II-induced Reactive Oxygen Species Production Research Articles

Sirt3 Protects Against Thoracic Aortic Dissection Formation by Reducing Reactive Oxygen Species, Vascular Inflammation, and Apoptosis of Smooth Muscle Cells.

Sirtuin3 (Sirt3) is a histone deacetylase involved in the regulation of many cellular processes. Sirt3 deficiency is known to increase oxidative stress. Reactive oxygen species (ROS) promote degradation of the extracellular matrix and vascular smooth muscle cell (VSMC) apoptosis. Reducing oxidative stress by Sirt3 overexpression could have therapeutic potential for limiting thoracic aortic dissection (TAD) development. We hypothesized that Sirt3 deficiency could increase the risk for TAD by decreasing ROS elimination and that Sirt3 overexpression (Sirt3OE) could provide an alternative option for TAD treatment. Mice with TAD had significantly lower Sirt3 expression than normal subjects. Sirt3 KO mice exhibit significantly increased TAD incidence rate and increased aortic diameters. Moreover, Sirt3 overexpression reduced Ang II-induced ROS production, NF-kB activation, and apoptosis in human aortic smooth muscle cells (HASMCs). Sirt3 overexpression attenuated aneurysm formation and decreased aortic expansion. In conclusion, our data showed that Sirt3 deficiency increases susceptibility to TAD formation by attenuating anti-ROS effects and increasing VSMC apoptosis and vascular inflammation.

ClC-3 promotes angiotensin II-induced reactive oxygen species production in endothelial cells by facilitating Nox2 NADPH oxidase complex formation.

Recent evidence suggests that ClC-3, a member of the ClC family of Cl- channels or Cl-/H+ antiporters, plays a critical role in NADPH oxidase-derived reactive oxygen species (ROS) generation. However, the underling mechanisms remain unclear. In this study we investigated the effects and mechanisms of ClC-3 on NADPH oxidase activation and ROS generation in endothelial cells. Treatment with angiotensin II (Ang II, 1 μmol/L) significantly elevated ClC-3 expression in cultured human umbilical vein endothelial cells (HUVECs). Furthermore, Ang II treatment increased ROS production and NADPH oxidase activity, an effect that could be significantly inhibited by knockdown of ClC-3, and further enhanced by overexpression of ClC-3. SA-β-galactosidase staining showed that ClC-3 silencing abolished Ang II-induced HUVEC senescence, whereas ClC-3 overexpression caused the opposite effects. We further showed that Ang II treatment increased the translocation of p47phox and p67phox from the cytosol to membrane, accompanied by elevated Nox2 and p22phox expression, which was significantly attenuated by knockdown of ClC-3 and potentiated by overexpression of ClC-3. Moreover, overexpression of ClC-3 increased Ang II-induced phosphorylation of p47phox and p38 MAPK in HUVECs. Pretreatment with a p38 inhibitor SB203580 abolished ClC-3 overexpression-induced increase in p47phox phosphorylation, as well as NADPH oxidase activity and ROS generation. Our results demonstrate that ClC-3 acts as a positive regulator of Ang II-induced NADPH oxidase activation and ROS production in endothelial cells, possibly via promoting both Nox2/p22phox expression and p38 MAPK-dependent p47phox/p67phox membrane translocation, then increasing Nox2 NADPH oxidase complex formation.

The inositol-requiring enzyme 1 (IRE1α) RNAse inhibitor, 4µ8C, is also a potent cellular antioxidant

