis after light-induced retinal damage medicated by p-ERK through cycle re-entry mechanism.

Honokiol Attenuates Choroidal Neovascularization by Inhibiting the Hypoxia-Inducible Factor-α/Vascular Endothelial Growth Factor Axis via Nuclear Transcription Factor-Kappa B Activation.

PURPOSE: Honokiol is a lignan isolated from Magnolia officinalis and exhibits anti-angiogenic properties. This study was conducted to investigate the role of honokiol in choroidal neovascularization. METHODS: C57BL/6 mice were treated with honokiol at 10-20 mg/kg by daily intraperitoneal injection from day 1 to 6 after laser photocoagulation. ARPE-19 cells were cultured under hypoxic conditions with or without the presence of honokiol. After laser photocoagulation and honokiol treatment, hematoxylin and eosin staining, immunofluorescence and fundus fluorescein angiography were used to analyze the effect of honokiol on choroidal neovascularization formation. Quantitative real-time PCR, western blot, enzyme-linked immunosorbent assay, immunofluorescence, luciferase assay, and chromatin immunoprecipitation were performed to explore the mechanism of honokiol in the pathological process of choroidal neovascularization. Finally, the role of honokiol on the human choroidal vascular endothelial cells was detected by using 5-ethynyl-20-deoxyuridine assay, Transwell and Tube formation assays. RESULTS: The results of hematoxylin and eosin staining and immunofluorescence suggested that honokiol reduced the thickness, length, and area of choroidal neovascularization lesions in laser-induced choroidal neovascularization mouse model. Fundus fluorescein angiography showed that choroidal neovascularization leakage was reduced in honokiol group and the concentration of 20 mg/kg showed better effects. Mechanism studies have shown that honokiol exerted inhibitory effects on choroidal neovascularization by inactivating hypoxia-inducible factor-1α/vascular endothelial growth factor axis through the nuclear transcription factor-kappa B signaling pathway. The same results were obtained in ARPE-19 cells under hypoxic conditions. Furthermore, the conditional medium of retinal pigmented epithelial cells promoted the proliferation, migration, and tube formation of human choroidal vascular endothelial cells, while honokiol reversed these. CONCLUSION: We demonstrated that honokiol attenuated choroidal neovascularization formation by inactivating the hypoxia-inducible factor-1α/vascular endothelial growth factor axis through nuclear transcription factor-kappa B signaling pathway.

Age-dependent impaired neurogenic differentiation capacity of dental stem cell is associated with Wnt/ß-catenin signaling.

Two kinds of dental stem cells (DSCs), dental pulp stem cells (DPSCs) and stem cells from human-exfoliated deciduous teeth (SHED), have been identified as novel populations of mesenchymal stem cells that can be induced to differentiate into osteoblasts, chondrocytes, adipocytes, and neuron-like cells in vitro. As we know, both of them originate from the neural crest, but have distinct characteristics and functions in vitro and in vivo. The regeneration potential of DSCs declines with advanced age; however, the mechanism of the impaired potential in DSCs has not been fully explored. In this study, we investigated whether declined neurogenic differentiation capacity is associated with an altered expression of Wnt signaling-related proteins in vitro. We compared stem cells isolated from human dental pulp in two age groups: the exfoliated deciduous teeth (5-12 years), and the third permanent teeth (45-50 years). We found that the expression levels of neuron markers, such as ßIII-tubulin, microtubule-associated protein 2(MAP2), tyrosine hydroxylase (TH), and Nestin were lower in the DPSCs group compared with that in the SHED group; however, in supplementation with human recombinant Wnt1 in the medium, the DPSCs were prone to neural differentiation and expressed higher levels of neurogenic markers. In summary, our study demonstrated that Wnt/ß-catenin signaling may play a vital role in the age-dependent neural differentiation of DSCs. Therefore, DSCs may provide an ideal source of stem cells that can further extend their therapeutic application in nerve injury and neurodegenerative diseases.

Association of angiotensin-converting enzyme gene 2350 G/A polymorphism with diabetic retinopathy in Chinese Han population.

The angiotensin-converting enzyme (ACE) gene is one of the most studied candidate genes related to diabetic retinopathy (DR). ACE 2350 G/A polymorphism (rs4343) is known among the polymorphisms of this gene to have the most significant effect on plasma ACE concentrations. The aim of the present study was to investigate the relationship between 2350 G/A polymorphism of ACE gene and the susceptibility of DR in Chinese Han population. A case-control study for 145 type 2 diabetes mellitus (DM) patients, including 63 type 2 DM without DR (NDR) and 82 type 2 DM with DR (DR), and 90 subjects of age, gender matched normal controls (NC group) was performed. ACE 2350 G/A genotypes were identified by polymerase chain reaction and restriction digestion in all study participants. The distribution of the ACE 2350 G/A genotypes (GG, GA, and AA) was 35.56, 45.55, and 18.89 % in the NC group, 28.57, 46.03, and 25.40 % in the NDR group, and 15.85, 46.34, and 37.81 % in the DR group, respectively. There were no significant differences in either genotype frequency distribution (P = 0.5266) or allele frequency distribution (P = 0.2425) between the NC group and NDR group. However, the distribution of genotype frequency (P = 0.0026) and allele frequency (P = 0.0003) in the DR group showed a significant difference when compared to that of NC group (P = 0.0075). Moreover, there was statistical difference in allele frequency distribution (P = 0.0328) between the DR group and the NDR group. No statistical differences were observed between ACE 2350 G/A polymorphism and the diabetes duration or types of DR. Results obtained in this study indicate that ACE 2350 G/A polymorphism is associated with DR in Han Chinese patients with type 2 DM.

p21CIP/WAF1 saRNA inhibits proliferative vitreoretinopathy in a rabbit model.

PURPOSE: Proliferative vitreoretinopathy (PVR) is a disease process resulting from proliferation of retinal pigment epithelial (RPE) cells in the vitreous and periretinal area, leading to periretinal membrane formation and traction and eventually to postoperative failure after vitreo-retinal surgery for primary rhegmatogenous retinal detachment (RRD). The present study was designed to test the therapeutic potential of a p21CIP/WAF1 (p21) inducing saRNA for PVR. METHODS: A chemically modified p21 saRNA (RAG1-40-53) was tested in cultured human RPE cells for p21 induction and for the inhibition of cell proliferation, migration and cell cycle progression. RAG1-40-53 was further conjugated to a cholesterol moiety and tested for pharmacokinetics and pharmacodynamics in rabbit eyes and for therapeutic effects after intravitreal administration in a rabbit PVR model established by injecting human RPE cells. RESULTS: RAG1-40-53 (0.3 mg, 1 mg) significantly induced p21 expression in RPE cells and inhibited cell proliferation, the progression of cell cycle at the G0/G1 phase and TGF-ß1 induced migration. After a single intravitreal injection into rabbit eyes, cholesterol-conjugated RAG1-40-53 exhibited sustained concentration in the vitreal humor beyond at least 8 days and prevented the progression of established PVR. CONCLUSION: p21 saRNA could represent a novel therapeutics for PVR by exerting a antiproliferation and antimigration effect on RPE cells.