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Abstract
Abstract Augmentation of nitric oxide (NO) production in vivo decreases lesions in a variety of models of arterial injury, and inhibition of NO synthase exacerbates experimental intimal lesions. Both vascular smooth muscle cell (VSMC) proliferation and migration contribute to lesion formation. Although NO inhibits VSMC proliferation, its effects on VSMC migration are unknown. To test the hypothesis that NO inhibits VSMC migration independent of inhibition of proliferation, we examined migration of rat aortic VSMCs after wounding of a confluent culture in the presence of chemical donors of NO. Hydroxyurea was used to eliminate any confounding effect of NO on proliferation. Three NO donors, diethylamine NONOate, spermine NONOate, and S-nitrosoglutathione, exhibited concentration-dependent inhibition of both number of migrating VSMCs and maximal distance migrated. Inhibition of migration was also seen with 8-Br-cGMP, suggesting that activation of guanylate cyclase may play a role in mediating the antimigratory effects of NO. Migration resumed after removal of NO donors, as evidenced by an increase in distance migrated. Measurement of VSMC protein synthesis and mitochondrial respiration indicated that inhibition of migration by NO donors was not due to metabolic cytostasis. These findings indicate that NO reversibly inhibits VSMC migration independent of proliferation or cytotoxicity, a novel mechanism by which both endogenous and pharmacological NO may alter vascular pathology.
Nitric oxide modulates several physiological processes in the vasculature, including vascular tone, platelet aggregation, and leukocyte adhesion to the endothelium. Augmentation of NO production under pathological conditions results in diminished neointimal lesion formation in several experimental models. Dietary supplementation with l-arginine, the precursor for NO, improves endothelium-dependent relaxation and diminishes intimal lesions after balloon angioplasty.1 2 Similar effects are seen after balloon catheter injury with infusion of a pharmacological NO donor3 or gene transfer of NO synthase to the injured artery.4 The benefit of increasing NO on intimal lesions has also been shown in other clinically relevant models of vessel injury, including cholesterol-induced atherogenesis5 and vein grafting into the arterial circulation.6 A role for endogenous NO in limiting neointimal hyperplasia is illustrated by in vivo studies demonstrating that treatment with inhibitors of NO synthase after balloon injury2 or cholesterol feeding7 increases neointimal hyperplasia.
Despite evidence that NO inhibits neointimal hyperplasia in several different experimental models, the mechanisms involved in this inhibition by NO are unclear. Formation of intimal lesions involves VSMC proliferation, migration of VSMCs through the internal elastic lamina into the intima, and deposition of extracellular matrix. NO donors have been shown to inhibit proliferation of cultured VSMCs,8 9 10 11 and decreased VSMC proliferation has been noted in vivo after NO augmentation by gene transfer.4 The effects of NO on other processes involved in neointima formation are less clear. Exogenous NO inhibits the production of collagen and total protein in vascular VSMCs,12 and inhibition of extracellular matrix deposition may contribute to the in vivo effects of NO on neointima formation. Inhibition of VSMC migration may be another mechanism by which NO inhibits neointima formation under physiological and pathological conditions. NO has divergent effects on cell migration that are cell-type specific. NO synthase inhibitors decrease migration of human neutrophils,13 and this effect appears to be mediated by decreasing levels of the second messenger cGMP. NO also has promigratory effects on capillary endothelial cells,14 which appear to be cGMP mediated. In contrast, exogenous NO inhibits migration of monocytes by increasing cGMP levels.15
The purpose of our study was to test the hypothesis that NO inhibits the migration of VSMCs in vitro and to examine the mechanisms involved in such inhibition. We used the well-characterized wounding model of in vitro cell migration16 17 to examine the effects of chemically derived NO on migration of cultured VSMCs. Our data demonstrate that multiple donors of NO inhibit the migration of VSMCs in a concentration-dependent, reversible, and noncytotoxic fashion. These findings delineate an additional mechanism that may explain the inhibitory effects of NO on neointimal hyperplasia in vivo.
