ABSTRACT
The roles of Src homology domain 2-containing protein tyrosine phosphatase 2 (SHP-2) and its signaling in atherosclerosis have not been explored. Therefore, we investigated the roles of SHP-2 in the movement of rat aortic smooth muscle cells (RASMCs) and in the neointima formation of the carotid artery. Platelet-derived growth factor (PDGF)-BB (1 - 20 ng/ml) increased the activity and phosphorylation of SHP-2 and migration in RASMCs and these were suppressed by SHP-2 inhibitor NSC-87877 (30 µM) and small interfering RNA of SHP-2. PDGF-BB increased the phosphorylations of spleen tyrosine kinase (Syk) and p38 mitogen-activated protein kinase (MAPK), which were recovered by inhibition of SHP-2. Moreover, PDGF-BB increased the levels of reactive oxygen species (ROS) and ROS inhibitors decreased PDGF-BB-increased migration. Treatment of RASMCs with H(2)O(2) (100 µM) increased cell migration and SHP-2 phosphorylation and also enhanced the phosphorylation levels of Syk and p38 MAPK. Oral administration of NSC-87877 (10 mg/kg) significantly suppressed neointima formation in a rat model of carotid artery injury. These results suggest that the activity of SHP-2 is controlled by ROS and is positively involved in the regulation of PDGF-BB-induced RASMC migration and neointima formation.
Subject(s)
Myocytes, Smooth Muscle/pathology , Neointima/physiopathology , Platelet-Derived Growth Factor/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Reactive Oxygen Species/metabolism , Animals , Aorta/cytology , Becaplermin , Carotid Arteries/pathology , Cell Movement/drug effects , Hydrogen Peroxide/pharmacology , Male , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/metabolism , Oxidants/pharmacology , Phosphorylation/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Proto-Oncogene Proteins c-sis , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effectsABSTRACT
The roles of Src homology domain 2-containing protein tyrosine phosphatase 2 (SHP-2) and its signaling in atherosclerosis have not been explored. Therefore, we investigated the roles of SHP-2 in the movement of rat aortic smooth muscle cells (RASMCs) and in the neointima formation of the carotid artery. Platelet-derived growth factor (PDGF)-BB (1 - 20 ng/ml) increased the activity and phosphorylation of SHP-2 and migration in RASMCs and these were suppressed by SHP-2 inhibitor NSC-87877 (30 µM) and small interfering RNA of SHP-2. PDGF-BB increased the phosphorylations of spleen tyrosine kinase (Syk) and p38 mitogen-activated protein kinase (MAPK), which were recovered by inhibition of SHP-2. Moreover, PDGF-BB increased the levels of reactive oxygen species (ROS) and ROS inhibitors decreased PDGF-BB-increased migration. Treatment of RASMCs with H2O2 (100 µM) increased cell migration and SHP-2 phosphorylation and also enhanced the phosphorylation levels of Syk and p38 MAPK. Oral administration of NSC-87877 (10 mg/kg) significantly suppressed neointima formation in a rat model of carotid artery injury. These results suggest that the activity of SHP-2 is controlled by ROS and is positively involved in the regulation of PDGF-BB-induced RASMC migration and neointima formation.
ABSTRACT
In this study, we clarified the intracellular mechanism of angiotensin II (Ang II) in promoting migration in rat aortic smooth muscle cells (RASMCs). RASMC migration was measured with the Boyden chamber assay, and the result was confirmed with an aortic sprout assay. The activities of kinases were investigated by western blot analysis. Ang II enhanced RASMC migration, which was chemotaxis directed, and induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), and heat shock protein 27 (Hsp27). Ang II-enhanced cell migration was inhibited by SB203580 (a p38 MAPK inhibitor) and piceatannol (a spleen tyrosine kinase inhibitor), but only partially by PD98059 (an ERK inhibitor) and PP2 (a Src inhibitor). The Ang II-stimulated phosphorylation of p38 MAPK and Hsp27 in RASMCs was inhibited by piceatannol and SB203580. The phosphorylation of ERK1/2 stimulated by Ang II was suppressed by PD98059, piceatannol, and PP2. Ang II increased the sprout outgrowth from aortic rings and this response was attenuated by pretreatment with SB203580, PD98059, PP2, or piceatannol. These results suggest that p38 MAPK contributes to the regulation of the Ang II-induced chemotactic migration of vascular smooth muscle cells, which is mediated by Hsp27 phosphorylation.
