Increased Plasminogen Activator Inhibitor-1 in Keloid Fibroblasts May Account for their Elevated Collagen Accumulation in Fibrin Gel Cultures
2003; Elsevier BV; Volume: 162; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)64292-7
ISSN1525-2191
AutoresTai‐Lan Tuan, Huayang Wu, Eunice Y. Huang, Sheree Chong, Walter E. Laug, Diana V. Messadi, Paul A. Kelly, Anh Lê,
Tópico(s)Periodontal Regeneration and Treatments
ResumoProteolytic degradation of the provisional fibrin matrix and subsequent substitution by fibroblast-produced collagen are essential features of injury repair. Immunohistochemical studies revealed that although dermal fibroblasts of normal scars and keloids expressed both urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1), keloid fibroblasts had a much higher PAI-1 expression. In long-term three-dimensional fibrin gel cultures (the in vitro fibroplasia model), normal fibroblasts expressed moderate and modulated activity levels of uPA and PAI-1. In contrast, keloid fibroblasts expressed a persistently high level of PAI-1 and a low level of uPA. The high PAI-1 activity of keloid fibroblasts correlated with their elevated collagen accumulation in fibrin gel cultures. Substituting collagen for fibrin in the gel matrix resulted in increased uPA activity and reduced collagen accumulation of keloid fibroblasts. Furthermore, decreasing PAI-1 activity of keloid fibroblasts in fibrin gel cultures with anti-PAI-1-neutralizing antibodies also resulted in a reduction in collagen accumulation by keloid fibroblasts. Cumulatively, these results suggest that PAI-1 overexpression is a consistent feature of keloid fibroblasts both in vitro and in vivo, and PAI-1 may play a causative role in elevated collagen accumulation of keloid fibroblasts. Proteolytic degradation of the provisional fibrin matrix and subsequent substitution by fibroblast-produced collagen are essential features of injury repair. Immunohistochemical studies revealed that although dermal fibroblasts of normal scars and keloids expressed both urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1), keloid fibroblasts had a much higher PAI-1 expression. In long-term three-dimensional fibrin gel cultures (the in vitro fibroplasia model), normal fibroblasts expressed moderate and modulated activity levels of uPA and PAI-1. In contrast, keloid fibroblasts expressed a persistently high level of PAI-1 and a low level of uPA. The high PAI-1 activity of keloid fibroblasts correlated with their elevated collagen accumulation in fibrin gel cultures. Substituting collagen for fibrin in the gel matrix resulted in increased uPA activity and reduced collagen accumulation of keloid fibroblasts. Furthermore, decreasing PAI-1 activity of keloid fibroblasts in fibrin gel cultures with anti-PAI-1-neutralizing antibodies also resulted in a reduction in collagen accumulation by keloid fibroblasts. Cumulatively, these results suggest that PAI-1 overexpression is a consistent feature of keloid fibroblasts both in vitro and in vivo, and PAI-1 may play a causative role in elevated collagen accumulation of keloid fibroblasts. Keloids are scars that show exuberant growth beyond the margins of the original wound that rarely regress throughout time. It is estimated that ∼15 to 20% of African-Americans, Hispanics, and Asians develop keloids with a suggested genetic predisposition to keloid formation.1Tuan TL DiCesare P Cheung D Nimni ME Keloids and hypertrophic scars.in: Nimni ME Kang AH Collagen: Pathobiochemistry. CRC Press, Boca Raton1991: 125-136Google Scholar, 2Tuan TL Nichter LS The molecular basis of keloid and hypertrophic scar formation.Mol Med Today. 