eNOS Ser1177 Phosphorylation Research Articles

Resveratrol rescues hyperglycemia-induced endothelial dysfunction via activation of Akt.

Resveratrol (RSV), a phytoalexin, has shown to prevent endothelial dysfunction and reduce diabetic vascular complications and the risk of cardiovascular diseases. The aim of this study was to investigate the signaling mechanisms underlying the protecting effects of RSV against endothelial dysfunction during hyperglycemia in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) were treated with RSV, and then exposed to high glucose (HG, 30 mmol/L). Akt-Ser473 phosphorylation, eNOS-Ser1177 phosphorylation, and PTEN protein levels in the cells were detected using Western blot. For in vivo studies, WT and Akt-/- mice were fed a normal diet containing RSV (400 mg·kg-1·d-1) for 2 weeks, then followed by injection of STZ to induce hyperglycemia (300 mg/dL). Endothelial function was evaluated using aortic rings by assessing ACh-induced vasorelaxation. RSV (5-20 μmol/L) dose-dependently increased Akt-Ser473 phosphorylation, accompanied by increased eNOS-Ser1177 phosphorylation in HUVECs; these effects were more prominent under HG stimulation. Transfection with Akt siRNA abolished RSV-enhanced eNOS phosphorylation and NO release. Furthermore, RSV (5-20 μmol/L) dose-dependently decreased the levels of PTEN, which was significantly increased under HG stimulation, and PTEN overexpression abolished RSV-stimulated Akt phosphorylation in HG-treated HUVECs. Moreover, RSV dramatically increased 26S proteasome activity, which induced degradation of PTEN. In in vivo studies, pretreatment with RSV significantly increased Akt and eNOS phosphorylation in aortic tissues and ACh-induced vasorelaxation, and improved diabetes-induced endothelial dysfunction in wild-type mice but not in Akt-/- mice. RSV attenuates endothelial function during hyperglycemia via activating proteasome-dependent degradation of PTEN, which increases Akt phosphorylation, and consequentially upregulation of eNOS-derived NO production.

Serine proteinases from Bothrops snake venom activates PI3K/Akt mediated angiogenesis

The discovery of rapid acting and powerful angiogenic proteins are of significant interest in the treatment of various human disorders associated with insufficient angiogenesis such as ischemia, menorrhagia and delayed wound healing. Snake venoms consist of a mixture of bioactive proteins and polypeptides and are rich sources of pharmacologically important molecules. Serine proteinases are one of the abundant proteins present in Bothrops snake venoms and possess multiple biological functions including the regulation of the blood coagulation cascade. In this study, serine proteinases from Bothrops atrox (B. atrox) and Bothrops brazili (B. brazili) that modulate angiogenesis were purified and characterized. Molecular size exclusion chromatography, affinity chromatography followed by ion exchange chromatography of the serine proteinases indicated molecular masses of around 32 kDa. Serine proteinases from both the species exhibited diverse catalytic activities such as the ability to induce amidolytic, fibrinogenolytic, gelatinolytic activities and also coagulated plasma with a minimal coagulation concentration of 2.4 μg/mL. Serine proteinases facilitated the sprouting of human umbilical vein endothelial cells (HUVEC) in three-dimensional culture systems and induced tubule formation in monolayer culture systems. Serine proteinase stimulated Aktser473 and eNOSser1177 phosphorylation in endothelial cells and addition of PI3K inhibitor LY294002 abrogated the effects of serine proteinases on sprout formation of endothelial cells in 3D collagen gels, suggesting that serine proteinase facilitated angiogenesis was mediated by PI3K/eNOS signaling axis. We also show in agarose plug assays using a mouse model, serine proteinases from Bothrops venoms significantly enhanced neovascularization. Our data suggests pro-angiogenic activity by the serine proteinases from B. atrox and B. brazili venom and further studies are warranted to explore the therapeutic applications.

Glycemic Control with Ipragliflozin, a Novel Selective SGLT2 Inhibitor, Ameliorated Endothelial Dysfunction in Streptozotocin-Induced Diabetic Mouse.

