Proteoglycans synthesized by cultured fibroblasts derived from normal and inflamed human gingiva

Summary The in vitro proliferations rates and ptoteoglycans synthesized by adult human gingival fibroblasts derived from six age- and sex-matched donors of healthy and chronically inflamed gingiva were analyzed. Fibroblasts from inflamed gingiva demonstrated a slower growth rate than cells from healthy tissue. The rate of incorporation of [³⁵S]sulfate into cell layer-associated proteoglycans and the release of these macromolecules into the culture medium did not differ appreciably between the two groups of cells. Similarly, no detectable differences in the overall charge of the proteoglycans synthesized by normal and inflamed gingival fibroblasts, as assessed by their elution from DEAE-Sephacel, were noted. However, sepharose CL-4B chromatography revealed that the medium-associated proteoglycans made by the inflamed tissue fibroblasts were depleted in one species of chondroitin sulfate proteoglycans and contained more dermatan sulfate than did control cells. In addition, the intracellular proteoglycan pool was found to be greatly diminished in the inflamed tissue fibroblast cell layers. Glycosaminoglycan analysis of the proteoglycans confirmed these observations. Compared to normal gingival fibroblasts, the inflamed tissue fibroblasts released less heparan sulfate into the medium. Additionally, increased levels of dermatan sulfate and depleted amounts of chondroitin sulfate in the medium of inflamed gingival cells were noted. The observed changes were stable through several transfers in culture and indicate that chronically inflamed tissue may contain fibroblasts mainfesting a heritable phenotype differing from fibroblasts in normal connective tissue. P. Mark Bartold was supported by a C. J. Martin Fellowship for the National Health and Medical Research Council of Australia. This work was also supported by grants DE-03301 and DE-02600 from the National Institutes of Health, Bethesda, MD.     

Distinctive fibroblastic subpopulations in skin and oral mucosa demonstrated by differences in glycosaminoglycan content

Summary The glycosaminoglycan (GAG) content of rabbit skin, oral mucosa, and cultured [³H]-glucosamine-labeled dermal and submucosal fibroblasts was compared. Skin contained predominantly dermatan sulfate (DS) and a small amount of hyaluronic acid (HA), whereas mucosa contained primarily keratan sulfate (KS) and smaller quantities of HA and DS. Culture medium from dermal and submucosal fibroblasts contained GAGs co-electrophoresing with DS, HA, and chondroitin sulfate (CS), although the relative proportions of these GAG differed. CS isolated from dermal and mucosal fibroblast culture medium co-electrophoresed with chondroitin 4-sulfate (C4-S) on cellulose acetate, whereas dermal medium CS was resistant to digestion by chondroitinase ABC, and mucosal medium CS was chondroitinase ABC-susceptible. The pericellular matrix of dermal fibroblasts contained primarily DS and C4-S/C6-S, as confirmed by chondroitinase ABC digestion; the corresponding fraction of mucosal fibroblasts contained HS and a GAG co-electrophoresing with a C6-S standard, yet resistant to digestion by chondroitinase ABC. Thus the GAG content of dermal and mucosal fibroblasts differed both qualitatively in terms of the type of GAG secreted into the culture medium and pericellular matrix, and quantitatively, in terms of the relative proportions of these GAGs in both fractions. These differences support the concept of distinctive fibroblastic subpopulations in skin and mucosal tissue, inasmuch as the cells were subjected to identical culturing conditions. This work was supported by research grant 15878 (C.N.B.) from the Shriners Hospitals for Crippled Children and DE 07803 (C.N.B.) from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD.     

Membrane function in cystic fibrosis. I. Putrescine transport in normal and cystic fibrosis fibroblasts

Putrescine transport was examined in normal and cystic fibrosis fibroblasts. No differences were observed in accumulation pattern, kinetics of uptake, or efflux between CF and normal cells. In both growing and growth-arrested CF and normal fibroblasts, exogenously supplied putrescine remained unchanged for at least 60 min. Some differences were observed in the response of CF and normal cells to environmental (media) changes.This research was supported by a grant from the Cystic Fibrosis Foundation and by a grant from the National Institutes of Health, Training Grant (GM01316 11 GNC).     

