scholarly journals In Vitro Studies of Synthesis and Release of Factor VIII Related Protein by Endothelial Cells

1977 ◽  
Author(s):  
E. G. D. Tuddenham ◽  
L. W. Hoyer
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Calf Serum ◽  
In Vivo Studies ◽  
Separate Experiment ◽  
Related Protein ◽  
Binding Studies ◽  
Radioactive Label

In vivo studies have shown that many stimuli such as epinephrine, exercise and pregnancy lead to a rise in factor VIII levels. However, the physiologic mechanisms controlling factor VIII levels are poorly, if at all, understood. Since endothelial cells synthesize factor VIII related protein (FVIII:RP) and can be grown in tissue culture, they provide a suitable in vitro model to study synthesis and release of FVIII:RP. Endothelial cells were harvested by collagenase digestion from human umbilical cords and grown in medium 199 supplemented with 20 to 30% pooled human serum. Confluent cultures were washed and then maintained in medium 199 supplemented with 20% fetal calf serum. Release of FVIII:RP into the medium was measured by immunoradiometric assay. Labeled amino acids were added to the medium for studies of FVIII:RP synthesis. Incorporation of radioactive label into FVIII:RP was measured in binding studies using a specific immunoadsorbent. Epinephrine in concentrations from 1 ng to 10 ug per ml had no effect on rate of release of FVIII:RP from cultured endothelial cells, suggesting that the in vivo effect of epinephrine is not due to a direct action on endothelial cells. In a separate experiment,exogenous FVIII:RP was added to the culture medium at a high concentration (2 units FVIII:RP per ml) along with 3H Leucine. A control without exogenous VIII:RP incorporated as much radioactivity into VIII:RP as did the culture with added FVIII:RP. This result suggests that there is no end product inhibition of FVIII:RP synthesis which operates on the endothelial cell.

Factor VIIa induces extracellular vesicles from the endothelium: A potential mechanism for its hemostatic effect

Blood ◽  
2021 ◽  
Author(s):  
Kaushik Das ◽  
Shiva Keshava ◽  
Shabbir A Ansari ◽  
Vijay Kumar Reddy Kondreddy ◽  
Charles Esmon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Factor Viia ◽  
Mutant Mice ◽  
In Vivo Studies ◽  
Cell Protein ◽  
Wild Type ◽  
Hemostatic Effect

Recombinant FVIIa (rFVIIa) is used as a hemostatic agent to treat bleeding disorders in hemophilia patients with inhibitors and other groups of patients. Our recent studies showed that FVIIa binds endothelial cell protein C receptor (EPCR) and induces protease-activated receptor 1 (PAR1)-mediated biased signaling. The importance of FVIIa-EPCR-PAR1-mediated signaling in hemostasis is unknown. In the present study, we show that FVIIa induces the release of extracellular vesicles (EVs) from endothelial cells both in vitro and in vivo. Silencing of EPCR or PAR1 in endothelial cells blocked the FVIIa-induced generation of EVs. Consistent with these data, FVIIa treatment enhanced the release of EVs from murine brain endothelial cells isolated from wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice. In vivo studies revealed that administration of FVIIa to wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice, increase the number of circulating EVs. EVs released in response to FVIIa treatment exhibit enhanced procoagulant activity. Infusion of FVIIa-generated EVs and not control EVs to platelet-depleted mice increased thrombin generation at the site of injury and reduced blood loss. Administration of FVIIa-generated EVs or generation of EVs endogenously by administering FVIIa augmented the hemostatic effect of FVIIa. Overall, our data reveal that FVIIa treatment, through FVIIa-EPCR-PAR1 signaling, releases EVs from the endothelium into the circulation, and these EVs contribute to the hemostatic effect of FVIIa.

scholarly journals Cardioprotective Activity of Selected Polyphenols Based on Epithelial and Aortic Cell Lines. A Review

Molecules ◽  
2020 ◽  
Vol 25 (22) ◽  
pp. 5343
Author(s):  
Michał Otręba ◽  
Leon Kośmider ◽  
Jerzy Stojko ◽  
Anna Rzepecka-Stojko
Keyword(s):  
Endothelial Cells ◽  
Tube Formation ◽  
Health Claims ◽  
In Vivo Studies ◽  
Positive Effects ◽  
Endothelial Tube Formation ◽  
Disease Therapy ◽  
Species Production

