Ang II-induced Reactive Oxygen Species Research Articles

763: AT1-AA increases AngII-induced ET-1 and ROS

Endothelin-1 (ET-1), reactive oxygen species (ROS) and agonistic autoantibodies to the angiotensin II (AngII) type I receptor (AT1-AA) may be important links between endothelial dysfunction and hypertension during preeclampsia. Although it is clear that activation of the AngII receptor increases endothelial cell production of ET-1 and vascular smooth muscle NADPH oxidase, it is unknown whether interaction of the AT1-AA with the AT1R enhances AngII-induced ET-1 or oxidative stress. Therefore, we tested the hypothesis that AT1-AA increases AngII-induced ET-1 and ROS release from vascular endothelial cells (HUVEC).

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.

NADPH oxidase-derived reactive oxygen species involved in angiotensin II-induced monocyte chemoattractant protein-1 expression in mesangial cells

To investigate the origin of oxidative stress induced by angiotensin II (AngII) in human mesangial cells and the role of reactive oxygen species (ROS) in AngII-induced monocyte chemoattractant protein-1 (MCP-1) expression. MCP-1 expression was determined by real time RT-PCR. ROS production was measured by DCFDA fluorescence. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was examined by lucigenin chemiluminescence. p47phox and p67phox translocation was assayed by Western blot. Twenty-four male mice were randomly divided into three groups: the control, the AngIIinfusion [AngII 400 ng/(kg.min)], and the apocynin treatment. AngII was infused by subcutaneously osmotic minipump for 14 days. Urinary albumin and 8-isoprostane excretion were measured by ELISA. In cultured human mesangial cells, AngII induced the MCP-1 expression in a dose-dependent manner with 3.56 fold increase as compared with the control. AngII increased intracellular ROS production as early as 3 min with the peak at 60 min and was in a time and dose-dependent. Incubation with different dosages of AngII (1 nmol/L, 10 nmol/L, and 100 nmol/L AngII) for 60 min, ROS production increased at 1.82, 2.92, and 4.08 folds respectively. AngII-induced ROS generation was sensitive to diphenyleneiodonium sulfate (DPI, 10 micromol/L) and apocynin (500 micromol/L), two structurally distinct NADPH oxidase inhibitors. In contrast, inhibitors of other oxidant-producing enzymes, including the mitochondrial complex Iinhibitor rotenone, the xanthine oxidase inhibitor allopurinol, the cyclooxygenase inhibitor indomethacin, the lipoxygenase inhibitor nordihydroguiaretic acid, the cytochrome P450 oxygenase inhibitor ketoconazole and the nitric oxide synthase inhibitor G-nitro-L-arginine methyl ester were without an effect. AngII-induced ROS generation was inhibited by the AT1 antagonist losartan (10 micromol/L) but not the AT2 antagonist PD123319 (10 micromol/L). AngII treatment induced translocation of cytosolic of p47phox and p67phox to the membrane. The antioxidants almost abolished AngII-induced MCP-1 expression. AngII infusion increased urinary and p67 translocation by 2.69-, 2.97-, and 2.67-fold, respectively. NADPH oxidase-derived ROS is involved in AngII-induced MCP-1 expression. Inhibition of NADPH oxidase alleviates AngII-induced renal injury.

Endothelin-1 Regulates Volume-Sensitive Chloride Current in Rabbit Atrial Myocytes via Reactive Oxygen Species from Mitochondria and NADPH Oxidase

