Overexpression of Inducible Nitric Oxide Synthase by Neointimal Smooth Muscle Cells

From the Center for Molecular Medicine, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden.

Correspondence to Dr Göran K. Hansson, Center for Molecular Medicine (L8:03), Karolinska Hospital, S-17176 Stockholm, Sweden. E-mail Goran.Hansson{at}cmm.ki.se

	Abstract

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Abstract—The formation of a neointima represents an important repair mechanism in response to vascular injury. It is associated with the expression of a specific set of genes by the intimal smooth muscle cells. Recently, expression of the inducible isoform of NO synthase (iNOS) has been identified in injured arteries during neointimal formation, suggesting that intimal SMCs have a unique mechanism for regulating NO production. Therefore, we have analyzed the expression of iNOS in intimal SMCs. Although first expressed in the media within 1 day after injury, iNOS was confined to neointimal smooth muscle cells at 1 to 2 weeks after injury. Isolated intimal SMCs were found to consistently reexpress iNOS in reaction to proinflammatory mediators. This was associated with a 5- to 8-fold higher output of NO in comparison with SMCs derived from the media of uninjured arteries. Western blot and Northern blot analyses likewise revealed that the high production of NO by intimal SMCs was due to overexpression of iNOS. Moreover, the same stimuli induced a higher transcriptional activity in intimal than in medial SMCs, as detected by transfection of a reporter gene under the iNOS promoter. Induction of iNOS led to a reduced proliferation in both medial and intimal SMCs. This inhibitory effect was, however, less pronounced in intimal than in medial SMCs. Similarly, intimal cells were less sensitive to NO-induced inhibition of mitochondrial respiration. When SMC clones were analyzed, there was no correlation between iNOS expression and growth pattern, suggesting that iNOS expression is independent of the morphological phenotype of SMCs. Together, our data show that the intimal SMC is the main iNOS-expressing cell type in the injured artery, that it responds more vividly to iNOS-inducing cytokines because of a more efficient activation of the iNOS promoter, and that it is more resistant to the actions of NO compared with medial SMCs. Intimal production of NO via the inducible pathway may be important for the restoration of vascular homeostasis after injury.

Key Words: nitric oxide • nitric oxide synthase (EC 1.14.13.39) • restenosis • vascular smooth muscle cell • transcriptional regulation

	Introduction

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Vascular smooth muscle cells form a neointima as a response to tissue injury. For instance, removal of the arterial endothelium and intima by a balloon catheter results in SMC migration from media to surface and, subsequently, an intense phase of cell proliferation and matrix synthesis in the developing neointima.^{1 2 3 4 5} Although the molecular mechanisms responsible for the formation of the neointima are far from elucidated, numerous studies have shown that the proliferative process is stimulated by the growth factors, basic fibroblast growth factor and platelet-derived growth factor.^{1 2 3 4 5}

A remarkable feature of the neointima is that, although lacking endothelium, it rapidly acquires two important features of the endothelialized artery, ie, a nonthrombogenic surface and a relaxed vascular tone. These two phenomena are mediated by endothelium-derived NO, and we have proposed that neointimal SMCs compensate for the loss of endothelium by producing their own NO. In support of this hypothesis, we have shown that an inducible isoform of NO synthase (iNOS) is rapidly induced in SMCs in vivo during the formation of the neointima.^{6 7} Furthermore, NO is produced in large amounts by the injured artery,^{6 8} and inhibition of NO production results in increased vascular tone and loss of nonthrombogenicity.⁷ Furthermore, iNOS may have important regulatory functions in the vessel wall, since NO can inhibit SMC proliferation⁹ and regulate the expression of several genes, including interleukin-12¹⁰ and the vascular cell adhesion molecule-1.¹¹

iNOS was originally identified in cytokine-activated macrophages.^{12 13} However, studies by us and others have shown that SMCs as well as several other cell types respond to proinflammatory cytokines by transcribing the same iNOS gene as the macrophage.^{14 15 16} Since iNOS is expressed during neointimal formation but not in the normal arterial wall and since intimal SMCs exhibit a different phenotype and express a set of genes that is not expressed by SMCs of the media,^{2 5 17} the capacity to express iNOS may be related to the acquisition of the neointimal phenotype.

In the present study, we have examined iNOS expression in neointimal SMCs in vivo and in vitro. Our data indicate that intimal SMCs produce higher levels of NO in response to proinflammatory cytokines than medial SMCs. This was due to an enhanced responsiveness of the iNOS promoter in the intimal cells, which were also relatively resistant to the actions of NO. We propose that NO production via the inducible pathway is a central part of the response-to-injury program of the arterial wall.

