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PubMed1997

Serum-free keloid fibroblast cell culture: an in vitro model for the study of aberrant wound healing.

The purpose of this study was to develop an in vitro serum-free keloid fibroblast model. Keloid formation remains a problem for every surgeon. Prior evaluations of fibroblast characteristics in vitro, especially those of growth factor measurement, have been confounded by the presence of serum-containing tissue culture media. The serum itself contains growth factors, yet has been a "necessary evil" to sustain cell growth. The design of this study is laboratory-based and uses keloid fibroblasts obtained from five patients undergoing facial (ear lobule) keloid removal in a university-affiliated clinic. Keloid fibroblasts were established in primary cell culture and then propagated in a serum-free environment. The main outcome measures included sustained keloid fibroblast growth and viability, which was comparable to serum-based models. The keloid fibroblast cell cultures exhibited logarithmic growth, sustained a high cellular viability, maintained a monolayer, and displayed contact inhibition. Demonstrating model consistency, there was no statistically significant difference between the mean cell counts of the five keloid fibroblast cell lines at each experimental time point. The in vitro growth of keloid fibroblasts in a serum-free model has not been done previous to this study. The results of this study indicate that the proliferative characteristics described are comparable to those of serum-based models. The described model will facilitate the evaluation of potential wound healing modulators, and cellular effects and collagen modifications of laser resurfacing techniques, and may serve as a harvest source for contaminant-free fibroblast autoimplants. Perhaps its greatest utility will be in the evaluation of endogenous and exogenous growth factors.

Cell Division↗

PubMed1998

Epidermal regulation of dermal fibroblast activity.

Although the association between delayed burn wound healing and subsequent hypertrophic scar formation is well-established, the mechanism for this relationship is unknown. Unhealed burn wounds lack an epidermis, suggesting a possible regulatory role for the epidermis in controlling dermal fibroblast matrix synthesis. Therefore, we examined the effect of epidermal cells and media conditioned by epidermal cells on fibroblast collagen synthesis and replication. Purified fibroblast and keratinocyte cell strains were developed from discarded normal adult human skin. Conditioned media were created by incubation of cytokine-free and serum-free medium with either confluent fibroblast or keratinocyte cultures for 18 hours (n = 3). Nearly confluent fibroblast cultures were exposed for 48 hours to graded concentrations of either unconditioned medium (control), conditioned medium, or varying numbers of keratinocytes. Replication was quantified by the incorporation of 3H-thymidine. Collagen synthesis was measured by the incorporation of 3H-proline into collagenase-sensitive protein. Data were compared using analysis of variance (ANOVA) and linear regression. Keratinocyte conditioned medium induced a significant increase in replication (n = 3) (p = 0.004) and a decrease in collagen synthesis (n = 6) (p < 0.001). In contrast, neither fibroblast conditioned medium nor control medium had an effect on fibroblast replication or collagen synthesis. Co-culture of fibroblast with a graded number of keratinocytes similarly decreased collagen synthesis (n = 6) (p < 0.001). Dermal fibroblast collagen synthesis appears to be regulated by a soluble keratinocyte product. This result suggests a mechanism for the clinical observation that unhealed burn wounds, which lack the epidermis, demonstrate excess collagen production and scar. Clinical strategies to decrease hypertrophic scar should include an attempt at early wound closure with skin grafting or the application of cultured epithelial autografts.

Adult↗

PubMed2000

Cellular signaling by tyrosine phosphorylation in keloid and normal human dermal fibroblasts.

Keloids represent a dysregulated response to cutaneous wounding that results in disfiguring scars. Unique to humans, keloids are characterized by an accumulation of extracellular matrix components. The underlying molecular mechanisms of keloid pathogenesis, however, remain largely uncharacterized. Similarly, cellular signaling mechanisms, which may indicate inherent differences in the way keloid fibroblasts and normal human dermal fibroblasts interact with extracellular matrix or other cells, have not been investigated. As part of a fundamental assessment of cellular response to injury in keloid fibroblasts, phosphorylation studies were performed using three different keloid (n = 3) and normal human dermal (n = 3) fibroblast cell lines. These studies were undertaken to elucidate whether keloid and normal human dermal fibroblasts exhibit different tyrosine kinase activity. Initially, distinct tyrosine phosphorylation patterns of keloid and normal human dermal fibroblasts were demonstrated. Next, the phosphorylation patterns were correlated with known molecules that may be important to keloid pathogenesis. On the basis of molecular weight, it was hypothesized that the highly phosphorylated bands seen in keloid fibroblasts represented epidermal growth factor receptor (EGFR); discoidin domain receptor 1 (DDR1); and Shc, an adaptor protein known to bind many tyrosine kinases, including EGFR and DDR1. Individual immunoblotting using EGFR, DDR1, and Shc antibodies revealed greater expression in keloid fibroblasts compared with normal human dermal fibroblasts. These data substantiate for the first time the finding of greater phosphorylation by the above-mentioned molecules, which may be important in keloid pathogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Investigation of the influence of keloid-derived keratinocytes on fibroblast growth and proliferation in vitro.

