Atherosclerosis, 85 (1990) 127-137 Elsevier Scientific

ATHERO

Publishers

Ireland,

127 Ltd.

04555

Increased degradation of low density lipoproteins by mononuclear leukocytes associated with coronary artery disease Fang Shi I, Paul G. Hurst * and Donald J. McNamara

1

’ Department of Nutrition and Food Science and Nurritional Sciences Program, University of Arizona, Tucson, AZ 85721 (U.S.A.) and ’ Deparlment ofInternal Medicine, Section of Cardiology, Arizona Health Sciences Center, Tucson, AZ 85721 (U.S.A.) (Received 14 May, 1990) (Revised, received 18 July, 1990) (Accepted 26 July, 1990)

Summary

Low density lipoprotein (LDL) and peripheral blood mononuclear leukocytes (MNL) were isolated from patients with (n = 11) and without (n = 11) angiographically documented coronary artery disease (CAD). LDL degradation rates in MNL were determined in vitro using both autologous and homologous LDL. The mean rate of LDL degradation was 1.7-fold higher in CAD-MNL than in control-MNL (P < 0.05), independent of the LDL source. The increased LDL degradation rate in CAD-MNL appeared to be due to an increased receptor-mediated LDL degradation rate in CAD-MNL and not to an increased CAD-LDL interaction with the receptor since LDL isolated from patients with and without CAD had similar in vitro degradation rates in HL-60 cells and 1,25-dihydroxyvitamin D,-induced HL-60 macrophages. An increased ratio of apo B to cholesterol, specifically apo B to cholesteryl ester, was observed in LDL isolated from patients with CAD. LDL particles isolated from CAD patients contained 14.8% less cholesteryl ester than LDL from control subjects (P < 0.01). The data suggest that CAD patients have an increased plasma LDL particle number even though they have similar plasma LDL-cholesterol levels as compared to control subjects. These data indicate that CAD patients with normal plasma LDL cholesterol levels have two metabolic abnormalities: an altered LDL composition resulting in particles with reduced cholesteryl ester content and an increased LDL catabolism resulting in an increased influx of LDL cholesterol into MNL; both of which may play a role in the development of coronary heart disease.

Key words: Coronary artery disease; Plasma lipoproteins; Low density lipoprotein degradation

Correspondence to; Donald J. McNamara, Ph.D., Department of Nutrition & Food Science, 309 Shantz Building, University of Arizona, Tucson, AZ 85721, U.S.A. Phone: (602) 621-1971. 0021-9150/90/$03.50

0 1990 Elsevier Scientific

Publishers

Ireland,

Mononuclear

leukocyte;

Apo B/E receptors;

Introduction

tein

Ltd.

Elevated plasma total and low density lipopro(LDL) cholesterol levels are associated with

128 increased risk for coronary heart disease (CHD); however, many patients with CHD are found to be normolipidemic [1,2] suggesting that abnormalities in lipoprotein metabolism, independent of elevated plasma LDL-cholesterol concentrations, may promote atherogenesis. Two types of abnormalities in LDL metabolism are associated with CHD incidence: one is defective catabolism of LDL either due to a deficiency of cell surface LDL (apo B/E) receptors or a defective apo B,, and the other is overproduction of LDL concomitant with either increased or decreased clearance rates. The clinical and metabolic consequences of a deficiency of LDL receptors have been extensively characterized by Goldstein and Brown in patients with familial hypercholesterolemia [3-51. Familial defective apo B,, is a genetic disorder in which a point mutation in the apo B gene causes a glutamine-for-arginine substitution at position 3500 in the protein [6]. This single amino acid substitution inhibits the binding of LDL to the LDL receptor [7]. As a consequence, patients with familial defective apo B,, have moderate to severe hypercholesterolemia [El. Increased LDL production with associated decreased LDL clearance results in elevated levels of plasma LDL cholesterol, whereas increased production and clearance would result in an increased net flux of LDL cholesterol into body tissues [l]. The LDL-receptor hypothesis predicts that most of the atherosclerosis in the general population is caused by high plasma levels of LDL resulting from failure to produce sufficient LDL receptors [9]. However, increased LDL fractional catabolic rates (FCR) have been reported for patients with CHD, primary hypertriglyceridemia and obesity [l,lO,ll]. The two conditions, decreased LDL receptor levels and increased LDL catabolism, seem to present a paradox. There is an apparent need to investigate the factors determining the increased FCR of LDL in patients with coronary artery disease (CAD) and the role that this increased catabolism plays in the development of atherosclerosis in these patients. The FCR of LDL is controlled by the cellular LDL receptor levels of the liver and peripheral tissues with the majority of LDL catabolism occurring in hepatic tissue via receptor mediated processes [12]. In addition, several studies [13,14] have found an increased

