scholarly journals Peroxide-induced membrane blebbing in endothelial cells associated with glutathione oxidation but not apoptosis

1999 ◽  
Vol 277 (1) ◽  
pp. C20-C28 ◽  
Author(s):  
Roosje M. A. van Gorp ◽  
Jos L. V. Broers ◽  
Chris P. M. Reutelingsperger ◽  
Nancy M. H. J. Bronnenberg ◽  
Gerard Hornstra ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Early Stage ◽  
Morphological Changes ◽  
Reductase Activity ◽  
Umbilical Vein ◽  
Cell Types ◽  
Surface Expression ◽  
Nuclear Condensation ◽  
Membrane Blebbing ◽  
Bleb Formation

Cells under oxidative stress induced by peroxides undergo functional and morphological changes, which often resemble those observed during apoptosis. Peroxides, however, also cause the oxidation of intracellular reduced glutathione (GSH). We investigated the relation between these peroxide-induced effects by using human umbilical vein endothelial cells (HUVEC) and two HUVEC-derived cell lines, ECRF24 and ECV304. With HUVEC, tert-butyl hydroperoxide ( tBH) or hydrogen peroxide application in the presence of serum induced, in a dose-dependent way, reorganization of the actin cytoskeleton, membrane blebbing, and nuclear condensation. These processes were accompanied by transient oxidation of GSH. With ECRF24 cells, this treatment resulted in less blebbing and a shorter period of GSH oxidation. However, repeated tBH addition increased the number of blebbing cells and prolonged the period of GSH oxidation. ECV304 cells were even more resistant to peroxide-induced bleb formation and GSH oxidation. Inhibition of glutathione reductase activity potentiated the peroxide-induced blebbing response in HUVEC and ECRF24 cells, but not in ECV304 cells. Neither membrane blebbing nor nuclear condensation in any of these cell types was due to apoptosis, as evidenced by the absence of surface expression of phosphatidylserine or fragmentation of DNA, even after prolonged incubations with tBH, although high tBH concentrations lead to nonapoptotic death. We conclude that, in endothelial cells, peroxide-induced cytoskeletal reorganization and bleb formation correlate with the degree of GSH oxidation but do not represent an early stage of the apoptotic process.

Stimulation of adhesion molecule expression in human endothelial cells (HUVEC) by adrenomedullin and corticotrophin

2004 ◽  
Vol 286 (2) ◽  
pp. C239-C246 ◽  
Author(s):  
Eleni Hagi-Pavli ◽  
Paula M. Farthing ◽  
Supriya Kapas
Keyword(s):  
Endothelial Cells ◽  
Adhesion Molecules ◽  
Umbilical Vein ◽  
Cell Types ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
A Cell ◽  
Response To Stress ◽  
Umbilical Vein Endothelial Cells ◽  
Adenylyl Cyclase Inhibitor

Adrenomedullin (AM) and corticotrophin (ACTH) are both vasoactive peptides produced by a variety of cell types, including endothelial cells. Although AM and ACTH are considered to be important in the control of blood pressure and the response to stress, respectively, their role in inflammation and the immune response has not been clarified. This study shows, with the use of a cell-based ELISA, that AM and ACTH induce cell surface expression of the adhesion molecules E-selectin, VCAM-1, and ICAM-1 on human umbilical vein endothelial cells (HUVEC). Furthermore, this effect appears to be mediated in part via elevation of cAMP, given that both peptides elevate cAMP, the cell-permeable cAMP analog dibutyryl cAMP is able to mimic induction of all three cell adhesion molecules and the effect of AM and ACTH is inhibited by the adenylyl cyclase inhibitor SQ-22536. These findings demonstrate a role for AM and ACTH in the regulation of the immune and inflammatory response.

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.

