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1.
Cytokine Growth Factor Rev ; 20(4): 305-17, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19656717

ABSTRACT

With the development of growth factors and growth factor modulators as therapeutics for a range of disorders, it is prudent to consider whether modulating the growth factor profile in a tissue can influence tumour initiation or progression. As recombinant human TGF-beta3 (avotermin) is being developed for the improvement of scarring in the skin it is important to understand the role, if any, of this cytokine in tumour progression. Elevated levels of TGF-beta3 expression detected in late-stage tumours have linked this cytokine with tumourigenesis, although functional data to support a causative role are lacking. While it has proved tempting for researchers to interpret a 'correlation' as a 'cause' of disease, what has often been overlooked is the normal biological role of TGF-beta3 in processes that are often subverted in tumourigenesis. Clarifying the role of this cytokine is complicated by inappropriate extrapolation of the data relating to TGF-beta1 in tumourigenesis, despite marked differences in biology between the TGF-beta isoforms. Indeed, published studies have indicated that TGF-beta3 may actually play a protective role against tumourigenesis in a range of tissues including the skin, breast, oral and gastric mucosa. Based on currently available data it is reasonable to hypothesize that administration of acute low doses of exogenous TGF-beta3 is unlikely to influence tumour initiation or progression.


Subject(s)
Neoplasms/metabolism , Transforming Growth Factor beta3/metabolism , Amino Acid Sequence , Animals , Humans , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Neoplasms/genetics , Neoplasms/pathology , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Sequence Homology, Amino Acid , Transforming Growth Factor beta3/chemistry , Transforming Growth Factor beta3/genetics
2.
Br J Ophthalmol ; 88(2): 169-73, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14736764

ABSTRACT

AIM: To determine the effects of single doses of beta radiation on the wound healing functions of human Tenon's capsule fibroblasts (hTf). METHODS: hTf were grown in tissue culture and irradiated with beta radiation using a strontium 90 source. The effects of beta radiation on fibroblast migration was studied using microporous transwell membranes. The effects of radiation on fibroblast contraction was investigated using a fibroblast populated collagen gels model. Production of extracellular matrix molecules (collagen I, collagen III, and fibronectin) by monolayers of irradiated fibroblasts was quantified for 14 days following single doses of beta radiation. RESULTS: Growth inhibiting doses of beta radiation did not inhibit fibroblast migration or contraction at any time point. Levels of soluble fibronectin from irradiated populations were significantly reduced after >500 cGy beta radiation. Collagen I and III levels were not reduced after any dose of radiation, and increased following treatment with 1000 cGy beta radiation. CONCLUSIONS: Growth arresting doses of beta radiation have unique effects on the wound healing behaviour of human Tenon's capsule fibroblasts. There was no significant effect on cellular migration or contraction, but ECM production was altered. Fibronectin production was inhibited following higher radiation doses, and collagen I and III production increased after 1000 cGy. The effects of single doses of beta radiation on ocular fibroblast wound healing behaviour are very different from those of 5-fluorouracil and mitomycin C, and these differences may be exploited clinically in the regulation of wound healing after glaucoma filtration surgery.


Subject(s)
Connective Tissue Cells/radiation effects , Eye/radiation effects , Fibroblasts/radiation effects , Wound Healing/radiation effects , Beta Particles , Cell Division/radiation effects , Cell Movement/radiation effects , Cell Size/radiation effects , Cells, Cultured , Collagen Type I/biosynthesis , Collagen Type III/biosynthesis , Connective Tissue Cells/metabolism , Culture Media, Conditioned , Dose-Response Relationship, Radiation , Extracellular Matrix/metabolism , Extracellular Matrix/radiation effects , Eye/metabolism , Fibroblasts/metabolism , Fibronectins/biosynthesis , Filtering Surgery , Humans , Strontium Radioisotopes
3.
Am J Pathol ; 159(4): 1555-66, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11583981

ABSTRACT

The most common cause of failure of retinal reattachment surgery is formation of fibrocellular contractile membranes on both surfaces of the neuroretina. This intraocular fibrosis, known as proliferative vitreoretinopathy, results in a blinding tractional retinal detachment because of the contractile nature of the membrane. Contractility is a cell-mediated event that is thought to be dependent on locomotion and adhesion to the extracellular matrix. Interactions between cells and the extracellular matrix can be influenced by matrix metalloproteinases (MMPs) and we investigated the role of MMPs in two in vitro models (two- and three-dimensional) of human retinal pigment epithelial (RPE) cell-mediated contraction. MMP activity was detected using enzyme-linked immunosorbent assays and zymography techniques that revealed MMP-1, -2, -3, and -9 positivity during the collagen matrix contraction assays. RPE-populated collagen matrix contraction (three-dimensional) was inhibited using a cocktail of anti-MMP antibodies and with Galardin (a broad-spectrum MMP inhibitor). Galardin inhibition was dose-dependent, reversible, and dependent on cell number. MMP inhibitors had no effect on contraction when RPEs were seeded on two-dimensional collagen matrices or on cellular adhesion to collagen type I. Our results suggest that MMP activity may be required for three-dimensional but not two-dimensional RPE-collagen matrix contraction.


