Study On The Relationship Between Plasma High - Density Lipoprotein Fraction And Inflammation In Patients With Coronary Heart Disease

Part 1. Correlations between plasma levels of inflammatory markers and HDL components in patients with coronary artery diseaseBackgroundIt has been widely accepted that inflammation is involved in coronary artery disease. Increasing number of observational studies confirmed the relationship between plasma inflammatory biomarkers and CAD. High plasma levels of these markers usually predicts poor prognosis in acute myocardial infarction. However, the correlations between these acute phase proteins/pro-inflammatory cytokines and stable coronary artery disease (sCAD) have not been identified. Even the mechanisms of inflammation contributing to CAD were not completely clarified. High-density lipoprotein (HDL) exerts atheroprotective effects through its cholesterol efflux promotion, anti-inflammation and direct endothelial protection. Low high-density lipoprotein cholesterol (HDL-C) level was related to higher risk of cardiovascular disease (CVD). However, recent data confirmed that patients with higher HDL-C levels were still at risk of acute myocardial infarction (AMI). and the value of plasma HDL-C predicting cardiovascular events in CAD patients was even lower. All of these results suggested attenuated protective effects of HDL in CAD patients. Since the long-term inflammatory state causes alterations in HDL levels and compositions, it is considered as an important cause leading to the dysfunctional HDL in CAD patients. As an acute phase protein mainly produced in the liver, levels of serum amyloid A (SAA) both in plasma and HDL compositions increase markedly when inflammation. The elevated levels of SAA within HDL may render it pro-inflammatory. Therefore, we supposed that HDL-associated SAA was related to the inflammatory markers in CAD patients, which has not been widely recognized.Objectives1. Compare plasma levels of acute phase protein and pro-inflammatory cytokines among patients with stable coronary artery disease, acute coronary syndrome and non-CAD controls.2. Analyse the associations between CAD and plasma levels of these inflammatory biomarkers.3. Assess the diagnostic value of these inflammatory biomarkers combined in CAD patients.4. Analyse the correlations between plasma HDL-C level or HDL-associated SAA and these plasma inflammatory markers.Methods1. Clinical data and plasma sample collection:Overall,116 CAD patients diagnosed by coronary angiography were enrolled in our study. Another 65 age and gender matched non-CAD controls were also enrolled. CAD patients with stable angina pectoris were enrolled in stable coronary artery disease subgroup (sCAD, n=42), and unstable angina pectoris or AMI patients were enrolled in acute coronary syndrome subgroup (ACS, n=74).2. Laboratory test:Fasting venous blood was drawn from all subjects. And HDL sample were isolated from fresh venous plasma through density ultracentrifugation. BCA method was used to test the protein amount, and SDS-PAGE was used to confirm the apoA-I abundance within HDL sample. ELISA method was used to detect the plasma levels of al-antitrypsin (AAT) and serum amyloid A (SAA). Plasma levels of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) were measured using liquid chip technology. And SAA contents within HDL sample were also detected using ELISA method.3. Data analysis:Differences in plasma levels of inflammatory markers among groups were compared using one-way ANOVA. Logistic regression analysis was performed to detect the association between CAD and these inflammatory biomarkers. Reveiver operating characteristic curves were constructed to evaluate the diagnostic value of combined or single inflammatory biomarker in CAD. Pearson or Spearman correlation models was used to analyse the correlations between plasma HDL-C or HDL-associated SAA levels with these inflammatory markers above.Results1. Compared with non-CAD controls, the plasma levels of hsCRP, AAT, SAA, IL-6 and MCP-1 were significantly higher in ACS patients [hsCRP:2.59 (1.07-5.31) mg/L vs.1.21 (0.70-2.07) mg/L,P<0.001; AAT:151.55±22.17mg/dl vs.137.35±12.67 mg/dl, P=0.005; SAA:9.54 (2.92-24.50)μg/ml vs.4.99(2.53-9.88)μg/ml, P<0.001; IL-6:2.37(1.57-6.30) pg/ml vs.1.77 (1.175-2.62) pg/ml, P<0.001; MCP-1:305.26 (244.96-373.29) pg/ml vs.281.29 (225.74-344.01) pg/ml, P=0.013]. Patients with sCAD exerted higher plasma levels of hsCRP, AAT, SAA and IL-6 [hsCRP:2.20 (1.27-3.45) mg/L vs.121 (0.70-2.07) mg/L,P=0.001; AAT:148.90±26.36 mg/dl vs.137.35±12.67 mg/dl, P=0.047; SAA:7.04 (4.26-15.63)μg/ml vs.4.99(2.53-9.88)μg/ml,P=0.004; IL-6:2.46 (1.66-4.01) pg/ml vs.1.77 (1.175-2.62) pg/ml, P=0.026]but not MCP-1 than the controls[264.15 (215.35-391.30) pg/ml vs.281.29 (225.74-344.01) pg/ml,P=0.213]. However, the difference between ACS and sCAD was not statistically significant (all P>0.05).2. Multiple varaibles Logistic regression analysis showed that plasma levels of IL-6 and SAA correlated with CAD risk after adjusting traditional cardiovascular risks (IL-6:OR=5.384, 