DAF-2 DA Research Articles

Aging in Normotensive and Spontaneously Hypertensive Rats: Focus on Erythrocyte Properties.

Erythrocyte deformability, crucial for oxygen delivery to tissues, plays an important role in the etiology of various diseases. As the factor maintaining the erythrocyte deformability, nitric oxide (NO) has been identified. Reduced NO bioavailability also plays a role in the pathogenesis of hypertension. Our aim was to determine whether aging and hypertension affect erythrocyte deformability and NO production by erythrocytes in experimental animals divided into six groups according to age (7, 20 and 52 weeks), labeled WKY-7, WKY-20 and WKY-52 for normotensive Wistar-Kyoto (WKY) rats, and SHR-7, SHR-20 and SHR-52 for spontaneously hypertensive rats (SHR). The filtration method for the determination of erythrocyte deformability and the fluorescent probe DAF-2 DA for NO production were applied. Deformability and NO production by erythrocytes increased at a younger age, while a decrease in both parameters was observed at an older age. Strain-related differences in deformability were observed at 7 and 52 weeks of age. SHR-7 had reduced deformability and SHR-52 had increased deformability compared with age-matched WKY. Changes in NO production under hypertensive conditions are an unlikely primary factor affecting erythrocyte deformability, whereas age-related changes in deformability are at least partially associated with changes in NO production. However, an interpretation of data obtained in erythrocyte parameters observed in SHRs of human hypertension requires precaution.

The lncRNA OIP5-AS1/miR-4500 axis targeting ARG2 modulates oxidative stress-induced premature senescence in endothelial cells: implications for vascular aging

ABSTRACT Background Endothelial senescence due to increased age or oxidative stress can cause endothelial dysfunction, which is strongly associated with the pathogenesis of cardiovascular diseases (CVDs). Research design and methods Hydrogen peroxide (H2O2) was used to induced human umbilical vein endothelial cells (HUVECs) senescence model. Cell senescence and cell proliferation were assessed by SA-β-gal staining and PCNA staining. Nitric oxide (NO) and reactive oxygen species (ROS) levels were detected by DAF-2 DA and DCFH-DA. Inflammatory indicators were quantified by qPCR. Meanwhile, western blot was used to examine the ARG2 protein. Finally, an aging mice model induced by H2O2 was established to confirm the role of OIP5-AS1/miR-4500/ARG2 in endothelial dysfunction in vivo. Results ARG2 was upregulated and miR-4500 was reduced in H2O2-induced HUVECs. MiR-4500 negatively regulates ARG2 expression, meanwhile ameliorating H2O2-induced ECs senescence and dysfunction. Targeted interactions among OIP5-AS1, miR-4500, and ARG2 were confirmed by dual-luciferase reporter assays. OIP5-AS1 as miR4500 sponge negatively mediates miR-4500 expression, and is upregulated upon H2O2 stimulation in HUVECs. OIP5-AS1 depletion shows the protective effects on H2O2-induced ECs senescence, dysfunction, and SASP. In vivo, a higher expression of OIP5-AS1 and ARG2 in the aortas of aged mice. Conclusions We disclosed a regulatory mechanism for OIP5-AS1/miR-4500/ARG2 in the regulation of oxidative stress-related ECs senescence and vascular aging.

In vivo imaging of nitric oxide in the male rat brain exposed to a shock wave.

While numerous studies have suggested the involvement of cerebrovascular dysfunction in the pathobiology of blast-induced traumatic brain injury (bTBI), its exact mechanisms and how they affect the outcome of bTBI are not fully understood. Our previous study showed the occurrence of cortical spreading depolarization (CSD) and subsequent long-lasting oligemia/hypoxemia in the rat brain exposed to a laser-induced shock wave (LISW). We hypothesized that this hemodynamic abnormality is associated with shock wave-induced generation of nitric oxide (NO). In this study, to verify this hypothesis, we used an NO-sensitive fluorescence probe, diaminofluorescein-2 diacetate (DAF-2 DA), for real-time in vivo imaging of male Sprague-Dawley rats' brain exposed to a mild-impulse LISW. We observed the most intense fluorescence, indicative of NO production, along the pial arteriolar walls during the period of 10-30 min post-exposure, parallel with CSD occurrence. This post-exposure period also coincided with the early phase of hemodynamic abnormalities. While the changes in arteriolar wall fluorescence measured in rats receiving pharmacological NO synthase inhibition by nitro-L-arginine methyl ester (L-NAME) 24 h before exposure showed a temporal profile similar to that of changes observed in LISW-exposed rats with CSD, their intensity level was considerably lower; this suggests partial involvement of NOS in shock wave-induced NO production. To the best of our knowledge, this is the first real-time in vivo imaging of NO in rat brain, confirming the involvement of NO in shock-wave-induced hemodynamic impairments. Finally, we have outlined the limitations of this study and our future research directions.

