Inflammation and oxidative stress are pathogenic mediators of many diseases, but therapeutic targets remain elusive. In the vasculature, abdominal aortic aneurysm (AAA) formation critically involves inflammaton and matrix degradation. Cyclophilin A (CyPA, encoded by Ppia) is highly expressed in vascular smooth muscle cells (VSMC), is secreted in response to reactive oxygen species (ROS), and promotes inflammation. Using the angiotensin II (AngII)-induced AAA model in Apoe−/− mice, we show that Apoe−/−Ppia−/− mice were completely protected from AngII–induced AAA formation, in contrast to Apoe−/−Ppia+/+ mice. Apoe−/−Ppia−/− mice showed decreased inflammatory cytokine expression, elastic lamina degradation, and aortic expansion. These features were not altered by reconstitution of bone marrow cells from Ppia+/+ mice. Mechanistic studies demonstrated that VSMC-derived intracellular and extracellular CyPA were required for ROS generation and matrix metalloproteinase-2 activation. These data define a novel role for CyPA in AAA formation and suggest CyPA is a new target for cardiovascular therapies.

Cyclophilin A promotes atherosclerosis in part by inducing reactive oxygen species and promoting endothelial cell apoptosis and macrophage recruitment into lesions.

Background— Oxidative stress, generated by excessive reactive oxygen species, promotes cardiovascular disease. Cyclophilin A (CyPA) is a 20-kDa chaperone protein secreted from vascular smooth muscle cells (VSMCs) in response to reactive oxygen species that stimulates VSMC proliferation and inflammatory cell migration in vitro; however, the role CyPA plays in vascular function in vivo remains unknown. Methods and Results— We tested the hypothesis that CyPA contributes to vascular remodeling by analyzing the response to complete carotid ligation in CyPA knockout mice, wild-type mice, and mice that overexpress CyPA in VSMC (VSMC-Tg). After carotid ligation, CyPA expression in vessels of wild-type mice increased dramatically and was significantly greater in VSMC-Tg mice. Reactive oxygen species–induced secretion of CyPA from mouse VSMCs correlated significantly with intracellular CyPA expression. Intimal and medial hyperplasia correlated significantly with CyPA expression after 2 weeks of carotid ligation, with marked decreases in CyPA knockout mice and increases in VSMC-Tg mice. Inflammatory cell migration into the intima was significantly reduced in CyPA knockout mice and increased in VSMC-Tg mice. Additionally, VSMC proliferation assessed by Ki67 + cells was significantly less in CyPA knockout mice and was increased in VSMC-Tg mice. The importance of CyPA for intimal and medial thickening was shown by strong correlations between CyPA expression and the number of both inflammatory cells and proliferating VSMCs in vivo and in vitro. Conclusions— In response to low flow, CyPA plays a crucial role in VSMC migration and proliferation, as well as inflammatory cell accumulation, thereby regulating flow-mediated vascular remodeling and intima formation.

