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Diastolic dysfunction (DD) underlies heart failure with preserved ejection fraction (HFpEF), a clinical syndrome associated with aging that is becoming more prevalent. Despite extensive clinical studies, no effective treatment exists for HFpEF. Recent findings suggest that oxidative stress contributes to the pathophysiology of DD, but molecular mechanisms underpinning redox-sensitive cardiac remodeling in DD remain obscure. Using transgenic mice with mitochondria-targeted NOX4 overexpression (Nox4TG618) as a model, we demonstrate that NOX4-dependent mitochondrial oxidative stress induces DD in mice as measured by increased E/E', isovolumic relaxation time, Tau Glantz and reduced dP/dt

AIMS: NADPH oxidase (NOX)-derived reactive oxygen species (ROS) are implicated in the pathophysiology of hypertension in chronic kidney disease patients. Genetic deletion of NOX activator 1 (Noxa1) subunit of NOX1 decreases ROS under pathophysiological conditions. Here, we investigated the role of NOXA1-dependent NOX1 activity in the pathogenesis of angiotensin II (Ang II)-induced hypertension (AIH) and possible involvement of abnormal renal function. RESULTS: NOXA1 is present in epithelial cells of Henle's thick ascending limb and distal nephron. Telemetry showed lower basal systolic blood pressure (BP) in Noxa1(−/−) versus wild-type mice. Ang II infusion for 1 and 14 days increased NOXA1/NOX1 expression and ROS in kidney of male but not female wild-type mice. Mean BP increased 30 mmHg in wild-type males, with smaller increases in Noxa1-deficient males and wild-type or Noxa1(−/−) females. In response to an acute salt load, Na(+) excretion was similar in wild-type and Noxa1(−/−) mice before and 14 days after Ang II infusion. However, Na(+) excretion was delayed after 1–2 days of Ang II in male wild-type versus Noxa1(−/−) mice. Ang II increased epithelial Na(+) channel (ENaC) levels and activation in the collecting duct principal epithelial cells of wild-type but not Noxa1(−/−) mice. Aldosterone induced ROS levels and Noxa1 and Scnn1a expression and ENaC activity in a mouse renal epithelial cell line, responses abolished by Noxa1 small-interfering RNA. INNOVATION AND CONCLUSION: Ang II activation of renal NOXA1/NOX1-dependent ROS enhances tubular ENaC expression and Na(+) reabsorption, leading to increased BP. Attenuation of AIH in females is attributed to weaker NOXA1/NOX1-dependent ROS signaling and efficient natriuresis. Antioxid. Redox Signal. 36, 550–566.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases regulate production of reactive oxygen species (ROS) that cause oxidative damage to cellular components but also regulate redox signaling in many cell types with essential functions in the cardiovascular system. Research over the past couple of decades has uncovered mechanisms by which NADPH oxidase (NOX) enzymes regulate oxidative stress and compartmentalize intracellular signaling in endothelial cells, smooth muscle cells, macrophages, cardiomyocytes, fibroblasts, and other cell types. NOX2 and NOX4, for example, regulate distinct redox signaling mechanisms in cardiac myocytes pertinent to the onset and progression of cardiac hypertrophy and heart failure. Heart failure with preserved ejection fraction (HFpEF), which accounts for at least half of all heart failure cases and has few effective treatments to date, is classically associated with ventricular diastolic dysfunction, i.e., defects in ventricular relaxation and/or filling. However, HFpEF afflicts multiple organ systems and is associated with systemic pathologies including inflammation, oxidative stress, arterial stiffening, cardiac fibrosis, and renal, adipose tissue, and skeletal muscle dysfunction. Basic science studies and clinical data suggest a role for systemic and myocardial oxidative stress in HFpEF, and evidence from animal models demonstrates the critical functions of NOX enzymes in diastolic function and several HFpEF-associated comorbidities. Here, we discuss the roles of NOX enzymes in cardiovascular cells that are pertinent to the development and progression of diastolic dysfunction and HFpEF and outline potential clinical implications.

Aging is characterized by increased aortic stiffness, an early, independent predictor and cause of cardiovascular disease. Oxidative stress from excess reactive oxygen species (ROS) production increases with age. Mitochondria and NADPH oxidases (NOXs) are two major sources of ROS in cardiovascular system. We showed previously that increased mitochondrial ROS levels over a lifetime induce aortic stiffening in a mouse oxidative stress model. Also, NADPH oxidase 4 (NOX4) expression and ROS levels increase with age in aortas, aortic vascular smooth muscle cells (VSMCs) and mitochondria, and are correlated with age-associated aortic stiffness in hypercholesterolemic mice. The present study investigated whether young mice (4 months-old) with increased mitochondrial NOX4 levels recapitulate vascular aging and age-associated aortic stiffness. We generated transgenic mice with low (Nox4TG605; 2.1-fold higher) and high (Nox4TG618; 4.9-fold higher) mitochondrial NOX4 expression. Young Nox4TG618 mice showed significant increase in aortic stiffness and decrease in phenylephrine-induced aortic contraction, but not Nox4TG605 mice. Increased mitochondrial oxidative stress increased intrinsic VSMC stiffness, induced aortic extracellular matrix remodeling and fibrosis, a leftward shift in stress-strain curves, decreased volume compliance and focal adhesion turnover in Nox4TG618 mice. Nox4TG618 VSMCs phenocopied other features of vascular aging such as increased DNA damage, increased premature and replicative senescence and apoptosis, increased proinflammatory protein expression and decreased respiration. Aortic stiffening in young Nox4TG618 mice was significantly blunted with mitochondrial-targeted catalase overexpression. This demonstration of the role of mitochondrial oxidative stress in aortic stiffness will galvanize search for new mitochondrial-targeted therapeutics for treatment of age-associated vascular dysfunction., Graphical abstract Image 1, Highlights • Aortic stiffness in aging is associated with increased mitochondrial NOX4 levels. • Young mitochondrial Nox4 overexpressing transgenic mice phenocopy aortic stiffness. • Nox4 transgenic mice show increased VSMC stiffness, aortic remodeling and fibrosis. • Nox4 transgenic mouse VSMC show DNA damage, senescence, apoptosis and inflammation. • High mitochondrial catalase levels blunt aortic stiffness in Nox4 transgenic mice.