Angiotensin II (AngII) signaling and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) are implicated in the activation of volume-sensitive Cl current (ICl,swell) by both beta1-integrin stretch and osmotic swelling. Because endothelin-1 (ET-1) is a potential downstream mediator of AngII and ET-1 blockade abrogates AngII-induced ROS generation, we studied how ET-1 signaling regulates ICl,swell. Under isosmotic conditions, ET-1 (10 nM) elicited an outwardly rectifying Cl current that was fully blocked by the highly selective ICl,swell inhibitor DCPIB (10 μM) and by osmotic shrinkage. Selective ETA (BQ-123, 1 μM) but not ETB blockade (BQ-788, 100 nM) fully suppressed ET-1-induced current. ET-1-induced ICl,swell also was abolished by inhibitors of EGFR kinase (AG1478, 10 μM) and PI-3K (LY294002, 20 μM; wortmannin, 500 nM), which also suppress stretch- and swelling-induced ICl,swell. ERK inhibitors (PD 98059, 10 μM; U0216, 1 μM) partially and fully blocked ET-1- and EGF- induced currents, respectively, but did not effect ICl,swell elicited by H2O2. ET-1 acts downstream from AngII. ETA blockade (BQ-123) abolished ICl,swell elicited by both AngII and osmotic swelling, whereas AT1 blockade (losartan, 5 μM) did not effect ET-1-induced ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes. Blocking NOX with apocynin (500 μM) or mitochondrial complex I with rotenone (10 μM) both completely suppressed ET-1-induced ICl,swell. In contrast, ICl,swell elicited by antimycin A (10 μM), which stimulates superoxide production by mitochondrial complex III, was insensitive to apocynin and the NOX fusion peptide inhibitor gp91ds-tat (500 nM). These data suggest that ET-1 and ETA receptors are required intermediates in AngII-, swelling-, and stretch-induced activation of ICl,swell. Moreover, enhancement of mitochondrial ROS production by ROS from NOX is likely to contribute to activation of ICl,swell by ET-1.

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.

NPRA-mediated suppression of AngII-induced ROS production contribute to the antiproliferative effects of B-type natriuretic peptide in VSMC

Excessive proliferation of vascular smooth cells (VSMCs) plays a critical role in the pathogenesis of diverse vascular disorders, and inhibition of VSMCs proliferation has been proved to be beneficial to these diseases. In this study, we investigated the antiproliferative effect of B-type natriuretic peptide (BNP), a natriuretic peptide with potent antioxidant capacity, on rat aortic VSMCs, and the possible mechanisms involved. The results indicate that BNP potently inhibited AngiotensinII (AngII)-induced VSMCs proliferation, as evaluated by [(3)H]-thymidine incorporation assay. Consistently, BNP significantly decreased AngII-induced intracellular reactive oxygen species (ROS) and NAD(P)H oxidase activity. 8-Br-cGMP, a cGMP analog, mimicked these effects. To confirm its mechanism, siRNA of natriuretic peptide receptor-A(NRPA) strategy technology was used to block cGMP production in VSMCs, and siNPRA attenuated the inhibitory effects of BNP in VSMCs. Taken together, these results indicate that BNP was capable of inhibiting VSMCs proliferation by NPRA/cGMP pathway, which might be associated with the suppression of ROS production. These results might be related, at least partly, to the anti-oxidant property of BNP.

Mitochondria-derived reactive oxygen species mediate sympathoexcitation induced by angiotensin II in the rostral ventrolateral medulla

Reactive oxygen species (ROS) in the central nervous system are thought to contribute to sympathoexcitation in cardiovascular diseases such as hypertension and heart failure. Nicotinamide adenine dinucleotide phosphate oxidase is a major source of ROS in the central nervous system, which acts as a key mediator (mediators) of angiotensin II (AngII). It is not clear, however, whether mitochondria-derived ROS in the central nervous system also participate in sympathoexcitation. In an in-vivo study, we investigated whether the AngII-elicited pressor response in the rostral ventrolateral medulla, which controls sympathetic nerve activity, is attenuated by adenovirus-mediated gene transfer of a mitochondria-derived antioxidant (Mn-SOD). In an in-vitro study, using differentiated PC-12 cells with characteristics similar to those of sympathetic neurons, we examined whether AngII increases mitochondrial ROS production. Overexpression of Mn-SOD attenuated the AngII-induced pressor response and also suppressed AngII-induced ROS production, as evaluated by microdialysis in the rostral ventrolateral medulla. Using reduced MitoTracker red, we showed that AngII increased mitochondrial ROS production in differentiated PC-12 cells in vitro. Overexpression of Mn-SOD and rotenone, a mitochondrial respiratory complex I inhibitor, suppressed AngII-induced ROS production. Depletion of extracellular Ca2+ with ethylene glycol bis-N,N,N',N'-tetraacetate (EGTA) and administration of p-trifluoromethoxycarbonylcyanide phenylhydrazone, which prevents further Ca2+ uptake into the mitochondria, blocked AngII-elicited mitochondrial ROS production. These results indicate that AngII increases the intracellular Ca2+ concentration and that the increase in mitochondrial Ca2+ uptake leads to mitochondrial ROS production.

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.

