The Study Of LCN2 In The Occurrence And Development Of Aortic Dissection/Aneurysm

BackgroundAortic dissection/ aneurysm refers to the disease which intima of vessels tears,stripsand extends,and then two cavities within the artery formation of true and false,at last leads to the dilatation even rupture of the aorta.Early after aortic dissection/ aneurysm,a number of inflammatory cells infiltrated into the site of injury to secret inflammatory factors and matrix metalloproteinases,causing damage to the vessel,such as Elastic fiber breaking,SMC apoptosis and collagen degradation and so on.With the occurrence and development of disease,on the one hand,the vascular adventitia fibroblasts are activated and secretion of extracellular matrix(ECM)increase;on the other hand,proinflammatory macrophages which infiltrated into the outer membrane in inflammatory reaction stage loss its activity and transform to repairphenotype which can secret collagen.These two aspects collectively promote the vascular restoration and evenmore prevent the dilatation even rupture of the aorta.Previous research has shown that LCN2 may also be involved in the process of AD/AA because serum LCN2 levels are significantly higher than those in healthy patients and mouse,and is mainly secreted by fibroblasts,macrophages and neutrophils,all this indicates that LCN2 may participate in the AD/AA development process.However,the specific function and mechanism of LCN2 in the occurrence and development of AD/AA is not clear.The deposition of ECM can be as the last line of defence to save dilatation and rupture after aortic dissection/aneurysm.Promotion of ECM in the adventitia has been considered as a promising therapeutic strategy for the treatment of aortic dissection/aneurysm.Therefore,a comprehensive understanding of ECM production of fibroblasts and the identification of macrophage polarization are essential in the prevention and treatment of aortic dissection/ aneurysm.LCN2 can secrete collagen to promote the aldosterone/ mineralocorticoid receptor mediated vascular fibrosis and promote macrophage polarization to anti-inflammatory phenotypein infectious diseases.However,the specific role of LCN2 in the deposition of ECM and macrophage polarization of AD/AA is still unknown.We hypothesized that aortic dissection/ aneurysm stimulated the expression of LCN2 in the vessel tissue.LCN2 regμlated the expression of genes involved in ECM and inflammation,and participates in the process of vessel injury、expansion and even rupture.Duringaortic dissection/ aneurysm,LCN2 deficiency inhibited the secretion of molecules related to ECM and promoted the infiltration of macrophages to inflammatory properties,and ultimately affected the vessel injury and repair of the pathological process.In this study,we used LCN2 gene knock mice as research objects,and focused on vessel injury and repair triggered by aortic dissection/ aneurysm.We explored the core event,ECM secreted by fibroblast and macrophage phenotype switch,in the purpose of elucidating pathogenesis of vessel injury and repair caused by aortic dissection/aneurysm.Our result provides a new target and early intervention for vessel remodeling and aortic dissection/ aneurysm induced vessel injury and repair.ObjectivesWe made the mouse model of aortic dissection/ aneurysm in WT and LCN2 KO mice by drink water involved BAPN and angiotensin II perfusion and then investigated vessel injury and repair after aortic dissection/aneurysm.We tested LCN2 expression after aortic dissection/aneurysm.We explored the destructive effect onthe development of aortic dissection/aneurysm caused by LCN2 deletion using LCN2 gene knockout mice and the protective effect by recombinant protein of LCN2.Furthermore,we used RNA seq、pathological staining and qRT-PCR method to confirm the effect of ECM deposition in the development of aortic dissection/ aneurysm.Moreover,we attempted to clarify the role of LCN2 in macrophage phenotype switch by means of trypsin perfusion model.We used pathological staining and qRT-PCR method to observe the expression of macrophage-related inflammatory molecules in impaired vessel.All this is to confirm the effect of LCN2 in macrophage phenotype switchin the development of aortic dissection/aneurysm.Part 1 Methods 1.Study groups: 77 Wild Type C57/BL6 mice male;41 LCN2 KO mice male,were randomly divided into groups.