Relation Of Monocyte/High-Density Lipoprotein Ratio With Coronary Artery Disease In Type 2 Diabetes Mellitus

Background The number of patients with impaired glucose tolerance and atherosclerotic cardiovascular disease is growing rapidly in China as a result of tremendous lifestyle change and the aging population.Type 2 diabetes mellitus is a chronic metabolic disease characteristic of macroangiopathy.It is indicated that diabetes mellitus is the main independent risk of coronary artery disease(CAD)by epidemiological investigation.Impaired glucose increases the risk of cardiovascular events,and diabetic patients have a worse outcome once the typical coronary artery disease symptoms appear.Atherosclerotic cardiovascular diseases are the leading cause of morbidity and mortality of patients with type 2 diabetes mellitus,and coronary atherosclerosis is the main cause of coronary artery disease,thus,early detection and intervention of CAD may improve the cardiovascular outcomes.However,most of the existing wildly used methods for detecting coronary artery disease are invasive and expensive,which may limit the application in clinical diagnosis.It is indicated by previous study that both atherosclerosis and diabetes mellitus are associated with inflammation,and inflammation plays important roles in endothelium dysfunction,atherosclerotic thrombosis and atherosclerotic plaque rupture.Monocyte,which accounts for 3% to 8% of leucocytes in peripheral blood,posses a key effect on inflammatory process.Activation of monocyte is the important step to initiate the process of atherosclerosis.Activated monocyte interacts with damaged vascular endothelial cell and leads to the overexpression of adhension molecules and pro-inflammation,which plays a crucial role in the development of vascular inflammation and atherosclerosis.High density lipoprotein(HDL)composed of phospholipid,apolipoprotein,cholesterol and fatty acid,is mainly synthesized in liver and partly produced from the metabolism of chylomicron emulsion in small intestine.HDL can help the chylomicron emulsion and very low density lipoprotein(VLDL)metabolize into cholesteryl ester in plasma through the activation of lipoprotein lipase(LPL)and lecithin-cholesterol acyltransferase(LCAT).HDL has anti-inflammatory,antioxidative effects,and it also indicates a role in endothelial function and the process of thrombogenesis.HDL molecules counteract the migration of macrophages and promote efflux of oxidized cholesterol from foamy cells,and inhibit the accumulation of cholesterol on artery walls through the process of reverse cholesterol transport(RCT),which helps prevent the process of atherosclerosis.It is demonstrated by previous evidence-based study that HDL is an anti-atherosclerosis factor and the incidence of CAD declines 2%-3% with the increment of HDL by every 1mg/d L.Low level of HDL is negatively associated with the incidence of atherosclerosis,and it is considered as an independent predictor of CAD.It is found that elevated monocyte to high-density lipoprotein ratio(MHR)may increase the incidence of CAD,but there is no report on the relation of MHR and CAD in patients of type 2 diabetes to date.It is assumed that MRH may have a more significant meaning than the respective monocyte and HDL-C level.Meanwhile,the Ras Homolog Gene Family Memmber A(Rho A)is a growing attractive factor when focused on the study of atherosclerosis.Rho A is a subgroup of the Ras superfamily of small guanine nucleotide-binding proteins and it regulates many cellular activities,including activation of endothelium and endothelium dysfunction.It can also increase the permeability of endothelium and lead to lipid accumulation.Rho A and its downstream effector can be activated in the pathophysiological process of atherosclerosis.Thus,based on the aforementioned pro-inflammation of monocyte and anti-inflammation of HDL-C and the close association between Rho A and atherosclerosis,the present study focuses on MHR and Rho A,investigates the relationof MHR and Rho A,and further investigates the relation of MHR to the incidence of CAD in type 2 diabetes patients based on a predictive model.The receiver operating characteristic(ROC)curve of MHR were used for predicting the presence of CAD in type 2 diabetic patients and it may provide a possible way for early detection for CAD in T2 DM patients.Objective To observe the Rho A express level in the diabetes-atherosclerosis rat model,and to observe the serum Rho A level in type 2 diabetes with CAD patients.To analyze the relation of MHR to Rho A through the method of linear regression,and to further investigate the relation of MHR with the presence of CAD in type 2 diabetes based on a predictive model and determine the cutoff point through ROC curve.Methods1.Experiments on animals(1)32 adult male Wister rats of pathogen-free were assigned to two groups randomly,with 10 in the control group(group A),22 in the observation group(group B).The A group was fed with normal diet and the B group was fed with high fat diet.Intraperitoneal injection of nicotinamide(NA)with dosage of 260mg/kg,caudal injection of STZ with dosage of 60mg/kg and a total dosage of 600,000 IU/kg vitamin D3 was fed to group B(fed in three respective days)to establish diabetic animal model.The group A received an equal dose of normal saline for intraperitoneal injection and tail vein injection.Ultrasound was used to detect the abdominal aorta atherosclerotic plaque to confirm the diabetic model with atherosclerosis at the end of 8th week.Body weight(BW)was measured and all rats were executed to detect the levels of Fasting plasma glucose(FPG),Fasting insulin(FINS),Triglyceride(TG),High-density lipoprotein(HDL),Low-density lipoprotein(LDL).Pathological HE staining and Masson staining were used to observe the changes of vascular wall.