Small-molecule cyclic αVβ3 antagonists inhibit sickle red cell adhesion to vascular endothelium and vasoocclusion

2007 ◽  
Vol 293 (2) ◽  
pp. H1038-H1045 ◽  
Author(s):  
Eileen M. Finnegan ◽  
Gilda A. Barabino ◽  
Xiao-du Liu ◽  
Hee-Yoon Chang ◽  
Alfred Jonczyk ◽  
...  
Keyword(s):  
Small Molecule ◽  
Vascular Endothelium ◽  
Cyclic Peptides ◽  
Therapeutic Strategy ◽  
Umbilical Vein ◽  
Platelet Activating Factor ◽  
Αvβ3 Integrin ◽  
Flow Conditions ◽  
Umbilical Vein Endothelial Cells ◽  
Adhesive Proteins

Abnormal adhesion of sickle red blood cells (SS RBCs) to vascular endothelium may play an important role in vasoocclusion in sickle cell disease. Accruing evidence shows that endothelial αVβ3-integrin has an important role in SS RBC adhesion because of its ability to bind several adhesive proteins implicated in this interaction. In the present studies, we tested therapeutic efficacy of small-molecule cyclic pentapeptides for their ability to block αVβ3-mediated SS RBC adhesion by using two well-established assay systems, i.e., cultured human umbilical vein endothelial cells (HUVEC) and artificially perfused mesocecum vasculature of the rat under flow conditions. We tested the efficacy of two RGD-containing cyclic pentapeptides, i.e., cRGDFV (EMD 66203) and cRGDF-ACHA (α-amino cyclohexyl carboxylic acid) (EMD 270179), based on their known ability to bind αVβ3. An inactive peptide, EMD 135981 (cRβ-ADFV) was used as control. Cyclization and the introduction of d-Phe (F) results in a marked increase in the ability of cyclic peptides to selectively bind αVβ3 receptors. In the mesocecum vasculature, both EMD 66203 and EMD 270179 ameliorated platelet-activating factor-induced enhanced SS RBC adhesion, postcapillary blockage, and significantly improved hemodynamic behavior. Infusion of a fluorescent derivative of EMD 66203 resulted in colocalization of the antagonist with vascular endothelium. Also, pretreatment of HUVEC with either αVβ3 antagonist resulted in a significant decrease in SS RBC adhesion. Because of their metabolic stability, the use of these cyclic αVβ3 antagonists may constitute a novel therapeutic strategy to block SS RBC adhesion and associated vasoocclusion under flow conditions.

PAF mediates neutrophil adhesion to thrombin or TNF-stimulated endothelial cells under shear stress

1995 ◽  
Vol 269 (1) ◽  
pp. C42-C47 ◽  
Author(s):  
D. Macconi ◽  
M. Foppolo ◽  
S. Paris ◽  
M. Noris ◽  
S. Aiello ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Platelet Activating Factor ◽  
Flow Chamber ◽  
Tnf Alpha ◽  
Image Analysis System ◽  
Flow Conditions ◽  
Necrosis Factor Alpha ◽  
Umbilical Vein Endothelial Cells ◽  
Static Conditions

Platelet-activating factor (PAF) is known to modulate polymorphonuclear leukocyte (PMN) adhesion to endothelial cells cultured under static conditions and activated by thrombin. In contrast, there are no data on the role of PAF in PMN adhesion to cells exposed to flow conditions and activated by stimuli other than thrombin. Here we used the PAF receptor antagonist L-659,989 to evaluate PMN adhesion to human umbilical vein endothelial cells (HUVEC) in basal conditions or upon challenge with thrombin or tumor necrosis factor-alpha (TNF-alpha). Experiments were performed under dynamic flow using a parallel-plate flow chamber and a computer-based image analysis system. Rolling and adhesion of PMNs to endothelial cells significantly increased upon stimulation with thrombin. Thrombin-stimulated HUVEC also synthesized higher amounts of PAF than untreated cells. Pretreatment of PMNs with L-659,989 significantly reduced their rolling and adhesion to thrombin-activated HUVEC. Stimulation of HUVEC with TNF-alpha significantly increased the number of rolling and adherent PMNs as compared with untreated cells. Adhesion of PMNs to and migration across TNF-alpha-stimulated HUVEC were reduced by L-659,989, whereas cell rolling was unchanged. We conclude that PAF mediates leukocyte interaction under flow conditions with HUVEC activated by inflammatory stimuli.

