Nox4 Protein Research Articles

Abstract 676: Hypoxia Upregulates NADPH Oxidase 4-Mediated Hydrogen Peroxide Release by a HIF-Independent Mechanism in Human Endothelial Cells

NADPH oxidases are important sources of reactive oxygen species in the vascular wall. Recent evidence supports a vasoprotective role of H 2 O 2 produced by the main endothelial isoform Nox4. The impact of hypoxia on NOX4 expression in human endothelial cells and the underlying mechanism remains to be elucidated. In this study, we show that NOX4 mRNA and protein expression was upregulated by hypoxia (1 % O 2 ) in human umbilical vein endothelial cells (HUVEC). Correspondingly, H 2 O 2 production was 2-fold elevated in HUVEC after hypoxia. In contrast to rotenone and oxypurinol, lentiviral downregulation via shNOX4 abolished the elevated hypoxic hydrogen peroxide levels to normoxic values. Hypoxia stabilized the hypoxia-inducible factor (HIF)-1α protein in endothelial cells. Furthermore, VEGF promoter activity and a control promoter containing 3 hypoxia-responsive elements (HRE) were induced by hypoxia. NOX4 promoter deletions up to -119/+239 had an increased basal activity compared to control vector. A full-length and a terminally deleted NOX4 promoter construct missing a putative HRE showed a comparable activity under hypoxic and normoxic conditions, suggesting that NOX4 is not induced on the transcriptional level by hypoxia in endothelial cells. In addition, stabilization of HIF-1α protein under normoxic conditions using DMOG did not change NOX4 mRNA expression in HUVEC. Furthermore, overexpression of HIF-1α did not alter NOX4 promoter activity. Blockade of active transcription by actinomycin D revealed an increased stability of the NOX4 mRNA under hypoxic conditions. In conclusion, this study demonstrates an HIF-independent upregulation of NOX4 as major source of endothelial hydrogen peroxide generation in response to hypoxia. Our data support as a novel mechanism an increased NOX4 mRNA stability under hypoxic conditions in human endothelial cells.

Beneficial Effect of Ocimum sanctum (Linn) against Monocrotaline-Induced Pulmonary Hypertension in Rats.

Background: The study was designed to explore any beneficial effect of Ocimum sanctum (Linn) (OS) in experimental pulmonary hypertension (PH) in rats. OS is commonly known as “holy basil” and “Tulsi” and is used in the Indian System of Medicine as antidiabetic, antioxidant, hepatoprotective, adaptogenic, and cardioprotective. Methods: Monocrotaline (MCT) administration caused development of PH in rats after 28 days and rats were observed for 42 days. Treatments (sildenafil; 175 µg/kg, OS; 200 mg/kg) were started from day 29 after the development of PH and continued for 14 days. Parameters to assess the disease development and effectiveness of interventions were echocardiography, right and left ventricular systolic pressures, and right ventricular end diastolic pressure, percentage medial wall thickness (%MWT) of pulmonary artery, oxidative stress markers in lung tissue, NADPH oxidase (Nox-1) protein expression in lung, and mRNA expression of Bcl2 and Bax in right ventricular tissue. Results: OS (200 mg/kg) treatment ameliorated increased lung weight to body weight ratio, right ventricular hypertrophy, increased RVSP, and RVoTD/AoD ratio. Moreover, OS treatment decreases Nox-1 expression and increases expression of Bcl2/Bax ratio caused by MCT. Conclusion: The present study demonstrates that OS has therapeutic ability against MCT-induced PH in rat which are attributed to its antioxidant effect. The effect of OS was comparable with sildenafil.

Possible Role of NADPH Oxidase 4 in Angiotensin II-Induced Muscle Wasting in Mice.

Background: Muscle wasting is a debilitating phenotype associated with chronic heart failure (CHF). We have previously demonstrated that angiotensin II (AII) directly induces muscle wasting in mice through the activation of NADPH oxidase (Nox). In this study, we tested the hypothesis that deficiency of NADPH oxidase 4 (Nox4), a major source of oxidative stress, ameliorates AII-induced muscle wasting through the regulation of redox balance.Methods and Results: Nox4 knockout (KO) and wild-type (WT) mice were used. At baseline, there were no differences in physical characteristics between the WT and KO mice. Saline (vehicle, V) or AII was infused via osmotic minipumps for 4 weeks, after which, the WT + AII mice showed significant increases in Nox activity and NOX4 protein compared with the WT + V mice, as well as decreases in body weight, gastrocnemius muscle weight, and myocyte cross-sectional area. These changes were significantly attenuated in the KO + AII mice (27 ± 1 vs. 31 ± 1 g, 385 ± 3 vs. 438 ± 13 mg, and 1,330 ± 30 vs. 2281 ± 150 μm2, respectively, all P < 0.05). The expression levels of phospho-Akt decreased, whereas those of muscle RING Finger-1 (MuRF-1) and MAFbx/atrogin-1 significantly increased in the WT + AII mice compared with the WT + V mice. Furthermore, nuclear factor erythroid-derived 2-like 2 (Nrf2) and the expression levels of Nrf2-regulated genes significantly decreased in the WT + AII mice compared with the WT + V mice. These changes were significantly attenuated in the KO + AII mice (P < 0.05).Conclusion: Nox4 deficiency attenuated AII-induced muscle wasting, partially through the regulation of Nrf2. The Nox4–Nrf2 axis may play an important role in the development of AII-induced muscle wasting.

