| Objective: The prevalence rate of CRS(chronic rhinosinusitis)in China is about 8%,which brings heavy economic and social burden.As a heterogeneous disease,the molecular mechanism of pathophysiology of CRS has not been fully elucidated.Clinically,CRS is often divided into two phenotypes: chronic sinusitis with polyps(chronic rhinosinusitis with nasalpolyps,CRSw NP)and chronic sinusitis without polyps(chronic rhinosinusitis without nasalpolyps,CRSs NP).CRSw NP is characterized by T helper 2(Th2)-skewed,eosinophilic inflammation with massive tissue edema and usually has severe clinical symptoms.According to the infiltration of eosinophils in polyps,CRSw NP can be divided into two subgroups: Eosinophilic CRSw NP(ECRSw NP)and noneosinophilic CRSw NP(non ECRSw NP).ECRSw NP is mainly Th2 type inflammatory reaction,and non-ECRSw NP is mainly Th1/Th17 type inflammatory.ECRSw NP has a high recurrence rate and poor prognosis,which is also known as the refractory sinusitis.There are many theories about the causes of nasal polyps in CRSw NP,including pseudocyst,edema and structural and functional changes of submucosal glands.Previous studies have shown that the relationship between epithelial dysfunction,type 2inflammation and fibrin deposition may partly explain the growth of nasal polyps.Mucosal edema,collagen deposition and inflammatory cells infiltration can be observed in nasal polyps,which promote the activation of thrombin and the lysis of fibrinogen in patients with CRS.Polyps express low levels of plasminogen activator,which reduces fibrinolysis and eventually leads to fibrin accumulation and polyp growth.In addition to the cellular and structural skeleton elements,the largest percentage of the dry weight of polyps is fibrin and the fluid in the fibrin network.The mechanism above seems to have relationship with both ECRSw NP and non ECRSw NP.The extravasation of intravascular substances such as inflammatory cells and plasma proteins into the tissue is mostly caused by the impairment of vascular barrier function in the inflammatory state,which is vascular endothelium dysfunction.Vascular endothelium is formed by monolayer vascular endothelial cells covering the inner surface of all types of blood vessels in the body,which plays a highly specialized role in protecting vascular wall and tissue under harmful stimulation.Vascular endothelium acts as the "interface" between circulation and surrounding tissue,and most of the communication between cells occurs in microvessels composed of arterioles,capillaries and venules.Vascular endothelium is not a passive element,on the contrary,it has the functions of secretion,synthesis,metabolism,immunity and surface expression,and plays a key role in regulating a variety of physiological processes,including the control of vascular tension,neovascularization and regulation of vascular permeability.Vascular endothelial dysfunction,which is more accurately described by the activation of vascular endothelial cells,is one of the prominent manifestations of many acute and chronic inflammatory diseases.During acute inflammation,such as sepsis,COVID19,or radiation exposure,a series of inflammatory events can change the phenotype of vascular endothelial cells.It can also appear in chronic inflammatory diseases,such as allergic rhinitis,chronic urticaria,rheumatoid arthritis and so on.It has been recognized that vascular endothelial cells play an important role in many aspects of inflammatory response,such as the production of pro-inflammatory mediators,the initiation of blood coagulation,the increasing of vascular permeability,leukocyte transport,angiogenesis and the repair of damaged organs.These functions make endothelial cells a key participant and potential therapeutic target in most inflammatory disease.It has been reported that inflammationinduced vascular endothelium dysfunction can be observed in CRSw NP,and its pathological changes include angiogenesis,enlargement of endothelial cell space,increased expression of mucin and so on.Previous studies have shown that after the activation of vascular endothelial cells,the gap widens and a large amount of plasma protein leaks out,resulting in a high degree of tissue edema,which is involved in the formation of nasal polyps.In other words,the vascular leakage that