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With the development of a wide range of new biomaterials for the sensing of different cell behaviour, it is important to consider whether the cells tested in vitro are in direct contact with the material or whether cell-biomaterial contact is mediated by an interfacial layer of proteins originating from the culture medium or from the cells themselves. Thus, this study describes the differences between the cell adhesion mediated by proteins originating from foetal bovine serum and without the presence of such proteins 2 h following cell seeding exemplarily with different cell types (an osteoblastic cell line, primary fibroblasts, and mesenchymal stem cells). Three of the examined cell types were found to react differently to differing conditions in terms of cell shape, area, and number. Nevertheless, the expression and localization of the various proteins involved in cell adhesion and signalling (CD44, vinculin, talin, actin, focal adhesion kinase, Rho-GTPases and extracellular signal-regulated kinases 1 and 2) were, in general, similar with respect to all the cell types tested, albeit varying according to the presence or absence of serum. Moreover, no classical focal adhesions were formed during cell adhesion without serum proteins, while different signalling pathways were involved in this process. The study systematically describes and discusses the cell adhesion of three different human cell types to a well-known substrate without the presence of external proteins and it is hoped that this knowledge will be subsequently applied in biomaterial applications in which the presence of external proteins is undesirable (e.g. for biosensing purposes).

Chirality is one of the basic, unique, and most appealing features of biological molecules; however, many intriguing chiral phenomena in biological world remains insufficiently revealed yet. In this research, we fabricated chiral surfaces by assembling natural chiral amino acids-cysteine of opposite configurations (D- and L-) onto gold surfaces, respectively, and investigated the adhesion of the L929 fibroblast on them. No significant differences were observed in the density of adherent cells under serum-free culture condition; while in serum-containing condition, significantly more cells adhered on the L-Cys assembled surfaces. This phenomenon suggested that serum protein might play an important role in mediating the selective adhesion of cells on chiral surfaces. Hence, we adopted both radiolabeling and surface plasmon resonance (SPR) techniques to monitor protein adsorption onto the above surfaces. The results evidently showed more proteins adsorbed onto surfaces assembled with L-Cys. We propose that the difference in protein adsorption on chiral surfaces as demonstrated in this paper might not only shed light on the ensuing investigation of bio-related chirality phenomena, but also provide a novel strategy for the rational design and fabrication of novel biomaterials and bio-related devices based on chiral effects., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: Lung inflammation is present in patients with systemic sclerosis (SSc) and interstitial lung disease (ILD), but the mechanisms linking inflammatory and fibrotic processes in ILD are unknown. Our aim was to investigate whether alveolar inflammation, reflected by increased alveolar concentration of exhaled nitric oxide (C(A)NO), is related to the ability of serum from patients with SSc to induce pulmonary fibroblast proliferation (PFP) and myofibroblast conversion., Methods: C(A)NO was measured in all subjects (37 patients with SSc and 10 healthy controls) whose sera were used to stimulate PFP (assessed by BrdU labeling index) and myofibroblast conversion (detected by alpha-smooth muscle actin expression). The PFP index in patients with SSc was compared to control values, and between patients with SSc who had elevated (> 4.3 ppb) and normal ( 4.3 ppb; n = 25, median 1.1, range 0.98-1.23) than in patients with SSc who had normal C(A)NO ( 4.3 ppb than in patients whose C(A)NO was

The fast development of tissue engineering and regenerative medicine drives the old biomaterials, for example, fibrin glue, to find new applications in these areas. Aiming at developing a commercially available hydrogel for cell entrapment and delivery, in this study we optimized the fabrication and gelation conditions of fibrin gel. Fibrinogen was isolated from human plasma by a freeze-thaw circle. Gelation of the fibrinogen was accomplished by mixing with thrombin. Absorbance of the fibrinogen/thrombin mixture at 550 nm as a function of reaction time was monitored by UV-VIS spectroscopy. It was found that the clotting time is significantly influenced by the thrombin concentration and the temperature, while less influenced by the fibrinogen concentration. After freeze-drying, the fibrin gel was characterized by scanning electron microscopy (SEM), revealing fibrous microstructure. Thermal gravimetric analysis found that the degradation temperature of the crosslinked fibrin gel starts from 288 degrees C, which is about 30 degrees C higher than that of the fibrinogen. The hydrogel has an initial water-uptake ratio of approximately 50, decreased to 30-40 after incubation in water for 11 h depending on the thrombin concentration. The fibrin gels lost their weights in PBS very rapidly, while slowly in DMEM/fetal bovine serum and DMEM. In vitro cell culture found that human fibroblasts could normally proliferate in the fibrin gel with spreading morphology. In conclusion, the fibrin gel containing higher concentration of fibrinogen (20 mg ml(-1)) and thrombin (5 U ml(-1)) has suitable gelation time and handling properties, and thus is applicable as a delivery vehicle for cells such as fibroblasts.

Objective: Cholesterol efflux is achieved by several mechanisms. This study examines contributions of these pathways to efflux to human serum., Methods and Results: Human fibroblasts were stably transfected with SR-BI while ABCA1 was upregulated. Quantitation of cholesterol efflux to human serum demonstrated that there was efflux from cells without either protein. Expression of ABCA1 produced a small increase in efflux, whereas SR-BI expression had a dramatic impact. To quantitate ABCA1 and SR-BI contribution, fibroblasts were pretreated with Probucol and BLT-1 to, respectively, inhibit these efflux proteins. Exposing SR-BI-expressing fibroblasts to BLT-1 inhibited efflux by 67%. Probucol pretreatment of ABCA1-expressing fibroblasts reduced efflux to serum by 26%. A large fraction of total efflux was uninhibited. For both J774 and mouse peritoneal macrophages, contributions of either ABCA1 or SR-BI to efflux to serum were low, with background/uninhibited efflux contributing from 70% to 90% of total efflux., Conclusions: We have shown that ABCA1-mediated efflux to serum responds to the pool of lipid-free/poor apolipoproteins, whereas phospholipid-containing particles mediate SR-BI efflux. Although SR-BI and ABCA1 contribute to efflux from fibroblasts and cholesterol-enriched macrophages, a large proportion of the total efflux to human serum is mediated by a mechanism that is neither SR-BI nor ABCA1.

1. Hereditary spastic paraplegia (HSP) is a genetically heterogeneous group of neurodegenerative disorders affecting 1 in 10,000 individuals. The present study was aimed to elucidate the role played by reactive oxygen species (ROS) in the pathogenesis of this disease. 2. To address this question we used 7-11 passaged fibroblasts from HSP patients to measure the extent of DNA damage induced by H2O2 treatment and to evaluate the JNK phosphorylation level after hydrogen peroxide and serum stimuli. 3. The present study demonstrates that HSP cells compared to controls are more sensitive to DNA damages induced by H2O2 treatment, and that JNK phosphorylation levels are increased in HSP fibroblasts compared to controls after hydrogen peroxide and serum stimuli. These results suggest a ROS-mediated pathogenetic mechanism for this disease.

