Radical Roles for RAGE in the Pathogenesis of Oxidative Stress in Cardiovascular Diseases and Beyond

Oxidative stress is a central mechanism by which the receptor for advanced glycation endproducts (RAGE) mediates its pathological effects. Multiple experimental inquiries in RAGE-expressing cultured cells have demonstrated that ligand-RAGE interaction mediates generation of reactive oxygen species (ROS) and consequent downstream signal transduction and regulation of gene expression. The primary mechanism by which RAGE generates oxidative stress is via activation of NADPH oxidase; amplification mechanisms in the mitochondria may further drive ROS production. Recent studies indicating that the cytoplasmic domain of RAGE binds to the formin mDia1 provide further support for the critical roles of this pathway in oxidative stress; mDia1 was required for activation of rac1 and NADPH oxidase in primary murine aortic smooth muscle cells treated with RAGE ligand S100B. In vivo, in multiple distinct disease models in animals, RAGE action generates oxidative stress and modulates cellular/tissue fate in range of disorders, such as in myocardial ischemia, atherosclerosis, and aneurysm formation. Blockade or genetic deletion of RAGE was shown to be protective in these settings. Indeed, beyond cardiovascular disease, evidence is accruing in human subjects linking levels of RAGE ligands and soluble RAGE to oxidative stress in disorders such as doxorubicin toxicity, acetaminophen toxicity, neurodegeneration, hyperlipidemia, diabetes, preeclampsia, rheumatoid arthritis and pulmonary fibrosis. Blockade of RAGE signal transduction may be a key strategy for the prevention of the deleterious consequences of oxidative stress, particularly in chronic disease.     

New In Vitro Model of Oxidative Stress: Human Prostate Cells Injured with 2,2-diphenyl-1-picrylhydrazyl (DPPH) for the Screening of Antioxidants

The antioxidant activity of natural compounds consists in their ability to modulate gene and protein expression, thus inducing an integrated cell protective response and repair processes against oxidative stress. New screening tools and methodologies are crucial for the actual requirement of new products with antioxidant activity to boost endogenous oxidative stress responsive pathways, Reactive Oxygen Species (ROS) metabolism and immune system activity, preserving human health and wellness. In this study, we performed and tested an integrated oxidative stress analysis, using DPPH assay and PNT2 cells injured with DPPH. We firstly investigated the mechanism of action of the oxidising agent (DPPH) on PNT2 cells, studying the variation in cell viability, oxidative stress genes, inflammatory mediator and ROS levels. The results reveal that DPPH activated ROS production and release of Prostaglandin E₂ in PNT2 at low and intermediate doses, while cells switched from survival to cell death signals at high doses of the oxidising agent. This new in vitro oxidative stress model was validated by using Trolox, β-carotene and total extract of the green microalga Testraselmis suecica. Only the T. suecica extract can completely counteract DPPH-induced injury, since its chemical complexity demonstrated a multilevel protecting and neutralising effect against oxidative stress in PNT2.     

Biomarkers and Mechanisms of Oxidative Stress—Last 20 Years of Research with an Emphasis on Kidney Damage and Renal Transplantation

Oxidative stress is an imbalance between pro- and antioxidants that adversely influences the organism in various mechanisms and on many levels. Oxidative damage occurring concomitantly in many cellular structures may cause a deterioration of function, including apoptosis and necrosis. The damage leaves a molecular “footprint”, which can be detected by specific methodology, using certain oxidative stress biomarkers. There is an intimate relationship between oxidative stress, inflammation, and functional impairment, resulting in various diseases affecting the entire human body. In the current narrative review, we strengthen the connection between oxidative stress mechanisms and their active compounds, emphasizing kidney damage and renal transplantation. An analysis of reactive oxygen species (ROS), antioxidants, products of peroxidation, and finally signaling pathways gives a lot of promising data that potentially will modify cell responses on many levels, including gene expression. Oxidative damage, stress, and ROS are still intensively exploited research subjects. We discuss compounds mentioned earlier as biomarkers of oxidative stress and present their role documented during the last 20 years of research. The following keywords and MeSH terms were used in the search: oxidative stress, kidney, transplantation, ischemia-reperfusion injury, IRI, biomarkers, peroxidation, and treatment.     