leads to the formation of nasal polyps is directly affected by the endothelial cells.However,at present,the panoramic study and significance of vascular dysfunction caused by inflammationmediated endothelial cell dysfunction in CRSw NP has not attracted more attention.As a group of classic pro-inflammatory molecules,arachidonic acid(AA)and its metabolites play key roles in many inflammatory diseases(such as asthma,arthritis,etc.),cardiovascular biology and cancer.AA can be metabolized by three different enzyme systems: lipoxygenase(LOXs),cyclooxygenase(COXs)and cytochrome P450(CYP).Among them,the product of arachidonic acid metabolism catalyzed by 5-lipoxygenase(5-LOX)pathway is leukotriene(LTs),which is a group of lipid pro-inflammatory mediators with high biological activity.In the LTs family,LTC4,LTD4 and LTE4 are collectively called cysteinyl leukotrienes(Cys LTs),which are mainly produced by stimulated eosinophils,mast cells and macrophages.Previous studies have shown that Cys LTs plays a biological role by activating its receptors Cys LT1 R and Cys LT2 R,which are widely expressed by vascular endothelial cells,bronchial smooth muscle cells and other cells in vivo.Cys LTs-activated receptors can cause pathophysiological reactions such as increased vascular permeability,inflammatory cell aggregation,increased glandular mucus secretion and airway smooth muscle spasm,and then participate in the occurrence and development of various inflammatory diseases,including asthma,allergic rhinitis,atopic dermatitis,allergic conjunctivitis,rheumatoid arthritis and so on.Cys LT1 R is sensitive to classical receptor antagonists such as montelukast sodium.In contrast,until recently,there has been no treatment for specific antagonism against Cys LT2 R.At present,the role of anti-leukotriene therapy targeting leukotriene and its receptors in the diagnosis and treatment of inflammatory rhinopathy has attracted more and more attention.For allergic rhinitis,many clinical trials have confirmed that anti-leukotriene therapy can improve the symptoms of nasal congestion,runny nose and sneezing by reducing vascular permeability,inhibiting glandular secretion and reducing inflammatory cell infiltration.For CRSw NP,especially in patients with ECRSw NP,up-regulation of leukotriene pathway caused by AA metabolic disorders has been detected in nasal polyps.It has been observed that the expression of mucin in nasal polyps decreased after the use of leukotriene receptor antagonist montelukast sodium,which makes montelukast sodium may be used as an adjuvant in the treatment of nasal polyps to promote the recovery of CRSw NP patients.However,there is no consensus among international scholars on this result.Therefore,the role of Cys LTs and its receptors in the pathogenesis of CRSw NP and its potential therapeutic significance need to be further studied.Therefore,this study intends to explore the effect of Cys LTs on vascular endothelial cells and its relationship with CRSw NP,so as to provide new scientific evidence for antileukotriene therapy in patients with CRSw NP.Materials and methods:1.A total of 17 patients with non ECRSw NP,11 patients with ECRSw NP and 5 patients as control were enrolled in the Department of Otorhinolaryngology,Head and Neck Surgery and Allergy,Beijing Tongren Hospital affiliated to Capital Medical University.2.Immunohistochemical method was used to detect the expression of Cys LT1 R and Cys LT2 R on the surface of vascular endothelial cells in nasal polyps and the expression of chemokine receptors on the surface of inflammatory cells related to vascular endothelial disorders.3.RT-qPCR assay was used to detect the m RNA expression of vascular endothelial dysfunction-related genes in human umbilical vein endothelial cell line(HUVEC)treated with Cys LTs family members LTD4,LTE4 and their receptor antagonists Montelukast,Bay Cys LT2 R and BAYu9773.4.The protein expression of vascular endothelial dysfunction-related factors was detected by ELISA method.5.HUVEC cell line was cultured on transwell to establish the model of endothelial barrier in vitro.after being treated with Cys LTs and its receptor antagonist,the transendothelial resistance(TEER)was detected by Ford ohmmeter and the permeability of FITC-dextran was detected by fluorescence reader