The Roles And Mechanisms Of Inflammatory Cells Activation In Arterial And Pulmonary Vascular Remodeling Under Specific Physical And Chemical Factors Exposure

Background:Cardiovascular diseases(CVDs),one of the primary causes of morbidity and mortality globally,seriously threaten public health and quality of life in patients.Classical CVDs’ risk factors include smoking,obesity,hypertension,hyperglycemia,high cholesterol and so on.Besides,long-term exposure to pernicious environmental factors(such as environmental pollutants,hypoxia,etc.)has also been reported to be closely related to cardiovascular morbidity and mortality,but the relevant mechanisms are still unrevealed.Bisphenol A(BPA)is one of the environmental pollutants,which is ubiquitous in our daily life.It is abundant in air,plastic products,food packaging bags and so on,and its health hazard has attracted great public concern.Chronic BPA exposure is closely associated with multiple diseases such as obesity,diabetes,CVDs,tumors,birth defects and so on.Previous studies have shown that chronic BPA exposure is closely related to the severity of atherosclerosis(AS),but the mechanisms and intervention strategies have not been reported yet.Chronic hypoxia exposure is another common special environment,which leads to pulmonary vascular disorders in individuals with the condition of pulmonary diseases and high-altitude sojourn.Chronic hypoxia exposure may lead to pulmonary vascular remodeling,resulting in increased pulmonary artery pressure,increased right ventricular afterload,and even right heart failure and death.However,patients with hypoxic pulmonary hypertension(HPH)have poor clinical prognosis and high mortality.Moreover,there are no guidelines recommending targeted drug,mainly because the pathogenesis of HPH has not been elucidated.These specific physical and chemical factors can widely cause the physiological and pathophysiological responses of the cardiovascular system,and even play a vital role in the occurrence and development of CVDs.However,it is extremely important to study the related mechanisms of vascular remodeling under the special environment beyond the classical risk factors of CVDs.Inflammatory cell activation plays an important role in vascular remodeling,but its roles and mechanisms in arterial and pulmonary vascular remodeling under specific physical and chemical factors exposure are still unclear.In this study,BPA and hypoxia are used to induce arterial and pulmonary vascular remodeling,we focus on inflammatory cell activation to explore the mechanisms of vascular remodeling,aiming to provide new theoretical basis and intervention targets for the prevention and treatment of CVDs related to specific physical and chemical factors.Objectives:1.To explore the characteristics and mechanisms of inflammatory cell activation in the formation of AS by chronic BPA exposure;2.To construct a inflammatory cell atlas of hypoxic pulmonary vascular remodeling based on single cell RNA sequencing(scRNA-seq)and single cell ATAC sequencing(scATAC-seq);3.To reveal the key molecules and regulatory mechanisms of inflammatory cell activation in hypoxic pulmonary vascular remodeling based on scRNA-seq and scATAC-seq.Methods:1.In order to construct the AS animal model,four-week-old Ldlr-/-C57BL/6 male mice with 250 mg/L BPA through drinking water for 30 weeks and with western diet and/or with resveratrol(RESV)for 12 weeks.Using western blotting and real-time quantitative PCR to detect the mRNA and protein expression levels of inflammatory cytokines,Sirtuin 1 and Nijmegen breakage syndrome 1(NBS1),the number of pro-inflammatory monocytes was determined by flow cytometry,the comet assay was used to evaluate DNA damage repair ability,and the histology and immunofluorescence were used to evaluate the severity of atherosclerotic lesions.2.In order to construct HPH animal model,8-10 weeks old C57BL/6 male mice with 10%oxygen for 5 weeks,with a weekly injection of SU5416(20mg/kg).Single-cell suspension was prepared from mouse lung tissue,then constructed a single-cell atlas of hypoxic pulmonary vascular remodeling based on scRNA-seq and scATAC-seq,and cell-cell interaction characteristics and the key molecules and signaling pathways involved in HPH were explored by differential expressed genes,cell interaction analysis and so on.3.ScRNA-seq and scATAC-seq were used to determine the characteristic cytological alteration of HPH,its key molecules and key transcription factors by cell reclustering analysis,KEGG,pseudo-time cell trajectory analysis,and ligand-receptor interaction.Based on the results of scRNA-seq and scATAC-seq,S100a9 knockout mice(S100a9-/-)were further used to define the role of S100a9 in the development of HPH.Pulmonary vascular remodeling was evaluated by H&E staining,αSMA immunofluorescence staining and pulmonary vascular imaging.Right heart hypertrophy and cardiomyocyte