Gelatinases Increase in Bleomycin-induced Systemic Sclerosis Mouse Model

Keywords: Bleomycin, Gelatinase, Matrix metalloproteinase 2, Matrix metalloproteinase 9, Systemic scleroderma


Systemic sclerosis is a fibrotic autoimmune disease in which aberrant remodeling of the extracellular matrix in organs disturbs their functionalities. The aim of this study was to investigate the expression of gelatinases on systemic sclerosis. Consequently, a mouse model of systemic sclerosis was employed and the gelatinolytic activity of gelatinases was evaluated on the fibrotic tissues of this model. Two groups of ten mice were considered in this work: a group of systemic sclerosis model and control group. For the generation of systemic sclerosis model, mice received bleomycin, while the control group was subjected to phosphate buffered saline (PBS) reception. Mice were tested for fibrosis by using trichrome staining, hydroxyproline measurement and α-SMA detection in tissue sections. Additionally, the gelatinolytic activity of matrix metalloproteinase 2 and matrix metalloproteinase 9 were measured using gelatin zymography in lungs and skin tissue homogenates. The obtained results indicated that subcutaneous injection of bleomycin-induced fibrosis in skin and lung tissues of mice. Pro and active forms of matrix methaloproteinase 9 were increased in fibrotic lung tissues (p<0.05 and p<0.01, respectively), while, the gelatinolytic activity of MMP2 was unaffected in these tissues. Additionally, in skin tissues of bleomycin-treated animals, both pro and active forms of MMP9 and MMP2 were increased (p<0.05). Pro and active forms of gelatinases increase differently in skin and lung tissues of bleomycin-induced scleroderma.

Author Biographies

Kazem Mousavizadeh, Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran

Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine

Hadi Poormoghim, Scleroderma Study Group, Firoozgar Hospital, Iran University of Medical Sciences, Tehran, Iran

Scleroderma Study Group, Firoozgar Hospital

Tahereh Musavi Shabestari, Institute of Immunology and Infectious Diseases, Antimicrobial Resistance Research Center, Iran University of Medical Sciences, Tehran, Iran

Institute of Immunology and Infectious Disease, Antimicrobial resistance research center

Sayed Ali Javadmoosavi, Air Pollution Research Centre, School of Public Health, Iran University of Medical Sciences, Tehran, Iran

Air Pollution Research Centre, School of Public Health


1. Ho, Y.Y., et al., Fibrosis—a lethal component of systemic sclerosis. Nature Reviews Rheumatology, 2014. 10(7): p. 390.

2. Cox, T.R. and J.T. Erler, Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer. Disease models & mechanisms, 2011. 4(2): p. 165-178.

3. Jinnin, M., Mechanisms of skin fibrosis in systemic sclerosis. The Journal of dermatology, 2010. 37(1): p. 11-25.

4. Nagase, H., R. Visse, and G. Murphy, Structure and function of matrix metalloproteinases and TIMPs. Cardiovascular research, 2006. 69(3): p. 562-573.

5. Galis, Z.S., et al., Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions. Annals of the New York Academy of Sciences, 1994. 748(1): p. 501-507.

6. Imada, K., et al., Hormonal regulation of matrix metalloproteinase 9/gelatinase B gene expression in rabbit uterine cervical fibroblasts. Biology of reproduction, 1997. 56(3): p. 575-580.

7. Rao, V.H., et al., Interleukin-1β upregulates MMP-9 expression in stromal cells of human giant cell tumor of bone. Journal of interferon & cytokine research, 1999. 19(10): p. 1207-1217.

8. Liang, K.C., et al., Interleukin‐1β induces MMP‐9 expression via p42/p44 MAPK, p38 MAPK, JNK, and nuclear factor‐κB signaling pathways in human tracheal smooth muscle cells. Journal of cellular physiology, 2007. 211(3): p. 759-770.