Lutein Protects RGC-5 Cells Against Hypoxia and Oxidative Stress

Retinal ischemia and oxidative stress lead to neuronal death in many ocular pathologies. Recently, we found that lutein, an oxy-carotenoid, protected the inner retina from ischemia/reperfusion injury. However, it is uncertain whether lutein directly protects retinal ganglion cells (RGCs). Here, an in vitro model of hypoxia and oxidative stress was used to further investigate the neuroprotective role of lutein in RGCs. Cobalt chloride (CoCl₂) and hydrogen peroxide (H₂O₂) were added to a transformed RGC cell line, RGC-5, to induce chemical hypoxia and oxidative stress, respectively. Either lutein or vehicle was added to cultured cells. A higher cell count was observed in the lutein-treated cells compared with the vehicle-treated cells. Our data from this in vitro model revealed that lutein might protect RGC-5 cells from damage when exposed to either CoCl₂-induced chemical hypoxia or H₂O₂-induced oxidative stress. These results suggest that lutein may play a role as a neuroprotectant.     

A systematic framework for the design,simulation and optimization of personalized healthcare: Making and healing blood

We review the key building blocks of a design framework for modeling and optimizing biomedical systems under development in the Biological Systems Engineering Laboratory and the Centre for Process Systems Engineering at Imperial College. The framework features the following components: (i) in vitro environment, where model parameters can be obtained and new setups can be tested; (ii) in silico environment, including a simulation module for representing relevant physical or biological processes, and an optimization module, for calculating improved in vitro or in vivo outcomes; (iii) in vivo environment, from which organ and patient-specific parameters are collected and which can also implement personalized suggestions for improved outcomes. Two applications in the area of healthy and diseased blood are thoroughly discussed to exemplify the framework's characteristics. We discuss progress in the different areas and the way in which they are connected and finally propose a hybrid in vitro/in silico/in vivo platform.     

Treatment of Oxidative Stress with Exosomes in Myocardial Ischemia

A thrombus in a coronary artery causes ischemia, which eventually leads to myocardial infarction (MI) if not removed. However, removal generates reactive oxygen species (ROS), which causes ischemia–reperfusion (I/R) injury that damages the tissue and exacerbates the resulting MI. The mechanism of I/R injury is currently extensively understood. However, supplementation of exogenous antioxidants is ineffective against oxidative stress (OS). Enhancing the ability of endogenous antioxidants may be a more effective way to treat OS, and exosomes may play a role as targeted carriers. Exosomes are nanosized vesicles wrapped in biofilms which contain various complex RNAs and proteins. They are important intermediate carriers of intercellular communication and material exchange. In recent years, diagnosis and treatment with exosomes in cardiovascular diseases have gained considerable attention. Herein, we review the new findings of exosomes in the regulation of OS in coronary heart disease, discuss the possibility of exosomes as carriers for the targeted regulation of endogenous ROS generation, and compare the advantages of exosome therapy with those of stem-cell therapy. Finally, we explore several miRNAs found in exosomes against OS.     

The Chemistry of Reactive Oxygen Species (ROS) Revisited: Outlining Their Role in Biological Macromolecules (DNA,Lipids and Proteins) and Induced Pathologies

Living species are continuously subjected to all extrinsic forms of reactive oxidants and others that are produced endogenously. There is extensive literature on the generation and effects of reactive oxygen species (ROS) in biological processes, both in terms of alteration and their role in cellular signaling and regulatory pathways. Cells produce ROS as a controlled physiological process, but increasing ROS becomes pathological and leads to oxidative stress and disease. The induction of oxidative stress is an imbalance between the production of radical species and the antioxidant defense systems, which can cause damage to cellular biomolecules, including lipids, proteins and DNA. Cellular and biochemical experiments have been complemented in various ways to explain the biological chemistry of ROS oxidants. However, it is often unclear how this translates into chemical reactions involving redox changes. This review addresses this question and includes a robust mechanistic explanation of the chemical reactions of ROS and oxidative stress.     

