- Email: [email protected]
Oxidation Decreases Low Density Lipoprotein Association with the Subendothelium Extracellular Matrix
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
245, 497–501 (1998)
RC988470
Oxidation Decreases Low Density Lipoprotein Association with the Subendothelium Extracellular Matrix Naphtali Savion, Orly Zavaro, and Shlomo Kotev-Emeth Goldschleger Eye Research Institute, Sackler Faculty of Medicine, Tel-Aviv University, Sheba Medical Center, Tel-Hashomer 52621, Israel
Received March 9, 1998
Atherosclerosis is initiated by accumulation of low density lipoprotein (LDL) in the subendothelium extracellular matrix (ECM) followed by oxidation and scavenger receptor mediated uptake by the vessel wall recruited macrophages. The effect of oxidation on LDL association with the ECM is not yet clear. In the present study we examined the hypothesis that excessive oxidation of LDL results in decreased LDL association with ECM, thereby increasing its accessibility to the scavenger receptor on macrophages. We have studied the relative association of Cu/2 ion oxidized LDL to native LDL with the native or oxidized bovine corneal endothelial cells ECM. Oxidation of LDL decreased its binding to the ECM. The kinetic of this process was characterized by approximately 1 h lag phase followed by a significant decreased binding of 80% after 6.5 h oxidation. This kinetic more closely resembled the pattern of the oxidation product dienals accumulation rather than thiobarbituric acid reactive substance formation. Oxidation of ECM-bound LDL resulted in an increased LDL release from the ECM as a function of Cu/2 ion concentration with a maximal increase of 2fold at 3 mM. ECM oxidation prior to exposure to LDL resulted in a 30% decrease in LDL binding to the ECM. In conclusion, these results suggest that oxidation processes in the vessel wall result in increased dissociation of ECM-bound LDL, which in turn makes this oxidized LDL more accessible for binding and uptake by macrophages leading to foam cell formation. q 1998 Academic Press
Development of atherosclerotic fatty streak lesion is initiated by accumulation of unesterified cholesterol in subendothelium extracellular matrix (ECM) (1-3). Low density lipoprotein (LDL) is transported across an intact endothelium where it is trapped in the extracellular spaces of the artery wall (4). Massive accumulation of LDL in the subendothelium is the result of the close association of LDL with ECM components rather than the increased rate of transport into the artery wall (5).
The ECM-bound and trapped LDL goes through a mild oxidative modification involving mostly lipid oxidation (6). The mildly oxidized LDL is responsible for monocyte recruitment to the developing lesion which then further oxidizes the trapped LDL at its lipid moiety, as well as at its protein portion. This latter process results in a loss of recognition by the LDL receptor and a shift to recognition by the macrophage/monocyte scavenger receptor (for review see Berliner et al. (7)). The direct binding of LDL with the ECM has been studied and a significant interaction with the heparan sulfate proteoglycans in the ECM demonstrated (8). However, the effect of oxidation, that occurs to the subendothelium trapped LDL, on its association with the ECM is not yet clear. In the present study, the hypothesis that excessive oxidation of LDL results in decreased LDL binding to ECM, thereby increasing its accessibility to the scavenger receptor on the monocyte/macrophage cells recruited to the fatty streak was examined. Using the previously described ECM model (8), the effect of oxidation of LDL or ECM on LDL binding to ECM and the effect of oxidation on the release of ECMbound LDL was studied. MATERIALS AND METHODS Materials Tissue culture media, sera and antibiotics were purchased from Biological Industries (Beit Haemek, Israel). Tissue culture 4-wells plates were obtained from Nunc (Roskilde, Denmark). Dextran T40, 2-thiobarbituric acid (TBA) and bovine serum albumin (BSA), fraction V were obtained from Sigma Chemicals Co. (St. Louis, MO). Carrier-free sodium [125I] iodide was purchased from Rotem Industries (Beer-Sheva, Israel). Malonaldehyde bis(dimethyl acetal) (MDA) was purchased from Aldrich Chemical Co., Inc., (Milwaukee, WI).
Preparation of ECM-Coated Plates Tissue culture 4-wells plates were coated with ECM according to the procedure described by Gospodarowicz et al. (9). Bovine corneal endothelial cells grown to confluency for 12 to 14 days in the presence of 4% Dextran T-40 were washed with saline and dissolved by expo-
497
0006-291X/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 8470
/
694f$$$841
03-31-98 11:41:18
bbrcg
AP: BBRC
Vol. 245, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
sure to 0.5% Triton X-100 and 0.1 M NH4OH solution followed by extensive washing with distilled water. The wet ECM-coated plates were sealed in plastic bags and maintained at 47C for up to 6 month before use.
Preparation and Iodination of Low Density Lipoproteins LDL was prepared according to Havel et al. (10). Briefly, it was separated by differential ultracentrifugal flotation from blood bank human plasma. The fraction with a density of 1.019õdõ1.063 g/ml was collected as LDL. Its protein concentration was determined by Markwell et al.’s modification (11) of the method of Lowry et al. (12). LDL was iodinated with iodine monochloride (13) as previously described (14), and the unbound 125I was removed by gel filtration on a Sephadex G-25 column. The specific activities of the labeled LDL ranged from 600-1200 cpm/ng protein.
