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Candidate Genes Related to Atherosclerosis

Identification of the Candidate Genes in the Progression of Atherosclerosis by Bioinformatics Analysis
Running title: Candidate genes related to atherosclerosis
Highlights:
A total of 670 DEGs in atherosclerotic samples were obtained.
KIAA0101 may be a crucial molecule in atherosclerosis by binding with PCNA.
TRAC may participate in atherosclerosis by binding with MHC antigen.
PLXNC1 and HLA-DQB1 may play a key role in atherosclerosis via immune system.
Abstract
Objective: The purpose of our study was to identify candidate genes in the progression of atherosclerosis. In addition, we aimed to explore the molecular mechanism of the development and progression of atherosclerosis.
Materials and Methods: The gene expression profile of GSE28829 was downloaded from Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) in early and advance atherosclerotic samples were analyzed with limma package. Cluster analysis of the screened DEGs was performed through affinity propagation cluster method (APCluster). The DEGs enrichment was obtained via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Finally, protein-protein interaction (PPI) network of the high relative genes was constructed using the Cytoscape software.
Results: Totally, 670 DEGs in atherosclerotic samples were obtained. After cluster analysis of DEGs, 28 genes were selected from the 69 clustering gene sets. The most significant gene was major histocompatibility complex, class II, DQ beta 1 (HLA-DQB1) in the 14 biological pathways based on the pathway enrichment analysis of KEGG. Totally, 3 DEGs of KIAA0101, plexin C1 (PLXNC1) and T cell receptor alpha constant (TRAC) were gained after the attributive analysis of protein-protein interaction (PPI) network.
Conclusion: KIAA0101, PLXNC1 and TRAC may be candidate genes for regulating the progression of atherosclerosis.
Keywords: atherosclerosis; bioinformatics analysis; differentially expressed genes
Introduction Atherosclerosis is an ongoing process which already starts in childhood [1]. It is a progressive multifaceted inflammatory disease that affects large- and medium-sized arteries by thickening and hardening these arteries [2-4]. Atherosclerosis can mainly induce myocardial infarction or ischemic stroke, which is considered as the most common reason of death worldwide [5]. Besides, numerous lines of evidence suggest that many other diseases, such as coronary heart disease, diabetes mellitus and dyslipidemia, are associated with atherosclerosis [6-8]. Thus, atherosclerosis has been a worldwide threat to public health.
Atherosclerosis has been reported to be a disorder with multiple genetic and environmental contributions [9]. Genetic-epidemiologic studies have identified the activation of protein kinase C (PKC) was induced by elevated levels of diacylglycerol, which resulted from a high concentration of glucose and nonesterified fatty acids. This event is considered to be a link between altered vascular cell signaling and abnormal metabolism, finally lead to atherosclerosis [10]. Furthermore, PKC isoforms can induce expression of pro-inflammatory cytokine and activation of nuclear factor ?B and NADPH oxidase, as well as increase signaling proteins, eg. extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), thus contribute to vascular inflammation and atherosclerosis [11]. Angiotensin II (ATII) has been demonstrated to have direct effects on development of atherosclerosis through stimulation of monocyte recruitment, activation of macrophages, and enhanced oxidative stress, all of which have been linked to increase of the atherogenic process [12-14]. On the other hand, angiotensin-(1-7) (AT-(1-7)) opposes AT II action to prevent atherosclerosis [15]. In spite of the expanded efforts to study the genetic bases of atherosclerosis, the molecular mechanisms of the development and progression still needed further study.
In the present study, we aimed to identify the differentially expressed genes (DEGs) from the early- and advanced-stage atherosclerotic samples and explore the molecular mechanisms in the onset and progression of atherosclerosis. Furthermore, the in-depth understanding of this disease can provide the basic for appropriate treatment.
Materials and methods
Data source
The atherosclerotic gene expression profiles of GSE28829 [16] was downloaded from Gene Expression Omnibus (GEO) database (http://www. ncbi.nlm.nih.gov/geo/) based on Affymetrix Human Genome U133 Plus 2.0 Array (GPL570 [HG-U133_Plus_2]). A total of 29 samples, consisting of 13 early atherosclerotic samples and 16 advanced atherosclerotic samples, were available in the present analysis.
Data pre-processing
The original probe-level data in CEL files were converted into expression measures using the affy package [17] in R language. Quality analysis was constructed to confirm the available chips. Background correction and quartile data normalization were performed by gcrma package [18] to obtain the expression profile data.