Chaperone-mediated autophagy (CMA) is a selective lysosomal protein degradative process that is activated in higher organisms under conditions of prolonged starvation and in cell culture by the removal of serum. Ketone bodies are comprised of three compounds (beta-hydroxybutyrate, acetoacetate, and acetone) that circulate during starvation, especially during prolonged starvation. Here we have investigated the hypothesis that ketone bodies induce CMA. We found that physiological concentrations of beta-hydroxybutyrate (BOH) induced proteolysis in cells maintained in media with serum and without serum; however, acetoacetate only induced proteolysis in cells maintained in media with serum. Lysosomes isolated from BOH-treated cells displayed an increased ability to degrade both glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A, substrates for CMA. Isolated lysosomes from cells maintained in media without serum also demonstrated an increased ability to degrade glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A when the reaction was supplemented with BOH. Such treatment did not affect the levels of lysosome-associated membrane protein 2a or lysosomal heat shock cognate protein of 70 kDa, two rate-limiting proteins in CMA. However, pretreatment of glyceraldehyde-3-phosphate and ribonuclease A with BOH increased their rate of degradation by isolated lysosomes. Lysosomes pretreated with BOH showed no increase in proteolysis, suggesting that BOH acts on the substrates to increase their rates of proteolysis. Using OxyBlot analysis to detect carbonyl formation on proteins, one common marker of protein oxidation, we showed that treatment of substrates with BOH increased their oxidation. Neither glycerol, another compound that increases in circulation during prolonged starvation, nor butanol or butanone, compounds closely related to BOH, had an effect on CMA. The induction of CMA by ketone bodies may provide an important physiological mechanism for the activation of CMA during prolonged starvation.

Coexpression of the serum and glucocorticoid inducible kinase 1 (SGK1) up-regulates Kv channel activity in Xenopus oocytes and human embryonic kidney cells. To investigate the physiological impact of SGK1 dependent Kv channel regulation, we recorded whole-cell currents in lung fibroblasts from SGK1 knockout mice (sgk1-/-) and wild-type littermates (sgk1+/+). Serum-grown mouse lung fibroblasts (MLF) from both genotypes exhibited voltage-gated outwardly rectifying K(+)-currents with time-dependent activation (tau(act) approximately 3 msec), slow inactivation (tau(inact) approximately 700 msec), use-dependent inactivation, and (partial) inhibition by K(+) channel blockers TEA, 4-AP, and margatoxin. In serum grown MLF peak Kv current density at +100 mV was significantly lower in sgk1-/- (14 +/- 2 pA/pF, n = 13) than in sgk1+/+ (31 +/- 4 pA/pF, n = 16). PCR amplification of different Kv1 and Kv3 subunits from mouse fibroblasts demonstrated the expression of Kv1.1-1.7, Kv3.1, and Kv3.3 mRNA in both sgk1+/+ and sgk1-/- cells. Upon serum deprivation Kv currents almost disappeared in sgk1+/+ (4 +/- 1 pA/pF, n = 11) but not in sgk1-/- (10 +/- 1 pA/pF, n = 6) MLF. Accordingly, following serum deprivation Kv current density was significantly lower in sgk1+/+ than in sgk1-/-. Stimulation of serum-depleted cells with dexamethasone (dex) (1 microM, 1 day), IGF-1 (6.7 microM, 4-6 h) or both, significantly activated Kv currents in sgk1+/+ but not in sgk1-/- MLF. In the presence of both, dex and IGF-1, the Kv current density was significantly larger in sgk1+/+ (27 +/- 3 pA/pF, n = 12) than in sgk1-/- (13 +/- 3 pA/pF, n = 10) cells. Similar to MLF, Kv currents were significantly higher in sgk1+/+ mouse tail fibroblasts (MTF). In sgk1+/+ but not sgk1-/- MTF the Kv currents were inhibited upon serum deprivation and reincreased after stimulation of serum deprived MTF with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h). According to Fura-2-fluorescence capacitative Ca(2+) entry was lower in sgk1-/- MTF compared to sgk1+/+ MTF. Upon serum deprivation capacitative Ca(2+) entry decreased significantly in sgk1+/+ but not in sgk1-/- MTF. Stimulation of depleted cells with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h) reincreased capacitative Ca(2+) entry in sgk1+/+ MTF, whereas in sgk1-/- cells it remained unchanged. In conclusion, lack of SGK1 does not abrogate Kv channel activity but abolishes regulation of those channels by serum, glucocorticoids and IGF-1, an effect influencing capacitative Ca(2+) entry., ((c) 2004 Wiley-Liss, Inc.)

The restriction point (R) separates the G1 phase of continuously cycling cells into two functionally different parts. The first part, G1-pm, represents the growth factor dependent post-mitotic interval from mitosis to R, which is of constant length (3-4 h). The second part, G1-ps, represents the growth factor independent, pre-S phase interval of G1 that lasts from R to S and that varies in time from 1 to 10 h. G1-pm cells rapidly exit (within 1 h) from the cell cycle and enter G0 as a response to serum withdrawal. The finding that R occurs at a set time after mitosis indicates that R may be related to the metabolic and/or structural changes that the cell underwent during the previous mitosis. We have recently shown that phosphorylation of the retinoblastoma tumor suppressor protein (pRb) is not the molecular mechanism behind R, as has been suggested previously. Here, we present an alternative explanation for R. In the present study, we applied a single cell approach using time-lapse analysis, which revealed that upon serum starvation the G1-pm cells rapidly underwent a transient change in cell shape from flat to spherical before exiting to G0. Platelet derived growth factor (PDGF) counteracted this change in shape and also prevented exit to G0 to the same extent. Furthermore epidermal growth factor (EGF) and insulin like growth factor (IGF-1), which only partially counteracted this change, only partially counteracts exit to G0. These data clearly indicate a direct link between change in cell shape and exit to G0 in G1-cells that have not passed R., (2004 Wiley-Liss, Inc.)

An after-cataract is caused by the proliferation of residual cells over the equator of the lens. These cells subsequently migrate to the posterior lens capsule, where they undergo aberrant differentiation into fiber-like cells or transdifferentiation into fibroblast-like cells. To study the precise molecular mechanisms of transdifferentiation, an attempt was made to establish an in vitro system, in which the lens epithelial cells (LECs) of the pre-equatorial zone could be transdifferentiated into fibroblast-like cells. The required conditions for culturing the LECs were identified as consisting of four phases; intact bovine explants, explant-cultured, serum-modulated and additionally modulated LECs. The LECs of each phase were compared by examining changes in the expression of the epithelial-mesenchymal transition (EMT)-related genes and changes in cellular morphology and adhesion. The explants that were cultured in a medium containing 10% fetal bovine serum (FBS) for 2 weeks, showed changes in the expression of the EMT-related genes, although the other explant-cultured cells maintained an epithelial morphology. To introduce a transition into mesenchymal cells, the explant cultures were subcultured in a medium containing 20% FBS for six passages. These cells displayed an elongated morphology and were able to grow and migrate in a similar way to fibroblast cells. The expression of the EMT-related genes, such as, extracellular matrix proteins and integrins, was altered. This was similar to the alteration of the 3-dimensional collagen gels model previously reported. During a further process of EMT by additional serum modulation, the inhibitory effect of disintegrin on cell adhesion was gradually decreased, integrin expression was differentially regulated and alpha-smooth muscle actin was post-translationally modified from the point of passage number six. Overall, it can be concluded that terminal transdifferentiation accompanies changes in the cytoskeletal proteins and cell surface molecules. These are modulated in systematic patterns of post-transcriptional and post-translational regulation and patterns of gene regulation, by the synergic effects of several transforming factors contained in serum. Therefore, posterior capsular opacification may also be accompanied by this molecular mechanism.