LDL Oxidation LDL stock solution contains EDTA and therefore was dialyzed against calcium magnesium free phosphate buffered saline (PBS) for 24 h at 47C to remove EDTA traces. The dialyzed lipoprotein was maintained at 47C for no more than 48 h and then subjected to oxidation at a concentration of 50 mg/ml for various time periods in the presence of 10 mM CuSO4 at 377C. Oxidation was terminated by adding EDTA to a final concentration of 0.1 mM.
FIG. 1. Effect of oxidation on LDL binding to ECM. 125I-LDL was oxidized for 0.25-6.5 h as described in Methods, and then incubated at a concentration of 2 mg/ml on ECM-coated wells for 3 h at 377C. The amount of specific binding was determined. The binding results represent mean { SD (in ng LDL per well) from two experiments carried out in duplicate. Results were compared to control (zero oxidation time) and statistical analysis was done using student’s paired t-test. *P õ 0.05 was considered as statistically significant.
Determination of Oxidation Level A. Determination of thiobarbituric acid reactive substances (TBARS). Oxidation of polyunsaturated fatty acids results in malondialdehyde (MDA) formation and its concentration was determined according to Buege and Aust (15). Briefly, the lipid sample was boiled under acidic conditions in the presence of thiobarbituric acid (TBA) formed upon reaction with MDA the TBARS. The concentration of TBARS was determined by measuring the absorbance at 535 nm and calculated as mmole/mg protein using a MDA standard curve. B. Determination of dienals. Lipid oxidation products include dienals. The dienals were determined by measuring the absorbance of the lipid samples at 245, 250 and 268 nm using a Uvikon 930 Spectrophotometer (Kontron, Basal, Switzerland) and calculating their concentration (in mM) according to Pinchuk and Lichtenberg (16), using the equation C(mM) Å O.D.245 1 2.76 0 O.D.250 1 8.8 / O.D.268 1 37.4.
Lipoprotein Binding to ECM LDL (native or oxidized) was placed on ECM-coated wells (0.5 ml/well) in PBS (containing magnesium and calcium) at different concentrations (0.6 to 3 mg/ml) and incubated at 377C for various time periods (15 min to 6.5 h). Non-specific binding, subtracted from the total binding, was determined by incubating the ECM with the iodinated ligand in the presence of a 20-fold excess of unlabeled lipoprotein. At the end of the binding period, plates were washed 10 times with PBS. The bound LDL was dissolved in 0.2 M NaOH (0.5 ml) for 20 min and the amount of ECM-associated radioactivity was determined in a gamma counter (Gammamatic-I, Kontron, Basel, Switzerland). The data are expressed as the specific binding of iodinated LDL in ng/well.
Lipoprotein Release from ECM LDL (2 mg/ml) binding to ECM was performed as described above. The ECM-coated wells were then washed 5 times with PBS and fresh PBS (0.5 ml; containing magnesium and calcium) was
added to the wells. The plates were further incubated for the indicated time periods at 377C in the absence or presence of Cu/2 ions. At the end of the incubation, the PBS solution was collected, the wells were washed 3 times with PBS and the ECM dissolved in NaOH (0.2 M) and collected. Both the PBS solution and the dissolved ECM were counted in a gamma counter and their sum presented the total ECM-bound LDL. The released lipoprotein was calculated as the percentage of radioactivity in the PBS solution of the total ECM-bound radioactivity.
ECM Oxidation ECM-coated wells were incubated with PBS (calcium magnesium free) containing CuSO4 at concentration range of 0.1-30 mM for various time periods (1.5 to 24 h) at 377C. At the end of the oxidation period, the PBS was removed and fresh PBS containing 0.1 mM EDTA was added, followed by washing the ECM 10 times with PBS. The oxidized ECM was immediately subjected to lipoprotein binding assay.
RESULTS Effect of LDL Oxidation on Its Binding to ECM LDL was oxidized by incubation with Cu/2 ions for various time periods as indicated in Fig. 1. The oxidation was terminated by addition of excess EDTA and the material was divided into three samples which were used to determine 125I-LDL binding to ECM, as well as TBARS (Fig. 2) and dienals accumulation in the LDL (Fig. 3). The amount of 125I-LDL binding to the ECM did not change during the first hour of oxidation. However, a significant, 55% decrease in 125I-LDL binding to the ECM was observed after 3 h of Cu/2-induced oxidation and a further decrease of up to 80% was observed after 6.5 h oxidation.
498
AID
BBRC 8470
/
694f$$$841
03-31-98 11:41:18
bbrcg
AP: BBRC
Vol. 245, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Effect of LDL oxidation time on TBARS concentration. I-LDL was oxidized as described in Fig. 1. Samples from each oxidation period containing 1-10 mg protein were used for TBARS determination as described in Methods. This experiment was repeated twice.
