Resveratrol Affects Sphingolipid Metabolism in A549 Lung Adenocarcinoma Cells

Resveratrol is a naturally occurring polyphenol which has various beneficial effects, such as anti-inflammatory, anti-tumor, anti-aging, antioxidant, and neuroprotective effects, among others. The anti-cancer activity of resveratrol has been related to alterations in sphingolipid metabolism. We analyzed the effect of resveratrol on the enzymes responsible for accumulation of the two sphingolipids with highest functional activity—apoptosis promoting ceramide (CER) and proliferation-stimulating sphingosine-1-phosphate (S1P)—in human lung adenocarcinoma A549 cells. Resveratrol treatment induced an increase in CER and sphingosine (SPH) and a decrease in sphingomyelin (SM) and S1P. Our results showed that the most common mode of CER accumulation, through sphingomyelinase-induced hydrolysis of SM, was not responsible for a CER increase despite the reduction in SM in A549 plasma membranes. However, both the activity and the expression of CER synthase 6 were upregulated in resveratrol-treated cells, implying that CER was accumulated as a result of stimulated de novo synthesis. Furthermore, the enzyme responsible for CER hydrolysis, alkaline ceramidase, was not altered, suggesting that it was not related to changes in the CER level. The enzyme maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was downregulated, and its expression was reduced, resulting in a decrease in S1P levels in resveratrol-treated lung adenocarcinoma cells. In addition, incubation of resveratrol-treated A549 cells with the SK1 inhibitors DMS and fingolimod additionally downregulated SK1 without affecting its expression. The present studies provide information concerning the biochemical processes underlying the influence of resveratrol on sphingolipid metabolism in A549 lung cancer cells and reveal possibilities for combined use of polyphenols with specific anti-proliferative agents that could serve as the basis for the development of complex therapeutic strategies.     

Chitin-Derived AVR-48 Prevents Experimental Bronchopulmonary Dysplasia (BPD) and BPD-Associated Pulmonary Hypertension in Newborn Mice

Bronchopulmonary dysplasia (BPD) is the most common complication of prematurity and a key contributor to the large health care burden associated with prematurity, longer hospital stays, higher hospital costs, and frequent re-hospitalizations of affected patients through the first year of life and increased resource utilization throughout childhood. This disease is associated with abnormal pulmonary function that may lead to BPD-associated pulmonary hypertension (PH), a major contributor to neonatal mortality and morbidity. In the absence of any definitive treatment options, this life-threatening disease is associated with high resource utilization during and after neonatal intensive care unit (NICU) stay. The goal of this study was to test the safety and efficacy of a small molecule derivative of chitin, AVR-48, as prophylactic therapy for preventing experimental BPD in a mouse model. Two doses of AVR-48 were delivered either intranasally (0.11 mg/kg), intraperitoneally (10 mg/kg), or intravenously (IV) (10 mg/kg) to newborn mouse pups on postnatal day (P)2 and P4. The outcomes were assessed by measuring total inflammatory cells in the broncho-alveolar lavage fluid (BALF), chord length, septal thickness, and radial alveolar counts of the alveoli, Fulton’s Index (for PH), cell proliferation and cell death by immunostaining, and markers of inflammation by Western blotting and ELISA. The bioavailability and safety of the drug were assessed by pharmacokinetic and toxicity studies in both neonatal mice and rat pups (P3-P5). Following AVR-48 treatment, alveolar simplification was improved, as evident from chord length, septal thickness, and radial alveolar counts; total inflammatory cells were decreased in the BALF; Fulton’s Index was decreased and lung inflammation and cell death were decreased, while angiogenesis and cell proliferation were increased. AVR-48 was found to be safe and the no-observed-adverse-effect level (NOAEL) in rat pups was determined to be 100 mg/kg when delivered via IV dosing with a 20-fold safety margin. With no reported toxicity and with a shorter half-life, AVR-48 is able to reverse the worsening cardiopulmonary phenotype of experimental BPD and BPD-PH, compared to controls, thus positioning it as a future drug candidate.     

