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At least 55 records · Page 3Linked to original sources

PubMed2006

Cell interactions between human gingival fibroblasts and monocytes stimulate the production of matrix metalloproteinase-1 in gingival fibroblasts.

BACKGROUND: Matrix metalloproteinase-1 (MMP-1) plays an important role in inflammatory diseases including periodontitis, which is characterized by tissue destruction and dense infiltration of mononuclear cells. OBJECTIVES: The aim of this study was to investigate the effect of cell interactions between human gingival fibroblasts and human monocytes on the production of MMP-1 in a coculture model. METHODS: The fibroblasts were cultured in either cell-to-cell contact with monocytes or in separated cocultures using a microporous membrane to prevent cell-to-cell contact. The mRNA expression of MMP-1 was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and the protein levels of MMP-1 in the cell medium were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: Coculturing gingival fibroblasts with monocytes in cell-to-cell contact increased the mRNA expression of MMP-1 in both fibroblasts and monocytes. The protein levels of MMP-1 increased in the culture media of the cocultures and correlated to the number of fibroblasts as well as to the number of monocytes. When fibroblasts were cultured with monocytes in separated cocultures, the mRNA expression and protein level of MMP-1 increased in the fibroblasts. In addition, treatment of fibroblasts with conditioned medium from monocytes also stimulated the production of MMP-1 in the fibroblasts. Moreover, the levels of the MMP-1 inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), increased in cocultures with cell-to-cell contact, but not in fibroblasts of separated cocultures. The glucocorticoid dexamethasone and the tetracycline doxycycline reduced the enhanced level of MMP-1 in the cocultures with cell-to-cell contact. CONCLUSION: The current study demonstrates that monocytes stimulate the production of MMP-1 in gingival fibroblasts by cell interactions, which may contribute to the maintenance of MMP-mediated tissue destruction in periodontitis.

Adolescent↗

PubMed1998

Continuity between wound macrophage and fibroblast phenotype: analysis of wound fibroblast phagocytosis.

Analysis of phagocytic activity in wound fibroblasts was chosen as a means to assess the possible continuity between macrophage and fibroblast phenotypes. Fibroblast phagocytosis of uncoated, IgG-coated, or collagen-coated fluorescent beads was analyzed by flow cytometry in vivo and in vitro. Phagocytosis of fluorescent beads by procollagen I-positive cells (fibroblasts) was evaluated in vivo by injecting beads into subcutaneously implanted sponge wounds in anesthetized Fisher rats. Phagocytic activity of a purified population of wound fibroblasts was measured in vitro and correlated with oxidation state using hydroethidium. In the wound environment, 50-60% of the cells that engulfed uncoated, IgG-coated, or collagen-coated beads were procollagen I-positive cells (i.e., fibroblasts). Procollagen I-positive cells engulfed uncoated and IgG-coated beads in preference to collagen-coated beads in vivo. Cultured wound fibroblasts engulfed uncoated, IgG-coated, and collagen-coated particles. The majority of fibroblasts that engulfed beads were in an elevated oxidation state. We conclude that substantial fibroblast phagocytosis occurs in the wound, but scavenger receptor-mediated fibroblast phagocytosis is different from that of macrophages. Additional markers will be helpful in defining the macrophage fibroblast continuum.

Animals↗

PubMed1988

Tumor necrosis factor interacts with interleukin-1 and interferons to inhibit fibroblast proliferation via fibroblast prostaglandin-dependent and -independent mechanisms.

Mononuclear cells are important regulators of fibroblast function. However, the soluble factors mediating these effects and the importance of intercytokine interactions in these regulating events remain poorly understood. We analyzed the effect of recombinant (r) interleukin-1-alpha (IL-1), tumor necrosis factor (TNF), and gamma, alpha, and beta 1 interferons (IFN), alone and in combination, on the proliferation of normal human lung fibroblasts. rIL-1 and rTNF weakly stimulated and the rIFNs inhibited fibroblast proliferation. Importantly, when rTNF was combined with rIL-1 or rIFN, synergistic inhibition of fibroblast proliferation was noted. The inhibition caused by rIFN-gamma plus rTNF was largely independent of fibroblast prostaglandin (PG) production because fibroblast PG levels were unaltered in the presence of these cytokines and blocking fibroblast PG production did not alter their inhibitory effect. In contrast, the inhibition caused by rIL-1 plus rTNF appeared to be at least partially mediated by fibroblast PG production because these cytokines acted synergistically to stimulate fibroblast PG production and blocking fibroblast PG production reversed the inhibition that they caused. These studies demonstrate that TNF can interact with IL-1 or IFN to inhibit the proliferation of normal diploid fibroblasts and that the inhibitory effects of these cytokine combinations are mediated by different mechanisms.

