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Effects of hypoxia on cholesterol metabolism in human monocyte-derived macrophages
Life Sciences 67 (2000) 2083Ð2091
Effects of hypoxia on cholesterol metabolism in human monocyte-derived macrophages Ken Matsumotoa, Takahiro Taniguchia,*, Yoshio Fujiokaa, Hiroshi Shimizua, Yuichi Ishikawab, Mitsuhiro Yokoyamaa a
The First Department of Internal Medicine, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan b Faculty of Health Sciences, Kobe University School of Medicine, Kobe 650-0017, Japan
Abstract We assessed the metabolism of low density lipoprotein (LDL) of human monocyte-derived macrophages under hypoxia. The speciÞc binding and association of 125I-labeled LDL (125I-LDL) were not changed under hypoxia compared to normoxia. However, the degradation of 125I-LDL under hypoxia decreased to 60%. The rate of cholesterol esteriÞcation under hypoxia was 2-fold greater on incubation with LDL or 25-hydroxycholesterol. The cellular cholesteryl ester content was also greater under hypoxia on incubation with LDL. Secretion of apolipoprotein E into the medium was not altered under hypoxia, suggesting that apolipoprotein E independent cholesterol efßux may be reduced under hypoxia. Thus, hypoxia affects the intracellular metabolism of LDL, stimulates cholesterol esteriÞcation, and enhances cholesteryl ester accumulation in macrophages. Hypoxia is one of the important factors modifying the cellular lipid metabolism in arterial wall. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Hypoxia; Macrophage; Low density lipoprotein; Cholesterol esteriÞcation; Cholesterol accumulation
Introduction The arterial wall has an anatomically speciÞc structure for its blood supply. The oxygen supply to the arterial wall is unique since it is regulated by the two different sources; the rate of oxygen supply to the inner third of the arterial wall is dependent solely on the radial diffusion from endothelium of the lumen which is in contact with highly oxygenated blood, while that to the outer two-thirds of the arterial wall is dependent on the supply from adventitial vasa vasorum (1). The avascular medial zone of the arterial wall is likely to be exposed to hy-
* Corresponding author. Tel.: 181-78-382-5846; fax: 181-78-382-5859. E-mail address: [email protected] (T. Taniguchi) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 7 9 4 -3
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poxia easily. Conditions that increase wall thickness and reduce oxygen diffusion or oxygen transmissibility also produce hypoxia and steep PO2 gradients within the wall (2). Several investigators have reported that it was 10Ð20 mmHg in experimental rabbit atheroma (3), the aorta of hypertensive rabbits (4) and diabetic rabbits (5). Deleterious effects of hypoxia on atherogenesis have been reported in cholesterol-fed rabbits and Watanabe heritable hyperlipidemic (WHHL) rabbits (6,7). Lorenzen et al. showed the development of severe gross arteriosclerosis in the rabbit aorta as a result of exposure to systemic hypoxia (8). Thus, the reduced oxygen supply to the arterial wall is expected to take part in atherogenesis. It has been also reported that the hypoxia affects the lipid metabolism in aortic tissue cultures (9,10), in aortic smooth muscle cells (11,12), in skin Þbroblasts (13,14) and in myocardial cells (15). The monocytes in circulating blood where the oxygen tension is high are recruited to the arterial walls where the oxygen tension is low. These microenvironmental changes may affect the LDL metabolism in human monocyte-derived macrophages (16). The oxygen consumption of foam cell fractions isolated from rabbit aortic intima-media was three times higher than that of smooth muscle cell fractions isolated from the same sample (17). Therefore, it is speculated that as the intimal thickness and the number of foam cells in the intima increases, the diffusion capacity of oxygen to the deeper parts of the arterial wall may decrease. The present study was designed to investigate the metabolism of LDL in human monocytederived macrophages under hypoxia and elucidate the effects of hypoxia on the atherogenesis. Materials and methods Materials Sodium [125I] iodide, [1-14C] oleic acid, and [1,2,6,7-3H] cholesteryl oleate were purchased from DuPont-New England Nuclear (Boston, MA). IODO-GEN was obtained from Pierce Chemical Co. (Rockford, IL). RPMI-1640 medium was purchased from Whittaker Bioproducts (Walkersville, MD). Culture dishes and plates were obtained from Corning (Iwaki Glass, Tokyo, Japan) and Becton Dickinson Labware (Lincoln Park, NJ). 25-hydroxycholesterol and essentially fatty acid free bovine serum albumin were obtained from Sigma (St.Louis, MO). All other chemicals were of commercially available reagent grade. Preparation of low density lipoprotein Low density lipoprotein (LDL, d51.020Ð1.060 g/ml) was prepared from human plasma of fasted normolipidemic volunteers and isolated by sequential ultracentrifugation as described previously (18). LDL was radio-iodinated using carrier-free Na125I and IODO-GEN (19). The speciÞc activity of the 125I-LDL was approximately 300Ð500 cpm/ng of protein. There was no difference in the electrophoretic mobility on agarose gel electrophoresis (Corning, Palo Alto, CA) among freshly isolated native LDL, LDL incubated under normoxia, and LDL incubated under hypoxia for 24 h. The contents of total cholesterol, triglyceride, and phospholipid in LDL were measured by enzymatic techniques. The content of lipid peroxide in LDL was determined by the TBA (thiobarbituric acid) and MCDP (10-N methylcarbamoyl-3, 7-dimethylamino-10h-phenothiazine) methods (20). There were no differences in the contents of cholesterol, triglyceride, phospholipid and lipid peroxide among LDLs. Protein
