ABSTRACT
Smooth muscle cell migration in response to platelet-derived growth factor (PDGF) is a key event in several vascular pathologies, including atherosclerosis and restenosis. PDGF increases intracellular levels of reactive oxygen species (ROS) in vascular smooth muscle cells (VSMCs), but the ROS sensitivity of migration and of the signaling pathways leading to migration are largely unknown. In VSMCs, PDGF dose-dependently increased migration compared with nonstimulated cells, with a maximum increase at 10 ng/mL. Pretreatment with the antioxidant N-acetyl-cysteine, the flavin-containing enzyme inhibitor diphenylene iodonium, or the glutathione peroxidase mimetic ebselen significantly attenuated migration (PDGF alone, 5.0+/-1.1-fold; NAC, 1.8+/-0.2-fold; diphenylene iodonium, 1.4+/-0.3-fold migration; and ebselen, 2.0+/-0.5-fold migration), as did overexpression of catalase. Pretreatment of VSMCs with the Src inhibitor PP1 or dominant-negative Rac adenovirus significantly inhibited migration, but only Src activation was attenuated by ROS inhibitors. Phosphorylation of the Src- and Rac-effector p21-activated protein kinase (PAK) 1 on Thr423 (the phosphoinositide-dependent kinase-1 [PDK1] site) was attenuated by ROS inhibition, and infection of VSMCs with dominant-negative PAK1 adenovirus attenuated migration. Moreover, kinase-inactive K111N-PDK1 inhibited PAK1 phosphorylation on Thr423, and both K111N-PDK1 and Y9F-PDK1 significantly inhibited VSMC migration. PDK1 tyrosine phosphorylation was also ROS dependent. These data indicate that PDGF-induced VSMC migration is ROS dependent and identify the Src/PDK1/PAK1 signaling pathway as an important ROS-sensitive mediator of migration. Such information is critical to understanding the role of ROS in vascular diseases in which migration of VSMCs is an important component.
Subject(s)
Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/enzymology , Platelet-Derived Growth Factor/pharmacology , Protein Serine-Threonine Kinases/physiology , Reactive Oxygen Species/pharmacology , src-Family Kinases/physiology , 3-Phosphoinositide-Dependent Protein Kinases , Acetylcysteine/pharmacology , Animals , Aorta, Thoracic/cytology , Azoles/pharmacology , Becaplermin , Cell Movement/drug effects , Cell Movement/physiology , Cells, Cultured/drug effects , Cells, Cultured/enzymology , Enzyme Activation/drug effects , Humans , Isoindoles , Mitogen-Activated Protein Kinase 1/physiology , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/physiology , Myocytes, Smooth Muscle/drug effects , Organoselenium Compounds/pharmacology , Phosphorylation/drug effects , Protein Processing, Post-Translational/drug effects , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-sis , Rats , Receptors, Platelet-Derived Growth Factor/drug effects , Receptors, Platelet-Derived Growth Factor/physiology , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/physiology , Signal Transduction/drug effects , p21-Activated Kinases , rac GTP-Binding Proteins/physiology , src-Family Kinases/antagonists & inhibitorsABSTRACT
OBJECTIVE: We have previously shown that macrophage colony stimulating factor (M-CSF), a potent survival and mitogenic factor for monocytes/macrophages (MM), enables MM to induce vascular smooth muscle cell (VSMC) apoptosis. The killing requires the binding of MM to VSMC via Mac-1 (CD11b/CD18) on MM and intracellular adhesion molecule-1 (ICAM-1) on VSMC. We hypothesized that, in addition to Mac-1 binding, the killing process requires the activation of the Fas-death receptor pathway, which can be blocked at the level of Fas-Fas ligand interaction. METHODS AND RESULTS: Human peripheral blood monocytes and VSMC were isolated and cultured as previously described. Soluble Fas (sFas) was overexpressed in VSMC by transduction using adenovirus specifying soluble Fas (Ad3hsFas). M-CSF markedly increased the expression of ICAM-1 in VSMC, resulting in enhanced clustering of MM on the surface of VSMC (>/=3 MM per VSMC). MM, but not VSMC, expressed Fas-ligand (FasL), and VSMC apoptosis was inhibited by secretion of sFas by VSMC upon Ad3sFas transduction. CONCLUSIONS: MM and M-CSF-induced VSMC killing requires MM binding to VSMC mediated by Mac-1 and ICAM-1, and Fas-FasL interaction.
