The Predictive Value Of Pericoronary Adipose Tissue Attenuation On Functional Coronary Stenosis And Its Association With Coronary Inflammation And Endothelial Function

Objective:The invasive measurement of coronary fractional flow reserve（FFR）is the gold standard for the diagnosis of functional ischemic stenosis.However,the application of FFR is limited by the invasive procedure and high cost.Traditional coronary CTA parameters,such as the stenosis severity and the plaque characteristics,have their limitations on the prediction of FFR abnormality.Peri-coronary adipose tissue（PCAT）is closely connected and interact with the coronary artery.Fat attenuation index（FAI）is believed to reflect the status of adipocytes in PCAT,thus indirectly providing clues to coronary inflammation.Studies have shown that FAI is related to culprit lesions in patients with acute coronary syndrome,and the increase of FAI during follow-up is related to the occurrence of major cardiovascular adverse events（MACE）.At present,research on the diagnostic value of lesion-specific FAI for FFR abnormalities was limited.Meanwhile,evidence of the relationship between FAI and the pathological changes in coronary artery&PCAT inflammation（beside adipocyte changes）and the correlation between FAI and the expression intensity of inflammatory factors in coronary artery&PCAT tissues was still in demanded.Therefore,this study was conducted to:（1）evaluate whether lesion-specific FAI could provide supplemental diagnostic value for abnormal FFR;（2）To explore the relationship between PCAT attenuation（FAI）and the pathological changes of coronary&PCAT inflammation,and the correlation between PCAT attenuation（FAI）with the expression intensity of inflammatory factors in coronary artery&PCAT tissues;（3）To explore the effect of coronary inflammation on endothelial function and its potential pathway in vitro.Methods:In the first part of this study,patients from multiple medical centers were included.All included patients were matched following criteria:（1）had intermediate coronary stenosis through invasive coronary angiography and the FFR testing were performed for these intermediate lesions;（2）coronary CTA was performed within one week before the invasive procedures.The lesion-specific FAI(FAI_lesion)and lesion-free FAI(FAI_nomal)of the PCAT were measured on coronary CTA images.Meanwhile,the total volume and mean attenuation of the epicardial adipose tissue（EAT）,as well as the plaque characteristics were measured.Functional stenosis was defined by FFR<0.8.Patients were divided into ischemic and non-ischemic groups;vessels were divided into ischemic and non-ischemic lesions.The association between FFR and each parameter was analyzed by logistic regression and the diagnostic performance was evaluated by receiver operating characteristic curve.In the second part of this study,we analyzed the correlation between CT attenuation（FAI）of the PCAT and the m RNA expression levels of inflammatory factors in PCAT tissues of patients with coronary heart disease and swine coronary inflammation models.PCAT from the proximal（10-50mm）right coronary artery（RCA）of patients who underwent coronary CTA within one week before coronary artery bypass grafting（CABG）was collected.FAI of proximal RCA was measured.Total RNA was extracted and the expression of IL-1β/IL-6/TNF-αm RNA was detected by RT-PCR.The correlation between FAI and IL-1β/IL-6/TNF-αm RNA expression was analyzed.Adult male Bama pigs were used to investigate whether PCAT attenuation could monitor the changes of coronary inflammation.The animals were divided into control group（n=4）and experimental group（group A n=4,group B n=4）,each experimental animal was provided with 2 coronary arteries for imaging and pathophysiological analysis.In the experimental group coronary inflammation model was established by percutaneous coronary balloon dilation of RCA and the proximal segment of left anterior descending（LAD）,while the control group only underwent coronary angiography.All animals were performed baseline coronary CTA within 24hours before percutaneous intervention/coronary angiography.In group A,coronary CTA was performed one week and four weeks after the intervention respectively,then the animals were executed and the inflammatory RCA/LAD segments