Synergetic Antioxidant and Vasodilatory Action of Carbon Monoxide in Angiotensin II–Induced Cardiac Hypertrophy
The aim of this study was to determine the effects of carbon monoxide (CO) at a nontoxic low concentration on the cardiac and vascular hypertrophic response and reactive oxygen species generation, compared with the action of a vasodilator, hydralazine. Twelve- to 16-week–old low-density lipoprotein receptor knockout mice were subjected to angiotensin II (Ang II) infusion using osmotic minipumps (Ang II group; n=11) for 2 weeks. Controls were administered saline (n=10). Animals were exposed to CO in a chamber at 60 ppm for 2 hours per day with or without Ang II infusion (Ang II+CO group, n=10; CO group, n=9). Hydralazine was administered with Ang II infusion (n=10). Animals exhibited elevated arterial carboxyhemoglobin after CO exposure. Although the CO exposure did not affect systolic blood pressure without Ang II infusion, the hypertensive response after Ang II infusion was significantly attenuated by CO. Accordingly, the mice in the Ang II+CO group showed lesser left ventricular hypertrophy compared with those in the Ang II group. CO treatment also attenuated aortic hypertrophy. Interestingly, these changes were accompanied by the reduction of reactive oxygen species production, p47phox and p67phox subunit expressions of reduced nicotinamide-adenine dinucleotide phosphate oxidase, and Akt phosphorylation. Although hydralazine showed stronger antihypertensive action, superior inhibition on cardiac hypertrophy was obtained by CO (P<0.05). Furthermore, Ang II–dependent myocardial reactive oxygen species generation was more effectively suppressed by CO. Low-dose exogenous CO treatment attenuates Ang II-dependent reactive oxygen species generation, suggesting that appropriate CO administration alleviates hypertension and reduces organ hypertrophy mediated by Ang II.
Carbon monoxide (CO), a low-molecular-weight diatomic gaseous molecule, has been considered a dangerous inhalation hazard, because CO binds to hemoglobin with high affinity.1 CO arises primarily from the large-scale environment and human activities, such as volcanic emissions, forest fires, plant metabolisms, industrial processes, and exhaust emissions.
However, recent studies have revealed that CO has profound effects on intracellular signaling processes. The physiological signaling effects of CO involve modulation of soluble guanylate cyclase and subsequent upregulation of cGMP production similar to those of NO. Additional mechanisms include the modulation of several mitogen-activated protein kinase activation pathways.2,3 CO is endogenously produced during the processes of heme catabolism via heme oxygenase (HO).4 Because protective roles of HO under various pathophysiological processes have been reported,5–7 the roles of CO under these processes have currently drawn attention.
The vasodilating properties of CO have been investigated in the cardiovascular system.4,8 CO also has antiapoptotic9 and anti-inflammatory10 potentials. However, because these studies involve extremely high exogenous CO exposure, which induces symptoms of CO intoxication in humans, it is still unknown whether lower concentrations of CO, which might be therapeutically applicable, possess such physiological properties.
In this study, we have developed a mouse model with hypertension and hyperlipidemia commonly found in a clinical setting, in which angiotensin II (Ang II) had been continuously infused in low-density lipoprotein (LDL) receptor knockout mice without high-fat diet feeding, because large-scale clinical trials such as the Framingham Study11 demonstrated that individuals with a greater number of risk factors, such as hypertension, hyperlipidemia, and diabetes, have a higher incidence of atherosclerotic diseases. We examined the effects of exogenous low-dose CO exposure on the cardiac and vascular hypertrophic response, reactive oxygen species (ROS) generation, and serine-threonine kinase Akt in LDL-receptor knockout mice after Ang II administration. We also compared the actions of CO with a nonspecific vasodilator, hydralazine.
Animals and Experimental Protocol
All of the animal experiments were conducted in accordance with the guidelines of Fukushima Medical University Animal Research Committee. Male LDL-receptor knockout mice with C57BL/6 background were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in a specific, pathogen-free environment. Standard sterilized laboratory chow containing 5% (wt/wt) fat, 0.075% (wt/wt) cholesterol, and 1.5% l-arginine (Oriental Bio Service Kanto Inc) and water were available ad libitum.
