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Inhibitory effect of high density lipoprotein subfractions on the in vitro binding of low density lipoproteins to arterial elastin
Atherosclerosis, 49 (1983) 171-175 Elsevier Scientific Publishers Ireland,
171 Ltd.
ATH 03397
Inhibitory Effect of High Density Lipoprotein Subfractions on the In Vitro Binding of Low Density Lipoproteins to Arterial Elastin Akio Noma ‘, Toyoko
Hirayama
2 and Akira Yachi 2
‘Division of Clinical Biochemisiry, Tokyo Metropolitan Geriatric Hospital, Tokyo, and 2Departmen! of Internal Medicine, Sapporo Medical College, Sapporo (Japan) (Received 20 May, 1983) (Revised, received 10 June, 1983) (Accepted 10 June, 1983)
Summary
The inhibitory effects of high density lipoprotein (HDL) subfractions on the in vitro complex formation between plasma low density lipoprotein (LDL) and arterial elastin were studied. (1) The inhibitory effects were significantly higher with HDL, than HDL,, and with HDL-without E than HDL-with E. (2) The inhibitory effect of a phospholipid complex with apoHDL, was higher than that with apoHDL,. (3) In contrast with the inhibitory effects, the binding abilities of HDL, and HDL-with E to elastin were significantly higher than those of HDL, and HDLwithout E. (4) These results suggest that the inhibitory effects of HDL subfractions are not due to competitive binding with arterial elastin. Key words: Complex formation - Elastin - High density lipoprotein subfractions Inhibition
-
- Low density lipoproteins
Correspondence: Dr. Akio Noma, Division of Clinical Hospital, 35, Sakae-cho, Itabashi-ku, Tokyo-173, Japan.
Biochemistry,
0021-9150/83/$03.00
Ireland,
0 1983 Elsevier Scientific
Publishers
Ltd.
Tokyo
Metropolitan
Geriatric
172
Introduction The significance of the selective interaction of plasma low density lipoproteins in the pathogenesis of (LDL) with arterial connective tissue macromolecules atherosclerosis is well recognized [l-5]. Studies in our laboratory [6,7] indicated the in vitro formation of a stable complex between LDL and delipidated arterial elastin, and the inhibition of the LDL binding to elastin in the presence of high density lipoproteins (HDL). The present study was undertaken to explore the inhibitory effects of HDL and HDL subfractions on the binding of LDL to delipidated arterial elastin. Materials and Methods Preparation of tissues and elastin Human aortas were obtained at autopsy, cleaned and stored as described previously [5]. Elastin preparations were isolated from the intimal-subintimal layers of human aortas by the previously described method [S]. The delipidation of elastin samples was also carried out as described elsewhere [6]. Preparation of lipoproteins LDL and HDL from fresh normal human plasma were isolated by preparative ultracentrifugation [8]. All centrifugal procedures were carried out at 10°C and 120000 x g in a Beckman L8-70 preparative ultracentrifuge using Type 70-Ti rotor. LDL, HDL, HDL, and HDL, were isolated at densities of 1.019-1.063, 1.063-1.21, 1.063-1.125 and 1.125-1.21 g/ml, respectively. After isolation, each lipoprotein fraction was dialyzed exhaustively against 0.15 M NaCl, containing 0.01% EDTA, at pH 7.0 and at 4°C. HDL-with E and HDL-without E were fractionated by heparin-Sepharose affinity chromatography as described by Weisgraber and Mahley [9], and the HDL subfractions were dialyzed against NaCl-EDTA solution at 4°C. Preparation of apolipoprotein-phospholipid complex An appropriate volume of 0.03 M phosphate buffer, pH 7.35, containing 0.15 M NaCl was added to a glass tube containing dipalmitoyl phosphatidylcholine (Grade I, Sigma Chemical Co.), and this was subjected to ultrasonic irradiation using a Branson Sonifier B-12. The sonication was carried out under N, gas for 5 periods of 1 min, during which time the solution was kept in an ice-water bath. The final concentration of phosphatidylcholine was 10 mg/ml. ApoHDL was added to the sonicated liposome and the mixture was sonicated for 5 or 10 periods of 1 min in an ice-water bath. Incubation The formation of insoluble LDL-elastin complex was studied as described previously [6,7]. Delipidated elastin samples (100 mg) were incubated in a shaking incubator in 50 ml glass tubes at 37°C for 18 h with 10 ml of incubation medium
