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PubMed1999

Proliferation and mitogenic response to PDGF-BB of fibroblasts isolated from chronic venous leg ulcers is ulcer-age dependent.

Several pathophysiologic mechanisms have been proposed to explain slow-healing leg ulcers, but little is known about the growth behavior of cells in these wounds. Platelet-derived growth factor-BB applied topically to chronic wounds has shown beneficial effects, although the effects have been less pronounced than would have been expected based on studies on acute wounds. The objective of this study was to compare fibroblasts in culture obtained from chronic wounds (non-healing chronic venous leg ulcers), acute wounds and normal dermis regarding growth, mitogenic response to platelet-derived growth factor-BB and levels ofplatelet-derived growth factor alpha-receptor and beta-receptor. Fibroblasts were obtained by an explant technique and expanded in vitro using fibroblast growth medium supplemented with 10% fetal bovine serum and used for the assays at their third passage. Growth of chronic wound fibroblasts (n = 8) was significantly (p < 0.05) decreased compared with those from acute wounds (n = 10) and normal dermis (n = 5). Fibroblasts from ulcers older than 3 y grew significantly (p < 0.01) slower than those from ulcers that had been present for less than 3 y. Morphology and size of fibroblasts from the oldest chronic wounds deviated substantially from those of acute wounds and normal dermis, and resembled in vitro aged or senescent fibroblasts. Mitogenic response of chronic wound fibroblasts to human recombinant platelet-derived growth factor-BB was also reduced with ulcer age. No significant differences were found in the amount of either platelet-derived growth factor alpha-receptor or beta-receptor among the three groups. The features decreased growth related to ulcer age, altered morphology, and reduced response to platelet-derived growth factor, indicating that fibroblasts in some chronic wounds have approached or even reached the end of their lifespan (phase III). This might provide one explanation for the non-healing state and therapy resistance to topical platelet-derived growth factor-BB of some venous leg ulcers.

Aged↗

PubMed2000

Keloid fibroblasts resist ceramide-induced apoptosis by overexpression of insulin-like growth factor I receptor.

Keloids are benign dermal tumors, characterized by overgrowth of lesions, invasiveness beyond the original boundary of the insult, and recurrence of lesions. The exact etiology is unknown, however. Our hypothesis is that keloids are acquired as a result of an abnormal or prolonged wound healing process, with persistent proliferation and extracellular matrix production of fibroblasts that should otherwise discontinue in normal wound healing. In this study, we examined the response of keloid fibroblasts to proapoptotic signaling. Cell-permeable ceramide, N-acetyl-D-sphingosine, induced apoptosis of dermal fibroblasts in a dose- and time-dependent manner, which was detected by phase contrast microscopy, fluorescent microscopy, the TUNEL method, flow cytometric analysis, and WST-1 assay. In contrast, keloid fibroblasts resisted apoptosis induced by N-acetyl-D-sphingosine (percent survival with 40 mM ceramide treatment for 12 h, normal versus keloid: 9.6% +/- 6.6% vs 66.8% +/- 5.5%). Western blotting analysis showed insulin-like growth factor I receptor overexpression in keloid fibroblasts, but not in normal fibroblasts. Exogenously added insulin-like growth factor I enhanced the resistance of keloid fibroblasts to ceramide-induced apoptosis. Wort- mannin, a phosphatidylinositol 3 kinase inhibitor, suppressed the antiapoptotic action of insulin-like growth factor I in keloid fibroblasts. Our results suggest that keloid fibroblasts overexpressing insulin-like growth factor I receptor are resistant to apoptosis, thus allowing persistent proliferation and production of excessive extracellular matrix. J Invest Dermatol 115:1065-1071 2000

Apoptosis↗

PubMed2001

Platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin cooperatively regulate fibroblast growth factor-induced modulations of adherens junction functions.

