**4. Cell Model**

vention of LDL modification may be an approach to anti-atherosclerosis therapy. The second approach may be selective removal of modified LDLs from blood (target 2). The third one may be based on prevention of modified LDL accumulation in arterial cells (target 3). Also one more approach is removal of excess lipids from foam cells (target 4). Figure 1 schematically represents these four approaches. We have used all of these approaches and now we believe that the most suitable approach is the third one, namely, the prevention of modified LDL accumulation in arterial cells. Bellow we describe the application of this ap‐

Agents capable of preventing atherogenesis are anti-atherogenic drugs, agents promoting the regression of atherosclerotic manifestations are anti-atherosclerotic drugs. Prevention of intracellular lipid accumulation accompanied by the stimulation of arterial cell proliferation and massive extracellular matrix production may be regarded as anti-atherogenic (preven‐ tive). In terms of arterial cells, any drug effect which does not prevent directly the conver‐ sion of the normal cell into an atherosclerotic one (foam cell) should be regarded as an indirect anti-atherogenic action. Only that drug which exhibits its preventive activity at the arterial level is a direct anti-atherogenic drug. At the arterial cell level, a drug with a direct anti-atherosclerotic action should induce the regression of the major cellular manifestations of atherosclerosis, i.e. reduce the intracellular lipid content, suppress cell proliferation and

proach for the development of anti-atherosclerotic therapy.

**Figure 1.** Targets of anti-atherosclerotic and anti-atherogenic drug actions

(see explanation in the text).


Solid circles, multiple modified LDL; open circles, native LDL

Thus, the drugs that affect atherosclerosis can be divided into 3 groups:

inhibit the extracellular matrix production.

190 Current Trends in Atherogenesis

The identification of anti-atherosclerotic or/and anti-atherogenic activities of a drug is asso‐ ciated with considerable difficulties. There are no simple and rapid techniques to estimate the anti-atherogenic/anti-atherosclerotic effect of a drug in an animal model or in clinical tri‐ als. That is why we employ a culture of human atherosclerotic vascular cells in the screening of potential drugs, investigation of their mechanisms of action and optimization of anti-athe‐ rosclerotic drug therapy.

We use human aortic cells to examine the effects of various agents on atherosclerosis-related features of cultured cells. Cells are isolated from the subendothelial part of the human aortic intima, i.e. from the part of aorta which is localized between the endothelial lining and the media [32]. The intima of adult human aorta is a well-defined structure. The thickness of a normal intima varies from 50 to 120 µm [32]. Sometimes a thickened intima is called a dif‐ fuse intimal thickening [32]. This term underlines its essential difference from a very thin in‐ tima of animal and adolescent aorta. Unaffected intima of adult human aorta contains 10-12 lines of subendothelial cells [32].

Using collagenase and elastase, cells are isolated from the subendothelial layer of the intima of both normal and atherosclerotic parts of the aorta [33-36]. This approach makes it possible to study a direct anti-atherosclerotic and anti-atherogenic action of a drug at the vascular cell level. An important advantage of this technique is that human material is used and thus, the results obtained are relevant to human atherosclerosis.

By the well-established criteria, the cells cultured from the intima can be classified as the cells of smooth muscle origin. These cells are stained with antibodies to smooth muscle myosin [33-35]. For further identification of cultured cells we have used a monoclonal antibody HHF-35 which reacts specifically with muscle α-actin and can reveal smooth muscle cells [37]. Accord‐ ing to our calculations, primary culture of subendothelial cells contains about 90% of smooth muscle cells interacting with HHF-35. In addition, cells cultured from subendothelial part of uninvolved (healthy) intima have the ultrastructural features characteristic of smooth mus‐ cle cells, namely: the basal membrane and filament bundles with dense bodies [33-36]. The culture on which our experiments are performed is represented by mixed population of typical and modified smooth muscle cells revealed in the human aorta earlier [32].

Cells of the subendothelial intima isolated from atherosclerotic lesions retain in primary cul‐ ture all major characteristics of atherosclerotic cells. Cells cultures from fatty streak and fatty infiltration zones have an enhanced proliferative activity [38]. These cells have a higher pro‐ liferative activity as compared with the cells cultured from unaffected intima [38,39].

Many cells cultured from atherosclerotic lesions are so called foam cell containing numerous inclusions filling the whole of the cytoplasm, these inclusions are lipid droplets [34]. The bulk of excess lipids in foam cells is represented by free cholesterol and cholesteryl esters [34]. It should be noted that the content and composition of lipids in cultured cells within the first 10-12 days in culture remain unchanged and correspond to the respective indices of freshly isolated cells [34-39].

**Agent References**

Use of Natural Products for Direct Anti-Atherosclerotic Therapy

http://dx.doi.org/10.5772/52967

193

Cyclic AMP elevators [39,42 - 45] Prostacyclin [39,46 - 50] Prostaglandin E2 [39,46, 51] Artificial HDL [52] Antioxidants [39]

Calcium antagonists [39,49, 50, 53-56]

Trapidil and trapidil derivatives [57, 58] Lipoxygenase inhibitors [51] Lipostabil [39] Mushroom extracts [59]

Beta-blockers [55,60] Thromboxane A2 [49,50] Phenothiazines [39]

Nitrates [55] Cholestyramine [39]

In addition to anti-atherosclerotic effects imitating the regression of atherosclerosis, antiatherogenic effects in culture imitating prevention of atherosclerosis were studied. Table 2 demonstrates the major differences between these two approaches. In the case of anti-athe‐ rosclerotic effect the regression of atherosclerosis is imitated, whereas in the case of antiatherogenic effect, the prevention of atherosclerosis is imitated. In the first case the cells obtained from an atherosclerotic plaque are used, while in the second type of experiments cells derived from unaffected intima are employed. When anti-atherosclerotic effect is exam‐ ined, cells are cultured in the presence of a standard fetal calf serum, while in the experi‐ ments on anti-atherogenic effect - atherogenic serum obtained from coronary heart disease patients is added to culture. This serum induces the accumulation of cholesterol and stimu‐ lates other atherogenic manifestations in cultured cells [61-64]. In the case of anti-athero‐ sclerotic effect the efficacy of a drug is judged upon by its ability to decrease an elevated content of cholesterol in cultured atherosclerotic cells but in the case of anti-atherogenic ef‐ fect, the efficacy of a drug is judged upon by the ability to prevent the deposition of intracel‐

**ANTI-ATHEROSCLEROTIC**

**PRO-ATHEROGENIC**

**INDIFFERENT**

**Table 1.** Substances tested on cellular model

lular cholesterol in normal cells.

Cells cultured from the subendothelial intima are capable of synthesizing collagen, proteo‐ glycans and other components of extracellular matrix [40,41].

Thus, the cells isolated from an atherosclerotic lesion of human aorta retain in culture all the main properties characteristic of atherosclerotic cells. They exhibit an enhanced proliferative activity, contain excess cholesterol in the form of intracellular inclusions and synthesize the extracellular matrix. This allows one to regard a culture of atherosclerotic cells as a conven‐ ient model for the investigation of the effects of various agents on atherosclerotic manifesta‐ tions [21]. Thus, the investigations in the cell culture model are carried out directly on exactly the same cells which require a therapeutic action in vivo.

Using this model, we have examined the effects of different drugs and chemicals. By now many substances have been tested [21]. The effects of several substances are summarized in Table 1. Some of them elicited anti-atherosclerotic effects in culture, some proved to be inef‐ fective in this respect, while others even stimulated the development of atherogenic process‐ es.
