Long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions

Summary The understanding of pathways associated with differentiated function in human epithelial cells has been enhanced by the development of methods for the short-term culture of human epithelial cells. In general these methods involve the use of serum. The subculture and maintenance of epithelial cells in long-term culture has been more problematic. A serum-free medium developed for human bronchial epithelial cells was slightly modified and found to be useful for the subculture and long-term maintenance of not only bronchial epithelial cells, but also tracheal, nasal polyp, and sweat gland epithelial cells from either normal or cystic fibrosis individuals. The cells maintained epithelial-specific characteristics after multiple subcultures. Monolayers of epithelial cells showed junctional complex formation, the presence of keratin, and micro villi. Functional studies with Ussing chambers showed short circuit current (I_sc) responses to isoproterenol, bradykinin, or calcium ionophore (A23187) in subcultured tracheal and bronchial cells. This work is supported by grants HL41928 and DK39619 (DCG), HL24136 (CBB), and HL42368 (JHW and DCG) from the National Institutes of Health, Bethesda, MD.     

In vitro differentiation of rabbit blastocyst cells

Summary Six- and seven-day post-coitus (p.c.) rabbit embryos have been cultured in an attempt to establish a trophectodermal cell line. Results indicate that cells with epithelial characteristics (i.e. positive staining for cytokeratin) will survive in culture until Passage 3. At that time a fibroblastlike cell becomes predominant. In addition, we have found that the presence of the inner cell mass is required for embryo explants often results in the development of cells that spontaneously contract. These cells stain positively for myosin, which indicates that they may be precardiac cells. Maximum diastolic potential was −59±1.2 mV and the threshold potential was −53±2.3 mV. Spontaneously contracting cells did not respond to atropine, acetylcholine, epinephrine, isoproterenol, or propranolol. Action potential seems to be a result of an inward calcium current, because the beating rate is decreased in a dose-related manner with the calcium channel blocker verapamil, whereas the voltage-sensitive sodium channel blocker tetrodotoxin was without effect. This work was supported by grants HD21302, HD07069, DK31091, and HL37320 from the National Institutes of Health, Bethesda, MD, with additional support from a University of Alabama at Birmingham Cardviovascular Research and Training Center Award.     

Opposite effects of monokines (interleukin-1 and tumor necrosis factor) on proliferation and heparin-binding (fibroblast) growth factor binding to human aortic endothelial and smooth muscle cells

Summary Heparin-binding (fibroblast) growth factors (HBGF) are mitogens for both human aortic endothelial and smooth muscle cells. Under similar conditions, both vascular cells display similar numbers of specific HBGF binding sites with similar apparent affinity for HBGF. The monokines, interleukin-1 and tumor necrosis factor, inhibit endothelial cell growth and stimulate smooth muscle cell growth. The opposite mitogenic effects correlate with reduction and increase in HBGF receptor number displayed by endothelial and smooth muscle cells, respectively. These results suggest that the two monokines may depress endothelial cell regeneration and augment smooth muscle cell hyperplasia by differential modulation of the HBGF receptor in the two vascular cell types. This work was supported by US Public Health Service grants DK35310 and HL33487. H. S. is a visiting scientist from Takeda Chemical Industries, Ltd., Central Research Division, Juso-Honmachi-2, Yodogawa-ku, Osaka 532, Japan.     

