Download Lysosomal Enzymes Are Released From Cultured Human

Lysosomal Enzymes Are Released From Cultured Human
Macrophages, Hydrolyze LDL In Vitro, and Are Present
Extracellularly in Human Atherosclerotic Lesions
Jukka K. Hakala, Riina Oksjoki, Petri Laine, Hong Du, Gregory A. Grabowski,
Petri T. Kovanen, Markku O. Pentikäinen
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Objective—Human atherosclerotic lesions have been shown to contain lipid droplets and vesicles resembling those of in
vitro enzymatically modified LDL. However, little is known about the hydrolytic enzymes in the arterial intima that
induce fusion of LDL particles and so produce lipid droplets or that induce foam cell formation.
Methods and Results—Human coronary atherosclerotic lesions obtained at surgery and at autopsy were stained for
lysosomal acid lipase and cathepsin D. The extracellular areas of macrophage-rich intimal regions of the atherosclerotic
lesions stained positively for both cathepsin D and lysosomal acid lipase, whereas normal arteries were negative. When
monocyte-derived macrophages were incubated with opsonized zymosan to stimulate the release of lysosomal enzymes
from the cells and LDL was incubated with the macrophage-conditioned media, the apolipoprotein B-100, cholesteryl
esters, and triacylglycerols of LDL were hydrolyzed. These hydrolytic modifications rendered the LDL particles
unstable and induced their fusion. Cultured macrophages and smooth muscle cells took up the hydrolase-modified LDL
particles avidly and were transformed into foam cells.
Conclusions—Our in vivo and in vitro results suggest that lysosomal enzymes released from macrophages may induce
hydrolytic modification of LDL and foam cell formation in the human arterial intima. (Arterioscler Thromb Vasc Biol.
2003;23:1430-1436.)
Key Words: macrophage 䡲 lysosomal enzymes 䡲 LDL 䡲 atherosclerosis
A
therogenesis is characterized by accumulation of LDLderived lipids in the extracellular matrix of the arterial
intima, recruitment of monocyte-derived macrophages, and
their transformation into lipid-laden foam cells.1 The lipid
accumulates extracellularly in atherosclerotic lesions in the
form of small lipid droplets and vesicles.2 Modification of
LDL in vitro by proteolysis, lipolysis, and oxidation has been
shown to produce particles resembling those observed in the
arterial intima.3–10 Lipid particles enriched in unesterified
cholesterol have been isolated from the human arterial intima,11 and evidence for the presence of the particles resembling in vitro enzymatically modified LDL3 has been provided by the immunohistochemistry.12 However, the enzymes
responsible for such LDL modification in the arterial intima
have remained uncharacterized.
100)13 and is found in atherosclerotic intima.14,15 The latter is
normally responsible for lysosomal hydrolysis of cholesteryl
esters and triacylglycerols of the endocytosed LDL particles.16 Extracellular discharge of lysosomal enzymes is a
common physiological response to a variety of inflammatory
stimuli.17,18 Under pathologic conditions, cathepsin D has
been found extracellularly in many tissues, eg, in the invading
edge of the rheumatoid synovium,19 in the brain of patients
suffering from Alzheimer disease,20 and in invasive human
carcinomas.21 Moreover, Briozzo et al22 showed that cathepsin D is a major acidic protease secreted by breast cancer
cells. LAL, in turn, is present in macrophage foam cells in the
subendothelial area and deep in the intimal fibrous tissue,23
but there is little in vivo evidence of secretion of LAL.
Recently, Buton et al24 suggested that LAL-mediated cholesteryl ester hydrolysis of extracellularly located and matrixretained and aggregated LDL starts during the prolonged
cell-surface contact, implying that enzymatically active LAL
may have been released into the pericellular areas of activated
macrophages.
In this study, we studied the presence and localization of
cathepsin D and LAL in normal and atherosclerotic arterial
See page 1312
Two enzymes that participate in the hydrolysis of endocytosed LDL in the lysosomal compartment of cells are cathepsin D and lysosomal acid lipase (LAL). The former initiates
the lysosomal degradation of apolipoprotein B-100 (apoB-
Received February 17, 2003; revision accepted April 17. 2003.
From the Wihuri Research Institute, Helsinki, Finland and Division of Human Genetics (H.D., G.A.G.), Cincinnati Children’s Hospital Research
Foundation, Cincinnati, Ohio.