173
containing 2.0-2.5 mg cholesterol of LDL, various amounts of HDL subfractions and antibiotics in 0.03 M phosphate buffer at pH 7.35. After incubation the washing, dehydration and lipid extraction were carried out as described elsewhere F-5971. Results and Discussion
When a definite amount of LDL was incubated with delipidated elastin in the presence of increasing concentrations of HDL, the binding of lipids to elastin decreased progressively. The magnitudes of the inhibitory effects were comparable for cholesterol, triacylglycerol and phospholipids. With the physiological molar ratio of both lipoproteins (HDL: LDL = 8 : 1, based on protein content), the binding of LDL cholesterol was reduced by 30% [6]. To obtain further information concerning this inhibitory action, we studied the effect of HDL subfractions on the binding ability of LDL. As shown in Fig. 1, inhibition was significantly higher with HDL, than with HDL,, and with HDLwithout E than with HDL-with E. Since little effect was observed with apolipoprotein alone, the effects of liposomes or a phospholipid complex with apoHDL, or apoHDL, were investigated in regard
Li----1.0
HDL-cholesterol
2.0
(mg)
Fig. 1. Effects of increasing concentrations of HDL subfractions on elastin-LDL binding. Incubations were carried out at 37OC for 18 h with 100 mg of delipidated elastin and 2.0 mg of LDL-cholesterol. Values given are means i-SD. Figures in parentheses are the number of experiments. A: 0 = HDL,, 0 = HDL,; B: n = HDL-with E, 0 = HDL-without E.
174 TABLE
1
INHIBITION Results
OF ELASTIN-LDL
are expressed
BINDING
BY APOLIPOPROTEIN
AND PHOSPHOLIPIDS
as mean + SD. Relative binding (W
LDL (LDL cholesterol; 2 mg) LDL + (PLC; 4 mg) LDL+(PLC; 4 mg+apoHDL,; LDL+(PLC; 4 mg+ apoHDL,;
100.0 236.7 + 6.9 (N = 4) T:.4+6.1 (N = 4) 59.6 + 2.7 (N = 5)
4 mg) 4 mg)
PLC = dipalmitoyl phosphatidylcholine; Sonifier; N = number of experiments.
PLC + apoHDL
= sonicated
at 4’C for 5 min using a Branson
to binding. As shown in Table 1, the binding ability of LDL was markedly activated by addition of phospholipid alone to the incubation medium. In contrast, it was significantly inhibited by addition of sonicated phospholipid-apolipoprotein complexes. It was noted that the inhibitory effect of phospholipid complex with apoHDL, was higher than with apoHDL,. The results of a kinetic study [6] suggested that the binding capacity of HDL to elastin was the lowest among the major lipoprotein fractions. It was of interest to compare the binding abilities of LDL and HDL subfractions with the same amounts of lipoprotein cholesterol. In contrast to the inhibitory effects, the binding abilities of HDL, and HDL-with E to delipidated elastin were significantly higher than those of HDL, and HDL-without E (Table 2). The inhibitory effects of HDL on the cell uptake of LDL have been explained by the competitive binding of apoE-containing HDL to the LDL receptors on the cell surface [lO,ll]. However, receptor-mediated binding seems an unlikely explanation in view of the binding of LDL to elastin at the molecular level. With regard to the binding of LDL at the molecular level, Bihari-Varga reported a relationship between
TABLE
2
BINDING Results
ABILITY
are expressed
OF HDL SUBFRACTIONS
TO DELIPIDATED
ELASTIN
as mean f SD.
Lipoprotein
N
Relative binding (I%)
LDL HDL, HDL, HDL-with E HDL-without E
Incubations delipidated
were carried elastin.
