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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:264.)
© 2004 American Heart Association, Inc.

Vascular Biology

Apolipoprotein C-I Induces Apoptosis in Human Aortic Smooth Muscle Cells via Recruiting Neutral Sphingomyelinase

Antonina Kolmakova; Peter Kwiterovich; Donna Virgil; Petar Alaupovic; Carolyn Knight-Gibson; Sergio F. Martin; Subroto Chatterjee

From the Lipid Research Atherosclerosis Division (A.K., P.K., D.V., S.F.M., S.C.) and the Department of Pediatrics (S.C.), Johns Hopkins University, Baltimore, MD; and the Oklahoma Medical Research Foundation (P.A., C.K.-G.), Oklahoma City, OK.

Correspondence to Subroto Chatterjee, 550 North Broadway, Suite 312, Baltimore, MD 21205. E-mail schatte2{at}jhmi.edu

	Abstract

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Objectives— Apolipoprotein C-I (apoC-I) influences lipoprotein metabolism, but little is known about its cellular effects in aortic smooth muscle cells (ASMC).

Methods and Results— In cultured human ASMC, apoC-I and immunoaffinity purified apoC-I–enriched high-density lipoproteins (HDL) markedly induced apoptosis (5- to 25-fold), compared with control cells, apoC-I–poor HDL, and apolipoprotein C-III (apoC-III) as determined by 4', 6-diamidino-2-phenylindole dihydrochloride staining and DNA ladder assay. Preincubation of cells with GW4869, an inhibitor of neutral sphingomyelinase (N-SMase), blocked apoC-I–induced apoptosis, an effect that was bypassed by C-2 ceramide. The activity of N-SMase was increased 2- to 3-fold in ASMC by apoC-I, apoC-I–enriched HDL, and tumor necrosis factor (TNF-) (positive control) after 10 minutes and then decreased over 60 minutes, which is a kinetic pattern not seen with controls, apoC-III, and apoC-I–poor HDL. ApoC-I and apoC-I–enriched HDL stimulated the generation of ceramide, the release of cytochrome c from mitochondria, and activation of caspase-3 greater than that found in controls, apoC-III, and apoC-I–poor HDL. GW4869 inhibited apoC-I–induced production of ceramide and cytochrome c release.

Conclusions— ApoC-I and apoC-I–enriched HDL activate the N-SMase-ceramide signaling pathway, leading to apoptosis in human ASMC, which is an effect that may promote plaque rupture in vivo.

Key Words: apolipoprotein C-I • apoptosis • sphingomyelinase • high-density lipoproteins • tumor necrosis factor-

	Introduction

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A polipoprotein C-I (apoC-I), a 6.6-kDa plasma protein, has a basic pI because of its high content of lysine (16 mol %) and contains no histidine, tyrosine, cysteine, or carbohydrate.^1,2 Residues 7 to 24 and 35 to 53 of apoC-I are important for binding to plasma lipids.^1,2 ApoC-I is a component of very-low-density (VLDL), intermediate density, and high-density lipoproteins (HDL). ApoC-I displaces apolipoprotein E (apoE) from VLDL and intermediate density, thereby decreasing their clearance from plasma.³ ApoC-I decreases the binding of ß-VLDL to a remnant receptor, the low-density lipoprotein (LDL) receptor-related protein (LRP),^4,5 and apoE-mediated binding of VLDL and intermediate density to the LDL receptor (LDLR).^6,7 ApoC-I inhibits cholesterol ester transfer protein⁸ and phospholipase A2 activity.⁹ ApoC-I stimulates lecithin cholesterol acyl transferase to 80% of that of apolipoprotein A-I (apoA-I).¹⁰

