eNOS Ser1177 Phosphorylation Research Articles

Fibrillin-1 deficiency perturbs aortic cholinergic relaxation and adrenergic contraction in a mouse model of early onset progressively severe Marfan syndrome.

The pathogenic role of nitric oxide (NO) signaling during development of thoracic aortic aneurysm (TAA) in Marfan syndrome (MFS) is currently unclear. We characterized vasomotor function and its relationship to the activity of the NO-generating enzymes in mice with early onset progressively severe MFS. Wire myography, immunoblotting, measurements of aortic NO and superoxide levels were used to compare vasomotor function, contractile-protein levels, and the activity of endothelial and inducible NO synthase (eNOS and iNOS, respectively) in ascending thoracic aortas of Fbn1mgR/mgR mice relative to wild type (WT) littermates. Isometric force measurements of aortic rings from 16-day-old male Fbn1mgR/mgR mice revealed a significant reduction in acetylcholine (ACh)-induced relaxation and increased phenylephrine (PE)-promoted contractility, associated with abnormally low eNOSSer1177 phosphorylation, decreased NO production and augmented superoxide levels. Greater aortic contractility was associated with α1-adrenoceptor upregulation and normal levels of contractile proteins. While iNOS inhibition had no effect on vasomotor functions, mutant aortic rings pre-incubated with a non-specific NOS inhibitor yielded a greater PE response, implying a significant contribution of endothelial dysfunction to aortic hypercontractility. Impaired eNOS signaling disrupts aortic cholinergic relaxation and adrenergic contraction in MFS mice with dissecting TAA.

LCZ696, an Angiotensin Receptor-Neprilysin Inhibitor, Ameliorates Endothelial Dysfunction in Diabetic C57BL/6 Mice.

LCZ696 (sacubitril/valsartan) exerts cardioprotective effects. Recent studies have suggested that it improves the endothelial function; however, the underlying mechanisms have not been thoroughly investigated. We investigated whether LCZ696 ameliorates diabetes-induced endothelial dysfunction. Diabetes was induced using streptozotocin in 8-week-old male C57BL/6 mice. Diabetic mice were randomly assigned to receive LCZ696 (100 mg/kg/day), valsartan (50 mg/kg/day), or a vehicle for three weeks. The endothelium-dependent and endothelium-independent vascular responses of the aortic segments were determined based on the response to acetylcholine and sodium nitroprusside, respectively. Human umbilical vein endothelial cells (HUVEC) and aortic segments obtained from C57BL/6 mice were used to perform in vitro and ex vivo experiments, respectively. LCZ696 and valsartan reduced the blood pressure in diabetic mice (P＜0.05). The administration of LCZ696 (P＜0.001) and valsartan (P＜0.01) ameliorated endothelium-dependent vascular relaxation, but not endothelium-independent vascular relaxation, under diabetic conditions. LCZ696, but not valsartan, increased eNOSSer1177 (P=0.06) and Akt (P＜0.05) phosphorylation in the aorta. In HUVEC, methylglyoxal (MGO), a major precursor of advanced glycation end products, decreased eNOSSer1177 phosphorylation (P＜0.05) and increased eNOSThr495 phosphorylation (P＜0.001). However, atrial natriuretic peptide (ANP) reversed these effects. ANP also ameliorated the MGO-induced impairment of endothelium-dependent vascular relaxation in the aortic segments (P＜0.05), although L-NAME completely blocked this effect (P＜0.001). LCZ696 ameliorated diabetes-induced endothelial dysfunction by increasing the bioavailability of ANP. Our findings suggest that LCZ696 has a vascular protective effect in a diabetic model and highlight that it may be more effective than valsartan.

Low density lipoprotein oxidized under lysosomal conditions decreases arterial vasodilatation

Endothelial dysfunction is a risk factor for atherosclerosis and includes impaired endothelium-dependent vasodilatation. We have shown previously that low density lipoprotein (LDL) can be oxidized by iron in the lysosomes of macrophages. Macrophage lysis in atherosclerotic lesions might expose endothelial cells to this oxidized LDL and adversely affect their function. LDL was oxidized by ferrous sulfate (5 µM) for 24 h at pH 4.5 at 37 °C. Aortas from male Wistar rats were cut into rings and subjected to wire myography for isometric tension recording. The rings were incubated with or without oxidized LDL (50 µg protein/ml) for one hour, constricted with 100 nM phenylephrine and relaxation to acetylcholine (1 nM − 3 µM) was measured. There was about 50% less relaxation in the presence of this oxidized LDL. Endothelial-independent vasodilatation induced by sodium nitroprusside was less affected by oxidized LDL. Oxidized LDL increased the formation of reactive oxygen species by the aortic rings and by cultured human aortic and dermal microvascular endothelial cells, which might have inactivated nitric oxide. Acetylcholine increased the activatory phosphorylation of eNOS (ser-1177), but oxidized LDL had little effect on this activation in cultured human aortic endothelial cells. These findings raise the possibility that LDL oxidized in lysosomes and released from lysed macrophages might decrease vasodilatation in atherosclerotic arteries.

Lipid Emulsions Inhibit Labetalol-Induced Vasodilation in the Isolated Rat Aorta.