Aims: Oxidative stress is implicated in cardiomyocyte cell death and cardiac remodeling in the failing heart. The role of NADPH oxidase 4 (NOX4) in cardiac adaptation to pressure overload is controversial, but its function in myocardial ischemic stress has not been thoroughly elucidated. This study examined the function of NOX4 in the pathogenesis of ischemic heart failure, utilizing mouse models, cell culture, and human heart samples. Results: Nox4(−/−) mice showed a protective phenotype in response to permanent left anterior descending coronary artery ligation with smaller infarction area, lower cardiomyocyte cross-sectional area, higher capillary density, and less cell death versus wild-type (WT) mice. Nox4(−/−) mice had lower activity of soluble epoxide hydrolase (sEH), a potent regulator of inflammation. Nox4(−/−) mice also showed a 50% reduction in the number of infiltrating CD68(+) macrophages in the peri-infarct zone versus WT mice. Adenoviral overexpression of NOX4 in cardiomyoblast cells increased sEH expression and activity and CCL4 and CCL5 levels; inhibition of sEH activity in NOX4 overexpressing cells attenuated the cytokine levels. Human hearts with ischemic cardiomyopathy showed adverse cardiac remodeling, increased NOX4 and sEH protein expression and CCL4 and CCL5 levels compared with control nonfailing hearts. Innovation and Conclusion: These data from the Nox4(−/−) mouse model and human heart tissues show for the first time that oxidative stress from increased NOX4 expression has a functional role in ischemic heart failure. One mechanism by which NOX4 contributes to ischemic heart failure is by increasing inflammatory cytokine production via enhanced sEH activity.

We recently reported that increased NADPH oxidase 4 (NOX4) expression and activity during aging results in enhanced cellular and mitochondrial oxidative stress, vascular inflammation, dysfunction, and atherosclerosis. The goal of the present study was to elucidate the molecular mechanism(s) for these effects and determine the importance of NOX4 modulation of proinflammatory gene expression in mouse vascular smooth muscle cells (VSMCs). A novel peptide-mediated siRNA transfection approach was used to inhibit Nox4 expression with minimal cellular toxicity. Using melittin-derived peptide p5RHH, we achieved significantly higher transfection efficiency (92% vs. 85% with Lipofectamine) and decreased toxicity (p < 0.001 vs. Lipofectamine in MTT and p < 0.0001 vs. Lipofectamine in LDH assays) in VSMCs. TGFβ1 significantly upregulates Nox4 mRNA (p < 0.01) and protein (p < 0.01) expression in VSMCs. p5RHH-mediated Nox4 siRNA transfection greatly attenuated TGFβ1-induced upregulation of Nox4 mRNA (p < 0.01) and protein (p < 0.0001) levels and decreased hydrogen peroxide production (p < 0.0001). Expression of pro-inflammatory genes Ccl2, Ccl5, Il6, and Vcam1 was significantly upregulated in VSMCs in several settings cells isolated from aged vs. young wild-type mice, in atherosclerotic arteries of Apoe−/− mice, and atherosclerotic human carotid arteries and correlated with NOX4 expression. p5RHH-mediated Nox4 siRNA transfection significantly attenuated the expression of these pro-inflammatory genes in TGFβ1-treated mouse VSMCs, with the highest degree of inhibition in the expression of Il6. p5RHH peptide-mediated knockdown of TGFβ-activated kinase 1 (TAK1, also known as Map3k7), Jun, and Rela, but not Nfkb2, downregulated TGFβ1-induced Nox4 expression in VSMCs. Together, these data demonstrate that increased expression and activation of NOX4, which might result from increased TGFβ1 levels seen during aging, induces a proinflammatory phenotype in VSMCs, enhancing atherosclerosis.

Increased reactive oxygen species (ROS) production and inflammation are key factors in the pathogenesis of atherosclerosis. We previously reported that NOX activator 1 (NOXA1) is the critical functional homolog of p67phox for NADPH oxidase activation in mouse vascular smooth muscle cells (VSMC). Here we investigated the effects of systemic and SMC-specific deletion of Noxa1 on VSMC phenotype during atherogenesis in mice. Neointimal hyperplasia following endovascular injury was lower in Noxa1-deficient mice versus the wild-type following endovascular injury. Noxa1 deletion in Apoe-/- or Ldlr-/- mice fed a Western diet showed 50% reduction in vascular ROS and 30% reduction in aortic atherosclerotic lesion area and aortic sinus lesion volume (P, Graphical abstract fx1, Highlights • NOXA1 is a VSMC-specific regulator of NADPH oxidase 1 activity and downstream cell signaling. • NOX1 NADPH oxidase-dependent ROS generation is required for VSMC proliferation and migration after endovascular injury. • NOXA1-dependent NOX1 activation of KLF4 in atherosclerotic lesions induces SMC phenotypic switch to macrophage-like cells. • Atherosclerotic lesion macrophage-like cells promote plaque inflammation, matrix remodeling and increase volume expansion.

The intracellular signaling events by which NADPH oxidase-generated reactive oxygen species (ROS) modulate vascular smooth muscle cell (VSMC) function and atherogenesis are yet to be entirely elucidated. We previously demonstrated that NADPH oxidase deficiency decreased atherosclerosis in apoE(-/-) mice and identified adhesion protein CD44 as an important ROS-sensitive gene expressed in VSMC and atherosclerotic lesions. Here, we examined the molecular mechanisms by which NADPH oxidase-generated ROS regulate the expression of CD44 and its principal ligand, hyaluronan (HA), and how CD44-HA interaction affects VSMC proliferation and migration and inflammatory gene expression in apoE(-/-) mice aortas. Thrombin-induced CD44 expression is mediated by transcription factor AP-1 in a NADPH oxidase-dependent manner. NADPH oxidase-mediated ROS generation enhanced thrombin-induced HA synthesis, and hyaluronan synthase 2 expression in VSMC. Hyaluronidase, which generates low molecular weight HA (LMW-HA), is induced in VSMC in a NADPH oxidase-dependent manner and LMW-HA stimulated ROS generation and cell proliferation in wild-type but not p47(phox-/-) VSMC, effects that were enhanced by thrombin pretreatment. Haptotactic VSMC migration toward HA was increased by thrombin in a CD44-dependent manner. HA expression in atherosclerotic lesions and plasma-soluble CD44 and HA levels were higher in apoE(-/-) compared with apoE(-/-)/p47(phox-/-) mice. HA-regulated pro-inflammatory gene expression was higher in apoE(-/-) than apoE(-/-)/p47(phox-/-) mouse aortas. GKT136901, a specific inhibitor of Nox1- and Nox4-containing NADPH oxidase activity, attenuated ROS generation and atherosclerosis and decreased CD44 and HA expression in atherosclerotic lesions. Together, these data suggest that increased CD44 and HA expression and CD44-HA-dependent gene regulation may play a role in atherosclerosis stimulated by NADPH oxidase activation.

