Differential Activation of Mitogenic Signaling Pathways in Aortic Smooth Muscle Cells Deficient in Superoxide Dismutase Isoforms
Objective— Reactive oxygen species (ROS) integrate cellular signaling pathways involved in aortic smooth muscle cell (SMC) proliferation and migration associated with atherosclerosis. However, the effect of subcellular localization of ROS on SMC mitogenic signaling is not yet fully understood.
Methods and Results— We used superoxide dismutase (SOD)–deficient mouse aortic SMCs to address the role of subcellular ROS localization on SMC phenotype and mitogenic signaling. Compared with wild-type, a 54% decrease in total SOD activity (≈50% decrease in SOD1 protein levels) and a 42% reduction in SOD2 activity (≈50% decrease in SOD2 protein levels) were observed in SOD1+/− and SOD2+/− SMCs, respectively. Consistent with this, basal and thrombin-induced superoxide levels increased in these SMCs. SOD1+/− and SOD2+/− SMCs exhibit increased basal proliferation and enhanced [3H]-thymidine and [3H]-leucine incorporation in basal and thrombin-stimulated conditions. Our results indicate preferential activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinases in SOD1+/− and janus kinase/signal transducer and activator of transcriptase (JAK/STAT) pathway in SOD2+/− SMCs. Pharmacological inhibitors of ERK1/2 p38 and JAK2 confirm the SOD genotype-dependent SMC proliferation.
Conclusions— Our results suggest that SOD1 and SOD2 regulate SMC quiescence by suppressing divergent mitogenic signaling pathways, and dysregulation of these enzymes under pathophysiological conditions may lead to SMC hyperplasia and hypertrophy.
Smooth muscle cells (SMCs) are characterized by marked plasticity in their proliferative potential and differentiation status. As the primary constituents of the arterial media, they provide mechanical support for blood vessels and participate in regulation of vasomotor tone. In addition, SMCs contribute to neointimal formation after vascular injury and during atherosclerotic lesion progression in humans. In advanced atherosclerotic lesions, SMCs are prone to apoptosis and release proteolytic enzymes such as matrix metalloproteinases that may contribute to plaque instability. The remarkable diversity of SMC phenotype and function is determined in large part through regulatory cues in the extracellular microenvironment and interactions among intracellular signaling pathways. In addition, SMC phenotypes are modified over time by the accumulation of injuries to cellular macromolecules.
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Reactive oxygen species (ROS) are key components for integration of SMC signaling events, whereas at the same time, they are major contributors to the degradation of cellular function through their interactions with proteins and structural components of the cell. Exogenous ROS are potent stimuli for activation of SMC signaling and mitogenesis,1 although in high (but physiological) concentrations, they may induce DNA damage and mitochondrial dysfunction.2 SMCs have several intrinsic mechanisms for generating ROS, the major one (at least with respect to intracellular signaling) being the SMC NAD(P)H oxidase.3 This oxidase contains a catalytic component, Nox1,4 and the cytoplasmic component p47phox, which is recruited to the active enzyme by growth factor stimulation and activation of the small GTPase Rac.5 The importance of the vascular NAD(P)H oxidase in general, and its activating component p47phox in particular, has been demonstrated in genetic studies, indicating that this oxidase is required for superoxide production in aortic explants and cultured SMCs.6,7 Pathophysiologically, this oxidase is required for regulation of blood pressure8 and progression of atherosclerotic lesions in apolipoprotein E−/− (apoE−/−) mice.7
Because vascular cells exist in a delicate oxidative balance, the systems that quench oxidative species are of equal importance in modulating SMC behavior. Indeed, exogenous antioxidants can inhibit growth factor–dependent proliferative and signaling responses in SMCs.5,9 The role of endogenous antioxidant defenses is less clear. Members of the superoxide dismutase (SOD) family react with superoxide ion to produce hydrogen peroxide, which is subsequently converted to water by catalase.10 There are 3 members of this family, which are enzymatically similar but distinguished by their localization. SOD1 (copper-zinc SOD) is present in the cytoplasm and nucleus, SOD2 (manganese SOD) is localized in the mitochondria, and SOD3 (extracellular SOD) is secreted to the extracellular space. We recently demonstrated that SOD2+/− mice develop arterial mitochondrial DNA damage.11 More importantly, a phenotype of accelerated atherogenesis at arterial branch points of apoE−/− mice is observed when they also lack a single copy of SOD2. These studies demonstrate the importance of oxidant scavenging in protection against oxidative injury and atherogenesis. On the basis of these observations, we examined the cellular phenotypes and signaling profiles of aortic SMCs derived from mice lacking a single copy of either the SOD1 or SOD2 gene. These studies provide further support for the importance of endogenous antioxidant systems in SMC function. Unexpectedly, divergent mitogenic signaling pathways are activated in response to deficiency of either SOD1 or SOD2 in SMCs, indicating that ROS-dependent signaling is compartmentalized and is regulated by ROS-scavenging enzymes in subcellular compartments.
