Basic mechanisms in atherosclerosis the role of calcium

Send Orders for Reprints to reprints@benthamscience.ae Medicinal Chemistry, 2016, 12, 103-113 103 Basic Mechanisms in Atherosclerosis: The Role of Calcium Aimilios Kalampogias#, Gerasimos Siasos#,*, Evangelos Oikonomou, Sotirios Tsalamandris, Konstantinos Mourouzis, Vasiliki Tsigkou, Manolis Vavuranakis, Thodoris Zografos, Spyridon Deftereos, Christodoulos Stefanadis and Dimitris Tousoulis 1st Cardiology Department, University of Athens Medical School, “Hippokration” Hospital, Athens, Greece Abstract: In the beginning, atherosclerosis was considered to be the result of passive lipid accumulation in the vascular walls After tremendous technological advancements in research, we are now able to almost admire the complexity of the atherosclerotic process Atherosclerosis is a chronicinflammatory condition that begins with the formation of calcified plaque, influenced by a number of different factors inside the vascular wall in large and mid-sized arteries Calcium mineralization of the lumen in the atherosclerotic artery promotes and solidifies plaque formation causing narrowing of the vessel Soft tissue calcification associated with tissue denegation or necrosis is a passive precipitation event The process of atherogenesis is mainly driven by CD4+ T cells, CD40L, macrophages, foam cells with elevated transcription of many matrix metalloproteinases, osteoblasts, cytokines, selectins, myeloperoxidases, vascular adhesion molecules (VCAM), and smooth muscle cells Our knowledge in the genesis of atherosclerosis has changed dramatically in the last few years New imaging techniques such as intravascular ultrasound or IVUS have made possible to investigate atherosclerosis in early stages Arterial calcification emerges from two different types, the medial-elastin dependent and the intimal, both of which are directly related to atherosclerosis due to osteoblast differentiation of vascular smooth muscle cells The deposition of minerals in the form of calcium (Ca2+) initially emerges from the inorganing mineral octacalcium phosphate [Ca8H2(PO4)6.5H2O] to the form of Hydroxylapatite [Ca10(PO4)6(OH)2] This review is devoted to broaden the understanding regarding atherosclerosis and the central role of calcium in the development of the condition Keywords: Atherosclerosis, calcium, cardiovascular disease, immune system, inflammation, osteoblasts, vascular endothelium INTRODUCTION In recent years, the attention of research has been motivated by the unique molecular machinery that gives rise to calcification in the vascular wall during plaque formation [16] Atherosclerosis is a disease in which lipids and other debris accumulate in the walls of large and medium arteries [7] The main risk factors include genetic predisposition, age, gender, life style, weight gain, hypertension, hypercholesterolemia, diabetes and smoking [8-11] A multifactorial course of events involves many stages in the plaque formation which by extent could impair cardiovascular hemodynamics [12, 13] Key elements from the immune system such as T-cells and monocytes in conjunction with oxidized LDL are responsible for vascular inflammation and a primary cause to endothelial dysfunction and atherosclerotic lesions, all causing calcification [8, 14-27] A process of pathobiological descent which shares common denominators with the embryonic bone formation, can sometimes get out of *Address correspondence to this author at the 1st Cardiology Department, University of Athens Medical School, “Hippokration” Hospital, Vas Sophias 114, 115 28, Athens, Greece; Tel: +30-210-7462719: Fax +30-2107462718; E-mail: gsiasos@med.uoa.gr # The first two authors contributed equally to this paper 1875-6638/16 $58.00+.00 hand and provide guidance to arterial wall mineralization with the help of osteoblasts, and a naturally occurring mineral; the hydroxylapatite [Ca10(PO4)6(OH)2] [5, 6, 28-35] Multiple pathological events could support the expression of atherosclerosis, like metabolic syndromes, renal complications, maladaptive immune response and cardiovascular conditions (Fig 1) Even though atherosclerosis is an asymptomatic at the begging disease the clinical diagnosis could fall far behind as the progression of the atheromatic plaque takes over the endothelium causing systemic imbalance [36] The aim of this review is to depict the main mechanisms in atherosclerosis and overview the significant role of calcium in atherosclerosis progression and cardiovascular disease manifestations MAIN MECHANISMS IN ATHEROSCLEROSIS Atherosclerosis is a chronic disease resulting in the buildup of plaque Atherosclerotic plaques develop and progress within the arterial wall long before they begin to encroach on the lumen [37-39] Because they not protrude into the lumen they escape detection by conventional diagnostic tools like angiography [40-42] Recent research has also shown that this early atherosclerotic plaques which are filled with lipids are more likely to rupture and cause coronary events than more stable advanced plaques that cause narrowing of © 2016 Bentham Science Publishers 104 Medicinal Chemistry, 2016, Vol 12, No Kalampogias et al       " # "  !  !   % ! %$%!!    9(66(/            &E                 ! ! !  !"!            Fig (1) Pathophysiology of atherosclerosis: The role of risk factors Aging act synergistically with multiple risk factors to cause endothelial dysfunction which in turn motivate a number of detrimental pathways finally causing calcium deposition in atherosclerotic plaques LDL: Low density lipoprotein, VSMC: Vascular smooth muscle cells, IL-1 : Interleukin-1 , MSC: mesenchymal stem cells the lumen [43-45] Atherosclerotic rupture events are a leading cause of morbidity and mortality worldwide, to reduce the extraordinary risk associated with such an event, prognosis and aggressive risk factor intervention are imperative for the evaluation of the state of the condition [46-48] Elevated low-density lipoprotein or LDL cholesterol is strongly associated with an increased risk of cardiovascular events (hyperlipidaemia) [49-52] due to the process of LDL oxidation Ox-LDL [53-56] As the majority of cholesterol in the circulation is found in LDL form which transports cholesterol from the liver to nerve tissues, cell membranes and other cells for metabolic purposes, the cholesterol in atherosclerotic plaques is derived mainly from excess of LDL cholesterol High-Density Lipoprotein or HDL functions as a retrieval service by removing cholesterol from the circulation to the liver for excretion Low levels of HDL and the LDLHDL cholesterol ratio are considered to be strong risk predictors for cardiovascular disease [57, 58] The normal artery wall is composed of three layers, the tunica intima the media and the adventitia The intima or the inner layer is a strip of a single layer of cells called the endothelium, this layer with a non - thrombogenic surface mainly acts as a semi selective barrier between the vessel and the lumen making it responsible for critical functionalities of the vascular biochemistry [59-62] It also initiates the stimulation of cytokines for production of NO by endothelial nitric oxide synthase in order to control vasoconstriction and vasodilation [63-66] Other characteristics are the properties of thrombosis and fibrinolysis plus the ability to facilitate cell proliferation and angiogenesis with the use of angiopoietin-2 in conjunction with vascular endothelial growth factor (VEGF) Interactions with pro - angiogenic properties of oestrogen could be protective in women without atherosclerotic disease but in the case of chronic vascular inflammation, plaque destabilization may occur [67, 68] The media is separated from the intima by the internal elastic membrane and contains smooth muscle cells that promote the vessel to dilate or constrict by increasing the elasticity Calcification occurs in the intimal and medial layers [62, 69] The adventitia or outer most layer is composed mostly of fibro-elastic tissue The adventitia is separated from the media by the external elastic membrane LDL is a macromolecule that can penetrate the artery wall, the atherosclerotic event begins when the LDL accumulates abnormally within the arterial wall at a rate determined by plasma concentrations of LDL and the physiological state of the endothelium Elevated LDL levels could have devastating consequences in the artery, as when the low lipoprotein macromolecules manage to penetrate the vascular wall and enter the intima they start to initiate the processes of oxidation The chemical form of oxidized LDL is toxic to the cells that are the part where the inflammatory event initiates [7072] In the initial stages, the atherosclerotic plaque forms beneath the intima media - inside the arterial wall and gets a crescent form (Fig 2) As the lipid core stacks increases in size, a fibrous capsule is then formed in the shape of a vascular growth causing the lumen to expand It has been found that the most frequent pathological cleavage points, which are prone to lead in rupture of the plaque, are based in the neck This site appears to be the most anatomically vulnerable due to maximum stress point of the friction separation of the blood flow (shear stress) [73] Plaques that are frequently subject to rupture are small with rich lipid, soft core with a thin fibrous capsule, while plaques