1998; 4: 19-24Abstract Full Text PDF PubMed Scopus (378) Google Scholar Before keloids form, the affected skin shows excessive and prolonged fibroplasia, and keloids differ from other scars by having multiple collagen nodules in the deep dermis. In vitro, keloid fibroblasts exhibit an altered phenotype of either intrinsic or growth factor-stimulated collagen, fibronectin, elastin, and proteoglycan accumulation.3Uitto J Perejda AJ Abergel RP Chu ML Ramirez F Altered steady-state ratio of type I/III procollagen mRNAs correlates with selectively increased type I procollagen biosynthesis in cultured keloid fibroblasts.Proc Natl Acad Sci USA. 1985; 82: 5935-5939Crossref PubMed Scopus (171) Google Scholar, 4Russell SB Trupin KM Rodriguez-Eaton S Russell JD Trupin JS Reduced growth-factor requirement of keloid-derived fibroblasts may account for tumor growth.Proc Natl Acad Sci USA. 1988; 85: 587-591Crossref PubMed Scopus (115) Google Scholar, 5Babu M Diegelmann R Oliver N Fibronectin is overproduced by keloid fibroblasts during abnormal wound healing.Mol Cell Biol. 1989; 9: 1642-1650Crossref PubMed Scopus (146) Google Scholar, 6Kischer CW Wagner Jr, HN Pindur J Holubec H Jones M Ulreich JB Scuderi P Increased fibronectin production by cell lines from hypertrophic scar and keloid.Connect Tissue Res. 1989; 23: 279-288Crossref PubMed Scopus (48) Google Scholar, 7Russell SB Trupin JS Myers JC Broquist AH Smith JC Myles ME Russell JD Differential glucocorticoid regulation of collagen mRNAs in human dermal fibroblasts. 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The Molecular and Cell Biology of Wound Repair. Plenum Press, New York1996: 22-32Google Scholar Organ fibrosis is typified by excessive collagen accumulation at the site of injury that involves excessive collagen synthesis or delayed collagen degradation. Therefore, molecules that promote collagen synthesis or inhibit its degradation must be involved. PAI-1 belongs to the serpin family and is the major inhibitor to urokinase-type and tissue-type plasminogen activators (uPA and tPA, respectively).22Plow EF Herren T Redlitz A Miles LA Hoover-Plow JL The cell biology of the plasminogen system.EMBO J. 1995; 9: 939-945Google Scholar Both uPA and tPA convert plasminogen into plasmin. Being the primary protease in fibrinolysis, plasmin also participates in the breakdown of other glycoproteins in the extracellular matrix (ECM), the activation of matrix metalloproteinases (MMPs) from their proenzyme form,23Parks WC Mecham RP Matrix Metalloproteinases. Academic Press, San Diego1998: 1-362Crossref Google Scholar and the release of transforming growth factor (TGF)-β from its latency-associated protein.24Rifkin DB Gleizes PE Harpel J Nunes I Munger J Mazzieri R Noguera I Plasminogen/plasminogen activator and growth factor activation.Ciba Found Symp. 1997; 212: 105-115PubMed Google Scholar In fact, targeted gene manipulations of MMPs display abnormalities in injury repair overlapping with that of the members of the plasminogen activator system.25Bullard KM Lund L Mudgett JS Mellin TN Hunt TK Murphy B Ronan J Werb Z Banda MJ Impaired wound contraction in stromelysin-1-deficient mice.Ann Surg. 1999; 230: 260-265Crossref PubMed Scopus (191) Google Scholar Furthermore, inhibition of both the plasminogen activator/plasmin and MMP systems leads to complete arrest of wound healing and wound closure.26Lund LR Romer J Bugge TH Nielsen BS Frandsen TL Degen JL Stephens RW Dano K Functional overlap between two classes of matrix-degrading proteases in wound healing.EMBO J. 1999; 18: 4645-4656Crossref PubMed Scopus (229) Google Scholar On the other hand, increased PAI-1 has been a hallmark of organ fibrosis in which it has been associated with thrombotic diseases and metabolic disorders that are linked with the development of arteriosclerosis and organ fibrosis in liver, lung, kidney, blood vessels, and skin.27Olman MA Mackman N Gladson CL Moser KM Loskutoff DJ Changes in procoagulant and fibrinolytic gene expression during bleomycin-induced lung injury in the mouse.J Clin Invest. 1995; 96: 1621-1630Crossref PubMed Scopus (157) Google Scholar, 28Eitzman DT McCoy RD Zheng X Fay WP Shen T Ginsburg D Simon RH Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene.J Clin Invest. 1996; 97: 232-237Crossref PubMed Scopus (528) Google Scholar, 29Higgins PJ Slack JK Diegelmann RF Staiano-Coico L Differential regulation of PAI-1 gene expression in human fibroblasts predisposed to a fibrotic phenotype.Exp Cell Res. 1999; 248: 634-642Crossref PubMed Scopus (32) Google Scholar, 30Zhang LP Takahara T Yata Y Furui K Jin B Kawada N Watanabe A Increased expression of plasminogen activator and plasminogen activator inhibitor during liver fibrogenesis of rats: role of stellate cells.J Hepatol. 