Comparison of tissue-cultured bovine endothelial cells from aorta and sphenous vein

Summary Endothelial cells were harvested from bovine aorta and saphenous vein with collagenase and cultured in McCoy's 5a medium (modified GIBCO) supplemented with 10% fetal bovine serum. The cells were subcultured through 17 passages over 4 to 5 months. The growth properties in culture of the two cell types were compared. Morphological comparisons included phase microscopy and scanning and transmission electron microscopy. Comparisons with cultured aortic smooth-muscle cells were made using phase and scanning electron microscopy. No differences were found between cultured endothelial cells from aorta and saphenous vein. Differences in growth patterns in culture clearly distinguished both endothelial cell types from smooth-muscle cells. The presence of Weibel-Palade bodies identified the cells from both sources as endothelial. This work was supproted by Grants HL-1330 and HL-17269 from NIH.     

Growth factor responses of human arterial endothelial cells in vitro

Summary Human arterial endothelial cells were cultured in vitro for up to 40 cumulative population doublings. Culture conditions similar to those required for long-term propagation of human umbilical vein endothelial cells were employed. These included fibronectin-coated culture vessels, 5 to 20% fetal bovine serum, endothelial cell growth factor, and heparin. Thoracic aorta endothelial cells were larger than iliac artery endothelial cells. Both cell types stained positively for Factor VIII antigen by immunofluorescence. A decrease in confluent density as a function of population doubling level was correlated with the appearance of large, senescent cells in the cultures. Serum growth factors to which the arterial endothelial cells responded included insulin, transferrin, epidermal growth factor, thrombin, and somatomedins. The effect of thrombin did not require the availabilty of the active site of the protease. The effect of the somatomedins was only seen in the presence of heparin. Neither platelet-derived growth factor nor hydrocortisone induced arteiral endothelial cell proliferation. These growth factor responses were also observed on the part of human umbilical vein endothelial cells. This work was supported in part by Public Health Service grants HL01030, HL01734, and AG00599.     

Vascular smooth muscle influences the release of endothelium-derived relaxing factor

Conditioned medium was collected from vascular smooth-muscle cells grown in culture to determine if these cells synthesize vasoactive substances. The medium caused a short-acting endothelium-independent constriction of rat aorta, followed by a prolonged, endothelium-dependent relaxation. This relaxation was mediated through the release of endothelium-derived relaxing factor (EDRF) as it was abolished by the addition of methylene blue (5 x 10(-6) M), haemoglobin (10(-6) M) or methyl arginine, but was not affected by indomethacin (10(-5) M). Smooth-muscle medium stimulated the production of EDRF from both rat and rabbit thoracic aortic rings as well as from cultured bovine pulmonary artery endothelial cells. The prolonged stimulation of EDRF by smooth-muscle medium was not mimicked by known physiological stimuli to EDRF release; EDRF-stimulating activity was not affected when smooth-muscle cells were grown in the presence of indomethacin (10(-5) M), although serum in the medium was required. The EDRF-stimulating substance(s) in the smooth-muscle medium was heat stable and associated with a high molecular mass (30,000 greater than Mr greater than 3500) water-soluble species that is as yet unidentified.     

Characterization of beta-adrenergic receptors in cultured human and bovine endothelial cells

We used radioligand binding methods to characterize beta-adrenergic receptors on endothelial cells cultured from adult human iliac vein (HIVE) and bovine fetal aorta (BFAE). For comparison, we also studied the well-characterized C6 glioma cell line (C6). Both human and bovine endothelial cells showed specific saturable binding of [125I]iodopindolol. There was no difference in the binding affinity (KD) of iodopindolol to membranes from the three cell types. However, the beta-receptor density (Bmax) was greater on HIVE cells and BFAE cells than on C6 cells. Displacement of ligand from HIVE and BFAE cells by zinterol or from BFAE cells by ICI 89,406 was consistent with binding to the beta 2-subtype. In contrast, displacement of ligand from C6 cells by zinterol or ICI 89,406 was consistent with binding to both beta 1- and beta 2-subtypes. Exposing BFAE cells in culture to 10 microM isoproterenol for 6 h resulted in a 55% decrease in Bmax without a change in KD. We conclude that 1) human and bovine endothelial cells in culture contain a substantial population of beta-adrenergic receptors, which are predominantly of the beta 2-subtype, and 2) endothelial beta-receptors exhibit downregulation by beta-agonists in culture.     

