The response of the vascular wall to mechanical injury resulting in the formation of an occlusive intimal lesion is the major complication of invasive vascular procedures, including percutaneous transluminal coronary angioplasty (1-4) and coronary bypass grafting (5,6). Smooth muscle cell (SMC) proliferation and migration are characteristic of the neointimal lesion formed after mechanical injury to the vessel wall (7,8). Reduction of SMC proliferation and migration may reduce the restenotic lesion after the vascular injury. Despite a variety of attempts with pharmacologic interventions and instrument techniques, restenosis after mechanical injury to the vessel wall remains a problem (9).
Minoxidil (2,4-diamino-6-piperidinopyrimidine 3-oxide, I) is a potent vasodilator and therefore has been used to reduce the blood pressure. The antihypertensive activity of minoxidil results from a decrease in peripheral resistance, due primarily to direct relaxation of SMCs (10-12). In some in vitro studies, minoxidil suppressed keratinocyte growth and human lymphocyte activities (13,14). An antiproliferative effect of minoxidil also has been observed in SMCs from chick aorta (15). However, the effect of minoxidil on SMC migration and its significance in inhibition of vascular lesions was poorly understood. Our studies examined the effects of minoxidil on SMC migration and proliferation in vitro and on neointimal formation in vivo after vascular injury.
MATERIALS AND METHODS
Minoxidil (U-10858) was generously provided by Pharmacia-Upjohn, Inc. (Kalamazoo, MI, U.S.A.). A stock solution containing 1% minoxidil for tissue culture was prepared by dissolving minoxidil in 20% ethanol. For in vivo studies, minoxidil was dissolved in tap water in the concentrations of 120 mg/L and 200 mg/L plus 250 mg/L of sodium hydrochlorothiazide, respectively (Sigma, St. Louis, MO, U.S.A.). Platelet-derived growth factor-BB (PDGF-BB) was purchased from R&D Systems (Minneapolis, MN, U.S.A.).
Male Wistar rats, aged 6 months, were killed by an overdose of sodium pentobarbital, and aortae, including thoracic and abdominal portions, were removed under sterile conditions. After rinsing several times in antibiotic-containing Hank's balanced salt solution (Gibco, Grand Island, NY, U.S.A.), the vessel was opened longitudinally, and the adventitia and intima were carefully removed. SMC cultures were established using the technique described previously (16). In brief, tissues were cut into small pieces and placed into 60-mm petri dishes with Dulbecco's Modified Eagle's Medium (DMEM; Gibco) containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml of penicillin, 100 μg/ml of streptomycin, 20 mM L-glutamine, 1 mM nonessential amino acids, 0.1 μg/ml amphotericin B, and 0.08 μg/ml sodium deoxycholate. Cells were detached by incubation with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution and reseeded for subcultures. The explants, and subsequently cells, were cultured in an incubator at 37°C, in 5% carbon dioxide and 95% air. Cells were fed twice a week with fresh culture medium. Third and fourth passage SMCs, verified by positive staining of α-smooth muscle actin, were used in this study.
SMCs were seeded at a density of 1.5 × 106 and fed with DMEM containing 10% FBS followed by serum-starvation for 48 h. The culture medium was replaced by medium containing 10% FBS and 0.24, 0.48, 0.96, 1.91, and 3.82 mM minoxidil for 18 h. The medium containing vehicle alone was used in the control group. Cells were detached by incubation with 0.25% trypsin, washed with PBS containing 1% bovine serum albumin, and fixed with 100% ethanol at 4°C for 1 h. After washing with PBS containing 1% bovine serum albumin, cells were treated with DAPI solution. The distribution of SMCs in different phases of the cell cycle was analyzed according to their DNA content determined by a flow cytometer. The distribution of cells in the cell cycle was calculated as the average of that from three different preparations of SMCs from a pooled cell line.
A modified Boyden chamber assay, as described elsewhere (17,18), was used for evaluation of SMC migration. In brief, PVPF filters with 8-mm pores (Nucleopore filters, Cambridge, MA, U.S.A.) were coated with 50 μl collagen I (100 μg/ml). The coated filter was allowed to dry overnight at room temperature. DMEM containing 100 ng/ml of PDGF-BB, as a chemoattractant, was loaded into the lower chamber. The coated filter then was placed into the Boyden chamber to separate the upper from the lower chamber. Serum-starved SMCs suspended in the media containing 0.01, 0.05, and 1.25 mg/ml of minoxidil were loaded into the upper chamber (final volume of 800 μl of cell suspension containing 2 × 105 cells). SMCs suspended in DMEM containing vehicle alone were used as the control. The chambers were placed in an incubator for 4 h at 37°C in 5% carbon dioxide and 95% air. The filters were removed from the chambers and fixed by HEMA fixative solution (Curtin Matheson Scientific Inc., Houston, TX, U.S.A.). Hematoxylin and eosin solutions (HEMA) were used to stain the cells that had migrated toward the chemoattractant and were attached to the lower side of the filter. From each filter, the cell number in eight adjacent fields was counted under a light microscope (×100), and the average was taken to represent the number of migrated cells. Five filters were counted from each group. The migration assay was repeated 3 times with different SMC preparations from the pooled cell lines.
