Exaggerated myocardial oxLDL amount and LOX-1receptor
over-expression associated with coronary microvesl inflammation in unstable angina
Gian Gastone Neri Serneri a,*,Mirella Coppo a,Manuela Bandinelli a,Paoletto Paoletti a, Thomas Toscano b,Ezio Micalizzi b,Marco Chiostri a,Maria Boddi a
我从草原来歌词a Department of Medical and Surgical Critical Care,University of Florence,Italy
b Department of Cardia
c Surgery,Ospedale Maggiore della Carita’,University of East Piedmont,Novara,Italy
a r t i c l e i n f o
小白理财Article history:
Received1June2012
Received in revid form
28October2012
Accepted6November2012 Available online21November2012
Keywords:
Inflammation
Coronary microvesls
该字笔顺oxLDL
Atherosclerosis a b s t r a c t
The pathophysiological relationship between coronary atherosclerosis and coronary microvesls remains undefined and the specific causative role of oxidatively modified low density lipoprotein(oxLDL)in human atherosclerosis is debated.The purpos of this study are to investigate whether coronary microvesls are involved in coronary atherosclerosis and whether incread myocardial oxLDL amount can be associated with coronary microvesl inflammation.A combination of immunohistochemical,RT-PCR and real-time PCR studies performed on myocardial biopsy specimens from patients with mitral stenosis(control hearts,CHs)and from unstable and stable angina patients(UAP and SAP),demonstrated that myocardial oxLDL was associated with a chronic l
ow-grade inflammation in SAP and with a vere high grade inflammation LDL amount was notably higher in UAP than in SAP and in UAP the high grade of inflammation was correlated with the incread amount of oxLDL in endothelial cells and macrophages. The exaggerated amount of oxLDL in UAP and the interaction of oxLDL with lectin-like oxLDL(LOX-1) receptor are amplified by the activation of transcriptional factor octamere1(OCT-1)with conquent activation of a ries of inflammatory endothelial feed-back mechanisms resulting in LOX-1gene over-expression,endothelial inflammation as well as uncontrolled nuclear factor kappa B(NFkB)activation. Moreover,in UAP genes for signal transducer and activator transcriptional factor1a(STAT1a),angiotensin converting enzyme(ACE)and numerous pro-inflammatory cytokines were over-expresd.The prent results may have clinical relevance becau they show that coronary atherosclerosis is a dia not confined to the large arteries but involving the whole coronary tree.In UAP the exaggerated amount of myocardial oxLDL is associated with widespread high grade microvesl inflammation.
Ó2012Elvier Ireland Ltd.All rights rerved.
温州大桥
1.Introduction
怎样避孕
Coronary vascular inflammation has a pivotal role in the devel-opment of acute coronary syndromes
and thefinding of a wide-spread coronary and myocardial inflammation was consistently reported[1].This diffu inflammation ems to be the major determinant of clinical instability,more than the vulnerable plaque inflammation.Indeed,early after acute ischemic injury,inflamma-tory alterations of microvesls may predict early adver outcome and facilitate myocardial susceptibility[2].In unstable angina(UA) an immune-mediated,ischemic independent widespread inflam-mation of coronary microvesls occurs that is further amplified by local cardiac renin-angiotensin system(RAS)activation[3,4].The arch for the candidate triggers of inflammation remains chal-lenging and the cau(s)and pathophysiological mechanisms underlying the development of the inflammatory process are not known.Experimental and human studies support the hypothesis that oxidized low density lipoproteins(oxLDL)are involved in the development of atherosclerosis[5e7].Evidence was shown that circulating levels of oxLDL are high in acute coronary syndromes (ACS)and,in particular,in UA[8]and are associated with macrophage-rich plaques with high concentration of oxLDL[9,10]. Incread plasma and plaque levels of oxLDL correlated with the vulnerability to rupture of atherosclerotic lesions.Interactions between oxLDL and its receptor,lectin-like-oxLDL receptor-1(LOX-1),appear to play key roles in oxLDL-induced vascular dysfunction, including cell apoptosis and matrix metalloproteina(MMP) production and activation,which evokes atherosclerotic plaque
哺乳时间*Corresponding author.Clinica Medica Generale e Cardiologia,Viale Morgagni 85,50134Firenze,Italy.Tel.:þ39(0)55411666.
