Basal Nitric Oxide Production Research Articles

TNF/iNOS/NO pathway mediates host susceptibility to endothelial-dependent circulatory failure and death induced by betacoronavirus infection

Poor disease outcomes and lethality are directly related to endothelial dysfunction in betacoronavirus infections. Here, we investigated the mechanisms underlying the vascular dysfunction caused by the betacoronaviruses MHV-3 and SARS-CoV-2. Wild-type C57BL/6 (WT) and knockout mice for inducible nitric oxide synthase (iNOS-/-) or TNF receptor 1 (TNFR1-/-) were infected with MHV-3, and K18-hACE2 transgenic mice expressing human ACE2 were infected with SARS-CoV-2. Isometric tension was used to evaluate vascular function. Protein expression was determined by immunofluorescence. Tail-cuff plethysmography and Doppler were used to assess blood pressure and flow, respectively. Nitric oxide (NO) was quantified with the DAF probe. ELISA was used to assess cytokine production. Survival curves were estimated using Kaplan-Meier. MHV-3 infection reduced aortic and vena cava contractility, arterial blood pressure, and blood flow, resulting in death. Resistance mesenteric arteries showed increased contractility. The contractility of the aorta was normalized by removing the endothelium, inhibiting iNOS, genetically deleting iNOS, or scavenging NO. In the aorta, iNOS and phospho-NF-kB p65 subunit expression was enhanced, along with basal NO production. TNF production was increased in plasma and vascular tissue. Genetic deletion of TNFR1 prevented vascular changes triggered by MHV-3, and death. Basal NO production and iNOS expression were also increased by SARS-CoV-2. In conclusion, betacoronavirus induces an endothelium-dependent decrease in contractility in macro-arteries and veins, leading to circulatory failure and death via TNF/iNOS/NO. These data highlight the key role of the vascular endothelium and TNF in the pathogenesis and lethality of coronaviruses.

Heterogenous improvements in endothelial function by sub-blood pressure lowering doses of ARBs result in major anti-aortic root remodeling effects

Low basal nitric oxide (NO) production is associated with a dysfunctional endothelium and vascular diseases. We have shown that some angiotensin II (AngII) receptor type 1 (AT1R) blockers (ARBs), a group of clinic-approved blood pressure (BP)-lowering medications, are also capable of activating endothelial function acutely and chronically, both ex vivo and in vivo, in pleiotropic, AngII-independent fashions, which suggested that endothelial function enhancement with ARBs may be independent of their well-documented BP lowering properties. Herein, we attempt to identify the most potent ARB at activating endothelial function when administered at sub-BP-lowering doses and determine its anti-aortic root remodeling properties in a model of Marfan syndrome (MFS). Amongst the 8 clinically available ARBs tested, only telmisartan and azilsartan induced significant (70% and 49%, respectively) NO-dependent inhibition of aortic contractility when administered for 4 weeks at sub-BP lowering, EC5 doses. Low-dose telmisartan (0.47 mg/kg) attenuated MFS-associated aortic root widening, medial thickening, and elastic fiber fragmentation to the same degree as high-dose telmisartan (10 mg/kg) despite wide differences in BP lowering between the two doses. Our study suggests that telmisartan is the most potent ARB at promoting increased endothelial function at low sub-BP doses and that it retained major aortic root widening inhibition activities. ARBs may enhance endothelial function independently from BP-lowering pathways, which could lead to new therapeutic approaches.

Nitric oxide synthase inhibition reveals differences in the nitric oxide pathway in previously laminitic ponies

Previously laminitic (PL) ponies are reported to have higher blood pressure than non laminitic (NL) ponies. This relative hypertension may be related to endothelial cell dysfunction, similar to humans with metabolic syndrome. To investigate the relationship between laminitis predisposition and endothelial dysfunction, the effect of nitric oxide synthase (NOS) inhibition on the change in circulating nitric oxide (NO) concentrations and systemic blood pressure (BP) was determined. An intravenous NO sensor was used to measure changes in blood NO concentration during and after intravenous infusion of the NOS inhibitor L-nitroarginine methyl ester (L-NAME; 4 mg/kg IV) in PL and NL ponies. NO concentrations decreased and BP increased in response to L-NAME infusion, with a significantly (p = 0.02) greater rate of increase in systolic BP in PL (14.62 ± 1.88 mmHg/h) compared to NL (8.54 ± 1.88 mmHg/h) ponies. This greater effect on BP in PL compared to NL ponies is consistent with higher basal NO production in PL ponies, suggesting that the relative hypertension previously seen in PL ponies results in increased basal NO production, rather than being caused by reduced NO production as hypothesised. Up-regulation of the NO system may be a compensatory mechanism stimulated by the higher resting BP in PL ponies. Further investigation of the mechanism underlying the relative hypertension seen in PL ponies is required.

