Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis.

Arterial calcification (AC) is generally regarded as an independent risk factor for cardiovascular morbidity and mortality. Matrix Gla protein (MGP) is a potent inhibitor of AC, and its activity depends on vitamin K (VK). In rats, inactivation of MGP by treatment with the vitamin K antagonist warfarin leads to rapid calcification of the arteries. Here, we investigated whether preformed AC can be regressed by a VK-rich diet. Rats received a calcification-inducing diet containing both VK and warfarin (W&K). During a second 6-week period, animals were randomly assigned to receive either W&K (3.0 mg/g and 1.5 mg/g, subsequently), a diet containing a normal (5 g/g) or high (100 g/g) amount of VK (either K 1 or K 2 ). Increased aortic calcium concentration was observed in the group that continued to receive W&K and also in the group changed to the normal dose of VK and AC progressed. Both the VK-rich diets decreased the arterial calcium content by some 50%. In addition, arterial distensibility was restored by the VK-rich diet. Using MGP antibodies, local VK deficiency was demonstrated at sites of calcification. This is the first study in rats demonstrating that AC and the resulting decreased arterial distensibility are reversible by high-VK intake. IntroductionArterial calcification is an important independent risk factor for the development of atherosclerosis, myocardial infarction, stroke, and renal disease. 1,2 Patients with manifest arterial calcification have an unfavorable prognosis compared with patients with no or mild calcification. 3,4 Therefore, the prevention or reversal of arterial calcification may lead to improved patient outcomes.For a long time it has been thought that calcification was a passive process and the end stage of cardiovascular disease. During the past 10 years, however, it has become clear that several osteoregulatory proteins, both stimulatory and inhibitory, are involved in the calcification of vascular tissue. [5][6][7][8] One of the strongest in vivo inhibitors of arterial calcification is matrix Gla protein (MGP). MGP was first discovered in bone, 9 but it is mainly produced by vascular smooth muscle cells and chondrocytes. Its function became clear in MGP-deficient mice, 10 which died within 6 to 8 weeks after birth as a result of rupture of the large arteries. Histochemical evaluation demonstrated complete calcification of the elastic fibers in the arterial vessels and a phenotypic change of smooth muscle cells into chondrocytes. MGP acts by direct inhibition of calcium crystal formation and regulates bone morphogenetic protein-2, a growth factor responsible for osteogenic differentiation. [11][12][13] Murshed et al 14 demonstrated that restoration of MGP exclusively in the vascular smooth muscle cells of the MGP-null mice completely rescued the vascular calcification phenotype. For this effect the MGP needed to be ␥-carboxylated because mutating the Gla residues into aspartic acid residues led to the synthesis of nonfunctional MGP and to the death of all animals.Vitamin K is an e...

For decades, oxidative stress has been discussed as a key mechanism of endothelial dysfunction and cardiovascular disease. However, attempts to validate and exploit this hypothesis clinically by supplementing antioxidants have failed. Nevertheless, this does not disprove the oxidative stress hypothesis. As a certain degree of reactive oxygen species (ROS) formation appears to be physiological and beneficial. To reduce oxidative stress therapeutically, two alternative approaches are being developed. One is the repair of key signalling components that are compromised by oxidative stress. These include uncoupled endothelial nitric oxide (NO) synthase and oxidized/heme-free NO receptor soluble guanylate cyclase. A second approach is to identify and effectively inhibit the relevant source(s) of ROS in a given disease condition. A highly likely target in this context is the family of NADPH oxidases. Animal models, including NOX knockout mice and new pharmacological inhibitors of NADPH oxidases have opened up a new era of oxidative stress research and have paved the way for new cardiovascular therapies.LINKED ARTICLESThis article is part of a themed issue on Vascular Endothelium in Health and Disease. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.164.issue-3

Abstract-The endothelial cytoskeleton plays a key role in arterial responses to acute changes in shear stress. We evaluated whether the intermediate filament protein vimentin is involved in the structural responses of arteries to chronic changes in blood flow (BF). In wild-type mice (Vϩ/ϩ) and in vimentin-deficient mice (VϪ/Ϫ), the left common carotid artery (LCA) was ligated near its bifurcation, and 4 weeks later, the structures of the occluded and of the contralateral arteries were evaluated and compared with the structures of arteries from sham-operated mice. 3 m 2 for LCA and RCA, respectively). In Vϩ/ϩ, LCA ligation eliminated BF in the occluded vessel (before ligation, 0.35Ϯ0.02 mL/min) and increased BF from 0.34Ϯ0.02 to 0.68Ϯ0.04 mL/min in the RCA. In VϪ/Ϫ, the BF change in the occluded LCA was comparable (from 0.38Ϯ0.05 mL/min to zero-flow rates), but the BF increase in the RCA was less pronounced (from 0.33Ϯ0.02 to 0.50Ϯ0.05 mL/min). In the occluded LCA of Vϩ/ϩ, arterial diameter was markedly reduced (Ϫ162 m), and CSAm was significantly increased (5ϫ10 3 m 2 ), whereas in the high-flow RCA of Vϩ/ϩ, carotid artery diameter and CSAm were not significantly modified. In the occluded LCA of VϪ/Ϫ, arterial diameter was reduced to a lesser extent (Ϫ77 m) and CSAm was increased to a larger extent (10ϫ10 3 m 2 ) than in Vϩ/ϩ. In contrast to Vϩ/ϩ, the high-flow RCA of VϪ/Ϫ displayed a significant increase in diameter (52 m) and a significant increase in CSAm (5ϫ10 3 m 2 ). These observations provide the first direct evidence for a role of the cytoskeleton in flow-induced arterial remodeling.