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1.
J Am Acad Dermatol ; 37(4): 586-9, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9344198

ABSTRACT

BACKGROUND: A keloid is a benign tumor that contains excess collagen, primarily type I collagen. A common therapy is intralesional injection of a glucocorticosteroid, such as triamcinolone acetonide (TA). Surgical excision is also common; often a glucocorticosteroid is injected weeks after excision when wound repair has already begun. OBJECTIVE: Our purpose was to determine the efficacy of TA in reducing the pro-alpha1(I) type I collagen mRNA in the dermis, when TA is injected into the wound bed immediately after surgical excision of the keloid. METHODS: Six patients with previously untreated keloids were studied. Three were treated with 10 mg/ml of TA immediately after excision of the keloid (experimental group); the other three patients were not treated with TA until 2 weeks after excision (control). Punch biopsy specimens were obtained from the TA-treated sites 2 weeks after removal of the keloid and from the wounds of the control group of patients before they were treated with TA. Sections were prepared for in situ hybridization analysis of the pro-alpha1(I) collagen mRNA, as well as for histochemical analysis of collagen fibers. RESULTS: All keloids showed greatly elevated levels of pro-alpha1(I) type I collagen mRNA in the dermis. Postsurgical wounds injected with TA after removal of the keloid expressed decreased pro-alpha1(I) collagen transcripts, compared with skin not treated with TA. The collagen bundles were also thinner and less dense in the TA-treated skin. CONCLUSION: Downregulation of the type I collagen gene expression is elicited by immediate TA injection after keloid excision. This suggests that prevention of recurrent keloid growth is possible if surgical excision is accompanied by immediate TA injection into the wound bed and that healing of the wound is not apparently compromised by inhibition of type I collagen gene expression.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Collagen/drug effects , Glucocorticoids/therapeutic use , Keloid/surgery , Procollagen/drug effects , Skin/drug effects , Triamcinolone Acetonide/therapeutic use , Administration, Topical , Adolescent , Adult , Anti-Inflammatory Agents/administration & dosage , Biopsy , Collagen/analysis , Collagen/genetics , Collagen/ultrastructure , Coloring Agents , Combined Modality Therapy , Down-Regulation , Ear Diseases/drug therapy , Ear Diseases/surgery , Ear, External/surgery , Gene Expression Regulation/drug effects , Glucocorticoids/administration & dosage , Humans , Immunohistochemistry , In Situ Hybridization , Injections, Intralesional , Procollagen/analysis , Procollagen/genetics , Procollagen/ultrastructure , RNA, Messenger/drug effects , RNA, Messenger/genetics , Thorax/pathology , Transcription, Genetic/drug effects , Triamcinolone Acetonide/administration & dosage , Wound Healing/drug effects
2.
Eur J Biochem ; 247(2): 503-10, 1997 Jul 15.
Article in English | MEDLINE | ID: mdl-9266691

ABSTRACT

Basic fibroblast growth factor (bFGF) is a pleiotropic factor that is implicated in tissue remodeling. The growth factor is capable of up-regulating the expression of the interstitial collagenase (matrix metalloproteinase-1 or MMP-1) gene. In this study, the full-length human MMP-1 promoter, spanning 4.3 kb, was sequenced and the regulatory control of its activity by bFGF was examined in NIH3T3 fibroblasts. Several regulatory sequences, including five activator protein-1 (AP-1), five activator protein-2 (AP-2), five glucocorticoid-response elements and multiple ets/polyoma enhancer-binding 3 elements, were identified. Deletion constructs were prepared and transiently transfected into fibroblast cultures incubated with and without bFGF. The results showed that bFGF enhanced the activity of the deletion promoter fragments and the full-length MMP-1 promoter by sixfold or more in the cell cultures. Stimulation of the MMP-1 promoter activity by bFGF was reflected in substantial increase of the collagenase mRNA levels. A bFGF-responsive element appeared to be the AP-1 consensus sequence. Mutation of the first AP-1 site resulted in major reduction of the basal level of the MMP-1 promoter activity, supporting the notion that the AP-1 consensus sequence is essential for the constitutive expression of the MMP-1 gene. Furthermore, bFGF induction of the activity of the promoter constructs containing a mutant AP-1 site was essentially absent, suggesting that the regulatory element is necessary for the induction of the promoter activity by the growth factor. Thus, bFGF up-regulates MMP-1 gene expression in NIH3T3 fibroblasts via induction of its promoter activity that is dependent on an AP-1 consensus sequence.


