HO-1 and CO Decrease Platelet-Derived Growth Factor-Induced Vascular Smooth Muscle Cell Migration Via Inhibition of Nox1
Objective— Heme oxygenase-1 (HO-1), via its enzymatic degradation products, exhibits cell and tissue protective effects in models of vascular injury and disease. The migration of vascular smooth muscle cells (VSMC) from the medial to the intimal layer of blood vessels plays an integral role in the development of a neointima in these models. Despite this, there are no studies addressing the effect of increased HO-1 expression on VSMC migration.
Results and Methods— The effects of increased HO-1 expression, as well as biliverdin, bilirubin, and carbon monoxide (CO), were studied in in vitro models of VSMC migration. Induction of HO-1 or CO, but not biliverdin or bilirubin, inhibited VSMC migration. This effect was mediated by the inhibition of Nox1 as determined by a range of approaches, including detection of intracellular superoxide, nicotinamide adenine dinucleotide phosphate oxidase activity measurements, and siRNA experiments. Furthermore, CO decreased platelet-derived growth factor-stimulated, redox-sensitive signaling pathways.
Conclusion— Herein, we demonstrate that increased HO-1 expression and CO decreases platelet-derived growth factor-stimulated VSMC migration via inhibition of Nox1 enzymatic activity. These studies reveal a novel mechanism by which HO-1 and CO may mediate their beneficial effects in arterial inflammation and injury.
Heme oxygenase (HO)-1 is an inducible stress protein that has cellular and tissue protective effects in vascular injury and disease.1,2 The tissue protection of HO-1 likely relates to the production of its enzymatic products, biliverdin/bilirubin and carbon monoxide (CO).2,3 Biliverdin and bilirubin are antioxidants that can provide protection against oxidative stress in cell culture and in vivo.3–5 Recent evidence also demonstrate antiinflammatory and antiproliferative properties of these pigments.6 Although toxic at high concentrations, low concentrations of CO confer anti-inflammatory, anti-apoptotic, antiproliferative, and vasodilatory effects.7 Both CO and biliverdin/bilirubin have been shown to inhibit vascular smooth muscle cell (VSMC) proliferation in vitro and neointima formation in response to vascular injury.6,8
In animal models of vascular injury, intimal and medial thickening is thought to be attributable not only to VSMC proliferation but also to migration of VSMC from the media to the intima.9,10 The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoform Nox1 and Nox4 were recently shown to play important roles in the migration of VSMC.11–14 The NADPH oxidase family of enzymes catalyzes the one-electron reduction of molecular oxygen to superoxide (O2−). Superoxide may be dismutated to hydrogen peroxide by various isoforms of superoxide dismutase (SOD). These reactive oxygen species (ROS) promote the migration of VSMC via activation of redox-sensitive kinases or inhibition of phosphatases.15
Previous studies suggest that CO or biliverdin/bilirubin may inhibit NADPH oxidase activity.16–18 Additionally, biliverdin/bilirubin may scavenge NADPH oxidase-derived ROS because of their antioxidant properties. Thus, in this study we hypothesized that increased expression of HO-1 and heme degradation products exhibit antimigratory properties in addition to their antiproliferative effects. We further hypothesized that the inhibition of NADPH oxidase activity or scavenging of ROS by these HO-1–derived products mediates this antimigratory effect.
Materials and Methods
For more complete methods please see online data supplement at http://atvb.ahajournals.org.
Tricarbonyldichlororuthenium (II) dimer (CORM-2), ruthenium (III) chloride hydrate (RuCl3), peg-SOD, platelet-derived growth factor BB (PDGF-BB), dihydroethidium, Triton X-100, dimethyl sulfoxide, and reduced NADPH were from Sigma (St. Louis, Mo). Diphenylene iodonium was from Calbiochem (San Diego, Calif). Lucigenin was from Alexis (San Diego, Calif). Antibodies against total and phospho-ERK, p42/p44, Jun NH2 terminal kinase, and p38 antibodies were from Cell Signaling Technology Inc. (Danvers, Mass). Co(III) protoporphyrin IX chloride was from Frontier Scientific (Logan, Utah).
