Inhibiting effect of procollagen peptides on collagen biosynthesis in fibroblast cultures.

NH2-terminal extension peptides of type I and type III procollagens were isolated from dermatosparactic and normal fetal calfskin, respectively. Cell culture experiments showed that the globular domains of the tested procollagen peptides were biologically active but that peptides from the helical region of collagen had no effect. The peptides were added to the incubation medium of calf fibroblasts along with radioactive precursor amino acids, and the amount of newly synthesized collagen was determined. The experiments indicated that procollagen peptides exerted a feedback-like inhibitory effect specific for the synthesis of collagen. Neither degradation of collagen, hydroxylation of collagen alpha chains, nor synthesis of noncollagenous proteins were affected. Synthesis of type II collagen by calf chondrocytes was not reduced. In addition, it was shown that procollagen peptides from calf were equally effective when added to human fibroblast cultures, an observation that could be of considerable medical interest.     

Stimulation of collagen synthesis in fibroblast cultures by superoxide.

Exposure of diploid fetal human fibroblasts (IMR-90) to superoxide generated by dihydroxyfumarate resulted in increased collagen synthesis. The synthesis of type III collagen was stimulated to a greater extent than the synthesis of type I collagen. The stimulation of collagen synthesis was abolished by superoxide dismutase. Our observations suggest that superoxide may play a role in the regulation of collagen synthesis and may modulate differential collagen gene expression. These observations may explain the increased synthesis of collagen in tissues following inflammation or exposure to oxidant conditions.     

A quantitative assay for collagen synthesis in microwell fibroblast cultures.

A new method for estimating collagen synthesis in microwell cultures of fibroblasts is presented, ³H-Labeled-proline-collagen was purified by successive salt precipitations at acid and neutral pH in the presence of carrier collagen. Variability between replicates was less than 10% (standard deviation) and recovery of labeled collage internal standards was greater than 90%. More than 90% of recoverable radioactivity was in collagen as demonstrated by polyacrylamide gel electrophoresis and carboxymethyl cellulose chromatography. The culture system is highly reproducible and allows use of a large number of separate cultures with uniformity of culture conditions and economy of reagents.     

Influence of type I collagen surface density on fibroblast spreading, motility, and contractility

We examine the relationships of three variables (projected area, migration speed, and traction force) at various type I collagen surface densities in a population of fibroblasts. We observe that cell area is initially an increasing function of ligand density, but that above a certain transition level, increases in surface collagen cause cell area to decline. The threshold collagen density that separates these two qualitatively different regimes, approximately 160 molecules/ microm(2), is approximately equal to the cell surface density of integrin molecules. These results suggest a model in which collagen density induces a qualitative transition in the fundamental way that fibroblasts interact with the substrate. At low density, the availability of collagen binding sites is limiting and the cells simply try to flatten as much as possible by pulling on the few available sites as hard as they can. The force per bond under these conditions approaches 100 pN, approximately equal to the force required for rupture of integrin-peptide bonds. In contrast, at high collagen density adhesion, traction force and motility are limited by the availability of free integrins on the cell surface since so many of these receptors are bound to the surface ligand and the force per bond is very low.     

Tripeptide analysis of collagen synthesized by silica-exposed fibroblast cultures

The effect of silica on collagen biosynthesis by confluent monolayers of WI-38 fibroblast cultures was examined by a more comprehensive method of analysis. The presence of the particulates had no direct effect on protein (collagen) synthesis, proline incorporation or prolyl hydroxylase activity; the latter is determined by the degree of hydroxylation. Silica, however, was highly toxic to the cells.     

High-performance liquid chromatographic quantitation of collagen biosynthesis in explant cultures

The capacity of lung explant cultures to synthesize collagen can be estimated by determining the content of [³H]hydroxyproline in protein following incubation with [³H]proline. The technique requires acid hydrolysis followed by quantitative separation of hydroxyproline from proline for scintillation counting and is often restricted to methods that can accommodate large samples because of relatively low specific radioactivity. A method which is useful for such samples, providing rapid separation of nonderivatized amino acids by ion-exchange HPLC, is described here. The HPLC system employs an HPX-87C cation-exchange column in 10 mm calcium acetate, pH 5.5, at 85°C. Under isocratic conditions hydroxyproline is completely resolved from proline with quantitative recovery of the ³H cpm applied to the column. Large amounts of material, equivalent to at least 150 mg wet wt of lung, can be applied without affecting resolution or recovery, and samples can be injected at intervals as short as 40 min. This method was used to study collagen biosynthesis in a model of pulmonary fibrosis induced in rabbits by the tumor-promoting agent, phorbol myristate acetate (PMA), and provides information concerning total protein synthesis as well as production of collagen. The data show a doubling in the rate of collagen production in lung explants prepared from animals treated with PMA compared with explants from control animals.     

