Abstract 1420: Mitochondrial Arginase II Constrains Endothelial NOS Activity

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Hyun Kyo LIM ◽  
Hyun Kyoung Lim ◽  
Sungwoo Ryoo ◽  
Alex Benjo ◽  
Karl Shuleri ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Vascular Stiffness ◽  
No Production ◽  
Vascular Endothelial ◽  
Knock Out ◽  
Enos Activity ◽  
Arginase Ii ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Background: Arginase II is known as a major isoenzyme in the vascular endothelial cells of humans and other species and modulates endothelial nitric oxide synthase (eNOS) activity by regulating intracellular L-arginine bioavailability. We tested under hypothesis that subcellular localization of arginase II is confined to the mitochondria and mitochondrial arginase II deficiency reciprocally regulates the vascular endothelial NO production, contributes to improve endothelial dysfunction and decrease vascular stiffness. Methods and Results: Western blot, immunocytochem with Mitotracker and immunoEM confirmed that arginase II is confined predominantly but not exclusively to the mitochondria. Arginase activity was significantly reduced, whereas NO production was significantly increased in aorta and isolated endothelial cells from arginase II knock out (ArgII−/−) compared to WT mice. Vasorelaxation to acetylcholine (ACh) was markedly enhanced and vasoconstriction to phenylephrine (PE) was impaired in ArgII−/− rings. Furthermore inhibition of eNOS by N G -nitro-L-arginine methyl ester (L-NAME) impaired ACh response and restored PE response. The baseline pulse wave velocity was significantly decreased in ArgII −/− mice (3.30 ± 0.21 vs. 3.64 ± 0.12 m/s, ArgII −/− vs. WT, *p<0.01), while 14 days of L-NAME treatment resulted in significant increase the PWV in both WT (4.23 ± 0.11 m/s, **p<0.001) and ArgII −/− mice (4.36 ± 0.20 m/s, **p<0.001). Conclusions: These data suggest that arginase II is predominantly confined to the mitochondria and this mitochondrial arginase II regulate the NO production, vascular endothelial function, and vascular stiffness by modulating eNOS activity.

scholarly journals Mitochondrial arginase II constrains endothelial NOS-3 activity

2007 ◽  
Vol 293 (6) ◽  
pp. H3317-H3324 ◽  
Author(s):  
Hyun Kyo Lim ◽  
Hyun Kyoung Lim ◽  
Sungwoo Ryoo ◽  
Alex Benjo ◽  
Karl Shuleri ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Vascular Stiffness ◽  
No Production ◽  
Vascular Endothelial ◽  
Vascular Endothelial Function ◽  
Vasoconstrictor Response ◽  
Arginase Ii ◽  
Endothelial Nitric Oxide ◽  
Arginase I

Emerging evidence supports the idea that arginase, expressed in the vascular endothelial cells of humans and other species, modulates endothelial nitric oxide (NO) synthase-3 (NOS-3) activity by regulating intracellular l-arginine bioavailability. Arginase II is thought to be expressed in the mitochondria of a variety of nonendothelial cells, whereas arginase I is known to be confined to the cytosol of hepatic and other cells. The isoforms that regulate NOS-3 and their subcellular distribution, however, remain incompletely characterized. We therefore tested the hypothesis that arginase II is confined to the mitochondria and that mitochondrial arginase II reciprocally regulates vascular endothelial NO production. Western blot analysis, immunocytochemistry with MitoTracker, and immunoelectron microscopy confirmed that arginase II is confined predominantly but not exclusively to the mitochondria. Arginase activity was significantly decreased, whereas NO production was significantly increased in the aorta and isolated endothelial cells from arginase II knockout (ArgII−/−) mice compared with wild-type (WT) mice. The vasorelaxation response to acetylcholine (ACh) was markedly enhanced and the vasoconstrictor response to phenylephrine (PE) attenuated in ArgII−/− in pressurized mouse carotid arteries. Furthermore, inhibition of NOS-3 by NG-nitro-l-arginine methyl ester (l-NAME) impaired ACh response and restored the PE response to that observed in WT vessels. Vascular stiffness, as assessed by pulse wave velocity (PWV), was significantly decreased in ArgII−/− compared with WT mice. On the other hand, 14 days of oral l-NAME treatment significantly increased PWV in both WT and ArgII−/− mice, such that they were not significantly different from one another. These data suggest that arginase II is predominantly confined to the mitochondria and that this mitochondrial arginase II regulates NO production, vascular endothelial function, and vascular stiffness by modulating NOS-3 activity.