that cause severe narrowing of the vessel lumen is often more fibrous and stable However, the vulnerability of the rupture depends mainly on the biological factors The on-site Calcium and Atherosclerosis Medicinal Chemistry, 2016, Vol 12, No 105 /XPHQ )RDP FHOO IRUPDWLRQ 3URJHQLWRU 2VWHREODVWV &\WRNLQHV 6FD 3')*5Į 2VWHREODVWLF  2VWHRFODVWLF SURJHQLWRU FHOOV 3URFRDJXODQW IDFWRUV 0DFURSKDJHV 3URWHDVHV %LQGLQJ /'/ WR VFDYHQJHU UHFHSWRU $FFHOHUDWHG YHVLFOH UHOHDVH &\WRNLQH VHFUHWLRQ 71) Į ,/ ,/ȕ 0DWUL[ YHVLFOHV RVWHRFODVWV 3KDJRF\WRVLV 0DWUL[ YHVLFOHV ,QWLPD 2VWHREODVWV &DOFLILFDWLRQ RVWHRFODVWV &DOFLILFDWLRQ $SRSWRVLV RI 960&¶V 3URJHQLWRU 2VWHRFODVWV 6\QWKHVL]HG 960&¶V 960& DGDSWDWLRQ )RDP FHOO 0HGLD 0LQHUDOLVLQJ PDWUL[ YHVLFOHV $SRSWRWLF ERG\ VHFUHWLRQ 9DVFXODU VPRRWK PXVFOH FHOOV 960&¶6 2[LGL]HG /'/ Fig (2) The complex interplay between multiple mechanism, molecules, cytokines and cells causing the formation of atherosclerotic plaques LDL: Low density lipoprotein, VSMC: Vascular smooth muscle cells, IL: Interleukin, TNF : Tumor necrosis factor alpha concentration of phagocytes and T - lymphocytes is an omen to rupture [74] This process could be likened to the skin response to entry of a foreign body, e.g a thorn; if the production of mast cells can entrench and organize according to the immune response then no acute inflammatory response can occur, the only side effect being a local induration However, if stimulated phagocytes colonize the site of infection, a necrotic environment will form around the foreign body Additionally the surrounding tissue becomes thin and erosion takes place driving the intruder outward In the case of the artery, the foreign body is the oxidized LDL and the site of infection is in the lumen of the vessel The consequences of plaque rupture systematically depend on thrombogenic factors such as the primary hypercoagulable potential of the blood and the depth-size ratio of the plaque [75] Small surface ruptures can cause the formation of fragile thrombus entrained by the bloodstream in contrast with big lacerations, which can cause ulcerations exposing collagen in the blood clotting factors forming clots that are not easily distracted In a case like this what matters, as the first line of defense, is the activity of natural thrombolytic proteins in the rupture phase The mechanism of thrombosis following the arterial injury (rupture of the plaque) is a product of thrombin that is a powerful stimulus of activation and aggregation of platelets converting fibrinogen into fibrin fibers along with the formation of transverse bridges THE ROLE OF CALCIUM IN ATHEROSCLEROSIS The implications of the calcium build up in the vascular cell wall may interpose multiple configurations that have been pervasive and hence far-fetched in the common knowledge of the atherosclerotic pathology Intricate molecular pathways derived from the process of bone formation, widely known as osteogenesis may and in the majority of the cases, will interfere with the immune system’s components causing imbalance in the natural continuum of the mineral deposition and resorption Drawing in the cause, factors from different sites such as interleukins, macrophages, monocytes, foam cells, and some cells from the bone formation and degradation system, like the mesenchymal precursor derived cells called osteoblasts and the osteoclasts arise especially from hematopoietic mononuclear phagocyte progenitors along with macrophage colony stimulating factor-1 (CSF-1) In addition, chondrocytes are the third cell type associated with the progression of calcification in the vascular wall, derived from mesenchymal cells with the main role to induce an initial cartilage template on which mineral deposition in the native crystalline form of calcium apatite [Ca10(PO4)6(OH)2] -hydroxyapatite (HA) may occur Calcium is an element considered necessary for the normal conduction of the biochemical events in the human physiology In a healthy organism, calcium is abundant in many different forms, as it drives many complicated molecular pathways Calcium ions [Ca2 +] hold a critical role in the normal management of the nervous system as they act as a secondary messenger, providing guidance to the ion influx from the voltage-gated calcium channels (VDCCs), hence being beneficially responsible for muscle contraction Ca2+ influx stimulates vesicles that contain the neurotransmitter acetylcholine guiding them towards the plasma membrane fusion with imminent release of the neurotransmitter into the extracellular space in the midst of the neuromuscular junction and the motor neuron terminals of the skeletal muscle fibres [76, 77] Migration of calcium ions from the extracellular