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

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.

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

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Kyung In Baek ◽  
René R Packard ◽  
Arian Saffari ◽  
Zhao Ma ◽  
Anh P Luu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Notch Signaling ◽  
Ultrafine Particles ◽  
Ultrafine Particle ◽  
Dominant Negative ◽  
Vascular Repair ◽  
Active Components ◽  
Aortic Endothelial Cells ◽  
Notch Activity ◽  
Differentiation And Proliferation

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.

Early Neuromotor Behavior in Craniosynostotic Rabbits

2003 ◽  
Vol 40 (5) ◽  
pp. 486-492 ◽  
Author(s):  
Ronal L. Mitchell ◽  
Timothy E. Barbano ◽  
H. Wolfgang Losken ◽  
Michael I. Siegel ◽  
Mark P. Mooney
Keyword(s):  
Early Onset ◽  
Rabbit Model ◽  
Zealand White Rabbit ◽  
Psychomotor Development ◽  
Activity Levels ◽  
Coronal Suture ◽  
Wild Type ◽  
Significant Group ◽  
Delayed Onset ◽  
Neuromotor Development

Objective Clinical studies have shown both abnormal and normal mental and psychomotor development in patients with craniosynostosis. However, a number of confounding variables make study comparisons difficult. For these reasons, the present study describes early neuromotor development in an homogeneous rabbit model of craniosynostosis. Design Fifty-three newborn New Zealand white rabbit kits were used: 13 were wild-type, normal control rabbits; 23 had delayed-onset coronal suture synostosis (onset is approximately 57 to 74 days post conception); and 17 had early-onset coronal suture synostosis (onset is approximately 21 to 25 days post conception). All rabbits were observed individually and blindly in an open field, daily for 2 minutes, from birth through the first 14 days of life. The first day of emergence of 10 different mature behaviors and developmental events (in developmental order of appearance: falling, righting, cliff avoidance, first sign of fur, body elevation, head elevation, circling, dragging, eye opening, and hopping) was recorded for each kit. Daily activity levels (grid crossing), and body weights were also recorded. Results Significant group (p < .05) differences were observed in 9 of 11 measures. Both synostosed groups had significantly (p < .05) accelerated onset of behavior in 8 of 9 measures, compared with wild-type controls. The early-onset synostosis group had significantly (p < .05) accelerated onset in five of eight measures, compared with wild-type controls, and three of eight measures, compared with the delayed-onset synostosis group. Conclusions Synostotic rabbits showed precocious neuromotor development possibly through frontal lobe constrictions and altered brain activity from increased intracranial pressure, although primary genetic effects cannot be ruled out.

scholarly journals Endothelial lumen signaling complexes control 3D matrix–specific tubulogenesis through interdependent Cdc42- and MT1-MMP–mediated events

Blood ◽  
2010 ◽  
Vol 115 (25) ◽  
pp. 5259-5269 ◽  
Author(s):  
Anastasia Sacharidou ◽  
Wonshill Koh ◽  
Amber N. Stratman ◽  
Anne M. Mayo ◽  
Kevin E. Fisher ◽  
...  
Keyword(s):  
Tube Formation ◽  
Dominant Negative ◽  
Collagen Matrices ◽  
Signaling Complex ◽  
Vascular Morphogenesis ◽  
Cytoplasmic Tails ◽  
3D Matrix ◽  
Dependent Signal ◽  
3D Collagen ◽  
Membrane Type

Abstract Here, we define an endothelial cell (EC) lumen signaling complex involving Cdc42, Par6b, Par3, junction adhesion molecule (Jam)–B and Jam-C, membrane type 1–matrix metalloproteinase (MT1-MMP), and integrin α2β1, which coassociate to control human EC tubulogenesis in 3D collagen matrices. Blockade of both Jam-B and Jam-C using antibodies, siRNA, or dominant-negative mutants completely interferes with lumen and tube formation resulting from a lack of Cdc42 activation, inhibition of Cdc42-GTP–dependent signal transduction, and blockade of MT1-MMP–dependent proteolysis. This process requires interdependent Cdc42 and MT1-MMP signaling, which involves Par3 binding to the Jam-B and Jam-C cytoplasmic tails, an interaction that is necessary to physically couple the components of the lumen signaling complex. MT1-MMP proteolytic activity is necessary for Cdc42 activation during EC tube formation in 3D collagen matrices but not on 2D collagen surfaces, whereas Cdc42 activation is necessary for MT1-MMP to create vascular guidance tunnels and tube networks in 3D matrices through proteolytic events. This work reveals a novel interdependent role for Cdc42-dependent signaling and MT1-MMP–dependent proteolysis, a process that occurs selectively in 3D collagen matrices and that requires EC lumen signaling complexes, to control human EC tubulogenesis during vascular morphogenesis.

scholarly journals Shear Stress–Activated Wnt-Angiopoietin-2 Signaling Recapitulates Vascular Repair in Zebrafish Embryos

