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Behavior and ultrastructure of in vitro ageing mouse embryo fibroblasts grown in collagen gels.

We have compared the behaviour and the ultrastructure of embryonic mouse fibroblasts embedded into collagen gels at early, middle and late population doubling levels (PDL). Late mouse fibroblasts were able to induce gel contraction with a greater efficiency than young cells. We did not find any differences in the organization of these gels. Electron microscope observations on gels containing fibroblasts of different PDL showed that the collagen lattice induced new specific and distinct phenotypes. The well-known ultrastructural differences between young and late fibroblasts grown on plastic substrates were less prominent when these cells were embedded into collagen gels. The late fibroblasts grown into gels kept their large size and their lobulated nuclei and resembled fibroblasts grown on plastic surfaces. However, dramatic changes were observed in their pattern of microfilaments, in the dispersion of their chromatin and in their ergastoplasmic structure; these characteristics observed in late fibroblasts grown into gels were close to those of young cells. The new phenotypes of young, middle-aged and late fibroblasts in the collagen gels seemed to be stable and did not display the characteristics of an older phenotype on continued incubation. When the fibroblasts left the gel, they returned to their initial phenotype.

Actin Cytoskeleton↗

Receptor-mediated endocytosis of fibroblast beta-glucuronidase by peritoneal macrophages.

beta-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of beta-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.

Alkaline Phosphatase↗

Selective assessment of in vitro radiosensitivity of tumour cells and fibroblasts from single tumour biopsies using immunocytochemical identification of colonies in the soft agar clonogenic assay.

The assumed selective growth of tumour cells has formed the basis for the use of the soft agar clonogenic assay to test in vitro radio- and chemosensitivity of tumours. However, recent studies have demonstrated that fibroblasts proliferate in soft agar in addition to tumour cells. The present study was initiated to quantify the contaminating growth of non-malignant cells in the modified form of the Courtenay-Mills soft agar assay, in order to establish a reliable assay for estimating tumour cell radiosensitivity in squamous cell carcinomas of the head and neck. DNA flow cytometry analysis confirmed that 'tumour fibroblasts' (fibroblasts obtained from tumour biopsies) grow in soft agar. In contrast, white blood cells did not form colonies. Different media were tested with soft agar, but a selective medium for tumour cells was not found. Therefore, a colony filter-technique combined with an immunocytochemical analysis was developed to quantify the number of tumour cell and fibroblast colonies. In 12 tumour biopsies, 2-33% of the colonies were Cytokeratin AE1-3 positive, whereas 83-100% of the colonies were 5B5 fibroblast antibody positive. The parameter normally reported, the overall SF2 (surviving cell fraction at 2 Gy) based on colonies in agar, was found to be statistically significantly correlated to the fibroblast SF2, but not to the tumour cell SF2. The overall SF2 was significantly different from the tumour cell SF2 in half of the tumours. Furthermore, the tumour cell SF2 was not correlated to fibroblast SF2. In consequence of our findings, correcting for fibroblast contamination is a necessity, when studying in vitro sensitivity of tumour cells. Combining the soft agar clonogenic assay with the new colony filter-technique and the immunocytochemical analysis appear to be useful for making this routine correction and for measuring the in vitro radiosensitivity of both tumour cells and fibroblasts from single tumour biopsies, which is of interest in future clinical studies on predictive assays.

Biopsy↗

Basic fibroblast growth factor-like immunoreactivity in Purkinje cells of the rat cerebellum.

An antiserum against basic fibroblast growth factor was characterized by immunoblot experiments and used to investigate immunohistochemically the projection fields and fine structures of basic fibroblast growth factor-containing cerebellar Purkinje cells. The antiserum demonstrated clearly purified basic fibroblast growth factor and basic fibroblast growth factor-like molecules of the same molecular weight in homogenates of the adult rat cerebellum. Light and electron microscopic immunohistochemistry revealed that a large number of Purkinje cells, if not all, send immunoreactive dendrites to the molecular layer and basic fibroblast growth factor-containing axons to the deep cerebellar and lateral vestibular nuclei, where basic fibroblast growth factor nerve terminals form synapses with the soma and dendrites of neurons labeled weakly with basic fibroblast growth factor. Nerve cells with basic fibroblast growth factor had immunoreaction deposits mainly in free ribosomes, those attached to the endoplasmic reticulum and in the nuclear euchromatin. These findings suggest that basic fibroblast growth factor is present in cerebellar Purkinje cells and undergoes two modes of transport, one to axon terminals and the other to nuclear euchromatin, known as the RNA transcription zone.

