Lipodermatosclerosis is Characterized by Elevated Expression and Activation of Matrix Metalloproteinases: Implications for Venous Ulcer Formation
1998; Elsevier BV; Volume: 111; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.1998.00369.x
ISSN1523-1747
AutoresYared Herouy, Gudula Pornschlegel, Christoph Stetter, Harald Grenz, Erwin Schöpf, Johannes Norgauer, Wolfgang Vanscheidt, Andreas E. May, Klaus T. Preissner,
Tópico(s)Cell Adhesion Molecules Research
ResumoLipodermatosclerosis refers to skin induration of the lower extremities and is associated with patients preceding venous ulcerations. To better understand the pathogenesis of ulcer formation we investigated the expression of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in lipodermatosclerosis. By preparing biopsies from healthy skin and liposclerotic lesions, MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 were analyzed by using reverse transcriptase-polymerase chain reaction, western blot, zymography, hydrolysis of [3H]labeled collagens, and immunohistochemistry. Our investigations provide evidence that mRNA and protein expression of MMP-1, MMP-2, and TIMP-1 were significantly increased in lipodermatosclerosis, whereas the total amount of MMP-9 and TIMP-2 mRNA and protein was not altered. Western blot of liposclerotic lesions revealed an inactive proMMP-1-TIMP-1 complex, whereas MMP-2 was prominent as an active 66 kDa band. Increased proteolytic activity of MMP-2 could be proven in lesional in comparison with healthy skin by zymography and [3H]collagen degradation. Increased diffuse staining was found for MMP-1 in the epidermis and dermis in comparison with controls. In lipodermatosclerosis, MMP-2 was predominantly localized in the basal and suprabasal layers of the epidermis, in perivascular regions, and in the reticular part of the dermis. Furthermore, MMP-2 was imbalanced by locally reduced expression of TIMP-2 in the basement membrane zone of lesional skin. Our findings indicate lipodermatosclerosis to be characterized by elevated matrix turnover. Lipodermatosclerosis refers to skin induration of the lower extremities and is associated with patients preceding venous ulcerations. To better understand the pathogenesis of ulcer formation we investigated the expression of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in lipodermatosclerosis. By preparing biopsies from healthy skin and liposclerotic lesions, MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 were analyzed by using reverse transcriptase-polymerase chain reaction, western blot, zymography, hydrolysis of [3H]labeled collagens, and immunohistochemistry. Our investigations provide evidence that mRNA and protein expression of MMP-1, MMP-2, and TIMP-1 were significantly increased in lipodermatosclerosis, whereas the total amount of MMP-9 and TIMP-2 mRNA and protein was not altered. Western blot of liposclerotic lesions revealed an inactive proMMP-1-TIMP-1 complex, whereas MMP-2 was prominent as an active 66 kDa band. Increased proteolytic activity of MMP-2 could be proven in lesional in comparison with healthy skin by zymography and [3H]collagen degradation. Increased diffuse staining was found for MMP-1 in the epidermis and dermis in comparison with controls. In lipodermatosclerosis, MMP-2 was predominantly localized in the basal and suprabasal layers of the epidermis, in perivascular regions, and in the reticular part of the dermis. Furthermore, MMP-2 was imbalanced by locally reduced expression of TIMP-2 in the basement membrane zone of lesional skin. Our findings indicate lipodermatosclerosis to be characterized by elevated matrix turnover. matrix metalloproteinases tissue inhibitors of metalloproteinases Connective tissue remodeling is a highly organized process. Principal healthy and disease-related pathways for metabolic degradation of the extracellular matrix involve the action of matrix metalloproteinases (MMP). These metallo-endopeptidases participate in tissue remodeling during morphogenesis, tumor progression, cell migration, angiogenesis, and wound healing (Woessner, 1991Woessner J.F. Matrix metalloproteinases and their inhibitors in connective tissue remodeling.Faseb J. 1991; 5: 2145-2154Crossref PubMed Scopus (3009) Google Scholar;Salo et al., 1994Salo T. Makela M. Kylmaniemi M. Autio-Harmainen H. Larjava H.H. Expression of matrix metalloproteinase-2 and -9 during early human wound healing.Lab Invest. 