GLP-1 promotes angiogenesis in human endothelial cells in a dose-dependent manner, through the Akt, Src and PKC pathways

The effects of mechanical strain on monocyte chemotactic protein-1 (MCP-1) secretion were examined on human endothelial cells (ECs) grown on a flexible membrane base. MCP-1 release into culture medium from strained ECs was demonstrated to be time and strain dose dependent. Northern blot analysis demonstrated a mainly serum-independent 1.8-fold induction of MCP-1 mRNA levels in ECs strained at 15 kPa compared with unstrained controls. ECs treated with actinomycin D abolished this strain-induced expression. Strained ECs at the periphery of wells showed higher MCP-1 gene expression than ECs at the center. Pretreatment of ECs with either cytochalasin D or phalloidin did not abolish strain-induced gene expression. ECs pretreated with stretch-activated ion channel blocker gadolinium or with ryanodine to deplete intracellular stored Ca2+ strongly inhibited the strain-induced MCP-1 levels. We conclude that 1) cyclical strain can modulate the secretion of MCP-1 in a dose-dependent manner, 2) strain-induced MCP-1 production is mediated by increasing MCP-1 mRNA levels via transcription, 3) cytoskeletal rearrangement is not essential for this strain-induced MCP-1 expression, and 4) both Ca2+ influx via stretch-activated ion channels and intracellular Ca2+ release contribute to the strain-induced effect. Such strain-induced MCP-1 secretion might contribute to the trapping of monocytes in the subendothelial space to initiate atherogenesis.

Download Full-text

Purification, molecular cloning and mechanism of action of graminelysin I, a snake-venom-derived metalloproteinase that induces apoptosis of human endothelial cells

Biochemical Journal ◽

10.1042/bj3570719 ◽

2001 ◽

Vol 357 (3) ◽

pp. 719-728 ◽

Cited By ~ 20

Author(s):

Wen Bin WU ◽

Shin C. CHANG ◽

Ming-Yi LIAU ◽

Tur-Fu HUANG

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Matrix Proteins ◽

Dependent Manner ◽

Single Chain ◽

Human Endothelial Cells ◽

Putative Precursor ◽

Induced Apoptosis ◽

Umbilical Vein Endothelial Cells ◽

Dose Dependent

Apoptosis, a programmed, physiological mode of cell death, is important in tissue homoeostasis. Here we report that a new metalloproteinase, graminelysin I, purified from Trimeresurus gramineus venom, induced apoptosis of human endothelial cells as examined by electrophoresis and flow cytometry. Graminelysin I contains only a metalloproteinase domain. It is a single-chain proteinase with a molecular mass of 27020Da. cDNA sequence analysis revealed that the disintegrin-like and cysteine-rich domains of the putative precursor protein of graminelysin I are likely to be processed post-translationally, producing the proteinase domain (graminelysin I). Graminelysin I cleaved the α chain of fibrinogen preferentially and cleaved the β chain either on longer incubation or at higher concentration. Graminelysin I inhibited the adhesion of human umbilical-vein endothelial cells (HUVECs) to immobilized fibrinogen and induced HUVECs detachment in a dose-dependent manner. These effects on HUVECs were abolished when graminelysin I was pretreated with EDTA. However, graminelysin I did not inhibit the adhesion of HUVECs to immobilized collagen. HUVECs were susceptible to death after treatment with graminelysin I when they were cultured on immobilized fibrinogen. In contrast, HUVECs were rather resistant to treatment with graminelysin I if they were cultured on immobilized collagen. Furthermore, graminelysin I induced apoptosis of HUVECs in a dose-dependent manner. Similarly, its apoptosis-inducing activity was blocked if it was treated with EDTA. These results suggest that the catalytic activity of graminelysin I on matrix proteins contributes to its apoptosis-inducing activity.

Download Full-text

Defibrotide Stimulates Expression of Thrombomodulin in Human Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642468 ◽

1994 ◽

Vol 71 (04) ◽

pp. 507-510 ◽

Cited By ~ 44

Author(s):

Quansheng Zhou ◽

Xiaohong Chu ◽

Changgeng Ruan

Keyword(s):