Correspondence to Petri T. Kovanen, M.D., Ph.D., Wihuri Research Institute, Kalliolinnantie 4, FIN-00140 Helsinki, Finland. E-mail
[email protected]
© 2003 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1430
DOI: 10.1161/01.ATV.0000077207.49221.06
Hakala et al
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Figure 1. Immunohistochemistry of human coronary arteries.
Cross-sections of grossly normal (left) and atherosclerotic (right)
human coronary artery from explanted hearts were immunostained for cathepsin D (Cath D, A and B), LAL (C and D), macrophages (Mf, E and F), and LAMP-1 (G and H). Note the paucity of macrophages and the weak staining for lysosomal
enzymes and LAMP-1 in the grossly normal sample and the
strong immunostaining for lysosomal enzymes and LAMP-1 in
the macrophage-rich area in the atherosclerotic coronary artery.
I indicates intima; m, media; and a, adventitia.
intima and, moreover, studied modifications of LDL by
lysosomal enzymes released by stimulated human
macrophages.
Methods
An online Methods section is available at http://atvb.ahajournals.org.
Results
The presence of the lysosomal enzymes cathepsin D and LAL
was studied by immunohistochemistry in normal and in
atherosclerotic human coronary arteries obtained from autopsied and explanted hearts and compared with that of a
lysosomal marker, the lysosome-associated membrane
protein-1 (LAMP-1). Figure 1 shows immunostaining pattern
that was typically found both in autopsy and transplant
specimens. The amounts of cathepsin D and LAL in the cells
of the normal arterial intima were so low that they were
barely detectable by immunohistochemistry (panels A and C).
Modification of LDL by Lysosomal Enzymes
1431
Figure 2. Modification of LDL in macrophage-conditioned
medium. 3H-LDL (20 ␮g) was incubated for the indicated times
in 96-well plates with 175 ␮L of macrophage-conditioned media
from Zop-stimulated (H-LDL) and unstimulated (control LDL)
cells in 200 ␮L of buffer A. After the incubations, proteolysis of
LDL was determined by measuring the TCA-soluble 3H radioactivity (A), the degree of lipolysis was determined by analyzing
the amount of free fatty acids (B), and the turbidity of the reaction mixture was measured at 340 nm (C). Data shown are
means of 3 independent experiments⫾SEM. *P⬍0.05.
However, as shown in panels B and D, in atherosclerotic
lesions that contained large number of macrophages, the
amounts of cathepsin D and LAL were dramatically increased, and both enzymes were colocalized with the macrophages (panel F). All of the sections stained with control
antibodies (mouse-irrelevant isotype-specific Igs and rabbit
IgG) were negative (not shown). Interestingly, in contrast to
the mainly intracellular staining of LAMP-1 (panel H),
cathepsin D and LAL were widely present extracellularly in
the intima.
Next, we studied whether lysosomal hydrolases released
from human monocyte-derived macrophages could modify
LDL in vitro. As a model of stimulation-induced secretion of
acid hydrolases, we incubated human macrophages with
zymosan A opsonized with fresh human serum (Zop).27
Incubation of LDL with conditioned media derived from
Zop-treated macrophages, but not from untreated control
macrophages, led to generation of TCA-soluble radioactivity
(Figure 2A) and free fatty acids (FFAs) (Figure 2B), indicating proteolysis and lipolysis of the LDL particles, respectively. This modification was not caused by oxidation, because the samples contained EDTA, the amount of
thiobarbituric acid reactive substances (TBARS) in H-LDL
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Arterioscler Thromb Vasc Biol.
August 2003
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Figure 3. Characterization of proteolytic
activity in macrophage-conditioned
media. A, 3H-LDL (20 ␮g, 2000 dpm/␮g)
in buffer A, 175 ␮L of conditioned
medium from unstimulated and from
Zop-stimulated macrophages, and 1 of
the inhibitors (pepstatin A [29 ␮mol/L],
LHVS [1 ␮mol/L], leupeptin [4 ␮mol/L],
␣2-macroglobulin [␣2-M, 60 nmol/L],
PMSF [1 mmol/L], EDTA [10 mmol/L], or
aprotinin [0.3 ␮mol/L]) were incubated at
⫹37°C for 6 hours. After the incubations,
the degrees of apolipoprotein B-100
degradation were determined by measuring the TCA-soluble 3H radioactivity.
