© 1995 American Heart Association, Inc.
Articles |
Binding and Phagocytosis of Apoptotic Vascular Smooth Muscle Cells Is Mediated in Part by Exposure of Phosphatidylserine
From the Department of Pathology (M.R.B., S.M.S.) and the Department of Laboratory Medicine (D.F.G., J.F.T.), University of Washington, Seattle.
Correspondence to Dr M.R. Bennett, Unit of Cardiovascular Medicine, University of Cambridge School of Clinical Medicine, Department of Medicine, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK. E-mail mrb@mole.bio.cam.ac.uk.
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Abstract |
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Abstract Apoptosis of vascular smooth muscle cells has recently been demonstrated to occur in vitro and in vivo. Uptake of apoptotic cells into adjacent normal cells appears to be rapid and specific. We have investigated binding and phagocytosis of apoptotic vascular smooth muscle cells by normal smooth muscle cell monolayers. Vascular smooth muscle cells were infected with the proto-oncogene c-myc or the adenovirus E1A gene, induced to undergo apoptosis in low-serum conditions, and then incubated with normal smooth muscle cells. Apoptosis was accompanied by a marked increase in exposure of phosphatidylserine on the outer surface of the cell, which was recognized by binding to annexin V. Liposomes containing phosphatidylserine but not phosphatidylinositol inhibited uptake of apoptotic cells in a dose-dependent manner to a maximum of 50% inhibition; annexin V also inhibited the uptake of apoptotic cells in a dose-dependent and calcium-dependent manner. Binding of apoptotic bodies did not appear to be mediated by endogenous annexin V, as evidenced by the inability of an antibody to annexin V to inhibit uptake. Smooth muscle cells were also able to recognize exposed phosphatidylserine on other cell types, as judged by their ability to bind erythrocytes having a high degree of exposed phosphatidylserine. We conclude that smooth muscle cells express phosphatidylserine during apoptosis, and this exposure partly mediates binding and phagocytosis of dead cells. This mechanism may be important in promoting rapid cell removal in the vessel wall.
Key Words: apoptosis • phosphatidylserine • vascular smooth muscle • annexin V • phagocytosis
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis, or programmed cell death, is a physiological mechanism of deleting cells from a wide variety of tissues. In particular, apoptosis plays an important role in embryogenesis, morphogenesis, normal cell turnover, and resolution of hyperplasia and neoplasia. In cells of the vessel wall, both VSMCs and endothelial cells undergo apoptosis in culture upon removal of serum survival cytokines, specifically insulin-like growth factor-1 and fibroblast growth factor, respectively.1 2 3 4 Although apoptosis can be demonstrated in normal vessel wall cells from both animals and humans, apoptosis is seen only at low rates in normal human vessels (References 5 and 65 6 and M.R. Bennett, S.M. Schwartz, unpublished data, 1995) Even in vessels that show physiological remodeling due to changes in blood flow after birth, where apoptotic death rates are of the order of 3% to 6% of cells, apoptosis is hard to demonstrate directly.7 This may be because the process occurs rapidly, typically lasting 2 to 4 hours in vitro, and cells are rapidly phagocytosed.3 4 Indeed, recognition and uptake of dying cells is an important component of the apoptotic process, preventing the release of toxic intracellular contents and the formation of an inflammatory infiltrate. The recent demonstration of high levels of apoptotic cells in atherosclerosis5 6 may thus reflect a failure of this uptake mechanism, which could contribute to the accumulation of inflammatory cells seen in this lesion.
The mechanisms by which apoptotic cells are recognized and
phagocytosed are incompletely understood and appear to vary according
to the cell type. The phagocytosis of cells before their lysis
indicates that some surface change occurs in the dying cell that can be
recognized by phagocytes. In some cell types (eg, thymocytes induced to
die by dexamethasone or hepatocytes), cells may
be recognized by means of specific carbohydrates on the surface of the
cell.8 9 10 In contrast, macrophage recognition of
peripheral blood lymphocytes11 or
thymocytes12 or recognition of apoptotic
neutrophils by fibroblasts13 occurs by means of the
macrophage 
vß3 integrin receptor.