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concentrations were determined by the method of Lowry et al. (21) using bovine serum albumin as a standard. Cells Human monocyte-derived macrophages (HMDM) were obtained from human peripheral blood according to the modiÞed method of Bšyum (22) with the Leuco PREP¨ tube (Becton Dickinson Labware. Lincoln Park, NJ). Cells were seeded at a concentration of 1Ð3 3 106 cells/ml of RPMI 1640 supplemented with penicillin (100 IU/ml) and streptomycin (100 mg/ ml), and cultured for 2 h at 378C in 5% CO2 and 95% air. Then the non-adherent cells were removed and the cells adhered to dishes were incubated with RPMI 1640 supplemented with 10% autologous serum, and the culture medium was changed every two days. The cells, adhered to each dish, were used as HMDM within 9 days after plating. There was no signiÞcant difference between the two incubation conditions in the light microscopic Þndings. The contents of cell protein and the numbers were not different between both incubations. Hypoxia 24 hours before each experiment, the cells were separated into the two incubation conditions (normoxia and hypoxia). Normoxic condition was performed at 378C in 95% air/5% CO2 and hypoxic condition was at 378C in 1.5% O2/5% CO2/balanced N2 with O2/CO2 multigas incubator BL-3200 (Astec, Fukuoka, Japan). Oxygen tension in the culture medium in each incubation condition was measured using PO2 monitor (PO2-100, Inter Medical, Aichi, Japan). Oxygen tension under hypoxia was approximately 10 mmHg, whereas that under normoxia was approximately 150 mmHg. The pH of the culture medium was measured by gas analyzer (model 1312-Blood Gas Manager, Instrumentation Laboratory, Milano, Italy). There was no signiÞcant difference in pH of both culture medium (hypoxia; 7.14260.002 and normoxia; 7.14460.005, n54). LDL binding, degradation, and association studies LDL binding assay of HMDM was performed at 48C essentially according to the method of Goldstein and Brown (23). After incubation for 24 h under normoxia and hypoxia, and pre-cooling at 48C for 30 min, the cells were incubated with RPMI 1640 containing various concentrations of 125I-LDL at 48C for 2 h. Thereafter, the cells were washed and dissolved in 0.5 ml of 0.5 N NaOH for measuring the radioactivities and protein concentrations. NonspeciÞc binding was determined in parallel incubations with a 20-fold excess of unlabeled LDL and speciÞc binding was calculated by subtracting nonspeciÞc binding from total binding. Degradation and association experiments were performed at 378C for 5 h under normoxia or hypoxia. Then, the medium was transferred to a glass tube containing trichloroacetic acid (TCA) to precipitate non-degraded 125I-LDL. TCA soluble supernatant was used for measuring the amounts of degraded radioactivity. On the other hand, the cells were washed, and dissolved with 0.5 N NaOH for measuring the amount of associated radioactivities and protein concentration. SpeciÞc degradation and association were determined as described above.
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Cellular cholesteryl ester synthesis Cholesteryl ester synthesis was determined by measuring the rate of the incorporation of [ C]-oleate into the cholesteryl ester of the cells, according to the method of Goldstein and Brown (23) with minor modiÞcations. HMDM were incubated in RPMI-1640 containing various concentrations of LDL or a mixture of 5 mg/ml 25-hydroxycholesterol and 10 mg/ml cholesterol which were dissolved in ethanol for 24 h (except for the time course experiment) under normoxia or hypoxia, and then pulse-labeled with 4 mM [14C]-oleate-albumin-complex for 6 h at 378C. Thereafter, cellular lipids were extracted in hexane-isopropyl alcohol (3:2 vol/vol), to which [1,2,6,7-3H] cholesteryl oleate was added as an internal standard, and then evaporated to dryness under nitrogen. The lipids were developed on silica gel plates with isooctane/diethyl ether/acetic acid (75/25/2 vol/vol/vol) and the cholesteryl ester spot was visualized with iodine vapor, cut from the chromatogram, and counted by an analytic liquid scintillation counter. 14
Measurement of cellular cholesterol content The intracellular free and esteriÞed cholesterol contents were measured by the method of Heider and Boyett (24). HMDM were incubated with RPMI-1640 containing various concentrations of LDL for 24 h under normoxia and hypoxia, and the cellular lipids extracted in hexane:isopropyl alcohol. The content of cholesteryl ester was calculated by subtracting the content of free cholesterol from that of total cholesterol. For the determination of cholesterol efßux, the cells were loaded with LDL by a 48-hour incubation in RPMI1640 containing autologus serum and LDL (100 mg/ml), incubated with lipoprotein deÞcient serum (LPDS) for 48 hours, and followed by measurement of the content of cholesteryl ester. Determination of apolipoprotein E (apo E) secretion Apo E in culture media from the experiments of determination of cholesterol efßux was quantiÞed using an enzyme-linked immunosorbent assay (ELISA) (25) with anti-apo E monoclonal antibody and puriÞed apo E as a standard (kindly gifted by Daiichi Chemicals, Tokyo, Japan). Northern blotting analysis Northern blotting of ACAT1 mRNA was performed according to the methods previously described (Ref. 25, the probes were kindly gifted by Dr. T.Y. Chang and Dr. S. Horiuchi). Statistics All data are presented as mean 6 SEM except Fig. 1. Paired and unpaired StudentÕs t tests were used to evaluate the statistical signiÞcance of difference. Results Effect of hypoxia on cellular binding and uptake of LDL The rates of binding of LDL to the cells at 48C were determined after 24 hours preincubation under normoxia and hypoxia, and there was no signiÞcant difference between the two incubation conditions (Fig. 1). Next, we studied the effect of hypoxia on cellular uptake of
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Fig. 1. Effects of hypoxia on binding of 125I-LDL to HMDM. Under normoxia or hypoxia, HMDM were cultured for 24 h, and thereafter incubated with indicated concentrations of 125I-LDL at 48C for 2 h. The amounts of 125 I-LDL bound to the cells (open bars, normoxic; Þlled bars, hypoxic) were determined and speciÞc binding was calculated as described in Materials and Methods. Results are representative of three experiments and each point represents the mean of duplicate dishes. N; normoxia, H; hypoxia.