	Materials and Methods

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Materials
Recombinant murine IFN- was purchased from PharMingen, and recombinant murine TNF- and IL-1ß were from Genzyme. Bacterial LPS (from Escherichia coli serotype O55:B5) and L-NMMA were from Sigma Chemical Co. All reagents, except the LPS itself, contained <0.012 ng/mL endotoxin by the Limulus amebocyte lysate assay.

Cell Culture and SMC Cloning
SMCs were isolated from the medial layer of the thoracic aorta of 6-week-old male Sprague-Dawley rats, and intimal SMCs were derived from the neointimal thickening of the aorta or carotid artery 2 weeks after balloon angioplasty.

Cell cultures were maintained in DMEM supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (DMEM/10% FCS), 1 mmol/L L-glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin. Cells from passages 5 to 10 were used for the present studies.

SMC clones were established from normal medial SMC cultures at passage 2 by limiting dilution (at an average of 0.25 cells per well) in 96-well plates in DMEM containing 20% FCS. All wells were inspected every other day for 2 weeks to remove any well suspected to contain more than one clone. Forty clones were selected and passed to 24-well plates and then to 6-well plates. For phenotypic characterization, cloned cells were grown on coverslips, fixed with methanol, and stained with a monoclonal antibody to -smooth muscle actin (Sigma) followed by FITC-labeled anti-mouse IgG.

Nitrite Assay
The accumulation of NO₂^-, a stable end product of NO formation, in conditioned medium was measured as an indicator of NO production by SMCs.¹⁴ NO₂^- in the samples was calculated from a standard curve of sodium nitrite. For comparison of NO production by intimal and medial SMC, NO₂^- values were normalized by the cell number assessed by cell counting in a Coulter counter after trypsinization of parallel cultures.

Immunohistochemistry
Male Sprague-Dawley rats (average weight, 400 g; 3 to 4 months old) were used in the experiments, which were approved by the regional ethical committee for animal research. The left carotid artery was denuded as described using a Fogarty 2F balloon catheter (Baxter Healthcare Corp).⁷ For studying expression and distribution of iNOS, animals were killed under fluanisone/fentanyl citrate anesthesia at the indicated time points after injury. Tissues were snap-frozen in liquid nitrogen and kept at -80°C for immunohistochemistry. Serial cryostat sections (10 µm) were air-dried and fixed for 10 minutes in cold acetone at -20°C. The sections were preincubated with 5% normal swine serum to block nonspecific antibody binding and then incubated overnight at 4°C with polyclonal rabbit antibodies against murine macrophage iNOS (Affiniti BioReagents) diluted 1:500 with PBS. Subsequently, sections were incubated with a biotinylated swine anti-rabbit IgG (Dako) for 30 minutes at room temperature. An avidin-biotin-immunoperoxidase system and 3,3'-diaminobenzidine (Vector Laboratories) were used to detect the antigen-antibody complexes.

Western Blot Analysis
SMCs were lysed with Laemmli sample buffer and denatured by boiling for 5 minutes. The protein concentration was determined using a bicinchonic acid kit (Pierce). For Western blot analysis, 10 µg of protein per lane was separated on 7.5% SDS-polyacrylamide gels and electroblotted to PVTC membranes (Amersham). The membrane was blocked in 5% nonfat dry milk dissolved in TTBS (150 mmol/L NaCl, 10 mmol/L Tris-HCl, and 0.1% Tween 20, pH 7.4) and subsequently incubated for 1 hour at room temperature with a monoclonal antibody against macrophage iNOS (Transduction Laboratory), followed by incubation for 1 hour with horseradish peroxidase–conjugated sheep anti-mouse Ig F(ab') fragments (Amersham). Immunoreactive bands were visualized using an enhanced chemiluminescence kit (Amersham). Quantification was by densitometric scanning of bands on the developed film.

RNA Isolation and Northern Blot
Total RNA was extracted from cells with an RNA isolation kit (Pharmacia). RNA was size-fractionated in 1% agarose gels containing 0.66 mol/L formaldehyde and transferred to Hybond-N nylon membranes (Amersham). A 4119-bp full-length iNOS cDNA¹⁶ was labeled with [-³²P]dCTP using a random-priming DNA labeling kit (Amersham). Filters were prehybridized for 2 to 5 hours at 42°C with a solution containing 5x SSPE (1x SSC contains 150 mmol/L sodium chloride, 10 mmol/L sodium phosphate, and 1 mmol/L EDTA), 5x Denhardt's solution, 50% deionized formamide, 100 µg/mL salmon sperm DNA, and 0.1% SDS and hybridized overnight at 42°C in the same buffer containing 1x10⁶ cpm/mL denatured probe. After hybridization, the filters were washed twice for 10 minutes at room temperature with 2x SSPE plus 0.1% SDS, for 20 minutes at 65°C with 1x SSPE plus 0.1% SDS, and for 15 minutes at 65°C with 0.1x SSPE plus 1% SDS before autoradiography. To normalize hybridization signals for variations in loading and/or transfer, filters were initially visualized for 18S rRNA by methylene blue staining.