Animals 12 to 16 weeks of age were subcutaneously implanted with an osmotic mini pump (Alzet model 2001, Alza Corp) to continuously infuse Ang II or saline described below after an intraperitoneal injection of pentobarbital (50 mg/kg). Ang II was administered at a dose of 1.0 mg/kg per day for 14 days. Mice infused with an identical volume of saline were used as controls. Hydralazine (250 mg/L) was administered in the drinking water for 2 days before surgery and continued for 14 days. CO was blended with air at 150 ppm by a commercial vendor (Yamasho Sanso, Fukushima, Japan). Gas was delivered into specially designed chambers in which mice were kept for 2 h/d. The concentrations of O2 and CO in the chambers were serially monitored. O2 was kept at 20% by volume, and CO was kept at 60 ppm. The concentration of carboxyhemoglobin (COHb) was measured by a hemoximeter (model OSM3, Radiometer Trading Co) using retro-orbital capillary blood. CO exposure was started 2 days before minipump operation and continued for 14 days during Ang II or saline infusion.
This study was performed with 5 groups (Figure 1). LDL-receptor knockout mice were subjected to Ang II infusion (1.0 mg/kg per day) using osmotic minipumps (Ang II group, n=11) for 2 weeks. Controls were administered saline (n=10). Animals were exposed to CO in a chamber at 60 ppm for 2 hours per day with or without Ang II infusion (Ang II+CO group, n=10; CO group, n=9). Hydralazine was administered in the drinking water with Ang II infusion (n=10).
Plasma Lipid Analysis
Fourteen days after operation, blood was collected from mice fasted overnight. Total plasma cholesterol, triglyceride, and high-density lipoprotein concentrations were determined enzymatically. Plasma lipid hydroperoxides were measured by the methylene blue hemoglobin method.12
Systolic Blood Pressure Measurement
At the end of this study, systolic blood pressure was measured by tail-cuff plethysmography (model BP98 A, Softron). All of the animals were habituated to the blood pressure measurement device for 7 days.
Assessment of Cardiac Hypertrophy
Transthoracic echocardiography was performed with a 30-MHz imaging transducer (model Vevo770, Visual Sonics) or a 12-MHz imaging transducer (model Aplio, Toshiba Co) after an intraperitoneal injection of pentobarbital (50 mg/kg). After a good-quality 2D image was obtained, M-mode images of the left ventricle were recorded. Intraventricular septum thickness, end-diastolic left ventricular diameter (EDD), end-systolic left ventricular diameter (ESD), and left ventricular posterior wall thickness were measured. Percentage of fractional shortening (%FS) was calculated as follows: %FS=[(EDD−ESD)/EDD]×100. After euthanasia, hearts were rapidly excised and weighed. The heart:body weight ratio was calculated using the whole ventricle weight (milligrams) over the body weight (grams). The observer was blinded to the treatment of the mice.
Assessment of Medial Hypertrophy in Aorta
Paraffin-embedded sections (4 μm) were obtained from the descending thoracic aorta and stained with hematoxylin-eosin. Radial thickness of the media and circumference of the internal elastic lamina were measured using Scion Image. The diameter of the lumen was calculated by dividing the circumference of the internal elastic lamina by π. Aortic medial hypertrophy was determined among groups by comparing the ratio of medial thickness:lumen diameter.
Assessment of ROS Generation
In situ detection of ROS formation in the aortic wall was measured by oxidative fluorescent microtopography as described previously13 with modification. Excised thoracic aortic ring segments were immediately frozen in optimal cutting temperature compound, cut into 30-μm–thick cross-sections and incubated with PBS (pH 7.4) containing 10 μm of dihydroethidium (Sigma-Aldrich), and cover slipped. Then, slides were placed in a light-protected humidified chamber at 37°C for 30 minutes. ROS generation was evaluated by the formation of ethidium bromide, which is the oxidative product of dihydroethidium by ROS. Ethidium bromide is excited at 488 nm with an emission spectrum of 610 nm. Images were obtained with a confocal fluorescent microscopy (Olympus Fluoview FV300) under identical laser settings. Fluorescence was detected with a 585-nm long-pass filter and quantitatively analyzed by Scion image software. Generations of ROS in aorta and heart were also assessed using the fluorescence probe 5- (and 6-)chloromethyl-2′, 7′-dichlorodihydro-fluorescein diacetate acetyl ester (Molecular Probes), which becomes highly fluorescent on oxidation by H2O2.14 Excised tissues were briefly washed with PBS, immediately resuspended in 50 mmol/L of HEPES buffer (pH 7.4), and incubated with 5- (and 6-)chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate acetyl ester (5 μmol/L) for 30 minutes at 37°C. Fluorescence was measured (excitation 485 nm; emission 515 nm) using a model SpectraMax M2 spectrophotometer (Molecular Devices).