Lipid emulsions are used as adjuvant drugs to alleviate intractable cardiovascular collapse induced by drug toxicity. We aimed to examine the effect of lipid emulsions on labetalol-induced vasodilation and the underlying mechanism in the isolated rat aorta. We studied the effects of endothelial denudation, NW-nitro-l-arginine methyl ester (l-NAME), calmidazolium, methylene blue, 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ), and lipid emulsions on labetalol-induced vasodilation. We also evaluated the effects of lipid emulsions on cyclic guanosine monophosphate (cGMP) formation, endothelial nitric oxide synthase (eNOS) phosphorylation, and endothelial calcium levels induced by labetalol. Labetalol-induced vasodilation was higher in endothelium-intact aortas than that in endothelium-denuded aortas. l-NAME, calmidazolium, methylene blue, and ODQ inhibited labetalol-induced vasodilation in endothelium-intact aortas. Lipid emulsions inhibited labetalol-induced vasodilation in endothelium-intact and endothelium-denuded aortas. l-NAME, ODQ, and lipid emulsions inhibited labetalol-induced cGMP formation in endothelium-intact aortas. Lipid emulsions reversed the stimulatory and inhibitory eNOS (Ser1177 and Thr495) phosphorylation induced by labetalol in human umbilical vein endothelial cells and inhibited the labetalol-induced endothelial calcium increase. Moreover, it decreased labetalol concentration. These results suggest that lipid emulsions inhibit vasodilation induced by toxic doses of labetalol, which is mediated by the inhibition of endothelial nitric oxide release and reduction of labetalol concentration.

Protein phosphatase 4 mediates palmitic acid-induced endothelial dysfunction by decreasing eNOS phosphorylation at serine 633 in HUVECs

Plasma saturated free fatty acid (FFA)-induced endothelial dysfunction (ED) contributes to the pathogenesis of atherosclerosis and cardiovascular diseases. However, the mechanism underlying saturated FFA-induced ED remains unclear. This study demonstrated that palmitic acid (PA) induced ED by activating the NADPH oxidase (NOX)/ROS signaling pathway to activate protein phosphatase 4 (PP4) and protein phosphatase 2A (PP2A), thereby reducing endothelial nitric oxide synthase (eNOS) phosphorylation at Ser633 and Ser1177, respectively. Okadaic acid (OA) and fostriecin (FST), which are inhibitors of PP2A, inhibited the PA-induced decreases in eNOS phosphorylation at Ser633 and Ser1177. The antioxidants N-acetylcysteine (NAC) and apocynin (APO) or knockdown of gp91phox or p67phox (NOX subunits) restored PA-mediated downregulation of PP4R2 protein expression and eNOS Ser633 phosphorylation. Knockdown of the PP4 catalytic subunit (PP4c) specifically increased eNOS Ser633 phosphorylation, while silencing the PP2A catalytic subunit (PP2Ac) restored only eNOS Ser1177 phosphorylation. Furthermore, PA dramatically decreased the protein expression of the PP4 regulatory subunit R2 (PP4R2) but not the other regulatory subunits. PP4R2 overexpression increased eNOS Ser633 phosphorylation, nitric oxide (NO) production, cell migration and tube formation but did not change eNOS Ser1177 phosphorylation levels. Coimmunoprecipitation (Co-IP) suggested that PP4R2 and PP4c interacted with the PP4R3α and eNOS proteins. In summary, PA decreases PP4R2 protein expression through the Nox/ROS pathway to activate PP4, which contributes to ED by dephosphorylating eNOS at Ser633. The results of this study suggest that PP4 is a novel therapeutic target for ED and ED-associated vascular diseases.

The Role of Ca2+/PI3K/Akt/eNOS/NO Pathway in Astragaloside IV-Induced Inhibition of Endothelial Inflammation Triggered by Angiotensin II.

Inflammation induced by angiotensin II (Ang II) is a key event in the progression of numerous cardiovascular diseases. Astragaloside IV (AS-IV), a glycoside extracted from Astragalus membranaceus Bunge, has been shown to inhibit Ang II-induced inflammatory responses in vivo. However, the mechanisms underlying the beneficial effects are still unclear. This study investigated whether AS-IV attenuates endothelial inflammation induced by Ang II via the activation of endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) pathway. Human umbilical vein endothelial cells (HUVECs) were cultured in the presence of AS-IV with or without the specific inhibitor of NOS or Ca2+- and phosphatidylinositol 3-kinase (PI3K)/Akt-dependent cascade prior to Ang II exposure. Incubation of HUVECs with AS-IV enhanced NO production and eNOSser1177 phosphorylation. These responses were abrogated by the inhibition of NOS or Ca2+- and PI3K/Akt-dependent pathway. In addition, preincubation of HUVECs with AS-IV inhibited Ang II-induced cytokine and chemokine production, adhesion molecule expression, monocyte adhesion, and nuclear factor kappa B (NF-κB) activation as evidenced by the attenuation of inhibitor of kappa B alpha phosphorylation and subsequent NF-κB DNA binding. These effects of AS-IV were abolished by the suppression of NOS or Ca2+- and PI3K/Akt-dependent cascade. Our findings indicate that AS-IV attenuates inflammatory responses triggered by Ang II possibly via the activation of Ca2+/PI3K/Akt/eNOS/NO pathway in endothelial cells.