Reactive oxygen species (ROS) have been implicated in vascular smooth muscle cell (VSMC) apoptosis, a hallmark of advanced atherosclerotic lesions. Transient oxidation and inactivation of protein-tyrosine phosphatases play a critical role in cellular response to ROS production. However, the function of leukocyte antigen-related (LAR) protein-tyrosine phosphatase in ROS signaling is not known. To determine the expression of LAR in ROS-induced apoptosis, we investigated hydrogen peroxide-induced cell death and signaling in aortic VSMCs from wild-type and LAR(-/-) mice. Histone-associated DNA fragmentation and caspase-3/7 activity were significantly enhanced, mitochondrial membrane integrity was compromised, and cell viability was significantly decreased following H(2)O(2) treatment in LAR(-/-) VSMCs compared with wild-type cells. Stronger and sustained increase in autophosphorylation and activity of Fyn, an Src family tyrosine kinase, was observed in LAR(-/-) cells compared with wild-type cells following H(2)O(2) treatment. LAR binds to activated Fyn in H(2)O(2)-treated VSMCs, and recombinant LAR dephosphorylates phosphorylated-Fyn in vitro. In addition, LAR deficiency enhanced H(2)O(2)-induced phosphorylation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and p38 mitogen-activated protein kinase (MAPK). PP2, a Fyn-specific inhibitor, blocked JAK2, STAT3, and p38 MAPK activation and significantly attenuated apoptosis induced by H(2)O(2). AG490, a JAK2-specific inhibitor, significantly attenuated H(2)O(2)-induced apoptosis, and blocked H(2)O(2)-induced activation of STAT3, but not p38 MAPK in both wild-type and LAR(-/-) VSMCs. Attenuation of Fyn expression by short hairpin RNA significantly decreased H(2)O(2)-induced downstream signaling and apoptosis in VSMCs. Together, these data indicate that LAR regulates Fyn/JAK2/STAT3 and Fyn/p38 MAPK pathways involved in ROS-induced apoptosis.

α 1 -Adrenoceptor–dependent proliferation of vascular smooth muscle cells (VSMCs) is strongly augmented by vascular injury, and may contribute to intimal growth and lumen loss. Because reactive oxygen species (ROS) are increased by injury and have been implicated as second messengers in proliferation of VSMCs, we investigated the role of ROS in catecholamine-induced VSMC growth. Rat aortae were isolated 4 days after balloon injury, maintained in organ culture under circumferential wall tension, and exposed to agents for 48 hours. The antioxidants N-acetylcysteine (NAC, 10 mmol/L) and Tiron (5 mmol/L) and the flavin-inhibitor diphenylene iodonium (DPI, 20 μmol/L) abolished norepinephrine-induced increases in protein synthesis and DNA content in media. In aortic sections, norepinephrine augmented ROS production (dihydroethidium confocal microscopy), which was dose-dependently inhibited by NAC, Tiron, and DPI. In cultured VSMCs, phenylephrine caused time- and dose-dependent ROS generation (aconitase activity), had similar efficacy to thrombin (1 U/mL), and was eliminated by the superoxide dismutase (SOD) mimetic Mn-(III)-tetrakis-(4-benzoic-acid)-porphyrin-chloride (200 μmol/L) and Tiron. Phenylephrine-induced ROS production and increases in DNA and protein content were blocked by prazosin (0.3 μmol/L) and abolished in p47 phox −/− cells. PEG-SOD (25 U/mL) had little effect, whereas PEG-catalase (50 U/mL) eliminated phenylephrine-induced proliferation in VSMCs. DPI (10 μmol/L) and apocynin (30 μmol/L) abolished phenylephrine-stimulated mitogenesis, whereas inhibitors of other intracellular ROS sources had not effect. Furthermore, PE increased p47 phox expression (RT-PCR). These data demonstrate that the trophic effect of catecholamines on vascular wall cells is dependent on a ROS-sensitive step that we hypothesize consists of activation of the NAD(P)H-dependent vascular oxidase.

Aims: Increased oxidative stress and vascular inflammation are implicated in increased cardiovascular disease (CVD) incidence with age. We and others demonstrated that NOX1/2 NADPH oxidase inhibition, by genetic deletion of p47phox, in Apoe−/− mice decreases vascular reactive oxygen species (ROS) generation and atherosclerosis in young age. The present study examined whether NOX1/2 NADPH oxidases are also pivotal to aging-associated CVD. Results: Both aged (16 months) Apoe−/− and Apoe−/−/p47phox−/− mice had increased atherosclerotic lesion area, aortic stiffness, and systolic dysfunction compared with young (4 months) cohorts. Cellular and mitochondrial ROS (mtROS) levels were significantly higher in aortic wall and vascular smooth muscle cells (VSMCs) from aged wild-type and p47phox−/− mice. VSMCs from aged mice had increased mitochondrial protein oxidation and dysfunction and increased vascular cell adhesion molecule 1 expression, which was abrogated with (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) treatment. NOX4 expression was increased in the vasculature and mitochondria of aged mice and its suppression with shRNA in VSMCs from aged mice decreased mtROS levels and improved function. Increased mtROS levels were associated with enhanced mitochondrial NOX4 expression in aortic VSMCs from aged subjects, and NOX4 expression levels in arterial wall correlated with age and atherosclerotic severity. Aged Apoe−/− mice treated with MitoTEMPO and 2-(2-chlorophenyl)-4-methyl-5-(pyridin-2-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione had decreased vascular ROS levels and atherosclerosis and preserved vascular and cardiac function. Innovation and Conclusion: These data suggest that NOX4, but not NOX1/2, and mitochondrial oxidative stress are mediators of CVD in aging under hyperlipidemic conditions. Regulating NOX4 activity/expression and using mitochondrial antioxidants are potential approaches to reducing aging-associated CVD. Antioxid. Redox Signal. 23, 1389–1409.

Activation of the human protease-activated receptor-1 (PAR-1) by thrombin leads to myriad functions essential for maintaining vascular integrity. Upregulation of PAR-1 expression is considered important in atherosclerosis, angiogenesis and tumor metastasis. In vitro analysis of the human PAR-1 promoter function revealed a positive regulatory element between −4.2 and −3.2 kb of the transcription start site. This element was examined in transgenic mice containing either 4.1 or 2.9 kb of the 5′ flanking sequence driving a LacZ reporter gene. Only the 4.1 kb PAR-1 transgene was expressed in vivo and only during embryonic development. The transgene expression was observed only in developing arteries and not in veins. Further examination of this putative regulatory sequence identified a novel noncoding RNA (ncR-uPAR:noncoding RNA upstream of the PAR-1) gene at −3.4 kb. The ncR-uPAR upregulated PAR-1-core promoter-driven luciferase activity and mRNA expression in vitro in a Pol II-dependent manner. This noncoding RNA appears to act in trans, albeit locally at the adjacent PAR-1 promoter. These data suggest that an untranslated RNA plays a role in PAR-1 gene expression during embryonic growth.

