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

Rationale: In diabetic animals as well as high glucose cell culture conditions, endothelial nitric oxide synthase (eNOS) is heavily O-GlcNAcylated, which inhibits its phosphorylation and nitric oxide (NO) production. It is unknown, however, whether varied blood flow conditions, which affect eNOS phosphorylation, modulate eNOS activity via O-GlcNAcylation-dependent mechanisms. Objective: The goal of this study was to test if steady laminar flow, but not oscillating disturbed flow, decreases eNOS O-GlcNAcylation, thereby elevating eNOS phosphorylation and NO production. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were exposed to either laminar flow (20 dynes/cm2 shear stress) or oscillating disturbed flow (4{plus minus}6 dynes/cm2 shear stress) for 24 hours in a cone-and-plate device. eNOS O-GlcNAcylation was almost completely abolished in cells exposed to steady laminar but not oscillating disturbed flow. Interestingly, there was no change in protein level or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway (HBP) activity, thereby reducing UDP-GlcNAc pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation, thereby increasing eNOS phosphorylation and NO production. Finally, we detected significantly higher O-GlcNAcylated proteins in endothelium of the inner aortic arch in mice, suggesting that disturbed flow increases protein O-GlcNAcylation in vivo. Conclusions: Our data demonstrate that steady laminar but not oscillating disturbed flow decreases eNOS O-GlcNAcylation by limiting glycolysis and UDP-GlcNAc substrate availability, thus enhancing eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli, relates flow-induced glycolytic reductions to macrovascular disease, and highlights targeting HBP metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent EC dysfunction in cardiovascular disease.

Abstract P262: Spike Protein 1 Of Sars-cov-2 Increases Interferon Stimulated Genes And Induces An Immune/inflammatory Responses In Human Endothelial Cells.

Introduction: Interferon (IFN) alpha (IFNα) and lambda3 (IFNL3) constitute the first line of immunity against SARS-CoV-2 infection by increasing interferon-stimulated genes (ISGs). IFNs influence the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. Here we hypothesized that in human microvascular endothelial cells (EC) IFNL3 and IFNα influence ACE2 and immune/inflammatory responses mediated by S1P. Methods: EC were stimulated with S1P of SARS-CoV-2 (1 μg/10^6 cells), IFNα (100 ng/mL) or IFNL3 (100 IU/mL). Because ACE2, metalloproteinase domain 17 (ADAM17) and type II transmembrane serine protease (TMPRSS2) are important for SARS-CoV-2 infection, cells were treated with inhibitors of ADAM17 (marimastat, 3.8nM and TAPI-1, 100nM), ACE2 (MLN4760, 440pM), and TMPRSS2 (camostat, 50μM). Expression of ISGs (ISG15, IFIT1, and MX1) was investigated by real-time PCR (5h) and protein expression by immunoblotting (24h). Results: EC stimulated with S1P increased expression of ISGs: ISG15 (2 fold), IFIT1 (6 fold), MX1 (6 fold) (n=12, p<0.05). EC exhibited higher responses to IFNα (ISG15: 16 fold, IFIT1: 21 fold, MX1: 31 fold) than to IFNL3 (ISG15: 1.7 fold, IFIT1: 1.9 fold, MX1: 1.7 fold) (p<0.05). S1P increased gene expression of IL-6 (1.3 fold), TNFα (6.2 fold) and IL-1β (3.3 fold), effects that were maximized 100% by IFNα. Only marimastat inhibited S1P effects. IL-6 was increased by IFNα (1230 pg/mL) and IFNL3 (1124 pg/mL) vs control (591pg/mL). IFNα increased expression of ACE2 (75 kDa) (63%), ADAM17 (36%), and TMPRSS2 (65%). This was associated with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). Nitric oxide production and eNOS phosphorylation (Ser1177) were reduced by IFNα and (40%) and IFNL3 (40%). Conclusions: In human microvascular endothelial cells, S1P, IFNα and IFNL3 induced an immune response characterized by increased expression of interferon-stimulated genes and IL-6 production, processes that involve ADAM17. Inflammation induced by S1P was amplified by IFNα. Our novel findings demonstrate that S1P induces an endothelial immune/inflammatory response that may be important in endotheliitis associated with COVID-19.

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.

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.

Distinct Signaling Functions of Rap1 Isoforms in NO Release From Endothelium

Small GTPase Rap1 plays a prominent role in endothelial cell (EC) homeostasis by promoting NO release. Endothelial deletion of the two highly homologous Rap1 isoforms, Rap1A and Rap1B, leads to endothelial dysfunction ex vivo and hypertension in vivo. Mechanistically, we showed that Rap1B promotes NO release in response to shear flow by promoting mechanosensing complex formation involving VEGFR2 and Akt activation. However, the specific contribution of the Rap1A isoform to NO release and the underlying molecular mechanisms through which the two Rap1 isoforms control endothelial function are unknown. Here, we demonstrate that endothelial dysfunction resulting from knockout of both Rap1A and Rap1B isoforms is ameliorated by exogenous L-Arg administration to rescue NO-dependent vasorelaxation and blood pressure. We confirmed that Rap1B is rapidly activated in response to agonists that trigger eNOS activation, and its deletion in ECs attenuates eNOS activation, as detected by decreased Ser1177 phosphorylation. Somewhat surprising was the finding that EC deletion of Rap1A does not lead to impaired agonist-induced vasorelaxation ex vivo. Mechanistically, the deletion of Rap1A led to elevated eNOS phosphorylation both at the inhibitory, T495, and the activating Ser1177 residues. These findings indicate that the two Rap1 isoforms act via distinct signaling pathways: while Rap1B directly positively regulates eNOS activation, Rap1A prevents negative regulation of eNOS. Notably, the combined deficiency of Rap1A and Rap1B has a severe effect on eNOS activity and NO release with an in vivo impact on endothelial function and vascular homeostasis.

