The Mechanism Of H3K18 Lactylation Promoting PASMCs Migration And Participating In The Progress Of Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension(PAH)is a progressive disease with poor prognosis,which is caused by various factors including cell proliferation,immune,inflammation and environmental factors.PAH lacks biomarkers for early diagnosis,which results in the delayed diagnosis for most patients.The treatment for PAH is the combination of targeted medicine,but the effect is limited on late stage PAH.Thus,the explore of new mechanism and target point for PAH is beneficial for the diagnosis and treatment.Histone lactylation is a newly discovered epigenetic modification,which is widely researched in cancers and macrophage-related diseases.These researches mainly focused on H3K18 lactylation,which can bind the promoter region of DNA and manipulate gene expression,but study of H3K18 lactylation is limited in PAH.Lactate serves as substrate of H3K18 lactylation,which is also accumulate in PAH lungs because of the hypoxic environment and the increase of Warburg effect.We hypothesis that H3K18 lactylation is elevated in PAH,and promoted the progress of PAH.Objective: To explore the effect H3K18 lactylation in PAH,and to confirm the mechanism of it.Methods:1.Use Alphaphold2 and AMBER 18 to predict and analyze the structure of H3K18 lactylation.2.Measure the pulmonary artery blood flow acceleration time(PAAT)and tricuspid annular plane systolic excursion(TAPSE)through echocardiography;measure the right ventricular systolic pressure(RVSP)through right heart catheter;assess the pathological change in pulmonary vessels by HE staining.Expolre the expression and location of H3K18 lactylation in PAH through western blot and immunofluorescence.3.Explore the binding patterns of H3K18 lactylation through Ch IPseq;explore the possible downstreame of H3K18 lactylation using GO analysis and KEGG analysis and verify the results by western blot.Using motif analysis to explore the binding sequence of H3K18 lactylation and possible transcription factor.4.Extract PASMCs and establish lactate and platelet-derived growth factor – BB(PDGF-BB)stimulated cell models to explore the effect of lactate and PDGF-BB on H3K18 lactylation.Using p300 inhibitor to verify that H3K18 lactylation promote PASMCs proliferation and migration through HIF-1α/MMP9 pathway.Results:1.The structure of H3K18 lactylation is different from wild type histone H3.Lactylation changed the structure of histone H3 and made it more stable than the wild type.2.The PAAT and TAPSE were declined in PAH rat models,while RVSE increased.HE stanning showed pulmonary vessel reconstruction especially in the tunica media,which indicated the success of the models.Besides,western blot and immunofluorescence showed H3K18 lactylation was highly expressed in the PASMCs.3.Ch IP-seq revealed H3K18 lactylation have different binding patterns in PAH models,including the location on chromosomes and gene regions.KEGG analysis revealed H3K18 lactylation related genes were enriched in HIF-1α pathway.Motif analysis uncovered several transcription factors participated in the interaction of H3K18 lactylation and DNA,which mainly include: Max,Myc and NFY4.Lactate and PDGF-BB could result in the elevation of H3K18 lactylation and p300 in a time-dependent manner.Wound healing and CCK8 experiment revealed p300 inhibitor could inhibited the elevation of H3K18 lactylation and PASMCs proliferation and migration;western blot found that H3K18 lactylation could participate in the regulation of HIF-1α/MMP-9 pathway.Conclusion:1.Lactylation changed the structure of histone H3 and made it more stable than the wild type.2.H3K18 lactylation is higly expressed in the PASMCs of PAH rat model.3.H3K18 lactylation have different binding patterns in PAH models,including the location on chromosomes and gene regions.4.H3K18 lactylation related genes were enriched in neuro regulation pathway and HIF-1-α pathway.5.H3K18 lactylation have specific binding position,which might interaction with transcription factors including: Max,Myc and NFY.6.Lactate and PDGF-BB could promote the elevation of p300 and H3K18 lactylation.7.H3K18 lactylation could promote PASMCs proliferation and migration through HIF-1α/MMP-9 pathway.