Elevation of serum apelin-13 associated with proliferative diabetic retinopathy in type 2 diabetic patients

AIM: To compare apelin-13, a ligand of G-proteincoupled receptor which has been shown to be involved in retinal angiogenesis, and vascular endothelial growth factor(VEGF) serum levels in type 2 diabetes mellitus(T2DM) with or without retinopathy, and to investigate the relationship between the serum concentration of apelin-13 and diabetes retinopathy.METHODS: Sixty-nine patients with T2 DM were enrolled.Of the 69 patients, 16 had proliferative diabetic retinopathy(PDR group), 23 had non-PDR(NPDR group)and 30 had no retinopathy(T2DM group). Subjects’ information, including demographics, medical history,and use of medications were recorded. Their serum samples were collected for measuring the levels of C-reactive protein(CRP), serum lipid and glycosylated hemoglobin. Apelin-13 and VEGF serum levels were measured by enzyme-linked immunosorbent assay.Kruskal-Wallis test and one-way ANOVA were used to compare the differences among these groups. Chi-square test was used to assess categorical variables.Correlations between variables were investigated by Spearman rho correlation test and stepwise regression analysis. All statistical analyses were performed through SPSS 17.0 software.RESULTS: Sex, age, body mass index(BMI), blood pressure, CRP, hemoglobin A1c(Hb A1c) have no significantly difference in the three groups. Serum level of apelin-13 was significantly elevated in PDR group as compared with T2 DM group(P =0.041). Differences of VEGF serum concentration in the three groups were statistically significant(P =0.007, P =0.007 and P 【0.001,respectively). Spearman rho correlation test showed that serum apelin-13 was positively correlated with BMI,serum triglycerides, VEGF, but not with age, duration of diabetes, blood pressure, CRP, Hb A1 c and total-cholesterol. Stepwise regression analysis showed that BMI also significantly associated with serum apelin-13(P =0.002), while VEGF and serum triglycerides were irrelevant. CONCLUSION: This study elucidated a positive association of apelin-13 serum level with PDR, but not with VEGF. Apelin-13 may influence the promotion of PDR but unrelated with VEGF.

Autophagy: a new mechanism for regulating VEGF and PEDF expression in retinal pigment epithelium cells

AIM: To investigate the regulation of vascular endothelial growth factors(VEGF) and pigment epithelium-derived factor(PEDF) expression by autophagy in retinal pigment epithelium(RPE) cells on exposure to hypoxia. METHODS: ARPE-19, an RPE cell line, was treated as following: the control group was kept in a normoxic incubator; the hypoxia group was incubated in a hypoxic incubator with 1% O_2/5% CO_2/94% N_2 for 24 h; the hypoxia + 3-methyladenine(3-MA) group was pretreated with 10 mmol/L 3-MA for 1 h and then in the hypoxic incubator for 24 h; and the hypoxia + chloroquine(CQ) group was pretreated with 50 μmol/L CQ for 1 h and then in the hypoxic incubator for 24 h. The morphology and ultrastructure of the cells was observed by an inverted microscope or a transmission electronic microscope(TEM). Western blot was performed to assay the expression of autophagy-associated markers, including microtubule associated protein 1 light chain 3 B(LC3 B), Beclin-1, Atg5 and p62. The concentration of VEGF and PEDF in the culture supernatant was determined by ELISA, and the ratio of VEGF/PEDF was calculated. RESULTS: There were no obvious differences in cell morphology among different groups and autolysosomes could be observed in the cytoplasm in all groups. Compared to the control cells, the LC3 B-II/I ratio and levels of Beclin-1 and Atg5 were significantly increased and p62 level was significantly decreased in the hypoxia group. With the increase of VEGF and decrease of PEDF concentration, the VEGF/PEDF ratio was significantly increased in the hypoxia group compared to the control cells. The LC3 B-II/I ratio was significantly reduced by 3-MA treatment and increased by CQ treatment. The expressions of Beclin-1 and Atg5 were significantly reduced by 3-MA or CQ treatment, while expression of p62 was increased in the 3-MA or CQ treated cells. The concentration of VEGF was significantly decreased and PEDF increased, thereby the VEGF/PEDF ratio was decreased in the hypoxia + 3-MA group and hypoxia + CQ group compared with that in the hypoxia group. CONCLUSION: Hypoxia leads to elevated autophagy in RPE cells, and expression of VEGF and PEDF might be regulated by autophagy on exposure to hypoxia to further participate in regulating the formation of retinal neovascularization.