125
The kinetic of LDL oxidation was monitored by measuring the concentrations of TBARS and dienals in the LDL as a function of exposure time to Cu/2 ions. The appearance of TBARS was characterized by a lag period of 3 h followed by a sharp increase between 3 to 5 h which then remained at the same level (Fig. 2). The accumulation of dienals in the LDL exposed to Cu/2 ions demonstrated a different kinetic. A close to linear increase during the first 3 h of oxidation was observed (Fig. 3). These experiments suggest that LDL oxidation is associated with a decreased binding to ECM and is correlated with the accumulation of dienals. The kinetic of native and oxidized LDL binding to ECM, as a function of LDL concentration, was studied
FIG. 3. Effect of LDL oxidation time on dienals concentration. I-LDL was oxidized as described in Fig. 1. Based on absorbance measurements of samples from each oxidation period, dienals concentration was calculated as described in Methods. This experiment was repeated twice.
125
FIG. 4. Binding of native and oxidized LDL to ECM as a function of concentration. 125I-LDL was oxidized for 3 h. Native or oxidized LDL were incubated on ECM at the indicated concentration range of 0.6-3 mg/ml for 0.5 h at 377C. Results represent a mean { SD of two experiments carried out in duplicate. Differences between the oxidized LDL and native LDL binding were statistically analyzed using student’s paired t-test *P õ 0.05 was considered statistically significant. For symbols without error bars, the SD was less than symbols size.
(Fig. 4). A linear increase in native LDL binding was observed while the oxidized LDL demonstrated a significantly lower binding to the ECM. Effect of Oxidation on Release of ECM-Bound LDL ECM-coated wells were allowed to bind 125I-LDL and then exposed to medium containing increasing concentrations of Cu/2 (Fig. 5). At the end of 90 min incubation, the amount of 125I-LDL released into the medium
FIG. 5. Effect of Cu/2 ions concentration on LDL release from ECM. The release of ECM-bound 125I-LDL to the incubation media, following exposure to increasing concentrations of Cu/2 ions for 1.5 hour, was studied as described in Methods. Results represent mean { SD of 1 experiment carried out in duplicate.
499
AID
BBRC 8470
/
694f$$$841
03-31-98 11:41:18
bbrcg
AP: BBRC
Vol. 245, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 6. Effect of ECM oxidation on LDL binding. ECM was oxidized by increasing concentrations of Cu/2 ions for 24 h and then exposed to 125I-LDL (2 mg/ml) and the specific binding was determined. Results represent a mean { SD of 5 experiments carried out in duplicate. Results were compared to control (zero Cu/2 ions) and statistical analysis was carried out using student’s paired t-test. *P õ 0.05 was considered as statistically significant.
was determined. The results demonstrated an increased LDL release from the ECM, as a function of the Cu/2 ions concentration, up to a concentration of 3 mM at which a 2-fold maximum increase in LDL release was achieved. Effect of ECM Oxidation on
125
I-LDL Binding
ECM-coated wells were exposed to increasing concentrations of Cu/2 ions for 24 h. Oxidation was then stopped and the treated ECM allowed to bind 125I-LDL. The 125I-LDL binding to the ECM decreased as a function of Cu/2 concentration. At its maximum a 30% decrease was observed at a concentration of 10 mM (Fig. 6). ECM oxidation was a time-dependent process with maximum decrease in 125I-LDL binding to the treated ECM observed after 3 h pre-incubation of the ECM with Cu/2 ions (Fig. 7).
tion of oxidized LDL with the ECM is limited and therefore, the present study was aimed at exploring the effect of oxidation on LDL interaction with the intact endothelial cell ECM. Recent studies have provided conflicting results. Oxidation has been shown to enhance LDL association with lipoprotein lipase anchored to endothelial cell ECM (18), as well as increase direct binding of oxidized LDL to macrophage-secreted ECM (19). However, another recent report indicated that oxidized LDL binds less well to arterial ECM proteoglycans than does native LDL which suggests that LDL might be released from the ECM due to oxidation (20). This study further demonstrates that oxidized LDL binds to smooth muscle cells ECM to a greater extent than native LDL. The findings of the present study correlate with the first part of this latter study and suggest that the total sum of the ongoing oxidation processes at the ECM level results in increasing the lipoprotein release rather than transporting it to other ECM components. Since this study used a more complete endothelial ECM system, which is closer to the in vivo subendothelium, it can be suggested that the induced release of LDL following oxidation may have a physiological relevance. Macrophages secrete oxidants, like other vascular cells, and may promote LDL release from ECM, making the oxidized LDL more accessible to binding and uptake by macrophages through the scavenger receptor, in an unregulated way, turning them into foam cells. The degree of LDL oxidation, as a function of oxidation time, was evaluated by measuring the formation of TBARS and dienals. The correlation of the kinetic of
DISCUSSION Oxidized LDL has been demonstrated in human atherosclerotic lesions and its role in the progression of these lesions has been clearly demonstrated (7, 17). It is involved in the recruitment process of monocytes to the vessel wall and in foam cell formation. It is well accepted that oxidation of LDL occurs mostly in the vessel wall rather than in the plasma. This process occurs following its transport into the vessel wall and its retention at this site. Retention of LDL in the artery wall is enhanced by the lipoprotein binding to ECM components as previously demonstrated (8). These previous observations may suggest that oxidation mostly occurs to ECM-bound LDL. Knowledge of the interac-
FIG. 7. Effect of ECM oxidation time on LDL binding. ECM was exposed to Cu/2 ions (10 mM) for the indicated time periods, then oxidation was terminated and the ECM was exposed to 125I-LDL (2 mg/ml) for a 90 min binding period. The specific binding of 125I-LDL was determined and presented as a mean { SD from 7 experiments carried out in duplicate. Results were compared to control (zero time) and statistical analysis was done using student’s paired t-test. *P õ 0.05 was considered as statistically significant.