Angelica gigas extract inhibits acetylation of eNOS via IRE1α sulfonation/RIDD-SIRT1-mediated posttranslational modification in vascular dysfunction.

Angelica gigas NAKAI (AG) is a popular traditional medicinal herb widely used to treat dyslipidemia owing to its antioxidant activity. Vascular disease is intimately linked to obesity-induced metabolic syndrome, and AG extract (AGE) shows beneficial effects on obesity-associated vascular dysfunction. However, the effectiveness of AGE against obesity and its underlying mechanisms have not yet been extensively investigated. In this study, 40 high fat diet (HFD) rats were supplemented with 100-300 mg/kg/day of AGE to determine its efficacy in regulating vascular dysfunction. The vascular relaxation responses to acetylcholine were impaired in HFD rats, while the administration of AGE restored the diminished relaxation pattern. Endothelial dysfunction, including increased plaque area, accumulated reactive oxygen species, and decreased nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) Ser1177 phosphorylation, were observed in HFD rats, whereas AGE reversed endothelial dysfunction and its associated biochemical signaling. Furthermore, AGE regulated endoplasmic reticulum (ER) stress and IRE1α sulfonation and its subsequent sirt1 RNA decay through controlling regulated IRE1α-dependent decay (RIDD) signaling, ultimately promoting NO bioavailability via the SIRT1-eNOS axis in aorta and endothelial cells. Independently, AGE enhanced AMPK phosphorylation, additionally stimulating SIRT1 and eNOS deacetylation and its associated NO bioavailability. Decursin, a prominent constituent of AGE, exhibited a similar effect in alleviating endothelial dysfunctions. These data suggest that AGE regulates dyslipidemia-associated vascular dysfunction by controlling ROS-associated ER stress responses, especially IRE1α-RIDD/sirt1 decay and the AMPK-SIRT1 axis.

A selective mineralocorticoid receptor blocker, esaxerenone, attenuates vascular dysfunction and inhibits atherogenesis in apolipoprotein e-deficient mice

Abstract Introduction Pharmacological blockade of mineralocorticoid receptors (MR) is a potential therapeutic approach to reduce cardiovascular complications because MR play a crucial role in cardiovascular regulation. Recent studies suggest that MR antagonists affect several extrarenal tissues, including vascular function. We investigated the effects of a novel non-steroidal selective MR blocker, esaxerenone (ESAX), recently approved for hypertension treatment in Japan, on vascular function and the development of atherosclerosis. Methods and results ESAX (3mg/kg/day) or vehicle was orally administered to western type diet-fed apolipoprotein E-deficient mice for 20 weeks or for 8 weeks to investigate the effects on atherogenesis or vascular dysfunction. Administration of ESAX for 20 weeks suppressed the development of atherosclerotic plaques in the aortic arch compared with the vehicle group (p<0.001) without alteration of blood pressure. In addition, ESAX reduced the expression of intercellular cell adhesion molecule-1 (p<0.05), macrophage infiltration (p<0.01), lipid deposition (p<0.05) and tended to decrease the expression of vascular cell adhesion molecule-1 as determined by immunohistochemical analysis. Moreover, phosphorylation of eNOSSer1177 (p<0.05) and Akt (p<0.05) were increased in the aorta of ESAX-treated mice. Administration of ESAX for 8 weeks attenuated western type diet-induced endothelial dysfunction (p<0.05) accompanied with the decrease in expression of ICAM1 (p<0.05) and phosphorylation of JNK (p<0.05) in the descending aorta. In in vitro experiment, aldosterone decreased the phosphorylation of eNOSSer1177 and increased the phosphorylation of eNOSThr495 in a dose dependent manner, which were attenuated by ESAX in HUVECs. In ex vivo vascular reactivity assay, ESAX diminished the aldosterone-induced endothelial dysfunction. Conclusion ESAX ameliorated hyperlipidemia-induced atherosclerosis and vascular dysfunction in mice, suggesting that ESAX is a promising therapeutic approach for cardiovascular complications.

Phosphorylated PKM2 regulates endothelium-dependent vasodilation in diabetes.

