Affect Nitric Oxide Production Research Articles

Endothelial TRPV4/Cx43 Signaling Complex Regulates Vasomotor Tone in Resistance Arteries.

S-nitrosylation of Cx43 gap junction channels critically regulates communication between smooth muscle cells and endothelial cells. This posttranslational modification also induces the opening of undocked Cx43 hemichannels. However, its specific impact on vasomotor regulation remains unclear. Considering the role of endothelial TRPV4 channel activation in promoting vasodilation through nitric oxide (NO) production, we investigated the direct modulation of endothelial Cx43 hemichannels by TRPV4 channel activation. Using the proximity ligation assay, we identify that Cx43 and TRPV4 are found in close proximity in the endothelium of resistance arteries. In primary endothelial cell cultures from resistance arteries (ECs), GSK-induced TRPV4 activation enhances eNOS activity, increases NO production, and opens Cx43 hemichannels via direct S-nitrosylation. Notably, the elevated intracellular Ca2+ levels caused by TRPV4 activation were reduced by blocking Cx43 hemichannels. In ex vivo mesenteric arteries, inhibiting Cx43 hemichannels reduced endothelial hyperpolarization without affecting NO production in ECs, underscoring a critical role of TRPV4/Cx43 signaling in endothelial electrical behavior. We perturbed the proximity of Cx43/TRPV4 by disrupting lipid rafts in ECs using β-cyclodextrin. Under these conditions, hemichannel activity, Ca2+ influx, and endothelial hyperpolarization were blunted upon GSK stimulation. Intravital microscopy of mesenteric arterioles in vivo further demonstrated that inhibiting Cx43 hemichannels activity, NO production and disrupting endothelial integrity reduce TRPV4-induced relaxation. These findings underscore a new pivotal role of Cx43 hemichannel associated with TRPV4 signaling pathway in modulating endothelial electrical behavior and vasomotor tone regulation.

Impact of different hormones on the regulation of nitric oxide in diabetes

Polymetabolic syndrome achieved pandemic proportions and dramatically influenced public health systems functioning worldwide. Chronic vascular complications are the major contributors to increased morbidity, disability, and mortality rates in diabetes patients. Nitric oxide (NO) is among the most important vascular bed function regulators. However, NO homeostasis is significantly deranged in pathological conditions. Additionally, different hormones directly or indirectly affect NO production and activity and subsequently act on vascular physiology. In this paper, we summarize the recent literature data related to the effects of insulin, estradiol, insulin-like growth factor-1, ghrelin, angiotensin II and irisin on the NO regulation in physiological and diabetes circumstances.

Effect of Latanoprostene Bunod on the Permeability of Trabecular Meshwork Cells

Purpose: To investigate the effects of latanoprostene bunod (LBN) on nitric oxide (NO) production and permeability in human trabecular meshwork cells (HTMC).Methods: HTMC were treated with 50 and 100 µM LBN and latanoprost free acid (LAT) for 30 minutes. Additionally, 100 µM LBN was co-exposed to 0.5 mM L-NAME (N-Nitroarginine methyl ester). Cellular viability and NO production were measured using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) and Griess assays. The permeability and resistance of the HTMC monolayer were evaluated by trans-endothelial electrical resistance (TEER) and carboxyfluorescein permeability.Results: Exposure to 100 µM LBN led to increased NO production, whereas co-exposure to L-NAME reduced NO production. Treatment with 100 µM LBN decreased the TEER of the HTMC monolayer. LBN exposure heightened carboxyfluorescein permeability, but co-exposure to 100 µM LBN and L-NAME reduced permeability. LAT treatment did not affect NO production or permeability.Conclusions: LBN increased the permeability of the HTMC monolayer and increased NO production. Therefore, LBN might increase trabecular outflow in addition to promoting uveoscleral outflow.

Cardioprotective Activity of Pharmacological Agents Affecting NO Production and Bioavailability in the Early Postnatal Period after Intrauterine Hypoxia in Rats.

