Nox4 mRNA Expression Research Articles

425 - A Potential Role of Nox1 Isoform of NADPH Oxidase in the Development of Nonalcoholic Steatohepatitis

Oxidative stress is known to play a critical role in the development of nonalcoholic steatohepatitis. To clarify the source of oxidative stress, we examined the expression of NADPH oxidase isoforms in the liver of high fat diet (HFD)-fed mice. The mRNA expression of Nox1, but not of Nox2 or Nox4 was significantly elevated after 8 weeks of HFD. Increased levels of serum alanine aminotransferase and hepatic cleaved caspase-3 in HFD-fed wild-type mice (WT) were significantly ameliorated in mice deficient in Nox1 (Nox1-KO). Increased nitrotyrosine adduct formation was observed in hepatic sinusoids of WT, which was significantly suppressed in Nox1-KO. The expression of Nox1 mRNA was much higher in the fractions of liver sinusoidal endothelial cells (LSECs) than in those of hepatocytes. In primary cultured LSECs, palmitic acid, a saturated free fatty acid, significantly up-regulated Nox1 mRNA. Accordingly, Nox1 in sinusoidal endothelial cells may be one of the sources of oxidative stress in the liver to promote peroxinitrite-induced hepatocellular injury.

105 - Antioxidant Reprogram Macrophages from Pro-Tumor M2 to Anti-Tumor M1

Macrophages (Mφ) have high plasticity and can polarize to two extremes, pro-inflammatory M1 Mφ and immuno-suppressive M2 Mφ. In breast cancer, tumor associated Mφ (TAM) have a pro-tumor M2-like phenotype and are associated with decreased survival. Thus, it is vital to develop treatments to reprogram TAM away from pro-tumor M2 Mφ toward anti-tumor M1 Mφ. A recent study reported a lipid peroxide scavenger selectively inhibits M2 Mφ markers and cytokines. However, little is known about the differences in reactive oxygen species (ROS) metabolism between M1 Mφ and M2 Mφ. Our hypothesis is pro-tumorigenic M2 Mφ possess a distinct profile of pro- and anti-oxidant enzymes, as well as a different sensitivity to ROS modulation versus the anti-tumor M1 Mφ. Our results show M2 Mφ, derived from primary human monocytes, have a significant 20-30% reduction in intracellular ROS levels versus M1 Mφ by DCFH and DHE staining. Extracellular ROS synthesis was dramatically reduced by ~70% in M2 Mφ, in part due to reduced mRNA expression of Nox2 and its accessory protein CYBA in M2 Mφ. Another contributing factor to lower ROS levels is that M2 Mφ have significantly increased Cu/ZnSOD and Gpx1 protein and activity, as well as increased Catalase mRNA expression. These data suggest M2 Mφ have increased ROS metabolism versus M1 Mφ, which may provide an advantage in the oxidative tumor microenvironment. Furthermore, EcSOD re-expression in MB231, a triple negative breast cancer cell line, inhibited the MB231-mediated increase in M2 Mφ markers and decrease in M1 Mφ markers. We moved forward utilizing Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE), a small molecule SOD mimetic, shown to have anti-tumor effects. MnTE treatment increased M1 Mφ markers, IL-12b and CD86, while concurrently decreasing M2 Mφ markers, IL-10, CD163, and C206. MnTE also inhibited the ability of M2 Mφ to inhibit T cell activation suggesting ROS is required for M2 Mφ polarization and function. These studies provide rationale for antioxidant treatment of breast cancer to reprogram TAM from pro-tumorigenic M2-like TAM phenotype to a more tumoricidal M1-like phenotype.

NOX1/NADPH Oxidase Expressed in Colonic Macrophages Contributes to the Pathogenesis of Colonic Inflammation in Trinitrobenzene Sulfonic Acid-Induced Murine Colitis.

NOX1/NADPH oxidase, a nonphagocytic isoform of reactive oxygen species-producing enzymes, is highly expressed in the colon, but the physiologic and pathophysiologic roles of this isoform are not fully understood. The present study investigated the role of NOX1 in the development of colonic inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model. Intrarectal injection of TNBS caused severe colitis accompanied by body weight loss, diarrhea, and increased myeloperoxidase (MPO) activity in wild-type (WT) mice. In contrast, the severity of colitis was significantly attenuated in NOX1-deficient (NOX1KO) mice (the inhibitions of macroscopic damage score, body weight loss, diarrhea score, and MPO activity were 73.1%, 36.8%, 83.3%, and 98.4%, respectively). TNBS-induced upregulation of inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-1β), chemokines (CXCL1 and CXLC2), and inducible nitric oxide synthase (iNOS) was also significantly less in NOX1KO than in WT mice (the inhibitions were 100.8%, 89.0%, 63.5%, 96.7%, and 97.1%, respectively). Expression of NOX1 mRNA was detected not only in the lamina propria but also in peritoneal macrophages isolated from WT mice. Increased expression of TNF-α, IL-1β, and iNOS in peritoneal macrophages exposed to lipopolysaccharide was significantly attenuated in macrophages isolated from NOX1KO mice (68.1%, 67.0%, and 79.3% inhibition, respectively). These findings suggest that NOX1/NADPH oxidase plays an important role in the pathogenesis of TNBS-induced colonic inflammation via upregulation of inflammatory cytokines, chemokines, and iNOS. NOX1 in colonic macrophages may become a potential target in pharmacologic intervention for inflammatory bowel disease.

