Recombinant Irisin Inhibits The Formation Of Athersclerosis And Its Underlying Mechanisms

Background:Atherosclerosis,a chronic progressive vascular disease,is the primary cause of coronary artery disease,stroke,and peripheral vascular disease,remains a leading cause of morbidity and mortality worldwide.Various risk factors can induce atherosclerosis,the major are hypertension,hyperlipidemia,smoking,diabetes,obesity,genetic factor and so on.The pathophysiological mechanisms of atherosclerosis are rather complex and yet not well clarified,among which endothelial dysfunction and inflammation play a critical role.Endothelial cells(ECs)with its intercellular tight junctional complexes function as a selectively permeable barrier between circulating blood and surrounding tissues,therefore their integrity is critical for the maintenance of blood vessel homeostasis.Normal endothelial cells can regulate the tone and structure of vascular.It also regulate vascular inflammation through secrete anticoagulant and anti-platelet agent and fibrinolytic protein to control the adhesion and gathering of leukocytes.When exposed to various noxious stimuli,focal areas of the endothelial monolayer are activated to secrete adhesion molecules,causing the adhesion of leukocytes,followed by the development of vascular lesions in the blood vessels.Various risk factors can induce endothelial injury which contributes to the initiation and progression of atherosclerosis.Many studies have demonstrated that endothelial dysfunction is not only an initiator,but could also be an important factor in the progression of atherosclerosis.ECs with adequate survival and proliferation capacities are essential for the maintenance of endothelial integrity and repair of endothelial damage.Therefore,maintain endothelial integrity,improving endothelial function could be an important therapeutic target in the treatment of atherosclerotic cardiovascular diseases.Skeletal muscle has been identified as an endocrine organ in recent years.Cytokines secreting by skeletal muscle during or immediately after exercise,known as myokines,mediate some of the beneficial effect of exercise on metabolism and the cardiovascular system.Irisin,a newly discovered myokine,is mainly secreted by skeletal muscle after exercise.It has a potent effect on the browning of white adipose tissues by stimulating expression of thermogenic genes including uncoupling protein 1,thus increasing energy expenditure and regulating glucose homeostasis,closely associated with obesity and insulin resistance.Recently,a growing number of studies have shown that irisin improves endothelial function in patients with obesity,type 2 diabetes and cardiovascular disease.However,the effects of irisin on the formation of atherosclerosis and its underlying mechanisms are not fully understood.Objective:1.Through use the mouse atherosclerosis model to test the effect of irisin on atherosclerosis lesion formation.2.To investigate the effect of irisin on endothelial cells proliferation,inflammation,and apoptosis and its associated molecular mechanisms.Materials and methods:1.Expression and purification of human irisin.2.Establish two atherosclerosis animal models,apolipoprotein E-deficient mice fed on a high-cholesterol diet and a mouse carotid partial ligation model.Apolipoprotein E-deficient mice fed on a high-cholesterol diet:sixteen 8 weeks old apolipoprotein E-deficient mice were randomly divided into two groups,control(n = 8)and irisin-treatment(n = 8).In the irisin treatment group,the mice were treated daily with purified irisin at a dose of 0.5 ?g/g body weight by intraperitoneal(i.p.)injection for the last 8 weeks of a 12-week HCD feeding program.In the control group,the mice were given normal saline in the same manner.At the end of irisin treatment,aortas and aortic roots were taken from the mice and then fixed in 4%paraformaldehyde.Mouse carotid partial ligation model:Partial ligation of the left common carotid artery was carried out in ten 8 weeks old apolipoprotein E-deficient mice,after partial ligation,the mice were randomly divided into two groups,control(n = 5)and irisin-treatment(n = 5).In the irisin treatment group,the mice were treated daily with purified