Subject(s)
Cicatrix/physiopathology , Matrix Metalloproteinases/physiology , Retina , Vitreous Body , Cell Adhesion/drug effects , Cell Survival/drug effects , Cells, Cultured , Cicatrix/pathology , Collagen/physiology , Dipeptides/pharmacology , Humans , Matrix Metalloproteinase Inhibitors , Pigment Epithelium of Eye/drug effects , Pigment Epithelium of Eye/pathology , Pigment Epithelium of Eye/physiopathology , Protease Inhibitors/pharmacology
4.
Br J Ophthalmol ; 84(5): 506-11, 2000 May.
Article in English | MEDLINE | ID: mdl-10781515

ABSTRACT

AIM: To assess clinical variables and vitreous protein as risk factors for the development of postoperative proliferative vitreoretinopathy (PVR). METHODS: A prospective study was conducted on 140 patients with a rhegmatogenous retinal detachment in whom a primary vitrectomy was performed. 12 clinical variables were recorded and vitreous samples obtained for measurement of protein concentration. Univariate and multivariate logistic regression analysis was used to determine the risk factors for PVR. RESULTS: Complete data were available for 136 of 140 patients. 40 of the 136 patients (29.4%) developed postoperative PVR. Univariate regression revealed that significant (p<0.05) risk factors included aphakia, presence of preoperative PVR, size of detachment, the use of silicone oil, and high vitreous protein level. Multivariate regression analysis revealed only aphakia (odds ratio 2.72), the presence of preoperative PVR (odds ratio 3.01), and high vitreous protein concentration (odds ratio 1.11) to be significant (p<0.05) independent, predictive risk factors for the development of PVR. CONCLUSIONS: This study has shown that the significant risk factors for PVR are preoperative PVR, aphakia, and high vitreous protein levels. Two models (clinical factors only and clinical factors and vitreous protein) were constructed to predict the probability of developing postoperative PVR and may be used to identify those at risk for possible intravitreal pharmacological treatment.


Subject(s)
Vitrectomy/adverse effects , Vitreoretinopathy, Proliferative/etiology , Adolescent , Adult , Aged , Aged, 80 and over , Eye Proteins/metabolism , Humans , Middle Aged , Multivariate Analysis , Prospective Studies , Risk Factors , Treatment Outcome , Visual Acuity , Vitreoretinopathy, Proliferative/metabolism , Vitreous Body/metabolism
5.
Br J Ophthalmol ; 84(1): 92-7, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10611106

ABSTRACT

AIM: To study how the delivery of 5-fluorouracil (5-FU) to ocular tissues is affected by altering delivery variables. METHOD: Sponge(s) soaked in radiolabelled 5-FU were placed between the conjunctiva and sclera of pig eyes. Application time, sponge size, sponge make (Altomed, Weck, Merocel), and 5-FU concentration were varied. Conjunctival and scleral tissue levels were determined in samples taken from the application site. RESULTS: Dose-response curves for scleral and conjunctival 5-FU levels against application time showed increasing tissue levels that reached a plateau after 2-3 minutes. Application beyond 3 minutes did not increase tissue levels. There was no difference in tissue levels between 7x4 and 3. 5x2 mm sponges. Altomed sponges produced 5-FU tissue levels that were twice as high as those obtained with Weck-cell (p<0.01) or Merocel (p<0.02) sponges. Changing the 5-FU concentration from 25 mg/ml to 6.25 mg/ml reduced the conjunctival concentration by a factor of 3.5 (p<0.003). CONCLUSION: Application time up to 3 minutes, sponge make, and 5-FU concentration can have a large effect on the tissue delivery of 5-FU. Application time beyond 3 minutes, using 3.5x2 mm or 7x4 mm sponges, and replacing sponges every minute did not have a significant effect on tissue levels. This study models the effect that different variables can have on the ocular tissue levels of an antimetabolite applied intraoperatively.


