Nitrotyrosine Staining Research Articles

Tissue-Resident TSP1-CD47 Signaling Promotes Sickle Cell-Associated Arterial Vasculopathy and Pulmonary Hypertension

Pulmonary hypertension (PH) is a chronic pulmonary complication of sickle cell disease (SCD) and a leading cause of death in adults. The exact molecular causes of SCD-associated PH are unknown although pathologic hemolysis related to chronic nitric oxide depletion and oxidative stress are recognized as contributing factors. We have reported that the protein thrombospondin-1 (TSP1) is elevated in the plasma of patients with SCD and by interacting with its receptor CD47 limits vasodilation of distal pulmonary arteries ex vivo . We hypothesized that the TSP1-CD47 interaction may promote PH in the BERK sickle mouse model. To test this hypothesis, we inquired whether TSP1 and CD47 are upregulated in the lungs of BERK mice and patients with SCD-associated PH. We found that lungs of age matched male 13-14 months old BERK sickle mice overexpressed TSP1 and CD47 as compared to both C57BL/6J and Hemi BERK mice and also confirmed our prior findings that sickle mice develop PH as they age. Immunohistochemistry of lung sections from 6 patients with SCD-associated PH revealed increased levels of CD47 as compared to sections from patients without PH or overt lung disease. We interrogated the TSP1-CD47 axis in vivo by generating chimeric animals with a sickle erythropoiesis on a CD47KO background by transplanting BERK sickle bone marrow into C57BL/6J (n=24) and CD47 knockout (CD47KO, n=27) mice. Fully engrafted chimeric mice underwent pulmonary hemodynamic assessment after 6 months. Right ventricular (RV) pressures were lower in Sickle-to-CD47KO chimeras as compared to Sickle-to-C57BL/6J chimeras as shown by the reduced maximum pressure of the RV (22.1 ± 3.6 vs. 28.1 ± 8.8 mmHg, p=0.013) and mean pulmonary artery pressure (15.3 ± 2.6 vs. 18.8 ± 5.7 mmHg, p=0.020). The afterload of the Sickle-to-CD47KO chimeras was lower compared to Sickle-to-C57BL/6J chimeras as shown by the diminished pulmonary vascular resistance (1.2 ± 0.7 vs. 2.4 ± 2.2 Wood units, p=0.024) and RV effective arterial elastance (1.5 ± 0.9 vs. 2.7 ± 2.6 mmHg/µL, p=0.052). The RV diastolic function as measured by the RV dP/dtmin also showed a trend towards improvement as compared to Sickle-to-C57BL/6J chimeras (-1297.0 ± 318.2 vs. -1604.0 ± 668.2 mmHg/s, p=0.059). Finally, the Sickle-to-CD47KO chimeras had a tendency for less RV hypertrophy as evidenced by lower RV free wall weight as compared to Sickle-to-C57BL/6J chimeras (21.45 ± 5.0 vs. 24.63 ± 6.0 mg, p=0.056). Interestingly, plasma levels of soluble TSP1 were reduced in Sickle-to-CD47KO chimeras (61.45 ± 10.11 vs. 76.89 ± 10.84 pg/mL, p=0.012). On myography, aortic segments from Sickle-to-CD47KO chimeras had improved relaxation to the endothelial activator acetylcholine. We hypothesized that in SCD TSP1-CD47 signaling promotes PH, in part, by increasing ROS generation. Treatment with exogenous TSP1 (2.2x10-9 M, a concentration found in the plasma of SCD patients) stimulated increased levels of superoxide and hydrogen peroxide in human pulmonary artery endothelial cells by cytochrome C and Amplex Red assays, respectively. Furthermore, lungs of CD47KO chimeras had decreased oxidative damage as measured by reduced 4-hydroxynonenal and 3-nitrotyrosine staining vs. Sickle-to-C57BL/6J chimeras. Finally, suppression of CD47 with a morpholino (10 mg/kg body weight i.p. weekly) for 8 weeks (n=8 animals) also reduced pulmonary pressures in BERK mice. Our results show that genetic deletion or suppression of CD47 ameliorates SCD-associated PH, which may be due, in part, to decreased ROS levels. Targeting TSP1-CD47 may provide a new molecular approach to the treatment of SCD-associated PH. DisclosuresIsenberg:Tioma Therapeutics: Equity Ownership; Radiation Control Technologies, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Abstract 238: S-nitrosoglutathione Reductase (GSNOR) Plays a Critical Role in Placental Vascularization Working Through the VEGF-NO Pathway