NADPH oxidase 2 knockout prevents chronic intermittent hypoxia induced sternohyoid muscle weakness in adult male mice

Obstructive sleep apnoea syndrome (OSAS) is the most prevalent form of sleep disordered breathing (SDB) which affects 1 in 5 people worldwide. Chronic intermittent hypoxia (CIH) is a hallmark feature of SDB as a consequence of repetitive upper airway occlusions in patients during sleep. These recurring hypoxia‐reoxygenation cycles result in the overproduction of reactive oxygen species (ROS), ultimately yielding a state of oxidative stress. Excessive levels of ROS have been associated with aberrant plasticity at multiple levels of the respiratory system including impaired upper airway muscle function. However, there is a paucity of information regarding the molecular mechanisms underlying these effects. The NADPH oxidase (NOX) family of enzymes have been linked to a multitude of CIH induced morbidities in a range of organs and systems with NOX subunits also having been identified in skeletal muscle.A mouse model of CIH was generated by the cycling of gas from normoxia (21% O2) for 210 seconds to hypoxia (5% O2 at the nadir) for 90 seconds 8 hr/day for 2 weeks during light hours. 10–11 week old male mice (C57BL/6J) were randomly assigned to one of 3 groups: Normoxic controls (sham; n=24), CIH exposed (n=24), and CIH + Apocynin (2 mM; n=24) treated throughout the gas exposure. 10–11 week old NOX2 null (B6.129S‐Cybbtm1Din/J) male mice were also assigned to a sham (n=12) or CIH (n=12) group. Following this, sternohyoid muscle contractile function was examined ex vivo. Gene expression of the entire family of NOX enzymes was examined by RT‐PCR. Western blot was used to quantify NOX2 and NOX4 protein expression. NOX enzyme activity was determined using a NOX activity assay. Data were expressed as mean±SD and were statistically compared by unpaired Student t‐test or two‐way ANOVA with Bonferroni post‐hoc test.2 weeks of exposure to CIH resulted in a significant decrease of ~45% in the peak specific force (Fmax) of the sternohyoid muscle when compared to normoxic controls (sham). CIH resulted in no significant alteration to the gene expression of any members of the NOX family of enzymes when compared to sham controls. There was no significant difference in NOX2 or NOX4 protein expression between CIH and sham groups. However, CIH exposure resulted in a significant increase in sternohyoid NOX enzyme activity compared to shams. Treatment with Apocynin (2 mM) throughout the 2 week CIH exposure prevented sternohyoid muscle weakness, restoring force to levels equivalent to controls. Furthermore, NOX2 knockout prevented CIH induced sternohyoid muscle weakness.Adult male C57BL/6J mice show signs of profound sternohyoid muscle dysfunction following exposure to 2 weeks of CIH when compared with sham animals. This occurs without alteration to the gene or protein expression of various NOX subunits. Increased NOX activity suggests a CIH induced increase in NOX enzyme assembly rather than abundance. Administration of the putative NOX inhibitor, Apocynin, prevents sternohyoid muscle weakness. NOX2 knockout also entirely prevents sternohyoid muscle weakness confirming its specific role in CIH induced muscle dysfunction. The striking muscle phenotype in the C57 mouse and its prevention in the absence of NOX2 provides a novel and robust platform for the study of mechanisms of hypoxia‐related muscle dysfunction, which may have relevance to respiratory conditions characterised by CIH, such as sleep apnoea.Support or Funding InformationDepartment of Physiology, University College Cork, Cork, IrelandThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aging and Endothelial Dysfunction: The Role of NADPH Oxidase and the Insufficient Inhibition by PKG