Materials and Methods
Mouse aortic SMCs were isolated from 4-month-old C57BL/6 SOD1+/− and SOD2+/− mice as described previously.12 SOD1+/− and SOD2+/− mice were back-crossed at least 8× into the C57BL/J6 background. Experiments were performed on multiple primary cultures derived from several mice. Cells were grown in DMEM as described previously.13 All experiments were conducted using SMCs between passage numbers 4 and 11 that were growth-arrested by incubation in DMEM containing 0.1% FBS for 72 hours.
Total and SOD2 activity were determined by measuring the ability of SOD to inhibit xanthine/xanthine oxidase–induced cytochrome c reduction.12 SOD2 activity was determined in the presence of 5 mmol/L potassium cyanide (KCN), which inhibits SOD1 and SOD3 activities.
Measurement of Superoxide Generation by SMCs
Intracellular O2 generation was measured by assaying the activity of aconitase.13 In situ O2 levels in SMCs were also detected by oxidative fluorescent probe dihydroethidium. Mitochondria were visualized with MitoTracker Green, whereas mitochondrial ROS production was visualized using reduced MitoTracker Red (see online supplement, available at http://atvb.ahajournals.org).
Cell Counts, [3H]-Thymidine, and [3H]-Leucine Incorporation Assays
Cell counts, [3H]-thymidine, and [3H]-leucine uptake experiments were performed in SMCs as described previously14 (see online supplement).
Preparation of protein extracts and Western blotting were performed as described14 (see online supplement).
Characterization of Primary SMCs With Haploinsufficiency of SOD Isoforms
To test the effects of impaired superoxide scavenging on SMC behavior, we characterized primary culture SMCs derived from SOD1+/− and SOD2+/− mice. Western blot analysis indicated that SOD1 protein levels were reduced by ≈50% in SOD1+/− mice without evidence of compensation by SOD2 (Figure 1A). Similarly, SOD2+/− mice had 50% less SOD2 protein, but SOD1 protein levels were not significantly different from wild-type SMCs (Figure 1B).
Total SOD activity was reduced by 54% in SOD1+/− SMCs (P<0.001 compared with wild-type SMCs; Figure 1C), consistent with the notion that this isoform accounts for the majority of cellular SOD activity.10 Total SOD activity was not significantly reduced in SOD2+/− SMCs. However, SOD2 activity in SOD2+/− mice was 42% of that in wild-type SMCs (P<0.01; Figure 1D), whereas SOD2 activity was unchanged in SOD1+/− SMCs. Decrease in the expression of SOD isoforms had no significant effect on catalase activity in SMCs (data not shown). Thus, our results show that deficiency in 1 SOD isoform does not lead to compensatory changes in protein levels or activity of the second SOD isoform and suggest that in all likelihood, the effects observed are attributable specifically to the absence of each isoform.
