Abstract 442: FOXO1/Notch Signaling Modulates Ambient Ultrafine Particle Impaired Vascular Repair

Introduction: Exposure to ultrafine particles (UFP, d < 0.1 μm), redox-active components of particular matter (PM 2.5 ), promotes endothelial dysfunction. Notch signaling in endothelial cells (EC) regulates differentiation and proliferation of vasculature. FOXO1 interacts with Notch signaling by enhancing assembly of activation complex during induction of Notch signaling. Whether UFP impair vascular repair by modulating FOXO1/Notch signaling axis remains elusive. Hypothesis: We hypothesized that UFP impairs vascular repair by attenuating Notch signaling via inhibition on FOXO1. Methods and Results: Control transgenic Tg(fli1:gfp) zebrafish embryos underwent tail amputation at 3 days post fertilization (dpf) developed complete vascular repair at 3 days post amputation (dpa), whereas exposure to UFP, or treatment with ADAM10 inhibitor to prevent Notch activation, or micro-injection of dominant negative(DN) Notch1b mRNA disrupted vascular network and impaired regeneration (* P < 0.05, n=20). By crossing the Notch reporter line Tg(tp1:gfp) with the Tg(flk1:mCherry) line, we demonstrated UFP inhibits endothelial Notch signaling on the amputated site at 3 dpa. Micro-injection of NICD mRNA only partially rescued endothelial Notch activity and impaired vascular repair in the presence of UFP (* P < 0.05, n=20). FOXO1 MO significantly inhibited Notch signaling, mimicking the UFP-impaired vascular repair. Injection of FOXO1 mRNA accentuated Notch activity and rescued UFP-impaired vascular repair. In human aortic endothelial cells, UFP suppressed FOXO1 expression and the co-localization with NICD, but not Master-Mind Like 1(MAML) or active NICD expression (* P < 0.05, n=3). As a corollary, UFP exposure induced dose and time-dependent reduction in Notch reporter activity, FOXO1 mRNA expression and the expression of Notch signaling related genes including the Notch ligand Dll4 and Notch target HES1. (* P < 0.05, n=3). Conclusions: In conclusion, UFP attenuated FOXO1/Notch cooperation to modulate Notch signaling and impaired vascular repair in embryonic zebrafish.

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Abstract 614: Shear Stress-Induced Glycolytic Metabolites Promote Vascular Repair

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.614 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Kyung In Baek ◽

Rongsong Li ◽

David Liem ◽

Tyler Beebe ◽

Jianguo Ma ◽

...

Keyword(s):

Shear Stress ◽

Rabbit Model ◽

Zealand White Rabbit ◽

Tube Formation ◽

Dominant Negative ◽

Vegf Receptor ◽

Vascular Repair ◽

Aortic Endothelial Cells ◽

Enhanced Tube ◽

Vegfr Inhibitor

Introduction: Hemodynamic shear stress is intimately linked with transcriptomic and epigenomic changes to maintain endothelial homeostasis. Metabolomics studies have led to emergent metabolic biomarkers and therapeutic targets. Whether shear stress modulates metabolomic pathway to promote vascular repair remains to be investigated. Hypothesis: We hypothesized that shear stress regulates VEGF receptor-PKCε-PFKFB3 signaling-mediated glycolytic metabolites to promote vascular repair. Method and Results: Both pulsatile (PSS: 23 ± 8 dyn·cm -2 at 1 Hz) and oscillatory shear stress (OSS: 0.1 ± 3 dyn·cm -2 at 1 Hz) up-regulated PKCε expressions and the activity (* P < 0.05, n =3), whereas silencing VEGFR2 with siRNA, or treating with VEGFR inhibitor, Cediranib, attenuated shear stress-mediated PKCε expression in human aortic endothelial cells(HAEC). Constitutively active (CA)-PKCε adenovirus infection enhanced tube formation assessed by Matrigel as well as significantly increased PFKFB3 expressions promoting glycolysis, whereas the dominant negative(DN) PKCε resulted in opposite effects. Co-localization of PKCε and PFKFB3 expression was demonstrated in the endothelium of aortic arch and thoracic aorta in a New Zealand White rabbit model. In the zebrafish tail amputation model, reduction of shear stress via GATA-1a morpholino oligonucleotide(MO) injection and inhibition of PKCε expression via PKCε MO impaired vascular repair between the dorsal aorta and the dorsal longitudinal anastomotic vessel at 3 days post amputation(dpa). PKCε mRNA rescued GATA-1a MO-mediated impairment of vascular repair (* P < 0.01, n =20, ** P < 0.05, n =5). Metabolomic analysis in HAEC applied to PSS and OSS revealed modulation of a number of metabolites including increased glycolytic metabolite dihydroxyacetone, which was blocked by PKCε siRNA. Treatment with dihydroxyacetone rescued PKCε-impaired vascular repair. Conclusion: In conclusion, shear stress-mediated VEGFR-PKCε-PFKFB3 signaling increased glycolytic metabolites to mediate vascular repair.

