Nitric Oxide Synthase Phosphorylation Research Articles

It's well and truly time to stop stating that AMPK regulates glucose uptake and fat oxidation during exercise.

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Angiotensin II inhibits DDAH1-nNOS signaling via AT1R and μOR dimerization to modulate blood pressure control in the central nervous system.

G protein-coupled receptors (GPCRs) are important drug targets. Blocking angiotensin II (Ang II) type 1 receptor signaling alleviates hypertension and improves outcomes in patients with heart failure. Changes in structure and trafficking of GPCR, and desensitization of GPCR signaling induce pathophysiological processes. We investigated whether Ang II, via induction of AT1R and μ-opioid receptor (μOR) dimerization in the nucleus tractus solitarius (NTS), leads to progressive hypertension. Ang II signaling increased μOR and adrenergic receptor α2A (α2A-AR) heterodimer levels and decreased expression of extracellular signal-regulated kinases 1/2T202/Y204, ribosomal protein S6 kinaseT359/S363, and nNOSS1416 phosphorylation. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) expression was abolished in the NTS of adult spontaneously hypertensive rats (SHRs). Endomorphin-2 was overexpressed in NTS of adult SHRs compared with that in 6-week-old Wistar-Kyoto rats (WKY). Administration of μOR agonist into the NTS of WKY increased blood pressure (BP), decreased nitric oxide (NO) production, and decreased DDAH1 activity. μOR agonist significantly reduced the activity of DDAH1 and decreased neuronal NO synthase (nNOS) phosphorylation. The AT1R II inhibitor, losartan, significantly decreased BP and abolished AT1R-induced formation of AT1R and μOR, and α2A-AR and μOR, heterodimers. Losartan also significantly increased the levels of nNOSS1416 phosphorylation and DDAH1 expression. These results show that Ang II may induce expression of endomorphin-2 and abolished DDAH1 activity by enhancing the formation of AT1R and μOR heterodimers in the NTS, leading to progressive hypertension.

Abstract A130: Cannabidiol overcomes oxaliplatin resistance by enhancing autophagy through reduction NOS3 and SOD2

Abstract Although oxaliplatin is an effective chemotherapeutic drug for colorectal cancer (CRC) treatment, patients often develop resistance to it. Therefore, a new strategy for CRC treatment is needed. The purpose of this study was to determine the effect of cannabidiol (CBD), one of the components of the cannabis plant, in overcoming oxaliplatin resistance in CRC cells. We established oxaliplatin-resistant cell lines, DLD-1 R and colo205 R, in CRC DLD-1 and colo205 cells. Autophagic cell death was induced when oxaliplatin-resistant cells were treated with both oxaliplatin and CBD. Additionally, phosphorylation of nitric oxide synthase 3 (NOS3) was increased in oxaliplatin-resistant cells compared to that in parent cells. Combined treatment with oxaliplatin and CBD reduced phospho-NOS3 levels and NO production and resulted in the production of reactive oxygen species (ROS) by reducing the levels of superoxide dismutase 2, an antioxidant present in the mitochondria, causing mitochondrial dysfunction. Taken together, these results suggest that elevated phosphorylation of NOS3 is essential for oxaliplatin resistance. The combination of oxaliplatin and CBD decreased NOS3 phosphorylation, which resulted in autophagy, by inducing the overproduction of ROS through mitochondrial dysfunction, thus overcoming oxaliplatin resistance. Citation Format: Bu Gyeom Kim, Soyeon Jeong, Dae Yeong Kim, Bo Ram Kim, Jung Lim Kim, Seong Hye Park, Yoo Jin Na, Min Jee Jo, Hye Kyeong Yun, Yoon A Jeong, Sun Il Lee, Yoon A Jeong, Hong Jun Kim, Han Do Kim, Dae Hyun Kim, Dae-Hee Lee, Sang Cheul Oh. Cannabidiol overcomes oxaliplatin resistance by enhancing autophagy through reduction NOS3 and SOD2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A130. doi:10.1158/1535-7163.TARG-19-A130

Abstract 195: Chronic Enhancement of Endogenous Nitric Oxide Production via Whole Body Periodic Acceleration (WBPA) Induces Cardioprotection in 15 Months Diabetic Mice