9. Zhang, W., et al., Matrix metalloproteinase-9 is up-regulated by CCL19/CCR7 interaction via PI3K/Akt pathway and is involved in CCL19-driven BMSCs migration. Biochemical and biophysical research communications, 2014. 451(2): p. 222-228.

10. Pai, R., et al., Prostaglandin E 2 transactivates EGF receptor: a novel mechanism for promoting colon cancer growth and gastrointestinal hypertrophy. Nature medicine, 2002. 8(3): p. 289.

11. Vu, T.H. and Z. Werb, Matrix metalloproteinases: effectors of development and normal physiology. Genes & development, 2000. 14(17): p. 2123-2133.

12. Gaffney, J., et al., Multilevel regulation of matrix metalloproteinases in tissue homeostasis indicates their molecular specificity in vivo. Matrix Biology, 2015. 44: p. 191-199.

13. Khorramizadeh, M.R., et al., Dermal wound fibroblasts and matrix metaloproteinases (MMPs): their possible role in allergic contact dermatitis. Iranian Journal of Allergy, Asthma and Immunology, 2004. 3(1): p. 7-11.

14. Taheri, F.H., et al., The effect of Candida albicans systemic infection on matrix metalloproteinases in breast cancer bearing BALB/c mice. Iranian Journal of Allergy, Asthma and Immunology, 2013. 12(1): p. 81.

15. Parks, W.C. and S.D. Shapiro, Matrix metalloproteinases in lung biology. Respiratory research, 2000. 2(1): p. 3.

16. Hadler‐Olsen, E., et al., Regulation of matrix metalloproteinase activity in health and disease. The FEBS journal, 2011. 278(1): p. 28-45.

17. Aimes, R.T. and J.P. Quigley, 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 ¾-and ¼-Length Fragments. Journal of Biological Chemistry, 1995. 270(11): p. 5872-5876.

18. Yu, Q. and I. Stamenkovic, Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-β and promotes tumor invasion and angiogenesis. Genes & development, 2000. 14(2): p. 163-176.

19. Imai, K., et al., Degradation of decorin by matrix metalloproteinases: identification of the cleavage sites, kinetic analyses and transforming growth factor-beta1 release. Biochemical Journal, 1997. 322(Pt 3): p. 809.

20. Kim, J.Y., et al., Early and late changes of MMP-2 and MMP-9 in bleomycin-induced pulmonary fibrosis. Yonsei medical journal, 2009. 50(1): p. 68-77.

21. Yaguchi, T., et al., Immunohistochemical and gelatin zymography studies for matrix metalloproteinases in bleomycin‐induced pulmonary fibrosis. Pathology international, 1998. 48(12): p. 954-963.

22. Yamamoto, T., et al., Animal model of sclerotic skin. I: Local injections of bleomycin induce sclerotic skin mimicking scleroderma. Journal of Investigative Dermatology, 1999. 112(4): p. 456-462.

23. Lam, A.P., et al., Distinct patterns of pulmonary injury and fibrosis induced by intratracheal and subcutaneous bleomycin in the mouse: Relevance for distinct forms of human lung fibrosis, in Cystic and Idiopathic Pulmonary Fibrosis: Risk Factors, Management and Long-Term Health Outcomes. 2016, Nova Science Publishers, Inc.

24. Toth, M. and R. Fridman, Assessment of gelatinases (MMP-2 and MMP-9 by gelatin zymography, in Metastasis research protocols. 2001, Springer. p. 163-174.

25. Varga, J. and D. Abraham, Systemic sclerosis: a prototypic multisystem fibrotic disorder. The Journal of clinical investigation, 2007. 117(3): p. 557-567.

26. Page-McCaw, A., A.J. Ewald, and Z. Werb, Matrix metalloproteinases and the regulation of tissue remodelling. Nature reviews Molecular cell biology, 2007. 8(3): p. 221.

27. Giannandrea, M. and W.C. Parks, Diverse functions of matrix metalloproteinases during fibrosis. Disease models & mechanisms, 2014. 7(2): p. 193-203.

