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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2648-2658

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2648.)
© 1999 American Heart Association, Inc.

Vascular Biology

Sphingomyelinase, an Enzyme Implicated in Atherogenesis, Is Present in Atherosclerotic Lesions and Binds to Specific Components of the Subendothelial Extracellular Matrix

Sudhir Marathe; George Kuriakose; Kevin Jon Williams; Ira Tabas

*    Abstract
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Abstract—Atherosclerotic lesions contain an extracellular sphingomyelinase (SMase) activity that hydrolyzes the sphingomyelin of subendothelial low density lipoprotein (LDL). This SMase activity may promote atherosclerosis by enhancing subendothelial LDL retention and aggregation, foam cell formation, and possibly other atherogenic processes. The results of recent cell-culture studies have led to the hypothesis that a specific molecule called secretory SMase (S-SMase) is responsible for the SMase activity known to be in lesions, although its presence in atheromata had not been examined directly. Herein we provide immunohistochemical and biochemical support for this hypothesis. First, 2 different antibodies against S-SMase detected extracellular immunoreactive protein in the intima of mouse, rabbit, and human atherosclerotic lesions. Much of this material in lesions appeared in association with the subendothelial matrix. Second, binding studies in vitro demonstrated that 125I-S-SMase adheres to the extracellular matrix of cultured aortic smooth muscle and endothelial cells, specifically to the laminin and collagen components. Third, in its bound state, S-SMase retains substantial enzymatic activity against lipoprotein substrates. Overall, these data support the hypothesis that S-SMase is an extracellular arterial wall SMase that contributes to the hydrolysis of the sphingomyelin of subendothelial LDL. S-SMase may therefore be an important participant in atherogenesis through local enzymatic effects that stimulate subendothelial retention and aggregation of atherogenic lipoproteins.


Key Words: sphingomyelinase • lipoproteins • atherosclerosis • extracellular matrix • collagen


*    Introduction
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 *Introduction
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Sphingomyelinase (SMase; sphingomyelin phosphodiesterase; EC 3.1.4.12) catalyzes the hydrolysis of sphingomyelin, a constituent of plasma lipoproteins and mammalian plasma membranes, to ceramide and choline phosphate.1 This enzymatic activity has been implicated in a myriad of physiological and pathophysiological pathways, including cell-signaling events that lead to cellular differentiation, apoptosis, and inflammatory responses2 3 4 as well as processes that are predicted to promote atherogenesis.5 6 7 8 9 10 11 12 13 Although several distinct mammalian SMase activities have been identified, only 2 classes of SMase have received much attention. One is an intracellular, neutral, magnesium-dependent SMase activity that is abundant in brain tissue.1 The cloning of an enzyme with neutral SMase activity has been reported,14 but this cloned enzyme exhibits several important differences from highly purified magnesium-dependent SMase from brain.15

The second class of mammalian SMases arises from the acid sphingomyelinase (acid SMase) gene.16 17 This gene gives rise to a single mRNA that encodes a single polypeptide that has 2 distinct fates during cellular trafficking: targeting to lysosomes or secretion.10 18 The lysosomal form (L-SMase) has high-mannose oligosaccharides and an N-terminus slightly foreshortened by a proteolytic clip; the secretory form (S-SMase) has complex sugars and an intact N'-terminus.18 Both forms are zinc-metalloenzymes, although most L-SMase becomes complexed with Zn2 inside the cell, whereas most S-SMase is secreted without Zn2 and thus requires physiological concentrations of Zn2+ in the extracellular space for enzymatic activity.18 While both forms of this SMase have an acid pH optimum, S-SMase has been shown to hydrolyze the sphingomyelin of certain atherogenic lipoproteins at neutral pH.11

There are several possible mechanisms whereby SMase may promote atherogenesis, including inducing subendothelial retention and aggregation of atherogenic lipoproteins,5 6 7 8 9 10 11 12 13 decreasing the efflux potential of HDL,19 -21 stimulating the esterification of cellular cholesterol,22 23 promoting apoptosis of arterial-wall cells,24 mediating oxidized LDL–induced proliferation of smooth muscle cells (SMCs),25 and promoting platelet aggregation.26 Note that most of these events would require an extracellular SMase. Because S-SMase is the only mammalian SMase identified to date that is extracellular, it is an attractive candidate to mediate these atherogenic effects.