Animals↗

Fast and widespread increase of basic fibroblast growth factor messenger RNA and protein in the forebrain after kainate-induced seizures.

Basic fibroblast growth factor promotes the survival and outgrowth of neurons and protects neurons from glutamate mediated excitotoxicity. The present study investigates the effects of kainate-induced epileptic seizures on the cellular expression of basic fibroblast growth factor messenger RNA and protein. Seizures were induced by injection of 12 mg/kg kainic acid. Rats were killed 3 h, 6 h, and 24 h after injection of the drug and analysed by radioactive and non-radioactive in situ hybridization as well as immunohistochemistry for glial fibrillary acidic protein and basic fibroblast growth factor. Radioactive in situ hybridization revealed a fast (6 h), strong (300-400% of control) and widespread increase of basic fibroblast growth factor messenger RNA after kainate-induced seizures. Non-radioactive in situ hybridization using digoxigenin-labeled riboprobes combined with glial fibrillary acidic protein immunohistochemistry showed that basic fibroblast growth factor messenger RNA was markedly increased in astroglial cells throughout the brain. Immunohistochemistry for basic fibroblast growth factor revealed labeling of nuclei in astrocytes in many forebrain areas and in neurons in area CA2 and fasciola cinereum. Kainate markedly increased basic fibroblast growth factor-like immunoreactivity in nuclei of astrocytes in several forebrain areas. This effect peaked 24 h after injection. It is concluded that basic fibroblast growth factor may play a neuroprotective role in kainate mediated excitotoxicity as seen from a massive and widespread astroglial increase in basic fibroblast growth factor messenger RNA and -like immunoreactivity. These effects may, to a large degree, be mediated through the excessive release of endogenous glutamate, induced by the epileptic seizures, leading to activation of glutamate receptors on astroglial cells through volume transmission, i.e. via diffusion of electrochemical signals in the extracellular fluid pathways.

Animals↗

Chronic continuous infusion of (-)nicotine reduces basic fibroblast growth factor messenger RNA levels in the ventral midbrain of the intact but not of the 6-hydroxydopamine-lesioned rat.

A negative correlation has been found between smoking and Parkinson's disease. There is evidence to suggest that this correlation appears to be associated with a neuroprotective role of nicotine for dopamine neurons at least in relation to mechanical injury. However, 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) neurotoxicity to dopamine neurons is enhanced by chronic continuous (-)nicotine. More recently, basic fibroblast growth factor has been found to possess neurotrophic activities for many nerve cells including the dopamine cells in vivo and in vitro. Therefore, it is of interest to explore a possible effect of nicotine on basic fibroblast growth factor expression in the ventral midbrain of intact and 6-hydroxydopamine-lesioned rats and how treatment with nicotine can alter the 6-hydroxydopamine-induced injury of the nigral dopamine nerve cells as evaluated by dopamine biochemistry. In the present paper, an analysis of the effects of chronic continuous infusion of (-)nicotine via minipumps was carried out on basic fibroblast growth factor expression in the vental midbrain of the intact male rat and of the 6-hydroxydopamine lesioned rat. A quantitative messenger RNA protection assay analysis was used as well as an immunocytochemical analysis in the substantia nigra. Our findings give evidence that a two-week continuous infusion with (-)nicotine in the intact rat leads to substantial and dose-related (0.03-0.3 mg/kg per h) reductions of basic fibroblast growth factor messenger RNA levels in the ventral midbrain. These changes are not associated with changes in neuronal and glial basic fibroblast growth factor immunoreactivity in this region with the antibodies used. However, a one-week continuous infusion with (-)nicotine (0.125 mg/kg per h) failed to significantly alter the basic fibroblast growth factor messenger RNA levels in the ventral midbrain of solvent and 6-hydroxydopamine-injected rats and thus also the 6-hydroxydopamine-induced increase of basic fibroblast growth factor messenger RNA levels in the ventral midbrain of the lesioned side observed at this time-interval and known to be of astroglial origin [Chadi G. et al. (1994) Neuroscience 61, 891-910]. In agreement, the 6-hydroxydopamine-induced depletion of dopamine in the neostriatum was unaltered by the nicotine treatment (0.125 mg/kg per h). Thus, chronic continuous (-)nicotine treatment may lead to a reduced basic fibroblast growth factor trophic tone in the ventral midbrain of the intact but not of the 6-hydroxydopamine-lesioned rat neither on the lesioned nor on the unlesioned side of the ventral midbrain. It seems possible that chronic nicotine treatment mainly reduces basic fibroblast growth factor messenger RNA levels of neuronal origin, since the astroglial messenger RNA levels dominate after the 6-hydroxydopamine-induced lesions.

