Diabetes and vascular disease: pathophysiology, clinical consequences, and medical therapy: part I

The reactive advanced glycation end product (AGE) precursor methylglyoxal (MGO) and MGO-derived AGEs are associated with diabetic vascular complications and also with an increase in oxidative stress. Glyoxalase-I (GLO-I) transgenic rats were used to explore whether overexpression of this MGO detoxifying enzyme reduces levels of AGEs and oxidative stress in a rat model of diabetes. Rats were made diabetic with streptozotocin, and after 12 weeks, plasma and multiple tissues were isolated for analysis of AGEs, carbonyl stress, and oxidative stress. GLO-I activity was significantly elevated in multiple tissues of all transgenic rats compared with wild-type (WT) littermates. Streptozotocin treatment resulted in a 5-fold increase in blood glucose concentrations irrespective of GLO-I overexpression. Levels of MGO, glyoxal, 3-deoxyglucosone, AGEs, and oxidative stress markers nitrotyrosine, malondialdehyde, and F2-isoprostane were elevated in the diabetic WT rats. In diabetic GLO-I rats, glyoxal and MGO composite scores were significantly decreased by 81%, and plasma AGEs and oxidative stress markers scores were significantly decreased by ∼50%. Hyperglycemia induced a decrease in protein levels of the mitochondrial oxidative phosphorylation complex in the gastrocnemius muscle, which was accompanied by an increase in the lipid peroxidation product 4-hydroxy-2-nonenal, and this was counteracted by GLO-I overexpression. This study shows for the first time in an in vivo model of diabetes that GLO-I overexpression reduces hyperglycemia-induced levels of carbonyl stress, AGEs, and oxidative stress. The reduction of oxidative stress by GLO-I overexpression directly demonstrates the link between glycation and oxidative stress.

HIF-1 Prevents Hematopoietic Cells from Cell Damage by Overproduction of Mitochondrial ROS after Cytokine Stimulation through Induction of PDK-1

