Increased Nox4 Expression Research Articles

Abstract 10: Adrenal Zona Glomerulosa Long Form Leptin Receptor (LepRb) Protects from Leptin-Mediated Vascular Disorders in Female Mice

Introduction: Previous work from our group demonstrated that leptin is a major contributor to obesity-associated cardiovascular disease, in females, by acting on the adrenal gland to promote aldosterone production. Hypothesis: This leptin-mediated aldosterone secretion is dependent on the presence of the long form leptin receptor (LepRb) in adrenal aldosterone synthase (AS) expressing cells. Methods: We generated AS LepRb deficient mice, ASKO and WT control (ASWT), by crossing LepRb flox/flox mice with AS-Cre mice to investigate the role of LepRb in AS expressing cells in leptin-mediated cardiovascular alteration in females and also generated db/AS + mice that have LepRb only in AS expressing cells by crossing LepR TBflox/flox (db/db) with AS-Cre mice to compare their phenotype. Results: Under baseline conditions, ASKO female mice exhibited no alteration in adrenal AS, plasma aldosterone, systolic blood pressure, and aortic endothelium-dependent relaxation. To mimic obese conditions, we submitted ASWT and ASKO to chronic leptin infusion for a week. Leptin increased adrenal AS expression, aldosterone levels, and blood pressure and impaired endothelial function, which were further elevated in ASKO compared to ASWT, suggesting protective effects of AS LeRb. Both mineralocorticoid receptor (MR) antagonist. eplerenone, or AS antagonist, fadrazole, rescued endothelial function in ASWT and ASKO mice treated with leptin, indicating that leptin-mediated endothelial dysfunction is still aldosterone-dependent in ASKO mice. Restoration of LepRb in AS expressing cells in db/db decreased adrenal AS and plasma aldosterone and improved endothelial function further indicating that AS LepRb blunts leptin-mediated aldosterone production. On a molecular level, leptin treatment elevated aorta NOX1 expression in ASWT, which was further increased in ASKO. Aldosterone treatment increased NOX1 expression in cultured microvascular endothelial cells. Incubation of aortic rings with a selective NOX1 inhibitor restored endothelial function in both ASWT and ASKO treated with leptin, suggesting leptin-mediated aldosterone increased NOX1 expression in the aorta, which causes endothelial dysfunction. Conclusion: These data indicate that AS LepRb protects from leptin-mediated aldosterone production and vascular dysfunction by mitigating the effects of leptin on aldosterone production and suggests that other isoforms of LepR are involved in stimulating aldosterone production.

Rolipram impacts on redox homeostasis and cellular signaling in an experimental model of abdominal aortic aneurysm

IntroductionCyclic nucleotide phosphodiesterases (PDEs) of the PDE4 subfamily are responsible for the hydrolysis and subcellular compartmentalization of cAMP, a second messenger that modulates vascular functionality. We had shown that PDE4B is induced in abdominal aortic aneurysms (AAA) and that PDE4 inhibition by rolipram limits experimental aneurysms. In this study we have delved into the mechanisms underlying the beneficial effect of rolipram on AAA. MethodsAAA were induced in ApoE−/− mice by angiotensin II (Ang II) infusion. Aneurysm formation was evaluated by ultrasonography. The expression of enzymes involved in redox homeostasis was analyzed by real-time RT-PCR and the activation of signaling pathways by Western blot. ResultsInduction of PDE4B in human AAA has been confirmed in a second cohort of patients. In Ang II-infused ApoE−/− mice, rolipram increased the percentage of animals free of aneurysms without affecting the percentage of aortic ruptures. Quantitative analyses determined that this drug significantly attenuated aortic collagen deposition. Additionally, rolipram reduced the increased Nox2 expression triggered by Ang II, exacerbated Sod1 induction, and normalized Sod3 expression. Likewise, PDE4 inhibition decreased the activation of both ERK1/2 and the canonical Wnt pathway, while AKT activity was not altered. ConclusionsThe inhibition of PDE4 activity modulates the expression of enzymes involved in redox homeostasis and affects cell signaling pathways involved in the development of AAA.

Investigation of the Effects of a Novel NOX2 Inhibitor, GLX7013170, against Glutamate Excitotoxicity and Diabetes Insults in the Retina.