Rat aortic smooth muscle cells (RASMC) were isolated by collagenase/elastase digestion and maintained in DMEM with 10% fetal bovine serum, 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. RASMC were used at passages 3 to 8.
Adenovirus expressing recombinant β-galactosidase and recombinant rat HO-1 have been described elsewhere.19
HO Activity Assay
The activity of HO enzymes was determined in cell extracts by measuring conversion of hemin to bilirubin as previously described.20
Wound Migration Assay
PDGF-induced migration of RASMC was measured by wounding a monolayer of cells and monitoring the decrease in area after 18 hours.
Measurement of Intracellular O2−
PDGF-induced intracellular O2− production was evaluated by measuring the conversion of dihydroethidium to hydroxyethidium in a fluorometer (TECAN infinite M200).
Preparation of 28 000g Membrane Fraction
Membrane fractions were prepared as previously described.21
NADPH Oxidase Activity in Cell Culture
NADPH oxidase activity was measured by monitoring lucigenin (5 μmol/L) chemiluminescence or cytochrome c reduction.21
Preparation of Aorta for NADPH Oxidase Activity
The medial layers of aorta were isolated, membrane fractions were prepared, and NADPH oxidase activity was measured by lucigenin (5 μmol/L) chemiluminescence.21
Statistics were performed using Graphpad Prizm software. Data were analyzed by 1-way ANOVA followed by Tukey test for multiple comparisons. For grouped analysis, data were analyzed by 2-way ANOVA followed by Bonferroni post hoc test.
HO-1/CO Inhibits RASMC Migration
Increased expression of HO-1 has been shown to modulate VSMC proliferation,8 but not VSMC migration. Therefore, we examined PDGF-induced migration of RASMC with or without previous treatment with HO-1 adenovirus or control β-galactosidase adenovirus. In all cases, cells were treated with adenovirus 24 hours before stimulation with PDGF. As seen in Figure 1B, overexpression of HO-1 mediated by infection of cells with HO-1 adenovirus resulted in decreased RASMC migration. The inhibition of VSMC migration correlated with both increased HO-1 protein expression and activity (Figure 1A). Similar results were obtained using the chemical inducer of HO-1, Co(III) protoporphyrin IX chloride (supplemental Figure I).
HO-1 catalyzes the breakdown of heme into CO, free iron, and biliverdin (and subsequently bilirubin). To determine which of these products are involved in the regulation of PDGF-induced RASMC migration by HO-1, we treated cells with CO gas (250 ppm), biliverdin (30 μmol/L), or bilirubin (10 μmol/L). Only the addition of CO to the cells decreased PDGF-induced migration (Figure 1C), providing evidence that CO is responsible for the antimigratory effects seen with increased HO-1 expression. As an alternative to CO gas, we also tested whether a CO-releasing molecule, CORM-2, could inhibit RAMSC migration. CORM-2 dose-dependently inhibited RASMC migration (Figure 1D). Importantly, RuCl3 did not affect PDGF-induced migration, demonstrating that the effect of CORM-2 was attributable to CO and not the presence of the ruthenium base compound. Similar results were obtained using the Boyden chamber assay (supplemental Figure II).
CO Inhibits PDGF-Stimulated Increases in O2−
We hypothesized that the antimigratory effects of CO might be mediated via inhibition of NADPH oxidase-derived ROS. Using quantitative reverse-transcription polymerase chain reaction, we confirmed the expression of Nox1 and Nox4, but not Nox2, in our RAMSC cultures (supplemental Figure III). Our first approach to elucidate a possible role for ROS and NADPH oxidase in the CO-dependent inhibition of VSMC migration was to evaluate levels of O2− in RASMC in response to PDGF. We observed that PDGF induced a rapid and sustained increase in O2− levels (supplemental Figure IV) as measured by dihydroethidium fluorescence. To determine if the ROS production was NADPH oxidase-dependent, we preincubated the cells with the nonspecific flavo-protein inhibitor diphenylene iodonium, which is often used as a preliminary indicator of NADPH oxidase activity. Diphenylene iodonium abolished PDGF-induced increases in ROS levels, suggesting a possible role for NADPH oxidase in PDGF-induced O2− production (Figure 2A). Peg-SOD (10 U/mL) was used to validate the specificity of the assay for O2−.
