Stimulation Of Nitric Oxide Production Research Articles

The Factors Affecting the Stability of IOP Homeostasis.

Shear-induced nitric oxide (NO) production by Schlemm's canal (SC) endothelial cells provides a fast, IOP-sensitive feedback signal that normally contributes to IOP homeostasis. Our goal was to analyze the response of this homeostatic system under constant flow perfusion (as occurs in vivo) vs. constant pressure perfusion (as typical for laboratory perfusions). A mathematical model of aqueous humor dynamics, including shear-mediated NO signaling, was formulated and analyzed for stability. The model includes Goldmann's equation, accounting for proximal and distal outflow resistance, and describes how elevated IOP causes narrowing of SC lumen that increases the shear stress on SC cells. Elevated shear stress stimulates NO production, which acts to reduce outflow resistance and relax trabecular meshwork cells to decrease trabecular meshwork stiffness, affecting the SC luminal caliber. During constant flow perfusion, the outflow system is typically stable, returning to baseline IOP after a perturbation. In contrast, during constant pressure perfusion, the outflow system can become unstable and exhibit a time-dependent change in outflow resistance that diverges from baseline. The stability of shear mediated IOP homeostasis is predicted to differ critically between constant flow vs. constant pressure perfusion. Because outflow facility is typically measured at a constant pressure in the laboratory, this instability may contribute to the characteristic time-dependent increase in outflow facility, known as washout, observed in many nonhuman species. Studies of IOP homeostasis should consider how the outflow system may respond differently under constant pressure vs. constant flow perfusion.

EGCG’s effects on Mean Arterial Pressure and Heart Rate Variability and Their Correlations with Metabolic and Hormonal Profiles in Obese Subjects

This study aimed to investigate the effects of epigallocatechin gallate (EGCG), a potent polyphenol found in green tea, on mean arterial pressure (MAP) and heart rate variability (HRV) in obese subjects along with their associations with metabolic factors. We hypothesized that EGCG treatment could significantly reduce MAP and sympathetic tone, represented by decreased low-frequency power (LF) and LF-to-high frequency power (HF) ratio. Furthermore, we expected that systolic blood pressure (SBP), diastolic blood pressure (DBP), and MAP would be positively correlated with obesity parameters, LF, and LF-to-HF ratio. This study is a double-blind, randomized controlled trial. Thai obese subjects with a body mass index (BMI) ≥ 25 kg/m2 (criteria for an Asian population) were randomly allocated into EGCG and placebo groups and received a capsule of 150 mg of EGCG or starch, respectively, twice a day without any dietary restrictions. Clinical and anthropometric data, as well as spontaneous 5-minute HRV measurements in both time and frequency domains, were assessed at baseline (week 0), week 4, and week 8. Additionally, 8-hour fasting blood samples were collected at weeks 0 and 8 to measure metabolic and hormonal levels. EGCG treatment for 8 weeks significantly reduced MAP compared to week 0 and week 4 (P < 0.05 for all). In the EGCG treatment group, there was a significant increase in the LF-to-HF ratio and a nonsignificant decrease in HF at week 8 compared to week 0. LF showed a trend toward increasing in week 8 of EGCG treatment compared to week 4. For correlation analysis at week 0, SBP had significant positive correlations with BMI, percent fat, fat mass, serum leptin and insulin levels, and the homeostatic model assessment for insulin resistance (HOMA-IR) (R = 0.468-0.593, p<0.05 for all) but had negative correlations with the quantitative insulin sensitivity check index (QUICKI) and HDL-cholesterol (R = (-0.437)-(-0.468), P <0.05 for all). DBP was positively correlated with age and HF in normal units (nu) (R = 0.412-0.568, p<0.05 for all), but was negatively correlated with height, HDL-cholesterol, LF in ms (R = (-0.379)-(-0.509), P <0.05 for all). MAP showed significant positive correlations with age, BMI, percent fat, fat mass, serum leptin levels, and HOMA-IR (R = 0.370-0.459, P < 0.05 for all), but had a negative correlation with HDL-cholesterol (R = -0.522, P = 0.004). EGCG treatment over an 8-week duration reduced MAP, a pivotal component of overall blood pressure in obese individuals, and exhibited a significant increase in the LF/HF ratio, tending toward an increase in LF and a non-significant decrease in HF. We postulate that the decrease in MAP may be attributed to EGCG's direct or indirect vasodilatory effects, potentially through the stimulation of nitric oxide production, reduced oxidative stress, and/or decreased levels of kisspeptin, a vasoconstrictive substance, as reported by previous studies. An increase in the LF/HF ratio could reflect a shift toward sympathetic nervous system dominance (LF) and/or a decrease in parasympathetic nervous system activity (HF) indicating a compensatory response to the reduced blood pressure post-vasodilation. HDL cholesterol showed negative correlations with SBP, DBP, and MAP, reflecting its protective benefit against high blood pressure. In summary, 8 weeks of EGCG treatment resulted in a significant decrease in MAP and an increase in the LF/HF ratio, reflecting EGCG’s primary vasodilatory effects and a compensatory increase in sympathetic nervous system activity. The research was funded by the Faculty of Medicine Siriraj Hospital Research Fund ((IO) R015932003, R0159333013 and R016132012). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Auxin acts upstream of nitric oxide to regulate cell wall xyloglucan and its aluminium-binding capacity in Arabidopsis thaliana.

