Major Source Of Oxidative Stress Research Articles

Protein Kinase C Beta II Inhibitor Conjugated with Myristic Acid and Trans-Activator of Transcription improves intracellular delivery and cardiovascular protection in porcine myocardial ischemia/reperfusion injury

Introduction: While timely reperfusion of oxygenated blood is critical during resuscitation of ischemic myocardium, oxidative stress during reperfusion still leads to ischemia/reperfusion (I/R) injury and ultimately, cardiomyocyte death. The major sources of oxidative stress targeted in this study are NADPH oxidase (NOX-2) and mitochondria, which are both activated by protein kinase C beta II (PKCβII). In previous studies with an ex-vivo rat heart I/R model, Myristic Acid (Myr) and Trans-Activator Transcription (Tat; YGRKKRRQRRR) conjugated PKCβII inhibitor (Myr-Tat-PKCβII-; N-Myr-Tat-CC-SLNPEWNET) showed cardioprotective effects and decreased infarct size. In this study, we hypothesized that Myr-Tat-PKCβII- will offer cardioprotective effects in comparison with a scrambled peptide control (Myr-Tat-PKCβII-scram; N-Myr-Tat-CC-WNPESLNTE) in an in-vivo porcine myocardial I/R model. Methods: Regional I(1 hour)/R(3 hours) was induced in Male Yorkshire pigs (38-50kg) by inflating a balloon to occlude the Left Anterior Descending Artery (LAD) at the level of the second diagonal branch (supplies 40% of anterior left ventricle) and then deflating it to allow for reperfusion. Then, a bolus of either Myr-Tat-PKCβII- or Myr-Tat-PKCβII-scram (20ng/kg; ~100pmol in blood) was immediately administered into the LAD. Cardiac function and injury were measured by monitoring changes in ejection fraction (EF) and through routine measurements of serum creatine phosphokinase (CPK), troponin I, and myoglobin, respectively. After I/R, the hearts were stained with 1% Evans Blue dye to identify the area at risk (AR) and 1% triphenyltetrazolium chloride to determine the area of necrosis (AN). Infarct size was then quantified (AN/AR) and all data was analyzed via Student’s t-test. Results: Analysis showed that Myr-Tat-PKCβII- significantly restored EF to within 1.40.7% of baseline compared to controls which only restored EF to within 6.42.1% (p<0.05) of baseline EF. Myr-Tat-PKCβII- showed a significant increase in serum myoglobin levels at 1 hr of reperfusion (1362.1 83.4 μg/mL, n=4) compared to scrambled control (202.1197.3 μg/mL, n=3 p<0.05). Myr-Tat-PKCβII- reduced infarct size to 10.0±2.8%; n=4; compared to scrambled control hearts (28.5±8.3%; n=6; p<0.05). CPK and Troponin I levels were comparable in both groups. These results suggest that Myr-Tat-PKCβII- can help prevent cardiac injury when given immediately after an ischemic event. Conclusions: Data from ex-vivo rat heart I/R model, coupled with data from this in-vivo porcine myocardial I/R model indicate the effcacy of Myr-Tat-PKCβII- in preventing oxidative stress-induced myocardial I/R injury when given at the beginning of reperfusion. These findings suggest that Myr-Tat-PKCβII- could be useful in the clinical setting when administered immediately after cardiac resuscitation. Future studies include the treatment of rat cardiomyocyte cells with Myr-Tat-PKCβII- prior to 24 hr hypoxia/24 hr reoxygenation, followed by cell viability assays compared to untreated control. This additional data can help determine the optimal dose to use in a 12 week survival study using the same porcine myocardial I/R protocol. Funding: This study was funded by the Philadelphia College of Osteopathic Medicine, Department of Biomedical Sciences, Division of Research, and the Center for Chronic Diseases of Aging, the National Heart, Lung & Blood Institute at NIH (grant # 1R43HL160338-01) and Young Therapeutics, LLC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Putative Molecular Mechanisms Underpinning the Inverse Roles of Mitochondrial Respiration and Heme Function in Lung Cancer and Alzheimer's Disease.

