This study was conducted to determine the expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in a time-dependent manner and the effect of extracellular-signal-regulated kinases-1/2 (ERK1/2) inhibition on the expressions of MMP-9, TIMP-1, and inflammatory biomarkers in the retinas of diabetic rats. The expression of MMP-9 was quantified by zymography, and the mRNA level of MMP-9 and TIMP-1 was quantified by RT-PCR. The expression of inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) was examined by Western blot analysis. MMP-9 expression was significantly higher in diabetic rat retinas compared to controls at all time points.TIMP-1 expression was nonsignificantly upregulated at 1week of diabetes and was significantly downregulated at 4 and 12 weeks of diabetes. Intravitreal administration of the ERK1/2 inhibitor U0126 prior to induction of diabetes decreased ERK1/2 activation, attenuated diabetes-induced upregulation of MMP-9, iNOS, IL-6, and TNF-α and upregulated TIMP-1 expression. In MMP-9 knockout mice, diabetes had no effect on retinal iNOS expression and its level remained unchanged. These data provide evidence that ERK1/2 signaling pathway is involved in MMP-9, iNOS, IL-6, and TNF-α induction in diabetic retinas and suggest that ERK1/2 can be a novel therapeutic target in diabetic retinopathy. 1. Introduction Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and remains one of the leading causes of blindness worldwide. DR is characterized by gradual progressive alterations in the retinal microvasculature, leading to loss of retinal capillary cells, disruption of vascular barrier, retinal nonperfusion, and preretinal neovascularization [1–4]. However, the exact molecular mechanisms, which mediate such response, remain largely unknown. In recent years, it has become evident that inflammatory mechanisms play an important role in the pathogenesis of DR, and proinflammatory mediators contribute significantly to the development and progression of DR [5–13]. Inflammation is a multistep process where proteases, growth factors, cytokines, and chemokines are released from retinal cells and interact with each other to promote inflammation in the diabetic retinal microenviroment. In the retina, it was shown that diabetes activates induction of proinflammatory mediators such as monocyte chemoattractant protein-1 (MCP-1) [8], interleukin-6 (IL-6) [9], intercellular adhesion molecule-1 (ICAM-1) [10], inducible nitric oxide
References
[1]
H. P. Hammes, Y. Feng, F. Pfister, and M. Brownlee, “Diabetic retinopathy: targeting vasoregression,” Diabetes, vol. 60, no. 1, pp. 9–16, 2011.
[2]
N. A. Calcutt, M. E. Cooper, T. S. Kern, and A. M. Schmidt, “Therapies for hyperglycaemia-induced diabetic complications: from animal models to clinical trials,” Nature Reviews Drug Discovery, vol. 8, no. 5, pp. 417–429, 2009.
[3]
R. N. Frank, “Diabetic retinopathy,” The New England Journal of Medicine, vol. 350, no. 1, pp. 48–58, 2004.
[4]
R. F. Gariano and T. W. Gardner, “Retinal angiogenesis in development and disease,” Nature, vol. 438, no. 7070, pp. 960–966, 2005.
[5]
A. M. El-Asrar, “Role of inflammation in the pathogenesis of diabetic retinopathy,” Middle East African Journal of Ophthalmology, vol. 19, no. 1, pp. 70–74, 2012.
[6]
S. Rangasamy, P. G. McGuire, and A. Das, “Diabetic retinopathy and inflammation: novel therapeutic targets,” Middle East African Journal of Ophthalmology, vol. 19, no. 10, pp. 52–59, 2012.
[7]
G. Mohammad, M. M. Siddiquei, A. Othman, M. Al-Shabrawey, and A. M. Abu El-Asrar, “. High-mobility group box-1 protein activates inflammatory signaling pathway components and disrupts retinal vascular-barrier in the diabetic retina,” Experimental Eye Research, vol. 107, pp. 101–109, 2012.
[8]
A. M. Abu El-Asrar, S. Struyf, D. Kangave, K. Geboes, and J. van Damme, “Chemokines in proliferative diabetic retinopathy and proliferative vitreoretinopathy,” The European Cytokine Network, vol. 17, no. 3, pp. 155–165, 2006.
