Mediated Reactive Oxygen Species Production Research Articles

Succinate dehydrogenase mediated ROS production contributes to ASIC1a-induced chondrocyte pyroptosis in rheumatoid arthritis

Our previous study showed that acidic stimuli activate acid-sensitive ion channel 1a (ASIC1a), resulting in chondrocyte destruction associated with rheumatoid arthritis (RA). However, the exact underlying processes remain unclear. Recent evidence suggests that the production of reactive oxygen species (ROS) mediated by succinate dehydrogenase (SDH), contributes to chondrocyte damage. The objective of this study was to investigate the involvement of SDH in ASIC1a-induced chondrocyte destruction in RA and to explore the associated mechanisms both in vivo and in vitro. Our findings revealed that the cartilage of mice with collagen-induced arthritis (CIA) and acid-treated chondrocytes exhibited a substantial increase in SDH expression. Furthermore, SDH inhibition attenuates acidosis-induced pyroptosis in chondrocytes. Notably, ASIC1a activation through acid stimuli increases SDH activity and pyroptosis through the Ca2+/CaMKK2/AMPK pathway in chondrocytes. Mechanistically, SDH assembly factor 2 (SDHAF2) was identified as a key modulator of SDH activity induced by ASIC1a in acid-stressed chondrocytes. Moreover, the expression of SDH in CIA mouse chondrocytes decreased and the histological characteristics of ankle joint damage were reduced by the ASIC1a-particular blocker PcTx-1. Overall, these observations suggest that ASIC1a activation under acidic conditions increases SDH activity and modulates SDHAF2, thereby promoting chondrocyte pyroptosis through the Ca2+/CaMKK2/AMPK pathway.

Abstract Tu128: p22 phox is a critical factor for the prevention of oxidation and stabilization of SERCA2a in the heart.

Introduction: Elevated oxidative stress linked with heart failure is a major cause for the downregulation of sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ ATPase 2a (SERCA2a). NADPH oxidase (NOX) complexes are a key source of reactive oxygen species (ROS) in cardiomyocytes. p22 phox , a transmembrane partner of NOX1-4, plays an essential role in mediating ROS production in multiple NOX complexes. We investigated the role of p22 phox in oxidative stress and SERCA2a levels during pressure overload (PO). Question: Does loss of p22 phox alleviate heart failure through reduction of oxidative stress and stabilization of SERCA2a levels during PO? Methods/approach: Cardiac-specific p22 phox knockout ( p22 phox -cKO) mice were subjected to sham operation or transverse aortic constriction (TAC). The p22 phox interactome was analyzed by co-immunoprecipitation and mass spectrometry. Protein cysteine oxidation was probed by biotinylated-iodoacetamide (BIAM) labelling. Results: The p22 phox -cKO mouse heart showed a 30% reduction in Dityrosine levels compared to WT after 4-week TAC (p<0.05) and 50% lower total tissue H 2 O 2 levels compared to WT after 1-week TAC (p<0.01). Unexpectedly, p22 phox -cKO mice had higher mortality (+20%, p<0.05), lung congestion (2-fold, p<0.05), and fibrosis (>40%, p<0.01) and a lower (40%, p<0.01) left ventricle ejection fraction than WT after TAC. p22 phox directly interacted with SERCA2a. Lack of p22 phox led to oxidation of SERCA2a (50% reduction in BIAM labelling, p<0.01) at cysteine 498 and promoted proteasome-mediated degradation of SERCA2a. The relative SERCA2a ATPase activity was unaltered in both WT and p22 phox -cKO mice. Furthermore, in the absence of p22 phox , SERCA2a interacted strongly with HMG-CoA reductase degradation protein 1 (Hrd1), an endoplasmic reticulum-associated degradation (ERAD)-specific E3 ubiquitin ligase. Knockdown of Hrd1 in the presence of p22 phox knockdown restored SERCA2a to 70% of WT levels (ns and p<0.5 vs p22 phox knockdown). SERCA2a C498S knock-in mice exhibited better cardiac function and stable SERCA2a protein levels after TAC compared to WT mice. Conclusion: The loss of p22 phox exacerbated heart failure through increased SERCA2a cysteine 498 oxidation and degradation through ERAD.

Thiotert Induces Myelodysplastic Syndromes Cells Apoptosis by Activating Oxidative Stress

To explore whether thiotert treatment can inhibit proliferation and induce apoptosis in myelodysplastic syndromes (MDS) cells. CCK-8 assay was used for determining the cytotoxicity of thiotert to MDS cell line SKM-1 and the reversal effect of GSH, NAC, and Z-VAD-FMK on thiotert-induced inhibition of cell viability. EdU assay was deployed to detect the cell proliferation ability. Intracellular reactive oxygen species (ROS) was measured by flow cytometry after DCFH-DA staining. The expression of DNA damage- and apoptosis-related proteins was detected by Western blot. Thiotert treatment significantly suppressed the cell viability and proliferation ability in SKM-1 cells. A large amount of ROS generation and markedly elevated C-PARP, C-Caspase 3, and γ-H2AX were observed after thiotert administration, while BCL-2 was significantly decreased. In addition, GSH, NAC, and Z-VAD-FMK were able to mitigate the cytotoxicity of thiotert on SKM-1 cells. Thiotert can promote MDS cell apoptosis by mediating ROS production and pro-apoptotic proteins expression.