LDL-apo B to LDL-cholesterol ratio in patients with CHD, suggesting the possibility that enhanced exposure of apo B on the surface of the LDL particle might alter LDL interactions with the receptor and hence degradation rates. The interrelationship between the reported changes in LDL catabolism and composition in CHD patients is currently unknown. Previous studies have demonstrated that peripheral mononuclear leukocytes (MNL) provide a useful model system for studies of the influence of dietary and pharmacologic interventions on cellular cholesterol homeostasis and LDL metabolism in humans [15-171. The major source of foam cells in atherosclerotic plaques is the blood monocytederived macrophages which play a unique role in deposition of cholesterol in the arterial wall [18211. It has been postulated that increased receptor-mediated degradation of LDL by MNL in the presence of high cellular sterol content may result from failure of the cells to down-regulate LDL receptor synthesis in some patients with high risk of atherosclerosis [22]. In the present study we have examined LDL degradation rates in MNL isolated from patients with and without CAD. The objectives of this study were: (a) to determine if different rates of LDL degradation by MNL existed in these 2 groups, using both autologous and homologous LDL; and (b) to examine the effect of the increased LDL-apo B/LDL-cholesterol ratio observed in CAD patients on in vitro LDL degradation rates, using cultured HL-60 cells and 1,25-dihydroxyvitamin D,-induced HL-60 macrophages as model systems. Our results indicate that MNL isolated from CAD patients have significantly higher rates of LDL degradation as compared to cells from controls, independent of the source of radiolabeled LDL, and that the increased apo B/cholesterol ratio of LDL isolated from CAD patients does not alter lipoprotein-receptor interactions and has no effect on LDL degradation rates in vitro. Materials and methods Materials

Cholesterol and triglyceride kits, horseradish peroxidase

enzymatic assay and cholesterol

129 oxidase were obtained from Boehringer Mannheim, Indianapolis, IN; Lymphoprep from Accurate Chemical and Scientific Corp, Westbury, NY; Na’251 was obtained from Amersham, Arlington Heights, IL; human AB serum, fetal calf serum and horse serum were purchased from Flow Labs, McLean, VA; HL-60 promyelocytic leukemia cells were purchased from American Type Culture Collection; isopropanol was spectrophotometric grade from Aldrich Chemical, Milwaukee, WI; 1,25(OH),-vitamin D, was kindly provided by Dr. Uskokovic, (Hoffman-La Roche, Inc., Nutley, NJ).

Patients

The study was carried out in 22 free-living outpatients who had undergone coronary artery catheterization for evaluation of chest pain or valvular heart diseases, 11 with proven coronary atherosclerosis defined as > 70% stenosis of two or more major coronary arteries (CAD patients: 7 males and 4 females), and 11 with normal coronary arteries (control subjects: 8 males and 3 females). No patient had a history of familial hyperlipidemia, diabetes mellitus, hypothyroidism or renal insufficiency. Patients receiving lipid lowering drugs or estrogen were excluded. Female patients were postmenopausal. All patients were on their home diets and no dietary interventions were initiated following the angiographic evaluation. Fasting blood samples were taken from each of the patients, and for 10 patients a second fasting blood sample was taken 3 weeks later in order to perform MNL degradation measurements using autologous LDL. All patients gave informed consent for participation in the study protocol which was approved by the Human Subjects Committee of the University of Arizona.

