Increased eNOS Phosphorylation Research Articles

Polyphenol-rich Aronia melanocarpa juice sustains eNOS activation through phosphorylation and expression via redox-sensitive pathways in endothelial cells.

A sustained formation of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is crucial to safeguard the vascular system against the development of cardiovascular diseases. This study investigated the prolonged phosphorylation and expression of eNOS induced by polyphenol-rich Aronia melanocarpa juice (AMJ), along with its underlying mechanisms. The findings revealed that AMJ triggered concentration- and time-dependent increases in eNOS phosphorylation and expression, leading to sustained NO production for 15h. Investigations with various enzymes and inhibitors revealed that the effect of AMJ was associated with redox sensitivity, activating the PI3-kinase/Akt, JNK, and p38 MAPK pathways. These pathways led to the inactivation of transcription factors FoxO1 and FoxO3a through phosphorylation, relieving their repression on eNOS expression. Therefore, the capability of AMJ to consistently trigger prolonged eNOS phosphorylation and expression via complex redox-sensitive pathways highlights its potential for maintaining vascular health and preventing cardiovascular diseases.

Subarachnoid hemorrhage-associated brain injury and neurobehavioral deficits are reversed with synthetic adropin treatment through sustained Ser1179 phosphorylation of endothelial nitric oxide synthase.

Subarachnoid hemorrhage (SAH) is a life-threatening vascular condition without satisfactory treatment options. The secreted peptide adropin is highly expressed in the human brain and has neuroprotective effects in brain injury models, including actions involving the cerebrovasculature. Here, we report an endothelial nitric oxide synthase (eNOS)-dependent effect of synthetic adropin treatment that reverses the deleterious effects of SAH. We tested the molecular, cellular, and physiological responses of cultured brain microvascular endothelial cells and two mouse models of SAH to treatment using synthetic adropin peptide or vehicle. SAH decreases adropin expression in cultured brain microvascular endothelial cells and in murine brain tissue. In two validated mouse SAH models, synthetic adropin reduced cerebral edema, preserved tight junction protein expression, and abolished microthrombosis at 1 day post-SAH. Adropin treatment also prevented delayed cerebral vasospasm, decreased neuronal apoptosis, and reduced sensorimotor deficits at seven days post-SAH. Delaying initial treatment of adropin until 24 h post-SAH preserved the beneficial effect of adropin in preventing vasospasm and sensorimotor deficits. Mechanistically, adropin treatment increased eNOS phosphorylation (Ser1179) at 1 & 7 days post-SAH. Treating eNOS-/- mice with adropin failed to prevent vasospasm or behavioral deficits, indicating a requirement of eNOS signaling. Adropin is an effective treatment for SAH, reducing cerebrovascular injury in both the acute (1 day) and delayed (7 days) phases. These findings establish the potential of adropin or adropin mimetics to improve outcomes following subarachnoid hemorrhage.

S-propargyl-cysteine promotes the stability of atherosclerotic plaque via maintaining vascular muscle contractile phenotype.

Introduction: Plaque rupture in atherosclerosis contributes to various acute cardiovascular events. As a new sulfide-containing donor, S-propargyl-cysteine (SPRC) has been reported to play a beneficial role in cardioprotection, potentially through its anti-inflammatory, anti-oxidative and anti-atherogenic activities. Our previous study observed an increase in eNOS phosphorylation in endothelial cells. However, it remains unclear whether SPRC influences vascular smooth muscle cells (VSMCs) within the plaque and if this effect contributes to plaque stabilization. Methods: An atherosclerotic unstable plaque mouse model was established by subjecting ApoE-/- mice to tandem stenosis of the right carotid artery along with a Western diet. Daily SPRC administration was conducted for 13weeks. Plaque morphology and stability were assessed using MRI scanning and histopathological staining. In our in vitro studies, we stimulated human artery vascular smooth muscle cells (HAVSMCs) with platelet-derived growth factor-BB (PDGF-BB), both with and without 100μM SPRC treatment. Cell phenotype was assessed using both Western blot and Real-time PCR. Cell proliferation was assessed using the BrdU cell proliferation kit and immunofluorescence of Ki-67, while cell migration was measured using scratch wound healing and transwell assay. MiR-143-3p overexpression and knockdown experiments were used to investigate whether it mediates the effect of SPRC on VSMC phenotype. Results and Discussion: SPRC treatment reduced plasma lipid levels, increased collagen content and decreased cell apoptosis in atherosclerotic plaques, indicating improved plaque stability. Both in vivo and in vitro studies elucidated the role of SPRC in preserving the contractile phenotype of VSMCs through up-regulation of miR-143-3p expression. Furthermore, SPRC suppressed the pro-proliferation and pro-migration effects of PDGF-BB on HAVSMCs. Overall, these findings suggest that the inhibitory effect of SPRC on phenotype switch from contractile to synthetic VSMCs may contribute to its beneficial role in enhancing plaque stability.

