Vulnerable areas of atherosclerotic plaques often contain lipid-laden macrophages and display matrix metalloproteinase activity. We hypothesized that reactive oxygen species released by macrophage-derived foam cells could trigger activation of latent proforms of metalloproteinases in the vascular interstitium. We showed that in vivo generated macrophage foam cells produce superoxide, nitric oxide, and hydrogen peroxide after isolation from hypercholesterolemic rabbits. Effects of these reactive oxygens and that of peroxynitrite, likely to result from simultaneous production of nitric oxide and superoxide, were tested in vitro using metalloproteinases secreted by cultured human vascular smooth muscle cells. Enzymes in culture media or affinitypurified (pro-MMP-2 and MMP-9) were examined by SDS-PAGE zymography, Western blotting, and enzymatic assays. Under the conditions used, incubation with xanthine/ xanthine oxidase increased the amount of active gelatinases, while nitric oxide donors had no noticeable effect. Incubation with peroxynitrite resulted in nitration of MMP-2 and endowed it with collagenolytic activity. Hydrogen peroxide treatment showed a catalase-reversible biphasic effect (gelatinase activation at concentrations of 4 M, inhibition at Ն 10-50 M). Thus, reactive oxygen species can modulate matrix degradation in areas of high oxidant stress and could therefore contribute to instability of atherosclerotic plaques. ( J. Clin. Invest. 1996. 98:2572-2579.)

Abstract-Diverse stimuli, including shear stress, cyclic strain, oxidized LDL, hyperglycemia, and cell growth, modulate endothelial nitric oxide synthase (eNOS) expression. Although seemingly unrelated, these may all alter cellular redox state, suggesting that reactive oxygen intermediates might modulate eNOS expression. Key Words: paraquat Ⅲ superoxide dismutase Ⅲ eNOS mRNA stability Ⅲ cultured endothelial cells A lthough endothelial nitric oxide synthase (eNOS) was originally thought to be a constitutively expressed enzyme, it has become clear in recent years that its expression can be modulated by a variety of chemical, physical, and developmental stimuli. 1 Regulation of eNOS expression occurs at both the transcriptional and post-transcriptional levels. For example, in cultured human and bovine endothelial cells, cyclic strain, 2 nordihydroguaiaretic acid (NDGA), 3 estrogens, 4 and the oxidized LDL (oxLDL) components 13-hydroperoxyoctadecadienoate 5 and lysophosphatidylcholine 6 have been shown to increase eNOS gene transcription. Likewise, transforming growth factor-␤ (TGF-␤) 7 and laminar shear stress 8 increased the transcriptional activity of the 5Ј-promoter region of the eNOS gene as assessed by transfection of bovine aortic endothelial cells (BAECs) with chimeric eNOS promoter/luciferase constructs. In contrast, stimuli such as cell growth 9 and the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, simvastatin 10 increase eNOS expression by prolonging the half-life of the eNOS mRNA.A common property of many stimuli that increase eNOS expression is the ability to increase the production of reactive oxygen intermediates (ROIs) in endothelial cells. Thus, laminar shear stress, 11 cyclic strain, 12 oxLDL, 13 high glucose, 14 TGF-␤ 15 and proliferation 16 have all been associated with an elevated production of ROIs in endothelial cells. Furthermore, for many of these stimuli, this increase in production of ROIs was shown to be important for regulating expression of other genes. For example, induction of heme-oxygenase-1, 17 c-fos, 18 monocyte chemotactic protein-1, 19 and intercellular adhesion molecule-1 (ICAM-1) 20,21 in human umbilical vein endothelial cells by shear stress and cyclic strain was inhibited by the antioxidants N-acetylcysteine (NAC) and catalase. Similarly, the phenolic antioxidants probucol and vitamin E have been shown to inhibit oxLDL-mediated induction of ICAM-1 and vascular cell adhesion molecule-1 expression. 22 Finally, transcriptional induction of macrophage-colony stimulating factor by TGF-␤ 1 was inhibited by catalase but not by superoxide dismutase (SOD), suggesting that H 2 O 2 , rather than superoxide anions (O 2 Ϫ⅐ ), mediates this effect. 23 These observations raise the possibility that ROIs represent common signaling molecules for modulating eNOS expression. Indeed, the human, bovine, and murine eNOS promoter

