| BackgroundClinical and epidemiological studies have shown that a strong inverse correlation between high-density lipoprotein-cholesterol (HDL-C) levels and coronary artery disease(CAD) , and for every 1 mg/dl increase in HDL-C, the predicted incidence of coronary events decreases by 2-3%. HDL as a multifunctional protein complexes is thought to protect against atherosclerosis by promoting reverse cholesterol transport (RCT) and potentially through anti-oxidative and anti-inflammatory capacities. However, many studies have confirmed HDL-C levels do not accurately represent the functions of HDL and quantifying HDL-C levels provide limited information about HDL’s cardioprotective effects:torcetrapib, an inhibitor of cholesteryl ester transfer protein (CETP) was associated with an increase in the number of cardiovascular events, despite a 72% increase in HDL-C levels. Furthermore, individuals with the apolipoprotein A-IMilano mutant have very low plasma HDL-C levels, but have very low incidence of CADt. Visibly, it is more important to pay close attention to HDL mass(functions) than HDL-C levels.Amit v. Khera, etc published an article in the New England journal of medicine in 2011:They measured cholesterol efflux capacity in 203 healthy volunteers who underwent assessment of carotid artery intima-media thickness,442 patients with angiographically confirmed coronary artery disease, and 351 patients without such angiographically confirmed disease. Linear regression was used to characterize the relationship between efflux capacity and carotid intima-media thickness(IMT). Cardiovascular disease risk factors, and levels of glycated hemoglobin, low-density lipoprotein (LDL) cholesterol, HDL cholesterol, and apolipoproteinA-I were included as covariates. No significant relationship between HDL cholesterol level and carotid intima-media thickness was noted; Cholesterol efflux capacity from macrophages, a metric of HDL function, has a strong inverse association with both carotid intima-media thickness and the likelihood of angiographic coronary artery disease, independently of the HDL-C level. Cholesterol efflux capacity is independently related to both the presence and the extent of atherosclerosis. These findings reinforce the concept that assessment of HDL function may prove informative in refining our understanding of HDL-mediated atheroprotection and predicting cardiovascular disease risk. In Addition, reports of marked heterogeneity in the particle composition and biologic properties of HDL have reinforced a need for assessment of HDL function.The latest basic and clinical studies showed the effect of HDL against AS is impaired and even present pro-atherosclerosis effect in chronic inflammation, acute phase responses and some metabolic diseases, which may be ralate to structural modification and compositional alteration of HDL particles as a result of chronic inflammation and acute phase responses may adversely affect or reverse their normal biological function. Shao et al demonstrated that oxidative damage impairs the ability of apolipoprotein A-I (apoA-I), the major HDL protein, to remove cholesterol from macrophages. Heinecke found HDL from CAD subjects also contained markedly elevated levels of chlorotyrosine and nitrotyrosine, two characteristic products of myeloperoxidase, indicating that oxidative damage might generate dysfunctional HDL.ACS constitutes a uniquely inflammatory milieu; for example, the proinflammatory cytokine CXCL16 is more highly expressed in subjects with acute myocardial infarction than those with chronic atherosclerosis. Under such in fl ammatory conditions, it is suggested that the protein and phospholipid. moieties of HDL are substantially altered, thereby modifying the functional characteristics of the HDL particles. Indeed, animal studies convincingly demonstrate that changes in proteins involved in HDL metabolism can promote atherosclerosis, even when plasma levels of HDL-C are elevated. A few, mostly small, studies have suggested that patients with underlying