| BackgroundCardiovascular diseases are the leading cause of morbidity and mortality in the global world, and atherosclerosis (As) is known to be the primary underlying factor responsible for the development of these diseases,accounting for 75%. As is considered to be a chronic, progressive, inflammatory disease, and revealing the pathogenesis of As is a fundamental measure for prevention and treatment of cardiovascular and cerebrovascular disease. The formation of atherosclerotic plaque is long and complex, now that the pathological process of As is participated by a variety of factors and inflammatory responses are throughout the entire process of the development and progression of As. With the role of a great many of cytokines, the permeability of endothelial cells are damaged and the counts of endothelial cells decrease, mononuclear cells invade into intima layer in the formation of macrophage that devour lipid cells into foam cells, smooth muscle cells proliferate and a great many extracellular matrixs are synthetized. The pathological changes form a composite process from fatty streaks, fibrous plaques, plaques to plaque rupture. Despite extensive research, the detailed molecular mechanisms underlying the development of atherosclerosis remain poorly understood and new findings are urgently needed to complement the current knowledge and to identify new drug targets. Rapid advances in biological technology, including DNA microarrays able to detect the expression levels of tens of thousands of genes simultaneously, might help to provide comprehensive insights into the pathogenesis of atherosclerosis.Clinical observation found that, the degree of stenosis dose not occur in parallel with acute cardiovascular and cerebrovascular diseases such as acute coronary syndrome, acute ischemic cerebral stroke. A growing number of studies suggest that, the occurrence of acute cardiovascular and cerebrovascular events depends not only on the degree of stenosis of the artery lumen, but also is more important in the formation of unstable atherosclerotic plaques. Unstable plaque is the rupture of plaque, also called vulnerable plaque, and its pathological characteristic is with collagen content reduced, the count of smooth muscle cells decreased, inflammatory cells such as macrophages, mast cells and T lymphocytes infiltrating into intima, thin fibrous cap, large lipid core, intraplaque hemorrhage, calcification, fissure plaque surface, and new born blood vessels appear in the plaque. Unstable plaque cause plaque rupture, thrombosis and occlusion of vascular lumen is the main reason causing acute cardiovascular events. At present, the pathophysiological process of unstable plaque mainly comprises the increase of inflammation, the formation of new blood vessels and the induction of cell apoptosis. But the molecular mechanism of pathological and physiological process of unstable plaque is not entirely revealed and is with important clinical significance in the prevention of the occurence of acute cardiovascular events.The expression of tens of thousands of genes could be simultaneously measured in the microarray technology,which can not only screen for differential expressed genes in relation with the disease or phenotype, but also can reveal the relationship between the expression changes of genes, providing a clue to study the relation between genes and diseases.The application of gene expression profiles in the field of atherosclerosis has already been a history of 14 years. During this period, some important genes and pathways are obtained form the domestic and foreign research with gene expression profile analysis, which identified some key diseased genes in the development of atherosclerosis. It has been made a tremendous contribution to deeply and comprehensively understand the molecular mechnism of disease. It has previously been used to detect altered gene expression in normal and diseased arteries, establish crucial players in disease progression, identify differentially expressed genes (DEGs) according to patient symptomatology, and discover pathways affected in coronary atherosclerosis. However, there are drawbacks associated with these types of comparisons. Differences in the cellular compositions and morphologies of plaques and normal arterial wall may lead to differences in gene expression that simply reflect this variation. In addition, irregular sample-collecting methods, for example, samples from different sites or sources or small sample sizes, may affect the reliabilities of the studies. Furthermore, a high degree of variability in plaque composition and gene expression between humans and animal models may limit the extension of cDNA array studies on animal material to clinical use.Features of unstable plaques, such as surface ulceration and rupture, may also occur in both asymptomatic and symptomatic patients, which may also confound studies that classify samples according to patient symptomatology. The relative lack of systematic bioinformatic analysis of cDNA microarrays limits the effective exploitation of gene-expression data sets. An integrated bioinformatic analysis based on cDNA microarray studies of human tissues, including large sample sizes, is thus urgently required to clarify the mechanisms underlying the development and progression of atherosclerosis.The aim of this study was to use an integrated bioinformatic method involving whole-transcriptome analysis to characterize the gene-expression signature of unstable plaque regions of carotid endarterectomy specimens, compared with ajacent regions of the same specimen. To interpret the biological relevance of these changes in gene expression, the microarray data were analyzed by gene-ontology methods and pathway analysis, thereby allowing the construction of interaction networks, which might identify novel prognostic markers and therapeutic targets. Real time PCR and Western blot are applied to our study to verify the expression of some differential expressed genes. Our study provided some clue on future study of unstable plaque in the area of pathogenesis of As, screening for early warning indicator and biochemical markers in the serum for the unstable plaque and providing theoretical guidance in screening for therapeutic targets in the clinic.Aims1. Get the differential expresson profile of unstable plaque, disease relevant genes and signaling pathways2. Exprimental verification of expression