Keloids are disfiguring, proliferative scars that represent a pathological response to cutaneous injury. The overabundant extracellular matrix formation, largely from collagen deposition, is characteristic of these lesions and has led to investigations into the role of the fibroblast in its pathogenesis. Curiously, the role of the epidermis in extracellular matrix collagen deposition of normal skin has been established, but a similar hypothesis in keloids has not been investigated. The aim of this study was to investigate the influence of keloid epithelial keratinocytes on the growth and proliferation of normal fibroblasts in an in vitro serum-free co-culture system. A permeable membrane separated two chambers; the upper chamber contained a fully differentiated stratified epithelium derived from the skin of excised earlobe keloid specimens, whereas the lower chamber contained a monolayer of normal or keloid fibroblasts. Both cell types were nourished by serum-free medium from the lower chamber. Epithelial keratinocytes from five separate earlobe keloid specimens were investigated. Four sets of quadruplicates were performed for each specimen co-cultured with normal fibroblasts or keloid-derived fibroblasts. Controls consisted of (1) normal keratinocytes co-cultured with normal fibroblasts, and (2) fibroblasts grown in serum-free media in the absence of keratinocytes in the upper chamber. Fibroblasts were indirectly quantified by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay, with results confirmed by DNA content measurement, at days 1 and 5 after the co- culture initiation.Significantly, increased proliferation was seen in fibroblasts co-cultured with keloid keratinocytes, as compared with the normal keratinocyte controls at day 5 (analysis of variance, p < 0.001). These results strongly suggest that the overlying epidermal keratinocytes of the keloid may have an important, previously unappreciated role in keloid pathogenesis using paracrine or epithelial-mesenchymal signaling.

Adolescent↗

PubMed2004

Prostaglandin E2 affects proliferation and collagen synthesis by human patellar tendon fibroblasts.

OBJECTIVE: To determine the effect of prostaglandin E2 on proliferation and collagen synthesis by human patellar tendon fibroblasts. DESIGN AND SETTING: Controlled laboratory study. METHODS: Human patellar tendon fibroblasts were treated with different concentrations (1, 10, 100 ng/mL) of prostaglandin E2 in cultures. Fibroblasts without prostaglandin E2 treatment were used as the control group. The fibroblast proliferation and collagen synthesis were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and Sircol collagen assay, respectively. MAIN OUTCOME MEASURED: Changes in proliferation and collagen production of human patellar tendon fibroblasts. RESULTS: : At 1 ng/mL of prostaglandin E2, there was no significant effect on fibroblast proliferation compared with the control group. At concentrations of 10 ng/mL and 100 ng/mL prostaglandin E2, however, fibroblast proliferation significantly decreased, by 7.3% (P = 0.002) and 10.8% (P < 0.0001), respectively, compared with the control group. At 1 ng/mL of prostaglandin E2, collagen production of the tendon fibroblasts was unaffected. However, at both 10 ng/mL and 100 ng/mL prostaglandin E2, collagen production was significantly decreased, by 45.2% (P < 0.0001) and 45.7% (P < 0.0001), respectively, compared with the control group. The levels of collagen production between these 2 dosages did not differ significantly. CONCLUSIONS: Prostaglandin E2 affects the proliferation of and collagen production by human patellar tendon fibroblasts in a dosage-dependent manner. CLINICAL RELEVANCE: Based on these in vitro findings, we speculate that production of prostaglandin E2 in tendons might play some role in the acellularity and matrix disorganization seen in exercise-induced tendinopathy.

Cells, Cultured↗

PubMed2004

Cellular origins of fibroblasts: possible implications for organ fibrosis in systemic sclerosis.