Subject(s)
Angiotensin II/pharmacology , Cell Movement/drug effects , Muscle, Smooth, Vascular/drug effects , p38 Mitogen-Activated Protein Kinases/metabolism , Actins/metabolism , Animals , Aorta/cytology , Aorta/drug effects , Aorta/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Imidazoles/pharmacology , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mitogen-Activated Protein Kinase 1/metabolism , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Phosphorylation/drug effects , Protein-Tyrosine Kinases/metabolism , Pyridines/pharmacology , Pyrimidines/pharmacology , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Stilbenes/pharmacology , Syk Kinase , Vasoconstrictor Agents/pharmacologyABSTRACT
Although spleen tyrosine kinase (Syk) has crucial roles in various cells, its function on vascular smooth muscle contraction has not been determined. In the present study, we performed experiments to determine if Syk contributes to the endothelin-1 (ET-1)-mediated contraction in rat aortic smooth muscle. ET-1-induced contraction of aortic strips was inhibited by piceatannol, PD98059, and SB203580, inhibitors of Syk, extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK), respectively. Piceatannol also attenuated high K(+)-induced contraction. ET-1 dose-dependently enhanced the activity of Syk and this was inhibited by piceatannol in both rat aortic strip and rat aortic smooth muscle cells. The phosphorylation of p38 MAPK and heat shock protein 27 (HSP27), but not that of ERK1/2, in response to ET-1 was inhibited by both piceatannol and SB203580. These results suggest that Syk may play an important role in the regulation of aortic smooth muscle contraction induced by ET-1, which may be mediated by the p38 MAPK/HSP27 signaling pathway.
Subject(s)
Endothelin-1/pharmacology , Intracellular Signaling Peptides and Proteins/metabolism , Muscle, Smooth, Vascular/drug effects , Protein-Tyrosine Kinases/metabolism , Vasoconstriction/drug effects , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Aorta/cytology , Aorta/drug effects , Aorta/physiology , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cells, Cultured , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Imidazoles/pharmacology , Immunoblotting , Immunoprecipitation , In Vitro Techniques , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Male , Muscle, Smooth, Vascular/physiology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Phosphorylation/drug effects , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , Stilbenes/pharmacology , Syk Kinase , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
OBJECTIVE: Here we investigated the role of spleen tyrosine kinase (Syk) in the migration induced by platelet-derived growth factor (PDGF) in rat aortic smooth muscle cells (RASMC). METHODS: Cell migration was determined using a Boyden chamber, by wound-healing, and by aortic ring assays. Activity of Syk, mitogen-activated protein kinase (MAPK), and heat shock protein 27 (HSP27) were tested using immunoblotting with kinase inhibitors and small interference RNAs. RESULTS: PDGF-BB induced binding of Syk to the PDGFbeta receptor and increased the phosphorylation of Syk and migration in RASMC. These effects of PDGF-BB were inhibited by piceatannol, an inhibitor of Syk. PDGF-BB increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and HSP27, which were significantly inhibited by piceatannol and in Syk-knockdown cells. The p38 MAPK inhibitor SB203580 and ERK1/2 inhibitor PD98059 inhibited the migration, which was further inhibited by the combination of these inhibitors. SB203580, but not PD98059, inhibited the phosphorylation of HSP27 induced by PDGF-BB in RASMC. PDGF-BB-induced migration was attenuated in HSP27-knockdown cells. Kinase inhibitors and Syk-knockdown diminished PDGF-BB-induced sprout outgrowth in the aortic ring assay. CONCLUSIONS: These results imply that Syk is an upstream signal of the p38 MAPK/HSP27 and ERK1/2 pathways that contributes to PDGF-BB-mediated migration in RASMC.