Angiotensin II-induced upregulation of MAP kinase phosphatase-3 mRNA levels mediates endothelial cell apotosis

Angiotensin II (Ang II) is central to the pathobiology of atherosclerosis. In endothelial cells (EC), Ang II induces apoptosis. The MAP kinase ERK1/2 plays a key role in regulating cell survival. We therefore investigated the effect of Ang II on ERK1/2. Incubation of EC with Ang II led to the dephosphorylation of ERK1/2 (43 % of control). To characterize the phosphatase involved, we investigated the effect of Ang II on MAP kinase phosphatase expression. Ang II induced MAP kinase phosphatase-3 (MKP-3) mRNA levels to about 2-fold, whereas MKP-1 expression was not affected. Transfection with a dominant negative MKP-3 construct (dnMKP-3mt) prevented the Ang II-induced ERK1/2 dephosphorylation and apoptosis in EC (p < 0.001). ERK1/2 inactivation has been shown to result in the dephosphorylation and proteasomal degradation of the antiapoptotic protein Bcl-2. Ang II induced the degradation of Bcl-2 wild type, whereas the dephosphorylation-resistant Bcl-2 construct mimicking phosphorylation by ERK1/2 was resistant to Ang II stimulation. These results indicate that Ang II-induced apoptosis signaling in human EC is mediated via MKP-3-dependent dephosphorylation of ERK1/2, which in turn leads to the degradation of Bcl-2. Received: 11 April 2001, Returned for revision: 9 May 2001, Revision received: 30 May 2001, Accepted: 1 June 2001     

Impaired angiotensin II-extracellular signal-regulated kinase signaling in failing human ventricular myocytes

Angiotensin II was reported to induce insulin-like growth factor-I and endothelin-1 gene expression and peptide release by ventricular cardiomyocytes. However, the progression from cardiac hypertrophy to failure in humans is characterized by a reduced myocyte expression of insulin-like growth factor-I and endothelin-1, notwithstanding the enhanced cardiac generation of angiotensin II. In the present study we investigated the functional status of the signaling pathways responsible for angiotensin II-induced endothelin-1 and insulin-like growth factor-I formation in human ventricular myocytes isolated from patients with dilated (n = 19) or ischemic (n = 14) cardiomyopathy and nonfailing donor hearts (n = 6).In human nonfailing ventricular myocytes, angiotensin II (100 nmol/l) induced insulin-like growth factor-I and endothelin-1 gene expression, and peptide release was mediated by extracellular signal-regulated kinase activation and inhibited by extracellular signal-regulated kinase antagonism (PD98059, 30 micromol/l), endothelin-1 formation being partially reduced also by c-Jun N-terminal kinase inhibition (SP600125, 10 micromol/l); insulin-like growth factor-I and endothelin-1 formations were unaffected by the inhibition of p38 mitogen-activated protein kinase (SB203580, 10 micromol/l) and Janus tyrosine kinase 2 (AG490, 10 micromol/l). In failing myocytes, angiotensin II failed to induce insulin-like growth factor-I and endothelin-1 formation; angiotensin II-induced extracellular signal-regulated kinase activation was significantly impaired (-88% vs. controls) although c-Jun NH2-terminal kinase activation was preserved. The impaired extracellular signal-regulated kinase phosphorylation in failing myocytes was associated with increased myocyte levels of mitogen-activated protein kinase phosphatases.Therefore, the altered growth factor production in failing myocytes is associated with a significant derangement in intracellular signaling.     

Angiotensin II-induced apoptosis in rat cardiomyocyte culture: a possible role of AT1 and AT2 receptors