Endothelial Cells ◽

Conditioned Medium ◽

Umbilical Vein ◽

Dependent Manner ◽

Human Endothelial Cells ◽

Antithrombotic Activity ◽

Beneficial Effects ◽

Chromogenic Assay ◽

Umbilical Vein Endothelial Cells ◽

Dose Dependent

SummaryCultured human umbilical vein endothelial cells were incubated with defibrotide at concentrations of 0, 5, 50 and 500 jxg/ml for 4 and 24 h respectively. Thrombomodulin activity and molecules on the surface of the cells were determined by chromogenic assay and radioimmunoassay, thrombomodulin antigen in endothelial cells and in conditioned medium of the cells was measured by immunoradioassay. Thrombomodulin mRNA within the cells was analysed by slot blot. After 24 h of incubation, the activity and molecules of thrombomodulin on the surface of endothelial cells, as well as the antigen and mRNA of thrombomodulin in the cells were significantly increased in a dose dependent manner. However, the level of thrombomodulin antigen in conditioned medium was about equal to that of the control. Our data indicate that defibrotide stimulates expression of thrombomodulin in human endothelial cells. These beneficial effects may play a role in antithrombotic activity of defibrotide.

Download Full-text

The RUNX1b Isoform Defines Hemogenic Competency in Developing Human Endothelial Cells

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.812639 ◽

2021 ◽

Vol 9 ◽

Author(s):

Sara Menegatti ◽

Bethany Potts ◽

Eva Garcia-Alegria ◽

Roberto Paredes ◽

Michael Lie-A-Ling ◽

...

Keyword(s):

Endothelial Cells ◽

Human Embryonic Stem Cell ◽

Embryonic Stem Cell Line ◽

Embryonic Stem ◽

Dependent Manner ◽

Human Endothelial Cells ◽

Stem Cell Line ◽

Cell Specification ◽

Dose Dependent

The transcription factor RUNX1 is a master regulator of blood cell specification. During embryogenesis, hematopoietic progenitors are initially generated from hemogenic endothelium through an endothelium-to-hematopoietic transition controlled by RUNX1. Several studies have dissected the expression pattern and role of RUNX1 isoforms at the onset of mouse hematopoiesis, however the precise pattern of RUNX1 isoform expression and biological output of RUNX1-expressing cells at the onset of human hematopoiesis is still not fully understood. Here, we investigated these questions using a RUNX1b:VENUS RUNX1c:TOMATO human embryonic stem cell line which allows multi-parameter single cell resolution via flow cytometry and isolation of RUNX1b-expressing cells for further analysis. Our data reveal the sequential expression of the two RUNX1 isoforms with RUNX1b expressed first in a subset of endothelial cells and during the endothelial to hematopoietic transition while RUNX1c only becomes expressed in fully specified blood cells. Furthermore, our data show that RUNX1b marks endothelial cells endowed with hemogenic potential and that RUNX1b expression level determines hemogenic competency in a dose-dependent manner. Together our data reveal the dynamic of RUNX1 isoforms expression at the onset of human blood specification and establish RUNX1b isoform as the earliest known marker for hemogenic competency.

Download Full-text

Hemoglobin Enhances the Binding of Bacterial Endotoxin to Human Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650565 ◽

1996 ◽

Vol 76 (02) ◽

pp. 258-262 ◽

Cited By ~ 12

Author(s):

Robert I Roth

Keyword(s):

Endothelial Cells ◽

Binding Protein ◽

Bacterial Endotoxin ◽

Dependent Manner ◽

Serum Free Medium ◽

Free Medium ◽

Human Endothelial Cells ◽

Serum Factors ◽

Serum Free ◽

Lps Binding

SummaryHuman endothelial cells, when incubated with bacterial endotoxin (lipopolysaccharide, LPS), modify their surface in association with prominent production of procoagulant tissue factor (TF) activity. This deleterious biological effect of LPS has been shown previously to be enhanced approximately 10-fold by the presence of hemoglobin (Hb), a recently recognized LPS binding protein that causes disaggregation of LPS and increases the biological activity of LPS in a number of in vitro assays. The present study was performed to test the hypothesis that Hb enhances the LPS-induced procoagulant activity of human umbilical vein endothelial cells (HUVEC) by increasing LPS binding to the cells. The binding of 3H-LPS to HUVEC was determined in the absence or presence of Hb or two other known LPS-binding proteins, human serum albumin (HSA) and IgG. LPS binding was substantially increased in the presence of Hb, in a Hb concentration-dependent manner, but was not increased by HSA or IgG. Hb enhancement of LPS binding was observed in serum-free medium, indicating that there was no additional requirement for any of the serum factors known to participate in the interaction of LPS with cells (e.g., lipopolysaccharide (LPS)-binding protein (LBP) and soluble CD14 (sCD14)). Hb enhancement of LPS binding also was observed in the more physiologic condition of 100% plasma. LPS-induced TF activity was stimulated by Hb, but not by HSA or IgG. In serum-free medium, TF activity was not stimulated under any of the conditions tested. Ultrafiltration of LPS was dramatically increased after incubation with Hb but not with HSA or IgG, suggesting that LPS disaggregation by Hb was responsible for the enhanced binding of LPS to HUVEC and the subsequent stimulation of TF activity.

