Induction and Suppression of Endothelial Cell Apoptosis by Sphingolipids: A Possible In Vitro Model for Cell-Cell Interactions Between Platelets and Endothelial Cells

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4293-4299 ◽  
Author(s):  
Nobuo Hisano ◽  
Yutaka Yatomi ◽  
Kaneo Satoh ◽  
Shigeo Akimoto ◽  
Masako Mitsumata ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Cell Interaction ◽  
Dependent Manner ◽  
Survival Factor ◽  
Highly Active ◽  
Sphingosine 1 Phosphate ◽  
Umbilical Vein Endothelial Cells

Because sphingosine (Sph) is actively incorporated into platelets and rapidly converted to sphingosine 1-phosphate (Sph-1-P), which is then released extracellularly, it is important to study the effects of Sph and Sph-1-P on endothelial cells from the viewpoint of platelet-endothelial cell interaction. In this study, we found that Sph, as well as ceramide, induces apoptosis in human umbilical vein endothelial cells (HUVECs). In contrast, Sph-1-P acts as a HUVEC survival factor; this bioactive lipid was shown to protect HUVECs from apoptosis induced by the withdrawal of growth factors and to stimulate HUVEC DNA synthesis. In metabolic studies, [3H]Sph, incorporated into HUVECs, was converted to [3H]Cer and further to [3H]sphingomyelin in a time-dependent manner, whereas [3H]Sph-1-P formation from [3H]Sph was weak and transient. These findings in HUVECs are very different from those of platelets, which possess a highly active Sph kinase but lack Sph-1-P lyase. As a result, platelets abundantly store Sph-1-P, whereas HUVECs contain much less Sph-1-P. Finally, HUVECs, in contrast to platelets, failed to release Sph-1-P extracellularly, indicating that HUVECs themselves are not able to supply the survival factor Sph-1-P, but receive it from activated platelets. Our results suggest that platelets may maintain the integrity of endothelial cells by incorporating Sph and releasing Sph-1-P.

Induction and Suppression of Endothelial Cell Apoptosis by Sphingolipids: A Possible In Vitro Model for Cell-Cell Interactions Between Platelets and Endothelial Cells

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4293-4299 ◽  
Author(s):  
Nobuo Hisano ◽  
Yutaka Yatomi ◽  
Kaneo Satoh ◽  
Shigeo Akimoto ◽  
Masako Mitsumata ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Cell Interaction ◽  
Dependent Manner ◽  
Survival Factor ◽  
Highly Active ◽  
Sphingosine 1 Phosphate ◽  
Umbilical Vein Endothelial Cells

Abstract Because sphingosine (Sph) is actively incorporated into platelets and rapidly converted to sphingosine 1-phosphate (Sph-1-P), which is then released extracellularly, it is important to study the effects of Sph and Sph-1-P on endothelial cells from the viewpoint of platelet-endothelial cell interaction. In this study, we found that Sph, as well as ceramide, induces apoptosis in human umbilical vein endothelial cells (HUVECs). In contrast, Sph-1-P acts as a HUVEC survival factor; this bioactive lipid was shown to protect HUVECs from apoptosis induced by the withdrawal of growth factors and to stimulate HUVEC DNA synthesis. In metabolic studies, [3H]Sph, incorporated into HUVECs, was converted to [3H]Cer and further to [3H]sphingomyelin in a time-dependent manner, whereas [3H]Sph-1-P formation from [3H]Sph was weak and transient. These findings in HUVECs are very different from those of platelets, which possess a highly active Sph kinase but lack Sph-1-P lyase. As a result, platelets abundantly store Sph-1-P, whereas HUVECs contain much less Sph-1-P. Finally, HUVECs, in contrast to platelets, failed to release Sph-1-P extracellularly, indicating that HUVECs themselves are not able to supply the survival factor Sph-1-P, but receive it from activated platelets. Our results suggest that platelets may maintain the integrity of endothelial cells by incorporating Sph and releasing Sph-1-P.

scholarly journals Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation

2018 ◽  
Vol 19 (9) ◽  
pp. 2753 ◽  
Author(s):  
Nina Zippel ◽  
Annemarieke Loot ◽  
Heike Stingl ◽  
Voahanginirina Randriamboavonjy ◽  
Ingrid Fleming ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Reactivity ◽  
Umbilical Vein ◽  
No Production ◽  
Dependent Manner ◽  
Calmodulin Binding ◽  
Umbilical Vein Endothelial Cells ◽  
Endothelial Nitric Oxide ◽  
Specific Deletion

