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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:19-27.)
© 1996 American Heart Association, Inc.

Articles

Apoptosis of Vascular Smooth Muscle Cells Induced by In Vitro Stimulation With Interferon-, Tumor Necrosis Factor–, and Interleukin-1ß

Yong-Jian Geng; Qi Wu; Maria Muszynski; Göran K. Hansson; Peter Libby

From the Vascular Medicine and Atherosclerosis Unit, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass, and the King Gustaf V Research Institute (G.K.H.), Karolinska Institute, Karolinska Hospital, Stockholm, Sweden.

Correspondence to Dr Peter Libby, Vascular Medicine and Atherosclerosis Unit, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave, Boston, MA 02115.

	Abstract

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Abstract Recent studies have documented evidence for the death of smooth muscle cells (SMCs) within advanced human atheroma. These lesions contain macrophages and T lymphocytes in addition to SMCs. We therefore investigated whether interferon- (IFN-), a cytokine secreted by T lymphocytes, or interleukin-1ß (IL-1ß) and tumor necrosis factor– (TNF-), two cytokines characteristically produced by activated macrophages, can trigger apoptosis of vascular SMCs. Simultaneous treatment with IFN- and TNF- and/or IL-1ß but not with each cytokine alone promoted death of human and rat SMCs. Exposure for 48 hours to a combination of IFN- (400 U/mL), TNF- (400 U/mL), and IL-1ß (100 U/mL) significantly (P<.001) increased the accumulation of oligonucleosomes comprising DNA fragments and histones in human SMCs. Electrophoresis of genomic DNA showed internucleosomal fragments of genomic DNA isolated from the cytokine-cotreated SMCs of both humans and rats. These cells exhibited morphological changes typical of apoptosis, including cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation. In situ 3' end labeling of DNA fragments with terminal transferase confirmed the fragmentation of genomic DNA in these cells. Simultaneous treatment with IFN- and TNF- or IL-1ß induced elaboration of nitrite, an end product of nitric oxide, in rat but not human SMCs. N^G-monomethyl-L-arginine inhibited nitrite accumulation and also partly blocked cytokine-induced apoptosis of rat SMCs but had little effect on human SMCs, suggesting operation of both nitric oxide–dependent and –independent mechanisms for cytokine-induced apoptosis in vascular SMCs. Production of immune cytokines by vascular cells and/or infiltrating leukocytes may regulate apoptotic death of SMCs during atherogenesis.

Key Words: smooth muscle cells • cell death • nitric oxide • cytokines • atherosclerosis

	Introduction

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In addition to lipids and connective tissue, atherosclerotic lesions consist of SMCs, macrophages, and T lymphocytes.¹ During the prolonged process of atherogenesis, complex interactions between the vascular cells and infiltrating immune cells yield local production of a great variety of biologically active substances such as cytokines and growth factors.^{2 3} These bioactive substances may in turn regulate cell functions, such as replication, differentiation, and death, in a paracrine or autocrine fashion and consequently influence cellularity and morphogenesis of the vessel wall.

The cytokines IFN-^{4 5} (secreted mainly by activated T cells) and IL-1^{6 7} and TNF-⁸ (products of activated macrophages as well as SMCs themselves) can regulate gene expression, differentiation, and growth of vascular SMCs in vitro and in vivo. Although IL-1⁷ and TNF-⁸ stimulate but IFN- inhibits SMC proliferation,^{4 9} these cytokines share many biological actions. For example, at least in rodent cells, IFN-, TNF-, and IL-1, together or alone, can induce expression of NOS.^{10 11} NO produced by the cytokine-inducible form of NOS exerts many well-known effects on cells, including the ability to kill microbial pathogens and tumor cells.¹²

Cell death occurs in advanced atherosclerotic lesions, often resulting in formation of hypocellular fibrous zones and a lipid-rich "necrotic" core. Recent reports provide evidence that apoptosis, a form of programmed cell death,¹³ can mediate some of the cell death in human atherosclerotic lesions.^{14 15 16} SMCs isolated from atherosclerotic arteries undergo apoptosis more frequently than do cells from normal vessels.¹⁷ In situ detection of DNA fragments demonstrates that many SMCs in plaques^{15 16} and in balloon-injured arteries^{16 18} bear this marker of apoptosis.