Intrauterine hypoxia in newborns leads to a multifaceted array of alterations that exert a detrimental impact on the cardiovascular system. The aim of this research was to assess the cardioprotective effects of modulators of the nitric oxide (NO) system, including L-arginine, Thiotriazoline, Angiolin, and Mildronate, during the early postnatal period following intrauterine hypoxia. Methods: The study involved 50 female white rats. Pregnant female rats were given a daily intraperitoneal dose of 50 mg/kg of sodium nitrite starting on the 16th day of pregnancy. A control group of pregnant rats received saline instead. The resulting offspring were divided into the following groups: Group 1-intact rats; Group 2-rat pups subjected to prenatal hypoxia (PH) and daily treated with physiological saline; and Groups 3 to 6-rat pups exposed to prenatal hypoxia and treated daily from the 1st to the 30th day after birth. Nitrotyrosine levels, eNOS, iNOS, and NO metabolites were evaluated using ELISA; to measure the expression levels of iNOS mRNA and eNOS mRNA, a PCR test was utilized. Results: Angiolin enhances the expression of eNOS mRNA and boosts eNOS activity in the myocardium of rats with ischemic conditions. Arginine and particularly Thiotriazoline exhibited a consistent impact in restoring normal parameters of the cardiac nitroxidergic system following PH. Mildronate notably raised iNOS mRNA levels and notably reduced nitrotyrosine levels, providing further support for its antioxidative characteristics.

The Effect of Aging on Nitric Oxide Production during Cerebral Ischemia and Reperfusion in Wistar Rats and Spontaneous Hypertensive Rats: An In Vivo Microdialysis Study.

Nitric oxide (NO) is involved in the pathogenesis of cerebral ischemic injury. Here, we investigated the effects of aging on NO production during cerebral ischemia-reperfusion (IR). Male Wister rats (WRs) were assigned to 12-month-old (older; n = 5) and 3-month-old (younger; n = 7) groups. Similarly, male spontaneous hypertensive rats (SHRs) were allocated to 12-month-old (older; n = 6) and 3-month-old (younger; n = 8) groups. After anesthesia, their NO production was monitored using in vivo microdialysis probes inserted into the left striatum and hippocampus. Forebrain cerebral IR injuries were produced via ligation of the bilateral common carotid arteries, followed by reperfusion. The change in the NO3- of the older rats in the SHR groups in the striatum was less compared to that of the younger rats before ischemia, during ischemia, and after reperfusion (p < 0.05). In the hippocampus, the change in the NO3- of the older rats in the SHR groups was lower compared to that of the younger rats after reperfusion (p < 0.05). There were no significant differences between the two WR groups. Our findings suggested that aging in SHRs affected NO production, especially in the striatum, before and during cerebral ischemia, and after reperfusion. Hypertension and aging may be important factors impacting NO production in brain IR injury.

Poly I:C and R848 facilitate nitric oxide production via inducible nitric oxide synthase in chicks

Nitric oxide (NO) is a gaseous bioactive molecule associated with many physiological functions including vasodilation and neurotransmission. NO also plays an important role in immune responses during viral infections in mammals. However, there is a paucity of knowledge regarding the involvement of NO in viral infections in birds. Therefore, the purpose of the present study was to determine if intraperitoneal (IP) injection of poly I:C and R848 (resiquimod), which are analogues of virus component, affects NO production in chicks (Gallus gallus) as a bird model. The involvement of inducible NO synthase (iNOS) in poly I:C- and R848-induced anorexia and corticosterone release was also investigated. These virus analogues significantly increased plasma NO metabolites (NOx) concentrations. IP injection of poly I:C and R848 significantly increased iNOS mRNA expression in several organs including the liver. On the other hand, poly I:C and R848 significantly decreased mRNA expressions of endothelial NOS and neural NOS in several organs, indicating that induction of iNOS might be responsible for increased NOx levels in plasma. This finding was further confirmed by using a selective iNOS inhibitor, S-methylisothiourea sulfate (SMT), which abolished the poly I:C- and R848-induced increase in plasma NOx concentration. In addition, SMT partly attenuated the poly I:C- and R848-induced increase in plasma corticosterone concentration, suggesting that corticosterone release induced by these virus analogues may be partly mediated by iNOS. Collectively, the present results suggest that viral infections facilitate NO production by inducing iNOS. The liver would play an important role in the NO production because the response in iNOS mRNA expression to poly I:C and R848 was remarkable. The present results also suggest that NO is associated with corticosterone release in birds under viral infection.