ROS Generation in SOD-Deficient SMCs
To correlate the haploinsufficiency of SOD isoforms with intracellular ROS production, we assayed O2 generation indirectly by measuring aconitase activity. In conformity with our previous results,7 thrombin significantly induced O2 generation in wild-type SMCs (Figure I, available online at http://atvb.ahajournals.org). In addition, basal- and thrombin-stimulated O2 levels were increased in SOD1+/− and SOD2+/− SMCs. Superoxide levels were significantly higher in SOD2+/− SMCs compared with SOD1+/− SMCs under quiescent and thrombin-stimulated conditions (P<0.05), which suggests that SOD2 scavenges a greater proportion of O2 despite its smaller proportion in total SOD activity. The reduction in aconitase activity was inhibited by pretreatment of cells with polyethylene-glycolated SOD (data not shown).
We also determined ROS levels directly in SMCs by staining mitochondria with MitoTracker Green and O2 production with red fluorescence dye dihydroethidium (Figure 2A). Superimposition of images from confocal microscopy revealed that SOD2+/− SMCs have the highest basal O2 levels of the 3 cell types as visualized by bright orange fluorescence, whereas yellow fluorescence of SOD1+/− SMCs indicates higher basal ROS levels than wild-type SMCs. Higher mitochondrial ROS production in SOD2+/− SMCs was confirmed with bright red fluorescence of cells stained with reduced MitoTracker Red, which accumulates in mitochondria and becomes fluorescent when oxidized (Figure 2B).
Proliferation and Hypertrophy of SOD-Deficient SMCs
Based on the established role of ROS as signaling intermediaries for mitogenic and hypertrophic stimuli in SMCs, we determined the role of the intracellular SOD isoforms on these events. In wild-type SMCs, thymidine incorporation was very low after serum deprivation and increased by 51% after thrombin stimulation (Figure 3A), consistent with previous observations.13 Surprisingly, SOD1+/− and SOD2+/− SMCs had elevated thymidine uptake after serum deprivation compared with wild-type SMCs (P<0.05), indicating that these SMCs are capable of mitogen-independent proliferation. This is consistent with the notion that intracellular oxidant species can play a direct role in mitogenesis. After treatment with thrombin, thymidine uptake was increased markedly and to equivalent degrees in SOD1+/− and SOD2+/− cells (118% and 119%, respectively: P<0.001 compared with untreated SMCs for each genotype). To confirm that changes in thymidine incorporation accurately reflected cell cycle entry, we measured cell numbers 2, 4, and 6 days after thrombin treatment. At each time point, cell counts were increased in SOD1+/− and SOD2+/− SMCs (Figure 3B), indicating that rates of proliferation were indeed increased. Because ROS are also required for SMC hypertrophy,15 we measured leucine uptake. As with their proliferative responses, leucine incorporation was increased under serum-deprived and thrombin-stimulated conditions in SOD1+/− and SOD2+/− SMCs (Figure 3C). These results are consistent with previous observations indicating a critical role for ROS in SMC proliferation and hypertrophy9,14,15 and indicate that lack of a single allele of either SOD1 or SOD2 enhances ROS generation. Interestingly, there does not seem to be a direct correlation between either absolute superoxide levels or total SOD activity and proliferation/hypertrophy, which indicates the absence of a linear relationship between ROS levels above a threshold and mitogenesis.
Mitogen-Activated Protein Kinase Activity in SOD1+/− and SOD2+/− SMCs
Mitogen-activated protein (MAP) kinase family members are key proliferative signaling intermediaries for many mitogens, and ROS have been implicated in activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAP kinase in SMCs.15,16 Thrombin induced phosphorylation of ERK1 and ERK2 in wild-type SMCs (2.8±0.5-fold increase at 5 minutes) and basal (2.1±0.3-fold increase versus wild-type SMCs), and thrombin-induced ERK phosphorylation (4.5±0.8-fold increase at 5 minutes) was consistently increased in SOD1+/− SMCs (Figure 4A). However, ERK1/2 phosphorylation was decreased in SOD2+/− SMCs under basal (0.7±0.1-fold) and thrombin-stimulated conditions (2.1±0.5-fold at 5 minutes) compared with wild-type SMCs. In concordance, the phosphorylated form of MEK1 (the upstream activator of ERK1/2) was thrombin inducible in wild-type SMCs (3.7±0.9-fold increase at 5 minutes), and phospho-MEK1 levels were much higher in SOD1+/− cells (2.7±0.3-fold increase at basal level and 5.0±0.5-fold increase at 5 minutes versus wild-type SMCs; Figure II, available online at http://atvb.ahajournals.org). Consistent with attenuated thrombin-inducible ERK1/2 activation in SOD2+/− cells, phospho-MEK1 levels were also lower under basal (0.7±0.1-fold) and stimulated (2.2±0.3-fold increase at 5 minutes) conditions.
