Interaction between Advanced Glycation End Products Formation and Vascular Responses in Femoral and Coronary Arteries from Exercised Diabetic Rats

Background: The majority of studies have investigated the effect of exercise training (TR) on vascular responses in diabetic animals (DB), but none evaluated nitric oxide (NO) and advanced glycation end products (AGEs) formation associated with oxidant and antioxidant activities in femoral and coronary arteries from trained diabetic rats. Our hypothesis was that 8-week TR would alter AGEs levels in type 1 diabetic rats ameliorating vascular responsiveness. Methodology/Principal Findings: Male Wistar rats were divided into control sedentary (C/SD), sedentary diabetic (SD/DB), and trained diabetic (TR/DB). DB was induced by streptozotocin (i.p.: 60 mg/kg). TR was performed for 60 min per day, 5 days/week, during 8 weeks. Concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP), phenylephrine (PHE) and tromboxane analog (U46619) were obtained. The protein expressions of eNOS, receptor for AGEs (RAGE), Cu/Zn-SOD and Mn-SOD were analyzed. Tissues NO production and reactive oxygen species (ROS) generation were evaluated. Plasma nitrate/nitrite (NOx-), superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS) and N-epsilon-(carboxymethyl) lysine (CML, AGE biomarker). A rightward shift in the concentration-response curves to ACh was observed in femoral and coronary arteries from SD/DB that was accompanied by an increase in TBARS and CML levels. Decreased in the eNOS expression, tissues NO production and NOx- levels were associated with increased ROS generation. A positive interaction between the beneficial effect of TR on the relaxing responses to ACh and the reduction in TBARS and CML levels were observed without changing in antioxidant activities. The eNOS protein expression, tissues NO production and ROS generation were fully re-established in TR/DB, but plasma NOx- levels were partially restored. Conclusion: Shear stress induced by TR fully restores the eNOS/NO pathway in both preparations from non-treated diabetic rats, however, a massive production of AGEs still affecting relaxing responses possibly involving other endothelium-dependent vasodilator agents, mainly in coronary artery.

Impact of exercise training on redox signaling in cardiovascular diseases

Reactive oxygen and nitrogen species regulate a wide array of signaling pathways that governs cardiovascular physiology. However, oxidant stress resulting from disrupted redox signaling has an adverse impact on the pathogenesis and progression of cardiovascular diseases. In this review, we address how redox signaling and oxidant stress affect the pathophysiology of cardiovascular diseases such as ischemia-reperfusion injury, hypertension and heart failure. We also summarize the benefits of exercise training in tackling the hyperactivation of cellular oxidases and mitochondrial dysfunction seen in cardiovascular diseases

Molecular mechanism of jet fuel induced immune suppression

Dermal exposure to jet fuel suppresses the immune response. Immune regulatory cytokines, and biological modifiers, including platelet activating factor, prostaglandin E2, and interleukin-10 have all been implicated in the pathway leading to immunosuppression. It is estimated that approximately 260 different hydrocarbons are found in JP-8 (jet propulsion-8) jet fuel, and the identity of the immunotoxic compound is not known. The recent availability of synthetic jet fuel (S-8), which is devoid of aromatic hydrocarbons, made it feasible to design experiments to test the hypothesis that the aromatic hydrocarbons are responsible for jet fuel induced immune suppression. Applying S-8 to the skin of mice does not up-regulate the expression of epidermal cyclooxygenase-2 nor does it induce immune suppression. Adding back a cocktail of 7 of the most prevalent aromatic hydrocarbons found in jet fuel to S-8 up-regulated cyclooxygenase-2 expression and induced immune suppression. Cyclooxygenase-2 induction can be initiated by reactive oxygen species (ROS). JP-8 treated keratinocytes increased ROS production, S-8 did not. Antioxidant pre-treatment blocked jet fuel induced immune suppression and cyclooxygenase-2 up-regulation. Accumulation of reactive oxygen species induces oxidant stress and affects activity of ROS sensitive transcription factors. JP-8 induced activation of NFκB while S-8 did not. Pre-treatment with antioxidants blocked activation of NFκB and parthenolide, an NFκB inhibitor, blocked jet fuel induced immune suppression and cyclooxygenase-2 expression in skin of treated mice. p65 siRNA transfected keratinocytes demonstrated NFκB is critically involved in jet fuel induced COX-2 expression. These findings clearly implicate the aromatic hydrocarbons found in jet fuel as the agents responsible for inducing immune suppression, in part by the production of reaction oxygen species, NFκB dependent up-regulation of cyclooxygenase-2, and the production of immune regulatory factors and cytokines. ^

Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia.

