II-induced Production Research Articles

Unique mode of binding between angiotensin II type 1 receptor and its blockers

ABSTRACTBACKGROUND: We hypothesized that the 5-oxo-1,2,4-oxadiazole moiety of azilsartan (AZL), which represents a small difference in the molecular structures of AZL and candesartan (CAN) [angiotensin II type 1 receptor (AT1R) blockers], may be responsible for the molecular effects of AZL. METHODS: We examined the binding affinities of AZL and CAN to AT1R, along with their ability to block receptor activity. A competition binding study, inositol phosphate (IP) production assay and extracellular signal-regulated kinase (ERK) assay were performed using wild-type (WT) and mutants AT1R-transfected cells. RESULTS: The binding affinities of CAN and AZL were reduced by > 5-fold for Y35F, W84F, R167K, K199Q and I288A compared with WT. In addition, AZL showed a > 5-fold reduction in its binding affinity to V108A. CAN and AZL exhibited > 20-fold and > 100-fold reductions in binding affinity to R167K, respectively. The loss of binding affinity of AZL to R167K was greater than that of CAN. CAN-7H exhibited a > 10-fold reduction in binding affinity to R167K compared with CAN. On the other hand, the binding affinity of AZL-7H to R167K was comparable to that of AZL. While 10-6M CAN and CAN-7H partly blocked Ang II-induced IP production in R167K, 10-6M AZL and AZL-7H did not. In addition, 10-6M CAN, but not 10-6M AZL, partly blocked Ang II-induced ERK activation in R167K. CONCLUSIONS: The interaction between 5-oxo-1,2,4-oxadiazole in AZL and Arg167 in the AT1R appears to be more important than the interaction between the tetrazole ring in CAN and Arg167.

Alpha-Asarone Protects Endothelial Cells from Injury by Angiotensin II.

α-Asarone is the major therapeutical constituent of Acorus tatarinowii Schott. In this study, the potential protective effects of α-asarone against endothelial cell injury induced by angiotensin II were investigated in vitro. The EA.hy926 cell line derived from human umbilical vein endothelial cells was pretreated with α-asarone (10, 50, 100 µmol/L) for 1 h, followed by coincubation with Ang II (0.1 µmol/L) for 24 h. Intracellular nitric oxide (NO) and reactive oxygen species (ROS) were detected by fluorescent dyes, and phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 was determined by Western blotting. α-Asarone dose-dependently mitigated the Ang II-induced intracellular NO reduction (P < 0.01 versus model) and ROS production (P < 0.01 versus model). Furthermore, eNOS phosphorylation (Ser1177) by acetylcholine was significantly inhibited by Ang II, while pretreatment for 1 h with α-asarone partially prevented this effect (P < 0.05 versus model). Additionally, cell viability determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (105~114.5% versus control, P > 0.05) was not affected after 24 h of incubation with α-asarone at 1–100 µmol/L. Therefore, α-asarone protects against Ang II-mediated damage of endothelial cells and may be developed to prevent injury to cardiovascular tissues.

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.

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 (&gt;80%) and leucine incorporation (&gt;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.

Abstract 1374: A Novel eNOS-Independent Protective Action of Statin against Angiotensin II-Induced Atrial Remodeling Via Attenuating Rac-1-Mediated Oxidative Stress

Activation of the renin-angiotensin system exacerbates atrial remodeling, leading to atrial fibrillation (AF) and thrombosis, especially in a condition with decreased nitric oxide bioavailability. Although antiarrhythmic and anticoagulation agents are used under these pathological conditions, these drugs are not able to ameliorate atrial remodeling. Recently, it has been reported that statins reduce the incidence of AF through attenuating atrial remodeling; however, the mechanisms have not been completely elucidated. This study was designed to determine the beneficial effect of pitavastatin (Pit) against angiotensin II (Ang II)-induced atrial remodeling and to elucidate its mechanism. eNOS knockout mice were sham-operated or infused with Ang II by an osmotic mini-pump for 2 weeks, and Ang II-infused mice were divided into 3 treatment groups: Pit, tempol, a free radical scavenger, or a vehicle. Echocardiography and electrocardiography showed that Ang II infusion increased left atrial volume and caused a high incidence of AF, whereas Pit and tempol treatment prevented Ang II-induced left atrial enlargement and AF. Histological analysis showed that acceleration of Ang II-induced interstitial fibrosis, perivascular fibrosis and cardiomyocyte hypertrophy in the atrium were all attenuated by Pit and tempol treatment. Immunohistochemistry showed that Ang II down-regulated thrombomodulin and up-regulated tissue factor and plasminogen activator inhibitor-1 in the left atrium and that Pit and tempol treatment corrected the Ang II-induced thrombogenic condition. Moreover, Pit and tempol reduced Ang II-induced atrial superoxide production, detected by dihydroethidium staining, and atrial TGF- β 1 expression and Smad 2/3 phosphorylation. Activity of Rac-1-GTPase involved in the activation of NADPH oxidase in the atrium was attenuated by Pit but not by tempol. Pit exerts eNOS-independent protective actions against Ang II-induced atrial structural and electrical remodeling with enhanced thrombogenicity through suppressing Rac-1-mediated oxidative stress, leading to suppression of the TGF- β 1/Smad pathway.

