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Chronic renal and vascular oxidative stress in association with an enhanced inflammatory burden are determinant processes in the development and progression of diabetic complications including cardiovascular disease (CVD), atherosclerosis and diabetic kidney disease (DKD). Persistent hyperglycaemia in diabetes mellitus increases the production of reactive oxygen species (ROS) and activates mediators of inflammation as well as suppresses antioxidant defence mechanisms ultimately contributing to oxidative stress which leads to vascular and renal injury in diabetes. Furthermore, there is increasing evidence that ROS, inflammation and fibrosis promote each other and are part of a vicious connection leading to development and progression of CVD and kidney disease in diabetes., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Cardiovascular disease, despite all the recent advances in treatment of the various risk factors, remains the major cause of mortality in both type 1 and type 2 diabetes. Experimental models of diabetes-associated atherosclerosis, despite their limitations in recapitulating the human context, have assisted in the elucidation of molecular and cellular pathways implicated in the development and progression of macrovascular injury in diabetes. Our own studies have emphasized the role of oxidative stress and advanced glycation and identified potential targets for vasoprotective therapies in the setting of diabetes. Furthermore, it has been clearly shown that previous episodes of hyperglycemia play a key role in promoting end-organ injury in diabetes, as shown in clinical trials such as the UK Prospective Diabetes Study (UKPDS), Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation Observational Study (ADVANCE-ON), and the Diabetes Control and Complications Trial/ Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC). The cause of this phenomenon, known as metabolic memory, remains to be elucidated, but it appears that epigenetic pathways, including glucose-induced histone methylation, play a central role. Further delineation of these pathways and their link to not only glucose but also other factors implicated in vascular injury should lead to more rational, potentially more effective therapies to retard diabetes-associated cardiovascular disease., (© 2018 by the American Diabetes Association.)

Significance: Intrarenal oxidative stress plays a critical role in the initiation and progression of diabetic kidney disease (DKD). Enhanced oxidative stress results from overproduction of reactive oxygen species (ROS) in the context of concomitant, insufficient antioxidant pathways. Renal ROS production in diabetes is predominantly mediated by various NADPH oxidases (NOXs), but a defective antioxidant system as well as mitochondrial dysfunction may also contribute. Recent Advances: Effective agents targeting the source of ROS generation hold the promise to rescue the kidney from oxidative damage and prevent subsequent progression of DKD. Critical Issues and Future Directions: In the present review, we summarize and critically analyze molecular and cellular mechanisms that have been demonstrated to be involved in NOX-induced renal injury in diabetes, with particular focus on the role of increased glomerular injury, the development of albuminuria, and tubulointerstitial fibrosis, as well as mitochondrial dysfunction. Furthermore, novel agents targeting NOX isoforms are discussed. Antioxid. Redox Signal. 25, 657-684.

Objective: Oxidative stress is considered a hallmark of atherosclerosis. In particular, the superoxide-generating type 1 NADPH oxidase (NOX1) has been shown to be induced and play a pivotal role in early phases of mouse models of atherosclerosis and in the context of diabetes mellitus. Here, we investigated the role of the most abundant type 4 isoform (NOX4) in human and mouse advanced atherosclerosis., Approach and Results: Plaques of patients with cardiovascular events or established diabetes mellitus showed a surprising reduction in expression of the most abundant but hydrogen peroxide (H2O2)-generating type 4 isoform (Nox4), whereas Nox1 mRNA was elevated, when compared with respective controls. As these data suggested that NOX4-derived reactive oxygen species may convey a surprisingly protective effect during plaque progression, we examined a mouse model of accelerated and advanced diabetic atherosclerosis, the streptozotocin-treated ApoE(-/-) mouse, with (NOX4(-/-)) and without genetic deletion of Nox4. Similar to the human data, advanced versus early plaques of wild-type mice showed reduced Nox4 mRNA expression. Consistent with a rather protective role of NOX4-derived reactive oxygen species, NOX4(-/-) mice showed increased atherosclerosis when compared with wild-type mice. Deleting NOX4 was associated with reduced H2O2 forming activity and attenuation of the proinflammatory markers, monocyte chemotratic protein-1, interleukin-1β, and tumor necrosis factor-α, as well as vascular macrophage accumulation. Furthermore, there was a greater accumulation of fibrillar collagen fibres within the vascular wall and plaque in diabetic Nox4(-/-)ApoE(-/-) mice, indicative of plaque remodeling. These data could be replicated in human aortic endothelial cells in vitro, where Nox4 overexpression increased H2O2 and reduced the expression of pro-oxidants and profibrotic markers. Interestingly, Nox4 levels inversely correlated with Nox2 gene and protein levels. Although NOX2 is not constitutively active unlike NOX4 and forms rather superoxide, this opens up the possibility that at least some effects of NOX4 deletion are mediated by NOX2 activation., Conclusions: Thus, the appearance of reactive oxygen species in atherosclerosis is apparently not always a nondesirable oxidative stress, but can also have protective effects. Both in humans and in mouse, the H2O2-forming NOX4, unlike the superoxide-forming NOX1, can act as a negative modulator of inflammation and remodeling and convey atheroprotection. These results have implications on how to judge reactive oxygen species formation in cardiovascular disease and need to be considered in the development of NOX inhibitory drugs., (© 2015 American Heart Association, Inc.)

