Deletion of p22phox-dependent oxidative stress in the hypothalamus protects against obesity by modulating ß3-adrenergic mechanisms.

A role for oxidative stress in the brain has been suggested in the pathogenesis of diet-induced obesity (DIO), although the underlying neural regions and mechanisms remain incompletely defined. We tested the hypothesis that NADPH oxidase-dependent oxidative stress in the paraventricular nucleus (PVN), a hypothalamic energy homeostasis center, contributes to the development of DIO. Cre/LoxP technology was coupled with selective PVN adenoviral microinjection to ablate p22phox , the obligatory subunit for NADPH oxidase activity, in mice harboring a conditional p22phox allele. Selective deletion of p22phox in the PVN protected mice from high-fat DIO independent of changes in food intake or locomotor activity. This was accompanied by ß3-adrenoceptor-dependent increases in energy expenditure, elevations in brown adipose tissue thermogenesis, and browning of white adipose tissue. These data reveal a potentially novel role for brain oxidative stress in the development of DIO by modulating ß3-adrenoceptor mechanisms and point to the PVN as an underlying neural site.

Adverse metabolic phenotype of female offspring exposed to preeclampsia in utero: a characterization of the BPH/5 mouse in postnatal life.

Preeclampsia (PE) is a devastating disorder of pregnancy that classically presents with maternal hypertension and proteinuria after 20 wk of gestation. In addition to being a leading cause of maternal and fetal morbidity/mortality, epidemiological and prospective studies have revealed long-term consequences for both the mother and baby of preeclamptic pregnancies, including chronic hypertension as well as other cardiovascular diseases and metabolic derangements. To better understand the effect of in utero exposure of PE on offspring, we utilized the BPH/5 mouse, a spontaneous model of the maternal and fetal PE syndrome. We hypothesized that young BPH/5 offspring would have altered metabolic and cardiovascular phenotypes. Indeed, BPH/5 growth-restricted offspring showed excess catch-up growth by early adulthood due to hyperphagia and increased white adipose tissue (WAT) accumulation, with inflammation markers isolated to the reproductive WAT depot only. Both excessive WAT accumulation and the inflammatory WAT phenotype were corrected by pair-feeding young BPH/5 female mice. We also found that young BPH/5 female mice showed evidence of leptin resistance. Indeed, chronic hyperleptinemia has been shown to characterize other rodent models of PE; however, the maternal metabolic profile before pregnancy has not been fully understood. Furthermore, we found that these mice show signs of cardiovascular anomalies (hypertension and cardiomegaly) and altered signaling within the reproductive axis in early life. Future studies will involve challenging the physiological metabolic state of BPH/5 mice through pair-feeding to reduce WAT before pregnancy and determining its causal role in adverse pregnancy outcomes.

Decidual Cox2 inhibition improves fetal and maternal outcomes in a preeclampsia-like mouse model.

Preeclampsia (PE) is a disorder of pregnancy that manifests as late gestational maternal hypertension and proteinuria and can be life-threatening to both the mother and baby. It is believed that abnormal placentation is responsible for the cascade of events leading to the maternal syndrome. Embryo implantation is critical to establishing a healthy pregnancy. Defective implantation can cause adverse "ripple effects," leading to abnormal decidualization and placentation, retarded fetal development, and poor pregnancy outcomes, such as PE and fetal growth restriction. The precise mechanism(s) of implantation defects that lead to PE remain elusive. BPH/5 mice, which spontaneously develop the cardinal features of PE, show peri-implantation defects including upregulation of Cox2 and IL-15 at the maternal-fetal interface. This was associated with decreased decidual natural killer (dNK) cells, which have important roles in establishing placental perfusion. Interestingly, a single administration of a Cox2 inhibitor (celecoxib) during decidualization restrained Cox2 and IL-15 expression, restored dNK cell numbers, improved fetal growth, and attenuated late gestational hypertension in BPH/5 female mice. This study provides evidence that decidual overexpression of Cox2 and IL-15 may trigger the adverse pregnancy outcomes reflected in the preeclamptic syndrome, underscoring the idea that Cox2 inhibitor treatment is an effective strategy for the prevention of PE-associated fetal and maternal morbidity and mortality.

