Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells.

Angiotensin II, a hypertrophic/anti-apoptotic hormone, utilizes reactive oxygen species (ROS) as growth-related signaling molecules in vascular smooth muscle cells (VSMCs). Recently, the cell survival protein kinase Akt/protein kinase B (PKB) was proposed to be involved in protein synthesis. Here we show that angiotensin II causes rapid phosphorylation of Akt/PKB (6- +/- 0.4-fold increase). Exogenous H(2)O(2) (50-200 microM) also stimulates Akt/PKB phosphorylation (maximal 8- +/- 0.2-fold increase), suggesting that Akt/PKB activation is redox-sensitive. Both angiotensin II and H(2)O(2) stimulation of Akt/PKB are abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitors wortmannin and LY294002 (2(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), suggesting that PI3-K is an upstream mediator of Akt/PKB activation in VSMCs. Furthermore, diphenylene iodonium, an inhibitor of flavin-containing oxidases, or overexpression of catalase to block angiotensin II-induced intracellular H(2)O(2) production significantly inhibits angiotensin II-induced Akt/PKB phosphorylation, indicating a role for ROS in agonist-induced Akt/PKB activation. In VSMCs infected with dominant-negative Akt/PKB, angiotensin II-stimulated [(3)H]leucine incorporation is attenuated. Thus, our studies indicate that Akt/PKB is part of the remarkable spectrum of angiotensin II signaling pathways and provide insight into the highly organized signaling mechanisms coordinated by ROS, which mediate the hypertrophic response to angiotensin II in VSMCs.     

Angiotensin II regulates phosphoinositide 3 kinase/Akt cascade via a negative crosstalk between AT1 and AT2 receptors in skin fibroblasts of human hypertrophic scars

Angiotensin II (Ang II) stimulation has been shown to regulate proliferation of skin fibroblasts and production of extracellular matrix, which are very important process in skin wound healing and scarring; however, the signaling pathways involved in this process, especially in humans, are less explored. In the present study, we used skin fibroblasts of human hypertrophic scar, which expressed both AT1 and AT2 receptors, and observed that Ang II increased Akt phosphorylation and phosphoinositide 3 kinase (PI 3-K) activity. In addition, the Ang II-induced Akt phosphorylation was blocked by wortmannin, a PI 3-K inhibitor. This Ang II-activated PI 3-K/Akt cascade was markedly inhibited by valsartan, an AT(1) receptor-specific blocker, whereas it was enhanced by PD123319, an AT(2) receptor antagonist. On the other hand, the Ang II- or EGF-induced activation of PI 3-K/Akt was strongly attenuated by AG1478, an inhibitor of epidermal growth factor (EGF) receptor kinase. Moreover, Ang II stimulated tyrosine phosphorylation of EGF receptor and p85alpha subunit of PI 3-K accompanied by an increase in their association, which was inhibited by valsartan, and enhanced by PD123319. The Ang II-induced transactivation of EGF receptor resulted in activation of extracellular signal-regulated kinase (ERK) that was also inhibited by valsartan, and enhanced by PD123319. Taken together, our results showed that AT(1) receptor-mediated activation of PI 3-K/Akt cascades occurs at least partially via the transactivation of EGF receptor, which is under a negative control by AT(2) receptor in hypertrophic scar fibroblasts. These findings contribute to understanding the molecular mechanism of human hypertrophic scar formation.     

Early endosomal antigen 1 (EEA1) is an obligate scaffold for angiotensin II-induced, PKC-alpha-dependent Akt activation in endosomes