Endothelial dysfunction caused by increased oxidative stress is a critical initiator of macro- and micro-vascular disease development in diabetic patients. Ipragliflozin, a selective sodium-glucose cotransporter 2 (SGLT2) inhibitor, offers a novel approach for the treatment of diabetes by enhancing urinary glucose excretion. The aim of this study was to examine whether ipragliflozin attenuates endothelial dysfunction in diabetic mice. Eight-week-old male C57BL/6 mice were treated with streptozotocin (150 mg/kg) by a single intraperitoneal injection to induce diabetes mellitus. At 3 days of injection, ipragliflozin (3 mg/kg/day) was administered via gavage for 3 weeks. Vascular function was assessed by isometric tension recording. Human umbilical vein endothelial cells (HUVEC) were used for in vitro experiments. RNA and protein expression were examined by quantitative RT-PCR (qPCR) and western blot, respectively. Oxidative stress was determined by measuring urine 8-hydroxy-2'-deoxyguanosine (8-OHdG) level. Ipragliflozin administration significantly reduced blood glucose level (P < 0.001) and attenuated the impairment of endothelial function in diabetic mice, as determined by acetylcholine-dependent vasodilation (P < 0.001). Ipragliflozin did not alter metabolic parameters, such as body weight and food intake. Ipragliflozin administration ameliorated impaired phosphorylation of Akt and eNOSSer1177 in the abdominal aorta and reduced reactive oxygen species generation as determined by urinary excretion of 8-OHdG in diabetic mice. Furthermore, qPCR analyses demonstrated that ipragliflozin decreased the expression of inflammatory molecules [e.g., monocyte chemoattractant protein-1 (MCP-1) vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule (ICAM)-1] in the abdominal aorta (P < 0.05). In in vitro studies, incubation with methylglyoxal, one of the advanced glycation end products, significantly impaired phosphorylation of Akt and eNOSSer1177 (P < 0.01) and increased the expression of MCP-1, VCAM-1, and ICAM-1 in HUVEC. Ipragliflozin improved hyperglycemia and prevented the development of endothelial dysfunction under a hyperglycemic state, at least partially by attenuation of oxidative stress.

High glucose induces the expression of osteopontin in blood vessels in vitro and in vivo

Osteopontin (OPN) is involved in mineral metabolism and the inflammatory response while diabetes mellitus is associated with severe and extensive vascular calcification. Therefore, we speculated that OPN could be a key factor in the calcification and dysfunction of blood vessels exposed to high glucose. To identify the relationship between high glucose and OPN, we used high glucose medium to stimulate smooth muscle cells (SMCs) and vascular endothelial cells (VECs) in vitro and diabetic rats for in vivo analyses. As assessed by flow cytometry and western blots, SMC and VEC apoptosis levels increased with high glucose. Potassium and calcium uptake by cells were also increased with high glucose. These findings demonstrated the relationship between mineral metabolism and high glucose. Western blot and quantitative real time polymerase chain reaction analyses demonstrated that OPN increased in vitro with high glucose stimulation. The inflammatory factor ICAM1 and the inhibitory phosphorylation of endothelial nitric-oxide synthase (eNOS) (Thr495) were also upregulated by high glucose. In contrast, the anti-inflammatory factor Nrf2 and the activating phosphorylation of eNOS (Ser1177) were downregulated. Similar to the change of OPN, phosphorylated P38 was increased with high glucose. SB203580, an inhibitor of P38 phosphorylation, downregulated the expression of OPN and related inflammatory factors. Additionally, OPN was increased in the aortas and plasma of diabetic rats. In conclusion, our findings demonstrate that high glucose can induce the expression of OPN, which may be a key factor in the calcification and dysfunction of the vascular wall in diabetes.

Carbon monoxide releasing molecule induces endothelial nitric oxide synthase activation through a calcium and phosphatidylinositol 3-kinase/Akt mechanism

The production of nitric oxide (NO) by endothelial NO synthase (eNOS) plays a major role in maintaining vascular homeostasis. This study elucidated the potential role of carbon monoxide (CO)-releasing molecules (CORMs) in NO production and explored the underlying mechanisms in endothelial cells. We observed that 25μM CORM-2 could increase NO production and stimulate an increase in the intracellular Ca2+ level. Furthermore, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid caused CORM-2-induced NO production, which was abolished by 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetraacetoxy-methyl ester (BAPTA-AM), indicating that intracellular Ca2+ release plays a major role in eNOS activation. The inhibition of the IP3 receptor diminished the CORM-2-induced intracellular Ca2+ increase and NO production. Furthermore, CORM-2 induced eNOS Ser1179 phosphorylation and eNOS dimerization, but it did not alter eNOS expression. CORM-2 (25μM) also prolonged Akt phosphorylation, lasting for at least 12h. Pretreatment with phosphatidylinositol 3-kinase inhibitors (wortmannin or LY294002) inhibited the increases in NO production and phosphorylation but did not affect eNOS dimerization. CORM-2-induced eNOS Ser1179 phosphorylation was intracellularly calcium-dependent, because pretreatment with an intracellular Ca2+ chelator (BAPTA-AM) inhibited this process. Although CORM-2 increases intracellular reactive oxygen species (ROS), pretreatment with antioxidant enzyme catalase and N-acetyl-cysteine did not abolish the CORM-2-induced eNOS activity or phosphorylation, signifying that ROS is not involved in this activity. Hence, CORM-2 enhances eNOS activation through intracellular calcium release, Akt phosphorylation, and eNOS dimerization.