To determine whether CO gas (250 ppm) or CORM-2 could inhibit PDGF-stimulated increases in O2−, RASMC were treated with CO gas or CORM-2, and 30 minutes later RASMC were stimulated with PDGF. Superoxide production was monitored by dihydroethidium as described. Both CO gas and CORM-2, but not RuCl3, decreased PDGF-induced O2− production (Figure 2B).
HO-1/CO Inhibits PDGF-Stimulated NADPH Oxidase Activity
To more directly test the hypothesis that HO-1/CO inhibits PDGF-induced NADPH oxidase activity, NADPH-dependent O2− production was measured in 28 000g membrane fractions from treated cells using lucigenin-enhanced chemiluminescence. Treatment of RASMC with PDGF caused a >2-fold increase in NADPH-dependent O2− production that was inhibited by diphenylene iodonium and scavenged by peg-SOD (Figure 3A). Importantly, l-NAME or allopurinol had no effect on NADPH-dependent superoxide production, providing evidence that the superoxide measured was not from uncoupled nitric oxide synthase or xanthine oxidase activity. The effect of diphenylene iodonium or peg-SOD on NADPH oxidase activity was further confirmed via measurement of O2− using cytochrome c reduction in similar experiments (Figure 3B). We next tested whether induction of HO-1 expression could inhibit PDGF-induced NADPH oxidase activity in RASMC. The increased HO-1 expression and activity attributable to HO-1 adenovirus resulted in significant inhibition of PDGF-induced NADPH oxidase activity (Figure 3C).
To determine whether the effect of HO-1 on NADPH oxidase activity was attributable to CO, RASMC were pretreated with different concentrations of CO gas (100 or 250 ppm) or 100 μmol/L CORM-2 and stimulated with PDGF. CO gas as well as CORM-2 decreased PDGF-induced NADPH oxidase activity (Figure 4A). In ex vivo experiments, strips of medial smooth muscle were preincubated with or without CORM-2 for 30 minutes, followed by an additional 45 minutes of incubation with PDGF. Membrane fractions from the treated medial strips were then prepared and NADPH oxidase activity measured. CORM-2, as well as peg-SOD, inhibited NADPH oxidase activity in the medial layer of the rat aorta (Figure 4B), providing evidence that CO could inhibit NADPH oxidase activity in an intact tissue.
We next sought to determine whether CO could directly inhibit NADPH oxidase activity. Figure 4C shows representative tracings of real-time lucigenin chemiluminescence from membrane fractions derived from control or PDGF-stimulated cells. Addition of NADPH to the membrane fraction from PDGF-stimulated cells results in a >2-fold increase in lucigenin chemiluminescence compared to membrane fraction from control cells. Addition of CO saturated buffer (final concentration of ≈9 μmol/L based on the solubility of CO in water) to the PDGF-treated sample after NADPH addition caused a rapid decrease in lucigenin chemiluminescence.
Effects of CO on PDGF-Induced Migration Are Mediated by Inhibition of Nox1
To test whether the inhibition of migration by HO-1/CO is mediated by inhibition of NADPH oxidase, we transiently transfected RASMC with siRNA against Nox1, Nox4, or a nontargeting siRNA. Transfection of RASMC with Nox1 or Nox4 siRNA decreased their mRNA levels by ≈85% and 60%, respectively, after 72 hours (supplemental Figure VA). Nox4, but not Nox1 or nontargeting siRNA, significantly inhibited basal NADPH oxidase activity measured in membrane fractions from unstimulated cells (Figure 5A). Alternatively, Nox1 siRNA, but not nontargeting or Nox4 siRNA, reduced PDGF-induced NADPH oxidase activity to basal levels as measured by lucigenin chemiluminescence in membrane fractions from PDGF-stimulated cells (Figure 5B). Importantly, CORM-2 inhibited PDGF-stimulated NADPH oxidase activity in Nox4, but not Nox1, siRNA-treated cells. Both Nox1 and Nox4 siRNA, but not nontargeting siRNA, significantly reduced PDGF-induced migration; however, only cells treated with nontargeting or Nox4 siRNA were still sensitive to inhibition of migration by CORM-2 (Figure 5C). Similar results were obtained using the Boyden chamber assay (supplemental Figure VB). These data strongly support the hypothesis that whereas Nox1 and Nox4 are both important in mediating RASMC migration, HO-1/CO inhibits RASMC migration via inhibition of Nox1.