Abstract 1423: Pitavastatin Ameliorates Angiotensin II-induced Cardiorenal Insufficiency In eNOS Knockout Mice Through Attenuating Rac-1-mediated Oxidative Stress

Background: It is well known that angiotensin II (Ang II) generates reactive oxygen species through NADPH oxidase activation, leading to promotion of cardiorenal diseases, especially in a condition with decreased nitric oxide (NO) bioavailability. In previous scientific sessions, we have shown that pitavastatin, an HMGCoA reductase inhibitor, ameliorates Ang II-induced cardiorenal damage in eNOS knockout (KO) mice through inhibition of the TGF-β-Smad2/3 pathway. However, the mechanism underlying eNOS-independent protective actions by pitavastatin has not been fully elucidated. Methods and Results: eNOS KO mice at 10 weeks of age were infused with Ang II (2.0 mg/kg/day) by an osmotic mini-pump for 2 weeks and they were divided into 3 treatment groups: pitavastatin (0.2 mg/kg/day), tempol, a free radical scavenger, (1.5 mg/kg/day), or a vehicle. Pitavastatin as well as tempol ameliorated Ang II-induced left ventricular hypertrophy with interstitial and pericoronary artery fibrosis and ameliorated diastolic dysfunction and they attenuated Ang II-induced glomerular damage, such as low glomerular filtration rate, increased albuminuria and PAS-positive glomerular depositions compared to those in vehicle-treated mice. Ang II-induced high mortality rate in eNOS KO mice was restored by pitavastatin as well as tempol treatment. Moreover, in eNOSKO mice, Ang II-induced cardiorenal superoxide production, detected with dihydroethidium staining, and Ang II-induced activity of rac-1-GTPase, a small G-protein involved in the activation of NADPH oxidase, were attenuated by pitavastatin treatment. Conclusions: Pitavastatin exerts an eNOS-independent protective action against Ang II-induced cardiorenal insufficiency through attenuating rac-1-GTPase-mediated oxidative stress. Our findings suggest that pitavastatin treatment is useful for caridorenal protection in patients with impaired NO bioavailability.

RETRACTED: Transcriptional regulation of the AT1 receptor gene in immortalized human trophoblast cells

Studies investigating the mechanisms that govern the expression of the human angiotensin II (Ang II) type 1 receptor (hAT1R) gene have progressed slowly due to the lack of human cell lines that express the AT1R. Recently, however, an immortalized human trophoblast cell line (HTR-8/SVNeo) was demonstrated to respond to Ang II. Therefore, we utilized this cell line to characterize the AT1R expressed on the cell surface and to investigate the mechanisms by which the hAT1R gene is regulated in these cells. HTR-8/SVNeo cells were shown to express functional high affinity AT1Rs having a Bmax value of 114+/-11 fmol/mg protein and a Kd value of 0.14+/-0.1 nM. Additionally, Ang II-induced IP3 production was mediated via the AT1R. Deletional analysis of the hAT1R promoter localized a major basal regulatory sequence within the -105 to -79 bp region, relative to the transcription start site, in HTR-8/SVNeo cells. Electrophoretic mobility shift assay (EMSA) and Chromatin Immunoprecipitation (ChIP) assay demonstrated that the transcription factors, Sp1 and Sp3, interact with this region of the hAT1R promoter in vitro and in vivo. Taken together, our data demonstrate that HTR-8/SVNeo cells express functional AT1Rs and that basal level expression of this gene is regulated, in part, by Sp1 and Sp3 in this cell line.

P2790 Mechanisms for angiotensin in II-induced superoxide production in conduit arteries in patients with cardiovascular disease

P2790 Mechanisms for angiotensin in II-induced superoxide production in conduit arteries in patients with cardiovascular disease

P38 MAP Kinase Regulates Vascular Smooth Muscle Cell (VSMC) Collagen Synthesis by Ang II in Shr But not in Wky.

P81 Vascular remodeling in hypertension is associated with cell growth and increased deposition of extracellular matrix components, particularly collagen. Molecular mechanisms underlying these processes are unclear, but MAP kinases, particularly ERK1/2 and p38, may be important. We studied the role of ERK1/2 and p38 in vascular growth effects mediated by Ang II in SHR. Cultured mesenteric VSMC from WKY and SHR were used. Phosphorylation of ERK1/2 and p38 was assessed by Western blot using phospho-specific antibodies. c-fos mRNA expression was determined by RT-PCR. Ang II-stimulated DNA, protein and proline synthesis were assessed as indices of VSMC proliferation, hypertrophy and collagen production and were determined by measuring incorporation of 3 H-thymidine, 3 H-leucine and 3 H-proline respectively. Ang II dose-and time-dependently increased ERK1/2 and p38 phosphorylation. Responses were increased (p c-fos mRNA expression in SHR (173 ±12 % of control). These actions were inhibited by PD98059, but not by SB202190. Ang II stimulated 3 H-thymidine, 3 H-leucine and 3 H-proline incorporation. Responses were enhanced (p 1 receptor antagonist, but not by PD123319, AT 2 receptor blocker. Thus 1)Ang II-mediated activation of vascular ERK1/2 and p38 is increased in SHR, 2) augmented c-fos and growth responses are generated primarily via ERK1/2, 3) p38 influences Ang II-induced collagen production in SHR but not in WKY. In conclusion, these results indicate differential roles of ERK1/2 and p38 in AT 1 -stimulated VSMC growth and collagen production, which may contribute to vascular remodeling in genetic hypertension. (Supported by a research grant from Bristol-Myers Squibb/Sanofi)