Cellular adherens junctions are formed by cadherins linked to proteins of the catenin family. In endothelial cells, not only vascular endothelial cadherin but also platelet endothelial cell adhesion molecule-1 localizes into junctions and associates with beta-catenin. To explore a putative cooperation of platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin, we analyzed transfectants expressing either platelet endothelial cell adhesion (CD31 cells) or vascular endothelial cadherin (CD144 cells) or both molecules (CD31/CD144 cells), and, for comparison, human umbilical vein endothelial cells. Basic fibroblast growth factor completely dissociated vascular endothelial cadherin/beta-catenin complexes and robustly moved beta-catenin into the nucleus in CD144 cells, whereas in CD31/CD144 cells as well as in human umbilical vein endothelial cells, fibroblast growth factor only partially dissociated the junctional complex followed by a significantly reduced nuclear translocation of beta-catenin. In contrast, in CD31 cells, the subcellular distribution of beta-catenin remained unaffected by fibroblast growth factor. As a functional consequence, fibroblast growth factor induced a complete collapse of the F-actin network in CD144 cells, a limited rearrangement of F-actin fibers in CD31/CD144 cells and no F-actin rearrangement in CD31 cells. We also analyzed the effect of fibroblast growth factor-induced rearrangement of junctions on junction permeability for leukocytes: in line with our observation that vascular endothelial cadherin was required for cells to respond to fibroblast growth factor, only in CD31/CD144 cells, but not in CD31 cells, leukocyte transmigration was significantly enhanced by fibroblast growth factor. In conclusion platelet endothelial cell adhesion molecule-1 cooperates with vascular endothelial cadherin in a mutual fashion; platelet endothelial cell adhesion molecule-1 reduces and temporarily limits fibroblast growth factor-induced dissociation of vascular endothelial cadherin/beta-catenin complexes, but requires vascular endothelial cadherin to control leukocyte transmigration in dependence of fibroblast growth factor.

Adherens Junctions↗

PubMed2001

Fibroblast matrix gene expression and connective tissue remodeling: role of endothelin-1.

This study examines endothelin-induced modulation of extracellular matrix synthesis and remodeling by fibroblasts, and its potential role in the pathogenesis of systemic sclerosis (scleroderma). Endothelin-1 promoted fibroblast synthesis of collagen types I and III, but not fibronectin, by a mechanism dependent upon both ETA and ETB receptors. Conversely, endothelin-1 inhibited both protein expression of matrix metalloproteinase 1 and zymographic activity exclusively via ETA receptors. A dual regulatory role for endothelin-1 in transcriptional regulation was suggested by the ability of endothelin-1 to enhance steady-state levels of collagen mRNA and activate the proalpha2(I) collagen (Col1a2) promoter, but in contrast to reduce matrix metalloproteinase 1 transcript expression and suppress transcription of a human matrix metalloproteinase 1 promoter reporter construct in transient transfection assays. Although endothelin-1 significantly enhanced remodeling of three-dimensional collagen lattices populated by normal fibroblasts, this was not observed for lattices populated by systemic sclerosis fibroblasts. Promotion of matrix remodeling was dependent upon ETA receptor expression and was blocked by specific inhibitors of tyrosine kinases or protein kinase C. Reverse transcriptase polymerase chain reaction, S1 nuclease, and functional cell surface binding studies showed that normal and systemic sclerosis fibroblasts express both ETA and ETB receptors (predominantly ETA), but that ETA receptor mRNA levels and ETA binding sites on fibroblasts cultured from systemic sclerosis skin biopsies are reduced by almost 50%. Endothelin-1 is thus able to induce a fibrogenic phenotype in normal fibroblasts that is similar to that of lesional systemic sclerosis fibroblasts. Moreover, reduced responsiveness to exogenous endothelin-1 in systemic sclerosis suggests that downstream pathways may have already been activated in vivo. These data further implicate dysregulated endothelin-receptor pathways in fibroblasts in the pathogenesis of connective tissue fibrosis.

Cells, Cultured↗

PubMed2003

Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone can produce type VII collagen at the basement membrane zone.