Despite the wealth of pre-clinical support for a role for reactive oxygen and nitrogen species (ROS/RNS) in the aetiology of diabetic complications, enthusiasm for antioxidant therapeutic approaches has been dampened by less favourable outcomes in large clinical trials. This has necessitated a re-evaluation of pre-clinical evidence and a more rational approach to antioxidant therapy. The present review considers current evidence, from both pre-clinical and clinical studies, to address the benefits of antioxidant therapy. The main focus of the present review is on the effects of direct targeting of ROS-producing enzymes, the bolstering of antioxidant defences and mechanisms to improve nitric oxide availability. Current evidence suggests that a more nuanced approach to antioxidant therapy is more likely to yield positive reductions in end-organ injury, with considerations required for the types of ROS/RNS involved, the timing and dosage of antioxidant therapy, and the selective targeting of cell populations. This is likely to influence future strategies to lessen the burden of diabetic complications such as diabetes-associated atherosclerosis, diabetic nephropathy and diabetic retinopathy.

The hemodynamic and nonhemodynamic effects of angiotensin II on diabetic complications are considered to be primarily mediated by the angiotensin II type 1 receptor subtype. However, its biological and functional effect mediated through the angiotensin II type 2 receptor subtype is still unclear. Activation of the angiotensin II type 2 receptors has been postulated to oppose angiotensin II type 1 receptor-mediated actions and thus attenuate fibrosis. This study aimed to elucidate the reno-protective role of the novel selective angiotensin II type 2 receptor agonist, Compound 21, in an experimental model of type 1 diabetic nephropathy. Compound 21 treatment significantly attenuated diabetes mellitus-induced elevated levels of cystatin C, albuminuria, mesangial expansion, and glomerulosclerosis in diabetic mice. Moreover, Compound 21 markedly inhibited the expression of various proteins implicated in oxidative stress, inflammation, and fibrosis, in association with decreased extracellular matrix production. These findings demonstrate that monotherapy of Compound 21 is protective against the progression of experimental diabetic nephropathy by inhibiting renal oxidative stress, inflammation, and fibrosis., (© 2015 American Heart Association, Inc.)