Abstract-In response to biological and mechanical injury, or in vitro culturing, vascular smooth muscle cells (VSMCs) undergo phenotypic modulation from a differentiated "contractile" phenotype to a dedifferentiated "synthetic" one. This results in the capacity to proliferate, migrate, and produce extracellular matrix proteins, thus contributing to neointimal formation. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP or cGMP, are critical in the homeostasis of cyclic nucleotides that regulate VSMC growth. Here, we demonstrate that PDE1A, a Ca 2ϩ -calmodulin-stimulated PDE preferentially hydrolyzing cGMP, is predominantly cytoplasmic in medial "contractile" VSMCs but is nuclear in neointimal "synthetic" VSMCs. Using primary VSMCs, we show that cytoplasmic and nuclear PDE1A were associated with a contractile marker (SM-calponin) and a growth marker (Ki-67), respectively. This suggests that cytoplasmic PDE1A is associated with the "contractile" phenotype, whereas nuclear PDE1A is with the "synthetic" phenotype. To determine the role of nuclear PDE1A, we examined the effects loss-of-PDE1A function on subcultured VSMC growth and survival using PDE1A RNA interference and pharmacological inhibition. Reducing PDE1A function significantly attenuated VSMC growth by decreasing proliferation via G 1 arrest and inducing apoptosis. Inhibiting PDE1A also led to intracellular cGMP elevation, p27Kip1 upregulation, cyclin D1 downregulation, and p53 activation. We further demonstrated that in subcultured VSMCs redifferentiated by growth on collagen gels, cytoplasmic PDE1A regulates myosin light chain phosphorylation with little effect on apoptosis, whereas inhibiting nuclear PDE1A has the opposite effects. These suggest that nuclear PDE1A is important in VSMC growth and survival and may contribute to the neointima formation in atherosclerosis and restenosis. (Circ Res. 2006;98:777-784.) Key Words: PDE Ⅲ smooth muscle cell Ⅲ growth Ⅲ apoptosis Ⅲ vascular injury V ascular smooth muscle cells (VSMCs) in response to injury and hormonal stimuli exhibit phenotypic plasticity, changing from a differentiated (quiescent, contractile) phenotype to a dedifferentiated (active, synthetic) one. 1 This process was originally defined as "phenotypic modulation." 2 Under normal conditions, VSMCs residing in the media of vessels are quiescent with a very low turnover rate. 3,4 Quiescent VSMCs are fully differentiated cells that possess the "contractile" phenotype and function principally to maintain vascular tone. If the vessel is injured or cells are placed in tissue culture, VSMCs respond by changing from the "contractile" to the "synthetic" phenotype. 4 Synthetic VSMCs contribute to neointima formation by downregulating contractile proteins and acquiring the capacity to proliferate, migrate, and produce extracellular matrix proteins. 5 Therefore, phenotypic modulation of VSMCs plays a key role in the pathogenesis of cardiovascular disorders such as atherosclerosis, postangioplasty restenosis, bypass vein graft failure, and ca...

Abstract-Intima-media thickening (IMT) in response to hemodynamic stress is a physiological process that requires coordinated signaling among endothelial, inflammatory, and vascular smooth muscle cells (VSMC). Axl, a receptor tyrosine kinase, whose ligand is Gas6, is highly induced in VSMC after carotid injury. Because Axl regulates cell migration, phagocytosis and apoptosis, we hypothesized that Axl would play a role in IMT. Vascular remodeling in mice deficient in Axl (Axl Ϫ/Ϫ ) and wild-type littermates (Axl ϩ/ϩ ) was induced by ligation of the left carotid artery (LCA) branches maintaining flow via the left occipital artery. Both genotypes had similar baseline hemodynamic parameters and carotid artery structure. Partial ligation altered blood flow equally in both genotypes: increased by 60% in the right carotid artery (RCA) and decreased by 80% in the LCA. There were no significant differences in RCA remodeling between genotypes. However, in the LCA Axl Ϫ/Ϫ developed significantly smaller intimaϩmedia compared with Axl Key Words: Axl Ⅲ flow Ⅲ carotid artery Ⅲ remodeling Ⅲ mouse Ⅲ apoptosis Ⅲ inflammation T he receptor tyrosine kinase Axl (also known as Ufo and Tyro7) belongs to a family of tyrosine receptors that includes Tyro3 (Sky) and Mer (Tyro12). 1,2 A common ligand for Axl family is Gas6 (Growth arrest-specific protein 6). 2,3 Important cellular functions of Gas6/Axl include cell adhesion, migration, phagocytosis, and inhibition of apoptosis. 4 -7 Gas6 and Axl family receptors are highly regulated in a tissue and disease specific manner. 8 -11 A recent genetic study found a significant association of a Gas6 mutation with stroke in humans. 12 Both Gas6 and Axl-family receptor knockout mice show abnormalities in platelet function, further supporting a vascular role for the Gas6/Axl pathway. 13,14 In human umbilical vein endothelial cells (HUVEC) and pulmonary artery EC, Gas6/Axl signaling promotes cell survival, 8,15 possibly via an autocrine pathway. 16 Gas6/Axlmediated survival signals include activation of Akt, phosphorylation of nuclear factor B (NF-B), increased expression of Bcl-2, and decreased caspase-3 activation. 17 Similarly, in vascular smooth muscle cells (VSMC), Gas6/Axl powerfully stimulates cell migration and cell survival. 18 We reported that Axl was highly regulated after rat carotid balloon injury consistent with a role in intimal VSMC proliferation. 19 Our recent findings showed that the Gas6/Axl/PI3K/Akt pathway inhibited VSMC apoptosis. 20 Moreover, Axl contributes to generation of reactive oxygen species in VSMC. 21 In addition, the Gas6/Axl pathway is important in the mammalian immune system. 7 Recent data suggest that the Axl family is a central regulator of macrophage activation and phagocytosis. 22 We developed a reproducible mouse model of flowdependent vascular remodeling that resembles human intimamedia thickening (IMT). 23 In response to decreased blood flow IMT occurs, which involves critical interactions among cells of the vessel wall. 23,24 Based on the significant rol...