ADAM10 as a major activator of reactive oxygen species production and klotho shedding in podocytes under diabetic conditions

Early stages of diabetes are characterized by elevations of insulin and glucose concentrations. Both factors stimulate reactive oxygen species (ROS) production, leading to impairments in podocyte function and disruption of the glomerular filtration barrier. Podocytes were recently shown to be an important source of αKlotho (αKL) expression. Low blood Klotho concentrations are also associated with an increase in albuminuria, especially in patients with diabetes. We investigated whether ADAM10, which is known to cleave αKL, is activated in glomeruli and podocytes under diabetic conditions and the potential mechanisms by which ADAM10 mediates ROS production and disturbances of the glomerular filtration barrier.In cultured human podocytes, high glucose increased ADAM10 expression, shedding, and activity, NADPH oxidase activity, ROS production, and albumin permeability. These effects of glucose were inhibited when cells were pretreated with an ADAM10 inhibitor or transfected with short-hairpin ADAM10 (shADAM10) or after the addition soluble Klotho. We also observed increases in ADAM10 activity, NOX4 expression, NADPH oxidase activity, and ROS production in αKL-depleted podocytes. This was accompanied by an increase in albumin permeability in shKL-expressing podocytes. The protein expression and activity of ADAM10 also increased in isolated glomeruli and urine samples from diabetic rats. Altogether, these results reveal a new mechanism by which hyperglycemia in diabetes increases albumin permeability through ADAM10 activation and an increase in oxidative stress via NOX4 enzyme activation. Moreover, αKlotho downregulates ADAM10 activity and supports redox balance, consequently protecting the slit diaphragm of podocyteσ under hyperglycemic conditions.

Roles of AhR/CYP1s signaling pathway mediated ROS production in uremic cardiomyopathy

PurposeUremic cardiomyopathy (UCM) is the leading cause of chronic kidney disease (CKD) related mortality. Uremic toxins including indoxyl sulfate (IS) play important role during the progression of UCM. This study was to explore the underlying mechanism of IS related myocardial injury. MethodsUCM rat model was established through five-sixths nephrectomy to evaluate its effects on blood pressure, cardiac impairment, and histological changes using echocardiography and histological analysis. Additionally, IS was administered to neonatal rat cardiomyocytes (NRCMs) and the human cardiomyocyte cell line AC16. DHE staining and peroxide-sensitive dye 2′,7′-dichlorofluorescein diacetate (H2DCFDA) was conducted to assess the reactive oxygen species (ROS) production. Cardiomyocyte hypertrophy was estimated using wheat germ agglutinin (WGA) staining and immunofluorescence. Aryl hydrocarbon receptor (AhR) translocation was observed by immunofluorescence. The activation of AhR was evaluated by immunoblotting of cytochrome P450 1 s (CYP1s) and quantitative real-time PCR (RT-PCR) analysis of AHRR and PTGS2. Additionally, the pro-oxidative and pro-hypertrophic effects were evaluated using the AhR inhibitor CH-223191, the CYP1s inhibitor Alizarin and the ROS scavenger N-Acetylcysteine (NAC). ResultsUCM rat model was successfully established, and cardiac hypertrophy, accompanied by increased blood pressure, and myocardial fibrosis. Further research confirmed the activation of the AhR pathway in UCM rats including AhR translocation and downstream protein CYP1s expression, accompanied with increasing ROS production detected by DHE staining. In vitro experiment demonstrated a translocation of AhR triggered by IS, leading to significant increase of downstream gene expression. Subsequently study indicated a close relationship between the production of ROS and the activation of AhR/CYP1s, which was effectively blocked by applying AhR inhibitor, CYP1s inhibitor and siRNA against AhR. Moreover, the inhibition of AhR/CYP1s/ROS pathway collectively blocked the pro-hypertrophic effect of IS-mediated cardiomyopathy. ConclusionThis study provides evidence that the AhR/CYP1s pathway is activated in UCM rats, and this activation is correlated with the uremic toxin IS. In vitro studies indicate that IS can stimulate the AhR translocation in cardiomyocyte, triggering to the production of intracellular ROS via CYP1s. This process leads to prolonged oxidative stress stimulation and thus contributes to the progression of uremic toxin-mediated cardiomyopathy.

