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Atherosclerosis is a global disease affecting the entire arterial system and it is a major cause of morbidity and mortality in all continents. It is the commonest form of arteriosclerosis, a term used to indicate thickening of the arterial walls.

Atherosclerosis derives from the Greek language: athere meaning soft paste-like material and sclero for hard which results from the proliferation of fibrous tissue and the presence of calcification.

Its pathogenesis is complex and multifactorial and several epidemiological studies have identified risk factors, which are associated with its severity and widespread involvement of the arterial tree. Smoking, hypertension, dyslipidemia, and diabetes mellitus have all been associated with atherosclerosis and the mechanisms leading to arterial injury became clear in extensive research conducted in the last two decades.

Atherosclerosis is a common process associated with human development and aging. Usually starts at arterial bifurcations and ostia, where it appears as a localized thickening or elevation of the inner side of the artery—atheromatous plaque—which proliferates in the sub-endothelial layer and determines narrowing of the artery. This atheromatous plaque can act as a focus for localized thrombosis—atherothrombosis—or as a source of distal embolization—atheroembolism. Necropsy studies conducted in young children [1] have documented the presence of lipid deposition in coronary arteries and aortas, suggesting that atherosclerotic changes may occur silently even in infants.

To better understand the pathogenesis of atherosclerosis and what could be achieved with modern imaging methods available in today’s clinical practice, from morphological to functional and molecular technologies, it is important to briefly review the basic structure and histology of the arterial system and the morphological changes produced by atherosclerosis.

Arterial Wall Structure and Function

Usually arteries are divided into larger or central arteries with predominant elastic component (like the aorta), medium size or muscular arteries, and small arteries and arterioles, distributing blood flow into the vast capillary network.

The wall of the arteries is composed of three layers separated by elastic laminas: intima, media, and adventitia. The inner layer—intima—is composed by a single layer of endothelial cells providing the interface with the circulating blood and an extracellular component with connective tissue, smooth muscle cells, and macrophages, within a biochemical complex matrix rich in proteoglycans occupying the sub-endothelial space from the endothelial cells to the internal lamina elastica which separates the intima from the media layer. Connective tissue, collagen fibrils, elastin, and smooth muscle cells essentially compose the media and its relative composition varies according to size a function of the arteries. Elastic fibers are dominant in large transport arteries like the aorta, and muscular fibers predominate in medium size arteries like the superficial femoral artery. The media is separated from the outer layer—adventitia—by the external lamina elastica. This external layer contains undifferentiated dendritic cells, collagen fibers, and smooth muscle cells through which penetrates a rich network of very small vessels—the vasa vasorum—responsible for cell nutrition in the arterial wall, plus multiple nerve fibers from the autonomic system which are essential to regulate vascular tone through muscle cell contraction. Thickness of these different layers varies according to the size and function of the arteries in the body and it is early affected by atherogenesis, a process that leads to a remarkable thickening of the arterial wall.

Endothelial cells are connected through junctional complexes, forming tight and gap junctions and they have a wide range of membrane receptors for circulating blood cells and plasma components [2–4]. Its functions are diverse and very important: barrier to penetration of plasma proteins and lipids, non-thrombogenic interface with circulating blood through the production of prostacyclin (PGI2) and thrombomodulin–thrombin complex preventing adhesion and aggregation of circulating cells and plasma components to the arterial wall, regulator of vascular tone by the production of nitric oxide which has a dilating effect and endothelin 1, a potent constrictor, modulation for immune and inflammatory reactions mediated through cytokines and growth factors, and production of extracellular matrix proteins including a monocyte chemoattractant protein MCP-1.

Endothelial dysfunction seems to be the initial trigger of a very complex process called atherogenesis leading to established arterial lesions.

Increased permeability of the endothelial cells and deposition of lipids within the extracellular matrix in the sub-endothelial space induce an inflammatory reaction in the arterial wall mediated by circulating monocytes and T-lymphocytes and leading to migration and proliferation of smooth muscle cells (SMCs) through known biochemical mediators. Evidence has been recently published associating both systemic and intraplaque markers of inflammation with symptomatic stroke and plaque instability [5].