Our laboratory has focused on LDL aggregation, which is a prominent event in the subendothelial space during atherogenesis,27 28 29 and on the potential role of S-SMase in mediating this aggregation.9 11 30 Arterial-wall macrophages loaded with large amounts of cholesteryl ester (macrophage foam cells) are thought to play a major role throughout atherogenesis.31 32 33 34 35 36 37 38 Studies with cultured macrophages have revealed that massive cholesteryl ester accumulation cannot simply be induced by many types of monomeric lipoproteins that are thought to be atherogenic, such as native LDL,39 certain forms of oxidized LDL,40 41 42 43 and lipoproteins from atherosclerotic apolipoprotein E–knockout (E0) mice44 (S.M. et al, unpublished data, 1999). The aggregated forms of these lipoproteins, however, lead to extensive foam cell formation7 8 45 46 47 48 (S.M. et al, unpublished data, 1999). Furthermore, aggregation itself may enhance the subendothelial retention of lipoproteins, which is a critical process in both the initiation and progression of atherosclerotic lesions.8 13 The mechanisms whereby lipoprotein aggregation leads to increased lipoprotein retention include size effects (ie, monomeric lipoproteins are small enough to enter the subendothelium but aggregated lipoproteins are too big to leave), mass effects (ie, aggregated material contains more lipid per retained particle than do monomeric forms), and enhanced affinity of aggregated lipoproteins for subendothelial proteoglycans.8 13

The evidence that SMase activity in general and S-SMase in particular is an important mediator of subendothelial LDL aggregation includes the following: (1) both bacterial SMase and S-SMase induce the formation of LDL aggregates that appear similar to those that occur in vivo and are capable of inducing foam cell formation7 11 12 30 ; (2) aggregated LDL isolated from human and animal lesions, but not unaggregated lesional LDL or plasma LDL, is enriched in ceramide,9 a nonexchangeable product of sphingomyelin hydrolysis; (3) LDL retained in strips of rabbit aorta ex vivo is hydrolyzed by a cation-dependent, extracellular SMase9 ; (4) certain atherogenic forms of lipoproteins, including human lesional LDL, are excellent substrates for S-SMase, even at neutral pH11 30 ; and (5) macrophages and especially endothelial cells are rich sources of S-SMase, and endothelium-derived S-SMase, which is partially active even in the absence of exogenous zinc, is further induced by exposure to cytokines known to be present in atherosclerotic lesions.10 12 Nevertheless, before the current study, the presence or absence of S-SMase from atherosclerotic lesions had not been directly examined.

Herein, using specific antibodies that recognize S-SMase, we show that there is abundant immunoreactive material in the intima of mouse, rabbit, and human atherosclerotic lesions, much of which appeared to be extracellular and associated with matrix. Matrix binding studies in vitro demonstrated substantial binding of 125I-S-SMase to the extracellular matrix (ECM) derived from SMC and endothelial cell, as well as to purified laminin and collagen, but not to arterial proteoglycans. These results lend further support to the proposal that S-SMase plays a role in the hydrolysis of subendothelial LDL sphingomyelin and possibly cellular sphingomyelin during atherogenesis.