Similarly, basal p38 phosphorylation was markedly increased in SOD1+/− SMCs compared with wild-type cells (4.1±0.2-fold increase) but was suppressed in SOD2+/− SMCs (Figure 4B). These results were paralleled by increased activation of MAP kinase kinase 3 (MKK3) (the direct upstream kinase of p38) in SOD1+/− SMCs (2.4±0.1-fold increase) and a concomitant attenuation of phospho-MKK3 levels in SOD2+/− cells (Figure II). In contrast to ERK1/2 and p38, JNK phosphorylation was inducible by thrombin to similar levels in SMCs regardless of genotype (data not shown). These results indicate that constitutive and thrombin-inducible MAP kinase activation in SOD1+/− SMCs may account for the increased proliferative potential of these cells; however, SOD2+/− SMCs must use a different proliferative mechanism.
Constitutive Activation of the JAK/STAT Pathway in SOD2+/− SMCs
Examination of MAP kinase activation provides a possible explanation for the proliferative advantage of SOD1+/− SMCs but leaves unexplained the more robust behavior of SOD2+/− cells. We demonstrated previously thrombin-induced, ROS-dependent Janus kinase/signal transducer and activator of transcription (JAK/STAT) activation in SMCs,14 so we considered that this signaling pathway might be constitutively activated in either SOD1+/− or SOD2+/− SMCs. Phosphorylated JAK2 levels were barely detectable in either wild-type or SOD1+/− SMCs but were markedly elevated in SOD2+/− cells (Figure 5A). Quantitative analysis indicated that activated JAK2 levels were 2.7-fold greater in SOD2+/− SMCs (P<0.05 compared with wild-type SMCs). Consistent with these observations, phospho-STAT3 levels were increased by 3.4-fold in SOD2+/− SMCs (P<0.05 compared with wild-type cells; Figure 5B). Increased mitochondrial ROS production in SOD2+/− SMCs and enhanced nuclear STAT3 levels (increased tyrosine phosphorylation) in SOD-deficient SMCs were confirmed in confocal laser microscopy by staining the cells with reduced MitoTracker Red and phosphospecific STAT3 antibody and a secondary antibody conjugated to a green fluorescent probe (Figure 5C).
Differential Effects of MAP Kinase and JAK2 Inhibitors on Cell Proliferation in SOD-Deficient SMCs
The disparities in signaling pathway activation between SOD1+/− and SOD2+/− SMCs was unanticipated and suggested that increased ambient ROS levels in different cellular compartments have the ability to activate divergent mitogenic pathways. We used specific inhibitors of ERK1/2 (UO126 and PD980589), p38 (SB203580), and JAK2 (AG490) to test the necessity of these enzymes in SOD genotype-dependent SMC proliferation. We performed these experiments under unstimulated conditions for 2 reasons. First, proliferation of SOD-deficient SMCs occurred in spite of serum deprivation (Figure 3A), indicating that the relevant signaling pathways are activated under these conditions. Second, multiple proliferative pathways are activated after mitogenic stimulation, and the consequent partial responses to inhibitors are less easily interpreted. As anticipated, thymidine incorporation in growth-arrested wild-type SMCs was unaffected by inhibition of MAP kinase or JAK/STAT signaling (Figure 6A) because activation of these pathways is minimal or absent under these conditions. In SOD1+/− SMCs, inhibitors of ERK1/2 (UO126 and PD980589) and p38 (SB203580) significantly inhibited thymidine uptake (P<0.001 for each compared with untreated cells; Figure 6B), whereas AG490 had no effect in these cells. In contrast, SOD2+/− SMCs were unaffected by ERK1/2 or p38 inhibitors, but thymidine uptake was significantly reduced by inhibition of JAK/STAT signaling (P<0.001 compared with untreated cells; Figure 6C). Thus, SOD1 and SOD2 deficiency results in elevated ambient O2 levels and increased proliferative capacity, but the signaling pathways activated are surprisingly distinct.