Several mechanisms have been considered responsible for endothelial injury leading to its dysfunction. Physical injury may be caused by hemodynamic flow disturbances resulting from turbulence, increased pulse pressure associated with arterial hypertension, low shear stress in arterial segments close to bifurcations and ostia, and low flow situations which increase contact between chemical agents affecting endothelial function [6, 7]. Chemical agents include lipids and oxidized lipid molecules, oxidative stress from exposure to oxygen-derived free radicals that inactivate nitric oxide and reduce its availability, and cytokines from damaged tissues or infection. These injuries promote coagulation changes leading to thrombosis and enhanced production of inflammatory mediators, which have been extensively studied and described [8–10]. Gene de-regulation has been implied in changes in nitric oxide synthesis, tissue-plasminogen activation, monocyte chemoattractant protein-1, and platelet-derived growth factor offering insight and possible explanation on the mechanisms of genetic susceptibility to atherosclerosis in some families and its increased incidence in younger age patients.

Endothelial dysfunction results in monocyte penetration and activation in the sub-endothelial space, thus initiating a complex inflammatory and proliferative reaction leading to thickening of the artery and narrowing of its lumen, reducing blood flow, and further enhancing hemodynamic changes that in turn aggravate cell damage within the arterial wall.

Smooth muscle cells (SMCs) have two different phenotypes: contractile and synthetic [11]. Contractile plays an essential role to maintain vascular tone regulation required for an effective blood flow distribution according to the needs of organs- and tissues: borrowing–lending effect. It is through the synthetic phenotype that SMCs are directly involved in atherogenesis, through migration from the media to the sub-endothelial matrix, proliferation in response to various mitogens, and synthesis of collagen, proteoglycans, and elastin.

Macrophages are closely associated with the changes in the arterial wall during atherogenesis and play a substantial role in the stability of established atheroma. These cells derive from circulating monocytes which following penetration of the endothelial layer differentiate into macrophages. They respond to several chemoattractants such as MCP-1, tumor necrosis factor (TNF) alpha, and interleukins. They are scavengers of lipids and cell debris resulting in the formation of the foam cell within the atherosclerotic plaque, but also secrete a large amount of enzymes such as proteases, hydrolases, elastase, and growth factors and act as modulators of immune responses [12, 13].

Atherogenesis and Evolution of Atheromatous Plaques

Atherosclerosis begins with deposition of cholesterol and lipoproteins and macrophages filled with lipid components—foam cells—in the sub-endothelial matrix without destruction of the endothelial layer and appearing in susceptible arterial segments. These changes are designated by fatty streak, produce a yellowish discoloration of the intima of the artery, and may remain quiescent or progress forming multiple cell layers in the intima, as described by Stary [14].

There is strong evidence that accumulation of plasma-derived lipoproteins initiates the formation of foam cells and this is the key element conducting the formation of the atherosclerotic lesion. The progression of the lesion is related to increased deposition and accumulation of extracellular lipids favored by local hemodynamic factors and endothelial injury as described previously. Cell necrosis in which debris tend to accumulate within the media layerand to form a core nucleus with deposits of lipids, cell degradation products, and calcium. The fibrous proliferation at the luminal surface constitutes a fibrous cap that protects the inner components of the lesion from the circulating blood and replaces the damaged endothelial layer.

This lesion is usually designated as fibrous plaque disrupting the normal architecture of the arterial wall with destruction of inner and outer elastic laminas.

On direct examination it protrudes in the arterial lumen causing localized narrowing stenosis. When it affects normal laminar flow and causes localized acceleration and turbulence it is considered to be hemodynamically significant. It determines high shear stress further increasing the endothelial injury. Areas of flow stagnation also coexist increasing endothelial exposure to chemical injuries from plasma components and promoting adhesion of blood cells and platelets to the arterial wall [15] a persistent mechanism that further damages the arterial wall.

These lesions can be visualized with high-definition B-mode echography (Figs. 1.1 and 1.2) and are markers of early atherosclerosis and increased risk of cardiovascular clinical events [16, 17]. Hemodynamic studies conducted in these group of patients with early atherosclerotic lesions suggest impairment of reactive hyperemia following induced ischemia, indicating associated endothelial dysfunction with reduced production of endothelial-derived relaxing factor [18].