matrix towards the intracellular space alters membrane 106 Medicinal Chemistry, 2016, Vol 12, No milieu This phenomenon is noticeable in the cardiac cycle, where the plateau phase of ventricular contraction takes place Due to ionic polarization/depolarization calcium is strongly related to the preservation of homeostasis in the normal cardiac rhythm The vascular system is the connective bridge between the bones and the body delivering hormones, nutrients and various other chemical signals to the bone marrow for the normal production of progenitor cells like the osteoblasts Once osteoblasts are activated they start to produce the organic component of bone, osteoid, which is predominantly made of collagen Minerals start to crystalize around the collagen scaffold to form hydroxyl apatite, the major inorganic constituent of bone which contains calcium phosphate Bone mineral density or BMD can be used to estimate the strength of bone and to assess the risk of fracture As osteoblasts form new bone tissue many become embedded within the matrix and differentiate into osteocytes The build-up of plaque is the deposit of the mineral hydroxylapatite [Ca10(PO4)6(OH)2] and is responsible for three main complications in atherosclerosis: aneurism ischemia and embolism [28, 33, 78] As the plaque weakens the vessel wall it becomes more prone to aneurism which may lead to internal haemorrhage if the aneurism ruptures Plaque and thrombi build up in the vessels can disturb the lamina flow of the blood causing inflammatory and coagulation factors to contact the injured site, causing further damage as it may further promote the occlusion of the vessel [38] A successful event like this will further advance into ischemia resulting into apoptosis and necrosis of tissue while in contrast of an event like the embolism of the plaque or coagulated thrombi where the thrombus forms on the atherosclerotic site and it may break off and occlude vessels in other sites of the circulatory system such as the coronary arteries or the carotid arteries The process of atherogenesis is mainly driven by CD4+ T cells, CD40L, macrophages, foam cells with elevated transcription of many matrix metalloproteinases, osteoblasts, cytokines, selectins, myeloperoxidases, vascular adhesion molecules (VCAM), and smooth muscle cells [4, 79-88] Through the process of mineralization divergent cell types can result, counting a vast majority of aortic smooth muscular cells which through differentiation can acquire osteoblast characteristics, this is considered to be the epitome in vascular calcification For the progression in development of osteoblasts many factors are essentially responsible for transcription, such as Cbfa1 (binding factor 1) [89] where is expressed in calcified areas by macrophages while MGP is missing [29, 90, 91] This event seems to share a considerable versatility in the fate of osteogenesis in the vascular wall [5] THE ORIGINS OF ENDOTHELIAL CELLS AND SMOOTH MUSCLE CELLS (SMC) IN PLAQUE FORMATION SMCs and endothelial cells in atherosclerotic plaques can originate from bone marrow [92] Active TGF released from the injured arteries recruits mesenchymal stem cells from the blood to the neointima, playing a key role in the Kalampogias et al development of osteogenic vascular calcification [93] The vascular system may contain in circulation, cells analogous to primary types such as osteoclasts, chondrocytes and osteoblasts associated in bone formation, holding different signal pathways for protein expression directly correlated with bone formation [94-98] Calcium deposition in regions where osteoblasts can be found, are highly correlated with plaque formation where matrix vesicles and bone matrix proteins are found [97, 99] MOLECULAR BIOLOGY OF ATHEROSCLEROSIS With the first glimpse at the progression of lesions in vessels, we come across with the key components of atherosclerotic plaque containing fatty streaks, foam cells and macrophages which are substantially recruited monocytes The more advanced atherosclerotic lesions adopt the form of a more vulnerable plaque structure as the result of continuous recruitment of monocytes and migration/ proliferation of smooth muscle cells Macrophages and smooth muscle cells migrate to the lumen of the vessel, wherein they are installed beneath the endothelial cells and proliferate As a result, the endothelium gradually projects into the lumen and loses the smooth surface Increased LDL can passively diffuse through the endothelial cell tight junctions and begin to accumulate in the intima, the retention of this LDL is mediated by interactions between matrix proteoglycans and apolipoprotein B The LDL in the intima is then