eNOS Coupling Research Articles

Exploring the effects of vasoactive constituents in large cardamom: implications for the anti-hypertensive effect via eNOS coupling pathway – an in-vitro study in rat endothelial cells

Endothelial dysfunction, linked to reduced eNOS expression and nitric oxide (NO) availability, contributes to cardiovascular diseases (CVDs). Large cardamom exhibits antihypertensive effects by augmenting NO levels and antioxidant activity. To decipher its mechanisms, selected constituents were docked with eNOS-associated target genes such as GTP cyclohydrolase I (GTPCH-1) and (dihydrofolate reductase [DHFR]). Endothelial damage induced by L-NAME and fructose was countered by assessing nitric oxide metabolites (NOx), tetrahydrobipterin (BH4 levels), GCH-I expression and super oxide dismutase (SOD) activity after constituent incubation. Cyanidin-3-O-glucoside and petunidin-3-O-glucoside notably restored impaired vascular markers in both models. These phytoconstituents are likely to activate GCH-BH4-eNOS pathways, upregulating SOD and NO expression, maintaining endothelial integrity. Large cardamom’s antihypertensive effects may stem from these components, synergistically enhancing endothelial NO release via the eNOS pathway.

SUMOylation and coupling of eNOS mediated by PIAS1 contribute to maintenance of vascular homeostasis.

Endothelial dysfunction (ED) is commonly considered a crucial initiating step in the pathogenesis of numerous cardiovascular diseases. The coupling of endothelial nitric oxide synthase (eNOS) is important in maintaining normal endothelial functions. However, it still remains elusive whether and how eNOS SUMOylation affects the eNOS coupling. In the study, we investigate the roles and possible action mechanisms of protein inhibitor of activated STAT 1 (PIAS1) in ED. Human umbilical vein endothelial cells (HUVECs) treated with palmitate acid (PA) invitro and ApoE-/- mice fed with high-fat diet (HFD) invivo were constructed as the ED models. Our invivo data show that PIAS1 alleviates the dysfunction of vascular endothelium by increasing nitric oxide (NO) level, reducing malondialdehyde (MDA) level, and activating the phosphatidylinositol 3-kinase-protein kinase B-endothelial nitric oxide synthase (PI3K-AKT-eNOS) signaling in ApoE-/- mice. Our invitro data also show that PIAS1 can SUMOylate eNOS under endogenous conditions; moreover, it antagonizes the eNOS uncoupling induced by PA. The findings demonstrate that PIAS1 alleviates the dysfunction of vascular endothelium by promoting the SUMOylation and inhibiting the uncoupling of eNOS, suggesting that PIAS1 would become an early predictor of atherosclerosis and a new potential target of the hyperlipidemia-related cardiovascular diseases.

Induction of KLF2 by Exercise Activates eNOS to Improve Vasodilatation in Diabetic Mice.

Exercise and statins restore the endothelial expression of Krüppel-like factor 2 (KLF2), which is diminished in diabetic db/db mice. Endothelium-specific overexpression of KLF2 improves endothelium-dependent relaxation and flow-mediated dilation through increasing nitric oxide bioavailability. KLF2 promotes endothelial nitric oxide synthase (eNOS) coupling and phosphorylation in addition to its known role in eNOS transcription. KLF2 upregulates the expression of several panels of genes that regulate eNOS activity.

Nitric oxide synthases in GtoPdb v.2023.1

Nitric oxide synthases (NOS, E.C. 1.14.13.39) are a family of oxidoreductases that synthesize nitric oxide (NO.) via the NADPH and oxygen-dependent consumption of L-arginine with the resultant by-product, L-citrulline. There are 3 NOS isoforms and they are related by their capacity to produce NO, highly conserved organization of functional domains and significant homology at the amino acid level. NOS isoforms are functionally distinguished by the cell type where they are expressed, intracellular targeting and transcriptional and post-translation mechanisms regulating enzyme activity. The nomenclature suggested by NC-IUPHAR of NOS I, II and III [12] has not gained wide acceptance, and the 3 isoforms are more commonly referred to as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) which reflect the location of expression (nNOS and eNOS) and inducible expression (iNOS). All are dimeric enzymes that shuttle electrons from NADPH, which binds to a C-terminal reductase domain, through the flavins FAD and FMN to the oxygenase domain of the other monomer to enable the BH4-dependent reduction of heme bound oxygen for insertion into the substrate, L-arginine. Electron flow from reductase to oxygenase domain is controlled by calmodulin binding to canonical calmodulin binding motif located between these domains. eNOS and nNOS isoforms are activated at concentrations of calcium greater than 100 nM, while iNOS shows higher affinity for Ca2+/calmodulin with great avidity and is essentially calcium-independent and constitutively active. Efficient stimulus-dependent coupling of nNOS and eNOS is achieved via subcellular targeting through respective N-terminal PDZ and fatty acid acylation domains whereas iNOS is largely cytosolic and function is independent of intracellular location. nNOS is primarily expressed in the brain and neuronal tissue, iNOS in immune cells such as macrophages and eNOS in the endothelial layer of the vasculature although exceptions in other cells have been documented. L-NAME and related modified arginine analogues are inhibitors of all three isoforms, with IC50 values in the micromolar range.