Effects of collagen density on cardiac fibroblast behavior and gene expression

Interactions between cells and the extracellular matrix (ECM) play essential roles in modulating cell behavior during development and disease. The myocardial ECM is composed predominantly of interstitial collagen type I and type III. The composition, organization, and accumulation of these collagens are altered concurrent with cardiovascular development and disease. Changes in these parameters are thought to play significant roles in myocardial function. While a number of studies have examined how changes in the ECM affect myocardial function as a whole, much less is known regarding the response at the cellular level to changes in the collagenous ECM. Experiments were carried out to determine the effects of alterations in collagen density and ECM stiffness on the behavior of isolated heart fibroblasts. In vitro bioassays were performed to measure the effects of changes in collagen concentration (0.75-1.25 mg/ml) on adhesion, migration, spreading, and gene expression by heart fibroblasts. Increased density of collagen in 3-dimensional gels resulted in more efficient adhesion, spreading, and migration by heart fibroblasts. These experiments indicated that the density of the collagen matrix has a significant impact on fibroblast function. These studies begin to elucidate the effects of ECM density at the cellular level in the myocardium.     

Histochemical demonstration of collagen fibers in ascorbic-acid-fed cell cultures.

Nine cultures of fibroblast cell types and 13 epithelial-like cell types were maintained for 1 week in media supplemented with L-asborbic acid (50 microgram per ml). All fibroblast-like cultures produced extracellular fibers that stained positively by a silver-impregnation reticulin stain. Nine of the 13 epithelial-like cultures produced fibers that stained positively for reticulin. Nearly all cultures not supplemented with ascorbic acid showed no fiber staining. Those few lines that stained positively for reticulin in the absence of ascorbic-acid supplementation demonstrated only slight reticulin formation. Reticulin from one fibroblast culture and one epithelial culture was examined by electron microscopy, and the silver-impregnated fibrils were morphologically identical to collagen. The reticulin was digestible with collagenase, providing further evidence that the silver-impregnation reticulin stain identifies collagen in culture. The demonstartion of collagen can be performed easily in histology laboratories using Formalin-fixed cells, and provides a means of assaying a functional property of cells in culture which is characteristic of connective tissue fibroblasts in general as well as certain specialized epithelia.     

Stimulation of collagenase production by collagen in mammalian cell cultures.

In this study, we investigated the possible regulatory role of collagen in collagenase production by cultured human skin fibroblasts and human and rabbit synovial cells. Addition of types I, II or III collagen in solution to the culture media markedly stimulated trypsin-activable collagenase activity in these cultures. In the human cell cultures the stimulatory effect of collagen was further enhanced by a soluble factor isolated from human monocyte culture media (Dayer, Russell and Krane, 1977). Both native and denatured forms of collagen stimulated enzyme production; their relative efficacy varied among the different types. The native form of both types I and II collagen showed a greater effect on collagenase production than the corresponding denatured form, whereas with type III collagen the denatured form was more effective.     

Influence of bcl-2 on antibody productivity in high cell density perfusion cultures of hybridoma

Apoptosis is an active, genetically determined death mechanism which can be induced by a wide range of physiological factors and by mild stress. It is the predominant form of cell death during the production of antibodies from murine hybridoma cell lines. A number of studies have now demonstrated that the suppression of this death pathway, by means of over-expression of survival genes such as bcl-2, results in improved cellular robustness and antibody productivity during batch culture. In the present study, the influence of bcl-2 expression on hybridoma productivity in two high density perfusion bioreactor systems was investigated. In the first system, a fixed-bed reactor, the DNA content in the spent medium was 25% higher in the control (TB/C3-pEF) culture than that found in the bcl-2 transfected (TB/C3-bcl2) cultures at all perfusion rates. This is indicative of a higher level of cell death in the control cell line. The average antibody concentration for the TB/C3-pEF cell line was 14.9 mg L-1 at perfusion rates of 2.6 and 5.2 d-1. However, for the TB/C3-bcl2 cell line it was 33 mg L-1 at dilution rates of 2 and 4 d-1. A substantial increase in antibody concentration was also found in the Integra Tecnomouse hollow fibre reactor. The antibody titre in the TB/C3-bcl2 cassette was nearly 100% higher than that in the TB/C3-pEF cassette during the cultivation period which lasted 6 weeks. Clearly, these results demonstrate the positive impact of bcl-2 over-expression on production of antibody in hybridoma perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of two cell harvesting methods on intracellular ATP and amino acid concentrations in human fibroblast cultures

The intracellular ATP and amino acid concentrations were determined in human fibroblast cultures reaching confluence. The values obtained were very different, depending on the cell harvesting method: trypsinization or scraping. Trypsinization appeared to be the better method for measuring the ATP concentrations (21.25 +/- 0.96 nmol per mg cell protein), this level being much lower with scraping. On the contrary, scraping was the most appropriate method for amino acid measurement. This work underlines the importance of harvesting methods for metabolic studies in human cell cultures.     

