Endothelial Nitric Oxide Synthase Inhibitor Research Articles

Pulmonary microvascular remodeling in chronic thrombo-embolic pulmonary hypertension.

Pulmonary vascular remodeling in pulmonary arterial hypertension involves perturbations in the nitric oxide (NO) and endothelin-1 (ET-1) pathways. However, the implications of pulmonary vascular remodeling and these pathways remain unclear in chronic thrombo-embolic pulmonary hypertension (CTEPH). The objective of the present study was to characterize changes in microvascular morphology and function, focussing on the ET-1 and NO pathways, in a CTEPH swine model. Swine were chronically instrumented and received up to five pulmonary embolizations by microsphere infusion, whereas endothelial dysfunction was induced by daily administration of the endothelial NO synthase inhibitor Nω-nitro-l-arginine methyl ester until 2 wk before the end of study. Swine were subjected to exercise, and the pulmonary vasculature was investigated by hemodynamic, histological, quantitative PCR, and myograph experiments. In swine with CTEPH, the increased right-ventricular afterload, decreased cardiac index, and mild ventilation-perfusion-mismatch were exacerbated during exercise. Pulmonary microvascular remodeling was evidenced by increased muscularization, which was accompanied by an increased maximal vasoconstriction. Although ET-1-induced vasoconstriction was increased in CTEPH pulmonary small arteries, the ET-1 sensitivity was decreased. Moreover, the contribution of the ETA receptor to ET-1 vasoconstriction was increased, whereas the contribution of the ETB receptor was decreased and the contribution of Rho-kinase was lost. A reduction in endogenous NO production was compensated in part by a decreased phosphodiesterase 5 (PDE5) activity resulting in an apparent increased NO sensitivity in CTEPH pulmonary small arteries. These findings suggest that pulmonary microvascular remodeling with a reduced activity of PDE5 and Rho-kinase may contribute to the lack of therapeutic efficacy of PDE5 inhibitors and Rho-kinase inhibitors in CTEPH.

RXFP1 Receptor Activation by Relaxin-2 Induces Vascular Relaxation in Mice via a Gαi2-Protein/PI3Kß/γ/Nitric Oxide-Coupled Pathway.

Background: Relaxins are small peptide hormones, which are novel candidate molecules that play important roles in cardiometablic syndrome. Relaxins are structurally related to the insulin hormone superfamily, which provide vasodilatory effects by activation of G-protein-coupled relaxin receptors (RXFPs) and stimulation of endogenous nitric oxide (NO) generation. Recently, relaxin could be demonstrated to activate Gi proteins and phosphoinositide 3-kinase (PI3K) pathways in cultured endothelial cells in vitro. However, the contribution of the Gi-PI3K pathway and their individual components in relaxin-dependent relaxation of intact arteries remains elusive.Methods: We used Gαi2- (Gnai2-/-) and Gαi3-deficient (Gnai3-/-) mice, pharmacological tools and wire myography to study G-protein-coupled signaling pathways involved in relaxation of mouse isolated mesenteric arteries by relaxins. Human relaxin-1, relaxin-2, and relaxin-3 were tested.Results: Relaxin-2 (∼50% relaxation at 10-11 M) was the most potent vasodilatory relaxin in mouse mesenteric arteries, compared to relaxin-1 and relaxin-3. The vasodilatory effects of relaxin-2 were inhibited by removal of the endothelium or treatment of the vessels with N (G)-nitro-L-arginine methyl ester (L-NAME, endothelial nitric oxide synthase (eNOS) inhibitor) or simazine (RXFP1 inhibitor). The vasodilatory effects of relaxin-2 were absent in arteries of mice treated with pertussis toxin (PTX). They were also absent in arteries isolated from Gnai2-/- mice, but not from Gnai3-/- mice. The effects were not affected by FR900359 (Gαq protein inhibitor) or PI-103 (PI3Kα inhibitor), but inhibited by TGX-221 (PI3Kβ inhibitor) or AS-252424 (PI3Kγ inhibitor). Simazine did not influence the anti-contractile effect of perivascular adipose tissue.Conclusion: Our data indicate that relaxin-2 produces endothelium- and NO-dependent relaxation of mouse mesenteric arteries by activation of RXFP1 coupled to Gi2-PI3K-eNOS pathway. Targeting vasodilatory Gi-protein-coupled RXFP1 pathways may provide promising opportunities for drug discovery in endothelial dysfunction and cardiometabolic disease.