Heme oxygenase (HO) catalyzes the rate-limiting step in the degradation of heme to biliverdin, which is reduced by biliverdin reductase to bilirubin. Heme oxygenase-1 (HO-1) is inducible not only by its heme substrate, but also by a variety of agents causing oxidative stress. Although much is known about the regulation of HO-1 expression, the functional significance of HO-1 induction after oxidant insult is still poorly understood. We hypothesize and provide evidence that HO-1 induction serves to protect cells against oxidant stress. Human pulmonary epithelial cells (A549 cells) stably transfected with the rat HO-1 cDNA exhibit marked increases of HO-1 mRNA levels which were correlated with increased HO enzyme activity. Cells that overexpress HO-1 (A549-A4) exhibited a marked decrease in cell growth compared with wild-type A549 (A549-WT) cells or A549 cells transfected with control DNA (A549-neo). This slowing of cell growth was associated with an increased number of cells in G0/G1 phase during the exponential growth phase and decreased entry into the S phase, as determined by flow cytometric analysis of propidium iodide-stained cells and pulse experiments with bromodeoxyuridine. Furthermore, the A549-A4 cells accumulated at the G2/M phase and failed to progress through the cell cycle when stimulated with serum, whereas the A549-neo control cells exhibited normal cell cycle progression. Interestingly, the A549-A4 cells also exhibited marked resistance to hyperoxic oxidant insult. Tin protoporphyrin, a selective inhibitor of HO, reversed the growth arrest and ablated the increased survival against hyperoxia observed in the A549-A4 cells overexpressing HO-1. Taken together, our data suggest that overexpression of HO-1 results in cell growth arrest, which may facilitate cellular protection against non-heme-mediated oxidant insult such as hyperoxia.

Susceptibility of cloned K+ channels to reactive oxygen species.

Free radical-induced oxidant stress has been implicated in a number of physiological and pathophysiological states including ischemia and reperfusion-induced dysrhythmia in the heart, apoptosis of T lymphocytes, phagocytosis, and neurodegeneration. We have studied the effects of oxidant stress on the native K+ channel from T lymphocytes and on K+ channels cloned from cardiac, brain, and T-lymphocyte cells and expressed in Xenopus oocytes. The activity of three Shaker K+ channels (Kv1.3, Kv1.4, and Kv1.5), one Shaw channel (Kv3.4), and one inward rectifier K+ channel (IRK3) was drastically inhibited by photoactivation of rose bengal, a classical generator of reactive oxygen species. Other channel types (such as Shaker K+ channel Kv1.2, Shab channels Kv2.1 and Kv2.2, Shal channel Kv4.1, inward rectifiers IRK1 and ROMK1, and hIsK) were completely resistant to this treatment. On the other hand tert-butyl hydroperoxide, another generator of reactive oxygen species, removed the fast inactivation processes of Kv1.4 and Kv3.4 but did not alter other channels. Xanthine/xanthine oxidase system had no effect on all channels studied. Thus, we show that different types of K+ channels are differently modified by reactive oxygen species, an observation that might be of importance in disease states.

4-Hydroxylation of estradiol by human uterine myometrium and myoma microsomes: implications for the mechanism of uterine tumorigenesis.