Akt/protein kinase B (PKB) activation/phosphorylation by angiotensin II (Ang II) is a critical signaling event in hypertrophy of vascular smooth muscle cells (VSMCs). Conventional wisdom asserts that Akt activation occurs mainly in plasma membrane domains. Recent evidence that Akt activation may take place within intracellular compartments challenges this dogma. The spatial identity and mechanistic features of these putative signaling domains have not been defined. Using cell fractionation and fluorescence methods, we demonstrate that the early endosomal antigen-1 (EEA1)-positive endosomes are a major site of Ang II-induced Akt activation. Akt moves to and is activated in EEA1 endosomes. The expression of EEA1 is required for phosphorylation of Akt at both Thr-308 and Ser-473 as well as for phosphorylation of its downstream targets mTOR and S6 kinase, but not for Erk1/2 activation. Both Akt and phosphorylated Akt (p-Akt) interact with EEA1. We also found that PKC-α is required for organizing Ang II-induced, EEA1-dependent Akt phosphorylation in VSMC early endosomes. EEA1 expression enables PKC-α phosphorylation, which in turn regulates Akt upstream signaling kinases, PDK1 and p38 MAPK. Our results indicate that PKC-α is a necessary regulator of EEA1-dependent Akt signaling in early endosomes. Finally, EEA1 down-regulation or expression of a dominant negative mutant of PKC-α blunts Ang II-induced leucine incorporation in VSMCs. Thus, EEA1 serves a novel function as an obligate scaffold for Ang II-induced Akt activation in early endosomes.     

TNF-alpha induces protein synthesis through PI3-kinase-Akt/PKB pathway in cardiac myocytes

The activation of phosphatidylinositol (PI) 3-kinase and Akt/protein kinase B (PKB) by tumor necrosis factor (TNF)-alpha and their roles on stimulation of protein synthesis were investigated in cultured neonatal rat cardiac myocytes. Treatment of cells with TNF-alpha resulted in enlargement of cell surface area and stimulation of protein synthesis without affecting myocyte viability. TNF-alpha induced marked activation of PI3-kinase and Akt/PKB, and the activation of PI3-kinase and Akt/PKB was rapid (maximal at 10 and 15 min, respectively) and concentration dependent. Akt/PKB activation by TNF-alpha was inhibited by a PI3-kinase-specific inhibitor LY-294002 and adenovirus-mediated expression of a dominant negative mutant of PI3-kinase, indicating that TNF-alpha activates Akt/PKB through PI3-kinase activation. Furthermore, TNF-alpha-induced protein synthesis was inhibited by pretreatment with LY-294002 and expression of a dominant negative mutant of PI3-kinase or Akt/PKB. These results indicate that activation of the PI3-kinase-Akt/PKB pathway plays an essential role in protein synthesis induced by TNF-alpha in cardiac myocytes.     

Nox4 NAD(P)H oxidase mediates Src-dependent tyrosine phosphorylation of PDK-1 in response to angiotensin II: role in mesangial cell hypertrophy and fibronectin expression

Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to hypertrophy and extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces an increase in PDK-1 (3-phosphoinositide-dependent protein kinase-1) kinase activity that required its phosphorylation on tyrosine 9 and 373/376. Introduction into the cells of PDK-1, mutated on these tyrosine residues or kinase-inactive, attenuates Ang II-induced hypertrophy and fibronectin accumulation. Ang II-mediated PDK-1 activation and tyrosine phosphorylation (total and on residues 9 and 373/376) are inhibited in cells transfected with small interfering RNA for Src, indicating that Src is upstream of PDK-1. In cells expressing oxidation-resistant Src mutant C487A, Ang II-induced hypertrophy and fibronectin expression are prevented, suggesting that the pathway is redox-sensitive. Ang II also up-regulates Nox4 protein, and siNox4 abrogates the Ang II-induced increase in intracellular reactive oxygen species (ROS) generation. Small interfering RNA for Nox4 also inhibits Ang II-induced activation of Src and PDK-1 tyrosine phosphorylation (total and on residues 9 and 373/376), demonstrating that Nox4 functions upstream of Src and PDK-1. Importantly, inhibition of Nox4, Src, or PDK-1 prevents the stimulatory effect of Ang II on fibronectin accumulation and cell hypertrophy. This work provides the first evidence that Nox4-derived ROS are responsible for Ang II-induced PDK-1 tyrosine phosphorylation and activation through stimulation of Src. Importantly, this pathway contributes to Ang II-induced MC hypertrophy and fibronectin accumulation. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal hypertrophy and fibrosis.     