Endothelin-1 acutely increases nitric oxide production via the calcineurin mediated dephosphorylation of Caveolin-1

Endothelin (ET)-1 is an endothelial-derived peptide that exerts biphasic effects on nitric oxide (NO) levels in endothelial cells such that acute exposure stimulates-while sustained exposure attenuates-NO production. Although the mechanism involved in the decrease in NO generation has been identified but the signaling involved in the acute increase in NO is still unresolved. This was the focus of this study. Our data indicate that exposing pulmonary arterial endothelial cells (PAEC) to ET-1 led to an increase in NO for up to 30min after which levels declined. These effects were attenuated by ET receptor antagonists. The increase in NO correlated with significant increases in pp60Src activity and increases in eNOS phosphorylation at Tyr83 and Ser1177. The ET-1 mediated increase in phosphorylation and NO generation were attenuated by the over-expression of a pp60Src dominant negative mutant. The increase in pp60Src activity correlated with a reduction in the interaction of Caveolin-1 with pp60Src and the calcineurin-mediated dephosphorylation of caveolin-1 at three previously unidentified sites: Thr91, Thr93, and Thr95. The calcineurin inhibitor, Tacrolimus, attenuated the acute increase in pp60Src activity induced by ET-1 and a calcineurin siRNA attenuated the ET-1 mediated increase in eNOS phosphorylation at Tyr83 and Ser1177 as well as the increase in NO. By using a Caveolin-1 celluSpot peptide array, we identified a peptide targeting a sequence located between aa 41–56 as the pp60Src binding region. This peptide fused to the TAT sequence was found to decrease caveolin-pp60Src interaction, increased pp60Src activity, increased eNOS pSer1177 and NO levels in PAEC and induce vasodilation in isolated aortic rings in wildtype but not eNOS knockout mice. Together, our data identify a novel mechanism by which ET-1 acutely increases NO via a calcineurin-mediated dephosphorylation of caveolin-1 and the subsequent stimulation of pp60Src activity, leading to increases in phosphorylation of eNOS at Tyr83 and Ser1177.

LncRNA HOXA11-AS promotes vascular endothelial cell injury in atherosclerosis by regulating the miR-515-5p/ROCK1 axis.

AimsLong non‐coding RNA HOXA11‐AS participated in heart disease. In this study, we aim to evaluate the potential roles of HOXA11‐AS in atherosclerosis and its underlying mechanisms.Methods and resultsThe expression levels of HOXA11‐AS in ox‐LDL‐treated HUVECs and arch tissues of high‐fat diet‐fed ApoE−/− mice (n = 10) were assessed by qRT‐PCR. The effects of HOXA11‐AS knockdown on the development of atherosclerosis were evaluated using in vitro and in vivo models. Luciferase reporter and RNA immunoprecipitation (RIP) assays verified the potential relationships between HOXA11‐AS or ROCK1 and miR‐515‐5p. The interactive roles between HOXA11‐AS and miR‐515‐5p and between miR‐515‐5p and ROCK1 were further characterized in ox‐LDL‐treated HUVECs. Our data showed that HOXA11‐AS was significantly up‐regulated (P < 0.001), whereas miR‐515‐5p was dramatically down‐regulated in AS mice tissues (P < 0.001) and ox‐LDL‐treated HUVECs (P < 0.01). Ox‐LDL could induce endothelial injuries by inhibiting cell proliferation (P < 0.001) and SOD synthesis (P < 0.001), promoting apoptosis (P < 0.01), ROS (P < 0.001), and MDA production (P < 0.001), increasing Bax (P < 0.001) and cleaved Caspase‐3 (P < 0.001), and decreasing Bcl‐2 (P < 0.001) and phosphorylated eNOS (P < 0.01). HOXA11‐AS knockdown attenuated endothelial injuries via increasing eNOS phosphorylation. Luciferase assay and RIP results confirmed that miR‐515‐5p is directly bound to HOXA11‐AS and ROCK1. HOXA11‐AS promoted ox‐LDL‐induced HUVECs injury by directly inhibiting miR‐515‐5p from increasing ROCK1 expression and subsequently decreasing the expression and phosphorylation of eNOS. MiR‐515‐5p mimics could partially reverse the effects of HOXA11‐AS knockdown.ConclusionsHOXA11‐AS contributed to atherosclerotic injuries by directly regulating the miR‐515‐5p/ROCK1 axis. This study provided new evidence that HOXA11‐AS might be a candidate for atherosclerosis therapy.

