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Effects of Cerivastatin on Human Arterial Smooth Muscle Cell Proliferation and Migration in Transfilter Cocultures

Axel, Dorothea I.; Riessen, Reimer; Runge, Heike; Viebahn, Richard*; Karsch, Karl R.

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Journal of Cardiovascular Pharmacology: April 2000 - Volume 35 - Issue 4 - p 619-629
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Abstract

Recently a variety of clinical studies have shown that HMG-CoA reductase inhibitors (statins) reduce the risk of cardiovascular events and coronary mortality (1,2), slow the progression, and even lead to a regression of coronary atherosclerosis (3). There is compelling evidence that the beneficial effects of statins are not only due to their lipid-lowering properties but also to the influence on several important cellular functions (4,5). The substrate of the HMG-CoA reductase, mevalonic acid, is a common product for both cholesterol biosynthesis and for numerous isoprenoids, which are precursors of a variety of factors interfering with cell growth (6,7). Previous evidence showed that isoprenylation of the ras oncogene-encoded protein (p21ras) (8) and G-proteins is an important mechanism that allows anchorage of these proteins to cell membranes (9). Because the p21ras protein has a regulatory role in the cell cycle (10,11), inhibition of isoprenylation by statins might be an interesting approach in the prevention of uncontrolled cell growth, such as smooth muscle cell proliferation in atherosclerosis and restenosis (12).

In vitro studies with cells from different tissues (13,14) demonstrated previously that predominantly highly lipophilic statins, such simvastatin or lovastatin, exert significant growth-inhibitory effects on cells in monocultures of both animal (15) and human origin (16,17). Besides these classic statins that demonstrated a potent lipid-lowering effect in numerous in vivo studies (18,19), an increasing number of new synthetic HMG-CoA reductase inhibitors have been available during the last 2 years. The "second generation" compound, atorvastatin (20,21), and predominantly the recently developed "third generation" product, cerivastatin (22), were shown to exert inhibitory effects on myocyte growth in monocultures at very low concentrations (17,23), which are nearly equivalent to serum levels found after oral administration in humans (24,25). However, nearly all in vitro studies were performed in monocultures of one single, isolated cell type, which was treated directly with increasing statin concentrations. The results of these monoculture experiments are of rather limited value because a variety of studies have already shown that the growth characteristics of vascular cells, especially of smooth muscle cells, is influenced significantly by adjacent cell types, such as endothelial cells or blood cells [e.g., by the release of growth factors or cytokines (26,27)]. Thus the characteristic complex morphology of the arterial vessel wall is not represented by mass cultures of one isolated cell type, and therefore results of these in vitro assays cannot be transferred directly to the in vivo situation (28). To overcome the obvious limitations of monocultured cells, we developed the transfilter coculture system in which human arterial smooth muscle cells (haSMCs) are coincubated with human arterial endothelial cells (haECs) (29,30) and in which test compounds were added exclusively to the endothelial side to imitate the in vivo application of drugs from the blood side of the vessel wall (31).

This study was designed to examine the third-generation compound cerivastatin in comparison with other classic statins in its effects on haSMC proliferation, migration, and the formation of fibromuscular proliferates.

METHODS

Cell separation and culture

Specimens of iliac arteries from human donors, which were discarded after liver transplantations, were used to isolate haSMCs and haECs, as described previously (32). After mechanical removal of haECs, subcultivation was performed in collagen-coated plastic flasks (collagen I from rat tail; Sigma, Deisenhofen, Germany) using the EGM-2 kit without heparin (Cell Systems, St. Katharinen, Germany). HaSMCs were obtained by the explant technique. Subcultivation of haSMCs was performed in plastic culture dishes with Waymouth's MB 752/1 and Nutrient Mixture Ham's F12 (1 + 1), supplemented with 10% fetal calf serum (FCS; PAA Laboratories, Cölbe, Germany) and 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco BRL).

Smooth muscle cell origin was proven immunocytochemically using specific antibodies against α-smooth muscle actin (Progen, Heidelberg, Germany) and by microscopic observation of the characteristic morphology and the "hill and valley" growth pattern. HaECs were identified by immunocytochemical staining against the von Willebrand factor (Boehringer Mannheim, Germany) and their characteristic "cobblestone" growth pattern with contact inhibition between cells. Both cell types were isolated from the same vessel specimen of the same donor to avoid immune reactions and used in the first three passages. Routine staining with the DNA dye DAPI (4′,6-diamidine-2-phenylindole-dihydrochloride; Boehringer Mannheim) was used to exclude mycoplasm contaminations.

