eNOS Gene Transfer Inhibits Smooth Muscle Cell Migration and MMP-2 and MMP-9 Activity

Materials

Chemicals and materials were obtained from the following sources: TRIzol, total RNA isolation reagent, and fibronectin (human plasma), GIBCO BRL; platelet-derived growth factor (PDGF), R&D Systems Inc; DETA NONOate, Cayman Chemicals; Dowex AG50W-X8 (200-mesh), Ready-gels, Bio-Rad; [³H]arginine (35 to 70 Ci/mmol) and [α³²-P]dCTP (3000 Ci/mmol), Amersham; random-primed DNA labeling kit, Boehringer Mannheim; 10% gelatin zymography precast gels, renaturing buffer, developing buffer, and Seablue molecular weight marker, NOVEX; Transwell migration assay dishes, Costar Inc; rabbit anti-eNOS polyclonal antibody, Santa Cruz Biotechnology; rabbit anti–TIMP-2 polyclonal antibody, Chemicon; chemiluminescence detection kit, Transduction laboratories; and goat anti–rabbit IgG horseradish peroxidase (HRP) conjugate, NADPH, FAD, tetrahydrobiopterin, calmodulin, nitro-l-arginine (LNA), 8-bromo-cGMP, and other chemicals and cell culture additives not listed were of the highest grade available from Sigma.

Cell Culture

Rats and guinea pigs used in the study were maintained and used in compliance with the principles set forth in the Guide for the Care and Use of Laboratory Animals and approved by the University of Iowa Animal Care and Use Committee.

Aortic SMCs were cultured from Wistar male rats (8 to 10 weeks old; 200 to 300 g body weight). Aorta was removed from rats anesthetized with ether and kept in Ham’s F12 culture medium.¹⁹ Guinea pig coronary artery cells were obtained from 4- to 6-month-old females, 600 to 800 g body weight. Hearts were removed from guinea pigs anesthetized with xylazine (1 mg/kg) and ketamine (80 mg/kg IP) and kept in Ham’s F12 culture medium.²⁰ SMCs were cultured according to procedures established in our laboratory.^{19 20} The purity of SMCs was confirmed by immunohistochemical localization of smooth muscle–specific actin by use of monoclonal antibody.^{19 20} Cells were subcultured weekly in DMEM high-glucose medium supplemented with 10% FBS and antibiotics (100 μg/mL streptomycin, 100 U/mL penicillin, and 2.5 μg/mL Fungizone) and were used between the third and seventh passages.

Adenovirus-Mediated Gene Transfer

We used replication-deficient recombinant adenovirus, Ad5/RSVeNOS, containing bovine aortic endothelial cell NOS cDNA²¹ or a reporter gene (green fluorescent protein, GFP).¹⁶ Adenoviral vectors were prepared by The Vector Core, The University of Iowa College of Medicine, as described elsewhere.^{16 18 22 23 24} The adenovirus vector has had the sequence in the E1A, E1B, and E3 regions deleted, impairing the ability of the virus construct containing eNOS and GFP cDNA to replicate in nonpermissive cells. We have shown earlier that infection of SMCs with adenovirus vectors carrying reporter or eNOS gene increases transgene expression in a dose-dependent manner.^{16 23} In addition, our previous studies demonstrated that 50 to 100 pfu of viral vector per cell (50 to 100 multiplicity of infection, MOI) produces optimal gene expression of both reporter and eNOS genes^{16 18 22 23} ; therefore, in this study we used 50 MOI of virus to transfer the eNOS gene. Confluent SMCs in 100-mm dishes were transfected by incubation with Ad5/RSVeNOS virus at 50 MOI (50 pfu/cell) in 2 mL of serum-free DMEM supplemented with 0.1% BSA and antibiotics for 3 hours. Then, 3 mL of serum-free DMEM/0.1% BSA was added to each dish of cultured cells and incubated for 24 hours. After 24 hours, virus-containing medium was removed, 4 mL of fresh serum-free DMEM/0.1% BSA was added, and cells were incubated for 24 hours. GFP gene–carrying control vector (Ad5/RSVGFP)-transfected cells were used as control. Expression of eNOS gene was confirmed by Northern and Western blot analysis. eNOS activity was measured by citrulline assay on cell homogenates according to established protocols.^{16 18} Nitrite levels were measured in conditioned medium with Griess reagent as described before.²⁵ The transfection efficiency of GFP protein was examined under a fluorescent microscope with a fluorescin filter, and eNOS protein was examined by immunohistochemistry followed by confocal microscopy as described earlier.^{16 18 22}