Glutamate excitotoxicity and oxidative stress represent two major pathological mechanisms implicated in retinal disorders. In Diabetic Retinopathy (DR), oxidative stress is correlated to NADPH oxidase (NOX), a major source of Reactive Oxygen Species (ROS), and glutamate metabolism impairments. This study investigated the role of NOX2 and the novel NOX2 inhibitor, GLX7013170, in two models of a) retinal AMPA excitotoxicity [AMPA+GLX7013170 (10-4 M, intravitreally)] and b) early-stage DR paradigm (ESDR), GLX7013170: 14-day therapeutic treatment (topically, 20 μL/eye, 10 mg/mL (300 × 10-4 M), once daily) post-streptozotocin (STZ)-induced DR. Immunohistochemical studies for neuronal markers, nitrotyrosine, micro/macroglia, and real-time PCR, Western blot, and glutamate colorimetric assays were conducted. Diabetes increased NOX2 expression in the retina. NOX2 inhibition limited the loss of NOS-positive amacrine cells and the overactivation of micro/macroglia in both models. In the diabetic retina, GLX7013170 had no effect on retinal ganglion cell axons, but reduced oxidative damage, increased Bcl-2, reduced glutamate levels, and partially restored excitatory amino acid transporter (EAAT1) expression. These results suggest that NOX2 in diabetes is part of the triad, oxidative stress, NOX, and glutamate excitotoxicity, key players in the induction of DR. GLX7013170 is efficacious as a neuroprotective/anti-inflammatory agent and a potential therapeutic in retinal diseases, including ESDR.

Particulate matter stimulates the NADPH oxidase system via AhR-mediated epigenetic modifications

Stimulation of human keratinocytes with particulate matter 2.5 (PM2.5) elicits complex signaling events, including a rise in the generation of reactive oxygen species (ROS). However, the mechanisms underlying PM2.5-induced ROS production remain unknown. Here, we show that PM2.5-induced ROS production in human keratinocytes is mediated via the NADPH oxidase (NOXs) system and the Ca2+ signaling pathway. PM2.5 treatment increased the expression of NOX1, NOX4, and a calcium-sensitive NOX, dual oxidase 1 (DUOX1), in human epidermal keratinocyte cell line. PM2.5 bound to aryl hydrocarbon receptor (AhR), and this complex bound to promoter regions of NOX1 and DUOX1, suggesting that AhR acted as a transcription factor of NOX1 and DUOX1. PM2.5 increased the transcription of DUOX1 via epigenetic modification. Moreover, a link between DNA demethylase and histone methyltransferase with the promoter regions of DUOX1 led to an elevation in the expression of DUOX1 mRNA. Interestingly, PM2.5 increased NOX4 expression and promoted the interaction of NOX4 and Ca2+ channels within the cytoplasmic membrane or endoplasmic reticulum, leading to Ca2+ release. The increase in intracellular Ca2+ concentration activated DUOX1, responsible for ROS production. Our findings provide evidence for a PM2.5-mediated ROS-generating system network, in which increased NOX1, NOX4, and DUOX1 expression serves as a ROS signal through AhR and Ca2+ activation.

ATM deficiency differentially affects expression of proteins related to fatty acid oxidation and oxidative stress in a sex-specific manner in response to Western-type diet prior to and following myocardial infarction

AimsPublished work has shown that ataxia-telangiectasia mutated kinase (ATM) deficiency is associated with cardioprotective effects in Western-type diet (WD)-fed female mice. This study assessed the expression of proteins related to fatty acid oxidation (FAO) and oxidative stress in WD-fed male and female mouse hearts, and investigated if sex-specific cardioprotective effects in WD-fed female ATM-deficient mice are maintained following myocardial infarction (MI). Main methodsWild-type (WT) and ATM-deficient (hKO) mice (both sexes) were placed on WD for 14 weeks. Myocardial tissue from a subset of mice was used for western blot analyses, while another subset of WD-fed mice underwent MI. Heart function was analyzed by echocardiography prior to and 1 day post-MI. Key findingsCPT1B (mitochondrial FAO enzyme) expression was lower in male hKO-WD, while it was higher in female hKO-WD vs WT-WD. WD-mediated decrease in ACOX1 (peroxisomal FAO enzyme) expression was only observed in male WT-WD. PMP70 (transports fatty acyl-CoA across peroxisomal membrane) expression was lower in male hKO-WD vs WT-WD. Catalase (antioxidant enzyme) expression was higher, while Nox4 (pro-oxidant enzyme) expression was lower in female hKO-WD vs WT-WD. Heart function was better in female hKO-WD vs WT-WD. However, post-MI heart function was not significantly different among all MI groups. Post-MI, CPT1B and catalase expression was higher in male hKO-WD-MI vs WT-WD-MI, while Nox4 expression was higher in female hKO-WD-MI vs WT-WD-MI. SignificanceIncreased mitochondrial FAO and decreased oxidative stress contribute towards ATM deficiency-mediated cardioprotective effects in WD-fed female mice which are abolished post-MI with increased Nox4 expression.

