Effects Of Doxazosin And Its Enantiomers On Cell Proliferation And Apoptosis In VSMCs Of The Rat Aorta And Related Mechanisms

Vascular smooth muscle cells (VSMCs) located in the middle layer of the arterial wall regulate blood pressure by contraction and relaxation in physiological condition. Migration from the media to the intima space and abnormal proliferation of VSMCs are the common pathological bases of atherosclerosis (AS), hypertension and vascular restenosis (RS). Injury of vascular endothelial cells causes production and activation of a variety of inflammatory cytokines and growth regulation factors, which induce a change in VSMCs phenotype, a migration of VSMCs from the media to the intima, and excessive proliferation as well as apoptosis inhibition. Therefore, protecting vascular endothelial cells and inhibiting proliferation and migration of VSMCs might prevent the diseases such as AS, RS and hypertension.(±)Doxazosin, a long-acting selectiveα1-adrenergic receptor antagonist, has been extensively used in the treatment of benign prostatic hyperplasia (BPH) complicated with lower urinary tract symptoms (LUTS). BPH/LUTS patients with hypertension treated with (±)doxazosin received two beneficial therapeutic actions. Recent studies demonstrated that (±)doxazosin markedly inhibited proliferation of the aortic VSMCs, and the inhibitory effects had characteristics of concentration-dependent andα1-receptor independent.In the late 20th century, the study of chiral drug became an important field of the drug development. It was reported that (-)doxazosin and (+)doxazosin were prepared using chiral mobile phase HPLC and high performance capillary electrophoresis. The blocking effect of (-)doxazosin onα1-adrenoceptor was significantly weaker than that of (±)doxazosin and (+)doxazosin in the isolated rabbit thoracic aorta, carotid artery and mesenteric artery. The effect of (-)doxazosin on arterial blood pressure in the anesthetized rats was also significantly weaker than that of (±)doxazosin and (+)doxazosin administered intravenously or intraduodenally. However, the effect of decreasing urinary bladder pressure by (-)doxazosin in the anesthetized rats and guinea-pig was the same as that of (±)doxazosin. These findings suggested that the pharmacological activity of (-)doxazosin had chiral selective effect between the cardiovascular system and lower urinary tract system. However, it remains to be clarified whether (+)doxazosin and (-)doxazosin have chiral selective effect on the cell proliferation and apoptosis of VSMCs and whether their effects are related toα1-adrenoceptor blockade. In the present study, MTT assay was used to determine the cell proliferation; and cell cycle distribution, cell apoptosis and apoptotic proteins (Bcl-2 and Bax) were analyzed using flow cytometry (FCM). The purpose of the study is to observe the effects of (±)doxazosin and its enantiomers on the cell proliferation and apoptosis in cultured VSMCs of the rat aorta and to analyze the related mechanisms.Part 1 Effects of doxazosin and its enantiomers on cell proliferation in VSMCs of the rat aorta and a relationship of the effects withα1-adrenoceptorVSMCs of the rat aorta were cultured, and MTT assay was used to determine the cell proliferation. The effects of (-)doxazosin, (+)doxazosin and (±)doxazosin on the cell proliferation of VSMCs and a relationship of the effects induced by (-)doxazosin and (+)doxazosin withα1-adrenoceptor were investigated.1 Effects of (±)doxazosin and its enantiomers on cell proliferation in VSMCs of the rat aortaTreatment with (-)doxazosin and (±)doxazosin at 10~30μmol·L-1 for 48h or 72h significantly inhibited cell proliferation in VSMCs of the rat aorta, but (+)doxazosin had an inhibitory effect only at 30μmol·L-1. When the VSMCs were incubated with the three agents for 96h, (-)doxazosin, (+) doxazosin and (±)doxazosin at 3~30μmol·L-1 inhibited the cell proliferation significantly. Treatments with (-)doxazosin, (+)doxazosin and (±)doxazosin at used concentrations for 48h, 72h or 96h inhibited cell proliferation of the VSMCs in a concentration-dependent manner. Statistic results with two-way ANOVA showed that the inhibition rate of cell proliferation by (-)doxazosin at 10μmol·L-1 was significantly higher than that by (+)doxazosin in VSMCs exposed to the drugs for 48h or 72h (P <0.05 ), and the inhibition rate of cell proliferation by (-)doxazosin at 3~30μmol·L-1 was significantly higher than that by (+)doxazosin in VSMCs exposed to the drugs for 