Download Full-text

Cyclooxygenase-2 Glycosylation Is Affected by Peroxynitrite in Endothelial Cells: Impact on Enzyme Activity and Degradation

Antioxidants ◽

10.3390/antiox10030496 ◽

2021 ◽

Vol 10 (3) ◽

pp. 496

Author(s):

Sonia Eligini ◽

Susanna Colli ◽

Aida Habib ◽

Giancarlo Aldini ◽

Alessandra Altomare ◽

...

Keyword(s):

Endothelial Cells ◽

De Novo ◽

Cyclooxygenase 2 ◽

Nitric Oxide Donors ◽

Metabolic Labeling ◽

Dependent Manner ◽

De Novo Synthesis ◽

Human Endothelial Cells ◽

Prolonged Incubation ◽

Cox 2

The exposure of human endothelial cells to 3-morpholinosydnonimine (SIN-1) induced the expression of cyclooxygenase-2 (COX-2) in a dose- and time-dependent manner. Interestingly, after a prolonged incubation (>8 h) several proteoforms were visualized by Western blot, corresponding to different states of glycosylation of the protein. This effect was specific for SIN-1 that generates peroxynitrite and it was not detected with other nitric oxide-donors. Metabolic labeling experiments using 35S or cycloheximide suggested that the formation of hypoglycosylated COX-2 was dependent on de novo synthesis of the protein rather than the deglycosylation of the native protein. Moreover, SIN-1 reduced the activity of the hexokinase, the enzyme responsible for the first step of glycolysis. The hypoglycosylated COX-2 induced by SIN-1 showed a reduced capacity to generate prostaglandins and the activity was only partially recovered after immunoprecipitation. Finally, hypoglycosylated COX-2 showed a more rapid rate of degradation compared to COX-2 induced by IL-1α and an alteration in the localization with an accumulation mainly detected in the nuclear membrane. Our results have important implication to understand the effect of peroxynitrite on COX-2 expression and activity, and they may help to identify new pharmacological tools direct to increase COX-2 degradation or to inhibit its activity.

Download Full-text

Biochemical and biological activity of arginine deiminase from Streptococcus pyogenes M22

Biochemistry and Cell Biology ◽

10.1139/bcb-2015-0069 ◽

2016 ◽

Vol 94 (2) ◽

pp. 129-137 ◽

Cited By ~ 2

Author(s):

Eleonora A. Starikova ◽

Alexey V. Sokolov ◽

Anna Yu. Vlasenko ◽

Larisa A. Burova ◽

Irina S. Freidlin ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Streptococcus Pyogenes ◽

Group A Streptococcus ◽

Bacterial Pathogen ◽

Arginine Deiminase ◽

Dependent Manner ◽

Group A ◽

Micro Organisms ◽

Dose Dependent

Streptococcus pyogenes (group A Streptococcus; GAS) is an important gram-positive extracellular bacterial pathogen responsible for a number of suppurative infections. This micro-organism has developed complex virulence mechanisms to avoid the host’s defenses. We have previously reported that SDSC from GAS type M22 causes endothelial-cell dysfunction, and inhibits cell adhesion, migration, metabolism, and proliferation in a dose-dependent manner, without affecting cell viability. This work aimed to isolate and characterize a component from GAS type M22 supernatant that suppresses the proliferation of endothelial cells (EA.hy926). In the process of isolating a protein possessing antiproliferative activity we identified arginine deiminase (AD). Further study showed that this enzyme is most active at pH 6.8. Calculating Km and Vmax gave the values of 0.67 mmol·L–1 and 42 s−1, respectively. A distinctive feature of AD purified from GAS type M22 is that its optimum activity and the maximal rate of the catalytic process is close to neutral pH by comparison with enzymes from other micro-organisms. AD from GAS type M22 suppressed the proliferative activity of endothelial cells in a dose-dependent mode. At the same time, in the presence of AD, the proportion of cells in G0/G1 phase increased. When l-Arg was added at increasing concentrations to the culture medium containing AD (3 μg·mL–1), the enzyme’s capacity to inhibit cell proliferation became partially depressed. The proportion of cells in phases S/G2 increased concomitantly, although the cells did not fully recover their proliferation activity. This suggests that AD from GAS type M22 has potential for the suppression of excessive cell proliferation.