with surrounding PCAT were collected.In group B,coronary CTA was performed one week after intervention and then the animals were sacrificed for sampling.The animals in the control group were performed coronary CTA at baseline,one week and four weeks as the same as group A,and then sacrificed for sampling.The attenuation of PCAT（proximal segment of RCA and LAD）of the injured segment in the experimental group and the corresponding segment in the control group were measured.The dynamic changes of PCAT attenuation at each time point in the control group and the experimental group A were analyzed.HE staining were performed on the tissue samples to analyze the pathological changes of coronary artery and PCAT,then immunohistochemical staining was performed to analyze the protein expression of IL-1β.The total RNA of coronary artery and PCAT were extracted,while the PCAT was divided into inner（distance to coronary adventitia≤vessel diameter）,middle（between the inner and outer layer）and outer（distance to coronary adventitia>2×vessel diameter）layer.The m RNA expression of IL-1β,TNF-α,IL6,MCP-1 was quantified by RT-PCR.Human artery endothelial cells（Telo HAEC）were treated with active recombined human IL-1βprotein（10ng/ml）,the RNA and protein were collected at 6h,12h,24h respectively.The m RNA and protein expression of e NOS,ET-1,ETAR,ACE,AT1R were analyzed by RT-PCR and Western-blot.Finally,STRING and KEGG databases were used to analyze the relevant pathways that may be involved in the intra-cellular（IL-1βtreated Telo HAEC cells）biological process and determine the expression changes of its key factors.Results:Part I:A total of 227 patients from seven centers were included.EAT volume（180±73ml vs.164±60ml,P=0.07）or attenuation（-84±6 HU vs.-83±7 HU,P=0.54）,traditional risk factors and FAI_normal（-82±11 HU vs.-81±11 HU,P=0.65）were not significantly different in patients with or without abnormal FFR.In contrast,lesions causing functional ischemia presented more severe stenosis（72±9%vs.56±12%,P<0.01）,greater plaque volume（157（102,248）mm³ vs.126（77,188）mm3,P<0.01）and higher FAI_lesion（-71±8 HU vs.-76±9 HU,P<0.01）.Additionally,the CTA-assessed stenosis severity（OR 1.06,95%CI 1.04–1.08,P<0.01）and FAI_lesion（OR 1.08,95%CI 1.04–1.12,P<0.01）were determined to be independent factors that could predict ischemia.The combination model of these two CTA parameters exhibited a diagnostic value similar to ICA-assessed stenosis severity（AUC:0.82 vs.0.84,P=0.39）.Part II:1.PCAT samples of RCA from 19 patients were prospectively collected and their FAI values were measured.The m RNA expression of IL-1β,IL-6,TNF-αwere analyzed by-ΔCt method.Pearson correlation analysis found that FAI was positively correlated with IL-1β（r=0.61,P<0.01）and IL-6（r=0.48,P=0.04） expression,the correlation between FAI and TNF-αwas non-significant（r=0.18,P=0.45）.2.The paraffin HE staining of the coronary artery-PCAT specimens of the swine showed that compared with the control group,there was a significant inflammatory reaction in the coronary artery and PCAT at 1 week after balloon dilation,and the inflammation was decreased at 4 weeks but still clearly for detecting.For specific,there was massive inflammatory cells infiltration in the coronary artery wall and remarkable inflammatory infiltration and fibrous hyperplasia extending from coronary artery into PCAT.In quantitative analysis,the thickness of coronary artery wall was significantly increased（386±42μm vs.731±148μm vs.528±131μm,P<0.01）.Meanwhile,the size of adipocytes around the coronary artery were significantly smaller（67±16μm vs.41±12μm vs.40±8μm,P<0.01）.Immunohistochemical staining showed that IL-1βprotein had significant positive expression in the coronary artery and PCAT in group B（one week）and lasted to the fourth week after operation（group A）.The results of integral optical density（IOD）indicated that the expression level of IL-1βin the coronary artery and PCAT of the control group,group B and group A were 1.22（0.69,1.66）×10⁴ vs.7.36（5.07,9.75）×10⁴ vs.6.82（5.94,8.23）×10⁴,P<0.01;0.25（0.06,0.80）×10⁴ vs.2.42（0.98,2.62）×10⁴ vs.1.00（0.76,1.60）×10⁴,P<0.01,respectively。3.The m RNA expression level of IL-1β,IL-6,i NOS and TNF-αin coronary artery and PCAT（inner,middle and outer layers）was analyzed by RT-PCR（-ΔCt method）.The results indicated that IL-1β（-8.03±1.85 vs.