Test compounds

Lovastatin and simvastatin were generously supplied by Merck, Sharp & Dohme (Munich, Germany). The inactive lactone forms were transformed into the active hydroxy acid forms by acidification, as described previously (33). Atorvastatin was a generous gift from Gödecke/Parke Davies (Freiburg, Germany) and dissolved in 100% absolute ethanol (Merck, Darmstadt, Germany). Cerivastatin was kindly supplied by Bayer (Leverkusen, Germany) and dissolved in sterile water. Mevalonic acid (Sigma, Deisenhofen, Germany) also was converted from the inactive lactone form to the active hydroxy acid form, as described earlier for statins. Native low-density lipoprotein (LDL) was obtained from hypercholesteremic patients undergoing LDL apheresis. The isolation method was based on an affinity chromatography with absorption columns. Venous blood was passed through the LDL apheresis system MA-01 (Kanegafuchi, Osaka, Japan), and cellular components were separated by a plasma filter (Sulfux-FS-05). The plasma runs over two absorption columns (Liposorber LA15) containing dextransulfate-cellulose beads. This method allowed a high selective binding of apolipoprotein B100, the main component of LDL. Isolated LDL was immediately used after the elution process. Oxidation of LDL was prevented by air and light protection. Nevertheless, LDL oxidation was measured by spectrophotometric determination of the degradation product malondialdehyde using the thiobarbituric acid test, according to the method of Mihara and Uchiyama (34). We found that no significant oxidation occurred during storage at 4°C for 1 week.

It was supplemented to culture media to a final concentration of 200 μg/ml to exclude LDL-deficiency effects. Previous studies showed that this concentration exerted no cytotoxic effects on haSMC and haEC growth (32). The EGM-2 medium used for standard endothelial cell culture was supplemented by 2% FCS containing 7.2 μg/ml cholesterol, whereas smooth muscle cell medium (Waymouth's and Ham's F12) was supplemented with 10% FCS containing 36 μg/ml cholesterol.

Stock solutions of simvastatin, lovastatin, and atorvastatin were diluted in culture medium in order to achieve final test concentrations between 0.1 and 50.0 μM. Cerivastatin was diluted to 10- to 100-fold lower concentrations between 0.001 and 5.0 μM. Reversal studies were performed by simultaneous supplementation of statin-treated cultures with mevalonic acid in different concentrations (50-1,000 μM). Each test consisted of six measurements for each concentration and each test compound.

Culture systems and determination of cell growth

Monocultures. To study cell proliferation with or without addition of statins, haSMCs or haECs were seeded onto microtiter plates at a density of 5 × 103 cells/cm2. After cell attachment (24 h), culture media were renewed, and test compounds were added to FCS-containing standard culture medium. After 3 days, two assays were performed: (a) MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) test, which measures the mitochondrial activity indicating cell viability and cell proliferation in a cultivation period of 3 days (35); and (b) BrdU-ELISA, which determines mitotic activity of cells (36) at a defined time point (after 3 days of cultivation) 18 h after labeling of cells with the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU; Colorimetric Cell Proliferation ELISA, Boehringer Mannheim). In each assay, controls using the highest dose of the vehicle ethanol were performed, added under the same cultivation conditions. Ethanol in concentrations ≤1% (dilution of 1:100, which corresponds to the ethanol concentration of the highest atorvastatin test dose of 50 μM) did not show any independent effect on haSMC or haEC proliferation, migration, or viability.

Pretests in which cells were deprived of serum for 3 days before the addition of 10% FCS showed comparable effects of all statins as in FCS-containing cultures. Thus the data obtained in serum-deprived cells are not shown. All results shown here were performed in the presence of FCS, which was continuously added during the entire cultivation period. The presence of serum should resemble "normal" in vivo conditions (allowing a better transfer to the patient) containing growth factors, lipoproteins, and small amounts of cholesterol to avoid stimulation of cellular cholesterol synthesis under serum-deprived conditions (23), as shown by Brown and Goldstein (6).