Thrombin is a potent vascular smooth muscle cell (VSMC) mitogen. Because recent evidence implicates reactive oxygen intermediates (ROI) in VSMC proliferation in general and atherogenesis in particular, we investigated whether ROI generation is necessary for thrombin-induced mitogenesis. Treatment of human aortic smooth muscle cells with thrombin increased DNA synthesis, an effect that was antagonized by diphenyleneiodonium but not by other inhibitors of cellular oxidase systems. This effect of thrombin was accompanied by increased O-2 and H2O2 generation and NADH/NADPH consumption. ROI generation in response to thrombin pretreatment could also be blocked by diphenyleneiodonium, suggesting that the NAD(P)H oxidase was necessary for ROI generation and thrombin-induced mitogenesis. Because of observed differences between the VSMC and neutrophil oxidase, we examined whether the cytosolic components of the phagocytic NAD(P)H oxidase were present in VSMC. p47(phox) and Rac2 were present in VSMC. Furthermore, thrombin increased expression of p47(phox) and Rac2 and stimulated their translocation to the cell membrane. We examined whether p47(phox) might be similarly regulated in vivo in a rat aorta balloon injury model and found that p47(phox) protein was increased after injury. Immunocytochemistry localized expression of p47(phox) to the neointima and media of injured arteries. Our data demonstrate that generation of O-2 and H2O2 is required for thrombin-mediated mitogenesis in VSMC and that p47(phox) is regulated by thrombin in vitro and is associated with vascular lesion formation in vivo.

To understand the mechanisms of prostaglandin F2alpha (PGF2alpha)-induced protein synthesis in vascular smooth muscle cells (VSMC), we have studied its effect on two major signal transduction pathways: mitogen-activated protein kinases and phosphatidylinositol 3-kinase (PI3-kinase) and their downstream targets ribosomal protein S6 kinase (p70(S6k)) and eukaryotic initiation factor eIF4E and its regulator 4E-BP1. PGF2alpha induced the activities of extracellular signal-regulated kinase 2 (ERK2) and Jun N-terminal kinase 1 (JNK1) groups of mitogen-activated protein kinases, PI3-kinase, and p70(S6k) in a time-dependent manner in growth-arrested VSMC. PGF2alpha also induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and basic fibroblast growth factor-2 (bFGF-2) expression in VSMC. Whereas inhibition of PI3-kinase by wortmannin completely blocked the p70(S6k) activation, it only partially decreased the ERK2 activity, and had no significant effect on global protein synthesis and bFGF-2 expression induced by PGF2alpha. Rapamycin, a potent inhibitor of p70(S6k), also failed to prevent PGF2alpha-induced global protein synthesis and bFGF-2 expression, although it partially decreased ERK2 activity. In contrast, inhibition of ERK2 activity by PD 098059 led to a significant loss of PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and bFGF-2 expression. PGF2alpha-induced phosphorylation of eIF4E and 4E-BP1 was also found to be sensitive to inhibition by both wortmannin and rapamycin. These findings demonstrate that 1) PI3-kinase-dependent and independent mechanisms appear to be involved in PGF2alpha-induced activation of ERK2; 2) PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation appear to be mediated by both ERK-dependent and PI3-kinase-dependent rapamycin-sensitive mechanisms; and 3) ERK-dependent eIF4E phosphorylation but not PI3-kinase-dependent p70(S6k) activation correlates with PGF2alpha-induced global protein synthesis and bFGF-2 expression in VSMC.

To understand the role of redox-sensitive mechanisms in vascular smooth muscle cell (VSMC) growth, we have studied the effect of N-acetylcysteine (NAC), a thiol antioxidant, and diphenyleneiodonium (DPI), a potent NADH/NADPH oxidase inhibitor, on serum-, platelet-derived growth factor BB-, and thrombin-induced ERK2, JNK1, and p38 mitogen-activated protein (MAP) kinase activation; c-Fos, c-Jun, and JunB expression; and DNA synthesis. Both NAC and DPI completely inhibited agonist-induced AP-1 activity and DNA synthesis in VSMC. On the contrary, these compounds had differential effects on agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression. NAC inhibited agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression except for platelet-derived growth factor BB-induced ERK2 activation. In contrast, DPI only inhibited agonist-induced p38 MAP kinase activation and c-Fos and JunB expression. Antibody supershift assays indicated the presence of c-Fos and JunB in the AP-1 complex formed in response to all three agonists. In addition, cotransfection of VSMC with expression plasmids for c-Fos and members of the Jun family along with the AP-1-dependent reporter gene revealed that AP-1 with c-Fos and JunB composition exhibited a higher transactivating activity than AP-1 with other compositions tested. All three agonists significantly stimulated reactive oxygen species production, and this effect was inhibited by both NAC and DPI. Together, these results strongly suggest a role for redox-sensitive mechanisms in agonist-induced ERK2, JNK1, and p38 MAP kinase activation; c-Fos, c-Jun, and JunB expression; AP-1 activity; and DNA synthesis in VSMC. These results also suggest a role for NADH/NADPH oxidase activity in some subset of early signaling events such as p38 MAP kinase activation and c-Fos and JunB induction, which appear to be important in agonist-induced AP-1 activity and DNA synthesis in VSMC.

Spatiotemporal regulation of the activity of a vast array of intracellular proteins and signaling pathways by reactive oxygen species (ROS) governs normal cardiovascular function. However, data from experimental and animal studies strongly support that dysregulated redox signaling, resulting from hyper-activation of various cellular oxidases or mitochondrial dysfunction, is integral to the pathogenesis and progression of cardiovascular disease (CVD). In this review, we address how redox signaling modulates the protein function, the various sources of increased oxidative stress in CVD, and the labyrinth of redox-sensitive molecular mechanisms involved in the development of atherosclerosis, hypertension, cardiac hypertrophy and heart failure, and ischemia–reperfusion injury. Advances in redox biology and pharmacology for inhibiting ROS production in specific cell types and subcellular organelles combined with the development of nanotechnology-based new in vivo imaging systems and targeted drug delivery mechanisms may enable fine-tuning of redox signaling for the treatment and prevention of CVD.