Isolation of mononuclear leukocytes

Peripheral blood MNL were isolated according to the method of Boyum [23] as modified by Ho et al. [24]. 54 ml fasting blood were drawn into a syringe containing 6 ml 20 mM disodium EDTA in 50 mM KHPO, (pH 6.6), placed on ice and used for isolating MNL and LDL within 3 h of phlebotomy. Blood samples were centrifuged at

400 x g for 30 min at 4’ C and the plasma used for analysis of plasma lipids and lipoproteins and for lipoprotein isolation. The cell pellet was mixed with 150 ml phosphate-buffered saline (PBS) containing 1 mM disodium EDTA (pH 7.4). 18 ml Lymphoprep was underlayered to 25 ml of the cell suspension, and the samples centrifuged at 400 x g for 30 min at 4” C. After centrifugation, the interface containing MNL was collected, mixed with PBS (pH 7.4) to a final volume of 50 ml and washed at 400 X g for 10 min at 4” C. This washing procedure was repeated twice. Finally, the MNL were resuspended in PBS and used for LDL degradation studies. An aliquot of the cell suspension was removed for cell counts by mixing with a myoperoxidase staining solution for 5 min at room temperature, and cell counts were performed by hemocytometer according to characteristic stains [25]. A smear of MNL was prepared by cytocentrifuge (Cytospin 2, Shandon Instruments, Sewickley, PA), stained with myeloperoxidase and differential counts determined by the percentage of cells with characteristic stains [25].

HL-60 cell culture and macrophage differentiation HL-60 promyelocytic leukemia cells were cul-

tured in 15% horse serum in RPMI-1640 media supplemented with NaHCO, (24 mM), r_-glutamine (2 mM), penicillin (100 units/ml) and streptomycin (100 pg/ml) at 37” C in a humidified incubator with 5% CO,. For LDL degradation studies, HL-60 cells were preincubated in RPMI1640 medium containing 15% lipoprotein depleted fetal calf serum (LPDFS) to induce LDL receptors. To prepare HL-60 macrophages, 3 x 10’ HL60 cells were transferred to 100 x 15 mm plastic petri dish containing 30 ml of 15% LPDFS in RPMI-1640 media, and 1,25-(OH),-vitamin D, was added to the media at a final concentration of 5.0 X lo--’ M. The cells were incubated for 48 h to induce macrophage differentiation [26]. Nonadherent cells were removed by gentle washing with PBS; adherent cells (macrophages) were detached following a 2 min incubation with 1 mM disodium EDTA in PBS, washed twice with PBS at 400 x g for 10 min at 4°C and resuspended in PBS for LDL degradation measurements. Cell protein was determined by a modified Lowry method [27].

130 Analysis of 12’I-LDL degradation rates

Degradation of rz51-labeled LDL was used as an index of LDL receptor activity. LDL (d = 1.019-1.063 g/ml) was isolated by sequential density ultracentrifugation, dialyzed [28] and radioiodinated according to the method of McFarlane [29] as modified by Goldstein et al. [30]. ‘*?-LDL preparations (250-400 cpm/ng protein) were used within 3 weeks following iodination. For analysis of LDL degradation rates either 2-5 X lo6 MNL or 0.4 x lo6 D,-induced HL-60 macrophages were incubated with 10 pg of ‘251-LDL in the absence (total degradation) or presence (non-specific degradation) of a 20-fold excess of unlabeled LDL in a final volume of 1 ml RPMI-1640 containing 50 PM CaCl, and 20% of either lipoprotein depleted human serum (LPDHS) for MNL or LPDFS for HL-60 cells and macrophages. The incubations were performed in microcentrifuge tubes for 4 h at 37 o C. Incubations were terminated by addition of 0.5 ml 50% (w/v) trichloroacetic acid (TCA) to precipitate cells and nondegraded LDL. To 1 ml of the TCA-soluble supemate, 0.25 ml silver nitrate (lo%, w/v) was added to precipitate free iodide [31]. Following centrifugation, TCA-soluble radioactivity was determined in a gamma-counter. Rates of “specific, receptor-mediated” LDL degradation were estimated by subtracting “nonspecific” LDL degradation rates (i.e., parallel incubation containing a 20-fold excess of unlabeled LDL) from the total amount of LDL degraded. Blank values were obtained by parallel cell-free incubations with I*?-LDL, and these were subtracted to give total cell mediated LDL degradation rates [32]. For autologous LDL degradation studies, MNL isolated from the second blood sample were incubated with the patients’ autologous ‘251-LDL prepared from the initial plasma sample. LDL degradation kinetics were determined using 1 x lo6 HL-60 cells/ml at ‘251-LDL concentrations of 5, 10, 15 and 25 pg/ml in the absence or presence of a 20-fold excess of unlabeled LDL. HL-60 cells (1 x lo6 cells/ml) were also used for LDL competition studies in which 10 pg ‘251-LDL/ml in the presence of unlabeled LDL at 0, 25, 50, 100 and 200 pg/ml were used. 1251LDL degradation rates are expressed as ng LDL

protein degraded protein per 4 h.

