Proteomic Study On Serum Protein Biomarkers Of Atherosclerosis And Relationship Of CDK9 With Atherosclerosis

Research purposesAtherosclerosis (Atherosclerosis, AS) is the pathological basis of a majority of cardiovascular diseases and the main causes of cardiovascular diseases deaths. Traditional biomarkers of atherosclerosis used in clinical detection are the total cholesterol, high or low density lipoprotein (HDL/LDL). However, atherosclerosis is a chronic and complex disease, involving multiple causes and lots of physiological, biochemical and pathological changes, that includes not only lipid infiltration, but also a series of progresses such as endothelial dysfunction, inflammation, oxidation stress, cell proliferation and apoptosis, platelet activating-blood clotting response, and so on. Nearly a decade of scientific research found that some markers like CRP (C-reactive protein) occurred in the whole process of atherosclerosis, some marker on the stage-specific expression, such as endothelial dysfunction (Intro-cellular adhesion factors-1, ICAM-1), White blood cell Recruitment and infiltration (Monocyte-chemoattractant protein-1,MCP-1), extracellular matrix degradation (MMPs), lipid oxidation (MPO),thrombosis (Fibrinogen,sCD40L), and so on. CRP, C-reactive protein, is the most studied biological markers of atherosclerosis, primarily produced by hepatocytes in infections, injuries and other conditions induced by IL-6 induction. In the presence of Ca2+, CRP can bind with damaged cells, activate the complement system, and promoted the formation of atherosclerosis. MCP-1 belongs to the chemokine family. Animal model studies indicate that MCP-1 can promote artery-forming and is massively expressed in atherosclerosis plague. Inhibiting MCP-1 activity reduced plaque formation. Others, such as PAPPA (Pregnancy-associated plasma protein-A), are a kind of syncytial trophoblast glycoprotein traditionally used for Down syndrome screening during pregnancy. PAPPA can cause complete rupture of atherosclerotic protection cap. Its expression increases were associated with atherosclerosis. Recent study found that cartilage glycoprotein YKL-40 level is elevated in connection with atherosclerosis.However, these biomarkers study only focus on single specific molecules. They have limitations due to that atherosclerosis is a pathological process involving many factors. Only by searching the entire system, not just a single molecule can possibly find more comprehensive and more accurate biological markers (or combination of markers), that are easier to duplicate and standardize. So, "omics" studies (genomics research) which explore biological systems in the overall level of composition and activity become the focus of the study on markers of disease. It can provide clues and basis for molecular diagnosis of cardiovascular diseases, help achieving goals of early diagnosis and individual treatment, is of great research and clinical value. Yet proteomics breakthrough on biomarkers, especially on atherosclerosis markers is rarely reported.In the different levels of proteomics study, searching for serum protein markers for disease has advantages:(A) easy sample access, serum protein group contains various types of protein information from cells, tissues, and organs; (B) certain cells secrete protein in abnormal body state, protein on cell surface can be "off into the environment and into the blood, so a lot of diseases show abnormal protein expression in blood. P-selectin, PAPPA and YKL-40 markers of atherosclerosis are glycoprotein; (C) blood contains over 106 kinds of proteins, concentration differences is at 1010 level, focusing on serum glycoprotein can reduce the complexity of the system.Based on the above analysis, serum protein biomarker studies of atherosclerosis is of great scientific and application value, but there are still many shortcomings in the current study, need further research and analysis on related fields. This study uses atherosclerotic cardiovascular medicine specimen, improved MALDI/TOF mass spectrometry based detection methods of protein group, aimed to screen and identify biological markers for atherosclerosis. The results would help to finally establish clinical applicable biomarkers, and provide clue and basis for elucidating the mechanisms for the development of cardiovascular diseases.Recent research showed that cell cycle dependent protein kinase 9 (CDK9, Cyclin dependent kinase 9) plays a key role in the regulation of cell cycle and monitoring the pro-inflammatory response gene activation. More and more CDK9 inhibitors, such as Flavopiridol (FLA), PHA 767491, as a target for inflammation and cancer, have been studied