Introduction: Preeclampsia (PE), a leading cause of maternal and fetal mortality and morbidity, is characterized by increased levels of reactive oxygen species (ROS) and S-nitrosylated protein, and decreased levels of the antioxidant, ascorbate (Asc), which is required for the release of nitric oxide (NO) from nitrosylated proteins. Mice lacking S-nitrosoglutathione reductase (GSNOR –/– ), a denitrosylase that regulates protein S-nitrosylation, exhibit a PE-like phenotype including maternal hypertension, cardiac concentric hypertrophy and impaired placental vascularization. We hypothesized that impaired placental vascularization, one of the primary causes of preeclampsia is mediated by alteration in S-nitrosylation of the VEGF-NO pathway, and ascorbate treatment rescues this pathologic phenotype. Methods: Pregnant GSNOR –/– and control (WT) mice (n=5-7) were studied at late pregnancy (day 17.5). Ascorbate was provided in drinking water beginning at day 0.5. Fetoplacental capillary density was determined from isolectin staining and reactive nitrosative stress determined from nitrotyrosine staining in placental sections. S-nitrosylation of VEGF was determined using SNO-Rac and eNOS levels by Western blot analysis. Results: Fetoplacental capillary density was reduced 19% in GSNOR –/– fetuses compared to WT (P<0.001). GSNOR –/– placentas exhibited higher nitrotyrosine staining than WT placentas, indicating the presence of nitrosative stress. These increases were associated with reduced level of eNOS protein (P<0.05) and decreased S-nitrosylation of VEGF (P<0.05) in the GSNOR –/– placentas as compared to WT. Ascorbate treatment decreased nitrotyrosine staining, and increased fetoplacental capillary density ~10% (P<0.001), eNOS protein levels (P<0.05) and S-nitrosylation of VEGF (P<0.05) in the GSNOR –/– placentas as compared to WT. Conclusion: These findings suggest that GSNOR plays an essential role in promoting placental vascularization in part working through the VEGF-NO pathway. Ascorbate treatment rescued the nitrosative stress and improved placental vascularization, suggesting that it can be used therapeutically to treat or prevent preeclampsia.

Conditional Deletion of Bmal1 Accentuates Microvascular and Macrovascular Injury

The brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1 constitutes a major transcriptional regulator of the circadian clock. Here, we explored the impact of conditional deletion of Bmal1 in endothelium and hematopoietic cells in murine models of microvascular and macrovascular injury. We used two models of Bmal1fx/fx;Tek-Cre mice, a retinal ischemia/reperfusion model and a neointimal hyperplasia model of the femoral artery. Eyes were enumerated for acellular capillaries and were stained for oxidative damage markers using nitrotyrosine immunohistochemistry. LSK (lineage-negative, stem cell antigen-1-positive, c-Kit-positive) cells were quantified and proliferation assessed. Hematopoiesis is influenced by innervation to the bone marrow, which we assessed using IHC analysis. The number of acellular capillaries increased threefold, and nitrotyrosine staining increased 1.5-fold, in the retinas of Bmal1fx/fx;Tek-Cre mice. The number of LSK cells from the Bmal1fx/fx;Tek-Cre mice decreased by 1.5-fold and was accompanied by a profound decrease in proliferative potential. Bmal1fx/fx;Tek-Cre mice also exhibited evidence of bone marrow denervation, demonstrating a loss of neurofilament-200 staining. Injured femoral arteries showed a 20% increase in neointimal hyperplasia compared with similarly injured wild-type controls. Our study highlights the importance of the circadian clock in maintaining vascular homeostasis and demonstrates that specific deletion of BMAL1 in endothelial and hematopoietic cells results in phenotypic features similar to those of diabetes.

C-X-C Chemokine Receptor Type 4 Plays a Crucial Role in Mediating Oxidative Stress-Induced Podocyte Injury.

Oxidative stress plays a role in mediating podocyte injury and proteinuria. However, the underlying mechanism remains poorly understood. In this study, we investigated the potential role of C-X-C chemokine receptor type 4 (CXCR4), the receptor for stromal cell-derived factor 1α (SDF-1α), in mediating oxidative stress-induced podocyte injury. In mouse model of adriamycin nephropathy (ADR), CXCR4 expression was significantly induced in podocytes as early as 3 days. This was accompanied by an increased upregulation of oxidative stress in podocyte, as demonstrated by malondialdehyde assay, nitrotyrosine staining and secretion of 8-hydroxy-2'-deoxyguanosine in urine, and induction of NOX2 and NOX4, major subunits of NADPH oxidase. CXCR4 was also induced in human kidney biopsies with proteinuric kidney diseases and colocalized with advanced oxidation protein products (AOPPs), an established oxidative stress trigger. Using cultured podocytes and mouse model, we found that AOPPs induced significant loss of podocyte marker Wilms tumor 1 (WT1), nephrin, and podocalyxin, accompanied by upregulation of desmin both in vitro and in vivo. Furthermore, AOPPs worsened proteinuria and aggravated glomerulosclerosis in ADR. These effects were associated with marked activation of SDF-1α/CXCR4 axis in podocytes. Administration of AMD3100, a specific inhibitor of CXCR4, reduced proteinuria and ameliorated podocyte dysfunction and renal fibrosis triggered by AOPPs in mice. In glomerular miniorgan culture, AOPPs also induced CXCR4 expression and downregulated nephrin and WT1. Innovation and Conclusion: These results suggest that chemokine receptor CXCR4 plays a crucial role in mediating oxidative stress-induced podocyte injury, proteinuria, and renal fibrosis. CXCR4 could be a new target for mitigating podocyte injury, proteinuria, and glomerular sclerosis in proteinuric chronic kidney disease. Antioxid. Redox Signal. 27, 345-362.