ALDH2 mutation results in excessive basal nitric oxide production and a delayed response to nitroglycerin

Abstract Background and purpose: Nitroglycerin tolerance is a common phenomenon in patients with coronary artery disease (CAD). Aldehyde dehydrogenase 2 (ALDH2) is the enzyme that metabolizes nitroglycerin to its active form nitric oxide (NO). Previous studies showed altered nitroglycerin in subjects with ALDH2 mutation, but the functional impact on endothelial cells is not fully understood. Methods: In the first step of in vitro experiments, we examined functional properties of induced pluripotent stem cell-derived CD144+ endothelial cells (iPSC-ECs) that expressed wildtype (WT) vs ALDH2+/− variant. In the second step of human studies, diameter of the left anterior descending (LAD) coronary artery was determined using angiography in 151 adult volunteers (111 with WT ALDH2, 32 with ALDH2+/− and 8 with ALDH2−/− genotype) prior to as well as after intracoronary injection of 200-μg nitroglycerin. Results: Briefly, the ALDH2+/− iPSC-ECs demonstrated impaired low-density lipoprotein (LDL) uptake, proliferation, migration, tube formation, oxidative stress resistance, and viability. In comparison to the WT control, the ALDH2+/− iPSC-ECs had elevated NO production under baseline conditions, but exhibited a delayed NO release after nitroglycerin treatment. Exposure to 10-μg/mL nitroglycerin for 2 h increased NO production in WT iPSC-ECs but 4-h exposure was required to stimulate NO production in the ALDH2+/− iPSC-ECs. In comparison to the WT control, the subjects carrying the ALDH2+/− variants had seemingly larger LAD coronary artery diameter (3.5 and 3.8 mm vs 3.4 mm in the WT control), but attenuated vasodilatory response to nitroglycerin (ALDH2MUT group vs the WT control, 7.1 ± 0.6% vs 10.1 ± 0.8%, P = 0.024). Conclusion: These findings indicated elevated NO production by endothelia cells under basal conditions but attenuated response to nitroglycerin upon ALDH2 mutation.

Cardiomyocyte depolarization triggers NOS-dependent NO transient after calcium release, reducing the subsequent calcium transient

Cardiac excitation–contraction coupling and metabolic and signaling activities are centrally modulated by nitric oxide (NO), which is produced by one of three NO synthases (NOSs). Despite the significant role of NO in cardiac Ca2+ homeostasis regulation under different pathophysiological conditions, such as Duchenne muscular dystrophy (DMD), no precise method describes the production, source or effect of NO through two NO signaling pathways: soluble guanylate cyclase-protein kinase G (NO-sGC-PKG) and S-nitrosylation (SNO). Using a novel strategy involving isolated murine cardiomyocytes loaded with a copper-based dye highly specific for NO, we observed a single transient NO production signal after each electrical stimulation event. The NO transient signal started 67.5 ms after the beginning of Rhod-2 Ca2+ transient signal and lasted for approximately 430 ms. Specific NOS isoform blockers or NO scavengers significantly inhibited the NO transient, suggesting that wild-type (WT) cardiomyocytes produce nNOS-dependent NO transients. Conversely, NO transient in mdx cardiomyocyte, a mouse model of DMD, was dependent on inducible NOS (iNOS) and endothelial (eNOS). In a consecutive stimulation protocol, the nNOS-dependent NO transient in WT cardiomyocytes significantly reduced the next Ca2+ transient via NO-sGC-PKG. In mdx cardiomyocytes, this inhibitory effect was iNOS- and eNOS-dependent and occurred through the SNO pathway. Basal NO production was nNOS- and iNOS-dependent in WT cardiomyocytes and eNOS- and iNOS-dependent in mdx cardiomyocytes. These results showed cardiomyocyte produces NO isoform-dependent transients upon membrane depolarization at the millisecond time scale activating a specific signaling pathway to negatively modulate the subsequent Ca2+ transient.

Endothelial-Specific Overexpression of Histone Deacetylase 2 Protects Mice against Endothelial Dysfunction and Atherosclerosis.

We recently described a novel regulatory role for histone deacetylase 2 (HDAC2) in protecting endothelial cells from oxidized low-density lipoprotein (OxLDL)-induced injury. In this study, we examined the effects of endothelial-specific HDAC2 overexpression on endothelial-dependent vasorelaxation and atherogenesis in vivo. Endothelial-specific HDAC2-overexpressing transgenic mice (HDAC2-Tg) were generated under control of the Tie2 promoter. An atherosclerosis model was produced by injecting HDAC2-Tg and wild-type (WT) mice with adeno-associated virus encoding a PCSK9 gain-of-function mutant under control of a liver-specific promoter and feeding them a high-fat diet for 12 weeks. Aortic stiffness in vivo was determined by measuring pulse wave velocity. Wire myography was used to measure endothelium dependent (acetylcholine) and independent (sodium nitroprusside) relaxation in isolated mice aortas. Atherosclerotic plaque burden in aortas was determined by Oil Red O staining and protein expression was determined by western blotting. At baseline, HDAC2-Tg mice had normal mean arterial blood pressure (MAP) and body weight, but pulse wave velocity (PWV), an inverse measure of vascular health and stiffness, was decreased, suggesting that their vessels were more compliant. Moreover, basal nitric oxide production was enhanced in the vessels of HDAC2-Tg mice as compared to that in WT controls, although no significant differences in acetylcholine (endothelial component)- or sodium nitroprusside (non-endothelial component)-mediated relaxation were observed. However, after exposure to OxLDL, aortas from HDAC2-Tg mice exhibited greater acetylcholine-induced relaxation than did those from WT mice. Thus, endothelial-specific vasodilator production was enhanced despite oxidative injury. Atherosclerosis induction in WT mice led to a significant increase in PWV, but in HDAC2-Tg mice, PWV and MAP remained unchanged. Further, aortic rings from HDAC2-Tg exhibited better endothelial-dependent vascular relaxation than did those from WT mice, but not when treated with nitric oxide synthase inhibitor L-NAME. Finally, plaque burden, determined by Oil red O staining, was significantly increased in WT, but not HDAC2-Tg mice, subjected to the atherogenic model. Deletion of endothelial HDAC2 led to impaired endothelial cell-dependent vascular relaxation and increased PWV, compared with those in littermate controls. HDAC2 protects against endothelial dysfunction and atherogenesis induced by oxidized lipids. Hence, overexpression or activation of HDAC2 represents a novel therapy for endothelial dysfunction and atherosclerosis. HDAC2-Tg mice provide an opportunity to determine the role of endothelial HDAC2 in vascular endothelial homeostasis.

