Abstract 361: Cyclophilin A Mediates Angiotensin II--Stimulated NADPH Oxidase Activation in Vascular Smooth Muscle Cells

Objective: Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses PPIase activity and scaffold function. CyPA regulates Angiotensin II (Ang II) induced reactive oxygen species (ROS) production in vascular smooth muscle cells. However, the mechanism of this CyPA regulation remains unclear. We hypothesized that CyPA regulates plasma membrane translocation of NADPH oxidase cytosolic subunit, p47phox, which is required for NADPH oxidase structural organization and activity. Methods and results: Immunofluorescence studies in rat aortic smooth muscle cells revealed that CyPA translocated from the cytosol to the plasma membrane in response to Ang II in a time dependent manner with a peak at 10min (46.4±5.4 fold increase). Mouse Aortic Smooth Muscle Cells (MASM) were isolated from mice lacking CyPA (CyPA-/-) and wild type controls (WT), treated with Ang II (100nM) and immunofluorescence analysis was performed. Ang II induced p47phox plasma membrane translocation at 10min in WT mice. However, p47 phox translocation was significantly inhibited in CyPA -/- MASM. CyPA and p47phox colocalized at the plasma membrane in response to Ang II. Further analysis using subcellular fractionation studies confirmed that Ang II induced p47phox plasma membrane translocation was inhibited in CyPA -/- MASM compared to WT (1.2±2.7 vs 4.3±3.4 fold increase). Coimmunoprecipitation analyses confirmed that Ang II increased CyPA association with p47phox in a time dependent manner (2.5±3.4 fold increase at 10min). Finally, pretreatment with the PPIase activity inhibitor, cyclosporine A (1uM), could not inhibit CyPA association with p47phox and CyPA mediated p47phox translocation to the plasma membrane. Conclusion: These data suggest that Ang II promotes an association between CyPA and p47phox that enhances plasma membrane translocation of p47phox. This is proposed to increase the NADPH oxidase activity thereby increasing cellular ROS production. This process is independent of the PPIase activity of CyPA. Therefore, inhibition of the CyPA and p47phox association could be a future therapeutic target for Ang II induced ROS regulated cardiovascular diseases such as atherosclerosis and abdominal aortic aneurysm formation.

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ANG II increases TIMP-1 expression in rat aortic smooth muscle cells in vivo

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00986.2001 ◽

2003 ◽

Vol 284 (2) ◽

pp. H635-H643 ◽

Cited By ~ 41

Author(s):

Giovanna Castoldi ◽

Cira R. T. di Gioia ◽

Federico Pieruzzi ◽

Cristina D'Orlando ◽

Willy M. M. van de Greef ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Vascular Wall ◽

Dependent Manner ◽

Ang Ii ◽

Maximal Increase ◽

Aortic Smooth Muscle Cells ◽

Aortic Smooth Muscle

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tissue remodeling processes. TIMP-1 is the main native inhibitor of MMPs and it contributes to the development of tissue fibrosis. It is known that ANG II plays a fundamental role in vascular remodeling. In this study, we investigated whether ANG II modulates TIMP-1 expression in rat aortic smooth muscle cells. In vitro, ANG II induces TIMP-1 mRNA expression in a dose-dependent manner. The maximal increase in TIMP-1 expression was present after 3 h of ANG II stimulation. The ANG II increase in TIMP-1 expression was mediated by the ANG type 1 receptors because it was blocked by losartan. The increase in TIMP-1 expression was present after the first ANG II treatment, whereas repeated treatments (3 and 5 times) did not modify TIMP-1 expression. In vivo, exogenous ANG II was administered to Sprague-Dawley rats (200 ng · kg−1· min−1sc) for 6 and 25 days. Control rats received physiological saline. After treatment, systolic blood pressure was significantly higher ( P < 0.01), whereas plasma renin activity was suppressed ( P < 0.01), in ANG II-treated rats. ANG II increased TIMP-1 expression in the aorta of ANG II-treated rats both at the mRNA ( P < 0.05) and protein levels as evaluated by Western blotting ( P < 0.05) and/or immunohistochemistry. Neither histological modifications at the vascular wall nor differences in collagen content in the tunica media were present in both the ANG II- and saline-treated groups. Our data demonstrate that ANG II increases TIMP-1 expression in rat aortic smooth muscle cells. In vivo, both short- and long-term chronic ANG II treatments increase TIMP-1 expression in the rat aorta. TIMP-1 induction by ANG II in aortic smooth muscle cells occurs in the absence of histological changes at the vascular wall.