Download Full-text

Glyoxalase I is a novel nitric-oxide-responsive protein

Biochemical Journal ◽

10.1042/bj3440837 ◽

1999 ◽

Vol 344 (3) ◽

pp. 837-844 ◽

Cited By ~ 19

Author(s):

Atsushi MITSUMOTO ◽

Kwi-Ryeon KIM ◽

Genichiro OSHIMA ◽

Manabu KUNIMOTO ◽

Katsuya OKAWA ◽

...

Keyword(s):

Nitric Oxide ◽

Culture Medium ◽

Molecular Mechanisms ◽

Peptide Mapping ◽

Glyoxalase I ◽

Dependent Manner ◽

Human Endothelial Cells ◽

Native Form ◽

No Donors ◽

Dose Dependent

To clarify the molecular mechanisms of nitric oxide (NO) signalling, we examined the NO-responsive proteins in cultured human endothelial cells by two-dimensional (2D) PAGE. Levels of two proteins [NO-responsive proteins (NORPs)] with different pI values responded to NO donors. One NORP (pI 5.2) appeared in response to NO, whereas another (pI 5.0) disappeared. These proteins were identified as a native form and a modified form of human glyoxalase I (Glox I; EC 4.4.1.5) by peptide mapping, microsequencing and correlation between the activity and the isoelectric shift. Glox I lost activity in response to NO, and all NO donors tested inhibited its activity in a dose-dependent manner. Activity and normal electrophoretic mobility were restored by dithiothreitol and by the removal of sources of NO from the culture medium. Glox I was selectively inactivated by NO; compounds that induce oxidative stress (H2O2, paraquat and arsenite) failed to inhibit this enzyme. Our results suggest that NO oxidatively modifies Glox I and reversibly inhibits the enzyme's activity. The inactivation of Glox I by NO was more effective than that of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), another NO-sensitive enzyme. Thus Glox I seems to be a novel NO-responsive protein that is more sensitive to NO than G3PDH.

Download Full-text

Calcium dependence of the thrombin-induced increase in endothelial albumin permeability

Journal of Applied Physiology ◽

10.1152/jappl.1989.66.3.1471 ◽

1989 ◽

Vol 66 (3) ◽

pp. 1471-1476 ◽

Cited By ~ 39

Author(s):

H. Lum ◽

P. J. Del Vecchio ◽

A. S. Schneider ◽

M. S. Goligorsky ◽

A. B. Malik

Keyword(s):

Endothelial Cells ◽

Endothelial Permeability ◽

Base Line ◽

Dependent Manner ◽

Extracellular Medium ◽

Influx Rate ◽

Before And After ◽

Dependent Inhibition ◽

Permeability Increase ◽

Dose Dependent

We examined whether the increase in endothelial albumin permeability induced by alpha-thrombin is dependent on extracellular Ca2+ influx. Permeability of 125I-albumin across confluent monolayers of cultured bovine pulmonary artery endothelial cells was measured before and after the addition of 0.1 microM alpha-thrombin. In the presence of normal extracellular Ca2+ concentration ([Ca2+]o, 1000 microM), alpha-thrombin produced a 175 +/- 10% increase in 125I-albumin permeability. At lower [Ca2+]o (100, 10, 1, or less than 1 microM), alpha-thrombin caused a 140% increase in permeability (P less than 0.005). LaCl3 (1 mM), which competes for Ca2+ entry, blunted 38% of the increase in permeability. Preloading endothelial monolayers with quin2 to buffer cytosolic Ca2+ (Cai2+) produced a dose-dependent inhibition of the increase in 125I-albumin permeability. Preincubation with nifedipine or verapamil was ineffective in reducing the thrombin-induced permeability increase. A 60 mM K+ isosmotic solution did not alter base-line endothelial permeability. alpha-Thrombin increased [Ca2+]i in a dose-dependent manner and the 45Ca2+ influx rate. Extracellular medium containing 60 mM K+ did not increase 45Ca2+ influx, and nifedipine did not block the rise in 45Ca2+ influx caused by alpha-thrombin. Ca2+ flux into endothelial cells induced by alpha-thrombin does not occur through voltage-sensitive channels but may involve receptor-operated channels. In conclusion, the increase in endothelial albumin permeability caused by alpha-thrombin is dependent on Ca2+ influx and intracellular Ca2+ mobilization.

Download Full-text