scholarly journals A Fifty Percent Reduction of Platelet Surface Glycoprotein Ib Does Not Affect Platelet Adhesion Under Flow Conditions

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2353-2359 ◽  
Author(s):  
G. Henrita van Zanten ◽  
Harry F.G. Heijnen ◽  
Yaping Wu ◽  
Karin M. Schut-Hese ◽  
Pieter J. Slootweg ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Surface Expression ◽  
Surface Glycoprotein ◽  
Flow Conditions ◽  
Platelet Surface ◽  
Microscopic Studies ◽  
Umbilical Vein Endothelial Cells ◽  
Adhesive Proteins

Glycoprotein (GP) Ib is an adhesion receptor on the platelet surface that binds to von Willebrand Factor (vWF). vWF becomes attached to collagens and other adhesive proteins that become exposed when the vessel wall is damaged. Several investigators have shown that during cardiopulmonary bypass (CPB) surgery and also during platelet activation in vitro by thrombin or thrombin receptor activating peptide (TRAP) GPIb disappears from the platelet surface. Such a disappearance is presumed to lead to a decreased adhesive capacity. In the present study, we show that a 65% decrease in platelet surface expression of GPIb, due to stimulation of platelets in Orgaran anticoagulated whole blood with 15 μmol/L TRAP, had no effect on platelet adhesion to both collagen type III and the extracellular matrix (ECM) of human umbilical vein endothelial cells under flow conditions in a single-pass perfusion system. In contrast to adhesion, ristocetin-induced platelet agglutination was highly dependent on the presence of GPIb. Immunoelectron microscopic studies showed that GPIb almost immediately returned to the platelet surface once platelets had attached to collagen. In a subsequent series of experiments, we showed that when less than 50% of GPIb was blocked by an inhibitory monoclonal antibody against GPIb (6D1), platelet adhesion under flow conditions remained unaffected.

scholarly journals A Fifty Percent Reduction of Platelet Surface Glycoprotein Ib Does Not Affect Platelet Adhesion Under Flow Conditions

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2353-2359 ◽  
Author(s):  
G. Henrita van Zanten ◽  
Harry F.G. Heijnen ◽  
Yaping Wu ◽  
Karin M. Schut-Hese ◽  
Pieter J. Slootweg ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Surface Expression ◽  
Surface Glycoprotein ◽  
Flow Conditions ◽  
Platelet Surface ◽  
Microscopic Studies ◽  
Umbilical Vein Endothelial Cells ◽  
Adhesive Proteins

AbstractGlycoprotein (GP) Ib is an adhesion receptor on the platelet surface that binds to von Willebrand Factor (vWF). vWF becomes attached to collagens and other adhesive proteins that become exposed when the vessel wall is damaged. Several investigators have shown that during cardiopulmonary bypass (CPB) surgery and also during platelet activation in vitro by thrombin or thrombin receptor activating peptide (TRAP) GPIb disappears from the platelet surface. Such a disappearance is presumed to lead to a decreased adhesive capacity. In the present study, we show that a 65% decrease in platelet surface expression of GPIb, due to stimulation of platelets in Orgaran anticoagulated whole blood with 15 μmol/L TRAP, had no effect on platelet adhesion to both collagen type III and the extracellular matrix (ECM) of human umbilical vein endothelial cells under flow conditions in a single-pass perfusion system. In contrast to adhesion, ristocetin-induced platelet agglutination was highly dependent on the presence of GPIb. Immunoelectron microscopic studies showed that GPIb almost immediately returned to the platelet surface once platelets had attached to collagen. In a subsequent series of experiments, we showed that when less than 50% of GPIb was blocked by an inhibitory monoclonal antibody against GPIb (6D1), platelet adhesion under flow conditions remained unaffected.