Data shown are means of 6 independent
experiments⫾SEM. B, Cathepsin D was
immunoprecipitated from macrophageconditioned medium. LDL was then incubated with either medium from unstimulated macrophages (control medium),
medium from Zop-stimulated macrophages (Mf medium), or immunodepleted
medium from Zop-stimulated macrophages (Cath D immunodepleted Mf
medium). After the incubations, the
degrees of apoB-100 proteolysis were
determined. Data shown are means of 4
independent experiments⫾SEM. C, 50
␮g of LDL was incubated for indicated
times with either commercial cathepsin D
(1.6 U/mL) or conditioned medium (450
␮L) from Zop-stimulated macrophages
(Mf medium). Reaction mixtures were then analyzed in SDS-PAGE, stained with Coomassie blue, and photographed. Data shown are
representative of 4 independent experiments. D, As controls, LDL was incubated with thrombin (15 U/mL), kallikrein (4.6 U/mL), and
pronase (20 ␮g/mL) and analyzed in SDS-PAGE. The control data shown are a representative of 3 independent experiments.
(⬍1 nmol/mg LDL) was low as compared with oxidized LDL
(22 nmol/mg LDL), and such modification was unaffected by
the presence of 20 ␮mol/L butylated hydroxytoluene. Incubation of LDL in culture medium with or without Zop
particles in the absence of macrophages failed to generate
TCA-soluble radioactivity, showing that proteolytic activity
was released from macrophages (not shown). The level of
lactate dehydrogenase activity in the control media
(6.1⫾3.5% of total cellular activity) and the conditioned
media from Zop-treated macrophages (6.5⫾0.3% of total
cellular activity) was low, suggesting that Zop did not exert
cytotoxic effects on macrophages. Similar proteolytic activity, albeit smaller in extent, was released from macrophages
when they were incubated with LDL immunocomplexes,
vortexed LDL, or zymosan particles (not shown).
Incubation of LDL with conditioned media from Zoptreated macrophages led to an increase in the turbidity of the
sample, indicating that the LDL particles had undergone
aggregation or fusion (Figure 2C). The morphology of
H-LDL was studied by electron microscopy. Treatment of
LDL with conditioned media from Zop-treated macrophages
led to the formation of lipid droplets, ie, it had induced fusion
of the LDL particles (Figure I, please see the online supplement at http://atvb.ahajournals.org).
The proteolytic activity in the macrophage-conditioned
medium was characterized with various protease inhibitors
(Figure 3A). We found that of the various protease inhibitors
studied, only pepstatin A, an inhibitor of aspartic proteinases,
fully abolished the proteolytic activity of the macrophage-
conditioned medium. We also found that immunodepletion of
the macrophage-conditioned media with antibody against
cathepsin D blocked LDL proteolysis (Figure 3B), demonstrating that hydrolysis of apoB-100 depends on cathepsin D
activity. Finally, the patterns of apoB-100 fragmentation of
LDL were compared in SDS-PAGE. As shown in Figures 3C
and 3D, only commercial cathepsin D produced the degradation pattern of LDL similar to the pattern induced by the
macrophage-conditioned media. Taken together, the data
suggest a major role for cathepsin D in the proteolytic
modifications of LDL.
Analysis of the lipid composition of the H-LDL particles
revealed that the amounts of cholesteryl esters (CEs) and
triacylglycerols (TGs) were significantly decreased and the
amounts of unesterified cholesterol (UC) and FFAs were
significantly increased in H-LDL (Figure 4A). No significant
hydrolysis of phosphatidylcholine or sphingomyelin was
found. To confirm that LDL lipids were hydrolyzed by LAL,
LAL was removed from the macrophage-conditioned medium by immunoprecipitation. It was found that immunodepletion of LAL from the conditioned medium totally
inhibited release of free fatty acids from the LDL lipids
(Figure 4B).
To study the forms of cathepsin D and LAL that are
secreted on treatment of human macrophages with Zop,
macrophage-conditioned media were studied by Western blot
analysis. As shown in Figure 5, the conditioned medium from
unstimulated macrophages contained a single immunopositive 52-kDa band corresponding to the pre-proform of ca-
Hakala et al
Modification of LDL by Lysosomal Enzymes
1433
Figure 4. Analysis of lipolytic activity in macrophageconditioned media. A, Lipid composition in native
and hydrolase-modified LDL was analyzed with
HPTLC. Data shown are means of 7 independent
experiments⫾SEM. *P⬍0.05, **P⬍0.01. B, LDL was
incubated with either medium from unstimulated
macrophages (control medium), conditioned medium
from Zop-treated macrophages (Mf medium), or LAL
immunodepleted medium from Zop-treated macrophages (LAL immunodepleted Mf medium). After the
incubation, the lipids were extracted and analyzed by
HPTLC. Data shown are means of 3 independent
experiments⫾SEM. TG indicates triacylglycerols; PC,
phosphatidylcholine; SM, sphingomyelin; and LPC,
lysophosphatidylcholine.
