Induction of heme oxygenase-1 is involved in anti-proliferative effects of paclitaxel on rat vascular smooth muscle cells

In this study, we evaluated the possibility that the anti-proliferative effects of paclitaxel on vascular smooth muscle cells (VSMCs) of the rat might be due to the induction of HO-1 gene expression. Treatment of the cells with paclitaxel resulted in marked time- and dose-dependent inductions of HO-1 mRNA, followed by corresponding increases in HO-1 protein expression and HO enzymatic activities. Furthermore, paclitaxel rapidly activated the JNK, ERK, and p38 mitogen-activated protein kinase pathways. A specific inhibitor of JNK, SP600125, abolished paclitaxel-induced HO-1 mRNA expression, whereas PD98059, a specific inhibitor of ERK, and SB203580, a specific inhibitor of p38, had no significant effect. Finally, the suppression of platelet-derived growth factor induced VSMC proliferation was abolished by the HO inhibitor, ZnPP, as well as by the CO scavenger, hemoglobin. These results demonstrated that paclitaxel induces the expression of HO-1 via the JNK pathway in VSMC and that HO-1 expression might be responsible for the anti-proliferative effect of paclitaxel on VSMC.     

Role of the actin cytoskeleton in angiotensin II signaling in human vascular smooth muscle cells

Angiotensin II (Ang II) regulates vascular smooth muscle cell (VSMC) function by activating signaling cascades that promote vasoconstriction, growth, and inflammation. Subcellular mechanisms coordinating these processes are unclear. In the present study, we questioned the role of the actin cytoskeleton in Ang II mediated signaling through mitogen-activated protein (MAP) kinases and reactive oxygen species (ROS) in VSMCs. Human VSMCs were studied. Cells were exposed to Ang II (10-7 mol/L) in the absence and presence of cytochalasin B (10-6 mol/L, 60 min), which disrupts the actin cytoskeleton. Phosphorylation of p38MAP kinase, JNK, and ERK1/2 was assessed by immuno blotting. ROS generation was measured using the fluoroprobe chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (4 micromol/L). Interaction between the cytoskeleton and NADPH oxidase was determined by evaluating the presence of p47phox in the Triton X-100 insoluble membrane fraction. Ang II significantly increased phosphorylation of p38MAP kinase, JNK, and ERK1/2 (two- to threefold above control, p < 0.05). Cytochalasin B pretreatment attenuated p38MAP kinase and JNK effects (p < 0.05) without altering ERK1/2 phosphorylation. ROS formation, which was increased in Ang II stimulated cells, was significantly reduced by cytochalasin B (p < 0.01). p47phox, critically involved in NADPH oxidase activation, colocalized with the actin cytoskeleton in Ang II stimulated cells. Our data demonstrate that Ang II mediated ROS formation and activation of p38MAP kinase and JNK, but not ERK1/2, involves the actin cytoskeleton in VSMCs. In addition, Ang II promotes interaction between actin and p47phox. These data indicate that the cytoskeleton is involved in differential MAP kinase signaling and ROS generation by Ang II in VSMCs. Together, these studies suggest that the cytoskeleton may be a central point of crosstalk in growth- and redox-signaling pathways by Ang II, which may be important in the regulation of VSMC function.     

Rosiglitzone Suppresses Angiotensin II-Induced Production of KLF5 and Cell Proliferation in Rat Vascular Smooth Muscle Cells

