Research Article
Matrix metalloproteinase 12–dependent cleavage of urokinase receptor in systemic sclerosis microvascular endothelial cells results in impaired angiogenesis
Silvia D'Alessio, Gabriella Fibbi, Marina Cinelli, Serena Guiducci, Angela Del Rosso, Francesca Margheri, Simona Serratì, Marco Pucci, Bashar Kahaleh, Pansheng Fan, Francesco Annunziato, Lorenzo Cosmi, Francesco Liotta, Marco Matucci-Cerinic, Mario Del Rosso,
Corresponding Author
Mario Del Rosso
University of Florence, Florence, Italy
Professor of General Pathology, Department of Experimental Pathology and Oncology, Viale G. B. Morgagni, 50, Florence 50134, ItalySearch for more papers by this authorSilvia D'Alessio, Gabriella Fibbi, Marina Cinelli, Serena Guiducci, Angela Del Rosso, Francesca Margheri, Simona Serratì, Marco Pucci, Bashar Kahaleh, Pansheng Fan, Francesco Annunziato, Lorenzo Cosmi, Francesco Liotta, Marco Matucci-Cerinic, Mario Del Rosso,
Corresponding Author
Mario Del Rosso
University of Florence, Florence, Italy
Professor of General Pathology, Department of Experimental Pathology and Oncology, Viale G. B. Morgagni, 50, Florence 50134, ItalySearch for more papers by this authorAbstract
Objective
Defective angiogenesis, resulting in tissue ischemia, is particularly prominent in the diffuse form of systemic sclerosis (SSc). The present study was undertaken to identify possible differences between normal and SSc microvascular endothelial cells (MVECs) in the expression of the cell-associated urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) system, which is critical in the angiogenic process.
Methods
MVECs were isolated from the dermis of healthy individuals and from the dermis of patients with diffuse SSc. The uPA/uPAR system was examined at the protein and messenger RNA levels. Angiogenesis was assayed on Matrigel-coated porous filters and plates to evaluate cell proliferation, invasion, and capillary morphogenesis. Cleavage of uPAR and the activity of matrix metalloproteinase 12 (MMP-12) were evaluated by Western blotting.
Results
Compared with MVECs from healthy skin, MVECs from SSc patients showed higher expression of uPAR. However, in SSc MVECs, uPAR undergoes truncation between domain 1 and domain 2, as shown by flow cytometry, enzyme-linked immunosorbent assay, and Western blotting, a cleavage that is known to impair uPAR functions. These properties of SSc MVECs were associated with poor spontaneous and uPA-dependent invasion, proliferation, and capillary morphogenesis. The uPAR cleavage occurring in SSc MVECs was associated with overexpression of MMP-12. SSc MVEC–conditioned medium impaired uPA-dependent proliferation and invasion as well as capillary morphogenesis in normal MVECs in vitro. Both a general hydroxamate inhibitor of MMP activity and anti–MMP-12 antibodies restored this SSc MVEC–induced impaired functioning.
Conclusion
Overproduction of MMP-12 by SSc MVECs accounts for the cleavage of uPAR and the impairment of angiogenesis in vitro and may contribute to reduced angiogenesis in SSc patients.
REFERENCES
- 1 Cutolo M, Grassi W, Matucci Cerinic M. Raynaud's phenomenon and the role of capillaroscopy. Arthritis Rheum 2003; 48: 3023–30.
- 2 Majewski S, Skopinska-Rozewska E, Jablonska S, Polakowski IJ, Pawinska M, Marczak M, et al. Modulatory effect of sera from scleroderma patients on lymphocyte-induced angiogenesis. Arthritis Rheum 1985; 28: 1133–9.
- 3 Polakowski IJ, Majewski S, Skopinska-Rozewska E, Zukowska M, Wlorarska B, Jablonska S. Modulatory effect of sera from scleroderma patients on lymphocyte-induced angiogenesis: II. Effector cells for the enhancing effect of acroscleroderma patients' sera. Arch Dermatol Res 1988; 280: 395–8.