Cell Division↗

PubMed1978

Contribution of human somatomedin activity to the serum growth requirement of human skin fibroblasts and chick embryo fibroblasts in culture.

Although purified human somatomedins and related peptides have been shown to stimulate growth-related processes in cultured human skin fibroblasts, it is unknown whether the serum macromolecules required for the routine growth of human fibroblasts in culture include the somatomedins. To evaluate this question we have obtained sera from five GH-deficient patients before and after GH treatment and have compared these pre- and post-GH sera for their ability to stimulate [3H]thymidine incorporation into DNA and cell multiplication in the patients' own fibroblasts in culture. Subconfluent human fibroblasts demonstrated a dose-dependent increase in thymidine incorporation and cell number when exposed to human sera. Pre- and post-GH sera were equipotent. Partial purification of somatomedin activity by boiling sera at pH 5.5 diminished the level of thymidine incorporation to 10-20% of the level achieved with unboiled sera but did not unmask a difference between pre- and post-GH sera. In contrast to the results in human fibroblasts, boiled post-GH sera were 2- to 5-fold more potent than pre-GH sera in stimulating thymidine incorporation in chick embryo fibroblasts. Mixing experiments failed to demonstrate inhibition of thymidine incorporation by boiled pre-GH sera. Boiled post-GH sera also were twice as active as boiled pre-GH sera in stimulating multiplication of chick embryo fibroblasts and confirmed that thymidine incorporation reflected DNA synthesis. GH added directly to chick embryo fibroblasts did not stimulate thymidine incorporation. We conclude that despite the presence of specific receptors for somatomedin-like peptides on human skin fibroblasts, somatomedins are not a major component of the serum macromolecules required for the routine growth of human fibroblasts in culture. In contrast, in chick embryo fibroblasts, somatomedins do appear to constitute an important part of the serum macromolecules supporting cell growth.

Animals↗

PubMed2005

Growth kinetics and integrin expression of fibroblasts infiltrating devitalised patellar tendons are different from those of intrinsic fibroblasts.

We compared the biological characteristics of extrinsic fibroblasts infiltrating the patellar tendon with those of normal, intrinsic fibroblasts in the normal tendon in vitro. Infiltrative fibroblasts were isolated from the patellar tendons of rabbits six weeks after an in situ freeze-thaw treatment which killed the intrinsic fibroblasts. These intrinsic cells were also isolated from the patellar tendons of rabbits which had not been so treated. Proliferation and invasive migration into the patellar tendon was significantly slower for infiltrative fibroblasts than for normal tendon fibroblasts. Flow-cytometric analysis indicated that expression of alpha5beta1 integrin at the cell surface was significantly lower in infiltrative fibroblasts than in normal tendon fibroblasts. The findings suggest that cellular proliferation and invasive migration of fibroblasts into the patellar tendon after necrosis are inferior to those of the normal fibroblasts. The inferior intrinsic properties of infiltrative fibroblasts may contribute to a slow remodelling process in the grafted tendon after ligament reconstruction.

Animals↗

PubMed1994

Subsets of murine lung fibroblasts express membrane-bound and soluble IL-4 receptors. Role of IL-4 in enhancing fibroblast proliferation and collagen synthesis.