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thepsin D. In addition to the 52-kDa band, conditioned
medium from Zop-treated macrophages contained 48-kDa
and 32-kDa bands corresponding to the catalytically active
intermediate proenzyme and the mature forms of cathepsin D,
respectively.28 Conditioned medium from Zop-treated macrophages also contained large amounts of LAL. In addition to
the 49-kDa band, LAL also appeared as the 41-kDa band,
representing glycosylated and deglycosylated forms of the
enzyme, respectively.29
Next we studied uptake of H-LDL by macrophages in
comparison with native LDL, acetylated LDL (acLDL), and
oxidized LDL (oxLDL). Figures 6A through 6D show mouse
peritoneal macrophages incubated for 18 hours with LDL,
acLDL, oxLDL, or H-LDL and then stained with Oil Red O.
No lipid staining was found in the LDL-treated cells (panel
A), whereas typical intracellular lipid droplets were found in
the macrophages incubated with acLDL (panel B), oxLDL
(panel C), and H-LDL (panel D). Analysis of the cholesterol
content of mouse macrophages (Figure 6E) showed that the
intracellular contents of both UC and CE were significantly
increased in H-LDL–treated cells (H-LDL) compared with
resting cells (Bl), whereas incubation of the cells with acLDL
significantly increased the intracellular content of CE but not
UC. Importantly, incubation of human macrophages with
H-LDL and acLDL (Figure 6F) also significantly increased
intracellular contents of both UC and CE. Macrophageconditioned medium, in the absence or presence of native
LDL, did not increase the cholesterol content of the macrophages (not shown). Electron microscopy (Figure II, please
see the online supplement at http://atvb.ahajournals.org) revealed that H-LDL–treated human macrophages had large
amounts of modified lipoproteins in compartments resembling phagosomes and also contained cytoplasmic lipid inclusions. The mechanisms of uptake of H-LDL by macrophages was studied by competition experiments. In the
presence of 20-fold excess of unlabeled lipoproteins, the
degradation of H-LDL by macrophages was inhibited by
53⫾12% with LDL, by 59⫾12% with acLDL, by 69⫾1%
with mildly (3-hour) oxidized LDL, by 57⫾35% with totally
(18-hour) oxidized LDL, and by 67⫾12% with the unlabeled
H-LDL. This suggests that the uptake of H-LDL occurs
through both scavenger receptors and the LDL receptor.
Finally, cultured human coronary artery smooth muscle
cells (HCASMCs) were incubated for 72 hours in the absence
or presence of LDL, acLDL, oxLDL, or H-LDL and stained
with Oil Red O (Figure 7). Numerous lipid droplets were seen
in the cytoplasm of H-LDL-treated HCASMCs (panel D) but
not in that of cells treated with LDL, acLDL, or oxLDL
(panels A, B, and C). The results are consistent with previous
studies showing little expression of scavenger receptors in
resting smooth muscle cells in culture and in normal arteries30,31 and suggest that in smooth muscle cells the uptake of
H-LDL is mediated by the LDL receptor.
Discussion
Figure 5. Western blot analysis of macrophage-conditioned
media. Human macrophages were incubated with (⫹Zop) or
without (control) Zop for 18 hours. After incubation, the conditioned media were collected and applied (35 ␮L) to a 15% SDSPAGE gel under reducing conditions and transferred to a nitrocellulose membrane. The membrane was blocked and probed
with antibodies against cathepsin D and LAL. The bands were
visualized with ECL. The results are representative of 3 independent experiments
The present study shows that 2 lysosomal enzymes, cathepsin
D and lysosomal acid lipase, are present in human atherosclerotic lesions. The lysosomal enzymes showed both punctate cellular and diffuse extracellular staining, whereas
LAMP-1 showed mainly punctate cellular staining. This
observation supports the notion that the enzymes were present not only in the lysosomes but also extracellularly in the
human atherosclerotic lesions. To exclude that this finding
results from postmortem release of these enzymes, not only
autopsy material but also samples obtained at surgery were
examined. The paucity of extracellular staining of LAMP-1
reveals that the lysosomes are intracellular and have remained
intact, suggesting that rather than being passively released
from dying cells, the lysosomal enzymes were secreted
actively by living cells. On the basis of their colocalization
with macrophage infiltrates, it is also evident that the lyso-
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August 2003
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Figure 7. Uptake of H-LDL by HCASMCs. HCASMCs were cultured on coverslips, and their growth was arrested for 48 hours.