and developed. However, there are few reports about CDK9 in the study of the inflammatory mechanism of atherosclerosis or coronary artery atherosclerosis.In the first part, the serum proteomics study showed that among several deferentially expressed protein molecules, CDK9 was significantly increased in the serum of patients with coronary atherosclerosis. Therefore, in the second part, CDK9 was further verified with validated serum sample, peripheral blood mononuclear cells and coronary plaque tissue from patients with coronary atherosclerosis; And furthermore, in the third part, the impact of CDK9 inhibition on monocyte activities was carried out through monocytic cell line THP-1 cell proliferation, cell cycle and apoptosis in order to explore the possibility of CDK9 as a predictive biomarker of risk for coronary atherosclerosis to provide basis for targeting CDK9 diagnosis and treatment of coronary atherosclerosis.PartⅠ Study of serum protein markers of coronary atherosclerosis with proteomic methodResearch PurposeImproving serum proteomics methods and exploring abnormal expression of serum proteins in atherosclerosis pathogenesis.Research methods1. Study populationFrom October 2012 to October 2014,43 cases of patients with coronary heart disease were selected from Shandong Provincial Hospital and Qilu Hospital of Shandong University, including 25 males and 18 females, aged 60 +3.3 years old. All the selected cases were in accordance with the diagnostic criteria of coronary heart disease established by WHO. Selected with the symptoms of angina pectoris,50% of one or more coronary artery stenosis confirmed by coronary angiography, without sign of cardiac shock and severe heart failure, serious ventricular arrhythmia, other serious diseases such as cancer, etc. and exclude hyperthyroidism, hypertension, rheumatic diseases, severe liver and kidney dysfunction and acute infection. In the same period, 38 healthy persons were selected as healthy control, including 17 males and 21 females, aged 58+11.1 years old. With no atherosclerosis and coronary vascular disease or inflammatory diseases; test concentrations of serum total cholesterol (TC), glycerin (TG), high density lipoprotein (HDL), low density lipoprotein (LDL) and blood glucose levels by normal methods, exclude hyperlipidemia, hypertension, and diabetes. Medical history inquiry, physical examination, electrocardiogram examination and other biochemical examination found normal. This study was approved by the ethics committee of Shandong University Medical College, the patients were informed consent.2. Serum collection and protein extractionAn aliquot of 5 mL from each peripheral blood sample was separated into serum fractions. Serum protein was harvested with the Albumin and IgG Removal Kit according to the manufacturer’s instructions, then processed using ReadyPrepTM 2-D Clean-up Kit to remove salt, lipids, nucleic acids and other interfering substances. The supernatant was subsequently collected and used for 2-D electrophoresis. Approximately 300 μg of protein was suspended in a rehydration solution and applied to 2D electrophoresis.3. Isoelectric focusingProtein sample is applied to 18 cm pH 3-10 non-linear ImmobilineTM DryStrip Gels for isoelectro-focusing. The gel strip groove is arranged on the horizontal plate with filter paper, adding urea rehydration solution containing the sample, then covered with Strip Dry solution and plastic cover. The gel strip groove is then arranged on the IPGphor electrode plate, and is parallel to the direction of the electrode. Set up the voltage program for serum sample. The isoelectro-focusing was performed using an Ettan IPGphorTM Instrument.4. Two-dimensional electrophoresisThe IPG strips were subsequently placed on a 12% uniform SDS-polyacrylamide gel. The electrophoresis program using in experiment (18cm, pH3-10, NL):5W/gel for approximately 40 minutes till bromophenol blue reach a straight line, then change to 15W/glue, stop running when the bromophenol blue reach bottom of the gel. The gels were then silver-stained and scanned with an Image Scanner in transmission mode, after which the image analysis was undertaken using 2-D PDQuest.5. In gel digestionUse silyanized tips to harvest proteins of different abundance at the 2D protein gel, process according to lab improved in gel digestion protocol, collect the protein powder.6. Mass spectrometry analysisDissolve the protein powder obtained through in gel digestion process in 3 μL 0.1%TFA. Take 1.5 μL mixture and directly drop on a sample target plate, when the sample is dried at room temperature, cover it with 1μL matrix. After the matrix is completely crystallized, detect the