PURPOSE OF REVIEW: There is an intense interest in the potential of circulating blood cells and epithelium-related nonfibroblast cells to change into matrix synthesizing fibroblasts and myofibroblasts. These sources of fibroblasts may have importance in systemic sclerosis (scleroderma). RECENT FINDINGS: Epithelial cells from different sources can transition into fibroblasts and myofibroblasts in response to transforming growth factor beta and other growth factors/cytokines. This is called epithelial-mesenchymal transition (EMT). EMT has been repeatedly demonstrated to occur in several models of renal fibrosis including lupus prone mice. Quite unexpectedly, bone morphogenic protein 7 prevents EMT and protects lupus mice and other renal fibrosis models from developing fibrosis in the kidneys. Human peripheral blood mononuclear cells under different conditions of culture give rise to several different types of fibroblast-like cells. In SSc, it has been observed that the sera have low levels of serum amyloid protein. Serum amyloid protein has been found to inhibit the generation of fibrocytes from CD14 precursors. The implications of these potential sources of fibroblasts and myofibroblasts in systemic sclerosis and related rheumatic diseases are discussed. SUMMARY: Fibroblasts and myofibroblasts in skin and internal organs of patients with systemic sclerosis and related diseases may possibly arise not only from the resident fibroblast population but from epithelial cells, pericytes, monocytes, and other progenitors from the circulating pool of hematopoietic cells and stem cells. These alternative sources of fibroblasts would best be treated by specifically targeting the transition or transdifferentiation process by which cells change into fibroblasts.

Fibroblasts↗

PubMed2006

Pulmonary fibroblasts stimulate the proliferation of cell lines from human lung adenocarcinomas.

Human lung cancer cell lines are widely used to test anticancer drugs. These in-vitro tests, however, preclude the detection of responses to paracrine factors from surrounding stroma. We have cocultured pulmonary fibroblasts CCD-19Lu, from a healthy donor, or HLF-A, from a patient with epidermoid carcinoma of the lung, with two human pulmonary adenocarcinoma cell lines to test the hypothesis that the fibroblasts stimulate the growth of the tumor cells. Both fibroblast cell lines significantly increased the proliferation of the pulmonary adenocarcinoma cell lines in 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assays, with HLF-A fibroblasts yielding the most pronounced responses. The proliferation of the pulmonary adenocarcinoma cell lines in coculture with fibroblasts was blocked by antibodies against the transforming growth factor-alpha and amphiregulin. In addition, reverse transcription-polymerase chain reaction showed expression of mRNA for amphiregulin and transforming growth factor-alpha in all cell lines, whereas mRNA for the epidermal growth factor was detected only in pulmonary adenocarcinoma cell lines. Western blot analysis revealed that medium containing growth factors released by each fibroblast cell line activated extracellular signal-regulated kinase 1/2 in the both tested pulmonary adenocarcinoma cell lines, but activated Akt kinase only in A549 cells. Assessment of protein levels for cyclin D1 and cyclin E by Western blots demonstrated pronounced increases of both proteins in each pulmonary adenocarcinoma cell line, whereas protein levels for cyclin-dependent kinase inhibitor p21 remained unchanged. Immunocytochemical analysis showed positive immunoreactivity for P-extracellular signal-regulated kinase 1/2, cyclin D1 and cyclin E in pulmonary adenocarcinoma cells cocultured with fibroblasts or exposed to fibroblast-conditioned media. Our data suggest that the growth of pulmonary adenocarcinoma is stimulated by amphiregulin and transforming growth factor-alpha released from pulmonary fibroblasts. This may contribute to the disappointing clinical responses to anticancer drugs, which have shown promise in tests with lung cancer cell lines.

Adenocarcinoma↗

PubMed2005

Hypertrophic scar fibroblasts have increased connective tissue growth factor expression after transforming growth factor-beta stimulation.