Baicalein Preconditioning Cardioprotection Involves Pro-Oxidant Signaling and Activation of Pyruvate Dehydrogenase.

Preconditioning has a powerful protective potential against myocardial ischemia-reperfusion injury (I/R). Our prior work demonstrated that baicalein, a flavonoid derived from the root of Scatellaria baicalensis Georgi (also known as Huangqin), confers this preconditioning protection. This study further explored the mechanisms of baicalein preconditioning (BC-PC) in mouse cardiomyocytes. Cells were treated with baicalein (10 μM) for a brief period of time (10 min) prior to simulated ischemia 90 min/reperfusion for 180 min. Baicalein triggered an induction of a small amount of mitochondrial reactive oxygen species (ROS) prior to the initiation of ischemia, assessed by 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate (6-carboxy-H2DCFDA). It also significantly increased cell viability measured by propidium iodide (PI) and lactate dehydrogenase and preserved mitochondrial membrane potential assessed by TMRM fluorescence intensity. Myxothiazol, a mitochondrial electron transport chain complex III inhibitor, partially blocked ROS generation induced by BC-PC and reduced cell viability. BC-PC increased phosphorylation of Akt (Thr308 and Ser473) and eNOS Ser1177, and nitric oxide (NO) production measured using 4,5-diaminofluorescein diacetate (DAF-2 DA, 1 μM). Akt inhibitor API-2 abolished Akt phosphorylation and reduced DAF-2 production and cell viability. In addition, BC-PC decreased phosphorylation of pyruvate dehydrogenase (PDH) reflecting upregulated PDH activity, and increased ATP production at 30 min during reperfusion. Taken together, baicalein preconditioning-induced cardioprotection involves pro-oxidant generation, activates survival signaling Akt/eNOS/NO, and improves metabolic recovery after I/R injury. Our work provides new perspectives on the effect of baicalein on cardiac preconditioning against I/R injury.

Lithium inhibits NF-\u03baB nuclear translocation and modulate inflammation profiles in Rift valley fever virus-infected Raw 264.7 macrophages

IntroductionRift Valley fever virus (RVFV) is a zoonotic life-threatening viral infection endemic across sub-Saharan African countries and the Arabian Peninsula; however, there is a growing panic of its spread to non-endemic regions. This viral infection triggers a wide spectrum of symptoms that span from fibril illnesses to more severe symptoms such as haemorrhagic fever and encephalitis. These severe symptoms have been associated with dysregulated immune response propagated by the virulence factor, non-structural protein (NSs). Thus, this study investigates the effects of lithium on NF-κB translocation and RFVF-induced inflammation in Raw 264.7 macrophages.MethodsThe supernatant from RVFV-infected Raw 264.7 cells, treated with lithium, was examined using an ELISA assay kit to measure levels of cytokines and chemokines. The H2DCF-DA and DAF-2 DA florigenic assays were used to determine the levels of ROS and RNS by measuring the cellular fluorescence intensity post RVFV-infection and lithium treatment. Western blot and immunocytochemistry assays were used to measure expression levels of the inflammatory proteins and cellular location of the NF-κB, respectively.ResultsLithium was shown to stimulate interferon-gamma (IFN-γ) production as early as 3 h pi. Production of the secondary pro-inflammatory cytokine and chemokine, interleukin-6 (IL-6) and regulated on activation, normal T cell expressed and secreted (RANTES), were elevated as early as 12 h pi. Treatment with lithium stimulated increase of production of tumor necrosis factor-alpha (TNF-α) and Interleukin-10 (IL-10) in RVFV-infected and uninfected macrophages as early as 3 h pi. The RVFV-infected cells treated with lithium displayed lower ROS and RNS production as opposed to lithium-free RVFV-infected control cells. Western blot analyses demonstrated that lithium inhibited iNOS expression while stimulating expression of heme oxygenase (HO) and IκB in RVFV-infected Raw 264.7 macrophages. Results from immunocytochemistry and Western blot assays revealed that lithium inhibits NF-κB nuclear translocation in RVFV-infected cells compared to lithium-free RVFV-infected cells and 5 mg/mL LPS controls.ConclusionThis study demonstrates that lithium inhibits NF-kB nuclear translocation and modulate inflammation profiles in RVFV-infected Raw 264.7 macrophage cells.