(1)BAPN/ A II model: 17 Wild Type C57/BL6 mice male;17 LCN2 KO mice male were randomly divided into 4 groups.WT mice group(ordinary water group,n=6);LCN2 KO mice group(ordinary water group,n=6);WT mice group(BAPN group,n=11);LCN2 KO mice group(BAPN group,n=11);(2)A II model: 12 Wild Type C57/BL6 mice male;12 LCN2 KO mice malewere randomly divided into 4 groups.WT mice group(NS group,n=6);LCN2 KO mice group(NS group,n=6);WT mice group(A II group,n=6);LCN2 KO mice group(A II group,n=6);(3)Reconbinant LCN2 in vivo: 12 LCN2 KO mice male were randomly divided into 2 groups,LCN2 KO mice group(NS group,n=6);LCN2 KO mice group(r LCN2 group,n=6).2.The model of aortic dissection/ aneurysm:(1)Three-week-old male mice on a C57B/L6 background were fed a regular dietand 3-aminopropionitrile fumarate salt(BAPN,Sigma-Aldrich)for 4 weeks.Freshly BAPN solution at a concentration of 1 g/kg/day was prepared everyday according to the body weight Allmice died before expected end time of this experiment were autopsied immediately;(2)To induce AAA formation in mice,malemice at the age of 10 to 12 weeks were infused with Ang II as previously described.Alzet model 1007 D,1002,or 1004 osmoticminipumps(DURECT Corp.)implanted subcutaneously were used todeliver Ang II(1000 ng/kg/min,Sigma-Aldrich)or vehicle(normalsaline)for 42 days,depending on the experimental design described in Results.Mice were anesthetized with ketamine(1.5mg/kg)and xylazine(0.3 mg/kg),which was followed by minipumpimplantation.To relieve pain from surgery,postoperative analgesiawas administered using intraperitoneal injection of buprenorphine(0.05–0.10 mg/kg administered every 8 to 12 hours)on the day ofsurgery and on postoperative days 1 to 3.3.The expression of LCN2 after aortic dissection/ aneurysm: Real-time PCR,ELISA and Immunohisochemical.4.Vessel diameter and elastic fibers grading: to test the diameter and elasticfibers by VVG staining.5.ECM deposition: to test the collagen deposition by Sirius Red.6.ECM-relatedmolecules:Real-time PCR and Immunohisochemical..7.The effect of LCN2 on fibroblasts: Mice fibroblasts were extracted and cultured for 3-4generations and were stimulated by recombinant LCN2,extracted cell RNA and protein,and the expression of ECM-related factors were detected by PCR Real-Time and Western Blot.8.Statistics: All data are presented as the mean ± standard error of the mean.Two group comparisons were analysed by unpaired t-test.Multi-group comparisons were performed using one-or two-way ANOVA.A P-value of<0.05 was considered statistically significant.Results 1.aortic dissection/aneurysm increases LCN2 at both mRNA and protein levels and in the WT vessel and serum.2.LCN2 was mainly produced by neutrophils/macrophages and fibroblasts after aortic dissection/ aneurysm.3.Compared with WT mice,LCN2 knockout significantly increased the post-aortic dissection/ aneurysm survival rate.4.Compared with WT mice,LCN2 knockout significantlyincreased the post-aortic dissection/ aneurysm vessel diameter and elastic fibers disorder.5.Compared with WT mice,LCN2 knockout significantly decreased the post-aortic dissection/ aneurysm ECM deposition.6.We extracted RNA from the vessel tissue of wild type and LCN2 knockout mouse after aortic dissection/aneurysm.RNA-sequencing was used to find compared with WT vessel,differentially expressed gene of thetranscriptome.These differentially expressed genes were grouped using Gene Ontology(GO)analysis,which identified ECM-related genes as the most significantly regulated gene function groups in LCN2-KO vessel.7.The ECM-relatedmolecules were tested in the WT and KO mice after aortic dissection/ aneurysm,and found that LCN2-KO vessels down-regulated several ‘pro-ECM’ genes.8.Compared with WT mice,LCN2 knockout significantly decreased the post-aortic dissection/ aneurysm ECM deposition by Sirius Red.9.Compared with WT mice,LCN2 knockout significantly decreased the post-aortic