Vol.
177,
May
31, 1991
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
582-587
Pages
GANGLIOSIDE
GM3 STIMULATES THE UPTAKE AND PROCESSING OF LOW DENSITY LIPOPROTEINS BY NACROPBAGES
Nina V.Prokazova,
Irina
A.Mikhailenko
and Lcv D.Bergelson
Institute
of Experimental Cardiology, Cardiology Research Center of the USSRAcademy of Medical Sciences, MOSCOW, USSR
Received
April
22,
1991
Preincubation of low densiy l’poproteins GM3 (1-2x 10 -5 M/ 2.5x10 the ganglioside increase of LDL-uptake, enhances cholesterol accumulation and cholesteryl At the same time the lysosomal degradation of ester formation by macrophages. LDL in macrophages was inhibited under these conditions. These effects depended on the ganglioside structure and concentration. It is suggested that the effects observed could be caused by GM3-induced modification of LDL to a form that becomes recognized by macrophages. 0 1991 Academic Press, Inc.
One
of
the
migration
earliest
of
differentiate esters
into
and other
lipids
which
plasma
low
are
several
which
to accumulate believed that LDL
into
However The can
a the
are
artificial
cause
The term blood by
recognized
"native
formation
LDL”
sequental
refers density
of
called
cells.
0006-291X/91 Copyright All rights
LDL-low
density
(1). occurs
in vivo
receptor
remains cultured
to LDL isolated ultracentrifugation
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reservea.
582
even
still
is
high shown into them
therefore
which on
LDL*
transforms macrophages.
unknown.
lipoprotein
macrophages
from
them
and cause It
the
induce
has been
in vitro
esters
high-affinity
lipoproteins;
native do not
It
The
from
that
particles include
normal human according to
standard methods. Abbreviations; Cer .
cholesteryl
that
macrophages
macrophages
they
foam cells.
fact and
is
where
to be derived
the
of LDL producing from
wall,
of LDL can transform
up by
modification
nodifications
foam cell
within
of cholesteryl by a
are
vitro with
of LDL-modification
of this
atherosclerosis
amounts
of the
in
incubated
taken
amounts
high
believed
modifications
rapidly
some type form
inspite
esters
artificial
massive
nature
(LDL) macrophages
of much cholesteryl
that
are
LDL when
of
arterial
cells
cells
lipoproteins by
the
lipid-loaded
in foam
of native
development
into
and accumulate
These
recognized
concentrations
particles
lipids.
the
blood
macrophages
density
accumulation
in
from
accumulate
weakly
however
events
monocytes
GM3- II3-NeuAc-Lac-
acetylation
that
Vol.
177,
No.
1, 1991
(2),
acetoacetylation
can
be
LDL-oxidation. complexes
excluded.
Recently stability
plasma,
and we
that
and are
of
LDL
with
gangliosides
hepatoaa
cells
uptake
and LDL-uptake
and degradation
MATERIALS
of GM3,
we (6)
in
by these main
by LDL
their
and others
reduced the
l), of
LDL
cells. ganglioside
of LDL by mouse peritoneal
are is
of human
of
plasma (7).
modified as
is
or plasma,
bound
in vitro
to
on formation
components
actively
hardly
in vivo
(reviewed based
of LDL as well Previously
influence
place
matrix
gangliosides
properties
the
to take
of LDL by gangliosides
(7).
investigate
on the
(6)
which leading
extensively
normal
apo-B
against
preincubation
fibroblasts study
space
are
(4)
of modification
intracellular
modification
COMMUNICATIONS
ralonaldehyde
type
studied
of the the
fluorescence
RESEARCH
of LDL- modification,
gangliosides
demonstrated
and
that
them,
intercellular
antibodies
with
Another
has been
components
because
we
BIOPHYSICAL
and more likely
pathways
Among
interest
and aorta1
with
process
biological
of LDL with
special
in vivo.
by macrophages
This
other
AND
and conjugation
to occur
LDL uptake
however
(5)
(3)
expected
enhanced
not
BIOCHEMICAL
size, interaction (9)
found
binding
to
In the of
present human
macrophages.