The cells were then incubated for 72 hours with 20 ␮g/mL of
native LDL (LDL), acetylated LDL (acLDL), LDL oxidized with 5
␮mol/L CuSO4 for 18 hours (oxLDL), or (H-LDL). After incubation, the cells on the coverslips were stained with Oil Red O and
counterstained with hematoxylin. The micrographs shown are
representative of 2 independent experiments.
Figure 6. Uptake of modified LDL by mouse peritoneal macrophages and analysis of the cholesterol content of H-LDL–treated
macrophages. Mouse macrophages were plated on glass coverslips and incubated for 18 hours in the presence of 40 ␮g/mL of
native LDL (LDL) (A), acLDL (B), LDL oxidized with 5 ␮mol/L
CuSO4 for 18 hours (oxLDL) (C), or LDL that was modified with
conditioned medium from Zop-treated macrophages (H-LDL)
(D). After incubation, the cells were stained with Oil Red O and
counterstained with hematoxylin. The micrographs shown are
representative of 4 independent experiments. Mouse peritoneal
macrophages (E) and human monocyte-derived macrophages
(F) were treated as described above, and after incubation, the
cellular lipids were extracted and analyzed by HPTLC. The
amounts of unesterified cholesterol and cholesteryl esters were
normalized to the total protein content of the cells. Data shown
are means of 4 to 7 independent experiments⫾SEM. *P⬍0.05
vs control cells (Bl), **P⬍0.01 vs Bl.
somal enzymes were synthesized and secreted chiefly by the
macrophages present in the lesions. Consistent with our
results, other lysosomal enzymes have also been found
extracellularly, ie, sphingomyelinase32 in human atherosclerotic lesions and cathepsins B and D20 in amyloid deposits in
Alzheimer brains. Moreover, cathepsins K, B, S, L, and D
have been shown to be secreted simultaneously by cultured
monocyte-derived macrophages.33 Taken together, the present and previous findings suggest that when the macrophages
appear and the atherosclerotic lesions start to develop, several
different lysosomal enzymes are present extracellularly in the
human arterial intima.
To be capable of modifying the LDL particles, the released
enzymes must be catalytically active. Most mammalian cells
release precursor forms of the enzymes that are ultimately
targeted to lysosomes. The precursors are taken up by the
secreting cells themselves or by the neighboring cells via
mannose-6-phosphate receptors located on the cell surface
and are then targeted to the lysosomes for final maturation.34
Importantly, specialized phagocytes, notably neutrophils and
macrophages, also release mature, ie, catalytically active,
lysosomal enzymes, a property related to their competence in
host defense and in the generation of local inflammation.34 In
our present in vitro system using human monocyte-derived
macrophages, both Western blot analysis and functional
studies with LDL indicated that cathepsin D and lysosomal
acid lipase were secreted in catalytically active form.
Various agents that have been shown to trigger the release
of lysosomal enzymes from macrophages, ie, asbestos,35
streptococcal cell wall substance,36 zymosan,37 and immuno-
Hakala et al
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complexes,38 all cause macrophages to also exert phagocytic
activity. In the present study, we used serum-opsonized
zymosan particles to induce secretion of enzymes by the
macrophages. Serum opsonization leads to coating of the
particles with complement iC3b and immunoglobulins.39 By
binding to their respective receptors (the complement receptor CR3 [CD11b/CD18] and the Fc␥ receptors on the macrophages), these ligands trigger phagocytosis with ensuing
efficient secretion of lysosomal enzymes. Importantly, immunoglobulins, in the form of LDL-immunocomplexes,40 and,
as a result of local complement activation, iC3b41 are deposited in the human atherosclerotic intima and are among the
potential triggers of release of lysosomal enzymes in the
lesions.
The pH in the normal arterial intima is near neutral,
whereas the pH in lysosomes and the optimum pH of
lysosomal enzymes is acidic. Accordingly, a major question
is whether the pH locally in atherosclerotic lesions allows
catalytic activity of the lysosomal enzymes. Macrophages are
able to acidify their pericellular environment with the aid of
proton pumps42 and by secreting lactic acid43 into the extracellular space.44 Atherosclerotic lesions are characterized by
chronic inflammation,45 and the pH of the extracellular fluid
in inflammatory sites is known to be acidic.42 Indeed, as signs
of an acidic environment, atherosclerotic lesions have been
shown to contain accumulations of lactic acid46 and hypoxia
has been demonstrated by the low oxygen tension,47 the
positive staining with the hypoxia marker theophylline,48 and
the presence of neovascularization.49 Thus, there is substantial evidence that the pH of the extracellular milieu may be
acidic in atherosclerotic lesions, at least in inflammatory
infiltrates, potentially allowing extracellular action of lysosomal enzymes.