Inositol-requiring enzyme 1 alpha (IRE1α) is an endoplasmic reticulum (ER)-transmembrane endonuclease that is activated in response to ER stress as part of the unfolded protein response (UPR). Chronic activation of the UPR has been implicated in the pathogenesis of many common diseases including diabetes, cancer, and neurological pathologies such as Huntington's and Alzheimer's disease. 7-Hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (4µ8C) is widely used as a specific inhibitor of IRE1α ribonuclease activity (IC50 of 6.89 µM in cultured cells). However, in this paper, we demonstrate that 4µ8C acts as a potent reactive oxygen species (ROS) scavenger, both in a cell-free assay and in cultured cells, at concentrations lower than that widely used to inhibit IRE1α activity. In vitro we show that, 4µ8C effectively decreases xanthine/xanthine oxidase catalysed superoxide production with an IC50 of 0.2 µM whereas in cultured endothelial and clonal pancreatic β-cells, 4µ8C inhibits angiotensin II-induced ROS production with IC50 values of 1.92 and 0.29 µM, respectively. In light of this discovery, conclusions reached using 4µ8C as an inhibitor of IRE1α should be carefully evaluated. However, this unexpected off-target effect of 4µ8C may prove therapeutically advantageous for the treatment of pathologies that are thought to be caused by, or exacerbated by, both oxidative and ER stress such as endothelial dysfunction and/or diabetes.

Human Endomyocardial Biopsy Specimen-Derived Stromal Cells Modulate Angiotensin II-Induced Cardiac Remodeling.

Despite effective pharmacological treatment with angiotensin II type I receptor antagonists or angiotensin II-converting enzyme inhibitors, morbidity and mortality associated with heart failure are still substantial, prompting the search of novel therapeutic strategies. There is accumulating evidence supporting the use of cell therapy for cardiac repair. This study demonstrates that cells derived from human endomyocardial biopsies, cardiac-derived adherent proliferating cells (CardAPs), have the potential to reduce angiotensin II-induced cardiac remodeling and improve left ventricular function in angiotensin II mice. The mechanism involves antifibrotic and antihypertrophic effects via paracrine actions and immunomodulatory properties. These findings support the potential of CardAPs for the treatment of heart failure.

TLR4 is a critical regulator of angiotensin II-induced vascular remodeling: the roles of extracellular SOD and NADPH oxidase.

Toll-like receptor 4 (TLR4) and angiotensin II (AngII) induce vascular remodeling through the production of reactive oxygen species (ROS). AngII has also been shown to increase antioxidant enzyme extracellular superoxide dismutase (ecSOD). However, the roles of TLR4 in Ang II-induced ROS production, vascular remodeling and hypertension remain unknown. Mice lacking TLR4 function showed significant inhibition of vascular remodeling in response to chronic AngII infusion, with no impact on blood pressure. The increases in ROS level and NADPH oxidase activity in response to AngII infusion were markedly blunted in TLR4-deficient mice. Similar effects were observed in wild-type (WT) mice treated with a sub-depressor dose of the AT1 receptor antagonist irbesartan, which had no effects on TLR4-deficient mice. Intriguingly, the AngII infusion-induced increases in ecSOD activity and expression were rather enhanced in TLR4-deficient mice compared with WT mice, whereas the expression of the proinflammatory chemokine MCP-1 was decreased. Importantly, AngII-induced vascular remodeling was positively correlated with NADPH oxidase activity, ROS levels and MCP-1 expression levels. Notably, chronic norepinephrine infusion, which elevates blood pressure without increasing ROS production, did not induce significant vascular remodeling in WT mice. Taken together, these findings suggest that ROS elevation is required for accelerating vascular remodeling but not for hypertensive effects in this model. We demonstrated that TLR4 plays a pivotal role in regulating AngII-induced vascular ROS levels by inhibiting the expression and activity of the antioxidant enzyme ecSOD, as well as by activating NADPH oxidase, which enhances inflammation to facilitate the progression of vascular remodeling.