Effects of aerobic exercise training on metabolism of nitric oxide and endothelin-1 in lung parenchyma of rats with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by vasoconstriction and proliferative obstruction of pulmonary vessels, which promotes a progressive increase in pulmonary vascular resistance (PVR). The effect of exercise training on oxidative stress, metabolism, and markers of nitric oxide (NO) and endothelin-1 (ET-1) was analyzed in the lung tissue of rats with PAH induced by monocrotaline (MCT).Twenty-four Wistar rats were divided into four groups (5-7 animals): sedentary control (SC), sedentary MCT (SM), trained control (TC), and trained MCT (TM). The TC and TM groups participated in a treadmill training protocol (60% VO2 max) for 5weeks, 3weeks of which were performed after the injection of MCT (60mg/kg i.p.) or saline. MCT administration promoted an increase in PVR and right ventricle hypertrophy, and reduction of right ventricle systolic function assessed by echocardiography. These changes were not improved by exercise training. The activity of NO synthase was reduced in the animals of the TC, TM, and SM groups. No significant differences were found in total nitrite concentration and expression of endothelial NO synthase. Moreover, the TM group showed strong staining for iNOS and nitrotyrosine, suggesting an increase in oxidative stress in these animals. In parallel, reduced expression of type B ET-1 receptors was noticed in the SM and TM groups in comparison to controls. In conclusion, the aerobic training protocol was unable to mitigate changes in the metabolism of NO and ET-1, probably because of the disease severity in these animals, especially in the TM group.

Moringa oleifera Seeds Attenuate Vascular Oxidative and Nitrosative Stresses in Spontaneously Hypertensive Rats.

Moringa oleifera (MOI) is a tree currently used in traditional medicine in tropical Africa, America, and Asia for therapeutic applications in several disorders including arterial hypertension. We previously described a cardiac protective role of a treatment with MOI seeds in spontaneously hypertensive rats (SHR). Here, we investigated the effects of this treatment on oxidative and nitrosative vascular stresses in SHR, with normotensive Wistar Kyoto rats used as controls. Oxidative and nitrosative stresses detected in SHR aortas using the fluorescent dye dihydroethidine and protein nitrotyrosine staining were reduced in MOI-treated SHR aortas. This was associated with a decrease of free 8-isoprostane circulating level, vascular p22phox and p47phox expressions, and SOD2 upregulation. Moreover, circulating nitrites and C-reactive protein, increased in SHR, were both reduced in SHR receiving MOI. This was associated to decrease iNOS and NF-κB protein expressions after MOI treatment. In functional studies, the endothelium-dependent carbachol-induced relaxation was improved in MOI-treated SHR resistance arteries. Oral administration of MOI seeds demonstrates vascular antioxidant, anti-inflammatory, and endothelial protective effects in SHR. Our data support the use of MOI seeds in diet against cardiovascular disorders associated with oxidative stress and inflammation such as hypertension, scientifically validating the use of these seeds in Malagasy traditional medicine.

Lack of the P2X7 receptor protects against AMD-like defects and microparticle accumulation in a chronic oxidative stress-induced mouse model of AMD

The P2X7 receptor (P2X7R) is an ATP-gated ion channel that is a key player in oxidative stress under pathological conditions. The P2X7R is expressed in the retinal pigmented epithelium (RPE) and neural retina. Chronic oxidative stress contributes to the pathogenesis of age-related macular degeneration (AMD). Mice lacking Cu, Zn superoxide dismutase (Sod1) developed chronic oxidative stress as well as AMD-like features, but whether the P2X7R plays a causative role in oxidative stress-induced AMD is unknown. Thus, the main purpose of this study was to test if concurrent knockout (KO) of P2X7R could block AMD-like defects seen in Sod1 KO mice. Using multiple approaches, we demonstrate that Sod1 KO causes AMD-like defects, including positive staining for oxidative stress markers, 3-nitrotyrosine and carboxymethyl lysine, thinning of the RPE and retina, thickening of Bruch's membrane, presence of basal laminar and linear deposits, RPE barrier disruption and accumulation of microglia/macrophages. Moreover, we find that Sod1 KO mice accumulate more microparticles (MPs) within RPE/choroid tissues. Concurrent KO of the P2X7R protects against AMD-like defects and MP accumulation in Sod1 KO mice. Together, we show for the first time, that deficiency of P2X7R prevents in vivo oxidative stress-induced accumulation of MPs and AMD-like defects. This work could potentially lead to novel therapies for AMD and other oxidative stress-driven diseases.