Activation of APJ Receptors by CMF‐019, but not Apelin, Causes Endothelium‐Dependent Relaxation of Spontaneously Hypertensive Rat Coronary Arteries

Hypertension is a major risk factor for coronary artery disease, but the pathophysiological mechanisms linking these diseases are not completely understood. Apelin is a novel vasoactive adipokine. Receptors for apelin (APJ receptors) are G‐protein‐coupled receptors and are widely expressed throughout the cardiovascular system. APJ receptors can also signal via G‐protein‐independent pathways, including G‐protein‐coupled‐receptor kinase 2 (GRK2), which inhibits nitric oxide synthase (eNOS) activity and nitric oxide (NO) production in endothelial cells. Apelin causes endothelium‐dependent, NO‐mediated relaxation of coronary arteries from normotensive animals, but the effects of activating APJ receptor signaling pathways in hypertensive coronary arteries are unknown. Since endothelium‐dependent relaxations are often blunted in arteries from hypertensive animals, we tested the hypothesis that apelin‐induced relaxation is impaired in coronary arteries from spontaneously hypertensive rats (SHR). Moreover, we hypothesized that endothelium‐dependent relaxations in response to CMF‐019, an APJ receptor G‐protein biased agonist with little effect on GRK2, would be unaffected in SHR coronary arteries. Western blot and RT‐qPCR analysis demonstrated that APJ receptor protein and mRNA expression did not differ between normotensive WKY (Wistar‐Kyoto) and SHR coronary arteries. eNOS protein expression was also similar between SHR and WKY coronary arteries, but GRK2 expression was significantly increased in SHR coronary endothelial cells, as compared to WKY. In myograph studies, apelin (10‐9‐10‐6 M) caused endothelium‐dependent relaxation of isolated WKY coronary arterial rings (pD2= 7.00 ± 0.11; Emax= 54 ± 4% relaxation; n=6), but had no effect in arteries from SHR. By contrast, CMF‐019 (10‐9‐10‐6 M)‐induced relaxation did not differ between normotensive and hypertensive coronary arteries. The relaxation response to CMF‐019 was abolished by the APJ receptor antagonist, F13A (10‐7 M), by removal of the endothelium, and by inhibition of eNOS with nitro‐ L‐arginine (3 x 10‐5 M). Moreover, the GRK2 inhibitor, CMPD 101 (3 x 10‐5 M), partially restored the relaxation response to apelin in SHR coronary arteries (pD2= 6.56 ± 0.22; Emax= 46 ± 6% relaxation; n=6). Immunoblot analysis of SHR coronary endothelial cells demonstrated that CMF‐019 (10‐7 M) significantly increased eNOS phosphorylation whereas apelin (10‐7 M) had no effect. We conclude that apelin failed to cause relaxation in hypertensive coronary arteries, likely due to impaired eNOS activity resulting from APJ receptor signaling via the GRK2 pathway and decreased eNOS phosphorylation. APJ receptor activation by the biased agonist, CMF‐019, caused similar endothelium‐dependent relaxations in WKY and SHR coronary arteries. Thus, APJ receptor‐biased agonists, such as CMF‐019, may be more effective than apelin in causing vasodilation of hypertensive coronary arteries.

