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PubMed1988

A defective cell surface collagen-binding protein in dermatosparactic sheep fibroblasts.

Fibroblasts from dermatosparactic sheep fail to contract collagen gels and show a reduced attachment to collagenous substrates. By comparing collagen-binding membrane proteins of normal (+/+), homozygote (-/-), and heterozygote (+/-) fibroblasts, we present evidence that the interaction of normal fibroblasts with native type I collagen involves a protein of apparent Mr = 34,000 which is absent from dermatosparactic fibroblasts and seems to be related to anchorin CII. This conclusion was reached from the following experiments: (a) On a blot of membrane proteins from normal fibroblasts radioactively labeled type I collagen bound predominantly to a protein band of 34 kD; dermatosparactic membranes revealed only a small amount of binding to a component with a molecular mass of 47 kD. (b) After separation of normal fibroblast membrane proteins on type I collagen-Sepharose, a collagen-binding component of 34 kD was found which was absent from the corresponding fraction of dermatosparactic membranes. (c) Antibodies to anchorin CII stained the surface of normal (+/+), but not of dermatosparactic (-/-) fibroblasts and labeled a 34-kD component after immunoblotting of normal fibroblast membrane proteins. (d) After metabolic labeling of fibroblasts with [35S]methionine and immunoprecipitation with anti-anchorin CII, 40- and 34-kD components were precipitated from extracts of normal fibroblasts, while the latter component was absent from affected cells. Similar differences were found after immunoblotting of membranes from whole normal or affected skin. These data indicate that dermatosparaxis of sheep involves a molecular defect of a collagen-binding protein. Therefore this disease represents a model to study the complex interaction of cells with the extracellular matrix on a molecular level.

Animals↗
PubMed1981

Serum-free growth of normal and transformed fibroblasts in milk: differential requirements for fibronectin.

Bovine milk may be used as a supplement for the serum-free growth of certain fibroblastic cells in culture. The growth properties of three representative cell types in milk-supplemented medium were examined; fibroblastic cell strains, fibroblastic cell lines, and transformed fibroblasts. Transformed fibroblasts, which included RNA and DNA tumor virus-transformed cells and carcinogen-transformed cells, grew in milk. Instead of growing attached to the culture dishes, as they normally do in serum, transformed fibroblasts grew in milk as large clusters in suspension. In contrast, nontransformed fibroblastic cell strains and cell lines did not grow in milk-supplemented medium. Fibroblasts transformed by a temperature-sensitive transformation mutant of Rous sarcoma virus were temperature-sensitive for growth in milk. The failure of cells to adhere to the substratum in milk-supplemented medium suggested that milk might be deficient in attachment factors for fibroblasts. When the attachment of fibroblastic cells in milk-supplemented medium was facilitated by pretreating culture dishes with fibronectin, (a) transformed cells grew attached rather than in suspension, (b) normal cell lines attached and grew to confluence, and (c) normal cell strains adhered and survived but did not exhibit appreciable cell proliferation.

Animals↗
PubMed1983

Role of fibronectin as a growth factor for fibroblasts.

Fibroblast replication is regulated by exogenous signals provided by growth factors, mediators that interact with the target cell surface and signal the cell to proliferate. A useful model of growth regulation, the "dual control model," suggests that growth factors can be grouped either as competence factors or as progression factors, and that optimal replication of fibroblasts requires the presence of both types of growth factors. Although most growth factors are soluble mediators, recent studies have demonstrated that, for some cell types, the extracellular matrix can replace the requirement for a competence factor. Since fibronectin is an important constituent of the extracellular matrix that interacts with specific domains on the fibroblast surface, we examined the ability of fibronectin to act as a competence factor to promote the growth of human diploid fibroblasts. To accomplish this, fibronectins purified from two sources, human plasma and human alveolar macrophages, were tested for their ability to (a) stimulate fibroblast replication in serum-free medium containing characterized progression factors (insulin or alveolar macrophage-derived growth factor); (b) provide a growth-promoting signal early in G1. Fibronectin stimulated fibroblast replication in a dose-dependent manner in the presence of a fixed dose of a progression factor. Conversely, fibronectin conferred on previously unresponsive fibroblasts the ability to replicate in a dose-dependent manner when cultured with increasing amounts of a progression factor. Moreover, fibronectin signaled growth-arrested fibroblasts to traverse G1 approximately 4 h closer to S phase. No differences were observed in the ability of plasma or macrophage fibronectins to provide a competence signal for fibroblast replication. Since fibronectin is a major component of the extracellular matrix, these observations suggest that it may provide at least one of the signals by which the matrix conveys the "competence" that permits fibroblasts to replicate in the presence of an appropriate progression signal.