Role of chemokine RANTES in the regulation of perivascular inflammation, T-cell accumulation, and vascular dysfunction in hypertension.

Recent studies have emphasized the role of perivascular inflammation in cardiovascular disease. We studied mechanisms of perivascular leukocyte infiltration in angiotensin II (Ang II)-induced hypertension and their links to vascular dysfunction. Chronic Ang II infusion in mice increased immune cell content of T cells (255 ± 130 to 1664 ± 349 cells/mg; P < 0.01), M1 and M2 macrophages, and dendritic cells in perivascular adipose tissue. In particular, the content of T lymphocytes bearing CC chemokine receptor (CCR) 1, CCR3, and CCR5 receptors for RANTES chemokine was increased by Ang II (CCR1, 15.6 ± 1.5% vs. 31 ± 5%; P < 0.01). Hypertension was associated with an increase in perivascular adipose tissue expression of the chemokine RANTES (relative quantification, 1.2 ± 0.2 vs. 3.5 ± 1.1; P < 0.05), which induced T-cell chemotaxis and vascular accumulation of T cells expressing the chemokine receptors CCR1, CCR3, and CCR5. Mechanistically, RANTES(-/-) knockout protected against vascular leukocyte, and in particular T lymphocyte infiltration (26 ± 5% in wild type Ang II vs. 15 ± 4% in RANTES(-/-)), which was associated with protection from endothelial dysfunction induced by Ang II. This effect was linked with diminished infiltration of IFN-γ-producing CD8(+) and double-negative CD3(+)CD4(-)CD8(-) T cells in perivascular space and reduced vascular oxidative stress while FoxP3(+) T-regulatory cells were unaltered. IFN-γ ex vivo caused significant endothelial dysfunction, which was reduced by superoxide anion scavenging. In a human cohort, a significant inverse correlation was observed between circulating RANTES levels as a biomarker and vascular function measured as flow-mediated dilatation (R = -0.3, P < 0.01) or endothelial injury marker von Willebrand factor (R = +0.3; P < 0.01). Thus, chemokine RANTES is important in the regulation of vascular dysfunction through modulation of perivascular inflammation.-Mikolajczyk, T. P., Nosalski, R., Szczepaniak, P., Budzyn, K., Osmenda, G., Skiba, D., Sagan, A., Wu, J., Vinh, A., Marvar, P. J., Guzik, B., Podolec, J., Drummond, G., Lob, H. E., Harrison, D. G., Guzik, T. J. Role of chemokine RANTES in the regulation of perivascular inflammation, T-cell accumulation, and vascular dysfunction in hypertension.

Role of decidual natural killer cells, interleukin-15, and interferon-γ in placental development and preeclampsia.

Preeclampsia is a hypertensive, proteinuric disease that affects 5-10% of all pregnancies and is a leading cause of maternal and perinatal morbidity/mortality (Soto et al., J Matern Fetal Neonatal Med 25: 498-507, 2011). The primary treatment for preeclampsia still is delivery of the fetus and placenta. The underlying mechanisms remain elusive. One possibility is inadequate uterine angiogenesis/vascularity (decidualization) at the time of implantation (Torry et al., Am J Reprod Immunol 51: 257-268, 2004). Here, we review evidence for dysregulation of decidual natural killer (dNK) cells, which secrete important angiogenic factors during decidualization, as a contributing factor in preeclampsia.

Role of vascular oxidative stress in obesity and metabolic syndrome.