Abstract PR031

Background & Objectives: The physiological mechanisms that regulate tissue blood flow sufficiently are involved in the control of endothelium-dependent vasodilation. Nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), acts as an endothelium-dependent vasodilator. eNOS activity is regulated by several mechanisms including intracellular calcium concentration ([Ca2+]i) and phosphorylation of eNOS. Phosphorylation of Ser1177 increases Ca2+ sensitivity of the enzyme (1). Endothelium-dependent vasodilation is impaired by sevoflurane (2). The interaction of desflurane locally with the action of endothelial cells remains unknown. This study was carried out to elucidate the effects of desflurane and sevoflurane on endothelium-dependent vasodilation and the mechanisms of regulation of eNOS activity. Materials & Methods: With institutional approval, a rat descending aorta with the endothelium was suspended in a temperature-controlled organ bath for measurement of isometric tension. Changes in tension in response to phenylephrine (3×10–7 M) followed by acetylcholine (10–6 M) were recorded in the presence or absence of desflurane (5.7%, 11.4%; 1, 2 MAC for normal rats) or sevoflurane (2.4%, 4.8%; 1, 2 MAC for normal rats) to test endothelium-dependent vasodilation. Cultured bovine aortic endothelial cells were treated with bradykinin (10–6 M) in the presence or absence of desflurane or sevoflurane (11.4% and 4.8%, respectively). After treatments, Western blotting was used to determine eNOS Ser1177 phosphorylation, and measurement of the fluorescence intensity of Fluo-3/AM, as a [Ca2+]i indicator, was used to study the changes of [Ca2+]i. Statistical analysis was performed using Kruskal-Wallis’ H-test followed by Newman-Keuls test for multiple comparisons. A P value < 0.05 was considered statistically significant. Results: Endothelium-dependent vasodilation of the rat aorta and production of NO was inhibited by both anesthetics. The inhibitory effect of desflurane was greater than that of sevoflurane (Figure 1A, 1B). Western blotting revealed that phosphorylation of eNOS Ser1177 was inhibited by sevoflurane but not by desflurane (Figure 1C). Live cell calcium imaging revealed that bradykinin-induced increase in [Ca2+]i was suppressed by desflurane but not by sevoflurane (Figure 1D).Conclusion: The findings suggest that sevoflurane inhibits endothelium-dependent vasodilation caused by inhibition of eNOS Ser1177 phosphorylation, whereas desflurane inhibits vasodilation by inhibition of calcium signal transduction of eNOS activity. This difference in inhibitory mechanisms might reflect the different degrees of inhibitory effects of sevoflurane and desflurane on endothelium-dependent vasodilation.

Tert-butylhydroquinone lowers blood pressure in AngII-induced hypertension in mice via proteasome-PTEN-Akt-eNOS pathway.

Tert-butylhydroquinone (tBHQ), as an antioxidant, has been widely used for many years to prevent oxidization of food products. The aim of this study was to investigate whether tBHQ activates endothelial nitric oxide synthase (eNOS) to prevent endothelial dysfunction and lower blood pressure. The role of Akt in tBHQ-induced eNOS phosphorylation was examined in human umbilical vein endothelial cells (HUVEC) or in mice. tBHQ treatment of HUVEC increased both Akt-Ser473 phosphorylation, accompanied with increased eNOS-Ser1177 phosphorylation and NO release. Mechanically, pharmacologic or genetic inhibition of Akt abolished tBHQ-enhanced NO release and eNOS phosphorylation in HUVEC. Gain-function of PTEN or inhibition of 26S proteasome abolished tBHQ-enhanced Akt phosphorylation in HUVEC. Ex vivo analysis indicated that tBHQ improved Ach-induced endothelium-dependent relaxation in LPC-treated mice aortic arteries, which were abolished by inhibition of Akt or eNOS. In animal study, administration of tBHQ significantly increased eNOS-Ser1177 phosphorylation and acetylcholine-induced vasorelaxation, and lowered AngII-induced hypertension in wildtype mice, but not in mice deficient of Akt or eNOS. In conclusion, tBHQ via proteasome-dependent degradation of PTEN increases Akt phosphorylation, resulting in upregulation of eNOS-derived NO production and consequent improvement of endothelial function in vivo. In this way, tBHQ lowers blood pressure in hypertensive mice.