Krüppel-like factor (KLF) 5, which initiates vascular smooth muscle cell (VSMC) proliferation, also participates in Angiotensin (Ang) II-induced vascular remodeling. The protective effect of rosiglitazone on vascular remodeling may be due to their impact on VSMC proliferation. However, the underlying mechanisms involved remain unclear. This study was designed to investigate whether the antiproliferation effects of rosiglitazone are mediated by regulating Ang II/KLF5 response. We found that, in aortas of Ang II-infused rats, vascular remodeling and KLF5 expression were markedly increased, and its target gene cyclin D1 was overexpressed. Co-treatment with rosiglitazone diminished these changes. In growth-arrested VSMCs, PPAR-γ agonists (rosiglitazone and 15d-PGJ2) dose-dependently inhibited Ang II-induced cell proliferation and expression of KLF5 and cyclin D1. Moreover, these effects were attenuated by the PPAR-γ antagonists GW9662, bisphenol A diglycidyl ether and PPAR-γ specific siRNA. Furthermore, rosiglitazone inhibited Ang II-induced phosphorylation of protein kinase C (PKC) ζ and extracellular signal-regulated kinase (ERK) 1/2 and activation of early growth response protein (Egr). In conclusion, in Ang II-stimulated VSMCs, rosiglitazone might have an antiproliferative effect through mechanisms that include reducing KLF5 expression, and a crosstalk between PPAR-γ and PKCζ/ERK_1/2/Egr may be involved in. These findings not only provide a previously unrecognized mechanism by which PPAR-γ agonists inhibit VSMC proliferation, but also document a novel evidence for the beneficial vascular effect of PPAR-γ activation.     

MicroRNA-199a-5p aggravates angiotensin II–induced vascular smooth muscle cell senescence by targeting Sirtuin-1 in abdominal aortic aneurysm

Vascular smooth muscle cells (VSMCs) senescence contributes to abdominal aortic aneurysm (AAA) formation although the underlying mechanisms remain unclear. This study aimed to investigate the role of miR-199a-5p in regulating VSMC senescence in AAA. VSMC senescence was determined by a senescence-associated β-galactosidase (SA-β-gal) assay. RT-PCR and Western blotting were performed to measure miRNA and protein level, respectively. The generation of reactive oxygen species (ROS) was evaluated by H2DCFDA staining. Dual-luciferase reporter assay was used to validate the target gene of miR-199a-5p. VSMCs exhibited increased senescence in AAA tissue relative to healthy aortic tissue from control donors. Compared with VSMCs isolated from control donors (control-VSMCs), those derived from patients with AAA (AAA-VSMCs) exhibited increased cellular senescence and ROS production. Angiotensin II (Ang II) induced VSMC senescence by promoting ROS generation. The level of miR-199a-5p expression was upregulated in the plasma from AAA patients and Ang II–treated VSMCs. Mechanistically, Ang II treatment significantly elevated miR-199a-5p level, thereby stimulating ROS generation by repressing Sirt1 and consequent VSMC senescence. Nevertheless, Ang II–induced VSMC senescence was partially attenuated by a miR-199a-5p inhibitor or Sirt1 activator. Our study revealed that miR-199a-5p aggravates Ang II–induced VSMC senescence by targeting Sirt1 and that miR-199a-5p is a potential therapeutic target for AAA.     

Ribozyme to human TGF-beta1 mRNA inhibits the proliferation of human vascular smooth muscle cells

Transforming growth factor-beta (TGF-beta) has been reported to be involved in the pathogenesis of cardiovascular proliferative diseases such as hypertensive vascular disease, atherosclerosis, and arterial restenosis after angioplasty. We designed a 38-base DNA-RNA chimeric hammerhead ribozyme to cleave human TGF-beta1 mRNA as a gene therapy for human arterial proliferative diseases. In the presence of MgCl(2), synthetic ribozyme to human TGF-beta1 mRNA cleaved the synthetic target RNA into two RNA fragments of predicted size. A control mismatch ribozyme, with one different base in the catalytic loop region, was inactive. DNA-RNA chimeric ribozyme (0. 01-1.0 microM) significantly inhibited angiotensin II (Ang II)-stimulated DNA synthesis in a dose-dependent manner in human vascular smooth muscle cells (VSMC). The mismatch ribozyme did not affect Ang II-stimulated DNA synthesis in the cells. DNA-RNA chimeric ribozyme (1.0 microM) inhibited the proliferation of human VSMC in the presence of Ang II. DNA-RNA chimeric ribozyme (1.0 microM) significantly inhibited Ang II-stimulated TGF-beta1 mRNA and protein expression in human VSMC. These results indicate that the designed DNA-RNA chimeric hammerhead ribozyme targeted to human TGF-beta1 mRNA can effectively and potentially inhibit growth of human VSMC by cleaving the TGF-beta1 mRNA. This finding suggests that this ribozyme will be useful in the gene therapy of arterial proliferative diseases.     