Eicosapentaenoic acid (EPA) reduces pulmonary endothelial dysfunction and inflammation due to changes in protein expression during exposure to particulate matter air pollution

AimsInhalation of air pollution small particle matter (PM) is a leading cause of cardiovascular (CV) disease. Exposure to PMs causes endothelial cell (EC) dysfunction as evidenced by nitric oxide (NO) synthase uncoupling, vasoconstriction and inflammation. Eicosapentaenoic acid (EPA) has been shown to mitigate PM-induced adverse cardiac changes in patients receiving omega-3 fatty acid supplementation. We set out to determine the pro-inflammatory effects of multiple PMs (urban and fine) on pulmonary EC NO bioavailability and protein expression, and whether EPA restores EC function under these conditions. Methods and resultsWe pretreated pulmonary ECs with EPA and then exposed them to urban or fine air pollution PMs. LC/MS-based proteomic analysis to assess relative expression levels. Expression of adhesion molecules was measured by immunochemistry. The ratio of NO to peroxynitrite (ONOO–) release, an indication of eNOS coupling, was measured using porphyrinic nanosensors following calcium stimulation. Urban/fine PMs also modulated 9/12 and 13/36 proteins, respectively, linked to platelet and neutrophil degranulation pathways and caused > 50% (p < 0.001) decrease in the stimulated NO/ONOO– release ratio. EPA treatment altered expression of proteins involved in these inflammatory pathways, including a decrease in peroxiredoxin-5 and an increase in superoxide dismutase-1. EPA also increased expression of heme oxygenase-1 (HMOX1), a cytoprotective protein, by 2.1-fold (p = 0.024). EPA reduced elevations in sICAM-1 levels by 22% (p < 0.01) and improved the NO/ONOO– release ratio by > 35% (p < 0.05). ConclusionThese cellular changes may contribute to anti-inflammatory, cytoprotective and lipid changes associated with EPA treatment during air pollution exposure.

Impact of l-citrulline on nitric oxide signaling and arginase activity in hypoxic human pulmonary artery endothelial cells.

Impaired nitric oxide (NO) signaling contributes to the development of pulmonary hypertension (PH). The l-arginine precursor, l-citrulline, improves NO signaling and has therapeutic potential in PH. However, there is evidence that l-citrulline might increase arginase activity, which in turn, has been shown to contribute to PH. Our major purpose was to determine if l-citrulline increases arginase activity in hypoxic human pulmonary artery endothelial cells (PAECs). In addition, to avoid potential adverse effects from high dose l-citrulline monotherapy, we evaluated whether the effect on NO signaling is greater using co-treatment with l-citrulline and another agent that improves NO signaling, folic acid, than either alone. Arginase activity was measured in human PAECs cultured under hypoxic conditions in the presence of l-citrulline (0-1 mM). NO production and endothelial nitric oxide synthase (eNOS) coupling, as assessed by eNOS dimer-to-monomer ratios, were measured in PAECs treated with l-citrulline and/or folic acid (0.2 μM). Arginase activity increased in hypoxic PAECs treated with 1 mM but not with either 0.05 or 0.1 mM l-citrulline. Co-treatment with folic acid and 0.1 mM l-citrulline increased NO production and eNOS dimer-to-monomer ratios more than treatment with either alone. The potential to increase arginase activity suggests that there might be plasma l-citrulline concentrations that should not be exceeded when using l-citrulline to treat PH. Rather than progressively increasing the dose of l-citrulline as a monotherapy, co-therapy with l-citrulline and folic acid merits consideration, due to the possibility of achieving efficacy at lower doses and minimizing side effects.

