INTRODUCTION

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INTIMAL HYPERPLASIA is a major clinical problem that limits the long-term efficacy of vascular procedures, such as balloon angioplasty and coronary bypass grafts, and a key component of this process is excessive vascular smooth muscle cell (SMC) proliferation (1, 38). The proliferative response evoked by vascular wall injury is preceded by a cascade of events, including synthesis of inducible nitric oxide (NO) synthase (iNOS), which likely modulates the extent of subsequent SMC proliferation. iNOS, like the constitutively expressed endothelial NOS (eNOS), produces NO via the five-electron oxidation of one of the guanidine nitrogens in L-arginine. However, distinct from eNOS, which is expressed in normal blood vessels and inhibits intimal proliferation evoked by vascular injury (34), it is not clear whether iNOS plays a pro- or antiproliferative role when expressed in injured blood vessels. NO, when added as a chemical NO donor or as NO gas, inhibits proliferation of SMC in vitro (18, 36, 33). However, NO has also been reported to stimulate SMC proliferation in vitro (20, 42), and neointima formation induced by mechanical injury is attenuated in iNOS knockout mice (13), suggesting iNOS may play a pro-proliferative role in this model.

Pro-inflammatory cytokines are the primary inducers of iNOS expression in many cell types, including rat aortic SMC. Among those that induce iNOS expression in rat aortic SMC in vitro, interleukin-1 (IL-1) is the most potent (6). Several lines of evidence suggest that SMC-derived IL-1 may be an important stimulus to iNOS expression in SMC. Vascular SMC synthesize both - and -forms of IL-1 when activated in vitro (7, 27, 44), and IL-1 is also synthesized by smooth musclelike cells in clinically relevant intimal hyperplastic lesions. IL-1 was detected by immunostaining in spindle-shaped cells of saphenous vein bypass grafts that had become stenotic, but not in internal mammary arteries that had remained patent, or in normal arteries and veins (12). Also, a recent study (5) indicates that low levels of IL-1 are biologically active when produced endogenously by human vascular SMC. IL-1 is synthesized as a 271-amino acid precursor molecule, which lacks a classical signal sequence (15), and is not efficiently released by cells, but nevertheless appears to be active in its cell-associated form. In several cell types, including SMC, IL-1 precursor is thought to associate with the plasma membrane in a form that can activate the type I IL-1 receptor on adjacent cells via juxtacrine mechanisms (2, 22, 28). Recent studies have suggested that the IL-1 precursor may also act within the cell, by a mechanism involving direct localization to the nucleus (21, 30, 32, 46).

The present study determined whether endogenous production of IL-1 can induce iNOS gene expression in rat aortic SMC. Rat aortic SMC were transiently transfected with expression plasmids, which direct the production of either the precursor or mature form of IL-1, and basal and inducible expression of iNOS was assessed. Because mature IL-1 lacks the nuclear localization sequence, and putative membrane localization signals that are present in the precursor molecule, and does not localize to the nucleus (5, 30) or associate with the plasma membrane (9, 11, 17), its expression should reveal the effects that occur after cellular release. To distinguish between autocrine versus juxtacrine actions of IL-1 precursor, immunostaining was used to separately assess iNOS expression in transfected cells themselves, and in those cells that contact a transfected cell. The results indicate that IL-1 precursor is an effective stimulus for iNOS gene expression in rat aortic SMC and can also augment iNOS expression induced by exogenous cytokines. In addition, juxtacrine effects appear to play a key role in the biological activity of IL-1 precursor.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Rat aortic SMC culture. SMC were isolated from rat thoracic aorta by enzymatic dissociation as described previously (8). SMC were cultured in growth media composed of DMEM supplemented with 10% FCS, glutamine, penicillin, and streptomycin. Cells were passaged by harvesting with tryspin-EDTA and used between passages 3 and 12. Rat aortic SMC expressed -actin, as determined by immunofluorescent staining using mouse anti--actin-FITC conjugate (Sigma).

IL-1 expression plasmids. IL-1 expression plasmids were constructed by PCR amplification of a plasmid containing human IL-1 cDNA, cloned from LPS-activated human peripheral blood cells (ATCC), as previously described (5), and cloned into pcDNA3. The IL-1 precursor expression plasmid encodes amino acids 1-271, whereas the mature IL-1 expression plasmid encodes amino acids 113-271, both cloned downstream of an introduced Kozak consensus sequence.