500
AID
BBRC 8470
/
694f$$$841
03-31-98 11:41:18
bbrcg
AP: BBRC
Vol. 245, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
LDL binding with the oxidation level of the lipoprotein indicates that the kinetic of dienals accumulation fits better with the reduction rate of LDL binding to ECM. Oxidation processes occurring in the vessel wall may oxidize soluble (non-bound) LDL, ECM-bound LDL, or may oxidize ECM components. The effects of oxidation at these three potential sites at the vessel wall were studied. Oxidation of free LDL in solution resulted in a decreased binding capacity to ECM. The oxidation of ECM-bound LDL resulted in a faster release of the bound LDL. This process may be caused by oxidation of either the bound LDL or ECM components. The latter possibly was studied and demonstrated reduced binding of LDL to Cu/2 ions treated ECM. It is not clear as to whether or not exposure of ECM bound LDL to Cu/2 ions results in LDL or ECM oxidation or both are oxidized. However, this process results in an increased dissociation of LDL from ECM. In conclusion, this study may suggest a new regulatory mechanism for LDL oxidation by enhancing the dissociation of ECM-bound LDL. This, in turn, makes this oxidized LDL more accessible for macrophages leading to foam cells formation. ACKNOWLEDGMENTS
2. Kruth, H. S. (1985) Atherosclerosis 57, 337–341. 3. Simionescu, N., Vasile, E., Lupu, F., Popescu, G., and Simionesco, M. (1986) Am. J. Pathol. 123, 1109–1125. 4. Nievelstein, P. F. E. M., Fogelman, A. M., Mottino, G., and Frank, J. S. (1991) Arterioscler. Thromb. 11, 1795–1805. 5. Schwenke, D. C., and Carew, T. E. (1989) Arteriosclerosis 9, 908– 918. 6. Witztum, J. L. and Steinberg, D. (1991) J. Clin. Invest. 88, 1785– 1792. 7. Berliner, J. A., Navab, M., Fogelman, A. M., Frank, J. S., Demer, L. L., Edwards, P. A., Watson, A. D., and Lusis, A. J. (1995) Circulation 91, 2488–2496. 8. Eisenberg, S., Schayek, E., Olivecrona, T., and Vlodavsky, I. (1992) J. Clin. Invest. 90, 2013–2021. 9. Gospodarowicz, D., Vlodavsky, I., and Savion, N. (1980) Endocrine Rev. 1, 201–207. 10. Havel, R. J., Eder, H. A., and Bragdon, J. H. (1955) J. Clin. Invest. 34, 1345–1353. 11. Markwell, M. A. K., Hass, S. M., Beiber, L. L., and Tolbert, N. E. (1978) Anal. Biochem. 87, 206–210. 12. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265–275. 13. MacFarlane, A. S. (1958) Nature 182, 53. 14. Savion, N., Laherty, R., Lui, G. M., and Gospodarowicz, D. (1981) J. Biol. Chem. 256, 12817–12822. 15. Buege, J. A., and Aust, S. D. (1976) Methods Enzymol. 52, 302– 310.
This research was supported by a grant from the Chief Scientist, Ministry of Health, Israel. We thank Dr. D. Lichtenberg, Sackler, Faculty of Medicine, Tel-Aviv University, for helpful discussions. This work is in partial fulfillment of the requirement for the M.Sc. degree of O. Zavaro from the Sackler Faculty of Medicine at Tel-Aviv University.
16. Pinchuk, I., and Lichtenberg, D. (1996) Free Rad. Res. 24, 351– 360. 17. Holvoet, P., and Collen, D. (1994) FASEB. J. 8, 1279–1284. 18. Auerbach, B. J., Bisgaier, C. L., Wolle, J., and Saxena, U. (1996) J. Biol. Chem. 271, 1329–1335. 19. Kaplan, M., and Aviram, M. (1997) Biochem. Biophys. Res. Commun. 237, 271–276.
REFERENCES 1. Frank, J. S. and Fogelman, A. M. (1989) J. Lipid. Res. 30, 967– 978.