Mitochondria are the powerhouse of the cell. Mitochondria serve as the major source of oxidative stress. Impaired mitochondria produce less adenosine triphosphate (ATP) but generate more reactive oxygen species (ROS), which could be a major factor in the oxidative imbalance observed in Alzheimer's disease (AD). Well-balanced mitochondrial respiration is important for the proper functioning of cells and human health. Indeed, recent research has shown that elevated mitochondrial respiration underlies the development and therapy resistance of many types of cancer, whereas diminished mitochondrial respiration is linked to the pathogenesis of AD. Mitochondria govern several activities that are known to be changed in lung cancer, the largest cause of cancer-related mortality worldwide. Because of the significant dependence of lung cancer cells on mitochondrial respiration, numerous studies demonstrated that blocking mitochondrial activity is a potent strategy to treat lung cancer. Heme is a central factor in mitochondrial respiration/oxidative phosphorylation (OXPHOS), and its association with cancer is the subject of increased research in recent years. In neural cells, heme is a key component in mitochondrial respiration and the production of ATP. Here, we review the role of impaired heme metabolism in the etiology of AD. We discuss the numerous mitochondrial effects that may contribute to AD and cancer. In addition to emphasizing the significance of heme in the development of both AD and cancer, this review also identifies some possible biological connections between the development of the two diseases. This review explores shared biological mechanisms (Pin1, Wnt, and p53 signaling) in cancer and AD. In cancer, these mechanisms drive cell proliferation and tumorigenic functions, while in AD, they lead to cell death. Understanding these mechanisms may help advance treatments for both conditions. This review discusses precise information regarding common risk factors, such as aging, obesity, diabetes, and tobacco usage.

Metal-induced oxidative stress and human plasma protein oxidation after SARS-CoV-2 infection

Pathogenesis of COVID-19 by SARS-CoV-2 resulted in a global pandemic and public health emergency in 2020. Viral infection can induce oxidative stress through reactive oxygen species (ROS). Inflammation and environmental stress are major sources of oxidative stress after infection. Micronutrients such as iron, copper, zinc, and manganese play various roles in human tissues and their imbalance in blood can impact immune responses against pathogens including SARS CoV-2. We hypothesized that alteration of free metal ions during infection and metal-catalyzed oxidation plays a critical role towards pathogenesis after infection. We analyzed convalescent and hospitalized COVID-19 patient plasma using orthogonal analytical techniques to determine redox active metal concentrations, overall protein oxidation, oxidative modifications, and protein levels via proteomics to understand the consequences of metal-induced oxidative stress in COVID-19 plasma proteins. Metal analysis using ICP-MS showed significantly greater concentrations of copper in COVID-19 plasma compared to healthy controls. We demonstrate significantly greater total protein carbonylation, other oxidative modifications, and deamidation of plasma proteins in COVID-19 plasma compared to healthy controls. Proteomics analysis showed that levels of redox active proteins including hemoglobulin were elevated in COVID-19 plasma. Molecular modeling concurred with potential interactions between iron binding proteins and SARS CoV-2 surface proteins. Overall, increased levels of redox active metals and protein oxidation indicate that oxidative stress-induced protein oxidation in COVID-19 may be a consequence of the interactions of SARS-CoV-2 proteins with host cell metal binding proteins resulting in altered cellular homeostasis.

Hyperkalemic or Low Potassium Cardioplegia Protects against Reduction of Energy Metabolism by Oxidative Stress.

Open-heart surgery is often an unavoidable option for the treatment of cardiovascular disease and prevention of cardiomyopathy. Cardiopulmonary bypass surgery requires manipulating cardiac contractile function via the perfusion of a cardioplegic solution. Procedure-associated ischemia and reperfusion (I/R) injury, a major source of oxidative stress, affects postoperative cardiac performance and long-term outcomes. Using large-scale liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics, we addressed whether cardioplegic solutions affect the baseline cellular metabolism and prevent metabolic reprogramming by oxidative stress. AC16 cardiomyocytes in culture were treated with commonly used cardioplegic solutions, High K+ (HK), Low K+ (LK), Del Nido (DN), histidine-tryptophan-ketoglutarate (HTK), or Celsior (CS). The overall metabolic profile shown by the principal component analysis (PCA) and heatmap revealed that HK or LK had a minimal impact on the baseline 78 metabolites, whereas HTK or CS significantly repressed the levels of multiple amino acids and sugars. H2O2-induced sublethal mild oxidative stress causes decreases in NAD, nicotinamide, or acetylcarnitine, but increases in glucose derivatives, including glucose 6-P, glucose 1-P, fructose, mannose, and mannose 6-P. Additional increases include metabolites of the pentose phosphate pathway, D-ribose-5-P, L-arabitol, adonitol, and xylitol. Pretreatment with HK or LK cardioplegic solution prevented most metabolic changes and increases of reactive oxygen species (ROS) elicited by H2O2. Our data indicate that HK and LK cardioplegic solutions preserve baseline metabolism and protect against metabolic reprogramming by oxidative stress.

