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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:420-428.)
© 1995 American Heart Association, Inc.

Articles

Effects of 26-Aminocholesterol, 27-Hydroxycholesterol, and 25-Hydroxycholesterol on Proliferation and Cholesterol Homeostasis in Arterial Myocytes

A. Corsini; D. Verri; M. Raiteri; P. Quarato; R. Paoletti; R. Fumagalli

From the Institute of Pharmacological Sciences, University of Milan, Milan, Italy.

Correspondence to Dr Alberto Corsini, Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy.

	Abstract

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Abstract The major relation existing between cell growth and cholesterol homeostasis prompted us to investigate the effect of 26-aminocholesterol (26-NH₂), 27-hydroxycholesterol (27-OH), and 25-hydroxycholesterol (25-OH) on these cellular events. To test this relation, we incubated human and rat arterial myocytes with the sterols for 72 hours. All the tested compounds (0.5 to 7.5 µmol/L) inhibited rat and human myocyte proliferation and cholesterol biosynthesis in a dose-dependent manner. 26-NH₂ was more potent than oxysterols in inhibiting human myocyte proliferation but equieffective in rat cells; 27-OH and 25-OH displayed similar activity in both cell lines. Inhibition of nuclear incorporation of thymidine in rat myocytes is consistent with decreased cell count. The antiproliferative effect of the tested sterols was reversible. The high inhibition (80%) of cholesterol biosynthesis necessary to induce a decrease in myocyte proliferation suggests a causal relation between the cholesterol synthetic pathway and these cellular processes. In addition, all the tested sterols were able to inhibit hydroxymethyl glutaryl–coenzyme A reductase activity in intact myocytes but not in cell-free extracts. The finding that 26-NH₂ but not 27-OH or 25-OH does not suppress LDL receptor activity in either human or rat myocytes supports the achievement of selectivity over the coordinately regulated LDL receptor gene. The ability of 26-NH₂ to interfere with myocyte proliferation and cholesterol synthesis without affecting the LDL receptor pathway confers at least in vitro a pharmacological interest on the compound in the process of atherogenesis.

Key Words: cholesterol synthesis • LDL receptor • oxysterols • smooth muscle cells • hydroxymethyl glutaryl–coenzyme A reductase

	Introduction

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Among the processes involved in the formation of atherosclerotic lesions, ultimately responsible for clinical sequelae, a pivotal role is played by migration and proliferation of arterial smooth muscle cells (SMCs, myocytes) and by accumulation of lipids in the arterial wall.^{1 2 3} In atherosclerotic plaques SMCs are the predominant cell type, and their accumulation is the key prerequisite leading to vascular occlusion.^{1 3 4 5 6} In addition to their capacity to synthesize large amounts of connective tissue,⁷ SMCs, together with macrophages, can also accumulate lipids and become foam cells.^{4 6 8} Factors affecting SMC migration and proliferation are believed to be important in controlling the development of the atherosclerotic process.^{9 10}

A major relation exists between the processes of cell growth and cholesterol synthesis and metabolism.^{11 12 13 14 15} Cholesterol is a major component of cell membranes, and an adequate supply of this sterol, derived either from endogenous synthesis or from exogenous sources (mainly via LDL uptake), is needed to support cell growth and proliferation.¹³ Both pathways are activated in rapidly growing cells.^{13 14 16 17} Conversely, when cells are cultured in serum-free or lipoprotein-free medium and cholesterol synthesis is inhibited, cell growth is blocked.^{13 18} Cholesterol biosynthesis is mainly regulated by the activity of 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase, which catalyzes the synthesis of mevalonic acid, a crucial intermediate in the formation of sterols and nonsterol isoprenoid compounds.¹³ While cholesterol seems to be required early in the cell cycle (G₁ phase),¹⁹ mevalonate itself and some of its nonsteroidal derivatives (isoprenoids) are determining factors in cell division and growth regulation.^{13 20} A number of studies have confirmed this interpretation. SMCs treated with specific competitive inhibitors of HMG-CoA reductase, such as simvastatin and fluvastatin, fail to grow unless sufficient amounts of mevalonate or geranylgeraniol are supplied.^{15 21} These findings clearly support a causal relation between the mevalonate synthetic pathway and cell proliferation.