Thrombin-Induced Responses via Protease-Activated Receptor 1 Blocked by the Endothelium on Isolated Porcine Retinal Arterioles

ABSTRACTPurpose: Thrombin, a serine protease, causes organ-specific responses to vessels. However, the mechanism by which thrombin affects the retinal microcirculation remains unclear. We examined the effects of thrombin on the retinal microvasculature and signaling mechanisms.Methods: Porcine retinal arterioles were isolated, cannulated, and pressurized (55 cmH2O) without flow in this in vitro study. Videomicroscopy techniques recorded changes in diameter in the retinal arterioles in response to thrombin at concentrations ranging from 0.001 to 20 mU/ml.Results: Extraluminal administration of thrombin induced concentration-dependent vascular responses, that is, vasoconstriction at low concentrations less than 5 mU/ml and vasorelaxation with high concentrations greater than 5 mU/ml. However, intraluminal administration of thrombin (5 mU/m) did not constrict the retinal arterioles; in denuded vessels, intraluminal administration constricted the retinal arterioles. Thrombin-induced vasoconstriction was significantly (p < 0.01) suppressed by pretreatment with a protein kinase C (PKC) inhibitor and a protease-activated receptor (PAR)-1 inhibitor but not by PAR-2 and PAR-4 inhibitors or denudation. A rho kinase (ROCK) inhibitor also suppressed thrombin-induced vasoconstriction (5 mU/ml) compared with sodium nitroprusside. Endothelial denudation and pretreatment with an endothelial nitric oxide (NO) synthase inhibitor suppressed vasorelaxation caused by a high concentration of thrombin.Conclusions: A low concentration of thrombin causes vasoconstriction of smooth muscles via PAR-1, PKC, and ROCK, and a high concentration of thrombin possibly causes vasorelaxation of the retinal arterioles via nitric oxide synthase activation in the endothelium. The vascular endothelium might block signaling of thrombin-induced vasoconstriction in the retinal arterioles when administered intraluminally.

Mechanisms Responsible for Serotonin Vascular Reactivity Sex Differences in the Internal Mammary Artery.

BackgroundThe increased adverse cardiac events in women undergoing coronary artery bypass grafting are multifactorial and may include clinical, psychosocial, and biological factors. Potential contributing biological factors could include vascular hyperreactivity of the internal mammary artery (IMA) to endogenous vasoconstrictors in women, resulting in a predilection to myocardial ischemia. This study evaluated sex differences in serotonin and thromboxane A2 dependent vasoconstriction in human isolated IMA, with the mechanistic role of (1) the endothelium, (2) nitric oxide (NO), (3) prostaglandins, and (4) receptor activity investigated for any observed sex difference.Methods and ResultsViable isolated human IMA segments were obtained from 116 patients (44 women [mean age, 66.8±12.2 years] and 72 men [mean age, 66.6±10.4 years]) undergoing coronary artery bypass grafting. Cumulative concentration‐response curves for serotonin and thromboxane A2 mimetic, U46619, were determined and revealed an increased sensitivity to serotonin but not U46619 in women. This sex difference to serotonin was further assessed by the following: (1) endothelial denudation, (2) endothelial NO synthase inhibition and NO quantification using electron paramagnetic resonance, (3) cyclooxygenase inhibition and prostaglandin metabolite quantification using mass spectrometry, and (4) quantification of receptor activity status. The female hyperreactivity to serotonin was (1) abolished by endothelial denudation; (2) unaffected by NO synthase inhibition, with no difference in electron paramagnetic resonance–assessed NO levels; (3) abolished by cyclooxygenase inhibition (quantification of prostaglandins in IMA revealed a trend towards reduced 6‐keto prostaglandin F1α in female IMA; P=0.08); and (4) unrelated to receptor activity.ConclusionsThese data indicate that female IMAs are hyperreactive to serotonin but not U46619, with the former attributable to an endothelium‐dependent cyclooxygenase pathway.

Measurement of flow-mediated dilation of mouse femoral artery in vivo by optical coherence tomography.

Flow-mediated vasodilation (FMD) is used for assessment of vascular endothelial function in humans as a predictor of cardiovascular events. It has been challenging to carry it on preclinical murine models due to the diminutive size of the femoral artery. Here, we present a new approach to accurately measure the blood velocity and femoral artery diameters of mice by acquiring Doppler optical coherence tomography and optical coherence tomography angiography continuously within 1 single experimental scanning protocol. Using the 3-dimensional imaging and new velocity algorithm, the measurement precision of diameter, blood flow, velocity and wall shear stress are improved to 0.91%, 11.0%, 10.7% and 14.0%, respectively. FMD of healthy mouse femoral artery measured by this method was 11.96% ± 0.98%, which was blunted to 5.69% ± 0.4% by intravenous administration of endothelial nitric oxide synthase inhibitor (L-NG -Nitroarginine methyl ester), in agreement with that reported in the literature.