Focused ultrasound-induced targeted delivery of miR-155-5p inhibitor employing biomimetic microbubbles reduces NADPH oxidase expression and inflammation in atherosclerotic ApoE-/- mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): UEFISCDI Background MicroRNAs are important biomarkers and potential therapeutic targets in atherosclerosis. MiR-155-5p amplifies inflammation in atherosclerosis by targeting negative regulators of NF-kB signaling pathway. Inhibition of pro-atherogenic miRNAs or mimicking the function of selected anti-atherogenic miRNAs have emerged as prospective oligonucleotide-base therapeutic interventions in order to correct the expression of key genes that are mechanistically linked to atherosclerotic lesion formation. Purpose The aim of this study was to develop a strategy to reduce oxidative stress and inflammation in atherosclerotic mice employing sterically stabilized biomimetic microbubbles (MB) as focused ultrasound-responsive targeted delivery systems of miR-155-5p inhibitor. Methods ApoE-/- mice were fed a normal (control) or a high-fat, cholesterol-rich diet for 14 weeks to accelerate de development of aortic atherosclerotic lesions. FAM-labelled miRNA negative control inhibitor, miRNA negative control inhibitor or miR-155-5p inhibitor were charge-coupled to VCAM1-targeted MB. Targeted MB were delivered to mice in a single dose via retro-orbital injection. Ultrasound imaging interrogation of atherosclerotic lesions in ApoE-/- mice was performed using a conventional 30 MHz transducer (Vevo 2100 System). The Sonidel SP100 sonoporator was used for ultrasound-induced targeted release of miRNA inhibitors in mice. The biodistribution of MB was monitored by high-resolution fluorescence imaging. Atherosclerotic lesions were assessed by Oil Red O staining. Real time PCR and Western blot were employed to determine the expression of NADPH oxidase (Nox) subtypes, a major source of oxidative stress, and markers of oxidative stress inflammation (4-HNE-protein adducts, inducible nitric oxide synthase/NOS2, CD68). Results Epi-fluorescence analysis demonstrated the enhanced uptake of FAM-labelled miRNA control inhibitor by lungs and the atherosclerotic aorta of ApoE-/- mice following ultrasound wave exposure (1 MHz, 2.0 w/cm2, 50% duty cycles, 15 minutes). The mRNA/protein levels of Nox1-4 subtypes, 4-HNE-protein adducts, NOS2, and macrophage infiltration marker CD68 were found significantly elevated in the atherosclerotic aorta of ApoE-/- mice. The expression levels of Nox isoforms, 4-HNE-protein adducts, NOS2, and CD68 were significantly decreased in the atherosclerotic aorta of ApoE-/- mice 1-week after administration of targeted MB carrying miR-155-5p inhibitor as compared with atherosclerotic ApoE-/- mice treated with targeted MB carrying miRNA negative control inhibitor. Conclusion Systemic administration of miR-155-5p inhibitor conjugated with VCAM1-targeted MB reduces the expression of oxidative stress and inflammation-related markers in experimental atherosclerosis. Ultrasound-induced targeted delivery of miRNA-oriented oligonucleotides could become an important theranostic strategy combining imaging and treatment procedures in clinical atherosclerosis.

Nox, Nox, Are You There? The Role of NADPH Oxidases in the Peripheral Nervous System.

Significance: Reactive oxygen species (ROS) contribute to multiple aspects of peripheral nervous system (PNS) biology ranging from physiological processes (e.g., axonal outgrowth and regeneration) to pathophysiology (e.g., nerve degeneration). Although ROS are derived from multiple sources, NADPH oxidase (Nox) family members are dedicated to ROS generation. Noxs are expressed in the PNS, and their overexpression is associated with detrimental effects on nerve function and contributes, at least in part, to peripheral neuropathies. Recent Advances: Of the seven members, studies mostly focused on Nox1, Nox2, and Nox4, which are expressed in the PNS in a cell-specific manner. We have also recently identified human Nox5 in sural nerve biopsies. When maintained at homeostatic levels, Noxs regulate several aspects of peripheral nerve health, most notably neurite outgrowth and axonal regeneration following nerve lesion. While Nox2 and Nox4 dysregulation is a major source of oxidative stress in PNS disorders, including neuropathic pain and diabetic peripheral neuropathy, recent evidence also implicates Nox1 and Nox5. Critical Issues: Although there is compelling evidence for a direct role of Noxs on nerve function, little is known about their subcellular localization, intercellular regulation, and interaction. These, together with redox signaling, are considered crucial components of nerve redox status. In addition, the lack of isoform-specific inhibitors limits conclusions about the physiological role of Noxs in the PNS and their therapeutic potential in peripheral neuropathies. Future Directions: Future research using isoform-specific genetic and pharmacological approaches are therefore needed to better understand the significance of Nox enzymes in PNS (patho) physiology. Antioxid. Redox Signal. 37, 613-630.