Auxin acts upstream of NO through NOA and XXT5 pathways to regulate the binding capacity of the root cell wall to Al. In our previous study, we identified an unknown mechanism by which 1-naphthaleneacetic acid (NAA) decreased the fixation of aluminum (Al) in the cell wall. Here, we observed that external application of the nitric oxide (NO) donor S-nitrosoglutathion (GSNO) increased the inhibition of Al on root elongation. Further analysis indicated that GSNO could induce Al accumulation in the roots and root cell walls, which is consistent with lower xyloglucan content. In comparison to the Columbia-0 (Col-0) wild type (WT), endogenous NO-reduced mutants noa1 (NOA pathway) and nia1nia2 (NR pathway) were more resistant to Al, with lower root Al content, higher xyloglucan content, and more Al accumulation in the root cell walls. By contrast, the xxt5 mutant with reduced xyloglucan content exhibited an Al-sensitive phenotype. Interestingly, Al treatment increased the endogenous auxin and NO levels, and the auxin levels induced under Al stress further stimulated NO production. Auxin application reduced Al retention in hemicellulose and decreased the xyloglucan content, similar to the effects observed with GSNO. In yucca and aux1-7 mutants, exogenous application of NO resulted in responses similar to those of the WT, whereas exogenous auxin had little effect on the noa1 mutant under Al stress. In addition, as auxin had similar effects on the nia1nia2 mutant and the WT, exogenous auxin and NO had little effect on the xxt5 mutant under Al stress, further confirming that auxin acts upstream of NO through NOA and XXT5 pathways to regulate the binding capacity of the root cell wall to Al.

Abstract 11818: The Beneficial Effects of Ceramide on Human Microvascular Endothelial Function is Dependent on S1P/S1PR1 Signaling Axis

Despite the known damaging effects of elevated intracellular ceramides on human microvascular function, our recent work showed that loss of ceramide-formation through neutral sphingomyelinase (NSmase) promotes endothelial dysfunction in arterioles from healthy adults. This presents as a switch in the endothelial-derived mediator responsible for eliciting dilation to flow (flow-induced dilation; FID), from the vasoprotective nitric oxide (NO) to pro-inflammatory hydrogen peroxide (H 2 O 2 ). The mechanism through which ceramide exerts beneficial effects on the vasculature remains poorly understood. One of ceramide’s metabolites, sphingosine-1-phosphate (S1P), has been shown to stimulate NO production through the activation of S1P receptor 1 (S1PR1). Thus, we hypothesize that ceramide promotes NO-mediated FID through its metabolism to S1P and activation of S1PR1. Arterioles (100-250μm) from healthy adults were dissected from discarded surgical adipose tissue. Vessels were cannulated in organ chambers, pre-constricted with endothelin-1, and changes in internal diameter in response to increases in flow or C2-ceramide was measured via videomicroscopy. Difference between mean % dilation ±SE with two-way ANOVA is reported. Agonism of S1PR1 (CYM5442, 1μM, 30min) during NSmase inhibition (GW4869, 4μM, 30min) restored dependency on NO for FID, as L-NAME (100μM; NO synthase inhibitor) diminished FID (GW+CYM, n=4 vs GW+CYM+L-NAME, n=4: -70.3±20.0, p=0.01). A similar switch to NO-mediated FID was seen with C2-ceramide (10μM)+GW4869 treatment (GW+C2, n=5 vs GW+C2+L-NAME, n=4: -52.5±5.9, p<0.001). Inhibition of S1PR1 (W146, 10μM, 30min) induced a switch from NO to H 2 O 2 mediated FID, as catalase (500U/ml; W146, n=5 vs W146+Catalase, n=4: -34.0±7.9; p=0.01) but not L-NAME (W146, n=5 vs W146+L-NAME, n=4; -1.4±9.9, p=0.89) impaired FID. Acute ceramide-induced dilation (1min) was diminished in the presence of L-NAME (C2, n=5 vs C2+L-NAME, n=4: -33.40±10.06, p=0.02) and W146 (C2, n=5 vs C2+W146, n=4; -39.7±5.9, p=0.01). Together, these data suggest that ceramide induces NO-signaling through the activation of S1PR1 and provide mechanistic insight into how ceramide may exert beneficial effects within the human microvascular endothelium.