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Mechanism of angiotensin II-induced arachidonic acid metabolite release in aortic smooth muscle cells: involvement of phospholipase D

Acta Endocrinologica ◽

10.1530/eje.0.1360207 ◽

1997 ◽

Vol 136 (2) ◽

pp. 207-212 ◽

Cited By ~ 6

Author(s):

Junji Shinoda ◽

Osamu Kozawa ◽

Atsushi Suzuki ◽

Yasuko Watanabe-Tomita ◽

Yutaka Oiso ◽

...

Keyword(s):

Arachidonic Acid ◽

Smooth Muscle ◽

Angiotensin Ii ◽

Smooth Muscle Cells ◽

Phospholipase D ◽

Muscle Cells ◽

Dependent Manner ◽

Ang Ii ◽

Aortic Smooth Muscle Cells ◽

Aortic Smooth Muscle

Abstract In a previous study, we have shown that angiotensin II (Ang II) activates phosphatidylcholinehydrolyzing phospholipase D due to Ang II-induced Ca2+ influx from extracellular space in subcultured rat aortic smooth muscle cells. In the present study, we have investigated the role of phospholipase D in Ang II-induced arachidonic acid (AA) metabolite release and prostacyclin synthesis in subcultured rat aortic smooth muscle cells. Ang II significantly stimulated AA metabolite release in a concentration-dependent manner in the range between 1 nmol/l and 0·1 μmol/l. d,l-Propranolol hydrochloride (propranolol), an inhibitor of phosphatidic acid phosphohydrolase, significantly inhibited the Ang II-induced release of AA metabolites. The Ang II-induced AA metabolite release was reduced by chelating extracellular Ca2+ with EGTA. Genistein, an inhibitor of protein tyrosine kinases, significantly suppressed the Ang II-induced AA metabolite release. 1,6-Bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a potent and selective inhibitor of diacylglycerol lipase, significantly inhibited the Ang II-induced AA metabolite release. Both propranolol and RHC-80267 inhibited the Ang II-induced synthesis of 6-keto-prostaglandin F1α, a stable metabolite of prostacyclin. The synthesis was suppressed by genistein. These results strongly suggest that the AA metabolite release induced by Ang II is mediated, at least in part, through phosphatidylcholine hydrolysis by phospholipase D activation in aortic smooth muscle cells. European Journal of Endocrinology 136 207–212

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Abstract 523: Acetylation of Cyclophilin A in Response to Oxidative Stress Enhances Its Expression and Secretion

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a523 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Nwe Nwe Soe ◽

Mark Sowden ◽

Bradford C Berk

Keyword(s):

Oxidative Stress ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Time Dependent ◽