Western Blot Analysis
For Western blot analysis of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase subunits, mouse aorta and heart were washed, lysed, and homogenized in 10 mmol/L of Tris-HCl (pH 7.4) containing 0.1% sodium dodecyl sulfate and a protease inhibitor mixture (Boehringer Mannheim). A total of 20 μg of tissue homogenates was electrophoresed on 10% SDS-PAGE, blotted onto polyvinylidene diflouride membranes (ATTO), blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, and incubated with polyclonal anti-NADPH oxidase (p67phox, Santa Cruz Biotechnology; p47phox, Upstate Biotechnology) or anti-Akt (total Akt and phospho-Akt, Cell Signaling) antibodies. Bound antibody was detected using the enhanced chemiluminescence detection system (Amersham Pharmacia). The protein content was determined using a DC protein assay kit (BioRad). Densitometric analysis was performed using Scion Image software.
All of the values are expressed as mean±SD. Significant difference was determined by 1-way ANOVA with the Fisher posthoc test. P<0.05 was considered statistically significant.
Effects of Exogenous CO Exposure on COHb and Saturation of Oxygen Levels in LDL Receptor Knockout Mice
Because clinical symptoms of CO poisoning appear when the COHb level in blood exceeds 10%,1 we tried to examine the biological effects of CO within a nontoxic level in our experiments. In preliminary experiments, we exposed LDL-receptor knockout mice to different concentrations of CO gas for 2 hours in a specially designed chamber, followed by the measurement of COHb in blood by a hemoximeter (data not shown). The concentration of CO in the chamber that gave the 10% of COHb in blood was 60 ppm (Figure 2A). The effect of CO exposure disappeared after 4 hours because of the discontinuation of CO exposure determined by serial COHb monitoring (Figure 2A). We also measured the saturation of oxygen after 60 ppm of CO exposure for 2 hours by oximetry (Figure 2B). Saturation of oxygen levels did not decrease under this CO exposure condition.
Ang II Infusion Elicits Hypercholesteremia and Plasma Lipid Hydroperoxide Elevation in LDL Receptor Knockout Mice
Table shows body weight, plasma lipids, and lipid hydroperoxide concentration of 12- to 16-week-old male mice fed a standard chow diet. Compared with C57BL/6J mice, LDL-receptor knockout mice showed significantly higher concentrations of plasma total cholesterol, triglycerides, high-density lipoprotein, and lipid hydroperoxides. These lipid profiles of LDL-receptor knockout mice were similar to those of the human hyperlipidemic state, although these mice showed higher high-density lipoprotein levels because of a lack of plasma cholesteryl ester transfer protein.
Significant elevations of plasma total cholesterol and lipid hydroperoxide were observed in the LDL-receptor knockout mice infused with Ang II (Table). Although there were no statistically significant changes in body weight, Ang II–infused LDL-receptor knockout mice showed a slight decrease in body weight compared with sham-operated LDL-receptor knockout mice. Interestingly, CO exposure suppressed Ang II–induced elevations of plasma total cholesterol and lipid hydroperoxide levels, although the significance was limited (Table).
Effects of CO Exposure in Ang II–Infused LDL Receptor Knockout Mice on Arterial Blood Pressure and Left Ventricular Hypertrophy
After 14-day Ang II infusion, mice exhibited significantly higher systolic pressure (Figure 3A). Although CO exposure for 14 days did not affect the systolic pressure level itself, it significantly lowered the Ang II–dependent systolic blood pressure elevation (Figure 3A).
Figure 3B shows representative charts of transthoracic M-mode echocardiograms. Chronic Ang II infusion induced hypertrophic changes of the left ventricle as judged from the thickening of the interventricular septum and posterior wall. Importantly, these hypertrophic changes were significantly attenuated by CO exposure (Figure 3B). In contrast, there were no differences in fractional shortening, suggesting that CO did not affect the resting left ventricular systolic function. The inhibitory effects of CO on the hypertrophic changes of left ventricles were also observed by the heart weight (milligrams):body weight (grams) ratio (Figure 3C).
