Genetic Deficiency Of Phactr1 Promotes Atherosclerosis Development Via Facilitating M1 Macrophage Polarization And Foam Cell Formation

Background:Genetic variants in phosphatase actin regulator-1(Phactr1)is reported to be associated with arteriosclerotic cardiovascular disease.However,the function of Phactr1in atherosclerosis remains unclear.At present,there are many studies on atherosclerosis and Phactr1,but the function of Phactr1 in atherosclerosis is still unclear.Clarifying the mechanism and effects of Phactr1 will promote our further understanding of atherosclerosis and may discover new therapeutic targets.Therefore,in-depth study of the clinical value of its mechanism is a significant subject.Objective:For better understand the role of Phactr1 in plaque stabilization and explore the potential mechanism of Phactr1 in atherosclerosis,we investigated ox-LDL-induced macrophage polarization,inflammation response and foam cell formation in Phactr1deficiency mice.Methods:Arteriosclerosis models were induced with Apoe knockout mice(Apoe^-/-),Phactr1 global knockout mice(Phactr1^-/-)mice,Phactr1^-/-Apoe^-/-mice(DKO)and Myeloid cells specific KLF4 overexpression with Phactr1^-/-Apoe^-/-mice(KLF4^?MC/Phactr1^-/-Apoe^-/-).Eight-week-old male DKO and Apoe^-/-mice were fed on high-fat diet(16%fat and 1.3%cholestero)for 16 weeks.Then we compare the plasma levels of triglyceride(TG),total cholesterol(TC),low density lipoprotein-cholesterol(LDL-C)and high density lipoprotein-cholesterol(HDL-C).After staining the mouse vascular tissue,we evaluate the lipid content in the cells,observe the morphology of the plaque,evaluate the plaque burden,quantificate the necrotic core and fibrous cap thickness.Phactr1 m RNA expression in mouse macrophages was detected by quantitative RT-PCR.Evaluate the degree of inflammation(TNF-?and IL-6)in mice by quantitative RT-PCR and ELISA analysis.Then BM transplantation strategy was used to evaluate the effect of hematopoietic Phactr1 deficiency on arteriosclerosis.The expressions of TNF-?,IL-6,Arg1,CD206 and KLF4 were detected by quantitative RT-PCR method.Representative immunoblotting and quantitative analysis showed the protein expression of LOX-1,SR-A,CD36,ABCA1 and ABCG1and KLF4.Representative immunoblotting and quantitative analysis showed the phosphorylation of p38MAPK and CREB.The luciferase activity of CREB responsive element was detected by Dual-Luciferase Reporter Assay system.Results:(1)Phactr1 fluorescence intensity was stronger in advanced atherosclerotic plaques and predominantly expressed in Mac2 positive cells.We also found a marked increase in Phactr1expression in circulating monocytes from Apoe^-/-mice with advanced lesions,whereas the expression of Phactr1 was virtually low in monocytes from Apoe^-/-mice with normal chow diet.(P<0.05)(2)After consumption of high-fat diet for 16 weeks,there was no difference in body weight and food intake between Phactr1^+/+and Phactr1^-/-mice.While Apoe^-/-mice on high-fat diet had high plasma levels of TC,TG and LDL-C,Phactr1^-/-Apoe^-/-mice retained comparable plasma levels of the lipid profile to Apoe^-/-mice.(P<0.05)(3)Oil Red O staining on aortic sinus cross-sections showed that Phactr1^-/-Apoe^-/-mice had stronger atherosclerotic plaques on the aortic sinus than those in Apoe^-/-mice.Moreover,Phactr1^-/-Apoe^-/-mice displayed larger area of necrotic core,a thinner fibrous cap and more accumulation of macrophages..Phactr1 deficiency led to robust decreases in collagen accumulation within atherosclerotic plaques on the aortic sinus in Apoe^-/-mice.(P<0.05)(4)Phactr1^-/-Apoe^-/-mice showed more abundance of TNF-?and IL-6 in the aorta.Plasma levels of TNF-?and IL-6 are also elevated in Phactr1^-/-Apoe^-/-mice.