[9]
O. Arjamaa, M. P？ll？nen, K. Kinnunen, T. Ryh？nen, and K. Kaarniranta, “. Increased IL-6 levels are not related to NF-κB or HIF-1α transcription factors activity in the vitreous ofproliferative diabetic retinopathy,” Journal of Diabetes and Its Complications, vol. 25, no. 6, pp. 393–397, 2011.
[10]
A. M. Joussen, V. Poulaki, M. L. Le et al., “A central role for inflammation in the pathogenesis of diabetic retinopathy,” FASEB Journal, vol. 18, no. 12, pp. 1450–1452, 2004.
[11]
A. M. Abu El-Asrar, S. Desmet, A. Meersschaert, L. Dralands, L. Missotten, and K. Geboes, “Expression of the inducible isoform of nitric oxide synthase in the retinas of human subjects with diabetes mellitus,” American Journal of Ophthalmology, vol. 132, no. 4, pp. 551–556, 2001.
[12]
N. Demircan, B. G. Safran, M. Soylu, A. A. Ozcan, and S. Sizmaz, “Determination of vitreous interleukin-1 (IL-1) and tumour necrosis factor (TNF) levels in proliferative diabetic retinopathy,” Eye, vol. 20, no. 12, pp. 1366–1369, 2006.
[13]
G. Mohammad and R. A. Kowluru, “Diabetic retinopathy and signaling mechanism for activation of matrix metalloproteinase-9,” Journal of Cellular Physiology, vol. 227, no. 3, pp. 1052–1061, 2012.
[14]
A. S. Yar, S. Menevse, I. Dogan et al., “Investigation of ocular neovascularization-related genes and oxidative stress in diabetic rat eye tissues after resveratrol treatment,” Journal of Medicinal Food, vol. 15, no. 4, pp. 391–398, 2012.
[15]
R. A. Kowluru, G. Mohammad, J. M. dos Santos, and Q. Zhong, “Abrogation of MMP-9 gene protects against the development of retinopathy in diabetic mice by preventing mitochondrial damage,” Diabetes, vol. 60, no. 11, pp. 3023–3033, 2011.
[16]
F. J. Descamps, E. Martens, D. Kangave et al., “The activated form of gelatinase B/matrix metalloproteinase-9 is associated with diabetic vitreous hemorrhage,” Experimental Eye Research, vol. 83, no. 2, pp. 401–407, 2006.
[17]
S. J. Giebel, G. Menicucci, P. G. McGuire, and A. Das, “Matrix metalloproteinases in early diabetic retinopathy and their role in alternation of the blood-retinal barrier,” Laboratory Investigation, vol. 85, no. 5, pp. 597–607, 2005.
[18]
H. Nagase and J. F. Woessner, “Matrix metalloproteinases,” Journal of Biological Chemistry, vol. 274, pp. 21491–21494, 1999.
[19]
S. Uemura, H. Matsushita, W. Li et al., “Diabetes mellitus enhances vascular matrix metalloproteinase activity role of oxidative stress,” Circulation Research, vol. 88, no. 12, pp. 1291–1298, 2001.
[20]
N. P. Kadoglou, S. S. Daskalopoulou, D. Perrea, and C. D. Liapis, “Matrix metalloproteinases and diabetic vascular complications,” Angiology, vol. 56, no. 2, pp. 173–189, 2005.
[21]
W. Cruz-Munoz and R. Khokha, “The role of tissue inhibitors of metalloproteinases in tumorigenesis and metastasis,” Critical Reviews in Clinical Laboratory Sciences, vol. 45, no. 3, pp. 291–338, 2008.
[22]
M. Jin, K. Kashiwagi, Y. Iizuka, Y. Tanaka, M. Imai, and S. Tsukahara, “Matrix metalloproteinases in human diabetic and nondiabetic vitreous,” Retina, vol. 21, no. 1, pp. 28–33, 2001.