Cardiac oxidative stress is an early event associated with diabetic cardiomyopathy, triggered by hyperglycemia. We tested the hypothesis that targeting left-ventricular (LV) reactive oxygen species (ROS) upregulation subsequent to hyperglycemia attenuates type 1 diabetes-induced LV remodeling and dysfunction, accompanied by attenuated proinflammatory markers and cardiomyocyte apoptosis. Male 6-week-old mice received either streptozotocin (55mg/kg/day for 5 days), to induce type 1 diabetes, or citrate buffer vehicle. After 4 weeks of hyperglycemia, the mice were allocated to coenzyme Q10 supplementation (10mg/kg/day), treatment with the angiotensin-converting-enzyme inhibitor (ACE-I) ramipril (3mg/kg/day), treatment with olive oil vehicle, or no treatment for 8 weeks. Type 1 diabetes upregulated LV NADPH oxidase (Nox2, p22(phox), p47(phox) and superoxide production), LV uncoupling protein UCP3 expression, and both LV and systemic oxidative stress (LV 3-nitrotyrosine and plasma lipid peroxidation). All of these were significantly attenuated by coenzyme Q10. Coenzyme Q10 substantially limited type 1 diabetes-induced impairments in LV diastolic function (E:A ratio and deceleration time by echocardiography, LV end-diastolic pressure, and LV -dP/dt by micromanometry), LV remodeling (cardiomyocyte hypertrophy, cardiac fibrosis, apoptosis), and LV expression of proinflammatory mediators (tumor necrosis factor-α, with a similar trend for interleukin IL-1β). Coenzyme Q10's actions were independent of glycemic control, body mass, and blood pressure. Coenzyme Q10 compared favorably to improvements observed with ramipril. In summary, these data suggest that coenzyme Q10 effectively targets LV ROS upregulation to limit type 1 diabetic cardiomyopathy. Coenzyme Q10 supplementation may thus represent an effective alternative to ACE-Is for the treatment of cardiac complications in type 1 diabetic patients., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Most diabetes-related complications and causes of death arise from cardiovascular disease and end-stage renal disease. Amongst the major complications of diabetes mellitus are retinopathy, neuropathy, nephropathy and accelerated atherosclerosis. Increased bioavailability of reactive oxygen species (ROS) (termed oxidative stress), derived in large part from the NADPH oxidase (Nox) family of free radical producing enzymes, has been demonstrated in experimental and clinical diabetes and has been implicated in the cardiovascular and renal complications of diabetes. The present review focuses on the role of Noxs and oxidative stress in some major complications of diabetes, including nephropathy, retinopathy and atherosclerosis. We also discuss Nox isoforms as potential targets for therapy.

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy., Competing Interests: HHHWS and KW declare a potential competing interest as shareholder and previous employee, respectively, of Vasopharm GmbH, which develops NADPH oxidase inhibitors such as VAS2870. All authors declare that they adhere to all PLoS Biology policies on sharing data and materials as detailed in the PLoS Biology guide for authors.

Activation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome has been reported in diabetic complications including diabetic kidney disease (DKD). However, it remains unknown if NLRP3 inhibition is renoprotective in a clinically relevant interventional approach with established DKD. We therefore examined the effect of the NLRP3-specific inhibitor MCC950 in streptozotocin-induced diabetic mice to measure the impact of NLRP3 inhibition on renal inflammation and associated pathology in DKD. We identified an adverse effect of MCC950 on renal pathology in diabetic animals. Indeed, MCC950-treated diabetic animals showed increased renal inflammation and macrophage infiltration in association with enhanced oxidative stress as well as increased mesangial expansion and glomerulosclerosis when compared with vehicle-treated diabetic animals. Inhibition of the inflammasome by MCC950 in diabetic mice led to renal up-regulation of markers of inflammation (Il1β, Il18 and Mcp1), fibrosis (Col1, Col4, Fn1, α-SMA, Ctgf and Tgfβ1) and oxidative stress (Nox2, Nox4 and nitrotyrosine). In addition, enhanced glomerular accumulation of pro-inflammatory CD68 positive cells and pro-oxidant factor nitrotyrosine was identified in the MCC950-treated diabetic compared with vehicle-treated diabetic animals. Collectively, in this interventional model of established DKD, NLRP3 inhibition with MCC950 did not show renoprotective effects in diabetic mice. On the contrary, diabetic mice treated with MCC950 exhibited adverse renal effects particularly enhanced renal inflammation and injury including mesangial expansion and glomerulosclerosis. [ABSTRACT FROM AUTHOR]