Materials and Methods
DMEM, trypsin-EDTA, glutamine, and penicillin/streptomycin were from GIBCO Labs. Fetal bovine serum was purchased from Hyclone. [3H]Thymidine and [3H]leucine were obtained from Amersham. GSNO was from Alexis Biochemicals. DEA NO and SP NO were purchased from Cayman Chemical. All other reagents were supplied by Sigma Chemical Co.
VSMC Culture and Migration Assay
Rat aortic VSMCs were derived from the thoracic aortas of adult male Sprague-Dawley rats by using standard enzymatic dissociation techniques and were characterized as previously described.18 Cells were plated and grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum, 1 mmol/L glutamine, and penicillin/streptomycin. VSMC migration was assessed with a razor blade wounding injury of confluent cultured cells.16 17 Migration from a wounded confluent culture is dependent on the density of the culture, and thus in all experiments, cells (passages 4 through 10) were plated into 100-mm dishes at a constant density of 7000 cells/cm2 and grown for 9 days before use. Medium was changed every other day for 9 days and then replaced with medium containing 5 mmol/L hydroxyurea. Hydroxyurea was added to eliminate any confounding effects of NO on cell proliferation, as both NO and exogenous cGMP inhibit proliferation of cultured VSMCs,8 and proliferation contributes to the number of migrating cells in scratch injury models of cell migration.16 Preliminary experiments showed that 24-hour treatment with 5 mmol/L hydroxyurea resulted in complete inhibition of cell proliferation (thymidine uptake <1% of untreated control cells) similar to previous studies, which have also shown that 5 mmol/L hydroxyurea does not alter VSMC migration compared with irradiated cells.16 After 24 hours of hydroxyurea treatment, the cultures were scraped with a single-edged razor blade. Care was taken to ensure that a discernible scratch resulted that could be found upon later examination. Cells were washed twice with PBS and placed in medium containing hydroxyurea and experimental agents. Whenever experimental agents were dissolved in buffers other than medium, control cells were treated with appropriate concentrations of buffer alone. Medium containing NO donors was changed every 12 hours (24 hours for 8-Br-cGMP), and 48 hours after the initial wounding, cells were washed twice with PBS, fixed with absolute ethanol, and stained with toluidine blue. Three microscopic fields (2-mm diameter) were evaluated for each wounding injury. The number of cells migrating across the wound edge and the maximum distance migrated (wound edge to nucleus of farthest cell) were determined in each field and averaged for each injury (see Fig 1⇓). Reversal experiments were performed in which VSMCs were treated with NO donors for 48 hours and then allowed to migrate for an additional 48 hours in medium without NO donors. The purpose of these experiments was to determine whether VSMCs inhibited from migrating by NO for 48 hours were still capable of migration or whether irreversible changes had occurred. After 48-hour exposure to 0.6 mmol/L DEA NO or SP NO, VSMCs were washed twice with PBS and placed in fresh medium containing hydroxyurea without NO donors for an additional 48 hours before assessment of migration, as described above. Migration of the reversal VSMCs was compared with identically treated VSMCs exposed to NO donors for 48 hours to evaluate whether migration resumed in the second 48-hour period without NO donors.
VSMC migration after removal of NO donors. Solid bars represent number of migrating VSMCs, and hatched bars represent maximum distance migrated. A, Bar graph illustrating VSMC migration during and after treatment with NO donors. VSMC cultures were wounded and exposed to the NO donors DEA NO and SP NO for 48 hours. The number of cells migrating across the wound edge (solid bars) and the farthest distance migrated (hatched bars) after 48 hours are shown (Control, DEA NO, and SP NO). Parallel cultures were treated identically with DEA NO or SP NO for 48 hours and then placed in medium without NO donors for an additional 48 hours. Migration was then assessed (DEA NO+removal, SP NO+removal). Bars are the mean±SEM of eight woundings (three fields averaged per wounding) from two separate experiments. B through D, Representative photomicrographs illustrating migration of VSMCs. B, Control VSMCs in hydroxyurea alone. C, Migration of VSMCs treated with 0.6 mmol/L DEA NO. Note both decreased cell number and decreased distance of migration compared with B. D, Cells treated with 0.6 mmol/L DEA NO for 48 hours and then allowed to migrate after DEA NO removal for an additional 48 hours. Note increased distance but cell number similar to that in C. Scale bar represents 200 μm. Magnification ×100. *P<.05 vs control; §P<.05 vs DEA NO; †P<.05 vs SP NO.