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Functional expression of Kir2.x in human aortic endothelial cells: the dominant role of Kir2.2

AJP Cell Physiology ◽

10.1152/ajpcell.00077.2005 ◽

2005 ◽

Vol 289 (5) ◽

pp. C1134-C1144 ◽

Cited By ~ 47

Author(s):

Yun Fang ◽

Gernot Schram ◽

Victor G. Romanenko ◽

Congzhu Shi ◽

Lisa Conti ◽

...

Keyword(s):

Endothelial Cells ◽

Dominant Role ◽

Functional Expression ◽

Inward Rectifier ◽

Dominant Negative ◽

Current Sensitivity ◽

Aortic Endothelial Cells ◽

Human Aortic Endothelial Cells ◽

Aortic Endothelial

Inward rectifier K+ channels (Kir) are a significant determinant of endothelial cell (EC) membrane potential, which plays an important role in endothelium-dependent vasodilatation. In the present study, several complementary strategies were applied to determine the Kir2 subunit composition of human aortic endothelial cells (HAECs). Expression levels of Kir2.1, Kir2.2, and Kir2.4 mRNA were similar, whereas Kir2.3 mRNA expression was significantly weaker. Western blot analysis showed clear Kir2.1 and Kir2.2 protein expression, but Kir2.3 protein was undetectable. Functional analysis of endothelial inward rectifier K+ current ( IK) demonstrated that 1) IK current sensitivity to Ba2+ and pH were consistent with currents determined using Kir2.1 and Kir2.2 but not Kir2.3 and Kir2.4, and 2) unitary conductance distributions showed two prominent peaks corresponding to known unitary conductances of Kir2.1 and Kir2.2 channels with a ratio of ∼4:6. When HAECs were transfected with dominant-negative (dn)Kir2.x mutants, endogenous current was reduced ∼50% by dnKir2.1 and ∼85% by dnKir2.2, whereas no significant effect was observed with dnKir2.3 or dnKir2.4. These studies suggest that Kir2.2 and Kir2.1 are primary determinants of endogenous K+ conductance in HAECs under resting conditions and that Kir2.2 provides the dominant conductance in these cells.

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High-Level Expression of Mastermind-Like 2 (MAML2) Contributes to Aberrant Activation of the NOTCH Signaling Pathway In Human Lymphomas

Blood ◽

10.1182/blood.v116.21.2685.2685 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2685-2685

Author(s):

Karl Köchert ◽

Stephan Kreher ◽

Jon C Aster ◽

Motoo Kitagawa ◽

Korinna Jöhrens ◽

...

Keyword(s):

Notch Signaling ◽

Signaling Pathway ◽

Conflicts Of Interest ◽

Gene Expression Signature ◽

Notch Signaling Pathway ◽

Dominant Negative ◽

Alternative Mechanism ◽

Aberrant Expression ◽

Notch Activity ◽

High Level

Abstract Abstract 2685 Inappropriate activation of the NOTCH signaling pathway, e.g. by activating mutations, contributes to the pathogenesis of various human malignancies. Here, we demonstrate that aberrant expression of an essential NOTCH co-activator of the Mastermind-like (MAML) family provides an alternative mechanism to activate NOTCH signaling in human lymphoma cells. We detected high-level MAML2 expression in several B cell-derived lymphoma types, including classical Hodgkin lymphoma (cHL) cells, relative to normal B cells. Inhibition of MAML protein activity by a dominant negative form of MAML or by shRNAs targeting MAML2 in cHL cells resulted in down-regulation of the NOTCH target genes HES7 and HEY1, which we identified as overexpressed in cHL cells, and in reduced proliferation. Furthermore, a NOTCH gene-expression signature in cHL cells confirmed their cell-autonomous NOTCH activity. Finally, in line with the essential role of MAML proteins for assembly and activity of the NOTCH transcriptional complex (NTC) we show that MAML-derived small-peptide constructs block NOTCH activity and disrupt NTC formation in vitro. These data strongly suggest direct targeting of the NTC as treatment strategy for NOTCH-dependent malignancies. Disclosures: No relevant conflicts of interest to declare.