Endothelium-dependent vasodilation dysfunction is the pathological basis of diabetic macroangiopathy. The utilization and adaptation of endothelial cells to high glucose determine the functional status of endothelial cells. Glycolysis pathway is the major energy source for endothelial cells. Abnormal glycolysis plays an important role in endothelium-dependent vasodilation dysfunction induced by high glucose. Pyruvate kinase isozyme type M2 (PKM2) is one of key enzymes in glycolysis pathway, phosphorylation of PKM2 can reduce the activity of pyruvate kinase and affect the glycolysis process of glucose. TEPP-46 can stabilize PKM2 in its tetramer form, reducing its dimer formation and phosphorylation. Using TEPP-46 as a tool drug to inhibit PKM2 phosphorylation, this study aims to explore the impact and potential mechanism of phosphorylated PKM2 (p-PKM2) on endothelial dependent vasodilation function in high glucose, and to provide a theoretical basis for finding new intervention targets for diabetic macroangiopathy. The mice were divided into 3 groups: a wild-type (WT) group (a control group, C57BL/6 mice) and a db/db group (a diabetic group, db/db mice), which were treated with the sodium carboxymethyl cellulose solution (solvent) by gavage once a day, and a TEPP-46 group (a treatment group, db/db mice+TEPP-46), which was gavaged with TEPP-46 (30 mg/kg) and sodium carboxymethyl cellulose solution once a day. After 12 weeks of treatment, the levels of p-PKM2 and PKM2 protein in thoracic aortas, plasma nitric oxide (NO) level and endothelium-dependent vasodilation function of thoracic aortas were detected. High glucose (30 mmol/L) with or without TEPP-46 (10 μmol/L), mannitol incubating human umbilical vein endothelial cells (HUVECs) for 72 hours, respectively. The level of NO in supernatant, the content of NO in cells, and the levels of p-PKM2 and PKM2 protein were detected. Finally, the effect of TEPP-46 on endothelial nitric oxide synthase (eNOS) phosphorylation was detected at the cellular and animal levels. Compared with the control group, the levels of p-PKM2 in thoracic aortas of the diabetic group increased (P<0.05). The responsiveness of thoracic aortas in the diabetic group to acetylcholine (ACh) was 47% lower than that in the control group (P<0.05), and that in TEPP-46 treatment group was 28% higher than that in the diabetic group (P<0.05), while there was no statistically significant difference in the responsiveness of thoracic aortas to sodium nitroprusside (SNP). Compared with the control group, the plasma NO level of mice decreased in the diabetic group, while compared with the diabetic group, the phosphorylation of PKM2 in thoracic aortas decreased and the plasma NO level increased in the TEPP-46 group (both P<0.05). High glucose instead of mannitol induced the increase of PKM2 phosphorylation in HUVECs and reduced the level of NO in supernatant (both P<0.05). HUVECs incubated with TEPP-46 and high glucose reversed the reduction of NO production and secretion induced by high glucose while inhibiting PKM2 phosphorylation (both P<0.05). At the cellular and animal levels, TEPP-46 reversed the decrease of eNOS (ser1177) phosphorylation induced by high glucose (both P<0.05). p-PKM2 may be involved in the process of endothelium-dependent vasodilation dysfunction in Type 2 diabetes by inhibiting p-eNOS (ser1177)/NO pathway.

Lipid Emulsion Inhibits Amlodipine-Induced Nitric Oxide-Mediated Vasodilation in Isolated Rat Aorta.

This study aimed to examine the effect of lipid emulsion on the vasodilation induced by a toxic dose of amlodipine in isolated rat aorta and elucidate its mechanism, with a particular focus on nitric oxide. The effects of endothelial denudation, NW-nitro-L-arginvine methyl ester (L-NAME), methylene blue, lipid emulsion, and linolenic acid on the amlodipine-induced vasodilation and amlodipine-induced cyclic guanosine monophosphate (cGMP) production were examined. Furthermore, the effects of lipid emulsion, amlodipine, and PP2, either alone or combined, on endothelial nitric oxide synthase (eNOS), caveolin-1, and Src-kinase phosphorylation were examined. Amlodipine-induced vasodilation was higher in endothelium-intact aorta than in endothelium-denuded aorta. L-NAME, methylene blue, lipid emulsion, and linolenic acid inhibited amlodipine-induced vasodilation and amlodipine-induced cGMP production in the endothelium-intact aorta. Lipid emulsion reversed the increased stimulatory eNOS (Ser1177) phosphorylation and decreased inhibitory eNOS (Thr495) phosphorylation induced via amlodipine. PP2 inhibited stimulatory eNOS, caveolin-1, and Src-kinase phosphorylation induced via amlodipine. Lipid emulsion inhibited amlodipine-induced endothelial intracellular calcium increase. These results suggest that lipid emulsion attenuated the vasodilation induced via amlodipine through inhibiting nitric oxide release in isolated rat aorta, which seems to be mediated via reversal of stimulatory eNOS (Ser1177) phosphorylation and inhibitory eNOS (Thr495) dephosphorylation, which are also induced via amlodipine.

Shear stress facilitates Akt activation in human pulmonary microvascular endothelial cells

