The peptide angiotensin II is the effector molecule of the reninangiotensin system. All the haemodynamic effects of angiotensin II, including vasoconstriction and adrenal aldosterone release, are mediated through a single class of cell-surface receptors known as AT1 (refs 1, 2). These receptors contain the structural features of the G-protein-coupled receptor superfamily. We show here that angiotensin II induces the rapid phosphorylation of tyrosine in the intracellular kinases Jak2 and Tyk2 in rat aortic smooth-muscle cells and that this phosphorylation is associated with increased activity of Jak2. The Jak family substrates STAT1 and STAT2 (for signal transducers and activators of transcription) are rapidly tyrosine-phosphorylated in response to angiotensin II. We also find that Jak2 co-precipitates with the AT1 receptor, indicating that G-protein-coupled receptors may be able to signal through the intracellular phosphorylation pathways used by cytokine receptors.

Vascular smooth muscle cells (VSMCs) proliferate in response to arterial injury. Recent findings suggest that, in addition to platelet-derived growth factors, growth factors from inflammatory cells and endothelial cells at the site of injury may contribute to VSMC proliferation. We hypothesized that a common mechanism by which endothelial cells and inflammatory cells stimulate VSMC growth could be the active oxygen species (i.e., 02, H202, and OH) generated during arterial injury. Using xanthine/ xanthine oxidase to generate active oxygen species, we studied the effects of these agents on VSMC growth. Xanthine/xanthine oxidase (100 ,M xanthine and 5 microunits/ml xanthine oxidase) stimulated DNA synthesis in growth-arrested VSMCs by 180%o over untreated cells. All tissues are constantly exposed to exogenous and endogenous oxidants.4"15 Vascular endothelial cells exhibit metabolic activities that may produce high concentrations of active oxygen species.16-'8 In fact, the physiological production of endothelium-derived relaxing factor necessarily involves generation of active oxygen species. Active oxygen species concentrations are increased in blood vessels and myocardium in response to a variety of injury-related conditions such as ischemia, thrombosis and reperfusion, and angioplasty.19'20 These same circumstances frequently are associated with intimal hyperplasia and accelerated atherosclerosis. Therefore, there may be a relation between arterial injury, active oxygen species production, and VSMC proliferation. To test this hypothesis, we studied the effects of active oxygen species on VSMC growth and c-myc and c-fos mRNA levels.2",22 In this work, we show that H202 specifically stimulates VSMC DNA synthesis and protooncogene expression. Materials and Methods Cell CultureVSMCs were isolated from the thoracic aortas of 200-250-g male Sprague-Dawley rats by enzymatic dissociation as described previously.5'6 Cells were grown in Dulbecco's modified Eagle's medium (DME) supplemented with 10% (vol/vol) heat-inactivated calf serum, 100 units/ml penicillin, and 100 ,ug/ml streptomycin.The cultures were maintained in humidified 95%

Abstract-Reactive oxygen species have been implicated in the pathogenesis of atherosclerosis, hypertension, and restenosis, in part by promoting vascular smooth muscle cell (VSMC) growth. Many VSMC growth factors are secreted by VSMC and act in an autocrine manner. Here we demonstrate that cyclophilin A (CyPA), a member of the immunophilin family, is secreted by VSMCs in response to oxidative stress and mediates extracellular signal-regulated kinase (ERK1/2) activation and VSMC growth by reactive oxygen species. Human recombinant CyPA can mimic the effects of secreted CyPA to stimulate ERK1/2 and cell growth. The peptidyl-prolyl isomerase activity is required for ERK1/2 activation by CyPA. In vivo, CyPA expression and secretion are increased by oxidative stress and vascular injury. These findings are the first to identify CyPA as a secreted redox-sensitive mediator, establish CyPA as a VSMC growth factor, and suggest an important role for CyPA and enzymes with peptidyl-prolyl isomerase activity in the pathogenesis of vascular diseases. (Circ Res. 2000;87:789 -796.)Key Words: oxidative stress Ⅲ cyclophilin Ⅲ secretion Ⅲ mitogen-activated protein kinase Ⅲ smooth muscle cells R eactive oxygen species (ROS) have been implicated in the pathogenesis of atherosclerosis, hypertension, and restenosis, in part by promoting vascular smooth muscle cell (VSMC) growth. [1][2][3][4] We have previously reported that ROS stimulate VSMC growth and DNA synthesis. 5 This proliferation was associated with stimulation of protein kinases, especially the extracellular signal-regulated kinases (ERK1/2, also termed p42/44 mitogen-activated protein kinases [MAPKs]). 4 ERK1/2 are stimulated by growth factors and cytokines and play pivotal roles in cell growth and differentiation. 6,7 Activation of ERK1/2 by ROS generators, such as the napthoquinolinedione LY83583, menadione, and xanthine/xanthine oxidase as well as H 2 O 2 , was biphasic; an early peak of ERK1/2 activity was present at 5 to 10 minutes, whereas a delayed ERK1/2 activation appeared at 2 hours. 8 A similar biphasic activation of ERK1/2 has been reported for mitogens such as fibroblast growth factor. 9 Recently, the delayed ERK1/2 activation has been reported to be mediated by different mechanisms than the early ERK1/2 activation and to be critical for cell cycle progression and cell proliferation. 9,10 Increasing evidence suggests that secretion of growth factors in response to VSMC agonists mediates their mitogenic activity. For example, epiregulin, an epidermal growth factor-related growth factor, is a potent VSMC-secreted mitogen whose expression is regulated by angiotensin II, endothelin-1, and thrombin. 11 These same agonists also stimulate secretion of other growth factors, including plateletderived growth factor 12,13 and transforming growth factor-␤. 14 However, no factors have been identified as mediators of VSMC proliferation in response to ROS.We hypothesized that in response to ROS, VSMCs may secrete factors that participate in autocrine and paracrine growth mecha...

Endothelial cells release nitric oxide (NO) more potently in response to increased shear stress than to agonists which elevate intracellular free calcium concentration ([Ca2+]i). To determine mechanistic differences in the regulation of endothelial constitutive NO synthase (ecNOS), we measured NO production by bovine aortic endothelial cells exposed to shear stress in a laminar flow chamber or treated with Ca2+ ionophores in static culture. The kinetics of cumulative NO production varied strikingly: shear stress (25 dyne/cm2) stimulated a biphasic increase over control that was 13-fold at 60 minutes, whereas raising [Ca2+]i caused a monophasic 6-fold increase. We hypothesized that activation of a protein kinase cascade mediates the early phase of flow-dependent NO production. Immunoprecipitation of ecNOS showed a 210% increase in phosphorylation 1 minute after flow initiation, whereas there was no significant increase after Ca2+ ionophore treatment. Although ecNOS was not tyrosine-phosphorylated, the early phase of flow-dependent NO production was blocked by genistein, an inhibitor of tyrosine kinases. To determine the Ca2+ requirement for flow-dependent NO production, we measured [Ca2+]i with a novel flow-step protocol. [Ca2+]i increased with the onset of shear stress, but not after a step increase. However, the step increase in shear stress was associated with a potent biphasic increase in NO production rate and ecNOS phosphorylation. These studies demonstrate that shear stress can increase NO production in the absence of increased [Ca2+]i, and they suggest that phosphorylation of ecNOS may importantly modulate its activity during the imposition of increased shear stress.