| BackgroundPercutaneous coronary intervention(PCI)has become one of the most effective intervensional techniques for treating coronary bifurcation lesions,but it often comes along with complex procedure,high risk and postoperative restenosis.Treatment of coronary bifurcation lesions with PCI is difficult and at high risk,because the release of stents to the main branches extrudes stenostic side branches,resulting in further stenosis of side-branch ostium,or even occlusion,which has become one of the decisive causes for the occurrence of major adverse cardiovascular events in the patients undergoing PCI.The main purpose of PCI for treating coronary bifurcation lesions is to ensure the patency of the main coronary arteries and side branches,reducing the occurrence of postoperative restenosis in the side branches.Clinically,the kissing balloon technique is a preferred technique for protecting the side branches of coronary arteries.For the rich and small target branches,however,it is difficult to achieve dilation of the stenostic segments by using plain balloon(PB),with low rate of procedural success.The above dilemma has allowed the development of cutting balloon(CB).With metal blades evenly distributing on the surface of the balloon,the CB can cut,separate and displace the plaque,preventing displaced plaque from occluding the vascular branches.Compared with PB,CB can make the stenostic segments of coronary artery regular in shape,minimize the damages to the intima and media in the process of stent expansion,so as to protect the side branches and diminish the incidence of perioperative complications.As a result,CB is particularly suitable for the treatment of the rich and small side branches.In addition,for coronary artery disease patients complicated with diabetes mellitus(DM),which is a common coexisting disease,coronary artery disease often manifests as multiple vessels,diffuse stenosis at many sites,and poor vascular compliance.Such patients are at higher risk in the case of PCI with PB.Nevertheless,few reports have concerned with CB use in such patients.Therefore,in the current study,we compared the efficacy of CB and PB for PCI in the management of coronary artery bifurcation lesions patients complicated with DM.PatientsFrom May 2015 through May 2016,a total of 88 patients undergoing PCI with diabetic mellitus and coronary artery bifurcation lesions,who were admitted to the Department of Cardiovascular Medicine in our hospital,were selected as subjects in this study.A total of 96 coronary bifurcation lesions of Stage D or F on Duke’s classification were identified.The patients were randomly divided into the CB group and the PB group.The patients were eligible for inclusion if they had coronary artery bifurcation lesions on coronary angiography,branch vessel diameter of 2-3 mm,with stenosis higher than 75%,side branches in need of protection as a result of extrusion of side branch ostium from stenting release.They were also requied to have DM at the intermediate or advanced stage,without severe complications including diabetic nephropathy,ocular complications or diabetic foot,with the blood glucose levels which could be stabilized after hypoglycemic therapy.The patients were excluded from the study if they had target lesions which were restenosis lesions or graft lesions with serious calcification,angulation and flexure,ST-segment elevation acute myocardial infarction,previous PCI procedures or contraindications to antiplatelet drugs.MethodsAll the patients underwent coronary angiography via the transfemoral or transradial approach.The angiograms with regard to the left and right main coronary arteries at different sites of the patients were acquired.Before and after the PCI procedure,they received quantitative measurements of the main and side branch arteries in size.Before the PCI procedure,they were given Antiplatelet aggregation drugs,statins lipid-lowering agents,and hypoglycemic therapy to stabilize blood glucose and pressure.During the PCI procedure,they received intravenous infusion of 100 U/kg heparin,and 1000 U heparin per hour was added when the PCI procedure was longer than 2 h.Stenting was performed for the main artery bifurcation