Human apoC-I–transgenic mice, with a wild-type background or with a knockout background for the LDLR or apoE, manifest a marked combined hyperlipidemia because of significantly delayed remnant clearance.^11–17 Free fatty acid levels are elevated because of reduced fatty acid uptake in peripheral tissues, which is an effect that is paradoxically associated with increased sensitivity to insulin and protection from obesity.^12,13 Of particular interest here, Conde-Knape et al,¹⁷ using a moderately expressing apoC-I transgenic on apoE-null background to study the effect of apoC-I independent of apoE, found a marked combined dyslipidemia that included an apoC-I–enriched HDL and increased atherosclerosis. ApoC-I–enriched HDL (but not VLDL) had a marked inhibitory effect on hepatic lipase.¹⁷ ApoC-I knockouts are normolipidemic rather than hypolipidemic.¹⁸ Cholesterol ester transfer protein-transgenic/apoC knockout mice manifest a markedly increased transfer of cholesteryl esters from HDL to VLDL.¹⁹

In humans, Bjorkegren et al reported a significant enrichment of apoC-I in VLDL remnants in normolipidemic patients with coronary artery disease and exaggerated postprandial triglyceridemia²⁰ and in healthy, normolipidemic men with early asymptomatic atherosclerosis.²¹

Because low-birth-weight is associated with increased risk for and death from cardiovascular disease in adults,²² we examined lipoprotein heterogeneity at birth in cord blood (unpublished data). We found, in agreement with others, that a significant proportion of infants had increased levels of large HDL particles^23–25 but, in addition, these particles were enriched with apoC-I. In pilot experiments, it was found that the apoC-I–enriched HDL, as well as highly purified apoC-I, promoted apoptosis in cultured human aortic smooth muscle cells (ASMC).

ASMC play a critical role in preventing the complications of atherosclerosis as part of the fibrous cap that sequesters the lipid core and prevents plaque rupture.²⁶ We report that purified apoC-I alone, and immunoaffinity purified apoC-I–enriched HDL particles, induces marked apoptosis in cultured human ASMC. Moreover, the mechanism of action of apoC-I appears to be mediated through the recruitment of N-SMase, generating ceramide and activating cytochrome c release as well as caspase. We speculate that this biochemical mechanism by which apoC-I promotes apoptosis in ASMC may contribute to an unstable plaque that is more likely to rupture, leading to thrombosis, myocardial infarction, and death.²⁶

	Methods

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Please see online Methods, available at http://atvb.ahajournals.org.

	Results

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ApoC-I and ApoC-I–Enriched HDL Particles Stimulate Apoptosis in ASMC
Cells incubated with apoC-I contained many apoptotic cells, as judged by white staining of the nucleus after treatment with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) reagent (Figure 1A). In contrast, cells incubated with apoC-III for 24 hours had only a few apoptotic cells (Figure 1A). After fluorescence microscopic quantitative analysis of DAPI staining of the nucleus in attached cells after treatment (Figure 1B), 2.24% of normal (control) cells, 4.78% of apoC-III–treated cells, and 26.19% of apoC-I–treated cells were apoptotic. In addition, 50% of ASMC incubated with apoC-I–enriched HDL particles were apoptotic; whereas only 2% of the cells incubated with apoC-I–poor HDL were apoptotic (Figure 1B; Table 1).

View larger version (61K): [in this window] [in a new window]   Figure 1. Effect of apolipoproteins on apoptosis in human ASMC. ASMC were grown on sterilized glass cover slips and incubated for 24 hours with media alone (control), apoC-III (negative control, 2.5 µg/mL medium), apoC-I (2.5 µg/mL medium), apoC-I–enriched HDL (1 µg apoA-I/mL medium), or apoC-I–poor HDL (5 µg apoA-I/mL medium) for 24 hours. Cells were then fixed with ethanol-acetic acid (3:1 volume/volume) and washed 3 times with phosphate-buffered saline. The cells were then stained with DAPI reagent, mounted on a glass cover slide, and subjected to fluorescence microscopy. A, The nucleus of normal cells stains blue and the nucleus of apoptotic cells stains is fragmented white. B, 300 to 500 cells in each sample were counted and the percent of apoptotic cells was plotted. Values are mean±SD; **P<0.01, ***P<0.001. C, DNA ladder assay. Confluent cultures of ASMC were incubated with control medium alone, apoC-I (2.5 µg/mL medium), apoC-III (negative control, 2.5 µg/mL medium), and TNF- (positive control, 20 ng/mL medium). Genomic DNA was extracted and 10 µg DNA was subjected to agarose gel electrophoresis. The gels were calibrated with commercially available DNA standards. After electrophoresis, the gels were stained with ethidium bromide and then photographed.