BACKGROUND: Hypertrophic scars and keloids respond to dermal disruption with excessive collagen deposition and increased transforming growth factor (TFG)-beta expression. Connective tissue growth factor (CTGF) is a downstream mediator of TGF-beta activity that is associated with scar and fibrosis. The authors hypothesize that there is increased expression of CTGF by hypertrophic scar and keloid fibroblasts in response to TGF-beta stimulation. METHODS: Primary fibroblasts were isolated in culture from human hypertrophic scar (n = 2), keloid (n = 2), and normal skin (n = 2). After 18 hours of serum starvation, the cells were stimulated with 10 ng/ml of TGF-beta1, TGF-beta2, and TGF-beta3 for 24 hours. Quantitative real-time polymerase chain reaction was performed on extracted RNA samples to assay for CTGF mRNA expression. RESULTS: Baseline CTGF expression was increased 20-fold in unstimulated hypertrophic scar fibroblasts and 15-fold in keloid fibroblasts compared with normal fibroblasts. CTGF expression increased greater than 150-fold when stimulated with TGF-beta1 (p < 0.002) and greater than 100-fold when stimulated by TGF-beta2 or TGF-beta3 compared with normal fibroblasts (p < 0.02 and p < 0.002, respectively). CTGF expression was greatest after TGF-beta1 stimulation in hypertrophic scar fibroblasts compared with TGF-beta2 (p < 0.04) and TGF-beta3 (p < 0.02). Keloid fibroblast CTGF expression also increased greater than 100-fold after stimulation with TGF-beta1 (p = 0.16) and greater than 75-fold after addition of TGF-beta2 and TGF-beta3 (p = 0.06 and p = 0.22, respectively). CONCLUSIONS: Hypertrophic scar fibroblasts have both intrinsic up-regulation of CTGF transcription and an exaggerated capacity for CTGF transcription in response to TGF-beta stimulation. These data suggest that blockage of CTGF activity may reduce pathologic scar formation.

Adult↗

PubMed2006

A comparison of human and porcine acellularized dermis: interactions with human fibroblasts in vitro.

BACKGROUND: Dermal substitutes derived from xenograft materials require elaborate processing at a considerable cost. Acellularized porcine dermis is a readily available material associated with minimal immunogenicity. The objective of this study was to evaluate acellularized pig dermis as a scaffold for human fibroblasts. METHODS: In vitro methods were used to evaluate fibroblast adherence, proliferation, and migration on pig acellularized dermal matrix. Acellular human dermis was used as a control. RESULTS: Pig acellularized dermal matrix was found to be inferior to human acellularized dermal matrix as a scaffold for human fibroblasts. Significantly more samples of human acellularized dermal matrix (83 percent, n = 24; p < 0.05) demonstrated fibroblast infiltration below the cell-seeded surface than pig acellularized dermal matrix (31 percent, n = 49). Significantly more (p < 0.05) fibroblasts infiltrated below the surface of human acellularized dermal matrix (mean, 1072 +/- 80 cells per section; n = 16 samples) than pig acellularized dermal matrix (mean, 301 +/- 48 cells per section; n = 16 samples). Fibroblasts migrated significantly less (p < 0.05) distance from the cell-seeded pig acellularized dermal matrix surface than in the human acellularized dermal matrix (78.8 percent versus 38.3 percent cells within 150 mum from the surface, respectively; n = 5). Fibroblasts proliferated more rapidly (p < 0.05) on pig acellularized dermal matrix (n = 9) than on the human acellularized dermal matrix (7.4-fold increase in cell number versus 1.8-fold increase, respectively; n = 9 for human acellularized dermal matrix). There was no difference between the two materials with respect to fibroblast adherence (8120 versus 7436 average adherent cells per section, for pig and human acellularized dermal matrix, respectively; n = 20 in each group; p > 0.05). CONCLUSION: Preliminary findings suggest that substantial differences may exist between human fibroblast behavior in cell-matrix interactions of porcine and human acellularized dermis.

Adolescent↗

PubMed2003

Dietary compounds inhibit proliferation and contraction of keloid and hypertrophic scar-derived fibroblasts in vitro: therapeutic implication for excessive scarring.

