Ϫ ) react with nitric oxide (NO) to form peroxynitrite (ONOO Ϫ ), a process that limits NO availability, results in NO synthase (NOS) uncoupling, and, through the action of ONOO Ϫ , leads to protein and thiol oxidation as well as tyrosine nitration. 1 Hydrogen peroxide (H 2 O 2 ), the dismutation product of O 2 Ϫ , also elicits multiple effects, among them smooth muscle cell hypertrophy, activation of metalloproteinases, and, in higher concentrations, NOS inhibition by phosphorylation of tyrosine 657 through the redox-activated tyrosine kinase Pyk2. 2 Interestingly, H 2 O 2 also induces positive endothelial effects because it can activate protein kinase-G I␣ by thiol oxidation and subsequent dimerization. 3 Moreover, H 2 O 2 induces as well as activates endothelial NOS (eNOS). 4

ROS are implicated in bone diseases. NADPH oxidase 4 (NOX4), a constitutively active enzymatic source of ROS, may contribute to the development of such disorders. Therefore, we studied the role of NOX4 in bone homeostasis. Nox4(-/-) mice displayed higher bone density and reduced numbers and markers of osteoclasts. Ex vivo, differentiation of monocytes into osteoclasts with RANKL and M-CSF induced Nox4 expression. Loss of NOX4 activity attenuated osteoclastogenesis, which was accompanied by impaired activation of RANKL-induced NFATc1 and c-JUN. In an in vivo model of murine ovariectomy–induced osteoporosis, pharmacological inhibition or acute genetic knockdown of Nox4 mitigated loss of trabecular bone. Human bone obtained from patients with increased osteoclast activity exhibited increased NOX4 expression. Moreover, a SNP of NOX4 was associated with elevated circulating markers of bone turnover and reduced bone density in women. Thus, NOX4 is involved in bone loss and represents a potential therapeutic target for the treatment of osteoporosis.

Abstract-Monoamine oxidases (MAOs) generate H 2 O 2 as a by-product of their catalytic cycle. Whether MAOs are mediators of endothelial dysfunction is unknown and was determined here in the angiotensin II and lipopolysaccharidemodels of vascular dysfunction in mice. Quantitative real-time polymerase chain reaction revealed that mouse aortas contain enzymes involved in catecholamine generation and MAO-A and MAO-B mRNA. MAO-A and -B proteins could be detected by Western blot not only in mouse aortas but also in human umbilical vein endothelial cells. Ex vivo incubation of mouse aorta with recombinant MAO-A increased H 2 O 2 formation and induced endothelial dysfunction that was attenuated by polyethylene glycol-catalase and MAO inhibitors. In vivo lipopolysaccharide (8 mg/kg IP overnight) or angiotensin II (1 mg/kg per day, 2 weeks, minipump) treatment induced vascular MAO-A and -B expressions and resulted in attenuated endothelium-dependent relaxation of the aorta in response to acetylcholine. MAO inhibitors reduced the lipopolysaccharide-and angiotensin II-induced aortic reactive oxygen species formation by 50% (ferrous oxidation xylenol orange assay) and partially normalized endothelium-dependent relaxation. MAO-A and MAO-B inhibitors had an additive effect; combined application completely restored endothelium-dependent relaxation. To determine how MAO-dependent H 2 O 2 formation induces endothelial dysfunction, cyclic GMP was measured. Histamine stimulation of human umbilical vein endothelial cells to activate endothelial NO synthase resulted in an increase in cyclic GMP, which was almost abrogated by MAO-A exposure. MAO inhibition prevented this effect, suggesting that MAO-induced H 2 O 2 formation is sufficient to attenuate endothelial NO release. Sturza et al MAOs in Endothelial Dysfunction 141MAO-A-mediated H 2 O 2 production has been shown to be relevant for ischemia-reperfusion injury of the kidney 11 and the heart. Moreover, MAO-A is thought to be involved in myocyte hypertrophy ex vivo 12,13 and also in maladaptive myocardial hypertrophy and transition to heart failure in vivo.14 The unfavorable effects of MAO activation are antagonized by a couple of relatively selective MAO inhibitors, which are either irreversible, such as clorgyline for MAO-A and selegiline for MAO-B, or reversible, such as moclobemide for MAO-A and lazabemide for MAO-B, respectively.8 Systemic MAO inhibition leads to the accumulation of catecholamines with subsequent increase in sympathetic activity and hypertension. 15 This aspect limits the use of MAO inhibitors in a vascular scenario, and therefore, MAO inhibitors have not been considered an approach to improve vascular function.Another prototypic hypertensive agent is angiotensin II (Ang II). Interestingly, potential interactions of Ang II and MAOs have been reported in the central nervous system. In coculture systems of hypothalamic and brain stem neurons, Ang II stimulated MAO activity, but the underlying mechanism was not studied. 16 More recently, it was reported that in...

Objective— Obesity is associated with hyperleptinemia but it is not clear whether leptin protects vascular function or promotes dysfunction. We therefore studied the consequences of hyperleptinemia in lean mice. Methods and Results— Wild-type and endothelial NO synthase (eNOS) −/− mice were infused with leptin (0.4 mg/kg per day, 7 days), and endothelium-dependent relaxation was studied in aortic segments. Leptin had no effect on acetylcholine-induced endothelium-dependent relaxation in normal wild-type mice but restored endothelium-dependent relaxation in wild-type mice treated with angiotensin II (0.7 mg/kg per day, 7 days) to induce endothelial dysfunction. Leptin also sensitized aortae from eNOS −/− mice to acetylcholine, an effect blocked by neuronal NOS (nNOS) inhibition and not observed in eNOS-nNOS double −/− mice. Consistent with these findings, leptin induced nNOS expression in murine and human vessels and human endothelial but not smooth muscle cells. Aortic nNOS expression was also induced in mice by a high-fat diet. Mechanistically, leptin increased endothelial Janus kinase 2 and signal transducer and activator of transcription 3 phosphorylation, and inhibition of Janus kinase 2 prevented nNOS induction in cultured cells and leptin-induced relaxations in eNOS −/− mice. Conclusion— Leptin induces endothelial nNOS expression, which compensates, in part, for a lack of NO production by eNOS to maintain endothelium-dependent relaxation.