The mechanisms that trigger apoptosis in atherosclerotic lesions remain unknown. Cytokines can promote apoptosis in some cell types: IL-1 induces apoptosis of pancreatic cells¹⁹ and chondrocytes²⁰ in an NO-dependent pattern; TNF- promotes apoptosis of endothelial cells,²¹ and IFN- triggers apoptosis of vascular SMCs, which overexpress functional c-myc after transfection with c-myc cDNA.²² Since cytokine-producing cells (eg, macrophages and T lymphocytes) abound in advanced atherosclerotic lesions,^{2 23} we hypothesized that cytokines might contribute to apoptosis of vascular SMCs during atherogenesis. The present study tested whether cultured SMCs undergo apoptosis when exposed to recombinant cytokines.

	Methods

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Reagents
The recombinant cytokines used in this study were rat IFN- (Holland Biotechnology), human IFN- (Genzyme Corp), murine TNF- (Genentech Inc), and human TNF- and IL-1ß (Endogen, Inc). Sulfanilamide and N-(1-naphthyl)ethylenediamine hydrochloride were obtained from Merck. L-NMMA was from Calbiochem Corp. Acridine orange, ethidium bromide, and propidium iodide were purchased from Sigma Chemical Co, and YOYO-1, a sensitive DNA-binding fluorochrome, was from Molecular Probes, Inc.

Vascular SMC Isolation and Culture
Human²⁴ and rat⁴ vascular SMCs were isolated from the tunica media of aortas of 35- to 45-year-old organ donors without overt atherosclerosis and 6-week-old Sprague-Dawley rats, respectively. The cells were cultured in Dulbecco's minimal essential medium (GIBCO) supplemented with 10% FCS and antibiotics.²⁴ Cells were identified as vascular SMCs by their characteristic hills–and-valleys growth pattern and by immunofluorescence with anti––smooth muscle actin monoclonal antibody. They were passed by trypsinization, plated at a density of 2x10⁴ cells/mL, and used for experiments at passages 2 through 7. Cells at a subconfluent density were treated with IFN-, IL-1ß, and TNF- alone or together. In some experiments, L-NMMA (500 µmol/L) was added together with the cytokines to block endogenous NO synthesis.

Cell Viability
Cell viability was determined by fluorochrome staining. SMCs (10⁴ cells/chamber) were cultured in eight-chamber slides. The cells were treated with 400 U/mL IFN-, 400 U/mL TNF-, and 100 U/mL IL-1ß for 24, 48, 72, or 96 hours. At the end of incubation, the nucleic acid–binding fluorescent dyes acridine orange and ethidium bromide (10 µg/mL each in culture medium) were added to the cultures. After the slides were stained, they were coverslipped and observed by fluorescent microscopy, and viable (green fluorescent nuclei) and nonviable (red or orange fluorescent nuclei) cells were counted. For each sample, at least 200 cells were counted in different high-power fields. The percentage of viable cells was determined by the following formula: percent cell viability=100x(number of viable cells)/(total number of cells).

Cell Morphology and Nuclear Staining
Cell morphology was examined by phase-contrast microscopy and recorded by microphotography during incubation with cytokines. For visualization of nuclei, cells were cultured in eight-chamber slides and treated with or without cytokines. At the end of incubation, cells were fixed in ice-cold 4% formaldehyde. After washing with PBS containing Tween 20 (0.5%), cells were stained with 1 µg/mL YOYO-1 in PBS for 5 minutes, mounted in 70% glycerol in PBS, and observed under an Olympus fluorescent microscope.