Comparison of the anti-inflammatory activities of furanocoumarins from the roots of Angelica dahurica

Background: The roots of Angelica dahurica Bentham et Hooker filius ex Franchet et Savatier (Apiaceae) have traditionally been used for inflammatory skin diseases. A. dahurica roots (Byakushi) contain furanocoumarins, such as imperatorin and byakangelicin. To elucidate which constituents are responsible for the anti-inflammatory effects, we evaluated the potency of crude A. dahurica root extract fractions by monitoring the production of the inflammatory mediator nitric oxide (NO) in hepatocytes.Methods: The dried roots of A. dahurica were collected in South Korea and extracted with methanol. The resulting extract was fractionated into ethyl acetate (EtOAc)-soluble, n-butanol-soluble, and water-soluble fractions. Primary cultured rat hepatocytes were treated with interleukin (IL)-1β and each fraction for 8 h, and then the NO production and lactate dehydrogenase activity in the medium were measured. The expression of inducible nitric oxide synthase (iNOS) was detected by Western blotting, and its mRNA expression level was measured by quantitative reverse transcription-polymerase chain reaction.Results: Among the three fractions, the EtOAc-soluble fraction markedly suppressed NO production without showing cytotoxicity and decreased iNOS expression in hepatocytes. From this hydrophobic fraction, we isolated five furanocoumarins: isoimperatorin, imperatorin, phellopterin, oxypeucedanin, and oxypeucedanin methanolate. Phellopterin and oxypeucedanin methanolate significantly suppressed NO production and reduced the mRNA expression of iNOS and tumor necrosis factor α. In contrast, the other three constituents did not affect NO production. Comparison of their chemical structures suggests that a methoxy group at carbon 5 and a side chain at carbon 8 in the furanocoumarin skeleton may be essential for NO production suppression.Conclusion: These data imply that phellopterin and oxypeucedanin methanolate, which are hydrophobic furanocoumarins, may contribute to the anti-inflammatory effects of A. dahurica roots by suppressing iNOS gene expression.Keywords: Inflammation, nitric oxide, hepatocyte, coumarin, Kampo medicine

Biological and Inflammatory Effects of Antigen 5 from Polybia paulista (Hymenoptera, Vespidae) Venom in Mouse Intraperitoneal Macrophages.

The social wasp Polybia paulista (Hymenoptera, Vespidae) is highly aggressive, being responsible for many medical occurrences. One of the most allergenic components of this venom is Antigen 5 (Poly p 5). The possible modulation of the in vitro immune response induced by antigen 5 from P. paulista venom, expressed recombinantly (rPoly p 5), on BALB/c mice peritoneal macrophages, activated or not with LPS, was assessed. Here, we analyzed cell viability changes, expression of the phosphorylated form of p65 NF-κB subunit, nitric oxide (NO), proinflammatory cytokines production, and co-stimulatory molecules (CD80, CD86). The results suggest that rPoly p 5 does not affect NO production nor the expression of co-stimulatory molecules in mouse peritoneal macrophages. On the other hand, rPoly p 5 induced an increase in IL-1β production in non-activated macrophages and a reduction in the production of TNF-α and MCP-1 cytokines in activated macrophages. rPoly p 5 decreased the in vitro production of the phosphorylated p65 NF-κB subunit in non-activated macrophages. These findings suggest an essential role of this allergen in the polarization of functional M2 macrophage phenotypes, when analyzed in previously activated macrophages. Further investigations, mainly in in vivo studies, should be conducted to elucidate Polybia paulista Ag5 biological role in the macrophage functional profile modulation.

Inhibition of Inducible Nitric Oxide Synthase Prevents IL-1β-Induced Mitochondrial Dysfunction in Human Chondrocytes.

Interleukin (IL)-1β is an important pro-inflammatory cytokine in the progression of osteoarthritis (OA), which impairs mitochondrial function and induces the production of nitric oxide (NO) in chondrocytes. The aim was to investigate if blockade of NO production prevents IL-1β-induced mitochondrial dysfunction in chondrocytes and whether cAMP and AMP-activated protein kinase (AMPK) affects NO production and mitochondrial function. Isolated human OA chondrocytes were stimulated with IL-1β in combination with/without forskolin, L-NIL, AMPK activator or inhibitor. The release of NO, IL-6, PGE2, MMP3, and the expression of iNOS were measured by ELISA or Western blot. Parameters of mitochondrial respiration were measured using a seahorse analyzer. IL-1β significantly induced NO release and mitochondrial dysfunction. Inhibition of iNOS by L-NIL prevented IL-1β-induced NO release and mitochondrial dysfunction but not IL-1β-induced release of IL-6, PGE2, and MMP3. Enhancement of cAMP by forskolin reduced IL-1β-induced NO release and prevented IL-1β-induced mitochondrial impairment. Activation of AMPK increased IL-1β-induced NO production and the negative impact of IL-1β on mitochondrial respiration, whereas inhibition of AMPK had the opposite effects. NO is critically involved in the IL-1β-induced impairment of mitochondrial respiration in human OA chondrocytes. Increased intracellular cAMP or inhibition of AMPK prevented both IL-1β-induced NO release and mitochondrial dysfunction.

