HMG-CoA Reductase Inhibitors Reduce Acetyl LDL Endocytosis in Mouse Peritoneal Macrophages
Abstract We previously reported that mevalonate starvation elicited by hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors reduced cholesterol accumulation promoted in murine macrophages by acetylated LDL (AcLDL). In the present study we investigated the cellular mechanism of this effect. Our results indicate that the HMG-CoA reductase inhibitors fluvastatin and simvastatin reduce, in a concentration-dependent manner, more then 50% of the 125I-AcLDL degradation by macrophages. This effect was not due to a decrease of lysosomal enzyme activity, and it was paralleled by the retention of AcLDL-associated cholesteryl ester in the incubation medium. The ability of fluvastatin to inhibit AcLDL degradation was completely overcome by mevalonate and its derivative geranylgeraniol. Evaluation at 4°C of 125I-AcLDL binding to plasma membrane suggested that the inhibitory effect of fluvastatin on lipoprotein catabolism was not due to a decreased expression of scavenger receptors. Fluorescent microscope analysis of cellular internalization of AcLDL labeled with the fluorochrome 3,3′-dioctadecyl indocarbocyanine demonstrated that fluvastatin inhibits lipoprotein endocytosis, an effect reversed by mevalonate. Studies performed with native 125I-LDL indicated that fluvastatin did not inhibit but rather increased the degradation of LDL taken up by the normal LDL receptor. These results exclude a generalized depression of the cellular endocytotic activity by the drug. The ability of fluvastatin to reduce AcLDL catabolism and cholesterol esterification was more pronounced in cholesterol-enriched macrophages compared with normal cells. In conclusion, the present results demonstrate that HMG-CoA reductase inhibitors may reduce the in vitro cholesterol accumulation in macrophages by inhibiting AcLDL endocytosis.
- Received May 31, 1994.
- Accepted June 9, 1995.
A major process in atheroma formation is the accumulation of cholesterol in macrophages present in the arterial wall. These cells have been demonstrated in vitro to accumulate an excess of cholesterol via scavenger receptors that mediate the internalization and degradation of modified lipoproteins such as AcLDL and oxidized LDL.1 2 The cholesteryl esters transported by these lipoproteins are hydrolyzed in the lysosomes, and free cholesterol is released to a pool(s) where it participates as a substrate for the esterification reaction catalyzed by the microsomal enzyme ACAT.3 Several compounds have been reported to reduce cholesterol esterification. Some act directly by inhibiting ACAT activity,4 others indirectly by reducing lipoprotein degradation and their cholesteryl ester hydrolysis in the lysosomes,5 6 slowing down intracellular cholesterol movement,7 8 and inhibiting scavenger receptor expression and cellular influx of modified lipoproteins.9 10 Depending on the mechanism involved, each agent may have different effects on cellular cholesterol content and localization and on the esterified-to–free form ratio. ACAT inhibitors may increase free cholesterol content of the plasma membrane, with a minor effect on total cellular cholesterol.11 Free cholesterol accumulation in lysosomes is observed with compounds active on intracellular cholesterol movement.7 8 Accumulation of cholesteryl esters in these organelles is achieved with agents inhibiting the activity of lysosomal enzymes.5 6 Finally, a decrease of total cellular cholesterol is observed with compounds that inhibit the expression of scavenger receptors.10
Previous studies demonstrated that HMG-CoA reductase inhibitors (vastatins) decreased cholesterol esterification and deposition in macrophages.12 13 This action did not occur in a cell-free homogenate12 and was observed only under conditions of simultaneous incubation of vastatins with AcLDL but not in cholesterol-preloaded cells,13 thus indicating that these drugs are not direct ACAT inhibitors. The inhibition of cholesterol esterification by vastatins was completely overcome by mevalonate and geranylgeraniol13 and took place in the presence of an excess of exogenous cholesterol.12 13 These results showed that vastatins did not effect esterification by preventing the intracellular formation of substrate for ACAT (ie, cholesterol) but rather by inhibiting the formation of nonsterol mevalonate product(s).
The cellular mechanism by which vastatins limit cholesterol esterification has not been elucidated. Since the blockade of cholesteryl ester formation is concomitant with a net loss in cellular cholesterol content,13 it can be inferred that vastatins either induce cholesterol efflux or reduce cholesterol uptake from AcLDL.
