**4.** *In vitro* **model**

*In vitro* model based on primary culture of human aortic wall cells was developed for screening of potential anti‐atherosclerotic substances. Cells were isolated from the subendothelial layer of healthy human aortic intima, the layer of the arterial wall, which is most severely affected in atherosclerosis [41]. The process of cell isolation from autopsy material using collagenase and elastase treatment has been described previously [9, 42–44]. The obtained cell population has been characterized using immunocytochemistry methods and was found to be heteroge‐ neous and containing smooth muscle cells (20–50%), pericytes (30–70%) and inflammatory cells and tissue macrophages (10%) (**Table 1**) [9, 43, 44].


**Table 1.** Proportion of cell types in primary culture cells isolated from human aortic subendothelial intima (% of positive cells for each marker).


**Table 2.** Substances that have been tested *in vitro* cell model.

the tissue functions. On the other hand, formation of fibrolipid plaques predisposed to rupture

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

**3. Evaluation of substances' anti‐atherosclerotic activity using cellular**

Preventive anti‐atherosclerotic therapy should be aimed at reduction of intracellular lipid accumulation [35]. Such reduction can be achieved by different approaches [36]. First, the therapy may decrease the level of circulating modified LDL. Second, it can target atherogenic modification of LDL in the bloodstream. Third, it can reduce lipid uptake and storage by the arterial wall cells. Finally, the therapy can be aimed at depletion of the existing intracellular lipid stores. All these approaches can be evaluated by measuring the reduction of intracellular lipid accumulation and the decrease of the intracellular pool of cholesterol esters [9, 37, 38]. A number of available medications can be used to decrease blood serum atherogenicity [9, 36, 38, 39], which is defined as the ability of blood serum to induce cholesterol accumulation in cultured cells. Blood serum from patients with coronary atherosclerosis usually has high atherogenicity [19]. Changes of blood serum atherogenicity reflect lipid accumulation in the arterial wall and are therefore relevant for the development of preventive therapy. Such changes can be detected using cultured cells as models of early stages of human atherogenesis [9, 38, 40]. Cellular models can be used for evaluation of anti‐atherosclerotic potential of different drugs and active substances, for screening of potential anti‐atherosclerotic agents and

*In vitro* model based on primary culture of human aortic wall cells was developed for screening of potential anti‐atherosclerotic substances. Cells were isolated from the subendothelial layer of healthy human aortic intima, the layer of the arterial wall, which is most severely affected in atherosclerosis [41]. The process of cell isolation from autopsy material using collagenase and elastase treatment has been described previously [9, 42–44]. The obtained cell population has been characterized using immunocytochemistry methods and was found to be heteroge‐ neous and containing smooth muscle cells (20–50%), pericytes (30–70%) and inflammatory

(unstable plaques) can have fatal consequences because of thrombus formation.

for the development of anti‐atherosclerotic therapy.

22 Cholesterol Lowering Therapies and Drugs

for evaluation of potential clinical efficacy of various molecules.

cells and tissue macrophages (10%) (**Table 1**) [9, 43, 44].

**models**

**4.** *In vitro* **model**

Smooth muscle cells and pericytes were positive for smooth muscle α‐actin. Pericytes had a distinct stellate shape and were identified using antibodies to 3G5 and 2A7 that are expressed by resting and activated pericytes, respectively. Together, smooth muscle cells and pericytes represented the majority of cell population in the obtained primary cultures. A smaller population consisted of the inflammatory cells that could be detected using antibodies to leukocyte‐specific marker CD45 and macrophage marker CD68 [45]. Cellular lipid accumula‐ tion was induced by incubation of cells with atherogenic serum obtained from patients with confirmed atherosclerosis. The increase of cellular cholesterol content reached as high as two folds after a 24‐h incubation with atherogenic serum.

Potential anti‐atherogenic substances were evaluated by concomitant incubation of cells with atherogenic serum and aqueous solutions of tested substances. Anti‐atherosclerotic effect was measured as a decrease in the levels of intracellular cholesterol in the cells with test substances compared to the control cells (treated with atherogenic serum only). The described model allowed evaluating a number of different drugs and substances and detecting several novel active molecules with anti‐atherosclerotic potential. Some substances were demonstrated to possess a pro‐atherogenic effect, enhancing intracellular cholesterol accumulation induced by atherogenic serum (**Table 2**).
