Dec 19, 1985 - Jerome and Lewis observed an increase in monocyte adhesion to the ...... Redgrave TG, Robert DCK, West CE: Separation of lipoproteins by .... Taylor RJ, Lewis JC: Monocyte adhesion and endothelial turnover in pellet vs ...
LDL Enhances
Monocyte Adhesion to Endothelial
Cells
in Vitro
LLOYD M. ALDERSON, DSc, GERDA ENDEMANN, PhD, SARALYN LINDSEY, BS, ANDY PRONCZUK, PhD, RICHARD L. HOOVER, PhD, and K. C. HAYES, DVM, PhD
From the Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts; the Foster Biomedical Research Laboratory, Brandeis University, Waltham, Massachusetts; and the Department of Pathology, Harvard Medical School, Boston, Massachusetts
Monocyte adhesion to the arterial endothelium is an early event in diet-induced atherogenesis. The possibility that low-density lipoprotein (LDL) may influence this adhesion was investigated by using an in vitro monolayer collection assay. Postprandial and fasting LDL was isolated from 12 normal adult human donors (8 male and 4 female) and incubated with primary cultures of bovine aortic endothelial cells (BAEC) for 6 hours. 31Cr-labeled mononuclear leukocytes (MNLs) were then added and incubated an additional 30 minutes. When results were expressed as the ratio of adherent counts per minute in
LDL-treated BAEC cultures to that in PBS-treated controls, 10 of the 16 LDL samples isolated from male donors induced a significant increase (P < 0.05) in MNL adhesion (1.06-1.27) attributable to esterase-positive cells. This increase was dose-dependent and maximal at 100 gg LDL protein/ml. The magnituJe of the response was significantly correlated with LDL composition (r = 0.857, P < 0.01) such that LDL rich in cholesterol and triglyceride relative to protein enhanced MNL adhesion, whereas lipid-poor LDL (typically isolated from the women) reduced adhesion. (Am J Pathol 1986, 123:334-342)
AN INCREASE in monocyte adhesion to the arterial endothelium is an early event in the development of diet-induced atherosclerosis in several animal models. 1-5 In the pig, Gerrity observed a dramatic increase in the number of blood monocytes adhering to the aortic endothelium 2 weeks after the animals began eating a high-fat diet.2 In subsequent weeks these adherent monocytes appeared to migrate between adjacent endothelial cells, become lodged in the intima, and accumulate lipid. A similar series of events was described by Faggiotto et al in the cholesterol-fed pigtail macaque.6 These studies and others7' 8 have implicated the blood monocyte as the major contributor to the intimal foam cell accumulation that characterizes the fatty
bit, monocyte adhesion to the regenerating endothelium was noted only in the hypercholesterolemic group.5 These findings suggest circulating lipoproteins may be involved in both monocyte adhesion and the lipid accumulation of foam cell development. In our study of atherosclerosis in cynomolgus monkeys, the concentration of intimal foam cells was correlated with total cholesterol and the ratio of total to high-density lipoprotein (HDL) cholesterol in plasma.9 These observations led us to consider the possibility that LDL mediates the enhancement of the endothelial cell-monocyte interaction that leads to fatty streak development in hypercholesterolemic man and animals. In the present study, the effects of LDL on monocyte adhesion to endothelial cells in vitro was investigated with the result that lipid-rich LDL induced a dose-dependent increase in monocyte adhesion.
streak lesion. Diet-induced increases in the adhesion of monocytes to the arterial endothelium are invariably associated with elevated circulating cholesterol concentrations, particularly low-density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol. In their study of atherosclerosis in young, cholesterol-fed pigeons, Jerome and Lewis observed an increase in monocyte adhesion to the arterial endothelium in the hypercholesterolemic birds relative to those with normocholesterolemia.4 Similarly, in the injured aorta of the rab-
Accepted for publication December 19, 1985. Address reprint requests to Dr. K. C. Hayes, Foster Biomedical Research Laboratory, Brandeis University, Waltham, MA 02254.