Debottlenecking of an In Vitro Enzyme Cascade Using a Combined Model- and Experiment-Based Approach

Designing a feasible in vitro enzyme cascade is a challenging task that is often preceded by optimization steps. Computational methods can increase the efficiency of the optimization process by providing a deeper insight into the system. In this study, bottlenecks of a farnesyl pyrophosphate-producing in vitro enzyme cascade were determined by a combination of in silico modeling and wet-lab experiments. ATP and the precursor-isomerizing enzyme isopentenyl diphosphate δ-isomerase were identified as limitations of the in vitro enzyme cascade.     

Insulin and α-Tocopherol Enhance the Protective Effect of Each Other on Brain Cortical Neurons under Oxidative Stress Conditions and in Rat Two-Vessel Forebrain Ischemia/Reperfusion Injury

Clinical trials show that insulin administered intranasally is a promising drug to treat neurodegenerative diseases, but at high doses its use may result in cerebral insulin resistance. Identifying compounds which could enhance the protective effects of insulin, may be helpful to reduce its effective dose. Our aim was thus to study the efficiency of combined use of insulin and α-tocopherol (α-T) to increase the viability of cultured cortical neurons under oxidative stress conditions and to normalize the metabolic disturbances caused by free radical reaction activation in brain cortex of rats with two-vessel forebrain ischemia/reperfusion injury. Immunoblotting, flow cytometry, colorimetric, and fluorometric techniques were used. α-T enhanced the protective and antioxidative effects of insulin on neurons in oxidative stress, their effects were additive. At the late stages of oxidative stress, the combined action of insulin and α-T increased Akt-kinase activity, inactivated GSK-3beta and normalized ERK1/2 activity in cortical neurons, it was more effective than either drug action. In the brain cortex, ischemia/reperfusion increased the lipid peroxidation product content and caused Na⁺,K⁺-ATPase oxidative inactivation. Co-administration of insulin (intranasally, 0.25 IU/rat) and α-T (orally, 50 mg/kg) led to a more pronounced normalization of the levels of Schiff bases, conjugated dienes and trienes and Na⁺,K⁺-ATPase activity than administration of each drug alone. Thus, α-T enhances the protective effects of insulin on cultured cortical neurons in oxidative stress and in the brain cortex of rats with cerebral ischemia/reperfusion injury.     

Antioxidant and Protective Mechanisms against Hypoxia and Hypoglycaemia in Cortical Neurons in Vitro

In the present work, we have studied whether cell death could be induced in cortical neurons from rats subjected to different period of O₂ deprivation and low glucose (ODLG). This “in vitro” model is designed to emulate the penumbra area under ischemia. In these conditions, cortical neurons displayed loss of mitochondrial respiratory ability however, nor necrosis neither apoptosis occurred despite ROS production. The absence of cellular death could be a consequence of increased antioxidant responses such as superoxide dismutase-1 (SOD1) and GPX3. In addition, the levels of reduced glutathione were augmented and HIF-1/3α overexpressed. After long periods of ODLG (12–24 h) cortical neurons showed cellular and mitochondrial membrane alterations and did not recuperate cellular viability during reperfusion. This could mean that therapies directed toward prevention of cellular and mitochondrial membrane imbalance or cell death through mechanisms other than necrosis or apoptosis, like authophagy, may be a way to prevent ODLG damage.     

