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Additive inhibitory effect of hydrocortisone and cyclosporine on low-density lipoprotein receptor activity in cultured HepG2 cells
Additive Inhibitory Effect of Hydrocortisone and Cyclosporine on Low-Density Lipoprotein Receptor Activity in Cultured HepG2 Cells OSAMA AL RAYYES, ANDERS WALLMARK,
Both glucocorticoids and cyclosporine are used to prevent rejection in organ transplant recipients. However, long-term treatment with these drugs is known to induce hyperlipidemia and premature development of atherosclerosis. In previous studies, we have shown that the immunosuppressive drug cyclosporine inhibits catabolism of low-density lipoproteins (LDL) mainly by reducing the expression of LDL-receptor messenger RNA (mRNA), thus explaining the increased plasma levels of LDL cholesterol observed in patients treated with cyclosporine. In the present study, our objective was to investigate the mechanism by which glucocorticoids increase plasma levels of LDL cholesterol. We studied the catabolism of LDL in the human hepatoma cell line HepG2. Our results show that hydrocortisone at physiologically relevant concentrations inhibits LDL binding, uptake, and degradation in a dose-dependent way. Moreover, hydrocortisone also reduces the expression of LDL-receptor mRNA in a dose-dependent way. Cyclosporine also has an additive inhibitory effect on hydrocortisone in the catabolism of LDL. The 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor fluvastatin reverses the inhibitory effect of both hydrocortisone and cyclosporine. We conclude that treatment with hydrocortisone and/or cyclosporine induces increased plasma levels of LDL cholesterol because of reduced hepatic LDL receptor activity. HMG-CoA reductase inhibitors reverse this undesirable effect and thus reduce the risk of the development of atherosclerosis in patients subjected to immunosuppressive treatment. (HEPATOLOGY 1997;26:967-971.) Hyperlipidemia is one of the most serious long-term complications occurring in successful organ transplantation. Transplant recipients are prone to cardiovascular disease as a result of atherosclerosis induced by high levels of lowdensity lipoprotein (LDL) cholesterol levels. Hydrocortisone and the synthetic glucocorticoids (e.g., prednisolone) are
Abbreviations: LDL, low-density lipoprotein; mRNA, messenger RNA; HMG-CoA reductase inhibitor, 3-hydroxy-3-methylglutaryl CoA reductase inhibitor; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate buffered saline; HSA, human serum albumin; LDFCS, lipoprotein deficient fetal calf serum; RT-PCR, reverse-transcription polymerase chain reaction; cDNA, complementary DNA; MuLV, murine leukemia virus. From the Department of Medicine and Wallenberg Laboratory, Malmo¨ University Hospital, Malmo¨, Sweden. Received January 29, 1997; accepted May 15, 1997. Address reprint requests to: Osama Al Rayyes, M.D., Wallenberg Laboratory, 2nd floor, Malmo¨ University Hospital, S-205 02 Malmo¨, Sweden. Fax: 46-40-33-7041. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2604-0026$3.00/0
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widely used to prevent organ rejection in transplant recipients in combination with cyclosporine and azathioprine. Several clinical studies have shown that glucocorticoids or cyclosporine, or both in combination, increase the development of atherosclerosis as a result of increased levels of LDL cholesterol.1-8 Other clinical studies have also shown that the use of glucocorticoids in chronic diseases, such as rheumatoid arthritis and systemic lupus erythematosus, increases the liability for development of atherosclerosis due to development of hyperlipidemia.9-11 The aim of the present study was to elucidate the mechanism by which glucocorticoids (e.g., hydrocortisone) elevate blood levels of LDL cholesterol. Because the liver is responsible for about two thirds of the catabolism of LDL (mainly by the LDL receptor pathway), we performed the experiments on the hepatoma cell line HepG2, a well-defined, human-derived cell line that has maintained secretory functions, such as lipoprotein secretion, as well as functional LDL receptors.12,13 In previous studies, we showed that cyclosporine reduces the uptake of LDL by HepG2 cells mainly because of decreased expression of LDL-receptor protein.14 We also found that the cyclosporine-induced reduction of LDL uptake can be reversed by 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors,15 thus verifying earlier clinical observations.16,17 In the present work, we studied the inhibitory effect of hydrocortisone on the catabolism of LDL and investigated whether there is an additive or synergistic effect between hydrocortisone and cyclosporine. We also evaluated whether HMG-CoA reductase inhibitors can reverse this inhibitory effect. MATERIALS AND METHODS Hydrocortisone, Cyclosporine, and HMG-CoA Reductase Inhibitors. Hydrocortisone solution (Sigma Chemical Co., St. Louis, MO) was prepared by dissolving 5 mg of the powder in 1 mL of 96% ethanol. Cyclosporine and fluvastatin were obtained as powders from Sandoz Pharm. Ltd. (Basel, Switzerland). The cyclosporine solution was prepared by dissolving 10 mg of the powder in 0.5 mL of 96% ethanol followed by addition of 0.5 mL of water. During the experimental procedures, the maximal final concentration of ethanol was less than 0.01% in the tissue culture medium, when both compounds were used alone or together. Cell Culture. The established hepatoblastoma cell line HepG2 was obtained from the American Tissue Type Culture Collection (Rockville, MD) and grown as previously described.14 Lipoprotein Isolation and Labeling. Blood was drawn from normocholesterolemic subjects into Vacutainer tubes (100 1 16 mm) containing 0.084 mL of 0.34 mol/L ethylenediaminetetraacetic acid (EDTA; Becton Dickinson Co., Rutherford, NJ). Thimerosal (so-
