| BackgroundThe same pathophysiologic basis of Coronary atherosclerotic heart disease (CAD) is the formation of atherosclerotic plaque (AS plaque). Acute coronary syndrome (ACS) is the critical type of CAD. Lots of studies have found that sudden changes of AS plaque may lead to most acute cardiovascular events. Therefore, the research on the stability of AS plaque and the risk assessment of ACS has gradually been a hotspot.In the progression of AS, the integrity of the fibrous cap rely most on the entracelluar matrix (ECM). As the dominant ingredient of ECM, collagen(Ⅰ/Ⅲ)’s reduced synthesis or excessive degradation may result in thin fibrous cap and vulnerable plaque. Prolyl 4-hydroxylase is the key enzyme of collagen synthesis, and a is the rate-limiting subunit. The recent study have found that TNFa may down-regulate Collagen via P4Hα1. P4Hal can either stable the mature plaque or accelerate the progression in the early stage. Argonaute (AGO) is a protein family which exists widely in organisms. The representative Ago2 is the major element of RNA-induced silencing complex (RISC), which can bind with mature microRNA (miRNA) and regulate the functions such as the silencing of targeted protein. In the peripheral blood, Ago2 can also be the carrier of circulating miRNAs in case being degraded by RNase. Several circulating and vascular related miRNAs have been found while the relationship of Ago2 and ACS has not been reported. Hank H et. have found that in 293ET cells and mouse embryonic fibroblast cells, Ago2 can be hydroxylated by P4Hal at proline 700 and the non-hydroxylated Ago2 shows reduced stability and activity. While the relation of P4Hα1 and Ago2 in human aortic smooth muscle cells (HASMCs), and whether Ago2 contributes to the P4Hα1-Collagen pathway are still to be investigated. On the other hand, the correlation of peripheral P4Hα1, Ago2 and the severity of ACS is also to be studied.Objectives1. To investigate the correlation of peripheral P4Hα1, Ago2 and the severity of ACS.2. To investigate the relation of P4Hal and Ago2 in HASMCs, and whether Ago2 participate in the TNFa-P4Hal-Collagen pathway.Methods1. We included 22 patients of acute myocardial infarction (AMI),22 patients of unstable angina (UA) and 22 normal healthy people from 2005.7.21 to 2005.11.23 in Qilu Hospital of Shandong University. The general information and blood samples are collected, in which the AMI team not only contains the 24h blood samples (24 MI), but also the samples of 7 days after AMI (7-day MI). The total sample number is 88. The samples were tested by enzyme linked immunosorbent assay (Elisa) and acquire the level of P4Hα1 and Ago2 in plasma. The data were analyzed by IBM SPSS 23. Numerical variable data were compared by analysis of variance (ANOVA) and linear correlation analysis. The level of statistical significance was set at p<0.05 and |r|<0.4 means low correlation,0.4≤|r|<0.7 means moderate correlation while |r|>0.7 represents significant correlation. We also used the ROC line to analyze the sensitivity and specificity of the target.2. The HASMCs were cultivated and the generation of 8-10 were selected. The group set is Lenti-P4Hα1 virus group, P4Hα1 siRNA group, N.C and blank group. We use western blot to test the protein level of P4Hα1 and Ago2.3. The HASMCs were cultivated and the generation of 8-10 were selected. The group set is Ago2 siRNA group, TNFa group and blank group. All the groups are transfected with Lenti-P4Hal virus. We use western blot to test the protein level of P4Hα1, Ago2 and collagen Ⅲ.ResultsThe results of blood samples1. The ELISA date of plasma Ago2 and P4Hα1 with linear correlation analysis With Kolmogorov-Smirnov test, the Ago2 level is in accordance with normal distribution (p=0.200,>0.05). The mean is 0.4085±0.008ng/ml. The median is 0.4089ng/ml. The range is 0.03ng/ml. The inter-quartile range is 0.01ng/ml. With Pearson linear correlation analysis, the plasma level of P4Hal and Ago2 is significant correlated (r=0.876, p<0.01).2. The general information analysis between different clinical types There are no significant difference in age, sex, Cr and systolic pressure (p>0.05) between different clinical types. There are no significant difference in TG, TC, LDL-C, HDL-C (p>0.05).3. The ANOVA of plasma level of P4Ha1 and Ago2 between different clinical types(1) Ago2:The MI level is significantly higher than the UA and the normal healthy people (p<0.01). The UA level is significantly higher than the normal healthy people (p<0.01). There are no significant difference between the 24h MI and the 7-days MI (p>0.05).(2) P4Hα1:The MI level is significantly higher than the UA and the normal healthy people (p<0.01). The UA level is significantly higher than the normal healthy people (p<0.01). The 24 MI level is significantly higher than the 7-day MI (p<0.01).4. The linear correlation analysis of Ago2 and GRACE risk score With Pearson linear correlation analysis, the plasma level of Ago2 moderately correlate with GRACE risk score (r=0.547, p<0.01).5. The linear correlation analysis of Ago2 and Gensini score With Pearson linear correlation analysis, there are no linear correlation between the plasma level of Ago2 and the GRACE risk score (p>0.05).6. The linear correlation analysis of Ago2 and popular serologic markers of CAD With Pearson linear correlation analysis, Ago2 moderately correlate with CK (r=0.451, p<0.01). Ago2 moderately correlate with CK-MB (r=0.441, p<0.01). Ago2 moderately correlate with LDH (r=0.575, p<0.01). Ago2 significantly correlate with NT-proBNP (r=0.782, p<0.01). Ago2 moderately correlate with cTnI(r=0.592,p<0.01).7. The sensitivity and specificity of Ago2 in MI diagnosis The underline area of the ROC line is 0.869. The sensitivity is 77.3% and the specificity is 86.4%. The critical value is 0.4135ng/ml.The results in HASMCs1. Western blot shows that in HASMCs, overexpression of P4Hal may lead to the up-regulation of Ago2 protein, while reduced P4Hal cause the limited Ago2 expression.(p<0.05)2. Western blot shows that in HASMCs, Ago2 siRNA may lead to reduced collagen Ⅲ while overexpression of P4Hal.(p<0.01)3. Western blot shows that in HASMCs, with Lenti-P4Hal transfected, TNFa 24h may lead to significant reduced P4Hα1, Ago2 and CollagenⅢ. (p<0.01)4. Western blot shows that in HASMCs, with TNF a 24h cultivated, Ago2 siRNA may lead to significant reduced Ago2 and CollagenⅢ. (p<0.01)Conclusions1. The ACS plasma level of Ago2 and P4Ha1 is higher than the healthy people while the MI is higher than the UA group. Ago2 and P4Ha1 is significantly correlated. Ago2 moderately correlate with CK, CK-MB, LDH, cTnI, and significantly correlate with NT-proBNP. All the results indicate the potential of Ago2 to ACS risk assessment.2. P4Hal may up-regulate Ago2 in HASMCs, and Ago2 participate in P4Ha-collagen regulation. Ago2 participate in the regulation of Collagen by TNF. |
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