Dapagliflozin-Induced Myocardial Flow Reserve Improvement is not Associated with HDL Ability to Stimulate Endothelial Nitric Oxide Production.

Sodium-glucose cotransporter-2 (SGLT2) inhibitors have shown controversial results in modulating plasma lipids in clinical trials. Most studies found slight increases in high-density lipoprotein (HDL) cholesterol but few have provided evidence on HDL functionality with disappointing results. However, there is broad agreement that these drugs provide cardiovascular protection through several mechanisms. Our group demonstrated that dapagliflozin improves myocardial flow reserve (MFR) in patients with type 2 diabetes (T2D) with coronary artery disease (CAD). The underlying mechanisms are still unknown, although in vitro studies have suggested the involvement of nitric oxide (NO). To investigate changes in HDL-mediated modulation of NO production with dapagliflozin and whether there is an association with MFR. Sixteen patients with CAD-T2D were enrolled and randomized 1:1 to dapagliflozin or placebo for 4weeks. Blood samples were collected before and after treatment for each group. The ability of HDL to stimulate NO production in endothelial cells was tested in vitro by incubating human umbilical vein endothelial cells (HUVEC) with apoB-depleted (apoB-D) serum of these patients. The production of NO was assessed by fluorescent assay, and results were expressed as fold versus untreated cells. Change in HDL-mediated NO production remained similar in dapagliflozin and placebo group, even after adjustment for confounders. There were no significant correlations between HDL-mediated NO production and MFR either at baseline or after treatment. No changes were found in HDL cholesterol in either group, while low-density lipoprotein cholesterol (LDL cholesterol) significantly decreased compared to baseline only in treatment group (p = 0.043). In patients with T2D-CAD, beneficial effects of dapagliflozin on coronary microcirculation seem to be unrelated to HDL functions. However, HDL capacity to stimulate NO production is not impaired at baseline; thus, the effect of drug treatments would be negligible. To conclude, we can assume that HDL-independent molecular pathways are involved in the improvement of MFR in this population. EudraCT No. 2016-003614-27; ClinicalTrials.gov Identifier: NCT03313752.

Nitric oxide regulates mitochondrial biogenesis in plants.

The site of nitric oxide (NO) production in mitochondrial cytochrome c oxidase and the role of NO in mitochondrial biogenesis are not known in plants. By imposing osmotic stress and recovery on Arabidopsis seedlings we investigated the site of NO production and its role in mitochondrial biogenesis. Osmotic stress reduced growth and mitochondrial number while increasing NO production. During the recovery phase the mitochondrial number increased and this increase was higher in wild type and the high NO-producing Pgb1 silencing line in comparison to the NO-deficient nitrate reductase double mutant (nia1/nia2). Application of nitrite stimulated NO production and mitochondrial number in the nia1/nia2 mutant. Osmotic stress induced COX6b- 3 and COA6-L genes encoding subunits of COX. The mutants cox6b-3 and coa6-l were impaired both in NO production and mitochondrial number during stress to recovery suggesting the involvement of these subunits in nitrite-dependent NO production. Transcripts encoding the mitochondrial protein import machinery showed reduced expression in cox6b-3 and coa6-l mutants. Finally, COX6b-3 and COA6-L interacted with the VQ27 motif-containing protein in the presence of NO. The vq27 mutant was impaired in mitochondrial biogenesis. Our results suggest the involvement of COX derived NO in mitochondrial biogenesis.