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dium ethylmercurithiosalicylate, Sigma Chemical Co.) was added to a final concentration of 25 mmol/L to inhibit proteolytic activity and bacterial growth. LDL was isolated and labeled with 125I as previously described.14 LDL Uptake and Degradation Assays. Subconfluent HepG2 cells were washed with phosphate-buffered saline (PBS) and grown in 2 mL RPMI 1640 containing 0.5% human serum albumin (HSA; Sigma Chemical Co.), 125I-LDL (4 mg LDL protein/mL), hydrocortisone (100 ng/mL), and cyclosporine (600 ng/mL) at 377C for 24 hours. Degradation and uptake were then determined as described previously.14 LDL Binding Assays. The cells were washed with PBS and grown at 377C for 24 hours in 2 mL RPMI 1640 medium containing 0.5% HSA and different combinations of hydrocortisone (20-100 ng/mL) and cyclosporine (600 ng/mL). HSA was substituted with 5% lipoprotein-deficient fetal calf serum (LDFCS) in cell experiments involving treatment with fluvastatin (400 ng/mL). Binding studies of LDL were performed by washing the cells with ice-cold PBS and cooling at 47C for 20 minutes in 2 mL of ice-cold medium. The cells were incubated at 47C for 2 hours with 125I-LDL (4 mg/ mL). The LDL receptor–bound LDL was then released after washing the cells with ice-cold PBS, followed by a second incubation of the cells at 47C for 1 hour with 2 mL dextran sulfate (10 mg/mL) in a rotatory shaker (Inter Med, immuno VIB4, Roskilde, Denmark) at 60 revolutions/min.18 The medium was then aspirated and subjected to measurement of radioactivity. RNA Isolation. Total RNA was prepared from HepG2 cells by a single-step guanidium thiocyanate extraction method19 and stored at 0807C until LDL receptor messenger RNA (mRNA) was quantified by reverse-transcription polymerase chain reaction (RT-PCR). The quantity of RNA was estimated by spectrophotometric readings at 260 and 280 nm. RT-PCR. RNA was reverse-transcribed into complementary DNA (cDNA) in a 20-mL reaction mixture containing 1 mL of total cellular RNA (100 ng/mL), 1 mL of internal control template pAW109 cRNA (3 1 105 molecules), 1 mL of RNAse inhibitor (20 U/mL), 1 mL of random hexamers (50 mmol/L), 1 mL of murine leukemia virus (MuLV) reverse transcriptase (50 U/mL), 2 mL of 101 PCR buffer (500 mmol/L KCl, 100 mmol/L TRIS-HCl, pH 8.3), 4 mL of MgCl2 (25 mmol/L), 8 mL of dNTP (2.5 mmol/L in respect to dATP, dCTP, dGTP and dTTP), and 1 mL of diethyl pyrocarbonate-treated distilled water. All reagents were purchased from Perkin Elmer (Go¨teborg, Sweden) except diethyl pyrocarbonate, which was purchased from Sigma Chemical Co. The mixture was overlaid with 70 mL of mineral oil, and target molecules were reverse-transcribed with a Perkin Elmer Cetus thermocycler at 427C for 15 minutes. The reaction was stopped by denaturation of the reverse transcriptase at 997C for 5 minutes and then cooled at 57C for 5 minutes. The PCR was performed in 100 mL of reaction mixture by adding 80 mL of a master mix to the cDNA mix. The master mix contained 0.5 mL of Taq DNA polymerase (5 U/mL, AmpliTaq, Perkin Elmer, Go¨teborg, Sweden), 8 mL of 101 PCR buffer, 4 mL of MgCl2 , 65.25 mL of distilled water, 1.25 mL of 5*-fluorescein-labeled LDL receptor 5* primer (15 mmol/L) and 1 mL of unlabeled LDL receptor 3*primer (15 mmol/L).20 The oligonucleotides were purchased from Pharmacia Biotech (Uppsala, Sweden). The amplification was performed with a Perkin Elmer Cetus thermocycler using the following cycle profile: denaturation at 957C for 1 minute, primer annealing and extension at 607C for 1 minute. The initial step was prolonged to 3 minutes, and after 30 cycles the reaction mixture was incubated at 727C for 7 minutes and then cooled to 47C. Quantitative Analysis of Messenger RNA. Twenty microliters of each PCR product was electrophoresed along with a DNA molecular weight marker (fX174 cleaved with HaeIII, Pharmacia Biotech, Uppsala, Sweden) in 4% agarose-sieving gel (2:3 [wt/wt] NuSieve Agarose and 1:3 [wt/wt] SeaKem LE Agarose, In Vitro AB, Stockholm, Sweden) in TAE (40 mmol/L Tris, 20 mmol/L sodium acetate, 1 mmol/L EDTA, pH 7.4) running buffer at 90 V for 3 to 4 hours at cold room temperature. The gel was scanned in a FluorImager
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FIG. 1. Effect of various concentrations of hydrocortisone on dextran sulfate releasable binding of 125I-LDL to HepG2 cells. Each point represents mean { SD of three dishes.
SI (Molecular Dynamics, Sunnyvale, CA) using an excitation wavelength of 488 nm (argon laser). Images were analyzed using ImageQuant software (Molecular Dynamics), and the signal intensity was calculated with the ‘‘integrated volume’’ method. The amount of LDL receptor PCR fragment was normalized to that of the internal standard. The values are expressed as percent of levels of LDLreceptor mRNA in control HepG2 cells. Cell Protein Determination. The protein content of LDL and cells was determined using the assay of Lowry et al. with HSA as a standard.21 Statistical Analysis. The differences in the means in experimental results were analyzed for their statistical significance with Student’s t test or with one-way analysis of variance (ANOVA) and Scheffe´ multiple range test with a Å .05.22 The software SPSS (Statistical Package for Social Sciences) for Windows, release 6.0, was used for the statistical calculations.23 RESULTS Effect of Hydrocortisone on LDL Binding to HepG2 Cells. Figure 1 shows that hydrocortisone reduces the LDL binding to HepG2 cells in a dose-dependent manner. LDL binding is reduced by about 22% (P õ .01, t test) at a concentration of 20 ng/mL medium and by about 33% at a concentration of 100 ng/mL. A time study showed that the maximal inhibitory effect of hydrocortisone occurred after 24 hours (data not shown). Simultaneous Treatment With Hydrocortisone and Cyclosporine Reduces the LDL Binding to HepG2 Cells. As Table 1 shows,
cyclosporine has an additive rather than a synergistic effect to the inhibitory effect of hydrocortisone on the LDL binding to HepG2 cells. One-way ANOVA shows significantly reduced binding at a hydrocortisone concentration of 20 mg/ mL or at a cyclosporine concentration of 600 ng/mL. Combining the two drugs results in further reduced binding of LDL to the cells. The inhibitory effect of the combined treat-
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TABLE 1. Combined Effect of Hydrocortisone and Cyclosporine on Dextran Sulfate Releasable Binding of 125I-LDL to Hep G2 Cells Cyclosporine 0 ng/mL medium 600 ng/mL medium ng LDL/mg Cellular protein
Relative %
ng LDL/mg Cellular protein
Relative %
Hydrocortisone (ng/mL medium) 0 5.00 { 0.01 100 { 0.1 3.95 { 0.15 79.0 { 3.0 20 3.74 { 0.35 74.8 { 7.0 3.11 { 0.24 62.2 { 4.9 100 2.44 { 0.16 48.9 { 3.2 1.92 { 0.07 38.5 { 1.4 NOTE. Data are expressed as mean { SD of three dishes. One-way ANOVA and Scheffe´ multiple range test (a Å .05) show significant differences between means of all test combinations and control cells.