Abstract 3635: Inhibition of Inhibitor Kappa B Kinase Beta Augments Endothelial Nitric Oxide Synthase Activity and Nitric Oxide Production in Aortic Endothelial Cells

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Sumathy Mohan ◽  
Ryzard Konopinski ◽  
Mohan Natarajan
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Vascular Endothelial Cells ◽  
No Production ◽  
Enos Activity ◽  
Hsp 90 ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hall-mark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein 90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases (inhibitor kappa B kinases α, β and γ) in non-vascular cells. In this study, we have investigated the interaction of Hsp-90-IKKβ in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKβ interacts with Hsp-90 and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (4.02 ± 0.81-folds) and translational (1.97 ± 0.17-fold) expression as well as the catalytic activity of IKKβ (2.04 ± 0.06-folds). Another important and novel finding is that both IKKβ and eNOS could be co-immunoprecipitated with Hsp-90 (Figures A & B ) thus indicating the possible existence of a complex of IKKβ and eNOS interacting with single pool of Hsp-90. Inhibition of Hsp-90 with geldanamycin (2μM) or Radicicol (20μM) mitigated (0.45 ± 0.04 -fold and 0.93 ± 0.16-fold, respectively) HG induced-IKKβ activity (2.5 ± 0.416-fold). Blocking of IKKβ expression by IKK inhibitor II (15μM wedelolactone) or siRNA improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.

Abstract P069: Microvesicles From Enos-suppressed Endothelial Cells Induce Endothelial Cell Dysfunction

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Vinicius P Garcia ◽  
Jamie G Hijmans ◽  
Kelly A Stockelman ◽  
Madden Brewster ◽  
Hannah Fandl ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
No Production ◽  
Cell Dysfunction ◽  
Enos Activity ◽  
Umbilical Vein Endothelial Cells ◽  
And Function ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Introduction: Endothelial nitric oxide synthase (eNOS) activity is critical to vascular health. Impaired eNOS activity and diminished NO production are common characteristics of a proatherogenic, dysfunctional endothelial phenotype that is associated with cardiovascular risk factors and disease. Extracellular microvesicles, particularly endothelial cell derived microvesicles (EMVs) represent novel mechanistic mediators of endothelial dysfunction and vascular disease. It is unknown whether eNOS suppression affects EMV number and function. We tested the following hypotheses: 1) eNOS blockade increases EMV release; and 2) EMVs derived from eNOS-suppressed cells adversely affect endothelial cell inflammation, apoptosis and NO production. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with the eNOS inhibitor, L-N G -Nitroarginine methyl ester (L-NAME; 300mM) for 24 h. EMVs (CD144 + ) released into the supernatant from cells treated with L-NAME or vehicle were isolated and quantified by flow cytometry. Fresh HUVECs were then treated with either L-NAME-derived or control EMVs for 24 h. To evaluate the role of endocytosis on the endothelial effects of EMVs, HUVECs were pre-incubated (12 h) with EIPA, filipin and chlorpromazine for 2 h, and all experiments repeated. Results: EMV release was markedly higher (~100%; P<0.05) in cells treated with L-NAME compared with control (81±6 vs. 40±7 EMV/μL). L-NAME-generated EMVs induced significantly higher release of IL-6 (38.4±5.1 vs. 21.0±1.9 pg/mL) and IL-8 (38.9±3.5 vs. 27.2±3.1 pg/mL) as well as greater active NF-κB p65 (Ser-536) (9.7±0.7 vs. 6.1±0.6 AU) expression than control EMVs. The expression of activated-caspase-3 was significantly higher in the cells treated with L-NAME (9.5±1.1 vs. 6.4±0.4 AU). Total eNOS (97.1±8.2 vs. 157.5±15.6 AU), activated eNOS (4.9±1.2 vs. 9.1±1.3 AU) and NO production (5.0±0.8 vs. 7.0±0.6 μmol/L) were significantly lower in endothelial cells treated with EMVs from eNOS suppressed cells. Endocytosis blockers mitigated the deleterious endothelial effects of EMVs. Conclusion: eNOS-suppression increases EMV release. Moreover, EMVs from eNOS-suppressed cells increase endothelial cell inflammation and apoptosis and decrease NO production.