Type VII collagen is synthesized and secreted by both human keratinocytes and fibroblasts. Although both cell types can secrete type VII collagen, it is thought that keratinocytes account for type VII collagen at the dermal-epidermal junction (DEJ). In this study, we examined if type VII collagen secreted solely by dermal fibroblasts could be transported to the DEJ. We established organotypic, skin-equivalent cultures composed of keratinocytes from patients with recessive dystrophic epidermolysis bullosa (RDEB) and normal dermal fibroblasts. Immuno-labeling of skin equivalent sections with the anti-type VII collagen antibody revealed tight linear staining at the DEJ. RDEB fibroblasts, were gene-corrected to make type VII collagen and used to regenerate human skin on immune-deficient mice. The human skin generated by gene-corrected RDEB fibroblasts or normal human fibroblasts combined with RDEB keratinocytes restored type VII collagen expression at the DEJ in vivo. Further, intradermal injection of normal human or gene-corrected RDEB fibroblasts into mouse skin resulted in the stable expression of human type VII collagen at the mouse DEJ. These data demonstrate that human dermal fibroblasts alone are capable of producing type VII collagen at the DEJ, and it is possible to restore type VII collagen gene expression in RDEB skin in vivo by direct intradermal injection of fibroblasts.

Animals↗

PubMedDate not supplied

Impaired proliferation and increased L-lactate production of dermal fibroblasts in the GK-rat, a spontaneous model of non-insulin dependent diabetes mellitus.

Intact fibroblast function is required for normal wound healing. Although healing is generally accepted to be disturbed in non-insulin dependent diabetes mellitus, the signals modulating this disturbance are not fully understood. Therefore, we studied dermal fibroblasts from the GK rat, a non-insulin dependent diabetes mellitus model, and the Wistar rat (control) regarding growth characteristics, and L-lactate production at 5.5 mM and 25.5 mM glucose in the absence or presence of protein kinase C-inhibition, or alpha-tocopherol acetate. In addition, growth and L-lactate responses to hyaluronic acid were assessed under normal glucose conditions. At 5.5 mM glucose, the fibroblasts from the GK rat showed a lower proliferation rate during the first 24 hours, measured as DNA content, as compared to Wistar rats, i.e. at 8 hours GK was 57% of control, p < 0.01, at 24 hours GK was 60% of control, p < 0.01. The GK rat fibroblasts accumulated higher L-lactate levels in the media at 24-96 hours. Addition of glucose at a concentration of 25.5 mM decreased the total DNA content in GK rat fibroblast cultures to 74% (p < 0.05) and in control to 87% (p < 0.05), and increased L-lactate levels, measured at 48 hours. A protein kinase C-inhibitor, bisindolylmaleimide IX, increased DNA content and decreased L-lactate in both cell types during culture in high glucose, but only affected GK rat fibroblasts during normal glucose. Hyaluronic acid, increased DNA content in both types of fibroblasts, GK: 139% (p < 0.05), control: 127% (p < 0.05) and reduced L-lactate production. The above observations indicate that GK rat fibroblast proliferation is suppressed when the cells are cultured in high glucose containing media. In addition, protein kinase C and hyaluronic acid might play a role as modulators of fibroblast proliferation during the diabetic state.

Adjuvants, Immunologic↗

PubMedDate not supplied

Phenotypic variation in the production of bioactive hepatocyte growth factor/scatter factor by oral mucosal and skin fibroblasts.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic growth factor produced principally by cells of mesenchymal origin. HGF/SF is an important mitogen, morphogen, and motogen and plays an important role in wound healing, tumorigenesis and particularly fetal development. Oral mucosal fibroblasts exhibit a fetal phenotype, including an increased extracellular matrix reorganizational ability, cellular migration and experimental wound repopulation in comparison to skin fibroblasts. In this study the expression, production, and bioactivity of HGF/SF by oral mucosal and skin fibroblasts was investigated. Although both oral mucosal and skin fibroblasts expressed HGF/SF, the oral mucosal fibroblasts produced significantly increased amounts of total HGF/SF (p < 0.01) as measured by enzyme-linked immunosorbent assay and bioactive HGF/SF as measured by cell scatter and cell-dissociation techniques (p < 0.01). The possible effect of increased HGF/SF in production mediating the previously described preferential responses of oral mucosal fibroblasts was studied in vitro. Reverse transcriptase-polymerase chain reaction-Western blotting and immunocytochemistry methods all showed that both oral mucosal and skin fibroblasts expressed and produced the c-Met receptor. Recombinant HGF (20-40 ng/mL) however, failed to affect fibroblast repopulation of monolayer wounds or cellular proliferation. In contrast, recombinant HGF significantly increased ECV304 wound repopulation. These studies provide direct evidence of another mechanism by which site-specific variations in fibroblast phenotype may contribute in a paracrine fashion to the rapid reepithelialization and revascularization of oral wounds.