Effects of selective inhibition of nNOS and iNOS on neuropathic pain in rats

Various animal models have been employed to understand the pathogenic mechanism of neuropathic pain. Nitric oxide (NO) is an important molecule in nociceptive transmission and is involved in neuropathic pain. However, its mechanistic actions remain unclear. The aim of this study was to better understand the involvement of neuronal and inducible isoforms of nitric oxide synthase (nNOS and iNOS) in neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats. We evaluated pain sensitivity (mechanical withdrawal thresholds using Randall and Selitto, and von Frey tests, and thermal withdrawal thresholds using Hargreaves test) prior to CCI surgery, 14 days post CCI and after intrathecal injections of selective nNOS or iNOS inhibitors. We also evaluated the distribution of NOS isozymes in the spinal cord and dorsal root ganglia (DRG) by immunohistochemistry, synthesis of iNOS and nNOS by Western blot, and NO production using fluorescent probe DAF-2 DA (DA). Our results showed higher number of nNOS and iNOS-positive neurons in the spinal cord and DRG of CCI compared to sham rats, and their reduction in CCI rats after treatment with selective inhibitors compared to non-treated groups. Western blot results also indicated reduced expression of nNOS and iNOS after treatment with selective inhibitors. Furthermore, both inhibitors reduced CCI-evoked mechanical and thermal withdrawal thresholds but only nNOS inhibitor was able to efficiently lower mechanical withdrawal thresholds using von Frey test. In addition, we observed higher NO production in the spinal cord and DRG of injured rats compared to control group. Our study innovatively shows that nNOS may strongly modulate nociceptive transmission in rats with neuropathic pain, while iNOS may partially participate in the development of nociceptive responses. Thus, drugs targeting nNOS for neuropathic pain may represent a potential therapeutic strategy.

Involvement of nitrate reductase-dependent nitric oxide production in magnetopriming-induced salt tolerance in soybean.

In the present study, experiments were performed to investigate the role of nitric oxide (NO) in magnetopriming-induced seed germination and early growth characteristics of soybean (Glycine max) seedlings under salt stress. The NO donor (sodium nitroprusside, SNP), NO scavenger (2-[4-carboxyphenyl]-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, CPTIO), inhibitors of nitrate reductase (sodium tungstate, ST) or NO synthase (N-nitro-L-Arg-methyl ester, LNAME) and NADPH oxidase inhibitor (diphenylene iodonium, DPI) have been used to measure the role of NO in the alleviation of salinity stress by static magnetic field (SMF of 200 mT, 1h). Salt stress (50 mM NaCl) significantly reduced germination and early growth of seedlings emerged from non-primed seeds. Pre-treatment of seeds with SMF positively stimulated the germination and consequently promoted the seedling growth. ST, LNAME, CPTIO and DPI significantly decreased the growth of seedling, activities of α-amylase, protease and nitrate reductase (NR), hydrogen peroxide (H2 O2 ), superoxide (O2 •- ) and NO content in roots of seedlings emerged from non-primed and SMF-primed seeds. However, the extent of reduction was higher with ST in seedlings of SMF-primed seeds under both conditions, whereas SNP promoted all the studied parameters. Moreover, the generation of NO was also confirmed microscopically using a membrane permanent fluorochrome (4-5-diaminofluorescein diacetate [DAF-2 DA]). Further, analysis showed that SMF enhanced the NR activity and triggered the NO production and NR was maximally decreased by ST as compared to LNAME, CPTIO and DPI. Thus, in addition to ROS, NO might be one of the important signaling molecules in magnetopriming-induced salt tolerance in soybean and NR may be responsible for SMF-triggered NO generation in roots of soybean.

Abstract 002: Macula Densa SGLT1-NOS1-TGF Pathway -- A New Mechanism for Glomerular Hyperfiltration in Diabetes