Transfilter cocultures. Transfilter cocultures were prepared according to techniques previously described (30,32). In brief, Nuclepore polycarbonate filters (PC MB 50 mm, pore size: 5 μm; Costar Scientific Corp., Bodenheim, Germany) were coated with collagen I (from rat tail; Sigma Chemicals Inc.) and inserted between two sterile polycarbonate frames. Then haECs were seeded on the "lower" filter side, and after 24 h, haSMCs were seeded on the "upper" filter side (2.5 × 104 cells/cm2). After a further 24 h, prediluted test compounds were added to the endothelial side. LDL was supplemented simultaneously to the endothelial side in a concentration of 200 μg/ml, according to results obtained in previous studies (32). At this time, both cell types were in the proliferative growth stage (log-phase) in which haECs stimulate haSMC proliferation and migration (27). Culture media in both compartments contained 10% FCS during the entire cultivation period to ensure a sufficient supply with nutrients, growth factors, and so on, and to avoid haSMC migration along a serum gradient to the other compartment. Media were changed every third day, and test compounds were renewed simultaneously. After 14 days' cocultivation time, cell numbers were determined on each filter side and related to control cultures treated with the corresponding vehicles. Each concentration of the four statins was tested in a total of three cocultures.

Determination of cytotoxicity

To define cytotoxicity of statins after different cultivation periods, a lactate dehydrogenase (LDH) assay (Boehringer Mannheim, Germany) (37) combined with an MTT test (see earlier) was carried out simultaneously. HaSMCs were seeded onto microtiter plates in a density of 15,000 cells/well for 24 h and cultured for a further 1, 2, or 3 days with standard culture medium containing 2% FCS and increasing statin concentrations. For LDH assays, cell-culture supernatants were collected cell free, whereas MTT tests were performed on the remaining attached cells. The low serum concentration of 2% was chosen because higher serum levels were found to exert extraordinarily high cell-independent, inherent LDH activity.

Statistics

All values are expressed as mean ± SEM (unless stated otherwise), whether in numbers or charted. Differences between groups were assessed by unpaired t test (two groups) or one-way analysis of variance (ANOVA; more than two groups) (38). Subsequent multiple comparisons for three or more groups were performed only if one-way ANOVA reached statistical significance (p < 0.05), using the Student-Newman-Keuls test to compare all pairs or Dunnett's post hoc test to compare each group against controls. All data satisfied the requirements of normally distributed samples of equal number between groups. Statistical calculations were carried out with GraphPad Prism 3.00 for Windows [GraphPad Software, San Diego, CA, U.S.A. (www.graphpad.com)]. The difference between means was considered statistically significant at p < 0.05 and highly significant at p < 0.01.

RESULTS

Effect of statins on monocultures of haSMCs and haECs

First the effects of the four different statins were investigated in haSMC and haEC monocultures in regard to their influence on DNA synthesis measured by the BrdU-ELISA and in regard to the mitochondrial activity determined by the MTT test.

Growth inhibition after treatment with cerivastatin. The newest HMG-CoA reductase inhibitor cerivastatin emerged as the strongest growth inhibitor among all statins, which was proven by both BrdU-ELISA (Fig. 1A) and MTT tests (Fig. 1B). The IC50 of haSMCs was determined at very low levels: at 0.037 μM in BrdU-ELISAs and 0.26 μM in MTT tests (Table 1). Furthermore, haEC mitosis was inhibited in similar concentration ranges as in haSMCs with an IC50 at 0.068 μM in BrdU-ELISA (Fig. 1A), whereas MTT tests showed that haECs reacted much more sensitively to cerivastatin treatment in regard to their mitochondrial activity, yielding an average IC50 at 0.019 μM(Fig. 1B). Light-microscopic observations of the morphology of haSMCs treated with hydrophobic statins (cerivastatin; Fig. 2B) in comparison with control cultures (Fig. 2A) demonstrated cell rounding and floating of cells at doses >1 μM for cerivastatin and >10 μM for the other statins.