Introduction: Pulmonary arterial obstruction due to acute thromboembolism causes local reduction or cessation of blood flow. Under normal conditions, pulmonary blood flow exerts shear stress on endothelial cells, known to play an important role in maintaining vascular homeostasis. We hypothesized that pulmonary microvascular endothelial cells exposed to abrupt reductions in shear stress would demonstrate attenuated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and phosphorylation of downstream effectors Akt and endothelial nitric oxide synthase (eNOS). Methods: We tested our hypothesis in primary cultures of human lung microvascular endothelial cells (hLMVECs). We cultured early passage (p≤8) hLMVECs from 5 unique donor lots in standard 6-well culture plates and exposed cells to shear stress using an orbital shaker. Using a fixed depth (volume) and viscosity of tissue culture media, we adjusted the magnitude of shear stress exerted on hLMVECs by changing the angular velocity of orbital rotation. We acclimatized hLMVECs to physiologically-relevant shear stress (~12 dyn/cm 2 ) for 24 hours, then exposed cells to low shear stress (~3 dyn/cm 2 ) or no shear stress (0 dyn/cm 2 ) for 30 minutes. Control cells were maintained at 12 dyn/cm 2 of shear stress. In some experiments, PI3K activity was inhibited throughout the acclimatization and shear stress manipulation period by including LY294002 (10 μM) or vehicle (DMSO, 0.1% v/v) in culture media. We prepared whole cell lysates and used western blot to evaluate the expression and phosphorylation of Akt (ser473) and eNOS (ser1177). Results: We found that phosphorylation of Akt, but not Akt expression, decreased significantly (p&lt;0.05) when shear stress was acutely reduced (~3 dyn/cm 2 ) or removed (0 dyn/cm 2 ). Inhibition of PI3K activity with LY294002 further diminished shear-mediated changes in Akt phosphorylation in all 3 groups. Conversely, eNOS phosphorylation was not modified by acute reductions in shear stress, but eNOS expression was markedly attenuated by PI3K inhibition under all conditions. Conclusions: Our results indicate that phosphorylation of Akt, but not its downstream target eNOS, is rapidly attenuated by abrupt reductions in shear stress in hLMVECs. However, shear stress appears to regulate eNOS expression in a PI3K-dependent manner. Taken together, these findings suggest that acute disruption of flow, as seen in pulmonary embolism, may disrupt human lung microvascular endothelial cell homeostasis and nitric oxide bioavailability via multiple PI3K-dependent signaling pathways. This work is supported by HL126514 and HL159906. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Selective Mineralocorticoid Receptor Blocker, Esaxerenone, Attenuates Vascular Dysfunction in Diabetic C57BL/6 Mice.

Pharmacological blockade of mineralocorticoid receptors (MRs) is a potential therapeutic approach to reduce cardiovascular complications since MRs play a crucial role in cardiovascular regulation. Recent studies suggest that MR antagonists affect several extrarenal tissues, including vessel function. We investigated the effect of a novel nonsteroidal selective MR blocker, esaxerenone, on diabetes-induced vascular dysfunction. Diabetes was induced by a single dose of streptozotocin in 8-week-old male C57BL/6 mice. Esaxerenone (3 mg/kg/day) or a vehicle was administered by gavage to diabetic mice for 3 weeks. Metabolic parameters, plasma aldosterone levels, and parameters related to renal function were measured. Endothelium-dependent or -independent vascular responses of the aortic segments were analyzed with acetylcholine or sodium nitroprusside, respectively. Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study. Induction of diabetes elevated plasma aldosterone level (P＜0.05) and impaired endothelium-dependent vascular relaxation (P＜0.05). The administration of esaxerenone ameliorated the endothelial dysfunction (P＜0.01) without the alteration of metabolic parameters, blood pressure, and renal function. Esaxerenone improved the eNOSSer1177 phosphorylation in the aorta obtained from diabetic mice (P＜0.05) compared with that in the vehicle-treated group. Furthermore, a major MR agonist, aldosterone, decreased eNOSSer1177 phosphorylation and increased eNOSThr495 phosphorylation in HUVECs, which recovered with esaxerenone. Esaxerenone ameliorated the endothelium-dependent vascular relaxation caused by aldosterone in the aortic segments obtained from C57BL/6 mice (P＜0.001). Esaxerenone attenuates the development of diabetes-induced endothelial dysfunction in mice. These results suggest that esaxerenone has potential vascular protective effects in individuals with diabetes.

Theophylline-induced endothelium-dependent vasodilation is mediated by increased nitric oxide release and phosphodiesterase inhibition in rat aorta.

This study aimed to examine the endothelial dependence of vasodilation induced by the phosphodiesterase inhibitor theophylline in isolated rat thoracic aortas and elucidate the underlying mechanism, with emphasis on endothelial nitric oxide (NO). The effects of various inhibitors and endothelial denudation on theophylline-induced vasodilation, and the effect of theophylline on vasodilation induced by NO donor sodium nitroprusside, cyclic guanosine monophosphate (cGMP) analog bromo-cGMP, and β-agonist isoproterenol in endothelium-denuded aorta were examined. The effects of theophylline and sodium nitroprusside on cGMP formation were also examined. We examined the effect of theophylline on endothelial nitric oxide synthase (eNOS) phosphorylation and intracellular calcium levels. Theophylline-induced vasodilation was greater in endothelium-intact aortas than that in endothelium-denuded aortas. The NOS inhibitor, NW-nitro-L-arginine methyl ester; non-specific guanylate cyclase (GC) inhibitor, methylene blue; and NO-sensitive GC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one inhibited theophylline-induced vasodilation in endothelium-intact aortas. Theophylline increased the vasodilation induced by sodium nitroprusside, bromo-cGMP, and isoproterenol. Theophylline increased cGMP formation in endothelium-intact aortas, and sodium nitroprusside-induced cGMP formation in endothelium-denuded aortas. Moreover, theophylline increased stimulatory eNOS (Ser1177) phosphorylation and endothelial calcium levels, but decreased the phosphorylation of inhibitory eNOS (Thr495). These results suggested that theophylline-induced endothelium-dependent vasodilation was mediated by increased endothelial NO release and phosphodiesterase inhibition.