Cytochemical localization of acid phosphatase in striated muscle

Summary Normal skeletal and cardiac striated muscle from adult rats was incubated for the cytochemical detection of acid phosphatase activity with cerium as the capture metal. Results from these experiments show that normal striated muscle has a greater number of acid phosphatase-positive structures, which are presumed to be lysosomes, than has been indicated by several previous cytochemical studies.Supported in part by grants AI 17945 and HL 17747 from the United States Public Health Service, National Institutes of Health     

Mechanical effects on endothelial cell morphology: In vitro assessment

Summary Endothelial cells are subjected to fluid mechanical forces which accompany blood flow. These cells become elongated and orient their long axes parallel to the direction of shear stress when the cultured cells are subjected to flow in an in vitro circulatory system. When the substrate is compliant and cyclically deformed, to simulate effects of pressure in the vasculature, the cells elongate an orient perpendicular to the axis of deformation. Cell shape changes are reflected in the alignment of microtubule networks. The systems described provide tools for assessing the individual roles of shear stress, pressure, and mechanical strain on vascular cell structure and function. This work was partially supported by grants HL 17437, HL 18072, and HL 23016 from the National Institutes of Health, Bethesda, MD, and grant C-938 from the Robert A. Welch Foundation.     

Toxicity to organ cultured heats of media prepared with disposable filter units

Summary Organ cultures of hearts maintained in media sterilized by filtration through different types of commercially available disposable membrane filter units displayed different beating rates and survival patterns. Media filtered through two different filter units sold by one company were severely toxic. Prewashing the filters removed most if not all of the toxins. This work was supported by grants from the National Heart, Lung and Blood Institute (HL14706) and the Moss Heart Fund.     

Cinematographic analysis of vascular smooth muscle cell interactions with extracellular matrix

Summary The interactions of vascular smooth muscle cells with growth modulators and extracellular matrix molecules may play a role in the proliferation and migration of these cells after vascular injury and during the development of atherosclerosis. Time-lapse cinematographic techniques have been used to study cell division and migration of bovine carotid artery smooth muscle cells in response to matrix molecules consisting of solubilized basement membrane (Matrigel) and type I collagen. When cells were grown adjacent to Matrigel, both migration and cell proliferation were increased and interdivision time was shortened. Cells grown in Matrigel or in type I collagen had markedly reduced migration rates but interdivision time was not altered. Further, diffusible components of the Matrigel were found to stimulate proliferation of the smooth muscle cells. This work was supported by grants HL35684 and SCOR HL14212 from the National Institutes of Health, Bethesda, MD.     

Effects of cyclic mechanical stimulation of the cellular components of the heart: In vitro

Summary The response of the cellular components of the heart to cyclic mechanical stimulation is of particular importance because these cells are continually subjected to mechanical forces as a result of changes in blood volume and pressure. To directly investigate how mechanical tension affects these cellular components of the heart, an in vitro system that exposes the particular cell type (cardiac myocytes, endothelial cells, or fibroblasts) to a calibrated increase in cyclical linear stretch was developed. Cells were grown on silastic membranes coated with laminin and subjected to a 10% cyclical distention 10 times a minute for 72 h. Within 24 h of being exposed to the mechanical stretch, the cells became elongated and oriented perpendicular to the direction of the stretch. These results indicate that cyclical mechanical stimulation directly influences the cellular organization of the heart cells in vitro. This work was supported in part by grants HL 33656 and HL 24935 from the National Institutes of Health, Bethesda, MD.     

Susceptibility of endothelial cells derived from different blood vessels to common viruses

Summary We examined whether endothelial cells derived from different blood vessels vary in their susceptibility to viral infection. Five common viral pathogens of humans (herpes simplex 1, measles, mumps, echo 9, and coxsackie B4 viruses) were evaluated for growth in endothelial cells derived from bovine fetal pulmonary artery thoracic aorta, and vena cava. All five viruses replicated in each type of endothelial cell. There were apparent differences in the quantities of measles and mumps viruses produced in pulmonary artery endothelium compared with thoracic aorta and vena cava when endothelial cells were obtained from different animals. However when pulmonary artery endothelial cells were compared with vena cava cells from the same animal, growth of each virus was similar in the two cell types. Four of the viruses replicated in the various endothelial cells without producing appreciable changes in cell morphology. These results indicate that endothelial cells from different blood vessels are equally susceptible to the human viruses evaluated, and that viral replication can occur without major alteration in cell morphology. Endothelial cells could serve as permissive cells permitting viruses to leave the circulation and initiate infection in adjacent tissues, including subendothelial smooth muscle cells. This work was supported by Public Health Service grants HL28220, HL 29492, and HL 24914 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