Distinct signals via Rho GTPases and Src drive shape changes by thrombin and sphingosine-1-phosphate in endothelial cells

2002 ◽  
Vol 115 (12) ◽  
pp. 2475-2484 ◽  
Author(s):  
Valérie Vouret-Craviari ◽  
Christine Bourcier ◽  
Etienne Boulter ◽  
Ellen Van Obberghen-Schilling
Keyword(s):  
Endothelial Cells ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Morphological Changes ◽  
Umbilical Vein ◽  
Cell Spreading ◽  
Actin Binding ◽  
Sphingosine 1 Phosphate ◽  
And Migration ◽  
Umbilical Vein Endothelial Cells

Soluble mediators such as thrombin and sphingosine-1-phosphate regulate morphological changes in endothelial cells that affect vascular permeability and new blood vessel formation. Although these ligands activate a similar set of heterotrimeric G proteins, thrombin causes cell contraction and rounding whereas sphingosine-1-phosphate induces cell spreading and migration. A functional requirement for Rho family GTPases in the cytoskeletal responses to both ligands has been established, yet the dynamics of their regulation and additional signaling mechanisms that lead to such opposite effects remain poorly understood. Using a pull-down assay to monitor the activity of Rho GTPases in human umbilical vein endothelial cells, we find significant temporal and quantitative differences in RhoA and Rac1 activation. High levels of active RhoA rapidly accumulate in cells in response to thrombin whereas Rac1 is inhibited. In contrast, sphingosine-1-phosphate addition leads to comparatively weak and delayed activation of RhoA and it activates Rac1. In addition, we show here that sphingosine-1-phosphate treatment activates a Src family kinase and triggers recruitment of the F-actin-binding protein cortactin to sites of actin polymerization at the rim of membrane ruffles. Both Src and Rac pathways are essential for lamellipodia targeting of cortactin. Further, Src plays a determinant role in sphingosine-1-phosphate-induced cell spreading and migration. Taken together these data demonstrate that the thrombin-induced contractile and immobile phenotype in endothelial cells reflects both robust RhoA activation and Rac inhibition, whereas Src- and Rac-dependent events couple sphingosine-1-phosphate receptors to the actin polymerizing machinery that drives the extension of lamellipodia and cell migration.

scholarly journals Hydrogel-Coated Textile Scaffolds as Three-Dimensional Growth Support for Human Umbilical Vein Endothelial Cells (HUVECs): Possibilities as Coculture System in Liver Tissue Engineering

2002 ◽  
Vol 11 (4) ◽  
pp. 369-377 ◽  
Author(s):  
Makarand V. Risbud ◽  
Erdal Karamuk ◽  
René Moser ◽  
Joerg Mayer
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Three Dimensional ◽  
Mesh Size ◽  
Cell Types ◽  
Cell Number ◽  
Human Umbilical Vein ◽  
Hydrogel Matrix ◽  
Umbilical Vein Endothelial Cells

Three-dimensional (3-D) scaffolds offer an exciting possibility to develop cocultures of various cell types. Here we report chitosan–collagen hydrogel-coated fabric scaffolds with defined mesh size and fiber diameter for 3-D culture of human umbilical vein endothelial cells (HUVECs). These scaffolds did not require pre-coating with fibronectin and they supported proper HUVEC attachment and growth. Scaffolds preserved endothelial cell-specific cobblestone morphology and cells were growing in compartments defined by the textile mesh. HUVECs on the scaffold maintained the property of contact inhibition and did not exhibit overgrowth until the end of in vitro culture (day 6). MTT assay showed that cells had preserved mitochondrial functionality. It was also noted that cell number on the chitosan-coated scaffold was lower than that of collagen-coated scaffolds. Calcein AM and ethidium homodimer (EtD-1) dual staining demonstrated presence of viable and metabolically active cells, indicating growth supportive properties of the scaffolds. Actin labeling revealed absence of actin stress fibers and uniform distribution of F-actin in the cells, indicating their proper attachment to the scaffold matrix. Confocal microscopic studies showed that HUVECs growing on the scaffold had preserved functionality as seen by expression of von Willebrand (vW) factor. Observations also revealed that functional HUVECs were growing at various depths in the hydrogel matrix, thus demonstrating the potential of these scaffolds to support 3-D growth of cells. We foresee the application of this scaffold system in the design of liver bioreactors wherein hepatocytes could be cocultured in parallel with endothelial cells to enhance and preserve liver-specific functions.