Cell Cycle↗
PubMed2003

Engineering of a dermal equivalent: seeding and culturing fibroblasts in PEGT/PBT copolymer scaffolds.

The engineering of dermal skin substitutes, using autologous fibroblasts, requires high seeding efficiencies, a homogeneous cell distribution in the scaffolds, and optimal culture conditions. Dynamic seeding in spinner flasks was used to seed and subsequently culture fibroblasts in three-dimensional scaffolds. Several seeding and culture variables were investigated. Simulation of medium movement with microspheres showed that three different regions existed in medium (outer, middle, and inner), where overall particle movement was different. In the middle region the flow was turbulent and scaffolds were best placed in this region. After fibroblast seeding, methylene blue staining and scanning electron microscopy analysis of the scaffolds showed that at a low stirring speed (20 rpm) fibroblasts attached mainly onto the upper part of the scaffold, and at 40 and 60 rpm fibroblasts attached and spread throughout the scaffolds. Measurements of total DNA content per scaffold showed that lower stirring speeds (20 and 40 rpm) resulted in significantly higher cell-seeding efficiencies (20 rpm, 99.8 +/- 11.3%; 40 rpm, 93.8 +/- 10.5%) compared with 60 rpm (85.9 +/- 5.3%). Seeding kinetics were comparable for all three speeds investigated. In subsequent studies, 40 rpm was chosen for seeding. Using initial cell numbers ranging from 0.3 x 10(6) to 1.5 x 10(6) fibroblasts per scaffold, seeding efficiencies higher than 85% were consistently found (n = 4). The culture of fibroblast-seeded scaffolds at different stirring speeds (10-80 rpm) showed that stirring speeds higher than 10 rpm significantly stimulated fibroblast proliferation and glycosaminoglycan and collagen deposition as compared with 10 rpm. After 21 days, scaffolds cultured at 80 rpm showed significantly more collagen deposition as compared with those maintained at lower speeds. In conclusion, to achieve high seeding efficiencies, uniform fibroblast distribution and tissue formation in a three-dimensional scaffold, fibroblasts can be dynamically seeded at 40 rpm and subsequently cultured at a stirring speed of 60-80 rpm in spinner flasks. This flexible system shows that it is feasible to tissue engineer autologous dermal substitutes in a clinically acceptable time frame.

Cell Culture Techniques↗
PubMed2005

Immunosuppression with either cyclosporine a or FK506 supports survival of transplanted fibroblasts and promotes growth of host axons into the transplant after spinal cord injury.