Redox-dependent MAP kinase signaling by Ang II in vascular smooth muscle cells: role of receptor tyrosine kinase transactivation

We investigated the role of receptor tyrosine kinases in Ang II-stimulated generation of reactive oxygen species (ROS) and assessed whether MAP kinase signaling by Ang II is mediated via redox-sensitive pathways. Production of ROS and activation of NADPH oxidase were determined by DCFDA (dichlorodihydrofluorescein diacetate; 2 micromol/L) fluorescence and lucigenin (5 micromol/L) chemiluminescence, respectively, in rat vascular smooth muscle cells (VSMC). Phosphorylation of ERK1/2, p38MAP kinase and ERK5 was determined by immunoblotting. The role of insulin-like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) was assessed with the antagonists AG1024 and AG1478, respectively. ROS bioavailability was manipulated with Tiron (10(-5) mol/L), an intracellular scavenger, and diphenylene iodinium (DPI; 10(-6) mol/L), an NADPH oxidase inhibitor. Ang II stimulated NADPH oxidase activity and dose-dependently increased ROS production (p < 0.05). These actions were reduced by AG1024 and AG1478. Ang II-induced ERK1/2 phosphorylation (276% of control) was decreased by AG1478 and AG1024. Neither DPI nor tiron influenced Ang II-stimulated ERK1/2 activity. Ang II increased phosphorylation of p38 MAP kinase (204% of control) and ERK5 (278% of control). These effects were reduced by AG1024 and AG1478 and almost abolished by DPI and tiron. Thus Ang II stimulates production of NADPH-inducible ROS partially through transactivation of IGF-1R and EGFR. Inhibition of receptor tyrosine kinases and reduced ROS bioavaliability attenuated Ang II-induced phosphorylation of p38 MAP kinase and ERK5, but not of ERK1/2. These findings suggest that Ang II activates p38MAP kinase and ERK5 via redox-dependent cascades that are regulated by IGF-1R and EGFR transactivation. ERK1/2 regulation by Ang II is via redox-insensitive pathways.     

Exendin-4 Prevents Vascular Smooth Muscle Cell Proliferation and Migration by Angiotensin II via the Inhibition of ERK1/2 and JNK Signaling Pathways

Angiotensin II (Ang II) is a main pathophysiological culprit peptide for hypertension and atherosclerosis by causing vascular smooth muscle cell (VSMC) proliferation and migration. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, is currently used for the treatment of type-2 diabetes, and is believed to have beneficial effects for cardiovascular diseases. However, the vascular protective mechanisms of GLP-1 receptor agonists remain largely unexplained. In the present study, we examined the effect of exendin-4 on Ang II-induced proliferation and migration of cultured rat aortic smooth muscle cells (RASMC). The major findings of the present study are as follows: (1) Ang II caused a phenotypic switch of RASMC from contractile type to synthetic proliferative type cells; (2) Ang II caused concentration-dependent RASMC proliferation, which was significantly inhibited by the pretreatment with exendin-4; (3) Ang II caused concentration-dependent RASMC migration, which was effectively inhibited by the pretreatment with exendin-4; (4) exendin-4 inhibited Ang II-induced phosphorylation of ERK1/2 and JNK in a pre-incubation time-dependent manner; and (5) U0126 (an ERK1/2 kinase inhibitor) and SP600125 (a JNK inhibitor) also inhibited both RASMC proliferation and migration induced by Ang II stimulation. These results suggest that exendin-4 prevented Ang II-induced VSMC proliferation and migration through the inhibition of ERK1/2 and JNK phosphorylation caused by Ang II stimulation. This indicates that GLP-1 receptor agonists should be considered for use in the treatment of cardiovascular diseases in addition to their current use in the treatment of diabetes mellitus.     