E-mail address:maria.boddi@unifi.it(M.
Boddi).Contents lists available at SciVer ScienceDirect Atherosclerosis
jo urn al homepag e:/locate/at hero
sclerosis
0021-9150/$e e front matterÓ2012Elvier Ireland Ltd.All rights rerved.
dx.doi/10.1016/j.atherosclerosis.2012.11.007
Atherosclerosis226(2013)476e482
rupture or erosion[11,12].Moreover,oxLDL/LOX-1receptor inter-action modulates cellular function[13]and induces NFkB activation in human endothelial cells and in monocytes of UA patients(UAP) [14].The activation of NFkB in inflammatory cells participates in up-regulating the expression of genes involved in the immune-mediated inflammatory reaction of UA.LOX-1expression
is induc-ible by pro-inflammatory and oxidative stimuli related to athero-genesis[14].Pro-inflammatory stimuli promote LDL oxidative modification,and oxLDL provokes inflammation,thus in arterial walls oxidative stress and inflammation are cloly linked.Finally, the transcriptor factor octamer(Oct)-1plays a major role in oxLDL-induced LOX-1promoter activation in human endothelial cells, especially when oxLDL and Ang II concur in inducing LOX-1 expression as in UA microvesl inflammation[3,4,15,16].With this knowledge,we designed our study to investigate whether incread myocardial oxLDL amount and LOX-1over-expression contribute to the coronary microvesl inflammation of angina patients.
2.Materials and methods
Additional information is included in the Online Data Supplement.
2.1.Study population
Sixteen patients with unstable angina(UA)defined as angina at rest,in class IIB(n¼7)and IIIB(n¼9)of the Braunwald classifi-cation and13patients with stable angina(SA)in Canadian Class II (n¼8)and III(n¼5)were investigated.Patients with UA(UAP)and SA(SAP)were considered for inclusion in the study if they had 2-vesl dia,one of which was the left anterior descendant (LAD)artery,and if t
hey had been scheduled for a coronary artery bypass graft(CABG),having been found unsuitable for percuta-neous transluminal angioplasty.More precily,11UAP had dia of LAD and coronary right artery(CRA),and5patients had dia of LAD and circumflex artery(LCX).In the group of SAP6had dia of LAD and CRA,5had dia of LAD and LCX and2had dia of LAD and obtu marginal artery.The distribution of coronary vesl dia and baline clinical characteristics of the study population are reported in Table1.No patient had heart failure,myocardial infarction or died before,during or after surgery. Four out of16UAP and2out of13SAP suffered anginal episodes in the interval between enrollment and surgery.More precily, among the4UAP who suffered anginal pain,2patients had1 episode and2patients had2episodes.No anginal episode lasted more than5min and no patients showed incread troponin-I levels.
Diagnosis and risk evaluation of UAP and SAP were performed according to the ACC/AHA guidelines[17,18].High risk patients and patients with recent myocardial within the preceding3months)were not accepted for the study.Only patients with recent ont(<5days before admission)who had at least2 episodes of angina at rest or1episode lasting more than20min during the preceding24h associated with transient ischemic ST gment changes and troponin T or I levels<0.1ng/mL(troponin negative)were included in the study.Patients who on admission had recent
infectious dia(within3weeks)or were affected by chronic infections were not accepted for the study.Likewi, patients with known or suspected neoplasms or with erythrocyte dimentation rate>20mm/h were considered not eligible for the study.All the UAP were on aspirin(200mg)and nitrates.In addi-tion to this therapy,the UAP in class IIIB and4patients in class IIB were treated with fractionated heparin.Twelve patients were on betablockers and4received immediate-relea dihydropyridine (DHP)calcium antagonists in the abnce of betablockers(for class IIIB)or immediate DHP in the prence of betablockers(for class IIB patients).No patient had been on statin treatment for at least3 months.The protocol of this study complies with the principles of the Helsinki declaration and was approved by the Ethical Committee of Ospedale Maggiore della Carita’,Novara.All patients gave their informed connt to participate and to have blood samples and biopsy specimens taken and ud for the study.