One of the challenges of manipulating genes in primary cells is that the cells have a finite proliferation capacity. This, combined with the lower gene targeting efficiency of somatic cells, makes identification of targeted clones very difficult. The objective of this study was to establish a system that allows porcine foetal fibroblasts to reach their maximal proliferation capacity in vitro. The influence of fibroblast origin, stage of foetal development, cell seeding densities and concentration of foetal bovine serum (FBS) on the population doublings, the percentage of beta-galactosidase-activity-positive cells and the genome stability of foetal fibroblasts during in vitro culture was investigated. It was found that porcine foetal fibroblasts could be cultured for over 80 population doublings in the appropriate culture system. Fibroblasts from earlier stages of foetal development were better candidate cells than those from the later stages. Cells from the heart were more actively proliferative and more resistant to replicative senescence than those from the liver. Compared to 10% FBS content, 15% FBS provided better homeostatic support, not only to proliferative performance, but also in maintaining a normal karyotype. In addition, the proliferative life span of porcine foetal fibroblasts is also dependent on seeding density of the culture.

Wound-induced injury of 3T6 fibroblast cultures initiated a repair process stimulated by fetal calf serum (FCS) that restored the integrity of cell cultures. In these experimental conditions, FCS induced arachidonic acid (AA) release and eicosanoid production. Our results show that the inhibition of the cyclooxygenase (COX) and/or cytochrome P-450 pathways significantly decreases the wound closure, whereas that of the lipoxygenase pathway does not modify the wound repair process. Both EP(1) and EP(4) receptors of prostaglandin E(2) (PGE(2)) mediate PGE(2) stimulated 3T6 fibroblast wound closure. Our data suggest that calcium and cAMP are involved in the signaling event induced by PGE(2) during the 3T6 fibroblast wound repair process. On the other hand, we show that ketoconazole, a cytochrome P-450 inhibitor, hinders the wound closure induced by FCS in wounded 3T6 fibroblast cultures. 12 and 20 Hydroxyeicosatetraenoic acids (HETEs), which are key AA metabolites synthesized by cytochrome P-450, partially revert the effects of ketoconazole on the wound repair process. Thus, the COX and cytochrome P-450 pathways of the arachidonate cascade are involved in 3T6 fibroblast wound closure., (Copyright 2003 Wiley-Liss, Inc.)

The allergic reaction begins with the antigen-induced aggregation of occupied high-affinity IgE receptors expressed on mast cell surface, their activation, and the release of proinflammatory mediators that cause the "early phase" of this process. In addition, mast cell activation induces the onset of a "late phase" reaction characterized by the tissue infiltration of inflammatory cells, mainly eosinophils. We have hypothesized that during the late phase mast cells interact with and are activated by eosinophils. Here we report that highly purified human lung mast cells became responsive to eosinophil major basic protein (MBP) when in coculture with human lung fibroblasts. In addition, cord blood-derived mast cells maintained in coculture with 3T3 fibroblasts released more histamine and prostaglandin D(2) (PGD(2)) compared with cells maintained in suspension. The fibroblast-derived membrane form of stem cell factor (SCF) was found to be involved in the mast cell increased responsiveness to MBP. In fact, cord blood-derived mast cells cocultured with 3T3 in the presence of antisense for SCF or cocultured with fibroblasts that do not express the membrane form of SCF were inhibited in their histamine-releasing activity toward MBP. In addition, this form of SCF induced the expression of a pertussis toxin-sensitive G(i) protein, G(i3) that interacts with MBP to trigger mast cell non-IgE-dependent activation in a manner similar to other cationic compounds such as compound 48/80. Mast cell responsiveness to eosinophil mediators is a potentially novel evidence for an alternative pathway of allergen-independent activation able to contribute to the perpetuation of allergy.

Modulation of ion permeability during the cell cycle is one of the key events in cell cycle progression. We have compared the effects of K+ and Cl- channel blockers on the cell cycle in synchronous and asynchronous NIH3T3 cells. The Cl- channel blocker 5-N-2-(3-phenylpropylamino) benzoic acid (NPPB; 0.2 mM) inhibited entry into S phase in synchronous cells but not in asynchronous cells, while the K+ channel blocker 4-aminopyridine (4-AP) showed similar inhibitory effects in both conditions. In NIH3T3 cells synchronized by serum deprivation/replenishment, G0-to-G1 transition occurred within 8 h after serum addition, and the G1/S checkpoint at 10-14 h. NPPB applied only at 0-8 or 8-14 h after serum addition inhibited entry into S phase. Cl- permeability measured as 125I efflux increased at 4 and 10 h after serum addition. Ki-67-negative cells, which represent quiescent G0 phase cells, progressively decreased in number until 8 h after serum addition. The Cl- channel blockers (NPPB and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid [DIDS]) but not the K+ channel blocker (4-AP) significantly decreased the rate of reduction in number of Ki-67-negative cells. These data indicate that an increase in Cl- permeability plays an important role in reentry of quiescent cells into the proliferating phase, in addition to the known effects on passage through the G1/S checkpoint., (Copyright 2003 Wiley-Liss, Inc.)

Fibroblasts form fibers when grown in culture medium containing native type 1 collagen. The contractile forces generated can be precisely quantified and used to analyze the signal transduction pathways regulating fibroblast contraction. Calf serum (30%) induces a sustained contraction that is accompanied by a transient increase in intracellular calcium ([Ca(2+)](i)). W-7, a calmodulin inhibitor, KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase, and ML-7, a myosin light-chain kinase inhibitor, had no effects on either the contraction or the [Ca(2+)](i) responses. Neither genistein, a tyrosine kinase inhibitor, nor calphostin C, a protein kinase C inhibitor, had major effects on force or [Ca(2+)](i). In contrast, the Rho kinase inhibitors (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and HA1077 depressed the contraction in a dose-dependent manner without affecting the [Ca(2+)](i) response. Stress fiber formation was also suppressed by Y-27632. Surprisingly, calf serum, Y-27632, and calf serum plus Y-27632 did not alter mono- or diphosphorylation of the myosin regulatory light chain (MRLC) compared with control untreated fibers. These results suggest that the sustained contraction of NIH 3T3 fibroblast fibers induced by calf serum is mediated by Rho kinase but is independent of a sustained increase in [Ca(2+)](i), calcium/calmodulin- or protein kinase C-dependent pathways, or increases in MRLC phosphorylation.