Abstract 13683: Eicosapentaenoic Acid (EPA) Decreases Endothelin Converting Enzyme-1 Expression and Improves Nitric Oxide Release in Pulmonary Endothelium Following Challenge With Air Pollutants

Background: Air pollution particulate matter (PM) contributes to global mortality and chronic conditions, especially cardiovascular (CV) disease. Exposure to PM and cytokines like IL-6 cause endothelial cell (EC) dysfunction, leading to nitric oxide synthase (eNOS) uncoupling. The omega-3 fatty acid EPA improves eNOS coupling and reduces vascular inflammation. REDUCE-IT showed that treatment with icosapent ethyl (IPE), a purified EPA ethyl ester, reduced composite CV events. We compared the effects of EPA on pulmonary EC (PEC) function and protein expression following challenge with PMs versus IL-6. Methods: PECs were treated with EPA (40 μM) or equivolume vehicle for 2 h and then challenged with IL-6 (12 ng/mL) or PM (50 μg/mL) collected from urban environments (“urban PM”) for up to 8 h. Proteomic analysis was performed using LC/MS to measure relative protein expression. Significant ( p &lt;0.05) changes between treatment groups (&gt;1-fold) were analyzed by bioinformatic packages. Biological pathways were analyzed by gene set enrichment analysis (GSEA). In parallel, cells were stimulated with calcium ionophore to elicit nitric oxide (NO) and peroxynitrite (ONOO – ) release as assayed by tandem porphyrinic nanosensors; the NO/ONOO – ratio indicates eNOS coupling efficiency. Results: Challenge with IL-6 and urban PM caused eNOS uncoupling as evidenced by a decreased NO/ONOO – ratio of 35% (1.43 ± 0.04 vs. 2.19 ± 0.02, p &lt;0.001) and 50% (1.22 ± 0.07 vs. 2.42 ± 0.15 , p &lt;0.001), respectively, compared to control. EPA increased the NO/ONOO – ratio relative to both IL-6 (39%; 1.99 ± 0.11, p &lt;0.001) and urban PM (39%; 1.69 ± 0.14 , p &lt;0.05). Proteomics revealed that EPA decreased expression of endothelin converting enzyme-1 (ECE1) relative to both IL-6 (1.2-fold, p = 0.017) and urban PM (1.1-fold, p = 0.045). GSEA showed significant modulations to 18 and 35 proteins within the neutrophil degranulation pathway (GO:0043312) with EPA treatment relative to IL-6 and urban PM ( p &lt;0.0001 in both comparisons). Conclusion: EPA favorably reversed EC dysfunction and expression of inflammatory proteins and pathways following challenge with air pollution PMs similar to IL-6. These broad anti-inflammatory actions may contribute to reduced CV risk with EPA as observed in outcome trials.

Eicosapentaenoic acid (EPA) increased endothelial nitric oxide synthase (eNOS) levels and proteins associated with cellular responses to oxidative stress during inflammation

Background and Aims : Eicosapentaenoic acid (EPA) administered as icosapent ethyl (IPE) reduced cardiovascular (CV) ischemic events (REDUCE-IT) in contrast to trials using docosahexaenoic acid (DHA)-containing formulations in statin-treated patients. EPA improves endothelial cell (EC) function and nitric oxide (NO) bioavailability, features that may reduce atherothrombotic events. We measured the effects of EPA versus DHA on eNOS expression and coupling efficiency.

Electronic cigarette exposure causes vascular endothelial dysfunction due to NADPH oxidase activation and eNOS uncoupling.