Magnoflorine from Tinospora crispa upregulates innate and adaptive immune responses in Balb/c mice

Magnoflorine shows a diverse range of pharmacological actions, including immunomodulatory, antioxidant and neuropharmacological activities. However, its effects on the immune responses in animal studies have not been reported. In this study, magnoflorine isolated from Tinospora crispa, at doses of 25, 50 and 100 mg/kg was administered to male Balb/c mice daily for 14 days to evaluate its effect on innate immune responses, while for evaluation of adaptive immune responses, on day 0 the mice were injected intraperitoneally with sheep red blood cells (sRBC) and treated orally with the various doses of magnoflorine for the same duration. The effects of magnoflorine on phagocytosis, myeloperoxidase (MPO) activity, lysozyme serum level, nitric oxide (NO) production, CD4+ and CD8+ cells population, T and B lymphocytes proliferation, activated T cells cytokines production, antibodies levels and delayed type hypersensitivity (DTH) were determined. Magnoflorine dose-dependently stimulated NO production, E. coli engulfment by neutrophils and peritoneal macrophages, MPO activity and lysozyme serum level in treated mice. Magnoflorine at 100 mg/kg exhibited comparable stimulation of B cell production compared to levamisole at 2.5 mg/kg. It also significantly increased CD4+ and CD8+ cells population, upregulated the Th1 (IFN-γ, IL-2 and TNF-α) and Th2 (IL-4 and IL-6) cytokines in a dose-dependent manner. At similar concentrations, magnoflorine also exhibited a strong dose-dependent stimulation on DTH reaction and upregulation of immunoglobulins (IgG and IgM) production in mice immunized with sRBC. The strong upregulation of innate and adaptive immune responses indicates that magnoflorine has potential to be developed into an effective immunostimulant.

Dual Strategy with Intracavernosal Implantation of Adipose-derived Stem Cells and Daily Oral L-arginine Optimize Recovery of and Cavernosal Functions after Cavernous Nerve Injury in Rats

ABSTRACT Introduction Radical prostatectomy for prostate cancer has a significant potential role in inducing cavernous nerve injury (CNI). Increasing hypoxia and fibrosis induces destructive changes within cavernous vasculature. Adipose-derived stem cells (ADSCs) can maintain self-renewal and enhanced multi-differentiation potential through the release of a variety of paracrine factors and extracellular vesicles, allowing to repair damaged tissues. Considerable attention has increasingly been paid to their application in tissue engineering and regeneration. In addition, ADSCs can be obtained with a minimally invasive procedure and easily expanded in vitro with low immunogenicity. Nitric oxide (NO) is synthesized from the amino acid L-arginine by a family of enzymes, the nitric oxide synthases (NOS). Orally administered L-arginine enhances the erectile response presumably by stimulating NO production by the corporal tissues increasing cGMP production. Objective To evaluate the efficacy and tolerability of ADSCs and daily oral L-arginine alone or combination in treating rats with bilateral CNI (BCNI)-induced ED. Methods Adult Sprague-Dawley rats (n = 35) were divided into five groups: 1) control; 2) BCNI (4-wk); 3) BCNI+ADSCs (1 × 106 cells by intracavernosal injection); 4) BCNI+ L-arginine (4wk,10 mg/kg/day, oral); and 5) combination treatment with ADSCs and L-arginine in BCNI. Rats in the ADSCs treatment groups were subjected to intracavernosal injection after BCNI. In vivo erectile response was measured by CN stimulation. The relaxant and contractile responses were also detected on in vitro preparations of corpus cavernosum (CC) strips. The expression and localization of neuronal (nNOS), endothelial NOS (eNOS) as well as hypoxia (hypoxia-inducible factor-1α, HIF-1α), fibrosis [transforming growth factor-beta 1 (TGF- β1)]and apoptosis (Bax and Bcl-2) markers were determined using Western blotting and immunohistochemistry. The relative area of smooth muscle to collagen was measured using Masson trichrome staining. Results Rats with CNI had the lowest ICP/MAP ratio at 63% and total ICP at 55% for 7.5 V (P < .001 vs controls) as well as in vitro electrical field stimulation-induced relaxant response at 10% (P < .05 vs controls) and endothelium-dependent acetylcholine-caused relaxation at 56% (P < .001 vs controls). The combined treatment showed more significantly improved erectile and relaxant responses more effectively than ADSCs or L-arginine treatment alone. Increased in the expressions of nNOS, HIF-1α, Bax, and Bcl-2 in the BCNI group were restored by all treatment regiments. eNOS and TGF- β1 protein expressions were similar among groups. Mono-treatment partially improved decreased smooth muscle mass while combined therapy completely recovered. Conclusions The intracavernous injection of ADSCs and oral administration of L-arginine, and, importantly, their combination could effectively protect against hypoxia-induced neurotoxicity, via their increasing endothelial cell function and smooth muscle content, anti-inflammatory, anti-apoptotic, anti-fibrotic properties in addition to reversing erectile function in the BCNI rat model. HIF-1α overexpression by the inhibition of the apoptotic pathway with the Bcl-2 family may have protective effects on the survival of cavernosal neurons. Disclosure No

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.