Impacto del tratamiento con rolipram sobre la homeostasis rédox y la señalización celular en un modelo experimental de aneurisma de aorta abdominal

IntroductionCyclic nucleotide phosphodiesterases (PDEs) of the PDE4 subfamily are responsible for the hydrolysis and subcellular compartmentalization of cAMP, a second messenger that modulates vascular functionality. We had shown that PDE4B is induced in abdominal aortic aneurysms (AAA) and that PDE4 inhibition by rolipram limits experimental aneurysms. In this study we have delved into the mechanisms underlying the beneficial effect of rolipram on AAA. MethodsAAA were induced in ApoE-/− mice by angiotensin II (Ang II) infusion. Aneurysm formation was evaluated by ultrasonography. The expression of enzymes involved in rédox homeostasis was analyzed by real-time RT-PCR and the activation of signaling pathways by Western blot. ResultsInduction of PDE4B in human AAA has been confirmed in a second cohort of patients. In Ang II-infused ApoE-/− mice, rolipram increased the percentage of animals free of aneurysms without affecting the percentage of aortic ruptures. Quantitative analyses determined that this drug significantly attenuated aortic collagen deposition. Additionally, rolipram reduced the increased Nox2 expression triggered by Ang II, exacerbated Sod1 induction, and normalized Sod3 expression. Likewise, PDE4 inhibition decreased the activation of both ERK1/2 and the canonical Wnt pathway, while AKT activity was not altered. ConclusionsThe inhibition of PDE4 activity modulates the expression of enzymes involved in rédox homeostasis and affects cell signaling pathways involved in the development of AAA.

Abstract 025: Role Of The C-C Motif Chemokine Ligand 5 (CCL5) And Its Receptor, C-C Motif Chemokine Receptor 5 (CCR5) In The Genesis Of Aldosterone-induced Hypertension, Vascular Dysfunction, And End-organ Damage

Background: Hyperaldosteronism (HA) is the most common cause of endocrine hypertension. CCL5 is a pro-inflammatory chemokine acting primarily via C-C Motif Chemokine Receptor 5 (CCR5). We hypothesize that HA leads to hypertension, vascular injury, and renal demage via CCL5/CCR5 and Nox1-dependent mechanisms. Methods: 10-12-weeks-old male wild type (CCR5+/+) and CCR5 receptor knockout (CCR5-/-) mice were infused with aldosterone for 14 days (600ug/Kg/day, osmotic mini-pump). Endothelial function was studied in aortae and blood pressure was analyzed by radiotelemetry. Mouse endothelial cells (MEC) were used to examine the molecular mechanisms. Results: HA increased circulating CCL5 levels [Vehicle: 7.41±1.6 vs. HA: 13.2±1.4 (pg/mL)] and reduced acetylcholine (ACH)-induced vasodilation (% relaxation) in CCR5+/+, which was abolished by inhibiting Nox1 (NOXA1ds, 10 μM) [Vehicle: 91.2±2.1 vs. HA: 42.7±3.9 vs. HA+NOXA1ds: 89.7±1.9]. To understand if CCL5 induces endothelial dysfunction via CCR5 and Nox1, aortae were incubated with CCL5 (100 ng/mL for 24 h) with vehicle or CCR5 antagonist (Maraviroc, 40 μM) or NOXA1ds and endothelial function and Nox1 expression were analyzed. CCL5 treatment induced endothelial dysfunction [ACH response, Vehicle: 90.1±1.9 vs. CCL5: 31.9±2.7] and increased Nox1 expression, which were blunted by Maraviroc or NOXA1ds, indicating that CCL5 affects endothelial function dependent on CCR5 and Nox1. We found that CCR5-/- mice are protected from HA-induced endothelial dysfunction, hypertension [mean arterial pressure (mmHg) - CCR5+/+: 154.8±3.7 vs. CCR5-/-: 104.5±2.9], and renal damage (analyzed by NGAL, Synaptopodin, and Colagen III gene expressions). On mechanistic levels, we found that CCL5 decreases nitric oxide production, leads to NF-κB activation, induces oxidative stress (elevated superoxide anion and H2O2) and ICAM and VCAM expressions in MEC, which were associated with higher macrophage adhesion, such effects were prevented by Maraviroc. Conclusion: These findings reveal a novel signaling pathway in HA-associated vascular and renal injuries and hypertension, CCL5 activates CCR5 stimulating NF-κB and Nox1 activities, and place CCR5 as a pharmacological target for cardiovascular outcomes in HA.

Obesity Contributes to Transformation of Myometrial Stem-Cell Niche to Leiomyoma via Inducing Oxidative Stress, DNA Damage, Proliferation, and Extracellular Matrix Deposition.