Subject(s)
Antimetabolites/administration & dosage , Conjunctiva/metabolism , Fluorouracil/administration & dosage , Sclera/metabolism , Administration, Topical , Animals , Antimetabolites/pharmacokinetics , Carbon Radioisotopes , Conjunctiva/chemistry , Fluorouracil/pharmacokinetics , Sclera/chemistry , Surgical Sponges , Swine , Time Factors , Tissue Distribution
6.
Exp Eye Res ; 69(1): 117-27, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10375456

ABSTRACT

The use of single five-minute applications of antimetabolites during glaucoma filtration surgery has significantly reduced the occurrence of post-operative scarring and bleb failure. However, surgery for some patients is still unsuccessful, despite the use of antiproliferative agents, due to formation of scar tissue at the drainage site. It is not known if cells growth arrested in the treated area with a single application of antimetabolites influence the activity of adjacent non-treated cells. We hypothesise that the activity of non-treated cells recruited to the wound site may be involved in post-operative scarring. The aim of this study was to investigate the effects of antimetabolite induced cellular growth arrest on cell-cell interactions using in vitro techniques.Tenon's capsule fibroblast cultures were growth arrested by exposure for 5 minutes to mitomycin-C (0.001, 0.01 and 0.1 mg ml-1), 5-fluorouracil (0.25, 2.5 and 25 mg ml-1) or phosphate buffered saline (PBS). Following a period of serum-starvation, conditioned media (CM) were subsequently collected from the cells at intervals up to 29 days post-treatment. Correction for cell number was made prior to supplementation of serum-free medium with CM. CM were assessed for ability to support or inhibit normal non-treated fibroblast proliferation, migration and collagen contraction. Conditioned media collected from cells growth arrested with MMC or 5FU stimulated normal fibroblast proliferation, migration and collagen contraction in excess of non-conditioned serum-free medium. Peaks of fibroblast activity in CM differed according to which drug and concentration had originally been given to the treated cells. This study has demonstrated that CM collected from fibroblasts treated for 5 minutes with a range of concentrations of antimetabolites can differentially influence normal non-treated fibroblast activity. This in vitro data suggests that despite entering growth arrest, fibroblasts may still influence the behaviour of other cells via soluble mediators. They may have implications in the clinical setting, in that it may not be sufficient to suppress proliferation alone to prevent fibroblast behaviour associated with scarring after glaucoma filtration surgery.


Subject(s)
Antimetabolites/pharmacology , Cell Communication/drug effects , Fibroblasts/drug effects , Cell Culture Techniques , Cell Division/drug effects , Chemotaxis/drug effects , Collagen/metabolism , Connective Tissue Cells/drug effects , Culture Media, Conditioned , Dose-Response Relationship, Drug , Eye/cytology , Eye/drug effects , Humans , Mitomycin/pharmacology , Nucleic Acid Synthesis Inhibitors/pharmacology
7.
Invest Ophthalmol Vis Sci ; 40(3): 705-12, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10067974

ABSTRACT

PURPOSE: Proliferative vitreoretinopathy (PVR) is a major cause of failure of retinal detachment surgery. It is believed to be a wound-healing process in the retina. Many of the cellular functions are influenced by cytokines and growth factors such as interleukins (ILs). The present study was conducted to investigate the presence of transforming growth factor-beta 2 (TGF-beta2), basic fibroblast growth factor (bFGF), IL-1beta, IL-6, and protein in the vitreous of patients with retinal detachment and to determine the value of these mediators in predicting the future development of PVR. METHODS: A prospective study was conducted in 140 consecutive patients with rhegmatogenous retinal detachment in whom vitrectomy was considered necessary. Vitreous samples were analyzed for the presence of TGF-beta2, bFGF, IL-1beta, IL-6, and protein. Patients were then followed up for 3 months for the development of postoperative PVR. RESULTS: The mean levels of TGF-beta2, bFGF, IL-1beta, and protein in the vitreous were significantly higher (P < 0.05) in patients with preoperative PVR compared with those without. The mean levels of TGF-beta2, bFGF, IL-6, and protein in the vitreous were significantly higher (P < 0.05) in patients who had postoperative PVR compared with those who did not. Multivariate logistic regression analysis showed IL-6 and protein to be significant (P < 0.05), independent, predictive risk factors for the development of PVR. CONCLUSIONS: The various cytokines may play a role in the pathobiology of PVR. High vitreous levels of IL-6 and protein were identified as significant risk factors for PVR. A model was developed to predict the probability of development of postoperative PVR in these patients, and it may be used to indicate intravitreal pharmacologic treatment for those at risk.