Glutathione reductase was purified from pea seedlings using a procedure that included 2′,5′-ADP Sepharose, fast protein liquid chromatography (FPLC)-anion exchange, and FPLC-hydrophobic interaction chromatography. The purified glutathione reductase was resolved into six isoforms by chromatofocusing. The isoform eluting with an isoelectric point of 4.9 accounted for 18% of the total activity. The five isoforms with isoelectric points between 4.1 and 4.8 accounted for 82% of the activity. Purified glutathione reductase from isolated, intact chloroplasts also resolved into six isoforms after chromatofocusing. The isoform eluting at pH 4.9 constituted a minor fraction of the total activity. By comparing the chromatofocusing profile of the seedling extract with that of the chloroplast extract, we inferred that the least acidic isoform was extraplastidic and that the five isoforms eluting from pH 4.1 to 4.8 were plastidic. Both the plastidic (five isoforms were pooled) and extraplastidic glutathione reductases had a native molecular mass of 114 kD. The plastidic glutathione reductase is a homodimer with a subunit molecular mass of 55 kD. Both glutathione reductases had optimum activity at pH 7.8. The Km for the oxidized form of glutathione (GSSG) was 56.0 and 33.8 μm for plastidic and extraplastidic glutathione reductase, respectively, at 25°C. The Km for NADPH was 4.8 and 4.0 μm for plastidic and extraplastidic isoforms, respectively. Antiserum raised against the plastidic glutathione reductase recognized a 55-kD polypeptide from purified antigen on western blots. In addition to the 55-kD polypeptide, another 36-kD polypeptide appeared on western blots of leaf crude extracts and the purified extraplastidic isoform. The lower molecular mass polypeptide might represent GSSG-independent enzyme activity observed on activity-staining gels of crude extracts or a protein that has an epitope similar to that in glutathione reductase. Fumigation with 75 nL L−1 ozone for 4 h on 2 consecutive days had no significant effect on glutathione reductase activity in peas (Pisum sativum L.). However, immunoblotting showed a greater level of glutathione reductase protein in extracts from ozone-fumigated plants compared with that in control plants at the time when the target concentration was first reached, approximately 40 min from the start of the fumigation, and 4 h on the first day of fumigation.

An oxidative chain reaction of sulfite initiated by the superoxide ion produced in the Mehler reaction has been implicated in the damage of plants exposed to sulfur dioxide. The toxicity of SO2 may be alleviated by free radical scavenging systems acting to terminate this chain reaction. Hence, the relative sensitivity of plants to SO2 toxicity could depend on differences in the responses of the levels of antioxidant metabolites and enzymes. The effect of SO2 exposure on glutathione and ascorbic acid contents, glutathione reductase, and superoxide dismutase activities was assayed in two cultivars (Progress, Nugget) of pea (Pisum sativum L.) in which apparent photosynthesis showed a differential sensitivity to 0.8 microliter per liter SO2 (R. Alscher, J. L. Bower, W. Zipfel [1987] J Exp Bot 38:99-108). Total and reduced glutathione increased more rapidly and to a greater extent in the insensitive Progress than in the sensitive Nugget, as did glutathione reductase activities. Superoxide dismutase activities increased significantly in Progress, whereas no such change was observed in Nugget as a result of SO2 exposure. This increase in superoxide dismutase activity was observed at 210 minutes after 0.8 microliter per liter SO2 concentration had been reached, in marked contrast to the increases in reduced glutathione content and glutathione reductase activity, which were apparent at the 90 minute time point. These data suggest that one basis for the relative insensitivity of the apparent photosynthesis of the pea cultivar Progress to SO2 is the enhanced response of glutathione reductase, superoxide dismutase activities, and glutathione content.

Objective—We previously showed that NAD(P)H oxidase deficiency significantly reduces atherosclerosis in apoE−/−mice. The present study was designed to determine the relative contribution of monocyte/macrophage versus vascular wall cell NAD(P)H oxidase to atherogenesis in this model.Methods and Results—Cell-specific NAD(P)H oxidase inhibition was achieved via allogenic, sex-mismatched bone marrow transplantation. Aortic atherosclerosis and superoxide production in apoE−/−mice (Control) with functional NAD(P)H oxidase in both monocytes/macrophages and vascular wall cells was compared with that in apoE−/−mice with nonfunctional monocyte/macrophage NAD(P)H oxidase (BMO) or nonfunctional vessel wall NAD(P)H oxidase (VWO). A significant decrease in superoxide production and atherosclerotic lesions was observed in BMO and VWO mice compared with control mice. Interestingly, BMO mice had significantly lower plasma oxidized LDL levels compared with control and VWO mice, whereas aortic sections of VWO mice showed decreased expression of cellular adhesion molecules compared with control and BMO mice. NAD(P)H oxidase deficiency also attenuated neointimal hyperplasia and mitogenic protein activation in apoE−/−mice after arterial injury.Conclusions—We conclude that (1) both monocyte/macrophages and vessel wall cells play critical roles in atherogenesis; (2) decrease in atherosclerosis results from attenuated superoxide generation in monocyte/macrophages or vessel wall cells; and (3) superoxide generation may impact atherosclerosis, in part, by activating smooth muscle cell mitogenic signaling pathways.

Increase in the expression of leukocyte antigen-related (LAR) protein causes insulin resistance, an important contributor to atherosclerosis. However, the function of LAR in atherosclerosis is not known. To address whether LAR is important in the response of vascular cells to atherogenic stimuli, we investigated cell proliferation, migration, and insulin-like growth factor-1 receptor (IGF-1R) signaling in wild-type and LAR(-/-) mouse vascular smooth muscle cells (VSMC) treated with IGF-1. Absence of LAR significantly enhanced proliferation and migration of VSMC compared with wild-type cells after IGF-1 treatment. U0126 and LY249002, specific inhibitors of MAPK/ERK kinase (MEK) and phosphoinositide 3-kinase, respectively, inhibited IGF-1-induced DNA synthesis and migration in both wild-type and LAR(-/-) VSMC. IGF-1 markedly enhanced IGF-1R phosphorylation in both wild-type and LAR(-/-) VSMC, but the phosphorylation was 90% higher in knock-out cells compared with wild-type cells. Absence of LAR enhanced phosphorylation of insulin receptor substrate-1 and insulin receptor substrate-1-associated phosphoinositide 3-kinase activity in VSMC treated with IGF-1. IGF-1-induced phosphorylation of ERK1/2 also increased significantly in LAR(-/-) VSMC compared with wild-type cells. Furthermore, LAR directly binds to IGF-1R in glutathione S-transferase-LAR pull-down and IGF-1R immunoprecipitation experiments and recombinant LAR dephosphorylates IGF-1R in vitro. Neointima formation in response to arterial injury and IGF-1R phosphorylation in neointima increased significantly in LAR(-/-) mice compared with wild-type mice. A significant decrease in body weight, fasting insulin, and IGF-1 levels were observed in LAR(-/-) mice compared with wild-type mice. Together, these data indicate that LAR regulates IGF-1R signaling in VSMC and dysregulation of this phosphatase may lead to VSMC hyperplasia.

