LKB1 Promotes Proliferation Of Vascular Smooth Muscle Cells And Graft Arteriosclerosis Via Suppression Of The GATA6/Fbln5 Signaling Axis

Objective Chronic rejection,a major obstacle to the long-term healthy survival of patients received an organ transplant,has a common pathological change in different transplanted organs,namely graft arteriosclerosis.During the formation of graft arteriosclerosis,vascular smooth muscle cells(VSMCs)proliferation and migration was significantly enhanced,with a large amount of extracellular matrix synthesized and secreted.Liver kinase B1(LKB1)is a serine/threonine protein kinase mainly distributed in the nucleus and can regulate cell proliferation.However,whether and how LKB1 regulating VSMCs proliferation is still unclear.This study aimed to investigate the effect and mechanism of LKB1 in graft arteriosclerosis and VSMCs proliferation.Method 1.A rat abdominal aorta transplantation model was established,using a smooth musclespecific LKB1-knockdown lentivirus.Immunohistochemical staining,H&E staining,and EVG staining were used to detect neointima formation and LKB1 phosphorylation;2.Isolated rat primary VSMCs was treated by sh RNA-LKB1 and platelet-derived growth factor-BB(PDGF-BB),whose proliferation,DNA synthesis and cell cycle progression were detected by CKK-8,Ed U,flow cytometry and Western blot(WB);3.RNA-sequencing,co-immunoprecipitation,quantitative mass spectrometry detection of non-labeled proteins and bioinformatics analysis were used to explore the mechanism of LKB1-mediated PDGF-BB-induced VSMCs proliferation.4.The mechanism that LKB1 regulating downstream gene transcription was demonstrated by co-immunoprecipitation,molecular docking,fluorescence colocalization,dualluciferase reporter assay system,and WB.5.Small molecule drugs that inhibit PDGF-BB were screened using CMap online database.LKB1-targeted drugs were predicted by Caver web server based on the protein structure prediction.Results Compared to that in donors and iosgrafts,LKB1 phosphorylation in allografts was increased.Smooth muscle-specific LKB1-knockdown inhibited neointima formation and luminal stenosis.In vitro,PDGF-BB-induced VSMCs proliferation was inhibited by LKB1 knockdown.The results of RNA-sequencing and analysis showed that the most significant enrichmented biological function of 664 LKB1-dependent differential genes was positively cell proliferation regulation.The transcription factors predicted based on these differential genes and the LKB1 co-immunoprecipitated products shared the transcription factor GATA binding protein 6(GATA6).Fluorescence colocalization,co-immunoprecipitation,and molecular docking confirmed the interaction between LKB1 and GATA6.GATA6 knockdown reverses the inhibition of VSMCs proliferation by LKB1 knockdown.The downstream genes of GATA6 and LKB1-dependent differential genes shared Fbln5,Fam174 b,Car3,Hmgcs2,and Angptl2.The dual-luciferase reporting result showed that GATA6 promoted Fbln5 promoter activity,and LKB1 overexpression inhibited the transcriptional promotion effect of GATA6.Overexpression of LKB1 enhanced PDGF-BBinduced proliferation,which was further inhibited after overexpression of Fbln5.The CMap database-screened small molecule compounds,such as Aminopurvalanol-a,Purvalanol-a,and JAK3-inhibitor-VI,may interfere with PDGF-BB.A variety of drugs,including fosaprepitant,lestaurtinib and emend,that may target LKB1 were further screen out.Conclusion Phosphorylation of LKB1 in the middle layer of the graft artery promoted neointima formation.By interacting with GATA6,LKB1 inhibited Fbln5 promoter activity and downregulated Fbln5 expression,mediating PDGF-BB-induced VSMCs proliferation and cell cycle progression.A variety of drugs that may target LKB1 have the potential to treat grafts arteriosclerosis.