The Study Of Correlation Between Several Inflammatory Factor Detection And Coronary Vulnerable Plaque

Background:During atherosclerosis initiation and progression, a common underlying cause of acute cardiovascular syndromes, such as myocardial infarction, is erosion or rupture of an unstable atherosclerotic plaque, which selectively increases circulating classical monocyte counts and induces phenotypic changes that facilitate their migration into atherosclerotic lesions.The inflammatory response continues as monocyte-derived macrophages, dendritic cells and a subset of T cells migrate into the subendothelial area.Inflammation in atherosclerosis and coronary plaque vulnerability plays a key role.Studies have shown that macrophages in atherosclerotic plaque composition within the lesion determines the process of atherosclerosis, plaque stability, closely related to the occurrence of thrombosis.Studies have also indicated that coronary atherosclerosis were inflammation and endothelial cell dysfunction of the initiating event. Involved a series of inflammatory cytokines such as IL-1β、IL-6, IL-18 and TNF-a;Matrix metallo protein as protein MMP-9;Chemokines:MCP-1, IL-8;Cell adhesion molecules:ICAM-1, VCAM-1; E-selectin; P-selectin.For the majority of the current detection biomarkers femoral artery sheath is used to evaluate blood inflammatory cytokines and ACS relevance. Because the peripheral blood of many confounding factors, leaving the level of various factors and not an accurate degree and extent of plaque vulnerability partial response of coronary artery disease.Objective:However, a variety of markers to predict plaque rupture and acute cardiovascular events were not satisfactory sensitivity and specificity.Although, we found many biomarkers, but not yet found a way to accurately determine the atherosclerotic disease and specific markers of vulnerable plaque.Methods:1. Serum levels of NFκB (p65) in patients with coronary artery disease1.1 In the present study,we recruited 105 consecutive patients who underwent elective CAG for suspected CAD at our department. Coronary artery blood samples were collected while CAG or PCI for NFκB (p65); hs-CRP and Lp-PLA2 measurements by ELISAs.1.2 In another pilot study,95 patients consecutively hospitalized in our department with a diagnosis of angina pectoris were included. Subgroup of CAD were categorized as UAP and SAP. Control subjects comprised of age-and sex-matched healthy volunteer blood donors. Blood levels of the NFκB (p65); hs-CRP and Lp-PLA2 were compared between the two groups and control group, respectively.1.3 A Pearson correlation analysis was performed to identify independently associated with the presence of coronary artery disease of blood levels of the NFκB (p65); hs-CRP and Lp-PLA2.1.4 Receiver operating curves were plotted to evaluate and compare the accuracy of NFκB (p65) and hs-CRP as diagnostic biomarkers to predict UAP.2. Biological effects of NFκB (p65)THP-1 macrophages were stimulated with different concentrations of NFκB (p65) (vehicle,50ng/1,100ng/1,500ng/l and 1000ng/l),supernatants were harvested at different time points (12 hours and 24 hours). THP-1 macrophages were pretreated with PTDC (10umol/l) for 30 minutes before incubating with NFκB (p65) at the concentration of 500ng/ml for 24 hours, supernatants were harvested for measurement. Levels of IL-1β,IL-6,IL-18,TNF-a,MMP-9 in supernatants were examined by ELISA. HUEVCs were stimulated with different concentrations of NFKB(p65)(vehicle,50ng/l, 100ng/l,500ng/l and 1000ng/l)12hours or 24 hours. Levels of MCP-1 and IL-8 in the supernatants were measured by using ELISAs (24hours). Total cellular expression of ICAM-1; VCAM-1; E-seletin and P-seletin were assessed by cellular ELISA. For chemotaxis assays, THP-1 cells were seeded at 2×105/ml into the upper well of the chambers. Increasing concentrations of NF-κB (vehicle,50ng/l,100ng/l,500ng/l and 1000ng/l) was added to the lower well of the chambers. Incubated and allowed to migrate through the membrane for 30 minutes in 5% CO2 at 37℃. Then the upper chambers were sequentially washed, fixed with 4% paraformaldehyde and stained with haematoxylin. Cells that had migrated through the membrane were counted using a microscope in five randomized high-power fields, and the results were given in mean number of cells per high-power field. In separated experiments,20ug/ml neutralizing antibody of anti-NFκB (p65) were added into the lower chambers before addition of NFκB (p65) (500ng/l) in the lower chamber and