Conditioning of Medium

After transfection, the cells were divided into unstimulated and stimulated groups. Two milliliters of serum-free DMEM/0.1% BSA was added to all cell culture dishes. Interleukin (IL)-1β was added to the stimulated group to make a final concentration of 10 ng/mL. For DETA NONOate and cGMP treatment, cells were serum-starved for 24 hours in 2 mL serum-free DMEM/0.1% BSA. Unstimulated cells were incubated with 2 mL serum-free DMEM/0.1% BSA. Cells were incubated for an additional 24 hours. After 24 hours, conditioned medium was collected, centrifuged to remove cell debris, and stored in aliquots at −70°C for future use.

Migration Assay

The chemotactic migration of eNOS gene– and GFP gene–transfected SMCs was measured with a transwell migration apparatus as described previously.^{11 26} PDGF-BB was diluted (10 ng/mL) in 0.6 mL DMEM/0.1% BSA and added to the lower wells of the chamber. The wells were covered with a PVP-free filter (Costar Inc) with 8-μm pores and coated on both sides with 6 μg/100 mm² fibronectin. Cells were trypsinized (0.01% trypsin/0.11 mmol/L EDTA), washed once in DMEM/0.1% BSA, and resuspended in DMEM/0.1% BSA at a density of ≈10⁶ cells/mL. SMCs (75 000 to 100 000 cells in 0.1 mL) were added into the upper wells of the transwell chamber. A group of eNOS gene–transfected cells were treated with 1 mmol/L of LNA 24 hours before and during the assay. The chambers were incubated for 6 hours at 37°C in an atmosphere of 95% air and 5% CO₂. At the end of incubation, cells were fixed and stained with hematoxylin. Nonmigrated cells on top of the filters were wiped off, filters were mounted, and migrated cells attached to the bottom of filter were counted in 6 random (×400 microscope) high-power fields.²⁶ Cell migration was calculated as difference between the number of migrated cells in the eNOS-treated and control groups.

Northern Blot Analysis

Total RNA was extracted from eNOS gene– and control vector–transfected cultured SMCs as described previously with a commercial guanidinium isothiocyanate reagent (TRIzol, GIBCO BRL).^{19 27} RNA was quantified by spectrophotometry, and equal amounts of denatured RNA samples were separated by electrophoresis on 1.2% agarose-formaldehyde gels and transferred to Nitran membrane. Prehybridization was done at 42°C with herring sperm DNA. Hybridization was done with cDNA probes: eNOS (2-kbp EcoRI/Hinc II, obtained from Dr Thomas Michel, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA).²⁸ Probes were radiolabeled by use of a random-primed DNA labeling kit. Blots were exposed to Kodak XAR-5 film with an intensifying screen for 24 to 48 hours at −70°C, developed, and photographed.

Zymography

Gelatinase activity in conditioned medium collected from cultures was measured by zymography.²⁹ This procedure has been shown to quantitatively estimate both proenzyme and activated MMP enzyme activity. Equal amounts of conditioned medium (5 μL) were subjected to electrophoresis with Novex 10% zymography gels containing 0.1% gelatin. Gels were washed with renaturing buffer (Novex) for 30 minutes and incubated at 37°C for 20 hours in developing buffer (Novex). After 20 hours, gels were stained with Coomassie blue. All gels were calibrated with Seablue molecular-weight marker (Novex).

