Clin Biochem, Vol. 25, pp. 345-349, 1992 Printedin the USA. All rightsreserved.

0009-9120/92 $5.00 + .00 Copyright© 1992 The Canadian Societyof ClinicalChemists.

Role of Cholesterol-Accumulating Macrophages on Vascular Smooth Muscle Cell Proliferation KOZO HAYASHI, EISUKE NISHIO, KOICHIRO NAKASHIMA, HIROSHI AMIOKA, JUN-ICHI KUROKAWA, and GORO KAJIYAMA First Department

of Internal Medicine, Hiroshima University School of Medicine, M i n a m i - k u , H i r o s h i m a 734, J a p a n

The proliferation of vascular smooth muscle cells (VSMC) was stimulated by co-incubation with macrophages. Further stimulation was observed when co-incubated macrophages were supplied with LDL or cholesterol. However, the stimulation of VSMC proliferation did not result from co-incubation with macrophages supplemented with acetylated LDL or delipidated LDL. The addition of anti-PDGF antibody partially abolished the stimulation of VSMC proliferation induced by co-incubation with macrophages supplemented with LDL or cholesterol. A high concentration of prostaglandin E2 inhibited the proliferation of VSMC stimulated by PDGF and plasma-derived serum when they were at the Go/G1 stage. However, the inhibitory effect of prostaglandin E2 on proliferation was not observed when cells were incubated with macrophages supplemented with LDL or cholesterol in spite of the promotion under these conditions of prostaglandin E2 production. These results suggest that cholesterol-accumulating macrophages may exert a regulatory effect on the proliferation of VSMC through the synthesis and secretion of platelet-derived growth factor (PDGF) and prostaglandin E2, besides foam-cell formation.

KEY WORDS: macrophage; vascular smooth muscle cell proliferation; platelet-derived growth factor; prostaglandin E2. Introduction

'acrophage-originated foam cells loaded with

M,cholesterol esters and proliferation of vascular smooth muscle cells (VSMC) in the subendothelial space are typical features of atherosclerotic lesions. Modified LDL, such as acetylated LDL and oxidized LDL, is known to accumulate in macrophages and to contribute to foam-cell formation (1,2). VSMC proliferation is stimulated by growth-regulatory molecules such as platelet-derived growth factor (PDGF), and platelets are an important source of PDGF (3). However, macrophage infiltration and VSMC proliferation have been demonstrated prior to platelet adhesion and aggregation to the vascular wall in a hypercholesterolemic animal model (4,5). The secretion of PDGF-B chain from macrophages in atherosclerotic lesions has also been suggested (6,7). Recent studies have demonstrated the possibility that

these macrophages regulate migration and proliferation of VSMC and play an important role in promoting development of typical atherosclerotic lesions. As fatty streaks are easily induced in hypercholesterolemic animal models and are the first lesions of atherosclerosis, stimulation of migration and proliferation of VSMC through synthesis and secretion of growth-regulatory molecules by macrophages accumulating cholesterol may be possible in the process of progression of complex atherosclerotic lesions. In the present study, we investigated the effect of atherogenic lipoproteins on the proliferation of VSMC co-incubated with macrophages as well as the role of atherogenic lipoproteins on prostaglandin E2 (PG-E2) and PDGF secretion in macrophages, because these molecules have been suggested to play an important role in the regulation of VSMC proliferation and development of atherosclerotic lesions (3,6-9). Materials and methods MATERIALS

PG-E2 was obtained from Wako Chemical Co. (Tokyo, Japan); [3H]thymidine and [3H]arachidonic acid were from Amersham; fetal bovine serum from Flow Laboratories Incorporated (Tokyo, Japan); anti-human PDGF polyclonal antibodies from Genzyme Corporation (Boston, MA, USA); and h u m a n PDGF from Collaborative Research Incorporated (Bedford, MA, USA). Millicell CM was obtained from Millipore Corporation. RABBIT V S M C

CLINICALBIOCHEMISTRY,VOLUME25, OCTOBER1992

AND

PERITONEAL

MACROPHAGES

Rabbit VSMC were prepared as described previously (10), and cells from passages 4 - 1 0 were used. Rabbit peritoneal macrophages were prepared as described previously (11). All cells were grown at 37 °C in an atmosphere of 5% C02 and 95% air. PREPARATION

Correspondence: Kozo Hayashi. Manuscript received March 17, 1992; revised May 19, 1992; accepted May 22, 1992.