2.2.Experimental procedures
Patients with the above mentioned clinical characteristics were enrolled in the study after coronary angiography which was per-formed from6to48h after ont of anginal attacks.Severity of coronary lesions and prence and grade of coronary collateral circulation were evaluated.No patients were suffering from angina when they underwent surgery.CABG was performed from8to14 days after coro
nary angiography.Immediately after sternotomy and before inducing cardioplegia,1transmural biopsy(10Â0.5mm) was taken from the anterolateral wall of the left ventricle clo to the apex in the distribution territory of the LAD,by a biopsy needle MN1416,diameter2.1mm;BIP(Gembh).Although this area may be potentially ischemic in veral patients,this eventuality is not troubling becau the immune-mediated microafter acute pha therapy vesl inflammation occurring in UAP is independent of myocardial ischemia and prents immunohistochemical features different from tho of the ischemia-reperfusion injury[3].More-over,veral specific histological and molecular characteristics differentiate the coronary microvesl inflammation of UAP from both the late pha of ischemia-preconditioning[19]and the hibernating myocardium[20].
As control hearts(CHs)we ud biopsy myocardial specimens obtained from the anterolateral wall of the left ventricle of6 patients(3men and3women aged between38and51)affected by mitral stenosis who underwent surgical valve replacement.All the patients with mitral stenosis were free from coronary risk factors (particularly hypertension,hypercholesterolemia and smoking) and non-invasive tests for myocardial ischemia were negative.
2.3.Immunohistochemical identification of inflammatory cells and morphometric analysis
Immunohistochemical identification of inflammatory cells and morphometric analysis were performed as previously described[4]. Table1
Principal demographic and clinical characteristics of anginal patients. Characteristics Stable angina Unstable angina
(n¼13)(n¼16)
Males/females8/511/5
Age,y64Æ859Æ13 Weight,kg79.3Æ9.677.4Æ8.2 Smoking,%8(61%)10(62.5%) Cholesterolemia,mmol/L 5.2Æ0.8 5.6Æ0.7
LDL cholesterol,mmol/L 3.7Æ0.5 4.0Æ0.6
HDL cholesterol,mmol/L 1.1Æ0.2 1.0Æ0.1 Diabetes,%3(26%)7(48%) Hypertension,%7(52%)10(62%)
Left ventricular mass,g/m2112Æ11113Æ13 LVEF,%54.6Æ5.853.2Æ5.3
Mean aortic pressure,mm Hg97.8Æ12.4100.9Æ12.5 Mean angiographic score a16.0Æ4.116.6Æ3.9 Coronary bloodflow mL/min80.3Æ4.681.9Æ12.7 CVR,mm Hg/mL per min 1.50Æ0.411 1.65Æ0.64
LVEF indicates left ventricular ejection fraction;CVR,coronary vascular resistance. Values are meanÆSD or number(percentage).
a According to American Heart Association.
4岁宝宝睡前故事G.G.Neri Serneri et al./Atherosclerosis226(2013)476e482477
2.4.Immunohistochemical identification and quantification of oxLDL,NFkB-p65subunit,and NADPH oxida p22-phox subunit Oxidized LDL was immunolabelled with an antihuman poly-clonal antibody(Immunodiagnostik AG,Benshein,Germany)that recognizes hypochlorite-modified LDL,a potentially proathero-genic LDL[21],becau it maintains the main proatherogenic activities of native LDL[5].Activated transcriptional factor NFkB was evaluated using antihuman polyclonal antibody against p65 subunit[22];the p22-phox subunit of NADPH oxida was immu-nolabelled with an antihuman polyclonal antibody(Santa Cruz, Biotechnology,CA,USA)with the aim to investigate whether the coronary microvesls are a/the site of LDL oxidation[23].The labeling was quantitatively evaluated using Scion Image for Windows(NIH,Bethesda,MD)according to Faussone et al.[24].
