| Background:With instinct characteristics of the onset but disastrous consequences,diabetes and its complications have posed major threats to human health.Among them,macrovascular complications including vascular calcification and atherosclerosis have become the most decisive factors that have led to patients' deaths or disabilities since they finally cause such severe consequences as myocardial infarction,heart failure and cerebrovascular accident.As one of the most important characteristics of vascular diseases of diabetes patients and one of the universally recognized independent risk factors,calcification is closely related to the risks of the occurrences of the above mentioned cardio-cerebrovascular accidents.However,the number of therapeutic measures of treating calcification is still rather limited.A large part of the cardiovascular accidents can still not be prevented despite the premise that both the therapy with interventional equipments and the existing medication with standardized and comprehensive dosages are applied.Now it is generally considered that the formation of calcification is a biological process similar to ostosisthat is active and can be regulated to a high extent.The formation of diabetes patients' vascular intimal calcificationis majorly associated withhyperglycemia,the elevation of the level of its advanced glycationend products(AGEs),inflammatory cells,oxidative stress as well as the intimal proliferation and migration of vascular smooth muscle cells and the subsequent phenotype inversion.Many researches have proven that the axis of AGEs-RAGE(receptor for AGEs)has taken part in the generation and development of thediabetes patients' vascular calcification.However,the specific molecular mechanism is still not clear.Endothelial progenitor cells(EPCs)are precursor cells used for helping form the vascular endothelial cells.Relevant researches have revealed that EPCs are able to infiltrate into the plaques and take part in the intimal calcification of arteriosclerosis after osteogenic differentiation occurs,indicating that EPCs are most probablyosteoid precursor cells.Stimulated by osteogenetic signals,EPCs are able to detect such calcification-related proteins as alkaline phosphatase(ALP)and Runt-related transcription factor 2(Runx2)at a high level so as to promote the development of the process of vascular calcification.Besides,EPCs also function to promote the osteogenic differentiation of bonemarrow mesenchymal stem cells(BMSCs).As is indicated in the researches,microRNA-133 a has played significant roles in the growth,development and functional changes of the skeletal muscles and cardiac muscles while Runx2 is one of its target proteins.In the environment of hyperglycemia,the level of miR-133 a decreaseswhile the level of Runx2 increases.The signal axis of miR-133a/Runx2 is closely associated with osteogenic differentiation.Objectives: Through case studies and comparisons,the differences of the degrees of calcification in the coronary arteries of both diabetes patients and non-diabetes patients are clarified while the expressive levels of mi R-133 ain the peripheral blood mononuclear cells(PBMC)are compared between the two groups of patients.In vivo,the diabetic mice model are established,which are fed with high-fat diet and help to clarify the osteogenic protein Runx-2 and BMP-2 expression levels in aorta arch.In vitro,AGEs are used to stimulate and intervene inEPCs so as to further prove that in the environment of diabetes,the accumulated advanced glycationend products(AGEs)exert influences on the migration,proliferation and apoptosis of EPCs as well as on the changes of the relevant osteogenic expressions so that it is clarified that AGEs are able to induce the osteogenic phenotype inversion of EPCs.The changes of Runx2-related microRNAs in EPCs are further detected after EPCs are stimulated by AGEs.The focus is positioned at the signal axis of miR-133a/Runx2 and then specific antibodies of RAGE are utilized to block the AGEs-RAGE axis.Then the changes of relative expressions of miR-133 a in EPCs are observed with the stimulation of AGEs after the AGEs-RAGE axis is blocked.The overexpressed plasmids of miR-133 a are constructed and further transfected.After the transfection,the expressive level of Runx2 is detected and a comparison is made between the groups.In this way,the mechanism can be further studied in which AGEs-RAGE axis promotes