Visfatin activates eNOS via Akt and MAP kinases and improves endothelial cell function and angiogenesis in vitro and in vivo: translational implications for atherosclerosis

2009 ◽  
Vol 296 (6) ◽  
pp. E1440-E1449 ◽  
Author(s):  
Fina Lovren ◽  
Yi Pan ◽  
Praphulla C. Shukla ◽  
Adrian Quan ◽  
Hwee Teoh ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Function ◽  
Vein Graft ◽  
Map Kinases ◽  
Enos Expression ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Enos Phosphorylation

Improving endothelial nitric oxide synthase (eNOS) bioactivity and endothelial function is important to limit native, vein graft, and transplant atherosclerosis. Visfatin, a NAD biosynthetic enzyme, regulates the activity of the cellular survival factor, Sirt1. We hypothesized that visfatin may improve eNOS expression, endothelial function, and postnatal angiogenesis. In human umbilical vein (HUVEC) and coronary artery endothelial cells, we evaluated the effects of recombinant human visfatin on eNOS protein and transcript expression and mRNA stability, in the presence and absence of visfatin RNA silencing. We also assessed visfatin-induced protein kinase B (Akt) activation and its association with src-tyrosine kinases, phosphorylation of Ser1177within eNOS in the presence and absence of phosphatidylinositol 3-kinase (PI 3-kinase) inhibition with LY-294002, and evaluated the contributory role of extracellular signal-regulated kinase 1/2. Finally, we determined the impact of visfatin on HUVEC migration, proliferation, inflammation-induced permeability, and in vivo angiogenesis. Visfatin (100 ng/ml) upregulated and stabilized eNOS mRNA and increased the production of nitric oxide and cGMP. Visfatin-treated HUVEC demonstrated greater proliferation, migration, and capillary-like tube formation but less tumor necrosis factor-α-induced permeability; these effects were decreased in visfatin gene-silenced cells. Visfatin increased total Akt and Ser473-phospho-Akt expression with concomitant rises in eNOS phosphorylation at Ser1177; these effects were blocked by LY-2940002. Studies with PP2 showed that the nonreceptor tyrosine kinase, src, is an upstream stimulator of the PI 3-kinase-Akt pathway. Visfatin also activated mitogen-activated protein (MAP) kinase through PI 3-kinase, and mitogen/extracellular signal-regulated kinase inhibition attenuated visfatin-elicited Akt and eNOS phosphorylation. Visfatin-filled Matrigel implants showed an elevated number of infiltrating vessels, and visfatin treatment produced significant recovery of limb perfusion following hindlimb ischemia. These results indicate a novel effect of visfatin to stimulate eNOS expression and function in endothelial cells, via a common upstream, src-mediated signaling cascade, which leads to activation of Akt and MAP kinases. Visfatin represents a translational target to limit endothelial dysfunction, native, vein graft and transplant atherosclerosis, and improve postnatal angiogenesis.

scholarly journals Rho GTPase/Rho Kinase Negatively Regulates Endothelial Nitric Oxide Synthase Phosphorylation through the Inhibition of Protein Kinase B/Akt in Human Endothelial Cells

2002 ◽  
Vol 22 (24) ◽  
pp. 8467-8477 ◽  
Author(s):  
Xiu-Fen Ming ◽  
Hema Viswambharan ◽  
Christine Barandier ◽  
Jean Ruffieux ◽  
Kozo Kaibuchi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Nitric Oxide Synthase ◽  
Rho Gtpase ◽  
Protein Kinase B ◽  
Rho Kinase ◽  
Enos Expression ◽  
Enos Phosphorylation ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

ABSTRACT Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.

scholarly journals Cadmium reduces nitric oxide production by impairing phosphorylation of endothelial nitric oxide synthase

2008 ◽  
Vol 86 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Syamantak Majumder ◽  
Ajit Muley ◽  
Gopi Krishna Kolluru ◽  
Samir Saurabh ◽  
K. P. Tamilarasan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Function ◽  
Egg Yolk ◽  
Cellular Migration ◽  
No Production ◽  
Enos Phosphorylation ◽  
Low Levels ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Cadmium (Cd) perturbs vascular health and interferes with endothelial function. However, the effects of exposing endothelial cells to low doses of Cd on the production of nitric oxide (NO) are largely unknown. The objective of the present study was to evaluate these effects by using low levels of CdCl2 concentrations, ranging from 10 to 1000 nmol/L. Cd perturbations in endothelial function were studied by employing wound-healing and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. The results suggest that a CdCl2 concentration of 100 nmol/L maximally attenuated NO production, cellular migration, and energy metabolism in endothelial cells. An egg yolk angiogenesis model was employed to study the effect of Cd exposure on angiogenesis. The results demonstrate that NO supplementation restored Cd-attenuated angiogenesis. Immunofluorescence, Western blot, and immuno-detection studies showed that low levels of Cd inhibit NO production in endothelial cells by blocking eNOS phosphorylation, which is possibly linked to processes involving endothelial function and dysfunction, including angiogenesis.