B-type natriuretic peptide inhibits angiotensin II-induced proliferation and migration of pulmonary arterial smooth muscle cells

Pulmonary vascular remodeling, characterized by disordered proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), is a pathognomonic feature of pulmonary arterial hypertension. Thus, pharmacologic strategy targeting on anti-proliferation and anti-migration of PASMCs may have therapeutic implications for PAH. Here we investigated the effects and underlying mechanisms of B-type natriuretic peptide (BNP) on angiotensin II (Ang II)-induced proliferation and migration of PASMCs. Proliferation and migration of PASMCs cultured from Wistar rats were induced by Ang II, with or without BNP treatment. In addition, potential underlying mechanisms including cell cycle progression, Ca(2+) overload, reactive oxygen species (ROS) production, signal transduction of MAPK and Akt, and the cGMP/PKG pathway were examined. We found that BNP inhibited Ang II-induced PASMCs proliferation and migration dose dependently. BNP could also arrest the cell cycle progression in the G0/G1-phase. In addition, BNP attenuated intracellular calcium overload caused by Ang II. Moreover, Ang II-induced ROS production was mitigated by BNP, with associated down-regulation of NAD(P)H oxidase 1 (Nox1) and reduced mitochondrial ROS production. Finally, Ang II-activated MAPKs and Akt were also counteracted by BNP. Of note, all these effects of BNP were abolished by a PKG inhibitor (Rp-8-Br-PET-cGMPS). In conclusion, BNP inhibits Ang II-induced PASMCs proliferation and migration. These effects are potentially mediated by decreased calcium influx, reduced ROS production by Nox1 and mitochondria, and down-regulation of MAPK and Akt signal transduction, through the cGMP/PKG pathway. Therefore, this study implicates that BNP may have a therapeutic role in pulmonary vascular remodeling.

Antihypertensive effect of gomisin A from Schisandra chinensis on angiotensin II-induced hypertension via preservation of nitric oxide bioavailability

Gomisin A (GA) is a small molecular weight lignan present in Schisandra chinensis, and has been demonstrated to have vasodilatory activity. In the present study, we investigated the effect of GA on blood pressure (BP) in angiotensin II (Ang II)-induced hypertensive mice. C57/BL6 mice infused subcutaneously with Ang II (1 and 2 μg kg−1 per min for 2 weeks) showed an increase in BP with a decrease in nitric oxide (NO) metabolites in plasma, and a negative correlation between these two parameters was demonstrated. In the thoracic aorta from Ang II-induced hypertensive mice, a decrease in vascular NO that was accompanied by a diminution of phosphorylated endothelial nitric oxide synthase (eNOS), as well as by increased reactive oxygen species (ROS) production, was demonstrated. These alterations in BP, eNOS phosphorylation and ROS production in the vasculature of Ang II-treated mice were markedly and dose-dependently reversed by simultaneous administration of GA (2 and 10 μg kg−1 per min). In addition, Ang II-induced ROS production in cultured vascular cells such as endothelial cells and vascular smooth muscle cells was markedly attenuated by GA. These results suggested that GA attenuated the increase in BP via preservation of vascular NO bioavailability not only by inhibiting ROS production but also by preventing the impairment of eNOS function in the vasculature of Ang II-induced hypertensive mice.

Angiotensin II type 2 receptor-coupled nitric oxide production modulates free radical availability and voltage-gated Ca2+ currents in NTS neurons.

The medial region of the nucleus tractus solitarius (mNTS) is a key brain stem site controlling cardiovascular function, wherein ANG II modulates neuronal L-type Ca(2+) currents via activation of ANG II type 1 receptors (AT(1)R) and production of reactive oxygen species (ROS). ANG II type 2 receptors (AT(2)R) induce production of nitric oxide (NO), which may interact with ROS and modulate AT(1)R signaling. We sought to determine whether AT(2)R-mediated NO production occurs in mNTS neurons and, if so, to elucidate the NO source and the functional interaction with AT(1)R-induced ROS or Ca(2+) influx. Electron microscopic (EM) immunolabeling showed that AT(2)R and neuronal NO synthase (nNOS) are coexpressed in neuronal somata and dendrites receiving synapses in the mNTS. In the presence of the AT(1)R antagonist losartan, ANG II increased NO production in isolated mNTS neurons, an effect blocked by the AT(2)R antagonist PD123319, but not the angiotensin (1-7) antagonist D-Ala. Studies in mNTS neurons of nNOS-null or endothelial NOS (eNOS)-null mice established nNOS as the source of NO. ANG II-induced ROS production was enhanced by PD123319, the NOS inhibitor N(G)-nitro-l-arginine (LNNA), or in nNOS-null mice. Moreover, in the presence of losartan, ANG II reduced voltage-gated L-type Ca(2+) current, an effect blocked by PD123319 or LNNA. We conclude that AT(2)R are closely associated and functionally coupled with nNOS in mNTS neurons. The resulting NO production antagonizes AT(1)R-mediated ROS and dampens L-type Ca(2+) currents. The ensuing signaling changes in the NTS may counteract the deleterious effects of AT(1)R on cardiovascular function.

