Change in the number of CD117+ stem cells, cytogenetic and cytokinetic parameters under the use of candesartan, candesartan cilexetil and resveratrol in vitro

Due to the increase in cardiovascular disease, it is urgent to research new effective and safe drugs and their combinations. Candesartan cilexetil, an angiotensin II receptor antagonist, is a precursor to the active form of candesartan. However, these antiischemic drugs have a cytotoxic effect, affecting the antioxidant system. Therefore, to prevent the cytotoxic effect is the need to use antioxidants. To study the effect of candesartan cilexetil, candesartan and resveratrol antioxidant in various doses and combinations on CD117+ stem cell mobilization, on the number of apoptotic and micronucleated cells and cell cycle parameters in vitro. Bone marrow cells isolated from C57Bl / 6 mice were selected for experiments. After incubation for 2 days with the means in different concentrations and combinations, the biological characteristics of the stem cells were determined. Flow cytometry was used to analyze the number of CD117 + stem cells, the ratio of apoptotic cells, cells with micronuclei and cell cycle parameters when using candesartan cilexetil, candesartan, and resveratrol in vitro. It was found that using candesartan cilexetil with resveratrol and candesartan with resveratrol promotes the formation of CD117 + stem cells from 1.2 times to almost 2 times compared with controls and 1.5 and 2.5 compared with cytostatics. Candesartan cilexetil and candesartan were cytotoxic, while resveratrol reduced the adverse effects of the substances in combination. Combination of candesartan cilexetil with resveratrol; Candesartan with resveratrol significantly increased CD117+ stem cell count and was not cytotoxic.

Introduction. The incidence of cardiovascular diseases has increased several times in many countries all over the world. So it's necessary to study new methods of treatment of cardiovascular diseases, investigate new effective and safe drugs and combinations thereof.
Candesartan cilexetil is an angiotensin II receptor antagonist. It is a pro-drug, which converted to the active form candesartan during absorption from the gastrointestinal tract. It is used as a long-term antihypertensive agent [1]. Candesartan cilexetil increases resistance to stress and endurance during exercise in people suffering from hypertension [2]. However, it is shown that candesartan has also a number of side effects, such as dizziness, weakness, headache. High doses of candesartan cilexetil influence the formation of separate subpopulations of cells in bone marrow [3]. Resveratrol, a naturally occurring polyphenol, shows pleiotropic health beneficial effects, including antioxidant, anti-inflammatory, anti-aging, cardioprotective and neuroprotective activities [4][5][6]. It's found that this substance decreases the synthesis of lipids in liver and eico-sanoids in leukocytes in animals, inhibits platelet activation/aggregation, decreases the activity of protein kinases, inhibits formation of reactive oxygen species [7].
At present stem cell technologies are widely applied for treatment of different pathologies and also cardiovascular diseases. It is shown that mobilized stem cells and endothelial progenitor cells have the capacity to migrate into the heart muscle and endothelium with the subsequent positive therapeutic effect [8][9][10].
In recent study we evaluate the effect of candesartan cilexetil, candesartan and safer resveratrol in different dosages and combinations on mobilization of CD117+ stem cells, on the number of apoptotic cells and micronucleated cells and parameters of cell cycle in vitro.

Materials and methods.
Bone marrow cells, isolated from C57Bl/6 mice were sampled for further experiments. Cells were inoculated into growth medium (90 % of DMEM medium ("Sigma-Aldrich"), 10 % of fetal bovine serum ("HyClone") with addition of 0.1 % antibiotics (Antibioticantimycotic solution, "Sigma-Aldrich") at the cell density 10 4 /cm 2 in 6-well plates. This culture was incubated in CO2-incubator (37 °C, 5 % CO2) and then candesartan cilexetil, candesartan and resveratrol (we used transresveratrol) were supplied to the cell culture. The medium was changed every 3-4 days. Cells were removed by 0.25 % trypsin/EDTA solution and washed in 0.1 % PBS buffer. We studied the next doses and combinations of substances: candesartan cilexetil at 1.5 µg/ml dose and 3 µg/ml dose, candesartan at 1.5 µg/ml dose and 3 µg/ml dose, resveratrol at 1, 5, 10, 30 and 50 µg/ml doses, combination of candesartan cilexetil at 1.5 µg/ml and resveratrol at 1, 5, 10, 30 and 50 µg/ml doses, combination of candesartan at 1.5 µg/ml and resveratrol at 1, 5, 10, 30 and 50 µg/ml doses. Flow cytometry method was used for the analysis of the number of CD117+ stem cells. For this purpose, monoclonal antimouse antibodies СD117 (Beckman Coulter, США) were used. The ratio of apoptotic cells and micrinucleated cells and parameters of cell cycle were also studied by flow cytometry method.
The results of the study are presented as mean±SEM. We used Student's t-test to compare 2 samples and oneway ANOVA for multiple comparisons followed by pair-wise comparison.
Results and discussion. We investigated the effect of candesartan cilexetil, candesartan and resveratrol on changes in the number of CD117+ stem cells. Cell surface marker CD117 (or c-kit) is a marker of endothelial progenitor cells, which is a cytokine receptor and KIT gene product.
It was shown that candesartan cilexetil at 1.5 µg/ml dose didn't stimulate generation of CD117+ stem cells in vitro as compared to the control. Candesartan cilexetil at 3 µg/ml dose also didn't promoted production of CD117+ stem cells (Table 1). It was demonstrated that candesartan at 1.5 µg/ml and 3 µg/ml doses stimulate the formation of endothelial progenitor cells as shown in the table 1. We found that resveratrol influenced the ratio of endothelial progenitor cells in cell culture. The obtained result was dose-depended. Resveratrol at 1 µg/ml, 5 µg/ml and 10 µg/ml doses didn't increase the amount of CD117+ stem cells in comparison with the control. Application of resveratrol at 30 µg/ml and 50 µg/ml doses significantly increased the number of CD117+ stem cells in vitro. The largest increment of the number of endothelial progenitor cells in cell culture was observed in variant with resveratrol at 50 µg/ml dose (Table 1).