Abstract 080: mTORC1 as a Novel Regulator of Vascular Endothelial Function in Obese Mice and Humans

Obesity-induced hypertension is associated with vascular endothelial dysfunction. Recently, our laboratory has demonstrated a critical role of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in cardiovascular regulation. Here, we tested the hypothesis that dysregulation of mTORC1 signaling is involved in the endothelial dysfunction associated with obesity in mice and humans. We found that diet-induced obese (DIO) mice that display vascular endothelial dysfunction as compared to lean controls have increased mTORC1 signaling in aortic lysates indicated by the elevated (p<0.05) phosphorylated levels of mTOR and its downstream signaling targets S6-kinase and the ribosomal S6 protein measured by Western blot. Increased vascular mTORC1 signaling in DIO mice was associated with increased aortic NOX2 mRNA expression (2.0±0.2 vs. 1.0±0.3AU in lean controls; p<0.05). Isolated abdominal subcutaneous adipose arterioles from non-diabetic obese (BMI ≥30 kg/m 2 ; n=4; age 51±6 yrs; BMI 54±3 kg/m 2 ) humans exhibited a strong trend towards increased phosphorylated S6 protein compared to normal-weight (BMI <30kg/m 2 ; n=3; age 44±15 yrs; BMI 26±1 kg/m 2 ) individuals (5.0±1.9 vs 0.8±0.4AU; p=0.12), suggesting increased vascular mTORC1 signaling in human obesity. Next, we used an adenoviral construct of a constitutively active (CA) S6-kinase (Ad-CAS6K) to enhance mTORC1 signaling. In mouse endothelial cells, Ad-CAS6K increased mRNA expression of oxidative stress (NOX1and NOX2) and inflammatory markers (IL-1β) and decreased endothelial NOS expression (p<0.05). Transfection of aortic rings with the Ad-CAS6K resulted in impairment in acetylcholine-induced relaxation (Max. relaxation: 67± 5 vs. 81 ±3%; p<0.05) without altering the relaxation evoked by sodium nitroprusside (Max. relaxation: 90±1% vs. 90±2%) recapitulating the vascular phenotype in obese mice. Taken together, our data demonstrate a novel role of the mTORC1 signaling pathway in the regulation of vascular endothelial function. Our data also implicate dysregulation of the endothelial mTORC1 signaling pathway in the endothelial dysfunction associated with obesity.

Coagulin-L ameliorates TLR4 induced oxidative damage and immune response by regulating mitochondria and NOX-derived ROS

Withanolides possess diverse biological and pharmacological activity but their immunomodulatory function is less realized. Hence, coagulin-L, a withanolide isolated from Withania coagulans Dunal has been studied for such an effect in human and murine cells, and mice model. Coagulin-L (1, 3, 10μM) exhibited immunomodulatory effect by suppressing TLR4 induced immune mediators such as cytokines (GMCSF, IFNα, IFNγ, IL-1α, IL-1Rα, IL-1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p40/p70), IL-13, IL-15, IL-17), chemokines (IL-8/CXCL8, MIG/CXCL9, IP-10/CXCL10, KC, MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5, eotaxin/CCL11), growth factors (FGF-basic, VEGF), nitric oxide and intracellular superoxide. Mechanistically, coagulin-L abrogated LPS induced total and mitochondrial ROS generation, NOX2, NOX4 mRNA expression, IRAK and MAPK (p38, JNK, ERK) activation. Coagulin-L also attenuated IκBα degradation, which prevented NFκB downstream iNOS expression and pro-inflammatory cytokine release. Furthermore, coagulin-L (10, 25, 50mg/kg, p.o.), undermined the LPS (10mg/kg, i.p.) induced endotoxemia response in mice as evinced from diminished cytokine release, nitric oxide, aortic p38 MAPK activation and endothelial tissue impairment besides suppressing NOX2 and NOX4 expression in liver and aorta. Moreover, coagulin-L also alleviated the ROS mediated oxidative damage which was assessed through protein carbonyl, lipid hydroperoxide, 8-isoprostane and 8-hydroxy-2-deoxyguanosine quantification. To extend, coagulin-L also suppressed carrageenan-induced paw edema and thioglycollate-induced peritonitis in mice. Therefore, coagulin-L can be of therapeutic importance in pathological conditions induced by oxidative damage.

