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%

12/15-lipoxygenase (12/15-LOX) is an enzyme, which oxidizes polyunsaturated fatty acids, particularly omega-6 and −3 fatty acids, to generate a number of bioactive lipid metabolites. A large number of studies have revealed the importance of 12/15-LOX role in oxidative and inflammatory responses. The in vitro studies have demonstrated the ability of 12/15-LOX metabolites in the expression of various genes and production of cytokine related to inflammation and resolution of inflammation. The studies with the use of knockout and transgenic animals for 12/15-LOX have further shown its involvement in the pathogenesis of a variety of human diseases, including cardiovascular, renal, neurological and metabolic disorders. This review summarizes our current knowledge on the role of 12/15-LOX in inflammation and various human diseases.

Neovascularization is a hallmark of neointimal formation in atherosclerotic plaques and restenotic lesions. Vascular endothelial growth factor (VEGF) promotes neovascular growth, whereas oxidative stress is a potent factor in vascular cell proliferation. To investigate the mechanisms of neovascular formation, we treated human and rat vascular smooth muscle cells (VSMCs) with H2O2. Northern blot analysis demonstrated a dose- and time-dependent increase in VEGF mRNA, with a maximum of 4-fold at 3 hours (200 mumol/L). As determined by immunoblotting and enzyme-linked immunosorbent assay, VEGF protein expression and secretion were similarly increased. Human umbilical vein endothelial cells were treated with conditioned medium from VSMCs incubated with 200 mumol/L H2O2. DNA synthesis, measured by thymidine incorporation, was increased 4-fold compared with control, an effect that was blocked by a neutralizing anti-VEGF antibody. The lipid peroxidation product 4-hydroxynonenal (1 mumol/L), an endogenous reactive oxygen species present in human atherosclerotic lesions, also increased VEGF secretion in VSMCs in a similar time-dependent fashion. Immunohistochemical staining and in situ hybridization of aortic sections from balloon-injured baboons demonstrated increased VEGF expression in discrete areas of the neointima and media compared with control sections, and expression correlated with the generation of 4-hydroxynonenal. Regulators of VEGF expression, such as reactive oxygen species, may enhance neovascularization of atherosclerotic and restenotic arteries.

It has recently been reported that protein-tyrosine kinase activity is required for thrombin-induced growth in vascular smooth muscle cells (VSMC). In the present study, we have identified several phosphoproteins that are tyrosine-phosphorylated in response to thrombin in quiescent VSMC. These proteins are insulin-like growth factor-1 receptor ␤-subunit (IGF-IR␤), insulin receptor substrate-1 (IRS-1), and phospholipase C-␥1 (PLC-␥1). Thrombin-stimulated phosphorylation of these proteins was rapid; it was maximal at 1 min and reduced thereafter. Thrombin also activated mitogen-activated protein kinases (MAPK) in quiescent VSMC in a biphasic manner with a rapid and larger peak at 10 min (6-fold) followed by a sustained smaller second peak at 2 h (2-fold). Inhibition of protein-tyrosine kinase activity by the use of two structurally different protein-tyrosine kinase inhibitors, genistein and herbimycin A, significantly blocked the thrombin-induced tyrosine phosphorylation of IGF-1R␤, IRS-1, and PLC-␥1 and decreased thrombin-stimulated DNA synthesis. In contrast, however, inhibition of protein-tyrosine kinase activity had no effect on thrombin activation of MAPK. Collectively, these findings suggest a role for tyrosine phosphorylation of IGF-IR␤, IRS-1, and PLC-␥1 in thrombin-induced mitogenic signaling events in VSMC. Furthermore, while protein tyrosine phosphorylation is essential for thrombin-induced DNA synthesis, it is not required for thrombin-stimulated MAPK activation. Since thrombin rapidly activated Src in VSMC, Src may be involved in the cross-talk between the G-protein-coupled receptor agonist and a tyrosine kinase receptor such as IGF-1R.

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These data demonstrate that HNE, one of several important lipid peroxidation products, induces rat aortic smooth muscle cell growth through redox-sensitive mechanisms and growth factor expression. These observations are consistent with a role for lipid peroxidation products in vascular smooth muscle cell growth in atherogenesis.

To understand the molecular mechanisms underlying 14,15-epoxyeicosatrienoic acid (14,15-EET)–induced angiogenesis, here we have studied the role of signal transducer and activator of transcription-3 (STAT-3). 14,15-EET stimulated the tyrosine phosphorylation of STAT-3 and its translocation from the cytoplasm to the nucleus in human dermal microvascular endothelial cells (HDMVECs). Adenovirus-mediated delivery of dominant negative STAT-3 substantially inhibited 14,15-EET–induced HDMVEC migration, and tube formation and Matrigel plug angiogenesis. 14,15-EET activated Src, as measured by its tyrosine phosphorylation and blockade of its activation by adenovirus-mediated expression of its dominant negative mutant, significantly attenuated 14,15-EET–induced STAT-3 phosphorylation in HDMVECs and the migration and tube formation of these cells and Matrigel plug angiogenesis. 14,15-EET induced the expression of vascular endothelial cell growth factor (VEGF) in a time- and Src-STAT-3–dependent manner in HDMVECs. Transfac analysis of VEGF promoter revealed the presence of STAT-binding elements and 14,15-EET induced STAT-3 binding to this promoter in vivo, and this interaction was inhibited by suppression of Src-STAT-3 signaling. Neutralizing anti-VEGF antibodies completely blocked 14,15-EET–induced HDMVEC migration and tube formation and Matrigel plug angiogenesis. These results reveal that Src-dependent STAT-3–mediated VEGF expression is a major mechanism of 14,15-EET–induced angiogenesis.

We have previously reported that nuclear factor of activated T cells (NFATs) play an important role in the regulation of vascular smooth muscle cell migration and proliferation by receptor tyrosine kinase and G proteincoupled receptor agonists, platelet-derived growth factor-BB and thrombin, respectively. To understand the role of NFATs in vascular disease, we have now studied the involvement of these transcription factors in neointima formation in a rat carotid artery balloon injury model. The levels of NFATc1 in injured right common carotid arteries were increased at 72 h, 1 week, and 2 weeks after balloon injury compared with its levels in uninjured left common carotid arteries. Intraperitoneal injection of cyclosporine A (CsA), a pharmacological inhibitor of the calcineurin-NFAT activation pathway, suppressed balloon injury-induced neointima formation by 40%. Similarly, adenoviral-mediated expression of GFPVIVIT, a competent peptide inhibitor of the calcineurin-NFAT activation pathway, in injured arteries also reduced neointima formation by about 40%. Furthermore, CsA and GFPVIVIT attenuated balloon injury-induced neointimal smooth muscle cell proliferation as determined by bromodeoxyuridine staining. Platelet-derived growth factor-BB induced the expression of COX-2 in cultured VSMC in a time-and NFAT-dependent manner. COX-2 expression was also increased in the right common carotid artery in a time-dependent manner after balloon injury as compared with its levels in uninjured left common carotid artery and both CsA and GFPVIVIT negated this response. Together these results for the first time demonstrate that NFATs play a critical role in neointima formation via induction of expression of COX-2.