Oxygenated derivatives of cholesterol (oxysterols) have long been known to exhibit a number of biological activities, including the inhibition of cholesterol biosynthesis and cell proliferation.^{13 22} These compounds decrease HMG-CoA reductase activity by suppressing gene transcription and by increasing enzyme degradation.¹³ There is some evidence that the antiproliferative effect might partly be explained by suppression of de novo sterol biosynthesis.^{18 22 23} 25-Hydroxycholesterol (25-OH)¹³ and 27-hydroxycholesterol (27-OH)²⁴ are among the most potent inhibitors of cholesterol biosynthesis and, in addition, they decrease the receptor-mediated LDL pathway in cultured cells,^{24 25 26 27} thus interfering with both the endogenous and exogenous supplies of cholesterol to the cells. 26-Aminocholesterol (26-NH₂), an analogue of 27-OH with the same polarity, displays similar ability in regulating cellular cholesterol homeostasis.²⁸ The possibility exists that 26-NH₂ as well as oxysterols could potentially affect SMC proliferation by suppressing the cholesterol synthetic pathway. Since 27-OH is synthesized in aortic endothelium and in human macrophages, there is a rationale for studying its effect and that of related sterols, such as 27-aminocholesterol, on SMC proliferation as related to cellular cholesterol homeostasis.^{29 30} (Recent publications have chosen to use 27-hydroxycholesterol rather than the conventional name 26-hydroxycholesterol to indicate that the mitochondrial enzyme stereospecifically hydroxylates only the methyl group in position C-27.³¹ )

	Methods

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Materials
Eagle's minimum essential medium (MEM), fetal calf serum (FCS), trypsin-EDTA, penicillin (10 000 U/mL), streptomycin (6.86 mmol/L), tricine buffer (1 mol/L, pH 7.4), and nonessential amino acid solution (100x) were purchased from GIBCO. Disposable culture flasks and Petri dishes were from Corning Glassworks (Corning), and filters were from Millipore. 25-OH, 27-OH, and 25(R),26-NH₂ were kindly provided by Fujimoto Pharmaceutical Corp and brought into solution by ethanol. Simvastatin in lactone form, kindly provided by Merck, Sharp & Dohme Research Laboratories, was brought into solution by 0.1 mol/L NaOH (MSD file) to give the active form, and the pH was adjusted to 7.4 by adding 0.1 mol/L HCl.¹⁵ [6-³H]Thymidine (specific activity, 2 Ci/mmol), Na¹²⁵I, [2-¹⁴C]acetate sodium salt 58.9 mCi/mmol, 3-hydroxy-3-methyl[3-¹⁴C]glutaryl–CoA 52 mCi/mmol, DL-[2-³H]mevalonate acid lactone 100 mCi/mmol, and [1,2(n)-³H]cholesterol 47.7 Ci/mmol were from Amersham. Silica gel G thin-layer chromatography products were from Merck. Brij 96 was from Sigma Chemical Co. Isoton II was purchased from Coulter Instruments. All reagents were analytical grade.

Cell Culture
SMCs were cultured according to the procedure of Ross³² from the intimal-medial layer of aortas of male Sprague-Dawley rats (200 to 250 g). Cells were grown in monolayers at 37°C in a humidified atmosphere of 5% CO₂ in MEM supplemented with 10% (vol/vol) FCS, 100 U/mL penicillin, 68.6 µmol/L streptomycin, 20 mmol/L tricine buffer, and 1% (vol/vol) nonessential amino acid solution.¹⁵ The medium was changed every third day. Cells were used between the fourth and 10th passages. Cell viability was assessed by trypan blue exclusion. SMCs were identified by growth behavior and morphology by using monoclonal antibody specific for -actin, the actin isoform typical of SMCs.³³ The cells grew out of explants after 12 to 16 days, piled up after confluency, and contained numerous myofilaments and dense bodies as observed by transmission electron microscopy.^{2 25} Human vascular myocytes (A 617 from human femoral artery) were grown in the same culture conditions.¹⁵

