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Abstract |
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Top Abstract Introduction Materials and Methods Results Discussion References |
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Key Words: -adrenergic • vascular smooth muscle • NAD(P)H oxidase • arterial injury • proliferation
Introduction |
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Top Abstract Introduction Materials and Methods Results Discussion References |
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Reactive oxygen species (ROS) such as superoxide anions (O2-·) and hydrogen peroxide (H2O2) are important second messengers in intracellular signal transduction pathways for several functions, including VSMC growth.10 In particular, ROS mediate proliferation by several GPCR agonists and growth factors in VSMCs, including angiotensin II,11 thrombin,12 platelet-derived growth factor (PDGF),13 and endothelin-1.14 The membrane-bound NAD(P)H oxidase is the major source of ROS production in both the intact vessel wall and cultured VSMCs.15–18 The enzyme generates O2-· by NAD(P)H-derived one-electron reduction of molecular oxygen, that subsequently may be rapidly dismutated to form H2O2.19 This "vascular" oxidase shows similarities with the better characterized "phagocytic" NAD(P)H oxidase. The phagocytic oxidase consists of a membrane-associated flavocytochrome (cytochrome b558) that is composed of two glycoproteins, gp91phox (Nox 2) and p22phox, and the cytosolic components, p47phox, p67phox, p40phox, and the G protein Rac1/2.19 Activation of the phagocytic NAD(P)H oxidase requires association of p47phox and p67phox, followed by their targeting to the plasma membrane by Rac-GTP.19 p47phox plays a key role in assembly and activation of the oxidase, because in its absence, p67phox fails to assemble with the membrane-bound subunits.19 In addition, p47phox regulates electron transfer from flavin adenine dinucleotide (FAD) to the heme center of cytochrome b558, leading to O2-· generation.19 Although the vascular NAD(P)H oxidase is believed to be similar to the phagocytic oxidase, isoforms of all the vascular subunits have not been identified and the mechanism of activation is not completely understood. Several other ROS-generating systems, including the mitochondrial electron transport chain, nitric oxide synthase, xanthine oxidase, and cyclooxygenase have also been implicated in intracellular signaling cascades leading to changes in cell structure, function, and proliferation.20 However, the NAD(P)H oxidase is a much larger source of induced ROS in the vascular wall than these other enzymes. Besides activation by certain extracellular agonists, mechanical and chemical injury of arteries also strongly increase NAD(P)H oxidase–associated ROS generation, VSMC proliferation, and neointimal formation that are inhibited by treatment with antioxidants.21–23
The responsible pathways mediating the proliferative, hypertrophic, and chemotactic actions of catecholamines in VSMCs are not well understood. Norepinephrine-induced proliferation of VSMCs is mediated by extracellular signal-regulated kinases (ERK) 1/2.4,24 These mitogen-activated protein kinases (MAPK) also appear to mediate 1-AR-stimulated hypertrophy in cultured cardiomyocytes.25 Although ROS generation is involved in ERK1/2-dependent hypertrophy of cultured cardiomyocytes by 1-AR stimulation,26 only one report suggests ROS may be involved in adrenergic proliferation of cultured VSMCs.27 Furthermore, no studies have examined this question in the intact vessel wall. Therefore, the purpose of the present study was to determine if ROS mediate 1-AR–induced VSMC growth in vitro and in the vascular wall ex vivo, and whether the NAD(P)H oxidase is the source of catecholamine-stimulated ROS generation.