Acute and Chronic Biological Effects of Copeptin on Vascular Function in Mice and Humans

BackgroundCopeptin, the 39 amino acid C‐terminal segment of the precursor protein of arginine vasopressin (AVP), is secreted from the posterior pituitary in a 1:1 ratio with AVP in response to osmotic and hemodynamic stimuli. Copeptin is significantly elevated in pregnant women with preeclampsia, and predicts the development of preeclampsia as early as the 6th week of gestation. Preliminary data in a recent prospective study demonstrated that higher copeptin was associated with lower endothelial function (brachial artery flow‐mediated dilation, FMD) during the 2nd trimester in pregnant women at high risk of preeclampsia. However, whether copeptin itself has any independent biological effects on the vasculature is completely unknown.Methods and ResultsChronic incubation of mouse aortic rings with copeptin (n=4 male mice, 1nM, 24 hours) had no effect on endothelium‐ dependent (acetylcholine) or ‐independent (sodium nitroprusside) vasodilatory responses, indicating the lack of effect on endothelial or vascular smooth muscle function. Similarly, circulating copeptin was not associated with brachial artery FMD in non‐pregnant young women (n=11, r= −0.08, P=0.81), postmenopausal women (n=15, r=0.06, P=0.82) or men (n=24, r=0.10, P=0.62). In contrast, acute incubation of aortic rings (n=6 male mice) with copeptin demonstrated a dose response vasodilation beginning at 1nM up to a maximum relaxation at 100nM (43.6 ± 15.5%, p&lt;0.05, n=6). Co‐incubation with the nitric oxide (NO) inhibitor of L‐NG‐Nitroarginine methyl ester (L‐NAME) significantly attenuated the max vasodilatory response to 12.5 ± 0.6%, indicating that the acute vascular response to copeptin is largely NO‐mediated. Incubation of aorta with copeptin (60 min, 0.5nM) caused a significant increase in protein kinase B (AktSer473) phosphorylation (Western blot) vs. control tissue (0.63 ± 0.19 vs 1.27 ± 0.6, p&lt;0.04, n=5) and a trend towards increased phosphorylation of eNOSSer1177 (0.69 ± 0.02 vs 0.91 ± 0.21, p&lt;0.3, n=2).ConclusionsThese data indicate that acute exposure of aorta of mice to high copeptin results in marked NO‐mediated vasodilation. However, chronic exposure to high copeptin does not alter vascular endothelial or smooth muscle function in mice nor is circulating copeptin associated with endothelial function in males or non‐pregnant females. This suggests that adverse vascular effects associated with chronically elevated copeptin concentrations in preeclampsia are more likely a result of the pathophysiological consequences of high AVP rather than copeptin.Support or Funding InformationAPS STRIDE Undergraduate Summer Research Fellowship;AHA 15SFRN23730000;University of Iowa Center for Research by Undergraduates (ICRU); University of Iowa Center for Hypertension Research.

Uric acid enhances PKC-dependent eNOS phosphorylation and mediates cellular ER stress: A mechanism for uric acid-induced endothelial dysfunction.

The mechanism by which hyperuricemia induced-endothelial dysfunction contributes to cardiovascular diseases (CVDs) is not yet fully understood. In the present study, we used uric acid (UA) to trigger endothelial dysfunction in cultured endothelial cells, and investigated the effects of induced reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress induction, and the protein kinase C (PKC)-dependent endothelial nitric oxide synthase (eNOS) signaling pathway. Human umbilical vein endothelial cells (HUVECs) were incubated with 6, 9 or 12 mg/dl UA, ROS scavenger polyethylene glycol-superoxide dismutase (PEG-SOD), ER stress inhibitor 4-phenylbutyric acid (4-PBA), and PKC inhibitor polymyxin B for 6–48 h. Nitric oxide (NO) production, eNOS activity, intracellular ROS, ER stress levels, and the interaction between eNOS and calmodulin (CaM) and cytosolic calcium levels were assessed using fluorescence microscopy and western blot analysis. Apoptosis was assessed by annexin V staining. UA increased HUVEC apoptosis and reduced eNOS activity and NO production in a dose- and time-dependent manner. Intracellular ROS was elevated after 3 h, while ER stress level increased after 6 h. UA did not alter intracellular Ca2+, CaM, or eNOS concentration, or eNOS Ser1177 phosphorylation. However, PKC-dependent eNOS phosphorylation at Thr495 was greatly enhanced, and consequently interaction between eNOS and CaM was reduced. Cellular ROS depletion, ER stress inhibition and PKC activity reduction inhibited the effect of UA on eNOS activity, NO release and apoptosis in HUVECs. Thus, we concluded that UA induced HUVEC apoptosis and endothelial dysfunction by triggering oxidative and ER stress through PKC/eNOS-mediated eNOS activity and NO production.

Impact of Lifestyle Intervention on HDL-Induced eNOS Activation and Cholesterol Efflux Capacity in Obese Adolescent.