Phosphatidylinositide 3-kinase regulates angiotensin II-induced cytosolic phospholipase A2 activity and growth in vascular smooth muscle cells

Angiotensin (Ang) II via the AT(1) receptor acts as a mitogen in vascular smooth muscle cells (VSMC) through stimulation of multiple signaling mechanisms, including tyrosine kinases and mitogen-activated protein kinase (MAPK). In addition, cytosolic phospholipase A(2)(cPLA(2))-dependent release of arachidonic acid (AA) is linked to VSMC growth and we have reported that Ang II stimulates cPLA(2) activity via the AT(1) receptor. The coupling of Ang II to the activation of cPLA(2) appears to involve mechanisms both upstream and downstream of MAPK such that AA stimulates MAPK activity which phosphorylates cPLA(2) to further enhance AA release. However, the upstream mechanisms responsible for activation of cPLA(2) are not well-defined. One possibility includes phosphatidylinositide 3-kinase (PI3K), since PI3K has been reported to participate in the upstream signaling events linked to activation of MAPK. However, it is not known whether PI3K is involved in the Ang II-induced activation of cPLA(2) or if this mechanism is associated with the Ang II-mediated growth of VSMC. Therefore, we used cultured rat VSMC to examine the role of PI3K in the Ang II-dependent phosphorylation of cPLA(2), release of AA, and growth induced by Ang II. Exposure of VSMC to Ang II (100 nM) increased [(3)H]thymidine incorporation, cell number, and the release of [(3)H]AA. Also, using Western analysis, Ang II increased the phosphorylation of MAPK and cPLA(2) which were blocked by the MAPK kinase inhibitor PD98059 (10 microM/L). Similarly, the PI3K inhibitor LY294002 (10 microM/L) abolished the Ang II-mediated increase in MAPK phosphorylation, as well as phosphoserine-PLA(2). Further, inhibition of PI3K blocked the Ang II-induced release of AA and VSMC mitogenesis. However, exogenous AA was able to restore VSMC growth in the presence of LY294002, as well as reverse the inhibition of MAPK and cPLA(2) phosphorylation by LY294002. Thus, it appears from these data that Ang II stimulates the PI3K-sensitive release of AA which stimulates MAPK to phosphorylate cPLA(2) and enhance AA release. This mechanism may play an important role in the Ang II-induced growth of VSMC.     

Calcineurin-mediated pathway involved in the differentiated phenotype of smooth muscle cells

The calcineurin-mediated pathway is involved in skeletal and cardiac hypertrophy and vascular development in vivo, but the relationship between this pathway and the phenotype of smooth muscle cells (SMCs) remains unknown. Using visceral SMCs in culture as a model system of differentiated SMCs, we investigated the role of the calcineurin-mediated pathway in maintaining the differentiated phenotype of SMCs, which depends on the insulin-like growth factor (IGF-I)-triggered activation of the phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB(Akt)) pathway. Treatment with calcineurin inhibitors, cyclosporin A or FK506, or the forced expression of the natural calcineurin inhibitor, CAIN, induced SMC dedifferentiation. Notably, suppression of the promoter activities of the SMC molecular markers caldesmon and alpha1 integrin by blocking the PI3-K/PKB(Akt) pathway was rescued by the forced expression of constitutively active calcineurin Aalpha, suggesting that the calcineurin-mediated pathway is critical for maintaining the differentiated phenotype of SMCs and works downstream of the PI3-K/PKB(Akt) pathway.     

Activation of PPARδ counteracts angiotensin II-induced ROS generation by inhibiting rac1 translocation in vascular smooth muscle cells

Angiotensin II (Ang II)-mediated modification of the redox milieu of vascular smooth muscle cells (VSMCs) has been implicated in several pathophysiological processes, including cell proliferation, migration and differentiation. In this study, we demonstrate that the peroxisome proliferator-activated receptor (PPAR) δ counteracts Ang II-induced production of reactive oxygen species (ROS) in VSMCs. Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly reduced Ang II-induced ROS generation in VSMCs. This effect was, however, reversed in the presence of small interfering (si)RNA against PPARδ. The marked increase in ROS levels induced by Ang II was also eliminated by the inhibition of phosphatidylinositol 3-kinase (PI3K) but not of protein kinase C, suggesting the involvement of the PI3K/Akt signalling pathway in this process. Accordingly, ablation of Akt with siRNA further enhanced the inhibitory effects of GW501516 in Ang II-induced superoxide production. Ligand-activated PPARδ also blocked Ang II-induced translocation of Rac1 to the cell membrane, inhibiting the activation of NADPH oxidases and consequently ROS generation. These results indicate that ligand-activated PPARδ plays an important role in the cellular response to oxidative stress by decreasing ROS generated by Ang II in vascular cells.     