Cultivation of microvascular endothelial cells from human preputial skin

Summary A procedure is described for the isolation and cultivation of microvascular endothelium from human skin. Neonatal foreskins are pooled, washed, minced, and dissociated by a mixture of collagenase and dispase. Microvascular endothelium, liberated in the form of intact capillary fragments, is incompletely separated from fibroblasts and epidermal cells by sieving through nylon mesh, followed by velocity sedimentation on 5% bovine serum albumin. The endothelium-enriched fraction has been maintained in primary culture for up to 3 weeks. The resulting epithelioid colonies have been characterized morphologically by both light and transmission electron microscopy and manifest all of the structural features that distinguish other, large-vessel endothelia in culture. In addition, immunohistochemical studies using an indirect fluorescent antibody technique demonstrate that these cells contain the endothelium-specific product, Factor VIII antigen. This work was supported by National Institutes of Health Grants AM18904 and AM20571, the RGK Foundation, the Charlotte and Sidney Lifschultz Foundation, the Juvenile Diabetes Foundation, and the South Carolina Geenral Medical Faculty Research Appropriation.     

Inborn errors of cobalamin metabolism: Effect of cobalamin supplementation in culture on methylmalonyl CoA mutase activity in normal and mutant human fibroblasts

We have examined the effect of addition of hydroxocobalamin to growth medium on the activity of the adenosylcobalamin-requiring enzyme methylmalonyl CoA mutase in normal human fibroblasts and in mutant human fibroblasts derived from patients with inherited methylmalonicacidemia. The mutant cell lines were assigned to four distinct genetic complementation groups (cbl A, cbl B, cbl C, and cbl D), each deficient in some step in the synthesis of adenosylcobalamin from hydroxocobalamin. After control cells were grown in cobalamin-supplemented medium, mutase holoenzyme activity increased markedly in a time- and concentration-dependent fashion. Growth in cobalamin-supplemented medium had no effect on mutase activity in some mutant lines belonging to the cbl B group, while activity increased severalfold in other cbl B mutants and in all cbl A, cbl C, and cbl D mutants examined, although mutase activity was still <10% of control. Comparison of mutase holoenzyme activity and total propionate pathway activity suggests that enhancement of mutase activity in mutant cells after cobalamin supplementation to values 5–10% of control may be sufficient to overcome the inherited metabolic block and to restore total pathway activity to normal.This work was supported in part by a research grant from the National Institutes of Health (AM 12579). H. F. W. is a recipient of a traineeship from the National Institutes of Health (T01-GM02299).     

A simple fractionation of chicken egg yolk yields a protein component that stimulates cell proliferation and differentiation in primary avian tendon cells

Summary An easily prepared and stable protein fraction from chick egg yolk promotes cell division (40 h generation time) and expression of procollagen (60% of total protein synthesis) in primary avian tendon cells in a serum-free medium. The activity of this yolk fraction (YF) is proteinaceous as reflected by its sensitivity to protease treatment. Yolk fraction is resolved into four major components on sodium dodecyl sulfate polyacrylamide gel electrophoresis with apparent molecular weights of 82,70,42,35 (× 10⁻³). Under nondenaturing conditions, YF runs as a mixture of high molecular weight aggregates on Sephacryl G-200. We postulate that the active part of YF could be the in ovo growth promoter for embryonic chick tendon cells. This work was supported in part by National Institutes of Health grant CA 37958 and in part by the Office of Health and Environmental Research, Office of Energy Research, U.S. Department of Energy, under Contract DE-AC03-76SF00098.     

