The Complex Cardiac Atherosclerotic Disorder : the Elusive Role of Genetics and the New Consensus of Systems Biology Approach

Introduction During the last two decades genetics focused on to scarce single-gene diseases encountered in less than 1% of the general population [1]. It was considered that the affected genes were responsible for highly penetrable mutations interfering with the normal function of the encoded proteins and were accountable for the appearance of modified phenotypes. Furthermore, a number of people were examined for their genotype, looking for correlations between single nucleotide polymorphisms (SNPs) and phenotypic features. However, complex cardiovascular diseases are multifaceted and are not explained by single-gene mutations. Unfortunately, the Human Genome Project proved unable to solve many of the questions related to health and disease. The novel genomewide case-control association approach which is designated as GWAS (Genome Wide Association Studies) proved effective in the identification of specific markers related to a disease [1]. Stranger et al [2] reported that over 700 of GWAS were published until 2010 for more than 400 diseases and traits. With GWAS, the identification of specific markers increases the prospects to enquire into complex diseases like coronary artery disease (CAD). However, with GWAS, the problem is to locate the correct combination among thousands of interconnections between SNPs and atherosclerotic risk factors that is meaningful for clinical purposes. The construction of functional networks between interconnections with statistical significance is very complicated. Cantor et al [3] indicate that “in GWAS, even for main effects, the number of predictors far exceeds the number of observations”.


Introduction
During the last two decades genetics focused on to scarce single-gene diseases encountered in less than 1% of the general population [1].It was considered that the affected genes were responsible for highly penetrable mutations interfering with the normal function of the encoded proteins and were accountable for the appearance of modified phenotypes.Furthermore, a number of people were examined for their genotype, looking for correlations between single nucleotide polymorphisms (SNPs) and phenotypic features.However, complex cardiovascular diseases are multifaceted and are not explained by single-gene mutations.Unfortunately, the Human Genome Project proved unable to solve many of the questions related to health and disease.The novel genomewide case-control association approach which is designated as GWAS (Genome Wide Association Studies) proved effective in the identification of specific markers related to a disease [1].Stranger et al [2] reported that over 700 of GWAS were published until 2010 for more than 400 diseases and traits.
With GWAS, the identification of specific markers increases the prospects to enquire into complex diseases like coronary artery disease (CAD).However, with GWAS, the problem is to locate the correct combination among thousands of interconnections between SNPs and atherosclerotic risk factors that is meaningful for clinical purposes.The construction of functional networks between interconnections with statistical significance is very complicated.Cantor et al [3] indicate that "in GWAS, even for main effects, the number of predictors far exceeds the number of observations".
Heritability is conveyed by the deoxyribonucleic acid (DNA) through SNPs, insertions and deletions and with epigenetic mechanisms [4].Postgenomics and epigenetics challenge the notion that the genome is the only source of information in health and disease.It is argued that there are significant gene-environment interrelations and interactions explaining the variance in complex diseases as the exposure to environmental risks could increase the level of complexity.
The central position of the genome as the only factor that affects the genesis of complex human diseases is disputed also by the new science of systems biology with its system-level approach.Systems biology is the concept that could be adapted in the field of clinical cardiology and explain in depth the complexity of human chronic heart diseases.Chronic cardiovascular diseases, like atherosclerosis and CAD, are complex diseases generated by the integration of various genetic and environmental factors.The evolvement of computational methodologies encouraged scientists and clinical practitioners to tackle the complex pathologies with the systems biology approach.Thus, we can widen and extend the interdisciplinary systems biology concept up to the level of clinical medicine as "systems medicine" or "systems cardiology".This way it is easier to explain the whole disease procedure and not only the pathology of the organs in isolation.The disease is considered as an "entity" and the clinical phenotype is an "emergent" clinical development.
In this review article the systems biology approach to explain the progression of CAD from the early atherosclerotic lesions to the level of symptomatic disease is underlined.In complex cardiac disorders the role of the causative genes is expressed only through systems genetics and epigenetic mechanisms that are constructing functional molecular networks.The integration of the genetic mechanisms with environmental risk factors is responsible for the whole spectrum of the clinical phenotypes (Fig 1).The search for individual genes responsible for complex clinical entities is limited and is replaced by investigation of multifaceted causes [5].The proposed new understanding of CAD through systems biology methodology is a more holistic conceptual approach.The atherosclerotic involvement of the coronary arteries should be considered as a network of closely connected patterns of self-regulated relationships.To the classical 'reductionist' approach of examining in isolation the genetic, molecular and organs' dysfunction during the pathological processes of CAD should be added the proposed 'holistic' view of the systems biology approach.A shortcoming of the systems biology approach is that we don't have yet all the necessary biological networks of the early stages of the atherosclerotic plaque.It is obvious, that the recognition of the missing biological networks will increase our understanding of the progression patterns of the clinical phenotypes.