In the present study we report that mevalonate starvation induced by vastatins does not influence cholesterol efflux but rather reduces cellular uptake of exogenous cholesterol by inhibiting AcLDL endocytosis.
DMEM, HEPES buffer solution (1 mol/L), penicillin-streptomycin (10 000 IU/mL to 10 000 UG/ml), and FCS were purchased from GIBCO Laboratories. EFAF-BSA was obtained from Sigma Chemical Co. Cell culture wells (35 mm, Multidish Nunclon) were obtained from NUNC.
Simvastatin, kindly provided by Merck Sharp & Dohme Research Laboratories, was added in the active form in 0.1 mol/L NaOH adjusted to pH 7.4 with HCl. Fluvastatin and S-58035, kindly provided by Sandoz Prodotti Farmaceutici, were added to monolayers in ethanol and DMSO, respectively, whose concentrations did not exceed 0.5% of culture medium; control dishes contained the same percentage of solvent. Farnesol and mevalonic acid lactone were also obtained from Sigma. (AII-trans)-geranylgeraniol was kindly provided by Prodotti Roche. [1-14C]oleic acid (56 mCi/mmol), [1a,2a(n)-3H]cholesteryl linoleate (40 Ci/mmol), and sodium [125I]iodide (17 mCi/mg) were from Amersham.
MPMs were obtained by peritoneal lavage from mice (BALB/c, Charles River, Calco, Italy) 3 days after intraperitoneal injection of thioglycollate. Cells (2 to 3×106) were plated in 35-mm wells with DMEM containing 10% FCS. After 3 hours, the dishes were washed to eliminate unattached cells and maintained in DMEM plus 10% FCS for 24 hours before use. Afterwards cell-plating experiments were performed at 37°C (except where indicated) in serum-free DMEM containing 0.2% EFAF-BSA plus the indicated additions.
In experiments conducted at 4°C, bicarbonate was substituted with HEPES 10 mmol/L. Protein was measured according to method reported by Lowry et al.14
Human LDL (d=1.019 to 1.063 g/mL) was isolated from plasma of healthy volunteers by sequential ultracentrifugation (Beckman L5-50).15 For acetylation, LDL was dialyzed against 0.15 mol/L NaCl, pH 7.4, diluted with an equal volume of saturated sodium acetate, and treated with acetic anhydride, according to technique used by Basu et al.16
For 125I-AcLDL and 125I-LDL preparations, lipoproteins were labeled with sodium [125I]iodide according to technique reported by Bilheimer et al,17 and desalted by gel filtration on Sephadex G-25 eluted with PBS. Specific activity was 100 to 200 cpm/ng protein. TCA nonprecipitable radioactivity was below 2% of total.
Cholesteryl linoleate–labeled AcLDL (specific activity, 20 disintegrations per minute/ng) was prepared by incubation of lipoproteins with 10 mCi/mg protein of [3H]cholesteryl linoleate in the presence of rabbit serum containing the cholesteryl ester transfer protein.18 Dil-AcLDL was kindly provided by Dr David Via (Baylor College of Medicine, Houston, Tx). All lipoproteins were sterile filtered.
Cellular Cholesterol Efflux
Cells were incubated for 24 hours with the tested compounds, followed by 24 hours in the same fresh medium with [3H]CE-AcLDL (25 μg/mL) added. Cells and medium were subsequently analyzed as described below for radioactivity content in free and esterified cholesterols. Free-cholesterol radioactivity in the medium indicated the amount of cholesterol efflux from cells.
Lipoprotein Degradation and Binding
Cells were incubated for 24 hours with the tested compounds. After substitution of the medium with an identical one, incubation was continued for an additional 24 hours. During this second incubation 125I-labeled lipoproteins were added at the times and concentrations indicated in the figures and tables. Then medium was removed and lipoprotein degradation was measured as the accumulation of noniodide TCA–soluble 125I in excess of that occurring in the absence of cells19 (less than 0.2% of total degradation of control). Lipoprotein binding was determined after incubation of cells at 4°C for 4 hours with 125I-AcLDL followed by extensive washing of the monolayers with albumin buffer and their digestion with 0.1 mol/L NaOH.20 The nonspecific lipoprotein binding was evaluated in the presence of a 50-fold excess of unlabeled lipoprotein.21 Nonspecific values were less than 15% of total binding.