Inactivation of Adenosine A2A Receptor Attenuates Basal and Angiotensin II-induced ROS Production by Nox2 in Endothelial Cells

Endothelial cells (ECs) express a Nox2 enzyme, which, by generating reactive oxygen species (ROS), contributes to EC redox signaling and angiotensin II (AngII)-induced endothelial dysfunction. ECs also express abundantly an adenosine A2A receptor (A2AR), but its role in EC ROS production remains unknown. In this study, we investigated the role of A2AR in the regulation of Nox2 activity and signaling in ECs with or without acute AngII stimulation. In cultured ECs (SVEC4–10), AngII (100 nm, 30 min) significantly increased Nox2 membrane translocation and association with A2AR. These were accompanied by p47phox, ERK1/2, p38 MAPK, and Akt phosphorylation and an increased ROS production (169 ± 0.04%). These AngII effects were inhibited back to the control levels by a specific A2AR antagonist (SCH58261), or adenosine deaminase, or by knockdown of A2AR or Nox2 using specific siRNAs. Knockdown of A2AR, as determined by Western blotting, decreased Nox2 and p47phox expression. In wild-type mouse aorta, SCH58261 significantly reduced acute AngII-induced ROS production and preserved endothelium-dependent vessel relaxation to acetylcholine. These results were further confirmed by using aortas from A2AR knock-out mice. In conclusion, A2AR is involved in the regulation of EC ROS production by Nox2. Inhibition or blockade of A2AR protects ECs from acute AngII-induced oxidative stress, MAPK activation, and endothelium dysfunction.

BAS/BSCR28 Blockade of adenosine A2A receptor attenuates angiotensin II-induced reactive oxygen species production and impairment of endothelium-dependent vessel relaxation in mouse aortas

The endothelium expresses abundantly an adenosine A2A receptor (A2AR) which has important roles in the regulation of vascular function. The endothelium expresses also the AT1-type receptor for angiotensin II (AngII)...

EGCG reduces Ang II‐induced NAD(P)H oxidase and NO synthase expression in HUVECs

Atherosclerosis is a chronic systemic disease of the vasculature with an inflammatory component. The impairment of vascular endothelial cell function which is an important determinant of atherosclerosis progression is in part due to increased release of reactive oxygen species (ROS). Angiotensin II (Ang II) causes endothelial dysfunction by increasing NAD(P)H oxidase‐mediated vascular superoxide production. Epigallocatechin‐3‐gallate (EGCG), the major bioactive polyphenol present in green tea and a potent antioxidant, has been reported to lower the risk of cardiovascular diseases such as atherosclerosis and hypertension. In this study, we investigated the effects of EGCG on Ang II‐induced expression of NAD(P)H oxidase and nitric oxide synthase (NOS), which may contribute to oxidative stress. We observed that Ang II stimulated mRNA and protein expression of NAD(P)H oxidase subunit and iNOS in human umbilical vein endothelial cells (HUVECs). EGCG inhibited Ang II‐induced expression of NAD(P)H oxidase subunit and iNOS in a concentration‐dependent manner. However, EGCG did not affect Ang II‐induced ROS production. Surprisingly, EGCG itself produced ROS in HUVECs. These results suggest that anti‐atherosclerotic activity of EGCG is likely not due to decreased ROS formation although there may be some benefit through inhibition of expression of NAD(P)H oxidase and NOS induced by Ang II.