A major determinant of the level of cellular superoxide anion (O 2 −• ) is the dismutation of O 2 −• to hydrogen peroxide by the enzyme superoxide dismutase (SOD). Three forms of SOD exist, but in endothelial cells, the major form outside of the mitochondria is the cytosolic copper/zinc-containing superoxide dismutase (Cu/Zn SOD). Since fluid shear stress is an important determinant of the function and structure of endothelial cells in vivo, we examined the effect of laminar shear stress on the expression of Cu/Zn SOD in cultured human aortic endothelial cells. Laminar shear stress of 0.6 to 15 dyne/cm 2 increased Cu/Zn SOD mRNA in a time- and dose-dependent manner in human aortic endothelial cells. Shear stress also increased both Cu/Zn SOD protein content and the enzyme activity. Nuclear run-on assays showed that nuclei from human aortic endothelial cells exposed to laminar shear stress had a 1.6-fold greater transcriptional activity of the Cu/Zn SOD gene compared with cells not exposed to shear, indicating that an increase in Cu/Zn SOD mRNA induced by laminar shear stress is at least in part mediated by increased transcription. In contrast, shear stress had no effect on Cu/Zn SOD mRNA levels in human aortic smooth muscle cells. These findings show that physiological levels of shear stress increase expression of Cu/Zn SOD in the endothelium. This adaptation to shear stress might augment the effect of locally produced NO • and thereby promote the antiatherogenic and anti-inflammatory properties of the endothelial cell.

We conclude that loss of one copy of the eNOS gene, as observed in heterozygotic animals, has no effect on vascular reactivity, blood pressure or eNOS protein expression. Isoforms of NOS, other than eNOS are unlikely involved in blood pressure regulation but may participate in heart rate control.

Liver function is crucial for maintaining metabolic homeostasis in mammals. Numerous genes must be properly regulated for the liver to develop and perform a variety of activities. Several recent gene-knockout studies in mice have clarified the roles of GATA6, HNF4alpha, and Foxa1/Foxa2 in early stages of liver formation. After the liver forms, transcriptional changes continue to occur; during the perinatal period, certain genes such as alpha-fetoprotein and H19 are silenced, others are activated, and position-dependent (or zonal) regulation is established. Zhx2 was recently identified as one factor involved in postnatal repression of alpha-fetoprotein and other genes. Furthermore, several studies indicate that negative regulation is involved in the zonal control of glutamine synthetase. Finally, exciting new evidence indicates that signaling through the Wnt/beta-catenin pathway is also involved in zonal regulation in the adult liver.

Protein levels and polymorphisms of p22(phox) have been suggested to modulate vascular NAD(P)H oxidase activity and vascular production of reactive oxygen species (ROS). We sought to determine whether increasing p22(phox) expression would alter vascular ROS production and hemodynamics by targeting p22(phox) expression to smooth muscle in transgenic (Tg) mice. Aortas of Tg(p22smc) mice had increased p22(phox) and Nox1 protein levels and produced more superoxide and H(2)O(2). Surprisingly, endothelium-dependent relaxation and blood pressure in Tg(p22smc) mice were normal. Aortas of Tg(p22smc) mice produced twofold more nitric oxide (NO) at baseline and sevenfold more NO in response to calcium ionophore as detected by electron spin resonance. Western blot analysis revealed a twofold increase in endothelial NO synthase (eNOS) protein expression in Tg(p22smc) mice. Both eNOS expression and NO production were normalized by infusion of the glutathione peroxidase mimetic ebselen or by crossing Tg(p22smc) mice with mice overexpressing catalase. We have previously found that NO stimulates extracellular superoxide dismutase (ecSOD) expression in vascular smooth muscle. In keeping with this, aortic segments from Tg(p22smc) mice expressed twofold more ecSOD, and chronic treatment with the NOS inhibitor N(G)-nitro-L-arginine methyl ester normalized this, suggesting that NO regulates ecSOD protein expression in vivo. These data indicate that chronic oxidative stress caused by excessive H(2)O(2) production evokes a compensatory response involving increased eNOS expression and NO production. NO in turn increases ecSOD protein expression and counterbalances increased ROS production leading to the maintenance of normal vascular function and hemodynamics.