to study the permeability of endothelial cells.6.Human peripheral blood eosinophils were purified by magnetic bead sorting technique,and the purity was detected by flow cytometry,and the adhesion chemotaxis between eosinophils and endothelial cells was detected by cell adhesion test.7.RNAseq technique was used to explore mechanisms of vascular endothelial dysfunction caused by LTD4,LTE4 and their receptor antagonists after treatment of HUVEC.8.Statistical analysis: each experiment was repeated for 3 times,and t-test,onewayANOVA test and Spearman correlation analysis were carried out by Graphpad9.1.2statistical software.Results:1.LTD4,LTE4 interact with vascular endothelial cells in nasal polyps: nasal polyps were fixed in paraffin,and immunohistochemical results showed that LTD4,LTE4 receptor Cys LT1 R and Cys LT2 R were expressed on the surface of vascular endothelial cells in nasal polyps.2.LTD4 and LTE4 could promote vascular endothelial cell dysfunction in CRSw NP by weakening tight junction and promoting leukocyte chemotaxis: q RT-PCR technique was used to screen the possible directions of the effects of LTD4 and LTE4 on endothelial cells from multiple subfunctions.3.LTD4 and LTE4 affected the tight junction function of endothelial cells: q RT-PCR results showed that the expression of Esam m RNA in HUVEC increased significantly after treatment with LTD4 and LTE4,and the TEER value of HUVEC decreased significantly after treatment with LTD4 and LTE4.The fluorescence intensity of FITC-dextran leakage of HUVEC increased significantly after stimulation of LTD4 and LTE4.4.LTD4 and LTE4 impaired tight junction of endothelial cells by binding to receptors Cys LT1 R and Cys LT2 R,and had a closer relationship with Cys LT1R:(1)After antagonizing Cys LT1 R and Cys LT2 R,the q RT-PCR results of HUVEC treated with LTD4 and LTE4 showed that the expression of Esam m RNA was significantly lower than that of non-antagonistic group(p < 0.05).After antagonizing Cys LT1 R and Cys LT2 R,the TEER value of HUVEC treated with LTD4 and LTE4 was higher than that of non-antagonistic group,and there was significant difference between Montelukast antagonistic LTD4 group,LTE4 antagonistic group and Bay Cys LT2 R antagonistic LTD4 group,while there was a downward trend in Bay Cys LT2 R antagonistic LTE4 group.(3)After antagonizing Cys LT1 R and Cys LT2 R,the fluorescence intensity of FITC-dextran leakage of HUVEC treated with LTD4 and LTE4 was lower than that of non-antagonistic group,and there was significant difference between Montelukast antagonistic LTD4 group,LTE4 treated group and Bay Cys LT2 R antagonistic LTE4 treated group,and there was a downward trend in Bay Cys LT2 R antagonistic LTD4 group.5.LTD4 and LTE4 affected the leukocyte chemotactic function of endothelial cells by upregulating the expression of CCL13,CXCL6 and chemokine receptors CCR2 and ACKR1:(1)The results of q RT-PCR showed that the m RNA expressions of Ccl13,Cxcl6,Ccr2 and Ackr1 in HUVEC were significantly increased after LTD4 and LTE4 treatment.(2)The result of ELISA showed that the protein expression of CCL13 and CXCL6 in HUVEC increased significantly after LTD4 and LTE4 treatment.(3)The cell adhesion test of purified human peripheral blood eosinophils showed that the eosinophil adhesion rate of HUVEC increased significantly after LTD4 and LTE4 treatment.6.LTD4 and LTE4 increased the chemotactic function of endothelial cells by binding to receptors Cys LT1 R and Cys LT2R:(1)After antagonizing Cys LT1 R and Cys LT2 R respectively,the q RT-PCR results of HUVEC treated with LTD4 and LTE4 showed that the m RNA expression of Ccl13,Cxcl6 and their receptors Ccr2 and Ackr1 was significantly lower than that of non-antagonistic group.(2)After antagonizing Cys LT1 R and Cys LT2 R,the ELISA results of HUVEC treated with LTD4 and LTE4 showed that the protein expression of CCL13 and CXCL6 was significantly lower than that of non-antagonistic group.(3)After antagonizing Cys LT1 R and Cys LT2 R,the eosinophil adhesion test of HUVEC treated with LTD4 and LTE4 showed that the eosinophil adhesion rate was significantly lower than that of non-antagonistic group.7.CCL13,CXCL6 and CCR2 played roles in the pathogenesis of CRSw NP,in which ECRSw NP is mainly related to CCL13 and non ECRSw NP is mainly related to CXCL6:(1)The q RT-PCR results of nasal polyps showed that the level of Ccl13 m RNA in ECRSw NP group was significantly higher than that in non ECRSw NP group,while the levels of Ackr1 and Cxcl6 m RNA were significantly decreased.There was no significant difference in Ccr2 m RNA level between the two groups.