MO761THE CONTRIBUTION OF HIGH-PHOSPHORUS IN EXTRACELLULAR MATRIX ACCUMULATION OF VALVULAR CALCIFICATION IN CHRONIC KIDNEY DISEASE

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Liting Wang ◽  
Yuxia Zhang ◽  
Yujia Wang ◽  
Rining Tang
Keyword(s):  
Chronic Kidney Disease ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Kidney Disease ◽  
Early Stage ◽  
Umbilical Vein ◽  
Akt Pathway ◽  
Protein Levels ◽  
Valvular Calcification ◽  
High Phosphorus

Abstract Background and Aims The characteristics of valvular calcification (VC) in early stage are extracellular matrix (ECM) accumulation and muti-cells activation. In our previous work, we found high-phosphorus (HP) diet aggravated ECM accumulation in both aortic valve and mitral valve in rats with chronic kidney disease (CKD). However, the underlying mechanism of HP contribution in ECM accumulation of CKD-induced VC is still unknown. Method canine valvular interstitial cells (VICs), valvular endothelial cells (VECs) and human umbilical vein endothelial cells (HUVECs) were used in this study. CKD mice (C57b and Tek-EGFP-PolyA) was build by 0.2% adenine-diet for 6 weeks and HP diet/NP diet for 10 weeks. Results As for VICs, HP induced qVICs transfer into aVICs, not oVICs, which was characterized with upregulated level of smoothelin and viemitin. There was no calcium accumulation was observed, suggesting that VICs do not have the ability to synthesize calcium crystals under pure HP intervention. As for VECs, aVICs activated by HP induced VECs EndMT in a transwell-assay, which was characterized with decreasing protein levels of endothelial markers (CD31, vWF, VE-cadherin) and increasing protein levels of mesenchymal makers (α-SMA, FSP1, N-cadherin). Then, IL-8 was found as the main factor releasing from VICs to induce VECs EndMT. In vitro, the concentration of IL-8 in the lower chamber could reach 2-4ng/ml. Reparixin was used to inhibit IL-8 receptor of VECs, which could relive aVICs-induced EndMT. In vivo, the expression of valve CXCL-2 (the mouse IL8 functional homolog) was increased in HP-diet compared with NP-diet, though the serum level of CXCL-2 was similar between two groups. AAV9-sm22a-CXCL-2 and Reparixin could inhibit VECs EndMT by inhibiting VICs relasing CXCL-2 and inhibiting VECs IL-8 receptor in CKD mice of Tek-EGFP-PolyA respectively. Then, IL-8 was found to induced VECs EndMT by miR-214-3p/PTEN/Akt pathway. Inhibiting EndMT by blocking IL-8/miR-214-3p could alleviate ECM accumulation. Conclusion HP could induce qVICs transfer into aVICs, and aVICs could cause VECs EndMT via IL-8/miR-214-3p/PTEN/Akt pathway. Both take part in ECM accumulation in CKD-induced VC.

scholarly journals Collagen scaffolds functionalized with triple-helical peptides support 3D HUVEC culture

2020 ◽  
Vol 7 (5) ◽  
pp. 471-482
Author(s):  
Jean-Daniel Malcor ◽  
Emma J Hunter ◽  
Natalia Davidenko ◽  
Daniel V Bax ◽  
Ruth Cameron ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Three Dimensional ◽  
Cell Types ◽  
Cross Linking ◽  
Collagen Scaffolds ◽  
Mechanical Stiffness ◽  
Helical Peptides ◽  
Surface Receptors ◽  
Engineered Tissues

Abstract Porous biomaterials which provide a structural and biological support for cells have immense potential in tissue engineering and cell-based therapies for tissue repair. Collagen biomaterials that can host endothelial cells represent promising tools for the vascularization of engineered tissues. Three-dimensional collagen scaffolds possessing controlled architecture and mechanical stiffness are obtained through freeze–drying of collagen suspensions, followed by chemical cross-linking which maintains their stability. However, cross-linking scaffolds renders their biological activity suboptimal for many cell types, including human umbilical vein endothelial cells (HUVECs), by inhibiting cell–collagen interactions. Here, we have improved crucial HUVEC interactions with such cross-linked collagen biomaterials by covalently coupling combinations of triple-helical peptides (THPs). These are ligands for collagen-binding cell-surface receptors (integrins or discoidin domain receptors) or secreted proteins (SPARC and von Willebrand factor). THPs enhanced HUVEC adhesion, spreading and proliferation on 2D collagen films. THPs grafted to 3D-cross-linked collagen scaffolds promoted cell survival over seven days. This study demonstrates that THP-functionalized collagen scaffolds are promising candidates for hosting endothelial cells with potential for the production of vascularized engineered tissues in regenerative medicine applications.