BACKGROUND: Keloid and hypertrophic scars commonly occur after injuries. Overproliferation of fibroblasts, overproduction of collagen, and contraction characterize these pathologic scars. Current treatment of excessive scars with intralesional corticosteroid injections used individually or in combination with other methods often have unsatisfactory outcome, frustrating both the patient and the clinician. The phytochemical compounds are well known as potential anticancer agents. We have investigated the inhibitory effects of compounds on keloid fibroblasts (KF) and hypertrophic scar-derived fibroblasts (HSF). METHODS: Fibroblasts were cultured from nontreated earlobe keloids and burn hypertrophic scars. Ten compounds (three hydroxybenzoic and four hydroxycinnamic acid derivatives, two flavonols [quercetin and kaempferol], and turmeric curcumin) were tested with fibroblasts. The inhibitory effects of compounds on fibroblasts was assessed by proliferation assays, fibroblast-populated collagen lattice (FPCL) contraction, and electron microscopy. RESULTS: The phytochemicals significantly inhibited KF and HSF proliferation in a dose- and time-dependent manner. In the hydroxybenzoic and flavonol groups, increasing inhibitory effects seemed to depend on increasing numbers of hydroxyl groups in their chemical structures. This phenomenon was not observed in the hydroxycinnamic acid group. The phytochemicals inhibited fibroblast proliferation by inducing cell growth arrest but not apoptosis. The reversibility of growth inhibition occurred when the compounds were removed from the culture and fresh media was replaced. Slower reversibility of growth inhibition was observed in the groups treated with quercetin, chlorogenic acid, or curcumin. The compounds quercetin, gallic acid, protocatechuic acid, and chlorogenic acid were the strongest inhibitors of FPLC contraction by HTFs. When the compounds were washed out of the lattices and replaced by fresh medium, the FPCL contraction was resumed. The resumption of FPCL contraction was slowest in the quercetin-treated group, indicating again the strong inhibitory effect of quercetin. CONCLUSION: From this in vitro study, quercetin seemed to have good potent effects to inhibit proliferation and contraction of excessive scar-derived fibroblasts.

Adolescent↗

PubMed2003

Evidence for recipient derived fibroblast recruitment and activation during the development of chronic cardiac allograft rejection.

BACKGROUND: Allograft fibrosis is a prominent feature of chronic rejection. Although intragraft fibroblasts contribute to this process, their origin and exact role remain poorly understood. METHODS: Using a rat model of chronic rejection, LEW to F344, cardiac fibroblasts were isolated at the point of rejection and examined in a collagen gel contraction assay to measure fibroblast activation. The allograft microenvironment was examined using immunohistochemistry for fibrogenic markers (transforming growth factor [TGF]-beta, platelet-derived growth factor [PDGF], tissue plasminogen activator [TPA], plasminogen activator inhibitor [PAI]-1, matrix metalloproteinase [MMP]-2, and tissue inhibitor of matrix metalloproteinase [TIMP]-2). The origin of intragraft fibroblasts was studied using female to male allografts followed by polymerase chain reaction [PCR] and in situ hybridization for the male sry gene. RESULTS: The cardiac fibroblasts isolated from allografts with chronic rejection exhibited higher gel contractibility (50.9% +/- 6.1% and 68.2% +/- 3.8% at 4 and 24 hr) compared with naive cardiac fibroblasts (30.7% +/- 3.5% and 55.3% +/- 6.6% at 4 and 24 hr; P<0.05 and <0.05, respectively). Immunostaining for TGF-beta, PDGF, TPA, PAI-1, MMP-2 and TIMP-2 was observed in all allografts at the time of rejection. In situ hybridization demonstrated the presence of sry positive cells in female allografts rejected by male recipients. Sixty-five percent of fibroblast colonies (55 of 85) isolated from female heart allografts expressed the male sry gene. CONCLUSION: Cardiac fibroblasts are activated and exist in a profibrogenic microenvironment in allografts undergoing chronic rejection. A substantial proportion of intragraft fibroblasts are recruited from allograft recipients in this experimental model of chronic cardiac allograft rejection.

Animals↗

PubMed1985

Biochemical composition of the connective tissue in keloids and analysis of collagen metabolism in keloid fibroblast cultures.

