Serial propagation of human endothelial cells in vitro

Human umbilical vein (HUV) endothelial cells were grown for 15 to 21 passages at a split ratio of 1:5 (at least 27 population doublings) on a human fibronectin (HFN) matrix in Medium 199 supplemented with fetal bovine serum (FBS) and endothelial-cell growth factor (ECGF). This system also permitted the growth of HUV endothelial cells at cell densities as low as 1.25 cells/cm2. In addition to delaying the premature senescence of HUV endothelial cells, ECGF also reduced the serum requirement for low-density HUV endothelial-cell growth; 2.5% serum and ECGF yields half-maximum growth as compared to high serum controls. Significant HUV endothelial-cell growth was also observed in medium supplemented with either ovine hypophysectomized (HYPOX) serum, plasma-derived serum (PDS), or HYPOX-PDS in the presence of ECGF, suggesting that neither the pituitary nor the platelet contributes to HUV endothelial-cell growth.     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

Mitogen-induced disorganization of capillary-like structures formed by human large vessel endothelial cells in vitro

Endothelial cells (EC) from human aorta, umbilical vein and pulmonary artery were grown in Medium 199 supplemented with 20% human serum (HS), endothelial cell growth factor (ECGF) from bovine and human brain (200 micrograms/ml) and heparin (100 micrograms/ml) in gelatin-coated flasks. Under these conditions cells rapidly proliferated and survived 15-25 passages (40-60 cumulative population doublings). When cells were cultured on plastic substrate and without growth factors a capillary-like network appeared after 3-4 weeks of growth. According to TEM, this network consisted of tubes with the lumen encircled by one or several cells. The reduction of serum concentration in the medium or the replacement of plasma-derived serum (PDS) for HS reduced the time of network formation to 3-5 days. S-180 conditioned medium mitogenic for EC induced a rapid spreading of the cells and a partial reversion to a two-dimensional monolayer structure. Trypsin inhibitor did not abolish the effect of tumour conditioned medium. Other EC mitogens, e.g. ECGF and fibroblast growth factor (FGF), also disorganized the capillary-like network. In a day or two the network was completely restored. In contrast, culturing EC on gelatin-coated substrate is a sufficient condition for monolayer formation from tubes and long-term maintenance. We suggest that mitogens can influence the EC morphology but that it is the nature of the substrate that determines the stage of large vessel EC differentiation.     

Human endothelial cells are chemotactic to endothelial cell growth factor and heparin

The response of human endothelial cell migration to various extracellular matrix components and growth factors has been assessed. Human endothelial cells demonstrate increased chemotaxis and chemokinesis when placed in a modified Boyden chamber with endothelial cell growth factor (ECGF) used at a concentration of 10(-9) M. Anti-ECGF antibody inhibits the chemotactic response. Heparin (10(-8) to 10(-10) M) was also chemotactic and was shown to potentiate the chemotactic activity of ECGF. Although laminin, fibronectin, the polypeptide (epidermal, fibroblast, and nerve) growth factors, and collagen types I, II, III, IV, and V demonstrate a chemotactic response, these activities were one third to one half less than observed with ECGF. These data suggest that ECGF and heparin may play a significant role as response modifiers of human endothelial cell migration which may be relevant to tumor metastasis, wound healing, and atherogenesis.     

Heparin and endothelial cell growth factor modulate collagen and proteoglycan production in human smooth muscle cells

The effects of heparin and endothelial cell growth factor (ECGF) on extracellular matrix production were examined in human iliac smooth muscle cells. The cells were grown in (a) medium supplemented with heparin (100 micrograms/ml) and ECGF (75 micrograms/ml), (b) medium supplemented with ECGF (75 micrograms/ml) alone, or (c) unsupplemented medium. In the presence of heparin and ECGF, collagen production was inhibited 91-95% as compared to cultures incubated with ECGF alone or without both supplemental factors. In contrast, the production of proteoglycans was elevated 2.5 fold in the presence of heparin and ECGF. Enzymatic digestion of the proteoglycans indicated that both large and small molecular weight chondroitin sulfate proteoglycans were markedly elevated, while dermatan sulfate and heparan sulfate proteoglycans were increased to a lesser extent. The results suggest that the combination of heparin and ECGF elicits potent modulation of extracellular matrix production, with divergent effects on collagen and proteoglycan synthesis.     