Patients with diabetic hypertensive nephropathy have accelerated disease progression. Diabetes and hypertension have both been associated with changes in renal catecholamines and reactive oxygen species. With a specific focus on renal catecholamines and oxidative stress we examined a combined model of hypertension and diabetes using normotensive BPN/3J and hypertensive BPH/2J Schlager mice. Induction of diabetes (5 × 55 mg/kg streptozotocin i.p.) did not change the hypertensive status of BPH/2J mice (telemetric 24 h avg. MAP, non-diabetic 131 ± 2 vs. diabetic 129 ± 1 mmHg, n.s at 9 weeks of study). Diabetes-associated albuminuria was higher in BPH/2J vs. diabetic BPN/3J (1205 + 196/-169 versus 496 + 67/-59 μg/24 h, p = 0.008). HPLC measurement of renal cortical norepinephrine and dopamine showed significantly greater levels in hypertensive mice whilst diabetes was associated with significantly lower catecholamine levels. Diabetic BPH/2J also had greater renal catecholamine levels than diabetic BPN/3J (diabetic: norepinephrine BPN/3J 40 ± 4, BPH/2J 91 ± 5, p = 0.010; dopamine: BPN/3J 2 ± 1; BPH/2J 3 ± 1 ng/mg total protein, p < 0.001 after 10 weeks of study). Diabetic BPH/2J showed greater cortical tubular immunostaining for monoamine oxidase A and cortical mitochondrial hydrogen peroxide formation was greater in both diabetic and non-diabetic BPH/2J. While cytosolic catalase activity was greater in non-diabetic BPH/2J it was significantly lower in diabetic BPH/2J (cytosolic: BPH/2J 127 ± 12 vs. 63 ± 6 nmol/min/ml, p < 0.001). We conclude that greater levels of renal norepinephrine and dopamine associated with hypertension, together with diabetes-associated compromised anti-oxidant systems, contribute to increased renal oxidative stress in diabetes and hypertension. Elevations in renal cortical catecholamines and reactive oxygen species have important therapeutic implications for hypertensive diabetic patients. [ABSTRACT FROM AUTHOR]

Oxidative stress is a consequence of up-regulation of pro-oxidant enzyme-induced reactive oxygen species (ROS) production and concomitant depletion of antioxidants. Elevated levels of ROS act as an intermediate and are the common denominator for various diseases including diabetes-associated macro-/micro-vascular complications and hypertension. A range of enzymes are capable of generating ROS, but the pro-oxidant enzyme family, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), are the only enzymes known to be solely dedicated to ROS generation in the vascular tissues, kidney, aortas and eyes. While there is convincing evidence for a role of NOX1 in vascular and eye disease and for NOX4 in renal injury, the role of NOX5 in disease is less clear. Although NOX5 is highly up-regulated in humans in disease, it is absent in rodents. Thus, so far it has not been possible to study NOX5 in traditional mouse or rat models of disease. In the present review, we summarize and critically analyse the emerging evidence for a pathophysiological role of NOX5 in disease including the expression, regulation and molecular and cellular mechanisms which have been demonstrated to be involved in NOX5 activation. [ABSTRACT FROM AUTHOR]

Adults with diabetes are 2-4 times more likely to suffer from heart disease or ischemic stroke than adults without diabetes, yet standard antiplatelet therapy, which is the cornerstone for primary and secondary prevention of cardiovascular disease, fails in many patients with diabetes. Three independent but often interrelated variables that contribute to platelet hyperreactivity-high blood glucose, oxidative stress, and elevated vascular shear forces-coexist in patients with diabetes, creating a perilous concurrence of risk factors for cardiovascular events. Recent research has focused attention on the platelet-specific collagen receptor glycoprotein VI (GPVI) as a potential antithrombotic target. Signaling events downstream of GPVI are influenced by hyperglycemia, oxidative stress, and shear stress. Importantly, drugs targeting these GPVI signaling pathways are already in existence. The potential to repurpose existing drugs is a high-gain strategy for yielding new antiplatelet agents and could have particular benefit in individuals with diabetes. [ABSTRACT FROM AUTHOR]

Oxidative stress and inflammation are central mediators of atherosclerosis particularly in the context of diabetes. The potential interactions between the major producers of vascular reactive oxygen species (ROS), NADPH oxidase (NOX) enzymes and immune-inflammatory processes remain to be fully elucidated. In the present study we investigated the roles of the NADPH oxidase subunit isoforms, NOX4 and NOX1, in immune cell activation and recruitment to the aortic sinus atherosclerotic plaque in diabetic ApoE-/- mice. Plaque area analysis showed that NOX4- and NOX1-derived ROS contribute to atherosclerosis in the aortic sinus following 10 weeks of diabetes. Immunohistochemical staining of the plaques revealed that NOX4-derived ROS regulate T-cell recruitment. In addition, NOX4-deficient mice showed a reduction in activated CD4+ T-cells in the draining lymph nodes of the aortic sinus coupled with reduced pro-inflammatory gene expression in the aortic sinus. Conversely, NOX1-derived ROS appeared to play a more important role in macrophage accumulation. These findings demonstrate distinct roles for NOX4 and NOX1 in immune-inflammatory responses that drive atherosclerosis in the aortic sinus of diabetic mice. [ABSTRACT FROM AUTHOR]