OBJECTIVES : To investigate the mechanism of angiotensin II-induced apoptosis in cultured cardiomyocytes by determining which receptor subtype is involved, and what is the relationship between intracellular Ca2+ changes and apoptosis. DESIGN AND METHODS : Neonatal rat cardiomyocytes were pretreated with either the AT1 antagonist irbesartan or the AT2 antagonist PD123319 before exposure to angiotensin II. Apoptosis was evaluated using morphological technique, staining nuclei by Feulgen and Hoechst methods followed by image analysis and by in situ terminal deoxynucleotidyl transferase nick-end (TUNEL) labelling. TUNEL-positive cardiocytes were distinguished from other cells by double staining with alpha-sarcomeric actin. Intracellular Ca2+ changes were assessed by indo-1 fluorescence microscopy, and the effect of Ca2+ on angiotensin II-induced apoptosis was tested using the calcium channel blocker verapamil. RESULTS : Exposure to angiotensin II (10 nmol/l) resulted in cell replication and a three-fold increase in programmed cell death (P < 0.05). Pretreatment with either irbesartan (an AT1receptor antagonist, 100 nmol/l) or PD123319 (an AT2 receptor antagonist, 1 micromol/l) prevented the angiotensin II-induced apoptosis, indicating the presence of both AT1 and AT2receptors on cardiomyocytes. Exposure of myocytes to angiotensin II caused an immediate and dose-dependent increase in the concentration of intracellular free Ca2+ that lasted 40-60 s. The effect was sustained in a Ca2+ free medium. Pretreatment of cells with irbesartan (100 nmol/l) and PD123319 (10 micromol/l) blocked Ca2+ elevation. Pretreatment with verapamil (10 micromol/l) prevented angiotensin II-induced apoptosis. CONCLUSIONS : Angiotensin II-induced apoptosis in rat cardiomyocytes is mediated through activation of both AT1 and AT2 receptors. The apoptotic mechanism is not related to the immediate angiotensin II-induced Ca2+ rise from intracellular stores. However, it is accompanied by cardiomyocyte proliferation and requires Ca2+ influx through L-type channel activity.     

Suppressor of cytokine signaling-3 Provides a novel interface in the cross-talk between angiotensin II and insulin signaling systems

Angiotensin II inhibits insulin-induced activation of phosphatidylinositol 3-kinase through a mechanism, at least in part, dependent on serine phosphorylation of the insulin receptor and insulin receptor substrates (IRS)-1/2. Recent evidence shows that suppressor of cytokine signaling-3 (SOCS-3) is induced by insulin and angiotensin II and participates in the negative control of further stimulation of each of these signaling systems independently. In the present study, we evaluated the interaction of angiotensin II-induced SOCS-3 with the insulin signaling pathway in the heart of living rats. A single iv dose of angiotensin II promotes a significant increase of SOCS-3 in heart, an effect that lasts up to 180 min. Once induced, SOCS-3 interacts with the insulin receptor, JAK-2, IRS-1, and IRS-2. The inhibition of SOCS-3 expression by a phosphorthioate-modified antisense oligonucleotide partially restores angiotensin II-induced inhibition of insulin-induced insulin receptor, IRS-1 and IRS-2 tyrosine phosphorylation, and IRS-1 and IRS-2 association with p85-phosphatidylinositol 3-kinase and [Ser473] phosphorylation of Akt. Moreover, the inhibition of SOCS-3 expression partially reverses angiotensin II-induced inhibition of insulin-stimulated glucose transporter-4 translocation to the cell membrane. These results are reproduced in isolated cardiomyocytes. Thus, SOCS-3 participates, as a late event, in the negative cross-talk between angiotensin II and insulin, producing an inhibitory effect on insulin-induced glucose transporter-4 translocation.     

Involvement of reactive oxygen species in angiotensin II-induced endothelin-1 gene expression in rat cardiac fibroblasts

OBJECTIVES: The aim of this study was to investigate the effects of angiotensin II (Ang II) on fibroblast proliferation and endothelin-1 (ET-1) gene induction, focusing especially on reactive oxygen species (ROS)-mediated signaling in cardiac fibroblasts. BACKGROUND: Angiotensin II increases ET-1 expression, which plays an important role in Ang II-induced fibroblast proliferation. Angiotensin II also stimulates ROS generation in cardiac fibroblasts. However, whether ROS are involved in Ang II-induced proliferation and ET-1 expression remains unknown. METHODS: Cultured neonatal rat cardiac fibroblasts were stimulated with Ang II, and then [(3)H]thymidine incorporation and the ET-1 gene expression were examined. We also examined the effects of antioxidants on Ang II-induced proliferation and mitogen-activated protein kinase (MAPK) phosphorylation to elucidate the redox-sensitive pathway in fibroblast proliferation and ET-1 gene expression. RESULTS: Both AT(1) receptor antagonist (losartan) and ET(A) receptor antagonist (BQ485) inhibited Ang II-increased DNA synthesis. Endothelin-1 gene was induced with Ang II as revealed by Northern blotting and promoter activity assay. Angiotensin II increased intracellular ROS levels, which were inhibited with losartan and antioxidants. Antioxidants further suppressed Ang II-induced ET-1 gene expression, DNA synthesis, and MAPK phosphorylation. PD98059, but not SB203580, fully inhibited Ang II-induced ET-1 expression. Truncation and mutational analysis of the ET-1 gene promoter showed that AP-1 binding site was an important cis-element in Ang II-induced ET-1 gene expression. CONCLUSIONS: Our data suggest that ROS are involved in Ang II-induced proliferation and ET-1 gene expression. Our findings imply that the combination of AT(I) and ET(A) receptor antagonists plus antioxidants may be beneficial in preventing the formation of excessive cardiac fibrosis.     