More recently, apoptotic cells have been shown in some
instances to expose PS on the external leaflet of their plasma
membrane.14 15 This PS, which is normally sequestered in
the internal leaflet of the plasma membrane, may be recognized by
macrophages.12 Although apoptotic cells
can be phagocytosed by macrophages, they can also be ingested
by cells of the same tissue of origin. Indeed, we have recently shown
that VSMCs rapidly ingest apoptotic VSMCs in
culture.3 4 Therefore, we have investigated the mechanism
by which apoptotic VSMCs are recognized and phagocytosed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant annexin V was expressed in Escherichia coli and purified by slight modifications of previous methods16 ; the final purity was
99%, and the protein
was functionally identical to native annexin V in its ability to bind
to phospholipids. Annexin V was labeled with 125I to a
specific activity of 2000 cpm/ng as previously
described.17 Phospholipid vesicles containing 20 mol%
diheptanoyl-PC, 60 mol% 1-palmitoyl-2-oleoyl-PC, and 20 mol% of
either bovine brain PS or bovine liver PI were prepared in a buffer
consisting of 50 mmol/L HEPES sodium, pH 7.4, and 100 mmol/L NaCl at
concentrations of 6 mmol/L (expressed as monomer), as previously
described18 ; all phospholipids were obtained from Avanti
Polar Lipids. Fresh human blood was obtained from normal volunteers by
venipuncture into EDTA anticoagulant; preserved whole blood
(4CPlus Normal Control) was from the Coulter Corp. The polyclonal
antiserum to placental annexin V was raised in rabbits, and the IgG
fraction was isolated. The IgG fraction was kept as a stock solution at
12.9 mg/mL in (mmol/L) Tris-HCl 50, pH 8.0, NaCl 100, and EDTA 1.
Cell Culture
VSMCs were isolated from thoracic aortic explants of
6-week-old Sprague-Dawley rats. Cells were cultured in
Waymouth medium containing 10% FCS (GIBCO) and 20 mmol/L HEPES (Flow)
and equilibrated with 95%/5% CO2. Subconfluent cells were
passaged by trypsinization in 0.05% trypsin in PBS and reseeded in
Waymouth's plus 10% FCS (normal culture medium). VSMCs were
identified by their typical hill-and-valley morphology in
culture and their characteristic immunocytochemical staining for
-smooth muscle actin (monoclonal anti–smooth muscle
-actin antibody, Sigma Chemical Co). Cells at passage 5 were
used for experiments and retrovirus infections.
Production of Retrovirus-Infected Cell Lines
The retrovirus constructs used to create VSMC cell lines
constitutively expressing c-myc or E1A were based on the
pDORneo retrovirus vectors.19 In these retrovirus vectors,
expression of the gene of interest is driven by the Moloney murine
leukemia virus long terminal repeat and expression of the neomycin
resistance gene selectable marker from the Simian virus 40 early
promoter. The c-myc construct encoded full-length human
c-myc, and the adenovirus E1A sequence encoded the 12S
subunit. VSMC cell lines constitutively expressing c-myc or
E1A were produced as previously described3 20 and
designated VSM-myc or VSM-E1A cells, respectively.
Detection of PS on Apoptotic Cells by Annexin V
Binding
VSM-myc or VSM-E1A cells were cultured in medium containing 10%
FCS. After 24 hours, 107 cells were washed three times
in PBS and cultured in medium containing 0% FCS. After a further 24
hours, cells in 10% FCS and 0% FCS were trypsinized, and both
monolayers and any cells in the culture supernatant were
centrifuged at 1000 rpm for 5 minutes. The cell pellet was
washed three times in PBS and then resuspended in PBS at 3 to
14x106 cells per milliliter. The binding assay was
performed as previously described17 ; 20 to 30 µL of cell
suspension was diluted to a final volume of 250 µL in a buffer
consisting of (mmol/L) HEPES sodium 10, pH 7.4, NaCl 136, KCl 2.7,
MgCl2 2, NaH2PO4 1, and glucose 5,
along with 5 mg/mL BSA, and incubated for 15 minutes at 37°C with 100
nmol/L [125I]annexin V and 2.5 mmol/L CaCl2.