Fibroblasts that have been genetically modified to secrete neurotrophins can stimulate axonal regeneration, rescue injured neurons, and improve function when grafted into a spinal cord injury site. These grafts are usually allografts that require immunosuppression to prevent rejection. In this study, we compared the effects of two immunophilin-ligands (cyclosporine A [CsA] and FK506) that are used clinically to prevent transplant rejection on protection of grafted fibroblasts. As there are risks associated with prolonged immunosuppression, we compared the effects of 2 or 8 weeks of administration of these drugs, in combination with our standard methylprednisolone protocol, in animals that survived for 8 weeks, to determine whether a shorter course of immunosuppression would be effective. Outcome measures included fibroblast survival, infiltration of activated macrophages and microglia into the graft, final lesion size, and growth of host axons into the graft. The graft consisted of a Vitrogen matrix into which fibroblasts were suspended; the graft was placed into a C3/C4 lateral funiculus lesion. The fibroblasts were isolated from a transgenic strain of Fischer rats that produce the marker alkaline phosphatase (Fb/AP). This enabled us to track the grafted fibroblasts and to evaluate the extent of their survival. The grafted matrix filled the lesion cavity. The density of fibroblasts within the matrix differed according to treatment. Fibroblast survival was most robust in animals that received 8 weeks of immunophilin-ligand treatment. FK506 supported greater Fb/AP survival than CsA. ED-1 immunostaining for activated microglia and macrophages showed an inverse correlation between AP immunoreactivity and the density of immune cells within the graft. Thus, prolonged administration of either FK506 or CsA was necessary for maximal fibroblast survival and for limiting the macrophage invasion of the graft. None of the FK506 or CsA protocols modified the size of the lesion, indicating that these immunophilin-ligands had little effect on secondary enlargement of the lesion and therefore little neuroprotective effect. Because immunophilin-ligands have been shown to be neurotrophic, we used RT-97 immunostaining for neurofilaments and calcitonin gene related protein (CGRP) staining for dorsal root axons to visualize axons that grew into the graft. Some axons grew into the matrix even in the absence of immunophilin-ligand treatment, suggesting that the Vitrogen matrix itself is permissive, but all of the immunophilin-ligand protocols were much more effective in eliciting axonal growth. Growth of axons into the transplants was equally increased by drug treatment for 2 or 8 weeks. Thus, both treatments improved fibroblast survival, diminished immune cell invasion, and promoted axonal growth, and a 2-week course of treatment with either immunophilin-ligand was as effective as 8 weeks in stimulating axonal growth.

Animals↗
PubMed2006

Repair of tendon defect with dermal fibroblast engineered tendon in a porcine model.

Harvesting autologous tenocytes for tendon engineering may cause secondary tendon defect at the donor site. Dermal fibroblasts are an easily accessible cell source and do not cause major donor site defect. This study aims to explore the possibility of tendon engineering using dermal fibroblasts. A total of 45 hybrid pigs were randomly divided into three groups: experimental group (n = 15)--repair of tendon defect with a dermal fibroblast engineered tendon; control group 1 (n = 15)--repair of defect with a tenocyte engineered tendon; and control group 2 (n = 15)-repair of defect with a scaffold alone. Both autologous dermal fibroblasts and tenocytes were seeded on polyglycolic acid (PGA) unwoven fibers to form a cell-scaffold construct and cultured in vitro for 7 days before in vivo implantation to repair a defect of flexor digital superficial tendon. Specimens were harvested at weeks 6, 14, and 26 for gross, histological, and mechanical analyses. Microscopy revealed good attachment of both dermal fibroblasts and tenocytes on PGA fibers and matrix production. In vivo results showed that fibroblast and tenocyte engineered tendons were similar to each other in their gross view, histology, and tensile strength. At 6 weeks, parallel collagen alignment was observed at both ends, but not in the middle in histology, with more cellular components than natural tendons. At weeks 14 and 26, both engineered tendons exhibited histology similar to that of natural tendon. Collagens became parallel throughout the tendon structure, and PGA fibers were completely degraded. Interestingly, dermal fibroblast and tenocyte engineered tendons did not express type III collagen at 26 weeks, which remained observable in normal pig skin and control group 2 tissue using polarized microscopy, suggesting a possible phenotype change of implanted dermal fibroblasts. Furthermore, both fibroblast and tenocyte engineered tendons shared similar tensile strength, about 75% of natural tendon strength. At 6 weeks in control group 2, neo-tissue was formed only at the peripheral area by host cells. A cord-like tissue was formed at weeks 14 and 26. However, the formed tissue was histologically disorganized and mechanically weaker than both cell-engineered tendons (p < 0.05). These results suggest that dermal fibroblasts may have the potential as seed cells for tendon engineering.