Subject(s)
Fibroblast Growth Factor 2/metabolism , Interleukin-1/metabolism , Interleukin-6/metabolism , Transforming Growth Factor beta/metabolism , Vitreoretinopathy, Proliferative/metabolism , Vitreous Body/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Middle Aged , Prospective Studies , Retinal Detachment/etiology , Retinal Detachment/metabolism , Retinal Detachment/surgery , Vitrectomy , Vitreoretinopathy, Proliferative/complications
8.
Microsc Res Tech ; 42(5): 317-33, 1998 Sep 01.
Article in English | MEDLINE | ID: mdl-9766427

ABSTRACT

In response to injury, the body usually initiates a full and swift wound healing response resulting in reconstructed, repaired tissue. In certain instances, due to a variety of factors, this may not happen, an example being chronic granulating venous leg ulcers. At the other extreme, the wound may heal excessively, producing disabling hypertrophic scarring such as can occur following large, deep burn injuries. Our group is interested in the surgical treatment of the eye disease glaucoma. As will be explained, the successful surgical treatment of this disease depends on a reduced scarring response at the end of wound healing. The purpose of this article is to give an overview of our microscopic and histological experimental work which has furthered our understanding of tissue repair, particularly the scarring response and its potential modification for successful glaucoma surgery.


Subject(s)
Antimetabolites/therapeutic use , Cicatrix/pathology , Cicatrix/prevention & control , Conjunctiva/pathology , Glaucoma/surgery , Cell Division , Extracellular Matrix/metabolism , Fibroblasts/cytology , Filtering Surgery , Humans , Wound Healing
9.
Wound Repair Regen ; 6(2): 157-66, 1998.
Article in English | MEDLINE | ID: mdl-9776859

ABSTRACT

Contraction and scarring of the cornea and conjunctiva following disease or injury are major causes of visual morbidity. The aim of this study was to identify any specific ultrastructural features of ocular fibroblast behavior in different collagen lattices in order to understand some of the mechanisms of cell-mediated contraction. Normal human Tenon's capsule fibroblasts were cultured within both restrained and floating collagen lattices for periods of up to 13 days and then analyzed using transmission electron microscopy. The contractile force of these fibroblasts was also tested using the culture force monitor, an instrument capable of measuring the minute forces exerted by cells within a collagen lattice. The results showed differences in the behavior of fibroblasts cultured in the two gel models. The features seen in restrained gels suggest that fibroblasts were actively migrating across and through the lattice. These migratory features were not seen to the same extent in untethered gels, which lack the inherent tension and support of the tethered model. We hypothesize that contraction of the collagen matrix in tethered lattices is due to cellular migration and that this fact cannot be ascertained from untethered gels. Both lattice models have experimental value, but it is important to appreciate what mechanical signals cells receive from the matrix in order to understand cellular behavior.


Subject(s)
Collagen/ultrastructure , Eye/cytology , Fibroblasts/ultrastructure , Cell Movement , Cells, Cultured , Conjunctiva/ultrastructure , Connective Tissue/ultrastructure , Humans , Microscopy, Electron, Scanning Transmission , Surface Properties
10.
Br J Ophthalmol ; 82(5): 554-60, 1998 May.
Article in English | MEDLINE | ID: mdl-9713065

ABSTRACT

AIM: To investigate the effects of single, short-term (5 or 30 minutes) exposures to thiotepa or 5-fluorouracil (5-FU) on collagen lattice contraction and retinal pigment epithelial (RPE) cell proliferation. METHODS: For collagen contraction studies, RPE cells seeded into free floating type I collagen lattices were exposed to single 5 or 30 minute treatments with thiotepa (0.06-4 mg/ml), or 5-FU (0.25-25 mg/ml), or phosphate buffered saline alone as a control. For proliferation studies, RPE cell monolayers were similarly exposed to these agents. The degree of contraction, effects on cell number, and viability were determined up to 14 days after treatment. RESULTS: Contraction of collagen lattices containing RPE cells and proliferation of RPE cells were significantly inhibited (p < 0.05) by thiotepa and 5-FU at concentrations above 0.06 mg/ml and 0.25 mg/ml respectively (for both 5 and 30 minute treatments), compared with controls. Cell death did not occur except for exposure of the RPE cells in collagen lattices to the highest concentration of thiotepa (4 mg/ml). CONCLUSION: It was concluded that single 5 or 30 minute exposures to thiotepa or 5-FU significantly inhibited collagen contraction and the proliferation of RPE cells. These findings suggest that short, single, non-toxic exposures to thiotepa or 5-FU which can be reproduced clinically may be useful in the modulation of proliferative vitreoretinopathy.


