The production and localization of laminin in cultured vascular and corneal endothelial cells

The production and localization of laminin, as a function of cell density (sparse versus confluent cultures) and growth stage (actively growing versus resting cultures), has been compared on the cell surfaces of cultured vascular and corneal endothelial cells. Comparison of the abilities of the two types of cells to secrete laminin and fibronectin into their incubation medium reveals that vascular endothelial cells can secrete 20-fold as much laminin as can corneal endothelial cells. In contrast, both cell types produce comparable amounts of fibronectin. Furthermore, if one compares the secretion of laminin and fibronectin as a function of cell growth, it appears that the laminin released into the medium by either vascular or corneal endothelial cells, is a function of cell density and cell growth, since this release is most pronounced when the cells are sparse and actively growing, and decreases by 10- and 30-fold, respectively, when either vascular or corneal endothelial cell cultures become confluent. With regard to fibronectin secretion, no such variation can be seen with vascular endothelial cell cultures, regardless of whether they are sparse and actively growing or confluent and resting. Corneal endothelial cell cultures, demonstrated a twofold increase in fibronectin production when they were confluent and resting as compared to when they were sparse and actively growing. When the distribution of laminin versus fibronectin within the apical and basal cell surfaces of cultured corneal and vascular endothelial cells is compared, one can observe that unlike fibronectin, which in sparse and subconfluent cultures can be seen to be associated with both the apical cell surface. In confluent cultures, laminin can be found associated primarily with the extracellular matrix beneath the cell monolayer, where it codistributes with type IV collagen.     

Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin

Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of being passage repeatedly while maintaining a high mitotic index. The stock cultures used for these studies have been passed weekly with a split ratio of 1 to 10 and are currently in their 30th passage. These cultures are indistinguishable from earlier passages when examined for the presence of Weibel-Palade bodies or Factor VIII antigen. We conclude that the use of FGF and thrombin can prevent the precocious senescence observed in most human endothelial cells cultures previously described.     

Human capillary endothelial cells from abdominal wall adipose tissue: Isolation using an anti-pecam antibody

Summary We have developed a novel isolation technique for harvesting human capillary endothelial cells. We compared the use of eitherUlex Europaeus Agglutinin (UEA) lectin or anti-platelet endothelial cell adhesion molecule (PECAM) antibody conjugated to magnetic beads for the ability to isolate and maintain pure cultures of human capillary endothelial cells. Cells isolated using either method actively scavenged DiI-acetylated-low density lipoprotein and expressed von Willebrand factor (vWf) up to four passages as assessed by immunofluorescent labeling. Endothelial cells isolated using the anti-PECAM antibody method maintained these endothelial-specific properties for up to 12 passages while the percentage of UEA selected cells expressing these properties decreased during increasing passage number. Furthermore, while both techniques yielded cells that bind UEA at Passage six, only the antibody selected cells expressed the normal pattern of endothelial-specific cellular adhesion molecules as assessed by flow cytometry. Both cell isolates were cultured within a three-dimensional matrix of type I collagen, the antibody selected cells formed tubelike structures within 2 days, while the lectin selected cells did not. The antibody selected capillary endothelial cells were transduced with a retroviral vector containing the human growth hormone cDNA and were found to secrete growth hormone from both two- and three-dimensional cultures. We propose that anti-PECAM antibodies linked to a solid support provide a highly selective step in the isolation and maintenance of pure populations of human capillary endothelial cells from abdominal wall liposuction remnants.     

COMPARISON OF THE GROWTH AND DIFFERENTIATION OF EPITHELIAL CELLS FROM NORMAL AND HYPERPLASTIC LENSES OF THE CHICK: STUDIES OF IN VITRO CELL CULTURES

Epithelial cells from hyperplastic lenses of a strain of chicks (Hy-1) selected for high growth rate were dissociated and cultured in vitro and compared with lens epithelial cells from a normal strain (N) in similar conditions. The hyperplastic lens cells showed remarkable motility and adhesiveness after dissociation and formed cell aggregates of various sizes before attaching to the substrate, giving a rather low plating efficiency. The lens structures (lentoid bodies) developed in partially confluent cultures of Hy-1 cells at least three days earlier than those in the cultures from normal control cells, in which the lens structures developed only after the cultures reached confluence. The results of culture at low cell density showed that the Hy-1 cell population consisted of at least two cell types different from each other in growth capacity. These striking differences in in vitro behaviour of dissociated cells from normal and hyperplastic lens epithelia and the results of clonal culture are discussed in relation to the possible mechanisms of abnormal morphogenesis and growth which are likely to be involved in the development of the hyperplastic lens in situ .     