Intensive blood glucose control can prevent the initiation and progression of diabetic complications. However, the impacts of intensive therapy against diabetic complications might be limited, because of difficulty in maintaining blood glucose concentrations close to the normal range or other unknown reasons. Thus, another approach based on the elucidation of mechanisms of diabetic complications might be required to prevent the progression of the complications. Production of reactive oxygen species (ROS) and lipid peroxidation are increased in diabetic patients, especially in those with poor glycemic control. Oxidative stress can be crucial for the development of diabetic vascular complications. Thus, there is interest in determining whether antioxidant therapy can complement intensive blood glucose control. In fact, a large number of studies evaluating the efficacy of antioxidants have been carried out. However, the efficacy of these antioxidant-based therapies is still uncertain in relation to preventing diabetic complications in clinical practice. Certainly, a number of experimental studies suggest that some natural antioxidants, such as α-tocopherol (vitamin E), ascorbate (vitamin C), coenzyme Q (CoQ), taurine, glutathione or α lipoic acid, showed beneficial effects on diabetic complications. However, the results from large, long-term clinical trials using α-tocopherol were disappointing. Among the various antioxidant-based therapies, only α-lipoic acid might be somewhat useful in preventing diabetic complications. Meta-analysis has provided evidence that intravenous treatment with 600 mg/day α-lipoic acid over 3 weeks significantly improves both positive neuropathic symptoms and neuropathic deficits in diabetic patients with symptomatic polyneuropathy. α-Lipoic acid is approved in Germany as an agent for the treatment of diabetic neuropathy. The effectiveness of natural antioxidants in preventing diabetic complications is still uncertain. Therefore, new strategies for controlling oxidative stress, such as development of new mechanism-based antioxidants, will be required to prevent diabetic complications. In addition, to develop these agents, we should investigate the mechanisms that underlie the association between diabetes and oxidative stress. There are several potential mechanisms by which hyperglycemia can lead to oxidative stress1: We previously reported that hyperglycemia could increase the production of ROS from the mitochondrial electron transport chain (mitochondrial ROS)2. In addition, the normalization of the mitochondrial ROS production prevented the glucose-induced activation of PKC and polyol pathway, and the formation of advanced glycation end-products (AGEs), all of which are known to be involved in the development of diabetic complications (Figure 1). Because the production of mitochondrial ROS is thought to be one of the key events in the pathogenesis of diabetic complications and other mitochondria-related diseases, mitochondria-targeted antioxidants, such as idebenone and mitoquinone, have been developed. Idebenone is a synthetic short chain analog of CoQ10, initially patented to provide a suitable medical composition for treating and improving the after-effects of cerebral infarction. It was reported that idebenone acts as an antioxidant and protects the mitochondrial membrane against lipid peroxidation. Interestingly, it was reported that in clinical trials, idebenone is useful in controlling cardiac hypertrophy in Friedreich's ataxia (FRDA), recovery of visual acuity in Leber's hereditary optic neuropathy (LHON), and improvement of mitochondrial oxidative metabolism in the brain of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Furthermore, a phase 3 study of idebenone in Duchenne muscular dystrophy (DMD) is ongoing now. Subcellular localization of idebenone is thought to be in mitochondria. However, because it distributes through the extracellular and intracellular compartments, its effectiveness might be uncertain. Thus, mitoquinone, which is a mitochondria-targeted antioxidant analog of idebenone, has been newly developed. Because mitoquinone possesses a terminal triphenylphosphonium group instead of a hydroxyl group, it is accumulated several hundred-fold within mitochondria, enhancing the protection of mitochondria from oxidative damage. A phase 2 study of mitoquinone in chronic hepatitis C virus (HCV) infection was carried out, and mitoquinone could decrease the liver damage associated with chronic HCV infection3. Disappointingly, to our knowledge, there is no clinical evidence of idebenone and mitoquinone in preventing diabetic complications. However, FRDA, LHON and MELAS are clinical syndromes of mitochondrial disorders, in which respiratory chain functions are defective. In addition, molecular pathology of DMD is reported to be associated with increased ROS production and mitochondrial dysfunction. Furthermore, in HCV infection, there is considerable evidence for increased mitochondrial ROS and damage leading to cell death and tissue fibrosis. Therefore, similar to those mitochondria-related diseases, both idebenone and mitoquinone might show benefits in preventing diabetic complications (Figure 1). By the way, in classical natural antioxidants, why could α-lipoic acid show some effectiveness in preventing diabetic complications when α-tocopherol could not? One possible explanation for the difference might be as a result of the difference in antioxidant capacity between α-lipoic acid and α-tocopherol. α-Lipoic acid is believed to be a powerful antioxidant compared with α-tocopherol. Another explanation might be that α-lipoic acid has additional effects in preventing diabetic complications. We previously reported that metformin and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) activate adenosine monophosphate-activated protein kinase (AMPK), normalize hyperglycemia-induced mitochondrial ROS production and promote mitochondrial biogenesis in cultured human umbilical vein endothelial cells. Because an overexpression of dominant negative AMPKα1 (T172A) attenuated metformin-induced and AICAR-induced inhibition of mitochondrial ROS and production of mitochondrial biogenesis, the effects of metformin and AICAR were dependent on the activation of AMPK4. AMPK might be one of the molecular targets for attenuating hyperglycemia-induced overproduction of mitochondrial ROS. Interestingly, it was recently reported that α-lipoic acid could activate AMPK in multiple peripheral tissues, including skeletal muscle, liver and adipocytes5. In addition, it was reported that α-lipoic acid improves mitochondrial dysfunction and oxidative damage in aging. Taking all this together, administrated α-lipoic acid might reduce hyperglycemia-induced mitochondrial ROS through AMPK activation in peripheral tissues, and prevent diabetic complications. Because oxidative stress is generally defined as an imbalance that favors the production of ROS over the antioxidant defense system, there is another strategy to reinforce the antioxidant defense system. Nuclear factor E2-related factor 2 (Nrf2) is one of the most important cellular defense mechanisms to cope with oxidative stress. Thus, Nrf2-targeted agents, such as bardoxolone methyl and sulforaphane, have been developed to prevent or slow down the progression of oxidative stress-related diseases. Nrf2 is a transactivator of genes containing an antioxidant response element (ARE) in their promoter. Such genes code for a number of antioxidative enzymes including NADPH: quinone oxidoreductase, glutathione S-transferases, aldo-keto reductases and heme oxygenase-1. Under normal physiological conditions, Nrf2 is anchored in the cytoplasm by binding to Kelchlike ECH-associated protein 1 (Keap1), which promotes the ubiquitination and subsequent proteolytic degradation of Nrf2; whereas both bardoxolone methyl and sulforaphane interact with cysteine residues on Keap1, allowing Nrf2 translocation to the nucleus and subsequent upregulation of a number of genes of antioxidative enzymes. Recently, the 52-Week Bardoxolene Methyl Treatment: Renal Function in CKD/Type 2 Diabetes (BEAM) study was carried out in patients with moderate to severe chronic kidney disease (CKD) and type 2 diabetes as a double-blind, randomized, placebo-controlled phase 2 clinical trial6. After 24 weeks of the treatment, the patients treated with bardoxolone methyl showed a significant increase in the mean estimated glomerular filtration rate compared with those treated with placebo. Although it is unknown whether bardoxolone methyl could reduce hyperglycemia-induced mitochondrial ROS, it was reported that heme oxygenase-1 regulates cardiac mitochondrial biogenesis through Nrf2-mediated transcriptional control of nuclear respiratory factor-1 (NRF-1). In addition, activation of Nrf2 by sulforaphane increased ARE-linked gene expression of transketolase and glutathione reductase, and ameliorated hyperglycemia-induced production of ROS, activation of hexosamine and PKC pathways, and prevented increased cellular accumulation and excretion of the glycating agent methylglyoxal. Furthermore, Nrf2 was reported to regulate promoter activity of the aldose reductase gene, which is the key enzyme of the polyol pathway. Because activation of Nrf2 might prevent hyperglycemia-induced ROS production and metabolic dysfunctions, such as activation of hexosamine, PKC and polyol pathways and accumulation of intracellular AGEs, bardoxolone methyl or sulforaphane might have promise for the treatment of diabetic complications (Figure 1). Considered together, although oxidative stress has been implicated in the pathology of diabetic complications, the efficacy of classical natural antioxidants in preventing diabetic complications is still uncertain. However, mechanism-based antioxidants, such as idebenone, mitoquinone, bardoxolone methyl and sulforaphane, have been developed. These mechanism-based strategies might suggest the potential for better treatment approaches to reduce the burden of oxidative stress and to prevent diabetic complications in clinical practice. In particular, because mitochondrial ROS production in response to hyperglycemia might be the central in the pathogenesis of diabetic complications, reduction of mitochondrial ROS might be the important therapeutic strategy to prevent diabetic complications.