Variations in protein levels of the apelinergic system in adipose tissue of hypertensive individuals with class 3 obesity

The aim of this study was to investigate the expression of apelin (APLN) and its receptor (APLNR) in visceral adipose tissue (VAT), and its effect on the downstream expression of endothelial nitric oxide synthase (eNOS) in individuals with class 3 obesity, with or without hypertension. Seventy-five unrelated individuals presenting obesity class 3 with or without hypertension were included. Gene expression of APLN, and APLNR were analyzed in VAT, by reverse transcription quantitative polymerase chain reaction. The APLN, APLNR and eNOS (total and phosphorylated) levels in VAT were evaluated by Western blot. Analysis of differences between groups of APLN, APLNR and eNOS were performed by a logistic regression adjusting by confounding factors. Forty-five individuals with hypertension formed the case group, and 30 individuals constituted the control group. The APLN mRNA and protein levels were higher in the group of individuals with hypertension versus individuals without hypertension (p = 0.027 and p = 0.036, respectively). Meanwhile, APLNR mRNA and protein levels in subjects with hypertension were lower versus the group of subjects without hypertension (p = 0.001 and p = 0.008, respectively). Further, the group with hypertension presented a lower level of phosphorylation of eNOS Ser1177, compared to the control group (p = 0.002). In conclusion, individuals with class 3 obesity and hypertension present a modified APLN/APLNR expression in visceral adipose tissue, which could be secondary to reduced eNOS phosphorylation.

Association between atherogenic risk-modulating proteins and endothelium-dependent flow-mediated dilation in coronary artery disease patients

PurposeEndothelial dysfunction is an early and integral event in the development of atherosclerosis and coronary artery disease (CAD). Reduced NO bioavailability, oxidative stress, vasoconstriction, inflammation and senescence are all implicated in endothelial dysfunction. However, there are limited data examining associations between these pathways and direct in vivo bioassay measures of endothelial function in CAD patients. This study aimed to examine the relationships between in vivo measures of vascular function and the expression of atherogenic risk-modulating proteins in endothelial cells (ECs) isolated from the radial artery of CAD patients.MethodsFifty-six patients with established CAD underwent trans-radial catheterization. Prior to catheterization, radial artery vascular function was assessed using a) flow-mediated dilation (FMD), and b) exercise-induced dilation in response to handgrip (HE%). Freshly isolated ECs were obtained from the radial artery during catheterization and protein content of eNOS, NAD(P)H oxidase subunit NOX2, NFκB, ET-1 and the senescence markers p53, p21 and p16 were evaluated alongside nitrotyrosine abundance and eNOS Ser1177 phosphorylation.ResultsFMD was positively associated with eNOS Ser1177 phosphorylation (r = 0.290, P = 0.037), and protein content of p21 (r = 0.307, P = 0.027) and p16 (r = 0.426, P = 0.002). No associations were found between FMD and markers of oxidative stress, vasoconstriction or inflammation. In contrast to FMD, HE% was not associated with any of the EC proteins.ConclusionThese data revealed a difference in the regulation of endothelium-dependent vasodilation measured in vivo between patients with CAD compared to previously reported data in subjects without a clinical diagnosis, suggesting that eNOS Ser1177 phosphorylation may be the key to maintain vasodilation in CAD patients.

Icariside II induces rapid phosphorylation of endothelial nitric oxide synthase via multiple signaling pathways.

Icariside II, as a favonoid compound derived from epimedium, has been proved to involed in a variety of biological and pharmacological effects such as anti-inflammatory, anti-osteoporosis, anti-oxidation, anti-aging, and anti-cancer but its mechanism is unclear, especially in terms of its effect on post-transcriptional modification of endothelial nitric oxide synthase (eNOS). Phosphorylation of eNOS plays an important role in the synthesis of nitric oxide in endothelial cells, which is closely related to erectile dysfunction, atherosclerosis, Alzheimer’s disease, and other diseases. Our study aims to investigate the effect and mechanism of Icariside II on the rapid phosphorylation of eNOS. In this study, human umbilical vein endothelial cells (HUVECs) were stimulated with Icariside II in the presence or absence of multiple inhibitors (1 µM), including LY294002 (PI3K-inhibitor), MK-2206 (AKT-inhibitor), Bisindolylmaleimide X (AMPK-inhibitor), H-89 (CaMKII-inhibitor), KN-62 (PKA-inhibitor), Dorsomorphin (PKC-inhibitor). The proliferation of HUVECs was assessed using cell counting kit-8 (CCK-8). The release of nitric oxide (NO) within HUVECs was detected via fluorescence probe (DAF-FM). Western blot was used to examine the effect of Icariside II on the expression of eNOS, phosphorylation of eNOS, and common signaling pathways proteins. In this study, Icariside II was found to promote the cell proliferation and rapid NO release in HUVECs. The phosphorylation of eNOS-Ser1177 was significantly increased after Icariside II stimulation and reached a peak at 10 min (p < 0.05). Meanwhile, the phosphorylation of eNOS-Thr495 was significantly decreased after 45 min of stimulation (p < 0.05). Following the intervention with multiple inhibitors, it was found that MK-2206 (AKT inhibitor), LY294002 (PI3K inhibitor), KN-62 (AMPK inhibitor), and Bisindolylmaleimide X (PKC inhibitor) could significantly inhibit the phosphorylation of eNOS-Ser1177 caused by Icariside II (p < 0.05), while MK-2206, LY294002, and Bisindolylmaleimide X reversed the alleviated phosphorylation of eNOS-Thr495. We concluded that Icariside can regulate rapid phosphorylation of eNOS- Ser1177 and eNOS-Thr495 via multiple signaling pathways, resulting in the up-regulation of eNOS and the increased release of NO.