per lo6 MNL or per mg cell

Plasma lipids analysis and isolation of lipoprotein depleted serum

Plasma total cholesterol (TC) and triglyceride (TG) were measured by enzymatic analysis [33,34]. Plasma HDL-cholesterol levels were determined by enzymatic analysis following precipitation of plasma very low density lipoprotein (VLDL) and LDL with dextran sulfate-Mg*+ [35]. Plasma LDL-cholesterol (LDL-C) was calculated as follows [36]: LDL-C = TC - (0.16 x TG + HDL-C). Lipoprotein-deficient serum (d > 1.25) was prepared from human AB serum (LPDHS) and fetal calf serum (LPDFS) by differential density ultracentrifugation at d = 1.25 g/ml [28]. Determination of LDL apo B and lipia3

LDL apo B concentrations were quantitated as the difference between total LDL protein and soluble proteins following isopropanol precipitation of apo B [37-391. Total LDL protein was measured by a modified Lowry procedure using 1% SDS in reagent A [27]. 1 ml LDL (containing 1.5 mg protein) was mixed with 1 ml isopropanol in a conical glass centrifuge tube, vigorously mixed (1 min), incubated overnight at room temperature and centrifuged at 1000 X g for 30 min. The isopropanol soluble protein was determined by the modified Lowry procedure [27], using bovine serum albumin standards containing the same amount of isopropanol as that of the samples. Over 97% of LDL protein was apo B. Total cholesterol and triglycerides in LDL were measured by enzymatic analysis. LDL free-cholesterol was determined by a similar enzymatic analysis except that cholesterol esterase was excluded from the incubation mix [40]. LDL cholesteryl-ester was calculated as the difference between total cholesterol and free cholesterol. Statistical analysis

Data are presented as mean f SD. Statistically significant differences were determined by Student’s unpaired t-test. Correlation coefficients were determined by linear regression analysis.

131 Results

TABLE

Plasma lipid levels in CAD and control subjects

RATIOS OF APO B TO LIPIDS IN LDL ISOLATED CAD PATIENTS AND CONTROL SUBJECTS

Plasma lipid concentrations and other relevant clinical data for CAD patients and control subjects are given in Table 1. No significant differences were found between CAD and control groups for total, LDL, or HDL cholesterol levels, or for the ratio of LDL to HDL cholesterol. Plasma triglyceride levels were significantly higher in CAD patients as compared to controls (P < 0.05). Composition of LDL from CAD and control patients

Similar percentages of apo B protein to total protein were found in LDL from patients with and without CAD (97.8 f 0.98, n = 11 and 97.8 + 0.68, n = 11). The ratios of LDL-apo B to LDL-total cholesterol were significantly higher in CAD patients as compared to controls (0.72 f 0.06, n = 11 vs. 0.66 + 0.05, n = 11; P < 0.02) (Table 2.). The LDL apo B/free cholesterol ratios were similar in both groups, whereas the ratios of apo B/cholesteryl ester were significantly higher in LDL from patients with CAD as compared to controls (1.23 _t 0.14, n = 11 vs. 1.07 + 0.09, n = 11; P < 0.01). There were no significant differences between CAD patients and control sub-

TABLE

2 FROM

LDL ratios

CAD(n=ll)

Control(n=ll)

Apo Apo Apo Apo

0.72 1.77 1.23 3.33

0.66 f 0.05 * 1.7010.15 1.07*0.09 ** 3.24 k 0.67

B/total cholesterol B/free cholesterol B/cholesteryl ester B/triglyceride

f 0.06 + 0.30 k 0.14 _+0.58

Values are significantly different r-test (*P -c0.02; * *P < 0.01).

for two groups

by Student’s

jects with regard to the apo B/triglyceride of LDL.

