The abnormal proliferation of smooth-muscle cells (SMCs) has received considerable attention. The importance of this issue has further increased because the drug has been implicated in some diseases such as atheroscle-rosis and hypertension (1).
It has been suggested that cyclosporin A (CsA) is able to potentiate graft vascular disease by stimulating SMC proliferation (2). However, both the in vitro and in vivo data in support of this suggestion are discordant, because both stimulatory and inhibitory effects of CsA on vascular SMC proliferation have been reported. Studies in humans have shown that long-term exposure to CsA accelerates SMC proliferation in graft vasculature (3). However, some animal experiments do not seem to corroborate this. Jonasson et al. (4) demonstrated that CsA reduces the number of SMCs and extracellular tissue in rat carotid. Despite this, the authors did not find any effect of CsA on cultured SMCs (3). Ferns et al. (5), by using rabbits as an in vivo experimental model, found no changes in the proliferative capacity of SMCs in the presence of CsA. The in vitro studies of this group showed that CsA has a small effect on inhibiting SMC proliferation. By contrast, the in vitro experiments of Leszczynski et al. (6) pointed to dose- and time-dependent effects of CsA: the drug stimulated the proliferation on day 3, but this effect disappeared by the day 6.
The aim of this work was to study the direct effects of CsA in cultured rat aortic SMCs. After a review of the literature addressing this issue, our attention focused on the concentration of CsA used. In this study, we used concentrations corresponding approximately to maximal plasma (10−6M) and trough (10−7M) values attained in patients treated over the long term with CsA.
Collagenase type IA, from Clostridium histolyticum, L-glutamine, sodium selenite, transferrin, and insulin (human) were purchased from Sigma (St. Louis, MO, U.S.A.). Penicillin was obtained from Laboratorios Level SA (Barcelona, Spain). DMEM, Ham's F-12, Hank's balanced salt solution, trypsin-EDTA solution and fetal calf serum were obtained from Whittaker Laboratories (Barcelona, Spain). [3H]thymidine was purchased from New England Nuclear (Bad Homburg, Germany). Crystal violet was obtained from Fluka (Buchs, Switzerland). Cyclosporin A was a kind gift from Sandoz, Switzerland. All the other reagents were of the highest grade available commercially.
Preparation of cyclosporin A
In previous experiments, when cyclosporin A was dissolved in ethanol, we observed the same effect described by some authors using this solvent: the development of cytoplasmic vacuoles, cell detachment, and morphologic alterations. To avoid these pitfalls, we used dimethyl sulfoxide (DMSO) as solvent; no morphologic alterations were observed in the cells.
Smooth-muscle cell cultures
Rat medial aortic SMCs were prepared from the thoracic aortas of Wistar rats weighing 300 g, by following the method of Alipui et al. (7) modified as follows: animals were killed by pentobarbital overdose, and a segment of the thoracic aorta was excised and cleaned of fat, connective tissue, and blood. The aortic segment was opened lengthwise and incubated at 37°C for 20 min in collagenase type IA solution. The vessel was then washed with Hank's solution and transferred to trypsin solution (1.5 mg/ml). The segment was then minced into 2-mm pieces, which were incubated with collagenase for 15 min. After this incubation, the supernatant was discarded, and the tissue was subjected to three successive 10-min digestions with trypsin (1.25 mg/ml) at 37°C. After each digestion, the supernatant was collected and centrifuged at 2,800 rev/min for 5 min. The SMC-containing pellet was washed in culture medium, resuspended in complete growth medium (DMEM + Ham's F-12, 10% FCS, 1 mM glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, 2.5 μg/ml amphotericin B, 5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml sodium selenite) and kept at 37°C until final plating. The resuspended pellets were pooled, counted, and sown at a density of 5 × 103 cells. The trypan blue dye-exclusion method was used to determine cell viability. The effect of CsA on the viability and growth of SMCs, as well as the effects of the solvents used, was investigated.
Cell proliferation was measured both by [3H]thymidine incorporation into DNA and by measuring viable cell number. For this purpose, cells were subcultured by treatment with 0.05% trypsin and 0.02% EDTA and plated in 6 × 4-well plates (Nunc, Denmark). Experiments were performed on cells from passages four through six.
SMCs were rendered quiescent by removal of their serum-containing growth media. Washed cells were left in the same culture medium for 2 days but only with 0.5% FCS and antibiotics. During this period, the cells incorporated a minimal amount of [3H]methylthymidine, indicating a quiescent state. At this point, cells were reactivated by exposure to the same culture medium and the respective agonists or antagonists. The conditions tested included the exposure of the cells to quiescent medium alone, as a negative control, and to medium containing 10% FCS as a positive control for proliferation. After 24 h of incubation with CsA (10−6 and 10−7M), cells were pulsed for 6 h with 1 μCi/ml of [3H]methylthymidine. Cell layers were washed with ice-cold phosphate-buffered saline and fixed with 1 ml of ice-cold 10% trichloroacetic acid for 15 min; the acid-insoluble material was solubilized with 1 ml of 0.1 M NaOH for 30 min at 60°C. After NaOH exposure, radioactivity was measured in a scintillation counter (Betamatic IV; Kontron, Montigny le Bretonneaux, France).
