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PubMed2003

Effects of cholesterol on the phenotype of rabbit bile duct fibroblasts.

AIM: To investigate how cholesterol (Ch) can affect the phenotype of bile duct fibroblasts of New Zealand rabbits. METHODS: 16 rabbits were divided randomly into two groups: the control group and the experiment group. The rabbits in experiment group were fed with hypercholesterol diet for 8 weeks. Bile duct was dissociated from rabbits and prepared for transmission electron microscopy. The purified bile duct fibroblasts were cultured and divided randomly into three groups: control group, Ch middle concentration group (0.6 g/L), Ch high concentration group (1.2 g/L). After incubated for 72 h, the fibroblasts were made into specimens for transmission electron microscopy. The expression of alpha-actin in bile duct fibroblasts was measured by means of laser scanning confocal microscopy. RESULTS: With the transmission electron microscopy, the normal bile duct fibroblasts were shuttle-shaped, and there were abundant rough endoplasmic reticulums (RER), but few mitochondria or microfilaments in cytoplasm. This is the typical phenotype of fibroblasts. Bile duct fibroblasts of hypercholesterolemic rabbits were observed. by the transmission electron microscopy Rough endoplasmic reticulums were significantly reduced, with a lot of microfilament bundles or stress fibers appeared in cytoplasm, especially under plasma membrane. Dense bodies were scattered within these bundles. Macula densas and discontinuous sarcolemma were found under plasma membrane. It suggested that the bile duct fibroblasts of hypercholesterolemic rabbits presented the phenotype of smooth muscle cell. The cultured bile duct fibroblasts also had typical phenotype of fibroblasts. After stimulated by middle concentration cholesterol (0.6 g/L) for 72 h, there appeared lots of microfilaments in cytoplasm, but without dense body, macula densa and discontinuous sarcolemma. Observed with confocal microscopy, there were many regular bundles of microfilaments in fibroblasts treated with middle concentration ch (0.6 g/L) and the expression of alpha-actin was significantly increased. The average fluorescence value of middle concentration group was 1 628+/-189 (P<0.01 vs control group). Microfilaments and the expression of alpha-actin were greatly decreased in fibroblasts of high concentration group (1.2 g/L). The average fluorescence value of high concentration group was 1 427+/-153 (P<0.05 vs middle concentration group). There were a lower expression of alpha-actin and few microfilaments in bile duct fibroblasts of control group with an average fluorescence value of 1 224+/-138. CONCLUSION: Cholesterol can make bile duct fibroblasts have the phenotypic characteristics of smooth muscle cell both in vitro and in vivo and this effect is more significant in vivo. The effect is probably associated with some other factors besides cholesterol.

Animals↗
PubMed2002

Expression of hepatocyte growth factor and its receptor (c-met) in skin fibroblasts from patients with systemic sclerosis.

OBJECTIVE: To determine the effect of excessive induction of hepatocyte growth factor (HGF)/c-met signaling in fibroblasts derived from patients with systemic sclerosis (SSc). METHODS: Fibroblasts were obtained from skin of patients with SSc and healthy controls. The 2.4 kb interleukin 1alpha (IL-1alpha) cDNA was subcloned into pcDNA3 expression vector, which was stably transfected into normal fibroblasts by lipofection. HGF production in cultured fibroblasts was measured by ELISA. C-met protein (a receptor for HGF) in cultured fibroblasts was evaluated by immunocytochemistry using polyclonal anti-c-met antibody. Production of procollagen type I was estimated by an ELISA system using antibodies against procollagen type I C-peptide. RESULTS: Cultured skin fibroblasts expressed mRNA and protein of HGF constitutively in both SSc and control cultures. However, HGF production in SSc fibroblasts was significantly higher than in normal fibroblasts. In both SSc and normal fibroblasts, HGF production was dose dependently increased by the addition of recombinant IL-1alpha. Immunocytochemical staining revealed that c-met was spontaneously expressed in SSc fibroblasts, whereas no expression of c-met was detected in normal fibroblasts. C-met mRNA was expressed in normal fibroblasts transfected with the IL-1alpha gene. Addition of recombinant HGF (100 ng/ml) to cultured SSc fibroblasts significantly decreased procollagen type I production. CONCLUSION: High concentration of HGF inhibited collagen production in cultured fibroblasts derived from patients with SSc. Overexpression of HGF/c-met appears to be a biological feedback response to the fibrotic process of SSc, suggesting that the antifibrotic effect of HGF might be used as a novel strategy for treatment of SSc.