AMP-activated protein kinase (AMPK) is frequently reported to phosphorylate Ser1177 of the endothelial nitric-oxide synthase (eNOS), and therefore, is linked with a relaxing effect. However, previous studies failed to consistently demonstrate a major role for AMPK on eNOS-dependent relaxation. As AMPK also phosphorylates eNOS on the inhibitory Thr495 site, this study aimed to determine the role of AMPKα1 and α2 subunits in the regulation of NO-mediated vascular relaxation. Vascular reactivity to phenylephrine and acetylcholine was assessed in aortic and carotid artery segments from mice with global (AMPKα−/−) or endothelial-specific deletion (AMPKαΔEC) of the AMPKα subunits. In control and AMPKα1-depleted human umbilical vein endothelial cells, eNOS phosphorylation on Ser1177 and Thr495 was assessed after AMPK activation with thiopental or ionomycin. Global deletion of the AMPKα1 or α2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPKα1 subunit attenuated phenylephrine-mediated contraction in an eNOS- and endothelium-dependent manner. In in vitro studies, activation of AMPK did not alter the phosphorylation of eNOS on Ser1177, but increased its phosphorylation on Thr495. Depletion of AMPKα1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPKα1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.

scholarly journals Thrombin-induced gap formation in confluent endothelial cell monolayers in vitro

Blood ◽  
1983 ◽  
Vol 62 (3) ◽  
pp. 549-556 ◽  
Author(s):  
M Laposata ◽  
DK Dovnarsky ◽  
HS Shin
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Gap Formation ◽  
Culture Dish ◽  
Cell Monolayers ◽  
Umbilical Vein Endothelial Cells

Abstract When thrombin is incubated with confluent monolayers of human umbilical vein endothelial cells in vitro, there is a change in the shape of the endothelial cells that results in gaps in the monolayer, disrupting the integrity of the endothelium and exposing the subendothelium. Using a grid assay to measure this phenomenon, we observed that up to 80% of the surface area once covered by cells was uncovered after a 15-min incubation with 10(-2) U/ml (10(-10)M) thrombin. The effect was apparent within 2 min and did not remove cells from the surface of the culture dish. The gaps in the monolayer completely disappeared within 2 hr after exposure to thrombin. The effect of thrombin was inhibited by preincubation of thrombin with hirudin or antithrombin III plus heparin or by preincubation of the monolayers with dibutyryl cyclic adenosine monophosphate (dbcAMP). Histamine also induced gap formation in endothelial cell monolayers. Both pyrilamine and cimetidine prevented the histamine-induced effect, but they had no effect on thrombin- induced gap formation. Intact monolayers were not disrupted by bradykinin, serotonin, C5a, or C3a. Our results suggest that small amounts of thrombin can induce repeated and transient exposure of the subendothelium, a situation believed to be conducive to atherogenesis and thrombosis.

scholarly journals Endothelialized silicone aneurysm models for in vitro evaluation of flow diverters

2020 ◽  
pp. neurintsurg-2020-016859
Author(s):  
Alyssa McCulloch ◽  
Ashley Turcott ◽  
Gabriella Graham ◽  
Sergey Frenklakh ◽  
Kristen O'Halloran Cardinal
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Flow Diverter ◽  
In Vitro Models ◽  
Cell Coverage ◽  
Platelet Endothelial Cell Adhesion ◽  
Umbilical Vein Endothelial Cells ◽  
Over Time

ObjectiveThe goal of this work was to endothelialize silicone aneurysm tubes for use as in vitro models for evaluating endothelial cell interactions with neurovascular devices. The first objective was to establish consistent and confluent endothelial cell linings and to evaluate the silicone vessels over time. The second objective was to use these silicone vessels for flow diverter implantation and assessment.MethodsSilicone aneurysm tubes were coated with fibronectin and placed into individual bioreactor systems. Human umbilical vein endothelial cells were deposited within tubes to create silicone vessels, then cultivated on a peristaltic pump and harvested at 2, 5, 7, or 10 days to evaluate the endothelial cell lining. A subset of silicone aneurysm vessels was used for flow diverter implantation, and evaluated for cell coverage over device struts at 3 or 7 days after deployment.ResultsSilicone vessels maintained confluent, PECAM-1 (platelet endothelial cell adhesion molecule 1) positive endothelial cell linings over time. These vessels facilitated and withstood flow diverter implantation, with robust cell linings disclosed after device deployment. Additionally, the endothelial cells responded to implanted devices through coverage of the flow diverter struts with increased cell coverage over the aneurysm seen at 7 days after deployment as compared with 3 days.ConclusionsSilicone aneurysm models can be endothelialized and successfully maintained in vitro over time. Furthermore, these silicone vessels can be used for flow diverter implantation and assessment.