Inactivation of catalytic and non-catalytic PI3 kinase gamma functions enhances hypoxia-induced pulmonary hypertension in mice

Abstract Rationale Pulmonary hypertension (PH) is a pulmonary vascular disease characterised by chronically elevated pulmonary arterial mean pressure, increased pulmonary vascular resistance and right ventricular (RV) dysfunction and hypertrophy. The pathogenesis includes vasoconstriction of pulmonary vessels and pulmonary vascular remodelling. Phosphatidylinositol 3'-kinase γ (PI3Kγ) is highly expressed in leukocytes, endothelial cells (EC) and cardiomyocytes, which are involved in the pathogenesis of PH. The catalytic function of PI3Kγ has been shown to be associated with numerous processes that are potentially important for both vascular remodelling and maladaptive cardiac hypertrophy, including leukocyte recruitment and endothelial cell proliferation and survival. However, inhibition of non-catalytic PI3Kγ function results in reduced nitric oxide (NO) production by endothelial NO synthase (eNOS), which may lead to increased vascular resistance. Therefore, the aim of our study was to investigate the role of both catalytic and non-catalytic PI3Kγ functions in the pathogenesis of PH. Methods The importance of PI3Kγ in the pathogenesis of PH was analysed in vivo using the hypoxia-induced mouse model of PH (21 days at 10% O2 hypoxia (HOX)). PI3Kγ knockout mice (PI3Kγ−/−), as well as mice expressing a catalytically inactive form of PI3Kγ (PI3KγKD/KD) were investigated. To specifically inhibit the non-catalytic function of PI3Kγ, C57BL/6N mice were treated with a blocking peptide via intratracheal instillation and studied in the same model. Subsequently, systolic right ventricular pressure (RVSP) was measured using a Millar pressure catheter inserted via the jugular vein. In vitro, western blotting was used to investigate the phosphorylation of eNOS (Ser1177) in pulmonary ECs from wild type (WT), PI3Kγ−/− and PI3KγKD/KD mice. Results PI3Kγ−/− mice showed significantly increased RVSP after three weeks of hypoxia compared with WT controls (HOX WT 34.16±3.47mmHg vs. HOX PI3Kγ−/− 37.04±2.43mmHg; p=0.005; n≥7). A significant increase in RVSP was also detected in PI3KγKD/KD and blocking peptide-treated mice (HOX WT 34.67±2.02mmHg vs. HOX PI3KγKD/KD 37.95±1.39mmHg; p=0.023; n≥5 and HOX vehicle 34.19±2.74mmHg vs. HOX blocking peptide 37.61±2.46mmHg; p=0.011; n≥9). Heart rate, as well as systemic blood pressure, remained unchanged. Under normoxic conditions, no difference in RVSP between the groups could be measured. Interestingly, western blot detected reduced phosphorylation of eNOS after stimulation with fetal calf serum in PI3Kγ−/− ECs compared to WT and PI3KγKD/KD. Conclusion These results show that blunting of both catalytic and non-catalytic functions of PI3Kγ in vivo do not prevent the pathogenesis of PH, but conversely enhance hypoxia-induced elevation of RVSP. In this context, reduced phosphorylation of eNOS may play a crucial role, leading to increased vasoconstriction. Additional studies are required to evaluate the underlying mechanisms in detail. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): DFG Deutsche Forschungsgemeinschaft

Dual effect of amitriptyline in the control of vascular tone: Direct blockade of calcium channel in smooth muscle cells and reduction of TLR4-dependent NO production in endothelial cells