Adult↗
PubMed1986

Antigen presentation by human dermal fibroblasts: activation of resting T lymphocytes.

We have shown that human dermal fibroblasts, exposed to interferon-gamma (IFN-gamma) to induce surface class II major histocompatibility complex (MHC) antigens, were capable of presenting tetanus toxoid (TT) antigen to human TT-specific T cell clones. Antigen presentation by fibroblasts was antigen dependent, required HLA-DR expression by fibroblasts, and was MHC restricted. In contrast, we now report that IFN-gamma-treated fibroblasts are unable to present TT antigen to purified resting T cells obtained from the peripheral blood of TT-immune donors. In addition, although IFN-gamma-treated fibroblasts were able to stimulate alloreactive T cell clones, they were unable by themselves to stimulate primary allogeneic responses in resting T cells. The failure of fibroblasts to stimulate resting T cells was not due to suppressor effects by fibroblasts, because induction of TT and alloantigen responses in resting T cells by monocytes was not inhibited by the presence of fibroblasts. On the contrary, IFN-treated fibroblasts were synergistic with small numbers of monocytes in activating resting T cells. In addition, the failure of antigen presentation by fibroblasts to resting T cells was reversed by the addition of recombinant human interleukin 2 (rIL 2) to cultures, but not of purified human interleukin 1 (IL 1). These results emphasize that the requirements for activation of resting T cells differ from those of T cell clones. Although fibroblasts can efficiently present antigen to T cell clones, antigen presentation by fibroblasts to resting T cells requires the addition of exogenous IL 2. It is postulated that fibroblasts differ from classical antigen-presenting cells in that fibroblasts are incapable of stimulating the production of IL 2 in resting T cells.

Adult↗
PubMed1995

Senescent human fibroblasts resist programmed cell death, and failure to suppress bcl2 is involved.

Programmed cell death (apoptosis) is an active process by which cells initiate their own self-destruction. Growing evidence shows that this event is controlled by the activation of unique gene expression; some function as survival genes, such as bcl2, and others as killer genes, such as ced3 or interleukin converting enzyme. Likewise, external factors, such as the presence or absence of stimuli in the microenvironment of a cell, play a key role in ushering it towards survival or suicidal fate. Previously, I and others have reported that withdrawal of serum from culture medium can induce contact-inhibited quiescent mouse 3T3 fibroblasts to undergo rapid programmed cell death, as evidenced by the presence of massive DNA fragmentation within 24 h. I now report that, although the same process of serum withdrawal is capable of inducing apoptotic death in quiescent young human fibroblasts, the process takes as long as 2 weeks. Repeated attempts at the same serum withdrawal with cultures of senescent human fibroblasts show that phenotypic signs of apoptosis, such as DNA fragmentation and loss of cell viability, are not observed for up to 4 weeks; I suggest that in vitro aged human fibroblasts are resistant to undergoing programmed cell death. I have investigated the level of bcl2 presence as a possible protector of senescent human fibroblasts from apoptotic death; biochemical characterization shows that in mouse as well as human fibroblasts, bcl2 is present as an easily extractable (0.1% Triton) cytoplasmic protein. bcl2 level is in inverse relationship with the ease of induction of apoptotic death between young and senescent human fibroblasts. Immunofluorescence staining shows that, in senescent human fibroblasts, bcl2 is present not only in the cytoplasmic punctate spots seen in both mouse and young human fibroblasts but also in the nuclei as well as large granules surrounding the nuclei. Upon serum deprivation, the bcl2 level is reduced to undetectable in mouse 3T3 fibroblasts within 24 h and in young and intermediate aged human fibroblasts within 2 weeks; however, it remains unchanged in senescent human fibroblasts after the deprivation of serum for 2 weeks. These findings lead me to conclude that senescent fibroblasts are resistant to the induction of apoptotic death by serum deprivation. Furthermore, I suggest that repeated serial passaging during the in vitro aging process has inadvertently instituted a molecular mechanism whereby the bcl2 level cannot be repressed upon serum deprivation, which may subsequently allow senescent fibroblasts to be long-lived and protected from self-destruction.

3T3 Cells↗
PubMedDate not supplied

In vitro analysis of fetal fibroblast collagenolytic activity.