Lipoprotein and Lipoprotein-Deprived Serum
Lipoproteins were prepared from the plasma of clinically healthy normolipidemic volunteers and from cholesterol-fed rabbits.³⁴ LDL (d=8.56 to 8.93 mol/L) and rabbit ß-VLDL (d<8.45 mol/L) were isolated by sequential preparative ultracentrifugation.³⁴ Lipoproteins were iodinated with ¹²⁵I by the McFarlane monochloride procedure³⁵ as modified for lipoproteins³⁶ and exhaustively dialyzed at 4°C against 0.15 mol/L NaCl and 0.3 mmol/L EDTA, pH 7.4.³⁷ Radioactive lipoproteins were used within 2 weeks from preparation and sterilized by passage through a Millipore filter (0.22-µm pore size) immediately before incubation with the cells. The final specific activity varied between 100 and 200 cpm/ng protein for LDL and 200 to 300 cpm/ng protein for ß-VLDL. Lipoprotein-deprived serum (LPDS) was prepared by ultracentrifugation of pooled human sera at d=10.5 mol/L, 40 000 rpm in a 50.2-Ti Beckman rotor for 72 hours.³⁸

Binding, Uptake, and Degradation of ¹²⁵I-LDL and ¹²⁵I–ß-VLDL
For determining cell surface binding at 4°C, the monolayers were directly digested in 0.1 mol/L NaOH after a standard washing procedure; one aliquot was counted for the cell-associated radioactivity as a measure of lipoprotein binding,³⁹ and another aliquot was used for cell protein assay.⁴⁰ No appreciable internalization and degradation of lipoprotein occurred at 4°C.⁴¹

The cell surface binding of ¹²⁵I-LDL was also determined at 37°C as heparin-releasable radioactivity.⁴¹ Cell monolayers were digested in 0.1 mol/L NaOH at room temperature overnight; one aliquot was counted for residual cell radioactivity as a measure of LDL internalization,^{41 42} and another aliquot was used for cell protein assay. For total uptake (binding plus internalization) of ß-VLDL, cell monolayers were directly digested in 0.1 mol/L NaOH after standard washing procedures.⁴³ The specific lipoprotein binding and uptake were computed by subtracting values observed in the presence of a 100-fold excess of unlabeled lipoprotein from those obtained in their absence. Lipoprotein degradation was measured from the accumulation of noniodide trichloroacetic acid–soluble ¹²⁵I in the incubation medium in excess of that occurring in the absence of cells.^{41 42} Each experimental point represents the average value of triplicate incubations.

Synthesis of Total Sterols
The synthesis of cholesterol was determined by measuring the incorporation of radioactive acetate into cellular total sterols.^{44 45} Cell monolayers after incubation with [2-¹⁴C]acetate (1 µCi/mL; specific activity, 0.9 µCi/µmol) for 72 hours were washed with phosphate-buffered saline and digested with 0.1 mol/L NaOH. Aliquots were saponified at 60°C for 1 hour in alcoholic NaOH after the addition of [1,2(n)-³H]cholesterol as an internal standard (0.04 µCi/sample). The unsaponifiable material was extracted with low–boiling point petrol ether and counted for radioactivity. To evaluate the incorporation of labeled acetate into cellular sterols, these were separated from the unsaponifiable fraction by thin-layer chromatography by using petroleum ether (boiling point, 40°C to 60°C)/diethyl ether/acetic acid (70:30:1). Radioactivity was measured with a lipoluma scintillator (Lumal). Proteins were determined according to the method of Lowry et al.⁴⁰

HMG-CoA Reductase Assay
HMG-CoA reductase activity was determined by measuring the rate of conversion of radioactive HMG-CoA into mevalonate in detergent-solubilized cell-free extract.^{41 45 46} Aliquots of the cell-free extracts (40 to 100 µg) were assayed in a buffer containing 0.25 mol/L K₂HPO₄ (pH 7.4), 100 mmol/L glucose-6-phosphate, 15 mmol/L NADP, 50 mmol/L dithiothreitol, and 110 µmol/L HMG-CoA (90 000 dpm/sample HM[¹⁴C]G-CoA) in a total volume of 200 µL. Microsomes were preincubated in the reaction buffer at 37°C for 10 minutes before the addition of HMG-CoA and then incubated for 120 minutes at 37°C with moderate shaking. The reaction was stopped by the addition of 20 µL of 5 mol/L HCl, and 90 000 dpm [³H]mevalonolactone standard was added to measure recovery. The reaction solution was then incubated at 37°C for 30 minutes to allow lactonization of the mevalonate. The mixture was extracted twice with 10 mL (20 mL total) of diethyl ether. The upper phase was transferred to a 50-mL conical tube, and the combined upper phases were dried; the residue was resuspended in acetone, spotted on a thin-layer chromatography plate, and chromatographed in acetone/benzene (1:1). Recovery of labeled mevalonolactone was more than 40%. The activity of HMG-CoA reductase was expressed as picomoles of mevalonate formed per milligram of detergent-solubilized protein per minute.

