The Role And Molecular Mechanism Of Chemokine Receptor CXCR2 Regulates Angiotensin Ⅱ-induced Myocardial Fibrosis And Remodeling

BackgroundMyocardial fibrosis is a hallmark of cardiac remodeling.Inflammation plays an important role in initiating cardiac remodeling and heart failure(HF).Chemokine-receptor signaling is a critical regulator that mediates the mobilization and migration of immune cells,includingmonocytes/macrophages and neutrophils,which play a fundamental role in various inflammatory-related diseases.However,the role of chemokine receptor CXCR2 in cardiac fibrosis and remodeling remains unknown.AimsTo elucidate the functional role of chemokine receptor CXCR2 in angiotensin II(Ang II)-induced myocardial fibrosis andremodeling in mice and the underlying molecular mechanism.To identify whether CXCR2 could be potential therapeutic target for the treatment of these diseases.Methods:1.AnimalsWild-type(WT)mice(C57BL/6J,male),CXCR2 knockout mice(CXCR2 KO,B6.129S2(C)-Cxcr2tm1Mwm/J),WT mice were treated with a CXCR2-specific antagonist SB265610(2mg/kg/day)and bone marrow chimeramice at 8-10 weeks of age were used to establish the cardiac remodeling model by subcutaneous infusion of angiotensin II(Ang II)for 1-14 days at a dose of 1000 ng/kg/min.2.Chemotaxis and co-culture assays in vitroBone marrow-derived macrophages were isolated from WT or CXCR2 KO mice,and detect the effect of chemokine CXCL1 on macrophage migration and activation was observed by Transwell migration assay.WT or CXCR2 KO macrophages were and co-cultured with WT neonatal rat fibroblasts(CFs)to examine the effect of macrophages on fibroblast differentiation.3.Measurement of blood pressure and echocardiographyThe diastolic and systolic blood pressure of male mice was detected by the tail-cuff system.Transthoracic echocardiography was performed with a high-resolution micro imaging system(Vevo1100;VisualSonics Inc.).To evaluate the precise cardiac function,these adjustments of both heart rate and body temperature in each group were made.M-mode cardiac images of the left ventricle(LV)from the short-axis view were used to assess end-diastolic LVPW thickness,LVAW thickness,LVIDd and LVIDs.4.Analysis of gene and protein expression,immune cells and tissuesThe gene expression of chemokines and inflammatory cytokines were examined by microarray analysis.The inflammatory cells in the heartwere analyzed by flow cytometry.Hematoxylin and eosin(H&E)staining was used to detect infiltration of myocardial inflammatory cells..Masson’s trichrome staining was conducted to analyze tissue interstitial and perivascular fibrosis.Immunohistochemistry was performed to analyze myofibroblast differentiation.The expression levels of gene markers and signaling proteins related to fibrosis,and inflammation were analyzed by quantitative real-time PCR or Western blot analysis.5.Human study populationsBlood samples and medical history were collected from 30 control subjects and 32patients with hypertensive HF.Flow cytometry analysis was performed to analyze subpopulations of immune cells in the blood.ELISA assay was used to evaluate the levels of chemokines in serum.6.Statistical analysisAll data are expressed as mean±SD.If data were normally distributed,the student t test was used to determine the significant difference between two groups.If the data were not normally distributed,the Mann-Whitney test was used.More than three groups of data were analyzed by ANOVA.Multivariable logistic regression models were used to evaluate the association of human blood CXCR2~+cells and CXCL1 levels with HF.Results1.Microarray assay showed that CXCL1 was the most markedly upregulated in Ang II-infused hearts compared with control hearts at day 1.After 1,3,7 and 14 days of Ang II infusion,the protein level of CXCR2 and CXCR2~+immune cells were time-dependently increased in Ang II-infused hearts compared with controls.2.Compared with Ang II-treated WT mice,CXCR2 knockout significantly reduced Ang II-induced the elevation of systolic blood pressure,cardiac contractile dysfunction,myocardial fibrosis and inflammatory cell infiltration.Moreover,Ang II-induced upregulation of CD45~+immunce cells including monocytes/macrophages and neutrophils was markedly reduced in CXCR2 KO mice.In addition,Ang II-induced activation of fibrotic and inflammatory signaling pathways including TGF-β/Smad2/3and NF-κB)was also inhibited in CXCR2 KOm mice.3.Therapeutic administration of CXCR2 inhibitor SB265610 blunts Ang II-induced hypertension,cardiac contractile dysfunction,fibrosis and inflammatory cell infiltration compared with vehicle-treated WT mice after Ang II infusion.4.Wt mice transplanted with CXCR2 KO bone marrow(BM)cells had a lower systolic blood pressure,and improvement of cardiac dysfunction,fibrosis and inflammatory cell infiltration compared with WT transplanted with WT BM cells.5.In vitro Trans-well migration assay showed that CXCL1 treatment significantly enhanced migration and activation of NF-κB in WT macrophages but not CXCR2KOcells.Co-culture of CXCR2 KO macrophages with fibroblasts(CFs)showed a significant reduction in myofibroblast differentiation and activation of TGF-β/p-Smad2/3 compared with WT macrophage.6.Circulating CXCR2~+monocytes and CXCL1 level were significantly higher in HF patients compared with control subjects,and there was a statistically significant association between the number of CXCR2~+cells or CXCL1 level with HF occurrence.ConclusionsThis study demonstrates a critical role of CXCR2~+monocytes/macrophages in Ang II-induced cardiac fibrosis and remodeling.Mechanistically,Ang II induces the expression of myocardial CXCL1,which promotes the infiltration of BM derived-CXCR2~+monocytes/macrophages into the heart and production of inflammatory factors that stimulates cardiac fibroblast differentiation thereby leading to cardiac fibrosis and remodeling.Thus,selective inhibition of CXCR2 represents an attractive new strategy for treating hypertensive HF.