*    Methods
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 *Methods
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Materials
The Falcon tissue-culture plastic ware used in these studies was purchased from Fisher Scientific Co. Tissue-culture media and other tissue-culture reagents were obtained from GIBCO BRL. FBS was obtained from Hyclone Laboratories and was heat-inactivated for 1 hour at 65°C. [9,10-3H]Palmitic acid (56 Ci/mmol) and Na125I (carrier free) were purchased from DuPont NEN. [N-Palmitoyl-9,10-3H]sphingomyelin was synthesized as previously described.10 49 50 (N,N)-Dimethylformamide; 1,3-dicyclohexylcarbodiimide; (N)-hydroxysuccinimide; and (N,N)-diisopropylethylamine were purchased from Aldrich Chemical Inc. Rabbit anti–S-SMase IgG and affinity-purified anti–S-SMase IgG were provided by Drs Henry Lichtenstein and G. Andrew Keesler (Amgen, Boulder, Colo), and goat anti-SMase antiserum was obtained from Dr Konrad Sandhoff (University of Bonn, Bonn, Germany). Peroxidase-conjugated goat anti-rabbit IgG and donkey anti-goat IgG were purchased from Pierce Chemical Co. S-SMase, which was purified to homogeneity by DEAE-Sepharose and concanavalin A chromatography of conditioned medium from SMase-transfected DG44 Chinese hamster ovary cells,10 was kindly provided by Dr Edward Schuchman (Mt. Sinai School of Medicine, New York, NY). LDL (density 1.020 to 1.063 g/mL) was isolated from fresh human plasma by preparative ultracentrifugation as described previously.51 Purified S-SMase and LDL were iodinated as follows. Solutions of S-SMase or LDL in 0.3 mol/L borate buffer, pH 9.0, were placed in Iodogen-coated tubes (Pierce), and 0.5 mCi of Na125I was added. The LDL plus VLDL fraction was isolated from the plasma of apolipoprotein E–knockout mice and labeled with [3H]sphingomyelin as described.11 Bovine plasma fibronectin, laminin from the basement membrane of Engelbreth-Holm-Swarm mouse sarcoma, collagen III from calf skin, and collagen VI from human placenta were purchased from Sigma Chemical Co. Purified collagen I (Vitrogen 100) was from Collagen Corporation. Heparin was obtained from Elkins-Sinn, Inc. Chondroitin sulfate proteoglycan and dermatan sulfate proteoglycan were prepared from cynomolgus monkey aorta52 and were generously supplied by Dr William D. Wagner (Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC). All other chemicals and reagents were from Sigma, and all organic solvents were from Fisher Scientific Co.

Cells
Bovine aortic endothelial cells (BAECs)53 and smooth muscle cells (BASMCs)8 were grown in Dulbecco’s modified Eagle’s medium containing 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine. To establish SMC-derived ECM, SMCs were grown in 3% serum for the first 5 days, and then the cells were maintained for 8 days in serum-free medium to arrest cellular proliferation and promote ECM proliferation. Unless indicated otherwise, the cells were plated in 24-well dishes until they were confluent. Cell culture medium was changed and fresh medium was added every 2 days.

Preparation of Histological Sections
Hearts from chow-fed wild-type mice (25% SV129/75% C57BL6 and purebred C57BL6 genetic backgrounds), acid SMase–knockout mice (25% SV129/75% C57BL6 genetic background),54 and 15- to 30-week-old chow-fed apolipoprotein E–knockout (E0) mice55 56 (pure C57BL6 genetic background) were perfused, embedded in OCT compound (Sakura Finetek), snap-frozen in an ethanol–dry ice bath, and stored at -70°C. New Zealand White male rabbits (2 to 3 kg) were fed either a standard rabbit chow diet (control) or cholesterol-enriched rabbit chow (2% cholesterol plus 10% soybean oil) diet for 4 to 8 weeks. The rabbits were then anesthetized with intravenous sodium pentobarbital (70 mg/kg), and the aortas were excised, embedded in OCT compound, and stored at -70°C. Segments of human atherosclerotic abdominal aorta from the Pathological Determinants of Atherosclerosis in Youth (PDAY) study,57 embedded in OCT compound, were obtained from Dr Gray T. Malcom (Department of Pathology, Louisiana State University Medical Center, New Orleans); the material came from a 34-year-old African-American female (ACC No. F53470).

SMase Immunohistochemistry of Murine, Rabbit, and Human Aortas
Multiple 8-µm-thick sections of murine, rabbit, and human aortas were cut on a cryostat, placed on poly-L-lysine–coated glass slides, and fixed in 10% buffered formalin for 5 minutes at room temperature. The sections were air-dried for 10 to 15 minutes, washed in PBS containing 0.1% Triton X-100 for 20 minutes, and rinsed in PBS for 5 minutes in PBS at room temperature. The sections were then preincubated with 2% normal serum in PBS for 1 hour at room temperature. Next, the sections were incubated with 2% donkey serum containing 10 to 30 µg/mL of rabbit anti-SMase IgG or a 1:500 dilution of goat anti-SMase antiserum for 2 to 6 hours at room temperature or for 16 hours at 4°C. In some control experiments, immunoaffinity-purified rabbit anti-SMase IgG was immunoabsorbed by S-SMase Affi-Gel chromatography. After the sections were washed in PBS for 5 minutes, the bound primary antibody was visualized using biotinylated secondary antibody, followed by streptavidin peroxidase (Vectastain Elite ABC peroxidase kit, Vector Laboratories Inc) and 3,3'-diaminobenzidine. The sections were counterstained with hematoxylin, rinsed, mounted in Permount, and viewed with an Olympus IX 70 inverted microscope with a 10x, 40x, or 100x objective.