CO Inhibits PDGF-Stimulated Redox-Sensitive Signaling Pathways
NADPH oxidase-derived ROS have been shown to activate pro-growth, pro-migratory pathways in RASMC.22,23 Serum-starved RASMC were stimulated with PDGF with or without previous treatment with CORM-2. Western blot analysis revealed that treatment of RASMC with CORM-2 resulted in decreased phosphorylation of ERK1/2, p38, Jun NH2 terminal kinase, and AKT when compared to control samples (Figure 6A–F). Each of these pathways is known to be involved in RASMC migration because specific inhibition of these pathways results in decreased migration. Finally, RuCl3 had no effect on these redox-sensitive signaling pathways (supplemental Figure VI).
The protective role of HO-1 has been studied in the context of various vascular diseases. In children lacking a functional HO-1 allele, atherosclerosis (hyperlipidemia, fatty streaks, and plaques) is increased.24 Moreover, HO-1 overexpression reduces lesional area in the aorta of apolipoprotein E−/− mice.25 HO-1 is induced after balloon angioplasty in rats,26 and neointimal hyperplasia is exacerbated in HO-1–null mice. Additionally, both CO8,27 and biliverdin28 have been shown to inhibit neointima formation. Interestingly, to date, there exists no study exploring the effect of HO-1/CO on vascular smooth muscle cell migration, an integral process to the development of atherosclerosis and restenosis after angioplasty.
Herein, we provide evidence that induction of HO-1 expression in RASMC inhibits PDGF-induced migration. CO mediated the inhibition of migration by HO-1 because CO gas or the CO releasing molecule, CORM-2, but not biliverdin or bilirubin, was able to inhibit RASMC migration. Therefore, we focused on the mechanism by which CO inhibits PDGF-induced RAMSC migration.
Many of the pro-migratory signaling pathways stimulated by PDGF are mediated by ROS.15 In VSMC, antioxidants block migration in response to PDGF.29 In contrast, VSMC extracted from Nox1 or p22phox-overexpressing mouse aortas exhibit an increase in PDGF-stimulated migration.30 These studies led us to hypothesize that CO inhibits RASMC migration via inhibition of NADPH oxidase activity. We demonstrate that CO inhibits PDGF-induced O2− production in intact cells as well as NADPH oxidase activity in membrane fractions from PDGF-stimulated cells. By isolating the medial layer of rat aorta, we were likewise able to demonstrate the inhibition of PDGF-stimulated NADPH oxidase activity in an ex vivo setting. Furthermore, direct addition of CO to the membrane fraction isolated from PDGF-treated cells rapidly decreased NADPH-stimulated O2− production. These data provide compelling evidence that CO inhibits NADPH oxidase activity in RASMC by directly interacting with the enzyme.
Aortic VSMC express both Nox1 and Nox4. Additionally, recent studies demonstrate an important role for either Nox1 or Nox4 in VSMC migration.11,12 In particular, Lee et al demonstrated that VSMC derived from Nox1-null mice exhibit decreased PDGF-induced migration, whereas VSMC derived from smooth muscle-specific Nox1-overexpressing mice exhibit enhanced migratory responses. In this study, the specific involvement of Nox1 in PDGF-stimulated NADPH oxidase activity was revealed by experiments demonstrating that this activity could be prevented by Nox1 siRNA but not Nox4 siRNA. Both Nox1 siRNA and Nox4 siRNA were able to prevent PDGF-induced migration to various extents; however, cells treated with Nox1 siRNA were resistant to further inhibition by CO, whereas Nox4-treated cells were not. From these observations, we draw the conclusion that CO inhibits Nox1-dependent ROS production, leading to inhibition of VSMC migration.