Absorbable Implants↗
PubMed1993

Different stability of AP1 proteins in human keratinocyte and fibroblast cells: possible role in the cell-type specific expression of human papillomavirus type 18 genes.

Human papillomaviruses (HPV) replicate in keratinocyte but not fibroblast cells. Several factors, including AP1 (Jun/Fos), contribute to the cell-type specific transcription of HPV genes. The binding of AP1 upstream of the HPV type 18 early gene E6 is essential for transcription of the early genes. Here we show that AP1 levels are low in early passage human fibroblast extracts. In contrast, human keratinocyte extracts contain high levels of AP1. In agreement with this, in vivo an AP1-dependent promoter is more active in keratinocytes than in fibroblasts. Pulse chase experiments indicated that Jun and Fos are relatively stable in human keratinocyte cells after serum induction, whereas in early passage human fibroblasts they are rapidly broken down. Nuclear extracts of these fibroblasts contain a cysteine proteinase which can degrade AP1. Furthermore, the activity of a cathepsin B-like cysteine proteinase is elevated in these human fibroblast extracts relative to other cell types. Interestingly, after several passages in culture the fibroblasts lose this proteinase activity and the amount of AP1 increases. Taken together, these results suggest that the quantitative difference in AP1 proteins between human keratinocytes and fibroblasts is due to a difference in protein stability. The cathepsin B-like cysteine proteinase is a candidate for a role in the unusually rapid breakdown of AP1 in early passage human fibroblast cells. Low levels of AP1 in the fibroblasts correlate with the low activity of AP1-dependent promoters, like that of HPV-18, in these cells.

Adult↗
PubMed1998

Analysis of cartilage oligomeric matrix protein (COMP) in synovial fibroblasts and synovial fluids.

We investigated the expression of cartilage oligomeric matrix protein (COMP) in normal and rheumatoid arthritis (RA) synovial fibroblasts. In situ hybridization (ISH) was conducted on synovial specimens from five RA patients applying specific probes for COMP or fibroblast collagen type I. ISH was combined with immunohistochemistry, applying antibodies to the macrophage marker CD68. Ribonuclease protection assay (RPA) and rapid amplification of 3'-cDNA ends (3'-RACE) were performed on total RNA from normal and RA synovial fibroblast cultures. Protein extracts from fibroblasts and culture supernatants were compared with synovial fluids and protein extracts from isolated chondrocytes by Western blot utilizing polyclonal and monoclonal antibodies (18-G3 mAb) to COMP. COMP mRNA was detected in fibroblasts of RA synovium by ISH, and in normal and RA synovial fibroblast cultures by RPA. 3'-RACE demonstrated sequence homology of chondrocyte and synovial fibroblast COMP along the coding sequence. COMP protein was detected in synovial fibroblasts and culture supernatants by immunoblot. Using polyclonal antibodies, the major portion of COMP from fibroblasts and culture supernatants was present as low-molecular-weight (LMW) bands, corresponding to those found in synovial fluids. These LMW COMP bands, however, were not detected in any of the cells or tissues tested using 18-G3 mAb. In protein extracts from chondrocytes and in COMP purified from cartilage, these LMW bands could not be detected. In conclusion, the data suggest that certain forms of COMP detected in synovial fluid are secreted from synovial fibroblasts and could be distinguished by specific mAbs from COMP secreted by chondrocytes.

Arthritis, Rheumatoid↗
PubMed1998

Potential role of fibroblast growth factor in enhancement of fracture healing.