Materials and Methods |
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Top Abstract Introduction Materials and Methods Results Discussion References |
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The oxidative fluorescent probe dihydroethidium (DHE) and confocal microscopy were used to detect in situ levels of ROS in aorta sections.29 Images were acquired by an observer blinded to the treatment groups. The aconitase assay was used to measure ROS generation in cultured cells.30 p47phox mRNA expression was measured by reverse transcription-PCR (RT-PCR).31
Data are expressed as mean±SEM. Statistical analysis was performed using Student’s t test, with significance defined as P<0.05.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Results |
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Top Abstract Introduction Materials and Methods Results Discussion References |
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Norepinephrine Augments ROS Production in Aorta Media
We next used in situ dihydroethidium (DHE) confocal microscopy to determine if NE augments ROS generation ). Norepinephrine enhanced DHE intensity in injured media by 167±13% of vehicle (average response of F, n=14). The increase was abolished by NAC and DPI and dose-dependently inhibited by Tiron. Tiron and NAC alone inhibited baseline activity (E), possibly reflecting an effect of injury itself. The increase in DHE intensity induced by NE was not blocked by inhibition of the MAPK (ERK1/2) kinase, MEK1/2, with PD-98059 or UO-126, or by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG-1478 (F). These results suggest that the ROS-dependent signaling step in the adrenergic trophic pathway is upstream of the EGFR-ERK1/2 pathway. PD-98059 and AG-1478 had small inhibitory effects alone, suggesting that injury itself may increase ROS and activate the EGFR-ERK pathway.
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To confirm that DHE fluorescence reflects ROS levels, sections from aortae previously treated for 48 hours with NE were preincubated for 5 minutes with high levels of DPI (50 µmol/L) or Tiron (20 mmol/L) before incubation with DHE. Both agents strongly reduced the NE signal by 84±3% (n=8) and 60±9% (n=5), respectively.
Norepinephrine Induces ROS-Dependent Proliferation in Adventitia
Like medial VSMCs, stimulation of 1-ARs on adventitial fibroblasts (AFBs) causes their proliferation and adventitial thickening, which are strongly increased after injury in both organ culture6 and in vivo.7 In the present study, results for adventitia were similar to media for all groups in . Norepinephrine tended to increase protein synthesis and DNA content by 140±18% and 118±10% of vehicle (P<0.11 and P<0.14, n=5 per group, respectively), and NAC prevented these increases (84±15% and 99±7%, respectively, n=6 per group). Tiron and DPI also inhibited adventitial growth by NE, and all three inhibitors had no significant effect alone. Norepinephrine caused an increase in DHE fluorescence over vehicle [7±1 (n=10) versus 12±2 (n=14), fluorescence units, P=0.02; ], and changes similar to those obtained for media were also obtained for adventitia for the other treatment groups shown in .
Norepinephrine Effects in Uninjured Aorta
Because the uninjured artery is less sensitive than the injured artery to the trophic effect of NE,6,7 we performed a more limited study of uninjured aorta and only examined media. Norepinephrine (1 µmol/L, 48 hours) caused a small nonsignificant increase in protein synthesis (112±11% of vehicle, n=6). Tiron (5 mmol/L) inhibited this response (78±4%, P<0.05, n=6) and had an inhibitory effect alone (59±5%, P<0.001, n=6) as reported in cell culture.33 Changes in DNA content mimicked these results. Norepinephrine increased DHE fluorescence (71±4) above vehicle (34±4, P<0.001, n=6), and this was partially inhibited by Tiron (51±4, P<0.01, n=6) but unaffected by AG-1478 (1 µmol/L) (62±7, n=6). Tiron and AG-1478 had no effects alone.
Phenylephrine Increases Intracellular ROS Production in Cultured VSMCs
To confirm the DHE results, intracellular levels of ROS were determined by their ability to inhibit aconitase activity in vitro.30 Stimulation of cultured VSMCs with the selective 1-AR agonist phenylephrine (PE) induced a time-dependent decline in aconitase activity, with a maximum response at 10 minutes (-45.3±8.5%) that remained reduced at 60 minutes (A). The 10-fold higher concentration of PE than NE used herein reflects PE’s known 10-fold lower potency for activation of 1-ARs. Maximal trophic concentrations of NE (1 µmol/L) and thrombin (1 U/mL), measured after 20 minutes exposure, were equi-effective: aconitase activity was decreased -37.2±5.5% by NE (n=4) and -34.2±7.6% by thrombin (n=4). Thrombin served as a positive control for generation of ROS in VSMCs.12 Inhibition of aconitase activity by PE (at 10 minutes) was concentration-dependent, with potent inactivation at 10 µmol/L PE (-61.8±3.9%) (B). Reduction in aconitase activity was inhibited by Tiron and the SOD mimetic MnTBAP (C). Tiron and MnTBAP had no effect alone, although there was a trend for MnTBAP to increase baseline activity (ie, to lower basal ROS), as reported by others.34
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Phenylephrine-Induced Growth of VSMCs Is Inhibited by Catalase
To determine the relative roles of O2-· and H2O2 in catecholamine-induced VSMC growth, PEG-SOD and PEG-catalase were examined. The increase in DNA and protein content induced by PE was little affected by PEG-SOD, but abolished by PEG-catalase ().