5 6 8 9
100.0 30.9 f 3.4 7.3 * 5.4 24.5 f 2.3 lti.2k5.3
out at 37°C for 18 h with 2.0 mg of lipoprotein-cholesterol
and 100 mg of
175
LDL and HDL in the formation of in vitro complexes with human aortic glycosaminoglycans [ 121. The present results indicate that the binding abilities to elastin are higher with HDL, and HDL-with E than with HDL, and HDL-without E, whereas the inhibitory effects on the LDL binding are higher with HDL, and HDL-without E than HDL, and HDL-with E, in spite of correction for their binding abilities. These results suggest that the inhibitory effects of HDL and HDL subfractions are not due to the competitive binding to elastin. However, the real mechanism of inhibition by HDL remains obscure at present. Regardless of the mechanism of inhibition, the results of the present in vitro experiment provide some possible interpretation for the anti-atherosclerotic effect of HDL. References 1 Kramsch,
2 3
7 8 9 10 11 12
D.M., Franzblau, C. and Hollander, W., The protein and lipid composition of arterial elastin and its relationship to lipid accumulation in the atherosclerotic plaque, J. Chn. Invest., 50 (1971) 1666. Smith, E.B., The relation between plasma and tissue lipids in human atherosclerosis, Adv. Lipid Res.. 12 (1974) 1. Hoff, H.F., Jackson, R.L., Mao, J.T. and Gotto, A.M., Localization of low density lipoproteins in arterial lesions from normolipidemics employing a purified fluorescent labeled antibody, B&him. Biophys. Acta, 351 (1974) 407. Srinivasan, S.R., Yost, C., Radhakrishnamurthy, B., Deleferes, Jr., E.R. and Berenson, G.S., Lipoprotein-hyaluronate associations in human aorta fibrous plaque lesions, Atherosclerosis, 36 (1980) 25. Noma, A., Takahashi, T., Yamada, K. and Wada, T., Elastin-lipid interaction in the arterial wall, Part 1 (Extraction of elastin from human aortic intima), Atherosclerosis, 33 (1979) 29. Noma, A., Takahashi, T. and Wada, T., Elastin-lipid interaction in the arterial wall, Part 2 (In vitro binding of lipoprotein-lipids to arterial elastin and the inhibitory effect of high density lipoproteins on the process), Atherosclerosis, 38 (1981) 373. Noma, A., Hirayama, T. and Yachi, A., Studies on the binding of plasma low density lipoproteins to arterial elastin, Corm. Tissue Res., 11 (1983) 123. Havel, R.J., Eder, H.A and Bragdon, J.H., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. Weisgraber, K.H. and Mahley, R.W., Subfractionation of human high density lipoproteins by heparine-Sepharose affinity chromatography, J. Lipid Res., 21 (1980) 316. Carew, T.E., Koschinsky, T., Hayes, S.B. and Steinberg, D., A mechanism by which high-density lipoproteins may slow the atherogenic process, Lancet, i (1976) 1315. Stein, 0. and Stein, Y., High density lipoproteins reduce the uptake of low density lipoproteins by human endothelial cells in culture, Biochim. Biophys. Acta, 431 (1976) 363. Bihari-Varga, M., Influence of serum high density lipoproteins on the low density lipoprotein-aortic glycosaminoglycan interaction, Artery, 4 (1978) 504.
171 Ltd.
ATH 03397
Inhibitory Effect of High Density Lipoprotein Subfractions on the In Vitro Binding of Low Density Lipoproteins to Arterial Elastin Akio Noma ‘, Toyoko
Hirayama
2 and Akira Yachi 2
‘Division of Clinical Biochemisiry, Tokyo Metropolitan Geriatric Hospital, Tokyo, and 2Departmen! of Internal Medicine, Sapporo Medical College, Sapporo (Japan) (Received 20 May, 1983) (Revised, received 10 June, 1983) (Accepted 10 June, 1983)
Summary
The inhibitory effects of high density lipoprotein (HDL) subfractions on the in vitro complex formation between plasma low density lipoprotein (LDL) and arterial elastin were studied. (1) The inhibitory effects were significantly higher with HDL, than HDL,, and with HDL-without E than HDL-with E. (2) The inhibitory effect of a phospholipid complex with apoHDL, was higher than that with apoHDL,. (3) In contrast with the inhibitory effects, the binding abilities of HDL, and HDL-with E to elastin were significantly higher than those of HDL, and HDLwithout E. (4) These results suggest that the inhibitory effects of HDL subfractions are not due to competitive binding with arterial elastin. Key words: Complex formation - Elastin - High density lipoprotein subfractions Inhibition
-
- Low density lipoproteins
Correspondence: Dr. Akio Noma, Division of Clinical Hospital, 35, Sakae-cho, Itabashi-ku, Tokyo-173, Japan.
Biochemistry,
0021-9150/83/$03.00
Ireland,
0 1983 Elsevier Scientific
Publishers
Ltd.