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinases/metabolism , Myocytes, Smooth Muscle/enzymology , Platelet-Derived Growth Factor/pharmacology , Protein-Tyrosine Kinases/metabolism , Animals , Aorta , Becaplermin , Cell Movement/drug effects , Cells, Cultured , Enzyme Activation , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Imidazoles/pharmacology , Immunoblotting , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/analysis , Intracellular Signaling Peptides and Proteins/genetics , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Myocytes, Smooth Muscle/cytology , Phosphorylation , Platelet-Derived Growth Factor/metabolism , Protein-Tyrosine Kinases/analysis , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins c-sis , Pyridines/pharmacology , RNA Interference , RNA, Small Interfering , Rats , Receptor, Platelet-Derived Growth Factor beta/metabolism , Stilbenes/pharmacology , Syk Kinase , Transfection/methodsABSTRACT
The role of mitogen-activated protein kinase (MAPK) in the decreased contractile response to phorbol ester in aortic smooth muscle strips from deoxycorticosterone acetate (DOCA)-salt hypertensive rats was examined. Norepinephrine (NE) evoked greater contractility in aortic strips from DOCA rats than in those of sham-operated rats. 12-Deoxyphorbol 13-isobutyrate (DPB) induced contraction in Ca2+-free medium, which was diminished in strips from DOCA rats compared to sham-operated rats. Vasoconstrictions induced by these stimulants were inhibited by SB203580 and PD098059, inhibitors of p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2, respectively, in both strips. The phosphorylation of p38 MAPK and ERK1/2 induced by NE was greater in strips from DOCA rats compared to those from sham-operated rats, and this phosphorylation was inhibited by the kinase inhibitors. DPB increased the phosphorylation of p38 MAPK and ERK1/2 in strips from both animals, and the increment of p38 MAPK phosphorylation by the stimulant was diminished in strips from DOCA rats compared to sham-operated rats. These findings suggest that the Ca2+-independent contraction evoked by DPB results from the activation of MAPKs in rat aortic smooth muscle and that the attenuated contractility by DPB in DOCA rat appears to be associated with diminished p38 MAPK activity.
Subject(s)
Hypertension/physiopathology , Muscle, Smooth, Vascular/drug effects , Phorbol Esters/pharmacology , p38 Mitogen-Activated Protein Kinases/physiology , Animals , Aorta, Thoracic/drug effects , Blood Pressure/drug effects , Body Weight/drug effects , Desoxycorticosterone , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Hypertension/chemically induced , Imidazoles/pharmacology , Isometric Contraction/drug effects , Male , Muscle Contraction/drug effects , Organ Size/drug effects , Phosphorylation , Protein Kinase C/metabolism , Pyridines/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
We investigated whether increased contractile responsiveness to epidermal growth factor (EGF) is associated with altered activation of mitogen-activated protein kinase (MAPK) in the aortic smooth muscle of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. EGF induced contraction and MAPK activity in aortic smooth muscle strips, which were significantly increased in tissues from the DOCA-salt hypertensive rats compared with those from sham-operated rats. AG1478, PD98059, and LY294002, inhibitors of EGF receptor (EGFR) tyrosine kinase, MAPK/extracellular signal-regulated kinase (ERK) kinase, and phosphatidylinositol 3-kinase (PI3K), respectively, inhibited the contraction and the activity of ERK1/2 that were elevated by EGF. Y27632 and GF109203X, inhibitors of Rho kinase and protein kinase C, respectively, attenuated EGF-induced contraction, with no diminution of ERK1/2 activity. Although EGF also elevated the activity of EGFR tyrosine kinase in both sham-operated and DOCA-salt hypertensive rats, the expression and the magnitude of activation did not differ between strips. These results strongly suggest that EGF induces contraction by the activation of ERK1/2, which is regulated by the PI3K pathway in the aortic smooth muscle of DOCA-salt hypertensive rats.
