Isolation and culture of human decidual capillary endothelial cells in serum-free medium supplemented with human uterine angiogenic factor

Human decidual capillary endothelial (HDCE) cells, obtained from decidual fragments of legally induced first-trimester terminations of pregnancies, were cultured in a serum-free medium supplemented with human uterine angiogenic factor (HUAF). The method of isolating the cells from the decidual tissue is described. Identification of the decidual endothelial cells was based on light- and electron-microscopic observations as well as on antifactor VIII immunoperoxidase-staining technique. The HDCE cell culture in the serum-free medium lasted for 25 weeks through eight subcultures. The cells frozen in glycerin yielded 80% viability after thawing. Electron-microscopic observations of the monolayer demonstrated Weibel-Palade bodies, desmosomes and tight junctions. HUAF is mitogenic to HDCE at 10-100 ng/ml. The dependency of HDCE cells grown in culture on HUAF may explain in part the mechanism involved in the dynamic vascular expansion occurring during gestation.     

Isolation and culture of rat microvascular endothelial cells

The purpose of this study is to identify the separation techniques that result in pure cultures of rat microvascular endothelial cells (MECs). A multistep process is used to optimize the separation of the cells from rat epididymal fat pads, obtaining as pure a culture as possible within a relatively short processing time. The process initially employs the digestion, filtration, and density gradient separation steps. We further describe the use of an attachment phase that allows the differential adherence of contaminating cell types. Immunomagnetic purification is the final step in the process and is performed using anti-PECAM-1 (CD31) monoclonal antibody-labeled DynaBeads.     

Isolation and culture of pulmonary artery endothelial cells.

It has become increasingly evident that endothelial cells function as far more than a mechanical barrier between blood and parenchyma. Endothelial cells from one vesicular bed are known to differ structurally from those of another, and it has been suggested that they may differ functionally. Further to test the hypothesis that endothelial cells from one site may differ in terms of function from those of another site, it is necessary to test endothelium from various source after having obtained these cells in pure, well-characterized cultures. To facilitate such studies, we herein describe in detail means for the isolation, culture and characterization of endothelial cells from calf pulmonary artery. These cells may be of major interest in terms of specific metabolic activities as it has become evident that the lungs play a prominent role in determining the hormonal composition of systemic arterial blood.     

Morphological behavior of cultured bovine adrenal medulla capillary endothelial cells.

Bovine adrenal medulla capillary endothelial cells were isolated and cloned, and their morphological behaviors in vitro were examined. In the culture of primary or early passage, one type of colony formed intracellular lumina both on the dish and in the three dimensional collagen gel. Another type proliferated well and showed morphology ranging from slender-shape to cobblestone shape, and were easily cloned. Cloned cells which showed slender-shapes formed tubular network on plastic dish after addition of PMA, OAG or vanadate, and these cells also formed multicellular tubules in the three dimensional collagen gel. However, the formation of diaphragmed fenestrae by these slender-shape clones was rare. One clone which showed cobblestone shape formed diaphragmed fenestrae, when cultured on collagen gel for more than one month. Isolated colonies or clones showed heterogeneity of cell shape, angiogenic behaviors and fenestrae formation.     

Long-term culture of human endothelial cells

Summary Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10³/cm²) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts. This research was supported by grants from the U.S. Public Health Service (AG 01732, HL 16387, and HL 07080), the Cystic Fibrosis Foundation, and the New York and American Heart Associations. Victor B. Hatcher is an Established Fellow of the New York Heart Association and a recipient of the Ann Weinberg Cystic Fibrosis Research Scholarship Award.     

Long-term culture of human endothelial cells

Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10(3)/cm2) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts.     

Isolation and characterization of human thyroid endothelial cells

From collagenase digests of human thyroid, endothelial cells were separated from follicular cells by their greater adherence to gelatin-coated plates. Endothelial cells were further purified using fluorescence-activated cell sorting, selecting for cells expressing factor VIII-related antigen. Isolated cells were negative for thyroglobulin and calcitonin when examined by immunostaining. The receptor for the angiopoietins, Tie-2, was expressed by the cells, and expression was increased by agents that elevate cAMP. Nitric oxide synthase (NOS) 3, the endothelial form of NOS, was expressed by the cells and similarly regulated. Cells responded strongly to the mitogen fibroblast growth factor (FGF)-2 in growth assays but only weakly to vascular endothelial growth factor (VEGF). VEGF was, however, able to stimulate nitric oxide release from the cells consistent with their endothelial origin. The FGF receptor (FGFR1) was full length (120 kDa) and immunolocalized to the cytosol and nucleus. Thyrotropin (TSH) did not regulate FGFR1, but its expression was increased by VEGF. Thrombospondin, a product of follicular cells, was a growth inhibitor, but neither TSH nor 3,5,3'-triiodothyronine had direct mitogenic effects. Thyroid follicular cell conditioned medium contained plasminogen activator activity and stimulated the growth of the endothelial cells, but when treated with plasminogen to produce the endothelial-specific inhibitor, angiostatin, growth was inhibited. Human thyroid endothelial cell cultures will be invaluable in determining the cross talk between endothelial and follicular cells during goitrogenesis.     