A rapid method for culturing guinea pig gastric mucous cell monolayers

Summary A method has been developed for growing confluent primary cultured monolayers of guinea pig gastric mucous cells suitable for in vitro electrophysiological, transport, and pharmacological studies. Isolated mucous cells were enriched on a one-step Percoll density gradient and plated on fibronectin-coated plastic dishes or in small cups with holes containing glutaraldehyde-fixed Vitrogen gels. These cups were designed to fit in Ussing chambers. Mucous cells attached, proliferated, and formed confluent monolayers in 3 d. The low cuboidal cells contained periodic acid Schiff-positive mucous granules that were negative by Bowie and indirect immunofluorescent staining for pepsinogen. Electron microscopy revealed polarized mucous cells with microvilli, mucous granules, microfilaments, small mitochondria, some vacuoles, and junctional complexes that excluded wheat germ agglutinin-peroxidase. No basal lamina was present. Monolayers could be maintained for over 2 wk but subcultures were not made. The cultures were virtually free of fibroblasts. Epithelial sheets produced by this simple and rapid method can be used for electrophysiological, ion transport, and pharmacological studies. This research was supported in part by National Institutes of Health grants GM7806, AM31158, AM 15681, and AM 30303.     

Glucagon and glicentin immunoreactive cells in human colon

Summary An immunohistochemical study of glucagon and glicentin immunoreactive endocrine cells in the human colon epithelium was performed. Serial sections and qualitative analysis show a cell population containing both immunoreactivities. However, there is another cell population exhibiting only an immunoreactivity with glicentin. The exact distribution of these immunoreactive endocrine cells within the colon crypt segments is also analysed. The significance of these findings concerning the synthesis of glucagon and glicentin and their function is discussed.Supported by a grant of the German Research Foundation and by a research grant AM 17537 from the National Institutes of Health, Bethesda, Maryland (USA) Dr. Colony was the recipient of a stipend for a research project in the Federal Republic of Germany from the Deutscher Akademischer Austauschdienst (DAAD)     

p-Nitrophenyl-β-d-xyloside modulates proteoglycan synthesis and secretory differentiation in mouse mammary epithelial cell cultures

Summary Primary cultures of mouse mammary epithelial cells synthesize significant quantities of chondroitin and heparan sulfate proteoglycans (16). Long term treatment of such cultures with p-nitrophenyl-β-D-xylopyranoside leads to a 10–20 fold increase in the synthesis and secretion of free chondroitin sulfate glycosaminoglycan (GAG) chains and assembly of a cell-associated matrix that is relatively enriched in heparan sulfate proteoglycan. This modulation of cell-synthesized proteoglycans leads to significant changes in cell morphology and cellular differentiation. Notably cells cultured on plastic culture dishes change from being flattened to cuboidal. The synthesis of the milk proteins α1, α2, and β-casein is also increases as is the formation of fat droplets and fat droplet membrane components. Promotion of differentiation increases with increasing xyloside concentration in the range 0–1.5 mM, but there may be a block in secretion at higher xyloside concentrations. While the detailed mechanisms remain to be elucidated, we conclude that the composition of proteoglycans incorporated into the matrix (and possibly the glycosaminoglycans secreted into the medium), may play a significant role in maintaining the phenotypic characteristics of terminally differentiated mammary epithelial cells. This research was supported by the Office of Health and Environmental Research, Office of Energy Research, U.S. Dept. of Energy under contract No. DEAC-03-76SF00098 and by National Institutes of Health Grant CA44398-01 (G. Parry) Editor's Statement Exogenous elements of extracellular matrix affect expression of cultured mammary cell function. This work reports manipulation of cell-derived endogenous matrix elements and shows correlative changes in cell functions.     

Carnitine transport in cultured muscle cells and skin fibroblasts from patients with primary systemic carnitine deficiency

Summary l-Carnitine transport was studied in cultured muscle cells and skin fibroblasts of patients with primary systemic carnitine deficiency and control subjects. In both cell culture types, two systems for carnitine transport were identified. The kinetic parameters for carnitine transport were remarkably similar in cultured muscle cells and skin fibroblasts. Normal rates and kinetic properties of carnitine transport were observed for both cell lines from patients with systemic carnitine deficiency. These studies do not rule out a defect in carnitine transport in vivo. This study was supported by research grants AM27451 and NS06277 from the National Institutes of Health and by a Research Center Grant from the Muscular Dystrophy Association.     