BackgroundNicotinamide adenine dinucleotide phosphate (NADPH) oxidase‐induced oxidative stress plays a critical role in the age‐related impairment of endothelium‐dependent dilation. However, the role of NADPH oxidase (i.e. which nitrogen oxides (NOX) may be involved) and the potential role of protein kinase G (PKG) inhibition of NADPH in human skeletal muscle feed arteries (SMFAs) has yet to be clarified. Therefore, this study sought to determine, in vitro, if treatment with NOX1 inhibitor (ML090), NOX2 inhibitor (gp91ds‐tat), and NOX4 inhibitor (plumbagin) could restore the age‐related endothelial dysfunction in human SMFAs.MethodsPKG, NOX1, NOX2, and NOX4 protein expression and cyclic guanosine monophosphate (cGMP) activity were analyzed in young (24±6 yrs, n=7) and old (77±8 yrs, n=8) subjects via Western blot analysis and an enzyme‐linked immunosorbent assay (ELISA). Using pressure myography, endothelium‐dependent and ‐independent vasodilation were assessed with and without ML090, gp91ds‐tat, and plumbagin in SMFAs obtained from 13 young and 16 old subjects in response to three stimuli 1) flow‐induced shear stress, 2) acetylcholine (ACh), and 3) sodium nitroprusside (SNP). Additionally, L‐NG‐monomethyl arginine citrate (L‐NMMA) was utilized to identify the impact of blocking nitric oxide (NO) synthase on these responses.ResultsNOX2 and NOX4, but not NOX1, protein expression were significantly increased in the old compared to the young SMFAs. In addition, cGMP activity and PKG protein expression were significantly lower in the old compared to the young SMFAs. Endothelial‐dependent vasodilatory dysfunction was evident in response to intraluminal flow (Young: 81 ± 15; Old: 27 ± 14%, P &lt; 0.05) and ACh (Young: 95 ± 17, Old: 34 ± 15 %, P &lt; 0.05). The NOX2 inhibitor, gp91ds‐tat, restored the vasodilation response in the old SMFAs to both flow and Ach (70 ± 11 and 89 ± 13 %, respectively, P &lt; 0.05), while the NOX4 inhibitor, plumbagin, partially restored the vasodilation response in the old SMFAs to flow and ACh (45 ± 15 %, P=0.08; 59 ± 13 %, P=0.07). However, the NOX1 inhibitor, ML090, had no effect. Endothelium‐independent vasodilation (SNP) was not affected by age and, therefore, all NOX inhibitors had no effect on SNP‐induced vasodilation. L‐NMMA negated the restorative effects of NOX2 and NOX4 blockade.ConclusionsBoth NOX2 and to a lesser extent NOX4 appear to play an important role in the age‐associated endothelial dysfunction exhibited by SMFAs. Furthermore, in combination, these findings suggest that there is likely an age‐related weakening of the NO‐initiated link between cGMP, PKG, and NADPH oxidase, with PKG demonstrating insufficient NADPH oxidase inhibition with advancing age and, ultimately, attenuated vascular function.Support or Funding Informationthe National Heart, Lung, and Blood Institute at the National Institute of Health (PO1 HL1091830) and the Veteran's Administration Rehabilitation Research and Development Service (E6910‐R, E1697‐R, E1433‐P, E9275‐L and E1572‐P).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GPER Attenuates Angiotensin II‐Induced Oxidative Stress via cAMP‐Mediated Regulation of NOX4

Our previous work shows that the G protein‐coupled estrogen receptor (GPER) is protective in the vasculature and kidneys during angiotensin (Ang) II‐dependent hypertension, in part by inhibiting oxidative stress. In addition, we find that acute estrogenic signaling via GPER involves activation of the cAMP signaling pathway in vascular smooth muscle cells. Ang II stimulates the production of reactive oxygen species (ROS) via NADPH oxidase (NOX) complex activation, but whether GPER interferes with NOX activation and whether this occurs through cAMP signaling is not known. Therefore, the goal of the current study was to assess the molecular mechanisms by which GPER attenuates Ang II‐induced oxidative stress in vitro. A7r5 rat aortic smooth muscle cells were treated overnight with 100 nM Ang II in the presence of vehicle or 100 nM of the GPER agonist G‐1. ROS was measured via electron spin resonance spectroscopy (e‐scan) and CellRox™ Green fluorescence. NOX activity and expression were measured by NADP/NADPH ratio, real time RT‐PCR, and immunoblotting. Both e‐scan and CellRox experiments showed that Ang II increased oxidative stress, while co‐administration of the GPER agonist G‐1 prevented this increase (4.3 × 105 vs 1.9 × 105 AU/mg protein, P&lt;0.001, n=7–8). Similarly, NOX activity was elevated in response to Ang II but normalized by G‐1 (189% vs. 83% RLU, P&lt;0.0001, n=5). When the production of cAMP was inhibited with the adenylyl cyclase inhibitor SQ22536, G‐1 was unable to block Ang II‐induced NOX activity (153% vs. 142% RLU, P=0.39, n=6). Ang II increased NOX4 mRNA over time, with a maximum increase at 4h (3.5‐fold change, P&lt;0.05, n=3). Ang II also increased NOX4 protein (1.2 vs 2.1 ADU/β‐actin, P&lt;0.01, n=2). In contrast, administration of G‐1 for 4h decreased NOX4 mRNA by 60% (P&lt;0.05, n=3) with no significant impact on protein expression (P=0.91, n=2). Preliminary studies using male wildtype and knockout mice show higher expression of NOX4 in response to genetic deletion of GPER. These experiments indicate that GPER inhibits NOX4 transcription and suppresses NOX activity, thereby limiting the production of ROS by Ang II. The impact of GPER on NOX activity was dependent on the production of cAMP. Taken together with our previous work, this study provides insight into how acute estrogen signaling provides cardiovascular protection during Ang II‐induced hypertension and potentially other diseases that are characterized by increased oxidative stress.Support or Funding InformationThis study was funded by National Institutes of Health grant HL133619 to S.H.L.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Renal protective effects of the flavonoid (−)‐Epicatechin in a chronic kidney disease (nephrectomy 5/6) model