ment of the cells is totally reversed by simultaneous treatment with fluvastatin (400 ng/mL), as shown in Fig. 2. Effect of Subsequent Treatment With Hydrocortisone, Cyclosporine, and Fluvastatin on LDL Binding. Incubation of HepG2
cells with fluvastatin (400 ng/mL) for 24 hours (Fig. 3A) increases the dextran sulfate releasable binding of radiolabeled LDL by 159% (P õ .001, t test). A second incubation for 24 hours with hydrocortisone (100 mg/mL) alone or hydrocortisone combined with cyclosporine (600 ng/mL) reduces the LDL binding by 14% (P Å .05, t test) and by 33% (P õ .001, t test), respectively. Supplying the compounds in the inverse order does not significantly alter the net effect on the LDL receptor (Fig. 3B).
FIG. 3. The resultant effect after incubation for 24 hours with various combinations of hydrocortisone (100 ng/mL), cyclosporine (600 ng/mL), and fluvastatine (400 ng/mL) on dextran sulfate releasable binding of 125ILDL to HepG2 cells pretreated for 24 hours with (A) fluvastatin (400 ng/ mL) or (B) hydrocortisone (100 ng/mL)/cyclosporine (600 ng/mL). Each point represents mean { SD of three dishes.
Effect of Hydrocortisone and Cyclosporine on LDL Uptake and Degradation in HepG2 Cells. Hydrocortisone (20 ng/mL) sig-
nificantly inhibits LDL uptake and degradation (one-way ANOVA) by 13% and 10%, respectively (Fig. 4). Combining hydrocortisone with cyclosporine (600 ng/mL) magnifies the inhibitory effect on LDL uptake as well as degradation to 29% and 36%, respectively. Effect of Hydrocortisone and Cyclosporine on Expression of LDLReceptor mRNA. The yields of total RNA prepared from
FIG. 2. The combined effect of hydrocortisone (100 ng/mL), cyclosporine (600 ng/mL) and fluvastatine (400 ng/mL) on dextran sulfate releasable binding of 125I-LDL to HepG2 cells. Each point represents mean { SD of three dishes.
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HepG2 cells subjected to the different treatments were estimated between 40 to 70 mg/mg total cellular protein. Figure 5 shows that hydrocortisone reduces the expression of LDL receptor mRNA in a dose-dependent way: 25 ng/mL medium of hydrocortisone reduces the expression by 12.8% (P ú .05, Student’s t test) and 100 ng/mL by 29.5% (P õ .05,
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Hydrocortisone (ng/mL medium) 0 100
0
1,000
100 { 24.4 60.7 { 18.8
61.4 { 1.6 56.9 { 13.5
NOTE. Values are expressed as a percentage of levels of LDL-receptor mRNA in nontreated HepG2 cells. Data are expressed as mean { SD of three dishes.
DISCUSSION
FIG. 4. 125I-LDL uptake and degradation in HepG2 cells treated with hydrocortisone (20 ng/mL) and/or cyclosporine (600 ng/mL) for 24 hours. Each point represents mean { SD of three dishes.
Student’s t test), respectively. As shown in Table 2, combined treatment of HepG2 cells with hydrocortisone (100 ng/mL) and cyclosporine (1,000 ng/mL) pronounces the inhibitory effect on the expression of LDL receptor mRNA.
FIG. 5. Effect of various concentrations of hydrocortisone on expression of the LDL receptor mRNA in HepG2 cells. The values are expressed as percent of levels of LDL receptor mRNA in nontreated cells. Each point represents mean { SD of four dishes.
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Glucocorticoids have an anti-inflammatory action and are for this reason used in the treatment of autoimmune diseases and for immunosuppression. Usually glucocorticoids and cyclosporine with addition of azathioprine are used to prevent organ rejection in transplant recipients. However, both glucocorticoids and cyclosporine can cause an elevation of LDL cholesterol levels,1-8 and we have shown earlier that the cause of elevated LDL-cholesterol levels in cyclosporinetreated patients is a down-regulation of hepatic LDL receptors.14 In this study, we extend the experiments to include glucocorticoids and to investigate what effect the combination of glucocorticoids and cyclosporine has on hepatic LDLreceptor activity. Our results show that hydrocortisone in physiological concentrations decrease LDL binding, uptake, and degradation, mainly by reducing the LDL-receptor activity. The down-regulatory effect of hydrocortisone started to appear after 4 hours and reached a maximum after 24 hours. Because the effect started to appear after 4 hours, it was plausible that hydrocortisone mediated its effect on synthesis of the LDL-receptor protein. By RT-PCR, we found that hydrocortisone decreased transcription of mRNA of the LDL receptor by about 40%. Moreover, in some experiments, we induced the LDL receptor by the HMG-CoA reductase inhibitor fluvastatin. The results of these experiments are consistent with the conclusion that hydrocortisone decreases hepatic LDL catabolism by reducing LDL-receptor activity. Our results partially agree with a previous study of cultured human fibroblasts and arterial smooth muscle cells, which showed that hydrocortisone decreased cellular LDL uptake and degradation but did not decrease binding.24 In our previous study, we showed that cyclosporine inhibits LDL catabolism in HepG2 cells by about 25%.14 This inhibition occurs mainly through down-regulation of LDL receptor activity by reduction of the expression of mRNA of the LDL receptor. Those results and the present ones show that both cyclosporine and hydrocortisone have the same effect on LDL catabolism. Because both cyclosporine and glucocorticoids are used to prevent organ rejection, we investigated whether the drugs have a synergistic or an additive effect on downregulation of LDL catabolism. We found an additive effect. Thus, in conclusion, this study shows that hydrocortisone decreases LDL catabolism mainly as a result of its downregulatory effect on LDL receptors by reduction of the expression of mRNA and that both hydrocortisone and cyclosporine have an additive hyperlipidemic effect that can be counteracted by HMG-CoA reductase inhibitors. These results support the trend observed at some transplantation centers toward use of HMG-CoA reductase inhibitors to treat the