Effect of Minoxidil on Trabecular Outflow via the Paracellular Pathway

PurposeTo investigate the pathway and effects of minoxidil on trabecular outflow in cultured human trabecular meshwork (TM) cells.MethodsAfter exposing primarily cultured TM cells to 0, 10, 50, or 100 µM minoxidil sulfate (MS), trabecular outflow was assessed by measuring TM cell monolayer permeability to carboxyfluorescein and transepithelial electrical resistance. To assess the pathway of permeability changes, caveolin-1, occludin, and claudin-5 levels were measured via western blot. Generation of reactive oxygen species (ROS) was measured using the dichlorofluorescein diacetate assay. To assess the involvement of nitric oxide (NO) in minoxidil-induced permeability increase, the degrees of endothelial nitric oxide synthase mRNA expression and NO production were measured with reverse transcription polymerase chain reaction and Griess assays, respectively. Permeability was also measured with co-exposure to 50 µM N-acetyl cysteine.ResultsMS significantly increased TM cell monolayer permeability (p < 0.05) and decreased transepithelial electrical resistance (p < 0.05). MS decreased the degree of endothelial nitric oxide synthase mRNA expression but did not affect NO production. MS decreased occludin and claudin-5 levels but did not affect caveolin-1 level. MS at 100 µM increased the generation of ROS, and MS-induced permeability increase was attenuated after co-exposure to 50 µM N-acetyl cysteine.ConclusionsMinoxidil may preferentially increase trabecular permeability via a paracellular pathway by downregulation of tight junction proteins. This minoxidil-induced permeability through the TM may be mediated by generation of ROS.

Chicken lines divergently selected on feather pecking differ in immune characteristics

It is crucial to identify whether relations between immune characteristics and damaging behaviors in production animals exist, as these behaviors reduce animal welfare and productivity. Feather pecking (FP) is a damaging behavior in chickens, which involves hens pecking and pulling at feathers of conspecifics. To further identify relationships between the immune system and FP we characterized high FP (HFP) and low FP (LFP) selection lines with regard to nitric oxide (NO) production by monocytes, specific antibody (SpAb) titers, natural (auto)antibody (N(A)Ab) titers and immune cell subsets. NO production by monocytes was measured as indicator for innate pro-inflammatory immune functioning, SpAb titers were measured as part of the adaptive immune system and N(A)Ab titers were measured as they play an essential role in both innate and adaptive immunity. Immune cell subsets were measured to identify whether differences in immune characteristics were reflected by differences in the relative abundance of immune cell subsets.Divergent selection on FP affected NO production by monocytes, SpAb and N(A)Ab titers, but did not affect immune cell subsets. The HFP line showed higher NO production by monocytes and higher IgG N(A)Ab titers compared to the LFP line. Furthermore the HFP line tended to have lower IgM NAAb titers, but higher IgM and IgG SpAb titers compared to the LFP line. Thus, divergent selection on FP affects the innate and adaptive immune system, where the HFP line seems to have a more responsive immune system compared to the LFP line. Although causation cannot be established in the present study, it is clear that relationships between the immune system and FP exist. Therefore, it is important to take these relationships into account when selecting on behavioral or immunological traits.

Aqueous and methanol extracts of Paullinia pinnata L. (Sapindaceae) improve inflammation, pain and histological features in CFA-induced mono-arthritis: Evidence from in vivo and in vitro studies.