Dependent Manner ◽

Ang Ii ◽

Aortic Smooth Muscle ◽

Dose Dependent ◽

Cypa Expression

Objective: Cyclophilin A (CyPA) is a Secreted OXidative stress-induced Factor (SOXF) secreted by cardiovascular cells in response to Angiotensin II (Ang II) and reactive oxygen species (ROS). Extracellular CyPA is a proinflammatory mediator that regulates vascular remodeling, abdominal aortic aneurysm, atherosclerosis and cardiac hypertrophy. Post-translational modification of CyPA by acetylation in response to ROS has been described. Moreover, acetylation of CyPA is important in HIV pathogenesis. The mechanism and regulation of CyPA acetylation as well as its role in cardiovascular diseases are currently unknown. We hypothesized that Ang II regulates oxidative stress-induced CyPA acetylation that alters its expression and/or secretion in vascular smooth muscle cells. Methods and results: Ang II (1μM) increased acetylation of CyPA (Acyl-CyPA) in a time dependent manner, with a peak at 8hr (3.5±0.6 fold increase) in rat aortic smooth muscle cells (RASMC) as shown by Western blot. Mouse aortic smooth muscle cells from mice lacking CyPA (CyPA-/-) and wild type controls (WT) confirmed that Ang II induced acetylation reactivity coincided exactly with CyPA reactivity. In AT1R and CyPA cotransfected HeLa cells, Ang II increased Acyl-CyPA in a time dependent manner consistent with that in RASMC. The ROS scavengers Tiron or N-acetylcysteine significantly inhibited Ang II induced Acyl-CyPA in a dose dependent manner in RASMC. Ang II-induced CyPA acetylation was enhanced by 2 hr pretreatment with histone deacetylase inhibitor trichostatin (TSA) or sirtinol in a dose dependent manner. Similarly, Ang II-induced CyPA secretion was enhanced by pretreatment with TSA (1μM) in a time dependent manner. Moreover, acetyltransferase p300 and PCAF (p300/CBP-asociated factor) inhibitor anacardic acid (6-nonadecyl salicylic acid) dramatically inhibited CyPA expression, and Ang II induced Acyl-CyPA in a dose dependent manner. Conclusion: These results suggest that Ang II-induced CyPA acetylation is oxidative stress dependent, and that acetylation enhanced CyPA expression and secretion. Detailed mechanistic studies of the regulation of CyPA acetylation will help to identify a future therapeutic target for CyPA regulated cardiovascular diseases.

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Interaction of vasopressin and angiotensin II in stimulation of prostacyclin synthesis in vascular smooth muscle cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1989.257.5.e617 ◽

1989 ◽

Vol 257 (5) ◽

pp. E617-E624

Author(s):

M. B. Vallotton ◽

C. Gerber-Wicht ◽

W. Dolci ◽

R. P. Wuthrich

Keyword(s):

Smooth Muscle ◽

Angiotensin Ii ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

Dependent Manner ◽

Ang Ii ◽

Prostacyclin Production ◽

Prostacyclin Synthesis

The effect of angiotensin II (ANG II) and arginine vasopressin (AVP) on prostacyclin production by vascular smooth muscle cells (VSMC) has been examined. Cultured rat aortic VSMC were studied during either static incubation in multiwell plates or during dynamic incubation in superfusion columns. Prostacyclin synthesis was assessed by radioimmunoassaying one of its stable metabolites, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha). Both ANG II and AVP stimulated the biosynthesis of prostacyclin in a concentration-dependent manner (10(-10) to 10(-5) M). ANG II (ED50 = 3 nM) displayed a higher potency than AVP (ED50 = 10 nM). ANG II was 4.4 times more potent than AVP at 10(-8) M. The effect of both peptides was inhibited selectively by antagonists. In the case of AVP (10(-8) M), a pure V1 antagonist (dEt2AVP) and the V2 agonist dDAVP, both at 10(-6) M, completely blocked the production of prostacyclin induced by AVP, whereas a mixed V1-V2 antagonist [d(CH2)5-D-Leu-VAVP] at 10(-6) M displaced the concentration-response curve by approximately two orders of magnitude to the right. Superfusion with a calcium-free medium containing ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid inhibited 89 +/- 3% of the ANG II- and 70 +/- 8% of the AVP-induced prostacyclin production, whereas nifedipine (10(-6) M) had no effect. A potentiating effect was observed when the stimulation with either ANG II or AVP was repeated two or three times. An even more marked potentiation resulted when the stimulation by ANG II (10(-8) M) followed stimulation by AVP (10(-8) M).(ABSTRACT TRUNCATED AT 250 WORDS)

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Stretch activates heparin-binding EGF-like growth factor expression in bladder smooth muscle cells

