Investigation Of The Role And Mechanism Of ANGPTL4 In Diabetic Retinopathy

Background:Diabetes mellitus(DM)is an increasing global disease resulting in many severe complications such as diabetic retinopathy(DR),which is a common cause of visual impairment affecting 93 million people globally.The pathogenesis of DR is a multifactorial process,the progressive effects of high glucose,hypoxia,inflammation and neuronal dysfunction can create a large signal network to result in the breakdown of blood-retinal barrier and retinal microvascular defects.Therefore,it is vital to explore the mechanisms involved in preventing or reversing the retinal microvascular dysfunction and blood-retinal barrier damage to prevent and treat DR.Angiopoietin-like protein 4(ANGPTL4)is a member of a family of angiopoietin-like proteins.Recent studies have shown that ANGPTL4 regulates tumorigenesis,angiogenesis,vascular permeability,glucose homeostasis,lipid metabolism,cell differentiation,energy homeostasis,inflammation,and redox regulation.Multiple developmental and pathological roles related to eye diseases are attributable to ANGPTL4 including the promotion of angiogenesis,vascular permeability,and inflammation.However,the study of ANGPTL4 functionality is in its infancy,leading to controversy and a lack of comprehensive investigation regarding the precise role(s)of ANGPTL4 in various disease processes.Hypoxia induced factor-1?(HIF-1?)recruits target genes immediately after hypoxic stimulation,including stromal-derived growth factor-1,platelet-derived growth factor-?,vascular endothelial growth factor(VEGF)and other cytokines,all of which are involved in cell migration,proliferation and neovascularization.HIF-1? is a known transcription regulator of ANGPTL4 in osteosarcoma cell lines and human umbilical vein endothelial cells.Based on the mentioned knowledge,we hypothesized that HIF-1? is also a key transcriptional factor that regulates ANGPTL4 expression in DR progression.Our study also concentrated on a member of janus-activated kinase-singal transducers and activators of transcriprion(JAK-STAT)pathway,the signal transducer and activator of transcription 3(STAT3).STAT3 is known to participate in cytokine signaling involved in inflammation and endothelial permeability.In wounded epithelia,nitric oxide synthesis was stimulated by ANGPTL4 through the integrin/JAK/STAT3 pathway.Interleukin 6(IL-6)-induced retinal endothelial permeability through STAT3 activation which reduced expression of ZO-1 and Occludin.Based on known data,we hypothesize that ANGPTL4 stimulates STAT3 activation and works together in regulating permeability,angiogenesis and inflammation of ARPE-19 cells.Objective:In this study,the expression of HIF-1? and ANGPTL4 were detected in the diabetic rat retinas and retinal pigment epithelium(RPE)cells.RPE cells were cultured under hypoxic conditions to simulate the DR environment in vitro,and I evaluated the changes in the expression of HIF-1?,ANGPTL4 and STAT3 and explored the regulatory relationship between them.Furthermore,I explored the effects of this mechanism on the function of human RPE cells under hypoxic conditions.Methods:1.Detecting the expression levels of HIF-1? and ANGPTL4 in the rats' retinasForty male 8 weeks Sprague-Dawley rats(180~220g)were randomized into 20 rats in diabetes group(DM)and 20 rats in negative control group(NC).The DM rats were injected with STZ [65mg/kg,dissolved in citrate buffer(pH 4.5)].NC rats were injected citrate buffer as the same volumes.Both groups were raised either for 4,8 or 12 weeks.Six eyeballs at different time points in each group were prepared for retinal tissue sections(4?m)to execute HE and immunohistochemistry assay.Protein and mRNA assay was performed from six eyes' retinal tissue at different time points in each group.2.Exploring the HIF-1? and ANGPTL4 expression in ARPE-19 cells under hypoxic conditionsARPE-19 cells were cultured under hypoxic conditions for 8,16,24 hours,and RT-qPCR and western blot were used to detect the changes in the expression of HIF-1 ? and ANGPTL4 as a function of the duration of exposure to hypoxia.3.Detecting the changes in the expression of HIF-1? and ANGPTL4 after transfection of small interfering RNA(siRNA)into ARPE-19 cellsARPE-19 cells cultrured under hypoxia were transfected with HIF-1? siRNA or ANGPTL4 siRNA,and RT-qPCR and western blot were performed to explore the effects of silencing HIF-1? and ANGPTL4.Furthermore,the mRNA and protein levels of ANGPTL4 were