YY1 Lactylation In Microglia Promotes Angiogenesis Through Transcription Activation-mediated Upregulation Of FGF2

Background Ocular pathological neovascularization is a leading cause of blindness.Hypoxia is a triggering factor for retinal neovascularization in ischemic retinopathy,which is affected by the changes in the microenvironment of hypoxic site.Retinal microglia has been implicated in hypoxia-induced angiogenesis and vasculopathy,but the underlying mechanisms remain largely unknown.Hypoxia is often accompanied by the production and accumulation of lactate.In addition to its original metabolic function,lactate can play additional non-metabolic effects,including regulation of macrophage polarization,T cell activation,and tumor growth.Lactylation is a novel lactate-derived posttranslational modification(PTM),which can directly regulate gene expression and play a key role in a variety of life processes,but whether it is involved in hypoxia-induced retinal angiogenesis has not reported yet.This study aimed to explore the role and mechanism of microglia lactylation modification in retinal neovascularization,and to provide new therapeutic target for retinal neovascularization diseases.PART ONE: HYPERLACTYLATION OF MICROGLIA CELLS PROMOTE ANGIOGENESIS IN VIVO AND IN VITROObjective1.To establish the OIR model in C57BL/6J mice,to explore the effect of lactate/lactylation of microglia on retinal neovascularization,and to explore the possibility of eliminating microglia(PLX3397)reversing retinal neovascularization of OIR.2.To establish the coculture system of HMC3 and HRMECs and to explore the effect of hypoxia-induced lactylation of human microglia on angiogenesis in vitro.Method1.Animal model establishment,male and female C57BL/6J pups were defined as P0 when they were born.The pups and suckling mice received food and water freely with a 12/12 hrs-day/night cycle in normal air for 7 days(P0-P7).On day P7,healthy pups and suckling mice were placed into a glass oxygen chamber together,with the oxygen flow controlled to 0.50-0.75 L/min and the oxygen concentration was maintained at 75±2%.The animals were kept in a glass oxygen chamber for five consecutive days(P7-P12).At P12,the pups were returned to a normal air environment and continually co-bred with the suckling mice for five days(P12-P17).PLX3397 treatment group completed the intraperitoneal injection at P10-P17.DCA,Rotenone and DMSO treatment group completed the intraperitoneal injection at P13-P16 of OIR,respectively.The pups were sacrificed at the age of P17,and the retinas were removed for subsequent experiments.Lactic acid Assay Kit was used to detect retinal lactate content,WB was used to detect Pan-Kla level and IF was used to observe retinal angiogenesis.2.HMC3 cells were treated with 1% O2 for 0 hr,12 hrs and 24 hrs.The lactate content at three time points was detected by Lactic acid Assay Kit,and the level of Pan-Kla was detected by WB.HMC3 treated with hypoxia and normoxia for 24 hrs was cocultured with HRMECs,and endothelial function was detected by three experiments including tube formation,migration,proliferation.Then DCA,Rotenone and DMSO were added in hypoxic HMC3 for 24 hrs,Lactic acid Assay Kit was used to detect the lactate content of the three groups,and WB was used to detect the level of Pan-Kla.Based on hypoxia for 24 hrs,HMC3 added with three groups of compounds was cocultured with HRMECs,and the endothelial function was detected by three experimental methods including tube formation,migration,proliferation.Result1.After modeling,retinal microglia proliferated a lot and gathered around the site of neovascularization in the OIR retina.Compared with the control group,the production of lactate and Pan-Kla levels were both upregulated on P17(the peak of neovascularization in OIR)in the OIR group,and lactylation colocalized with microglia in the retina.After compound(DCA,Rotenone)treatment,lactate and lactylation levels of retina were reduced in the DCA group,but increased in the Rotenone group,we observed that retinal neovascular disease was alleviated to a certain extent after DCA treatment and aggravated in the Rotenone group.After administration of PLX3397,the