Abstract 012: Involvement of Drp1, A Mitochondrial Fission Inducer, in Angiotensin Ii-induced Hypertensive Vascular Remodeling in vitro and in vivo

Mitochondrial dysfunction has been implicated in various types of cardiovascular diseases which may involve overload and de-compensation in mitochondrial quality/quantity control. However, limited mechanistic insight is available regarding the contribution and mechanism of mitochondrial quality control in hypertension. In the present study, we tested our hypothesis that enhancement of mitochondrial fission via Drp1 activation in vascular smooth muscle cells (VSMCs) is involved in hypertensive vascular remodeling. Rat aortic VSMCs pretreated with adenovirus encoding Drp1 siRNA (Ad-siDrp1) or control non-silencing RNA (100 moi) were stimulated with 100 nM angiotensin II (AngII) up to 72 h. 8 week old male C57/BL6 mice were infused with (1000 ng/kg/min) for 2 weeks with or without treatment of Drp1 inhibitor mdivi1 (25 mg/kg ip every other day). In VSMCs, AngII induced transient mitochondrial fission (max at 2-4 h assessed by mito-tracker staining) associated with Drp1 phosphorylation at Ser616 (10-30 min). Pretreatment of ad-siDrp1 (100 moi) or mdivi1 (5 μM) attenuated AngII-induced mitochondrial fission. Ad-siDrp1 or mdivi1 also attenuated AngII-induced enhancements of mitochondrial reactive oxygen species (ROS) generation, total cell protein, cell volume and extracellular collagen content. In mice, mdivi1 significantly suppressed vascular hypertrophy and perivascular fibrosis induced by AngII in aorta, heart and kidney. mdivi1 also inhibited AngII-induced left ventricular hypertrophy assessed by heart weight body weight ratio (mg/g: 7.8±0.9 vs 6.3±0.2 p<0.01) as well as by echocardiogram. However, mdivi1 did not affect hypertension induced by AngII assessed by telemetry (mean arterial pressure: sham 150±8 vs mdivi1 155±7 mmHg). KDEL and nitro-tyrosine staining of the heart and kidney suggest attenuation of vascular ER stress and oxidative stress, respectively. In conclusion, this data suggests that Drp1-dependent mitochondrial fission contributes to AngII-induced cardiovascular remodeling independently of hypertension via enhancement of mitochondrial ROS and ER stress in target organs.

Endoplasmic reticulum stress inhibition reduces hypertension through the preservation of resistance blood vessel structure and function.

Our purpose was to determine if endoplasmic reticulum stress inhibition lowers blood pressure (BP) in hypertension by correcting vascular dysfunction. The spontaneously hypertensive rat (SHR) was used as a model of human essential hypertension with its normotensive control, the Wistar Kyoto rat. Animals were subjected to endoplasmic reticulum stress inhibition with 4-phenylbutyric acid (4-PBA; 1 g/kg per day, orally) for 5 weeks from 12 weeks of age. BP was measured weekly noninvasively and at endpoint with carotid arterial cannulation. Small mesenteric arteries were removed for vascular studies. Function was assessed with a Mulvany-Halpern style myograph, and structure was assessed by measurement of medial-to-lumen ratio in perfusion fixed vessels as well as three-dimensional confocal reconstruction of vessel wall components. Endoplasmic reticulum stress was assessed by quantitative real time-PCR and western blotting; oxidative stress was assessed by 3-nitrotyrosine and dihydroethidium staining. 4-PBA significantly lowered BP in SHR (vehicle 206.1 ± 4.3 vs. 4-PBA 178.9 ± 3.1, systolic) but not Wistar Kyoto. 4-PBA diminished contractility and augmented endothelial-dependent vasodilation in SHR small mesenteric arteries, as well as reducing media-to-lumen ratio. 4-PBA significantly reduced endoplasmic reticulum stress in SHR resistance vessels. Normotensive resistance vessels, treated with the endoplasmic reticulum stress-inducing agent, tunicamycin, show decreased endothelial-dependent vasodilation; this was improved with 4-PBA treatment. 3-Nitrotyrosine and dihydroethidium staining indicated that endoplasmic reticulum stress leads to reactive oxygen species generation resolvable by 4-PBA treatment. Endoplasmic reticulum stress caused endothelial-mediated vascular dysfunction contributing to elevated BP in the SHR model of human essential hypertension.

Vascular endothelial-cadherin downregulation as a feature of endothelial transdifferentiation in monocrotaline-induced pulmonary hypertension.

