The Role And Mechanism Of Insulin-like Growth Factor Binding Protein 1 In The Development Of Aging Related Atherosclerosis

Background:Coronary heart disease is the leading cause of morbidity and mortality in the world.The main pathological characteristic of coronary heart disease is coronary atherosclerosis.Aging is an independent risk factor of atherosclerosis.Age related vascular remodeling which attributes to mediate vascular cells’ biological function facilitates the progress of atherosclerosis.Increasing age participant in the initiation and development of atherosclerosis by accelerating endothelial cell senescence and apoptosis.Several signaling pathways are involved in age-related vascular remodeling.Investigations have revealed a potential link between Notch signaling and the abnormity of age-related tissue repair,thus the growth and development associated Notch signaling may play a certain role in aging.We have made a preliminary research to make sure the role of Notch signaling in regulating the proliferation and senescence of vascular cells.In the study we found that the expression of jagged1,one kind of Notch ligands was significantly reduced in senescent endothelial cell,meanwhile the downregulation of jagged1 contribute to the decreased secretion of Insulin-like growth factor-binding protein 1（IGFBP-1）.IGFBP-1 is a protein produced mainly in the liver,and it belongs to insulin-like growth factor binding protein（IGFBPs）family.IGFBPs family consists of six structurally highly homologous proteins.IGFBPs play a pivotal role in modulating IGFs bioactivity.IGFBPs have also been shown to have IGF-independent actions.The effect of IGFBP-1 in the progress of atherosclerosis mainly depends on its acute regulation of IGF-1 bioactivity,however it also take part in the development of atherosclerosis in the absence of IGF-1.The role of IGFBP-1 in atherosclerosis by IGF-independent action is still unclear so far.Several studies found that IGFBP-1 is inversely associated with established cardiovascular risk factors,including high body mass index（BMI）,hyperglycemia,unfavorable lipid profile.Low levels of IGFBP-1 predicted fatal ischemic heart disease events.Other studies found that high levels of IGFBP-1 were associated with increased cardiovascular mortality.In this study,we measured the level of serum IGFBP-1 to investigate the relationship among IGFBP-1,age and the severity of coronary artery stenosis.We further observed its influence on proliferation,senescence and apoptosis of HCAEC to explore the mechanism of IGFBP-1 in age-enhanced atherosclerosis.Objective:This study exploded the relationship between level of serum IGFBP-1 and age,the correlation between level of serum IGFBP-1 and severity of coronary artery stenosis.Further study was made to observe the effect of IGFBP-1 on proliferation,senescence and apoptosis of HCAEC,to clarify the influence and mechanism of IGFBP-1 to age-related atherosclerosis.Methods:Part 1:1.A total of 112 patients,who were suspected acute coronary syndrome for the symptoms of acute chest pain and underwent coronary angiography between July 2014 and July 2015 in our hospital were enrolled in the study.Those with diabetes mellitus,malignant tumor,valvular heart disease,aortic aneurysm,stable angina,autoimmune diseases,severe hepatic and renal dysfunction were excluded.those who had undertook Percutaneous intracoronary arterial stenting or coronary artery bypass grafting were excluded.2.The basic characteristics and biochemical indexes of the patients which including age,sex,BMI,smoking history,hypertension,hyperlipidemia,Medication,systolic blood pressure（SBP）,diastolic blood pressure（DBP）,total cholesterol（TC）,total triglyceride（TG）,apolipoprotein B（ApoB）,high density lipoprotein（HDL）,low density lipoprotein（LDL）,neutrophil to lymphocyte ratio（NLR）and monocytes to lymphocyte