Leiomyomas (fibroids) are monoclonal tumors in which myometrial stem cells (MSCs) turn tumorigenic after mutation, abnormal methylation, or aberrant signaling. Several factors contribute to metabolic dysfunction in obesity, including abnormal cellular proliferation, oxidative stress, and DNA damage. The present study aims to determine how adipocytes and adipocyte-secreted factors affect changes in MSCs in a manner that promotes the growth of uterine leiomyomas. Myometrial stem cells were isolated from the uteri of patients by fluorescence-activated cell sorting (FACS) using CD44/Stro1 antibodies. Enzyme-linked immunosorbent assay (ELISA), Western blot, and immunocytochemistry assays were performed on human adipocytes (SW872) co-cultured with MSCs and treated with leptin or adiponectin to examine the effects of proliferation, extracellular matrix (ECM) deposition, oxidative damage, and DNA damage. Co-culture with SW872 increased MSC proliferation compared to MSC culture alone, according to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) results. The expressions of PCNA and COL1A increased significantly with SW872 co-culture. In addition, the expression of these markers was increased after leptin treatment and decreased after adiponectin treatment in MSCs. The Wnt/β-catenin and TGF-β/SMAD signaling pathways promote proliferation and ECM deposition in uterine leiomyomas. The expression of Wnt4, β-catenin, TGFβ3, and pSMAD2/3 of MSCs was increased when co-cultured with adipocytes. We found that the co-culture of MSCs with adipocytes resulted in increased NOX4 expression, reactive oxygen species production, and γ-H2AX expression. Leptin acts by binding to its receptor (LEP-R), leading to signal transduction, resulting in the transcription of genes involved in cellular proliferation, angiogenesis, and glycolysis. In MSCs, co-culture with adipocytes increased the expression of LEP-R, pSTAT3/STAT3, and pERK1/2/ERK/12. Based on the above results, we suggest that obesity may mediate MSC initiation of tumorigenesis, resulting in leiomyomas.

Contemporary Antiretroviral Therapy Dysregulates Iron Transport and Augments Mitochondrial Dysfunction in HIV-Infected Human Microglia and Neural-Lineage Cells.

HIV-associated cognitive dysfunction during combination antiretroviral therapy (cART) involves mitochondrial dysfunction, but the impact of contemporary cART on chronic metabolic changes in the brain and in latent HIV infection is unclear. We interrogated mitochondrial function in a human microglia (hμglia) cell line harboring inducible HIV provirus and in SH-SY5Y cells after exposure to individual antiretroviral drugs or cART, using the MitoStress assay. cART-induced changes in protein expression, reactive oxygen species (ROS) production, mitochondrial DNA copy number, and cellular iron were also explored. Finally, we evaluated the ability of ROS scavengers or plasmid-mediated overexpression of the antioxidant iron-binding protein, Fth1, to reverse mitochondrial defects. Contemporary antiretroviral drugs, particularly bictegravir, depressed multiple facets of mitochondrial function by 20-30%, with the most pronounced effects in latently infected HIV+ hμglia and SH-SY5Y cells. Latently HIV-infected hμglia exhibited upregulated glycolysis. Increases in total and/or mitochondrial ROS, mitochondrial DNA copy number, and cellular iron accompanied mitochondrial defects in hμglia and SH-SY5Y cells. In SH-SY5Y cells, cART reduced mitochondrial iron-sulfur-cluster-containing supercomplex and subunit expression and increased Nox2 expression. Fth1 overexpression or pre-treatment with N-acetylcysteine prevented cART-induced mitochondrial dysfunction. Contemporary cART impairs mitochondrial bioenergetics in hμglia and SH-SY5Y cells, partly through cellular iron accumulation; some effects differ by HIV latency.

Aggravation of TGFβ1-Smad Pathway and Autoimmune Myocarditis by Fungicide (Tebuconazole) Exposure.

Myocarditis is an inflammatory cardiac disorder and the primary cause of heart failure in young adults. Its origins can be attributed to various factors, including bacterial or viral infections, exposure to toxins or drugs, endocrine disruptors (EDs), and autoimmune processes. Tebuconazole (TEB), which is a member of the triazole fungicide family, is utilized to safeguard agricultural crop plants against fungal pathogens. Although TEB poses serious threats to mammal health, the information about how it induces toxic effects through various pathways, particularly in autoimmune diseases, are still limited. Thus, the aim of this paper was to evaluate the effect of TEB exposure in autoimmune myocarditis (AM). To induce AM, rats were immunized with porcine cardiac myosin and exposed to TEB for 21 days. Thereafter, animals were sacrificed, and histological, biochemical, and molecular analyses were performed. TEB exposure increased heart weight, systolic blood pressure and heart rate already augmented by AM. Additionally, it significantly increased creatine phosphokinase heart (CK-MB), creatine phosphokinase (CPK), cardiac troponin T (cTnT), and cardiac troponin I (cTnI), as compared to the control. From the histological perspective, TEB exacerbates the histological damage induced by AM (necrosis, inflammation and cell infiltration) and increased fibrosis and collagen deposition. TEB exposure strongly increased pro-inflammatory cytokines and prooxidant levels (O2-, H2O2, NO2-, lipid peroxidation) and reduced antioxidant enzyme levels, which were already dysregulated by AM. Additionally, TEB increased NOX-4 expression and the TGFβ1-Smads pathway already activated by AM. Overall, our results showed that TEB exposure strongly aggravated the cardiotoxicity induced by AM.

Inhibition of NADPH Oxidase (NOX) 2 Mitigates Colitis in Mice with Impaired Macrophage AMPK Function.