scholarly journals Insights into the arginine paradox: evidence against the importance of subcellular location of arginase and eNOS

2013 ◽  
Vol 305 (5) ◽  
pp. H651-H666 ◽  
Author(s):  
Shawn Elms ◽  
Feng Chen ◽  
Yusi Wang ◽  
Jin Qian ◽  
Bardia Askari ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Subcellular Location ◽  
No Production ◽  
Simple Relationship ◽  
Intracellular Location ◽  
Enos Activity ◽  
Arginase Ii ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Arginase I

Reduced production of nitric oxide (NO) is one of the first indications of endothelial dysfunction and precedes overt cardiovascular disease. Increased expression of Arginase has been proposed as a mechanism to account for diminished NO production. Arginases consume l-arginine, the substrate for endothelial nitric oxide synthase (eNOS), and l-arginine depletion is thought to competitively reduce eNOS-derived NO. However, this simple relationship is complicated by the paradox that l-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis. One mechanism proposed to explain this is compartmentalization of intracellular l-arginine into distinct, poorly interchangeable pools. In the current study, we investigated this concept by targeting eNOS and Arginase to different intracellular locations within COS-7 cells and also BAEC. We found that supplemental l-arginine and l-citrulline dose-dependently increased NO production in a manner independent of the intracellular location of eNOS. Cytosolic arginase I and mitochondrial arginase II reduced eNOS activity equally regardless of where in the cell eNOS was expressed. Similarly, targeting arginase I to disparate regions of the cell did not differentially modify eNOS activity. Arginase-dependent suppression of eNOS activity was reversed by pharmacological inhibitors and absent in a catalytically inactive mutant. Arginase did not directly interact with eNOS, and the metabolic products of arginase or downstream enzymes did not contribute to eNOS inhibition. Cells expressing arginase had significantly lower levels of intracellular l-arginine and higher levels of ornithine. These results suggest that arginases inhibit eNOS activity by depletion of substrate and that the compartmentalization of l-arginine does not play a major role.

scholarly journals High glucose-induced IKK-Hsp-90 interaction contributes to endothelial dysfunction

2009 ◽  
Vol 296 (1) ◽  
pp. C182-C192 ◽  
Author(s):  
Sumathy Mohan ◽  
Ryszard Konopinski ◽  
Bo Yan ◽  
Victoria E. Centonze ◽  
Mohan Natarajan
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
High Glucose ◽  
Vascular Endothelial Cells ◽  
Heat Shock Protein 90 ◽  
No Production ◽  
Enos Activity ◽  
Hsp 90 ◽  
Endothelial Nitric Oxide

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hallmark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein-90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases [inhibitor κB kinases (IKK)α, β, and γ] in nonvascular cells. In this study, we have investigated the interaction of Hsp-90-IKKβ in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKβ interacts with Hsp-90, and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (3.5 ± 0.65-fold) and translational (1.97 ± 0.17-fold) expression as well as the catalytic activity of IKKβ (2.45 ± 0.4-fold). Both IKKβ and eNOS could be coimmunoprecipitated with Hsp-90. Inhibition of Hsp-90 with geldanamycin (2 μM) or Radicicol (20 μM) mitigated (0.45 ± 0.04-fold and 0.93 ± 0.16-fold, respectively) HG induced-IKKβ activity (2.5 ± 0.42-fold). Blocking of IKKβ expression by IKK inhibitor II (15 μM wedelolactone) or small interferring RNA (siRNA) improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.

scholarly journals Telmisartan Activates Endothelial Nitric Oxide Synthase via Ser1177 Phosphorylation in Vascular Endothelial Cells

PLoS ONE ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. e96948 ◽  
Author(s):  
Masahiro Myojo ◽  
Daisuke Nagata ◽  
Daishi Fujita ◽  
Arihiro Kiyosue ◽  
Masao Takahashi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide
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