95%CI:1.194-24.283,P=0.029;SAA:OR=5.205,95%CI:1.326-20.423, P=0.018).3. Plasma levels of hsCRP, IL-6 and SAA were associated with the severity of coronary leision in CAD patients (hsCRP:r=0.274,P=0.004; IL-6:r=0.210,P=0.027; SAA:r=0.205,P=0.025).4. Combined these inflammatory biomarkers detection had a medium value in CAD diagnosis[area under the curve (AUC)=0.849,95%CI:0.776-0.906] with a sensitivity of 63.3%, and specificity of 88.2%.5. Correlation between plasma HDL-C and hsCRP was only observed in the general study population instead of in CAD patients (in general population:r=-0.192, P=0.011; in CAD patients:r=-0.132, P=0.165). However, HDL-associated SAA levels correlated with all of these inflammatory markers above in CAD patients (hsCRP:r=0.584, P<0.001; AAT:r=0.221, P <0.001; SAA:r=0.740,P<0.001; IL-6:r=0.412, P<0.001; MCP-1:r=-0.242, P=0.009).ConclusionsCAD patients exerts chronic inflammatory state. Both sCAD patients and ACS patients display significantly higher levels of plasma pro-inflammatory cytokines and acute phase proteins. Elevated plasma levels of IL-6 and SAA independently correlate with the risk of CAD. Combined detection of multiple inflammatory biomarkers might improve the diagnostic value in CAD. HDL-associated SAA levels shows a better correlation with inflammatory markers, indicating that HDL compositional alterations associated with chronic inflammation and might be a novel target in CAD diagnosis and treatment.Part 2. HDL from CAD patients promotes inflammatory activation in endothelial cellsBackgroundPlasma high-density lipoprotein (HDL) has always been considered as the most important cardioprotective lipoprotein. HDL from healthy individuals effectively prevents endothelial injury and inflammation caused by atherogenic factors. Physiological functions of HDL depends on its normal components and contents. However, recent data confirmed that plasma HDL exerted decreased protection when some diseases. And inflammation may be one of the reasons. Coronary artery disease (CAD) is an inflammatory disease. HDL isolated from CAD patients could not protect endothelium effectively, but the mechanism has not been clarified. Compositional modifications within HDL associated with chronic inflammatory state may partly explain. As an acute phase protein, serum amyloid A (SAA) was confirmed to be upregulated both in plasma and HDL components in our previous studies. Therefore, we speculated that SAA accumulation within HDL may contribute to its decreased endothelial protection in CAD patients.Objectives1. Compare the differences of plasma HDL isolated from CAD patients and non-CAD controls in activating endothelial inflammation.2. Explore the possible signalling pathways contributing to HDL pro-inflammation in CAD patients.3. Assess the role of SAA accumulated within HDL components contributing to HDL pro-inflammation in CAD patients.MethodsTotally,12 confirmed CAD patients and another 10 age and gender matched subjects without CAD were enrolled in our study. HDL was isolated from fresh plasma of both CAD patients (HDLCAD) and non-CAD controls (HDLcontrol) using the ultracentrifugation technique. Serum amyloid A (SAA) abundant HDL (HDL-bound-SAA) was prepared by incubation plasma from controls with recombinant SAA (rSAA) and then ultracentrifugation. Human umbilical vein endothelial cells (HUVECs) were respectively exposed to 100μg/ml HDLCAD, HDLcontrol and HDL-bound-SAA. And the inflammatory activations of endothelial cells were observed. (1)Nuclear translocation of nuclear factor-KB (NF-κB) p65 was assessed by laser scanning confocal microscope. (2)Realtime-PCR and ELISA were used to assess the expression and secretion of pro-inflammatory cytokines such as interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1).(3)Realtime-PCR and Western blot were used to assess the expression of intercellular adhesion molecule-1 (ICAM-1) and Toll-like receptor 2 (TLR2). To further explore the associated mechanisms of the pro-inflammatory effects of HDLCAD, specific TLR2 neutralizing antibody and p38MAPK inhibitor SB203580 were used to block the intracellular activity of TLR2 and p38MAPK pathway, respectively.Results1. Different from non-CAD controls, HDL isolated from CAD patients induced nuclear translocation of NF-κB p65, significantly upregulated the synthesis and secretion of IL-6 and MCP-1 and the expression of ICAM-1 in cultured HUVECs.2. Both TLR2 neutralizing antibody and p38MAPK inhibitor SB203580 effectively inhibited the synthesis and secretion of IL-6 and MCP-1 and the expression of ICAM-1 induced by HDLCAD, indicating that the pro-inflammatory effects of HDL in CAD patients were mediated by TLR2 and p38MAPK signaling pathway.3. Different from HDLcontroi, HDL-bound-SAA stimulated endothelial IL-6 and MCP-1 secretion and ICAM-1 expression which was also associated with TLR2 and p38MAPK.ConclusionsHDL from CAD patients itself activates endothelial inflammation independent exogenous stimuli, which was associated with SAA accumulation and partly mediated through TLR2 and p38MAPK signalling pathway.