Altered l-arginine metabolism results in increased nitric oxide release from uraemic endothelial cells

2002 ◽  
Vol 103 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Raj C. THURAISINGHAM ◽  
Norman B. ROBERTS ◽  
Mark WILKES ◽  
David I. NEW ◽  
A. Claudio MENDES-RIBEIRO ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
In Vivo Model ◽  
No Production ◽  
Arginine Metabolism ◽  
Enos Expression ◽  
Nitric Oxide Release ◽  
Excessive Consumption ◽  
And Control

Results regarding the nitric oxide (NO) system in uraemia are contradictory. l-Arginine, the precursor of NO, is also metabolized by arginase to form ornithine and urea. In the present study, endothelial NO production and arginine metabolism in uraemia were assessed. In addition an in vivo model was used to examine excess consumption of NO in uraemia. NO and amino acid measurements were made from basal and stimulated (by bradykinin) uraemic and control endothelial cells. Reverse-transcriptase PCR was used to assess endothelial NO synthase (eNOS) and inducible NOS (iNOS) expression. Finally, aortae of uraemic rats were stained for nitrotyrosine (a marker of peroxynitrite). Basal uraemic cells produced more NO than the control cells. l-Arginine levels were greater in uraemic (supernatants/cells), but ornithine levels were higher in control (supernatants/cells). Following stimulation, NO levels in supernatants were similar, but the rise in NO production was greater in control compared with uraemic cells; l-arginine levels still remained higher in uraemic supernatants/cells. Differences in ornithine concentration (supernatants/cells) disappeared following bradykinin stimulation, due to a rise in ornithine levels in the uraemic group. There was no difference in eNOS expression, nor was iNOS detected in either group. Only aortae from uraemic rats showed evidence for nitrotyrosine staining. These studies demonstrated increased basal NO release in uraemic endothelial cells, perhaps by inhibition of arginase and hence diversion of arginine to the NO pathway. The increased NO produced under basal conditions may be inactive due to excessive consumption, resulting in peroxynitrite formation. Interestingly, bradykinin appears to restore arginase activity in uraemia, resulting in normalization of NO production.

Deferiprone increases endothelial nitric oxide synthase phosphorylation and nitric oxide production

2018 ◽  
Vol 96 (9) ◽  
pp. 879-885 ◽  
Author(s):  
Thanaporn Sriwantana ◽  
Pornpun Vivithanaporn ◽  
Kittiphong Paiboonsukwong ◽  
Krit Rattanawonsakul ◽  
Sirada Srihirun ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Endothelial Cells ◽  
Endothelial Function ◽  
Healthy Subjects ◽  
Iron Chelation ◽  
No Production ◽  
Hemoglobin E ◽  
Enos Phosphorylation

Iron chelation can improve endothelial function. However, effect on endothelial function of deferiprone has not been reported. We hypothesized deferiprone could promote nitric oxide (NO) production in endothelial cells. We studied effects of deferiprone on blood nitrite and blood pressure after single oral dose (25 mg/kg) in healthy subjects and hemoglobin E/β-thalassemia patients. Further, effects of deferiprone on NO production and endothelial NO synthase (eNOS) phosphorylation in primary human pulmonary artery endothelial cells (HPAEC) were investigated in vitro. Blood nitrite levels were higher in patients with deferiprone therapy than those without deferiprone (P = 0.023, n = 16 each). Deferiprone increased nitrite in plasma and whole blood of healthy subjects (P = 0.002 and 0.044) and thalassemia patients (P = 0.003 and 0.046) at time 180 min (n = 20 each). Asymptomatic reduction in diastolic blood pressure (P = 0.005) and increase in heart rate (P = 0.009) were observed in healthy subjects, but not in thalassemia patients. In HPAEC, deferiprone increased cellular nitrite and phospho-eNOS (Ser1177) (P = 0.012 and 0.035, n = 6) without alteration in total eNOS protein and mRNA. We conclude that deferiprone can induce NO production by enhancing eNOS phosphorylation in endothelial cells.

scholarly journals Adrenomedullin increases the expression of calcitonin-like receptor and receptor activity modifying protein 2 mRNA in human microvascular endothelial cells

2006 ◽  
Vol 190 (2) ◽  
pp. 505-514 ◽  
Author(s):  
Nele Schwarz ◽  
Derek Renshaw ◽  
Supriya Kapas ◽  
Joy P Hinson
Keyword(s):  
Endothelial Cells ◽  
Kinase Inhibitor ◽  
Receptor Activity ◽  
Microvascular Endothelial Cells ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Microvascular Endothelial