View this table: [in this window] [in a new window]   Lipid and Apolipoprotein Composition of ApoC-Enriched HDL and ApoC-I–Poor HDL

Apoptotic cells undergo endonucleosomal cleavage, resulting in the fragmentation of DNA into 180 to 200 base-pair fragments that resolve as a ladder on agarose gel electrophoresis. Cells incubated with tumor necrosis factor (TNF-) (positive control), or with apoC-I, exhibited DNA laddering. In contrast, control cells, and those incubated with apoC-III, did not exhibit significant DNA laddering (Figure 1C).

Neutral Sphingomyelinase Inhibitor, GW4869, Abrogates ApoC-I–Induced Apoptosis
As shown in Figure 2A and the corresponding densitometric scan (Figure 2B), N-SMase inhibitor, GW4869 (20 µmol/L), completely abrogated the apoC-1–induced apoptosis (12% compared with control 2%), an effect that was also observed using a lower dose (10 µmol/L) of GW4869. However, when C2-ceramide was added to the ASMC with apoC-I and GW4869, the inhibitory effect of GW4869 on N-SMAse was bypassed and apoptosis was restored (Figure 2A and B) (12.5% of apoptotic cells compared with control of 2.2% apoptotic cells).

View larger version (34K): [in this window] [in a new window]   Figure 2. Effect of apoC-I, the N-SMase inhibitor, GW4869, and C-2 ceramide on apoptosis in cultured human ASMC. A, ASMC (1 x103) were seeded on sterilized glass cover slips and grown in tissue culture medium with 10% fetal calf serum for 48 hours. Next, fresh serum-free medium, with and without GW4869 (20 µmol/L in DMSO), was added to some dishes. Thirty minutes later, vehicle DMSO, apoC-I (2.5 µg/mL), or C2-ceramide (10 µmol/L) were added to dishes and the incubation continued for 24 hours. The cells were then fixed with ethanol-acetic acid (3:1 v/v), stained with DAPI reagent, assessed by fluorescent microscopy, and photographed. Top to bottom: control cells; cells incubated with apoC-I ± the N-SMase inhibitor, GW4869; and cells incubated with apoC-I, GW4869, and C2-ceramide. B, The DAPI-stained ASMC from the four experimental conditions described was analyzed for quantitative apoptosis. Mean (SD) from 4 experiments. *P<0.05.

ApoC-I and ApoC-I–Enriched HDL Particles Stimulate the Activity of N-SMase in Cultured ASMC
Within 5 minutes of incubation of cells with TNF- (positive control) and apoC-I, there was 2-fold increase in the activity of N-SMase that reached a maximum at 10 minutes and then decreased to baseline by 30 to 60 minutes (Figure 3A). Such a pattern was not seen in the control cells or in those incubated with apoC-III. The stimulation of N-SMase activity with apoC-I–enriched HDL particles was even more pronounced than that seen with TNF- and apoC-I (Figure 3B). At 5 minutes, there was a 2.6-fold stimulation of activity of N-SMase that reached a maximum of 2.7-fold stimulation at 10 minutes. In contrast to TNF- and apoC-I, the stimulation of N-SMase activity with apoC-I-enriched HDL was still manifested (2.2-fold) at 30 minutes and then approached baseline at 60 minutes (Figure 3B). ApoC-I–poor HDL also stimulated N-SMase activity in a similar pattern, but to a considerably lesser extent than apoC-I–enriched HDL (Figure 3B). ApoC-I exerted a concentration-dependent increase in the activity of N-SMase in ASMC (Figure 3C), with a maximum increase in N-SMase activity (1.5-fold) at a concentration of apoC-I of 2.5 µg/mL medium). In additional experiments to examine the specificity of this inhibitor, we found that GW4869 did not alter the acid sphingomyelinase activity (control: 400 nmol/mg protein; GW4869: 340 nmol/mg protein) in cultured human ASMC.