Obesity is associated with vascular diseases that are often attributed to vascular oxidative stress. We tested the hypothesis that vascular oxidative stress could induce obesity. We previously developed mice that overexpress p22phox in vascular smooth muscle, tg(sm/p22phox), which have increased vascular ROS production. At baseline, tg(sm/p22phox) mice have a modest increase in body weight. With high-fat feeding, tg(sm/p22phox) mice developed exaggerated obesity and increased fat mass. Body weight increased from 32.16 ± 2.34 g to 43.03 ± 1.44 g in tg(sm/p22phox) mice (vs. 30.81 ± 0.71 g to 37.89 ± 1.16 g in the WT mice). This was associated with development of glucose intolerance, reduced HDL cholesterol, and increased levels of leptin and MCP-1. Tg(sm/p22phox) mice displayed impaired spontaneous activity and increased mitochondrial ROS production and mitochondrial dysfunction in skeletal muscle. In mice with vascular smooth muscle-targeted deletion of p22phox (p22phox(loxp/loxp)/tg(smmhc/cre) mice), high-fat feeding did not induce weight gain or leptin resistance. These mice also had reduced T-cell infiltration of perivascular fat. In conclusion, these data indicate that vascular oxidative stress induces obesity and metabolic syndrome, accompanied by and likely due to exercise intolerance, vascular inflammation, and augmented adipogenesis. These data indicate that vascular ROS may play a causal role in the development of obesity and metabolic syndrome.

Arterial stiffening precedes systolic hypertension in diet-induced obesity.

Stiffening of conduit arteries is a risk factor for cardiovascular morbidity. Aortic wall stiffening increases pulsatile hemodynamic forces that are detrimental to the microcirculation in highly perfused organs, such as the heart, brain, and kidney. Arterial stiffness is associated with hypertension but presumed to be due to an adaptive response to increased hemodynamic load. In contrast, a recent clinical study found that stiffness precedes and may contribute to the development of hypertension although the mechanisms underlying hypertension are unknown. Here, we report that in a diet-induced model of obesity, arterial stiffness, measured in vivo, develops within 1 month of the initiation of the diet and precedes the development of hypertension by 5 months. Diet-induced obese mice recapitulate the metabolic syndrome and are characterized by inflammation in visceral fat and aorta. Normalization of the metabolic state by weight loss resulted in return of arterial stiffness and blood pressure to normal. Our findings support the hypothesis that arterial stiffness is a cause rather than a consequence of hypertension.

Role of the NADPH oxidases in the subfornical organ in angiotensin II-induced hypertension.

Reactive oxygen species and the NADPH oxidases contribute to hypertension via mechanisms that remain undefined. Reactive oxygen species produced in the central nervous system have been proposed to promote sympathetic outflow, inflammation, and hypertension, but the contribution of the NADPH oxidases to these processes in chronic hypertension is uncertain. We therefore sought to identify how NADPH oxidases in the subfornical organ (SFO) of the brain regulate blood pressure and vascular inflammation during sustained hypertension. We produced mice with loxP sites flanking the coding region of the NADPH oxidase docking subunit p22(phox). SFO-targeted injections of an adenovirus encoding cre-recombinase markedly diminished p22(phox), Nox2, and Nox4 mRNA in the SFO, as compared with a control adenovirus encoding red-fluorescent protein injection. Increased superoxide production in the SFO by chronic angiotensin II infusion (490 ng/kg min(-1) × 2 weeks) was blunted in adenovirus encoding cre-recombinase-treated mice, as detected by dihydroethidium fluorescence. Deletion of p22(phox) in the SFO eliminated the hypertensive response observed at 2 weeks of angiotensin II infusion compared with control adenovirus encoding red-fluorescent protein-treated mice (mean arterial pressures=97 ± 15 versus 154 ± 6 mm Hg, respectively; P=0.0001). Angiotensin II infusion also promoted marked vascular inflammation, as characterized by accumulation of activated T-cells and other leukocytes, and this was prevented by deletion of the SFO p22(phox). These experiments definitively identify the NADPH oxidases in the SFO as a critical determinant of the blood pressure and vascular inflammatory responses to chronic angiotensin II, and further support a role of reactive oxygen species in central nervous system signaling in hypertension.

T lymphocytes and vascular inflammation contribute to stress-dependent hypertension.