白藜芦醇对大鼠脑缺血再灌注氧化应激损伤的影响

目的探讨白藜芦醇(Resveratrol,Res)对大鼠脑缺血再灌注氧化应激损伤的影响。方法将SD大鼠随机分为假手术组(S组)、缺血再灌注组(I/R组,线栓法复制大鼠右侧大脑中动脉栓塞模型)、Res低剂量组(15 mg/kg,I/R+R1组)和Res高剂量组(30 mg/kg,I/R+R2组),于缺血2 h再灌注24 h进行神经功能缺损评分;化学比色法测定大鼠血清和脑组织中丙二醛(Malondialdehyde,MDA)含量及超氧化物歧化酶(Superoxide dismutase,SOD)活性;TTC法测定脑梗死体积;干湿重法测定脑含水量,HE染色观察脑组织的病理改变。结果与I/R组相比,Res能改善大鼠脑缺血再灌注损伤后的神经功能缺失(P<0.01),降低血清及脑组织中MDA的含量(P<0.01),提高SOD活性(P<0.01),缩小脑梗死体积(P<0.01),降低损伤侧脑含水量(P<0.01),改善脑组织的病理变化,且呈剂量依赖性。结论 Res对大鼠局灶性脑缺血再灌注氧化应激损伤具有良好的保护作用,其机制可能与清除自由基,减轻氧化性损伤有关。     

Alzheimer’s Disease-Associated Neurotoxic Peptide Amyloid-β Impairs Base Excision Repair in Human Neuroblastoma Cells

Alzheimer’s disease (AD) is the leading cause of dementia in developed countries. It is characterized by two major pathological hallmarks, one of which is the extracellular aggregation of the neurotoxic peptide amyloid-β (Aβ), which is known to generate oxidative stress. In this study, we showed that the presence of Aβ in a neuroblastoma cell line led to an increase in both nuclear and mitochondrial DNA damage. Unexpectedly, a concomitant decrease in basal level of base excision repair, a major route for repairing oxidative DNA damage, was observed at the levels of both gene expression and protein activity. Moreover, the addition of copper sulfate or hydrogen peroxide, used to mimic the oxidative stress observed in AD-affected brains, potentiates Aβ-mediated perturbation of DNA damage/repair systems in the “Aβ cell line”. Taken together, these findings indicate that Aβ could act as double-edged sword by both increasing oxidative nuclear/mitochondrial damage and preventing its repair. The synergistic effects of increased ROS production, accumulated DNA damage and impaired DNA repair could participate in, and partly explain, the massive loss of neurons observed in Alzheimer’s disease since both oxidative stress and DNA damage can trigger apoptosis.     

Renal Ischemia/Reperfusion Early Induces Myostatin and PCSK9 Expression in Rat Kidneys and HK-2 Cells

During visceral interventions, the transient clampage of supraceliac aorta causes ischemia/reperfusion (I/R) in kidneys, sometime resulting in acute renal failure; preclinical studies identified redox imbalance as the main driver of I/R injury. However, in humans, the metabolic/inflammatory responses seem to prevail on oxidative stress. We investigated myostatin (Mstn) and proprotein convertase subtilisin/kexin type 9 (PCSK9), proatherogenic mediators, during renal I/R. Compared to sham-operated animals, the kidneys of rats who had experienced ischemia (30 min) had higher Mstn and PCSK9 expression after 4 h of reperfusion. After 24 h, they displayed tubular necrosis, increased nitrotyrosine positivity, and nuclear peroxisome proliferator-activated receptor gamma coactivator-1alpha relocation, markers of oxidative stress and mitochondria imbalance. Mstn immunopositivity was increased in tubuli, while PCSK9 immunosignal was depleted; systemically, PCSK9 was higher in plasma from I/R rats. In HK-2 cells, both ischemia and reperfusion enhanced reactive oxygen species production and mitochondrial dysfunction. H₂O₂ upregulated Mstn and PCSK9 mRNA after 1 and 3.5 h, respectively. Accordingly, ischemia early induced Mstn and PCSK9 mRNA; during reperfusion Mstn was augmented and PCSK9 decreased. Mstn treatment early increased PCSK9 expression (within 8 h), to diminish over time; finally, Mstn silencing restrained ischemia-induced PCSK9. Our study demonstrates that renal I/R enhances Mstn and PCSK9 expression and that Mstn induces PCSK9, suggesting them as therapeutic targets for vascular protection during visceral surgery.     