Macrophage adenosine monophosphate-activated protein kinase (AMPK) limits the development of experimental colitis. AMPK activation inhibits NADPH oxidase (NOX) 2 expression, reactive oxygen species (ROS) generation, and pro-inflammatory cytokine secretion in macrophages during inflammation, while increased NOX2 expression is reported in experimental models of colitis and inflammatory bowel disease (IBD) patients. Although there are reductions in AMPK activity in IBD, it remains unclear whether targeted inhibition of NOX2 in the presence of defective AMPK can reduce the severity of colitis. Here, we investigate whether the inhibition of NOX2 ameliorates colitis in mice independent of AMPK activation. Our study identified that VAS2870 (a pan-Nox inhibitor) alleviated dextran sodium sulfate (DSS)-induced colitis in macrophage-specific AMPKβ1-deficient (AMPKβ1LysM) mice. Additionally, VAS2870 blocked LPS-induced TLR-4 and NOX2 expression, ROS production, nuclear translocation of NF-κB, and pro-inflammatory cytokine secretion in bone marrow-derived macrophages (BMDMs) from AMPKβ1LysM mice, whereas sodium salicylate (SS; AMPK β1 activator) did not. Both VAS2870 and SS inhibited LPS-induced NOX2 expression, ROS production, and pro-inflammatory cytokine secretions in bone marrow-derived macrophages (BMDMs) from wildtype (AMPKβ1fl/fl) mice but only VAS2870 inhibited these effects of LPSs in AMPKβ1LysM BMDMs. Furthermore, in macrophage cells (RAW 264.7), both SS and VAS2870 inhibited ROS production and the secretion of pro-inflammatory cytokines and reversed the impaired autophagy induced by LPSs. These data suggest that inhibiting NOX2 can reduce inflammation independent of AMPK in colitis.

Supraphysiological levels of testosterone induces endothelial injury via increasing generation of reactive oxygen species and ERK1/2 activation

Introduction: High levels of testosterone (Testo) is associated with higher cardiovascular risk by positively modulate the generation of reactive oxygen species (ROS) and inflammatory response. Excessive ROS generation by NADPH oxidases (NOX, major source of ROS in the vasculature) leads to endothelial dysfunction, apoptosis, and inflammation. However, the intracellular mechanisms whereby supraphysiological levels of Testo promote cardiovascular damage are still ill defined. We sought to determine whether supraphysiological levels of Testo induces endothelial cell dysfunction via ROS generation and ERK1/2 activation. Methods: To study whether Testo present the same effects independent on endothelial cell type, we used Primary Human Mesenteric vascular Endothelial Cells (HMEC) and Human Umbilical Vein Endothelial Cells (HUVEC). Cells were stimulated with Testo (10 −7 M, for different time points) in the presence or absence of superoxide dismutase mimetic (Tempol, 10 -4 M, for 30 min). NOX activity, gene expression, and protein expression were determined pharmacological assays and immunoblotting, respectively. Results: Testo increased superoxide anion (O 2 − ) generation in HMEC and HUVEC at short (10 min) stimulation times, which was abolished by superoxide dismutase (SOD) mimetic [(% of control) Ctrl: 100.0 ± 0.0 n=5 vs Testo: 363.2 ± 19.77 n=6 vs. Tempol+Testo: 156.7 ± 12.12 n=6, *P<0.05]. Furthermore, long-term treatment (24 h) of endothelial cells with Testo increased Nox4 expression at gene (2.44-Fold change) and protein levels [(% of control) Ctrl: 100.0 ± 0.0 n=4 vs. Testo: 157.3 ± 4.49 n=4, *P<0.05]. Testo did not affect Nox1, Nox2, and Nox5 expressions. Furthermore, Testo increased ERK1/2 phosphorylation at 10 minutes and promoted endothelial cell inflammation at 8h, characterized by increased ICAM-1(6.49-Fold changes) and VCAM-1 [(1.42-Fold changes) gene expressions. To confirm that Testo-induced ROS production is leading to endothelial injury via ERK1/2, we treated endothelial cells with Tempol prior Testo treatment. Interestingly, superoxide dismutase mimetic prevented ERK1/2 phosphorylation induced by Testo [(% of control) Testo: 161.2 ± 11.39 n=5 vs. Tempol+Testo: 91.45 ± 9.61 n=5, *P<0.05]. Conclusion: These data indicate that supraphysiological levels of testosterone induce endothelial dysfunction via ROS generation and inflammatory response. Our data helps understanding the intracellular mechanisms that justify the cardiovascular damage caused by supraphysiological doses of testosterone and places antioxidant therapy as a possible pharmacological approach to treat patients with dysregulated testosterone levels. 2022/00103-3, São Paulo Research Foundation (FAPESP). NHLBI-R00 (R00HL14013903), AHA-CDA (CDA857268), Vascular Medicine Institute, the Hemophilia Center of Western Pennsylvania Vitalant, and Children's Hospital of Pittsburgh of the UPMC Health System. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Increased NOX2 expression in astrocytes leads to eNOS uncoupling through dihydrofolate reductase in endothelial cells after subarachnoid hemorrhage.

