Role Of TXNIP On Proliferative Diabetic Retinopathy

Objectives: Diabetes' s main ocular complication-Diabetic retinopathy(DR)now become the leading cause of blindness among working-aged adults around the world.Proliferative diabetic retinopathy(PDR)is the late stage of DR which characterized by abnormal angiogenesis in the retina.PDR is one of the main reason that causes vision loss in diabetic patients.About 1/3 diabetes patients will develop to PDR stage after 20 years' duration.It was recognized that hyperglycemia plays the key role in the development of PDR.High glucose can induce several intracellular damages like protein kinase C(PKC)activation,increased flux through the polyol pathway,increased hexosamine biosynthesis pathway activity and overproduction of advanced glycation end products(AGEs).Moreover,hyperglycemia-induced damage of these pathways can lead to alteration of oxidative stress.Additionally,it was reported that high glucose itself is a promoter of angiogenesis in retinal endothelial cells.Intraocular vascular endothelial growth factor(VEGF)over-accumulation is critical for retinal neovascularization in PDR.VEGF can induce endothelial cell proliferation,migration,tube formation and finally leads to angiogenesis.Abnormal ocular angiogenesis can cause vitreous hemorrhage,retinal detachment,neovascular glaucoma and lead to visual impairment even with medical intervention.Anti-VEGF drugs(e.g.Bevacizumab,Ranibizumab,Aflibercept,Conbercept)now generally used in treating diabetic macular edema,also over-label used in PDR.However,long-term intraocular injection of the anti-VEGF drug could potentially cause ocular and systemic adverse effect,like hypertension,thrombogenesis and the decrease of wound healing,particularly in long-term diabetic patients.So,the new therapy strategy for pathological angiogenesis of diabetic retinopathy still needs to be explored.Thioredoxin-interacting protein(TXNIP)also known as vitamin D3 up-regulated protein-1(VDUP-1),which is the inhibitor of reactive oxygen species(ROS)-scavenging protein thioredoxin(TRX),can be induced by high glucose in several retinal cells includes retina endothelial cells,Müller cells,and Pericyte cells.Several studies showed that TXNIP is highly expressed in retinal endothelial cells under diabetic conditions.Also,TXNIP has been shown to translocate to the plasma membrane from the nucleus after treatment with oxidative stress inducers and interacts with VEGF receptor 2(VEGFR2)to enhance human umbilical vein endothelial cells(HUVEC)survival.Therefore,the role of TXNIP in pathological angiogenesis of DR is yet not to be fully investigated.In the present study,we investigated the overexpression of TXNIP in high glucose-treated human retinal endothelial cells(HRMECs)and the effects of knockdown of TXNIP with siRNA on MHG or VEGF-induced HRMECSs angiogenesis in vitro.Furthermore,we studied the effect of TXINP on mice retina angiogenesis in ex-vivo retinal angiogenesis assay.Methods:1 The effect of high glucose on HRMECs proliferation,migration and tube formationHRMECs were inoculated for serial passage in 75 cm2 flasks with ECM medium containing 5% fetal bovine serum,1% endothelial cell growth supplement(ECGS),100 U/ml penicillin,and 100 ?g/ml streptomycin in a humidified 5% CO2 atmosphere at 37°C.HRMECs were used between the 4 to 6 in vitro passage.(1)Effect of high glucose on HRMECs proliferation assessed by CCK-8 assay: HRMECs cells were stimulated with normal glucose(Control,5.6 mM),MHG(moderately high glucose,MHG,15 mM)and high glucose(HG,30 mM).After incubated for 24 h,10 ?l CCK-8 was added to each well followed by incubation for an additional 2 h.When the media changed from red to yellow,the absorbance value at a wavelength of 450 nm was detected.(2)Effect of high glucose on HRMECs migration assessed by scratch wound migration assay: HRMECs were placed on a 24-well plate and cultured to 90% confluence.After 6 h starvation in serum-free medium,the monolayer cells were scratched with a 1ml yellow pipette tip.Then,the cells were washed with PBS three times to remove the floating cells,and exposed to different stimulating factors.The images of the migration monolayer were photographed at 0 h and 18 h.Migration distance was quantized by Image J analysis software.