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.     

Purinergic Signaling and Inflammasome Activation in Psoriasis Pathogenesis

Psoriasis is a chronic inflammatory disease of the skin associated with systemic and joint manifestations and accompanied by comorbidities, such as metabolic syndrome and increased risk of cardiovascular disease. Psoriasis has a strong genetic basis, but exacerbation requires additional signals that are still largely unknown. The clinical manifestations involve the interplay between dendritic and T cells in the dermis to generate a self-sustaining inflammatory loop around the TNFα/IL-23/IL-17 axis that forms the psoriatic plaque. In addition, in recent years, a critical role of keratinocytes in establishing the interplay that leads to psoriatic plaques’ formation has re-emerged. In this review, we analyze the most recent evidence of the role of keratinocytes and danger associates molecular patterns, such as extracellular ATP in the generation of psoriatic skin lesions. Particular attention will be given to purinergic signaling in inflammasome activation and in the initiation of psoriasis. In this phase, keratinocytes’ inflammasome may trigger early inflammatory pathways involving IL-1β production, to elicit the subsequent cascade of events that leads to dendritic and T cell activation. Since psoriasis is likely triggered by skin-damaging events and trauma, we can envisage that intracellular ATP, released by damaged cells, may play a role in triggering the inflammatory response underlying the pathogenesis of the disease by activating the inflammasome. Therefore, purinergic signaling in the skin could represent a new and early step of psoriasis; thus, opening the possibility to target single molecular actors of the purinome to develop new psoriasis treatments.     

Effect of Quercetin and Fingolimod,Alone or in Combination,on the Sphingolipid Metabolism in HepG2 Cells

Combinations of anti-cancer drugs can overcome resistance to therapy and provide new more effective treatments. In this work we have analyzed the effect of the polyphenol quercetin and the anti-cancer sphingosine analog fingolimod on the sphingolipid metabolism in HepG2 cells, since sphingolipids are recognized as mediators of cell proliferation and apoptosis in cancer cells. Treatment of hepatocellular carcinoma HepG2 cells with quercetin and fingolimod, alone or in combination, induced different degrees of sphingomyelin (SM) reduction and a corresponding activation of neutral sphingomyelinase (nSMase). Western blot analysis showed that only treatments containing quercetin induced up-regulation of nSMase expression. The same treatment caused elevation of ceramide (CER) levels, whereas the observed alterations in sphingosine (SPH) content were not statistically significant. The two tested drugs induced a reduction of the pro-proliferative sphingolipid, sphingosine 1 phosphate (S1P), in the following order: quercetin, fingolimod, quercetin + fingolimod. The activity of the enzyme responsible for CER hydrolysis, alkaline ceramidase (ALCER) was down-regulated only in the incubations involving quercetin and fingolimod did not affect this activity. The enzyme, maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was down-regulated by incubations in the following order: quercetin, fingolimod, quercetin + fingolimod. Western blot analysis showed down-regulation in SK1 expression upon quercetin but not upon fingolimod treatment. Studies on the effect of quercetin and fingolimod on the two proteins associated with apoptotic events, AKT and Bcl-2, showed that only quercetin, alone or in combination, down-regulated the activity of the two proteins. The reported observations provide information which can be useful in the search of novel anti-tumor approaches, aiming at optimization of the therapeutic effect and maximal preservation of healthy tissues.     