Background: Diabetes mellitus (DM) is a major risk factor for the development of cardiovascular disease. Over time, diabetic patients develop cardiomyopathy, referred to as diabetic cardiomyopathy (DCM). The etiology of DCM in part relates to the duration of hyperglycemia. We have previously shown that type 1 diabetic mice (T1D) have abnormally elevated cardiomyocyte diastolic [Ca 2+ ] d , sodium ([Na + ] d ) after 3 months of diabetes. Whole body periodic acceleration (WBPA) is the motion of the supine body headward to footward in a sinusoidal fashion to induce pulsatile shear stress, increasing expression and phosphorylation of endothelial-derived nitric oxide synthase (eNOS, p-eNOS). We have previously shown that WBPA decreases [Ca 2+ ] d , sodium ([Na + ] d in various models of cardiomyopathy. We hypothesized that WBPA might reverse ion dyshomeostasis in the long term (15-months) hyperglycemia (glucose>250mg/dl) mice. Methods: 15 C57BL/6J (CONT) and diabetic mice (T1D) were randomized to receive WBPA (480 rpm, 1 hr daily for 20 days). Diastolic [Ca 2+ ] d , ([Na + ] d ) (selective microelectrodes), and ROS production (fluorescent) were determined in isolated cardiomyocytes at day 0 and at upon completion of the treatment (day 20). Results: Hyperglycemia produced an increase in [Ca 2+ ] d , [Na + ] d and ROS production in cardiomyocytes. 20 days of WBPA treatment in established DCM significantly decreased [Ca 2+ ] d , [Na + ] d , and ROS production toward normal values. Conclusions: These findings suggest that WBPA may be a therapeutic strategy to reverse ion dyshomeostasis and oxidative stress in a very established(15mos) DCM

Quiescence/Mobilization of Hematopoietic Immature Cells Induced By Polyphenols through Modulation of APC/EPCR/PAR-1 Axis

The mechanism underlying quiescence and/or mobilization of hematopoietic stem cells and their bone marrow progenitors (HSPC) into circulation are tightly regulated for the continuous supply of peripheral blood cells; however, non-physiological or stress conditions, such as infections, can accelerate these mechanisms. Our results have shown that polyphenols modulate quiescence/mobilization of HSPC, but do not affect mature populations. Thrombin has been reported to induce the rapid HSPC mobilization through coagulation thrombin/PAR-1 axis, and quiescence is maintained across the APC/EPCR/PAR-1 axis (Nat. Med. 2015, 21:1307-17). Our objective was to investigate the effect of polyphenols on thrombin/PAR-1 and APC/EPCR/PAR-1 axis. C57BL/6J mice (6-8 weeks old) were treated with polyphenols from green tea extract (250 mg/kg body weight) orally (gavage) once every seven days and injected (i.p.) at day 7 with lipopolysaccharide (LPS) (100μg;Sigma) (n=6). The control group received vehicle and was injected with LPS (n=6). After 24h of LPS injection, mice were anesthetized for blood collection, and then sacrificed for bone marrow collection. PAR-1 and EPCR expression, nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) phosphorylation were evaluated in HSPC by flow cytometry. The functional ability of HSC was assessed by competitive repopulation assay. Vascular permeability was studied using Evans blue. After LPS injection, mice showed reduced expression of EPCR in bone marrow LSK parallel to an increase of PAR-1 expression in circulating immature and mature cells. Treatment of these mice with polyphenols partially prevented the reduced expression of EPCR in bone marrow LSK (13±3 vs 54±12; p<0.05), but did not affect the increased PAR-1 expression in circulating immature and mature cells. Evans blue assay revealed a reduction in the vascular permeability of the bone marrow of LPS-injected mice treated with polyphenols (3.9±0.5 vs 2.1±0.1; p<0.05). To assess whether polyphenols altered NO production, we measured NO levels and eNOS phosphorylation in immature LSK EPCRhigh (or LT-HSC) cells. NO production is activated by eNOS phosphorylation at Ser1177 and negatively regulated by eNOS phosphorylation at Thr495. LPS injection rapidly increased NO levels and eNOS phosphorylation at Ser1177 in bone marrow LSK of mice. Treatment of these mice with polyphenols reduced the percentage of bone marrow LSK EPCRhigh cells with higher intracellular NO (52±2.8 vs 28±5.6; p<0.01) and increased eNOS phosphorylation at Thr495 in immature LSK. In order to evaluate the action of polyphenols on the functional ability of HSC, a competitive bone marrow repopulation assay was performed. Donor mice (C57BL/6J) received or not polyphenols followed by LPS injection (treated group: Polyphenols+LPS; control group: LPS), and bone marrow cells were transplanted (1:1) together with bone marrow cells of competitors (B6.SJL-PtprcaPepcb/BoyJ) in lethally irradiated recipients (B6.SJL-PtprcaPepcb/BoyJ). Mice were followed for 16 weeks and hematological analysis revealed no difference in circulating leukocytes, platelets or hemoglobin levels. Transplanted mice (recipients) presented a higher percentage of CD45+ cells from Polyphenols+LPS donors (33.7±13 vs 78.6±0.9; p<0.05) in the peripheral blood, as well as increased number of T lymphocytes (6.7±4.5 vs 37.2±2.9; p<0.05) and myeloid cells (68.5±1.7 vs 82.5±3.5; p<0.05) from Polyphenols+LPS group. After 16 weeks, mice were euthanized and a higher percentage of LSK (or HSC) and LSK EPCRhigh (or LT-HSC) cells from Polyphenols+LPS donors were detected in the bone marrow, although only the percentage of LSK EPCRhigh was statistically different (0.0014±0.0001 vs 0.0032±0.001; p<0.05). Taken together, our results indicate that polyphenols increased the functional ability of HSC in LPS-injected mice showing increased percentage of bone marrow LSK EPCRhigh cells, which are the most quiescent stem cells with strong self-renewal ability. Polyphenols reduced EPCR expression and NO production in immature cells of LPS-injected mice, exhibiting an anti-inflammatory effect that leads to the maintenance of barrier integrity and quiescence of cells, which was corroborated by reducing vascular permeability in the bone marrow. Thus, polyphenols appear to modulate quiescence/mobilization of HSPC through APC/EPCR/PAR-1 axis. Disclosures No relevant conflicts of interest to declare.

