Peroxynitrite Levels Research Articles

Local Peroxynitrite Impairs Endothelial Transient Receptor Potential Vanilloid 4 Channels and Elevates Blood Pressure in Obesity.

Impaired endothelium-dependent vasodilation is a hallmark of obesity-induced hypertension. The recognition that Ca2+ signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca2+ signaling is compromised during obesity, but the underlying abnormality is unknown. In this regard, transient receptor potential vanilloid 4 (TRPV4) ion channels are a major Ca2+ influx pathway in endothelial cells, and regulatory protein AKAP150 (A-kinase anchoring protein 150) enhances the activity of TRPV4 channels. We used endothelium-specific knockout mice and high-fat diet-fed mice to assess the role of endothelial AKAP150-TRPV4 signaling in blood pressure regulation under normal and obese conditions. We further determined the role of peroxynitrite, an oxidant molecule generated from the reaction between nitric oxide and superoxide radicals, in impairing endothelial AKAP150-TRPV4 signaling in obesity and assessed the effectiveness of peroxynitrite inhibition in rescuing endothelial AKAP150-TRPV4 signaling in obesity. The clinical relevance of our findings was evaluated in arteries from nonobese and obese individuals. We show that Ca2+ influx through TRPV4 channels at myoendothelial projections to smooth muscle cells decreases resting blood pressure in nonobese mice, a response that is diminished in obese mice. Counterintuitively, release of the vasodilator molecule nitric oxide attenuated endothelial TRPV4 channel activity and vasodilation in obese animals. Increased activities of inducible nitric oxide synthase and NADPH oxidase 1 enzymes at myoendothelial projections in obese mice generated higher levels of nitric oxide and superoxide radicals, resulting in increased local peroxynitrite formation and subsequent oxidation of the regulatory protein AKAP150 at cysteine 36, to impair AKAP150-TRPV4 channel signaling at myoendothelial projections. Strategies that lowered peroxynitrite levels prevented cysteine 36 oxidation of AKAP150 and rescued endothelial AKAP150-TRPV4 signaling, vasodilation, and blood pressure in obesity. Peroxynitrite-dependent impairment of endothelial TRPV4 channel activity and vasodilation was also observed in the arteries from obese patients. These data suggest that a spatially restricted impairment of endothelial TRPV4 channels contributes to obesity-induced hypertension and imply that inhibiting peroxynitrite might represent a strategy for normalizing endothelial TRPV4 channel activity, vasodilation, and blood pressure in obesity.

Djulis (Chenopodium formosanum) and its bioactive compounds affect vasodilation, angiotensin converting enzyme activity, and hypertension

Djulis (Chenopodium formosanum) and its bioactive compounds' effect on vasodilation, angiotensin-converting enzyme activity (ACE) and hypertension, in vitro and in vivo, were evaluated. Rutin, betanin and another 13 compounds were observed in the water extracts of djulis (WECF) using HPLC-DAD and HPLC-MS/MS analysis. WECF, rutin and betanin showed significant induction of nitric oxide and prostacyclin as well as expression of eNOS and COX-2. In addition, WECF, rutin and betanin showed a marked inhibitory effect on ROS generation, peroxynitrite levels and ACE activity. Oral administration of WECF, rutin and betanin lowered systolic and diastolic blood pressures of spontaneously hypertensive rats. The ACE activity in the plasma of WECF-, betanin- and rutin-administrated groups was lower than the untreated group. Overall, WECF bioactive compounds could potentially be used as functional ingredients for preventing hypertension.

Mito-Specific Ratiometric Terbium(III)-Complex-Based Luminescent Probe for Accurate Detection of Endogenous Peroxynitrite by Time-Resolved Luminescence Assay.