Effect of different mitogens and serum concentration on HUVEC morphology and characteristics: implication on use of higher passage cells

Human umbilical vein endothelial cells (HUVEC) were cultured in two different media, viz. the commonly used M199 containing 20% fetal bovine serum (FBS) and endothelial cell growth factor and a defined media EGM-2 containing 2% FBS along with growth supplements in known concentrations. The purpose of this study was to determine the effect of different media on the growth potential and cell morphology in subsequent passages.We have established that a dual coating of gelatin and human fibronectin extracellular matrix provides optimal cell attachment. Growth rate for primary culture was almost double in defined media. For secondary culture a two fold higher proliferation rate was observed in defined EGM-2 media. Histological studies were done using phase contrast, confocal and scanning electron microscopy which showed that cells cultured in M199 started losing their morphological characteristic from 3rd passage and after 6th passage appeared to come in senescent stage, while in case of defined media there was no change observed in the cells up to 10th passage. A significant difference was found in the expression of soluble intracellular adhesion molecule-1 (sICAM-1) which is an endothelial cell marker on cells cultured in different media. Additionally it was observed that exposure duration to trypsin-EDTA during cell detachment also plays an important role in maintaining cell morphological characteristics.These results show that significant morphological changes appear in higher order passages if cells are grown in routine medium for a long time and therefore may not be suitable for cell signaling experiments.     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

Human and bovine vascular endothelial cells. Comparative effect on cell growth and longevity of an eye-derived growth factor (EDGF) and of extracellular matrix

Human and bovine vascular endothelial cells from the umbilical vein and the aorta, respectively, were cultured in the presence of EDGF (a growth factor prepared from bovine retina) on plastic or on extracellular matrix (ECM). Both EDGF and ECM are required to allow the maximal proliferation of human cells and their organization in a typical monolayer. Conversely, bovine aortic endothelial cells grow perfectly in the absence of both factors in 6% fetal calf serum. However, a requirement for EDGF can also be demonstrated in low serum conditions, or in cells at high passage number. ECM had no growth promoting activity by itself. Thrombin acts similarly to EDGF on bovine serum-starved cells. EDGF prolongs the in vitro lifespan of both types of cells. Cells at all stages still synthesize factor VIII antigen as revealed by immunofluorescence. Thus EDGF, like other growth factors from brain, FGF or ECGF, may have an important role in angiogenesis, a critical problem in pathological retinas.     

人隐静脉血管内皮细胞的分离、培养及鉴定

利用冠脉搭桥术后遗弃的隐静脉段获取内皮细胞,采用消化酶消化收集内皮细胞,扩增、冻存、复苏,在体外建立内皮细胞系。此方法简便易行,能在体外获得大量生物学特性保持良好的内皮细胞,为临床血管内皮化研究提供新的细胞来源。     

Production of 5-hydroxyindoleacetic acid from serotonin by cultured endothelial cells

