The Effects And Mechanism Of FGFR1 On Inflammation And Cell Foamation In Atherosclerotic Progress

Atherosclerosis(AS)is a lipid driven chronic inflammatory disease.It is also an important cardiovascular disease,caused by lipid metabolism disorders.It is the main cause of morbidity and mortality resulting from cardiovascular diseases.Atherosclerosis is a complex inflammatory disease process,the main feature of which is the deposition of lipids in certain parts of the intima,and the proliferation of smooth muscle cells and fiber matrix.Gradually this causes the formation of atherosclerotic plaque(s).Abundant evidence have not only revealed the crital role of vascular wall cells in the progression of atherosclerotic plaque,but also emphasized the indispensable effect of peripheral circulating cells,such as macrophage,lymphocyte and eosnophils.Type I fibroblast growth factor receptor(FGFR1)belongs to the FGFR family and is activated through binding with fibroblast growth factors(FGFs).Subsequently,the dimerization and phosphorylation of FGFR1 triger downstream FGF receptor substrate 2(FRS2)and phospholipase C-y(PLCy)signal transduction pathway and play a role in cellular differentiation,proliferation and survival.FGFR1 can not only interact with fibroblast growth factor(FGF),but also with other molecules,including heparin sulfate proteoglycan(HSPGs),heparin and neural cell adhesion molecules.Clinical studies have found that multiple cancers are associated with FGFR1 overexpression or mutation activation in tumor tissues to promote tumor angiogenesis and tumor development.Therefore,FGFR1 is also widely considered as an important target for anti-tumor drugs.Now,three FGFR1 small molecule inhibitors have entered the phase 2 clinical trial.In recent years,it has been reported that FGFR1 has been involved in non-tumor diseases.At present,the role of FGFR1 in cardiovascular disease is rarely reported.Only two reports found,1)elevated FGFR1 gene expression in mouse atherosclerotic plaque;2)oral FGFR1 inhibitor SU5402 can delay the process of atherosclerosis in apolipoprotein E knockout(ApoE-/-)mice.It’s reported that FGFR1 may be involved in the regulation of atherosclerosis,but the authors did not clarify the regulation process and mechanism of FGFR1.Therefore,the role of FGFR1 in the development of atherosclerosis is not clear.In this study,we used ApoE-/-mice and FGFR1 small molecule inhibitor AZD4547 to study the role of FGFR1 in atherosclerosis.A possible signal transduction pathway is also discussed.The following three parts give a brief account of the methods,results and conclusions of this studyPart 1:Abnormal expression of FGFR1 in atherosclerosisObjective:To observe the expression and activation of FGFR1 in atherosclerosis.Methods:High fat diet(HFD)induced atherosclerosis in ApoE-/-mice,and the mice were sacrificed after 16 weeks.The protein levels of p-FGFR and FGFR1 in the aorta were detected by Western Blot and immunohistochemical;The FGFR1 localized at the atherosclerotic plaques in aortas of the ApoE-/-mice on LFD or HFD by colocalization immunofluorescence staining:CD31/CD68/SMA and FGFR1 using the frozen section.With density gradient centrifugation,peripheral blood mononuclear cells were obtained from atherosclerosis patients group(Atherosclerosis Patients)and normal group(normal subjects),and mRNA and total protein were extracted.Real-time fluorescence quantitative PCR(RT-PCR)was used for detecting the mRNA levels of FGFR1 and Western Blot was used for detecting the protein levels of FGFR1Results:FGFR1 activation in the aorta of ApoE-/-mice fed with HFD increased significantly,and colocalization immunofluorescence staining showed that the rising rate of the FGFR1 express of macrophages in atherosclerotic plaques of high-fat fed ApoE-/-mice was significantly higher than that of endothelial cells and smooth muscle cells.In the collection of clinical specimens,the results showed that the protein and mRNA levels of FGFR1 in the mononuclear cells of the atherosclero sis patients were significantly increased.Conclusion:FGFR1 was significantly activated in atherosclerosis,and we further found that the expression of macrophage FGFR1 is associated with atherosclerosis.Part 2:FGFR1 