ANG II induces c-Jun NH2-terminal kinase activation and proliferation of human mesangial cells via redox-sensitive transactivation of the EGFR

We previously showed that ANG II induces mesangial cell (MC) proliferation via the JNK-activator protein-1 pathway. The present study attempted to determine the upstream mediators of JNK activation, with emphasis on reactive oxygen species (ROS) and the epidermal growth factor (EGF) receptor (EGFR). In cultured human MCs (HMCs), as early as 3 min, ANG II time dependently increased intracellular ROS production, which was sensitive to 10 microM diphenyleneiodonium sulfate and 500 microM apocynin, two structurally distinct NADPH oxidase inhibitors. In contrast, inhibitors of other oxidant-producing enzymes, including the mitochondrial complex I inhibitor rotenone, the xanthine oxidase inhibitor allopurinol, the cyclooxygenase inhibitor indomethacin, the lipoxygenase inhibitor nordihydroguiaretic acid, the cytochrome P-450 oxygenase inhibitor ketoconazole, and the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester, were without effect. ANG II-induced ROS generation was inhibited by the angiotensin type 1 receptor antagonist losartan (10 muM) but not the angiotensin type 2 receptor antagonist PD-123319 (10 microM). ANG II induced translocation of p47(phox) and p67(phox) from the cytosol to the membrane. The antioxidants almost abolished the ANG II mitogenic response, as assessed by [(3)H]thymidine incorporation and cell number, associated with a remarkable blockade of the activation of EGFR (90% inhibition) and JNK (83% inhibition). The EGFR inhibitor AG-1478 was able to mimic the effect of antioxidants, in that it inhibited the mitogenic response and the JNK activation following ANG II treatment. Together, these data suggest that the ROS-EGFR-JNK pathway is involved in transducing the proliferative effect of ANG II in cultured HMCs.

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).

Role of mitochondria in angiotensin II-induced reactive oxygen species and mitogen-activated protein kinase activation

Peptide hormone Angiotensin II (Ang II) activates NAD(P)H oxidase, via AT1 receptors leading to increased generation of reactive oxygen species (ROS), such as the superoxide anion (O(2)(-)). As an important intracellular second messenger, ROS can activate many downstream signaling molecules, including mitogen-activated protein kinases (MAPK), protein tyrosine phosphatases, protein tyrosine kinases, and transcriptional factors. Activation of these signaling cascades is highly related to risk for cardiovascular diseases. Accumulating evidence reveals that membrane-bound NAD(P)H oxidase is the main source responsible for Ang II-induced ROS generation. However, recent novel findings suggest that Ang II stimulation induces opening of mitochondrial K(ATP) channels, depolarizes mitochondrial potential (DeltaPsi(M)), and further amplifies ROS generation from mitochondria, resulting in redox-sensitive activation of MAPK. In this review, we discuss the possible mechanisms of Ang II-induced cardiac pharmacological preconditioning (PC), and focus on the role of mitochondrial K(ATP) channels, mitochondrial ROS production, and MAPK activation in response to Ang II stimulation.

Pigment Epithelium-Derived Factor (PEDF) Blocks Angiotensin IIInduced T Cell Proliferation by Suppressing Autocrine Production of Interleukin-2

Angiotensin II (Ang II) elicits numerous inflammatory-proliferative responses in vascular cells, thereby being involved in atherosclerosis. We have previously shown that pigment epithelium-derived factor (PEDF) blocks the Ang II-induced endothelial cell activation, thus suggesting that PEDF may play a role in atherosclerosis. However, effects of PEDF on T cell activation, another key steps of atherosclerosis, remain to be elucidated. In this study, we examined whether PEDF could inhibit the Ang II-induced MOLT-3 T cell proliferation in vitro and the way that it might achieve this effect. Ang II significantly stimulated DNA synthesis in MOLT-3 T cells, which was inhibited by PEDF, olmesartan, an Ang II type I receptor blocker, an anti-oxidant N-acetylcysteine (NAC), or antibodies directed against IL-2. PEDF or NAC suppressed gene expression of interleukin-2 (IL-2) in Ang II-exposed MOLT-3 T cells. Furthermore, PEDF blocked the Ang II-induced reactive oxygen species (ROS) generation and NADPH oxidase activity in MOLT-3 T cells. These results demonstrate that PEDF inhibits the Ang II-induced T cell proliferation by blocking autocrine production of IL-2 via suppression of NADPH oxidase-mediated ROS generation. Blockade by PEDF of T cell activation may become a novel therapeutic target for atherosclerosis.