LDL at 378C. There were measurable and speciÞc uptake of 125I-LDL in both conditions. We found no signiÞcant difference in cellular association of 125I-LDL between the two conditions (Fig. 2A), however, degradation of 125I-LDL was suppressed under hypoxia compared to that under normoxia (Fig. 2B). Effect of hypoxia on cholesteryl ester synthesis To test whether hypoxia affect cholesteryl ester synthesis in HMDM, the rate of incorporation of 14C-oleate into cellular cholesteryl ester was studied. The rate of basal cholesterol es-
Fig. 2. Effects of hypoxia on association(A) and degradation(B) of 125I-LDL in HMDM. Under normoxic (open bars) or hypoxic (Þlled bars) condition, HMDM were cultured for 24 h and thereafter incubated with indicated concentrations of 125I-LDL at 378C for 5 h, and the amounts of associated 125I-LDL and degraded 125I-LDL from TCA soluble fraction of medium were determined as described in Methods. Results are representative of four experiments and each point represents the mean 6 SEM of three dishes (* p,0.05, ** p,0.005; compared to normoxia).
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Fig. 3. Effects of hypoxia on cholesteryl ester synthesis in HMDM. Under normoxic (open bars and squares) or hypoxic (Þlled bars and squares) condition, HMDM were incubated with indicated concentrations of LDL for 24 h (A) or incubated with 100 mg/ml of LDL for indicated hours (B). After incubation, the incorporation of 14C-oleate into cellular cholesteryl ester was measured as described in Methods. Results are representative of Þve (A) or three (B) experiments and each value represents the mean 6 SEM of Þve dishes (* p,0.05, ** p,0.005; compared to normoxia).
teriÞcation (without LDL) under hypoxia was twice higher than that under normoxia (Fig. 3A). This result is compatible with the data observed in smooth muscle cells and Þbroblasts (13,14). Hypoxia induced twice greater rate of cholesterol esteriÞcation in HMDM under incubation with LDL (Fig. 3A). Greater rate of cholesterol esteriÞcation of hypoxia was also found in time-dependent fashion in HMDM (Fig. 3B). Addition of 25-hydroxycholesterol, oxysterols taken up via the LDL-receptor-independent pathway, led to a signiÞcant increase of cholesteryl 14C-oleate formation under both normoxia and hypoxia (Fig. 4). Effect of hypoxia on cholesteryl ester accumulation To test whether the stimulated cholesteryl ester synthesis in hypoxic condition resulted in intracellular cholesteryl ester accumulation, we incubated the cells with indicated concentration of LDL for 24 hours, and measured cellular total-, free- and esteriÞed-cholesterol. Cellular free-cholesterol content increased slightly, but not signiÞcantly, under both normoxic and hypoxic conditions (data not shown). However, the increase in cellular esteriÞed-cholesterol content under hypoxia was signiÞcantly larger than that under normoxia (Fig. 5). Effect of hypoxia on cholesterol efßux and apo E secretion To elucidate the effect of hypoxia on cholesterol efßux and apo E secretion, we measured the amount of cellular cholesterol under normoxic and hypoxic condition with lipoprotein deÞcient serum. There was no difference in free cholesterol contents between normoxia and hypoxia. The decrease in the content of cholesteryl ester was smaller under hypoxia (Fig. 6A), though it was not signiÞcant. There was no difference in the secretion of apo E under both conditions (Fig. 6B). These results suggest that, under hypoxia, the efßux of cholesterol may be suppressed by apo E-independent mechanism.
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Fig. 4. Effects of hypoxia on cholesteryl ester synthesis in HMDM incubated with 25-hydroxycholesterol. Under normoxia or hypoxia, HMDM were cultured for 24 h and thereafter incubated with culture medium containing 14 C-oleate-albumin complex with (1) or without (2) a mixture of 5 mg/ml of 25-hydroxycholesterol and 10 mg/ ml of cholesterol. After 6 h incubation, the incorporation of 14C-oleate into cellular cholesteryl ester was measured as described in Materials and Methods. Results are representative of four experiments and each value represents the mean 6 SEM of Þve dishes. (* P,0.005; compared to normoxia).
Discussion We have found in this study that the degradation of LDL in HMDM under hypoxia was lower than that under normoxia, though there was no differences in binding and association of LDL between hypoxic and normoxic incubations. Albers et al. (12) reported that in cultured human arterial smooth muscle cells the binding of 125I-LDL under hypoxic incubation
Fig. 5. Effects of hypoxia on the content of cholesteryl ester. Under normoxia or hypoxia, HMDM were incubated for 24 h with indicated concentrations of LDL and thereafter the cellular contents of total and free cholesterol were determined. Cholesterol ester was calculated as described in Methods. Results are representative of three experiments and each value represents the mean 6 SEM of Þve dishes (* p,0.05; compared to normoxia).