Inhibition of histone deacetylase reduces transcription of NADPH oxidases and ROS production and ameliorates pulmonary arterial hypertension

Excessive levels of reactive oxygen species (ROS) and increased expression of NADPH oxidases (Nox) have been proposed to contribute to pulmonary artery hypertension (PAH) and other cardiovascular diseases (CVD). Nox enzymes are major sources of ROS but the mechanisms regulating changes in Nox expression in disease states remain poorly understood. Epigenetics encompasses a number of mechanisms that cells employ to regulate the ability to read and transcribe DNA. Histone acetylation is a prominent example of an epigenetic mechanism regulating the expression of numerous genes by altering chromatin accessibility. The goal of this study was to determine whether inhibition of histone deacetylases (HDAC) affects the expression of Nox isoforms and reduces pulmonary hypertension. In immune cells, we found that multiple HDAC inhibitors robustly decreased Nox2 mRNA and protein expression in a dose-dependent manner concomitant with reduced superoxide production. This effect was not restricted to Nox2 as expression of Nox1, Nox4 and Nox5 was also reduced by HDAC inhibition. Surprisingly, Nox promoter-luciferase activity was unchanged in the presence of HDAC inhibitors. In macrophages and lung fibroblasts, ChIP experiments revealed that HDAC inhibitors block the binding of RNA polymerase II and the histone acetyltransferase p300 to the Nox2, Nox4 and Nox5 promoter regions and decrease histones activation marks (H3K4me3 and H3K9ac) at these promoter sites. We further show that the ability of CRISPR-ON to drive transcription of Nox1, Nox2, Nox4 and Nox5 genes is blocked by HDAC inhibitors. In a monocrotaline (MCT) rat model of PAH, multiple HDAC isoforms are upregulated in isolated pulmonary arteries, and HDAC inhibitors attenuate Nox expression in isolated pulmonary arteries and reduce indices of PAH. In conclusion, HDAC inhibitors potently suppress Nox gene expression both in vitro and in vivo via epigenetically regulating chromatin accessibility.

Bilateral neuroinflammatory processes in visual pathways induced by unilateral ocular hypertension in the rat.

BackgroundGlaucoma is one of the leading causes of irreversible blindness in the world. The major risk factor is elevated intraocular pressure (IOP) leading to progressive retinal ganglion cell (RGC) death from the optic nerve (ON) to visual pathways in the brain. Glaucoma has been reported to share mechanisms with neurodegenerative disorders. We therefore hypothesize that neuroinflammatory mechanisms in central visual pathways may contribute to the spread of glaucoma disease. The aim of the present study was to analyze the neuroinflammation processes that occur from the pathological retina to the superior colliculi (SCs) in a rat model of unilateral ocular hypertension induced by episcleral vein cauterization (EVC).ResultsSix weeks after unilateral (right eye) EVC in male Long-Evans rats, we evaluated both the neurodegenerative process and the neuroinflammatory state in visual pathway tissues. RGCs immunolabeled (Brn3a+) in ipsilateral whole flat-mounted retina demonstrated peripheral RGC loss associated with tissue macrophage/microglia activation (CD68+). Gene expression analysis of hypertensive and normotensive retinas revealed a significant increase of pro-inflammatory genes such as CCL2, IL-1β, and Nox2 mRNA expression compared to naïve eyes. Importantly, we found an upregulation of pro-inflammatory markers such as IL-1β and TNFα and astrocyte and tissue macrophage/microglia activation in hypertensive and normotensive RGC projection sites in the SCs compared to a naïve SC. To understand how neuroinflammation in the hypertensive retina is sufficient to damage both right and left SCs and the normotensive retina, we used an inflammatory model consisting in an unilateral stereotaxic injection of TNFα (25 ng/μl) in the right SC of naïve rats. Two weeks after TNFα injection, using an optomotor test, we observed that rats had visual deficiency in both eyes. Furthermore, both SCs showed an upregulation of genes and proteins for astrocytes, microglia, and pro-inflammatory cytokines, notably IL-1β. In addition, both retinas exhibited a significant increase of inflammatory markers compared to a naïve retina.ConclusionsAll these data evidence the complex role played by the SCs in the propagation of neuroinflammatory events induced by unilateral ocular hypertension and provide a new insight into the spread of neurodegenerative diseases such as glaucoma.

Globular adiponectin attenuates LPS-induced reactive oxygen species production in HepG2 cells via FoxO3A and HO-1 signaling

AimsAdiponectin has been shown to possess potent anti-oxidative properties in various experimental conditions. However, its anti-oxidative effects and underlying mechanisms have not been reported in liver cells. Herein, we investigated the effects of globular adiponectin (gAcrp) on LPS-stimulated reactive oxygen species (ROS) production and its mechanisms underlying in human hepatic cells (HepG2). Main methodsIntracellular ROS production was determined by fluorescence of 5-chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). NADPH oxidase-dependent ROS formation was determined by lucigenin-derived chemiluminescence. Messenger RNA expression level of target genes was determined by quantitative RT-PCR and protein expression was measured by Western blot analysis. Key findingsLPS-induced increase in ROS production was prevented by pretreatment with gAcrp in HepG2 cells. Furthermore, gAcrp treatment suppressed LPS-induced activation of NADPH oxidase and increase in mRNA and protein expression of Nox-4. We also found that adiponectin increased expression of FoxO3A and HO-1 and ablation of either of these genes partially restored suppression of LPS-induced ROS production and NADPH oxidase activation by gAcrp, indicating the vital role of FoxO3A and HO-1 signaling in the inhibition of ROS production and NADPH oxidase activation by gAcrp. SignificanceThese results suggest that gAcrp prevents LPS-induced ROS production and NADPH oxidase activity in HepG2 cells via FoxO3A and HO-1 signaling-dependent mechanisms. The present study demonstrated a suppressive effect of adiponectin on ROS production in liver cells and presented a novel mechanism underlying suppression of ROS production by adiponectin.

