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.

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Preeclampsia complicates 5–8% of all pregnancies worldwide, and although its pathophysiology remains obscure, placental oxidative stress and mitochondrial abnormalities are considered to play a key role. Mitochondrial abnormalities in preeclamptic placentae have been described, but the extent to which mitochondrial content and the molecular pathways controlling this (mitochondrial biogenesis and mitophagy) are affected in preeclamptic placentae is unknown. Therefore, in preeclamptic (n = 12) and control (n = 11) placentae, we comprehensively assessed multiple indices of placental antioxidant status, mitochondrial content, mitochondrial biogenesis, mitophagy, and mitochondrial fusion and fission. In addition, we also explored gene expression profiles related to inflammation and apoptosis. Preeclamptic placentae were characterized by higher levels of oxidized glutathione, a higher total antioxidant capacity, and higher mRNA levels of the mitochondrial-located antioxidant enzyme manganese-dependent superoxide dismutase 2 compared to controls. Furthermore, mitochondrial content was significantly lower in preeclamptic placentae, which was accompanied by an increased abundance of key constituents of glycolysis. Moreover, mRNA and protein levels of key molecules involved in the regulation of mitochondrial biogenesis were lower in preeclamptic placentae, while the abundance of constituents of the mitophagy, autophagy, and mitochondrial fission machinery was higher compared to controls. In addition, we found evidence for activation of apoptosis and inflammation in preeclamptic placentae. This study is the first to comprehensively demonstrate abnormalities at the level of the mitochondrion and the molecular pathways controlling mitochondrial content/function in preeclamptic placentae. These aberrations may well contribute to the pathophysiology of preeclampsia by upregulating placental inflammation, oxidative stress, and apoptosis.

Abstract-The renin-angiotensin system has been implicated in obesity-related hypertension and insulin resistance. We examined whether locally produced components of the renin-angiotensin system in adipose tissue and skeletal muscle play an endocrine role in vivo in humans. Furthermore, the effects of ␤-adrenergic stimulation on plasma concentrations and tissue release of renin-angiotensin system components were investigated. Systemic renin-angiotensin system components and arteriovenous differences of angiotensin II (Ang II) and angiotensinogen (AGT) across abdominal subcutaneous adipose tissue and skeletal muscle were assessed in combination with measurements of tissue blood flow before and during systemic ␤-adrenergic stimulation in 13 lean and 10 obese subjects. Basal plasma Ang II and AGT concentrations were not significantly different between lean and obese subjects. Ang II concentrations were increased in obese compared with lean subjects during ␤-adrenergic stimulation (12.6Ϯ1.5 versus 8.1Ϯ1.0 pmol/L; Pϭ0.04), whereas AGT concentrations remained unchanged. Plasma renin activity increased to a similar extent in lean and obese subjects during ␤-adrenergic stimulation (both PϽ0.01). No net Ang II release across adipose tissue and skeletal muscle could be detected in both groups of subjects. However, AGT was released from adipose tissue and muscle during ␤-adrenergic stimulation in obese subjects (both PϽ0.05). In conclusion, locally produced Ang II in adipose tissue and skeletal muscle exerts no endocrine role in lean and obese subjects. In contrast, AGT is released from adipose tissue and muscle in obese subjects during ␤-adrenergic stimulation, which may contribute to the increased plasma Ang II concentrations during ␤-adrenergic stimulation in obese subjects. Key Words: angiotensin Ⅲ ␤-adrenergic receptors Ⅲ obesity Ⅲ renin Ⅲ sympathetic nervous system A bdominal obesity plays a central role in the metabolic syndrome and is a major risk factor for chronic diseases, such as type 2 diabetes mellitus and cardiovascular disease. 1 Although abdominal obesity is frequently accompanied by hypertension, the mechanisms by which an excess fat mass may lead to hypertension are not fully understood. The reninangiotensin system (RAS) has been established as a major determinant of blood pressure and cardiovascular disease. 2 Furthermore, recent clinical trials suggest that blockade of RAS may reduce the incidence of type 2 diabetes. 3 Angiotensin II (Ang II), the main effector component of RAS, is produced in the circulation from angiotensinogen (AGT) because of the action of the enzymes renin and angiotensinconverting enzyme. In addition, it has become evident that several components of RAS are present in a variety of tissues, including adipose tissue and skeletal muscle (reviewed in Reference 4), implying that these tissues have the ability to produce Ang II.There is substantial evidence that circulating RAS components are increased in (hypertensive) obese subjects. [5][6][7][8] In line with this, it has been shown t...

Objective-Tissue kallikrein (TK) participates in acute flow-induced dilatation (FID) of large arteries. We investigated whether TK deficiency blunts FID and alters chronic flow-related arterial structural and functional changes in resistance-sized muscular arteries. Methods and Results-Vasomotor responses and structural parameters were determined in uterine arteries isolated from nonpregnant, 18-to 19-day pregnant, and 7-day postpartum TK Ϫ/Ϫ and TK ϩ/ϩ littermate mice. In TK Ϫ/Ϫ mice, values of diameter, medial cross-sectional area (CSA), myogenic tone, and dilatation in response to acetylcholine were comparable to those values in TK ϩ/ϩ mice, but FID (0 to 100 L/min) was significantly reduced (55Ϯ4% versus 85Ϯ4% in TK ϩ/ϩ mice). In both mouse strains, pregnancy resulted in significant increases in diameter and medial CSA and in the N w -nitro-L-arginine methyl ester-sensitive component of FID. By 7 days after pregnancy, uterine arterial diameter and CSA values no longer differed from nonpregnant values, and FID was markedly reduced in TK Ϫ/Ϫ and TK ϩ/ϩ mice. Conclusions-These observations (1) confirm at the level of resistance arteries the key role of TK in FID and (2) Pressure and flow are hemodynamic determinants of circumferential wall stress and wall shear stress (WSS) in the arterial system. On an acute basis, they modulate arterial smooth muscle tone: an increase in transmural pressure triggers a myogenic contractile response, 6 -8 whereas an increase in flow induces an endothelium-dependent FID. 8 -11 Long-term changes in blood flow lead to arterial structural adaptations to normalize WSS. [12][13][14][15][16][17] In general, chronic blood flow elevations lead to a widening of lumen diameter, whereas sustained reductions in blood flow lead to a narrowing of lumen diameter. These structural arterial adaptations have been shown to be endothelium dependent. 18 Pregnancy may be considered a model of physiological flow-related remodeling of uterine arteries. It has previously been studied in rats, 19 guinea pigs, 20 and ewes. 21 During pregnancy, blood flow through the uterine circulation increases substantially. 22,23 To accommodate this increase in uterine blood flow, the uterine vasculature undergoes luminal expansion and an increase in wall mass, 19 a process termed outward hypertrophic remodeling or arteriogenesis. The driving force for this remodeling is increased WSS. Previous studies have demonstrated that chronic elevations in blood flow lead to an upregulation of FID, which is mainly due to an enhanced production of endothelial factors, such as NO and prostaglandins.The purpose of the present study was to investigate whether TK deficiency alters FID and chronic flow-related arterial structural and functional alterations in resistance-sized muscular arteries. Vasomotor responses and structural parameters were determined in uterine arteries isolated from nonpregnant, latepregnant, and postpartum TK Ϫ/Ϫ and TK ϩ/ϩ mice. Methods Mice Lacking TKTK-null mice were obtained by targeted disruption of the...