1994; 70: 176-182PubMed Google Scholar;Birkedal-Hansen, 1995Birkedal-Hansen H. Proteolytic remodeling of extracellular matrix.Curr Opin Cell Biol. 1995; 7: 728-735Crossref PubMed Scopus (961) Google Scholar;Zucker et al., 1995Zucker S. Conner C. DiMassmo B.I. Ende H. Drews M. Seiki M. Bahou W.F. Thrombin induces the activation of progelatinase A in vascular endotlial cells. Physiologic regulation of angiogenesis.J Biol Chem. 1995; 270: 23730-23738Crossref PubMed Scopus (166) Google Scholar;Powell and Matrisian, 1996Powell W.C. Matrisian L.M. Complex roles of matrix metalloproteinases in tumor progression. Attempts to understand metastasis formation.in: Gunthert U. Schlag P.M. Birchmeier W. Metastasis Related Molecules. Springer-Verlag, Heidelberg, Heidelberg1996: 1-21Google Scholar). They belong to a growing family of at least 14 soluble and membrane-bound zinc-dependent endopeptidases (Sato et al., 1994Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. A matrix metalloproteinase expressed on the surface of invasive tumor cells.Nature. 1994; 370: 61-65Crossref PubMed Scopus (2321) Google Scholar). Proteolytic properties of these enzymes are controlled by transcriptionally regulated protein synthesis as well as by post-translational modification of the synthesized proteins. After release in zymogen forms (proMMP), these proteases can be extracellularly activated by cleavage of the propeptide region from the residual protein. In addition, the activity of MMP is controlled by specific inhibitors, so-called tissue inhibitors of metalloproteinases (TIMP) (Matrisian, 1990Matrisian L.M. Metalloproteinases and their inhibitors in matix remodeling (review).Trends Genet. 1990; 6: 121-125Abstract Full Text PDF PubMed Scopus (1505) Google Scholar). Inhibition of the proteolytic activity of these enzymes occurs by preferential complex formation between MMP-1 with TIMP-1 and MMP-2 with TIMP-2 (Howard et al., 1991Howard E.W. Bullen E.C. Banda M.J. Preferential inhibition of 72- and 92-kDaa gelatinases by tissue inhibitor of metalloproteinase-2.J Biol Chem. 1991; 266: 13070-13075Abstract Full Text PDF PubMed Google Scholar). Lipodermatosclerosis is associated with venous circulatory disorders of the lower extremities preceding chronic venous ulceration. The scleroderma-like hardening of lipodermatosclerosis is characterized by fibrous scar tissue of the reticular dermis built up of collagen bundles, degraded elastic fibers, and loss of cellular components. Additional features are perivascular fibrin cuffs surrounding dermal capillary vessels and loss of papillary structures (Kirsner et al., 1993Kirsner R.S. Pardes J.B. Eaglstein W.H. Falanga V. The clinical spectrum of lipodermatosclerosis.J Am Acad Dermatol. 1993; 28: 623-627Abstract Full Text PDF PubMed Scopus (121) Google Scholar). Histochemical analysis of skin lesions of lipodermatosclerosis revealed matrix structures of the deeper reticular dermis and subcutaneous tissues to consist of predominantly type I and III collagens (Herrick et al., 1992Herrick S.E. Sloan P. McGurk M. Freak L. McCollum C.N. Ferguson MwJ Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers.Am J Pathol. 1992; 141: 1085-1095PubMed Google Scholar). The perivascular fibrin cuffs are found to be highly organized structures containing fibrin, fibronectin, and denatured type IV collagen (Neumann and Van den Broek, 1991Neumann H.A. Van den Broek M.J. Increased collagen IV layer in the basal membrane area of the capillaries in severe chronic venous insufficiency.Vasa. 1991; 20: 26-29PubMed Google Scholar;Herrick et al., 1992Herrick S.E. Sloan P. McGurk M. Freak L. McCollum C.N. Ferguson MwJ Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers.Am J Pathol. 