Subject(s)
Alkylating Agents/pharmacology , Antimetabolites/pharmacology , Fluorouracil/pharmacology , Pigment Epithelium of Eye/drug effects , Thiotepa/pharmacology , Cell Culture Techniques , Cell Division/drug effects , Cell Survival/drug effects , Collagen/drug effects , Dose-Response Relationship, Drug , Drug Administration Schedule , Humans , Pigment Epithelium of Eye/cytology , Vitreoretinopathy, Proliferative/drug therapy
11.
Invest Ophthalmol Vis Sci ; 39(8): 1524-9, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9660504

ABSTRACT

PURPOSE: The migration, proliferation, differentiation, and adhesion of cells and other cellular functions are influenced by the surrounding extracellular matrix, in normal and wound-healing conditions. The matrix metalloproteinases (MMPs) are a family of enzymes that degrade and remodel the extracellular matrix and, thus, play a central role in the wound-healing process. Proliferative vitreoretinopathy (PVR), a wound-healing process in the retina, is a major cause of the failure of retinal detachment surgery. The role of MMPs in the pathobiology of PVR is unknown. We have investigated the presence of MMPs in the vitreous of patients with retinal detachment and the predictive value of MMPs for the future development of PVR. METHODS: A prospective study was conducted on 140 consecutive patients with a rhegmatogenous retinal detachment in whom vitrectomy was considered necessary because of a giant retinal tear and the presence of preoperative PVR, among other reasons. Vitreous samples were obtained and analyzed by zymography for the presence of MMPs. The patients were then followed up for the development of postoperative PVR (mild and severe). RESULTS: Two species of MMPs were detected in the vitreous: MMP-2 and MMP-9. MMP-2 was detected in all of the vitreous samples obtained, whereas MMP-9 was found in only 64 (47%) of 136 samples. The levels of MMPs detected were not significantly associated with the presence of preoperative PVR (P > 0.05), but they were significantly associated (P < 0.05) with the development of postoperative PVR (mild and severe). CONCLUSIONS: The results from this prospective study suggest that MMPs may be an important predictor and may also play a role in the development of postoperative PVR.


Subject(s)
Metalloendopeptidases/metabolism , Vitreoretinopathy, Proliferative/enzymology , Adolescent , Adult , Aged , Aged, 80 and over , Collagenases/metabolism , Extracellular Matrix/enzymology , Female , Follow-Up Studies , Gelatinases/metabolism , Humans , Male , Matrix Metalloproteinase 2 , Matrix Metalloproteinase 9 , Middle Aged , Molecular Weight , Prospective Studies , Retinal Detachment/etiology , Retinal Detachment/surgery , Vitrectomy , Vitreoretinopathy, Proliferative/complications , Vitreoretinopathy, Proliferative/surgery , Vitreous Body/enzymology
12.
Br J Ophthalmol ; 82(4): 448-52, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9640199

ABSTRACT

AIMS/BACKGROUND: Antimetabolites are increasingly used to manipulate the healing response after filtration surgery, but problems with thin cystic blebs have been encountered with the liquid agents commonly used such as 5-fluorouracil and mitomycin C. beta Radiation appears to be a useful adjuvant treatment for preventing scarring after trabeculectomy, resulting in diffuse rather than cystic bleb formation, but much of the basic cell biology of the ocular fibroblast response to beta radiation remains unclear. The effects of beta radiation on ocular fibroblast proliferation and cell cycling were investigated to determine the nature and duration of these effects on these cells. METHODS: In vitro cell culture techniques were used to investigate fibroblast proliferation. Cell viability was studied using trypan blue dye exclusion. The effect of radiation on cell cycling was investigated using bromodeoxyuridine uptake. p53 expression was demonstrated using immunocytochemistry. RESULTS: beta Radiation inhibited fibroblast proliferation in a dose dependent manner. Early cell death was not a prominent feature, but irradiated fibroblasts demonstrated a rapid onset and sustained period of growth arrest. p53 expression was found to be increased in irradiated cells. CONCLUSIONS: Single doses of beta radiation significantly inhibit Tenon's capsule fibroblast proliferation in vitro over a 28 day period. This inhibition is the result of a rapid onset and sustained period of growth arrest in irradiated cells. Irradiated fibroblasts show an increase in p53 expression, a nuclear phosphoprotein which has been associated with control of the cell cycle. Single applications of beta radiation may be an effective treatment for the prevention of bleb failure as a result of prolonged growth arrest of Tenon's capsule fibroblasts.