Estradiol is converted to catechol estrogens via 2- and 4-hydroxylation by cytochrome P450 enzymes. 4-Hydroxyestradiol elicits biological activities distinct from estradiol, most notably an oxidant stress response induced by free radicals generated by metabolic redox cycling reactions. In this study, we have examined 2- and 4-hydroxylation of estradiol by microsomes of human uterine myometrium and of associated myomata. In all eight cases studied, estradiol 4-hydroxylation by myoma has been substantially elevated relative to surrounding myometrial tissue (minimum, 2-fold; mean, 5-fold). Estradiol 2-hydroxylation in myomata occurs at much lower rates than 4-hydroxylation (ratio of 4-hydroxyestradiol/2-hydroxyestradiol, 7.9 +/- 1.4) and does not significantly differ from rates in surrounding myometrial tissue. Rates of myometrial 2-hydroxylation of estradiol were also not significantly different from values in patients without myomata. We have used various inhibitors to establish that 4-hydroxylation is catalyzed by a completely different cytochrome P450 than 2-hydroxylation. In myoma, alpha-naphthoflavone and a set of ethynyl polycyclic hydrocarbon inhibitors (5 microM) each inhibited 4-hydroxylation more efficiently (up to 90%) than 2-hydroxylation (up to 40%), indicating > 10-fold differences in Ki (<0.5 microM vs. > 5 microM). These activities were clearly distinguished from the selective 2-hydroxylation of estradiol in placenta by aromatase reported previously (low Km, inhibition by Fadrozole hydrochloride or ICI D1033). 4-Hydroxylation was also selectively inhibited relative to 2-hydroxylation by antibodies raised against cytochrome P450 IB1 (rat) (53 vs. 17%). These data indicate that specific 4-hydroxylation of estradiol in human uterine tissues is catalyzed by a form(s) of cytochrome P450 related to P450 IB1, which contribute(s) little to 2-hydroxylation. This enzyme(s) is therefore a marker for uterine myomata and may play a role in the etiology of the tumor.

Oxidative Status in Epileptic Children Using Carbamazepine

Background: There is an increasing attention towards the relationship between oxidative stress and epilepsy. The effect of antiepileptic drugs on oxidant status is of major interest. Antiepileptic drugs can increase levels of free radicals, which consequently might lead to seizures. Carbamazepine (CBZ) is an antiepileptic drug commonly used in childhood and adolescence. Objectives: Therefore we aimed to investigate the effects of CBZ on total antioxidant status, total oxidant stress, and oxidative stress index. Patients and Methods: The study included 40 epileptic patients and 31 healthy children between 4 and 12 years of age. Serum CBZ level, total antioxidant capacity and total oxidant status were measured. Oxidative stress index was also calculated both in controls and patients. Results: In the epileptic group, decreased levels of total antioxidant capacity, increased total oxidative stress and oxidative stress index levels were found. Positive correlation between plasma CBZ levels and total oxidant status was observed. Conclusions: Antioxidant action could not be playing any role in antiepileptic effect of CBZ. Furthermore, increased oxidative stress induced by CBZ could be the cause of CBZ-induced seizures. Therefore combining CBZ with antioxidants could be beneficial.

Atmospheric pressure gas plasma-induced colorectal cancer cell death is mediated by Nox2-ASK1 apoptosis pathways and oxidative stress is mitigated by Srx-Nrf2 anti-oxidant system

Atmospheric pressure gas plasma (AGP) generates reactive oxygen species (ROS) that induce apoptosis in cultured cancer cells. The majority of cancer cells develop a ROS-scavenging anti-oxidant system regulated by Nrf2, which confers resistance to ROS-mediated cancer cell death. Generation of ROS is involved in the AGP-induced cancer cell death of several colorectal cancer cells (Caco2, HCT116 and SW480) by activation of ASK1-mediated apoptosis signaling pathway without affecting control cells (human colonic sub-epithelial myofibroblasts; CO18, human fetal lung fibroblast; MRC5 and fetal human colon; FHC). However, the identity of an oxidase participating in AGP-induced cancer cell death is unknown. Here, we report that AGP up-regulates the expression of Nox2 (NADPH oxidase) to produce ROS. RNA interference designed to target Nox2 effectively inhibits the AGP-induced ROS production and cancer cell death. In some cases both colorectal cancer HT29 and control cells showed resistance to AGP treatment. Compared to AGP-sensitive Caco2 cells, HT29 cells show a higher basal level of the anti-oxidant system transcriptional regulator Nrf2 and its target protein sulfiredoxin (Srx) which are involved in cellular redox homeostasis. Silencing of both Nrf2 and Srx sensitized HT29 cells, leads to ROS overproduction and decreased cell viability. This indicates that in HT29 cells, Nrf2/Srx axis is a protective factor against AGP-induced oxidative stress. The inhibition of Nrf2/Srx signaling should be considered as a central target in drug-resistant colorectal cancer treatments.