1999; 31: 703-711Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar Definitive proof of the plasminogen activator system involvement in ECM metabolism during tissue injury repair comes from targeted gene interruption or overexpression in mice. In this regard, it has been shown that bleomycin-induced pulmonary fibrosis is more severe in transgenic mice overexpressing PAI-1 or in mice deficient of plasminogen, uPA, or tPA.28Eitzman DT McCoy RD Zheng X Fay WP Shen T Ginsburg D Simon RH Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene.J Clin Invest. 1996; 97: 232-237Crossref PubMed Scopus (528) Google Scholar, 31Swaisgood CM French EL Noga C Simon RH Ploplis VA The development of bleomycin-induced pulmonary fibrosis in mice deficient for components of the fibrinolytic system.Am J Pathol. 2000; 157: 177-187Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar Administration of uPA into the lungs of wild-type or PAI-1 transgenic mice after bleomycin injury decreases lung fibrosis.32Sisson TH Hattori N Xu Y Simon RH Treatment of bleomycin-induced pulmonary fibrosis by transfer of urokinase-type plasminogen activator genes.Hum Gene Ther. 1999; 10: 2315-2323Crossref PubMed Scopus (91) Google Scholar Accordingly, PAI-1 knockout mice are protected against bleomycin-induced pulmonary fibrosis.33Hattori N Degen JL Sisson TH Liu H Moore BB Pandrangi RG Simon RH Drew AF Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice.J Clin Invest. 2000; 106: 1341-1350Crossref PubMed Scopus (232) Google Scholar Furthermore, plasminogen knockout mice exhibit delayed skin wound repair,34Romer J Bugge TH Pyke C Lund LR Flick MJ Degen JL Dano K Impaired wound healing in mice with a disrupted plasminogen gene.Nat Med. 1996; 2: 287-292Crossref PubMed Scopus (485) Google Scholar and PAI-1-deficient mice show accelerated wound closure.35Chan JC Duszczyszyn DA Castellino FJ Ploplis VA Accelerated skin wound healing in plasminogen activator inhibitor-1-deficient mice.Am J Pathol. 2001; 159: 1681-1688Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar These observations suggest that members of the plasminogen activator system play an essential role in ECM metabolism during wound repair. Increased PAI-1, which inhibits PA activity and the extent of plasminogen activation, may result in fibrin accumulation at the site of tissue injury and in fibrosis. In contrast to acute wounds, chronic wounds contain very high levels of active uPA and multiple species of gelatin- and casein-degrading proteases.36Wysocki AB Kusakabe AO Chang S Tuan TL Temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor and gelatinase-B in chronic wound fluid switches from a chronic to acute wound profile with progression to healing.Wound Repair Regen. 1999; 7: 154-165Crossref PubMed Scopus (97) Google Scholar Furthermore, the activity of these proteases seems to regress as the chronic wound heals.36Wysocki AB Kusakabe AO Chang S Tuan TL Temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor and gelatinase-B in chronic wound fluid switches from a chronic to acute wound profile with progression to healing.Wound Repair Regen. 1999; 7: 154-165Crossref PubMed Scopus (97) Google Scholar Therefore, abnormal healing may result from changes in either the activity of proteases, their inhibitors, or in the regulatory machinery that controls the expression of these molecules. The complex nature of the repair process and a lack of proper model systems have made it difficult to investigate the underlying mechanism(s) of fibrosis after injury repair in humans. We have developed an in vitro three-dimensional fibrin matrix gel culture system for this purpose.18Tuan TL Song A Chang S Younai S Nimni ME In vitro fibroplasia: matrix contraction, cell growth, and collagen production of fibroblasts cultured in fibrin gels.Exp Cell Res. 1996; 223: 127-134Crossref PubMed Scopus (168) Google Scholar This system models the key features of fibroplasia, ie, cell proliferation, fibrin reorganization and degradation, and collagen synthesis and deposition. It is suitable for the cellular and molecular studies of the intricate interactions between the cell and its surrounding ECM in wound repair. In continued experiments we demonstrated that PAI-1 overexpression is a consistent feature of keloid fibroblasts both in vitro and in vivo. We also studied collagen production and uPA/PAI-1 expression of normal and keloid