Subject(s)
Collagenases/biosynthesis , Collagenases/genetics , Fibroblast Growth Factor 2/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Promoter Regions, Genetic/drug effects , 3T3 Cells , Animals , Base Sequence , Consensus Sequence , DNA Primers , Enzyme Induction/drug effects , Humans , Kinetics , Matrix Metalloproteinase 1 , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Regulatory Sequences, Nucleic Acid , Transcription, Genetic , Transfection
3.
J Cell Biochem ; 65(1): 32-41, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9138078

ABSTRACT

Basic fibroblast growth factor (bFGF) is a mitogenic factor that is implicated in smooth muscle cell growth in atherosclerosis and vascular restenosis. In this study, we examined the effect of bFGF on the expression of the interstitial collagenase gene in human vascular smooth muscle cells. Results from Northern transfer analysis showed that bFGF increased collagenase mRNA levels greater than threefold as early as 24 h. Collagenase pre-mRNA levels were elevated approximately threefold by bFGF, according to RT-PCR analysis. Transient transfections of the smooth muscle cells with a 4.4-kb human collagenase promoter-CAT reporter gene, however, failed to show upregulation of the promoter activity by bFGF. Interestingly, transfections with deleted fragments containing promoter sequences from -1047 to -2271 resulted in modest stimulation of the collagenase-CAT promoter activity by bFGF, bFGF did not alter the stability of the collagenase mRNA, as demonstrated by degradation studies. The enhanced collagenase mRNA levels elicited by bFGF were reflected in increased amounts of collagenase protein that were detected by Western blot analysis. In summary, bFGF upregulates the interstitial collagenase expression, resulting in turnover of the extracellular matrix, an event that could facilitate smooth muscle cell migration and proliferation during the early stages of atherosclerosis and restenosis.


Subject(s)
Collagenases/genetics , Fibroblast Growth Factor 2/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Cells, Cultured , Collagenases/metabolism , Humans , Muscle, Smooth, Vascular , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism
4.
Biochem J ; 310 ( Pt 2): 585-8, 1995 Sep 01.
Article in English | MEDLINE | ID: mdl-7654198

ABSTRACT

Platelet-derived growth factor (PDGF) is a mitogen associated with tissue repair, a process involving collagen synthesis and remodelling by interstitial collagenase. This study examines and compares the regulation of interstitial collagenase and collagen gene expression by PDGF-AA and -BB in human fibroblasts. Time-course analysis showed that neither PDGF-AA or -BB had a consistent effect on the expression of pro-alpha 1(I) or pro-alpha 2(I) type-I collagen genes. In contrast, interstitial collagenase gene expression was found to be consistently up-regulated severalfold by PDGF-BB. Enhanced expression of the collagenase gene was not apparently due to up-regulation of its promoter activity in human dermal fibroblasts, as indicated by transient and stable transfection experiments. Unlike PDGF-BB, PDGF-AA did not alter collagenase mRNA levels under low-serum culture conditions. Thus, the biological activities of the PDGF homodimers are different, with PDGF-BB being clearly more potent than PDGF-AA in its regulation of collagenase gene expression.