Fig. 1.1
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(a) Ultrasonographic morphology of the normal arterial wall with quantification of the intima–media thickness (IMT). (b) Increased IMT with destruction of the normal artery morphology

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Fig. 1.2
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Progression from the normal arterial wall morphology to atheromatous plaque

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More advanced (Figs. 1.2 and 1.3) or complicated lesions result from persistence of the injury mechanisms to the arterial wall, promoting an inflammatory reaction with development of neovascularization from the vasa vasorum which can also be detected in vivo by enhanced color-flow Doppler imaging technologies using microparticles [19] or by using micro-CT assessment of symptomatic coronary arteries [20]. Extensive plaque neovascularization in carotid endarterectomy specimens was predictor of late cardiovascular events [21]. This process is associated with higher production of several proteases and other enzymes inducing cell destruction and loss of tensile strength of the plaque by the hemodynamic stress resulting from flow disturbances which lead to rupture of the fibrous cap, intraplaque hemorrhage, and thrombosis inside the lesion and at its surface [22–24] as shown in Fig. 1.3b, c.

Fig. 1.3
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(a) Stable carotid plaque. (b) Carotid lesion with intraplaque hemorrhage. (c) Carotid plaque showing extensive surface thrombosis (atherothrombosis)

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These morphologic changes lead to an increase in the volume of the lesion, rupture of the plaque with extrusion of material from its core nucleus, associated thrombosis and increased severity of the stenosis, or complete arterial occlusion.

It is important to understand the underlying mechanisms that increase plaque progression and its biological markers in order to improve our capability for its recognition.

The concept of vulnerable plaque introduced by Fuster and others [25, 26] shifted the emphasis from degree of stenosis to plaque composition and structure, which increases the risk of rupture and associated clinical acute events.

The major determinants of plaque’s vulnerability to rupture may be listed as follows [26]:

  • Size and location of the core nucleus rich in lipids and cell debris.

  • Thickness and collagen content of the fibrous cap overlying the core.

  • Distance from the core to the lumen [27, 28].

  • Exposure of plaque components to circulating blood, which have a potent thrombogenic effect.

  • Active inflammatory and immunological processes resulting from increased deposition and activation of macrophages, T lymphocytes, and mast cells.

  • Local flow disturbances usually associated with severity of stenosis [29, 30].

  • Systemic thrombotic propensity from the individual patient.

In vivo detection of plaque morphology, recognition of individual inflammatory activity, and immunological status plus increased propensity to intravascular thrombosis completely changed the scope of diagnostic methods in atherosclerosis. The challenge is no longer the assessment of severity of stenosis and its hemodynamic effect but the identification of these potentially dangerous lesions, which are associated with increased risk of adverse clinical events.

The new developments on imaging technology, both morphological and functional, will be briefly dealt subsequently.

Imaging Technologies

Arteriography

Introduced by Egas Moniz in 1926 [31] for the diagnosis of brain tumors it provided the first clinical correlations between ischemic stroke and occlusion of extracraneal internal carotid [32]. It was subsequently extended to visualize the abdominal aorta and peripheral arteries [33]. Selective intra-arterial catheterization by Seldinger [34] provided an extraordinary expansion of the arteriographic method, which for decades was only available method to study atherosclerosis in vivo (Fig. 1.4).

Fig. 1.4
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Arteriography of the carotid bifurcation with tight stenosis in the origin of the internal carotid artery

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The development of arterial reconstructive surgery, from endarterectomy introduced by Cid dos Santos in 1946 [35] and bypass techniques popularized by M.E. DeBakey in the early 1950s, to present-day endovascular intervention rested upon arteriography.

Arteriography is a luminography: its images are of the contrasted blood flow and indentations upon the circulating flow produced by the diseased arterial wall. It does not provide any image of the vessel wall and nature of the disease, but only its effect upon blood flow.

It does not also provide accurate information on plaque morphology characteristics relevant to clinical management as previously described, although the presence of irregularities and/or ulceration in carotid arteriography performed within the scope of the ECST trial were markers of higher risk for stroke in the medical arm of the study [36].