of TYROBP, CXCR4 and VCL in human carotid vessels.Methods1. Download the raw data of gene expression profileDownload the E-MTAB-2055 raw data set from the ArrayExpress database. Signal values at the probe level are quality controlled, variation of stability was analyzed, background was corrected, data was normalized and genes were annotated. Through above analysis, the raw data was eventually changed into the value at the level of gene expression.2. Identification of differentially expressed genesT test and the fold change methods are jointly applied to identify the differential expressed genes.3. Enrichment analysis of differential expressed genesThrough the DAVID website (Database for Annotation, Visualization and Integrated Discovery), we submitted the differential expressed genes to the website, and GO terms and KEGG pathways enrichment analysis were conducted.4. Protein-protein interaction network constructionFrom the string (version 9.1) web site, we download all human protein protein interaction list. The protein-protein interaction data were preprocessed, including removing redundancy and self-loops, and converted into Entreze Gene. The DEGs were mapped into the PPI network to determine the molecular mechanisms of atherosclerosis in terms of protein-protein interactions. We extracted the nodes and relationships between DEGs to construct a network. We then uniformly converted all Entreze Genes in the constructed network into Gene Symbols. The biological graph-visualization tool Cytoscape was then used to construct the PPI network, in which proteins/genes were represented with nodes, while interactions derived from experimental repositories and computational prediction methods between any two proteins/genes were represented as edges. The hub proteins/genes in the network were the nodes with the highest number of interactions. Cfinder was used to find disease-related modules and the Cytoscape Network Analysis plugin was applied to calculate the degrees of the nodes in the network.5. Obtain the stable and unstable carotid plaque specimensWe obtained the plaques tissues of carotid artery at vascular surgical operation room of the Nanfang Hospital of Southern medical university after the operation of carotid endarterectomy. Before collection, color Doppler ultrasound were conducted to distinguish the characteristic of carotid artery plaque, selecting the unstable plaques based on its characteristics and the adjacent tissues are used as stable plaque.6. Detection the expression of TYROBP, CXCR4 and VCL of human carotid artery tissue (stable plaques and unstable plaques) at the transcriptional level.7. Detection the expression of TYROBP, CXCR4 and VCL of human carotid artery tissue (stable plaques and unstable plaques) at the protein level.statistical analysis1. We used T test and FC method to obtain differentially expressed genes. The screening thresholds of differential expressed genes were set as p-value<=0.01 and |logFC|>l. The hypergeometric test was used in the enrichment analysis, which the screening threshold for GO enrichment is FDR<=0.01 and for KEGG enrichment is p-value<=0.05.2. All data are representative of 3 repeat experiments, with a mean ± standard deviation. All the statistical analysis was processed by SPSS 19.0 statistical software package, which the Mann-whitney U test were used to obtain the statistical significance of expression of differential expressed genes between unstable plaques and stable plaques. The threshold of the statistical test:alpha=0.05, P<0.05.Result1. Identification of differentially expressed genes439 differentially expressed genes were obtained in this study, including 232 up regulated genes and 207 down regulated genes. The most up regulated differential expressed gene is heme oxygenase 1 (HMOX1, logFC=3.1), and the most down regulated differential expressed genes is alpha-actin (ACTC1, logFC=-2.2).2. The enrichment analysis for differentially expressed genesGO enrichment results show that immune response, immune defense, inflammatory response, cell adhesion, cell factor were significantly enriched. Pathway enrichment results showed a total of 9 pathways meet the enrichment threshold requirements, namely,lysosomal pathway, cytokine signaling, PPAR signaling pathway, cytokine cytokine interaction pathway, white cell transmembrane migration pathway of complement, coagulation cascade pathway, hematopoietic cell lineage and tyrosine metabolism pathways were significantly enriched.3. Protein protein interaction network analysisIn PPI network, TYROBP, CTSS, C1QB, PLEK, IGSF6, HCK, VCL, LAPTM5, CXCR4 and FCER1G is at the central location, which play an important role in biological networks. Our study obtained four risk modules related to unstable plaque and TYROBP, CXCR4, VCL and APOE respectively occupy the center position of four modules, which play a key role in the modules.4. Compared with the stable plaque, the expression of TYROBP and CXCR4 were up regulated in the unstable plaque tissues, and the Mann-whitney U test showed the difference with statistical significance; The expression of VCL in unstable plaque tissue was down regulated, but with no statistical significance.5. Compared with the stable plaque tissue, the protein expression level of TYROBP and CXCR4 were increased in unstable plaques; The protein expression of VCL in unstable plaque tissues was decreased.Conclusion1. The immune related pathways and inflammatory responses play an important role in the process of unstable plaque;2.Gene expression profile analysis showed that TYROBP and CXCR4 were up regulated, while VCL was down regulated, which may play important roles in the process of unstable plaques;3. At the level of mRNA, the expression of CXCR4 and TYROBP in unstable plaque were up regulated with statistical significance between the two group. The expression of VCL in unstable plaques showed downward trend, but with no statistical significance; The protein expression of CXCR4 and TYROBP in unstable plaque tissue were significantly increased, and VCL protein expression decreased obviously in the unstable plaque tissues; Above results were basicly consistent with the microarray analysis;4. TYROBP, CXCR4 and VCL may become new molecular markers in the development and progression of unstable plaques as well as prognostic markers and new therapy targets to reverse the process of unstable plaque. |
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