Western Blot Analysis

Control and eNOS gene–transfected cells were serum-starved for 48 hours, then treated with IL-1β (final concentration of 10 ng/mL in DMEM/0.1% BSA) and incubated for 24 hours. After 24 hours, conditioned medium was collected, and cells were rinsed with ice-cold PBS and then lysed with 1 mL lysis buffer^{16 18 22} on a rocking platform for 30 minutes at 4°C. The lysates were centrifuged at 14 000g for 10 minutes at 4°C. The supernatants were collected and total proteins quantified with BIO-RAD reagents. One hundred microliters of supernatant from each sample was mixed with 2× sample buffer and boiled for 5 minutes for immunoblot analysis. Equal amounts of total protein (20 μg) from each sample were run on an SDS-PAGE gel. Similarly, conditioned medium (25 μL) from each sample was mixed with 2× or 6× sample buffer and boiled for 5 to 10 minutes, and equal amounts of each sample were run on a 10% SDS-PAGE gel. The samples were then electroblotted onto Immobilon-P membranes. Membranes were then serially incubated, first with blocking buffer containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.5), 0.2% (vol/vol) Tween 20, and 5% (wt/vol) nonfat milk for 1 hour. The next incubation was performed with primary antibody diluted 1:3000 in blocking buffer. A final incubation was carried out with anti–rabbit IgG HRP diluted 1:10 000 in blocking buffer. Immunoreactive bands were visualized with a chemiluminescence detection kit (Transduction Laboratories).

Data Analysis

Zymograms and Northern and Western blots were scanned, and the relative intensity of bands was determined by densitometry. Statistical analysis was carried out by use of a commercially available program (STATVIEW, Cricket Software Inc), and differences were considered significant at P<0.05. The results are presented as mean±SEM, and n in parentheses represents the number of separate experiments.

eNOS Gene Transfer

Northern and Western analysis demonstrated expression of eNOS mRNA and protein in Ad5/RSVeNOS-transfected cells (Figure 1⇓, A and B). Calcium-dependent NOS activity in eNOS gene–transfected cells was ≈225 pmol citrulline · mg⁻¹ · min⁻¹, compared with no activity in GFP gene–transfected cells (Figure 1C⇓). The NOS activity of eNOS gene–transfected cells was completely calcium-dependent and was inhibited by 1 mmol/L LNA. eNOS gene transfer increased PDGF-BB–stimulated NO production by 4- to 6-fold over GFP-transfected cells (Figure 1D⇓). We have demonstrated earlier that adenoviral vectors produce >80% gene transfer of both reporter gene and eNOS gene in vascular cells.^{16 18 22 23} Similar transfection efficiency was observed in SMCs incubated with 50 MOI of viral constructs in this study (data not shown). These results demonstrate that transfected eNOS gene in rat aortic SMCs is functional and can be used to study the effect of NO on cell migration and regulation of MMP activity.

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Figure 1.

Adenovirus-mediated transfer of eNOS gene in SMCs. Cells were infected for 3 hours with 50 pfu/cell of Ad5/RSVeNOS vector carrying bovine eNOS gene. Northern (A) and Western (B) blot analysis 48 hours after gene transfer showed robust expression of eNOS mRNA and protein, respectively, in eNOS gene–transfected cells vs none in cells transfected with control vector (GFP reporter gene). Citrulline assay (C) demonstrates calcium-dependent NOS activity (pmol citrulline · mg⁻¹ · min⁻¹) in eNOS gene–transfected cells compared with no detectable activity in GFP-transfected cells. Nitrite assay (D) showing increased production of nitrite with or without PDGF-BB stimulation in eNOS gene–transfected cells compared with the control group. *Significant (P<0.05) increase in NOS activity and NO₂ production in eNOS gene–transfected cells vs GFP-transfected cells.

Effect of eNOS Gene Transfer on SMC Migration

PDGF released from restenotic or atherosclerotic lesions is a chemoattractant and proliferative agent for SMCs.²⁷ It has also been observed that cultured SMCs express predominantly PDGF-BB receptors.³⁰ Therefore, we used PDGF-BB (10 ng/mL) to test the effect of eNOS gene transfer on SMC migration in a modified Boyden chamber. eNOS gene transfer significantly (P<0.05, n=8) inhibited PDGF-BB–induced chemotactic migration of rat aortic SMCs. There was a 30% decrease in migration in eNOS gene–transfected cells compared with GFP gene–transfected cells (Figure 2A⇓). Treatment of eNOS gene–transfected SMCs with 1 mmol/L LNA, a specific inhibitor of eNOS, completely reversed this inhibitory effect, showing that inhibition of SMC migration is due to increased NO production in these cells. As in rat aortic SMCs, eNOS gene transfer significantly inhibited (40% to 50%) migration of guinea pig coronary artery SMCs (Figure 2B⇓). Treatment of cells with control vector carrying the GFP gene in place of the eNOS gene had no effect on SMC migration (Figure 2⇓, A and B), indicating that inhibition of cell migration in eNOS gene–transfected cells is not due to the viral vector used. In addition, the extent of inhibition of cell migration by eNOS gene transfer was comparable to that produced by 500 μmol/L DETA NONOate treatment of coronary artery SMCs (Figure 2B⇓). These results would suggest that eNOS gene transfer inhibits SMC migration by increasing local NO production in SMCs.