Supplementation With the Sialic Acid Precursor N-Acetyl-D-Mannosamine Breaks the Link Between Obesity and Hypertension

Obesity-related hypertension is a common disorder, and attempts to combat the underlying obesity are often unsuccessful. We previously revealed that mice globally deficient in the inhibitory immunoglobulin G (IgG) receptor FcγRIIB are protected from obesity-induced hypertension. However, how FcγRIIB participates is unknown. Studies were designed to determine if alterations in IgG contribute to the pathogenesis of obesity-induced hypertension. Involvement of IgG was studied using IgG μ heavy chain-null mice deficient in mature B cells and by IgG transfer. Participation of FcγRIIB was interrogated in mice with global or endothelial cell-specific deletion of the receptor. Obesity was induced by high-fat diet (HFD), and blood pressure (BP) was measured by radiotelemetry or tail cuff. The relative sialylation of the Fc glycan on mouse IgG, which influences IgG activation of Fc receptors, was evaluated by Sambucus nigra lectin blotting. Effects of IgG on endothelial NO synthase were assessed in human aortic endothelial cells. IgG Fc glycan sialylation was interrogated in 3442 human participants by mass spectrometry, and the relationship between sialylation and BP was evaluated. Effects of normalizing IgG sialylation were determined in HFD-fed mice administered the sialic acid precursor N-acetyl-D-mannosamine (ManNAc). Mice deficient in B cells were protected from obesity-induced hypertension. Compared with IgG from control chow-fed mice, IgG from HFD-fed mice was hyposialylated, and it raised BP when transferred to recipients lacking IgG; the hypertensive response was absent if recipients were FcγRIIB-deficient. Neuraminidase-treated IgG lacking the Fc glycan terminal sialic acid also raised BP. In cultured endothelial cells, via FcγRIIB, IgG from HFD-fed mice and neuraminidase-treated IgG inhibited vascular endothelial growth factor activation of endothelial NO synthase by altering endothelial NO synthase phosphorylation. In humans, obesity was associated with lower IgG sialylation, and systolic BP was inversely related to IgG sialylation. Mice deficient in FcγRIIB in endothelium were protected from obesity-induced hypertension. Furthermore, in HFD-fed mice, ManNAc normalized IgG sialylation and prevented obesity-induced hypertension. Hyposialylated IgG and FcγRIIB in endothelium are critically involved in obesity-induced hypertension in mice, and supportive evidence was obtained in humans. Interventions targeting these mechanisms, such as ManNAc supplementation, may provide novel means to break the link between obesity and hypertension.