Redox Homeostasis in Cardiovascular Disease: The Role of Mitochondrial Sirtuins.

Cardiovascular disease (CVD) is still the leading cause of death worldwide. Despite successful advances in both pharmacological and lifestyle strategies to fight well-established risk factors, the burden of CVD is still increasing. Therefore, it is necessary to further deepen our knowledge of the pathogenesis of the disease for developing novel therapies to limit even more its related morbidity and mortality. Oxidative stress has been identified as a common trait of several manifestations of CVD and could be a promising target for innovative treatments. Mitochondria are a major source of oxidative stress and sirtuins are a family of enzymes that generate different post-translational protein modifications, thus regulating important cellular processes, including cell cycle, autophagy, gene expression, and others. In particular, three sirtuins, SIRT3, SIRT4, and SIRT5 are located within the mitochondrial matrix where they regulate energy production and antioxidant pathways. Therefore, these sirtuins are strongly involved in the balance between oxidant and antioxidant mechanisms. In this review, we summarize the activities of these sirtuins with a special focus on their role in the control of oxidative stress, in relation to energy metabolism, atherosclerosis, and CVD.

Neutrophil-mediated oxidative stress and albumin structural damage predict COVID-19-associated mortality.

Human serum albumin (HSA) is the frontline antioxidant protein in blood with established anti-inflammatory and anticoagulation functions. Here, we report that COVID-19-induced oxidative stress inflicts structural damages to HSA and is linked with mortality outcome in critically ill patients. We recruited 39 patients who were followed up for a median of 12.5 days (1-35 days), among them 23 had died. Analyzing blood samples from patients and healthy individuals (n=11), we provide evidence that neutrophils are major sources of oxidative stress in blood and that hydrogen peroxide is highly accumulated in plasmas of non-survivors. We then analyzed electron paramagnetic resonance spectra of spin-labeled fatty acids (SLFAs) bound with HSA in whole blood of control, survivor, and non-survivor subjects (n=10-11). Non-survivors' HSA showed dramatically reduced protein packing order parameter, faster SLFA correlational rotational time, and smaller S/W ratio (strong-binding/weak-binding sites within HSA), all reflecting remarkably fluid protein microenvironments. Following loading/unloading of 16-DSA, we show that the transport function of HSA may be impaired in severe patients. Stratified at the means, Kaplan-Meier survival analysis indicated that lower values of S/W ratio and accumulated H2O2 in plasma significantly predicted in-hospital mortality (S/W≤0.15, 81.8% (18/22) vs. S/W>0.15, 18.2% (4/22), p=0.023; plasma [H2O2]>8.6 μM, 65.2% (15/23) vs. 34.8% (8/23), p=0.043). When we combined these two parameters as the ratio ((S/W)/[H2O2]) to derive a risk score, the resultant risk score lower than the mean (<0.019) predicted mortality with high fidelity (95.5% (21/22) vs. 4.5% (1/22), log-rank χ2=12.1, p=4.9×10-4). The derived parameters may provide a surrogate marker to assess new candidates for COVID-19 treatments targeting HSA replacements and/or oxidative stress.

Role of Natural Bioactives and their Nanocarriers for Overcoming Oxidative Stress Induced Cancer.

Oxidative stress and free radicals are harmful to human health. Reactive oxygen species are the major source of oxidative stress and are one of the major causes of cancer development. Cancer is one of the leading causes of death worldwide. Prolonged use of synthetic chemotherapeutic due to their low bioavailability leads to systemic toxic side effects. To surmount this problem, the use of antioxidants is recommended as they have the ability to counteract oxidative stress and mitigate its effects on human health. They inhibit various pathways that are involved with the initiation and progression of cancer. Various nanoformulations have been used to deliver these antioxidants (curcumin, mangiferin, quercetin) in the treatment of various cancer for overcoming oxidative stress. The main focus of this review article is to illustrate various studies performed using nanocarriers of natural bioactives to overcome oxidative stress and cancer associated with it. It also describes pathways associated with the induction, initiation and progression of cancer due to reactive oxidative species. Research articles that focused on the use of natural bioactives and their nanoformulations for the treatment of various cancers induced due to oxidative stress, were collected from various search engines like PubMed, Science Direct and Google Scholar, using keywords like oxidative stress, antioxidants, cancers, ROS, etc. Conclusion: Natural bioactives have shown great potential in overcoming oxidative stress for the treatment of various cancers. However, extensive research is required so that these antioxidants and their nanocarriers can be used for the welfare of mankind, in the treatment of various cancers, in the near future.

Ineffective Erythropoiesis in β-Thalassaemia: Key Steps and Therapeutic Options by Drugs.