Identification of differentially expressed genes (DEGs)
The Affymetrix Microarray Suite 5 (mas5) calls was applied to get the expressive gene in at least one chip by gene screening. After that, differentially expressed genes (DEGs) were identified through the contrastive analysis of early- and advanced -stage samples using the limma package [19]. Fold change value (|log2FC|) of DEGs larger than 2.0 and P-value less than 0.05 were used as the cut-off criterion.
Cluster analysis of DEGs
The genes in relation to atherosclerosis were downloaded from Online Mendelian Inheritance in Man (OMIM). Then, cluster analysis of the screened DEGs was performed through affinity propagation cluster method (APCluster) [20], which works based on consideration of all the data points as potential cluster centers [21].
Functional enrichment analysis
For the screened DEGs, functional analysis of GO (gene ontology) term, using biological process (BP) GO term, was firstly performed using the online DAVID (database for annotation, visualization, and integrated discovery) [22], GOEAST (Gene Ontology Enrichment Analysis Software Toolkit) [23] and Toppgene [24].Reliable results were obtained via the comprehensive analysis of the three results. Then, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of the DEGs enrichment were also obtained using the online DAVID. P-value less than 0.05 and false discovery rate (FDR) less than 0.05 were considered as the cut-off criterion.
Protein-protein interaction (PPI) network construction
The screened DEGs were further selected combining with the known atherosclerosis genes. PPI data were downloaded from human PPI datasets, such as HPRD (Human Protein Reference Database) [25], BioGRID (Biological General Repository for Interaction Datasets) [26], MINT (Molecular INTeraction database ) [27], InAct [28] through which the highest reliability PPI data was obtained. The PPI networks were constructed using the Cytoscape software [29] based on the PPI relationship.
Results Identification of DEGs
We used Limma package in R to construct the contrastive analysis between the early- and late-stage samples. According to the cut-off criteria of |log2FC| > 2.0 and P-value < 0.05, we finally gained 670 DEGs.
Cluster analysis of DEGs
There were 4 atherosclerotic genes from OMIM (table 1), and only arachidonate 5-lipoxygenase (ALOX5) was one of the screened DEGs. After cluster analysis of DEGs, a total of 69 clustering gene sets were gained, and the clustering heat map shown in Fig 1. Finally, 28 genes were selected from the 69 clustering gene sets based on whether ALOX5 was in the clustering gene sets (table 2).
Functional enrichment analysis
A total of 187 DEGs were obtained based on a P < 0.05 after the functional enrichment analysis of BP GO terms. Following the pathway enrichment analysis of KEGG, 14 biological pathways (shown in table 3) were chosen according to FDR < 0.05. The most significant enrichment pathway in atherosclerosis was systemic lupus erythematosus, and the most significant gene was major histocompatibility complex, class II, DQ beta 1 (HLA-DQB1).
PPI network
The resources of PPI networks were showed in table 4. The PPI network was obtained with 4,888 nodes and 88,423 edges. Then, the node degree, betweenness centrality and closeness centrality in the PPI network were calculated to select high relative genes. The top 10 key genes in each group were showed in table 5. Totally, there were 17 genes including 3 DEGs of KIAA0101, plexin C1 (PLXNC1) and T cell receptor alpha constant (TRAC) and 1 known relative gene of estrogen receptor 1 (ESR1). At last, a key sub-network, consisting of 3,859 nodes and 75,916 interaction edge, was constructed using the 17 genes and their neighbor nodes.
Discussion Generally, atherosclerosis is a systematic and widespread disease [30]. It is frequently associated with chronic heart disease of the arteries with high death rates all over the world [31]. Nowadays, the diagnosis of atherosclerosis still remains difficult for its asymptomatic early-stage [32]. Therefore, genetic therapies or new drug targets in the early detection of atherosclerosis is urgently needed based on the molecular pathogenesis. In the current study, we analyzed 670 DEGs upon gene expression profile of early and advance stage atherosclerosis by bioinformatics analysis. Finally, 3 DEGs including KIAA0101, PLXNC1 and TRAC was significantly identified. By analysis of the 14 biological pathways enriched by DEGs, we found that HLA-DQB1 might be also an important gene associated with atherosclerotic development.