Background and purposeAmitriptyline (AM) is a classical and typical tricyclic antidepressant drug. Despite its well-known effects on the nervous system, it has been described to work as a TLR4 antagonist and several clinical works suggested some unexpected cardiovascular effects. The role of amitriptyline on vascular tone is not clear, thus we hypothesized that amitriptyline has a double effect on vascular tone by both endothelial TLR4-dependent nitric oxide down-regulation and calcium channel blockade in smooth muscle cells. Experimental approachChanges in isometric tension were recorded on a wire myograph. NO production was evaluated by fluorescence microscopy and flow cytometry in the mouse aorta and EAhy926 cells using DAF fluorescence intensity. Calcium influx was evaluated in A7r5 cells by flow cytometry. Western blot was used to analyze eNOS and nNOS phosphorylation. Key resultsAM reduced PE-induced contraction by calcium influx diminution in smooth muscle cells (F/F0 = 225.6 ± 15.9 and 118.6 ± 17.6 to CT and AM, respectively). AM impaired Ach-dependent vasodilation (Emax = 95.8 ± 1.4; 78.1 ± 1.8; 60.4 ± 2.9 and −7.4 ± 1.0 for CT, 0.01, 0,1 and 1 μmol/L AM, respectively) through reduction of calcium influx and NO availability and TLR4 antagonism in a concentration-dependent manner. AM or TLR4 gene deletion significantly reduced NO production (Fluorescence = 9503 ± 871.7, 2561 ± 282, 4771 ± 728 and 1029 ± 103 to CT, AM, TLR4−/− and AM + TLR4−/−, respectively) by an increase in nNOSser852 and reduction in eNOSser1177 phosphorylation in endothelial cells. Conclusions and implicationsOur data show that amitriptyline impaired vascular function through two different mechanisms: blockade of TLR4 in endothelial cells and consequent decrease in NO production and calcium influx reduction in smooth muscle and endothelial cells. We also suggest, for the first time, nNOS activity reduction by AM in non-neuronal cells.

Apelin-Induced Relaxation of Coronary Arteries Is Impaired in a Model of Second-Hand Cigarette Smoke Exposure.

Apelin, an endogenous ligand for APJ receptors, causes nitric oxide (NO)-dependent relaxation of coronary arteries. Little is known about the effects of apelin/APJ receptor signaling in the coronary circulation under pathological conditions. Here, we tested the hypothesis that the vasorelaxing effect of apelin is impaired by cigarette smoke extract (CSE), an established model for second-hand smoke exposure. Isolated rat coronary arteries were treated with 2% CSE for 4 hours. Apelin-induced relaxation of coronary arteries was abolished by CSE exposure, while relaxations to acetylcholine (ACh) (endothelium-dependent relaxation) and to diethyl amine NONOate (NO donor) were similar in control and CSE-treated arteries. Immunoblot analysis demonstrated that apelin increased eNOS ser1177 phosphorylation under control conditions but had no effect after exposure to CSE. Moreover, GRK2 expression was increased in CSE-exposed coronary endothelial cells. Pretreatment with CMPD101, a GRK2 inhibitor, improved the relaxation response to apelin in CSE-exposed coronary arteries. CSE treatment failed to inhibit relaxations evoked by CMF-019, an APJ receptor biased agonist that has little effect on GRK2. In arteries exposed to CSE, apelin impaired the response to ACh but not to diethyl amine NONOate. ACh-induced relaxation was unaffected by CMF-019 in either control or CSE-treated coronary arteries. The results suggest that APJ receptor signaling using the GRK2 pathway contributes to both loss of relaxation to apelin itself and the ability of apelin to inhibit endothelium-dependent relaxation to ACh in CSE-exposed coronary arteries, likely because of impaired production of NO from endothelial cells. These changes in apelin/APJ receptor signaling under pathological conditions (eg, exposure to second-hand smoke) could create an environment that favors increased vasomotor tone in coronary arteries.

IGFBP5 antisense and short hairpin RNA (shRNA) constructs improve erectile function by inducing cavernosum angiogenesis in diabetic mice.

The incidence of diabetic erectile dysfunction (ED) is rapidly increasing, and due to the severe angiopathy caused by diabetes, current drugs are ineffective at treating ED. Insulin-like growth factor-binding protein 5 (IGFBP5) promotes cell death and induces apoptosis in various cell types. To evaluate the effectiveness of IGFBP5 knockdown in improving erectile function in diabetic mice. Diabetes was induced by injecting streptozotocin (STZ) intraperitoneally into male 8-week-old C57BL/6 mice. Eight weeks after diabetes induction, mice were divided into four groups: a nondiabetic control group and three STZ-induced diabetic mice groups, which were administered intracavernous injections of phosphate buffered saline, scrambled control shRNA, or shRNA targeting mouse IGFBP5 (shIGFBP5) lentivirus particles. Two weeks later, we measured erectile function by electrically stimulating the bilateral cavernous nerve. To mimic diabetic angiopathy, primary cavernous endothelial cells (MCECs) from healthy mice were cultured and treated with glucose. IGFBP5 expression in MCECs or cavernous tissues were significantly increased under diabetic conditions, and knockdown of IGFBP5 induced MCECs angiogenic activity under high-glucose conditions. STZ-induced diabetic mice had reduced erectile function, but shIGFBP5 treatment resulted in significant improvements (to 90% of the nondiabetic control group level). Furthermore, in diabetic mice, numbers of cavernous endothelial cells, pericytes, and neuronal cells were increased by shIGFBP5 treatment, which also increased eNOS Ser1177 phosphorylation, decreased permeability and apoptosis of cavernous endothelial cells. In addition, IGFBP5 was found to mediate the AKT, ERK, p38 signaling pathways. Knockdown of IGFBP5 improved erectile function in diabetic mice by promoting cell proliferation and reducing apoptosis and permeability. Local inhibition of IGFBP5 expression may provide a new treatment strategy for diabetic ED and other ischemic vascular or neurological diseases.