The level of peroxynitrite (ONOO-) is closely related to the pathogenesis of a series of diseases. Hence, the accurate sensing of ONOO- is very important for a better understanding of its biological functions. Here, a time-resolved ratiometric nanoprobe (TD-DC) composed of an energy donor unit (Tb(DPA)3, where DPA signifies a dipicolinate dianion ligand) and an energy receptor unit (DC) was designed. The response of TD-DC toward ONOO- in vitro was assessed by luminescence intensity and lifetime. The results demonstrated that TD-DC could precisely (39 nM) detect ONOO- with high selectivity and efficiency (29 s). TD-DC was further successfully employed in sensing endogenous ONOO- in A-375 cells. Moreover, it could specifically localize in the mitochondrion, where endogenous ONOO- is mainly generated. The above results revealed that TD-DC is efficient and useful for real-time detection of ONOO- at subcellular levels.

Electrochemical Signature of Peroxynitrite on Modified Boron-Doped Diamond Electrodes

Endogenous and exogenous reactive oxygen and nitrogen species such as peroxynitrite have serious damaging effects on cell components and cell function. Peroxynitrite has emerged as a reactive species that is linked with oxidation and/or nitration of biomolecules. In biological context, peroxynitrite is the product of the combination between superoxide and nitric oxide radicals through a diffusion-limited reaction. The short lifespan and fast reactivity of peroxynitrite are adding more challenges to measure its concentration under physiologic conditions. Although several methods have been established for peroxynitrite detection in aqueous solutions, they are still facing the problem of real-time quantification. In this paper, the electrochemical behavior of peroxynitrite has been studied boron-doped diamond electrodes (BDD). Recently, BDD have been successfully worked as platforms for electrochemical detection of various analytes in biological and environmental systems. BDD electrodes provide a wide potential window and a low background current that creates an excellent platform to fine-tune the surface for optimized detection of peroxynitrite. In this research study, the direct electrochemical oxidation of peroxynitrite on modified BDD electrodes was recorded and investigated. A wide range of physicochemical characterizations were conducted to study the catalytic oxidation of peroxynitrite on modified BDD electrodes. The same modified BBD platform is used to develop a selective peroxynitrite sensor that can further be developed into a tool that can be used for both electrical stimulation and monitoring of peroxynitrite levels under physiologic conditions.

Protective effect of piceatannol and bioactive stilbene derivatives against hypoxia-induced toxicity in H9c2 cardiomyocytes and structural elucidation as 5-LOX inhibitors

Stilbenes with well-known antioxidant and antiradical properties are beneficial in different pathologies including cardiovascular diseases. The present research was performed to investigate the potential protective effect of resveratrol (1) and piceatannol (2), against hypoxia-induced oxidative stress in the H9c2 cardiomyoblast cell line, and the underlying mechanisms. Compounds 1 and 2 significantly inhibited the release of peroxynitrite and thiobarbituric acid levels at na no- or submicromolar concentrations, and this effect was more evident in piceatannol-treated cells, that significantly increased MnSOD protein level in a concentration dependent manner. Furthermore, since piceatannol, which is far less abundant in natural sources, displayed a higher bioactivity than the parent compound, we hereby report on a very fast synthesis and detailed structure-based design of a focused stilbene library. Finally, taking into account that hypoxia-induced ROS accumulation also increases expression and activity of 5-lipoxygenase (5-LOX) with production of leukotrienes, we have disclosed structural key factors crucial for 5-LOX activity. Among the synthesized analogues ( 3–7), compound 7 was the most effective in improving cardiomyocytes viability and in 5-LOX inhibition. In conclusion, modeling and experimental studies provided the basis for further optimization of stilbene analogues as multi-target inhibitors of the inflammatory and oxidative pathway.

Sesame Oil Attenuates Renal Oxidative Stress Induced by a High Fat Diet.