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Aldosterone Induces Superoxide Generation via Rac1 Activation in Endothelial Cells

Endocrinology ◽

10.1210/en.2007-0864 ◽

2007 ◽

Vol 149 (3) ◽

pp. 1009-1014 ◽

Cited By ~ 83

Author(s):

Fumiko Iwashima ◽

Takanobu Yoshimoto ◽

Isao Minami ◽

Maya Sakurada ◽

Yuki Hirono ◽

...

Keyword(s):

Endothelial Cells ◽

G Protein ◽

Mineralocorticoid Receptor ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Oxidase Inhibitor ◽

Dominant Negative ◽

Superoxide Generation ◽

Cellular Mechanisms ◽

Mineralocorticoid Receptor Antagonist ◽

Aortic Endothelial Cells

Currently, aldosterone is believed to be involved in the development of cardiovascular injury as a potential cardiovascular risk hormone. However, its exact cellular mechanisms remain obscure. This study was undertaken to examine the effect of aldosterone on superoxide production in cultured rat aortic endothelial cells with possible involvement of the small GTP-binding (G) protein Rac1. The aldosterone levels showed a time-dependent (6–24 h) and dose-dependent (10−8 to 10−6m) increase in superoxide generation, whose effect was abolished by mineralocorticoid receptor antagonist (eplerenone), Src inhibitor (PP2), and reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase inhibitor (apocynin). Aldosterone activated NADP(H) oxidase and Rac1, whose effects were abolished by eplerenone. The aldosterone-induced superoxide generation was abolished either by nonselective small G protein inhibitor (Clostridium difficile toxin A) or dominant-negative Rac1. Dominant-negative Rac1 also inhibited aldosterone-induced ACE gene expression. Thus, the present study is the first to demonstrate that aldosterone induces superoxide generation via mineralocorticoid receptor-mediated activation of NAD(P)H-oxidase and Rac1 in endothelial cells, thereby contributing to the development of aldosterone-induced vascular injury.

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Endothelial-to-hematopoietic transition is induced by Notch glycosylation and upregulation of Mycn

10.1101/2020.09.13.295238 ◽

2020 ◽

Author(s):

Briane Laruy ◽

Irene Garcia-Gonzalez ◽

Veronica Casquero-Garcia ◽

Rui Benedito

Keyword(s):

Cell Cycle ◽

Endothelial Cells ◽

Stem Cell ◽

Notch Signaling ◽

Hematopoietic Stem Cell ◽

Molecular Mechanisms ◽

Subsequent Development ◽

Hematopoietic Stem ◽

Notch Activity ◽

Arterial Endothelial Cells

AbstractA better understanding of the molecular mechanisms driving hematopoietic stem cell (HSC) specification and expansion may enable better pharmacological strategies to produce them in sufficient numbers for transplantation. In the embryo, HSCs arise from a defined subset of arterial endothelial cells (ECs) located in the aorta–gonad–mesonephros (AGM) region that undergo endothelial-to-hematopoietic transition (EHT). Arterialization and HSC development are generally believed to require the action of Notch. Here we show that although Notch activity is initially required for arterialization, it is detrimental to subsequent EHT. Mechanistically, we show that effective EHT depends on a Mfng-induced decrease in Jag1-Notch signaling in hemogenic ECs. This causes upregulation of Mycn, an important metabolic and cell-cycle regulator that we found to be required for EHT. During the subsequent development of hematopoietic lineages, Mycn expression decreases and its function is taken on by the homologous Myc gene.