Transfection of rat aortic SMC by electroporation. Rat aortic SMC (5 × 10⁶/condition) were transfected by electroporation with pcDNA3 alone or encoding IL-1 precursor or mature IL-1 at 300 V and 960 µF (Genepulser, Bio-Rad). Cells were then plated and incubated overnight in media supplemented with sodium butyrate (5 mM) and then washed, and fresh media were added to the cells. In some studies, stable transfectants expressing the neomycin resistance gene in pcDNA3 were selected by supplementing the growth media with 200 µg/ml of Geneticin (Sigma; St. Louis, MO). Experiments were conducted after 4 wk of selection.

IL-1 enzyme immunometric assay. Cell lysates were prepared by three successive freeze-thaw cycles in buffer containing 10 mM sodium phosphate, 0.15 M NaCl, 0.25% BSA, and 0.05% sodium azide and were cleared by centrifugation at 500 g for 10 min. Immunoreactive IL-1 in cell lysates was measured by an enzyme immunometric assay (EIA) that is specific for both the precursor and mature forms of human IL-1 (Cayman Chemical; Ann Arbor, MI).

RT-PCR analysis. Total RNA was isolated by using RNAzol B (Biotecx Laboratories; Houston, TX). RNA (2 µg) was reverse-transcribed for 1 h at 37°C with 200 units of Moloney murine leukemia virus RT (GIBCO-BRL), by using an oligo (dT) primer and the buffer supplied by the manufacturer, in a total volume of 20 µl. The reaction was terminated by heating to 95°C for 5 min, and 2 µl of this first strand cDNA was added to each PCR reaction. The PCR mixes contained 50 mM KCl, 10 mM Tris · HCl (pH 8.3), 1 mM MgCl₂, 0.01 mg/ml gelatin, 200 µM of each dNTP, 250 nM of each primer, and 1 U Taq DNA polymerase (GIBCO-BRL) in a 20 µl volume. The primers used to amplify iNOS mRNA were 5'-TAGAGGAACATCTGGCCAGG-3' and 5'-TGGCCGACCTGATGTTGCCA-3' (sense and antisense, respectively). The primers for the housekeeping gene -actin were 5'-TCCTAGCACCATGAAGATC-3' and 5'-AAACGCAGCTCAGTAACAG-3'. Amplification was performed as previously described (7), with annealing temperatures of 57 and 50°C for iNOS and -actin primers, respectively. Each sample was amplified for 25-30 cycles with iNOS primers, and for 18-20 cycles with -actin primers (within the exponential range of amplification for each product). Products were size separated by electrophoresis in a 5% polyacrylamide gel and visualized by ethidium bromide staining.

Nitrite assay. Aliquots of cell supernatants were assayed for nitrite by using a standard protocol in which samples were mixed with an equal volume of Greiss reagent (0.05% N-(1-Naphthyl)ethylenedamine dihydrochloride, 0.5% sulfanilamide, and 2.5% phosphoric acid) and incubated at room temperature for 10 min (19). The absorbance at 550 nm was measured, and nitrite concentration was determined by using sodium nitrite diluted in media as standards. Nitrite concentrations of the media were normalized to cell number, as determined by Coulter counter analysis.

Localization of iNOS expression in nontransfected and transfected rat aortic SMC cocultures. Rat aortic SMC were cotransfected, as described above for single plasmid transfections, with 10 µg pEGFP (Clontech), which encodes green fluorescent protein (GFP) and serves as a marker of transfected cells, and 20 µg pcDNA3 alone or encoding IL-1 precursor or mature IL-1. After electroporation was completed, 50,000 nontransfected SMC and 50,000 transfected SMC were plated together in 60-mm plates containing glass coverslips. Cells were then incubated overnight in media supplemented with sodium butyrate, 24 h in fresh media alone, and an additional 24 h in media with or without IL-1 (2 ng/ml). iNOS protein was localized in SMC cocultures by indirect immunofluorescence staining. Cells were washed in PBS, fixed in 3.7% formaldehyde (15 min), and permeabilized in 0.2% Triton X-100/PBS (5 min). Nonspecific binding sites were blocked with 10% normal horse serum, and the cells were incubated for 1 h at 37°C with polyclonal rabbit antiserum specific for murine macrophage iNOS (Cayman Chemical; Ann Arbor, MI) and then 45 min at room temperature with Texas-Red-coupled donkey anti-rabbit IgG (Jackson Immunoresearch). Cells were then incubated with Hoechst 33342 to stain cell nuclei, mounted in 90% glycerol/PBS, and observed through a microscope (Diaphot-TMD, Nikon) equipped with epifluorescence. The presence of iNOS was evaluated in transfected cells (GFP positive), in nontransfected cells (GFP negative) that were in direct contact with a transfected cell, and in cells that were neither transfected nor in direct contact with any other cell. For each treatment group, 300 cells (100 of each subgroup) were analyzed. The observer performing the analysis was blinded with respect to the specimen treatment.