Ripasudil Attenuates Lipopolysaccharide (LPS)-Mediated Apoptosis and Inflammation in Pulmonary Microvascular Endothelial Cells via ROCK2/eNOS Signaling.

BackgroundMicrovascular endothelial inflammation and apoptosis are responsible for septic acute lung injury (ALI). Ripasudil is a novel Rho/Rho kinase (ROCK) inhibitor which shows therapeutic effects on several vascular diseases. The aim of this study was to investigate the protective effects and correlated molecular mechanisms of ripasudil on lipopolysaccharide-induced inflammation and apoptosis of pulmonary microvascular endothelial cells (PMVECs).Material/MethodsCultured PMVECs were exposed to lipopolysaccharide (LPS). Ripasudil at various concentrations was used to treat the cells. Several cells were also co-administrated with the endothelial nitric oxide synthase (eNOS) inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME). Cell viability was assessed by MTT assay. Terminal dUTP transferase nick-end labeling (TUNEL) assay was used to detect the apoptosis. The colorimetric method was used to measure the activity of eNOS and ROCK2. Protein phosphorylation and expression were assessed by Western blotting.ResultsRipasudil attenuated the LPS-induced inflammation and apoptosis in PMVECs, which was reversed by L-NAME. Ripasudil suppressed ROCK2 activity and further increased the eNOS activity. Ripasudil treatment increased the phosphorylation of eNOS, increased the expression level of Bcl2, and decreased the expression level of active caspase3 in LPS-treated PMVECs. Moreover, the ripasudil treatment also inhibited the nuclear translocation of NF-κB and further suppressed the levels of interleukin (IL) 6 and tumor necrosis factor (TNF) α. The co-treatment with L-NAME, however, impaired the anti-apoptotic and anti-inflammatory effects of ripasudil on PMVECs without affecting ROCK2.ConclusionsThe novel ROCK2 inhibitor ripasudil suppressed LPS-induced apoptosis and inflammation in PMVECs by regulating the ROCK2/eNOS signaling pathway.

Differential Respiratory Response of Isolated Cardiac Mitochondria from Young and Aged Mice towards Nitric Oxide Synthase Inhibitors

Mitochondrial respiratory dysfunction is proposed to be an important underlying mechanism in the aging‐induced cardiac abnormalities. Identifying the mechanisms involved in the development of aging‐induced respiratory dysfunction in cardiac mitochondria could be useful to develop therapeutic strategies to reduce the risk of developing cardiovascular diseases. Role of mitochondrial nitric oxide synthase (NOS) to aging‐induced respiratory changes in cardiac mitochondria are not studied previously. Here we treated the isolated cardiac mitochondria from young (3 months) and old mice (18 months) hearts with neuronal NOS inhibitor (nNOS, ARL 17477 dihydrochloride or ARL) and endothelial NOS inhibitor (eNOS, N5‐(1‐Iminoethyl)‐L‐ornithine or NIO) and studied the respiratory characteristics using Seahorse Xfe24 analyzer.ResultsMitochondria from the old mice hearts exhibited significantly increased basal respiration (24.7%, 169.0±14.0 vs 135.5±5.1, p≤0.05) with unchanged ATP production. They also showed a significant decrease in state IIIu respiration (23%, 635.4±40.2 vs 826.4±49.2, p≤0.05) and also the membrane potential. Both ARL and NIO treatments significantly decreased state IIIu respiration (22.1%, 643.7±34.4 vs 826.4±49.2, p≤0.05 &amp; 23.9%, 448±18.5 vs 587.1±34.8, p≤0.05 respectively) in mitochondria from young mice hearts without affecting membrane potential and oxidative stress parameters. Interestingly, ARL and NIO neither induced similar responses in the mitochondria from the old mice hearts nor they corrected the aging‐induced decline in state IIIu respiration and membrane potential. Mitochondria from the old mice hearts have increased nNOS protein levels (421%, 1.98±0.80 vs 0.38±0.20, p≤0.05) with decreased protein S‐nitrosylation (46.25%, 8.3±1.5 vs 15.44±3.2, p≤0.07). The observed differential respiratory responses of cardiac mitochondria from young and old mice following nNOS and eNOS inhibitors were associated with reduced protein S‐nitrosylation in mitochondria from young mice hearts (64.2%, 5.53±0.35 vs 15.44±3.2, p≤0.05 &amp; 65.7%, 5.3±0.84 vs 15.44±3.2, p≤0.05 respectively) with unaltered nitrosylation in mitochondria from old mice hearts.ConclusionsThus the functional loss of mitochondrial nNOS and eNOS in the aged hearts accompanied by the reduced NO‐mediated regulation of mitochondrial proteins contributes to the aging‐induced mitochondrial dysfunction.Support or Funding InformationAmerican Heart Association (PVG: 14SDG20490359 and VNS: 16PRE31450006) and National Institute of Health: National Institute of Neurological Disorders and Stroke and National Institute of General Medical Sciences (PVK: R01NS094834).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CD70 Modulates the Role of eNOS In Endothelial Cells