β-thalassaemia is a rare genetic condition caused by mutations in the β-globin gene that result in severe iron-loading anaemia, maintained by a detrimental state of ineffective erythropoiesis (IE). The role of multiple mechanisms involved in the pathophysiology of the disease has been recently unravelled. The unbalanced production of α-globin is a major source of oxidative stress and membrane damage in red blood cells (RBC). In addition, IE is tightly linked to iron metabolism dysregulation, and the relevance of new players of this pathway, i.e., hepcidin, erythroferrone, matriptase-2, among others, has emerged. Advances have been made in understanding the balance between proliferation and maturation of erythroid precursors and the role of specific factors in this process, such as members of the TGF-β superfamily, and their downstream effectors, or the transcription factor GATA1. The increasing understanding of IE allowed for the development of a broad set of potential therapeutic options beyond the current standard of care. Many candidates of disease-modifying drugs are currently under clinical investigation, targeting the regulation of iron metabolism, the production of foetal haemoglobin, the maturation process, or the energetic balance and membrane stability of RBC. Overall, they provide tools and evidence for multiple and synergistic approaches that are effectively moving clinical research in β-thalassaemia from bench to bedside.

8‐oxoguanine glycosylase (OGG1) is a determinant of endothelial dysfunction via calcium and nitric oxide signaling

Diabetes mellitus is a chronic disease that hinders the body's ability to produce or respond to the hormone insulin, resulting in abnormal metabolism and elevated glucose levels. Type II diabetes has been characterized by a two-to-four-fold increase in cardiovascular disease, such as carotid artery stenosis, generally attributed to the adverse effects of hyperglycemia and oxidative stress on vascular biology. With it being a very common and significant health complication, understanding the pathology and the mechanisms by which diabetes-induced oxidative stress serves as a precursor to several notable vascular diseases is paramount, and is the overall objective of this study. Endothelial dysfunction (ED) is a vascular phenomenon attributed to a reduction in nitric oxide (NO) bioactivity and an increase in oxygen free radical formation with subsequent oxidative stress, thus leading to impaired vasodilation, and compromised vascular function. ED tends to be a consistent finding in diabetes. A major source of oxidative stress is the abnormal production of reactive oxygen species (ROS) in the context of diabetic inflammation. Increased ROS production leads to mitochondrial DNA (mt DNA) damage and aberrant cell signaling. Typically, mt DNA damage is repaired through the base excision repair (BER) pathway. However, during chronic inflammation in diabetes, this repairing capability is ineffective due to downregulation of key BER enzymes, namely 8-oxoguanine glycosylase (OGG1). Given this rational, we hypothesized that the BER pathway is an important determinant of endothelial dysfunction via impaired calcium and NO signaling. To test this hypothesis, we examined the effect of BER defects on endothelial calcium signaling and NO production using carotid arteries isolated from mice deficient in OGG1. Opened artery measurements of endothelial calcium and NO signaling were performed using a custom-designed parallel plate flow chamber. Briefly, segments of carotid arteries (~200 µm in diameter) were isolated from OGG1 deficient (KO) and wild type (WT) mice and cut open lengthwise to expose the endothelial surface. The isolated vessels were then stained with a loading solution composed of Pluronic acid (Fischer), Cal-520 (AAT bio) calcium indicator dye and DAR-4M (Calbiochem), a fluorescent probe for NO. Time lapse signal measurements were collected via confocal microscopy and were then analyzed and processed with S8, a custom automated solution to signal analysis based on noise filtering and object tracking, which computed dynamic amplitude, frequency, duration, and spatial spread. We discovered that although the number of calcium signals detected within the measurement interval were similar between KO and WT (10 ± 3 vs 9 ± 3, µ ± SEM, respectively), the spatial spread of signals within the KO (19.95 ± 3.78 µm2, µ ± SEM) was greater than WT (9.65 ± 2.7 µm2, p = 0.057 via Welch's unpaired t-test). These data suggest that OGG1 may be a determinant of normal endothelial calcium signaling and might serve as a therapeutic target for preventing the adverse effects of endothelial dysfunction present in diabetes.

Sidestream Smoke Affects Dendritic Complexity and Astrocytes After Model Mild Closed Head Traumatic Brain Injury.

Mild traumatic brain injuries can have long-term consequences that interfere with the life of the patient and impose a burden on our health care system. Oxidative stress has been identified as a contributing factor for the progression of neurodegeneration following TBI. A major source of oxidative stress for many veterans is cigarette smoking and second-hand smoke, which has been shown to have an effect on TBI recovery. To examine the potential influences of second-hand smoke during recovery from TBI, we utilized a mouse model of closed head injury, followed by repeated exposure to cigarette smoke and treatment with a neuroprotective antioxidant. We found that neither the mild injuries nor the smoke exposure produced axonal damage detectable with amino cupric silver staining. However, complexity in the dendritic arbors was significantly reduced after mild TBI plus smoke exposure. In the hippocampus, there were astrocytic responses, including Cyp2e1 upregulation, after the injury and tobacco smoke insult. This study provides useful context for the importance of lifestyle changes, such as reducing or eliminating cigarette smoking, during recovery from TBI.