NO Donors
The nucleophilic adducts of NO, SP NO and DEA NO, release free NO in a pH-dependent fashion, with release initiated on mixing of stock solution (pH >8.5) with culture medium (pH 7.4).19 Stock solutions (100 mmol/L) were prepared in phosphate buffer (50 mmol/L Na2HPO4, pH 8.5) and stored for no more than 3 days at −70°C. Dissociation of nucleophilic adducts of NO follows first-order kinetics, with complete dissociation occurring within 1 hour, and thus media containing DEA NO or SP NO were changed every 12 hours during migration studies. The antimigratory properties of diethylamine, the non–NO-releasing analogue of DEA NO, were evaluated to confirm that the effects of DEA NO were due to NO release. In other experiments, VSMCs were treated with SP NO that had been preincubated with medium for 12 hours at 37°C, which causes both complete dissociation of NO from the nucleophilic complex (SP NO half-life=47 minutes at pH 7.4) and conversion of the labile NO to its biologically inactive metabolite nitrite. The nitrosothiol NO donor GSNO20 was dissolved in PBS (50 mmol/L) immediately before application to cells. A stable cell-permeable analogue of cGMP, 8-Br-cGMP, was dissolved in medium and applied every 24 hours after wounding of cultures.
Cytotoxicity Assays
After 48-hour treatment with 1 mmol/L DEA NO or SP NO, cells were washed twice with PBS and exposed to 0.1% trypan blue for 5 minutes and the percentage of trypan blue–positive cells determined. For evaluation of mitochondrial respiration in VSMCs treated with NO donors, the MTT reduction technique21 was used. MTT serves as a substrate for mitochondrial dehydrogenases, which reduce MTT to insoluble formazan. VSMCs were pretreated with hydroxyurea (5 mmol/L) and treated with DEA NO (0.6 mmol/L), SP NO (0.6 mmol/L), or 8-Br-cGMP (1 mmol/L) in identical fashion to cells in migration assays. Control cells were exposed to hydroxyurea alone to match control cells in migration assays. Four hours after the last medium change (total exposure to NO donors was 40 hours), MTT (0.3 mg/mL) was added for 2 additional hours. Cells were washed with PBS and then solubilized in isopropanol containing 0.1 mol/L HCl and 1% Triton X-100. The solubilized formazan was measured by determining A570, and background (A690) was subtracted for each sample. For measurement of VSMC protein synthesis, cells were treated with NO donors or hydroxyurea alone, as described for mitochondrial respiration assays, and then [3H]leucine (5 μCi/mL) was added for 1 hour, beginning 4 hours after the last addition of NO donors. Incorporation of radioleucine into protein was determined by a standard technique12 of fixing cells with cold methanol after 1 hour, precipitating protein with three washes with cold trichloroacetic acid (10%), and solubilizing precipitates with 0.3 mol/L sodium hydroxide before scintillation counting. Incorporation of radioleucine measured by this technique was inhibited by cycloheximide in a concentration-dependent fashion (EC50=0.03 μg/mL), confirming that incorporation reflected protein synthesis.
Data Analysis and Statistics
Three 2-mm fields were evaluated for each wounding, and the number of migrating cells and the farthest distance migrated were determined. These three values were averaged to give a mean for each wounding. These mean values (n=8 to 22 from 2 to 5 separate experiments) were each then normalized to appropriately treated control plates for each experiment to allow comparison between experiments and then averaged to give the data shown (mean±SEM). Differences between groups were analyzed with one-way analysis of variance with Bonferroni correction for repeated comparisons where appropriate. If analysis of variance demonstrated significant differences between groups, then individual differences were analyzed with a two-tailed unpaired t test. Differences were considered significant when P<.05.