- 4 Marczak M, Majewsk S, Skopinska-Rozewska E, Polakowski I, Jablonka S. Enhanced angiogenic capability of monocyte-enriched mononuclear cell suspension from patients with systemic scleroderma. J Invest Dermatol 1986; 86: 355–8.
- 5 Kaminski MJ, Majewski S, Jablonska S, Pawinska M. Lowered angiogenic capability of peripheral blood lymphocytes in progressive systemic sclerosis (scleroderma). J Invest Dermatol 1984; 82: 239–43.
- 6 Koch AE, Litvak MA, Burrows JC, Polverini PJ. Decreased monocyte-mediated angiogenesis in scleroderma. Clin Immunol Immunopathol 1992; 64: 153–60.
- 7 Ribatti D, Cantatore FP, Vacca A, D'Amore M, Ria R, Roncali L, et al. Systemic sclerosis stimulates angiogenesis in the chick embryo chorioallantoic membrane. Clin Rheumatol 1998; 17: 115–20.
- 8 Hebbar M, Peyrat JP, Hornez L, Hatron PY, Hachulla E, Devulder B. Increased concentrations of the circulating angiogenesis inhibitor endostatin in patients with systemic sclerosis. Arthritis Rheum 2000; 43: 889–93.
- 9 Mazar AP, Henkin J, Goldfarb RH. The urokinase plasminogen activator system in cancer: implications for tumor angiogenesis and metastasis. Angiogenesis 1999; 3: 15–32.
- 10 Koolwijk P, van Erck MG, de Vree WJ, Vermeer MA, Weich HA, Hanemaaijer R, et al. Cooperative effect of TNFα, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix: role of urokinase activity. J Cell Biol 1996; 132: 1177–88.
- 11 Fibbi G, Caldini R, Chevanne M, Pucci M, Schiavone N, Morbidelli L, et al. Urokinase-dependent angiogenesis in vitro and diacylglycerol production are blocked by antisense oligonucleotides against the urokinase receptor. Lab Invest 1998; 78: 1109–19.
- 12 Kroon ME, Koolwijk P, van Goor H, Weidle UH, Collen A, van der Pluijm G, et al. Role and localization of urokinase receptor in the formation of new microvascular structures in fibrin matrices. Am J Pathol 1999; 154: 1731–42.
- 13 Koolwijk P, Sidenius N, Peters E, Sier CF, Hanemaaijer R, Blasi F, et al. Proteolysis of the urokinase-type plasminogen activator receptor by metalloproteinase-12: implication for angiogenesis in fibrin matrices. Blood 2001; 97: 3123–31.
- 14 Fibbi G, Pucci M, Grappone C, Pellegrini G, Salzano R, Casini A, et al. Function of the fibrinolytic system in human Ito cells and its control by basic fibroblast and platelet-derived growth factor. Hepatology 1999; 29: 868–78.
- 15 Scott PA, Bicknell R. The isolation and culture of microvascular endothelium. J Cell Sci 1993; 105: 269–73.
- 16 Manconi F, Markham R, Fraser IS. Culturing endothelial cells of microvascular origin. Method Cell Sci 2000; 22: 89–99.
- 17 Annunziato F, Romagnani P, Cosmi L, Beltrame C, Steiner BH, Lazzeri E, et al. MDC and ELC attract human thymocytes in different stages of development and are produced by distinct subsets of medullary epithelial cells: possible implications for negative selection. J Immunol 2000; 165: 238–46.
- 18 Romagnani P, Annunziato F, Lazzeri E, Cosmi L, Beltrame C, Lasagni L, et al. Interferon-inducible protein 10, monokine induced by interferon γ, and interferon-inducible T-cell chemoattractant are produced by thymic epithelial cells and attract T-cell receptor (TCR) αβ CD8+ single-positive T cells, TCRγδ T cells, and natural killer-type cells in human thymus. Blood 2001; 97: 601–7.
- 19 Fibbi G, Barletta E, Dini G, Del Rosso A, Pucci M, Cerletti M, et al. Cell invasion is affected by differential expression of the urokinase plasminogen activator/urokinase plasminogen activator receptor system in muscle satellite cells from normal and dystrophic patients. Lab Invest 2001; 81: 27–39.