Endothelial cells cells from bovine aorta and human umbilical vein and fibroblasts from human foreskin were cultured and subsequently evaluated for ability to metabolize serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA). Cells were incubated for three hours with 4 X 10(-6) M [14C] 5-HT creatinine sulfate. [14C] 5-HIAA was separated from labeled 5-HT by column chromatography and measured for scintillation counting. Production of 5-HIAA by bovine aorta cells was 39.0+/-7.5 (S.E.M., n=6) nmoles per 10(9) cells per hour. Production of 5-HIAA was markedly inhibited by the presence of 10(-4) M iproniazid (an inhibitor of monoamine oxidase) or 10(-4) M imipramine (an inhibitor of amine transport). 5-HIAA was the only product of 5-HT metabolism detected by thin layer chromatography. Production of 5-HIAA by human umbilical vein endothelial cells was 5.4+/-2.0 nmoles per 10(9) cells per hour (n=5) and by human foreskin fibroblasts was 3.9+/-1.4 nmoles per 10(9) cells per hour (n=5). The results obtained during incubation in the presence and absence of inhibitors indicate that bovine aorta endothelial cells maintained in tissue culture are able to transport serotonin with subsequent production of 5-HIAA. By contrast, human umbilical vein endothelial cells and fibroblasts exhibited relatively low rates of 5-HT uptake and metabolism.     

Lymphatic endothelial and smooth-muscle cells in tissue culture

Summary Endothelial and smooth-muscle cells from bovine mesenteric lymphatic vessels have been collected and cultured in vitro. The endothelial cells grew as a monolayer exhibiting a “cobblestone” appearance with individual cells tending to be more flattened at confluence than their blood vascular counterparts. Approximately 30% of these cells expressed Factor VIII antigen compared with bovine mesenteric artery or human umbilical-vein endothelium in which the majority of cells were positive. The lymphatic smooth-muscle cells exhibited focal areas of multilayering and were Factor VIII negative. The availability of lymphatic endothelial and smooth-muscle cells in culture will provide a new tool for the investigation of the biological properties of the lymphatic vessels and their role in homeostasis. Supported by the Medical Research Council of Canada, Grant MA-7925     

Macro- and microvascular endothelial cells in vitro: Maintenance of biochemical heterogeneity despite loss of ultrastructural characteristics

Summary Microvascular endothelial cells from bovine adrenal medulla and brain and macrovessel endothelial cells from bovine aorta were isolated and cultured under similar conditions in order to determine morphologic and biochemical heterogeneity in vitro. All three cell types exhibited nearly identical ultrastructural morphology and two-dimensional gel protein patterns of³⁵S-methionine-labeled whole cells. Two-dimensional gel analysis of³⁵S-methionine-labeled plasma membrane proteins however, revealed two-dimensional gel protein patterns unique to the tissue type from which the endothelial cells were isolated. This suggests that the functional significance of these specific endothelial cell types is manifested primarily in surface-associated proteins and that many of the differences are sustained in culture. To determine the potential of aorta, brain, and adrenal medulla endothelial cell (EC) cultures to respond to developmentally significant signals, morphology, growth pattern, and cell surface proteins were monitored in the presence and absence of growth factors. A 17 to 26% increase in cell density as well as an increase in the number of elongated and overlapping cells resulted when all three EC types were exposed to a mitogenic medium. Additionally, expression of specific glycoprotein profiles, as determined by Concanavalin A Western blotting of two-dimensional gels, was dependent on the presence or absence of growth factors in the medium. The ability to induce this morphologic and biochemical variation in the three endothelial cell types was maintained into later passage. Taken together, these data imply that endothelial cells isolated from different tissues exhibit and maintain biochemical heterogeneity and do not completely dedifferentiate into a common endothelial cell type in culture. Furthermore, expression of specific subsets of cell surface proteins is dependent on environmental conditions, and in some cases is both cell-type and media-type dependent. Thus, even though endothelial cells are considered terminally differentiated cells, there exists additional or “latent” heterogeneity in the ability of these different cells to respond to “developmental signals” (i.e. mitogenic medium) in vitro. This work was supported in part by a grant from the American Heart Association (860929), NIH (GM29127), and a Biomedical Research Grant from the University of Massachusetts.     