Adolescent↗

PubMed2001

Highly proliferative intratumoral fibroblasts and a high proliferative microvessel index are significant predictors of tumor metastasis in T3 ulcerative-type colorectal cancer.

Fibroblast and endothelial cell mitotic figures are seen in some areas of colorectal cancers, and the purpose of this study was to investigate whether the proliferative activity of fibroblasts and endothelial cells plays an important role in tumor progression of T3 ulcerative-type colorectal cancer. The tumor area of 157 colorectal cancers was divided into marginal elevated area and central depressed area (CDA), and at half the depth of the depression the CDA was in turn divided into CDA upper area (CDAU) and CDA lower area (CDAL). The proliferative activity of the tumor cells, fibroblasts, and endothelial cells was assessed immunohistochemically by double CD31/MIB-1 (anti--Ki-67 antigen) staining. The proliferative microvessel index was estimated as the percentage of microvessels lined by MIB-1-positive endothelial cells relative to the total microvessel count. Proliferative activities of tumor cells showed significant associations with those of fibroblasts and the proliferative microvessel indices in all of the corresponding areas. Proliferative activities of fibroblasts also showed significant associations with proliferative microvessel indices in all of the corresponding areas. Colorectal cancers with nodal metastasis showed significantly higher proliferative activities of fibroblasts in the CDAU than those without nodal metastasis (P <.001). The high proliferative activities of fibroblasts and proliferative microvessel indices in the CDAU showed significant associations with short distant organ metastasis-free periods in colorectal cancers without nodal metastasis (P <.001 and P =.010, respectively) and those with nodal metastasis (P =.024 and P =.036, respectively). Multivariate analysis showed that the highly proliferative fibroblasts in the CDAU significantly increased hazard rates of distant organ metastasis of colorectal cancer patients with nodal metastasis (P =.018). Proliferative activities of fibroblasts and endothelial cells in the CDAU are useful parameters for predicting tumor metastasis in patients with T3 ulcerative-type colorectal cancer. HUM PATHOL 32:401-409.

Cell Division↗

PubMed1999

Differential responses of collagen and glycosaminoglycan syntheses and cell proliferation to exogenous transforming growth factor beta 1 in the developing mouse skin fibroblasts in culture.

Skin fibroblast cultures were established from mouse foetuses at days 14, 15, 16 and 18 of gestation and from newborn mice. Modulations of mitotic and biosynthetic phenotypes of the fibroblasts by transforming growth factor beta1 (TGFbeta1) were studied. Treatment of the fibroblasts with TGFbeta1 at doses of 1 and 10 ng/ml for 48 h resulted in significant stimulation of cell proliferation in the 15-, 16- and 18-day foetal fibroblasts and a slight stimulation in the 14-day foetal fibroblasts. Treatment with TGFbeta1 resulted in stimulation of collagen synthesis approximately 2-fold in the 18-day foetal and newborn fibroblasts, but failed to stimulate it in the 14-, 15- and 16-day foetal fibroblasts. TGFbeta1 stimulated glycosaminoglycan (GAG) synthesis throughout all developmental stages approximately 1.8-2.6 fold. Histological study demonstrated that skin wounds made at day 16 of gestation were replaced with normal-appearing dermis, but at day 18 the wounds left dermal fibrosis and lack of hair follicles. These results indicate that the modulations of fibroblast phenotypes (proliferation and syntheses of collagen and GAG) in response to TGFbeta1 occur at different stages of gestation. Ontogenic transitions of skin wound healing and collagen synthetic phenotype with TGFbeta1 treatment in cultured fibroblasts occurred between days 16 and 18 of gestation, suggesting that the unresponsiveness of collagen synthesis to exogenous TGFbeta1 in cell culture may be related to the phenomenon of scarless wounds in the foetus.