Abstract-The expression of the endothelial NO synthase (eNOS) is dramatically influenced by the state of cell growth.In proliferating cells, mRNA levels are increased 4-fold compared with postconfluent, nonproliferating cells. Nuclear run-on analysis indicated that there is no difference in the transcriptional rate of eNOS in proliferating versus postconfluent cells. The half-life of eNOS mRNA, measured after actinomycin D transcriptional arrest, was 3-fold greater in preconfluent compared with confluent endothelial cells. Using UV-cross-linking analysis, a cytoplasmic protein with an apparent molecular mass of 51 kDa was found to bind to terminal 545-nt eNOS mRNA 3-fold more in confluent cells than in proliferating cells. 4 Although eNOS is constitutively expressed, it has become clear that its expression is subject to modest, but likely important, degrees of regulation. In cultured cells, eNOS expression is increased by shear stress, 5 cyclic strain, 6 lysophosphatidylcholine, 7 low concentrations of oxidized LDL, 8 oxidized linoleic acid, 9 and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. 10 In vivo, exercise training in dogs increases eNOS expression and the ability of blood vessels to dilate in response to agonists that release endogenous nitric oxide (NO⅐). 11 In contrast, exposure of cultured endothelial cells to tumor necrosis factor (TNF)-␣, 12 hypoxia, 13 and high concentrations of oxidized LDL 8,14 decreases eNOS levels. In these latter conditions, posttranscriptional changes in mRNA half-life play an important role in downregulation of eNOS. [12][13][14] Earlier, our laboratory found that eNOS expression is rather dramatically influenced by the state of endothelial cell proliferation. The mRNA for eNOS was Ϸ4-to 6-fold greater in proliferating cells as compared with cells several days after confluence. 15 In addition, protein expression (by Western analysis) and enzyme activity (by arginine-to-citrulline conversion) were similarly increased in proliferating versus confluent cells. 15 In this study, the mechanisms responsible for the increase in eNOS mRNA expression during cell growth were not defined. Thus, the purpose of the present study was to examine factors responsible for changes in eNOS levels during endothelial cell proliferation. Nuclear run-on assays and studies of mRNA stability were used to examine both transcriptional and posttranscriptional mechanisms, respectively. On the basis of these results, we further examined the role of protein/RNA interactions and the role of specific portions of the 3Ј-untranslated region (UTR) of the eNOS mRNA in regulation of eNOS expression during cell growth. Our findings demonstrate a novel mechanism for control of eNOS levels in endothelial cells. Materials and Methods Cell CultureBovine aorta endothelial cells (BAECs) were harvested and cultured in medium (M199; Gibco Laboratories) containing 10% FCS (Hyclone Laboratories) as described earlier. 9 Experiments were conducted on cells between passages 3 and 9. Cells were split at a ratio of...

ObjectiveThe purpose of this review is to evaluate the impact of recent epidemiologic literature on the National Research Council (NRC) assessment of the lung and bladder cancer risks from ingesting low concentrations (< 100 μg/L) of arsenic-contaminated water.Data sources, extraction, and synthesisPubMed was searched for epidemiologic studies pertinent to the lung and bladder cancer risk estimates from low-dose arsenic exposure. Articles published from 2001, the date of the NRC assessment, through September 2010 were included. Fourteen epidemiologic studies on lung and bladder cancer risk were identified as potentially useful for the analysis.ConclusionsRecent epidemiologic studies that have investigated the risk of lung and bladder cancer from low arsenic exposure are limited in their ability to detect the NRC estimates of excess risk because of sample size and less than lifetime exposure. Although the ecologic nature of the Taiwanese studies on which the NRC estimates are based present certain limitations, the data from these studies have particular strengths in that they describe lung and bladder cancer risks resulting from lifetime exposure in a large population and remain the best data on which to conduct quantitative risk assessment. Continued follow-up of a population in northeastern Taiwan, however, offers the best opportunity to improve the cancer risk assessment for arsenic in drinking water. Future studies of arsenic < 100 μg/L in drinking water and lung and bladder cancer should consider adequacy of the sample size, the synergistic relationship of arsenic and smoking, duration of arsenic exposure, age when exposure began and ended, and histologic subtype.