Modulation of Heat-Shock Proteins Mediates Chicken Cell Survival against Thermal Stress.

Simple SummaryThe impact of heat stress is a major challenge causing economical losses to the poultry industry. Heat stress exerts a damaging effect on physiological responses such as immunity, oxidative stress, and intestinal and muscular functions. In this review, we highlight the potential cytoprotective effects of heat-shock proteins to antagonize the adverse effects of heat shock, and how we can modulate their functions with pharmacological and nutritional interventions.Heat stress is one of the most challenging environmental stresses affecting domestic animal production, particularly commercial poultry, subsequently causing severe yearly economic losses. Heat stress, a major source of oxidative stress, stimulates mitochondrial oxidative stress and cell dysfunction, leading to cell damage and apoptosis. Cell survival under stress conditions needs urgent response mechanisms and the consequent effective reinitiation of cell functions following stress mitigation. Exposure of cells to heat-stress conditions induces molecules that are ready for mediating cell death and survival signals, and for supporting the cell’s tolerance and/or recovery from damage. Heat-shock proteins (HSPs) confer cell protection against heat stress via different mechanisms, including developing thermotolerance, modulating apoptotic and antiapoptotic signaling pathways, and regulating cellular redox conditions. These functions mainly depend on the capacity of HSPs to work as molecular chaperones and to inhibit the aggregation of non-native and misfolded proteins. This review sheds light on the key factors in heat-shock responses for protection against cell damage induced by heat stress in chicken.

NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein.

Background: The superoxide-generating enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2 or gp91phox, the phagocytic isoform) was reported as a major source of oxidative stress in various human diseases. Genetic deletion is widely used to study the impact of NOX2-derived reactive oxygen species (ROS) on disease development and progression in various animal models. Here, we investigate why NOX2 knockout mice show no NOX2 activity but express NOX2 mRNA and protein. Methods and Results: Oxidative burst (NOX2-dependent formation of ROS) was measured by L-012-based chemiluminescence and was largely absent in whole blood of NOX2 knockout mice. Protein expression was still detectable in different tissues of the NOX2 knockout mice, at the expected and a slightly lower molecular weight (determined by Western blot). The NOX2 gene was even largely enhanced at its expressional level in NOX2 knockout mice. RNA sequencing revealed a modified NOX2 mRNA in the knockout mice that is obviously translated to a truncated inactive mutant enzyme. Conclusion: Although the commercial NOX2 knockout mice display no considerable enzymatic NOX2 activity, expression of the NOX2 gene (when using standard primers) and protein (when using antibodies binding to the carboxy-terminal end) can still be detected, which may lead to confusion among investigators.

Auranofin Attenuates Non-Alcoholic Fatty Liver Disease by Suppressing Lipid Accumulation and NLRP3 Inflammasome-Mediated Hepatic Inflammation In Vivo and In Vitro.

Non-alcoholic fatty liver disease (NAFLD) causes liver dysfunction and is associated with obesity and type 2 diabetes. Chronic inflammation is associated not only with the development of NAFLD, but also with hepatic diseases, including steatohepatitis, cirrhosis, and hepatocellular carcinoma. Auranofin is a treatment for rheumatoid arthritis and has recently been reported to have potential effects against a variety of diseases, including inflammation, cancer, and viral infection. In this study, auranofin may be considered as a new treatment for the management of metabolic syndrome, as well as in the treatment of NAFLD through immunomodulation. To determine the effect of auranofin on NAFLD, C57BL/6 mice were randomly grouped, fed a regular diet or a high fat diet (HFD), and injected with normal saline or auranofin for 8 weeks. Auranofin significantly decreased the body weight, epididymal fat weight, serum aspartate aminotransferase (AST), and glucose, as well as the serum triglyceride, cholesterol, and low-density lipoprotein cholesterol levels as compared to the HFD group. We also observed that hepatic steatosis was increased in the HFD group and was suppressed by auranofin treatment. In addition, auranofin suppressed the expressions of interleukin (IL)-1β, IL-18, caspase-1, and the NOD-like receptor family pyrin domain containing 3 (NLRP3) in the liver tissue. Furthermore, the expression of NADPH oxidase 4 and peroxisome proliferator-activated receptor γ (PPARγ), which are a major source of oxidative stress and a regulator of adipogenesis, respectively, were also decreased by auranofin. In addition, primary mouse hepatocytes were incubated with lipopolysaccharide (LPS) and palmitic acid (PA) to induce lipid accumulation and hepatic inflammation for an in vitro model. Auranofin could significantly inhibit LPS- and PA-induced inflammatory activity including nitric oxide and NLRP3 inflammasome-mediated cytokines. The results of this study demonstrate that auranofin treatment inhibits the characteristics of NAFLD through the inhibition of NLRP3 inflammasome. Therefore, auranofin may have potential as a candidate for improving NAFLD symptoms.