28. Manicone, A.M., I. Huizar, and J.K. McGuire, Matrilysin (matrix metalloproteinase-7) regulates anti-inflammatory and antifibrotic pulmonary dendritic cells that express CD103 (αEβ7-integrin). The American journal of pathology, 2009. 175(6): p. 2319-2331.

29. Yamashita, C.M., et al., Matrix metalloproteinase 3 is a mediator of pulmonary fibrosis. The American journal of pathology, 2011. 179(4): p. 1733-1745.

30. Hayashi, T., et al., Immunohistochemical study of metalloproteinases and their tissue inhibitors in the lungs of patients with diffuse alveolar damage and idiopathic pulmonary fibrosis. The American journal of pathology, 1996. 149(4): p. 1241.

31. Selman, M., et al., TIMP-1,-2,-3, and-4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment? American Journal of Physiology-Lung Cellular and Molecular Physiology, 2000. 279(3): p. L562-L574.

32. SUGA, M., et al., Characteristic elevation of matrix metalloproteinase activity in idiopathic interstitial pneumonias. American journal of respiratory and critical care medicine, 2000. 162(5): p. 1949-1956.

33. Lemjabbar, H., et al., Overexpression of alveolar macrophage gelatinase B (MMP-9) in patients with idiopathic pulmonary fibrosis: effects of steroid and immunosuppressive treatment. American journal of respiratory cell and molecular biology, 1999. 20(5): p. 903-913.

34. Andersen, G.N., et al., Bronchoalveolar matrix metalloproteinase 9 relates to restrictive lung function impairment in systemic sclerosis. Respiratory medicine, 2007. 101(10): p. 2199-2206.

35. Betsuyaku, T., et al., Gelatinase B is required for alveolar bronchiolization after intratracheal bleomycin. The American journal of pathology, 2000. 157(2): p. 525-535.

36. Cabrera, S., et al., Overexpression of MMP9 in macrophages attenuates pulmonary fibrosis induced by bleomycin. The international journal of biochemistry & cell biology, 2007. 39(12): p. 2324-2338.

37. Solli, A.I., et al., Tissue-and cell-specific co-localization of intracellular gelatinolytic activity and matrix metalloproteinase 2. Journal of Histochemistry & Cytochemistry, 2013. 61(6): p. 444-461.

38. Mattila, L., et al., Activation of tissue inhibitor of metalloproteinases-3 (TIMP-3) mRNA expression in scleroderma skin fibroblasts. Journal of Investigative Dermatology, 1998. 110(4): p. 416-421.

39. Fakhoury, H., M. Hillarby, and J. Weiss, Increased gelatinase activity in systemic sclerosis dermal fibroblast cultures with unaltered gelatinase A mRNA expression. Journal of dermatological science, 2002. 29(1): p. 62-69.

40. Kirk, T.Z., et al., Myofibroblasts from scleroderma skin synthesize elevated levels of collagen and tissue inhibitor of metalloproteinase (TIMP-1) with two forms of TIMP-1. Journal of Biological Chemistry, 1995. 270(7): p. 3423-3428.

41. Kim, W.-U., et al., Elevated matrix metalloproteinase-9 in patients with systemic sclerosis. Arthritis Res Ther, 2004. 7(1): p. R71.

42. Yazawa, N., et al., Serum levels of tissue inhibitor of metalloproteinases 2 in patients with systemic sclerosis. Journal of the American Academy of Dermatology, 2000. 42(1): p. 70-75.

43. Meng, C., et al., Expression of MMP-9 and TIMP-1 in lesions of systemic sclerosis and its implications. Journal of Huazhong University of Science and Technology [Medical Sciences], 2008. 28(4): p. 480-482.

How to Cite
Vafashoar F, Mousavizadeh K, Poormoghim H, Tavasoli A, Musavi Shabestari T, Javadmoosavi S, Mojtabavi N. Gelatinases Increase in Bleomycin-induced Systemic Sclerosis Mouse Model. Iran J Allergy Asthma Immunol. 18(2):182-189.
Original Article(s)