Minoxidil treatment and vascular injury
Twenty-eight male, 4-month-old Wistar rats (300 g) were randomly divided into three groups. A group of rats (n = 8) received minoxidil, 120 mg/L in their drinking water. Another group of rats (n = 10) received drinking water containing 200 mg/L minoxidil. In both concentrations, 250 mg/L of sodium hydrochlorothiazide was added to minimize sodium and water retention, side effects of minoxidil (19). A group of 10 rats, given tap water without minoxidil, served as controls. In a separate group of rats (n = 5), the carotid arteries were injured, and minoxidil (200 plus 250 mg/L sodium hydrochlorothiazide) was given in the drinking water from days 1 to 14 after vascular injury. All animals were fed with normal rat chow and tap water or minoxidil-containing water ad libitum for 3 weeks before vascular injury. The animal care complied with the "Principles of Laboratory Animal Care (formulated by the National Society for Medical Research) and the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, revised 1985).
Vascular injury was effected by the technique described by Clowes et al. (20). In brief, rats were anesthetized by intraperitoneal injection with 40 mg/kg of sodium pentobarbital and 8 mg/kg of xylazine. The left carotid artery was surgically separated, and a small incision was made on the external carotid artery with ophthalmologic scissors. A 2F Fogarty arterial embolectomy balloon catheter (Baxter, McGraw Park, IL, U.S.A.) was inserted through the incision in the external carotid artery into the common carotid artery. The catheter was inflated with 30 μl of saline and withdrawn along the common carotid artery. The effective passing distance of the balloon catheter was 2.5-3.0 cm (i.e., the entire length of the common carotid artery). The ballooning procedure was repeated 3 times to ensure a complete endothelial denudation. The external carotid artery was ligated, and the animals were allowed to recover. Minoxidil-treated groups continued to receive minoxidil in their drinking water for another 2 weeks after surgery.
Animals were killed by an overdose of sodium pentobarbital, and 4% neutralized formalin was infused into the circulation for in situ fixation. The infusion pressure was maintained constantly at 100 mm Hg. Morphometric evaluation was carried out with a computerized imaging analysis system equipped with an Axioplan microscope (Zeiss, Germany) and a black-and-white MTI video camera (Dage-MTI Inc.). Video images were enhanced by normalization and dynamic thresholding and analyzed by IBAS 2.0 software (IBAS, Kontron Elektronik).
For determination of neointimal area, images were acquired from Movat pentachrome-stained slides under ×100 magnification. The neointimal area was defined as that between the internal elastic lamina and the lumen surface. Five images were obtained from different segments harvested from each injured artery, and the average was taken to represent the neointimal area of the vessel. The average neointimal area was calculated from each group and compared statistically among the groups. All the morphometric measurements were carried out by individuals blinded as to the drug treatment each rat received.
Data are presented as mean ± standard error of the mean. Distribution of SMCs in the cell cycle was analyzed by one-way analysis of variance (ANOVA). All other data were statistically analyzed by one-way ANOVA followed by Tukey's Honest Significant Difference (HSD) test for multiple comparisons. Significance was considered only when the p value was <0.05.
Minoxidil inhibits SMC proliferation and migration
SMCs were allowed to reach a quiescent stage by incubation in a serum-starved state for 48 h. The culture media was then switched to DMEM containing 10% fetal bovine serum. SMCs in the control group started to grow and nearly reach confluence in the culture dish (Fig. 1A). However, the density of SMCs cultured with minoxidil-containing media was greatly reduced. The reduction of SMC density caused by minoxidil was concentration dependent (Fig. 1 B-F). No evidence of toxicity was observed after treatment with any the concentrations of minoxidil.
Minoxidil altered the distribution of SMCs in various phases of the cell cycle. In the absence of minoxidil, an average of 64.13% SMCs distributed in the G1 phase, 12.87% in the G2 phase, and 23% in the S phase (Fig. 2). In a concentration-related manner, minoxidil increased the number of SMCs in G1 and G2 phases and decreased the number of SMCs in S phase (Fig. 2). Low concentrations of minoxidil (0.24 mM) did not affect the cell-cycle distribution of the SMCs, whereas 3.82 mM minoxidil significantly increased the number of SMCs that remained in G1 phase and reduced the number of SMCs in S phase.
An inhibitory effect of minoxidil on SMC migration was observed in the modified Boyden chamber assay. Minoxidil-induced inhibition of SMC migration was dose dependent (Fig. 3): the number of SMCs that migrated was reduced 13.5% by 0.01 mg/ml of minoxidil (p < 0.05); 16.8% by 0.05 mg/ml (p < 0.01); 40.47% by 0.25 mg/ml (p < 0.001); and 65.87% by 1.25 mg/ml (p < 0.001) of minoxidil added to the medium.