We recently reported a mouse model of chronic electronic cigarette (e-cig) exposure-induced cardiovascular pathology, where long-term exposure to e-cig vape (ECV) induces cardiac abnormalities, impairment of endothelial function, and systemic hypertension. Here, we delineate the underlying mechanisms of ECV-induced vascular endothelial dysfunction (VED), a central trigger of cardiovascular disease. C57/BL6 male mice were exposed to ECV generated from e-cig liquid containing 0, 6, or 24 mg/mL nicotine for 16 and 60 wk. Time-dependent elevation in blood pressure and systemic vascular resistance were observed, along with an impairment of acetylcholine-induced aortic relaxation in ECV-exposed mice, compared with air-exposed control. Decreased intravascular nitric oxide (NO) levels and increased superoxide generation with elevated 3-nitrotyrosine levels in the aorta of ECV-exposed mice were observed, indicating that ECV-induced superoxide reacts with NO to generate cytotoxic peroxynitrite. Exposure increased NADPH oxidase expression, supporting its role in ECV-induced superoxide generation. Downregulation of endothelial nitric oxide synthase (eNOS) expression and Akt-dependent eNOS phosphorylation occurred in the aorta of ECV-exposed mice, indicating that exposure inhibited de novo NO synthesis. Following ECV exposure, the critical NOS cofactor tetrahydrobiopterin was decreased, with a concomitant loss of its salvage enzyme, dihydrofolate reductase. NADPH oxidase and NOS inhibitors abrogated ECV-induced superoxide generation in the aorta of ECV-exposed mice. Together, our data demonstrate that ECV exposure activates NADPH oxidase and uncouples eNOS, causing a vicious cycle of superoxide generation and vascular oxidant stress that triggers VED and hypertension with predisposition to other cardiovascular disease.NEW & NOTEWORTHY Underlying mechanisms of e-cig-induced vascular endothelial dysfunction are delineated. e-cig exposure activates and increases expression of NADPH oxidase and disrupts activation and coupling of eNOS, leading to a vicious cycle of superoxide generation and peroxynitrite formation, with tetrahydrobiopterin depletion, causing loss of NO that triggers vascular endothelial dysfunction. This process is progressive, increasing with the duration of e-cig exposure, and is more severe in the presence of nicotine, but observed even with nicotine-free vaping.

Abstract P509: Novel Protein Kinase C Epsilon Inhibitor Attenuates Uncoupled Endothelial Nitric Oxide Synthase And Vascular Endothelial Dysfunction

Endothelial nitric oxide synthase (eNOS) uncoupling reduces nitric oxide (NO) production when tetrahydrobiopterin (BH 4 ) is oxidized to dihydrobiopterin (BH 2 ), resulting in endothelial dysfunction and leukocyte adherence. Renovascular damage following shockwave lithotripsy (SWL) may occur via the potentiation of this mechanism. We aim to modulate uncoupled eNOS activity with myristoylated protein kinase C epsilon inhibitor ( myr -PKCε–; N- myr -EAVSLKPT) and activator ( myr -PKCε+; N- myr -HDAPIGYD). We hypothesize that myr -PKCε– with uncoupled eNOS (i.e., bound to BH 2 ) should reduce leukocyte-endothelial interactions in postcapillary venules and H 2 O 2 levels after SWL. Myr -PKCε+ should promote similar effects when eNOS is coupled (i.e., bound to BH 4 ). Mesenteric venules of anesthetized male SD rats (300g) were superfused for 120 min with 100 μM BH 2 and test solutions (Krebs’ buffer, 10 μM myr -PKCε+, or 10 μM myr -PKCε–) with or without 100 μM BH 4 . Leukocyte activity was evaluated via intravital microscopy. In separate SD rats, SWL was performed on left kidneys (16 kV, 500 shocks at 60 shocks/min and at 120 shocks/min). Then, normal saline or test solution cocktail ( myr -PKCε+ [0.9 mg/kg]/ myr -PKCε- [0.8 mg/kg] with BH 2 [2mg/kg] or BH 4 [0.8 mg/kg]) was given i.v. NO or H 2 O 2 levels were measured with 100 μm microsensors in left renal veins at baseline, end of SWL, and 5-min intervals for 30 min post-SWL. Data were analyzed using ANOVA Student-Newman-Keuls test. BH 2 -induced leukocyte rolling, adherence, and transmigration were significantly increased by myr -PKCε+ ( n = 6) and attenuated with myr- PKCε– ( n = 5) compared to control (n=5) at 120 min (p&lt;0.01). Following SWL, all treatments except myr -PKCε+ with BH 2 significantly reduced H 2 O 2 ( n = 5, p &lt; .01) and increased NO levels ( n = 5, p &lt; .01) compared to control. NO and H 2 O 2 levels following administration of myr -PKCε+ with BH 2 were similar to SWL control. Results suggest that myr -PKCε– attenuates uncoupled eNOS activity and H 2 O 2 release in rat renal veins, after SWL. Promoting coupled eNOS activity with BH 4 yields similar results. Promoting eNOS coupling with BH 4 or inhibiting uncoupled eNOS with myr- PKCε– attenuates oxidative stress and endothelial dysfunction following SWL and BH 2 -induced inflammation.