Ionizing radiation induces early, sustained increases in collagen biosynthesis: a 48-week study in mouse skin and skin fibroblast cultures

Groups of 10 CF1 female mice, irradiated to the thorax with a dual-head 137Cs gamma-RAY source, received single doses of 0, 5, 10, 15, or 25 Gy. One to forty-eight weeks later collagen synthesis was measured in minced skin specimens incubated in medium containing [3H]proline and then assayed for radioactive hydroxyproline. A progressive, generally dose-dependent increase in collagen biosynthesis, up to 50% above control sites, was found 1, 4, and 12 weeks after radiation exposure. These changes showed further small fluctuations at 12-36 weeks, increasing again at the 48-week interval. At the same times throughout the study fibroblasts were cultured from skin explants. Following the second subculture, these cells were also incubated in medium containing [3H]proline, and collagen synthesis was again determined by [3H]hydroxyproline assay. At all radiation dose levels studied, collagen production increased threefold by 12 weeks postradiation and remained elevated for the 48-week duration of the study. In vitro radiation dose response differences were not observed.     

Reversible release of chick embryo fibroblast cultures from density dependent inhibition of growth.

Initiation of proliferation in density-inhibited chick embryo fibroblast cultures induced by insulin or trypsin was partially reversed by replacing the medium with supernatants from parallel non-stimulated cultures. Growth stimulation by neuraminidase, pokeweed mitogen, bacterial lipopolysaccharides or purified tuberculin was less, or not at all, affected by this procedure. Medium change per se caused some proliferation in non-stimulated cultures. Increased rate of sugar uptake in insulin-stimulated cultures returned to the level of that in non-stimulated cultures within a few hours after medium change. This reversion took place apparently irrespective of the phase of the cell cycle. Replacing the medium with supernatant from non-stimulated cultures induced a rapid decline in subsequent thymidine incorporation during the first S-phase, and completely abolished the second peak of DNA synthesis. The fraction of cells irreversibly committed to mitosis increased when the time after stimulation increased. Less than three hours' incubation with insulin or trypsin was needed to initiate proliferation of a significant fraction of the cell population. It is concluded that reversion of the initiated cycle of a given cell is no more possible after the cell has entered the S-phase.     

Cell density affects cell motility in P. carterae cultures

The swimming, calcifying alga P. carterae provides an excellent model to study the relationship of biomineralization and morphology to motility and gravitaxis. In preparation for a spaceflight experiment, cell motility was analyzed in cultures grown under various conditions. Cells in young growing cultures with low cell concentration swam randomly. As cell density increased, bioconvection was established, with distinct areas of cells swimming up, narrow streams of cells moving down, and random cell movement in less concentrated pockets of the culture. Aging of the culture may increase cell adhesiveness and sedimentation that depletes the number of swimmers in the population. Motility was maintained for two months in some of the conditions tested, indicating that an equilibrium that allows swimming behavior studies can be established in a closed system. Cell concentration seems to be responsible for the oriented movement in P. carterae, due to its effect on the establishment of bioconvection.     

Lysine hydroxylation of collagen in a fibroblast cell culture system

The lysine (Lys) hydroxylation pattern of type I collagen produced by human fibroblasts in culture was analyzed and compared. Fibroblasts were cultured from normal human skin (NSF), keloid (KDF), fetal skin (FDF), and skin tissues of Ehlers-Danlos syndrome type VIA and VIB patients (EDS-VIA and -VIB). The type I collagen alpha chains with or without non-helical telopeptides were purified from the insoluble matrix and analyzed. In comparison with NSFs, KDF and FDF showed significantly higher Lys hydroxylation, particularly in the telopeptide domains of both alpha chains. Both EDS-VIA and -VIB showed markedly lower Lys hydroxylation in the helical domains of both alpha chains whereas that in the telopeptides was comparable with those of NSFs. A similar profile was observed in the tissue sample of the EDS-VIB patient. These results demonstrate that the Lys hydroxylation pattern is domain-specific within the collagen molecule and that this method is useful to characterize the cell phenotypes in normal/pathological connective tissues.