Background: To gain insight into the mechanisms responsible for tissue neutrophil immigration in sinusitis, primary nasal fibroblasts are analyzed for synthesizing and delivering neutrophil chemokines., Methods: Primary nasal fibroblast cell culture was treated with tumor necrosis factor (TNF)-alpha concentrations of 20 and 200 ng/ml for 2, 8, 24 and 72 h. Chemokine concentrations in supernatants were determined by enzyme-linked immunoassay (ELISA) and chemokine mRNA expression in fibroblasts was measured by reverse transcriptase polymerase chain reaction (RT-PCR). Biological chemotactic activity was identified by three-step high-performance liquid chromatography (HPLC) and by bioassay measuring neutrophil chemotaxis in a single Boyden chamber system., Results: Interleukin (IL)-8 and growth-related oncogene (GRO)-alpha were induced in nasal fibroblast culture by proinflammatory stimulus. After 24 h of stimulation neutrophil chemotactic activity only was detected for IL-8. Granulocyte chemotactic protein (GCP)-2 mRNA was already significantly up-regulated after 2 h of stimulation., Conclusion: Induction of IL-8 protein dominates chemokine synthesis 24 and 72 h after stimulation, whereas induction of GCP-2 mRNA seems to have a role in the early phase after 2 h of exposition with TNF-alpha.

When c-Src and v-Src were immunoprecipitated together from hamster fibroblasts transformed by Rous sarcoma virus containing v-src oncogene, the total Src activity was almost threefold higher compared to c-Src activity in the control cells. The activity of v-Src immunoprecipitated separately, however, accounting for only 40% of the total Src activity, indicating that c-Src is activated upon transformation. An increased activity of Csk was also found in RSV-transformed cells. It decreased upon serum stimulation in parallel with an increase in Src kinase activity. In nontransformed cells, serum stimulation induced an enhanced Csk activity, but no changes in c-Src activity were observed. This may suggest that Csk may have more functions in hamster fibroblasts, in addition to its inhibitory effect on c-Src.

The capacity to generate circadian rhythms endogenously and to confer this rhythmicity to other cells was compared in immortalized cells derived from the suprachiasmatic nucleus (SCN) and a fibroblast line to differentiate SCN pacemaker properties from the oscillatory behavior of non-clock tissues. Only SCN2.2 cells were capable of endogenously generating circadian rhythms in 2-deoxyglucose uptake and Per gene expression. Similar to SCN function in vivo, SCN2.2 cells imposed rhythms of metabolic activity and Per gene expression on cocultured NIH/3T3 fibroblasts via a diffusible signal. The conferred rhythms in NIH/3T3 cells were phase delayed by 4-12 hr relative to SCN2.2 circadian patterns, thus resembling the phase relationship between SCN and peripheral tissue rhythms in vivo. Sustained metabolic rhythmicity in NIH/3T3 cells was dependent on continued exposure to SCN2.2-specific outputs. In response to a serum shock the NIH/3T3 fibroblasts exhibited recurrent oscillations in clock gene expression, but not in metabolic activity. These molecular rhythms in serum-shocked fibroblasts cycled in a phase relationship similar to that observed in the SCN in vivo; peak Per1 and Per2 mRNA expression preceded the rhythmic maxima in Cry1 and Cry2 mRNA levels by 4 hr. Despite these clock gene oscillations the serum-shocked NIH/3T3 cells failed to drive circadian rhythms of Per1 and Per2 expression in cocultures of untreated fibroblasts, suggesting that expression and circadian regulation of the Per and Cry genes are not sufficient to confer pacemaker function. Therefore, SCN-specific outputs are necessary to drive circadian rhythms of metabolic activity, and these output signals are not a direct product of clock gene oscillations.

Serum has been known to stimulate collagen production by dermal fibroblasts. As part of an ongoing study of the molecular mechanisms of collagen production, we have investigated transcriptional regulation of the human alpha2(I) collagen gene by serum in human dermal fibroblasts. Serum responsive elements were mapped by deletion analysis between bp -353 and -264, and between -148 and -108 in the alpha2(I) collagen promoter. Further functional analysis of the alpha2(I) collagen promoter containing various substitution mutations revealed that serum stimulation of this promoter is mediated equally by a GC-rich region located between bp -303 and -271 and by the TCCTCC motif located between bp -123 and -128, both of which constitute binding sites for transcription factor Spl and Sp3. No differences were observed in electrophoretic mobility shift assays between unstimulated and serum stimulated fibroblasts. The Spl inhibitor mithramycin blocked stimulation of the alpha2(I) collagen promoter activity by serum. Furthermore, immunoprecipitation analysis showed that serum stimulation increased Spl phosphorylation. In conclusion, this study characterized response elements that mediate serum stimulation of the human alpha2(I) collagen promoter and suggests that serum stimulation was mediated via Sp1/Sp3 binding sites in this promoter.

Recently, we suggested that arachidonic acid and/or its cyclooxygenase pathway metabolites may be involved in regulating 3T6 fibroblast proliferation. In the present study we evaluate the role of high-molecular phospholipase A2 (PLA2) enzymes in the 3T6 fibroblast growth. Our results demonstrate that the cytosolic PLA2 inhibitor, arachidonyl trifluoromethylketone and the cytosolic calcium-independent PLA2 (iPLA2) inhibitor, bromoenol lactone, decrease arachidonic acid release and prostaglandin E2 production in 3T6 fibroblast cultures stimulated by fetal calf serum. These effects were correlated with the impairment of 3T6 fibroblast proliferation and DNA synthesis at the S/G2 boundary, which prolongs the S phase. These data suggest a role of iPLA2 in the control of 3T6 fibroblast growth.

Eosinophil infiltration occurs in a variety of allergic and inflammatory diseases. The release of preformed mediators from eosinophils may contribute to inflammatory responses. We investigated the ability of eosinophil-derived major basic protein and eosinophil-derived neurotoxin to stimulate production of IL-8 from intestinal myofibroblasts. Intestinal myofibroblasts (18-Co cells) were incubated with major basic protein, eosinophil-derived neurotoxin, or a synthetic analogue of major basic protein, poly-L-arginine. Immunoreactive IL-8 was measured by ELISA and IL-8 mRNA levels were analysed by Northern blot or reverse transcription-polymerase chain assay. Major basic protein induced IL-8 mRNA production and release of significant levels of IL-8 immunoreactive protein. By contrast, eosinophil-derived neurotoxin stimulated little IL-8 release. The induction of IL-8 mRNA by poly-L-arginine was significantly inhibited by actinomycin D. These findings demonstrate a novel interaction between eosinophils and intestinal fibroblasts that may be involved in the pathogenesis of diseases associated with tissue eosinophilia.