FIG. 1
FIG. 1:
The effects of increasing concentrations of the new lipophilic compound cerivastatin on human arterial smooth muscle cells (haSMCs) and human arterial endothelial cells (haECs) growth. A: Results of the 5-bromo-2′-deoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA) after 3 days' treatment with cerivastatin compared with controls, indicating DNA synthesis activity of cells. B: Results of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test after 3 days' treatment with cerivastatin compared with controls, which indicate indirectly the extent of cell proliferation and cell viability. Even the inhibitory effects of the highest dose could be abolished totally (5.0 + mev.) by supplementation of mevalonate (*p < 0.05, **p < 0.01 vs. controls).
TABLE 1
TABLE 1:
Comparison of the IC50 of different statins in monocultures of haECs and haSMCs: BrdU ELISA and MTT test after 3 days
FIG. 2
FIG. 2:
The effect of nonstop incubation with 5.0 μM cerivastatin on human arterial smooth muscle cells (haSMCs) morphology after 3 days' treatment. Phase-contrast reflection micrographs. A: Control culture with elongated haSMCs forming a confluent monolayer with the typical "hill and valley" growth pattern. B: After high-dose cerivastatin treatment, cell numbers were reduced significantly. Some haSMCs showed a round cell shape and were floating from the culture dish. Scale bars represent 20 μm.

Growth inhibition after treatment with simvastatin, lovastatin, and atorvastatin. Simvastatin, lovastatin, and atorvastatin also showed a dose-dependent inhibition of haSMC growth after 3 days cultivation time, but in 10- to 30-fold higher doses than cerivastatin. The IC50 in BrdU-ELISAs was calculated at 0.38, 0.36, and 0.57 μM, respectively, and in MTT tests at 5.0, 6.86, and 7.50 μM, respectively (Table 1). Nevertheless, haSMC viability was first affected by 10-fold higher statin doses than cell mitosis. All three statins yielded maximal growth-inhibitory effects (Cmax) on haSMC proliferation in doses between 5.0 and 50.0 μM. The Cmax was calculated at 10.0 and 20.0 μM in BrdU-ELISA and MTT tests, respectively (39).

In addition, haEC growth also was dose-dependently inhibited by the three statins after 3 days of cultivation time with the calculated IC50 in BrdU-ELISAs of 0.17, 0.28, and 0.33 μM, respectively. Viability studies with the MTT reagent showed that haECs reacted more sensitively to statin treatment than did haSMCs, with the IC50 determined at 0.28, 0.38, and 0.55 μM, respectively (Table 1). The Cmax for the three statins was determined at four- to 10-fold lower concentrations than described for haSMCs (1.0 μM in BrdU-ELISAs and 5.0 μM in MTT tests) (39).

Cytotoxic effects of hydrophobic statins. Time-matched cytotoxicity tests were performed after 1, 2, and 3 days of statin treatment. After periods >3 days, LDH assays could not be interpreted correctly, because cytotoxicity increases as a result of serum depletion. In addition, MTT tests were performed in the presence of 10% FCS up to 7 days. The LDH assay results showed that LDH liberation of haSMCs was dose and time dependent after treatment with the various statins. The highest LDH levels were found after 3 days of treatment. However, compared with the simultaneously performed MTT test, an increase of cytotoxicity was not induced as early as the decrease of mitochondrial activity. MTT tests showed that cytotoxicity did not increase any more after 7 days of statin treatment. Figure 3 shows some representative results after treatment of haSMCs for 3 days. Whereas the mitochondrial activity was reduced >40% after treatment with 0.01 μM cerivastatin, no significant increase of LDH release was observed at this concentration. The MTT results of haSMCs cultured with only 2% FCS showed also that serum-depleted haSMCs reacted ∼20% more sensitively to statin treatment than in the presence of 10% FCS.

FIG. 3
FIG. 3:
The effects of increasing cerivastatin concentrations on cell viability and lactate dehydrogenase (LDH) liberation. A: Results of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test after 3 days' cultivation with serum-reduced culture medium (2% fetal calf serum) showing cell viability in comparison to placebo controls. B: Results of the simultaneously performed LDH assay after 3 days' cultivation time, showing percentage cytotoxicity versus controls and totally lysed cells with Triton X-100. (*p < 0.05, **p < 0.01 vs. controls).

Effects of statins on haSMCs in transfilter cocultures with haECs

Cocultivation of haSMCs with proliferating haECs in transfilter cultures resulted in a significant stimulation of haSMC migration from the upper to the lower filter side when compared with transfilter cultures lacking haECs (Fig. 4A: second pair of bars, 73.2 ± 6.9% vs. first pair of bars, 37.5 ± 0.4%; p < 0.01). An additional but not statistically significant stimulation also was induced by LDL (Fig. 4A: third pair of bars vs. second pair of bars: 100.0 ± 12.1% vs. 73.2 ± 6.9%, respectively; p = NS). Previous studies measuring LDL cholesterol in conditioned media by the Cholesterol Monotest (Boehringer, Mannheim) have shown that LDL cholesterol diffuses to the opposite compartment. Furthermore, lipid stainings showed that cells on both filter sides were filled with high amounts of lipids in their cytoplasm (32).