Preparation of Plates With Whole ECM and Individual ECM Components
The ECMs of confluent BAECs and BASMCs in 16-mm wells were prepared by treating the cell monolayers with 0.5% Triton X-100 (3 minutes), followed by 25 mmol/L NH4OH in PBS (3 minutes) and then PBS (5 washes).58 59 In some cases, the exposed ECMs were treated for 18 hours with chondroitinase ABC (2.0 U/mL) or heparitinase I (4.0 U/mL) in a buffer containing 10 mmol/L HEPES, pH 7.4, 140 mmol/L NaCl, 10 µmol/L CaCl2, and 0.4% BSA (binding buffer). After the 18-hour incubation the enzymes were removed, and the wells were washed 3 times with binding buffer before proceeding with the binding studies. Wells of 16-mm tissue-culture dishes were coated with heparin, fibronectin, or laminin by incubation with the compounds (10 µg/mL in borate buffer, pH 10) for 16 hours at 37°C. The solutions were then removed and replaced with binding buffer before proceeding with the binding studies. Coating with dermatan sulfate proteoglycan or chondroitin sulfate proteoglycan was achieved by adding 1 mL of 10 µg/mL solutions of the compounds in PBS and then air-drying the dishes at 37°C. The wells were then washed as above. Wells were coated with collagen types I, III, and VI by adding 1 mL of the collagen solutions (10 µg/mL in PBS) for 60 minutes at 37°C and then leaving the plates uncovered in a laminar-flow hood overnight to allow drying.60 The collagen coatings were then hydrated in PBS for 1 hour before proceeding with the binding protocol.

ECM Binding Studies
The wells coated with ECM or ECM components were incubated with binding buffer for 1 hour at 37°C to saturate nonspecific binding sites and then incubated with 100 pmol (in 0.5 mL of binding buffer) of either 125I-S-SMase or 125I-LDL for 16 hours at 37°C. The wells were then washed 4 times with binding buffer without BSA, and the bound 125I-labeled material was dissolved in 0.2 mmol/L NaOH and counted in an LKB gamma counter.

Statistics
Unless otherwise indicated, results are given as mean±SD (n=3); absent error bars in figures indicate SD values smaller than the symbols.


*    Results
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 *Results
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Immunohistochemical Staining of SMase in Murine, Rabbit, and Human Aortas
Formalin-fixed sections of proximal aortas from chow-fed control mice and E0 mice with various stages of atherosclerosis were incubated with goat polyclonal anti-SMase antiserum; all mice were in the C57BL/6J genetic background. The bound antibody was visualized using biotinylated secondary antibody, streptavidin peroxidase, and 3,3'-diaminobenzidine, which yielded a brown reaction product. As demonstrated previously,12 the aortas of control mice showed prominent SMase staining that was localized mostly to the endothelium (Figure 1BDown); very little SMase staining was in the media, while the adventitia (far right edge of Figure 1BDown) showed some diffuse staining. The staining was entirely specific, as demonstrated by the absence of brown material in a section from an acid SMase–knockout mouse (Figure 1ADown).



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  		Figure 1. SMase immunohistochemistry of normal and atherosclerotic mouse proximal aorta. Serial 8-µm sections of proximal aorta from an acid SMase–knockout mouse (A, x1000), a normolipidemic, nonatherosclerotic wild-type mouse (B, x1000), a 15-week-old, chow-fed E0 mouse (C and D, x400), and a 30-week-old chow-fed E0 mouse (E and F, x400) were either stained with the goat anti-SMase antiserum (A, B, D, and F) or incubated with secondary antibody alone (C and E). Similar results were obtained with the rabbit anti-SMase IgG (not shown). The arrows in panel D mark the accumulation of SMase staining in acellular areas. Lumen (L), endothelium (E), intima (I), and media (M) are marked in panels A, C, and E.