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MONOCYTE ADHESION TO
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Materials and Methods Endothelial Cell Cultures Bovine aortic endothelial cells (BAECs) were isolated according to the method of Booyse and co-workers.10 Briefly, 3-5-inch segments of thoracic aorta were excised from calves within 15 minutes of sacrifice and placed on ice. They were then immersed in cold phosphate-buffered saline (PBS) containing 5 .g/ml amphotericin B and 0.1% glucose, and the surrounding connective tissue was removed. The aortas were incubated in Opti-MEM medium (GIBCO Laboratories, Grand Island, NY) containing 1.0 mg/ml collagenase (Worthington Biochemical Co., Freehold, NJ), 100 U/ml penicillin, and 100 gg/ml streptomycin (pen/ strep) for 25 minutes at 37 C. Following incubation, 25 ml of medium was flushed through each segment to dislodge the endothelial cells. The cells were washed twice with medium and suspended in growth medium (Opti-MEM, 10% fetal bovine serum, pen/strep, and amphotericin B). The cells from one aortic segment were plated on two 24-well tissue culture plates. The cells were incubated in a 5%o CO2 atmosphere of 37 C. Growth medium was changed every other day until the cells reached confluence (5-7 days). All adhesion assays were done within 6-8 days of plating the cells, at which time the cell density was 0.8-1.2 x 105 cells/sq cm. The identity of these cells was confirmed by the cobblestone appearance of confluent cultures under light microscopy10 and positive immunofluorescent staining for Factor
Vill.,1 Isolation of Lipoproteins Lipoproteins were isolated by discontinuous gradientdensity ultracentrifugation according to the method of Redgrave and co-workers.12 Two whole blood samples (one fasting and one 120 minutes postprandial) were obtained by venipuncture from 12 adult human donors (8 male, 4 female). Four mililiters of serum were adjusted to a density of 1.21 g/ml with KBr and overlaid with three NaBr solutions (3 ml of d = 1.060, 3 ml of d = 1.020, and 2 ml of d = 1.006). The tubes were centrifuged in a swinging-bucket rotor (Model SW41, Beckman Instruments, Palo Alto, Calif) for 44 hours at 37,000 rpm at 20 C. LDL was harvested as a narrow band in the 1.020-1.040 density range, and lipoproteindeficient serum (LPDS) from the bottom of the tube (d > 1.21). LDL and LPDS were dialyzed against 0.9q% NaCl and 0.02%o EDTA (50 volumes x 4) followed by PBS and pen/strep (50 volumes x 1) at 4 C. Protein concentrations in the lipoprotein solutions were determined by the method of Kashyap and co-workers. 13 The
ENDOTHELIAL CELLS
335
purity of LDL samples was confirmed by SDS polyacrylamide gel electrophoresis (7%). Cholesterol and triglyceride concentrations were determined enzymatically (Sigma Diagnostic Kits 350 and 90, Sigma Chemical Company, St. Louis, Mo). Phospholipids were determined by the method of Bartlett.14 Isolation of Mononuclear Cells Mononuclear leukocytes (MNLs) were isolated from the peripheral blood of normal adults according to the method of Boyum.15 Whole blood was collected into EDTA vacutainers and diluted 1:1 with PBS containing 1 mm EDTA and 0.5 mm sodium citrate. Thirtyeight-milliliter aliquots of this diluted blood was layered over 12 ml of Ficoll-Hypaque (lymphocyte separation medium, Litton Bionetic Co., Rockville, Md) in a 50ml conical tube and centrifuged (570g for 40 mins) at 20 C. The leukocyte layer was washed twice with PBS containing EDTA and sodium citrate for removal of platelets. Platelets were isolated from whole blood according to the method of Born.16 Labeling of Mononuclear Cells The mononuclear leukocytes and platelets were labeled with 51Cr according to the method of Gallin and co-workers.17 Briefly, the cells were suspended in 1.0-1.5 ml of medium to which 0.1 ml 51Cr (1.0 mCi/ml as sodium chromate in normal saline, New England Nuclear Corp., Boston, Mass) was added. The suspension was incubated for 60 minutes at 4 C. Following incubation, the cells were washed with medium and suspended in 5-10 ml of medium for a final concentration of 1.0-2.0 x 106 cells/ml. The Adhesion Assay
Mononuclear leukocyte adhesion was quantitated with a monolayer collection assay similar to that described by Walther and co-workers.18 BAEC cultures were preincubated for 18 hours in medium containing 10% LPDS and pen/strep. Cells were then rinsed with 0.5 ml medium and incubated with 300 gl of medium plus 100 gl PBS or 100 gl PBS containing LDL (final concentration, 100 gg protein/ml unless stated otherwise). After 6 hours, 100 gl of the 51Cr-labeled MNL suspension was added to each well, and cells were incubated an additional 30 mins. Nonadherent MNLs were washed off by rinsing the cultures once with 0.5 ml of medium. The cultures were then solubilized in 0.2 M NaOH and neutralized with 2 M HCI. Adherent
336
ALDERSON ET AL
AJP * May 1986
treated controls. Analysis of variance was used to determine whether monocyte donor had a significant effect on monocyte adhesion.