Synergic effect of atorvastatin and ambrisentan on sinusoidal and hemodynamic alterations in a rat model of nash

Background & Aims: In non-alcoholic steatohepatitis (NASH) decreased nitric oxide and increased endothelin-1 (ET-1) released by sinusoidal endothelial cells (LSEC), induce hepatic stellate cells (HSC) contraction and contribute to portal hypertension (PH). Statins improve LSEC function, while ambrisentan is a selective endothelin-receptor-A antagonist. We aimed to analyse the combined effects of atorvastatin and ambrisentan on liver histopathology and hemodynamics, together with assessing the underlying mechanism in a rat NASH model. Methods: Diet-induced NASH rats were treated with atorvastatin (10 mg/kg/day), ambrisentan (30 mg/kg/day or 2 mg/kg/day) or the combination for 2 weeks. Hemodynamic parameters were registered and liver histology and serum biochemical determinations analysed. Expression of proteins were studied by immunoblots. Conditioned media experiments were performed in LSEC. HSC were characterized by RT-PCR and a collagen lattice contraction assay was performed. Results: Atorvastatin and ambrisentan act synergistically in combination to completely normalize liver hemodynamic and reverse histological NASH by 75%. Atorvastatin reversed the sinusoidal contractile phenotype improving endothelial function, while ambrisentan prevented the contractile response in HSC by blocking ET-1 response. Additionally, ambrisentan also increased eNOS phosphorylation levels in LSEC, via facilitating the stimulation of endothelin-receptor-B in these cells. Furthermore, the combined treated group reduced to normal serum ALT levels and showed restoration of the HSC quiescent phenotype. Conclusions: Combination of atorvastatin and ambrisentan remarkably improves liver histology and PH in a diet-induced NASH model. By recovering LSEC function, together with inhibiting the activation and contraction of HSC, this combined treatment may be an effective treatment for patients with NASH.

β-Cyclodextrin Inhibits Monocytic Adhesion to Endothelial Cells through Nitric Oxide-Mediated Depletion of Cell Adhesion Molecules

Cyclodextrins (CDs) are used as drug delivery agents. In this study, we examined whether CDs have an inflammatory effect on endothelial cells. First, we found that β-CD promoted cell proliferation in bovine aortic endothelial cells and elevated nitric oxide (NO) production through dephosphorylation of threonine-495 (T-495) in endothelial nitric oxide synthetase (eNOS). Dephosphorylation of T-495 is known to activate eNOS. Phosphorylation of T-495 was found to be catalyzed by protein kinase Cε (PKCε). We then found that β-CD inhibits binding of PKCε to diacylglycerol (DAG) via formation of a β-CD-DAG complex, indicating that β-CD inactivates PKCε. Furthermore, β-CD controls activation of PKCε by reducing the recruitment of PKCε into the plasma membrane. Finally, β-CD inhibits expression of intercellular and vascular cell adhesion molecule-1 by increasing NO via control of PKCε/eNOS and suppression of THP-1 cell adhesion to endothelial cells. These findings imply that β-CD plays an important role in anti-inflammatory processes.

Clinically relevant high levels of human C-reactive protein induces endothelial dysfunction and hypertension by inhibiting the AMPK-eNOS axis

Successful treatment of resistant hypertension accompanied by elevated human C-reactive protein (hCRP) remains a key challenge in reducing the burden of cardiovascular diseases. It is still unclear whether clinically relevant high-level hCRP is merely a marker or a key driver of hypertension. Here, we investigated the role and mechanism of clinically relevant high level of hCRP in hypertension. Elevated blood pressure was observed in all three hCRP overexpression models, including adeno-associated virus 9 (AAV9)-transfected mice, AAV9-transfected rats and hCRP transgenic (hCRPtg) rats. hCRPtg rats expressing clinically relevant high-level hCRP developed spontaneous hypertension, cardiac hypertrophy, myocardial fibrosis and impaired endothelium-dependent relaxation. Mechanistically, studies in endothelial nitric oxide (NO) synthase (eNOS) knockout mice transfected with AAV9-hCRP and phosphoproteomics analysis of hCRP-treated endothelial cells revealed that hCRP inhibited AMP-activated protein kinase (AMPK)-eNOS phosphorylation pathway. Further, activation of AMPK by metformin normalized endothelial-dependent vasodilation and decreased the blood pressure of hCRPtg rats. Our results show that clinically relevant high-level hCRP induces hypertension and endothelial dysfunction by inhibiting AMPK-eNOS signaling, and highlight hCRP is not only an inflammatory biomarker but also a driver of hypertension. Treatment with metformin or a synthetic AMPK activator may be a potential strategy for vaso-dysfunction and hypertension in patients with high hCRP levels.