Current treatments for diabetic nephropathy (DN) only result in slowing its progression, thus highlighting a need to identify novel targets. Increased production of reactive oxygen species (ROS) is considered a key downstream pathway of end-organ injury with increasing data implicating both mitochondrial and cytosolic sources of ROS. The enzyme, NADPH oxidase, generates ROS in the kidney and has been implicated in the activation of protein kinase C (PKC), in the pathogenesis of DN, but the link between PKC and Noxderived ROS has not been evaluated in detail in vivo. In this study, global deletion of a NADPH-oxidase isoform, Nox4, was examined in mice with streptozotocin-induced diabetes (C57Bl6/J) in order to evaluate the effects of Nox4 deletion, not only on renal structure and function but also on the PKC pathway and downstream events. Nox4 deletion attenuated diabetes-associated increases in albuminuria, glomerulosclerosis, and extracellular matrix accumulation. Lack of Nox4 resulted in a decrease in diabetes-induced renal cortical ROS derived from the mitochondria and the cytosol, urinary isoprostanes, and PKC activity. Immunostaining of renal cortex revealed that major isoforms of PKC, PKC-α and PKC-β1, were increased with diabetes and normalized by Nox4 deletion. Downregulation of the PKC pathway was observed in tandem with reduced expression of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β1 and restoration of the podocyte slit pore protein nephrin. This study suggests that deletion of Nox4 may alleviate renal injury via PKC-dependent mechanisms, further strengthening the view that Nox4 is a suitable target for renoprotection in diabetes. [ABSTRACT FROM AUTHOR]

The development of macro- and microvascular complications is accelerated in diabetic patients. While some therapeutic regimes have helped in delaying progression of complications, none have yet been able to halt the progression and prevent vascular disease, highlighting the need to identify new therapeutic targets. Increased oxidative stress derived from the NADPH oxidase (Nox) family has recently been identified to play an important role in the pathophysiology of vascular disease. In recent years, specific Nox isoforms have been implicated in contributing to the development of atherosclerosis of major vessels, as well as damage of the small vessels within the kidney and the eye. With the use of novel Nox inhibitors, it has been demonstrated that these complications can be attenuated, indicating that targeting Nox derived oxidative stress holds potential as a new therapeutic strategy. [ABSTRACT FROM AUTHOR]

NADPH oxidase (Nox) isoforms have been implicated in contributing to diabetic microvascular complications, but the functional role of individual isoforms in diabetic kidney are unclear. Nox2, in particular, is highly expressed in phagocytes and may play a key inflammatory role in diabetic kidney disease. To determine the role of Nox2, we evaluated kidney function and pathology in wild-type (WT; C57BL/6) and Nox2 knockout (KO) mice with type 1 diabetes. Diabetes was induced in male Nox2 KO and WT mice with a multiple low-dose streptozotocin protocol. Groups were studied for kidney disease after 8 and 20 wk of diabetes. Hyperglycemia and body weights were similar in WT and Nox2 KO diabetic mice. All functional and structural features of early and later stage diabetic kidney disease (albuminuria, mesangial matrix, tubulointerstitial disease, and gene expression of matrix and transforming growth factor-β) were similar in both diabetic groups compared with their respective nondiabetic groups, except for reduction of macrophage infiltration and monocyte chemoattractant protein-1 in the diabetic Nox2 KO mice. Systolic blood pressure by telemetry was surprisingly increased in Nox2 KO mice; however, the systolic blood pressure was reduced in the diabetic WT and Nox2 KO mice by tail-cuff. Interestingly, diabetic Nox2 KO mice had marked upregulation of renal Nox4 at both the glomerular and cortical levels. The present results demonstrate that lack of Nox2 does not protect against diabetic kidney disease in type 1 diabetes, despite a reduction in macrophage infiltration. The lack of renoprotection may be due to upregulation of renal Nox4. [ABSTRACT FROM AUTHOR]