Subject(s)
Fibroblasts/radiation effects , Trabeculectomy , Wound Healing/radiation effects , Beta Particles , Bromodeoxyuridine/metabolism , Cell Division/radiation effects , Cells, Cultured , Dose-Response Relationship, Radiation , Fibroblasts/cytology , Fibroblasts/metabolism , Humans
13.
J Hand Surg Am ; 23(2): 266-73, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9556267

ABSTRACT

Injured and surgically repaired tendons heal with the formation of scar tissue. Scar tissue represents 1 of the most unpredictable factors contributing to postoperative morbidity. The main cell involved in scar formation is the fibroblast. The relative activity of fibroblasts from the fibro-osseous sheath (the tissue surrounding the tendon in zone II) and the endotenon (the core of the tendon) with respect to proliferation and the ability to contract a collagen lattice were compared in vitro. The fibroblasts derived from the fibro-osseous sheath were more active in both these respects. In addition, the amount of matrix metalloproteinase activity was found to be greater for the fibro-osseous sheath fibroblasts, implying a greater capacity to degrade and disorganize connective tissue and thus migrate. These results imply that the fibro-osseous fibroblasts represent a more active population of cells compared with endotenon fibroblasts, and perhaps should be specifically targeted in future modes of therapy.


Subject(s)
Collagen/ultrastructure , Fibroblasts/cytology , Synovial Membrane/cytology , Tendons/cytology , Analysis of Variance , Animals , Bone and Bones/cytology , Cell Division , Cell Movement , Cicatrix/pathology , Collagen/metabolism , Connective Tissue/metabolism , Fibroblasts/enzymology , Metalloendopeptidases/metabolism , Rabbits , Wound Healing
14.
Invest Ophthalmol Vis Sci ; 39(2): 449-54, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9478007

ABSTRACT

PURPOSE: To determine whether treatment with mitomycin-c and 5-fluorouracil induces apoptotic death in cultured subconjunctival fibroblasts. METHODS: Cultured human subconjunctival Tenon's capsule fibroblasts were exposed to 5-minute applications of mitomycin-C (up to 1 mg/ml) or 5-fluorouracil (up to 50 mg/ml) or phosphate-buffered saline solution (PBS). Fibroblast apoptosis was determined by cell morphology, apoptosis-specific protein expression, and DNA fragmentation by TdT-mediated dUTP nick-end labeling (TUNEL). In addition, apoptosis was quantified by direct cell counts based on morphology or lactate dehydrogenase release. RESULTS: Morphologic changes characteristic of apoptosis included nuclear and cytoplasmic condensation and occasional nuclear fragmentation while the plasma membrane remained intact. Apoptosis-specific protein expression and DNA fragmentation was observed in fibroblasts 48 hours after mitomycin-C treatment but not in control PBS-treated fibroblasts. The amount of apoptosis induced was dose dependent and partially inhibited by the addition of fetal calf serum to growth medium immediately after treatment. CONCLUSIONS: Mitomycin-C and high-dose 5-fluorouracil induce apoptosis in cultured Tenon's fibroblasts. Mitomycin-C-induced apoptosis is inhibited by fetal calf serum, indicating that exogenous factors influence the susceptibility of a fibroblast population to apoptosis. The induction and regulation of fibroblast apoptosis provides a novel target for the potential regulation of scarring.


Subject(s)
Antimetabolites/pharmacology , Apoptosis/drug effects , Fibroblasts/drug effects , Fluorouracil/pharmacology , Mitomycin/pharmacology , Cell Count , Cells, Cultured , Connective Tissue/drug effects , Connective Tissue/enzymology , Connective Tissue/pathology , DNA Fragmentation/drug effects , Dose-Response Relationship, Drug , Fibroblasts/enzymology , Fibroblasts/ultrastructure , Humans , L-Lactate Dehydrogenase/metabolism
15.
Invest Ophthalmol Vis Sci ; 38(10): 1998-2007, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9331263