(3)Effect of high glucose on HRMECs migration assessed by Transwell assay: HRMECs were cultured under various conditions for 24 h in a 5% CO2 incubator.Afterward,cells were seeded at 1 × 105 cells/well in the upper chamber of Transwell plates(8 ?m pore size,Costar,Lowell,MA,USA)with 200 ?l serum-free medium.A volume of 600 ?l of different medium was added to the lower chamber and incubated at 37°C for 6 h.At the end of the assay,the cells were fixed in 4% paraformaldehyde and stained with crystal violet.The upper surface of the Transwell membrane was scraped with a cotton swab to remove un-migrating cells.The membrane was photographed,and the number of cells migrated to the bottom side of the insert was determined by counting five random microscope fields.(4)Effect of high glucose on HRMECs tube formation assessed by Matrigel tube formation assay: Pre-treated HRMECs(5×104 cells per well)were immediately seeded on the surface of Matrigel and cultured for another 6 h to allow the formation of a capillary-like structure.The total tube length of each field was quantified assessed using Image-Pro Plus 6.0 software.(5)Effect of MHG on HRMECs intracellular ROS: Intracellular ROS was measured using the fluorescence dye 2'7'-dichlorofluorescein diacetate(DCFH-DA),cells were grown to 60–70% confluence,and then incubated under the different experimental conditions in 24-well plates for 5min to 24 h.The media were then replaced with serum-free media containing 10 ?M DCHF-DA at 37°C for 30 min.After that,cells were washed,trypsinized,and suspended in 500 ?l PBS,and the fluorescence intensity was measured immediately using a flow cytometer.(6)Effect of MHG on HRMECs VEGF secretion: VEGF secreted by HRMECs in supernatants from different groups of cells was evaluated by using commercially available ELISA kit according to the manufacturer's instructions.(7)Effect of MHG on HRMECs Akt/mTOR pathway: Western blot was used to detect the expression of Akt and mTOR in HRMECs under MHG condition.2 The effect of TXNIP on MHG-induced HRMECs migration and tube formation.To evaluate the TXNIP change of HRMECs,we use Western blot to detect the TXNIP expression level after incubated by MHG.To knockdown TXNIP,HRMECs were transfected with a specific TXNIP siRNA(100 nM)or Scramble siRNA(Scr siRNA)(100 nM)using FuGENE-HD transfection reagent according to the manufacturer's instructions.Forty-eight hours after transfection,the cells were incubated with various stimulating factors for further analysis.Transfected HRMECs were incubated in normal glucose or MHG condition,migration and tube formation were assessed by Scratch wound migration assay,Transwell assay,and Matrigel tube formation assay as above-mentioned.HRMECs Akt/mTOR pathway changes were assessed by Western blot as above-mentioned after transfected by TXNIP siRNA or Scr si RNA.We also used N-Acetyl-L-cysteine(NAC,10mM)to evaluate its effect on MHG induced HRMECs migration and tube formation.3 The effect of TXNIP on VEGF-induced HRMECs angiogenic response.To evaluate the effect of TXNIP on VEGF-induced HRMECs angiogenic response,we used VEGF(20 ng/ml)to stimulate HRMECs transfected by TXNIP si RNA or Scr siRNA as above-mentioned.Then,we used CCK-8 assay,Scratch wound migration assay,Transwell migration assay and Matrigel tube formation assay to assess the effect of TXNIP on VEGF induced HRMECs angiogenic response.We also used NAC(10mM)to evaluate its effect on VEGF induced HRMECs migration and tube formation.Western blot was used to analyze the expression of VEGFR2 and its downstream signal Akt and mTOR.4 The effect of TXNIP knockout on mice retina ex-vivo angiogenesis.All experiments were performed using 6-week old male C57Bl/6(Wild-type,WT)mice and same C57BL/6 background TXNIP-/-(TXNIP knockout,TKO)mice,TKO mice were generated by transcription activator-like effector nucleases(TALEN)technique.All experimental animals were kept under same conditions with standard temperatures and lighting.After full anesthesia has been reached,the retina was dissected from enucleated eye and cut with forceps to obtain four fragments and incubated in serum-free medium for 2 h.200 ?l of Matrigel was added to each well of a 48-well tissue culture plate and solidify the Matrigel for 1 h at 