Taken together, the present study reveals the possibility
that the lysosomal enzymes cathepsin D and LAL released
from macrophages participate in the modification of LDL in
atherosclerotic lesions. These observations suggest novel
candidate enzymes for generating hydrolase-modified LDL in
atherosclerotic lesions.12 Such hydrolytically modified LDL
particles may generate extracellular lipid droplets and vesicles and cause both extracellular and intracellular accumulation of lipid in the arterial intima.
Acknowledgments
The Wihuri Research Institute is maintained by the Jenny and Antti
Wihuri Foundation. This study was also supported by grants from the
Academy of Finland, the Sigrid Juselius Foundation, the Federation
of Finnish Insurance Companies (P.T.K.) and by grant No. QLG11999-01007 from the European Commission to the consortium
Macrophage Function and Stability of the Atherosclerotic Plaque
(MAFAPS) as part of the Fifth Framework Program of the European
Union. The excellent assistance of Laura Vatanen, Mari Jokinen, and
Suvi Mäkinen is gratefully acknowledged. The authors also acknowledge Drs Martin Goddard and Andrew Ritchie for collecting
coronary samples as part of the MAFAPS project.
References
1. Lusis AJ. Atherosclerosis. Nature. 2000;407:233–241.
2. Pasquinelli G, Preda P, Vici M, Gargiulo M, Stella A, D’Addato M,
Laschi R. Electron microscopy of lipid deposits in human atherosclerosis.
Scanning Microsc. 1989;3:1151–1159.
Modification of LDL by Lysosomal Enzymes
1435
3. Bhakdi S, Dorweiler B, Kirchmann R, Torzewski J, Weise E, Tranum J,
Walev I, Wieland E. On the pathogenesis of atherosclerosis: enzymatic
transformation of human low density lipoprotein to an atherogenic
moiety. J Exp Med. 1995;182:1959 –1971.
4. Kokkonen JO, Kovanen PT. Accumulation of low density lipoproteins in
stimulated rat serosal mast cells during recovery from degranulation. J
Lipid Res. 1989;30:1341–1348.
5. Piha M, Lindstedt L, Kovanen PT. Fusion of proteolyzed LDL in the fluid
phase: a novel mechanism generating atherogenic lipoprotein particles.
Biochemistry. 1995;34:10120 –10129.
6. Suits AG, Chait A, Aviram M, Heinecke JW. Phagocytosis of aggregated
lipoprotein by macrophages: low density lipoprotein receptor-dependent
foam-cell formation. Proc Natl Acad Sci U S A. 1989;86:2713–2717.
7. Xu XX, Tabas I. Sphingomyelinase enhances low density lipoprotein
uptake and ability to induce cholesteryl ester accumulation in macrophages. J Biol Chem. 1991;266:24849 –24858.
8. Öörni K, Hakala JK, Annila A, Ala-Korpela M, Kovanen PT. Sphingomyelinase induces aggregation and fusion, but phospholipase A2 only
aggregation, of low density lipoprotein (LDL) particles: two distinct
mechanisms leading to increased binding strength of LDL to human
aortic proteoglycans. J Biol Chem. 1998;273:29127–29134.
9. Hakala JK, Öörni K, Ala-Korpela M, Kovanen PT. Lipolytic modification
of LDL by phospholipase A2 induces particle aggregation in the absence
and fusion in the presence of heparin. Arterioscler Thromb Vasc Biol.
1999;19:1276 –1283.
10. Pentikäinen MO, Lehtonen EMP, Kovanen PT. Aggregation and fusion of
modified low density lipoprotein. J Lipid Res. 1996;37:2638 –2649.
11. Chao FF, Amende LM, Blanchette-Mackie EJ, Skarlatos SI, Gamble W,
Mergner WT, Kruth HS. Unesterified cholesterol-rich lipid particles in
atherosclerotic lesions of human and rabbit aortas. Am J Pathol. 1988;
131:73– 83.