Modulation Of PGI2-Like Activity Of Human Endothelial Cells By An Extracellular Collagen Matrix

1981 ◽  
Author(s):  
K M Spiegel ◽  
S F Mohammad ◽  
H Y K Chuang ◽  
R G Mason
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Collagen Matrix ◽  
Significant Loss ◽  
Experimental Conditions ◽  
Cell Lysates ◽  
Collagen Coating ◽  
Umbilical Vein Endothelial Cells

Human umbilical vein endothelial cells were grown on several artificial matrices including collagen and on unmodified plastic dishes. Freeze thaw preparations of confluent monolayer endothelial cell cultures were assayed for PGI2-like activity by testing for inhibition of platelet a88regation. The cells grown on a collagen matrix expressed slightly less PGI2-like activity initially compared to the cells grown on plastic. When the PGI2like activity of the cell lysates was examined over a period of three hours after the initial preparation, endothelial cells grown on a collagen matrix exhibited a more rapid loss of activity. Preliminary quantitative evaluations suggest that lysates derived from cells grown on unmodified plastic retained 75% PGI2 like activity at 15 min, at which point collagen grown cells exhibited essentially no PGI2-like activity. Furthermore, as shown in the figure, under identical experimental conditions, cell lysates obtained from endothelial cells grown on unmodified plastic continued to express approximately 50% of the initial PGI2-like activity after one hour. Since addition of purified PGI2 or cell lysates in vitro to the collagen coating used as tissue culture substrate failed to cause any significant loss of platelet aggregation inhibitory activity beyond the normal rate of decay of PGI2 it appears likely that the reduction of PGI2-like activity may be associated with changes in the substrate collagen, possibly effected by the endothelial cell layer. Alternatively, the reduction of activity may be related to differences in the endothelial cells caused by growth on the collagen substrate.

scholarly journals Ethyl Acetate Fraction of Teucrium Polium Extract Abolishes Human Umbilical Vein Endothelial Cells (HUVEC) Tubulogenesis in Collagen Bed through Suppression of Cell Proliferation/VEGF Secretion

2019 ◽  
Author(s):  
Vahide Askari ◽  
Somayeh Shamlou ◽  
Ali Mostafaie ◽  
Sara Khaleqi
Keyword(s):  
Endothelial Cells ◽  
Ethyl Acetate ◽  
Umbilical Vein ◽  
Ethyl Acetate Fraction ◽  
Dependent Manner ◽  
Angiogenic Effect ◽  
Teucrium Polium ◽  
Umbilical Vein Endothelial Cells ◽  
Dose Dependent

Angiogenesis has essential role in growth and metastasis of tumors. Development of therapies aimed to suppress angiogenesis using medicinal plants is one of the effective approaches for prevention/treatment of cancer. The current study was performed to investigate in vitro anti-angiogenic effect of Teucrium Polium (TP) extract and its fractions. The aerial part of Teucrium Polium was powdered and extracted with 50% ethanol. The extract was fractionated in to aqueous (AQ), n-butanol (BU), ethyl acetate (EA) and n-hexane (HE) fractions. Anti-angiogenic effect of TP was examined on human umbilical vein endothelial cells (HUVECs) in three-dimensional collagen matrix. The endothelial cells form capillary-like branches that can be visualized using phase contrast microscope and the number of tube-like structures can be quantified as a measure of in vitro angiogenesis. Furthermore, anti-proliferative and vascular endothelial growth factor(VEGF )suppressive effect of TP as important factors in the process of angiogenesis were assessed using3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)and quantitative ELISA, respectively. Based on our findings, among the TP fractions, EA fraction showed the highest inhibitory activity on angiogenesis. This fraction with IC50: 68 µg/mL, inhibited angiogenesis at 60 µg/mL. The crude extract and other fractions of TP inhibited angiogenesis in a dose-dependent manner at doses higher concentrations than EA fraction, significantly.TP extract and EA fraction were able to inhibit proliferation of HUVEC and inhibited VEGF secretion in a dose dependent manner. The ethyl acetate fraction at 60 µg/ml inhibited VEGF secretion perfectly. Our data indicated that ethyl acetate fraction of Teucrium Polium could be a potential candidate for the prevention of angiogenesis in cancer and other related disorders. However, this suggestion needs more quantitative and in vivo investigations for confirmation.

scholarly journals Immunosuppressive nano-therapeutic micelles downregulate endothelial cell inflammation and immunogenicity