Regulation of in vitro capillary tube formation by anti-integrin antibodies

Human endothelial cells are induced to form an anastomosing network of capillary tubes on a gel of collagen I in the presence of PMA. We show here that the addition of mAbs, AK7, or RMAC11 directed to the alpha chain of the major collagen receptor on endothelial cells, the integrin alpha 2 beta 1, enhance the number, length, and width of capillary tubes formed by endothelial cells derived from umbilical vein or neonatal foreskins. The anti-alpha 2 beta 1 antibodies maintained the endothelial cells in a rounded morphology and inhibited both their attachment to and proliferation on collagen but not on fibronectin, laminin, or gelatin matrices. Furthermore, RMAC11 promoted tube formation in collagen gels of increased density which in the absence of RMAC11 did not allow tube formation. Neither RMAC11 or AK7 enhanced capillary formation in the absence of PMA. Lumen structure and size were also altered by antibody RMAC11. In the absence of antibody the majority of lumina were formed intracellularly from single cells, but in the presence of RMAC11, multiple cells were involved and the lumen size was correspondingly increased. Endothelial cells were also induced to undergo capillary formation in fibrin gels after PMA stimulation. The addition of anti-alpha v beta 3 antibodies promoted tube formation in fibrin gels and inhibited EC adhesion to and proliferation on a fibrinogen matrix. The enhancement of capillary formation by the anti- integrin antibodies was matrix specific; that is, anti-alpha v beta 3 antibodies only enhanced tube formation on fibrin gels and not on collagen gels while anti-alpha v beta 1 antibodies only enhanced tubes on collagen and not on fibrin gels. Thus we postulate that changes in the adhesive nature of endothelial cells for their extracellular matrix can profoundly effect their function. Anti-integrin antibodies which inhibit cell-matrix interactions convert endothelial cells from a proliferative phenotype towards differentiation which results in enhanced capillary tube formation.     

人脐静脉内皮细胞的原代培养和鉴定

目的：探索建立稳定有效的脐静脉内皮细胞体外培养方法。方法：用0．1％II型胶原酶消化分离人脐静脉肉皮细胞,加入含内皮细胞生长因子的M199完全培养液中培养,用胰蛋白酶-EDTA进行消化传代培养,用光镜和免疫组化方法对培养的细胞进行形态观察和鉴定。结果：原代培养的内皮细胞在接种后24h后完全贴壁生长,第445天后融合呈铺路石样镶嵌排列,免疫组化可见胞浆中第Ⅷ因子相关抗原呈阳性反应,证实培养的细胞为内皮细胞。结论：脐静脉灌注II型胶原酶消化法配合M199完全培养液可获得高纯度的内皮细胞,细胞可传代5—6次,但细胞产出量不高,不能传10代以上,5代以后细胞形态变化较大,对于复杂的基础研究应用受限。     

Autocrine Hyaluronan Influences Sprouting and Lumen Formation During HUVEC Tubulogenesis In Vitro

Although many studies have focused on a role for hyaluronan (HA) of interstitial extracellular matrix (presumably produced by non-vascular “stromal” cells) in regulating vascular growth, we herein examine the influence of “autocrine HA” produced by vascular endothelial cells themselves on tubulogenesis, using human umbilical vein endothelial cells (HUVECs) in angiogenic and vasculogenic three-dimensional collagen gel cultures. Relative to unstimulated controls, tubulogenic HUVECs upregulated HAS2 mRNA and increased the synthesis of cell-associated HA (but not HA secreted into media). Confocal microscopy/immunofluorescence on cultures fixed with neutral-buffered 10% formalin (NBF) revealed cytoplasmic HAS2 in HUVEC cords and tubes. Cultures fixed with NBF (with cetylpyridinium chloride added to retain HA), stained for HA using “affinity fluorescence” (biotinylated HA-binding protein with streptavidin-fluor), and viewed by confocal microscopy showed HA throughout tube lumens, but little/no HA on the abluminal sides of the tubes or in the surrounding collagen gel. Lumen formation in angiogenic and vasculogenic cultures was strongly suppressed by metabolic inhibitors of HA synthesis (mannose and 4-methylumbelliferone). Hyaluronidase strongly inhibited lumen formation in angiogenic cultures, but not in vasculogenic cultures (where developing lumens are not open to culture medium). Collectively, our results point to a role for autocrine, luminal HA in microvascular sprouting and lumen development. (J Histochem Cytochem 69: 415–428, 2021)     