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(2)O(2) increased Akt activation. The Akt inhibitor SH6 blocked Ang II-, ArAc-, or H(2)O(2)-induced Akt activation, as did inhibition of phosphoinositide 3-kinase (PI(3)K). 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 (3)H-thymidine incorporation induced by all three agonists. Thus, Ang II-induced ArAc release and ROS production leads to the PI(3)K-dependant activation of Akt and VSMC growth.     

Activation of PPARδ counteracts angiotensin II-induced ROS generation by inhibiting rac1 translocation in vascular smooth muscle cells

Abstract

Angiotensin II (Ang II)-mediated modification of the redox milieu of vascular smooth muscle cells (VSMCs) has been implicated in several pathophysiological processes, including cell proliferation, migration and differentiation. In this study, we demonstrate that the peroxisome proliferator-activated receptor (PPAR) δ counteracts Ang II-induced production of reactive oxygen species (ROS) in VSMCs. Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly reduced Ang II-induced ROS generation in VSMCs. This effect was, however, reversed in the presence of small interfering (si)RNA against PPARδ. The marked increase in ROS levels induced by Ang II was also eliminated by the inhibition of phosphatidylinositol 3-kinase (PI3K) but not of protein kinase C, suggesting the involvement of the PI3K/Akt signalling pathway in this process. Accordingly, ablation of Akt with siRNA further enhanced the inhibitory effects of GW501516 in Ang II-induced superoxide production. Ligand-activated PPARδ also blocked Ang II-induced translocation of Rac1 to the cell membrane, inhibiting the activation of NADPH oxidases and consequently ROS generation. These results indicate that ligand-activated PPARδ plays an important role in the cellular response to oxidative stress by decreasing ROS generated by Ang II in vascular cells.     

A novel phosphoinositide 3-kinase-dependent pathway for angiotensin II/AT-1 receptor-mediated induction of collagen synthesis in MES-13 mesangial cells

Chronic activation of the angiotensin II (ANG II) type 1 receptor (AT-1R) is critical in the development of chronic kidney disease. ANG II activates mesangial cells (MCs) and stimulates the synthesis of extracellular matrix components. To determine the molecular mechanisms underlying the induction of MC collagen, a mouse mesangial cell line MES-13 was employed. ANG II treatment induced an increase in collagen synthesis, which was abrogated by co-treatment with losartan (an AT-1R antagonist), wortmannin (a phosphoinositide 3-kinase (PI3K) inhibitor), an Akt inhibitor, and stable transfection of dominant negative-Akt1. ANG II induced a significant increase in PI3K activity, which was abolished by co-treatment with losartan or 2',5'-dideoxyadenosine (2',5'-DOA, an adenylyl cyclase inhibitor) but not by PD123319 (an AT-2R antagonist) or H89 (a protein kinase A (PKA) inhibitor). The Epac (exchange protein directly activated by cAMP)-specific cAMP analog, 8-pHPT-2'-O-Me-cAMP, significantly increased PI3K activity, whereas a PKA-specific analog, 6-benzoyladenosine-cAMP, showed no effect. The ANG II-induced increase in PI3K activity was also blocked by co-treatment with PP2, an Src inhibitor, or AG1478, an epidermal growth factor receptor (EGFR) antagonist. ANG II induced phosphorylation of Akt and p70S6K and EGFR, which was abrogated by knockdown of c-Src by small interference RNA. Knockdown of Src also effectively abolished ANG II-induced collagen synthesis. Conversely, stable transfection of a constitutively active Src mutant enhanced basal PI3K activity and collagen production, which was abrogated by AG1478 but not by 2',5'-DOA. Moreover, acute treatment with ANG II significantly increased Src activity, which was abrogated with co-treatment of 2',5'-DOA. Taken together, these results suggest that ANG II induces collagen synthesis in MCs by activating the ANG II/AT-1R-EGFR-PI3K pathway. This transactivation is dependent on cAMP/Epac but not on PKA. Src kinase plays a pivotal role in this signaling pathway between cAMP and EGFR. This is the first demonstration that an AT1R-PI3K/Akt crosstalk, along with transactivation of EGFR, mediates ANG II-induced collagen synthesis in MCs.     