Time-Dependent Changes in Hepatic Sphingolipid Accumulation and PI3K/Akt/mTOR Signaling Pathway in a Rat Model of NAFLD

Increased lipid bioavailability in a diet favors lipid accumulation, enhancing hepatic lipotoxicity and contributing to insulin resistance (IR) development. The aim of our study was to examine time-dependent alterations in the intrahepatic content of sphingolipids and insulin signaling pathway in rats fed a high-fat diet (HFD). The experiment was conducted on male Wistar rats receiving a standard diet or HFD for five weeks. At the end of each experimental feeding week, liver sphingolipids were determined using high-performance liquid chromatography. The expression of proteins from the sphingolipid pathway and glucose transporter expression were assessed by Western blot. The content of phosphorylated form of proteins from the insulin pathway was detected by a multiplex assay kit. Our results revealed that HFD enhanced hepatic ceramide deposition by increasing the expression of selected proteins from sphingomyelin and salvage pathways in the last two weeks. Importantly, we observed a significant inhibition of Akt phosphorylation in the first week of HFD and stimulation of PTEN and mTOR phosphorylation at the end of HFD. These changes worsened the PI3K/Akt/mTOR signaling pathway. We may postulate that HFD-induced reduction in the insulin action in the time-dependent matter was exerted by excessive accumulation of sphingosine and sphinganine rather than ceramide.     

Mesenchymal Stem Cells and Formyl Peptide Receptor 2 Activity in Hyperoxia-Induced Lung Injury in Newborn Mice

Formyl peptide receptor (FPR) 2 is known to play a critical role in regulating inflammation, including either the pro-inflammatory or pro-resolving effects. However, its role in neonatal hyperoxia-induced lung injury has not been delineated. In this study, we investigate whether mesenchymal stem cells (MSCs) attenuate hyperoxia-induced neonatal lung injury by regulating FPR2 activity. We observed a significant increase in FPR2 levels in alveolar macrophages (RAW264.7 cells) after H₂O₂-induced stress, which decreased after MSC treatment. In the H₂O₂-induction model, increased levels of inflammatory cytokines (IL-1α and TNF-α) were significantly reduced in RAW264.7 cells after treatment with WRW4, an inhibitor of FPR2, or MSCs. Viability of lung epithelial cells and endothelial cells was significantly improved when cultured in the conditioned media of RAW264.7 cells treated with WRW4 or MSCs, compared to when cultured in the conditioned media of control RAW265.7 cells exposed to H₂O₂. For the in vivo study, wild-type and FPR2 knockout (FPR2^−/−) C57/BL6 mouse pups were randomly exposed to 80% oxygen or room air from postnatal day (P) 1 to P14. At P5, 2 × 10⁵ MSCs were transplanted intratracheally. MSCs reduced the elevated FPR2 activity at P7 and improved the decreased FPR2 activity as well as the increased immuno-stained FPR2 activity in alveolar macrophages in hyperoxic lungs at P14. Both FPR2^−/− and MSCs similarly attenuated impaired alveolarization and angiogenesis, and increased apoptosis and inflammation of hyperoxic lungs without synergistic effects. Our findings suggest that the protective effects of MSCs in hyperoxic lung injury might be related to indirect modulation of FPR2 activity, at least of alveolar macrophages in neonatal mice.     

Lung Surfactant Decreases Biochemical Alterations and Oxidative Stress Induced by a Sub-Toxic Concentration of Carbon Nanoparticles in Alveolar Epithelial and Microglial Cells

Carbon-based nanomaterials are nowadays attracting lots of attention, in particular in the biomedical field, where they find a wide spectrum of applications, including, just to name a few, the drug delivery to specific tumor cells and the improvement of non-invasive imaging methods. Nanoparticles inhaled during breathing accumulate in the lung alveoli, where they interact and are covered with lung surfactants. We recently demonstrated that an apparently non-toxic concentration of engineered carbon nanodiamonds (ECNs) is able to induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells. Therefore, the complete understanding of their “real” biosafety, along with their possible combination with other molecules mimicking the in vivo milieu, possibly allowing the modulation of their side effects becomes of utmost importance. Based on the above, the focus of the present work was to investigate whether the cellular alterations induced by an apparently non-toxic concentration of ECNs could be counteracted by their incorporation into a synthetic lung surfactant (DPPC:POPG in 7:3 molar ratio). By using two different cell lines (alveolar (A549) and microglial (BV-2)), we were able to show that the presence of lung surfactant decreased the production of ECNs-induced nitric oxide, total reactive oxygen species, and malondialdehyde, as well as counteracted reduced glutathione depletion (A549 cells only), ameliorated cell energy status (ATP and total pool of nicotinic coenzymes), and improved mitochondrial phosphorylating capacity. Overall, our results on alveolar basal epithelial and microglial cell lines clearly depict the benefits coming from the incorporation of carbon nanoparticles into a lung surfactant (mimicking its in vivo lipid composition), creating the basis for the investigation of this combination in vivo.     

