Clinical And Basic Study Of The Influence On The Stability Of Atherosclerosis Plaque Caused By Decrease Of Tissue Factor Pathway Inhibitor 2 Expression

BackgroundAtherosclerotic disease has become one of the major diseases that threats to people’s health, the instability of atherosclerosis plaque is the main cause of unstable angina, acute myocardial infarction, and sudden cardiac death. Now the main hypothesis is that during the rupture of atherosclerotic plaque, the thrombogenic lipid core has exposed, resulting in thrombosis and ischemic cardiac events, such as acute coronary syndrome. The unstable fibrous cap of the plaque plays an important role in plaque development and disruption. The structural integrity of fibrous cap seems to depend on a balance between synthesis and degradation of the extracellular matrix depends on the (ECM). Matrix metalloproteinases (MMPs) are important proteinases which regulated the synthesis and degradation of ECM, so they are closely related to the instability of atherosclerotic plaque.Tissue factor pathway inhibitor 2 (TFPI-2) is a Kunitz-type serine protease inhibitor, mainly existed in endothelial cells and fibroblasts extracellular matrix (ECM). TFPI-2 is a strong endogenous inhibitor of MMPs, which plays a key role in maintain and regulate the extracellular matrix remodeling. TFPI-2 could directly inhibit the activity of MMP infront of other protease inhibitors; meanwhile it could also prevent pro-matrix metalloproteinase (proMMPs) that exist in the ECM turns into MMPs through inhibiting plasmin and trypsin; that is an indirect way to regulate MMPs. Our clinical studies in the past suggest that TFPI-2 level is lower in the peripheral blood of patients with acute coronary syndrome, and we found that in the animal experiment TFPI-2 expresses lower in the shoulder area of the vulnerable plaque, which has a negative correlation with MMPs.We speculated that the decrease of expression of TFPI-2 may be one of the pathophysiological basis of atherosclerotic plaque development. So we designed both clinical and basic studies to investigate the relationship between TFPI-2 level and atherosclerotic plaque stability and trying to clarify its mechanism.Part I:Clinical study of the association between TFPI-2 gene polymorphisms and acute coronary syndromeObjective Investigation of the relationship between TFPI-2 gene polymorphism and acute coronary syndrome (ACS) in Chinese Han population.Method The polymorphism of TFPI-2 gene was detected by PCR-sequencing in all people recruited, and a case control study was carried out in three groups, the ACS group consisted of 140 ACS patients (including 68 unstable angina patients and 72 acute myocardial infarction patients), the SA group included 273 SA patients, and the control group consisted of 306 healthy people.Results:The study enrolled a total of 719 cases, including 140 cases of ACS group (including 68 cases of unstable angina and 72 cases of acute myocardial infarction); 273 cases of stable angina group; 306 cases of control group. Eight single nucleotide polymorphisms of TFPI-2 gene were found in all the objects, they were rs3763473, rs59805398, rs60215632, rs59999573, rs59740167, rs34489123, rs4517, rs4264. Which rs59805398 C allele was statistically significant differences (p= 0.0001; p<0.05) between ACS group and control group; so is rs34489123 G allele between ACS and control groups (p=0.015; p<0.05); rs59805398 C allele frequency had statistically significant differences (p= 0.0001; p<0.05) between the SA group and the control group, so is the rs34489123 G allele (p= 0.0001; p<0.05). rs3763473, rs59999573, rs59740167, rs34489123 show linkage disequilibrium in the ACS group, associated analysis with rs59805398 suggest two main CCGGG and TCGGGhaplotypes.rs59999573, rs59740167, rs34489123show linkage disequilibrium in the SA group, association analysis with rs59805398, there suggest two haplotypes:CGGG and GAAA.There were no significant differences between the ACS group and the SA group.Conclusions:There are 8 SNPs have been detected from all the cases.C allele in rs59805398 and A allele in rs34489123 have correlation with CHD, implying that they might be a higher risk factor of CHD among Chinese Han population. There is apparent linkage disequilibrium among SNPs of TFPI-2 gene in CHD patients.Part II Experimental study of the influence on atherosclerotic plaque stability caused by down regulation of tissue factor pathway inhibitor 2 expression1. The establishment of vascular endothelial cell TFPI-2 conditional knockout mice modelObjective To construct the vascular endothelial cell TFPI-2 conditional knockout mice model for the studying of TFPI-2 and the relationship with atherosclerosis plaque stability.Methods Recombined tissue-specific Cre/LoxP system is used to construct a vascular endothelial cell TFPI-2 conditional knockout mouse model. Extract the