The purpose of this study was to determine whether or not membrane-bound and soluble forms of IL-4 receptors are expressed by isolated subsets of murine lung fibroblasts and to evaluate the potential functional consequences of IL-4 receptor triggering. Recent studies demonstrate that IL-4-synthesizing Th2 cells and mast cells are present in increased numbers in the lung during inflammation and fibrosis, suggesting that IL-4 may play a regulatory role in these events. We hypothesize that pulmonary fibroblasts and subsets thereof are intimately involved in this inflammatory response and that IL-4 is an active player in stimulating fibroblast collagen synthesis and hyperproliferation, creating a fibrotic environment in the lung. The fibroblast subsets used in these experiments differ not only in surface expression of the thymocyte-1 (Thy-1) Ag, but also in function and morphology. We now report the novel finding that IL-4 receptors are present at discordant levels on Thy-1+ and Thy-1- lung fibroblasts. IL-4R level and affinity were analyzed using a monoclonal anti-IL-4R Ab and equilibrium binding analysis with 125I-labeled IL-4. Reverse transcriptase PCR demonstrated the presence of mRNA for membrane-bound and soluble IL-4R. Lung fibroblast subsets secrete soluble IL-4R protein at dramatically different levels, as detected by an ELISA. Thy-1+ and Thy-1- lung fibroblasts were treated with IL-4 to determine whether this cytokine was profibrotic. Thy-1+ fibroblasts responded to IL-4 by proliferating and up-regulating collagen production. In contrast, Thy-1- fibroblasts proliferate to a lesser degree than Thy-1+ fibroblasts and were not stimulated to secrete increased levels of collagen. Overall, these results suggest that elevated levels of IL-4 at a site of injury could result in the development of fibrosis by enhancing fibroblast subset proliferation and collagen synthesis.

Animals↗

PubMed1996

Effect of human basic fibroblast growth factor on fibroblast proliferation, cell volume, collagen lattice contraction: in comparison with acidic type.

Human recombinant basic fibroblast growth factor (bFGF) is a potent mitogen for normal human dermal fibroblasts in the presence of heparin which binds to and stabilizes it. The optimal mitotic response is obtained with a concentration of 1 ng/ml of bFGF in monolayer cultures (non-differentiated fibroblasts) as well as in the better-differentiated fibroblasts obtained through the 'collagen lattices' culture system (fibroblasts embedded in a 3-dimensional collagen gel) achieving a doubling of the cell number in 8 days. Despite increasing the number of cells, bFGF decreases the ability of fibroblasts to contract collagen fibers. This inhibition is concentration-dependent and reaches a plateau at a dose of about 1 ng/ml. This effect is associated with a bFGF-induced decrease of fibroblast volume. Various dosing regimens indicate that although the highest response was obtained by daily dosing nearly optimal response was obtained either by early daily dosing or short intermittent treatment. Interestingly, the fibroblast mitotic response to bFGF decreases steadily when fibroblasts mature in collagen gels. The mitogenic properties of bFGF associated to its ability to inhibit fibroblasts contraction, if demonstrated in vivo, may be of interest in the management of wound healing.

Cell Division↗

PubMed1996

Interaction of substance P with epidermal growth factor and fibroblast growth factor in cyclooxygenase-dependent proliferation of human skin fibroblasts.

Substance P (SP), fibroblast growth factor (FGF), and epidermal growth factor (EGF) are mitogens for fibroblasts. EGF acts as a progression factor, whereas FGF and SP have competence factor activity. The ability of eicosanoids to regulate proliferation of fibroblasts and the increased production of prostaglandins by fibroblasts in response to the growth factors, led us to investigate the involvement of cyclooxygenase-dependent arachidonic acid metabolites in the mitogenic response of serum-starved human skin fibroblasts to SP, FGF, and EGF. We tested the interaction of a submaximal concentration of SP(10(-9)M) with baFGF(40 micrograms/ml) and EGF(0.01 microgram/ml) both on fibroblast proliferation and release of arachidonic acid metabolites. A combination of SP and EGF synergistically stimulated fibroblast proliferation and prostaglandin E2 release, whereas addition of SP to FGF-containing cultures did not affect cell growth. Inhibition of cyclooxygenase by acetylsalicylic acid augmented the growth response of fibroblasts to all: SP, FGF, and EGF. In the presence of acetylsalicylic acid, SP combined with FGF enhanced fibroblasts proliferation, whereas a combination with EGF inhibited cellular growth with respect to growth induced by EGF alone. Thus, interactions of SP with FGF and EGF differently affected the mitogenic response depending on the formation of arachidonic acid metabolites. The findings indicate that eicosanoids may be important mediators of competence and progression factor activities that may determine the effects of substance P on fibroblast proliferation in a cytokine network.

Animals↗

PubMed1995

Co-existence of NADPH-diaphorase, fibroblast growth factor-2 and fibroblast growth factor receptor in spinal autonomic system suggests target-specific actions.