Effect of Empagliflozin on Sphingolipid Catabolism in Diabetic and Hypertensive Rats

The profile of sphingomyelin and its metabolites shows changes in the plasma, organs, and tissues of patients with cardiovascular, renal, and metabolic diseases. The objective of this study was to investigate the effect of empagliflozin on the levels of sphingomyelin and its metabolites, as well as on the activity of acid and neutral sphingomyelinase (aSMase and nSMase) and neutral ceramidase (nCDase) in the plasma, kidney, heart, and liver of streptozotocin-induced diabetic and Angiotensin II (Ang II)-induced hypertension rats. Empagliflozin treatment decreased hyperglycemia in diabetic rats whereas blood pressure remained elevated in hypertensive rats. In diabetic rats, empagliflozin treatment decreased sphingomyelin in the plasma and liver, ceramide in the heart, sphingosine-1-phosphate (S1P) in the kidney, and nCDase activity in the plasma, heart, and liver. In hypertensive rats, empagliflozin treatment decreased sphingomyelin in the plasma, kidney, and liver; S1P in the plasma and kidney; aSMase in the heart, and nCDase activity in the plasma, kidney, and heart. Our results suggest that empagliflozin downregulates the interaction of the de novo pathway and the catabolic pathway of sphingolipid metabolism in the diabetes, whereas in Ang II-dependent hypertension, it only downregulates the sphingolipid catabolic pathway.     

The Role of Mitochondria Dysfunction in Inflammatory Bowel Diseases and Colorectal Cancer

Inflammatory bowel disease (IBD) is one of the leading gut chronic inflammation disorders, especially prevalent in Western countries. Recent research suggests that mitochondria play a crucial role in IBD development and progression to the more severe disease—colorectal cancer (CRC). In this review, we focus on the role of mitochondrial mutations and dysfunctions in IBD and CRC. In addition, main mitochondria-related molecular pathways involved in IBD to CRC transition are discussed. Additionally, recent publications dedicated to mitochondria-targeted therapeutic approaches to cure IBD and prevent CRC progression are discussed.     

水杨酸在诱导系统获得抗性中的信号传导作用

水杨酸(SA)是植物的一种内源信号传导物质,外源SA能诱导植物防卫基因的表达并对病害产生部分保护效果;内源SA水平增加与防卫基因和抗病性发展一致,因此,SA是植物产生系统获得抗性(SAR)必不可少的条件,但SA不是诱导SAR产生的唯一条件,SAR的产生是SA与其他物质共同作用的结果。一旦SAR诱导植物在侵染点产生一种信号,经过某些信号物质的传导最终可系统性地传播到整株植物,SA可能是这种信使,但这种信号的化学本质和分子机制尚缺乏实验证据。     

The Role of mTOR and eIF Signaling in Benign Endometrial Diseases

Adenomyosis, endometriosis, endometritis, and typical endometrial hyperplasia are common non-cancerous diseases of the endometrium that afflict many women with life-impacting consequences. The mammalian target of the rapamycin (mTOR) pathway interacts with estrogen signaling and is known to be dysregulated in endometrial cancer. Based on this knowledge, we attempt to investigate the role of mTOR signaling in benign endometrial diseases while focusing on how the interplay between mTOR and eukaryotic translation initiation factors (eIFs) affects their development. In fact, mTOR overactivity is apparent in adenomyosis, endometriosis, and typical endometrial hyperplasia, where it promotes endometrial cell proliferation and invasiveness. Recent data show aberrant expression of various components of the mTOR pathway in both eutopic and ectopic endometrium of patients with adenomyosis or endometriosis and in hyperplastic endometrium as well. Moreover, studies on endometritis show that derangement of mTOR signaling is linked to the establishment of endometrial dysfunction caused by chronic inflammation. This review shows that inhibition of the mTOR pathway has a promising therapeutic effect in benign endometrial conditions, concluding that mTOR signaling dysregulation plays a critical part in their pathogenesis.     