PFKFB3 Mediated Changes in ROS/NO Balance Contribute to Endothelial Dysfunction in Obesity

Obesity is a strong risk factor for cardiovascular disease, in large part due to an altered metabolic state. One of the earliest consequences of cardiovascular dysfunction in obesity is the loss of endothelial function and impaired nitric oxide (NO) signaling. In blood vessels, NO is synthesized by endothelial nitric oxide synthase (eNOS). The biological actions of NO can be compromised by rapid inactivation by reactive oxygen species (ROS) such as superoxide that is mainly generated by NADPH oxidases (NOXs). However, the signals coordinating the increased superoxide production leading to loss of NO with altered metabolism remain unknown.We have found that the expression of 6‐phosphofructo‐2‐kinase/fructose‐2, 6‐biphosphatase 3 (PFKFB3), which is a key glycolytic regulatory enzyme, is significantly increased in endothelial cells of obese animals. Overexpression of PFKFB3 within the endothelium of blood vessels is accompanied by the loss of vascular relaxation and in decreased NO production as a result of increased eNOS phosphorylation on the inhibitory T495 residue. PFKFB3 overexpression also blunted Akt‐S473 phosphorylation, reducing stimulus‐dependent phosphorylation and activation of eNOS at S1177. While PFKFB3 exerted negative effects on NO signaling, it increased the activity and mRNA levels of NOX1, a major contributor to endothelial dysfunction. Further, we have also found that pharmacological inhibition of NOX1 attenuates the decreased vasodilator responses in PFKFB3 overexpressing aortic rings.These results demonstrate a novel functional relationship between endothelial metabolism, ROS and NO production that may contribute to endothelial dysfunction in obesity.

Fustin, a Flavanonol, Synergically Potentiates the Anticancer Effect of Green Tea Catechin Epigallocatechin-3-O-Gallate with Activation of the eNOS/cGMP Axis.

Epigallocatechin-3-O-gallate (EGCG), a catechin present in green tea, selectively elicits apoptosis in multiple myeloma cells by activating the endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) axis. However, the effects of EGCG alone are limited. Herein, we revealed that fustin, a flavanonol, enhances the EGCG-elicited activation of the cGMP/eNOS axis in multiple myeloma cells. Isobologram analysis demonstrated that EGCG/fustin synergistically elicited cell death in multiple myeloma cells. Importantly, this chemical combination significantly promoted cell death without affecting the normal cells. To assess the effects of EGCG and fustin in vivo, female BALB/c mice were inoculated with multiple myeloma MPC11 cells and then treated with each compound. The combination of EGCG/fustin suppressed tumor growth in vivo without affecting alanine aminotransferase/aspartate aminotransferase levels, the dose-limiting toxicity of EGCG. Consistent with in vitro findings, this combination increased eNOS phosphorylation at Ser1177 in the tumor. Collectively, fustin amplified EGCG-induced activation of the eNOS/cGMP axis.

Anti-VEGF Effect of Bioactive Indolic Compounds and Hydroxytyrosol Metabolites.

Angiogenesis is a key process involved in both cancer and cardiovascular diseases, the vascular endothelial growth factor (VEGF) and its VEGF receptor-2 (VEGFR-2) being the main triggers. The aim of this study was to determine the molecular mechanism underlying the potent inhibition of VEGF signaling by hydroxytyrosol (HT) metabolites and indolic compounds and establish a relation between their structure and bioactivity. Experiments involved the evaluation of their potential to inhibit VEGF on human umbilical vein endothelial cells (HUVECs) by ELISA assay and their subsequent effect on the downstream signaling pathway (PLCγ1, Akt, and endothelial nitric oxide synthetase (eNOS)) by Western blot. Respectively, 3,4-dihydroxyphenylacetaldehyde (DOPAL) (100 µM) and indole pyruvic acid (IPy) (1 mM) were capable of inhibiting VEGFR-2 activation with an IC50 value of 119 µM and 1.037 mM. The anti-angiogenic effect of DOPAL and IPy is mediated via PLCγ1. Additionally, DOPAL significantly increases eNOS phosphorylation, while IPy maintained it. These data provide for the first time evidence of the anti-angiogenic effect of DOPAL and IPy for future use as potential bioactive food ingredients.