In spite of the long-standing association of SMC proliferation and ROS derived from exogenous or endogenous sources, the role of endogenous ROS-scavenging systems in vascular function has received limited attention. In the present report, we characterize in detail the phenotypes of SMCs derived from mice lacking a single copy of the genes encoding the cytoplasmic or mitochondrial isoforms of SOD. Heterozygous mice are ideal to study the relative importance of these isoforms in vascular pathology because SOD2−/− mice die within weeks of birth. The consequences of SOD genetic modifications are much less subtle than might otherwise be expected and indicate that function of this enzyme within the cytoplasmic and mitochondrial compartments is crucial for tuning ambient ROS levels and modulating SMC proliferation. Whereas other studies have emphasized the importance of ROS generation (in particular, by the vascular NAD(P)H oxidase) in these processes,3,5 the present experiments reinforce the importance of ROS-scavenging systems in modulation of SMC phenotypes.
In SMC mitogenic cascades, several signaling pathways are downstream of ROS generated by the vascular NAD(P)H oxidase. ROS generation is a relatively proximal event in response to growth factors such as thrombin and angiotensin II, insofar as growth factor–induced MAP kinase activity and JAK/STAT signaling are ROS dependent.14,17,18 Although absolute ROS levels have been considered a major determinant of kinase cascade activation, the studies presented here indicate that mitogen-dependent signaling must have other determinants. In particular, our studies indicate that impaired cytosolic SOD activity preferentially activates the ERK1/2 and p38 MAP kinases, whereas JAK/STAT activation occurs when mitochondrial SOD activity is decreased (Figures 4 and 5⇑). We also infer from these studies that suppression of MAP kinases and the JAK/STAT pathway by SOD1 and SOD2, respectively, may keep SMCs in quiescent state in normal conditions, and that deficiency/overwhelming of these enzymes under pathophysiological conditions may be linked mechanistically to SMC hyperplasia and hypertrophy.
We consider at least 2 explanations for this divergence in signaling pathway activation. One possibility is that activation of these 2 pathways is dose dependent; we observe higher superoxide levels in SOD2+/− than in SOD1+/−- SMCs (Figure 2), so it is possible that the JAK/STAT pathway has a higher threshold for activation. However, this would not explain either the suppression of ERK1/2 and p38 MAP kinase phosphorylation in SOD2+/− SMCs (Figure 4) or the absence of a dose response in signaling pathway activation. It is also possible that SOD1 and SOD2 have functions other than O2 scavenging that account for divergent signaling cascade activation.
A second possibility, and the one we favor, is that the location of ROS is critical for activation of specific mitogenic signaling events. To our knowledge, such compartmentalization of ROS-dependent signaling has not been reported. Our linkage of SOD1 and MAP kinase activity is supported by the observation that overexpression of SOD1 decreases phospho-ERK1/2 levels and attenuates neuronal cell death after transient focal cerebral ischemia.19 Although we have not tested this possibility, SOD1 deficiency can alter the redox status of the cell and activate apoptosis signal-regulated kinase 1 and its downstream effector p38 MAP kinase.20 An inverse relationship between phospho-ERK1/2 and p38 MAP kinase levels and SOD1 protein expression in endothelial cells also complements our observation.21 In contrast, a specific linkage between SOD2 function and JAK/STAT activation has not been reported previously, although STAT3 activation by mitochondrial ROS generation was reported.22 Alternatively, overexpression of constitutively active STAT3 (caSTAT3) significantly suppresses ROS generation and augments SOD2 activity, and dominant-negative STAT3 suppresses SOD2 expression,23 suggesting that increased STAT3 activation in SOD2+/− SMCs in the present investigation may reflect a compensatory response to decreased SOD2 levels.