Oxidative Stress and Thrombosis during Aging: The Roles of Oxidative Stress in RBCs in Venous Thrombosis.

Mid-life stage adults are at higher risk of developing venous thrombosis (VT)/thromboembolism (VT/E). Aging is characterized by an overproduction of reactive oxygen species (ROS), which could evoke a series of physiological changes involved in thrombosis. Here, we focus on the critical role of ROS within the red blood cell (RBC) in initiating venous thrombosis during aging. Growing evidence has shifted our interest in the role of unjustifiably unvalued RBCs in blood coagulation. RBCs can be a major source of oxidative stress during aging, since RBC redox homeostasis is generally compromised due to the discrepancy between prooxidants and antioxidants. As a result, ROS accumulate within the RBC due to the constant endogenous hemoglobin (Hb) autoxidation and NADPH oxidase activation, and the uptake of extracellular ROS released by other cells in the circulation. The elevated RBC ROS level affects the RBC membrane structure and function, causing loss of membrane integrity, and decreased deformability. These changes impair RBC function in hemostasis and thrombosis, favoring a hypercoagulable state through enhanced RBC aggregation, RBC binding to endothelial cells affecting nitric oxide availability, RBC-induced platelet activation consequently modulating their activity, RBC interaction with and activation of coagulation factors, increased RBC phosphatidylserine exposure and release of microvesicles, accelerated aging and hemolysis. Thus, RBC oxidative stress during aging typifies an ultimate mechanism in system failure, which can affect major processes involved in the development of venous thrombosis in a variety of ways. The reevaluated concept of the critical role of RBC ROS in the activation of thrombotic events during aging will help identify potential targets for novel strategies to prevent/reduce the risk for VT/E or VT/E recurrences in mid-life stage adults.

Both metal-chelating and free radical-scavenging synthetic pentapeptides as efficient inhibitors of reactive oxygen species generation.

Reactive oxygen species (ROS) are major sources of oxidative stress playing prominent roles in the development of several pathologies including cardiovascular and neurodegenerative diseases or cancers. The presence of transition biometal ions, specifically copper and iron, induces ROS formation by catalyzing the reduction of molecular oxygen to superoxide anion (O2˙-), hydrogen peroxide (H2O2) and hydroxyl (HO˙) radical. To limit ROS production and their detrimental effects, we report on the synthesis, physicochemical studies and antioxidant assays of an innovative series of synthetic pentapeptides exhibiting a dual direct/indirect mode of action, both as iron(iii)-chelators and as radical scavengers. These combined effects lead to a drastic reduction of in vitro reactive oxygen species production up to 95% for the more reactive hydroxyl radical.

The Potent Antioxidant MitoQ Protects Against Preeclampsia During Late Gestation but Increases the Risk of Preeclampsia When Administered in Early Pregnancy.

Aims: Although preeclampsia (PE) has been attributed to excessive oxidative stress (OS) in the placenta, mild antioxidants failed to prevent PE in clinical trials. As mitochondria are a major source of OS, this study assessed the potential of a potent mitochondria-targeting antioxidant MitoQ in the prevention of PE. Results: Placentas from women with PE and from reduced uterine perfusion pressure (RUPP) mice demonstrated significantly higher OS, along with increased mitochondrial damage and compromised glutathione peroxidase (GPx) activities. MitoQ administration during late gestation alleviated RUPP-induced PE; whereas early-pregnancy MitoQ treatment not only exacerbated blood pressure, fetal growth restriction, and proteinuria but also reduced the labyrinth/spongiotrophoblast ratio and blood sinuses in the labyrinth. Invasion (Matrigel transwell) and migration (wound healing assay) of trophoblasts were greatly improved by 1 μM hydrogen peroxide (H2O2), but this improvement was abolished by MitoQ or MitoTempo. Mild OS enhanced the expression of miR-29b-3p, which regulates five genes involved in viability and mobility, in HTR8-S/Vneo cells. Innovation and Conclusions: Although the potent mitochondrial-targeting antioxidant MitoQ protects against hypertension and kidney damage induced by RUPP in mice when administered in late gestation, it exacerbates the PE-like phenotype when given in early gestation by interfering with placenta formation because mild OS is required to stimulate trophoblast proliferation, invasion, and migration. Eliminating trophoblastic OS during early pregnancy may lead to compromised placentation and a risk of diseases of placental origin. Therefore, antioxidant therapy for pregnant women should be carefully considered.