Fibroblast growth factors are present in significant amounts in bone and several studies have suggested that they may be involved in normal fracture healing. It is well established that fibroblast growth factors have mitogenic and angiogenic activity on mesoderm and neuroectoderm derived cells. Of particular interest as a member of the fibroblast growth factor family, basic fibroblast growth factor stimulates mitogenesis, chemotaxis, differentiation, and angiogenesis. It also plays an important role in the development of vascular, nervous, and skeletal systems, promotes the maintenance and survival of certain tissues, and stimulates wound healing and tissue repair. Animal studies have shown that the direct injection of fibroblast growth factor into fresh fractures stimulates callus formation, which provides mechanical stability to the fracture, accelerates healing, and restores competence. The matrix used to present the fibroblast growth factor at the fracture site plays a critical role in the effectiveness of the treatment. The evaluation of injectable basic fibroblast growth factor in a sodium hyaluronate gel for its effectiveness in stimulating fracture healing is described. When applied directly into a freshly created fracture in the rabbit fibula, a single injection of the basic fibroblast growth factor and hyaluronan results in the stimulation of callus formation, increased bone formation, and earlier restoration of mechanical strength at the fracture site. The hyaluronan gel serves as a reservoir that sequesters the basic fibroblast growth factor at the injection site for the length of time necessary to create an environment conducive to fracture healing. It is concluded that basic fibroblast growth factor and sodium hyaluronate act synergistically to accelerate fracture healing and that the combination is suitable for clinical evaluation as a therapy in fracture treatment.

Adjuvants, Immunologic↗
PubMed2004

Anoxia-reoxygenation stimulates collagen type-I and MMP-1 expression in cardiac fibroblasts: modulation by the PPAR-gamma ligand pioglitazone.

BACKGROUND: Cardiac remodeling after ischemic injury is a major cause of heart failure. In this process, fibroblast growth and collagen synthesis and degradation play a critical role. Recent studies indicate that ligands of the peroxisome proliferator-activated receptors-gamma (PPAR-gamma) alter cardiac remodeling during chronic ischemia. This study was designed to investigate if the PPAR-gamma ligand pioglitazone would modulate fibroblast growth and collagen type-I synthesis (and expression) in cardiac fibroblasts exposed to anoxia-reoxygenation (A-R). METHODS AND RESULTS: Cardiac fibroblasts were exposed to anoxia (95% N2/5% CO2) and then reoxygenation (95% air/5% CO2). A-R increased fibroblast growth (MTT assay) as well as collagen type-I synthesis (H-proline incorporation) and protein expression (Western analysis). Concurrently, there was a parallel increase in the expression of matrix metalloproteinase-1 (MMP-1) in fibroblasts. Pretreatment of cardiac fibroblasts with pioglitazone (10 M) reduced all these effects of A-R. Further, A-R stimulated intracellular reactive oxygen species (ROS) generation and activated the redox-sensitive transcription factor NF-kappaB (both P < 0.05). Both these phenomena were inhibited by pretreatment of cells with pioglitazone. CONCLUSION: Thus, it appears that A-R stimulates fibroblast cell growth, collagen type-I synthesis, and MMP-1 expression in cardiac fibroblasts, most likely a result of ROS generation. Inhibition of ROS generation and induction of NF-kappaB in cardiac fibroblasts during A-R may be a mechanism of action of pioglitazone.

Animals↗
PubMed2004

Ectopic expression of tyrosinase increases melanin synthesis and cell death following UVB irradiation in fibroblasts from familial atypical multiple mole and melanoma (FAMMM) patients.

Patients with familial atypical multiple mole and melanoma (FAMMM) [so-called familial dysplastic naevus syndrome (FDNS)] have a high risk for the development of malignant melanoma. The underlying gene defect has an autosomal dominant inheritance with variable expression and incomplete penetrance. Fibroblasts derived from FAMMM patients have high sensitivity to UVC and mutagens, e.g. 4-nitroquinoline-1-oxide. We were interested in identifying how the combination of inherent sensitivity to UV light and abnormal melanin synthesis interacts in the development of melanoma in FAMMM patients. Intermediates of melanin synthesis produce free radicals that are toxic to cells. Atypical moles (dysplastic naevi) are engaged in the biosynthesis of abnormal melanin pigments. This study examined whether there was any abnormal melanin pigmentation or cell damage after the ectopic expression of tyrosinase in fibroblasts from FAMMM patients when compared with fibroblasts from normal subjects. Fibroblasts from FAMMM patients (3012T and 3072T) were associated with a higher sensitivity than normal human fibroblasts to the toxicity of UVB. When cells were infected with tyrosinase-expressing adenovirus (Ad-HT) and irradiated with UVB, FAMMM fibroblasts showed higher tyrosinase activity, produced more melanin pigments and were degraded more significantly than normal human fibroblasts. Western blot analysis revealed that Ad-HT-infected 3072T produced a larger amount of tyrosinase protein than did Ad-HT-infected normal fibroblasts after UVB irradiation. Our findings suggest: (1) that FAMMM fibroblasts have an unknown machinery which enhances tyrosinase expression by UVB irradiation; and (2) that the resulting increase in melanin synthesis affects the cytotoxicity of UVB to FAMMM fibroblasts. All of these processes may be involved in the genomic instability and development of melanoma in FAMMM patients.