Animals↗

Differential expression of CRABP-II in fibroblasts derived from dermis and subcutaneous fat.

We have shown previously that fibroblasts derived from fat or dermal tissue differ in their functional properties, such as proliferation rate and contractile properties. To study these differences further, two-dimensional electrophoresis (2D PAGE) was performed on proteins isolated from cultured subcutaneous fat and dermal fibroblasts. The 2D gels were screened for proteins that were differentially expressed in all donors (n = 5). Five protein spots were subjected to further analysis by mass spectrometry. Two proteins could be identified: brain acid soluble protein 1 (BASP1) and cellular retinoic acid binding protein-II (CRABP-II). CRABP-II is of interest in terms of re-epithelialisation and was clearly expressed in dermal fibroblasts but not in fat fibroblasts. Real time PCR was performed to confirm the 2D data on CRABP-II. The CRABP-II mRNA level was significantly increased in dermal tissue and cultured dermal fibroblasts compared to fat tissue and cultured fat-derived fibroblasts, respectively. The mode of action of CRABP-II in skin is to mediate retinoic acid activity. Retinoic acid is known to inhibit migration and to stimulate differentiation of keratinocytes. The expression of CRABP-II by dermal fibroblasts implicates a role for these fibroblasts in wound re-epithelialisation, in contrast to subcutaneous fat-derived fibroblasts.

Adipose Tissue↗

Potential regulatory elements of the constitutive up-regulated alpha2(I) collagen gene in scleroderma dermal fibroblasts.

The promoter activity of the full-length alpha2(I) collagen gene is higher in scleroderma fibroblasts, when compared to normal fibroblasts. In this study, to investigate the molecular mechanisms up-regulating the expression of the alpha2(I) collagen gene in scleroderma dermal fibroblasts more clearly, we compared promoter activities of serial 5'-deletion mutants and the substitution mutants of the alpha2(I) collagen promoter constructs between normal and scleroderma fibroblasts. The transient transfection assays using a series of 5'-deletions of the promoter revealed that the up-regulated fold-increase in scleroderma fibroblasts relative to that in normal fibroblasts was significantly decreased by the removal of bp -353 to -264 fragment or bp -264 to -186 fragment. The substitution mutations introduced into binding sites of Sp1 (bp -303 and -271), Ets1 (bp -285 and -282), as well as Smad (bp -263 and -258) also abrogated the fold-increase in promoter activity in scleroderma fibroblasts synergistically. A DNA affinity precipitation assay showed that the binding activity of Ets1 as well as Smad3 to their binding sites was increased in scleroderma fibroblasts compared with normal cells. Taken together, our promoter analysis emphasized that Ets1 form a transcriptionally active complex with Smad and Sp1 by autocrine transforming growth factor (TGF)-beta signaling, leading to the intrinsic up-regulation of alpha2(I) collagen promoter activity in scleroderma fibroblasts. The blockade of autocrine TGF-beta signaling is thought to be one of the most reliable approaches in the treatment of scleroderma, and further study targeting Ets1, Smad or Sp1 may contribute to this blockade.

Collagen↗

Fibroblasts improve performance of cultured composite skin substitutes on athymic mice.