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hyperlipidemia induced by both glucocorticoids and cyclosporine.16,17 The results also provide a rationale for treating patients (e.g., patients with rheumatoid arthritis or systemic lupus erythematosus) who are put on long-term therapy with glucocorticoids with HMG-CoA reductase inhibitors. However, caution is advised, because rhabdomyolysis has been described in cases in which cyclosporine is combined with the HMG-CoA reductase inhibitor lovastatin.25 Acknowledgment: Supported by grants from the Ernhold Lundstro¨m Foundation, the Swedish Medical Research Council, the Pa˚hlsson Foundation and the Research Foundation of Malmo¨ University Hospital. The authors gratefully acknowledge Camilla Orbjo¨rn for her excellent technical assistence. REFERENCES 1. Bittar AE, Ratcliffe PJ, Richardson AJ, Raine AE, Jones L, Yudkin PL, Carter R, Mann JI, Morris PJ. The prevalence of hyperlipidemia in renal transplant recipients. Associations with immunosuppressive and antihypertensive therapy. Transplantation 1990;50:987-992. 2. Yoshimura N, Oka T, Okamoto M, Ohmori Y. The effects of pravastatin on hyperlipidemia in renal transplant recipients. Transplantation 1992; 53:94-99. 3. Hricik DE, Bartucci MR, Mayes JT, Schulak JA. The effects of steroid withdrawal on the lipoprotein profiles of cyclosporine-treated kidney and kidney-pancreas transplant recipients. Transplantation 1992;54: 868-871. 4. Ingulli E, Tejani A, Markell M. The beneficial effects of steroid withdrawal on blood pressure and lipid profile in children posttransplantation in the cyclosporine era. Transplantation 1993;55:1029-1033. 5. Montagnino G, Tarantino A, Aroldi A, Banfi G, Cesana B, Ponticelli C. Lipid profile in renal transplant recipients under various immunosuppressive regimens. Transplant Proc 1994;26:2634-2636. 6. Armstrong VW, Kaltefleiter M, Luy Kaltefleiter M, Schutz E, Wieland E, Loss M, Winkler M, Ringe B, Oellerich M. Metabolic liver function and lipoprotein metabolism after orthotopic liver transplantation in patients on immunosuppressive therapy with FK 506 or cyclosporine. Transplant Proc 1995;27:1201-1203. 7. Gunnarsson R, Lo¨fmark R, Nordlander R, Nyquist O, Groth C-G. Acute myocardial infarction in renal transplant recipients: incidence and prognosis. Eur Heart J 1984;5:218-221.
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8. Lo´pez-Miranda J, Pe´rez-Jime´nez F, Torres A, Espino-Montoro A, Gomez P, Hidalgo-Rojas L, Ordovas MJ, et al. Effects of cyclosporine on plasma lipoproteins in bone marrow transplantation patients. Clin Biochem 1992;25:379-386. 9. Spiera H and Rothenberg RR. Myocardial infarction in four young patients with SLE. J Rheumatol 1983;10:464-466. 10. Nashel DJ. Is atherosclerosis a complication of long-term corticosteroid treatment. Am J Med 1986;80:925-929. 11. Ettinger WH, Goldberg AP, Applebaum-Bowden D. Dyslipoproteinemia in systemic lupus erythematosus. Am J Med 1987;83:503-508. 12. Havel RJ, Hamilton RL. Hepatocytic lipoprotein receptors and intracellular lipoprotein catabolism. HEPATOLOGY 1988;8:1689-1704. 13. Javitt NB. HepG2 cells as a resource for metabolic studies: lipoproteins, cholesterol, and bile acids. FASEB J 1990;4:161-168. 14. Al Rayyes O, Wallmark A, and Flore´n C-H. Cyclosporine inhibits catabolism of low-density lipoproteins in HepG2 cells by 25%. HEPATOLOGY 1996;24:613-619. 15. Al Rayyes O, Wallmark A, Flore´n C-H. Reversal of cyclosporine inhibited low density lipoprotein receptor activity in HepG2 cells by 3-hydroxy-3 methylglutaryl coenzyme A reductase inhibitors. HEPATOLOGY 1997;25:991-994. 16. Kandus A, Kovac D, Kveder R and Bren AF. Lovastatin treatment of hyperlipidemia in kidney transplant recipients on cyclosporine immunosuppression. Transplant Proc 1994;26:2642-2643. 17. Vanhaecke J, Cleemput JV, Lierde JV, Daenen W, and Geest HD. Safety and efficacy of low-dose simvastatin in cardiac transplant recipients treated with cyclosporine. Transplantation 1994;58:42-45. 18. Salter AM, Saxton J, Brindley D. Characterization of the binding of human low-density lipoprotein to primary monolayer cultures of rat hepatocytes. Biochem J 1986;240:549-557. 19. Ausubel MA, Bernt R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Preparation and analysis of RNA. In: Current Protocols in Molecular Biology. Cambridge: Green and Wiley, 1993:1:4.0.1-4.10.9. 20. Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A 1989;86:9717-9721. 21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurements with folin phenol reagents. J Biol Chem 1951;193:265-275. 22. Afifi AA, Azen SP. Statistical Analysis. A Computer Oriented Approach (2nd ed.). New York: Academic, 1977. 23. Norusis MJ. SPSS for Windows, rel. 6.0. SPSS Inc., Chicago, IL, 1993. 24. Henze K, Chait A, Albers JJ, Bierman EL. Hydrocortisone decreases the internalization of low density lipoprotein in cultured human fibroblasts and arterial smooth muscle cells. Eur J Clin Invest 1983;13:171-177. 25. Corpier CL, Jones PH, Suki WN, Lederer ED, Quinones MA, Schmidt SW, Young JB. Rhabdomyolysis and renal injury with lovastatin use. JAMA 1988;260:239-241.
