Inhibition of Vascular Smooth Muscle Cell Migration by Cytochrome P450 Epoxygenase-Derived Eicosanoids
Vascular smooth muscle cell (SMC) migration and proliferation contribute to neointimal hyperplasia and restenosis after vascular injury. The epoxyeicosatrienoic acids (EETs), which are products of cytochrome P450 (CYP) epoxygenases, possess vasodilatory, antiinflammatory, and fibrinolytic properties. To determine whether these compounds also possess antimigratory and antiproliferative properties, we stimulated rat aortic SMCs with either 20% serum or platelet-derived growth factor (PDGF-BB, 20 ng/mL). In a concentration-dependent manner, treatment with EETs, particularly 11,12-EET, inhibited SMC migration through a modified transwell filter by 53% to 60%. EETs, however, have no inhibitory effects on PDGF-stimulated SMC proliferation. Adenoviral-mediated overexpression of the CYP isoform, CYP2J2, in SMCs also inhibited serum- and PDGF-induced SMC migration by 32% and 26%, respectively; both effects of which were reversed by the CYP inhibitors SKF525A or clotrimazole, but not by the KCa channel blocker, charybdotoxin, or the cyclooxygenase inhibitor, diclofenac. The effect of EETs correlated with increases in intracellular cAMP levels. Indeed, forskolin and 8-bromo-cAMP exert similar inhibitory effects on SMC migration as 11,12-EET and the effects of 11,12-EET were blocked by cAMP and protein kinase A (PKA) inhibitors. These findings indicate that EETs possess antimigratory effects on SMCs through the cAMP-PKA pathway and suggest that CYP epoxygenase-derived eicosanoids may play important roles in vascular disease and remodeling.
The cis-epoxyeicosatrienoic acids (EETs) are vasoactive eicosanoid products of cytochrome P450 (CYP) epoxygenases.1–3⇓⇓ The EETs have properties similar to those of endothelium-derived hyperpolarizing factor (EDHF) because they hyperpolarize and relax vascular smooth muscle cells (SMCs) by activating calcium-sensitive potassium (KCa) channels.4,5⇓ Recently, several CYP epoxygenases, including members of the CYP2B, CYP2C, and CYP2J subfamilies, have been identified in vascular endothelial cells. However, their relative importance in endothelial EET biosynthesis has not been determined.1,6–8⇓⇓⇓ Treatment of porcine coronary artery endothelial cells in vitro with either β-naphthoflavone or nifedipine induces CYP2C expression, increases 11,12-EET biosynthesis, and enhances bradykinin-induced coronary artery relaxation via SMC membrane hyperpolarization.1,9⇓ Moreover, transfection of endothelial cells with an antisense oligonucleotide to CYP2C8/9 or treatment with the selective CYP2C9 inhibitor, sulfaphenazole, attenuates EDHF-mediated vascular responses, thus providing supporting evidence that the EDHF synthase in the porcine vascular bed may be a CYP2C isoform.1,9⇓
In contrast to their vasodilatory effects, EETs have also been shown to possess important nonvasodilatory actions within the vascular system. For example, EETs which are produced by a member of the CYP2J family, CYP2J2, inhibit cytokine-induced endothelial cell adhesion molecule expression by inhibiting the proinflammatory transcriptional factor, NF-κB.6 Furthermore, in vascular endothelial cells, addition of physiologically relevant concentrations of EETs or overexpression of CYP2J2 increases tissue plasminogen activator (tPA) expression and fibrinolytic activity via a Gαs-dependent, cAMP-mediated mechanism.10 However, neither the antiinflammatory nor the fibrinolytic actions of the EETs were blocked by KCa channel inhibitors, indicating that these EET effects were independent of their membrane-hyperpolarizing effects.6,10⇓ Munzenmaier and Harder11 have recently shown that CYP epoxygenase products are necessary for cerebral microvascular endothelial cells to form tubular structures in vitro, suggesting a role of these eicosanoids in angiogenesis. More recently, we showed that overexpression of CYP2J2 or treatment with synthetic EETs protects endothelial cells against hypoxia-reoxygenation injury.12 However, CYPs are an important source of not only EETs, but also, 20-HETEs, 13 and are a major source of reactive oxygen species in coronary endothelial cells.14 Taken together, these studies indicate that CYP-derived eicosanoids exert multiple homeostatic effects on the vasculature, in addition to their vasodilatory actions.
