**5. Cardiovascular Drugs**

Three classes of cardiovascular drugs: calcium antagonists, beta-blockers and nitrates have been tested on our cellular model. These drugs are widely used in clinic for therapy of various disorders resulting from atherosclerosis of different arteries. We attempted to find out how calcium antagonists, beta-blockers and nitrates affect atherosclerotic indices of arterial cells.

First, we examined the effects of calcium antagonists on major atherosclerotic indices. It has been found that calcium antagonist, verapamil, has a positive effect on all atherosclerotic cellular indices. Within 48 hrs, verapamil added to culture reduced total intracellular choles‐ terol level by 3-fold, sharply decreased the [3 H]thymidine incorporation into cellular DNA, i.e. suppressed cell proliferative activity, and inhibited the collagen synthesis by cultured cells [38,39]. Thus, this drug has a direct anti-atherosclerotic effect at the arterial cell level.

Several calcium antagonists: nifedipine, darodipine, isradipine, nicardipine, nitrendipine, fe‐ lodipine, tiapamil, gallopamil, diltiazem, papaverin, nicardipine, and others were also test‐ ed. Verapamil and nifedipine proved to be the most effective [49,55]. Within 24 hrs of incubation with cultured cells all calcium antagonists substantially inhibited [3 H] thymidine incorporation and reduced intracellular cholesterol level [54,55]. Thus, calcium antagonists produce a direct anti-atherosclerotic effect on the vascular cells normalizing the major athe‐ rosclerotic cell parameters.


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

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

Cells cultured from the subendothelial intima are capable of synthesizing collagen, proteo‐

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

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‐

Three classes of cardiovascular drugs: calcium antagonists, beta-blockers and nitrates have been tested on our cellular model. These drugs are widely used in clinic for therapy of various disorders resulting from atherosclerosis of different arteries. We attempted to find out how calcium antagonists, beta-blockers and nitrates affect atherosclerotic indices of arterial cells.

First, we examined the effects of calcium antagonists on major atherosclerotic indices. It has been found that calcium antagonist, verapamil, has a positive effect on all atherosclerotic cellular indices. Within 48 hrs, verapamil added to culture reduced total intracellular choles‐

i.e. suppressed cell proliferative activity, and inhibited the collagen synthesis by cultured cells [38,39]. Thus, this drug has a direct anti-atherosclerotic effect at the arterial cell level.

Several calcium antagonists: nifedipine, darodipine, isradipine, nicardipine, nitrendipine, fe‐ lodipine, tiapamil, gallopamil, diltiazem, papaverin, nicardipine, and others were also test‐ ed. Verapamil and nifedipine proved to be the most effective [49,55]. Within 24 hrs of

incorporation and reduced intracellular cholesterol level [54,55]. Thus, calcium antagonists produce a direct anti-atherosclerotic effect on the vascular cells normalizing the major athe‐

incubation with cultured cells all calcium antagonists substantially inhibited [3

H]thymidine incorporation into cellular DNA,

H] thymidine

glycans and other components of extracellular matrix [40,41].

exactly the same cells which require a therapeutic action in vivo.

freshly isolated cells [34-39].

192 Current Trends in Atherogenesis

**5. Cardiovascular Drugs**

rosclerotic cell parameters.

terol level by 3-fold, sharply decreased the [3

es.

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‐ lular cholesterol in normal cells.

Four-hour preincubation of cultured cells with verapamil led to complete prevention of the serum atherogenic effect [65]. Thus, verapamil possesses not only an atherosclerotic effect in culture causing the regression of atherosclerotic manifestations at the cellular level but also elicits an anti-atherogenic, i.e. preventive effect, eliminating atherogenic potential of the serum.

action of beta-blockers can be inhibited if a beta-blocker is used in combination with a calci‐ um antagonist or nitrate. This finding allows to hoping that in the nearest future it will be

Use of Natural Products for Direct Anti-Atherosclerotic Therapy

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

195

Thus, three classes of cardiovascular drugs reveal different influence on cellular manifesta‐ tion of atherosclerosis. Calcium antagonists exhibit anti-atherosclerotic action. On the con‐ trary, beta-blockers are atherogenic. Nitrates are neutral, indifferent in this respect. Our data obtained on cellular model were supported by results of clinical study. Loaldi et al. have re‐ ported that long-term per oral administration of propranolol aggravates coronary athero‐ sclerosis in patients with angina of effort as compared with the calcium antagonists, nifedipiene, and isorobide dinitrate [68]. Nifedipine produced the best effect on coronary atherosclerosis by suppressing the development of existing and preventing the appearance of new atherosclerotic lesions. Isosorbide dinitrate was less effective in this respect, while with propranolol therapy the situation was the worst. These clinical observations encourage

All the above conclusions and hypotheses are based on the data obtained in in vitro experi‐ ments. Obviously, the question arises, whether anti-atherosclerotic effects of calcium antago‐ nists and atherogenic effects of beta-blockers can be manifested in vivo and what is the

To optimize anti-atherosclerotic and anti-atherogenic drug therapy, ex vivo model was de‐ veloped. In case of ex vivo model not drug but blood serum taken from patients after oral