A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures

Summary Normal colonic epithelial cell cultures of mammalian origin are required to facilitate the study of both normal cellular functions as well as pathogenesis of certain (human) colonic diseases. To date, little information is available regarding the growth requirements of colonic epithelial cells in culture of eitehr animal or human origin. Such data would enable the development of a long-term culture system for these cells. In this study, we present methodology that results in the establishment of homogeneous cultures of adult rabbit colonic epithelial reproducibly, quickly, and in quantity. The epithelial nature of the cultures is unambiguously established by intermediate filament typing using antikeratin antibodies. Such culutres can now be used for a variety of functional studies as well as to investigate the growth requirements of colonic epithelial in culture. This work was supported by the Blinder Foundation for Crohn’s Disease Research, Harbor UCLA IBD Center (AM 36200) and grant AM 27806 from the National Institutes of Health, Bethesda, MD.     

Clonal growth and culture life span of bovine adrenocortical cells in a serum-free medium

Summary The life span and growth from clonal density of bovine adrenocortical cell cultures were studied in serum-supplemented medium and a serum-free defined medium, which supported sustained cell proliferation and steroid production. The total culture life span was 79 population doublings in serum-supplemented medium with fibroblast growth factor (FGF) and 36 population doublings in the defined medium without serum. Older passage cell cultures grown in the defined medium progressively lost the ability to produce 11β- and 21-hydroxylated steroids, which was observed previously for cultures in serum-supplemented medium, and also had a decline of 17α-hydroxylated steroid production. The cloning efficiency in the defined medium was 12.2% as compared to 24% in serum-supplemented medium with FGF. Five isolated clonal cell lines grown in the defined medium were characterized for steroid function in response to steroidogenic agents. All five clonal cell lines had stimulated steroid production with 8-bromo-cAMP, but only four of the clonal lines were stimulated also by adrenocorticotropin. None of the clonal cell lines produced 11β-, 21- or 17α-hydroxylated steroids in response to treatment with either steroidogenic agent, results that were similar to data obtained from older mass cultures. The apparent deficiency of the defined medium as compared to serum-supplemented medium for maximum support of the culture life span and cloning efficiency may be useful in studies of cellular aging and its relation to differentiated function for this cell culture system. This study was supported by the Iowa Diabetes and Endocrinology Research Center (grant AM25295 from the National Institutes of Health, Bethesda, MD). D.A.F. was supported by a National Research Service Award from the National Institutes of Health (grant HL07485).     

The effect of donor age on the in vitro life span of cultured human arterial smooth-muscle cells

Summary The number of population doublings of cultured human arterial smooth-muscle cells decreased as a function of donor age (0.5 to 82 years). Cells from older donors als showed longer latent periods for outgrowth from explants. These results extend other comprable observations with human skin fibroblasts to another cell type, and may have relevance to the pathogenesis of atherosclerosis with aging in vivo. This study was supported by a reserach program project grant (AG00299) from the National Institutes of Health.     

In vitro modulation of antioxidant enzymes in normal and malignant renal epithelium

Summary The activities of three antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, were monitored in isolated human renal adenocarcinoma tissues and in cultured human renal adenocarcinoma cells. The results were compared to the activities of these enzymes in the proposed cell of origin, isolated human proximal tubular tissues, and cultured proximal tubular epithelial cells. Strong modulation of these enzymes by culture conditions was observed in normal cells but not in carcinoma cells. Low levels of cellular lipid peroxidation, as assessed by levels of malondialdehyde (MDA), were observed in adenocarcinoma cells under the culture conditions tested with one exception: greatly elevated MDA was observed in renal adenocarcinoma cells growth on plastic in serum-free, chemically defined medium. This increased lipid peroxidation correlated with a loss of cell viability under these conditions. This work was supported by a grant from the Veterans Adminsitration (T. D. O.) and by grant 1 R01 CA 41267 from the National Institutes of Health (L. W. O.), Bethesda, MD.     