1500
Results
1000 E
cL
-41 -P
500
0
5 10
20 30
60 Time in Minutes
!20
Figure 1-The control time course of s'Cr-labeled MNL adhesion to PBSincubated BAECs in vitro is plotted. Subsequently, all adhesion assays were stopped 30 minutes after adding MNLs.
MNLs were quantitated by liquid scintillation counting. Esterase activity was determined in adherent MNLs with the a-naphthyl acetate esterase reaction (Sigma Chemical Co., St. Louis, Mo). The results were expressed as the ratio of the adherent mononuclear cells in the lipoprotein-treated BAEC cultures to that in the PBS-treated cultures. Six replicates were done for each treatment.
Statistics The student t-test was used to determine if MNL adhesion in LDL-treated cultures differed from PBS-
A) Monocyte Adhesion to BAEC 1.30
MNL Adhesion to BAEC To establish a time course for MNL adhesion to BAEC monolayers, we followed MNL adhesion (as determined from 5"Cr counts per minute over a 2-hour period (Figure 1). Adhesion appeared to be maximal at 30 minutes. Therefore, all subsequent adhesion assays were stopped at 30 minutes after the addition of labelled MNLs. More than 9007o of the adherent MNLs were esterase-positive. Effect of LDL on MNL Adhesion The effect of preincubating BAEC cultures for 6 hours with LDL (postpraiudial from a male donor), BSA, or LPDS (all at 100 g1 protein/ml) was assessed (Figure 2). To control for interassay variation, the data are expressed as a ratio of MNL adhesion to treated BAEC cultures relative to PBS-treated controls. LDL induced a significant increase in MNL adhesion, whereas BSA and LPDS at the same protein concentration had no effect. MNL adhesion to LDL-treated BAECs that were washed with protein-free medium before adding the labeled MNLs was less than adhesion to unwashed cultures, but still greater than to PBS-
B) Monocyte Adhesion to Plastic
1-
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0 60 L Figure 2-Monocyte adhesion to BAEC monolayers (A) and tissue culture plastic (B) is depicted. The results are expressed as the ratio of adherent counts per minute relative to the PBS control. LDL was isolated from a postprandial serum sample for donor Ml. LDL, BSA, or LPDS were added to BAEC monolayers 6 hours before adding labeled MNLs. In one group (LDL + wash), the incubation medium containing LDL was replaced by fresh medium containing PBS just before the labeled MNLs were added.
M(ONOCYTE ADHESION TO ENDOTHELIAL CELLS
Vol. 123 * No. 2
treated controls. LDL also induced a significant reduction in MNL adhesion to plastic. In order to test whether LDL was enhancing monocyte adhesion specifically, simultaneous experiments were conducted in which MNL adhesion was quantitated from 51Cr counts per minute and from counts of adherent esterase-positive cells under light microscopy. The LDL-induced increase in MNL adhesion was comparable for both assays (Figure 3). A dose-response curve for MNL adhesion to determine the effect of preincubating BAEC cultures with different concentrations of LDL is shown in Figure 4. LDL induced a dose-dependent increase in adhesion which was maximal at 100 gig LDL protein/ml. The effect of preincubating BAECs with LDL for increasing amounts of time is shown in Figure 5. LDL had no effect on MNL adhesion when added simultaneously with the labeled cells, whereas 30 minutes of preincubation resulted in a maximal response.
in
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0 10 25
100 75 50 Concentration of LDL
150
200
(/.Lg Protein/ml)
Figure 4-The effect of increasing concentrations of LDL (postprandial from donor M5) on monocyte adhesion to BAEC monolayers in vitro is plotted. Adhesion to BAECs appeared to be maximal between 75 and 100 gg LDL protein/ml and was significantly greater than with PBS alone (P< 0.05).
co a.