Glomerular hyperfiltration is a common observation in early diabetes and considered as risk factor for diabetic nephropathy. The mechanisms underlying glomerular hyperfiltration have not been fully clarified. In the present study, we tested a novel SGLT1-NOS1 -TGF (sodium-glucose cotransporter 1 - neuronal nitric oxide synthase – tubuloglomerular feedback) pathway with a hypothesis that tubular glucose is sensed by SGLT1 of the macula densa, which enhances macula densa NOS1 activity and blunts TGF response, therefore increasing glomerular filtration rate (GFR). NO generation by the macula densa, measured by DAF-2 DA in isolated perfused juxtaglomerular apparatus (JGA), increased by 78.4±9.1% when tubular glucose (glucose) was increased from 100 mg/dl to 300 mg/dl (n=4, p<0.01 vs 100 mg/dl). A selective SGLT1 inhibitor KGA-2727 (10 -6 M) blocked this glucose-induced NO generation. Human kidney biopsy tissues were cultured in normal glucose (100 mg/dl) and high glucose (300 mg/dl) media for 30 minutes with hanging drop technique. Protein levels of P-NOS1 ser1417 increased by 63.1±9.2% and P-NOS1 ser847 decreased by 47.4±5.9% (n=4, p<0.01 vs normal glucose) with high glucose. TGF response was significantly reduced from 3.8±0.2 μm to 2.4±0.2 μm (n=4, p<0.05 vs 100 mg/dl), measured in isolated and double perfused JGAs when tubular glucose was increased from 100 mg/dl to 300 mg/dl. The glucose-induced TGF inhibition was blocked in the presence of SGLT1 inhibition (n=4). GFR, measured in conscious mice with a single bolus injection of FITC-inulin, increased by 19.1±3.5% in response to an acute intravenous injection of 50 μl 2M glucose in C57BL/6 mice (n=4, p<0.01 vs baseline). Then, we repeated the experiments in macula densa specific NOS1 KO (KO) mice. Both glucose-induced TGF (ΔP sf was from 7.8±1.3 to 7.3±2.1 mmHg) and GFR (from 223±15.7 to 240±6.9 μl/min) (n=5) were blocked in the KO mice. We conclude that increase of tubular glucose concentration activates NOS1 by phosphorylation of NOS1 ser1417 via SGLT1 in the macula densa, which increases GFR by inhibiting TGF response.

Curcumin prevents strokes in stroke-prone spontaneously hypertensive rats by improving vascular endothelial function

BackgroundAntioxidants have shown great promise in stroke prevention. Diarylheptanoids (also known as diphenylheptanoids) are a small class of plant secondary metabolites that possess antioxidant activity greater than that of α-tocopherol. Curcumin is the best known member and is mainly extracted from turmeric. This study aimed to explore whether curcumin has a preventive effect on stroke.MethodsStroke-prone spontaneously hypertensive rats (SHRsp) were randomly divided into control group (n = 10) and curcumin group (n = 10), and saline or curcumin (100 mg/kg/day) was administrated daily. Vascular endothelial function was examined by the relaxation of the artery in response to acetylcholine (ACH). The levels of reactive oxygen species (ROS) and nitric oxide (NO) were measured by using dihydroethidium (DHE) and 4, 5-diaminofluorescein (DAF-2 DA), respectively. The expression of uncoupling protein 2 (UCP2) was examined by RT-PCR and immunoblotting.ResultsAdministration of curcumin significantly delayed the onset of stroke and increased the survival of SHRsp, which was ascribed to decreased ROS and improved endothelial dependent relaxation of carotid arteries. In the presence of UCP2 inhibitor genipin, both curcumin-mediated decrease of ROS and increase of NO production were blocked.ConclusionOur study suggests that curcumin exerts a stroke preventive effect by attenuating oxidative stress to improve vascular endothelial function, which might be associated with UCP2 signaling.

The aflatoxin B1‐fumonisin B1 toxicity in BRL‐3A hepatocytes is associated to induction of cytochrome P450 activity and arachidonic acid metabolism

Human oral exposure to aflatoxin B1 (AFB1 ) and fumonisin B1 (FB1 ) is associated with increased hepatocellular carcinoma. Although evidence suggested interactive AFB1 -FB1 hepatotoxicity, the underlying mechanisms remain mostly unidentified. This work was aimed at evaluating the possible AFB1 -FB1 interplay to induce genetic and cell cycle toxicities in BRL-3A rat hepatocytes, reactive oxygen species (ROS) involvement, and the AFB1 metabolizing pathways cytochrome P450 (CYP) and arachidonic acid (ArAc) metabolism as ROS contributors. Flow cytometry of stained BRL-3A hepatocytes was used to study the cell cycle (propidium iodide), ROS intracellular production (DCFH-DA, HE, DAF-2 DA), and phospholipase A activity (staining with bis-BODIPY FL C11-PC). The CYP1A activity was assessed by the 7-ethoxyresorufin-O-deethylase (EROD) assay. Despite a 48-h exposure to FB1 (30μM) not being genotoxic, the AFB1 (20μM)-induced micronucleus frequency was overcome by the AFB1 -FB1 mixture (MIX), presumably showing toxin interaction. The mycotoxins blocked G1/S-phase, but only MIX caused cell death. Overall, the oxidative stress led these alterations as the pretreatment with N-acetyl-l-cysteine reduced such toxic effects. While AFB1 had a major input to the MIX pro-oxidant activity, with CYP and ArAc metabolism being ROS contributors, these pathways were not involved in the FB1 -elicited weak oxidative stress. The MIX-induced micronucleus frequency in N-acetyl-l-cysteine pretreated cells was greater than that caused by AFB1 without antioxidants, suggesting enhanced AFB1 direct genotoxicity probably owing to the higher CYP activity and ArAc metabolism found in MIX. The metabolic pathways modulation by AFB1 -FB1 mixtures could raise its hepatocarcinogenic properties.