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Fig. 6. A; The change of the content of cholesteryl ester of lipid-loaded HMDM before and after incubation in the medium containing LPDS under normoxia or hypoxia. HMDM were incubated for 24 h with LDL (100mg/ml), and then incubated for 48 hours with LPDS. The percentage compared to control condition (before incubation with LPDS) was calculated from the cellular contents of cholesteryl ester determined as described in Methods. Results are the mean 6 SEM of three experiments. B; The concentration of secreted apo E in medium. Lipidloaded HMDM were incubated for 48 h with LPDS under normoxia or hypoxia, and thereafter the concentration of apo E were determined by ELISA as described in Methods. Results are representative of three experiments and each value represents the mean 6 SEM of three dishes.
was similar to that under normoxic incubation. They also demonstrated that the degradation of 125I-LDL under hypoxic incubation was less as compared to that under normoxic incubation. Our results agree with their Þndings, and support the idea that hypoxia did not change the afÞnity and the number of LDL receptor on the cell surface. Tsukitani et al. reported that ACAT activity increased in a time-dependent manner and cholesteryl ester increased under hypoxic incubation in cultured rabbit skin Þbroblasts (13). In this study, we obtained the similar results in HMDM. Additionally, we showed that 25-hydroxycholesterol, which cells take up via LDL receptor-independent pathway, stimulated cholesteryl ester synthesis more extensively under hypoxic incubation. Mukodani showed the efßux of cholesterol was suppressed under hypoxic incubation in cultured rabbit skin Þbroblasts (14). In HMDM, we showed that there was reduced efßux of cellular esteriÞed cholesterol in incubation with lipoprotein deÞcient serum under hypoxia. This is considered to be due to the changes of the LDL metabolism in HMDM, not to be due to the changes of characteristics of LDL particles as mentioned already or apo E secretion (26). We have examined Northern blot analysis for ACAT1 (25,27), resulting in no alteration in hypoxia (data not shown). Though ACAT1 could be predominant in macrophages (28), it is important to identify which acyltransferase enzyme(s) is affected under hypoxia. Taken together, we speculate the mechanism of reduced LDL degradation and increased cholesteryl ester synthesis as follows; First, decreased degradation under hypoxic condition may be due to reduced rate of the transport of degraded lipoprotein into the culture medium or lowered activities of lysosomal enzymes. Hypoxia might modulate the transport of cholesterol and protein in the cytosol. As a result, the amount of microsomal cholesterol available for esteriÞcation may increase and cholesteryl ester synthesis may be stimulated, resulting in the reduction of cholesterol efßux under hypoxia. Second, as the rate of cholesterol esteriÞcation increased under hypoxic condition without the addi-
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tion of LDL or 25-hydroxycholesterol as shown in Fig. 3A and 4, hypoxia may directly stimulate cholesterol ester synthesis, though it remains still unclear what kind of mechanisms operate cholesteryl ester synthesis under hypoxia. It is also necessary to investigate the comparison with modiÞed LDL like oxidation, and the cholesterol efßux in incubation with high density lipoproteins. Moreover, in intact animal experiments, hypoxia could induce physical stress and increase the catecholamines level that might accelerate the atherosclerosis. Hypoxia could affect expression of speciÞc genes associated with vascular tone and remodeling (29), and production of oxyradical formation and tissue damage (2). Further investigations are needed to clarify which mechanism could be operated to the cellular cholesterol metabolism by hypoxia. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
E. GEIRINGER, J. Pathol. Bacteriol. 63 201Ð211 (1951). D.W. CRAWFORD, and D.H. BLANKENHORN, Atherosclerosis 89 97Ð108 (1991). C. HEUGHAN, J. NIINIKOSKI, and T.K. HUNT, Atherosclerosis 17 361Ð367 (1973). S.M. SANTILLI, V.D. FIEGEL, and D.R. KNIGHTON, Hypertension 19 33Ð39 (1992). S.M. SANTILLI, V.D. FIEGEL, and D.R. KNIGHTON, J. Vasc. Surg. 18 227Ð233 (1993). K. KJELDSEN, J. WANSTRUP, and P. ASTRUP, J. Atherosclerosis Res. 8 835Ð845 (1968). R. OKAMOTO, M. HATANI, M. TSUKITANI, A. SUEHIRO, M. FUJINO, N. IMAI, S. TAKANO, Y. WATANABE, and H. FUKUZAKI, Atherosclerosis 47 47Ð53 (1983). I. LORENZEN, and P. HELIN, Acta Path. et Microbiol. Scandinav. 69 158Ð159(1967). I. FILIPOVIC, and M. RUTEM…LLER, Atherosclerosis 24 457Ð469 (1976). C.F. HOWARD, Jr., Atherosclerosis 15 359Ð369 (1972). M. TSUKITANI, R. OKAMOTO, and H. FUKUZAKI, Atherosclerosis 52 167Ð174 (1984). J.J. ALBERS, and E.L. BIERMAN, Biochim. Biophys. Acta. 424 422Ð429 (1976). M. TSUKITANI, R. OKAMOTO, Y. ISHIKAWA, J. MUKODANI, N. WATANABE, A. SUEHIRO, M. HATANI, M. FUJINO, S. TAKANO, Y. WATANABE, and H. FUKUZAKI, J. Clin. Biochem. Nutr. 2 279Ð286 (1987). J. MUKODANI, Y. ISHIKAWA, and H. FUKUZAKI, Arteriosclerosis 10 106Ð110 (1990). M. HOLLENBERG, Circ. Res. 28 148Ð157 (1971). M.S. BROWN, Y.K. HO, and J.L. GOLDSTEIN, J. Biol. Chem. 255 9344Ð9352 (1980). T. BJORNHEDEN, and G. BONDJERS, Arteriosclerosis 7 238Ð247 (1987). R.J. HAVEL, H.A. ELDER, and J.H. BRAGDON, J. Clin. Invest. 