Gene mutations and atherosclerosis
There is increased risk of early atherosclerotic development in some families with a genetic condition called homozygous familial hypercholesterolaemia (FH).The FH disorder is an autosomal dominant inherited condition characterized by defects in the low-density lipoprotein (LDL) receptor induced by mutations in the gene encoding the LDL receptor.The FH is a classical mendelian disorder and usually has low prevalence.In extended families specific loci have been recognized in people with a high probability of early CAD but with a low chance to identify specific genes responsible for atherosclerosis [6].In a database for FH patients more than 1000 mutations in the LDL receptor gene were described [7].These mutations involve different proteins of the LDL receptor like the gene encoding protein 6 of the LDL receptor (LRP6) in a family with autosomal dominant premature CAD and metabolic syndrome [8].
Nevertheless, despite the description of mutations in many genes predisposing to atherosclerosis, the majority of those are rare and their genetic contribution is limited.Regardless of the number of the involved genes the significance of each one of those is restricted.The methodology applied in explaining singlegene disorders is insufficient for the genetic enquiry of complex and multilevel cardiac diseases as the task of recognizing the polymorphisms is difficult under the current technology [9].Prediction of the phenotypic characteristics is even more difficult as the accurate gene interdependence is unknown.

Complex cardiac atherosclerotic disorder: genomewide association studies
The GWAS approach is used to identify and compare SNPs in specific chromosomal locations scattered along the human genome of a known population.At first, it was thought that the GWAS would be able to track down statistically significant relationships between SNPs and atherosclerosis.That was based on the fact that SNPs related to atherosclerosis were documented more frequently in patients with CAD compared with control individuals.Thus, the GWAS approach was proposed to analyze genomic DNA markers in a vast number of unassociated people using the new statistical methodology of GWAS.But, soon it was realized that modern genomic technologies like SNP array, gene expression microarray and microRNA array were not sufficient to explain the genetic inclination in patients with CAD.Thus, GWAS showed that only 10.6% of people with atherosclerosis had a probable heritable genetic factor [10,11].In the remaining cases epigenetic studies identified specific CAD risk loci with the DNA methylation status to play significant role [10,11].
Bjorkegren et al [12], using network models based on genetic and genomic datasets, are proposing the systems genetics approach to explain CAD heritability and etiology.They argue that GWAS recognized 153 possible CAD loci with 46 of those having genome-wide importance.But, these loci account for <10% of the genetic variance in CAD while the remaining 90% of CAD missing heritability-not elucidated by the known GWA loci-is due to environmental factors and other risk variants.They suggest that a lot of "information on the heritability of complex disease remains hidden in GWA datasets" and that this "information can be revealed" in order to "identify the disease-driving molecular processes" [12].They emphasized the importance of the "genetics of gene expression studies (GGES) of multiple tissues, namely the STAGE (Stockholm Atherosclerosis Gene Expression) and STARNET (Stockholm Tartu Atherosclerosis Reverse Network Engineering Task) studies" [12,13].
The above approach is significant and in the near future probably will give some answers for the genetic basis of the complex CAD.But, at the present, the missing point from the perspective of clinical cardiology is the complicated clinical phenotypes of CAD in the everyday clinical practice.The GGES should be accompanied by studies referring to the clinical pictures emerging at the level of clinical modules (e.g.degree of coronary occlusion or collaterals) and clinical phenotypes (e.g.acute coronary syndromes or myocardial infarction).It is imperative to recognize the factors that are shaping the emergent clinical manifestations of the CAD like the degree of the arterial occlusion or the reasons of the sudden appearance of the acute coronary syndrome.
Over the last few years a number of GWAS scheduled to identify CAD risk-related genetic variants has been accomplished.As an example, the first identified genetic risk DNA variant was located on 9p21 risk region, followed by the discovery of a small number of other loci implicated in atherosclerosis and/or thrombosis [14].Lusk et al [15] believe that the genotype variation in the chromosome 9p21 region probably is related to classical cardiovascular risk factors in some subgroups of patients.The majority of the remaining genetic loci were associated to CAD through an unrecognized yet molecular pathway [14].
Epidemiological studies have supported the relationship of the ABO blood groups and the novel locus ADAMTS7 with the development of coronary atherosclerosis and myocardial infarction [16].Two large prospective cohort studies, the Nurses' Health Study and the Health Professionals Follow-up Study supported that ABO blood groups are strongly related to the CAD risk while individuals with O blood group have lower CAD risk [17].