Differential stress response and susceptibility to oxidative injury in alveolar macrophages from C57BL/6J and C3H/HeJ mice following oxidant exposure

Studies have demonstrated a variable response to ozone among individuals and animal species and strains. For instance, C57BL/6J mice have a greater inflammatory response to ozone exposure than C3H/HeJ mice. In these studies, I utilized these strain differences in an effort to derive a mechanistic explanation to the variable strain sensitivity to ozone exposure. Therefore, alveolar macrophages (AM) from C57BL/6J and C3H/HeJ mice were exposed in vitro to hydrogen peroxide ($\rm H\sb2O\sb2$), heat and acetyl ceramide or in vivo to ozone. Necrosis and DNA fragmentation in macrophages from the two murine strains were determined to assess cytotoxicity following these treatments. In addition, synthesis and expression of the stress proteins, stress protein 72 (SP72) and heme oxygenase (HO-1), were examined following treatments. The in vitro experiments were conducted to eliminate the possibility of in vivo confounders (i.e., differences in breathing rates in the two strains) and thus directly implicate some inherent difference between cells from the two murine strains. $\rm H\sb2O\sb2$ and heat caused greater cytotoxicity in AM from C57BL/6J than C3H/HeJ mice and DNA fragmentation was a particularly sensitive indicator of cell injury. Similarly, AM from C57BL/6J mice were more sensitive to ozone exposure than cells from C3H/HeJ mice. Exposure to either 1 or 0.4 ppm ozone caused greater cytotoxicity in macrophages from C57BL/6J mice compared to macrophages from C3H/HeJ mice. The increased sensitivity of AM to injury was associated with decreased synthesis and expression of stress proteins. AM from C57BL/6J mice synthesized and expressed significantly less stress proteins in response to heat and ozone than AM from C3H/HeJ mice. Heat treatment resulted in greater synthesis and expression of SP72. In addition, macrophages from C57BL/6J mice expressed lower amounts of HO-1 than macrophages from C3H/HeJ mice following 0.4 ppm ozone exposure. Therefore, AM from C57BL/6J mice are more susceptible to oxidative injury than AM from C3H/HeJ mice which might be due to differential expression of stress proteins in these cells. ^

Obesity and hypertension have differing oxidant handling molecular pathways in age-related chronic kidney disease

Chronic kidney disease (CKD) in ageing is a burden on health systems worldwide. Rat models of age-related CKD linked with obesity and hypertension were used to investigate alterations in oxidant handling and energy metabolism to identify gene targets or markers for age-related CKD. Young adult (3 months) and old (21–24 months) spontaneously-hypertensive (SHR), normotensive Wistar-Kyoto (WKY) and Wistar rats (normotensive, obese in ageing) were compared for renal functional and physiological parameters, renal fibrosis and inflammation, oxidative stress (hemeoxygenase-1/HO-1), apoptosis and cell injury (including Bax:Bcl-2), phosphorylated and non-phosphorylated forms of oxidant and energy sensing proteins (p66Shc, AMPK), signal transduction proteins (ERK1/2, PKB), and transcription factors (NF-κB, FoxO1). All old rats were normoglycemic. Renal fibrosis, tubular epithelial apoptosis, interstitial macrophages and myofibroblasts (all p < 0.05), p66Shc/phospho-p66 (p < 0.05), Bax/Bcl-2 ratio (p < 0.05) and NF-κB expression (p < 0.01) were highest in old obese Wistars. Expression of phospho-FoxO/FoxO was elevated in old Wistars (p < 0.001) and WKYs (p < 0.01). SHRs had high levels in young and old rats. Expression of PKB, phospho-PKB, ERK1/2 and phospho-ERK1/2 were significantly elevated in all aged animals. These results suggest that obesity and hypertension have differing oxidant handling and signalling pathways that act in the pathogenesis of age-related CKD

Phosphorylation of caveolin-1 is anti-apoptotic and promotes cell attachment during oxidative stress of kidney cells