pathological retinal angiogenesis was greatly suppressed along with the depletion of microglia and the level of Pan-Kla in the retina was relatively downregulated with the use of PLX3397.2.As the degree of hypoxia increased,the lactate content of HMC3 cells increased,and the level of lactylation was also elevated,especially the protein in the range of 55-70 k D.As the hypoxia time of HMC3 cells increased,the tube formation,migration and proliferation of HRMECs were crucially enhanced.HMC3 cells were treated with DCA and Rotenone.The lactate/lactylation levels of HMC3 cells were decreased in the DCA group and increased in the Rotenone group.We then cocultured HMC3 microglial cells from the DCA/DMSO/Rotenone groups with HRMECs.The tube formation,migration and proliferation abilities of HRMECs cocultured with HMC3 cells in the DCA group were weakened,while they were enhanced in the Rotenone group.ConclusionThese results demonstrate elevated lactate and lactylation levels in microglia contribute to retinal neovascularization.PART TWO: MICROGLIA ENHANCES ANDIOGENESIS VIA LACTATE/P300/YY1 LACTYLATION/FGF2 SIGNALING PATHWAYObjective1.To explore the key protein with altereing level of lactylation in microglia under hypoxic condition according to lactylome analysis.2.To explore the important role of YY1 lactylation of microglia in regulating angiogenesis.3.To explore the targeted factors of YY1 lactylation to promote angiogenesis.4.To explore the lactylation-modifying enzymes affecting the lactylation of YY1 regulate angiogenesis in vitro and in vivo.Method1.We treated HMC3 cells with normoxia or hypoxia for 24 hrs and then applied lactylome analysis by the 4D label-free platform method to identify the differentially lactylated proteins.HMC3 cells were treated with hypoxia and normoxia for 24 hrs respectively,IP YY1 of these two groups and then to detect the relative lactylation level of YY1 by IB.We mutated lysine(K)183 of HMC3 to arginine(R)by transfecting HMC3 cells with lentivirus containing c DNA of the Flag-tagged YY1 WT or YY1K183 R mutant.The mutant group(Hypoxia+K183R)and the control group(Hypoxia+WT)were cocultured with HRMECs,then to observe whether the phenotypes were rescued according to tube formation,migration and proliferation capabilities of HRMECs after coculturing with mutant group.2.To detect the expressions of classic angiogenic-related genes VEGF,FGF2,MMP9,MMP2,ANGPTL6 in vitro and in vivo by use of rt-PCR.Hypoxia,drug(PLX3397)and compounds(DCA,DMSO,ROT)treatment were used then to detect the expression levels of FGF2 protein and Pan-Kla in vitro and in vivo by WB.Three databases of TRANSFAC,Harmonizome and Cistrome Data Browser were used to explore the potential target gene of YY1.To verify the regulatory effects of YY1 lactylation on the potential target gene FGF2 by use of Chip-q PCR,WB and dual-luciferase reporter system..3.The expression levels of known acylation modification writers(TIP60,p300 and PCAF)and erasers(HDAC6 and SIRT1)were detected by WB after HMC3 hypoxia treatment.IP YY1 and these several classical acylation-modified enzymes and then to explore whether there is a combination between classical acylation-modified enzymes and YY1 by IB.Double-labeled immunofluorescence was used to prove YY1 was co-combined with p300;HMC3 cells were treated with p300 inhibitor(A-485)and p300 overexpression lentivirus to establish p300 underexpression and overexpression models,which divided into Hypoxia+Control group,Hypoxia+A-485(20μM)group,Control group,oep300-WT group.Subsequently,the levels of Pan-Kla in each group were measured by WB and the relative levels of Pan-Kla in each treatment group were determined by IB after IP YY1 of each group.At P14 of OIR,A-485(1μl,200 μM)/DMSO(1μl)was injected into the vitreous cavity of the left or right eye,respectively.Retinal samples were collected at P17.The levels of retinal Pan-Kla,FGF2 and YY1 Pan-Kla were detected by WB,IP and IB experiments.Retinal angiogenesis and microglia lactylation levels were observed by IF.Result1.77 sites of 67 proteins with increased lactylation and 190 sites of162 proteins with decreased lactylation under hypoxic conditions as differentially