Roberts (1), in a review article exploring 50 genetic risk variants associated with CAD, suggested that these variants were common and more than half of those were present in >50% of the general population, raising very little the relative risk for CAD.The 50 genetic risk variants are responsible for only 15% to 20% of the verified heritability while the expected heritability for CAD is calculated at 40% to 60% [10].Prins et al [18], in a review article, emphasized and described "the type of genetic variants potentially underlying the missing heritability of CAD" and "proposed a systems genetics approach in the post-GVA study era".The difference between the expected and the verified heritability is recorded as the missing heritability phenomenon [19].The small relative risk for CAD that is carried out by the genetic variants and identified by the GWAS is prompted novel research strategies with extended sequencing techniques concerning the whole exome-genome, in order to unravel missing heritability [19].
The GWAS increased the spectrum of human quantitative genetics but they did not solve the problem of the genetic variation of CAD.The GWAS only explain partially the genetic variation of complex traits even if all the measured SNPs are used [20].The remaining heritability is not missing, but most probably is not detected due to the fact that individual effects are too small to be recorded with the stringent significance tests [21].Roberts [1], speculates that the "discovery of more common genetic variants with more appropriate calculations will probably account for the missing heritability".Therefore, the GWAS approach has not succeeded to detect and follow up the pathological and clinical changes of CAD.The CAD loci recognized by the GWAS are mainly related to the initial atherosclerotic process and not to the later stages of the clinical disease.The GWAS failed to recognize the progressive nature of atherosclerosis from one clinical state to the next and to predict modulation and clinical phenotypes.

Systems biology approach
In this state of knowledge, systems biology approach is compatible with the classical method of research but it sounds more appealing to give some answers to the complex problem of atherosclerosis and to coronary artery involvement.Despite the advances made in the fields of mathematics and computing two basic requirements remain: a) how the information embedded in biological networks could be transferred in a progressively complex disease from network to network and how clinical phenotypes (in this paper the term "models" and "phenotypes" are used interchangeably) can be predicted; b) how the interconnected networks between molecules, cells, tissues and organs can explain the complex pathophysiology of a heart disease [21].
In each level of complexity the biological elements are organized in specific interacting entities, the networks.The interactions are taking place between networks within each level or between networks in different levels.The advent of whole genome sequencing made possible the construction of genome-scale networks and the integration of molecular and cellular elements in biological functional networks [22].The construction of similar networks in atherosclerosis and CAD will increase the knowledge of the basic underlying molecular and cellular mechanisms responsible for the progression of the atherosclerotic plaque to clinical phenotypes.
In systems biology the physical and biological facts of atherosclerosis and CAD are studied by two methods or directions, bottom-up and top-down directions.The bottom-up direction is reductionist and examines/composes the biological facts from genetics to phenotypes, and the top-down direction is holistic and examines/ decomposes the phenotype to modules, networks, cellular functioning and genetics.The two directions are realistic and indispensable in their concept as both of them are important for research and treatment.The bottom-up direction is clinically useful because it explores the lower levels for appropriate diagnostic biomarkers or drugs and the top-down direction dictates the method of diagnosis and treatment of the phenotype as a whole entity.
Atherosclerosis and CAD are generated by the integration of multiple contributing causes [23].The involved environmental risk factors could be internal to the patient (e.g.hypertension, diabetes, dyslipidemia, inflammatory diseases) or external to him (poverty, poor hygiene, level of education, smoking habit, sedentary way of living).The atherosclerotic process of a patient could be represented by the equation: AP=f(PG, E), where the AP (atherosclerotic process) is a function of the PG (patient's genomic behavior) and his E (patient's environment).The genetic and environmental factors involved in the integrating procedure are preventable to a significant degree.
The study of the atherosclerotic process with the systems biology approach can follow certain steps in four conceptual areas of interest: 1) the two previously mentioned potential directions of study of the atherosclerotic phenomenon, functional composition (bottom-up direction) and functional decomposition (top-down direction); 2) the disciplines or levels of complexity and integration of the atherosclerotic process: the genetic and genomics level, the cellular and molecular level, the modular level and the model level (clinical phenotypes); 3) the concept of network construction extending across the discipline levels of the atherosclerotic process; 4) the atherosclerotic plaque development and progression across the discipline levels of the atherosclerotic process (Table 1).This methodology of approaching the process of atherosclerosis with the concept of interacting links between the biological and clinical patterns is meaningful.