The number of viable cells in each well was measured by using the calorimetric method based on the staining of cell nuclei (8). In brief, cells subcultured to subconfluence in 24-well plates were incubated for 24 h under the previously described experimental conditions. Incubation periods lasted 6, 24, and 48 h. Then cells were fixed with 1% glutaraldehyde over 10 min and washed twice with Hank's solution. Cell nuclei were dyed by incubating the cells for 30 min in a 1% crystal violet solution. Cells were then washed exhaustively with distilled water and left to dry overnight. Finally, 2 ml of 10% acetic acid was added to each well. The optical density at 595 nm was proportional to the number of viable cells in each well.
Evaluation of cellular lysis
To determine cellular lysis, LDH enzyme activity (lactate dehydrogenase EC 126.96.36.199) was quantified in the cell-culture medium after incubation with CsA.
SMCs subcultured to subconfluence in 24-well plates were incubated for 6, 24, and 48 h under the experimental conditions described. The supernatants of the cultures were collected, and LDH activity was determined by an enzymatic method (Boehringer Mannheim, Barcelona, Spain).
Data are shown as means ± SEM of four to 12 experiments, each carried out in triplicate. Statistically significant differences with respect to basal values were analyzed by one-way analysis of variance and Scheffé's multiple-comparisons test.
The structure of SMCs grown in 0.5 or 10% FCS appeared normal, as judged from their appearance under phase-contrast microscopy. Moreover, incubation with CsA revealed no changes in cellular structure as compared with untreated SMCs. No cell detachment from the plastic surface of the culture dishes was observed (data not shown).
In the presence of 0.5 and 10% FCS medium, both concentrations of CsA increased [3H]thymidine incorporation by SMCs (Fig. 1). With 10% serum medium, this increase was higher for the lowest CsA concentration (10−7M). With 0.5% FCS, [3H]thymidine incorporation into DNA was similar for both concentrations. As measured by the number of viable cells, cell proliferation showed that the increase in [3H]thymidine incorporation induced by 10−6M CsA in 0.5% serum medium did not result in increased cell numbers (Fig. 2). The numbers of cells after 6, 24, and 48 h of incubation with CsA were not statistically different from control values (Fig. 2). However, the increase in [3H]thymidine uptake induced by 10−7M CsA in 0.5% FCS medium was seen to be correlated with a clear induction of SMC proliferation at 48 h of incubation.
Experiments performed with the lowest concentration of CsA in 10% serum medium afforded results similar to those obtained with 0.5% FCS. With 10−6M CsA, no differences were found with respect to control cell number at 6 and 24 h. In contrast, differences were seen at 48 h, although the increase was less than that obtained with 10−7M CsA (Fig. 2). To exclude the possibility that the failure of cell number to increase after treatment with 10−6M CsA might be the result of simultaneous cell proliferation and a toxic effect of the drug, with consequent cell lysis, LDH enzymatic activity was measured in the culture medium. However, no increases in LDH levels were observed with respect to control conditions with either of the CsA concentrations (Fig. 3).
Our results demonstrate that CsA affects rat aortic SMC proliferation in different ways, depending on the concentration used: 10−7M CsA induced DNA synthesis and cell proliferation, whereas 10−6M CsA induced DNA synthesis but not cell proliferation. Previous reports on the effects of CsA on SMC proliferation are contradictory. Such discrepancies can be explained because some authors used very high (10 μg/ml, 10−5M) or very low (0.1 ng/ml, 10−10M) concentrations (5,6). With such extreme concentrations, results pointing to no effect or toxic effects would be expected. Moreover, those concentrations are quite different from the levels reached in the plasma of patients treated over the long term with CsA. Some studies with animal models have attempted to use doses equivalent to the CsA plasma concentration (5). In this study, we used two concentrations: one equivalent to the transitory plasma concentration (the maximal concentration peak) and another that persists in the plasma for a long time and thus has a greater potential to elicit vascular damage (the trough concentration).
It is known that many circulating vasoactive agents modulate vascular SMC growth and that FCS contains many of these substances. Our experiments were therefore performed with smooth muscle grown in a low concentration (0.5%) of FCS with a view to detecting factors that determine cell growth rates rather than only the rate of mitosis (growth-initiating factors; 9). Under these conditions, 10−6M CsA induced an increase in DNA synthesis with no increases in cell numbers, whereas 10−7M CsA elicited an increase in both DNA synthesis and cell numbers. The presence of 10% FCS did not change the type of response of SMC cultures in the presence of CsA. Measurement of LDH activity revealed no statistically significant differences between the control and treated cells, suggesting that the CsA concentrations used were not toxic for the cells.
The dose-dependent increase in [3H]thymidine incorporation, with no direct correlation with cell growth, suggests that CsA interferes in the cell cycle. Thus 10−6M CsA might drive cells from the G0 to G1 phase, but it would prevent them from completing the cell cycle.
However, in the presence of 10% FCS, an increase in DNA synthesis occurs. CsA at 10−7M might induce some, but not all, cells to complete the cell cycle, resulting in cell proliferation. This was suggested by the observation that the increase in [3H]thymidine uptake in the presence of 10% FCS was 3 times higher than that seen with 0.5% FCS. However, the increase in the numbers of cells induced by 10−7M CsA was similar with both 10 and 0.5% FCS, suggesting that the presence of FCS in CsA-induced SMC growth contributes to increasing DNA synthesis but not to potentiating CsA-induced proliferation.
In conclusion, CsA showed a differential effect on SMC proliferation, depending on the concentration used: at both concentrations used (10−6 and 10−7M), the drug increased DNA replication in cultured rat aortic SMCs, whereas only the lower dose (10−7M) induced proliferation in this cellular type.
Acknowledgment: We are grateful to Sandoz, Inc., for their kind collaboration in this project. We thank Nicholas Skinner for his help with the English translation.
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