Interestingly, endogenous vasodilators such as nitric oxide (NO) also possess antiinflammatory,15 antithrombotic,16 antiproliferative, and antimigratory properties.17,18⇓ Indeed, endothelial dysfunction gives rise to vasoconstriction, smooth muscle cell proliferation and migration, inflammation, and thrombosis.19 In particular, proliferation and migration of vascular SMCs are important processes in neointima formation after coronary angioplasty.20,21⇓ Although EETs have been shown to exert potent mitogenic effects in renal epithelial and glomerular mesangial cells,22,23⇓ it remains to be determined whether they could also stimulate vascular SMC migration and proliferation. Thus, the purpose of this study was to investigate whether EETs could regulate the proliferation and/or migration of SMCs, and if so, to determine the mechanism(s) involved.
Materials and Methods
All standard culture reagents were obtained from JRH Bioscience. Platelet-derived growth factor (PDGF-BB) was from Upstate Biotechnologies. SKF525A and charybdotoxin were purchased from Sigma. EETs were obtained from Cayman Chemical. 8-Bromo-cAMP, forskolin, the cAMP Rp-isomer (Rp-cAMP), and the selective PKA inhibitory peptide (PKI) were obtained from Calbiochem. [3H]thymidine was purchased from NEN. The PKA assay kit was purchased from MBL International. The chemiluminescence detection kit was obtained from Amersham Pharmacia Biotech.
Rat aortic SMCs were prepared from thoracic aorta of 8- to 10-week old Wister male rats (Taconic Farms, Germantown, NY). The guidelines on handling and care of these rats were approved by our institutional Animal Care Committee. The cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 200 mmol/L l-glutamine. The purity and identity of SMCs were confirmed by immunohistochemical staining for smooth muscle α-actin. For all experiments, early passaged SMCs (ie, less than 4 passages) were grown to 60% to 70% confluence and rendered quiescent by serum starvation (0.1% BSA for at least 16 hours). When used, EETs were added 1 hour before the addition of growth factor or serum.
SMCs were grown to 80% confluence and growth-arrested by incubation in DMEM containing 0.4% fetal calf serum (FCS) for 48 hours for synchronization to G0. Cells were preincubated with the indicated agents for 1 hour. The media was then replaced with growth medium (Dulbecco’s modified Eagle’s medium) containing 6 ng/mL of PDGF-BB, and the indicated reagents were simultaneously added. After 24 hours, [3H]thymidine (10 μCi/mL) was added, and the cells were incubated an additional 24 hours. Incorporated radioactivity in cell lysates was determined using a liquid scintillation counter (Beckman LS 6000IC).
Western Blot Analysis
SMCs were lysed in 1% SDS, 0.1% Triton X-100, 10 mmol/L Tris-HCl (pH 7.4), centrifuged at 12 000 rpm for 10 minutes, and the insoluble fraction was discarded. Proteins (80 μg per lane) were separated on SDS-12% Tris-Glycine precast gels (Invitrogen, Carlsbad, Calif), and the resolved proteins were transferred to nitrocellulose membranes. The membranes were then immunoblotted with a rabbit polyclonal antibody to COX-1 (Santa Cruz Biotechnology) at 1:500 dilution or a CYP2J2-specific peptide (HMDQNFGNRPVTPMR; anti-CYP2J2pep1) at 1:2000 dilution followed by incubation with a goat anti-rabbit IgG conjugated to horseradish peroxidase (BioRad, Hercules, Calif). Blots were visualized using the enhanced chemiluminescence detection system (Amersham) as described.12,24⇓
The migration of SMCs was examined according to the procedure described by Law et al.25 Briefly, the cells were suspended in DMEM with 0.4% FCS at a concentration of 2×105 cells/mL. The cells were preincubated with EETs for 1 hour at 37°C, and 0.1-mL aliquots of the cell suspension (2×104 cells) were added to the top chamber of gelatin-treated transwell PVF-free membrane with 8-μm pores in 24-well plates (Costar Inc). The lower transwell compartment contained 0.6 mL of DMEM, 0.4% FBS containing migration factors such as FCS or PDGF-BB. Incubation was continued for 4 hours at 37°C, after which the adherent cells were washed extensively, fixed with methanol, and then stained with hematoxylin. The number of cells that migrated to the lower surface of each filter was counted in different high-power fields (HPFs) at a magnification of 320×. Experiments were performed in triplicate. In the experiments to determine the effects of EETs, cAMP, or PKA inhibitors on SMC migration, these agents were incubated with the cell suspension in the upper well of the chamber.