Tissue repair in the rabbit fetus is remarkably rapid and occurs without significant inflammation or excessive collagen deposition. The objective of this study was to compare the ability of rabbit fetal and adult fibroblasts to express the matrix metalloproteinases which are thought to be critical to scar tissue remodeling. In vitro, both fetal and adult rabbit fibroblasts express procollagenase messenger RNA in a constitutive manner. Mechanical disruption of fetal fibroblast monolayers caused a twofold increase in procollagenase mRNA. In contrast, the adult rabbit fibroblast procollagenase mRNA remained unchanged. The mRNA data correlated well with enzyme protein levels. Quantitation by immunoprecipitation showed a 2.3-fold increase in fetal fibroblast procollagenase protein after mechanical injury, whereas the level in adult rabbit fibroblasts remained unchanged. However, it was noted that the constitutive levels of procollagenase mRNA and protein were higher in adult fibroblasts. Analysis of enzyme activity, by means of a fluorogenic substrate, showed that adult fibroblasts had 2.2 times more collagenase activity compared with fetal cells. After mechanical injury, the fetal fibroblast collagenase activity increased 1.3-fold compared with 1.7-fold in the adult fibroblasts. In contrast, fetal fibroblast gelatinase activity was 1.25 times greater than in adult cells and increased 1.4-fold after mechanical injury, whereas the adult profile remained unchanged. Immunolocalization studies indicated that 1 hour after mechanical injury, procollagenase was produced primarily by fibroblasts along the mechanical injury ridge. By 4 hours after injury, the ridge cells began to migrate out into the open area and procollagenase was noted in adjacent cells of both adult and fetal origin. By 12 and 18 hours, all cells throughout the monolayer were expressing procollagenase. These findings show that in vitro fetal fibroblasts actually had lower levels of procollagenase, but higher levels of gelatinase compared with adult fibroblasts. The increased gelatinase expression may explain why fetal wounds do not have excessive collagen accumulation and heal without a visible scar.

Journal Article↗
PubMedDate not supplied

Autocrine growth factor production by fetal, keloid, and normal dermal fibroblasts.

OBJECTIVE: To evaluate differences in fibroblast autocrine growth factor production by human fetal, keloid, and normal adult dermal fibroblasts. DESIGN: Serum-free cell lines of fetal, keloid, and normal adult dermal fibroblasts were established. Cell counts were performed and supernatants collected at 4, 24, and 72 hours. Cell-free supernatants were quantitatively assayed for transforming growth factor beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF). RESULTS: Population doubling times for fetal, keloid, and normal adult fibroblasts were 120.0, 88.1, and 128.4 hours, respectively. Differences in population doubling times did not reach statistical significance. Statistically significant differences between TGF-beta1 levels from fetal and normal adult fibroblasts were seen at 24 and 72 hours. Significant differences between TGF-beta1 levels from keloid and normal adult fibroblasts were also seen at 24 and 72 hours. Fetal fibroblasts demonstrated higher levels of bFGF than normal adult fibroblasts at each time point, but these differences were not statistically significant. No significant differences were observed between keloid and normal adult bFGF levels. CONCLUSIONS: Both fetal and keloid fibroblasts produce significantly more TGF-beta1 than normal adult fibroblasts. Our data and the data of others suggest that fetal fibroblasts produce more bFGF than adult fibroblasts. The serum-free model we describe can be used to quantitatively measure autocrine growth factor production by cells that underlie clinically different types of wound healing. This model provides information that may allow us to better treat and prevent undesirable scarring.

Adolescent↗
PubMed2026

Senescent fibroblasts drive CD8+ T cell dysfunction in colorectal cancer via CD36-mediated lipid transfer and peroxidation.