ABSTRACT

PURPOSE: To determine the long-term effects of single, 5-minute exposures to 5-fluorouracil (5FU) and mitomycin-C (MMC) on Tenon's capsule fibroblast migration, growth factor production, growth factor receptor expression, and extracellular matrix (ECM) production. METHODS: Monolayer cultures and the overlying growth medium of Tenon's capsule fibroblasts exposed to 5FU (0.25 to 25 mg/ml) or MMC (0.001 to 0.1 mg/ml) were harvested up to 48 days after treatment. The expression of growth factors and growth factor receptors, including transforming growth factor beta (TGFbeta), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF), and ECM molecules (collagen type I, collagen type III, and fibronectin) were quantitated at the mRNA and protein levels. The ability of fibroblasts exposed to 5FU and MMC to migrate to fetal calf serum was also investigated up to 48 days after treatment. RESULTS: Control cultures were found to produce the growth factors TGFbeta and bFGF but not EGF. Exposure to 5FU or MMC resulted in an initial significant increase (P < 0.05) in the production of TGFbeta and bFGF, with levels then decreasing toward those of controls. Cells exposed to 5FU or MMC exhibited an initial significant decrease (P < 0.05) in the number of TGFbeta, bFGF, and EGF growth factor receptors, with subsequent recovery toward control levels by day 48 after treatment. Both 5FU and MMC caused a significant reduction (P < 0.05) in collagen type I and fibronectin production compared to controls throughout the 48-day culture period. The production of collagen type III was initially elevated (P < 0.05) compared to controls after exposure to 5FU or MMC, production then decreasing toward control levels over the remainder of the 48-day culture period. The migration of cells exposed to 5FU or MMC was significantly reduced (P < 0.05) compared to controls up to 48 days after treatment; these cells exhibited a partial recovery of migratory ability throughout this period. CONCLUSIONS: Fibroblasts whose growth was arrested using single, short exposures to 5FU or MMC appear to be capable of performing several crucial aspects of wound healing, including the expression of growth factors and receptors and ECM molecules and the ability to migrate. These findings may help explain why in some patients treated with antiproliferatives, glaucoma filtration surgery fails because of scarring.


Subject(s)
Antimetabolites/pharmacology , Eye/drug effects , Fibroblasts/drug effects , Fluorouracil/pharmacology , Mitomycin/pharmacology , Wound Healing/drug effects , Cell Movement/drug effects , Cells, Cultured , Child, Preschool , Connective Tissue/drug effects , Connective Tissue/metabolism , Connective Tissue Cells , Extracellular Matrix Proteins/metabolism , Eye/cytology , Eye/metabolism , Fibroblasts/metabolism , Growth Substances/metabolism , Humans , Male , RNA, Messenger/metabolism , Receptors, Growth Factor/metabolism
16.
Plast Reconstr Surg ; 99(2): 465-71, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9030156

ABSTRACT

After injury, adhesions may develop between the digital flexor tendons and their sheaths. Fibroblasts are key cells in this fibrotic adhesive process, and two possible sources for these cells are the synovial sheath and the endotenon tissue (tendon core). Fibroblasts seeded into a collagen lattice will contract the collagen. This fibroblast-populated collagen lattice contraction was used to investigate the ability of the fibroblasts from the synovial sheath and endotenon to reorganize collagen (an important function in the formation of adhesions). Endotenon and synovial fibroblasts isolated from 30 animals were used in the study. Synovial fibroblasts produced significantly greater collagen lattice contraction compared with endotenon fibroblasts (p < 0.05). The possibility of preventing collagen lattice contraction with a single, nontoxic 5-minute treatment of the fibroblast-populated collagen lattices with the antimetabolite 5-fluorouracil was investigated. Compared with controls the degree of fibroblast-populated collagen lattice contraction was significantly inhibited (p < 0.05) with the use of 5-fluorouracil for endotenon and synovial cells. These results demonstrate the potential for locally targeted therapy in tendon healing. Because of the different contractile properties of the two cell lines, a change in the balance between intrinsic and extrinsic healing might be achieved with this method of therapy; in turn, this might lead to better functional results following surgery.


Subject(s)
Antimetabolites/administration & dosage , Cicatrix/prevention & control , Collagen/drug effects , Collagen/physiology , Fibroblasts/drug effects , Fluorouracil/administration & dosage , Tendon Injuries/complications , Animals , Cells, Cultured , Cicatrix/etiology , Cytoskeleton/drug effects , Fibroblasts/physiology , Rabbits , Tendon Injuries/pathology
18.
Br J Cancer ; 72(1): 155-9, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7599046

ABSTRACT

Both cutaneous and uveal melanoma undergo haematogenous dissemination. Detection of tyrosinase mRNA by reverse transcription-polymerase chain reaction (RT-PCR) has been described as an extremely sensitive way of detecting circulating viable melanoma cells in the peripheral venous blood, and this technique may be of value in the early detection of dissemination. Also, it has been suggested that surgical manipulation of the eye, such as occurs during enucleation, can provoke uveal melanoma dissemination. The purpose of this study was to evaluate whether tyrosinase mRNA is detectable in the peripheral blood of patients with uveal and cutaneous melanoma and in patients with uveal melanoma undergoing surgical procedures on the eye harbouring the tumour. Venous blood samples from 36 patients diagnosed as having active uveal melanoma and from six patients with advanced metastatic cutaneous melanoma were analysed. In addition, blood samples were spiked with known numbers of cells from three cell lines and four primary uveal melanoma cultures. The reported sensitivity of the technique was confirmed, with an ability to detect down to one cell per ml of blood. All 51 blood samples from the 36 patients with uveal melanoma were negative, and this included 20 perioperative blood samples. The test was also negative for the six patients with advanced cutaneous melanoma. There were two positives among 31 control samples analysed. This study demonstrates that there are far fewer circulating viable melanocytes than has been previously supposed in patients with melanoma and that the RT-PCR is of no clinical value in detecting metastatic melanoma disease. There was no evidence for surgery causing a bolus of melanoma cells to enter the peripheral circulation.