Tokyo
Metropolitan
Geriatric
172
Introduction The significance of the selective interaction of plasma low density lipoproteins in the pathogenesis of (LDL) with arterial connective tissue macromolecules atherosclerosis is well recognized [l-5]. Studies in our laboratory [6,7] indicated the in vitro formation of a stable complex between LDL and delipidated arterial elastin, and the inhibition of the LDL binding to elastin in the presence of high density lipoproteins (HDL). The present study was undertaken to explore the inhibitory effects of HDL and HDL subfractions on the binding of LDL to delipidated arterial elastin. Materials and Methods Preparation of tissues and elastin Human aortas were obtained at autopsy, cleaned and stored as described previously [5]. Elastin preparations were isolated from the intimal-subintimal layers of human aortas by the previously described method [S]. The delipidation of elastin samples was also carried out as described elsewhere [6]. Preparation of lipoproteins LDL and HDL from fresh normal human plasma were isolated by preparative ultracentrifugation [8]. All centrifugal procedures were carried out at 10°C and 120000 x g in a Beckman L8-70 preparative ultracentrifuge using Type 70-Ti rotor. LDL, HDL, HDL, and HDL, were isolated at densities of 1.019-1.063, 1.063-1.21, 1.063-1.125 and 1.125-1.21 g/ml, respectively. After isolation, each lipoprotein fraction was dialyzed exhaustively against 0.15 M NaCl, containing 0.01% EDTA, at pH 7.0 and at 4°C. HDL-with E and HDL-without E were fractionated by heparin-Sepharose affinity chromatography as described by Weisgraber and Mahley [9], and the HDL subfractions were dialyzed against NaCl-EDTA solution at 4°C. Preparation of apolipoprotein-phospholipid complex An appropriate volume of 0.03 M phosphate buffer, pH 7.35, containing 0.15 M NaCl was added to a glass tube containing dipalmitoyl phosphatidylcholine (Grade I, Sigma Chemical Co.), and this was subjected to ultrasonic irradiation using a Branson Sonifier B-12. The sonication was carried out under N, gas for 5 periods of 1 min, during which time the solution was kept in an ice-water bath. The final concentration of phosphatidylcholine was 10 mg/ml. ApoHDL was added to the sonicated liposome and the mixture was sonicated for 5 or 10 periods of 1 min in an ice-water bath. Incubation The formation of insoluble LDL-elastin complex was studied as described previously [6,7]. Delipidated elastin samples (100 mg) were incubated in a shaking incubator in 50 ml glass tubes at 37°C for 18 h with 10 ml of incubation medium
173
containing 2.0-2.5 mg cholesterol of LDL, various amounts of HDL subfractions and antibiotics in 0.03 M phosphate buffer at pH 7.35. After incubation the washing, dehydration and lipid extraction were carried out as described elsewhere F-5971. Results and Discussion
When a definite amount of LDL was incubated with delipidated elastin in the presence of increasing concentrations of HDL, the binding of lipids to elastin decreased progressively. The magnitudes of the inhibitory effects were comparable for cholesterol, triacylglycerol and phospholipids. With the physiological molar ratio of both lipoproteins (HDL: LDL = 8 : 1, based on protein content), the binding of LDL cholesterol was reduced by 30% [6]. To obtain further information concerning this inhibitory action, we studied the effect of HDL subfractions on the binding ability of LDL. As shown in Fig. 1, inhibition was significantly higher with HDL, than with HDL,, and with HDLwithout E than with HDL-with E. Since little effect was observed with apolipoprotein alone, the effects of liposomes or a phospholipid complex with apoHDL, or apoHDL, were investigated in regard
Li----1.0
HDL-cholesterol
2.0
(mg)
Fig. 1. Effects of increasing concentrations of HDL subfractions on elastin-LDL binding. Incubations were carried out at 37OC for 18 h with 100 mg of delipidated elastin and 2.0 mg of LDL-cholesterol. Values given are means i-SD. Figures in parentheses are the number of experiments. A: 0 = HDL,, 0 = HDL,; B: n = HDL-with E, 0 = HDL-without E.
174 TABLE
1
INHIBITION Results
OF ELASTIN-LDL
are expressed
BINDING
BY APOLIPOPROTEIN
AND PHOSPHOLIPIDS
as mean + SD. Relative binding (W
LDL (LDL cholesterol; 2 mg) LDL + (PLC; 4 mg) LDL+(PLC; 4 mg+apoHDL,; LDL+(PLC; 4 mg+ apoHDL,;
100.0 236.7 + 6.9 (N = 4) T:.4+6.1 (N = 4) 59.6 + 2.7 (N = 5)
4 mg) 4 mg)
PLC = dipalmitoyl phosphatidylcholine; Sonifier; N = number of experiments.