Directions of systems biology in atherosclerosis
The classical systems biology approach to a complex and dynamic biological phenomenon follows the methodology of the two complementary directions: the top-down direction and the bottom-up direction [24].The systems biology approach of the two directions is applicable to atherosclerosis and its progression from the early atherosclerotic plaque to the development of clinical phenotypes (Fig 2).Both directions are vital in order to understand the causation of the hierarchical development and to schedule novel ways for research in physiology and drug therapy.

Bottom-up direction
In the bottom-up direction of the atherosclerotic process, a hierarchy of dynamic networks is assembled from genomics to clinical phenotypes.This self-erected biological build-up is constructed by the continuous incorporation of added information from each level to the next level of the ladder.In each level is integrated the information coming from both opposite directions, bottomup and top-down.The atherosclerotic progression in successive pathological steps and the interexchange of information are both responsible for the emergence of new properties in each step of the ladder.An appropriate example of interaction in the higher levels of this ladder is the relation between a thrombus occluding a coronary artery and the repercussions of this occlusion.In the bottom-up direction, an arterial obstruction (modular level) can lead to angina pectoris or myocardial infarction (modeling or phenotype level).
The pathological variables in the modular level, like the size of the occluding thrombus or the diameter of the occluded artery, are affecting the outcome in the higher modeling level of phenotype leading to the clinical appearance of angina or myocardial infarction.Nevertheless, the resulting pathological/clinical picture, size of infarction or frequency of angina attacks and the method of therapy are unpredicted and variable.This variability is not predicted by the pathological data of the modular level but further information is needed from the same or higher level.Therefore, the size of the infarction or the frequency of the angina attacks or method of therapy depend also from other factors that were pre-existent or developed after the acute coronary obstruction.These factors could be for example the extent of collaterals or the presence of arrhythmias or the arterial or myocardial remodeling mechanism.Thus, the bottom-up direction alone is not adequate to explain the whole pathological/clinical relationship or to make questions of clinical significance.But the bottom-up direction of research is important to explore novel diagnostic biomarkers and drugs or to schedule preventive invasive methods.This position has important repercussions to the prognosis and therapy of the atherosclerotic process and CAD.

Top-down direction
The top-down causation is particularly significant for systems biology approach in two topics of interest, medical/healthcare and education [25].The top-down causation is needed in biological complex systems or diseases as an important source of information which "cannot be derived in a bottom-up way, because it implicitly embodies information about environmental niches" and "higher level conditions influence what happens at the lower levels, even if the lower levels do the work" [25].It is imperative to comprehend the top-down causation and the influence that the higher level order exercises to the pattern of processes in the lower levels [26].The lower level outcome depends on the nature of the medical constraints imposed to it by the higher level systems.The term medical constraints in reality are decreased degrees of freedom imposed by the higher level.For example the size of the myocardial infarction (phenotype) will impose the extent of the post-infarction collaterals and the size of the myocardial remodeling (modular lower level).This way, the size of the myocardial infarction modifies the boundaries (imposes constrains) of collateral extension or of the degree of myocardial remodeling.Thus, atherosclerosis is characterized by multiple constraints energized at all the discipline levels from phenotypes to cellular and genomic levels.
The integrating process of the two directions is translated as constraints imposed on the lower level systems.In reality, in each disease level there is integration of the information that is coming from both directions which modifies the therapeutic management.For the top-down causation process and information interexchange between levels various mechanisms are proposed [25].When adjusted, these mechanisms are applicable to human atherosclerotic process and constitute a field of research and scientific thinking.These mechanisms are: 1) the existence of higher level structural, boundary (constrains) and initial conditions that influence lower level dynamics; 2) the presence of genetically determined homeostatic systems in accord with the concept of feedback mechanisms; 3) the presence of an adaptive mechanism which represents the integration of information from both bottom-up and top-down Table 1.directions; 4) the presence of complex adaptive systems where feedback mechanisms are installed between level states; 5) the presence of another adapting higher level mechanism which is selecting lower level elements for use [25].

