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.     

Immunohistochemical evidence for the heterogeneity of maternal and fetal vascular endothelial cells in human full-term placenta

The heterogeneity of endothelial cell surface antigen expression was studied in 5 human full-term placentae by means of indirect immunohistochemistry using 9 monoclonal antibodies and by staining with fluorescent-conjugated Ulex europaeus lectin, both of which are widely used endothelial cell markers. (1) A highly specific, homogeneous staining of fetal and maternal placental vessels of all sizes and anatomical regions was observed by the monoclonal antibodies PAL-E, QBEND10 and 1F10. These antibodies were even more specific than Ulex europaeus lectin, factor VIII antibody and von Willebrand factor antibody, which cross-reacted with some non-endothelial cells and structures. The reactivity of PAL-E, QBEND10 and 1F10 with residual surface cells of the basal plate strongly suggests an endothelial origin of these cells. (2) In contrast to other organs, PAL-E, QBEND10 and HM 15/3 strongly stained endothelial cells of the macrovascular system in the human placenta. This might indicate an organ-associated heterogeneity of fetal endothelial cells. (3) Monoclonal antibodies against receptors for transferrin and IgG (FcRII) labeled the endothelial cells of fetal placental vessels with increasing intensity distal to the insertion of the umbilical cord. The vessels of the umbilical cord itself were unreactive. This might suggest a heterogeneity of macro- and microvascular endothelial cells.     

Immunohistochemical localization of endothelin in human vascular endothelial cells

The cellular localization of endothelin (ET), a novel vasoconstrictor peptide, was studied in human vascular tissues by immunohistochemistry. Distinct and diffuse staining for ET-like immunoreactivity was demonstrated in the cytoplasm of vascular endothelial cells, but not in smooth muscle cells or adventitial fibroblasts. The specificity was confirmed by the negative results following immunoabsorption. These findings suggest that human vascular endothelial cells function as an endocrine and/or paracrine cells for ET secretion.     

Virus induced adherence of monocytes to endothelial cells

Abstract In this study we have demonstrated that infection of human umbilical vein endothelial cells (HUVEC) with Herpes simplex virus type 1 (HSV-1) resulted in an increased adherence of monocytes (MC). This enhanced adherence occurred at 3 h post infection (p.i.) when about 20% of the monolayer is infected and when there is no cytopathic effect observable in the monolayer. The adherence of human MC to virus-infected HUVEC monolayers proved to be effective and reproductible if a multiplicity of infection (MOI) of ten and a ratio of number of MC to number of HUVEC of 5 was used. The increased adherence was also induced by incubating non-infected HUVEC with the 'supernatant medium' of the HSV-1 infected cells, showing that soluble factors induced by viral infection are responsible for the increased adherence. The augmentation of MC adherence to infected endothelium was sensitive to tunicamycin treatment, suggesting that the MC adherence is probably mediated by glycoproteins expressed on the HUVEC membranes by virus infection.     

Feedback modulation of endothelial cells promotes epithelial-mesenchymal transition and metastasis of osteosarcoma cells by Von Willebrand Factor release

Endothelial cells (ECs), as a tumor niche cell, generate and secrete Von Willebrand factor (VWF) that is linked to osteosarcoma (OS) progression. However, the role and regulatory mechanisms of VWF that underpin OS progression remain unclear. Here, using a coculture system ex vivo, we showed that ECs promoted the epithelial-mesenchymal transition (EMT) process in OS cells via enhanced VWF secretion. VWF secreted by ECs directly contributed to OS EMT and metastasis by activating NF-κB signaling. In addition, OS cells exerted a feedback effect on ECs to promote VWF release via activation of phospholipase D 1 signaling, through which enhanced VWF secretion results in further tumor deterioration. To conclude, ECs served as a modulator and an effector of OS, accelerating OS exacerbation by VWF release.     

Subcellular localization of leukotriene receptors in human endothelial cells

Leukotriene B₄ (LTB₄), a potent chemotactic and immune-modulating mediator, signals via two receptors, BLT₁ and BLT₂. Recently, we reported that BLT₁ is the predominating BLT expressed on human umbilical vein endothelial cells (HUVEC), and that BLT₁ mediated functions are enhanced by LTB₄ and lipopolysaccharide (LPS), but not by TNFα. Here, we demonstrate that BLT₁ is found on the outer cell membrane of HUVECs but also in intracellular granules, co-localized with monocyte chemotactic protein-1 and P-selectin, but not with interleukin-8 and von Willebrand factor. Upon stimulation with LTB₄ or LPS, more BLT₁ protein is found, now evenly distributed over the cytoplasm and in the cell nucleus, but less on the cell surface. An MAP kinase inhibitor prevented this enhancement and translocation, suggesting this signaling pathway to be crucial. Thus, BLT₁, a G-protein-coupled 7-transmembrane receptor, is located in various subcellular compartments in endothelial cells, which may have implications for cellular LT dependent responses and target accessibility for BLT₁ antagonists.     