Extremely low frequency electromagnetic stimulation reduces ischemic stroke volume by improving cerebral collateral blood flow

Extremely low frequency electromagnetic stimulation (ELF-EMS) has been considered as a neuroprotective therapy for ischemic stroke based on its capacity to induce nitric oxide (NO) signaling. Here, we examined whether ELF-EMS reduces ischemic stroke volume by stimulating cerebral collateral perfusion. Moreover, the pathway responsible for ELF-EMS-induced NO production was investigated. ELF-EMS diminished infarct growth following experimental stroke in collateral-rich C57BL/6 mice, but not in collateral-scarce BALB/c mice, suggesting that decreased lesion sizes after ELF-EMS results from improved collateral blood flow. In vitro analysis demonstrated that ELF-EMS increased endothelial NO levels by stimulating the Akt-/eNOS pathway. Furthermore, ELF-EMS augmented perfusion in the hind limb of healthy mice, which was mediated by enhanced Akt-/eNOS signaling. In healthy C57BL/6 mouse brains, ELF-EMS treatment increased cerebral blood flow in a NOS-dependent manner, whereas no improvement in cerebrovascular perfusion was observed in collateral-sparse BALB/c mice. In addition, ELF-EMS enhanced cerebral blood flow in both the contra- and ipsilateral hemispheres of C57BL/6 mice subjected to experimental ischemic stroke. In conclusion, we showed that ELF-EMS enhances (cerebro)vascular perfusion by stimulating NO production, indicating that ELF-EMS could be an attractive therapeutic strategy for acute ischemic stroke by improving cerebral collateral blood flow.

Melatonin promotes the normal cellular mitochondrial function of lotus seeds through stimulating nitric oxide production

Melatonin (MT) plays a vital role in diverse physiological processes of postharvest fruit and vegetables but information on the involvement of nitric oxide (NO) in the MT pathway is limited. The effect of NO, MT and MT combined with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (MT + cPTIO) on the cellular mitochondrial function of lotus seeds was investigated. The browning of lotus seeds was delayed by NO and MT treatments but aggravated by cPTIO treatment. Reactive oxygen species in the mitochondria of NO and MT-treated seeds were efficiently scavenged by antioxidant enzymes stimulated by the treatment, thus inhibiting malondialdehyde production and oxidative damage of the mitochondria. The activities of energy metabolism-related enzymes involved in the respiratory chain were also increased by NO and MT treatments, thereby enhancing ATP production in the lotus seeds. This resulted in increased mitochondrial membrane potential and oxygen consumption during respiratory metabolism. However, the MT + cPTIO treatment produced the opposite effect to MT alone that increased NOS activity but showed no consistent effect on NR activity. These findings suggested that exogenous MT treatment maintained normal mitochondrial function in lotus seeds by inducing endogenous NO production, which could be resulted from the increased NOS activity.

Antileishmanial Activity of Ziziphus spina-christi Leaves Extract and Its Possible Cellular Mechanisms.

This experimental investigation was designed to assess the in vitro and in vivo antileishmanial effects of Z. spina-christi methanolic extract (ZSCME) and also aims to assess some of the antileishmanial mechanisms such as the NO production, apoptosis, and plasma membrane permeability. We assessed the in vitro leishmanicidal effects of ZSCME (10–200 µg/mL) against intracellular amastigote stage of the Leishmania major (MRHO/IR/75/ER) and, then, in vivo examined male BALB/c mice infected by L. major. In addition, the rate of infectivity, Caspase 3 activity, nitric oxide (NO) production, the plasma membrane permeability, and the cytotoxic effects of ZSCME were studied. The primary phytochemical analysis of ZSCME revealed the existence of high amounts of flavonoids, tannins, glycosides, alkaloids, and saponin in this plant. The findings exhibited that ZSCME meaningfully (p < 0.001) reduced the viability of amastigotes of L. major, whereas it prompted the creation and release of NO, apoptosis, and the plasma membrane permeability (p < 0.05) and indicated no cytotoxicity in macrophage cells. The in vivo results also demonstrated that ZSCME significantly decreased the parasite load and the diameter of the lesions in the infected mice. Our results demonstrate the promising in vitro and in vivo antileishmanial effects of ZSCME against of L. major. Although the findings of the present study showed some possible antileishmanial mechanisms of ZSCME, such as stimulating NO production, apoptosis, and increasing plasma membrane permeability, additional investigations are required to confirm these results.