Aortic Medial Hypertrophy and ROS Generation in Ang II–Infused LDL Receptor Knockout Mice
Figure 4A presents representative microphotographs of thoracic aorta in the Ang II–infused LDL-receptor knockout mice. Mice treated with Ang II exhibited significant medial hypertrophy as judged from the mean medial:lumen ratio (Figure 4B). CO exposure effectively suppressed these hypertrophic changes (Figure 4B).
To determine whether ROS is involved in the Ang II-dependent aortic medial hypertrophy, we next examined the superoxide generation in thoracic aorta using dihydroethidium staining (Figure 4C). Ang II infusion dramatically increased the ROS generation in the thoracic aorta, whereas CO exposure suppressed it (Figure 4D). By an alternative method using 5- (and 6-)chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate acetyl ester, similar attenuation of Ang II–dependent ROS generation in both the heart and aortic tissue was observed by CO treatment (Figure S1, available at http://hyper.ahajournals.org).
CO Suppresses Ang II–Induced NADPH Oxidase Expression and Akt Phosphorylation
To examine the involvement of NADPH oxidase in Ang II–induced ROS generation, we performed Western blot analyses using homogenates from the heart (Figure 5A) and aorta (Figure 5B) of LDL-receptor knockout mice. Ang II infusion increased p47phox and p67phox subunits of NADPH oxidase in both heart and aortic tissues (Figure 5). Although CO exposure itself did not have significant effects on such NADPH oxidase subunit expression, it significantly attenuated the Ang II–induced p47phox and p67phox expressions, suggesting the possibility that the inhibitory effect of CO on Ang II–induced ROS generation is mediated via the NADPH oxidase–dependent pathway. We next examined the effect of CO exposure on Akt phosphorylation, because involvement of the Akt signaling pathway in Ang II–dependent ROS generation has been reported.15,16 CO exposure significantly attenuated Akt phosphorylation elicited by Ang II both in the heart (Figure 6A) and the aorta (Figure 6B).
Synergetic Antioxidant and Vasodilatory Action of CO Is More Effective Than Hydralazine Against Ang II–Induced Cardiac Hypertrophy
To characterize the biological action of CO treatment on cardiac hypertrophy, we next compared its action with hydralazine in Ang II-infused LDL-receptor knockout mice (Figure 7). Mice administered hydralazine (250 mg/L in drinking water) showed stronger antihypertensive effects than those exposed to exogenous CO (Figure 7A). However, mice administered hydralazine showed more severe cardiac hypertrophy as judged by the ratio of heart weight:body weight compared with those exposed to CO (Figure 7B). Figure 7C shows ROS generation measured with a fluorescent probe, 5- (and 6-)chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate acetyl ester, in the heart tissues of Ang II–infused LDL-receptor knockout mice. CO exposure strongly inhibited the ROS generation compared with hydralazine treatment. In addition, CO exposure more effectively inhibited expression of NADPH oxidase subunits, p67phox and p47phox (Figure 7D).
In this study, we developed a mouse model of combined hypertension and hyperlipidemia, using Ang II–infused LDL-receptor knockout mice. From a chow diet, this model had moderate levels of hyperlipidemia, which were similar to those of human disorders. Continuous administration of Ang II for 14 days induced significant cardiac and aortic medial hypertrophy in these mice with increased accumulation of ROS. With this model, we examined the effects of low-dose exogenous CO administration. Different from conventional CO exposure conditions at 250 to 1000 ppm3,10,17,18 that clinically induce CO intoxication, we used a lower and shorter-time CO exposing condition at 60 ppm for 2 hours daily. This clinically applicable CO treatment significantly attenuated Ang II–induced hypertension and cardiac and aortic hypertrophic responses accompanied with reduced ROS accumulation in these tissues. CO exposure also alleviated Ang II-induced hyperlipidemia.
Because CO binds with hemoglobin with 240 times higher affinity than molecular oxygen, excess exogenous CO exposure causes intoxication, resulting in headaches, dizziness, vomiting, and, ultimately, death. Clinical symptoms of CO poisoning have been reported to begin at ≈10% of blood COHb.1 Therefore, we first set the CO exposure condition so as not to exceed this threshold. In virtually all of the in vivo studies reported thus far, animals have been exposed to a CO concentration of >250 ppm for several hours or days before a stressful condition.10,17,18 These studies showed that the levels of COHb rose to ≈20% after 250-ppm CO exposure. Therefore, we performed all of our experiments with CO at 60 ppm. As shown in Figure 2, the concentration of blood COHb did not exceed 10% COHb, which is considered the threshold level of clinical CO poisoning.