the osteogenic differentiation of EPCs by means of miR-133a/Runx2.This research aims to preliminarily enunciate that in the environment of diabetes,the AGEs accumulated in the locally impaired vascular intima are able to activate the signal axis of miR-133a/Runx2 through RAGE and to induce the osteogenic differentiation of EPCs,thus taking part in the formation and development of diabetes patients' vascular intimal calcification.Methods:1)30 cases of diabetes patients are selected who underwent the examination of coronary artery CTA while hospitalized in our department from February 2014 to March 2015.30 cases of non-diabetes patients are also selected who underwent the examination of coronary artery CTA while hospitalized in our department during the same time period.Baseline clinical data as well as such indices as blood fat,blood sugar and blood pressure are collected and compared.And then the coronary artery calcification scores(CACS)and the expressive levels of miR-133 a in PBMCs are compared.2)The diabetic mice model are established by peritoneal injection of STZ and are fed with high-fat diet.The mice are sacrificed after 12 weeks and the protein expression levels of Runx-2 and BMP-2 in aorta arch are detected by immunohistochemical staining.3)Isolated and cultured from PBMCs,the EPCs are identified through the observation of cellular morphology and examination of the surface markers by means of flow cytometer.The migration,proliferation and apoptosis of EPCs in the control groups and intervention groups intervened in by different levels of density and grads of Carboxy-methyl-lysine(CML)-BSA are respectively observed by means of proliferation kits,Annexin-V apoptosis detection kits and TRANSWELL chamber migration experiments.And besides,the expressions of ALP,osteocalcin(OC),Runx2 and bone morphogenetic protein(BMP2)are also detected so as to specify the influences on osteogenic differentiation of EPCs.4)After isolated,cultured and identified,EPCs are intervened in by CML-BSA with the density of 80?g/mL.And then the relative expressions of Runx2-related microRNAs in EPCs are detected by means of qPCR which include miR-133 a,miR-204,miR-433,miR-335 and miR-211.After specific antibodies are added,the expressions of miR-133 a in EPCs are detected after stimulated by CML which are to be compared with each of the control groups.Then the overexpressed plasmids of miR-133 a are constructed and transfected.The expressions of Runx2 mRNA in EPCs are detected by means of qPCR after stimulated by CML.And then the level of protein is detected by means of Western Blot.Results:1)Between the two groups of diabetes patients and non-diabetes patients,there are no statistic differences in terms of age,sex,BMI and blood pressure.In terms of medication,33.3% of the diabetes patients are taking statins.Compared with the percentage(13.3%)of non-diabetes patients who are taking statins,P<0.05 and there are statistic differences.There are no statistic differences in the taking of other drugs such as aspirin,ACEI/ARB and calcium antagonist.Between the two groups,there are no statistic significances(P>0.05)in comparing the levels and indices of total cholesterol,low-density lipoprotein,high-density lipoprotein,alanine aminotransferase(ALT),glutamic oxalacetic transaminase(GOT)and serum creatinine.In comparing the levels of fasting blood-glucose(6.47±2.88mmol/L VS 5.18±1.52mmol/L)and triglyceride(1.98±1.09mmol/L VS 1.60±0.48mmol/L),the levels of the diabetes patients group are obviously higher than those of the non-diabetes patients group and therefore there are statistic significances(P<0.05).In the two groups of examinees,the lowest calcification score is 0 while the highest is 3120.Between the two groups,the calcification score of the diabetes patients group is 388±556 while the calcification score of the non-diabetes patients group is 96±130.As is seen from this comparison,the calcification score of the diabetes patients group is obviously higher than that of non-diabetes patients group.And besides,in terms of stratification of risks,the percentage of the diabetes patients with high(or above high)risks of cardiovascular accidents is obviously higher than that in the non-diabetes patients group.The relative expression(0.42 ± 0.09)of miR-133 a in PBMC in the diabetes patients group is obviously lower than that(1.02±0.17)of miR-133 a in PBMC in the non-diabetes patients group and this