Transfection and CAT Assay
For transfection of SMCs with iNOS promoter constructs, cells (50% confluent) were preincubated in OptiMEM medium (Life Technologies) for 2 hours at 37°C. Cells were transfected with 2 µg of an iNOS promoter–CAT reporter plasmid DNA (piNOS-CAT, Oxford Biomedical Research, Inc) or 2 µg of pCAT 3–basic vector (Promega) by using lipofectin (Life Technologies). pSVß-galactosidase vector (2 µg, Promega) was cotransfected to all samples as an internal control for the transfection efficiency. Twenty hours after transfection, cells were treated for 20 hours with a combination of 100 U/mL IFN- and 10 µg/mL LPS in DMEM with 0.4% FCS. Cell extracts were prepared by three cycles of freeze-thawing and applied for measurement of CAT activity by thin-layer chromatography. ß-Galactosidase activity was measured spectrophotometrically at 420 nm by the generation of o-nitrophenol from o-nitrophenyl-ß-D-galactopyranoside. All data were normalized as CAT activity units/ß-galactosidase activity.

Proliferation Assay
Cells were plated at 2x10⁴ per well in a 24-well plate and allowed to attach in DMEM/10% FCS. After attachment, they were washed with PBS and growth-arrested by replacing the medium with DMEM/0.5% FCS. After 48 hours of growth arrest, cells were released from G₀ by the addition of DMEM/10% FCS and simultaneously exposed to IFN- or a combination of IFN-+TNF-. At the indicated time points, cells were released from the dish by trypsinization, and the cell number was determined by a Coulter counter.

MTT Assay
For evaluation of mitochondrial respiration in SMCs, the MTT reduction assay was performed. SMCs (7x10³ per well) were seeded in 96-well plates containing 100 µL of DMEM/10% FCS. MTT (0.1 mg/mL) was added to each well and incubated for 2 hours at 37°C. Thereafter, the culture medium was removed, and the cells were solubilized in 100 µL dimethyl sulfoxide. The extent of reduction of MTT to formazan within cells was quantified at spectrophotometrically at 540 nm and taken as an indicator of cellular respiration. The average absorption in three wells with 7x10³ SMCs treated with medium alone was referred to as 100% conversion of MTT. Viability was assessed by trypan blue staining of parallel cultures.

Statistical Analysis
Results are reported as mean±SEM. Student's t test was used to evaluate the statistical differences between means, and values of P<.05 were considered significant. Nitrite production by SMC clones was analyzed by testing differences between median values using Fisher's exact test.

	Results

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Expression of iNOS in the Injured Artery In Vivo
Arterial injury was induced by balloon catheter denudation of the rat common carotid artery, and iNOS expression in the injured artery was followed by immunohistochemistry. Enzyme protein was detectable in medial SMCs as early as 1 day after injury (Fig 1a). When SMCs migrated from media to intima during the first week, iNOS expression was similarly translocated from media to intima (Fig 1b). By 2 weeks after injury, iNOS expression was still intense and confined to the neointimal thickening (Fig 1c). Staining intensity decreased by 4 weeks and was localized to the innermost layer of the neointima, adjacent to the lumen of the artery (Fig 1d). These data imply that the expression of iNOS is closely correlated with the development of the neointima; thus, they suggest that SMCs composing the neointima constitute the major cell population capable of expression of iNOS in the injured artery.

View larger version (128K): [in this window] [in a new window]   Figure 1. Expression of iNOS in the injured carotid artery. iNOS protein (immunoperoxidase, brown color) was detected by immunohistochemistry 1 day (a), 1 week (b), 2 weeks (c), and 4 weeks (d) after balloon injury. Nuclei were counterstained with hematoxylin. iNOS protein was rapidly induced in the media after injury (a) and then relocalized to the intima in parallel with the development of the neointimal thickening (b and c). By 4 weeks after injury (d), iNOS expression had declined and was limited to the "pseudoendothelial" surface–forming intimal SMCs. Arrowheads indicate the internal elastic lamina. Magnification x200.