Cholesterol Esterification Assay
Cells were incubated for 24 hours with the drug, followed by 24 hours in the same fresh medium added with 5 μg/mL of AcLDL. Cholesterol esterification was measured after addition of [1-14C]oleic acid (0.68 mCi sample) complexed with BSA during the last 2 hours of incubation and subsequent determination of radioactivity associated with cellular cholesteryl esters.22
Cholesterol Enrichment of Cells
Where indicated, cells were enriched with cholesterol by 24-hour incubation with 10 to 25 μg/mL of AcLDL before drug addition and experimental determinations.
Lipoprotein–Cholesteryl Ester Hydrolysis
After the 24-hour drug treatment, cells were incubated in the same fresh medium with [3H]CE-AcLDL (25 μg/mL) added and the ACAT inhibitor S-58035.23 Cells and media were then assayed for radioactivity content of the free and esterified cholesterol fractions. The percentage of radioactivity associated with free cholesterol (cell plus medium) indicated the amount of AcLDL-derived cholesteryl ester hydrolyzed by cells.
Determination of Free and Esterified Cholesterol
After the indicated incubations cells were washed with PBS and extracted with hexane/isopropanol (3:2, vol/vol). Media were extracted with chloroform/methanol (2:1 vol/vol).
After solvent removal, free and esterified cholesterol was partitioned by thin-layer chromatography (isooctane/diethyl ether/acetic acid, 75:25:2 by volume).21 Cholesterol mass or radioactivity of the spots was determined by an enzymatic method (Boeringher Mannheim)13 24 or by liquid scintillation counting (Lipoluma Lumac, Landgraf), respectively.
The cells to be used for fluorescence microscopy were seeded (106 cells/sample) on 18×24-mm coverslips. After treatment with the tested compounds as described above, cells were added with Dil-AcLDL (5 μg/mL) and incubated for an additional 5 hours. Monolayers were subsequently washed and fixed with 3% formaldehyde. Fluorescent photomicrographs were made on a Zeiss Axiovert 100 with an excitation filter for rhodamine using Kodak TMY film (ASA 400). Since fluvastatin was more active in cholesterol-loaded cells (see Fig 4⇓) the reported results were obtained in this experimental condition. Consistent results were observed in unloaded cells (data not shown).
To test whether the activity of HMG-CoA reductase inhibitors on cellular cholesterol metabolism could involve a stimulatory effect on cholesterol efflux, MPMs were incubated with [3H]CE-AcLDL. Incubation of cells with 25 μg/mL [3H]CE-AcLDL induced the accumulation of 20 μg/mg cell proteins of AcLDL-derived cholesterol, two thirds in the esterified form. Treatment of cells with fluvastatin reduced the amount of radioactive cholesterol esters by 60%, with minor changes of free [3H]cholesterol content (Table 1⇓). Analysis of radioactive cholesterol present in the incubation medium revealed that the decrease of [3H]cholesteryl esters in cells was paralleled by an increase of extracellular radioactivity associated with the esterified fraction of cholesterol, whereas the content of labeled free cholesterol was unaffected by the drug (Table 1⇓). All these effects were abolished by mevalonate (data not shown).
We next evaluated whether HMG-CoA reductase inhibitors could influence cellular catabolism of AcLDL. Fluvastatin and simvastatin were tested for their actions on specific degradation of 125I-AcLDL (50 μg/mL) by MPMs. Data reported in Fig 1⇓ demonstrate that both drugs inhibited in a dose-dependent manner up to 50% 125I-AcLDL degradation by macrophages. The inhibitory effects of both drugs were overcome by the addition of 100 μmol/L mevalonate. Mevalonate alone did not affect this parameter (Fig 1⇓).
In addition to mevalonate, fluvastatin inhibitory activity on lipoprotein degradation was fully prevented by geranylgeraniol and only slightly by farnesol (Table 2⇓). The activity of fluvastatin was also investigated on the catabolism by macrophages of native LDL. The effects of the drug on 125I-LDL or 125I-AcLDL cell degradation were compared in identical experimental conditions. Fluvastatin, despite its ability to inhibit 125I-AcLDL degradation, did not reduce degradation of 125I-LDL. On the contrary, the drug showed a slight but significant stimulatory effect on 125I-LDL degradation (Table 3⇓). To evaluate the effect of fluvastatin on AcLDL specific binding to plasma membrane, cells were treated for 44 hours with the drug, followed by a 4-hour incubation with 125I-AcLDL (40 μg/mL) at 4°C. The results indicate a lack of effect of fluvastatin in these experimental conditions (539±21 and 545±12 ng/mg cell protein of specific binding in control and treated cells, respectively).