Diabetes mellitus (DM) is a common metabolic disease, representing a serious risk factor for the development of cardiovascular complications, such as coronary heart disease, peripheral arterial disease and hypertension. Oxidative stress (OS), a feature of DM, is defined as an increase in the steady-state levels of reactive oxygen species (ROS) and may occur as a result of increased free radical generation and/or decreased anti-oxidant defense mechanisms. Increasing evidence indicates that hyperglycemia is the initiating cause of the tissue damage in DM, either through repeated acute changes in cellular glucose metabolism, or through long-term accumulation of glycated biomolecules and advanced glycation end products (AGEs). AGEs are formed by the Maillard process, a non-enzymatic reaction between ketone group of the glucose molecule or aldehydes and the amino groups of proteins that contributes to the aging of proteins and to the pathological complications of DM. In the presence of uncontrolled hyperglycemia, the increased formation of AGEs and lipid peroxidation products exacerbate intracellular OS and results in a loss of molecular integrity, disruption in cellular signaling and homeostasis, followed by inflammation and tissue injury such as endothelium dysfunction, arterial stiffening and microvascular complications. In addition to increased AGE production, there is also evidence of multiple pathways elevating ROS generation in DM, including; enhanced glucose auto-oxidation, increased mitochondrial superoxide production, protein kinase C-dependent activation of NADPH oxidase, uncoupled endothelial nitric oxide synthase (eNOS) activity, increased substrate flux through the polyol pathway and stimulation of eicosanoid metabolism. It is, therefore, not surprising that the correction of these variables can result in amelioration of diabetic cardiovascular abnormalities. A linking element between these phenomena is cellular redox imbalance due to glycoxidative stress (GOS). Thus, recent interest has focused on strategies to prevent, reverse or retard GOS in order to modify the natural history of diabetic cardiovascular abnormalities. This review will discuss the links between GOS and diabetes-induced cardiovascular disorders and the effect of antioxidant therapy on altering the development of cardiovascular complications in diabetic animal models.