INTERACTION OF S PROTEIN/VITRONECTIN WITH CULTURED ENDOTHELIAL CELLS: PROMOTION OF ATTACHMENT AND SPECIFIC BINDING

1987 ◽  
Author(s):  
K T Preissner ◽  
E Anders ◽  
G Müller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Cell Attachment ◽  
Specific Binding ◽  
Umbilical Vein ◽  
Attachment Site ◽  
Cell Types ◽  
S Protein ◽  
Specific Association ◽  
Umbilical Vein Endothelial Cells ◽  
Adhesive Proteins

The interaction of the complement inhibitor S protein, which is identical to the serum spreading factor, vitronectin, with cultured human endothelial cells of macro- and microvas- cular origin was investigated. Purified S protein, coated for 2 h on polystyrene petri dishes, induced concentration- and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVEC) as well as human omental tissqe microvasular endothelial cells (HOTMEC) at 37°C. With 3 × 105 cells/ml (final concentration) more than 50% of the cells attached within 2 h incubation at 0.3 - 3 μg/ml S protein. The effect of S protein was specific, since only monospecific antibodies against S protein prevented attachment of cells, while antibodies against fibronectin, fibrinogen or von Wille-brand factor were uneffective. The pentapeptide Gly-Arg-Gly-Asp-Ser, which contains the cell-attachment site of these adhesive proteins including S protein, inhibited the activity of S protein to promote attachment of endothelial cells in a concentration-dependent fashion; at 200 μM peptide, less than 10% of the cells became attached. Direct binding of S protein to HUVEC and HOTMEC was studied with cells in suspension at a concentration of 1 × 106 cells/ml in the presence of 1% (w/v) human serum albumin and 1 mM CaCl2 and was maximal after 120 min. Both cell types bound S protein in a concentration-dependent fashion with an estimated dissociation constant KD=0.2pM. More than 80% of bound radiolabelled S protein was displaced by unlabelled S protein, whereas binding was reduced to about 50% by the addition in excess of either fibronectin, fibrinogen, von Willebrand factor or the pentapeptide. These findings provide evidence for the specific association of S protein with endothelial cells, ultimately leading to attachment and spreading of cells. Although the promotion of attachment was highly specific for S protein, other adhesive proteins than S protein, also known to associate with endothelial cells, may in part compete with direct S protein binding.

scholarly journals Rapamycin Inhibited Pyroptosis and Reduced the Release of IL-1β and IL-18 in the Septic Response

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Luo Zhuo ◽  
Xiaobing Chen ◽  
Yan Sun ◽  
Yanli Wang ◽  
Yuanfeng Shi ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Genetic Manipulation ◽  
Umbilical Vein ◽  
Disease Model ◽  
Positive Control ◽  
Sepsis Model ◽  
Autophagy Induction ◽  
Immune Imbalance ◽  
Umbilical Vein Endothelial Cells

Pyroptosis, an inflammatory form of programmed cell death, is the initiating event of sepsis and results in immune imbalance by releasing IL-1β and IL-18 in the early stages. Studies show that enhancing autophagy via genetic manipulation can inhibit pyroptosis and prolong the survival of a sepsis animal model, indicating a possible therapeutic strategy against sepsis. However, almost no study so far has achieved pyroptosis inhibition via pharmacological autophagy induction in a sepsis disease model. To this end, we established an in vitro sepsis model by stimulating primary human umbilical vein endothelial cells (HUVECs) with lipopolysaccharide (LPS), and analyzed the effect of the autophagy agonist rapamycin (RAPA) on pyroptosis. Phorbol 12-myristate 13-acetate- (PMA-) activated human THP-1 cells were used as the positive control. LPS significantly increased the levels of the pyroptotic protein Gasdermin D (GSDMD), cysteinyl aspartate-specific proteinase 1 (caspase-1), secreted LDH, IL-1β, and IL-18. RAPA treatment downregulated the above factors and enhanced autophagy in the LPS-stimulated HUVECs and THP-1 cells. This study shows that RAPA abrogates LPS-mediated increase in IL-1β and IL-18 by inhibiting pyroptosis and enhancing autophagy.

scholarly journals Targeting PELP1 Attenuates Angiogenesis and Enhances Chemotherapy Efficiency in Colorectal Cancer

Cancers ◽  
2022 ◽  
Vol 14 (2) ◽  
pp. 383
Author(s):  
Jianlin Zhu ◽  
Lu Wang ◽  
Fan Liu ◽  
Jinghua Pan ◽  
Zhimeng Yao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Solid Tumors ◽  
Therapeutic Strategy ◽  
Umbilical Vein ◽  
Defined Contribution ◽  
Cancer Types ◽  
Control Tumor ◽  
Umbilical Vein Endothelial Cells ◽  
Oncogenic Protein