Ethnopharmacological relevancePaullinia pinnata L. (Sapindaceae) is an African woody vine, traditionally used for the treatment of itch and pain-related conditions such as rheumatoid arthritis. AimThis work evaluates, in vitro and in vivo, the anti-inflammatory and analgesic effects of aqueous (AEPP) and methanol (MEPP) extracts from Paullinia pinnata leaves. MethodsAEPP and MEPP (100, 200 and 300 mg/kg/day) were administered orally in monoarthritic rats induced by a unilateral injection of 50 μl of Complete Freund's Adjuvant (CFA) in the ankle joint. During the 14 days of treatment, pain and inflammation were evaluated alternatively in both ankle and paw of the CFA-injected leg. Malondialdehyde (MDA) and glutathione (GSH) levels were assessed in serum and spinal cord. Histology of soft tissue of the ankle was also analyzed. For in vitro studies, AEPP and MEPP (10, 30 and 100 μg/ml) were evaluated against nitric oxide (NO) production by macrophages that were either non-stimulated or stimulated with LPS, 8-Br-AMPc and the mixture of both substances after 8 h exposure. These extracts were also evaluated on TNF-α and IL-1β production in cells stimulated with LPS for 8 h. Finally, the ability of the extracts to bind to neuroactive receptors was evaluated in vitro using competitive binding assays with >45 molecular targets. ResultsAEPP and MEPP significantly reduced by 20–98% (p < 0.001) the inflammation and pain sensation in both the ankle and paw. AEPP significantly increased glutathione levels (p < 0.05) in serum. Both extracts reduced MDA production in serum and spinal cord (p < 0.001), and significantly improved tissue reorganization in treated arthritic rats. P. pinnata extracts did not affect NO production in non-stimulated macrophages but significantly reduced it by 47–88% in stimulated macrophages. AEPP and MEPP also significantly inhibited TNF-α (35–68%) and IL-1β (31–36%) production in LPS stimulated macrophages. No cytotoxic effect of plant extracts was observed. MEPP showed concentration-dependent affinity for Sigma 2 receptors with an IC50 of 50 μg/ml. ConclusionThese results demonstrate the analgesic and anti-inflammatory effects of P. pinnata extracts on monoarthritis and further support its traditional use for pain and inflammation. These activities are at least partly due to the ability of these extracts to inhibit the production of NO, TNF-α, IL-1β and to likely modulate Sigma 2 receptors.

Involvement of exosomes in dopaminergic neurodegeneration by microglial activation in midbrain slice cultures

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of dopamine neurons in the substantia nigra. Microglial activation is frequently observed in the brains of patients with PD and animal models. Interferon-γ (IFN-γ)/lipopolysaccharide (LPS) treatment triggers microglial activation and the reduction of dopamine neurons in midbrain slice cultures. We have previously reported that nitric oxide (NO) is mainly involved in this dopaminergic degeneration. However, this degeneration was not completely suppressed by the inhibition of NO synthesis, suggesting that factors other than NO also contribute to dopaminergic neurodegeneration. Exosomes are extracellular vesicles with diameters of 40–200 nm that contain various proteins and micro RNAs and are regarded as a novel factor that mediates cell-to-cell interactions. Previous studies have demonstrated that exosome release is enhanced by microglial stimulation and that microglia-derived exosomes increases neuronal apoptosis. In the present study, we investigated whether exosomes are involved in dopaminergic neurodegeneration triggered by microglial activation in midbrain slice cultures. IFN-γ/LPS treatment to the midbrain slice cultures activated microglia, increased exosomal release, and decreased dopamine neurons. GW4869, an inhibitor of a neutral sphingomyelinase 2, decreased exosomal release and significantly prevented dopaminergic neurodegeneration by IFN-γ/LPS without affecting NO production. In contrast, D609, an inhibitor of sphingomyelin synthase and NO synthase, did not affect dopaminergic neurodegeneration, although it strongly inhibited NO production. The protective effect mediated by inhibition of NO synthase would be counteracted by enhanced exosomal release caused by D609 treatment. In addition, dopaminergic neurodegeneration is triggered by the treatment of exosomes isolated from culture media of IFN-γ/LPS-treated slices. These results suggest that exosomes are involved in dopaminergic neurodegeneration by microglial activation.

The root cause of Duchenne muscular dystrophy is the lack of dystrophin in smooth muscle of blood vessels rather than in skeletal muscle per se