120 F-
0
.' 1.15 F
LDL Composition Fasting and postprandial LDL were isolated from 12 different donors (8 male and 4 female) and tested for their ability to alter MNL adhesion to BAEC (Table 1). Postprandial LDL from men induced a significant increase in MNL adhesion when compared to fasting LDL, whereas fasting and postprandial LDL from female donors tended to reduce MNL adhesion relative to PBS-treated controls (Figure 6). Human LDL
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Figure 3-Monocyte adhesion assessed by 5'Cr counts per minute and that assessed by esterase-positive cells by light microscopy were com-
parable.
338
ALDERSON ET AL
AJP * May 1986
Table 1-Monocyte Adhesion to Endothelial Cells Treated With Fasting and Postprandial LDL From Human Male (M1-8) and Female (F1-4) Donors (Relative to PBS-Incubated Control) Donor Fasting Postprandial Ml M2 M3 M4 M5 M6 M7 M8 Fl F2 F3 F4 *
1.02 1.18 1.03 1.06 1.03 0.96 1.09 0.98 0.95 1.00 0.89 0.98
+ + + + + + + + + + + +
0.02 0.02* 0.01 0.02* 0.03 0.03 0.02* 0.02 0.01 0.03 0.03* 0.03
1.27 1.23 1.12 1.09 1.15 1.07 1.00 1.18 0.93 0.92 0.98 1.01
+ + + + + + + + + + + +
0.03* 0.03* 0.05* 0.02* 0.02* 0.01* 0.01 0.02 0.01 * 0.02* 0.03 0.02
Normocholesterolemic Human LDL 1.2 r W
m
*
a-
LDL and Platelet Adhesion Because MNL isolated with Ficoll-Hypaque are contaminated with platelets and lymphocytes, the effects of preincubating BAEC with LDL on platelet adhesion was examined. LDL had no effect on the adhesion of 51Cr-labeled platelets (1.00 ± 0.04), whereas this same LDL induced a significant increase in adherent counts per minute when labeled MNLs were added (1.22 + 0.03). Discussion
The results from these experiments demonstrate that LDL particles rich in cholesterol and triglyceride relative to protein induce a dose-dependent increase in
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Figure 6-Overall comparison of LDL from fasted or fed male donors (n = 8) and equivalent female donors (n = 4) in their ability to induce MNL adhesion indicates that only postprandial male LDL collectively caused a significant increase in monocyte adhesion.
monocyte adhesion to aortic endothelial cells in vitro. This finding is consistent with the many reports of an increase in monocyte adhesion to the arterial en-
dothelium of animals with high concentrations of LDL cholesterol. Rudel and co-workers have observed that hypercholesterolemic monkeys have enlarged LDL particles that are rich in cholesterol relative to protein.19 A change in LDL structure to a larger, more lipid-rich particle in fat-fed animals may therefore be an important determinant of the intimal monocytosis that several investigators have described as an early event in atherogenesis.
-8
The lipid-rich LDL particles that
Table 2-Composition of LDL From Human Male and Female Donors Protein Phospholipid Triglyceride Donor status (% + SEM) (% ± SEM) (% ± SEM)
Female Fasting Postprandial
p(O.025
0
0
BAEC Density The effects of LDL on MNL adhesion were determined at four different BAEC densities ranging from soon after a continuous monolayer was established (1.0 x 105 cells/sq cm) to a more quiescent monolayer (3.0 x 105 cells/sq cm). MNL adhesion to PBS-control and LDL-treated cultures (ie, total counts per minute of 51Cr) was higher only at the lowest density (Figure 8).
Postprandial
Females
to
Value different from 1.00 (P < 0.05).
Male Fasting
Males
Cholesterol ± SEM)
(%
were
particularly
Chol
+
TG/protein
(ratio ± SEM)
24.1 + 1.3 22.1 ± 1.6
26.1 ± 0.9 24.0 ± 1.1
6.1 ± 0.4 13.4 ± 4.5
43.7 ± 1.8 40.5 + 3.6
2.15 ± 0.23 2.60 ± 0.31
28.8 ± 0.4' 26.4 ± 1.4
26.0 ± 1.6 27.6 ± 1.5
5.7 ± 0.6 7.3 ± 2.4
39.5 ± 1.5 38.8 ± 2.1
1.58 + 0.05 1.77 ± 0.13
'Differs from fasting male (P < 0.025).
MONOCYTE ADHESION TO
Vol. 123 * No. 2 1-.
CL)
m 0CD
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339
CELLS
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0
Figure 7-The correlation between MNL adhesion and LDL neutral-lipid concentration (relative to LDL protein) is depicted. LDL with a large concentration of cholesterol and triglyceride enhanced MNL adhesion, whereas lipid-poor LDL either decreased adhesion or had no effect.