Regulation of Ang2 release by PTEN/PI3-kinase/Akt in lung microvascular endothelial cells

Angiopoietin-2 (Ang2) is a Tie-2 ligand that destabilizes vascular structures, allowing for neovascularization or vessel regression depending on local vascular endothelial cell growth factor (VEGF) concentrations. Although various stimuli have been shown to affect Ang2 expression, information on the underlying mechanisms involved in Ang2 production in endothelial cells (EC) is just beginning to emerge. In the present study, we have used adenovirus-mediated gene transfer and pharmacological inhibitors to examine the role of the PTEN/PI3-K/Akt pathway on Ang2 release. Inhibition of PI3-kinase with wortmannin led to a stimulation of basal Ang2 release in EC, while overexpression of an active form of Akt reduced Ang2. In addition, adenovirus-mediated gene transfer of the phosphatase PTEN stimulated Ang2 release. Incubation of the cells with Ang1, an agent that activates the PI3-K/Akt pathway in EC, reduced Ang2 release. This effect of Ang1 could be prevented by wortmannin and LY-294002 pretreatment. Similarly, in VEGF-treated EC the increase in Ang2 production observed was greater in the presence of a PI3-K inhibitor. Our observations that PTEN acts as a positive modulator of Ang2 release, while activation of the PI3-K/Akt pathway downregulates Ang2, reveal an additional mechanism through which the PTEN/PI3-K/Akt pathway could affect the angiogenic process.     

The T-cell receptor regulates Akt (protein kinase B) via a pathway involving Rac1 and phosphatidylinositide 3-kinase

The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. Here we report that the small GTPase Rac1 is implicated in this pathway, connecting the receptor with the lipid kinase. We show that in Jurkat cells, activated forms of Rac1 or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rac1 but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutive active PI 3-kinase is unaffected when dominant negative Rac1 is coexpressed, placing Rac1 upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, we found that Rac1 acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. Our results reveal a previously unrecognized role of the GTPase Rac1, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rac1 as a downstream transducer for Akt/PKB activation.     

Expression and mechanism of spleen tyrosine kinase activation by angiotensin II and its implication in protein synthesis in rat vascular smooth muscle cells

Syk, a 72-kDa tyrosine kinase, is involved in development, differentiation, and signal transduction of hematopoietic and some non-hematopoietic cells. This study determined if Syk is expressed in vascular smooth muscle cells (VSMC) and contributes to angiotensin II (Ang II) signaling and protein synthesis. Syk was found in VSMC and was phosphorylated by Ang II through AT1 receptor. Ang II-induced Syk phosphorylation was inhibited by piceatannol and dominant negative but not wild type Syk mutant. Syk phosphorylation by Ang II was attenuated by cytosolic phospholipase A(2) (cPLA(2)) inhibitor pyrrolidine-1 and retrovirus carrying small interfering RNAs (shRNAs) of this enzyme. Arachidonic acid (AA) increased Syk phosphorylation, and AA- and Ang II-induced phosphorylation was diminished by inhibitors of AA metabolism (5,8,11,14-eicosatetraynoic acid) and lipoxygenase (LO; baicalein) but not cyclooxygenase (indomethacin). AA metabolites formed via LO, 5(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acids, which activate p38 MAPK, increased Syk phosphorylation. p38 MAPK inhibitor SB202190, and dominant negative p38 MAPK mutant attenuated Ang II- and AA-induced Syk phosphorylation. Adenovirus dominant negative c-Src mutant abolished Ang II - and AA-induced Syk phosphorylation and SB202190, and dominant negative p38 MAPK mutant inhibited Ang II-induced c-Src phosphorylation. Syk dominant negative mutant but not epidermal growth factor receptor blocker AG1478 also inhibited Ang II-induced VSMC protein synthesis. These data suggest that Syk expressed in VSMC is activated by Ang II through p38 MAPK-activated c-Src subsequent to cytosolic phospholipase A(2) and generation of AA metabolites via LO, and it mediates Ang II-induced protein synthesis independent of epidermal growth factor receptor transactivation (Ang II --> cPLA(2) --> AA metabolites of LO --> p38 MAPK --> c-Src --> Syk --> protein synthesis).     

Intracellular signaling of angiotensin II-induced p70 S6 kinase phosphorylation at Ser(411) in vascular smooth muscle cells. Possible requirement of epidermal growth factor receptor, Ras, extracellular signal-regulated kinase, and Akt

Activation of p70 S6 kinase (p70(S6K)) by growth factors requires multiple signal inputs involving phosphoinositide 3-kinase (PI3K), its effector Akt, and an unidentified kinase that phosphorylates Ser/Thr residues (Ser(411), Ser(418), Ser(424), and Thr(421)) clustered at its autoinhibitory domain. However, the mechanism by which G protein-coupled receptors activate p70(S6K) remains largely uncertain. By using vascular smooth muscle cells in which we have demonstrated Ras/extracellular signal-regulated kinase (ERK) activation through Ca(2+)-dependent, epidermal growth factor (EGF) receptor transactivation by G(q)-coupled angiotensin II (Ang II) receptor, we present a unique cross-talk required for Ser(411) phosphorylation of p70(S6K) by Ang II. Both p70(S6K) Ser(411) and Akt Ser(473) phosphorylation by Ang II appear to involve EGF receptor transactivation and were inhibited by dominant-negative Ras, whereas the phosphorylation of p70(S6K) and ERK but not Akt was sensitive to the MEK inhibitor. By contrast, the phosphorylation of p70(S6K) and Akt but not ERK was sensitive to PI3K inhibitors. Similar inhibitory pattern on these phosphorylation sites by EGF but not insulin was observed. Taken together with the inhibition of Ang II-induced p70(S6K) activation by dominant-negative Ras and the MEK inhibitor, we conclude that Ang II-initiated activation of p70(S6K) requires both ERK cascade and PI3K/Akt cascade that bifurcate at the point of EGF receptor-dependent Ras activation.     

Epigallocathechin-3 gallate inhibits cardiac hypertrophy through blocking reactive oxidative species-dependent and -independent signal pathways

Cardiac hypertrophy is a major cause of morbidity and mortality worldwide. Recent in vitro and in vivo studies have suggested that reactive oxygen species (ROS) may play an important role in cardiac hypertrophy. It was therefore thought to be of particular value to examine the effects of antioxidants on cardiac hypertrophy. Epigallocatechin-3-gallate (EGCG) is a major bioactive polyphenol present in green tea and a potent antioxidant. The current study was designed to test the hypothesis that EGCG inhibits cardiac hypertrophy in vitro and in vivo. In this study, we investigated the effects of EGCG on angiotensin II- (Ang II) and pressure-overload-induced cardiac hypertrophy. Our results showed that EGCG attenuated Ang II- and pressure-overload-mediated cardiac hypertrophy. Both reactive oxygen species generation and NADPH oxidase expressions induced by Ang II and pressure overload were suppressed by EGCG. The increased hypertension by pressure overload was almost completely blocked after EGCG treatment. Further studies showed that EGCG inhibited Ang II-induced NF-kappaB and AP-1 activation. Inhibition of the activity of NF-kappaB was through blocking ROS-dependent p38 and JNK signaling pathways, whereas inhibition of AP-1 activation was via blocking EGFR transactivation and its downstream events ERKs/PI3K/Akt/mTOR/p70(S6K). The combination of these actions resulted in repressing the reactivation of ANP and BNP, and ultimately preventing the progress of cardiac hypertrophy. These findings indicated that EGCG prevents the development of cardiac hypertrophy through ROS-dependent and -independent mechanisms involving inhibition of different intracellular signaling transductional pathways.     

Calcium and integrin binding protein 1 (CIB1) induces myocardial fibrosis in myocardial infarction via regulating the PI3K/Akt pathway