View larger version (20K): [in this window] [in a new window]   Figure 3. Effects of apoC-I, apoC-III, TNF-, apoC-I–enriched HDL, and apoC-I–poor HDL on the activity of N-SMase in ASMC. Confluent cultures of ASMC were preincubated for 30 minutes with serum-free (control) medium, then apoC-I (2.5 µg/mL medium), apoC-III (2.5 µg/mL medium), TNF- (20 ng/mL medium) (A, top), or apoC-I–enriched HDL (1 µg apoA-I/mL medium), or apoC-I-poor HDL (5 µg apoA-I/mL medium) (B, middle) were added individually to cells. The cells were then incubated and harvested at the indicated time points, and the activity was of N-SMase determined using 14C sphingomyelin as substrate (see Methods). The N-SMase activity in control cells was 22 nmol/h per milligram cell protein and this value was considered as 100% activity. In a separate experiment (C, bottom), cells (0.5 x 105) were seeded in P60 Petri dishes and grown in DMEM supplemented with 10% fetal bovine serum for 5 days. After preincubation of cells for 30 minutes in serum-free medium, increasing amounts of apoC-I were added. After incubation for 10 minutes, cells were harvested and N-SMase activity measured as described in Methods.

Antibody Against N-SMase Abrogates apoC-I–Enriched HDL-Induced N-SMase Activity in Cultured ASMC
Previous studies in human renal proximal tubular cells²⁷ and neuronal cells²⁸ have shown that N-SMase is localized on the outer leaflet of plasma membrane. Using N-SMase antibody and FITC- conjugated secondary antibody, we made similar observations in human ASMC (data not shown). We therefore preincubated ASMC with an antibody (IgG) against N-SMase (1:500 dilution) and found that anti-N-SMase, but not rabbit IgG (control), inhibited the stimulation of N-SMase activity by apoC-I–enriched HDL by 77%. This result suggested that apoC-I mediated the increased activity of N-SMase in human ASMC by the apoC-I–enriched HDL particle at the cell surface.

ApoC-I and ApoC-I–Enriched HDL Particles Stimulate the Generation of Ceramide in Cultured Human ASMC
ApoC-I exerted a time-dependent increase in ceramide levels, which reached a maximum effect (1.7-fold) by 5 minutes and then decreased to a value similar to that in control cells by 10 minutes (Figure 4A). Incubation of ASMC with apoC-I–enriched HDL particles with time reached a maximum increase (1.7-fold) in the level of ceramide by 30 minutes, compared with control cells, whereas apoC-I–poor HDL only stimulated the ceramide level slightly (Figure 4B). GW4869 (20 µmol/L) significantly decreased the cellular level of ceramide induced by apoC-I after 5 minutes and 6 hours of incubation. GW4869 completely inhibited apoC-I–induced ceramide production after 12 hours of incubation but reversed it to a high level 24 hours later (Figure 4C). Using an anti-ceramide antibody, cells treated with apoC-I reacted strongly, as judged by immunofluorescence microscopy, compared with control cells (Figure 4D). Most fluorescence was localized in the perinuclear area, an effect of apoC-I on the ceramide level that was eliminated by GW 4869, an inhibitor of N-SMase.