expressed lactylated proteins(DELPs).Cluster analysis of the identified DELPs was performed to characterize the function of these lactylated proteins.The classification analysis indicated that most of the DELPs exert functions in the nucleus and have a potential role in regulating DNA transcription.Using the Search Tool for the Retrieval of Interacting Genes/Proteins(STRING)database,the protein interaction networks of DELPs suggested that the DELPs mainly function in the DNA binding process,which was brought to our attention of YY1.The band size of YY1 is 68 k D,which is consistent with the range(55-70 k D)that is mainly regulated by hypoxia or treatment with DCA and Rotenone according to our previous results.The proteomic analysis identified only one lactylated lysine residue in YY1(K183).YY1 lactylation was largely increased under hypoxia with no difference in YY1 expression level.And we observed colocalization of YY1 and Pan-Kla with double-labeled immunofluorescence in hypoxic HMC3 cells by IF.Immunoprecipitation of Flag-YY1 followed by immunoblotting for Pan-Kla confirmed that YY1 was lactylated,and that YY1 lactylation levels were decreased in K183 R mutant hypoxia-treated HMC3 cells.Compared with the WT group,HRMECs cocultured with K183 R mutant HMC cells showed weakened tube formation,migration and proliferation capabilities.2.The FGF2 and VEGF mRNA expression levels were significantly elevated in OIR mice compared with the control group,while only FGF2 levels decreased notably at both the m RNA and protein levels after administration of PLX3397.Similarly,hypoxia increased FGF2 m RNA expression within 12 hrs of hypoxia exposure,and the levels remained elevated until 24 hrs in HMC3 cells.In the OIR model treated with DCA and Rotenone,the expression of FGF2 was decreased in the DCA group,but increased in the Rotenone group at P17,similar results were found in HMC3 cells exposed to hypoxia with DCA or Rotenone.After the lactylation site of YY1 was mutated,we detected lower expression of FGF2 at both the m RNA and protein levels following decreased lactylation levels of YY1.We found that YY1 may directly bind to the promoter of FGF2 according to the three databases.We found three potential YY1 binding sites in the FGF2 promoter from the JASPAR website,and Ch IP-q PCR showed that YY1 can bind to the region of-1336to-1172 bp upstream of the transcription start site of FGF2.A dual-luciferase reporter system was applied to verify the result that YY1 directly promotes the transcription of FGF2 under hypoxia and that mutation at the lactylation site of YY1 resulted in lower transcription ability.3.p300,HDAC6 and SIRT1 levels were significantly upregulated and TIP60 levels were slightly upregulated in HMC3 cells under hypoxia.Only p300 combined with the target protein YY1 according to the Co-IP data and the result of double-labeled immunofluorescence.We overexpressed p300 in HMC3 cells and found that the lactylation level was significantly increased,and the expression of FGF2 was also upregulated.After A-485 treatment,the lactylation of HMC3 was significantly reduced,and the expression level of FGF2 was also downregulated.As IB detected,p300 overexpression increased the lactylation level of YY1,while decreased lactylation levels of YY1 were observed in the A-485 group.The tube formation,migration and proliferation abilities of HRMECs cocultured with p300-overexpressing HMC3 cells were enhanced but weakened in the A-485 group compared with the respective control.We found that the expression of p300 was upregulated in the OIR retina,while no difference in YY1 was observed.IB after IP showed hyperlactylation of YY1 in OIR retinas.Intravitreal administration of A-485 was applied at P14.Lactylation and FGF2 expression were both reduced after administration.YY1 lactylation was decreased after treatment with A-485 in OIR as detected by IB.ConclusionOur findings present a previously undefined regulatory mechanism in angiogenesis,which hypoxia leads to an increase in the lactylation of YY1 in microglia by up-regulating the expression of p300.Upregulation of YY1 lactylation enhances FGF2 expression,thereby promotes the angiogenesis of HRMECs.