Background:The dystrophin protein is part of the dystrophin associated protein complex (DAPC) linking the intracellular actin cytoskeleton to the extracellular matrix. Mutations in the dystrophin gene cause Duchenne and Becker muscular dystrophy (D/BMD). Neuronal nitric oxide synthase associates with dystrophin in the DAPC to generate the vasodilator nitric oxide (NO). Systemic dystrophin deficiency, such as in D/BMD, results in muscle ischemia, injury and fatigue during exercise as dystrophin is lacking, affecting NO production and hence vasodilation. The role of neuregulin 1 (NRG) signaling through the epidermal growth factor family of receptors ERBB2 and ERBB4 in skeletal muscle has been controversial, but it was shown to phosphorylate α-dystrobrevin 1 (α-DB1), a component of the DAPC. The aim of this investigation was to determine whether NRG signaling had a functional role in muscular dystrophy.Methods:Primary myoblasts (muscle cells) were isolated from conditional knock-out mice containing lox P flanked ERBB2 and ERBB4 receptors, immortalized and exposed to Cre recombinase to obtainErbb2/4double knock-out (dKO) myoblasts where NRG signaling would be eliminated. Myotubes, thein vitroequivalent of muscle fibers, formed by fusion of the lox P flankedErbb2/4myoblasts as well as theErbb2/4dKO myoblasts were then used to identify changes in dystrophin expression.Results:Elimination of NRG signaling resulted in the absence of dystrophin demonstrating that it is essential for dystrophin expression. However, unlike the DMD mouse model mdx, with systemic dystrophin deficiency, lack of dystrophin in skeletal muscles ofErbb2/4dKO mice did not result in muscular dystrophy. In these mice, ERBB2/4, and thus dystrophin, is still expressed in the smooth muscle of blood vessels allowing normal blood flow through vasodilation during exercise.Conclusions:Dystrophin deficiency in smooth muscle of blood vessels, rather than in skeletal muscle, is the main cause of disease progression in DMD.

The root cause of Duchenne muscular dystrophy is the lack of dystrophin in smooth muscle of blood vessels rather than in skeletal muscle per se

Background:The dystrophin protein is part of the dystrophin associated protein complex (DAPC) linking the intracellular actin cytoskeleton to the extracellular matrix. Mutations in the dystrophin gene cause Duchenne and Becker muscular dystrophy (D/BMD). Neuronal nitric oxide synthase associates with dystrophin in the DAPC to generate the vasodilator nitric oxide (NO). Systemic dystrophin deficiency, such as in D/BMD, results in muscle ischemia, injury and fatigue during exercise as dystrophin is lacking, affecting NO production and hence vasodilation. The role of neuregulin 1 (NRG) signaling through the epidermal growth factor family of receptors ERBB2 and ERBB4 in skeletal muscle has been controversial, but it was shown to phosphorylate α-dystrobrevin 1 (α-DB1), a component of the DAPC. The aim of this investigation was to determine whether NRG signaling had a functional role in muscular dystrophy.Methods:Primary myoblasts (muscle cells) were isolated from conditional knock-out mice containing lox P flanked ERBB2 and ERBB4 receptors, immortalized and exposed to CRE recombinase to obtainErbb2/4double knock-out (dKO) myoblasts where NRG signaling would be eliminated. Myotubes, thein vitroequivalent of muscle fibers, formed by fusion of the lox P flankedErbb2/4myoblasts as well as theErbb2/4dKO myoblasts were then used to identify changes in dystrophin expression.Results:Elimination of NRG signaling resulted in the absence of dystrophin demonstrating that it is essential for dystrophin expression. However, unlike the DMD mouse model mdx, with systemic dystrophin deficiency, lack of dystrophin in skeletal muscles ofErbb2/4dKO mice did not result in muscular dystrophy. In these mice, ERBB2/4, and thus dystrophin, is expressed in the smooth muscle of blood vessels allowing normal blood flow through vasodilation during exercise.Conclusions:Dystrophin deficiency in smooth muscle of blood vessels, rather than in skeletal muscle, is the main cause of disease progression in DMD.

Vasoprotective Effects of Vildagliptin in Mice Is Masked by Metformin—Overlapping Mechanisms Implied

Preclinical and small clinical studies have demonstrated the direct cardiovascular protective effects of dipeptidyl peptidase 4 inhibitors (DPP-4Is). However, cardiovascular benefits have not been proven in large clinical trials that utilized multiple antidiabetic agents. We hypothesized that the cardiovascular protective effects of the first-line antidiabetic agent metformin (Met) affects those of DPP-4Is, so we evaluated the vasoprotective effects of vildagliptin (Vil) in combination with Met in mice. Methods: We randomly assigned C57BL6 and diabetic db/db mice (8 weeks old) to treatment with vehicle, Vil (3 mg/kg/d), Met (400 mg/kg/d), or the combination of Vil and Met. The mice were subjected to femoral artery wire injury to induce arterial remodeling. Four weeks after the injury, the femoral arteries were collected. Human umbilical vein endothelial cells (HUVECs) were used in the in vitro experiments. Results: In wild type mice, the biological parameters were similar among the groups. Both Vil and Met treatment reduced neointimal area and vascular cell proliferation to a similar extent (40% reduction) compared to that of the vehicle, and combining the two drugs did not further reduce those values. Similarly, both Vil and Met treatment reduced neointimal area in db/db mice (50% reduction) without affecting biological parameters, but no additive effect was found. In wild type mice, cotreatment with the nitric oxide (NO) synthase inhibitor completely abolished the vascular effects of both Vil and Met, suggesting that Vil and Met had common mechanisms that are dependent on endothelial NO. In HUVECs, Vil did not directly affect NO production. In contrast, glucagon like peptide (GLP)-1 did stimulate NO production. Met also stimulated NO production. However, the combination of GLP-1 and Met did not increase NO production. Conclusions: Metformin masks the vasoprotective effects of vildagliptin, possibly because of overlapping mechanisms that are dependent on endothelial NO. Disclosure H. Kushima: None. Y. Mori: None. M. Koshibu: None. M. Hiromura: None. K. Kohashi: None. M. Terasaki: None. T. Hirano: Speaker's Bureau; Self; Novo Nordisk Inc., AstraZeneca.