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Both glucocorticoids and cyclosporine are used to prevent rejection in organ transplant recipients. However, long-term treatment with these drugs is known to induce hyperlipidemia and premature development of atherosclerosis. In previous studies, we have shown that the immunosuppressive drug cyclosporine inhibits catabolism of low-density lipoproteins (LDL) mainly by reducing the expression of LDL-receptor messenger RNA (mRNA), thus explaining the increased plasma levels of LDL cholesterol observed in patients treated with cyclosporine. In the present study, our objective was to investigate the mechanism by which glucocorticoids increase plasma levels of LDL cholesterol. We studied the catabolism of LDL in the human hepatoma cell line HepG2. Our results show that hydrocortisone at physiologically relevant concentrations inhibits LDL binding, uptake, and degradation in a dose-dependent way. Moreover, hydrocortisone also reduces the expression of LDL-receptor mRNA in a dose-dependent way. Cyclosporine also has an additive inhibitory effect on hydrocortisone in the catabolism of LDL. The 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor fluvastatin reverses the inhibitory effect of both hydrocortisone and cyclosporine. We conclude that treatment with hydrocortisone and/or cyclosporine induces increased plasma levels of LDL cholesterol because of reduced hepatic LDL receptor activity. HMG-CoA reductase inhibitors reverse this undesirable effect and thus reduce the risk of the development of atherosclerosis in patients subjected to immunosuppressive treatment. (HEPATOLOGY 1997;26:967-971.) Hyperlipidemia is one of the most serious long-term complications occurring in successful organ transplantation. Transplant recipients are prone to cardiovascular disease as a result of atherosclerosis induced by high levels of lowdensity lipoprotein (LDL) cholesterol levels. Hydrocortisone and the synthetic glucocorticoids (e.g., prednisolone) are
Abbreviations: LDL, low-density lipoprotein; mRNA, messenger RNA; HMG-CoA reductase inhibitor, 3-hydroxy-3-methylglutaryl CoA reductase inhibitor; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate buffered saline; HSA, human serum albumin; LDFCS, lipoprotein deficient fetal calf serum; RT-PCR, reverse-transcription polymerase chain reaction; cDNA, complementary DNA; MuLV, murine leukemia virus. From the Department of Medicine and Wallenberg Laboratory, Malmo¨ University Hospital, Malmo¨, Sweden. Received January 29, 1997; accepted May 15, 1997. Address reprint requests to: Osama Al Rayyes, M.D., Wallenberg Laboratory, 2nd floor, Malmo¨ University Hospital, S-205 02 Malmo¨, Sweden. Fax: 46-40-33-7041. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2604-0026$3.00/0
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widely used to prevent organ rejection in transplant recipients in combination with cyclosporine and azathioprine. Several clinical studies have shown that glucocorticoids or cyclosporine, or both in combination, increase the development of atherosclerosis as a result of increased levels of LDL cholesterol.1-8 Other clinical studies have also shown that the use of glucocorticoids in chronic diseases, such as rheumatoid arthritis and systemic lupus erythematosus, increases the liability for development of atherosclerosis due to development of hyperlipidemia.9-11 The aim of the present study was to elucidate the mechanism by which glucocorticoids (e.g., hydrocortisone) elevate blood levels of LDL cholesterol. Because the liver is responsible for about two thirds of the catabolism of LDL (mainly by the LDL receptor pathway), we performed the experiments on the hepatoma cell line HepG2, a well-defined, human-derived cell line that has maintained secretory functions, such as lipoprotein secretion, as well as functional LDL receptors.12,13 In previous studies, we showed that cyclosporine reduces the uptake of LDL by HepG2 cells mainly because of decreased expression of LDL-receptor protein.14 We also found that the cyclosporine-induced reduction of LDL uptake can be reversed by 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors,15 thus verifying earlier clinical observations.16,17 In the present work, we studied the inhibitory effect of hydrocortisone on the catabolism of LDL and investigated whether there is an additive or synergistic effect between hydrocortisone and cyclosporine. We also evaluated whether HMG-CoA reductase inhibitors can reverse this inhibitory effect. MATERIALS AND METHODS Hydrocortisone, Cyclosporine, and HMG-CoA Reductase Inhibitors. Hydrocortisone solution (Sigma Chemical Co., St. Louis, MO) was prepared by dissolving 5 mg of the powder in 1 mL of 96% ethanol. Cyclosporine and fluvastatin were obtained as powders from Sandoz Pharm. Ltd. (Basel, Switzerland). The cyclosporine solution was prepared by dissolving 10 mg of the powder in 0.5 mL of 96% ethanol followed by addition of 0.5 mL of water. During the experimental procedures, the maximal final concentration of ethanol was less than 0.01% in the tissue culture medium, when both compounds were used alone or together. Cell Culture. The established hepatoblastoma cell line HepG2 was obtained from the American Tissue Type Culture Collection (Rockville, MD) and grown as previously described.14 Lipoprotein Isolation and Labeling. Blood was drawn from normocholesterolemic subjects into Vacutainer tubes (100 1 16 mm) containing 0.084 mL of 0.34 mol/L ethylenediaminetetraacetic acid (EDTA; Becton Dickinson Co., Rutherford, NJ). Thimerosal (so-