Role of microRNA and Oxidative Stress in Influenza A Virus Pathogenesis

MicroRNAs (miRNAs) are non-coding RNAs that regulate diverse cellular pathways by controlling gene expression. Increasing evidence has revealed their critical involvement in influenza A virus (IAV) pathogenesis. Host–IAV interactions induce different levels of oxidative stress (OS) by disrupting the balance between reactive oxygen species (ROS) and antioxidant factors. It is thought that miRNA may regulate the expression of ROS; conversely, ROS can induce or suppress miRNA expression during IAV infection. Thus, miRNA and OS are the two key factors of IAV infection and pathogenesis. Accordingly, interactions between OS and miRNA during IAV infection might be a critical area for further research. In this review, we discuss the crosstalk between miRNAs and OS during IAV infection. Additionally, we highlight the potential of miRNAs as diagnostic markers and therapeutic targets for IAV infections. This knowledge will help us to study host–virus interactions with novel intervention strategies.     

One Week of CDAHFD Induces Steatohepatitis and Mitochondrial Dysfunction with Oxidative Stress in Liver

The prevalence of nonalcoholic fatty liver disease (NAFLD) has been rapidly increasing worldwide. A choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) has been used to create a mouse model of nonalcoholic steatohepatitis (NASH). There are some reports on the effects on mice of being fed a CDAHFD for long periods of 1 to 3 months. However, the effect of this diet over a short period is unknown. Therefore, we examined the effect of 1-week CDAHFD feeding on the mouse liver. Feeding a CDAHFD diet for only 1-week induced lipid droplet deposition in the liver with increasing activity of liver-derived enzymes in the plasma. On the other hand, it did not induce fibrosis or cirrhosis. Additionally, it was demonstrated that CDAHFD significantly impaired mitochondrial respiration with severe oxidative stress to the liver, which is associated with a decreasing mitochondrial DNA copy number and complex proteins. In the gene expression analysis of the liver, inflammatory and oxidative stress markers were significantly increased by CDAHFD. These results demonstrated that 1 week of feeding CDAHFD to mice induces steatohepatitis with mitochondrial dysfunction and severe oxidative stress, without fibrosis, which can partially mimic the early stage of NASH in humans.     

Signaling Role of Mitochondrial Enzymes and Ultrastructure in the Formation of Molecular Mechanisms of Adaptation to Hypoxia

This study was the first comprehensive investigation of the dependence of mitochondrial enzyme response (catalytic subunits of mitochondrial complexes (MC) I-V, including NDUFV2, SDHA, Cyt b, COX1 and ATP5A) and mitochondrial ultrastructure in the rat cerebral cortex (CC) on the severity and duration of in vivo hypoxic exposures. The role of individual animal’s resistance to hypoxia was also studied. The respiratory chain (RC) was shown to respond to changes in environmental [O₂] as follows: (a) differential reaction of mitochondrial enzymes, which depends on the severity of the hypoxic exposure and which indicates changes in the content and catalytic properties of mitochondrial enzymes, both during acute and multiple exposures; and (b) ultrastructural changes in mitochondria, which reflect various degrees of mitochondrial energization. Within a specific range of reduced O₂ concentrations, activation of the MC II is a compensatory response supporting the RC electron transport function. In this process, MC I develops new kinetic properties, and its function recovers in hypoxia by reprograming the RC substrate site. Therefore, the mitochondrial RC performs as an in vivo molecular oxygen sensor. Substantial differences between responses of rats with high and low resistance to hypoxia were determined.     