Experimental Protocol
Proliferation of and Cholesterol Synthesis in SMCs
Cells were seeded at various densities for rat (2x10⁵) and human (5x10⁴) myocytes per Petri dish (35 mm) and incubated with MEM supplemented with 10% FCS.¹⁵ Twenty-four hours later the medium was changed to one containing 0.4% FCS to stop cell growth, and the cultures were incubated for 48 hours. At this time (time 0) the medium was replaced by one containing 10% FCS in the presence or absence of known concentrations of the tested compounds, and incubations were continued for a further 72 hours at 37°C. At time 0, just before the addition of the substances to be tested, three Petri dishes were used for cell counting. Cell proliferation was evaluated by cell count after trypsinization of the monolayers using a Coulter counter model ZM.¹⁵ In a separate set of Petri dishes cholesterol synthesis was estimated under the same experimental conditions by measuring the incorporation of [¹⁴C]acetate into cellular sterols. The amount of sterols required to inhibit one half of cholesterol synthesis and of cell proliferation was calculated by linear regression analysis of the logarithm of the concentrations (in micromoles per liter) versus probits and read from a probit transformation table. In another set of experiments cell proliferation was estimated by nuclear incorporation of [³H]thymidine that had been incubated with cells (1 µCi/mL medium) for 3 hours according to the method of Corsini et al.²¹ Radioactivity was measured with filter-count scintillation cocktail. The reversibility of the inhibitory effect of sterols on cell growth was also investigated. Arterial myocytes were treated with the tested compounds for 72 hours, after which the incubation medium was removed and replaced with fresh culture medium for a further 48 hours. Cell proliferation was then evaluated. Cell viability, assessed by trypan blue exclusion and lactate dehydrogenase leakage,⁴⁷ was found to be higher than 90% at the drug concentrations used.

HMG-CoA Reductase Assay in Cell-Free SMC Extracts
After incubation in the same experimental conditions described for cell proliferation in the presence of the tested compounds, SMCs were washed twice with 3 mL of 50 mmol/L Tris-HCl and 150 mmol/L NaCl (pH 7.4) and then scraped into 1 mL of the same buffer. The suspension was centrifuged at low speed, the supernatant was discarded, and the cell pellet was frozen in liquid nitrogen and stored until the time of the assay. Cell pellets were extracted with 100 µL of 50 mmol/L potassium phosphate, 5 mmol/L dithiothreitol, 1 mmol/L disodium EDTA, 200 mmol/L KCl, and 0.0025 Brij 96.^{41 46} The rate of conversion of HM[¹⁴C]G–CoA to [¹⁴C]mevalonate was then measured. In another set of experiments microsomes were prepared by confluent cells preincubated for 24 hours at 37°C in a medium containing 10% LPDS to induce HMG-CoA reductase activity.^{13 16 41} Reductase activity was then performed at 37°C for 2 hours under standard conditions except for the addition of the tested compounds in the incubation assays.

LDL Receptor–Mediated Lipoprotein Catabolism
For all experiments, cells were seeded in 35-mm dishes at various densities for rat (2x10⁵) and human (5x10⁴) myocytes and used just before reaching confluency, usually 5 days after plating.

Confluent monolayers were preincubated for 24 hours at 37°C in a medium containing 5% LPDS to upregulate apolipoprotein B,E LDL receptors⁴¹ in the presence or absence of different concentrations of the tested sterols dissolved in ethanol. Control dishes contained the same volume of the solvent. After this time a fixed concentration of ¹²⁵I-LDL (12.5 nmol/L) or ¹²⁵I–ß-VLDL (1 nmol/L) was added to the cells, which were then incubated at 37°C or 4°C for an additional 5 and 3 hours, respectively.^{26 41 42 43} (This assumes a molecular weight of 3x10⁶ [of which 20% is protein] for LDL and 10x10⁶ [of which 10% is protein] for ß-VLDL, respectively.) In the latter case, HEPES pH 7.4 is substituted for NaHCO₃.³⁷

Statistical data are expressed as mean±SD. The effects of the tested compounds versus control on the different parameters were analyzed by two-tailed Student's t test for unpaired data.