FIG. 4
FIG. 4:
The effects of lipophilic hydroxy-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors on human arterial smooth muscle cells (haSMCs) proliferation on the upper filter side (dark bars) and haSMC migration through the 5 μm-wide filter pores (hatched bars) of transfilter cocultures with proliferating human arterial endothelial cells (haECs) after 14 days' cocultivation time. HaECs were seeded first on the lower filter side. After 24 h, haSMCs were added to the opposite filter side. After a further 24 h when haSMCs were attached and spread on the membrane, increasing statin concentrations were supplemented to the endothelial side (lower compartment) and renewed simultaneously with medium changes every third day. A: Results of cell counting after treatment with atorvastatin for 14 days. Migration of haSMCs was stimulated by proliferating haECs (second pair of bars) when compared with haSMC monocultures, as shown in the first pair of bars (SMC). Addition of 200 μg/ml low-density lipoprotein (LDL; control 2) on the lower filter side showed a further slight stimulatory effect on haSMC migration when compared with controls without LDL (control 1). The inhibitory effects could just be prevented partially by the addition of 50 μM mevalonate (50 + mev.). B: Results of cell counting 14 days after treatment with cerivastatin, showing a dose-dependent inhibition of both proliferation and migration of haSMCs in extremely low concentrations, which could be abolished completely by the addition of 5.0 μM mevalonate (5.0 + mev.) Results of cell counting after treatment with lovastatin (C) and simvastatin (D) for 14 days, showing a dose-dependent inhibition of haSMC proliferation and migration but in 10-fold higher concentrations than those of cerivastatin. The inhibitory effects could be prevented by addition of mevalonate. (*p < 0.05, **p < 0.01 vs. controls).

Inhibition of haSMC proliferation and migration by cerivastatin. The addition of cerivastatin to the endothelial side (the lower side of the filter) resulted in a dose-dependent inhibition of haSMC proliferation on the upper filter side and also of haSMC migration from the upper to the lower filter side after 14 days' cocultivation time in LDL-containing standard culture medium. Specific inhibitory effects were determined at extremely low cerivastatin doses. Significant inhibition was observed at concentrations ≥0.005 μM for haSMC proliferation and ≥0.05 μM for haSMC migration (Fig. 4B). The IC50 was 0.064 and 0.04 μM, respectively (Table 2). In contrast to the other three hydrophobic statins, cerivastatin doses ≥1.0 μM were found to be toxic for both haSMCs and haECs.

TABLE 2
TABLE 2:
Comparison of the IC50 of different statins in transfilter cocultures: haSMC proliferation and migration in coculture with proliferating haECs after 14 days

Inhibition of haSMC proliferation and migration by simvastatin, lovastatin, and atorvastatin. Simvastatin, lovastatin, and atorvastatin caused dose-dependent inhibitory effects on both haSMC proliferation and migration (Fig. 4A, C, and D) but with significant effects in 10- to 100-fold higher doses than cerivastatin (0.05-50 μM,Table 2). The IC50 for the inhibition of haSMC proliferation by simvastatin was 0.65 μM and for lovastatin, 4.01 μM, which is about five- to 20-fold lower when compared with atorvastatin with a calculated IC50 at 17.5 μM. However, the IC50 for the inhibition of haSMC migration was found to be in a comparable concentration range between 0.77 and 5.75 μM for all three statins (Table 2).