Na(+)/K(+)/Cl(-) cotransporter activity is stimulated in early G(1) phase of the cell cycle and this stimulation was shown to be an essential event in fibroblast cell proliferation. In order to elucidate further the role of the Na(+)/K(+)/Cl(-) cotransporter in cell proliferation, we overexpressed the gene encoding the Na(+)/K(+)/Cl(-) cotransporter in mouse fibroblasts, and analyzed cellular phenotypic changes. Mouse Balb/c 3T3 cells were stably transfected with the cDNA of the shark rectal gland Na(+)/K(+)/Cl(-) cotransporter gene (NKCC1), and expressed in a mammalian vector under the cytomegalovirus promoter (Balb/c-NKCC1 cells). The transfected cells exhibited up to 10-fold greater bumetanide-sensitive Rb(+) influx compared to the control cells. The Balb/c-NKCC1 cells have acquired a typical transformation phenotype indicated by: (1) Loss of contact inhibition exhibited by growth to a higher cell density in confluent cultures, and formation of cell foci; (2) proliferation in low serum concentrations; and (3) formation of cell colonies in soft agar. The control cells transfected with the NKCC1 gene inserted in the opposite orientation in the vector retained their normal phenotype. Furthermore, the two specific inhibitors of the Na(+)/K(+)/Cl(-) cotransporter activity; bumetanide and furosemide inhibited the clonogenic efficiency in the NKCC1 transfected cells. These control experiments indicate that the apparent transformation phenotype acquired by the Balb/c-NKCC1 cells was not merely associated with the process of transfection and selecting for the neomycin-resistant clones, but rather with the overexpression of the Na(+)/K(+)/Cl(-) cotransporter gene. In order to ascertain that the regulated and normal expression of the Na(+)/K(+)/Cl(-) cotransporter control cell proliferation, the effect of bumetanide a specific inhibitor of the cotransporter, was tested on Balb/c 3T3 cell proliferation, induced by fibroblasts growth factor (FGF) and fetal calf serum (FCS). Bumetanide inhibited synchronized Balb/c 3T3 cell exit from the G(0)/G(1) arrest and entering S-phase. The inhibition was reversible, as removal of bumetanide completely released cell proliferation. Taken together, these results propose that the NKCC1 gene is involved in the control of normal cell proliferation, while its overexpression results in apparent cell transformation, in a manner similar to some protooncogenes., (Copyright 2000 Wiley-Liss, Inc.)

Objective: To investigate scleroderma fibroblast behavior, and the effect of different human sera on fibroblast behavior, we established a model using the newly developed electrical biosensor, electrical cell-substrate impedance sensing (ECIS)., Methods: Cellular locomotion, defined as cellular micromotion, measured by ECIS indicates the dynamic vertical motion of a given group of cells. The junctional resistance (Rb) between adjacent cells and the average height (h) between basal cell surface and substratum derived from ECIS were quantified for dermal fibroblasts obtained from patients with scleroderma and normal controls. The cellular micromotions of both scleroderma and normal control fibroblasts were compared in the presence of different human sera, including those from patients with scleroderma, systemic lupus erythematosus (SLE), and Sjögren's syndrome (SS) and from normal individuals., Results: The Rb and h levels for scleroderma and normal fibroblasts were 2.7 omega.cm2, 150 nm and 0.8 omega.cm2, 303 nm respectively. The micromotions of scleroderma fibroblasts were more active than those of normal fibroblasts. Sera from patients with scleroderma can inhibit the micromotions of normal fibroblasts but not those of scleroderma fibroblasts, while sera from patients with SLE and SS have no inhibitory effect on either normal or scleroderma fibroblast micromotions., Conclusion: We have demonstrated the previously unrecognized characteristics of dermal fibroblasts and sera derived from patients with scleroderma. It is possible that in vivo activities cause scleroderma fibroblasts to display active cellular micromotions, while sera from patients with scleroderma inhibit the micromotions of normal fibroblasts. The use of ECIS technology has also provided a new approach to the study of scleroderma.

Tenascin is an extracellular matrix glycoprotein which affects cell behavior such as cell migration. This study was undertaken to investigate the time of appearance of tenascin (TN) in human gingival fibroblasts (HGF) and how it was affected by the surface topography of the titanium substratum or by serum concentration in the medium. HGF were cultured for 4 to 24 h and then processed for confocal immunofluorescence microscopy. Very few cells stained positive for TN 4 h after plating, but the number of TN-positive HGF gradually increased between 8 and 18 h after plating. The increase in the rate of the proportion of TN-positive cells on the grooved surface lagged behind that of HGF cultured on the smooth surface. The number of TN-positive cells in medium + 15% serum was significantly greater than that of cells in 5% serum or serum-free medium. The number of TN-positive cells was greater on the smooth titanium surface than on the grooved titanium surface in both 15% serum and 5% serum-containing medium. These findings suggest that TN production by fibroblasts in vitro can be modulated by factors in serum and by the surface topography of the substratum., (Copyright 1998 Academic Press.)

Certain concentrations of the antiseptic AgNO3, a potent broad-spectrum antimicrobial agent, exert cytotoxic effects on fibroblasts and endothelial cells which are directly related to the wound-healing process. In vitro assessment of human fibroblast cytotoxicity has proved to be a useful method for characterizing cell toxicity mechanisms of topically-applied antiseptics. In the present study human dermal fibroblasts were exposed to AgNO3 at concentrations of 4.12-82.4 microM for 8 and 24 h. Silver ions greatly inhibited fibroblast proliferation and prolonged AgNO3 exposure produced Ag-dependent cell loss. In the sequence of events occurring during our in vitro experimental model, the inhibitory action on DNA synthesis was the primary event in AgNO3 cytotoxicity, associated with significant loss of cell protein. Both parameters decreased as the content of fetal calf serum (FCS) in the exposure medium was gradually reduced from 10 to 2%. The parallel study of DNA synthesis and cell protein content suggests that the toxic damage produced by silver in different phases of the cell cycle may lead to destruction of the entire cell population and therefore hinder the tissue regeneration process. A concentration- and time-dependent depletion of intracellular ATP content is also caused by ionic silver, thereby compromising the cell energy charge which precedes human dermal fibroblast death. Concomitant incorporation of FCS to the medium always attenuated Ag+ cytotoxicity curves in a concentration-dependent fashion and maximum protection was observed at 10% in situations closely resembling physiological conditions.

The effect of burn patient serum on fibroblast and keratinocyte cell morphology in culture was investigated using the scanning electron microscope. Serum was taken from five patients with burn injuries ranging from 8 to 65 per cent TBSA (10-65 per cent full-thickness). One patient had superficial burns. Pooled serum from 23 volunteers was used as the control serum. The cells were seeded onto collagen-coated glass coverslips and incubated for 5 days with culture medium containing 10 per cent (v/v) control serum or patient serum taken during the early postburn period. Scanning electron micrographs demonstrated a reduction in fibroblast cell density with serum from patients with full-thickness burns. Furthermore, the spindle shape of the fibroblast cell was greatly exaggerated compared with control cultures. The integrity of the keratinocyte sheet was destroyed when keratinocyte cells were incubated with serum fron patients with full-thickness burns. Globular-like structures or membrane protrusions were present in concentrated areas on keratinocyte cells which were not present in control cultures. This study demonstrated the vulnerability of cutaneous cells to systemic factors present in the early postburn serum. The extent of the effect appears to be related to the presence of full-thickness injury. This effect may further explain the frequent aberrant wound healing response to burn injury.