To investigate the presence of SMase in atherosclerotic aorta, multiple sections of proximal aortas from 15- and 30-week-old, chow-fed, E0 mice were studied. Aortic sections from a 15-week-old E0 mouse demonstrated a hypercellular, thickened intima (Figures 1CUp and 1DUp) that stained heavily with oil red O (data not shown). Figure 1DUp shows that SMase is present throughout the thickened intima, and Figure 1CUp is the corresponding control without primary antibody. A significant portion of the staining in Figure 1DUp (arrows) appeared to have accumulated in regions without any nuclei and most likely represents extracellular pools of SMase bound and retained on the subendothelial matrix. This is an important observation, because the antiserum used in these studies cannot distinguish L-SMase from S-SMase, and so the location of the enzyme (ie, extracellular versus intracellular) is the only way of determining whether S-SMase might be present. Aortic sections from a 30-week-old E0 mouse are shown in Figure 1EUp, the control without primary antibody, and 1F, stained with anti-S-SMase antiserum. The intimas of these sections were almost completely devoid of nuclei, and they contained extracellular lipid, cholesterol crystals, and fibrous tissue. The prominent SMase staining seen in Figure 1FUp is therefore mostly extracellular and appears associated with the intimal matrix. Whether the source of the extracellular SMase in this section is S-SMase and/or L-SMase released from necrotic cells38 cannot be determined from these data.

To further demonstrate the specificity of the anti-SMase antibody staining, a separate experiment was conducted in which 1 section was stained with rabbit immunoaffinity-purified anti-SMase IgG (Figure 2BDown), and the control section was stained with the same IgG adsorbed with purified S-SMase (Figure 2ADown). As discussed above, intimal staining was seen in the experimental section but not in the control section.



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  		Figure 2. Specific immunohistochemical staining with immunoaffinity-purified anti–S-SMase IgG. Serial sections of proximal aorta from a 15-week-old, chow-fed E0 mouse were either incubated with rabbit immunoaffinity-purified anti–S-SMase IgG (B, x100) or with the IgG preabsorbed with purified S-SMase (A, x100). Lumen (L), intima (I), and media (M) are marked in panel A.

We next analyzed the pattern of SMase staining in sections of aorta excised from chow-fed or cholesterol-fed rabbits. As observed with murine aortas (above), normal rabbit aortas showed abundant SMase staining in the endothelium (compare Figures 3ADown and 3BDown). Aortic sections from rabbits fed cholesterol for 4 weeks (Figures 3CDown and 3DDown) and for 8 weeks (Figures 3EDown and 3FDown) demonstrated a markedly thickened and hypercellular intima, and, as with mouse lesions, prominent intimal SMase staining was observed (Figures 3DDown and 3FDown). Careful inspection of Figure 3FDown shows some acellular areas that stained with SMase, thus indicating the presence of extracellular SMase.



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  		Figure 3. SMase immunohistochemistry of normal and atherosclerotic proximal rabbit aortas. Serial 8-µm sections of proximal aorta from a wild-type rabbit (A and B, x1000), from a rabbit fed a high-cholesterol diet 4 weeks (C and D, x400), and from a rabbit fed a high-cholesterol diet for 8 weeks (E and F, x400) were either stained with the goat anti-SMase antibody (B, D, and F) or with secondary antibody alone (A, C, and E). Similar results were obtained with the rabbit anti-SMase antibody (not shown). Lumen (L), endothelium (E), intima (I), and media (M) are marked in panels A, C, and E.

Figure 4Down shows 2 adjacent sections of an atherosclerotic abdominal aorta from a 34-year-old human female. As with murine and rabbit lesion, anti-SMase antibody detected significant levels of SMase in the thickened intima, and much of the staining appeared extracellular (compare Figures 4ADown and 4BDown). Note, however, that the SMase staining does not occur throughout the entire intima but rather in a fibrous, acellular strip running through the middle of the intima.