AND METHODS
Ganglioside GM3 was isolated from human liver (10). [l’C]Oleic acid 50-60 Ci/maol was recieved from Amersham. Human LDL (d-1.019-1.063 g/ml) were obtained from fresh plasma of normal volunteers by sequential ultracentrifugation as described in (11). [1251J-LDL was prepared by the iodine monochloride method (12), specific activity was 50-100 cpn/ng protein. Association of gangliosides with LDL was achieved by incubation of LDL (2.5 uM) with 50 uM gangliosides for 0.5-2 h at 37’C. Mouse peritoneal macrophages were collected from unstimulated mice in 199 medium containing 20 units heparin/ml (13). Macrophages from 20 mice (5x10’ cells) were pooled and collected by low speed centrifugation at 1000 rpm for 5 min at room temperature. The cells were then resuspended in 199 medium containing 20% bovine serum, 100 ug/ ml of penicillin and 100 ug of streptomycin. Cells (1~10~ per well) were dispensgd into 24-well tissue culture cluster (Costar, USA) with a density of 10 cells/ well and allowed to adhere for 4-6 h at 37’C in a humidified 5% C02/95% air atmosphere. Then the cells were washed twice with 199 medium to remove nonadherent cells. The 199 medium containing 20% human lipoprotein-deficient serum was added to each well after which the macrophages were used for experiments. LDL binding and degradation were performed as described in (14). The were nacrophage monolayers incubated in presence of [1251]-LDL (100 ug or LDL-ganglioside associates at 37’C for 3 h. The cell protein/ml) monolayers were washed three times with 199 medium containing 20% bovine serum albunine. The radioactivity associated with the cells was measured in a gamma-counter (LKB, Bromna, Sweden, model 1282). The proteolytic degradation and LDL-ganglioside associates was measured by assaying the of [ 1251]-LDL amount of [1251]-labeled trichloracetic acid-soluble material formed by the nacrophages and excreted into the culture medium. For this aim the nacrophage monolayers were incubated for 24 b as described for binding experiments. The medium removed after incubation was used to study LDL proteolytic degradation. Incorporation of [l* Cl-oleate by macrophages into cholesteryl esters and triglycerides was determined as follows (14). The macrophage monolayers were incubated with 0.3 ml 199 medium containing 10% of lipid-deficient serum, various amounts of native LDL or LDL-ganglioside associates and 0.2 urol/ml 583
Vol.
177,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
[14C]-oleic acid/albumine (M/M) at 31’ for 24 h. After incubation the monolayers were washed with PBS and the total lipids were extracted in situ The cell residues were used to determine with hexan/isopropyl alcohol (3:2). the cellular protein (15). The extracts of nacrophage monolayers from three wells were combined and the cholesteryl esters and triglycerides were isolated by silica gel thin-layer chromatography using the solvent system The cholesteryl ester and hexan/ diethyl ether/ acetic acid (85:15:1 v/v). triglyceride bands were visualized with iodine vapor, scraped into vials and counted in a Racbeta 1250 beta-counter. The incorporation of chlesterol by nacrophages was determined as described early (14). The macrophage monolayers were incubated with 0.3 ml of 199 medium containing 20% human lipoprotein-deficient serum and several amounts of LDL-ganglioside assosiates or native LDL for 24 b at 37’C. The cells were The total washed with PBS, and lipids were extracted as described above. cholesterol content in lipid extracts was determined using Boehringer Mannheim Monotest Cholesterol CHOD-PAP-Method ( Cat. no 290 319, Boehringer, FRG).
RESULTS AND DISCUSSION As it
can
be
seen
macrophages
increases
effect
the ganglioside
of
concentrations
of
data
of Table of the
was concentration exceeding
GM3
1, the uptake
lipoproteins
dependent
2~10-~
M.
had no influence
with and
Further
on the
of LDL GM3.
This
saturable
at
increase
uptake
by
of
in
the
lipoproteins
by
in
macrophages,
as
in
the
cells. At
the
same
determined medium, the
the
preincubation
concentration
ganglioside the
after
from
by
time
the
lysosomal
the appearance
was inhibited
in
the
ganglioside
concentration.
