Experimental Study On Imaging And Inflammatory Features Of Artetiosclerosis And Immune Intervention

Objective:Atherosclerosis（AS）is a leading cause of vascular disease worldwide,accounting for about 17.6 million deaths annually,and is expected to rise to 23.6 million by 2030.Atherosclerosis is the pathological mechanism of cardiovascular diseases.Arterial embolism from rupture of vulnerable plaques is a direct cause of cardiovascular events.Although many treatments were developed to treat cardiovascular disease,its mortality rate is still increasing.Therefore,to clarify the characteristics of the occurrence and development of AS and its underlying mechanisms,and to give hint to new interventions are the primary issues to be urgently addressed to reduce cardiovascular morbidity and mortality in high-risk populations.Inflammation plays as a key factor in plaque rupture and acute cardiovascular events.Although imaging techniques can help identifying vulnerable atherosclerotic plaques,there are still inevitable limitations of the technique.Non-invasive methods,such as plasma inflammatory markers,will be very helpful to identify the exist of vulnerable plaques.Cytokines/Chemokines play a key role in multiple inflammatory diseases,in which interleukin 1（IL-1）,as a major regulator of inflammation,triggers a series of inflammatory mediators by activating IL-1 receptors（IL-1R）.The classical IL-1-IL-6-CRP inflammatory pathway is one of the important mechanisms involving in AS,and IL-6 has been shown to be closely associated with the risk of cardiovascular events and stroke.Macrophage is an important component of AS lesion.The migration,activation,infiltration and proliferation of macrophages and secretion of a large number of proteases lead to the formation and rupture of AS plaques.Microenvironment factors affect the phenotype and function of monocytes/macrophages,thereby promoting the progression of AS lesions.Cytokines related to monocytes/macrophages,such as MCP-1/CCL2,M-CSF,and MIP-1β,are closely related to cardio-cerebrovascular embolism events.In clinical,angiography is the gold standard for the diagnosis of AS,but is not widely used due to invasion,radiation,complexity and high cost.Ultrasound,a simple and feasible examination method,has become the first choice for screening.Carotid Intima and Media Thickness（c IMT）and Plaque indicate systemic AS,c IMT/Plaque is an independent risk factors for cardiovascular disease,shows better value in assessment of AS than traditional risk factors such as smoking,hypertension,hyperlipidemia and others.Ultrasonography indicators and serum biomarkers can reflect the progression of AS from different perspectives,but the current studies on the combination of ultrasound indicators with serum cytokines and other indicators are not comprehensive,and the combination of the two has a great significance for early diagnosis and intervention of AS and prevention of clinical events in high-risk patients.At present,statins are the common treatment for AS mainly by reducing blood lipid,however some people can’t benefit from it,also there are inevitable side effects from long-term drug administration.Over the past two decades,the viewpoint of atherosclerosis has been replaced gradually by a lipid driven,chronic,low-grade inflammatory disease of the arterial wall.Current treatment of atherosclerosis is focused on limiting its risk factors,such as hyperlipidemia or hypertension.However,treatment targeting the inflammatory nature of atherosclerosis is still very limited and deserves further attention to fight atherosclerosis successfully.More recently,the CANTOS trial showed that directly reducing inflammation with canakinumab,an interleukin（IL）-1βneutralizing monoclonal antibody,could reduce IL-6 and CRP level,also reduce secondary cardiovascular event rates in patients with myocardial infarction.There are few studies about IL-1βmonoclonal antibody on peripheral circulating lipid,cytokines,immune cells and plaque composition and still need to be further studied.Vitamin D（VD）is widely known as part of bone pathology/physiology,involved in regulating calcium,phosphorus and bone remodeling.In terms of cardiovascular diseases,VD deficiency is associated the occurrence of AS,supplementation of VD can reduce the risk of embolic events.Other studies have suggested that VD may increase the degree of calcification in the vessel wall and the risk of embolism.In addition,VD can affect the phenotype and function of some immune cells through epigenetic regulation,and monocytes are most significantly affected.Whether supplemental VD has a positive effect on AS and its effect it has on the phenotype and function of innate immune cells during the disease course of AS has not been determined yet,and further studies are still needed.The objective of this subject is to discover the characteristics of ultrasound indicators and circulating cytokines in different stages of carotid artery atherosclerosis（CAS）,and to observe effects of IL-1βantibody and VD on regulating peripheral lipids,monocyte and formation of plaques on established AS mouse model,aiming to provide more information for early diagnosis and treatment assessment.Methods:1.We recruited 140 volunteers who underwent carotid artery ultrasound examination in the First Affiliated Hospital of China Medical University from January to December,2019,include 78 males and 62 females,the average age is 62.58±10.30.Age,gender,body mass index（BMI）,history of smoking and alcohol,systolic and diastolic pressure.Exclusion criteria:（1）hemorrhagic stroke;（2）arterial dissection;（3）arteritis:such as polynodular arteritis,systemic lupus erythematosus;（4）diseases of the blood system:polycythemia eukaryocytosis,leukemia,etc.;（5）history of carotid artery surgery.2.Peripheral blood indicators:triglyceride（TG）,total cholesterol（TC）,low density lipoprotein（LDL）,high density lipoprotein（HDL）,C-reactive