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Figure 2.

Effect of eNOS gene transfer on PDGF-BB–stimulated chemotactic migration of rat aortic (A) and guinea pig coronary (B) SMCs. Cells grown to confluence in 100-mm culture dishes were incubated with 50 pfu/cell of Ad5/RSVeNOS or control vector (Ad5/RSVGFP) for 3 hours. Cell migration was measured 48 hours after transfection by a modified Boyden chamber assay as described in Methods section. Multiple high-power (×400) fields were counted and data expressed as mean±SEM (n=8). The y axis represents cells migrated vs control (GFP-transfected cells). LNA (1 mmol/L) was added to a group of eNOS gene–transfected cells 24 hours before and during cell migration to demonstrate that the inhibition of cell migration by eNOS gene transfer was due to NO production. eNOS gene transfer inhibited SMC migration compared with GFP-transfected cells in rat aortic SMCs (A). Inhibition of migration due to eNOS gene transfer was reversed by addition of LNA to the transfected cells. Similarly, transfer of eNOS gene and treatment of cells with 500 μmol/L DETA NONOate inhibited cell migration in guinea pig coronary SMCs vs control and GFP-transfected groups (B). *Significant (P<0.05) decrease in cell migration in eNOS gene–transfected or DETA NONOate–treated cells vs GFP-transfected or control cells.

Effect of eNOS Gene Transfer on MMP-2 and MMP-9 Activities

MMP-2 and MMP-9 play an important role in the migration of cells^{6 7} and are increased after balloon injury.⁹ Therefore, using gelatin zymography, we examined whether local NO production by eNOS gene transfer to SMCs had any effect on MMP-2 and MMP-9 activities. eNOS gene transfer to SMCs significantly (P<0.05, n=8) reduced the activated MMP-2 band compared with GFP gene transfection. There was a 40% decrease in the activated MMP-2 band in the eNOS gene–transfected SMCs compared with control cells transfected with GFP, whereas the pro-MMP-2 band was not altered by eNOS gene transfer (Figure 3A⇓). This was not unexpected, because MMP-2 is constitutively expressed in SMCs and is regulated mainly at the posttranslational level.^{8 31}

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Figure 3.

Effect of eNOS gene transfer on the MMP activity in conditioned medium collected from unstimulated (A) and IL-1β–treatment (B) groups. Confluent SMCs in 100-mm culture dishes were transfected with Ad5/RSVeNOS or control vector as described in Figure 1⇑. After 48 hours of gene transfer, 2 mL of fresh medium containing 0.1% BSA was added, and conditioned medium was collected after 24 hours. Equal amounts of conditioned medium (5 μL) were resolved on 10% gelatin zymography gels containing 0.1% gelatin (Novex). Zymograms were scanned, and gelatinase activity was quantified. Data are presented as mean±SEM. Compared with cells transfected with the control vector, eNOS gene–transfected cells showed a decrease in the activated MMP-2 band in the unstimulated group (n=8) and MMP-9 and activated MMP-2 activities in cells stimulated with 10 ng/mL of IL-1β (n=5). *Significant (P<0.05) decrease in MMP-2 and MMP-9 activities in eNOS gene–transfected cells vs GFP-transfected cells.

Because vessel injury results in release of cytokines, which activate MMP-2 and induce MMP-9,⁸ we tested whether NO had any effect on MMP-2 and MMP-9 activities in response to IL-1β stimulation. We stimulated SMCs with 10 ng/mL IL-1β for 24 hours and collected the conditioned medium for zymography. Compared with GFP gene transfection, eNOS gene–transfected SMCs showed a significant (P<0.05, n=5) decrease in activated MMP-2 and pro-MMP-9 activities in response to IL-1β stimulation. Densitometric analysis showed a 25% to 30% decrease in activated MMP-2 and pro-MMP-9 activities in eNOS gene–transfected cells compared with those transfected with GFP (Figure 3B⇑).