ratio（MLR）were collected.All the participants underwent coronary angiography.The severity of coronary artery stenosis is determined by the Syntax Score which according to the outcome of coronary angiography,meanwhile the risk stratification is determined by the GRACE score.The blood sample were collected and the leve of fasting serum IGFBP-1 was determined by enzyme-linked immunosorbent assay（ELISA）.SPSS 20.0 was used for statistics analyzing.Part 2:1.Firstly transfected si-Jagged1 into cultured HCAEC,then Real-time fluorescent quantitative PCR,western blot and ELISA were applied to measure the expression of Jagged1 and IGFBP-1.2.HCAEC was cultured with different concentrations of IGFBP-1（0ng/ml,5ng/ml,50ng/ml and100ng/ml）,HCAEC proliferation was analyzed by cck-8 and 3H-Td R incorporation.3.According to the passage of HCAEC and if it was treated with IGFBP-1 or H₂O₂,HCAEC was divided into 6 groups: passage 5 group,passage 5+IGFBP-1 group,passage 20 group,passage 20+IGFBP-1 group,H₂O₂ group,H₂O₂ +IGFBP-1 group.SA-β-gal staining was used to test the senescence of HCAEC4.Cells were divided into 4 groups（control group,IGFBP-1 group,H₂O₂ group,and H₂O₂ + IGFBP-1 group）according to if HCAEC was treated with IGFBP-1 or H₂O₂.Cell apoptosis was analyzed by AnnexinV-FITC/PI.5.The expression of p-AKT,AKT was determined by western-blot to observe the effect of IGFBP-1 on the expression of p-AKT and AKT in HCAEC.6.In order to explore if AKT signaling is invoved in IGFBP-1 meidated HCAEC biological function,HCAEC was cultured with AKT inhibitor LY294002 and IGFBP-1.Western blot was used to test the influence of LY294002 on p-AKT and AKT expression in HCAEC.The proliferation of HCAEC was measured by cck-8 and 3H-Td R incorporation.The senescence of HCAEC was analyzed by senescence associate-β-gal staining.The apoptosis of HCAEC was tested by Annexin V-FITC/PI staining.Results:Part 1:1.The correlations of IGFBP-1,age and the severity of coronary artery stenosis: The present study included 112 cases of subjects,including young group of 50 patients（age≤60）and old group of 62 patients（age>60）.When compared with the youth group,the concentration of IGFBP-1 in the old group is higher（P=0.001）.IGFBP-1 was positively correlated with age（r=0.505;P<0.001）.According to the coronary angiography results,all the participants are divided into two group,the diameter stenosis<50% group（n=45）and the diameter stenosis≥50% group（n=67）.The level of IGFBP-1was significant higher in the diameter stenosis≥50% group when compared to the diameter stenosis<50% group.the diameter stenosis≥50% group was group into two different sub-group: the mild stenosis group（Syntax score ≤ 22）and the severe stenosis group（Syntax score ≥ 23）;The concentration of IGFBP-1was significantly elevated in severe stenosis group when compared to mild stenosis group（P<0.001）.The level of serum IGFBP-1 in multi-vessel lesion group is higher than single-vessel lesion group（P=0.002）.IGFBP-1 was positively correlated with Syntax Score（r=0.409,P=0.001）.IGFBP-1 was also positively correlated with GRACE Score（r=0.452,P<0.001）.2.The prediction of IGFBP-1 in coronary artery diameter stenosis≥50%:Logistic regression which used to analysis the association between IGFBP-1 and the severity of coronary artery stenosis,it revealed that the increase in IGFBP-1concentration would elevate the occurrence probability of the coronary artery diameter stenosis ≥ 50%.Receiver’s operating characteristic curve（ROC）analysis revealed the areas under curves（AUC）for IGFBP-1 predicting diameter stenosis≥50% were 0.719（P<0.001）,and the sensitivity and specificity were 0.448 and 0.933.The curves（AUC）for IGFBP-1 combined HDL predicting diameter stenosis≥50% were 0.865（P<0.001）,and the sensitivity and specificity were 0.821 and 0.800.Part 2:1.The m RNA and protein expression of IGFBP-1 in HCAEC:Down-regulated