Membrane Potential Changes Visualized in Complete Growth Media through Confocal Laser Scanning Microscopy of bis-Oxonol-Loaded Cells

Confocal laser scanning microscopy (CLSM) was employed to visualize and measure membrane potential changes in several types of cultured adherent cells, such as human fibroblasts, mouse mammary tumor C127 cells, and human saphenous vein endothelial cells, preloaded with the anionic dye bis-1,3,-diethylthiobarbituratetrimethineoxonol (bis-oxonol). The fluorescence of cell-associated bis-oxonol was detected in a single confocal plane. An original flow-chamber apparatus was employed to replace the extracellular medium, avoiding alterations of the plane selected for observation. In all the cell types and the experimental situations tested the intracellular distribution of the dye was typical; perinuclear zones accumulated the dye which, conversely, was excluded by the nucleus. Fluorescence was calibrated versus the membrane potential by varying the extracellular concentration of sodium in the presence of gramicidin. With this approach membrane potential was measured (i) in cultured human fibroblasts incubated under anisotonic conditions, (ii) in heterogeneous cell populations which respond unevenly to potential perturbing conditions, and (iii) in human macrovascular endothelial cells maintained in high-serum, complete growth medium. The results obtained indicate that CLSM can be successfully employed to measure changes of membrane potential in single, bis-oxonol-loaded adherent cells under experimental conditions which severely hinder conventional spectrofluorimetric approaches.     

GTSF-2: A new, versatile cell culture medium for diverse normal and transformed mammalian cells

Summary The aim of this study was to test the versatility of a new basal cell culture medium, GTSF-2. In addition to traditional growth-factors, GTSF-2 contains a blend of three sugars (glucose, galactose, and fructose) at their physiological levels. For these studies, we isolated normal endothelial cells from human, bovine, and rat large blood vessels and microvessels. In addition, GTSF-2 was also tested as a replacement for high-glucose-containing medium for PC12 pheochromocytoma cells and for other, transformed cell lines. The cell growth characteristics were assessed with a novel cell viability and proliferation assay, which is based on the bioreduction of the fluorescent dye, Alamar Blue. After appropriate calibration, the Alamar Blue assay was found to be equivalent to established cell proliferation assays. Alamar Blue offers the advantage that cell proliferation can be measured in the same wells over an extended period of time. For some of the cell types (e.g., endothelial cells isolated from the bovine aorta, the rat adrenal medulla, or the transformed cells), proliferation in unmodified GTSF-2 was equivalent to that in the original culture media. For others cell types (e.g., human umbilical vein endothelial cells and PC12 cells), GTSF-2 proved to be a superior growth medium, when supplemented with simple additives, such as endothelial cell growth supplement (bFGF) or horse serum. Our results suggest that GTSF-2 is a versatile basal medium that will be useful for studying organ-specific differentiation in heterotypic coculture studies.     

大鼠主动脉内皮细胞和平滑肌细胞的原代培养及生物学特性比较

目的培养大鼠主动脉平滑肌细胞和内皮细胞,细胞纯化与鉴定,比较生物学特性的差异。方法采用血管环贴壁法培养动脉内皮细胞,组织块贴壁法培养动脉平滑肌细胞,并采用有限稀释法挑选内皮细胞单克隆,免疫细胞荧光鉴定二者的特异性标志,相差显微镜观察二者单个细胞及细胞群体在形态上的差异性,CCK-8试剂盒检测细胞的增殖,比较二者对胰酶消化,粘附,冻存后复苏的情况。结果血管环贴壁法成功培养血管内皮细胞,组织块培养法成功培养出血管平滑肌细胞,内皮细胞能够形成单克隆集落,培养的细胞均表达相应的特异性标志,内皮细胞增殖速度和平滑肌细胞有差异,内皮细胞对胰酶的耐受性较差,内皮细胞粘附所需时间短,对冻存后的耐受性较好。结论组织块贴壁法适合内皮细胞和平滑肌细胞的培养,有限稀释法能够纯化原代培养的内皮细胞,大鼠主动脉平滑肌细胞和内皮细胞在细胞形态、增殖、粘附、对胰酶的反应、冻存后复苏均存在差异。     