Angiotensin II–Induced Oxidative Stress Resets the Ca 2+ Dependence of Ca 2+ –Calmodulin Protein Kinase II and Promotes a Death Pathway Conserved Across Different Species

Angiotensin (Ang) II-induced apoptosis was reported to be mediated by different signaling molecules. Whether these molecules are either interconnected in a single pathway or constitute different and alternative cascades by which Ang II exerts its apoptotic action, is not known. To investigate in cultured myocytes from adult cat and rat, 2 species in which Ang II has opposite inotropic effects, the signaling cascade involved in Ang II-induced apoptosis. Ang II (1 micromol/L) reduced cat/rat myocytes viability by approximately 40%, in part, because of apoptosis (TUNEL/caspase-3 activity). In both species, apoptosis was associated with reactive oxygen species (ROS) production, Ca(2+)/calmodulin-dependent protein kinase (CaMK)II, and p38 mitogen-activated protein kinase (p38MAPK) activation and was prevented by the ROS scavenger MPG (2-mercaptopropionylglycine) or the NADPH oxidase inhibitor DPI (diphenyleneiodonium) by CaMKII inhibitors (KN-93 and AIP [autocamtide 2-related inhibitory peptide]) or in transgenic mice expressing a CaMKII inhibitory peptide and by the p38MAPK inhibitor, SB202190. Furthermore, p38MAPK overexpression exacerbated Ang II-induced cell mortality. Moreover, although KN-93 did not affect Ang II-induced ROS production, it prevented p38MAPK activation. Results further show that CaMKII can be activated by Ang II or H(2)O(2), even in the presence of the Ca(2+) chelator BAPTA-AM, in myocytes and in EGTA-Ca(2+)-free solutions in the presence of the calmodulin inhibitor W-7 in in vitro experiments. (1) The Ang II-induced apoptotic cascade converges in both species, in a common pathway mediated by ROS-dependent CaMKII activation which results in p38MAPK activation and apoptosis. (2) In the presence of Ang II or ROS, CaMKII may be activated at subdiastolic Ca(2+) concentrations, suggesting a new mechanism by which ROS reset the Ca(2+) dependence of CaMKII to extremely low Ca(2+) levels.

Isoproterenol Inhibits Angiotensin II-Stimulated Proliferation and Reactive Oxygen Species Production in Vascular Smooth Muscle Cells through Heme Oxygenase-1

Heme oxygenase (HO)-1 is a well-known cytoprotectant against oxidative stress and exhibits an antiproliferative effect in vascular smooth muscle cells (VSMCs). The purpose of the present study was to test whether isoproterenol, one of the synthetic catecholamines having beta-adrenergic activity, affected angiotensin II (Ang II)-induced cell proliferation and reactive oxygen species (ROS) production. Also, the presumptive underlying signaling pathways in VSMCs were studied. Aortic VSMCs from 11-week-old male Sprague-Dawley rats were used. Isoproterenol dose-dependently increased HO-1 expression through beta(2)-adrenoceptor (AR) and protein kinase A (PKA) pathway, and isoproterenol concentration-dependently increased beta(2)-AR mRNA expression. Isoproterenol attenuated Ang II-induced cell proliferation, as evidenced by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. This effect of isoproterenol was inhibited by pretreatment of the cells with beta(2)-AR antagonist butoxamine, PKA inhibitor H-89 and HO inhibitor Tin Protoporphyrin IX (SnPP IX), respectively. Isoproterenol inhibited phosphorylation level of Ang II-induced extracellular signal-regulated kinase (ERK1/2). Isoproterenol significantly inhibited Ang II-induced ROS production through the ERK1/2 pathway. These findings suggest that isoproterenol, via induction of HO-1, inhibits Ang II-stimulated proliferation and ROS production in cultured VSMCs.