34 1345Ð1352 (1955). P.R.P. SALACINSKI, C. MCLEAN, J.E.C. SYKES, V.V. CLEMENT-JONES, and P.J. LOWRY, Anal. Biochem. 117 136Ð146 (1981). N. OHISHI, H. OHKAWA, A. MIIKE, T. TAKANO, and K. YAGI, Biochem. Int. 10 205Ð211(1985). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR, and R.J. RANDALL, J. Biol. Chem. 193 265Ð275 (1951). A. B…YUM, Scand. J. Clin. Lab. Invest. 21(suppl.97) 77Ð89 (1968). J.L. GOLDSTEIN, S.K. BASU, and M.S. BROWN, Methods in Enzymology 98 241Ð260 (1983). J.G. HEIDER, and R.L. BOYETT, J. Lipid Res. 19 514Ð518 (1978). H. MATSUDA, H. HAKAMATA, A. MIYAZAKI, M. SAKAI, C.C.Y. CHANG, T.Y. CHANG, S. KOBORI, M. SHICHIRI, and S. HORIUCHI, Biochim. Biophys. Acta. 1301 76Ð84 (1996). W-Y. ZHANG, P.M. GAYNOR, and H.S. KRUTH, J. Biol. Chem. 271 28641Ð28646 (1996). O. LEE, C.C.Y. CHANG, W. LEE, and T.Y. CHANG, J. Lipid Res. 39 1722Ð1727 (1998). C. JOYCE, K. SKINNER, R.A. ANDERSON, and L.L. RUDEL, Curr. Opin. Lipidol. 10 89Ð95 (1999). D.V. FALLER, Clin. Exp. Pharmaco. Physiol. 26 74Ð84 (1999).
Effects of hypoxia on cholesterol metabolism in human monocyte-derived macrophages Ken Matsumotoa, Takahiro Taniguchia,*, Yoshio Fujiokaa, Hiroshi Shimizua, Yuichi Ishikawab, Mitsuhiro Yokoyamaa a
The First Department of Internal Medicine, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan b Faculty of Health Sciences, Kobe University School of Medicine, Kobe 650-0017, Japan
Abstract We assessed the metabolism of low density lipoprotein (LDL) of human monocyte-derived macrophages under hypoxia. The speciÞc binding and association of 125I-labeled LDL (125I-LDL) were not changed under hypoxia compared to normoxia. However, the degradation of 125I-LDL under hypoxia decreased to 60%. The rate of cholesterol esteriÞcation under hypoxia was 2-fold greater on incubation with LDL or 25-hydroxycholesterol. The cellular cholesteryl ester content was also greater under hypoxia on incubation with LDL. Secretion of apolipoprotein E into the medium was not altered under hypoxia, suggesting that apolipoprotein E independent cholesterol efßux may be reduced under hypoxia. Thus, hypoxia affects the intracellular metabolism of LDL, stimulates cholesterol esteriÞcation, and enhances cholesteryl ester accumulation in macrophages. Hypoxia is one of the important factors modifying the cellular lipid metabolism in arterial wall. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Hypoxia; Macrophage; Low density lipoprotein; Cholesterol esteriÞcation; Cholesterol accumulation
Introduction The arterial wall has an anatomically speciÞc structure for its blood supply. The oxygen supply to the arterial wall is unique since it is regulated by the two different sources; the rate of oxygen supply to the inner third of the arterial wall is dependent solely on the radial diffusion from endothelium of the lumen which is in contact with highly oxygenated blood, while that to the outer two-thirds of the arterial wall is dependent on the supply from adventitial vasa vasorum (1). The avascular medial zone of the arterial wall is likely to be exposed to hy-
* Corresponding author. Tel.: 181-78-382-5846; fax: 181-78-382-5859. E-mail address: [email protected] (T. Taniguchi) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 7 9 4 -3
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poxia easily. Conditions that increase wall thickness and reduce oxygen diffusion or oxygen transmissibility also produce hypoxia and steep PO2 gradients within the wall (2). Several investigators have reported that it was 10Ð20 mmHg in experimental rabbit atheroma (3), the aorta of hypertensive rabbits (4) and diabetic rabbits (5). Deleterious effects of hypoxia on atherogenesis have been reported in cholesterol-fed rabbits and Watanabe heritable hyperlipidemic (WHHL) rabbits (6,7). Lorenzen et al. showed the development of severe gross arteriosclerosis in the rabbit aorta as a result of exposure to systemic hypoxia (8). Thus, the reduced oxygen supply to the arterial wall is expected to take part in atherogenesis. It has been also reported that the hypoxia affects the lipid metabolism in aortic tissue cultures (9,10), in aortic smooth muscle cells (11,12), in skin Þbroblasts (13,14) and in myocardial cells (15). The monocytes in circulating blood where the oxygen tension is high are recruited to the arterial walls where the oxygen tension is low. These microenvironmental changes may affect the LDL metabolism in human monocyte-derived macrophages (16). The oxygen consumption of foam cell fractions isolated from rabbit aortic intima-media was three times higher than that of smooth muscle cell fractions isolated from the same sample (17). Therefore, it