irisin at a dose of 0.5 ?g/g body weight by i.p.injection for 4 weeks.In the control group,the mice were given NS in the same manner.At the end of irisin treatment,the segments of the left carotid artery just proximal to the ligation were excised and then fixed in 4%paraformaldehyde.3.Oil-Red O staining to quantification the effect of irisin on atherosclerotic lesion formation in the aorta and aortic root;HE staining to quantification the effect of irisin on neointima formation in the carotid artery.4.CD31 and Ki67 labelled immunohistofluorescence staining to quantification the effect of irisin on endothelial cell proliferation.5.HUVEC culture.6.BrdU incorporation assay,CCK-8 assay,MTT assay to determine the effect of irisin on HUVEC proliferation.7.microRNA(miRNA)expression profile of HUVECs was assessed following stimulation with irisin using miRNA microarray analysis.8.RNA isolation from HUVECs and carotid arteries,qRT-PCR to measure the expression of related microRNA and mRNA.9.Western blot to measure the expression of DLK1,p-ERK,p-eNOS,and inflammation,apoptosis related protein in HUVECs after irisin treatment.10.Immunohistochemical staining to test the effect of irisin on lesional macrophage area.11.ELISA to test the effect of irisin on serum concentrations of inflammation factors in mice.12.TUNEL staining to detect the effect of irisin on endothelial cell apoptosis in partial ligated carotid arteries.13.Flow cytometry was used to detect the effect of irisin on HUVEC apoptosis.14.Using Lipofectamine 2000 transfected the microRNA126-5p(miR126-5p)inhibitor and the Dlkl siRNA to HUVECs.15.Fifty ?L F-127 pluronic gel containing 0.1 mg antagomir against miR126-5p was applied to the adventitia around ligated artery segments.16.Statistical analysis:All experiments were performed at least three times.Data are expressed as mean ± SEM.Statistical comparisons were made between two groups using Student t test and between multiple groups by ANOVA followed by Student-Newman-Keuls post hoc test.P values<0.05 were considered statistically significant.Results:1.Irisin inhibits atherosclerotic lesion formation:(1)Apo E-deficient mice were treated with irisin for the last 8 weeks of a 12-week HCD feeding program,Oil-Red O staining showed that irisin reduced atherosclerotic lesions on the surface of en face prepared aorta and cross sections of the aortic root.(2)Apo E-deficient mice were treated with or without irisin for 4 weeks after carotid artery partial ligation;HE staining showed that irisin inhibited neointima formation in partially ligated carotid artery.2.Irisin promotes vascular EC proliferation:Immunofluorescent staining showed that the number of Ki67-positive ECs in partially ligated carotid artery samples was increased after irisin treatment.3.Irisin promotes HUVEC proliferation through up-regulating the expression of miR126-5p:(1)BrdU incorporation assay showed that irisin can promote HUVEC proliferation.(2)CCK-8 assay showed that following irisin treatment,the HUVEC viability was 1.26-fold higher than in the control group.(3)miRNA microarray analysis and qRT-PCR showed that miR126-5p expression was significant up-regulated by irisin treatment in HUVECs.(4)Cellular miR126-5p was inhibited by the miR126-5p inhibitor,BrdU incorporation assay and CCK-8 assay showed that inhibition of miR126-5p reduced HUVEC proliferation activity induced by irisin treatment.4.Irisin induces miR126-5p expression through the ERK signaling pathway:(1)Western blot showed that phosphorylated ERK was significantly increased after treatment with irisin and was abolished by pretreatment with ERK inhibitor U0126.(2)qRT-PCR showed that irisin induced miR126-5p expression was down-regulated after pretreatment with ERK inhibitor U0126.(3)Western blot showed that the miR126-5p inhibitor did not affect irisin-induced ERK phosphorylation.5.Dlk1 is the target gene of miR126-5p in irisin-treated HUVECs:(1)qRT-PCR showed that Dlk1 was significantly decreased after irisin treatment,whereas ADAM9,MMP7,and SDF-1? were unaffected.(2)Western blot showed that Dlkl protein expression was significantly reduced in HUVECs treated with irisin.However,Dlk1 expression was restored by the miR126-5p inhibitor after the HUVECs were treated with irisin.