(2)Immunohistochemical staining was used to determine the Rho A expression level in rats' abdominal aorta,and computer image analysis system was used to calculate the Integral optical density(IOD)of Rho A.2.Clinical research A total of consecutive 458 individuals were enrolled from The Second Affiliated Hospital of Anhui Medical University,comprising of 178 type 2 diabetic patients,124 type 2 diabetes with CAD,and 156 healthy volunteers as the healthy controls.Demographic,clinical and laboratory characteristics of the study population were recorded,including age,gender,BW,body mass index(BMI),FPG,TCH,TG,LDL,HDL,Hb A1 c,monocyte count.MHR was calculated and Roh A was measured through the method of Enzyme linked immunosorbent assay(ELISA).The relation of MHR with Rho A was analyzed and a multivariable logistic regression model was used to evaluate the relationship between the MHR and CAD in type 2 diabetes,and the receiver operating characteristic(ROC)curve of MHR were used for predicting the presence of CAD in type 2 diabetic patients.3.Statistical analysis Statistical analysis was performed using SPSS software version 16.0.The Kolmogorov-Smirnov method was used to test the distribution pattern.Participants' clinical and biochemical indices were expressed as mean ±SD.The t-test was used to analyze data from two groups and one way ANOVA was used to compare data from three or more sets of data.A multivariate logistic regression model analysis and ROC analysis were used to analyze for MHR as a predictor of the presence of CAD in type 2diabetes,the relationship strength is represented by HR(hazard ratio)value and its 95%confidence interval.A P value <0.05 was considered statistically significant.Results1.Results of the study in rats(1)The level of BW,FPG,TG,LDL and FINS was higher in group B than in the group A,and HDL-C was lower in group B than in group A(P <0.05).(2)Morphology HE staining: In group A,clear demarcations of tunica intima,tunica media and tunica adventitia were observed in the vessel wall of abdominal aorta.The vascular endothelium was smooth,the smooth muscle cells were lined up in media,the structure of the elastic fiber layer was clear and complete,and the outer membrane was loose connective tissue.Contrarily,in group B,obvious intimal hyperplasia and derangement of smooth muscle cells were observed.Degeneration and disintegration of elastic fibers occurred,and pathological change of atherosclerosis appeared.Masson staining: Collagen fiber and elastic fiber were blue,cytoplasms were red and cell nucleuses were blue-brown.In group A,vascular elastic membrane was thin and elastic fibers were equally distributed.However,in group B,vascular elastic membrane appeared thicker,elastic fibers presented derangement distribution with partly rupture.Additionally,extracellular collagen fibers increased obviously.Immumohistochemical staining: Rho A expressed in rat abdominal aorta presented yellow-brown particles after binding to specific antibody.Rho A expression level in group B was higher than that in group A(238.68 ±4.37 vs.769.42 ±6.57,P<0.05).Pearson correlation analysis indicated that there was a negative relationship between Rho A expression level and FPG(r=0.452,P< 0.05),and positive relationship between Rho A expression level and HDL(r=-0.33,P< 0.05).2.Results of the clinical research(1)Compared with DM group and HC group,patients in DM with CAD group had a older age and had a more higher percentage of hypertension,monocyte counts were higher,HDL-C level was lower,MHR and Rho A level was higher(P <0.001).(2)Univariable logistic regression analysis indicated that age,history of hypertension,BMI,MHR and Rho A were related to CAD in type 2 diabetes.(3)When these parameters were included in a multivariate logistic regression analysis,MHR,age and Rho A were independently and significantly associated with CAD(P<0.001),and there was a Linear correlation between MHR and Rho A(in DM group,r=0.2832,P< 0.05,in DM with CAD group,r=0.3204,P< 0.05),and the correlation between Rho A and age was not observed(P> 0.05).MHR is an independent predictive marker for the CAD in type 2 diabetes,and the strength of association between MHR and CAD was represented by hazard ratios and their accompanying95 % confidence intervals.The ROC analysis was performed to determine the cutoff value of MHR(>8.2)in predicting the presence of CAD in type 2 diabetes.The sensitivity value was 83.74%,specificity value was 62.15%,and area under the ROC curve value was 0.795(95%CI: 0.745-0.840;P <0.001).Conclusion1.There is a close relationship between Rho A and atherosclerosis and the Rho A is highly expressed in rat of diabetes with atherosclerosis and in patients of type 2 diabetes with CAD.2.MHR and Rho A is positively related and MHR is an independent indicator for predicting CAD in type 2 diabetes.MRH is a cheap and easily accessible,and might be a potential biomarker to predict the presence of CAD in type 2 diabetic patients.