Leptin activates the JAK/STAT pathway to promote angiogenesis in RF/6A cells in vitro

AIM: To investigate the effect of leptin on the angiogenesis of RF/6 A cells(monkey retinal choroidal endothelial cells) in vitro and test the cellular signaling in the mechanism.METHODS: RF/6 A cells were cultured in vitro and randomly divided into four groups: normal control, with leptin at 50, 100, 200 ng/m L for cell counting kit-8(CCK8). RF/6 A cell proliferation and migration were examined by Transwell assays, while RF/6 A cell tube formation by Matrigel assay. JAK2, p-JAK2, STAT3, and p-STAT3 protein expression was measured by Western blotting. Cells were then divided into the following treatment groups: control, 100 ng/m L leptin and AG-490(100 ng/m L leptin+10 μmol/L AG-490) for examinations of RF/6 A cellular behaviour again. Analysis of differences was carried out using one-way ANOVA and least significant difference(LSD).RESULTS: RF/6 A cell proliferation, migration and cell tube formation were promoted significantly by leptin in a dose-dependent manner(P<0.05). Western blotting showed that leptin up-regulated p-JAK2 and p-STAT3 expression levels. Treatment with the JAK/STAT pathway inhibitor, AG-490, decreased leptin-induced p-JAK2 and p-STAT3 expression, and inhibited cell proliferation, migration and cell tube formation induced by leptin(P<0.05). CONCLUSION: Leptin can promote RF/6 A cell angiogenesis in vitro via activation of the JAK2/STAT3 signaling pathway.

Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia

AIM： To explore the state of autophagy and related mechanisms in the murine retinal microvascular endothelial cells（RMECs） under hypoxia stimulation.METHODS： The murine RMECs were primarily cultured and randomly divided into three groups： hypoxia group（cultured in 1% O_2 environment）, hypoxia＋autophagy inhibition group [pretreated with 5 mmol/L 3-methyladenine（3-MA） for 4 h followed by incubation in 1% O_2] and control group（cultured under normoxic condition）. The state of autophagy in RMECs was examined by assaying the turnover of light chain 3 B（LC3BB） and expression of Beclin-1, Atg3 and Atg5 proteins with Western blotting, by detecting formation of autophagosomes with transmission electron microscopy（TEM） and by counting the number of GFP＋ puncta in RMECs. The protein levels of AMPK, P-AMPK, Akt, P-Akt, m-TOR and P-m TOR were also assayed by Western blotting.RESULTS： Primary murine RMECs were successfully cultured. Under hypoxic conditions, the ratio of LC3BB-Ⅱ/Ⅰ and the expression of Beclin-1, Atg3 and Atg5 proteins were increased when compared with the control group. In addition, the numbers of autophagosome and the GFP＋ puncta were also increased under hypoxia. However, pretreatment with 3-MA obviously attenuated these changes in autophagy in RMECs under hypoxia. Protein expression of P-Akt and P-AMPK was increased but P-m TOR level was decreased in cells exposed to hypoxia. CONCLUSION： In murine RMECs autophagy is activated under hypoxia possibly through activation of the AMPK/mTOR signaling pathway.

Autophagy: a potential target for the treatment of intraocular neovascularization

The formation of neovascularization is a common pathological feature of many ocular vascular diseases, and is an important cause of vision loss in patients. Neovascularization can cause retinal hemorrhage, vitreous hemorrhage, and other serious complications, leading to loss of vision. The treatment of intraocular neovascularization is the focus of ophthalmology research. In recent years, some studies have found that autophagy is closely related to vascular endothelial growth factor and the formation of neovascularization. Autophagy is expected to become a new target for the treatment of intraocular neovascularization. Therefore, this article reviews the research on autophagy and the formation of intraocular neovascularization.

Autophagy dysregulation mediates the damage of high glucose to retinal pigment epithelium cells

AIM:To observe the role and mechanism of autophagy in retinal pigment epithelial cell(RPE)damaged by high glucose,so as to offer a new idea for the treatment of diabetic retinopathy(DR).METHODS:ARPE-19,a human RPE cell line cultured in vitro was divided into the normal control(NC),autophagy inhibitor 3-methyladenine(3-MA),high-glucose(HG),and HG+3-MA groups.Cell viability was detected by CCK-8 assay and the apoptosis rate was measured by flow cytometry.The protein expressions of apoptosis markers,including Bax,Bcl-2,and Caspase-3,as well as autophagy marker including microtubule-related protein 1 light chain 3(LC3),p62,and mechanistic target of rapamycin(m TOR)were detected by Western blotting.Autophagic flux was detected by transfection with Ad-m Cherry-GFP-LC3 B.RESULTS:Under high glucose conditions,the viability of ARPE-19 was decreased,and the apoptosis rate increased,the protein expressions of Bax,Caspase-3,and LC3-II/LC3-I were all increased and the expressions of Bcl-2,p62 and p-m TOR decreased,and autophagic flux was increased compared with that of the controls.Treatment with 3-MA reversed all these changes caused by high glucose.CONCLUSION:The current study demonstrates the mechanisms of cell damage of ARPE-19 through high glucose/m TOR/autophagy/apoptosis pathway,and new strategies for DR may be developed based on autophagy regulation to manage cell death of RPE cells.