| Objectives:Inflammation is one of the main pathophysiological mechanisms of coronary artery disease(CAD).The neovascularizations and lymphangiogenesis of epicardial adipose tissue(EAT)may play important roles in the course of CAD.EAT secrete a large number of adipocytokines,including pro-inflammatory factors:complement-Clq TNF-related protein(CTRP)1,chitinase-3-like protein 1(YKL-40),secreted frizzled-related protein 4(SFRP-4),Salusin-β,tumor necrosis factor-α(TNF-α);anti-inflammatory factors:CTRP9,meteorin-like(Metrn1),Salusin-α,adiponectin(ADP)and vascular lymphangiogenesis regulator:vascular endothelial growth factor(VEGF)-C,VEGF-D,vascular endothelial growth factor receptor 3(VEGFR-3).The study explored the correlation of adipocytokines serum levels,and the expression levels of adipocytokines,neovascularizations and lymphangiogenesis biomarkers in EAT and subcutaneous adipose tissue(SAT)with CAD.Methods:A total of 26 inpatients underwent elective coronary artery bypass grafting and 20 control inpatients needing heart valve replacement were enrolled in non-CAD(NCAD)group at the cardiac surgery department in our hospital from March to September 2019.The serum,EAT,SAT samples were collected.The serum inflammatory adipokines levels of Salusin-β,CTRP1,YKL-40,SFRP-4,TNF-α,CTRP9,Metrnl,Salusin-α,ADP were examined by enzyme-linked immunosorbent assay(ELISA)kit.CTRP1,YKL-40,CTRP9 protein expression in EAT and SAT were measured by Western Blot.The CTRP1,YKL-40,SFRP-4,CTRP9,Metrnl,VEGF-C,VEGF-D,VEGFR-3 gene expression in EAT and SAT were analyzed by quantitative real-time polymerase chain reaction(RT-qPCR).The morphology of EAT and SAT cells were compared via hematoxylin-eosin(HE)staining.Immunohistochemical was used to measure macrophages CD68+,CD206+,CD11c+ infiltration in EAT and SAT,and podoplanin protein(PDPN),lymphatic vessel endothelial hyaluronan receptor 1(LYVE-1)expression levels.CD31+,CD34+expression levels in EAT and SAT were determined by immunofluorescence.Results:1.Clinical features:In CAD group,the prevalences of chest pain,hypertension and diabetes were significantly higher than NCAD group[76.9%(20/26)vs 10.0%(2/20),61.5%(16/26)vs 25.0%(5/20),42.3%(11/26)vs 10.0%(2/20),all P<0.05].2.Inflammatory adipocytokines serum levels were measured by ELISA:2.1 Adipocytokines serum levels:pro-inflammatory adipocytokines TNF-α,CTRP1,Salusin-β,YKL-40,SFRP-4 serum levels were significantly higher in CAD group than NCAD group:125.90(112.00,146.80)ng/L vs 95.96(90.12,117.80)ng/L,60.10(50.50,71.57)ng/L vs 45.61(42.74,54.35)ng/L,8.42(7.90,10.90)ng/L vs 8.02(6.30,9.25)ng/L,54.03(48.89,63.42)ng/L vs 46.71(40.59,53.99)ng/L,41.13(42.51,56.19)ng/L vs 31.96(28.15,45.62)ng/L,respectively(all P<0.05).The serum levels of anti-inflammatory adipocytokines ADP,CTRP9,Salusin-α,Metrnl were significantly lower in CAD group than NCAD group:323.1(282.2,356.1)ng/L vs 365.8(311.0,463.9)ng/L,17.09(16.14,18.92)ng/L vs 19.92(16.91,23.95)ng/L,63.3±14.0 ng/L vs 77.76±31.4 ng/L,166.8±32.9 ng/L vs 206.9±42.6 ng/L,respectively(all P<0.05).2.2 ROC curve analysis of serum adipocytokines:the area under the curve(AUC)of pro-inflammatory factors TNF-α,CTRP1,Salusin-β,YKL-40,and SFRP-4 were 0.764(95%CI:0.617-0.911,P=0.006),0.785(95%CI:0.643-0.927,P=0.003),0.685(95%CI:0.508-0.862,P=0.048),0.759(95%CI:0.603-0.916,P=0.008),0.832(95%CI:0.699-0.965,P<0.001),respectively,and