CD70 has traditionally been identified on antigen presenting cells and interacts with CD27 on T cells, promoting proliferation and memory cell formation. We have recently shown that CD70 is important in the formation of effector memory T cells in hypertension. However, the precise cell types that express CD70 and the role of CD70 on non‐immune cells has not been investigated. Here we describe a new role of CD70 in endothelial function. We found that human umbillical vein endothelial cells express CD70 mRNA and that this is increased by &gt; 30 fold by laminar (15 dynes/cm2) compared to oscillatory shear. Studies of mesenteric vascular reactivity showed that CD70−/− mice have markedly impaired endothelium‐dependent vasodilation to acetylcholine compared to WT mice (45± 5 vs 25 ± 4 %) at baseline. In contrast, there were no differences in relaxation responses to sodium nitroprusside. Pre‐incubating vessels with the endothelial nitric oxide synthase (eNOS) inhibitor L‐NAME blocked endothelium‐dependent vasodilatation to acetylcholine in WT mice nearly 75%; in contrast, L‐NAME had no effect on endothelial vessel relaxation in CD70−/− mice. This suggests that vessels of CD70−/− mice lack the capacity to produce nitric oxide (NO). Western blot for the endothelial nitric oxide synthase (eNOS) showed a marked reduction of this enzyme in the aortas of CD70−/− as compared to wild type mice. We found that CD70 and eNOS interact by co‐immunoprecipitation, however this association is abolished by siRNA against CD70. In additional experiments, aortas from CD70−/− did demonstrate expression of both neuronal NOS (nNOS) and inducible NOS (iNOS). In order to assess functional effects of this expression, nNOS NO production was blocked with LVNIO, resulting in further impairment in vasodilation of CD70−/− mesenteric vessels (−logEC50 of 5.4 after treatment, compared to 6.2 before treatment); however blockade of iNOS with 1400 W resulted in apparent recovery of acetylcholine‐induced vasodilation. These data indicate that loss of CD70−/− leads to destabilization of the eNOS protein in endothelial cells resulting in endothelial dysfunction. In the absence of eNOS in CD70−/− vessels, iNOS contributes to endothelial dysfunction, while nNOS has a compensatory role. These studies have identified a new role of the immune co‐stimulatory protein CD70 in modulating endothelial eNOS protein levels and vascular function.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial modulation of a nitric oxide donor complex-induced relaxation in normotensive and spontaneously hypertensive rats

We hypothesized that endothelium modulates relaxation induced by a nitric oxide (NO) donor ruthenium complex (TERPY, [Ru(terpy)(bdq)NO]3+) in mesenteric arteries of normotensive and spontaneously hypertensive (SHR) rats in different ways. We analyzed the mechanism involved in TERPY-induced relaxation in the second and third branches of mesenteric arteries and investigated how endothelium contributes to the TERPY vasodilator effect on SHR blood vessels. TERPY induced concentration-dependent relaxation in endothelium-denuded (E−) and endothelium-intact (E+) mesenteric arteries of normotensive rats and SHR. Pretreatment with ODQ (which inhibits soluble guanylyl cyclase) or TEA (tetraethylammonium, which blocks potassium channels) significantly reduced the TERPY vasodilator effect on E− mesenteric arteries of normotensive rats and SHR. The presence of endothelium shifted the concentration–effect curves for TERPY in E+ mesenteric arteries of normotensive rats to the right. Conversely, the presence of endothelium shifted the concentration–effect curves for TERPY in the case of SHR E+ mesenteric arteries to the left, which suggested increased potency. L-NNA, a more selective endothelial NO synthase (eNOS) inhibitor, reduced TERPY potency in SHR. The presence of endothelium and notably of NOS contributed to the TERPY vasodilator action in SHR: TERPY promoted eNOS Ser1177 phosphorylation with consequent NO production and increased soluble guanylyl cyclase activity, which may have directly activated potassium channels.