Use of commercial serum replacements for the culture of insect cells

Summary Two commercially available serum replacements developed for use in the culture of hybridoma and other mammalian cells were tested for their suitability as replacements for fetal bovine serum in insect cell culture medium. CPSR-1 and CPSR-3 both supported growth of the insect cell line IPLB-SF-21AE. CPSR-3 supported adequate growth, but cells in medium supplemented with CPSR-1 grew much slower and achieved only about half the final cell density of either FBS or CPSR-3 supplemented medium. This work was supported in part by grant 187159 from the Juvenile Diabetes Foundation and BRSG RR05876 from the National Institutes of Health, Bethesda, MD.     

Urinary excretion of glycosaminoglycans and albumin in experimental diabetes mellitus

Diabetes mellitus was induced in one group of rats by a single injection of streptozotocin. The glycemia, the body weight, and the blood systolic pressure were measured every week, and the 24 h urine volume and urinary excretions of creatinine, albumin and glycosaminoglycans were measured every 2 weeks. At the end of the experiment (12 weeks) the weight and the glycosaminoglycan composition of the kidneys were determined. All the diabetic animals were hyperglycemic, hypertense, and did not gain weight during all the experimental period. Albuminuria appeared from the second week on. Rat urine was shown to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate, and the glycosaminoglycan excretion decreased in all diabetic animals. The onset of the change in glyco-samino-glycan excretion rate was a very early event, appearing in the second week after diabetes induction. The main glycosaminoglycan found in normal rat kidney was heparan sulfate and, in contrast to the urine, the total kidney glycosaminoglycans increased in diabetic kidney, due to chondroitin sulfate and dermatan sulfate accumulation. The heparan sulfate concentration (per tissue dry weight) did not change. Our results suggest that quantification of urinary glycosaminoglycans may be a useful tool for the early diagnosis of diabetic nephropathy.     

Isolation,characterization, and long-term culture of fetal bovine tracheal epithelial cells

Summary Epithelial cells were isolated from fetal bovine trachea by exposing and stripping the mucosal epithelium from the adjacent connective tissue. The tissue was minced and enzymically dissociated in Ca-Mg-free medium containing dispase and dithiothreitol. The stripping procedure and selective trypsinization produced epithelial cell cultures free of fibroblasts. Seeded on plastic, the plating efficiency was 21.5% with a doubling time of 24 h. Dome formation, evidence of occluding junctions and active ion transport characteristic of epithelial cells, was common. Growth of the cells on glass, collagen, and Engelbreth-Holm-Swarm (EHS) substrate demonstrated a striking difference in morphology. Cells grown on EHS presented a more distinctly three-dimensional growth pattern and many more microvilli when compared to cells grown on glass or collagen. The cells retained their epithelioid characteristics through more than 30 passages as shown by the presence of distinct apical and basolateral membranes, tight junctions, and positive keratin staining. This study was supported in part by grants BRSG S07 RR05408-25, Biomedical Research Support Grant Program, Division of Research Resources, by ES 00159, Center Grant, National Institutes of Environmental Health Sciences, by R23-HL37621, New Investigator Award, National Heart, Lung and Blood Institutes, National Institutes of Health, and by the Health Effects Institute, an organization jointly funded by the U.S. Environmental Protection Agency (Assistance Agreement X-8120059) and automotive manufactures. The contents do not necessarily reflect the views of policies of HEI, EPA, or automotive manufacturers.     

Turnover, change of composition with rate of cell growth and effect of phenylxyloside on synthesis and structure of cell surface sulfated glycosaminoglycans of normal and transformed cells