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  		Figure 4. SMase immunohistochemistry of human atherosclerotic abdominal aorta. Serial 8-µm sections of abdominal aorta from a 34-year-old African-American female were either stained with the goat anti-SMase antibody (B) or with secondary antibody alone (A). Similar results were obtained with the rabbit anti-SMase antibody (not shown). Lumen (L), intima (I), and media (M) are marked in panel A.

In summary, SMase is present in both normal and atherosclerotic aortas. The staining in the normal aorta is limited mostly to the endothelium, whereas in the atherosclerotic aorta, the abnormal, thickened intima is heavily stained. Significant portions of SMase staining were found in acellular regions of the diseased intima, and most likely represent extracellular SMase bound to the subendothelial matrix.

Interaction of Purified 125I-S-SMase With ECM In Vitro
The immunohistochemical data described above indicate that at least a portion of extracellular SMase is associated with the subendothelial matrix. To explore the mechanism of this association, we assessed the binding of 125I-S-SMase in vitro to ECM derived from either BASMCs or BAECs. As shown in Figure 5Down, both SMC-derived (A) and endothelium-derived (B) ECM bound {approx}5 times as much 125I-S-SMase as did the no-matrix control dishes. Importantly, the number of picomoles of specific 125I-S-SMase binding was >2-fold higher than the binding of 125I-LDL to the same respective ECM preparations (Figure 6Down) and {approx}60% of the level of 125I-LDL bound in the presence of lipoprotein lipase (data not shown), which is thought to facilitate the interaction of LDL with subendothelial matrix.61



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  		Figure 5. S-SMase binding to SMC- and endothelium-derived ECM. 125I-S-SMase was incubated for 16 hours at 37°C in 16-mm tissue culture-dishes coated with BSA alone (no matrix) or with ECM derived from BASMCs (A) or BAECs (B). At the end of the incubation period, the dishes were washed to remove nonbound 125I-S-SMase, and bound 125I-S-SMase was quantified as described under Methods.



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  		Figure 6. Effect of glycosaminoglycan-hydrolyzing enzymes on S-SMase binding to the ECM. 125I-SMase was incubated for 16 hours at 37°C in 16-mm tissue-culture dishes coated with BSA alone (no matrix), ECM derived from BAECs (ECM), or ECM treated with chondroitinase ABC (ECM Chon’ase) or heparitinase I (ECM Hep’ase), as described under Methods. At the end of the incubation period, bound 125I-SMase was quantified. Insert, a similar experiment was conducted using 125I-LDL.

SMCs and endothelial cells secrete a variety of ECM components, including proteoglycans, various glycoproteins, and collagen, that assemble into a complex, biomechanically active network. To investigate whether SMase interacts with the glycosaminoglycan side chains of proteoglycans, endothelium-derived matrix was pretreated with chondroitinase ABC or with heparitinase I (Figure 6Up). Interestingly, rather than diminishing 125I-S-SMase-binding, these glycosaminoglycanases slightly enhanced it. In contrast, the interaction of 125I-LDL with the ECM, which is known to involve chondroitin sulfate and heparan sulfate glycosaminoglycans,5 was substantially diminished by these treatments (Figure 6Up, inset). These data indicate that S-SMase interacts with 1 or more nonglycosaminoglycan components of the ECM that may be partially obscured by nearby glycosaminoglycan chains.62

To test this idea further, we assayed the binding of 125I-S-SMase to individual matrix components. As shown in Figure 7Down, S-SMase bound relatively poorly to dermatan sulfate proteoglycan, chondroitin sulfate proteoglycan, and heparin compared with the BSA control, consistent with the data in Figure 6Up, and the binding to fibronectin was also low. The binding of S-SMase to laminin, however, was 3-fold higher than control, and this interaction was specific in the sense that 125I-LDL interacted poorly with laminin (Figure 7Down, inset). S-SMase was also found to bind very well to 3 types of fibrillar collagens found in the vessel wall, types I, III, and VI,63 whereas 125I-LDL bound poorly to these collagens (Figure 7Down, inset).