Motion of polymerized albumin particles in a model arteriole in the presence of red blood cells

Polymerized albumin particles (poly Alb) with recombinant glycoprotein Ibα (rGPIbα-poly Alb) are a promising candidate for a platelet substitute. Thus, we focused on the lateral motion of poly Alb in the presence of red blood cells, because the lateral motion plays an important role in aggregate formation. We visualized the microscopic motion of poly Alb toward the immobilized ligand (von Willebrand factor, VWF) surface in a model arteriole with red blood cells with a high-speed camera. At a higher shear rate of 1,500 s⁻¹, the concentration profile of poly Alb appeared to peak near the wall. This profile enhances the interaction between the particles and wall. Particularly the migration angle, being the angle of the poly Alb velocity vector, was enlarged near the wall and contributed to transfer of poly Alb toward the immobilized VWF surface. This tendency is desirable to achieve the adhesion of particles on the wall.     

Isolation of endothelial cells from human placental microvessels: effect of different proteolytic enzymes on releasing endothelial cells from villous tissue

Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary.     

High glucose accelerates MCP-1 production via p38 MAPK in vascular endothelial cells

In diabetes mellitus (DM), hyperglycemia causes cardiovascular lesions through endothelial dysfunction. Monocyte chemoattractant protein-1 (MCP-1) is implicated in the pathogenesis of cardiovascular lesions. By using human umbilical vein endothelial cells, we investigated the effect of hyperglycemia on MCP-1 production and its signaling pathways. Chronic incubation with high glucose increased mRNA expression and production rate of MCP-1 in a time (1-7 days)- and concentration (10-35 mM)-dependent manner. Chronic exposure to high glucose resulted in enhancement of generation of reactive oxygen species (ROS), as determined by increasing level of 2,7-dichlorofluorescein (DCF), and subsequent activation of p38 mitogen-activated protein kinase (MAPK). Neither c-Jun NH(2)-terminal kinase nor extracellular signal-regulated kinase1/2 was affected. SB203580 or FR167653, p38 MAPK specific inhibitors, completely suppressed MCP-1 expression. Catalase suppressed p38 MAPK phosphorylation and MCP-1 expression. These results indicate that hyperglycemia can accelerate MCP-1 production through the mechanism involving p38 MAPK, ROS-sensitive signaling pathway, in vascular endothelial cells.     

Biochemical properties of vascular endothelial cells

Present knowledge in the field of vascular endothelial cells is reviewed. The role of endothelial cells in the synthesis of matrix proteins and glycosaminoglycans is described. Endothelial cells play a considerable role in the processes of coagulation and fibrinolysis. They also interact with neurotransmitters and vasomotoric substances, and participate in inflammation and immunological responses. They procuce several different growth factors. Their role in lipoprotein metabolism is of special importance to research into atherosclerosis.     

High-density lipoprotein endocytosis in endothelial cells

AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.     

Production of recombinant Von Willebrand factor by CHO cells cultured in macroporous microcarriers

Recombinant Chinese hamster ovary cells producing Von Willebrand factor have been successfully grown in gelatin macroporous microcarriers (Cultispher-G). Serum-free cultures were maintained in 1, 4, and 10 liter fermentors for more than two months. Comparative studies with Cytodex-3 microcarriers have been performed in 1 liter fermentors. The lower specific Von Willebrand factor productivity of CHO cells cultivated on Cultispher-G were offset by higher cell densities (10⁷–2×10⁷ cells/ml). Volumetric Von Willebrand factor productivity was influenced by oxygen concentration, and remained stable during scale-up from 1 to 10 liter fermentors.     

Neurogenic effect of vascular endothelial growth factor during germ layer formation of human embryonic stem cells

Vascular endothelial growth factor (VEGF), a potent mitogen for vascular endothelial cells, has been suggested as a modulator that is involved in neurogenesis as well as angiogenesis. Here, we directly examined the effect of VEGF on neuroectodermal differentiation using human embryonic stem cells (hESCs). VEGF treatment upregulated the expression of neuroectodermal genes (Sox1 and Nestin) during germ layer formation in embryoid bodies (EBs) and efficiently increased the number of neural rosettes expressing both Pax6 and Nestin. The neural progenitors generated from VEGF-treated EBs further differentiated into cells that showed a similar pattern of gene expression observed in the development of dopaminergic neurons upon terminal differentiation. These results support the neurogenic effect of VEGF on hESC differentiation.     