lesions,and stenting was also applicable for the side and main coronary artery if necessary.Appropriate balloons were selected based on lesion length and the ratio of balloon diameter to reference diameter(1.0-1.1:1.0).The patients in the CB group received pre-dilation of artery coronary bifurcation lesions with CB alone whereas those in the PB group received pre-dilation of the lesions with PB alone.After full pre-dilation of bifurcation lesions with balloons,stents were implanted into the main vessels.The treatment regimen decision,primarily kissing-balloon dilatation technique or stent implantation,was made on the basis of the degree of extrusion to the side branch from the implanted stent at the main artery.When the side branches showed beneficial hemodynamics and stenosis diameter of less than 50%,the patient needed close observation but no treatment.In case of evident abnormal hemodynamics in the side branch,with thrombolysis in myocardial infarction(TIMI)flow lower than Grade 2,and stenosis dimeter no less than 50%,kissing-balloon dilatation or stent implantation was performed based on the dissection and occlusion profiles.In the process of pre-dilation with CB,CB was sure to maintain in a negative pressure state,without containing any gas or liquid and with the blades uniformly distributing on the surface of the balloon.To ensure a good support,the CB was delivered by appropriate catheters or guide wires.Pressure in the course of CB predilation should be gradually increased,then released and removed.Side branch extrusion and occlusion and perioperative complications were observed.Results1.Clinical data of patients at baselineThe clinical data including sex,age,comorbidities of hyperlipidemia and hypertension,left ventricular ejection fraction,DM stage,fasting blood glucose,fasting insulin and glycosylated hemoglobin were compared between the two groups,indicating no striking differences(P>0.05).The number of diseased vessels,bifurcation site,lesion classification were compared between the two groups,indicating no striking differences(P>0.05).2.Comparison of MLD of main branch vessels and side branch vessels in all patients before and after PCI between the two groupsThe MLD of the main branch vessels in all patients after PCI was higher than that before PCI,but there was no significant difference in the MLD difference between the two groups(P>0.05).The MLD of side branch vessels in all patients after PCI was higher than that before PCI,and the MLD difference in CB group was more significant than that in PB group(P<0.05).3.Comparison of the incidence of side branch vessels extrusion and occlusion after PCI between the two groupsThe immediate postoperative angiography of the CB group showed that the rates of side branch extrusion and occlusion were substantially lower,but the rate of unaffected side branches was markedly higher compared to the PB group(P<0.05).4.Comparison of Perioperative complications in all patients between the two groupsAs for complications,no death,arrhythmia and heart failure occurred among all patients in the perioperative period,and fewer episodes of perioperative myocardial infarction occurred in the CB group versus the PB group(P<0.05).Conclusion:CB pre-dilation of bifurcation lesions applied in PCI for artery bifurcation lesions patients with DM is associated with better protection of side branches,reduction in side branch occlusion and perioperative complications.Thus,CB is of greater significance in clinical practice than PB.BackgroundCoronary heart disease,also known as coronary artery disease(CHD),is caused by the formation of plaque in coronary arteries that are responsible for the supply of oxygen-rich blood to heart muscle.Blockage of coronary arteries leads to insufficient nutrients,blood,and oxygen for the heart.Therefore,CHD is also called ischemic heart disease.CHD is a group of cardiovascular diseases including stable angina,unstable angina,myocardial infarction,and even sudden cardiac death.It is the leading cause of death worldwide,especially in developing countries such as China.Over 40%of all deaths are related to CHD.Even worse,the incidence of this disease is predicted to continuously