Adrenomedullin (AM) is a multifunctional peptide hormone, which plays a significant role in vasodilation and angiogenesis, implicating it in hypertension as well as in carcinogenesis. AM exerts its effects via the calcitonin receptor-like receptor (CRLR, now known as CL) complexed with either receptor activity modifying protein (RAMP) 2 or 3. We have investigated the effect of AM on immortalized human microvascular endothelial cells 1, since endothelial cells are a major source as well as a target of AM actions in vivo. Cells treated with AM showed elevated cAMP in a time (5–45 min)-dependent and dose (10−6–10−14 M)-dependent manner. Pre-treatment with the AM receptor antagonist AM22–52 partially suppressed the AM-induced increase in cAMP levels. An increase in extracellular signal-regulated kinase 1/2 phosphorylation was observed after 5 min of treatment with 10−8 M AM. This phosphorylation was specific, since we were able to block the AM-induced effect with 1 μM U0126, a specific mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor. Using real-time PCR, we were able to show for the first time that AM upregulates peptide and mRNA expression of vascular endothelial growth factor (VEGF). However, AM treatment of cells did not result in increased cell proliferation. Instead, we observed that AM and VEGF induced cell migration, which could be inhibited by the AM22–52 and anti-VEGF antibody respectively. AM also significantly elevated mRNA levels of CL (after 2 and 24 h treatment) and RAMP2 (after 1 and 24 h treatment). The upregulation of the AM receptor at two time points reflects possibly different cellular responses to short- and long-term exposure to AM.

scholarly journals The Effect of Race and Shear Stress on CRP-Induced Responses in Endothelial Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Chenyi Ling ◽  
Marc D. Cook ◽  
Heather Grimm ◽  
Maitha Aldokhayyil ◽  
Dulce Gomez ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Function ◽  
Racial Differences ◽  
Umbilical Vein ◽  
Receptor Expression ◽  
No Production ◽  
Laminar Shear Stress ◽  
Enos Expression

Background. C-reactive protein (CRP) is an independent biomarker of systemic inflammation and a predictor of future cardiovascular disease (CVD). More than just a pure bystander, CRP directly interacts with endothelial cells to decrease endothelial nitric oxide synthase (eNOS) expression and bioactivity, decrease nitric oxide (NO) production, and increase the release of vasoconstrictors and adhesion molecules. Race is significantly associated with CRP levels and CVD risks. With aerobic exercise, the vessel wall is exposed to chronic high laminar shear stress (HiLSS) that shifts the endothelium phenotype towards an anti-inflammatory, antioxidant, antiapoptotic, and antiproliferative environment. Thus, the purpose of this study was to assess the racial differences concerning the CRP-induced effects in endothelial cells and the potential role of HiLSS in mitigating these differences. Methods. Human umbilical vein endothelial cells (HUVECs) from four African American (AA) and four Caucasian (CA) donors were cultured and incubated under the following conditions: (1) static control, (2) CRP (10 μg/mL, 24 hours), (3) CRP receptor (FcγRIIB) inhibitor followed by CRP stimulation, (4) HiLSS (20 dyne/cm2, 24 hours), and (5) HiLSS followed by CRP stimulation. Results. AA HUVECs had significantly higher FcγRIIB receptor expression under both basal and CRP incubation conditions. Blocking FcγRIIB receptor significantly attenuated the CRP-induced decrements in eNOS expression only in AA HUVECs. Finally, HiLSS significantly counteracted CRP-induced effects. Conclusion. Understanding potential racial differences in endothelial function is important to improve CVD prevention. Our results shed light on FcγRIIB receptor as a potential contributor to racial differences in endothelial function in AA.

scholarly journals Phosphorylation of Phosphoprotein Enriched in Astrocytes (PEA-15) Regulates Extracellular Signal-regulated Kinase-dependent Transcription and Cell Proliferation

2005 ◽  
Vol 16 (8) ◽  
pp. 3552-3561 ◽  
Author(s):  
Joseph Krueger ◽  
Fan-Li Chou ◽  
Angela Glading ◽  
Erik Schaefer ◽  
Mark H. Ginsberg
Keyword(s):  
Cell Proliferation ◽  
Nuclear Localization ◽  
Cell Cycle Progression ◽  
Map Kinases ◽  
Mammary Epithelium ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Cell cycle progression is dependent on the nuclear localization and transcriptional effects of activated extracellular signal-regulated kinase (ERK)1 and ERK2 mitogen-activated protein (MAP) kinases (ERK1/2). Phosphoprotein enriched in astrocytes (PEA-15) binds ERK1/2 and inhibits their nuclear localization, thus blocking cell proliferation. Here, we report that phosphorylation of PEA-15 blocks its interaction with ERK1/2 in vitro and in vivo and that phosphorylation of both Ser104 and Ser116 is required for this effect. Using phosphomimetic and nonphosphorylatable mutants of PEA-15, we found that PEA-15 phosphorylation abrogates its capacity to block the nuclear localization and transcriptional activities of ERK1/2; this phosphorylation therefore enables the proliferation of cells that express high levels of PEA-15. Additionally, we report that PEA-15 phosphorylation can modulate nontranscriptional activities of ERK1/2, such as the modulation of the affinity of integrin adhesion receptors. Finally, we used a novel anti-phospho-specific PEA-15 antibody to establish that PEA-15 is phosphorylated in situ in normal mammary epithelium. These results define a novel posttranslational mechanism for controlling the subcellular localization of ERK1/2 and for specifying the output of MAP kinase signaling.

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