BackgroundChronic kidney disease (CKD) is a worldwide public health problem affecting 10% of the adult population. One of the most important factors in the progression of CKD from early stages is oxidative stress. Currently there is limited efficacy to treat this problem so the objective of this study was to test Epicatechin (Epi), effects in a mice model of CKDMethodsA nephrectomy 5/6 (Nf) in C‐57BL6 mice model was implemented and Epi treatment (14 days doses doses of 0.01, 0.1 and 1 mg/kg) was provided by gavage. Changes in Systolic blood pressure (SBP), serum urea and creatinine, renal cortex morphology, serum tetrahydrobiopterin (BH4), in 3‐nitrotyrosine as oxidative damage marker and of Angiotensin II AT1 receptor (AGTR1) and NOX‐4 protein expression were evaluatedResultsSBP increased in the Nf group and decrease significantly in Nf+ Epi groups. Serum urea and creatinine levels increased in the Nf group Epi treatment induced a significant decrease. Epi treatment induced a dose dependent increase in serum BH4 in Nf animals. Normal renal cortex morphology was conserved at 1 m/kg Epi dose. The expression of AGTR1 and NOX‐4 was increased in the Nf group, Epi treatment in a dose dependent manner decrease the AGTR1 and NOX‐4 expression.ConclusionThe present findings suggest that Epi could prevent the progression of CKD and may be used as complement for the modulation of blood pressure and f renal dysfunction.Support or Funding InformationConacyt 253769This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of NADPH oxidase 1 and 4 blockade in activated human retinal endothelial cells.

Over-production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS-generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans. Differential Nox enzyme expression was studied by real-time-quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 μM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2',7'-dichlorofluorescein. DMOG-stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production. Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG-induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth. Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases.

Early onset of the pathological phenotype in line 61 α-synuclein transgenic mice

In this study, we report a paediatric patient with a lethal phenotype of respiratory distress, failure to thrive, pancreatic insufficiency, liver dysfunction, hypertrophic cardiomyopathy, bone marrow suppression, humoral and cellular immune deficiency. To identify the genetic basis of this unusual clinical phenotype and potentially make available the option of future prenatal testing, whole exome sequencing (WES) was used followed by functional studies in a bid to confirm pathogenicity. The WES we identified a homozygous novel variant, AK298328; c.9_10insGAG; p.[Glu3dup], in NOX4 in the proband, and parental heterozygosity for the variant (confirmed by Sanger sequencing). NADPH Oxidase 4 NOX4 (OMIM 605261) encodes an enzyme that functions as the catalytic subunit of the NADPH oxidase complex. NOX4 acts as an oxygen sensor, catalysing the reduction of molecular oxygen, mainly to hydrogen peroxide (H2O2).However, although, our functional data including 60% reduction in NOX4 protein levels and a 75% reduction in the production of H2O2 in patient fibroblast extracts compared to controls was initially considered to be the likely cause of the phenotype in our patient, the potential contribution of the NOX4 variant as the primary cause of the disease was clearly excluded based on following pieces of evidence. First, Sanger sequencing of other family members revealed that two of the grandparents were also homozygous for the NOX4 variant, one of who has fibromuscular dysplasia. Second, re-evaluation of more recent variant databases revealed a high allele frequency for this variant.Our case highlights the need to re-interrogate bioinformatics resources as they are constantly evolving, and is reminiscent of the short-chain acyl-CoA dehydrogenase deficiency (SCADD) story, where a functional defect in fatty acid oxidation has doubtful clinical ramifications.

Deletion of NADPH oxidase 4 reduces severity of traumatic brain injury.