1992; 141: 1085-1095PubMed Google Scholar). Cleavage of type I and III collagen at a single site resulting in two fragments has been reported to be specific for MMP-1 (Birkedal-Hansen, 1995Birkedal-Hansen H. Proteolytic remodeling of extracellular matrix.Curr Opin Cell Biol. 1995; 7: 728-735Crossref PubMed Scopus (961) Google Scholar). Matrix metalloproteinase-2 (MMP-2) cleaves type I and III collagens at multiple sites, continuously processing the generated products into smaller fragments (Aimes and Quigley, 1995Aimes R.T. Quigley J.P. Matrix metalloproteinase-2 is an interstitial collagenase. Inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments.J Biol Chem. 1995; 270: 5872-5876Crossref PubMed Scopus (803) Google Scholar). Matrix proteins such as type IV collagen, elastin, fibronectin, laminin, and components of anchoring fibrils of hemidesmosomes, represent additional substrates of MMP-2 (Collier et al., 1988Collier I.E. Wilhelm S.M. Eisen A.Z. et al.H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen.J Biol Chem. 1988; 263: 6579-6587Abstract Full Text PDF PubMed Google Scholar). Recently, increased expression and activity of MMP-2 has been reported in chronic venous ulcer exudates (Weckroth et al., 1996Weckroth M. Vaheri A. Lauharanta J. Sorsa T. Konttinen Y.T. Matrix metalloproteinases, gelatinase and collagenase in chronic leg ulcers.J Invest Dermatol. 1996; 106: 1119-1124Crossref PubMed Scopus (205) Google Scholar). It has been suggested that the elevated MMP-2 activity may play an important role in impeding wound healing and thereby maintaining venous ulcers. In spite of accumulating evidence indicating the importance of MMP in the development of chronic venous ulcers, the participation of these endopeptidases in ulcer preceding stages has not been examined. To better understand the pathogenesis of venous ulcer formation, we here analyzed the expression and state of activity of specific MMP and TIMP in skin lesions of lipodermatosclerosis. Patients with chronic ulcers have been characterized according to the international clinical CEAP classification of chronic venous insufficiency. The diagnosis was confirmed clinically by duplex scanning, by Doppler sonography, and by photoplethysmography. Punch biopsies of liposclerotic skin extending to the subcutaneous fat of the lower portion of the leg were obtained 1 cm distance from the ulcer edge. These sites were scored as extensive according to the CEAP classification (LDS: clinical score = 2). Biopsy specimens were snap-frozen in liquid nitrogen and stored at –70°C until further processing. Lipodermatosclerosis was proven by conventional histology. Control biopsies from healthy volunteers were taken from the lower portion of the leg. All subjects provided informed written consent, and procedures involving humans were approved by the Ethics Committee of the Health Authority. Monoclonal antibodies (mouse IgG1) against MMP-1 (Ab41–1E5), MMP-2 (Ab42–5D11), MMP-9 (Ab6–6B), TIMP-1 (Ab7–6C1), and TIMP-2 (AbT2–101) were purchased from Oncogene Science (Cambridge, MA). The mRNA expression was analyzed semiquantitatively by reverse transcription and polymerase chain reaction (Grewe et al., 1994Grewe M. Gyufko K. Schöpf E. Krutmann J. Lesional expression of interferon-gamma in atopic eczema.Lancet. 