The antioxidative effects of the bioactive compounds enriched sesame oil (e.g. lignans and tocopherols) are well established. This study aims to elucidate whether sesame oil could reduce renal oxidative stress induced by a high fat diet (HFD). Mice received HFD for 12 weeks (n=7 per group), which was prepared by adding 20% (w/w) lard (lard group) or sesame oil (sesame group) to the chow diet, respectively. Compared with mice in the lard group, renal lipid levels of those in the sesame group were reduced, shown by decreases in protein expression of transcription factors and enzymes involved in fatty acid synthesis (sterol regulatory element-binding protein-1 and acetyl coenzyme A carboxylase α) and an increase in β-oxidation (peroxisome proliferator-activated receptor α and carnitine palmitoyltransferase I) (P<0.05). In the sesame group, levels of peroxynitrite and thiobarbituric acid reactive substances were also reduced, whereas the level of glutathione was increased. In addition, there was elevated protein expression levels of antioxidant enzymes regulated by nuclear factor-like 2, such as superoxide dismutase, glutathione peroxidase, and glutathione S-transferase (P<0.05), and decreased expression for nuclear factor kappa B and cyclooxygenase 2 (P<0.05). These results suggest that sesame oil could ameliorate HFD-induced renal damage by suppressing oxidative stress and inflammation.

Enhanced nitric oxide generation mitigates cadmium toxicity via superoxide scavenging leading to the formation of peroxynitrite in barley root tip

The aim of this study was to observe the possible function of increased superoxide and NO production in the response of barley root tip to the harmful level of Cd. While superoxide generation was detected only in the transition zone, the formation of NO was observed in the apical elongation zones of the control root tips. However, the root region with the superoxide generation was also associated with peroxynitrite specific fluorescence signal. Superoxide, H2O2 and peroxynitrite generation increased with Cd treatment in a dose-dependent manner. In turn, NO level increased at low 10–20 μM but decreased at high 50–60 μM Cd concentrations in comparison with the control. While co-treatment of roots with rotenone markedly attenuated the Cd-induced superoxide generation and lipid peroxidation, it increased the level of NO in the root tips. Although rotenone did not influence the Cd-induced increase of GPX activity at 10–30 μM Cd concentrations, it markedly reversed the high 40–60 μM Cd concentrations-induced decline of GPX activity. Cd-induced cell death was associated with robust superoxide generation, but not with a high level of peroxynitrite. The Cd-evoked inhibition of root growth was significantly reversed by a strong antioxidant N-acetyl cysteine but not by a peroxynitrite scavenger uric acid, suggesting that similarly to Cd-induced cell death, an imbalance in the ROS homeostasis and not an enhanced level of peroxynitrite is responsible for the Cd-induced root growth inhibition. Based on these findings, it can be assumed that NO acts mainly in the regulation of superoxide level in the tips of root. Under Cd stress, the enhanced NO level is involved in the scavenging of highly toxic superoxide through the formation of peroxynitrite, thus reducing the superoxide-mediated cell death in barley root.

Tyrosine nitrations impaired intracellular trafficking of FSHR to the cell surface and FSH-induced Akt-FoxO3a signaling in human granulosa cells.

Many infertile women suffered from poor ovarian response, and increased reactive oxygen species with age might mediate the poor ovarian response to FSH. In this study, we collected follicular fluids and isolated granulosa cells from female patients. Increased levels of peroxynitrite, tyrosine nitrations of FSH receptor (FSHR) and apoptosis were obviously detectable with decreased FSHR protein expressions in granulosa cells of the poor ovarian responders. In KGN (a human ovarian granulosa cell line) cells, exogenous peroxynitrite could sequester FSHR in the cytoplasm, and these dislocated FSHR might suffer from proteasome-mediated degradations. Here, we identified four peroxynitrite-mediated nitrated tyrosine residues of FSHR. Site-directed mutagenesis of FSHR revealed that Y626 was pivotal for intracellular trafficking of FSHR to the cell surface. Akt-induced inactivation of FoxO3a was required for the repression of FSH on granulosa cell apoptosis. However, peroxynitrite impaired FSH-induced Akt-FoxO3a signaling, while FSHR-Y626A mutant took similar effects. In addition, FoxO3a knockdown indeed impaired FSH-mediated cell survival, while FoxO3a-S253A mutant reversed that significantly.