Traumatic brain injury (TBI) contributes to over 30% of injury-related deaths and is a major cause of disability without effective clinical therapies. Oxidative stress contributes to neurodegeneration, neuroinflammation, and neuronal death to amplify the primary injury after TBI. NADPH oxidase (NOX) is a major source of reactive oxygen species following brain injury. Our current study addresses the functional role of the NOX4 isoform in the damaged cortex following TBI. Adult male C57BL/6 J and NOX4-/- mice received a controlled cortical impact and lesion size, NOX4 expression, oxidative stress, neurodegeneration, and cell death were assessed in the injured cerebral cortex. The results revealed that NOX4 mRNA and protein expression were significantly upregulated at 1-7 days post-TBI in the injured cerebral cortex. Expression of the oxidative stress markers, 8-OHdG, 4-HNE, and nitrotyrosine was upregulated at 2 and 4 days post-TBI in the WT injured cerebral cortex, and nitrotyrosine primarily colocalized with neurons. In the NOX4-/- mice, expression of these oxidative stress markers, 8-OHdG, 4-HNE, and nitrotyrosine were significantly attenuated at both timepoints. In addition, examination of NOX4-/- mice revealed a reduced number of apoptotic (TUNEL+) and degenerating (FJB+) cells in the perilesional cortex after TBI, as well as a smaller lesion size compared with the WT group. The results of this study implicate a functional role for NOX4 in TBI induced oxidative damage and neurodegeneration and raise the possibility that targeting NOX4 may have therapeutic efficacy in TBI.

Maternal diabetes up-regulates NOX2 and enhances myocardial ischaemia/reperfusion injury in adult offspring.

Offspring of diabetic mothers are at risk of cardiovascular diseases in adulthood. However, the underlying molecular mechanisms are not clear. We hypothesize that prenatal exposure to maternal diabetes up‐regulates myocardial NOX2 expression and enhances ischaemia/reperfusion (I/R) injury in the adult offspring. Maternal diabetes was induced in C57BL/6 mice by streptozotocin. Glucose‐tolerant adult offspring of diabetic mothers and normal controls were subjected to myocardial I/R injury. Vascular endothelial growth factor (VEGF) expression, ROS generation, myocardial apoptosis and infarct size were assessed. The VEGF‐Akt (protein kinase B)‐mammalian target of rapamycin (mTOR)‐NOX2 signalling pathway was also studied in cultured cardiomyocytes in response to high glucose level. In the hearts of adult offspring from diabetic mothers, increases were observed in VEGF expression, NOX2 protein levels and both Akt and mTOR phosphorylation levels as compared to the offspring of control mothers. After I/R, ROS generation, myocardial apoptosis and infarct size were all significantly higher in the offspring of diabetic mothers relative to offspring of control mothers, and these differences were diminished by in vivo treatment with the NADPH oxidase inhibitor apocynin. In cultured cardiomyocytes, high glucose increased mTOR phosphorylation, which was inhibited by the PI3 kinase inhibitor LY294002. Notably, high glucose‐induced NOX2 protein expression and ROS production were inhibited by rapamycin. In conclusion, maternal diabetes promotes VEGF‐Akt‐mTOR‐NOX2 signalling and enhances myocardial I/R injury in the adult offspring. Increased ROS production from NOX2 is a possible molecular mechanism responsible for developmental origins of cardiovascular disease in offspring of diabetic mothers.

Cigarette Smoke Initiates Oxidative Stress-Induced Cellular Phenotypic Modulation Leading to Cerebral Aneurysm Pathogenesis.

Cigarette smoke exposure (CSE) is a risk factor for cerebral aneurysm (CA) formation, but the molecular mechanisms are unclear. Although CSE is known to contribute to excess reactive oxygen species generation, the role of oxidative stress on vascular smooth muscle cell (VSMC) phenotypic modulation and pathogenesis of CAs is unknown. The goal of this study was to investigate whether CSE activates a NOX (NADPH oxidase)-dependent pathway leading to VSMC phenotypic modulation and CA formation and rupture. In cultured cerebral VSMCs, CSE increased expression of NOX1 and reactive oxygen species which preceded upregulation of proinflammatory/matrix remodeling genes (MCP-1, MMPs [matrix metalloproteinase], TNF-α, IL-1β, NF-κB, KLF4 [Kruppel-like factor 4]) and downregulation of contractile genes (SM-α-actin [smooth muscle α actin], SM-22α [smooth muscle 22α], SM-MHC [smooth muscle myosin heavy chain]) and myocardin. Inhibition of reactive oxygen species production and knockdown of NOX1 with siRNA or antisense decreased CSE-induced upregulation of NOX1 and inflammatory genes and downregulation of VSMC contractile genes and myocardin. p47phox-/- NOX knockout mice, or pretreatment with the NOX inhibitor, apocynin, significantly decreased CA formation and rupture compared with controls. NOX1 protein and mRNA expression were similar in p47phox-/- mice and those pretreated with apocynin but were elevated in unruptured and ruptured CAs. CSE increased CA formation and rupture, which was diminished with apocynin pretreatment. Similarly, NOX1 protein and mRNA and reactive oxygen species were elevated by CSE, and in unruptured and ruptured CAs. CSE initiates oxidative stress-induced phenotypic modulation of VSMCs and CA formation and rupture. These molecular changes implicate oxidative stress in the pathogenesis of CAs and may provide a potential target for future therapeutic strategies.