A gradient of responsiveness to the growth-promoting activity of ZPA (zone of polarizing activity) in the chick limb bud

Limb bud mesoderm of stage 22-23 embryos was dissected into four pieces along the anteroposterior axis and dissociated cells of each region were separately cultured under various conditions. When the cells were cultured in medium containing 0.1% fetal calf serum (serum-poor medium) only a slight increase in cell number occurred in the cultures of all four regions. However, when the cells were cultured in medium containing 10% FCS, only cells of two central regions proliferated rapidly, and no growth promotion was observed in cells in the most anterior and posterior regions. Using the serum-poor medium, we examined the growth-promoting effects of cocultured limb bud fragments and of some growth factors on the cells of four regions. Anterior, distal, and proximal fragments promoted cell proliferation and their promotive effect on the cells of each region was equal. On the other hand, posterior fragments (containing ZPA) showed stronger promotive effects on preaxial cells than on postaxial cells. For comparison with the growth-promotive effect of the posterior fragment, fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin, and retinoic acid were tested in cell culture. FGF showed position-dependent growth promotion, while EGF and insulin promoted growth in the cells of all four regions to a similar degree. Retinoic acid showed no effect on cell growth at low concentrations, and was rather toxic at high concentrations. These results suggest that the cells of the posterior region secrete an FGF-like growth factor(s), which controls normal limb development and experimental duplicate formation.     

The relationship of the saturation density of multilayer cell cultures to their mass exchange with the medium

Chinese hamster fibroblasts (CHF) and NIH 3T3 cells were cultured on a glass substrate at different distances from the porous membrane separating the cells from the perfusing medium. It is shown that with perfusion of medium above the membrane there is no movement of the medium near the cells. In both the types of culture, the cells grow in multilayers, however the multilayer character of growth in CHF is more pronounced than in NIH 3T3 cells. The saturation density of the cultures depends on the cell-membrane separation, and at separations of no more than 0.2 mm exceeds the saturation density in the monolayer by 8-10 fold. The dependences of the saturation density on separation are different for CHE and NIH 3T3 cells, indicating qualitative differences in the inhibition of cell growth in monolayers between these cultures. The results obtained indicate that the inhibition of cell growth in monolayer is due to mass exchange limitations, rather than to intercellular contact interactions.     

Growth requirements of low-density rabbit costal chondrocyte cultures maintained in serum-free medium

The factors required for the active proliferation of low-density rabbit costal chondrocytes exposed to 9:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium have been defined. Low-density primary cultures of rabbit costal chondrocytes proliferated actively when the medium was supplemented with high-density lipoprotein (300 micrograms/ml), transferrin (60 micrograms/ml), fibroblast growth factor (FGF) (1 ng/ml), hydrocortisone (10(-6) M), and epidermal growth factor (EGF) (30 ng/ml). Insulin, although it slightly decreased the final cell density, was required for reexpression of the cartilage phenotype at confluence. Optimal proliferation of low-density chondrocyte cultures was only observed when dishes were coated with an extracellular matrix (ECM) produced by cultured corneal endothelial cells, but not on plastic. Furthermore, serum-free chondrocyte cultures seeded at low density and maintained on ECM-coated dishes gave rise to a homogeneous cartilage-like tissue composed of spherical cells. These chondrocytes therefore seem to provide a good experimental system for analyzing factors involved in supporting proliferation of chondrocytes and their phenotypic expression.     

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.     

Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum-free medium

Experimental conditions have been defined that allow bovine corneal endothelial (BCE) cells to grow in the complete absence of serum. Low density BCE cell cultures maintained on extracellular matrix (ECM)-coated dishes and plated in the total absence of serum proliferate actively when exposed to a synthetic medium supplemented with high density lipoprotein (HDL 500 μg protein/ml), transferrin (10 μg/ml), insulin (5 μg/ml), and fibroblast (FGP) or epidermal growth factor (EGF) added at concentrations of 100 or 50 ng/ml, respectively. Omission of any of these components results in a lower growth rate and/or final cell density of the cultures. BCE cell cultures plated on plastic dishes and exposed to the same synthetic medium grow very poorly. The longevity of BCE cultures maintained on plastic versus ECM and exposed to serum-free versus serum-containing medium has been studied. The use of ECM-coated dishes extended the life span of BCE cultures maintained in serum-supplemented medium to over 120 generations, as compared to less than 20 generations for cultures maintained on plastic. Likewise, BCE cells maintained on ECM and exposed to a synthetic medium supplemented with optimal concentrations of HDL, transferrin, insulin, and FGF underwent 85 generations, whereas control cultures maintained on plastic could not be passaged. The enhancing effect of ECM on BCE cell growth and culture longevity clearly illustrates the importance of the cell substrate in the control of proliferation of these 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.     

Control of lens epithelial cell survival

We have studied the survival requirements of developing lens epithelial cells to test the hypothesis that most cells are programmed to kill themselves unless they are continuously signaled by other cells not to do so. The lens cells survived for weeks in both explant cultures and high-density dissociated cell cultures in the absence of other cells or added serum or protein, suggesting that they do not require signals from other cell types to survive. When cultured at low density, however, they died by apoptosis, suggesting that they depend on other lens epithelial cells for their survival. Lens epithelial cells cultured at high density in agarose gels also survived for weeks, even though they were not in direct contact with one another, suggesting that they can promote one another's survival in the absence of cell- cell contact. Conditioned medium from high density cultures promoted the survival of cells cultured at low density, suggesting that lens epithelial cells support one another's survival by secreting survival factors. We show for the first time that normal cell death occurs within the anterior epithelium in the mature lens, but this death is strictly confined to the region of the anterior suture.     

Glycated type 1 collagen induces endothelial dysfunction in culture

Hyperglycemia-induced protein glycation is thought to be implicated in the diabetic vasculopathy. In this study, we cultured vascular endothelial cells on native or glycated collagen matrix and compared their growth and functional characteristics. At lower plating density, the cells grew equally well on both substrata; however, at higher planting density, the cells plated on glycated collagen grew slower and reached a lower confluent density compared to that of the native collagen-based cultures. Confluent cell layers formed on glycated collagen exhibited a lower diffusion barrier function and a less response to epidermal growth factor stimulated prostacyclin production, compared to their native collagen-cultured counterparts.     

Channel open time of acetylcholine receptors on Xenopus muscle cells in dissociated cell culture

The kinetics of acetylcholine (ACh) receptor channels on cultured myotomal muscle cells from Xenopus embryos were studied by analyzing focally recorded membrane currents. The mean open time for receptor channels on embryonic muscle cells grown in dissociated cell cultures showed a time-dependent decrease similar to that seen in vivo. The changes in power density spectra are consistent with the hypothesis that the decrease results from the appearance of a class of ACh receptor with a short mean channel open time (0.7 msec) and a decrease in the proportion of receptors with a long mean channel open time (3 msec). The addition of dissociated neural tube cells to muscle cell cultures resulted in an unexpected increase in mean channel open time for ACh receptors in both synaptic and nonsynaptic regions. These studies demonstrate that ACh receptor function may be altered in cultured muscle cells.     

Cultured endothelial cells derived from the human iliac arteries

Cells derived from the endothelium of human iliac arteries were cultured in vivo. The cells were isolated, grown, and subcultured in HEPES buffered Medium 199 supplemented with 20% heat inactivated human whole blood serum, human alpha-thrombin, and commercial endothelial cell growth supplement derived from bovine brain. The cells were viable in culture for 8 to 10 passages at a split ratio of 1:3. After the 10th passage, the cells began to enlarge and their growth rate was reduced. No cultures were viable after the 12th passage. The cells were determined to be of endothelial origin by their morphology at confluence; their ultrastructural characteristics, including the presence of Weibel-Palade bodies; the production and release of factor VIII-related antigen; and by their maintenance of a surface that prevented platelet attachment. The cultured arterial endothelial cells released prostacyclin in response to challenge with thrombin and protamine sulfate but not in response to bradykinin or the platelet-derived growth factor. Although the cultures described in this report were derived from patients with varying degrees of atherosclerotic disease, there were no significant differences in morphological or physiological parameters among these cultures or in comparison with commonly studied cells derived from human umbilical veins.     