Polyphenols have recently gained popularity among the general public as products and diets classified as healthy and containing naturally occurring phenols. Many polyphenolic extracts are available on the market as dietary supplements, functional foods, or cosmetics, taking advantage of clients’ desire to live a healthier and longer life. However, due to the difficulty of discovering the in vivo functions of polyphenols, most of the research focuses on in vitro studies. In this review, we focused on the cardioprotective activity of different polyphenols as possible candidates for use in cardiovascular disease therapy and for improving the quality of life of patients. Thus, the studies, which were mainly based on endothelial cells, aortic cells, and some in vivo studies, were analyzed. Based on the reviewed articles, polyphenols have a few points of action, including inhibition of acetylcholinesterase, decrease in reactive oxygen species production and endothelial tube formation, stimulation of acetylcholine-induced endothelium-derived mediator release, and others, which lead to their cardio- and/or vasoprotective effects on endothelial cells. The obtained results suggest positive effects of polyphenols, but more long-term in vivo studies demonstrating effects on mechanism of action, sensitivity, and specificity or efficacy are needed before legal health claims can be made.

IN VIVO STUDIES ON RED CELLS AND PLATELETS STORED IN HALFSTRENGTH CITRATE

1987 ◽  
Author(s):  
C Prewse ◽  
K Bell ◽  
B Griffin
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Red Cells ◽  
In Vivo Studies ◽  
Dramatic Improvement ◽  
Half Strength ◽  
Survival Studies ◽  
Cellular Components

We have previously shown that donation of blood into anticoagulants containing half the normal amount of citrate results in a dramatic improvement in the stability of coagulation factor VIII and has no adverse effect on the in vitro qualities of red cells or platelets during storage. To confirm the viability of stored cellular components we are now performing autologous survival studies in healthy volunteers using radiolabelled cells from red cells and platelets stored for 35 and 5 days respectively. Results to date indicate a 24 hour survival of 80% for red cells stored at a haematocrit of 0.70 for 35 days. Infusion of Ill-In oxine labelled platelets after storage for 5 days in full or half-strength citrate gave recoveries of 40% and survivals of 7 days. These encouraging results suggest use of halfstrength citrate may be a route to increasing factor VIII supply without any additional donor recruitment. Further in vitro studies have also been performed on cellular components and reveal adequate in vitro quality for half-strength citrate blood held at room temperature for 20 hours prior to component preparation.

scholarly journals SCIDOT-28. TARGETED NANOPARTICLES FOR IMPROVED DRUG DELIVERY TO MEDULLOBLASTOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi277-vi277
Author(s):  
Joelle P Straehla ◽  
Natalie Boehnke ◽  
Tamara G Dacoba ◽  
Paula T Hammond
Keyword(s):  
Drug Delivery ◽  
Endothelial Cells ◽  
Side Effects ◽  
Tumor Cells ◽  
Chemical Engineering ◽  
Xenograft Model ◽  
In Vivo Studies ◽  
Layer By Layer

Abstract Platinum-based agents remain a key component of therapy for children with medulloblastoma, despite significant systemic side effects and only modest blood-brain barrier (BBB) penetration. Cisplatin has a cerebrospinal fluid-to-plasma ratio <5% and dose-limiting side effects of nephrotoxicity, ototoxicity, and myelosuppression. Improving delivery of cisplatin across the BBB and selectively accumulating in tumors could improve its therapeutic index. To this end, we are leveraging chemical engineering techniques to rationally design cisplatin nanoparticles (NPs) to cross the BBB and preferentially enter medulloblastoma tumor cells. Using the layer-by-layer (LbL) platform to ‘wrap’ polyelectrolytes around a NP core by iterative electrostatic adsorption, we screened six negatively charged polypeptide and polysaccharide outer layers in medulloblastoma cell lines. Poly-L-aspartic acid (PLD) layered NPs had significant accumulation in tumor cells after 24 hours incubation, with an uptake index of 18±4 over unlayered control NPs. Next, we generated propargyl-functionalized PLD and used click chemistry to covalently conjugate the BBB shuttle ligands glutathione, angiopep-2, and transferrin, which have been shown to mediate transcytosis across brain endothelial cells. PLD layered NPs functionalized with angiopep-2 and transferrin had enhanced uptake in medulloblastoma tumor cells and NPs functionalized with glutathione were non-inferior to PLD layered NPs. After incubation with endothelial cells in vitro, all three BBB shuttle ligands enhanced uptake of PLD layered NPs over unlayered and non-functionalized control NPs. We then incorporated cisplatin into the nanoparticle core of this platform. Cisplatin-loaded NPs with PLD layering and ligand functionalization were more effective than free cisplatin as measured by IC50 over 72 hours in culture, and led to faster apoptosis as assessed by flow cytometry with annexin V and propidium iodide staining. In summary, functionalized nanoparticles are a promising platform to modulate drug delivery to medulloblastoma. In vivo studies using an orthotopic xenograft model are underway to investigate biodistribution, efficacy, and toxicity.