Although human exposure to airborne particulate matter (PM) is associated with adverse cardiovascular effects (Pope et al. 2004), the specific causative components or sources have not been identified. Ambient PM is physicochemically heterogeneous and contains significant quantities of metals, including iron, aluminum, silica, zinc, and copper; the levels of these elements vary depending on the geographic location and the local sources (Harrison and Yin 2000; Kodavanti et al. 2005; Ostro et al. 2007; Schwar et al. 1988). Zinc is a ubiquitous PM metal reaching nearly 27 μg/m3 airborne concentration in industrial areas of developing countries (Harrison and Yin 2000). Tire- and brakewear may also contribute to the near-road atmospheric concentration of zinc (Adachi and Tainosho 2004; Councell et al. 2004). Zinc is an essential nutrient required for the maintenance of cell growth, immune maturation, and reproduction and is known to function as an antioxidant via induction of metallothioneins (Maret 2000, 2004; Maret and Sandstead 2006). Although milligram quantities of zinc are ingested daily in foods and with vitamin supplements at slightly higher than physiologic levels, zinc causes cardiovascular and neuronal toxicity (Dineley et al. 2003; Maret 2000, 2006). Although few reports suggest zinc-mediated cardiac toxicity (Evangelou and Kalfakakou 1993; Klevay et al. 1994), the multifaceted effects of zinc on mitochondrial respiration (Ye et al. 2001), calcium homeostasis (Hershfinkel et al. 2001; Maret 2001), sulfur-metal coordination (Maret 2004), intracellular signaling (Samet et al. 2003), myocyte ion channels (Graff et al. 2004), and competition with other essential metals (Klevay 2000; Labbe and Fischer 1984) are well documented and may portend a risk of toxicity. PM-associated water-soluble metals, including zinc, can be absorbed via the pulmonary vasculature upon deposition, potentially reaching cardiac tissue at high concentrations (Gilmour et al. 2006a; Wallenborn et al. 2007) before it is sequestered in the liver. We recently reported that long-term, episodic inhalation exposure to zinc-enriched combustion particles, similar to some ambient PM (Adamson et al. 2000), caused myocardial injury in the Wistar Kyoto rat (Kodavanti et al. 2003). We further demonstrated that a bolus pulmonary exposure to zinc causes marked changes in cardiac gene expression reflective of the impairment in mitochondrial respiration, cell signaling, Ca2+ homeostasis, and ion channel function (Gilmour et al. 2006b). In the present study we have hypothesized that particle-associated, water-soluble zinc is one of the causative PM components responsible for myocardial effects, including oxidative stress and altered cell signaling resulting from protracted exposures. We compared the toxicities of PM with or without zinc, the water-soluble fraction of zinc-containing PM, and soluble zinc alone. Particle exposures with or without zinc increased the incidence of cardiac lesions to a small extent, perhaps as a result of long-term pulmonary inflammation. However, only the soluble zinc, and to some extent zinc-containing PM suspension, or the leachate fraction caused moderate inhibition of cardiac total aconitase activity, mitochondrial DNA damage, and changes in cardiac gene expression. These changes in gene expression are consistent with alterations in cell growth, signaling, mitochondrial fatty acid metabolism, ion channel function, and overall oxidative stress. Thus, PM-associated zinc may be one of the causative components of ambient PM responsible for cardiac effects.

To characterize novel signaling pathways that underlie NAD(P)H oxidase–mediated signaling in atherosclerosis, we first examined differences in thrombin-induced gene expression between wild-type and p47phox −/− (NAD[P]H oxidase–deficient) VSMC. Of the 9000 genes analyzed by cDNA microarray method at the G 1 /S transition point, 76 genes were similarly and significantly modulated in both the cell types, whereas another 22 genes that encompass various functional groups were regulated in NAD(P)H oxidase–dependent manner. Among these 22 genes, thrombin-induced NAD(P)H oxidase–mediated regulation of Klf15, Igbp1, Ak4, Adamts5, Ech1, Serp1, Sec61a2, Aox1, Aoh1, Fxyd5, Rai14, and Serpinh1 was shown for the first time in VSMC. The role of NAD(P)H oxidase in the regulation of a subset of these genes (CD44, BMP4, Id1, and Id3) was confirmed using modulators of reactive oxygen species (ROS) generation, a ROS scavenger and in gain-of-function experiments. We then characterized regulation of these genes in restenosis and atherosclerosis. In both apoE −/− mice and in a mouse vascular injury model, these genes are regulated in NAD(P)H oxidase–dependent manner during vascular lesion formation. Based on these findings, we propose that NAD(P)H oxidase–dependent gene expression in general, and the CD44 and BMP4-Id signaling pathway in particular, is important in restenosis and atherosclerosis.

The French paradox has been attributed to the antioxidant properties of flavonoids present in the red wine. Quercetin, a bioflanoid present in the human diet, is known to inhibit angiotensin II-induced hypertrophy and serum-induced smooth muscle cell proliferation. However, it is not known whether quercetin exerts similar cardioprotective effects in cells treated with TNF-alpha. In this study, we investigated whether quercetin exerts the multiple suppressive effects on cytokine TNF-alpha-induced human aortic smooth muscle cells (HASMC). Treatment of quercetin showed potent inhibitory effects on the DNA synthesis of cultured HASMC in the presence of TNF-alpha. These inhibitory effects were associated with reduced extracellular signal-regulated kinase (ERK)1/2 activity and G1 cell-cycle arrest. Treatment of quercetin, which induced a cell-cycle block in G1-phase, induced down-regulation of cyclins and CDKs and up-regulation of the CDK inhibitor p21 expression, whereas up-regulation of p27 or p53 by quercetin was not observed. Because anti-atherogenic effects need not be limited to antiproliferation, we decided to examine whether quercetin exerted inhibitory effects on matrix metalloproteinase-9 (MMP-9) activity in TNF-alpha-induced HASMC. Quercetin inhibited TNF-alpha-induced MMP-9 secretion on HASMC in a dose-dependent manner. This inhibition was characterized by down-regulation of MMP-9, which was transcriptionally regulated at NF-kappaB site and activation protein-1 (AP-1) site in the MMP-9 promoter. These findings indicate the efficacy of quercetin in inhibiting cell proliferation, G1- to S-phase cell-cycle progress, and MMP-9 expression through the transcription factors NF-kappaB and AP-1 on TNF-alpha-induced HASMC.