protein（CRP）and creatinine.3.Carotid artery ultrasound was performed to measure IMT on long axis plane at 1 cm to the carotid artery bifurcation,carotid artery bifurcation and internal carotid artery,the average was calculated.Echo intensity of plaque was assessed,volume and stenosis area were calculated after the plaque size was measured on double section.According to the degree of carotid atherosclerosis,it is classified into grade I:no AS occurs;Ⅱ:increased IMT but no plaques;Ⅲ:plaque presentation,but no obvious carotid artery stenosis（stenosis rate<30%）.According to the degree of carotid artery stenosis,it was further divided into mild stenosis（<30%）;Moderate stenosis（30-50%）;Severe stenosis（>50%）.Normal IMT<1.0mm;intimal-media thicken:1.0mm≤IMT<1.5mm,plaque:IMT≥1.5mm.According to the echo of plaques,the plaques were classified into low,medium,high and mixed echo intensity.4.48 cytokines/chemokines such as IL-1β,TNF-αwere detected by Luminex technique.5.Apo E^-/-C57BL/6 mice were given a high-fat diet（Western diet）to establish the AS mouse model,the lipid level was tested and aorta arch was observed by HE staining.6.At 4/14/20th weeks after given high-fat diet,part of Apoe^-/-mice received IL-1 beta antibodies intervention（300 ug,i.p.,qod,3 times）,another part of Apoe^-/-mice received VD therapy（0.1 mu g/only,the p.o,day 1,3）from the third week of Western diet.At 6/16/22nd week,peripheral blood and aortic arch were collected,lipid levels were detected,the phenotype of monocytes was detected by flow cytometry.Monocytes were labeled with APC-CY7-anti-CD11b and PE-anti-CD115,and patrol or inflammatory monocytes were differentiated by BV510-anti-LY6C.Ly6C^lo represents patrolling monocytes,non-classical monocytes with low expression of APC-anti-CCR2 and high expression of PE-CY7-anti-CX3CR1.Ly6C^hi represents inflammatory monocytes,while high expression of CCR2 and low expression of CX3CR1 represent classical monocytes.ALEXA-488-anti-CD36 labeled cells foam-like.and plaques formation were observed by HE staining.IL-6 and IL-1 expression of aorta and i NOS⁺/Arg⁺macrophage were assessed by IHC.Results:1.Peripheral IL–1β,IL-6 and TNF-αof CAS patients were higher than normal group.In addition the proinflammatory factors（IL-6,IL-1αetc）increased gradually during AS progression.The anti-inflammation factors IL-4,IL-10 increased in the early stage of AS defined as intimal thickness by ultrasound,so as monocyte chemokines MCP-1,MCP-3,however MIP-1α,M-CSF increased in severe stenosis of carotid artery.2.All of the ultrasonographic parameters were correlated with IL-6,IL-13 and TRAIL,the intima thickness was positively correlated with IL-6,IL-2RA,MIP-1B,IL-1RA,IL-8,MCP-1,G-CSF,HGF,IL-7,IL-12 and TNF-α,and significantly correlated with IL-6 level.The echo intensity of carotid plaque was negatively correlated with IP-10,IL-13 and TRAIL,and positively correlated with IL-6.The degree of carotid artery stenosis was positively correlated with IL-6 and TNF-α,and negatively correlated with IL-13 and TRAIL.The number of carotid plaques was positively correlated with IL-6,and negatively correlated with GM-CSF,TRAIL and IL-13.Carotid plaque volume was positively correlated with IL-6,IL-12 and TNF-α,and negatively correlated with IL-13and TRAIL.3.Apo E^-/-C57BL/6J mice were given the Western diet for 6 weeks,the peripheral HDL-C,LDL-CT,TCHO,TG were significantly increased,the proportion of Ly6C^hi monocytes was increased,the aortic arch pathology showed obvious plaque formation,and the infiltration of immune cells.4.After anti-IL-1βtreatment,TG,HDL,LDL,and TCHO in peripheral blood of mice were significantly decreased compared with the non-intervention group,the overall number of monocytes was decreased,the proportion of CCR2⁺Ly6C^hi monocytes was decreased,CX3CR1⁺Ly6C^lo monocytes were increased,and the expression of CD36 on monocytes was upregulated.Pathology of aortic arch showed no obvious plaque formation and no obvious inflammatory cell infiltration,IHC showed decreased production of both IL-1 and IL-6,also the proportion of i NOS⁺and Arg I⁺macrophages was significantly decreased.5.After VD treatment,LDL,TCHO and TG decreased from the 16^th week and increased at the 22^ndweek.Compared to non-intervention group,the proportion of monocytes in PBMC were higher at the 6^th week but lower at the 22^nd week.The proportion of CCR2⁺Ly6C^hiand CX3CR1⁺Ly6C^lo monocytes were lower than non-intervention group but increased at the 22^nd week,and the expression of CD36 on monocytes were significantly lower than non-intervention group but increased after the 16^th week.HE staining of aortic arch showed less lipid components and inflammatory cells infiltrated in the plaques compared to Western diet fed mice,but the wall of artery was thicker and stiffer than normal group,IHC showed lower IL-1 but no significantly difference of IL-6 between VD group and non-intervention group,the proportion of i NOS⁺and Arg⁺macrophage were decreased in VD group.Conclusion:1.The peripheral cytokines levels increased earlier than the appearance of clinical symptoms,even earlier than the formation of plaques,there was a unique pattern of secretion during the progression of AS.Moreover,they were well correlated with ultrasound indicators,showing consistency in assessment of AS progression,which can provide simpler and more scientific information for the prediction of the occurrence and development of AS.2.In atherosclerosis mouse model,IL-1βantibody and VD could reduce the formation of plaque,regulate the level of lipid,increased the proportion of anti-inflammatory monocytes,lowered the inflammatory level of artery wall,and comprehensively inhibited the infiltration of immune cells into the subepithelial layer.In addition,both IL-1βantibody and VD could up-regulate the level of CD36 in monocytes,so that helped to reduce the level of blood lipid in the precursor of anti-inflammatory environment.