Enzyme Immunoassay of Oligonucleosomes
Internucleosomal DNA fragmentation characteristic of apoptosis occurs several hours before breakdown of the plasma membrane of apoptotic cells. Enrichment of mononucleosomes or oligonucleosomes in cytokine-treated and untreated SMCs was quantitatively determined by sandwich-enzyme immunoassay with a cell-death detection ELISA kit purchased from Boehringer Mannheim Corp. Briefly, 5x10⁴ cells cultured in 24-well plates were treated with IFN-, TNF-, and IL-1ß for 48 hours. At the end of the incubation, culture medium was removed to an Eppendorf tube and centrifuged at 20 000g. The resulting pellets contained oligonucleosomal fragments. Meanwhile, adherent SMCs were permeabilized in the sample buffer containing Tween 20. The supernatants containing the cytoplasmic oligonucleosomes released from SMC nuclei were combined with the oligonucleosomal fragments in medium. The combined supernatants (100 µL) were transferred to a 96-well microplate precoated with monoclonal antibody against histone-4. After incubation for 1 hour at room temperature and washing, oligonucleosomal DNA fragments bound to the microplate were detected by using monoclonal anti-DNA antibody conjugated with the enzyme peroxidase. 2,2'-Azinodi-[3-ethylbenzthiazoline sulfonate] was used as the enzyme substrate, and colored product was measured by spectrophotometry at 405 nm.

In Situ Detection of Apoptotic Cells
Cells undergoing apoptosis can accumulate internucleosomal DNA fragments in their nuclei. In situ detection of apoptotic cells was performed by using TUNEL with an ApoTag in situ apoptosis detection kit (Oncor Inc). Briefly, cells were cultured in eight-chamber slides and treated with or without cytokines for 72 hours. After cytokine treatment, cells were washed in PBS, fixed, incubated with 5 µg/mL proteinase K for 10 minutes, and then labeled with digoxigenin-conjugated dUTP and the enzyme terminal deoxyribonucleotide transferase. Labeled DNA fragments were stained with anti-digoxigenin monoclonal antibody linked with peroxidase. The chromogenic substrate diaminobenzidine was used as the substrate for peroxidase.

Flow Cytometry
SMCs were cultured in six-well plates in Dulbecco's minimal essential medium containing 10% FCS. After incubation with or without cytokines and trypsinization, cells were suspended in fresh medium containing 50 µg/mL propidium iodide for 5 minutes at 37°C and then subjected to flow cytometry on a fluorescence-activated cell sorter flow cytometer immediately after incubation. A light-scatter gate was set up to eliminate cell debris from analysis. Cellular propidium iodide fluorescence signal was recorded on the FL2 channel and analyzed by using Lysys II software.

DNA Fragmentation Analysis
Cells (5x10⁶) were lysed in 1 mL DNA extraction solution containing 20 mmol/L Tris-HCl, pH 7.4, 0.1 mol/L NaCl, 5 mmol/L EDTA, and 0.5% sodium dodecyl sulfate. The lysates were incubated with 100 µg/mL proteinase K at 37°C for 16 hours. After incubation, 1 mL of phenol/chloroform (1:1) was mixed well with the enzyme-digested cell lysates, and the mixture was centrifuged at 20 000g for 20 minutes. DNA in the upper (aqueous) phase was incubated with 5 µg/mL DNase-free RNase A at 37°C for 1 hour and extracted with phenol/chloroform again. DNA was collected by precipitation with 1 mL isopropanol and 0.1 mL of 5 mol/L NaCl at -20°C overnight. After centrifugation, the resulting DNA pellets were washed with 75% ethanol and air dried. DNA was dissolved in 10 mmol/L Tris-HCl and 1 mmol/L EDTA, and its concentration was determined at 260 nm by spectrophotometry. DNA electrophoresis was carried out in 1.5% agarose gels containing 1 µg/mL ethidium bromide, and DNA fragments were visualized by exposing the gel to UV light.

Nitrite Assay
In cell cultures, the majority of NO produced by cytokine-stimulated SMCs is converted into nitrite by reaction with ambient oxygen. We therefore measured nitrite accumulation in SMCs by using the Griess reagent, composed of 2.9 mmol/L sulfanilic acid and 0.2 mmol/L N-(1-naphthyl)ethylenediamine hydrochloride in 5% phosphatic acid.^{10 11} Briefly, culture medium was incubated with an equal volume of the Griess reagent at room temperature for 30 minutes. The colored product of the diazotization reaction was spectrophotometrically quantified at 540 nm by using a microplate ELISA reader. Sodium nitrite dissolved in the same medium was used as standard.