Subject(s)
Macrophage Activation , Macrophage Colony-Stimulating Factor/pharmacology , Macrophage-1 Antigen/metabolism , Muscle, Smooth, Vascular/pathology , fas Receptor/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Analysis of Variance , Apoptosis , Caspase 3 , Caspase Inhibitors , Coculture Techniques , Enzyme Inhibitors/pharmacology , Fas Ligand Protein , Flow Cytometry , Humans , Intercellular Adhesion Molecule-1/metabolism , Membrane Glycoproteins/metabolism , Muscle, Smooth, Vascular/metabolism , Protein Binding , Stimulation, Chemical , Transduction, Genetic , fas Receptor/geneticsABSTRACT
Angiotensin II (Ang II)-stimulated hypertrophy of vascular smooth muscle cells is mediated by reactive oxygen species (ROS) derived from NAD(P)H oxidases. The upstream signaling mechanisms by which Ang II activates these oxidases are unclear but may include protein kinase C, tyrosine kinases, phosphatidylinositol-3-kinase, and Rac, a small molecular weight G protein. We found that Ang II-stimulated ROS production is biphasic. The first phase occurs rapidly (peak at 30 seconds) and is dependent on protein kinase C activation. The larger second phase of ROS generation (peak at 30 minutes) requires Rac activation, because inhibition of Rac by either Clostridium difficile toxin A or dominant-negative Rac significantly inhibits Ang II-induced ROS production. Phosphatidylinositol-3-kinase inhibitors (wortmannin or LY294002) and the epidermal growth factor (EGF) receptor kinase blocker AG1478 attenuate both Rac activation and ROS generation. The upstream activator of EGF receptor transactivation, c-Src, is also required for ROS generation, because PP1, an Src kinase inhibitor, abrogates the Ang II stimulation of both responses. These results suggest that c-Src, EGF receptor transactivation, phosphatidylinositol-3-kinase, and Rac play important roles in the sustained Ang II-mediated activation of vascular smooth muscle cell NAD(P)H oxidases and provide insight into the integrated signaling mechanisms whereby Ang II stimulation leads to activation of the growth-related NAD(P)H oxidases.
Subject(s)
Angiotensin II/pharmacology , Muscle, Smooth, Vascular/drug effects , NADH, NADPH Oxidoreductases/metabolism , Androstadienes/pharmacology , Animals , Cells, Cultured , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , ErbB Receptors/metabolism , Flow Cytometry , Hydrogen Peroxide/metabolism , Indoles/pharmacology , Male , Maleimides/pharmacology , Morpholines/pharmacology , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , NADH, NADPH Oxidoreductases/drug effects , NADPH Oxidases , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Time Factors , Wortmannin , rac GTP-Binding Proteins/metabolism , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/metabolismABSTRACT
BACKGROUND: Plaque disruption is the inciting event for coronary thrombosis and acute coronary syndromes. Multiple factors influence plaque rupture, including the loss of vascular smooth muscle cells (VSMCs). We hypothesized that monocytes/macrophages (MMs) activated by macrophage colony-stimulating factor (M-CSF) are responsible for VSMC death. METHODS AND RESULTS: VSMC apoptosis was markedly increased in the presence of both M-CSF and MMs (58.8+/-3.3%) compared with VSMCs plus M-CSF without MMs (15.7+/-1.5%, P< or =0.00005), VSMCs plus MMs without M-CSF (22.7+/-3.7%, P< or =0.0001), or control VSMCs alone (13.2+/-2.1%, P< or =0.0001). MM cell contact was required for M-CSF-stimulated killing of VSMCs, and MMs displayed an M-CSF concentration-dependent killing effect. Abciximab binds Mac-1 (CD11b/CD18) on MMs. When added to VSMCs exposed to MMs and M-CSF, abciximab (7 microg/mL) significantly reduced VSMC apoptosis (19.1+/-2.2%, P< or =0.0003). Therapeutic doses of tirofiban (0.35 microg/mL) and eptifibatide (5 microg/mL), which inhibit platelet glycoprotein (GP) IIb/IIIa but not Mac-1, did not block activated MM-induced VSMC apoptosis (65.0+/-3.4% and 51.3+/-2.5%, respectively). A recombinant anti-CD-18 antibody had an effect similar to that of abciximab (16.5+/-0.4%). CONCLUSIONS: These data suggest that monocytes and physiological concentrations of M-CSF trigger VSMC apoptosis. Abciximab and specific inhibitors of the Mac-1 receptor can antagonize this process.