P5995A novel biased S1P1 agonist improves renal and cardiac functions in ZSF1 rats, a model of metabolic syndrome-associated Heart Failure with preserved Ejection Fraction

Abstract Background/Introduction Heart Failure with preserved ejection fraction (HFpEF) is a major cause of death worldwide with currently no approved treatment. Diastolic dysfunction, dyspnea, intolerance to effort, high cardiac filling pressure, and lung congestion coexist with normal ejection fraction in this clinical syndrome. Ageing, obesity, type 2 diabetes, hypertension and renal dysfunction are the main comorbidities found in this heterogeneous group of patients. Microvascular endothelial dysfunction, driven by these risk factors, may be a common link with other aspects of the HFpEF pathogenesis that include oxidative stress, inflammation, cardiomyocyte stiffness/hypertrophy, and myofibroblast accumulation. Sphingosine-1-phosphate type-1 receptor (S1P1), a G protein–coupled receptor highly expressed in endothelial cells, regulates vascular integrity, vascular development and immune cell trafficking. Compound A is a novel G protein-biased S1P1 agonist that lacks functional antagonism and has endothelial-protective properties. Purpose S1P1 activation could promote phosphorylation of endothelial nitric oxide synthase, restoration of endothelial structure and function, and thus diminish cardiac and vascular stiffness, hypertrophy and fibrosis. The aim of this study was to investigate if compound A could improve renal and cardiac functions in a rat model of HFpEF with metabolic syndrome. Methods 65-week-old obese ZSF1 rats were fed a chow diet containing compound A (8 mg/kg/day) or no compound for 4 weeks. Lean ZSF1 and Wistar rats were included in the study as control groups. Urinary protein/creatinine ratio was measured as an index of glomerular injury. Cardiac hypertrophy and function were assessed by two-dimensional and Doppler echocardiography. Total cardiac and atrial weights and pulmonary edema were assessed. Results The obese ZSF1 rat was confirmed as a relevant model of HFpEF with advanced renal dysfunction. These rats showed severe glomerular filtration impairment, left ventricular and atrial hypertrophy and pulmonary edema. Cardiac systolic function, cardiac output and chamber volumes were preserved, diastolic function was impaired, and left ventricular posterior walls and septal thicknesses were increased compared to control groups. Four weeks of compound A treatment reduced urinary protein/creatinine ratio, blunted cardiac and atrial hypertrophy, and partially restored diastolic function. Circulating lymphocytes were not reduced by compound A, confirming that these pharmacological effects were not associated with S1P1 desensitization. Conclusion Compound A, a novel S1P1 agonist with endothelial properties, improves cardiac and renal functions in a rat model of metabolic syndrome-associated HFpEF. Sustained S1P1 activation with compound A may be a promising strategy for HFpEF treatment.

Aspirin-triggered lipoxins modulate phosphorylation of endothelial nitric oxide synthase and protein nitration of endothelial cells stimulated with polyclonal IgG from patients with antiphospholipid syndrome

Aspirin-triggered lipoxins modulate phosphorylation of endothelial nitric oxide synthase and protein nitration of endothelial cells stimulated with polyclonal IgG from patients with antiphospholipid syndrome

MiR-181a affects myocardial ischemia-reperfusion injury in rats via regulating akt signaling pathway.

The aim of this study was to explore the influence of the micro ribonucleic acid (miR)-181a on myocardial ischemia-reperfusion injury (MIRI) in rats by regulating the protein kinase B (Akt) signaling pathway. A total of 30 male Sprague-Dawley rats were randomly divided into three groups, including: sham operation group (Sham group), ischemia-reperfusion group (I/R group), and miR group (MiR-181a group). The model of myocardial ischemia-reperfusion was successfully established in rats. The concentration of blood nitric oxide (NO) was detected by the relative kits. Myocardial apoptosis in rats of the three groups was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Furthermore, the expressions of myocardial cell apoptosis-related proteins and tumor necrosis factor-α (TNF-α), and the degree of Akt phosphorylation were determined by Western blotting. Compared with Sham group and miR-181a group, I/R group exhibited significantly elevated left ventricular end-diastolic pressure (LVEDP) (p<0.05). However, the left ventricular end-systolic pressure (LVESP), stroke work (SW), differential pressure (DP), end-systolic pressure-volume relationship (ESPVR), and end-diastolic pressure-volume relationship (EDPVR) significantly decreased in the I/R group (p<0.05). In comparison with miR-181a group, the apoptosis index of myocardial cells was remarkably elevated in the I/R group, showing statistically significant differences (p<0.05). The protein bands were analyzed using the Quantity One detection software. The results demonstrated that, compared with the Sham group, I/R group showed significantly elevated expressions of cysteine-aspartic protease (Caspase)-3 and TNF-α in rat myocardial tissues (p<0.05). However, the protein levels of Akt and endothelial NO synthase (eNOS) phosphorylation and NO in rat myocardial cells were significantly down-regulated (p<0.05). MiR-181a activates Akt to promote the phosphorylation of its downstream protein eNOS, inhibit the apoptosis of myocardial cells, and alleviate MIRI.