Identification of human thrombin-activatable fibrinolysis inhibitor in vascular and inflammatory cells

2011 ◽  
Vol 105 (06) ◽  
pp. 999-1009 ◽  
Author(s):  
Joellen Lin ◽  
Mathieu Garand ◽  
Branislava Zagorac ◽  
Steven Schadinger ◽  
Corey Scipione ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Umbilical Vein ◽  
Inflammatory Cells ◽  
Cell Types ◽  
Rt Pcr ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Gene Encoding ◽  
Fibrinolysis Inhibitor

SummaryTAFI (thrombin-activatable fibrinolysis inhibitor) is a carboxypeptidase zymogen originally identified in plasma. The TAFI pathway helps to regulate the balance between the coagulation and fibrinolytic cascades. Activated TAFI (TAFIa) can also inactivate certain pro-inflammatory mediators, suggesting that the TAFI pathway may also regulate communication between coagulation and inflammation. Expression in the liver is considered to be the source of plasma TAFI. TAFI has also been identified in platelets and CPB2 (the gene encoding TAFI) mRNA has been detected in megakaryocytic cell lines as well as in endothelial cells. We have undertaken a quantitative analysis of CPB2 mRNA and TAFI protein in extrahepatic cell types relevant to vascular disease. Using RT-PCR and quantitative RT-PCR, we detected CPB2 mRNA in the human megakaryoblastic cell lines MEG-01 and Dami, the human monocytoid cell line THP-1 as well as THP-1 cells differentiated into a macrophage-like phenotype, and in primary human umbilical vein and coronary artery endothelial cells. CPB2 mRNA abundance in MEG-01, Dami, and THP-1 cells was modulated by the state of differentiation of these cells. Using a recently developed TAFIa assay, we detected TAFI protein in the lysates of the human hepatocellular carcinoma cell line HepG2 as well as in MEG-01 and Dami cells and in the conditioned medium of HepG2 cells, differentiated Dami cells, and THP-1 macrophages. We have obtained clear evidence for extrahepatic expression of TAFI, which has clear implications for the physiological and pathophysiological functions of the TAFI pathway.

scholarly journals Liraglutide Inhibits Endothelial-to-Mesenchymal Transition and Attenuates Neointima Formation after Endovascular Injury in Streptozotocin-Induced Diabetic Mice

Cells ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 589 ◽  
Author(s):  
Tzu-Hsien Tsai ◽  
Chien-Ho Lee ◽  
Cheng-I Cheng ◽  
Yen-Nan Fang ◽  
Sheng-Ying Chung ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Morphological Changes ◽  
Umbilical Vein ◽  
Vascular Complications ◽  
Specific Marker ◽  
Neointima Formation ◽  
Diabetic Mice ◽  
Mesenchymal Transition ◽  
Compound C ◽  
Endothelial To Mesenchymal Transition

Hyperglycaemia causes endothelial dysfunction, which is the initial process in the development of diabetic vascular complications. Upon injury, endothelial cells undergo an endothelial-to-mesenchymal transition (EndMT), lose their specific marker, and gain mesenchymal phenotypes. This study investigated the effect of liraglutide, a glucagon-like peptide 1 (GLP-1) receptor agonist, on EndMT inhibition and neointima formation in diabetic mice induced by streptozotocin. The diabetic mice with a wire-induced vascular injury in the right carotid artery were treated with or without liraglutide for four weeks. The degree of neointima formation and re-endothelialisation was evaluated by histological assessments. Endothelial fate tracing revealed that endothelium-derived cells contribute to neointima formation through EndMT in vivo. In the diabetic mouse model, liraglutide attenuated wire injury-induced neointima formation and accelerated re-endothelialisation. In vitro, a high glucose condition (30 mmol/L) triggered morphological changes and mesenchymal marker expression in human umbilical vein endothelial cells (HUVECs), which were attenuated by liraglutide or Activin receptor-like 5 (ALK5) inhibitor SB431542. The inhibition of AMP-activated protein kinase (AMPK) signaling by Compound C diminished the liraglutide-mediated inhibitory effect on EndMT. Collectively, liraglutide was found to attenuate neointima formation in diabetic mice partially through EndMT inhibition, extending the potential therapeutic role of liraglutide.

scholarly journals Soluble CD14 participates in the response of cells to lipopolysaccharide.