Animals↗

PubMed2002

Metabolic regulation of collagen I in fibroblasts isolated from normal peritoneum and adhesions by dichloroacetic acid.

OBJECTIVE: We have previously demonstrated that collagen I, a major component of postoperative adhesions, is differentially regulated in fibroblasts isolated from normal human peritoneum and adhesions. Collagen I messenger RNA (mRNA) levels are significantly lower in fibroblasts from normal peritoneum compared with levels from adhesion fibroblasts. This variation is further accentuated by hypoxia. Because adhesions provide a means of supplying oxygen and nutrients to postsurgical ischemic tissue, we sought to examine the role of aerobic metabolism in the differential expression of collagen I. To examine this issue, we used a compound, dichloroacetic acid (DCA), that stimulates pyruvate dehydrogenase, causing pyruvate to be metabolized in the Kreb's cycle rather than converted into lactate, thereby switching anaerobic to aerobic metabolism. Specifically, we have exposed human fibroblast cultures from normal peritoneum and adhesions to DCA (0, 50, and 100 microg/mL) for 24 hours under normal and hypoxic (2% oxygen) conditions. STUDY DESIGN: Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) of collagen I and beta-actin was performed by using mRNA extracted from all treatment points. Analysis of PCR-amplified products was performed by fractionation over a 2% agarose gel, followed by ethidium bromide staining of DNA bands. A scanning densimeter was used to determine the ratio of intensity of each band relative to beta-actin. Densimetric analysis of gel bands was performed by using the National Institutes of Health image analysis program. RESULTS: Although DCA stimulated peritoneal fibroblast collagen I mRNA expression under normoxic conditions, its expression was reduced during hypoxia. In adhesion fibroblasts, DCA treatment consistently lowered collagen I mRNA expression; this effect was manifested to a greater extent under hypoxic conditions. CONCLUSION: In summary, these findings confirm that fibroblasts from adhesions are characterized by excessive collagen I production, which is further accentuated by hypoxia. These observations are extended to show the stimulation of oxidative metabolism by DCA increases collagen I production; in contrast DCA inhibits collagen I production by normoxic adhesion fibroblasts as well as under hypoxic conditions in both types of fibroblasts. Thus, regulation of metabolic activity of peritoneal cells may provide a target for future interventions for reduction of postoperative adhesions.

Cells, Cultured↗

PubMed2002

Platelet-derived growth factor-AA-mediated functional angiogenesis in the rat epigastric island flap after genetic modification of fibroblasts is ischemia dependent.

BACKGROUND: The aim of this study was to induce therapeutic angiogenesis in ischemically challenged flap tissue by means of gene transfer. METHODS: Isogenic rat fibroblasts were retrovirally transfected to produce platelet-derived growth factor (PDGF)-AA. Stable gene expression was monitored by PDGF-AA enzyme-linked immunosorbent assay. Eighty animals were divided into 2 groups (1 and 2), each with 4 subgroups. The angiogenic target was a 7 x 7-cm epigastric island flap used as a necrosis model. Group 1 received flap treatment 1 week before flap elevation: 10(7) genetically modified fibroblasts, expressing PDGF-AA (genetically modified fibroblasts) plus 1 mL of Dulbecco's modified Eagle's medium (DMEM) (1A), 10(7) nonmodified fibroblasts (NMFB) plus 1 mL of DMEM (1B), 1 mL of DMEM (1C), and 1 mL of sodium chloride 0.9% (1D). All substances were injected at evenly distributed spots into the panniculus carnosus of the entire flap. Group 2 had the same flap treatment at the day of flap elevation. All flaps were sutured back. Seven days later, the flaps were harvested and examined both clinically, histologically, and immunohistochemically. RESULTS: In vitro, the GMFB produced up to 117.9 +/- 57.2 ng of PDGF-AA/mL medium during a 4-day period, compared with 0.7 +/- 0.6 ng of PDGF-AA/mL medium produced by NMFB in the same time period. In vivo production of PDGF-AA in flaps amounted to 1.3 +/- 0.7 ng of PDGF-AA/1 microL flap tissue for group 1A and 1.7 +/- 1.1 ng of PDGF-AA/1 microL flap tissue for group 2A seven days after cell transplantation. Fibroblasts persisted in all flaps from groups 1A, 1B, 2A, and 2B without major inflammatory reaction. Clinically, group 2A developed significantly less flap necrosis compared with all other groups, including group 1A. Accordingly, only group 2A gave significant histologic and immunohistochemical evidence for enhanced angiogenesis within the flap tissue. CONCLUSIONS: After retroviral gene transfer, isogenic rat fibroblasts produce high amounts of PDGF-AA in vitro. In vivo, PDGF-AA can be detected in flaps receiving genetically modified fibroblasts, which suggests survival of the implanted fibroblasts in this model. PDGF-AA produced by GMFB can induce flap angiogenesis only under ischemic conditions in this model. Transplantation of PDGF-AA-overexpressing fibroblasts results in higher flap survival in this model.