Pretreatment
of LDL with
binding
but
also
inferred
from
the
presence formation
the greatly
of
2).
cholesteryl
Table
1. and
presence
This
of GM3.
appears
esters
results
of
not cells
by
GM3 on the macrophages
by
could
on
be
uptake
1251-LDL pg cell protein
1.2620.40 1.42+0,52 2.10+0.48
1.64+0.21 1.22+0.0s 0.91+0.08
in
LDL-induced
macrophages
degraded
584
it
accumulation also
bound
3 experiments.
again
LDL-macrophage
as
cholesterol
ganglioside of LDL
incubation
depended
only
of GM3 stimulated
ng/
-
effect
and triglycerides
of Influence the degradation
10 20
by the
intracellular
The presence
GM3 nmol/mg protein
The
to stimulate
of lipoproteins
enhanced
of GM3 (Table
of LDL products
GM3
uptake
degradation
of [1251]-labeled
in
a
Vol.
177,
No.
1, 1991
BIOCHEMICAL
Table 2. LOL-induced
AND
Influence accumulation
of of
Lipoproteins
BIOPHYSICAL
ganglioside cholesterol
Cholesterol
concentration
dependent
manner
(Table
the
chemical
structure
of the
ganglioside:
GDla
which
is both
cholesteryl The
ester
leukemia by
underlying
of cells
the
part
LDL
and that
formation molecular
immunoreactivity
of apo-B of LDl
Khoo
(18)
et al
by
Table
macrophages
to GM3,
ganglioside
a
differ
(200) (300)
nyelogenous not
able
LDL (5).
to like
native
density,
seem to be caused Brown
of cholesteryl
ganglioside and triglycerides
GM3
esters
in
and partly
and Goldstein be active ester
(17)
inductors accumulation
content protein) Triglycerides
3.65+0.83 10.13+0.20 11.05+0.80 29.3422.73
of
their size,
apolipopropteins,
on formation in macrophages acid cell
results LDL in
changes
LDL might
of
particle
and
aggregated
Recently
amounts
gangliosides
lipids
585
as well
we should
high
from
floating
0.03+0.01 0.15+0.01 0.14+0.07 0.22+0.07
lipid
be caused
(16) but
with
LDL with
[14C]-oleic (pmoles/mg
(300) (100)
(with
relatevelly
of gangliosides. and enhence
shown).
and
could
differentiation
associated
which
as:
not
as
latter.
is
These
observed
of evidence
bind
surface
that
component,
(data
cells
on
as well
At the moment we are
the
to
such (7).
ganglioside accumulation
recipient
of lack
complexes of
minor
cell
Cholesteryl native LOL LOL+GMS LOL+GM3 LOL+GMB
in contrast
of native
3. Influence of cholesteryl esters
Lipoproteins (pg protein/ml)
strongly
The effects the
of
able
in presence
suggested
depended
of GM3 on LDL uptake
favor
properties
aggregation
effects
influence with
incubation
organization
These
by macrophages
gangliosides
of LDL-ganglioside
physico-chemical
& 0.47 + 0.94
as
of LDL.
were
protein)
formation
because
of plasma
mg cell
18.35 32.9
macrophage-like
in
on the macropbages
cholesterol
unknown.
modification
that
gangliosides
are
possibility
some arguments
showed
LDL-uptake
the
GM3 induces
first
The major
only
the ganglioside
GMS-induced
to consider
we
serum intracellular
by macrophages
by interaction
discuss
in
and triglyceride
mechanisms
accumulation
as
present LDL-induced
3).
COMMUNICATIONS
GM3
in
(pg/
native LOL LOL+GMS
inhibited
RESEARCH
by and of in
Vol.
177,
these
No.