Background. Endothelial dysfunction occurs in obese children and adolescent and is regarded as a key step in the development of atherosclerosis. Important components for the development of endothelial dysfunction are reduced activity of endothelial nitric oxide synthase (eNOS) and an increase in cholesterol deposition in the vessel wall, due to reduced reverse cholesterol transport (RCT) activity. High density lipoprotein (HDL) exhibits antiatherosclerotic properties including modulation of eNOS activity and cholesterol efflux capacity. Lifestyle intervention programs can modify endothelial dysfunction in obese adolescents, but their impact on HDL-mediated eNOS activation and RCT is unknown so far. Methods. Obese adolescents (15 ± 1 years, BMI > 35 kg/m2) where randomized either to an intervention group (IG, n = 8; restricted diet and exercise) or to a usual care group (UC, n = 8). At the beginning and after 10 months of treatment HDL-mediated eNOS phosphorylation and cholesterol efflux capacity were evaluated. Results. Ten months of treatment resulted in a substantial weight loss (−31%), an improvement of endothelial function, and an increase in HDL-mediated eNOS-Ser1177 phosphorylation and RCT. A correlation between change in eNOS-Ser1177 phosphorylation or RCT and change in endothelial function was noted. Conclusion. A structured lifestyle intervention program improves antiatherosclerotic HDL functions, thereby positively influencing endothelial function.

Abstract 13192: Dipeptidyl Peptidase-4 Inhibitor, Linagliptin, Ameliorates Endothelial Dysfunction and Atherogenesis in Apolipoprotein-e Deficient Mice Through GLP-1 Dependent and Independent Manners

Background: The dipeptidyl peptidase-4 (DDP-4) inhibitors are a novel class of drug for the treatment of diabetes mellitus. Several studies suggested that their anti-inflammatory effects contribute to the prevention of cardiovascular complication of diabetes. This study aimed to investigate whether linagliptin, a xanthine-based DPP-4 inhibitor, attenuates endothelial dysfunction and prevents subsequent atherogenesis in non-diabetic apolipoprotein-E deficient (ApoE KO) mice. Methods and Results: Linagliptin (10 mg/kg/day) was administered orally to 8-week-old ApoE KO mice for 20 weeks. Linagliptin administration reduced atherosclerotic lesion progression in the aortic arch as determined by en-face Sudan IV staining compared with the control group (P&lt;0.05) without the alteration of metabolic parameters including blood glucose level. Plasma active GLP-1 was significantly higher in the treated group. Histological and quantitative RT-PCR analyses demonstrated that linagliptin decreased macrophages accumulation and the expression of inflammatory molecules (e.g; MCP-1 and VCAM-1) in atherosclerotic aorta (P&lt;0.05, respectively). Furthermore, linagliptin significantly attenuated the impairment of endothelial function in ApoE KO mice, as determined by acetylcholine-dependent vasodilation. These vasoprotective effects of linagliptin were associated with the decreased ROS generation as determined by urinary 8-OHdG level and the expression of NOX-2, in the abdominal aorta. Incubation of aortic segments from C57BL/6 mice with exendin-4 (Ex-4), a GLP-1 analogue, ameliorated palmitic acid (PA)-induced impairment of endothelium-dependent vasodilation. Ex-4 ameliorated impaired phosphorylation of Akt and eNOSSer1177 caused by PA in human umbilical vein endothelial cells (HUVECs). In addition, linagliptin directly attenuated PA-induced and eNOSSer1177 phosphorylation in HUVECs. Conclusion: Linagliptin inhibited the development of atherogenesis in non-diabetic ApoE KO mice. Our results suggested that linagliptin ameliorated endothelial dysfunction and oxidative stress through GLP-1 dependent and independent manners

Increased resting tension abolishes endothelial anti-contractile effect induced by phenylephrine in renal hypertensive rat aorta

The role of mechanical stress for vascular contraction is still unclear in hypertensive animals. This study aimed to evaluate the role of the rest tension of 1.5 g and 3.0 g on the endothelial nitric oxide synthase (eNOS) activity for contraction induced by the α1-adrenergic agonist phenylephrine (PE) in isolated intactendothelium aorta (E+) from renal hypertensive rat (2 K-1C) as compared to normotensive rat (2 K). Concentration-effect curves for PE were constructed in E+ under resting tension of 1.5 g or 3.0 g, in the absence or after incubation for 30 min with the NOS inhibitor (L-NAME 100 μM). The potency (pD2) and efficacy (ME) of PE were evaluated. eNOS residue Ser phosphorylation activates its enzyme. Phosphorylated Ser (P-Ser) expression was evaluated by Western Blot. This study was approved by the Ethics Committee of the University of Sao Paulo (156/2009). ME induced by PE on tension of 1.5 g was lower in 2 K-1C (1.2 ± 0.2 g, n = 7; p b 0.001) than in 2 K (2.2 ± 0.1 g; pD2:7.44 ± 0.03; n = 5), which was normalized by L-NAME. PE induced higher P-Ser-eNOS in 2 K-1C than in 2 K aorta. But under tension of 3.0 g, PE-induced contraction was similar in 2 K-1C and 2 K aorta. Therefore, these results indicate that the anti-contractile effect induced by PE in 2 K-1C E+ aorta under tension of 1.5 g is due to NO through eNOSSer phosphorylation that is abolished by the increased tension to 3.0 g. Supported by FAPESP, CNPq and CAPES.