inhibitor effectively attenuates the high-fat-diet induced atherosclerosis in ApoE-/-miceObjective:To observe the effect of FGFR1 inhibitor on the development of high-fat-diet(HFD)induced atherosclerosis in ApoE-/-mice.Methods:HFD induced atherosclerosis in ApoE-/-mice,and HFD-induced ApoE-/-mice were given FGFR1 inhibitor AZD4547 at the doses of 10mg/kg/day or 20mg/kg/day(once in other day)with gavage treatment.Then the mice were killed after 16 weeks and the mRNA and the whole aorta were extracted.Oil red O staining was used to detect the aortic atherosclerosis development and the degree of lipid accumulation;Immunohistochemistry and RT-PCR were used to detect the protein and mRNA levels of CD36 in the artery;RT-PCR was used to detect mRNA levels of the inflammatory factors(IL-6 and TNF-a)in the artery;Immunohistochemical was used to detect the expression of inflammatory factors and infiltration of macrophages in mice.The degree of oxidative stress in the arteries of mice was observed by DHE staining and 3-NT immunohistochemical staining.Results:FGFR1 inhibitor AZD4547 attenuates atherosclerotic development and lipid accumulation in HFD-induced ApoE-/-mice,and inhibits the expression of CD36 in the aorta;FGFR1 inhibitor AZD4547 can reduce the aortic inflammatory factor levels and increased infiltration of macrophage in HFD-induced ApoE-/-mice;FGFR1 inhibitor AZD4547 can significantly relieve the aortic oxidative stress in HFD-induced ApoE-/-mice.Conclusion:FGFR1 inhibitor AZD4547 attenuates the development of atherosclerosis by reducing inflammation and relieving the oxidation reaction in ApoE-/-mice.It shows that FGFR1 can mediate the development and progression of inflammation and oxidative stress in atherosclerosis.Part 3:The study of the role and mechanism of FGFR1 in macrophagesObjective:To explore the role and mechanism of FGFR1 in macrophagesMethods:The primary macrophages(MPMs)were extracted and cultured.Pretreatment with FGFR1 inhibitor AZD454,MPMs were stimulated by ox-LDL,and 15 minutes later,MPMs were lysed and the total proteins were extracted,Western Blot was used to detect the expression of p-FGFRl and FGFR1.Pretreatment with siLoxl,MPMs were stimulated by ox-LDL,and 15 minutes later,MPMs were lysed and the total protein were extracted,Western Blot was used to detect the expression of p-FGFRl and FGFR1.Pretreatment with FGFR1 inhibitor AZD4547,MPMs were stimulated by ox-LDL for 24 hours and oil red O staining was used to detect cellular lipid accumulation,immunofluorescence,flow cytometry and Western Blot were used to detect the expression of CD36 in macrophages.Silencing FRS2 and PLCy or pretreating with agonists and inhibitors of peroxisome proliferator-activated receptor y(PPARy),macrophages were stimulated by ox-LDL for 24 hours,lipid uptake was determined by immunofluorescence.After silenc:ing FRS2 and PLCy,macrophages were stimulated by ox-LDL,the mRNA level and secretion of inflammatory factors(IL-6 and TNF-a)were detected;DHE and DCF-DA staining were used to detect oxidative stress reaction of macrophages.Results:Ox-LDL could induce FGFR1 phosphorylation and lipid accumulation in macrophages.After pretreatment with FGFR1 inhibitor AZD4547,the phosphorylation of FGFR1 and lipid accumulation in macrophages decreased.Silencing Lox1 inhibited ox-LDL-stimulated FGFR1 activation;AZD4547 reduced ox-LDL-induced by lipid accumulation of macrophage,and AZD4547 and silencing CD36 by siRNA can effectively reduce ox-LDL-induced lipid accumulation.Inhibiting the expression of FRS2 and PLCy by siRNA and pretreating with agonist and inhibitor of PPARγ,the results showed that inhibition of PPARγ could reduce the lipid accumulation of macrophages.Inhibiting the expression of FRS2,PLCγ and PPARγ by siRNA and pretreating with agonist and inhibitor of PPARγ,the results showed that inhibition of PLC gamma can effectively reduce ox-LDL-induced inflammation and oxidative stress in macrophage.Conclusion:FGFR1 inhibitor AZD4547 can reduce inflammation,oxidation reaction and lipid accumulation in ox-LDL stimulated macrophage.It indicates that FGFR1 can play an important role in the pathogenesis of atherosclerosis,which mediated inflammation by PLCγ,and lipid accumulation by PPARγ/CD36.