Abdomen↗

PubMed2002

Long-term effects of surgical angiogenic therapy with fibroblast growth factor 2 protein.

OBJECTIVE: The long-term effects of surgical fibroblast growth factor 2 therapy are examined. METHODS: In a randomized, double-blind study, fibroblast growth factor 2 (10 microg or 100 microg) or placebo (n = 8 each) was delivered in the ungraftable myocardial territory of patients concomitantly undergoing coronary artery bypass grafting. Patients were followed up to 32.2 +/- 6.8 months postoperatively with clinical assessment and nuclear perfusion imaging. RESULTS: Baseline patient characteristics were similar between the 3 groups. There were 2 late deaths, one of pancreatic cancer and one of undetermined cause (both in the 100-microg fibroblast growth factor 2 group). Two patients (both in the control group) underwent a total of 6 repeat cardiac catheterizations for recurrent coronary events. Mean Canadian Cardiovascular Society angina class improved at late follow-up from baseline in all groups (P < or = .02); however, patients treated with either dose of fibroblast growth factor 2 had significantly more freedom from angina recurrence than those treated with placebo (P =.03). Late nuclear perfusion scans revealed a persistent reversible or a new, fixed perfusion defect in the ungraftable territory of 4 of 5 patients who received placebo versus only 1 of 9 patients treated with fibroblast growth factor 2 (P =.02). The overall sum of left ventricular stress perfusion defect scores was also lower in fibroblast growth factor 2-treated patients than in control subjects (1.3 +/- 1.4 vs 3.9 +/- 2.1, respectively; P =.04). A trend toward a higher late left ventricular ejection fraction was noted in fibroblast growth factor 2-treated patients (55.1% +/- 14.6% vs 44.3% +/- 6.5%, fibroblast growth factor 2-treated patients versus control subjects; P =.12). CONCLUSIONS: These data suggest that surgical angiogenic therapy with sustained-release fibroblast growth factor 2 may result in a prolonged myocardial revascularization effect that could translate into clinical benefit.

Coronary Artery Bypass↗

PubMed2001

Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair.

Nerve growth factor (NGF) is a polypeptide which, in addition to its effect on nerve cells, is believed to play a role in inflammatory responses and in tissue repair. Because fibroblasts represent the main target and effector cells in these processes, to investigate whether NGF is involved in lung and skin tissue repair, we studied the effect of NGF on fibroblast migration, proliferation, collagen metabolism, modulation into myofibroblasts, and contraction of collagen gel. Both skin and lung fibroblasts were found to produce NGF and to express tyrosine kinase receptor (trkA) under basal conditions, whereas the low-affinity p75 receptor was expressed only after prolonged NGF exposure. NGF significantly induced skin and lung fibroblast migration in an in vitro model of wounded fibroblast and skin migration in Boyden chambers. Nevertheless NGF did not influence either skin or lung fibroblast proliferation, collagen production, or metalloproteinase production or activation. In contrast, culture of both lung and skin fibroblasts with NGF modulated their phenotype into myofibroblasts. Moreover, addition of NGF to both fibroblast types embedded in collagen gel increased their contraction. Fibrotic human lung or skin tissues displayed immunoreactivity for NGF, trkA, and p75. These data show a direct pro-fibrogenic effect of NGF on skin and lung fibroblasts and therefore indicate a role for NGF in tissue repair and fibrosis.