Subject(s)
Epidermal Growth Factor/pharmacology , Hypertension/enzymology , Mitogen-Activated Protein Kinases/physiology , Phosphatidylinositol 3-Kinases/metabolism , Vasoconstriction/drug effects , Animals , Aorta/cytology , Desoxycorticosterone/administration & dosage , Desoxycorticosterone/toxicity , Dose-Response Relationship, Drug , ErbB Receptors/antagonists & inhibitors , Hypertension/chemically induced , Hypertension/physiopathology , Male , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Muscle, Smooth, Vascular/drug effects , Rats , Rats, Sprague-Dawley , Sodium Chloride, Dietary/toxicityABSTRACT
The cardioprotective effects of the novel sodium/hydrogen exchanger-1 (NHE-1) inhibitor KR-32560 {[5-(2-methoxy-5-fluorophenyl)furan-2-ylcarbonyl]guanidine} were studied in an anesthetized rat model of 30-min ischemia / 2.5-h reperfusion heart injury. KR-32560 (0.01 - 1 microM) dose-dependently inhibited NHE-1-mediated rabbit platelet swelling induced by intracellular acidification. KR-32560 at 0.1 and 1.0 mg/kg (i.v. bolus, given 10 min before ischemia) reduced infarct size from 65.9% (control) to 49.7% and 32.7%, respectively, while reducing the extension of myocardial injury (mm(3)/g of left heart weight) from 405.1 (control) to 302.9 and 185.4, respectively (all P<0.05 vs control). KR-32560 dose-dependently reduced the total number of ventricular premature beats (VPBs) during ischemia from 510.2 (control) to 353.8 and 134.2 beats (all P<0.05, n = 6), while reducing ventricular tachycardia (VT) incidence from 49.3 (control) to 26.8 and 4.3 and VT duration from 249.2 s (control) to 150.5 and 26.7 s (all P<0.05, n = 6). KR-32560 dose-dependently reduced ventricular fibrillation (VF) incidence from 19.0 (control) to 9.2 and 1.2 and VF duration from 88.0 s to 34.5 and 2.8 s (all P<0.05, n = 6). KR-32560 also exerted similar effects on reperfusion arrhythmias, except for VPBs. These results indicate that KR-32560 may exert significant cardioprotective effects in ischemia/reperfusion heart injury.
Subject(s)
Arrhythmias, Cardiac/prevention & control , Guanidines/pharmacology , Myocardial Infarction/prevention & control , Myocardial Reperfusion Injury/prevention & control , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Animals , Arrhythmias, Cardiac/etiology , Arrhythmias, Cardiac/physiopathology , Cardiotonic Agents/pharmacology , Disease Models, Animal , Dose-Response Relationship, Drug , Guanidines/chemistry , Heart Ventricles , Male , Molecular Structure , Myocardial Infarction/pathology , Myocardial Reperfusion Injury/pathology , Rabbits , Rats , Rats, Sprague-Dawley , Time FactorsABSTRACT
The effects of a novel sodium/hydrogen exchanger-1 (NHE-1) inhibitor, KR-32568, were studied in an anesthetized rat model of 30 min ischemia/2.5 h reperfusion heart injury. KR-32568 dose-dependently inhibited NHE-1-mediated rabbit platelet swelling induced by intracellular acidification. In our anesthetized rat model, KR-32568 reduced infarct size from 67 (control) to 43 and 24% at 0.1 and 1.0 mg/kg (i.v. bolus, given 10 min before ischemia), respectively. KR-32568 at the same doses also significantly reduced the total number of ventricular premature beats during ischemia/reperfusion from 530 (control) to 266 and 115 beats, ventricular tachycardia (VT) incidence from 51 (control) to 21 and 8, VT duration from 238 s (control) to 63 and 33 s, ventricular fibrillation (VF) incidence from 17 (control) to 8 and 0, and VF duration from 85 s to 18 and 1 s. These results indicate that KR-32568 may exert potent cardioprotective effects in rats via inhibition of sodium/hydrogen exchanger-1.