Endothelium adjustments to acute resistance exercise are intensity-dependent in healthy animals.

We evaluated the acute effects of different intensities of resistance exercise over endothelium-dependent vasodilatation, eNOSser1177 phosphorylation level and endothelial production of NO in superior mesenteric artery of healthy rats. Groups: control (Ct), resistance exercise in the intensities of 30% (Ex30%), 50% (Ex50%) and 70% (Ex70%) of the maximal load established by the maximal repetition test (1RM). Exercise protocol: 15 sets of 10 repetitions. The rings of mesenteric artery were mounted in an isometric system or were prepared for further implementation of Western blot and DAF-FM techniques. The maximal response of the relaxation induced by insulin was not altered in the animals of the Ex30% group when compared to the Ct group. However, the animals of the Ex50% and Ex70% groups presented an increase in this response when compared to the Ct group. The eNOSser1177 phosphorylation levels showed an increase in Ex50% and Ex70% groups when compared to the Ct (1.6-fold and 3.3-fold, respectively). In the endothelial production of NO, it was observed that the Ex30% group did not show alteration in the NO production when compared to the Ct group. On the other hand, the animals exercised in the Ex50% and Ex70% groups showed increase in the NO synthesis when compared to the animals in the Ct group. Our results suggest that the magnitude of these vascular endothelium adjustments is strongly related to the increase of the resistance exercise intensity from the intensity of 50% of 1 RM.

CCN1 acutely increases nitric oxide production via integrin αvβ3–Akt–S6K–phosphorylation of endothelial nitric oxide synthase at the serine 1177 signaling axis

Although CCN1 (also known as cysteine-rich, angiogenic inducer 61, CYR61) has been reported to promote angiogenesis and neovascularization in endothelial cells (ECs), its effects on endothelial nitric oxide (NO) production have never been studied. Using human umbilical vein ECs, we investigated whether and how CCN1 regulates NO production. CCN1 acutely increased NO production in a time- and dose-dependent manner, which was accompanied by increased phosphorylation of endothelial NO synthase (eNOS) at serine 1177 (eNOS-Ser1177), but not that of eNOS-Thr495 or eNOS-Ser114. The level of total eNOS expression was unaltered. Treatment with either LY294002, a selective inhibitor of phosphoinositide 3-kinase known as an upstream kinase of Akt, or H-89, an inhibitor of protein kinase A, mitogen- and stress-activated protein kinase 1, Rho-associated protein kinase 2, and ribosomal protein S6 kinase (S6K), inhibited CCN1-stimulated eNOS-Ser1177 phosphorylation and subsequent NO production. Ectopic expression of small interfering RNA against Akt and S6K significantly inhibited the effects of CCN1. Consistently, CCN1 increased the phosphorylation of Akt-Ser473 and S6K-Thr389. However, CCN1 did not alter the expression or secretion of VEGF, a known downstream factor of CCN1 and a potential upstream factor of Akt-mediated eNOS-Ser1177 phosphorylation. Furthermore, neutralization of integrin αvβ3 with corresponding antibody completely reversed all of the observed effects of CCN1. Moreover, CCN1 increased acetylcholine-induced relaxation in the rat aortas. Finally, we also found that CCN1-stimulated eNOS-Ser1177 phosphorylation and NO production are true for other types of EC tested. In conclusion, CCN1 acutely increases NO production via activation of a signaling axis in integrin αvβ3–Akt–S6K–eNOS-Ser1177 phosphorylation, suggesting an important role for CCN1 in vasodilation.

Dipeptidyl peptidase-4 inhibitor, linagliptin, ameliorates endothelial dysfunction and atherogenesis in normoglycemic apolipoprotein-E deficient mice