Cigarette Smoke Extract and Its Cytotoxic Factor Acrolein Inhibit Nitric Oxide Production in Human Vascular Endothelial Cells.

Acrolein (ACR), a highly reactive α,β-unsaturated aldehyde, is a major cytotoxic factor in nicotine- and tar-free cigarette smoke extract (CSE). There are conflicting results regarding endothelial functions despite the fact that both CSE and ACR cause cellular damage. Several lines of evidence indicate that CSE impairs endothelium-derived nitric oxide (NO)-dependent vasodilation by reducing the activity and protein expression of endothelial NO synthase (eNOS), whereas ACR elicits endothelium-dependent vasorelaxation by increasing the production of NO and expression of eNOS. To clarify whether CSE and its cytotoxic factor ACR cause endothelial dysfunction, this study examined the effects of CSE and ACR on human vascular endothelial EA.hy926 cells. CSE and ACR reduced the phosphorylation of eNOS at serine (Ser)1177 and total expression of eNOS. The CSE- and ACR-induced decrease in the phosphorylation and expression of eNOS was counteracted by glutathione (reduced form), an antioxidant. Basal NO production was inhibited by CSE, ACR, NG-nitro-L-arginine methyl ester (a competitive eNOS inhibitor), and nominally Ca2+-free solution supplemented with BAPTA-AM (a membrane permeable Ca2+ chelator). These results indicate that CSE and ACR increase oxidative stress, and reduce NO production by reducing the activity and total protein level of eNOS.

Combined aerobic and low-intensity resistance exercise training increases basal nitric oxide production and decreases arterial stiffness in healthy older adults.

Meta-analyses have concluded that combined aerobic and high-intensity or moderate-intensity resistance exercise training has no effects on arterial stiffness. However, a recent study demonstrated that combined aerobic training and resistance training using rubber bands increases basal nitric oxide (NO) production and decreases arterial stiffness with marked reduction of body weight in obese adolescent girls. To investigate whether combined aerobic and low-intensity resistance training increases basal NO production and decreases arterial stiffness without body weight reduction in older adults, 27 healthy older individuals participated in a 6-week program as a part of the training group (mean body mass index, 21 kg/m2; walking and resistance training using one’s body weight) or the control group (22 kg/m2, asked not to modify their lifestyle). The exercise intervention increased aerobic capacity, muscle strength, and plasma concentrations of nitrite/nitrate (end products of NO) and decreased pulse wave velocity (an index of arterial stiffness) without changes in body weight. In the control group, there were no differences in these measures before and after the study period. These results suggest that combined aerobic and low-intensity resistance exercise training increases basal NO production and decreases arterial stiffness in healthy older adults.

Sildenafil Prevents Marfan-Associated Emphysema and Early Pulmonary Artery Dilation in Mice

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in fibrillin-1 (Fbn1). Although aortic rupture is the major cause of mortality in MFS, patients also experience pulmonary complications, which are poorly understood. Loss of basal nitric oxide (NO) production and vascular integrity has been implicated in MFS aortic root disease, yet their contribution to lung complications remains unknown. Because of its capacity to potentiate the vasodilatory NO/cyclic guanylate monophosphate signaling pathway, we assessed whether the phosphodiesterase-5 inhibitor, sildenafil (SIL), could attenuate aortic root remodeling and emphysema in a mouse model of MFS. Despite increasing NO-dependent vasodilation, SIL unexpectedly elevated mean arterial blood pressure, failed to inhibit MFS aortic root dilation, and exacerbated elastic fiber fragmentation. In the lung, early pulmonary artery dilation observed in untreated MFS mice was delayed by SIL treatment, and the severe emphysema-like alveolar destruction was prevented. In addition, improvements in select parameters of lung function were documented. Subsequent microarray analyses showed changes to gene signatures involved in the inflammatory response in the MFS lung treated with SIL, without significant down-regulation of connective tissue or transforming growth factor-β signaling genes. Because phosphodiesterase-5 inhibition leads to improved lung histopathology and function, the effects of SIL against emphysema warrant further investigation in the settings of MFS despite limited efficacy on aortic root remodeling.