20. Chait, A., Chang, M. Y., Olin, K., and Wight, T. (1997) Atherosclerosis 134, 197.
501
AID
BBRC 8470
/
694f$$$841
03-31-98 11:41:18
bbrcg
AP: BBRC
245, 497–501 (1998)
RC988470
Oxidation Decreases Low Density Lipoprotein Association with the Subendothelium Extracellular Matrix Naphtali Savion, Orly Zavaro, and Shlomo Kotev-Emeth Goldschleger Eye Research Institute, Sackler Faculty of Medicine, Tel-Aviv University, Sheba Medical Center, Tel-Hashomer 52621, Israel
Received March 9, 1998
Atherosclerosis is initiated by accumulation of low density lipoprotein (LDL) in the subendothelium extracellular matrix (ECM) followed by oxidation and scavenger receptor mediated uptake by the vessel wall recruited macrophages. The effect of oxidation on LDL association with the ECM is not yet clear. In the present study we examined the hypothesis that excessive oxidation of LDL results in decreased LDL association with ECM, thereby increasing its accessibility to the scavenger receptor on macrophages. We have studied the relative association of Cu/2 ion oxidized LDL to native LDL with the native or oxidized bovine corneal endothelial cells ECM. Oxidation of LDL decreased its binding to the ECM. The kinetic of this process was characterized by approximately 1 h lag phase followed by a significant decreased binding of 80% after 6.5 h oxidation. This kinetic more closely resembled the pattern of the oxidation product dienals accumulation rather than thiobarbituric acid reactive substance formation. Oxidation of ECM-bound LDL resulted in an increased LDL release from the ECM as a function of Cu/2 ion concentration with a maximal increase of 2fold at 3 mM. ECM oxidation prior to exposure to LDL resulted in a 30% decrease in LDL binding to the ECM. In conclusion, these results suggest that oxidation processes in the vessel wall result in increased dissociation of ECM-bound LDL, which in turn makes this oxidized LDL more accessible for binding and uptake by macrophages leading to foam cell formation. q 1998 Academic Press
Development of atherosclerotic fatty streak lesion is initiated by accumulation of unesterified cholesterol in subendothelium extracellular matrix (ECM) (1-3). Low density lipoprotein (LDL) is transported across an intact endothelium where it is trapped in the extracellular spaces of the artery wall (4). Massive accumulation of LDL in the subendothelium is the result of the close association of LDL with ECM components rather than the increased rate of transport into the artery wall (5).
The ECM-bound and trapped LDL goes through a mild oxidative modification involving mostly lipid oxidation (6). The mildly oxidized LDL is responsible for monocyte recruitment to the developing lesion which then further oxidizes the trapped LDL at its lipid moiety, as well as at its protein portion. This latter process results in a loss of recognition by the LDL receptor and a shift to recognition by the macrophage/monocyte scavenger receptor (for review see Berliner et al. (7)). The direct binding of LDL with the ECM has been studied and a significant interaction with the heparan sulfate proteoglycans in the ECM demonstrated (8). However, the effect of oxidation, that occurs to the subendothelium trapped LDL, on its association with the ECM is not yet clear. In the present study, the hypothesis that excessive oxidation of LDL results in decreased LDL binding to ECM, thereby increasing its accessibility to the scavenger receptor on the monocyte/macrophage cells recruited to the fatty streak was examined. Using the previously described ECM model (8), the effect of oxidation of LDL or ECM on LDL binding to ECM and the effect of oxidation on the release of ECMbound LDL was studied. MATERIALS AND METHODS Materials Tissue culture media, sera and antibiotics were purchased from Biological Industries (Beit Haemek, Israel). Tissue culture 4-wells plates were obtained from Nunc (Roskilde, Denmark). Dextran T40, 2-thiobarbituric acid (TBA) and bovine serum albumin (BSA), fraction V were obtained from Sigma Chemicals Co. (St. Louis, MO). Carrier-free sodium [125I] iodide was purchased from Rotem Industries (Beer-Sheva, Israel). Malonaldehyde bis(dimethyl acetal) (MDA) was purchased from Aldrich Chemical Co., Inc., (Milwaukee, WI).
Preparation of ECM-Coated Plates Tissue culture 4-wells plates were coated with ECM according to the procedure described by Gospodarowicz et al. (9). Bovine corneal endothelial cells grown to confluency for 12 to 14 days in the presence of 4% Dextran T-40 were washed with saline and dissolved by expo-
497
0006-291X/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 8470
/
694f$$$841
03-31-98 11:41:18
bbrcg
AP: BBRC
Vol. 245, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
sure to 0.5% Triton X-100 and 0.1 M NH4OH solution followed by extensive washing with distilled water. The wet ECM-coated plates were sealed in plastic bags and maintained at 47C for up to 6 month before use.
Preparation and Iodination of Low Density Lipoproteins LDL was prepared according to Havel et al. (10). Briefly, it was separated by differential ultracentrifugal flotation from blood bank human plasma. The fraction with a density of 1.019õdõ1.063 g/ml was collected as LDL. Its protein concentration was determined by Markwell et al.’s modification (11) of the method of Lowry et al. (12). LDL was iodinated with iodine monochloride (13) as previously described (14), and the unbound 125I was removed by gel filtration on a Sephadex G-25 column. The specific activities of the labeled LDL ranged from 600-1200 cpm/ng protein.