Abnormal angiogenesis is one of the important hallmarks of colorectal cancer as well as other solid tumors. Optimally, anti-angiogenesis therapy could restrain malignant angiogenesis to control tumor expansion. PELP1 is as a scaffolding oncogenic protein in a variety of cancer types, but its involvement in angiogenesis is unknown. In this study, PELP1 was found to be abnormally upregulated and highly coincidental with increased MVD in CRC. Further, treatment with conditioned medium (CM) from PELP1 knockdown CRC cells remarkably arrested the function of human umbilical vein endothelial cells (HUVECs) compared to those treated with CM from wildtype cells. Mechanistically, the STAT3/VEGFA axis was found to mediate PELP1-induced angiogenetic phenotypes of HUVECs. Moreover, suppression of PELP1 reduced tumor growth and angiogenesis in vivo accompanied by inactivation of STAT3/VEGFA pathway. Notably, in vivo, PELP1 suppression could enhance the efficacy of chemotherapy, which is caused by the normalization of vessels. Collectively, our findings provide a preclinical proof of concept that targeting PELP1 to decrease STAT3/VEGFA-mediated angiogenesis and improve responses to chemotherapy due to normalization of vessels. Given the newly defined contribution to angiogenesis of PELP1, targeting PELP1 may be a potentially ideal therapeutic strategy for CRC as well as other solid tumors.

Abstract 14993: Dysfunctional Extracellular Microvesicles in Andean Highlanders With Excessive Erythrocytosis

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Madden Brewster ◽  
Anthony R Bain ◽  
Vinicius P Garcia ◽  
Hannah K Fandl ◽  
Rachel Stone ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Vascular Endothelium ◽  
Cardiovascular Health ◽  
Vascular Dysfunction ◽  
Umbilical Vein ◽  
No Production ◽  
Intracellular Expression ◽  
Cerro De Pasco ◽  
Umbilical Vein Endothelial Cells ◽  
Cell Expression

Background: Chronic mountain sickness, a maladaptation to high altitude (>2,500 m) characterized by excessive erythrocytosis (EE) and often severe hypoxemia, is prevalent in Andean highlanders. EE increases the risk of cardiovascular events and contributes to vascular dysfunction. Circulating extracellular microvesicles (MVs) are key mediators of cardiovascular health and disease through their interaction with the vascular endothelium. The experimental aim of this study was to determine the effects of MVs isolated from adults with EE on endothelial cell inflammation, oxidative stress, apoptosis and nitric oxide (NO) production. Methods: Twenty-six male residents of Cerro de Pasco, Peru (4,340 m) were studied: 12 highlanders without EE (healthy; age: 40±4 yr; BMI: 26.4±1.7; Hb: 17.4±0.5 g/dL, SpO 2 : 86.9±1.0%) and 14 highlanders with EE (EE: 43±4 yr; 26.2±0.9; 24.4±0.4 g/dL; 79.7±1.6%). MVs were isolated from plasma by flow cytometry. Human umbilical vein endothelial cells were cultured and treated with MVs from either healthy or EE men. Results: MVs from highlanders with EE induced significantly higher release of interleukin (IL)-6 (89.8±2.7 vs 77.1±1.9 pg/mL) and IL-8 (62.0±2.7 vs 53.3±2.2 pg/mL) compared with MVs from healthy highlanders. Although intracellular expression of total NF-κB p65 (65.3±6.0 vs 74.9±7.8.9 AU) was not significantly affected, MVs from EE men resulted in ~25% higher (P<0.05) expression of p-NF-κB p65 (Ser536; active NF-κB) (173.6±14.3 vs 132.8±12.2 AU). Additionally, cell expression of the anti-inflammatory miR-146a and miR-181b were significantly suppressed by EE MVs. Cell oxidative stress and apoptotic susceptibility were not significantly affected by MVs from EE men. However, eNOS activation (231.3±15.5 vs 286.6±23.0 AU) and NO production (8.3±0.6 vs 10.7±0.7 μM/L) were significantly lower in cells treated with MVs from EE vs healthy men. Conclusion: Increased inflammation and decreased eNOS activity and NO production renders the vascular endothelium highly susceptible to atherosclerosis and thrombosis. Andean highlanders with EE exhibit dysfunctional circulating extracellular MVs that induce a proatherogenic endothelial phenotype contributing to their increased cardiovascular risk.

