| Part 1 Potential associations of circulating growth differentiation factor-15 with sex hormones in male patients with coronary atherosclerotic diseaseBackground and Objective:There is a mutual interaction between inflammation and endocrine disorders in the development of coronary atherosclerotic disease(CAD).Growth differentiation factor15(GDF-15)belongs to transforming growth factor β superfamily.It is involved in the regulation of apoptosis,oxidative stress,inflammation and other responses,and is a promising biomarker for the diagnose of cardiovascular diseases.The effects of testosterone and estradiol on CAD as reported in literature have been considered as anti-atherosclerotic.Therefore,this part is aimed to study the changes and the possible association of serum GDF-15 levels with sex hormones in male patients with CAD.Methods:1.A total of 485 male patients were recruited from the Department of Cardiology of Renmin Hospital of Wuhan University,in China.All patients received coronary angiography,and 253 patients were recruited as CAD cases,205 patients were enrolled as controls.2.Serum concentrations of total cholesterol(TC),triglycerides(TG),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),serum creatinine(SCr)and glucose were measured using AD VIA 2400(Siemens,Germany).3.Serum concentrations of testosterone and estradiol were measured by AD VIA Centaur XP(Siemens Germany).4.Levels of the secretory form of human GDF-15 in crude serum samples were measured using a commercial enzyme-linked immunosorbent assay(ELISA)kit(Quantikine,R&D Systems,USA).Results:1.Compared with controls,levels of GDF-15 were significantly increased in CAD patients,whereas the levels of testosterone,estradiol and the ratio of testosterone to estradiol were significantly decreased(all P<0.001).And the changes were related to the severity of the disease.2.In CAD patients with high GDF-15 levels,they were older and had higher Gensini score,hs-CRP and SCr levels,and lower testosterone and the ratio of testosterone to estradiol than patients with low GDF-15 levels.3.According to the partial correlation analysis,GDF-15 levels were inversely associated with both testosterone levels(r=-0.339)and the ratio of testosterone to estradiol(r=-0.365)(both P<0.001).4.In the multivariable linear regression analysis,GDF-15 levels were inversely associated with the ratio of testosterone to estradiol(β-coefficient=-0.442,P=0.015 corrected FDR).Conclusions:These results suggest that upregulation of GDF-15 in the presence of low sex hormone levels may contribute to CAD.Thus,restoring levels of testosterone and estradiol to inhibit the effects of GDF-15 may serve as a promising therapeutic strategy for the treatment of CAD.Part 2 The mechanism of the synergistic effects of testosterone and estradiol on GDF-15Background and Objective:Atherosclerosis is the major risk factor for cardiovascular events such as acute coronary syndrome and sudden cardiac death.Inflammation involves in the whole process,from the injury of endothelial cells to the occurrence and development of atherosclerosis.Growth differentiation factor-15(GDF-15)is a pressure-induced factor that plays a promoting role in the early stage of atherosclerosis.And sex hormones can regulate GDF-15 secretion,thus affecting its corresponding biological effects.In addition,low levels of sex hormones are correlated with the occurrence of coronary atherosclerotic disease(CAD).This part of the research is aimed to explore the inhibiting effect of sex hormones on GDF-15 induced inflammation in primary human umbilical vein endothelial cells(HUVEC s).Methods:1.Primary HUVECs were treated with Interleukin-6(IL-6)and soluble interleukin-6 receptor α(sIL-6Rα)to simulate inflammation.Testosterone and estradiol were used for treatments.Western blot was used to detect the expression of GDF-15,and ELISA was used to assay the levels of GDF-15.2.HUVECs were treated with GDF-15,testosterone and estradiol were used for treatments.Western blot was used to detect the expression of relevant inflammatory factors and apoptotic proteins.3.The inhibitors of androgen and estrogen were used to verify whether the regulations of testosterone and estradiol on GDF-15 through their receptors.Results:1.Testosterone combined with estradiol downregulated the expression and secretion of GDF-15,induced by IL-6 and sIL-6 Rα.2.When HUVECs were treated with recombinant GDF-15 protein for 12 h,the expression of ICAM-1,VCAM-1,caspase-3,and Bax were significantly increased.And testosterone combined with estradiol significantly inhibited the above biological effects of GDF-15.3.Testosterone and estradiol are involved in the regulation of GDF-15 through their respective receptors.Conclusions:According to the results,testosterone combined with