Keloids are histologically characterized by an abundance of the extracellular matrix of connective tissue. In the present study, we examined the connective tissue composition of keloids, and analyzed the details of collagen metabolism utilizing fibroblast cultures established from keloid tissue. Quantitative connective tissue analyses indicated that collagen was the predominant extracellular matrix component in keloids. The ratio of genetically distinct collagens type I/III was significantly increased, as compared to normal human skin. Collagen biosynthesis was measured in fibroblast cultures by the formation of radioactive hydroxyproline: 5 of 9 keloid cell cultures studied demonstrated increased procollagen production in comparison to age-, sex-, and passage-matched control skin fibroblast lines, while the remaining 4 cell lines were within the control range. Keloid fibroblast cultures which were high collagen producers also demonstrated elevated prolyl hydroxylase activity. The mechanisms of increased procollagen production in fibroblast cultures were first examined by assaying the abundance of type I procollagen-specific mRNA utilizing dot blot hybridizations with a pro alpha 2(I)-chain-specific cDNA. The type I procollagen mRNA levels were significantly increased in 4 keloid fibroblast lines, and a good correlation between the mRNA levels and the rate of procollagen production in the same cultures was noted. These observations suggest regulation of the collagen gene expression on the transcriptional level. The catabolic pathway of collagen metabolism in fibroblast cultures was examined by determining the degradation of newly synthesized procollagen polypeptides through assay of radioactive hydroxyproline in small-molecular-weight peptide fragments. In 3 keloid cell cultures, the degradation of newly synthesized collagen polypeptides was below the range of normal controls. These findings suggest that a reduced degradation of newly synthesized polypeptides might contribute to the accumulation of procollagen in some keloid fibroblast cultures. The results of this study suggest two possible mechanisms for deposition of collagen in keloid lesions in vivo: first, the growth of the lesions may result from a localized loss of control of the extracellular matrix production by fibroblasts; secondly, reduced degradation of the newly synthesized procollagen polypeptides may contribute to collagen deposition in some keloids.

Adult↗

PubMed1996

Elevated levels of plasminogen activator inhibitor-1 may account for the altered fibrinolysis by keloid fibroblasts.

Using a 3-dimensional fibrin gel model system simulating fibroplasia of wound repair, we investigated the interaction between keloid fibroblasts and fibrin matrix and compared it with that of normal fibroblasts. Normal skin fibroblasts caused fibrin gel degradation under serum-free conditions, whereas keloid fibroblasts did not cause microscopically detectable gel degradation. Fibrin gel degradation occurred through plasmin-mediated fibrinolysis, which was initiated by fibroblasts exhibited high uPA but low plasminogen activator inhibitor-1 (PAI-1) activities, and transforming growth factor-beta 1 prevented fibrinolysis of normal fibroblasts by upregulating PAI-1 while downregulating uPA activities. In contrast, keloid fibroblasts exhibited an intrinsically high level of PAI-1 and a low level of uPA. This change in the ratio of activator and inhibitor activities was attributed to altered fibrin degradation by keloid fibroblasts. The PAI-1 increase was also demonstrated at the RNA level by Northern analysis. In terms of the pivotal role of the plasmin/plasminogen activator system in matrix remodeling, the elevated PAI-1 level exhibited by keloid fibroblasts may have significant consequences not only in altered fibrin degradation, but also in subsequent repair steps that lead to keloids and fibrosis.

Fibrin↗

PubMed1987

Identification of a protein in sera of normal humans that inhibits fibroblast chemotactic and random migration in vitro.

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

Blood Proteins↗

PubMed1987

Retinoid treatment of human psoriatic fibroblasts induces an increase in cyclic AMP-dependent protein kinase activity.

We recently showed a deficiency of cyclic AMP (cAMP)-dependent protein kinases in psoriatic cells. In this work the effects of retinoids on cAMP-dependent protein kinases of fibroblasts from 7 normal subjects and 7 psoriatic patients were studied. The levels of RI and RII (two forms of the cAMP-dependent protein kinases) present in control and retinoic acid-treated cells were quantitated by photoaffinity labeling with [8-azido-32P]cAMP. In psoriatic fibroblasts the levels of RII are decreased or undetectable compared with those of normal fibroblasts both in the cytosolic and membrane fractions. The amount of RI was normal in the cytosol of fibroblasts of 5 out of 7 patients and decreased in 2 patients. Membrane-associated levels of RI were decreased in 5 patients and normal in 2 patients. Retinoic acid treatment induces an increase in the amount of RI and RII regulatory subunits when they are deficient in the cytosolic and membrane fractions of psoriatic fibroblasts. Retinoic acid had no effect on RI and RII in normal fibroblasts. In addition, with in vitro retinoic acid treatment the cAMP-dependent protein kinase activity, measured in the fibroblasts of 4 psoriatic patients, was increased in the cytosol in 2 patients and in the membranes in all 4 patients. In these studies, comparable results were obtained with fibroblasts cultured from involved and uninvolved skin. This in vitro effect of retinoids on cAMP-dependent protein kinases in psoriatic fibroblasts may help to explain some of the in vivo therapeutic effects of retinoids.