During wound repair, fibroblasts accumulate in the injured area until any defect is filled with stratified layers of cells and matrix. Such fibroplasia also occurs in many fibrotic disorders. Transforming growth factor-beta (TGF-beta), a promotor of granulation tissue in vivo and extracellular matrix production in vitro, is expressed during the active fibroplasia of wound healing and fibroproliferative diseases. Under usual tissue culture conditions, normal fibroblasts grow to confluence and then cease proliferation. In this study, culture conditions with TGF-beta 1 have been delineated that promote human fibroblasts to grow in stratified layers mimicking in vivo fibroplasia. When medium supplemented with serum, ascorbate, proline, and TGF-beta was added thrice weekly to normal human dermal fibroblasts, the cells proliferated and stratified up to 16 cell layers thick within the culture dish, producing a tissue-like fibroplasia. TGF-beta stimulated both DNA synthesis as measured by 3H-thymidine uptake and cell proliferation as measured by a Hoechst dye DNA assay in these postconfluent cultures. The stratification was dependent on fibronectin assembly, as demonstrated by anti-fibronectin antibodies which inhibited both basal and TGF-beta-stimulated cell proliferation and stratification. Suppression of collagen matrix assembly in cell layers with beta-amino-proprionitrile (BAPN) did not inhibit basal or TGF-beta stimulated in vitro fibroplasia. BAPN did not interfere with fibronectin matrix assembly as judged by immunofluorescence microscopy. Thus, in concert with serum factors, TGF-beta stimulates postconfluent, fibronectin matrix-dependent, fibroblast growth creating a fibroplasia-like tissue in vitro.

Hyaluronic acid (HA)-dependent pericellular matrices (PCM) play a role in embryonic differentiation of mesodermal cells. Fetal fibroblasts have significantly larger PCMs than postnatal fibroblasts. To determine if this property is intrinsic to fetal fibroblasts or induced by factors in the fetal environment, we studied the effect of fetal bovine serum (FBS) of varying gestational age on human fetal, newborn, and adult fibroblast PCM formation. Cultured human fetal, newborn, and adult fibroblasts were plated in triplicate at a density of 1 x 10(5) cells and incubated in medium alone, medium containing 10% pooled FBS, or FBS from the first, second, or third trimesters. The cells were photographed and morphometric analysis of PCM was performed by the erythrocyte exclusion technique. PCM size was expressed as a ratio of the maximal width of the cell matrix to the maximal width of the cell. The unpaired Student's t test was used for statistical analysis. The earlier the gestational age of FBS used, the larger the PCM observed in fetal and newborn fibroblasts. The PCM of fetal fibroblasts was significantly larger (P < 0.001) than that of newborn and adult fibroblasts at each gestational age of FBS tested (fetal >> newborn > adult). Medium containing pooled FBS caused a significant (P < 0.001) increase in PCM size in all cell lines compared with serum-free medium. There are both intrinsic and extrinsic factors which affect PCM size. These factors which affect HA-dependent PCM size may contribute to a permissive microenvironment for cell migration, proliferation, and development which may be important for scarless fetal wound repair.

We studied whether serum basic proteins (BPs) produce abnormal changes in the mass of cellular lipids in fibroblasts from patients with hyperapobetalipoproteinemia (hyperapoB) and if inhibition or stimulation of protein kinase C affects these processes. In normal cells, BP I increased the mean mass of triglycerides about twofold, whereas there was significantly less stimulation in hyperapoB cells (P < .005). The increase in the mass of cell cholesteryl esters seen in normal cells with BP I was also significantly reduced in hyperapoB cells (P < .005). In contrast, BP II abnormally stimulated the mass of cell cholesteryl esters sixfold in hyperapoB cells (P < .005). BP I also stimulated the mass of total phospholipids about twofold in normal cells, an effect that was reduced by about one third in hyperapoB cells (P = .08). No abnormality was found in hyperapoB cells with BP III. H-7, an inhibitor of protein kinase C, decreased the effects of BP I and BP II in normal and hyperapoB cells. C:8, an analogue of diacylglycerol, activated protein kinase C and stimulated triglyceride formation in normal (fourfold) and hyperapoB (fivefold) cells in the absence of BP I. When added with C:8, BP I further increased triglyceride production 1.5-fold in normal cells but not in hyperapoB cells. Two cellular abnormalities in lipid metabolism in hyperapoB fibroblasts were found, one with BP I, another with BP II. Protein kinase C activity was not deficient in hyperapoB cells, and the defect(s) may occur at another, perhaps earlier, step in the pathway.

Defensins are a family of structurally homologous peptides contained within phagocytic cells. Although these peptides are best known for their broad spectrum antimicrobial properties, they also inhibit ACTH (corticotropin) stimulated corticosterone production, chemoattract monocytes, and lyse mammalian cells. We now report that these peptides are potent mitogens in vitro in the same concentration range that they display potent antimicrobial activity in vitro. These concentrations are in the same range as those expected to be present in vivo during the wound healing process. All defensins tested were stimulatory for epithelial cells and fibroblasts and acted synergistically with insulin. These are the first data to disclose the strong growth-promoting effects of this unique family of peptides and point to another basic mechanism whereby the macrophage and neutrophil may participate in a variety of trophic, physiologic, and pathologic processes.

The effects of various glycosaminoglycans (GAGs) on the growth rate of normal fibroblasts and a fibrosarcoma cell line (HT 1080) were examined. Cells were grown in 96-well microplates in the absence or presence of serum mitogens, epidermal (EGF), platelet-derived (PDGF), acidic fibroblast (aFGF), or basic fibroblast growth factor (bFGF). Cell number was measured by using crystal violet to stain cell nuclei (Westergren-Thorsson, G., Onnervik, P.-O., Fransson, L.-A., and Malmström, A. J. Cell. Phys. 147, 523-530, 1991) and also by using a Coulter counter. In the presence of serum mitogens, L-iduronate (IdoA)-rich GAGs, such as dermatan sulfate, heparin, and highly sulfated heparan sulfate, inhibited proliferation of normal cells (25-35%), whereas HT 1080 cells were unaffected or slightly stimulated. Ham's F-12 supplemented with insulin and transferrin but without growth factors was able to support growth of both cell types. Under these conditions, the IdoA-rich GAGs still suppressed growth of normal cells (40-55%), whereas HT 1080 cells again responded poorly. When growth factors were added proliferation of normal fibroblasts was further stimulated, EGF being the most effective. In the presence of either EGF, PDGF, or bFGF, IdoA-rich GAGs had a sustained inhibitory effect on normal fibroblasts (30-50% at concentrations at or above 10 micrograms/ml). However, in the presence of aFGF, both IdoA-rich and IdoA-poor heparan sulfates enhanced growth (nearly twofold after prolonged exposure) suggesting a stabilization of this growth factor. In general, IdoA-rich GAGs appear to inhibit proliferation of normal cells irrespective of the type of growth factor used. Therefore, GAGs are likely to act directly on cell-derived regulatory components, either before or after internalization. As fibrosarcoma cells were much less sensitive to growth inhibition, they may contain altered receptors for GAGs.