[23]
A. M. Abu El-Asrar, L. Dralands, M. Veckeneer et al., “Gelatinase B in proliferative vitreoretinal disorders,” The American Journal of Ophthalmology, vol. 125, no. 6, pp. 844–851, 1998.
[24]
R. A. Kowluru, “Role of matrix metalloproteinase-9 in the development of diabetic retinopathy and its regulation by H-Ras,” Investigative Ophthalmology and Visual Science, vol. 51, no. 8, pp. 4320–4326, 2010.
[25]
P. Liu and M. J. Wilson, “miR-520c and miR-373 upregulate MMP9 expression by targeting mTOR and SIRT1, and activate the Ras/Raf/MEK/Erk signaling pathway and NF-κB factor in human fibrosarcoma cells,” Journal of Cellular Physiology, vol. 227, no. 2, pp. 867–876, 2012.
[26]
S. S. Lakka, S. L. Jasti, C. Gondi et al., “Downregulation of MMP-9 in ERK-mutated stable transfectants inhibits glioma invasion in vitro,” Oncogene, vol. 21, no. 36, pp. 5601–5608, 2002.
[27]
A. Cho, J. Graves, and M. A. Reidy, “Mitogen-activated protein kinases mediate matrix metalloproteinase-9 expression in vascular smooth muscle cells,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 20, no. 12, pp. 2527–2532, 2000.
[28]
X. Ye, G. Xu, Q. Chang et al., “ERK1/2 signaling pathways involved in VEGF release in diabetic rat retina,” Investigative Ophthalmology and Visual Science, vol. 51, no. 10, pp. 5226–5233, 2010.
[29]
F. C. Barouch, K. Miyamoto, J. R. Allport et al., “Integrin-mediated neutrophil adhesion and retinal leukostasis in diabetes,” Investigative Ophthalmology and Visual Science, vol. 41, no. 5, pp. 1153–1158, 2000.
[30]
T. Qaum, Q. Xu, A. M. Joussen et al., “VEGF-initiated blood-retinal barrier breakdown in early diabetes,” Investigative Ophthalmology and Visual Science, vol. 42, no. 10, pp. 2408–2413, 2001.
[31]
B. Dubois, S. Masure, U. Hurtenbach et al., “Resistance of young gelatinase B-deficient mice to experimental autoimmune encephalomyelitis and necrotizing tail lesions,” Journal of Clinical Investigation, vol. 104, no. 11, pp. 1507–1515, 1999.
[32]
J. T. Neary, “MARK cascades in cell growth and death,” News in Physiological Sciences, vol. 12, no. 6, pp. 286–293, 2007.
[33]
R. H. Chen, C. Sarnecki, and J. Blenis, “Nuclear localization and regulation of erk- and rsk-encoded protein kinases,” Molecular and Cellular Biology, vol. 12, no. 3, pp. 915–927, 1992.
[34]
P. Lenormand, C. Sardet, G. Pages, G. L'Allemain, A. Brunet, and J. Pouyssegur, “Growth factors induce nuclear translocation of MAP kinases (p42(mapk) and p44(mapk)) but not their activator MAP kinase kinase (p45(mapkk)) in fibroblasts,” Journal of Cell Biology, vol. 122, no. 5, pp. 1079–1088, 1993.
[35]
A. M. Abu El-Asrar, L. Dralands, L. Missotten, I. A. Al-Jadaan, and K. Geboes, “Expression of apoptosis markers in the retinas of human subjects with diabetes,” Investigative Ophthalmology and Visual Science, vol. 45, no. 8, pp. 2760–2766, 2004.
[36]
Z. A. Khan and S. Chakrabarti, “Cellular signaling and potential new treatment targets in diabetic retinopathy,” Experimental Diabetes Research, vol. 2007, article 31867, 2007.
[37]
G. Mohammad and M. M. Siddiquei, “Role of matrix metalloproteinase-2 and -9 in the development of diabetic retinopathy,” Journal of Ocular Biology, Diseases, and Informatics, vol. 5, no. 1, pp. 1–8, 2012.