2.5.Real-time and rever transcripta PCR analysis for expression levels of mRNAs
Real-time PCR for receptor LOX-1,angiotensin converting enzyme(ACE)and Oct-1and RT-PCR analysis for STAT1-alfa,tumor necrosis factor(TNF)alfa,interleukin(IL)-12,IL-18,IL-10and interferon(INF)-gamma were performed.Total RNA was extracted, transcribed and quantitative gene expression analysis was per-formed by real-time PCR on Rotorgene6600real time analyzer (Mortlake,Australia)as previously described.Detailed methods are reported in the online ction.
2.6.Statistical analysis
Data are expresd as meanÆSD.Comparison between groups was made with Student’s test for paired and unpaired data.We ud1-way analysis of variance(ANOVA)followed by Tukey multiple-range comparison test,as appropriate,to examine the differences among the3groups(SAP,UAP and CHs).The relation-ship between the amount of oxLDL and grade of inflammation was evaluated using linear regression analysis.We ud Graph Pad prism for Windows5.00(Graph Pad Inc.,La Jolla CA,USA).Statis-tical significance was taken as p<0.05for all calculations.
3.Results
3.1.OxLDL in myocardium and morphometry
OxLDL was substantially undetectable in CHs,whereas it was detectable both in SAP and UAP myocardial biopsies,although in markedly different amounts.OxLDL was localized almost exclu-sively around microvesls and specifically in the endothelial cells and macrophages(Fig.1).The amount of oxLDL in UAP was significantly higher than in SAP(p<0.001)(Fig.1
).
Fig.1.OxLDL identification in myocardium.Immunohistochemical staining for oxLDL in myocardial biopsies from CHs(A),SAP(B)and UAP(C,D,E).OxLDL signal is undetectable in CHs(A),weak in SAP(B)and in marked amount in UAP(C,D,E).Adjacent ctions of a UAP myocardial biopsy:arrows point at oxLDL(E)and macrophages(F).Positive signal is revealed by red staining(magnificationÂ200).OxLDL quantification is reported as pixel/0.1mm2(G).*p<0.005vs CHs,x p<0.005vs SAP.
G.G.Neri Serneri et al./Atherosclerosis226(2013)476e482
478
3.2.NFkB and NADPH oxida activation and quanti fication and immunohystochemical identi fication of in flammatory cells
No signi ficant activation of NFkB-p65subunit was detectable in CHs (Fig.2),whereas the prence of NFkB-p65was evident both in the SAP and UAP myocardial biopsies where it is local-ized in endothelial cells and macrophages (Fig.2).However,the amount of p65subunit was signi ficantly high
er in biopsy speci-mens from UAP than in tho from SAP (p <0.001)(Fig.2).No evident signal of p22-phox without signi ficant activation of NADPH oxida was detectable in CHs,SAP and UAP myocardial biopsies.
Numerous cells expressing HLA-DR molecules were detected in biopsy specimens from UAP;in contrast DR þcells were rare in SAP and abnt in CHs (Fig.2).
3.3.Expression levels of mRNAs for LOX-1,OCT-1and ACE
Real-time PCR showed that the LOX-1mRNA was expresd both in biopsy specimens from CH and angina patients (Fig.3).LOX-1mRNA expression levels were signi ficantly higher in angina patients than in CHs and,most importantly,they were higher in UAP than SAP (Fig.3).Likewi,OCT-1mRNA was expresd both in CHs and in myocardial biopsies from SAP and UAP.OCT-1mRNA expression levels were signi ficantly higher in angina patients than
in CHs (p <0.001)but the difference between UAP and SAP was not signi ficant (Fig.3).The expression levels of ACE gene in UAP biopsy samples were signi ficantly higher (p <0.001)than in tho from SAP or CHs (Fig.3).
