Research Mechanism Of The Synergizes With Testosterone And Estradiol In Increasing The GAS6/Axl Singnal Pathway In Endothelial Cells And The Anti-Atherosclerosis Effect

Part 1 The Reaserach on the Correlation between Serum Sex Hormone and GAS6 Level in Male Coronary Heart Hisease Backgroud and Objective:Epidemiological studies have found that the serum testosterone levels is significantly decreased in men with cardiovascular disease.Moreover,the tyrosine kinase receptor Axl,its specific ligand is Growth arrest specifc protein 6(GAS6),is widely expressed in the vascular system.Studies have shown that serum GAS6 levels are associated with endothelial cell dysfunction and cardiovascular events.Testosterone and estradiol can regulate GAS6 gene transcription,and play cardioprotective function.Suggesting that decreased levels of sex hormones,resulting in lower levels of serum GAS6 may be a risk factor for coronary heart disease(CHD)in men.Therefore,this part is designed to study the sex hormones and GAS6 levels in male patients with CHD,and to analyze the relationship between sex hormones,GAS6 and CHD risk factors.Methords:A total of 327patients were enrolled in the Department of Cardiology,Renmin Hospital of Wuhan University.All cases underwent coronary angiography.Individuals found to have ?70%occlusion of at least one major coronary artery were defned as having CHD,Control subjects exhibited completely normal coronary arteries.Levels of serum GAS6 were measured by ELISA.Serum testosterone and estradiol levels were measured by immunoelectrochemical luminescence.Serum lipids,C-reactive protein and other biochemical factors were measured by automatic biochemical analyzer.Statistical analysis of the correlation between the indicators with CHD.Results:Serum testosterone level was 432.69 ± 14.40ng/dl in the healthy controls,was decreased to 300.76 ± 6.23ng/dl in CHD and the T/E2 ratio decreased from 12.96 to 9.83.The level of GAS6 was 16.20 ± 0.31ng/ml in the healthy controls,was decreased to 12.51 ± 0.19ng/ml in CHD.Spearman correlation analysis showed that serum GAS6 levels were positively correlated with serum testosterone,T/E2 ratio and hs-CRP in CHD(P<0.05).Logit regression analysis showed that serum testosterone and GAS6 were independent risk factors for CHD(P<0.01).Multiple linear regression analysis showed that the level of serum GAS6 was linear with testosterone(?-coefficient = 0.511,P<0.001).Conclusions:This study confirmed that serum testosterone,T/E2 ratio and GAS6 levels were decreased significantly in male patients with CHD.Serum GAS6 levels were positively correlated with testosterone.Multiple linear regression analysis showed that GAS6 levels were linearly related to testosterone,indicating that low level of serum testosterone may be decrease serum GAS6 levels,and reducing the protective effect of GAS6/Axl signaling pathways.GAS6/Axl signaling system is associated with endothelial cell dysfunction,and testosterone maybe play an important role in the development of atherosclerosis by influencing the GAS6/Axl signaling pathway.Part 2 The Synergetic Effects of Estrogen and Testosterone Increasing GAS6/Axl Signaling and Impact of Apoptosis in Endothelial CellsBackgroud and Objective:Growth arrest-specific protein 6(GAS6)is a vitamin K-dependent protein molecular.GAS6 interacts with specific tyrosine kinase receptor Axl,and activate the GAS6/Axl signaling pathway,which involved in processes rlated to proliferation,differentiation,adhesion and inflammation in vascular endothelial cells,vascular smooth muscle cells.The previous research has confirmed that serum testosterone and GAS6 levels were significantly decreased in male patients with CHD,and associated with CHD propress.Vascular endothelial cell injury is the initial initiation factor of atherosclerosis.GAS6/Axl signaling system plays an important role in the maintenance of vascular endothelial cell function.In the healthy human,serum androgen and estrogen was at the normal concentration level and proportion,to maintain the normal physiological