Systems biology approach of coronary artery disease
The causative principle of the top-down approach is helpful to analyze the hierarchical construction of CAD phenotypes.This is important for research, teaching and clinical evaluation in different stages of the CAD.In the previous example (described in the chapter of bottom-up direction) of coronary occlusion the size or the location of the myocardial infarction (phenotype level) lead to a better evaluation/therapy of the culprit preexistent coronary occlusion.This evaluation is leading to the most appropriate therapy, medical or invasive.The invasive therapy intends to eliminate or reduce the size of the occlusion via coronary angioplasty of the culprit lesion or the entire coronary system would be evaluated and treated surgically.
Therefore, the whole phenotypic spectrum of the CAD and its management is an interplay of medical interventions that take into account all the clinical facts as well as all the pre-or postocclusive modular factors.

Disciplines of systems biology in atherosclerosis
In the biological world the hierarchical organization and leveled construction are responsible for the phenotypic development.
Ellis [25] outlines the characteristics of this hierarchy with the following expressions: "the basis of complexity is modular hierarchical structures, leading to emergent levels of structure and function based on lower level networks" and that "each of the different levels of the hierarchy function according to laws of behavior appropriate to that level".
During the atherosclerotic process of the coronary arteries are distinguished four discipline levels or system-oriented domains that characterize the progressive nature of the disease (Table 1).

Models or clinical phenotypes
In atherosclerosis, significant processes from genetics to environmental risk factors are accountable for the variety of the phenotypes seen in clinical practice.The genetic and other risk factors are interacted and integrated forming large-scale patterns like complex networks.The merging incorporated information gives rise to novel emergent properties and produces the variety of atherosclerotic cardiovascular phenotypes.Furthermore, a complex clinical picture is created in every patient after further inter-phenotypic integration which modifies individual clinical progression.In the phenotypes of CAD are included the stable and unstable angina/ non-ST-Elevation Myocardial Infarction (NSTEMI), the ST-Elevation Myocardial Infarction (STEMI) and the ischemic cardiomyopathy.The phenotypic variation of CAD is increased when the complications of the disease or the modification of the original clinical picture after therapeutic interventions are included.The phenotypic variation incorporates modifications produced by post-infarction coronary or myocardial remodeling and/or by the presence of collateral coronary circulation.The clinical phenotypes are entities which can be changed if the lower levels of the atherosclerotic processes are amended through intervention, medical or invasive.A better knowledge of the atherosclerotic process across all the lower levels from genetics to modules will provide stronger predictive and therapeutic power to the clinical practitioner.That is despite the fact that the information included at the levels of "cells" and "genomics" is not able to predict with accuracy the two higher levels of "models" and "modules".

Modules
The term of modularity is referring to the existence of functional structures called "modules" that are robustly connected internally and interacting with each other.In this paper, the term "module" is restricted to only one level of complexity which describes the pathological changes, arterial or myocardial, that have clinical significance to CAD.Thus, the term of "modularity" is addressed to the variety of morphological endovascular appearances of the atherosclerotic plaque and its repercussions.In the above terminology of modularity are included various arterial and myocardial changes that affect the clinical picture, like the degree of artery obstruction, the collateral circulation and the myocardial or arterial remodeling.The term of modularity as it is described here has a close connection with discrete clinical cardiovascular phenotypes.The activation of the atherosclerotic plaque to the unstable status denotes the progression from the level of modularity to the phenotypic level.It is now accepted that the pathogenesis of acute coronary syndromes is related to the activation of inflammatory cells in the culprit lesion which becomes vulnerable and causes coronary instability [27,28].
Only with the early detection of the vulnerable plaques, can therapeutic strategies be applied and prevent plaque formation and rupture [29].Recently, Arbab-Zadeh and Fuster [30], are challenging the notion of the "vulnerable plaque" and give more attention to "the atherosclerotic disease burden than on features of individual plaques".