Abstract 12659: Hepatoma-Derived Growth Factor Related Protein-3 as a Novel Endothelial Ligand

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Michelle LeBlanc ◽  
Weiwen Wang ◽  
Feiye Guo ◽  
Chen Shen ◽  
Rui Chen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Umbilical Vein ◽  
Growth Effect ◽  
Human Aorta ◽  
Related Protein ◽  
And Migration ◽  
Endothelial Ligands

Background: Endothelial ligands extrinsically regulate a broad spectrum of vascular functions with therapeutic potentials, but are traditionally identified on a case-by-case basis with technical challenges. We recently developed open reading frame phage display (OPD) for unbiased identification of phagocytosis ligands. In this study, we identified hepatoma-derived growth factor related protein-3 (HRP-3) as a putative endothelial ligand by OPD. We hypothesized that HRP-3 is a novel endothelial growth factor, capable of promoting endothelial cell (EC) growth and migration. Methods and Results: We performed 3 rounds of in vivo phage binding selection in mice with an OPD library, screened enriched phage clones by next generation DNA sequencing, and identified HRP-3 as one of the putative endothelial ligands. To confirm the finding, clonal phages displaying HRP-3, VEGF and GFP were generated and analyzed for their binding to human umbilical vein endothelial cells (HUVECs). The results show that HRP-3-Phage and VEGF-Phage had significantly higher binding to HUVECs than GFP-Phage. Functional analysis showed that purified recombinant HRP-3 significantly increased the proliferation of HUVECs at 24 and 48 h, whereas VEGF induced significant growth only at 48 h. Consistent with these findings, HRP-3 significantly stimulated cell proliferation by MTT assay. In vitro wound-healing assay indicated that both HRP-3 (500 ng/ml) and VEGF (50 ng/ml) significantly promoted the migration of HUVECs into the denuded area. To dissect the downstream signaling pathway, we demonstrated that HRP-3 significantly induced ERK1/2 phosphorylation in HUVECs after 10 min treatment. Similar effects of HRP-3 and VEGF on EC growth, migration, and ERK activation were also verified using human aorta endothelial cells. Conclusions: Our findings demonstrate that HRP-3 is a novel ligand, capable of promoting proliferation and migration of ECs. The pro-growth effect of HRP-3 is at least partially mediated through ERK pathway activation. These results in turn support the broad applicability of OPD for the systematic discovery of endothelial ligands.

Circulating CD62E Occurs in Soluble Form as Well as Bound to Endothelial Microparticles (EMP): Functional Differences in Platelet Aggregation in Thrombotic Thrombocytopenic Purpura (TTP).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2624-2624
Author(s):  
Joaquin J. Jimenez ◽  
Wenche Jy ◽  
Lucia M. Mauro ◽  
Michael N. Markou ◽  
George W. Burke ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Platelet Aggregation ◽  
Acute Phase ◽  
Critical Role ◽  
In Vivo Studies ◽  
Soluble Form ◽  
Dependent Manner