Mixed Aqueous Extract of Salvia Miltiorrhiza Reduces Blood Pressure through Inhibition of Vascular Remodelling and Oxidative Stress in Spontaneously Hypertensive Rats

Background/Aims: Salvia miltiorrhiza (SM) contains four major aqueous active ingredients, which have been isolated, purified and identified as danshensu (DSS), salvianolic acid A (Sal-A), salvianolic acid B (Sal-B) and protocatechuic aldehyde (PAL), totally abbreviated as SABP. Although SM is often used to treat various cardiovascular diseases in traditional Chinese medicine, the efficacy and function of optimal compatibility ratio of SM's active ingredients (SABP) in the prevention and treatment of cardiovascular diseases remain uncertain. This study investigated antihypertensive effect and underlying mechanisms of SABP vs. SM lyophilized powder (SMLP) in spontaneously hypertensive rats (SHR) and to establish the ratio of the optimal compatibility of DSS, Sal-A, Sal-B and PAL in improving cardiovascular functions. Methods: The SHRs were treated with either SABP or SMLP and their systolic blood pressures (SBP) were monitored. The isolated thoracic aorta of SHRs was segregated for immunohistochemistry, Hematoxylin-Eosin stain and mRNA and protein expression of NOX4, TGF-β1, Col-I, ET-1, α-SMA and Smad7. Moreover, the adventitial fibroblasts (AFs) were isolated and cultured from SD rats' aorta and the reactive oxygen species (ROS) production was determined after SABP or SMLP treatment. Results: SABP, but not SMLP, significantly reduced SBP, which were accompanied by the inhibited morphological changes in the thoracic aorta and the reduced mRNA and protein expression of NOX4, TGF-β1, Col-I, ET-1 and α-SMA, but the increased Smad 7 expression in SHRs. Moreover, SABP also resulted in a decreased ROS production in AFs of SD rats. Conclusions: These results indicate that SABP, but not SMLP, treatment potently inhibits hypertension through improvements of vascular remodeling and oxidative stress. The present study provides new evidence that the efficacy and function from optimal compatibility ratio of SM active ingredients is much better than its lyophilized powder, which represents a strategy to develop SM's new beneficial effect in improving cardiovascular functions.

NADPH oxidase 2 does not contribute to early reperfusion-associated reactive oxygen species generation following transient focal cerebral ischemia.

Excess production of reactive oxygen species (ROS) critically contributes to occurrence of reperfusion injury, the paradoxical response of ischemic brain tissue to restoration of cerebral blood flow. However, the enzymatic sources of ROS generation remain to be unclear. This study examined Nox2-containing NADPH oxidase (Nox2) expression and its activity in ischemic brain tissue following post-ischemic reperfusion to clarify the mechanism of enzymatic reaction of ROS. Male Sprague-Dawley rats were subjected to 90-minute middle cerebral artery occlusion, followed by 3 or 22.5 hours of reperfusion. Quantitative reverse transcriptase PCR and western blot assay were performed to measure mRNA and protein expression of Nox2. Lucigenin fluorescence assays were performed to assess Nox activity. Our data showed that Nox2 mRNA and protein expression levels were significantly increased (3.7-fold for mRNA and 3.6-fold for protein) in ischemic brain tissue at 22.5 hours but not at 3 hours following post-ischemic reperfusion. Similar results were obtained for the changes of NADPH oxidase activity in ischemic cerebral tissue at the two reperfusion time points. Our results suggest that Nox2 may not contribute to the early burst of reperfusion-related ROS generation, but is rather an important source of ROS generation during prolonged reperfusion.

Myocardin-related Transcription Factor Regulates Nox4 Protein Expression: LINKING CYTOSKELETAL ORGANIZATION TO REDOX STATE