1, 1991
cells.
arterial
On the other wall,
ganglioside regions
BIOCHEMICAL
and that
Therefore
it
in atherosclerotic
was
at least
atherosclerotic
may well
ganglioside residing
complexes within
be
that
muscle
cells
the lesions,
(21)
times
RESEARCH
that
in the atherosclerotic
lesions
of
higher
in a relativelly concentration
both
the
than
were exceptionally
in such cases
COMMUNICATIONS
LDL
intima,
the unaffected
in rich
in GM3 (6).
and
arterial
high ganglioside used in the presente
growing cells actively release gangliosides be possible that in damaged regions of the are
Such mechanism could
three plaques
associated macrophages reside corresponding to the ganglioside into account that fast medium (19) it seems to
BIOPHYSICAL
hand, we demonstrated
especially
content
AND
also
modify
formed with are rapidly giving rise to formation explain LDL
to
how arterial a
form
taken up by of foam cells.
endothelial that
becomes
wall
environment work. Taking into aorta
the LDL-
phagocytes
(20) and smooth recognized
by
macrophages. The ganglioside-induced modification of LDl seems to be essential but not sufficient for transformation of macrophages into foam modification cells. In any case, the idea that ganglioside-induced of LDL and/or the effects of ganglioside on macrophages associated with wessel wall may play
a role
in the pathogenesis
of
atherosclerosis,
deserves
further
investigation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Steinberg, D., Pathasarathy, S., Carew, T.E., Khoo, J.C. and Witztum, J.L. (1989) New England j. Med. 320, 915-924. Goldstein, J.L., Ho, Y.K., Basu, S.K., Brown, M.S. (1979) Proc. Natl.Acad.Sci. USA 76, 333-337 Mahley, R.W., Innerarity, T.L., Weisgraber, K.H., Oh, S.Y. (1979) J. Clin. Invest. 64, 743-750. Shechter, I., Seager, J., Hokom, M., Child, J.S., Pogelman, A.M., Edwards, P.A. (1980) Proc. Natl. Acad. Sci. USA 77, 2214-2218. Senn, H.-J., Orth, M., Fitzke, E., Wieland. H. and Gerok, W. (1989) Eur. J. Biochen. 181, 657-662. Prokazova, N.V., Orekhov. A.N., Mukhin, D.N.. Wikhailenko, I.A., Gladkaya, E.M., Kogtev, L.S., Sadovskaya, V.L. and Bergelson, L.D. (1986) Bioorganich. Khim. USSR 12. 1405-1412. Mikhailenko, I.A., Dubrovskaya, S.A., Korepanova, O.B.,Morozkin, A.D., Prokazova, N.V. and Bergelson. L.D. (1991) in press. Wikhailenko, I.A., Ivanov, Prokazova, N.V., Preobrazhensky, S.N., V.O., Pokrovsky, S.N., Timofeeva, N.G.,Martynova, M.A., Repin, V.S. and Bergelson, L.D. (1986) Glycoconjugate J. 3, 273-286. Biophys. Filipovic, J., Schwarzman, G., Buddecke, E. (1981). Biochim. Acta 647, 112-118. Ando, S. and Yu, R.K. (1978) J.Lipid Res. 19, 538Seyfried, T.N., 543. 388, Bierman. E.L. and Albers, J.J. (1975) Biochim. Biophys. Acta 198-202. (1961) in: Metabolism of Plasma Proteins in McFarlane, A.B. Manmalians, Academic Press, New York, pp.331-338. CellEdelson, P.J. and Cohn, Z.A. (1976) in: In Vitro Methods in ( Bloom, B.R. and David, J.R., Mediated and Tumor Immunity eds.),Academic Press, New York, pp. 333-340. 586
Vol.
177,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
14. Goldstein, J.L., Basu, S.K., Brown, M.S. (1983) Methods Enzymol. 98, 241-260. 15. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 16. Saito, M. (1989) Develop. Growth 2. Diff. 31, 509-522. 17. Brown, M.S. and Goldstein, J.L. (1983) Ann. Rev. Biochem. 52, 223261. 18. Khoo, J.C.. Miller, E.. McLaughlin, P. and Steinberg, D. (1988) Arteriosclerosis 8, 348-358. 19. Shaposhnikova, G.I., Prokazova, N.V.,Buznikov, G.A., Zvezdina, N.D., Teplitz, N.A. and Bergelson, L.D. (1984) Eur. J. Biochem. 140, 567-570. 20. Henriksen, T., Mahoney, E.M. and Steinberg, D (1981) Proc. Natl. Acad. Sci. USA 78, 6499-6503. 21. Henriksen, T., E.M. Mahoney, and Steinberg, D. (1983) Arteriosclerosis 3, 149-159.