Pathogenesis of Target Organ Damage in Hypertension: Role of Mitochondrial Oxidative Stress

Hypertension causes target organ damage (TOD) that involves vasculature, heart, brain and kidneys. Complex biochemical, hormonal and hemodynamic mechanisms are involved in the pathogenesis of TOD. Common to all these processes is an increased bioavailability of reactive oxygen species (ROS). Both in vitro and in vivo studies explored the role of mitochondrial oxidative stress as a mechanism involved in the pathogenesis of TOD in hypertension, especially focusing on atherosclerosis, heart disease, renal failure, cerebrovascular disease. Both dysfunction of mitochondrial proteins, such as uncoupling protein-2 (UCP2), superoxide dismutase (SOD) 2, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), calcium channels, and the interaction between mitochondria and other sources of ROS, such as NADPH oxidase, play an important role in the development of endothelial dysfunction, cardiac hypertrophy, renal and cerebral damage in hypertension. Commonly used anti-hypertensive drugs have shown protective effects against mitochondrial-dependent oxidative stress. Notably, few mitochondrial proteins can be considered therapeutic targets on their own. In fact, antioxidant therapies specifically targeted at mitochondria represent promising strategies to reduce mitochondrial dysfunction and related hypertensive TOD. In the present article, we discuss the role of mitochondrial oxidative stress as a contributing factor to hypertensive TOD development. We also provide an overview of mitochondria-based treatment strategies that may reveal useful to prevent TOD and reduce its progression.     

Increased Post-Hypoxic Oxidative Stress and Activation of the PERK Branch of the UPR in Trap1-Deficient Drosophila melanogaster Is Abrogated by Metformin

Hypoxia is known to impair mitochondrial and endoplasmic reticulum (ER) homeostasis. Post-hypoxic perturbations of the ER proteostasis result in the accumulation of misfolded/unfolded proteins leading to the activation of the Unfolded Protein Response (UPR). Mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) is reported to preserve mitochondrial membrane potential and to impede reactive oxygen species (ROS) production thereby protecting cells from ER stress as well as oxidative stress. The first-line antidiabetic drug Metformin has been attributed a neuroprotective role after hypoxia. Interestingly, Metformin has been reported to rescue mitochondrial deficits in fibroblasts derived from a patient carrying a homozygous TRAP1 loss-of-function mutation. We sought to investigate a putative link between Metformin, TRAP1, and the UPR after hypoxia. We assessed post-hypoxic/reperfusion longevity, mortality, negative geotaxis, ROS production, metabolic activity, gene expression of antioxidant proteins, and activation of the UPR in Trap1-deficient flies. Following hypoxia, Trap1 deficiency caused higher mortality and greater impairments in negative geotaxis compared to controls. Similarly, post-hypoxic production of ROS and UPR activation was significantly higher in Trap1-deficient compared to control flies. Metformin counteracted the deleterious effects of hypoxia in Trap1-deficient flies but had no protective effect in wild-type flies. We provide evidence that TRAP1 is crucially involved in the post-hypoxic regulation of mitochondrial/ER stress and the activation of the UPR. Metformin appears to rescue Trap1-deficiency after hypoxia mitigating ROS production and downregulating the pro-apoptotic PERK (protein kinase R-like ER kinase) arm of the UPR.     

Roles of Oxidative Stress, Apoptosis, PGC-1α and Mitochondrial Biogenesis in Cerebral Ischemia