BackgroundDipeptidyl peptidase-4 (DPP-4) inhibitors have vasoprotective effects. This study investigated whether a recently approved DPP-4 inhibitor, linagliptin (Lina), suppresses atherogenesis in non-diabetic apolipoprotein-E deficient (ApoE−/−) mice, and examined its effects on endothelial function. Methods and resultsLina (10mg/kg/day) was administered orally to ApoE−/− mice for 20weeks. Lina reduced atherogenesis without alteration of metabolic parameters including blood glucose level compared with control (P<0.05). Results of immunohistochemical analyses and quantitative RT-PCR demonstrated that Lina significantly decreased inflammatory molecule expression and macrophage infiltration in the atherosclerotic aorta. Lina administration to ApoE−/− mice for 9weeks ameliorated endothelium-dependent vasodilation compared with that in untreated mice. Plasma active glucagon-like peptide-1 (GLP-1) level was significantly higher in the treated group (P<0.05). Exendin-4 (Ex-4), a GLP-1 analog, ameliorated endothelium-dependent vasodilation impaired by palmitic acid (PA) in wild-type mouse aortic segments. Ex-4 promoted phosphorylation of eNOSSer1177 and Akt, both of which were abrogated by PA, in human umbilical vein endothelial cells. In addition, Lina administration to ApoE−/− mice decreased oxidative stress, as determined by urinary 8-OHdG secretion and NADPH oxidase subunit expression in the abdominal aorta. ConclusionLina inhibited atherogenesis in non-diabetic ApoE−/− mice. Amelioration of endothelial dysfunction associated with a reduction of oxidative stress by GLP-1 contributes to the atheroprotective effects of Lina.

Salidroside improves endothelial function and alleviates atherosclerosis by activating a mitochondria-related AMPK/PI3K/Akt/eNOS pathway

Salidroside (SAL) is a phenylpropanoid glycoside isolated from the medicinal plant Rhodiola rosea. A recent study has reported that SAL can efficiently decrease atherosclerotic plaque formation in low-density lipoprotein receptor-deficient mice. This study was to investigate the molecular mechanism of antiatherogenic effects of SAL. Given the importance of endothelial nitric oxide synthase (eNOS) in atherosclerosis, we sought to elucidate whether SAL could stimulate eNOS activation and also to explore its upstream signaling pathway. Six-week old apoE−/− male mice were fed a high-fat diet for 8weeks and then were administered with SAL for another 8weeks. SAL significantly improved endothelial function associated with increasing eNOS activation, thus reduced the atherosclerotic lesion area. SAL increased eNOS-Ser1177 phosphorylation and decreased eNOS-Thr495 phosphorylation, indicative of eNOS activation in endothelium. The aortic sinus lesions in SAL treated mice displayed reduced inflammation. SAL significantly activated AMP-activated protein kinase (AMPK). Both AMPK inhibitor and AMPK small interfering RNA (siRNA) abolished SAL-induced Akt-Ser473 and eNOS-Ser1177 phosphorylation. In contrast, LY294002, the PI3k/Akt pathway inhibitor, abolished SAL-induced phosphorylation and expression of eNOS. High performance liquid chromatography (HPLC) analysis revealed that SAL decreased cellular ATP content and increased the cellular AMP/ATP ratio, which was associated with the activation of AMPK. SAL was found to decrease the mitochondrial membrane potential (ΔΨm), which is a likely consequence of reduced ATP production. The action of SAL to reduce atherosclerotic lesion formation may at least be attributed to its effect on improving endothelial function by promoting nitric oxide (NO) production, which was associated with mitochondrial depolarization and subsequent activation of the AMPK/PI3K/Akt/eNOS pathway. Taken together, our data described the effects of SAL on mitochondria, which played critical roles in improving endothelial function in atherosclerosis.

Liposomal tetrahydrobiopterin preserves eNOS coupling in the post-ischemic heart conferring in vivo cardioprotection

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase (NOS), and reduced BH4 availability leads to endothelial NOS (eNOS) uncoupling and increased reactive oxygen species (ROS) generation. Questions remain regarding the functional state of eNOS and role of BH4 availability in the process of in vivo myocardial ischemia–reperfusion (I/R) injury. Rats were subjected to 60min of in vivo left coronary artery occlusion and varying periods of reperfusion with or without pre-ischemic liposomal BH4 supplementation (1mg/kg, iv). Myocardial infarction was correlated with cardiac BH4 content, eNOS protein level, NOS enzyme activity, and ROS generation. In the vehicle group, 60-min ischemia drastically reduced myocardial BH4 content in the area at risk (AAR) compared to non-ischemic (NI) area and the level remained lower during early reperfusion followed by recovery after 24-h reperfusion. Total eNOS, activated eNOS protein level (eNOS Ser1177 phosphorylation) and NOS activity were also significantly reduced during ischemia and/or early reperfusion, but recovered after 24-h reperfusion. With liposomal BH4 treatment, BH4 levels were identical in the AAR and NI area during ischemia and/or early reperfusion, and were significantly higher than with vehicle. BH4 pre-treatment preserved eNOS Ser1177 phosphorylation and NOS activity in the AAR, and significantly reduced myocardial ROS generation and infarction compared to vehicle. These findings provide direct evidence that in vivo I/R induces eNOS dysfunction secondary to BH4 depletion, and that pre-ischemic liposomal BH4 administration preserves eNOS function conferring cardioprotection with reduced oxidative stress.