ratios

Receptor-mediated LDL degradation rates in MNL of CAD and control subjects

The results of analysis of lZSI-LDL degradation by MNL from CAD patients and control subjects are presented in Fig. 1. The data demonstrate that CAD-MNL expressed receptor mediated LDL degradation rates 1.7-fold higher than control-MNL, independent of the LDL source. In order to test whether the compositional difference between the LDL from CAD patients and controls exerted an

1

CLINICAL DATA AND TIENTS AND CONTROL

PLASMA LIPIDS SUBJECTS

64

Body mass index

25.6+

Blood pressures Systolic Diastolic

PA-

Control (n =ll)

CAD (n =11) Age (yrs)

OF CAD

+ 7 3.3

61

*lo

25.3+

3.3

(mm Hg) 127 68

Plasma lipids (mg/dl) Total cholesterol LDL-cholesterol HDL-cholesterol LDL/HDL Triglyceride * Values between I-test.

groups

*19 *12

220 *51 145 k48 43 k18 3.8rf: 1.6 202 f70 significantly

130 *25 78 f 8

211 rt49 139 +46 48 f 9 3.15 1.2 135 rt28 * different

(P < 0.05) by

CAD LDL

Control

LDL

Fig. 1. Receptor mediated degradation of ‘251-LDL by MNL from CAD patients and control subjects. Freshly isolated MNL from CAD patients (0, 0) and control subjects (A, A) were incubated for 4 h with 10 pg/rnl of either CAD-‘251-LDL or control-“‘I-LDL in the absence and presence of a 20-fold excess of unlabeled LDL to determine receptor mediated degradation. The mean rates of receptor mediated degradation (ng/4 h per lo6 cells) of CAD-LDL and of control-LDL were significantly higher by CAD-MNL (CAD-LDL: 0.46*0.17. n = 11, and control-LDL: 0.53 kO.20, n = 9) as compared to control-MNL (CAD-LDL: 0.30+_0.13, n =I1 and controlLDL: 0.27kO.14, n=12). * PcO.05, **P iO.01.

132

influence on the receptor-mediated LDL degradation by MNL, cells isolated from the same individuals were incubated concurrently with CAD‘251-LDL in one assay and control-‘251-LDL in a second assay to determine the effect of difference in LDL composition on receptor-mediated degradation rates by MNL. As shown in Fig. 2, CAD-MNL degraded more LDL, whether isolated from CAD patients or control subjects, while control-MNL degraded less LDL whether from CAD patients or control subjects. The data indicate that MNL isolated from each individual study subject degraded CAD-LDL and control-LDL to the same extent. For some study subjects degradation assays of autologous lz51-LDL were performed. Autologous LDL degradation rates were also 1.7-fold higher for CAD-MNL as compared to controls (0.42 + 0.10, n = 5 vs. 0.24 _t 0.08, n = 5 ng/4 h per lo6 MNL, P -C0.02). There was no significant difference for the rates of non-receptor mediated LDL degradation between CAD-MNL and control-MNL. Differential cell counts indicated that the mean percentage of mor:*cvtes in isolated MNL were

^D E

0

300.

CA0 LOL

A Control LDL

0

5

10

15

LDL CONCENTRATION

20

25

D

(pg/ml)

Fig. 3. Kinetic analysis of CAD-LDL and control-LDL degradation rates in HL-60 cells. HL-60 cells were incubated for 4 h with t2’I-LDL isolated from CAD patients (0) and control subjects (A) over a concentration range of 5-25 ag/ml in the absence and presence of a 20-fold excess of unlabeled LDL to determine receptor mediated degradation. Each point represents the average of values obtained from LDL preparations from 7 CAD patients and 7 control subjects.

18 + 7% (n = 11) and 20 + 8% (n = 11) for CADMNL and control-MNL, respectively. Thus, the observed increase in LDL degradation rates by CAD-MNL cannot be attributed to an increased percentage of monocytes in the MNL isolated from C * n DP+‘- .LS.

1.0 0 CAD MNL A Control MNL

0.8

LDL degradation rate: in HL-60 cells and macrophages

0.4.

