The Role Of ANGPTL-4 In The Pathogenesis Of Diabetic Retinopathy

Objective: To explore the role of Angiopoietin-like protein 4(ANGPTL-4)in the development of diabetic retinopathy(DR).Method:(1)Vitreous and serum samples were collected and divided into 4 groups: idiopathic macular hole(IMH)group(Control),nondiabetic retinopathy(NDR)group,non-proliferative diabetic retinopathy(NPDR)group and PDR group.The expression levels of ANGPTL-4 and vascular endothelial growth factor(VEGF)were detected via enzyme linked immunosorbent assay(ELISA).(2)Human retinal microvascular endothelial cells(HRMECs)were incubated in normal glucose(5 mM;NG),NG plus recombinant adenovirus encoding ?-galactosidase control(AdLacZ),NG plus AdHIF-1?,NG plus recombinant human angiopoietin-like protein 4(rhANGPTL-4),NG plus small interfering RNA for ANGPTL-4(siANGPTL-4),NG plus recombinant human VEGF protein(rhVEGF),NG plus small interfering RNA for VEGF(siVEGF),NG plus specific HIF-1? inhibitor Doxorubicin(DOX),high glucose(30 mM;HG),HG plus DOX,HG plus rhANGPTL-4,HG plus siANGPTL-4,HG plus rhVEGF,HG plus siVEGF with M199 medium respectively.(3)Male Sprague-Dawley rats were randomly assigned into 7 groups: control rats(Con),Con plus siANGPTL-4,diabetic mellitus rats(60 mg/kg Streptozotocin,intraperitoneal injection;DM),DM plus small interfering RNA for HIF-1?(siHIF-1?),DM plus rmANGPTL-4,DM plus siANGPTL-4,DM plus DOX(5 mg/kg/day;Drinking water).(4)The permeability of HRMECs was detected by the fluorescein isothiocyanate(FITC)-conjugated dextran assay.(5)The protein and mRNA expression levels of ANGPTL-4,HIF-1?,VEGF,interleukin 1 beta(IL-1?),IL-6,intercellular adherent molecule 1(ICAM-1),BAX and p53 in HRMECs and rat retinas were detected via Western blot and Real-time PCR.Meanwhile,the concentration of ANGPTL-4 and VEGF in HRMECs supernatants were measured via ELISA.(6)Immunofluorescence was utilized to detect the expression of HIF-1? in HRMECs and rat retinas and immunohistochemistry staining was utilized to detect the expression of ANGPTL-4 in rat retinas.(7)The apoptosis levels of HRMECs were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL).(8)The invasion,migration,proliferation and tube formation of HEMECs were measured by matrigel.(9)The results are expressed as the means ± standard deviation.The data were normally distributed by the Kolmogorov–Smirnov test.Group means were compared by oneway ANOVA using GraphPad Prism 4.0(GraphPad,San Diego,CA).Pearson's correlation tests were also performed to test relationships between ANGPTL-4 and BMI,HbA1 c and the expression of serum lipids.Additionally,multiple linear regression analyses were performed to show confounders.In all comparisons,a value of P < 0.05 was considered to indicate a statistically significant difference.Results:(1)Compared with the IMH group,the expression levels of ANGPTL-4 and VEGF in the vitreous fluid and serum of patients with PDR were significantly increased(P < 0.01).Meanwhile,close relationships between ANGPTL-4 and VEGF were detected via Pearson's correlation tests(P < 0.01).(2)ANGPTL-4 overexpression significantly decreased the level of apoptosis in HRMECs detected via TUNEL(P < 0.01).Meanwhile,ANGPTL-4 gene silencing displayed reverse results(P < 0.01).(3)ANGPTL-4 overexpression significantly promoted the cell invasion,migration,proliferation,and tube formation of HRMECs under HG condition(P < 0.01).Meanwhile,ANGPTL-4 gene silencing displayed reverse results(P < 0.01).(4)ANGPTL-4 overexpreesion significantly promoted the permeability of HRMECs while ANGPTL-4 gene silencing reduced it(P < 0.01).The mediation of ANGPTL-4 to permeability of HRMECs was regulated via HIF-1?.Both siHIF-1? and DOX could decrease the expression of ANGPTL-4 via inhibiting HIF-1?.(5)ANGPTL-4 overexpression significantly decreased the expression levels of BAX and p53 while increased the level of VEGF in DM retinas under HG condition(P < 0.05).Meanwhile,ANGPTL-4 gene silencing displayed reverse results(P < 0.05).(6)The protein and mRNA expression levels of inflammatory molecules,such as IL-1?,IL-6 and ICAM-1 in DM retinas were significantly decreased via ANGPTL-4 gene silencing(P < 0.05).Conclusion:(1)The ANGPTL-4 and VEGF expression levels were markedly elevated and the ANGPTL-4 expression was directly correlated with the VEGF expression in the vitreous and serum of patients with PDR.(2)The activation of ANGPTL-4 due to HG in vivo and in vitro that hinges on the HIF-1? activation which is also triggered by HG.Meanwhile,DOX and HIF-1? gene silencing both effectively inhibited the activation of ANGPTL-4 via inhibiting HIF-1?.(3)HIF-1? critically mediates the hyperpermeability resulting from HG via the ANGPTL-4/VEGF pathway.DOX plays a protective role on HRMECs and retinas in HG condition via blocking HIF-1? pathway.(4)The activation of ANGPTL-4 strengthens the inflammation,dampens the apoptosis,and enhances the angiogenesis under HG condition,suggesting the potential of ANGPTL-4 in the targeted therapy of DR.