Proteomic Analysis Of High-density Lipoprotein In Non-diabetic Patients With Early-Onset Coronary Heart Disease

BackgroundIn recent years, there has been a growing interest in applying proteomics to clinical diagnostics and predictive medicine. Protein biomarkers can assist in diagnosing a disease at the biochemical level or discriminating the responses of different patients to the same medical treatment. As a comparative proteomic analysis technique,2-DE provides an insight into intact and fragmented protein species and also provides information regarding post-translational modifications.2-DE, therefore, has a significant advantage over mass spectrometry based techniques which are based on peptide or peak analysis, where information on the mass and post-translational modifications of the intact protein is lost. 2D-DIGE incorporates the use of fluorescent molecules (CyDyes), that are used to prelabel samples prior to separation by 2-DE. The internal standard is a pool of all samples used in the experiment and is used not only to normalize the Cy3- and Cy5-labeled samples, but also to compare across the other 2-D gels in the experiment. This significantly reduces the intergel variability that plagues other 2-D gel-based analyses.Coronary heart disease (CHD) is a multifactorial disease having environmental and genetic components. Traditional risk factors, such as the male gender, hypertension, diabetes mellitus, family history of premature CHD, elevated plasma levels of low-density lipoprotein cholesterol (LDL-C) and decreased high density lipoprotein cholesterol (HDL-C) concentrations have been shown to be independent predictors of CHD in numerous studies performed in different settings. Among the risk factors identified by epidemiological studies, low plasma HDL-C concentration is one of the strongest. For any given LDL concentration, the HDL-C concentration is inversely correlated with the risk of CHD and stroke. But total plasma HDL-C level is a crude measure of their protective effects, as numerous studies have revealed differences between HDL subclasses and properties such as reverse cholesterol transport (RCT), anti-inflammatory and antioxidant properties. The difference of cardioprotective effects of HDL depend on the types of particles generated in vivo and that HDL in humans with established CAD is dysfunctional. Knowledge of the molecular speciation of HDL and of the protein constituents of HDL particles is crucial for our understanding of HDL metabolism and identify the precise mechanisms by which HDL may exert protective effects against atherosclerosis.Early-onset CHD, a special form of CHD, is defined by onset in males aged<55 years and females aged<65 years. The concentration of HDL correlates inversely with the development of arly-onset CHD within populations. In this study, we quantifyed the protein composition of HDL in early-onset CHD and compared with young control group and late-onset CHD group, which might provide insights into the change of antiatherogenic and antiinflammatory properties of HDL in arly-onset CHD.Part 1:Clinical, biochemical and angiographic characteristics of Chinese patients with early onset coronary heart diseaseObjectivesThe purpose of this study was to investigate clinical, angiographic, and biochemical features in Chinese patients with early onset coronary heart disease.Subjects and medthodsFour hundreds and twenty-three were selected from those who admitted to FuWai cardiovascular disease hospital from November 2008 to June 2009. All patients underwent coronary angiography. One hundred and nine consecutive patients with clinical onset of CHD disease at age≤45 years,238 older patients with clinical onset of disease at≥55 years and 76 age matched healthy subjects took part in this study. Angiographic features, clinical and biochemical risk factors were compared between these groups.ResultsCompared with healthy subjects, early onset CHD patients showed more prevalence in hypertension, diabetes, smoking and family history of coronary artery disease. The young patients had lower high density lipoprotein cholesterol (HDL-C) and apolipoproteinA-I (apoA-I) levels, higher triglyceride, lipoprotein(a) and higher high-sensitivity C-reactive protein (hsCRP) level.Compared with late onset CHD group,36.1% of patients presented with acute myocardial infarction in early onset CHD (p<0.001), but the number of coronary artery involved, the prevalence of different branches involved and Gensini score did not show significant difference. As to the clinical and biochemical risk factors, only prevalence of