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...

Axl is a receptor tyrosine kinase originally identified as a transforming gene product in human myeloid leukemia cells. Cultured rat vascular smooth muscle cells also express Axl, where it has been proposed that Axl may play a role in cell proliferation. In the current study, we tested the hypotheses that Axl expression would parallel neointima formation in balloon-injured rat carotid, and that Axl expression would be regulated by growth factors present at sites of vascular injury. Ribonuclease protection assay showed dynamic increases in Axl mRNA in vessels, with peak expression 7 and 14 days after injury. Immunohistochemical analysis confirmed these results and demonstrated that Axl protein expression was localized primarily to cells of the neointima after injury. Northern blot analysis indicated increased mRNA expression for the secreted Axl ligand, Gas6, in injured carotids, with a time course paralleling that of Axl upregulation. Axl and Gas6 expression were temporally correlated with neointima formation, suggesting a role for Axl signaling in this process. Other studies, performed in cultured rat vascular smooth muscle cells, revealed positive regulation of Axl mRNA expression by thrombin or angiotensin II but not by basic fibroblast growth factor, platelet-derived growth factor-BB, or transforming growth factor-ss1. Western blot analysis confirmed these results, showing that Axl protein expression was specifically increased by thrombin or angiotensin II. Our results implicate Axl as a potential mediator of vascular smooth muscle migration and proliferation caused by vascular injury and G protein-coupled receptor agonists.