sample plate by spectrometer. Use Matrix-assisted laser desorption ionization-Time of flight/Mass spectrometry (MALDI-TOF/MS) to identify enzymatic hydrolysates using reflection, ion chromatography, and automatically fetch data mode. Instruments choose Mass Standards Kit for the 4700 Proteomics Analyzer, analyze peptide by enzymatic hydrolysis for external standard calibration, ion delayed extraction 100m, mass spectrometry signals single scans 500 times, laser frequency 20.0 Hz, PMF quality scanning range of matrix and sample 100-3000u.7. Data analysisUsing Mascot software to analyze collected data from mass spectrometry, search the matched protein in NCBI-human protein database. Identify the protein according to search results along with iso-electric point and molecular weight of analyzed protein gel spot.Results1. Improvement of proteomic methods on searching for serum protein biomarkers1.1 Removal of high abundance proteins (albumin and IgG) and desalt in serum samplesIn view of the serum albumin and immunoglobulin accounted for about 90% of the total serum protein, and disease serum protein markers is usually low abundance proteins, high abundance proteins can seriously interfere with the detection of low abundance proteins, therefore we use Albumin and IgG Removal Kit (Pierce, Thermo Scientific, Rockford, IL, USA) successfully remove serum albumin and immunoglobulin (two kinds of high abundance proteins) from the samples, and set the base to detect disease-related low abundance proteins. Due to the interference by salts, lipids, nucleic acids and other impurities, we use ReadyPrep "2-D cleanup kit to process serum samples after removal of high abundance protein, this step effectively increased the detection sensitivity.1.2 To optimize several steps for the research method of serum proteomicsProblems still remains after removal of high abundance protein, including that less protein spots in 2-DE map, horizontal and vertical stripes and other problems. In this study, we made a series of explorations in pH range of SDS-PAGE samples, SDS concentration, IEF electrophoresis conditions and other procedures. After removal of salts, lipids, nucleic acids and other interfering substances by 2D-clean up kit, improved IEF voltage program and SDS-PAGE conditions, eventually we get the ideal protein separation of two-dimensional electrophoresis. This study studied on multiple steps in optimizing serum samples, exploring isoelectric focusing voltage program, to improve in-gel digestion method and process, finally established a more simple and rapid serum protein group study method, and a more suitable serum samples IEF condition.2.Identify abnormal serum protein expression in the course of atherosclerosis.Spot analysis detected 509 ±31 spots per patient sample and 565 ± 29 spots per healthy control sample. Thirty-three differentially expressed proteins between the atherosclerotic CAD patient and healthy control were excised from the 2-D gels, digested in the gel and applied to a sample template for MALDI-TOF mass spectrometry. Twenty-seven protein spots were successfully identified with Mascot using peptide mass fingerprinting data. Among 27 proteins identified,15 of them, including CDK9, were increased, whereas 12 proteins were decreased in atherosclerotic serum samples compared with the control group.3. Functional classification of differentially expressed proteins. All 27 identified proteins were further classified into 6 different groups based on cell proliferation and apoptosis, inflammation factor, immune factor, energy metabolism and signaling pathway and other molecules such as angiotensin II receptor according to known or postulated functions and pathways. CDK9 was highly expressed in highly expressed protein molecules found in the serum samples of atherosclerosis patients. So it was chosen as the focus of the next study.Part Ⅱ Expression of CDK9 in coronary atherosclerosisResearch PurposeTo further explore the expression of CDK9 in coronary atherosclerosis patients’serum, mononuclear cells and coronary atherosclerotic plaque tissue to provide a basis for exploring CDK9 as a possible serum biomarkers in atherosclerosis, and for diagnosis and targeting treatment of coronary atherosclerotic diseases.Research Methods1. Study population:Same as Part 1.2. Detection of the CDK9 in serum of patients with coronary atherosclerosis and healthy controls.CDK9 protein expression in serum of patients with coronary atherosclerosis and healthy controls was detected by Western blot, and serum CDK9 level was detected by enzyme-linked immuno-sorbent assay (ELISA) in patients with coronary atherosclerosis and healthy controls.3. Detection of CDK9 