fibroblasts in long-term fibrin matrix cultures, and investigated the effect of PAI-1 overproduction on collagen accumulation of keloid fibroblasts. Fibroblasts were established from donors of human normal skin, scar, and keloid using the explant method. The protocol for skin and scar collections was approved by the institutional review boards of both the Childrens Hospital Los Angeles and the Charles R. Drew University of Medicine and Science. The raised core region of keloid scars was used for fibroblast isolation. Fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc., Grand Island, NY) containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum (Life Technologies, Inc.). Cultures were incubated in a humidified incubator in an atmosphere of 5% CO2 and 95% air. Fibroblasts were harvested from cultures using 0.25% trypsin containing 0.05% ethylenediaminetetraacetic acid in Hanks' solution (Life Technologies, Inc.) and passaged once a week. Early passages (2 to 10) of fibroblasts were used in the experiments. Cell passage is defined as weekly expansion of cells from primary cultures. The source of each strain of fibroblasts used in the study is listed in Table 1.Table 1Normal Skin, Scar, and Keloid Fibroblasts Used in the StudyTypeCell strainEthnic backgroundGenderAgeAnatomical siteNormal skinN65HispanicM8EarNormal skinN77African-AmericanM4EarNormal skinN86African-AmericanM28EarNormal skinN123CaucasianF23NK*NK, not known.Normal skinN127IranianM33Post earNormal skinN141CaucasianMNewbornForeskinNormal skinN143CaucasianMNewbornForeskinNormal skinN144CaucasianMNewbornForeskinNormal skinK7NAfrican-AmericanNormal scarNSC14HispanicF25ElbowNormal scarNS70CaucasianM8FootNormal scarNS75African-AmericanM4CheekKeloidK9African-AmericanFNK*NK, not known.Ear lobeKeloidK10African-AmericanFNK*NK, not known.FlankKeloidK74African-AmericanM28Ear lobeKeloidK76African-AmericanM4EarKeloidK80HispanicM5Supra-pubicKeloidK86African-AmericanM28EarKeloidK109African-AmericanM14EarKeloidK134AsianF6ChestKeloidK135HispanicF6HandKeloidK139African-AmericanF47EarKeloidK142African-AmericanF7Sternal areaKeloidK147African-AmericanM18EarKeloidK148African-AmericanM14EarKeloidK150cAfrican-AmericanF23ChestKeloidK165HispanicM5Ear* NK, not known. Open table in a new tab Human fibrinogen (Calbiochem, San Diego, CA) was used for the preparation of fibrin gels. Fibrinogen was reconstituted in distilled H2O, adjusted to 10 mg/ml, and stored at −20°C. The clottability of fibrinogen was determined by mixing 1 to 5 mg/ml of fibrinogen with 1 to 2 U/ml of human thrombin and incubating for 30 minutes at 37°C. The clots that formed were detached from test tube walls. Tubes were centrifuged at 12,000 × g for 15 minutes to pellet the clot and to collect soluble fibrinogen. The soluble nonclottable fibrinogen remaining in the supernatant was determined by protein concentration at OD280. All fibrinogen used was 95 to 98% clottable. The method for fibrin gel preparation has been described in a previous publication.17Tuan TL Grinnell F Fibronectin and fibrinolysis are not required for fibrin gel contraction by human skin fibroblasts.J Cell Physiol. 1989; 140: 577-583Crossref PubMed Scopus (36) Google Scholar Briefly, human skin fibroblasts in DMEM were added to a fibrinogen solution at 24°C. Final concentrations of fibrinogen and fibroblasts were 2.5 mg/ml and 0.5 × 106 cells/ml, respectively. Aliquots (180 μl) of the fibroblast/fibrinogen mixtures were placed in wells of 24-well tissue culture plates (Costar, Cambridge, MA) with 1 U of thrombin per sample. Each aliquot occupied an area outlined by a 16-mm-diameter circular score within the well. The preparations were incubated at 37°C for 1 hour in a humidified incubator containing 5% CO2 to ensure polymerization of fibrin. At the end of the incubation period, 1.0 ml of DMEM containing 10% fetal calf serum was added to each well to cover the gel. Samples selected for uPA and PAI studies were first thoroughly rinsed (five times) with DMEM and incubated in DMEM for an additional 24 hours. Conditioned culture media were collected and subjected to fibrin overlay and reverse fibrin overlay assays. Collagen gels were prepared according to the method previously described by Tuan and colleagues37Tuan TL Keller LC Sun D Nimni