Cultured endothelial cells increase their capacity to synthesize prostacyclin following the formation of a contact inhibited cell monolayer

The synthesis of the prostaglandins (PG), prostacyclin (PGI2), PGE2, and thromboxane A2 (TXA2), has been investigated in actively growing and contact-inhibited bovine aortic endothelial cell cultures. Cells were stimulated to synthesize prostaglandins by exposure to exogenous arachidonic acid or to the endoperoxide PGH2 and by the liberation of endogenous arachidonic acid from cellular lipids with melittin or ionophore A23187. Increased capacity of the cells to synthesize PGI2 and PGE2 was observed as a function of time in culture, regardless of the type of stimulation. TXA2 production increased with time only upon stimulation of the cells with ionophore A23187. This increased PG synthetic capacity was independent of cell density since it was mainly observed in confluent, nondividing endothelial cell cultures. The fact that increased PGI2 production in confluent cells was also observed with PGH2, a direct stimulator of PGI2 synthetase, implies that this process is independent of the arachidonate concentration within the cells or in the culture medium. This increased capacity is likely to reflect an increased activity of the PG synthetase system associated with the formation of a contact inhibited endothelial cell monolayer. A similar time-dependent increase in the PGI2 production capacity was also observed during growth of cultured bovine corneal endothelial cells.     

Expression of simple epithelial cytokeratins in bovine pulmonary microvascular endothelial cells.

Polypeptides of bovine aortic, pulmonary artery, and pulmonary microvascular endothelial cells, as well as vascular smooth muscle cells and retinal pericytes were evaluated by two-dimensional gel electrophoresis. The principal cytoskeletal proteins in all of these cell types were actin, vimentin, tropomyosin, and tubulin. Cultured pulmonary microvascular endothelial cells also expressed 12 unique polypeptides including a 41 kd acidic type I and two isoforms of a 52 kd basic type II simple epithelial cytokeratin microvascular endothelial cell expression of the simple epithelial cytokeratins was maintained in cultured in the presence or absence of retinal-derived growth factor, and regardless of whether cells were cultured on gelatin, fibronectin, collagen I, collagen IV, laminin, basement membrane proteins, or plastic. Cytokeratin expression was maintained through at least 50 population doublings in culture. The expression of cytokeratins was found to be regulated by cell density. Pulmonary microvascular endothelial cells seeded at 2.5 X 10(5) cell/cm2 (confluent seeding) expressed 3.5 times more cytokeratins than cells seeded at 1.25 X 10(4) cells/cm2 (sparse seeding). Vimentin expression was not altered by cell density. By indirect immunofluorescence microscopy it was determined that the cytokeratins were distributed cytoplasmically at subconfluent cell densities but that cytokeratin 19 sometimes localized at regions of cell-cell contact after cells reached confluence. Vimentin had a cytoplasmic distribution regardless of cell density. These results suggest that pulmonary microvascular endothelial cell have a distinctive cytoskeleton that may provide them with functionally unique properties when compared with endothelial cells derived from the macrovasculature. In conjunction with conventional endothelial cell markers, the presence of simple epithelial cytokeratins may be an important biochemical criterion for identifying pulmonary microvascular endothelial cells.     