Statistical Analysis
Differences between means were evaluated by using two-tailed Student's t tests. ANOVA in cell viability was carried out by using the ANOVA program in Excel (Microsoft Co). Significance was established when the probability value was less than .05.

	Results

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Death of Vascular SMCs Exposed to Cytokines
We examined the viability of cultured human and rat vascular SMCs by staining the cells with the fluorochromes acridine orange and ethidium bromide. Viable cells excluded ethidium bromide but were permeable to acridine orange, which reacts with DNA to yield green nuclear fluorescence and with RNA to produce red fluorescence in the cytoplasm (Fig 1). Nonviable cells show red ethidium bromide fluorescence in their nuclei due to ethidium bromide entry (Fig 1d). Under basal conditions, cultures of both human and rat SMCs contained few nonviable cells (Fig 1a and 1e). Treatment with either IFN- (400 U/mL), TNF- (400 U/mL), or IL-1ß (100 U/mL) alone for 24 hours did not increase the percentage of dead cells, and prolongation of the cytokine incubation time to 48 and 72 hours slightly increased the number of dead cells in the single cytokine–treated SMCs (Figs 1 and 2). However, a combination of these cytokines induced much higher levels of cell death compared with the control cultures or those treated with a single cytokine. Simultaneous exposure to IFN-, TNF-, and IL-1ß caused higher levels of cell death than did the double treatments (Fig 2).

View larger version (53K): [in this window] [in a new window]   Figure 1. Photomicrographs of human (a through d) and rat (e and f) SMCs cultured in eight-well chamber slides and treated with IFN- and TNF-. After incubation, cells were stained with acridine orange and ethidium bromide at 50 µg/mL each and examined by fluorescent microscopy. a, Untreated human control (arrow shows dividing cells); b, IFN- 400 U/mL; c, TNF- 400 U/mL; d, IFN- with TNF- (arrow shows a dead cell); e, untreated rat control; and f, IFN- with TNF- (original magnification x200).

View larger version (17K): [in this window] [in a new window]   Figure 2. Line graph. Human SMCs cultured in medium containing 10% FCS were treated with IFN- (400 U/mL), TNF- (400 U/mL), and IL-1ß (100 U/mL) alone or in combination. Viable cells were enumerated based on fluorochrome staining with 50 µg/mL each acridine orange and ethidium bromide. Dead cells showed red nuclear fluorescence (Fig 1). Data represent means of four cultures (coefficient of variation <10%). *P<.05 vs untreated controls.

Flow cytometric determination of cell viability with the DNA-binding dye propidium iodide further demonstrated similar cytokine effects on rat SMCs, which are more easily detached during apoptosis than are human SMCs. Few rat SMCs died when cultured in normal medium containing either no cytokines or an individual cytokine (Fig 3). More than 95% of unstimulated rat SMCs excluded propidium iodide, and exposure to either IFN- or TNF- alone did not reduce cell viability (Fig 3). However, simultaneous treatment with both IFN- and TNF- at 400 U/mL each for 72 hours reduced cell viability from 96±1% to 48±8% (P<.01), indicating the synergy in induction of cell death between the two cytokines (Fig 3). Nonviable cells exhibited high but varying levels of propidium iodide fluorescent intensity (Fig 3), suggesting that DNA degradation might occur in these nonviable cells, leading to a wide range of their cellular DNA content.

View larger version (24K): [in this window] [in a new window]   Figure 3. Rat SMCs were treated with IFN- and/or TNF- (400 U/mL each) for 72 hours. In some experiments, L-NMMA (500 µmol/L) was added together with the cytokines. After incubation, cells were collected by trypsinization, stained in 50 µg/mL propidium iodide for 5 minutes, and analyzed by flow cytometry.