	Results

Top Abstract Introduction Methods Results Discussion References

 
The effects of 26-NH₂, 27-OH, and 25-OH on the proliferation of rat and human arterial myocytes as related to cholesterol homeostasis were investigated.

Myocyte Proliferation and Cholesterol Biosynthesis
Most reported studies on the in vitro inhibition of cholesterol biosynthesis by oxysterols have been performed in cells incubated either in a medium containing lipoprotein-deficient serum or in a serum-free medium, ie, in experimental conditions in which cholesterol synthesis is stimulated.¹³ Since myocyte proliferation occurs in the presence of FCS, we chose to study the effect of sterols under the same experimental conditions. All the tested sterols inhibited rat and human myocyte proliferation and cholesterol biosynthesis in a dose-dependent manner (Figs 1 and 2). Similar results on cell proliferation are reported in rabbit SMCs with 25-OH.⁴⁸ 26-NH₂ was more potent than oxysterols in inhibiting human SMC proliferation but equieffective in rat myocytes; 25-OH and 27-OH displayed similar activity on both cell lines. The concentrations of sterols required to halve proliferation and cholesterol synthesis (IC₅₀) are summarized in Table 1. To assess the effect of sterols on SMC proliferation in another way, the nuclear incorporation of [³H]thymidine was measured in rat myocytes. All the tested compounds inhibited thymidine incorporation in a dose-dependent manner (Fig 3). The results are consistent with those obtained by cell counting. Sterol-mediated inhibition of myocyte proliferation was not the result of cytotoxicity. In fact, when human myocytes were treated with sterols for 72 hours, and the incubation medium was removed and replaced with fresh medium, cells were able to recover from the inhibitory effect of sterols (Fig 4). Similar results have been obtained in rat myocytes (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 1. Line graphs showing effects of sterols on proliferation (solid symbols) and cholesterol synthesis (open symbols) of rat aorta myocytes. Cells were seeded (2x105/dish) and incubated with Eagle's minimum essential medium supplemented with 10% fetal calf serum (FCS); 24 hours later the medium was changed to one containing 0.4% FCS to stop cell growth, and the cultures were incubated for 48 hours. At this time (time 0) the medium was replaced with one containing 10% FCS and the reported concentrations of sterols, and the incubation was continued for a further 72 hours at 37°C. [14C]Acetate incorporation was used to assay cholesterol synthesis, and cell number provided an index of the effect of cell replication. Each point represents mean±SD of triplicate dishes. The mean value of control (1.0) for cell number was 832±41x103 cells/plate and for cholesterol synthesis was 29.2±4.2 pmol/mg cell protein per hour. Where error bars are not shown, they are within the symbol limits. *P<.05, **P<.01, ***P<.001 sterols vs control by Student's t test.

View larger version (22K): [in this window] [in a new window]   Figure 2. Line graphs showing effects of sterols on proliferation (solid symbols) and cholesterol synthesis (open symbols) of human arterial myocytes. Cells were seeded at the density of 5x104/dish. Experimental conditions are as in Fig 1. Each point represents mean±SD of triplicate dishes. The mean value of control (1.0) for cell number was 1446±100x103 cells/plate. The mean value of control for cholesterol synthesis was 2.6±0.5 pmol/mg cell protein per hour. Where error bars are not shown, they are within the symbol limits. *P<.05, **P<.01, ***P<.0001 sterols vs control by Student's t test.

View this table: [in this window] [in a new window]   Table 1. Potencies of Different Sterols in Inhibiting Proliferation and Cholesterol Synthesis in Arterial Myocytes

View larger version (31K): [in this window] [in a new window]   Figure 3. Line graph showing effect of sterols on nuclear incorporation of [3H]thymidine by rat aorta myocytes. Experimental conditions are as in Fig 1; labeled thymidine was added to the medium after 72 hours, and incubation was continued for a further 3 hours. Each point represents mean±SD of triplicate dishes. The mean value of control (1.0) was 61±7x103 dpm/mg cell protein. *P<.05, **P<.01, ***P<.001 sterols vs control by Student's t test.