Exogenous xanthine promotes neutrophil adherence to cultured endothelial cells

1997 ◽  
Vol 273 (2) ◽  
pp. G342-G347
Author(s):  
H. Ichikawa ◽  
R. E. Wolf ◽  
T. Y. Aw ◽  
N. Ohno ◽  
L. Coe ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Injury ◽  
Umbilical Vein ◽  
Platelet Activating Factor ◽  
Adhesion Assay ◽  
Neutrophil Adherence ◽  
Paf Receptor ◽  
Umbilical Vein Endothelial Cells ◽  
Adhesive Effect ◽  
Paf Receptor Antagonist

Oxidants generated by endothelial xanthine oxidase (XO) can help trigger free radical-mediated tissue injury. An important event in oxidant-mediated tissue injury is neutrophil-endothelial adhesion. Although activation of endothelial XO increases adhesion, little is known about xanthine in the adhesive effect of XO. This study examined administered xanthine on the adhesion of neutrophils. Endothelial [human umbilical vein endothelial cells (HUVEC)] monolayers were exposed to xanthine (15 min), and neutrophils were allowed to adhere to HUVEC in an adhesion assay. Adhesion was dose dependently increased by xanthine (3-100 microM). Either catalase (1,000 U/ml), oxypurinol (XO inhibitor; 100 microM), or platelet-activating factor (PAF) receptor antagonist (WEB 2086; 10 microM) reduced neutrophil adhesion. Superoxide dismutase (1,000 U/ml) had no effect. Pretreatment of HUVEC with 50 microM tungsten also blocked xanthine-induced adherence. Adhesion was also inhibited by preincubation with 100 U/ml heparin. Finally, anti-P-selectin antibody (PB1.3; 20 micrograms/ml) attenuated adhesion. Our results indicate that xanthine may promote neutrophil-endothelial adhesion via a hydrogen peroxide- and PAF-mediated P-selectin expression.

Plateiet-Activating Factor (1-0-Alkyl-2-Acetyl-sn-Glyceryl-3-Phosphorycholine) Stimulates Prostacyclin Synihesis By Cultured Human Endothelial Cells

1981 ◽  
Author(s):  
S T Test ◽  
N U Bang
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Platelet Rich Plasma ◽  
Umbilical Vein ◽  
Platelet Activating Factor ◽  
Biological Properties ◽  
Feedback Mechanism ◽  
Negative Feedback Mechanism ◽  
Cell Layers ◽  
Umbilical Vein Endothelial Cells

l-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (Ac-GEPC) has recently been established to possess biological properties identical to those of platelet-activating factor (PAF) released from stimulated mast cells and leucocytes (Demopoulos et al, J. Biol. Chem. 254:9355, 1979). Shaw et al (J. Imuunol. 121:1939, 1978) reported that PAF is capable of stimulating platelet thranbaxane A2 synthesis. We examine here whether synthetic Ac-GEPC stimulates PGI2 synthesis in human endothelial cell cultures. Human umbilical vein endothelial cells during the third passage were grown to confluency in 35 x 10 ran dishes and challenged with Na arachidonate(NaA), 8 x 10-5M or Ac-GEPC, 5 x 10-% (a concentration which produced optimal platelet aggregation in human platelet-rich plasma). PGI2 production was monitored through radioimmunoassays of its metabolite, 6-keto-PGF1α. After washing endothelial cell layers twice, the agonists were added in tris-Tyrodes buffer, pH 7.2 containing 2.5 g/L bovine serum albumin. Representative timed experiments produced the following results expressed in pM 6-keto-PGF1α/105 cells for NaA and Ac-GEPC respectively after the noted times of incubation: 10s: 274/467, 20s: 630/1136, 30s: 1111/1005, 60s: 1757/1217, 90s: 1842/1272, 120s: 1115/698, 180s: 1188/453. These data indicate that Ac-GEPC stimulates the production of PGI2 at a rate roughly comparable to that achieved by stimulation with NaA, reaching a maximum at 60 and 90s in these experiments. The decrease, compared to peak levels, in iranunoreactive 6-keto-PGF1α at 120 and 180s of 23% and 36% for NaA stimulated cells and 45% and 65% for Ac-GEPC stimulated cells suggests that enzymes are activated during the experiment which cause conversion of PGI2 into metabolites other than 6-keto-PGF1α (e.g. 6-keto-PGE1). These results suggest another theoretically important negative feedback mechanism for a potent platelet proaggregatorv substance.

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