is speculated that as the intimal thickness and the number of foam cells in the intima increases, the diffusion capacity of oxygen to the deeper parts of the arterial wall may decrease. The present study was designed to investigate the metabolism of LDL in human monocytederived macrophages under hypoxia and elucidate the effects of hypoxia on the atherogenesis. Materials and methods Materials Sodium [125I] iodide, [1-14C] oleic acid, and [1,2,6,7-3H] cholesteryl oleate were purchased from DuPont-New England Nuclear (Boston, MA). IODO-GEN was obtained from Pierce Chemical Co. (Rockford, IL). RPMI-1640 medium was purchased from Whittaker Bioproducts (Walkersville, MD). Culture dishes and plates were obtained from Corning (Iwaki Glass, Tokyo, Japan) and Becton Dickinson Labware (Lincoln Park, NJ). 25-hydroxycholesterol and essentially fatty acid free bovine serum albumin were obtained from Sigma (St.Louis, MO). All other chemicals were of commercially available reagent grade. Preparation of low density lipoprotein Low density lipoprotein (LDL, d51.020Ð1.060 g/ml) was prepared from human plasma of fasted normolipidemic volunteers and isolated by sequential ultracentrifugation as described previously (18). LDL was radio-iodinated using carrier-free Na125I and IODO-GEN (19). The speciÞc activity of the 125I-LDL was approximately 300Ð500 cpm/ng of protein. There was no difference in the electrophoretic mobility on agarose gel electrophoresis (Corning, Palo Alto, CA) among freshly isolated native LDL, LDL incubated under normoxia, and LDL incubated under hypoxia for 24 h. The contents of total cholesterol, triglyceride, and phospholipid in LDL were measured by enzymatic techniques. The content of lipid peroxide in LDL was determined by the TBA (thiobarbituric acid) and MCDP (10-N methylcarbamoyl-3, 7-dimethylamino-10h-phenothiazine) methods (20). There were no differences in the contents of cholesterol, triglyceride, phospholipid and lipid peroxide among LDLs. Protein
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concentrations were determined by the method of Lowry et al. (21) using bovine serum albumin as a standard. Cells Human monocyte-derived macrophages (HMDM) were obtained from human peripheral blood according to the modiÞed method of Bšyum (22) with the Leuco PREP¨ tube (Becton Dickinson Labware. Lincoln Park, NJ). Cells were seeded at a concentration of 1Ð3 3 106 cells/ml of RPMI 1640 supplemented with penicillin (100 IU/ml) and streptomycin (100 mg/ ml), and cultured for 2 h at 378C in 5% CO2 and 95% air. Then the non-adherent cells were removed and the cells adhered to dishes were incubated with RPMI 1640 supplemented with 10% autologous serum, and the culture medium was changed every two days. The cells, adhered to each dish, were used as HMDM within 9 days after plating. There was no signiÞcant difference between the two incubation conditions in the light microscopic Þndings. The contents of cell protein and the numbers were not different between both incubations. Hypoxia 24 hours before each experiment, the cells were separated into the two incubation conditions (normoxia and hypoxia). Normoxic condition was performed at 378C in 95% air/5% CO2 and hypoxic condition was at 378C in 1.5% O2/5% CO2/balanced N2 with O2/CO2 multigas incubator BL-3200 (Astec, Fukuoka, Japan). Oxygen tension in the culture medium in each incubation condition was measured using PO2 monitor (PO2-100, Inter Medical, Aichi, Japan). Oxygen tension under hypoxia was approximately 10 mmHg, whereas that under normoxia was approximately 150 mmHg. The pH of the culture medium was measured by gas analyzer (model 1312-Blood Gas Manager, Instrumentation Laboratory, Milano, Italy). There was no signiÞcant difference in pH of both culture medium (hypoxia; 7.14260.002 and normoxia; 7.14460.005, n54). LDL binding, degradation, and association studies LDL binding assay of HMDM was performed at 48C essentially according to the method of Goldstein and Brown (23). After incubation for 24 h under normoxia and hypoxia, and pre-cooling at 48C for 30 min, the cells were incubated with RPMI 1640 containing various concentrations of 125I-LDL at 48C for 2 h. Thereafter, the cells were washed and dissolved in 0.5 ml of 0.5 N NaOH for measuring the radioactivities and protein concentrations. NonspeciÞc binding was determined in parallel incubations with a 20-fold excess of unlabeled LDL and speciÞc binding was calculated by subtracting nonspeciÞc binding from total binding. Degradation and association experiments were performed at 378C for 5 h under normoxia or hypoxia. Then, the medium was transferred to a glass tube containing trichloroacetic acid (TCA) to precipitate non-degraded 125I-LDL. TCA soluble supernatant was used for measuring the amounts of degraded radioactivity. On the other hand, the cells were washed, and dissolved with 0.5 N NaOH for measuring the amount of associated radioactivities and protein concentration. SpeciÞc degradation and association were determined as described above.