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Prazosin Blocks Phenylephrine-Induced ROS Production and Growth of VSMCs
To confirm our previous studies showing that catecholamine trophic effects are 1-AR dependent3–8 and to determine if their ROS dependence, shown herein, is also 1-AR dependent, the 1-AR antagonist prazosin was examined. Prazosin inhibited both ROS production (aconitase inhibition; A) and VSMC growth (B).
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Phenylephrine-Stimulated ROS Generation and Proliferation Are Abolished in p47phox-/- VSMCs
To examine if catecholamine-stimulated ROS production and VSMC growth are mediated by the NAD(P)H oxidase, aortic VSMCs from p47phox-null mice were studied. As in rat aortic VSMCs, PE caused a reduction in aconitase activity in wild-type cells that was abolished in p47phox-/- cells (A). Thrombin served as a positive control for p47phox-dependent reduction in aconitase activity.31 Baseline levels of ROS were not lower in p47phox-/- cells (ie, basal aconitase activity was not greater) than in wild-type cells (0.31±0.01 versus 0.34±0.02 µmol/L NADPH/min per µg protein, respectively), in agreement with one35 but not another report.31 Elimination of PE-induced reduction in aconitase activity in p47phox-/- cells was associated with inhibition of PE-mediated growth (B). Furthermore, stimulation of wild-type VSMCs by PE for 1 hour promoted enhanced transcript levels of p47phox. The expected absence of expression in p47phox-/- cells served as a negative control (C).
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Inhibitors of NAD(P)H Oxidase but not of Other Cellular ROS Sources Inhibit PE-Induced Proliferation
Because additional enzymes besides NAD(P)H oxidase are known to generate low levels of ROS,20 we tested inhibitors of them on PE-induced proliferation. In contrast to DPI and the more specific NAD(P)H-oxidase inhibitor, apocynin, that abolished PE-stimulated proliferation (A), inhibitors of nitric oxide synthase (L-NAME), cyclooxygenase (indomethacin), xanthine oxidase (allopurinol), and cytochrome P450 oxidases (17-octadecynoic acid) had no effect on the PE-mediated increase in DNA content (B). These inhibitors had no effect alone. At a concentration that reduces oxidative phosphorylation (30 µmol/L),36 the NADH dehydrogenase inhibitor rotenone also had no effect. However, a higher concentration (100 µmol/L) did reduce the DNA increase by PE (C). Data for protein content (not shown) mimicked these DNA results.