Atrial endothelial cells senescence promotes thrombogenicity, inflammation and extracellular matrix remodeling: Role of the local Ang II/AT1 receptor pathway

Ageing promotes atrial remodeling that paves way to atrial fibrillation and thrombogenicity. Preclinical studies on endothelial atrial cells are lacking. This study aims to characterize phenotypical changes associated with atrial endothelial cells senescence and to decipher the link between ageing and thrombogenicity. Atrial endothelial cells (AEC) were obtained from freshly harvested porcine left atria. Endothelial senescence was assessed by senescence-associated beta-galactosidase activity (SA-β-gal), using flow cytometry, protein expression by Western blot analysis and platelet aggregation using an aggregometer. Replicative senescence was induced by passaging AEC from passage P1 to P4, and premature endothelial cell senescence by exposing AEC to L-NAME, an endothelial NO synthase (eNOS) inhibitor or H 2 O 2 . AEC senescence was characterized by an increase in SA-β-gal activity and an up-regulation of p53, a key regulator of cellular senescence, and of p21 and p16, key cyclin-dependent kinase inhibitors. Senescent AEC phenotype was characterized by: – cell thrombogenicity through an up-regulation of tissue factor expression, shedding of procoagulant microparticles, eNOS down-regulation and reduced NO-mediated inhibition of platelet aggregation; – cell adhesion through up-regulation of ICAM-1; – proteolysis and fibrosis remodeling through MMP-2, 9 and TGF-β1 expression; – up-regulation of the local Ang II system through enhanced AT1 receptors (AT1R) and angiotensin-converting enzyme (ACE) expression. Losartan, an AT1 receptor antagonist, and Perindoprilat, an ACE inhibitor, prevented atrial endothelial cell senescence. Thus atrial endothelial senescence promotes thrombogenicity, inflammation, matrix remodeling and the up-regulation of the local Ang II system. They further suggest that targeting the Ang II/AT1R pathway may be a promising therapeutic strategy to delay atrial endothelial ageing.

Vascular Protection by Ethanol Extract of Morus alba Root Bark: Endothelium-Dependent Relaxation of Rat Aorta and Decrease of Smooth Muscle Cell Migration and Proliferation.

Morus alba (white mulberry) is native to the northern part of Korea and popularly used as a traditional medicine due to its numerous health benefits against human's disease. However, the possibility that M. alba may also affect the cardiovascular system remains unexplored. This study sought to investigate the vascular protective effects of the root bark extract of M. alba (MAE). Vascular reactivity was performed in organ baths using isolated rat thoracic aorta, while platelet derived growth factor (PDGF) induced proliferation and migration of vascular smooth muscle cells (VSMCs) were studied by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and wound healing assay, respectively. MAE evoked a concentration dependent vasorelaxation following endothelium-dependent pathway. However, vessel relaxations in response to MAE were markedly reduced after endothelium removal; treatment of endothelial nitric oxide synthase inhibitor, guanylyl cyclase inhibitor, and nonspecific potassium channel inhibitor, however, was not altered by cyclooxygenase inhibitor. Furthermore, MAE also significantly blunted contractile response to vasoconstrictor agent, phenylephrine. Taken together, the current evidence revealed that MAE is a potent endothelium-dependent vasodilator and this effect was involved in, at least in part, nitric oxide cyclic-guanosine monophosphate (NO-cGMP) pathway in combination with potassium (K+) channel activation. Moreover, MAE inhibited proliferation and migration of VSMCs induced by PDGF. Therefore, MAE could be a promising candidate of natural medicine for preventing and controlling cardiovascular diseases linked with endothelial dysfunction.

A Reliable Mouse Model of Hind limb Gangrene

Lack of a reliable hind limb gangrene animal model limits preclinical studies of gangrene, a severe form of critical limb ischemia. We develop a novel mouse hind limb gangrene model to facilitate translational studies. BALB/c, FVB, and C57BL/6 mice underwent femoral artery ligation (FAL) with or without administration of NG-nitro-L-arginine methyl ester (L-NAME), an endothelial nitric oxide synthase inhibitor. Gangrene was assessed using standardized ischemia scores ranging from 0 (no gangrene) to 12 (forefoot gangrene). Laser Doppler imaging (LDI) and DiI perfusion quantified hind limb reperfusion postoperatively. BALB/c develops gangrene with FAL-only (n=11/11, 100% gangrene incidence), showing mean limb ischemia score of 12 on postoperative days (PODs) 7 and 14 with LDI ranging from 0.08 to 0.12 on respective PODs. Most FVB did not develop gangrene with FAL-only (n=3/9, 33% gangrene incidence) but with FAL and L-NAME (n=9/9, 100% gangrene incidence). Mean limb ischemia scores for FVB undergoing FAL with L-NAME were significantly higher than for FVB receiving FAL-only. LDI score and capillary density by POD 28 were significantly lower in FVB undergoing FAL with L-NAME. C57BL/6 did not develop gangrene with FAL-only or FAL and L-NAME. Reproducible murine gangrene models may elucidate molecular mechanisms for gangrene development, facilitating therapeutic intervention.