expression in peripheral blood mononuclear cells (PBMCs)3.1 Whole blood samples were collected from atherosclerotic CAD patients and healthy control groups. PBMCs separation was facilitated with Ficoll-Hypaque gradient centrifugation. PBMCs were further separated into monocytes and lymphocytes through in vitro adherent culture.3.2 CDK9 mRNA expression was detected in peripheral blood mononuclear cells by RT-PCR:Total RNA was extracted from isolated PBMCs, lymphocytes and monocytes. Specific primers for human CDK9 were used.3.3 Western detection of CDK9 protein expression in lymphocytes and monocytes among PDMCs. The total proteins were extracted. After 10% SDS-PAGE, Western blot was used to detect the expression of CDK9 protein in different subsets of cells with specific anti-CDK9 antibody.4. Detection of CDK9 expression in coronary plaques of atherosclerosis patients by Immunohistochemical staining and immunofluorencence stainingCollected coronary plaque tissue samples from Rabbit atherosclerosis model and patients with coronary atherosclerosis, Hematoxylin and eosin (H&E) staining of plaque tissue sections were performed according to standard procedures. Immunohistochemical staining was used to detect the expression CDK9 in rabbit model with primary antibody against rabbit CDK9, and Immunohistochemical staining/Immunofluorecence staining of CDK9/CD14 in the coronary artery plaque tissue with application of primary antibody against human CDK9 or CD 14. The slides were viewed under microscope to define sub-cellular localization of the CDK9 and CD 14.5. Statistical analysisStatistical analysis was done with vll.5 SPSS. GraphPad Prism 5 software (San Diego CA) was used for data processing. Continuous variables with average standard error, with one-way ANOVA, paired or non paired t test analysis. P<0.05 believes that there were significant differences.Results1. High CDK9 expression was detected in serum of patients with coronary atherosclerosis:Western Blot results showed that CDK9 expression was significantly increased in sera of patients with coronary atherosclerosis compared with the healthy control, p< 0.01; ELISA results showed that, 149.70+ 105.22 U/L CDK9 detected in sera of in serum of patients, while 64.51+ 25.94 U/L in healthy controls. The two groups showed significant difference, p< 0.01.2. CDK9 expression was increased in PBMCs of patients with coronary atherosclerosis. PBMCs were isolated from atherosclerotic patients and healthy controls to measure CDK9 expression. Both mRNA and protein levels of CDK9 were found to be significantly increased in PBMCs of atherosclerotic patients compared with healthy controls. In addition, CDK9 was higher expressed in monocyte subpopulations than in lymphocyte subpopulations in PBMCs of atherosclerotic patients compared with healthy controls (p< 0.01).3. CDK9 expression was increased in artery plaque tissue:To further investigate whether CDK9 was increased in atherosclerotic process, rabbit and human artery plaque tissue sections were analyzed by immunohistochemistry staining and immunoflluorecence staining. Compared with non-plaque tissue, plaque tissue showed irregular intimal thickening, calcification, and significant atherosclerotic plaque formation, along with infiltration of abundant inflammatory cells. CDK9 positive expression was found in atherosclerotic plaque intima mainly located within nucleus. Furthermore, the CD14 (monocyte/macrophage surface marker) immunohistochemistry and immnofluorecence staining showed positive staining within atherosclerotic plaques which represented the majority of inflammatory infiltration cells. Moreover, the CD14+ cells showed increased CDK9 levels in atherosclerotic plaques, which indicated the importantant role of CDK9 in monocyte infiltration during atherosclerosis.Part III Effects of CDK9 inhibition on atherosclerotic inflammatory monocytesResearch PurposesTo study the effect of inhibiting CDK9 on CDK9 isoform expression, proliferation, cell cycle, apoptosis and apoptosis related proteins in monocytes under the stimulation of inflammatory factors.Research Methods1. Culture and Processing of THP-1 cell line.The monocytic acute leukemia cell line THP-1 cells were cultured and treated with different concentrations of CDK9 inhibitor FLA (50 and 100 nM) for 6 h and 24 h with or without TNFa (50 ng/mL) existence. Apoptosis and proliferation effect by inhibiting CDK9 was measured using the FACSCalibur flow cytometry and Western Blot.2. RT-PCR:Same as Part 2.3. Western blot:Same as Part 2.4. Cell proliferation by CCK-8 methods:Cell Counting Kit-8, follow according to the instructions of the manufacturer.5. Cell cycle:Using Cell Cycle and