Statistical analysis. The significance of treatment-induced differences was determined either by Student's t-test or by ANOVA, followed by Dunnett's procedure to compare multiple means with a similar control value. P < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

iNOS gene expression is enhanced in rat aortic SMC transfected with IL-1 expression plasmids. Transient overexpression of either IL-1 precursor or mature IL-1 induced iNOS gene expression in rat aortic SMC. iNOS mRNA was not detectable by RT-PCR analysis in SMC transfected with vector alone, either 72 or 96 h after transfection (Fig. 1A). In contrast, iNOS mRNA was present in SMC that had been transfected with either IL-1 precursor or mature IL-1 expression plasmids. At both time points, the levels of iNOS mRNA were greater in SMC overexpressing IL-1 precursor compared with SMC overexpressing mature IL-1. To assess whether increased levels of iNOS mRNA were associated with increased iNOS activity, the levels of nitrite, a stable NO oxidation product, were measured in the supernatants, which overlayed the cells from 72-96 h after transfection. Nitrite production was significantly elevated in rat aortic SMC overexpressing either form of IL-1 relative to those transfected with vector alone, and SMC overexpressing IL-1 precursor produced more nitrite than SMC overexpressing mature IL-1 (Fig. 1B). The levels of nitrite produced by SMC overexpressing IL-1 precursor were low, however, accumulating to only 2-3 µM in 24 h relative to levels of 20-35 µM, which are reached after stimulation with exogenous IL-1 (6). Nitrite production was also increased in SMC that were stably transfected with IL-1 precursor expression plasmid (4.45 ± 1.32 µM/24 h per 10⁵ cells) relative to SMC stably transfected with vector alone (0.81 ± 0.29 µM/24 h per 10⁵ cells; P < 0.05).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Inducible nitric oxide sythase (iNOS) gene expression is enhanced in rat aortic smooth muscle cells (SMC) transiently transfected with interleukin (IL)-1 expression plasmids. A: levels of iNOS mRNA in SMC transfected with pcDNA3 alone, or encoding IL-1 precursor (Prec) or mature (Mat) IL-1. RNA was prepared 72 or 96 h after transfection, and levels of iNOS mRNA determined by RT-PCR (25 cycles). B: accumulation of nitrite in supernatants of rat aortic SMC, 48-72 or 72-96 h after transfection. Media were replaced every 24 h, and the nitrite content of the 72 and 96 h conditioned media was assessed and normalized to total cell counts, which were determined at the same time points (n = 4 for each group). Levels of iNOS mRNA and nitrite accumulation are higher in SMC overexpressing IL-1 Prec compared with SMC overexpressing IL-1 Mat. *P < 0.05, significantly different from SMC transfected with pcDNA3. +P < 0.05, significantly greater than SMC transfected with Mat IL-1 expression plasmid.