1-2-3 Kasumi,

OF LIPOPROTEINS

H u m a n or rabbit plasma was separated from blood collected into tubes containing 0.15% (w/v) 345

HAYASHI, NISHIO, NAKASHIMA, AMIOKA, KUROKAWA, AND KAJIYAMA

EDTA from normolipidemic fasting volunteers or rabbits. LDL (1.019 < d < 1.063) was isolated by a modification of the procedure of Havel et al. (12). Delipidated LDL and acetylated LDL were prepared as described previously (13,14). PREPARATION OF PLASMA-DERIVED SERUM FREE SERUM)

(PDGF

Plasma-derived serum and lipoprotein-deficient serum were prepared from healthy volunteers or rabbits as described previously (15,16). ASSAY OF

PG-E 2 SYNTHESIS

After the cells had been incubated as described in the Figure Legends, the growth medium was removed and each dish received 2 mL of medium A (RPMI 1640 medium containing 100 units/mL penicillin, 100 ~g/mL streptomycin) supplemented with 10% (v/v) fetal bovine serum and 0.25 ~Ci/mL [ZH]arachidonic acid, and the cells were incubated for 4 h. The medium was then removed, the cells were washed, and each dish received 1 mL of medium A in the absence or presence of native LDL, acetylated LDL or cholesterol. After 12 h of incubation, 500 ~L of medium was collected from each sample and 20 ~L of i M HC1 and 50 ~g of PG-E2 were added. The acidified medium was then extracted with chloroform/methanol (2:1, v/v) as described previously (17). PG-E2 was separated by thin-layer chromatography on a silica gel G plate using ethyl acetate/chloroform/ethanol]acetic acid (200/200/40/ 10, v/v). After visualization by iodine vapor, the areas corresponding to authentic PG-E2 were scraped off and their radioactivity was determined by liquid scintillation counting. ASSAY FOR D N A SYNTHESIS BY V S M C

This was assayed by measuring the incorporation of [3H]thymidine into acid-insoluble materials (18). VSMC were seeded at a density of 5 × 104 cells/dish and synchronized at the Go/G1 phase of the cell cycle by incubation in medium A containing 0.5% (v/v) fetal bovine serum for 3 days. The medium was then removed and the cells were stimulated to proliferate in medium A containing 5 half-maximal units/mL of h u m a n PDGF and 1% (v/v) li:poprotein-deficient plasma-derived serum, and [°H]thymidine was added (5 ~Ci/mL). At 8 h after stimulation, PG-E2 was added. [3H]Thymidine incorporation was measured over a 24-h period after the cells had been stimulated. When the effect of co-incubated macrophages on VSMC proliferation was investigated, cells synchronized at the Go/G1 phase were coincubated with macrol~hages seeded in Millicell CM at a density of 5 x 10 ° cells/dish in medium A cont a i n i n g 1% (v/v) lipoprotein-deficient plasmaderived serum and [°H]thymidine (5 ~Ci/mL) supplemented or not with each lipoprotein or choles346

terol for 24 h. [3H]Thymidine incorporation into the VSMC was then assayed as described above. INCORPORATION OF [14C]0LEATE INT0 CELLULAR CHOLESTERYL [14C]oLEATE

Cholesteryl [14C]oleate accumulation into macrophages incubated in medium A supplemented with [14C]oleate/albumin and each lipoprotein was measured (19).

Results EFFECT OF MACROPHAGES AND LIPOPROTEINS ON PROLIFERATION OF V S M C

When VSMC were incubated with LDL or acetylated LDL in a medium containing lipoproteindeficient plasma-derived serum, [3H]thymidine incorporation into DNA of the cells was not stimulated compared with the control (Figure 1). [3H]Thymidine incorporation into DNA of VSMC was stimulated by co-incubation with macrophages in this medium, and further stimulation was observed following co-incubation with macrophages and LDL but not acetylated LDL (Figure 1). This stimulation did not result from co-incubation with macrophages and delipidated LDL (Figure 1). Marked stimulation of [ZH]thymidine incorporation into DNA of VSMC was obtained by co-incubation with macrophages and cholesterol (Figure 1). Addition of anti-PDGF-B chain antibody diminished [ZH]thymidine incorporation into DNA of VSMC s t i m u l a t e d by coincubation with macrophages and LDL or cholesterol (Figure 1). INCORPORATION OF [14C]OLEATE INTO CHOLESTERYL [14C]OLEATE IN M A C R O P H A G E S

This was stimulated by incubation with LDL (Table 1). M a r k e d a c c u m u l a t i o n of c h o l e s t e r y l [14C]oleate in macrophages was obtained following incubation with acetylated LDL (Table 1). PG-E 2 SYNTHESIS AND RELEASE FROM MACROPHAGES This was stimulated by incubation with LDL, acetylated LDL, or cholesterol (Figure 2). EFFECT OF PG-E2 ON PROLIFERATIONOF VSMC PG-E2 inhibited in a dose-dependent manner the incorporation of [SH]thymidine into DNA of VSMC stimulated to proliferate by PDGF and lipoproteindeficient plasma-derived serum (Figure 3).