In the rat spinal cord, we found substantial co-existence of fibroblast growth factor-2, fibroblast growth factor receptor (type-1 or flg) immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity (a histochemical marker for neuronal nitric oxide synthase) in preganglionic autonomic cell groups of intermediate layers VI, VII and X. Anti-fibroblast growth factor-2 and anti-nitric oxide synthase binding sites were confined to the cytoplasm of reactive neurons as judged by immunogold electron microscopy. Within the major autonomic nucleus, i.e. intermediolateral column, three different populations were identified: (i) fibroblast growth factor and fibroblast growth factor receptor, (ii) fibroblast growth factor/NADPH-diaphorase and (iii) NADPH-diaphorase-only stained cell groups. Sympathoadrenal neurons were prelabelled with fluorescent tracer Fast Blue and co-stained for fibroblast growth factor-like protein and NADPH-diaphorase, suggesting heterologous diversification of neuronal phenotypes and functional organization in the spinal autonomic system. Our findings suggest intriguing roles for nitric oxide and fibroblast growth factor-2 cytokine in the preganglionic sympathetic spinal cord system: The "short-term" diffusible messenger nitric oxide may act as "tonic" and/or "phasic" signal within rostrocaudally oriented function-specific preganglionic units necessary for integrated target control. The "long-term" messenger fibroblast growth factor-2 may be involved in, for example, cytokine-dependent regulation of neuronal NADPH-diaphorase/nitric oxide synthase. Furthermore, co-existence of NADPH-diaphorase, fibroblast growth factor-2 and receptor in sympathoadrenal neurons suggest mutual target-specific regulatory functions, e.g. hormone release and blood perfusion or maintenance of phenotype and plasticity responsiveness of adrenal medullary tissue.

Animals↗

PubMed1999

Cell-to-cell communication and expression of gap junctional proteins in human diabetic and nondiabetic skin fibroblasts: effects of basic fibroblast growth factor.

Wound healing involves the interactions of many cell types, and is controlled in part by growth factors. Intercellular communication mediated by gap junctions is considered to play an important role in the coordination of cellular metabolism duringthe growth and development of tissues and organs. Basic fibroblast growth factor (bFGF), known to be important in wound healing, has been found to increase Cx43 expression and intercellular communication in endothelial cells and cardiac fibroblasts. It has been proposed that an increased coupling is necessary for the coordination of these cells in wound healing and angiogenesis, and that one of the actions of bFGF is to modulate intercellular communication. The aim of our study was to evaluate the effects of bFGF on gap junctional intercellular communication (GJIC) in vitro, and the presence of gap junctional proteins connexin (Cx) 26, Cx32, and Cx43 in fibroblasts of diabetic and nondiabetic individuals. Fibroblast cell lines (n = 10) were cultured for 3 d in serum-free media with or without bFGF (3 ng/mL). Cells were evaluated for the rate of GJIC by using laser cytometry, and for the presence of Cx26, Cx32, and Cx43 by immunohistochemical and Western analyses. All cell types communicated via contact-dependent mechanisms. The rate of GJIC was greater (p < 0.01) for diabetic than for nondiabetic fibroblasts (4.1 +/- 0.01 vs 3.3 +/- 0.01%/min). bFGF increased (p < 0.01) the rate of GJIC for diabetic (4.9 +/- 0.01 vs 4.1 +/- 0.01%) and nondiabetic (4.1 +/- 0.01 vs 3.3 +/- 0.01%) fibroblasts. Immunohistochemistry identified Cx26 in the cytoplasm, Cx32 was not detected, and Cx43 was present on the cellular borders in all cultures. Image analysis of immunofluorescent staining demonstrated that bFGF increased (p < 0.05) Cx43 expression in diabetic and nondiabetic fibroblasts. Western immunoblot analysis revealed bands at 43-46 kD that were similar in volume for diabetic and nondiabetic fibroblasts. Thus, gap junctions involving Cx43 and GJIC among fibroblasts appear to be targets for bFGF. Fibroblasts of diabetic individuals appear to have an increased rate of cell-cell coupling, correlating with a decreased rate of proliferation.

Adult↗

PubMed2000

Increased expression of IGF-binding protein-5 in Duchenne muscular dystrophy (DMD) fibroblasts correlates with the fibroblast-induced downregulation of DMD myoblast growth: an in vitro analysis.

