Pathway Analysis Of Gene Expression Profiling And Gene Regulatory Network Construction In Stanford Type A Aortic Dissection

Part Ⅰ Gene Expression Profiling and Pathway Analysis of Stanford Type A Aortic Dissection[Objects] Acute aortic dissection is a serious and fatal medical condition that is the consequence of damage to the aortic wall. Some risk factors associated with aortic dissection have been identified, including genetic connective tissue disease. However, most of the molecular features underlying the pathogenesis of aortic dissection, especially those without genetic background, remain unclear yet. We aimed to study the gene expression profiling and analyze the biological pathways in Stanford type A aortic dissection.[Methods] Ascending aorta specimens from patients with acute Stanford type A dissection were taken during surgery and compared with those from normal ascend ing aorta from organ donors using the Whole-Genome Gene Expression microarray. Data were analyzed with GenomeStudio Gene Expression Module v1.0. All differentially expressed genes were submitted for pathway analysis using KEGG database.[Results] 1) 1309 genes differently expressed in dissected and control aorta specimens, including 630 up-regulated and 679 down-regulated in aortic dissection, were detected by t-Test unpaired unequal variance (Welch) algorithm.2) Compared with the control group, patients with aortic dissection had 72 different gene-expression pathways (p<0.05), among which 37 pathways were significantly different (p<0.01) including Focal Adhesion, Vascular Smooth Muscle Contraction, Regulation of Actin Cytoskeleton and ECM-receptor Interaction pathways.3) Focal Adhesion pathway has 201 genes, among which 30 genes had different expression. Changes were seen in several genes of integrin alpha, e.g., ITGA2, ITGA5, ITGA7 and ITGA8. The expression of caveolin, another important transmembrane protein in Focal Adhesion pathway, was down-regulated, as well as the adapter proteins including actinin, vinculin and filamin. Additionally, Integrin-linked kinase also had lower expression in patients with aortic dissection.4) Vascular Smooth Muscle Contraction pathway has 115 genes, among which 17 genes had different expression. All the 17 genes were down-regulated in patients with aortic dissection. Genes related to potassium channel and its receptors on the membrane of vascular smooth muscle cells, such as KCNMB1, KCNMA1, ADRA1B, AGTR1, RAMP1 and RAMP3 were down-regulated; enzyme related genes including PLCB4, ADC Y9, PPP1R12A, PPP1R12B and PPP1R14A were down-regulated; genes encoding actin and myosin, such as ACTG2, MYL9, MYH11 and MYLK were down-regulated.5) Regulation of Actin Cytoskeleton pathway has 216 genes, among which 22 genes had different expression, including those also contained in above mentioned 2 pathways, e.g., ITGA2, ITGA5, ITGA7, ITGA8, ACTN4, VCL, MYL9, MYLK, PPP1R12A, PAK7, PIK3R1, PIK3R2 and PDGFD. Additionally, CFL2 and GSN, which encoding cofilin-2 and gelsolin respectively, had lower expression in patients with aortic dissection.6) ECM-receptor Interaction pathway has 84 genes, among which 16 genes had different expression. Gene expression of collagen Ⅰ, Ⅲ, Ⅴ and Ⅵ was up-regulated in patients with aortic dissection, including COL1A1, COL3A1, COL5A1, COL5A2 and COL6A3. The gene expression of laminin, another extra cellular protein, also changed. LAMA4, LAMB1 and LAMB3 were up-regulated, while LAMA3 was down-regulated.7) Among the 37 pathways with different expression, there were some pathways relevant to inflammation, such as Cytokine-cytokine Receptor Interaction, Chemokine Signaling pathway and JAK/STAT Signaling pathway.[Conclusions] Our data of microarray showed there were numbers of genes differentially expressed in patients having acute aortic dissection without genetic background. Pathway analysis indicated the abnormality was mainly in Focal Adhesion, Vascular Smooth Muscle Contraction, Regulation of Actin Cytoskeleton and ECM-receptor Interaction pathways of vascular smooth muscle cells. Our study suggested the vascular smooth muscle cells in aortic tunica media may have played an important role to the occurrence and development of acute aortic dissection.Part II Gene Regulatory Network Construction of Stanford Type A Aortic Dissection[Objects] We used Gene Set Enrichment Analysis and Expression Module Analysis as well as STRING database to further study the gene expression and construct the gene regulatory network in Stanford type A aortic dissection for well understanding the pathogenesis of acute aortic dissection.[Methods] Differentially expressed genes were found and expression modules were established through Gene Set Enrichment Analysis and the innovative Expression Module Analysis. JAK2-centered inflammation network was constructed with STRING database. Network Ontology Analysis was used to determine the key function of JAK2 inflammation network. The expression level of IL-6, KITLG and CCL2 were verified by RT-PCR.[Results] 1) The expression profiles of the top 25 genes were studied. Among them, CDC45L, ST14, CKAP2L, TIMP1, PHLDA1, LHFPL2, PCSK1, GINS2, TMEM158, TSPANS, ECT2, CCDC34, VARS, CDC7, NT5DC2, LOC646993 and PPIF were up-regulated, while RYR2, C5orf24, JAK2, REEP1, THSD4, BRP44L, RNF115 and BAMBI were down-regulated.2) Eight hotspots called Expression Modules were identified by a seed gene with its corresponding gene symbol:these are PER2, CNN1, MCM2, APITD1, JAK2, HIF1A, IL1R1 and CDC45L.3) In JAK2 ExpMod, JAK2 defined the hub of this module, and formed dense subnetworks with other differentially expressed genes e.g.IL6/IL6R, CCL2, IFNGR1/2, EPORand KITLG.4) In the protein-protein interaction network constructed by STRING, JAK2 also acted as the key regulatory factor of the network, and had predicted association with the other genes including IL-6/IL-6R, CCL2, IFNGR1/2, EPOR and KITLG.5) RT-PCR confirmed the significant upregulation of IL-6, CCL2 and KITLG.6) Network Ontology Analysis showed the function of the network was activation of JAK2 kinase activity and regulation of tyrosine phosphorylation of STAT protein.[Conclusions] We constructed the JAK2-centered network using different methods with different databases and got similar findings. Our study also shows that IL-6/IL-6R, CCL2, IFNGR, EPOR and KITLG were all up-regulated in patients with aortic dissection. All these genes concentrated to JAK2, forming the JAK2-centered regulation network. Our results indicated in acute aortic dissection, the abnormality in the extra-cellular ligands such as IL-6, CCL2, IFN-γ, EPO, KITLG and there relevant receptors had interaction with JAK2 and impacted the cell function through JAK/STAT signal pathway.