Results
Migration of VSMCs for 48 hours in control cultures resulted in 68±3 cells crossing the wound edge, with a maximal distance migrated of 428±14 μm (mean±SEM, n=53 to 56 woundings). The addition of the NO donors SP NO, DEA NO, and GSNO caused similar concentration-dependent inhibition of both the number of VSMCs migrating across the wound edge and the farthest distance migrated (Fig 2A⇓ through 2C). Inhibition of migration was statistically significant (P<.05) beginning at a concentration of 0.1 mmol/L for all three NO donors. The maximal inhibition achieved with the nucleophilic adducts of NO (SP NO and DEA NO) was greater (≈80%) than that obtained with the nitrosothiol GSNO (≈50%) at similar concentrations (1 mmol/L).
Bar graphs showing the effect of increasing concentrations of the NO donors DEA NO (top), SP NO (middle), and GSNO (bottom) on VSMC migration. Solid bars represent number of migrating VSMCs, and hatched bars represent maximum distance migrated. VSMC cultures were pretreated with hydroxyurea, wounded, and treated with the indicated concentrations of NO donors every 12 hours for 48 hours. Three 2-mm fields were evaluated for each wounding and averaged. Each bar represents the mean±SEM of 8 to 22 (average, 12) individual culture woundings (each evaluated in triplicate) from two to five separate experiments. *P<.05 vs control.
The non–NO-releasing analogue of DEA NO, diethylamine (1 mmol/L), had no significant effect on either number of VSMCs migrating or distance migrated, whereas DEA NO (1 mmol/L) inhibited both parameters of migration (Fig 3⇓). Treatment of VSMCs with fresh SP NO (0.6 mmol/L) resulted in inhibition of ≈60% of VSMC migration (Figs 2B⇑ and 3B⇓) over 48 hours, whereas VSMCs treated with SP NO (0.6 mmol/L) that had been preincubated at 37°C for 12 hours resulted in significantly less inhibition of migration (Fig 3⇓). Placement of VSMCs treated with 0.6 mmol/L DEA NO or SP NO for 48 hours in fresh medium for an additional 48 hours resulted in an almost twofold increase in distance of VSMC migration relative to migration during the NO treatment period (Fig 1A⇑), although the number of VSMCs migrating did not significantly increase during the 48-hour recovery period (Fig 1A⇑). Increasing concentrations of 8-Br-cGMP showed progressive inhibition of both the number of migrating VSMCs and distance of migration (Fig 4⇓); these differences, however, reached statistical significance only at the maximal concentration tested (1 mmol/L).
Bar graph showing effect of inactive analogue of DEA NO and preincubated SP NO on VSMC migration. Solid bars represent number of migrating VSMCs, and hatched bars represent maximum distance migrated. Shown is a comparison between the antimigratory effects of the NO donor DEA NO (1 mmol/L) and its non–NO-releasing analogue diethylamine (DEA) (1 mmol/L). Bars are the mean±SEM of 11 to 12 woundings from three separate experiments. Also shown is a comparison of the antimigratory effects of SP NO (0.6 mmol/L) either added immediately to cells (Fresh SP NO) or preincubated with medium for 12 hours at 37°C before addition to cells (Stale SP NO). Bars are the mean±SEM of 8 woundings from two separate experiments. *P<.05 vs control; §P<.05 vs DEA NO; †P<.05 vs Fresh SP NO.
Bar graph showing effect of increasing concentrations of the cGMP analogue 8-Br-cGMP on VSMC migration over 48 hours. Solid bars represent number of migrating VSMCs, and hatched bars represent maximum distance migrated. VSMC cultures (hydroxyurea treated) were wounded and treated with the indicated concentrations of 8-Br-cGMP every 24 hours for 48 hours. Each bar represents the mean±SEM of 11 to 12 individual culture woundings from three separate experiments. *P<.05 vs control.