-3.30±3.84 vs.-4.30±2.07,P<0.01）,IL-6（-8.50±2.48 vs.-2.96±0.61 vs.-5.43±2.08,P<0.01）,i NOS（-6.99±1.77 vs.-2.27±3.77 vs.-4.50±1.76,P<0.01）and TNF-α（-7.40±3.01 vs.-1.15±3.44 vs.-5.25±1.16,P<0.01）m RNA expression of the coronary artery were significantly higher than those in the control group at one week after intervention,and decreased at 4 weeks.In the inner PCAT,the changes of IL-1βand IL-6 m RNA expression were consistent with that in coronary artery tissue.Moreover,the m RNA expression of IL-1β（r=0.44,P=0.03）,IL-6（r=0.78,P<0.01）and TNF-α（r=0.43,P=0.03）in the coronary artery wall and the inner PCAT were significantly correlated.By analyzing the m RNA expressionof IL-1βand IL-6 in the inner,middle and outer layers of PCAT,it was found that there was no different in the IL-1βand IL-6 m RNA expression among each layer of PCAT in the control group.However,the m RNA expression of IL-1β（-5.48±1.39 vs-6.91±0.56 vs-7.28±1.69,P=0.03）was significantly higher in the inner PCAT than that of middle or outer layers,this difference disappeared at 4 weeks after intervention.The expression of IL-6 m RNA also showed this trend,but the difference was not statistically significant.4.There is no significant difference in baseline PCAT attenuation among the three groups of animals（-90.01±5.57 HU vs.-86.37±6.63 HU vs.-87.98±6.19 HU,P=0.51）.In experimental group A（8 vessels of 4 animals）PCAT attenuation was measured at baseline,one week and four weeks after intervention.The results indicated that the PCAT attenuation was significantly elevated at one week after intervention and partially recovered at 4 weeks,but it was still significantly higher than the baseline（-86.37±6.63 HU vs.-53.11±12.71 HU vs.-66.51±6.69 HU,P<0.01）.However,the attenuation of PCAT in the control group did not change among each CTA measurement（-90.01±5.57 HU vs.-87.94±5.92 HU vs.-88.78±5.68 HU,P=0.41）.5.We further investigated the relationship between the PCAT attenuation and the m RNA expression of inflammatory factors in coronary artery and PCAT.The results indicated that the PCAT attenuation was positively correlated with IL-1β and IL-6 m RNA expression whether in the coronary artery（IL-1β:r=0.61,P<0.01;IL-6:r=0.73,P<0.01）or in the PCAT（IL-1β:r=0.50,P=0.01;IL-6:r=0.56,P<0.01）.6.Human aortic endothelial cells（telo HAEC）were treated with active IL-1βprotein（10ng/ml）.In the IL-1βtreated endothelial cells,the expression of e NOS protein and m RNA which contributed to Vaso-dilation was decreased（the protein expression changed significantly at 12h and 24h after treatment）;The expression of endothelin-1（ET-1）and angiotensin-converting enzyme（ACE）protein and m RNA that contributed to Vaso-constriction were increased（ET-1 protein changed significantly at 12h and 24h,ACE protein changed significantly at 12h）,and the m RNA expression of ETAR and AT1R were also increased significantly.Through STRING and KEGG database,it suggested that the hypoxia inducible factor-1（HIF-1）pathway might be involved in the changes of these genes.Then we analyze the expression of the key factor of the HIF-1 pathway（HIF-1α）.The results indicated that HIF-1αprotein and m RNA expression were both significantly increased（the expression of protein increased significantly at 6h after treatment and lasted to 24h）,and the m RNA expressions of downstream genes in HIF-1 pathway（HMOX-1,VEGF and TF）were also increased significantly.Conclusions:1.In patients with intermediate stenosis,EAT volume and mean attenuation could not identify functional ischemic stenosis.However,as an independent predictor of FFR abnormality,the lesion-specified FAI would provide supplemental diagnostic value of functional ischemia in combination with stenosis severity.2.In the animal model of coronary artery inflammation,PCAT attenuation changed covariantly with the m RNA expression inflammatory factors and pathological changes of coronary-PCAT tissue.The FAI（PCAT attenuation）was correlated with the m RNA expression of IL-1βand IL-6 in human（animal）specimens.3.Inflammatory reaction mediated by IL-1βcould affect the balance of vasodilator and constrictor in arterial endothelial cells and tilt them towards the direction of Vaso-constriction.HIF-1 pathway might participate in this pathophysiological process.