Induction of iNOS in Intimal SMCs
To examine the hypothesis that intimal SMCs are a major source of NO, SMCs were isolated from the neointima of the thoracic aorta and carotid arteries 2 weeks after injury. First, we determined whether intimal SMCs were still able to express iNOS in vitro. Inducible expression of iNOS in intimal SMC cultures from three separate isolates was tested from passages 4 to 7. As shown in Fig 2A, intimal SMCs did not produce NO under baseline conditions. They were, however, able to produce large amounts of NO on stimulation with IL-1ß or a combination of IFN- and LPS, indicating that intimal SMCs preserve the ability to reexpress iNOS in vitro. Northern analysis using rat SMC iNOS cDNA¹⁶ as a probe revealed a 4.1-kb mRNA species (Fig 2B) expressed in the intimal SMC cultures treated with the above-mentioned cytokines. However, no apparent iNOS mRNA or activity was detected in the intimal cultures in the absence of cytokine stimulation (Fig 2A and 2B). This confirms the fact that intimal SMCs do not express iNOS constitutively in culture.

View larger version (25K): [in this window] [in a new window]   Figure 2. Reexpression of iNOS by intimal SMCs in vitro. A, Kinetics of NO production by intimal SMCs in response to proinflammatory mediators. Intimal SMCs derived from three separate preparations were tested. Subcultured intimal SMCs from passage 4 to 5 were treated with IL-1ß (10 ng/mL) or a combination of IFN- (300 U/mL) and LPS (10 µg/mL) for the indicated time. Conditioned media were analyzed for nitrite. Data are presented as mean±SEM of three separate experiments. B, Expression of iNOS mRNA (top) and 18S rRNA (bottom) by intimal SMCs stimulated with IFN- (100 U/mL) and LPS (10 µg/mL) or IL-1ß (5 ng/mL). Lanes are as follows: 1, untreated intimal SMCs; 2 and 3, IFN- and LPS for 6 or 24 hours, respectively; and 4, IL-1ß for 6 hours. Total RNA (15 µg) was subjected to Northern blot analysis of iNOS mRNA as described; 18S rRNA was visualized by methylene blue staining.

Comparison of iNOS Expression in Intimal and Medial SMCs
To investigate whether intimal SMCs could be the major cellular source of iNOS, we compared the regulation of iNOS in intimal SMCs with that of medial SMCs derived from uninjured vessels. Intimal SMCs produced markedly higher nitrite levels than did medial SMCs on stimulation with IFN- and LPS (Fig 3A). Moreover, LPS alone was able to induce a moderate iNOS expression in the intimal SMCs but had no effect on the medial SMCs (Fig 3B), implying qualitative differences between the two cell types in the signal transduction machinery necessary for iNOS induction. Treatment with IFN- alone was, however, not sufficient to induce iNOS expression in either cell type (data not shown). The high iNOS activity in intimal SMCs detected by the nitrite assay was corroborated by the results of Western analysis, which showed a 3-fold larger accumulation of immunoreactive iNOS protein in intimal SMCs than in medial SMCs after stimulation with IFN- and LPS (Fig 4, top). Northern analysis demonstrated that intimal SMCs also expressed more iNOS mRNA than did medial SMCs on identical stimulation (Fig 4, bottom). Finally, a single stimulus, ie, IL-1ß, was sufficient to induce iNOS mRNA in intimal SMCs, whereas medial SMCs required the synergistic action of IFN- and either IL-1ß, TNF-, or LPS (Fig 2B and data not shown). Taken together, these results reveal that intimal SMCs express significantly more iNOS in response to proinflammatory mediators.

View larger version (11K): [in this window] [in a new window]   Figure 3. iNOS activity in intimal and medial SMCs. Intimal SMCs and medial SMCs were treated with a combination of IFN- (100 U/mL) and LPS (10 µg/mL) (A) or LPS (10 µg/mL) alone (B) for the indicated time. Conditioned media were subsequently isolated and analyzed for nitrite. Results are mean±SEM of three separate experiments. *P<.05 vs medial SMCs.