Inhibitory Effect of Ethanol Extract from Folium et Ramulus Securinegae on TNF‐α induced vascular inflammation in Human Umbilical Vein Endothelial Cells

Folium et Ramulus Securinegae (FRS) is known as a drug that protects and strengthen kidney and spleen by improving the blood circulation and relaxing muscles and tendons. This study investigated whether FRS has protective effect of inhibiting the vascular inflammation in human umbilical vein endothelial cells (HUVECs) by inducing nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) coupling pathway. Results showed that FRS suppressed TNF-α (Tumor necrosis factor–α) induced protein and mRNA levels of cell adhesion molecules such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and Interleukin-6 (IL-6). Pretreatment of HUVEC with FRS decreased the adhesion of HL-60 cells to Ox-LDL (Oxidized Low-Density-Lipoprotein)-induced HUVEC. Moreover, FRS inhibited TNF-α induced intracellular reactive oxygen species (ROS) production. FRS also inhibited phosphorylation of IκB-α in cytoplasm and translocation of NF-κB (Nuclear factor-kappa B) p65 to the nucleus. FRS increased NO production, as well increased the phosphorylation of eNOS and Akt (protein kinase B) which are related with NO production. In addition, FRS increased the protein expression of GTPCH (Guanosine triphosphate cyclohydrolase Ⅰ) and the production of BH4 in HUVEC which are related with eNOS coupling pathway. Overall, the present data suggest that FRS has a protective effect against vascular inflammation and can be a potential therapeutic agent for early atherosclerosis.

Effects of Graft Preservation Conditions on Coronary Endothelium and Functional Recovery in a Rat Model of Donation after Circulatory Death

Purpose Donation after circulatory death (DCD) could significantly improve cardiac graft availability. However, DCD hearts are subjected to potentially damaging conditions prior to circulatory arrest and during warm ischemia and reperfusion. Although the feasibility of DCD heart transplantation has been demonstrated, protocols for graft preservation and storage require optimization to ensure graft quality. Therefore, we aimed to compare the current clinical conditions used for heart preservation (St. Thomas n°2) with a potentially more suitable solution for DCD hearts, evaluating endothelial and cardiac function to investigate differences in cardioprotective effects. Methods Following anaesthesia, diaphragm transection, and circulatory arrest in male Wistar rats to simulate DCD conditions, hearts were subjected to either 0 or 18 minutes of warm, in-situ ischemia. At end-ischemia, hearts were perfused with either: St. Thomas n°2 cardioplegia at 4°C as in current clinical practice, or normokalemic, adenosine-lidocaine cardioplegia (A-L) at 4°C or 22°C. Afterwards, hearts were stored for 15 min in their respective solutions, and then reperfused ex-situ for either 30 min (to assess endothelial function) or 60 min (to assess cardiac function and biochemical parameters). Results A-L cardioplegia at 22°C better preserves vascular function as compared to cold A-L or St. T cardioplegia, as demonstrated by greater eNOS coupling (p Conclusion Use of A-L and avoidance of profound hypothermia and hyperkalemic cardioplegia could improve endothelial and contractile function, potentially through the promotion of eNOS dimerization and activation of the RISK pathway; these approaches should help to optimize clinical protocols in DCD heart transplantation and facilitate its widespread adoption in clinical practice.

How does ascorbate improve endothelial dysfunction? - A computational analysis

Low levels of ascorbate (Asc) are observed in cardiovascular and neurovascular diseases. Asc has therapeutic potential for the treatment of endothelial dysfunction, which is characterized by a reduction in nitric oxide (NO) bioavailability and increased oxidative stress in the vasculature. However, the potential mechanisms remain poorly understood for the Asc mitigation of endothelial dysfunction. In this study, we developed an endothelial cell based computational model integrating endothelial cell nitric oxide synthase (eNOS) biochemical pathway with downstream reactions and interactions of oxidative stress, tetrahydrobiopterin (BH4) synthesis and biopterin ratio ([BH4]/[TBP]), Asc and glutathione (GSH). We quantitatively analyzed three Asc mediated mechanisms that are reported to improve/maintain endothelial cell function. The mechanisms include the reduction of •BH3 to BH4, direct scavenging of superoxide (O2•−) and peroxynitrite (ONOO−) and increasing eNOS activity. The model predicted that Asc at 0.1–100 μM concentrations improved endothelial cell NO production, total biopterin and biopterin ratio in a dose dependent manner and the extent of cellular oxidative stress. Asc increased BH4 availability and restored eNOS coupling under oxidative stress conditions. Asc at concentrations of 1–10 mM reduced O2•− and ONOO− levels and could act as an antioxidant. We predicted that glutathione peroxidase and peroxiredoxin in combination with GSH and Asc can restore eNOS coupling and NO production under oxidative stress conditions. Asc supplementation may be used as an effective therapeutic strategy when BH4 levels are depleted. This study provides detailed understanding of the mechanism responsible and the optimal cellular Asc levels for improvement in endothelial dysfunction.