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dium ethylmercurithiosalicylate, Sigma Chemical Co.) was added to a final concentration of 25 mmol/L to inhibit proteolytic activity and bacterial growth. LDL was isolated and labeled with 125I as previously described.14 LDL Uptake and Degradation Assays. Subconfluent HepG2 cells were washed with phosphate-buffered saline (PBS) and grown in 2 mL RPMI 1640 containing 0.5% human serum albumin (HSA; Sigma Chemical Co.), 125I-LDL (4 mg LDL protein/mL), hydrocortisone (100 ng/mL), and cyclosporine (600 ng/mL) at 377C for 24 hours. Degradation and uptake were then determined as described previously.14 LDL Binding Assays. The cells were washed with PBS and grown at 377C for 24 hours in 2 mL RPMI 1640 medium containing 0.5% HSA and different combinations of hydrocortisone (20-100 ng/mL) and cyclosporine (600 ng/mL). HSA was substituted with 5% lipoprotein-deficient fetal calf serum (LDFCS) in cell experiments involving treatment with fluvastatin (400 ng/mL). Binding studies of LDL were performed by washing the cells with ice-cold PBS and cooling at 47C for 20 minutes in 2 mL of ice-cold medium. The cells were incubated at 47C for 2 hours with 125I-LDL (4 mg/ mL). The LDL receptor–bound LDL was then released after washing the cells with ice-cold PBS, followed by a second incubation of the cells at 47C for 1 hour with 2 mL dextran sulfate (10 mg/mL) in a rotatory shaker (Inter Med, immuno VIB4, Roskilde, Denmark) at 60 revolutions/min.18 The medium was then aspirated and subjected to measurement of radioactivity. RNA Isolation. Total RNA was prepared from HepG2 cells by a single-step guanidium thiocyanate extraction method19 and stored at 0807C until LDL receptor messenger RNA (mRNA) was quantified by reverse-transcription polymerase chain reaction (RT-PCR). The quantity of RNA was estimated by spectrophotometric readings at 260 and 280 nm. RT-PCR. RNA was reverse-transcribed into complementary DNA (cDNA) in a 20-mL reaction mixture containing 1 mL of total cellular RNA (100 ng/mL), 1 mL of internal control template pAW109 cRNA (3 1 105 molecules), 1 mL of RNAse inhibitor (20 U/mL), 1 mL of random hexamers (50 mmol/L), 1 mL of murine leukemia virus (MuLV) reverse transcriptase (50 U/mL), 2 mL of 101 PCR buffer (500 mmol/L KCl, 100 mmol/L TRIS-HCl, pH 8.3), 4 mL of MgCl2 (25 mmol/L), 8 mL of dNTP (2.5 mmol/L in respect to dATP, dCTP, dGTP and dTTP), and 1 mL of diethyl pyrocarbonate-treated distilled water. All reagents were purchased from Perkin Elmer (Go¨teborg, Sweden) except diethyl pyrocarbonate, which was purchased from Sigma Chemical Co. The mixture was overlaid with 70 mL of mineral oil, and target molecules were reverse-transcribed with a Perkin Elmer Cetus thermocycler at 427C for 15 minutes. The reaction was stopped by denaturation of the reverse transcriptase at 997C for 5 minutes and then cooled at 57C for 5 minutes. The PCR was performed in 100 mL of reaction mixture by adding 80 mL of a master mix to the cDNA mix. The master mix contained 0.5 mL of Taq DNA polymerase (5 U/mL, AmpliTaq, Perkin Elmer, Go¨teborg, Sweden), 8 mL of 101 PCR buffer, 4 mL of MgCl2 , 65.25 mL of distilled water, 1.25 mL of 5*-fluorescein-labeled LDL receptor 5* primer (15 mmol/L) and 1 mL of unlabeled LDL receptor 3*primer (15 mmol/L).20 The oligonucleotides were purchased from Pharmacia Biotech (Uppsala, Sweden). The amplification was performed with a Perkin Elmer Cetus thermocycler using the following cycle profile: denaturation at 957C for 1 minute, primer annealing and extension at 607C for 1 minute. The initial step was prolonged to 3 minutes, and after 30 cycles the reaction mixture was incubated at 727C for 7 minutes and then cooled to 47C. Quantitative Analysis of Messenger RNA. Twenty microliters of each PCR product was electrophoresed along with a DNA molecular weight marker (fX174 cleaved with HaeIII, Pharmacia Biotech, Uppsala, Sweden) in 4% agarose-sieving gel (2:3 [wt/wt] NuSieve Agarose and 1:3 [wt/wt] SeaKem LE Agarose, In Vitro AB, Stockholm, Sweden) in TAE (40 mmol/L Tris, 20 mmol/L sodium acetate, 1 mmol/L EDTA, pH 7.4) running buffer at 90 V for 3 to 4 hours at cold room temperature. The gel was scanned in a FluorImager
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FIG. 1. Effect of various concentrations of hydrocortisone on dextran sulfate releasable binding of 125I-LDL to HepG2 cells. Each point represents mean { SD of three dishes.
SI (Molecular Dynamics, Sunnyvale, CA) using an excitation wavelength of 488 nm (argon laser). Images were analyzed using ImageQuant software (Molecular Dynamics), and the signal intensity was calculated with the ‘‘integrated volume’’ method. The amount of LDL receptor PCR fragment was normalized to that of the internal standard. The values are expressed as percent of levels of LDLreceptor mRNA in control HepG2 cells. Cell Protein Determination. The protein content of LDL and cells was determined using the assay of Lowry et al. with HSA as a standard.21 Statistical Analysis. The differences in the means in experimental results were analyzed for their statistical significance with Student’s t test or with one-way analysis of variance (ANOVA) and Scheffe´ multiple range test with a Å .05.22 The software SPSS (Statistical Package for Social Sciences) for Windows, release 6.0, was used for the statistical calculations.23 RESULTS Effect of Hydrocortisone on LDL Binding to HepG2 Cells. Figure 1 shows that hydrocortisone reduces the LDL binding to HepG2 cells in a dose-dependent manner. LDL binding is reduced by about 22% (P õ .01, t test) at a concentration of 20 ng/mL medium and by about 33% at a concentration of 100 ng/mL. A time study showed that the maximal inhibitory effect of hydrocortisone occurred after 24 hours (data not shown). Simultaneous Treatment With Hydrocortisone and Cyclosporine Reduces the LDL Binding to HepG2 Cells. As Table 1 shows,
cyclosporine has an additive rather than a synergistic effect to the inhibitory effect of hydrocortisone on the LDL binding to HepG2 cells. One-way ANOVA shows significantly reduced binding at a hydrocortisone concentration of 20 mg/ mL or at a cyclosporine concentration of 600 ng/mL. Combining the two drugs results in further reduced binding of LDL to the cells. The inhibitory effect of the combined treat-
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TABLE 1. Combined Effect of Hydrocortisone and Cyclosporine on Dextran Sulfate Releasable Binding of 125I-LDL to Hep G2 Cells Cyclosporine 0 ng/mL medium 600 ng/mL medium ng LDL/mg Cellular protein
Relative %
ng LDL/mg Cellular protein
Relative %
Hydrocortisone (ng/mL medium) 0 5.00 { 0.01 100 { 0.1 3.95 { 0.15 79.0 { 3.0 20 3.74 { 0.35 74.8 { 7.0 3.11 { 0.24 62.2 { 4.9 100 2.44 { 0.16 48.9 { 3.2 1.92 { 0.07 38.5 { 1.4 NOTE. Data are expressed as mean { SD of three dishes. One-way ANOVA and Scheffe´ multiple range test (a Å .05) show significant differences between means of all test combinations and control cells.