1994; 343: 25-26Abstract PubMed Scopus (371) Google Scholar). Briefly, total mRNA was isolated by guanidinium-phenol chloroform extraction. The cDNA was obtained with the first strand cDNA synthesis kit from Pharmacia (Freiburg, Germany) using mRNA. All oligonucleotides were designed to recognize a unique sequence exclusive for each cDNA. The control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as reference. Specific primers were designed as follows: MMP-1, 5′-ATCCAAGCCATATATGGACGT-3′ (sense), 5′-TCTGAGAGAGTCAAAATT-CTCTT-3′ (anti-sense); MMP-2, 5′-ACAAAGAGTGGCAGTGCAAT-3′ (sense), 5′-GTGCTCGT-TTCCGTAGTAGG-3′ (anti-sense); MMP-9, 5′-AGATGTGGAGTGCCAGATGT-3′ (sense), 5′-AT-CTGACGATGGTAGGCAGG-3′ (anti-sense); TIMP-1, 5′-AGTCAACCAGACCACCTTATA-3′ (sense), 5′-TTCAGAGCCTTGGAGGAGC-3′ (anti-sense); TIMP-2, 5′-AAGTGGACTCTGGAA-ACGAC-3′ (sense), 5′-CTCGATGTCGAGAAACTCCT-3′ (anti-sense); and GAPDH, 5′-CCACCC-ATGGCAAATTCCATGGCA-3′ (sense), 5′-TCTAGACGGCAGGTCAGGTCCACC-3′ (anti-sense). Polymerase chain reaction was performed using a GeneAmp PCR thermal cycler (Perkin Elmer, Weiterstadt, Germany) and consisted of cycles of denaturation (94°C, 1 min), ramped annealing (60°C for 30 s), and extension (72°C, 1 min). The generated products were subjected to electrophoresis on a 3% agarose gel and were visualized by ethidium bromide staining. To compare the mRNA expression of different biopsies, signals of MMP or TIMP were normalized to GAPDH and a ratio was calculated. In order to assure linear cDNA amplification in our experiments, different amplifying cycles (22–36) were checked. Linear amplification was obtained between 22 and 32 cycles. The specifities of the generated products were proven by sequencing after cloning using pCRII vectors. Protein analysis of MMP-1, MMP-2, and MMP-9 and their inhibitors TIMP-1 and TIMP-2 were performed by western blot. Skin samples were pulverized with a microdismembrator from Braun Biotech International (Melsungen, Germany). Proteins were solubilized in electrophoresis sample buffer and samples with equal protein content were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electroblotted to nitrocellulose membrane and were stained with indicated antibodies using peroxidase-conjugated secondary antibodies and the ECL chemiluminescence system from Amersham (Braunschweig, Germany). Gelatinase activity (MMP-2 and MMP-9) in liposclerotic lesions and in healthy skin samples was evaluated by zymography as previously described (Hibbs et al., 1985Hibbs M.S. Hasty K.A. Seyer J.M. Kang A.H. Mainardi C.L. Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase.J Biol Chem. 1985; 260: 2493-2500Abstract Full Text PDF PubMed Google Scholar). Homogenized skin samples were added to buffer containing 10% glycerol, 2% SDS, and 1% bromophenol blue. Equal amounts of total protein were loaded on SDS gel containing 0.5 mg gelatin per ml and electrophoresis was performed. Gelatinolytic activity of MMP-2 and MMP-9 was detected as transparent bands on blue background of Coomassie brillant blue stained gels. The hydrolytic activity of MMP in tissue extracts was investigated using N-[propionate-2,3–3H]-labeled type I and N-[propionate-2,3–3H]-labeled type IV collagens as substrates (Hu et al., 1985Hu C.L. Crombie F. Franzblau C. A new assay for collagenolytic activity.Anal Biochem. 1985; 88: 638-643Crossref Scopus (54) Google Scholar;Galis et al., 1994Galis Z.S. Sukhova G.K. Lark M.W. Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques.J Clin Invest. 1994; 94: 2493-2503Crossref PubMed Scopus (2122) Google Scholar). Briefly, radiolabeled collagens were incubated with 50 μg tissue extracts for 24 h. The digests were centrifuged at 20,000 × g for 10 min and radioactivity of the supernatants was determined in a liquid scintillation counter. Immunohistochemical staining was performed as previously described (Weyl et al., 1996Weyl A. Vanscheidt W. Weiss J.M. Peschen M. Schöpf E. Simon J. Expression of the adhesion molecules ICAM-1,VCAM-1, and E-selectin and their ligands VLA-4 and LFA-1 in chronic venous leg ulcers.J Am Acad Dermatol. 1996; 34: 418-423Abstract Full Text PDF PubMed Scopus (59) Google Scholar). Briefly, specimens were embedded in tissue freezing medium supplied by Jung (Nussloch, Germany). Serial sections (5 μm) were prepared using a Cryocut 3750 from Reichert & Jung (Nussloch, Germany). Cryosections were fixed in acetone supplied by Merck (Darmstadt, Germany) and rinsed in phosphate-buffered saline. Sections were incubated with indicated primary antibodies, with biotinylated secondary antibodies, and with peroxidase-labeled streptavidine from DAKO (Glostrup, Denmark). Staining procedures were evaluated using an Axioskop microscope, equipped with a MC100 camera system from Cark Zeiss (Obercochem, Germany). The intensity of bands and blots was quantitated by measuring the optical density with an OneDscan computer software package. Data were analyzed by an unpaired student’s t test. Differences were considered significant at p < 0.05. All values were expressed as means ± SEM. The mRNA expression of MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 in biopsies of liposclerotic lesions and controls was analyzed semiquantitatively by reverse transcriptase-polymerase chain reaction Figure 1a. These studies revealed increased expression of MMP-1, MMP-2, and TIMP-1 mRNA in lesions of lipodermatosclerosis in comparison with healthy controls. In contrast, mRNA levels of MMP-9, TIMP-2, and the control gene GAPDH were not altered in comparison with controls. The quantitation performed by determination of the relative expression normalized to the expression of GAPDH demonstrated significant differences for MMP-2 (p < 0.001), MMP-1 (p < 0.01), and TIMP-1 (p < 0.01) Figure 1b. The protein expression was analyzed by western blot. Extracts of liposclerotic tissue and control tissue samples showed three different immunoreactive bands for MMP-1 of ≈42 kDa and 52 kDa, as well as in the 85 kDa range (Figure 2a, b). The 42 kDa band corresponds to the active form, the 52 kDa form represents the inactive zymogen, and immunoreactivity in the 85 kDa range represents the proMMP-1-TIMP-1-complex. Immunoblots of MMP-2 showed immunoreactive bands of ≈66 kDa, 72 kDa, and 93 kDa, representing the active form, the inactive zymogen, and the proMMP-2-TIMP-2 complex, respectively. Immunoblotting with antibodies against MMP-9 displayed immunoreactivity for the proenzyme form as a minor band at 92 kDa. The 83 kDa active form of this protein was not detected. Free and complexed TIMP-1 protein was found in the 28 kDa and 85 kDa range, respectively. In addition, staining with antibodies against TIMP-2 revealed weak immunoreactivity for the free and complexed form in the 21 and 93 kDa range, respectively. Densitometric evaluation of band intensities between skin lesions of lipodermatosclerosis and healthy controls displayed significant differences for the active (p < 0.001) and zymogen form (p < 0.05) of MMP-2 Figure 2c and the proMMP-1-TIMP-1 complex (p < 0.001) Figure 2b. In addition, significantly enhanced protein expression in liposclerotic skin lesions in comparison with healthy skin was also found for the active form of MMP-1 (p < 0.01) and free TIMP-1 (p < 0.05) Figure 2b. No statistical differences were found for zymogens of MMP-1, free TIMP-2, and the proMMP-2-TIMP-2 complex between liposclerotic lesions and healthy skin (Figure 2c, d). The gelatinolytic activities of MMP-2 and MMP-9 were evaluated by SDS-PAGE zymography Figure 3a. In zymogen gel the latent (proMMP-2) and activated form of MMP-2 and the latent (proMMP-9) and activated form of MMP-9 were detected. Skin lesions of lipodermatosclerosis displayed strongly increased gelatinolytic activity for the active 66 kDa MMP-2, which was prominent in gel staining. Densitometric evaluation revealed significant differences of the active 66 kDa form of MMP-2 (p < 0.001) in lesional tissues of lipodermatosclerosis in comparison with healthy skin Figure 3b. No significant alterations were found for proMMP-2, proMMP-9, and active MMP-9. To assure higher functional activities of MMP in lesions of lipodermatosclerosis in comparison with healthy skin, degradation assays with N-[propionate-2,3–3H]-labeled type I and N-[propionate-2,3–3H]-labeled IV collagens were performed using tissue extracts Figure 4. Consistently higher proteolytic activities for MMP degrading type I collagen (p < 0.001) and type IV collagen (p < 0.01) were found in skin lesions of lipodermatosclerosis in comparison with healthy skin. Liposclerotic altered skin is characterized by atrophy of epidermis and loss of papillary structures at the dermo–epidermal junction zone. The underlying dermis usually contains numerous small and medium-sized capillary vessels surrounded by thick organized fibrin cuffs. Cellular components in liposclerotic dermis are decreased in comparison with healthy skin. Liposclerotic lesions show diffuse immunoreactive staining for MMP-1 in the epidermis and dermis in comparison with healthy controls Figure 5a. In contrast to MMP-1, staining of liposclerotic lesions with anti-MMP-2 displayed a cuffed pattern of vessel staining Figure 5b and an intense signal around collagen bundles in the deeper reticular dermis Figure 5c. There was also an intense immunoreactive staining for MMP-2 in the basal and suprabasal layer of epidermis Figure 5d. Instead, controls displayed very weak epidermal staining of MMP-2. In healthy skin TIMP-2 was localized in a sharp continuous line along the basement membrane Figure 5e, which was altered in the sense of weak immunoreactivity in liposclerotic lesions. Lipodermatosclerosis is highly associated with venous hypertension preceding venous ulceration of the lower extremities. Main histologic features are loss of papillary structures in the dermo–epidermal junction zone, dermal pericapillary fibrin cuffs, and fibrosis of the reticular dermis, whereas venous ulcers are characterized by total loss of epidermal and partly dermal cellular and matrix tissue components (Herrick et al., 1992Herrick S.E. Sloan P. McGurk M. Freak L. McCollum C.N. Ferguson MwJ Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers.Am J Pathol. 1992; 141: 1085-1095PubMed Google Scholar). The underlying mechanisms leading to such drastic cellular and matrix changes of venous ulcerations have been a matter of debate. Pressure-damaged capillary vessels with leakage of fibrinogen (Browse and Burnand, 1982Browse N.L. Burnand K.G. The cause of venous ulceration.Lancet. 1982; 2: 243-245Abstract PubMed Scopus (444) Google Scholar) or release of toxic metabolites by accumulated leukocytes (Coleridge Smith et al., 1988Coleridge Smith P.D. Thomas P. Acurr J.H. Dormandy J.A. Causes of venous ulceration: a new hypothesis.Br Med J. 1988; 296: 1726-1727Crossref PubMed Scopus (475) Google Scholar) or cytokine-mediated fibrin cuff formation (Herrick et al., 1992Herrick S.E. Sloan P. McGurk M. Freak L. McCollum C.N. Ferguson MwJ Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers.Am J Pathol. 1992; 141: 1085-1095PubMed Google Scholar) have been suggested. Impairment of gas and nutrient exchange between blood and dermis has been supposed to be common features provoking ulcer formation. Recently, altered expression of metalloproteinases and their inhibitors has been supposed to highly contribute to skin and other tissue alterations as found in lichen planus, skin aging, different bullous dermatosis, and corneal ulcers (Saarialho-Kere et al., 1995Saarialho-Kere U.K. Vaalamo M. Airola K. Niemi K.M. Oikarinen A.I. Parks W.C. Interstitial collagenase is expressed by keratinocytes that are actively involved in reepithelialization in blistering skin disease.J Invest Dermatol. 1995; 104: 982-988Crossref PubMed Scopus (61) Google Scholar;Fini et al., 1996Fini M.E. Parks W.C. Rinehart W.B. et al.Role of matrix metalloproteinases in failure to re-epithelialize after corneal injury.Am J Pathol. 1996; 149: 1287-1302PubMed Google Scholar;Fisher et al., 1996Fisher G.J. Datta S.C. Talwar H.S. Wang Z.Q. Varani J. Kang S. Voorhees J.J. Molecular basis of sun induced premature skin ageing and retinoid anatgonism.Nature. 