Extracellular Ca2+ promotes nitric oxide production via Ca2+-sensing receptor-Gq/11 protein-endothelial nitric oxide synthase signaling in human vascular endothelial cells

This study examined the possible involvement of Ca2+-sensing receptor (CaSR) in nitric oxide (NO) production in human vascular endothelial cells. Extracellular Ca2+ elevated the intracellular Ca2+ concentration, the endothelial NO synthase (eNOS) phosphorylation level, and NO release from the cells. These responses were inhibited by a CaSR antagonist and a Gq/11 protein inhibitor. Application of an endothelial cell suspension induced vasorelaxation in isolated rat thoracic aorta precontracted by phenylephrine. Adding an NO scavenger to the organ bath abolished this vasorelaxation response. These results suggest that extracellular Ca2+ promotes NO generation via CaSR- and Gq/11 protein-mediated eNOS activation.

Cannabidiol Overcomes Oxaliplatin Resistance by Enhancing NOS3- and SOD2-Induced Autophagy in Human Colorectal Cancer Cells.

Although oxaliplatin is an effective chemotherapeutic drug for colorectal cancer (CRC) treatment, patients often develop resistance to it. Therefore, a new strategy for CRC treatment is needed. The purpose of this study was to determine the effect of cannabidiol (CBD), one of the components of the cannabis plant, in overcoming oxaliplatin resistance in CRC cells. We established oxaliplatin-resistant cell lines, DLD-1 R and colo205 R, in CRC DLD-1 and colo205 cells. Autophagic cell death was induced when oxaliplatin-resistant cells were treated with both oxaliplatin and CBD. Additionally, phosphorylation of nitric oxide synthase 3 (NOS3) was increased in oxaliplatin-resistant cells compared to that in parent cells. Combined treatment with oxaliplatin and CBD reduced phospho-NOS3 levels and nitric oxide (NO) production and resulted in the production of reactive oxygen species (ROS) by reducing the levels of superoxide dismutase 2, an antioxidant present in the mitochondria, causing mitochondrial dysfunction. Taken together, these results suggest that elevated phosphorylation of NOS3 is essential for oxaliplatin resistance. The combination of oxaliplatin and CBD decreased NOS3 phosphorylation, which resulted in autophagy, by inducing the overproduction of ROS through mitochondrial dysfunction, thus overcoming oxaliplatin resistance.

Inositol 1,4,5-Trisphosphate Receptors in Endothelial Cells Play an Essential Role in Vasodilation and Blood Pressure Regulation.

BackgroundEndothelial NO synthase plays a central role in regulating vasodilation and blood pressure. Intracellular Ca2+ mobilization is a critical modulator of endothelial NO synthase function, and increased cytosolic Ca2+ concentration in endothelial cells is able to induce endothelial NO synthase phosphorylation. Ca2+ release mediated by 3 subtypes of inositol 1,4,5‐trisphosphate receptors (IP 3Rs) from the endoplasmic reticulum and subsequent Ca2+ entry after endoplasmic reticulum Ca2+ store depletion has been proposed to be the major pathway to mobilize Ca2+ in endothelial cells. However, the physiological role of IP 3Rs in regulating blood pressure remains largely unclear.Methods and ResultsTo investigate the role of endothelial IP 3Rs in blood pressure regulation, we first generated an inducible endothelial cell–specific IP 3R1 knockout mouse model and found that deletion of IP 3R1 in adult endothelial cells did not affect vasodilation and blood pressure. Considering all 3 subtypes of IP 3Rs are expressed in mouse endothelial cells, we further generated inducible endothelial cell–specific IP 3R triple knockout mice and found that deletion of all 3 IP 3R subtypes decreased plasma NO concentration and increased basal blood pressure. Furthermore, IP 3R deficiency reduced acetylcholine‐induced vasodilation and endothelial NO synthase phosphorylation at Ser1177.ConclusionsOur results reveal that IP 3R‐mediated Ca2+ release in vascular endothelial cells plays an important role in regulating vasodilation and physiological blood pressure.

Adenosine A1 receptors and mitochondria: targets of remote ischemic preconditioning.