The gene of KIAA0101 located in nucleus and mitochondrion [33, 34]. It encodes a 15 kDa protein, which is related to the proliferative activity of human atherosclerotic lesions [35, 36]. The KIAA0101 protein can bind with conserved proliferating cell nuclear antigen (PCNA) competitively with p21WAF, a cell cycle regulator [37]. PCNA has been reported to play a key role in DNA replication and damage repair as an indispensable factor for DNA polymerase, that is, the DNA repair, apoptosis and cell cycle progression is partly attributed to PCNA [33, 38, 39]. In addition, PCNA, as an essential component of the DNA synthesis machinery, promotes the proliferation human vascular smooth muscle cells [36]. To the best of our knowledge, atherosclerotic lesions is fundamentally characterized by the accumulation of cells within the intima [40]. Therefore, KIAA0101 may be a crucial molecule in the progression of atherosclerosis by binding with PCNA.
PLXNC1 encodes plexin C1 (a member of the plexin family), which is one of the semaphorins (SEMA) receptors, and the semaphorins is known as a large family including transmembrane and secreted signalling proteins [41]. Particularly, plexins are receptors for transmembrane semaphorins, which are frequently involved in immune response, from initiation to terminal inflammatory processes [42]. Immune responses consisting of adaptive and innate immunity have been evidenced to tightly participate in regulation during the progression of atherosclerosis [43], as immune activation is part of the disease process [44]. Besides, plexin can interact with some semaphorins on monocytes [45, 46]. As a result, monocytes are exposed to soluble SEMA4D/CD100 (a critical semaphorin in the immunoregulation), representing a significant down-modulation in pro-inflammatory cytokine production and leading to immune response [47]. These lead us to hypothesis that PLXNC1 may play an important role in the development of atherosclerosis through modulation in immune response.
TRAC, another significant DEG in our study, is a protein-coding gene. The TRAC protein can bind major histocompatibility complex (MHC) antigen. Aberrant MHC antigen expression in smooth muscle and endothelial cells may active T lymphocytes. Meanwhile, the activated T lymphocytes may modulate the functions of other cells in atherosclerotic plaque and the significant amounts of T lymphocytes are also an important cause of atherosclerosis [48]. In addition, overexpression of MHC antigen may also participate in the perpetuation of the atherogenetic autoimmune reaction [49]. Therefore, TRAC may participate in the development of atherosclerosis via binding MHC antigen.
Besides, in the present study, HLA-DQB1 was shown as the most significant gene involved in several biological pathways. HLA-DQB1 belongs to the histocompatibility leukocyte antigen (HLA) class II beta chain paralogs. The protein encoded by HLA-DQB1 is essential for constructing the DQ heterodimer, which is a cell surface receptor playing a central role in the immune system [50, 51]. Moreover, HLA-DQ may contribute to the presentation of antigen to suppressor systems. The antigen-presenting function is possibly related to inflammatory mechanisms in atherosclerosis [50]. Thus, HLA-DQB1 may be a significative gene in the development of atherosclerosis involving in the immune system.
As a result of this preliminary study, the 3 DEGs of KIAA0101, PLXNC1 and TRAC may be candidate genes that tightly associated with the development and progression of atherosclerosis. In addition, HLA-DQB1 involved in biological pathways may be also an important gene that plays a pivotal role in atherosclerotic development. These findings may provide possible molecular mechanism for well treatment of atherosclerosis. However, further study is warranted to verify our conclusions with more genetic experiments of DEGs as no experiments is performed in the present study.

Atrial Fibrillation and Cardiac Arrhythmia

Introduction Atrial fibrillation is the most common form of cardiac arrhythmia; it involves the two upper chambers of the heart known as the atria. During atrial fibrillation the normal pulses generated by the sinoatrial node are overcome by the electrical pulses that are generated in the atria and pulmonary veins, which leads to irregular impulses being conducted to the ventricles, and therefore irregular heartbeats are generated.
AF is identified by rapid and oscillatory waves that vary in amplitude, shape and timing instead of regular P-waves. Electrocardiograms are therefore used commonly to diagnose AF in patients.
Arterial Fibrillation can present asymptomatically meaning that it can present in a patient but show no symptoms, it is considered to be non life threatening in many cases although it can result in heart palpitations, fainting, chest pain and in chronic cases congestive heart failure. The risk of stroking is also increased due to the fact that blood may pool and form clots in poorly contracting atria. Patients with AF are usually given blood-thinning medication such as warfarin to stop clots forming. Atrial fibrillation can occur in the absence of structural heart disease, known as lone AF, although this only occurs in approx. 15% of cases. Commonly AF is associated with hypertension, diabetes, obesity, coronary artery disease, pulmonary disease, valvular heart disease and coronary heart failure.