BACKGROUND: Psychological stress is a significant risk factor for hypertension and also directly affects the immune system. We have previously reported that T lymphocytes are essential for development of hypertension and that the central nervous system contributes to peripheral T-lymphocyte activation and vascular inflammation in this disease; however, the role of T-cell activation in stress-related hypertension remains unclear. METHODS: Wild-type and T-cell-deficient (RAG-1(-/-)) mice were subjected to daily episodes of stress and blood pressure was measured. Circulating T-cell activation markers and vascular infiltration of immune cells were analyzed, as were stress hormone levels and gene expression changes in the brain. The effects angiotensin II infusion in the presence of chronic stress was also studied. RESULTS: Repeated daily stress contributed to acute elevations in blood pressure that were associated with increased activation of circulating T cells and increased vascular infiltration of T cells. Repeated stress increased blood pressure in wild-type but not RAG-1(-/-) mice. Adoptive transfer of T cells to RAG-1(-/-) mice restored blood pressure elevation in response to stress. Stress-related hypertension and vascular infiltration of T cells was markedly enhanced by angiotensin II. Moreover, angiotensin II-infused mice exposed to chronic stress exhibited greater blood pressure reactivity to an episode of acute stress. CONCLUSIONS: These data demonstrate that stress-dependent hypertension triggers an inflammatory response that raises blood pressure at baseline and augments the hypertension caused by angiotensin II. These data provide insight as to how psychological stress contributes to hypertension.

Role of vascular extracellular superoxide dismutase in hypertension.

Previous studies indicate that superoxide is important in the modulation of blood pressure but have not specifically identified the cell types or organs involved. We created mice with loxP sites flanking the extracellular superoxide dismutase (SOD3) gene. These mice were crossed with mice expressing inducible Cre-recombinase driven by the smooth muscle myosin heavy chain promoter allowing tissue-specific deletion of SOD3. Deletion of SOD3 increased vascular superoxide and reduced vascular NO levels as detected by electron spin resonance. Despite these changes in NO and superoxide, we did not observe increases in vascular inflammation caused by angiotensin II. Moreover, deletion of vascular SOD3 did not augment hypertension in response to angiotensin II. In additional studies, we also deleted SOD3 from the circumventricular organs by intracerebroventricular injection of an adenovirus encoding Cre-recombinase. Although this raised blood pressure and augmented the hypertension caused by angiotensin II, these responses were not further increased by vascular deletion of SOD3. These data suggest that the extracellular superoxide dismutase in vascular smooth muscle is not involved in the genesis of angiotensin II-induced hypertension and further emphasize the role of central SOD3 in the modulation of blood pressure.

Role of interleukin 17 in inflammation, atherosclerosis, and vascular function in apolipoprotein e-deficient mice.

OBJECTIVE: Interleukin 17A (IL17A) is involved in many inflammatory processes, but its role in atherosclerosis remains controversial. We examined the role of IL17A in mouse and human atherosclerosis. METHODS AND RESULTS: Atherosclerosis was induced in apolipoprotein E (ApoE)(-/-) and IL17A/ApoE(-/-) mice using high-fat feeding, angiotensin II infusion, or partial carotid ligation. In ApoE(-/-) mice, 3 months of high-fat diet induced interferon-γ production by splenic lymphocytes, and this was abrogated in IL17A/ApoE(-/-) mice. IL17A/ApoE(-/-) mice had reduced aortic superoxide production, increased aortic nitric oxide levels, decreased aortic leukocyte and dendritic cell infiltration, and reduced weight gain after a high-fat diet compared with ApoE(-/-) mice. Despite these favorable effects, IL17A deficiency did not affect aortic plaque burden after a high-fat diet or angiotensin II infusion. In a partial carotid ligation model, IL17A deficiency did not affect percentage of stenosis but reduced outward remodeling. In this model, neutralization of the related isoform, IL17F, in IL17A/ApoE(-/-) mice did not alter atherosclerosis. Finally, there was no correlation between IL17A levels and carotid intima-media thickness in humans. CONCLUSIONS: IL17 contributes to vascular and systemic inflammation in experimental atherosclerosis but does not alter plaque burden. The changes in plaque composition caused by IL17 might modulate plaque stability.