Apoptosis Analysis Kit, according to the instructions of the manufacturer.6. Apoptosis detection:Annexin V-FITC apoptosis detection kit, according to the instruction of the manufacturer.7. Statistical analysis:Using vll.5 SPSS for statistical analysis. Continuous variables with average standard error, with one-way ANOVA, paired or non paired t test analysis. P<0.05 believes that there were significant differences. GraphPad Prism 5 software (San Diego CA) was used for data processing.Results1. FLA inhibited expression of isoform CDK9 43kD in TNFα mediated THP-1 monocytic cell lineCDK9 inhibitor FLAlOOnM treatment 6 h significantly inhibited CDK9 expression in human acute monocyte leukemia cell line-THP-1 cells with TNFa (50ng/ml) stimulation, especially affect expression of isoform CDK9 43kD,without apparently effect on isoform CDK9 55kD.2. Inhibition of CDK9 can significantly inhibit TNFa-stimulated THP-1 monocytic cell proliferationCCK-8 cell proliferation analysis showed that FLA 50nM and 100nM treatment of THP-1 cells for 6 and 24h can significantly inhibit the proliferation of THP-1 cells; the role of high concentration of FLA is more obviously.3. Inhibition of CDK9 lead to TNFa-stimulated G2/M phase arrest of THP-1 monocytic cellAnd moreover,6h 100nM FLA treatment can lead to G2/M phase arrest (the percentage of G2/M cells rose from 10.9% to 12.6%). Under the stimulation of TNFa, FLA treatment can make the percentage of G2/M phase cells reach 15.2%. But there was no significant change in the percentage of G0/G1 or S phase cells.4. Inhibition of CDK9 can significantly promote TNFa-stimulated apoptosisFLA50 and 100nM treatment of THP-1 cells for 6h, the early apoptosis and later apoptosis of THP-1 were significantly increased, p<0.05, especially under TNFa stimulation.5. Inhibition of CDK9 can significantly promote TNFα-stimulated apoptosis-related protein expressionFLA100nM treatment of THP-1 cells for 6h and 24h, expression of apoptosis protein Bax of THP-1 were significantly increased, especially 24h under TNFα stimulation without apparently change for expression of anti-apoptosis protein Bcl-2.ConclusionThis thesis use 2DE-MALDI-TOF/MS proteomic technologies to conduct the comparative study of coronary atherosclerosis patients and healthy controls on the serum differential protein expression, and further verification of the correlation with candidate cyclin dependent protein kinase 9 and atherosclerotic disease, and has achieved the following innovative points,1. The abnormal serum protein expression was found in serum of patients with atherosclerosis.27 proteins deferentially expressed proteins between coronary atherosclerosis patients and normal controls were identified, which can be further divided into 6 different functional groups, mainly related to inflammation and cell activities.2. Expression of CDK9 in serum, peripheral blood mononuclear cells, and arterial plaque tissue expression was significantly increased in rabbit and Human coronary atherosclerotic plaque CDK9 expression was positively correlated with the high infiltration of CD 14+monocyte/macrophage.3. The investigation of CDK9 inhibition on TNFa-stimulated monocyte with human monocytic acute leukemia cell line-THP-1 showed that it could inhibit the TNFα-stimulated expression of isoform CDK943 and cell proliferation, promote TNFα-stimulated apoptosis and apoptosis-related protein expression, and result in TNFα-stimulated cell cycle G2/M arresting.The results suggested that there is differential expressed proteins within sera of patients with atherosclerotic CAD;CDK9 may be of great significance in the pathogenesis of coronary atherosclerosis, and CDK9 might be a potential biomarker of atherosclerotic inflammation and offer insights into the pathophysiology clarify and targeting diagnosis/therapy of atherosclerotic CAD.Innovation points1. The abnormal expression of serum protein in patients with coronary artery atherosclerosis by proteomic analysis was studied, and 27 kinds of differential proteins were obtained and further classified by functions.2. Study shows the high expression of CDK9 in the serum, peripheral blood mononuclear cells, especially in mononuclear cells of patients with coronary atherosclerosis. Further, CDK9 is found highly expressed in atherosclerotic plaques, and CD 14 expression was positively correlated. The result shows CDK9 may be a potential serum biomarker of coronary atherosclerosis.3. Study shows that CDK9 inhibition can result in inhibition of CDK943 isoform expression in TNFa-stimulated human monocytic cell line THP-1 cells, inhibit proliferation, cause G2/M arrest, and promote apoptosis and apoptosis-related protein expression.