Annexin V at 100 nmol/L is a near-saturating concentration, which
allows a single point measurement of PS exposure. Bound and free ligand
were separated by centrifugation (3 minutes at
7300g) through a barrier consisting of a mixture of silicone
oils of two different densities (DC550/DC200, 85.3%/14.7% [wt/wt];
obtained from William F. Nye). The number of annexin V molecules bound
to cells in the pellet was then determined from the radioactivity in
the cell pellet and the known specific radioactivity of the labeled
protein. Assays were performed in triplicate; nonspecific binding was
measured in the presence of 5 mmol/L EDTA and was always <1% of total
binding.
DNA Assay
Because of the difficulty of counting apoptotic cell
suspensions accurately, we used DNA content as a more reliable
indicator of cell number in the binding experiments. During binding
experiments, aliquots of cells were processed in parallel through the
oil-separation step. The cell pellets were then suspended in 2 mL
of 0.05 mol/L Na2HPO4, pH 7.4, and 2
mol/L NaCl, sonicated briefly to disrupt the cells and liberate the
DNA, and then analyzed fluorometrically for DNA content in
triplicate with fluorescent dye Hoechst 33258 according to
Labarca and Paigen.21 Calf thymus DNA, obtained from Sigma
and quantified by absorbance at 260 nm, was used as the standard.
Phagocytosis Assay
Phagocytosis of apoptotic VSMCs was assessed by using a
modification of previously described assays.12 14 22
VSM-myc cells were cultured in medium containing 10% FCS in the
continual presence of 10 µmol/L BrdU over a number of passages. This
concentration of BrdU did not affect proliferation or induce death of
VSMCs (data not shown). Approximately 107 cells were then
washed three times in PBS and transferred to medium containing 0% FCS.
After 24 hours, there was substantial cell death in the culture, with
apoptotic bodies present in the culture supernatant. These
apoptotic bodies were centrifuged at 1000 rpm for 5
minutes and then layered onto near-confluent monolayers of normal
rat VSMCs in four-well Tissue Tek (Nunc) chamber slides at
106 bodies per well. For experiments using the
addition of liposomes or annexin V, these agents were added to the
apoptotic bodies 30 minutes before addition of the
apoptotic bodies to the normal VSMCs. After 2 hours, cells were
washed three times with PBS and fixed in 4%
paraformaldehyde for 15 minutes. Cells were then
processed for immunocytochemistry, as previously
described,3 using a mouse monoclonal anti-BrdU antibody at
a dilution of 1 in 500 (Sera Lab) and a peroxidase-conjugated
anti-mouse secondary antibody at a dilution of 1 in 200 and a
peroxidase detection system (Vector Laboratories). Cell monolayers were
then counterstained with methyl green. The number of apoptotic
bodies present per 100 normal VSMCs was measured by an observer
blind to treatment conditions. All measurements were performed in
duplicate, and the results presented are a minimum of
triplicate experiments. For experiments using red blood cells,
106 cells were layered onto normal rat VSMCs, and
the assay was performed as described above.
Electron Microscopy
Preparation of smooth muscle cell cultures for examination by
electron microscopy was as described before4 at 2 and 24
hours after layering of the apoptotic bodies onto the VSMC
monolayer.
Statistical Analysis
The means of the number of apoptotic bodies per 100
normal VSMCs were analyzed by ANOVA for multiple comparisons.