2014 ◽  
Vol 34 (10) ◽  
pp. 2268-2275 ◽  
Author(s):  
Rongsong Li ◽  
Tyler Beebe ◽  
Nelson Jen ◽  
Fei Yu ◽  
Wakako Takabe ◽  
...  
Keyword(s):  
Shear Stress ◽  
Zebrafish Embryos ◽  
Vascular Repair ◽  
Angiopoietin 2

Endothelial albumin permeability is shear dependent, time dependent, and reversible

1991 ◽  
Vol 260 (6) ◽  
pp. H1992-H1996 ◽  
Author(s):  
H. Jo ◽  
R. O. Dull ◽  
T. M. Hollis ◽  
J. M. Tarbell
Keyword(s):  
Shear Stress ◽  
Endothelial Cell ◽  
Fluorescein Isothiocyanate ◽  
Vascular Wall ◽  
Laminar Shear Stress ◽  
Aortic Endothelial Cells ◽  
Transendothelial Permeability ◽  
Steady Laminar Shear Stress ◽  
Laminar Shear

Altered permeability of vascular endothelium to macromolecules may play a role in vascular disease as well as vascular homeostasis. Because the shear stress of flowing blood on the vascular wall is known to influence many endothelial cell properties, an in vitro system to measure transendothelial permeability (Pe) to fluorescein isothiocyanate conjugated bovine serum albumin under defined physiological levels of steady laminar shear stress was developed. Bovine aortic endothelial cells grown on polycarbonate filters pretreated with gelatin and fibronectin constituted the model system. Onset of 1 dyn/cm2 shear stress resulted in a Pe rise from 5.1 +/- 1.3 x 10(-6) cm/s to 21.9 +/- 4.6 X 10(-6) cm/s at 60 min (n = 6); while 10 dyn/cm2 shear stress increased Pe from 4.8 +/- 1.5 X 10(-6) cm/s to 50.2 +/- 6.8 X 10(-6) cm/s at 30 min and 49.6 +/- 8.9 X 10(-6) cm/s at 60 (n = 9). Pe returned to preshear values within 120 and 60 min after removal of 1 and 10 dyn/cm2 shear stress, respectively. The data show that endothelial cell Pe in vitro is acutely sensitive to shear stress.

scholarly journals Adenosine triphosphate is a critical determinant for VEGFR signal during hypoxia

2016 ◽  
Vol 311 (6) ◽  
pp. C985-C995 ◽  
Author(s):  
Abdullah Al Mamun ◽  
Hisaki Hayashi ◽  
Miho Sakima ◽  
Motohiko Sato
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Signal Propagation ◽  
Tube Formation ◽  
Activation Process ◽  
Vegf Receptor ◽  
Vegf Mrna ◽  
Critical Determinant ◽  
Hypoxia Exposure ◽  
Vegf Signaling

Hypoxia induces angiogenesis through the VEGF signaling pathway; however, signal propagation of VEGF in hypoxia is not fully understood. In this study, we examined alterations in VEGF signaling during hypoxia conditions and its determinant in endothelial cells. To analyze VEGF signaling during hypoxia, human umbilical vein endothelial cells (HUVECs) were exposed to 3 h of hypoxia (1% O2) followed by 3 h of reoxygenation or 12 h of hypoxia. Hypoxia induced expression of VEGF mRNA, but it was not associated with an increase in tube formation by HUVECs. During 3 h of hypoxia, VEGF-induced phosphorylation of VEGF receptor-2 (VEGFR-2) and downstream molecules were significantly inhibited without a change in VEGFR-2 expression, but it was completely restored after reoxygenation. VEGF-mediated VEGFR-2 phosphorylation is associated with a reduction in cellular ATP in hypoxia conditions (65.93 ± 8.32% of normoxia, means ± SE, P < 0.01). Interestingly, attenuation of VEGFR-2 phosphorylation was restored by addition of ATP to prepared membranes from cells that underwent 3 h of hypoxia. In contrast to 3 h of hypoxia, exposure of cells to 12 h of hypoxia decreased VEGFR-2 expression and VEGF-mediated VEGFR-2 phosphorylation. The magnitude of VEGFR-2 phosphorylation was not fully restored by addition of ATP to prepared membranes from cells exposed to 12 h of hypoxia. These data indicate that ATP is an important determinant of VEGF signaling in hypoxia and suggest that the activation process of VEGFR-2 was modified by sustained hypoxia. These observations contribute to our understanding of signal alterations in VEGF in endothelial cells during hypoxia.