Reduced Basal Nitric Oxide Production Induces Precancerous Mammary Lesions via ERBB2 and TGF\u03b2

One third of newly diagnosed breast cancers in the US are early-stage lesions. The etiological understanding and treatment of these lesions have become major clinical challenges. Because breast cancer risk factors are often linked to aberrant nitric oxide (NO) production, we hypothesized that abnormal NO levels might contribute to the formation of early-stage breast lesions. We recently reported that the basal level of NO in the normal breast epithelia plays crucial roles in tissue homeostasis, whereas its reduction contributes to the malignant phenotype of cancer cells. Here, we show that the basal level of NO in breast cells plummets during cancer progression due to reduction of the NO synthase cofactor, BH4, under oxidative stress. Importantly, pharmacological deprivation of NO in prepubertal to pubertal animals stiffens the extracellular matrix and induces precancerous lesions in the mammary tissues. These lesions overexpress a fibrogenic cytokine, TGFβ, and an oncogene, ERBB2, accompanied by the occurrence of senescence and stem cell-like phenotype. Consistently, normalization of NO levels in precancerous and cancerous breast cells downmodulates TGFβ and ERBB2 and ameliorates their proliferative phenotype. This study sheds new light on the etiological basis of precancerous breast lesions and their potential prevention by manipulating the basal NO level.

Free radical scavenging activities of phytochemical mixtures and aqueous methanolic extracts recovered from processed coffee leaves

SummaryIt is well known that the choice of extraction solvents greatly affects both phytochemical composition and related bioactivity of plant extract. Herein, we show that coffee leaf extracts derived from 80% methanol have no effect on basal nitric oxide production in Raw 264.7 cells, whereas, a different result was observed with a previous hot water extract. Phytochemical mixtures formulated to contain equivalent amounts of CQAs, mangiferin and rutin know to present in coffee leaves showed a relatively low (3.5–18.5%, TPC; 17.6–31.6%, ABTS; 7.9–13.6%, DPPH; and 8.5–29.7%, ORAC) contribution to antioxidant activities when compared to the coffee leaf extracts. We conclude that the solvent polarity is particularly important to the unique phytochemical mixture recovered, which in turn governs the bioactivity. This study also demonstrated that other phytochemicals in coffee leaves that were not measured in this study are important in assessing total antioxidant activity of this potential plant beverage.

Abstract 17140: Nitric Oxide Production is Decreased in the Left Ventricle of Diabetic Subjects Undergoing Coronary Artery Bypass Grafting

Introduction: Dysregulation of endothelial nitric oxide synthase (eNOS) and generation of nitric oxide (NO) are critical early indicators for diabetes-induced endothelial dysfunction and cardiovascular complications. We hypothesize that levels of NO production and eNOS expression by endothelial cells are decreased in DM subjects when compared to non-DM subjects. Methods: The study cohort consisted of 9 non-DM subjects and 6 DM subjects undergoing myocardial biopsy at the time of coronary artery bypass grafting surgery. The non-myocyte cell suspensions from the left ventricle (LV), right atrial appendages (RAA), and skeletal muscle (SKM) tissue were analyzed by flow cytometry to measure production of nitric oxide in subpopulations of endothelial and non-endothelial cells. Cells in suspension were incubated with DAF-2DA in the presence or absence of NO synthase inhibitor, L-NAME. Flow cytometry was used to determine production of NO in subpopulations of endothelial and non-endothelial cells from biopsies. Measurements of eNOS and phospho-eNOS (ser1177) were performed using western blot. Results: Basal Nitric oxide production was measurable in non-diabetic subjects \ in 55%, 80% and 65% of unstimulated endothelial cells obtained from RAA, LV and SKM biopsies, compared to 40%, 40%, and 66%, respectively in diabetic subjects ( P < 0.02, DM vs Non-DM). No differences were found in the number of NO-producing non-endothelial cells between DM and non-DM subjects. The level of eNOS showed a trend towards decreased protein expression in DM subjects compared to non-DM. Conclusions: Generation of NO by endothelial cells and level of eNOS expression are decreased in left ventricular endothelial cells of DM patients compared to non-DM. Left ventricular biopsies can be used safely for assessment of NO dysregulation and endothelial dysfunction, and whether these can be improved with interventions targeting diabetic cardiovascular disease.