Confluent SMCs were pretreated with 3-isobutyl-1-methyl-xanthine (IBMX, 0.5 mmol/L) for 30 minutes before stimulation with EETs. Cells were scraped on ice, pelleted, and resuspended in ice-cold IBMX (0.5 mmol/L), boiled for 3 minutes, and frozen at −70°C. The intracellular cAMP level was determined using a radioimmunoassay kit with radiolabeled cAMP (Amersham Pharmacia Biotech). A standard curve was constructed using increasing amounts of unlabeled cAMP.
PKA Activity Assay
PKA activity assay was performed essentially according to the manufacturer’s protocol. Briefly, SMCs were grown to confluence in 6-well plates. After 48 hours incubation with Dulbecco’s modified Eagle’s medium containing 0.4% FCS, the cells were treated with agonists as described and then washed and lysed in radioimmunoprecipitation assay (RIPA) buffer. Lysates were added to 96-well plates coated with the immobilized PKA substrate peptide (RFARKGSLRQKNV) for 10 minutes. This reaction was stopped by adding 20% H3PO4. Phosphorylation was quantified by adding a biotinylated antiphosphorylated PKA substrate peptide antibody 2B9, followed by peroxidase-conjugated streptavidin. Color development was achieved by o-phenylenediamine and hydrogen peroxide, and optical density was measured at 492 nm in a spectrophotometer.26
Generation of Adenovirus Containing CYP2J2
Adenoviral constructs (serotype 5; E1, E3-deleted) containing either the LacZ cDNA (Ad-LacZ) or a cDNA encoding human CYP2J2 (AdCYP2J2) were generated for these studies. These transgene cassettes employ the cytomegalovirus (CMV) immediate-early promoter and β-globin polyadenylation signals. Adenoviral vector preparations were titered by standard plaque assay on human 293 cells. Recombinant viral stocks were kindly provided by the Vector Core Laboratory of the Harvard Gene Therapy Initiative. Exposure of SMCs to adenoviral preparations was performed at a multiplicity of infection (MOI) of 200 for 4 hours, followed by multiple rinses and return to normal growth medium. Prior experience with infection of SMCs with Ad-LacZ using this protocol has yielded >95% transduction efficiency by β-galactosidase staining (data not shown).
Data are expressed as mean±SEM. The statistical significance of differences was assessed by Student’s t tests or ANOVA, as appropriate; a value of P<0.05 was considered statistically significant.
Relatively pure (>98%) rat aortic SMC cultures were confirmed by their morphological features using phase-contrast microscopy and immunofluorescent staining with antibodies to smooth muscle α-actin (data not shown). There were no observable adverse effects of EETs alone or in combination with PDGF-BB, charybdotoxin, Rp-cAMP, PKI, or SKF525A on cell viability and trypan blue exclusion.
Effects of EETs on SMC Proliferation
To investigate whether the 4 possible EET regioisomers affect PDGF-stimulated SMC DNA synthesis, quiescent cells were treated with EETs (1 nmol/L to 1 μmol/L) for 24 hours and [3H]thymidine incorporation was assessed. Treatment with EETs (1 μmol/L) had no effect on PDGF-induced SMC DNA synthesis (Figure 1A) and cell number (data not shown). Furthermore, addition of EETs alone did not affect basal SMC DNA synthesis. These results indicate that in contrast to other cell types where EETs are known to increase cell proliferation,22,23⇓ EETs have little or no effect on SMC proliferation.