0.0

0.2

0.4

CONTROL (ng/4

0.6

0.6

LOL DEGRADATION hr-106

cells)

Fig. 2. Receptor mediated degradation of CAD-LDL and control-LDL by MNL isolated from CAD patients and control subjects. Freshly isolated MNL from CAD patients (0) and control subjects (A) were incubated concurrently for 4 h with 10 pg/ml of CAD-1251-LDL in one assay and control-‘2SI-LDL in a second assay in the absence and presence of a 20-fold excess of unlabeled LDL to determine receptor mediated LDL degradation rates for each LDL preparation. The line of identity shown on the figure represents points of identical rates of degradation of control-LDL (x-axis) and CAD-LDL (y-axis). There was a significant correlation between the degradation rates of CAD-LDL and control-LDL by the isolated MNL (r = 0.87, n = 18; P < 0.001).

In order to determin: whether the incrc--nd rate of LDL degradation by CAD-MNL v : dr to an increased LDL receptor level of MNL or to an increased receptor affinity for LDL from CAD patients, cultured I-IL-60 cell and 1,25-(OH),vitamin D,-induced HL-60 macrophages were used to test the effect of LDL composition differences on in vitro LDL kinetics and rates of degradation. Fig. 3 presents the degradation kinetics of 1251LDL from CAD patients and control subjects in HL-60 cells and demonstrates that both sources of LDL had similar degradation rates over a concentration range of ‘251-LDL of 5, 10, 15, and 25 pg/ml. Fig. 4 presents the results of competition studies using CAD-LDL and control-LDL to compete with either CAD-‘251-LDL or control‘251-LDL in HL-60 cells. Both sources of LDL had similar affinity and competition for the LDL receptor. The results of analysis of LDL degradation by 1,25-(OH),-vitamin D,-induced HL-60

133 1000

Discussion

Competitor

” e E

75.

8

‘,

s

O-0

CAD

A---A

Control

LDL LDL

#a

g 5

“0

50

9 oz

!b\

-y&__

25-

0z

_

_

----___

P

i 0-r 0

50

100

UNLABELED

150

LDL CONCENTRATION

200

@g/ml)

Fig. 4. Competition by CAD-LDL and control-LDL for LDL degradation in HL-60 cells. HL-60 cells were incubated for 4 h 12’I-LDL in the absence and presence of with 10 pg/ml (0) and convarying concentrations of unlabeled CAD-LDL trol-LDL (A). Each point represents the average value obtained from LDL preparations from 6 CAD patients and 6 control subjects.

macrophages are presented in Fig. 5; there were no significant differences between CAD-LDL and control-LDL, though the degradation rate of control-LDL by macrophages was slightly, but not significantly, higher than CAD-LDL (168 + 33 vs. 144 _t 22 ng/4 h per mg protein; P > 0.05).

3

E !

f \

250

200 P

CAD

LDL

Control

LDL

LDL SOURCE

Fig. 5. Degradation of CAD-LDL and control-LDL in Ds-induced HL-60 macrophages. HL-60 cells were induced to macrophage differentiation using 1,25-(OH),-vitamin Da and incubated for 4 h with 10 pg/ml of either CAD-‘2SI-LDL or conrrol-‘251-LDL in the absence and presence of a 20-fold excess of unlabeled LDL to determine receptor mediated degradation. There was no significant difference between the degradation rates of CAD-LDL (n = 6) and control-LDL (n = 6) (144 f 22 vs. 168 f 33 ng/4 h per mg protein).

Numerous studies have documented a positive relationship between elevated plasma LDL cholesterol levels and increased risk of CAD [41]; however, many patients develop CAD in the absence of hypercholesterolemia suggesting that abnormalities of lipoprotein metabolism may play a role in accelerated atherosclerosis. Two metabolic abnormalities associated with CHD incidence in patients with normal LDL cholesterol levels are an increased FCR of LDL-apo B [l] and an increased LDL apo B to cholesterol ratio indicative of changes in LDL composition [13]. Kesaniemi and Grundy [l] reported that LDL synthesis rates were increased 55% and the FCR by 43% in normocholesterolemic males with CHD as compared to a matched set of controls. These authors also reported that the LDL from CHD patients had an increased LDL protein/ cholesterol ratio as previously reported by Sniderman et al. [13]. These two abnormalities of LDL metabolism, increased catabolism and altered composition, suggest that the development of CHD in normocholesterolemic subjects may result from alterations of the interaction between a modified LDL particle and its receptor and/or an altered regulation of the LDL (apo B/E) receptor in vivo. The present studies were carried out to determine whether freshly isolated blood MNL from normocholesterolemic CAD patients exhibit hyperactive receptor mediated endocytosis of LDL particles and to determine the effects of altered LDL composition on in vitro ligand-receptor interactions using a human monocytic cell line, HL60, and 1,25-dihydroxyvitamin D, induced HL-60 macrophages. Previous studies have demonstrated that isolated MNL can be used as a marker of changes in rates of endogenous cholesterol synthesis [15-17,42,43] and of LDL catabolism [22,44461. In the present study we determined the rates of receptor-mediated LDL degradation in MNL from patients with angiographically proven CAD as compared to patients with documented normal coronary arteries. Patients with normal coronary arteries were recruited as control subjects to ensure that misclassifications of controls due to the absence of clinical symptoms was minimized. Both