Rac regulates endothelial morphogenesis and capillary assembly

Endothelial cells undergo branching morphogenesis to form capillary tubes. We have utilized an in vitro Matrigel overlay assay to analyze the role of the cytoskeleton and Rho GTPases during this process. The addition of matrix first induces changes in cell morphology characterized by the formation of dynamic cellular protrusions and the assembly of discrete aggregates or cords of aligned cells resembling primitive capillary-like structures, but without a recognizable lumen. This is followed by cell migration leading to the formation of a complex interconnecting network of capillary tubes with readily identifiable lumens. Inhibition of actin polymerization or actin-myosin contraction inhibits cell migration but has no effect on the initial changes in endothelial cell morphology. However, inhibition of microtubule dynamics prevents both the initial cell shape changes as well as cell migration. We find that the small GTPase Rac is essential for the matrix-induced changes in endothelial cell morphology, whereas p21-activated kinase, an effector of Rac, is required for cell motility. We conclude that Rac integrates signaling through both the actin and microtubule cytoskeletons to promote capillary tube assembly.     

Morphological change in the early stages of the mating process of Rhodosporidium toruloides.

The events which occur in the early stages of the mating process of the yeast Rhodosporidium toruloides between strains M-919 (mating type A) and M-1057 (mating type a) were investigated. In preliminary experiments we determined the frequency of mating by two newly designed methods: the liquid culture method and the membrane-filter microculture method. The mating frequencies of strains M-919 and M-1057 were 89% in the liquid culture method and 62% in the membrane-filter microculture method. The early stages in the mating process included the following events: (i) M-919 cells produce constitutively the extracellular inducing substance (A factor), (ii) M-1057 cells receive A factor, and in response to it they form mating tubes and secrete another inducing substance (a factor), (iii) M-919 cells receive a factor, and in response to it they form mating tubes, (iv) mating tubes elongate to the cells or the tubes of mating partner, (v) tips of the growing tubes recognize the opposite mating type cells or their tubes, followed by cell-to-cell fusion.     

Increased thyrotropin binding in hyperfunctioning thyroid nodules

The object of this study was to investigate TSH receptors in hyperfunctioning thyroid nodules (HFN). In HFN, obtained from seven patients, 125-I-TSH binding as determined by equilibrium binding analysis on particulate membrane preparations, was found to be significantly increased as compared with normal thyroid tissues (five patients; P less than 0.001). Scatchard analysis of TSH-binding revealed two kinds of binding sites for both normal thyroid tissue and HFN, and displayed significantly increased association constants of high- and low-affinity binding sites in HFN (Ka = 11.75 +/- 6.8 10(9) M-1, P less than 0.001 and Ka = 2.1 +/- 1.0 10(7) M-1, P less than 0.025; x +/- SEM) as compared with normal thyroid tissue (Ka = 0.25 +/- 0.06 10(9) M-1, Ka = 0.14 +/- 0.03 10(7) M-1; x +/- SEM). The capacity of the high-affinity binding sites in HFN was found to be decreased (1.8 +/- 1.1 pmol/mg protein, x +/- SEM) in comparison with normal thyroid tissue (4.26 +/- 1.27 pmol/mg protein; x +/- SEM). TSH-receptor autoradiography applied to cryostatic tissue sections confirmed increased TSH binding of the follicular epithelium in HFN. These data suggest that an increased affinity of TSH-receptor sites in HFN in iodine deficient areas may be an important event in thyroid autonomy.     

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.     

Early post-larval development of the endoparasitic platyhelminth Mesocestoides corti: trypsin provokes reversible tegumental damage leading to serum-induced cell proliferation and growth

Mesocestoides corti is a suitable in vitro model for studying the development of human endoparasitic platyhelminthes. Treatment with trypsin, supplemented with fetal bovine serum (FBS), induces M. corti development from larvae (tetrathyridia) to segmented adult worm; however, the role of this protease and of FBS in post-larval development induction remains unknown. To characterize the participation of trypsin enzymatic activity and of FBS in the induction of tetrathyridia growth and development, both stimuli were added to the larvae either together or sequentially. Additionally, specific inhibition of trypsin activity was also monitored. Finally, the effect of the enzyme on the parasite tegument as well as the proliferative activity and location of proliferating cells after induction of tetrathyridia development were also studied. We conclude that trypsin-induced tetrathyridia development to adult worm is FBS-dependent and that the effect of serum factors is dependent upon a previous trypsin-induced reversible damage to the larva tegument. In dividing and non-dividing tetrathyridia, proliferative activity of cells is mainly located within the apical massif in the anterior region and nerve cords of larvae, respectively. In tetrathyridia stimulated to develop to adult worms, an intense proliferative activity is evident along the nerve cords. Our results suggest that in natural infections the tetrathyridia tegument is temporally made permeable to growth factors by proteolytic enzyme activity in the intestine juice of the definitive host, thus leading to development to adult worms.     