Increased Generation of Vascular Reactive Oxygen Species by Ang II Is Mediated Via Src-Dependent Pathways in Essential Hypertension.

42 We tested the hypothesis that augmented Ang II-induced vascular smooth muscle cell (VSMC) growth in human hypertension is mediated via Src-dependent pathways that generate reactive oxygen species (ROS). VSMCs from arteries of normotensive and hypertensive subjects were studied. Production of ROS was measured by fluorescence digital imaging using dichlorofluorescin diacetate (6 μM). The roles of Src and NADH/NADPH oxidase were assessed with the specific inhibitors, PP2 (10 μM) and diphenylene iodinium (DPI) (10 μM) respectively. c-Src phosphorylation was determined by western blot and kinase activity was assessed by measuring enolase phosphorylation. Ang II increased DCFDA fluorescence. This effect was inhibited by catalase, indicating that the signal was derived predominantly from H2O2. Ang II increased H2O2production within 40 minutes. Responses were greater (p<0.05) in cells from hypertensive patients (Emax=82±nM) than normotensive subjects (Emax= 67±nM). DPI and PP2, but not PP3 (inactive analogue) attenuated (p<0.05) Ang II-induced H2O2 production. PP2 effects were greater in cells from hypertensive patients (delta H2O2, 28±5nM) vs controls (delta H2O2, 16±2nM). Ang II increased c-Src phosphorylation and activity, with responses 3-4 fold higher in hypertensives. DPI and PP2 (p<0.01) attenuated Ang II-induced DNA and protein synthesis, as measured by 3H-thymidine and 3H-leucine incorporation respectively. Growth responses in hypertensive patients were normalized by PP2. In VSMCs from hypertensive patients, Ang II-induced generation of ROS and growth are augmented. These effects are mediated, in part, by Src-dependent, NADH/NADPH oxidase-dependent cascades. Thus increased Src activity may be an upstream modulator of redox-sensitive pathways that regulate vascular growth and remodeling in essential hypertension.

P38 MAP Kinase Regulates Vascular Smooth Muscle Cell (VSMC) Collagen Synthesis by Ang II in Shr But not in Wky.

P81 Vascular remodeling in hypertension is associated with cell growth and increased deposition of extracellular matrix components, particularly collagen. Molecular mechanisms underlying these processes are unclear, but MAP kinases, particularly ERK1/2 and p38, may be important. We studied the role of ERK1/2 and p38 in vascular growth effects mediated by Ang II in SHR. Cultured mesenteric VSMC from WKY and SHR were used. Phosphorylation of ERK1/2 and p38 was assessed by Western blot using phospho-specific antibodies. c-fos mRNA expression was determined by RT-PCR. Ang II-stimulated DNA, protein and proline synthesis were assessed as indices of VSMC proliferation, hypertrophy and collagen production and were determined by measuring incorporation of 3 H-thymidine, 3 H-leucine and 3 H-proline respectively. Ang II dose-and time-dependently increased ERK1/2 and p38 phosphorylation. Responses were increased (p&lt;0.01) 3-4 fold in SHR. PD98059, selective MEK1/2 inhibitor, and SB202190, selective p38 inhibitor, abolished Ang II-induced ERK1/2 and p38 activation respectively. Ang II increased (p&lt;0.05) c-fos mRNA expression in SHR (173 ±12 % of control). These actions were inhibited by PD98059, but not by SB202190. Ang II stimulated 3 H-thymidine, 3 H-leucine and 3 H-proline incorporation. Responses were enhanced (p&lt;0.02) 2-3 fold in SHR. PD98059 attenuated Ang II-induced growth effects and normalized responses in SHR. SB212190 did not alter Ang II-elicited DNA synthesis, but reduced (P&lt;0.05) collagen production in SHR. Effects were inhibited by irbesartan, AT 1 receptor antagonist, but not by PD123319, AT 2 receptor blocker. Thus 1)Ang II-mediated activation of vascular ERK1/2 and p38 is increased in SHR, 2) augmented c-fos and growth responses are generated primarily via ERK1/2, 3) p38 influences Ang II-induced collagen production in SHR but not in WKY. In conclusion, these results indicate differential roles of ERK1/2 and p38 in AT 1 -stimulated VSMC growth and collagen production, which may contribute to vascular remodeling in genetic hypertension. (Supported by a research grant from Bristol-Myers Squibb/Sanofi)