Exogenous IL-1 receptor antagonist abolishes induction of iNOS in rat aortic SMC overexpressing IL-1 precursor. The greater efficacy of IL-1 precursor, compared with mature IL-1, may be attributable to localization of the precursor molecule to the nucleus or to the plasma membrane, properties that are not shared by the mature IL-1 molecule. Effects of membrane-associated IL-1 precursor are mediated by the type I IL-1 receptor and are inhibited by high concentrations of exogenous IL-1 receptor antagonist (IL-1RA; 22), in contrast to the direct nuclear actions of IL-1, which are thought to occur independently of this receptor (30, 32). To distinguish between these two mechanisms, the ability of exogenous IL-1RA to attenuate iNOS induction in rat aortic SMC overexpressing IL-1 precursor was assessed. Addition of exogenous IL-1RA (10 µg/ml) to the cell culture media immediately after transfection prevented the subsequent induction of iNOS by expression of IL-1 precursor. iNOS mRNA was not detectable by RT-PCR in SMC overexpressing IL-1 precursor and incubated in the presence of exogenous IL-1RA, whereas iNOS PCR product was apparent in SMC overexpressing IL-1 precursor and incubated without IL-1RA (Fig. 2A). Also, nitrite production was not enhanced in SMC overexpressing the IL-1 precursor that were incubated in the presence of IL-1RA but was significantly enhanced in SMC overexpressing IL-1 precursor incubated in the absence of IL-1RA (Fig. 2B). These results indicate that IL-1 precursor acts extracellularly to stimulate iNOS gene expression via activation of the type I IL-1 receptor.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Exogenous IL-1 receptor antagonist (RA) prevents iNOS induction in rat aortic SMC transfected with IL-1 expression plasmids. SMC were transfected with pcDNA3 alone, or encoding IL-1 Prec or Mat IL-1, or not transected (NT). Cells were then plated in media alone (Con) or with IL-1RA (10 µg/ml). A: RNA was prepared 96 h after transfection, and levels of iNOS mRNA and -actin were determined by RT-PCR (26 and 20 cycles, respectively). B: fresh media with or without IL-1RA (10 µg/ml) were added after 72 h, and the nitrite content of the media was determined 24 h later (n = 4 cultures for each group, representative of 3 independent experiments). *P < 0.05, significantly different from SMC transfected with pcDNA3. +P < 0.05, significantly greater than SMC transfected with Mat IL-1 expression plasmid.

Increased sensitivity to exogenous IL-1 and TNF- in rat aortic SMC overexpressing IL-1. Exogenous IL-1 and TNF- can act synergistically to induce iNOS expression in rat aortic SMC (6). Therefore, the ability of IL-1 produced by rat aortic SMC to act synergistically with exogenous IL-1 or TNF- and induce iNOS activity was assessed. Exposure to exogenous IL-1 induced NO production in nontransfected and pcDNA3-transfected rat aortic SMC; however, a high concentration of IL-1 (2,000 pg/ml) was required to significantly stimulate nitrite production in both groups (Fig. 3A), as previously described (6). SMC transfected with IL-1 precursor expression plasmids produced significant nitrite in the absence of exogenous cytokine stimulation, as described above (Figs. 1B and 2B), and nitrite production was further enhanced by exposure to exogenous IL-1, even at concentrations as low as 20 pg/ml. Although SMC overexpressing mature IL-1 did not produce nitrite in the absence of exogenous IL-1 stimulation, sensitivity to exogenous IL-1 was enhanced relative to SMC transfected with vector alone. SMC overexpressing mature IL-1 were not as sensitive to exogenous IL-1 as cells that overexpressed IL-1 precursor; significant stimulation of nitrite production required exposure to 200 pg/ml IL-1, 10-fold more exogenous IL-1 than that required to stimulate nitrite production in rat aortic SMC overexpressing IL-1 precursor.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   IL-1- and tumor necrosis factor - (TNF-)-induced nitrite production are markedly enhanced in rat aortic SMC overexpressing IL-1 Prec and modestly enhanced in SMC overexpressing Mat IL-1. SMC were transiently transfected with pcDNA3 alone, or encoding IL-1 Prec or Mat IL-1. Fresh media were added after 72 h, with or without IL-1 (A) or TNF- (B) at the indicated concentrations, and nitrite content of the media determined 24 h later. Nitrite production was normalized to cell counts (n = 4 for each group). *P < 0.05, significantly different from SMC transfected with same vector and incubated without exogenous IL-1 or TNF- + P < 0.05, significantly greater than SMC transfected with pcDNA3.