ME Cheung D Dermal fibroblasts activate keratinocyte outgrowth on collagen gels.J Cell Sci. 1994; 107: 2285-2289PubMed Google Scholar using Vitrogen (Cohesion Technologies, Inc., Palo Alto, CA), a preparation of predominantly type I collagen. Briefly, the collagen was adjusted to physiological ionic strength and pH with 10× minimum essential medium (Sigma Chemical Co., St. Louis, MO) and 0.1 N of NaOH at 4°C. The final collagen concentration was 1.5 mg/ml. Fibroblasts were incorporated into the reconstituted collagen at a final concentration of 0.5 × 106 cells/ml. Samples of the collagen/fibroblast suspension were dispensed into 24-well culture plates. Each 180-μl aliquot was contained within a circle of 16-mm diameter scored onto the base of the well. The culture plates were then placed in an incubator at 37°C with 5% CO2 for 45 minutes to allow collagen to polymerize. Gels were prepared by mixing fibrinogen and collagen in different ratios (fibrin:collagen, 100%:0%; 50%:50%; 0%:100%). Fibroblasts were incorporated into the matrix at a final density of 0.5 × 106 cells/ml. Aliquots (180 μl) of gel-fibroblast mixtures were placed in wells of 24-well tissue-culture plates with 1 U of thrombin per sample following a similar format described above. Briefly, aliquots (25 μl) of serum-free conditioned culture media were subjected to electrophoresis using a 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS, Sigma). The gel was washed for 1 hour at room temperature in 2.5% Triton X-100 to remove SDS. After a brief rinse in distilled water, the gel was placed on an indicator gel layer (fibrin overlay assay for PA detection) that contained 1% low-temperature gelling agarose, human plasminogen (9 μg/ml, Sigma), thrombin (0.7 U/ml, Sigma), and fibrinogen (2 mg/ml). To detect PAI, SDS-polyacrylamide gels were washed in 2.5% Triton X-100 for 1 hour at room temperature and placed on top of a substrate gel similar to the indicator gel (above) with the addition of uPA (0.2 U/ml, Sigma) (reverse fibrin overlay assay). Both preparations were placed in a humidified chamber at 37°C. Activity of PA appeared as clear zones in the opaque fibrin indicator layer indicating fibrinolysis. Activity of PAI appeared as opaque zones in a cleared reverse overlay substrate layer indicating inhibition of fibrinolysis. The results were photographed. A two-stage, indirect enzymatic assay, Spectrolyse (pL) PAI (no. 101201; American Diagnostica, Greenwich, CT), was used for the quantitative determination of PAI-1 activity in plasma. In stage one, a fixed amount of tissue plasminogen activator (tPA) was added to the sample and allowed to react with PAI-1 present. The sample was then acidified to destroy α-2-anti-plasmin and other potential plasmin inhibitors that would otherwise interfere with the tPA assay. In stage two, the residual tPA activity was measured by adding the sample to a mixture of Glu-plasminogen, poly-d-lysine and chromogenic substrate at neutral pH. The residual tPA activity in the sample catalyzes the conversion of plasminogen to plasmin, which in turn hydrolyzes the chromogenic substrate. The amount of color developed is proportional to the amount of tPA activity in the sample. Poly-d-lysine is present as a stimulator of the tPA-catalyzed conversion of plasminogen to plasmin. The PAI content of the sample is then identified as the difference between the amount of tPA added and the amount of tPA recovered. One U of PAI activity (U) is defined as the amount of PAI that inhibits 1 IU of a human single chain tPA as calibrated against the International Standard for tPA lot 86/670 distributed by NIBSAC, Holly Hill, London, UK. [3H]Proline was used to label newly synthesized collagen by fibroblasts. Samples in triplicates were labeled for 48 hours with l-(5-[3H]proline) (50 μCi/ml; Amersham, Arlington Heights, IL) in DMEM-10% fetal calf serum supplemented with β-aminoproprionitrile (62.5 μg/ml). At the end of labeling, all samples were adjusted to 0.5 mol/L acetic acid and treated with 1 mg/ml of pepsin (PM grade; Worthington, Freehold, NJ) for 24 hours at 4°C to digest proteins other than intact collagen. Pepsin was inactivated by adding Tris to 50 mmol/L and titration to pH 7.4. Collagen was purified by sequential neutral salt (4 mol/L