Background—: In diabetes mellitus, vascular complications such as atherosclerosis are a major cause of death. The key underlying pathomechanisms are unclear. However, hyperglycemic oxidative stress derived from NADPH oxidase (Nox), the only known dedicated enzyme to generate reactive oxygen species appears to play a role. Here we identify the Nox1 isoform as playing a key and pharmacologically targetable role in the accelerated development of diabetic atherosclerosis. Methods and Results—: Human aortic endothelial cells exposed to hyperglycemic conditions showed increased expression of Nox1, oxidative stress, and proinflammatory markers in a Nox1-siRNA reversible manner. Similarly, the specific Nox inhibitor, GKT137831, prevented oxidative stress in response to hyperglycemia in human aortic endothelial cells. To examine these observations in vivo, we investigated the role of Nox1 on plaque development in apolipoprotein E–deficient mice 10 weeks after induction of diabetes mellitus. Deletion of Nox1, but not Nox4, had a profound antiatherosclerotic effect correlating with reduced reactive oxygen species formation, attenuation of chemokine expression, vascular adhesion of leukocytes, macrophage infiltration, and reduced expression of proinflammatory and profibrotic markers. Similarly, treatment of diabetic apolipoprotein E–deficient mice with GKT137831 attenuated atherosclerosis development. Conclusions—: These studies identify a major pathological role for Nox1 and suggest that Nox1-dependent oxidative stress is a promising target for diabetic vasculopathies, including atherosclerosis.

Glutathione peroxidases have been thought to function in cellular antioxidant defense. However, some recent studies on Gpx1 knockout (−/−) mice have failed to show a role for Gpx1 under conditions of oxidative stress such as hyperbaric oxygen and the exposure of eye lenses to high levels of H₂O₂. These findings have, unexpectedly, raised the issue of the role of Gpx1, especially under conditions of oxidative stress. Here we demonstrate a role for Gpx1 in protection against oxidative stress by showing that Gpx1 (−/−) mice are highly sensitive to the oxidant paraquat. Lethality was already detected within 24 h in mice exposed to paraquat at 10 mg·kg⁻¹ (approximately the LD₅₀of wild-type controls). The effects of paraquat were dose-related. In the 30 mg·kg⁻¹-treated group, 100% of mice died within 5 h, whereas the controls showed no evidence of toxicity. We further demonstrate that paraquat transcriptionally up-regulatesGpx1 in normal cells, reinforcing a role forGpx1 in protection against paraquat toxicity. Finally, we show that cortical neurons from Gpx1 (−/−) mice are more susceptible to H₂O₂; 30% of neurons fromGpx1 (−/−) mice were killed when exposed to 65 μm H₂O₂, whereas the wild-type controls were unaffected. These data establish a function for Gpx1 in protection against some oxidative stressors and in protection of neurons against H₂O₂. Further, they emphasize the need to elucidate the role of Gpx1 in protection against different oxidative stressors and in different disease states and suggest thatGpx1 (−/−) mice may be valuable for studying the role of H₂O₂ in neurodegenerative disorders.

Background— Recent clinical studies have suggested a major protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx1) in diabetes-associated atherosclerosis. We induced diabetes in mice deficient for both GPx1 and apolipoprotein E (ApoE) to determine whether this is merely an association or whether GPx1 has a direct effect on diabetes-associated atherosclerosis. Methods and Results— ApoE-deficient (ApoE ^−/− ) and ApoE/GPx1 double-knockout (ApoE ^−/− GPx1 ^−/− ) mice were made diabetic with streptozotocin and aortic lesion formation, and atherogenic pathways were assessed after 10 and 20 weeks of diabetes. Aortic proinflammatory and profibrotic markers were determined by both quantitative reverse-transcription polymerase chain reaction analysis after 10 weeks of diabetes and immunohistochemical analysis after 10 and 20 weeks of diabetes. Sham-injected nondiabetic counterparts served as controls. Atherosclerotic lesions within the aortic sinus region, as well as arch, thoracic, and abdominal lesions, were significantly increased in diabetic ApoE ^−/− GPx1 ^−/− aortas compared with diabetic ApoE ^−/− aortas. This increase was accompanied by increased macrophages, α-smooth muscle actin, receptors for advanced glycation end products, and various proinflammatory (vascular cell adhesion molecule-1) and profibrotic (vascular endothelial growth factor and connective tissue growth factor) markers. Quantitative reverse-transcription polymerase chain reaction analysis showed increased expression of receptors for advanced glycation end products (RAGE), vascular cell adhesion molecule-1, vascular endothelial growth factor, and connective tissue growth factor. Nitrotyrosine levels were significantly increased in diabetic ApoE ^−/− GPx1 ^−/− mouse aortas. These findings were observed despite upregulation of other antioxidants. Conclusions— Lack of functional GPx1 accelerates diabetes-associated atherosclerosis via upregulation of proinflammatory and profibrotic pathways in ApoE ^−/− mice. Our study provides evidence of a protective role for GPx1 and establishes GPx1 as an important antiatherogenic therapeutic target in patients with or at risk of diabetic macrovascular disease.