Critical examination of mechanisms underlying the reduction in heart failure events with SGLT2 inhibitors: identification of a molecular link between their actions to stimulate erythrocytosis and to alleviate cellular stress.

Sodium-glucose co-transporter 2 (SGLT2) inhibitors reduce the risk of serious heart failure events, even though SGLT2 is not expressed in the myocardium. This cardioprotective benefit is not related to an effect of these drugs to lower blood glucose, promote ketone body utilization or enhance natriuresis, but it is linked statistically with their action to increase haematocrit. SGLT2 inhibitors increase both erythropoietin and erythropoiesis, but the increase in red blood cell mass does not directly prevent heart failure events. Instead, erythrocytosis is a biomarker of a state of hypoxia mimicry, which is induced by SGLT2 inhibitors in manner akin to cobalt chloride. The primary mediators of the cellular response to states of energy depletion are sirtuin-1 and hypoxia-inducible factors (HIF-1α/HIF-2α). These master regulators promote the cellular adaptation to states of nutrient and oxygen deprivation, promoting mitochondrial capacity and minimizing the generation of oxidative stress. Activation of sirtuin-1 and HIF-1α/HIF-2α also stimulates autophagy, a lysosome-mediated degradative pathway that maintains cellular homoeostasis by removing dangerous constituents (particularly unhealthy mitochondria and peroxisomes), which are a major source of oxidative stress and cardiomyocyte dysfunction and demise. SGLT2 inhibitors can activate SIRT-1 and stimulate autophagy in the heart, and thereby, favourably influence the course of cardiomyopathy. Therefore, the linkage between erythrocytosis and the reduction in heart failure events with SGLT2 inhibitors may be related to a shared underlying molecular mechanism that is triggered by the action of these drugs to induce a perceived state of oxygen and nutrient deprivation.

Role of Deranged Energy Deprivation Signaling in the Pathogenesis of Cardiac and Renal Disease in States of Perceived Nutrient Overabundance.

Sodium-glucose cotransporter 2 inhibitors reduce the risk of serious heart failure and adverse renal events, but the mechanisms that underlie this benefit are not understood. Treatment with SGLT2 inhibitors is distinguished by 2 intriguing features: ketogenesis and erythrocytosis. Both reflect the induction of a fasting-like and hypoxia-like transcriptional paradigm that is capable of restoring and maintaining cellular homeostasis and survival. In the face of perceived nutrient and oxygen deprivation, cells activate low-energy sensors, which include sirtuin-1 (SIRT1), AMP-activated protein kinase (AMPK), and hypoxia inducible factors (HIFs; especially HIF-2α); these enzymes and transcription factors are master regulators of hundreds of genes and proteins that maintain cellular homeostasis. The activation of SIRT1 (through its effects to promote gluconeogenesis and fatty acid oxidation) drives ketogenesis, and working in concert with AMPK, it can directly inhibit inflammasome activation and maintain mitochondrial capacity and stability. HIFs act to promote oxygen delivery (by stimulating erythropoietin and erythrocytosis) and decrease oxygen consumption. The activation of SIRT1, AMPK, and HIF-2α enhances autophagy, a lysosome-dependent degradative pathway that removes dangerous constituents, particularly damaged mitochondria and peroxisomes, which are major sources of oxidative stress and triggers of cellular dysfunction and death. SIRT1 and AMPK also act on sodium transport mechanisms to reduce intracellular sodium concentrations. It is interesting that type 2 diabetes mellitus, obesity, chronic heart failure, and chronic kidney failure are characterized by the accumulation of intracellular glucose and lipid intermediates that are perceived by cells as indicators of energy overabundance. The cells respond by downregulating SIRT1, AMPK, and HIF-2α, thus leading to an impairment of autophagic flux and acceleration of cardiomyopathy and nephropathy. SGLT2 inhibitors reverse this maladaptive signaling by triggering a state of fasting and hypoxia mimicry, which includes activation of SIRT1, AMPK, and HIF-2α, enhanced autophagic flux, reduced cellular stress, decreased sodium influx into cells, and restoration of mitochondrial homeostasis. This mechanistic framework clarifies the findings of large-scale randomized trials and the close association of ketogenesis and erythrocytosis with the cardioprotective and renoprotective benefits of these drugs.