Molecules and cells
The vascular biology of the coronary atherosclerosis is characterized by a progressively worsening natural life plaque history passing from different phases: extracellular low density lipoprotein (LDL) accumulation in the intima; triggered inflammatory response; excessive accumulation of oxidized substances (mainly LDL); complex plaque involving smooth muscle cell migration, accumulation of extracellular matrix macromolecules, angiogenesis and calcification.Skogsberg et al [31], in atherosclerosis-prone mice revealed that the "atherosclerotic lesions progressed slowly at first, then expanded rapidly, and plateaued after advanced lesions formed".After the use of cholesterol-lowering drugs they identified 37 cholesterol-responsive genes which intervened and prevented the formation of advanced plaques [31].
The unraveling of related functional networks at the cellular postgenomic level would elucidate the interaction between environmental and genetic risk factors.

Genomics, epigenetics and postgenomics
The complexity of the atherosclerotic process could be partly explained by the scientific fields of genomics, epigenetics and postgenomics.Felsenfeld [32], argues that the term of "epigenetics" was originally used to explain how "a fertilized zygote developed into a mature, complex organism" and later on was changed to "which heritable traits can be associated not with changes in nucleotide sequence, but with chemical modifications of DNA, or of the structural and regulatory proteins bound to it".Richardson and Stevens [33], describe as "epigenetics" "the study of mechanisms that regulate gene expression in response to environmental signals".With the term "postgenomics" they are indicating the period after the achievement of the sequencing of the human genome that involves "rapids shifts in research methodology, funding, scientific labor, and disciplinary structures" [33].They suggest that "postgenomics are transforming our understanding of disease and health" and "forces a rethinking of the genome itself" [33].Wild [34], conceived the term "exposome" focused on the interaction between environmental and genetic factors and to "the critical need for more complete environmental exposure assessment in epidemiological studies".
A new stimulating field of research is the epigenetic domain of miRNAs (microribonucleic acids) with their vital role as posttranscriptional regulators of gene expression in the majority of the human cells.There are indications for miRNAs participation in the pathogenesis and rupture of the atherosclerotic plaque [35].The epigenetic field of miRNAs is raised to an important area of research and invention of biomarkers and drugs in atherosclerosis.
The translation of genotype to phenotype is better explained with the concept of epigenetics and DNA methylation.The epigenetic modifications are genetically programmed and affect gene expression.Methylation is an epigenetic modification of the genome and converts cytosine to 5-methylcytosine.The role of epigenetics in human atherosclerosis and the study of the methylation status in the arterial wall will unlock the prospects of modification of this status for therapeutic reasons.The methylation process does not change the basic genetic sequence, but closes down accessible genes which are not temporarily required.The epigenetic modification of DNA may occur through DNA methylation with the bondage of a methyl group to cytosine-guanosine (CpG) dinucleotides.The epigenetics and the DNA methylation participate in many biological activities and in the atherosclerotic procedure [36].Aavik et al [37] recognized in atherosclerotic plaques genomic hypomethylation that activated gene cluster in chromosomal locus 14q32 involving many clustered miRNAs that were up-regulated.The above authors concluded that epigenetic changes are implicated in the atherosclerotic process and provide novel potential therapeutic targets [37].