Morphological Characterization of Vascular SMCs Undergoing Apoptosis
Phase-contrast microscopy showed morphological changes characteristic of apoptosis in cytokine-treated human and rat SMCs. When exposed to IFN-, TNF-, and IL-1ß, some human SMCs shrank and retracted from their neighbor cells. Their membranes appeared blebbed, and the cytoplasm condensed (Fig 4). The surviving cells also showed certain degrees of morphological change, with an elongated and bipolar appearance (Figs 1 and 4). We used the ultrasensitive DNA-binding dye YOYO-1, which discloses nuclear morphology better than does acridine orange or ethidium bromide, to examine human SMCs treated with or without the cytokines. Staining with YOYO-1 clearly visualized fragmented nuclei with condensed chromatin, and some nuclei fragmented and formed apoptotic bodies (Fig 5). The morphological changes induced in SMCs by cytokine stimulation were distinguishable from those caused by treatment with high concentrations of H₂O₂, which kills SMCs very rapidly. In contrast to the cells treated with cytokines, SMCs exposed to H₂O₂ appeared swollen rather than shrunken, but their connections with adjacent cells remained intact (Fig 4h). In contrast to cytokine-treated cells, H₂O₂-treated SMCs showed a relatively normal nuclear morphology, as illustrated by YOYO-1 staining (Fig 5d). These observations suggest that cytokine treatment induces SMC death via apoptosis, whereas at high concentrations, H₂O₂ causes SMC death via primary necrosis or oncosis, a form of nonprogrammed cell death.²⁵ Rat SMCs similarly treated developed pyknotic nuclei as well (Fig 5f).

View larger version (135K): [in this window] [in a new window]   Figure 4. Photomicrographs. a through d, Human SMCs cultured in medium containing 10% FCS were incubated with or without IFN- (500 U/mL), TNF- (500 U/mL), and IL-1ß (100 U/mL). After 72 hours, cells were observed by phase-contrast microscopy. b and d, Higher magnification of squares in a and c, respectively (original magnification x100 [a and c]; x400 [b and d]). e through h, Rat SMCs cultured in medium containing 10% FCS were incubated with or without IFN- and TNF- (400 U/mL each) or with 0.1% H2O2. After incubation, cells were observed by phase-contrast microscopy (original magnification x400 [e through h]).

View larger version (98K): [in this window] [in a new window]   Figure 5. Photomicrographs showing nuclear morphology of cytokine-treated human and rat SMCs stained with YOYO-1. Human (a through d) and rat (e and f) SMCs cultured in eight-chamber slides were incubated with or without IFN- (400 U/mL), TNF- (400 U/mL), and IL-1ß (100 U/mL). After 72 hours, cells were fixed, permeabilized with 0.5% Tween 20, and stained with 1 µg/mL YOYO-1. Arrows indicate fragmented nuclei (original magnification x1000).

Accumulation of Oligonucleosomes in Vascular SMCs Undergoing Apoptosis
Cells undergoing apoptosis can release mononucleosomes or oligonucleosomes comprising DNA fragments and histones from their nuclei into the cytoplasm or even into the extracellular compartment.^{26 27} We examined the accumulation of oligonucleosomes in the cytokine-treated cells by using ELISA with anti-histone and anti-DNA antibodies to verify the occurrence of apoptosis. We observed a significantly increased accumulation of oligonucleosomes in human SMCs treated simultaneously with IFN-, TNF-, and IL-1ß for 48 hours (Fig 6). In contrast, few oligonucleosomes accumulated in the untreated cultures (Fig 6). In the presence of TNF- (400 U/mL) and IL-1ß (100 U/mL), IFN- increased accumulation of oligonucleosomes in human SMCs in a concentration-dependent manner (Fig 7).

View larger version (15K): [in this window] [in a new window]   Figure 6. Bar graph. Human SMCs were cultured in 24-well plates and treated with IFN- (400 U/mL), TNF- (400 U/mL), and IL-1ß (100 U/mL). After treatment, cells were permeabilized and centrifuged at 20 000g. Oligonucleosomes in the supernatants were determined by ELISA. Data are mean±SEM of four to six cultures. *P<.05; **P<.01.