MicroRNA‑146a/NAPDH oxidase4 decreases reactive oxygen species generation and inflammation in a diabetic nephropathy model.

The present study investigated the role of microRNA (miR)‑146a in a diabetic nephropathy (DN) model, and its molecular mechanism. DN mice were given intraperitoneal injections of streptozotocin (55mg/kg/day) for 5 consecutive days as an invivo DN model. The HK‑2 human kidney cell line were exposed to 45% D‑glucose as an invitro DN model. Firstly, it was demonstrated that miR‑146a expression was inhibited and NAPDH oxidase 4 (Nox4) was increased in DN mice. In HK‑2 cells, overexpression of miR‑146a inhibited Nox4 protein expression and decreased reactive oxygen species (ROS) generation, oxidative stress and inflammation, and suppressed vascular cell adhesion molecule‑1 (VCAM‑1) and intracellular adhesion molecule‑1 (ICAM‑1) protein expression. Nacetylcysteine, a Nox4 inhibitor, was demonstrated to inhibit ROS generation, suppress VCAM‑1 and ICAM‑1 protein expression, and decrease oxidative stress and inflammation in HK‑2 cells following overexpression of miR‑146a. In conclusion, these results indicated that miR‑146a/Nox4 decreases ROS generation and inflammation and prevents DN. Therefore, miR‑146a may represent a novel anti‑inflammatory and ‑oxidative modulator of DN.

NADPH oxidase 4 promotes cisplatin-induced acute kidney injury via ROS-mediated programmed cell death and inflammation

The goal of this study was to elucidate the functional role of Nox4 during acute kidney injury (AKI). NADPH oxidases are a major source of reactive oxygen species (ROS) in the kidney in normal and pathological conditions. Among NADPH oxidase isoforms, NADPH oxidase4 (Nox4) is highly expressed in the kidney and has an important role in kidney diseases, such as diabetic nephropathy and renal carcinoma. We previously found that Nox4 expression significantly increased in the toxic AKI model. However, its functional role and mechanism of action in AKI are still unknown. We scavenged ROS with apocynin in vitro and in vivo and found it attenuated cisplatin-triggered renal function decline. It also alleviated programmed cell death and renal inflammation, indicating a critical role for ROS in mediating AKI. Nox4 protein and mRNA levels were substantially upregulated by cisplatin in vivo and in vitro. Nox4 knockdown alleviated cisplatin-induced cell death and inflammatory response, while Nox4 overexpression aggravated them. Moreover, N-acetyl-L-cysteine (NAC)-mediated inhibition of ROS suppressed cell injury led by Nox4 overexpression, indicating Nox4-mediated ROS generation may be the key mediator in cisplatin-induced nephrotoxicity. Mechanistically, excessive expression of Nox4 induced programmed cell death, especially RIP-mediated necroptosis. Finally, we tested whether Nox4 is a potential therapeutic target using an AKI mouse model by injecting a lentivirus-packaged Nox4 shRNA plasmid through tail vein. Disruption of Nox4 led to renal function recovery, kidney damage relief and reduced inflammation. We conclude that Nox4 aggravates cisplatin-induced nephrotoxicity by promoting ROS-mediated programmed cell death and inflammation. Thus Nox4 may serve as a potential therapeutic target in the treatment of AKI.

TRPC1 Deficiency Exacerbates Cerebral Ischemia/Reperfusion-Induced Neurological Injury by Potentiating Nox4-Derived Reactive Oxygen Species Generation

Background/Aims: Transient receptor potential cation channel 1 (TRPC1)-mediated the calcium (Ca²⁺) influx plays an important role in several brain disorders. However, the function of TRPC1 in ischemia/reperfusion (I/R)-induced neurological injury is unclear. Methods: Wild-type or TRPC1 knockout mice underwent middle cerebral artery occlusion for 90 min followed by 24 h of reperfusion. In an in vitro study, neuronal cells were treated with oxygen–glucose deprivation and reoxygenation (OGD/R) to mimic I/R. The intracellular Ca²⁺ concentration [Ca²⁺]_i was measured by Fura 2-AM under a microscope. Cerebral infarct volume was measured by triphenyltetrazolium chloride staining. Neurological function was examined by neurological severity score, Morris water maze test, rotarod test and string test. Oxidative parameters were detected by malondialdehyde, glutathione peroxidase, and superoxide dismutase commercially available kits. The protein expression levels of TRPC1, Nox4, p22phox, p47phox, and p67phox were analyzed by western blotting. Results: Brain tissues from cerebral I/R mice showed decreased TRPC1 expression. Similarly, TRPC1 expression was reduced in HT22 cells upon exposure to OGD/R treatment, followed by decreased Ca²⁺ influx. However, TRPC1 overexpression reversed the OGD/R-induced decrease in [Ca²⁺]_i. TRPC1 knockout significantly exacerbated I/R-induced brain infarction, edema, neurological severity score, memory impairment, neurological deficits, and oxidative stress. In contrast, TRPC1 upregulation inhibited the increase in reactive oxygen species (ROS) generation induced by OGD/R. Analysis of key subunits of the Nox family and mitochondrial ROS revealed that the effects of TRPC1 downregulation on oxidative stress were associated with activation of Nox4-containing NADPH oxidase. TRPC1 interacted with Nox4 and facilitated Nox4 protein degradation under OGD/R conditions. In addition, TRPC1 inhibition potentiated the OGD/R-induced translocation of p47phox and p67phox as well as the interaction between Nox4 and p47phox or p67phox, whereas TRPC1 overexpression had the opposite effects. Conclusion: TRPC1 deficiency potentiates ROS generation via Nox4-containing NADPH oxidase, which exacerbates cerebral I/R injury. TRPC1 may be a promising molecular target for the treatment of stroke.

