Evangeline D. Motley

Involvement of Rho-Kinase in Angiotensin II–Induced Hypertrophy of Rat Vascular Smooth Muscle Cells

Angiotensin II (Ang II) is now believed to play a critical role in the pathogenesis of hypertrophy and/or hyperplasia of vascular smooth muscle cells (VSMCs). Several G(i)- and G(q)-coupled receptors, including the Ang II type 1 (AT(1)) receptor, activate Rho and Rho-associated kinase in Swiss 3T3 cells and cardiac myocytes. However, little is known about the role of Rho-kinase in Ang II-induced vascular hypertrophy in VSMCs. In the present study, we explored the role of Rho and Rho-kinase in Ang II-induced protein synthesis in VSMCs. In unstimulated cells, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to Ang II (100 nmol/L). This effect was completely blocked by the AT(1) receptor blocker candesartan but not by the Ang II type 2 (AT(2)) receptor antagonist PD123319. Botulinum C(3) exoenzyme, which inactivated RhoA, attenuated Ang II-induced [(3)H]leucine incorporation. The specific Rho-kinase inhibitor, Y-27632, dose-dependently abolished Ang II-induced protein synthesis and also suppressed Ang II-induced c-fos mRNA expression. On the other hand, Y-27632 had no effect on Ang II-stimulated phosphorylation of p70 S6 kinase and extracellular signal-regulated kinase 1/2, which are reported to be involved in Ang II-induced protein synthesis, nor had it any effect on the Ang II-induced phosphorylation of PHAS-I, a heat- and acid-stable eIF-4E-binding protein. The phosphorylation of PHAS-I is regulating for translation initiation. These observations suggest that the Rho, Rho-kinase, and c-fos pathways may play a role in Ang II-induced hypertrophic changes of VSMCs through a novel pathway.

Involvement of PYK2 in Angiotensin II Signaling of Vascular Smooth Muscle Cells

-PYK2, a recently identified Ca2+-sensitive tyrosine kinase, has been implicated in extracellular signal-regulated kinase (ERK) activation via several G protein-coupled receptors. We have reported that angiotensin II (Ang II) induces Ca2+-dependent transactivation of the epidermal growth factor receptor (EGFR) which serves as a scaffold for preactivated c-Src and downstream adaptors (Shc/Grb2), leading to ERK activation in cultured rat vascular smooth muscle cells (VSMC). Herein we demonstrate the involvement of PYK2 in this cascade. Ang II rapidly induced tyrosine phosphorylation of PYK2, whose effect was completely inhibited by an AT1 receptor antagonist and an intracellular Ca2+ chelator. A Ca2+ ionophore also induced PYK2 tyrosine phosphorylation to a level comparable with that by Ang II, whereas phorbol ester-induced phosphorylation was less than that by Ang II. Moreover, PYK2 formed a complex coprecipitable with catalytically active c-Src after Ang II stimulation. Although a selective EGFR kinase inhibitor completely abolished Ang II-induced recruitment of Grb2 to EGFR and markedly attenuated Ang II-induced ERK activation, it had no effect on Ang II-induced PYK2 tyrosine phosphorylation or its association with c-Src and Grb2. These data suggest that the AT1 receptor uses Ca2+-dependent PYK2 to activate c-Src, thereby leading to EGFR transactivation, which preponderantly recruits Grb2 in rat VSMC.

Involvement of Reactive Oxygen Species in the Activation of Tyrosine Kinase and Extracellular Signal-Regulated Kinase by Angiotensin II.

Reactive oxygen species (ROS) have been proposed to mediate vascular hypertrophy induced by angiotensin II (Ang II). Recently, we and others have shown that growth-promoting signals by Ang II involve protein tyrosine kinase (PTK) and extracellular signal-regulated kinase (ERK). However, whether ROS contribute to the Ang II-induced PTK and/or ERK activation in vascular smooth muscle cells (VSMCs) remains largely unclear. Here, we have investigated the possible involvement of ROS in Ang II-induced PTK and ERK activation. In the presence of a NADH/NADPH oxidase inhibitor, diphenyleneiodonium (DPI) or an antioxidant,α -tocopherol, Ang II-induced protein tyrosine phosphorylation of two major proteins (p120, p70) and ERK activation were markedly reduced, whereas ERK activation by epidermal growth factor was unaffected. DPI also inhibited Ang II-induced H2O2 production and PTK activation. In this regard, H2O2 and a membrane permeable thiol-oxidizing agent, diamide, stimulated protein tyrosine phosphorylation of ...