ENDOTHEUIAL
1.00 F-
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effective in our assay have been shown to be atherogenic in other studies. St. Clair and co-workers have shown that larger, cholesterol-rich LDL isolated from hypercholesterolemic monkeys cause more cholesterol ester to accumulate in smooth muscle cells in vitro than LDL isolated from normolipemic monkeys.20'21 Similar results were reported by Chen and Fischer-Dzoga for LDL isolated from hypercholesterolemic rabbits.22 The results from our study predict that the lipid-rich LDL particles described in the latter two reports would induce a significant increase in monocyte adhesion to the arterial endothelium. An interaction between LDL and the cultured endothelial cell appears to be necessary to induce an increase in monocyte adhesion. When LDL was added simultaneously with the radiolabeled MNLs, no effect on monocyte adhesion was seen; whereas preincubation of the endothelial cell cultures with LDL for 30 minutes resulted in a maximal response. The effects of LDL we report cannot, therefore, be attributed to LDLinduced changes in the monocyte or monocyte-lymphocyte interactions alone. Reckless and co-workers have described two classes of LDL binding sites on the endothelial cell surface, a high-affinity, low-capacity site and a low-affinity site which was not saturable.23 Our results do not support the involvement of either of these sites in LDL-induced changes in monocyte adhesion. The high-affinity LDL receptor becomes saturated at an LDL concentration of 20 .g protein/ml, well below the concentration at which the effect of LDL on monocyte adhesion was evident. The low-affinity site does not saturate even at LDL
concentrations exceeding 500 gg protein/ml. Again, this is inconsistent with our results. Endothelial cells also express binding sites for 3VLDL,24 which may be involved in this LDL-enhanced monocyte adhesion. Although ,B-VLDL isolated from cholesterol-fed animals has the highest affinity for this receptor,25 LDL24 and postprandial VLDL from normal human donors26 are also good ligands. Thus the ,B-VLDL receptor may mediate the effects of LDL on monocyte adhesion for three reasons. First, the concentration of LDL at which we saw a maximal response in monocyte adhesion (75-100 .g LDL protein/ml) corresponds to the saturation point for f-VLDL receptors on both the endothelial cell (60 jg protein/ml)24 and 0.15 r 0
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Figure 8-Monocyte adhesion per endothelial cell was significantly reduced from the earliest stage of endothelial confluency to later stages, where cells are more confluent.
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ALDERSON ET AL
monocyte-derived macrophage (100-120 ig protein/ml).26 Second, only the larger, lipid-rich LDL induced a significant increase in monocyte adhesion. The composition of these particles more closely approximates 3-VLDL particles, which have a higher affinity for the receptor, than the smaller, lipid-poor LDL. Finally, I-VLDL isolated from hypercholesterolemic cebus monkeys also induced a significant increase in monocyte adhesion to endothelial cells, whereas LDL and VLDL from normolipemic monkeys had no effect (unpublished data). It is unclear from our experiments whether internalization of LDL by the endothelial cell is necessary to alter monocyte adhesion. Washing the endothelial cell cultures prior to adding the labeled MNLs markedly reduced the LDL effect on adhesion when compared with cultures in which LDL was not removed. This suggests that LDL-enhanced monocyte adhesion may not depend on lipoprotein uptake, and that LDL must be present in the endothelial cell-monocyte incubation medium in order for us to see a maximal effect. If internalization is not necessary, it is possible that LDL enhances monocyte adhesion by interacting with both cell types simultaneously. We found the adhesion of monocytes to tissue culture plastic was reduced by the same LDL that increased monocyte adhesion to endothelial cells, which suggests that LDL is interacting with sites on the surface of monocytes and endothelium that mediate adhesion. By interacting with both cell types simultaneously, large, lipid-rich LDL could enhance monocyte adhesion by acting as a bridge, a postulate previously proffered by others.1 LDL appears to be just one of several factors that can influence monocyte adhesion. Recent work by Valente and co-workers27 demonstrates that blood proteins such as fibrinogen can dramatically increase monocyte adhesion to endothelial cells in vitro. These investigators also found that albumin (2 mg/ml) can inhibit monocyte adhesion by as much as 50%. In our system albumin at 100 gg/ml had no effect on monocyte adhesion. Another important variable may be the monocyte itself. Gerrity and co-workers have described significant morphologic heterogeneity among monocytes isolated from hypercholesterolemic pigs.28 Among normolipemic male donors, we did not observe a significant effect of monocyte donor on adhesion to BAEC cultures. However, the fact that a large proportion of monocytes adhere to BAEC without LDL present suggests that LDL may be acting on a functionally distinct subgroup of monocytes that binds lipid-rich lipoproteins. Studies conducted both in vivo and in vitro have shown that the rate of endothelial cell turnover may