The vascular endothelium is a dynamic interface between the blood vessel and circulating factors and, as such, plays a critical role in vascular events like inflammation, angiogenesis, and hemostasis. Whereas specific protein tyrosine kinases have been identified in these processes, less is known about their protein tyrosine phosphatase (PTP) counterparts. We utilized a RT-PCR/differential hybridization assay to identify PTP-epsilon as a highly abundant endothelial cell PTP. PTP-epsilon mRNA expression is growth factor responsive, suggesting a role for this enzyme in endothelial cell proliferation. Overexpression of PTP-epsilon decreases proliferation by 60% in human umbilical vein endothelial cells (HUVEC) but not in smooth muscle cells or fibroblasts. In contrast, overexpression of PTP-epsilon (D284A), a catalytically inactive mutant, has no significant effect on HUVEC proliferation. These data provide the first functional characterization of PTP-epsilon in endothelial cells and identify a novel pathway that negatively regulates endothelial cell growth. Such a pathway may have important implications in vascular development and angiogenesis.

Abstract —Reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ) activate intracellular signal transduction pathways implicated in the pathogenesis of cardiovascular disease. H 2 O 2 is a mitogen for rat vascular smooth muscle cells (VSMCs), and protein tyrosine phosphorylation is a critical event in VSMC mitogenesis. Therefore, we investigated whether the mitogenic effects of H 2 O 2 , such as stimulation of extracellular signal–regulated kinase (ERK)2, are mediated via activation of cytoplasmic Janus tyrosine kinases (JAKs). JAK2 was activated rapidly in VSMCs treated with H 2 O 2 , and signal transducers and activators of transcription (STAT) STAT1 and STAT3 were tyrosine-phosphorylated and translocated to the nucleus in a JAK2-dependent manner. Inhibition of JAK2 activity with AG-490 partially inhibited H 2 O 2 -induced ERK2 activity, suggesting that JAK2 is upstream of the Ras/Raf/mitogen-activated protein kinase–ERK/ERK mitogenic pathway. Because heat-shock proteins (HSPs) can protect cells from ROS, we investigated the effect of H 2 O 2 on HSP expression. H 2 O 2 stimulated HSP70 expression in a time-dependent manner, and AG-490 abolished H 2 O 2 -induced HSP70 expression. H 2 O 2 activated the HSP70 promoter via enhanced binding of STATs to cognate binding sites in the promoter. Regulation of chaperones such as HSP70 via activation of the JAK/STAT pathway suggests that in addition to its growth-promoting effects, this pathway may help VSMCs adapt to oxidative stress.

nadph oxidase (NOX)-mediated generation of reactive oxygen species (ROS) was first described in phagocytic cells, and for many years it was assumed that the primary role of NADPH oxidase activation was bacteriocidal; phagocytes generated a burst of ROS following activation. More recently, it has become clear that not only do multiple homologous NADPH oxidase subunits exist but that the expression of these and the constitution of functional NADPH oxidases vary under different conditions and in different cell types and tissues. The ever-growing family of NADPH oxidases is comprised of a mix of homologous subunits that not only vary by cell type but also depend on the physiological setting. In non-phagocytic cells, multi-subunit NADPH oxidases provide for much more subtle alterations in cellular oxidative stress, resulting in the initiation of intracellular signals that regulate processes including cell migration, growth, and physiological responses to stress. The study of Judkins et al. (6), reported in this issue of the American Journal of Physiology-Heart and Circulatory Physiology, is the most recent of a number of reports focused on the role of NADPH oxidase in vascular dysfunction and atherogenesis. The principal finding of their study is that deficiency of the catalytic component of NADPH oxidase, Nox2 (previously called gp91phox), results in reduced atherosclerosis in apolipoprotein E-null (ApoE−/−) mice. To put this observation into context, it is useful to restate the enormous growth in our understanding of the composition and function of NADPH oxidases in recent years. The phagocytic NADPH oxidase was the first to be characterized biochemically with the conclusion that gp91phox was a unique and required component for oxidase activity. The discovery of Nox1 in 1999, a homologue of gp91phox, provided the first unambiguous hint that the subunit composition of NADPH oxidases might vary in different cells (11). Since that time, a total of seven Nox homologues have been identified, along with numerous homologues of regulatory NADPH oxidase subunits. At least four Nox homologues—Nox1, Nox2, Nox4, and, most recently, Nox5—are expressed in cultured vascular cells and in intact blood vessels, all with subtly different expression patterns and functions. Nox1 is expressed primarily in vascular smooth muscle cells (VSMCs) and is inactive under basal conditions. Nox2 is present in endothelial cells, adventitial fibroblasts, and in circulating macrophages. Like Nox1, Nox2 is upregulated under pathophysiological conditions. The functional Nox2-NADPH oxidase consists of, in addition to Nox2, membrane-bound p22phox and cytosolic regulatory subunits, p47phox, p67phox, and Rac. Nox4 is highly expressed in all vascular cells—endothelial cells, VSMCs, and adventitial cells—and appears to be constitutively active. While the precise characterization of subunits of Nox1- and Nox4-NADPH oxidases remains elusive, it is clear that in Nox1-NADPH oxidase, p67phox, is likely replaced by NoxA1 (1, 9). Recent reports suggest that polymerase delta-interacting protein 2 is a novel cytosolic and nuclear regulator of the activity of Nox4, which had previously been thought to solely rely on its association with p22phox for activity (8). Taken together, these data indicate that an ever-increasing level of complexity is involved in NADPH oxidase function. These data are, however, consistent with the notion that at least one mechanism for NADPH oxidase-mediated ROS production is regulated expression of specific Nox isoforms. In their sophisticated series of experiments, Judkins et al. (6) demonstrated that Nox2 deficiency in ApoE−/− mice results in significantly less atherosclerosis from arch to the iliac bifurcation than in ApoE−/− mice with normal levels of Nox2 expression. This decrease in atherosclerosis is associated with decreased aortic ROS production and increased nitric oxide bioavailability. While not fitting the generally accepted dogma in vascular pathology, the finding that Nox2-NADPH oxidase is important in early atherosclerosis in ApoE−/− mice is not entirely unexpected. Several years ago we reported that ApoE−/− mice lacking p47phox (a requisite component of Nox2-NADPH oxidase) had a marked reduction of atherosclerosis in the descending aorta (3). Although the diversity of Nox homologs was not known at the time, further support for a role of Nox2-NADPH oxidase in atherogenesis came from our subsequent finding that the protective effect of p47phox deficiency in an ApoE−/− background was due to both a decrease in Nox2-NADPH oxidase activity in vessel wall cells as well as in circulating macrophages (12). Several lines of evidence suggest that multiple NADPH oxidases are likely involved in atherogenesis. The fact that p47phox is also required for Nox1-NADPH oxidase activity (9) is consistent with a role for Nox1. Reports that a deficiency of Nox1 protects mice from an angiotensin II-induced increase in blood pressure (4) and injury-induced neointima formation (7) support a role for Nox1-NADPH oxidase. It may well be that ROS produced by the Nox4-NADPH oxidase present in vascular cells contribute to atherogenesis. Relevant to this, Judkins et al. (6) found that in contrast to Nox2, the expression of which is increased in the aortas of ApoE−/− (proatherosclerotic) mice, Nox4 levels did not significantly vary between ApoE−/− versus wild-type mice. Nox4-NADPH oxidase regulates adipogenesis, a risk factor for atherosclerosis, by mediating preadipocyte differentiation in response to insulin treatment (10), and cytosolic regulatory factors such as polymerase delta-interacting protein 2 activate Nox4 and modulate VSMC migration (8), which provide support for the theme that a diversity of NADPH oxidases are important in the complex vascular phenotype of atherosclerosis. An additional, important finding of Judkins et al. (6) has to do with the subtleties of atherosclerosis in the ApoE−/− mouse model. Judkins et al. (6) found, as we have (3), an important effect of ROS generation throughout the aorta, with the exception of the dense atherosclerosis that occurs just above the aortic valve. Atherosclerotic lesions develop rapidly in this region—in fact, out of proportion to the development of atherosclerosis elsewhere. Their data add to the now increasing evidence in the literature that atherosclerosis at the level of the aortic valve in ApoE−/− mice may involve multiple processes and is not representative of inflammatory processes that lead to atherosclerosis in humans. In fact, the progression of atherosclerosis at the level of the aortic valve is very rapid, so much so that studies on factors that may impact atherogenesis cannot be adequately assessed in these very proximal lesions. Lastly, it is important to consider whether ROS generation and NADPH oxidase activity vary with aging. Age itself is an important risk factor for atherosclerosis. Adults over age 65 yr are four times more likely to suffer from coronary heart disease than those in the 40–49-yr age group. Using the same mouse models of atherosclerosis, numerous groups have demonstrated an effect of aging. Our own studies indicate that it is possible to recapitulate the effect of aging on atherosclerosis in mouse models. NADPH oxidase activity is important in these models. It also appears that mitochondrial ROS production is also of importance. We previously demonstrated that ApoE−/− mice deficient in manganese superoxide dismutase, a mitochondrial antioxidant enzyme, exhibited increased early atherosclerosis, associated with early increases in markers of oxidative stress (2). Our unpublished data indicate that atherosclerosis in these mice is exacerbated with aging and that the protective effect of decreased Nox2-NADPH oxidase activity (by knockout of p47phox) becomes less important in these aged mice. An important but unanswered question is whether NADPH oxidase-mediated and mitochondrial-mediated ROS generation both are important in atherogenesis with aging. One must not forget, however, that to date, clinical studies on antioxidant therapies have been disappointingly negative. Given the results of Judkins et al. (6) and others (3), it is attractive to speculate that selective and specific small molecule inhibitors of various Nox will have a salutary effect on at least premature atherosclerosis. Such inhibitors are currently being investigated (5). Whether or not these inhibitors are clinically useful, however, they will be immensely useful in characterizing the downstream molecular and biochemical pathways stimulated by Nox activation in the aorta and small blood vessels during atherogenesis. Over the past fifteen years, much has been learned about the role of NADPH oxidases in many areas, including in atherosclerosis. The report of Judkins et al. (6) published here represents an important advance in our knowledge in this field. With the development of ever more sophisticated animal models of atherosclerosis and the advent of specific NADPH oxidase inhibitors, the future offers opportunities in fundamental biology and potential therapy directed at ROS generation and modulation in the vasculature.