comparison between the two groups embodies significant statistic differences(P<0.05).2)Compared with the control group,the random blood glucose levels of diabetic mice are significantly increased(P<0.05).The diabetic mice demonstrate a significant body weight loss at 12 weeks(22.4±1.87 g VS 24.8±1.46 g,P<0.05).Immunohistochemical staining shows significantly elevated protein expression levels of Runx-2 and BMP-2 in aorta arch of diabetic mice.3)Compared with the BSA control group,the abilities of EPCs in proliferating and migrating are significantly inhibited by the stimulation of CML-BSA.Furthermore,this stimulation has increased the cellular apoptosis and promoted the osteogenic differentiation(P<0.05).Among different levels of density,CML-BSA with the density of 80?g/m L exerts the most obvious influences(P<0.05).Compared with the BSA control group,the stimulation of CML-BSA have significantly increased the expressions of ALP,Runx2,BMP2 and OC which are EPCs-related osteogenic genes.Besides,they increase by degrees of density dependency with the changes being the most obvious when the density is 80?g/mL(P<0.05)and they are respectively 11.97±0.25,10.04±0.47,10.04±0.47 and 9.28±0.32.However,they are on the trend of decline when the density is 100?g/mL(P<0.05)and they are respectively 6.01±0.12,3.11±0.01,2.60±0.20 and 3.15±0.13.4)After CML with the density of 80?g/mL is given,compared with BSA control group,the expressions of miR-133 a,miR-204 and mi R-211 obviously decrease(P<0.05)in terms of the relative expressions of Runx2-related microRNAs in EPCs and they are respectively 0.35±0.07,0.67±0.15 and 0.58±0.02.Between the two groups,there are no obvious differences in terms of the expressions of miR-433 and miR-335.After RAGE is blocked and the stimulation of CML with the density of 80?g/m L is given,the relative expression of miR-133 a in EPCs in RAGE+ CML group obviously increases(P<0.05)compared with that of miR-133 a in EPCs in Scramble+CML group and they are respectively 0.82±0.17 and 0.42±0.03.After miR-133 a is overexpressed,CML with the density of 80?g/mL is given to stimulate.Compared with the scramble+CML group,the expression of Runx2 protein of miR-133 a obviously decreases(P<0.05)in the mimic+CML group.This difference features statistic significances.Conclusions:1)Compared with the non-diabetes patients group,the coronary artery calcification is severer in the group of diabetes patients.Besides,the relative expression of miR-133 a in PBMC in the diabetes patients group is obviously lower than that of miR-133 a in PBMC in the non-diabetes patients group,indicating that mi R-133 a is probably associated with the vascular calcification of the diabetes patients.2)The protein expression levels of Runx-2 and BMP-2 are significantly elevated in aorta arch of diabetic mice fed with high-fat diet,indicating disorders of calcium and phosphorus metabolism as well as osteogenic differentiation.3)The stimulation and intervention of CML-BSA have impaired EPCs in terms of its proliferative and migratory functions while the apoptosis increases.Besides,the expressive levels of relevant osteogenic proteins increase which include BMP2,Runx2,OC and ALP.The accumulation of AGEs affects the functions of EPCs and promotes its osteogenic differentiation.This might be one of the important mechanisms in the forming and development of the vascular intimal calcification of the diabetes patients.4)After CML-BSA intervenes,the expression of miR-133 a in EPCs obviously decreases.But after RAGE is blocked,the influences of CML on miR-133 a are inhibited.The overexpression of miR-133 a has led to the decline of the expressive level of the protein of Runx2 and this decline is more obvious compared with the decline of Runx2 mRNA.These results support the theory that miR-133 a inhibits the expression of the protein of Runx2 as its target gene through the regulation in the condition of Runx2 mRNA being transcribed,thus exerting the role of reversely regulating the expression of the protein of Runx2.This has further enunciated the theory that in the environment of diabetes the locally accumulated AGEs are able to activate the signal axis of mi R-133a/Runx2 through RAGE and induce the osteogenic differentiation of EPCs in the process of taking part in the formation and development of vascular intimal calcification of diabetes patients. |
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