Abstract 17721: Loss of p22 phox Exacerbates Heart Failure Through Increased Oxidation and Proteasomal Degradation of SERCA2a

Introduction: Downregulation of sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ ATPase 2a (SERCA2a) accentuated by oxidative stress is a major driver of heart failure. NADPH oxidase (NOX) complexes are the key sources of reactive oxygen species (ROS) in cardiomyocytes. p22 phox , a transmembrane partner of NOX1-4, plays an essential role in mediating ROS production in multiple NOX complexes. We investigated the role of p22 phox in oxidative stress and SERCA2a levels during pressure overload (PO). Question: Does loss of p22 phox alleviate heart failure through reduction of oxidative stress and stabilization of SERCA2a levels during PO? Methods: Cardiac-specific p22 phox knockout ( p22 phox -cKO) mice were subjected to sham operation or transverse aortic constriction (TAC). The p22 phox interactome was analyzed by co-immunoprecipitation and mass spectrometry. Protein cysteine oxidation was probed by biotinylated iodoacetamide labelling. Results: The p22 phox -cKO mouse heart showed a 50% reduction in H 2 O 2 levels compared to WT after 1-week TAC (p<0.01). Unexpectedly, p22 phox -cKO mice had 20% higher mortality (p<0.05), lung congestion (2-fold, p<0.05), and fibrosis (>40%, p<0.01) and a lower (40%, p<0.01) left ventricle ejection fraction than WT after TAC. p22 phox directly interacted with SERCA2a. Lack of p22 phox led to oxidation of SERCA2a at cysteine 498 and promoted degradation of SERCA2a. SERCA2a C498S knock-in mice have better cardiac function and stable SERCA2a protein levels after TAC compared to WT mice. Further, p22 phox aided thioredoxin 1 (TRX-1) interaction with SERCA2a and prevented cysteine oxidation. SERCA2a was co-immunoprecipitated with Smad ubiquitination regulatory factor 1 (Smurf1) and HMG-CoA reductase degradation protein 1 (Hrd1) E3 ubiquitin ligases in the absence of p22 phox or TRX-1. Conclusion: p22 phox recruits TRX-1 to SERCA2a, prevents SERCA2a cysteine 498 oxidation, promotes its stability, and maintains cardiac function.

Human blood neutrophils generate ROS through FcγR-signaling to mediate protection against febrile P. falciparum malaria

Blood phagocytes, such as neutrophils and monocytes, generate reactive oxygen species (ROS) as a part of host defense response against infections. We investigated the mechanism of Fcγ-Receptor (FcγR) mediated ROS production in these cells to understand how they contribute to anti-malarial immunity. Plasmodium falciparum merozoites opsonized with naturally occurring IgG triggered both intracellular and extracellular ROS generation in blood phagocytes, with neutrophils being the main contributors. Using specific inhibitors, we show that both FcγRIIIB and FcγRIIA acted synergistically to induce ROS production in neutrophils, and that NADPH oxidase 2 and the PI3K intracellular signal transduction pathway were involved in this process. High levels of neutrophil ROS were also associated with protection against febrile malaria in two geographically diverse malaria endemic regions from Ghana and India, stressing the importance of the cooperation between anti-malarial IgG and neutrophils in triggering ROS-mediated parasite killing as a mechanism for naturally acquired immunity against malaria.

PiRNA hsa_piR_019914 Promoted Chondrocyte Anabolic Metabolism By Inhibiting LDHA-Dependent ROS Production.

Osteoarthritis (OA) is the most common joint disease. The occurrence and progression of OA are regulated by epigenetics. A large number of studies have shown the important regulatory role of noncoding RNAs in joint diseases. As the largest class of noncoding small RNAs, the importance of piRNAs in many diseases, especially cancer, has been increasingly recognized. However, few studies have explored the role of piRNAs in OA. Our study showed that hsa_piR_019914 decreased significantly in OA. This study aimed to demonstrate the role of hsa_piR_019914 as a potential biological target of OA in chondrocytes. The GEO database and bioinformatics analysis were used for a series of screenings, and the OA model using human articular chondrocytes (C28/I2 cells), SW1353 cells under inflammatory factor stimulation was used to determine that hsa_piR_019914 was significantly downregulated in OA. Overexpression or inhibition of hsa_piR_019914 in C28/I2 cells was achieved by transfecting mimics or inhibitors. The effect of hsa_piR_019914 on the biological function of chondrocytes was verified by qPCR, flow cytometry, and colony formation assays in vitro. The target gene of hsa_piR_019914, lactate dehydrogenase A (LDHA), was screened by small RNA sequencing and quantitative polymerase chain reaction (qPCR), LDHA was knocked out in C28/I2 cells by the transfection of siRNA LDHA, and the relationship between hsa_piR_019914, LDHA, and reactive oxygen species (ROS) production was verified by flow cytometry. The piRNA hsa-piR-019914 was significantly downregulated in osteoarthritis (OA). Hsa-piR-019914 reduced inflammation-mediated chondrocyte apoptosis and maintained cell proliferation and clone formation in vitro. Hsa-piR-019914 reduced the production of LDHA-dependent ROS through targeted regulation of LDHA expression, maintained chondrocyte-specific gene expression of ACAN and COL2, and inhibited the gene expression of MMP3 and MMP13. Collectively, this study showed that hsa_piR_019914 was negatively correlated with the expression of LDHA, which mediates ROS production. Under the stimulation of inflammatory factors, overexpression of hsa_piR_019914 had a protective effect on chondrocytes in vitro, and the absence of hsa_piR_019914 exacerbated the negative effect of inflammation on chondrocytes. Studies on piRNAs provide new therapeutic interventions for OA.