ment of the cells is totally reversed by simultaneous treatment with fluvastatin (400 ng/mL), as shown in Fig. 2. Effect of Subsequent Treatment With Hydrocortisone, Cyclosporine, and Fluvastatin on LDL Binding. Incubation of HepG2
cells with fluvastatin (400 ng/mL) for 24 hours (Fig. 3A) increases the dextran sulfate releasable binding of radiolabeled LDL by 159% (P õ .001, t test). A second incubation for 24 hours with hydrocortisone (100 mg/mL) alone or hydrocortisone combined with cyclosporine (600 ng/mL) reduces the LDL binding by 14% (P Å .05, t test) and by 33% (P õ .001, t test), respectively. Supplying the compounds in the inverse order does not significantly alter the net effect on the LDL receptor (Fig. 3B).
FIG. 3. The resultant effect after incubation for 24 hours with various combinations of hydrocortisone (100 ng/mL), cyclosporine (600 ng/mL), and fluvastatine (400 ng/mL) on dextran sulfate releasable binding of 125ILDL to HepG2 cells pretreated for 24 hours with (A) fluvastatin (400 ng/ mL) or (B) hydrocortisone (100 ng/mL)/cyclosporine (600 ng/mL). Each point represents mean { SD of three dishes.
Effect of Hydrocortisone and Cyclosporine on LDL Uptake and Degradation in HepG2 Cells. Hydrocortisone (20 ng/mL) sig-
nificantly inhibits LDL uptake and degradation (one-way ANOVA) by 13% and 10%, respectively (Fig. 4). Combining hydrocortisone with cyclosporine (600 ng/mL) magnifies the inhibitory effect on LDL uptake as well as degradation to 29% and 36%, respectively. Effect of Hydrocortisone and Cyclosporine on Expression of LDLReceptor mRNA. The yields of total RNA prepared from
FIG. 2. The combined effect of hydrocortisone (100 ng/mL), cyclosporine (600 ng/mL) and fluvastatine (400 ng/mL) on dextran sulfate releasable binding of 125I-LDL to HepG2 cells. Each point represents mean { SD of three dishes.
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HepG2 cells subjected to the different treatments were estimated between 40 to 70 mg/mg total cellular protein. Figure 5 shows that hydrocortisone reduces the expression of LDL receptor mRNA in a dose-dependent way: 25 ng/mL medium of hydrocortisone reduces the expression by 12.8% (P ú .05, Student’s t test) and 100 ng/mL by 29.5% (P õ .05,
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HEPATOLOGY October 1997 TABLE 2. Combined Effect of Hydrocortisone and Cyclosporine on Expression of LDL-Receptor mRNA in HepG2 Cells Cyclosporine (ng/mL medium)
Hydrocortisone (ng/mL medium) 0 100
0
1,000
100 { 24.4 60.7 { 18.8
61.4 { 1.6 56.9 { 13.5
NOTE. Values are expressed as a percentage of levels of LDL-receptor mRNA in nontreated HepG2 cells. Data are expressed as mean { SD of three dishes.
DISCUSSION
FIG. 4. 125I-LDL uptake and degradation in HepG2 cells treated with hydrocortisone (20 ng/mL) and/or cyclosporine (600 ng/mL) for 24 hours. Each point represents mean { SD of three dishes.
Student’s t test), respectively. As shown in Table 2, combined treatment of HepG2 cells with hydrocortisone (100 ng/mL) and cyclosporine (1,000 ng/mL) pronounces the inhibitory effect on the expression of LDL receptor mRNA.
FIG. 5. Effect of various concentrations of hydrocortisone on expression of the LDL receptor mRNA in HepG2 cells. The values are expressed as percent of levels of LDL receptor mRNA in nontreated cells. Each point represents mean { SD of four dishes.
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Glucocorticoids have an anti-inflammatory action and are for this reason used in the treatment of autoimmune diseases and for immunosuppression. Usually glucocorticoids and cyclosporine with addition of azathioprine are used to prevent organ rejection in transplant recipients. However, both glucocorticoids and cyclosporine can cause an elevation of LDL cholesterol levels,1-8 and we have shown earlier that the cause of elevated LDL-cholesterol levels in cyclosporinetreated patients is a down-regulation of hepatic LDL receptors.14 In this study, we extend the experiments to include glucocorticoids and to investigate what effect the combination of glucocorticoids and cyclosporine has on hepatic LDLreceptor activity. Our results show that hydrocortisone in physiological concentrations decrease LDL binding, uptake, and degradation, mainly by reducing the LDL-receptor activity. The down-regulatory effect of hydrocortisone started to appear after 4 hours and reached a maximum after 24 hours. Because the effect started to appear after 4 hours, it was plausible that hydrocortisone mediated its effect on synthesis of the LDL-receptor protein. By RT-PCR, we found that hydrocortisone decreased transcription of mRNA of the LDL receptor by about 40%. Moreover, in some experiments, we induced the LDL receptor by the HMG-CoA reductase inhibitor fluvastatin. The results of these experiments are consistent with the conclusion that hydrocortisone decreases hepatic LDL catabolism by reducing LDL-receptor activity. Our results partially agree with a previous study of cultured human fibroblasts and arterial smooth muscle cells, which showed that hydrocortisone decreased cellular LDL uptake and degradation but did not decrease binding.24 In our previous study, we showed that cyclosporine inhibits LDL catabolism in HepG2 cells by about 25%.14 This inhibition occurs mainly through down-regulation of LDL receptor activity by reduction of the expression of mRNA of the LDL receptor. Those results and the present ones show that both cyclosporine and hydrocortisone have the same effect on LDL catabolism. Because both cyclosporine and glucocorticoids are used to prevent organ rejection, we investigated whether the drugs have a synergistic or an additive effect on downregulation of LDL catabolism. We found an additive effect. Thus, in conclusion, this study shows that hydrocortisone decreases LDL catabolism mainly as a result of its downregulatory effect on LDL receptors by reduction of the expression of mRNA and that both hydrocortisone and cyclosporine have an additive hyperlipidemic effect that can be counteracted by HMG-CoA reductase inhibitors. These results support the trend observed at some transplantation centers toward use of HMG-CoA reductase inhibitors to treat the