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Discussion |
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Top Abstract Introduction Materials and Methods Results Discussion References |
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We previously found that NE induces growth of medial VSMCs in rat aorta maintained in organ culture, and that this is strongly augmented by balloon injury 4 or 12 days earlier in vivo.6 This mitogenic effect is mediated by the 1A-AR subtype, but not by the 1D-, 2D-, or ß-ARs that instead are vasoactive in this vessel. Furthermore, 1-AR–induced growth contributes significantly to restenosis after rat carotid balloon injury7,8 and injury-induced hypertrophic outward remodeling in mice.37 Results consistent with these observations were obtained in the present study, where NE caused significant proliferation of VSMCs in rat aorta studied in organ culture after receiving balloon injury 4 days earlier in vivo. This was associated with increased ROS generation (detected by DHE fluorescence). Both proliferation and ROS generation were abolished by Tiron, NAC, and DPI (), suggesting that the adrenergic trophic pathway includes a ROS-sensitive step(s). Tiron, NAC, and DPI alone had minimal-to-no inhibitory effects. This likely reflects the effect of injury itself to increase cytokines and growth factors, some of which may activate ROS, together with reduction in the extracellular concentration of these mediators by diffusion into the large (200 mL) tissue culture bath. Although no in vitro system can fully preserve in vivo conditions, the preloaded organ culture model permits VSMCs to be studied in the intact vascular wall.6
The source of catecholamine-induced ROS generation was not macrophages or neutrophils that are recruited to the vascular wall after injury. Rather, NE induced a mostly uniform increase in intracellular DHE fluorescence across the media (Figure 2C). We and others have shown that the media is composed almost entirely of cells expressing -smooth muscle actin, when examined 4 days after balloon injury.6 In addition, PE also caused cultured VSMCs to generate ROS, and with an efficacy comparable to thrombin (A).
To examine the source of ROS, we tested DPI and a more specific inhibitor of NAD(P)H oxidase, apocynin, on catecholamine-stimulated proliferation of cultured VSMCs. Both abolished PE-induced increases in DNA and protein content (Figure 7A).
Because DPI inhibits several flavin-containing enzymes that generate ROS, besides NAD(P)H oxidase, because apocynin may also have other actions, and because few specific inhibitors of the oxidase are available (p22phox-antisense, gp91ds-tat and dominant-negative p67phox), we examined primary cultures of aortic VSMCs obtained from mice genetically deficient in p47phox, a key regulatory subunit of NAD(P)H oxidase. ROS generation and proliferation induced by PE in wild-type cells were both abolished in p47phox-/- cells . Consistent with increase in ROS production, PE caused increased p47phox transcription levels at 1 hour .
Although these findings suggest that the NAD(P)H oxidase mediates the trophic action of catecholamines on VSMCs, we examined whether induction of other DPI-sensitive ROS-generating enzymes might participate. Phenylephrine-induced proliferation was unaffected by inhibition of nitric oxide synthase (NOS), cyclooxygenase, xanthine oxidase, or cytochrome P450 oxidases (Figure 7B). Although inhibition of NOS with L-NAME can inhibit VSMC proliferation by PDGF,38 our studies suggest that the pathway mediating 1-AR induced proliferation does not involve nitric oxide or the above oxidases. Interestingly, L-NAME also had no effect on 1-AR–mediated hypertrophy of cardiomyocytes.26 The NADH dehydrogenase inhibitor, rotenone (30 µmol/L), also did not inhibit PE-induced proliferation. However, a higher concentration of rotenone (100 µmol/L) caused a decrease in PE proliferation. This may reflect a reduction in mitochondrial oxidative phosphorylation36 and stimulation of apoptosis,39 an effect that is suggested by the decrease in baseline DNA content caused by this concentration (C).