Duox mediated ROS production inhibited WSSV replication in Eriocheir sinensis under short-term nitrite stress

Nitrite stress and white spot syndrome virus (WSSV) infection are major problems threatening the sustainable and healthy development of Eriocheir sinensis. Some studies have found that nitrite stress can lead to the production of reactive oxygen species (ROS), whereas synthetic ROS plays a vital role in the signaling pathway. However, whether nitrite stress influences the infection of crabs by WSSV remains unclear. NADPH oxidases, including NOX1-5 and Duox1-2, are important for ROS production. In the present study, a novel Duox gene (designated as EsDuox) was identified from E. sinensis. The studies found that nitrite stress could increase the expression of EsDuox during WSSV infection and decrease the transcription of the WSSV envelope protein VP28. Moreover, nitrite stress could increase the production of ROS, and the synthesis of ROS relied on EsDuox. These results indicated a potential “nitrite stress–Duox activation–ROS production” pathway that plays a negative role in WSSV infection in E. sinensis. Further studies found that nitrite stress and EsDuox could promote the expression of EsDorsal transcriptional factor and antimicrobial peptides (AMPs) during WSSV infection. Moreover, the synthesis of AMPs was positively regulated by EsDorsal in the process of WSSV infection under nitrite stress. Furthermore, EsDorsal played an inhibitory role in the replication of WSSV under nitrite stress. Our study reveals a new pathway for “nitrite stress–Duox activation–ROS production–Dorsal activation–AMP synthesis” that is involved in the defense against WSSV infection in E. sinensis during short-term nitrite stress.

Identification of CDPKs involved in TaNOX7 mediated ROS production in wheat.

As the critical sensors and decoders of calcium signal, calcium-dependent protein kinase (CDPK) has become the focus of current research, especially in plants. However, few resources are available on the properties and functions of CDPK gene family in Triticum aestivum (TaCDPK). Here, a total of 79 CDPK genes were identified in the wheat genome. These TaCDPKs could be classified into four subgroups on phylogenesis, while they may be classified into two subgroups based on their tissue and organ-spatiotemporal expression profiles or three subgroups according to their induced expression patterns. The analysis on the signal network relationships and interactions of TaCDPKs and NADPH (reduced nicotinamide adenine dinucleotide phosphate oxidases, NOXs), the key producers for reactive oxygen species (ROS), showed that there are complicated cross-talks between these two family proteins. Further experiments demonstrate that, two members of TaCDPKs, TaCDPK2/4, can interact with TaNOX7, an important member of wheat NOXs, and enhanced the TaNOX7-mediated ROS production. All the results suggest that TaCDPKs are highly expressed in wheat with distinct tissue or organ-specificity and stress-inducible diversity, and play vital roles in plant development and response to biotic and abiotic stresses by directly interacting with TaNOXs for ROS production.

Actin remodeling mediates ROS production and JNK activation to drive apoptosis-induced proliferation.

Stress-induced cell death, mainly apoptosis, and its subsequent tissue repair is interlinked although our knowledge of this connection is still very limited. An intriguing finding is apoptosis-induced proliferation (AiP), an evolutionary conserved mechanism employed by apoptotic cells to trigger compensatory proliferation of their neighboring cells. Studies using Drosophila as a model organism have revealed that apoptotic caspases and c-Jun N-terminal kinase (JNK) signaling play critical roles to activate AiP. For example, the initiator caspase Dronc, the caspase-9 ortholog in Drosophila, promotes activation of JNK leading to release of mitogenic signals and AiP. Recent studies further revealed that Dronc relocates to the cell cortex via Myo1D, an unconventional myosin, and stimulates production of reactive oxygen species (ROS) to trigger AiP. During this process, ROS can attract hemocytes, the Drosophila macrophages, which further amplify JNK signaling cell non-autonomously. However, the intrinsic components connecting Dronc, ROS and JNK within the stressed signal-producing cells remain elusive. Here, we identified LIM domain kinase 1 (LIMK1), a kinase promoting cellular F-actin polymerization, as a novel regulator of AiP. F-actin accumulates in a Dronc-dependent manner in response to apoptotic stress. Suppression of F-actin polymerization in stressed cells by knocking down LIMK1 or expressing Cofilin, an inhibitor of F-actin elongation, blocks ROS production and JNK activation, hence AiP. Furthermore, Dronc and LIMK1 genetically interact. Co-expression of Dronc and LIMK1 drives F-actin accumulation, ROS production and JNK activation. Interestingly, these synergistic effects between Dronc and LIMK1 depend on Myo1D. Therefore, F-actin remodeling plays an important role mediating caspase-driven ROS production and JNK activation in the process of AiP.