Abstract Injured endothelial cells (EC) are believed to play a critical role in the pathophysiology of TTP. Soluble markers of endothelial disturbance measured by enzyme-linked immunoassay (ELISA) have been found elevated in TTP. We have recently demonstrated an increase in the release of CD31/42b- EMP, and CD62E+ EMP. Moreover, we have observed that CD62E+ EMP also express vWF. The aim of this study was to quantitate soluble (s) vs. EMP-bound CD62E (bCD62E) in vitro and in vivo, in relation to the functional activity of vWF+ EMP. METHODS: Brain and renal microvascular endothelial cells (MVEC) were cultured and treated with 10ng/mL TNF-α to induce activation, or deprived of serum and growth factors (GFD) to induce apoptosis. Culture supernatants were collected and evaluated in a time-dependent manner. For in vivo studies, platelet-poor plasma was obtained from 4 TTP patients during the acute phase and upon remission. Filtration through 0.1μm, which retains most EMP, was employed to discriminate between (s) and bCD62E. sCD62E was measured by ELISA post-filtration and bCD62E by ELISA pre-filtration. Additionally, CD62E+ and CD62E+/vWF+ EMP were measured by flow cytometry. To assess pro-aggregatory function, EMP were added to washed platelets in the presence of 1 mg/mL ristocetin and aggregates were measured by flow cytometry. RESULTS: In vitro: Activation did not induce release of sCD62E at 3 hours, although bCD62E was present (1.5±0.5X106 EMP/mL). At 6 hours, some sCD62E was detected in the filtrate (0.09±0.02 ng/mL), but most was present in the unfiltered medium (3.5±0.85 ng/mL), signifying that the majority was bCD62E, confirmed by a doubling of CD62E+ EMP (3.0±0.6X106/mL). Subsequently, sCD62E levels were 1.0±0.2 ng/mL at 12 hr, 3.5±0.7 ng/mL at 18 hr, and 5±0.9 ng/mL at 24 hr. In contrast, EMP counts at 12, 18 and 24 hours were 4.6±1, 7±1.3 and 9±1.8 X106/mL (p=0.01, p=0.01, p=0.02, respectively). For all time periods, 40-60% of CD62E were positive for vWF. In control or GFD cultures, there was not a significant increase in sCD62E or CD62E+ EMP at any time period. MVEC from renal gave similar results. In acute TTP plasma samples, CD62E measured by ELISA was significantly increased (65±22 ng/mL) vs. remission (30±6 ng/mL). bCD62E accounted for 50% in acute and 15% in remission. CD62E+/vWF+ EMP were significantly elevated in plasma from acute TTP patients vs. remission (15±4.5 vs. 3±0.5, p=0.01). Sample filtration resulted in a decrease of &gt;95% EMP in both acute and remission TTP plasma. MVEC-derived CD62E+/vWF+ EMP resulted in a dose-dependent increase in platelet aggregation. Additionally, plasma from 4 TTP patients with elevated CD62E+/vWF+ EMP obtained during the acute phase enhanced the formation of platelet aggregates by 48±12% (p=0.02) above remission plasma with low EMP counts. CONCLUSIONS: The results demonstrate that CD62E heretofore regarded as a soluble marker of endothelial dysfunction, in reality exists in both a soluble and EMP-bound form. Indeed, this distinction is highly relevant because CD62E+ EMP also express vWF and are pro-aggregatory to platelets. These EMP have been shown to be elevated during the acute phase of TTP and decrease upon remission. Thus, CD62E+/vWF+ EMP may be active participants in the formation of platelet-rich thrombi in TTP.

An in vitro model of ischemia/reperfusion-induced microvascular injury

1990 ◽  
Vol 259 (1) ◽  
pp. G134-G139 ◽  
Author(s):  
W. Inauen ◽  
D. N. Granger ◽  
C. J. Meininger ◽  
M. E. Schelling ◽  
H. J. Granger ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
In Vitro Model ◽  
Cell Injury ◽  
Ischemia Reperfusion ◽  
In Vivo Studies ◽  
Cell Detachment ◽  
51Cr Release ◽  
Vitro Model