Role of nitric oxide on zymosan-induced inhibition of crop emptying in chicks

Zymosan, a component of yeast cell walls, reduces feed passage through the digestive tract in chicks (Gallus gallus), although the mechanism mediating this effect is poorly understood. Nitric oxide (NO) is associated with a variety of biological actions including effects on the immune system. In addition, it has been suggested that NO is involved in relaxation of the digestive tract and affects feed passage in mammals. It is therefore possible that NO might be related to zymosan-induced reduction of feed passage in chicks. However, the role of NO on the effect of zymosan feed passage has not been clarified yet. Thus, the purpose of the present study was to investigate whether NO is associated with zymosan-induced alteration of feed passage in chicks. Intraperitoneal (IP) injection of zymosan significantly increased plasma nitrate and nitrite (NOx) concentrations at 6 h after injection. Zymosan-induced elevation of plasma NOx concentration was abolished by co-injection of S-methylisothiourea (SMT), a selective inhibitor for inducible NO synthase (iNOS), indicating that zymosan facilitated the induction of iNOS. Furthermore, because zymosan increased iNOS mRNA expression in the digestive tract, NO is likely associated with the effect of zymosan on the digestive tract. IP injection of NO donors significantly decreased crop emptying rate, suggesting that NO functions as an inhibitor of crop emptying. This result implied that zymosan stimulates NO production by the induction of iNOS in the digestive tract and thereby inhibits crop emptying rate. However, the co-injection of SMT did not attenuate the inhibitory effect of zymosan on crop emptying. The present study provides evidence that some changes in the digestive tract caused by zymosan are mediated by iNOS-induced NO in chicks, but NO does not mediate the effect of zymosan on feed passage through the crop.

Estrogen enhances the cytotoxicity of PARP inhibitors on breast cancer cells through stimulating nitric oxide production

Inhibition of Poly(ADP-ribose) polymerase (PARP) is effective for breast cancer susceptibility genes 1 (BRCA1)-deficient breast cancers. Although hormones play critical roles on the occurrence as well as being used in conventional therapies of breast cancer, their impacts on PARP-targeted therapy have been poorly addressed. Here, we showed that addition of estrogen could enhance the cytotoxicity of PARP inhibitors on estrogen receptor (ER)-positive breast cancer cells, causing significant suppression of cell growth. Further analysis revealed that the impact was due to estrogen’s stimulating the production of nitric oxide (NO), which could be abrogated when blocking NO formation. Moreover, the effect of estrogen can be resembled by two exogenous nitric oxide donors (SNAP and GSNO). Using ER-negative cell line MDA-MB231, estrogen could not enhance the cell killing of PARP inhibitors any more, but addition of NO donors re-established the enhancing effects. The increased NO level led to accumulation of DNA double strand breaks (DSBs) based on the formation of H2AX foci. Consistent with earlier studies, we demonstrated that NO suppressed the expression of BRCA1, a key player involved in DSB recombination repair. Taken together, these data reveal an important role of estrogen on the treatment of PARP inhibitors, which may affect its clinical treatment and should be considered in precision therapies for ER-positive and negative cancers.

Diphlorethohydroxycarmalol Isolated from Ishige okamurae Exerts Vasodilatory Effects via Calcium Signaling and PI3K/Akt/eNOS Pathway.

Nitric oxide (NO) is released by endothelial cells in the blood vessel wall to enhance vasodilation. Marine polyphenols are known to have protective effects against vascular dysfunction and hypertension. The present study is the first to investigate how diphlorethohydroxycarmalol (DPHC) isolated from Ishige okamurae affects calcium levels, resulting in enhanced vasodilation. We examined calcium modulation with the well-known receptors, acetylcholine receptor (AchR) and vascular endothelial growth factor 2 (VEGFR2), which are related to NO formation, and further confirmed the vasodilatory effect of DPHC. We confirmed that DPHC stimulated NO production by increasing calcium levels and endothelial nitric oxide synthase (eNOS) expression. DPHC affected AchR and VEGFR2 expression, thereby influencing transient calcium intake. Specific antagonists, atropine and SU5416, were used to verify our findings. Furthermore, based on the results of in vivo experiments, we treated Tg(flk:EGFP) transgenic zebrafish with DPHC to confirm its vasodilatory effect. In conclusion, the present study showed that DPHC modulated calcium transit through AchR and VEGFR2, increasing endothelial-dependent NO production. Thus, DPHC, a natural marine component, can efficiently ameliorate cardiovascular diseases by improving vascular function.