Alterations in NO- and PGI2- dependent function in aorta in the orthotopic murine model of metastatic 4T1 breast cancer: relationship with pulmonary endothelial dysfunction and systemic inflammation

BackgroundPatients with cancer develop endothelial dysfunction and subsequently display a higher risk of cardiovascular events. The aim of the present work was to examine changes in nitric oxide (NO)- and prostacyclin (PGI2)-dependent endothelial function in the systemic conduit artery (aorta), in relation to the formation of lung metastases and to local and systemic inflammation in a murine orthotopic model of metastatic breast cancer.MethodsBALB/c female mice were orthotopically inoculated with 4T1 breast cancer cells. Development of lung metastases, lung inflammation, changes in blood count, systemic inflammatory response (e.g. SAA, SAP and IL-6), as well as changes in NO- and PGI2-dependent endothelial function in the aorta, were examined 2, 4, 5 and 6 weeks following cancer cell transplantation.ResultsAs early as 2 weeks following transplantation of breast cancer cells, in the early metastatic stage, lungs displayed histopathological signs of inflammation, NO production was impaired and nitrosylhemoglobin concentration in plasma was decreased. After 4 to 6 weeks, along with metastatic development, progressive leukocytosis and systemic inflammation (as seen through increased SAA, SAP, haptoglobin and IL-6 plasma concentrations) were observed. Six weeks following cancer cell inoculation, but not earlier, endothelial dysfunction in aorta was detected; this involved a decrease in basal NO production and a decrease in NO-dependent vasodilatation, that was associated with a compensatory increase in cyclooxygenase-2 (COX-2)- derived PGI2 production.ConclusionsIn 4 T1 metastatic breast cancer in mice early pulmonary metastasis was correlated with lung inflammation, with an early decrease in pulmonary as well as systemic NO availability. Late metastasis was associated with robust, cancer-related, systemic inflammation and impairment of NO-dependent endothelial function in the aorta that was associated with compensatory upregulation of the COX-2-derived PGI2 pathway.

Shear-Mediated Erythrocyte Nitric Oxide Production Is Differentially Regulated in Patients with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is an inherited hemoglobinopathy in which hemoglobin polymerizes when deoxygenated, causing increased red blood cell (RBC) stiffness and RBC sickling. This leads to microcirculatory slowing and occlusion, in turn causing vaso-occlusive crises acutely and microcirculatory damage chronically. RBC are known to signal blood vessels in the microcirculation, regulating vessel tone. Nitric oxide (NO) is a potent vasodilator and key regulator of vascular smooth muscle tone throughout the vasculature. However, the possibility that RBC make NO and that this might contribute to vascular tone, particularly in arterioles is controversial. Modulation of RBC NO production due to deformation of RBC by shear stress could have important physiological consequences in SCD where RBC deformability is decreased and dependent on the polymerization state of hemoglobin S.Objective: To determine if shear stress induced deformation induces change in NO production in individual RBC and if the magnitude and time course of this NO differs between RBC from patients with sickle cell disease and normal controls.Methods: NO production was measured using imaging microscopy of fluorescently labeled RBC exposed to shear after being allowed to adhere to a flow chamber attached to the microscope stage. Four SCD patients on simple chronic transfusion therapy, 4 SCD patients not on chronic transfusion therapy and 4 healthy control subjects were recruited from the hematology clinics at Children's Hospital Los Angeles under an IRB approved protocol. RBC underwent histopaque separation then washed 3x and resuspended in KRP buffer. The RBC were then incubated with 4 micromolar DAF-FM Diacetate (4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate, ThermoFisher Scientific), a fluorescent indicator related to NO production. RBC were then exposed to three experimental conditions: 1. buffer alone; 2. 1 milimolar arginine (substrate for NOS NO production); 3. 1 milimolar arginine and 1 milimolar L-NAME (a non-specific NOS inhibitor). The RBC were placed in poly L-lysine coated microfluidic chambers and placed under no shear conditions for 30 minutesk, then sheared at 0.5Pa for 30minutes. Individual cell analysis was performed using Image J analysis software (FIJI).Results: There is a slow baseline rise of NO fluorescent signal without exposure to shear and there is a large, acute increase in NO fluorescent signal with exposure to shear stress. (figure 1) The baseline fluorescent rate of rise is increased in non-transfused sickle cell subjects (p<0.0001) compared to transfused SCD patients and healthy control subjects, suggesting higher basal rate of NO production. (figure 2) We found a significant increase in fluorescence in response to shear in all three groups, with the transfused SCD patients showing a higher rate of change and higher magnitude fluorescence response. (figure 1) Addition of arginine increased the shear induced NO production in all three groups (P<0.05). The baseline NO related fluorescence was blunted with the addition of L-NAME in healthy subjects, whereas it was unchanged in both the transfused and non-transfused SCD groups. There was no significant change in shear-mediated NO related DAF fluorescence after L-NAME was added.Discussion: The results here indicate that RBC produce NO upon shear and the basal rate of NO production is higher in non-transfused patients with sickle cell disease. Addition of arginine increases both the magnitude and rate of NO production in all three groups. SCD patients on chronic transfusion therapy demonstrate a larger increase in NO response to shear. The increased response of NO related fluorescence with the addition of arginine suggests NOS has a role in shear mediated RBC NO production; however, L-NAME blockade does not significantly blunt the response in the presence of arginine. Shear induced RBC NO production may play an important role in regulation of microvascular profusion and would likely be modulated by the RBC deformability differences due to HbS polymerization.Conclusion: There is basal and shear dependent NO production in both AA and SCD RBC, and RBC NOS is differentially regulated in SCD and AA RBC. [Display omitted] [Display omitted] DisclosuresWood:Ionis Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy; Vifor: Consultancy; Vifor: Consultancy; World Care Clinical: Consultancy; World Care Clinical: Consultancy; Celgene: Consultancy; Celgene: Consultancy; AMAG: Consultancy; AMAG: Consultancy; Apopharma: Consultancy; Apopharma: Consultancy; Biomed Informatics: Consultancy; Biomed Informatics: Consultancy.

