Hydrogen peroxide stimulates exocytosis of von Willebrand factor in human umbilical vein endothelial cells

The aim of our research was to study the influence of hydrogen peroxide on the exocytosis of von Willebrand factor (vWF) in human umbilical vein endothelial cells (HUVEC). We have found that H₂O₂ at a non-toxic concentration (100 μM) increases the amount of vWF secreted by HUVEC by 43 ± 14% over control (p < 0.03) and elevates total exposition of vWF on cell surface by 89 ± 5% (p < 0.01). Analysis of immunofluorescent images of HUVEC with CellProfiler program revealed that the average number of antigen positive structures on the single cell surface increases from 11.4 ± 0.16 in control up to 17.5 ± 0.21 after incubation with H₂O₂ (p < 0.01). vWF is exposed on the cell surface as dots with the average sizes around 1–3 μm. H₂O₂ causes an increase in the number of these dots and the appearence of the strings of vWF which are absent in control HUVEC. It is suggested that H₂O₂ may serve as a messenger which stimulates vWF exocytosis.     

Isolation of a storage and secretory organelle containing Von Willebrand protein from cultured human endothelial cells

Von Willebrand protein was synthesized and secreted by human endothelial cells in culture. Ca²⁺ ionophore A23187 and phorbol myristate acetate stimulated the release of Von Willebrand protein from the cultured cells. Stimulated release was accompanied by the disappearance of rod-like structures from the cultured endothelial cells immunostained for Von Willebrand protein, suggesting the existence of a storage organelle for Von Willebrand protein in these cells (Loesberg, C., Gonsalves, M.D., Zandbergen, J., Willems, C., Van Aken, W.G., Stel, H.V., Van Mourik, J.A. and De Groot, P.G. (1983) Biochim. Biophys. Acta 763, 160–168). Cultured human endothelial cells were fractionated on a density gradient of colloidal silica. Von Willebrand protein was found in two organelle populations: a buoyant one sedimenting with a variety of cell organelle marker enzymes, including those of the Golgi apparatus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum and plasma membrane fragments (peak density of this fraction: 1.08 g·ml^?1), and a dense one with a peak density of 1.12 g·ml^?1. The dense organelles containing Von Willebrand protein were apparently free of other organelles. Stimulating Von Willebrand protein release with phorbol myristate acetate or Ca²⁺ ionophore A23187 resulted in a decrease or even complete disappearance of Von Willebrand protein from the high-density organelle fraction, implying a role of this organelle in the stimulus-induced release of Von Willebrand protein. The Von Willebrand protein content of the buoyant fraction was lowered to some extent or did not change upon incubation of the cells with ionophore A23187 and phorbol myristate acetate. Restoration of Von Willebrand protein content of the dense organelle fraction after stimulation occurred within 2 days; this was accompanied by recurrence of immunostaining of rod-shaped structures in cells and an increase in cellular Von Willebrand protein. The excretion of restored Von Willebrand protein could be stimulated again.     

Surface glycoproteins of cultured human umbilical vein endothelial cells

Radioactive surface-specific and metabolic labeling techniques were used to characterize the surface glycoprotein pattern of cultured human endothelial cells. Electrophoretic analysis of whole cells, surface labeled either by the galactose oxidase/sodium borotritide or the periodate/sodium borotritide method, revealed several major polypeptides in the Mr region of ca 40-220. During primary culture, the surface labeling pattern showed no changes related to cell density or to the establishment of confluence. A slightly different polypeptide profile was, however, seen when primary culture cells were labeled as an intact monolayer and not in suspension. On the other hand, in cells from later passages, when compared to their parental cells of early passages, there was a distinct intensification of polypeptides with Mr 155 and 90.     

Proteoglycans from human umbilical vein endothelial cells

Human umbilical vein endothelial cells were incubated with [35S]sulphate and investigated for their proteoglycan production. By gel chromatography, ion-exchange chromatography and CsCl density-gradient centrifugation we obtained preparative amounts of the endothelial proteoheparan sulphate HSI and of proteochondroitin sulphate from the conditioned medium of mass-cultured human umbilical vein endothelial cells. Approximately 90% of the 35S-labeled material in the endothelial cell conditioned medium was proteochondroitin sulphate. This molecule, with a molecular mass of 180-200 kDa, contains four side-chains of 35-40 kDa and a core protein of 35-40 kDa. Two proteoheparan sulphate forms (HSI and HSII) from the conditioned medium were distinguished by molecular mass and transport kinetics from the cell layer to the medium in pulse-chase experiments. One major form (HSI), with an approximate molecular mass of 160-200 kDa a core protein of 55-60 kDa and three to four polysaccharide side-chains of 35 kDa each, was found enriched in the cellular membrane pellet. Another proteoheparan sulphate (HSII), with polysaccharide moieties of 20 kDa, is enriched in the subendothelial matrix (substratum).     