modified by enzymes through the processes of oxidation, lipolysis and proteolysis Major pathophysiology altering components include myeloperoxidases lipoxygenases NADPH oxidases and nitricoxidesyntases In addition, oxidized and partially oxidized LDL damages the endothelial cells causing them to produce adhesion molecules [CAM’s] such as interferon gamma, macrophage chemotactic protein-1, integrins, selectins, and macrophage colony stimulating factors all of which attract monocytes from the blood to the lesion site [100, 101] This forces monocytes in the blood stream to undergo marginalization, endothelial rolling, adhesion and then migration through the endothelium [102, 103] This intricate process is mediated by selectins integrins and vascular adhesion molecule 1, starting with monocyte migration [104] through the endothelium into the sub-endothelial space in the intima- via the junctional adhesion molecule A (JAMs), in conjunction with platelet adhesion molecules which are expressed on leucocytes and concentrated at endothelial cellcell junctions Intravascular production of M-CSF in the arterial wall initiates the differentiation of the monocytes into macrophages, driving the phagocytosis of the oxidized LDL molecules mediated by scavenger receptors- SR-A and CD36 leading to foam cell formation Foam cells cannot break down the lipids therefore act as measures of restriction engulfing the OxLDL, while macrophages introduce onsite the enzyme myeloperoxidase (MPO) that produces hypochlorous acid (HOCl) which bears the potential of modifying and oxidizes lipids in the intima therefore attracting more monocytes to the lesion site As the progression of the event continues, more factors like vessel active substances including cytokines and growth factors force the smooth muscle cells to migrate and proliferate from the media to the damaged area forming a layer These VSMC’s further contribute to the fibrotic cap formation Under the fibrotic cap a layer of ne- Calcium and Atherosclerosis crotic core develops consisting of mostly foam cells lipids, and connective tissue This forms as macrophages release metalloproteinase which degrades the extracellular matrix and causes the macrophages to go in apoptosis This remarkable action is the main cause that keeps feeding the chain reaction toward the formation of the plaque [100, 105-110] THE IMMUNE SYSTEM IN ATHEROSCLEROSIS AND THE IMPORTANCE OF CYTOKINES The chronic inflammatory situation in atherosclerosis is mediated by the activation of both the adaptive and the innate parts of the immune system Hematopoietic stem cells (HSCs) have the potential to differentiate into many different cell types such as smooth muscle cells and monocytes, participating in the pathogenesis of atherosclerosis [111] Monocytes have the primary function of consuming foreign or damaging substances and by extension participating in the atherosclerotic process, taking up residence within the vessel wall and consuming the plethora of lipids [8, 112-115] Monocytes respond by migrating from the circulation into the artery wall, when they mount the inflammatory response, now called macrophages they engulf the cholesterol rich oxidized LDL and become foam cells Monocyte migration to atherosclerotic plaque depends on many chemokines such as CCL2, CX3 CL1, and CCL5 [116-120] After settling down, monocytes could turn into macrophages or dendritic cells [18] On the other hand, macrophages are an end effector cells that maintain the health of the tissues At sites of inflammation, monocytes and macrophages produce cytokines responsible for inflammation, which can drive forward the progression of the disease Macrophages cells can also produce pro-healing cytokines Furthermore, macrophages introduce antigens to the cells and can prime T cells [121] With the understanding of the monocyte molecular nature, new therapeutic opportunities may rise Research on animal subjects shows that local immune response and by extent, inflammatory events are triggered after the infiltration of CD4+ T cells at the lesion site during the progression of atherosclerosis, therefore participating in the formation of lesions in genetically hypercholesterolemic apoE -/- mice [122, 123] oxLDL was found to trigger the inducible factor (HIF-1) attenuate inflammation by regulating redox dependent mechanisms The (HIF-1) is also responsible for the attenuation of inflammation by T cell activation and cytokine production [124-126] The super family of cytokines includes chemokines, interleukins, interferons, lymphokines, and tumour necrosis factors which are mainly responsible for the signalling between the various cells in the human body Chemokines hold the key