In this study, we used Ang II–infused LDL-receptor knockout mice to observe the cardiovascular effects of CO on hypertension accompanied with hyperlipidemia. Most of the previous studies using LDL-receptor knockout mice were conducted under extreme hyperlipidemic conditions with high-fat diet feeding, in which plasma total cholesterol levels were >1000 mg/dL.19 Under such extreme hyperlipidemic conditions, Ang II–infused LDL-receptor knockout mice have been reported to show an abdominal aortic aneurysm formation, as well as hypertension and atherosclerotic lesion formation in aortic sinus.20,21 However, these studies do not seem to have observed the effects of Ang II on lipid metabolism, because LDL-receptor knockout mice exhibit extreme hyperlipidemia after high-fat diet feeding.20 Therefore, we fed these mice a chow diet to prevent extreme hyperlipidemia during 14-day Ang II infusion. The LDL-receptor knockout mice fed the chow diet showed plasma total cholesterol of ≈240 to 300 mg/dL, which was comparable to that in human hypercholesteremia. Nevertheless, LDL-receptor knockout mice exhibited significantly higher plasma cholesterol levels compared with a congenic strain C57BL/6J, which is susceptible to atherosclerosis. A significantly higher plasma lipid hydroperoxide level was confirmed even in LDL-receptor knockout mice fed the chow diet, suggesting that these mice were still exposed to stronger oxidative stress compared with C57BL/6J. Interestingly, Ang II infusion in LDL-receptor knockout mice fed the chow diet resulted in a 1.8-fold elevation of plasma total cholesterol and a 3.0-fold elevation of plasma lipid hydroperoxide (Table). These results suggest the detrimental effects of Ang II on the lipid metabolism. Further studies are warranted to elucidate the underlying mechanisms given the few investigations of the involvement of renin-angiotensin-aldosterone systems in the lipid metabolism. Also, a significant elevation of blood pressure was induced in this model. Hence, our model reflects the hyperlipidemia and hypertension common to a clinical condition. Formation of abdominal aortic aneurysm was not observed in our mice model (data not shown).
Ang II–infusion rodent models have been widely used in studies of cardiac hypertrophy,22,23 vascular medial hypertrophy,24,25 or kidney injury26 with hypertension. The findings have indicated that Ang II–mediated organ damage involves pro-oxidant, as well as hypertensive, effects.27,28 In the present study, continuous Ang II administration for 14 days induced a significant increase of ROS generation in the heart and aorta with increased expression of NADPH oxidase subunits p47phox and p67phox, as well as hypertension, cardiac hypertrophy, and aortic medial hypertrophy.
With this model, we observed that chronic CO exposure has significant effects to alleviate Ang II–dependent hypertension, cardiac and aortic medial hypertrophy, and ROS generation in cardiovascular tissues. These effects may in part be explained via the vasodilating effects of CO.29 CO has been reported to activate the guanylate cyclase/cGMP pathway and big-conductance calcium-activated K channels, which serves to lessen vascular smooth muscle tone, although a complex interaction between CO and NO has also been reported.4,30 Interestingly, CO exposure in the in vivo condition significantly reduced Ang II–induced hypertension. In contrast, CO exposure did not significantly affect blood pressure in sham mice. This different response may be partly explained by the alternative vasodilating role of CO instead of NO, which is converted into peroxynitrite via the reaction between NO and superoxide after Ang II infusion. In contrast, CO has little vasodilatory effect in the steady-state condition, in which NO highly activates soluble guanylate cyclase more than CO.31
We observed that CO exposure has significant inhibitory effects on Ang II–induced cardiac and aortic medial hypertrophy. This inhibition was mediated via the vasodilatory effects of CO, however, the antioxidative effects of CO seem to play a greater role. Involvement of ROS, as well as pressure overload, has been indicated in cardiac and aortic medial hypertrophy as benevolent molecules in cell signaling processes.32,33 CO may attenuate Ang II–induced cardiac and aortic medial hypertrophy via the inhibitory action on ROS generation. Ang II–dependent hypertrophic responses have been reported to be mediated via the mitogen-activated protein kinase pathway.34 Moreover, it is reported that anti-inflammatory effects of CO are mediated through p38 mitogen-activated protein kinase3 and Jun N-terminal kinase35 pathways in mononuclear cells. CO may similarly affect the cell-signaling pathway during hypertrophic responses in the cardiovascular system.