TGFβ-induced expression of the NADPH oxidase Nox4 is essential for fibroblast-myofibroblast transition. Rho has been implicated in Nox4 regulation, but the underlying mechanisms are largely unknown. Myocardin-related transcription factor (MRTF), a Rho/actin polymerization-controlled coactivator of serum response factor, drives myofibroblast transition from various precursors. We have shown that TGFβ is necessary but insufficient for epithelial-myofibroblast transition in intact epithelia; the other prerequisite is the uncoupling of intercellular contacts, which induces Rho-dependent nuclear translocation of MRTF. Because the Nox4 promoter harbors a serum response factor/MRTF cis-element (CC(A/T)6GG box), we asked if MRTF (and thus cytoskeleton organization) could regulate Nox4 expression. We show that Nox4 protein is robustly induced in kidney tubular cells exclusively by combined application of contact uncoupling and TGFβ. Nox4 knockdown abrogates epithelial-myofibroblast transition-associated reactive oxygen species production. Laser capture microdissection reveals increased Nox4 expression in the tubular epithelium also during obstructive nephropathy. MRTF down-regulation/inhibition suppresses TGFβ/contact disruption-provoked Nox4 protein and mRNA expression, Nox4 promoter activation, and reactive oxygen species production. Mutation of the CC(A/T)6GG box eliminates the synergistic activation of the Nox4 promoter. Jasplakinolide-induced actin polymerization synergizes with TGFβ to facilitate MRTF-dependent Nox4 mRNA expression/promoter activation. Moreover, MRTF inhibition prevents Nox4 expression during TGFβ-induced fibroblast-myofibroblast transition as well. Although necessary, MRTF is insufficient; Nox4 expression also requires TGFβ-activated Smad3 and TAZ/YAP, two contact- and cytoskeleton-regulated Smad3-interacting coactivators. Down-regulation/inhibition of TAZ/YAP mitigates injury-induced epithelial Nox4 expression in vitro and in vivo. These findings uncover new MRTF- and TAZ/YAP-dependent mechanisms, which link cytoskeleton remodeling and redox state and impact epithelial plasticity and myofibroblast transition.

Reactive Oxygen Species Can Provide Atheroprotection via NOX4-Dependent Inhibition of Inflammation and Vascular Remodeling.

Oxidative stress is considered a hallmark of atherosclerosis. In particular, the superoxide-generating type 1 NADPH oxidase (NOX1) has been shown to be induced and play a pivotal role in early phases of mouse models of atherosclerosis and in the context of diabetes mellitus. Here, we investigated the role of the most abundant type 4 isoform (NOX4) in human and mouse advanced atherosclerosis. Plaques of patients with cardiovascular events or established diabetes mellitus showed a surprising reduction in expression of the most abundant but hydrogen peroxide (H2O2)-generating type 4 isoform (Nox4), whereas Nox1 mRNA was elevated, when compared with respective controls. As these data suggested that NOX4-derived reactive oxygen species may convey a surprisingly protective effect during plaque progression, we examined a mouse model of accelerated and advanced diabetic atherosclerosis, the streptozotocin-treated ApoE(-/-) mouse, with (NOX4(-/-)) and without genetic deletion of Nox4. Similar to the human data, advanced versus early plaques of wild-type mice showed reduced Nox4 mRNA expression. Consistent with a rather protective role of NOX4-derived reactive oxygen species, NOX4(-/-) mice showed increased atherosclerosis when compared with wild-type mice. Deleting NOX4 was associated with reduced H2O2 forming activity and attenuation of the proinflammatory markers, monocyte chemotratic protein-1, interleukin-1β, and tumor necrosis factor-α, as well as vascular macrophage accumulation. Furthermore, there was a greater accumulation of fibrillar collagen fibres within the vascular wall and plaque in diabetic Nox4(-/-)ApoE(-/-) mice, indicative of plaque remodeling. These data could be replicated in human aortic endothelial cells in vitro, where Nox4 overexpression increased H2O2 and reduced the expression of pro-oxidants and profibrotic markers. Interestingly, Nox4 levels inversely correlated with Nox2 gene and protein levels. Although NOX2 is not constitutively active unlike NOX4 and forms rather superoxide, this opens up the possibility that at least some effects of NOX4 deletion are mediated by NOX2 activation. Thus, the appearance of reactive oxygen species in atherosclerosis is apparently not always a nondesirable oxidative stress, but can also have protective effects. Both in humans and in mouse, the H2O2-forming NOX4, unlike the superoxide-forming NOX1, can act as a negative modulator of inflammation and remodeling and convey atheroprotection. These results have implications on how to judge reactive oxygen species formation in cardiovascular disease and need to be considered in the development of NOX inhibitory drugs.

Abstract 16267: NADPH Oxidase Nox4 Up-regulation Contributes to the Worsening of Pressure Overload-induced Cardiac Dysfunction in Angiopoeitin-like 2 Knockdown Mice

Background: Angiopoietin-like 2 (angptl2) is a circulating protein promoting vascular inflammation and endothelial dysfunction in mice, but little is known on its impact on cardiac function. Our previous results suggest that knocking down angptl2 in mice (KD) worsens pressure overload-induced cardiac dysfunction while preserving cerebral artery structure and endothelial function. As NADPH oxidase NOX4 is known to produce H2O2, a deleterious hypertrophic stimulus in cardiomyocytes but also a vasodilatory factor, we hypothesized that increased expression of NOX4 contributes to the aggravated cardiac dysfunction observed in KD mice. Methods/Results: Cardiac function was measured in vivo by Millar catheter in KD mice versus their wild-type (WT) littermates in response to a 6-week pressure overload induced by transverse aortic constriction (TAC). Concomitantly to a worsened cardiac remodeling and an aggravated cardiac dysfunction compared to WT-TAC mice, only KD-TAC mice displayed an increase in cardiac mRNA and protein expression of NOX4 (p<0.05 vs. WT, n=8). To specifically decrease cardiac NOX4 expression, we performed, two weeks after TAC surgery, a single i.v. injection of cardiac specific associated adenovirus AAV9 expressing a NOX4-targeted shRNA or a scrambled shRNA. In KD-TAC mice, the AAV9-shNOX4 limited cardiac hypertrophy as evidenced by a smaller heart/tibia ratio (Table, * p<0.05). Moreover, cardiac function in KD-TAC-AAV9-shNOX4 mice was partly prevented by maintaining contraction and relaxation maximal velocities and by limiting the abnormal rise of minimal pressure and end diastolic pressure caused by TAC (Table, * p<0.05). Conclusion: Angptl2 knockdown worsens cardiac hypertrophy and contractile dysfunction induced by pressure overload: cardiac up-regulation of NOX4 could contribute to these deleterious effects in angptl2 KD mice.