IL-1 precursor stimulates iNOS expression in rat aortic SMC via juxtacrine effects. Studies with IL-1RA indicated that the action of IL-1 precursor was extracellular and involved the type I IL-1 receptor. To assess the role of juxtacrine effects in the action of IL-1 precursor, immunostaining studies were conducted to localize iNOS expression in cocultures of SMC, which contained a mixture of transfected and nontransfected SMC. Rat aortic SMC were cotransfected with an IL-1 expression plasmid and an expression plasmid encoding GFP, as a marker of transfected cells. In preliminary studies, iNOS protein was not detectable by indirect immunofluorescence staining in SMC overexpressing IL-1 precursor in the absence of stimulation with exogenous cytokine, even though the cells expressed detectable iNOS mRNA and produced detectable nitrite (Figs. 1-3). The absence of detectable iNOS protein may be due to a lower sensitivity of the immunostaining procedure compared with the higher sensitivities of the RT-PCR and nitrite assays. This hypothesis was supported by subsequent experiments, in which iNOS protein was detectable in SMC exposed to exogenous IL-1, a stimulus that induces higher levels of nitrite production than overexpression of IL-1 precursor (6). Surprisingly, however, <10% of nontransfected SMC expressed detectable iNOS protein after exposure to exogenous IL-1 (1 ng/ml), indicating an inherent variability of rat aortic SMC subpopulations in their sensitivity to exogenous IL-1.

View larger version (63K): [in this window] [in a new window]   Fig. 4.   Enhanced iNOS expression in rat aortic SMC that contact SMC transfected with IL-1 precursor expression plasmids. Rat aortic SMC were cotransfected with pEGFP, as a marker of transfected cells, and IL-1 precursor expression plasmid, and plated with nontransfected SMC. Cells were then incubated 24 h in fresh media alone, and an additional 24 h in media supplemented with IL-1 (1 ng/ml), then iNOS expression was localized by indirect immunofluorescence staining. Shown are representative patterns of immunoreactive iNOS (red) and green fluorescent protein (GFP) expression in cocultures containing SMC cotransfected with IL-1 precursor and GFP expression plasmids.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Scoring of the presence or absence of iNOS protein in rat aortic SMC subpopulations. SMC were cotransfected with pEGFP and either pcDNA3 alone or pcDNA3 encoding IL-1 precursor or mature IL-1, as described in Fig. 4. Percentage of cells expressing iNOS are shown for transfected cells (GFP positive; autocrine), nontransfected cells (GFP negative) that contacted transfected cells (juxtacrine), and nontransfected cells that did not contact any other cells (isolated). Immunoreactive iNOS was significantly increased in SMC transfected with IL-1 precursor expression plasmids, and in cells in contact with SMC overexpressing IL-1 precursor. Values are means ± SE, and were obtained in 4 independent experiments. *P < 0.05, significantly different from same subgroup of pcDNA3-transfected SMC cocultures.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The proliferation and function of cells in animal tissues are controlled by several forms of intercellular communication. The ability of diffusible polypeptide factors to activate membrane receptors and induce signals that influence cell function is well established. Membrane-anchored ligands can also activate receptors; however, this form of communication requires direct cell-cell contact (10). IL-1 precursor is one example of a polypeptide factor that has been proposed to act as a membrane-associated ligand that activates cells via cell-cell contact (2, 17, 22, 25, 28). The present studies have three principal findings. First, they indicate that endogenous production of IL-1 precursor, as a sole stimulus, can induce iNOS gene expression and NO production in rat aortic SMC. Second, endogenous production of IL-1 precursor also sensitizes rat aortic SMC to the stimulatory effects of exogenous proinflammatory cytokines, including IL-1 and TNF-. Finally, the primary mechanism of IL-1 precursor action involves juxtacrine effects, which are exerted via cell-cell contact, and involves activation of the type I IL-1 receptor.

Endogenous production of IL-1 precursor was a sufficient stimulus to induce detectable expression of iNOS in rat aortic SMC. Both iNOS mRNA and the release of nitrite, a stable NO oxidation product, were detectable in rat aortic SMC overexpressing IL-1 precursor either transiently or stably; however, the level of extracellular nitrite accumulation was low (~2 µM) relative to the nitrite levels found when rat aortic SMC are stimulated with exogenous IL-1 (~25 µM; Ref. 6). In previous studies (6), IL-1 and TNF-, when added as soluble factors, acted synergistically to induce iNOS gene expression. In the present study, low levels of IL-1 and TNF- (pg/ml) stimulated NO production in rat aortic SMC, which were producing IL-1 precursor. In contrast, higher levels of exogenous IL-1 (ng/ml) were required to induce NO production in the absence of endogenous IL-1 synthesis, and high concentrations of TNF- were not effective. These results indicate that IL-1 precursor that is produced endogenously by rat aortic SMC can synergize with exogenous IL-1 or TNF- to induce iNOS gene expression in SMC.