R59949, a diacylglycerol kinase inhibitor, inhibits inducible nitric oxide production through decreasing transplasmalemmal L-arginine uptake in vascular smooth muscle cells.

Although diacylglycerol kinase (DGK) is known to be expressed in vascular smooth muscle cell, its functional significance remains to be clarified. We hypothesized that DGK is involved in the pathway of cytokine-induced nitric oxide (NO) production in vascular smooth muscle cells. The purpose of this study was to investigate the effects of R59949, a diacylglycerol kinase inhibitor, on inducible nitric oxide production in vascular smooth muscle cell. Cultured rat aortic smooth muscle cells (RASMCs) were used to elucidate the effects of R59949 on basal and interleukin-1β (IL-1β)-induced NO production. The effects of R59949 on protein and mRNA expression of induced nitric oxide synthase (iNOS) and on transplasmalemmal L-arginine uptake were also evaluated using RASMCs. Treatment of RASMCs with R59949 (10μM) inhibited IL-1β (10ng/ml)-induced NO production but not basal NO production. Neither protein nor mRNA expression level of iNOS after stimulation with IL-1β was significantly affected by R59949. Estimated enzymatic activities of iNOS in RASMCs were comparable in the absence and presence of R59949. Stimulation of RASMCs with IL-1β caused a marked increase in transplasmalemmal L-arginine uptake into RASMCs. L-Arginine uptake in the presence of IL-1β was markedly inhibited by R59949, while basal L-arginine uptake was not significantly affected by R59949. Both IL-1β-induced NO production and L-arginine uptake were abolished in the presence of cycloheximide (1μM). The results indicate that R59949 inhibits inducible NO production through decreasing transplasmalemmal L-arginine uptake. DGK is suggested to be involved in cytokine-stimulated L-arginine transport and regulate its intracellular concentration in vascular smooth muscle cell.

Testosterone as a regulator of immune system via modulation of toll-like receptor 4/extracellular signal-regulated kinase signaling pathway

There is a growing body of evidence supporting the immunomodulation effects of testosterone. Previous researches have focused on its direct blunt inflammatory effect on mediation of cytokines secretion through down-regulated expression of toll-like receptor 4 (TLR4). However, how testosterone modulates immune responses via mechanisms of TLR4 downstream molecules has not yet been elucidated. Recently, we have firstly confirmed that testosterone deficiency is the main reason that caused the exacerbate inflammation status in rat spleen. Orchidectomy in rats resulted in a markedly enhance of spleen weight ( splenomegaly ) and basal production of nitric oxide (NO) from splenocytes. Moreover, lipopolysaccharide (LPS) amplified proliferation rate of splenocytes and the production of tumor necrosis factor-alpha (TNF-α) following castration. Extracellular signal-regulated kinase (ERK) is a critical mediator of TLR4 cascades, and we further examined whether absence of endogenous testosterone affects ERK expression. As anticipated, orchidectomized rats manifested an increased phosphorylation of ERK. Furthermore, testosterone administration was demonstrated to be associated with a diminished LPS-evoked TNF-α and NO secretion in a dose-dependent manner. In the present study, we answered how testosterone withdrawal affects downstream signaling cascades of TLR4 and supports that testosterone might potentially ameliorate inflammatory responses. Our findings mention the possibility that testosterone functions might serve as a useful endogenous regulator of immune responses.

DMBT1 promotes basal and meconium-induced nitric oxide production in human lung epithelial cells in vitro

Meconium aspiration syndrome (MAS) is characterized by surfactant inactivation and inflammation. As lung epithelial cells up-regulate nitric oxide (NO) in response to inflammation, the NO production following meconium exposition was examined in relation to expression of Deleted in Malignant Brain Tumors 1 (DMBT1), a protein with functions in innate immunity and inflammatory regulation. Here, DMBT1 expression was analyzed by immunohistochemistry in postmortem lung sections from patients with MAS. The lung epithelial cell line A549, stably transfected with a DMBT1 (DMBT1+ cells) expression plasmid or with an empty expression plasmid (DMBT1- cells), was exposed to meconium. NO was determined in dependence of aminoguanidine (inducible NO synthase inhibitor), steroids and lipopolysaccharide (LPS). DMBT1 is highly expressed in lungs with MAS. In the absence of meconium, DMBT1+ cells showed a higher NO production than the DMBT1- cells (p=0.0090). Meconium led in DMBT1- and DMBT1+ cells to elevated NO levels (p<0.0001), but with a higher NO level in DMBT1+ cells (p<0.0001). Aminoguanidine, an iNOS inhibitor, reduced the higher NO production in DMBT1+ cells (p=0.0476), but NO levels remained above NO production from DMBT1- cells (p=0.0289). Dexamethasone diminished NO production in DMBT1+ cells after meconium exposition (p=0.0076). Combined addition of LPS and meconium significantly increased NO production in both cell types (p<0.0001). In comparison to exposure with only meconium, the combined addition of LPS and meconium to the cells increased NO levels in both DMBT1- cells (p=0.0030) and DMBT1+ cells (p=0.0028). In conclusion, basal and meconium-induced NO production in lung epithelial cells is positively regulated by DMBT1.