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Abstract 565: Ambient Ultrafine Particle Digestion Alters Gut Microbiota in Association With Increased Atherogenic Lipid Metabolites

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.565 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Rongsong Li ◽

Jieping Yang ◽

Arian Saffari ◽

kyung Baek ◽

Jonathan Jacobs ◽

...

Keyword(s):

Gut Microbiota ◽

Ultrafine Particles ◽

Ultrafine Particle ◽

Density Lipoprotein ◽

Neutrophil Infiltration ◽

Microbiota Composition ◽

Active Components ◽

Lipid Metabolites ◽

Gut Microbiota Composition ◽

Atherogenic Lipid

Ambient particulate matter (PM) exposure is associated with atherosclerosis and inflammatory bowel disease. Ultrafine particles (UFP, d p < 0.1-0.2 μm) are redox active components of PM. We hypothesized that orally ingested UFP promoted atherogenic lipid metabolites in both the intestine and plasma via altered gut microbiota composition. Low density lipoprotein receptor-null ( Ldlr -/- ) mice on a high-fat diet were orally administered with vehicle control or UFP (40 μg/mouse/day) for 3 days a week. After 10 weeks, UFP ingested mice developed macrophage and neutrophil infiltration in the intestinal villi, accompanied by elevated cholesterol but reduced coprostanol levels in the cecum, as well as elevated atherogenic lysophosphatidylcholine (LPC 18:1) and lysophosphatidic acids (LPAs) in the intestine and plasma. At the phylum level, Principle Component Analysis revealed significant segregation of microbiota compositions which was validated by Beta diversity analysis. UFP-exposed mice developed increased abundance in Verrocomicrobia but decreased Actinobacteria, Cyanobacteria, and Firmicutes as well as a reduced diversity in microbiome. Spearman’s analysis negatively correlated Actinobacteria with cecal cholesterol, intestinal and plasma LPC18:1, and Firmicutes and Cyanobacteria with plasma LPC 18:1. Thus, ultrafine particles ingestion alters gut microbiota composition, accompanied by increased atherogenic lipid metabolites. These findings implicate the gut-vascular axis in a atherosclerosis model.

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Comparison of red wine extract and polyphenol constituents on endothelin-1 synthesis by cultured endothelial cells

Clinical Science ◽

10.1042/cs103s072s ◽

2002 ◽

Vol 103 (s2002) ◽

pp. 72S-75S ◽

Cited By ~ 40

Author(s):

Noorafza Q. KHAN ◽

Delphine M. LEES ◽

Julie A. DOUTHWAITE ◽

Martin J. CARRIER ◽

Roger CORDER

Keyword(s):

Coronary Heart Disease ◽

Endothelial Cells ◽

Heart Disease ◽

Red Wine ◽

Antioxidant Properties ◽

Active Components ◽

Aortic Endothelial Cells ◽

Endothelin 1 ◽

Dependent Inhibition ◽

Wine Polyphenols

Regular consumption of red wine reduces mortality from coronary heart disease. This observation has been attributed to the anti-thrombotic effects of ethanol and to the antioxidant properties of polyphenolic compounds present in red wine. Here we show that an extract of red wine polyphenols causes a concentration-dependent inhibition of endothelin-1 synthesis in cultured bovine aortic endothelial cells. This action was associated with modifications in phosphotyrosine staining, indicating that the active components of red wine cause specific modifications of tyrosine kinase signalling. Thus inhibition of endothelin-1 synthesis by red wine may reduce the development of atherosclerosis, and hence decrease coronary heart disease.

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Adaptor Protein Crk Is Required for Ephrin-B1-induced Membrane Ruffling and Focal Complex Assembly of Human Aortic Endothelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e02-04-0181 ◽

2002 ◽

Vol 13 (12) ◽

pp. 4231-4242 ◽

Cited By ~ 66

Author(s):

Ken-Ichiro Nagashima ◽

Akira Endo ◽

Hisakazu Ogita ◽

Akiko Kawana ◽

Akiko Yamagishi ◽

...