inflammatory conditions tend to have HDL that has less anti-inflammatory capacity. Therefore, we speculate that ACS may transform functional HDL to dysfunctional HDL according to triggering an inflamatory response which substantially change the the protein cargoes of HDL particle.Structure of proteins determines the function. Application of proteomics technique research provides a new method and train of thought for understanding HDL function. More recent studies suggest that the HDL proteome is implicated in HDL functionality, identifying HDL-associated proteins involved in lipid metabolism, complement activation, acute phase response protein, and proteolysis regulation, indicating alteration in HDL proteome may play a key role in HDL function. According to density-gradient ultracentrifugation method, the HDL particles by its density can be divided into HDL1, HDL2 and HDL3 three sub-types, and HDL in human plasma consists of two main subfractions HDL2 and HDL3. HDL2 are considerably larger and less dense than HDL3. Numerous population studies have suggested that HDL2 may be more cardioprotective than HDL3 (Miller NE. Am Heart J,1987113,589-597), however, there are also inconsistencies with reports suggesting that HDL2 and HDL3 are equally cardioprotective. In recent years, the studies on HDL subtractions proteomics were focused on HDL3, especially apoL1, PON1 and PON3 which is associated with the ability of HDL3 preventing LDL oxidated. Studies confirm HDL subfractions proteomics is the basis of determining HDL function. Presently, there is no report about the relation between different protein composition and function of HDL (reverse cholesterol transport, anti-inflammatory, antioxidant). Whether ACS could remodel HDL subfractions proteomics which substantially modifies the HDL subfractions functions is still blank.ObjectiveAccording to assess promoting reverse cholesterol transport (RCT) and antiinflammatory/antioxidant properties of HDL subclasses(HDL2 and HDL3) from acute coronary syndromes(ACS) patients and healthy subjects, to explicit whether ACS impairs HDL subfractions functions and makes functional HDL subclasses shift to dysfunctional HDL subfractions; And meanwhile to explore the possible proteomics mechanisms of the occurrence of dysfunctional HDL subfractions by comparing the proteomics differences between ACS patients and healthy subjects.MethodsForty ACS patients hospitalized in Nanfang Hospital from Jan.2011 to Jan. 2012 (ACS group) and 40 subjects simultaneously undergone health examination (control group) were enrolled in present study. Plasma lipid and hypersensitive C reactive protein (hs-CRP) levels, HDL subclasses-mediated cholesterol efflux rate, HDL subclasses inflammatory index (HII), paraoxonase-1 (PON1) activity and lipid hydroperoxides (LOOH) levels in both groups were measured. HDL subfractions were isolated by ultracentrifugation and the proteins were separated with 2D-DIGE and identified with mass spectrometry.1. The detection of basic clinical data both in ACS patients and healthy subjects(1) Serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol(HDL-C) concentrations were measured with an automated biochemical analyzer.(2) The hsCRP concentrations were determined by nephelometry.2. The detection of HDL subfractions functions both in ACS patients and healthy subjects(1) Plasma anticoagulated with EDTA was collected after an overnight fast from 40 subjects with established ACS (ACS group) and from 40 apparently healthy people(control group), and HDL2 and HDL3 were isolated by a density-gradient ultracentrifugation method.(2) Measuring PON1 activity of HDL2 and HDL3 by a phenyl acetate method.(3) HDL-mediated cholesterol efflux rate from macrophages were measured by liquid scintillation spectrometry.(4) HDL subclasses inflammatory index (HⅡ) were measured by a Cell-Free Assay.(5) HDL subclasses LOOH levels were measured by the ferrous oxidation-xylenol orange assay.3. Analyzing the the proteomics differences between ACS patients and healthy subjects.