ZmRop1 participates in maize defense response to the damage of Spodoptera frugiperda larvae through mediating ROS and soluble phenol production.

As plant-specific molecular switches, Rho-like GTPases (Rops) are vital for plant survival in response to biotic and abiotic stresses. However, their roles in plant defense response to phytophagous insect's damage are largely unknown. In this study, the expression levels of nine maize RAC family genes were analyzed after fall armyworm (FAW) larvae infestation. Among the analyzed genes, ZmRop1 was specifically and highly expressed, and its role in maize response to FAW larvae damage was studied. The results showed that upon FAW larvae infestation, salicylic acid and methyl jasmonate treatment ZmRop1 gene transcripts were all down-regulated. However, upon mechanical injury, the expression level of ZmRop1 was up-regulated. Overexpression of ZmRop1 gene in maize plants could improve maize plant resistance to FAW larvae damage. Conversely, silencing of ZmRop1 increased maize plant susceptibility to FAW larvae damage. The analysis of the potential anti-herbivore metabolites, showed that ZmRop1 promoted the enzyme activities of catalase, peroxidase and the expression levels of ZmCAT, ZmPOD, ZmRBOHA and ZmRBOHB, thereby enhancing the reactive oxygen species (ROS) production, including the content of O2- and H2O2. In addition, overexpression or silencing of ZmRop1 could have influence on the content of the total soluble phenol through mediating the activity of polyphenol oxidase. In summary, the results illuminated our understanding of how ZmRop1 participate in maize defense response to FAW larvae damage as a positive regulator through mediating ROS production and can be used as a reference for the green prevention and control of FAW larvae.

The Atrophic Effect of 1,25(OH)2 Vitamin D3 (Calcitriol) on C2C12 Myotubes Depends on Oxidative Stress.

Dysfunctional mitochondrial metabolism has been linked to skeletal muscle loss in several physio-pathological states. Although it has been reported that vitamin D (VD) supports cellular redox homeostasis by maintaining normal mitochondrial functions, and VD deficiency often occurs in conditions associated with skeletal muscle loss, the efficacy of VD supplementation to overcome muscle wasting is debated. Investigations on the direct effects of VD metabolites on skeletal muscle using C2C12 myotubes have revealed an unexpected pro-atrophic activity of calcitriol (1,25VD), while its upstream metabolites cholecalciferol (VD3) and calcidiol (25VD) have anti-atrophic effects. Here, we investigated if the atrophic effects of 1,25VD on myotubes depend on its activity on mitochondrial metabolism. The impact of 1,25VD and its upstream metabolites VD3 and 25VD on mitochondria dynamics and the activity of C2C12 myotubes was evaluated by measuring mitochondrial content, architecture, metabolism, and reactive oxygen species (ROS) production. We found that 1,25VD induces atrophy through protein kinase C (PKC)-mediated ROS production, mainly of extramitochondrial origin. Consistent with this, cotreatment with the antioxidant N-acetylcysteine (NAC), but not with the mitochondria-specific antioxidant mitoTEMPO, was sufficient to blunt the atrophic activity of 1,25VD. In contrast, VD3 and 25VD have antioxidant properties, suggesting that the efficacy of VD supplementation might result from the balance between atrophic pro-oxidant (1,25VD) and protective antioxidant (VD3 and 25VD) metabolites.

DRD4 (Dopamine D4 Receptor) Mitigate Abdominal Aortic Aneurysm via Decreasing P38 MAPK (mitogen-activated protein kinase)/NOX4 (NADPH Oxidase 4) Axis-Associated Oxidative Stress.

[Figure: see text].

CD36 promotes NLRP3 inflammasome activation via the mtROS pathway in renal tubular epithelial cells of diabetic kidneys