Its invasiveness, toxicity associated with contrast injection, X-ray exposure, and high costs have reduced its scope for diagnostic purposes. In today’s clinical practice conventional or digital arteriography for management of atherosclerotic patients is part of the interventional treatment procedure aimed to correct both occlusive and dilating disease.

CT Angiography and Magnetic Resonance Angiography

New developments in computed tomography technology including multi-detector CT and ECG-gated CT for accurate visualization of coronary circulation provided an useful tool to study atherosclerosis by combining flow assessment with arterial wall imaging (Fig. 1.5a, b).

Fig. 1.5
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(a) CT-angio of the supra-aortic trunks (calcified carotid plaques). (b) CT-angio of the abdominal vessels (aortic thrombosis)

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Identification of plaque morphology in carotid bifurcation disease using CT angiography (CTA) provided identification of intraplaque hemorrhage and ulceration confirmed by histology [37].

For coronary circulation CTA has been correlated with conventional arteriography and used to quantify the calcium content of the lesions (calcium score) and to correlate with symptoms.

A recent trial [38] using a 64 row multi-detector CTA confirmed its accuracy for identification of coronary stenosis and to assess disease severity in symptomatic patients with calcium score <600, but could not replace conventional coronary angiography because of negative predictive value of 83% and positive predictive value of 91%.

Failure to use this technology to identify vulnerable plaques and to discriminate between fibrous tissue and lipids was reported [39], although experimental evidence for the detection of macrophages in a rabbit model of atherosclerosis [40].

For peripheral lower limb arterial disease CTA provides acceptable visualization of the arteries useful for planning the treatment particularly in the absence of extensive calcification.

Although less invasive than conventional arteriography, CTA requires iodine contrast use and radiation exposure which limits its use.

Magnetic resonance imaging is safer, without conventional radiation and less toxic contrast agents. It provides flow images equivalent to conventional arteriography, with better visualization of low flow perfusion situations, like in critical lower limb ischemia, but it has limitations for the assessment of coronary circulation due to its low specificity [41]. Technical refinements provided suitable information to assess arterial remodeling in the presence of moderate stenosis [42] and to characterize unstable fibrous caps in advanced carotid atherosclerosis [43].

New molecular imaging tools and the use of nanotechnology are promising developments for MR imaging of atherosclerotic lesions and to provide more objective identification of markers of plaque vulnerability [44, 45] both in animal models and in humans.

However, this is an expensive technology and time-consuming, which reduces its applicability for routine clinical use.

B-Mode High-Definition Ultrasound and Color-Flow Duplex Scan

Color-flow duplex scan (CF DS) provides accurate visualization of the arterial system, from extracraneal to intracranial vessels from abdominal aorta and its major visceral branches to the arteries of both upper and lower limbs. It provides visualization of the arterial wall and its changes as well as the flow abnormalities directly induced by the lesions.

Extensive literature has been published to validate objective criteria from flow velocity changes to quantify the degree of stenosis [46–49] from carotids to renal and mesenteric circulation to peripheral limb occlusive disease.

Accurate quantification of stenosis severity is a key factor for clinical management in carotid bifurcation disease and it is extensively dealt in another chapter.

However high-definition B-mode echography offers an objective, noninvasive, reliable, and inexpensive tool to identify early atherosclerotic lesions (Fig. 1.1) and to measure intima–media thickness (IMT) a powerful marker of late occurrence of cardiovascular events. It also provides an objective quantification of the total atherosclerotic burden by using as windows to the arterial system, both the common carotid and femoral bifurcations [50, 51].

By using computerized technology and image standardization of carotid bifurcation plaques (Fig. 1.6a, b) it is possible to quantify the echogenicity of the plaque, to assess its homogenous or heterogeneous quality, and to identify markers of plaque vulnerability and obtain correlations with its biochemical composition and clinical outcomes [28, 30, 52–60].

Fig. 1.6
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(a) Homogenous carotid plaque visualized by high-definition ultrasonography. (b) Computer-assisted plaque analysis. (c) Activity index

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In a previously published report using computer-assisted HD echography we described ultrasonographic equivalents to histological markers of lesion instability and showed a good separation between symptomatic and asymptomatic lesions and also a positive correlation with ipsilateral brain hemispheric infarcts [28, 61, 62].