BACKGROUND: This study investigated the impact of adding human fibroblasts to a cultured composite skin substitute model of cultured human keratinocytes and acellular human dermis. METHODS: Skin substitutes were prepared by seeding human keratinocytes on the papillary side of acellular dermis with or without seeding fibroblasts on the reticular side. Performance of the grafts was compared both in vitro by histology and in vivo on surgically created full-thickness wounds on athymic mice. Graft size and contraction were measured and immunohistochemical stains were done to reveal vascularization. RESULTS: Skin substitutes with fibroblasts formed thicker epidermis than skin substitutes without fibroblasts. When transplanted onto athymic mice, skin substitutes with fibroblasts maintained their original size with only 2% contraction. In contrast, skin substitutes without fibroblasts showed 29% contraction. Vascular basement membrane specific mouse CD31staining and endothelial cell specific mouse collagen type IV staining revealed vascularization as early as 1 week posttransplant in grafts with fibroblasts, and was significantly higher than grafts without fibroblasts at 2 weeks. CONCLUSIONS: Addition of fibroblasts to keratinocyte based composite skin substitutes improves epidermis formation, enhances vascularization and reduces contraction.

Animals↗

Pentoxifylline inhibits mature burn scar fibroblasts in culture.

Fibroblasts are thought to be (in part) responsible for the persisting contractile forces that result in burn contractures. Using monolayer and fibroblast populated collagen lattice (FPCL) models we subjected burn scar fibroblasts to the anti-fibrinolytic agent Pentoxifylline (PFX) in an attempt to reduce proliferation and contraction of these cells. Fibroblasts were isolated from mature burn scars at reconstructive surgery. Fibroblasts were grown in monolayer or incorporated into FPCL's and exposed to PFX. Fibroblast numbers and FPCL surface areas were calculated using digital photography and image analysis. PFX showed a dose-dependent inhibition of contraction and reduced proliferation of burn scar fibroblasts. In monolayer, cell number proliferation was markedly reduced. FPCL's containing 0, 0.25, 0.5, 1, and 2 mg/ml of PFX had relative surface areas of 31, 40, 43, 59, and 85%, respectively. One and 2 mg/ml FPCL's contracted significantly less than controls (p < 0.0001). This is the first study to show the dose-dependent effects of Pentoxifylline on the proliferation and contraction of burn scar fibroblasts. This study suggests that Pentoxifylline has a direct effect on inhibiting burn scar fibroblasts. Further study of PFX on burn scars will provide opportunities to reduce burn scar contractures in vivo.

Burns↗

Effect of oxidized regenerated cellulose (Interceed) on the expression of tissue plasminogen activator and plasminogen activator inhibitor-1 in human peritoneal fibroblasts and mesothelial cells.

OBJECTIVE: To characterize the molecular changes that occur in normal fibroblasts, adhesion fibroblasts, and mesothelial cells as a result of exposure to oxidized regenerated cellulose (Interceed; Johnson & Johnson Medical, Inc., New Brunswick, NJ). DESIGN: Control and Interceed-treated normal peritoneal fibroblasts, adhesion fibroblasts, and mesothelial cells in culture were assessed for messenger RNA levels of molecules known to be associated with adhesion development, using multiplex reverse transcriptase polymerase chain reaction (n = 4). SETTING: University research laboratory. PATIENT(S): Normal and adhesion fibroblasts and mesothelial cells. INTERVENTION(S): Exposure of cells, normal fibroblasts, adhesion fibroblasts, and mesothelial cells to oxidized regenerated cellulose. MAIN OUTCOME MEASURE(S): Real-time reverse transcriptase polymerase chain reaction expression of messenger RNA tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), and tPA-PAI-1 ratio, an indicator of overall fibrinolytic activity. RESULT(S): Interceed treatment of normal peritoneal fibroblasts, adhesion fibroblasts, and mesothelial cells results in an increased expression of tPA in mesothelial cells and an increase in the tPA-PAI-1 ratio, signifying an overall increase in fibrinolytic activity. CONCLUSION(S): Interceed, which has been shown in multiple human in vivo studies to decrease postoperative adhesion development, increases the expression of tPA and the tPA-PAI-1 ratio (an indicator of overall fibrinolytic activity), thereby promoting dissolution of fibrin and healing without adhesion development. Thus, the ability of Interceed to reduce postoperative adhesion development may be derived from both a barrier and biologic effect.