3.4.Expression levels of mRNAs for STAT1a and cytokines
RT-PCR showed that mRNA for STAT1a was expresd both in CHs and in biopsy specimens obtained from the angina patients.STAT1a mRNA expression levels in SAP were not signi ficantly different from tho of CHs,whereas STAT1a mRNA expression levels in UAP were signi ficantly higher than in SAP and CHs (about twofold,p <0.001)(Fig.4).mRNA expression levels of INF-gamma and TNF-alfa were over-expresd in UAP compared with SAP and CHs biopsy specimens (p <0.005).RT-PCR showed that the genes for IL-12,IL-18and IL-10were expresd in biopsy specimens ob-tained from both CHs and from angina patients.However,the expression levels were signi ficantly different among the 3groups and among the various cytokines (Fig.4).The expression level of IL-12gene in SAP was similar to that of CHS,but in UAP was signi fi-cantly higher than in CHs (p <0.005)and in SAP (p <0.05).The IL-18gene in UAP biopsies was over-expresd when compared with that of CHs (p <0.05)and SAP (p <0.05),even if IL-18gene level of SAP was signi ficantly higher than CHs (p <0.05).In contrast,the gene expression for IL-10was signi ficantly lower in UAP than in
SAP
普通碳素结构钢
Fig.2.NFkB-p65identi fication in myocardium and relationship between amount of oxLDL and intensity of in flammation measured by immunohystochemical identi fication of in flammatory cells.Immunohistochemical staining for NFkB-p65in myocardial biopsies from CHs (A),SAP (B)and UAP (C).NFkB-p65quanti fication is reported as pixel/0.1mm 2(D).*p <0.005vs CHs,x p <0.005vs SAP.Immunohistochemical staining for HLA-DR þcells in myocardial biopsies from CHs (E),SAP(F)and UAP(G).A,E magni fication Â100;B,C,F,G magni fication Â200.Positive signal is revealed by red staining.Bar graphs show mean number ÆSD of DR þcells in CHs,SAP and UAP (H).Panels I and L show the relationship between amount of oxLDL and intensity of in flammation.Linear regression analysis showed that the amount of oxLDL distinctly differentiates 3groups of patients in relation to the number of DR þcells or to the amount of NFkB-p65subunit (r 2¼0.95;p <0.001).
G.G.Neri Serneri et al./Atherosclerosis 226(2013)476e 482479
and CHs biopsy specimens (p <0.05for both)without any differ-ence between the two groups.
3.5.Relationship between amount of oxLDL and intensity of in flammation
The relationship between amount of oxLDL found on micro-vesls and intensity of microvesl in fla
mmation was investigated using linear regression analysis.The indices of the intensity of in flammation were considered by the number of DR þin flammatory cells including endothelial cells and by the amount of NFkB-p65subunit.Linear regression analysis showed that the amount of oxLDL distinctly differentiates the 3groups of patients in relation to the number of DR þcells or to the amount of NFkB-p65subunit (Fig.2)(r 2¼0.95;p <0.001).4.Discussion
This study provides evidence that microvesls are involved in coronary atherosclerosis both in SAP and UAP and that oxLDL may play a decisive role in eliciting the in flammatory process in
coronary microvesls.The results support the concept that coronary atherosclerosis must not be considered a dia limited to the large and medium-size arteries but rather extended to the whole coronary tree.Microvesl in flammation shows veral features peculiar to atherosclerosis such as endothelial cell activa-tion and proliferation,the prence of DR positive cells as well as endothelial cells and macrophages containing oxLDL.However,the other typical characteristics of atherosclerotic coronary plaques such as atheroma,cluster of foam cells,smooth muscle cell prolif-eration and migration are not prent since microvesl anatomy prevents them from forming.In SAP the amount of oxLDL and the number of in flammatory cells were lower than in UAP and among the various in flammatory factors investigated only the pro-in flammatory NFkB was activated and only T
NF a gene was over-expresd.Altogether the findings fit with a chronic low-grade in flammation.In contrast,in UAP microvesl in flammation the amount of oxLDL and the number of in flammatory cells,particu-larly of tho expressing HLA-DR molecules,were notably higher than in SAP in flammation.Moreover both the pro-in flammatory factors NFkB and STAT-1a were activated and the genes of all the in flammatory cytokines investigated were over-expresd,thus suggesting an acute,high grade in flammation.