Endothelial cell adhesion, signaling, and morphogenesis in fibroblast-derived matrix

Extracellular matrix plays a critical role in cellular development by providing signaling cues that direct morphogenesis. In order to study both the cues that natural matrix provides and endothelial cell responses to that information, human fetal lung fibroblasts were used to produce a fibrous three-dimensional matrix. Following the removal of the fibroblasts by detergent extraction, protein and proteoglycan constituents of the remaining matrix were identified by immunofluorescence and immunoblotting. Matrix components included fibronectin, tenascin-C, collagen I, collagen IV, collagen VI, versican, and decorin. Colocalization analysis suggested that fibronectin was a uniquely distributed matrix protein. Morphology, three-dimensional matrix adhesions, and integrin-mediated signaling during vasculogenesis were then studied in human endothelial cells seeded onto the fibroblast-derived matrix. Elongated morphology and decreased cell area were noted, as compared with cells on fibronectin-coated coverslips. Cell-matrix adhesions contained vinculin, pY397-FAK, and pY410-p130Cas, and all of these colocalized more with fibronectin than tenascin-C, collagen I, or collagen VI. Additionally, the endothelial cells remodeled the fibroblast-derived matrix and formed networks of tubes with demonstrable lumens. Matrix adhesions in these tubes also predominantly colocalized with fibronectin. The pattern of membrane type 1 matrix metalloprotease expression in the endothelial cells suggested its involvement in the matrix remodeling that occurred during tubulogenesis. These results indicated that information in fibroblast-derived matrix promoted vasculogenic behavior.     

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 microvascular endothelial cell-extracellular matrix interaction in cellular growth state determination

We introduce two methods, both of which are based on cellular-extracellular matrix interaction, which will facilitate the study of human microvascular endothelial cells. One method describes the means to obtain a G1 population baseline in human microvasclular endothelial cells. Because of the contribution of the extracellular matrix in endothelial cell growth, synchronization in G1 was possible only after the incorporation of angiostatic levels of heparin and hydrocortisone into the extracellular matrix. In the second method, we demonstrate that selective perturbation of human microvascular endothelial cell-extracellular matrix interactions results in the induction of a transitional growth state, between proliferative and differentiated growth states, in human microvascular endothelial cells. In the functional, microtubule formation assays, transitional growth state endothelial cells display rates that are indermediate between those obtained from differentiated and proliferative endothelial cells. Our results demonstrate the importance of the human microvascular endothelial cell-extracellular matrix interaction in the determination of cellular growth state. Our findings also imply that responsiveness of microvascular endothelial cells to their cellular-extracellular matrix environs is highest during the differentiated growth state.     

Antiproteinases protect cultured lung endothelial cells from endotoxin injury

To determine whether the effects of endotoxin on cultured lung endothelium involve proteolytic mechanisms, we incubated bovine pulmonary arterial endothelial cells with endotoxin in medium 199 + 10% fetal bovine serum (FBS) in the presence and absence of several proteinase inhibitors. Three chloromethyl ketone (CK) derivatives [N-tosyl-L-lysine (CK)-(TLCK), N-tosyl-L-phenylalanine CK(TPCK), methoxysuccinyl-Ala-Ala-Pro-Val CK(SPCK)] and a single synthetic proteinase substrate [N-alpha-p-tosyl-L-arginine methyl ester hydrochloride (TAME)] attenuated endotoxin-induced cytotoxicity (lactate dehydrogenase release) and prostacyclin production in a dose-related fashion. The most effective inhibitors of endotoxin-induced cytotoxicity were TLCK and TPCK. TLCK and TAME most effectively attenuated endotoxin-stimulated prostacyclin production. Two chemically unrelated substances, soybean trypsin inhibitor and alpha 1 proteinase inhibitor also attenuated the endotoxin response. In the absence of FBS or in the presence of 10% heat-inactivated FBS, antiproteases attenuated endotoxin-induced prostacyclin production but had less effect on cytotoxicity than with 10% FBS. We also measured the capacity of the CK inhibitors to scavenge superoxide radicals generated in a cell-free xanthine/xanthine oxidase system by measuring inhibition of cytochrome c reduction. Percent scavenging of superoxide by these inhibitors was as follows: TLCK, 62.7 +/- 5.8 (SE); TPCK, 83.9 +/- 7.7; TAME, 24.5 +/- 6.4; SPCK, 0. We conclude that certain proteinase inhibitors attenuate endotoxin-induced endothelial cytotoxicity and prostacyclin production and that direct scavenging of superoxide radicals fails to explain the protective effects of proteinase inhibition. We speculate that the effects of endotoxin on lung endothelium may involve proteolytic mechanisms even in the absence of neutrophils.     