We next investigated whether the reduced lipoprotein degradation could involve a nonspecific cloroquine-like inhibitory action of fluvastatin on lysosomal enzyme activity. To answer this question we performed experiments incubating the cells with [3H]CE-AcLDL (25 μg/mL) in the presence of the specific ACAT inhibitor S-58035. The blockade of intracellular re-esterification of cholesterol allowed us to assess the ability of lysosomes to hydrolyze cholesteryl esters transported by AcLDL. In the presence of S-58035 ≈85% of the radioactive cholesteryl ester released to cells by [3H]CE-AcLDL was hydrolyzed (Table 4⇓). The addition of fluvastatin only slightly influenced this result and did not induce any increase of radioactivity in the esterified fraction, suggesting a minor effect on the cellular hydrolysis of esterified cholesterol. By evaluating cellular cholesterol mass we observed that the ability of fluvastatin to reduce total cellular cholesterol was unaffected by S-58035 (Fig 2⇓).
We further investigated the effect of HMG-CoA reductase inhibition on AcLDL catabolism by studying the effect of fluvastatin on lipoprotein endocytosis. For this purpose we evaluated with fluorescence microscopy the action of the drug on the internalization by MPMs of Dil-AcLDL. As expected25 incubation of MPMs with Dil-AcLDL resulted in intense perinuclear fluorescence (Fig 3A⇓). The fluorescence was reduced in cells exposed to fluvastatin compared with control cells (Fig 3B⇓). The addition of mevalonate alone did not influence Dil-AcLDL uptake (data not shown), but in fluvastatin-treated cells it restored a pattern of fluorescence similar to that of control cells (Fig 3C⇓).
Since the action of fluvastatin on AcLDL catabolism appears to be linked to the inhibition of HMG-CoA reductase, and since the activity of this enzyme is partially suppressed in cholesterol-loaded cells, we explored the effect of fluvastatin in cholesterol-enriched macrophages compared with unloaded cells. Data reported in Fig 4⇓ show that fluvastatin was able to inhibit AcLDL degradation with an IC50 of about 2 μmol/L and 0.08 μmol/L in normal and cholesterol-loaded cells, respectively. The efficacy of fluvastatin was greater in loaded cells than in normal cells (48% versus 32% inhibition). In the same experimental conditions a similar pattern of activity was observed when the drug was tested on cholesterol esterification (Fig 5⇓).
Data presented in this study show that HMG-CoA reductase inhibitors reduce in vitro cholesterol accumulation elicited by AcLDL in MPMs by inhibiting the endocytosis of these lipoproteins by the cells. The experiments performed with [3H]CE-AcLDL indicate that fluvastatin does not increase the concentration of free cholesterol in the incubation medium; since no extracellular esterification activity was detected under our experimental conditions (data not shown), an effect of the drug on cholesterol efflux from cells can be excluded. The decrease of labeled cholesterol esters in cells elicited by fluvastatin is concomitant with an increase of radioactive cholesteryl ester in the incubation medium, suggesting its reduced influx into the cells. Altogether these results indicate that fluvastatin inhibition of cellular accumulation of esterified cholesterol elicited by AcLDL may involve a decreased influx of these lipoproteins into cells, with the consequent reduction of AcLDL-derived cholesterol release to ACAT. This hypothesis is consistent with our previous finding13 that vastatins inhibited cholesterol esterification when added to macrophages before AcLDL but not in cholesterol-preloaded cells, ie, when AcLDL-derived cholesterol has already been released to the substrate pool of ACAT. Confirmatory data of a decreased cellular influx of AcLDL are provided by the observation that fluvastatin inhibits the degradation of 125I-AcLDL by MPMs. This inhibitory effect was dose dependent in the same range of concentrations effective on cholesterol esterification and deposition. This effect was shared by simvastatin, suggesting that this property is related to the class of these drugs.