Characterization of endocrine gland-derived vascular endothelial growth factor signaling in adrenal cortex capillary endothelial cells.

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) has been recently identified as a mitogen specific for the endothelium of steroidogenic glands. Here we report a characterization of the signal transduction of EG-VEGF in a responsive cell type, bovine adrenal cortex-derived endothelial (ACE) cells. EG-VEGF led to a time- and dose-dependent phosphorylation of p44/42 MAPK. This effect was blocked by pretreatment with pertussis toxin, suggesting that G alpha(i) plays an important role in mediating EG-VEGF-induced activation of MAPK signaling. The inhibitor of p44/42 MAPK phosphorylation PD 98059 resulted in suppression of both proliferation and migration in response to EG-VEGF. EG-VEGF also increased the phosphorylation of Akt in a phosphatidylinositol 3-kinase-dependent manner. Consistent with such an effect, EG-VEGF was a potent survival factor for ACE cells. We also identified endothelial nitric-oxide synthase as one of the downstream targets of Akt activation. Phosphorylation of endothelial nitric-oxide synthase in ACE cells was stimulated by EG-VEGF with a time course correlated to the Akt phosphorylation. Our data demonstrate that EG-VEGF, possibly through binding to a G-protein coupled receptor, results in the activation of MAPK p44/42 and phosphatidylinositol 3-kinase signaling pathways, leading to proliferation, migration, and survival of responsive endothelial cells.     

Human microvessel endothelial cells: Isolation,culture and characterization

Summary Over recent years, interest in endothelial cell biology has increased dramatically with our ability to grow and study endothelial cellsin vitro. While large veins and arteries remain a quick and convenient source of endothelial cells, the great morphological, biochemical and functional heterogeneity that endothelial cells express has necessitated the development of techniques to isolate microvessel endothelial cells from different tissues to create more realisticin vitro models. The majority of isolation procedures employ selective methods to enrich microvessel endothelial cells from tissue homogenates directly, or after a period in culture. These include sieving/filtration, manual weeding, isopycnic centrifugation, selective growth media, and the use of flow cytometry or magnetic beads coupled with specific endothelial cell markers. The establishment of pure endothelial cell populations is important for studying their biochemistry and physiology and there are many morphological, immunological and biochemical criteria which can be used to characterize human endothelial cells. These range from classical markers such as von Willebrand Factor and angiotensin-converting enzyme to novel markers like platelet endothelial cell adhesion molecule-1 (CD31) and the expression of E-selectin on cytokine-activated endothelial cells.     

Cloning and expression of mannosylphospho dolichol synthase from bovine adrenal medullary capillary endothelial cells

Mannosylphospho dolichol synthase (DPMS) is a critical enzyme in the biosynthesis of lipid-linked oligosaccharide (LLO; Glc₃Man₉GlcNAc₂-PP-Dol), a pre-requisite for asparagine-linked (N-linked) protein glycosylation. We have shown earlier that DPMS is important for angiogenesis, i.e., endothelial cell proliferation. This is true when cAMP is used for intracellular signaling. During cAMP signaling, DPMS is activated and ER stress is reduced. To understand the activation of DPMS at the molecular level we have isolated a cDNA clone for the DPMS gene (bDPMS) from the capillary endothelial cells of bovine adrenal medulla. DNA sequencing and the deduced amino acid sequence have established that bDPMS has a motif to be phosphorylated by cAMP-dependent protein kinase (PKA). Based on the sequence information Serine 165 has been found to be the phosphorylation target in bDPMS. Hydropathy Index when plotted against amino acid number indicates the presence of a hydrophobic region around the amino acid residues 120–160, supporting that bDPMS has one membrane spanning region. The recombinant bDPMS has now been purified as His-tag protein with an apparent molecular weight of M _r 33 kDa. Additionally, we show here that overexpression of DPMS is indeed angiogenic. The capillary endothelial cells proliferate at a higher rate carrying the DPMS overexpression plasmid over the parental cells or the vector.     