Tubulointerstitial inflammation plays a key role in the pathogenesis of diabetic nephropathy (DN). Interleukin-1β (IL-1β) is the key proinflammatory cytokine associated with tubulointerstitial inflammation. The NLRP3 inflammasome regulates IL-1β activation and secretion. Reactive oxygen species (ROS) represents the main mediator of NLRP3 inflammasome activation. We previously reported that CD36, a class B scavenger receptor, mediates ROS production in DN. Here, we determined whether CD36 is involved in NLRP3 inflammasome activation and explored the underlying mechanisms. We observed that high glucose induced-NLRP3 inflammasome activation mediate IL-1β secretion, caspase-1 activation, and apoptosis in HK-2 cells. In addition, the levels of CD36, NLRP3, and IL-1β expression (protein and mRNA) were all significantly increased under high glucose conditions. CD36 knockdown resulted in decreased NLRP3 activation and IL-1β secretion. CD36 knockdown or the addition of MitoTempo significantly inhibited ROS production in HK-2 cells. CD36 overexpression enhanced NLRP3 activation, which was reduced by MitoTempo. High glucose levels induced a change in the metabolism of HK-2 cells from fatty acid oxidation (FAO) to glycolysis, which promoted mitochondrial ROS (mtROS) production after 72 h. CD36 knockdown increased the level of AMP-activated protein kinase (AMPK) activity and mitochondrial FAO, which was accompanied by the inhibition of NLRP3 and IL-1β. The in vivo experimental results indicate that an inhibition of CD36 could protect diabetic db/db mice from tubulointerstitial inflammation and tubular epithelial cell apoptosis. CD36 mediates mtROS production and NLRP3 inflammasome activation in db/db mice. CD36 inhibition upregulated the level of FAO-related enzymes and AMPK activity in db/db mice. These results suggest that NLRP3 inflammasome activation is mediated by CD36 in renal tubular epithelial cells in DN, which suppresses mitochondrial FAO and stimulates mtROS production.

Consumption of the Fish Oil High-Fat Diet Uncouples Obesity and Mammary Tumor Growth through Induction of Reactive Oxygen Species in Protumor Macrophages.

Obesity is associated with increased risk of many types of cancer and can be induced by various high-fat diets (HFD) from different fat sources. It remains unknown whether fatty acid composition in different HFD influences obesity-associated tumor development. Here we report that consumption of either a cocoa butter or fish oil HFD induced similar obesity in mouse models. While obesity induced by the cocoa butter HFD was associated with accelerated mammary tumor growth, consumption of the fish oil HFD uncoupled obesity from increased mammary tumor growth and exhibited a decrease in protumor macrophages. Compared with fatty acid (FA) components in both HFDs, n-3 FA rich in the fish oil HFD induced significant production of reactive oxygen species (ROS) and macrophage death. Moreover, A-FABP expression in the protumor macrophages facilitated intracellular transportation of n-3 FA and oxidation of mitochondrial FA. A-FABP deficiency diminished n-3 FA-mediated ROS production and macrophage death in vitro and in vivo. Together, our results demonstrate a novel mechanism by which n-3 FA induce ROS-mediated protumor macrophage death in an A-FABP-dependent manner. SIGNIFICANCE: This study provides mechanistic insight into dietary supplementation with fish oil for breast cancer prevention and advances a new concept that not all HFDs leading to obesity are tumorigenic. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/12/2564/F1.large.jpg.

P0068APABETALONE, A CLINICAL STAGE BET INHIBITOR, REDUCES THE PRO-INFLAMMATORY RESPONSE IN PBMCS FROM FABRY DISEASE PATIENTS ON ERT THERAPY

Abstract Background and Aims Fabry disease (FD) is a rare X-linked recessive genetic disorder caused by mutations in the α-galactosidase A gene. Absence or defects of this lysosomal enzyme lead to globotriaosylceramide (Gb3) accumulation. Gb3 disrupts basic cellular metabolic processes, inducing oxidative stress and promoting inflammation. Systemically, Gb3 accumulation leads to organ damage throughout the body with an increase in renal, cardiac, cerebrovascular and skin complications. Enzyme replacement therapy (ERT) is current standard of care. This therapy may reduce Gb3 deposits but the impact on cardiac and kidney function is unclear based on the limited studies. Further, ERT is less effective in the late phases of FD, partially due to the development of uncontrolled inflammation and fibrosis. Apabetalone is a clinical stage orally administered bromo and extra terminal domain inhibitor (BETi) that has beneficial effects on vascular inflammation and vascular calcification through an epigenetic pathway. Based on its mechanism of action and impact on multiple disease drivers in both preclinical and clinical studies, apabetalone has therapeutic potential to modulate inflammation in FD patients on ERT therapy. Method PBMCs and neutrophils were isolated from fresh blood of FD patients ± ERT therapy using density gradient centrifugation. The baseline immune status of PBMCs was compared in naïve FD patients (without ERT, n=3) and ERT treated patients (n=8) via flow cytometry. PBMCs or neutrophils from ERT patients were treated ex vivo with 5µM or 20µM apabetalone ± LPS stimulation. Expression of key pro-inflammatory mediators was analyzed with real time quantitative PCR. The production of reactive oxygen species (ROS) in neutrophils was assessed by flow cytometry. Results The PBMC inflammatory profile in baseline showed monocytes from ERT patients produced lower amounts of pro-inflammatory mediators (TNFα and IL6) than naïve patients (∼60%, trending p≤0.06 and p≤0.08 respectively). However, surface abundance of CCR2, a chemokine receptor for the MCP1 chemokine that promotes monocyte recruitment to local inflamed tissue during inflammatory process, was ∼2-fold greater on monocytes from ERT subjects than the naïve controls (trending p≤0.06). Gene expression of MCP1 was approximately 5-fold higher in PBMC from ERT patients versus naïve controls (p=0.01). In response to ex vivo LPS stimulation, PBMCs from FD patients on ERT showed a robust induction of pro-inflammatory responses with increases in gene expression of MCP1, IL12B, TNFA and IL6. Ex vivo treatment with apabetalone (5µM) blocked the LPS-induction of MCP1 gene expression (∼90%, p<0.0001), and suppressed the upregulation of IL12B, TNFA and IL6 by 85%, 27% and 17%, respectively (p<0.0001,p=0.04,p=0.004). In response to LPS stimulation, neutrophils induced reactive oxygen species (ROS), an indicator of oxidative damage caused by intracellular Gb3 deposit in FD patients, by ∼6 fold. Apabetalone treatment reduced this induction by 32% and 62% at 5µM and 20 µM, respectively (p=0.01, p=0.0007). Conclusion Monocytes from FD patients on ERT therapy display an enhanced expression of the CCR2-MCP-1 axis as compared to naïve controls, indicating the potential for immune cell tissue infiltration and local inflammation. Apabetalone counters this increase by preventing MCP1 gene transcription. Apabetalone also reduces PBMC mediated pro-inflammatory gene transcription (TNFA, IL12B, IL6) and neutrophil mediated ROS production in response to LPS stimulation. Therefore, apabetalone treatment may reduce pathological inflammation in FD patients and thus complement ERT to optimize patient outcomes, which will be tested further with warranted clinical studies.