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  		Figure 7. S-SMase binding to individual matrix components. 125I-SMase was incubated for 16 hours at 37°C in 16-mm tissue-culture dishes coated with BSA alone (no matrix), dermatan sulfate proteoglycan, chondroitin sulfate proteoglycan, heparin (Hep’n), fibronectin (Fbrnctn), laminin (Lam’n), collagen I (Coll I), collagen III (Coll III), or collagen VI (Coll VI), as described under Methods. At the end of the incubation period, bound 125I-SMase was quantified. Insert, a similar experiment was conducted in which 125I-LDL binding to laminin, collagen I, collagen III, and collagen VI was determined.

To determine whether collagen-bound S-SMase was enzymatically active, purified enzyme was allowed to bind to collagen type VI, and after rinsing away unbound enzyme, the dish was incubated with [3H]sphingomyelin-labeled LDL plus VLDL from E0 mice at pH 7.4 for 48 hours.11 The data in Figure 8Down show that collagen-bound SMase hydrolyzed 3.5-fold more lipoprotein sphingomyelin compared with collagen not incubated with exogenous S-SMase.



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  		Figure 8. Matrix-bound S-SMase is enzymatically active. First, 16-mm tissue-culture dishes coated with collagen VI were incubated in the absence (open bars) or presence (solid bars) of unlabeled S-SMase for 16 hours at 37°C. Then, [3H]sphingomyelin-labeled LDL+VLDL from E0 mice was incubated with these dishes in pH 7.4 buffer at 37°C. After 48 hours, [3H]sphingomyelin hydrolysis was assayed as described under Methods.


*    Discussion
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 up arrowMethods
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 *Discussion
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Previous work from our laboratory has provided evidence for the presence of an extracellular SMase activity in atherosclerotic lesions.9 This lesional SMase, by acting on subendothelial atherogenic lipoproteins and possibly by acting on the sphingomyelin of arterial-wall cells, may lead to important atherogenic effects, such as subendothelial lipoprotein retention and aggregation, macrophage foam cell formation, apoptosis, and platelet aggregation (see the introduction). S-SMase has become a leading candidate for this arterial-wall enzyme because of its extracellular location, its secretion by cultured macrophages and endothelial cells, and its ability to hydrolyze the sphingomyelin of certain atherogenic lipoproteins.10 11 12 In fact, we and others have been unable to identify any other SMase activity secreted from cultured mammalian cells.10 Nonetheless, to support the hypothesis that S-SMase is indeed the lesional SMase discussed above, it was critical that we directly show that immunoreactive protein actually resides in lesions.

Several pieces of evidence strongly support the presence of S-SMase in normal and atherosclerotic arteries. First, using 2 different anti–S-SMase antibodies, we were able to show heavy staining in the endothelium of normal mouse and rabbit aortas and in the intimas of atherosclerotic lesions from mouse, rabbit, and human aortas. A key point in the interpretation of these data are related to the specificity of the antibodies. L-SMase and S-SMase originate from the same gene, mRNA, and polypeptide precursor, and the 2 enzymes differ structurally only in their carbohydrate moieties and in the absence of 6 amino acids from the N-terminus of the lysosomal enzyme.18 Therefore, it might be expected that antibodies against 1 form would recognize the other form with essentially the same sensitivity. In reality, the rabbit antibody used in this study, which was made against authentic human S-SMase secreted by cultured cells, recognizes S-SMase substantially better than it does L-SMase on immunoblots.12 18 Although we have not directly tested the specificity of the goat antiserum for L-SMase versus S-SMase, this preparation was raised against human urinary SMase,64 which may be a form of S-SMase. Moreover, the 2 antibodies gave identical staining patterns whenever we applied them to the same tissue, consistent with similar specificities for S-SMase over L-SMase. Second, the staining pattern of SMase in the cellular regions of both the normal aorta (ie, the endothelium) and lesions (ie, the intima) was not typical of 1 that was solely lysosomal, which usually appears as a perinuclear vesicular pattern.65 Rather, some of the stain appeared on the outer edges of cells or bound to the pericellular matrix. In fact, even though our data suggest that the media of lesions is rather devoid of S-SMase, it is notable that this region showed poor staining of intracellular lysosomal SMase, which is an enzyme presence in all cells, including SMCs. It is possible that this observation arises from the specificity of the antibodies discussed above and from the type of tissue preparation used in this study, which was not optimized for intracellular staining. Third, there was heavy SMase staining in areas that were clearly devoid of cells. Because the antibodies are not absolutely specific for S-SMase (see above), it is possible that some of the SMase staining in these areas represented L-SMase released from necrotic cells.38 Nonetheless, because L-SMase and S-SMase appear to have very similar enzymatic properties,10 18 released L-SMase may be an important additional source of extracellular SMase in advanced lesions. Such SMase, which is presumably already activated by zinc,18 may accelerate the growth of already established, advanced lesions.