Aims: Caveolin-1 (cav1) is reported to have both cell survival and pro-apoptotic characteristics. This may be explained by its localisation or phosphorylation in injured cells. This study investigated the role of cav1 in kidney cells of different nephron origin and developmental state after oxidative stress. Methods: Renal MCDK distal tubular, HK2 proximal tubular epithelial cells and HEK293T renal embryonic cells were treated with 1mM hydrogen peroxide. Apoptosis, loss of cell adhesion, and cell survival were compared with expression of cav1 in its non-phosphorylated and phosphorylated (p-cav1) forms. Cav1 was transfected into the HEK293T cells, or caveolae were disrupted with filipin or nystatin in HK2 cells, to investigate functions of cav1 and p-cav1. Results: Oxidative stress induced more apoptosis in HK2s than MDCKs (p<0.05). HK2s had lower endogenous cav1 and p-cav1 than MDCKs (p<0.05). Both cell lines had increased p-cav1, but not cav1, with oxidative stress. This increase was greatest in MDCKs (p<0.01). Cav1 was located mainly in the plasma membrane of untreated cells and translocated to the cytoplasm with oxidative stress in both cell lines, more so in MDCKs. Disruption of caveolae caused cytoplasmic translocation of cav1 in HK2s, but did not alter high levels of oxidative stress-induced apoptosis. When HEK293Ts lacking endogenous cav1 were transfected with cav1, oxidant-induced apoptosis and loss of cell adhesion was decreased (p<0.01), and p-cav1 was induced by treatment. Conclusion: Cav1 expression and localisation in kidney cells is not anti-apoptotic, but increased expression of p-cav1 may promote cell survival after oxidative stress. © 2008 Royal College of Pathologists of Australasia.

No indications of persistent oxidative stress in response to an ironman triathlon

Introduction: Training for and competing in ultraendurance exercise events is associated with an improvement in endogenous antioxidant defenses as well as increased oxidative stress. However, consequences on health are currently unclear. Purpose: We aimed to examine the impact of training- and acute exercise-induced changes in the antioxidant capacity on the oxidant/antioxidant balance after an ironman triathlon and whether there are indications for sustained oxidative damage. Methods: Blood samples were taken from 42 well-trained male triathletes 2 d before an ironman triathlon, then immediately postrace, 1, 5, and 19 d later. Blood was analyzed for conjugated dienes (CD), malondialdehyde (MDA), oxidized low-density lipoprotein (oxLDL), oxLDL:LDL ratio, advanced oxidation protein products (AOPP), AOPP:total protein (TP) ratio, Trolox equivalent antioxidant capacity (TEAC), uric acid (UA) in plasma, and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in erythrocytes. Results: Immediately postrace, there were significant increases in CD, AOPP, TEAC, UA (for all P < 0.001), and AOPP:TP (P < 0.01). MDA rose significantly (P < 0.01) 1 d postrace, whereas CD (P < 0.01), AOPP (P = 0.01), AOPP:TP (P < 0.05), and TEAC (P < 0.001) remained elevated. OxLDL:LDL trended to increase, whereas oxLDL significantly (P < 0.01) decreased 1 d postrace. Except for GSH-Px (P = 0.08), activities of SOD (P < 0.001) and CAT (P < 0.05) significantly decreased postrace. All oxidative stress markers had returned to prerace values 5 d postrace. Furthermore, several relationships between training status and oxidative stress markers, TEAC, and antioxidant enzyme activities were noted. Conclusions: This study indicates that despite a temporary increase in most (but not all) oxidative stress markers, there is no persistent oxidative stress in response to an ironman triathlon, probably due to training- and exercise-induced protective alterations in the antioxidant defense system.

Modulation of redox regulatory molecules and electron transport chain activity in muscle of air breathing fish Heteropneustes fossilis under air exposure stress

Responses of redox regulatory system to long-term survival (> 18 h) of the catfish Heteropneustes fossilis in air are not yet understood. Lipid and protein oxidation level, oxidant (H2O2) generation, antioxidative status (levels of superoxide dismutase, catalase, glutathione peroxidase and reductase, ascorbic acid and non-protein sulfhydryl) and activities of respiratory complexes (I, II, III and IV) in mitochondria were investigated in muscle of H. fossilis under air exposure condition (0, 3, 6, 12 and 18 h at 25 A degrees C). The increased levels of both H2O2 and tissue oxidation were observed due to the decreased activities of antioxidant enzymes in muscle under water deprivation condition. However, ascorbic acid and non-protein thiol groups were the highest at 18 h air exposure time. A linear increase in complex II activity with air exposure time and an increase up to 12 h followed by a decrease in activity of complex I at 18 h were observed. Negative correlation was observed for complex III and V activity with exposure time. Critical time to modulate the above parameters was found to be 3 h air exposure. Dehydration induced oxidative stress due to modulation of electron transport chain and redox metabolizing enzymes in muscle of H. fossilis was clearly observed. Possible contribution of redox regulatory system in muscle tissue of the fish for long-term survival in air is elucidated. Results of the present study may be useful to understand the redox metabolism in muscle of fishes those are exposed to air in general and air breathing fishes in particular.