Effect of 3-caffeoyl, 4-dihydrocaffeoylquinic acid from Salicornia herbacea on endothelial nitric oxide synthase activation via calcium signaling pathway.

3-Caffeoyl-4-dicaffeoylquinic acid (CDCQ) is a natural chlorogenic acid isolated from Salicornia herbacea that protects against oxidative stress, inflammation, and cancer. Nitric oxide (NO) plays a physiologically beneficial role in the cardiovascular system, including vasodilation, protection of endothelial cell function, and anti-inflammation. However, the effect of CDCQ on NO production and eNOS phosphorylation in endothelial cells is unclear. We investigated the effect of CDCQ on eNOS phosphorylation and NO production in human endothelial cells, and the underlying signaling pathway. CDCQ significantly increased NO production and the phosphorylation of eNOS at Ser1177. Additionally, CDCQ induced phosphorylation of PKA, CaMKII, CaMKKβ, and AMPK. Interestingly, CDCQ increased the intracellular Ca2+ level, and L-type Ca2+ channel (LTCC) blockade significantly attenuated CDCQ-induced eNOS activity and NO production by inhibiting PKA, CaMKII, CaMKKβ, and AMPK phosphorylation. These results suggest that CDCQ increased eNOS phosphorylation and NO production by Ca2+-dependent phosphorylation of PKA, CaMKII, CaMKKβ, and AMPK. Our findings provide evidence that CDCQ plays a pivotal role in the activity of eNOS and NO production, which is involved in the protection of endothelial dysfunction.

Beyond Thermal Sensation: Roles of Transient Receptor Potential Vanilloid Subfamily Member 1 and Spicy Food in Cardiometabolic Diseases

Beyond Thermal Sensation: Roles of Transient Receptor Potential Vanilloid Subfamily Member 1 and Spicy Food in Cardiometabolic Diseases

Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity.

[Figure: see text].

Rutaecarpine Increases Nitric Oxide Synthesis via eNOS Phosphorylation by TRPV1-Dependent CaMKII and CaMKKβ/AMPK Signaling Pathway in Human Endothelial Cells.

Rutaecarpine (RUT) is a bioactive alkaloid isolated from the fruit of Evodia rutaecarpa that exerts a cellular protective effect. However, its protective effects on endothelial cells and its mechanism of action are still unclear. In this study, we demonstrated the effects of RUT on nitric oxide (NO) synthesis via endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells and the underlying molecular mechanisms. RUT treatment promoted NO generation by increasing eNOS phosphorylation. Additionally, RUT induced an increase in intracellular Ca2+ concentration and phosphorylation of Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and Ca2+/calmodulin-dependent kinase II (CaMKII). Inhibition of transient receptor potential vanilloid type 1 (TRPV1) attenuated RUT-induced intracellular Ca2+ concentration and phosphorylation of CaMKII, CaMKKβ, AMPK, and eNOS. Treatment with KN-62 (a CaMKII inhibitor), Compound C (an AMPK inhibitor), and STO-609 (a CaMKKβ inhibitor) suppressed RUT-induced eNOS phosphorylation and NO generation. Interestingly, RUT attenuated the expression of ICAM-1 and VCAM-1 induced by TNF-α and inhibited the inflammation-related NF-κB signaling pathway. Taken together, these results suggest that RUT promotes NO synthesis and eNOS phosphorylation via the Ca2+/CaMKII and CaM/CaMKKβ/AMPK signaling pathways through TRPV1. These findings provide evidence that RUT prevents endothelial dysfunction and benefit cardiovascular health.