Axl, a receptor tyrosine kinase, is involved in cell survival, proliferation, and migration. We have shown that Axl expression increases in the neointima of ballooninjured rat carotids. Because oxidative stress is known to play a major role in remodeling of injured vessels, we hypothesized that H 2 O 2 might activate Axl by promoting autophosphorylation. H 2 O 2 rapidly stimulated Axl tyrosine phosphorylation in rat vascular smooth muscle cells within 1 min that was maximal at 5 min (6-fold).

Background The G-protein-coupled receptor (GPCR)-kinase interacting protein-1 (GIT1) is a multi-domain scaffold protein that participates in many cellular functions including receptor internalization, focal adhesion remodeling, and signaling by both GPCRs and tyrosine kinase receptors. However, there have been no in vivo studies of GIT1 function to date. Methods and Results To determine essential functions of GIT1 in vivo, we generated a traditional GIT1 knockout (KO) mouse. GIT1 KO mice exhibited ∼60% perinatal mortality. Pathologic examination showed that the major abnormality in GIT1-KO mice was impaired lung development characterized by markedly reduced numbers of pulmonary blood vessels and increased alveolar spaces. Since vascular endothelial growth factor (VEGF) is essential for pulmonary vascular development, we investigated the role of GIT1 in VEGF signaling in the lung and cultured endothelial cells (EC). Because activation of phospholipase-Cγ (PLCγ and ERK1/2 by angiotensin II requires GIT1, we hypothesized that GIT1 mediates VEGF-dependent pulmonary angiogenesis by modulating PLCγ and ERK1/2 activity in EC. In cultured EC, knockdown of GIT1 decreased VEGF-mediated phosphorylation of PLCγ and ERK1/2. PLCγ and ERK1/2 activity in lungs from GIT1 KO mice was reduced postnatally. Conclusions Our data support a critical role for GIT1 in pulmonary vascular development by regulating VEGF-induced PLCγ and ERK1/2 activation.

Objective Cyclophilin A (CyPA, encoded by Ppia) is a pro-inflammatory protein secreted in response to oxidative stress in mice and humans. We recently demonstrated that CyPA increased angiotensin II (AngII)-induced reactive oxygen species (ROS) production in the aortas of Apoe−/− mice. In this study we sought to evaluate the role of CyPA in AngII–induced cardiac hypertrophy. Methods and Results Cardiac hypertrophy was not significantly different between Ppia+/+ and Ppia−/− mice infused with AngII (1000 ng/min/kg for 4 weeks). Therefore, we investigated the effect of CyPA under conditions of high ROS and inflammation using the Apoe−/− mice. In contrast to Apoe−/− mice, Apoe−/− Ppia−/− mice exhibited significantly less AngII-induced cardiac hypertrophy. Bone marrow cell transplantation showed that CyPA in cells intrinsic to the heart plays an important role in the cardiac hypertrophic response. AngII-induced ROS production, cardiac fibroblast proliferation and migration were markedly decreased in Apoe−/− Ppia−/− cardiac fibroblasts. Furthermore, CyPA directly induced the hypertrophy of cultured neonatal cardiac myocytes. Conclusions CyPA is required for AngII-mediated cardiac hypertrophy by directly potentiating ROS production, stimulating the proliferation and migration of cardiac fibroblasts, and promoting cardiac myocyte hypertrophy.