AJP Cell Physiology ◽

10.1152/ajpcell.1998.275.5.c1247 ◽

1998 ◽

Vol 275 (5) ◽

pp. C1247-C1254 ◽

Cited By ~ 120

Author(s):

John M. Park ◽

Joseph G. Borer ◽

Michael R. Freeman ◽

Craig A. Peters

Keyword(s):

Smooth Muscle ◽

Growth Factor ◽

Smooth Muscle Cells ◽

Fold Increase ◽

Muscle Cells ◽

Mechanical Stretch ◽

Mrna Levels ◽

Ang Ii ◽

Heparin Binding ◽

Bladder Smooth Muscle

Cultured rat bladder smooth muscle cells (SMC) were grown on collagen-coated silicone membranes and subjected to continuous cycles of stretch-relaxation. Semiquantitative RT-PCR analysis revealed a time-dependent increase in heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) mRNA levels after stretch, with maximal levels appearing after 4 h. Immunostaining for proHB-EGF revealed higher levels of HB-EGF protein in the stretched than in the nonstretched SMC. The ANG II receptor type 1 antagonist losartan markedly suppressed stretch-activated HB-EGF expression. ANG II levels were 3.3-fold higher in the stretch- than in the non-stretch-conditioned media. Stretch stimulation of bladder SMC that had been transiently transfected with an HB-EGF promoter-luciferase expression construct resulted in an 11-fold increase in reporter activity. Mechanical stretch induced a 4.7-fold increase in tritiated thymidine incorporation rate, and this was reduced by 25% in the presence of losartan. We conclude that mechanical stretch activates HB-EGF gene expression in bladder SMC and that this is mediated in part by autocrine ANG II secretion.

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IGF-1 receptor transactivation mediates Src-dependent cortactin phosphorylation in response to angiotensin IIThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y09-052 ◽

2009 ◽

Vol 87 (10) ◽

pp. 805-812 ◽

Cited By ~ 10

Author(s):

Peter Zahradka ◽

Benjamin Storie ◽

Brenda Wright

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Kinase Inhibitor ◽

Src Kinase ◽

Muscle Cells ◽

Dependent Manner ◽

Ang Ii ◽

Intracellular Signals ◽

And Migration ◽

Receptor Transactivation

Release of angiotensin II (Ang II) after vascular injury promotes tissue repair by stimulating phenotypic modulation of smooth muscle cells, which enables cell proliferation and migration. This process requires cytoskeleton remodeling, which involves cortactin, a scaffold protein that is phosphorylated by Src kinase in response to Ang II. Since insulin-like growth factor (IGF)-1 receptor transactivation mediates intracellular signals originating from the Ang II type 1 (AT1) receptor in a Src kinase-dependent manner, we examined whether IGF-1 receptor transactivation was also required for cortactin phosphorylation. Treatment of quiescent smooth muscle cells with Ang II resulted in both cortactin phosphorylation and its translocation to the plasma membrane. Both events were prevented by 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo(3,4-d)pyrimidin-4-amine (PP1), a Src kinase inhibitor, and by AG1024, an inhibitor of the IGF-1 receptor tyrosine kinase. Additionally, PP1 and AG1024 blocked the association of cortactin with actin-related protein (Arp) 3, an actin nucleation factor. These results indicate that Src kinase and the IGF-1 receptor kinase are necessary for activating cortactin. Phosphorylation of Src kinase in Ang II-treated cells was subsequently examined and was shown to be prevented by AG1024. Furthermore, Src kinase phosphorylation was blocked by inhibitors of protein kinase C (PKC), but not by inhibitors of phosphatidylinositol (PI) 3-kinase. These data establish that IGF-1 receptor transactivation is required for Src kinase-mediated cortactin phosphorylation and cytoskeletal reorganization in response to Ang II.

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Activation of Rho/Rho-Associated Kinase Pathway Is Involved in the Induction of Vascular Fibrogenesis.