The synthesis of sulfated glycosaminoglycans was analysed in mouse fibroblasts during the transition from exponential growth to quiescent monolayers. 'Normal' Swiss 3T3 fibroblasts were compared with SV40 transformed 3T3, C6, ST1 and HeLa cells. p-Nitrophenyl-beta-D-xyloside, an artificial acceptor for glycosaminoglycans synthesis, was used as a probe. Exponentially growing 'normal' 3T3 cells synthesized both dermatan sulfate and chondroitin 4-sulfate, retaining the latter and releasing the former to the medium. Upon reaching quiescence these cells switched to retention of dermatan sulfate and release of chondroitin 4-sulfate. SV3T3 cells synthesized several fold less sulfated glycosaminoglycans than 'normal' 3T3. Even though SV3T3 cells are able to synthesize dermatan sulfate, they only retained chondroitin 4-sulfate, never switching to retention of dermatan sulfate. These results indicated that the transition from rapidly proliferating to resting G0 state in normal cells is accompanied by a switch from chondroitin-sulfate rich to dermatan-sulfate-rich cells. This switching was not observed with transformed cells, which are unable to enter the G0 state. Phenylxyloside caused a several fold increase in glycosaminoglycans released to the medium in both cell types, but it did not interfere with either growth rate or cell morphology. Particularly the phenylxyloside treatment led to an increase of more than 10-fold in production of dermatan and chondroitin sulfate by SV3T3, C6, ST1 and HeLa cells. This demonstrated that transformed cells have a high capacity for glycosaminoglycan synthesis. Analysis of enzymatic degradation products of glycosaminoglycans, synthesized in the presence of phenylxyloside, by normal and transformed cells, led to the finding of 4- and 6-sulfated iduronic and glucuronic acid-containing disaccharides. This result indicated that the xyloside causes the synthesis of a peculiar chondroitin sulfate/dermatan sulfate, in both normal and transformed cells.     

Membrane-associated chondroitin sulfate proteoglycans of human lung fibroblasts

Cultured human fetal lung fibroblasts produce some chondroitin sulfate proteoglycans that are extracted as an aggregate in chaotropic buffers containing 4 M guanidinium chloride. The aggregated proteoglycans are excluded from Sepharose CL4B and 2B, but become included, eluting with a Kav value of 0.53 from Sepharose CL4B, when Triton X-100 is included in the buffer. Conversely, some of the detergent-extractable chondroitin sulfate proteoglycans can be incorporated into liposomes, suggesting the existence of a hydrophobic membrane-intercalated chondroitin sulfate proteoglycan fraction. Purified preparations of hydrophobic chondroitin sulfate proteoglycans contain two major core protein forms of 90 and 52 kD. A monoclonal antibody (F58-7D8) obtained from the fusion of myeloma cells with spleen cells of BALB/c mice that were immunized with hydrophobic proteoglycans recognized the 90- but not the 52-kD core protein. The epitope that is recognized by the antibody is exposed at the surface of cultured human lung fibroblasts and at the surface of several stromal cells in vivo, but also at the surface of Kupffer cells and of epidermal cells. The core proteins of these small membrane-associated chondroitin sulfate proteoglycans are probably distinct from those previously identified in human fibroblasts by biochemical, immunological, and molecular biological approaches.     

Regulation of cell-substrate adhesion by proteoglycans immobilized on extracellular substrates

We have demonstrated previously that chick embryo fibroblasts synthesize and secrete a large chondroitin sulfate proteoglycan (designated PG-M) that binds to fibronectin. We now report the possibility that PG-M interactions with cell surfaces can modulate cell-substrate adhesion. When PG-M was added to the medium, various types of trypsinized cells failed to adhere not only to fibronectin-coated substrates but also to collagen- or vitronectin-coated substrates. Adhesion of the cells to laminin or glycyl-arginyl-glycyl-aspartyl-serine derivatized serum albumin (arginyl-glycyl-aspartic acid-containing molecules with no capacity to bind PG-M) was also inhibited by PG-M. Treatment of the proteoglycan with either proteolytic enzymes or chondroitinase abolished its inhibitory effects on the cell adhesion. These results suggest that direct binding between PG-M and fibronectin, if any, is not a cause of the inhibition by PG-M and that only the proteoglycan form is responsible for the activity. When the immobilization of added PG-M to available plastic surfaces of coated dishes was blocked by pretreating the dishes with serum albumin, the inhibitory effect of PG-M was abolished, suggesting that the immobilized fraction of PG-M can act as a cell adhesion inhibitor. In immobilized form, both cartilage chondroitin sulfate proteoglycan (designated PG-H) and chondroitin sulfate-derivatized serum albumin also inhibited cell adhesion. In contrast, heparan sulfate proteoglycan form LD and heparan sulfate-derivatized serum albumin had far lower inhibitory activities, indicating that the active site for the interaction between cells and PG-M is on the chondroitin sulfate chains.