The Role And Mechanism Of C-Fos In The Formation Of Vascular Smooth Muscle Cell-derived Foam Cell And Atherosclerotic Lesion

Objective:The morbidity and mortality of cardiovascular diseases caused by atherosclerosis(AS)remain high,which is a serious threat to human health.As an important pathologicalmarker for the formation of atherosclerotic lesion,foam cells are widely present in all stages of the lesions and play a crucial role in the initiation and development of AS.Although vascular smooth muscle cell(VSMC)is a main source of foam cells,the mechanisms of VSMC-derived foam cell formation are not yet completely understood.c-Fos has been shown to regulate lipid metabolism,and is associated with the accumulation of intracellular lipid droplets.However,few studies have focused on whether c-Fos is involved in the formation of VSMC-derived foam cells.Therefore,our study aims to explore the role and molecular mechanism of c-Fos in the formation of VSMC-derived foam cells and atherosclerotic lesion by establishing western type diet(WTD)-induced mouse models for AS and a VSMC-derived foam cell model induced by oxidized low-density lipoprotein(ox LDL).Methods:1.To establish AS models in Apo E^-/-and LDLR^-/-mice fed with WTD,real-time polymerase chain reaction(RT-PCR)and Western blot experiments were used to detect c-Fos expression in the aorta.The expression of c-Fos protein was determined by immunohistochemistry in the atherosclerotic lesions and by double immunofluorescence staining in VSMCs in the atherosclerotic lesions.After AS models in Apo E^-/-SM22?Cre^-c-Fos^flox/floxand Apo E^-/-SM22?Cre⁺c-Fos^flox/floxmice fed with WTD were established,the changes in blood lipid were analyzed by colorimetry,and Oil red O and hematoxylin-eosin staining were conducted to detect the lipidaccumulation and the areas in the atherosclerotic lesions.2.VSMCs were stimulated by different types of lipoproteins or ox LDL at different doses or for different time points,and then cholesteryl ester(CE)and free cholesterol(FC)quantitative assays,Filipin fluorescence staining and oil red O staining wereemployed to determine the lipid accumulation in VSMCs and the formation of VSMC-derived foam cells.The protein expression and phosphorylation levels of c-Fos protein were measured by Western blot.RNA interference technology was used to silence c-Fos expression and the technology of site-directed mutations ofphosphorylation sites technology was utilized to suppress the phosphorylation of c-Fos at Ser32 and Thr232,and then the lipid accumulation in VSMCs and the formation of VSMC-derived foam cells were determined.3.RT-PCR and Western blot experiments were conducted to measure the m RNA and protein expressions of CD36,lectin like oxidized low density lipoprotein receptor 1(LOX1)and scavenger receptor-A(SR-A)in ox LDL-induced VSMC-derived foam cells.LOX1 was knocked down by the specific si RNA,and then the lipidaccumulation in VSMCs and the formation of VSMC-derived foam cells were determined.Double immunofluorescence staining was utilized to observe the expression of LOX1 in VSMCs in the atherosclerotic lesions in Apo E^-/-mice.After the knockdown of c-Fos by the specific si RNA,the expressions of the m RNA and protein of LOX1 in ox LDL-induced VSMC-derived foam cells were detected by RT-PCR and Western blot experiments.Double immunofluorescence staining was used to observe the expressions of LOX1 in VSMCs in the atherosclerotic lesions in Apo E^-/-SM22?Cre^-c-Fos^flox/floxand Apo E^-/-SM22?Cre⁺c-Fos^flox/floxmice.The distribution of c-Fos in VSMCs was determined by immunofluorescence staining.The binding of c-Fos to the promoter region of LOX1 in VSMCs was detected by chromatin immunoprecipitation assay.Dual luciferase reporter gene assay was conducted to evaluate the transcriptional activity of c-Fos on LOX1.4.Mito SOX Red fluorescent probe was used to detect the content of mitochondrial reactive oxygen species(mt ROS)in VSMCs stimulated with ox LDL.After pretreated with Mito-TEMPO,VSMCs were stimulated with ox LDL,and then the lipid content in VSMCs and the formation of VSMC-derived foam cells were determined,Western blot was conducted to detect the protein expressions of c-Fos and LOX1,immunofluorescence staining was employed to determine the distribution c-Fos protein,and dual luciferase reporter gene assay was conducted to evaluate the transcriptional activity of c-Fos on LOX1.Results:1.The expression levels of c-Fos m RNA and protein were obviously increased in the aorta in Apo E^-/-and LDLR^-/-mice fed with WTD(P<0.001),and c-Fos protein was also significantly upregulated in the atherosclerotic lesions and VSMCs in the lesions in Apo E^-/-and LDLR^-/-mice fed with WTD.The blood lipid level was significantly ameliorated(P<0.05),and the lipid accumulation and the area in the atherosclerotic lesions were remarkably reduced in Apo E^-/-SM22?Cre⁺c-Fos^flox/floxmice fed withWTD(P<0.001).2.Ox LDL can induce lipid accumulation in VSMCs,and also upregulate the expression and phosphorylation levels of c-Fos protein in a dose-and time-dependent manner(P<0.05).After the silence of c-Fos or the suppression of the phosphorylation of c-Fos at Ser32 and Thr232,the role of ox LDL in the formation of VSMC-derived foam cells was significantly inhibited(P<0.05).3.The expression levels of LOX1 m RNA and protein were remarkably upregulated in ox LDL-induced VSMC-derived foam cells(P<0.001).The silence of LOX1 significantly inhibited ox LDL-induced formation of VSMC-derived foam cells(P<0.001).The expression of LOX1 protein in VSMCs was increased in theatherosclerotic lesions in Apo E^-/-mice.The suppression of c-Fos expression dramatically decreased the expressions of LOX1 m RNA and protein in ox LDL-induced VSMC-derived foam cells(P<0.001).The expression level of LOX1 protein in VSMCs in the atherosclerotic lesions in ApoE^-/-SM22?Cre⁺c-Fos^flox/floxmice was obviously reduced.Ox LDL promoted the increase of c-Fos protein in VSMC nuclei(P<0.05).The binding of c-Fos to LOX1promoter was significantly increased(P<0.001),and the transcriptional regulation of LOX1 by c-Fos was markedly enhanced(P<0.001).4.Ox LDL can obviously increase the content of mt ROS in VSMCs(P<0.01).After the elimination of mt ROS,the effect of ox LDL on the formation of VSMC-derived foam cells was dramatically suppressed(P<0.01),and the expression levels of c-Fos and LOX1 proteins were also obviously reduced(P<0.001),and c-Fos protein was significantly decreased in VSMC nuclei(P<0.001),and the transcriptional regulation of c-Fos on LOX1 was also obviously inhibited(P<0.001).Conclusion:c-Fos activated by mt ROS promotes the formation of VSMC-derived foam cells induced by ox LDL through regulating the transcription of LOX1,thereby facilitating the formation of atherosclerotic lesions.