function.Studies have reported that sex hormones(testosterone and estradiol)regulate GAS6 gene transcription directly,and then activate the GAS6/Axl signaling pathway to protect the endothelial cell function.However,different ratio of T/E2 is not yet clear to regulate GAS6 express,to activate GAS6/Axl signaling pathways,and play an endothelial cell protective effect.In this part,human umbilical vein endothelial cells(HUVECs)were used as models to study the expression of GAS6 in different T/E2 ratios,and to explore the optimal ratio of T/E2 to induce GAS6 protein expression.We use TNF-a to stimulate HUVECs to inducing inflammatory model,and explore the protection mechanism of optimal ratio of T/E2 to inducing GAS6 expression,activation of GAS6/Axl signaling pathway to repair and anti-apoptotic in HUVECs inflammatory.Methords:The HUVECs was chosed as the research subject in the second part.The different ratio of T/E2 was observed to induce GAS6 protein expression in HUVECs,the GAS6 protein was detected by Western blot.Then,we use TNF-a(lOng/ml)to induce the inflammatory model in HUVECs,to explor the optimal ratio T/E2 repare inflammation and anti-apoptotic protective effect.The expression of GAS6,VCAM-1,ABCG1,apoptosis-related protein and PI3K/Akt signaling protein were detected by Western blot.The reactive oxygen species(ROS)and apoptotic cell were detected by flow cytometry.Results:When the testosterone(20×10-8mol/L)combined with estradiol(1×10-8mol/L),the ratio of T/E2 was 20:1,the GAS6 protein expression level was increased mostly in HUVECs Testosterone or Estradiol can be inhibited by the androgen receptor inhibitor(flutamide)or estrogen receptor inhibitor(fulvestrant)to induce GAS6 expression in HUVECs.When HUVECs were treated with testosterone(20×10-8mol/L)combined with estradiol(1×10-8mol/L),ROS and apoptotic cells was significantly lower than that in TNF-a group,and was significantly better than testosterone alone.Western blot analysis showed that the apoptotic protein Bax increased and the anti-apoptotic protein Bcl-2 was significantly decreased in the TNF-a stimulated group,and treated with testosterone(20×10-8mol/L)and estradiol(1×10-8mol/L),the apoptosis protein Bax was significantly decreased,the anti-apoptotic protein Bcl-2 was increased,the repair effect was better than that of the testosterone alone.The ABCG1 wsa decreased,while VCAM-1 was significantly increased,HUVECs was treated with TNF-a.However,testosterone(20×10-8 mol/L)and estradiol(1×10-8mol/L)were treated,ABCG1 was significantly increased,VCAM-1 significantly decreased,and the pretection was significantly better than testosterone alone.The levels of GAS6 were significantly higher than those in testosterone alone.The phosphorylation level of Axl was significantly increased,and the phosphorylation levels of PI3K and Akt were also increased.Conlusion:The results showed that testosterone combined with estradiol induced GAS6 expression,activation of GAS6/Axl signaling pathway,decrease ROS and apoptosis cells,decreased expression of VCAM-1,elevated ABCG1,in TNF-a-induced endothelial cell injury.The protective effect on endothelial cells and anti-apoptotic effect was significantly better than the testosterone alone.The phosphorylation levels of PI3K,Akt were increased.Studies have confirmed that synergism testosterone combined and estradiol induced GAS6 expression,the specific receptor Axl phosphorylation,and then activate PI3K/Akt signaling pathway to play the role of anti-endothelial cell apoptosisPart 3 The Synergetic Effects of Estrogen and Testosterone Inducing GAS6 Protein expression and anti-Arteriosclerosis in ApoE-/-MiceBackgroud and Objective:Testosterone replacement therapy(TRT)was used to prevent coronary heart disease in men,but TRT was forbid to the use for serious side-effects.In recent years,studies have shown that estrogen has cardiovascular protection role,and anti-inflammation,to reduce the side-effects of androgen.In healthy people,androgen and estrogen were at a certain concentration and optimal ratio in serum,to maintain