One mechanism by which IL-1 could exert local effects on gene expression is by acting as a soluble mediator after release from the cell. IL-1 precursor and mature IL-1 are equipotent agonists of the type I IL-1 receptor when presented to IL-1 receptor expressing cells as soluble factors (35). Also, both the precursor and mature forms of IL-1 can be released from cells (26, 39, 45), although both forms lack a signal sequence for secretion via classical pathways, and the secretory mechanism for both proteins is unknown. However, mature IL-1 appears to be the preferentially released form, on the basis of studies with human monocytes and bladder carcinoma cells (39, 45). Therefore, if IL-1 acted as a soluble extracellular factor after release from rat aortic SMC, then one would predict that overexpression of mature IL-1 would induce iNOS gene expression more effectively than overexpression of IL-1 precursor. In contrast, in the present studies overexpression of IL-1 precursor consistently induced higher levels of iNOS gene expression than overexpression of mature IL-1. Therefore, the enhanced effectiveness of IL-1 precursor observed in this study argues against a primary mechanism involving cellular release of soluble IL-1.

A second mechanism by which IL-1 could induce iNOS gene expression in rat aortic SMC is as a membrane-associated ligand. IL-1 precursor localizes to the plasma membrane after synthesis in LPS-stimulated cells (14, 41, 49), where it remains associated with the cell surface and is thought to activate the type I IL-1 receptor on adjacent cells via juxtacrine mechanisms (2, 17, 22, 25, 28). In contrast, mature IL-1 does not associate with the cell surface (9, 11, 17). In the present study, addition of IL-1RA, a competitive antagonist of IL-1 binding to the type I IL-1 receptor (31), abolished expression of iNOS in SMC transfected with IL-1 precursor expression plasmids. These results support an extracellular site of action involving the type I IL-1 receptor. Immunostaining studies also supported the hypothesis that IL-1 associates with the surface of rat aortic SMC in a form that can activate type I IL-1 receptors via cell-to-cell contact. Cells in contact with IL-1-precursor overexpressing cells demonstrated induction of iNOS, whereas isolated cells did not, indicating an important role of juxtacrine stimulation. iNOS expression was also enhanced in those SMC that were transfected with IL-1 precursor expression plasmids, however, only when the cell was in direct contact with another cell. It is possible that this effect involves two-way communication between SMC, which are in contact with each other. Because IL-1 is known to induce its own synthesis (43), membrane expression of IL-1 in the transfected cell could induce membrane expression of IL-1 in an adjacent cell, which in turn induces expression of iNOS in the transfected cell. Alternatively, it is possible that binding of IL-1 precursor to the type I IL-1 receptor on the adjacent cell elicits signal transduction events in the IL-1 precursor expressing cells as well as in the adjacent cell. This possibility has been proposed for juxtacrine mediators, which contain transmembrane domains (10). However, IL-1 precursor lacks a transmembrane domain, thus a mechanism by which IL-1 binding to its receptor could influence the IL-1 precursor expressing cell is not clear.