Subject(s)
Collagen/biosynthesis , Collagenases/biosynthesis , Gene Expression Regulation/drug effects , Platelet-Derived Growth Factor/pharmacology , Procollagen/biosynthesis , Skin/metabolism , Becaplermin , Cells, Cultured , Dose-Response Relationship, Drug , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Humans , Keloid/metabolism , Kinetics , Promoter Regions, Genetic , Proto-Oncogene Proteins c-sis , Recombinant Proteins/pharmacology , Reference Values , Time Factors
5.
Exp Cell Res ; 209(2): 200-7, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8262136

ABSTRACT

Basic fibroblast growth factor (bFGF) is a multipotential heparin-binding factor that belongs to the fibroblast growth factor (FGF) family. The FGFs demonstrate a wide spectrum of biologic activities in vivo and in vitro. In this study, we investigated the potential of bFGF to regulate the expression of various dermal extracellular matrix proteoglycans and type I collagen mRNAs in cultured human fibroblasts from keloid, which is a prototype of dermal fibrosis, and normal skin tissue. We report that bFGF upregulates the expression of the decorin gene in normal and keloid fibroblasts. In contrast, the expression of biglycan is downregulated by bFGF. The mRNA steady-state level of versican, a large proteoglycan, is not altered by bFGF. Type I collagen gene expression is downregulated substantially in keloid and normal fibroblasts by bFGF. The results suggest that the expression of the proteoglycan genes are uncoordinately regulated and that the gene expression of type I collagen and biglycan is coordinately downregulated. The results also demonstrate that keloid fibroblasts respond similarly as do normal fibroblasts to bFGF in the regulation of proteoglycan and collagen expression.


Subject(s)
Chondroitin Sulfate Proteoglycans/genetics , Fibroblast Growth Factor 2/pharmacology , Fibroblasts/metabolism , Proteoglycans/genetics , Biglycan , Cells, Cultured , Chondroitin Sulfate Proteoglycans/metabolism , Collagen/genetics , Decorin , Extracellular Matrix Proteins , Gene Expression/drug effects , Humans , In Vitro Techniques , Keloid/metabolism , Lectins, C-Type , Proteoglycans/metabolism , RNA, Messenger/genetics , Versicans
6.
Am J Pathol ; 142(2): 463-70, 1993 Feb.
Article in English | MEDLINE | ID: mdl-7679551

ABSTRACT

The effects of acidic and basic fibroblast growth factors (FGFs) on collagen expression by keloid fibroblasts were examined in the absence and presence of heparin. Collagen biosynthesis and gene expression of type I collagen were down-regulated by the FGFs in the presence of heparin. Acidic FGF, in a concentration range of 0.4 to 50 ng/ml, had little or no effect on collagen synthesis after a 4-day incubation. However, in the presence of heparin (100 micrograms/ml) acidic FGF, in concentrations ranging from 2 to 50 ng/ml, decreased [3H]hydroxyproline synthesis by 44 to 68%, compared with untreated control cultures. Total [3H]hydroxyproline synthesis was similar between control and heparin-treated cultures. Basic FGF (2.0 to 50 ng/ml) was effective in suppressing [3H]hydroxyproline synthesis by 50 to 90% after a 4-day incubation without heparin in keloid and normal fibroblast cultures. The steady-state levels of type I collagen messenger RNA were significantly decreased by acidic FGF in the presence of heparin, as well as by basic FGF without heparin. The data suggest that the FGFs are effective in down-regulating excess collagen production by keloid fibroblasts and that this inhibitory effect is apparently associated with pretranslational events. Moreover, acidic FGF is apparently dependent on heparin, whereas basic FGF is not, for potentiation of the down-regulatory effects of the FGFs.


Subject(s)
Collagen/genetics , Fibroblast Growth Factor 1/pharmacology , Fibroblast Growth Factor 2/pharmacology , Gene Expression/drug effects , Keloid/genetics , Skin/physiopathology , Collagen/metabolism , Dose-Response Relationship, Drug , Fibroblasts/metabolism , Fibroblasts/physiology , Fluorescent Antibody Technique , Heparin/pharmacology , Homeostasis , Humans , Hydroxyproline/biosynthesis , Keloid/metabolism , Keloid/pathology , RNA, Messenger/metabolism , Skin/metabolism , Skin/pathology , Time Factors
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