- 20 Kubota Y, Kleinman HK, Martin GR, Lawley TJ. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J Cell Biol 1988; 107: 1589–98.
- 21 Grant DS, Tashiro K, Segui-Real B, Yamada Y, Martin GR, Kleinman HK. Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell 1989; 58: 933–43.
- 22 Hoyer-Hansen G, Ploug M, Behrendt N, Ronne E, Dano K. Cell-surface acceleration of urokinase-catalyzed receptor cleavage. Eur J Biochem 1997; 243: 21–6.
- 23 Hoyer-Hansen G, Ronne E, Solberg H, Behrendt N, Ploug M, Lund LR, et al. Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain. J Biol Chem 1992; 267: 18224–9.
- 24 Behrendt N, Ploug M, Patthy L, Houen G, Blasi F, Dano K. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator. J Biol Chem 1991; 266: 7842–7.
- 25 Ploug M, Ellis V. Structure-function relationships in the receptor for urokinase-type plasminogen activator: comparison to other members of the Ly-6 family and snake venom α-neurotoxins. FEBS Lett 1994; 349: 163–8.
- 26 Ploug M, Ronne E, Behrendt N, Jensen AL, Blasi F, Dano K. Cellular receptor for urokinase plasminogen activator: carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol. J Biol Chem 1991; 266: 1926–33.
- 27 Wei Y, Waltz DA, Rao N, Drummond RJ, Rosenberg S, Chapman HA. Identification of the urokinase receptor as cell adhesion receptor for vitronectin. J Biol Chem 1994; 269: 32380–8.
- 28 Hoyer-Hansen G, Behrendt N, Ploug M, Dano K, Preissner KT. The intact urokinase receptor is required for efficient vitronectin binding: receptor cleavage prevents ligand interaction. FEBS Lett 1997; 420: 79–85.
- 29 Montuori N, Carriero MV, Salzano S, Rossi G, Ragno P. The cleavage of the urokinase receptor regulates its multiple functions. J Biol Chem 2002; 277: 46932–9.
- 30 Sidenius N, Blasi F. The urokinase plasminogen activator system in cancer: recent advances and implication for prognosis and therapy. Cancer Metastasis Rev 2003; 22: 205–22.
- 31 Del Rosso M, Fibbi G, Pucci M, D'Alessio S, Del Rosso A, Magnelli L, et al. Multiple pathways of cell invasion are regulated by multiple families of serine proteases. Clin Exp Metastasis 2002; 19: 193–207.
- 32 LeRoy EC. Systemic sclerosis: a vascular perspective. Rheum Dis Clin North Am 1996; 22: 675–94.
- 33 Reynolds TY, Rockwell S, Glazer PM. Genetic instability induced by the tumor microenvironment. Cancer Res 1996; 56: 5754–7.
- 34 Hill RP. Tumor progression: potential role of unstable genomic changes. Cancer Metastasis Rev 1990; 9: 137–47.
- 35 Subarsky P, Hill RP. The hypoxic tumour microenvironment and metastatic progression. Clin Exp Metastasis 2003; 20: 237–50.
- 36 Ishikawa O, Ishikawa H. Macrophage infiltration in the skin of patients with systemic sclerosis. J Rheumatol 1992; 19: 1202–6.
- 37 Stacey KJ, Fowles LF, Colman MS, Ostrowski MC, Hume DA. Regulation of urokinase-type plasminogen activator gene transcription by macrophage colony-stimulating factor. Mol Cell Biol 1995; 15: 3430–41.
- 38 Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C. Macrophages and angiogenesis. J Leukoc Biol 1994; 55: 410–22.
- 39 Dekkers PE, ten Hove T, te Velde AA, van Deventer SJ, van Der Poll T. Upregulation of monocyte urokinase plasminogen activator receptor during human endotoxemia. Infect Immun 2000; 68: 2156–60.
- 40 Graham CH, Fitzpatrick TE, McCrae KR. Hypoxia stimulates urokinase receptor expression through a heme protein-dependent pathway. Blood 1998; 9: 3300–7.