Bile acids alter the subcellular localization of CNT2 (concentrative nucleoside cotransporter) and increase CNT2-related transport activity in liver parenchymal cells

PI3K (phosphoinositide 3-kinase) activity is involved in Ang (angiotensin) II-stimulated VSMC (vascular smooth muscle cell) growth and hypertrophy. In the present study, we demonstrate that the inhibition of PI3K in VSMCs by expression of a dominant-negative p85alpha mutant lacking the p110-binding domain (Deltap85), or by treatment of cells with LY294002, inhibited Ang II-stimulated PAI-1 (plasminogen activator inhibitor-1) mRNA expression. Using a GST (glutathione S-transferase) fusion protein containing the p85 N-terminal SH2 (Src homology 2) domain as 'bait' followed by MS/MS (tandem MS), we identified a 70 kDa fragment of the p70 PDGFR-beta (platelet-derived growth factor receptor-beta) as a signalling adapter that is phosphorylated and recruits the p85 subunit of PI3K after Ang II stimulation of AT1 (Ang II subtype 1) receptors on VSMCs. This fragment of the PDGFR-beta, which has a truncation of its extracellular domain, accounted for approx. 15% of the total PDGFR-beta detected in VSMCs with an antibody against its cytoplasmic domain. Stimulation of VSMCs with Ang II increased tyrosine-phosphorylation of p70 PDGFR-beta at Tyr751 and Tyr1021 and increased its binding to p85. PDGF also induced phosphorylation of p70 PDGFR-beta, a response inhibited by the PDGF tyrosine kinase selective inhibitor, AG1296. By contrast, Ang II-induced phosphorylation of the 70 kDa receptor was not affected by AG1296. Ang II-stimulated phosphorylation of the p70 PDGFR-beta was blocked by the AT1 receptor antagonist, candesartan (CV 11974) and was partially inhibited by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine}, an Src family kinase inhibitor. Our result suggests that the p70 PDGFR-beta functions as an adapter that recruits PI3K to the membrane upon AT1 receptor stimulation.     

High glucose augments the angiotensin II-induced activation of JAK2 in vascular smooth muscle cells via the polyol pathway

Angiotensin II (Ang II), protein kinase C (PKC), reactive oxygen species (ROS) generated by NADPH oxidase, the activation of Janus kinase 2 (JAK2), and the polyol pathway play important parts in the hyperproliferation of vascular smooth muscle cells (VSMC), a characteristic feature of diabetic macroangiopathy. The precise mechanism, however, remains unclear. This study investigated the relation between the polyol pathway, PKC-beta, ROS, JAK2, and Ang II in the development of diabetic macroangiopathy. VSMC cultured in high glucose (HG; 25 mm) showed significant increases in the tyrosine phosphorylation of JAK2, production of ROS, and proliferation activities when compared with VSMC cultured in normal glucose (5.5 mm (NG)). Both the aldose reductase specific inhibitor (zopolrestat) or transfection with aldose reductase antisense oligonucleotide blocked the phosphorylation of JAK2, the production of ROS, and proliferation of VSMC induced by HG, but it had no effect on the Ang II-induced activation of these parameters in both NG and HG. However, transfection with PKC-beta antisense oligonucleotide, preincubation with a PKC-beta-specific inhibitor (LY379196) or apocynin (NADPH oxidase-specific inhibitor), or electroporation of NADPH oxidase antibodies blocked the Ang II-induced JAK2 phosphorylation, production of ROS, and proliferation of VSMC in both NG and HG. These observations suggest that the polyol pathway hyperactivity induced by HG contributes to the development of diabetic macroangiopathy through a PKC-beta-ROS activation of JAK2.     