OP.8A.06] PULSATILE STRETCH ALTERS AMYLOID PRECURSOR PROTEIN EXPRESSION AND NITRIC OXIDE SIGNALLING IN HUMAN CEREBRAL ENDOTHELIAL CELLS PRE-EXPOSED TO GLYCOSPHINGOLIPID INHIBITION

Objective: Amyloid β (Aβ) deposits that arise from amyloid precursor protein (APP) are a hallmark of Alzheimer's disease (AD). Elevated pulsatility of arterial pressure and deregulation of ceramide and nitric oxide (NO) metabolism are associated with AD. Glycosphingolipid inhibitors that inhibit the production of the enzyme glucosylceramide synthase by blocking ceramide glycosylation are known to increase gamma secretase-mediated-Aβ release. This study investigates whether pulsatile stretch of human cerebral microvascular endothelial cells (hCMEC) pre-treated with the glycosphingolipid inhibitor, D-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), alters expression and/or phosphorylation of APP and endothelial NO synthase (eNOS). Design and method: hCMECs were pre-treated with 20 μM D-PDMP for 2 hours prior to 15% cyclic stretch for 18 hours at 1 Hz (n = 7–8). Protein expression and phosphorylation (phospho-eNOS, Ser1177) were quantified using western blots. Results were analyzed using one way ANOVA (mean ± SEM, % control). Results: APP expression increased significantly in response to stretch in ECs pre-treated with D-PDMP (170.2 ± 27%, p < 0.01) compared to cells stretched without pre-treatment (114.5 ± 23%). eNOS phosphorylation was down-regulated in ECs subjected to stretch (9.9 ± 4%) compared to vehicle control (100.0 ± 0%, p < 0.0001) and D-PDMP-treated-static control (107.0 ± 17%, p < 0.01). In ECs pre-treated with D-PDMP prior to stretch, phospho-eNOS levels decreased significantly (40.3 ± 15%) compared to vehicle control (100.0 ± 0%, p < 0.05) and PDMP-treated-static control (107.0 ± 17%, p < 0.01). Conclusions: APP expression increased in cerebral ECs pre-treated with D-PDMP after being exposed to cyclic stretch. eNOS phosphorylation was down-regulated by cyclic stretch. D-PDMP pre-treatment appeared to have a restoring effect on phospho-eNOS, although it did not statistically significance. Glycosphingolipid inhibition with D-PDMP and cyclic stretch may lead to ceramide accumulation thereby increasing the Aβ load. This can affect NO production and modulation of Aβ deposits.

Abstract 059: miR-29 is Required for Normal Endothelium-dependent Vasodilation and Protects Against Hypertension