View larger version (54K): [in this window] [in a new window]   Figure 4. Expression of iNOS mRNA and protein in intimal SMCs. Top, iNOS protein expression was assessed by Western blot analysis with a monoclonal anti-iNOS antibody. Intimal and medial SMCs derived from two separate isolations were tested. Protein was extracted from cells exposed for 24 hours to IFN- (100 U/mL) plus LPS (10 µg/mL). Protein (8 µg) was loaded in each lane. Lanes are as follows: 1, 2, 5, and 6, intimal SMCs; 3, 4, 7, and 8, medial SMCs. Bottom, iNOS mRNA was analyzed by Northern blot as described in the legend to Fig 2B. Intimal and medial SMCs were maintained in quiescence medium (DMEM plus 0.5% FBS) and exposed to IFN- (100 U/mL) plus LPS (10 µg/mL) for 6 hours. Lanes 1 and 3 show untreated medial and intimal SMCs, respectively; lanes 2 and 4 show treated medial and intimal SMCs, respectively. 18S rRNA was visualized by methylene blue staining. Data illustrated are representative of three separate experiments.

Induction of iNOS in SMC Clones
Several lines of evidence suggest that the arterial media is composed of a heterogeneous population of SMCs, with some SMC cultures derived from the media exhibiting many similarities to intimal SMCs.^{17 18} We thought that intimal SMCs might be recruited from a subpopulation of SMCs in the media; this would explain the rapid expression of iNOS in the media after injury. To investigate this, we established 42 clones from normal medial SMC cultures using a limiting dilution approach. On the basis of cell morphology and growth pattern, the clones could be divided into three categories, with cobblestone-shaped, spindle-shaped, or senescent features (Table). Both the cobblestone-shaped and the spindle-shaped clones expressed -smooth muscle actin, indicating that they were of smooth muscle origin. Induction of iNOS in these clones was followed for three passages to determine the stability and reproducibility in the expression of this gene in response to IFN- and LPS. The senescent clones, which were highly vacuolated and often multinucleated, grew too slowly to permit further characterization As shown in Fig 5a, the majority of spindle-shaped clones showed low iNOS activity on stimulation, ranging from 0 to 10 nmol/10⁶ cells after 24 hours of exposure to IFN- (50 U/mL) and LPS (5 µg/mL). Among cobblestone-shaped clones, there was substantial variation in NO production (Fig 5b), but median values of nitrite were not significantly different between the two clonal phenotypes (10.5 versus 9.5 nmol/10⁶ cells for spindle-shaped versus cobblestone-shaped clones). However, cobblestone-shaped clones appeared to be composed of two types of cells, one characterized by high and the other one by low expression of iNOS (Fig 5b).

View larger version (26K): [in this window] [in a new window]   Figure 5. Induction of iNOS in Cloned SMCs. SMC clones were established from a polyclonal population of normal arterial SMCs. Cloned SMCs were exposed to IFN- (50 U/mL) and LPS (5 µg/mL), and nitrite levels were determined in conditioned media 24 hours after treatment. Results are representative of three separate experiments. a, Histogram showing iNOS activity in spindle-shaped clones. b, Histogram showing iNOS activity in cobblestone-shaped clones. c, Western blot analysis of iNOS protein in cloned SMCs. Protein was extracted from six clones treated for 24 hours with IFN- (50 U/mL) plus LPS (5 µg/mL), and 6 µg of protein was loaded in each lane. SMC indicates polyclonal medial SMC exposed for 24 hours to IFN- (100 U/mL) plus LPS (10 µg/mL) serving as a positive control.

The heterogeneity of intimal SMCs with regard to iNOS induction was further supported by Western blot analysis. As shown in Fig 5c, there was substantial variation in iNOS protein between SMC clones treated identically with IFN- and LPS. These data suggest that the capacity to express high levels of iNOS on stimulation is not linked to a specific morphological phenotype of SMCs in the media.

Activity of iNOS Promoter/Enhancer in Intimal and Medial SMCs
Given the observations that intimal SMCs demonstrated high iNOS expression and that LPS alone was able to induce iNOS in intimal SMCs, we presumed that intimal SMCs may differ from medial SMCs with regard to transcriptional regulation of the iNOS gene. To test this possibility, intimal and medial SMCs were transfected with a plasmid construct containing 1749 bp of the 5' flanking region of the murine iNOS gene fused to a CAT reporter gene.¹⁹ This region of the iNOS gene contains the basal promoter/enhancer region, with several NF-B and IFN- response elements.¹⁹ Transfected cells were stimulated with IFN- and LPS, and CAT activity was assessed to evaluate promoter/enhancer activity.

As shown in Fig 6, both intimal and medial SMCs transfected with iNOS/CAT displayed CAT activity after stimulation with IFN- and LPS. However, the promoter activity in the intimal SMCs was 3-fold higher than that of the medial SMCs. The promoter activity in the respective cell type was thus compatible with the activity of the endogenous iNOS gene (Fig 4). The difference in promoter activity between the two cell types was unlikely to be due to differences in transfection efficiency, since no significant differences in the transfection efficiency of iNOS/CAT or other reporters were detected.