Regulation of Akt by arachidonic acid and phosphoinositide 3-kinase in angiotensin II-stimulated vascular smooth muscle cells

Angiotensin (Ang) II stimulates cytosolic phospholipase A2(cPLA 2)-dependent release of arachidonic acid (ArAc) in vascular smooth muscle cells (VSMC). ArAc release and production of reactive oxygen species (ROS) lead to the activation of downstream kinases resulting in VSMC growth. To determine the role of Akt in this pathway, we used VSMC to link Ang II-induced ArAc release and ROS production to the activation of Akt and VSMC growth. We observed that Ang II, ArAc, or H 2O 2 increased Akt activation. The Akt inhibitor SH6 blocked Ang II-, ArAc-, or H 2O 2-induced Akt activation, as did inhibition of phosphoinositide 3-kinase (PI 3K). Inhibition of cPLA 2 blocked Ang II effects, while the ROS scavenger NaC decreased Ang II- and ArAc-induced Akt activation. Inhibition of Akt blocked the 3H-thymidine incorporation induced by all three agonists. Thus, Ang II-induced ArAc release and ROS production leads to the PI 3K-dependant activation of Akt and VSMC growth.

Tetramethylpyrazine downregulates angiotensin II‐induced endothelin‐1 gene expression in vascular endothelial cells

1. Tetramethylpyrazine (TMP) is one of the active ingredients of the Chinese herb Ligusticum wallichii Franchat. It is well documented that TMP exerts a cardiovascular protective effect. The aims of the present study were to examine whether TMP alters angiotenisn (Ang) II-induced endothelin (ET)-1 gene expression and to identify the putative underlying signalling pathways in vascular endothelial cells. 2. Cultured vascular endothelial cells were pre-incubated with TMP, stimulated with AngII and ET-1 gene expression was then examined. The effects of TMP pretreatment on AngII-induced extracellular signal-regulated kinase (ERK) phosphorylation were investigated to elucidate the intracellular mechanism responsible for the effects of TMP on ET-1 gene expression. 3. Tetramethylpyrazine inhibited AngII-induced ET-1 gene expression, as revealed by nothern blotting and a promoter activity assay. Tetramethylpyrazine also inhibited the AngII-induced increase in intracellular reactive oxygen species (ROS), as measured by the redox sensitive fluorescent dye 2' 7'-dichlorofluorescin diacetate and ERK phosphorylation. 4. In summary, we have demonstrated, for the first time, that TMP inhibits AngII-induced ROS generation, ERK phosphorylation and ET-1 gene expression in vascular endothelial cells. Thus, the present study delivers important new insights into the molecular pathways that may contribute to the proposed beneficial effects of TMP in the cardiovascular system.

Role of Reactive Oxygen Species in Angiotensin II-Mediated Renal Growth, Differentiation, and Apoptosis

Angiotensin II (ANG II) induces cell-cycle arrest of cultured proximal tubular cells, resulting in cellular hypertrophy. This ANG II-mediated hypertrophy is associated with the induction of p27(Kip1), an inhibitor of G1 phase cyclin-dependent kinase cyclin complexes. We have recently demonstrated that ANG II-mediated expression of p27(Kip1) and induction of cellular hypertrophy depend on the generation of reactive oxygen species (ROS). The effects of ROS are mediated by stimulation of mitogen-activated protein (MAP) kinases. p44/42 MAP kinase directly phosphorylates p27(Kip1) at serine-threonine residues and increases thereby its half-life time. AT2-receptor activation has been implicated in apoptosis and/or cell differentiation. Recent studies, however, revealed a more indirect role of hypoxia in the antiproliferative effects of ANG II transduced through AT2 receptors. We found that SM-20 is down-regulated in ANG II-stimulated PC12 cells that express only AT2 receptors. It turned out that SM20 is the rat homologue of a dioxygenase that regulates hypoxia-inducible factor 1 (HIF-1). ANG II induces HIF-1alpha by a posttranscriptional mechanism suggesting that SM20 down-regulation leads to stabilization of HIF-1. Thus, ANG II-induced ROS generation plays a pivotal role in several pathophysiological situations, leading to renal growth regulation and remodeling after injury.