Paired analysis between two groups (eg, between cells treated
with inhibitors of phagocytosis) was performed by using
Student's t test where ANOVA indicated significance for the
multiple comparison.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have assessed the ability of rat VSMCs to recognize and phagocytose apoptotic VSMCs under defined culture conditions. We have produced rat VSMCs that undergo rapid and reproducible apoptosis upon removal of serum survival factors by stable expression of the proto-oncogene c-myc or the adenovirus E1A gene after retrovirus-mediated gene transfer. We have previously shown that these VSM-myc or VSM-E1A cells undergo apoptosis upon transfer to low-serum culture conditions, identified by characteristic morphology on time-lapse videomicroscopy and electron microscopy (Fig 1
) and DNA fragmentation
patterns.3 20 Apoptotic bodies generated from
VSMCs were used to identify the mechanisms by which normal VSMCs
recognize and phagocytose apoptotic VSMCs.
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Exposure of PS on the Surface of Apoptotic
VSMCs
Binding measurements with radiolabeled annexin V, a
calcium-dependent phospholipid-binding protein, were used to
assess PS exposure. We have previously shown that this assay can detect
changes in PS exposure in erythrocytes and
platelets,17 23 24 and more recently, annexin V
binding has been used to identify apoptotic lymphocytes and
neutrophils.15 25 Rat VSMCs containing a transfected
c-myc or E1A gene were induced to undergo apoptosis
by removal of serum survival factors. Apoptosis was rapid, and
40% to 50% of the cells had died by 24 hours, similar to previous
observations made when using these cell lines20 ; in
contrast, there was <2% apoptosis in control cells
(VSM-vector cells) cultured in 0% FCS. Annexin V binding was
10-fold higher for serum-starved VSM-myc cells and 3-fold higher
for serum-starved VSM-E1A cells than for comparable cells in 0%
FCS (Table 1). VSM-vector cells showed
minimal increase in annexin V binding when transferred to low-serum
conditions, confirming that the increase in annexin V binding occurring
in VSM-myc or VSM-E1A cells was not due to transfer to low serum per
se. Because apoptosis was associated with fragmentation of
cells into multiple apoptotic bodies, the actual number of
cells present at the end of the 24-hour period was impossible to
assay. Values given are therefore normalized to the amount of DNA
present, because this gives a more reliable indication of the
number of cells present.
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Effect of PS Exposure on Binding/Phagocytosis of
VSMCs
We have previously shown by time-lapse videomicroscopy that
phagocytosis of apoptotic VSMCs is a rapid
process.3 In the present study, we demonstrate that
within 2 hours of incubation of apoptotic bodies with a normal
VSMC monolayer, bodies are visibly being engulfed by the normal VSMCs.
To determine whether PS exposure can promote binding/phagocytosis of
apoptotic VSMCs, as it does for other cell types, we assayed
binding/phagocytosis of dead VSMCs by normal (untransfected) VSMCs.
VSM-myc cells were induced to die by transfer to low-serum
conditions for 24 hours. Apoptotic bodies were collected from
the culture supernatant and layered onto the normal VSMC monolayer (Fig 2). After 2 hours of incubation with
apoptotic bodies from VSM-myc cells, normal VSMCs bound 230
bodies per 100 cells. Prior incubation of the bodies with PS-containing
liposomes inhibited binding/phagocytosis of VSMCs in a
dose-dependent fashion (Fig 3).
However, no effect was observed when control anionic liposomes
containing an equimolar amount of PI were used in place of PS (Fig 3), demonstrating that this effect was specific to the
PS head group.
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Effect of Annexin V on Binding/Phagocytosis of VSMCs
If exposure of PS is required for binding and/or phagocytosis of
apoptotic bodies, covering the PS with annexin V should prevent
its recognition by the phagocytic cell and thus block binding of the
apoptotic bodies. Recombinant annexin V was incubated with the
apoptotic bodies 30 minutes before their addition to the normal
VSMC monolayer. As shown in Fig 4, annexin V inhibited
the binding/uptake of apoptotic bodies by VSMCs in a
dose-dependent manner; this effect could be abolished by chelation
of calcium by prior incubation with 5 mmol/L EDTA. Since binding of
annexin V to phospholipids is strictly calcium dependent, it is likely
that annexin-PS binding was mediating this inhibitory
effect. Because it has also been suggested recently that annexin V may
be involved in apoptosis of endothelial
cells,26 we also investigated whether exogenous annexin V
would promote apoptosis of VSMCs. However, addition of annexin
V to VSM-myc cells or VSM-E1A cells in a concentration range from 5
nmol/L to 5 µmol/L had no effect on apoptosis of these cells
(data not shown).