Actins↗

PubMed1976

Calcium-dependent regulatory protein of cyclic nucleotide metabolism in normal and transformed chicken embryo fibroblasts.

The concentration of a calcium-binding protein modulator of 3':5'-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17; 3':5'-cyclic-nucleotide 5'-nucleotidohydrolase) activity is increased in chicken embryo fibroblasts upon transformation by Rous sarcoma virus. This modulator protein from fibroblasts, which has roughly the same molecular size, charge, and functional properties as that isolated from chicken brain, comprises approximately 1.32% of the soluble protein in homogenates of fibroblasts infected and transformed by Rous sarcoma virus. In comparison, the modulator comprises approximately 0.30% of the soluble protein in homogenates of normal fibroblasts from confluent cultures and 0.36% of the soluble protein in homogenates of fibroblasts infected with a transformation-defective mutant of Rous sarcoma virus. Modulator levels in normal fibroblasts at subconfluent cell densities are 0.42-0.76% of the homogenate soluble protein, i.e., between that found in confluent normal fibroblasts and in fibroblasts transformed by Rous sarcoma virus. These observations suggest that the levels of the modulator protein are elevated under conditions in which chicken embryo fibroblasts are undergoing rapid growth and have decreased adenosine 3':5'-cyclic monophosphate levels.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1984

Interactions between human tumor cells and fibroblasts stimulate hyaluronate synthesis.

Several types of tumors contain high concentrations of hyaluronate, yet isolated tumor cells in culture often produce little glycosaminoglycan. To explore the possibility that interactions between tumor cells and host fibroblasts stimulate hyaluronate synthesis, human tumor cells were grown separately from and in coculture with normal human fibroblasts. Stimulation was observed with each of the three types of tumor cells used: LX-1 lung carcinoma, DAN pancreatic carcinoma, and TRIG melanoma. The interaction between LX-1 cells and fibroblasts was studied in detail. Under serum-free conditions, cocultures of LX-1 and fibroblasts synthesized 3-fold more hyaluronate than the sum of that produced by LX-1 and fibroblast cultures grown separately. This stimulation was linear over 72 hr and hyaluronate represented 80% of the glycosaminoglycan synthesized. Maximum stimulation occurred at a ratio of fibroblasts to LX-1 cells of 1-2:1. Quantitation of unlabeled glycosaminoglycans by HPLC analysis of disaccharides generated by digestion with chondroitin ABC and AC lyases (EC 4.2.2.4 and 4.2.2.5) demonstrated that net accumulation of hyaluronate increased 2-fold and that hyaluronate represented 80% of total chondroitinase-sensitive glycosaminoglycan produced by the cocultures. The disaccharide patterns obtained showed that accumulations of chondroitin-4- and chondroitin-6-sulfates were stimulated proportionately to that of hyaluronate in these cocultures. Similar levels of stimulation due to coculture were obtained in serum-containing and serum-free media. Stimulation was not effected by addition of LX-1-conditioned medium to fibroblast cultures or by culturing LX-1 and fibroblasts under conditions where they shared the same medium but were physically separated. Cell contact between LX-1 and fibroblasts thus appears to be necessary for the stimulation of hyaluronate synthesis.

Blood↗

PubMed1984

Increased adhesiveness of Down syndrome fetal fibroblasts in vitro.