Adenosine is involved in classic preconditioning in most species and acts especially through adenosine A1 and A3 receptors. The aim of the present study was to evaluate whether remote ischemic preconditioning (rIPC) activates adenosine A1 receptors and improves mitochondrial function, thereby reducing myocardial infarct size. Isolated rat hearts were subjected to 30 min of global ischemia and 60 min of reperfusion [ischemia-reperfusion (I/R)]. In a second group, before isolation of the heart, a rIPC protocol (3 cycles of hindlimb I/R) was performed. Infarct size was measured with tetrazolium staining, and Akt/endothelial nitric oxide (NO) synthase (eNOS) expression/phosphorylation and mitochondrial function were evaluated after ischemia at 10 and 60 min of reperfusion. As expected, rIPC significantly decreased infarct size. This beneficial effect was abolished only when 8-cyclopentyl-1,3-dipropylxanthine (adenosine A1 receptor blocker) and NG-nitro-l-arginine methyl ester (NO synthesis inhibitor) were administered during the reperfusion phase. At the early reperfusion phase, rIPC induced significant Akt and eNOS phosphorylation, which was abolished by the perfusion with an adenosine A1 receptor blocker. I/R led to impaired mitochondrial function, which was attenuated by rIPC and mediated by adenosine A1 receptors. In conclusion, we demonstrated that rIPC limits myocardial infarct by activation of adenosine A1 receptors at early reperfusion in the isolated rat heart. Interestingly, rIPC appears to reduce myocardial infarct size by the Akt/eNOS pathway and improves mitochondrial function during myocardial reperfusion. NEW & NOTEWORTHY Adenosine is involved in classic preconditioning and acts especially through adenosine A1 and A3 receptors. However, its role in the mechanism of remote ischemic preconditioning is controversial. In this study, we demonstrated that remote ischemic preconditioning activates adenosine A1 receptors during early reperfusion, inducing Akt/endothelial nitric oxide synthase phosphorylation and improving mitochondrial function, thereby reducing myocardial infarct size.

The Effects of Morinda citrifolia (Noni) Fruit Juice on the Prevention of Stroke by Promoting Production of Nitric Oxide through the Brain of the Spontaneously Hypertensive Stroke Prone (SHRSP) Rats

Morinda citrifolia (Noni) is a traditional folk medicinal plant and has a long history of use as a food and medicine. In order to reveal the effects of Noni fruit juice (NFJ) on stroke prevention, we performed experiments using spontaneously hypertensive stroke prone (SHRSP) rats. NFJ did not change rat body weight, food intake, and water intake. However, both systolic blood pressure (SBP) and diastolic blood pressure (DBP) were significantly decreased after NFJ treatment in SHRSP rats. Furthermore, NFJ significantly increased the survival rate, urinary nitric oxide (NO) concentration was significantly higher in the NFJ group, and endothelial NO synthase (eNOS) phosphorylation levels increased in the brain after NFJ treatment. Two pathways regulate eNOS phosphorylation: the insulin-dependent pathway and the insulin-independent pathway. For the insulin-dependent pathway, phosphorylation of insulin receptor substrate 1 (IRS1) and protein kinase B (Akt) did not change in the NFJ group. For the insulin-independent pathway, expression of adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, liver kinase B 1 (LKB1), and silent information regulator 1 (Sirt1) significantly increased in the brain of SHRSP rats after NFJ treatment. These data suggested that NFJ prevented stroke by improved blood circulation, increased NO production, and elevated eNOS phosphorylation by stimulating the insulin-independent pathway (Sirt1-LKB1-AMPK-eNOS).

GATA4 protects against hyperglycemia‑induced endothelial dysfunction by regulating NOX4 transcription.

Endothelial dysfunction is one of the most common complications associated with diabetes and may lead to atherosclerosis. Conflicting reports indicate that NADPH oxidase 4 (NOX4) induces hydrogen peroxide production and cytotoxicity, but also has a protective effect on endothelial dysfunction. The present study aimed to identify the transcription factor responsible for NOX4 expression using a transcription factor activation profiling plate array and chromatin immunoprecipitation. Data from these analyses indicated that GATA-binding protein 4 (GATA4) was able to mediate NOX4 transcription and is downregulated in human umbilical vein endothelial cells (HUVECs) that were exposed to hyperglycemic conditions as well as in the endothelial cells of a mouse diabetes model. Overexpression of GATA4 was demonstrated to lead to increased expression of NOX4 mRNA and protein. Furthermore, GATA4 overexpression resulted in increased nitric oxide (NO) production through the upregulation of endothelial NO synthase phosphorylation. Treatment with simvastatin, a drug known to preserve endothelial function through an unknown mechanism, improved endothelial cell function by upregulating GATA4 expression in HUVECs exposed to hyperglycemia. Results from these experiments demonstrated that GATA4 may inhibit diabetes-induced endothelial dysfunction by acting as a transcription factor for NOX4 expression and increasing NO production. Thus, the present study revealed a novel molecular mechanism underlying endothelial dysfunction in diabetes and identified GATA4 as a potential therapeutic target.