Confluent skin fibroblasts from normal humans were incubated in serum free medium with up to 100 nmole/mL eicosapentaenoic acid (EPA; bound to albumin in a 4.6:1 ratio) and compared with cells incubated with oleic acid (OA) at similar concentrations. The rate of [14C]OA incorporation into triacylglycerol (TG) (nmol/mg/h) was approximately 5-fold greater than that of [14C]EPA. The mass of TG formed after incubation of fibroblasts with EPA was also significantly lower than that formed with OA (43.2 +/- 9.3 vs. 59.5 +/- 6.6 micrograms/mg cell protein, respectively, P = 0.006). The addition of excess, unlabeled EPA reduced the rate of incorporation of [14C]OA into TG whereas unlabeled OA stimulated incorporation of [14C]EPA into TG. When the cells were preincubated with human serum basic proteins (BP I, II and III), the mass of TG formed (compared to baseline) was significantly higher with the basic proteins whether OA or EPA was studied. Only BP I significantly stimulated the mass of cell phospholipids, an effect that occurred with either OA or EPA in the medium. The results suggest that in cultured normal human fibroblasts, OA is a better substrate for TG synthesis than EPA and that this effect may be accentuated by the presence of the basis proteins.

Scleroderma (progressive systemic sclerosis; PSS) is a connective tissue disorder in which excessive collagen is deposited in the skin and internal organs. Mediators of abnormal fibroblast function in PSS have not yet been identified. Our objective was to examine the possibility that factors present in serum from patients with PSS, or in culture medium conditioned by PSS fibroblast growth, serve to regulate fibroblast function. Fibroblasts from affected and unaffected skin sites of patients with PSS and from normal adult skin were cultured in the presence of various human sera or conditioned media. Results indicate that (1) the proliferative influence of serum of patients with PSS is not different from that of normal donors, (2) increased proliferation and procollagen gene expression are not linked in affected or unaffected dermal fibroblasts, and (3) affected PSS fibroblasts produce a stimulatory factor(s) for procollagen gene expression to which they are differentially sensitive.

Rat-1 fibroblasts were transduced to express Drosophila choline acetyltransferase. The presence of an active enzyme in these cells (Rat-1/dChAT) was confirmed using various methods. Rat-1/dChAT fibroblasts released acetylcholine (ACh) into the culture medium. Moreover, intra- and extracellular levels of ACh could be increased by adding exogenous choline chloride. In addition, serum starvation or confluence-induced quiescence caused an 80% decrease in recombinant choline acetyltransferase activity (compared with actively growing cells). ACh release was also repressed in quiescent fibroblast cultures. Exogenous choline could mitigate the decrease in ACh release. These results indicate that Rat-1 fibroblasts can be genetically modified to produce ACh and that ACh release can be controlled by introducing choline into the culture medium. Furthermore, these data demonstrate that the expression of the retroviral promoter used in this study decreases with the onset of quiescence; however, exogenous choline can increase the amount of ACh released by quiescent fibroblasts.

The production of growth factors by endothelial cells (EC) and other vascular cells may regulate the migration and proliferation of smooth muscle cells in normal and pathological vessel wall processes. We have previously shown that EC production of platelet-derived growth factor-like protein (PDGFc) is regulatable, and in particular is inhibited by specific lipids and by lipid-containing complexes, e.g. oxidized low density lipoproteins and fish oil emulsions. In this report we show that the inhibitory activity of the fish oils is in turn regulated by a component present in serum. Addition of the fish oil extract MaxEPA to bovine aortic EC, in serum-free medium, reduced PDGFc secretion to about 30% that of control cells. Addition of calf serum to the medium almost completely suppressed this inhibitory activity of MaxEPA, while in contrast, fetal calf serum augmented the activity. The suppressor activity in calf serum was dose-dependent, with a half-maximal suppression of about 0.1% serum at a MaxEPA concentration of 50 micrograms/ml. Adult human serum was observed to have a quantitatively similar suppressor activity. The suppressor activity in human serum was identified as ceruloplasmin since: 1) purified ceruloplasmin also suppressed the activity of fish oil, and at concentrations comparable to the inhibitory levels in serum, and 2) removal of ceruloplasmin from human plasma-derived serum by immunoprecipitation restored the inhibitory activity of MaxEPA. These results may have implications in the effectiveness of fish oils as a therapeutic agent for the reduction of intimal thickening in atherosclerosis.

Fibroblast populated collagen lattices (FPCL) have facilitated the in vitro study of wound contraction and scar contracture. Mixing fibroblasts, serum containing culture medium and soluble collagen, together and then incubating the mixture at 37 degrees C produces a FPCL. The fibroblasts elongate and spread within the collagen matrix, and by forces associated with cell locomotion they reorganize the collagen fibers. The reorganization of the collagen produces a reduction in size of the FPCL, called lattice contraction. It was also found that dialyzed fetal bovine serum did not support lattice contraction. Supplementing dialyzed serum with fatty acids accelerated lattice contraction. The fatty acid composition of the fibroblast plasma membrane influences that membrane fluidity. These studies demonstrated that lattice contraction was enhanced by the additions of saturated fatty acids in the order of laurate (C-12), palmitic (C-16), and stearate (C-18). With unsaturated fatty acids additions, the order of enhanced lattice contraction was arachidonate (4 C = C), linoleate (2 C = C) and oleate (1 C = C). The addition of dialyzed serum with or without fatty acids neither altered ATP-induced cell contraction activity nor cell proliferation. It was concluded that free fatty acid additions do not modulate FPCL contraction by enhancing microfilaments contraction or increasing cell numbers. The mechanism of action was proposed to be by altering cell membrane fluidity. This finding further supports the theory that the mechanism for lattice contraction is cell locomotion, rather than cell contraction.

We have compared the expression and localization of carbohydrate binding protein 35 (CBP35) in human SL66 fibroblasts of different replicative capacities. When serum-starved, quiescent young (passage 11, corresponding to approximately 18 cumulative population doublings) SL66 cells were treated with serum, there was a marked stimulation in the expression of CBP35. This was revealed both by an increase in the percentage of cells positively stained with anti-CBP35 under immunofluorescence and by an increase in the amount of the protein in immunoblots, as well as by an increase in the level of accumulated mRNA in Northern blots. The rise in the expression of CBP35 in proliferating cells was manifested most clearly in the nuclear fraction, with elevation in the levels of the nonphosphorylated (pI8.7) protein, as well as the phosphorylated (pI8.2) derivative. In contrast, older (passage 27-35, 55-68 cumulative population doublings) cultures of SL66 fibroblasts appear to have lost the normal proliferation-dependent regulation of CBP35 expression. The level of CBP35 was high in quiescent high-passage cells and decreased somewhat after serum stimulation. Furthermore, the unphosphorylated (pI 8.7) form of the lectin could not be detected in either the nucleus or the cytoplasm of high-passage SL66 cells. Finally, the level of the mRNA for CBP35 was high in quiescent cultures of high-passage cells, but undetectable 17 h after serum stimulation. These results establish that the expression of CBP35 becomes altered as human fibroblasts acquire reduced replicative capacity.