Anti-Vascular Inflammatory Effect of Ethanol Extract from Securinega suffruticosa in Human Umbilical Vein Endothelial Cells.

Securiniga suffruticosa is known as a drug that has the effect of improving the blood circulation and relaxing muscles and tendons, thereby protects and strengthen kidney and spleen. Therefore, in this study, treatment of Securiniga suffruticosa showed protective effect of inhibiting the vascular inflammation in human umbilical vein endothelial cells (HUVECs) by inducing nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) coupling pathway. In this study, Securiniga suffruticosa suppressed TNF-α (Tumor necrosis factor–α) induced protein and mRNA levels of cell adhesion molecules such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and Interleukin-6 (IL-6). Pretreatment of HUVEC with Securiniga suffruticosa decreased the adhesion of HL-60 cells to Ox-LDL (Oxidized Low-Density-Lipoprotein)-induced HUVEC. Moreover, Securiniga suffruticosa inhibited TNF-α induced intracellular reactive oxygen species (ROS) production. Securiniga suffruticosa also inhibited phosphorylation of IκB-α in cytoplasm and translocation of NF-κB (Nuclear factor-kappa B) p65 to the nucleus. Securiniga suffruticosa increased NO production, as well increased the phosphorylation of eNOS and Akt (protein kinase B) which are related with NO production. In addition, Securiniga suffruticosa increased the protein expression of GTPCH (Guanosine triphosphate cyclohydrolase Ⅰ) and the production of BH4 in HUVEC which are related with eNOS coupling pathway. In conclusion, Securiniga suffruticosa has a protective effect against vascular inflammation and can be a potential therapeutic agent for early atherosclerosis.

Acute ischemic stroke induces magnetic resonance susceptibility signs dominated by endothelial nitric oxide synthase activation.

Acute ischemic stroke induces deoxyhemoglobin accumulation around the ischemic region while activating endothelial nitric oxide synthase (eNOS) coupling and the subsequent release of nitric oxide (NO). Because deoxyhemoglobin is a natural NO spin trap, its interplay with NO could be prominent during acute stroke. Its interaction with NO has been shown to induce overt paramagnetic signals in vitro; our goal was to investigate whether this interplay can be detected using MRI. To verify the in vivo image effects using the deoxyhemoglobin-NO interaction during acute stroke, eNOS states were manipulated in an animal model of acute ischemia, and the susceptibility signals, cerebral perfusion, and infarction were assessed noninvasively via MR susceptibility weighted imaging (SWI). Occlusion of the right middle cerebral artery increased eNOS coupling and susceptibility signals in the ischemic cortex while abolishing regional cerebral blood flow. Pharmacological eNOS blockage led to weakened susceptibility signals in the ischemic cortex as well as worsened tissue survival. Consistently, abolishment of eNOS coupling through genetic editing reduced the regional susceptibility signals in the ischemic cortex, causing large infarcts. Upregulation of eNOS during acute ischemia sustains tissue viability through the interaction between NO and deoxyhemoglobin. This interplay can be traced in vivo using SWI and can be considered a sensitive marker revealing the delicate oxygenation status of the ischemic tissue, therefore, guiding the management of acute stroke in clinical settings.

The correlation of IRE1α oxidation with Nox4 activation in aging-associated vascular dysfunction

Oxidative stress attributable to the activation of a Nox4-containing NADPH oxidase is involved in aging-associated vascular dysfunction. However, the Nox4-induced signaling mechanism for the vascular alteration in aging remains unclear. In an aged aorta, the expression of Nox4 mRNA and protein by Nox family of genes was markedly increased compared with a young aorta. Nox4 localization mainly to ER was also established. In the aorta of Nox4 WT mice aged 23–24 months (aged), reactive oxygen species (ROS) and endoplasmic reticulum (ER)/oxidative stress were markedly increased compared with the counter KO mice. Furthermore, endothelial functions including eNOS coupling process and acetylcholine-induced vasodilation were significantly disturbed in the aged WT, slightly affected in the counter KO aorta. Consistently, in d-galactose-induced in vitro aging condition, ER-ROS and its associated ER Nox4 expression and activity were highly increased. Also, in chronic d-galactose-treated condition, IRE1α phosphorylation and XBP-1 splicing and were transiently increased, but IRE1α sulfonation was robustly increased in the aging Nox4 WT condition when compared to the counter KO condition. In vitro D-gal-induced aging study, the phenomenon were abrogated with Nox4 knock-down condition and was significantly decreased in GKT, Nox4 inhibitor and 4-PBA, ER chemical chaperone-treated human umbilical vein endothelial cells. The state of Nox4-based ER redox imbalance/ROS accumulation is suggested to determine the pathway “the UPR; IRE1α phosphorylation and XBP-1 splicing and the UPR failure; IRE1α cysteine-based oxidation, especially sulfonation, finally controlling aging-associated vascular dysfunction.