Minoxidil reduced injury-induced neointimal lesions
Injury-induced neointimal formation is initiated from the penetration of blood-borne growth factors through the denuded intima into the media. Stimulated by these growth factors, the medial SMCs enter the cell cycle 1-3 days after the vascular injury (21). Although the SMCs continue to proliferate above basal levels for up to months, the peak of SMC proliferation occurs between 10 and 14 days, resulting in neointimal mass formation (22). In our study, animals received minoxidil in their drinking water. In both minoxidil-treated groups, the daily water consumption was 40-60 ml per rat. Therefore, the amount of minoxidil received by each rat was 16-24 mg/kg/day (120 mg/L) and 26-40 mg/kg/day (200 mg/L), respectively.
The vessels from both minoxidil-treated and control groups were harvested 14 days after injury (Fig. 4). In the carotid artery of control rats, an extensive neointimal lesion had formed by this time. The area of neointimal lesion was significantly reduced by both 120 and 200 mg/L of minoxidil treatment (Fig. 5). Treatment with 120 or 200 mg/L of minoxidil reduced the neointimal lesion by 37% (p < 0.01) and 44% (p < 0.001), respectively (Fig. 6). In the group of rats that received minoxidil treatment from days 1-14 after vascular injury, the area of neointima was not different compared with the control group (data not shown).
SMC proliferation and migration are the major factors that contribute to neointimal lesion formation in fibroproliferative vascular diseases, such as restenosis and atherosclerosis (7,8). Strategies to suppress SMC proliferation and migration therefore facilitate the reduction of vascular lesions. Our study demonstrates, for the first time, that minoxidil is an agent that can effectively reduce SMC migration and division. Evidence for this was observed both in vitro (i.e., the inhibitory effect of minoxidil on SMC proliferation and migration), as well as in vivo on the formation of neointimal lesion after mechanical injury to the vessel wall.
The mechanism by which minoxidil inhibits SMC proliferation and migration remains unclear. However, it has been found that minoxidil stimulates elastin synthesis in chick embryonic vascular SMCs (15) and also enhances the mRNA of lysyl oxidase, an extracellular enzyme that contributes to cross-linking of collagen and elastin in the extracellular matrix (23-25). Resultant changes in the extracellular matrix effected by minoxidil may inhibit or limit SMC proliferation and migration. Minoxidil is also an SMC relaxant that causes vasodilation. It acts as a K+ channel agonist to enhance K+ permeability, which results in decrease of cytoplasmic free Ca2+ concentration (26,27). A recent study has shown that intracellular Ca2+ concentration is a key factor in the ability of SMCs to migrate across a type I collagen membrane toward PDGF-BB (28). The inhibitory effect of minoxidil on SMC migration observed in our study may be due, at least partly, to a reduction in intracellular Ca2+ concentration. Furthermore, because minoxidil was found as a potential immunosuppressive agent (14), immunosuppressive activity of minoxidil might also contribute to the inhibition of SMC proliferation and migration, as well as neointimal formation after vascular injury.
Administration of minoxidil in drinking water has been previously used in long-term (10 weeks) treatment of high blood pressure in the spontaneously hypertensive rats (29). In our study, ≤200 mg/L of minoxidil was given to rats in drinking water for 5 weeks, and the maximal amount of minoxidil was 26-40 mg/kg/day. No evidence of toxicity was observed in the rats treated with minoxidil. It has been reported that rats safely received 30 mg/kg of minoxidil for a year (30). In addition, it has been noticed that minoxidil may induce sodium and fluid retention, as a side effect (31,32). The degree of sodium and water retention correlates with dose and time of administration of minoxidil, and also is related to the degree of impairment of renal function (19). To avoid such side effects, in our study and others (12,28), sodium hydrochlorothiazide, a diuretic agent, has been given along with the administration of minoxidil. Tsoporis et al. (33) have shown that minoxidil alone increased the medial area of the superior mesenteric artery in normotensive rats, but this increase was prevented by the combination of hydrochlorothiazide and minoxidil. This result suggests that the use of hydrochlorothiazide has been necessary to nullify minoxidil-induced abnormal alterations in the vessel wall. Future experiments may be required to determine the role of hydrochlorothiazide in SMC proliferation and migration, as well as whether the diuretic alone affects neointimal formation after balloon injury.
Minoxidil induces directly relaxation of smooth muscle; therefore it reduces vascular tone and lowers blood pressure (10-12). In this study, the blood pressure was not measured. However, any significant hemodynamic changes caused by minoxidil might have contributed to the diminished neointimal proliferative response, and thus might also be a mechanism of minoxidil-induced decrease in neointimal formation observed in our study.
Minoxidil treatment from days 1-14 after injury did not inhibit the neointimal formation. The major reason was probably that, in this regimen, minoxidil did not achieve concentration within the vessel wall that could effectively inhibit SMC proliferation and migration; thus pretreatment with minoxidil was used.
In summary, the presence of minoxidil in culture medium inhibits the proliferation and migration of vascular SMCs. Minoxidil also reduces the area of neointimal lesion formed after mechanical injury to the vessel wall. The antiproliferative and migratory effects of minoxidil suggest a new application for this agent (i.e., to reduce the extent of neointimal lesion formation in vascular diseases, especially restenosis after angioplasty).
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