determined with knockdown of HIF-1?.4.Detecting the changes in cellular functions of ARPE-19 cells influenced by HIF-1? / ANGPTL4 signaling pathwayARPE-19 cells cultured under hypoxic conditions were transfected with HIF-1? siRNA or ANGPTL4 siRNA.Transwell assay,wound healing assay and monolayer permeability assay were used to explore the changes of ARPE-19 cells migratory ability and permeability.5.Detecting the changes in the expression of tight junction proteins and inflammatory factors after transfection of ANGPTL4 siRNA into ARPE-19 cells and inhibition of STAT3 activation by WP1066ANGPTL4 siRNA was transfected or WP1066 was added into ARPE-19 cells cultured under hypoxic conditions to inhibit the expression of ANGPTL4 or STAT3 activation,and the expression of corresponding protein was detected by western blot.RT-qPCR,western blot or ELISA assay were used to detect the expression of Occludin,ZO-1,tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?).6.Detecting the changes in the expression of VEGF and the effect on human retinal endothelial cells angiogenic ability after transfection of ANGPTL4 siRNA into ARPE-19 cells and inhibition of STAT3 activation by WP1066ANGPTL4 siRNA was transfected or WP1066 was added into ARPE-19 cells cultured under hypoxic conditions to inhibit the expression of ANGPTL4 or STAT3 activation.Western blot was used to detect the expression of VEGF.Tube formation assay was used to measure the changes in human retinal endothelial cells angiogenesis.Results:1.HE staining of diabetic retinas showed that the ganglion cell layer(GCL)and nerve fiber layer(NFL)arranged disordered after 4 weeks of modeling,and deteriorated after 8 weeks and 12 weeks.Edema,vacuolar degeneration and the inner plexiform layer(IPL)thinner were discovered in 8 weeks and 12 week DM retina.RT-qPCR and western blot observed that the expression of both HIF-1? and ANGPTL4 increased with DR progression.Immunohistochemistry observed that HIF-1? and ANGPTL4 were weakly expressed in all layers of NC retina.In diabetic retinas the expression of HIF-1? and ANGPTL4 were upregulated in the NFL,GCL,IPL,the outer plexus layer(OPL)and RPE layer.These results suggested that HIF-1 ? and ANGPTL4 play a role in the early stages of diabetic retinopathy,which in turn exacerbates DR development.2.The mRNA and protein expression of HIF-1? and ANGPTL4 increased steadily in ARPE-19 cells with increasing exposure to hypoxia.3.HIF-1? siRNA and ANGPTL4 siRNA transfection in ARPE-19 cells could downregulate the hypoxia-induced high expression of corresponding genes.Furthermore,HIF-1? siRNA transfection in ARPE-19 cells could downregulate the hypoxia-induced high expression of ANGPTL4.4.The results of the transwell assay and wound healing assay showed that cell migration was significantly enhanced in the hypoxic condition and the hypoxia-stimulated increase in ARPE-19 cells migration and permeability could be inhibited by blocking the HIF-1? /ANGPTL4 signaling pathway.5.After ANGPTL4 siRNA transfection in ARPE-19 cells cultured in hypoxia,STAT3 activation was significantly inhibited,while mRNA and protein levels of TNF-? and IL-1? were also significantly decreased,protein levels of Occludin and ZO-1 were also significantly increased.WP1066 was added in ARPE-19 cells cultured in hypoxia.WP1066,STAT3 hosphorylation inhibitor,can also effectively inhibited the expression levels of TNF-? and IL-1?,while the expression levels of Occludin and ZO-1 were increased.6.After ANGPTL4 siRNA transfection in ARPE-19 cells cultured in hypoxia,STAT3 activation was significantly inhibited,then VEGF expression level was also significantly reduced and the angiogenic ability of human retinal endothelial cells was inhibited.WP1066,STAT3 hosphorylation inhibitor,can also effectively inhibited the expression level of VEGF and the angiogenic ability of human retinal endothelial cells.Conclusion:In the present study,the role and regulation mechanism of ANGPTL4 were investigated in DR.We find that the expression of HIF-1? and ANGPTL4 was increased with DR progression both in vivo and in vitro.Hypoxia-induced upregulation of ANGPTL4 in ARPE-19 cells is mediated by transcription factor HIF-1?.The HIF-1?/ANGPTL4 signaling may be involved in mediating the monolayer permeability and the migratory ability of ARPE-19 in vitro.ANGPTL4 regulates ARPE-19 cells permeability,inflammation and angiogenesis by,at least partly,activating STAT3 in vitro,which together contribute towards the dysfunction of DR.