Effects of EETs on SMC Migration
The effects of EETs on SMC migration in response to either PDGF or FCS were investigated using a modified transwell apparatus. Stimulation of SMC with PDGF-BB (20 ng/mL) caused a 5.3-fold increase in SMC migration (6±3 to 33±7 cells per field, P<0.05) (Figure 1B). At a concentration of 1 μmol/L, 11,12-EET significantly inhibited the PDGF-stimulated SMC migration by 60%. In contrast, the 5,6-EET and 14,15-EET regioisomers exhibited modest but significant inhibitory effects on PDGF-stimulated SMC migration, whereas 8,9-EET had no effect. These results suggest that there is some degree of regioselectivity in the antimigratory effects of the EETs. 11,12-EET and 14,15-EET also inhibited SMC migration in response to 20% FCS (53% and 28% decrease, respectively, P<0.05 for both).
In a concentration-dependent manner, 11,12-EET inhibited the PDGF- and FCS-stimulated SMC migration with an IC50 of 0.5 μmol/L (Figure 2A). At 10 μmol/L, 11,12-EET inhibited PDGF-BB- and FCS-induced SMC migration by 73% and 65%, respectively. Importantly, significant effects on SMC migration were also observed at concentrations as low as 0.1 μmol/L (even in the presence of serum, which tends to bind a great proportion of EETs). The KCa channel blocker, charybdotoxin, had no effect on 11,12-EET–mediated inhibition on SMC migration, suggesting that the antimigratory effect of 11,12-EET is independent of its membrane hyperpolarizing effect. Furthermore, the precursor, arachidonic acid (1 μmol/L), had no effect, and the 11,12-EET enantiomers, 11S,12R-EET and 11R,12S-EET, have similar inhibitory effect on PDGF- and FCS-induced SMC migration (Figure 2B). These findings suggest that the inhibitory effect of EET on SMC migration is regioselective, but not stereospecific.
Effects of CYP2J2 on SMC Migration
To determine whether endogenously produced EETs can also inhibit SMC migration, we generated a recombinant adenovirus for delivery of the CYP2J2 cDNA (Ad-CYP2J2) into SMCs. Infection of SMCs with Ad-CYP2J2 at a MOI of 200 resulted in robust expression of a 56- to 57-kDa band in rat SMCs corresponding to recombinant CYP2J2 (Figure 3A). In contrast, no expression of CYP2J2 was observed in control Ad-LacZ–infected SMCs as demonstrated by Western blot analysis. Transfection of CYP2J2 inhibited both FCS- and PDGF-induced SMC migration by 26% and 32%, respectively, compared with LacZ or empty vector. Treatment of CYP2J2-transfected cells with the CYP inhibitors, SKF525A (100 μmol/L) or clotrimazole (3 μmol/L), completely reversed the inhibitory effect of CYP2J2 gene transfer on PDGF- and FCS-induced SMC migration. These results suggest that endogenously-derived EETs are effective in inhibiting SMC migration, although other products of CYP2J2 may also be contributing to the antimigratory effects of CYP2J2.
To determine whether other arachidonic acid metabolites such as prostaglandins could be mediating the inhibitory effects of EETs on SMC migration, we investigated whether EETs can increase the expression and/or activity of cyclooxygenase (COX). Treatment of SMCs with increasing concentrations of 11,12-EET did not affect the expression of COX-1 (Figure 3B), and the expression of COX-2 was not detected under our experimental conditions (data not shown). Furthermore, the COX inhibitor, diclofenac (3 μmol/L), did not affect the inhibitory effect of 11,12-EET on SMC migration. These findings suggest that the effect of EETs on SMC migration is not mediated by prostaglandins.