587
177,
May
31, 1991
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
582-587
Pages
GANGLIOSIDE
GM3 STIMULATES THE UPTAKE AND PROCESSING OF LOW DENSITY LIPOPROTEINS BY NACROPBAGES
Nina V.Prokazova,
Irina
A.Mikhailenko
and Lcv D.Bergelson
Institute
of Experimental Cardiology, Cardiology Research Center of the USSRAcademy of Medical Sciences, MOSCOW, USSR
Received
April
22,
1991
Preincubation of low densiy l’poproteins GM3 (1-2x 10 -5 M/ 2.5x10 the ganglioside increase of LDL-uptake, enhances cholesterol accumulation and cholesteryl At the same time the lysosomal degradation of ester formation by macrophages. LDL in macrophages was inhibited under these conditions. These effects depended on the ganglioside structure and concentration. It is suggested that the effects observed could be caused by GM3-induced modification of LDL to a form that becomes recognized by macrophages. 0 1991 Academic Press, Inc.
One
of
the
migration
earliest
of
differentiate esters
into
and other
lipids
which
plasma
low
are
several
which
to accumulate believed that LDL
into
However The can
a the
are
artificial
cause
The term blood by
recognized
"native
formation
LDL”
sequental
refers density
of
called
cells.
0006-291X/91 Copyright All rights
LDL-low
density
(1). occurs
in vivo
receptor
remains cultured
to LDL isolated ultracentrifugation
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reservea.
582
even
still
is
high shown into them
therefore
which on
LDL*
transforms macrophages.
unknown.
lipoprotein
macrophages
from
them
and cause It
the
induce
has been
in vitro
esters
high-affinity
lipoproteins;
native do not
It
The
from
that
particles include
normal human according to
standard methods. Abbreviations; Cer .
cholesteryl
that
macrophages
macrophages
they
foam cells.
fact and
is
where
to be derived
the
of LDL producing from
wall,
of LDL can transform
up by
modification
nodifications
foam cell
within
of cholesteryl by a
are
vitro with
of LDL-modification
of this
atherosclerosis
amounts
of the
in
incubated
taken
amounts
high
believed
modifications
rapidly
some type form
inspite
esters
artificial
massive
nature
(LDL) macrophages
of much cholesteryl
that
are
LDL when
of
arterial
cells
cells
lipoproteins by
the
lipid-loaded
in foam
of native
development
into
and accumulate
These
recognized
concentrations
particles
lipids.
the
blood
macrophages
density
accumulation
in
from
accumulate
weakly
however
events
monocytes
GM3- II3-NeuAc-Lac-
acetylation
that
Vol.
177,
No.
1, 1991
(2),
acetoacetylation
can
be
LDL-oxidation. complexes
excluded.
Recently stability
plasma,
and we
that
and are
of
LDL
with
gangliosides
hepatoaa
cells
uptake
and LDL-uptake
and degradation
MATERIALS
of GM3,
we (6)
in
by these main
by LDL
their
and others
reduced the
l), of
LDL
cells. ganglioside
of LDL by mouse peritoneal
are is
of human
of
plasma (7).
modified as
is
or plasma,
bound
in vitro
to
on formation
components
actively
hardly
in vivo
(reviewed based
of LDL as well Previously
influence
place
matrix
gangliosides
properties
the
to take
of LDL by gangliosides
(7).
investigate
on the
(6)
which leading
extensively
normal
apo-B
against
preincubation
fibroblasts study
space
are
(4)
of modification
intracellular
modification
COMMUNICATIONS
ralonaldehyde
type
studied
of the the
fluorescence
RESEARCH
of LDL- modification,
gangliosides
demonstrated
and
that
them,
intercellular
antibodies
with
Another
has been
components
because
we
BIOPHYSICAL
and more likely
pathways
Among
interest
and aorta1
with
process
biological
of LDL with
special
in vivo.
by macrophages
This
other
AND
and conjugation
to occur
LDL uptake
however
(5)
(3)
expected
enhanced
not
BIOCHEMICAL
size, interaction (9)
found
binding
to
In the of
present human
macrophages.