View larger version (15K): [in this window] [in a new window]   Figure 7. Bar graph. Human SMCs were cultured in 24-well plates and treated with IFN- at 0, 50, 100, 200, 400, or 800 U/mL in the presence of TNF- (400 U/mL) and IL-1ß (100 U/mL). After treatment, cells were permeabilized, and media were collected and centrifuged at 20 000g. Oligonucleosomes in the combined supernatants were determined by ELISA. Data are mean±SEM of four to six cultures. *P<.05; **P<.01.

We further visualized DNA fragments in situ by using the TUNEL technique. Few cells (<5%) in the cultures with or without each cytokine alone showed TUNEL staining (Fig 8). However, 34±4% of human SMCs in the cultures cotreated with IFN-, TNF-, and IL-1ß for 48 hours showed positive staining with TUNEL (P<.05 versus untreated controls, n=3) (Fig 8). Some cells with TUNEL-positive nuclei also showed TUNEL staining in their cytoplasm, indicating the presence of cytoplasmic DNA fragments in the cells undergoing apoptosis (Fig 8).

View larger version (143K): [in this window] [in a new window]   Figure 8. Photomicrographs of in situ detection of DNA fragments. Human SMCs treated with IFN- (400 U/mL), TNF- (400 U/mL), and IL-1ß (100 U/mL) were fixed, washed, and stained by TUNEL. The brown color as developed in a peroxidase substrate system represents the positive areas. Quantification of nuclear labeling was performed by counting the percentage of TUNEL-positive cells in 200 cells in random fields for each condition. Arrows indicate TUNEL-positive nuclei in c and d (original magnification x400).

We also evaluated internucleosomal DNA fragmentation, a biochemical marker for apoptosis, by assessing the size of genomic DNA isolated from the cytokine-treated and untreated SMCs by using agarose gel electrophoresis (Fig 9). Most DNA extracted from both human (Fig 9A) and rat (Fig 9B) SMCs showed a high molecular weight (>20 kb). Internucleosomal DNA fragments at 180 to 200 bp or multiples appeared in SMCs treated simultaneously with IFN-, TNF-, and/or IL-1ß (Fig 9), indicating the occurrence of apoptosis in these cells. In contrast, no appreciable levels of DNA fragmentation existed in the untreated control cells (Fig 9).

View larger version (37K): [in this window] [in a new window]   Figure 9. Electrophoretic analysis of internucleosomal DNA fragmentation in cytokine-treated human and rat SMCs. Genomic DNA was isolated by phenol/chloroform extraction from human (A) and rat (B) SMCs treated with or without IFN- (500 U/mL), TNF- (500 U/mL), IL-1ß (100 U/mL), L-NMMA appears in both panels, but the original legend specifies the amount only for panel B.and/or L-NMMA (500 µmol/L) for 72 hours. DNA (10 µg/lane) was loaded into 1.5% agarose gels containing ethidium bromide. After electrophoresis, DNA bands were visualized under UV light. + and indicate presence and absence, respectively. Lane 7 in B shows DNA size markers.

NO Synthesis and Apoptosis in Vascular SMCs Stimulated With Cytokines
The cytokines IFN-, TNF-, and IL-1ß can together elicit expression of the inducible form of NOS by vascular SMCs.¹⁰ Such augmented NO synthesis can mediate apoptosis of murine macrophages²⁸ and human chondrocytes.²⁹ We therefore tested whether enzymatically synthesized NO mediates cytokine-induced apoptosis in vascular SMCs. In rat SMCs, stimulation with a combination of IFN- (400 U/mL) and TNF- (400 U/mL) induced high levels of nitrite accumulation (Fig 10). However, human SMCs so treated did not produce substantial amounts of NO in response to the same cytokines (Fig 10).

View larger version (30K): [in this window] [in a new window]   Figure 10. Bar graph. Rat and human vascular SMCs were incubated with IFN- and TNF- (400 U/mL each) for 48 hours. After incubation, 100 µL medium of the cultures was mixed with an equal volume of Griess reagent. Nitrite concentration in the medium was determined spectrophotometrically at 540 nm with sodium nitrite as standard. Data are mean±SEM of four cultures. *P<.05; **P<.01.