hypertrophy were evaluated by Fulton index and WGA staining.Pulmonary artery pressure were assessed by right heart catheterization and right heart function were evaluated by echocardiogram.Results:1.Chronic BPA exposure significantly aggravated atherosclerosis,enhanced the production of inflammatory cytokines but not lipid levels,promoted macrophage infiltration into plaque areas.Moreover,peritoneal macrophages isolated from BPA-exposed mice exhibited a more pro-inflammatory phenotype in response to cholesterol crystal treatment than those from control mice.The comet assay revealed that the DNA repair capacity of BPA-exposed macrophages was impaired,and western blotting showed that sirtuin 1 and Nijmegen breakage syndrome 1(NBS1)expression was reduced.However,restoring sirtuin 1 by RESV administration significantly blocked the BPA-induced decrease in NBS1 and subsequently attenuated the BPA-induced impairment of DNA repair and apoptosis,as indicated by phosphorylated H2AX expression and staining and PARP expression.Moreover,RESV administration significantly ameliorated BPA-aggravated NOD-like receptor pyrin domain 3 and caspase 1 activation and interleukin-1b production,which were abolished by NBS1 knockdown.Furthermore,RESV administration prevented BPA-induced aggravation of atherosclerosis.2.We constructed a single-cell transcriptome atlas and a single-cell chromatin accessibility atlas for a mouse model of HPH disease.Nine subsets of cells were identified,including granulocytes,monocyte-macrophages,NK/T cells,endothelial cells,B lymphocytes,fibrocytes,dendritic cells,smooth muscle cells,as well as epithelial cells.We found that hypoxia exposure led to a significant increase in the number of granulocytes and monocytes-macrophages in lung tissue and their interaction with other cells.Moreover,we used SCENIC to construct a transcriptional interaction map,and find that Statl,Spi1,Irf1 were the top transcription regulators for upregulated differential expressed genes(DEGs),whereas Jund and Etsl were the key transcriptional regulators for downregulated DEGs,DEGs.These genes were mainly involved in inflammation-related signaling pathway activation.Interestingly,hypoxia-inducing factor(HIF)pathway was activated mainly in granulocytes and monocytes-macrophages after hypoxia exposure.3.Granulocytes were further divided into five cell subsets by reclustering analysis:basophils,Hybrid granulocytes,IL1α+ neutrophils,Ngp-IL1α-neutrophils and Ngp+neutrophils,and monocyte-macrophages were further divided into nine cell subsets:CCR2+pro-inflammatory macrophages,M2-like macrophages,Adgre4+pro-inflammatory macrophages,F10+monocytes,classical monocytes,M1/M2-like macrophages,proliferative macrophages,non-classical monocytes,and CCL2+macrophages.After chronic hypoxia exposure,the changes of granulocyte were mainly characterized by increased Ngp-IL1α-neutrophils and Ngp+ neutrophils,while the changes of monocyte-macrophage were mainly represented by increased CCR2+pro-inflammatory macrophages and a significant decrease in M2-like macrophages.Chronic hypoxia exposure significantly increased the expression of S100a9 in CCR2+ pro-inflammatory macrophages.Moreover,chronic hypoxia exposure increased the accessibility of S100a9 in the promoter region and transcriptional start site,which could be bound by Id1.Then,we constructed the S100a9 knockout mice(S100a9-/-).In S100a9+/+ mice,the pulmonary vascular wall was significantly incrassated,the number and extent of distal pulmonary arteries increased,fewer pulmonary vascular branches and connections,the mean pulmonary artery pressure increased significantly,but the fraction of right ventricular area change significantly decreased after chronic hypoxia exposure,these changes were alleviated in S100a9-/-mice.Conclusion:1.Macrophage inflammatory aggravates AS plaque formation during chronic BPA exposure,where sirtuin 1 mediated-DNA damage repair by regulating NBS1 expression plays an important role in aggravating AS plaque formation and macrophage inflammatory response by chronic BPA exposure,and the Sirtuin 1 agonist RESV may be a potential therapeutic agent against BPA-related CVDs.2.We constructed a single-cell transcriptome map and a single-cell chromatin accessibility map for a mouse model of HPH disease.During HPH development,HIF pathway was activated in granulocytes and monocytes-macrophages.Moreover,Activations of inflammatory cells and inflammatory signaling pathway were found,and may be the main mechanism of HPH.3.We analyzed the change characteristics,interactions and molecular regulation mechanisms of the main inflammatory cells in the process of HPH,the gene knockout technology was used to clarify that S100a9 is one of the key regulators of HPH,moreover,we found that Id1 may be the key transcription factors for CCR2+macrophage activiation during the development of HPH by regulating the transcription of S100a9.