Long non-coding RNA-ROR aggravates myocardial ischemia/reperfusion injury

Long non-coding RNAs (lncRNAs) play an important role in the pathogenesis of cardiovascular diseases, especially in myocardial infarction and ischemia/reperfusion (I/R). However, the underlying molecular mechanism remains unclear. In this study, we determined the role and the possible underlying molecular mechanism of lncRNA-ROR in myocardial I/R injury. H9c2 cells and human cardiomyocytes (HCM) were subjected to either hypoxia/reoxygenation (H/R), I/R or normal conditions (normoxia). The expression levels of lncRNA-ROR were detected in serum of myocardial I/R injury patients, H9c2 cells, and HCM by qRT-PCR. Then, levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) were measured by kits. Cell viability, apoptosis, apoptosis-associated factors, and p38/MAPK pathway were examined by MTT, flow cytometry, and western blot assays. Furthermore, reactive oxygen species (ROS) production was determined by H2DCF-DA and MitoSOX Red probes with flow cytometry. NADPH oxidase activity and NOX2 protein levels were measured by lucigenin chemiluminescence and western blot. Results showed that lncRNA-ROR expression was increased in I/R patients and in H/R treatment of H9c2 cells and HCM. Moreover, lncRNA-ROR significantly promoted H/R-induced myocardial injury via stimulating release of LDH, MDA, SOD, and GSH-PX. Furthermore, lncRNA-ROR decreased cell viability, increased apoptosis, and regulated expression of apoptosis-associated factors. Additionally, lncRNA-ROR increased phosphorylation of p38 and ERK1/2 expression and inhibition of p38/MAPK, and rescued lncRNA-ROR-induced cell injury in H9c2 cells and HCM. ROS production, NADPH oxidase activity, and NOX2 protein levels were promoted by lncRNA-ROR. These data suggested that lncRNA-ROR acted as a therapeutic agent for the treatment of myocardial I/R injury.

3,3′-Diindolylmethane enhances apoptosis in docetaxel-treated breast cancer cells by generation of reactive oxygen species

Context: A major problem in the treatment of cancer is the development of toxic side effects and resistance to chemotherapy. The use of plant compounds to overcome resistance and prevent toxicity is a potential strategy for treatment.Objective: We evaluated whether 3,3′-diindolylmethane (DIM) enhanced the sensitivity of breast cancer cells to docetaxel (DOC).Materials and methods: MDA-MB231 and Sk-BR-3 cells were treated with and without 25 or 50 µM of DIM and 1 nM of DOC for 48 and 72 h, respectively. MTT assay was used to measure cell survival. Apoptosis and intracellular reactive oxygen species (ROS) were determined by flow cytometry. The expression of proteins regulating ROS production and apoptosis was evaluated by immunoblotting technique.Results: Combining 25 µM of DIM with 1 nM DOC decreased cell survival by 42% in MDA-MB231 cells and 59% in Sk-BR-3 cells compared to control, DIM, or DOC (p ≤ 0.05). The combination treatment increased apoptosis over 20% (p ≤ 0.01) in both cell lines, which was associated with decreased Bcl-2, increased Bax, cleaved PARP and activated JNK (p ≤ 0.01). ROS production increased by 46.5% in the MDA-MB231 and 29.3% in Sk-BR-3 cells with the combination compared to DIM or DOC alone. Pretreating cells with N-acetyl-cysteine or Tiron abrogated the anti-survival effect of the combination. The increase in ROS was associated with a 54% decrease in MnSOD and 47% increase in NOX2 protein compared to the other groups.Conclusions: Our findings indicated that DIM enhances the sensitivity of breast cancer cells to DOC treatment by increasing ROS, which led to decreased cell survival and apoptosis.

Interaction between p22phox and Nox4 in the endoplasmic reticulum suggests a unique mechanism of NADPH oxidase complex formation.