Myocardial infarction (MI) is a severe coronary artery disease resulted from substantial and sustained ischemia. Abnormal upregulation of calcium and integrin binding protein 1 (CIB1) has been found in several cardiovascular diseases. In this study, we established a mouse model of MI by permanent ligation of the left anterior descending coronary artery. CIB1 was upregulated in the heart of MI mice. Notably, CIB1 knockdown by intramuscular injection of lentivirus-mediated short hairpin RNA (shRNA) targeting Cib1 improved cardiac function and attenuated myocardial hypertrophy and infarct area in MI mice. MI-induced upregulation of α-SMA, vimentin, Collagen I, and Collagen III, which resulted in collagen production and myocardial fibrosis, were regressed by CIB1 silencing. In vitro, cardiac fibroblasts (CFs) isolated from mice were subjected to angiotensin II (Ang II) treatment. Inhibition of CIB1 downregulated the expression of α-SMA, vimentin, Collagen I, and Collagen III in Ang II-treated CFs. Moreover, CIB1 knockdown inhibited Ang II-induced phosphorylation of PI3K-p85 and Akt in CFs. The effect of CIB1 knockdown on Ang II-induced cellular injury was comparable to that of LY294002, a specific inhibitor of the PI3K/Akt pathway. We demonstrated that MI-induced cardiac hypertrophy, myocardial fibrosis, and cardiac dysfunction might be attributed to the upregulation of CIB1 in MI mice. Downregulation of CIB1 alleviated myocardial fibrosis and cardiac dysfunction by decreasing the expression of α-SMA, vimentin, Collagen I, and Collagen III via inhibiting the PI3K/Akt pathway. Therefore, CIB1 may be a potential target for MI treatment.     

Protein kinase B/Akt regulates coxsackievirus B3 replication through a mechanism which is not caspase dependent

The role of signaling pathways including the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) during viral infection has gained much recent attention. Our laboratory reported on an important regulatory role for extracellular signal-regulated kinases (ERK1/2), subfamily members of the MAPKs, during coxsackievirus B3 (CVB3) infection. However, the role of the PI3K pathway in CVB3 infection has not been well characterized. CVB3 is the most common known viral infectant of heart muscle that directly injures and kills infected cardiac myocytes during the myocarditic process. In the present study, we investigated the role of protein kinase B (PKB) (also known as Akt), a general downstream mediator of survival signals through the PI3K cascade, in regulating CVB3 replication and virus-induced apoptosis in a well-established HeLa cell model. We have demonstrated that CVB3 infection leads to phosphorylation of PKB/Akt on both Ser-473 and Thr-308 residues through a PI3K-dependent mechanism. Transfection of HeLa cells with a dominant negative mutant of Akt1 or pretreatment of wild-type HeLa cells with the specific PI3K inhibitor LY294002 significantly suppresses viral RNA expression, as reflected in diminished viral capsid protein expression and viral release. Dominant negative Akt1 and LY294002 also increase apoptosis in infected cells, which can be reversed by addition of the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk). Interestingly, blocking of apoptosis by zVAD.fmk does not reverse the viral RNA translation blockade, indicating that the inhibitory effect of dominant negative Akt1 on viral protein expression is not caspase dependent. In addition, we showed that the attachment of virus to its receptor-coreceptor complex is not sufficient for PKB/Akt activation and that postentry viral replication is required for Akt phosphorylation. Taken together, these data illustrate a new and imperative role for Akt in CVB3 infection in HeLa cells and show that the PI3K/Akt signaling is beneficial to CVB3 replication.     

Rho plays an important role in angiotensin II-induced hypertrophic responses in cardiac myocytes

Angiotensin II (Ang II) evokes a variety of hypertrophic responses such as activation of protein kinases, reprogramming of gene expressions and an increase in protein synthesis in cardiac myocytes. In this study, we examined the role of Rho family small GTP binding proteins (G proteins) in Ang II-induced cardiac hypertrophy. Ang II strongly activated extracellular signal-regulated protein kinases (ERKs) in cardiac myocytes of neonatal rats. Although Ang II-induced activation of ERKs was completely suppressed by an Ang II type 1 receptor antagonist, CV-11974, this activation was not inhibited by the pretreatment with C3 exoenzyme, which abrogates Rho functions. Overexpression of Rho GDP dissociation inhibitor (Rho-GDI), dominant negative mutants of Rac1 (D.N.Rac1), or D.N.Cdc42 had no effects on Ang II-induced activation of transfected ERK2. The promoter activity of skeletal a-actin and c-fos genes was increased by Ang II, and the increase was partly inhibited by overexpression of Rho-GDI and the pretreatment with C3 exoenzyme. Ang II increased phenylalanine incorporation into cardiac myocytes by approximately 1.4 fold as compared with control, and this increase was also significantly suppressed by the pretreatment with C3 exoenzyme. These results suggest that the Rho family small G proteins play important roles in Ang II-induced hypertrophic responses in cardiac myocytes.