Changes in cation composition of aortic endothelial cells in culture

As a first step in the study of membrane transport characteristics of aortic endothelial cells the content of the two main cations, Na and K, was determined in cultured cells from bovine and porcine origins. The Na and K contents of cultured endothelial cells, dissociated by scraping or trypsin and collagenase treatment and subsequently separated through oil (25% dodecyl-, 75% dibutyl-phthalate), were more than 20-fold higher and five-fold lower, respectively, than those of undissociated cells. Based on daily determination of cell Na, K, and protein contents, the following findings were made. (1) Steady-state levels of Na and K were not reached in subconfluent, confluent or post-confluent monolayers. Instead, intracellular K content varied by up to two-fold, and intracellular Na by more than six-fold with marked 'peaks' after confluency. (2) Increasing the number of passages decreased cellular Na but not K content. (3) In cells cultured with 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) the protein content was decreased by five-fold. (4) The K/Na ratio was dependent on the number of passages and buffers used and varied daily. (5) Cell Na decreased and K increased exponentially with the seeding density. These data not only reveal significant changes of ion transport parameters during manipulations of endothelial cell cultures, but moreover suggest unsynchronized development of ion transport systems and/or their intermittent activation and deactivation as reflected in the variations observed in cellular cation composition.     

Regulation of angiotensin I-converting enzyme activity in serially cultivated bovine endothelial cells

Angiotensin I-converting enzyme (ACE) activity was measured in lysates of cloned and uncloned cultures of bovine fetal aortic endothelial cells. The expression of ACE activity in these cells was complex, and influenced by subcultivation, cell density, serum, cumulative population doublings, and clonal heterogeneity. The ACE specific activity at any point in the in vitro lifespan was determined, at least in part, by interaction of these culture variables. After subcultivation to subconfluent densities, cellular ACE specific activity decreased markedly and did not reach detectable levels until cells attained confluent densities. The use of different suppliers' lots of serum in the growth medium resulted in different cellular ACE specific activities. The ACE specific activity decreased as cultures were serially subcultivated, but remained detectable throughout the lifespan, suggesting a linkage between the proliferative history of an endothelial cell and its remaining capacity to express ACE. Increased ACE activity was observed when cells at the end of their lifespan were cultured at high densities. Cloned strains behaved similarly to the uncloned parent culture, except that they exhibited a wide range of ACE specific activities.     

Cultured endothelial cells derived from the human iliac arteries

Summary Cells derived from the endothelium of human iliac arteries were cultured in vivo. The cells were isolated, grown, and subcultured in HEPES buffered Medium 199 supplemented with 20% heat inactivated human whole blood serum, human alpha-thrombin, and commercial endothelial cell growth supplement derived from bovine brain. The cells were viable in culture for 8 to 10 passages at a split ratio of 1:3. After the 10th passage, the cells began to enlarge and their growth rate was reduced. No cultures were viable after the 12th passage. The cells were determined to be of endothelial origin by their morphology at confluence; their ultrastructural characteristics, including the presence of Weibel-Palade bodies; the production and release of factor VIII-related antigen; and by their maintenance of a surface that prevented platelet attachment. The cultured arterial endothelial cells released prostacyclin in response to challenge with thrombin and protamine sulfate but not in response to bradykinin or the platelet-derived growth factor. Although the cultures described in this report were derived from patients with varying degrees of atherosclerotic disease, there were no significant differences in morphological or physiological parameters among these cultures or in comparison with commonly studied cells derived from human umbilical veins. The above work was supported by Grant CA28540 from the National Institutes of Health and by a grant from The Council for Tobacco Research, USA.     

Human angiogenin is rapidly translocated to the nucleus of human umbilical vein endothelial cells and binds to DNA

Human angiogenin is translocated to the nucleus of human umbilical vein endothelial cells in a time-dependent manner. Exogenous angiogenin appears in the nucleus in 2 min, reaches saturation in 15 min when 85% of the internalized angiogenin is in the nuclei, and remains associated with the nucleus for at least 4 h. Endothelial cells cultured at low density have a much higher capacity to translocate angiogenin to the nucleus than do those cultured at high density. This observation is consistent with previous findings that both the ability of endothelial cells to proliferate in response to angiogenin and the expression of an angiogenin receptor on the cell surface depend on cell density. Nuclear (125)I-angiogenin is not degraded and is neither spontaneously dissociated nor replaced by unlabeled angiogenin. It is, however, released by deoxyribonuclease I, but not by ribonuclease A, suggesting that angiogenin binds to DNA in the nucleus. These results suggest that in addition to acting as a ribonuclease, angiogenin may play a role in regulating gene expression by direct binding to DNA.