The p22phox protein is an essential component of the phagocytic- and inner ear NADPH oxidases but its relationship to other Nox proteins is less clear. We have studied the role of p22phox in the TGF-β1-stimulated H2O2 production of primary human and murine fibroblasts. TGF-β1 induced H2O2 release of the examined cells, and the response was dependent on the expression of both Nox4 and p22phox. Interestingly, the p22phox protein was present in the absence of any detectable Nox/Duox expression, and the p22phox level was unaffected by TGF-β1. On the other hand, Nox4 expression was dependent on the presence of p22phox, establishing an asymmetrical relationship between the two proteins. Nox4 and p22phox proteins localized to the endoplasmic reticulum and their distribution was unaffected by TGF-β1. We used a chemically induced protein dimerization method to study the orientation of p22phox and Nox4 in the endoplasmic reticulum membrane. This technique is based on the rapamycin-mediated heterodimerization of the mammalian FRB domain with the FK506 binding protein. The results of these experiments suggest that the enzyme complex produces H2O2 into the lumen of the endoplasmic reticulum, indicating that Nox4 contributes to the development of the oxidative milieu within this organelle.

Resveratrol attenuates hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats: Role of ROS and ROS-mediated cell signaling

Resveratrol, a natural polyphenolic compound has been reported to attenuate angiotensin II -induced vascular smooth muscle cell (VSMC) hypertrophy; however, whether resveratrol could also inhibit hyperproliferation of VSMC from spontaneously hypertensive rats (SHR) is unexplored. The present study investigates the effect of resveratrol on hyperproliferation of VSMC from SHR and the underlying molecular mechanisms responsible for this response. For these studies, aortic VSMC from SHR and Wistar-Kyoto (WKY) rats were used. The proliferation of VSMC was determined by [3H] thymidine incorporation and the levels of proteins were determined by Western blotting. The enhanced proliferation exhibited by VSMC from SHR was attenuated by resveratrol. In addition, resveratrol attenuated the overexpression of cyclin D1, cyclin E, cyclin dependent kinase 4 (Cdk4), Cdk2, phosphorylated retinoblastoma protein (pRb), Giα proteins and enhanced phosphorylation of ERK1/2 and AKT in VSMC from SHR. Furthermore, the overproduction of superoxide anion, increased NADPH oxidase activity, overexpression of Nox2, Nox4 and p47phox proteins, increased phosphorylation of EGFR, IGF-IR, and c-Src were all abrogated by resveratrol. These results suggest that resveratrol attenuates the hyperproliferation of VSMC from SHR through the inhibition of ROS, c-Src, growth factor receptor activation, MAPK/PI3K, Giα and cell cycle proteins that are implicated in VSMC hyperproliferation.

Natriuretic peptide receptor-C-mediated attenuation of vascular smooth muscle cell hypertrophy involves Gqα/PLCβ1 proteins and ROS-associated signaling.

Hypertension is associated with vascular remodeling due to hyperproliferation and hypertrophy of vascular smooth muscle cells (VSMC). Recently, we showed the implication of enhanced expression of Gqα and PLCβ1 proteins in hypertrophy of VSMCs from 16‐week‐old spontaneously hypertensive rats (SHR). The aim of this study was to investigate whether C‐ANP4‐23, a natriuretic peptide receptor‐C (NPR‐C) ligand that was shown to inhibit vasoactive peptide‐induced enhanced protein synthesis in A10 VSMC could also attenuate hypertrophy of VSMC isolated from rat model of cardiac hypertrophy and to further explore the possible involvement of Gqα/PLCβ1 proteins and ROS‐mediated signaling in this effect. The protein synthesis and cell volume, markers of hypertrophy were significantly enhanced in VSMC from 16‐week‐old SHR compared with age‐matched WKY rats and C‐ANP4‐23 treatment attenuated both to WKY levels. In addition, C‐ANP4‐23 treatment also attenuated the enhanced expression of AT1 receptor, Gqα, PLCβ1, Nox4, and p47phox proteins, the enhanced activation of EGFR, PDGFR, IGF‐1R, enhanced phosphorylation of ERK1/2/AKT and c‐Src in VSMC from SHR. Furthermore, the enhanced levels of superoxide anion and NADPH oxidase activity exhibited by VSMC from SHR were also attenuated to control levels by C‐ANP4‐23 treatment. These results indicate that C‐ANP4‐23 via the activation of NPR‐C attenuates VSMC hypertrophy through decreasing the overexpression of Gqα/PLCβ1 proteins, enhanced oxidative stress, increased activation of growth factor receptors, and enhanced phosphorylation of MAPK/AKT signaling pathways. Thus, it can be suggested that C‐ANP4‐23 may be used as a therapeutic agent for the treatment of vascular complications associated with hypertension and atherosclerosis.