Basic Pathophysiology of Atrial Fibrillation Atrial fibrillation usually begins with increased premature atrial contractions (ectopic beats) progressing to brief runs of atrial fibrillation usually that are usually self-terminating, over time these episodes can increase in duration and sometimes become persistent. During this progression structural changes in the atria occur as well as biochemical changes in the atrial myocytes. Pathophysiological adaptation of the atria to fibrillation has been broadly termed remodeling. More specifically, the changes primarily affecting the excitability and electrical activity of the atrial myocytes have been termed electrophysiological remodeling.
The primary change in the structure of the atria is fibrosis, which is usually considered to be due to the atrial dilation, although in some cases genetic influences and inflammation can also be a cause. In 1990 Sanfilippo stated that atrial dilation was not a consequence of AF although more recently in 2005 Osranek stated that atrial dilation was not a consequence of AF.
Dilation is due to almost any structural abnormality of the heart, such as hypertension, valvular heart disease and congestive heart failure; this structural abnormality causes a rise in intra-cardiac pressures. Demonstrating the strong relationship between atrial fibrillation and structural heart disease. Once dilation does occur it begins sequences of events that lead to the activation of the rein aldosterone angiotensin system and a subsequent increase in matrix metaloproteinases and disintegrin, leading to remodeling of the atria and fibrosis. Fibrosis is not limited to muscle mass of the atria, it can occur in sinus node and atroventricular node also, relating to sinus node dysfunction (sick sinus syndrome).
During normal electrical conduction of the heart the SA node generates a pulse that propagates to and stimulates the muscle of the heart (myocardium), when stimulated the myocardium contracts. The order of stimulation is what causes correct contraction of the heart, allowing the heart to function correctly. During atrial fibrillation the impulse produced by the SA node is overcome by rapid electrical discharges produced in the atria and adjacent parts of the pulmonary veins. When AF progresses from paroxysmal to persistent the sources of these conflictions increase and localise in the atria.
Principles of Catheterization and Ablation The fundamental aim of catheter ablation is to eliminate ectopic beats that arise most often in the pulmonary veins and less often in the superior vena cova and coronary sinus. This is accomplished through catheter insertion into blood veins in order to reach the heart, isolation of abnormal heart tissue and ablation of this abnormal heart tissue through the use of radiofrequency, cryoblation or high intensity focused ultrasound.
Rate Control and Rhythm Control Despite ablative techniques and antiarrhythmic drugs available, management of common rhythm disturbance remains a problem. Rate control is the preferred treatment for permanent atrial fibrillation and for some patients with persistent atrial fibrillation, if they are either over 65 years of age or have coronary heart disease. Rate control is usually done through the use of pharmaceutical drugs (usually beta blockers or rate limiting calcium channel blockers) in order to slow ventricular heart rate and stop the atria from fibrillating. Rhythm control is most commonly used for the treatment of paroxysmal atrial fibrillation and in some cases of persistent atrial fibrillation if the patient is either less than 65 years of age, has lone atrial fibrillation or congestive heart failure. Rhythm control is usually achieved through the use of either a cardioversion (electrically or pharmacological) or the use of pharmaceutical drugs (usually beta blockers) in order to maintain sinus rhythm. This treatment is needed for a longer time in order to stop reoccurrence of atrial fibrillation. [http://www.cks.nhs.uk/atrial_fibrillation/management/detailed_answers/first_or_new_presentation_of_af/rate_or_rhythm_control#-391784). Atrial fibrillation is treated most commonly pharmaceutically although if the drugs cannot control the AF or if the patient is having a bad reaction to the medication, catheter ablation therapy allows for greater control of heart rate and rhythm than drug therapy although it does present more risk to the patient.