Nox (NADPH oxidase)-derived ROS (reactive oxygen species) have been implicated in the development of diabetic nephropathy. Of the Nox isoforms in the kidney, Nox4 is important because of its renal abundance. In the present study, we tested the hypothesis that GKT136901, a Nox1/4 inhibitor, prevents the development of nephropathy in db/db (diabetic) mice. Six groups of male mice (8-week-old) were studied: (i) untreated control db/m, (ii) low-dose GKT136901-treated db/m (30 mg/kg of body weight per day), (iii) high-dose GKT136901-treated db/m (90 mg/kg of body weight per day), (iv) untreated db/db; (v) low dose GKT136901-treated db/db; and (vi) high-dose GKT136901-treated db/db. GKT136901, in chow, was administered for 16 weeks. db/db mice developed diabetes and nephropathy as evidenced by hyperglycaemia, albuminuria and renal injury (mesangial expansion, tubular dystrophy and glomerulosclerosis). GKT136901 treatment had no effect on plasma glucose or BP (blood pressure) in any of the groups. Plasma and urine TBARSs (thiobarbituric acid-reacting substances) levels, markers of systemic and renal oxidative stress, respectively, were increased in diabetic mice. Renal mRNA expression of Nox4, but not of Nox2, increased, Nox1 was barely detectable in db/db. Expression of the antioxidant enzyme SOD-1 (superoxide dismutase 1) decreased in db/db mice. Renal content of fibronectin, pro-collagen, TGFß (transforming growth factor ß) and VCAM-1 (vascular cell adhesion molecule 1) and phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2) were augmented in db/db kidneys, with no change in p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase). Treatment reduced albuminuria, TBARS and renal ERK1/2 phosphorylation and preserved renal structure in diabetic mice. Our findings suggest a renoprotective effect of the Nox1/4 inhibitor, possibly through reduced oxidative damage and decreased ERK1/2 activation. These phenomena occur independently of improved glucose control, suggesting GKT136901-sensitive targets are involved in complications of diabetes rather than in the disease process. [ABSTRACT FROM AUTHOR]

Advanced glycation end products (AGEs) are important mediators of diabetic nephropathy that act through the receptor for AGEs (RAGE), as well as other mechanisms, to promote renal inflammation and glomerulosclerosis. The relative contribution of RAGE-dependent and RAGE-independent signaling pathways has not been previously studied in vivo. In this study, diabetic RAGE apoE double-knockout (KO) mice with streptozotocin-induced diabetes were treated with the AGE inhibitor, alagebrium (1 mg/kg/day), or the ACE inhibitor, quinapril (30 mg/kg/day), for 20 weeks, and renal parameters were assessed. RAGE deletion attenuated mesangial expansion, glomerular matrix accumulation, and renal oxidative stress associated with 20 weeks of diabetes. By contrast, inflammation and AGE accumulation associated with diabetes was not prevented. However, treatment with alagebrium in diabetic RAGE apoE KO mice reduced renal AGE levels and further reduced glomerular matrix accumulation. In addition, even in the absence of RAGE expression, alagebrium attenuated cortical inflammation, as denoted by the reduced expression of monocyte chemoattractant protein-1, intracellular adhesion molecule-1, and the macrophage marker cluster of differentiation molecule 11b. These novel findings confirm the presence of important RAGE-independent as well as RAGE-dependent signaling pathways that may be activated in the kidney by AGEs. This has important implications for the design of optimal therapeutic strategies for the prevention of diabetic nephropathy. [ABSTRACT FROM AUTHOR]

OBJECTIVE--To investigate the effect of the GPx1-mimetic ebselen on diabetes-associated atherosclerosis and renal injury in a model of increased oxidative stress. RESEARCH DESIGN AND METHODS--The study was performed using diabetic apolipoprotein E/GPx1 (ApoE-/-GPx1-/-)-double knockout (dKO) mice, a model combining hyperlipidemia and hyperglycemia with increased oxidative stress. Mice were randomized into two groups, one injected with streptozotocin, the other with vehicle, at 8 weeks of age. Groups were further randomized to receive either ebselen or no treatment for 20 weeks. RESULTS--Ebselen reduced diabetes-associated atherosclerosis in most aortic regions, with the exception of the aortic sinus, and protected dKO mice from renal structural and functional injury. The protective effects of ebselen were associated with a reduction in oxidative stress (hydroperoxides in plasma, 8-isoprostane in urine, nitrotyrosine in the kidney, and 4-hydroxynonenal in the aorta) as well as a reduction in VEGF, CTGF, VCAM-1, MCP-1, and Nox2 after 10 weeks of diabetes in the dKO aorta. Ebselen also significantly reduced the expression of proteins implicated in fibrosis and inflammation in the kidney as well as reducing related key intracellular signaling pathways. CONCLUSIONS--Ebselen has an antiatherosclerotic and renoprotective effect in a model of accelerated diabetic complications in the setting of enhanced oxidative stress. Our data suggest that ebselen effectively repletes the lack of GPx1, and indicate that ebselen may be an effective therapeutic for the treatment of diabetes-related atherosclerosis and nephropathy. Furthermore, this study highlights the feasibility of addressing two diabetic complications with one treatment regimen through the unifying approach of targeted antioxidant therapy. Diabetes 59:3198-3207, 2010 [ABSTRACT FROM AUTHOR]