High doses of sodium nitrate prior to exhaustive exercise increases plasma peroxynitrite levels in well-trained subjects: randomized, double-blinded, crossover study.

The aim of this study was to investigate the effect of different doses of pre-workout sodium nitrate supplementation on nitric oxide, peroxynitrite levels, and performance parameters. Ten well-trained male subjects participated in a randomized, double-blinded, crossover study. They ingested 8, 16, and 24 mmol sodium nitrate or placebo (NaCl) dissolved in water at 2.5 h before an incremental exercise test. Respiratory gases (oxygen consumption, carbon dioxide production, respiratory exchange ratio) were measured throughout the exercise trials and 3 blood samples (pre-ingestion, 2.5 h post-ingestion and postexercise) were taken to analyze nitrate/nitrite (NOx) and peroxynitrite levels. Data were analyzed using repeated-measures ANOVA at significance level of P < 0.05. NOx levels significantly increased following sodium nitrate ingestion compared with placebo (placebo: 40.86 ± 10.7 μmol/L, 8 mmol: 203.69 ± 25.1 μmol/L, 16 mmol: 289.41 ± 30.1 μmol/L, and 24 mmol: 300.95 ± 42.4 μmol/L, respectively) (P = 0.0001). However, this did not induce any significant change in oxygen consumption (P = 0.351), blood lactate concentration (P = 0.245), and time-to-exhaustion (P = 0.147). Peroxynitrite levels were similar compared with placebo when participants ingested 8 and 16 mmol of inorganic nitrate but a significant increase was observed after exercise at maximal intensity when participants were supplemented with 24 mmol (mean = 14.60 ± 1.3 μmol/L, P = 0.001). Pre-workout ingestion of high dose of sodium nitrate (24 mmol) induced peroxynitrate formation, a marker of oxidative stress. Caution must be taken regarding administration of higher doses before benefits or adverse effects are established in this population.

Ablation of B1- and B2-kinin receptors causes cardiac dysfunction through redox-nitroso unbalance

AimsB1- and B2-kinin receptors play a major role in several cardiovascular diseases. Therefore, we aimed to evaluate cardiac functional consequences of B1- and B2-kinin receptors ablation, focusing on the cardiac ROS and NO generation. Main methodsCardiac contractility, ROS, and NO generation, and protein expression were evaluated in male wild-type (WT), B1- (B1−/−) and B2-kinin (B2−/−) knockout mice. Key findingsImpaired contractility in B1−/− and B2−/− hearts was associated with oxidative stress through upregulation of NADPH oxidase p22phox subunit. B1−/− and B2−/− hearts presented higher NO and peroxynitrite levels than WT. Despite decreased sarcoplasmic reticulum Ca2+ ATPase pump (SERCA2) expression, nitration at tyrosine residues of SERCA2 was markedly higher in B1−/− and B2−/− hearts. SignificanceB1- and B2-kinin receptors govern ROS generation, while disruption of B1- and B2-kinin receptors leads to impaired cardiac dysfunction through excessive tyrosine nitration on the SERCA2 structure.

Peroxynitrite Impairs Caveolin‐1‐TRPV4 Channel Signaling in Pulmonary Hypertension