Experiments performed in the presence of the ACAT inhibitor S-58035 showed that fluvastatin does not induce the deposition of esterified cholesterol, indicating that the drug has a negligible effect on lysosomal esterase activity. Moreover, the ability of fluvastatin to reduce cellular cholesterol content in MPMs incubated with AcLDL was unaffected by the presence of the ACAT inhibitor. This observation indicates that the reduction of cellular cholesterol deposition elicited by vastatins is not related to ACAT activity and is consistent with an inhibitory effect on cholesterol delivery to cells. The results obtained with Dil-AcLDL, a means to visualize AcLDL internalization,25 offer direct evidence that fluvastatin inhibits AcLDL catabolism by preventing its endocytosis.
The inhibition by vastatins of AcLDL-induced cholesterol esterification in human macrophages was previously reported by Kempen et al.12 These authors reported a slight but not statistically significant effect of these drugs on AcLDL degradation, and concluded that the inhibitory effect of vastatins on cholesterol esterification could not be attributed to a decrease in receptor-mediated uptake or degradation of AcLDL. The reasons for this discrepancy are not clear. A species difference is possible; however, the results reported by Kempen et al, indicating a net reduction of total cellular cholesterol content in human macrophages (see Table 1⇑ in Reference 1212 ), are consistent with our observations. In any case, our results do not exclude additional mechanisms (such as an interference with the intracellular cholesterol trafficking)12 that may contribute to the ability of vastatins to reduce cholesterol esterification. This possibility is supported by the observation that in our experiments the inhibition of cholesterol esterification by fluvastatin was greater than that elicited on AcLDL degradation (compare Figs 4⇑ and 5⇑).
The mechanism involved in fluvastatin action on AcLDL endocytosis needs clarification. The effect is not related to a decreased expression of the scavenger receptors, since no decrease of cellular binding at 4°C could be detected. Although we cannot exclude an inhibitory effect of vastatins on the endocytosis of other ligands, the effect on AcLDL seems not to be due to a nonspecific depression of cellular endocytotic functions, since in the condition in which AcLDL degradation is reduced, a slight but significant increase in native LDL degradation is observed. A slight but consistent increase of receptor-mediated catabolism of LDL in cultured macrophages by HMG-CoA reductase inhibitors has been recently reported.26 Our results show that the inhibition by fluvastatin of AcLDL catabolism is reversed by mevalonate and its isoprenoid derivative geranylgeraniol. This result suggests the involvement of nonsterol products of the mevalonate pathway in AcLDL endocytosis. For example, geranylgeranylated proteins such as rab4 and rab5 may play a regulatory role in receptor-mediated endocytosis and recycling.27 28 Interestingly, we also observed that fluvastatin is more effective in cholesterol-loaded cells. These results may be explained when one considers that in cholesterol-rich cells, HMG-CoA reductase activity is already largely reduced compared with the activity in unloaded cells.29
The fluvastatin IC50s of ≈2 and 0.1 μmol/L, evaluated in normal and loaded cells respectively, are in the range of the reported therapeutic concentrations of the drug.30 The accumulation of cholesterol in macrophages may result from an imbalance between cholesterol influx and cholesterol efflux from cells.3 Vastatin inhibition of AcLDL endocytosis might therefore affect foam cell formation. In vivo evidence indicates that HMG-CoA reductase inhibitors may have beneficial effects on atheroma generation not only by reducing plama cholesterol levels but also by directly acting on the arterial wall.31 32
In conclusion the present study demonstrates that HMG-CoA reductase inhibitors may reduce cholesterol accumulation in macrophages by inhibiting lipoprotein endocytosis through the scavenger receptors. This effect is related to the depletion of nonsterol products of the mevalonate pathway. The present results may help in the understanding of the mechanism of cholesterol accumulation in the arterial wall. The observation that fluvastatin is more active on cholesterol-loaded macrophages (ie, foam cells) suggests the possibility that the mevalonate pathway in the arterial lesions may represent a selective target for pharmacological intervention.
Selected Abbreviations and Acronyms
|ACAT||=||acyl-CoA cholesterol acyltransferase|
|DMEM||=||Dulbecco’s minimal essential medium|
|EFAF-BSA||=||essentially fatty acid–free bovine serum albumin|
|FCS||=||fetal calf serum|
|[3H]CE-AcLDL||=||AcLDL labeled with [3H]cholesteryl ester|
|HMG-CoA||=||hydroxymethyl glutaryl coenzyme A|
|MPM(s)||=||mouse peritoneal macrophage(s)|
The research was partially supported by MPI and CNR (Italian Government). We are indebted to Dr David Via for providing Dil-AcLDL. The secretarial help of Laura Mozzarelli is also acknowledged.
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