(1) Finding differentially expressed protein spots from different HDL2 samples or HDL3 samples by 2D-DIGE.(2) Peptide mass fingerprinting (PMF) obtained through MS were retrieved by using Mascot software, and meanwhile using MS and MS/MS combined pattern searched Swissprot databases to identify proteins.(3) To validate the ability of our proteomic approach to identify protein, we validated the relevant significant proteins by western blot and ELISA.Results1. Comparison of basic clinical data between ACS group and control groupPatients with ACS had significantly increased levels of LDL-C and high sensitivity C-reactive protein (hs-CRP) relative to controls (t=2.972, P=0.004 or t=9.175, P<0.001). However, there was no significant difference in TG, TC and HDL-C levels(P>0.05) between ACS group and control group.2. Effects of ACS on HDL subfractions functionsACS group VS control group:HDL2-PON1 activity:(77.58±14.88)U/mg VS (110.00±14.95) U/mg; HDL3-PON1 activity:(75.83±14.97) U/mg VS (104.57±13.16) U/mg; HDL2-cholesterol efflux rate:(20.6±3.7)%VS (27.7±5.5)%; HDL3-cholesterol efflux rate:(18.4±3.8)% VS (24.7±6.2%)%; HDL2-HⅡ:(1.25±0.27) VS (0.48±0.12); HDL3-HⅡ:(1.32±0.27)VS(0.62±0.15); HDL2-LOOH:(18.46±4.7) ngLOOH/(ug.chole) VS (9.09±2.27) ngLOOH/(ug.chole); HDL3-LOOH:(21.3±5.64) ngLOOH/(ug.chole) VS (11.44±2.81) ngLOOH/(ug.chole).The statistical results showed compared with control group, HDL subfractions in ACS group showed decreased cholesterol efflux rate and PON1 activity (P< 0.001), and increased HII and HDL-LOOH levels (P<0.001).3. Comparison of HDL subfractions functions in the same groupHDL3 had lower cholesterol efflux capacity [control group:HDL2 VS HDL3: (27.7±5.5)% VS (24.7±6.2)%, P=0.027; ACS group HDL2 VS HDL3:(20.6±3.7)% VS (18.4±3.8)%, P=0.012]and higher LOOH levels (P<0.001 or P=0.017) compared with HDL2 in both ACS group and control group[control group:HDL2 VS HDL3: (9.09±2.27) ngLOOH/(ug.chole) VS (11.44±2.81) ngLOOH/(ug.chole), P<0.001; ACSgroup:(18.46±4.7) ngLOOH/(ug.chole) VS (21.3±5.64) ngLOOH/(ug.chole), P=0.017].4. Effects of ACS on HDL subfractions proteomicsProteomic analysis revealed that 48 spots were differentially expressed in HDL subfractions between ACS and control group. By mass spectrometry, we demonstrated that compared with controls, HDL3 from ACS group was selectively enriched in 9 proteins (apolipoproteinA-I, apolipoproteinA-IV, apolipoproteinE, apolipoproteinLl, paraoxonase, alpha-1B-glycoprotein, serum amyloid P-component, vitamin D-binding protein, fibrinogen gamma chain), and meanwhile ras-related protein Rab-7b was decreased in HDL3. Additionly,12 proteins were lower in HDL2 from ACS subjects (apoA-I, apoE, PON, apoA-IV, apoLl, haptoglobin, hemopexin, serotransferrin, complement factorB, ras-related protein Rab-7b, fibrinogen gamma chain, Ig gamma-1 chain C region), and meanwhile 4 proteins were increased in HDL2(serum amyloid P-component, alpha-1-antitrypsin, acid ceramidase, vitamin D-binding protein). To validate the ability of our proteomic approach to identify protein, we validated the significant proteins by western blot, and meanwhile we also measured the concentration of serum amyloid P-component (SAP) by ELISA. The result showed that patients with ACS had higher SAP level[(3.53±0.41)lg (ng/ml)] compared with that of controls[(3.11±0.48) 1g(ng/ml)](t=4.173,P<0.001).Conclusions(1) ACS could impair the functions of HDL subfractions, and makes functional HDL subfractions with anti-atherosclerosis shift to dysfunctional HDL subclasses with pro-atherosclerosis.(2) Mature HDL2 possesses better RCT and antioxidant functions than HDL3, may play more cardioprotective effect to the patients.(3) ACS could alter the protein composition, and the change of HDL subfractions proteomics may be a important mechanism of the occurrence of dysfunctional HDL subclasses. In addition, this research reveals new proteins both in HDL2 and HDL3; serum amyloid P-component and ras-related protein Rab-7b. |
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