The molecular events that determine the divergence between JAK/STAT activation by mitochondrial ROS and MAP kinase activation by cytosolic ROS are unclear, but it is interesting to note that apoptotic signaling can similarly occur through mitochondrial-dependent and -independent pathways.24 Alternative mitochondrial arms of general signaling pathways may therefore be more common than is typically appreciated. Such a model may also explain how deficiencies in SOD isoforms have different physiological consequences; for example, postischemic recovery of cardiac contractile function is impaired in SOD2-deficient but not SOD1-deficient mice.25
Regardless of the source, the activity of ROS as signaling molecules is generally considered dose responsive.26 The impressive phenotypes of SOD1+/− or SOD2+/− SMCs described here indicate that the ROS-quenching activity within either the mitochondrial or cytoplasmic compartment is equally dosage sensitive. Vascular ROS concentrations must therefore be finely tuned at the cellular and subcellular level. This may be pathophysiologically relevant in the context of vascular disease. For example, SOD2 expression is decreased under proatherogenic low shear stress conditions in vascular cells.27 Together with the present data, these observations may indicate that increased mitochondrial ROS generation at low shear sites, such as arterial bifurcations, serves as a stimulus for the vasculoproliferative response that initiates atherogenesis. In fact, we demonstrated that apoE−/− mice deficient in SOD2 exhibit early increases in mitochondrial DNA damage and a phenotype of accelerated atherogenesis at arterial branch points.11 This also raises the question whether the current data portend other in vivo vascular phenotypes. We reported previously that SOD2+/− mice exhibit a potent deficit in postischemic myocardial function compared with wild-type hearts, whereas SOD1+/− hearts were more susceptible to tachychardia and arrhythmias during reperfusion.25 It is noteworthy that differential activation of STAT and MAP kinase proteins was demonstrated in ischemic heart disease and dilated cardiomyopathy.28 A comprehensive characterization of vascular phenotype of SOD2+/− and SOD1+/− mice with aging and under various stress conditions is currently in progress in our laboratory.
Given the diverse roles of ROS in vascular cell function10 and the tight cellular balance that must be maintained between oxidative signaling and oxidative damage, it is not surprising that systems in addition to SOD exist to quench ROS in vascular cells. For example, glutathione (GSH) in its reduced form can accept electrons from H2O2 and hydroxyl ions. In addition, the balance between GSH and the oxidized form regulates thiolation of many signaling molecules, including tyrosine phosphatases, ion channels, and G-proteins, which in turn affects their function in the regulation of cell signaling.10 Interestingly, the SOD and GSH antioxidant systems may be regulated in parallel. For example, SMCs derived from aged mouse aortas have decreased SOD2 (but not SOD1) levels and less free GSH.12 The interactions between these 2 antioxidant pathways may account for ROS accumulation and dysregulated cell signaling that accompany the aging process and the accelerated risk of vasculoproliferative disease in aging populations.29 The present study provides a basis for understanding the complicated interactions between antioxidant systems, cell signaling events, and vascular pathophysiology.
This work was supported by National Institutes of Health grants HL-57352 to M.S.R. and AG-21096 to C.P.
N.R.M. and S.-K.M. contributed equally to this work.
- Received February 11, 2004.
- Accepted February 21, 2005.
Madamanchi NR, Li S, Patterson C, Runge M. S. Reactive oxygen species regulate heat-shock protein 70 via the JAK/STAT pathway. Arterioscler Thromb Vasc Biol. 2001; 21: 321–326.
Ballinger SW, Patterson C, Yan CN, Doan R, Burow DL, Young CG, Yakes FM, Van Houten B, Ballinger CA, Freeman BA, Runge MS. Hydrogen peroxide- and peroxynitrite-induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells. Circ Res. 2000; 86: 960–966.
Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994; 74: 1141–1148.
Patterson C, Ruef J, Madamanchi NR, Barry-Lane P, Hu Z, Horaist C, Ballinger CA, Brasier AR, Bode C, Runge MS. Stimulation of a vascular smooth muscle cell NADPH oxidase by thrombin: evidence that p47phox may participate in forming this oxidase in vitro and in vivo. J Biol Chem. 1999; 274: 19814–19822.