Three electrophysiological phenotypes of cultured human umbilical vein endothelial cells

The conventional whole cell patch-clamp technique was used to measure the resting membrane conductance and membrane currents of nonstimulated cultured human umbilical vein endothelial cells (HUVECs) in different ionic conditions. Three electrophysiological phenotypes of cultured HUVECs (n = 122) were determined: first, 20% of cells as type I mainly displaying the inwardly rectifying potassium current (IKi); second, 38% of cells as type II in which IKi was super-posed on a TEA-sensitive, delayed rectifying current; third, 27% of cells as type III predominantly displaying the outwardly rectifying current which was sensitive to TEA and slightly inhibited by a chloride channel blocker niflumic acid (N.A.). In cells of type I, the mean zero-current potential (V0) was dependent on extracellular K+ ([K+]o) but not on Cl-, indicating major permeability to K+. Whereas V0 of type II was also affected by extracellular Cl- ([Cl-]o), indicating the contribution of an outward Cl- current in setting V0. The cells of type III were not sensitive to decrease of [Cl-]o and the outward current was activated in a relative stable voltage range. This varying phenotypic expression and multipotential behavior of HUVECs suggests that the electrical features of HUVEC may be primarily determined by embryonic origin and local effect of the microenvironment. This research provided the detailed electrophysiological knowledge of the endothelial cells.     

Thrombin-induced vimentin phosphorylation in cultured human umbilical vein endothelial cells

Cultured human umbilical vein endothelial cells were stimulated with thrombin (1 unit/ml) for 15-30 s and then lysed with a solution of Triton X-100 containing [gamma-32P]adenosine triphosphate. Thrombin-stimulated human umbilical vein endothelial cells showed an enhanced incorporation of 32P into at least 12 different proteins as compared to control cells treated similarly. The observed enhanced phosphorylation required the active site of thrombin because diisopropylphosphoryl-thrombin had no effect on the level of phosphorylation. The molecular weight of one of the phosphoproteins was similar to that of the intermediate filament protein vimentin (55-60 kDa), a major protein in endothelial cells. This 59-kDa protein was Triton X-100-insoluble and reacted on a Western blot with antibody raised in guinea pig against Chinese hamster ovary cell vimentin. Addition of the anti-vimentin antibody to the thrombin-stimulated, phosphorylated lysate immuno-precipitated a single 32P-labeled protein (59 kDa). These results demonstrate that thrombin rapidly stimulates the phosphorylation of vimentin in cultured endothelial cells and links thrombin stimulation to the phosphorylation of a cytoskeletal protein.     

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.     

Isolation of human umbilical vein endothelial cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.     

Calcium-mobilizing agonists stimulate anion fluxes in cultured endothelial cells from human umbilical vein

Intracellular ion concentrations were determined in split skins of Rana pipiens using the technique of electron microprobe analysis. Based on the 1 min Br uptake from the apical bath, two types of mitochondria-rich (MR) cells could be distinguished: active cells which rapidly exchanged their anions with the apical bath and inactive cells which did not. Br uptake and frequency of active MR cells were closely correlated with the skin conductance, g _t. Replacing Cl in the apical bath with an impermeant anion significantly lowered g _t and the Br uptake and Na concentration of active cells. Even larger reductions were observed after apical amiloride (0.1 mm). The inhibition of the Br uptake was reversible by voltage clamping (100 mV, inside positive). Cl removal and amiloride also led to some shrinkage of active cells. The results suggest that the active cell is responsible for a large part of g _t. Inactive MR cells had much lower Br and Na concentrations which were not significantly affected by Cl removal, amiloride, or voltage clamping. Principal cells, which represent the main cell type of the epithelium, showed only a minimal Br uptake from the apical side which was not correlated with g _t. Moreover, Cl removal had no effect on the Na, Br, and Cl concentrations of principal cells.I wish to thank Cathy Langford for her excellent technical assistance. Financial support was provided by National Institutes of Health grants DK35717 and 1S10-RR0-234501.     

Histamine stimulates prostacyclin synthesis in cultured human umbilical vein endothelial cells

Human venous endothelial cells synthesize prostacyclin (PGI₂) in response to treatment with histamine. The amount of PGI₂ produced is proportional to the histamine concentration over the range of 10^?7 to 10^?5 M, with a maximal response at 2–5 × 10^?6 M. PGI₂ synthesis occurs as a burst lasting less than 3 minutes after histamine addition. The H1 histamine receptor antagonist pyrilamine causes an 87% inhibition of PGI₂ synthesis, whereas the H2 antagonist cimetidine gives no significant inhibition, suggesting that PGI₂ synthesis in response to histamine is mediated by an H1 receptor.     