(P<0.05)(5)Apoe^-/-mice receiving Phactr1^-/-Apoe^-/-BM cells developed more aggressive plaques as compared with those receiving Apoe^-/-BM cells.Immunofluorescent staining showed an increase in macrophage burden in Apoe^-/-mice receiving Phactr1^-/-Apoe^-/-BM cells.(P<0.05)(6)The expression of TNF-?and IL-6 was significantly increased and plasma levels of TNF-?and IL-6 were elevated in Apoe^-/-mice receiving Phactr1^-/-Apoe^-/-BM cells as well.(P<0.05)(7)The expression of Phactr1 was upregulated in the presence of ox-LDL and reached the peak at 12-hour treatment The.ox-LDL induced Phactr1 expression,at least in part,through Lox-1/NF-?B pathway in macrophages.(8)Loss of Phactr1 prominently promoted the expression of M1 macrophage markers such as TNF-?and IL-6 in BMDMs in the presence of LPS and IFN-?,while the expression of M2 phenotype markers such as Arg1 and CD206 was dramatically decreased in Phactr1^-/-BMDMs induced by IL-4.An enhanced proportion of M1 macrophages in Phactr1^-/-BMDMs induced by LPS and IFN-?,and a suppressed proportion of M2 macrophages.(P<0.05)(9)The higher levels of proatherogenic cytokines,including TNF-?,IL-6,RANTES,CCL2,IL-1?,IFN-?and M-CSF,in Phactr1^-/-BMDMs as compared to those in Phactr1^+/+BMDMs.The silence of Phactr1 in BMDMs enhanced the expression of these cytokines induced by ox-LDL.(P<0.05)(10)Loss of Phactr1 induced a significant increase in intracellular lipid content stained by Oil Red O when compared to that in Phactr1^+/+BMDMs.The intracellular cholesterol content in Phactr1^-/-BMDMs was substantially higher than that in Phactr1^+/+BMDMs.(P<0.05)(11)Compared with Phactr1^+/+BMDMs,Phactr1 knockout led to increase levels of LOX-1,SR-A and CD36 in BMDMs,whereas Phactr1 knockout had no marked effect on the expression of ABCA1 and ABCG1,the hallmark of cholesterol efflux.(P<0.05)(12)Phactr1 and CREB appeared to physically bind together in BMDMs.The direct interaction between Phactr1 and CREB was further confirmed by co-transfection of Flag-Phactr1 and HA-CREB and co-immunoprecipitation in HEK293T cells.Functionally,overexpression of Phactr1 augmented the activity of CREB responsive element measured by Luciferase assay.(P<0.05)(13)The expression of KLF4 in Phactr1^-/-BMDMs is decrease.Ch IP-q PCR conducted at the promoter of KLF4 using CREB antibody pulldown demonstrated that depletion of Phactr1 blocked the abundance of CREB at the promoter of KLF4(CREB binding motif within Fragment 1)in BMDMs.Dual Luciferase assay further confirmed that the transcription activity of KLF4 was substantially strengthen after Phactr1 overexpression in HEK293T cells.(P<0.05)(14)Overexpression of KLF4 via transduction of KLF4 adenovirus(Ad-KLF4)alleviated the expression of M1 markers induced by loss of Phactr1 but restored the expression of M2 markers.(P<0.05)(15)Overexpression of KLF4 mitigated the increased expression of scavenger receptors,Lox-1,CD36 and SR-A in Phactr1-deficient BMDMs.(P<0.05)(16)The content of intracellular cholesterol in Phactr1^-/-BMDMs was significantly reduced by KLF4 upregulation.Results of Oil Red O staining indicated that transduction of Ad-KLF4 led to a decrease in foam cell formation induced by Phactr1 deficiency).Measurements of Oil Red O-stained areas displayed that Phactr1^-/-Apoe^-/-mice receiving KLF4^?MC/Phactr1^-/-Apoe^-/-BM cells had decreased plaque areas as compared with those in Phactr1^-/-Apoe^-/-mice receiving Phactr1^-/-Apoe^-/-BM cells.(P<0.05)Conclusions:(1)Phactr1 expression is increased in macrophages during murine atherogenesis.(2)Phactr1 inhibits M1 proinflammatory phenotype.Loss of Phactr1 accelerates the progression toward a vulnerable plaque phenotype.Phactr1 can postpone the development of atherosclerosis.(3)Phactr1 is a critical mediator of macrophage-mediated inflammation,macrophage polarization and foam cell formation.(4)KLF4 overexpression is able to partly counteract the aggravated effect of Phactr1deficiency on atherosclerotic plaques.