Astragaloside IV prevents endothelial dysfunction by improving oxidative stress in streptozotocin‑induced diabetic mouse aortas

Oxidative stress serves a role in endothelial dysfunction exhibited by patients with diabetes mellitus. Astragaloside IV (AS-IV) is a major active ingredient of Radix Astragali, which is considered to exhibit vasoprotective effects through unknown mechanisms. Thus, the current study was performed to investigate the protective effects of AS-IV in streptozotocin (STZ)-induced endothelial dysfunction and to explore whether antioxidant mechanisms were involved. The protective effects of AS-IV on the endothelium-dependent relaxation and contraction of aortic rings were determined by isometric tension recordings. NADPH subunits and endothelial nitric oxide synthase (eNOS) expression was identified via western blotting. Superoxide dismutase and malondialdehyde levels were assayed using ELISA. Furthermore, the generation of reactive oxygen species (ROS) and nitric oxide (NO) was detected via dihydroethidium and 4,5-diaminofluorescein diacetate staining, respectively. The results revealed that STZ-injected mice exhibited increased aortic endothelium-dependent vasoconstriction and decreased vasorelaxation to acetylcholine. However, AS-IV treatment reversed these effects. NG-nitro-L-arginine was subsequently used to completely inhibit impaired relaxation. Accordingly, impaired NO generation was restored following AS-IV treatment by increasing eNOS phosphorylation levels. Furthermore, ROS formation was also depressed following AS-IV treatment compared with that in STZ-injected mice. AS-IV also decreased the expression of various NADPH subunits, including human neutrophil cytochrome b light chain, neutrophil cytosolic factor 1, NADPH oxidase (NOX)2, NOX4 and Rac-1. The results of the current study may provide novel evidence that diabetes-induced vascular injury arises from either the inhibition of eNOS or the activation of NOX-derived ROS generation. In addition, the results warrant further investigation into the application of AS-IV treatment, leading to the improvement of oxidative stress, in patients with diabetes exhibiting endothelial dysfunction.

LncRNA ENSMUST00000155383 is Involved in the Improvement of DPP-4 Inhibitor MK-626 on Vascular Endothelial Function by Modulating Cacna1c-Mediated Ca2+ Influx in Hypertensive Mice.

Objective: This study investigated the protective effects of dipeptidyl peptidase-4 inhibitor MK-626 on vascular endothelial function by regulating lncRNAs in hypertensive vasculature. Methods: Angiotensin Ⅱ (Ang Ⅱ)-loaded osmotic pumps were implanted in mice with or without MK-626 administration. GLP-1 levels in plasma were measured by ELISA. Aortic rings were suspended in myograph for tension measurement. Microarray was performed to analyze lncRNA and mRNA expression profiles. Protein expression and phosphorylation were examined by Western blot. The differentially expressed (DE)-genes were validated by qRT-PCR. The intracellular Ca2+ concentration was detected by laser confocal system. Results: MK-626 elevated plasma GLP-1 level, increased eNOS phosphorylation, improved endothelium-dependent relaxations, and reduced systolic blood pressure in Ang Ⅱ-induced hypertensive mice. Microarray revealed the dysregulations of 723 lncRNAs and 742 mRNAs were reversed by MK-626 in hypertensive mouse aortae. qRT-PCR validation showed that 13 DE-lncRNAs and eight dysregulated mRNAs in both hypertensive mouse aortae and mouse aortic endothelial cells (MAECs) were rescued by MK-626. Among them, four mRNAs (Cacna1C, Itgav, Itga8, and Npnt) were co-expressed with lncRNA ENSMUST00000155383. Cacna1C protein expression was reduced in the ECs but was elevated in smooth muscle cells from Ang Ⅱ-infused mice, which were both reversed by MK-626. Knockdown of lncRNA ENSMUST00000155383 suppressed the increased Cacna1c protein and mRNA expression, elevated Ca2+ level, and enhanced eNOS phosphorylation induced by MK-626 in the hypertensive mouse ECs. Conclusion: The dysregulations of lncRNA ENSMUST00000155383-associated genes might play crucial roles in hypertension-induced endothelial dysfunction through affecting calcium pathway. MK-626 might ameliorate endothelial dysfunction by upregulating lncRNA ENSMUST00000155383, enhancing Ca2+ concentration, and subsequently restoring eNOS activity in hypertension.

Glossogyne tenuifolia Extract Increases Nitric Oxide Production in Human Umbilical Vein Endothelial Cells.