An additional issue is the origin of lesional S-SMase. While it is possible that some of the enzyme originates from serum, which has S-SMase activity66 (S.M. et al, unpublished data, 1999), we propose that most of the intramural enzyme originates directly from arterial-wall cells. As stated above, S-SMase is secreted by arterial endothelial cells and macrophages in culture, and a sizeable portion of the enzyme from cultured endothelial cells is secreted basolaterally.10 12 As demonstrated here, aortic endothelium and cells within the lesional intima, which are probably mostly macrophage foam cells, stain for the SMase gene product. An important finding in this report was the large amount of immunoreactive extracellular SMase in lesions compared with that in normal aorta. This phenomenon may be a consequence of the secretion of S-SMase by intimal macrophages recruited into the arterial wall after lesion initiation, the release of L-SMase from necrotic cells (see above), and the local influence of inflammatory cytokines, which we have shown enhances the secretion of S-SMase by endothelial cells both in vitro12 and in vivo (Wong M-L, Xie B, Beatini N, Phu P, Marathe S, Johns A, Hirsch E, Wiliams KJ, Licinio J, Tabas I. Acute systemic inflammation up-regulates secretory sphingomyelinase in vivo: a possible new link between inflammatory cytokines and the processes of ceramide signaling and atherogenesis. Submitted for publication). Furthermore, matrix alterations during atherosclerotic lesion development, such as a rise in arterial collagen content,63 may facilitate the trapping of locally produced S-SMase (see below). Interestingly, we have found that several types of cultured SMCs do not secrete much S-SMase (S.M. et al, unpublished data, 1999), an observation that may explain the poor staining in the media of lesions.

This study has addressed 1 possible fate of S-SMase after its secretion into the subendothelial space, namely, binding to the subendothelial matrix. The vascular ECM is a dynamically changing network of matrix components that, in addition to providing an architectural framework, selectively binds growth factors, cytokines, and enzymes affecting both their availability and biological activity.67 68 In this respect, the fate of S-SMase resembles that of another secreted arterial-wall enzyme, soluble nonpancreatic phospholipase A2 (snpPLA2).68 Unlike snpPLA2, however, S-SMase binds to nonproteoglycan components of the ECM, at least as assessed by in vitro binding studies. These binding studies suggest that S-SMase may bind to laminin and collagen, both of which are present in the normal arterial subendothelium and in the intima of atherosclerotic lesions.63 69 The exact nature of this binding, particularly whether S-SMase has specific domains that recognize these molecules, remains to be determined. Interestingly, we found that S-SMase bound to monomeric collagen to a similar degree as to fibrillar collagen (data not shown), perhaps suggesting that exposure of the critical S-SMase binding sites on collagen does not require collagen fibril formation. S-SMase is enzymatically active when bound to collagen (Figure 8Up), but it is more active free in solution (data not shown). This observation raises the intriguing possibility that release of S-SMase from collagen, for example by matrix metalloproteinases known to be present and enzymatically active in lesions,70 may promote S-SMase activity.

In conclusion, the data presented in this report, together with previous work from our laboratory, are consistent with a model in which relatively small amounts of endothelium-derived S-SMase play a role in lesion initiation by promoting the earliest stages of atherogenic lipoprotein retention and aggregation. Then, in response to these retained and modified lipoproteins,5 6 the arterial wall accumulates macrophages and inflammatory cytokines, which amplify the production of S-SMase in the intima, facilitating further lipoprotein retention, modification, and lesion growth.


*    Acknowledgments
 
This study was supported by National Institutes of Health grant HL56984 (to I.T. and K.J.W.) and by a research grant from Berlex Biosciences (to I.T.). The authors would like to thank the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group for supplying the specimen for this study.

Received February 11, 1999; accepted March 30, 1999.


*    References
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