Heat stress is an environmental factor that causes oxidative stress. We found previously that acute heat stress stimulates the production of reactive oxygen species (ROS) in the skeletal muscle mitochondria of birds, and that this was accompanied by an increase of the mitochondrial membrane potential (ΔΨ) due to increased substrate oxidation by the electron transport chain. We also showed that avian uncoupling protein (avUCP) expression is decreased by the heat exposure. The present study clarifies whether ΔΨ is a major determinant of the overproduction of ROS due to acute heat stress, and if the decrease in avUCP expression is responsible for the elevation in ΔΨ. Control (24°C) and acute heat-stressed (34°C for 12 h) birds exhibited increased succinate-driven mitochondrial ROS production as indicated by an elevation of ΔΨ, with this increase being significantly higher in the heat-stressed group compared with the control group. In glutamate/malate-energized mitochondria, no difference in the ROS production between the groups was observed, though the mitochondrial ΔΨ was significantly higher in the heat-stressed groups compared with the control group. Furthermore, mitochondria energized with either succinate/glutamate or succinate/malate showed increased ROS production and ΔΨ in the heat-stressed group compared with mitochondria from the control group. These results suggest that succinate oxidation could play an important role in the heat stress-induced overproduction of mitochondrial ROS in skeletal muscle. In agreement with the notion of a decrease in avUCP expression in response to heat stress, proton leak, which was likely mediated by UCP (that part which is GDP-inhibited and arachidonic acid-sensitive), was reduced in the heat-exposed group. We suggest that the acute heat stress-induced overproduction of mitochondrial ROS may depend on ΔΨ, which may in turn result not only from increased substrate oxidation but also from a decrease in the mitochondrial avUCP content.

Pharmacological mechanisms of Taohe Chengqi decoction in diabetic cardiovascular complications: A systematic review, network pharmacology and molecular docking

Introduction

Vascular complications in diabetes are a significant source of human morbidity and mortality, affecting multiple organ systems and persisting despite tight glucose control. Many of these complications can be linked to impairments in vasculogenesis, the process by which circulating and bone marrow-derived endothelial progenitor cells (EPCs) contribute to new vessel formation. Recent evidence suggests that hyperglycemia alone, through the mitochondrial overproduction of reactive oxygen species (ROS), can induce changes in gene expression and cellular behavior in diabetes. In this review, we examine how hyperglycemia-induced overproduction of ROS could explain EPC impairments observed in diabetes. Experimentally, impairments in EPC function prevent new blood vessel growth and are potentially reversible by manipulations to decrease ROS. Novel strategies aimed at reducing hyperglycemia-induced ROS may be a useful adjuvant to antihyperglycemic therapies in the restoration of vasculogenesis and the prevention of diabetic complications.