Organization and signaling of endothelial cell-to-cell junctions in various regions of the blood and lymphatic vascular trees

Adhesive intercellular junctions between endothelial cells are formed by tight junctions and adherens junctions. In addition to promoting cell-to-cell adhesion, these structures regulate paracellular permeability, contact inhibition of endothelial cell growth, cell survival, and maintenance of cell polarity. Furthermore, adherens junctions are required for the correct organization of new vessels during embryo development or during tissue proliferation in the adult. Extensive research on cultured epithelial and endothelial cells has resulted in the identification of many molecular components of tight junctions and adherens junctions. Such studies have revealed the complexity of these structures, which are formed by membrane-associated adhesion proteins and a network of several intracellular signaling partners. This review focuses on the structural organization of junctional structures and their functional interactions in the endothelium of blood vessels and lymphatics. We emphasize the way that these structures regulate endothelial cell homeostasis by transferring specific intracellular signals and by modulating activation and signaling of growth factor receptors. This work was supported by the Associazione Italiana per la Ricerca sul Cancro, Association for International Cancer Research, European Community (Integrated Project Contract no. LSHG-CT-2004–503573; NoE MAIN 502935; NoE EVGN 503254; EUSTROKE consortium; Angioscaff consortium; Optistem consortium), Istituto Superiore di Sanità, Italian Ministry of Health, MIUR (COFIN prot: 2006058482_002), and Fondation Leducq Transatlantic Network of Excellence (E.D.). Additional support came from US National Institutes of Health grants HL24136 and HL59157 from the National Heart, Lung, and Blood Institute and CA82923 from the National Cancer Institute and AngelWorks Foundation (D.McD.).     

Serum glucocorticoids have persistent and controlling effects on insulinlike growth factor i action under serum-free assay conditions in cultured human fibroblasts

Summary The biological actions of insulinlike growth factor I (IGF-I) measured under serum-free assay conditions were found to be significantly influenced by prior subculture conditions for adult human fibroblasts. Glucocorticoids seemed to be the major medium variable affecting IGF-I action. IGF-I added to serum-free cultures had little or no effect on [¹⁴C]aminoisobutyric acid uptake or [³H]thymidine incorporation in human fibroblasts previously maintained in media containing serum with low glucocorticoid levels or in serum stripped of endogenous steroids. However, a 48-h preincubation with dexamethasone resulted in a marked synergistic increase in IGF-I stimulation of [¹⁴C]aminoisobutyric acid uptake and [³Hthymidine incorporation in these cultures. In contrast, IGF-I in serum-free medium seemed to be a potent mitogenic and metabolic stimulus for human fibroblasts which had been subcultured in media with a high glucocorticoid content, either endogenous, or supplemented. After these culture conditions, a 48-h preexposure to dexamethasone had no further enhancing effect on IGF-I action. Dexamethasone also potentiated IGF-I, insulin, and epidermal growth factor stimulation of fibroblast replication depending on the earlier subcultivation conditions. Thus, glucocorticoids are important modulators of IGF-I bioactivity in cultured human fibroblasts. Serum glucocorticoids can exert a profound influence on the biological phenomena measured in cell culture, even when the serum has been removed before the actual experiment, and must be carefully taken into account for accurate evaluation of the biological function of IGF-I and other growth factors. This work was supported in part by grants DK28229, DK36054, CA42106, RCDA01275, and DK24085 from the National Institutes of Health, Bethesda, MD.     