96h (P<0.01). when the VSMCs were treated with the three agents at 30μmol·L-1 for 48h, 72h and 96h, the inhibition rates of cell proliferation in VSMCs were 27.13%, 38.05% and 58.87% by (-)doxazosin, 24.39%, 33.43% and 46.10% by (+)doxazosin, and 24.29%, 36.74% and 52.35% by (±)doxazosin, respectively. The inhibition rates of cell proliferation in VSMCs incubated with the three agents for 96h were significantly higher than those for 48h (p<0.05 and 0.01) and 72h (p<0.05 and 0.01). When the VSMCs were exposed to (-)doxazosin, (+)doxazosin and (±)doxazosin at different concentrations for 96h, the concentrations required for 40-percent inhibition (IC40) of the cell proliferation were 10.2±1.3μmol·L-1, 20.9±2.2μmol·L-1 and 12.1±2.6μmol·L-1, respectively. The IC40 value of (+)doxazosin was significantly higher than that of (±)doxazosin or (-)doxazosin (P<0.01).2α1-Adrenoceptor-independent effects of (±)doxazosin and its enantiomers on cell proliferation in VSMCs of the rat aortaThe value of cell proliferation in sub-control group (0.001% alcohol) of solventⅡgroup or in sub-control group (phenoxybenzamine) of phenoxybenzamine group was not significantly different from that in sub-control group (distilled water) of solventⅠgroup (P>0.05). In solventⅠgroup, the proliferation of VSMCs was significantly inhibited after 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin at 30μmol·L-1 (P<0.01), and the inhibition rates were 33.00±2.79%, 26.19±5.74% and 31.76±4.29%, respectively. The inhibition rate of (+)doxazosin was significantly lower than that of (-)doxazosin (P<0.05), but was not significantly different from that of (±)doxazosin. In solventⅡgroup and phenoxybenzamine group, the proliferation of VSMCs was significantly inhibited after 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin as well (P<0.01). Although the inhibition rate of VSMCs proliferation by (+)doxazosin was lower than that by (-)doxazosin or (±)doxazosin, there was no statistical differences among the three agents. In comparison with the sub-group [(-)doxazosin] of solventⅠgroup, the inhibition of VSMCs proliferation by (-)doxazosin in solventⅡgroup and in phenoxybenzamine group was not significantly different (P>0.05); and the same results were obtained in the experiments treated with (+)doxazosin and (±)doxazosin.3α1-Adrenoceptor-dependent effects of (±)doxazosin and its enantiomers on cell proliferation in VSMCs of the rat aortaPhenylephrine (10μmol·L-1) was able to stimulate VSMCs proliferation of the rat thoracic aorta, and the extent of cell proliferation promoted by phenylephrine was similar to that by 10% fetal bovine serum contained in DMEM. The VSMCs proliferation stimulated by phenylephrine was significantly inhibited after 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin at 30μmol·L-1 (P<0.01). The inhibition rate of VSMCs proliferation by (+)doxazosin was significantly lower than that by (-)doxazosin (P<0.05), but was to the same extent as (±)doxazosin.Part 2 Effects of doxazosin and its enantiomers on cell cycle and apoptosis in VSMCs of the rat aortaEffects of doxazosin and its enantiomers on cell cycle and apoptosis in cultured VSMCs of the rat thoracic aorta were investigated. Morphological changes in VSMCs were analyzed using Giemsa staining method, and the cell cycle, cell apoptosis and apoptotic proteins (Bcl-2 and Bax) were detected by FCM in order to investigate mechanisms of antiproliferation and induction of apoptosis b (±)doxazosin and its enantiomers in VSMCs of the rat thoracic aorta.1 Morphological changes in VSMCs of the rat aorta induced by (±)doxazosin and its enantiomers Morphological changes in cultured VSMCs of the rat thoracic aorta incubated with (-)doxazosin, (+)doxazosin and (±)doxazosin at 30μmol.L-1 for 96h were seen under an ordinary optical microscope. The overall cell shrinkage, chromatin condensation, nuclear margination toward the nuclear membrane, nuclear shrinkage, and apoptotic bodies of various sizes in the cytoplasm were observed,and the nucleus of alive normal cells uniformly stained blue or violet-blue.2 Effects of (±)doxazosin and its enantiomers on cell cycle in VSMCs of the rat aortaCell cycle distribution and proliferation index (PI) of the rat aortic VSMCs changed significantly after incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin at 25μmol·L-1 for 72h. In the VSMCs treated with (±)doxazosin or (-)doxazosin, the proportion of cells in G0/G1-phase was significantly increased; and the proportions of cells in