The endothelium is an important regulator of vascular resistance and overall pulmonary arterial pressure (PAP). Endothelial dysfunction in small, resistance pulmonary arteries (PAs) leads to impaired vasodilation, increased PAP, and the development of pulmonary hypertension (PH). However, the mechanisms leading to endothelial dysfunction in PH remain unknown. We recently identified endothelial TRPV4 (transient receptor potential vanilloid 4‐TRPV4EC) channels as important regulators of endothelium‐dependent vasodilation in small PAs, and showed that localized Ca2+ influx events through TRPV4EC channels activate an endothelial nitric oxide (eNOS)‐dependent vasodilatory pathway. Furthermore, inducible endothelium‐specific TRPV4 knockout (TRPV4EC−/−) mice have impaired vasodilation and elevated PAP, revealing TRPV4EC channels as critical regulators of resting PAP. However, the signaling pathways that regulate TRPV4EC channels and how they may be impaired in PH remain unknown. Caveolin‐1 (Cav‐1EC) is an important structural protein that is known to associate with TRPV4 channels in cultured endothelial cells, and global Cav‐1−/− mice were shown to have high PAP. We, therefore, hypothesized that impairment in Cav‐1EC‐TRPV4EC signaling regulation contributes to endothelial dysfunction in PH. PH was induced using a three week‐chronic hypoxia (CH) model. TRPV4EC channel function was studied using high‐speed Ca2+ imaging, which records Ca2+ influx events through TRPV4EC channels (TRPV4EC sparklets) in the intact endothelium of small PAs. Pressure myography in small PAs was used to determine the functional outcome of TRPV4EC channel signaling. Right ventricular systolic pressure (RVSP) was measured as an indicator of PAP. In normal PAs, Cav‐1EC anchoring of protein kinase C (PKC) increased TRPV4EC sparklet activity and TRPV4EC‐‐dependent vasodilation. However, in CH, decreased Cav‐1EC‐PKC localization resulted in impaired TRPV4EC sparklet activity and endotheliumdependent vasodilation. Elevated peroxynitrite (PN) levels have been shown to contribute to the development of PH. PN levels, as indicated by nitrotyrosine staining, were elevated in PAs from CH mice. PN scavenger, FeTPPS, rescued TRPV4EC sparklet activity and vasodilation in PAs obtained from CH mice. Furthermore, acute injection of FeTPPS significantly lowered PAP in CH mice. Exogenous PN significantly decreased TRPV4EC sparklet activity and vasodilation in control mice but had no effect on PAs from inducible Cav‐1EC−/− mice, indicating that the effect of PN on TRPV4EC channel activity is Cav‐1EC‐dependent. Our results support the idea that elevated PN levels in PH impairs Cav‐1EC‐PKC‐dependent potentiation of TRPV4EC channel activity. Inhibition of TRPV4EC channel activity by PN contributes to impaired vasodilation and elevated PAP observed in PH.Support or Funding Information17PRE33660762, HL138496This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Avocado Oil Alleviates Renal Damage and decreases NADPH Oxidase Activity, Peroxynitrite Production and Mitochondrial Calcium Uptake in Hypertension Rats

Hypertension (HT) has been associated with mitochondrial dysfunction due to both nitrosative and oxidative stresses that are generated by increased activity of NADPH oxidase (Nox), defective calcium transport and enhanced ROS generation by reverse electron transfer (RET) at complex I. These factors contribute to the pathogenesis of endothelial dysfunction and to the progress of hypertensive complications. Previously, we have shown that avocado oil, a rich source of bioactive compounds and antioxidants, decreases blood pressure and ameliorates both mitochondrial dysfunction and oxidative stress in kidney from hypertensive rats. However, it is unknown whether the effects of avocado oil on renal mitochondria are attributable to decreased activity of NADPH oxidase, lowered levels of reactive nitrogen species (RNS) and improved calcium uptake by mitochondria. Thus, we analyzed Nox activity and peroxynitrite (ONOO‐) levels in kidney cytosolic fractions of L‐NAME – induced hypertensive rats as well as mitochondrial calcium uptake and complex I activity. After 60‐days of L‐NAME administration, the hypertensive rats exhibited enhanced Nox activity and ONOO‐ levels, as well as increased complex I activity and excessive calcium uptake. All these alterations were prevented by co‐administration of L‐NAME with avocado oil. Moreover, the kidneys from hypertensive rats shown histological manifestations of renal damage that were also corrected by avocado oil. These data suggest that avocado oil could be beneficial to decrease hypertensive nephropathy progression by improving mitochondrial function, diminishing RNS production and normalizing Nox activity.Support or Funding InformationThis work was supported by a Coordinación de la Investigación Científica‐UMSNH grant (to CCR)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hesperidin modulates dextran sulfate sodium-induced ulcerative colitis in rats: Targeting sphingosine kinase-1- sphingosine 1 phosphate signaling pathway, mitochondrial biogenesis, inflammation, and apoptosis.