Abstract 1357: PGC-1 α as a Novel Regulator for Angiotensin II-induced Reactive Oxygen Species Production and Hypertrophy in Vascular Smooth Muscle Cells

Angiotensin II (Ang II) increases H 2 O 2 production and vascular smooth muscle cell (VSMCs) hypertrophy, in part through redox-sensitive PI3K/Akt, which is inhibited by catalase overexpression. The relevant molecular mechanism remains unclear. Peroxisome proliferator-activated receptor gamma coactivator-1 α (PGC-1 α ) is reported to protect from oxidative stress by regulating expression of antioxidant enzymes such as catalase. We thus hypothesized that PGC-1 α may be important mediator for Ang II-induced H2O2 production and vascular hypertrophy. Here we show that Ang II stimulation increases serine phosphorylation of PGC-1 α (2.2 folds) with a peak at 15 min, which is inhibited by LY294002, a specific PI3 kinase inhibitor (98% decrease), and by Akt inhibitor-2/Triciribine (95% decrease). Ang II promotes PGC-1 α phosphorylation mainly at Ser 570 in an Akt-dependent manner. Ang II significantly suppresses Gal4-fused PGC-1 α transcriptional activity in a dose dependent manner, which is partially reversed by PI3K/Akt inhibition. Chromatin immunoprecipitation (ChIP) assay shows that PGC-1 α associates with the catalase promoter and this association is blocked by Ang II in a PI3K/Akt-dependent manner. Consistent with these results, Ang II stimulation time-dependently decreases endogenous catalase expression at both messenger RNA and protein levels. Ang II-induced downregulation of catalase at protein level at 24 hrs is prevented by Akt inhibitor (86%) and by overexpression of PGC-1 α S570A, an Akt phosphorylation site mutant, (75%). Moreover, overexpression of PGC-1 α S570A significantly inhibits Ang II-induced increase in H2O2 production (>80%) and leucine incorporation (>90%) as measured at 12 and 24 hrs, respectively. In summary, Akt-dependent serine phosphorylation of PGC-1 α by Ang II plays an important role for Ang II-induced downregulation of catalase, thereby increasing H2O2 production, which may contribute to ROS-dependent VSMC hypertrophy. These findings provide insight into a novel mechanisms by which Ang II promotes long-term H2O2 production to increase oxidative stress via targeting PGC-1alpha, and mediates metabolic abnormalities.

Sex differences in angiotensin signaling in bulbospinal neurons in the rat rostral ventrolateral medulla

Sex differences may play a significant role in determining the risk of hypertension. Bulbospinal neurons in the rostral ventrolateral medulla (RVLM) are involved in the tonic regulation of arterial pressure and participate in the central mechanisms of hypertension. Angiotensin II (ANG II) acting on angiotensin type 1 (AT(1)) receptors in RVLM neurons is implicated in the development of hypertension by activating NADPH oxidase and producing reactive oxygen species (ROS). Therefore, we analyzed RVLM bulbospinal neurons to determine whether there are sex differences in: 1) immunolabeling for AT(1) receptors and the key NADPH oxidase subunit p47 using dual-label immunoelectron microscopy, and 2) the effects of ANG II on ROS production and Ca(2+) currents using, respectively, hydroethidine fluoromicrography and patch-clamping. In tyrosine hydroxylase-positive RVLM neurons, female rats displayed significantly more AT(1) receptor immunoreactivity and less p47 immunoreactivity than male rats (P < 0.05). Although ANG II (100 nM) induced comparable ROS production in dissociated RVLM bulbospinal neurons of female and male rats (P > 0.05), an effect mediated by AT(1) receptors and NADPH oxidase, it triggered significantly larger dihydropyridine-sensitive long-lasting (L-type) Ca(2+) currents in female RVLM neurons (P < 0.05). These observations suggest that an increase in AT(1) receptors in female RVLM neurons is counterbalanced by a reduction in p47 levels, such that ANG II-induced ROS production does not differ between females and males. Since the Ca(2+) current activator Bay K 8644 induced larger Ca(2+) currents in females than in male RVLM neurons, increased ANG II-induced L-type Ca(2+) currents in females may result from sex differences in calcium channel densities or dynamics.