Generation of ROS by PE was rapid, persisted for at least 60 minutes, was dose-dependent, and extended over the same dose range observed for NE-evoked contraction (). We have shown in organ culture that 1-AR–mediated proliferation also extends over this same range.6 Importantly, 1-adrenergic constriction is not associated with altered ROS activity and is not affected by inhibition of the NAD(P)H oxidase or ROS.40 These correlations strengthen the concept that the signaling pathway used by catecholamines to stimulate VSMCs growth in vivo involves the ROS-sensitive pathway identified in the present study. In addition, the maximum inactivation of aconitase after 10 minutes exposure to PE (40% to 60%), which we used to measure ROS production, is similar to that reported for thrombin31 and other studies using this assay.41 To validate this assay, we demonstrated that PE inhibition of aconitase activity was abolished by concomitant treatment with Tiron or MnTBAP (C). We also found that NE showed the same maximal efficacy as the 1-AR agonist PE, indicating that simultaneous stimulation of 2D- and ß-ARs that are also present does not influence ROS generation. This is in agreement with the effect of prazosin to inhibit ROS production and VSMC growth () and our previous results showing no involvement of 2D- and ß-ARs in catecholamine-induced growth of the intact aorta and carotid artery studied in organ culture6 and in vivo.7 The maintained ROS generation by PE (A) is similar to the action of certain other ROS-generating mitogenic factors. For example, angiotensin II induces biphasic production of ROS in VSMCs, involving a rapid early peak at 30 seconds, followed by sustained generation for at least 6 hours.11,42 Likewise, induction of ROS by thrombin was evident at 15 minutes, elevated further at 1 hour, and remained above baseline for at least 6 hours.12
In most cell types, O2-· generated by NAD(P)H oxidase is rapidly converted by SOD to H2O2. In cultured VSMCs, H2O2 mediates the mitogenic effects of angiotensin II,43 thrombin,12 and PDGF,13 and is itself mitogenic.44 In agreement with these studies, we found that proliferation of VSMCs by PE was abolished by membrane permeant PEG-catalase (). In contrast, PEG-SOD had minimal if any effect. This suggests that SOD is not limiting in the presence of PE stimulation, and also that O2-· itself or other ROS arising upstream of SOD (eg, peroxynitrite) have little or no direct role in mediating adrenergic proliferation. Similar results were reported for thrombin.12
We have evidence that activation of the intrinsic tyrosine kinase activity of EGFR and its known downstream activation of the raf1-MEK1/2-ERK1/2 pathway are required for 1-AR–induced growth of VSMCs in rat aorta studied in organ culture (unpublished data, H. Zhang, J.E. Faber, 2003). The EGFR tyrosine kinase inhibitor, AG-1478, and the MEK1/2 inhibitors, PD-98059 and UO-126, each abolished catecholamine-induced protein synthesis and DNA accumulation (while having no effects alone). This was associated with augmented phosphorylation of EGFR and ERK1/2 as assessed by immunoblot that was sustained for the duration of PE exposure. To begin to examine whether the ROS-sensitive step in the adrenergic trophic pathway is upstream or downstream of EGFR activation, in the present study, we determined the effect of these above-mentioned agents on ROS generation by DHE fluorescence (F). In contrast to their inhibitory effects on adrenergic growth, none had a significant effect on NE-induced ROS activity. This suggests that the ROS-sensitive step resides upstream of EGFR and ERK activation. Norepinephrine-induced ERK activation in cardiomyocytes has also been shown to be dependent on a ROS-sensitive step. In contrast, endothelin-114 and angiotensin II45 activation of ERK in VSMCs is not ROS-dependent; although there is controversy on this point for angiotensin II.46
is a proposed signaling pathway for catecholamine-mediated VSMC growth, based on our present results. Stimulation of 1-ARs by blood-borne NE or by increased release of NE from vascular nerves induced by injury47 is proposed to activate NAD(P)H oxidase–dependent generation of O2-· and in turn H2O2. Hydrogen peroxide causes activation of the EGFR-ERK1/2 pathway and subsequent induction of protein synthesis and proliferation. Although identification of the steps that are proximal and distal to the indicated mediators in this pathway await investigation, the pathway suggests a direct link or risk factor status may exist between catecholamines, injury, and the progression of vascular disease. It is well known that O2-· inactivates nitric oxide that serves as a tonically active, antiproliferative signal. It is also well known that promoters of vascular injury and disease augment ROS activity. This effect was recently underscored by the identification of an apparently crucial role for NAD(P)H oxidase in neointimal response to rat carotid balloon injury.48 The evidence we present herein for dependence of adrenergic growth on a ROS second messenger, together with the effects of injury on ROS and ROS on nitric oxide availability, may explain why injury so strongly amplifies the mitogenic action of catecholamines: an effect that our recent in vivo evidence suggests contributes to neointimal growth and hypertrophic remodeling in animal models of vascular disease.7,8 As such, therapeutic agents that reduce oxidative stress or interfere with generation of intracellular ROS may derive their salutary effect, in part, by amelioration of the hypertrophic effects of catecholamines induced by vascular injury and disease.
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Acknowledgments |
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References |
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