Lavigne MC, Malech HL, Holland SM, Leto TL. Genetic demonstration of p47phox-dependent superoxide anion production in murine vascular smooth muscle cells. Circulation. 2001; 104: 79–84.
Landmesser U, Cai H, Dikalov S, McCann L, Hwang J, Jo H, Holland SM, Harrison DG. Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension. 2002; 40: 511–515.
Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science. 1995; 270: 296–299.
Wolin MS. Interactions of oxidants with vascular signaling systems. Arterioscler Thromb Vasc Biol. 2000; 20: 1430–1442.
Ballinger SW, Patterson C, Knight-Lozano CA, Burow DL, Conklin CA, Hu Z, Reuf J, Horaist C, Lebovitz R, Hunter GC, McIntyre K, Runge MS. Mitochondrial integrity and function in atherogenesis. Circulation. 2002; 106: 544–549.
Moon SK, Thompson LJ, Madamanchi N, Ballinger S, Papaconstantinou J, Horaist C, Runge M. S, Patterson C. Aging, oxidative responses, and proliferative capacity in cultured mouse aortic smooth muscle cells. Am J Physiol Heart Circ Physiol. 2001; 280: H2779–H288.
Gardner PR, Nguyen DDH, White CW. Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs. Proc Natl Acad Sci U S A. 91: 12248–12252.
Madamanchi NR, Li S, Patterson C, Runge MS. Thrombin regulates vascular smooth muscle cell growth and heat shock proteins via the JAK-STAT pathway. J Biol Chem. 2001; 276: 18915–18924.
Zafari AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG, Taylor WR, Griendling KK. Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy. Hypertension. 1998; 32: 488–495.
Rao GN, Katki KA, Madamanchi NR, Wu Y, Birrer MJ. JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G-protein–coupled receptor agonists in smooth muscle cells. J Biol Chem. 1999; 274: 6003–6010.
Amiri F, Venema VJ, Wang X, Ju H, Venema RC, Marrero MB. Hyperglycemia enhances angiotensin II-induced janus-activated kinase/STAT signaling in vascular smooth muscle cells. J Biol Chem. 1999; 274: 32382–32386.
Simon AR, Rai U, Fanburg BL, Cochran BH. Activation of the JAK-STAT pathway by reactive oxygen species. Am J Physiol. 1998; 275: C1640–C1652.
Noshita N, Sugawara T, Hayashi T, Lewen A, Omar G, Chan PH. Copper/zinc superoxide dismutase attenuates neuronal cell death by preventing extracellular signal-regulated kinase activation after transient focal cerebral ischemia in mice. J Neurosci. 2002; 22: 7923–7930.
Dorion S, Lambert H, Landry J. Activation of the p38 signaling pathway by heat shock involves the dissociation of glutathione S-transferase Mu from Ask1. J Biol Chem. 2002; 277: 30792–30797.
Waris G, Huh KW, Siddiqui A. Mitochondrially associated hepatitis B virus X protein constitutively activates transcription factors STAT-3 and NF-kappa B via oxidative stress. Mol Cell Biol. 2001; 21: 7721–7730.
Negoro S, Kunisada K, Fujio Y, Funamoto M, Darville MI, Eizirik DL, Osugi T, Izumi M, Oshima Y, Nakaoka Y, Hirota H, Kishimoto T, Yamauchi-Takihara K. Activation of signal transducer and activator of transcription 3 protects cardiomyocytes from hypoxia/reoxygenation-induced oxidative stress through the upregulation of manganese superoxide dismutase. Circulation. 2001; 104: 979–981.
Asimakis GK, Lick S, Patterson C. Postischemic recovery of contractile function is impaired in SOD2(+/−) but not SOD1(+/−) mouse hearts. Circulation. 2002; 105: 981–986.
Topper JN, Cai J, Falb D, Gimbrone MA Jr. Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress. Proc Natl Acad Sci U S A. 1996; 93: 10417–10422.
Ng DC, Court NW, dos Remedios CG, Bogoyevitch MA. Activation of signal transducer and activator of transcription (STAT) pathways in failing human hearts. Cardiovasc Res. 2003; 57: 333–346.