Abstract 13480: High Density Lipoprotein Promotes Angiogenesis by Suppressing Mir-24-3p Expression

Introduction: Previous studies showed that high density lipoprotein (HDL) can stimulate angiogenesis. However, the mechanisms by which HDL promotes angiogenesis remains unclear. Hypothesize: HDL may promote angiogenesis by regulating miRNAs expression. Methods: HDL was isolated from healthy subjects. Human umbilical vein endothelial cells (HUVECs) were cultured with vehicle or HDL (100 μg/ml), and the differential miRNAs expression were indentified by miRNA array and verified by qRT-PCR. HUVECs were treated with vehicle or HDL (100 μg/ml) with or without miRNAs mimic, endothelial cells proliferation, migration and tube formation were detected. The production of nitric oxide(NO) was measured. The expression and phosphorylation of endothelial nitric oxide synthase (eNOS) was determined. Results: The miRNAs profile of HDL-treated HUVECs is significantly different from control group. HDL significantly downregulated miR-24-3p expression. HDL significantly promoted HUVECs proliferation, migration and tube formation. HDL also significantly stimulated NO production and up-regulated the expression and phosphorylation of eNOS. However, HDL did not stimulated HUVECs proliferation, migration, tube formation and NO production as well as the expression and phosphorylation of eNOS after pretreated with miR-24-3p mimic. Conclusions: HDL can promote angiogenesis by suppressing miR-24-3p expression.

Fluoride-induced hepatotoxicity is prevented by L-Arginine supplementation via suppression of oxidative stress and stimulation of nitric oxide production in rats

Concerns over fluoride toxicity have increased in recent times due to high exposures from various anthropogenic sources such as industrial sites, fluoride-containing pesticides, drugs, dental products, refridgerants and fire extinguishers. There is, therefore, continued search for agents that could ameliorate the toxicity of this chemical in various body organs. In this study, we sought to investigate the protective effects of L-Arginine (L-Arg), a nitric oxide donor, on liver toxicity induced by sodium fluoride (NaF) in rats. Rats received NaF (300 mg L−1) in drinking water alone or in co-treatment with L-Arg at two different doses, 100 and 200 mg kg−1, by oral gavage, for 7 days. Markers of hepatotoxicity, oxidative stress and antioxidant status were thereafter assessed. NaF caused marked increase in serum transaminases: alanine aminotransferase, Aspartate aminotransferase and Alkaline phosphatase, along with atrophy of the centri-lobular hepatic cords and dilatation of the sinusoids. Moreover, NaF stimulated increases in hepatic contents of hydrogen peroxide (H2O2), nitric oxide (NO), protein carbonyls, malondialdehyde and advanced oxidation protein products. NaF also inhibited the activities of antioxidant enzymes, Glutathione peroxidase and Superoxide dismutase. However, L-Arg supplementation caused significant alleviation of NaF hepatotoxicity by reducing lipid and protein oxidation indices, stimulation of antioxidant systems along with increased production of NO. L-Arg showed promise as a potential protective agent against NaF-induced hepatotoxicity via restoration of oxidant-antioxidant balance. Further studies are required to understand the involvement of NO signaling in the protective effects of L-Arg against fluoride toxicity.