A unique family of endothelial cell polypeptide mitogens: the antigenic and receptor cross-reactivity of bovine endothelial cell growth factor, brain-derived acidic fibroblast growth factor, and eye-derived growth factor-II

Bovine brain, hypothalamus, pituitary, and retina contain potent anionic polypeptide mitogens for endothelial cells. Immunological assays using murine monoclonal antibodies against bovine endothelial cell growth factor (ECGF) and radioreceptor assays using [125I]ECGF were performed to determine the cross-reactivity of ECGF with bovine acidic pI brain-derived fibroblast growth factor (acidic FGF) and bovine eye-derived growth factor-II [EDGF-II). We observed that acidic FGF and EDGF-II are recognized by anti-ECGF monoclonal antibodies and compete with [125I] ECGF for receptor occupancy. Furthermore, the biological activity of ECGF, acidic FGF, and EDGF-II is potentiated by the glycosaminoglycan, heparin. These results argue that ECGF, acidic FGF, and EDGF-II belong to a common family of polypeptide growth factors.     

Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures

We have defined a signal responsible for the morphological differentiation of human umbilical vein and human dermal microvascular endothelial cells in vitro. We find that human umbilical vein endothelial cells deprived of growth factors undergo morphological differentiation with tube formation after 6-12 wk, and that human dermal microvascular endothelial cells differentiate after 1 wk of growth factor deprivation. Here, we report that morphological differentiation of both types of endothelial cells is markedly accelerated by culture on a reconstituted gel composed of basement membrane proteins. Under these conditions, tube formation begins in 1-2 h and is complete by 24 h. The tubes are maintained for greater than 2 wk. Little or no proliferation occurs under these conditions, although the cells, when trypsinized and replated on fibronectin-coated tissue culture dishes, resume division. Ultrastructurally, the tubes possess a lumen surrounded by endothelial cells attached to one another by junctional complexes. The cells possess Weibel-Palade bodies and factor VIII-related antigens, and take up acetylated low density lipoproteins. Tubule formation does not occur on tissue culture plastic coated with laminin or collagen IV, either alone or in combination, or on an agarose or a collagen I gel. However, endothelial cells cultured on a collagen I gel supplemented with laminin form tubules, while supplementation with collagen IV induces a lesser degree of tubule formation. Preincubation of endothelial cells with antibodies to laminin prevented tubule formation while antibodies to collagen IV were less inhibitory. Preincubation of endothelial cells with synthetic peptides derived from the laminin B1 chain that bind to the laminin cell surface receptor or incorporation of these peptides into the gel matrix blocked tubule formation, whereas control peptides did not. These observations indicate that endothelial cells can rapidly differentiate on a basement membrane-like matrix and that laminin is the principal factor in inducing this change.     

Inhibitory effect of human recombinant interleukin-1 alpha and beta on growth of human vascular endothelial cells

Endothelial cell growth factor (ECGF) is a potent polypeptide mitogen which stimulates the growth of endothelial cells. The mitogenic effect of ECGF was inhibited by addition of recombinant interleukin-1 (rIL-1) alpha or beta in a concentration dependent manner. The morphological change was not observed distinctly. In the condition without ECGF, both types of rIL-1 enhanced [3H]-thymidine uptake slightly, but failed to increase cell numbers. These data suggest the possibility that the effect of rIL-1 on EC is modulated by the presence of ECGF.