Fibroblasts↗

PubMed1994

Regulation of gelatinase production in metastatic renal cell carcinoma by organ-specific fibroblasts.

We have recently established a human renal cell carcinoma KG-2 line that is tumorigenic in the subcutis (ectopic) and kidney (orthotopic) of nude mice but spontaneously metastasizes to the lung only after orthotopic implantation. KG-2 cells growing in the kidney (orthotopic) and lung metastases secreted higher levels of gelatinase than did cells growing in the subcutis (ectopic). We examined whether organ-specific fibroblasts play a role in the regulation of gelatinase production and invasion by renal carcinoma cells. The gelatinase level in the culture supernatants of KG-2 cells was increased by their cultivation with mouse kidney or lung fibroblasts. In contrast, cocultivation of KG-2 cells with mouse skin fibroblasts resulted in a significant reduction of gelatinase activity. Similar results were obtained by culturing KG-2 cells in the media conditioned by the different mouse fibroblasts. We, therefore, investigated effects on KG-2 cells of cytokines and growth factors known to be produced by fibroblasts of various origins. Of ten cytokines and growth factors tested, basic fibroblast growth factor, hepatocyte growth factor, and transforming growth factor-beta 1 (TGF-beta 1) stimulated gelatinase expression by the cultured KG-2 cells. Parallel immunohistochemical analyses revealed that mouse kidney and lung fibroblasts produced higher levels of TGF-beta 1 than did skin fibroblasts. These results indicate that gelatinase production by KG-2 renal cell carcinoma cells is influenced by the organ microenvironment. Specifically, organ-specific fibroblasts regulate the production of degradative enzymes by KG-2 cells and, hence, profoundly influence their invasive and metastatic capacity.

Animals↗

PubMed1996

Primary human fibroblasts induce diverse tumor invasiveness: involvement of HGF as an important paracrine factor.

Fibroblasts have been considered to play an important role in tumor progression. In order to evaluate the contribution of fibroblasts to tumor invasion, TE2-NS, an esophageal cancer cell line, was cultured on collagen gel containing primary fibroblasts derived from normal esophageal submucosa or cancerous tissues of seven esophageal cancer patients. TE2-NS showed diverse invasiveness into the underlying gel containing fibroblasts, but did not invade the gel not containing fibroblasts. The invasiveness of TE2-NS, which expressed hepatocyte growth factor (HGF) receptor, was well-correlated with the concentration of HGF in conditioned medium. Administration of neutralizing antibody against HGF effectively suppressed the invasion, but application of recombinant HGF without fibroblasts induced little invasion into the gel. Fibroblasts from non-cancerous tissue generally secreted a larger amount of HGF and induced tumor invasion to a greater extent than those from cancer tissue, with large diversity. Interestingly, HGF secretion of fibroblasts from non-cancerous tissue was stimulated by co-culture with TE2-NS in two lines, but not in the other four. These results indicate that HGF is an important paracrine factor which induces tumor cell invasion, and the diversity of HGF production by fibroblasts might suggest different potentiality to induce tumor invasion among patients.

Cadherins↗

PubMed1981

Increased haemopoietic cell survival in vitro induced by a human marrow fibroblast factor.

Human marrow fibroblasts were grown in vitro and examined for effects on human and mouse haemopoietic cells. When human marrow cells were incubated with fibroblasts or with fibroblast-conditioned medium for 1 week and then assayed for committed granulocyte/monocyte (CFU-c) and erythroid (BFU-e) progenitor cells, the numbers of CFU-c and BFU-e were considerably increased compared with controls. In contrast, human marrow-fibroblast-conditioned medium, when added directly to CFU-c or BFU-e assay cultures, had no effect on colony formation by these progenitor cells. As these results suggest that the fibroblast-derived factor may be acting on a relatively primitive progenitor cell, possibly a pluripotent haemopoietic stem cell, the effect of this factor on mouse pluirpotent haemopoietic stem cells (CFU-s) was examined. Human marrow-fibroblast-conditioned medium considerably enhanced CFU-s survival after a 24 h incubation and increased the proportion of CFU-s in cell-cycle. The increase in CFU-s survival depended on the concentration of the fibroblast-conditioned medium but not on the age of the fibroblast culture. The evidence suggests therefore that human marrow fibroblasts produce a factor that acts on a human myeloid progenitor cell more primitive than BFU-e and CFU-c, possibly the pluripotent haemopoietic stem cell.

Animals↗