hypertension, HDL-C and hsCRP showed significant different.Part 2:Proteomic analysis of high-density lipoprotein in Early-Onset Coronary Heart Disease using two-dimensional difference gel electrophoresis and mass spectrometryObjectivesHigh density lipoprotein cholesterol (HDL-C) levels are a strong, independent inverse predictor of coronary heart disease (CHD). The protein composition of HDL altered might affect the antiatherogenic and antiinflammatory properties of HDL. Identifying the difference of HDL-associated proteins is essential with regards to understanding the role of HDL played in CHD at the molecular level. In this study, we investigated the modification in the HDL of early-onset CHD.SubjectsWe investigated the HDL proteome in 2 groups of CAD subjects selected from those who admitted the Fuwai cardiovascular disease hospital from November 2008 to June 2009.The first group (15 control subjects and 15 subjects with early-onset CHD) was used for proteomics analysis of total HDL isolated by ultracentrifugation.The second group (30 control subjects and 30 subjects with early-onset CHD) was used for quantification of the protein differentially expressed in HDL biochemically.MedthodsTwo-dimensional difference gel electrophoresis (2D DIGE) technique, in combination with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), was used to determine the differentially expressed protein in HDL isolated from 15 early-onset CHD and 15 healthy control subjects. To confirm the 2D-DIGE protein expression data, validation of the results was carried out using Western blotting and ELISA in the original pilot cohort and also an independent serum seriesResultsImage analysis showed 5 spots were significantly up-regulated whereas 28 spots were down-regulated in the early-onset CHD group. MALDI-TOF MS results shwed 2 identified proteins were increased:A-SAA and apolipoproteinLl (apoLl),2 proteins were decreased:apoA-Ⅰand apolipoproteinA-IV (apoA-IV). ApoA-I, apoA-IV and A-SAA have undergone extensive validation in original cohort and also from a larger independent cohort of patients.Part 3:Comprehensive Evaluation of the Diagnostic Value of HDL Related Factors for Early-onset Coronary Heart Disease by ROC Curve and Logistic RegressionObjectivesTo explore the diagnostic value of HDL related factors for Early-onset CHD by receiver operating characteristic curve (ROC curve) and a model of logistic regression.SubjectsSixty consecutive patients with clinical onset of CHD at age≤45 years, and 59 age matched healthy subjects took part in this study. HDL related factors, apoA-I, apoA-IV and SAA and other biochemical risk factors were compared between these groups.MedthodsAfter analyzing all risk factors with logistic stepwise regression, we screened multiple diagnostic parameters for breast cancer and established a mathematical model for diagnosis. Then we assessed the diagnostic efficacy of the model and calculated the diagnostic cut-off points for early-onset CHD breast cancer using the ROC curve.ResultsThe area under ROC curve (AUC) of the HDL-C, apoA-I, SAA and hsCRP was greater than that of either parameter alone (P<0.05). According to the regression equation P=1/[1+e-(2.267-4.868·apoA-I+1.889·SAA-4.897·HDL-C+0.694·hsCRP)],the cut-off point, sensitivity and specificity of the combined parameters for diagnosing early-onset CHD were0.468,82.46% and 84.75%, respectively.Conclusion1. Hypertension, diabetes, heavily smoking, family history, high plasma triglyceride, lowHDL-C levels and apoA-Ⅰare associated with early onset CHD, the reduction of HDL-C and apoA-I may be the most important lipid risk factor in development of early onset CHD.2. In our study, we found 40 proteins in HDL, most of them wre apolipoproteins, inflammatory response related proteins, furthermore, we found a number of proteins not previously known to reside in HDL.3. We identified four proteins apoA-I, apoL1, apoA-IV and A-SAA differentially expressed between early-onset CHD patients and healthy control group, which might take part in the present of early-onset CHD.4. Although further investigations are needed to fully determine the changes of apoA-I, apoA-IV and SAA expression in large group of patients and their role to HDL function, our preliminary study clearly demonstrated that the levels of the three proteins may serve as potential biomarkers for HDLfuncition and early-onset CHD.5. The area under ROC curve (AUC) of the HDL-C, apoA-I, SAA and hsCRP was greater than that of either parameter alone. Application of logistic regression and ROC curve increase diagnostic accuracy in early-onset CHD.