Heparin and acidic fibroblast growth factor interact to decrease prostacyclin synthesis in human endothelial cells by affecting both prostaglandin H synthase and prostacyclin synthase

Prostaglandin production by cultured human endothelial cells varies with growth conditions. We observed a marked diminution in both spontaneous and inducible production of prostacyclin (PGI2) by human umbilical vein and saphenous vein endothelial cells when they were cultured in the presence of the heparin-binding growth factor, acidic fibroblast growth factor (aFGF) and heparin, compared with PGI2 production during culture in medium lacking these factors. Decreased PGI2 production was related to duration of exposure of the cells to aFGF and heparin and depended on the concentration of both substances. Heparin (1-100 micrograms/ml) strongly potentiated the effects of aFGF but had a limited and variable effect alone. The decrease in PGI2 production correlated with a reduction in the cellular content of immunoreactive prostaglandin H synthase and prostacyclin synthase. Arachidonate deacylation was not decreased. In addition, the eicosanoid profile of endothelial cells was changed by exposure to aFGF and heparin. These studies indicate that heparin acts as a modulator of prostaglandin synthesis in endothelial cells through its interaction with aFGF, mediated by alterations in two key enzymes in the arachidonate metabolic pathway.     

Isolation and primary culture of viable multicellular endothelial isolates from hard corals

Summary Conditions for the primary culture of branching scleractinian coral (Acropora micropthalma and Pocillopora damicornis) cells were established with a calcium-free seawater cell dissociation method. Cells were isolated and cultured in supple-mented Dulbecco’s modified Eagle media with heat-inactivated fetal bovine serum, antibiotics, and sterile seawater. Among the isolated cell types, large (60–100 μm) multicellular endothelial isolates (MEIs) were seen in high numbers. These isolates were observed to continually spin for up to 300 h without media change. The following parameters were optimized: media, serum, light, trace elements, and growth factor supplements. Rotations per minute were calculated to determine MEI motility in relation to size. Finally, analyses of external and internal structures were conducted with scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. Additional coral species, Montipora digitata, Stylophora pistillata, Seriatopora hystrix and Porites sp. were also cultured to determine the applicability of isolation techniques. The relatively long survival time of MEIs in primary culture makes them ideal candidates for in vitro studies examining coral disease processes (e.g., mode of infection and intracellular effects of disease-causing agents) as well as aspects of general coral growth and health (e.g., trace element requirements and transfer of products between host cell and zooxanthellae).     

Cytosolic-free calcium in smooth-muscle and endothelial cells in an intact arterial wall from rat mesenteric artery in vitro

The regulation of cytosolic-free calcium concentration of smooth-muscle and endothelial cells was mainly studied on cultured cells where the cross talk between these two coupled cell types is lost. In the present study, the cytosolic-free calcium concentration in the endothelial and the smooth-muscle cells was examined in an intact arterial wall in vitro. Strips of the main branch of rat mesenteric artery were used. Cytosolic-free calcium concentration [Ca2+]i was estimated by determining the fluorescence ratio of the two calcium probes, Fluo-4 and Fura red. The emitted fluorescence of both probes was measured with a confocal microscope. We showed that potassium and phenylephrine, which increase the cytosolic -free calcium concentration of the smooth-muscle cells, also indirectly influence the calcium concentration in the endothelial cells. By simultaneously determining [Ca2+]i in the endothelial and the smooth-muscle cells of an arterial strip, we observed that when calcium increases in the endothelial cells in response to acetylcholine, it slightly decreases in the smooth-muscle cells. We conclude that the regulation of [Ca2+]i in the arterial endothelial cell, depends according to the stimuli either upon the endothelial cells themselves, or upon the coupled smooth-muscle cells.     