Trypan blue exclusion was used to evaluate whether treatment with NO donors caused increased cell death. Exposure of VSMCs to 0.1% trypan blue after 48-hour treatment with DEA NO and SP NO (1 mmol/L) consistently resulted in exclusion of trypan blue by >99% of cells, which was similar to control VSMCs. Measurement of mitochondrial respiration demonstrated (Fig 5⇓) that there was no inhibition of VSMC mitochondrial respiration by NO donors at concentrations (0.6 mmol/L) that significantly inhibited migration (Fig 2A⇑ and 2B⇑). In protein synthesis experiments, control VSMCs incorporated 0.253±0.009 cpm/cell (mean±SEM, n=6) of radioleucine in the 1-hour incubation period. The rate of protein synthesis was decreased in cells treated with SP NO by 28% but not significantly altered in cells exposed to DEA NO (Fig 5⇓). Treatment with 8-Br-cGMP increased both protein synthesis and mitochondrial respiration relative to control VSMCs (Fig 5⇓).
Bar graph showing mitochondrial respiration and protein synthesis in VSMCs treated with NO donors and 8-Br-cGMP. VSMCs were pretreated with hydroxyurea and exposed to NO donors or 8-Br-cGMP for 40 hours. Mitochondrial respiration and protein synthesis were determined as described in the text. Data are the mean±SEM of six determinations from two experiments. *P<.05 vs control.
Discussion
Our findings demonstrate that exogenous, chemically derived NO inhibits migration of VSMCs independent of effects on proliferation. This inhibition was not due to suppression of VSMC proliferation, as all studies were done in the presence of hydroxyurea block, a technique designed to detect antimigratory effects of agents such as heparin16 that also inhibit VSMC proliferation. Three experimental findings strongly support the concept that the inhibitory effects noted in our studies were due to NO: (1) Inhibition of VSMC migration occurred at similar concentrations with three structurally different NO donors (Fig 2A⇑ through 2C), including donors that release NO via different mechanisms, ie, nucleophilic NO adducts (DEA NO and SP NO) and a nitrosothiol (GSNO). (2) Inhibition of VSMC migration by SP NO was abolished by preincubating SP NO with medium (Fig 3⇑), demonstrating lability of the inhibitory factor, consistent with the biology of NO. (3) Inhibition of migration by DEA NO was not mimicked by its inactive analogue diethylamine, which lacks a nucleophilic complex with NO (Fig 3⇑). As previously described by other investigators,8 12 the “stale SP NO” experiment (Fig 3⇑) also excludes the possibility that the effect of SP NO is not a direct effect on VSMCs but rather a degradation by NO of promigratory factors present in the serum or medium.
Significant inhibition of VSMC migration in our studies by all three NO donors occurred at concentrations (0.1 to 1 mmol/L) similar to those that inhibit proliferation8 9 10 11 and collagen synthesis of VSMCs in vitro.12 Furthermore, these concentrations of NO donors are the same as those that inhibit the in vitro migration of monocytes15 and augment neutrophil migration.13 A concern regarding these concentrations of NO donors is that they are several orders of magnitude greater than concentrations required to affect vasoreactivity and platelet aggregation in vitro.20 The development of neointimal hyperplasia, involving VSMC proliferation, migration, and matrix deposition, and the inhibition of these processes by NO in vivo occur over weeks. Higher concentrations of exogenous NO are required to demonstrate inhibition of these cellular functions in short-term in vitro experiments with cultured VSMCs8 9 10 11 12 as well as other cell types.13 15
Perhaps most importantly, VSMC migration resumed after removal of NO from the culture medium, demonstrating that the inhibition is reversible. Removal of NO donors in concentrations higher than those used in this study resulted in reversal of inhibition of VSMC collagen and protein synthesis,12 confirming that inhibitory effects of exogenous NO on VSMCs are not simply due to cytotoxicity. In the reversal experiments (Fig 1⇑), removal of NO donors resulted in a subsequent increase in distance migrated but not number of migrating cells. This finding suggests that prolonged treatment of VSMCs with NO donors affects cells that have begun migrating different from cells that did not initially migrate while treated with NO donors. An alternative explanation for this effect, which was seen with both DEA NO and SP NO, is that heterogeneity exists within the population of cultured VSMCs with respect to sensitivity to NO. Our studies also did not examine whether the inhibitory effects of NO donors on VSMC migration are lost after 48 hours. Further studies will be needed to address these issues.