LDL Oxidation LDL stock solution contains EDTA and therefore was dialyzed against calcium magnesium free phosphate buffered saline (PBS) for 24 h at 47C to remove EDTA traces. The dialyzed lipoprotein was maintained at 47C for no more than 48 h and then subjected to oxidation at a concentration of 50 mg/ml for various time periods in the presence of 10 mM CuSO4 at 377C. Oxidation was terminated by adding EDTA to a final concentration of 0.1 mM.
FIG. 1. Effect of oxidation on LDL binding to ECM. 125I-LDL was oxidized for 0.25-6.5 h as described in Methods, and then incubated at a concentration of 2 mg/ml on ECM-coated wells for 3 h at 377C. The amount of specific binding was determined. The binding results represent mean { SD (in ng LDL per well) from two experiments carried out in duplicate. Results were compared to control (zero oxidation time) and statistical analysis was done using student’s paired t-test. *P õ 0.05 was considered as statistically significant.
Determination of Oxidation Level A. Determination of thiobarbituric acid reactive substances (TBARS). Oxidation of polyunsaturated fatty acids results in malondialdehyde (MDA) formation and its concentration was determined according to Buege and Aust (15). Briefly, the lipid sample was boiled under acidic conditions in the presence of thiobarbituric acid (TBA) formed upon reaction with MDA the TBARS. The concentration of TBARS was determined by measuring the absorbance at 535 nm and calculated as mmole/mg protein using a MDA standard curve. B. Determination of dienals. Lipid oxidation products include dienals. The dienals were determined by measuring the absorbance of the lipid samples at 245, 250 and 268 nm using a Uvikon 930 Spectrophotometer (Kontron, Basal, Switzerland) and calculating their concentration (in mM) according to Pinchuk and Lichtenberg (16), using the equation C(mM) Å O.D.245 1 2.76 0 O.D.250 1 8.8 / O.D.268 1 37.4.
Lipoprotein Binding to ECM LDL (native or oxidized) was placed on ECM-coated wells (0.5 ml/well) in PBS (containing magnesium and calcium) at different concentrations (0.6 to 3 mg/ml) and incubated at 377C for various time periods (15 min to 6.5 h). Non-specific binding, subtracted from the total binding, was determined by incubating the ECM with the iodinated ligand in the presence of a 20-fold excess of unlabeled lipoprotein. At the end of the binding period, plates were washed 10 times with PBS. The bound LDL was dissolved in 0.2 M NaOH (0.5 ml) for 20 min and the amount of ECM-associated radioactivity was determined in a gamma counter (Gammamatic-I, Kontron, Basel, Switzerland). The data are expressed as the specific binding of iodinated LDL in ng/well.
Lipoprotein Release from ECM LDL (2 mg/ml) binding to ECM was performed as described above. The ECM-coated wells were then washed 5 times with PBS and fresh PBS (0.5 ml; containing magnesium and calcium) was
added to the wells. The plates were further incubated for the indicated time periods at 377C in the absence or presence of Cu/2 ions. At the end of the incubation, the PBS solution was collected, the wells were washed 3 times with PBS and the ECM dissolved in NaOH (0.2 M) and collected. Both the PBS solution and the dissolved ECM were counted in a gamma counter and their sum presented the total ECM-bound LDL. The released lipoprotein was calculated as the percentage of radioactivity in the PBS solution of the total ECM-bound radioactivity.
ECM Oxidation ECM-coated wells were incubated with PBS (calcium magnesium free) containing CuSO4 at concentration range of 0.1-30 mM for various time periods (1.5 to 24 h) at 377C. At the end of the oxidation period, the PBS was removed and fresh PBS containing 0.1 mM EDTA was added, followed by washing the ECM 10 times with PBS. The oxidized ECM was immediately subjected to lipoprotein binding assay.
RESULTS Effect of LDL Oxidation on Its Binding to ECM LDL was oxidized by incubation with Cu/2 ions for various time periods as indicated in Fig. 1. The oxidation was terminated by addition of excess EDTA and the material was divided into three samples which were used to determine 125I-LDL binding to ECM, as well as TBARS (Fig. 2) and dienals accumulation in the LDL (Fig. 3). The amount of 125I-LDL binding to the ECM did not change during the first hour of oxidation. However, a significant, 55% decrease in 125I-LDL binding to the ECM was observed after 3 h of Cu/2-induced oxidation and a further decrease of up to 80% was observed after 6.5 h oxidation.
498
AID
BBRC 8470
/
694f$$$841
03-31-98 11:41:18
bbrcg
AP: BBRC
Vol. 245, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Effect of LDL oxidation time on TBARS concentration. I-LDL was oxidized as described in Fig. 1. Samples from each oxidation period containing 1-10 mg protein were used for TBARS determination as described in Methods. This experiment was repeated twice.