In DMD the progressive loss of muscle ability and concomitant increasing fibrosis might originate from, besides other causes, the fibroblast paracrine inhibition of satellite cell "growth." In this study we report that in myoblast/fibroblast coculture experiments, the presence of DMD fibroblasts negatively interfered with DMD myoblast growth to an extent directly proportional to the percentage of DMD fibroblasts present in the mixed-cell cultures. Moreover, the observation that media conditioned with proliferating DMD fibroblasts inhibited the growth of DMD myoblasts more seriously than did control fibroblast-conditioned media suggested a paracrine effect by diffusible factors. IGF-binding proteins could act as such diffusible factors; in fact, IGFBP-5 transcript increased threefold in DMD fibroblasts proliferating in DMD muscle extracts, whereas IGFBP-3 mRNA decreased. In addition, high levels of IGFBP-5 protein were detected in DMD fibroblast-conditioned media. In neutralizing IGFBP-5 in DMD fibroblast-conditioned media by means of specific antibodies, or inhibiting IGFBP-5 gene expression in DMD fibroblasts by means of oligo antisense, the fibroblast-conditioned media lost inhibitory power over DMD myoblast proliferation.

Cell Communication↗

PubMed2003

Increased CD40 expression in skin fibroblasts from patients with systemic sclerosis (SSc): role of CD40-CD154 in the phenotype of SSc fibroblasts.

Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology that is characterized by tissue fibrosis, which may result from the activation of lesional fibroblasts exhibiting excessive production of extracellular matrix components. However, it has yet to be determined how SSc fibroblasts are activated. CD40 is a cell surface molecule expressed on various cells that is important for the response to activated T cells through CD154. CD40 mRNA was found to be constitutively expressed in both SSc and normal fibroblasts by reverse transcription PCR. Expression of CD40 protein was increased on SSc fibroblasts compared to normal fibroblasts as measured by flow cytometry. Ligation of CD40 by recombinant human CD154 (0.5-2 microg/ml) resulted in increased production of IL-6, IL-8, and monocyte chemoattractant protein-1 in SSc fibroblasts in a dose-dependent manner, whereas these phenomena were not shown in normal fibroblasts even with the addition of CD154. CD80, a costimulatory molecule, was also induced on SSc fibroblasts by CD40 ligation. In the present study, our findings suggest the possibility of a cell-mediated response between fibroblasts and T cells in the lesional skin of SSc patients. Since it is suggested that the CD40-CD154 system may play a crucial role in the aberrant production of immune mediators by SSc fibroblasts, blockage of CD40-CD154 may be a novel therapeutic strategy for SSc.

Adult↗

PubMed1995

Differential TNF secretion by wound fibroblasts compared to normal fibroblasts in response to LPS.

Fibroblasts cultured from wound sites have been shown to have an altered phenotype compared to normal dermal fibroblasts and are generally regarded as target cells of the cytokine response at sites of injury. This study was undertaken to determine whether wound fibroblasts can contribute to proinflammatory cytokine production in wounds and, in particular, whether they are capable of secreting TNF. Wound fibroblasts were cultured from polyvinyl alcohol sponges implanted subcutaneously for 2 weeks in Balb/c mice. Fibroblasts harvested from the skin and subcutaneous tissue of untreated mice served as a control population of cells. All cells were passaged at least twice and then stimulated with a dose range of LPS. Supernatants were harvested 8 hr following stimulation and TNF was assayed using a standard L929 cell-killing assay. There was a significant TNF response to LPS by wound fibroblasts, evident as early as 4 hr following exposure to LPS and associated with an upregulation of TNF mRNA. Normal dermal fibroblasts did not secrete any measurable amounts of TNF in response to LPS. The results indicate that wound fibroblasts generate a brisk TNF response to stimulation with LPS, in contrast to normal subcutaneous fibroblasts. These data reveal an additional unique property of wound-harvested fibroblasts and suggest a possible contributing mechanism to disordered wound healing in the face of infection or conditions characterized by excessive fibrosis.

Animals↗

PubMed1999

Psoriatic fibroblasts secrete lower amounts of IL-6 than healthy fibroblasts before and after stimulation with TNF-alpha.