Increased pulmonary vascular resistance in pulmonary hypertension (PH) is caused by vasoconstriction and obstruction of small pulmonary arteries by proliferating vascular cells. In analogy to cancer, subsets of proliferating cells may be derived from endothelial cells transitioning into a mesenchymal phenotype. To understand phenotypic shifts transpiring within endothelial cells in PH, we injected rats with alkaloid monocrotaline to induce PH and measured lung tissue levels of endothelial-specific protein and critical differentiation marker vascular endothelial (VE)-cadherin. VE-cadherin expression by immonoblotting declined significantly 24 h and 15 days postinjection to rebound to baseline at 30 days. There was a concomitant increase in transcriptional repressors Snail and Slug, along with a reduction in VE-cadherin mRNA. Mesenchymal markers α-smooth muscle actin and vimentin were upregulated by immunohistochemistry and immunoblotting, and α-smooth muscle actin was colocalized with endothelial marker platelet endothelial cell adhesion molecule-1 by confocal microscopy. Apoptosis was limited in this model, especially in the 24-h time point. In addition, monocrotaline resulted in activation of protein kinase B/Akt, endothelial nitric oxide synthase (eNOS), nuclear factor (NF)-κB, and increased lung tissue nitrotyrosine staining. To understand the etiological relationship between nitrosative stress and VE-cadherin suppression, we incubated cultured rat lung endothelial cells with endothelin-1, a vasoconstrictor and pro-proliferative agent in pulmonary arterial hypertension. This resulted in activation of eNOS, NF-κB, and Akt, in addition to induction of Snail, downregulation of VE-cadherin, and synthesis of vimentin. These effects were blocked by eNOS inhibitor N(ω)-nitro-l-arginine methyl ester. We propose that transcriptional repression of VE-cadherin by nitrosative stress is involved in endothelial-mesenchymal transdifferentiation in experimental PH.

Impact of maternal cigarette smoke exposure on brain inflammation and oxidative stress in male mice offspring.

Maternal cigarette smoke exposure (SE) during gestation can cause lifelong adverse effects in the offspring’s brain. Several factors may contribute including inflammation, oxidative stress and hypoxia, whose changes in the developing brain are unknown. Female Balb/c mice were exposed to cigarette smoke prior to mating, during gestation and lactation. Male offspring were studied at postnatal day (P) 1, P20 and 13 weeks (W13). SE dams had reduced inflammatory mediators (IL-1β, IL-6 and toll like receptor (TLR)4 mRNA), antioxidant (manganese superoxide dismutase (MnSOD)), and increased mitochondrial activities (OXPHOS-I, III and V) and protein damage marker nitrotyrosine. Brain hypoxia-inducible factor (HIF)1α and its upstream signalling molecule early growth response factor (EGR)1 were not changed in the SE dams. In the SE offspring, brain IL-1R, IL-6 and TLR4 mRNA were increased at W13. The translocase of outer mitochondrial membrane, and MnSOD were reduced at W13 with higher nitrotyrosine staining. HIF-1α was also increased at W13, although EGR1 was only reduced at P1. In conclusion, maternal SE increased markers of hypoxia and oxidative stress with mitochondrial dysfunction and cell damage in both dams and offspring, and upregulated inflammatory markers in offspring, which may render SE dams and their offspring vulnerable to additional brain insults.

Hydrogen-rich water ameliorates bronchopulmonary dysplasia (BPD) in newborn rats.

Bronchopulmonary dysplasia (BPD) is characterized by developmental arrest of the alveolar tissue. Oxidative stress is causally associated with development of BPD. The effects of hydrogen have been reported in a wide range of disease models and human diseases especially caused by oxidative stress. We made a rat model of BPD by injecting lipopolysaccharide (LPS) into the amniotic fluid at E16.5. The mother started drinking hydrogen-rich water from E9.5 and also while feeding milk. Hydrogen normalized LPS-induced abnormal enlargement of alveoli at P7 and P14. LPS increased staining for nitrotyrosine and 8-OHdG of the lungs, and hydrogen attenuated the staining. At P1, LPS treatment decreased expressions of genes for FGFR4, VEGFR2, and HO-1 in the lungs, and hydrogen increased expressions of these genes. In contrast, LPS treatment and hydrogen treatment had no essential effect on the expression of SOD1. Inflammatory marker proteins of TNFα and IL-6 were increased by LPS treatment, and hydrogen suppressed them. Treatment of A549 human lung adenocarcinoma epithelial cells with 10% hydrogen gas for 24 hr decreased production of reactive oxygen species in both LPS-treated and untreated cells. Lack of any known adverse effects of hydrogen makes hydrogen a promising therapeutic modality for BPD. Pediatr Pulmonol. 2016; 51:928-935. © 2016 Wiley Periodicals, Inc.