AJP * May 1986
also be an important determinant of monocyte adhesion. Experiments in the pigeon,29 rabbit,30'31 and dog32 demonstrate a correlation between areas of accelerated endothelial cell growth and the concentration of adherent monocytes. DiCorleto and de la Motte33 have drawn similar conclusions from in vitro studies in which U937 cells, a monocyte-like cell line, preferentially adhered to proliferating endothelial cells on the growing edge of cultures that had been partially denuded. These studies suggest that monocytes recognize and adhere to proliferating endothelial cells. In our cultures monocyte adhesion to endothelial cells was measured soon after cells had reached confluence (0.8-1.2 x 105 cells/sq cm). Baker et al reported that primary BAEC cultures continue dividing until they reach a cell density of 4.0-5.0 x 105 cells/sq cm.24 We also measured MNL adhesion to endothelial cells at a higher density (up to 3.0 x 105 cells/sq cm) and found a significant reduction of adherent leukocytes per endothelial cell, with increasing cell density. It is unclear whether this reduction reflects a change in the expression of binding sites on the endothelial cell surface with which the leukocytes can interact or simply a reduction in accessible cell surface area associated with a higher density. By using the lower cell density in our experiments, we modeled the in vivo replicating endothelial cell associated with atherogenesis rather than the quiescent, nonreplicating endothelium that typically overlies nonlesioned areas of arterial intima.3' LDL has been reported to be cytotoxic to endothelial cells in vitro,34-36 and this should be considered in the interpretation of our results. Monocytes can recognize dying endothelial cells and appear to have an increased affinity for them.38 However, it is unlikely that the effects of LDL on monocyte adhesion can be explained by its cytotoxic properties. Morel and co-workers35 demonstrated that LDL cytotoxicity was largely due to lipid peroxidation and was not seen with LDL isolated in the presence of an antioxidant. In our study, LDL was isolated in the presence of EDTA and used shortly after isolation. Henrikson and co-workers36 reported that LDL cytotoxicity was observed only in endothelial cell cultures exposed to a much higher concentration (approximately 500 ig LDL protein/ml) for a much longer time (48 hours) than in our study. Furthermore, the addition of BHT to our lipoprotein isolates has no effect on adhesion (unpublished data). It is noteworthy that the effects of LDL on monocyte adhesion cannot be attributed to the amount of lipid added (as LDL) per se. For instance, even when the concentrations of triglyceride, phospholipid, and cholesterol were higher with the lipid-poor LDL particles at 200 gg protein/ml than with the lipid-rich LDL particles at 100 1.g protein/ml, the lipid-poor LDL in-
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duced a significant decrease in monocyte adhesion. The composition of LDL is therefore more important to its ability to alter monocyte adhesion than quantitative changes in the concentration of LDL components in the incubation medium. The correlation between monocyte adhesion and LDL composition was observed, even with our small sample size, because of the heterogeneity of LDL. This was due not only to individuals within the group of subjects, but also to variation in their LDL under different metabolic circumstances. For example, 4 of the 8 male donors accumulated large amounts of triglyceride in their LDL postprandially. In all 4 cases this accumulation was associated with an increase in monocyte adhesion relative to their fasting LDL. Deckelbaum and coworkers described elevated LDL triglyceride in patients with familial hypertriglyceridemia.39 Our observations suggest that these triglyceride-rich LDLs found either transiently in the postprandial state or chronically in genetic abnormalities may enhance monocyte-endothelial cell interactions and thus be more atherogenic. This is consistent with epidemiologic studies that have linked elevated plasma triglyceride with coronary heart disease4 and the hypothesis of Zilversmit that postprandial lipoproteins are more atherogenic than those circulating postabsorptively.41 Although only 4 women were studied, results obtained with their fasting and postprandial LDL are also consistent with epidemiologic studies indicating that premenopausal women are at lower risk of coronary heart disease than men.42 None of the LDLs from women induced an increase in monocyte adhesion, and four of them significantly decreased monocyte adhesion. In summary, LDL rich in cholesterol and triglyceride can enhance monocyte adhesion to endothelial cells in culture. This may reflect an interaction with binding sites on one or both cell types. Further studies are needed to confirm the trends reported and to define the nature of this endothelial cell-monocyte-LDL interaction. However, the inference is that small changes in LDL composition may have a significant impact on the interaction of cells thought to be involved in the development of atherosclerotic lesions.