Ectopic Fatty Acid-Binding Protein 4 Expression in the Vascular Endothelium is Involved in Neointima Formation After Vascular Injury.

BackgroundFatty acid‐binding protein 4 (FABP4) is expressed in adipocytes, macrophages, and endothelial cells of capillaries but not arteries. FABP4 is secreted from adipocytes in association with lipolysis, and an elevated circulating FABP4 level is associated with obesity, insulin resistance, and atherosclerosis. However, little is known about the link between FABP4 and endovascular injury. We investigated the involvement of ectopic FABP4 expression in endothelial cells in neointima hyperplasia after vascular injury.Methods and ResultsFemoral arteries of 8‐week‐old male mice were subjected to wire‐induced vascular injury. After 4 weeks, immunofluorescence staining showed that FABP4 was ectopically expressed in endothelial cells of the hyperplastic neointima. Neointima formation determined by intima area and intima to media ratio was significantly decreased in FABP4‐defficient mice compared with that in wild‐type mice. Adenovirus‐mediated overexpression of FABP4 in human coronary artery endothelial cells (HCAECs) in vitro increased inflammatory cytokines and decreased phosphorylation of nitric oxide synthase 3. Furthermore, FABP4 was secreted from HCAECs. Treatment of human coronary smooth muscle cells or HCAECs with the conditioned medium of Fabp4‐overexpressed HCAECs or recombinant FABP4 significantly increased gene expression of inflammatory cytokines and proliferation‐ and adhesion‐related molecules in cells, promoted cell proliferation and migration of human coronary smooth muscle cells, and decreased phosphorylation of nitric oxide synthase 3 in HCAECs, which were attenuated in the presence of an anti‐FABP4 antibody.ConclusionsEctopic expression and secretion of FABP4 in vascular endothelial cells contribute to neointima formation after vascular injury. Suppression of ectopic FABP4 in the vascular endothelium would be a novel strategy against post‐angioplasty vascular restenosis.

REM sleep deprivation induces endothelial dysfunction and hypertension in middle-aged rats: Roles of the eNOS/NO/cGMP pathway and supplementation with L-arginine.

Sleep loss can induce or aggravate the development of cardiovascular and cerebrovascular diseases. However, the molecular mechanism underlying this phenomenon is poorly understood. The present study was designed to investigate the effects of REM sleep deprivation on blood pressure in rats and the underlying mechanisms of these effects. After Sprague-Dawley rats were subjected to REM sleep deprivation for 5 days, their blood pressures and endothelial function were measured. In addition, one group of rats was given continuous access to L-arginine supplementation (2% in distilled water) for the 5 days before and the 5 days of REM sleep deprivation to reverse sleep deprivation-induced pathological changes. The results showed that REM sleep deprivation decreased body weight, increased blood pressure, and impaired endothelial function of the aortas in middle-aged rats but not young rats. Moreover, nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) concentrations as well as endothelial NO synthase (eNOS) phosphorylation in the aorta were decreased by REM sleep deprivation. Supplementation with L-arginine could protect against REM sleep deprivation-induced hypertension, endothelial dysfunction, and damage to the eNOS/NO/cGMP signaling pathway. The results of the present study suggested that REM sleep deprivation caused endothelial dysfunction and hypertension in middle-aged rats via the eNOS/NO/cGMP pathway and that these pathological changes could be inhibited via L-arginine supplementation. The present study provides a new strategy to inhibit the signaling pathways involved in insomnia-induced or insomnia-enhanced cardiovascular diseases.

Low shear stress induces endothelial reactive oxygen species via the AT1R/eNOS/NO pathway.