View larger version (84K): [in this window] [in a new window]   Figure 6. Activation of the iNOS gene promoter in intimal and medial SMCs by IFN- and LPS. Intimal SMCs (lanes 1 to 4) and medial SMCs (lanes 5 to 8) were transiently transfected with piNOS-CAT and then exposed for 20 hours to 100 U/mL IFN- and 10 µg/mL LPS. All constructs were cotransfected with pSVß-galactosidase vector to correct for transfection efficiency. CAT activity was assayed by thin-layer chromatography. Data shown are representive of three experiments.

Effects of Endogenous NO on the Proliferation and Mitochondrial Respiration of SMCs
To evaluate the effect of endogenous NO generated from iNOS on the expressing cell, iNOS was induced by stimulating SMCs with IFN- and TNF-, and cell proliferation was determined in the presence or absence of the NO synthase inhibitor, L-NMMA. IFN-+TNF- significantly reduced the proliferation of both intimal and medial SMCs (Fig 7). The growth inhibition of medial cells could be partially prevented by the NO synthase inhibitor, L-NMMA, indicating that iNOS-derived NO inhibits the growth of these cells. In contrast, L-NMMA did not affect the proliferation of cytokine-treated intimal SMCs (Fig 7). This implies that in the latter cells, NO does not inhibit growth. Instead, the antiproliferative effect of IFN-+TNF- for these cells is likely due to NO-independent mechanisms such as the strong antiproliferative effect of IFN-.²⁰ This is supported by the observation that the addition of TNF- did not further inhibit the proliferation of IFN-–treated intimal SMCs (Fig 7). Taken together, these results suggest that intimal SMCs may be more resistant to the action of NO than medial SMCs.

View larger version (42K): [in this window] [in a new window]   Figure 7. Effect of autocrine NO production on the proliferation of intimal and medial SMCs. Intimal and medial SMCs were synchronized for 48 hours in starvation medium containing 0.1% FCS and then incubated for 48 hours with medium containing 10% FCS and the indicated additives. The cell number was determined by Coulter counting; 100% represents the growth rate of untreated intimal and medial SMCs. Each datum point is mean±SEM of three experiments performed with duplicate wells. *P<.05 for intimal vs medial SMCs under identical conditions. #P<.05 for identical culture treated with or without L-NMMA.

To further confirm this notion, mitochondrial function was determined in intimal and medial SMCs after a 48-hour exposure to IFN- and LPS. Mitochondrial respiration was reduced in both cell types but twice as much in medial compared with intimal SMCs (P<.05, Fig 8, top). In both intimal and medial SMCs, the antirespiratory effect could be ascribed to NO, since L-NMMA, an inhibitor of NO synthase, could prevent the inhibitory effect of IFN- and LPS (Fig 8, top). The relative resistance of intimal SMCs to NO occurred in spite of the fact that intimal SMCs concomitantly produced significantly more NO than did medial SMCs (Fig 8, bottom). These data thus show that intimal SMCs are equipped not only with a higher capacity to express iNOS on stimulation but also with a relative resistance to the product of this enzyme.

View larger version (28K): [in this window] [in a new window]   Figure 8. Effect of autocrine NO production on mitochondrial respiration. Intimal and medial SMCs were incubated for 48 hours with IFN- and LPS in the presence or absence of L-NMMA (3 mmol/L). Mitochondrial respiration for each cell culture was assessed by MTT assay of the cell pellet, and the corresponding medium was collected for measurement of nitrite concentration. Alterations of mitochondrial respiration in response to stimulation with IFN- and LPS are illustrated in the top panel, and the corresponding nitrite concentrations in conditioned media are shown in the bottom panel. Results are mean±SEM of three separate experiments. *P<.05 for intimal vs medial SMCs; #P<.05 for identical cultures treated with vs without L-NMMA.

	Discussion

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NO is an important mediator in the physiological regulation of vascular tone as well as in pathophysiological processes such as ischemia/reperfusion, atherosclerosis, and acute infections.^{21 22 23} In the latter case, iNOS gene expression is turned on, and the ensuing high output of NO may account for many events associated with inflammation. A better knowledge of iNOS gene expression in SMCs is therefore crucial to our understanding not only of the physiological importance of NO in response to certain regulatory signals but also of the pathological implications of its prolonged production. The findings reported in the present study suggest that intimal SMCs have unique features of iNOS regulation. First, intimal SMCs are capable of overexpressing iNOS and produce large amounts of NO in response to proinflammatory mediators. Second, this is due to a higher activity of the iNOS promoter/enhancer in intimal compared with medial SMCs, resulting in a lower requirement of external signals for the induction of iNOS transcription. Third, intimal SMCs, compared with medial SMCs, are relatively resistant to the antiproliferative and cytotoxic effects of NO. Thus, intimal SMCs are uniquely suited for producing high levels of NO during the response to injury and disease.