Angiotensin II-induced protein phosphorylation in the hypertrophic heart of the Dahl rat.

Angiotensin II-induced phosphorylation of proteins was examined in isolated myocytes from hearts of Dahl rats. A high salt diet induced cardiac hypertrophy in Dahl salt-sensitive rats. Angiotensin II-induced phosphorylation of a 42-kd protein (pp42) was detected by two-dimensional electrophoresis in hypertrophic but not normal ventricular myocytes. Angiotensin II stimulation was time-dependent, with a peak effect at 30 minutes. The half-maximal and maximal concentrations of angiotensin II that stimulated pp42 phosphorylation were 1 and 10 nM, respectively. Phosphorylation of pp42 was a function of cardiac hypertrophy. Phorbol 12-myristate 13-acetate-induced phosphorylation of pp42 indicates the possibility of an association between protein kinase C and the signal transduction pathway of angiotensin II-induced pp42 phosphorylation. Ionomycin and A23187 (both at 1 microM) did not stimulate phosphorylation of pp42. Angiotensin II produced a small increase in the synthesis of myocyte proteins in both normal and hypertrophic cells as shown by [35S]methionine incorporation. However, this increase could not account for the increase in the phosphate content of pp42. This protein was not an isoform of actin nor was it of platelet origin. These results raise the possibility that angiotensin II may play a role in the activation of factors in hypertrophic myocytes; however, further study is required to define a link between phosphorylation of pp42 and the hypertrophic process.     

The regulation of MAPKs in Y1 mouse adrenocortical tumor cells

The regulation of the MAPKs, Erk(1) and Erk(2), and the MAPK kinase, Mek, were examined in the Y1 mouse adrenocortical tumor cell line and in the protein kinase A-defective mutant, Kin-8. ACTH and basic fibroblast growth factor each increased Mek phosphorylation and stimulated Mek activity in both cell lines and also activated the Erks at concentrations that paralleled their effects on Mek. The specific Mek inhibitor, PD98059, blocked the activation of the Erks by ACTH and basic fibroblast growth factor, indicating that Mek is the upstream activator of Erk. PD98059 did not block the phosphorylation of Mek, as might have been expected from previous studies; instead PD98059 inhibited the activity of the activated enzyme. In ACTH-stimulated, mutant Kin-8 cells, PD98059 paradoxically increased the amount of phosphorylated Mek, while preventing the activation of Erk. These results are interpreted as reflecting the loss of a protein kinase A-mediated inhibitory influence on Mek phosphorylation and activation.     

Angiotensin II increases expression of IP-10 and the renin-angiotensin system in endothelial cells

Angiotensin II promotes vascular inflammation, which plays important roles in vascular injury. In this study, we found that angiotensin II-stimulated human endothelial cells increased the release of a CXC chemokine, IP-10, according to an antibody array. IP-10 expression was higher in the endothelium of coronary blood vessels in mice infused with angiotensin II than in control. Quantitative real-time PCR analysis revealed that angiotensin II significantly increased IP-10 mRNA expression compared to control. Pretreatment with valsartan, but not with PD123319, blocked angiotensin II-induced IP-10 mRNA expression. IP-10 levels in conditioned media detected by ELISA increased in response to angiotensin II compared to control, which was blocked by the pretreatment with valsartan. These data indicate that angiotensin II stimulates IP-10 production from endothelial cells via angiotensin II type 1 receptors. In endothelial cells, IP-10 significantly increased mRNA expression of renin, angiotensin-converting enzyme, and angiotensinogen. IP-10 also increased angiotensin II levels in conditioned media compared to control. Angiotensin II significantly increased mRNA expression of renin, angiotensin converting enzyme and angiotensinogen, which was blocked by neutralization of IP-10 with antibody in endothelial cells. IP-10 neutralization with antibody blocked angiotensin II-induced apoptosis and cell senescence in endothelial cells. These data indicate that IP-10 is involved not only in leukocyte-endothelial interaction but also in the circuit of endothelial renin-angiotensin system activation that potentially promotes atherosclerosis.     