Copper-transporting ATPases ATP7A and ATP7B: cousins, not twins

Copper plays an essential role in human physiology and is indispensable for normal growth and development. Enzymes that are involved in connective tissue formation, neurotransmitter biosynthesis, iron transport, and others essential physiological processes require copper as a cofactor to mediate their reactions. The biosynthetic incorporation of copper into these enzymes takes places within the secretory pathway and is critically dependent on the activity of copper-transporting ATPases ATP7A or ATP7B. In addition, ATP7A and ATP7B regulate intracellular copper concentration by removing excess copper from the cell. These two transporters belong to the family of P₁-type ATPases, share significant sequence similarity, utilize the same general mechanism for their function, and show partial colocalization in some cells. However, the distinct biochemical characteristics and dissimilar trafficking properties of ATP7A and ATP7B in cells, in which they are co-expressed, indicate that specific functions of these two copper-transporting ATPases are not identical. Immuno-detection studies in cells and tissues have begun to suggest specific roles for ATP7A and ATP7B. These experiments also revealed technical challenges associated with quantitative detection of copper-transporting ATPases in tissues, as illustrated here by comparing the results of ATP7A and ATP7B immunodetection in mouse cerebellum. This work was supported by the National Institute of Health grants PO1 GM 067166–01 and DK R01 DK071865 to S.L.     

Mitogens and hepatocyte growth control in vivo and in vitro

This work was supported by grants from the NIH [DK28215 (H. L. L.), GM41804 (D. A. B.), AI22166 (G. H. F.)], NSF [DCB86-16740 (H. L. L.)], ACS [IN93R (K. S. K.)], Veterans Administration (D. A. B.), and the Children's Liver Foundation (G. H. F.).     

The prepupal salivary glands of Drosophila melanogaster

Evidence is presented in support of the concept that the larval salivary gland of Drosophila melanogaster continues to function as an important secretory organ throughout prepupal stages and after pupation. Just after puparium formation, and at other later periods, the glands appear to be in the process of disintegration, but each time they recover until after pupation. Nuclear blebbing occurs through the time of survival of the glands, but is shown not to involve transport of RNA out of the nucleus. Transport in and out of the nucleus is clearly rapid and in a steady state as compared to the massive and intermittent export of cytoplasmic substance into the lumen of the gland.This work was supported by grants from the National Science Foundation (GB-23343, PCH-02044).     

Culture of human neoplastic gastrointestinal smooth muscle cells

Summary Due to limited growth potential of primary cultures and the absence of continuous lines of healthy enteric smooth muscle, we have studied the culture behavior of neoplastic gastrointestinal smooth muscle cells. Forty-six human enteric smooth muscle neoplasms (leiomyomas and leiomyosarcomas) were studied while fresh and/or after culture in vitro and growth in vivo in athymic nude mice, with assessments made of morphology, growth characteristics, and biochemical markers of differentiation. The state of differentiation of the tumors varied, with well-differentiated tumors tending to express binding sites for the gastrointestinal hormone cholecystokinin, whereas less well-differentiated tumors did not. Poorly differentiated tumors were the easiest to establish in culture in vitro and to grow in vivo in nude mice. When the cells placed directly into culture proliferated to confluent density, they underwent morphologic differentiation from a spread, fibroblastlike shape to a slender spindle morphology, with these cells possessing fewer biosynthetic organelles and arranging themselves in characteristic “hill and valley” arrays. However, the highly differentiated characteristics of expression of desmin or cholecystokinin-binding sites were not observed in cultured cells. In contrast, cells that had been passaged in nude mice before culture displayed a proliferative phenotype and failed to undergo morphologic differentiation on reaching confluent density. Four human enteric smooth muscle cell lines (documented by chromosomal analysis) originating in stomach, jejunum, ileum, and rectum were established using this strategy. This work was supported by grants DK32878 and DK34988 from the National Institutes of Health, Bethesda, MD.     

Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium

Summary The analysis of lipoprotein synthesis and secretion in primary hepatocytes has been restricted by the short-term viability and low proliferative response of hepatocytes in vitro. During this investigation a serum-free medium formulation was developed that supports long-term maintenance (>70 d) and active proliferation of primary baboon hepatocytes. Examination of proliferating cells by electron microscopy revealed a distinctive hepatocyte ultrastructure including intercellular bile canaliculi and numerous surface microvilli. High levels of secreted apolipoproteins A-I and E were detected in the tissue culture medium by gel electrophoresis and immunoblot analysis. Immunoprecipitation of proteins from [³⁵S]-methionine labeled tissue culture medium revealed the synthesis and secretion of numerous plasma proteins. Metabolic labeling of cells with [³⁵S]-methionine followed by single-spin density gradient flotation of the media demonstrated that apolipoproteins were being secreted in the form of lipoprotein particles with buoyant densities corresponding to the very low density lipoprotein and low density lipoprotein range, and to the high density lipoprotein range. The labeled apolipoproteins included B _h , E, and A-I. This system for primary hepatocyte culture should prove very useful in future investigations on the regulation of lipoprotein production by hepatocytes. This investigation was supported in part by a research grant from the Southwest Foundation Forum, by program project HL 28972 from the National Heart, Lung and Blood Institute, Bethesda, MD, and by grants to R. V. H. from the National Institutes of Health (HL 15062), the American Heart Association, and the Louis Block Fund.     

GTP-binding proteins regulate high conductance anion channels in rat bile duct epithelial cells

Summary Epithelial cells from the intrahepatic bile duct contribute to bile formation, but little is known of the cellular mechanisms responsible. In these studies, we have characterized the endogenous GTP-binding proteins (G proteins) present in these cells and evaluated their role in regulation of high conductance anion channels. G proteins were identified in purified plasma membranes of isolated bile duct epithelial cells using specific antisera on Western blots, and ion channel activity was measured in excised inside-out membrane patches using patch-clamp recording techniques. In patches without spontaneous channel activity, addition of cholera toxin to the cytoplasmic surface had no effect (n=10). Addition of pertussis toxin caused an activation of channels in 13/34 (38%) attempts, as detected by an increase in channel open probability. Activated channels were anion selective (gluconate/Cl^– permeability ratio of 0.17±0.04) and had a unitary conductance of 380 pS. Channel open probability was also increased by the nonhydrolyzable GDP analogue guanosine 5-0-(2-thiodiphosphate) in 8/14 (57%) attempts. In contrast, channel open probability was rapidly and reversibly decreased by the nonhydrolyzable analogue of GTP 5guanylylimidodiphosphate in 7/9 (78%) attempts. Western blotting with specific antisera revealed that both G_i –2 and G_i –3 were present in significant amounts, whereas G_i –1 and G_o were not detected. These studies indicate that in bile duct epithelial cells, high conductance anion channels are inhibited, in a membrane-delimited manner, by PTX-sensitive G proteins.We gratefully acknowledge the assistance of Marwan Farouk, M.D. in the preparation of bile duct epithelial cells, Lucy Seger in the identification of the G proteins, C.F. Starmer in channel analysis, and P.J. Casey for the gift of bacteria expressing the different G-protein -subunits. This work was supported in part by grants from the National Institutes of Health DK43278 (to J.G.F.), DK42486 (to T.W.G.), and DK07568 (to J.M.M.); American Gastroenterological Association/G.D. Searle Research Scholar Award (to J.G.F.) and an American Gastroenterological Association Advanced Research Training Award (J.M.M.).     