134 groups were normocholesterolemic and had similar plasma total and LDL-cholesterol levels. Analysis of LDL degradation rates in MNL from patients with CAD demonstrated enhanced LDL degradation relative to controls and supports the original observation of Kasimeimi and Grundy [l] that patients with CHD have increased LDL FCR in vivo. The data also demonstrate that LDL from CAD patients had a higher apo B/ cholesterol ratio than LDL from controls without CAD. The observation that patients with CAD had higher rates of receptor-mediated LDL degradation by MNL has three potential explanations: (a) an increased LDL receptor expression in MNL; (b) an increased receptor affinity for CADLDL since a high ratio of apo B/cholesteryl ester of CAD-LDL may enhance exposure of apo B on the surface of LDL particles; or (c) an increased percentage of monocytes in the isolated CADMNL preparations which would result in an increased rate of LDL degradation [32]. To test these various possibilities, analysis of LDL degradation rates in MNL was carried out using autologous and homologous LDL sources with a single patient’s MNL. Both autologous and homologous LDL degradation rates were determined to evaluate differences in receptor mediated LDL degradation versus changes in the LDL particle composition. Studies of the effects of different LDL sources on degradation rates were also carried out in cultured HL-60 cells and 1,25(OH),-vitamin D, induced HL-60 macrophages. Both HL-60 cells and D,-induced macrophages express the LDL receptor [47] and provide a useful model for comparisons of the effects of alterations of LDL composition on in vitro LDL catabolism. Our results indicate that LDL from patients with and without CAD had similar degradation rates in MNL, HL-60 cells, and HL-60 macrophages and had a similar ability to compete for LDL degradation by HL-60 cells. Based on these data it is unlikely that the enhanced LDL degradation rates by MNL from CAD patients result from the higher ratio of apo B/cholesteryl ester of CAD-LDL. It can be concluded from these data that the altered LDL composition does not result in a significant change in LDL-apo B/E receptor interaction.

MNL isolated by density fractionation through Lymphoprep are a mixture of lymphocytes and monocytes. Monocytes have a higher rate of LDL degradation as compared to lymphocytes [32] and differences in the relative proportion of these 2 cell types could explain the increased rate of LDL degradation by MNL from patients with CAD. The results of differential cell counts using specific staining characteristics indicate that the MNL populations obtained from patients with and without CAD had identical percentages of monocytes and therefore the observed increased LDL catabolism is not the result of an increased number of monocytes in the MNL preparations. It is also unlikely that differences in dietary patterns makes a significant contribution to the observed differences. Intake of very high cholesterol diets (1000-2000 mg/day) have been reported to decrease the rate of LDL degradation in freshly isolated MNL [48,49]; however, the observed differences in LDL degradation rates between CAD patients and control subjects cannot readily be ascribed to differences in dietary cholesterol intake. First, the study populations were on their habitual diet without special attention to a low-fat, low-cholesterol diet following angiography; second, it is highly unlikely that the control subjects were consuming diets containing 1000 mg of cholesterol daily and studies by Ginsberg et al. [50] have shown that moderate changes in dietary cholesterol intake do not affect LDL turnover in vivo; and finally, preliminary studies in a similar patient population have shown that an increase in dietary cholesterol intake from < 300 to 800 mg/day does not decrease the rate of LDL degradation in MNL from either CAD or control patients (McNamara, Shi, DOS Santos and Lowell, unpublished observations). The mechanisms involved in this enhanced receptor mediated LDL degradation by MNL from CAD patients are currently unknown. Both groups had similar total and LDL cholesterol levels and no patients were receiving cholesterol lowering medications. Although more patients in the CAD group, as compared to the control group, were receiving beta blockers or aspirin, there were no statistically significant differences in LDL degradation rates by MNL or in plasma lipids levels between patients within the same group receiving