MicroRNA miR-29 is down-regulated in Dahl salt-sensitive (SS) rats compared to salt-insensitive SS-13 BN rats. We investigated the role of miR-29 in endothelial function and the development of hypertension. Using TALENs (Transcriptional Activator-Like Effector Nucleases) we deleted 4 nucleotides from Mir29b-1 gene in SS-13 BN rats. The targeted mutation resulted in reduced abundance of miR-29b-3p and the co-transcribed miR-29a-3p in arteriolar endothelial cells. When stimulated with acetylcholine to induce endothelium-dependent vasodilation (EDVD), gluteal arterioles from Mir29b-1 -/- rats were only able to dilate to an average 47% of maximum diameter while wild-type littermates dilated to 77% (N=7 and 9, respectively, p&lt;0.05). The development of hypertension was significantly exacerbated in Mir29b-1 -/- compared to Mir29b-1 +/+ littermates. Mean arterial blood pressure was 129 ± 2 mmHg in Mir29b-1 +/+ rats after 2 weeks of 4% NaCl diet and 140 ± 5 mmHg in Mir29b-1 -/- rats (N=6, 10, respectively, p&lt;0.05). Gluteal arterioles from Mir29b-1 -/- rats exhibited significantly reduced nitric oxide (NO) levels compared to Mir29b-1 +/+ littermates as measured by DAF2-DA intensity (N=6, 11, respectively). Mutation of the Mir29b-1 gene resulted in preferential differential expression of genes in arterioles related to the regulation of NO levels including Lypla1. Lypla1 is a direct target of miR-29 and could abrogate the effect of miR-29 in promoting NO production. Reduction of LYPLA1 by transfection of pre-miR-29b or a Lypla1 siRNA resulted in a significant increase in NO as measured by DAF2-DA in cultured human dermal microvascular endothelial cells. Taken together, we have shown that targeted mutation of Mir29b-1 reduces EDVD and exacerbates the development of hypertension. We have identified a mechanism by which miR-29 affects NO production. These findings indicate miR-29 is required for normal endothelial function in rats and has therapeutic potential for hypertension.

Immunostimulatory Activity of Opuntia ficus-indica var. Saboten Cladodes Fermented by Lactobacillus plantarum and Bacillus subtilis in RAW 264.7 Macrophages.

To increase the functionality of Opuntia ficus-indica var. saboten cladodes, it was fermented by Lactobacillus plantarum and Bacillus subtilis. Eighty percent methanol extracts were investigated for their effects on nitric oxide (NO) production, cytokine secretion, nuclear factor-κB (NF-κB) activity, and mitogen-activated protein kinase (MAPK) phosphorylation in RAW 264.7 cells. Methanol extracts of L. plantarum culture medium (LPCME) and B. subtilis culture medium (BSCME) did not affect lipopolysaccharide (LPS)-induced NO production but, at 500 μg/mL, increased interferon (IFN)-γ-induced NO production by 55.2 and 66.5 μM, respectively, in RAW 264.7 cells. In RAW 264.7 cells not treated with LPS and IFN-γ, LPCME did not affect NO production, but BSCME increased NO production significantly in a dose-dependent manner. In addition, BSCME induced the expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW 264.7 cells in a dose-dependent manner. BSCME at 500 μg/mL increased TNF-α and IL-1β mRNA levels by 83.8% and 82.2%, respectively. BSCME increased NF-κB-dependent luciferase activity in a dose-dependent manner; 500 μg/mL BSCME increased activity 9.1-fold compared with the control. BSCME induced the phosphorylation of p38, c-JUN NH2-terminal protein kinase (JNK), and extracellular signal-regulated kinase (ERK) in a dose-dependent manner, but did not affect total ERK levels. In conclusion, BSCME exerted immunostimulatory effects, which were mediated by MAPK phosphorylation and NF-κB activation, resulting in increased TNF-α and IL-1β gene expression in RAW 264.7 macrophages. Therefore, BSCM shows promise for use as an immunostimulatory therapeutic.

Modeling Disease Progression: Angiotensin II Indirectly Inhibits Nitric Oxide Production via ADMA Accumulation in Spontaneously Hypertensive Rats.

Nitric oxide (NO) production impairment is involved in the onset and development of hypertension. Although NO production impairment in spontaneously hypertensive rat (SHR) has been reported in a variety of researches, the time course of this progressive procedure, as well as its relationship with asymmetric dimethylarginine (ADMA) and angiotensin II (Ang II), has not been quantified. The aim of this research is to establish a mechanism-based disease progression model to assess Ang II and ADMA's inhibition of NO production in SHR's disease progression with/without ramipril's intervention. SHR were randomly divided into three groups: one disease group (n = 8) and two treatment groups (n = 8 for each group): standard treatment group (receiving ramipril 2 mg/kg*day) and intensive treatment group (receiving ramipril 10 mg/kg*day). ADMA, Ang II, NO, and SBP were determined weekly. Intensive treatment with ramipril was found to have no further attenuation of plasma NO and ADMA than standard treatment beyond its significantly stronger antihypertensive effects. Four linked turnover models were developed to characterize the profiles of ADMA, Ang II, NO, and SBP during hypertensive disease progression with/without ramipril intervention. Our model described Ang II and ADMA's contribution to NO production impairment and their responses to ramipril treatment throughout the disease progression in SHR. Model simulations suggested that Ang II affected NO production mainly through inhibiting ADMA elimination rather than affecting nitric oxide synthase (NOS) directly.