PLC + apoHDL
= sonicated
at 4’C for 5 min using a Branson
to binding. As shown in Table 1, the binding ability of LDL was markedly activated by addition of phospholipid alone to the incubation medium. In contrast, it was significantly inhibited by addition of sonicated phospholipid-apolipoprotein complexes. It was noted that the inhibitory effect of phospholipid complex with apoHDL, was higher than with apoHDL,. The results of a kinetic study [6] suggested that the binding capacity of HDL to elastin was the lowest among the major lipoprotein fractions. It was of interest to compare the binding abilities of LDL and HDL subfractions with the same amounts of lipoprotein cholesterol. In contrast to the inhibitory effects, the binding abilities of HDL, and HDL-with E to delipidated elastin were significantly higher than those of HDL, and HDL-without E (Table 2). The inhibitory effects of HDL on the cell uptake of LDL have been explained by the competitive binding of apoE-containing HDL to the LDL receptors on the cell surface [lO,ll]. However, receptor-mediated binding seems an unlikely explanation in view of the binding of LDL to elastin at the molecular level. With regard to the binding of LDL at the molecular level, Bihari-Varga reported a relationship between
TABLE
2
BINDING Results
ABILITY
are expressed
OF HDL SUBFRACTIONS
TO DELIPIDATED
ELASTIN
as mean f SD.
Lipoprotein
N
Relative binding (I%)
LDL HDL, HDL, HDL-with E HDL-without E
Incubations delipidated
were carried elastin.
5 6 8 9
100.0 30.9 f 3.4 7.3 * 5.4 24.5 f 2.3 lti.2k5.3
out at 37°C for 18 h with 2.0 mg of lipoprotein-cholesterol
and 100 mg of
175
LDL and HDL in the formation of in vitro complexes with human aortic glycosaminoglycans [ 121. The present results indicate that the binding abilities to elastin are higher with HDL, and HDL-with E than with HDL, and HDL-without E, whereas the inhibitory effects on the LDL binding are higher with HDL, and HDL-without E than HDL, and HDL-with E, in spite of correction for their binding abilities. These results suggest that the inhibitory effects of HDL and HDL subfractions are not due to the competitive binding to elastin. However, the real mechanism of inhibition by HDL remains obscure at present. Regardless of the mechanism of inhibition, the results of the present in vitro experiment provide some possible interpretation for the anti-atherosclerotic effect of HDL. References 1 Kramsch,
2 3
7 8 9 10 11 12
D.M., Franzblau, C. and Hollander, W., The protein and lipid composition of arterial elastin and its relationship to lipid accumulation in the atherosclerotic plaque, J. Chn. Invest., 50 (1971) 1666. Smith, E.B., The relation between plasma and tissue lipids in human atherosclerosis, Adv. Lipid Res.. 12 (1974) 1. Hoff, H.F., Jackson, R.L., Mao, J.T. and Gotto, A.M., Localization of low density lipoproteins in arterial lesions from normolipidemics employing a purified fluorescent labeled antibody, B&him. Biophys. Acta, 351 (1974) 407. Srinivasan, S.R., Yost, C., Radhakrishnamurthy, B., Deleferes, Jr., E.R. and Berenson, G.S., Lipoprotein-hyaluronate associations in human aorta fibrous plaque lesions, Atherosclerosis, 36 (1980) 25. Noma, A., Takahashi, T., Yamada, K. and Wada, T., Elastin-lipid interaction in the arterial wall, Part 1 (Extraction of elastin from human aortic intima), Atherosclerosis, 33 (1979) 29. Noma, A., Takahashi, T. and Wada, T., Elastin-lipid interaction in the arterial wall, Part 2 (In vitro binding of lipoprotein-lipids to arterial elastin and the inhibitory effect of high density lipoproteins on the process), Atherosclerosis, 38 (1981) 373. Noma, A., Hirayama, T. and Yachi, A., Studies on the binding of plasma low density lipoproteins to arterial elastin, Corm. Tissue Res., 11 (1983) 123. Havel, R.J., Eder, H.A and Bragdon, J.H., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. Weisgraber, K.H. and Mahley, R.W., Subfractionation of human high density lipoproteins by heparine-Sepharose affinity chromatography, J. Lipid Res., 21 (1980) 316. Carew, T.E., Koschinsky, T., Hayes, S.B. and Steinberg, D., A mechanism by which high-density lipoproteins may slow the atherogenic process, Lancet, i (1976) 1315. Stein, 0. and Stein, Y., High density lipoproteins reduce the uptake of low density lipoproteins by human endothelial cells in culture, Biochim. Biophys. Acta, 431 (1976) 363. Bihari-Varga, M., Influence of serum high density lipoproteins on the low density lipoprotein-aortic glycosaminoglycan interaction, Artery, 4 (1978) 504.