135 these drugs and those not on medication (data not shown). Therefore, it is unlikely that variations in medication for some individuals could account for the observed differences between CAD patients and control subjects. Plasma LDL concentrations are regulated by a balance between the rate of synthesis and clearance of LDL, and clearance is related to LDL receptor expression [51]. Turner et al. [44] reported a positive relationship between the in vivo FCR of apo B and the in vitro rate of LDL degradation by MNL in healthy subjects. In vivo isotope kinetic studies [1,11,52,53] have demonstrated that overproduction of apo B may be seen in patients who have premature CHD. Thus, the enhanced LDL degradation rates in MNL from CAD patients in this study suggest that an increased receptor mediated catabolism of LDL may contribute to the maintenance of normal plasma LDL-cholesterol levels. LDL is a spherical particle with a core composed primarily of cholesteryl esters and triglyceride and a single molecule of apo B,, [54-561. Thus, the concentration of LDL-apo B reflects the number of circulating LDL particles. In this study the CAD-LDL has 10% less cholesterol relative to apo B than LDL from controls. Therefore, at similar concentrations of LDL-cholesterol, CAD patients will have a 10 percent increase in LDL particle number as compared to control subjects. The data indicate that the major change in composition is a 14.8% decrease in cholesteryl ester content per LDL particle. The LDL particle of patients with CAD would be predicted to be smaller in size and relatively denser than control LDL [57]. Patients with hyperapobetalipoproteinemia have an increased plasma apo B concentration yet the plasma LDL cholesterol levels are normal. These patients have been reported to frequently develop accelerated coronary atherosclerosis [13,14]. The decreased cholesterol content of CADLDL, while not modifying LDL interactions with the apo B/E receptor, may play a role in the increased expression of the LDL receptor in MNL and in the increased incidence of atherosclerosis. Since each CAD-LDL particle has 10% less cholesterol than control LDL, each cycle of the receptor delivers 10% less cholesterol to the cell

which could result in the observed 1.7-fold increase in LDL receptor expression. Preliminary studies suggest that CAD-LDL has a decreased ability to down-regulate the LDL receptor and activate acyl-CoA : cholesterol acyltransferase (ACAT) activity of cultured HL-60 cells [58]. From the cited studies and the data presented here, one hypothesis is that the increased LDL receptor-mediated degradation may function to protect against hypercholesterolemia induced by overproduction of LDL in some CAD patients. This protection may be achieved through an increase in LDL receptor expression in hepatic and peripheral tissues. However, this mechanism of protection may promote lipid-laden foam cell formation if interstitial macrophages also exhibit increased LDL-receptor expression. In conclusion, our findings demonstrate that normocholesterolemic CAD patients have an increased rate of LDL degradation in MNL and an increased LDL apo B/cholesteryl ester ratio and that the abnormal LDL composition does not alter the interactions between LDL and the apo B/E receptor. The data suggest that an increased LDL influx to MNL, as evidenced by an enhanced LDL degradation rate, and possibly to other cells in the body, and changes in LDL particle number and composition in patients with CAD may in part explain the accelerated development of CAD in these patients. The data also suggest that analysis of LDL degradation rates in MNL may be a useful marker for diagnosis of patients at risk for CAD due to increased fractional catabolism of LDL. Acknowledgements

We thank Ms. Melinda Milander for her assistance in collecting blood samples and Ms. Janet Sabb for her excellent technical assistance. These studies were supported in part by a Grant-in-Aid from the American Heart Association, Arizona Affiliate to Paul G. Hurst, M.D.; grants from the National Dairy Council, National Live Stock and Meat Board, and South Eastern Poultry & Egg Association; and funds from the University of Arizona Biomedical Research Support Grant Program. Paper No. 7080 from the University of Arizona Agricultural Experiment Station.

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