BACKGROUND: Functional exhaustion of tumor-infiltrating CD8+ T cells represents a hallmark of colorectal cancer (CRC) immunosuppression, though its mechanistic drivers remain elusive. Given the established correlation between CRC progression and stromal senescence characterized by pathological lipid accumulation and impaired immunity, we investigated whether and how senescent fibroblasts actively regulate CD8+ T cell dysfunction. METHODS: Single-cell RNA sequencing (scRNA-seq) analysis was conducted to unveil the diverse fibroblast populations and the significant lipid metabolism changes between senescent fibroblasts and non-senescent fibroblasts in human CRC specimens and adjacent normal mucosa. Machine-learning identified senescent fibroblasts with a distinct gene signature. Cell-cell communication analysis was used to evaluate the interactions between senescent fibroblasts and CD8+ T cells in colorectal cancer. Co-culture experiments were conducted among senescent fibroblasts, CD8+ T cells and patient-derived organoids of CRC (CRC-PDOs), with the results evaluated with high-content imaging and propidium iodide/Hoechst 33,342 staining. Flow cytometry, ELISA and lipid pulse-chase with BODIPY FL C16 were performed to detect the alterations of CD8+ T cell cytotoxic function and metabolic status. AOM/DSS-induced CRC mouse model was used to conduct in vivo validation to evaluate whether senolytics could suppress CRC progression. Patients from the Cancer Genome Atlas colorectal cancer cohort were stratified into CD36-high and CD36-low groups by median expression, and drug sensitivity for GDSC2 compounds was predicted computationally using the oncoPredict R package. RESULTS: ScRNA-seq demonstrated the specific cell population presence and divergence of senescent fibroblasts between neoplastic and histologically normal adjacent cell clusters in CRC. Random Forest was employed for cell senescence classification. Feature importance analysis identified five genes as key contributors to the model&#x2019;s decision process. Cell-cell communication analysis revealed enhanced interactions between senescent fibroblasts and CD8+ T cells in CRC. Co-culture of senescent fibroblasts significantly impaired the cytotoxic functions of CD8+ T cells on CRC-PDOs, which was reflected by the declined proportions of granzyme B (GZMB) + and interferon gamma (IFN&#x3b3;) + CD8+ T cells and enhanced viability of CRC-PDOs. Mechanistically, the co-culture with senescent fibroblasts promoted the lipid shuttling into CD8+ T cells to induce lipid peroxidation and downstream impairment of cytotoxicity. Furthermore, the inhibition of CD36, the specific scavenger receptor for lipid uptake of CD8+ T cells, effectively suppressed lipid transfer and peroxidation thereby preserving the effector functions of CD8+ T cells and ultimately promoting tumor apoptosis. Complementarily, in vivo senolytic treatment significantly suppressed CRC progression in AOM-DSS CRC mouse models. Top 12 therapeutic agents were identified significantly enhanced predicted efficacy in CD36-high tumors. CONCLUSIONS: Our study identified a substantial population of senescent fibroblasts in human CRC through single cell transcriptomics, machine-learning and clinical biopsies. These senescent fibroblasts impair CD8+ T cell-mediated killing of CRC-PDOs via CD36-dependent lipid transfer, suggesting senolytic targeting of stromal cells as a promising immunotherapeutic strategy for CRC.

Colorectal Neoplasms↗
PubMed1996

Conversion of dermal fibroblasts to a myogenic lineage is induced by a soluble factor derived from myoblasts.

The limb and axial skeletal muscles of mammals originate from somitic dermomyotome, which during early development separates to form two discrete structures, the dermatome and the myotome. The latter cell mass gives rise to the muscle-forming lineage while cells of the dermatome will form the skin dermal fibroblast population of the dorsal regions of the body. It has been generally accepted for some time that myotome-derived myoblasts were the sole source of muscle fibre nuclei, but evidence has recently been presented from several laboratories that fibroblasts can fuse with myoblasts to contribute active nuclei to the resulting myotubes. We report here an investigation into the myogenic capacity of fibroblasts. Confluent monocultures of mouse dermal fibroblasts, muscle fibroblasts, and C2C12 myoblasts each retain their individual phenotype when maintained for periods up to 7 days in culture. We also grew isolated colonies of fibroblasts and myoblasts in an arrangement which allowed free exchange of tissue culture medium between the 2 cell types. We found evidence of the conversion of dermal fibroblasts to a myogenic lineage as measured by the appearance of MyoD-positive cells expressing the muscle-specific intermediate filament desmin. In addition, dermal fibroblast cultures contained multinucleate syncytia positive for MyoD and containing sarcomeric myosin heavy chain. In contrast, muscle-derived fibroblasts showed no evidence of myogenic conversion when maintained in identical culture conditions. We prepared conditioned medium from confluent cultures of C2C12 myoblasts and added this material to confluent monocultures of either dermal or muscle fibroblasts. While muscle fibroblasts showed no phenotypic alterations, cultures of dermal fibroblasts responded to myoblast conditioned medium by converting to a myogenic lineage as judged by expression of MyoD and desmin. We conclude that a proportion of dermal fibroblasts retain a myogenic capacity into stages well beyond their early association with myoblasts in the dermomyotome.

Animals↗
PubMed1998

Transcriptional activation of the alpha1(I) procollagen gene and up-regulation of alpha1(I) and alpha1(III) procollagen messenger RNA in dermal fibroblasts from tight skin 2 mice.