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hyperlipidemia induced by both glucocorticoids and cyclosporine.16,17 The results also provide a rationale for treating patients (e.g., patients with rheumatoid arthritis or systemic lupus erythematosus) who are put on long-term therapy with glucocorticoids with HMG-CoA reductase inhibitors. However, caution is advised, because rhabdomyolysis has been described in cases in which cyclosporine is combined with the HMG-CoA reductase inhibitor lovastatin.25 Acknowledgment: Supported by grants from the Ernhold Lundstro¨m Foundation, the Swedish Medical Research Council, the Pa˚hlsson Foundation and the Research Foundation of Malmo¨ University Hospital. The authors gratefully acknowledge Camilla Orbjo¨rn for her excellent technical assistence. REFERENCES 1. Bittar AE, Ratcliffe PJ, Richardson AJ, Raine AE, Jones L, Yudkin PL, Carter R, Mann JI, Morris PJ. The prevalence of hyperlipidemia in renal transplant recipients. Associations with immunosuppressive and antihypertensive therapy. Transplantation 1990;50:987-992. 2. Yoshimura N, Oka T, Okamoto M, Ohmori Y. The effects of pravastatin on hyperlipidemia in renal transplant recipients. Transplantation 1992; 53:94-99. 3. Hricik DE, Bartucci MR, Mayes JT, Schulak JA. The effects of steroid withdrawal on the lipoprotein profiles of cyclosporine-treated kidney and kidney-pancreas transplant recipients. Transplantation 1992;54: 868-871. 4. Ingulli E, Tejani A, Markell M. The beneficial effects of steroid withdrawal on blood pressure and lipid profile in children posttransplantation in the cyclosporine era. Transplantation 1993;55:1029-1033. 5. Montagnino G, Tarantino A, Aroldi A, Banfi G, Cesana B, Ponticelli C. Lipid profile in renal transplant recipients under various immunosuppressive regimens. Transplant Proc 1994;26:2634-2636. 6. Armstrong VW, Kaltefleiter M, Luy Kaltefleiter M, Schutz E, Wieland E, Loss M, Winkler M, Ringe B, Oellerich M. Metabolic liver function and lipoprotein metabolism after orthotopic liver transplantation in patients on immunosuppressive therapy with FK 506 or cyclosporine. Transplant Proc 1995;27:1201-1203. 7. Gunnarsson R, Lo¨fmark R, Nordlander R, Nyquist O, Groth C-G. Acute myocardial infarction in renal transplant recipients: incidence and prognosis. Eur Heart J 1984;5:218-221.
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8. Lo´pez-Miranda J, Pe´rez-Jime´nez F, Torres A, Espino-Montoro A, Gomez P, Hidalgo-Rojas L, Ordovas MJ, et al. Effects of cyclosporine on plasma lipoproteins in bone marrow transplantation patients. Clin Biochem 1992;25:379-386. 9. Spiera H and Rothenberg RR. Myocardial infarction in four young patients with SLE. J Rheumatol 1983;10:464-466. 10. Nashel DJ. Is atherosclerosis a complication of long-term corticosteroid treatment. Am J Med 1986;80:925-929. 11. Ettinger WH, Goldberg AP, Applebaum-Bowden D. Dyslipoproteinemia in systemic lupus erythematosus. Am J Med 1987;83:503-508. 12. Havel RJ, Hamilton RL. Hepatocytic lipoprotein receptors and intracellular lipoprotein catabolism. HEPATOLOGY 1988;8:1689-1704. 13. Javitt NB. HepG2 cells as a resource for metabolic studies: lipoproteins, cholesterol, and bile acids. FASEB J 1990;4:161-168. 14. Al Rayyes O, Wallmark A, and Flore´n C-H. Cyclosporine inhibits catabolism of low-density lipoproteins in HepG2 cells by 25%. HEPATOLOGY 1996;24:613-619. 15. Al Rayyes O, Wallmark A, Flore´n C-H. Reversal of cyclosporine inhibited low density lipoprotein receptor activity in HepG2 cells by 3-hydroxy-3 methylglutaryl coenzyme A reductase inhibitors. HEPATOLOGY 1997;25:991-994. 16. Kandus A, Kovac D, Kveder R and Bren AF. Lovastatin treatment of hyperlipidemia in kidney transplant recipients on cyclosporine immunosuppression. Transplant Proc 1994;26:2642-2643. 17. Vanhaecke J, Cleemput JV, Lierde JV, Daenen W, and Geest HD. Safety and efficacy of low-dose simvastatin in cardiac transplant recipients treated with cyclosporine. Transplantation 1994;58:42-45. 18. Salter AM, Saxton J, Brindley D. Characterization of the binding of human low-density lipoprotein to primary monolayer cultures of rat hepatocytes. Biochem J 1986;240:549-557. 19. Ausubel MA, Bernt R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Preparation and analysis of RNA. In: Current Protocols in Molecular Biology. Cambridge: Green and Wiley, 1993:1:4.0.1-4.10.9. 20. Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A 1989;86:9717-9721. 21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurements with folin phenol reagents. J Biol Chem 1951;193:265-275. 22. Afifi AA, Azen SP. Statistical Analysis. A Computer Oriented Approach (2nd ed.). New York: Academic, 1977. 23. Norusis MJ. SPSS for Windows, rel. 6.0. SPSS Inc., Chicago, IL, 1993. 24. Henze K, Chait A, Albers JJ, Bierman EL. Hydrocortisone decreases the internalization of low density lipoprotein in cultured human fibroblasts and arterial smooth muscle cells. Eur J Clin Invest 1983;13:171-177. 25. Corpier CL, Jones PH, Suki WN, Lederer ED, Quinones MA, Schmidt SW, Young JB. Rhabdomyolysis and renal injury with lovastatin use. JAMA 1988;260:239-241.
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