Reactive oxygen species (ROS) contribute to many aspects of physiological and pathological cardiovascular processes. However, the underlying mechanism of ROS induction by low shear stress (LSS) remains unclear. Accumulating evidence has shown that the angiotensin II type 1 receptor (AT1R) is involved in inflammation, apoptosis, and ROS production. Our aim was to explore the role of AT1R in LSS-mediated ROS induction. We exposed human umbilical vein endothelial cells (HUVECs) to LSS (3 dyn/cm2 ) for different periods of time. Western blotting and immunofluorescence showed that LSS significantly induced AT1R expression in a time-dependent manner. Using immunohistochemistry, we also noted a similar increase in AT1R expression in the inner curvature of the aortic arch compared to the descending aorta in C57BL/6 mice. Additionally, HUVECs were cultured with a fluorescent probe, either DCFH, DHE or DAF, after being subjected to LSS. Cell chemiluminescence and flow cytometry results revealed that LSS stimulated ROS levels and suppressed nitric oxide (NO) generation in a time-dependent manner, which was reversed by the AT1R antagonist Losartan. We also found that Losartan markedly increased endothelial NO synthase (eNOS) phosphorylation at Ser(633,1177) and dephosphorylation at Thr(495), which involved AKT and ERK. Moreover, the ROS level was significantly reduced by endogenous and exogenous NO donors (L-arginine, SNP) and increased by the eNOS inhibitor L-NAME. Overall, we conclude that LSS induces ROS via AT1R/eNOS/NO.

Arginase-2 mediates renal ischemia-reperfusion injury.

Novel therapeutic interventions for preventing or attenuating kidney injury following ischemia-reperfusion injury (IRI) remain a focus of significant interest. Currently, there are no definitive therapeutic or preventive approaches available for ischemic acute kidney injury (AKI). Our objective is to determine 1) whether renal arginase activity or expression is increased in renal IRI, and 2) whether arginase plays a role in development of renal IRI. The impact of arginase activity and expression on renal damage was evaluated in male C57BL/6J (wild type) and arginase-2 (ARG2)-deficient (Arg2-/- ) mice subjected to bilateral renal ischemia for 28 min, followed by reperfusion for 24 h. ARG2 expression and arginase activity significantly increased following renal IRI, paralleling the increase in kidney injury. Pharmacological blockade or genetic deficiency of Arg2 conferred kidney protection in renal IRI. Arg2-/- mice had significantly attenuated kidney injury and lower plasma creatinine and blood urea nitrogen levels after renal IRI. Blocking arginases using S-(2-boronoethyl)-l-cysteine (BEC) 18 h before ischemia mimicked arginase deficiency by reducing kidney injury, histopathological changes and kidney injury marker-1 expression, renal apoptosis, kidney inflammatory cell recruitment and inflammatory cytokines, and kidney oxidative stress; increasing kidney nitric oxide (NO) production and endothelial NO synthase (eNOS) phosphorylation, kidney peroxisome proliferator-activated receptor-γ coactivator-1α expression, and mitochondrial ATP; and preserving kidney mitochondrial ultrastructure compared with vehicle-treated IRI mice. Importantly, BEC-treated eNOS-knockout mice failed to reduce blood urea nitrogen and creatinine following renal IRI. These findings indicate that ARG2 plays a major role in renal IRI, via an eNOS-dependent mechanism, and that blocking ARG2 activity or expression could be a novel therapeutic approach for prevention of AKI.

Adiponectin improves endothelial function in mesenteric arteries of rats fed a high-fat diet: role of perivascular adipose tissue.

Adiponectin, the most abundant peptide secreted by adipocytes, is involved in the regulation of energy metabolism and vascular physiology. Here, we have investigated the effects of exogenous administration of adiponectin on metabolism, vascular reactivity and perivascular adipose tissue (PVAT) of mesenteric arteries in Wistar rats fed a high-fat diet. The effects of adiponectin on NO-dependent and independent vasorelaxation were investigated in isolated mesenteric arteries from 12-month-old male Wistar rats (W12m) fed a high-fat diet (HFD) for 4months and compared with those from age-matched rats given a control diet. Adiponectin ((96μg·day-1 ) was administered by continuous infusion with a minipump, implanted subcutaneously, for 28days. Chronic adiponectin treatment reduced body weight, total cholesterol, free fatty acids, fasting glucose and area under the curve of intraperitoneal glucose tolerance test, compared with HFD rats. It also normalized NO-dependent vasorelaxation increasing endothelial NO synthase (eNOS) phosphorylation in mesenteric arteries of HFD rats. In PVAT from aged (W12m) and HFD rats there was increased expression of chemokines and pro-inflammatory adipokines, the latter being important contributors to endothelial dysfunction. Infusion of adiponectin reduced these changes. Adiponectin normalized endothelial cell function by a mechanism that involved increased eNOS phoshorylation and decreased PVAT inflammation. Detailed characterization of the adiponectin signalling pathway in the vasculature and perivascular fat is likely to provide novel approaches to the management of atherosclerosis and metabolic disease. This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue - Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc.