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• W2000230823 abstract "Patients with localized scleroderma receiving topical photodynamic therapy with 5-aminolevulinic acid show a reduction in skin tightness, suggesting that this therapy reduces skin sclerosis. To investigate potential mechanisms, the effects of 5-aminolevulinic acid and light on collagen metabolism were studied in vitro. Normal and scleroderma fibroblasts were treated with sublethal doses of 5-aminolevulinic acid and red light and transferred to three-dimensional collagen lattices. Cell supernatants were taken 6–72 h after photodynamic therapy to determine protein levels of the matrix metalloproteinases 1, 2, and 3, and of their inhibitors, tissue inhibitor of metalloproteinase 1 and 2 by enzyme-linked immunosorbent assay. Cellular mRNA expression of these proteins and of collagen type I and III was measured by quantitative real-time polymerase chain reaction. A significant, time-dependent induction of matrix metalloproteinase 1 (up to 2.4-fold after 48 h) and matrix metalloproteinase 3 (up to 4.3-fold after 48 h) protein levels was seen after 5-aminolevulinic acid-photodynamic therapy. Irradiation with ultraviolet A light, used as a positive control, showed a similar induction of matrix metalloproteinase 1 (2.3-fold after 48 h). The mRNA levels of matrix metalloproteinase 1 and matrix metalloproteinase 3 were significantly increased 12 h after irradiation, whereas collagen type I mRNA was strongly decreased already 6 h following irradiation. Collagen type III, tissue inhibitor of metalloproteinase 1, and matrix metalloproteinase 2 did not change after photodynamic therapy. Addition of nontoxic concentrations of sodium azide, a singlet-oxygen quencher, significantly inhibited induction of matrix metalloproteinase 1 by 5-aminolevulinic acid and light. These data show that 5-aminolevulinic acid and light induce matrix metalloproteinase 1 and matrix metalloproteinase 3 expression in normal and scleroderma fibroblasts in a singlet oxygen-dependent way while reducing collagen type I mRNA expression. Induction of collagen-degrading enzymes together with reduction of collagen production might be responsible for the anti-sclerotic effects of 5-aminolevulinic acid-photodynamic therapy observed in vivo. Patients with localized scleroderma receiving topical photodynamic therapy with 5-aminolevulinic acid show a reduction in skin tightness, suggesting that this therapy reduces skin sclerosis. To investigate potential mechanisms, the effects of 5-aminolevulinic acid and light on collagen metabolism were studied in vitro. Normal and scleroderma fibroblasts were treated with sublethal doses of 5-aminolevulinic acid and red light and transferred to three-dimensional collagen lattices. Cell supernatants were taken 6–72 h after photodynamic therapy to determine protein levels of the matrix metalloproteinases 1, 2, and 3, and of their inhibitors, tissue inhibitor of metalloproteinase 1 and 2 by enzyme-linked immunosorbent assay. Cellular mRNA expression of these proteins and of collagen type I and III was measured by quantitative real-time polymerase chain reaction. A significant, time-dependent induction of matrix metalloproteinase 1 (up to 2.4-fold after 48 h) and matrix metalloproteinase 3 (up to 4.3-fold after 48 h) protein levels was seen after 5-aminolevulinic acid-photodynamic therapy. Irradiation with ultraviolet A light, used as a positive control, showed a similar induction of matrix metalloproteinase 1 (2.3-fold after 48 h). The mRNA levels of matrix metalloproteinase 1 and matrix metalloproteinase 3 were significantly increased 12 h after irradiation, whereas collagen type I mRNA was strongly decreased already 6 h following irradiation. Collagen type III, tissue inhibitor of metalloproteinase 1, and matrix metalloproteinase 2 did not change after photodynamic therapy. Addition of nontoxic concentrations of sodium azide, a singlet-oxygen quencher, significantly inhibited induction of matrix metalloproteinase 1 by 5-aminolevulinic acid and light. These data show that 5-aminolevulinic acid and light induce matrix metalloproteinase 1 and matrix metalloproteinase 3 expression in normal and scleroderma fibroblasts in a singlet oxygen-dependent way while reducing collagen type I mRNA expression. Induction of collagen-degrading enzymes together with reduction of collagen production might be responsible for the anti-sclerotic effects of 5-aminolevulinic acid-photodynamic therapy observed in vivo. 5-aminolevulinic acid matrix metalloproteinase tissue inhibitor of metalloproteinase photodynamic therapy 2,3-bis-(2-methoxy-4-nitro-5-sulfenyl)-(2H)-tetrazolium-5-carboxanilide assay Photodynamic therapy (PDT) is a therapeutic approach currently under investigation in clinical studies for the treatment of superficial skin tumors (Morton et al., 1996Morton C.A. Whitehurst C. Moseley H. McColl J.H. Moore J.V. Mackie R.M. Comparison of photodynamic therapy with cryotherapy in the treatment of Bowen's disease.Br J Dermatol. 1996; 135: 766-771Crossref PubMed Scopus (249) Google Scholar;Szeimes et al., 2002Szeimes R.M. Karrer S. Radakovic-Fijan S. et al.Photodynamic therapy using topical 5-aminolevulinate compared with cryotherapy for actinic keratosis: A prospective, randomized study.Am Acad Dermatol. 2002; 47: 258-262Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar) and chronic inflammatory dermatoses, e.g., psoriasis (Boehncke et al., 1994Boehncke W.H. Sterry W. Kaufmann R. Treatment of psoriasis by topical photodynamic therapy with polychromatic light.Lancet. 1994; 343: 801Abstract PubMed Scopus (121) Google Scholar). Topical PDT using 5-aminolevulinic acid (ALA) is based on the photosensitization of diseased tissue by ALA-induced porphyrins and subsequent irradiation with red light (Kennedy and Pottier, 1992Kennedy J.C. Pottier R.H. Endogenous protoporphyrin IX, a clinical useful photosensitizer for photodynamic therapy.J Photochem Photobiol B Biol. 1992; 14: 275-292Crossref PubMed Scopus (1073) Google Scholar). The excitation of the photosensitizer results in the generation of reactive oxygen species, particularly singlet oxygen. Reactive oxygen species mediate cellular, e.g., lipid peroxidation, and vascular effects resulting in direct or indirect cytotoxic effects on the treated cells (Aveline, 2001Aveline B.M. Primary processes in photosensitization mechanisms.in: Calzavara-Pinton P. Szeimies R.M. Ortel B. Photodynamic Therapy and Fluorescence Diagnosis in Dermatology. Elsevier Science, Amsterdam2001: 17-37Crossref Scopus (21) Google Scholar). Photochemically generated singlet oxygen is mainly responsible for cytotoxicity induced by PDT. If targeted cells are not disintegrated, photo-oxidative stress leads to transcription and translation of various stress response and cytokine genes. Tumor necrosis factor and interleukins 1 and 6 have been shown to be induced by PDT, supporting inflammatory action and immunologic response (Kick et al., 1995Kick G. Messer G. Goetz A. Plewig G. Kind P. Photodynamic therapy induces expression of interleukin 6 by activation of AP-1 but not NF-kappa B DNA binding.Cancer Res. 1995; 55: 2373-2376PubMed Google Scholar). Thus far, however, the exact underlying mechanisms of PDT regarding cellular responses and gene regulation are poorly understood.Table ITable ISummary of study resultsNormal fibroblasts (maximal increase or decrease after PDT as compared with the untreated control)Scleroderma fibroblasts (maximal increase or decrease after PDT as compared with the untreated control)MMP-1 protein⇑⇑(max. after 48 h)(max. after 48–72 h)MMP-2 protein⇔⇔MMP-3 protein⇑⇑(max. after 48 h)(max. after 48 h)TIMP-1 protein⇔⇔TIMP-2 protein⇔⇔MMP-1 mRNA⇑⇑(max. after 12 h)(max. after 12 h)MMP-3 mRNA⇑⇑(max. after 12 h)(max. after 12 h)TIMP-1 mRNA⇔⇔Collagen type I mRNA⇓⇓(max. after 24 h)(max. after 24 h)Collagen type III mRNA⇔⇔⇑Significant increase after PDT as compared with untreated control.⇓Significant decrease after PDT as compared with untreated control.⇔No significant change after PDT as compared with untreated control. Open table in a new tab ⇑Significant increase after PDT as compared with untreated control. ⇓Significant decrease after PDT as compared with untreated control. ⇔No significant change after PDT as compared with untreated control. Recently, five patients with recalcitrant localized scleroderma have been successfully treated by topical PDT using 5-aminolevulinic acid. After several treatments with low doses of ALA and light a marked softening of the sclerotic plaques was observed in all patients (Karrer et al., 2000Karrer S. Abels C. Landthaler M. Szeimies R.M. Topical photodynamic therapy for localized scleroderma.Acta Dermatol Venereol. 2000; 80: 26-27Crossref PubMed Scopus (112) Google Scholar). Localized scleroderma is an inflammatory disorder that manifests itself as excessive sclerosis of the skin. It is generally accepted that dermal fibroblasts are the key in the pathogenesis of skin sclerosis by synthesizing increased amounts of collagen type I and III, whereas collagen degrading enzymes [matrix metallo-proteinase (MMP)-1, MMP-2, MMP-3] are decreased (Kähäri et al., 1988Kähäri V.M. Sandberg M. Kalimo H. Vuorio T. Vuorio E. Identification of fibroblasts responsible for increased collagen production in localized scleroderma by in situ hybridization.J Invest Dermatol. 1988; 90: 664-670Abstract Full Text PDF PubMed Google Scholar;Petersen et al., 1992Petersen M.J. Hansen C. Craig S. Ultraviolet A irradiation stimulates collagenase production in cultured human fibroblasts.J Invest Dermatol. 1992; 99: 440-444Abstract Full Text PDF PubMed Google Scholar;Hunzelmann et al., 1998Hunzelmann N. Scharffetter Kochanek K. Hager C. Krieg T. Management of localised scleroderma.Semin Cutan Med Surg. 1998; 17: 34-40Crossref PubMed Scopus (40) Google Scholar;Hawk and English, 2001Hawk A. English F.C. Localized and systemic scleroderma.Semin Cutan Med Surg. 2001; 20: 27-37Crossref PubMed Scopus (52) Google Scholar). At present, photochemotherapy using photosensitizing psoralen derivatives and ultraviolet (UV) A (PUVA) is one of the most effective and widely applied therapies for localized scleroderma (Morison, 1997Morison W.L. Psoralen UVA therapy for linear and generalized morphea.J Am Acad Dermatol. 1997; 37: 657-659Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). PUVA, however, is associated with an increased risk of squamous cell carcinoma and malignant melanoma (Stern et al., 1997Stern R.S. Nichols K.T. Väkevä L.H. Malignant melanoma in patients treated for psoriasis with methoxsalen (psoralen) and ultraviolet A radiation (PUVA).N Engl J Med. 1997; 336: 1041-1045Crossref PubMed Scopus (543) Google Scholar;Stern and Lunder, 1998Stern R.S. Lunder E.J. Risk of squamous cell carcinoma and methoxsalen (psoralen) and UV-A radiation (PUVA). A meta-analysis.Arch Dermatol. 1998; 134: 1582-1585Crossref PubMed Scopus (193) Google Scholar). Recently, also UVA1 phototherapy has been shown to improve or clear localized scleroderma markedly (Stege et al., 1997Stege H. Berneburg M. Humke S. et al.High-dose UVA1 radiation therapy for localized scleroderma.J Am Acad Dermatol. 1997; 36: 938-944Abstract Full Text PDF PubMed Scopus (178) Google Scholar). Induction of MMP is supposed to be the mechanism by which UVA light exhibits its anti-sclerotic effects (Herrmann et al., 1993Herrmann G. Wlaschek M. Lange T.S. Prenzel K. Goerz G. Scharffetter-Kochanek K. UVA irradiation stimulates the synthesis of various matrix-metalloproteinases (MMP) in cultured human fibroblasts.Exp Dermatol. 1993; 2: 92-97Crossref PubMed Scopus (148) Google Scholar). MMP are a growing family of zinc-dependent endopeptidases that are characterized by their ability to degrade various extracellular matrix components (Woessner, 1991Woessner J.F. Matrix metalloproteinases and their inhibitors in connective tissue remodelling.FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3009) Google Scholar). At present, there are more than 20 human MMP described. The family includes collagenases, gelatinases, stromelysins, metalloelastases, and membrane type metalloproteinases. MMP are expressed by various cell types during the process of development, as well as during certain physiologic and pathologic processes. MMP-1 (interstitial collagenases) degrades extracellular fibers comprised of types I, II, III, IX, and XI collagen. MMP-2 (72 kDa gelatinase) degrades types IV, V, and VII collagen. MMP-3 (stromelysin-1) has a broad spectrum of proteolytic activity, including degradation of proteoglycans and fibronectin as well as native types III, IV, and V collagen. MMP are regulated by: (i) cytokines, growth factors, and cell–cell and cell–matrix interactions that control gene expression; (ii) activation of their proenzyme form; and (iii) the presence of MMP inhibitors (tissue inhibitors of metallopro-teinases: TIMP) (Raza and Cornelius, 2000Raza S.L. Cornelius L.A. Matrix metalloproteinases: pro- and anti-angiogenic activities.J Invest Dermatol Symp Proc. 2000; 5: 47-54Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Similar to MMP, TIMP are synthesized by ordinarily resident dermal fibroblasts. TIMP-1 binds to MMP-1 and MMP-2 and inhibits their activities forming a 1 : 1 stochiometric complex (Gomez et al., 1997Gomez D.E. Alonso D.F. Yoshiji H. Thorgeirsson U.P. Tissue inhibitors of metallo-proteinases: structure, regulation and biological functions.Eur J Cell Biol. 1997; 74: 111-122PubMed Google Scholar). TIMP-2 preferentially binds to a distinct domain of the active site of MMP-2. Any imbalance between the expression of extracellular matrix components and their degrading factors could potentially lead to abnormal accumulation of extracellular matrix as found in scleroderma. Although UV light therapies are effective in the treatment of localized scleroderma by inducing MMP, ALA-PDT has the advantage of not being carcinogenic, as the DNA is not a target for cytotoxicity in PDT (Berg, 1996Berg K. Mechanism of cell damage in photodynamic therapy.in: Hönigsmann H. Jori G. Young A.R. The Fundamental Bases of Phototherapy. OEMF Spa, Milano1996: 181-207Google Scholar). The aim of this study was to investigate the mechanisms by which ALA and light might generate its anti-sclerotic effects by analyzing the expression of MMP and their counteracting inhibitors in normal human fibroblasts and scleroderma fibroblasts in comparison with UVA light and by investigating the effect of ALA-PDT on collagen type I and type III metabolism in vitro. Primary normal human dermal fibroblast strains were established by outgrowth from skin biopsies of healthy human donors or from involved skin of patients with localized scleroderma after written informed consent and local Institutional Review Board approval. A total of six cell strains (three scleroderma fibroblasts and three adult dermal fibroblasts) were used. The fibroblasts were maintained in Dulbecco modified Eagle's medium supplemented with 10% fetal bovine serum, 1% HEPES (1 mol per liter), 100 U per ml penicillin, and 100 μg streptomycin per ml, and grown as monolayers on plastic Petri dishes in a humidified atmosphere of a CO2 incubator at 37°C. Fibroblast cultures were subcultured by trypsinization and used between the third and tenth passages. After the cells in monolayer culture had grown to confluence medium was removed, serum-free medium containing ALA (75 μmol per liter; Merck AG, Darmstadt, Germany) was added, and cells were allowed to take up ALA for 24 h. The medium containing ALA was removed, the cells were rinsed and then submerged with phosphate-buffered saline. Irradiation was performed immediately afterwards. The light dose and ALA dose applied resulted in a 10% cell death (≃LD10) compared with the untreated control as determined by a tetrazolium salt assay, sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT test; Sigma Chemie, Deisenhofen, Germany) to measure cell viability. To investigate the role of singlet oxygen, sodium azide (Sigma Chemie), a potent chemical quencher that is specific for singlet oxygen, was added to the cell culture at a concentration of 50 mmol per liter in phosphate-buffered saline. Sodium azide was applied to the cells after they had been allowed to take up ALA for 24 h and immediately prior to the irradiation procedure and removed again immediately after irradiation. The sodium azide dose used was not toxic to the cells as confirmed by the XTT test. Supernatants were taken 24 h, 48 h, and 72 h following irradiation. The control group was also treated with 50 mmol per liter sodium azide in phosphate-buffered saline, but received no ALA or light. Irradiation of the cells within monolayer culture was performed using an incoherent light source with a 1200 W metal halogen lamp (PDT 1200L, Waldmann-Medizintechnik, Villingen-Schwenningen, Germany, emission wavelength λem 580–740 nm), with a light intensity of 40 mW per cm2 and a fluence of 24 J per cm2 (Szeimies et al., 1994Szeimies R.M. Hein R. Bäumler W. Heine A. Landthaler M. A possible new incoherent lamp for photodynamic treatment of superficial skin lesions.Acta Derm Venereol (Stockh). 1994; 74: 117-119PubMed Google Scholar). UVA light was used as a positive control (UVA lamp 700, Waldmann-Medizintechnik, emission wavelength λem 340–460 nm, 10 J per cm2, 60 mW per cm2). Seven treatment groups were formed: (i) the first group of fibroblasts served as a control and received neither sensitizer nor irradiation; (ii) group 2 received ALA only; (iii) group 3 was irradiated only with red light; (iv) group 4 received ALA and red light; (v) group 5 was treated with UVA light only (positive control); (vi) group 6 received sodium azide only; and (vii) group 7 was treated with ALA and light with the addition of sodium azide prior to irradiation. Collagen type I from rat tail tendon was prepared and used for lattice preparation according to the following conditions: 0.45 ml of a sterile solution of type I collagen was mixed with 1.4 ml of Dulbecco modified Eagle's medium with fetal bovine serum, and transferred into a 35 mm diameter Petri dish. Immediately after irradiation viable fibroblasts (2.5×106 viable cells per dish) were placed in the collagen lattices and incubated for several time periods between 6 h and 72 h at 37°C. Once embedded in the gel the fibroblasts contract the collagen to form a very dense matrix. The circular shape retained during contraction and the diameter of the contracting fibroblast populated collagen lattice was measured at each time-point and the conditioned medium surrounding the lattices was harvested from n=3 individual fibroblast populated collagen lattices for further analysis by enzyme-linked immunosorbent assay (ELISA). Then cell viability was determined using the XTT test and cells were snap frozen in liquid nitrogen and stored at –80°C until required for RNA extraction and quantitative reverse transcriptase–polymerase chain reaction (reverse transcriptase–PCR). Viability of cells after ALA and/or light treatment was assessed by means of the XTT assay (Roehm et al., 1991Roehm N.W. Rodgers G.H. Hatfield S.M. Glasebrook A.L. An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT.J Immunol Methods. 1991; 142: 257-265Crossref PubMed Scopus (824) Google Scholar) (Roche Diagnostics, Mannheim, Germany). All ELISA results were referred to the number of viable cells as measured by the XTT test. Human MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 ELISA kits and the monoclonal antibodies against these molecules were obtained from Amersham Pharmacia Biotech (Freiburg, Germany). For time-course experiments supernatants were collected 0, 6, 12, 24, 48, and 72 h after irradiation. The concentration of the enzymes was determined using the above-mentioned ELISA kits. To differentiate between active and other forms of MMP-1 an activity assay system for MMP-1 was additionally used (Biotrak® MMP-1 activity assay; Amersham Pharmacia Biotech). Total RNA was isolated from cells using the NucleoSpin® RNA II kit from Macherey-Nagel (Düren, Germany). Total RNA was stored at -80°C until use in reverse transcriptase–PCR analysis. A LightCycler assisted PCR approach was used to measure MMP-1, MMP-3, TIMP-1, collagen type I, and collagen type III mRNA. The LightCycler PCR and detection system (Roche Diagnostics, Mannheim, Germany) was used for amplification and online quantification. Specific oligonucleotide primers for MMP-1, MMP-3, TIMP-1, collagen type I [a1(I) collagen], and collagen type III [a1(III) collagen] were used in PCR reactions with total cDNA from cells. β-actin served as an internal standard. Quantification was performed by online monitoring for identification of the exact time point at which the logarithmic linear phase could be distinguished from the background (crossing point). The cycle numbers of the logarithmic linear phase were plotted against the logarithm of the concentration of template DNA. Each quantitative PCR was performed at least in duplicate. Independent experiments were performed at least in triplicate. Statistical significance was evaluated in paired analyses using the Student's paired t test or the U test (nonparametric), depending on the data distribution. Data values are expressed as mean±SEM. Statistical significance was defined as a p≤0.05. Constitutive MMP-1 production in the collected medium as measured by ELISA was 2.44-fold higher for untreated normal fibroblasts as compared with untreated scleroderma fibroblasts (scleroderma fibroblasts: 1.297±0.24 ng per ml; normal fibroblasts: 3.165±0.49 ng per ml; per 250,000 cells, p<0.05). Also constitutive MMP-3 production was 2.1-fold higher for normal fibroblasts as compared with scleroderma fibroblasts (p<0.05). Protein levels of MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 were measured by ELISA in cell supernatants 6, 12, 24, 48, and 72 h following treatment with ALA and light of scleroderma fibroblasts and of normal human dermal fibroblasts. In addition to the MMP-1 ELISA, an MMP-1 activity assay was performed. A time-dependent significant increase of protein levels of MMP-1 after PDT was detected (Fig 1A,B). MMP-1 (active and total MMP-1) gradually increased in a time-dependent manner with a maximal induction (up to 3.7-fold as compared with the control) at 48 h following PDT, the active form of MMP-1 presumably being the biologically relevant measure. There was no statistically significant difference regarding the induction of MMP-1 between scleroderma and normal fibroblasts. Irradiation of fibroblasts with sublethal doses of UVA light (10 J per cm2) used in this study as a positive regulator of MMP-1 production in this cell type, led also to a significant time-dependent increase of MMP-1 production (Fig 1A,B). There was no significant influence of red light alone or ALA alone on the expression of MMP-1 as compared with the untreated controls. MMP-3 protein levels showed a very similar behavior after PDT as compared with MMP-1. Also, MMP-3 increased after PDT in a time-dependent manner with a maximal induction (about 4.3-fold as compared with the control) at 48 h after treatment with ALA and light (Fig 2). Red light alone or ALA alone had no influence on the expression of MMP-3 as compared with the untreated controls. MMP-2, TIMP-1, and TIMP-2 protein levels remained unaltered by light, ALA and, in contrast to MMP-1 and MMP-3, also after ALA-PDT (data not shown). Total RNA was isolated 0, 4, 12, and 24 h after irradiation of scleroderma and normal fibroblasts and subjected to quantitative real-time PCR using the LightCycler. MMP-1 and MMP-3 mRNA expression was significantly increased as compared with the untreated controls and to the cells treated with ALA alone or with light alone in a time-dependent manner in response to the treatment with 75 μmol per ml ALA and red light in both normal and scleroderma fibroblasts (Fig 3A,B). An increase occurred as soon as 4 h after irradiation and was significant 12 h after treatment. At 24 h post-PDT mRNA levels were already returning to the levels of the untreated controls. Control fibroblasts receiving no ALA and light were often beyond detection limits. Collagen type I mRNA, measured 12, 24, and 72 h after PDT by real-time PCR, significantly decreased in normal and in scleroderma fibroblasts after PDT showing a maximal decrease 24 h following PDT (Fig 4). TIMP-1 mRNA and collagen type III mRNA levels did not change up to 72 h following PDT (data not shown). Addition of sodium azide (50 mmol per liter) prior to irradiation led to a significant inhibition of MMP-1 increase 24, 48, and 72 h after treatment by ALA plus red light in normal and in scleroderma fibroblasts (Fig 5A,B). Thus, the induction of MMP-1 by ALA and light could be abrogated by the addition of a singlet oxygen quencher prior to illumination. The addition of 50 mmol per liter sodium azide without ALA and light did not influence cell viability as measured by the XTT test nor did it result in any change of MMP-1 levels as compared with the untreated controls. In this study we present experimental evidence that ALA and light induced MMP-1 and MMP-3 synthesis in human dermal fibroblasts cultivated within a three-dimensional collagen gel while reducing collagen type I mRNA expression. ALA and light affected the regulation of MMP at various levels, including the pretranslational level with increased amounts of MMP-1 and MMP-3 mRNA at 12 h following irradiation. ELISA showed an induction of MMP-1 and MMP-3 protein levels with a maximum at 48 h following irradiation, thus indicating that the PDT-induced specific mRNA of distinct MMP are translated and actively secreted into the supernatants. There was no significant difference between normal human fibroblasts and scleroderma