Cells, Cultured↗

Wound fluid inhibits wound fibroblast nitric oxide synthesis.

BACKGROUND: Fibroblast-derived nitric oxide (NO) is an autocrine stimulator of collagen synthesis by wound fibroblasts. Little is known about the in vivo regulation of wound fibroblast NO synthesis. We investigated the net effect of wound environment on wound fibroblast NO production and characterized a soluble factor mediating this effect. MATERIALS AND METHODS: Wound fibroblasts and acellular wound fluid (pool of 100 Lewis rats) were isolated from subcutaneously implanted polyvinyl alcohol sponges harvested 10 days post-wounding. Fibroblasts were incubated in the presence of 10% (v/v) wound fluid. Nitrite, an index of NO synthesis, was measured in supernatants by Griess reagent. RESULTS: Wound fibroblasts spontaneously synthesize large amounts of NO. Spontaneous NO synthesis was further increased by LPS + IFN-gamma (P < 0.001). Wound fluid significantly inhibited both spontaneous and LPS plus IFN-gamma-stimulated NO synthesis (by 88 and 55%, respectively; P < 0.01). Wound fluid from 5- to 35-day-old wounds equally suppressed NO synthesis. Separation by Sephadex G-100 gel filtration identified the active factor in wound fluid to have a molecular weight of about 100 kDa. Characterization of this factor showed it to be a heat-resistant (56 degrees C, 30 min), trypsin-sensitive, and neuraminidase-resistant protein (ammonium sulfate precipitation). The isoelectric point appeared to be 7.0, as determined by ion exchange chromatography. Addition of high arginine did not restore the effect of wound fluid on fibroblast NO synthesis, suggesting that substrate is not a limiting factor. CONCLUSION: Our data demonstrate that following postoperative day 5 the wound environment contains a high molecular weight protein that inhibits NO synthesis by wound fibroblasts.

Animals↗

Age-related changes in focal adhesions lead to altered cell behavior in tendon fibroblasts.

During aging the increase in collagen cross-linking and total amount of collagen in tendon leads to a decline in both its flexibility and its ability to heal after injury. Fibroblasts are responsible for the synthesis of the macromolecules that constitute tendonous tissue. The ability of fibroblasts to maintain tissue homeostasis is compromised with increasing age underlying many of the age-related pathologies of the musculoskeletal system. This leads to a slowdown in connective tissue healing. Whether these deficits are due to changes in connective tissue, structure or to changes in tendon fibroblast function is unknown. We show that tendon fibroblasts from old mice have an altered morphology, reduced level of function, and exhibit changes in protein transport, compared to fibroblasts from young mice. The fibroblasts from old mice are not senescent, they are distinct phenotypes. Achilles tendon fibroblasts from old mice have low motility and proliferation, a poorly organised actin cytoskeleton and a different localisation of key focal adhesion proteins compared to the same cells from young mice. Additionally we found more of the protein misfolding indicator protein, GADD 153, in fibroblasts from old tendon. These results indicate that changes in tendon fibroblast function may well explain the age-related decline in tendon healing.

Achilles Tendon↗

Normal human fibroblasts enable melanoma cells to induce angiogenesis in type I collagen.