The major objective of this study was to develop an in vitro model of ischemia/reperfusion (I/R)-induced microvascular injury. Cultured venular endothelial cells were grown to confluency, labeled with 51Cr, and exposed to different durations of anoxia (0.5, 1, 2, 3, and 4 h). 51Cr release and cell detachment (indexes of cell injury) were determined at different times after reoxygenation (1, 2, 4, 6, 8, and 18 h). Because in vivo studies have implicated neutrophils in I/R injury, in some experiments human neutrophils were added to the endothelial cells upon reoxygenation. Periods of anoxia greater than or equal to 2 h resulted in 70-80% 51Cr release and 80-95% cell detachment upon reoxygenation. Under these conditions (near maximal injury), the addition of neutrophils produced negligible effects. Periods of anoxia less than or equal to 1 h resulted in 30-40% 51Cr release and 50-60% cell detachment. Under these conditions (moderate cell injury), addition of neutrophils enhanced endothelial cell injury. Using a 30-min period of anoxia, we also assessed the effects of superoxide dismutase (SOD; 300 U/ml) and allopurinol (20 microM) on anoxia/reoxygenation (A/R)-induced injury in the presence or absence of neutrophils. In the absence of neutrophils, SOD or allopurinol did not protect against A/R-induced injury. However, in the presence of neutrophils, both SOD and allopurinol attenuated the increases in 51Cr release. The results derived using this in vitro model of I/R injury are largely consistent with published in vivo studies. Thus this in vitro model may provide further insights regarding the mechanisms involved in I/R injury.

scholarly journals In Vitro Investigation of Vascular Permeability in Endothelial Cells from Human Artery, Vein and Lung Microvessels at Steady-State and Anaphylactic Conditions

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 439
Author(s):  
Katrine T. Callesen ◽  
Alma Yuste-Montalvo ◽  
Lars K. Poulsen ◽  
Bettina M. Jensen ◽  
Vanesa Esteban
Keyword(s):  
Endothelial Cells ◽  
Steady State ◽  
Vascular Permeability ◽  
Receptor Expression ◽  
In Vivo Studies ◽  
Human Artery ◽  
In Vitro Investigation ◽  
Fast Acting

Human anaphylactic reactions largely involve an increase in vascular permeability, which is mainly controlled by endothelial cells (ECs). Due to the acute and serious nature of human anaphylaxis, in vivo studies of blood vessels must be replaced or supplemented with in vitro models. Therefore, we used a macromolecular tracer assay (MMTA) to investigate the EC permeability of three phenotypes of human ECs: artery (HAECs), vein (HSVECs) and microvessels from lung (HMLECs). ECs were stimulated with two fast-acting anaphylactic mediators (histamine and platelet-activating factor (PAF)) and one longer-lasting mediator (thrombin). At steady-state conditions, HSVEC monolayers were the most permeable and HMLEC the least (15.8% and 8.3% after 60 min, respectively). No response was found in ECs from artery or vein to any stimuli. ECs from microvessels reacted to stimulation with thrombin and also demonstrated a tendency of increased permeability for PAF. There was no reaction for histamine. This was not caused by missing receptor expression, as all three EC phenotypes expressed receptors for both PAF and histamine. The scarce response to fast-acting mediators illustrates that the MMTA is not suitable for investigating EC permeability to anaphylactic mediators.

scholarly journals P-selectin and platelet-activating factor mediate initial endotoxin-induced neutropenia.

1994 ◽  
Vol 179 (1) ◽  
pp. 329-334 ◽  
Author(s):  
A F Coughlan ◽  
H Hau ◽  
L C Dunlop ◽  
M C Berndt ◽  
W W Hancock
Keyword(s):  
Endothelial Cells ◽  
Platelet Activating Factor ◽  
Surface Expression ◽  
Rapid Onset ◽  
In Vivo Studies ◽  
Polymorphonuclear Neutrophil ◽  
Initial Adhesion ◽  
Kidney Liver

Polymorphonuclear neutrophil (PMN) accumulation within damaged tissues, a hallmark of acute inflammation, is dependent upon initial adhesion to endothelial cells. In vitro studies suggest that P-selectin and platelet activating factor (PAF) are key molecules in this process by promoting the initial adhesion of PMN to endothelial cells. We report in vivo studies in which intravenous administration of lipopolysaccharide (LPS) to anesthetized rats caused a very rapid onset (&lt; 5 min) of neutropenia, in association with induction of surface expression of P-selectin on microvascular endothelial cells in kidney, liver and lung; analogous induction of P-selectin expression by cultured endothelial cells was observed in response to LPS stimulation in vitro. In addition, treatment with an antibody (Ab) to P-selectin (or use of a PAF antagonist) blocked development of neutropenia in vivo for at least 15 min post-LPS injection, and Ab treatment was shown to block PMN accumulation in tissues. These studies document roles for P-selectin and PAF in the early adhesion of PMN to endothelial cells in vivo.

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