Effects of 11,12-EET on Intracellular cAMP Level and PKA Activity
To determine the mechanism by which 11,12-EET inhibits SMC migration, we studied the effects of EETs on cAMP levels because the cAMP/PKA pathway has been shown to regulate SMC migration.27,28⇓ To test the hypothesis that the inhibitory effect of EETs on SMC migration are mediated through the cAMP/PKA pathway, we first examined the effect of 11,12-EET on intracellular cAMP levels and PKA activity in SMCs. Treatment of SMCs with 11,12-EET (1 μmol/L) produced a 4-fold increase in intracellular cAMP levels within 5 minutes, which was sustained for more than 4 hours (Figure 4A). In a concentration-dependent manner, 11,12-EET increased intracellular cAMP level with an EC50 of 70 nmol/L (Figure 4B). At 1 μmol/L, 11,12-EET caused the most substantial increase in intracellular cAMP levels, followed by 14,15- and 5,6-EET, whereas 8,9-EET had little or no effect. These results correlated with the regioselective inhibition of SMC migration by EETs (Figure 1B) and suggest that cAMP is an important mediator of the inhibitory effect of EET on SMC migration. Because PKA is a major downstream target of cAMP, we investigated the effect of EETs on PKA activity in SMCs. Indeed, treatment with forskolin or 11,12-EET (1 μmol/L) increased PKA activity in SMCs by 41±8% and 37±7%, respectively (n=4, P<0.05). Taken together, these data suggest that 11,12-EET activates the cAMP/PKA pathway in SMCs.
cAMP/PKA Pathway Mediates the Inhibitory Effects of EETs on SMC Migration
To demonstrate that the activation of the cAMP/PKA pathway by 11,12-EET contributes to the inhibitory effect of 11,12-EET on SMC migration, we first examined the effect of the cAMP analogue, 8-bromo-cAMP, on FCS- and PDGF-stimulated SMC migration. In a concentration-dependent manner, 8-bromo-cAMP inhibited both FCS- and PDGF-stimulated SMC migration at concentrations of 0.1 mmol/L and 1 mmol/L (Figure 5A). Similarly, treatment of SMCs with forskolin, a direct activator of adenylyl cyclase, inhibited FCS- and PDGF-stimulated SMC migration in a concentration-dependent manner (Figure 5B). At 1 μmol/L, forskolin inhibited SMC migration by 55%, although complete inhibition was observed at a concentration of 10 μmol/L. At concentration of forskolin (0.1 to 10 μmol/L), which inhibits SMC migration, forskolin had no effect on SMC proliferation as determined by thymidine incorporation (Figure 5C). Higher concentrations of forskolin (25 and 50 μmol/L), however, did inhibit SMC proliferation. These findings suggest that lower levels of cAMP are required to inhibit SMC migration as opposed to the higher levels of cAMP that may be required to inhibit SMC proliferation.
To determine whether the inhibitory effect of 11,12-EET on SMC migration requires the activation of cAMP/PKA pathway, we studied the effect of agents that block either cAMP or PKA. A competitive inhibitor of cAMP, Rp-cAMP, 29 at a concentration of 10 μmol/L, completely reversed the inhibitory effect of 11,12-EET on PDGF- and FCS-induced SMC migration (Figure 6A). Rp-cAMP, however, did not affect the basal rate of SMC migration (data not shown). Similarly, in a concentration-dependent manner, a selective cell-permeable peptide inhibitor of PKA, myristoylated PKI,30 also reversed the inhibitory effect of 11,12-EET on SMC migration without affecting basal migration (Figure 6B). These findings suggest that the antimigratory effect of EET is mediated through the cAMP/PKA pathway.
We have shown that exogenous EETs, particularly 11,12-EET, inhibit SMC migration in response to growth factors. Furthermore, overexpression of CYP2J2 in SMCs attenuated SMC migration, which was reversed by the CYP inhibitors, SKF525A and clotrimazole, but not by the KCa blocker, charybdotoxin. Interestingly, there was no effect of EETs on SMC proliferation. This is in contrast to the effects of EETs in other cell types where they have been found to be mitogenic. For example, Harris et al22 have shown that EETs increase thymidine incorporation in rat glomerular mesangial cells, and Chen et al23 have shown that EETs are mitogenic in proximal tubular epithelial cells. Thus, the antiproliferative, and possibly, the antimigratory effects of EETs may be tissue-specific. The mechanism by which EETs inhibit SMC migration is due, in part, to the activation of the cAMP-dependent PKA pathway. Indeed, treatment with 11,12-EET increased intracellular cAMP levels and PKA activity in SMCs, and inhibitors of cAMP and PKA reversed the antimigratory effects of 11, 12-EET. However, the antimigratory IC50s of EETs were much greater than their corresponding EC50s for cAMP generation, although for a given regioisomer of EET, the IC50 correlated inversely with the EC50. These findings suggest that pathways other than cAMP may also mediate the antimigratory effects of EETs.