BACKGROUND: We previously reported that fibroblasts induce human microvascular endothelial cells (HMVECs) to differentiate from monolayer to capillarylike morphology. We now test the hypothesis that fibroblasts modulate angiogenesis in melanoma cells. METHODS: We tested 12 human melanoma lines (2 radial growth phase (RGP), 3 vertical growth phase (VGP), and 7 metastatic (MM)) for ability to induce HMVECs to invade/migrate into collagen and form capillarylike networks. HMVEC monolayers were overlaid with 3-dimensional collagen gels embedded with melanoma cells alone (M), fibroblasts alone (F), or a 1:1 mixture of the 2 cells (M+F). After 5 days, gels were removed, fixed, and HMVEC networks were quantified by von Willebrand's factor (vWF) immunofluorescence. The influence of soluble factors on HMVEC invasion/migration into collagen was assessed with the use of acellular 3-D collagen gels overlaid on HMVEC monolayers, cultured with conditioned media (CM) derived from monolayers of M, F, or M+F. Angiogenic growth factors involved in the observed invasion/migration were identified with the use of a RayBio Cytokine Antibody Array (RayBiotech, Norcross, Ga). RESULTS: Cell line-specific variability in melanoma-supported angiogenesis was observed only when in combination with fibroblasts (analysis of variance [ANOVA], P < .01). Melanoma plus fibroblasts uniformly resulted in a significantly higher angiogenic response than melanoma alone (P < .05). One vertical growth phase and one metastatic melanoma line, while weakly angiogenic alone, induced significantly higher angiogenesis than either fibroblast or melanoma alone (P < .05) when combined with fibroblasts. CM from M or M+F induced significantly less HMVEC invasion/migration into collagen than CM from fibroblasts alone. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 were identified as significantly elevated in the media derived from M+F cultures, compared with either cell type alone. CONCLUSION: To our knowledge, this is the first report demonstrating that melanoma-supported angiogenesis in collagen is more significantly influenced by normal skin-derived fibroblasts than by the intrinsic biology of the melanoma cell type. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 are implicated as potential paracrine factors regulating this observed effect.

Angiogenic Proteins↗

Quantitative analysis of the cardiac fibroblast transcriptome-implications for NO/cGMP signaling.

Cardiac fibroblasts regulate tissue repair and remodeling in the heart. To quantify transcript levels in these cells we performed a comprehensive gene expression study using serial analysis of gene expression (SAGE). Among 110,169 sequenced tags we could identify 30,507 unique transcripts. A comparison of SAGE data from cardiac fibroblasts with data derived from total mouse heart revealed a number of fibroblast-specific genes. Cardiac fibroblasts expressed a specific collection of collagens, matrix proteins and metalloproteinases, growth factors, and components of signaling pathways. The NO/cGMP signaling pathway was represented by the mRNAs for alpha(1) and beta(1) subunits of guanylyl cyclase, cGMP-dependent protein kinase type I (cGK I), and, interestingly, the G-kinase-anchoring protein GKAP42. The expression of cGK I was verified by RT-PCR and Western blot. To establish a functional role for cGK I in cardiac fibroblasts we studied its effect on cell proliferation. Selective activation of cGK I with a cGMP analog inhibited the proliferation of serum-stimulated cardiac fibroblasts, which express cGK I, but not higher passage fibroblasts, which contain no detectable cGK I. Currently, our data suggest that cGK I mediates the inhibitory effects of the NO/cGMP pathway on cardiac fibroblast growth. Furthermore the SAGE library of transcripts expressed in cardiac fibroblasts provides a basis for future investigations into the pathological regulatory mechanisms underlying cardiac fibrosis.

Adaptor Proteins, Signal Transducing↗

Matrix contraction by dermal fibroblasts requires transforming growth factor-beta/activin-linked kinase 5, heparan sulfate-containing proteoglycans, and MEK/ERK: insights into pathological scarring in chronic fibrotic disease.

Scarring is characterized by excessive synthesis and contraction of extracellular matrix. Here, we show that fibroblasts from scarred (lesional) areas of patients with the chronic fibrotic disorder diffuse scleroderma [diffuse systemic sclerosis (dSSc)] show an enhanced ability to adhere to and contract extracellular matrix, relative to fibroblasts from unscarred (nonlesional) areas of dSSc patients and dermal fibroblasts from normal, healthy individuals. The contractile abilities of normal and dSSc dermal fibroblasts were suppressed by blocking heparin sulfate-containing proteoglycan biosynthesis or antagonizing transforming growth factor-beta receptor type I [activin-linked kinase (ALK5)] or ras/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK). Compared with both normal and nonlesional fibroblasts, lesional dSSc fibroblasts overexpressed the heparin sulfate-containing proteoglycan syndecan 4. We also found that the procontractile signals from transforming growth factor (TGF)-beta were integrated through syndecan 4 and MEK/ERK because the ability of TGFbeta to induce contraction of dermal fibroblasts was prevented by MEK antagonism. TGFbeta could not induce a contractile phenotype or phosphorylate ERK in syndecan 4(-/-) dermal fibroblasts. These results suggest that integrating TGFbeta and ERK signals via syndecan 4 is essential for the contractile ability of dermal fibroblasts. We conclude that antagonizing MEK/ERK, TGFbeta1/ALK5, or syndecan 4 may alleviate scarring in chronic fibrotic disease.