Angiotensin converting enzyme-2 confers endothelial protection and attenuates atherosclerosis

The endothelium plays a central role in the maintenance of vascular homeostasis. One of the main effectors of endothelial dysfunction is ANG II, and pharmacological approaches to limit ANG II bioactivity remain the cornerstone of cardiovascular therapeutics. Angiotensin converting enzyme-2 (ACE2) has been identified as a critical negative modulator of ANG II bioactivity, counterbalancing the effects of ACE in determining net tissue ANG II levels; however, the role of ACE2 in the vasculature remains unknown. In the present study, we hypothesized that ACE2 is a novel target to limit endothelial dysfunction and atherosclerosis. To this aim, we performed in vitro gain and loss of function experiments in endothelial cells and evaluated in vivo angiogenesis and atherosclerosis in apolipoprotein E-knockout mice treated with AdACE2. ACE2-deficient mice exhibited impaired endothelium-dependent relaxation. Overexpression of ACE2 in human endothelial cells stimulated endothelial cell migration and tube formation, and limited monocyte and cellular adhesion molecule expression; effects that were reversed in ACE2 gene silenced and endothelial cells isolated from ACE2-deficient animals. ACE2 attenuated ANG II-induced reactive oxygen species production in part through decreasing the expression of p22phox. The effects of ACE2 on endothelial activation were attenuated by pharmacological blockade of ANG-(1-7) with A779. ACE2 promoted capillary formation and neovessel maturation in vivo and reduced atherosclerosis in apolipoprotein E-knockout mice These data indicate that ACE2, in an ANG-(1-7)-dependent fashion, functions to improve endothelial homeostasis via a mechanism that may involve attenuation of NADPHox-induced reactive oxygen species production. ACE2-based treatment approaches may be a novel approach to limit aberrant vascular responses and atherothrombosis.

Essential Role of Angiotensin II Type 1a Receptors in the Host Vascular Wall, but Not the Bone Marrow, in the Pathogenesis of Angiotensin II−Induced Atherosclerosis

The angiotensin II (Ang II) type 1a (AT1a) receptor is expressed on multiple cell types in atherosclerotic lesions, including bone marrow-derived cells and vascular wall cells, and mediates inflammatory and proliferative responses. Indeed, Ang II infusion accelerates atherogenesis in hyperlipidemic mice by recruiting monocytes and by activating vascular wall cells. Here, we investigated the relative roles of AT1a receptors in the bone marrow vs. the vascular wall in Ang II-induced atherogenesis. Apolipoprotein E-knockout (ApoE(-/-)) mice with or without bone marrow AT1a receptor were generated by experimental bone marrow transplantation using AT1a(+/+) or AT1a(-/-) recipients. In these mice, 28-d Ang II infusion induced significant atherosclerosis in the aorta, and the severity of plaque formation was not affected by the absence of bone marrow AT1a receptor. We then generated AT1a(-/-)ApoE(-/-) mice with or without bone marrow AT1a receptor. Ang II-induced plaque formation was blunted irrespective of the presence of bone marrow AT1a receptor. Host AT1a receptor deficiency was found to suppress Ang II-induced reactive oxygen species production. In addition, AT1a receptor deficiency also impaired monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in the arterial wall 7 d after Ang II initiation. These molecules normally initiate later macrophage-mediated inflammation in the vascular wall. By contrast, AT1a receptor deficiency in the bone marrow did not affect MCP-1-induced monocyte chemotaxis in vitro. In conclusion, AT1a receptors in the host vascular wall, but not in the bone marrow, are essential in Ang II-induced atherogenesis.