OBJECTIVE: To investigate the levels of expression of type I and type III collagen genes in dermal fibroblasts from tight skin 2 (Tsk2) and normal mice and to examine the transcriptional regulation of the alpha1(I) procollagen gene (COL1A1) in these cells. METHODS: Dermal fibroblasts from Tsk2 mice and from normal age- and sex-matched control mice were studied. Steady-state levels of alpha1(I) and alpha1(III) procollagen messenger RNA (mRNA) were evaluated by Northern and dot-blot hybridization analyses. The transcriptional regulation of COL1A1 was examined by transient transfection experiments with deletion constructs containing portions of the COL1A1 promoter ligated to the chloramphenicol acetyltransferase reporter gene. To identify DNA binding proteins that interact with regulatory elements within the COL1A1 promoter, gel mobility shift assays were performed with nuclear extracts prepared from normal and Tsk2 dermal fibroblasts. RESULTS: Synthesis of collagen was almost 100% higher in Tsk2 dermal fibroblasts than in control fibroblasts. Up-regulation of mRNA for 2 extracellular matrix proteins was observed in the Tsk2 dermal fibroblasts compared with the normal cells: the alpha1(I) procollagen mRNA steady-state levels were 50% higher, and those of the alpha1(III) procollagen mRNA 100% higher, in Tsk2 cells. The results of transient transfection experiments with COL1A1 promoter constructs demonstrated that the elevated levels of alpha1(I) collagen mRNA in Tsk2 cells were largely due to increased transcriptional activity of the corresponding gene. Electrophoretic mobility shift assays performed with a probe encompassing a relevant COL1A1 promoter region revealed increased DNA-protein binding activities in nuclear extracts prepared from Tsk2 fibroblasts compared with normal fibroblasts. Competition experiments using consensus Spl and nuclear factor 1 (NF-1) oligonucleotides and supershift experiments using anti-Sp1 and anti-NF-1 antibodies indicated that at least 2 transcription factors, Sp1 and NF-1, or their homologs are involved in the up-regulated transcriptional activity of the COL1A1 promoter in Tsk2 fibroblasts. CONCLUSION: Dermal fibroblasts from Tsk2 mice display increased collagen synthesis and up-regulation of alpha1(I) and alpha1(III) procollagen mRNA in vitro. The data also directly demonstrate the transcriptional activation of COL1A1 in dermal fibroblasts from Tsk2 mice and suggest that the transcription factors Sp1 and NF-1 or their homologs play an important role in the upregulated expression of this gene in Tsk2 fibroblasts. These findings are similar to those described for fibroblasts from humans with systemic sclerosis and validate the use of Tsk2 as a model for the study of the connective tissue alterations in this disease.

Animals↗
PubMed2001

Decreased susceptibility to Fas-induced apoptosis of systemic sclerosis dermal fibroblasts.

OBJECTIVE: To determine whether dysregulated apoptosis of systemic sclerosis (SSc) fibroblasts contributes to progressive fibrosis by promoting fibroblast longevity. METHODS: We examined the pattern of fibroblast proliferation and apoptosis in SSc skin lesions and the susceptibility of cultured SSc dermal fibroblasts to apoptosis. Skin biopsy samples from SSc patients and control subjects were used to establish fibroblast cultures and were examined histologically. In skin sections, apoptosis was examined by TUNEL, and proliferation by immunostaining for proliferating cell nuclear antigen. Susceptibility of fibroblasts to apoptosis induced in vitro by different stimuli was studied by TUNEL. Expression of Bcl-2, Bcl-x, and Bax proteins in cultured fibroblasts was studied by Western blotting. RESULTS: Proliferation of dermal fibroblasts was not observed in normal skin but was present in skin from patients with SSc and other inflammatory skin diseases. Apoptosis of fibroblasts in SSc fibrotic skin lesions was not observed. In vitro, SSc fibroblasts were specifically resistant to apoptosis induced by Fas receptor stimulation but had normal susceptibility to apoptosis induced by nonspecific stimuli (protein kinase inhibition or serum withdrawal). Decreased susceptibility to Fas stimulation was not caused by decreased levels of surface Fas receptor. In SSc fibroblasts, quiescence induced by confluence and serum starvation was followed by an abnormal down-regulation of proapoptotic Bax protein. Up-regulation of the Bax:Bcl-2 ratio in SSc fibroblasts by Bcl-2 antisense oligonucleotides restored their susceptibility to Fas-mediated apoptosis. CONCLUSION: Our findings suggest that abnormal apoptotic regulation in fibroblasts can contribute to the pathogenesis of progressive fibrosis in SSc. Modulation of Bcl-2-related proteins appears to be a potential target for the development of apoptosis-based antifibrotic strategies.

Adult↗
PubMed2002

Increased transcriptional activities of transforming growth factor beta receptors in scleroderma fibroblasts.