Activin Receptors, Type I↗

Constitutive thrombospondin-1 overexpression contributes to autocrine transforming growth factor-beta signaling in cultured scleroderma fibroblasts.

The extracellular matrix (ECM) glycoprotein thrombospondin-1 (TSP-1) has been reported to activate the latent complex of transforming growth factor-beta (TGF-beta), the major effects of which in mesenchymal cells is stimulation of the synthesis of ECM. Previous reports suggested the involvement of an autocrine TGF-beta loop in the pathogenesis of scleroderma. In this study, we examined whether TSP-1 plays a role in maintaining the autocrine TGF-beta loop in scleroderma. TSP-1 expression was increased in scleroderma patients compared with in healthy controls in vivo and in vitro. TGF-beta blocking antibody or TGF-beta1 antisense oligonucleotide markedly reduced the up-regulated TSP-1 expression in scleroderma fibroblasts but had little effect on normal fibroblasts. The expression of TSP-1 is up-regulated in scleroderma fibroblasts, possibly at the post-transcriptional level just like in normal fibroblasts stimulated with exogenous TGF-beta1. TSP-1 blocking peptide or antisense oligonucleotide had an inhibitory effect on the up-regulated alpha2I collagen and phosopho-Smad3 levels in scleroderma fibroblasts but had little effects on normal fibroblasts. The transient overexpression of TSP-1 up-regulated alpha2I collagen and phospho-Smad3 levels in normal fibroblasts but had no major effect on scleroderma fibroblasts. Furthermore, these effects of transiently overexpressed TSP-1, which possibly occurred via the activation of latent TGF-beta1, were abolished by the TGF-beta1 antisense oligonucleotide. These results indicate that the constitutive overexpression of TSP-1 may play an important role in autocrine TGF-beta signaling and accumulation of ECM in scleroderma fibroblasts.

Autocrine Communication↗

Thy-1 expression in human fibroblast subsets defines myofibroblastic or lipofibroblastic phenotypes.

Fibroblasts represent a dynamic population of cells, exhibiting functional heterogeneity within and among tissues. Fibroblast heterogeneity also results from phenotypic differences and may arise from activation or differentiation processes taking place in the cells. We previously reported that human fibroblasts were heterogeneous with respect to surface Thy-1 expression and that separation into Thy-1(+) and Thy-1(-) subsets resulted in functionally distinct subpopulations, leading to the concept of fibroblast subset specialization. In this report we investigated whether Thy-1(+) and/or Thy-1(-) fibroblasts were capable of differentiating into myofibroblasts or lipofibroblasts. Fibroblast subsets were used from human myometrium and orbit to test this hypothesis. Only Thy-1(+) human myometrial and orbital fibroblasts were capable of myofibroblast differentiation after treatment with TGFbeta or platelet concentrate supernatant, assessed by alpha smooth muscle actin expression. Interestingly, only Thy-1(-), but not Thy-1(+) subsets differentiated to lipofibroblasts, as determined by the accumulation of cytoplasmic lipid droplets after treatment with 15-deoxy-Delta(12, 14)-PGJ(2) or ciglitazone. We propose that fibroblast Thy-1 display pre-determines lineage to a contractile or lipid-like phenotype in the human myometrium and orbit. This additional distinction between Thy-1(+) and Thy-1(-) human fibroblast subtypes has important consequences in normal tissue homeostasis and in pathogenesis of orbital and myometrial diseases characterized by persistent myofibroblasts or fat accumulation, such as occurs in Graves' ophthalmopathy, tissue fibrosis, abnormal wound healing, and scarring.

Actins↗