(3)CCK-8 assay showed that silencing of Dlk1 restored the inhibitory effect of the miR126-5p inhibitor on irisin-induced HUVEC proliferation.6.Inhibition of miR126-5p abrogates irisin-mediated inhibition of neointima formation and pro-proliferation of ECs in apoE-deficient mice after carotid artery partial ligation.(1)qRT-PCR showed that the miR126-5p level in carotid arteries of apoE-deficient mice after treatment with irisin for 4 weeks was 2.78-fold higher than in the control group.Treatment of mice with the miR126-5p antagomir caused reduction of the irisin-induced miR126-5p expression in the carotid artery.(2)Apo E-deficient mice were treated with or without irisin for 4 weeks after carotid artery partial ligation,HE staining showed that the inhibitory effect of irisin on neointima formation was abrogated by miR126-5p antagomir treatment.(3)Immunofluorescent staining showed that blocking miR1 26-5p expression abrogated irisin-mediated increase of the Ki67-positive ECs number.7.Lesional macrophage area is decreased in irisin-treated mice:Immunohistochemistry staining showed that the CD68-positive macrophage content and CD3-positive T lymphocyte content in the lesions of irisin injected mice were significantly reduced.8.Irisin suppresses inflammatory genes expression in partial ligated carotid arteries:(1)qRT-PCR showed that IL-6,MCP-1,ICAM-1,and VCAM-1 mRNA expression in plaque lesions of the partial ligated carotid artery were significantly decreased in irisin-treated mice.(2)ELISA showed that the protein levels of serum IL-6,MCP-1,ICAM-1,and VCAM-1 were significantly decreased in irisin-treated mice.9.Irisin inhibits ox-LDL-induced endothelial inflammation by suppressing the ROS/p38 MAPK/NF-?B pathway.(1)qRT-PCR showed that irisin significantly suppressed ox-LDL-induced up-regulation of IL-6,MCP-1,ICAM-1,and VCAM-1 mRNA expression in HUVECs.(2)Western blot showed that irisin treatment significantly suppressed the ox-LDL-induced activation of phosphorylated p38 MAPK.(3)Western blot showed that irisin also decreased both the phosphorylated and total NF-?B p65 expression,which was activated by ox-LDL.The ratio of phosphorylated-over total-NF-?B p65 was significantly decreased after irisin treatment,compared with the ox-LDL treated group.(4)Western blot showed that irisin treatment significantly decreased the expression of NF-?B p65 in the nucleus and increased its expression in the cytosol compared with the ox-LDL treated group,suppressed ox-LDL induced nuclear translocation of NF-?B as the percentage of NF-?B p65 in the nucleus was decreased.(5)DCFH-DA labeling showed that irisin significantly reduced ox-LDL induced intracellular ROS accumulation in HUVECs.10.Irisin inhibits cell apoptosis in partial ligated carotid arteries:TUNEL staining showed that irisin treatment significantly decreased the cell apoptotic rate in atherosclerotic lesions.11.Irisin inhibits ox-LDL-induced apoptosis in HUVECs:Flow cytometry results demonstrated that ox-LDL-induced cell apoptosis was effectively attenuated after being exposed irisin.12.Irisin up-regulates Bcl-2 and down-regulates Bax and caspase-3 expression:Western blot showed that irisin pretreatment increased Bcl-2 and decreased Bax and caspase-3 expression.Conclusion:1.Irisin treatment significantly reduced the severity of aortic atherosclerosis in apolipoprotein E-deficient mice fed on a high-cholesterol diet and suppressed carotid neointima formation in a carotid partial ligation model.2.Irisin can promote endothelial cell proliferation through up-regulating miR126-5p both in vivo and in vitro.3.Irisin induced miR126-5p through the ERK signaling pathway.Furthermore,irisin promoted HUVEC proliferation through miR126-5p by suppressing Dlkl.4.Irisin decreases inflammatory cell content and suppresses inflammatory genes IL-6,MCP-1,ICAM-1,and VCAM-1expression in partial ligated carotid arteries.5.Irisin inhibits ox-LDL-induced endothelial inflammation by suppressing the ROS/p38 MAPK/NF-?B pathway.6.Irisin inhibits endothelial cell apoptosis both in vivo and in vitro.7.Irisin reduces the ox-LDL-induced HUVEC apoptosis via up-regulated Bcl-2 expression and down-regulated Bax and caspase-3 expression.