Inhibition of endothelial nitric oxide synthase decreases breast cancer cell MDA-MB-231 adhesion to intact microvessels under physiological flows.

Nitric oxide (NO) at different concentrations may promote or inhibit tumor growth and metastasis under various conditions. To test the hypothesis that tumor cells prefer to adhere to the locations with a higher endothelial NO production in intact microvessels under physiological flows and to further test that inhibiting NO production decreases tumor cell adhesion, we used intravital fluorescence microscopy to measure NO production and tumor cell adhesion in postcapillary venules of rat mesentery under normal and reduced flow conditions, and in the presence of an endothelial nitric oxide synthase (eNOS) inhibitor, N(G)-monomethyl-l-arginine (l-NMMA). Rats (SD, 250-300 g) were anesthetized. A midline incision (∼2 inch) was made in the abdominal wall, and the mesentery was taken out from the abdominal cavity and spread over a coverslip for the measurement. An individual postcapillary venule (35-50 μm) was first loaded with 4,5-diaminofluorescein diacetate (DAF-2 DA), a fluorescent indictor for NO. Then the DAF-2 intensity was measured for 30 min under a normal or reduced flow velocity, with and without perfusion with MDA-MB-231 breast cancer cells, and in the presence of l-NMMA. We found that tumor cells prefer to adhere to the microvessel locations with a higher NO production such as curved portions. Inhibition of eNOS by l-NMMA attenuated the flow-induced NO production and reduced tumor cell adhesion. We also found that l-NMMA treatment for ∼40 min reduced microvessel permeability to albumin. Our results suggest that inhibition of eNOS is a good approach to preventing tumor cell adhesion to intact microvessels under physiological flows.

Oxidative Stress-Dependent Coronary Endothelial Dysfunction in Obese Mice.

Obesity is involved in several cardiovascular diseases including coronary artery disease and endothelial dysfunction. Endothelial Endothelium vasodilator and vasoconstrictor agonists play a key role in regulation of vascular tone. In this study, we evaluated coronary vascular response in an 8 weeks diet-induced obese C57BL/6 mice model. Coronary perfusion pressure in response to acetylcholine in isolated hearts from obese mice showed increased vasoconstriction and reduced vasodilation responses compared with control mice. Vascular nitric oxide assessed in situ with DAF-2 DA showed diminished levels in coronary arteries from obese mice in both basal and acetylcholine-stimulated conditions. Also, released prostacyclin was decreased in heart perfusates from obese mice, along with plasma tetrahydrobiopterin level and endothelium nitric oxide synthase dimer/monomer ratio. Obesity increased thromboxane A2 synthesis and oxidative stress evaluated by superoxide and peroxynitrite levels, compared with control mice. Obese mice treated with apocynin, a NADPH oxidase inhibitor, reversed all parameters to normal levels. These results suggest that after 8 weeks on a high-fat diet, the increase in oxidative stress lead to imbalance in vasoactive substances and consequently to endothelial dysfunction in coronary arteries.

Changing shear stress‐induced ATP release from erythrocytes increases endothelial calcium and vascular permeability independent from nitric oxide production in intact rat venules (672.2)

Disturbed shear stress (SS) promotes atherosclerosis, thrombosis, and inflammation of the vessel wall. Endothelial cell (EC) barrier dysfunction has been implicated to play important roles in the pathogenesis of many cardiovascular diseases. However, how the disturbed blood flow affects EC barrier function in vivo and the involved cellular mechanisms remain to be identified. Our study investigated changing SS‐induced endothelial signaling and permeability in intact rat venules. EC [Ca2+]i and NO were measured in Fura‐2 AM and DAF‐2 DA loaded venules. EC eNOS activation was evaluated by immunofluorescence staining with confocal imaging. ATP released from red blood cells (RBCs) in response to SS is measured by bioluminescent assay. In response to a sudden change of SS, transient shear magnitude‐dependent increases in EC [Ca2+]i occurred only in vessels perfused with whole blood or perfusate containing RBCs, which was correlated with EC gap formation illustrated by fluorescent microsphere accumulation. Carbenoxolone, a Pannexin 1 inhibitor, significantly reduced shear magnitude‐dependent ATP release from RBCs and also abolished SS‐induced increases in EC [Ca2+]i. However, both plasma and whole blood perfusion induced shear magnitude‐dependent NO production and eNOS‐ser1177 phosphorylation. These results indicated that in intact venules ATP released from RBCs in response to the sudden change of SS is accountable for the increases in EC [Ca2+]i and vascular permeability, while SS‐induced NO production can be independent of increased EC [Ca2+]i.Grant Funding Source: Supported by HL56237, HL084338 and AHA 12PRE11470010