Characterization of the receptor to vasculotropin on bovine adrenal cortex-derived capillary endothelial cells

Recently a new growth factor was purified to homogeneity, and its bioactivity seemed to be restricted to vascular endothelial derived cells. As it was also angiogenic in vivo, it was provisionally named vasculotropin (VAS). As an iodination procedure used to label VAS did not damage the molecule, it was possible to undertake binding studies. The binding of iodinated vasculotropin to bovine adrenal cortex-derived capillary endothelial cells was saturable at 250 pM, and half-maximal binding occurred at 47 pM. Scatchard's analysis of the data demonstrated two apparent classes of binding sites with apparent dissociation constants of 2 and 82 pM displaying 280 and 3400 binding sites, respectively. The binding was specific; half-displacement was observed with a 2-fold excess of unlabeled VAS. The structurally related platelet-derived growth factor did not compete in a radioreceptor assay. 125I-VAS was displaced by suramin and not by heparin. 125I-VAS was covalently cross-linked to its cell surface receptor on intact bovine adrenal cortex-derived capillary endothelial cells using the homobifunctional agents ethylene glycol bis(succinimidyl succinate) or disuccinimidyl tartarate. A major macromolecular species with an apparent molecular mass of 230,000 Da was labeled under reducing and nonreducing conditions. These data demonstrate the existence of a specific binding protein for VAS and an estimation of the size at 185,000 Da.     

Isolation and culture of human intestinal smooth muscle cells

Intestinal smooth muscle cells were isolated from human bowel and maintained in culture through several passages. These cells were obtained by enzyme digestion of slices taken from the circular layer of the muscularis propria of human jejunum. When subcultured, they initially flattened out and then began proliferating after 3 days. After 3 weeks in culture, they began aggregating into ridges. Fluorohistochemical staining revealed numerous prominent actin stress fibers. When these cells were exposed to the C-terminal octapeptide of cholecystokinin they contracted in a dose-dependent fashion. The availability of human intestinal smooth muscle cells in culture will considerably enhance our ability to study the contractile, proliferative and connective tissue responses of the smooth muscle of the human gastrointestinal tract.     

Lung endothelial cells strengthen,but brain endothelial cells weaken barrier properties of a human alveolar epithelium cell culture model

The blood–air barrier in the lung consists of the alveolar epithelium, the underlying capillary endothelium, their basement membranes and the interstitial space between the cell layers. Little is known about the interactions between the alveolar and the blood compartment. The aim of the present study was to gain first insights into the possible interplay between these two neighbored cell layers. We established an in vitro Transwell model of the alveolar epithelium based on human cell line H441 and investigated the influence of conditioned medium obtained from human lung endothelial cell line HPMEC-ST1.6R on the barrier properties of the H441 layers. As control for tissue specificity H441 layers were exposed to conditioned medium from human brain endothelial cell line hCMEC/D3. Addition of dexamethasone was necessary to obtain stable H441 cell layers. Moreover, dexamethasone increased expression of cell type I markers (caveolin-1, RAGE) and cell type II marker SP-B, whereas decreased the transepithelial electrical resistance (TEER) in a concentration dependent manner. Soluble factors obtained from the lung endothelial cell line increased the barrier significantly proven by TEER values and fluorescein permeability on the functional level and by the differential expression of tight junctional proteins on the molecular level. In contrast to this, soluble factors derived from brain endothelial cells weakened the barrier significantly. In conclusion, soluble factors from lung endothelial cells can strengthen the alveolar epithelium barrier in vitro, which suggests communication between endothelial and epithelial cells regulating the integrity of the blood–air barrier.     

Variant endothelial cells from human carotid artery in culture

Summary Endothelial cells were cultured from the carotid artery with thickened intima comprised of two to five layers of smooth muscle cells, isolated from a 19-yr-old female, who died from a accident. The cells were grown and subcultured in Medium 199 supplemented with 20% heat inactivated fetal bovine serum. The cells are still viable at present, the 22nd passage. The cultured cells were found to have the following characteristics: existence of Factor VIII-related surface angiten and prostacyclin synthesis slightly less than that for typical endothelial cells. The most outstnading feature was the formation by an individual cell of a single ring, and composite ring formed by two to five cells. Neither the synthesis of an angiotensin converting enzyme nor that of a Weibel-Palade body could be detected by electron microscopy. The cultured cells possessed only a few characteristics specific for typical endothelial cells and were designated as variant endothelial cells.     