The goal of our research is to understand reciprocal relationships between cell function and tissue organization. We studied the regulation of fibroblast activity in an in vitro culture model that recapitulates in continuous fashion the cycle of events occurring during connective tissue repair. We present evidence that concomitant with spatial reorganization of the extracellular matrix, there was a dramatic decline in extracellular matrix synthesis and cell proliferation. Therefore, spatial reorganization was a crucial turning point for fibroblast activity. Factors that regulated the timing of spatial reorganization included serum, transforming growth factor beta, and fibronectin. By accelerating spatial reorganization of the cultures, transforming growth factor beta led to a relative decrease in cell proliferation and extracellular matrix synthesis. By retarding spatial reorganization of the cultures, fibronectin led to a relative increase in cell proliferation and extracellular matrix synthesis. The results indicate that spatial information in the three-dimensional cell-matrix interaction permits higher order, tissue-level regulation of fibroblast function.

In both experimental and spontaneous tumors, c-myc expression is often enhanced following its amplification or its rearrangement adjacent to a strong promotor/enhancer. However, c-myc by itself does not induce foci efficiently in fibroblast cultures. The effect of high levels of c-myc expression from a retroviral construct was investigated in several rodent fibroblast cell lines grown in medium containing 10% fetal calf serum or in serum-free PC-1 medium. c-myc-infected NIH3T3 clone 7 cells exhibited efficient quantitative focus formation when grown in PC-1 medium, whereas foci were not detected when grown in serum-supplemented medium. NIH3T3 clone 7 was the only cell line found to be sensitive to c-myc; other clones of NIH3T3 or other rodent fibroblast cell lines proved to be resistant to c-myc focus formation. At least two major types of morphologically distinct c-myc-induced foci were observed; the first was similar to ras-transformed foci induced in NIH3T3 and other fibroblast cell lines, and the second type was composed of adipocyte-like cells similar to NIH3T3 L1 cells. The c-myc infected cells cloned from these two types of foci expressed high levels of retrovirus-derived c-myc RNA and exhibited elevated levels of immunoreactive myc protein, as detected by immunofluorescent staining with an anti-myc polyclonal antibody. c-myc-transformed clones displayed only a limited ability to grow in soft-agar in the presence of serum and were not tumorigenic in nude mice. Focus formation by c-myc was quantitatively inhibited by the addition of interferon alpha + beta (INF alpha, beta), tumor necrosis factor alpha (TNF alpha) or transforming growth factor beta 1 (TGF beta 1) to the serum-free PC-1 medium, and, in correlation, NIH3T3 clone 7 cells produced the lowest level of endogenous TGF beta of the various cell lines tested.

The growth-stimulating activity of sera from 10 patients with von Recklinghausen neurofibromatosis (NF-1) was compared with that of sera from 10 age- and sex-matched healthy donors. Cell cultures derived from peripheral neurofibromas and from skin biopsies of healthy persons were used as indicator cells. Stimulation of 3H-thymidine incorporation increased with the clotting time of the blood samples, reaching a plateau at about 35 h with no decline of activity during the following 48 h. There was no difference between the stimulating activity of sera from the NF-1 patients and that of sera from the healthy donors with either type of indicator cells. This held true when the indicator cells were stimulated during the exponential growth phase or when serum-starved confluent cells were exposed to the various sera. Three NF-1 strains used as indicator cells during this investigation showed a significantly less vigorous response to both types of human sera and to fetal calf serum than the respective control strains. All human sera collected after 42 h clotting time were superior to fetal calf serum with both types of indicator cells.

Calf serum as well as rat and mouse sera has a factor that stimulates hyaluronic acid synthesis in cultured rat fibroblasts. Such a factor was partially purified from calf serum and characterized. It has a molecular weight of approximately 150,000. The activity of the factor is lost by treatment with pronase and by periodate oxidation. It is suggested, therefore, that the factor is a glycoprotein. Its susceptibility to alpha-mannosidase and affinity for Con A-Sepharose may suggest that the factor contains a mannose residue(s) which is essential for the activity to induce hyaluronic acid synthesis.

Multiplication-stimulating activity (MSA), a protein which stimulates DNA synthesis and growth of chicken embryo fibroblasts, was purified from serum-free medium conditioned by the growth of a rat liver cell line. Purified MSA was shown to rapidly stimulate ouabain-sensitive Na+, K+-ATPase activity as measured by both enzyme assay and rate of 86Rubidium uptake. Labeled ouabain binding was also shown to increase after stimulation of quiescent cells by serum or purified MSA. Conditions which interfere with the ability of the cells to accumulate potassium, such as the presence of the specific inhibitor, ouabain; incubation in potassium-free medium; or the presence of the potassium ionophore, valinomycin, were all demonstrated to inhibit the stimulation of DNA synthesis by serum or purified MSA. These results suggest that an early event in the stimulation of DNA synthesis by purified MSA is an activation of membrane Na+, K+-ATPase with a resulting accumulation of potassium ions inside the cell.

Previous work performed with mouse fibroblast-like L cells has shown that the level of expression of NGF gene is modulated in these transformed cells by the composition of the growth medium. Glucocorticoids were found to exert a down-regulation on NGF production, while serum stimulated the synthesis of the factor. The contrasting effects of serum and dexamethasone were further investigated in cultures of primary rat fibroblasts or in iris transplants. ELISA assays of NGF released by fibroblasts or by transplanted irides showed that both experimental systems responded to dexamethasone by a 4-5-fold decrease of the amounts of secreted factor. Half-maximal effect took place at a concentration of 3-5 X 10(-9) M, a value close to the dissociation constant of the glucocorticoid receptor in fibroblasts. The glucocorticoid did not influence the secretion of macromolecules. Assays of NGF mRNA performed at a concentration of 10(-7) M dexamethasone indicated that the steroid decreased the pool of NGF transcripts in either experimental systems. In contrast to dexamethasone, serum induced a 4-fold enhancement of the amounts of factor secreted by fibroblasts. This effect was reproduced with serum that was previously heat-treated at mild acidic pH, or with a macromolecular fraction of this heat-treated serum which contains an effector promoting NGF synthesis in L cells. The fact that promotion of NGF synthesis takes place in primary cells raises the possibility that this process may also occur in vivo, for instance following disruption of vasculature, as a part of a wound mechanism. Data collected with iris transplants provide some support to this interpretation.(ABSTRACT TRUNCATED AT 400 WORDS)

Normal human serum contains a 230,000 Mr protein that inhibits fibroblast chemotactic and random migration. This serum inhibitor of fibroblast migration (SIFM) is a heat-stable, trypsin-sensitive protein with a pI of 4.8 that reversibly inhibits the random and chemotactic migration of fibroblasts in vitro. Although SIFM effectively inhibits the chemotaxis of fibroblasts to interstitial collagens, fibronectin, lymphocyte-derived chemotactic factor for fibroblasts, and serum-derived chemotactic factor, it does not alter the chemotactic migration of human peripheral blood neutrophils or monocytes, and does not act as a cytotoxin to human dermal fibroblasts. The SIFM appears to act through a cell-directed mechanism to alter the fibroblast's ability to migrate. Serum inhibitor of fibroblast migration may function in vivo to modulate fibroblast migration under physiologic and pathologic conditions.