We have previously demonstrated that arachidonic acid activates extracellular signal-regulated protein kinases (ERKs) group of mitogen-activated protein kinases (MAPKs) in vascular smooth muscle cells (VSMC). To understand the role of arachidonic acid in cellular signaling events, we have now studied its effect on jun N-terminal kinases (JNKs) group of MAPKs in VSMC. Arachidonic acid activated JNK1 in a time- and concentration-dependent manner with maximum effects at 10 min and 50 microM. Induced activation of JNK1 by arachidonic acid is specific as other fatty acids such as linoleic and stearic acids had no such effect. Indomethacin and nordihydroguaiaretic acid (NDGA), potent inhibitors of the cyclooxygenase (COX) and the lipoxygenase (LOX)/monooxygenase (MOX) pathways, respectively, had no effect on arachidonic acid activation of JNK1 suggesting that the observed phenomenon is independent of its metabolism through either pathway. However, 12-hydroperoxyeicosatetraenoic acid (12-HpETE), the LOX metabolite of arachidonic acid significantly induced JNK1 activity. Protein kinase C (PKC) depletion by prolonged treatment of VSMC with phorbol 12-myristate 13-acetate (PMA) resulted in partial decrease in the responsiveness of JNK1 to arachidonic acid suggesting a role for both PKC-dependent and -independent mechanisms in the activation of JNK1 by this important fatty acid. On the other hand, the responsiveness of JNK1 to 12-HpETE was completely abolished in PKC-depleted cells, suggesting a major role for PKC in 12-HpETE-induced JNK1 activation. IL-1beta and TNF-alpha activated JNK1 in a time-dependent manner with maximum effect at 10 min. Desensitization of JNK1 by arachidonic acid significantly reduced its responsiveness to both the cytokines. In addition, 4-bromophenacyl bromide (4-BPB), a potent and selective inhibitor of phospholipase A2 (PLA2), significantly attenuated the cytokine-induced activation of JNK1. Together, these results show that (1) arachidonic acid and its LOX metabolite, 12-HpETE, activate JNK1 in VSMC, (2) PKC-dependent and -independent mechanisms play a role in the activation of JNK1 by arachidonic acid and 12-HpETE, and (3) arachidonic acid mediates, at least partially, the cytokine-induced activation of JNK1.

Pea cultivars Progress and Nugget have been shown previously to be differentially sensitive with respect to apparent photosynthesis in a short-term exposure to 0.8 microliters/l SO2. One possible contributing factor to the relative insensitivity of apparent photosynthesis of Progress to SO2 is an increase in superoxide dismutase (SOD) activities. We show here that both chloroplastic and cytoplastic Cu,Zn-SOD proteins increased in Progress on exposure to sulfur dioxide whereas both proteins decreased in Nugget. The increase in cytosolic Cu,Zn-SOD protein was greater than that of chloroplastic Cu,Zn-SOD protein. Using a gene-specific probe for the plastid SOD, northern blot analysis revealed an initial decrease in transcript abundance of the chloroplastic Cu,Zn-SOD gene in Progress on exposure to SO2 with an eventual recovery to pre-exposure levels. The transcript levels of the chloroplastic Cu,Zn-SOD decreased in Nugget over the time period of the exposure. These results suggest that a combination of translational and post-translational mechanisms may be involved in SO2-induced changes in cytosolic and plastidic Cu,Zn-SODs in pea.