IL-1 precursor has also been proposed to act within the cell in some cell types, via a mechanism that is independent of the type I IL-1 receptor and involves direct localization to the nucleus (21, 30, 32, 46). In human vascular SMC and endothelial cells transfected with the corresponding expression plasmid, IL-1 precursor localizes to the nucleus, whereas mature IL-1 remains in the cytosol (5, 30, 46). The action of IL-1 precursor was linked to its nuclear localization in two studies with human endothelial cell lines. In one study, stable expression of IL-1 precursor stimulated expression of IL-1-inducible genes and inhibited cellular proliferation, whereas stable expression of mature IL-1 was ineffective (30). In the second study, stable expression of IL-1 precursor inhibited cell migration, whereas stable expression of mature IL-1, or a nuclear localization deficient mutant of IL-1 precursor, stimulated cell migration (32). In both studies, the fact that exogenous IL-1RA did not reverse the effects of IL-1 precursor expression was used as evidence that IL-1 precursor acts within cells. In the present study, exogenous IL-1 receptor antagonist abolished iNOS expression in rat aortic SMC overexpressing IL-1 precursor, arguing against a role of nuclear localization in IL-1 precursor induced iNOS gene expression. The ineffectiveness of IL-1RA in previous studies could be due to the fact that lower IL-1RA concentrations were used (100 ng/ml). The present study used 10 µg/ml IL-1RA on the basis of evidence that higher concentrations are required to inhibit membrane-associated IL-1 activity (22). Alternatively, the short incubation with exogenous IL-1RA (20-24 h) may not have been sufficient time to reverse the long-term effects in stable transfectants that had been producing IL-1 precursor for many weeks. In support of this interpretation, the mitogenic effects of long term IL-1 exposure in human SMC are only partially reversed by a 72-h exposure to IL-1RA (5). It is possible that localization to the plasma membrane, rather than to the nucleus, also accounts for the actions of IL-1 precursor in human endothelial cells (30, 32).

Several lines of evidence indicate that SMC-derived iNOS contributes to the modulation of SMC proliferation in pathophysiological states. However, iNOS may limit or promote intimal hyperplasia, depending on the pathological condition involved. Vascular SMC within balloon-injured rat carotid arteries express iNOS (47), and targeted deletion of the iNOS gene in mice is associated with a decrease in neointimal thickening after mechanical injury to the carotid artery (13), consistent with a pro-proliferative role of iNOS. In distinct contrast, other studies indicate that iNOS limits SMC proliferation, which occurs in coronary vessels after cardiac transplantation. iNOS is expressed by SMC within coronary arteries of transplanted human hearts that exhibit accelerated graft arteriosclerosis (37). Also, the accelerated arteriosclerosis that occurs after allogeneic cardiac transplantation is exacerbated in iNOS-deficient mice (24), indicating a protective role of the enzyme in limiting excessive SMC proliferation in the setting of transplant rejection. iNOS is also expressed by SMC within atherosclerotic lesions of the human aorta (29). However, whether iNOS plays a protective or deleterious role in atherosclerosis remains to be established.

Expression of iNOS in vascular SMC has also been proposed to contribute to vasodilatation that occurs during sepsis (8). Exposure of intact rat aortic segments to bacterial lipopolysaccharide (LPS) or IL-1 in vitro markedly inhibits vascular contraction, an effect that is independent of the endothelium and involves expression of iNOS (3, 4, 6, 16). iNOS is also expressed in rat aorta after injection of bacterial LPS in vivo (6); however, immunostaining studies indicated that iNOS protein is expressed primarily in adventitial fibroblasts, whereas iNOS expression was not detectable in medial SMC (48). The adventitia may also be the primary source of NO in intact rat aortic segments exposed to LPS in vitro, on the basis of evidence that intact rat aortic segments produce more NO after LPS stimulation than do segments of rat aortic media from which the adventitia was removed (23). In the present study, rat aortic SMC were obtained by enzymatic digestion of the aortic media after separation from the adventitia, and a subpopulation of cells that subsequently grew in culture expressed high levels of iNOS protein after stimulation with exogenous IL-1. Another subpopulation of rat aortic SMC showed high levels of iNOS protein when activated by two stimuli, soluble IL-1 and membrane-associated IL-1 precursor. SMC derived from enzymatic digestion of the media of many blood vessels, including the rat aorta (40), are known to be heterogeneous with respect to phenotype. Another aspect of this heterogeneity may be the endogenous production of IL-1, or alternatively IL-1 or TNF-. The present study indicates that differential endogenous production of IL-1 in rat aortic SMC subpopulations may account for differential sensitivity to exogenous cytokines.

The present study provides evidence that endogenous production of IL-1 precursor can induce low-level iNOS gene expression in rat aortic SMC and can act synergistically with exogenous IL-1 and TNF- to induce high-level iNOS expression. A primary mechanism of IL-1 precursor action involves juxatacrine effects that are exerted via cell-cell contact and are mediated via activation of the type I IL-1 receptor. SMC-derived IL-1 precursor may be a clinically relevant stimulus to iNOS expression in SMC and may contribute to the development of intimal hyperplastic lesions in injured blood vessels or transplanted hearts.