1992 ◽  
Vol 176 (6) ◽  
pp. 1665-1671 ◽  
Author(s):  
E A Frey ◽  
D S Miller ◽  
T G Jahr ◽  
A Sundan ◽  
V Bazil ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Leukocyte Adhesion ◽  
Umbilical Vein ◽  
Cell Types ◽  
Mononuclear Phagocytes ◽  
Soluble Cd14 ◽  
Bovine Endothelial Cells ◽  
Endothelial Leukocyte Adhesion Molecule ◽  
Umbilical Vein Endothelial Cells ◽  
Serum Dependent

CD14 is a 55-kD protein found both as a glycosylphosphatidyl inositol-linked protein on the surface of mononuclear phagocytes and as a soluble protein in the blood. CD14 on the cell membrane (mCD14) has been shown to serve as a receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein, but a function for soluble CD14 (sCD14) has not been described. Here we show that sCD14 enables responses to LPS by cells that do not express CD14. We have examined induction of endothelial-leukocyte adhesion molecule 1 expression by human umbilical vein endothelial cells, interleukin 6 secretion by U373 astrocytoma cells, and cytotoxicity of bovine endothelial cells. None of these cell types express mCD14, yet all respond to LPS in a serum-dependent fashion, and all responses are completely blocked by anti-CD14 antibodies. Immunodepletion of sCD14 from serum prevents responses to LPS, and the responses are restored by addition of sCD14. These studies suggest that a surface anchor is not needed for the function of CD14 and further imply that sCD14 must bind to additional proteins on the cell surface to associate with the cell and transduce a signal. They also indicate that sCD14 may have an important role in potentiating responses to LPS in cells lacking mCD14.

scholarly journals Attachment of cultured human endothelial cells is promoted by specific association with S protein (vitronectin) as well as with the ternary S protein-thrombin-antithrombin III complex

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1581-1589 ◽  
Author(s):  
KT Preissner ◽  
E Anders ◽  
J Grulich-Henn ◽  
G Muller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Antithrombin Iii ◽  
Cell Attachment ◽  
Umbilical Vein ◽  
Cell Types ◽  
Human Endothelial Cells ◽  
S Protein ◽  
Major Vessel ◽  
Specific Association ◽  
Dose Dependent

Abstract The interaction of the multifunctional S protein (vitronectin) with cultured human endothelial cells of macrovascular and microvascular origin was investigated. Purified S protein, coated on polystyrene Petri dishes, induced dose-dependent and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVECs) as well as human omental tissue microvascular endothelial cells (HOTMECs) at 37 degrees C. Not only isolated S protein, but also the ternary S protein- thrombin-antithrombin III (STAT) complex promoted attachment of approximately 90% of the cells within 2 hours at an S protein concentration of 0.13 mumol/L. Inhibition of attachment in these experiments was achieved by the addition of the cell-attachment pentapeptide Gly-Arg-Gly-Asp-Ser and by monospecific antibodies against S protein, whereas nonrelated peptides or antibodies against fibronectin, fibrinogen, or von Willebrand factor (vWF) were ineffective. Direct binding of S protein to HUVECs and HOTMECs was studied with cells in suspension at a density of 1 x 10(6) cells/mL and was maximal after 120 minutes. S protein bound to both cell types in a dose-dependent fashion with an estimated dissociation constant Kd = 0.2 mumol/L. At a 200-fold to 500-fold molar excess of unlabeled S protein, greater than 80% of bound radiolabeled S protein was displaceable, whereas binding was reduced to 30% to 50% by addition of the pentapeptide, the STAT complex, or by physiologic concentrations of fibrinogen or vWF as well as Fab fragments of anti(human S protein)IgG, but not by Fab rabbit IgG. These findings present evidence for the specific association of S protein with endothelial cells ultimately leading to attachment and spreading of cells. Moreover, a novel function for the ternary STAT complex, which induced endothelial cell attachment and spreading virtually identical to free S protein, is described. These data further suggest a possible role for S protein during coagulation as major vessel wall-related adhesive protein at sites of vascular injury.

Sign in / Sign up

Export Citation Format

Share Document