Regulation of cardiac nitric oxide signaling by nuclear β-adrenergic and endothelin receptors

At the cell surface, βARs and endothelin receptors can regulate nitric oxide (NO) production. β-adrenergic receptors (βARs) and type B endothelin receptors (ETB) are present in cardiac nuclear membranes and regulate transcription. The present study investigated the role of the NO pathway in the regulation of gene transcription by these nuclear G protein-coupled receptors. Nitric oxide production and transcription initiation were measured in nuclei isolated from the adult rat heart. The cell-permeable fluorescent dye 4,5-diaminofluorescein diacetate (DAF2 DA) was used to provide a direct assessment of nitric oxide release. Both isoproterenol and endothelin increased NO production in isolated nuclei. Furthermore, a β3AR-selective agonist, BRL 37344, increased NO synthesis whereas the β1AR-selective agonist xamoterol did not. Isoproterenol increased, whereas ET-1 reduced, de novo transcription. The NO synthase inhibitor l-NAME prevented isoproterenol from increasing either NO production or de novo transcription. l-NAME also blocked ET-1-induced NO-production but did not alter the suppression of transcription initiation by ET-1. Inhibition of the cGMP-dependent protein kinase (PKG) using KT5823 also blocked the ability of isoproterenol to increase transcription initiation. Furthermore, immunoblotting revealed eNOS, but not nNOS, in isolated nuclei. Finally, caged, cell-permeable isoproterenol and endothelin-1 analogs were used to selectively activate intracellular β-adrenergic and endothelin receptors in intact adult cardiomyocytes. Intracellular release of caged ET-1 or isoproterenol analogs increased NO production in intact adult cardiomyocytes. Hence, activation of the NO synthase/guanylyl cyclase/PKG pathway is necessary for nuclear β3ARs to increase de novo transcription. Furthermore, we have demonstrated the potential utility of caged receptor ligands in selectively modulating signaling via endogenous intracellular G protein-coupled receptors.

TRPV1-mediated UCP2 upregulation ameliorates hyperglycemia-induced endothelial dysfunction

BackgroundDiabetic cardiovascular complications are characterised by oxidative stress-induced endothelial dysfunction. Uncoupling protein 2 (UCP2) is a regulator of mitochondrial reactive oxygen species (ROS) generation and can antagonise oxidative stress, but approaches that enhance the activity of UCP2 to inhibit ROS are scarce. Our previous studies show that activation of transient receptor potential vanilloid 1 (TRPV1) by capsaicin can prevent cardiometabolic disorders. In this study, we conducted experiments in vitro and in vivo to investigate the effect of capsaicin treatment on endothelial UCP2 and oxidative stress. We hypothesised that TRPV1 activation by capsaicin attenuates hyperglycemia-induced endothelial dysfunction through a UCP2-mediated antioxidant effect.MethodsTRPV1-/-, UCP2 -/- and db/db mice, as well as matched wild type (WT) control mice, were included in this study. Some mice were subjected to dietary capsaicin for 14 weeks. Arteries isolated from mice and endothelial cells were cultured. Endothelial function was examined, and immunohistological and molecular analyses were performed.ResultsUnder high-glucose conditions, TRPV1 expression and protein kinase A (PKA) phosphorylation were found to be decreased in the cultured endothelial cells, and the effects of high-glucose on these molecules were reversed by the administration of capsaicin. Furthermore, high-glucose exposure increased ROS production and reduced nitric oxide (NO) levels both in endothelial cells and in arteries that were evaluated respectively by dihydroethidium (DHE) and DAF-2 DA fluorescence. Capsaicin administration decreased the production of ROS, restored high-glucose-induced endothelial dysfunction through the activation of TRPV1 and acted in a UCP2-dependent manner in vivo. Administration of dietary capsaicin for 14 weeks increased the levels of PKA phosphorylation and UCP2 expression, ameliorated the vascular oxidative stress and increased NO levels observed in diabetic mice. Prolonged dietary administration of capsaicin promoted endothelium-dependent relaxation in diabetic mice. However, the beneficial effect of capsaicin on vasorelaxation was absent in the aortas of UCP2 -/- mice exposed to high-glucose levels.ConclusionTRPV1 activation by capsaicin might protect against hyperglycemia-induced endothelial dysfunction through a mechanism involving the PKA/UCP2 pathway.