Exercise facilitates early recognition of cardiac and vascular remodeling in chronic thromboembolic pulmonary hypertension in swine.

Chronic thromboembolic pulmonary hypertension (CTEPH) develops in 4% of patients after pulmonary embolism and is accompanied by an impaired exercise tolerance, which is ascribed to the increased right ventricular (RV) afterload in combination with a ventilation/perfusion (V/Q) mismatch in the lungs. The present study aimed to investigate changes in arterial Po2 and hemodynamics in response to graded treadmill exercise during development and progression of CTEPH in a novel swine model. Swine were chronically instrumented and received multiple pulmonary embolisms by 1) microsphere infusion (Spheres) over 5 wk, 2) endothelial dysfunction by administration of the endothelial nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester (L-NAME) for 7 wk, 3) combined pulmonary embolisms and endothelial dysfunction (L-NAME + Spheres), or 4) served as sham-operated controls (sham). After a 9 wk followup, embolization combined with endothelial dysfunction resulted in CTEPH, as evidenced by mean pulmonary artery pressures of 39.5 ± 5.1 vs. 19.1 ± 1.5 mmHg (Spheres, P < 0.001), 22.7 ± 2.0 mmHg (L-NAME, P < 0.001), and 20.1 ± 1.5 mmHg (sham, P < 0.001), and a decrease in arterial Po2 that was exacerbated during exercise, indicating V/Q mismatch. RV dysfunction was present after 5 wk of embolization, both at rest (trend toward increased RV end-systolic lumen area, P = 0.085, and decreased stroke volume index, P = 0.042) and during exercise (decreased stroke volume index vs. control, P = 0.040). With sustained pulmonary hypertension, RV hypertrophy (Fulton index P = 0.022) improved RV function at rest and during exercise, but this improvement was insufficient in CTEPH swine to result in an exercise-induced increase in cardiac index. In conclusion, embolization in combination with endothelial dysfunction results in CTEPH in swine. Exercise increased RV afterload, exacerbated the V/Q mismatch, and unmasked RV dysfunction. NEW & NOTEWORTHY Here, we present the first double-hit chronic thromboembolic pulmonary hypertension swine model. We show that embolization as well as endothelial dysfunction is required to induce sustained pulmonary hypertension, which is accompanied by altered exercise hemodynamics and an exacerbated ventilation/perfusion mismatch during exercise.

The Curcumin-Induced Vasorelaxation in Rat Superior Mesenteric Arteries

Curcumin (Cur) is a natural lipophilic polyphenol compound extracted from the rhizome of turmeric. Recently, protective effect of Cur on cardiovascular system is paid close attention. Some researches demonstrated that Cur could induce vascular relaxation in many arterial beds. However, relaxant effect of Cur on rat superior mesenteric artery is not clear. This present study will investigate the vasorelaxant effect of Cur on rat superior mesenteric arteries and the mechanisms involved. The isometric tension of rat superior mesenteric arterial rings was recorded by a sensitive myograph system invitro. The vasodilation of Cur at various concentrations (range: 10-8-10-4 M) on potassium chloride (KCl; 60mmol/L)-precontracted or phenylephrine hydrochloride (PE; 10μmol/L)-precontracted arterial rings were observed. We also observed vasorelaxant effect of Cur on KCl (60mM)-preconstricted rat superior mesenteric arterial rings after incubating the inhibitors of endothelial mechanism, including the endothelial nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester, the guanylate cyclase inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, and the cyclooxygenase inhibitor indomethacin, and inhibitors of potassium ion channel, including 4-aminopyridine (Voltage-sensitive K+ channel blockers), and tetraethylammonium chloride (Ca2+ activated K+ channel blockers), and BaCl2 (Inward rectifying K+ channel blockers), and glibenclamide (ATP -sensitive K+ channel blockers), respectively. The effects of Cur are expressed as percentage of relaxation from the precontraction induced by KCl (60mmol/L) or PE (10μmol/L). The Emax value refers to the maximum relaxation. The pD2 value refers to the negative logarithmic value of the drug concentration that produces 50% Emax. Cur concentration dependently relaxed the superior mesenteric artery rings with endothelium precontracted by PE (Emax=84.33±1.11 and pD2=5.03±0.02) or KCl (Emax=80.96±2.12% and pD2=4.32±0.01). The vasorelaxant effect of Cur on the superiormesenteric artery rings relied on endothelium partially. Indomethacin (5μM) significantlyinhibited the effect. However, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (10μM) and Nω-nitro-L-arginine methyl ester (100μM) had no effect on the action. In artery rings without endothelium, vasorelaxation induced by Cur was attenuated by 4-aminopyridine (100μM). However, barium chloride dehydrate (10μM), glibenclamide (10μM), and traethylammonium chloride (1mM) did not affect vasorelaxation induced by Cur. Moreover, Cur also significantly inhibited contraction induced by increasing external calcium in Ca2+-free medium plus K+ (60mM) and releasing intracellular Ca2+ in the Ca2+-free solution. Our results suggested that Cur induces relaxation in superior mesenteric arterial rings through an endothelium-dependent pathway, involving prostanoid, and also through an endothelium-independent pathway, opening K+ channel, and blockade of Ca2+ influx and intracellular Ca2+ release.