Reversal of the inhibitory effects by mevalonate and LDL cholesterol

To prove whether the inhibitory effects of the statins on haEC and haSMC proliferation and migration were caused by a lack of mevalonic acid, the reversibility was analyzed of the effects by the addition of different amounts of mevalonic acid (50-1,000 μM) to cultures treated with the highest test concentrations of each statin. The lowest concentration of 50 μM mevalonic acid was not able to reverse the statin-induced inhibitory effects totally, but each concentration >100 μM abolished the effects nearly totally (>80%), with an optimum at 200 μM (data not shown). Whereas the effects of lovastatin, simvastatin, and cerivastatin on both DNA synthesis and mitochondrial activity (Fig. 1), as well as on cell numbers in transfilter cocultures (Fig. 4B-D) were reversed nearly completely by addition of mevalonic acid, inhibitory effects of atorvastatin on haSMC growth could be counteracted only partially to 35% in monocultures (Fig. 5), and 60% in transfilter cocultures (Fig. 4A). Control cultures with addition of 1,000 μM mevalonic acid alone showed that mevalonic acid itself exerted no independent inhibitory or stimulatory effect on haSMC and haEC growth (Fig. 5). The last bar of each figure shows the percentage of reversibility by mevalonic acid substitution in each culture with the highest statin concentration (cerivastatin, 5.0 μM; the other statins, 50 μM). In contrast to mevalonate, coaddition of LDL cholesterol (200 μg/ml) could not prevent the inhibition of both haEC and haSMC proliferation and migration induced by the statins. As described in previous studies (32), LDL cholesterol itself exerted slight growth-stimulatory effects on haSMC growth. Control cultures in Fig. 4A show a significant stimulatory effect of LDL cholesterol predominantly on haSMC migration of 27% (control 2) compared with controls without LDL (control 1). However, both supplementation of LDL cholesterol and addition of 10% serum did not significantly influence the statin-induced inhibitory or toxic effects. The presence of additional LDL cholesterol (besides the low concentration of 36 μg/ml found in FCS-containing medium) was not necessary to obtain the described results (e.g., to reduce sensitivity of haSMCs or haECs for toxic effects), as pretests showed, in which the same experiments were performed after addition of high LDL cholesterol levels (200 μg/ml). An increase of toxicity was exclusively found after >3 days serum deprivation (change of cell shape, increase of cell detachment), which was caused by a lack of growth factors and not by cholesterol deficiency (data not shown).

FIG. 5
FIG. 5:
Results of the several studies with increasing mevalonic acid doses supplemented to human arterial smooth muscle cells (haSMCs) treated with 50.0 μM atorvastatin, which was the highest concentration tested in each assay. Treated cultures were compared with placebo controls (control) and with high-dose mevalonic acid-treated cultures without atorvastatin (1,000 μM mev).

DISCUSSION

The results of this study demonstrate that cerivastatin is the most potent inhibitor of both proliferation and migration of human arterial smooth muscle cells in monocultures and in transfilter cocultures among all studied statins, even when applied exclusively to the endothelial side distant from the in vitro media and even when cholesterol is freely available.

Besides lipid deposition, proliferation and migration of smooth muscle cells as well as matrix formation are equally prominent features of both atherogenesis and restenosis (40). For this reason, several in vitro studies in smooth muscle cell monocultures from rat, porcine, and human arteries including ours were performed during recent years to detect the direct growth inhibitory effects of the first-generation compounds pravastatin, simvastatin, lovastatin, and fluvastatin (4,41,42). In our study, these experiments were extended in several ways: (a) to the synthetic, recently developed lipophilic compound cerivastatin; (b) to human arterial endothelial cells, which are very important for the integrity of the arterial vessel surface; and (c) to a much more complex coculture model that was established to imitate more closely the morphology of the human arterial vessel wall and to consider cell-to-cell interactions involved in vascular biology and pathology (27,32,43).

Both the described monoculture assays and the studies in our transfilter coculture system demonstrated that haSMC growth was inhibited to different extents by the 3-day treatment with the five different statins. In a potential inhibition of restenosis in humans, a therapy with the newest synthetic compound cerivastatin seems to be the most promising strategy because it was found to inhibit both proliferation and migration in transfilter cocultures with human vascular cells in ∼10- to 100-fold lower concentrations than the other statins. Simvastatin, lovastatin, and atorvastatin showed a dose-dependent inhibition of haSMC proliferation and migration with an IC50 in a concentration range of 0.65-17.5 μM, which cannot be achieved in serum of patients (maximum, 0.01-0.5 μM) after oral administration of the highest dosage and after liver passage (24,44,45). As already alluded to, exclusively low-dose cerivastatin treatment caused significant inhibitory effects on haSMC growth at concentrations ≥0.005 μM, which corresponds nearly exactly with serum levels in humans (0.002-0.03 μM) after treatment with oral or i.v. doses (25). However, it should be pointed out that we have no data about real intracellular statin concentrations in vitro in comparison with those achieved in vivo after cellular uptake, which do actually not correspond with serum levels because the cellular compartments are much smaller. Furthermore, long-term effects after long-term statin treatment and potential effects of metabolic products in vivo (e.g., after liver passage) cannot adequately be mimicked by the 14-day cocultivation period in vitro. Nevertheless, the accordance of the efficient in vitro doses with basal serum level achieved after statin treatment in vivo might be important because it might determine the diffusion gradient or the equilibrium between the extra- and intracellular compartments.