AND METHODS
Ganglioside GM3 was isolated from human liver (10). [l’C]Oleic acid 50-60 Ci/maol was recieved from Amersham. Human LDL (d-1.019-1.063 g/ml) were obtained from fresh plasma of normal volunteers by sequential ultracentrifugation as described in (11). [1251J-LDL was prepared by the iodine monochloride method (12), specific activity was 50-100 cpn/ng protein. Association of gangliosides with LDL was achieved by incubation of LDL (2.5 uM) with 50 uM gangliosides for 0.5-2 h at 37’C. Mouse peritoneal macrophages were collected from unstimulated mice in 199 medium containing 20 units heparin/ml (13). Macrophages from 20 mice (5x10’ cells) were pooled and collected by low speed centrifugation at 1000 rpm for 5 min at room temperature. The cells were then resuspended in 199 medium containing 20% bovine serum, 100 ug/ ml of penicillin and 100 ug of streptomycin. Cells (1~10~ per well) were dispensgd into 24-well tissue culture cluster (Costar, USA) with a density of 10 cells/ well and allowed to adhere for 4-6 h at 37’C in a humidified 5% C02/95% air atmosphere. Then the cells were washed twice with 199 medium to remove nonadherent cells. The 199 medium containing 20% human lipoprotein-deficient serum was added to each well after which the macrophages were used for experiments. LDL binding and degradation were performed as described in (14). The were nacrophage monolayers incubated in presence of [1251]-LDL (100 ug or LDL-ganglioside associates at 37’C for 3 h. The cell protein/ml) monolayers were washed three times with 199 medium containing 20% bovine serum albunine. The radioactivity associated with the cells was measured in a gamma-counter (LKB, Bromna, Sweden, model 1282). The proteolytic degradation and LDL-ganglioside associates was measured by assaying the of [ 1251]-LDL amount of [1251]-labeled trichloracetic acid-soluble material formed by the nacrophages and excreted into the culture medium. For this aim the nacrophage monolayers were incubated for 24 b as described for binding experiments. The medium removed after incubation was used to study LDL proteolytic degradation. Incorporation of [l* Cl-oleate by macrophages into cholesteryl esters and triglycerides was determined as follows (14). The macrophage monolayers were incubated with 0.3 ml 199 medium containing 10% of lipid-deficient serum, various amounts of native LDL or LDL-ganglioside associates and 0.2 urol/ml 583
Vol.
177,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
[14C]-oleic acid/albumine (M/M) at 31’ for 24 h. After incubation the monolayers were washed with PBS and the total lipids were extracted in situ The cell residues were used to determine with hexan/isopropyl alcohol (3:2). the cellular protein (15). The extracts of nacrophage monolayers from three wells were combined and the cholesteryl esters and triglycerides were isolated by silica gel thin-layer chromatography using the solvent system The cholesteryl ester and hexan/ diethyl ether/ acetic acid (85:15:1 v/v). triglyceride bands were visualized with iodine vapor, scraped into vials and counted in a Racbeta 1250 beta-counter. The incorporation of chlesterol by nacrophages was determined as described early (14). The macrophage monolayers were incubated with 0.3 ml of 199 medium containing 20% human lipoprotein-deficient serum and several amounts of LDL-ganglioside assosiates or native LDL for 24 b at 37’C. The cells were The total washed with PBS, and lipids were extracted as described above. cholesterol content in lipid extracts was determined using Boehringer Mannheim Monotest Cholesterol CHOD-PAP-Method ( Cat. no 290 319, Boehringer, FRG).
RESULTS AND DISCUSSION As it
can
be
seen
macrophages
increases
effect
the ganglioside
of
concentrations
of
data
of Table of the
was concentration exceeding
GM3
1, the uptake
lipoproteins
dependent
2~10-~
M.
had no influence
with and
Further
on the
of LDL GM3.
This
saturable
at
increase
uptake
by
of
in
the
lipoproteins
by
in
macrophages,
as
in
the
cells. At
the
same
determined medium, the
the
preincubation
concentration
ganglioside the
after
from
by
time
the
lysosomal
the appearance
was inhibited
in
the
ganglioside
concentration.