Propofol ameliorates endothelial inflammation induced by hypoxia/reoxygenation in human umbilical vein endothelial cells: Role of phosphatase A2

Hypoxia/reoxygenation (H/R) induces endothelial inflammation with augmentation of endothelial adhesion molecules over-expression. Propofol was reported to attenuate endothelial adhesion molecule expression in some situations. Here, we examined the molecular mechanism for how propofol restored H/R-mediated up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells (HUVECs). Compared with the control group, H/R up-regulated expression of Pin-1 and PP2A, increased p66Shc–Ser36 phosphorylation, induced p66Shc mitochondrial translocation, O2− accumulation and NF-κB activation, and decreased eNOS–Ser1177 phosphorylation and nitric oxide (NO) production, thus up-regulating expression of endothelial adhesion molecules and increasing mononuclear-endothelial interaction. More importantly, except that propofol had no effect on H/R-induced p66Shc–Ser36 phosphorylation, most of H/R-mediated changes were alleviated by propofol, resulting in the reduction of endothelial adhesion molecules expression and mononuclear-endothelial adhesion. Moreover, we demonstrated the protective effect of propofol on H/R-induced endothelial inflammation was similar to that of calyculin A, an inhibitor of PP2A. In contrast, FTY720, an activator of PP2A, antagonized the effect of propofol. Our data indicated that propofol down-regulated PP2A expression, leading to reduced dephosphorylation of p66Shc–Ser36 and eNOS–Ser1177, which is associated with ROS accumulation and NO reduction, resulting in inhibition of endothelial adhesion molecule expression and mononuclear-endothelial interaction.

Novel role for retinol-binding protein 4 in the regulation of blood pressure.

Elevated levels of serum retinol-binding protein 4 (RBP4) contribute to insulin resistance and correlate with increased prevalence of hypertension and myocardial infarction. We sought to determine whether lowering RBP4 would improve blood pressure (BP) and protect against obesity- or angiotensin (Ang)-II-induced hypertension. Systolic and diastolic BP were lower in the RBP4-knockout (RBP4-KO) mice and higher in the RBP4-overexpressing (RBP4-Tg) mice compared with BP in the wild-type (WT) littermates. Carbachol-induced vasodilatation was increased in arteries from the RBP4-KO compared with the WT mice and was impaired in the RBP4-Tg mice. Aortic eNOS(Ser1177) phosphorylation was enhanced ∼50% in the RBP4-KO mice, with no change in total eNOS protein. Feeding a high-fat diet increased BP in the RBP4-KO mice only to the level in the WT mice fed chow and had no effect on aortic eNOS(Ser1177) phosphorylation. Ang-II infusion resulted in 22 mmHg lower systolic BP in the RBP4-KO than in the WT mice, although the relative BP increase over saline infusion was ∼30% in both. Ang-II treatment decreased aortic eNOS(Ser1177) phosphorylation in the WT and RBP4-KO mice, but phosphorylation remained higher in the RBP4-KO mice. Cardiac hypertrophy with Ang-II treatment was diminished by 56% in the RBP4-KO mice. Thus, elevated serum RBP4 raises BP and lack of RBP4 reduces it, with commensurate changes in aortic eNOS(Ser1177) phosphorylation. Lowering RBP4 may reduce BP through enhanced eNOS-mediated vasodilatation and may be a novel therapeutic approach for hypertension.

Roles of ROS and PKC-βII in ionizing radiation-induced eNOS activation in human vascular endothelial cells

Vascular endothelial cells can absorb higher radiation doses than any other tissue in the body, and post-radiation impaired endothelial nitric oxide synthase (eNOS) function may be developed as a potential contributor to the pathogenesis of vascular injury. In this study, we investigated early alterations of eNOS signaling in human umbilical venous endothelial cells (HUVECs) exposed to X-ray radiation. We found that ionizing radiation increased eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in HUVECs in a dose-dependent (≤20Gy) and time-dependent (6–72h) manner. The total expression levels of eNOS were unchanged by radiation. Although a transient but significant increase in extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation and a biphasic decline in Akt phosphorylation were observed after irradiation, these inhibitors were without effect on the radiation-induced changes in eNOS phosphorylation. There was an increase in protein kinase C-βII (PKC-βII) expression and the ablation of PKC-βII by small interfering RNA (siRNA) negated the radiation effect on the two eNOS phosphorylation events. Furthermore, when the radiation-induced increase in reactive oxygen species (ROS) generation was prevented by the anti-oxidant N-acetyl-l-cysteine, eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation in irradiated HUVECs were significantly reduced. However, transfection of PKC-β siRNA did not alter ROS production after irradiation, and NAC failed to block the radiation-induced increase in PKC-βII expression. Taken together, our results suggest that ionizing radiation-induced eNOS activation in human vascular endothelial cells is attributed to both the up-regulation of PKC-βII and the increase in ROS generation which were independent of each other.