Altered function of the fibroblasts is thought to contribute to the pathogenesis of psoriasis. To further elucidate this point, we compared the ability of fibroblasts from psoriatic lesions and of fibroblasts from healthy individuals to produce interleukin-6 (IL-6). The IL-6 levels were measured by ELISA in serum-free culture medium before and after stimulation of monolayer fibroblasts with various concentrations of tumour necrosis factor-alpha (TNF-alpha), platelet-derived growth factor (PDGF), and interferon-gamma (IFN-gamma), alone and in different combinations. The production of IL-6 in the fibroblast cultures was stimulated by TNF-alpha (0.01-10 nm/ml medium) in a dose-dependent way. Fibroblasts from psoriatic lesions produced lower amounts of IL-6 than fibroblasts from healthy individuals both before and after stimulation with the different concentrations of TNF-alpha (P = 0.012). The ratios between the IL-6 concentrations before and after stimulation with TNF-alpha were similar in both types of fibroblasts, indicating that the capacity to produce IL-6 is reduced in psoriatic fibroblasts compared with healthy ones. The production of IL-6 was not influenced by either PDGF or IFN-gamma. These findings support the view that the phenotype of the fibroblast is altered in psoriasis.

Adult↗

PubMed2005

Eosinophil-fibroblast interactions induce fibroblast IL-6 secretion and extracellular matrix gene expression: implications in fibrogenesis.

BACKGROUND: Eosinophils are frequently associated with tissue remodeling and fibrosis in allergic and other diseases and animal models. Their close physical proximity to fibroblasts at sites of tissue remodeling strongly implicates them in fibrogenesis, including subepithelial fibrosis and airway remodeling characteristic of asthma. OBJECTIVE: To identify the mediators and characterize the mechanisms underlying the fibrogenic activities of eosinophils. METHODS: A coculture system of blood eosinophils or eosinophil cell lines with normal fibroblasts was used to assess their ability to induce a fibrogenic fibroblast phenotype, including IL-6 secretion and mRNA expression, and induction of genes involved in extracellular matrix production and homeostasis. The mediators of these responses were identified by using transwell barrier cocultures, eosinophil-conditioned media, and cytokine-specific antibody neutralization. RESULTS: Eosinophil-fibroblast coculture induced potent fibroblast IL-6 secretion and mRNA expression, responses further enhanced by IL-5. The soluble nature of the eosinophil-derived mediators was demonstrated by using eosinophil-fibroblast coculture in the presence of permeable transwell barriers, and fibroblast culture in eosinophil-conditioned media, indicating that cell contact was not required. Induction of fibroblast IL-6 expression was accompanied by increased expression of fibronectin and the extracellular matrix regulatory genes plasminogen activator inhibitor 1 and tissue inhibitor of metalloproteinase 1. Antibody neutralization identified the principal eosinophil-derived mediator of fibroblast IL-6 expression as IL-1beta (>60%), with lesser contributions from IL-1alpha, IL-4, and TGF-beta (10% to 20%). CONCLUSION: Eosinophils express at least 2 potent mediators (IL-1beta and TGF-beta) that induce a fibrogenic fibroblast phenotype, strongly supporting a role for the eosinophil in the dysregulation of extracellular matrix homeostasis and consequent tissue remodeling and fibrosis in eosinophil-associated diseases.

Cell Communication↗

PubMed2002

Sustained ability for fibroblast outgrowth from stored neonatal foreskin: a model for studying mechanisms of fibroblast outgrowth.

Fibroblast cultures derived from neonatal foreskin explants have been an important model for understanding the basic mechanisms of fibroblast function and activity. Neonatal foreskin has been routinely used for establishing such fibroblast cultures in vitro. In general, tissue explants and fibroblast cultures are established from freshly harvested neonatal foreskin tissue. It is unknown whether prolonged storage of tissue, even in optimal growth media and conditions would still result in successful explant outgrowth. Specifically, no studies have properly examined the maximal duration of foreskin storage that can produce viable fibroblasts with normal growth and synthetic characteristics. We, therefore, set out to isolate and propagate fibroblast cultures from neonatal foreskin tissue that had been kept in complete culture media at 4 degrees C for various periods of time. We found that outgrowth and propagation of viable fibroblasts in vitro occurred with foreskin samples obtained within 24 days after surgical harvesting. The morphology, cellular proliferative capacity (measured by [3H]-thymidine uptake), steady state levels of alpha1(I) procollagen mRNA, and collagenous protein synthesis were comparable among fibroblast cultures derived from foreskin explants established 3 days (control) and up to 24 days (but not 38 days) after tissue harvesting. These studies demonstrate that viable and functional fibroblasts, with apparently similar in vitro characteristics, can be isolated and propagated from explants of neonatal foreskin tissue that have undergone prolonged storage. Moreover, these findings may be useful in understanding the lack of fibroblast growth in such conditions as found in delayed wound healing and aging.