125
The kinetic of LDL oxidation was monitored by measuring the concentrations of TBARS and dienals in the LDL as a function of exposure time to Cu/2 ions. The appearance of TBARS was characterized by a lag period of 3 h followed by a sharp increase between 3 to 5 h which then remained at the same level (Fig. 2). The accumulation of dienals in the LDL exposed to Cu/2 ions demonstrated a different kinetic. A close to linear increase during the first 3 h of oxidation was observed (Fig. 3). These experiments suggest that LDL oxidation is associated with a decreased binding to ECM and is correlated with the accumulation of dienals. The kinetic of native and oxidized LDL binding to ECM, as a function of LDL concentration, was studied
FIG. 3. Effect of LDL oxidation time on dienals concentration. I-LDL was oxidized as described in Fig. 1. Based on absorbance measurements of samples from each oxidation period, dienals concentration was calculated as described in Methods. This experiment was repeated twice.
125
FIG. 4. Binding of native and oxidized LDL to ECM as a function of concentration. 125I-LDL was oxidized for 3 h. Native or oxidized LDL were incubated on ECM at the indicated concentration range of 0.6-3 mg/ml for 0.5 h at 377C. Results represent a mean { SD of two experiments carried out in duplicate. Differences between the oxidized LDL and native LDL binding were statistically analyzed using student’s paired t-test *P õ 0.05 was considered statistically significant. For symbols without error bars, the SD was less than symbols size.
(Fig. 4). A linear increase in native LDL binding was observed while the oxidized LDL demonstrated a significantly lower binding to the ECM. Effect of Oxidation on Release of ECM-Bound LDL ECM-coated wells were allowed to bind 125I-LDL and then exposed to medium containing increasing concentrations of Cu/2 (Fig. 5). At the end of 90 min incubation, the amount of 125I-LDL released into the medium
FIG. 5. Effect of Cu/2 ions concentration on LDL release from ECM. The release of ECM-bound 125I-LDL to the incubation media, following exposure to increasing concentrations of Cu/2 ions for 1.5 hour, was studied as described in Methods. Results represent mean { SD of 1 experiment carried out in duplicate.
499
AID
BBRC 8470
/
694f$$$841
03-31-98 11:41:18
bbrcg
AP: BBRC
Vol. 245, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 6. Effect of ECM oxidation on LDL binding. ECM was oxidized by increasing concentrations of Cu/2 ions for 24 h and then exposed to 125I-LDL (2 mg/ml) and the specific binding was determined. Results represent a mean { SD of 5 experiments carried out in duplicate. Results were compared to control (zero Cu/2 ions) and statistical analysis was carried out using student’s paired t-test. *P õ 0.05 was considered as statistically significant.
was determined. The results demonstrated an increased LDL release from the ECM, as a function of the Cu/2 ions concentration, up to a concentration of 3 mM at which a 2-fold maximum increase in LDL release was achieved. Effect of ECM Oxidation on
125
I-LDL Binding
ECM-coated wells were exposed to increasing concentrations of Cu/2 ions for 24 h. Oxidation was then stopped and the treated ECM allowed to bind 125I-LDL. The 125I-LDL binding to the ECM decreased as a function of Cu/2 concentration. At its maximum a 30% decrease was observed at a concentration of 10 mM (Fig. 6). ECM oxidation was a time-dependent process with maximum decrease in 125I-LDL binding to the treated ECM observed after 3 h pre-incubation of the ECM with Cu/2 ions (Fig. 7).
tion of oxidized LDL with the ECM is limited and therefore, the present study was aimed at exploring the effect of oxidation on LDL interaction with the intact endothelial cell ECM. Recent studies have provided conflicting results. Oxidation has been shown to enhance LDL association with lipoprotein lipase anchored to endothelial cell ECM (18), as well as increase direct binding of oxidized LDL to macrophage-secreted ECM (19). However, another recent report indicated that oxidized LDL binds less well to arterial ECM proteoglycans than does native LDL which suggests that LDL might be released from the ECM due to oxidation (20). This study further demonstrates that oxidized LDL binds to smooth muscle cells ECM to a greater extent than native LDL. The findings of the present study correlate with the first part of this latter study and suggest that the total sum of the ongoing oxidation processes at the ECM level results in increasing the lipoprotein release rather than transporting it to other ECM components. Since this study used a more complete endothelial ECM system, which is closer to the in vivo subendothelium, it can be suggested that the induced release of LDL following oxidation may have a physiological relevance. Macrophages secrete oxidants, like other vascular cells, and may promote LDL release from ECM, making the oxidized LDL more accessible to binding and uptake by macrophages through the scavenger receptor, in an unregulated way, turning them into foam cells. The degree of LDL oxidation, as a function of oxidation time, was evaluated by measuring the formation of TBARS and dienals. The correlation of the kinetic of
DISCUSSION Oxidized LDL has been demonstrated in human atherosclerotic lesions and its role in the progression of these lesions has been clearly demonstrated (7, 17). It is involved in the recruitment process of monocytes to the vessel wall and in foam cell formation. It is well accepted that oxidation of LDL occurs mostly in the vessel wall rather than in the plasma. This process occurs following its transport into the vessel wall and its retention at this site. Retention of LDL in the artery wall is enhanced by the lipoprotein binding to ECM components as previously demonstrated (8). These previous observations may suggest that oxidation mostly occurs to ECM-bound LDL. Knowledge of the interac-
FIG. 7. Effect of ECM oxidation time on LDL binding. ECM was exposed to Cu/2 ions (10 mM) for the indicated time periods, then oxidation was terminated and the ECM was exposed to 125I-LDL (2 mg/ml) for a 90 min binding period. The specific binding of 125I-LDL was determined and presented as a mean { SD from 7 experiments carried out in duplicate. Results were compared to control (zero time) and statistical analysis was done using student’s paired t-test. *P õ 0.05 was considered as statistically significant.