mice genomic from tail tissue; identify the genotype of mice through PCR. Finally we get 6 TFPI-2-/+/Tek-cre mice as the experimental group, the control group contains 6 C57BL/6J mice which have the same background and 6 ApoE-/-mice; every two weeks all the three groups of mice take 200ul blood each and separation for the serum to test the level of TFPI-2.The 20 weeks old mice are sacrificed to get aortic vascular tissue, the expression level of TFPI-2 was detected by western blot to verify the knockout efficiency.Results We successfully get 6 vascular endothelial TFPI-2 conditional knockout mice model, which genotypes is TFPI-2fl/-/Tek, the serum TFPI-2 level of this group is about 300-400pg/ml, significantly lower than the C57BL/6J and ApoE-/-group (P <0.05). Western blot show that vascular TFPI-2 expression levels of TFPI-2fl/-/Tek group is significantly lower than the C57BL/6J and ApoE-/-group, and the vascular TFPI-2 expression levels of ApoE-/-group is lower than the C57BL/6J group.Conclusions The vascular endothelial TFPI-2 conditional knockout mouse model was successfully established.2. The impact to the stability of atherosclerotic plaques on the vascular endothelial cell TFPI-2 gene conditional knockout mice.Objective Using the method of high-fat diet feeding induces atherosclerosis on the vascular endothelial cell TFPI-2 conditional knockout mice, analyze the morphological indicators of the stability of atherosclerotic plaques.Methods Six 4-6 weeks old ApoE-/-mice,6 C57 mice and 6 TFPI-2fl/-/Tek mice were divided into three groups, feeding with high-fat diet to induce atherosclerosis. The mice were sacrificed after 20 weeks old, morphological indicators of plaque instability are detected with HE staining, elastic fiber staining of aortic plaque tissue sections, to make clear the impact of AS plaque instability after the contional knockout of TFPI-2 gene..Results Three groups of mice show no difference in body weight, but the TC TG、 LDL-C、HDL-C level of ApoE-/-group was significantly higher than C57BL/6J group and TFPI-2+/-group(P<0.05); there is no difference in blood lipids between the C57BL/6J group and TFPI-2+/-group, the inducement of atherosclerosis mice model is successful. Plaque stability analysis showed:TFPI-2+/-group and ApoE-/-group was significantly higher than C57BL/6J group(P<0.05)in the lumen area, plaque area, lipid core size, fibrous cap layers and the number of plaque rupture; while the fibrous cap thickness of this tow group is less than the C57BL/6J group (P<0.05); Campared with ApoE-/-group, TFPI-2+/-group has small plaque area, lipid core size, less fibrous cap layers and number of plaque rupture, but has higher fibrous cap thickness(P<0.05), therefore TFPI-2+/-plaque is much stable than the ApoE-/-group, but less than the C57BL/6J group.Conclusions Compared with ApoE-/-group, downregulate the TFPI-2 level of vascular endothelial cell can increase the instability of atherosclerotic plaque.3. A preliminary research on the impact of mice atherosclerotic plaques instability caused by TFPI-2 expression downregulation and its mechanism.Objective The vascular endothelial cells TFPI-2 conditional knockout mice were induced with atherosclerosis, its atherosclerotic plaque stability are between the ApoE-/-mice and C57BL/6J mice.By detecting the expression of MMPs and TFPI-2 in the plaque with IHC and western blot, we want to explore the mechanism of the impact on plaque instability caused by TFPI-2 downregulation.Method The expression levels of MMPs (MMP-2, MMP-9) in the aorta specimens of 3 groups are detected by immunohistochemical staining; western-blot are used to detect activation level of 3 pathway which close to plaque stability:NF-K B, PPAR-γ and PI3Ky/PKB.Results The average OD of TFPI-2 in AS plaques of TFPI-2-/+group are 992±72, which are significantly lower than the ApoE-/-group 1336±171 and C57BL/6J group 1609±210, with statistical difference (P<0.05); The ApoE-/-group is also lower than the C57BL/6J group, with statistical difference (P<0.05). Meanwhile, the average OD of MMP-2 in AS plaques of TFPI-2-/+group are13260±1844, significantly higher than the ApoE-/-group 10385±1183 and C57BL/6J group 10087±1344 (P<0.05), the average OD of MMP-9 in AS plaques of TFPI-2-/±group are 11098±1186, significantly higher than the ApoE-/-group 9048±952 and C57BL/6J group 9184±1297(P<0.05). Western blot shows that the phosphorylation levels of pathway PPAR-y is decreased in TFPI-2-/-group compared with ApoE-/-and C57BL/6J group, while PI3Ky/PKB, NF-κB pathway activity did not change significantly. Which remind us downregulate of TFPI-2 levels inhibit the activity of PPAR-y pathway.Conclusions The mechanism of the increasing atherosclerotic plaque instability caused by downregulation of TFPI-2 levels in the mice endothelial cells may be related to it’s weaken inhibition of MMP-2,9 and suppression of PPAR-y pathway.