fibroblasts, indicating that these responses are not specific for fibroblasts derived from patients with localized scleroderma, but occur also in normal human fibroblasts in vitro. As scleroderma fibroblasts exhibited more than 2-fold lower constitutive levels of MMP-1 and MMP-3 as compared with normal fibroblasts, sublethal PDT enhanced MMP levels of scleroderma fibroblasts to the physiologic level of normal fibroblasts. The fibroblast-populated collagen lattice model was used to study the behavior of normal and scleroderma fibroblasts and their interactions with the extracellular matrix, because dermal fibroblasts embedded in this collagen gel function in a more in vivo-like environment than do monolayer cultures (Bell et al., 1983Bell E. Sher S. Hull B. et al.The reconstitution of living skin.J Invest Dermatol. 1983; 81: 2-10Crossref PubMed Scopus (429) Google Scholar;Mauch et al., 1988Mauch C. Hatamochi A. Scharffetter K. Krieg T. Regulation of collagen synthesis in fibroblasts within a three-dimensional collagen gel.Exp Cell Res. 1988; 178: 493-503Crossref PubMed Scopus (202) Google Scholar;Fertin et al., 1991Fertin C. Nicolas J.F. Gillery P. Kalis B. Banchereau J. Maquart F.X. Interleukin-4 stimulates collagen synthesis by normal and scleroderma fibroblasts in dermal equivalents.Cell Mol Biol. 1991; 37: 823-829PubMed Google Scholar;Serpier et al., 1992Serpier H. Gillery P. Polette M. et al.Modulation of the organization of the extracellular matrix and the production of collagen by interferon gamma in three-dimensional cultures of normal and sclerodermic fibroblasts.Pathol Biol. 1992; 40: 865-870PubMed Google Scholar). Once embedded in the collagen lattice, fibroblasts reorganize and contract the collagen fibers to form a very dense matrix resembling authentic connective tissue. There is a difference, however, between the behavior of fibroblasts in this rapidly remodeling culture and the behavior of fibroblasts in dermis, so that this dynamic model cannot exactly mimic normal dermis. As MMP and TIMP production is influenced by cell/extracellular matrix interactions and mechanical forces (Stephens et al., 2001Stephens P. Davies K.J. Occleston N. et al.Skin and oral fibroblasts exhibit phenotypic differences in extracellular matrix reorganization and matrix metalloproteinases activity.Br J Dermatol. 2001; 144: 229-237Crossref PubMed Scopus (106) Google Scholar;Scott et al., 1998Scott K.A. Wood E.J. Karran E.H. A matrix metalloproteinase inhibitor which prevents fibroblast-mediated collagen lattice contraction.FEBS Lett. 1998; 441: 137-140Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), the three-dimensional extracellular matrix environment was nevertheless considered a valuable model to study collagen metabolism of fibroblasts after PDT. Remodeling of the extracellular matrix plays a pivotal role during many biologic and pathologic processes, as diverse as embryonal development, wound healing, tumor invasion, and fibrosis in scleroderma. The effect of solar irradiation (315–800 nm) on the steady-state levels of the mRNA of type I and III collagens and their degrading enzymes (MMP-1 and MMP-3) were measured in human dermal fibroblasts cultured in a three-dimensional collagen gel (Le-Tallec et al., 1998Le-Tallec L. Zmimowska C.K. Adolphe M. Effects of simulated solar radiation on type I and type III collagens, collagenase (MMP-1) and stromelysin (MMP-3) gene expression in human dermal fibroblasts cultured in collagen gels.J Photochem Photobiol B Biol. 1998; 42: 226-232Crossref PubMed Scopus (15) Google Scholar). Exposure to low levels of solar irradiation (315–800 nm, 0–10 J per cm2 in the UVA doses) caused a transient decrease in type I procollagen mRNA, an increase in MMP mRNA, and no change in type III procollagen mRNA steady-state levels. These results are similar to our results with ALA and light inducing MMP-1 and MMP-3 and reducing collagen type I mRNA expres" @default.
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• W2000230823 title "Influence of 5-Aminolevulinic Acid and Red Light on Collagen Metabolism of Human Dermal Fibroblasts" @default.
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• W2000230823 doi "https://doi.org/10.1046/j.1523-1747.2003.12037.x" @default.
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