The vascular nitric oxide (NO) system has a protective effect in atherosclerosis. NO is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of endothelial NO synthase (eNOS). Compounds with the effect of enhancing eNOS expression are considered to be candidates for the prevention of atherosclerosis. In this study, extracts from the aerial, root, and whole plant of Glossogyne tenuifolia (GT) were obtained with ethanol, n-hexane, ethyl acetate (EA), and methanol extraction, respectively. The effects of these GT extracts on the synthesis of NO and the expression of eNOS in human umbilical vein endothelial cells (HUVECs) were investigated. NO production was determined as nitrite by colorimetry, following the Griess reaction. The treatment of HUVECs with EA extract from the root of GT and n-hexane, methanol, and ethanol extract from the aerial, root, and whole plant of GT increased NO production in a dose-dependent manner. When at a dose of 160 μg/mL, NO production increased from 0.9 to 18.4-fold. Among these extracts, the methanol extract from the root of GT (R/M GTE) exhibited the most potent effect on NO production (increased by 18.4-fold). Furthermore, using Western blot and RT–PCR analysis, treatment of HUVECs with the R/M GTE increased both eNOS protein and mRNA expression. In addition, Western blot analysis revealed that the R/M GTE increased eNOS phosphorylation at serine1177 as early as 15 min after treatment. The chemical composition for the main ingredients was also performed by HPLC analysis. In conclusion, the present study demonstrated that GT extracts increased NO production in HUVECs and that the R/M GTE increased NO production via increasing eNOS expression and activation by phosphorylation of eNOS at serine1177.

A20/TNFAIP3 Increases ENOS Expression in an ERK5/KLF2-Dependent Manner to Support Endothelial Cell Health in the Face of Inflammation.

Rationale: Decreased expression and activity of endothelial nitric oxide synthase (eNOS) in response to inflammatory and metabolic insults is the hallmark of endothelial cell (EC) dysfunction that preludes the development of atherosclerosis and hypertension. We previously reported the atheroprotective properties of the ubiquitin-editing and anti-inflammatory protein A20, also known as TNFAIP3, in part through interrupting nuclear factor-kappa B (NF-κB) and interferon signaling in EC and protecting these cells from apoptosis. However, A20's effect on eNOS expression and function remains unknown. In this study, we evaluated the impact of A20 overexpression or knockdown on eNOS expression in EC, at baseline and after tumor necrosis factor (TNF) treatment, used to mimic inflammation.Methods and Results: A20 overexpression in human coronary artery EC (HCAEC) significantly increased basal eNOS mRNA (qPCR) and protein (western blot) levels and prevented their downregulation by TNF. Conversely, siRNA-induced A20 knockdown decreased eNOS mRNA levels, identifying A20 as a physiologic regulator of eNOS expression. By reporter assays, using deletion and point mutants of the human eNOS promoter, and knockdown of eNOS transcriptional regulators, we demonstrated that A20-mediated increase of eNOS was transcriptional and relied on increased expression of the transcription factor Krüppel-like factor (KLF2), and upstream of KLF2, on activation of extracellular signal-regulated kinase 5 (ERK5). Accordingly, ERK5 knockdown or inhibition significantly abrogated A20's ability to increase KLF2 and eNOS expression. In addition, A20 overexpression in HCAEC increased eNOS phosphorylation at Ser-1177, which is key for the function of this enzyme.Conclusions: This is the first report demonstrating that overexpression of A20 in EC increases eNOS transcription in an ERK5/KLF2-dependent manner and promotes eNOS activating phosphorylation. This effect withstands eNOS downregulation by TNF, preventing EC dysfunction in the face of inflammation. This novel function of A20 further qualifies its therapeutic promise to prevent/treat atherosclerosis.

Vascular damage effect of circulating microparticles inpatients with ACS is aggravated by type2 diabetes.