12. Torzewski M, Klouche M, Hock J, Messner M, Dorweiler B, Torzewski
J, Gabbert HE, Bhakdi S. Immunohistochemical demonstration of enzymatically modified human LDL and its colocalization with the terminal
complement complex in the early atherosclerotic lesion. Arterioscler
Thromb Vasc Biol. 1998;18:369 –378.
13. van der Westhuyzen DR, Gevers W, Coetzee GA. Cathepsin-Ddependent initiation of the hydrolysis by lysosomal enzymes of apoprotein B from low-density lipoproteins. Eur J Biochem. 1980;112:
153–160.
14. Kaesberg B, Harrach B, Dieplinger H, Robenek H. In situ immunolocalization of lipoproteins in human arteriosclerotic tissue. Arterioscler
Thromb. 1993;13:133–146.
15. Jormsjö S, Wuttge DM, Sirsjo A, Whatling C, Hamsten A, Stemme S,
Eriksson P. Differential expression of cysteine and aspartic proteases
during progression of atherosclerosis in apolipoprotein E-deficient mice.
Am J Pathol. 2002;161:939 –945.
16. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol
homeostasis. Science. 1986;232:34 – 47.
17. Schnyder J, Baggiolini M. Secretion of lysosomal hydrolases by stimulated and nonstimulated macrophages. J Exp Med. 1978;148:435– 450.
18. McCarthy K, Musson RA, Henson PM. Protein synthesis-dependent and
protein synthesis-independent secretion of lysosomal hydrolases from
rabbit and human macrophages. J Reticuloendothel Soc. 1982;31:
131–144.
19. Poole AR, Hembry RM, Dingle JT, Pinder I, Ring EF, Cosh J. Secretion
and localization of cathepsin D in synovial tissues removed from rheumatoid and traumatized joints: an immunohistochemical study. Arthritis
Rheum. 1976;19:1295–1307.
20. Cataldo AM, Nixon RA. Enzymatically active lysosomal proteases are
associated with amyloid deposits in Alzheimer brain. Proc Natl Acad Sci
U S A. 1990;87:3861–3865.
21. Poole AR, Tiltman KJ, Recklies AD, Stoker TA. Differences in secretion
of the proteinase cathepsin B at the edges of human breast carcinomas and
fibroadenomas. Nature. 1978;273:545–547.
22. Briozzo P, Morisset M, Capony F, Rougeot C, Rochefort H. In vitro
degradation of extracellular matrix with Mr 52, 000 cathepsin D secreted
by breast cancer cells. Cancer Res. 1988;48:3688 –3692.
23. Davis HR, Glagov S, Zarins CK. Role of acid lipase in cholesteryl ester
accumulation during atherogenesis: correlation of enzyme activity with
acid lipase-containing macrophages in rabbit and human lesions. Atherosclerosis. 1985;55:205–215.
24. Buton X, Mamdouh Z, Ghosh R, Du H, Kuriakose G, Beatini N,
Grabowski GA, Maxfield FR, Tabas I. Unique cellular events occurring
during the initial interaction of macrophages with matrix-retained or
1436
25.
26.
27.
28.
29.
30.
31.
Downloaded from http://atvb.ahajournals.org/ by guest on April 29, 2017
32.
33.
34.
35.
Arterioscler Thromb Vasc Biol.
August 2003
methylated aggregated low density lipoprotein (LDL): prolonged cellsurface contact during which LDL-cholesteryl ester hydrolysis exceeds
LDL protein degradation. J Biol Chem. 1999;274:32112–32121.
Deleted in proof.
Deleted in proof.
Imort M, Zuhlsdorf M, Feige U, Hasilik A, von Figura K. Biosynthesis
and transport of lysosomal enzymes in human monocytes and macrophages: effects of ammonium chloride, zymosan and tunicamycin.
Biochem J. 1983;214:671– 678.
Heinrich M, Wickel M, Schneider-Brachert W, Sandberg C, Gahr J,
Schwandner R, Weber T, Saftig P, Peters C, Brunner J, Kronke M,
Schutze S. Cathepsin D targeted by acid sphingomyelinase-derived
ceramide. EMBO J. 1999;18:5252–5263.
Sando GN, Rosenbaum LM. Human lysosomal acid lipase/cholesteryl
ester hydrolase: purification and properties of the form secreted by fibroblasts in microcarrier culture. J Biol Chem. 1985;260:15186 –15193.
Mietus-Snyder M, Gowri MS, Pitas RE. Class A scavenger receptor
up-regulation in smooth muscle cells by oxidized low density lipoprotein:
enhancement by calcium flux and concurrent cyclooxygenase-2
up-regulation. J Biol Chem. 2000;275:17661–17670.