The impaired glycocalyx increases the endothelial susceptibility to shear stress resulting in augmented nitric oxide production in microvessels of diabetic rats

Glycocalyx (GCX), a layer of proteoglycans covering the endothelium, has been implicated as a mechanical sensor for changes in shear stress (SS) in vitro. Though acute hyperglycemia has been reported to impair GCX in human by indirect volume estimation, the direct experimental evidence and how the impaired GCX affects SS‐mediated endothelial function in vivo remain to be demonstrated. The objective of this study is to identify the changes in GCX in microvessels of streptozotocin‐induced diabetic rats and evaluate the associated changes in sensing SS and SS‐induced nitric oxide (NO) production in individually perfused venules of diabetic rats. GCX is visualized either by electron microscope (EM) or by lectin fluorescence labeling. EC NO production was quantified in DAF‐2 DA loaded vessels using fluorescence imaging. Microvessels of diabetic rats showed degraded GCX by both EM and confocal images, a similar observation found in pronase (0.1 mg/ml, 3 min) treated microvessels. Unexpectedly, the EC NO production in response to blood flow‐generated changes in SS in diabetic vessels was significantly higher than that in normal vessels. The NO production rate in normal vessels were 0.13 ± 0.02 and 0.24 ± 0.02 AU/min at 11 and 32 dyn/cm2. In contrast, the NO production rate of diabetic vessel in response to the same magnitude changes of SS were 0.25 ± 0.05 and 0.37 ± 0.03 AU/min, respectively. These findings were also supported by an increased NO production observed in pronase treated normal venules. Our results indicate that the impaired GCX in diabetic microvessels enhances EC response to mechanical force and potentiates NO production. Supported by HL56237, HL084338 and AHAPRE11470010.

바지락 (Ruditapes philippinarum) 혈구의 일산화질소 (nitric oxide) 정량

Nitric oxide (NO) is an important intra-intercellular signaling molecule that regulates many physiological processes and participates in the development some pathological conditions in animals. In this study, we compared different methods for determining NO concentration in the hemocytes of Manila clam Ruditapes philippinarum. For measuring the intracellular NO levels, we used the specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA), and the quantification methods that were compared were based on image analysis, spectrophotometry, and flow cytometry. NO concentration could be determined using all the 3 methods, and the concentration varied significantly depending upon the presence of NO regulators in the hemocytes; NO concentration increased in the presence of L-arginine, while it decreased in the presence of N-nitro-L-arginine methyl ester. In particular, it is found that estimation of NO using a flowcytometry is more economical, reliable and accurate compared to image analysis and spectrophotometry. Accordingly we believe that determining NO concentration by using flowcytometry will be useful in evaluating physiological and pathological conditions in marine bivalves.

Generation and characterization of a spontaneously immortalized endothelial cell line from mice microcirculation

Endothelial cells from microvasculature are directly involved in a large number of vascular diseases; however, culture of these cells is problematic, since most methodologies employ proteolytic enzymes or mechanical techniques, leading to cell damage and contamination of endothelial cultures with other cellular types. Besides, primary cultured cells have a short life span in vitro and undergo replicative senescence after 3–4 passages, limiting long-term studies. In the present work we report the generation of a spontaneously immortalized endothelial culture obtained from mice pulmonary capillaries. Firstly, primary (third passage) and immortalized (100th) cultures were established. Further, monoclonal populations were obtained by serial dilutions from immortalized cultures. Cells were analyzed according to: (1) morphological appearance, (2) expression of specific endothelial markers by fluorescent staining [von Willebrand Factor (vWF), endothelial nitric oxide synthase (eNOS), angiotensin converting enzyme (ACE) and Ulex europaeus (UEA-1)] and by flow cytometry (endoglin, VE-cadherin and VCAM-1), and (3) release of nitric oxide (NO), assessed by the specific fluorescent dye DAF-2 DA, and prostacyclin (PGI2), quantified by enzyme immune assay. In both cultures cells grew in monolayers and presented cobblestone appearance at confluence. Positive staining for vWF, eNOS, ACE and UEA-1 was detected in cloned as well as in early-passage cultured cells. Similarly, cultures presented equal expressions of endoglin, VE-cadherin and VCAM-1. Values of NO and PGI2 levels did not differ between cultures. From these results we confirm that the described spontaneously immortalized endothelial cell line is capable of unlimited growth and retains typical morphological and functional properties exhibited by primary cultured cells. Therefore, the endothelial cell line described in the present study can become a suitable tool in the field of endothelium research and can be useful for the investigation of production of endothelial mediators, angiogenesis and inflammation.