Obesity, Inflammation, and Exercise Training: Relative Contribution of iNOS and eNOS in the Modulation of Vascular Function in the Mouse Aorta.

Background: The understanding of obsesity-related vascular dysfunction remains controversial mainly because of the diseases associated with vascular injury. Exercise training is known to prevent vascular dysfunction. Using an obesity model without comorbidities, we aimed at investigating the underlying mechanism of vascular dysfunction and how exercise interferes with this process.Methods: High-sugar diet was used to induce obesity in mice. Exercise training was performed 5 days/week. Body weight, energy intake, and adipose tissues were assessed; blood metabolic and hormonal parameters were determined; and serum TNFα was measured. Blood pressure and heart rate were assessed by plethysmography. Changes in aortic isometric tension were recorded on myograph. Western blot was used to analyze protein expression. Nitric oxide (NO) was evaluated using fluorescence microscopy. Antisense oligodeoxynucleotides were used for inducible nitric oxide synthase isoform (iNOS) knockdown.Results: Body weight, fat mass, total cholesterol, low-density lipoprotein cholesterol fraction, insulin, and leptin were higher in the sedentary obese group (SD) than in the sedentary control animals (SS). Exercise training prevented these changes. No difference in glucose tolerance, insulin sensitivity, blood pressure, and heart rate was found. Decreased vascular relaxation and reduced endothelial nitric oxide synthase (eNOS) functioning in the SD group were prevented by exercise. Contractile response to phenylephrine was decreased in the aortas of the wild SD mice, compared with that of the SS group; however, no alteration was noted in the SD iNOS−/− animals. The decreased contractility was endothelium-dependent, and was reverted by iNOS inhibition or iNOS silencing. The aortas from the SD group showed increased basal NO production, serum TNFα, TNF receptor-1, and phospho-IκB. Exercise training attenuated iNOS-dependent reduction in contractile response in high-sugar diet–fed animals, decreased iNOS expression, and increased eNOS expression.Conclusion: Obesity caused endothelium dysfunction, TNFα, and iNOS pathway up-regulation, decreasing vascular contractility in the obese animals. Exercise training was an effective therapy to control iNOS-dependent NO production and to preserve endothelial function in obese individuals.

34 Decreased dilation of hippocampal arterioles in a rat model of preeclampsia

Introduction Women with preeclampsia (PE) and eclampsia have increased incidence of white matter lesions in the brain and self-report cognitive impairment later in life, suggesting seizures during PE may lead to long-lasting brain injury. The hippocampus, a brain region central to cognition, is more susceptible to seizure-induced injury due to less effective functional hyperemia. How PE affects the function and structure of the hippocampal vasculature remains unknown, but could further limit functional hyperemia and potentiate seizure-induced injury. Objective We tested the hypothesis that hippocampal arterioles (HpAs) in PE have impaired responses to mediators of dilation generated during seizure including extracellular potassium (K+) and nitric oxide (NO) and undergo inward remodeling that limits blood flow to the hippocampus during seizure. Methods Sprague Dawley rats were nonpregnant (NP; n = 6), pregnant (P; d20; n = 6) or with PE (induced by a high cholesterol diet gestational days 7–20; n = 6). HpAs were isolated and pressurized to 60 mmHg in an arteriograph chamber. The function of HpAs was investigated by measuring changes in lumen diameter in response to the small- and intermediate-conductance calcium-activated potassium (SK/IK) channel agonist NS309, and in response to increasing K+ concentration from 3–15 mM to activate inward rectifier potassium (KIR) channels. Basal production of NO and the sensitivity of vascular smooth muscle to NO were investigated using the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10-3M) and the NO donor sodium nitroprusside (SNP; 10-5M), respectively. HpA structure and distensibility were determined by measuring lumen diameter and wall thickness of fully relaxed arterioles. Data are mean ± SEM and comparisons made using a one-way ANOVA with a post-hoc Bonferroni test. Results HpAs from PE rats dilated less when SK/IK channels were activated with NS309 compared to P and NP rats (55 ± 18% vs. 88 ± 2% and 90 ± 4%; p = 0.053). Activation of KIR with 15 mM K+ caused less dilation in HpAs from PE rats compared to P and NP rats, although this did not reach statistical significance (9 ± 8% vs. 61 ± 27% and 20 ± 11%; p = 0.13). NOS inhibition with L-NAME did not constrict any HpAs, suggesting there was no basal NO production. HpAs from PE rats were less sensitive to NO, with less reactivity to SNP compared to P and NP rats (22 ± 10% vs. 30 ± 10% and 38 ± 8%; p > 0.05). Structurally, lumen diameters were smaller in fully relaxed HpAs from PE rats than NP rats (Fig. 1), and vessel walls were thinner in PE and P rats compared to NP rats (7.7 ± 0.6 μm and 8.3 ± 0.7 μm vs. 11.5 ± 0.7 μm at 60 mmHg; p p Conclusion HpAs from PE rats had impaired dilatory responses, were less distensible, and underwent inward, hypotrophic remodeling making them structurally smaller than the NP and P states. This combination of impaired vasodilation and structural remodeling of HAs in PE could limit blood flow to the hippocampus during seizure, resulting in ischemic neuronal injury.