Abstract 318: Eicosapentaenoic Acid Improved Nitric Oxide Bioavailability and Reduced Nitroxidative Stress in Human Endothelial Cells in Contrast to Arachidonic Acid In Vitro

Treatment with prescription, high dose, stable icosapent ethyl (IPE), which is eicosapentaenoic acid (EPA), an omega-3 fatty acid (O3FA), significantly reduced clinical events in high-risk patients with diabetes and other risk factors or cardiovascular disease (REDUCE-IT). Previous studies suggest that the benefits of EPA correlate positively with its levels and ratio to arachidonic acid (AA) in circulation. Unlike EPA, AA is an omega-6 fatty acid (O6FA) that, along with its metabolites, contributes to inflammation and diabetes. One mechanism of benefit of an increased EPA to AA ratio may be improved endothelial cell (EC) function, as evidenced by increased nitric oxide (NO) release and decreased nitroxidative (ONOO – ) stress. In this study, human umbilical vein endothelial cells (HUVECs) were pretreated with EPA or AA at equimolar levels (10 μM) at various time points (4-24 hr) in 5% FBS. Following treatment, the cells were stimulated with calcium ionophore and assayed for the ratio of NO and ONOO – release, an indicator of eNOS coupling, using tandem porphyrinic nanosensors. ECs treated with EPA had significantly greater NO release following stimulation compared with vehicle at all time points, including 17% and 21% at 4 and 24 hr, respectively (p&lt;0.05 and p&lt;0.01) without changes in eNOS expression. By contrast, AA did not significantly improve NO production. ECs treated with EPA also showed a non-significant reduction in ONOO - release by 10% at 4 hr and 14% at 24 hr. EPA, but not AA, increased NO/ONOO - release ratio by 42% (4.03 ± 0.06 vs 2.83 ± 0.05; p &lt;0.01) by 24 hr. Thus, EPA increased NO bioavailability in human ECs, unlike AA, due to improved eNOS coupling and reduced oxidative stress. These findings support a preferential benefit of EPA on endothelial function as compared to AA and supports further investigation.

Mechanisms underlying selective coupling of endothelial Ca2+ signals with eNOS vs. IK/SK channels in systemic and pulmonary arteries.

Endothelial cell TRPV4 (TRPV4EC ) channels exert a dilatory effect on the resting diameter of resistance mesenteric and pulmonary arteries. Functional intermediate- and small-conductance K+ (IK and SK) channels and endothelial nitric oxide synthase (eNOS) are present in the endothelium of mesenteric and pulmonary arteries. TRPV4EC sparklets preferentially couple with IK/SK channels in mesenteric arteries and with eNOS in pulmonary arteries. TRPV4EC channels co-localize with IK/SK channels in mesenteric arteries but not in pulmonary arteries, which may explain TRPV4EC -IK/SK channel coupling in mesenteric arteries and its absence in pulmonary arteries. The presence of the nitric oxide-scavenging protein, haemoglobin α, limits TRPV4EC -eNOS signalling in mesenteric arteries. Spatial proximity of TRPV4EC channels with eNOS and the absence of haemoglobin α favour TRPV4EC -eNOS signalling in pulmonary arteries. Spatially localized Ca2+ signals activate Ca2+ -sensitive intermediate- and small-conductance K+ (IK and SK) channels in some vascular beds and endothelial nitric oxide synthase (eNOS) in others. The present study aimed to uncover the signalling organization that determines selective Ca2+ signal to vasodilatory target coupling in the endothelium. Resistance-sized mesenteric arteries (MAs) and pulmonary arteries (PAs) were used as prototypes for arteries with predominantly IK/SK channel- and eNOS-dependent vasodilatation, respectively. Ca2+ influx signals through endothelial transient receptor potential vanilloid 4 (TRPV4EC ) channels played an important role in controlling the baseline diameter of both MAs and PAs. TRPV4EC channel activity was similar in MAs and PAs. However, the TRPV4 channel agonist GSK1016790A (10nm) selectively activated IK/SK channels in MAs and eNOS in PAs, revealing preferential TRPV4EC -IK/SK channel coupling in MAs and TRPV4EC -eNOS coupling in PAs. IK/SK channels co-localized with TRPV4EC channels at myoendothelial projections (MEPs) in MAs, although they lacked the spatial proximity necessary for their activation by TRPV4EC channels in PAs. Additionally, the presence of the NO scavenging protein haemoglobin α (Hbα) within nanometer proximity to eNOS limits TRPV4EC -eNOS signalling in MAs. By contrast, co-localization of TRPV4EC channels and eNOS at MEPs, and the absence of Hbα, favour TRPV4EC -eNOS coupling in PAs. Thus, our results reveal that differential spatial organization of signalling elements determines TRPV4EC -IK/SK vs. TRPV4EC -eNOS coupling in resistance arteries.