View larger version (37K): [in this window] [in a new window]   Figure 4. Bar graph showing reversibility of the antiproliferative effect of sterols (3 µmol/L) on human smooth muscle cell growth. Experimental conditions are as in Fig 1. Sterol-containing media were removed after 72 hours, and new media (without sterols) supplemented with 10% fetal calf serum were added for an additional 48 hours. Data are mean±SD of triplicate dishes. *P<.001 sterol vs control by Student's t test.

HMG-CoA Reductase Activity
The experiment shown in Table 2 revealed that under conditions in which sterols inhibited cholesterol synthesis in intact rat and human myocytes (Figs 1 and 2), the activity of HMG-CoA reductase as measured in cell-free extracts decreased progressively. Despite the similar inhibition of cholesterol synthesis achieved by simvastatin¹⁵ and by the tested sterols in intact cells, extracts of the simvastatin-treated myocytes showed a much higher HMG-CoA reductase activity than the sterol-treated myocytes. Similar results have been reported in human fibroblasts incubated with compactin, another competitive inhibitor of the reductase.⁴⁴ We then investigated the ability of the tested sterols to directly interfere with HMG-CoA reductase activity in cell-free extracts. Microsomes were prepared from rat myocytes preincubated with 10% LPDS for 24 hours to induce HMG-CoA reductase activity.^{13 16 41} We chose rat cells as our source of microsomes since a higher enzymatic activity was detected (Table 2) compared with human SMCs, and similar responses to sterols were observed in both cell lines. As shown in Table 3, all the tested sterols failed to inhibit HMG-CoA reductase activity when added directly to cell-free extracts. As expected,⁴⁴ simvastatin showed a potent inhibitory effect on HMG-CoA reductase under the same experimental conditions (Table 3).

View this table: [in this window] [in a new window]   Table 2. Effect of Preincubation With Sterols and Simvastatin on HMG-CoA Reductase Activity in Arterial Myocytes

View this table: [in this window] [in a new window]   Table 3. Effect of Sterols and Simvastatin on HMG-CoA Reductase Activity in Rat Arterial Myocyte-Free Extracts

LDL Receptor–Mediated Lipoprotein Metabolism
In a first set of experiments the tested sterols were investigated on rat aortic myocytes. Since human LDL is poorly recognized by rat cells,^{49 50 51} we used ß-VLDL from cholesterol-fed rabbits as ligand for the rat apolipoprotein B,E LDL receptor. 25-OH and 27-OH downregulated the LDL receptor–mediated pathway in a dose-dependent manner, while 26-NH₂ did not affect or slightly increased, at the highest concentration, the uptake and degradation of ¹²⁵I–ß-VLDL by rat myocytes (Fig 5). The effect of the compounds tested on cell surface binding of ¹²⁵I–ß-VLDL was also investigated at 4°C to minimize or abolish the internalization process. Specific binding of lipoproteins was decreased by increasing concentrations of oxysterols, while 26-NH₂ was ineffective on this parameter (Fig 6).

View larger version (21K): [in this window] [in a new window]   Figure 5. Line graphs showing effects of sterols on 125I–ß-VLDL uptake and degradation by rat aorta myocytes. Confluent cells were preincubated for 24 hours in the medium supplemented with 5% lipoprotein-deprived serum and increasing concentrations of sterols. 125I–ß-VLDL protein (1 nmol/L · mL-1) was then added and the incubation was performed at 37°C for a further 5 hours. Uptake and degradation were measured as described in "Methods" and were corrected for nonspecific values observed in the presence of a 100-fold excess of unlabeled ß-VLDL. Each point represents mean±SD of triplicate dishes. *P<.02, **P<.01, ***P<.001 sterols vs control by Student's t test.

View larger version (27K): [in this window] [in a new window]   Figure 6. Line graph showing effect of sterols on 125I–ß-VLDL binding to rat aorta myocytes. Experimental conditions are as in Fig 5 except that the incubation with 125I–ß-VLDL was performed at 4°C for 3 hours. Binding was measured as described in "Methods" and was corrected for nonspecific binding observed in the presence of a 100-fold excess of unlabeled ß-VLDL. Values are mean±SD of triplicate dishes. *P<.02, **P<.01, ***P<.001 sterols vs control by Student's t test.