Bovine brain and pituitary fibroblast growth factors: comparison of their abilities to support the proliferation of human and bovine vascular endothelial cells

The mitogenic effects of brain and pituitary fibroblast growth factors (FGF) on vascular endothelial cells derived from either human umbilical vein or bovine aortic arch have been compared. Both brain and pituitary FGF are mitogenic for low density human umbilical endothelial (HUE) cell cultures maintained on either fibronectin- or laminin-coated dishes or on biomatrices produced by cultured cells such as bovine corneal endothelial cells or the teratocarcinoma cell line PF-HR-9. Pituitary FGF triggered the proliferation of HUE cells at concentrations as low as 0.25 ng/ml, with a half-maximal response at 0.55 ng/ml and optimal effect at 2.5 to 5 ng/ml. It was 50,000-fold more potent than commercial preparations of endothelial cell growth factor and 40 times more potent than commercial preparations of pituitary FGF. Similar results were observed when the effect of pituitary FGF was tested on low density cultures of adult bovine aortic endothelial cells. When the activity of brain and pituitary FGF on low density HUE cell cultures was compared, both mitogens were active. To confirm the presence in brain extract of both acidic and neutral, as well as of basic mitogen, for HUE cells, brain tissues were extracted at acidic (4.5), neutral (7.2), and basic (8.5) pH. The three types of extracts were equally potent in supporting the proliferation of either HUE or adult bovine aortic endothelial cells. When the various extracts were absorbed at pH 6.0 on a carboxymethyl Sephadex C-50 column, the neutral and basic extracts had an activity after adsorption similar to that of unadsorbed extracts. In contrast, extracts prepared at pH 4.5 lost 90-95% of their activity which was recovered in the adsorbed fraction containing FGF.     

Culture of human blood vessel endothelial cells--a simple and inexpensive culture method

The purpose of this review is to introduce a simple and inexpensive culture method for human umbilical blood vessel endothelial cells. The medium used is MCDB-104 supplemented with 10% fetal bovine serum, 70 ng/ml endothelial cell growth factor from new-born bovine brains, 10 ng/ml murine epidermal growth factor, and 100 micrograms/ml heparin. The culture dishes are coated with gelatin. Under these conditions, endothelial cells from human vessels were grown with doubling times of 18-22 hrs and reached saturation densities of 8-12 x 10(4) cells/cm2. To determine the lifespan of the endothelial cells, the cells were serially subcultivated weekly at an inoculum size of 1,000 cells/cm2. Human endothelial cells from umbilical vein and artery were grown for 21 to 37 passages with 55 to 125 population doublings. This culture method seems to be useful for studying cell proliferation and functions of human endothelial cells.     

Neovascular responses induced by cultured aortic endothelial cells

Neovascularization was studied in the chorioallantoic membrane of the chick embryo after implantation of bovine aortic endothelial and smooth muscle cells, Swiss and BALB/c 3T3 cells and human diploid fibroblasts cultured separately on microcarrier beads. Quantitative analysis of neovascularization indicated a 3 1/2-fold increase in the number of blood vessels responding to endothelial cells while smooth muscle cells induced a twofold increase when compared to the response of beads without cells. Skin fibroblasts and Swiss 3T3 cells did not elicit a comparable response. The marked angiogenic response induced by endothelial cells was characterized by a 137% increase in total vessel length and a 35% increase in average vessel area when compared to controls. Two of the properties required for an angiogenesis factor--stimulation of cellular migration and proliferation--can also be demonstrated using endothelial cell-conditioned medium in cell culture systems. Medium from cultured bovine aortic endothelium stimulates DNA synthesis, proliferation, and migration of smooth muscle cells. In addition, conditioned media from both endothelial cells and smooth muscle cells produced an angiogenic response in the chorioallantoic membrane assay, which was comparable to that produced by intact cells growing on microcarrier beads. Similar responses were not evident with medium conditioned by other cell types. These results indicate the potential importance of endothelial cells and endothelial cell products in regulating blood vessel growth.