to the inflammatory response, initiating numerous members of the CC chemokine family, MCP-1/CCL2, CXC, IL-8/CCL8, SDF-1/CXCL12, IP10/CXCL10, fractalkine/CX3CL1, and CXCR6 all of which have shown implication in atherosclerosis [127] By blocking chemokine receptor interactions, a potential therapeutic solution may rise to treat atherosclerosis Interleukins are secreted proteins and signalling molecules in which serum levels have found to be positively associated with coronary arterial disease Having part in the pro - atherogenic event by upregulating adhesion molecules on endothelium, activate macrophages and the proliferation of the smooth muscle cells A challenging but promising hypothesis is that anti- Medicinal Chemistry, 2016, Vol 12, No 107 inflammatory interleukins may hold the potential for administration of decoys like antibodies against the counter proinflammatory interleukins [128, 129] Interferons like the type I (IFN and IFN ) are known for pro- or –antiinflammatory potential in numerous pathological conditions [130-133] Lymphokines typically produced by T cells with the role of directing the immune response by attracting other immune cells on the site of infection-lesion, therefore playing a central role in the inflammation Important lymphokines secreted by Th cells are the IL2, IL3, Il4, Il5, Il6, granulocyte-macrophage colony stimulating factor and interferon gamma The variation between pro-inflammatory and antiinflammatory cytokines in the immune system and key elements of the lipid metabolism, presents outstanding importance regarding the pathophysiology and the progression of the atherosclerotic disease but also gives to modern day research the opportunity to investigate the potential as biomarkers for early prognosis Certain bone-related cytokines like Osteopontin (OPN) have shown pro-atherogenic potential during experimentation in hyperlipidemic mice [134, 135] with OPN deficiency the progression of atherosclerosis was effectively decreased IL-17 seems to be controlling the action OPN also mediates vascular smooth muscle cell proliferation in response to periodic high glucose levels [136138] The gene expression of inflammatory molecules, the production of vasoactive growth factor, surface receptors and fibrinolytic agents has been shown to be affected by the form of the parietal stress accorded to endothelial cells [139] Specifically one atheroprotective endothelial phenotype requires a level parietal stress Simply putting endothelial cells to function normally must be under pressure Unlike an atherogenic phenotype leading to production of proinflammatory molecules and proliferative growth factors manifested in low or abnormal parietal stress points The cellular mechanoreceptors of such changes has been supported by some observations that are in cytoskeleton, while other studies suggest that the increase in endothelial permeability triggers these changes [140, 141] NADPH OXIDASES IN VASCULAR PATHOLOGY The NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) is the predominant enzyme superoxide anion that is produced in the vascular wall Found in all cells both the vascular wall and in polymorph nuclear infiltrating it in the course vascular inflammation It consists of complex individual proteins During phase resting two proteins, a glycoprotein family Nox [non-phagocytic NAD (P) oxidase: non phagocytic oxidase NADPH] that varies depending on the type of cell and a second protein, located on the p22phox cell membrane and mutually supportive The other proteins such as p40phox, the p47phox, the p67phox, and rac located in the cytoplasm During the stimulation phase caused by various endogenous substances such as angiotensin II, endothelin-1 [142], the TNF-a (Tumour Necrosis Factor-a: tumour necrotizing factor-a), and PDGF (Platelet Derived Growth Factor: Platelet Derived Growth Factor), all proteins present in the cytoplasm mobilize and run the cell membrane where they join the existing protein complex NOx and p22phox With the formation of this new complex, NADPH oxidase is activated and produces oxidative radical superoxide anion triggering the vascular oxidative stress Other fac- 108 Medicinal Chemistry, 2016, Vol 12, No Kalampogias et al tors that activate the NADPH oxidase are the angiotensin II, endothelin-1, the TNF-a, and PDGF (Platelet Derived Growth Factor) [142-148] Epigenetics can be defined as the external factor which can enforce aberrant gene expression changes characterizing the progression, the functionality of specific genes and by extent the development of a biological system Essential genes that are epigenetically modified through the process of the atherogenesis by methylation alterations