Characterization of vascular smooth muscle cell phenotype in long-term culture

Summary Studies of bovine carotid artery smooth muscle cells, during long-term in vitro subcultivation (up to 100 population doublings), have revealed phenotypic heterogeneity among cells, as characterized by differences in proliferative behavoir, cell morphology, and contractile-cytoskeletal protein profiles. In vivo, smooth muscle cells were spindle-shaped and expressed desmin and alpha-smooth muscle actin (50% of total actin) as their predominant cytoskeletal and contractile proteins. Within 24 h of culture, vimentin rather than desmin was the predominant intermediate filament protein, with little change in alpha-actin content. Upon initial subcultivation, all cells were flattened and fibroblastic in appearance with a concommitant fivefold reduction in alpha-actin content, whereas the beta and gamma nonmuscle actins predominated. In three out of four cell lines studied, fluctuations in proliferative activity were observed during the life span of the culture. These spontaneous fluctuations in proliferation were accompanied by coordinated changes in morphology and contractile-cytoskeletal protein profiles. During periods of enhanced proliferation a significant proportion of cells reverted to their original spindle-shaped morphology with a simultaneous increase in alpha-actin content (20 to 30% of total actin). These results suggest that in long-term culture smooth muscle cells undergo spontaneous modulations in cell phenotype and may serve as a useful model for studying the regulation of intracellular protein expression. This work was supported by grants from from National Institutes of Health, Bethesda, MD, to DMW (HL35684), JW (HL36412), and JM and RL (SCOR HL 14212).     

Analysis for possible linkage between the loci for the Waardenburg syndrome and various blood groups and serological traits

Summary Linkage was sought between the Waardenburg syndrome locus and the loci for various genetic markers segregating in a single family. Close linkage was shown to be unlikely with the loci for Rh, MN, Ag, ADA, HL-A, and Gm. Evidence obtained is consistent with the possibility of linkage with the locus for the AB0 blood group, but study of additional families will be required to provide a definite answer.

---

Zusammenfassung In einer Familie wurde nach Genkopplung zwischen dem locus für das Waardenburg-Syndrom und verschiedenen genetischen Markern gefahndet. Für die loci für Rh, MN, Ag, ADA, HL-A und Gm wurde enge Kopplung als unwahrscheinlich erwiesen. Dagegen lassen die Daten die Annahme einer Kopplung mit dem AB0-locus zu. Für eine endgültige Entscheidung müßten zusätzliche Familien untersucht werden.

---

---

Research supported by grants No. HD 04134, HL 09011, and HL 08630 from the National Institutes of Health.     

Isolation and monolayer culture of guinea pig pancreatic duct epithelial cells

Summary Monolayers of cultured epithelial cells have been prepared from fragments of guinea pig pancreatic excretory ducts isolated by a simple procedure employing collagenase digestion and manual selection, through which virtually all of the ductal system can be recovered. The isolated fragments were cultured in enriched Waymouth's medium on extracellular matrices of various composition and thickness, including: thin (<5 μm) and thick (0.5 mm) layers of rat tail collagen; thin layers of human placental collagen; thin layers of Matrigel (a reconstituted basement membrane material); uncoated tissue culture plastic; and the cellulose ester membranes of Millipore Millicells. Cells spread rapidly from duct fragments cultured on uncoated plastic or on plastic coated with thin layers of rat tail collagen or human placental collagen and formed epithelial monolayers. However, these cells were squamous and lacked the abundant basolateral membrane amplification and apical microvilli characteristic of freshly isolated duct epithelial cells. Cells did not spread from duct fragments cultured on Matrigel. In contrast, when fragments of pancreatic ducts were explanted onto either a thick layer of rat tail collagen or onto Millicell membranes, cells readily spread and formed confluent monolayers of cuboidal epithelial cells characterized by abundant mitochondria, apical microvilli, and basolateral plasma membrane elaboration. These results demonstrate that different forms of extracellular matrix modulate the growth and differentiation of pancreatic duct epithelial cells, and that culture on a permeable substrate markedly enhances the maintenance of differentiated characteristics in this cell type. The monolayers formed on Millicell membranes should provide a useful model system for physiologic analysis of the regulation of electrolyte secretion by this epithelium. This research was supported by grants DK32994 and DK35912 from the National Institutes of Health, Bethesda, MD.