Investigation Of The Role And Regulation Mechanism Of SP1 On Robo4 In Diabetic Retinopathy

It is known that diabetic retinopathy(DR)is one of serious microvascular complications of diabetes,the pathogenesis of which is extremely complicated,implicating many different mechanisms.Previous studies proved that consistently elevated blood glucose in retina could cause oxidative stress,inflammation,neuronal dysfunction and epigenetic modifications.All these mechanisms can interact closely and create a large signal network to result in the common pathological changes in retinal vessels,including the loss of retinal endothelial cells or structural damage,apoptosis of pericytes,disruption of vascular integrity,increased vascular permeability and retinal neovascularization.Therefore,preventing or reversing the retinal microvascular dysfunction has become the focus of a large part of the research on DR.Roundabout4(Robo4)is a cell surface protein that is specifically expressed in endothelial cells and is involved in the angiogenesis and stability of blood vessels.Research relevant to DR showed that the expression level of Robo4 increased significantly in human retinal endothelial cells(HREC)under hypoxic conditions,and RNA interference of Robo4 expression could inhibit abnormities in HREC proliferation and migration induced by hypoxia.Also in diabetic rat models,there was a gradual increase in the expression of Robo4 in all retinal layers,concomitant with the development of DR.All these reports indicate that Robo4 plays a significant role in DR generation and development.However,the mechanisms that regulate the expression of Robo4 are still unclear.Research into the transcriptional mechanism of Robo4 gene,NeuralNetwork Promoter Prediction software was used to analyze the promoter region of Robo4,and detected multiple binding sites which could bind specificity protein 1(SP1),predicting that SP1 might be the transcription factor that regulates the activation of Robo4.SP1 is one of the sequence specific DNA binding proteins that contains C2H2-type zinc finger and binds to GC-box promoter elements.It can activate or repress the transcription of various genes to affect the occurrence and development of DR.So I hypothesized that SP1 was the essential transcription factor that directly regulated Robo4 expression in the pathogenetic process of DR.In this study,the expression of SP1 and Robo4 were detected in fibrovascular membranes from patients with proliferative diabetic retinopathy(PDR).HREC were cultured under hyperglycemic conditions to simulate the DR environment in vitro,and I evaluated the changes in the expression of SP1 and Robo4 and explored the regulatory relationship between them.Vascular endothelial cellular functions were analyzed to demonstrate the role of this mechanism during the occurrence and development of DR.Methods1.Detecting the SP1 and Robo4 expression in fibrovascular membranes from patients with PDR Fibrovascular membranes were obtained from patients with PDR who accepted pars plana vitrectomy with membrane peeling.The hematoxylin-eosin staining was performed to the membrane tissues to observe the pathological changes.Immunohistochemistry and double immunofluorescence were done to analyze the colocalization of SP1 and Robo4 in the fibrovascular membranes.2.Detecting the SP1 and Robo4 expression in HREC under hyperglycemic conditionsHREC were cultured under hyperglycemic conditions in vitro,and RT-q PCR and western blot were performed to evaluate the changes in the expression of SP1 and Robo4 as a function of the duration of exposure to hyperglycemia.3.Detecting the changes in the expression of SP1 and Robo4 influenced by small interfering RNA(si RNA)Si RNA was used to silence SP1 or Robo4 successively in HREC under hyperglycemic conditions,and the expression of both genes at the m RNA and protein levels was measured by RT-q PCR and western blotting,respectively.4.Detecting the regulatory relationship between SP1 and Robo4 HREC were cultured under normal or hyperglycemic conditions,and the effect of transcription factor SP1 on Robo4 promoter activity and the location of SP1 binding sites were investigated using chromatin immunoprecipitation(Ch IP)and luciferase assay.5.Detecting the changes in cellular functions of HREC influenced by SP1/Robo4 signaling pathway HREC cultured under hyperglycemic conditions were transfected with SP1 si RNA or Robo4 si RNA,and transwell assay,wound healing assay,monolayer permeability assay and tube formation assay were performed to measure the changes in HREC migratory ability,monolayer permeability and angiogenesis.Results1.There were four histopathologic types were observed in fibrovascular membranes from PDR patients,including the cellular proliferation,neovascularization,hemorrhage and fibroplasia.SP1 and Robo4 were overexpressed in all these membranes,and they were colocalized in microvascular endothelial cells.2.The m RNA and protein expression of SP1 and Robo4 increased steadilyin HREC with the increasing exposure time in hyperglycemia.3.SP1 si RNA transfection in HREC could downregulate the hyperglycemia-induced high expression of SP1 and Robo4 to normal levels,but Robo4 si RNA decreased Robo4 expression alone and had no effect on SP1.4.Results from Ch IP showed that the Robo4 promoter region(-2400/-2700bp)contained an SP1 binding site in HREC under normal condition,while under hyperglycemic condition,there was one more binding site in the promoter region(-1800/-2100bp).Results from luciferase reporter assays showed that SP1 binding site in the region of-2452/-2448 bp was conserved and was required for the normal transcription of Robo4,while in contrast,the additional SP1 binding site in the region of-1912/-1908 bp mediated the hyperglycemic effect.5.The results of the transwell assay and wound healing assay showed that cell migration was significantly enhanced in the high glucose condition and the hyperglycemia-stimulated increase in HREC migration could be inhibited by blocking the SP1/Robo4 signaling pathway.Monolayer permeability and tube formation assays yielded similar results:repressing the SP1/Robo4 pathway could mitigate the increase of HREC permeability and the inhibition of angiogenic ability induced by hyperglycemia.Conclusion In the present study,the role and regulation mechanism of SP1 on Robo4 were investigated in DR.Results showed that SP1 and Robo4 were colocalized in fibrovascular membranes from patients with PDR,suggesting that both SP1 and Robo4 may have vital roles during the formation of fibrovascular membranes.And studies on regulatory mechanism between SP1 and Robo4 revealed that hyperglycemia-induced overexpression of Robo4 in HREC was mediated by transcription factor SP1,for which,there were two binding sites in the promoter region of Robo4 that enhanced thetranscriptional level of Robo4.The SP1/Robo4 signaling pathway could regulate the migratory ability,monolayer permeability and angiogenesis of HREC in vitro,suggesting that it may be contributing significantly to the microvascular dysfunction of DR.