We compared the in vitro rate of divalent cation-independent aggregation of fibroblasts derived from abortuses with normal karyotypes and with trisomy 21 (Down syndrome). Fibroblasts from five lung and two of three cardiac cultures from subjects with Down syndrome aggregated more rapidly than matched fibroblasts from normal controls or lung fibroblasts from an abortus with trisomy 13. In contrast, skin fibroblasts derived from the trisomy 21 subjects had low rates of aggregation. The high rates of aggregation of trisomy 21 lung fibroblasts were not affected by hyaluronidase treatment. Lung fibroblasts from both normal and Down syndrome subjects had similar membrane polarization values in an assay using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Thus, the increased aggregation rate we observed for trisomy 21 fibroblasts was restricted to specific fibroblast cultures, was not mediated by hyaluronic acid or gross membrane lipid alterations, and was specific for trisomy of chromosome 21. As illustrated in computer simulations presented elsewhere, increased intercellular adhesiveness during organogenesis could explain the frequent occurrence of malformations, including pulmonary hypoplasia and congenital heart defects, in Down syndrome.

Cell Adhesion↗

PubMed1995

Cloning and expression of glucocorticoid-induced genes in fetal rat lung fibroblasts. Transforming growth factor-beta 3.

Glucocorticoids have been shown to accelerate fetal lung type II cell maturation, and this effect appears, in part, to be mediated via fibroblasts. To identify glucocorticoid induced genes in fetal lung fibroblasts, we screened a cDNA library from cortisol-treated fetal lung fibroblasts with a subtracted cDNA probe which was enriched for sequences specific for cortisol-treated fetal lung fibroblasts. Fifty-seven clones were isolated from the cDNA library. One cDNA represented approximately 30% of the 57 clones. Analysis of DNA sequence homology suggested that this cDNA encodes the rat transforming growth factor-beta 3 (TGF beta 3). We found that TGF beta 3 mRNA was expressed in fetal lung fibroblasts but not epithelial cells. Expression of message in fetal lung fibroblasts was developmentally regulated. TGF beta 3 mRNA levels were low during the pseudoglandular stage (day 18), peaked during the early canalicular stage of lung development (day 19), then fell again at days 20 and 21 (term = 22 days). Exposure of fetal lung fibroblasts to cortisol increased TGF beta 3 mRNA expression in a time- and dose-dependent manner. Maternal administration of dexamethasone also enhanced mRNA expression of TGF beta 3 in fetal lung fibroblasts. These data suggest that glucocorticoids may mediate their stimulatory effect on lung maturation by inducing TGF beta 3 expression in fetal lung fibroblasts.

Amino Acid Sequence↗

PubMed1999

Furin-independent pathway of membrane type 1-matrix metalloproteinase activation in rabbit dermal fibroblasts.

We investigated the gene expression and intracellular activity of processing protease furin and its involvement in the process of membrane type 1-matrix metalloproteinase (MT1-MMP) activation in rabbit dermal fibroblasts. When the rabbit fibroblasts were treated with concanavalin A (ConA), pro-MMP-2 was converted to an active 62-kDa MMP-2 through the appearance of a 64-kDa intermediate MMP-2. The ConA-induced pro-MMP-2 activation resulted from increasing the gene expression and production of MT1-MMP in the rabbit fibroblasts. Reverse transcriptase-polymerase chain reaction demonstrated that in rabbit dermal fibroblasts furin mRNA was detected and, unlike MT1-MMP, was not increased by ConA. These findings are further supported by the fact that the intracellular furin activity also was constitutively detected and was unchanged by the ConA treatment. Very similar phenomena were also observed in human uterine cervical fibroblasts, which are known to produce MT1-MMP by ConA stimulation. These results suggest that the expression of the furin gene and the intracellular activity are not regulated by ConA. On the other hand, neither a synthetic furin inhibitor, decanoyl-RVKR-CH(2)Cl (25-100 microM) nor a furin antisense oligonucleotide (40 microM) inhibited the MT1-MMP-mediated pro-MMP-2 activation in ConA-treated rabbit dermal fibroblasts, whereas these compounds interfered with pro-MMP-2 activation in ConA-treated human uterine cervical fibroblasts. Nonetheless, the furin antisense oligonucleotide completely suppressed furin gene expression in both rabbit and human fibroblasts. These results suggest that furin does not participate in the process of MT1-MMP activation induced by ConA in rabbit dermal fibroblasts.

Animals↗