THP-1 cells in the upper chambers. Incubated and allowed to migrate through the membrane for 30 minutes in 5% CO2 at 37℃.Then stained and counted according to procedure above-mentioned.Results:1. Compared with control group, the level of NFκB(p65), Lp-PLA2 and hs-CRP in CAD patient group were significantly higher. The level of NFκB (p65) levels were 89.92±29.67ng/1 vs 477.77±108.63ng/1, P<0.001; in control and CAD patients group. The level of Lp-PLA2 in normal control group compared with patients with CAD were 12.31±5.17 IU/ml vs 18.00±6.22 IU/ml, P<0.05; hs-CRP levels in the serum levels of CAD patients and controls were 4.90± 0.97mg/1 vs 1.84±0.35mg/1, P<0.0035.2. The level of serum NFκB (p65); hs-CRP and Lp-PLA2 levels were significantly increased in UAP and SAP compared with healthy control group. Particularly in the UAP group the most significantly increased. The level of NFκ(p65) in SAP was 345.2±96.30 ng/1, the level of NFκB(p65) in UAP was 565.0±128.1ng/1, and the level of control group was 98.9±44.9 ng/1, P<0.001. The level of Lp-PLA2 in SAP group was 15.23+7.38 IU/ml. The level of Lp-PLA2 was 19.87 ±8.12IU/ml in UAP group. The level of Lp-PLA2 in control group wasl2.89± 6.15 IU/ml, P<0.001. The level of hs-CRP in SAP group was 2.43±3.31mg/1; the level of hs-CRP in UAP group was 6.62±13.24mg/1; the level of hs-CRP in the control group was 1.85±1.68mg/1,P<0.001.3. NFκB (p65) with hs-CRP levels showed a significant positive correlation in UAP patients (r= 0.473, P-0.001). However, NFκB (p65) levels with hs-CRP but no significant correlation in patients with SAP(r=0.287, P=0.185). Lp-PLA2 levels showed no correlation with both NFκB (p65) and hs-CRP in UAP and SAP.4. The area under the ROC curve analysis of NFκ(p65) were 0.93 (95% CI: 0.89-0.97, P<0.001) and hs-CRP 0.80 (95% CI:0.69-0.89, P<0.001). But compared to hs-CRP is concerned, NFκB (p65) are more accuracy in the diagnosis of UAP respect, NFκB (p65) AUC-hs-CRP AUC= 0.145, P=0.002). In this study,427.6 ng/1 of NFκB (p65) concentration can be used as a diagnostic value of UAP (cutoff-value), the serum NFκB (p65) diagnosis UAP sensitivity and specificity were 85.5% and 82.8%.5. In the THP-1 macrophages, as NFκB (p65) concentration gradually increased stimulation, cell supernatants IL-1β, IL-6, IL-18, TNF-a,MMP-9 level also increased gradually, and this effect is to stimulate the most significant 24 hours. NFκB (p65) inhibitor PTDC (10umol/1) can effectively suppress or even completely eliminate the NFκB (p65) induced THP-1 macrophages supernatant secretion of IL-1 p, IL-6, IL-18, TNF-a,MMP-9 (P<0.05).6. In the THP-1 macrophages, as NFκB (p65) concentration gradually increased stimulation, cell supernatants MCP-1 and IL-8 level also increased gradually. NFκB (p65) induced a dose-dependent expression of ICAM-1 on HUEVCs surface. NFκB (p65) for the regulation of VCAM-1 also has a similar trend.HUEVCs cellular protein levels of E-seletin with NFκB (p65) concentration increased with increasing application of different concentrations of NFκB (p65) stimulation. Similarly, the P-seletin secretion HUEVCs also having NFκB (p65) concentration dependence.7. Transwel experiment results showed that the different concentrations of Nuclear transcription factor NFκB (p65) on THP-1 monocytes (from 50ng/1-1000ng/1) were significant chemotactic activity (P<0.05). NFκB (p65) neutralizing antibody (final concentration 20ug/ml) was added to the lower chamber is able to completely block the NFκB (p65) (500ng/1) induced chemotaxis of THP-1 cells (P<0.05).Conclusion:1. NFκB (p65),hs-CRP and lp-LPA2 was upregulate in the atherosclerotic plaque raised locally both in patients with UAP and SAP, particularly in the UAP group.2. NFκB (p65) regulated the expression of inflammatory cytokines such as IL-1P; IL-6; IL-18; TNF-a in THP-1 macrophages.3. NFκB (p65) induced secretion and expression of endothelial cell chemistry chemokine (MCP-1, IL-8) and adhesion molecules (ICAM-1, VCAM-1, E-seletin and P-seletin) in HUVECs,chemically enhanced monocyte chemotaxis and secretion mechanism of regulation of monocyte/macrophage matrix metalloproteinase MMP-9, and the activation of endothelial cells and monocytes/ macrophages, enhancing inflammation and extracellular matrix degradation, involved atherosclerosis and plaque destabilization process.4. NFκB (p65) can be used as a biological marker to predict the short-term prognosis in patients with unstable angina.5. NFκB (p65) can be used as a new target for the treatment of coronary heart disease.