(2)The results of searching the GEO database published data GSE72713 showed that the gene expression of Ccl13 in ECRSw NP was higher than that in non-polyp control group(FPKM log2(foldchange)=5.98484),and that in ECRSw NP was higher than that in non-ECRSw NP(FPKM log2(foldchange)= 9.02834).The gene expression of Cxcl6 in non-ECRSw NP was higher than that in non-polyp control group(FPKM log2(foldchange)= 2.02165),and that in ECRSw NP was lower than that in non-ECRSw NP(FPKM log2(foldchange)=-2.8006).The gene expression of Ccr2 in ECRSw NP was higher than that in non-polyp control group(FPKM log2(foldchange)= 1.1532).(3)Immunohistochemical results of nasal polyps showed that CCR2 and ACKR1 were expressed on the surface of eosinophils and other inflammatory cells,and there was significant difference between ECRSw NP and non ECRSw NP expression.Semi-quantitative analysis showed that CCR2 positive eosinophils in nasal polyps in ECRSw NP patients were significantly higher than those in non ECRSw NP patients,and ACKR1 positive eosinophils in non ECRSw NP patients were significantly higher than those in ECRSw NP patients.(4)The results of searching the GEO database published data(GSE3982)showed that CCR2 and ACKR1 were expressed in many kinds of human peripheral blood inflammatory cells such as eosinophils.(5)Correlation analysis of Spearman in clinical cases showed that the level of Ccl13 in nasal polyps was positively correlated with the percentage of eosinophils in peripheral blood,neutrophils and monocytes,but negatively correlated with peripheral blood lymphocytes,and positively correlated with Lund-Kennedy endoscopic mucosal morphology score.The m RNA level of nasal polyps was positively correlated with the percentage of eosinophil,neutrophils and monocytes,but negatively correlated with peripheral blood lymphocytes,and positively correlated with Lund-Kennedy endoscopic mucosal morphology score.(6)Spearman correlation analysis showed that the level of Cxcl6 in nasal polyps was positively correlated with the percentage of eosinophils,monocytes and lymphocytes,but negatively correlated with neutrophils in peripheral blood.Cxcl6 levels in nasal polyps were positively correlated with the percentage of eosinophils in peripheral blood,monocytes and lymphocytes,but negatively correlated with neutrophils,and positively correlated with Lund-Kennedy endoscopic mucosal morphology score.8.RNA was extracted from HUVEC treated with LTD4,LTE4 and their receptor antagonists for RNAseq: the whole transcriptional group of HUVEC treated with LTD4,LTE4 and their receptor antagonists was analyzed by principal component analysis(PCA).It was found that there were significant differences in the characteristics of the whole transcriptional group between the stimulant group and the antagonist group,suggesting that LTD4,LTE4 and their antagonists could lead to different biological changes of endothelial cells.The genes obviously up-regulated in(LTD4 / LTE4 VS Control)and down-regulated in(LTD4 / LTE4+ antagonist VS LTD4 / LTE4)were screened and their functions were annotated.It was found that the functions of these genes were mainly related to lipid metabolism,cell connection,intercellular signal transduction and so on.These results were also verified by gene enrichment analysis(GSEA).There was gene enrichment of Apelin signal pathway in both LTD4 and LTE4 groups.It is suggested that LTD4 and LTE4 may act on HUVEC through Apelin signal pathway to produce leukocyte chemokines and adhesion molecules,thus affecting endothelial cell barrier function 。Conclusion: In CRSw NP,LTD4 and LTE4 act to Cys LT1 R and Cys LT2 R on the surface of vascular endothelial cells,which not only affect the stability of vascular endothelial tight junction,but also affect the chemotaxis of endothelial cells to leukocytes,resulting in vascular endothelium dysfunction and articipate in the occurrence and development of nasal polyps. |
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