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In an attempt to discriminate between apoptotic bodies that
were just bound to the surface of cells in the monolayer and bodies
that were actually being internalized, we screened assay slides by
electron microscopy for evidence of bodies being engulfed (Fig 1
) or
present intracellularly.4 Using 106
bodies per monolayer, we found that at 2 hours after incubation, 5%
of bound bodies (VSM-myc cells) showed evidence of being engulfed or
were fully internalized (300 bodies per slide analyzed).
Although electron microscopy is too unwieldy to allow extensive
quantification of numbers of bodies being internalized with each
treatment, prior incubation with 1 µmol/L PC/PS liposomes (but not
PC/PI liposomes) or 5 µmol/L annexin V, inhibited internalization by
50% in each case (not shown). In addition, to assess whether PC/PS
liposomes or annexin V inhibited binding/phagocytosis of
apoptotic bodies when death occurred in situ, we cocultured
VSM-myc cells with normal smooth muscle cells in a 1:1 ratio for 24
hours until the monolayer was near confluent. The cells were then
placed in 0% FCS for a further 26 hours, in the presence or absence of
1 µmol/L PC/PS or PI liposomes or 5 µmol/L annexin V, and the
number of bound/phagocytosed apoptotic bodies was assessed.
These data are shown in Table 2 and indicate that
coincubation with PC/PS liposomes or annexin V, but not PC/PI
liposomes, inhibited binding/phagocytosis of apoptotic bodies
in situ.
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Effect of Annexin V Antibody on Binding/Phagocytosis of
Apoptotic Bodies
The results outlined above suggest that endogenous
annexin V may play a role in mediating binding/phagocytosis of
apoptotic cells. To investigate this possibility, we incubated
apoptotic bodies with the IgG portion of a rabbit polyclonal
antibody to placental annexin V. This antibody was shown to block the
binding of 10 nmol/L [125I]annexin V to human
erythrocytes in a dose-dependent manner (50% inhibition of binding
at 130 µg/mL IgG). Prior incubation of apoptotic bodies
derived from VSM-myc cells with this antibody did not affect
binding/phagocytosis of apoptotic bodies, up to a concentration
of 500 µg/mL of IgG (not shown).
Binding of Erythrocytes to VSMC Monolayers
We have previously demonstrated that PS exposure in erythrocytes
can be measured with annexin V binding; the level of PS exposure is
very low in fresh normal blood samples but is considerably higher in a
commercially available preparation of stabilized preserved blood or in
stored erythrocytes.17 We hypothesized that if PS exposure
was involved in binding or phagocytosis of dead cells by VSMCs,
phagocytosis should be demonstrable at low levels with normal
erythrocytes and at higher levels with preserved cells. Fresh blood
from healthy volunteers or preserved blood was incubated with a VSMC
monolayer for 2 hours, and bound erythrocytes were counted. Fig 5B shows that fresh erythrocytes bound at very low
levels to VSMCs; preserved erythrocytes showed 10-fold higher
binding (Fig 5A, Table 3). The uptake
of preserved erythrocytes was similar to the uptake of
apoptotic VSMCs, ie, an average of 2.5 particles bound per
VSMC (compare with Fig 2 and Table 3). This suggests that although
erythrocytes are significantly smaller than apoptotic bodies
derived from smooth muscle cells, with a correspondingly smaller
surface area, these two cell types may have very similar local
concentrations of PS on the surface of the cell membrane.