The effect of sterols on LDL receptor–mediated catabolism was also investigated in human arterial myocytes. Oxysterols caused a slight decrease in LDL internalization and degradation; 26-NH₂ enhanced lipoprotein binding and internalization without affecting degradation (Fig 7).

View larger version (20K): [in this window] [in a new window]   Figure 7. Line graphs showing effects of sterols on binding, internalization, and degradation of 125I-LDL by human arterial myocytes. Confluent cells were preincubated for 24 hours in the medium supplemented with 5% lipoprotein-deprived serum and increasing concentrations of sterols. 125I-LDL protein (12.5 nmol/L · mL-1) was then added, and the incubation was performed at 37°C for a further 5 hours. Binding, internalization, and degradation were measured as described in "Methods" and were corrected for nonspecific values observed in the presence of a 100-fold excess of unlabeled LDL. Each point represents mean±SD of triplicate dishes. *P<.05, **P<.02, ***P<.01, ****P<.001 sterols vs control by Student's t test.

Similar results were obtained with the tested sterols on LDL uptake and degradation by human fibroblasts under the same experimental conditions (data not shown).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Proliferation of SMCs in the medial layer of the arterial wall followed by their migration and further proliferation in the intimal layer are major mechanisms involved in atherogenesis.^{2 3} The observation that treatment of arterial SMCs with inhibitors of HMG-CoA reductase such as vastatins and oxygenated sterols results in growth arrest^{15 22 48} prompted us to investigate the effect of 26-NH₂, an analogue of 27-OH with a similar capacity to modulate cholesterol homeostasis,²⁸ on the proliferation of arterial myocytes. The results show that 26-NH₂ as well as 25-OH and 27-OH reduce SMC proliferation in a dose-dependent manner. The mechanism whereby these compounds exert these effects remains to be addressed. There is some evidence that the antiproliferative effect of oxysterols might be partially explained by the inhibitory effect on cholesterol biosynthesis, since this pathway is believed to be essential for DNA synthesis via nonsteroidal metabolites of mevalonate.^{13 18 22 23} The observation that the inhibitory effect of all the tested sterols on cell proliferation occurred when cholesterol synthesis was suppressed by more than 80% supports a causal relation between inhibition of cholesterol synthesis and these cellular events. Furthermore, similar results have been reported for HMG-CoA reductase inhibitors,¹⁵ suggesting that a strong inhibition of mevalonate synthesis might impede sufficient formation of endogenously derived products (dolichol, ubiquinone, prenylated proteins) to support cell proliferation.^{13 14} However, this cannot be the only explanation since the antiproliferative effects of sterols, in contrast to those observed with vastatins,¹⁵ cannot be prevented by the addition of mevalonate, farnesol, or geranylgeraniol to the culture medium (data not shown). Mechanistic studies with sterols, and in particular 25-OH, have shown that sterol-dependent regulation of the mevalonate pathway occurs at both the transcriptional and posttranscriptional levels.¹³ A 10–base pair element in the 5' flanking region of the LDL receptor, HMG-CoA reductase and HMG-CoA synthase and protein interacting with this element^{52 53 54 55} appears to be responsible for sterol-dependent control of gene transcription.¹³ In addition, a control at the transcriptional level of farnesyl diphosphate synthase and squalene synthase genes by 25-OH has been reported.^{56 57} Oxygenated sterols can also affect HMG-CoA reductase activity at the posttranscriptional level by increasing enzyme degradation.¹³ The fact that 26-NH₂, 27-OH, and 25-OH inhibited HMG-CoA reductase activity in intact myocytes but not in cell-free extracts suggests a similar behavior of the investigated sterols in the modulation of enzyme activity. Finally, the possibility that the inhibitory action of the tested sterols on de novo sterol biosynthesis occurs also at earlier⁵⁸ or later steps of the mevalonate pathway, including transformation of C₃₀ sterol to C₂₇ sterols and enzymes affecting C₂₇ sterol transformation,^{22 59 60 61} cannot be ruled out. Altogether, the complex mechanisms by which sterols modulate the mevalonate pathway underline the difficulty in establishing a relation between inhibition of cholesterol synthesis and cell proliferation.