Early studies on Nox2 (ie, gp91phox, cytochrome b558) used CO as a tool to study the 2 heme moieties within the protein.31,32 These in vitro studies using partially purified enzyme generally concluded that CO binds the heme group in Nox2 poorly, if at all. One inherent drawback of those studies, however, is that partially purified Nox2 was used, which likely contained only the membrane components of the enzyme complex. At the time, it was not known that additional cytosolic subunits, such as p47 and p67, are required in the enzymatic complex. This leaves open the possibility that the conformation of Nox2 may be altered when bound to these subunits, which could allow for interaction of CO with the heme group. More recent studies examining the effect of CO on Nox2 have demonstrated changes in the heme absorbance spectra in response to CO.16,17 Despite this demonstration, these studies did not address whether CO could directly inhibit NADPH oxidase enzymatic activity. Rather, these studies demonstrate decreased NADPH oxidase activity in intact cells treated with CO and thus could not distinguish whether CO was inhibiting signaling processes leading to the activation of Nox2 or directly inhibiting Nox2 activity. Our studies demonstrating a direct effect of CO on NADPH oxidase enzymatic activity are unique in this respect. Taken together, our studies along with those showing alteration of the heme spectra of Nox2 suggest that CO may inhibit NADPH oxidase enzymatic activity via binding to 1 or both of the heme groups in the Nox subunit of the enzyme complex.
As discussed, many of the pro-migratory pathways stimulated by PDGF are redox-sensitive.15 One important mediator of growth factor responses in VSMC is Akt. ROS sensitivity of Akt is conferred by the phosphorylation of mitogen-activated protein kinase APK-2 by p38, a redox-sensitive kinase.33 This leads to recruitment of mitogen-activated protein kinase APK-2 to an Akt–p38 mitogen-activated protein kinase complex and phosphorylation of Akt.34 Besides p38 mitogen-activated protein kinase, other mitogen-activated protein kinases are sensitive to ROS. The Jun NH2 terminal kinase activation in response to angiotensin II is blocked by antioxidants.35 ERK1/2 was the earliest discovered redox-sensitive kinase having been shown to be activated by direct addition of hydrogen peroxide to cells.22 Additionally, Janus tyrosine kinases activate ERK1/2 in VSMC, and Janus tyrosine kinase-2 activation in response to angiotensin II was shown to be attenuated by NADPH oxidase inhibitors.36 In agreement with these findings, we were able to demonstrate that, in RASMC, inhibition of Nox1 activity by CO correlates with decreased phosphorylation of AKT, as well as the mitogen-activated protein kinases, p38, ERK1/2, and Jun NH2 terminal kinase-1.
The use of CORM-2 in this study raises some question as to whether the effects seen are attributable to direct effects of CO or secondary effects of CORM-2. In particular, CORM-2 may induce HO-1 and thus contribute to the effects of CORM-2 in the wound migration assay. However, our studies show an effect of CO gas in this model of migration assay. Furthermore, CORM-2 also inhibits RASMC migration in the Boyden chamber assay of migration. The time frame of this assay (4 hours) is such that one would not expect increased HO expression to be a factor. In terms of NADPH oxidase activity, these assays are all on a short time scale, and thus induction of HO-1 by CORM-2 cannot be a factor. Concerns that the ruthenium metal center might be mediating the effects of CORM-2 were assuaged by the demonstration that ruthenium chloride had no effects in the models studied.
In conclusion, our studies demonstrate that in RASMC CO inhibits Nox1-dependent migration stimulated by PDGF. Furthermore, we show that CO inhibits Nox1 activity, likely via direct interaction with the enzyme complex. These studies reveal a novel mechanism by which increased HO-1 expression and activity and HO-1-derived CO may mediate their beneficial effects in arterial inflammation and injury.
Sources of Funding
This work was supported by NIH HL085134 to P.M.B.
A.I.R. and A.G. contributed equally to this work.
Received May 29, 2009; revision accepted October 15, 2009.
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