OBJECTIVE: To investigate the molecular mechanism of the overexpression of transforming growth factor beta receptors (TGF(beta)Rs) in dermal fibroblasts from patients with systemic sclerosis (SSc). METHODS: Dermal fibroblasts from 7 patients with diffuse SSc of recent onset and from 7 healthy individuals were studied. The expression of TGF(beta)R type I (TGF(beta)RI), TGF(beta)RII, and type I collagen proteins in dermal fibroblasts was determined by immunoblotting. TGF(beta)RI, TGF(beta)RII, and alpha2(I) collagen messenger RNA (mRNA) were evaluated by Northern blot analysis. The transcriptional activities of the TGF(beta)RI and TGF(beta)RII genes were examined by luciferase assay. RESULTS: SSc fibroblasts expressed increased levels of TGF(beta)RI and TGF(beta)RII protein and mRNA, as well as increased levels of type I collagen protein and alpha2(I) collagen mRNA. Moreover, the half-lives of TGF(beta)RI and TGF(beta)RII mRNA in SSc fibroblasts did not change compared with those in control dermal fibroblasts. The promoter activities of the TGF(beta)RI and TGF(beta)RII genes were both significantly increased in SSc fibroblasts compared with those in control fibroblasts. Calphostin C, a specific inhibitor of protein kinase C (PKC), inhibited TGF(beta)RI promoter activity in SSc fibroblasts, and LY294002, an inhibitor of phosphoinositide 3-kinase (PI 3-kinase), inhibited TGF(beta)RII promoter activity in SSc fibroblasts. Moreover, calphostin C and LY294002 inhibited the up-regulation of TGF(beta)RI and TGF(beta)RII mRNA, respectively, in SSc fibroblasts. CONCLUSION: These results suggest that increased levels of TGF(beta)Rs in SSc fibroblasts play a role in excessive collagen production, and that up-regulation of TGF(beta)R expression might occur at the transcriptional levels. PKC and/or PI 3-kinase might contribute to the up-regulation of TGF(beta)R expression in SSc fibroblasts.

Activin Receptors, Type I↗
PubMed2001

Fetal and adult human skin fibroblasts display intrinsic differences in contractile capacity.

One of the differences between fetal and adult skin healing is the unique ability of fetal wounds to heal without contracture and scar formation. Studies have shown that the ratio between the three isoforms of TGFbeta is different in adult and fetal wounds. Thus, we analyzed the capacity of adult and fetal human skin fibroblasts to contract collagen gels after stimulation with TGFbeta isoforms. In control medium, fetal fibroblasts had a contractile capacity similar to that of adult fibroblasts. However, the growth capacity of fetal fibroblasts was completely inhibited, in contrast to adult fibroblasts. When cells were treated with TGFbeta, fetal fibroblasts showed an inhibition of their contractile capacity whereas adult fibroblasts further contracted gels. The contractile response was similar for all isoforms of TGFbeta although TGFbeta3 always had the strongest effect. We considered that the regulation of cell contractile capacity by TGFbeta may be dependent on receptor expression for this cytokine, on myofibroblast differentiation of the cells, or in cell links with matrix. Since TGFbeta receptor analysis did not show differences in receptor affinity, we studied the expression of alpha-smooth muscle (SM) actin, a fibroblast contractile marker and of three integrins, the cell surface receptors specific of the attachment of the fibroblasts with collagen matrix. We observed that the expression of alpha-SM actin and alpha3 and beta1 integrin subunits was increased when TGFbeta was added to the medium of adult fibroblasts whereas the levels of the alpha1 and alpha2 subunits were unchanged. In contrast, fetal fibroblasts treated with TGFbeta showed a decrease of alpha1, alpha2, and beta1 integrin expression but no change in alpha3 integrin and in alpha-SM actin expression. These results indicate that intrinsic differences between fetal and adult fibroblasts might explain their opposite responses to TGFbeta stimuli. The variations in their alpha-SM actin and integrin expression patterns represent potentially important mechanisms used by fetal fibroblasts to regulate their response to cytokines, and likely contribute to the resultant differences in the quality of wound repair.

Actins↗
PubMedDate not supplied

Different effects of growth factors on proliferation and matrix production of normal and fibrotic human lung fibroblasts.