The Potential Role of Flavonoids in Ameliorating Diabetic Cardiomyopathy via Alleviation of Cardiac Oxidative Stress,Inflammation and Apoptosis

Diabetic cardiomyopathy is one of the major mortality risk factors among diabetic patients worldwide. It has been established that most of the cardiac structural and functional alterations in the diabetic cardiomyopathy condition resulted from the hyperglycemia-induced persistent oxidative stress in the heart, resulting in the maladaptive responses of inflammation and apoptosis. Flavonoids, the most abundant phytochemical in plants, have been reported to exhibit diverse therapeutic potential in medicine and other biological activities. Flavonoids have been widely studied for their effects in protecting the heart against diabetes-induced cardiomyopathy. The potential of flavonoids in alleviating diabetic cardiomyopathy is mainly related with their remedial actions as anti-hyperglycemic, antioxidant, anti-inflammatory, and anti-apoptotic agents. In this review, we summarize the latest findings of flavonoid treatments on diabetic cardiomyopathy as well as elucidating the mechanisms involved.     

The Role of Neurotrophin Signaling in Age-Related Cognitive Decline and Cognitive Diseases

Neurotrophins are a family of secreted proteins expressed in the peripheral nervous system and the central nervous system that support neuronal survival, synaptic plasticity, and neurogenesis. Brain-derived neurotrophic factor (BDNF) and its high affinity receptor TrkB are highly expressed in the cortical and hippocampal areas and play an essential role in learning and memory. The decline of cognitive function with aging is a major risk factor for cognitive diseases such as Alzheimer’s disease. Therefore, an alteration of BDNF/TrkB signaling with aging and/or pathological conditions has been indicated as a potential mechanism of cognitive decline. In this review, we summarize the cellular function of neurotrophin signaling and review the current evidence indicating a pathological role of neurotrophin signaling, especially of BDNF/TrkB signaling, in the cognitive decline in aging and age-related cognitive diseases. We also review the therapeutic approach for cognitive decline by the upregulation of the endogenous BDNF/TrkB-system.     

A Rheostat of Ceramide and Sphingosine-1-Phosphate as a Determinant of Oxidative Stress-Mediated Kidney Injury

Reactive oxygen species (ROS) modulate sphingolipid metabolism, including enzymes that generate ceramide and sphingosine-1-phosphate (S1P), and a ROS-antioxidant rheostat determines the metabolism of ceramide-S1P. ROS induce ceramide production by activating ceramide-producing enzymes, leading to apoptosis, while they inhibit S1P production, which promotes survival by suppressing sphingosine kinases (SphKs). A ceramide-S1P rheostat regulates ROS-induced mitochondrial dysfunction, apoptotic/anti-apoptotic Bcl-2 family proteins and signaling pathways, leading to apoptosis, survival, cell proliferation, inflammation and fibrosis in the kidney. Ceramide inhibits the mitochondrial respiration chain and induces ceramide channel formation and the closure of voltage-dependent anion channels, leading to mitochondrial dysfunction, altered Bcl-2 family protein expression, ROS generation and disturbed calcium homeostasis. This activates ceramide-induced signaling pathways, leading to apoptosis. These events are mitigated by S1P/S1P receptors (S1PRs) that restore mitochondrial function and activate signaling pathways. SphK1 promotes survival and cell proliferation and inhibits inflammation, while SphK2 has the opposite effect. However, both SphK1 and SphK2 promote fibrosis. Thus, a ceramide-SphKs/S1P rheostat modulates oxidant-induced kidney injury by affecting mitochondrial function, ROS production, Bcl-2 family proteins, calcium homeostasis and their downstream signaling pathways. This review will summarize the current evidence for a role of interaction between ROS-antioxidants and ceramide-SphKs/S1P and of a ceramide-SphKs/S1P rheostat in the regulation of oxidative stress-mediated kidney diseases.     

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.