S-phase and G2/M-phase, and PI were significantly decreased (P<0.01). In the (+)doxazosin group, however, only the proportion of cells in S-phase was decreased significantly (P<0.01). The proportions of cells in G0/G1-phase, S-phase and G2/M-phase, and PI value in the VSMCs treated with (-)doxazosin were significantly different from that treated with (+)doxazosin (P<0.05 and 0.01).3 Effects of (±)doxazosin and its enantiomers on the cell apoptosis in VSMCs of the rat aortaIn the VSMCs incubated with (-)doxazosin, (+)doxazosin and (±)doxazosin at 25μmol·L-1 for 72h, the cell apoptosis was induced by (-)doxazosin and (±)doxazosin (P<0.01), but not by (+)doxazosin (P>0.05). The apoptotic rates of the cultured VSMCs treated with (-)doxazosin and (±)doxazosin were 6.06±0.31% and 3.63±0.99%, respectively. The induction of VSMCs apoptosis induced by (-)doxazosin was significantly stronger than that by (±)doxazosin (P<0.01).4 Effects of (±)doxazosin and its enantiomers on the expression of apoptosis protein Bcl-2 and Bax in VSMCs of the rat aorta After incubation of VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin at 25μmol·L-1 for 72h, the expression of antiapoptotic protein Bcl-2 and proapoptotic protein Bax did not change significantly in comparison with solvent-treated VSMCs (P>0.05). On the other hand, the ratios of Bcl-2 protein/Bax protein in the cultured VSMCs treated with (-)doxazosin, (+)doxazosin and (±)doxazosin were 0.68±0.01, 0.92±0.03 and 0.82±0.10, respectively. The ratios of Bcl-2 protein/Bax protein in the cultured VSMCs were significantly reduced by (-)doxazosin and (±)doxazosin in comparison with the VSMCs treated with solvent (P<0.05 and 0.01), but (+)doxazosin did not significantly affect the ratio of Bcl-2 protein/Bax protein (P>0.05). The ratio of Bcl-2 protein/Bax protein in VSMCs treated by (-)doxazosin was significantly lesser than that by (+)doxazosin (P<0.01).Part 3 Effects of doxazosin and its enantiomers on cell proliferation of the rat aortic VSMCs induced by different stimulatorsEffects of (±)doxazosin and its enantiomers on cell proliferation determined by MTT assay in the rat aortic VSMCs stimulated by platelet-derived growth factor-BB (PDGF-BB), angiotensin II, high concentration glucose and thrombin.1 Effects of (±)doxazosin and its enantiomers cell proliferation of the rat aortic VSMCs induced by PDGF-BBPDGF-BB (1nmol·L-1) induced cell proliferation of the rat aortic VSMCs, and the extent of cell proliferation promoted by PDGF-BB (1nmol·L-1) was significantly stronger than that by 10% fetal bovine serum contained in DMEM (P<0.01). Incubation of the rat aortic VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin (3～30μmol·L-1) for 48h inhibited the cell proliferation stimulated by PDGF-BB significantly (P<0.01). In the culture medium of DMEM containing 10% fetal bovine serum, the maximal inhibition rates of VSMCs proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from 24.29±3.72% to 27.13±2.41%, in the culture medium of DMEM containing 0.4% fetal bovine serum and 1nmol·L-1PDGF-BB, however, the maximal inhibition rates of cell proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from 95.21±6.05% to 99.67±1.99%. Statistic results with two-way ANOVA showed that the inhibition rate of cell proliferation by (+)doxazosin at 3μmol·L-1 was significantly lower than that by (±)doxazosin at the same concentration (P<0.05). A concentration required for 50-percent inhibition (IC50) of the VSMCs proliferation by (-)doxazosin, (+)doxazosin or (±)doxazosin was 5.44±3.16, 7.50±3.40 or 4.98±4.52μmol·L-1.2 Effects of (±)doxazosin and its enantiomers cell proliferation of the rat aortic VSMCs induced by angiotensin IIAngiotensin II (100 nmol·L-1) induced cell proliferation of the rat aortic VSMCs, and the extent of cell proliferation promoted by angiotensin II (100 nmol·L-1) was significantly stronger than that by 10% fetal bovine serum contained in DMEM (P<0.01). Incubation of the rat aortic VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin (3～30μmol·L-1) for 48h inhibited the cell proliferation stimulated by angiotensin II significantly (P<0.01). The inhibition rates of the VSMCs proliferation by (-)doxazosin, (+)doxazosin and (±)doxazosin were increased with the increase in concentrations used (0.3～30μmol·L-1). Statistic results with two-way ANOVA showed that the inhibition rate of VSMCs proliferation by 48h-incubation with (-)doxazosin was not significantly different from that with (+)doxazosin or (±)doxazosin (P>0.05). In the culture medium of DMEM containing 0.4% fetal bovine serum and 100 nmol·L-1 angiotensin II, the maximal inhibition rates of cell proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from 93.01±3.23% to 94.61±3.11%. A concentration required for 50-percent inhibition (IC50) of the VSMCs proliferation by (-)doxazosin, (+)doxazosin or (±)doxazosin was 13.38±2.50, 14.24±2.47 or 13.71±2.89μmol·L-1.3 Effects of (±)doxazosin and its enantiomers cell proliferation of the rat aortic VSMCs induced by thrombinThrombin (1μmol·L-1) induced cell proliferation of the rat aortic VSMCs, and the extent of cell proliferation promoted by thrombin (1μmol·L-1) was similar to that by 10% fetal bovine serum contained in DMEM. Incubation of the rat aortic VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin (3～30μmol·L-1) for 48h inhibited the cell proliferation stimulated by angiotensin II significantly (P<0.01). The inhibition rates of the VSMCs proliferation by (-)doxazosin, (+)doxazosin and (±)doxazosin were increased with the increase in concentrations used (0.3～30μmol·L-1). Statistic results with two-way ANOVA showed that the inhibition rate of VSMCs proliferation by 48h-incubation with (-)doxazosin was not significantly different from that with (+)doxazosin or (±)doxazosin (P>0.05). In the culture medium of DMEM containing 0.4% fetal bovine serum and 1μmol·L-1 thrombin, the maximal inhibition rates of cell proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from from 91.87±5.13% to 94.63±3.33%. A concentration required for 50-percent inhibition (IC50) of the VSMCs proliferation by (-)doxazosin, (+)doxazosin or (±)doxazosin was 13.52±2.87, 14.60±2.95 or 14.32±2.75μmol·L-1.4 Effects of (±)doxazosin and its enantiomers cell proliferation of the rat aortic VSMCs induced by a high concentration of glucoseA high concentration of glucose (25.6mmol·L-1) induced cell proliferation of the rat aortic VSMCs, and the extent of cell proliferation promoted by the high concentration of glucose (25.6mmol·L-1) was similar to that by 1μmol·L-1 thrombin contained in DMEM. Incubation of the rat aortic VSMCs with (-)doxazosin, (+)doxazosin and (±)doxazosin (3～30μmol·L-1) for 48h inhibited the cell proliferation stimulated by the high concentration of glucose significantly (P<0.01). Statistic results with two-way ANOVA showed that the inhibition rate of VSMCs proliferation by 48h-incubation with 3μmol·L-1 (-)doxazosin was significantly higher than that with (+)doxazosin at the same concentration (P<0.05). In the culture medium of DMEM containing 0.4% fetal bovine serum and 25.6mmol·L-1 glucose, the maximal inhibition rates of cell proliferation by 48h-incubation with (-)doxazosin, (+)doxazosin and (±)doxazosin were from 94.17±4.75% to 96.16±3.09%. A concentration required for 50-percent inhibition (IC50) of the VSMCs proliferation by (-)doxazosin, (+)doxazosin or (±)doxazosin was 14.20±6.34, 14.10±3.29 or 13.98±3.53μmol·L-1.Conclusion1 (-)Doxazosin, (+)doxazosin and (±)doxazosin inhibit the rat aortic VSMCs proliferation induced by 10% fetal bovine serum, and the extent of inhibition of cell proliferation by (-)doxazosin is stronger than that by (+)doxazosin, suggesting that (±)doxazosin and its enantiomers have chirally selective effect on cell proliferation in the used experimental condition.2Αnα1-adrenoceptor-dependent mechanism is involved in the anti-proliferation of the rat aortic VSMCs by (±)doxazosin and its enantiomers besides a knownα1-adrenoceptor-independent mechanism.3 (-)Doxazosin and (±)doxazosin are able to inhibit cell proliferation by a cell-cycle arrest in G0/G1 phase and a decrease in PI. The effect on cell cycle by (-)doxazosin is significantly stronger than that by of (+)doxazosin.4 (-)Doxazosin and (±)doxazosin are able to induce cell apoptosis in the rat aortic VSMCs, and the proapoptotic effects are partially related to a reduced ratio of Bcl-2 protein/Bax protein, indicating that (-)doxazosin might be the main component of (±)doxazosin to induce cell apoptosis in the rat aortic VSMCs.5 (±)Doxazosin and its enantiomers significantly inhibit cell proliferation in the rat aortic VSMCs stimulated by PDGF-BB, angiotensin II, thrombin and high concentration of glucose without chirally selective effects. The inhibition extent of cell proliferation by (-)doxazosin is much stronger in the VSMCs stimulated with PDGF-BB, angiotensin II, thrombin or high concentration of glucose than that in the VSMCs stimulated with fetal bovine serum or phenylephrine.