Many factors that inhibit SMC migration act through pathways involving cAMP and PKA. For example, stimulation of rat aortic SMCs with adrenomedullin increases cAMP levels and inhibits SMC migration.27 Phorbol esters, which activate PKC, decrease SMC migration in response to PDGF.28 Furthermore, the inhibitory effect of PDGF-AA on SMC chemotaxis is mediated, in part, through cAMP.31 Indeed, in our study, we found that activators of cAMP or stable cAMP analogues stimulate PKA and inhibit SMC migration. cAMP is generated from intracellular ATP by adenylyl cyclase, a family of membrane-bound enzymes that is regulated by the heterotrimeric G proteins, Gαs and Gαi. Interestingly, EETs have been shown to stimulate Gαs and adenylyl cyclase.10,32⇓
The precise mechanism by which 11,12-EET activates Gαs, however, is not known, but may involve ADP-ribosylation. For example, 11,12-EET stimulates ADP-ribosylation of a number of intracellular proteins in SMCs including Gαs.33,34⇓ Alternatively, EETs may elicit their biological effects through cell surface receptors. Indeed, the binding of 14,15-EET to a putative receptor on U937 cells leads to increased intracellular cAMP levels.35 An important downstream cellular target of cAMP is PKA. PKA contains 2 inhibitory regulatory subunits, which when bound to cAMP, dissociate from and derepress the active catalytic subunits, leading to increased activity.36 The mechanism by which PKA inhibits SMC migration remains to be determined, although elevation of cAMP is associated with disruption of the actin filaments,37 and several downstream targets of PKA are cytoskeletal-associated elements.
EETs possess vasodilatory, antiinflammatory, and fibrinolytic properties. The vasodilatory effect of EETs, which resemble those of endothelium-derived hyperpolarizing factor, is mediated by activation of KCa channels and subsequent hyperpolarization of SMC membranes.4,5⇓ The antiinflammatory effects of EETs involve the downregulation of cellular adhesion molecule expression via inhibition of NF-κB and IκB kinase activities.6 The fibrinolytic effect of EETs involves a Gαs-dependent, cAMP-mediated mechanism,10 similar to the pathway that mediates inhibition of SMC migration. Our present study, therefore, adds to the growing list of vascular-protective effects of EETs. In this respect, EETs are similar to endothelium-derived NO, which relaxes vascular smooth muscle, 38 inhibits NF-κB,15 and attenuates SMC migration.17,18⇓ However, in contrast to NO, EETs do not inhibit SMC proliferation.
The migration of medial SMCs into the intima is an important process in various vascular proliferative diseases.20,21⇓ The finding that 11,12-EET inhibits SMC migration, therefore, may have important clinical implications. Migration of SMC from the media to the intima gives rise to the neointima, which constitutes the bulk of the lumenal obstruction after arterial injury or transplant-associated arteriopathy.21 Our results, therefore, suggest that EETs may lower the risks of certain types of vascular diseases by inhibiting SMC migration. Although EETs have been reported to be present in the bloodstream of healthy humans and rats, their plasma concentrations (≈30 nmol/L)39 are substantially lower than the concentrations that were required to inhibit SMC migration in vitro. However, it should be noted that EET levels in human and rat heart tissues are estimated to be 6- to 7-fold higher40 and may be elevated during vascular injury and inflammatory conditions. Indeed, increased EET biosynthesis occurs after cholesterol feeding in rabbits and treatment of human endothelial cells with oxidized LDL.41,42⇓ Thus, it is possible that 11,12-EET antagonizes the development of vascular lesions by acting as a local antimigratory factor for SMCs. Further studies with vascular CYPs such as CYP2J2 or CYP2C8/9 should yield further insights into the role of EETs in the vascular wall.
This work was supported by grants from the National Institutes of Health (HL-52233, HL-48743, and HL-04189), the NIEHS Division of Intramural Research, and the American Heart Association Bugher Foundation Award. J.K.L. is an Established Investigator of the American Heart Association. We thank the Vector Core Laboratory of the Harvard Gene Therapy Initiative for providing the recombinant viral stocks.
Original received January 15, 2002; revision received March 5, 2002; accepted March 28, 2002.
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