Angiotensin II stimulates endothelial NO synthase phosphorylation in thoracic aorta of mice with abdominal aortic banding via type 2 receptor

Abdominal aortic banding in mice induces upregulation of angiotensin II (Ang II) type 2 (AT2) receptors in the pressure-overloaded thoracic aorta. To clarify mechanisms underlying the vascular AT2 receptor-dependent NO production, we measured aortic levels of endothelial NO synthase (eNOS), eNOS phosphorylated at Ser633 and Ser1177, protein kinase B (Akt), and Akt phosphorylated at Ser473 in thoracic aortas of mice after banding. Total eNOS, both forms of phosphorylated eNOS, Akt, and phosphorylated Akt levels, as well as cGMP contents, were significantly increased 4 days after banding. The administration of PD123319 (an AT2 receptor antagonist) or icatibant (a bradykinin B2 receptor antagonist) abolished the banding-induced upregulation of both forms of phosphorylated eNOS, as well as elevation of cGMP, but did not affect the upregulation of eNOS, Akt, and phosphorylated Akt. In the in vitro experiments using aortic rings prepared from banded mice, Ang II produced significant increases in both forms of phosphorylated eNOS, as well as cGMP, and these effects were blocked by PD123319 and icatibant. Ang II-induced eNOS phosphorylation and cGMP elevation in aortic rings were inhibited by protein kinase A (PKA) inhibitors H89 and KT5720 but not by phosphatidylinositol 3-kinase inhibitors wortmannin and LY24002. The contractile response to Ang II was attenuated in aortic rings from banded mice via AT2 receptor, and this attenuation was blocked by PKA inhibitors. These results suggest that the activation of AT2 receptor by Ang II induces phosphorylation of eNOS at Ser633 and Ser1177 via a PKA-mediated signaling pathway, resulting in sustained activation of eNOS.     

Contribution of PI 3-kinase isoforms to angiotensin II- and alpha-adrenoceptor-mediated signalling pathways in cardiomyocytes

OBJECTIVE: Angiotensin II stimulation increases the formation of reactive oxygen species (ROS), the phosphorylation of p38 mitogen-activated protein kinase (MAPK), and the expression of transforming growth factor beta (TGFbeta) in adult cardiomyocytes. The aim of this study was to determine the involvement of PI 3-kinase and to specify the participation of different isoforms in the angiotensin II-induced formation of ROS in comparison to the hypertrophic pathway triggered by alpha-adrenoceptor stimulation. METHODS: Freshly isolated myocytes were used to examine formation of ROS via H(2)DCF fluorescence. p38 MAPK phosphorylation, p70(S6)-kinase phosphorylation, PI 3-kinase, and TGFbeta expression were measured by Western blotting. Sense and antisense oligonucleotides were used to down-regulate diverse PI 3-kinase isoforms. Hypertrophy was measured by (14)C-phenylalanine incorporation and cell volume. RESULTS: Inhibition of PI 3-kinase by Ly294002 or wortmannin, two inhibitors, decreased formation of ROS, phosphorylation of p38 MAPK, and TGFbeta expression. Down-regulation of the p110beta isoform by antisense oligonucleotides inhibited the angiotensin II-induced signalling pathway but not the alpha-adrenoceptor-mediated hypertrophic growth of cardiomyocytes. In contrast, down-regulation of the p110alpha isoform decreased the alpha-adrenoceptor-mediated hypertrophic growth of cardiomyocytes but did not affect the angiotensin II-mediated signalling pathway. CONCLUSION: Thus, our study identifies an involvement of PI 3-kinase in the angiotensin II-induced formation of ROS and provides a biochemical basis for ligand-specific responses for angiotensin II and alpha-adrenoceptor stimulation as relates to hypertrophy.     