Endothelial nitric oxide synthase (eNOS) uncoupling plays a significant role in acute vasoconstriction during early brain injury (EBI) after subarachnoid hemorrhage (SAH). Astrocytes in the neurovascular unit extend their foot processes around endothelia. In our study, we tested the hypothesis that increased nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) expression in astrocytes after SAH leads to eNOS uncoupling. We utilized laser speckle contrast imaging for monitoring cortical blood flow changes in mice, nitric oxide (NO) kits to measure the level of NO, and a co-culture system to study the effect of astrocytes on endothelial cells. Moreover, the protein levels were assessed by Western blot and immunofluorescence staining. We used CCK-8 to measure the viability of astrocytes and endothelial cells, and we used the H2O2 kit to measure the H2O2 released from astrocytes. We used GSK2795039 as an inhibitor of NOX2, whereas lentivirus and adeno-associated virus were used for dihydrofolate reductase (DHFR) knockdown in vivo and in vitro. The expression of NOX2 and the release of H2O2 in astrocytes are increased, which was accompanied by a decrease in endothelial DHFR 12 h after SAH. Moreover, the eNOS monomer/dimer ratio increased, leading to a decrease in NO and acute cerebral ischemia. All of the above were significantly alleviated after the administration of GSK2795039. However, after knocking down DHFR both in vivo and in vitro, the protective effect of GSK2795039 was greatly reversed. The increased level of NOX2 in astrocytes contributes to decreased DHFR in endothelial cells, thus aggravating eNOS uncoupling, which is an essential mechanism underlying acute vasoconstriction after SAH.

Polydatin Ameliorates High Fructose-Induced Podocyte Oxidative Stress via Suppressing HIF-1α/NOX4 Pathway.

Long-term high fructose intake drives oxidative stress, causing glomerular podocyte injury. Polydatin, isolated from Chinese herbal medicine Polygonum cuspidatum, is used as an antioxidant agent that protects kidney function. However, it remains unclear how polydatin prevents oxidative stress-driven podocyte damage. In this study, polydatin attenuated high fructose-induced high expression of HIF-1α, inhibited NOX4-mediated stromal cell-derived factor-1α/C-X-C chemokine receptor type 4 (SDF-1α/CXCR4) axis activation, reduced reactive oxygen species (ROS) production in rat glomeruli and cultured podocytes. As a result, polydatin up-regulated nephrin and podocin, down-regulated transient receptor potential cation channel 6 (TRPC6) in these animal and cell models. Moreover, the data from HIF-1α siRNA transfection showed that high fructose increased NOX4 expression and aggravated SDF-1α/CXCR4 axis activation in an HIF-1α-dependent manner, whereas polydatin down-regulated HIF-1α to inhibit NOX4 and suppressed SDF-1α/CXCR4 axis activation, ameliorating high fructose-induced podocyte oxidative stress and injury. These findings demonstrated that high fructose-driven HIF-1α/NOX4 pathway controlled podocyte oxidative stress damage. Intervention of this disturbance by polydatin could help the development of the therapeutic strategy to combat podocyte damage associated with high fructose diet.

Impact of NOX4 Knockout by CRISPR/Cas9 on the MCF-7, HCA-7 and UM-RC-6 Cancer Cells.

The second most common cause of mortality is cancer. Increased NOX4 expression is linked to cancer development and metastasis. However, the significance of NOX4 in cell growth and assault, remains unclear. This study aimed to evaluate the effect of NOX4 knockouts in MCF7, UM-RC-6, HCA-7 cell lines. The NOX4 gene was knocked out in MCF7, UM-RC-6, and HCA-7 cell lines through using CRISPR Cas-9 genetic engineering techniques. After transfection, the CRISPR Cas-9 cassette, the T7 endonuclease I, qPCR, and western blotting assay detected the NOX4 knockouts. MTT and Annexin assessed the percentage of cell proliferation and apoptosis. Real-time PCR was used to measure the expression of pro- and anti-apoptotic genes. Occurrence of NOX4 gene knockout in the examined cell lines, was confirmed by q-PCR and Western blot (P<0.001). The NOX4-deleted cell lines with increased sub-G1 caused lowered cell proliferation and population at S / G2/ M phases. In Vitro, NOX4 silencing caused lowered expressions of anti-apoptosis genes BCL-2 and SURVIVIN(P<0.0001), leading to increased tendency of apoptosis in the cell lines (P<0.0001) of the apoptotic genes BAX, P53, FAS. Additionally, the MTT and Annexin results of the target gene NOX4 knockout inhibited proliferation, increased mortality rates (P<0.01), and increased apoptosis. The findings of this study indicate that using NOX4 as a target can have therapeutic value for creating potential treatments against breast, colorectal, and kidney cancers which shows a need for a deeper understanding of the biology of these cancers with direct clinical outcomes for developing novel treatment strategies.

