**2. Atherosclerotic plaque development**

**1. Introduction**

18 Cholesterol Lowering Therapies and Drugs

effectiveness by clinical studies.

Atherosclerosis remains one of the most challenging problems of modern medicine. Epide‐ miological data on atherosclerosis and cardiovascular diseases are frequently updated and demonstrate an increase in overall mortality, partly because of the ageing of human popula‐ tion, especially in favourable economic conditions [1]. In developed countries, cardiovascular diseases remain the primary cause of overall morbidity and mortality [2]. Atherosclerotic lesions develop in the walls of large arteries and cause occlusion of blood vessels as a result of either arterial wall thickening or thrombus formation on the surface of unstable plaques. This latter condition is especially dangerous, since it can lead to a sudden and often fatal thromboembolia, which represents the first clinical manifestation of atherosclerosis in many patients. By contrast, early stages of the disease usually pass unnoticed. Recent studies have demonstrated that asymptomatic atherosclerosis is, in fact, a widespread condition among young adults [2–5]. In this cohort of subjects, the incidence of atherosclerotic lesions reaches

The development of atherosclerosis is a complex process, which, despite the significant progress made during the last decade, still remains to be fully understood. Atherosclerosis and related cardiovascular disorders are associated with several known risk factors, including

Modern atherosclerosis prevention strategies are largely based on elimination or attenuation of relevant risk factors, which slows down the atherosclerotic plaque progression in an indirect way [8]. For instance, statins are commonly used for plasma cholesterol reduction and attenuation of atherosclerosis progression. However, limited indications and serious side effects make statins unsuitable for preventive therapy of atherosclerosis, which has to be long‐ term. Currently, there exists no widespread "direct" anti‐atherosclerotic therapy that could be suitable for treatment of the early, subclinical stages of the disease. Such therapy should target the molecular and cellular mechanisms of atherogenesis at the level of blood vessel wall and should result in prevention of *de novo* lesion formation or regression of existing plaques [8– 10]. Natural agents appear to be attractive candidates for preventive anti‐atherosclerosis therapy because of their favourable safety profile and low cost. Because of their complex composition, biologically active substances of botanical origin and their combinations may have a wider range of effects than synthetic drugs, targeting several atherosclerosis risk factors simultaneously. It is therefore possible that the botanical substances can possess both direct and indirect anti‐atherosclerotic effects, such as protective activity at the cellular level com‐ bined with cholesterol lowering and hypotensive activity. Current knowledge of cardiopro‐ tective effects of natural agents and nutraceuticals is rather limited, although they have been actively studied by several groups during the recent years [11–17]. It is important to establish novel anti‐atherosclerotic preventive therapies based on natural products and confirm their

The search for potential anti‐atherosclerotic agents and evaluation of their activity requires adequate test models. Lipid accumulation is one of the most prominent features of athero‐ sclerotic lesions. Lipid uptake and storage are performed by several cell types of the arterial

100%, although no clinical manifestations can be observed [3–5].

elevated plasma cholesterol level, diabetes, tobacco smoking and others [6, 7].

According to the classic lipid theory of atherogenesis, atherosclerotic lesion development is caused by extracellular and intracellular lipid accumulation in the intimal layer of the arterial wall [20, 21]. It has been shown that the major source of lipid accumulation in the intimal cells is circulating LDL, especially its atherogenic forms, such as chemically modified and aggre‐ gated LDL. Chemical modification of lipoprotein particles appears to be necessary for the atherogenic effect, since native (non‐modified) LDL added to cultured cells could not induce significant lipid accumulation. Atherogenic modifications of LDL in the bloodstream include desialylation, acquisition of negative charge and increase of the particle hydrated density (small dense LDL formation). All these modifications can be accompanied by oxidation [22– 25]. Study of the atherogenic LDL modification in the bloodstream currently remains chal‐ lenging. Different laboratory methods of LDL isolation, quantification and analysis deliver different results, which hinders direct comparison of studies employing different methods and protocols. For instance, analysing LDL size and density by ultracentrifugation in different buffers will give slightly different outcome. Moreover, no consensus has been reached so far on the classification of LDL subfractions [22]. It is likely that LDL particles undergo multiple atherogenic modification in human plasma, but the resulting products are differently evalu‐ ated by different methods from several laboratories [26–28]. One of the earliest atherogenic modifications demonstrated to occur in human bloodstream is desialylation. The removal of sialic acid residues from the carbohydrate components of LDL particles is performed by trans‐ sialidase, which is active in the bloodstream. Increased level of circulating modified LDL leads to aggregation of the particles, which is facilitated by increased surface charge. The resulting large complexes have especially high atherogenic potential. Moreover, modified forms of LDL can induce formation of autoantibodies triggering inflammatory response and giving rise to circulating immune complexes. Another feature that can significantly increase atherogenic potential of modified LDL is its ability to associate with the components of extracellular matrix proteins in the subendothelial space of the arterial wall, which prolongs its residence time and facilitates lipid accumulation. Unlike native LDL, which is internalized by cells via receptor‐ mediated uptake, modified LDL complexes enter the cells through uncontrolled phagocytosis and follow a distinct metabolic pathway [29]. This can explain the rapid accumulation of atherogenic modified LDL in cellular cytoplasm, mostly in the form of lipid droplets. Cells containing large amounts of lipid inclusions in the cytoplasm are called "foam cells" because of their microscopic appearance. Such cells commonly occur in atherosclerotic lesions.