Networks of atherosclerotic process across disciplines
The integration of the molecular elements in networks and the interaction between intercellular and intracellular networks are underlying the cellular functions in health and disease.
The emphasis is given on patterns of organization based on the interconnection and interdependence of self-regulated networks.Weiss et al [38] described the systems biology approach to cardiovascular metabolism as a system composed of modular and spatially extended networks made up of nodes and links.In a biological network the genes, proteins and metabolites are referred to as "nodes" and their interactions are called "links" or "edges".Richardson and Stevens [33], emphasize "that network thinking is holistic in going beyond the reduction of biological systems to component parts".Important cellular functions can be revealed by the discovery of signaling and regulatory networks, and by the description of networks of highly connected proteins [39,40].
The presence of the biological networks is not limited to the level of cellular interactions but is extended to tissues and organs (Fig 2).The CAD is an integrated whole better comprehended in terms of interrelated network structures (Fig 1).The implication of the biological networks in clinical cardiology increased the interest for the concepts of network medicine and network cardiology [41,42].To analyze a network, criterion is the physical proximity of the various components and their functional interactions [43].Functional interactions are for example "energy-generating systems in a cardiomyocyte" including "glycolytic enzymes, glycogenolytic enzymes and oxidative phosphorylation" [43].
Novel experimental networks are constructed which are "based on experimental perturbations such as after a chemical treatment or genetic alteration" [43].As an example, networks are identified for cholesterol related genes responsible for the growth of atherosclerotic plaques in mice and for the endothelial inflammatory process linked to atherosclerosis [44].Transcriptional profiling in atherosclerosis-prone mice identified a network of atherosclerosis genes which responded to cholesterol lowering agents and prevented the formation of complex plaques [31].Gargalovic et al [45], demonstrated genetic variation in inflammatory responses to oxidized lipids of cultured aortic endothelial cells (ECs) derived from transplant donors.Data expression array analysis of the above aortic ECs cultures recognized that more than 1000 genes involved in the inflammatory response were controlled by oxidized phospholipids.They, also, "constructed a gene coexpression network comprised of 15 modules of highly connected genes" [45].Macrophage activation is associated with atherosclerosis through a transcriptional network mediated by Toll-like receptors (TLRs).Macrophages through their TLRs are able to recognize pathogen molecular systems like flagellin, lipopolysaccharides, and double-stranded RNA46.The activation program of TLR4 macrophages, after lipopolysaccharide stimulation, is started by a transcriptional regulatory network that involves more than 1000 genes and transcriptional factors [43].However, the present knowledge of all interconnected networks is limited and there is only a rough estimation of the whole pattern.The identification of the vulnerable nodes in each network and their close association with other networks and with the patient clinical status is critical.

Atherosclerotic plaque progression across discipline levels
Progression of the plaque, from an early endothelial to a complex encapsulated and obstructive lesion, embraces all the changes that are taking place in each discipline level (Fig 2).The CAD is characterized by a sigmoidal (S-shaped) curve of development with a slow initial growth period of 30-50 years, followed by a fast expanding asymptomatic period of 10 years, and eventually by a final period with clinical symptoms [12,47].
A strong physical connection exists between concentration of circulating plasma low density lipoprotein, in specific areas of the coronary arteries (like bifurcations or curbs) with turbulent flow, and development of atherosclerosis.In a computational fluid dynamic analysis digitized images of arterial post-mortem segments were examined and the critical role of the low local wall static pressure was emphasized for the pathogenesis of coronary atherosclerosis [48].In a top-down direction, in areas of bifurcation or trifurcation, the wall shear stress (WSS) is changing endothelial cells' genes expression, which, in a bottom-up direction, promotes arterial remodeling and atherosclerosis [49].
Arterial WSS is lower on the lateral walls of bifurcation during the systolic rather than the diastolic period while the distribution of low WSS is compatible with the ordinary location of the atherosclerotic plaques [50].

Conclusion
With the methodology of systems biology this review investigates the atherosclerotic process in coronary arteries and analyzes the complexity of atherosclerosis and CAD.With the systems biology approach four conceptual areas of study are described: the two directions that investigate the course of atherosclerosis and CAD, the four disciplines or levels of complexity, the construction of biological networks and the atherosclerotic plaque development and progression.In the present review the concept of the systems biology was proposed as a novel method able to revise the follow-up of complex diseases like atherosclerosis, in molecular and clinical level.

Figure 1 .
Figure 1.Coronary artery disease: The network complexity between systems biology discipline levels and environmental factors.

1 .
Directions of systems biology a. Bottom-up direction (functional composition) b.Top-down direction (functional decomposition) 2. Disciplines of systems biology a. Models or clinical phenotypes b.Modules c. Cellular and molecular field d.Genomics, postgenomics, epigenetics 3. Networks of atherosclerotic process across disciplines 4. Atherosclerotic plaque progression across disciplines