increase due to changes in life style,such as the popularity of Western-style diet.Although progress has been made in treatment of CHD,outcomes are usually poor.Therefore,in-depth investigation of the mechanism underlying this disease may improve the survival of CHD patients.As a transforming growth factor,TGF-β1 plays pivotal roles in various normal physiological and pathological processes through the regulation of cell proliferation,growth,differentiation,and apoptosis.The involvement of TGF-β1 in vascular morphogenesis and dysfunction has been extensively studied previously and TGF-βsignaling has been proved to be crucial in cardiovascular disorders.Increased expression level of TGF-β1 was observed during the development of a variety of diseases,including CHD.Sphingosine kinases,including SPHK1 and TIMP-1,also play pivotal roles in vascular development.SPHK1 interacts with different signaling molecules,such as vascular endothelial growth factor,to regulate angiogenesis.Secretion of TIMP-1 promotes the formation of new vessels.However,the function of those proteins in CHD and the underlying molecular mechanism remain unclear.Therefore,our study was carried out to investigate the role of these proteins in CHD and to explore the possible mechanism.Materials and methodsSubjectsA total of 60 patients with CHD were selected from January 2016 to January 2017 in Qilu Hospital of Shandong University.Those patients were diagnosed using coronary angiography according to the criteria proposed by the American College of Cardiology(ACC)/American Heart Association(AHA).Patients with other types of cardiovascular diseases,malignancies,or other severe diseases,as well as those with serious infections within 6 weeks before the admission or with active chronic inflammatory disease,were excluded.The enrolled patients included 33 males and 27 females,age 32-73 years,with an average age of 51±6.9 years.At the same time,54 healthy people with similar age and sex distributions were selected to serve as a control group.Study methods1.Serum preparation and enzyme-linked immunosorbent assayWhole blood(80ml)was extracted from each participant on the day of admission.Blood samples were kept at room temperature for 2h,followed by centrifugation at 2000rpm for 30 min to separate serum.Then,TGF-β1 ELISA Kit(R&D Systems,Inc.,Minneapolis,MN)was used to measure serum TGF-β1 level with recombinant human TGF-β1 as calibrator.Acid activation was performed to release biologically active TGF-β1.Serum samples were diluted in DPBS buffer with a ratio of 1:75 and were directly added into the ELISA plate to measure the concentration of active TGF-β1.Each measurement was performed 3 times and the mean value was calculated.2.Cell cultureHuman primary coronary artery endothelial cells(HCAEC)were obtained from ATCC(ATCC(?)PCS-100-020TM).Cells culture was performed according to ATCC protocol.Serum-free culture medium was used in drug treatment.Cells were collected during logarithmic growth phase for subsequent experiments.3.TransfectionTGF-β1 siRNA(catalog#AM 16708),TIMP-1 siRNA(catalog#AM 16708),and Silencer(?)Negative Control#1 siRNA(catalog#AM4611)were provided by Thermo Fisher Scientific.EcoRI-EcoRI fragments containing full-length TGF-β1 gene cDNA,SPHK1 gene cDNA,or TIMP-1 cDNA were inserted into pIRSE2-EGFP(Clontech,Palo Alto,CA,USA)vectors to establish TGF-β1 SPHK1,and TIMP-1 expression vectors,respectively.Empty pIRSE2-EGFP vector was used as a negative control.Before transfection,HCAECs were cultured overnight to reach 70-80%confluence.Lipofectamine 2000 transfection reagent(11668-019,Invitrogen,Carlsbad,USA)was used to transfect 50 nM siRNA or 10 nM vector into 5×106 cells.4.MTT assayAfter transfection,cells were cultured in culture medium containing 10 mM tetraethylammonium(TEA)at a density of 5×104 cells/ml to induce cell apoptosis.Then,100μl cell suspension(5×103 cells)was added into each well of 96-well plate.Cells were cultured at an incubator(37℃,5%CO2)for 6 h,followed by the addition of 10 μL of MTT.After that,cell culture was performed for another 4 h,and optical density was measured at 570 nm using a FisherbrandTM AccuSkanTM GO UV/Vis Microplate Spectrophotometer(Fisher