Von Willebrand factor targets IL-8 to Weibel-Palade bodies in an endothelial cell line

Vascular endothelial cells are able to store the chemotactic cytokine interleukin-8 (IL-8) in specialized storage vesicles, Weibel-Palade bodies, together with von Willebrand factor (VWF) and P-selectin. We investigated whether VWF plays a role in the sorting of IL-8 into these organelles. We examined the effect of VWF expression on IL-8 targeting in an endothelial cell line (EC-RF24). This cell line has retained the typical phenotypic characteristics of primary endothelial cells but has lost the capacity to produce VWF in appreciable amounts. EC-RF24 cells were retrovirally transduced with a vector encoding a VWF-green fluorescent protein chimera (VWF-GFP). This approach enables direct visualization of the cellular distribution and secretory behavior of the VWF-GFP hybrid. Expression of VWF-GFP resulted in the generation of Weibel-Palade body-like organelles as shown by the colocalization of VWF-GFP and P-selectin. VWF-GFP expressing EC-RF24 cells also showed significant colocalization of VWF-GFP with IL-8 in these storage vesicles. Live cell imaging revealed that the number of VWF-GFP-containing granules decreased upon cell stimulation. These observations indicate that VWF plays an active role in sequestering IL-8 into Weibel-Palade bodies.     

Phenotypic heterogeneity within clones of fetal human cells.

The heterogeneity of cell morphology characteristics of some colonies of human fetal kidney and amniotic fluid cells has been analyzed by biochemical and cell-cloning techniques. All the presumed subclones derived from dimorphic colonies were initially epithelioid, but some cells became fibroblastlike as the cell density increased. To determine if the observed heterogeneity occurred within clonal populations of cells, we determined the isozyme phenotype of dimers from renal cells heterozygous for glucose-6-phosphate dehydrogenase (G6PD). Colonies showing mixed cellular morphology expressed only a single G6PD isozyme, thus revealing their single-cell origin. Our results indicate that cell morphology is influenced by the cellular density within the clone, and that a single human renal cell in vitro can yield progeny of two morphological types.     

Expression of TRPC homologs in endothelial cells and smooth muscle layers of human arteries

TRPC channels are a group of Ca²⁺-permeable nonselective cation channels that mediate store-operated and/or agonist-stimulated Ca²⁺ influx in a variety of cell types. In this study, we extensively examined the expression patterns of TRPC homologs in human vascular tissues. RT-PCR amplified cDNA fragments of TRPC1 (505 bp), TRPC3 (372 bp), TRPC4 (499 bp), TRPC5 (325 bp), TRPC6 (509 bp), and TRPC7 (187 bp) from RNA isolated from cultured human coronary artery endothelial cells. In situ hybridization yielded strong labeling of TRPC1,3–6 in the endothelial and smooth muscle cells of human coronary and cerebral arteries. TRPC7 labeling was exclusively found in endothelial cells but not in smooth muscle cells. Results from immunohistochemical staining were consistent with those from in situ hybridization. Similar expression patterns of TRPC homologs were also observed in arterioles and vaso vasora. In conclusion, our study indicates that TRPC homologs are widely expressed in human vessels of all calibers, including medium-sized coronary arteries and cerebral arteries, smaller-sized resistance arteries, and vaso vasora. These results suggest a ubiquitous role of TRPC homologs in regulating blood supply to different regions and in controlling arterial blood pressure.     

Isolation of the factor VIII–von Willebrand factor complex directly from plasma by gel filtration

A high capacity gel filtration system was developed with the purpose of isolating factor VIII (FVIII) and von Willebrand factor (vWF) directly from plasma in significantly higher yields than obtained by cryoprecipitation, the technique most commonly used to recover FVIII–vWF from human plasma. After laboratory-scale gel filtration of plasma, a FVIII-containing fraction was collected containing about 90% of FVIII in the applied plasma and with almost tenfold higher purity than that obtained by cryoprecipitation. The gel filtration step has been scaled up for use as the initial step in the manufacturing process for a FVIII preparation (Nordiate).     

Aberrant expression of histo-blood group A type 3 antigens in vascular endothelial cells in inflammatory sites.

Histo-blood group ABH antigens are widely distributed in human tissues. The epitopes of ABH antigens are carried by at least four different peripheral core isotypes of internal carbohydrate backbones (type 1-4). Each type of ABH antigen is expressed tissue specifically, and aberrant expression of ABH antigens is often observed during oncogenesis. We immunohistochemically examined the expression of A type 3 antigens in wounded and diseased skin tissues (A and AB blood groups). In uninjured skin, the expression of A type 3 antigens was restricted to the eccrine sweat gland. In addition to the sweat glands, A type 3 antigens were found in vascular endothelial cells of the wound sites. The extent of A type 3 antigens expression related to postinfliction intervals. A significantly higher expression rate of A type 3 antigens in endothelial cells was also observed in diseased skin, suggesting that inflammation might induce A type 3 antigen expression in endothelial cells. Double-color immunofluorescence staining of the specimens showed that von Willebrand factor (vWF) was a core-protein of A type 3 determinants aberrantly expressed in endothelial cells in inflamed tissues, suggesting that aberrant expression of A type 3 antigens is involved in stabilization of vWF in inflammation.