Adenoviridae↗
PubMed2003

Effects of subepithelial fibroblasts on epithelial differentiation in human skin and oral mucosa: heterotypically recombined organotypic culture model.

The stratified squamous epithelia differ regionally in their patterns of morphogenesis and differentiation. Although some reports suggested that the adult epithelial phenotype is an intrinsic property of the epithelium, there is increasing evidence that subepithelial connective tissue can modify the phenotypic expression of the epithelium. The aim of this study was to elucidate whether the differentiation of cutaneous and oral epithelia is influenced by underlying mesenchymal tissues. Three normal skin samples and three normal buccal mucosa samples were used for the experiments. Skin equivalents were constructed in four ways, depending on the combinations of keratinocytes (cutaneous or mucosal keratinocytes) and fibroblasts (dermal or mucosal fibroblasts), and the effects of subepithelial fibroblasts on the differentiation of oral and cutaneous keratinocytes were studied with histological examinations and immunohistochemical analyses with anti-cytokeratin (keratins 10 and 13) antibodies. For each experiment, three paired skin equivalents were constructed by using single parent keratinocyte and fibroblast sources for each group; consequently, nine (3 x 3) organotypic cultures per group were constructed and studied. The oral and cutaneous epithelial cells maintained their intrinsic keratin expression. The keratin expression patterns in oral and cutaneous epithelia of skin equivalents were generally similar to their original patterns but were partly modified exogenously by the topologically different fibroblasts. The mucosal keratinocytes were more differentiated and expressed keratin 10 when cocultured with dermal fibroblasts, and the expression patterns of keratin 13 in cutaneous keratinocytes cocultured with mucosal fibroblasts were different from those in keratinocytes cocultured with cutaneous fibroblasts. The results suggested that the epithelial phenotype and keratin expression could be extrinsically modified by mesenchymal fibroblasts. In epithelial differentiation, however, the intrinsic control by epithelial cells may still be stronger than extrinsic regulation by mesenchymal fibroblasts.

Cell Differentiation↗
PubMed1989

Long-term culture of fibroblasts in contracted collagen gels: effects on cell growth and biosynthetic activity.

The purpose of these studies was to analyze the consequences of long-term collagen gel contraction on fibroblast growth and metabolic activity. After 4 weeks, floating gels were 98% contracted, and attached gels were 94% contracted. During this culture period, fibroblasts in floating gels regressed significantly compared to fibroblasts in attached gels, although the cells remaining in the floating gels were viable. In attached gels, fibroblasts were bipolar; whereas in floating gels, fibroblasts were stellate. Therefore, differences between survival of fibroblasts in attached and floating collagen gels might depend on cell shape. Similarly, extracellular matrix organization and its influence on cell shape might control fibroblast proliferation in granulation tissue. During long-term culture of fibroblasts in contracted collagen gels, 70%-80% of the starting collagen was degraded. Collagen synthesized by cells in 4-d cultures was mostly procollagen secreted into the medium. On the other hand, collagen synthesized in 4-week cultures was processed to alpha (I) chains and incorporated into the matrix. There also were other differences between the proteins synthesized by fibroblasts after short-term and long-term culture in contracted gels. These findings show that fibroblasts in long-term collagen gel cultures express unique growth and biosynthetic characteristics.