Ulcerative colitis (UC) is a chronic gastrointestinal disorder interfering with life quality. A total of 60 male Wistar rats were divided into four equal groups: Control (group I), hesperidin only (group II), UC untreated (group III), and UC treated with hesperidin (group IV). Hesperidin had modulatory effects on UC pathogenesis, which might be through alleviating colonic sphingosine phosphate phosphatase 2 messenger RNA expression and sphingosine kinase-1 levels, thus suppressing the subsequent downstream inflammatory and apoptotic cascades represented by decreased macrophage inflammatory protein-1α and enhancement of B-cell lymphoma 2 immunohistochemistry expression. Also, it improved mitochondrial biogenesis by increasing the peroxisome proliferator-activated receptor-gamma-coactivator 1-α level. It successfully restored redox potential as evidenced by marked alleviations of the nitric oxide and peroxynitrite levels, increasing total antioxidant capacity, and activating the superoxide dismutase enzyme. Also, hesperidin alleviated the UC disease activity index and improved the histopathological picture. These findings may offer a new therapeutic strategy for UC treatment.

Exogenous ascorbic acid is a pro-nitrant in Arabidopsis thaliana

Due to the intensified production of reactive nitrogen species (RNS) proteins can be modified by tyrosine nitration (PTN). Examination of PTN is a hot topic of plant biology, especially because the exact outcome of this modification is still pending. Both RNS and ascorbic acid (AsA) are redox-active molecules, which directly affect the redox state of cells. The possible link between RNS-dependent PTN and AsA metabolism was studied in RNS (gsnor1-3, nia1nia2) and AsA (vtc2-3) homeostasis Arabidopsis mutants. During physiological conditions, intensified PTN was detected in all mutant lines compared to the wild-type (WT); without altering nitration pattern. Moreover, the increased PTN seemed to be associated with endogenous peroxynitrite (ONOO-) levels, but it showed no tight correlation with endogenous levels of nitric-oxide (NO) or AsA. Exogenous AsA caused intensified PTN in WT, vtc2-3 and nia1nia2. In the background of increased PTN, significant NO and ONOO- accumulation was detected, indicating exogenous AsA-induced RNS burst. Interestingly, in AsA-triggered stress-situation, changes of NO levels seem to be primarily connected to the development of PTN. Our results point out for the first time that similarly to human and animal systems exogenous AsA exerts pro-nitrant effect on plant proteome.

Anti-inflammatory Role of Carotenoids in Endothelial Cells Derived from Umbilical Cord of Women Affected by Gestational Diabetes Mellitus.

Diabetes is associated with vascular inflammation, endothelial dysfunction, and oxidative stress, promoting the development of cardiovascular diseases (CVD). Several studies showed that a carotenoid-rich diet is associated to a reduced cardiovascular risk in healthy and diabetic subjects, although the mechanisms of action are still unknown. Here, the potential role of β-carotene (BC) and lycopene (Lyc) in human endothelial cells isolated from human umbilical cord vein (HUVECs) of women with gestational diabetes (GD) and respective controls (C) has been investigated. Results showed that BC and Lyc reduced the tumor necrosis factor alpha- (TNF-α-) stimulated monocyte-endothelium interaction (adhesion assay), membrane exposure (flow cytometry), and total expression levels (Western blot) of VCAM-1 and ICAM-1 in both cell types. Moreover, the treatment with BC and Lyc reduced the TNF-α-induced nuclear translocation of NF-κB (image flow cytometry) by preserving bioavailability of nitric oxide (NO, flow cytometry, and cGMP EIA kit assay), a key vasoactive molecule. Notably, BC and Lyc pretreatment significantly reduced peroxynitrite levels (flow cytometry), contributing to the redox balance protection. These results suggest a new mechanism of action of carotenoids which exert vascular protective action in diabetic condition, thus reinforcing the importance of a carotenoid-rich diet in the prevention of diabetes cardiovascular complications.