the cutoff values for the best diagnosis were 99.03 ng/L,49.06 ng/L,7.72 ng/L,47.71 ng/L,35.75 ng/L,respectively.The AUC of anti-inflammatory factors ADP,CTRP9,Salusin-α,Metrnl was 0.731(95%CI:0.569-0.894,P=0.014),0.708(95%CI:0.545-0.872,P=0.023),0.672(95%CI:0.492-0.851,P=0.065),0.776(95%CI:0.629-0.923,P=0.004),respectively.And the cut-off values for the above markers was 417.4 ng/L,18.89 ng/L,71.76 ng/L,152 ng/L,respectively.2.3 The correlation among serum inflammatory adipocytokines:pro-inflammatory adipocytokines CTRP1,Salusin-β,YKL-40,SFRP-4 are positively correlated with TNF-α,the r are 0.357,0.332,0.383,0.473,respectively(all P<0.05).CTRP1 and Salusin-β are positively correlated with SFRP-4,the r are 0.509 and 0.334,respectively(both P<0.05).YKL-40 correlated with CTRP1,Salusin-β and SFRP-4 positively,the r are 0.710,0.494,0.573,respectively(all P<0.05).SFRP-4 is negatively correlated with ADP,the r is-0.358(P<0.05).Metrnl correlated with CTRP9,Salusin-α,and ADP positively,the r are 0.377,0.348,0.406,respectively(all P<0.05).3.RT-qPCR measured the gene expression levels of adipocytokines in EAT and SAT:3.1 Gene expression levels of adipocytokines in EAT and SAT:the mRNA expression levels of pro-inflammatory adipocytokines CTRP1,YKL-40,SFRP-4 in EAT were significantly higher in CAD group than in NCAD group after log10 logarithmic conversion:-1.21 ±0.51 vs.-1.71 ±0.59,-1.94(-2.20,-1.51)vs-2.30(-2.67,-1.91),-1.44(-1.817,-1.15)vs-2.07(-1.817,-1.15),respectively(all P<0.05).In the CAD group,the mRNA expression levels of anti-inflammatory adipocytokines CTRP9 and Metrnl in EAT were significantly lower than NCAD group after log 10 logarithmic transformation:-2.25(-2.52,-1.65)vs-1.57(-2.23,-1.16),-1.40±0.39 vs-1.04±0.54(all P<0.05).The mRNA expression levels of VEGF-C,VEGF-D,VEGFR-3 in EAT were significantly higher in CAD group than in NCAD group after log 10 log conversion:-1.11(-1.41,-0.90)vs-1.51(-1.88,-1.29),-0.93(-1.50,-0.53)vs-1.52(-2.01,-0.98),-1.48(-2.00,-1.12)vs-2.14(-2.64,-1.84),respectively(all P<0.05).The mRNA expression levels of CTRP1,YKL-40,SFRP-4,CTRP9,Metml,VEGF-C,VEGF-D and VEGFR-3 in SAT were converted by log 10 were not statistically different between CAD and NCAD groups(all P>0.05).3.2 The correlation of adipocytokines mRNA expression levels in EAT:CTRP1,SFRP-4,YKL-40,CTRP9,VEGF-D,VEGFR-3 and VEGF-C are positively correlated,the r are 0.564,0.440,0.561,0.457,0.821,0.369,respectively(all P<0.05).CTRP1,SFRP-4,YKL-40,CTRP9,VEGFR-3 are positively correlated with VEGF-D,the r are 0.573,0.446,0.517,0.391,0.526,respectively(all P<0.05).CTRP1,SFRP-4,YKL-40,Metrnl are positively correlated with VEGFR-3,the r are 0.741,0.752,0.584,0.452,respectively(all P<0.05).CTRP1 is positively correlated with SFRP-4,YKL-40 and Metrnl,the r are 0.651,0.751,0.435,respectively(all P<0.05).CTRP9 is positively correlated with Metrnl,the r is 0.519(P<0.05).SFRP-4 is positively correlated with YKL-40,the r is 0.706(P<0.05).4.The protein expression levels of inflammatory adipocytokines in EAT and SAT:the protein expression levels of pro-inflammatory factors CTRP1 and YKL-40 in EAT were significantly higher in the CAD group than in the NCAD group:1.51(0.96,2.69)vs 0.51(0.29,1.24),2.02(1.01,3.93)vs 0.60(0.34,0.94)(both