The chronic low-grade in flammation of SAP provides evidence that oxLDL “per ”is injurious for coronary microvesls without the need to form complexes with subintimal proteoglycans.Thus,our results demonstrate that “in vivo,in humans ”oxLDL can induce microvesl in flammation with the features of a chronic athero-sclerotic in flammation.In UAP oxLDL are signi ficantly higher than in SAP,but this fact per is not suf ficient to demonstrate that oxLDL is the cau of the UAP acute microvesl in flammation.Indeed,UAP in flammation is also characterized by the prence of a marked immunological component that indicates that unknown antigen(s)might be responsible for the immune-mediated reaction.In this context oxLDL accumulation might be the conquence rather than the cau of in flammation and could contribute to heighten the level of in flammation.The pathophysiological mech-anisms that sustain the chronic low-grade in flammation of SAP,consist in t
he activation of NFkB,OCT-1factor and TNF a induced by interaction between oxLDL and LOX-1[14,25]and are revealed both by the prence of subunit p65into the macrophages and endo-thelial cells and TNF a gene over-expression.
The triggers of the immunological reaction underlying unstable angina are as yet unknown.Several studies suggest that the antigen triggers of unstable angina should be antigens of new formation [26]and with a particular molecular pro file.The pathophysiological mechanisms that underlie the acute microvesl in flammation of UAP are numerous and intricate,becau the immune-mediated reaction and the oxLDL accumulation synergistically activate veral feed-back mechanisms that further amplify the in flamma-tory process .The immune-mediated reaction is elicited by the ligature of antigen to the antigen-receptor of T cells [27]resulting in activating a cascade of various cytokines.In UAP the gene expres-sion levels of all investigated cytokines were higher than in SAP with the exception of IL-10which was higher in SAP than in UAP.This last finding when associated with the over-expression of IL-12and IL-18found in UAP,indirectly indicates a Th-1lymphocyte respon [28].Moreover,IL-12activates STAT4and drives the differentiation of naive T cells into Th-1cells that produce IFN-gamma resulting in a positive feed-back loop [29].Indeed,the incread production of INF g by lymphocytes augments STAT-1a gene expression,improves ef ficiency of anti
gen prentation by macrophages and increas synthesis of TNF a and IL-1cytokines [30].All the effects result in a further ampli fication of the in flammatory respons becau TNF a and INF g in turn activate NFkB and STAT-1a ,respectively.Remarkably,the
oxLDL
Fig.4.Cytokine gene expression in myocardium.RT-PCR for GAPDH,STAT1-alfa,TNF-alfa,INF-gamma,IL-12,IL-18,IL-10expression of mRNAs in biopsy specimens from CHs,SAP and UAP (A).Bar graphs show densitometric quanti fication of RT-PCR products obtained in CHs,SAP and UAP (B).Data are prented as mean ÆSD.*p <0.05vs CHs,**p <0.001vs CHs,x p <0.005vs SAP,#p <0.05vs SAP.
CHs LOX-1OCT-13
2-ΔC t
(ΔC t = C t T a r g e t -C t β-a c t i n )
X -4
ACE
Fig.3.LOX-1,ACE and OCT-1gene expression in myocardium and mRNAs expression for LOX-1,OCT-1and ACE in biopsy specimens from CHs,SAP and UAP assayed by quantitative real-time PCR calculated as 2ÀD Ct (D Ct ¼C t of the target gene minus C t of alfa-actin).Data are prented as mean ÆSD.*p <0.001vs CHs,x p <0.01vs SAP.
G.G.Neri Serneri et al./Atherosclerosis 226(2013)476e 482
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