Interestingly, although erythrocytes were bound to the surface of
VSMCs, on electron microscopy we could not demonstrate internalization
of these cells, in contrast to apoptotic VSMCs; this may
indicate that additional cell-surface elements, in addition
to PS exposure, are needed to promote phagocytosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis has been proposed to be a physiological process whereby the mass of VSMCs in the arterial wall is controlled.7 In particular, the process is characterized by its rapidity and the rapid uptake of dead cells into adjacent normal VSMCs.3 20 The apoptotic bodies produced generally have an intact cell membrane upon phagocytosis.3 20 However, if failure of uptake occurs, there is eventual breakdown of membrane integrity, with release of intracellular contents (M.R. Bennett, S.M. Schwartz, unpublished data, 1995, and Fig 1
). The release of intracellular
proteases, growth factors, toxic cationic proteins, and oxidizing
molecules, if it were to occur in the arterial wall, could
provoke inflammation, both by direct toxicity and by the generation of
chemotactic factors and mitogens from extracellular matrix
molecules.27 The resulting inflammation could, in turn,
hinder processes such as arterial remodeling. Uptake of
apoptotic VSMCs must therefore be rapid and specific. We have
investigated the mechanisms by which dead VSMCs are phagocytosed by
otherwise normal VSMCs. We have used the binding of apoptotic cells to annexin V to assess exposure of membrane phospholipid PS. We are confident that this assay measures PS exposure on the surface of apoptotic cells for a number of reasons. First, so far, only PS has been identified as a ligand for annexin V with the nanomolar binding affinity that is required to register in this assay. Second, there is fairly extensive evidence from our earlier studies on the binding of annexin V to platelets, erythrocytes, and ovarian carcinoma cells that the ligand of annexin V is PS, and the [125I]annexin V binding assay correlates well with other methods of assessing PS binding, specifically with PS-dependent prothrombinase activity.17 23 28 Third, binding of annexin V to apoptotic bodies was always reversible upon chelation of Ca2+, suggesting that annexin V was not getting trapped within cells. Finally, our assay depends upon centrifugation of cells or apoptotic bodies through an oil barrier. If annexin V was binding only to random membrane fragments, these are not dense enough to be registered on the assay.
We find that VSMCs undergoing apoptosis have increased exposure of PS on their surface. Cells undergoing apoptosis induced by two different gene products, c-myc and the adenovirus gene product E1A, both show increased PS exposure, as assessed by the number of annexin V binding sites per picogram DNA. Because apoptotic bodies are much smaller than their parent cells, with little loss of DNA, the surface area of such cells is small relative to their DNA content. Thus, the PS exposure we measure is actually an underestimate of the PS exposure per unit surface area of membrane. Although there were quantitative differences between annexin V binding sites in VSM-myc cells versus VSM-E1A cells, these differences can be partly explained by more pronounced apoptosis in the particular clone of VSM-myc cells used (51% of cells died versus 39% of the VSM-E1A cells in 24 hours).
PS exposure appears to be a requirement for recognition and uptake of apoptotic VSMCs, as apoptotic body binding/phagocytosis can be partially inhibited by prior incubation of bodies with liposomes containing PS. This recognition of apoptotic VSMCs is not due to nonspecific factors, such as a change in surface negative charge, or hydrophobic effects, because control anionic liposomes lacking PS did not inhibit. Furthermore, calcium-dependent blockade of PS with added annexin V could suppress phagocytosis, suggesting that recognition of apoptotic cells is mediated by PS receptors on the surface of the normal VSMCs. Our data are consistent with earlier studies demonstrating that macrophages can recognize apoptotic cells by PS exposure and that recognition is stereospecific and not inhibited by other anionic phospholipids.14
Although the phagocytosis assay does not discriminate between the binding of apoptotic cells and internalization, assessment by electron microscopy indicates that internalization is also suppressed by PS-containing liposomes, or exogenous annexin V. In fact, the low level of intracellular apoptotic bodies seen at 2 hours by electron microscopy suggests that internalization may be the rate-limiting step for phagocytosis rather than binding to the cell surface. However, although binding of apoptotic bodies may be a prerequisite for internalization and PS exposure is involved in binding of bodies to VSMCs, we present evidence that PS exposure may not in itself be sufficient to allow phagocytosis of nonapoptotic cells to occur. Erythrocytes with a high level of PS exposure were efficiently bound to VSMCs but were not seen to be internalized. In contrast, apoptotic VSMCs were rapidly phagocytosed, appearing as intracellular apoptotic bodies, in some cases within 2 hours of incubation. Thus, binding to PS receptors may be a requirement for internalization but may not be sufficient per se for it to occur. This is consistent with recent studies indicating that PS exposure mediates binding of erythrocytes to macrophages but was insufficient for internalization.29 However, we have not excluded the possibility that binding and phagocytosis of apoptotic VSMCs can also be mediated by other pathways. In fact, we were unable to completely inhibit binding of apoptotic VSMCs, even at high concentrations of PS-containing liposomes. This hypothesis is consistent with studies indicating that phagocytic uptake of apoptotic cells of a specific lineage can be mediated by at least two separate pathways.12 13 Along these lines, we have assessed the role of signaling via ß3 integrin, which has been implicated in mediating phagocytosis of other cell types.11 12 13 In preliminary studies, we could not demonstrate any effect of a rat polyclonal ß3 antibody on binding/phagocytosis of apoptotic smooth muscle cells.