normal physiological function.GAS6 is a vitamin K-dependent protein,whici interacts with receptor tyrosine kinases of the Axl.GAS6/Axl system has been implicated to maintain physiological function of vascular endothelial cells.Studies have found t the promoter region of the GAS6 gene has a sex hormone binding area,hat testosterone and estradiol regulate GAS6 gene transcription.Indicating that the in vivo testosterone and estrogen may activate GAS6/Axl signal to maintain the normal physiological function.Previous study has found that serum sex hormones and GAS6 levelsin were significantly decreased in male patients with CHD,and the reduce GAS6/Axl signal pathway was the risk factor of CHD.Meanwhile,we found that synergistic testosterone and estrogen have been reduced ROS and apoptosis protein production in HUVECs model by inducing GAS6 expression,play anti-inflammatory and anti-endothelial cell apoptosis.In this part,the orchiectomy male ApoE-/-mice was used to esearch the synergistic testosterone and estrogen on prevention of atherosclerosis.To expore the testosterone and estrogen induce GAS6 protein expression in the mice to anti-atherosclerosis,and to give us a prevention or treatment basis for new strategiesMethords:Eight-week-old male ApoE-/-mice were randomly divided into control group(n =10)and experimental group(n = 30).The experimental group was treated with orchiectomy for establishment of atherosclerotic mouse model of sex hormone deficiency All mice was divided into 4 groups:control group,model group,testosterone group alone(19?/d),and testosterone combined estrogen group(1?g/d estradiol +19?g/d testosterone).After 120 days of feeding,collecting the mice blood,and the mice were sacrificed to obtain organ tissue.The aorta and aortic atherosclerotic plaques were analyzed by oil red O staining.Aortic valve cell components and aortic smooth muscle hyperplasia were detected by immunofluorescence.Serum GAS6,inflammatory cytokines TNF-a,IL-6 and anti-inflammatory cytokine IL-10 levels were measured by enzyme-linked immunosorbent assay(ELISA).Results:Compared with the control group,there were more atherosclerotic plaques in aorta of the model group,lipid deposition and macrophage infiltration were increased in the plaque,and highly proliferation of aortic vascular smooth muscle cell.GAS6 pretion expression was decreased.Serum TC,TG and LDL-C levels in the model group were significantly higher thancontrol group(P<0.05).Serum IL-6 and TNF-a levels were increased(P<0.05),and anti-inflammatory cytokines IL-10 and GAS6 decreased(P<0.05).Compared with the model group,the number of whole aortic atherosclerotic plaques decreased significantly,and the lipid and macrophage infiltration were decreased,the GAS6 protein expression increased,inhibition vascular smooth muscle cell proliferation(P<0.05),Serum TC?TG?LDL-C levels were decreased(P<0.05),and HDL-C was significantly increased(P<0.05),inflammatory factors IL-6 and TNF-a were decreased(P<0.05),anti-inflammatory factors IL-10 and GAS6 increased(P<0.05).Conlusion:Synergistic testosterone and estrogen treated with orchiectomy mice,the atherosclerotic plaque significantly reduced,lipid deposition and macrophage infiltration were decreased,inhibition of foam cell formation,endothelial cell damage and vascular smooth muscle cell proliferation.Serum inflammatory cytokines IL-6 and TNF-alevels were decreased,serum anti-inflammatory factors IL-10 and GAS6 levels were significantly increased.Serum TG,TC,LDL-C were decreased,HDL-C levels were increased.The results suggest that the synergistic testosterone and estrogen may inhibit the development of atherosclerosis by inducing GAS6 protein expression,activating the GAS6/Axl signaling pathway,to reducing the inflammatory cytokines and increasing the anti-inflammatory cytokine levels.Therefore,the animal experiments further confirmed that the synergistic testosterone and estrogen may enhance the GAS6/Axl signal to inhibit atherosclerosis progress,and to provide TRT theoretical basis for atherosclerosis-related diseases.