LED-based red light photostimulation improves short-term response of cooled boar semen exposed to thermal stress at 37°C.

Pre-treatment of boar semen with a red light photostimulation procedure increases its "in vivo" fertilising ability. However, "in vitro" conducted studies shown contradictory results regarding the ability of photostimulated spermatozoa to react against strong stress and to achieve the capacitation status. The aim here was to determine the effect of photostimulation on the response to short-term moderate thermal stress of boar semen. Boar semen was exposed to red LED light regime emitting a 620-630nm during 10min of light, 10min of rest and 10min of light after 3hr since semen was collected. An aliquot without photostimulation was included as a control. After the photostimulation, the sperm cells were incubated for 15min at 37°C. Afterwards, motility, viability, intracellular Ca2+ level and production of reactive oxygen species (ROS) and peroxynitrite were analysed. The results showed that the photostimulated group maintained total motility throughout the time, whereas a significant decrease in total motility was observed in the nonphotostimulated control group. Furthermore, for kinetic parameters of motility, a significant increase was observed in LIN, STR and WOB in photostimulated spermatozoa. Peroxynitrite production was significantly increased in the photostimulated spermatozoa, whereas viability, ROS production and intracellular Ca2+ levels were not affected by photostimulation. In conclusion, photostimulation of commercial boar semen has a positive effect on motility of spermatozoa subjected to a short-term moderate thermal stress, which was concomitant with an increase in peroxynitrite production.

THE CHANGES IN DYNAMICS OF ORNITHINE CYCLE COMPONENTS LEVELS DURING THE ACUTE PERIOD OF POLYTRAUMA

Introduction: Polytrauma or multiple organ damage is associated with shock and lead to systemic inflammation, oxidative stress and endothelial dysfunction. A severe mechanical injury causes an increased proinflammatory mediators and cytokines levels. Among them, the overproduction of nitric oxide and its oxidation products play a key role in tissue damage. The aim: To evaluate the changes in dynamics of some ornithine cycle components levels during acute period of polytrauma. Materials and methods: We measured standard biomechanical parameters and serum levels of NO, sum of nitrite and nitrate (NOx), L-arginine, arginase, and peroxynitrite. According to the ISS, the study included patients with moderate (n=15) to severe (n=15) polytrauma. Results: In 24 hours after polytrauma on the background of intensive care, it was observed significant increasing of NO, NOx, and arginase levels (severe cases) with decreasing of L-arginine and peroxynitrite levels. Conclusions: Elevated NO and NOx serum levels in patients with polytrauma is associated with increasing of arginase activity with decreasing of L-arginine and peroxynitrite levels on the background of intensive care.

218 - Tyrosine nitration regulates signaling pathways involved in Neurofibromatosis type 2-associated Schwannoma growth