We provide evidence that VSMCs possess PS receptors by demonstrating that erythrocytes bind to VSMCs according to the number of PS sites that they possess. A PS receptor has been postulated to exist in earlier studies demonstrating that macrophages can bind erythrocytes depending upon the presence of PS, both in vitro and in the circulation, and that this binding is inhibited by PS-containing liposomes.30 31 Macrophage recognition of tumor cells is also dependent on the expression of PS in the outer membrane of the tumor cell line.32 Furthermore, uptake of liposomes by the reticuloendothelial system has been shown to be dependent on the presence of PS.33 The macrophage scavenger receptor may function in this role, because it can bind to several lipids,34 although whether it binds negatively charged lipids is more controversial.34 35 More recently, oxidized erythrocytes have been shown to be bound and phagocytosed by mouse macrophages via a scavenger receptor that is distinct from the acetyl low-density lipoprotein receptor.36 This receptor, which has also been shown to recognize oxidized low-density lipoprotein, has recently been shown to be a 94- to 97-kD protein in the macrophage membrane.29 37
The mechanism of exposure of PS in apoptotic cells has not yet been fully characterized. It is generally accepted that alterations in the fraction of PS in the outer leaflet of the plasma membrane reflect changes in the distribution of existing phospholipids rather than changes in the synthesis or degradation of PS itself.38 39 Membrane asymmetry of phospholipids is maintained by an aminophospholipid translocase, which transports PS, and to some extent by phosphatidylethanolamine, from the outer to the inner leaflet.40 A reduction in activity of the enzyme thus results in an increased PS level in the outer leaflet.41 42 This enzyme is ATP and magnesium dependent and is inhibited by calcium.40 43 Indeed, in erythrocytes and platelets, a rise in cytosolic calcium can induce a rapid scrambling of membrane phospholipids, which may be dependent in addition on the presence of phosphatidylinositol 4,5-diphosphate.44 45 46 Whether this enzyme is inhibited in apoptosis has not been determined, but in some cell types (eg, thymocytes), apoptosis is accompanied by an early sustained rise in intracellular calcium.47 Therefore, the possibility exists that a rise in intracellular calcium in apoptosis is associated with inhibition of the translocase and exposure of PS on the surface of the apoptotic cell.
In conclusion, we have demonstrated that VSMCs expose PS on the outer surface of their plasma membranes during apoptosis. This PS exposure appears to mediate, at least in part, binding and phagocytosis of the apoptotic cell by adjacent nonapoptotic VSMCs. This mechanism may be important in the rapid uptake of apoptotic cells in the normal arterial wall, and failure of this mechanism may contribute to the accumulation of apoptotic cells seen in atherosclerosis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL-18641 (Dr Schwartz) and HL-47151 (Dr Tait). Dr Bennett was supported by a British Heart Foundation Clinical Scientist Fellowship.
Received April 17, 1995; accepted August 28, 1995.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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