dissection/ aneurysm Collagen I/ Tnc expression by Immunohisochemical.10.We isolated the fibroblasts of mice aorta and the cells were stimulated by rmLCN2.qRT–PCR showed that rm LCN2 treatment promoted the expression of Collagen I/ Tnc in fibroblasts.11.LCN2 knockout mice that underwent aortic dissection/ aneurysm induction were administered arecombinant LCN2 or isotype control(2 μg/mouse.day)via the enterocoelia injection.The injections were on the first day of BAPN drinking and every two days afterwards.Recombinant LCN2 administration markedly attenuated the aortic dissection/ aneurysm-induced mortality,incidence,vessel diameter and increased the deposition of ECM.Part 2 Methods 1.Study groups: Using trypsin perfusion of aorta abdominalis to establish abdominal aortic aneurysm(AAA)model.The mice were randomly divided into the following groups(n=10),WT mice group(trypsin perfusion group,n=10);LCN2 KO mice group(trypsin perfusion group,n=10).2.Bone marrow monocytes culture: Using lymphocyte separation medium to culture monocytes.3.The model of aortic dissection/ aneurysm: Eight-to 12-week-old male mice were injected with intraperitonealketamine solution,perfused with elastase,and harvested as previously described,and the procedure was performed by Johnstonet al.The aortas(or aneurysms,when present)were harvested,and either 1)snap-frozen in liquid nitrogen for analyses by real-timepolymerase chain reaction(PCR)or protein extraction,or 2)incubatedovernight for histology or immunohistochemistry.4.Detection inflammatory factors of vessel and macrophage: Using real-time PCR to detect inflammatory factors and immunohistochemical staining to observe macrophage infiltration.5.Detection inflammatory factors level in serum and cell supertant: Using each ELISAs testing methods.6.Statistical analysis: See part 1.Results: 1.LCN2 promoted macrophage polarization into pro-inflammatory properties in aorta abdominalis by pathological staining.2.Compared with WT-type,LPS induced more M1-type biomarkers(IL-1β,IL-6,TNF-a,MCP-1)expression and less M2-type biomarkers(IL-10)by real-time PCR and ELISA.3.Culture bone marrow macrophages in vitro,recombinant LCN2 can inhibit M1-type biomarkers(IL-1β,IL-6,TNF-a,MCP-1)expression and pomote M2-type biomarkers(IL-10)by real-time PCR and ELISA.Conclusions 1.LCN2 expression was increased and was mainly produced by neutrophils/ macrophages and fibroblasts in aortic dissection/ aneurysm.2.Compared with wild type mice,the mortality,vessel diameter and elastic fibers disorder of LCN2 knockout mice were worse in the model of aortic dissection/ aneurysm,indicating that LCN2 participated in the pathological process of aortic dissection/ aneurysm.3.Vessel tissue RNA transcriptome sequencing and qRT-PCR analysis showed that compared with wild type mice LCN2 gene knockout mice showed an ECM-deposition properties.4.LCN2 recombinant protein promoted the deposition of ECM during the development of aortic dissection/ aneurysm.5.LCN2 deficiency promoted inflammatory response by increasing M1 macrophage-related cytokines production into the vessel.Taken together,this study showed that aortic dissection/ aneurysm markedly up-regulated LCN2 expression in fibroblasts.LCN2 deficiency induced by genetic or approaches accelerated the development of aortic dissection/ aneurysm and increased the mortality,vessel diameter and elastic fibers disorder and decreased the ECM deposition.LCN2 recombinant protein can improve the ECM deposition.ALL this is by up-regulating ECM-related molecules and down-regulating macrophage switching into M1-type.Thus,LCN2 is a potential therapeutic target for aortic dissection/ aneurysm.This study demonstrated for the first time that aortic dissection/ aneurysm activates LCN2 expression in neutrophils/ macrophages and fibroblasts,regμlating the properties of macrophage polarization,and promoting ECM secretion in fibroblasts,eventually leading to the development of aortic dissection/ aneurysm.This study provides novel evidence that the regulation of LCN2 in vivo and in vitro may become a new therapeutic target for target organ damage caused by aortic dissection/ aneurysm.