OBJECTIVE--Activation of the receptor for advanced glycation end products (RAGE) in diabetic vasculature is considered to be a key mediator of atherogenesis. This study examines the effects of deletion of RAGE on the development of atherosclerosis in the diabetic apoE[sup -/-] model of accelerated atherosclerosis. RESEARCH DESIGN AND METHODS--ApoE[sup -/-] and RAGE[sup -/-] apoE[sup -/-] double knockout mice were rendered diabetic with streptozotocin and followed for 20 weeks, at which time plaque accumulation was assessed by en face analysis. RESULTS--Although diabetic apoE[sup -/-] mice showed increased plaque accumulation (14.9 ± 1.7%), diabetic RAGE[sup -/-]apoE[sup -/-] mice had significantly reduced atherosclerotic plaque area (4.9 +0.4%) to levels not significantly different from control apoE[sup -/-] mice (4.3 ± 0.4%). These beneficial effects on the vasculature were associated with attenuation of leukocyte recruitment; decreased expression of proinflammatory mediators, including the nuclear factor-κB subunit p65, VCAM-1, and MCP-1; and reduced oxidative stress, as reflected by staining for nitrotyrosine and reduced expression of various NADPH oxidase subunits, gp91phox, p47phox, and rac-1. Both RAGE and RAGE ligands, including S100A8/A9, high mobility group box 1 (HMGB1), and the advanced glycation end product (AGE) carboxymethyllysine were increased in plaques from diabetic apoE[sup -/-] mice. Furthermore, the accumulation of AGEs and other ligands to RAGE was reduced in diabetic RAGE[sup -/-]apoE[sup -/-] mice. CONCLUSIONS--This study provides evidence for RAGE playing a central role in the development of accelerated atherosclerosis associated with diabetes. These findings emphasize the potential utility of strategies targeting RAGE activation in the prevention and treatment of diabetic macrovascular complications. Diabetes 57:2461-2469, 2008 [ABSTRACT FROM AUTHOR]

Simple Summary: The progressive nature of type 2 diabetes mellitus (T2DM) leads to micro- and macro-vascular complications, including renal and cardiovascular disease. These alone, or in combination, are a major cause of premature morbidity and mortality in diabetic patients. Despite advances in glucose lowering treatments, these diabetic complications are still inadequately prevented or reversed. This ongoing cardiovascular–renal burden in diabetes poses a heavy cost on the health care system. Therefore, there is an urgent need to develop more effective treatments. In this review, we discuss how oxidative stress and inflammation induce and perpetuate the renal and cardiovascular complications of diabetes. It is particularly important to understand these driving mechanisms in order to elucidate pharmacological targets and mechanism-based future drug therapies. Oxidative stress and inflammation are considered major drivers in the pathogenesis of diabetic complications, including renal and cardiovascular disease. A symbiotic relationship also appears to exist between oxidative stress and inflammation. Several emerging therapies target these crucial pathways, to alleviate the burden of the aforementioned diseases. Oxidative stress refers to an imbalance between reactive oxygen species (ROS) and antioxidant defenses, a pathological state which not only leads to direct cellular damage but also an inflammatory cascade that further perpetuates tissue injury. Emerging therapeutic strategies tackle these pathways in a variety of ways, from increasing antioxidant defenses (antioxidants and Nrf2 activators) to reducing ROS production (NADPH oxidase inhibitors and XO inhibitors) or inhibiting the associated inflammatory pathways (NLRP3 inflammasome inhibitors, lipoxins, GLP-1 receptor agonists, and AT-1 receptor antagonists). This review summarizes the mechanisms by which oxidative stress and inflammation contribute to and perpetuate diabetes associated renal and cardiovascular disease along with the therapeutic strategies which target these pathways to provide reno and cardiovascular protection in the setting of diabetes. [ABSTRACT FROM AUTHOR]