expression of Jagged1 in HCAEC could inhibit the expression of IGFBP-1.The RQ of IGFBP-1 RNA expression in si-Control group was higher than si-Jagged1 group（1.006±0.133 vs 0.474±0.029;P=0.002）.Downregulation of Jagged1 in HCAEC inhibited the secretion of IGFBP 1.The level of IGFBP-1 in supernatant of si-Control group and si-Jagged1 group were（197.933±20.805）pg/ml and（86.933±13.352）pg/ml,respectively（P=0.001）;Downregulation of Jagged1 in HCAEC inhibited IGFBP-1 protein synthesis for that the IGFBP-1/β-actin in si-Control group and si-Jagged1 group were 0.498±0.060 and 0.330±0.019,respectively（P= 0.009）.2.The effect of IGFBP-1 on the proliferation of HCAEC: IGFBP-1 could promote the proliferation of HCAEC in a dose-dependent way and the best concentration was 100ng/ml.When treated with different concentration of IGFBP-1（0ng/ml,5ng/ml,50ng/ml,100ng/ml）,the HCAEC was measured to test the proliferation ability.The OD levels of these groups were 1.000±0.031,1.080±0.015,1.219±0.029 and 1.293±0.023 in CCK-8 assay（P<0.001）.As in 3H-Td R incorporation method,relative 3H-Td R incorporation of those groups were 1.000±0.027,1.089±0.012,1.456±0.074 and 1.526±0.046,respectively（P<0.001）.3.The effect of IGFBP-1 on the senescence of HCAEC: The proportion of aging cells increased along with the number of cell passages,as well as age-related β-gal staining positive rate.The senescence associate-β-gal staining positive rate of HCAEC at passage 20（P20）were higher than those at passage 5（P5）（30.200±4.817 vs 5.400±2.074;P <0.001）;IGFBP-1（50ng/ml）can be reduced HCAEC（P20）aging to some extent.The age-related positive rates in β-gal staining of P20 group and P20 + IGFBP-1 group were 29.600±5.272 and 12.800±3.347,respectively（P<0.001）;IGFBP-1（50ng/ml）inhibited H₂O₂-induced senescence on HCAEC partly.The age-related β-gal staining positive rate of H₂O₂ group and H₂O₂ + IGFBP-1 group were 84.600±6.149 and 44.000±4.301,respectively（P<0.001）.4.The effect of IGFBP-1 on the apoptosis of HCAEC: IGFBP-1（50ng/ml）could inhibit H₂O₂-induced apoptosis of HCAEC to a certain extent.The apoptosis rate of H₂O₂ group and H₂O₂+ IGFBP-1 group were 35.050±1.344 and 19.210±1.952,respectively（P<0.001）.5.The protein expression of p-AKT in HCAEC: IGFBP-1 could promote the phosphorylation of AKT in HCAEC.After HCAEC treated with different concentration of IGFBP-1（0ng/ml,5ng/ml,50ng/ml,100ng/ml）,the p-AKT/β-actin ratios of those groups were 0.168±0.007,0.240±0.015,0.297±0.012 and 0.426±0.034,respectively（P <0.01）.6.The effect of AKT inhibitor LY294002 on IGFBP-1 mediated proliferation,senescence and apoptosis of HCAEC: The AKT inhibitor LY294002 could partly decrease the proliferation of HCAEC which is induced by IGFBP-1.In CCK-8 assay,relative OD values of IGFBP-1 group and IGFBP-1+LY294002 group were 1.000±0.024 and 0.751±0.021,respectively（P<0.001）.IGFBP-1 group and IGFBP-1+LY294002 group were 1.000±0.024 and 0.751±0.021,respectively（P<0.001）.The relative 3H-TdR incorporation of IGFBP-1 group and IGFBP-1+LY294002 group were 1.000±0.503 and 0.798±0.612,respectively（P=0.012）.LY294002 could partly reduce the inhibition of IGFBP-1 on HCAEC senescence.The rates of senescence cells in the IGFBP-1 group and IGFBP-1+LY294002 group were 13.400±3.209 and 23.200±5.404,respectively（P=0.008）.The rates of senescence cells in the H₂O₂ + IGFBP-1 group and H₂O₂ + IGFBP-1 + LY294002 group were 41.200±6.419 and 68.200±7.727,respectively（P<0.001）.LY294002 could partly suppress the inhibition of IGFBP-1on H₂O₂-induced apoptosis of HCAEC.The rates of apoptosis in H₂O₂ + IGFBP-1 group and H₂O₂ + IGFBP-1 + LY294002 group were 19.390±2.215 and 30.167±2.554,respectively（P<0.001）.Conclusion:IGFBP-1 affects the development of atherosclerosis in aging,which might be in part due to its regulations on the proliferation,senescence and apoptosis of vascular endothelial cells.