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Circulation Research. 1995;77:1136-1142

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(Circulation Research. 1995;77:1136-1142.)
© 1995 American Heart Association, Inc.

Articles

Binding and Phagocytosis of Apoptotic Vascular Smooth Muscle Cells Is Mediated in Part by Exposure of Phosphatidylserine

M.R. Bennett, D.F. Gibson, S.M. Schwartz, J.F. Tait

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.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {alpha}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.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Recombinant annexin V was expressed in Escherichia coli and purified by slight modifications of previous methods16 ; the final purity was {approx}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 {approx}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 {alpha}-smooth muscle actin (monoclonal anti–smooth muscle {alpha}-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, {approx}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 {approx}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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 1Down) 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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  		Figure 1. Electron microscopic appearance of apoptotic VSMCs in culture. A, An apoptotic body binding to the surface of a VSMC of normal morphology. This apoptotic body is in the process of breaking down, as shown by the loss of membrane continuity (magnification x10 000). B, An apoptotic body being engulfed by a cell of normal morphology (magnification x10 000).

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 {approx}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 {approx}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 1Down). 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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  		Table 1. Annexin V Binding Sites in Cells in 10% or 0% FCS

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 2Down). After 2 hours of incubation with apoptotic bodies from VSM-myc cells, normal VSMCs bound {approx}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 3Down). However, no effect was observed when control anionic liposomes containing an equimolar amount of PI were used in place of PS (Fig 3Down), demonstrating that this effect was specific to the PS head group.



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  		Figure 2. Apoptotic bodies derived from VSM-myc cells (dark blue spheres) bound to a near-confluent monolayer of normal VSMCs (spindle-shaped cells).



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  		Figure 3. Effect of liposomes containing PS/PC or control liposomes containing PI/PC on the number of bound/phagocytosed apoptotic bodies. Liposomes were added to the apoptotic bodies 30 minutes before addition to the normal cell monolayer. Phospholipid concentrations are given as concentration of monomer. Values given are means, and error bars represent SEM. *P<.05, **P<.01 vs PI liposome treatment (n=3).

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 4Down, 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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  		Figure 4. Effect of addition of annexin V on the number of bound/phagocytosed apoptotic bodies in the absence or presence of 5 mmol/L EDTA. Values given are means, and error bars represent SEM. **P<.01 vs no addition of annexin V (n=3).

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 1Up) or present intracellularly.4 Using 106 bodies per monolayer, we found that at 2 hours after incubation, {approx}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 {approx}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 2Down 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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  		Table 2. Number of Bound/Phagocytosed Apoptotic Bodies After Apoptosis In Situ

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 5BDown shows that fresh erythrocytes bound at very low levels to VSMCs; preserved erythrocytes showed {approx}10-fold higher binding (Fig 5ADown, Table 3Down). The uptake of preserved erythrocytes was similar to the uptake of apoptotic VSMCs, ie, an average of {approx}2.5 particles bound per VSMC (compare with Fig 2Up and Table 3Down). 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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  		Figure 5. Erythrocytes bound to a VSMC monolayer. A, Preserved erythrocytes. B, Fresh erythrocytes.


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  		Table 3. Erythrocyte Binding and Annexin V Binding Sites of Fresh and Preserved Cells


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
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 1Up). 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.


*    Selected Abbreviations and Acronyms
 
BrdU = bromodeoxyuridine
PC = phosphatidylcholine
PI = phosphatidylinositol
PS = phosphatidylserine
VSM-E1A cell = VSMC infected with E1A
VSM-myc cell = VSMC infected with c-myc
VSM-vector = VSMC infected with retrovirus vector alone
VSMC = vascular smooth muscle cell


*    Acknowledgments
 
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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Araki S, Shimada Y, Kaji K, Hayashi H. Apoptosis of vascular endothelial cells by fibroblast growth factor deprivation. Biochem Biophys Res Commun. 1990;168:1194-1200. [Medline] [Order article via Infotrieve]
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