A number of studies have shown that the major source of Ang II-stimulated ROS generation in the cardiovascular system is derived from the NADPH oxidase system.32,33,36,37 Consequently, we examined whether the antioxidative action of CO is mediated via the inhibitory effects on NADPH oxidase. We observed that low CO exposure significantly suppresses the activation of p47phox and p67phox subunits of NADPH oxidase, although the precise mechanism remains unclear. This is, to our knowledge, the first in vivo observation of the antioxidative action of CO using an animal model. Our observation in this study may support the previous in vitro observation that HO-1, the sole CO producing enzyme, inhibits NADPH oxidase activity in cultured macrophages.38
Activation of serine-threonine kinase Akt is known to play an important role in various cellular processes, including cell death, survival, proliferation, differentiation, and cell size.39 Because Akt is considered as one of the downstream signaling targets of Ang II via ROS production,15 we examined the involvement of Akt phosphorylation in the biological effects of CO. Importantly, CO significantly attenuated Ang II-dependent Akt phosphorylation both in the heart and aorta.
In an additional experiment, we compared the effects of CO and hydralazine, a vasodilating agent, on Ang II–induced hypertrophic responses and ROS generation in the heart. Although hydralazine showed more powerful antihypertensive effects compared with those of CO, the inhibitory effects of CO on cardiac hypertrophy were superior to greater than those of hydralazine. Furthermore, Ang II–dependent myocardial ROS generation and NADPH oxidase expression were more effectively suppressed by CO exposure than hydralazine. These data suggest that the CO has significant antioxidative, as well as vasodilating, action. Although the vasodilating action of hydralazine also suppressed cardiac hypertrophy and ROS generation to some extent, other additional mechanisms to inhibit these pathological processes seem to be required.
After a series of experimental reports showing the cytoprotective effects of HO-1 and CO, we are in a situation to pursue the further possibilities of HO-mediated gene therapy or pharmacotherapy using an agent that releases an adequate amount of CO in clinical settings. This study demonstrates that CO exposure even at low concentration was enough to suppress Ang II–mediated cardiovascular hypertrophic responses and ROS generation. However, the development of simple and reliable CO monitoring methods using blood samples (COHb) or a percutaneous route (saturation pulse CO) will be crucial in avoiding a clinically significant adverse effect of CO. In addition, the development of CO pollution control equipment will be necessary to avoid exposure of the medical staff even at low CO concentrations. It is also important to examine the applicable pathophysiological cardiovascular conditions other than Ang II–mediated refractory hypertension and resultant cardiac and aortic hypertrophy and damage.
Sources of Funding
This study was supported by grants from the Takeda Science Foundation and the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to K.I.).
- Received June 26, 2007.
- Revision received July 16, 2007.
- Accepted September 11, 2007.
Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol Rev. 2006; 86: 583–650.
Poss KD, Tonga S. Reduced stress defense in heme oxygenase 1-deficient cells. Proc Natl Acad Sci U S A. 1997; 94: 10925–10930.
Brouard S, Otterbein LE, Anrather J, Tobiasch E, Bach FH, Choi AM, Soares MP. Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med. 2000; 192: 1015–1026.
Otterbein LE, Zuckerbraun BS, Haga M, Liu F, Song R, Usheva A, Stachulak C, Bodyak N, Smith RN, Csizmadia E, Tyagi S, Akamatsu Y, Flavell RJ, Billiar TR, Tzeng E, Bach FH, Choi AM, Soares MP. Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nature Med. 2003; 9: 183–190.
Garcia MJ, Mcnamara PM, Gordon T, Kannell WB. Morbidity and mortality in diabetics in the Framingham population: sixteen year follow-up study. Diabetes. 1974; 23: 105–111.
Miller FJ, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998; 82: 1298–1305.
Hempel SL, Buettner GR, O’Malley YQ, Wessels DA, Flaherty DM. Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2′,7′-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, and dihydrorhodamine. Free Radic Biol Med. 1999; 27: 146–159.
Ushio-Fukai M, Alexander RW, Akers M, Yin Q, Fujio Y, Walsh K, Griendling KK. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem. 1999; 274: 22699–22704.