View larger version (47K): [in this window] [in a new window]   Figure 4. Effect of apoC-I, apoC-I–enriched HDL, and apoC-I–poor HDL on the level of ceramide in ASMC. Confluent ASMC were incubated with apoC-I (2.5 µg/mL) with time (A, upper left) or with apoC-I–enriched HDL (1 µg apoA-I/mL medium) or apoC-I–poor HDL (5 µg apoA-I/mL medium) (B, upper right) for 30 minutes. C, The cells were incubated with apoC-I alone (2.5 µmol/L) for 5 minutes or preincubated with GW4869 (20 µmol/L) for 30 minutes. After addition of apoC-I (2.5 µg/mL), incubation was continued for 6 hours, 12 hours, and 24 hours. After incubation cells were harvested and the levels of ceramide determined using the diacylglycerol kinase assay (see Methods). The level of phosphate was measured and the data (mean±SD) were expressed as percentage of the total ceramide present in untreated cells (vehicle DMSO). *P<0.05. In a separate experiment (D, right), confluent cells were grown on cover slips and pretreated with serum-free medium for 30 minutes. The cells were then incubated with vehicle DMSO alone (control), apoC-I (2.5 µg/mL), or apoC-I (2.5 µg/mL) plus the N-SMase inhibitor, GW4869 (20 µmol/L), for 24 hours. The cells were then washed, treated with a primary anti-ceramide antibody, washed with phosphate-buffered saline, and treated with a fluorescein isothiocyanate-conjugated anti-mouse IgG as described in Methods (C, lower panel).

ApoC-I and ApoC-I–Enriched HDL Particles Stimulate the Release of Cytochrome c From Mitochondria in ASMC
The release of cytochrome c from mitochondria into cytosol is a key pro-apoptotic event. ApoC-I stimulated the release of cytochrome c 4.1-fold. ApoC-III and apoC-I–enriched HDL particles also showed some stimulation of cytochrome c (2.3- and 1.2-fold, respectively) (Figure 5 A and B). Using immunofluorescence, a marked increase in the release of cytochrome c from mitochondria into the cytoplasm with apoC-I (even greater than that with TNF-) was noted, an effect that was inhibited by GW4869 (Figure 5C).

View larger version (41K): [in this window] [in a new window]   Figure 5. Effect of apoC-I, apoC-III, apoC-I–enriched HDL, and GW4869 on cytochrome c release in ASMC. After treatment of confluent cells for 24 hours with apoC-I (2.5 µg/mL medium), apoC-III (2.5 µg/mL medium), or apoC-I–enriched HDL (1 µg apoA-I/mL medium), the cells were lysed. The cell lysates was used as the source of cytochrome c, and the pellet was used as a source of caspase-3. The cell lysates were separated by SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and cytochrome c detected using a polyclonal antibody against cytochrome c (A, top panel). The gels were then subjected to densitometric scanning (B, middle panel). In a separate experiment (C, right), confluent cells were grown on cover slips and pretreated with serum-free medium for 30 minutes. The cells were then incubated with control medium, apoC-I (2.5 µg/mL medium), TNF- (20 ng/mL), or apoC-I (2.5 µg/mL) + GW4869 (20 µmol/L) for 24 hours. The cells were then washed, treated with a primary anti-cytochrome c antibody, washed with phosphate-buffered saline, and treated with a fluorescein isothiocyanate-conjugated anti-rabbit IgG as described in Methods (C, lower panel).

ApoC-I and ApoC-I–Enriched HDL Particles Stimulate the Expression of Caspase-3 in Human ASMC
ApoC-I stimulated the caspase-3 level 4-fold compared with control medium. ApoC-I–enriched HDL stimulated caspase-3 expression in ASMC 1.7-fold, whereas apoC-I–poor HDL did not. ApoC-III also stimulated caspase-3 expression, but to a lesser extent than apoC-I–enriched HDL (data not shown).