Besides these growth inhibitory effects, we found some cell rounding and detachment of both haSMCs and haECs at cerivastatin doses >0.2 μM, which also was reflected by the increase of LDH liberation at these high doses. In addition, TUNEL stainings, the end-labeling method of DNA fragments, indicated that the specific inhibitory effects might be partially overlapped by apoptosis, the programmed cell death (unpublished data), as described for lovastatin in murine B lymphocytes (46). A concentration-dependent number of floating, rounded cells also was found after high-dose treatment (>20 μM) with the other statins in our studies, as well as in others, which differed for various statins, cell types, and cell species. In rat smooth muscle cells, no morphologic change and no cell floating was observed using doses ≤30 μM lovastatin (47), whereas human smooth muscle cells from mammary arteries were detached after treatment with lipophilic statins >5 μM, and of human umbilical vein endothelial cells (HUVECs) at doses >1 μM(17). In the latter study, the authors explained this phenomenon as an direct effect of statins on cell adhesion. Discrepancies with our studies in regard to the critical toxic concentration are most probably due to differences in cell types, culture conditions, and that, as done in our studies, haECs were cultured on the extracellular matrix protein collagen I. Nevertheless, endothelial cell detachment might have some relevance in regard to the in vivo reendothelialization after interventional treatment of atherosclerotic coronary arteries. However, serum levels in the micromolar range cannot be achieved in patients after oral administration with the recommended standard dosages [e.g., used for serum cholesterol reduction (48)].

Despite the great number of studies with statins in the last decade, it is not fully understood by which mechanisms the growth-inhibitory effects of statins, including cerivastatin, are mediated and how differences between statins can be explained. The exact mechanism by which statins exert cell growth inhibition, favored by most investigators, is that mevalonate generates not only cholesterol, but also several other mediators, predominantly isoprenoids, as well as dolichol and isopentenyladenine (6). Prenylation by these mediators helps to attach lipophilic anchors to key proteins, as for example, to members of the p21ras and p21rho family, which is an important process for molecular function (12). However, data in rat smooth muscle cells showed that initial mevalonate derivatives that are more proximal in the cholesterol biosynthesis pathway and distinct from more distally synthesized isoprenoids may be also involved in cell proliferation (47). Conversely, lovastatin treatment of fibroblasts and rat smooth muscle cells demonstrated that DNA replication also can be affected directly by statins through inhibition of cell-signaling mechanisms [e.g., of the phosphatidylinositol-3-kinase (PI-3-kinase), which is required for growth factor-induced mitogenesis, for example, after receptor-binding of PDGF (15,49)].

The results of the described transfilter coculture studies showed also that all statins inhibited haSMC migration even when the test compound was applied exclusively to the endothelial side, which requires first diffusion across the filter pores to the haSMC layers. It is difficult to separate clearly the statin-induced effects on haSMC proliferation from those on haSMC migration in the complex transfilter coculture system. However, previous studies showed that the main peak of haSMC migration to the endothelial side occurred very early, starting within the first 24 h after seeding of cells. At this time, cell numbers were still not reduced significantly. After 7 and 14 days, the total number of haSMCs on the opposite side that are available for migration was reduced compared with controls as a result of the specific inhibition of proliferation. However, at these later times, endothelial cells did not divide any more because they had formed a confluent monolayer. Thus after 7 days' cocultivation, haSMC migration stopped because of the lack of the paracrine stimulatory effects exerted by dividing haECs, as shown previously (27), as well as because of the mechanical barrier formed by cells covering the filter surface. It could be assumed that in the first days of statin treatment, reduction of subendothelial haSMCs is caused by the inhibition of migration, and at later times, probably by a direct inhibitory effect on proliferation of those subendothelial haSMCs, which were already migrated. Thus differences in the number of cells on the endothelial side between statin-treated cultures and control cultures reflected mostly effects on haSMC migration. In addition to our studies, inhibition of rat smooth muscle cell migration was described in monocultures in response to fibrinogen as a chemotactic factor, and this effect was shown to be mevalonate dependent (50). Furthermore, intracellular Ca2+ mobilization in response to PDGF was shown to be impaired by statins (51). As an explanation for the underlying mechanism, inhibition of protein prenylation (e.g., of the ras-like small G protein rho) was discussed (12).