As a common factor of both type 2 diabetes mellitus (T2DM) and acute coronary syndrome (ACS), circulating microparticles (MPs) may provide a link between these two diseases. The present study compared the content and function of MPs from patients with ACS with or without T2DM. MPs from healthy subjects (n=20), patients with ACS (n=24), patients with T2DM (n=20) and patients with combined ACS and T2DM (n=24) were obtained. After incubating rat thoracic tissue with MPs, the effect of MPs on endothelial-dependent vasodilatation, expression of caveolin-1 and endothelial nitric oxide synthase (eNOS), phosphorylation of eNOS at the S1177 and T495 sites and its association with heat shock protein 90 (Hsp90), and the generation of NO and superoxide anion (O2˙-) were determined. MP concentrations were higher in patients with T2DM and patients with ACS with or without T2DM than in healthy subjects. Moreover, MPs from patients with T2DM or ACS led to impairment in endothelial-dependent vasodilatation, decreased expression of NO, as well as eNOS and its phosphorylation at Ser1177 and association with Hsp90, but increased eNOS phosphorylation at T495, caveolin-1 expression and O2˙- generation. These effects were strengthened by MPs from patients with ACS combined with T2DM. T2DM not only increased MP content but also resulted in greater vascular impairment effects in ACS. These results may provide novel insight into the treatment of patients with ACS and T2DM.

Endothelial-transcytosed myeloperoxidase activates endothelial nitric oxide synthase via a phospholipase C-dependent calcium signaling pathway

During vascular inflammation, the leukocyte-derived enzyme myeloperoxidase (MPO) is transcytosed across the endothelium and into the sub-endothelial extracellular matrix, where it promotes endothelial dysfunction by catalytically consuming nitric oxide (NO) produced by endothelial NO synthase (eNOS). In the presence of chloride ions and hydrogen peroxide (H2O2), MPO forms the oxidant hypochlorous acid (HOCl). Here we examined the short-term implications of HOCl produced by endothelial-transcytosed MPO for eNOS activity. Incubation of MPO with cultured aortic endothelial cells (ECs) resulted in its transport into the sub-endothelium. Exposure of MPO-containing ECs to low micromolar concentrations of H2O2 yielded enhanced rates of H2O2 consumption that correlated with HOCl formation and increased eNOS enzyme activity. The MPO-dependent activation of eNOS occurred despite reduced cellular uptake of the eNOS substrate l-arginine, which involved a decrease in the maximal activity (Vmax), but not substrate affinity (Km), of the major endothelial l-arginine transporter, cationic amino acid transporter-1. Activation of eNOS in MPO-containing ECs exposed to H2O2 involved a rapid elevation in cytosolic calcium and increased eNOS phosphorylation at Ser-1179 and de-phosphorylation at Thr-497. These signaling events were attenuated by intracellular calcium chelation, removal of extracellular calcium and inhibition of phospholipase C. This study shows that stimulation of endothelial-transcytosed MPO activates eNOS by promoting phospholipase C-dependent calcium signaling and altered eNOS phosphorylation at Ser-1179 and Thr-497. This may constitute a compensatory signaling response of ECs aimed at maintaining eNOS activity and NO production in the face of MPO-catalyzed oxidative stress.

Activation of AT2 receptors prevents diabetic complications in female db/db mice by NO-mediated mechanisms.

The AT2 receptor plays a role in metabolism by opposing the actions triggered by the AT1 receptors. Activation of AT2 receptors has been shown to enhance insulin sensitivity in both normal and insulin resistance animal models. In this study, we investigated the mechanism by which AT2 receptors activation improves metabolism in diabetic mice. Female diabetic (db/db) and non-diabetic (db/+) mice were treated for 1 month with the selective AT2 agonist, compound 21 (C21, 0.3 mg·kg-1 ·day-1 , s.c.). To evaluate whether the effects of C21 depend on NO production, a subgroup of mice was treated with C21 plus a sub-pressor dose of the NOS inhibitor l-NAME (0.1 mg·ml-1 , drinking water). C21-treated db/db mice displayed improved glucose and pyruvate tolerance compared with saline-treated db/db mice. Also, C21-treated db/db mice showed reduced liver weight and decreased hepatic lipid accumulation compared with saline-treated db/db mice. Insulin signalling analysis showed increased phosphorylation of the insulin receptor, Akt and FOXO1 in the livers of C21-treated db/db mice compared with saline-treated counterparts. These findings were associated with increased adiponectin levels in plasma and adipose tissue and reduced adipocyte size in inguinal fat. The beneficial effects of AT2 receptors activation were associated with increased eNOS phosphorylation and higher levels of NO metabolites and were abolished by l-NAME. Chronic C21 infusion exerts beneficial metabolic effects in female diabetic db/db mice, alleviating type 2 diabetes complications, through a mechanism that involves NO production.