Oxidative stress is a hallmark of metabolism-related diseases and a risk factor for atherosclerosis. FoxO factors have been shown to play a key role in vascular endothelial development and homeostasis. Foxo3a can protect quiescent cells from oxidative stress through the regulation of detoxification genes such as sod2 and catalase. Here we show that Foxo3a is a direct transcriptional regulator of a group of oxidative stress protection genes in vascular endothelial cells. Importantly, Foxo3a activity requires the transcriptional co-activator PGC-1␣, because it is severely curtailed in PGC-1␣-deficient endothelial cells. Foxo3a and PGC-1␣ appear to interact directly, as shown by co-immunoprecipitation and in vitro interaction assays, and are recruited to the same promoter regions. The notion that Foxo3a and PGC-1␣ interact directly to regulate oxidative stress protection genes in the vascular endothelium is supported by the observation that PGC-1␣ transcriptional activity at the sod2 (manganese superoxide dismutase) promoter requires a functional FoxO site. We also demonstrate that Foxo3a is a direct transcriptional regulator of PGC-1␣, suggesting that an auto-regulatory cycle regulates Foxo3a/PGC-1␣ control of the oxidative stress response.A central question in cardiovascular medicine is how metabolic dysfunctions affect the vascular system and in particular how they give rise to endothelial oxidative stress. Reactive oxygen species (ROS) 6 are normally produced in the mitochondria as a byproduct of aerobic metabolism. Low levels of ROS have been shown to play a role in cell signaling (reviewed in Ref.

Abstract-Endothelial dysfunction is characterized by an impaired vasodilatory response to endothelial agonists as well as by alterations in adhesion and coagulation processes. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) have been shown to be useful in the reversal of endothelial dysfunction, an effect that may be independent of the reduction in cholesterol levels. Both the L-arginine-nitric oxide-cGMP and endothelin pathways are involved in the regulation of vascular tone. Here, we show that the basal transcription rate of the preproendothelin-1 gene was decreased by simvastatin (10 mol/L) in bovine aortic endothelial cells. Transfection studies with the preproendothelin-1 gene promoter showed that mevalonate (100 mol/L) was able to prevent the inhibitory effect mediated by simvastatin. Protein geranylgeranylation, but not farnesylation, proved to be crucial for a correct expression of the preproendothelin-1 gene. The C3 exotoxin from Clostridium botulinum that selectively inactivates Rho GTPases, the processing of which involves geranylgeranylation, reproduced the inhibitory effect of simvastatin on the expression of preproendothelin-1. Overexpression of dominant-negative mutants of RhoA and RhoB led to a significant reduction in the preproendothelin-1 promoter activity, whereas the expression of wild-type and constitutively active forms of these proteins resulted in an increase, in support that Rho proteins are required for the basal expression of the preproendothelin-1 gene. Finally, we show that the Rho-dependent activation of the preproendothelin-1 gene transcription was inhibited by simvastatin. Thus, the control of vascular tone and proliferative response mediated by endothelin-1 is regulated at multiple levels, among which the Rho proteins play an essential role. (Circ Res. 2000;87:616-622.)

In damaged or proliferating endothelium, production of nitric oxide (NO) from endothelial nitric oxide synthase (eNOS) is associated with elevated levels of reactive oxygen species (ROS), which are necessary for endothelial migration. We aimed to elucidate the mechanism that mediates NO induction of endothelial migration. NO downregulates expression of peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC-1␣), which positively modulates several genes involved in ROS detoxification. We tested whether NO-induced cell migration requires PGC-1␣ downregulation and investigated the regulatory pathway involved. PGC-1␣ negatively regulated NO-dependent endothelial cell migration in vitro, and inactivation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, which is activated by NO, reduced NO-mediated downregulation of PGC-1␣. Expression of constitutively active Foxo3a, a target for Akt-mediated inactivation, reduced NO-dependent PGC-1␣ downregulation. Foxo3a is also a direct transcriptional regulator of PGC-1␣, and we found that a functional FoxO binding site in the PGC-1␣ promoter is also a NO response element. These results show that NO-mediated downregulation of PGC-1␣ is necessary for NO-induced endothelial migration and that NO/protein kinase G (PKG)-dependent downregulation of PGC-1␣ and the ROS detoxification system in endothelial cells are mediated by the PI3K/Akt signaling pathway and subsequent inactivation of the FoxO transcription factor Foxo3a.Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) plays critical roles in the physiology of endothelial cells (5), regulating vascular tone (19), endothelial cell growth (10), migration (22), survival under stress (4), and the levels of reactive oxygen species (ROS) (32). NO-induced angiogenesis requires a NO-dependent increase in ROS (2, 25), and although the molecular mechanisms linking NO and ROS homeostasis have been a matter of intense research, they are still only partly understood (reviewed in reference 20). NOdependent induction of angiogenesis requires activation of the soluble guanylyl cyclase (sGC)/protein kinase G (PKG)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway (13). However, whether and how this pathway is associated with the increase in ROS levels are still unclear.It has been proposed that endothelial angiogenesis requires the inactivation of FoxO transcription factors by Akt (11,26). Akt directly phosphorylates and inactivates members of the FoxO family, which are consequently translocated from the nucleus to the cytoplasm (12). The FoxO factors Foxo3a and Foxo4 protect cells from oxidative stress (14), although the significance of this process in normal vascular homeostasis is still unknown.Peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC-1␣) is a master regulator of cellular energy metabolism (8) and a positive and direct regulator of a set of genes whose products protect against oxidative stress (29, 31). PGC-1␣ is inactivated by phosphorylation by Akt...

Matrix metalloproteinases (MMPs) are synthesized in response to diverse stimuli, including cytokines, growth factors, hormones, and oxidative stress. Here we show that the nitric oxide (NO) donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO) and NO from murine macrophages transcriptionally regulate MMP-13 expression in vascular endothelial cells (BAEC). The cGMP analog, 8-bromo-cGMP (8-Br-cGMP) mimicked the effect of NO, whereas incubation with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, or the cGMP-dependent protein kinase (PKG) inhibitor phenyl-1,N 2 -etheno-8-bromoguanosine-3Ј,5Ј-cyclic monophosphorothioate, Rp-isomer (PET) reduced the stimulatory effect of DEA-NO on the activation of the MMP-13 promoter. Overexpression of the catalytic subunit of PKG1-␣ resulted in a 5-to 6-fold increase of the MMP-13 regulatory region over control cells. On the other hand, incubation with the mitogen-activated protein/ extracellular signal-regulated kinase inhibitor 2Ј-amino-3Ј-methoxyflavone (PD98059) significantly reduced DEA-NO and 8-Br-cGMP promoter activation and mRNA expression of MMP-13 in transfected BAEC. Moreover, a complex between PKG1-␣ and the G-protein Raf-1, an upstream activator of the extracellular signal-regulated kinase signaling pathway, was detected in cells overexpressing PKG1-␣ or treated either with DEA-NO or 8-Br-cGMP. Thus, we propose that the NO-cGMP-PKG pathway enhances MMP-13 expression by the activation of ERK 1,2. This effect of NO may be the result of pathophysiological importance in the context of inflammation or atherogenesis.