are the genes that encode for the enzyme nitric oxide synthase NOS, oestrogen receptors, collagen type XV alpha (co-l15a1), ten eleven translocation, VEGF, and are strictly associated with endothelial dysfunction While at the other side of the problem, atherosclerotic inflammatory process gains support from epigenetically modified gamma interferons (IFN- ), forkhead box p3 and TNF- Even though the understanding of the development of the disease has brought tremendous discoveries of the molecular machine that underlie the cause of atherosclerosis, epigenetics is the key player on the subject Gene regulation holds the key to the implications of extracellular matrix formation, proliferation and inflammation which are the guidelines for atherosclerosis DNA methylation can reduce the transcription of the genes by DNA methyltransferases causing histone modifications such as the processes of acetylation/deacetylation and creating disturbances in chromatin structure, changing the fate of transcriptional regulators The best illustrated example of an epigenetic modification is the NOS3 gene which translates the enzyme eNOS The chromatin structure at endothelial cells (hypomethylated) becomes transcriptionally permissive in comparison with the non-endothelial cells, which turns repressive (downregulated) Other epigenetic events that characterize the gene expression apart from methylation are phosphorylation and SUMOylation, which are directly correlated with cell proliferation in the endothelium and differentiation of VSMC and inflammation There is also a hypothesis that some matrix mettaloproteinases (MMP-2, MMP-7, MMP-9, and TIMP-3) are epigenetically controlled through methylation Even though this theory has only been tested on cancer cells it might hold a great potential [149-151] observed symptoms are shortness of breath and reduced exercise tolerance The severity of the stenosis can be determined echocardiographically measuring blood flow velocity through the aortic valve, the pressure gradients and the surface of the aortic valve [41, 42, 152-158] When the foam cells die they release their lipid content creating what is known as the lipid core, as the phases of the atheromatosis continues, a fibrous cap consisting largely of collagens and elastin forms over the lipid core, the fibrous cap represents an attempt of the body to heal the lesion while continued plaque growth caused by accumulation of LDL within the intima forcing the external elastic membrane to expand, this compensatory enlargement known as arterial remodelling allows the vessel to maintain an adequate if not normal lumen area affecting the haemodynamics of the vascular system, for this reason angiography which visualises only those plaques that encroach upon the lumen underrepresents the extent of atherosclerosis However, as the burden of plaque increases the artery cannot longer compensate by expanding outward and the plaque begins to protrude into the lumen this generally occurs when plaque involvement reaches about 40% of the vessel circumference Under certain conditions and by being prone to rupture, biomechanical or haemodynamic stress could cause a disruption of the plaques prone to rupture including those that contain a large lipid core covered by a thin fibrous cap Often these plaques have not penetrated the luminal area and hence there are not visible angiographically When a plaque ruptures the lipid core comes in contact with the blood [159-161] This sets the stage for the formation of a thrombus, the thrombus or clot may partially or totally block an artery causing an abrupt reduction in blood flow, followed by partial blockage of the lumen that may cause the symptoms of angina Complete blockage of the vessel lasting more than – hours can cause an acute event such as myocardial infraction Healing may also take place, in fact, plaque rupture with subsequent healing now believed to be the major mechanism by which atherosclerotic lesions progress and narrow the lumen [162] Plaques that heal generally have a higher fibrotic composition than before making them more stable and less prone to future rupture Calcium mineral deposits that periodically followed by an atherosclerotic event are accessible for quantification of the damage by radiography, serving as an alternate marker for higher prediction in the case of MI CLINICAL DIAGNOSIS CONCLUSION The intensity of vascular calcification, displaying reduced elasticity through calcium deposition, will predict the cardiovascular outcome of atherosclerotic patients Atherosclerosis begins in adolescence or adulthood, as progressive changes in the intima of the arteries occurs In practice, atherosclerosis can be diagnosed