Pretreatment
of LDL with
binding
but
also
inferred
from
the
presence formation
the greatly
of
2).
cholesteryl
Table
1. and
presence
This
of GM3.
appears
esters
results
of
not cells
by
GM3 on the macrophages
by
could
on
be
uptake
1251-LDL pg cell protein
1.2620.40 1.42+0,52 2.10+0.48
1.64+0.21 1.22+0.0s 0.91+0.08
in
LDL-induced
macrophages
degraded
584
it
accumulation also
bound
3 experiments.
again
LDL-macrophage
as
cholesterol
ganglioside of LDL
incubation
depended
only
of GM3 stimulated
ng/
-
effect
and triglycerides
of Influence the degradation
10 20
by the
intracellular
The presence
GM3 nmol/mg protein
The
to stimulate
of lipoproteins
enhanced
of GM3 (Table
of LDL products
GM3
uptake
degradation
of [1251]-labeled
in
a
Vol.
177,
No.
1, 1991
BIOCHEMICAL
Table 2. LOL-induced
AND
Influence accumulation
of of
Lipoproteins
BIOPHYSICAL
ganglioside cholesterol
Cholesterol
concentration
dependent
manner
(Table
the
chemical
structure
of the
ganglioside:
GDla
which
is both
cholesteryl The
ester
leukemia by
underlying
of cells
the
part
LDL
and that
formation molecular
immunoreactivity
of apo-B of LDl
Khoo
(18)
et al
by
Table
macrophages
to GM3,
ganglioside
a
differ
(200) (300)
nyelogenous not
able
LDL (5).
to like
native
density,
seem to be caused Brown
of cholesteryl
ganglioside and triglycerides
GM3
esters
in
and partly
and Goldstein be active ester
(17)
inductors accumulation
content protein) Triglycerides
3.65+0.83 10.13+0.20 11.05+0.80 29.3422.73
of
their size,
apolipopropteins,
on formation in macrophages acid cell
results LDL in
changes
LDL might
of
particle
and
aggregated
Recently
amounts
gangliosides
lipids
585
as well
we should
high
from
floating
0.03+0.01 0.15+0.01 0.14+0.07 0.22+0.07
lipid
be caused
(16) but
with
LDL with
[14C]-oleic (pmoles/mg
(300) (100)
(with
relatevelly
of gangliosides. and enhence
shown).
and
could
differentiation
associated
which
as:
not
as
latter.
is
These
observed
of evidence
bind
surface
that
component,
(data
cells
on
as well
At the moment we are
the
to
such (7).
ganglioside accumulation
recipient
of lack
complexes of
minor
cell
Cholesteryl native LOL LOL+GMS LOL+GM3 LOL+GMB
in contrast
of native
3. Influence of cholesteryl esters
Lipoproteins (pg protein/ml)
strongly
The effects the
of
able
in presence
suggested
depended
of GM3 on LDL uptake
favor
properties
aggregation
effects
influence with
incubation
organization
These
by macrophages
gangliosides
of LDL-ganglioside
physico-chemical
& 0.47 + 0.94
as
of LDL.
were
protein)
formation
because
of plasma
mg cell
18.35 32.9
macrophage-like
in
on the macropbages
cholesterol
unknown.
modification
that
gangliosides
are
possibility
some arguments
showed
LDL-uptake
the
GM3 induces
first
The major
only
the ganglioside
GMS-induced
to consider
we
serum intracellular
by macrophages
by interaction
discuss
in
and triglyceride
mechanisms
accumulation
as
present LDL-induced
3).
COMMUNICATIONS
GM3
in
(pg/
native LOL LOL+GMS
inhibited
RESEARCH
by and of in
Vol.
177,
these
No.