Abstract 19752: Nox4 Expression Regulates Inflammation in Tgfβ-treated Vascular Smooth Muscle Cells

NADPH oxidases (NOX) are a major source of reactive oxygen species in the cardiovascular system, contributing to the pathogenesis of disease. We recently reported that increased NOX4 expression in aging results in enhanced cellular and mitochondrial oxidative stress, vascular inflammation, dysfunction, and atherosclerosis. However, the role of NOX4 in the activation of inflammatory molecular pathways in vascular smooth muscle cells (VSMC) under pathophysiological conditions has not been elucidated. In this study, we investigated whether inhibition of NOX4 expression modulates proinflammatory gene expression in mouse VSMC by suppressing its expression with a novel peptide-mediated siRNA transfection approach. Using melittin derived peptide p5RHH, we achieved significantly higher transfection efficiency (92% vs 85% with Lipofectamine) and decreased toxicity (p<0.01 vs Lipofectamine in MTT assay). TGFβ, a proinflammatory cytokine, known to induce NOX4 expression significantly upregulated NOX4 mRNA (p<0.01) and protein expression (p<0.01) in VSMC. p5RHH mediated NOX4 siRNA transfection attenuated TGFβ induced upregulation of NOX4 mRNA (p<0.01) and protein (p<0.01) levels. Analysis of inflammatory marker genes, CCL2, CCL5, IL6, and VCAM-1, showed that NOX4 knockdown significantly attenuated the expression of these genes in TGFβ-treated VSMC, with the highest inhibition seen in the expression of IL6. Congruent with this, inflammatory marker gene expression, which increases with aging, was significantly lower in VSMC isolated from aged (16 months old) NOX4 knockout mice compared with the aged wild-type. TGFβ-activated kinase 1 (TAK1, also known as MAP3K7) is an upstream regulator of inflammatory signaling in cells treated with cytokines. p5RHH mediated TAK1 siRNA transfection downregulated AP1 and NFκB activation and inhibited NOX4 expression in wild-type VSMC, indicating that TAK1 regulates NOX4 expression via regulation of AP1 and NFκB. Because plasma TGFβ levels increase with aging, it may be of one of the factors contributing to increased NOX4 expression with aging. Our data indicate that NOX4 is a potential therapeutic target for modulation of inflammation in aging and aging-associated cardiovascular disease.

32 Premature vascular ageing in hypertension: role of aldosterone and nadph oxidases

In hypertension, the vasculature undergoes premature ageing. The role of aldosterone and NADPH oxidases (Nox’s) in the molecular mechanisms of age-associated vascular damage is unclear. We hypothesise that premature ageing in hypertension is due to increased aldosterone-induced Nox activation. We assessed vascular ageing in resistance arteries and vascular smooth muscle cells (VSMCs) from adult WKY (18 weeks), aged WKY (52 weeks) and adult stroke-prone spontaneously hypertensive (SHRSP) rats. Blood pressure (BP) was measured by tail-cuff. Vascular structure and function were analysed by pressure and wire myography. Gene and protein expression were assessed by qPCR and immunoblotting. We observed elevated BP, endothelial dysfunction and hypercontractility in vessels from SHRSP (vs. WKY). Vascular contraction in aged WKY rats was similar to SHRSP rats. Increased stiffness was observed in mesenteric arteries from aged WKY and SHRSP rats (vs. WKY). Nox2, NoxA1 and NoxO1 mRNA expression was increased (p

Curcuminoids Modulate the PKCδ/NADPH Oxidase/Reactive Oxygen Species Signaling Pathway and Suppress Matrix Invasion during Monocyte-Macrophage Differentiation.