37°C.The fragments were placed in the Matrigel-coated wells,covered with an additional 200 ?l of Matrigel and allowed to solidification for another 1 h at 37°C.Subsequently,200 ?l of ECM Medium containing different stimulators(VEGF(20 ng/ml),MHG(15 mM))were added to the top of the well and replaced each 2 days.After 10 days' incubation,retina sprouts were photographed by microscope.The total sprouts number were scored.VEGF level of mouse vitreous was evaluated by using commercially available ELISA kit(Mouse VEGF ELISA Kit,Abcam),according to the manufacturer's instructions.Results:1 Moderately high-glucose induced migration and tube formation in HRMECs by activation of Akt/mTOR pathway.1)We assessed the effect of different concentration of high-glucose on HRMECs proliferation by CCK-8 assay.It was found that neither MHG nor HG show the significant difference in the proliferation rate(P>0.05).2)To analyzed the effect of high-glucose on HRMECs migratory properties,we used Scratch wound migration assay and Transwell migration assay.MHG but not HG can significant enhance the migratory ability of HRMEC(P<0.01).3)MHG increased in total tube length compared with normal glucose by Matrigel tube formation assay(P<0.01).In contrast,HG still had no effect HRMECs tube formation capabilities(P>0.05).4)To analyze MHG's effect on the level of HRMECs derived VEGF,we measured supernatants from different groups of HRMECs by using ELISA kit.We found VEGF level was relatively low in both group,and MHG did not affect the HRMECs VEGF level(P>0.05).5)MHG active HRMECs Akt/mTOR pathway assessed by Western blot(P<0.05).2 Gene silencing of TXNIP impaired MHG-induced migration and tube formation in HRMECs by blocking Akt/mTOR pathway activation.1)TXNIP expression was assessed by Western blot.After stimulated by MHG,TXNIP was significant highly expressed from 2 hours to 6 hours compare with baseline(P<0.05 or P<0.01).2)The effect of MHG on HRMECs intracellular ROS was assessed by using DCFH-DA fluorescent ROS indicator.MHG can only induce short-time increasing of intracellular ROS in HRMECs(P<0.05 or P<0.01).ROS level quickly back to baseline after 1 h stimulated by MHG.3)TXNIP knocks down in HRMECs impaired MHG-induced migration and tube formation compared with Scrambled si RNA group(P<0.01).4)Silencing of TXNIP altered MHG-induced Akt/mTOR pathway in HRMECs assessed by Western blot(P<0.05 or P<0.01)..5)NAC(10mM)can block MHG-induced HRMECs migration and tube formation(P<0.01).3 Gene silencing of TXNIP impaired VEGF-induced angiogenic response in HRMECs by blocking VEGFR2 pathway activation.1)TXNIP knocks down in HRMECs impaired VEGF-induced proliferation migration and tube formation compared with Scrambled siRNA group(P<0.01).2)NAC(10mM)can block VEGF-induced HRMECs proliferation,migration and tube formation(P<0.01).3)Silencing TXNIP reduced VEGFR2 phosphorylation after VEGF(20ng/ml)stimulation(P<0.01).4)Silencing TXNIP reduced Akt and mTOR phosphorylation after VEGF(20ng/ml)stimulation(P<0.01).4 TKO mice have defective capacity of ex vivo retinal angiogenesis.1)MHG+VEGF induced more retinal microvascular sprouts compared with VEGF group(P<0.05).Retinal microvascular sprouts were very low without VEGF stimulation.2)TKO mice retina showed lower retinal microvascular sprouts number in either condition(P<0.01).3)Knockout TXNIP did not affect mice vitreous VEGF concentration assessed by using ELISA kit(P>0.05).Conclusions:1 MHG-induced HRMECs migration and tube formation is due to activation of Akt/mTOR pathway.It was not caused by the increasing of HRMECs endocrine VEGF level.MHG also could short time increasing HRMECs intracellular ROS level.2 MHG-induce HRMECs TXNIP expression.Silencing TXNIP in HRMECs can reduce the effect of MHG induced HRMECs migration,tube formation by blunt the Akt/mTOR pathway.NAC also could block MHG-induced HRMECs migration and tube formation.3 Silencing TXNIP in HRMECs can block the effect of VEGF-induced HRMECs angiogenic response by reducing the VEGFR2 phosphorylation and blunt its downstream Akt/mTOR pathway.4 MHG+VEGF induced more retinal microvascular sprouts compared with VEGF group,and TKO mice have a defective capacity of ex vivo retinal angiogenesis.TKO mice vitreous VEGF level had no different with WT mice.