Despite these unique mechanisms, which seem to be shared by all statins, there are marked differences in the growth-inhibitory potency between cerivastatin and the other statins. Recent work by Negrè-Aminou et al. (17) in monocultures of human arterial smooth muscle cells showed that cerivastatin displayed an equal cholesterol synthesis inhibitory potency to lovastatin, simvastatin, atorvastatin, and fluvastatin, with an IC50 of ∼0.002 μM (values were extracted from inhibition curves). When compared with the IC50 for haSMC growth inhibition, which was ∼0.04-0.06 μM (in accordance with our results), just a slight difference to the IC50 of cholesterol synthesis was found (20-fold), whereas a 250- to 1,500-fold difference was determined for the other lipophilic statins (IC50, 0.5-3.0 μM). These data indicate that the growth-inhibitory potency of cerivastatin seems to be correlated much more to the inhibition potency on cholesterol synthesis than is the case for the other statins, from which much higher doses are required to achieve 100% growth inhibition than to block cholesterol synthesis totally. In addition, our described reversal studies with mevalonic acid and LDL supplementation to cerivastatin-treated haSMCs and haECs confirmed that the absence of mevalonate, but not cholesterol depletion, is the main cause for cell growth inhibition because growth-inhibitory effects in both mono- and cocultures could be prevented nearly 100% when abundant levels of mevalonate were present in the culture medium. These results indicate that the growth inhibition induced by cerivastatin results nearly entirely from the interference with the mevalonate synthesis pathway. In contrast, the inhibitory effects of atorvastatin seems to be partially based on other mechanisms, as they could not be reversed completely by the addition of mevalonate. Thus we conclude that the stronger antiproliferative effect of cerivastatin at ultra-low doses compared with other statins seems to be based on its much higher inhibitory potency on the HMG-CoA reductase. This hypothesis also is confirmed by the pharmacologic data provided for cerivastatin by Bischoff et al. (52), showing a >100-fold higher Ki value for the isolated, membrane-bound HMG-CoA reductase of rat liver cells and a 10-fold lower IC50 for cholesterol synthesis inhibition in ovarian PA-1 cells than measured for lovastatin. The 100-fold higher IC50 values of pravastatin compared with simvastatin, lovastatin, and atorvastatin, as well the 1,000- to 10,000-fold difference from cerivastatin reflects its different chemical nature, not a reduced HMG-CoA reductase activity per se. Pravastatin penetrates poorly across cellular membranes of nonhepatic cells because of its hydrophilic character. Despite a variety of positive in vivo studies with the classic statins in normocholesterolemic rabbits (53), as well as the first animal study with the third-generation compound cerivastatin (54), the effects of statins on the incidence of restenosis in humans after balloon angioplasty are still controversial. Some trials showed that lovastatin (55) reduced restenosis, especially of high-grade stenotic lesions after percutaneous transluminal coronary angioplasty (PTCA) in humans, whereas recent data failed to demonstrate the efficacy of lovastatin (18,56). Other studies (e.g., the FLARE Study with fluvastatin just published), showed negative results in regard to restenosis prevention but a reduction in mortality and myocardial infarction 40 weeks after PTCA (57). The controversial results might be explained by the fact that the efficient in vitro doses of the classic statins pravastatin, lovastatin, simvastatin, or fluvastatin between 0.5 and 50.0 μM could not be achieved as steady-state levels in human plasma (58). However, it remains to be determined if the very potent third-generation compound cerivastatin, which is well tolerated by patients (59), will be efficient enough to reduce restenosis in humans.

Acknowledgment: We thank Dr. I. Spyridopoulos for assistance in the statistical analysis. This work was supported by a grant of the Foundation for the support and investigation of substitution and supplementation methods reducing animal studies (Stiftung zur Förderung und Erforschung von Ersatz und Ergänzungsmethoden zur Einschränkung von Tierversuchen) and by a grant of the Federal Ministry of Education, Science, Research, and Technology (Fö. 01KS9602) and the Interdisciplinary Clinical Research Center (IZKF) Tübingen.

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Keywords:

HMG-CoA reductase inhibitors; Cerivastatin; Lipid metabolism; Arterial smooth muscle cell growth; Coculture; Restenosis

© 2000 Lippincott Williams & Wilkins, Inc.