Extending further the analysis an indicator of plaque activity was obtained [63]—Activity Index—which had an excellent correlation with appropriate neurological symptoms and was a predictor of late ipsilateral cerebrovascular events in asymptomatic patients followed for a period of 4 years (Fig. 1.6c).

Recently the ACSRS trial was published [64] providing evidence on the value of plaque morphology analysis to stratify clinical and neurological risk in asymptomatic patients with carotid bifurcation stenosis >50%.

Also the efficacy of lipid-lowering agents like statins can be monitored by changes in echogenicity of the plaque and increased thickness of the fibrous cap [65].

There is now substantial evidence pointing to the possibility of this simple, noninvasive, and inexpensive technology to provide adequate imaging for atherosclerotic lesions in accessible sites, accurate measurements of lesion severity, and its direct hemodynamic repercussion and to obtain reliable information on plaque structure to identify vulnerable/unstable plaques.

Using a 2 MHz probe and injecting echogenic particles it has been possible to identify increased vascularization in segments of unstable carotid plaques, which could be a marker of active inflammation [66].

Intravascular Ultrasound

This is an invasive technology catheter-based providing two-dimensional and cross-section images of the arteries, thus providing accurate measurement of stenosis severity and plaque volume. It has been used associated with catheter-based techniques to improve selection of lesions for treatment and for completion assessment following interventional procedures [67].

Advances in intravascular ultrasound (IVUS) technology with spectral analysis of the signals provide plaque characterization and evaluation of its composition, which has been referred as Virtual Histology [68].

Several studies have provided validation of this technology. However it is invasive, expensive, and not suitable for routine clinical use and epidemiological studies.

Optical Coherence Tomography

It is an optical analogue of intravascular ultrasound with excellent spatial resolution and providing also a cross-sectional image of the artery [69].

Its clinical advantage seems to provide completion assessment following angioplasty and stent deployment in both coronary and carotid arteries and to identify intimal hyperplasia in early re-stenosis.

Fluorescence Imaging

This technology is available and provides the visualization of the inflammatory reaction within the plaque. [70] and visualize the inflammatory reaction within the plaque. It targets molecules and follows its incorporation and degradation in the structures. Its use has been experimental to test new imaging tools, but the recent developments with catheter technology allowed incorporation of a fluorescence probe which can be directed to coronary or other arterial bed. By injecting fluorochromes and activating them with the laser probe it is feasible to track down enzymes or other molecules involved in plaque instability [71].

Nuclear Imaging PET-SCAN

Single photon emission computed tomography (SPECT) and PET imaging of vascular structures have improved their sensitivity and accuracy by accurate anatomic location of the radiation source. Using 18 FDG (fluorodeoxyglucose), which is taken by active cells, has provided interesting images of carotid plaques correlating with neovascularization [72, 73]. It targets individual molecules involved in different metabolic pathways (VCAM-1), which adhere to endothelial cell in early atherosclerosis.

These technologies are promising tools for research, but its use in clinical practice requires further improvements on its sensitivity and specificity and also easier and less expensive instrumentation.

We have briefly described several technologies, some already tested in clinical studies and used in practice, and others still within the realm of experimentation and animal models.

There is a fundamental need to achieve better diagnosis of the unstable plaque, which often is not associated with severe stenosis and is a major cause of atherothrombosis and leads to acute cardiovascular events.

Some of the technologies provide only morphological imaging, but a new era of functional biological imaging is beginning with new optical devices and nuclear-based technology often used with combined MR and CT Imaging to achieve better resolution and accuracy. They offer new ways to visualize the ongoing metabolic process that leads to the morphological changes that are markers of plaque instability.

Also a new area of clinical research will be the combined use of these technologies with biomarkers of inflammation like CPR or others, which may help to identify the vulnerable lesions and the patients at a higher risk of cardiovascular events.

The road from arteriography to molecular and biological imaging is fascinating and provides a good example of translational research from the bench and animal experiments to the bedside, by allowing a better selection of patients who really need of interventional treatment and thus saving a relevant number from unnecessary procedures.