See related article, pages 1058–1065 Cardiovascular complications are a major culprit in the pathogenesis of obesity and diabetes. The increased prevalence of cardiovascular complications in diabetes is attributed to the disturbed balance between increased endothelial injury and hampered endothelial repair processes.1 Retinopathy, a blinding disease, is a major morbidity in diabetic patients. Diabetic retinopathy is initiated by hyperglycemia-induced endothelial cell injury, retinal vessel loss, and the adaptation of an inflammatory phenotype by the endothelium. Secretion of proangiogenic factors and proinflammatory cytokines from the ischemic retina lead to an exacerbated compensatory neovascularization, resulting in abnormal retinal neovascularization.2 Hence, the primary treatment of retinopathy aims to preserve vision through the inhibition of excessive vascularization and reduction of vascular damage. Interestingly, endothelial dysfunction, hampered endothelial repair, and pathological neovascularization during retinopathy reflect similar molecular alterations partially related to a state of mild chronic inflammation, ie, overproduction of inflammatory cytokines and reactive oxygen species, increased leukocyte extravasations, and vascular leakage.3 The traditional role attributed to fat tissue is energy storage; however, a change in perspective has risen since the discovery of the adipokines. Advances in the biology of the adipose tissue has revealed that it is not simply an energy storing organ but also secretes a variety of molecules, termed adipokines, that affect processes beyond metabolic regulation.4 Bioactive substances secreted by adipocytes, the adipokines, include proinflammatory cytokines, such as leptin, angiotensinogen, plasminogen activator inhibitor 1, interleukin (IL)-6, and tumor necrosis factor (TNF)α, and could contribute to the complications of obesity and diabetes through the amplification of inflammatory responses.5 In contrast, the adipokine adiponectin (APN) is unique in its antiinflammatory actions, and plasma levels of APN are paradoxically decreased in obese and diabetic subjects.6 In this issue of Circulation Research …

Necroptosis is a type of programmed necrosis regulated by receptor interacting protein kinase 1 (RIP1) and RIP3. Necroptosis is found to be accompanied by an overproduction of reactive oxygen species (ROS), but the role of ROS in regulation of necroptosis remains elusive. In this study, we investigated how shikonin, a necroptosis inducer for cancer cells, regulated the signaling leading to necroptosis in glinoma cells in vitro. Treatment with shikonin (2-10 μmol/L) dose-dependently triggered necrosis and induced overproduction of intracellular ROS in rat C6 and human SHG-44, U87 and U251 glioma cell lines. Moreover, shikonin treatment dose-dependently upregulated the levels of RIP1 and RIP3 and reinforced their interaction in the glioma cells. Pretreatment with the specific RIP1 inhibitor Nec-1 (100 μmol/L) or the specific RIP3 inhibitor GSK-872 (5 μmol/L) not only prevented shikonin-induced glioma cell necrosis but also significantly mitigated the levels of intracellular ROS and mitochondrial superoxide. Mitigation of ROS with MnTBAP (40 μmol/L), which was a cleaner of mitochondrial superoxide, attenuated shikonin-induced glioma cell necrosis, whereas increasing ROS levels with rotenone, which improved the mitochondrial generation of superoxide, significantly augmented shikonin-caused glioma cell necrosis. Furthermore, pretreatment with MnTBAP prevented the shikonin-induced upregulation of RIP1 and RIP3 expression and their interaction while pretreatment with rotenone reinforced these effects. These findings suggest that ROS is not only an executioner of shikonin-induced glioma cell necrosis but also a regulator of RIP1 and RIP3 expression and necrosome assembly.