Genipin Inhibits TNF-α-Induced Vascular Smooth Muscle Cell Proliferation and Migration via Induction of HO-1

Vascular smooth muscle cell (VSMC) proliferation and migration triggered by inflammatory stimuli contributes importantly to the pathogenesis of atherosclerosis and restenosis. On the other hand, genipin, an aglycon of geniposide, exhibits diverse pharmacological functions such as antitumor and anti-inflammatory effects. The protective effects of genipin on the cardiovascular system have also been reported. However, the molecular mechanism involved remains unknown. This study aimed to elucidate the precise function of genipin in VSMCs, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. We found that pretreatment of genipin induced HO-1 mRNA and protein levels, as well as its activity in VSMCs. Genipin inhibited TNF-α-induced VSMC proliferation and migration in a dose-dependent manner. At the molecular level, genipin prevented ERK/MAPK and Akt phosphorylation while left p38 MAPK and JNK unchanged. Genipin also blocked the increase of ROS generation induced by TNF-α. More importantly, the specific HO-1 siRNA partially abolished the beneficial effects of genipin on VSMCs. These results suggest that genipin may serve as a novel drug in the treatment of these pathologies by inducing HO-1 expression/activity and subsequently decreasing VSMC proliferation and migration.     

Anti-proliferative effect of rosiglitazone on angiotensin II-induced vascular smooth muscle cell proliferation is mediated by the mTOR pathway

VSMC (vascular smooth muscle cell) proliferation contributes significantly to intimal thickening in atherosclerosis, restenosis and venous bypass graft diseases. Ang II (angiotensin II) has been implicated in VSMC proliferation though the activation of multiple growth-promoting signals. Although TZDs (thiazolidinediones) can inhibit VSMC proliferation and reduce Ang II-induced fibrosis, the mechanism underlying the inhibition of VSMC proliferation and fibrosis needs elucidation. We have used primary cultured rat aortic VSMCs and specific antibodies to investigate the inhibitory mechanism of rosiglitazone on Ang II-induced VSMC proliferation. Rosiglitazone treatment significantly inhibited Ang II-induced rat aortic VSMC proliferation in a dose-dependent manner. Western blot analysis showed that rosiglitazone significantly lowered phosphorylated ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt (also known as protein kinase B), mTOR (mammalian target of rapamycin), p70S6K (70 kDa S6 kinase) and 4EBP1 (eukaryotic initiation factor 4E-binding protein) levels in Ang II-treated VSMCs. In addition, PPAR-γ (peroxisome-proliferator-activated receptor γ) mRNA increased significantly and CTGF (connective tissue growth factor), Fn (fibronectin) and Col III (collagen III) levels decreased significantly. The results demonstrate that the rosiglitazone directly inhibits the pro-atherosclerotic effect of Ang II on rat aortic VSMCs. It also attenuates Ang II-induced ECM (extracellular matrix) molecules and CTGF production in rat aortic VSMCs, reducing fibrosis. Importantly, PPAR-γ activation mediates these effects, in part, through the mTOR-p70S6K and -4EBP1 system.     

The GTPase ARF6 Controls ROS Production to Mediate Angiotensin II-Induced Vascular Smooth Muscle Cell Proliferation

High reactive oxygen species (ROS) levels and enhanced vascular smooth muscle cells (VSMC) proliferation are observed in numerous cardiovascular diseases. The mechanisms by which hormones such as angiotensin II (Ang II) acts to promote these cellular responses remain poorly understood. We have previously shown that the ADP-ribosylation factor 6 (ARF6), a molecular switch that coordinates intracellular signaling events can be activated by the Ang II receptor (AT₁R). Whether this small GTP-binding protein controls the signaling events leading to ROS production and therefore Ang II-dependent VSMC proliferation, remains however unknown. Here, we demonstrate that in rat aortic VSMC, Ang II stimulation led to the subsequent activation of ARF6 and Rac1, a key regulator of NADPH oxidase activity. Using RNA interference, we showed that ARF6 is essential for ROS generation since in conditions where this GTPase was knocked down, Ang II could no longer promote superoxide anion production. In addition to regulating Rac1 activity, ARF6 also controlled expression of the NADPH oxidase 1 (Nox 1) as well as the ability of the EGFR to become transactivated. Finally, ARF6 also controlled MAPK (Erk1/2, p38 and Jnk) activation, a key pathway of VSMC proliferation. Altogether, our findings demonstrate that Ang II promotes activation of ARF6 to controls ROS production by regulating Rac1 activation and Nox1 expression. In turn, increased ROS acts to activate the MAPK pathway. These signaling events represent a new molecular mechanism by which Ang II can promote proliferation of VSMC.     