Cell Division↗

PubMed2000

Response of periodontal ligament fibroblasts and gingival fibroblasts to pulsating fluid flow: nitric oxide and prostaglandin E2 release and expression of tissue non-specific alkaline phosphatase activity.

The capacity of the periodontal ligament to alter its structure and mass in response to mechanical loading has long been recognized. However, the mechanism by which periodontal cells can detect physical forces and respond to them is largely unknown. Besides transmission of forces via cell-matrix or cell-cell interactions, the strain-derived flow of interstitial fluid through the periodontal ligament may mechanically activate the periodontal cells, as well as ensure transport of cell signaling molecules, nutrients and waste products. Mechanosensory cells, such as endothelial and bone cells, are reported to respond to a flow of fluid with stimulated prostaglandin E2 (PGE2) and nitric oxide production. Therefore, we examined the PGE2 and nitric oxide response of human periodontal ligament and gingival fibroblasts to pulsating fluid flow and assessed the expression of tissue non-specific alkaline phosphatase activity. Periodontal ligament and gingival fibroblasts were subjected to a pulsating fluid flow (0.7 +/- 0.02 Pa, 5 Hz) for 60 min. PGE2 and nitric oxide concentrations were determined in the conditioned medium after 5, 10, 30 and 60 min of flowing. After fluid flow the cells were cultured for another 60 min without mechanical stress. Periodontal ligament fibroblasts, but not gingival fibroblasts, responded to fluid flow with significantly elevated release of nitric oxide and decreased expression of tissue non-specific alkaline phosphatase activity. In both periodontal ligament and gingival fibroblasts, PGE2 production was significantly increased after 60 min of flowing. Periodontal ligament fibroblasts, but not gingival fibroblasts, produced significantly higher levels of PGE2 during the postflow culture period. We conclude that human periodontal ligament fibroblasts are more responsive to pulsating fluid flow than gingival fibroblasts. The similarity of the early nitric oxide and PGE2 responses to fluid flow in periodontal fibroblasts with bone cells and endothelial cells suggests that these three cell types possess a similar sensor system for fluid shear stress.

Adolescent↗

PubMed2000

Modification of mitochondrial metabolism in fibroblasts from mice with a skeletal muscle mutation (muscular dysgenesis). Evidence of embryonic communication between myoblasts and fibroblasts.

Muscle development during embryogenesis is a complex process involving many mechanisms. It requires a close communication among the different cellular types of the muscle, especially the fibroblasts and myoblasts. Indeed, any abnormality in one cell type might influence the differentiation of the other. Thus, any disturbance altering the metabolism of the myoblasts might lead to modifications in the fibroblasts. To study this phenomenon, we used the dysgenic mouse (mdg-"muscular dysgenesis") carrying a homozygous recessive lethal mutation expressed only in skeletal muscle cells. First, we found that fibroblasts isolated from such mutant muscle (and not from mutant skin tissue) and grown in culture exhibited an altered metabolism. Secondly, muscle fibroblasts showed a lower capacity for proliferation. We also observed that respiration and ATP synthesis of dysgenic muscle fibroblasts were deficient, while respiratory chain enzymatic activities were normal. Finally, intracellular [Ca2+] levels of dysgenic fibroblasts are 50% of those of normal fibroblasts. These results support the hypothesis that certain characteristics of fibroblasts are determined by the surrounding cellular environment during embryonic organogenesis, and that such modifications are stable when the fibroblasts are isolated in vitro. Since fibroblast differentiation was disrupted permanently, this suggests, in the case of myopathies, that the modified cells, surrounding the muscle tissue, could contribute to the muscle pathology. Synergistic activities of this type should be considered when studying the course of pathologies in different types of muscle diseases.

Adenosine Triphosphate↗