Keyword(s):

Endothelial Cells ◽

Adaptor Protein ◽

Dominant Negative ◽

Aortic Endothelial Cells ◽

Induced Membrane ◽

Membrane Ruffling ◽

Focal Complex ◽

Human Aortic Endothelial Cells ◽

Aortic Endothelial

Endothelial cell migration is an essential step in vasculogenesis and angiogenesis, in which receptor tyrosine kinases play a pivotal role. We investigated the mechanism by which ephrin-B1 promotes membrane ruffling in human aortic endothelial cells, because membrane ruffling heralds cell body migration. We especially focused on the role of Crk adaptor protein in EphB-mediated signaling. Using DsRed-tagged Crk and a fluorescent time-lapse microscope, we showed that Crk was recruited to the nascent focal complex after ephrin-B1 stimulation. Furthermore, we found that p130Cas, but not paxillin, recruited Crk to the nascent focal complex. The necessity of Crk in ephrin-B1–induced membrane ruffling was shown both by the overexpression of dominant negative Crk mutants and by the depletion of Crk by using RNA interference. Then, we examined the role of two major downstream molecules of Crk, Rac1 and Rap1. The dominant negative mutant of Rac1 completely inhibited ephrin-B1–induced membrane ruffling and focal complex assembly. In contrast, rap1GAPII, a negative regulator of Rap1, did not inhibit ephrin-B1–induced membrane ruffling. However, in rap1GAPII-expressing cells, ephrin-B1 did not induce membrane spreading, probably due to instability of the focal complex. These results indicated that Crk plays a critical role in Rac1-induced membrane ruffling and Rap1-mediated nascent focal complex stabilization contributing to ephrin-B1–induced human aortic endothelial cells migration.

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Ornithine Decarboxylase Activity in Cultured Endothelial Cells Stimulated by Serum, Thrombin and Serotonin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646709 ◽

1978 ◽

Vol 39 (02) ◽

pp. 496-503 ◽

Cited By ~ 8

Author(s):

P A D’Amore ◽

H B Hechtman ◽

D Shepro

Keyword(s):

Endothelial Cells ◽

Ornithine Decarboxylase ◽

Decarboxylase Activity ◽

Aortic Endothelial Cells ◽

Ornithine Decarboxylase Activity ◽

Rate Limiting Step ◽

Bovine Aortic Endothelial Cells ◽

Limiting Step ◽

Rate Limiting ◽

Serum Serotonin

SummaryOrnithine decarboxylase (ODC) activity, the rate-limiting step in the synthesis of polyamines, can be demonstrated in cultured, bovine, aortic endothelial cells (EC). Serum, serotonin and thrombin produce a rise in ODC activity. The serotonin-induced ODC activity is significantly blocked by imipramine (10-5 M) or Lilly 11 0140 (10-6M). Preincubation of EC with these blockers together almost completely depresses the 5-HT-stimulated ODC activity. These observations suggest a manner by which platelets may maintain EC structural and metabolic soundness.

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Effects of Dipyrone on Prostaglandin Production by Human Platelets and Cultured Bovine Aortic Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657338 ◽

1983 ◽

Vol 49 (02) ◽

pp. 132-137 ◽

Cited By ~ 16

Author(s):

A Eldor ◽

G Polliack ◽

I Vlodavsky ◽

M Levy

Keyword(s):

Endothelial Cells ◽

Arachidonic Acid ◽

Competitive Inhibition ◽

Inhibitory Effect ◽

Human Platelets ◽

Ionophore A23187 ◽

Aortic Endothelial Cells ◽

Bovine Aortic Endothelial Cells ◽

Aortic Endothelial

SummaryDipyrone and its metabolites 4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoan- tipyrine inhibited the formation of thromboxane A2 (TXA2) during in vitro platelet aggregation induced by ADP, epinephrine, collagen, ionophore A23187 and arachidonic acid. Inhibition occurred after a short incubation (30–40 sec) and depended on the concentration of the drug or its metabolites and the aggregating agents. The minimal inhibitory concentration of dipyrone needed to completely block aggregation varied between individual donors, and related directly to the inherent capacity of their platelets to synthesize TXA2.Incubation of dipyrone with cultured bovine aortic endothelial cells resulted in a time and dose dependent inhibition of the release of prostacyclin (PGI2) into the culture medium. However, inhibition was abolished when the drug was removed from the culture, or when the cells were stimulated to produce PGI2 with either arachidonic acid or ionophore A23187.These results indicate that dipyrone exerts its inhibitory effect on prostaglandins synthesis by platelets or endothelial cells through a competitive inhibition of the cyclooxygenase system.

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