when arterial stenosis or aneurysms create a discomforting situation in the circulation, narrowing or significantly decreasing the normal flow of blood By using imaging methods including capital position held by angiography, computed tomography, magnetic resonance imaging, and the echocardiography (echocardiography) The clinical symptoms vary based on the degree of obstruction, the body type of the patient and the degree of physical activity The classic symptoms are usually heart failure, syncope and angina However, the most commonly Cardiovascular disease is a leading cause of death worldwide covering a range of conditions with atherosclerosis being implicated as the main cause for the vast majority of the events The consequence of atherosclerotic plaque formation and by extension rupture is by far the most common cause of CVD, from acute coronary syndromes, cerebrovascular disease to transient ischemic attacks and ischemic strokes The atherosclerotic development can be defined as the complex inflammatory process characterized by lipid and macrophage rich plaque formation, a consequence of endothelial - arterial wall injury Many variables can damage the arterial lining, from sear mechanical stresses, for example turbulent blood flow caused by hypertension to biochemical and immunological factors Emerging calcification in atherosclerotic lesions is the most common form of EPIGENETIC SCLEROSIS REPROGRAMMING IN ATHERO- Calcium and Atherosclerosis vasculopathic diseases and is linked mostly with cases of dyslipidaemia [163-165] It develops as a result of osteogenic differentiation of vascular cells driven by cytokines and modified lipoproteins and a variant of other inflammatory factors which can be traced in deposits of atheromatous plaque [165-167] Following endothelial damage there is an increased permeability of the vessel wall to plasma proteins and predominantly of lipoproteins into the intima, macrophages then engulf the lipoproteins and form lipid laden foam cells which are seen as fatty streaks With cytokine release further migration and proliferation of macrophages and smooth muscle cells a growing fibro-lipid plaque forms With plaque progression any further damage to the lumen causes platelet aggregation and thrombus formation and as blood pools into the ruptured plaque it becomes unstable growing in size to reduce vessel’s circumference [168-172] When the diameter of the lumen is halved hemodynamically significant losses occurs, which causes ischaemia with further increased oxygen demand Total occlusion results in infraction distally.Unstable plaques can also rupture and form emboli at distant sites Atherosclerosis is inevitable as we age and the pathobiological process could take the long run before we are able to identify any of the symptoms, however if we recognise the factors that contribute to the development we can evaluate ways to slowdown the progression Modifiable factors like blood pressure, smoking, diet, diabetes are the ones that can be influenced in a way by our obedience, in contrast ofcourse with the factors that cannot be influenced like the increasing age, gender, genetic predisposition of hyperlipidaemias, and family history [173] New imaging techniques such as intravascular ultrasound or IVUS have made possible to investigate atherosclerosis in early stages [174-176] The notion that these molecular and multifactorial harmonic events of atherosclerosis that is characterized by osteogenesis tied-up with inflammatory responses defines the atherosclerotic event as one of the biggest problems of the new – technologically advanced world Leaving no room for argument about the disastrous consequences of the patient’s condition Vascular calcification is immensely considered as a compelling topic due to the lack of research on the field As many questions remain unanswered, these interactive modalities are about to reach an end as a unified theory will bring together the myriads of the key elements that drive this exquisite process Medicinal Chemistry, 2016, Vol 12, No [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest [23] [24] ACKNOWLEDGEMENTS G.S received scholarship from” George D Behrakis Research Fellowship Program” The scholarship had no involvement in article design; in the writing of the manuscript; and in the decision to submit the article for publication [25] [26] REFERENCES [1] [2] Nicoll, R.; Henein, M.Y Arterial calcification: friend or foe? 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Following endothelial damage there is an increased permeability of the vessel wall to plasma proteins and predominantly of lipoproteins into the intima, macrophages then engulf the lipoproteins