It is now increasingly appreciated that inflammation is not limited to the control of pathogens by the host, but rather that sterile inflammation which occurs in the absence of viral or bacterial pathogens, accompanies numerous disease states, none more so than the complications that arise as a result of hyperglycaemia. Individuals with type 1 or type 2 diabetes mellitus (T1D, T2D) are at increased risk of developing cardiac and vascular complications. Glucose and blood pressure lowering therapies have not stopped the advance of these morbidities that often lead to fatal heart attacks and/or stroke. A unifying mechanism of hyperglycemia-induced cellular damage was initially proposed to link elevated blood glucose levels with oxidative stress and the dysregulation of metabolic pathways. Pre-clinical evidence has, in most cases, supported this notion. However, therapeutic strategies to lessen oxidative stress in clinical trials has not proved efficacious, most likely due to indiscriminate targeting by antioxidants such as vitamins. Recent evidence now suggests that oxidative stress is a major driver of inflammation and vice versa, with the latest findings suggesting not only a key role for inflammatory pathways underpinning metabolic and haemodynamic dysfunction in diabetes, but furthermore that these perturbations are driven by activation of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. This review will address these latest findings with an aim of highlighting the interconnectivity between oxidative stress, NLRP3 activation and inflammation as it pertains to cardiac and vascular injury sustained by diabetes. Current therapeutic strategies to lessen both oxidative stress and inflammation will be emphasized. This will be placed in the context of improving the burden of these diabetic complications.

Glutathione peroxidase is an antioxidant enzyme that is involved in the control of cellular oxidative state. Recently, unregulated oxidative state has been implicated as detrimental to neural cell viability and involved in both acute and chronic neurodegeneration. In this study we have addressed the importance of a functional glutathione peroxidase in a mouse ischemia/reperfusion model. Two hours of focal cerebral ischemia followed by 24 h of reperfusion was induced via the intraluminal suture method. Infarct volume was increased three‐fold in the glutathione peroxidase‐1 (Gpx‐1) –/– mouse compared with the wild‐type mouse; this was mirrored by an increase in the level of apoptosis found at 24 h in the Gpx‐1 –/– mouse compared with the wild‐type mouse. Neuronal deficit scores correlated to the histologic data. We also found that activated caspase‐3 expression is present at an earlier time point in the Gpx‐1 –/– mice when compared with the wild‐type mice, which suggests an enhanced susceptibility to apoptosis in the Gpx‐1 –/– mouse. This is the first known report of such a dramatic increase, both temporally and in level of apoptosis in a mouse stroke model. Our results suggest that Gpx‐1 plays an important regulatory role in the protection of neural cells in response to the extreme oxidative stress that is released during ischemia/reperfusion injury.

Although reactive oxygen species have been proposed to play a major role in the aging process, the exact molecular mechanisms remain elusive. In this study we investigate the effects of a perturbation in the ratio of Cu/Zn-superoxide dismutase activity (Sod1 dismutases ^. O ₂⁻ to H ₂ C ₂ ) to glutathione peroxidase activity (Gpx1 catalyses H ₂ O ₂ conversion to H ₂ O) on cell growth and development. Our data demonstrate that Sod1 transfected cell lines that have an elevation in the ratio of Sod1 activity to Gpx1 activity produce higher levels of H ₂ O ₂ and exhibit well characterised markers of cellular senescence viz . slower proliferation and altered morphology. On the contrary, Sod1 transfected cell lines that have an unaltered ratio in the activity of these two enzymes, have unaltered levels of H ₂ O ₂ and fail to show characteristics of senescence. Furthermore, fibroblasts established from individuals with Down syndrome have an increase in the ratio of Sod1 to Gpx1 activity compared with corresponding controls and senesce earlier. Interestingly, cells treated with H ₂ O ₂ also show features of senescence and/or senesce earlier. We also show that Cip1 mRNA levels are elevated in Down syndrome cells, Sod1 transfectants with an altered Sod1 to Gpx1 activity ratio and those treated with H ₂ O ₂ , thus suggesting that the slow proliferation may be mediated by Cip1. Furthermore, our data demonstrate that Cip1 mRNA levels are induced by exposure of cells to H ₂ O ₂ . These data give valuable insight into possible molecular mechanisms that contribute to cellular senescence and may be useful in the evolution of therapeutic strategies for aging.