Role of Pin1 in neointima formation: Down-regulation of Nrf2-dependent heme oxygenase-1 expression by Pin1

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intima formation after stenting and balloon angioplasty. Pin1, a peptidyl prolyl isomerase recognizing phosphorylated Ser/Thr–Pro, isomerizes the peptide bond. Because Pin1 overexpression is associated with transformation and the uncontrolled cell growth of tumors, we hypothesized that Pin1 functions as a chronic stimulator of VSMC proliferation. Pin1-positive smooth muscle cells were seen in the neointimal region of the femoral artery after guidewire injury. Exposure of VSMCS to platelet-derived growth factor (PDGF) increased Pin1 expression in a concentration-dependent manner. Basal cell growth rate and cyclin D1 expression were enhanced in Pin1-overexpressing VSMCs (Pin1-VSMCs). Moreover, PDGF-induced production of reactive oxygen species (ROS) in Pin1-VSMCs was higher than in control VSMCs. In Pin1-VSMCs, heme oxygenase-1 (HO-1) induction in response to nitric oxide donor was suppressed compared to control VSMCs. Nuclear translocation of nuclear factor E2-related factor-2 (Nrf2) was also diminished in Pin1-VSMCs. In contrast, the activity of the inducible minimal antioxidant response element (ARE) was potentiated in Pin1-null mouse embryonic fibroblasts (MEFs), compared to Pin1-wild-type MEFs. Moreover, Nrf2 ubiquitination was stimulated by Pin1 overexpression. Intraperitoneal injection of juglone (a Pin1 inhibitor) for 3 weeks (1 mg/kg, two times a week) significantly suppressed neointimal formation induced by wire injury. In conclusion, Pin1 induction during neointimal formation may be associated with ROS-mediated VSMC proliferation via down-regulation of Nrf2/ARE-dependent HO-1 expression. Pin1 may be a novel therapeutic target for several vascular diseases including atherosclerosis and stenosis.     

NS-398, a selective COX-2 inhibitor, inhibits proliferation of IL-1beta-stimulated vascular smooth muscle cells by induction of HO-1

We investigated whether NS-398, a selective inhibitor of COX-2, induces HO-1 in IL-1β-stimulated vascular smooth muscle cells (VSMC). NS-398 reduced the production of PGE₂ without modulation of expression of COX-2 in IL-1β-stimulated VSMC. NS-398 increased HO-1 mRNA and protein in a dose-dependent manner, but inhibited proliferation of IL-1β-stimulated VSMC. Furthermore, SnPPIX, a HO-1 inhibitor, reversed the effects of NS-398 on PGE₂ production, suggesting that COX-2 activity can be affected by HO-1. Hemin, a HO-1 inducer, also reduced the production of PGE₂ and proliferation of IL-1β-stimulated VSMC. CORM-2, a CO-releasing molecule, but not bilirubin inhibited proliferation of IL-1β-stimulated VSMC. NS-398 inhibited proliferation of IL-1β-stimulated VSMC in a HbO₂-sensitive manner. In conclusion, NS-398 inhibits proliferation of IL-1β-stimulated VSMC by HO-1-derived CO. Thus, NS-398 may facilitate the healing process of vessels in vascular inflammatory disorders such as atherosclerosis.