500
AID
BBRC 8470
/
694f$$$841
03-31-98 11:41:18
bbrcg
AP: BBRC
Vol. 245, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
LDL binding with the oxidation level of the lipoprotein indicates that the kinetic of dienals accumulation fits better with the reduction rate of LDL binding to ECM. Oxidation processes occurring in the vessel wall may oxidize soluble (non-bound) LDL, ECM-bound LDL, or may oxidize ECM components. The effects of oxidation at these three potential sites at the vessel wall were studied. Oxidation of free LDL in solution resulted in a decreased binding capacity to ECM. The oxidation of ECM-bound LDL resulted in a faster release of the bound LDL. This process may be caused by oxidation of either the bound LDL or ECM components. The latter possibly was studied and demonstrated reduced binding of LDL to Cu/2 ions treated ECM. It is not clear as to whether or not exposure of ECM bound LDL to Cu/2 ions results in LDL or ECM oxidation or both are oxidized. However, this process results in an increased dissociation of LDL from ECM. In conclusion, this study may suggest a new regulatory mechanism for LDL oxidation by enhancing the dissociation of ECM-bound LDL. This, in turn, makes this oxidized LDL more accessible for macrophages leading to foam cells formation. ACKNOWLEDGMENTS
2. Kruth, H. S. (1985) Atherosclerosis 57, 337–341. 3. Simionescu, N., Vasile, E., Lupu, F., Popescu, G., and Simionesco, M. (1986) Am. J. Pathol. 123, 1109–1125. 4. Nievelstein, P. F. E. M., Fogelman, A. M., Mottino, G., and Frank, J. S. (1991) Arterioscler. Thromb. 11, 1795–1805. 5. Schwenke, D. C., and Carew, T. E. (1989) Arteriosclerosis 9, 908– 918. 6. Witztum, J. L. and Steinberg, D. (1991) J. Clin. Invest. 88, 1785– 1792. 7. Berliner, J. A., Navab, M., Fogelman, A. M., Frank, J. S., Demer, L. L., Edwards, P. A., Watson, A. D., and Lusis, A. J. (1995) Circulation 91, 2488–2496. 8. Eisenberg, S., Schayek, E., Olivecrona, T., and Vlodavsky, I. (1992) J. Clin. Invest. 90, 2013–2021. 9. Gospodarowicz, D., Vlodavsky, I., and Savion, N. (1980) Endocrine Rev. 1, 201–207. 10. Havel, R. J., Eder, H. A., and Bragdon, J. H. (1955) J. Clin. Invest. 34, 1345–1353. 11. Markwell, M. A. K., Hass, S. M., Beiber, L. L., and Tolbert, N. E. (1978) Anal. Biochem. 87, 206–210. 12. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265–275. 13. MacFarlane, A. S. (1958) Nature 182, 53. 14. Savion, N., Laherty, R., Lui, G. M., and Gospodarowicz, D. (1981) J. Biol. Chem. 256, 12817–12822. 15. Buege, J. A., and Aust, S. D. (1976) Methods Enzymol. 52, 302– 310.
This research was supported by a grant from the Chief Scientist, Ministry of Health, Israel. We thank Dr. D. Lichtenberg, Sackler, Faculty of Medicine, Tel-Aviv University, for helpful discussions. This work is in partial fulfillment of the requirement for the M.Sc. degree of O. Zavaro from the Sackler Faculty of Medicine at Tel-Aviv University.
16. Pinchuk, I., and Lichtenberg, D. (1996) Free Rad. Res. 24, 351– 360. 17. Holvoet, P., and Collen, D. (1994) FASEB. J. 8, 1279–1284. 18. Auerbach, B. J., Bisgaier, C. L., Wolle, J., and Saxena, U. (1996) J. Biol. Chem. 271, 1329–1335. 19. Kaplan, M., and Aviram, M. (1997) Biochem. Biophys. Res. Commun. 237, 271–276.
REFERENCES 1. Frank, J. S. and Fogelman, A. M. (1989) J. Lipid. Res. 30, 967– 978.
20. Chait, A., Chang, M. Y., Olin, K., and Wight, T. (1997) Atherosclerosis 134, 197.
501
AID
BBRC 8470
/
694f$$$841
03-31-98 11:41:18
bbrcg
AP: BBRC