Ultrafine Diesel Exhaust Particles Induce Apoptosis of Oligodendrocytes by Increasing Intracellular Reactive Oxygen Species through NADPH Oxidase Activation.

Diesel exhaust particles (DEPs) are a main contributor to air pollution. Ultrafine DEPs can cause neurodegenerative diseases by increasing intracellular reactive oxygen species (ROS). Compared with other cells in the brain, oligodendrocytes responsible for myelination are more susceptible to oxidative stress. However, the mechanisms underlying ROS generation in oligodendrocytes and the susceptibility of oligodendrocytes to ROS by ultrafine DEPs remain unclear. Herein, we examined the effects of excessive ROS generated by NOX2, an isoform of the NADPH oxidase family, after exposure to ultrafine DEPs (200 μg/mL) on the survival of two types of oligodendrocytes—oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes (mOLs)––isolated from the brain of neonatal rats. In addition, mice were exposed to ultrafine DEP suspension (20 μL, 0.4 mg/mL) via the nasal route for 1 week, after which the expression of NOX2 and cleaved caspase-3 was examined in the white matter of the cerebellum. Exposure to DEPs significantly increased NOX2 expression and ROS generation in OPCs and mOLs. OPCs and mOLs clearly exhibited viability reduction, and a significant change in p53, Bax, Bcl-2, and cleaved caspase-3 expression, after DEP exposure. In contrast, treatment with berberine (BBR), an NOX2 inhibitor, significantly mitigated these effects. In mice exposed to DEP, the presence of NOX2-positive and cleaved caspase-3-positive oligodendrocytes was demonstrated in the cerebellar white matter; NOX2 and cleaved caspase-3 expression in the cerebellum lysates was significantly increased. BBR treatment returned expression of these proteins to control levels. These results demonstrate that the susceptibility of OPCs and mOLs to ultrafine DEPs is, at least in part, caused by excessive ROS produced by NOX2 and the sequential changes in the expression of p53, Bax, Bcl-2, and cleaved caspase-3. Overall, NOX2 inhibitor enhances the survival of two types of oligodendrocytes.

Abstract P173: The Impact Of Cigarette Smoking-oral Contraceptive Combination On Cardiac Remodeling In Premenopausal Female Mice

Introduction: Tobacco smoke is a major risk factor for coronary artery diseases (CAD) and chronic tobacco smoking (CS) increase the risk of CAD by 2 to 4 folds. CS effect on cardiac homeostasis has never been evaluated in premenopausal females taking oral contraceptive (OC). This study investigates CS effect on cardiac remodeling in female mice in the presence or absence of Ethinyl Estradiol (EE). Methods: Blood pressure and echocardiography were recorded for female C57BL/6J mice on EE, EE-CS, vehicle, and vehicle-CS at baseline and after 8-weeks of exposure. Cardiac inflammatory cytokines and oxidative stress markers were evaluated by real time PCR and western blots. Interstitial collagen deposition was assessed by Masson Trichrome staining. Results: Eight weeks of EE-CS treated mice showed no effect on BP but significant adverse structural and functional cardiac effects, represented by increased systolic (0.981 ± 0.035 N=11 (p&lt;0.0332)) and diastolic (1.450 ± 0.037 N=11 (p&lt;0.0002)) areas when compared to the vehicle and EE groups, along with increased systolic (1.981 ± 0.109 N=11 (p&lt;0.033)) and diastolic ( 3.911 ± 0.159 N=11 (p&lt;0.033)) volumes when compared to all groups. Functional changes were accompanied with a decreased fractional shortening (9.194 ± 1.006 N=11 (p&lt;0.033)) when compared to vehicle and EE groups with no changes in ejection fraction parameter. Moreover EE-CS treatment significantly increased NOX-4 expression (2.297 ± 0.643 N=5 (p&lt;0.033)) when compared to EE treatment alone, along with an increased pro-inflammatory profile including IL-1β (0.750 ±0.171N=5 (p&lt;0.033)) and IL-4 (4.110 ± 0.623 N= 5 (p&lt;0.002)) when compared to all groups, and IL-6 (3.045±0.910 N=5 (p&lt;0.002)) and IL-13 (2.686 ± 0.648 N=5 (p&lt;0.033)) when compared to vehicle-CS group. Morphologically, EE-CS exhibited a significant increase in interstitial fibrosis (1.186± 0.020 N=5, 3.2285 ± 0.683 N=5, (p&lt;0.033)) when compared to the vehicle-CS group. Conclusion: This study provides clear evidence that CS-exposed premenopausal female mice on OC regimen exhibited significant adverse cardiac events that could be classified under cardiac dysfunction with preserved ejection fraction. Additional experiments are warranted to further elaborate on these findings.