T a b l e 1. The influence of combination of candesartan cilexetil, candesartan and resveratrol on the number of CD117+ cells in vitro
It was demonstrated that combination of candesartan cilexetil at 1.5 µg/ml dose and resveratrol at 1 µg/ml, 5 µg/ml and 10 µg/ml doses didn't stimulate the formation of CD117+ stem cells, whereas with resveratrol at 30 µg/ml and 50 µg/ml doses significantly increased the ratio of endothelial progenitor cells in cell culture as compared to the control. It was found that combination of candesartan cilexetil at 1.5 µg/ml dose and resveratrol at 30 µg/ml and 50 µg/ml doses was effective in the mobilization of CD117+ stem cells (p < 0.05) ( Table 1).
Changing of the count of endothelial progenitor cells under using of candesartan at 1.5 µg/ml with resveratrol at 1, 5, 10, 30 and 50 µg/ml doses was studied. The effect was dose-dependent. The obtained data were also significantly higher than those recorded for individual compounds ( Table 1).
The next step was investigation of the number of apoptotic cells under using candesartan, candesartan cilexetil and resveratrol in vitro. It was shown that candesartan cilexetil at high dose increased the amount of apoptotic cells in comparison with the control. Candesartan cilexetil at 1.5 µg/ml dose didn't change the number of apoptotic cells as compared to the control (Table 2). Candesartan at 1.5 µg/ml and 3 µg/ml doses significantly increased the count of studied cells (p<0.05). It was found that the num-ber of apoptotic cells didn't change under using resveratrol at 1, 5, 10, 30 and 50 µg/ml doses (Table 2).

Samples
The number of apoptotic cells, % It was also recorded that candesartan cilexetil didn't influence the amount of cells with micronuclei, whereas candesartan increased the contents of micronucleated cells as compared to the control result. Resveratrol didn't raise the number of test cells in vitro ( Table 2).

Distribution of cells at the stages of the cell cycle
Combination of candesartan cilexetil and resveratrol didn't change the amount of cells with DNA damage. It was found that resveratrol in the combination with candesartan significantly decreased cytotoxic effect of second one. Resveratrol at 50 µg/ml dose was more effective in the complex (Table 2).
We also investigated distribution of cells at stages of cell cycle. It was shown that studied substances didn't change proliferation of cells, as shown in the Table 2.
Thus, the results obtained by our combined use of antiischemic agents with resveratrol in the primary culture of MSC indicate an increase in the content of immobilized CD117 positive cells. Also in recent years, transplanted MSCs have been intensively used in regenerative medicine. [12]. The transplantation of mesenchymal stromal cells (MSCs) has emerged as an effective strategy to protect against tissue and organ injury [13].According to the ISCT criteria, MSCs must have multipotency to differentiate into somatic cells, including osteocytes, adipose cells, and chondrocytes. MSCs with multilineage potential exist prevalently in almost all tissues, and they are promising cell sources for treating multiple diseases without ethical issues [14] Also date some authors demonstrated that various dosages of resveratrol (1, 5, and 10 mg/kg) significantly decreased myocardial lesions by increasing myocardial AKT expression and decreasing caspase-3 activity during carbon monoxide-induced cardiotoxicity in rats in a dose-dependent manner [15].

Conclusions.
It was found that active form candesartan stimulated formation of CD117+ stem cells in vitro, whereas pro-drug candesartan cilexetil decreased the number of studied cells, and both substances were cyto-toxic. It was shown that resveratrol increased the number of endothelial progenitor cells and was safe for cell culture. Combination of candesartan cilexetil and resveratrol didn't change the number of apoptotic cells and cells with micronuclei. Application of resveratrol with candesartan reduced cytotoxic effect of last one. It should be noted that combinations of candesartan cilexetil with resveratrol and candesartan with resveratrol significantly increased the count of CD117+ stem cells. The obtained data was better when resveratrol was used in higher dosages. These results are important to develop new complex drug to stimulate mobilization of endothelium progenitor cells and stimulate reparative processes.