RSC Advances ◽  
2015 ◽  
Vol 5 (54) ◽  
pp. 43552-43562 ◽  
Author(s):  
Satish N. Nadig ◽  
Suraj K. Dixit ◽  
Natalie Levey ◽  
Scott Esckilsen ◽  
Kayla Miller ◽  
...  
Keyword(s):  
Immune Response ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Human Umbilical Vein ◽  
Perfusion Solution ◽  
Umbilical Vein Endothelial Cells

Targeted micelles containing rapamycin (TRaM) suppressed the immune response of IL-8 in oxidatively stressed human umbilical vein endothelial cellsin vitro(a) and accumulated in aorta grafts for transplantation after 6 hours in cold perfusion solution (b).

scholarly journals Autocrine VEGF and IL-8 Promote Migration via Src/Vav2/Rac1/PAK1 Signaling in Human Umbilical Vein Endothelial Cells

2017 ◽  
Vol 41 (4) ◽  
pp. 1346-1359 ◽  
Author(s):  
Li Ju ◽  
Zhiwen Zhou ◽  
Bo Jiang ◽  
Yue Lou ◽  
Xirong Guo
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Endothelial Cell Migration ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Human Umbilical Vein ◽  
Rac1 Activity ◽  
Umbilical Vein Endothelial Cells

Background/Aims: Pro-angiogenic factors VEGF and IL-8 play a major role in modulating the migratory potential of endothelial cells. The goal of this study was to investigate the effect of autocrine VEGF and IL-8 in the form of self-conditioned medium (CM) on human umbilical vein endothelial cells (HUVECs). Methods: Enzyme-linked immunosorbent assay (ELISA) examined the automatic secretion of VEGF and IL-8 protein by HUVECs. Western blot, small interfering RNA (siRNA), pulldown and Transwell assays were used to explore the role and the mechanism of autocrine VEGF and IL-8 in migration of HUVECs. Results: Neutralizing VEGF and IL-8 in CM significantly abrogated CM-induced migration of HUVECs. Autocrine VEGF and IL-8 increased Src phosphorylation, Rac1 activity and PAK1 phosphorylation in a time dependent manner. Additionally, blocking Rac1 activity with Rac1 siRNA largely abolished autocrine VEGF and IL-8-induced cell migration. Vav2 siRNA suppressed autocrine VEGF and IL-8-induced Rac1 activation and cell migration. Furthermore, blocking Src signaling with PP2, a specific inhibitor for Src, markedly prevented autocrine VEGF and IL-8-induced Vav2 and Rac1 activation as well as consequently cell migration. PAK1 siRNA also significantly abolished autocrine VEGF and IL-8-induced cell migration. Conclusions: We demonstrated for the first time that autocrine VEGF and IL-8 promoted endothelial cell migration via the Src/Vav2/Rac1/PAK1 signaling pathway. This finding reveals the molecular mechanism in the increase of endothelial cell migration induced by autocrine growth factors and cytokines, which is expected to provide a novel therapeutic target in vascular diseases.

scholarly journals Thrombin-induced gap formation in confluent endothelial cell monolayers in vitro

Blood ◽  
1983 ◽  
Vol 62 (3) ◽  
pp. 549-556 ◽  
Author(s):  
M Laposata ◽  
DK Dovnarsky ◽  
HS Shin
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Gap Formation ◽  
Culture Dish ◽  
Cell Monolayers ◽  
Umbilical Vein Endothelial Cells

When thrombin is incubated with confluent monolayers of human umbilical vein endothelial cells in vitro, there is a change in the shape of the endothelial cells that results in gaps in the monolayer, disrupting the integrity of the endothelium and exposing the subendothelium. Using a grid assay to measure this phenomenon, we observed that up to 80% of the surface area once covered by cells was uncovered after a 15-min incubation with 10(-2) U/ml (10(-10)M) thrombin. The effect was apparent within 2 min and did not remove cells from the surface of the culture dish. The gaps in the monolayer completely disappeared within 2 hr after exposure to thrombin. The effect of thrombin was inhibited by preincubation of thrombin with hirudin or antithrombin III plus heparin or by preincubation of the monolayers with dibutyryl cyclic adenosine monophosphate (dbcAMP). Histamine also induced gap formation in endothelial cell monolayers. Both pyrilamine and cimetidine prevented the histamine-induced effect, but they had no effect on thrombin- induced gap formation. Intact monolayers were not disrupted by bradykinin, serotonin, C5a, or C3a. Our results suggest that small amounts of thrombin can induce repeated and transient exposure of the subendothelium, a situation believed to be conducive to atherogenesis and thrombosis.

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