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Cellular cholesteryl ester synthesis Cholesteryl ester synthesis was determined by measuring the rate of the incorporation of [ C]-oleate into the cholesteryl ester of the cells, according to the method of Goldstein and Brown (23) with minor modiÞcations. HMDM were incubated in RPMI-1640 containing various concentrations of LDL or a mixture of 5 mg/ml 25-hydroxycholesterol and 10 mg/ml cholesterol which were dissolved in ethanol for 24 h (except for the time course experiment) under normoxia or hypoxia, and then pulse-labeled with 4 mM [14C]-oleate-albumin-complex for 6 h at 378C. Thereafter, cellular lipids were extracted in hexane-isopropyl alcohol (3:2 vol/vol), to which [1,2,6,7-3H] cholesteryl oleate was added as an internal standard, and then evaporated to dryness under nitrogen. The lipids were developed on silica gel plates with isooctane/diethyl ether/acetic acid (75/25/2 vol/vol/vol) and the cholesteryl ester spot was visualized with iodine vapor, cut from the chromatogram, and counted by an analytic liquid scintillation counter. 14
Measurement of cellular cholesterol content The intracellular free and esteriÞed cholesterol contents were measured by the method of Heider and Boyett (24). HMDM were incubated with RPMI-1640 containing various concentrations of LDL for 24 h under normoxia and hypoxia, and the cellular lipids extracted in hexane:isopropyl alcohol. The content of cholesteryl ester was calculated by subtracting the content of free cholesterol from that of total cholesterol. For the determination of cholesterol efßux, the cells were loaded with LDL by a 48-hour incubation in RPMI1640 containing autologus serum and LDL (100 mg/ml), incubated with lipoprotein deÞcient serum (LPDS) for 48 hours, and followed by measurement of the content of cholesteryl ester. Determination of apolipoprotein E (apo E) secretion Apo E in culture media from the experiments of determination of cholesterol efßux was quantiÞed using an enzyme-linked immunosorbent assay (ELISA) (25) with anti-apo E monoclonal antibody and puriÞed apo E as a standard (kindly gifted by Daiichi Chemicals, Tokyo, Japan). Northern blotting analysis Northern blotting of ACAT1 mRNA was performed according to the methods previously described (Ref. 25, the probes were kindly gifted by Dr. T.Y. Chang and Dr. S. Horiuchi). Statistics All data are presented as mean 6 SEM except Fig. 1. Paired and unpaired StudentÕs t tests were used to evaluate the statistical signiÞcance of difference. Results Effect of hypoxia on cellular binding and uptake of LDL The rates of binding of LDL to the cells at 48C were determined after 24 hours preincubation under normoxia and hypoxia, and there was no signiÞcant difference between the two incubation conditions (Fig. 1). Next, we studied the effect of hypoxia on cellular uptake of
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Fig. 1. Effects of hypoxia on binding of 125I-LDL to HMDM. Under normoxia or hypoxia, HMDM were cultured for 24 h, and thereafter incubated with indicated concentrations of 125I-LDL at 48C for 2 h. The amounts of 125 I-LDL bound to the cells (open bars, normoxic; Þlled bars, hypoxic) were determined and speciÞc binding was calculated as described in Materials and Methods. Results are representative of three experiments and each point represents the mean of duplicate dishes. N; normoxia, H; hypoxia.
LDL at 378C. There were measurable and speciÞc uptake of 125I-LDL in both conditions. We found no signiÞcant difference in cellular association of 125I-LDL between the two conditions (Fig. 2A), however, degradation of 125I-LDL was suppressed under hypoxia compared to that under normoxia (Fig. 2B). Effect of hypoxia on cholesteryl ester synthesis To test whether hypoxia affect cholesteryl ester synthesis in HMDM, the rate of incorporation of 14C-oleate into cellular cholesteryl ester was studied. The rate of basal cholesterol es-
Fig. 2. Effects of hypoxia on association(A) and degradation(B) of 125I-LDL in HMDM. Under normoxic (open bars) or hypoxic (Þlled bars) condition, HMDM were cultured for 24 h and thereafter incubated with indicated concentrations of 125I-LDL at 378C for 5 h, and the amounts of associated 125I-LDL and degraded 125I-LDL from TCA soluble fraction of medium were determined as described in Methods. Results are representative of four experiments and each point represents the mean 6 SEM of three dishes (* p,0.05, ** p,0.005; compared to normoxia).
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Fig. 3. Effects of hypoxia on cholesteryl ester synthesis in HMDM. Under normoxic (open bars and squares) or hypoxic (Þlled bars and squares) condition, HMDM were incubated with indicated concentrations of LDL for 24 h (A) or incubated with 100 mg/ml of LDL for indicated hours (B). After incubation, the incorporation of 14C-oleate into cellular cholesteryl ester was measured as described in Methods. Results are representative of Þve (A) or three (B) experiments and each value represents the mean 6 SEM of Þve dishes (* p,0.05, ** p,0.005; compared to normoxia).
teriÞcation (without LDL) under hypoxia was twice higher than that under normoxia (Fig. 3A). This result is compatible with the data observed in smooth muscle cells and Þbroblasts (13,14). Hypoxia induced twice greater rate of cholesterol esteriÞcation in HMDM under incubation with LDL (Fig. 3A). Greater rate of cholesterol esteriÞcation of hypoxia was also found in time-dependent fashion in HMDM (Fig. 3B). Addition of 25-hydroxycholesterol, oxysterols taken up via the LDL-receptor-independent pathway, led to a signiÞcant increase of cholesteryl 14C-oleate formation under both normoxia and hypoxia (Fig. 4). Effect of hypoxia on cholesteryl ester accumulation To test whether the stimulated cholesteryl ester synthesis in hypoxic condition resulted in intracellular cholesteryl ester accumulation, we incubated the cells with indicated concentration of LDL for 24 hours, and measured cellular total-, free- and esteriÞed-cholesterol. Cellular free-cholesterol content increased slightly, but not signiÞcantly, under both normoxic and hypoxic conditions (data not shown). However, the increase in cellular esteriÞed-cholesterol content under hypoxia was signiÞcantly larger than that under normoxia (Fig. 5). Effect of hypoxia on cholesterol efßux and apo E secretion To elucidate the effect of hypoxia on cholesterol efßux and apo E secretion, we measured the amount of cellular cholesterol under normoxic and hypoxic condition with lipoprotein deÞcient serum. There was no difference in free cholesterol contents between normoxia and hypoxia. The decrease in the content of cholesteryl ester was smaller under hypoxia (Fig. 6A), though it was not signiÞcant. There was no difference in the secretion of apo E under both conditions (Fig. 6B). These results suggest that, under hypoxia, the efßux of cholesterol may be suppressed by apo E-independent mechanism.
