VEGF And AIMP1 Targeted Intervention Study In The Diabetic Retinopathy

OBJECTIVE: To elucidate the role of anti-vascular endothelial growth factor(VEGF)agents and Aminoacyl-t RNA synthetase-interacting multi-functional protein 1(AIMP1)pathway in diabetic retinopathy(DR)by interfering VEGF and AIMP1.METHODS:(1)156 eyes of 138 patients with severe PDR who underwent vitrectomy were enrolled,of whom,patients who received intravitreal injection of Ranibizuman(IVR,0.5 mg)were assigned to group A(59 eyes),and those who did not receive IVR were assigned to group B(97eyes).Demographic data and both intraoperative and postoperative findings were recorded.(2)35 eyes of 35 patients with PDR who underwent vitrectomy were enrolled.Patients who received IVR were defined as IVR group(9 eyes)and those who received intravitreal injection of Conbercept(IVC)were defined as IVC group(9 eyes).Untreated PDR patients were defined as DR group(8 eyes).Non-diabetic patients with idiopathic macular hole(i MH)were defined as normal glucose control group(NG,9 eyes).Difference in protein profiling in vitreous humor between DR and NG group,DR and IVR group,DR and IVC group,IVR and IVC group were measured by liquid chromatography-mass spectrometry(LC-MS/MS).Gene Ontology(GO)and REACTOME pathway analysis were performed to get a functional overview on the differential expressed proteins.The intravitreal level of specific candidate proteins was verified by enzyme-linked immuno sorbent assay(ELISA).(3)(1)Diabetic AIMP1-specific knockout(KO)C57 mouse model was cultured.(2)Human retinal microvascular endothelial cells(HRMECs)were respectively incubated with normal glucose(NG,5m M),high glucose(HG,30 m M)and HG+AIMP1-small interfering RNA(si RNA).(3)The expression of AIMP1,Toll-like receptor 2/4(TLR2 / 4),NOD-like receptor family,pyrin domain containing 3(NLRP3)inflammasome,interleukin(IL)-18,and caspase3 et al.in diabetic mice retina and HRMECs were measured by Western blot and polymerase chain reaction(PCR).(4)The apoptosis of HRMECs was detected by terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL)assay.(5)The level of AIMP1 in vitreous humor and serum was determined by ELISA.The correction between intravitreal level of AIMP1 and blood glucose,glycosylated hemoglobin Hb A1 C,intravitreal level of IL-1?,caspase 3 was evaluated.(6)SPSS software was used to statistical analysis.RESULTS:(1)Postoperative best-corrected visual acuity(BCVA)in group A was more than that in group B(log MAR units: 0.427±0.43 versus 0.691±0.59,P<0.05),similarly,there were significant difference in improved BCVA(91.5% versus 71.1%;P<0.05),unchanged BCVA(5.1% versus 15.5%;P<0.05),and worse BCVA(3.4% versus 13.4%;P<0.05)respectively.Postoperative early vitreous hemorrhage occurred significantly less frequently in group A(8 of 59 eyes,13.6%)than that in group B(32 of 97 eyes,33.0%;p < 0.01).Importantly,the incidence of postoperative NVG in group A was significantly less than that in group B(1.7 versus 12.4%;P<0.05).(2)(1)480 differentially expressed proteins were identified between DR and NG gourp.The most notable GO annotation was “platelet degranulation” and “proteolysis”.The most notable pathway was “platelet degranlulation”.(2)339 differentially expressed proteins were identified between DR and IVR gourp.Among these proteins,203 proteins were decreased in response to IVR.The most notable GO annotation and pathway were both “platelet degranlulation”;136 proteins were increased.The most notable GO annotation was “innate immune response” and “scavenging of heme from plasma”.(3)307 differentially expressed proteins were identified between DR and IVC gourp.Among these proteins,218 proteins were decreased in response to IVC.The most notable GO annotation was “innate immune response” and the most notable pathway was “platelet degranlulation”.89 proteins were increased.The most notable GO annotation was “receptor-mediated endocytosis” and the most notable pathway was “scavenging of heme from plasma”.(4)269 differentially expressed proteins were identified between IVR and IVC group.Amont these proteins,162 proteins were increased in IVR group compared to IVC.The most notable GO annotation was “innate immune response” and the most notable pathway was “amyloid fiber formation”.107 proteins were increased in IVC group compared to IVR.The most notable GO annotation and pathway were both “platelet degranulation”.(5)In IVR group,intravitreal level of Apolipoprotein-CI(APOC1),Keratin 1(KRT1),Serine protease inhibitors A5(SERPINA5)were decreased and the level of tissue inhibitor of metalloproteinase 2(TIMP2)was increased compared to those in DR group(P<0.01).In IVC group,intravitreal level of Apolipoprotein-II(APOA2)and ceruloplasmin(CP)were decreased compared to those in DR group(P<0.01).(3)(1)The expression of AIMP1 and inflammatory,apoptotic cytokines were significantly increased in retinal tissue of diabetic mice and HRMECs incubated with high glucose compared to normal glucose group.While the expression of these proteins was inhibited in AIMP1 KO mouse and HRMECs incubated with AIMP1-si RNA.(2)The apoptosis of HRMECs was higher than that of normal glucose control group,and was decreased in AIMP1-si RNA group.(3)The intravitreal level of AIMP1 in DR patients was significantly higher than that of non-diabetic patients(P<0.01).There was a positive correlation between intravitreal level of AIMP1 and Hb A1 C,intravitreals level of IL-1?and caspase 3(P<0.05).CONCLUSIONS:(1)Preoperative IVR treatment for patients with severe PDR contributes to the decreased occurance of NVG and improved surgical outcomes.(2)In addition to decreasing VEGF level,anti-VEGF treatment causes change of human vitreous protein profile in patients with PDR,in which the differential proteins are involved in immune,platelet degranulation,complement activation,apoptosis,inflammation,angiogenesis etc,suggesting that these pathways as well as the identified proteins such as APOC1?APOA2?TIMP2?SERPINA5?KRT1 and CP are involved in the mechanism of the effect of anti-VEGF treatment.(3)High glucose state can induce increased expression of AIMP1 in HRMECs and in retina from diabetic C57 mice,thereby increase the expression of inflammatory and apoptotic cytokines by the activation of NLRP3 and TLR2/4,promoting the progress of DR.While the decrease of AIMP1 expression can prevent the development of DR through inhibiting the activation of inflammatory and apoptotic signaling.AIMP1 is an effective interfering target in the prevention and treatment of DR.