Racial Differences in p66shc Activity in Activated Endothelial Cells

BackgroundOxidative stress plays a pivotal role in the development of endothelial dysfunction and the pathophysiology of atherosclerosis. P66shc is an adaptor protein that acts as a redox‐sensitive enzyme involved in mitochondrial reactive oxygen species (ROS) production. Upon endothelial cell activation, protein kinase C‐β2 (PKC‐β2) mediates Ser36 phosphorylation of p66shc, thereby allowing p66shc translocation from the cytosol to the mitochondria. Furthermore, we have shown that African American (AA) endothelial cells display higher levels of inflammation and oxidative stress markers compared to Caucasian American (CA) endothelial cells. Therefore, the purpose of this study was to investigate racial differences in p66shc activity in response to tumor necrosis factor‐α (TNF‐α)‐induced endothelial cell activation.MethodsHuman umbilical vein endothelial cells (HUVECs) from three AA donors and three CA donors were cultured and incubated with TNF‐α (10ng/ml, 4hr) to assess racial differences in p66shc, PKC‐β2 and endothelial nitric oxide synthase (eNOS) protein levels and bioactivity.ResultsEnzymatically active eNOS (p‐eNOSS1177) was significantly lower in AA HUVECs under both basal and TNF‐α incubation conditions (p= 0.01, Fig. 1A). Additionally, p‐Ser36 p66shc/total p66shc was significantly lower in AA HUVECs compared to CA HUVECs (p= 0.04, Fig. 3C). However, there was no group or treatment effect on any on the other variables measured.Conclusionp66shc represents an important molecular pathway that mediates ROS production and plays an integral role in the development of cardiovascular disease. According to our results, racial differences in endothelial function cannot be explained by p66shc. However, future research should focus on assessing cytosolic vs. mitochondrial p66shc levels in AA endothelial cells compared to CA.eNOS protein expression and bioactivity in CA and AA HUVECs after 4 hours of incubation with TNF‐α (10ng/ml).A) Densiometric quantification and representative Western blot of p‐eNOS. B) Densiometric quantification and representative Western blot of eNOS. C) p‐eNOS bioactivity. (* p= .01 vs. CA HUVECs, n= 4 independent experiments. Data are shown as mean ± SEM).Figure 1p66shc protein expression and bioactivity in CA and AA HUVECs after 4 hours of incubation with TNF‐α (10ng/ml).A) Densiometric quantification and representative Western blot of p‐p66shc. B) Densiometric quantification and representative Western blot of p66shc. C) p‐p66shc bioactivity. (* p= .04 vs. CA HUVECs, n= 4 independent experiments. Data are shown as mean ± SEM).Figure 2

IL‐6 Increases ROS via Rac1, Leading to Mineralocorticoid Receptor and ENaC Activation