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Fig. 4. Effects of hypoxia on cholesteryl ester synthesis in HMDM incubated with 25-hydroxycholesterol. Under normoxia or hypoxia, HMDM were cultured for 24 h and thereafter incubated with culture medium containing 14 C-oleate-albumin complex with (1) or without (2) a mixture of 5 mg/ml of 25-hydroxycholesterol and 10 mg/ ml of cholesterol. After 6 h incubation, the incorporation of 14C-oleate into cellular cholesteryl ester was measured as described in Materials and Methods. Results are representative of four experiments and each value represents the mean 6 SEM of Þve dishes. (* P,0.005; compared to normoxia).
Discussion We have found in this study that the degradation of LDL in HMDM under hypoxia was lower than that under normoxia, though there was no differences in binding and association of LDL between hypoxic and normoxic incubations. Albers et al. (12) reported that in cultured human arterial smooth muscle cells the binding of 125I-LDL under hypoxic incubation
Fig. 5. Effects of hypoxia on the content of cholesteryl ester. Under normoxia or hypoxia, HMDM were incubated for 24 h with indicated concentrations of LDL and thereafter the cellular contents of total and free cholesterol were determined. Cholesterol ester was calculated as described in Methods. Results are representative of three experiments and each value represents the mean 6 SEM of Þve dishes (* p,0.05; compared to normoxia).
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Fig. 6. A; The change of the content of cholesteryl ester of lipid-loaded HMDM before and after incubation in the medium containing LPDS under normoxia or hypoxia. HMDM were incubated for 24 h with LDL (100mg/ml), and then incubated for 48 hours with LPDS. The percentage compared to control condition (before incubation with LPDS) was calculated from the cellular contents of cholesteryl ester determined as described in Methods. Results are the mean 6 SEM of three experiments. B; The concentration of secreted apo E in medium. Lipidloaded HMDM were incubated for 48 h with LPDS under normoxia or hypoxia, and thereafter the concentration of apo E were determined by ELISA as described in Methods. Results are representative of three experiments and each value represents the mean 6 SEM of three dishes.
was similar to that under normoxic incubation. They also demonstrated that the degradation of 125I-LDL under hypoxic incubation was less as compared to that under normoxic incubation. Our results agree with their Þndings, and support the idea that hypoxia did not change the afÞnity and the number of LDL receptor on the cell surface. Tsukitani et al. reported that ACAT activity increased in a time-dependent manner and cholesteryl ester increased under hypoxic incubation in cultured rabbit skin Þbroblasts (13). In this study, we obtained the similar results in HMDM. Additionally, we showed that 25-hydroxycholesterol, which cells take up via LDL receptor-independent pathway, stimulated cholesteryl ester synthesis more extensively under hypoxic incubation. Mukodani showed the efßux of cholesterol was suppressed under hypoxic incubation in cultured rabbit skin Þbroblasts (14). In HMDM, we showed that there was reduced efßux of cellular esteriÞed cholesterol in incubation with lipoprotein deÞcient serum under hypoxia. This is considered to be due to the changes of the LDL metabolism in HMDM, not to be due to the changes of characteristics of LDL particles as mentioned already or apo E secretion (26). We have examined Northern blot analysis for ACAT1 (25,27), resulting in no alteration in hypoxia (data not shown). Though ACAT1 could be predominant in macrophages (28), it is important to identify which acyltransferase enzyme(s) is affected under hypoxia. Taken together, we speculate the mechanism of reduced LDL degradation and increased cholesteryl ester synthesis as follows; First, decreased degradation under hypoxic condition may be due to reduced rate of the transport of degraded lipoprotein into the culture medium or lowered activities of lysosomal enzymes. Hypoxia might modulate the transport of cholesterol and protein in the cytosol. As a result, the amount of microsomal cholesterol available for esteriÞcation may increase and cholesteryl ester synthesis may be stimulated, resulting in the reduction of cholesterol efßux under hypoxia. Second, as the rate of cholesterol esteriÞcation increased under hypoxic condition without the addi-
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tion of LDL or 25-hydroxycholesterol as shown in Fig. 3A and 4, hypoxia may directly stimulate cholesterol ester synthesis, though it remains still unclear what kind of mechanisms operate cholesteryl ester synthesis under hypoxia. It is also necessary to investigate the comparison with modiÞed LDL like oxidation, and the cholesterol efßux in incubation with high density lipoproteins. Moreover, in intact animal experiments, hypoxia could induce physical stress and increase the catecholamines level that might accelerate the atherosclerosis. Hypoxia could affect expression of speciÞc genes associated with vascular tone and remodeling (29), and production of oxyradical formation and tissue damage (2). Further investigations are needed to clarify which mechanism could be operated to the cellular cholesterol metabolism by hypoxia. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
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