Radiofrequency Catheter Ablation Electrically isolating arhythmogenic thoracic veins is the most important aspect of this procedure. The application of radiofrequency energy to an endocardial surface is used to cause cellular electrical destruction with the loss of cellular electrical properties, essentially the destruction of abnormal electrical activity [39,40]. This technique can be enhanced through the use of larger ablation electrodes, [41-46] allowing the creation of deeper lesions. During the procedure a physician will map the area to locate abnormal electrical activity, this is facilitated through the use of electroanatomic mapping system (fig 2) allowing for better navigation when the catheter is inserted into the artery. Reported success of radiofrequency ablation is dependent on the severity of the condition and ranges from 65% to 85% and patients presenting with complications is 5%.[cryostat]
Cryoblation The most used format of cryoblation is the cryoballoon approach. This involves a deflectable a deflectable over-the-wire catheter with an inner and outer balloon inserted, allowing for anatomical variance this balloon is available in two sizes (23mm and 28mm). The guidewire is positioned in the distal part of the pulmonary vein, the deflated balloon is then progressed to the pulmonary vein ostium. Using the central balloon marker the balloon position is then estimated before inflation, once the desired position is found the balloon is inflated; pressurized N20 is then delivered to the tip of the catheter via an ultrafine injection tube down a central lumen in the inner balloon, working like an expansion chamber. Sudden expansion of the liquid gas causes evaporation and absorption of heat from tissue and low temperatures are then achieved (Approx -80dc). An occlusion angiogram is then performed in the central lumen of the catheter to ensure good balloon pulmonary vein contact. Cryoblation is then started for at least five minutes under the condition that optimum pulmonary venous occlusion is achieved. The most important issue when using this technique is to establish optimum contact between the pulmonary vein antrum and the balloon.
High Intensity Focused Ultrasound (HIFU) High intensity ultrasound is used in percutaneous ablation of atrial fibrillation through the use of a steerable balloon catheter. The high intensity focused ultrasound balloon is positioned at the ostium of the pulmonary veins and forms a sonicating ring to ablate pulmonary vein antrum when high intensity focused ultrasound is delivered. An arrhythmia-free rate of 59%-75% was achieved by HIFU balloon in several studies investigating its effectiveness in atrial fibrillation ablation.15-17
Commercially Available Devices and Systems Medtronic GENius Multichannel RF Generator
This generator is used for the creation of endocardial lesions during cardiac ablation procedures for the treatment of supraventricular arrhythmias. The generator delivers temperature-controlled radiofrequency energy, utilizing five radiofrequency energy mode selections: bipolar only, unipolar only, and combination energy mode selections of 4:1, 2:1, and 1:1. This system must be used with a catheter that is single use and sold separately to the device. The generator automatically recognizes the attached Cardiac Ablation Catheter and loads preset default temperature, time, and energy mode setting parameters. Ablation parameters such as ablation duration, energy mode, target temperature and channels can also be manually selected.
Medtronic Cardiac CryoAblation Device
The CryoConsole contains both electrical and mechanical components as well as exclusive software for controlling and recording a cryotherapy procedure. This system requires catheters that are purchased separately such as Medtronics Artic front cryoablation catheter (Fig. 3). This system stores and controls the delivery of the liquid refrigerant through the coaxial umbilical to the catheter, recovers the refrigerant vapor from the catheter under constant vacuum, and disposes of the refrigerant through the hospital scavenging system. Multiple features are built into both the CryoConsole system and catheters to ensure safety.
Epicor™ Cardiac Ablation System Price
The Epicor™ LP Cardiac Ablation System delivers High Intensity Focused Ultrasound using algorithms designed to precisely deliver energy up to 10mm. Unlike the other treatments high intensity focused ultrasound has the ability to create lesions from the inside out, depositing energy at the endocardium first and then building the lesion back up to the surface. The ability to focus HIFU cardiac ablation energy helps reduce the risk of tissue disruption, charring and collateral damage as well as overcome procedural limitations that have historically been associated with other ablation technologies.
Conclusion In terms of ablation the umbrella terminology of Atrial Fibrillation does not take into account the complex nature of the disease. If a patient presents with paroxysmal atrial fibrillation they may only require a single catheter to be used, however if this condition becomes more continuous/chronic the patient may require multiple catheters and 3D navigational software. The three techniques described in this report appear to be similar in terms of their success rate, radiofrequency and cryoablation have a success rate of approx. 65-85% while High intensity focused ultrasound has a success rate of approx. 59-75%, this perhaps indicates that high intensity focused ultrasound may not be as effective in treating atrial fibrillation as radiofrequency and cryoablation although it is worth noting that these figures are taken from different research studies at different times and involve different patients that could be presenting greater or lesser a severity of atrial fibrillation.

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