OBJECTIVES AND METHODS: In idiopathic pulmonary fibrosis (IPF), proliferation of fibroblasts and increased matrix deposition result in pulmonary damage and respiratory insufficiency. We cultured human fibroblasts from lung biopsies of healthy adults and of three patients with IPF (histologically usual interstital pneumonitis, UIP) in order to compare proliferation ([(3)H]thymidine incorporation, cell count) and matrix protein expression (immune fluorescence, quantification of fibronectin synthesis using time-resolved immune fluorescence) of normal and UIP fibroblasts in response to various growth factors. FINDINGS: The growth factors platelet-derived growth factor-BB (PDGF), epidermal growth factor (EGF), insulin growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), tumor necrosis factor alpha (TNFalpha), Transforming growth factor-beta (TGFbeta(1)), and fibroblast growth factor-2 (FGF-2) stimulate proliferation of normal lung fibroblasts significantly more than proliferation of UIP fibroblasts. Immunofluorescence reveals extensive expression of collagen I, collagen III, and fibronectin induced by serum, TGFbeta(1), and TNFalpha. This expression is more pronounced in UIP fibroblasts than in normal fibroblasts. Quantification of fibronectin synthesis reveals an enhanced fibronectin synthesis by UIP fibroblasts in response to PDGF, EGF, IGF-1, IGF-2, TNFalpha, TGFbeta(1), and FGF-2). CONCLUSIONS: Fibroblasts from normal and UIP lungs differ in their response to growth factors: Whereas normal fibroblasts show a predominantly proliferative response, UIP fibroblasts show an enhanced synthetic activity. Different fibroblast responses may contribute to progressive pulmonary fibrosis in patients with UIP.

Actins↗
PubMed1998

Contact-dependent inhibition of angiogenesis by cardiac fibroblasts in three-dimensional fibrin gels in vitro: implications for microvascular network remodeling and coronary collateral formation.

Angiogenesis and coronary artery collateral formation can improve blood flow and thereby prevent myocardial ischemia. The role of perivascular fibroblasts in neovascularization remains incompletely understood. Here we investigated the effects of epicardial and myocardial fibroblasts on angiogenesis in vitro by using a serum-free microcarrier-based fibrin gel angiogenesis system. To clearly distinguish between different cell types, we either stained endothelial cells or fibroblasts in the living with 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine-perchlorate (DiI). In cocultures, low numbers of heart fibroblasts stimulated endothelial sprouting, and capillary growth was also induced by fibroblast-conditioned media, indicating a paracrine mechanism. Capillary formation was decreased by increasing the density of fibroblasts in the cocultures, indicating contact-dependent inhibition. Using time-lapse studies, it turned out that close contacts between fibroblasts and endothelial cells resulted in rapid retraction of endothelial cells or, rarely, in cell death. Depending on the local ratio of fibroblasts to endothelial cell numbers, fibroblasts determined the location of capillary growth and the size of developing capillaries and thereby contributed to capillary network remodeling. In contrast to primary heart fibroblasts, NIH 3T3 fibroblasts did not display contact-dependent inhibition of endothelial sprouts. NIH fibroblasts were frequently seen in close association with endothelial capillaries, resembling pericytes. Contact-dependent inhibition of angiogenesis by epicardial fibroblasts could not be reversed by addition of neutralizing anti-TGF-beta1 antibodies, by addition of serum, of medium conditioned by hypoxic tumor cells or myocardium, by various cytokines or by growing cocultures under hypoxic conditions. Our results implicate a pivotal role of periendothelial mesenchymal cells for the regulation of microvascular network remodeling and collateral formation.

Animals↗
PubMed1985

Comparative observation of fibroblasts derived from the papillary and reticular dermis of infants and adults: growth kinetics, packing density at confluence and surface morphology.

We have confirmed the reports of Harper and Grove (Science, 204 (1979) 526-527), and Azzarone and Macierira-Coehlo (J. Cell Sci., 57 (1982) 177-187) that fibroblasts derived from the papillary dermis have greater in vitro growth potential and longer replicative lifespans than genomically identical fibroblasts derived from the reticular dermis. In addition we demonstrate that the kinetics of cell replication differ for papillary and reticular fibroblasts derived from infant and adult donors. Infant papillary fibroblasts replicate at faster rates than reticular fibroblasts throughout the growth cycle. Adult papillary and reticular fibroblasts replicate at similar rates at low cell densities, but exponential growth of reticular fibroblasts slows at lower cell densities than papillary fibroblasts suggesting that they are more sensitive to density-dependent inhibition of replication. The surface morphologies of reticular fibroblasts and papillary fibroblasts at confluence correlate with their growth kinetics. The decreased cell yields of reticular fibroblasts appears related to the spreading behaviors of individual cells which stretch and occupy more area of the growth surface than do papillary fibroblasts. These data and the reports cited clearly show that one must account for the presence of at least two distinct populations of dermal fibroblasts when examining their biological properties in vitro.

