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.

Abstract-Mechanical forces are important modulators of cellular function in many tissues and are particularly important in the cardiovascular system. The endothelium, by virtue of its unique location in the vessel wall, responds rapidly and sensitively to the mechanical conditions created by blood flow and the cardiac cycle. In this study, we examine data which suggest that steady laminar shear stress stimulates cellular responses that are essential for endothelial cell function and are atheroprotective. We explore the ability of shear stress to modulate atherogenesis via its effects on endothelial-mediated alterations in coagulation, leukocyte and monocyte migration, smooth muscle growth, lipoprotein uptake and metabolism, and endothelial cell survival. We also propose a model of signal transduction for the endothelial cell response to shear stress including possible mechanotransducers (integrins, caveolae, ion channels, and G proteins N umerous studies suggest that normal functioning of the endothelium is critical in limiting the development of atherosclerosis, as illustrated by the correlation between risk factors for atherosclerosis (smoking, high cholesterol, high homocysteine, decreased estrogen, increasing age, and hypertension) and endothelial dysfunction.1 Endothelial cells play a critical role in vascular homeostasis by performing many functions. They sense and integrate hemodynamic and hormonal stimuli and effect alterations in vascular function through the secretion of various mediator proteins and molecules.2 As a result of these properties, endothelial cells modulate biological processes related to the blood vessel wall, including regulation of the permeability of plasma lipoproteins, adhesion of leukocytes, and release of prothrombotic and antithrombotic factors, growth factors, and vasoactive substances.3 Impairment of these endothelial cell-mediated processes has been postulated to play a central role in the pathogenesis of atherosclerosis. 1Just as other tissues have developed mechanisms to detect changes in the physiological conditions to which they are exposed, endothelial cells respond not only to humoral factors in the circulation but also to the mechanical conditions created by blood flow and the cardiac cycle. 4 As a result of their unique location, endothelial cells experience three primary mechanical forces: pressure, created by the hydrostatic forces of blood within the blood vessel; circumferential stretch or tension, created as a result of defined intercellular connections between the endothelial cells that exert longitudinal forces on the cell during vasomotion; and shear stress, the dragging frictional force created by blood flow. Of these forces, shear stress appears to be a particularly important hemodynamic force because it stimulates the release of vasoactive substances and changes gene expression, cell metabolism, and cell morphology. 4 The nature and magnitude of shear stress plays an important role in long-term maintenance of the structure and function of the blood vessel. T...

Vascular smooth muscle cells (VSMC) exhibit several growth responses to agonists that regulate their function including proliferation (hyperplasia with an increase in cell number), hypertrophy (an increase in cell size without change in DNA content), endoreduplication (an increase in DNA content and usually size), and apoptosis. Both autocrine growth mechanisms (in which the individual cell synthesizes and/or secretes a substance that stimulates that same cell type to undergo a growth response) and paracrine growth mechanisms (in which the individual cells responding to the growth factor synthesize and/or secrete a substance that stimulates neighboring cells of another cell type) are important in VSMC growth. In this review I discuss the autocrine and paracrine growth factors important for VSMC growth in culture and in vessels. Four mechanisms by which individual agonists signal are described: direct effects of agonists on their receptors, transactivation of tyrosine kinase-coupled receptors, generation of reactive oxygen species, and induction/secretion of other growth and survival factors. Additional growth effects mediated by changes in cell matrix are discussed. The temporal and spatial coordination of these events are shown to modulate the environment in which other growth factors initiate cell cycle events. Finally, the heterogeneous nature of VSMC developmental origin provides another level of complexity in VSMC growth mechanisms.

To investigate the role of vasoconstrictor hormones in vascular smooth muscle cell growth we have studied the effects of the potent vasoconstrictor angiotensin II on cell growth in a cultured rat aortic cell model. Angiotensin II was not mitogenic for these cells, as assessed by determining cell number, nor was it synergistic in this regard with 10% calf serum. However, 24-hour exposure to 100 nM angiotensin II caused an 80% increase in protein synthesis (compared with 0.4% increase with serum control) as measured by tritiated leucine incorporation. This was a "hypertrophic" response as indicated by a 30% increase in protein content and a 45% increase in cell volume. Angiotensin O-induced smooth muscle cell hypertrophy was maximal at 100 nM, had In hypertensive models such as aortic coarctation and experimental injury models of atherosclerosis, VSMC migration into and proliferation (hyperplasia) in the intima is the most dramatic pathological feature. Received March 28, 1988; accepted November 18, 1988. 107-111-day-old SHR that VSMC proliferation rather than hypertrophy or hyperploidy appeared to account for the increase in VSMC mass. Several factors have been implicated in growth of VSMC in these models. In experimental injury models, endothelial dysfunction or denudation may result in abnormal interactions between elements in the blood (platelets, polymorphonuclear leukocytes, and monocytes) and the vessel wall that lead to sustained release of a variety of growth factors including platelet-derived growth factor. These growth factors are both chemotactic and mitogenic for VSMC 67 and thus may contribute to VSMC migration and proliferation in this model. In certain hypertensive models the increased blood pressure may result in altered mechanical stress that stimulates VSMC growth in a manner analogous to the effects of stretch on skeletal muscle protein synthesis.8 This explanation is supported by data that demonstrate a significant correlation between blood pressure and aortic VSMC hypertrophy and polyploidy. 6 -7 Furthermore, reduction of blood pressure in the SHR model can decrease VSMC polyploidy and hypertrophy.

Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. Because mitogen-activated protein (MAP) kinases have been shown to be activated by physical forces, we measured the phosphorylation and enzyme activity of MAP kinase to identify the signal events involved in the endothelial cell response to fluid shear stress. Flow at physiological shear stress (3.5 to 117 dynes/cm2) activated 42-kD and 44-kD MAP kinases present in cultured bovine aortic endothelial cells, with maximal effect at 12 dynes/cm2. Activation of a G protein was necessary, as demonstrated by complete inhibition by the nonhydrolyzable GDP analog GDP-beta S. Activation of protein kinase C (PKC) was required, as shown by inhibiting PKC with staurosporine or downregulating PKC with phorbol 12,13-dibutyrate. Both Ca(2+)-dependent and -independent PKC activity, measured by translocation and substrate phosphorylation, increased in response to flow. However, MAP kinase activation was not dependent on Ca2+ mobilization, since Ca2+ chelation had no inhibitory effect. On the basis of these findings, it is proposed that flow activates two signal-transduction pathways in endothelial cells. One pathway is Ca2+ dependent and involves activation of phospholipase C and increases in intracellular Ca2+. A new pathway, described in the present study, is Ca2+ independent and involves a G protein and increases in PKC and MAP kinase activity.