Involvement of atrial natriuretic peptide in abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart.

Nitric oxide (NO) is an effective mediator of ischemic preconditioning (IPC)-induced cardioprotection. Atrial natriuretic peptide (ANP) is downregulated after ovariectomy, which results in reduction in the level of NO. The present study deals with the investigation of the role of ANP in abrogated cardioprotective effect of IPC in the ovariectomized rat heart. Heart was isolated from ovariectomized rat and mounted on Langendorff's apparatus, subjected to 30 min of ischemia and 120 min of reperfusion. IPC was given by four cycles of 5 min of ischemia and 5 min of reperfusion with Krebs-Henseleit solution. The myocardial infract size was estimated employing triphenyltetrazolium chloride stain, and coronary effluent was analyzed for creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) release to consider the degree of myocardial injury. The cardiac release of NO was estimated by measuring the level of nitrite in coronary effluent. IPC-mediated cardioprotection was significantly attenuated in ovariectomized rat as compared to normal rat, which was restored by perfusion with ANP. However, this observed cardioprotection was significantly attenuated by perfusion with L-NAME, an endothelial nitric oxide synthase inhibitor, and Glibenclamide, a KATP channel blocker, alone or in combination noted in terms of increase in myocardial infract size, release of CK-MB and LDH, and also decrease in release of NO. Thus, it is suggested that ANP restores the attenuated cardioprotective effect of IPC in the ovariectomized rat heart which may be due to increase in the availability of NO and consequent increase activation of mitochondrial KATP channels.

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.

Insufficient sleep is associated with impaired nitric oxide-mediated endothelium-dependent vasodilation

Background and aimsHabitual short nightly sleep duration is associated with increased atherosclerotic cardiovascular disease risk and morbidity. Vascular endothelial dysfunction represents an important mechanism that may underlie this heightened cardiovascular risk. Impaired endothelium-dependent vasodilation, particularly NO-mediated vasodilation, contributes to the development and progression of atherosclerotic vascular disease and acute vascular events. We tested the hypothesis that chronic insufficient sleep is associated with impaired NO-mediated endothelium-dependent vasodilation in middle-aged adults. MethodsThirty adult men were studied: 15 with normal nightly sleep duration (age: 58 ± 2 y; sleep duration: 7.7 ± 0.2 h/night) and 15 with short nightly sleep duration (55 ± 2 y; 6.1 ± 0.2 h/night). Forearm blood flow (FBF) responses to intra-arterial infusion of acetylcholine, in the absence and presence of the endothelial NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA), as well as responses to sodium nitroprusside, were determined by strain-gauge venous occlusion plethysmography. ResultsThe FBF response to acetylcholine was lower (∼20%; p<0.05) in the short sleep duration group (from 4.6 ± 0.3 to 11.7 ± 1.0 ml/100 ml tissue/min) compared with normal sleep duration group (from 4.4 ± 0.3 to 14.5 ± 0.5 ml/100 ml tissue/min). L-NMMA significantly reduced the FBF response to acetylcholine in the normal sleep duration group (∼40%), but not the short sleep duration group. There were no group differences in the vasodilator response to sodium nitroprusside. ConclusionsThese data indicate that short nightly sleep duration is associated with endothelial-dependent vasodilator dysfunction due, in part, to diminished NO bioavailability. Impaired NO-mediated endothelium-dependent vasodilation may contribute to the increased cardiovascular risk with insufficient sleep.

Saponin extract from Panaxnotoginseng promotesangiogenesis through AMPK‑ and eNOS‑dependent pathways in HUVECs.