Signal-Crosstalk Between Rho/ROCK and c-Jun NH2-Terminal Kinase Mediates Migration of Vascular Smooth Muscle Cells Stimulated by Angiotensin II

Background—Rho and its effector Rho-kinase/ROCK mediate cytoskeletal reorganization as well as smooth muscle contraction. Recent studies indicate that Rho and ROCK are critically involved in vascular remodeling. Here, we tested the hypothesis that Rho/ROCK are critically involved in angiotensin II (Ang II)-induced migration of vascular smooth muscle cells (VSMCs) by mediating a specific signal cross-talk. Methods and Results—Immunoblotting demonstrated that Ang II stimulated phosphorylation of a ROCK substrate, regulatory myosin phosphatase targeting subunit (MYPT)-1. Phosphorylation of MYPT-1 as well as migration of VSMCs induced by Ang II was inhibited by dominant-negative Rho (dnRho) or ROCK inhibitor, Y27632. Ang II–induced c-Jun NH2-terminal kinase (JNK) activation, but extracellular signal-regulated kinase (ERK) activation was not mediated through Rho/ROCK. Thus, infection of adenovirus encoding dnJNK inhibited VSMC migration by Ang II. We have further demonstrated that the Rho/ROCK activation by Ang II requires protein kinase C-&dgr; (PKC&dgr;) and proline-rich tyrosine kinase 2 (PYK2) activation, but not epidermal growth factor receptor transactivation. Also, VSMCs express PDZ-Rho guanine nucleotide exchange factor (GEF) and Ang II stimulated PYK2 association with tyrosine phosphorylated PDZ-RhoGEF. Conclusions—PKC&dgr;/PYK2-dependent Rho/ROCK activation through PDZ-RhoGEF mediates Ang II–induced VSMC migration via JNK activation in VSMCs, providing a novel mechanistic role of the Rho/ROCK cascade that is involved in vascular remodeling.

Mechanism of Endothelial Nitric Oxide Synthase Phosphorylation and Activation by Thrombin

Thrombin has been shown to activate endothelial NO synthase (eNOS) leading to endothelium-dependent vasorelaxation. In addition to its activation by Ca2+/calmodulin, eNOS has several regulatory sites. Ser1179 phosphorylation of eNOS by the phosphatidylinositol 3-kinase-dependent Akt stimulates its catalytic activity. In this study, we have elucidated the signaling mechanism of thrombin-induced phosphorylation of eNOS in the regulation of NO production. Immunoblot analysis showed that thrombin rapidly phosphorylates eNOS at Ser1179 in cultured bovine aortic endothelial cells. Also, thrombin was unable to stimulate eNOS if the Ser1179 was mutated to Ala. Akt is phosphorylated in response to thrombin at Ser473 at a later time point than eNOS. In this regard, a phosphatidylinositol 3-kinase inhibitor, LY294002, blocked Akt phosphorylation without affecting eNOS phosphorylation and cGMP production by thrombin. The Ca2+ ionophore A23187 stimulated eNOS phosphorylation, as well as cGMP production, and pretreatment with intracellular or extracellular Ca2+ chelators inhibited thrombin-induced eNOS phosphorylation and cGMP production. Moreover, infection of bovine aortic endothelial cell with adenovirus encoding dominant-negative mutants of protein kinase C (PKC)&agr; and PKC&dgr; or pretreatment of bovine aortic endothelial cells with PKC inhibitors revealed that PKC&dgr; is indispensable for thrombin-induced eNOS phosphorylation and activation. From these data, we concluded that thrombin induces the Ser1179 phosphorylation-dependent eNOS activation through a Ca2+-dependent, PKC&dgr;-sensitive, but phosphatidylinositol 3-kinase/Akt-independent pathway.