Monocyte recruitment and invasion play critical roles in the initiation and progression of atherosclerosis. The reduction in monocyte adhesion and infiltration is thought to exert antiatherosclerotic effects. Curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are the major active components of curcuminoids and exhibit several biological activities, including anti-inflammatory, anticarcinogenic, and hypocholesterolemic activities. The aim of this study was to investigate the antiatherogenic effects and mechanisms of curcuminoids during monocyte to macrophage differentiation. The results showed that curcumin, DMC, and BDMC (20 μM) suppressed matrix invasion from 100.0 ± 5.0% to 24.8 ± 1.4%, 26.6 ± 2.9%, and 33.7 ± 1.7%, respectively, during PMA-induced THP-1 differentiation. We found that curcuminoids significantly reduced PMA-induced CD11b and MMP-9 expression by THP-1 cells. Production of reactive oxygen species (ROS) induced by PMA (126.7 ± 2.1%) was markedly attenuated by curcumin, DMC, and BDMC to 99.5 ± 7.8%, 87.8 ± 8.2%, and 89.8 ± 7.6%, respectively, resulting in the down-regulation of CD11b and MMP-9 expression. We demonstrated that curcuminoids inhibited NADPH oxidase through the down-regulation of NOX2 expression and the reduction of p47phox membrane translocation. Moreover, we found involvement of PKCδ in the PMA-induced NOX2, CD11b, and MMP-9 mRNA expression. Curcumin, DMC, and BDMC decreased the active form of PKCδ protein stimulated by PMA in THP-1 cells. Overall, our results reveal that curcuminoids suppress matrix invasion through the inhibition of the PKCδ/NADPH oxidase/ROS signaling pathway during monocyte-macrophage differentiation.

Abstract P097: Molecular Mechanisms of VEGFR Inhibition-induced Endothelial Cell Damage

VEGF/VEGFR inhibitors, used as anti-angiogenic drugs to treat cancer, induce severe hypertension. Molecular mechanisms whereby VEGF inhibitors cause hypertension are unclear, but nitric oxide (NO) and oxidative stress may be involved. We questioned whether reactive oxygen species (ROS) and Ang II, important regulators of vascular function in hypertension, also play a role in VEGF inhibitor-induced vascular dysfunction. Human microvascular endothelial cells (HMECs) were stimulated with vatalanib (VAT-VEGFR inhibitor) and gefitinib (GEF-EGFR inhibitor) in the absence/presence of Ang II. Activation of eNOS and MAPKs were assessed by immunoblotting. Antioxidant enzyme mRNA was analysed by qPCR. Microparticle levels were measured by flow cytometry. Endothelial microparticles, biomarkers of endothelial damage, tend to increase in subjects treated with VEGFR inhibitors. Phosphorylation of eNOS activation site (Ser1177) (28.3% ± 7.1) was decreased by VAT, while no changes were observed after exposure of HMECs to GEF (p<0.05). VAT decreased mRNA expression of Nox4 (0.5 ± 0.2) and H2O2-regulating antioxidants enzymes such as catalase (0.4 ± 0.1) and glutathione peroxidase (0.4 ± 0.1), while increased mRNA levels of Nox5 (3.35±1.1) (p<0.05 vs. veh). Ang II stimulation increased eNOS (171.2% ± 17.4) and ERK1/2 (177.5% ± 38.5) activation (p<0.05); all effects were blocked only by GEF. Inhibition of VEGFR also blocked Ang II effects on SOD1 (1.33 ± 0.1), HO-1 (1.6 ± 0.3) and NQO1 (1.6 ± 0.3) mRNA levels (p<0.05). In addition, Ang II increased Nox4 mRNA expression through VEGFR-dependent mechanisms. VEGFR1/2 and AT2R, but not AT1R, were expressed in HMEC. Ang II effects on eNOS phosphorylation were inhibited by PD123319 (AT2R antagonist) but not by losartan (AT1R antagonist). In conclusion, our data identify novel mechanisms whereby AngII, possibly through AT2R-dependent VEGFR transactivation, regulates eNOS activation, MAPK signalling and H2O2-related antioxidant enzymes. In addition to changes in NO availability, VEGFR inhibition may interfere with the redox status of endothelial cells, leading to vascular dysfunction and hypertension.

Abstract 097: Vascular Aging in Aldosterone Associated Hypertension: Role of NADPH Oxidase 1

The vascular phenotype in hypertension is characterised by features typically observed in the ageing vasculature. Pathophysiological processes underlying premature vascular aging in hypertension remains unclear but aldosterone (aldo) and oxidative stress may be important. We postulated that physiological aging is amplified in hypertension due to increased aldo-induced Nox activation and redox signalling. We used arteries from adult WKY (18 weeks), aged WKY (52 weeks) and adult stroke-prone spontaneously hypertensive (SHRSP) rats. Blood pressure was measured by tail-cuff. Vascular function/structure was analysed by myography. Gene level was assessed by qPCR and protein by immunoblotting. BP was increased in SHRSP (180.7±2.5 vs. 127±2.7 mmHg, p<0.05). Endothelial dysfunction was observed in vessels from SHRSP. Increased vascular contraction in aged WKY rats was similar to SHRSP rats (p<0.05 vs WKY). Increased vascular stiffness was observed in arteries from aged WKY and SHRSP compared to WKY rats. Nox2 (0.82±0.4/2.4±0.9 vs 0.22±0.2), NoxA1 (4.9±2/9.5±5 vs 1±0.3) and NoxO1 (1.9±0.6/4.1±1 vs 1±0.4) mRNA was increased (p<0.05; SHRSP/aged WKY vs WKY). Nox1 mRNA (2.3±0.8 vs 1.1±0.4) was only increased in SHRSP rats (p<0.05; vs WKY). Similarly, mRNA levels of MCP-1 (2.3±0.5/3.9±1.9 vs 0.3±0.1) and RANTES (7.4±2/6.3±1.7 vs 1.1±0.2), aging-related inflammatory markers, and cell cycle inhibitors, p21 (3.2±1.1/3.1±0.7 vs 1±0.1) and p27 (2.2±0.7/2±0.8 vs 0.4±0.1), were increased in SHRSP and aged WKY rats (p<0.05; SHRSP/aged WKY vs WKY). ROS production (VSMC: 1.74±0.4 AU/protein), H2AX (DNA damage; 1.3±0.1) and aldosterone (plasma; 99.5±19 pg/mL) levels were increased in SHRSP rats (p<0.05; vs WKY). Aldo-induced Nox1 mRNA expression and p66SHC activation was exacerbated in VSMCs from SHRSP rats; an effect blocked by ML171 (a Nox1 inhibitor) and blunted in VSMCs from Nox1 KO mice. In conclusion, endothelial dysfunction and vascular remodelling in hypertension are associated with increased aldo-mediated activation of pro-inflammatory and redox-sensitive pathways. These processes involve Nox1. Our findings identify an important role for aldo/Nox1/ROS in molecular processes underlying vascular changes of ageing in hypertension.