**Figure 1** shows the development of atherosclerotic lesions and the main stages of the athero‐ genesis [30]. According to the current knowledge, atherosclerotic lesion initiation is dependent on two conditions: the presence of modified atherogenic LDL in the bloodstream in sufficient quantities and the internalization of LDL by the arterial wall cells. The latter is usually triggered by local disturbance of endothelial function that causes increased permeability of the endo‐ thelial lining allowing modified LDL to penetrate into the intimal layer of the arterial wall. Atherogenic modification of LDL may also occur in the intimal layer, after the particles have crossed the endothelial barrier. Local disturbances of endothelial function frequently take place in certain parts of the vascular system, such as branching points and bends, where laminar blood flow is altered [31]. Sites of the arterial wall that are especially vulnerable are marked by altered morphology of endothelial cells and presence of enlarged multinucleated cells. The pre‐existent mosaicism of the endothelial lining may explain the focal development of atherosclerotic lesions. However, more studies are needed to determine the mechanisms of endothelial dysfunction leading to atherosclerosis.

Focal lipid infiltration into the arterial wall intima marks the early stages of atherosclerotic lesion development. Apparently, several cell types of the arterial wall participate in lipid accumulation. Cells populating the intimal layer can be either resident mesenchymal cells,

**Figure 1.** Scheme showing the consecutive events in the development of atherosclerotic lesions. Reproduced with per‐ mission from [30].

such as smooth muscle cells, or inflammatory cells, such as monocytes/macrophages, that can be recruited from the bloodstream in large numbers by a local inflammatory response. Along with macrophages, smooth muscular cells also take part in lipid uptake and can be transformed into foam cells. While native LDL particles are metabolized by intimal cells through a well‐ developed and controlled receptor‐mediated endocytosis, it is likely that the LDL associations are recognized by macrophages as pathogens that have to be cleared by phagocytosis [32]. Such clearance is accompanied by secretion of signalling molecules that attract immune cells to the developing lesion site and therefore initiation of the inflammatory process [33]. Phago‐ cytosis‐mediated lipid accumulation in atherosclerosis can therefore be regarded as a variation of innate immune response. Enhanced phagocytosis followed by lipid accumulation and foam cell formation contributes to lesion development. Lipid accumulation affects intercellular contacts that are essential for proper function of intimal wall resident cells [34]. On the other hand, lipid accumulation also triggers processes that are typical for the reparative phase of inflammation, such as proliferation and extracellular matrix synthesis leading to the fibrosis. In favourable conditions, these reparation processes rapidly lead to formation of areas with increased cellularity and extracellular matrix deposition. Gradual development of such focal lesion areas leads to a diffuse intimal thickening, which is frequently observed in adult arteries. However, the inflammatory response can become chronic, with continuous local lipid infiltration, increased cellularity due to the proliferation of cells in the lesion site and enhanced fibrosis.

proteins in the subendothelial space of the arterial wall, which prolongs its residence time and facilitates lipid accumulation. Unlike native LDL, which is internalized by cells via receptor‐ mediated uptake, modified LDL complexes enter the cells through uncontrolled phagocytosis and follow a distinct metabolic pathway [29]. This can explain the rapid accumulation of atherogenic modified LDL in cellular cytoplasm, mostly in the form of lipid droplets. Cells containing large amounts of lipid inclusions in the cytoplasm are called "foam cells" because of their microscopic appearance. Such cells commonly occur in atherosclerotic lesions.

**Figure 1** shows the development of atherosclerotic lesions and the main stages of the athero‐ genesis [30]. According to the current knowledge, atherosclerotic lesion initiation is dependent on two conditions: the presence of modified atherogenic LDL in the bloodstream in sufficient quantities and the internalization of LDL by the arterial wall cells. The latter is usually triggered by local disturbance of endothelial function that causes increased permeability of the endo‐ thelial lining allowing modified LDL to penetrate into the intimal layer of the arterial wall. Atherogenic modification of LDL may also occur in the intimal layer, after the particles have crossed the endothelial barrier. Local disturbances of endothelial function frequently take place in certain parts of the vascular system, such as branching points and bends, where laminar blood flow is altered [31]. Sites of the arterial wall that are especially vulnerable are marked by altered morphology of endothelial cells and presence of enlarged multinucleated cells. The pre‐existent mosaicism of the endothelial lining may explain the focal development of atherosclerotic lesions. However, more studies are needed to determine the mechanisms of

Focal lipid infiltration into the arterial wall intima marks the early stages of atherosclerotic lesion development. Apparently, several cell types of the arterial wall participate in lipid accumulation. Cells populating the intimal layer can be either resident mesenchymal cells,

**Figure 1.** Scheme showing the consecutive events in the development of atherosclerotic lesions. Reproduced with per‐

endothelial dysfunction leading to atherosclerosis.

20 Cholesterol Lowering Therapies and Drugs

mission from [30].

**Figure 2.** Scheme showing the delicate balance between infiltrative and reparative phases in fatty atherosclerotic lesion. Reproduced with permission from [30].

Atherosclerotic plaques can be protected from the bloodstream by formation of a fibrous cap, which serves as a barrier for lipoproteins and inflammatory cells. Such isolation of the local inflammatory site has a protective role, suppressing the inflammatory response and restoring the tissue functions. On the other hand, formation of fibrolipid plaques predisposed to rupture (unstable plaques) can have fatal consequences because of thrombus formation.

In fibrolipid plaques, two opposing processes are likely to take place: infiltration and repara‐ tion that exist in a state of unstable equilibrium (**Figure 2**). Shifting the balance towards reparation leads to the formation of fibrous plaques, which is a favourable outcome from the clinical point of view. Inefficient reparation and continuous lipid infiltration cause plaque rupture with possible thrombus formation. Lipidosis plays therefore a crucial role in athero‐ sclerotic lesion development at cellular and tissue levels and represents an important target for the development of anti‐atherosclerotic therapy.