Ets-1 Is a Critical Transcriptional Regulator of Reactive Oxygen Species and p47 phox Gene Expression in Response to Angiotensin II

Angiotensin (Ang) II is a potent mediator of vascular inflammation. A central mechanism by which Ang II promotes inflammation is through the generation of reactive oxygen species (ROS). In the current study, we investigated the role of the transcription factor Ets-1 in regulating Ang II-induced ROS generation. ROS generation was measured in the thoracic aorta of Ets-1(-/-) mice compared with littermate controls after continuous infusion of Ang II. H2O2 and superoxide anion (O2(-)) production were significantly blunted in the Ets-1(-/-) mice. Inhibition of Ets-1 expression by small interfering RNA in primary human aortic smooth muscle cells also potently inhibited ROS production and the induction of the NAD(P)H oxidase subunit p47(phox) in response to Ang II. To evaluate the therapeutic potential of inhibiting Ets-1 in wild-type mice, dominant negative Ets-1 membrane-permeable peptides were administered systemically. Ang II-induced ROS production and medial hypertrophy in the thoracic aorta were markedly diminished as a result of blocking Ets-1. In summary, Ets-1 functions as a critical downstream transcriptional mediator of Ang II ROS generation by regulating the expression of NAD(P)H oxidase subunits such as p47(phox).

Receptor-independent intracellular radical scavenging activity of an angiotensin II receptor blocker

Angiotensin II plays a crucial role in the induction of oxidative stress and the pathogenesis of cardiovascular and renal diseases, and the beneficial mechanisms of angiotensin II receptor 1 blockers (ARBs) are multifactorial. We investigated the receptor-independent protective role of an ARB using primary-cultured mesangial cells from angiotensin II receptor 1 knockout or wild-type mice and a highly lipophilic ARB, telmisartan. Intracellular reactive oxygen species were estimated using a fluorogenic probe, CM-H2DCFDA. Non-angiotensin II-induced reactive oxygen species production was generated by exposing cells to hydrogen peroxide alone or after treatment with telmisartan. Flow cytometry analysis showed that angiotensin II induced an increase in oxidant production in a dose-dependent manner in wild-type cells, but not in knockout cells. In contrast, hydrogen peroxide induced oxidative stress in both wild-type and knockout cells. Interestingly, telmisartan attenuated the oxidative stress induced by hydrogen peroxide in both cells, suggesting that it acted via a receptor-independent antioxidant effect. Intracellular concentrations of telmisartan were confirmed by high-performance liquid chromatography analysis. Expression of plasminogen activator inhibitor 1, which is stimulated by oxidative stress, was also attenuated by telmisartan in a receptor-independent as well as receptor-dependent manner. Telmisartan did not change expression levels of antioxidative enzymes such as catalase or glutathione peroxidase. Furthermore, the amelioration of oxidative stress by telmisartan did not involve the peroxisome proliferator-activated receptor-gamma pathway. Telmisartan inhibits intracellular oxidative stress, at least in part, in a receptor-independent manner, possibly owing to its lipophilic and antioxidant structure.

Anti-oxidative effect of Klotho on endothelial cells through cAMP activation

Klotho, a regulatory factor implicated in countering the aging process, has been reported to ameliorate endothelial dysfunction in vivo. To clarify whether Klotho protein directly affects endothelial cell function, we studied the effects of membrane-form Klotho on manganese superoxide dismutase (Mn-SOD) expression and nitric oxide production in human umbilical vein endothelial cells (HUVEC). We incubated HUVEC with conditioned medium from COS-1 cells transfected with expression vector, pCAGGS-klotho (Klotho-CM) or a recombinant, purified 6His-tagged Klotho protein. Both Klotho-CM and 6His-tagged Klotho protein enhanced Mn-SOD expression by approximately two-fold, partially via activation of the cAMP signaling pathway. Furthermore, Klotho-CM increased nitric oxide production, which also contributed to the up-regulation of Mn-SOD. Using the oxidation-sensitive dye dihydroethidium, we found that Klotho inhibited angiotensin II-induced reactive oxygen species production in HUVEC. These findings provide new insights into the mechanisms of Klotho action and support the therapeutic potential of membrane-form Klotho to regulate endothelial function.