Topiroxostat Showed Renoprotection via Various Mechanisms Including Suppression of Xanthine Oxidoreductase-Induced-Oxidative Stress and Inflammation in db/db Mice

Background: Xanthine oxidoreductase (XOR) produces superoxide along with uric acid. This reactive oxygen species induces organ damage. Therefore, we aimed to investigate whether XOR-induced superoxide is associated with renal abnormality in diabetic mice. Methods: Db/db mice (9-week-old) were fed standard diets with or without topiroxostat (1, 3, and 10 mg/kg/day) or febuxostat (3, 10, 30, and 45 mg/kg/day) for 4 weeks. Furthermore, topiroxostat (1 and 3 mg/kg/day) and febuxostat (3 mg/kg/day) were administered for 7 days or 8 weeks. The levels of urinary albumin excretion (UAE), monocyte chemoattractant protein-1 (MCP-1), and angiotensin II were determined. For renal pathological or mechanistic analysis, periodic acid-Schiff (PAS), nitrotyrosine, and MCP-1 staining or renal gene expression, XOR activity, and purine body levels were evaluated. Results: Both topiroxostat and febuxostat suppressed UAE : topiroxostat 3 mg/kg/day, 11.5 ± 33 μg/day versus vehicle 253.7 ± 69 μg/day, p<0.05; febuxostat 3 mg/kg/day, 255 ± 92 μg/day versus vehicle 634 ± 101 μg/day, p=0.05, for 4 weeks, respectively, and reduced immunohistological staining of enhanced oxidative stress and inflammation markers without affecting glycated hemoglobin. Topiroxostat but not febuxostat decreased urinary MCP-1 and angiotensin II levels and attenuated morphological hypertrophy in the glomerulus and epithelial cell enlargement (diameter and height) in the proximal tubules. In the early stage, topiroxostat and febuxostat decreased renal XOR activity in response to increased purine body levels (topiroxostat 3 mg/kg/day, 2.25 ± 0.19 mg/g tissue, p=0.07; febuxostat 3 mg/kg/day, 2.34 ± 0.09 mg/g tissue, p<0.05 to vehicle 2.01 ± 0.12 mg/g tissue, for 7 days) relative to the vehicle. Moreover, febuxostat upregulated the gene expression of renin 1 (Ren1) and downregulated that of adenine phosphoribosyltransferase (APRT) and hypoxanthine guanine phosphoribosyltransferase (HPRT) compared with that of the diabetic control. Conclusion: Topiroxostat and febuxostat exhibited renoprotective effects by suppressing oxidative stress and inflammation, and enhancing the salvage pathway via XOR inhibition.

Abstract 12177: Important Roles of Endothelial Caveolin-1/eNOS Complex in Maintaining Microvascular Homeostasis Through Endothelium-dependent Hyperpolarization Mediated Mechanism

Background: The endothelium plays important roles in maintaining cardiovascular homeostasis by synthesizing and releasing several vasodilators, including prostacyclin (PGI2), nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) factor. It is widely accepted that EDH plays important roles especially in microcirculation. We have previously demonstrated that endothelial NO synthase (eNOS) plays diverse roles depending on vessel size, as a NO generating system in conduit arteries and an EDH-mediated system in resistance arteries. Recently, we also have reported that caveolin-1 is involved in enhanced EDH-mediated mechanisms in resistance arteries through functional inhibition of eNOS. However, the detailed functions of caveolin-1 in the endothelium remain to be elucidated. Material and Results: We newly generated endothelium-specific caveolin-1-knockout mice (eCav-1-KO) under the control of Tie2 promoter. We confirmed that caveolae is absent in endothelial cells of those mice. Co-immunoprecipitation experiments showed the eNOS/Cav-1 complex formation in control mice, but not in eCav-1-KO mice. Western blot experiments demonstrated that the extent of eNOS phosphorylation at Ser1177 (stimulatory site) was comparable, whereas that at Thr495 (inhibitory site) was significantly reduced in mesenteric arteries from eCav-1-KO mice as compared with control mice, demonstrating hyperactivation of eNOS. Isometric tension experiments and Langendorff experiments showed that NO-mediated responses were significantly enhanced, whereas EDH-mediated responses were significantly reduced in microcirculations in eCav-1-KO mice. Importantly, eCav-1-KO mice showed altered endothelial functions and cardiac hypertrophy despite normal blood pressure level. Furthermore, the hearts from eCav-1-KO mice showed enhanced staining for nitrotyrosine, a marker of nitrosative stress in microvessels and epicardial arteries, whereas those from control mice only showed mild staining in epicardial arteries alone. Conclusions: These results indicate that endothelial Cav-1/eNOS complex plays important roles in maintaining microvascular homeostasis through EDH-mediated mechanisms in mice in vivo.