Co-distribution of von Willebrand factor and fibronectin in cultured rhesus endothelial cells

Summary The synthesis and secretion of von Willebrand factor (VWF, or Factor VIII-related antigen) and fibronectin by cultured endothelial cells from rhesus monkey choroid retina were demonstrated by immunofluorescence, immunoperoxidase and single radial immunodiffusion techniques. Both VWF and fibronectin are localized in intracellular granules and extracellular fibrils. The results of double immunofluorescence staining and post-embedding immunoelectron microscopy showed that there was a co-distribution of VWF and fibronectin not only in pericellular fibrils where they co-aligned with each other to be the components of extracellular matrix, but also in intracellular granules, suggesting they were synthesized or translocated in the same compartment.     

Vascular endothelial growth factor upregulates follistatin in human umbilical vein endothelial cells

Vascular endothelial growth factor (VEGF), plays a key role in angiogenesis. Many endogenous factors can affect angiogenesis in endothelial cells. VEGF is known to be a strong migration, sprouting, survival, and proliferation factor for endothelial cells during angiogenesis in endothelial cells. Searching for novel genes, involved in VEGF signaling during angiogenesis, we carried out differential display polymerase chain reaction on RNA from VEGF-stimulated human umbilical vein endothelial cells (HUVECs). In this study, follistatin (FS) differentially expressed in VEGF-treated HUVECs, compared with controls. Addition of VEGF (10 ng/mL) produced an approximately 11.8-fold increase of FS mRNA. FS or VEGF produced approximately 1.8- or 2.9-fold increases, respectively, in matrix metalloproteinase-2 (MMP-2) secretion for 12 h, compared to the addition of a control buffer. We suggest that VEGF may affect the angiogenic effect of HUVECs, through a combination of the direct effects of VEGF itself, and the indirect effects mediated via induction of FSin vitro.     

Amadori-glycated phosphatidylethanolamine induces angiogenic differentiations in cultured human umbilical vein endothelial cells

Glycation has been implicated in the endothelial dysfunction that contributes to both diabetes- and aging-associated vascular complications. The aim of the present study was to determine whether Amadori-glycated phosphatidylethanolamine (Amadori-PE), a lipid-linked glycation compound that is formed at an increased rate in hyperglycemic states, affected proliferation, migration and tube formation of cultured human umbilical vein endothelial cells (HUVEC). Amadori-PE at a low concentration of less than 5 microM significantly enhanced these three factors involved in angiogenesis. Furthermore, stimulation of HUVEC with Amadori-PE resulted in secretion of matrix metalloproteinase 2 (MMP-2), a pivotal enzyme in the initial step of angiogenesis. Our results demonstrated for the first time that Amadori-PE may be an important compound that promotes vascular disease as a result of its angiogenic activity on endothelial cells. We also demonstrated that MMP-2 is a primary mediator of Amadori-PE-driven angiogenesis.     

The binding of plasminogen fragments to cultured human umbilical vein endothelial cells.

Glu-plasminogen, kringle 1-5, kringle 1-3, and miniplasminogen exhibited strong binding to human umbilical vein endothelial cells (HUVEC). On the other hand, no significant binding was obtained with microplasminogen and kringle 4. Kringle 1-5 and miniplasminogen, which both contained kringle 5, specifically inhibited the binding of plasminogen to HUVEC while kringle 1-3 did not. The results implied plasminogen molecule contained at least two binding sites, with which it interacted HUVEC. The stronger binding site was located in kringle 5 and the weaker one was in kringle 1-3. Kringle 4 and the active site domain exhibited no significant binding to HUVEC. The interaction of plasminogen with HUVEC is mainly through binding site on kringle 5.     

Perturbation of cultured human vascular endothelial cells by phorbol ester or thrombin alters the cellular von Willebrand factor distribution