Cells, Cultured↗
PubMed1996

c-myb proto-oncogene is expressed by quiescent scleroderma fibroblasts and, unlike B-myb gene, does not correlate with proliferation.

Systemic sclerosis (scleroderma) is characterized by excessive deposition of extracellular matrix constituents. Although it has been proposed that tissue fibrosis is due to increased fibroblast synthesis of various collagen polypeptides, there is some experimental evidence that patients with systemic sclerosis have a defect in the control of fibroblast growth. The myb family of genes includes, among others, the c-myb proto-oncogene and the structurally related gene, B-myb, which are both implicated in the regulation of differentiation and/or proliferation of hematopoietic and nonhematopoietic cells. To elucidate the molecular basis responsible for scleroderma fibroblast proliferation, we therefore elected to investigate the expression of c-myb and B-myb genes in scleroderma and control cells. Using the reverse transcriptase polymerase chain reaction technique, we detected c-myb transcripts in scleroderma skin fibroblasts rendered quiescent by serum deprivation. Under the same experimental conditions, c-myb message was not found in normal skin fibroblasts, but, after serum stimulation, c-myb RNA was clearly evident from 3 to 72 h in both normal and pathologic cells. Treatment of these cells with c-myb antisense oligonucleotides caused downregulation of c-myb expression, and the inhibition of scleroderma fibroblast proliferation was 42%, whereas in normal fibroblasts the inhibition was weaker (22%). In contrast to c-myb, in normal and scleroderma fibroblasts the level of expression of B-myb correlated with cell proliferation assessed by cell count, and densitometric analysis showed that B-myb message was 1.5-5 times higher in most of pathologic cells studied. The antisense B-myb oligonucleotides had a weaker antiproliferative effect compared with antisense c-myb, inhibiting scleroderma and normal fibroblasts by 23% and 13%, respectively. These data suggest that the B-myb and c-myb genes may play a role in scleroderma fibroblast proliferation and function.

Aged↗
PubMed1991

Stimulation of collagen and glycosaminoglycan production in cultured human adult dermal fibroblasts by recombinant human interleukin 6.

Interleukin (IL) 6 is a pleiotropic cytokine synthesized by fibroblasts in response to many stimuli, including IL-1 beta. To evaluate the possibility that previously observed stimulation of fibroblast biosynthetic functions by IL-1 beta may be mediated by autocrine IL-6, we investigated the effect of recombinant human (rh) IL-6 on the connective tissue-related biosynthetic functions of three lines of cultured human adult dermal fibroblasts. We found that rhIL-6 mimicked some of the activities of IL-1 beta, as 24-96-h treatment of confluent fibroblast cultures with rhIL-6 caused concentration (10 to 1000 ng/ml)-dependent increases in the production of collagen and the glycosaminoglycans (GAG), hyaluronic acid and chondroitin-4/6-sulfates, but had little effect on fibronectin or total protein production. Although the effective stimulating concentrations of IL-6 were within the range (approximately 100 ng/ml) we found produced by rhIL-1 beta-treated fibroblast cultures, rhIL-1 beta at 0.2-1.0 ng/ml induced significantly greater amounts of collagen and GAG than the maximum effective concentrations of IL-6. Moreover, an anti-rhIL-6 antibody, which effectively neutralized the fibroblast-stimulating activities of rhIL-6, only fractionally blocked the fibroblast-stimulating actions of rhIL-1 beta, suggesting autocrine IL-6 only partially mediates the effects of IL-1 beta on fibroblasts. Conversely, the fibroblast-stimulating effects of rhIL-6 are unlikely due to autocrine IL-1 beta, as an anti-rhIL-1 beta antibody had only minimal inhibitory action on rhIL-6-treated fibroblast cultures. Overall these results suggest that IL-6 could function as a paracrine/autocrine regulator of dermal fibrotic repair.

Adult↗
PubMed1983

The capacity of retracting a collagen matrix is lost by dermatosparactic skin fibroblasts.