Tadao Nakago, 1933-2016

Tadao Nakago died of bladder cancer on February 12, 2016, just 2 days before his 83rd birthday. He was born on February 14, 1933, in Sanda, north of the Rokko Mountains in Kobe, Japan. He graduated from Osaka University Dental School in 1957 and became a dentist. He chose orthodontics as his specialty and earned a PhD degree from Osaka University in 1965. His academic interest in the early phase of his career was the cephalometric assessment of craniofacial development in the Japanese population to qualify desirable tooth movement. He became a founding professor of orthodontics at Matsumoto Dental College in 1972. He moved to Okayama University in 1980 as a founding director of the dental school-affiliated hospital and served as president of the hospital until 1988. In 1982, he successfully moved the government of Japan to include, for the first time, orthodontic treatment for craniofacial anomalies, such as cleft palate, on the list for national health insurance reimbursement. He served as dean of the dental school from 1992 to 1994. He survived a bitter administrative storm at Okayama University in 1996 and restored its honor at the law court in 2002. He loved to educate young people and tried to disseminate practical thinking about the standards of orthodontic treatment and care. He translated Graber's Textbook of Orthodontics into Japanese, and it became a best-seller. He liked to organize laboratory studies to explain the mechanism of tooth movement. He designed a device to apply cyclic mechanical stretching to cultured cells in a Petri dish.1Matsuo T. Uchida H. Matsuo T. Bovine and porcine trabecular cells produce prostaglandin F2α in response to cyclic mechanical stretching.Jpn J Ophthalmol. 1996; 40: 289-296PubMed Google Scholar The cell-stretching machine was manufactured in 1983 at the workshop affiliated with the Faculty of Engineering of Okayama University. This unique original device preceded commercially available apparatuses and contributed to new knowledge in other fields of medicine.2Okada Y. Matsuo T. Ohtsuki H. Bovine trabecular cells produce TIMP-1 and MMP-2 in response to mechanical stretching.Jpn J Ophthalmol. 1998; 42: 90-94Crossref Scopus (44) Google Scholar, 3Sato Y. Matsuo T. Ohtsuki H. A novel gene (oculomedin) induced by mechanical stretching in human trabecular cells of the eye.Biochem Biophys Res Commun. 1999; 259: 349-351Crossref Scopus (30) Google Scholar, 4Yamaguchi K. Matsuo T. Shiraga F. Ohtsuki H. TIMP-1 production by bovine retinal pigment epithelial cells increases in response to cyclic mechanical stretch.Jpn J Ophthalmol. 2001; 45: 470-474Crossref Scopus (5) Google Scholar, 5Yamaoka A. Matsuo T. Shiraga F. Ohtsuki H. TIMP-1 production by human scleral fibroblast decreases in response to cyclic mechanical stretching.Ophthalmic Res. 2001; 33: 98-101Crossref Scopus (22) Google Scholar, 6Namba M. Matsuo T. Shiraga F. Ohtsuki H. Retinal cells produce TIMP-1 and TIMP-2 in response to cyclic mechanical stretching.Ophthalmic Res. 2001; 33: 163-169Crossref Scopus (11) Google Scholar His last work was focused on the threshold of hydraulic pressure to induce cellular response in periodontal ligament fibroblasts.7Nakago-Matsuo C. Matsuo T. Nakago T. Intracellular calcium response to hydraulic pressure in human periodontal ligament fibroblasts.Am J Orthod Dentofacial Orthop. 1996; 109: 244-248Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 8Nakago-Matsuo C. Matsuo T. Nakago T. Basal nitric oxide production is enhanced by hydraulic pressure in cultured human periodontal ligament fibroblasts.Am J Orthod Dentofacial Orthop. 2000; 117: 474-478Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar The threshold value was consistent with the force needed to move teeth in a clinical orthodontic setting. The work was continued by his daughter, an orthodontist, in collaboration with her husband, an ophthalmologist.9Matsuo T. Matsuo N. Intracellular calcium response to hydraulic pressure in human trabecular cells.Br J Ophthalmol. 1996; 80: 561-566Crossref Scopus (27) Google Scholar, 10Matsuo T. Basal nitric oxide production is enhanced by hydraulic pressure in cultured human trabecular cells.Br J Ophthalmol. 2000; 84: 631-635Crossref Scopus (15) Google Scholar Dr Nakago's funeral was held on his 83rd birthday, with family and friends present at the Buddhist temple where his wife's father had served as a priest.