Plasmalogen modulation attenuates atherosclerosis in ApoE- and ApoE/GPx1-deficient mice

Background and aimWe previously reported a negative association of circulating plasmalogens (phospholipids with proposed atheroprotective properties) with coronary artery disease. Plasmalogen modulation was previously demonstrated in animals but its effect on atherosclerosis was unknown. We assessed the effect of plasmalogen enrichment on atherosclerosis of murine models with differing levels of oxidative stress. Methods and resultsSix-week old ApoE- and ApoE/glutathione peroxidase-1 (GPx1)-deficient mice were fed a high-fat diet with/without 2% batyl alcohol (precursor to plasmalogen synthesis) for 12 weeks. Mass spectrometry analysis of lipids showed that batyl alcohol supplementation to ApoE- and ApoE/GPx1-deficient mice increased the total plasmalogen levels in both plasma and heart. Oxidation of plasmalogen in the treated mice was evident from increased level of plasmalogen oxidative by-product, sn-2 lysophospholipids. Atherosclerotic plaque in the aorta was reduced by 70% (P = 5.69E-07) and 69% (P = 2.00E-04) in treated ApoE- and ApoE/GPx1-deficient mice, respectively. A 40% reduction in plaque (P = 7.74E-03) was also seen in the aortic sinus of only the treated ApoE/GPx1-deficient mice. Only the treated ApoE/GPx1-deficient mice showed a decrease in VCAM-1 staining (−28%, P = 2.43E-02) in the aortic sinus and nitrotyrosine staining (−78%, P = 5.11E-06) in the aorta. ConclusionPlasmalogen enrichment via batyl alcohol supplementation attenuated atherosclerosis in ApoE- and ApoE/GPx1-deficient mice, with a greater effect in the latter group. Plasmalogen enrichment may represent a viable therapeutic strategy to prevent atherosclerosis and reduce cardiovascular disease risk, particularly under conditions of elevated oxidative stress and inflammation.

Effect of soluble guanylyl cyclase activator and stimulator therapy on nitroglycerin-induced nitrate tolerance in rats

article Chronic nitroglycerin (GTN) anti-ischemic therapy induces side effects such as nitrate tolerance and endothelial dysfunction. Both phenomena could be based on a desensitization/oxidation of the soluble guanylyl cyclase (sGC). Therefore, the present study aims at investigating the effects of the therapy with the sGC activator BAY 60-2770 and the sGC stimulator BAY 41-8543 on side effects induced by chronic nitroglycerin treatment. Male Wistar rats were treated with nitroglycerin (100 mg/kg/d for 3.5 days, s.c. in ethanol) and BAY 60-2770 (0.5 or 2.5 mg/kg/d) or BAY 41-8543 (1 and 5 mg/kg/d) for 6 days. Therapy with BAY 60-2770 but not with BAY 41-8543 improved nitroglycerin-triggered endothelial dysfunction and nitrate tolerance, corrected the decrease in aortic nitric oxide levels, improved the cGMP dependent activation of protein kinase I in aortic tissue and reduced vascular, cardiac and whole blood oxidative stress (fluorescence and chemiluminescence assays; 3- nitrotyrosine staining). In contrast to BAY 41-8543, the vasodilator potency of BAY 60-2770 was not impaired in isolated aortic ring segments from nitrate tolerant rats. sGC activator therapy improves partially the adverse effects of nitroglycerin therapy whereas sGC stimulation has only minor beneficial effects pointing to a

Neonatal exposure to mild hyperoxia causes persistent increases in oxidative stress and immune cells in the lungs of mice without altering lung structure

Preterm infants often require supplemental oxygen due to lung immaturity, but hyperoxia can contribute to an increased risk of respiratory illness later in life. Our aim was to compare the effects of mild and moderate levels of neonatal hyperoxia on markers of pulmonary oxidative stress and inflammation and on lung architecture; both immediate and persistent effects were assessed. Neonatal mice (C57BL6/J) were raised in either room air (21% O2), mild (40% O2), or moderate (65% O2) hyperoxia from birth until postnatal day 7 (P7d). The mice were killed at either P7d (immediate effects) or lived in air until adulthood (P56d, persistent effects). We enumerated macrophages in lung tissue at P7d and immune cells in bronchoalveolar lavage fluid (BALF) at P56d. At P7d and P56d, we assessed pulmonary oxidative stress [heme oxygenase-1 (HO-1) and nitrotyrosine staining] and lung architecture. The data were interrogated for sex differences. At P7d, HO-1 gene expression was greater in the 65% O2 group than in the 21% O2 group. At P56d, the area of nitrotyrosine staining and number of immune cells were greater in the 40% O2 and 65% O2 groups relative to the 21% O2 group. Exposure to 65% O2, but not 40% O2, led to larger alveoli and lower tissue fraction in the short term and to persistently fewer bronchiolar-alveolar attachments. Exposure to 40% O2 or 65% O2 causes persistent increases in pulmonary oxidative stress and immune cells, suggesting chronic inflammation within the adult lung. Unlike 65% O2, 40% O2 does not affect lung architecture.

Cinnamon and Its Metabolite Sodium Benzoate Attenuate the Activation of p21rac and Protect Memory and Learning in an Animal Model of Alzheimer's Disease.