When cultivated within a matrix made of reconstituted collagen fibers, fibroblasts derived from skin, tendon, vena cava, and aorta of a normal (N) calf retract the lattice. This effect progresses with time and is related to the density of the cells included in the lattice. Under similar conditions, fibroblasts derived from the skin of 2 dermatosparactic (D) calves do not contract the lattice. Fibroblasts from D tendon and cells from D vena cava and aorta contract the lattice at the same rate and to the same extent as do their normal counterparts. In the lattice, N skin fibroblasts are elongated along the collagen fibers while D skin fibroblasts remain round and develop little cell processes. N skin fibroblasts do not multiply in the lattice while D skin fibroblasts increase in number by a factor of 3 in 5 days. The addition of N skin fibroblasts, in an amount insufficient to retract the lattice, to D skin fibroblasts does not correct their defective capacity. It is suggested that the disturbed relationship between the D skin fibroblasts and collagen fibers is responsible for the lack of architectural organization of the bundles of collagen polymers in the D skin.

Animals↗
PubMed2004

Protease-activated receptor-2 (PAR-2) expression in human fibroblasts is regulated by growth factors and extracellular matrix.

Many cell types express a membrane receptor, activated by trypsin-like proteases, termed protease-activated receptor-2 (PAR-2). Previous studies describing PAR-2 expression on fibroblasts have been conflicting. In this report, we investigated in vitro PAR-2 expression on several fibroblast cell lines using flow cytometry, immunohistology, and immunoblots of cell lysates. Consistent PAR-2 expression was detected in cultured fibroblasts, although we observed heterogeneity of cellular expression among the cell lines. Some fibroblast lines expressed PAR-2 predominantly as an intracellular protein with differing cytoplasmic staining patterns, whereas other fibroblast lines displayed PAR-2 primarily as a cell surface receptor. Immunoblots of cell lysates with polyclonal anti-PAR-2 demonstrated a 44 kDa band, the predicted molecular weight for the PAR-2 core protein. Furthermore, we noted that expression of PAR-2 was subject to regulation. Fibroblasts grown within a collagen matrix downregulated receptor expression whereas increased PAR-2 expression was observed by the addition of fibroblast growth factors PDGF-BB and TGF-beta. This study may explain the previous inconsistencies in PAR-2 expression observed on tissue fibroblasts. Results indicate that the degree of fibroblast proliferation, attenuated by extracellular matrix and upregulated by growth factors, influences whether fibroblasts express PAR-2 and, thus, would be responsive to protease signaling.

Becaplermin↗
PubMed2005

Constitutive phosphorylation of focal adhesion kinase is involved in the myofibroblast differentiation of scleroderma fibroblasts.

Most of the cultured scleroderma fibroblasts have been reported to be myofibroblasts that have the ability to express alpha smooth muscle actin (alphaSMA). It is reported that, in human lung fibroblasts, alphaSMA is induced by transforming growth factor-beta (TGF-beta), which requires focal adhesion kinase (FAK) phosphorylation on its Tyr-397 site. In this study, we investigated how alphaSMA expression is upregulated in cultured scleroderma fibroblasts. 4-amino-5-(4-chlorophenyl)-7-(butyl)pyrazolo[3,4-d]pyrimidine, which is a pharmacologic inhibitor of FAK/Src, markedly diminished upregulated alphaSMA expression in scleroderma fibroblasts as well as in normal fibroblasts stimulated with TGF-beta. Likewise, alphaSMA expression was significantly reduced in sclerderma fibroblasts transfected with kinase-deficient FAK mutant. FAK phosphorylation levels on Tyr-397 in scleroderma fibroblasts were significantly higher than those in normal fibroblasts. Both alphaSMA expression and FAK phosphorylation levels in scleroderma fibroblasts were markedly diminished by the treatment with TGF-beta antisense oligonucleotide. These results indicate that the constitutive phosphorylation of FAK, which is possibly because of the autocrine TGF-beta signaling, may play an important role in alphaSMA expression in scleroderma fibroblasts.

Actins↗