Plants are an important source of pharmacologically active compounds. In the present work, we characterize the impact of black cumin (Nigella sativa L.) aqueous extracts on a yeast model of p53-dependent apoptosis. To this end, the Saccharomyces cerevisiae recombinant strain over-expressing p53 was used. The over-expression of p53 triggers the expression of apoptotic markers: the externalization of phosphatidylserine, mitochondrial defect associated with cytochrome-c release and the induction of DNA strand breaks. These different effects were attenuated by Nigella sativa L. aqueous extracts, whereas these extracts have no effect on the level of p53 expression. Thus, we focus on the anti-apoptotic molecules present in the aqueous extract of Nigella sativa L. These extracts were purified and characterized by complementary chromatographic methods. Specific fluorescent probes were used to determine the effect of the extracts on yeast apoptosis. Yeast cells over-expressing p53 decrease in relative size and have lower mitochondrial content. The decrease in cell size was proportional to the decrease in mitochondrial content and of mitochondrial membrane potential (ΔΨm). These effects were prevented by the purified aqueous fraction obtained by fractionation with different columns, named C4 fraction. Yeast cell death was also characterized by reactive oxygen species (ROS) overproduction. In the presence of the C4 fraction, ROS overproduction was strongly reduced. We also noted that the C4 fraction promotes the cell growth of control yeast cells, which do not express p53, supporting the fact that this purified extract acts on cellular mediators activating cell proliferation independently of p53. Altogether, our data obtained on yeast cells over-expressing p53 demonstrate that anti-apoptotic molecules targeting p53-induced apoptosis associated with mitochondrial dysfunction and ROS overproduction are present in the aqueous extracts of Nigella seeds and in the purified aqueous C4 fraction.

Reactive oxygen species (ROS) overproduction plays an essential role in the etiology of ischemic/hypoxic retinopathy caused by acute glaucoma. NADPH oxidase (NOX) 4 was discovered as one of the main sources of ROS in glaucoma. However, the role and potential mechanisms of NOX4 in acute glaucoma have not been fully elucidated. Therefore, the current study aims to investigate the NOX4 inhibitor GLX351322 that targets NOX4 inhibition in acute ocular hypertension (AOH)-induced retinal ischemia/hypoxia injury in mice. Herein, NOX4 was highly expressed in AOH retinas, particularly the retinal ganglion cell layer (GCL). Importantly, the NOX4 inhibitor GLX351322 reduced ROS overproduction, inhibited inflammatory factor release, suppressed glial cell activation and hyperplasia, inhibited leukocyte infiltration, reduced retinal cell senescence and apoptosis in damaged areas, reduced retinal degeneration and improved retinal function. This neuroprotective effect is at least partially associated with mediated redox-sensitive factor (HIF-1α, NF-κB, and MAPKs) pathways by NOX4-derived ROS overproduction. These results suggest that inhibition of NOX4 with GLX351322 attenuated AOH-induced retinal inflammation, cellular senescence, and apoptosis by inhibiting the activation of the redox-sensitive factor pathway mediated by ROS overproduction, thereby protecting retinal structure and function. Targeted inhibition of NOX4 is expected to be a new idea in the treatment of acute glaucoma.

Chronic Myeloid Leukemia Stem Cells (LSCs) and Leukemia Progenitor Cells (LPCs) Display Overlapping and Unique Mechanisms of Genomic Instability: The Role of PI3k-AKT and PI3k-Rac2-PAK Pathways

One of the main causes of cardiovascular complications in diabetes is the hyperglycaemia-induced cell injury, and mitochondrial fission has been implicated in the apoptotic process. We investigated the role of mitochondrial fission in high glucose-induced cardiovascular cell injury. We used several types of cultured mouse, rat, and bovine cells from the cardiovascular system, and evaluated mitochondrial morphology, reactive oxygen species (ROS) levels, and apoptotic parameters in sustained high glucose incubation. Adenoviral infection was used for the inhibition of the fission protein DLP1. We found that mitochondria were short and fragmented in cells incubated in sustained high glucose conditions. Under the same conditions, cellular ROS levels were high and cell death was increased. We demonstrated that the increased level of ROS causes mitochondrial permeability transition (MPT), phosphatidylserine exposure, cytochrome c release, and caspase activation in prolonged high glucose conditions. Importantly, maintaining tubular mitochondria by inhibiting mitochondrial fission in sustained high glucose conditions normalized cellular ROS levels and prevented the MPT and subsequent cell death. These results demonstrate that mitochondrial fragmentation is an upstream factor for ROS overproduction and cell death in prolonged high glucose conditions. These findings indicate that the fission-mediated fragmentation of mitochondrial tubules is causally associated with enhanced production of mitochondrial ROS and cardiovascular cell injury in hyperglycaemic conditions.

Protein kinase C in enhanced vascular tone in diabetes mellitus