HTN is an inflammatory disease characterized by excessive sodium (Na+) reabsorption and increased pro‐inflammatory cytokines, such as interleukin 6 (IL‐6). Aldosterone (Aldo) is the primary ligand for the mineralocorticoid receptor (MR), which is expressed in the aldosterone‐sensitive portion of the distal nephron (ASDN). Although studies show benefits of MR inhibition in HTN, Aldo levels are not always increased, implying an alternate MR activation pathway. The epithelial sodium channel (ENaC) is in both the late distal convoluted tubule (DCT2) and cortical collecting duct (CCD), and is a major Na+ transport protein. We have shown that IL‐6 activates the MR in vitro, and that pharmacological Rac inhibition reduces mineralocorticoid response element (MRE) transcription. We have also shown that IL‐6 increases reactive oxygen species (ROS). Therefore, we hypothesized that IL‐6 activates the MR through increases in ROS generation and Rac1 activity, leading to increased ENaC activity.Since our data show that IL‐6 induces nuclear MR translocation, we tested if Rac1 affects IL‐6 mediated MR translocation. We co‐transfected MR‐eGFP tagged constructs and Rac1 expression vectors into murine distal convoluted tubule cells (mDCT15). We found IL‐6 mediated MR translocation decreased after Rac1 inhibition, or inhibition of MR activation; therefore, we investigated the role of Rac1 in IL‐6 mediated ENaC activity in the CCD and DCT2. We measured transepithelial voltage and resistance using a voltohmmeter, and calculated current in both mDCT15 and cortical collecting duct cells (mpkCCD) (n=4–15/group). Cells were transfected with vectors containing wild‐type Rac1 (WT), Rac1 dominant‐negative (DN Rac1) or constitutively active Rac1 (CA) and then treated with vehicle, Aldo [100nM], or IL‐6 [100ng/mL]. Data are shown as a fold change of current before and after treatments (1hr). We observed a significant increase in amiloride‐sensitive current in both mDCT2 and mpkCCD cells following both Aldo and IL‐6 treatments. Interestingly, the current was higher in the DCT2 than in the CCD (10.04 vs.1.98‐fold change/baseline). We also found that DN Rac1 completely inhibited IL‐6‐induced Na+ current (−1.16‐fold change/baseline) and that IL‐6 did not further increase current in cells with Rac1 CA (See Figure 1). Our data are the first to investigate ENaC activity in DCT2. Additionally, we show that IL‐6 is a strong stimulus for ENaC activity in both the DCT2 and CCD.Since IL‐6 increases ROS production in the DCT2, we investigated the role of Rac1 in this mechanism. mDCT15 cells were transfected with Rac1 inhibition and treated with IL6 [100ng/mL]. Cells were stained with dihydroethidium (DHE, ROS stain) and with confocal microscopy, we visualized ROS production. Our preliminary data show that Rac1 knockdown inhibits IL‐6 mediated ROS production (832.5±18 vs. 1073±29 mean DHE fluorescence, p=0.001).Together, our data show that IL‐6 may play an important role in MR activation, coupled with Rac1 and ROS‐generation in the distal nephron. Our data reveal a potential mechanism of MR activation during HTN, without increased Aldo levels.Support or Funding InformationDK115660, ASN Gottschalk Award to BMW; DK110409 to DCERac1 Inhibition Reduced IL‐6‐Mediated ENaC Current in mDCT15 cell line.Data are shown as fold‐changes in current from baseline (n=4–15, *p<0.05 ANOVA, Kruskal‐Wallis).Figure 1

Reactive oxygen species production by BP-1,6-quinone and its effects on the endothelial dysfunction: Involvement of the mitochondria

Strong epidemiological evidence supports the association between increased air pollution and the risk of developing atherosclerotic cardiovascular diseases (CVDs). However, the mechanism remains unclear. As an environmental air pollutant and benzo-a-pyrene (BP) metabolite, BP-1,6-quinone (BP-1,6-Q) is present in the particulate phase of air pollution. This study was undertaken to examine the redox activity of BP-1,6-Q and mechanisms associated with it using EA.hy926 endothelial cells. BP-1,6-Q at 0.01–1 μM significantly stimulated the production of reactive oxygen species (ROS)·in intact cells and isolated mitochondria. Furthermore, BP-1,6-Q-induced ROS was altered by mitochondrial electron transport chain (METC) inhibitors of complex I (rotenone) and complex III (antimycin A), denoting the involvement of mitochondrial electron transport chain (METC) in BP-1,6-Q mediated ROS production. In METC deficient cells, interestingly, BP-1,6-Q-mediated ROS production was enhanced, suggesting that overproduction of ROS by BP-1,6-Q is not only produced from mitochondria but can also be from the cell outside of mitochondria (extramitochondrial). BP-1,6-Q also triggered endothelial-monocyte interaction and stimulated expression of vascular adhesion molecule-1 (VCAM-1). In conclusion, these results demonstrate that BP-1,6-Q can generate ROS within both mitochondria and outside of mitochondria, resulting in stimulation of adhesion of monocytes to endothelial cells, a key event in the pathogenesis of atherosclerosis.