Rooperol as an antioxidant and its role in the innate immune system: An in vitro study

Ethnopharmacological relevanceBiologically active rooperol is formed when the glucose subunits of the nontoxic glycoside, hypoxoside, are cleaved by β-glucosidase. Hypoxoside is isolated from Hypoxis, a medicinal plant genus frequently used by the indigenous people of South Africa as an immune system booster. The aim of this study was to investigate rooperol's antioxidant and anti-inflammatory properties using the ferric reducing ability of plasma (FRAP) assay, NO and ROS production, and phagocytosis. Materials and methodsDifferentiation of human promonocytic U937 leukemia cells to monocyte–macrophages was induced using 10–100nM 1,25(OH)2D3 and PMA over 72h. Differentiation was confirmed by light microscopy and flow cytometry. Undifferentiated and/or differentiated cells were treated with DMSO (0.25v/v%, vehicle control), hypoxoside (50μg/mL), rooperol (20μg/mL) or PMA (10/20nM, positive control). ROS production was measured in undifferentiated and differentiated monocyte-macrophages using DCFH-DA and flow cytometry. Phagocytosis of pHrodo™ Escherichia coli BioParticles® was measured using pre-treated monocyte-macrophage differentiated U937 cells. NO production was measured in monocyte–macrophage differentiated U937 cells using DAF-2 DA and flow cytometry. ResultsRooperol was shown to have similar or greater antioxidant potential than ascorbic acid. Differentiation of human promonocytic U937 leukemia cells to monocyte–macrophages were confirmed morphologically (cell attachment, clump- and pseudopodia-formation) and biochemically (CD11b and CD14 cell surface marker expression). Rooperol significantly increased ROS and NO production, and phagocytosis in undifferentiated and/or differentiated human promonocytic U937 leukemia cells. Hypoxoside had no or very little effect on ROS and NO production, and phagocytosis. ConclusionThis study confirms previous reports that hypoxoside has to be converted to rooperol to be biologically active. The FRAP assay confirms the antioxidant capacity of rooperol seen in previous studies, whereas rooperol's induction of ROS and NO production, and phagocytosis constitute novel findings. Possible mode(s) of action for the in vitro anti-inflammatory activities of rooperol may be explained by ROS and NO production, and phagocytosis.

DAF-fluorescence without NO: Elicitor treated tobacco cells produce fluorescing DAF-derivatives not related to DAF-2 triazol

Diaminofluorescein-dyes (DAFs) are widely used for visualizing NO production in biological systems. Here it was examined whether DAF-fluorescence could be evoked by other means than nitrosation. Tobacco (Nicotiana tabacum) suspension cells treated with the fungal elicitor cryptogein released compound(s) which gave a fluorescence increase in the cell-free filtrate after addition of DAF-2 or DAF-FM or DAR-4M. DAF-reactive compounds were relatively stable and identified as reaction products of H2O2 plus apoplastic peroxidase (PO). CPTIO prevented formation of these products. Horseradish-peroxidase (HR-PO) plus H2O2 also generated DAF-fluorescence in vitro. Using RP-HPLC with fluorescence detection, DAF derivatives were further analyzed. In filtrates from cryptogein-treated cells, fluorescence originated from two novel DAF-derivatives also obtained in vitro with DAF-2+HR-PO+H2O2. DAF-2T was only detected when an NO donor (DEA-NO) was present. Using high resolution mass spectrometry, the two above-described novel DAF-reaction products were tentatively identified as dimers.In cells preloaded with DAF-2 DA and incubated with or without cryptogein, DAF-fluorescence originated from a complex pattern of multiple products different from those obtained in vitro. One specific peak was responsive to exogenous H2O2, and another, minor peak eluted at or close to DAF-2T.Thus, in contrast to the prevailing opinion, DAF-2 can be enzymatically converted into a variety of highly fluorescing derivatives, both inside and outside cells, of which none (outside) or only a minor part (inside) appeared NO dependent. Accordingly, DAF-fluorescence and its prevention by cPTIO do not necessarily indicate NO production.