Lysophosphatidylcholine Stimulates MAP Kinase Activity in Rat Vascular Smooth Muscle Cells

Lysophosphatidylcholine (lyso-PC) has been implicated in atherogenesis and the inflammatory process. Although lyso-PC has been reported to contribute to the mitogenic effect of oxidized LDL on rat cultured vascular smooth muscle cells (VSMCs), the signaling mechanisms by which lyso-PC promotes its proliferation are poorly characterized. Mitogen-activated protein (MAP) kinases are important mediators involved in the intracellular network of interacting proteins that transduces extracellular cues to intracellular responses. We therefore examined the effect of lyso-PC on MAP kinase activation, proto-oncogene expression, and AP-1 binding activity using cultured rat VSMC. Marked activation of MAP kinase occurred within 10 minutes of lyso-PC treatment, whereupon rapid inactivation ensued. MAP kinase activation by lyso-PC was concentration-dependent (6.25 to 25 micromol/L). Pertussis toxin treatment did not affect lyso-PC-induced MAP kinase phosphorylation. Lyso-PC (25 micromol/L) also increased the mRNA expression of c-fos and c-jun genes. An electrophoretic mobility shift assay showed that AP-1 binding activity was enhanced by lyso-PC. To examine the upstream signaling of MAP kinase, we used several inhibitors on MAP kinase activation induced by lyso-PC. Although lyso-PC induced sustained increase in intracellular Ca2+ concentration, EGTA had no effect on MAP kinase activation induced by lyso-PC. However, protein kinase C inhibitor GF109203X and downregulation of protein kinase C activity by prolonged treatment with phorbol ester inhibited lyso-PC-induced MAP kinase activation. These data suggest that lyso-PC transmits its mitogenic activity through a MAP kinase-AP-1 pathway, which exists downstream of its protein kinase C activation in VSMCs.

Metalloprotease inhibitor blocks angiotensin II-induced migration through inhibition of epidermal growth factor receptor transactivation.

In vascular smooth muscle cells (VSMCs), angiotensin II (AngII) induces transactivation of the EGF receptor (EGFR) which involves a metalloprotease that stimulates processing of heparin-binding EGF from its precursor. However, the identity and pharmacological sensitivity of the metalloprotease remain unclear. Here, we screened the effects of several metalloprotease inhibitors on AngII-induced EGFR transactivation in VSMCs. We found that an N-phenylsulfonyl-hydroxamic acid derivative [2R-[(4-biphenylsulfonyl)amino]-N-hydroxy-3-phenylpropinamide] (BiPS), previously known as matrix metalloprotease (MMP)-2/9 inhibitor, markedly inhibited AngII-induced EGFR transactivation, whereas the MMP-2 or -9 inhibition by other MMP inhibitors failed to block the transactivation. BiPS markedly inhibited AngII-induced ERK activation and protein synthesis without affecting AngII-induced intracellular Ca2+ elevation. VSMC migration induced by AngII was also inhibited not only by an EGFR inhibitor but also by BiPS. Thus, BiPS is a specific candidate to block AngII-induced EGFR transactivation and subsequent growth and migration of VSMCs, suggesting its potency to prevent vascular remodeling.

Insulin-Induced Akt Activation Is Inhibited by Angiotensin II in the Vasculature Through Protein Kinase C-α