Mitogen-activated protein kinase-activated protein kinase 2 in angiotensin II-induced inflammation and hypertension: regulation of oxidative stress

Vascular oxidative stress and inflammation play an important role in angiotensin II-induced hypertension, and mitogen-activated protein kinases participate in these processes. We questioned whether mitogen-activated protein kinase-activated protein kinase 2 (MK2), a downstream target of p38 mitogen-activated protein kinase, is involved in angiotensin II-induced vascular responses. In vivo experiments were performed in wild-type and Mk2 knockout mice infused intravenously with angiotensin II. Angiotensin II induced a 30 mm Hg increase in mean blood pressure in wild-type that was delayed in Mk2 knockout mice. Angiotensin II increased superoxide production and vascular cell adhesion molecule-1 in blood vessels of wild-type but not in Mk2 knockout mice. Mk2 knockdown by small interfering RNA in mouse mesenteric vascular smooth muscle cells caused a 42% reduction in MK2 protein and blunted the angiotensin II-induced 40% increase of MK2 expression. Mk2 knockdown blunted angiotensin II-induced doubling of intracellular adhesion molecule-1 expression, 2.4-fold increase of nuclear p65, and 1.4-fold increase in Ets-1. Mk2 knockdown abrogated the angiotensin II-induced 4.7-fold and 1.3-fold increase of monocyte chemoattractant protein-1 mRNA and protein. Angiotensin II enhanced reactive oxygen species levels (by 29%) and nicotinamide adenine dinucleotide phosphate oxidase activity (by 48%), both abolished by Mk2 knockdown. Reduction of MK2 blocked angiotensin II-induced p47phox translocation to the membrane, associated with a 53% enhanced catalase expression. Angiotensin II-induced increase of MK2 was prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor Nox2ds-tat. Mk2 small interfering RNA prevented the angiotensin II-induced 30% increase of proliferation. In conclusion, MK2 plays a critical role in angiotensin II signaling, leading to hypertension, oxidative stress via activation of p47phox and inhibition of antioxidants, and vascular inflammation and proliferation.     

Regulation of sarcolemmal Na(+)/H(+) exchanger activity by angiotensin II in adult rat ventricular myocytes: opposing actions via AT(1) versus AT(2) receptors

Increased sarcolemmal Na(+)/H(+) exchanger activity has been implicated as a mediator of the cardiac actions of angiotensin II. We studied the receptor subtypes and signaling pathways involved in the regulation of sarcolemmal Na(+)/H(+) exchanger activity by angiotensin II in adult rat ventricular myocytes. Cells were loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1, and acid efflux rates estimated during recovery from intracellular acidosis were used to quantify exchanger activity. Sarcolemmal Na(+)/H(+) exchanger activity was not affected by angiotensin II alone but was increased by angiotensin II plus PD123319 (AT(2) antagonist). In contrast, angiotensin II plus losartan (AT(1) antagonist) or CGP42112A (AT(2) agonist) did not affect exchanger activity. The increase in Na(+)/H(+) exchanger activity induced by angiotensin II plus PD123319 was blocked by losartan, PD98059 (extracellular signal-regulated kinase inhibitor), GF109203X (protein kinase C inhibitor), and tyrphostin AG1478 (epidermal growth factor receptor kinase inhibitor). Extracellular signal-regulated kinase phosphorylation and activity, measured by immunoblot analysis and an immune-complex kinase assay, respectively, were increased significantly by angiotensin II plus PD123319; these increases were blocked by losartan and PD98059. The increase in extracellular signal-regulated kinase phosphorylation induced by angiotensin II plus PD123319 was blocked also by GF109203X and tyrphostin AG1478. These data show that AT(1) stimulation increases sarcolemmal Na(+)/H(+) exchanger activity in adult rat ventricular myocytes and that this response requires extracellular signal-regulated kinase activation through a protein kinase C- and epidermal growth factor receptor-mediated mechanism. The positive effect of AT(1) stimulation on Na(+)/H(+) exchanger activity is counteracted by simultaneous AT(2) stimulation through a mechanism that does not involve direct inhibition of the exchanger or attenuation of extracellular signal-regulated kinase activation.