Angiotensin II–Induced Vascular Smooth Muscle Cell Migration and Growth Are Mediated by Cytochrome P450 1B1–Dependent Superoxide Generation

Abstract

Abstract-Cytochrome P450 1B1, expressed in vascular smooth muscle cells, can metabolize arachidonic acid in vitro into several products including 12-and 20-hydroxyeicosatetraenoic acids that stimulate vascular smooth muscle cell growth. This study was conducted to determine whether cytochrome P450 1B1 contributes to angiotensin II-induced rat aortic smooth muscle cell migration, proliferation, and protein synthesis. Angiotensin II stimulated migration of these cells, measured by the wound healing approach, by 1. Metabolism of arachidonic acid to 5-, 12-, 15-, and 20-hydoxyeicosatetraenoic acids in these cells was not altered, but angiotensin II-and arachidonic acid-induced reactive oxygen species production and extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase activity were inhibited by 2,4,3Ј,5Ј-tetramethoxystilbene and cytochrome P450 1B1 small hairpin RNA (shRNA) and by Tempol, which inactivates reactive oxygen species. Tempol did not alter cytochrome P450 1B1 activity. These data suggest that angiotensin II-induced vascular smooth muscle cell migration and growth are mediated by reactive oxygen species generated from arachidonic acid by cytochrome P450 1B1 and activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. (Hypertension. 2010;55:1461-1467.) Key Words: angiotensin II Ⅲ CYP1B1 Ⅲ vascular smooth muscle cell growth Ⅲ ROS T he renin-angiotensin system is one of the major components of the mechanisms that contribute to the regulation of blood volume and vascular resistance. 1 Angiotensin II (Ang II), the main biologically active agent of this system, also stimulates vascular smooth muscle cell (VSMC) hypertrophy and/or hyperplasia and inflammation and contributes to the development of hypertension, atherosclerosis, heart failure, and restenosis after vascular injury. [1][2][3][4][5][6] The pathophysiological actions of Ang II are mediated by activation of Ն1 serine-threonine and tyrosine kinase, generation of oxygen radicals, 7-9 and/or release of arachidonic acid (AA) by cytosolic phospholipase A 2 (cPLA 2 ) and production of its metabolites, 12-hydroxyeicosatetraenoic acid (12-HETE) and 20-HETE, generated via lipoxygenase and/or cytochrome P450 (CYP) 4A, respectively. 10 -18 Both 12-and 20-HETE promote VSMC migration, hyperplasia, and/or hypertrophy. 11,19 -22 CYP enzymes that metabolize xenobiotics, including polycyclic aromatic hydrocarbons and endobiotics, such as fatty acids and retinoids, are also expressed in extrahepatic tissues, including the cardiovascular system. 23-27 CYP1A1-encoded enzymes are expressed in vascular endothelium and smooth muscle cells, with much higher levels of activity in endothelial cells, whereas CYP1B1 is highly expressed in VSMCs and, to a lesser degree, in endothelial cells, 28,29 but shear stress upregulates mRNA and protein levels of CYP1A1 and CYP1B1 in endothelial cells. 30 Whether CYP1A1 and CYP1B1 contribute to the vascular function is not known. Recombinant CYP1B1 has been shown to me...

Cytochrome P450 1B1 Is Critical for Neointimal Growth in Wire‐Injured Carotid Artery of Male Mice

“…Among many agents, including cytokines, Ang II, and growth factors, PDGF is considered to be a primary stimulator of VSMC migration and proliferation, acting on the PDGF‐β receptor in medial SMCs and leading to neointimal growth 7, 20. Ang II– and PDGF‐BB–induced VSMC migration and proliferation in vitro are mediated via generation of ROS 6, 21. Ang II stimulates ROS production in VSMCs via activation of nicotinamide adenine dinucleotide phosphate (NADPH oxidase), which is dependent on arachidonic acid (AA) 22.…”

“…Previously, we have reported that Ang II–induced VSMC migration and proliferation are mediated by ROS generated from the metabolism of AA by CYP1B1, resulting in stimulation of ≥1 signaling molecules 6. In the present study, the PDGF‐BB–induced increase in ROS, measured as H 2 O 2 in VSMCs, and their migration and proliferation were attenuated by Cyp1b1 gene disruption and by Tempol, which does not alter CYP1B1 activity6 and inactivates superoxides as well as H 2 O 2 by superoxide dismutase– and catalase‐like actions, respectively 9. This suggested that these effects of PDGF‐BB are also dependent on CYP1B1.…”

“…In the present study, PDGF‐BB did not alter CYP1B1 protein expression or its activity, and Cyp1b1 gene disruption also did not alter expression of PDGF‐B in VSMCs. CYP1B1 is constitutively active and requires substrate to generate ROS 6. Therefore, the demonstration that PDGF‐BB activates cPLA 2 (cytosolic phospholipase A 2 ) and releases AA, which participates in VSMC proliferation23 and migration,24 suggests that PDGF‐BB most likely also generates ROS via CYP1B1‐catalyzed AA metabolism, similar to Ang II 6.…”

“…CYP1B1 is constitutively active and requires substrate to generate ROS 6. Therefore, the demonstration that PDGF‐BB activates cPLA 2 (cytosolic phospholipase A 2 ) and releases AA, which participates in VSMC proliferation23 and migration,24 suggests that PDGF‐BB most likely also generates ROS via CYP1B1‐catalyzed AA metabolism, similar to Ang II 6. The mechanism by which AA metabolism by CYP1B1 leads to the activation of NADPH oxidase still remains to be elucidated and is one of the limitations in our study.…”

“…CYP1B1 also mediates angiotensin II (Ang II)–induced migration, proliferation, and growth of rat VSMCs, via generation of reactive oxygen species (ROS) 6. The present study was conducted to determine if CYP1B1 also contributes, via ROS production, to the action of platelet‐derived growth factor‐BB (PDGF‐BB) on VSMC migration and proliferation.…”