Adult↗
PubMed1979

Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin.

Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secretd spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblasts attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kideny cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells into the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-in-soluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.

Animals↗
PubMed1993

Basic fibroblast growth factor promotes the survival of embryonic ventral mesencephalic dopaminergic neurons--I. Effects in vitro.

We have studied the effects of basic fibroblast growth factor on rat embryonic mesencephalic neurons in vitro. Basic fibroblast growth factor promotes the survival of dopaminergic neurons in vitro, the effect increasing with dose and reaching a maximum at 10 ng/ml. In the absence of basic fibroblast growth factor the number of tyrosine hydroxylase-stained (tyrosine hydroxylase positive) neurons declines to almost zero within 14 days, whereas in the presence of basic fibroblast growth factor numbers remain almost constant from three to 28 days in vitro. This effect of basic fibroblast growth factor is abolished by preventing non-neuronal cells from appearing in the cultures, apart from a basic fibroblast growth factor-mediated increase in the numbers of tyrosine hydroxylase-positive cells during the first two days in vitro. The presence or absence of non-neuronal cells also influences dopaminergic neuronal morphology, the neurons having more, longer, and more varicose processes in the absence of astrocytes. Survival of dopaminergic neurons in vitro in the absence of basic fibroblast growth factor is very dependent on plating cell density, but in the presence of basic fibroblast growth factor this dependency vanishes. It is also possible to make survival independent of plating density by growing the cultures on inverted coverslips, which have the effect of concentrating secreted molecules in the thin layer of medium between coverslip and dish. Our conclusions from these experiments on plating density are that astrocytes probably constitutively secrete a small amount of a trophic factor which promotes survival of dopaminergic neurons, and that the rate of production of this factor is greatly increased by basic fibroblast growth factor. If basic fibroblast growth factor is withdrawn from cultures after two or seven days the dopaminergic neurons soon die. However, if basic fibroblast growth factor is withdrawn after 14 days, after the period of naturally occurring cell death of these neurons, there is no increase in dopaminergic neuronal death compared to controls in which basic fibroblast growth factor treatment is maintained. If basic fibroblast growth factor is used to improve the survival of dopaminergic neurons grafted in vivo, it should therefore be sufficient to treat the grafts for 14 days.

Animals↗
PubMed1996

Expression and localization of basic fibroblast growth factor (bFGF) in the repair process of rat liver injury.

BACKGROUND/AIMS: To clarify the expression and localization of basic fibroblast growth factor in the repair process of liver injury, acute liver injury was induced by administration of carbon tetrachloride, D-glactosamine hydrochloride, or dimethylnitrosamine to rats. METHODS: We measured basic fibroblast growth factor protein in the liver tissue by radioimmunoassay, evaluated the expression of basic fibroblast growth factor mRNA by the reverse transcriptase polymerase chain reaction, and identified basic fibroblast growth factor-positive cells by immunostaining. RESULTS: In the carbon tetrachloride injured liver, the basic fibroblast growth factor protein contents began to increase 2 days after administration when liver injury was most marked, and reached a peak after 4 days, decreasing thereafter. In the carbon tetrachloride-injured liver, basic fibroblast growth factor mRNA expression was observed from 12 h after administration, prior to an increase in the protein content. In the D-galactosamine hydrochloride-injured liver, basic fibroblast growth factor protein also increased. On the other hand, in the dimethylnitrosamine-injured liver, the basic fibroblast growth factor protein content decreased 2 days after administration when liver injury was marked, but increased after 7 days. In the regenerating liver after partial hepatectomy, the basic fibroblast growth factor protein content did not increase. Among cell fractions, the Ito cell fraction obtained from the carbon tetrachloride-injured liver after 4 days showed expression of basic fibroblast growth factor mRNA. In cells cultured for 24 h, this fraction was immunopositive for basic fibroblast growth factor. Ito cells in the liver tissue markedly increased in the carbon tetrachloride-injured liver and increased after 7 days in the dimethylnitrosamine-injured liver. CONCLUSIONS: This study confirmed basic fibroblast growth factor production in the liver tissue in the repair process of liver injury. Our results suggest that basic fibroblast growth factor is primarily produced in Ito cells, acts on sinusoidal wall cells including Ito cells by the autocrine and paracrine mechanisms, and promotes extracellular matrix production and vascularization, involving the repair process of liver injury.

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