Discussion The role of intimal macrophages and lipid-laden foam cells present in atherosclerotic lesions on the progression of atherosclerosis has been investigated, and g r o w t h - r e g u l a t o r y molecules for v a s c u l a r CLINICALBIOCHEMISTRY,VOLUME25, OCTOBER1992

MACROPHAGES

STIMULATE V A S C U L A R S M O O T H M U S C L E CELL PROLIFERATION

delipidated LDL •

macrophage (+) anti-PDGF antibody (+)

• •

macrophage (+) macrophage (-)

acetylated LDL cholesterol

f / / / ~

none

0

5000

10000

15000

[3H]Thymidine incorporation into DNA in vascular smooth muscle cells (dpm/mg cell prot./24h)

Figure 1 - - Stimulation of [SH]thymidine incorporation into DNA of VSMC by co-incubation with macrophages and the effect of lipoproteins and anti-PDGF-B chain antibody. VSMC, synchronized at the Go/Gz phase of the cell cycle, were co-incubated with macrophages with or without supplementation with lipoproteins (200 ~g proteirgmL), cholesterol (0.1 tLmol/mL) or anti-PDGF-B chain antibody (50 ~g/mL) in a medium containing 1% (v/v) lipoprotein-deficient plasmaderived serum. [3H]Thymidine incorporation into DNA of VSMC was measured over a 24-h period after the cells had been stimulated, as described in Materials and Methods. Each value represents the mean _+ SD for triplicate determinations. smooth muscle cells derived from macrophages have been demonstrated (3,6,7,20-28). In this study we investigated the metabolism of PDGF and PG-E2 in macrophages in response to incubation with lipoproteins and cholesterol and its effect on the proliferation of VSMC induced by incubation with macrophages in vitro. [3H]Thymidine incorporation into DNA of VSMC stimulated by co-incubation with macrophages was partially inhibited by anti-PDGF-B chain antibody (Figure 1). This indicates that P D G F contributes to the stimulation of VSMC proliferation. As the addition of LDL promoted the stimulation of [3H]thymidine incorporation into DNA of VSMC co-incubated with macrophages and this was inhibited by antiPDGF-B chain antibody (Figure 1), LDL seems to accelerate VSMC proliferation through PDGF secretion by macrophages. J u d g i n g from the facts that the use of delipidated LDL did not lead to the accumulation of cholesterol esters in macrophages (Table 1) and did not result in additional stimulation of TABLE 1 Incorporation of [z4C]Oleate Into Cellular Cholesteryl [z4C]Oleate by Macrophages

[SH]thymidine incorporation into DNA of VSMC coincubated with macrophages (Figure 1), and that a marked stimulation of [3H]thymidine incorporation into DNA of VSMC, which was inhibited by antiP D G F - B c h a i n a n t i b o d y , w a s i n d u c e d b y coincubation with macrophages and cholesterol (Figure 1), the accumulation of LDL-cholesterol seems to contribute to VSMC proliferation through P D G F secretion. If the cholesterol accumulation in macrophages is linked to PDGF secretion, the P D G F secretion by macrophages must be stimulated by incubation with acetylated LDL that is accumulated in macrophages through scavenger receptors (Table 1). However, no stimulation of [~H]thymidine incor100 At -

._=

Lipoproteins Added to Medium a Delipidated LDL LDL Acetylated LDL

8.1 -+ 0.8 40.1 _+ 2.4 423.1 _+ 14.9

80

v. ®

60

~.

"~ =>,

~' I

Incorporation of [z4C]Oleate Into Cholesteryl [z4C]Oleate (nmol/mg Cell Protein/10 h)

•,=.

~,7 m

40

20

~oE~ o flOl'~

Incorporation of [z4C]oleate into cellular cholesteryl [z4C]oleate was determined (19) after incubation in medium A containing 0.2 mM [z4C]oleate/albumin for 10 h at 37 °C. Each value represents the mean -+ SD for triplicate determinations. ~200 ~g protein/mI., CLINICAL BIOCHEMISTRY.VOLUME 25. OCTOBER 1992

LDL

acetylated

cholesterol

LDL

Figure 2 - - Stimulation of PG-E 2 synthesis by macrophages by lipoproteins and cholesterol. Macrophages were prelabeled with [3H]arachidonic acid (0.25 ~Ci/mL) and then challenged with lipoproteins (200 ~g protein/mL) or cholesterol (0.1 tLmol/mL) for 24 h. The amount of radioactivity released into the medium and co-chromatographed with PG-E2 was measured as described in Materials and Methods. Each value represents the mean -+ SD for triplicate determinations. 347

HAYASHI, NISHIO, NAKASHIMA,AMIOKA,KUROKAWA,AND KAJIYAMA

Role of cholesterol-accumulating macrophages on vascular smooth muscle cell proliferation.

The proliferation of vascular smooth muscle cells (VSMC) was stimulated by co-incubation with macrophages. Further stimulation was observed when co-in...
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