Calcium antagonists, verapamil and nifedipine, and beta-blockers, propranolol and pindo‐ lol, were examined using ex vivo model. Within 2-4 hrs after nifedipine or verapamil admin‐ istration, the patient's serum had anti-atherosclerotic properties, i.e. it was able to cause a fall in the intracellular cholesterol and inhibited atherosclerotic cell proliferation [55,56]. On the contrary, the serum of patients who received propranolol or pindolol was pro-athero‐ genic. Its pro-atherogenic properties manifested themselves at the arterial cell level in the rise of intracellular cholesterol and stimulation of cell proliferation [55,56]. This finding al‐ lows to assuming that not only in vitro, but in vivo as well, calcium antagonists and beta-

The effect of nifedipine on serum properties during a prolonged course was assessed. A pa‐ tient was on nifedipine for 7 days. He received 20 mg doses three times a day with an 8-hr interval. Twenty-eight days after regular nifedipine therapy the initial atherogenicity of the patient's serum was substantially lower than at the beginning. Directly after a dose of nifedi‐ pine the atherogenicity was practically completely eliminated [65]. On the contrary, as a re‐ sult of a prolonged therapy with a beta-blocker, propranolol, patient's serum acquired stable atherogenic properties. At the beginning of the course the serum of this patient was nona‐

optimal anti-atherosclerotic therapy based on calcium antagonists and other drugs.

possible to develop beta-blockers devoid of atherogenic side effects.

us to develop anti-atherosclerotic therapy using our cell culture model.

blockers are anti-atherosclerotic and atherogenic drugs, respectively.

**6. Ex Vivo Model**

drug administration is added to cultured cells.


**Table 2.** Anti-atherosclerotic and anti-atherogenic drug effects in culture

The effect of several calcium antagonists on primary cholesterol accumulation in cultured cells induced by the patients' serum was tested. Verapamil and nifedipine completely inhib‐ ited the accumulation of intracellular cholesterol induced by the serum while other calcium antagonists: diltiazem, nicardipine, isradipine, darodipine rather substantially reduced cho‐ lesterol accumulation [65]. As it is known, the examined calcium antagonists manifested an‐ ti-atherogenic action in vivo inhibiting the development of experimental atherosclerosis in animals [66,67]. Thus, our in vitro data obtained on cellular model correspond to the in vivo observations. One can conclude that calcium antagonists elicit not only anti-atherosclerotic but also anti-atherogenic, i.e. preventive effect at the arterial cell level.

Nitrates and beta-blockers have been tested to reveal their action on atherosclerotic cellular indices. Nitrates had no effect on proliferative activity of atherosclerotic cells and practically did not affect the cholesterol level [55]. On the other hand, all the examined beta-blockers, propranolol, alprenolol, metoprolol, pindolol, and timolol, more or less increased athero‐ sclerotic manifestations, i.e. all of these drugs exhibited atherogenic activity in culture [55,60]. If beta-blockers manifest a similar action in vivo, one may assume that these drugs are atherogenic and realize the atherogenic action at the arterial cell level. Apparently, ni‐ trates are neutral, indifferent in this respect.

The influence of cardiovascular drugs on atherosclerosis-related effects of each other was studied. The study was focused on metoprolol, nifedipine and nitroglycerin, the drugs widely used in clinic [55]. Metoprolol caused an elevation of intracellular cholesterol, nifedi‐ pine reduced the cholesterol level while nitroglycerin was without effect on this index. The use of nifedipine on the background of metoprolol did not modify the anti-atherosclerotic action of the calcium antagonist. In this combination atherogenic action of metoprolol was not revealed. The application of metoprolol in combination with nitroglycerin led to the elimination of an atherogenic effect of the beta-blocker. Nifedipine used together with meto‐ prolol and nitroglycerin was just as effective as in the absence of these drugs. Thus, nifedi‐ pine produces its anti-atherosclerotic effects both by itself and in combination with widely used nitrates and beta-blockers. These data suggest one important conclusion. Atherogenic action of beta-blockers can be inhibited if a beta-blocker is used in combination with a calci‐ um antagonist or nitrate. This finding allows to hoping that in the nearest future it will be possible to develop beta-blockers devoid of atherogenic side effects.

Thus, three classes of cardiovascular drugs reveal different influence on cellular manifesta‐ tion of atherosclerosis. Calcium antagonists exhibit anti-atherosclerotic action. On the con‐ trary, beta-blockers are atherogenic. Nitrates are neutral, indifferent in this respect. Our data obtained on cellular model were supported by results of clinical study. Loaldi et al. have re‐ ported that long-term per oral administration of propranolol aggravates coronary athero‐ sclerosis in patients with angina of effort as compared with the calcium antagonists, nifedipiene, and isorobide dinitrate [68]. Nifedipine produced the best effect on coronary atherosclerosis by suppressing the development of existing and preventing the appearance of new atherosclerotic lesions. Isosorbide dinitrate was less effective in this respect, while with propranolol therapy the situation was the worst. These clinical observations encourage us to develop anti-atherosclerotic therapy using our cell culture model.