Central and peripheral mechanisms of T-lymphocyte activation and vascular inflammation produced by angiotensin II-induced hypertension.

RATIONALE: We have previously found that T lymphocytes are essential for development of angiotensin II-induced hypertension; however, the mechanisms responsible for T-cell activation in hypertension remain undefined. OBJECTIVE: We sought to study the roles of the CNS and pressure elevation in T-cell activation and vascular inflammation caused by angiotensin II. METHODS AND RESULTS: To prevent the central actions of angiotensin II, we created anteroventral third cerebral ventricle (AV3V) lesions in mice. The elevation in blood pressure in response to angiotensin II was virtually eliminated by AV3V lesions, as was activation of circulating T cells and the vascular infiltration of leukocytes. In contrast, AV3V lesioning did not prevent the hypertension and T-cell activation caused by the peripheral acting agonist norepinephrine. To determine whether T-cell activation and vascular inflammation are attributable to central influences or are mediated by blood pressure elevation, we administered hydralazine (250 mg/L) in the drinking water. Hydralazine prevented the hypertension and abrogated the increase in circulating activated T cells and vascular infiltration of leukocytes caused by angiotensin II. CONCLUSIONS: We conclude that the central and pressor effects of angiotensin II are critical for T-cell activation and development of vascular inflammation. These findings also support a feed-forward mechanism in which modest degrees of blood pressure elevation lead to T-cell activation, which in turn promotes inflammation and further raises blood pressure, leading to severe hypertension.

Induction of hypertension and peripheral inflammation by reduction of extracellular superoxide dismutase in the central nervous system.

The circumventricular organs (CVOs) lack a well-formed blood-brain barrier and produce superoxide in response to angiotensin II and other hypertensive stimuli. This increase in central superoxide has been implicated in the regulation of blood pressure. The extracellular superoxide dismutase (SOD3) is highly expressed in cells associated with CVOs and particularly with tanycytes lining this region. To understand the role of SOD3 in the CVOs in blood pressure regulation, we performed intracerebroventricular injection an adenovirus encoding Cre-recombinase (5x10(8) particles per milliliter) in mice with loxP sites flanking the SOD3 coding region (SOD3(loxp/loxp) mice). An adenovirus encoding red-fluorescent protein was injected as a control. Deletion of CVO SOD3 increased baseline blood pressure modestly and markedly augmented the hypertensive response to low-dose angiotensin II (140 ng/kg per day), whereas intracerebroventricular injection of adenovirus encoding red-fluorescent protein had minimal effects on these parameters. Adenovirus encoding Cre-recombinase-treated mice exhibited increased sympathetic modulation of heart rate and blood pressure variability, increased vascular superoxide production, and T-cell activation as characterized by increased circulating CD69(+)/CD3(+) cells. Deletion of CVO SOD3 also markedly increased vascular T-cell and leukocyte infiltration caused by angiotensin II. We conclude that SOD3 in the CVO plays a critical role in the regulation of blood pressure, and its loss promotes T-cell activation and vascular inflammation, in part by modulating sympathetic outflow. These findings provide insight into how central signals produce vascular inflammation in response to hypertensive stimuli, such as angiotensin II.

Interleukin 17 promotes angiotensin II-induced hypertension and vascular dysfunction.