In rat SMCs cotreated with IFN- and TNF-, the addition of the NOS inhibitor L-NMMA (500 µmol/L) significantly increased the number of viable cells as demonstrated by flow cytometry (Fig 3) and fluorescent microscopy (Table) but diminished nitrite accumulation (Fig 10). L-NMMA also inhibited internucleosomal DNA fragmentation in the cytokine-cotreated rat SMCs (Fig 9B). However, this NOS inhibitor did not completely block the cytokine-induced apoptosis. Furthermore, L-NMMA did not appear to alter the viability (Table) of the cytokine-treated human vascular SMCs, which produced little nitrite. This result suggests that cytokines induce apoptosis of vascular SMCs by both NO-dependent and -independent pathways.

View this table: [in this window] [in a new window]   Table 1. Cell Viability of Human and Rat SMCs Treated With IFN- and TNF- in the Presence or Absence of L-NMMA

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Apoptosis appears to require an exogenous signal and a complex endogenous gene-controlled apoptotic machinery.^{30 31} We sought here to test whether cytokines produced by inflammatory cells trigger death of vascular SMCs. By using a combination of techniques for analyzing markers for apoptosis, we now demonstrate that when combined, the cytokines IFN-, TNF-, and IL-1ß can induce apoptosis of cultured human and rat SMCs. The data support the notion that SMCs in atherosclerotic lesions may undergo apoptosis in response to the proinflammatory cytokines produced locally by activated macrophages and T lymphocytes as a consequence of the ongoing local immune and inflammatory response characteristic of atherogenesis.¹⁵

Vascular SMCs exhibit heterogeneity both in vivo and in vitro. Normal arterial intima contain very small numbers of SMCs. During atherogenesis, increased numbers of SMCs exist in the thickening intima; these SMCs show substantial differences in gene expression and function from medial SMCs. However, many in vitro and in vivo studies^{2 3} argue that in in vitro culture, medial SMCs can undergo modification during passages and gain some of the biological properties of neointimal SMCs during atherogenesis; thus they are widely used as target cells in in vitro experiments.

It is likely that different subpopulations of SMCs may respond differently to factors such as cytokines. We found that some SMCs survived cytokine treatment even at high concentrations while their neighbor cells died and that the surviving cells showed a morphology distinct from that of untreated cells. Recent studies on the effects of IFN- on c-myc–transfected rat SMCs demonstrate that both apoptotic and mitotic figures can exist in the same cell cultures.²² These findings indicate that cytokines may selectively act on certain subpopulations of SMCs, thus leading to depletion of these cells from tissues or modulating their phenotype.

Prolonged exposure to IL-1 alters the morphology and growth pattern of human SMCs, resulting in accumulation of cells with an elongated, bipolar shape.⁷ Treatment with IFN- suppresses expression of –smooth muscle actin in rat SMCs⁹ while augmenting the expression of major histocompatibility complex class II genes in both rat⁴ and human³² SMCs. In atherosclerosis, SMCs in the intimal lesions differ from those of the tunica media in phenotype, but the underlying mechanism remains uncertain. Cytokines produced from and evoked by infiltrating immune cells are likely involved in phenotypic change of SMCs by inducing apoptosis of certain subtypes of SMCs that are sensitive to the cytokines.

Our experimental results indicate that the individual cytokines alone do not suffice to cause apoptosis of either rat or human SMCs. Exposure to either IL-1^{6 7} or TNF-⁸ can stimulate SMC proliferation, whereas IFN-^{4 5 9} inhibits SMC proliferation. These findings suggest that these cytokines may activate different signal transduction pathways that synergize or antagonize each other in regulation of cell turnover.