1996; 379: 335-339Crossref PubMed Scopus (1120) Google Scholar;Giannelli et al., 1996Giannelli G. Brassard J. Foti C. et al.Altered expression of basement membrane proteins and their integrin receptors in lichen planus: Possible pathogenetic role of gelatinase A and B.Lab Invest. 1996; 74: 1091-1104PubMed Google Scholar). In chronic venous ulcer exsudate enhanced MMP-1, MMP-2 expression and activity have been reported by early workers (Wysocki et al., 1993Wysocki A.B. Staiano-Coico L. Grinnell F. Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9.J Invest Dermatol. 1993; 101: 64-68Abstract Full Text PDF PubMed Google Scholar;Weckroth et al., 1996Weckroth M. Vaheri A. Lauharanta J. Sorsa T. Konttinen Y.T. Matrix metalloproteinases, gelatinase and collagenase in chronic leg ulcers.J Invest Dermatol. 1996; 106: 1119-1124Crossref PubMed Scopus (205) Google Scholar). They presumed that these proteolytic enzymes contribute to delayed wound healing maintaining chronic ulcerations. Our results provide direct evidence of alterations in the expression and functional state of metalloproteinases and their inhibitors in the ulcer preceding stage of lipodermatosclerosis. Elevated expression on mRNA and protein levels of MMP-1, MMP-2, and TIMP-1 could be proven using semiquantitative reverse transcriptase-polymerase chain reaction and western blot in comparison with healthy skin. No differences were found for MMP-9 and TIMP-2. In addition, in lipodermatosclerosis immunoblotting revealed immunoreactivity at a molecular weight of 85 kDa, strongly assuming proMMP-1 to form a proteolytically inactive complex with TIMP-1. In lesional skin MMP-2 was significantly expressed in its unbound 66 kDa active form in comparison with healthy skin. Furthermore, enhanced proteolytic activity could be demonstrated in liposclerotic lesions by zymography and collagen degradation assays. By immunohistochemistry there was an increased diffuse immunoreactive staining with anti-MMP-1 antibodies in the epidermis and dermis in comparison with healthy controls. In contrast, MMP-2 was localized in perivascular regions, in the reticular dermis, and predominantly in basal and suprabasal layers of the epidermis in liposclerotic lesions. The perivascular fibrin cuffs surrounding dermal capillary vessels are assumed to highly impair nutrients and gas exchange between blood and dermis. These cuffs are well-organized structures containing, apart from fibrin, fibronectin, type I collagens, and type III collagens (Herrick et al., 1992Herrick S.E. Sloan P. McGurk M. Freak L. McCollum C.N. Ferguson MwJ Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers.Am J Pathol. 1992; 141: 1085-1095PubMed Google Scholar). Because MMP-2 is able to digest type I collagens, type III collagens, and fibronectin, perivascularly localized MMP-2 may participate in the dissolution of fibrin cuff components. In contrast, MMP-2 is able to degrade elastic fibers that apart from the collagen network provide structural integrity and elasticity to human skin (Senior et al., 1991Senior R.M. Griffin G.L. Fliszar C.J. Shapiro S.D. Goldberg G.I. Welgus H.G. Human 92- and 72 kilodalton tpye IV collagenase are elastases.J Biol Chem. 1991; 266: 7870-7875Abstract Full Text PDF PubMed Google Scholar). Elastic fibers are found to be fragmented in the reticular dermis of liposclerotic lesions. It is tempting to speculate that enhanced MMP-2 protein expression in the reticular dermis could also contribute to the clinical findings of hardening and fragility of liposclerotic lesions. In addition, MMP-2 is also capable of processing extracellular matrix proteins of the basement membrane, such as type IV collagen and anchoring fibrils involved in hemidesmosomes. Enhanced expression of MMP-2 predominantly in the basal and suprabasal epidermal layer of liposclerotic lesions may result in the dissolution of epidermal matrix and cellular components. Furthermore, we reported here that the TIMP-2 expression is locally diminished along the basement membrane zone in lesional