Abstract 087: Mitochondrial Fission Inducer, Dynamin-Related Protein 1 (DRP1), is Required for Endothelial Inflammatory Responses

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Steven J Forrester ◽  
Tatsuo Kawai ◽  
Katherine J Elliott ◽  
Kunie Eguchi ◽  
Victor Rizzo ◽  
...  
Keyword(s):  
Er Stress ◽  
Inflammatory Responses ◽  
Vascular Dysfunction ◽  
Mitochondrial Fission ◽  
Critical Role ◽  
Dominant Negative ◽  
Low Grade ◽  
Aortic Endothelial Cells ◽  
Endothelial Inflammation ◽  
Inflammatory Stimuli

Among various cardiovascular diseases, hypertension (HTN) is considered to be a disease plagued by chronic low-grade inflammation associated with endothelial dysfunction. Interestingly, recent studies have identified mitochondrial adaptation and/or dysfunction as components to hypertensive vascular dysfunction. While mitochondria are indispensable to maintain cellular metabolism, they also participate in adaptive and maladaptive cell/tissue responses via several retro grade signaling pathways. DRP1 plays a major role in mitochondrial quality control. However, whether DRP1 is involved in mitochondrial dysfunction and endothelial inflammation during development of HTN remains unknown. In the present study, we tested the hypothesis that inflammatory stimuli, through DRP1-dependent mitochondrial alteration, enhance endothelial inflammation. In cultured rat aortic endothelial cells (RAECs), TNFα (10 μg/mL) transiently induced mitochondrial fission maximally at 3h which was inhibited using a mitochondrial fission inhibitor, Mdivi1 (10 μM) (0.16±0.04 vs 0.10±0.02 mitochondria fragmentation count with MitoTracker, p<.01 ). TNFα and FCCP (a fission agonist, 10 μM) increased THP-1 monocyte adhesion to RAECs, which was also inhibited with Mdivi1 (256±17 vs 139±16 for TNFα, 238±30 vs 156±14 for FCCP, attached cells per field scanned, p<.01 ). Likewise, mdivi1 and adenoviruses encoding siRNA for DRP1 or dominant-negative K38A DRP1 (50 moi) attenuated TNFα-induced VCAM-1 induction in RAECs. TNFα increased aerobic respiration, which was prevented by mdivi1 or ER stress inhibitor PBA (10 mM). Inhibition of ER stress, glycolysis or mitochondrial respiration using PBA, 2-DG (1 mg/mL) or oligomycin (1 μM) prevented VCAM-1 induction. However, suppression of TNFα-induced mitochondrial ROS production by mito-Tempo (25 nM) was unable to prevent VCAM-1 induction. In C57BL6 mice receiving AngII (1000 ng/kg/min, 2 weeks) infusion, treatment with Mdivi-1 (25 mg/kg ip every other day) or PBA (1g/kg/day) prevented vascular VCAM-1 induction. In conclusion, our data suggests a critical role for ER stress and subsequent functional and structural remodeling of mitochondria induced by DRP1 in mediating endothelial inflammatory activation in HTN.

Abstract 11: Thioredoxin-Interacting Protein Is Required for VEGF-Induced Angiogenesis Through Regulation of VEGFR2 Internalization

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Shin-Young Park ◽  
Chen Yan ◽  
Bradford C Berk
Keyword(s):  
Umbilical Vein ◽  
Fold Increase ◽  
Tube Formation ◽  
In Vivo Studies ◽  
Vegf Receptor ◽  
Cell Fractionation ◽  
Interacting Protein ◽  
Thioredoxin Interacting Protein

Introduction— Thioredoxin-interacting protein (TXNIP) is an arrestin-like scaffold protein. We have shown previously that it is necessary for the transactivation of the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) as well as promoting the migration and survival of endothelial cells (ECs). However, its roles in VEGF-induced angiogenesis and in vivo studies of TXNIP function have not been elucidated. Hypothesis— TXNIP regulates VEGF-mediated angiogenesis through modulation of angiogenic signaling pathways in ECs. Methods and Results— To determine the functions of TXNIP in ECs, we generated endothelial-specific TXNIP knockout (EC-TXNIP KO) mice (TXNIPflox/flox: Tie2-Cre/+). These mice displayed impaired capillary growth of the retinal vasculature compared to control mice. Furthermore, aortic rings from EC-TXNIP KO mice exhibited fewer and shorter vascular sprouts than those in control mice. To investigate the role of TXNIP in the regulation of VEGF-induced angiogenesis, we determined the subcellular localization of TXNIP in human umbilical vein EC (HUVEC). Immunofluorescence and cell fractionation studies revealed that upon VEGF stimulation (10ng/ml). TXNIP translocated from cytoplasm to the plasma membrane. There was a 9 fold increase of membrane associated TXNIP with a peak at 15 minutes compared to non-VEGF treatment cells. We hypothesized that membrane associated TXNIP may modulate VEGFR2 internalization and thereby affect VEGF-induced signaling and angiogenesis. To investigate this, we performed in vitro cell surface biotinylation assays in HUVEC. VEGFR2 internalization was decreased by 65% in TXNIP siRNA knockdown cells compared to control siRNA treated cells following VEGF stimulation. Consistent with this result, VEGF-induced phosphorylation of VEGFR2, PLCγ and ERK1/2 was decreased by knockdown of TXNIP. Significantly, TXNIP knockdown inhibited VEGF-induced proliferation and tube formation in vitro. Conclusion— Our results suggest that TXNIP can modulate VEGF-induced angiogenesis and signaling by regulation of VEGFR2 internalization.

Sign in / Sign up

Export Citation Format

Share Document