Interaction of urokinase with specific receptors stimulates mobilization of bovine adrenal capillary endothelial cells

On the basis of 125I-labeled plasminogen activator binding analysis we have found that bovine adrenal capillary endothelial cells have specific receptors for human urinary-type plasminogen activator on the cell membrane. Each cell exposes about 37,000 free receptors with a Kd of 0.8958 x 10(-9) M [corrected]. A monoclonal antibody against the 17,500 proteolytic fragment of the A chain of the plasminogen activator, not containing the catalytic site of the enzyme, impaired the specific binding, thus suggesting the involvement of a sequence present on the A chain in the interaction with the receptor, as previously shown in other cell model systems. Both the native molecule and the A chain are able to stimulate endothelial cell motility in the Boyden chamber, when used at nanomolar concentrations. The use of the same monoclonal antibody that can inhibit ligand-receptor interaction can impair the plasminogen activator and A-chain-induced endothelial cell motility, suggesting that under the conditions used in this in vitro model system, the motility of bovine adrenal capillary endothelial cells depends on the specific interaction of the ligand with free receptors on the surface of endothelial cells.     

Binding and endocytosis of heparin by human endothelial cells in culture

Binding of heparin and low molecular weight heparin fragments (CY 222, Mr range 1500-8000) to human vascular endothelial cells was studied. Primary culture of human umbilical vein endothelial cells and either 125I or 3H-labeled heparin or [125I]CY 222 were used. Slow, saturable and specific binding was found. No other tested glycosaminoglycan, excepting a highly sulfated heparan fraction, was able to compete for heparin binding. Two groups of binding sites for [3H]heparin could be distinguished: one with high affinity (Kd = 0.12 microM) and another with lower affinity (Kd = 1.37 microM) and a relative large capacity of binding (1.16 X 10(7) molecules/cell) was calculated. The Kd for unlabeled heparin, as calculated from competition experiments, was 0.23 microM. Much lower affinity was calculated for unlabeled low molecular weight heparin fragments CY 222 (Kd = 4.3 microM) from competition experiments with [125I]CY 222. The binding reversibility was only partial for unfractionated heparin. Even by chasing with unlabeled compound, a fraction of 25-30% was not dissociable from endothelial cells. This fraction was much lower if incubation was carried out at 4 degrees C. The addition of basic proteins (histones) to the incubation medium greatly enhanced the undissociable binding at 37 degrees C, but not at 4 degrees C. The undissociable fraction of heparin was not available to degradation by purified microbial heparinase. These results suggest that a fraction of bound heparin is internalized by the vascular endothelium.     

Proteomic study of human umbilical vein endothelial cells in culture

The endothelium is a single layer of cells lining the inside face of all blood vessels. It constitutes a major metabolic organ which is critically involved in the generation and the regulation of multiple physiological and pathological processes such as coagulation, hemostasis, inflammation, atherosclerosis, angiogenesis and cancerous metastasis dissemination. In order to increase our knowledge about the protein content and the main biological pathways of human vascular endothelial cells, we have undertaken the proteomic analysis of the most explored present endothelial cell model, i.e. primocultures of human umbilical vein endothelial cells (HUVECs). Using low levels of protein loads (~ 30 nug), the association of two-dimensional electrophoresis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry, liquid chromatography-tandem mass spectrometry and database interrogations allowed us to identify 53 proteins of suspected endothelial origin in quiescent HUVECs. Beside cytoskeletal proteins such as actin, tubulin, tropomyosin and vimentin, we identified various proteins more especially implicated in cellular motility and plasticity (e.g. cofilin, F-actin capping protein and prefoldin), in regulation of apoptosis and senescence (protease inhibitor 9, glucose related proteins, heat shock proteins, thioredoxin peroxidase, nucleophosmin) as well as other proteins implicated in coagulation (annexin V, high mobility group protein), antigen presentation (valosin containing protein and ubiquitin carboxyl terminal hydrolase isozyme L1) and enzymatic capabilities (glutathione-S-transferase, protein disulfide isomerases, lactate deshydrogenase). The presented annotated 2-D maps of HUVECs will be soon available on the web at http://www. huvec.com.     

Biological properties of human melanoma cells in culture

Summary Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary. THis work was supported in part by USPHS Grant No. 5 T01 AI00332-06 from the National Institutes of Health, Contract E73-2001-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, and USPHS Grant No. 0H00714-02 from the National Institute for Occupational Safety and Health.