Neurofibromatosis type 2 (NF2) is a genetic disorder of the nervous system caused by inactivation of the merlin tumor suppressor gene. NF2 patients develop bilateral vestibular schwannomas (VS) and other nervous system tumors throughout their life for which there is no effective drug treatment. Production of the oxidant peroxynitrite occurs in pathological conditions and leads to tyrosine (Tyr) nitration of proteins. Although Tyr nitration is found in multiple tumor types, its role in tumorigenesis is unknown. We discovered that VS and human merlin-deficient (MD) Schwann cells show significantly increased levels of peroxynitrite and Tyr nitration compared to normal Schwann cells. Notably, prevention of tyrosine nitration selectively decreases MD-Schwann cell survival. Our goal is to identify nitrated proteins that support VS growth as new potential tumor-specific therapeutic targets, enabling the development of safe pharmacological strategies for NF2 schwannomas. This is the first focused effort to determine the signaling pathways regulated by tyrosine nitration in pathological conditions. By performing immunoprecipitations using a polyclonal anti-nitrotyrosine antibody followed by mass spectrometry analysis and western blot, we identified a limited number of proteins endogenously nitrated in VS from NF2 patients, and in mouse and human MD-Schwann cells. A group of the identified proteins play a role in pathways that are dysregulated in NF2. Further, using complementary phospho-kinase arrays, we found that prevention of tyrosine nitration using urate and edaravone has an impact on several of these key signaling pathways. The identification of the specific nitrated proteins that promote schwannoma growth could provide exceptional novel targets for the treatment of NF2 and possibly other tumors of the nervous system as well. Supported by DoD, NRP, NIA W81XWH-17-1-0409 (to MCF).

116 - Fluorescence visualization of peroxisome peroxynitrite in live cells and vivo

Liver injury can cause liver failure and mortality, which have attracted a considerable concern in modern medicine. Peroxisomes are known to decompose lipid and produce lipid radicals which are involved in liver injury. Meantime, these organelles also generate reactive oxygen and nitrogen specie. To date, the role of lipid radicals in peroxisome in the process of liver injury has been researched extensively, but a rare effort is dedicated to study the effects of reactive oxygens and reactive nitrogen in peroxisomes on the occurrence and development of liver injury. Recently, a lot of works have reported that the high level of peroxynitrite (ONOO-) are observed in liver injury. So, we developed a new peroxisome-targeted and two-photon fluorescent probe for detecting ONOO-. The probe, named PX-1, was successfully applied to imaging ONOO- in vivo. And the experimental results showed that PX-1 can real-time monitor the level of ONOO- in peroxisomes with high specificity and sensitivity. Moreover, ONOO- was found to be elevated in mice with carbon tetrachloride-induced liver injury. We believe that PX-1 can be used as a potentially useful tool to study the mechanism of liver injury related to ONOO- in peroxisomes.

The Effect of Endothelial Cells on UVB-induced DNA Damage and Transformation of Keratinocytes In 3D Polycaprolactone Scaffold Co-culture System.

Nitric oxide ( ) plays an important role in the regulation of redox balance in keratinocytes post-UVB exposure. Since endothelial cells releases for a prolonged time post-UVB, we determined whether human umbilical vein endothelial cells (HUVEC) could have an effect on UVB-induced DNA damage and transformation of their adjacent keratinocytes (HaCaT) using a 3D cell co-culturing system. Our data show that the levels of DNA breaks and/or cyclobutane pyrimidine dimer (CPD) along with γH2AX are higher in the co-cultured than in the mono-cultured keratinocytes post-UVB. The level in the co-cultured cells is increased approximately 3-fold more than in mono-cultured HaCaT cells within 1-hour post-UVB but then is reduced quickly in co-cultured HaCaT cells comparing to mono-cultured cells from 6 to 24 h post-UVB. However, the peroxynitrite (ONOO- ) level is higher in the co-cultured than in the mono-cultured HaCaT cells in whole period post-UVB. Furthermore, while expression level of inducible nitric oxide synthase (iNOS) is increased, the ratio of coupled/uncoupled eNOS is reduced in co-cultured HaCaT cells compared to mono-cultured HaCaT cells. Finally, the co-cultured cells have a significantly increased transformation efficiency after repeating UVB exposure compared to mono-culture HaCaT cells. Our results suggest that endothelial cells could enhance /ONOO- imbalance and promote transformation of adjacent keratinocytes.