We have studied the influence of perturbation of cultured human umbilical vein endothelial cells on the distribution of the von Willebrand factor. As shown previously, short-term (less than 1 hr) treatment of endothelial cells with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) or thrombin resulted in the release of cellular stored von Willebrand factor. Long-term treatment with PMA or thrombin evoked a distinct change in the endothelial cell distribution of von Willebrand factor, evident 24 to 48 hrs after exposure. Whereas the contents of the von Willebrand factor storage sites in the cells were gradually restored within 48 hrs, enhanced amounts of von Willebrand factor were secreted into the medium. However, PMA did not increase the endothelial cell contents of mRNA encoding for von Willebrand factor. The number as well as the size of von Willebrand factor storage granules in the endothelial cells increased after exposure to the phorbol ester, as determined by immunofluorescence microscopy. A second treatment with PMA or thrombin, 48 hrs after cells had been stimulated with these agents, resulted again in the instantaneous release of von Willebrand factor. PMA and thrombin caused a decrease in the von Willebrand factor contents of the extracellular matrix. Pulse-chase experiments revealed that PMA blocked the deposition of von Willebrand factor in the subendothelium, whereas PMA did not affect the degradation of matrix von Willebrand factor. Thus, perturbation of endothelial cells changes the cellular distribution of von Willebrand factor.     

Incorporation of arachidonic and linoleic acid hydroperoxides into cultured human umbilical vein endothelial cells

The current study assessed the differential incorporation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), arachidonic acid (AA), 12-hydroxyeicosatetraenoic acid (12-HETE) and the linoleic acid (LA) oxidation products, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), into human umbilical vein endothelial cells (HUVEC). Approximately 80-90% of AA (10(-8)-10(-5)M) and 80% of LA (10(-8)-10(-5)M) were incorporated into HUVEC within 12h, while less than 50% of the hydroxy metabolites (12-HETE, 12-HPETE, 13-HODE, 13-HPODE) were incorporated into HUVEC over 48h. Further, treatment of HUVEC with either 12-HPETE or 13-HPODE (concentrations of 10(-5)M) had no effect on cell number at a 48h time point when compared with control. These results demonstrate that exogeneous hydroxy metabolites are incorporated into HUVEC to a lesser degree than were endogenous fatty acids. Further, we speculate that 12-HPETE and 13-HPODE are rapidly metabolized to substances without significant cytotoxic effects.     

Polar secretion of von Willebrand factor by endothelial cells

Human umbilical vein endothelial cells cultured on a collagen lattice were used to study the polarity of von Willebrand factor (vWF) secretion. Endothelial cells cultured under these conditions allow direct measurements of substances released at both the apical and basolateral surface. The constitutive secretion of vWF was compared to the release of vWF from their storage granules after stimulation (regulated secretion). The basal, constitutive release of vWF occurs into both the apical and subendothelial direction. The rate of accumulation of vWF to the subendothelial direction is about three times higher than the amount of vWF secreted into the lumenal medium per unit of time. However, upon stimulation of confluent endothelial cell monolayers with phorbol myristate acetate, endothelial cells predominantly secrete vWF at the lumenal surface. Under these conditions, vWF does not accumulate in the collagen matrix. Thus, endothelial cells are able to organize themselves into a polarized monolayer, in such a way that vWF secreted by the regulated pathway accumulates at the lumenal site, whereas resting endothelial cells release vWF predominantly at the opposite, basolateral surface.     

Release of annexin V-binding membrane microparticles from cultured human umbilical vein endothelial cells after treatment with camptothecin

Background

Elevated plasma counts of endothelial microparticles (MP) have been demonstrated in various diseases with a vascular injury component. We used flow cytometry to study the MP-release from cultured human umbilical vein endothelial cells (HUVEC) stimulated by various agonists. MP-release by a topoisomerase I inhibitor camptothecin has been studied in detail.

Results

Overnight stimulation of HUVEC with either LPS or TNFα, or 30 min stimulation with thrombin, phorbol-myristate-acetate, tissue plasminogen activator, or angiotensin-II did not cause a significant release of annexin V-binding MP. In contrast, induction of apoptosis with 5 μM camptothecin, documented by 60–70% desquamation of HUVEC culture, annexin V-binding to the cells and DNA-fragmentation, led to a release of annexin V-binding microparticles (~80,000 MP/10³ cells). This microparticle-release was prevented by Z-Val-Ala-Asp-fluoromethyl-ketone (ZVAD). Lower concentration of camptothecin (500 nM) induced comparable microparticle-release without loss of the culture confluence and without increase in annexin V-binding to the cells or DNA-fragmentation. Analyzed microparticles were free of nucleic acids and 95% of microparticles were 0.3–1 μm in size. Double-labeling flow cytometry assay showed that all annexin V-binding Microparticles expressed CD59 but only approximately 50% of these also expressed CD105.

Conclusions

Camptothecin treated HUVEC released different populations of annexin V-binding membrane microparticles at early stage after proapoptotic stimulation before detection of phosphatidylserine exposure on the cells or DNA fragmentation. The microparticle-release was ZVAD sensitive but was not enhanced at the executive phase of apoptosis. These observations offer a new insight into microparticle-release as a marker of endothelial stimulation and injury.