Suppression of cell proliferation and cholesterol synthesis may be separated in some cases.²² A direct effect of sterols on cell membrane structure and function has also been postulated and may be responsible in part for the antiproliferative effects of these compounds.^{22 62} 25-OH is reported to potentiate serum-induced arachidonic acid release and prostaglandin biosynthesis, an effect possibly involved in inhibition of cell proliferation.⁶³ Other authors suggest the probability that specific oxysterol binding protein(s)^{64 65 66} or an antiestrogen-binding site may be involved in mediating the antiproliferative effect of oxysterols.⁶⁷ Finally, the toxicity of oxysterols has been proposed as being responsible for the antiproliferative effect.²² However, none of the sterols used in our experimental conditions were cytotoxic: the cells excluded trypan blue, did not release lactate dehydrogenase, and started growing again after removal of the sterols.

Rapidly growing cells require an adequate supply of cholesterol, derived either from endogenous synthesis or from exogenous sources.^{13 17} Our results show some discrepancy between the effects of oxysterols and 26-NH₂ on cholesterol synthesis and cell proliferation. To learn whether LDL receptor activity, which is enhanced in rapidly growing cells,^{13 16 17} is involved, the effect of sterols on the LDL pathway was examined. While 26-NH₂ slightly increases LDL receptor activity in both human and rat SMCs to a similar extent, the opposite effect is seen with oxysterols, which are more potent in rat than in human SMCs. The reduced supply of exogenous cholesterol via the LDL pathway together with the almost suppressed cholesterol synthesis could explain the more pronounced inhibitory effect of oxysterols on proliferation of rat SMCs.

The finding that 26-NH₂ does not suppress LDL receptor activity in SMCs, although not consonant with a recent report of downregulation of the LDL receptor in human fibroblasts and the hepatoma cell line HepG2,²⁸ supports the achievement of selectivity over the coordinately regulated LDL receptor gene. This derivative should prove useful for critically testing the role of a specific sterol regulatory element binding protein(s) in the regulation of the gene for the LDL receptor and for enzymes of the mevalonate pathway.^{52 53 54 55 68 69} The capacity of 26-NH₂ to suppress HMG-CoA reductase activity without affecting LDL receptor activity clearly supports the recent hypothesis that the sterol-mediated control mechanisms for LDL receptor and HMG-CoA reductase genes are distinct.^{54 68} The possibility exists, however, that the mechanism by which 26-NH₂ modulates LDL receptor expression and reductase activity might be different. 26-NH₂ could also act at the later steps of the cholesterol synthesis by interfering with enzymes whose genes do not contain any sterol regulatory element. This would explain the fact that the expression of the LDL receptor is unchanged. For example, the target of 26-NH₂ might be squalene synthase or oxidosqualene lanosterol cyclase. Several studies have shown that certain amino derivatives of squalene are potent inhibitors of these enzymes.^{70 71} Finally, the basic property of the amino group present on the 26-NH₂ molecule could be responsible for the different effect on the LDL receptor activity; a similar explanation has been proposed for calcium antagonists.⁷²

In summary, our data indicate that 26-NH₂, 27-OH, and 25-OH are able to inhibit SMC proliferation and cholesterol synthesis in a dose-dependent manner. The marked inhibition of cholesterol synthesis necessary to induce a decrease in SMC growth supports the link between the mevalonate synthetic pathway and cell proliferation.

The ability of 26-NH₂ to interfere with myocyte proliferation and cholesterol synthesis without affecting the LDL receptor pathway confers in vitro specificity and pharmacological interest on the compound in the process of atherogenesis. Whether these in vitro effects have any relation to in vivo reality remains untested.

	Acknowledgments

 
Research was partially supported by the Italian government program MURST and the Fujimoto Pharmaceutical Corp, Osaka, Japan. The authors are grateful to Professor Norman B. Javitt (New York University, New York) for helpful discussions, to Professor G. Gabbiani (University of Geneva, Switzerland) for providing the human femoral artery cell line A617, and to Laura Mozzarelli for secretarial help.

Received April 5, 1994; accepted December 2, 1994.

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