Inlet flow rate of perfusion bioreactors affects fluid flow dynamics, but not oxygen concentration in 3D-printed scaffolds for bone tissue engineering: Computational analysis and experimental validation

Fluid flow dynamics and oxygen-concentration in 3D-printed scaffolds within perfusion bioreactors are sensitive to controllable bioreactor parameters such as inlet flow rate. Here we aimed to determine fluid flow dynamics, oxygen-concentration, and cell proliferation and distribution in 3D-printed scaffolds as a result of different inlet flow rates of perfusion bioreactors using experiments and finite element modeling. Pre-osteoblasts were treated with 1 h pulsating fluid flow with low (0.8 Pa; PFFlow) or high peak shear stress (6.5 Pa; PFFhigh), and nitric oxide (NO) production was measured to validate shear stress sensitivity. Computational analysis was performed to determine fluid flow between 3D-scaffold-strands at three inlet flow rates (0.02, 0.1, 0.5 ml/min) during 5 days. MC3T3-E1 pre-osteoblast proliferation, matrix production, and oxygen-consumption in response to fluid flow in 3D-printed scaffolds inside a perfusion bioreactor were experimentally assessed. PFFhigh more strongly stimulated NO production by pre-osteoblasts than PFFlow. 3D-simulation demonstrated that dependent on inlet flow rate, fluid velocity reached a maximum (50–1200 μm/s) between scaffold-strands, and fluid shear stress (0.5–4 mPa) and wall shear stress (0.5–20 mPa) on scaffold-strands surfaces. At all inlet flow rates, gauge fluid pressure and oxygen-concentration were similar. The simulated cell proliferation and distribution, and oxygen-concentration data were in good agreement with the experimental results. In conclusion, varying a perfusion bioreactor's inlet flow rate locally affects fluid velocity, fluid shear stress, and wall shear stress inside 3D-printed scaffolds, but not gauge fluid pressure, and oxygen-concentration, which seems crucial for optimized bone tissue engineering strategies using bioreactors, scaffolds, and cells.

Immunoglobulins G modulate endothelial function and affect insulin sensitivity in humans

Background and aimsData from animals suggest that immunoglobulins G (IgG) play a mechanistic role in atherosclerosis and diabetes through endothelial dysfunction and insulin resistance. Patients with common variable immunodeficiency (CVID), who have low circulating levels of IgG and are treated with intravenous polyclonal IgG (IVIgG), may provide an ideal model to clarify whether circulating IgG modulate endothelial function and affect insulin sensitivity in humans. Methods and resultsWe studied 24 patients with CVID and 17 matched healthy controls (HC). Endothelial function was evaluated as flow mediated dilation (FMD) of the brachial artery at baseline and 1, 7, 14, and 21 days after IVIgG infusion in the CVID patients. We measured also plasma glucose, insulin, and calculated the HOMA-IR index. We also investigated the role of human IgG on the production of Nitric Oxide (NO) in vitro in Human Coronary Artery Endothelial Cells (HCAEC).Compared to HC, FMD of CVID patients was significantly impaired at baseline (9.4 ± 0.9 and 7.6 ± 0.6% respectively, p < 0.05) but rose above normal levels 1 and 7 days after IVIgG infusion to return at baseline at 14 and 21 days. Serum insulin concentration and HOMA-IR index dropped by 50% in CVID patients after IVIgG (p < 0.002 vs. baseline). In vitro IgG stimulated NO production in HCAEC. ConclusionsReduced IgG levels are associated with endothelial dysfunction and IVIgG stimulates endothelial function directly while improving insulin sensitivity. The current findings may suggest an anti-atherogenic role of human IgG.

Enterococcus faecalis and Staphylococcus aureus stimulate nitric oxide production in macrophages and fibroblasts in vitro

Aim: Nitric oxide (NO) is an important mediator related to damage of the pulp tissue and at the same time to regenerative pulp processes. However, it is not clear how common endodontic microorganisms can regulate this mediator. This study aimed to investigate NO production by macrophages and fibroblasts against Enterococcus faecalis- and Staphylococcus aureus-antigens. Methods: RAW 264.7 macrophages and L929 fibroblast cell lines were stimulated with different heat-killed (HK) antigen concentrations (105-108 colony forming units - CFU) from E. faecalis and S. aureus with or without interferon-gamma (IFN-γ). Cell viability by MTT colorimetric assay and NO production from the culture supernatants were evaluated after 72 h. Results: Data here reported demonstrated that none of the antigen concentrations decreased cell viability in macrophages and fibroblasts. The presence of HK-S. aureus and HK-E. faecalis antigen- stimulated NO production with or without IFN-γ on RAW 264.7. The HK-S. aureus antigen stimulated NO production in L929 fibroblasts with or without IFN-γ, and the highest concentration of HK-E. faecalis with IFN-γ also stimulated NO production by these cells. Conclusion: The amount of NO produced by macrophages and fibroblasts may be involved in the concentration and type of prevalent endodontic microorganisms, generating new answers for the understanding of pulpal revascularization/regeneration processes.