Heat-stress and light-stress induce different cellular pathologies in the symbiotic dinoflagellate during coral bleaching

Coral bleaching is a significant contributor to the worldwide degradation of coral reefs and is indicative of the termination of symbiosis between the coral host and its symbiotic algae (dinoflagellate; Symbiodinium sp. complex), usually by expulsion or xenophagy (symbiophagy) of its dinoflagellates. Herein, we provide evidence that during the earliest stages of environmentally induced bleaching, heat stress and light stress generate distinctly different pathomorphological changes in the chloroplasts, while a combined heat- and light-stress exposure induces both pathomorphologies; suggesting that these stressors act on the dinoflagellate by different mechanisms. Within the first 48 hours of a heat stress (32°C) under low-light conditions, heat stress induced decomposition of thylakoid structures before observation of extensive oxidative damage; thus it is the disorganization of the thylakoids that creates the conditions allowing photo-oxidative-stress. Conversely, during the first 48 hours of a light stress (2007 µmoles m−2 s−1 PAR) at 25°C, condensation or fusion of multiple thylakoid lamellae occurred coincidently with levels of oxidative damage products, implying that photo-oxidative stress causes the structural membrane damage within the chloroplasts. Exposure to combined heat- and light-stresses induced both pathomorphologies, confirming that these stressors acted on the dinoflagellate via different mechanisms. Within 72 hours of exposure to heat and/or light stresses, homeostatic processes (e.g., heat-shock protein and anti-oxidant enzyme response) were evident in the remaining intact dinoflagellates, regardless of the initiating stressor. Understanding the sequence of events during bleaching when triggered by different environmental stressors is important for predicting both severity and consequences of coral bleaching

Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency

Background: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial oxidative stress and hypertrophic remodeling. Up-regulation of the cardiomyocyte adrenomedullin (AM) / intermedin (IMD) receptor signaling cascade is also apparent in NO-deficient cardiomyocytes: augmented expression of AM and receptor activity modifying proteins RAMP2 and RAMP3 is prevented by blood pressure normalization while that of RAMP1 and intermedin (IMD) is not, indicating that the latter is regulated by a pressure-independent mechanism. Aims: to verify the ability of an anti-oxidant intervention to normalize cardiomyocyte oxidant status and to investigate the influence of such an intervention on expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes. Methods: NO synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 35mg/kg/day) was given to rats for 8 weeks, with/without con-current administration of antioxidants (Vitamin C (25mg/kg/day) and Tempol (25mg/kg/day)). Results: In left ventricular cardiomyocytes isolated from L-NAME treated rats, increased oxidative stress was indicated by augmented (3.6 fold) membrane protein oxidation, enhanced expression of catalytic and regulatory subunits of pro-oxidant NADPH oxidases (NOX1, NOX2) and compensatory increases in expression of anti-oxidant glutathione peroxidase and Cu/Zn superoxide dismutases (SOD1, SOD3). Vitamin C plus Tempol did not reduce systolic blood pressure but normalized augmented plasma levels of IMD, but not of AM, and in cardiomyocytes: (i) abolished increased membrane protein oxidation; (ii) normalized augmented expression of prepro-IMD and RAMP1, but not prepro-AM, RAMP2 and RAMP3; (iii) attenuated (by 42%) increased width and normalized expression of hypertrophic markers, skeletal-�-actin and prepro-endothelin-1 similarly to blood pressure normalization but in contrast to blood pressure normalization did not attenuate augmented brain natriuretic peptide (BNP) expression. Conclusion: normalization specifically of augmented IMD/RAMP1 expression in NO-deficient cardiomyocytes by antioxidant intervention in the absence of blood pressure reduction indicates that these genes are likely to be induced directly by myocardial oxidative stress. Although oxidative stress contributed to cardiomyocyte hypertrophy, induction of IMD and RAMP1 is unlikely to be secondary to cardiomyocyte hypertrophy.