Significance: Oxidative stress plays a critical role in the development and progression of serious micro- and macrovascular complications of diabetes. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)-derived reactive oxygen species (ROS) significantly contribute to oxidative stress-associated inflammatory pathways that lead to tissue damage of different organs, including the kidneys, retina, brain, nerves, and the cardiovascular system. Recent Advances: Preclinical studies, including genetic-modified mouse models or cell culture models, have revealed the role of specific NOX isoforms in different diabetic complications, and suggested them as a promising target for the treatment of these diseases. Critical Issues: In this review, we provide an overview of the role of ROS and oxidative stress in macrovascular complications, such as stroke, myocardial infarction, coronary artery disease, and peripheral vascular disease that are all mainly driven by atherosclerosis, as well as microvascular complications, such as diabetic retinopathy, nephropathy, and neuropathy. We summarize conducted genetic deletion studies of different Nox isoforms as well as pharmacological intervention studies using NOX inhibitors in the context of preclinical as well as clinical research on diabetic complications. Future Directions: We outline the isoforms that are most promising for future clinical trials in the context of micro- and macrovascular complications of diabetes. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) have been mainly viewed as unwanted by-products of cellular metabolism, oxidative stress, a sign of a cellular redox imbalance, and potential disease mechanisms, such as in diabetes mellitus (DM). Antioxidant therapies, however, have failed to provide clinical benefit. This paradox can be explained by recent discoveries that ROS have mainly essential signaling and metabolic functions and evolutionally conserved physiological enzymatic sources. Disease can occur when ROS accumulate in nonphysiological concentrations, locations, or forms. By focusing on disease-relevant sources and targets of ROS, and leaving ROS physiology intact, precise therapeutic interventions are now possible and are entering clinical trials. Their outcomes are likely to profoundly change our concepts of ROS in DM and in medicine in general. Lack of comprehensive understanding of DM and its complications, together with its increasing prevalence, represent a major unmet medical need. There is quite a lot known about its mechanisms, but more is needed. Reactive oxygen species (ROS) may hold the answer to this need, but long-standing misconceptions about oxidative stress and the inefficacy of antioxidants have prevented clinical breakthroughs. A new understanding of ROS as both essential signaling molecules and pathomechanism have led to the identification, at different stages of diabetes, of different therapeutically relevant sources and targets of ROS. Network pharmacology and precision diagnostics, currently in advanced stages of clinical development, in conjunction with essential lifestyle changes, will revolutionize the therapy and prevention of diabetes and its end-organ damage. [ABSTRACT FROM AUTHOR]

[Display omitted] • Urinary biomarker detection without filtration steps using FTIR spectroscopy. • Quantification of urinary 8-isoprostane using fourth derivative spectroscopy. • Identification of the main 8-isoprostane bands in urine. Diabetes mellitus is a chronic disease leading to renal and cardiovascular complications associated with high personal and societal costs. Mouse models are routinely used to assess the mechanisms of disease and disease status, by monitoring biomarkers, including 8-isoprostane, which is a marker of oxidative stress. Here we report that transflection FTIR spectroscopy coupled with partial least squares regression is able to predict urinary 8-isoprostane concentrations in various murine models of type 1 and type 2 diabetes. Regression analysis produced linear correlations, with R2 values of correlation between 0.6 and 0.8, and RMSECV (%) values between 12.3 and 29.4%, between known (measured by enzyme-linked immunosorbent assay) and predicted urinary 8-isoprostane concentration, based on the fourth derivative spectra. This approach has significant advantages over the current methods utilised, namely it has higher throughput and requires minimal sample preparation, compared to enzyme-linked immunosorbent assay and gas- or liquid-chromatography coupled with mass spectrometry. Furthermore, it has the potential to be expanded into analysis of other biomarkers and human samples in a point-of-care context. [ABSTRACT FROM AUTHOR]

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 Noxl 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 Noxl, oxidative stress, and proinflammatory markers in a Noxl-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 Nox 1 on plaque development in apolipoprotein E-deficient mice 10 weeks after induction of diabetes mellitus. Deletion of Noxl, 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 Noxl and suggest that Nox I-dependent oxidative stress is a promising target for diabetic vasculopathies, including atherosclerosis. (Circulation. 2013;127:1888-1902.) [ABSTRACT FROM AUTHOR]