Panax notoginseng saponins (PNS) are among the most important compounds extracted from Panax notoginseng root, and have long been used in traditional Chinese medicine to control bleeding. PNS have recently garnered attention for the treatment of circulatory system diseases. The present study aimed to evaluate the effects of PNS on angiogenesis in vitro and to explore the molecular mechanisms underlying their actions. The present results demonstrated that the proliferative ability of human umbilical vein endothelial cells (HUVECs) was augmented following treatment with PNS. In addition, wound healing and Boyden chamber assays indicated that PNS may enhance HUVEC motility and increase the number of capillary-like tube branches in HUVECs. These effects were suppressed by 5′ adenosine monophosphate-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS) inhibitors. Furthermore, western blot analysis demonstrated that PNS stimulated the phosphorylation of AMPK and eNOS at Thr-172 and Ser-1179, respectively. These results suggested that PNS may promote tube formation in endothelial cells through AMPK- and eNOS-dependent signaling pathways.

Endothelial nitric oxide synthase inhibition triggers inflammatory responses in the brain of male rats exposed to ischemia-reperfusion injury.

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) plays a role in preserving and maintaining the brain's microcirculation, inhibiting platelet aggregation, leukocyte adhesion, and migration. Inhibition of eNOS activity results in exacerbation of neuronal injury after ischemia by triggering diverse cellular mechanisms, including inflammatory responses. To examine the relative contribution of eNOS in stroke-induced neuroinflammation, we analyzed the effects of systemic treatment with l-N-(1-iminoethyl)ornithine (L-NIO), a relatively selective eNOS inhibitor, on the expression of MiR-155-5p, a key mediator of innate immunity regulation and endothelial dysfunction, in the cortex of male rats subjected to transient middle cerebral artery occlusion (tMCAo) followed by 24 hr of reperfusion. Inducible NO synthase (iNOS) and interleukin-10 (IL-10) mRNA expression were evaluated by real-time polymerase chain reaction in cortical homogenates and in resident and infiltrating immune cells isolated from ischemic cortex. These latter cells were also analyzed for their expression of CD40, a marker of M1 polarization of microglia/macrophages.tMCAo produced a significant elevation of miR155-5p and iNOS expression in the ischemic cortex as compared with sham surgery. eNOS inhibition by L-NIO treatment further elevated the cortical expression of these inflammatory mediators, while not affecting IL-10 mRNA levels. Interestingly, modulation of iNOS occurred in resident and infiltrating immune cells of the ischemic hemisphere. Accordingly, L-NIO induced a significant increase in the percentage of CD40+ events in CD68+ microglia/macrophages of the ischemic cortex as compared with vehicle-injected animals. These findings demonstrate that inflammatory responses may underlie the detrimental effects due to pharmacological inhibition of eNOS in cerebral ischemia.

Abstract 623: Hyperhomocysteinemia Potentiates Inflammatory Monocyte Differentiation and Vascular Dysfunction in Type 2 Diabetic Mice

Hyperhomocysteinemia (HHcy) is associated with increased diabetic cardiovascular diseases. In this study, we investigated the effects of type 2 diabetes mellitus (T2DM), HHcy and their combination on inflammatory monocyte (MC) differentiation and vascular function. We established an atherosclerosis-susceptible T2DM+HHcy compound mouse model. T2DM was induced in db/db mice by spontaneous mutation (average blood glucose 493 mg/dL). HHcy was established by feeding the mice a high fat+high methionine diet (HF+HM, 8 weeks). Methionine is the homocysteine (Hcy) precursor, which elevated plasma Hcy level to 129 μM. Plasma Hcy level was lowered by vitamin therapy employing a HF+HM+high vitamin diet (HF+HM+HV), which reduced plasma Hcy levels from 129 μM to 42 μM. HHcy aggravated T2DM-impaired endothelial-dependent vessel relaxation to acetylcholine, which was completely abolished by endothelial nitric oxide synthase (eNOS) inhibitor N G -nitro-L-arginine methyl ester. HHcy potentiated T2DM-induced inflammatory monocytes (CD45 + CD11b + Ly6C + MCs) in aorta isolated from compound mice. Severe HHcy- and T2DM-induced mononuclear cells (MNCs), CD11b + MCs, CD11b + Ly6C middle+high inflammatory MCs and M1 macrophages (MØs) were potentiated by the combination of HHcy and T2DM in mouse bone marrow (BM), peripheral blood, and spleen. Folate based Hcy-lowering therapy (HF+HM+HV) reversed systemic MNC, MC, inflammatory MC and MØ and aortic inflammatory MC increases in T2DM mice. Finally, bone marrow transplantation (BMT) using lentivirus-shLy6C transduced BM cells, which decreased blood Ly6C + inflammatory MCs, resulted in ameliorated endothelial-dependent vessel relaxation. In conclusion, our data suggested that HHcy accelerated T2DM-induced systemic inflammatory MC and MØ differentiation, aortic inflammatory MC infiltration, and vascular dysfunction.