The primary physiological function of mitochondria is to generate adenosine triphosphate through oxidative phosphorylation via the electron transport chain. Overproduction of reactive oxygen species (ROS) as byproducts generated from mitochondria have been implicated in acute brain injuries such as stroke from cerebral ischemia. It was well-documented that mitochondria-dependent apoptotic pathway involves pro- and anti-apoptotic protein binding, release of cytochrome c, leading ultimately to neuronal death. On the other hand, mitochondria also play a role to counteract the detrimental effects elicited by excessive oxidative stress. Recent studies have revealed that oxidative stress and the redox state of ischemic neurons are also implicated in the signaling pathway that involves peroxisome proliferative activated receptor-γ (PPARγ) co-activator 1α (PGC1-α). PGC1-α is a master regulator of ROS scavenging enzymes including manganese superoxide dismutase 2 and the uncoupling protein 2, both are mitochondrial proteins, and may contribute to neuronal survival. PGC1-α is also involved in mitochondrial biogenesis that is vital for cell survival. Experimental evidence supports the roles of mitochondrial dysfunction and oxidative stress as determinants of neuronal death as well as endogenous protective mechanisms after stroke. This review aims to summarize the current knowledge focusing on the molecular mechanisms underlying cerebral ischemia involving ROS, mitochondrial dysfunction, apoptosis, mitochondrial proteins capable of ROS scavenging, and mitochondrial biogenesis.     

Achyranthes bidentata Polypeptides Reduces Oxidative Stress and Exerts Protective Effects against Myocardial Ischemic/Reperfusion Injury in Rats

Achyranthes bidentata, a Chinese medicinal herb, is reported to be neuroprotective. However, its role in cardioprotection remains largely unknown. Our present study aimed to investigate the effects of Achyranthes bidentata polypeptides (ABPP) preconditioning on myocardial ischemia/reperfusion (MI/R) injury and to test the possible mechanisms. Rats were treated with ABPP (10 mg/kg/d, i.p.) or saline once daily for one week. Afterward, all the animals were subjected to 30 min of myocardial ischemia followed by 4 h of reperfusion. ABPP preconditioning for one week significantly improved cardiac function following MI/R. Meanwhile, ABPP reduced infarct size, plasma creatine kinase (CK)/lactate dehydrogenase (LDH) activities and myocardial apoptosis at the end of reperfusion in rat hearts. Moreover, ABPP preconditioning significantly inhibited superoxide generation, gp91^phox expression, malonaldialdehyde formation and enhanced superoxide dismutase activity in I/R hearts. Furthermore, ABPP treatment inhibited PTEN expression and increased Akt phosphorylation in I/R rat heart. PI3K inhibitor wortmannin blocked Akt activation, and abolished ABPP-stimulated anti-oxidant effect and cardioprotection. Our study demonstrated for the first time that ABPP reduces oxidative stress and exerts cardioprotection against MI/R injury in rats. Inhibition of PTEN and activation of Akt may contribute to the anti-oxidant capacity and cardioprotection of ABPP.     

Effect of Fiber Length on Carbon Nanotube-Induced Fibrogenesis

Given their extremely small size and light weight, carbon nanotubes (CNTs) can be readily inhaled by human lungs resulting in increased rates of pulmonary disorders, particularly fibrosis. Although the fibrogenic potential of CNTs is well established, there is a lack of consensus regarding the contribution of physicochemical attributes of CNTs on the underlying fibrotic outcome. We designed an experimentally validated in vitro fibroblast culture model aimed at investigating the effect of fiber length on single-walled CNT (SWCNT)-induced pulmonary fibrosis. The fibrogenic response to short and long SWCNTs was assessed via oxidative stress generation, collagen expression and transforming growth factor-beta (TGF-β) production as potential fibrosis biomarkers. Long SWCNTs were significantly more potent than short SWCNTs in terms of reactive oxygen species (ROS) response, collagen production and TGF-β release. Furthermore, our finding on the length-dependent in vitro fibrogenic response was validated by the in vivo lung fibrosis outcome, thus supporting the predictive value of the in vitro model. Our results also demonstrated the key role of ROS in SWCNT-induced collagen expression and TGF-β activation, indicating the potential mechanisms of length-dependent SWCNT-induced fibrosis. Together, our study provides new evidence for the role of fiber length in SWCNT-induced lung fibrosis and offers a rapid cell-based assay for fibrogenicity testing of nanomaterials with the ability to predict pulmonary fibrogenic response in vivo.