in contrast to healthy skin. Regarding TIMP-2 as a protective factor, developing epidermal defects in liposclerotic lesions could be understood as a result of uninhibited proteolytical activity of MMP-2 released from basal and suprabasal epidermal cells. Currently, the mechanisms provoking this imbalance in liposclerotic lesions are purely a matter of speculation. Gene expression of MMP-2 and TIMP-2 is regulated by different cytokines and growth factors. In liposclerotic skin adjacent to venous ulcers, enhanced levels of transforming growth factor β1 (TGF-β1) were reported (Higley et al., 1995Higley H.R. Ksander G.A. Gerhardt C.O. Falanga V. Extravasation of macromolecules and possible trapping of transforming growth factor-beta in venous ulceration.Br J Dermatol. 1995; 132: 79-85Crossref PubMed Scopus (115) Google Scholar). This cytokine stimulates the production of MMP-2 in human fibroblasts (Overall et al., 1991Overall C.M. Wrana I.L. Sodek J. Transcriptional and posttranscriptional regulation of 72 kDaa gelatinase/type IV collagenase by transforming growth factor-beta 1 in human fibroblasts. Comparison with collagenase and tissue inhibitors of metalloproteinase gene expression.J Biol Chem. 1991; 266: 14064-14071Abstract Full Text PDF PubMed Google Scholar). Thus, one could assume that the herein reported enhanced expression of MMP-2 in liposclerotic lesions could be induced by transforming growth factor β1 (TGF-β1). On the other hand, in dermal fibroblasts reactive oxygen species induce the expression of MMP-2 and inhibit the synthesis of TIMP-2 (Kawagushi et al., 1996Kawagushi Y. Tanaka H. Okada T. Konishi H. Takahashi M. Ito M. Asai J. The effects of ultraviolet A and reactive oxygen species on the mRNA expression of 72 kDaa type IV collagenase and its tissue inhibitor in cultured human dermal fibroblasts.Arch Dermatol Res. 1996; 288: 39-44Crossref PubMed Scopus (118) Google Scholar). Reactive oxygen species produced by macrophages had also been reported to modulate the activity of vascular MMP-2 in vitro (Rajagopalan et al., 1996Rajagopalan S. Meng X.P. Ramasamy S. Harrison D.G. Galis Z.S. Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro.J Clin Invest. 1996; 98: 2572-2579Crossref PubMed Scopus (973) Google Scholar). Therefore, it is conceivable that the imbalance of MMP-2/TIMP-2 in the reticular dermis of lipodermatosclerosis could also be a result of perivascular trapped leukocytes releasing those oxidative metabolites. Obviously, further investigations will be required to clarify detailed pathophysiologic mechanisms underlying lipodermatosclerosis. In summary, our data indicate lipodermatosclerosis to be characterized by increased matrix turnover. The clinical impression and histologic findings of skin hardening with elevated collagen deposition stand in sharp contrast to the dynamic picture presented in lipodermatosclerosis, which reveals intense ongoing collagen degradation by elevated MMP-2 activity. These data suggest that the imbalance between MMP-2 and TIMP-2 on mRNA and protein level implies a disturbance in the dynamic balance of matrix synthesis and breakdown. Ulcer formation therefore may be favored by enhanced turnover of the extracelluar matrix mediated by an unrestrained matrix metalloproteinase activity. Targeting this protease activity may provide a potential therapeutic strategy in the management of patients with advanced complications of chronic venous insufficiency. This work was supported by grants from the Deutsche Gesellschaft für Phlebologie (DGP) and the Deutsche Forschungsgemeinschaft no. 266 2/1 (DFG). Andreas E. May is a recipient of a fellowship from the Deutsche Gesellschaft für Kardiologie (DGK). This work in part represents the thesis of Gudula Pornschlegel. We thank equally all contributors of this work, especially W. Dieterle for statistical advice and H. Blume for critically reading the manuscript.
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