This study underlines the importance of cinnamon, a commonly used natural spice and flavoring material, and its metabolite sodium benzoate (NaB) in attenuating oxidative stress and protecting memory and learning in an animal model of Alzheimer’s disease (AD). NaB, but not sodium formate, was found to inhibit LPS-induced production of reactive oxygen species (ROS) in mouse microglial cells. Similarly, NaB also inhibited fibrillar amyloid beta (Aβ)- and 1-methyl-4-phenylpyridinium(+)-induced microglial production of ROS. Although NaB reduced the level of cholesterol in vivo in mice, reversal of the inhibitory effect of NaB on ROS production by mevalonate, and geranylgeranyl pyrophosphate, but not cholesterol, suggests that depletion of intermediates, but not end products, of the mevalonate pathway is involved in the antioxidant effect of NaB. Furthermore, we demonstrate that an inhibitor of p21rac geranylgeranyl protein transferase suppressed the production of ROS and that NaB suppressed the activation of p21rac in microglia. As expected, marked activation of p21rac was observed in the hippocampus of subjects with AD and 5XFAD transgenic (Tg) mouse model of AD. However, oral feeding of cinnamon (Cinnamonum verum) powder and NaB suppressed the activation of p21rac and attenuated oxidative stress in the hippocampus of Tg mice as evident by decreased dihydroethidium (DHE) and nitrotyrosine staining, reduced homocysteine level and increased level of reduced glutathione. This was accompanied by suppression of neuronal apoptosis, inhibition of glial activation, and reduction of Aβ burden in the hippocampus and protection of memory and learning in transgenic mice. Therefore, cinnamon powder may be a promising natural supplement in halting or delaying the progression of AD.

The anti-inflammatory effects of palmitoylethanolamide (PEA) on endotoxin-induced uveitis in rats

The aim of this study was to investigate the effects of palmitoylethanolamide (PEA), an endogenous fatty acid amide belonging to the family of the N-acylethanolamines (NAEs), in rats subjected to endotoxin-induced uveitis (EIU). EIU was induced in male rats by a single footpad injection of 200μg lipopolysaccharide (LPS). PEA was administered intraperitoneally at 1h before and 7h after injection of LPS. Another group of animals was treated with vehicle. Dexamethasone (DEX) was administered as a positive control. Rats were sacrificed 16h after injection and the eyes tissues were collected for histology, immunohistochemical and western blot analyses. The histological evaluation of the iris–ciliary body showed an increase of neutrophilic infiltration and nuclear modification of vessel of endothelial cells. PEA treatment decreased the inflammatory cell infiltration and improved histological damage of eye tissues. In addition, PEA treatment reduced pro-inflammatory tumor necrosis factor (TNF-α) levels, protein extravasion and lipid peroxidation. Immunohistochemical analysis for intracellular adhesion molecule (ICAM)-1 and nitrotyrosine showed a positive staining from LPS-injected rats. The degree of staining for ICAM-1 and nitrotyrosine was significantly reduced in eye sections from LPS-injected rats treated with PEA. In addition, an increase of inducible nitric oxide synthase (iNOS) and nuclear factor (NF-κB) was also evaluated in inflammed ocular tissues by western blot. PEA strongly inhibited iNOS expression and nuclear NF-κB translocation. Thus, in this study we demonstrated that PEA reduces the degree of ocular inflammation in a rat model of EIU.

Effect of soluble guanylyl cyclase activator and stimulator therapy on nitroglycerin-induced nitrate tolerance in rats

Chronic nitroglycerin (GTN) anti-ischemic therapy induces side effects such as nitrate tolerance and endothelial dysfunction. Both phenomena could be based on a desensitization/oxidation of the soluble guanylyl cyclase (sGC). Therefore, the present study aims at investigating the effects of the therapy with the sGC activator BAY 60-2770 and the sGC stimulator BAY 41-8543 on side effects induced by chronic nitroglycerin treatment. Male Wistar rats were treated with nitroglycerin (100mg/kg/d for 3.5days, s.c. in ethanol) and BAY 60-2770 (0.5 or 2.5mg/kg/d) or BAY 41-8543 (1 and 5mg/kg/d) for 6days. Therapy with BAY 60-2770 but not with BAY 41-8543 improved nitroglycerin-triggered endothelial dysfunction and nitrate tolerance, corrected the decrease in aortic nitric oxide levels, improved the cGMP dependent activation of protein kinase I in aortic tissue and reduced vascular, cardiac and whole blood oxidative stress (fluorescence and chemiluminescence assays; 3-nitrotyrosine staining). In contrast to BAY 41-8543, the vasodilator potency of BAY 60-2770 was not impaired in isolated aortic ring segments from nitrate tolerant rats. sGC activator therapy improves partially the adverse effects of nitroglycerin therapy whereas sGC stimulation has only minor beneficial effects pointing to a nitroglycerin-dependent sGC oxidation/inactivation mechanism contributing to nitrate tolerance.