	Discussion

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The most striking phenotypic change in ASMC treated with apoC-I or apoC-I–enriched HDL particles was apoptosis that accompanied the activation of the sphingomyelinase signal transduction cascade. In additional studies, we observed that apoC-I induced the expression of Lyn-kinase protein (data not shown). Moreover, TNF-–induced N-SMase activation in ASMC was inhibited by the use of tyrosine kinase inhibitors. Therefore, we postulate that by stimulating one or more membrane-bound protein kinases, apoC-I may activate N-SMase in human ASMC. Both apoC-I and apoC-I–enriched HDL particles stimulated the activity of N-SMase in ASMC in a time- and concentration-dependent fashion. Moreover, the kinetics of N-SMase activation by these two agonists was similar to that of TNF-, in this study and in our previous studies.²⁹ The tenet that apoC-I induced N-SMase activation and the subsequent production of ceramide were essential steps in the signaling cascade leading to apoptosis was substantiated by the following: (1) N-SMase inhibitor, GW4869, abrogated apoC-I–induced ceramide generation, cytochrome c release, and apoptosis; (2) preincubation of cells with antibody against N-SMase (but not the control rabbit IgG) prevented apoC-I–induced N-SMase activation and apoptosis; (3) incubation of cells with apoC-III, which has some structural homology with apoC-I, did not stimulate N-SMase activation and apoptosis; and (4) incubation of cells with apoC-I–poor HDL particles did not elevate N-SMase activity and apoptosis to the same extent as apoC-I–enriched HDL. These studies were performed in vitro, and future studies will be necessary to determine the effect of apoC-I on ASMC in vivo.

Recently, the use of GW4869, an N-SMase inhibitor, was shown in human breast cancer cells, MCF-7, to reduce agonist (TNF-)-induced apoptosis.³⁰ Using molecular cloning, this inhibitor was shown to be a target for N-SMase.³¹ Furthermore, recombinant acid sphingomyelinase activity and the activity of serine palmitoyltransferase (SPT) involved in de novo ceramide biosynthesis were shown to be unaffected in MCF-7 cells by GW4869 treatment.³⁰ In addition, we also found that GW4869 does not alter the acid sphingomyelinase activity in cultured human ASMC. In our study, GW4869 (at 10 µmol/L and 20 µmol/L) completely inhibited apoC-I– induced apoptosis and apoC-I–induced ceramide generation (Figure 4C and D), suggesting that apoC-I may preferentially activate N-SMase to generate ceramide and induce apoptosis. Moreover, the increase in the cell level of ceramide after 24 hours of treatment with either apoC-I or GW4869 suggests that by this time, this inhibitor no longer prevented the apoC-I–induced increase in the cell ceramide level. Our findings in human ASMC are similar to those published by others using breast cancer cells.³⁰ Clearly, additional studies are necessary to delineate further the biochemical pathways by which apoC-I can generate ceramide and induce apoptosis.

HDL particles contain several other apolipoproteins, as well as lipids, in addition to apoC-I (Table 1). We therefore cannot exclude the possibility that other apolipoprotein and lipid moieties, independently or collectively, contribute to N-SMase activation and apoptosis. In addition, the presence of oxidized cholesterol in caveolae may activate a signaling pathway leading to apoptosis.³² However, our studies included a control, namely immunoaffinity-purified apoC-I–poor HDL particles, whose lipid composition was quite similar to that of the immunoaffinity-purified apoC-I–enriched HDL (Table 1).

Although several studies point to the role of oxidized LDL and its components in inducing apoptosis in cells of the vascular wall, little is known about various apolipoproteins and their effect on apoptosis. Our previous study showed that apolipoproteins B, A-I, A-II, and E did not alter apoptosis in cultured human ASMC. Only very high doses of LDL (>200 µg/mL medium) induced cell death, an action mostly caused by a necrotic/cytotoxic effect. HDL had either no effect on cell death or rescued cells from LDL induced necrosis.^33,34

In humans, apoC-I may be atherogenic by delaying the receptor-mediated uptake of remnants from triglyceride-rich lipoproteins.^20,21 Based on our present report, we postulate that increased plasma levels of apoC-I, perhaps associated with HDL, might also contribute to the complications of atherosclerosis by inducing apoptosis in ASMC, a biochemical mechanism that might contribute to plaque rupture and coronary artery disease.

Received October 20, 2003; accepted December 1, 2003.

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