iNOS Expression and SMC Phenotype
The iNOS gene is rapidly turned on and remains expressed in the neointima during several weeks after arterial injury. When intimal SMCs are put into culture, iNOS rapidly disappears. However, it is avidly reexpressed on stimulation with proinflammatory mediators. The fact that iNOS disappears but can be reinduced suggests that its expression in vivo is not a mandatory component of a differentiation program leading to the intimal SMC phenotype. It may rather be due to the combined effect of a reduced threshold for iNOS induction and the presence of inducing stimuli in the neointima of the injured artery. Therefore, whereas the capacity to overexpress iNOS may be linked to SMC differentiation in response to injury, the actual transcription of the iNOS gene is not constitutive but requires stimuli that are present in the neointima and can be induced by the addition of proinflammatory cytokines and IFN- to cultured intimal SMCs.

A series of studies show that medial SMCs are heterogeneous with regard to morphology, gene expression, and growth pattern in culture.^{2 18 24 25} Two major cell types that differ in growth pattern, growth rate, and the expression of contractile proteins have been identified.^{17 18} These differences are maintained with subculturing, implying that these SMC phenotypes may represent stable differentiation stages. Intimal SMCs isolated from injured arteries are highly similar to one of these subpopulations, which is characterized by a cobblestone-like growth pattern and the lack of myosin heavy chain expression.¹⁸ In view of our observation that many intimal but only few medial SMCs express iNOS in the injured artery (References 6 and 7^{6 7} and the present study), we speculated that the capacity to express iNOS may be part of the "intimal phenotype."

Therefore, SMC clones were established from the arterial media, and iNOS expression and NO production were followed in "intima-like" cobblestone-shaped clones as well as "media-like" spindle-shaped clones. Whereas most of the spindle-shaped SMCs responded rather modestly to iNOS-inducing stimuli, cobblestone-shaped clones were highly heterogeneous. Some cobblestone-shaped SMCs exhibited strong iNOS induction, but there was no significant difference when the median NO production was compared between the two phenotypes. Therefore, the morphology and growth pattern do not, per se, predict the capacity of SMCs to express iNOS, and the vasoregulatory activity in inflammation is not directly linked to the cobblestone-like growth pattern. Instead, a strong iNOS response may be associated with a subpopulation of the cobblestone-shaped SMCs and/or with features of intimal SMCs that are not present in those media-derived SMCs that grow in an intima-like pattern.

Intimal SMCs Are Less Sensitive to NO Than Are Their Medial Counterparts
NO has several negative effects on SMCs. It inhibits their proliferation,⁹ switches their energy metabolism into the glycolytic pathway by blocking mitochondrial respiration,¹⁴ and can, at high concentrations, induce apoptosis.²⁶ These activities may be related, since inhibition of energy metabolism and mitochondrial function have been shown to be related to apoptosis.²⁷ On the molecular level, it is likely that these effects are exerted by reactions between the NO radical and Fe(II)-containing prosthetic groups of several enzymes, including ribonucleotide reductase,²⁸ aconitase, and complexes I and II of the mitochondrial respiratory chain.^{14 29} These enzymes play critically important roles in the nucleotide and energy metabolism, respectively, of all living cells.

One would therefore expect that intimal SMCs, which have a high proliferative and metabolic rate, should be more sensitive than medial SMCs to NO. It appeared paradoxical that intimal SMCs express more iNOS and produce more NO than do medial SMCs. This stimulated us to compare intimal and medial SMCs with regard to their sensitivity to NO. It was found that intimal SMCs are less sensitive both to the antiproliferative and antimitochondrial effects of iNOS-induced NO, in spite of a larger production of NO. This explains why intimal SMCs survive and grow under conditions of high NO output. Furthermore, it suggests that the major targets of intima-derived NO are not the intimal SMCs. In line with this, we⁷ have recently found that NO produced in the neointima in vivo inhibits the adhesion of platelets and reduces the arterial tone (which is controlled by medial SMCs). Finally, it implies that important differences in gene regulation may exist between the two kinds of SMCs.