Hypertension ◽

10.1161/hyp.36.suppl_1.688 ◽

2000 ◽

Vol 36 (suppl_1) ◽

pp. 688-688

Author(s):

Pierre-Louis Tharaux ◽

Sophie Vandermeersch ◽

Christos Chatziantoniou ◽

Jean-Claude Dussaule

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Gene Activation ◽

Fold Increase ◽

Muscle Cells ◽

Gene Activity ◽

Collagen I ◽

Luciferase Reporter ◽

Ang Ii ◽

I Gene

56 Abnormal extracellular accumulation of collagens and reorganization of the contractile cytoskeleton network in the vascular smooth muscle cells are characteristic features of angiosclerosis and end-organ damage during cardiovascular diseases. Rho-associated kinase (ROCK) activation is a key factor controling myosin phosphoryation and thus, its availability to form stress fibers. We have found previously that vasoconstrictor agents, such as angiotensin II (Ang II) or endothelin-1 (ET-1) play a major role in the activation of collagen I gene expression and synthesis within blood vessels . In the present study we investigated whether ROCK activation is involved in the profibrotic action of these peptides. Experiments were performed in transgenic mice harboring the luciferase reporter gene under the control of the collagen I- chain α2 promoter. Bolus iv. administration of pressive doses of Ang II or ET-1 induced an early (1 h) two-fold increase of collagen I gene activity in aortas. Co-administration of Y-27632, a selective inhibitor of ROCK, blocked the vasoconstrictor peptide-induced collagen I gene activation. Similar effects were obtained in freshly isolated aortas in vitro: Y-27632 blunted the Ang II- and ET-1-induced collagen I gene activation and the clustering of contractile cytoskeleton as evidenced by immunicytochemistry and confocal microscopy. Cytochalasin D, a potent inhibitor of actin polymerization also prevented the effect of Ang II and ET-1 on collagen I gene. In addition, aortic smooth muscle cells transfected with a constitutively active ROCK exhibited a two-fold increase in collagen I gene activity vs. control. These data suggest that ROCK is a major intracellular signaling pathway required for the early in vivo activation of collagen I gene induced by vasoactive peptides such as Ang II and ET-1 within the vasculature. It would be interesting to pursue in chronic studies whether ROCK inhibitors can be effeciently used as drugs against the development of vascular fibrogenesis.

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Probucol decreases homocysteine-stimulated CRP production in rat aortic smooth muscle cells via regulating HO-1/NADPH oxidase/ROS/p38 pathway

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmaa163 ◽

2020 ◽

Author(s):

Yuxia Li ◽

Qun Zhao ◽

Yuan Cao ◽

Jigang Si ◽

Jing Li ◽

...

Keyword(s):

Smooth Muscle ◽

Nadph Oxidase ◽

Smooth Muscle Cells ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Muscle Cells ◽

Heme Oxygenase 1 ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Aortic Smooth Muscle Cells ◽

Aortic Smooth Muscle

Abstract The elevated homocysteine level is an independent risk factor for atherosclerosis, which is characterized as a chronic inflammatory disease associated with oxidative stress. We have confirmed that homocysteine can stimulate the production of C-reactive protein (CRP) in rat aortic smooth muscle cells (RASMCs). In the present study, we investigated the role of probucol in homocysteine-induced CRP expression in cultured RASMCs and high-methionine-diet-induced hyperhomocysteinemic rats. The results showed that probucol decreased homocysteine-induced CRP mRNA and protein expression in RASMCs in a concentration-dependent manner. In addition, the animal experiment showed that probucol not only inhibited CRP expression in the vessel wall but also reduced the circulating CRP level in hyperhomocysteinemic rats. Further investigations revealed that probucol markedly increased heme oxygenase-1 activity, suppressed nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, diminished superoxide anion generation, and decreased p38 phosphorylation in RASMCs and hyperhomocysteinemic rat aorta. These data demonstrate that probucol can inhibit homocysteine-induced CRP generation by interfering with the NADPH oxidase/p38 signal pathway in RASMCs, which will provide new evidence for the anti-inflammatory and anti-atherosclerotic effects of probucol.

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