Subject(s)
Melanoma/diagnosis , Monophenol Monooxygenase/genetics , Neoplastic Cells, Circulating , Polymerase Chain Reaction , RNA, Messenger/analysis , Base Sequence , Humans , Molecular Sequence Data , Sensitivity and Specificity , Skin Neoplasms/diagnosis , Uveal Neoplasms/diagnosis
19.
Invest Ophthalmol Vis Sci ; 35(10): 3681-90, 1994 Sep.
Article in English | MEDLINE | ID: mdl-8088956

ABSTRACT

PURPOSE: To determine the effects of short-duration treatments with 5-fluorouracil (5FU) and mitomycin-c (MMC) on "activated" and "nonactivated" ocular fibroblasts in collagen lattices. METHODS: Activated and nonactivated ocular fibroblasts seeded in collagen lattices were exposed to single 5-minute treatments with 5FU (0.01 to 25 mg/ml) and MMC (0.01 to 1 mg/ml). The effects of these treatments on lattice contraction, cellularity, cellular viability, cellular structure, and actin distribution were investigated. RESULTS: Treatment with 5FU (0.01 to 25 mg/ml) or MMC (0.1 to 1 mg/ml) significantly inhibited (P < 0.001 and P < 0.0001, respectively) lattice contraction compared to water controls. The degree of inhibition was greater in lattices containing nonactivated cells than in those containing activated cells. Activated cell viability and cellularity, unlike their nonactivated counterparts, were not significantly affected (P > 0.0083) by treatment with 5FU at high concentrations (25 mg/ml). MMC treatment had significant effects on cell viability and cellularity (P < 0.0001). Treatment with 5FU and MMC also affected cellular structure and actin distribution compared to water controls. CONCLUSIONS: Single, short exposures to 5FU or MMC inhibit ocular fibroblast-mediated collagen contraction. MMC causes cell death and a decrease in cellularity at high concentrations. The results also indicate that collagen lattices seeded with activated and nonactivated fibroblasts are differentially affected by short-term exposures to 5FU or MMC. These findings may have important clinical implications regarding the concentrations of these agents used in the treatment of different patient groups.


Subject(s)
Collagen/metabolism , Fascia/cytology , Fascia/metabolism , Fluorouracil/pharmacology , Mitomycins/pharmacology , Actins/metabolism , Cell Count , Cell Line , Cell Survival , Cells, Cultured , Fascia/drug effects , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/ultrastructure , Fluorescent Antibody Technique , Humans , Microscopy, Electron, Scanning
20.
Eur J Cancer ; 30A(1): 93-6, 1994.
Article in English | MEDLINE | ID: mdl-7511403

ABSTRACT

An important role in differentiation and proliferation has been demonstrated for the 20 cytokeratin (CK) polypeptides. The serum of 24 patients with biopsy-proven non-small cell lung cancer (NSCLC) and a similar number of controls was examined for evidence of CK8 and CK18. Using enzyme-linked immunosorbent assay (ELISA), all the control sera were negative, but 9 of the 24 patients were positive (mean 2.62 ng/ml; range 1.4-5.8; P = 0.0036). Western blotting confirmed the results of the ELISA in all cases, and indicated full size CK polypeptides. Advanced stage disease patients were more likely to be seropositive (P = 0.00024). Biopsy specimens showed CK8 expression in all 24 cases by immunochemistry and CK18 in 22 cases. This is the first study to demonstrate that a subgroup of NSCLC patients have intact CK8 and CK18 peptides in their serum, and their detection may correlate with advanced disease.


Subject(s)
Carcinoma, Non-Small-Cell Lung/blood , Keratins/blood , Lung Neoplasms/blood , Neoplasm Proteins/blood , Adenocarcinoma/chemistry , Aged , Aged, 80 and over , Blotting, Western , Cell Death/physiology , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoenzyme Techniques , Male , Middle Aged , Tumor Cells, Cultured
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