Transcriptional Regulation of iNOS in Intimal SMCs
Studies using immunohistochemistry, in situ hybridization, reverse-transcriptase polymerase chain reaction, and activity analyses have conclusively shown that iNOS is expressed in the intimal SMCs of the injured artery (References 6⁶ to 8 and the present study). This could be due to either the presence of iNOS-inducing stimuli in the neointima or a continuous expression of iNOS as a component of the intimal SMC phenotype. Our current observation that iNOS rapidly disappears in culture supports the former possibility. However, the stimuli required for iNOS induction differed between intimal and medial SMCs, suggesting that an increased capacity to express iNOS may be part of the "intimal phenotype."

Both qualitative and quantitative differences were observed between intimal and medial SMCs with regard to iNOS induction. When identical stimuli were applied to both cultures, intimal SMCs expressed more iNOS mRNA and protein than did medial SMCs. This was reflected in an 8- to 10-fold higher nitrite production in the intimal cells. The difference was explained by the finding that the iNOS promoter activity, as assessed by a reporter gene construct, was 3-fold higher in the local environment of an intimal compared with a medial SMC. Since there was no evidence for defects in the receptors for IFN-, proinflammatory cytokines, or LPS, it is likely that this difference was due to a more efficient signal transduction machinery in the intimal SMCs.

There were, however, also qualitative differences between intimal and medial SMCs in the response to iNOS. In the former, a proinflammatory cytokine (or LPS) alone was sufficient to induce iNOS, whereas synergistic activation by this type of stimulus and IFN- was needed in medial SMCs. Since IL-1, TNF, and LPS all use the NF-B signal transduction pathway, induction of this pathway was needed in both cell types. In contrast, activation of the IFN- response pathway was not mandatory in intimal SMCs, although it increased the expression level significantly.

Surprisingly, the iNOS/CAT promoter/reporter construct was only activated by synergistic stimulation with IFN- along with IL-1, TNF, or LPS. In contrast, expression of the endogenous iNOS gene could be induced by stimulation with one of the latter alone. This discrepancy could be due to differences in the sensitivity of the detection methods but also to regulatory elements outside the 1749-bp promoter/enhancer sequence used in the reporter construct.

The observation that NF-B–inducing stimuli are sufficient to induce iNOS expression in intimal SMCs could explain the strong iNOS expression in the neointima, where IFN-–producing T cells are sparse. It has recently been shown that TNF- is expressed after arterial injury and also in atherosclerotic plaques.^{30 31} Interestingly, NO inhibits NF-B by inducing IB,³² and it is therefore possible that proinflammatory cytokines, NO, and NF-B are components of a network that controls inflammation in the injured artery by feedback control.

Perspectives
SMCs of the neointima produce large amounts of NO via the inducible pathway, in spite of the fact that autocrine NO production can cause apoptosis of SMCs. The present data clarify that intimal SMCs are less sensitive to the antiproliferative and antimitochondrial effects of NO, which explains how they can survive under conditions of high NO production in the neointima. Furthermore, our data show that compared with medial SMCs, intimal SMCs are less rigorous in their demand for iNOS inducers and express more iNOS in response to a given concentration of stimuli. These differences are maintained in culture and must therefore be linked to a stable intimal SMC phenotype.

In the neointima, the intimal SMC, a cell type with a high capacity to produce NO on stimulation, appears in an environment where several different iNOS-inducing stimuli, such as proinflammatory cytokines and oxidative stress, are present. This explains why iNOS is rapidly induced and large amounts of NO are produced in the injured artery. At this location, NO synthesis is likely to be part of a defense mechanism to protect the tissue from thrombosis and ischemia. Whether iNOS-derived NO actually protects or damages the neointima may depend on the amount of NO produced, ie, on the levels of iNOS-inducing factors in the local milieu.

	Selected Abbreviations and Acronyms

 

CAT = chloramphenicol acetyltransferase IFN- = interferon gamma IL-1ß = interleukin-1ß iNOS = inducible NO synthase L-NMMA = NG-monomethyl-L-arginine LPS = lipopolysaccharide MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium NF-B = nuclear factor-B SMC = vascular smooth muscle cell TNF- = tumor necrosis factor-

	Acknowledgments

 
This study was supported by the Swedish Medical Research Council (project 6816), the Swedish Heart-Lung Foundation, the Swedish Cancer Society, the Axel and Margaret Johnson Foundation, the Nanna Svartz Fund, the Knut and Alice Wallenberg Foundation, and the King Gustaf V 80th Anniversary Fund. The authors are grateful to Drs Allan Sirsjö and Per Eriksson for advise on transfection experiments. We also thank Ingrid Törnberg for technical assistance.

Received May 13, 1997; accepted October 8, 1997.

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