Combined l-citrulline and tetrahydrobiopterin therapy improves NO signaling and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn pigs.

Newborn pigs with chronic hypoxia-induced pulmonary hypertension (PH) have evidence of endothelial nitric oxide synthase (eNOS) uncoupling. In this model, we showed that therapies that promote eNOS coupling, either tetrahydrobiopterin (BH4), a NOS cofactor, or l-citrulline, a NO-l-arginine precursor, inhibit PH. We wanted to determine whether cotreatment with l-citrulline and a BH4 compound, sapropterin dihydrochloride, improves NO signaling and chronic hypoxia-induced PH more markedly than either alone. Normoxic (control) and hypoxic piglets were studied. Some hypoxic piglets received sole treatment with l-citrulline or BH4, or were cotreated with l-citrulline and BH4, from day 3 through day 10 of hypoxia. Catheters were placed for hemodynamic measurements, and pulmonary arteries were dissected to assess eNOS dimer-to-monomer ratios and NO production. In untreated hypoxic piglets, pulmonary vascular resistance (PVR) was higher and NO production and eNOS dimer-to-monomer ratios were lower than in normoxic piglets. Compared with the untreated hypoxic group, PVR was lower in hypoxic piglets cotreated with l-citrulline and BH4 and in those treated with l-citrulline alone but not for those treated solely with BH4. NO production and eNOS dimer-to-monomer ratios were greater for all three treated hypoxic groups compared with the untreated group. Notably, greater improvements in PVR, eNOS dimer-to-monomer ratios, and NO production were found in hypoxic piglets cotreated with l-citrulline and BH4 than in piglets treated with either alone. Cotreatment with l-citrulline and BH4 more effectively improves NO signaling and inhibits chronic hypoxia-induced PH than either treatment alone. Combination therapies may offer enhanced therapeutic capacity for challenging clinical conditions, such as chronic neonatal PH.

CR6-interacting factor 1 deficiency reduces endothelial nitric oxide synthase activity by inhibiting biosynthesis of tetrahydrobiopterin

Downregulation of CR6 interacting factor 1 (CRIF1) has been reported to induce mitochondrial dysfunction, resulting in reduced activity of endothelial nitric oxide synthase (eNOS) and NO production in endothelial cells. Tetrahydrobiopterin (BH4) is an important cofactor in regulating the balance between NO (eNOS coupling) and superoxide production (eNOS uncoupling). However, whether the decreased eNOS and NO production in CRIF1-deficient cells is associated with relative BH4 deficiency-induced eNOS uncoupling remains completely unknown. Our results showed that CRIF1 deficiency increased eNOS uncoupling and depleted levels of total biopterin and BH4 by reducing the enzymes of BH4 biosynthesis (GCH-1, PTS, SPR, and DHFR) in vivo and vitro, respectively. Supplementation of CRIF1-deficient cells with BH4 significantly increased the recovery of Akt and eNOS phosphorylation and NO synthesis. In addition, scavenging ROS with MitoTEMPO treatment replenished BH4 levels by elevating levels of GCH-1, PTS, and SPR, but with no effect on the level of DHFR. Downregulation of DHFR synthesis regulators p16 or p21 in CRIF1-deficient cells partially recovered the DHFR expression. In summary, CRIF1 deficiency inhibited BH4 biosynthesis and exacerbated eNOS uncoupling. This resulted in reduced NO production and increased oxidative stress, which contributes to endothelial dysfunction and is involved in the pathogenesis of cardiovascular diseases.