estradiol inhibited the biological effects of GDF-15 through their receptors,and then inhibited the expression of its downstream inflammatory factors and apoptotic proteins to prevent the development of endothelial cell inflammation.Part 3 The inhibitory mechanism of the synergistic effects of testosterone and estradiol on atherosclerosis through GDF15 in ApoE-/-miceBackground and Objective:Atherosclerosis is a chronic inflammatory disease,according to the study of epidemiological investigation,older men and postmenopausal women are prone to develop atherosclerosis.It means higher age and low sex hormones are risk factors for coronary atherosclerotic diseases(CAD).And some studies showed that,low ratio of testosterone to estradiol was associated with both increased systemic inflammation and plaque instability,as well as adverse cardiovascular outcome.This suggested that the interaction between testosterone and estradiol may better reflect normal balance of physiology.Growth differentiation factor-15(GDF-15)is widely expressed in various cells and plays an important regulatory role in the initial stage of atherosclerosis.Our previous results have confirmed that the levels of testosterone and the ratio of testosterone to estradiol were decreased in male CAD patients and negatively correlated with GDF-15.And it has been confirmed in HUVECs that testosterone and estradiol synergistically regulated the biological effects of GDF-15 through androgen receptor and estrogen receptor.In this part of the study,we are aimed to explore the regulatory effect of testosterone and estradiol on GDF-15 and the mechanism of antiatherosclerotic effects to provide guidance for the prevention and treatment of CAD,as well as the development of new biomarkers.Methods:1.8-week-old male ApoE-/-mice were randomly divided into control group and experimental group.The experimental group underwent orchiectomy to establish sex hormones deficiency mouse model.Two weeks later,ApoE-/-mice were treated with Western diet and supplemented with testosterone,estradiol for 12 weeks.And GDF-15 for 4 weeks.2.After completed the modeling,we assayed serum levels of TC,TG,HDL-C and LDL-C by an automatic biochemical analyzer.3.Oil red O staining was performed to observe the lipid deposition and plaque formation in the aorta of each group of mice.4.HE staining was performed to analyze the endothelial cell damage in mouse aorta.5.The expression of SMA-α and CD68 was detected by immunofluorescence technology to evaluate the distribution and expression of macrophages in atherosclerotic plaques,proliferation of smooth muscle cells,and plaque stability.Results:1.The results of oil red O staining showed that,compared with the group of model,the group which treated with exogenous administration of recombinant GDF-15 protein had more lipid deposition and plaque.And the synergistic effects of testosterone and estradiol significantly inhibited the formation of plaque and lipid deposition.2.Compared with the group of model,after exogenous administration of recombinant GDF-15 protein,serum levels of TC,and LDL-C were increased.And testosterone combined with estradiol decreased the elevated levels of TC,and LDL-C.3.The HE staining results showed that compared with the model,more vacuole cells and plaques appeared in the group treated with GDF-15 recombinant protein,and the gap between the endothelial cells of the vascular intima increased or broken cell connections were found.The morphology of endothelial cells in the endometrium of the blood vessel improved after the treatment of testosterone and estradiol;4.Immunofluorescence technology were used to detect the expression of SMA-α and CD68,and the results showed that compared with the model group,the fluorescence intensity of CD68 was significantly enhanced and the content of macrophages in plaques was significantly increased.Whereas,the proliferation of smooth muscle cells in mouse plaques was significantly reduced.After supplemented with testosterone and estradiol,the proliferation of smooth muscle cells in plaques of mice were significantly improved,and the number of macrophages in the plaques were significantly decreased.Conclusions:These results showed that the administration of recombinant GDF-15 protein promoted the formation and progression of atherosclerotic plaques in mice.Testosterone combined with estradiol reduced the number of macrophages in the plaque,and increased the proliferation of smooth muscle cells,thus,reduced the deposition of lipids on the vascular wall and delayed the progression of atherosclerosis.These results provide a theoretical basis for the combined application of testosterone and estradiol to interfere with the progression of atherosclerosis. |
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