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II. Migration of foam cells from atherosclerotic lesions. Am J Pathol 1981, 103:191-200 Jerome WG, Lewis JC: Early atherogenesis in white carneau pigeons: I. Leukocyte margination and endothelial alterations at the celiac bifurcation. Am J Pathol 1984, 116:56-68 Walker LN, Bowyer DE: Endothelial healing in the rabbit aorta and the effect of risk factors for atherosclerosis. Arteriosclerosis 1984, 4:479-489 Faggiotto A, Ross R, Harker L: Studies of hypercholesterolemia in the nonhuman primate: I. Changes that lead to fatty streak formation. Arteriosclerosis 1984, 4:323-341 Schaffner T, Taylor K, Bartucci EJ, Fischer-Dzoga K, Beesor JH, Glagov S, Wissler RW: Arterial foam cells with distinctive immunomorphologic and histochemical features of macrophages. Am J Pathol 1980, 100:57-73 Gerrity RG, Naito HK: Ultrastructural identification of monocyte-derived foam cells in fatty streak lesions. Artery 1980, 8:208-214 Alderson LM, Hayes KC, Nicolosi RJ: Peanut oil reduces diet-induced atherosclerosis in cynomolgus monkeys.
(Manuscript submitted)
10. Booyse FM, Sedlak B, Rafelson N: Culture of arterial endothelial cells. Characterization and growth of bovine aortic cells. Thromb Diath Haemorrh 1975, 34:825-839 11. Jaffe EA, Hoyer LW, Nachman R: Synthesis of antihemophilic factor antigen by cultured human endothelial cells. J Clin Invest 1973, 52:2757-2764 12. Redgrave TG, Robert DCK, West CE: Separation of lipoproteins by density gradient ultracentrifugation. Anal Biochem 1975, 65:42-49 13. Kashyap ML, Hynd BA, Robinson K: A rapid and simple method for measurement of total protein in very low density lipoproteins by the Lowry method assay. J Lipid Res 1980, 21:491-495 14. Bartlett GR: Phosphorous assay in column chromatography. J Biol Chem 1959, 234:466-468 15. Boyum A: Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 1968, 97 (Suppl 21): 77-89 16. Born GVR: Quantitative investigation into aggregation of blood platelets. J Physiol 1962, 162:67-83 17. Gallin J, Clark RC, Kimball HP: Granulocyte chemotaxis: an improved in vitro assay employing 51Cr-labeled granulocytes. J Immunol 1973, 110:233-240 18. Walther BT, Ohman R, Roseman S: A quantitative assay for intracellular adhesion. Proc Nat Acad Sci USA 1973, 70:1569-1573 19. Rudel LL, Pitts, LL, Nelson CA: Characterization of plasma low density lipoproteins of nonhuman primates fed dietary cholesterol. J Lipid Res 1977, 18:211-222 20. St. Clair RW, Leight MA: Differential effects of isolated lipoproteins from normal and hypercholesterolemic rhesus monkeys on cholesterol esterification and accumulation in arterial smooth muscle cells in culture. Biochim Biophys Acta 1978, 530:279-291 21. St. Clair RW, Greenspan P, Leight M: Enhanced cholesterol delivery to cells in culture by LDL from hypercholesterolemic monkeys: Correlation of cellular cholesterol and LDL molecular weight. Arteriosclerosis 1983, 3:77-86 22. Chen RM, Fischer-Dzoga K: Effects of hyperlipemic serum lipoproteins on the lipid accumulation and cholesterol flux of rabbit aortic medial cells. Atherosclerosis 1977, 28:339-353 23. Reckless JPD, Weinstein WB, Steinberg D: Lipoprotein and cholesterol metabolism in rabbit arterial endothelial cells in culture. Biochim Biophys Acta 1978, 529:475-487 24. Baker DP, Van Lentin BJ, Fogelman AM, Edwards PA, Kean C, Berliner JA: LDL, scavenger and I-VLDL recep-
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25. 26.
27.
28. 29.
30. 31.
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