Effect of DETA NONOate and cGMP on MMP-2 and MMP-9 Activities

To confirm that eNOS gene transfer–mediated effects on MMP activity is due to the NO/cGMP pathway, we tested the effect of the NO donor DETA NONOate (half-life 18 hours at 37°C) and 8-bromo-cGMP on MMP-2 and MMP-9 activities. Conditioned medium collected from cells treated for 24 hours with 500 μmol/L DETA NONOate and 100 μmol/L 8-bromo-cGMP showed a decrease in activated MMP-2 by 45% to 50% in unstimulated and IL-1β–stimulated groups but had no effect on pro-MMP-2 bands (Figure 4⇓, A, B, and C). In addition, treatment of SMCs with DETA NONOate and 8-bromo-cGMP decreased MMP-9 activity by 80% to 90% in the IL-1β–stimulated group (Figure 4D⇓). These results confirm our eNOS gene transfer experiments and demonstrate that NO/cGMP pathway activation leads to a decrease in activated MMP-2 and induction of MMP-9 by inflammatory cytokines.

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Figure 4.

Effect of DETA NONOate and cGMP on MMP activity. Cells were treated with 500 μmol/L DETA NONOate or 100 μmol/L 8-bromo-cGMP for 24 hours. In the stimulated group, 10 ng/mL of IL-1β was added simultaneously with DETA NONOate or 8-bromo-cGMP. Conditioned medium was resolved on zymography gels as described in Figure 3⇑, and the gels were scanned and the relevant bands quantified. A, Representative zymogram showing the pro-MMP-9, pro-MMP-2, and activated MMP-2 bands (arrows). There was a decrease in activated MMP-2 in DETA NONOate– and cGMP-treated cells (B, n=3). Similarly, there was a decrease in activated MMP-2 and pro-MMP-9 levels in cells stimulated with IL-1β and treated with DETA NONOate or 8-bromo-cGMP (C and D, n=3). *Significant (P<0.05) decrease in MMP-2 and MMP-9 activities in DETA NONOate– or 8-bromo-cGMP–treated cells vs untreated control cells.

Effect of eNOS Gene Transfer on TIMP-2 Expression

MMP activity is modulated in vivo by TIMPs. Overexpression of TIMPs inhibits SMC migration in vitro and neointima formation in vivo.¹⁰ Also, TIMP-2 has been shown to inhibit MMP-2 and MMP-9 activities³² and is involved in regulating MMP-2 activation.^{31 33 34} Therefore, using Western blot analysis of the conditioned medium, we investigated whether eNOS gene transfection altered TIMP-2 secretion. Densitometric analysis of Western blots (Figure 5A⇓, representative blot) showed a significant increase (P<0.05, n=8) in the levels of TIMP-2 in the conditioned medium collected from eNOS gene–transfected cells compared with control (Figure 5⇓, A and B). eNOS gene transfer increased TIMP-2 secretion by almost 100% compared with GFP gene–transfected cells (Figure 5B⇓). A significant difference (P<0.05, n=6) was also observed in TIMP-2 levels in conditioned medium of eNOS and GFP gene–transfected cells in response to IL-1β stimulation (Figure 5⇓, A and B).

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Figure 5.

Effect of eNOS gene transfer on TIMP-2 secretion. SMCs were transfected with eNOS gene or control vector, and conditioned medium was collected from control and IL-1β cells as described in Figure 3⇑. Western blot analysis of the conditioned medium was performed, and A shows a representative blot. Data are presented as mean±SEM (n=8). Conditioned medium collected from eNOS gene–transfected SMCs showed a significant increase in TIMP-2 levels in both unstimulated [(a)*] and IL-1β–stimulated [(b)*] groups compared with conditioned medium collected from cells transfected with the control vector carrying genes. *Significant (P<0.05) increase in TIMP-2 level in eNOS gene–transfected cells vs GFP-transfected cells.