PP38 - PDIA1 overexpression activates acutely Nox1 NADPH oxidase in VSMC

Mechanisms regulating NADPH oxidase remain open and include the redox chaperone protein disulfide isomerase (PDI). We described previously that PDI transient overexpression in VSMC enhances basal Nox1 mRNA expression and activity, without any further response for AngII stimulus in ROS production. Here we investigated whether Nox1 activation mediated by PDI overexpression may involve AngII receptor (AT1R).We developed rabbit aortic VSMC with doxicycline inducible system for rat PDI (VSMC-PDI), which showed 3,5 fold increased total PDI expression after 48h of doxicycline addition. VSMC-PDI showed increased superoxide production by NADPH-triggered enriched membrane fraction (1 vs. 2,5 ± 0,03 RLU/mg protein, lucigenin reduction), which was totally inhibited by Nox1/4 inhibitor (GKT136901, 100uM). VSMC-PDI showed higher migration trajectories vs. VSMC with no stimulus (1 vs. 1,75 ± 0,04 pixels, end distance from origin by single cell migration assay for 16h). Although inhibitors of AT1R (losartan and candesartan, 10-100nM for 16h) decreased intracellular superoxide (2-hydroxyethidium quantification by HPLC) or extracellular H2O2 (AmplexRed assay) productions mediated by PDI transient overexpression, there is no direct PDI interaction with AT1R, based on several confocal microscopy analyzes in both VSMC and HEK293 cells transfected with AT1R-GFP. Since AT1R activates β-arrestin1/2 signaling, we silenced β-arrestin1/2 with further PDI induction in VSMC-PDI. Preliminary results suggest no changes in Nox1 NADPH activity. Finally, chronic VSMC-PDI (obtained after antibiotic selection of VSMC transiently transfected, 4 fold increased PDI expression) showed no more increased intracellular superoxide or Nox1 NADPH oxidase activation compared to basal VSMC. The mechanisms underlying Nox1 acute activation by PDI overexpression in VSMC does not involve direct interaction with AT1R, and might not involve β-arrestin1/2. Chronic PDI overexpression may induce VSMC redox adaptations, since Nox1 NADPH oxidase activity is decayed to basal levels.

Protective effect of allicin on high glucose/hypoxia-induced aortic endothelial cells via reduction of oxidative stress

This study was designed to explore the protective effect of allicin on aortic endothelial cell injury induced by high glucose/hypoxia and to investigate the corresponding mechanisms. The primary-cultured murine aortic endothelial cells were subcultured. The third passage of cells was adopted and randomly divided into five groups: The normal group (NG), the mannitol group (MG), the high-glucose/hypoxia group (HG), the allicin group (AG) and the protein kinase C (PKC) inhibitor group (GG). The general morphology was observed under an inverted phase-contrast microscope and cell viability was assessed using the MTT assay. Intracellular reactive oxygen species (ROS) levels in the endothelial cells were quantified using dihydroethidium staining. The levels of 8-hydroxydeoxyguanosine (8-OHdG), nuclear factor-κB (NF-κB), NADPH oxidase 4 (Nox4) and hypoxia-inducible factor-1α (HIF-1α) and the activity of PKC were measured using ELISA. A quantitative polymerase chain reaction (qPCR) was adopted to evaluate the mRNA expression of Nox4, HIF-1α and NF-κB. The altered cell morphology observed in HG was notably ameliorated in the AG and GG. The protein levels of 8-OHdG, NF-κB, Nox4, HIF-1α and PKC in the HG were higher than those in the other groups. Furthermore, the cell viability in the AG was significantly increased and the protein levels of 8-OHdG, NF-κB, Nox4, HIF-1α and PKC were significantly decreased compared with those in the HG. The ROS production was found to be increased in the HG cells, while there was a significant decrease in the AG cells. These data indicate that allicin exerts a protective effect against high glucose/hypoxia-induced injury in aortic endothelial cells through its antioxidative action, which may involve the inhibition of the PKC pathway and regulation of HIF-1α.