P<0.05).The anti-inflammatory factor CTRP9 protein expression level in EAT was significantly lower in the CAD group than in the NCAD group:0.76(0.60,1.85)vs 1.53(0.88,3.96)(P<0.05).The protein expression levels of pro-inflammatory factors CTRP1,YKL-40 and anti-inflammatory factor CTRP9 in SAT were not significantly different between the two groups(P>0.05).5.The morphology of EAT and SAT cells measured by HE staining:The area of EAT cells in the CAD group was significant smaller than the area of SAT cells:3241.9(3158.9,3693.9)vs 5545.9(4962.9,6394.5)(P<0.05).The EAT cells area in the NCAD group was also significantly smaller than SAT cells:3046.5(2673.7,3211.4)vs 4777.2(3785.1,5348.1).6.Macrophage infiltration in EAT and SAT measured by immunohistochemical:The CD68+,CD11c+,CD11c+/CD206+in EAT were significantly higher in CAD group than NCAD group:0.61±0.08 vs 0.48±0.06,0.62(0.57,0.64)vs 0.49(0.45,0.54),1.96(1.86,1.98)vs 1.33(1.24,1.45)(all P<0.05).The CD206+in EAT was significantly lower in CAD group than NCAD group:0.32(0.28,0.33)vs 0.36(0.33,0.39)(P<0.05).The CD68+in SAT was significantly higher in CAD group than NCAD group:0.60(0.54,0.70)vs 0.53(0.47,0.55)(P<0.05).The difference of CD11c+,CD206+,CD11c+/CD206+between CAD and NCAD groups in the SAT were not significantly(all P>0.05).7.The expression of CD31+,CD34+and the number of vessels in EAT and SAT measured by immunohistochemical:7.1 The blood vessels number in EAT and SAT:the blood vessels number in EAT was higher in CAD group than NCAD group:4.0(3.0,5.5)vs 2.5(2.0,3.0)(P<0.05).The number of blood vessels in SAT was not significantly different between the 2 groups(P>0.05).In CAD patients,the number of blood vessels was higher in EAT than SAT:4.0(3.0,5.5)vs 2.0(1.0,3.0)(P<0.05).The number of blood vessels in NCAD patients were not significantly different between EAT and SAT(P>0.05).7.2 CD31+and CD34+expression levels:CD31+and CD34+expression levels in EAT were significantly higher in CAD group compared to NCAD group:0.90(0.77,0.96)vs 0.72(0.70,0.75);0.87(0.80,0.96)vs 0.69(0.62,0.77)(all P<0.05).The expression of CD34+in SAT was significantly higher in CAD group than the NCAD group:0.98(0.95,1.01)vs 0.75(0.74,0.88)(P<0.05).The expression of CD31+in SAT was not significantly different between CAD group and NCAD group(P>0.05).8.The expression of lymphangiogenesis markers PDPN and LYVE-1 in EAT and SAT measured by immunofluorescence:The expression of PDPN and LYVE-1 in EAT were significantly higher in CAD group compared to NCAD group:0.59(0.55,0.63)vs 0.53(0.48,0.57);0.57(0.54,0.61)vs 0.47(0.38,0.52)(both P<0.05).The expression of PDPN and LYVE-1 in SAT was not significantly different between CAD group and NCAD group(all P>0.05).Conclusions1.EAT inflammation leads to imbalance secretion of pro-inflammatory and anti-inflammatory adipokines are related to the prevalence of CAD,which is an important pathogenesis of CAD.2.Neovascularization and lymphangiogenesis increased in EAT are related to the prevalence of CAD and is an important pathogenesis of CAD.3.Novel serum inflammatory adipokines CTRP1,Salusin-β,YKL-40,SFRP-4,CTRP9,Metrnl may be used as important biomarkers in the diagnosis of CAD.4.The biomarkers secreted by EAT may be new targets for the treatment of CAD. |
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