We have shown previously that T cells are required for the full development of angiotensin II-induced hypertension. However, the specific subsets of T cells that are important in this process are unknown. T helper 17 cells represent a novel subset that produces the proinflammatory cytokine interleukin 17 (IL-17). We found that angiotensin II infusion increased IL-17 production from T cells and IL-17 protein in the aortic media. To determine the effect of IL-17 on blood pressure and vascular function, we studied IL-17(-/-) mice. The initial hypertensive response to angiotensin II infusion was similar in IL-17(-/-) and C57BL/6J mice. However, hypertension was not sustained in IL-17(-/-) mice, reaching levels 30-mm Hg lower than in wild-type mice by 4 weeks of angiotensin II infusion. Vessels from IL-17(-/-) mice displayed preserved vascular function, decreased superoxide production, and reduced T-cell infiltration in response to angiotensin II. Gene array analysis of cultured human aortic smooth muscle cells revealed that IL-17, in conjunction with tumor necrosis factor-alpha, modulated expression of >30 genes, including a number of inflammatory cytokines/chemokines. Examination of IL-17 in diabetic humans showed that serum levels of this cytokine were significantly increased in those with hypertension compared with normotensive subjects. We conclude that IL-17 is critical for the maintenance of angiotensin II-induced hypertension and vascular dysfunction and might be a therapeutic target for this widespread disease.

Calcium-dependent NOX5 nicotinamide adenine dinucleotide phosphate oxidase contributes to vascular oxidative stress in human coronary artery disease.

OBJECTIVES: This study sought to examine the expression and activity of the calcium-dependent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in human atherosclerotic coronary arteries. BACKGROUND: The NOX-based NADPH oxidases are major sources of reactive oxygen species (ROS) in human vessels. Several NOX homologues have been identified, but their relative contribution to vascular ROS production in coronary artery disease (CAD) is unclear; NOX5 is a unique homolog in that it is calcium dependent and thus could be activated by vasoconstrictor hormones. Its presence has not yet been studied in human vessels. METHODS: Coronary arteries from patients undergoing cardiac transplantation with CAD or without CAD were studied; NOX5 was quantified and visualized using Western blotting, immunofluorescence, and quantitative real-time polymerase chain reaction. Calcium-dependent NADPH oxidase activity, corresponding greatly to NOX5 activity, was measured by electron paramagnetic resonance. RESULTS: Both Western blotting and quantitative real-time polymerase chain reaction indicated a marked increase in NOX5 protein and messenger ribonucleic acid (mRNA) in CAD versus non-CAD vessels. Calcium-dependent NADPH-driven production of ROS in vascular membranes, reflecting NOX5 activity, was increased 7-fold in CAD and correlated significantly with NOX5 mRNA levels among subjects. Immunofluorescence showed that NOX5 was expressed in the endothelium in the early lesions and in vascular smooth muscle cells in the advanced coronary lesions. CONCLUSIONS: These studies identify NOX5 as a novel, calcium-dependent source of ROS in atherosclerosis.

Loss of extracellular superoxide dismutase leads to acute lung damage in the presence of ambient air: a potential mechanism underlying adult respiratory distress syndrome.

The extracellular superoxide dismutase 3 (SOD3) is highly expressed in both blood vessels and lungs. In different models of pulmonary injury, SOD3 is reduced; however, it is unclear whether this contributes to lung injury. To study the role of acute SOD3 reduction in lung injury, the SOD3 gene was deleted in adult mice by using the Cre-Lox technology. Acute reduction of SOD3 led to a fivefold increase in lung superoxide, marked inflammatory cell infiltration, a threefold increase in the arterial-alveolar gradient, respiratory acidosis, histological changes similar to those observed in adult respiratory distress syndrome, and 85% mortality. Treatment with the SOD mimetic MnTBAP and intranasal administration of SOD-containing polyketal microparticles reduced mortality, prevented the histological alterations, and reduced lung superoxide levels. To understand how mice with the SOD3 embryonic deletion survived without lung injury, gene array analysis was performed. These data demonstrated the up-regulation of 37 genes and down-regulation of nine genes, including those involved in cell signaling, inflammation, and gene transcription in SOD3-/- mice compared with either mice with acute SOD3 reduction or wild-type controls. These studies show that SOD3 is essential for survival in the presence of ambient oxygen and that acute loss of this enzyme can lead to severe lung damage. Strategies either to prevent SOD3 inactivation or to augment its levels might prove useful in the treatment of acute lung injury.