NO activates smooth muscle guanylate cyclase, and treatment of cultured rat VSMCs with NO donors causes rapid increases in intracellular cGMP.11 Inhibition of VSMC migration by 8-Br-cGMP (Fig 4⇑) indicates that cGMP elevation by NO may play a role in the inhibition of VSMC migration noted with NO donors (Fig 2A⇑ through 2C), as has been shown for NO modulation of migration of other cell types.13 15 We did not use agents such as methylene blue to block activation of guanylate cyclase, as methylene blue releases oxygen radicals that bind and inactivate nitric oxide extracellularly,22 a confounding factor that does not allow these agents to be used to determine the role of guanylate cyclase in mediating the biological activity of exogenous NO. The maximal inhibition of migration obtained with cGMP (20% to 30%) was less than that obtained with NO donors (80% to 90%). A similar discrepancy between the efficacies of exogenous NO and exogenous 8-Br-cGMP is seen in inhibition of VSMC proliferation8 10 and may indicate that other signal transduction pathways are involved in mediating these inhibitory effects of NO on VSMCs.
We examined the effects of two NO donors on mitochondrial respiration and protein synthesis because NO can inactivate iron-containing enzymes of the mitochondrial electron transport chain as well as the enzyme aconitase in the citric acid cycle, leading to energy depletion and decreased protein synthesis.23 Because cell motility has been shown to require protein synthesis,24 we examined the possibility that the inhibition of VSMC migration seen with NO donors was associated with significant inhibition of protein synthesis or mitochondrial respiration. Although SP NO decreased protein synthesis by 28% (Fig 5⇑), DEA NO had no effect. Since both DEA NO and SP NO have similar potencies in inhibiting VSMC migration (Fig 2A⇑ and 2B⇑), the differences in protein synthesis suggest that this is an effect that is specific for SP NO and thus is not the mechanism by which exogenous NO inhibits VSMC migration.
No inhibition of mitochondrial respiration was noted with either NO donor (Fig 5⇑), and we found that 8-Br-cGMP (1 mmol/L), which inhibited VSMC migration (Fig 4⇑), increased both protein synthesis and mitochondrial respiration relative to control cells (Fig 5⇑). Although we cannot define the mechanism by which 8-Br-cGMP increases these two cellular functions, the opposite effects of exogenous cGMP on VSMC migration versus respiration and protein synthesis further demonstrate that the inhibition of migration by NO and cGMP is not due to inhibition of global cellular function.
Our studies illustrate that NO inhibits chemokinesis, or random migration of VSMCs. Further studies are needed to determine whether NO inhibits chemotaxis, or directed migration of VSMCs. The relevance of inhibition of chemokinesis of VSMCs in the wounded culture model is illustrated by the use of this model to demonstrate that heparin inhibits VSMC migration independent of proliferation in vitro.16 These findings were reflected in vivo, where heparin inhibits the migration of nondividing VSMCs to the intima after arterial injury.25
NO donors have been shown to inhibit VSMC proliferation in vitro8 11 as well as VSMC collagen production in vitro.12 These previous studies illustrate two possible mechanisms by which augmentation of NO production in vivo inhibits intimal lesion formation in different vascular injury models.1 2 5 6 Our observations delineate a third potential mechanism to explain how NO limits development of neointimal hyperplasia after vessel injury, namely, by inhibition of VSMC migration from the medium to the developing lesion in the intima.
Selected Abbreviations and Acronyms
| DEA NO | = | diethylamine NONOate |
| GSNO | = | S-nitrosoglutathione |
| MTT | = | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
| NO | = | nitric oxide |
| SP NO | = | spermine NONOate |
| VSMC(s) | = | vascular SMC(s) |
Acknowledgments
Dr Sarkar was supported by a National Research Service Award (F32 HL-08677). Eric Meinberg was a Student Research Fellow of the American Heart Association of Michigan. Additional support was provided by National Institutes of Health grants HL-18575 (to Dr Webb) and HL-02816 (to Dr Stanley) and by the Conrad Jobst Research Laboratories.
- Received June 5, 1995.
- Accepted October 27, 1995.
- © 1996 American Heart Association, Inc.
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