Li F, Malik KU. Angiotensin II-induced Akt activation is mediated by metabolites of arachidonic acid generated by CaMKII-stimulated Ca2+-dependent phospholipase A2. Am J Physiol. 2005; 288: H2306–H2316.
Fujimoto H, Ohno M, Ayabe S, Kobayashi H, Ishizaka N, Kimura H, Yoshida K, Nagai R. Carbon monoxide protects against cardiac ischemia-reperfusion injury in vivo via MAPK and Akt-eNOS pathways. Arterioscler Thromb Vasc Biol. 2004; 24: 1848–1853.
Mazzola S, Forni M, Albertini M, Bacci ML, Zannoni A, Gentilini F, Lavitrano M, Bach FH, Otterbein LE, Clement MG. Carbon monoxide pretreatment prevents respiratory derangement and ameliorates hyperacute endotoxic shock in pigs. FASEB J. 2005; 19: 2045–2047.
Daugherty A, Cassis LA. Mouse models of abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol. 2004; 24: 429–434.
Izumiya Y, Kim S, Izumi Y, Yoshida K, Yoshiyama M, Matsuzawa A, Ichijo H, Iwao H. Apoptosis signal-regulating kinase 1 plays a pivotal role in angiotensin II-induced cardiac hypertrophy and remodeling. Circ Res. 2003; 93: 874–883.
Ishizaka N, Aizawa T, Mori I, Taguchi J, Yazaki Y, Nagai R, Ohno M. Heme oxygenase-1 is upregulated in the rat heart in response to chronic administration of angiotensin II. Am J Physiol. 2000; 279: H672–H678.
Liu J, Yang F, Yang Xp, Jankowski M, Pagano PJ. NAD(P)H oxidase mediates angiotensin II–induced vascular macrophage infiltration and medial hypertrophy. Arterioscler Thromb Vasc Biol. 2003; 23: 776–782.
Ishizaka N, León HD, Laursen JB, Fukui T, Wilcox JN, Keulenaer GD, Griendling KK, Alexander RW. Angiotensin II–induced hypertension increases heme oxygenase-1 expression in rat aorta. Circulation. 1997; 96: 1923–1929.
Aizawa T, Ishizaka N, Taguchi J, Nagai R, Mori I, Tang SS, Ingelfinger JR, Ohno M. Heme oxygenase-1 is upregulated in the kidney of angiotensin II-induced hypertensive rats: possible role in renoprotection. Hypertension. 2000; 35: 800–806.
Berk BC. Vascular smooth muscle growth: autocrine growth mechanisms. Physiol Rev. 2001; 81: 999–1030.
Ushio-Fukai M, Zafari AM, Fukui T, Ishizaka N, Griendling KK. p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II induced hypertrophy in vascular smooth muscle cells. J Biol Chem. 1996; 271: 23317–23321.
Yang L, Quan S, Nasjletti A, Laniado-Schwartzman M, Abraham NG. Heme oxygenase-1 gene expression modulates angiotensin II-induced increase in blood pressure. Hypertension. 2004; 43: 1221–1226.
Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury: part I: basic mechanisms and in vivo monitoring of ROS. Circulation. 2003; 108: 1912–1916.
Nakamura K, Fushimi K, Kouchi H, Mihara K, Miyazaki M, Ohe T, Namba M. Inhibitory effects of antioxidants on neonatal rat cardiac myocyte hypertrophy induced by tumor necrosis factor-alpha and angiotensin II. Circulation. 1998; 98: 794–799.
Morse D, Pischke SE, Zhou Z, Davis RJ, Flavell RA, Loop T, Otterbein SL, Otterbein LE, Choi AM. Suppression of inflammatory cytokine production by carbon monoxide involves the JNK pathway and AP-1. J Biol Chem. 2003; 278: 36993–36998.
Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res. 2002; 91: 406–413.
Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. Am J Physiol. 2000; 279: L1005–L1028.
Taillé C, El-Benna J, Lanone S, Dang M, Ogier-Denis E, Aubier M, Boczkowski J. Induction of heme oxygenase-1 inhibits NAD(P)H oxidase activity by down-regulating cytochrome b558 expression via the reduction of heme availability. J Biol Chem. 2004; 279: 28681–28688.
Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev. 1999; 13: 2905–2927.