Zingg JM, Ricciarelli R, Andorno E, Azzi A. Novel 5⬘ exon of scavenger
receptor CD36 is expressed in cultured human vascular smooth muscle
cells and atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2002;
22:412– 417.
Marathe S, Kuriakose G, Williams KJ, Tabas I. Sphingomyelinase, an
enzyme implicated in atherogenesis, is present in atherosclerotic lesions
and binds to specific components of the subendothelial extracellular
matrix. Arterioscler Thromb Vasc Biol. 1999;19:2648 –2658.
Punturieri A, Filippov S, Allen E, Caras I, Murray R, Reddy V, Weiss SJ.
Regulation of elastinolytic cysteine proteinase activity in normal and
cathepsin K-deficient human macrophages. J Exp Med. 2000;192:
789 –799.
Hasilik A. The early and late processing of lysosomal enzymes: proteolysis and compartmentation. Experientia. 1992;48:130 –151.
Davies P, Allison AC, Ackerman J, Butterfield A, Williams S. Asbestos
induces selective release of lysosomal enzymes from mononuclear
phagocytes. Nature. 1974;251:423– 425.
36. Davies P, Page RC, Allison AC. Changes in cellular enzyme levels and
extracellular release of lysosomal acid hydrolases in macrophages
exposed to group A streptococcal cell wall substance. J Exp Med. 1974;
139:1262–1282.
37. Dean RT, Hylton W, Allison AC. Lysosomal enzyme secretion by macrophages during intracellular storage of particles. Biochim Biophys Acta.
1979;584:57– 65.
38. Cardella CJ, Davies P, Allison AC. Immune complexes induce selective
release of lysosomal hydrolases from macrophages. Nature. 1974;247:
46 – 48.
39. Cheson BD, Morris SE. The role of complement and IgG on zymosan
opsonization. Int Arch Allergy Appl Immunol. 1981;66:48 –54.
40. Ylä-Herttuala S, Palinski W, Butler SW, Picard S, Steinberg D, Witztum
JL. Rabbit and human atherosclerotic lesions contain IgG that recognizes
epitopes of oxidized LDL. Arterioscler Thromb. 1994;14:32– 40.
41. Seifert PS, Hansson GK. Complement receptors and regulatory proteins
in human atherosclerotic lesions. Arteriosclerosis. 1989;9:802– 811.
42. Leake DS. Does an acidic pH explain why low density lipoprotein is
oxidised in atherosclerotic lesions? Atherosclerosis. 1997;129:149 –157.
43. Newsholme P, Gordon S, Newsholme EA. Rates of utilization and fates
of glucose, glutamine, pyruvate, fatty acids and ketone bodies by mouse
macrophages. Biochem J. 1987;242:631– 636.
44. Loike JD, Kaback E, Silverstein SC, Steinberg TH. Lactate transport in
macrophages. J Immunol. 1993;150:1951–1958.
45. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999;
340:115–126.
46. Levin M, Evaldsson M, Wiklund O, Bondjers G, Björnheden T. Evidence
of ATP and glucose depleted areas within the atherosclerotic plaque in
vivo. Atherosclerosis. 2000;151:55. Abstract.
47. Hajjar DP, Farber IC, Smith SC. Oxygen tension within the arterial wall:
relationship to altered bioenergetic metabolism and lipid accumulation.
Arch Biochem Biophys. 1988;262:375–380.
48. Björnheden T, Levin M, Evaldsson M, Wiklund O. Evidence of hypoxic
areas within the arterial wall in vivo. Arterioscler Thromb Vasc Biol.
1999;19:870 – 876.
49. Barger AC, Beeuwkes R, Lainey LL, Silverman KJ. Hypothesis: vasa
vasorum and neovascularization of human coronary arteries. A possible
role in the pathophysiology of atherosclerosis. N Engl J Med. 1984;310:
175–177.
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Lysosomal Enzymes Are Released From Cultured Human Macrophages, Hydrolyze LDL
In Vitro, and Are Present Extracellularly in Human Atherosclerotic Lesions
Jukka K. Hakala, Riina Oksjoki, Petri Laine, Hong Du, Gregory A. Grabowski, Petri T.
Kovanen and Markku O. Pentikäinen
Arterioscler Thromb Vasc Biol. 2003;23:1430-1436; originally published online May 15, 2003;
doi: 10.1161/01.ATV.0000077207.49221.06
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