BackgroundThis　study　aims　to　elucidate　the　effect　and　mechanism　of　phospholipid　transfer　protein　(PLTP)　on　vascular　dysfunction　in　DR　and　explore　the　molecular　mechanism　of　abnormal　PLTP　expression　based　on　DNA　methylation.MethodsHuman　retinal　microvascular　endothelial　cells　(HRMECs)　cultured　in　high　glucose　(HG)　and　streptozotocin-treated　mice　were　used　as　DR　models　to　detect　and　screen　the　key　genes　with　abnormal　promoter　DNA　methylation.　Single-cell　sequencing,　tube　formation　and　migration　assays　were　employed　to　verify　the　relationship　between　PLTP　and　vascular　function.　Additionally,　siRNA　and　luciferase　reporter　assay　were　used　to　study　the　key　enzymes　regulating　the　DNA　methylation　of　PLTP.　Transcriptome　sequencing,　coimmunoprecipitation　and　GSK3　beta　inhibitor　were　utilized　to　identify　and　validate　the　key　downstream　pathways　of　PLTP.ResultsDR　models　exhibited　DNA　hypermethylation　and　decreased　expression　of　PLTP.　Abnormal　PLTP　expression　was　implicated　in　vascular　dysfunction,　and　PLTP　overexpression　reversed　HG-induced　effects　on　the　migration　and　tube　formation　of　endothelial　cells.　The　siDNMT3B　and　luciferase　reporter　assay　indicated　that　DNMT3B　is　the　primary　enzyme　affecting　abnormal　methylation.　Interestingly,　PLTP　promoted　the　phosphorylation　of　AKT　and　GSK3　beta,　indicating　that　PLTP　modulates　angiogenesis　via　the　AKT/GSK3　beta　signaling　pathway.ConclusionsPLTP　regulates　the　proliferation,　migration　and　tube　formation　of　HRMECs,　and　is　involved　in　maintaining　vascular　function　via　the　AKT/GSK3　beta　signaling　pathway.　In　HG　environment,　increased　DNMT3B　expression　upregulates　DNA　methylation　of　the　PLTP　promoter,　downregulating　PLTP　expression　and　leading　to　vascular　dysfunction　in　DR.Graphical　abstractPLTP　promotes　the　phosphorylation　of　AKT　and　GSK3　beta,　leading　to　the　enhancement　of　endothelial　cell　proliferation,　migration　and　tube　formation,　thereby　maintaining　vascular　function.　In　HG　environment,　increased　DNMT3B　expression　results　in　abnormally　increased　DNA　methylation　of　the　PLTP　promoter,　leading　to　decreased　PLTP　expression　and　subsequent　vascular　dysfunction.