Scientific).Cell apoptosis rate was normalized to the one with the highest apoptotic rate.This experiment was performed in triplicate.5.Western blotRIPA solution(Fisher Scientific)was used to extract total protein from in vitro cultured cells,and protein samples were quantified by BCA method.Then,20 μg protein was subjected to 10%SDS-PAGE gel electrophoresis,then transferred to a PVDF membrane.Blocking was performed by incubating the membrane with 5%skimmed milk.After that,membranes were washed 3 times with PBS,10 min each time,followed by incubation with primary antibodies,including rabbit anti-TGF-β1(1:2000,ab92486,Abeam),anti-SPHK1(1:2000,ab46719,Abcam),anti-TIMP-1(1:2000,ab61224,Abcam),and anti-GAPDH(1:1000,ab9845,Abcam)overnight at 4℃.The next day,membranes were washed 3 times with PBS,10 min each time,followed by incubation with anti-rabbit IgG-HRP secondary antibody(1:1000,MBS435036,MyBioSource)at room temperature for 1 h.After washing with PBS for 15 min,ECL method(Sigma-Aldrich,USA)was used for signal development.Relative expression levels of TGF-β1,SPHK1,and TIMP-1 were normalized to endogenous control GAPDH using Image J software.This experiment was performed in triplicate.Results1.Comparison of serum TGF-β1 level between CHD patients and healthy controls and the diagnostic valueNo significant differences in serum levels of total TGF-β1 were found between CHD patients and healthycontrols.However,serum levels of active TGF-β1 were significantly higher in CHD patients than those in controls(P<0.05).ROC curve analysis was performed to evaluate the diagnostic values of serum levels of total TGF-β1 and active TGF-β1 for CHD.While,the area under the curve(AUC)of serum levels of total TGF-β1 in the diagnosis of CHD was 0.5109(P=0.8429).AUC of serum levels of active TGF-β1 in the diagnosis of CHD was 0.9627(P<0.0001).These data suggest that active TGF-β1 but not total TGF-β1 may serve as a potential diagnostic biomarker for CHD.2.Effects of TGF-β1 siRNA silencing and overexpression on apoptosis of HCAECMTT assay was performed to explore the effects of TGF-β1 siRNA silencing and overexpression on apoptosis of HCAEC.TGF-β1 siRNA significantly inhibited cell apoptosis compared with control HCAEC(P<0.01).In contrast,TGF-β1 overexpression significantly promoted cell apoptosis(P<0.01).These data suggest that TGF-β1 signaling promotes apoptosis of HCAEC.3.TGF-β1 is a positive upstream regulator of SPHK1 and TIMP-1 in HCAECTGF-β1 overexpression significantly upregulated the expression of SPHK]and TIMP-1 in HCAEC(P<0.05).In contrast,SPHK1 and TIMP-1 overexpression showed no significant effects on TGF-β1 expression(P>0.05).These data suggest that TGF-β1 is a positive upstream regulator of SPHK1 and TIMP-1 in HCAEC.4.SPHK1 is a positive upstream regulator of TIMP-1 in HCAECSPHK1 overexpression significantly upregulated the expression of TIMP-1 in HCAEC(P<0.05).In contrast,TIMP-1 overexpression showed no significant effects on SPHK1 expression(P>0.05).These data suggest that SPHK1 is a positive upstream regulator of TIMP-1 in HCAEC.5.SPHK1 inhibitor and TIMP-1 silencing can reduce the apoptotic effect of overexpression of TGF-β1SPHK 1 Inhibitor 5C treatment for 1 h or TIMP-1 silencing significantly reduced the enhancing effects of TGF-β1 overexpression on cell apoptosis(P<0.05).In addition,cell apoptosis showed a similar pattern to the expression pattern of TIMP-1.ConclusionSerum level of TGF-β1 was higher in patients with CHD than in healthy controls.Serum level of active TGF-β1 but not total TGF-β1 can be employed to effectively diagnose CHD.TGF-β1 overexpression significantly promoted the apoptosis of HCAEC and TGF-β1 siRNA silencing significantly inhibited the apoptosis of HCAEC.TGF-β1 overexpression significantly increased the expression levels of SPHK1 and TIMP-1.SPHK1 overexpression upregulated the expression of TIMP-1.SPHK1 inhibitor and TIMP-1 silencing reduced the enhancing effects of TGF-β1 overexpression on cell apoptosis.Therefore,we conclude that TGF-β1 can promote CHD through the induction of cell apoptosis by upregulating SPHK1 expression and further upregulating its downstream TIMP-1. |
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