DPP4 inhibitor reduces portal hypertension in cirrhotic rats by normalizing arterial hypocontractility

AimsDipeptidyl peptidase-4 inhibitor (DPP4i), a new antidiabetic agent, is reported to affect the progression of chronic liver diseases. The study aims to investigate the effects of DPP4i on contractile response, splanchnic hemodynamics, and portal pressure in cirrhotic rats. Materials and methodsA rat model of carbon tetrachloride-induced cirrhosis was used in this study. Sixteen rats with cirrhosis were treated with DDP4i sitagliptin for 5 consecutive days. Portal and systemic pressures and portal blood flow were measured. Mesenteric arterioles were isolated, and concentration-response curves to norepinephrine (NE) were evaluated. The expression of NADPH oxidase (Nox)1, Nox2, Nox4, and soluble epoxide hydrolase (sEH) were detected. Reactive oxygen species (ROS) and epoxyeicosatrienoic acid (EET) levels in mesenteric arteries were also measured. Key findingsIn cirrhotic rats, sitagliptin significantly reduced portal blood flow and portal pressure without effects on systemic pressure and reversed the decreased response of mesenteric arterioles to NE in an endothelium-dependent manner. Sitagliptin suppressed the increased Nox4 expression and ROS production. In vitro studies showed that Nox4 inhibitor enhanced arteriolar response to NE and reduced hydrogen peroxide (H2O2) level in cirrhotic rats. Sitagliptin also reduced EET levels and increased sEH expression of mesenteric vessels. Pre-incubation with sEH inhibitor in vitro reversed sitagliptin-induced augmentation of response to NE in cirrhotic rats. SignificanceDPP4 inhibition by sitagliptin in vivo has beneficial effects on portal hypertension in cirrhotic rats through normalizing arterial hypocontractility. DDP4 inhibitor may be a novel strategy in the treatment of patients with cirrhosis and portal hypertension.

Hyperphosphatemia-Induced Oxidant/Antioxidant Imbalance Impairs Vascular Relaxation and Induces Inflammation and Fibrosis in Old Mice.

Aging impairs vascular function, but the mechanisms involved are unknown. The aim of this study was to analyze whether aging-related hyperphosphatemia is implied in this effect by elucidating the role of oxidative stress. C57BL6 mice that were aged 5 months (young) and 24 months (old), receiving a standard (0.6%) or low-phosphate (0.2%) diet, were used. Isolated mesenteric arteries from old mice showed diminished endothelium-dependent vascular relaxation by the down-regulation of NOS3 expression, increased inflammation and increased fibrosis in isolated aortas, compared to those isolated from young mice. In parallel, increased Nox4 expression and reduced Nrf2, Sod2-Mn and Gpx1 were found in the aortas from old mice, resulting in oxidant/antioxidant imbalance. The low-phosphate diet improved vascular function and oxidant/antioxidant balance in old mice. Mechanisms were analyzed in endothelial (EC) and vascular smooth muscle cells (SMCs) treated with the phosphate donor ß-glycerophosphate (BGP). In EC, BGP increased Nox4 expression and ROS production, which reduced NOS3 expression via NFκB. BGP also increased inflammation in EC. In SMC, BGP increased Collagen I and fibronectin expression by priming ROS production and NFκB activity. In conclusion, hyperphosphatemia reduced endothelium-dependent vascular relaxation and increased inflammation and vascular fibrosis through an impairment of oxidant/antioxidant balance in old mice. A low-phosphate diet achieved improvements in the vascular function in old mice.

Carboxyl group-modified α-lactalbumin induces TNF-α-mediated apoptosis in leukemia and breast cancer cells through the NOX4/p38 MAPK/PP2A axis

To clarify the mechanism of semicarbazide-modified α-lactalbumin (SEM-LA)-mediated cytotoxicity, we investigated its effect on human U937 leukemia cells and MCF-7 breast cancer cells in the current study. SEM-LA induced apoptosis in U937 cells, which showed increased NOX4 expression, procaspase-8 degradation, and t-Bid production. FADD depletion inhibited SEM-LA-elicited caspase-8 activation, t-Bid production, and cell death, indicating that SEM-LA activated death receptor-mediated apoptosis in U937 cells. SEM-LA stimulated Ca2+-mediated Akt activation, which in turn increased Sp1- and p300-mediated NOX4 transcription. The upregulation of NOX4 expression promoted ROS-mediated p38 MAPK phosphorylation, leading to protein phosphatase 2A (PP2A)-regulated tristetraprolin (TTP) degradation. Remarkably, TTP downregulation increased the stability of TNF-α mRNA, resulting in the upregulation of TNF-α protein expression. Abolishment of Ca2+-NOX4-ROS axis-mediated p38 MAPK activation attenuated SEM-LA-induced TNF-α upregulation and protected U937 cells from SEM-LA-mediated cytotoxicity. The restoration of TTP expression alleviated the effect of TNF-α upregulation and cell death induced by SEM-LA. Altogether, the data in this study demonstrate that SEM-LA activates TNF-α-mediated apoptosis in U937 cells through the NOX4/p38 MAPK/PP2A axis. We think that a similar pathway can also explain the death of MCF-7 human breast cancer cells after SEM-LA treatment.