Abstract—Insulin resistance is an important risk factor in the development of cardiovascular diseases such as hypertension and atherosclerosis. However, the specific role of insulin resistance in the etiology of these diseases is poorly understood. Angiotensin (Ang) II is a potent vasculotrophic and vasoconstricting factor. We hypothesize that in vascular smooth muscle cells (VSMCs), Ang II interferes with insulin action by inhibiting Akt, a major signaling molecule implicated in the biological actions of insulin. By immunoblotting with a phospho-specific antibody for Akt, we found that Ang II inhibits insulin-induced Akt phosphorylation in a time- and concentration-dependent manner. The inhibitory effect of Ang II was blocked by a Ang II type 1 receptor antagonist, RNH6270. A protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, also inhibited insulin-induced Akt phosphorylation. PKC inhibitors, including Go6976 (specific for &agr;- and &bgr;-isoforms), blocked the Ang II– and PMA-induced inhibition of Akt phosphorylation by insulin. Moreover, overexpression of PKC-&agr; but not PKC-&bgr; isoform by adenovirus inhibited insulin-induced Akt phosphorylation. By contrast, an epidermal growth factor receptor inhibitor (AG1478), a p42/44 mitogen-activated protein kinase (MAPK) kinase inhibitor (PD 598,059), and a p38 MAPK inhibitor (SB 203,580) did not block the Ang II–induced inhibition of Akt phosphorylation. From these data, we conclude that Ang II negatively regulates the insulin signal, Akt, in the vasculature specifically through PKC-&agr; activation, providing an alternative molecular mechanism that may explain the association of hyperinsulinemia with cardiovascular diseases.

Lysophosphatidylcholine Activates Extracellular Signal-Regulated Kinases 1/2 Through Reactive Oxygen Species in Rat Vascular Smooth Muscle Cells

Lysophosphatidylcholine (lysoPC) acts on vascular smooth muscle cells (VSMCs) to produce a mitogenic response through the activation of extracellular signal-regulated kinases 1/2 (ERK1/2). In the present study, we examined the importance of reactive oxygen species (ROS) in lysoPC-stimulated ERK1/2 activation in cultured rat VSMCs. Treatment with lysoPC for 3 minutes caused a 2-fold increase in intracellular ROS that was blocked by the NADH/NADPH oxidase inhibitor, diphenylene iodonium (DPI). Antioxidants, N-acetyl-l-cysteine, glutathione monoester, or &agr; -tocopherol, inhibited ERK1/2 activation by lysoPC. Almost identical results were obtained in the VSMC line A10. Pretreatment of VSMCs with DPI but not allopurinol or potassium cyanide (KCN) abrogated the activation of ERK1/2. The Flag-tagged p47phox expressed in A10 cells was translocated from the cytosol to the membrane after 2 minutes of stimulation with lysoPC. The overexpression of dominant-negative p47phox in A10 cells suppressed lysoPC-induced ERK activation. The ROS-dependent ERK activation by lysoPC seems to involve protein kinase C- and Ras-dependent raf-1 activation. Induction of c-fos expression and enhanced AP-1 binding activity by lysoPC were also inhibited by DPI and NAC. Taken together, these data suggest that ROS generated by NADH/NADPH oxidase contribute to lysoPC-induced activation of ERK1/2 and subsequent growth promotion in VSMCs.

Hydrogen peroxide inhibits insulin signaling in vascular smooth muscle cells.

Both insulin resistance and reactive oxygen species (ROS) have been reported to play essential pathophysiological roles in cardiovascular diseases, such as hypertension and atherosclerosis. However, the mechanistic link between ROS, such as H2O2 and insulin resistance in the vasculature, remains undetermined. Akt, a Ser/Thr kinase, mediates various biological responses induced by insulin. In this study, we examined the effects of H2O2 on Akt activation in the insulin-signaling pathway in vascular smooth muscle cells (VSMCs). In VSMCs, insulin stimulates Akt phosphorylation at Ser473. Pretreatment with H2O2 concentration- and time-dependently inhibited insulin-induced Akt phosphorylation with significant inhibition observed at 50 μM for 10 min. A ROS inducer, diamide, also inhibited insulin-induced Akt phosphorylation. In addition, H2O2 inhibited insulin receptor binding partially and inhibited insulin receptor autophosphorylation almost completely. However, pretreatment with a protein kinase C inhibitor, GF109203X (2 μM), for 30 min did not block the inhibitory effects of H2O2 on insulin-induced Akt phosphorylation, suggesting that protein kinase C is not involved in the inhibition by H2O2. We conclude that ROS inhibit a critical insulin signal transduction component required for Akt activation in VSMCs, suggesting potential cellular mechanisms of insulin resistance, which would require verification in vivo.