Several different signal transduction pathways may mediate the induction of apoptosis by these proinflammatory cytokines. Among them, the L-arginine/NOS pathway may serve as a potential trigger of apoptosis, as many studies have demonstrated that IFN-, TNF-, and IL-1, alone or synergistically, induce synthesis of NO from L-arginine in various types of rodent cells, including vascular SMCs.¹² We observed that cytokine-stimulated, NO-generating rat SMCs died via apoptosis, and addition of the NOS inhibitor L-NMMA could partially block these cytokine effects. This result agrees well with the data from recent studies showing that NO mediates apoptosis of murine macrophages^{28 33} and their tumor target cells,³⁴ rat pancreatic beta-cells,¹⁹ and human chondrocytes.²⁹

However, it remains unclear how activation of NO synthesis leads to apoptosis in the cytokine-treated cells. We have shown that NO can form iron (II)–nitrosyl complexes with respiratory enzymes containing nonheme iron–sulfur clusters in mitochondria of rat SMCs and consequently inhibit respiration and ATP synthesis in cytokine-treated SMCs.^{10 11} NO also inactivates enzymes important for DNA synthesis and repair.¹² Recently, NO generated by cytokine-inducible NOS was found to stimulate the expression of the tumor suppressor gene p53 in RAW 264.7 macrophages and pancreatic RINm5F cells before apoptosis.³⁵ Expression of p53 can lead to cell death via apoptosis in various normal and malignant cell types.³⁵ These observations suggest that high levels of NO production catalyzed by cytokine-inducible NOS may promote apoptosis by multiple mechanisms.

Under the same experimental conditions, however, human vascular SMCs do not produce any appreciable levels of NO in response to IFN-, TNF-, and IL-1ß, in accord with recent work from other laboratories.³⁶ A similar situation exists in cultured human macrophages, which, unlike rodent macrophages, produce little NO when exposed to the cytokines.^{37 38 39} However, human hepatocytes⁴⁰ and chondrocytes⁴¹ can express inducible NOS and synthesize considerable amounts of NO in response to cytokine stimulation. Apparently, differences in cytokine induction of NO exist between species and cell types. The relatively high capacity for NO synthesis in rodent SMCs may partly explain why the rodent cells die via apoptosis when exposed to cytokines more readily than do human SMCs, which require more stringent conditions for NO production. Nonetheless, stimulation with the cytokines can provoke apoptosis of human SMCs even in the absence of NO production, and blocking of NO synthesis with L-NMMA cannot completely abolish apoptosis of rat SMCs induced by cytokines. It is likely that both NO-dependent and -independent mechanisms mediate the cytokine-induced apoptosis of vascular SMCs.

Much prior work on the cell biology of atherosclerosis has focused on SMC proliferation.³ During atherogenesis, arterial SMCs migrate from the tunica media to the intima, where they proliferate and synthesize extracellular matrix, contributing to focal thickening of the intima. In this context, apoptosis may serve as an adaptive mechanism to limit excessive cell replication and thickening of the intima. However, in advanced atheroma, SMC synthesis of extracellular matrix may actually stabilize the plaque structure. A high level of apoptotic cell death might impair maintenance of this matrix scaffolding of the fibrous cap of the plaque and thus predispose to plaque rupture, a major cause of acute atherosclerotic syndromes such as acute myocardial infarction or unstable angina. Therefore, dysregulation of apoptosis may influence both formation and complication of atherosclerosis and thereby provide a new therapeutic target.

	Selected Abbreviations and Acronyms

 

ELISA = enzyme-linked immunosorbent assay FCS = fetal calf serum IFN- = interferon- IL = interleukin L-NMMA = NG-monomethyl-L-arginine NO = nitric oxide NOS = nitric oxide synthase PBS = phosphate-buffered saline SMC = smooth muscle cell TNF- = tumor necrosis factor– TUNEL = terminal deoxyribonucleotide transferase–mediated dUTP nick end labeling YOYO-1 = benzoxazolium-4-quinolinium dimer

	Acknowledgments

 
This work was supported by National Institutes of Health grant HL-34636, the Swedish Medical Research Council (project No. 6816), and the Swedish Medical Heart-Lung Foundation. Yong-Jian Geng is a recipient of the ICRETT Award granted by the International Union Against Cancer, Geneva, Switzerland.

Received July 7, 1995; accepted September 15, 1995.

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