1, 1991
cells.
arterial
On the other wall,
ganglioside regions
BIOCHEMICAL
and that
Therefore
it
in atherosclerotic
was
at least
atherosclerotic
may well
ganglioside residing
complexes within
be
that
muscle
cells
the lesions,
(21)
times
RESEARCH
that
in the atherosclerotic
lesions
of
higher
in a relativelly concentration
both
the
than
were exceptionally
in such cases
COMMUNICATIONS
LDL
intima,
the unaffected
in rich
in GM3 (6).
and
arterial
high ganglioside used in the presente
growing cells actively release gangliosides be possible that in damaged regions of the are
Such mechanism could
three plaques
associated macrophages reside corresponding to the ganglioside into account that fast medium (19) it seems to
BIOPHYSICAL
hand, we demonstrated
especially
content
AND
also
modify
formed with are rapidly giving rise to formation explain LDL
to
how arterial a
form
taken up by of foam cells.
endothelial that
becomes
wall
environment work. Taking into aorta
the LDL-
phagocytes
(20) and smooth recognized
by
macrophages. The ganglioside-induced modification of LDl seems to be essential but not sufficient for transformation of macrophages into foam modification cells. In any case, the idea that ganglioside-induced of LDL and/or the effects of ganglioside on macrophages associated with wessel wall may play
a role
in the pathogenesis
of
atherosclerosis,
deserves
further
investigation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Steinberg, D., Pathasarathy, S., Carew, T.E., Khoo, J.C. and Witztum, J.L. (1989) New England j. Med. 320, 915-924. Goldstein, J.L., Ho, Y.K., Basu, S.K., Brown, M.S. (1979) Proc. Natl.Acad.Sci. USA 76, 333-337 Mahley, R.W., Innerarity, T.L., Weisgraber, K.H., Oh, S.Y. (1979) J. Clin. Invest. 64, 743-750. Shechter, I., Seager, J., Hokom, M., Child, J.S., Pogelman, A.M., Edwards, P.A. (1980) Proc. Natl. Acad. Sci. USA 77, 2214-2218. Senn, H.-J., Orth, M., Fitzke, E., Wieland. H. and Gerok, W. (1989) Eur. J. Biochen. 181, 657-662. Prokazova, N.V., Orekhov. A.N., Mukhin, D.N.. Wikhailenko, I.A., Gladkaya, E.M., Kogtev, L.S., Sadovskaya, V.L. and Bergelson, L.D. (1986) Bioorganich. Khim. USSR 12. 1405-1412. Mikhailenko, I.A., Dubrovskaya, S.A., Korepanova, O.B.,Morozkin, A.D., Prokazova, N.V. and Bergelson. L.D. (1991) in press. Wikhailenko, I.A., Ivanov, Prokazova, N.V., Preobrazhensky, S.N., V.O., Pokrovsky, S.N., Timofeeva, N.G.,Martynova, M.A., Repin, V.S. and Bergelson, L.D. (1986) Glycoconjugate J. 3, 273-286. Biophys. Filipovic, J., Schwarzman, G., Buddecke, E. (1981). Biochim. Acta 647, 112-118. Ando, S. and Yu, R.K. (1978) J.Lipid Res. 19, 538Seyfried, T.N., 543. 388, Bierman. E.L. and Albers, J.J. (1975) Biochim. Biophys. Acta 198-202. (1961) in: Metabolism of Plasma Proteins in McFarlane, A.B. Manmalians, Academic Press, New York, pp.331-338. CellEdelson, P.J. and Cohn, Z.A. (1976) in: In Vitro Methods in ( Bloom, B.R. and David, J.R., Mediated and Tumor Immunity eds.),Academic Press, New York, pp. 333-340. 586
Vol.
177,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
14. Goldstein, J.L., Basu, S.K., Brown, M.S. (1983) Methods Enzymol. 98, 241-260. 15. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 16. Saito, M. (1989) Develop. Growth 2. Diff. 31, 509-522. 17. Brown, M.S. and Goldstein, J.L. (1983) Ann. Rev. Biochem. 52, 223261. 18. Khoo, J.C.. Miller, E.. McLaughlin, P. and Steinberg, D. (1988) Arteriosclerosis 8, 348-358. 19. Shaposhnikova, G.I., Prokazova, N.V.,Buznikov, G.A., Zvezdina, N.D., Teplitz, N.A. and Bergelson, L.D. (1984) Eur. J. Biochem. 140, 567-570. 20. Henriksen, T., Mahoney, E.M. and Steinberg, D (1981) Proc. Natl. Acad. Sci. USA 78, 6499-6503. 21. Henriksen, T., E.M. Mahoney, and Steinberg, D. (1983) Arteriosclerosis 3, 149-159.
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