NaCl) and acid salt (2 mol/L NaCl) precipitation as described previously.18Tuan TL Song A Chang S Younai S Nimni ME In vitro fibroplasia: matrix contraction, cell growth, and collagen production of fibroblasts cultured in fibrin gels.Exp Cell Res. 1996; 223: 127-134Crossref PubMed Scopus (168) Google Scholar The final collagen pellet was rinsed in 50 mmol/L of Tris and 40% ethanol and dissolved in 0.5 mol/L of acetic acid. Samples were subjected to SDS-polyacrylamide gel electrophoresis and followed by fluorography. Samples designated for cell count were treated with trypsin and collagenase, and viable cell numbers were estimated using a hemocytometer in the presence of Trypan Blue. Purified collagen was expressed as cpm/cell. Data presented is an average of three replicate samples. Statistical differences between and within groups were assessed using one-way analysis of variance. Standard Northern blot analysis was used to study RNA expression.38Sambrook J Fritsch EF Maniatis T Molecular Cloning.A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York1989Google Scholar Briefly, RNA samples were extracted using guanidinium thiocyanate and separated by centrifugation through cesium chloride. Total RNA (20 μg/lane) was separated by electrophoresis, transferred to nylon filters, and baked at 80°C under vacuum for 2 hours. After prehybridization, the radioactive-labeled DNA probes were hybridized to filters for 20 hours at 40°C, washed, and visualized by exposure to X-ray film at −70°C. The cDNA probes were labeled according to the method of Feinberg and Vogelstein.39Feinberg AP Vogelstein B A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.Addendum Anal Biochem. 1984; 137: 266-267Crossref PubMed Scopus (5191) Google Scholar All samples were standardized to the level of expression of β-actin in each cell strain. Specific human cDNA probes for uPA nucleotides 623 to 1039 and PAI-1 cDNA (full length) were used as hybridization probes.40Laug WE Cao XR T'Ang A Pasquale S Mundy R Coensgen-Luna M Weissman B Complex expression of the genes coding for plasminogen activators and their inhibitors in HeLa-smooth muscle cell hybrids.Cell Growth Differ. 1992; 3: 191-197PubMed Google Scholar Freshly collected skin and scar samples were rinsed in ice-cold phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (pH 7.5, Sigma) at 4°C for 24 hours. Samples were treated with 70% ethanol for 24 hours before dehydration. After dehydration, samples were embedded in paraffin (60°C), and 5-μm-thick sections were prepared using a microtome. Sections were rehydrated and treated with H2O2. To minimize nonspecific binding, sections were first treated with 1.5% bovine serum albumin/PBS for 30 minutes at room temperature. Mouse monoclonal antibody against human uPA at 1:50 dilution (no. 3698 and no. 394, American Diagnostica Inc.) and murine monoclonal antibody against human PAI-1 at 1:25 dilution (no. 3785, American Diagnostica Inc.) were used to detect uPA and PAI-1, respectively. After primary antibody treatment, sections were washed three times with PBS and incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech Limited, Buckinghamshire, UK) for 50 minutes. After thorough rinsing with PBS, sections were treated with 3,3′-diaminobenzidine (Sigma) to reveal antibody-antigen reaction. Sections were also stained lightly with hematoxylin for nuclear staining. Keloid fibroblasts exhibit elevated PAI-1 expression in culture.20Tuan TL Zhu JY Sun B Nichter LS Nimni ME Laug WE Elevated levels of plasminogen activator inhibitor-1 may account for the altered fibrinolysis by keloid fibroblasts.J Invest Dermatol. 1996; 106: 1007-1011Crossref PubMed Scopus (80) Google Scholar, 29Higgins PJ Slack JK Diegelmann RF Staiano-Coico L Differential regulation of PAI-1 gene expression in human fibroblasts predisposed to a fibrotic phenotype.Exp Cell Res. 1999; 248: 634-642Crossref PubMed Scopus (32) Google Scholar To examine if PAI-1 overexpression also occurs in vivo, protein expressions of both PAI-1 and uPA were studied in keloid lesions (n = 5) using antibodies against PAI-1 or uPA in immunohistochemistry. The results were compared with normal skin (n = 3) and normal scar (n = 3) samples. Keloids are
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