[38]
G. Mohammad and R. A. Kowluru, “The role of Raf-1 kinase in diabetic retinopathy,” Expert Opinion on Therapeutic Targets, vol. 15, no. 4, pp. 357–364, 2011.
[39]
S. K. Jo, W. Y. Cho, S. A. Sung, H. K. Kim, and N. H. Won, “MEK inhibitor, U0126, attenuates cisplatin-induced renal injury by decreasing inflammation and apoptosis,” Kidney International, vol. 67, no. 2, pp. 458–466, 2005.
[40]
M. R. Junttila, S. P. Li, and J. Westermarck, “Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival,” FASEB Journal, vol. 22, no. 4, pp. 954–965, 2008.
[41]
V. C. Ardi, T. A. Kupriyanova, E. I. Deryugina, and J. P. Quigley, “Human neutrophils uniquely release TIMP-free MMP-9 to provide a potent catalytic stimulator of angiogenesis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 51, pp. 20262–20267, 2007.
[42]
V. C. Ardi, P. E. van den Steen, G. Opdenakker, B. Schweighofer, E. I. Deryugina, and J. P. Quigley, “Neutrophil MMP-9 proenzyme, unencumbered by TIMP-1, undergoes efficient activation in vivo and catalytically induces angiogenesis via a basic fibroblast growth factor (FGF-2)/FGFR-2 pathway,” Journal of Biological Chemistry, vol. 284, no. 38, pp. 25854–25866, 2009.
[43]
S. D. Kim, S. I. Yang, H. C. Kim, C. Y. Shin, and K. H. Ko, “Inhibition of GSK-3β mediates expression of MMP-9 through ERK1/2 activation and translocation of NF-κB in rat primary astrocyte,” Brain Research, vol. 1186, no. 1, pp. 12–20, 2007.
[44]
L. Zheng, S. J. Howell, D. A. Hatala, K. Huang, and T. S. Kern, “Salicylate-based anti-inflammatory drugs inhibit the early lesion of diabetic retinopathy,” Diabetes, vol. 56, no. 2, pp. 337–345, 2007.
[45]
Y. Du, V. P. Sarthy, and T. S. Kern, “Interaction between NO and COX pathways in retinal cells exposed to elevated glucose and retina of diabetic rats,” American Journal of Physiology—Regulatory Integrative and Comparative Physiology, vol. 287, no. 4, pp. R735–R741, 2004.
[46]
A. Carmo, J. G. Cunha-Vaz, A. P. Carvalho, and M. C. Lopes, “Effect of cyclosporin-A on the blood-retinal barrier permeability in streptozotocin-induced diabetes,” Mediators of Inflammation, vol. 9, no. 5, pp. 243–248, 2000.
[47]
L. Zheng, C. Szabó, and T. S. Kern, “Poly(ADP-ribose) polymerase is involved in the development of diabetic retinopathy via regulation of nuclear factor-κB,” Diabetes, vol. 53, no. 11, pp. 2960–2967, 2004.
[48]
A. M. Joussen, V. Poulaki, W. Qin et al., “Retinal vascular endothelial growth factor induces intercellular adhesion molecule-1 and endothelial nitric oxide synthase expression and initiates early diabetic retinal leukocyte adhesion in vivo,” The American Journal of Pathology, vol. 160, no. 2, pp. 501–509, 2002.
[49]
A. M. Joussen, V. Poulaki, N. Mitsiades et al., “Nonsteroidal anti-inflammatory drugs prevent early diabetic retinopathy via TNF-alpha suppression,” The FASEB Journal, vol. 16, no. 3, pp. 438–440, 2002.
[50]
K. Arai, S. R. Lee, and E. H. Lo, “Essential role for ERK mitogen-activated protein kinase in matrix metalloproteinase-9 regulation in rat cortical astrocytes,” GLIA, vol. 43, no. 3, pp. 254–264, 2003.
[51]
L. Wang, G. Z. Zheng, L. Z. Rui et al., “Matrix metalloproteinase 2 (MMP2) and MMP9 secreted by erythropoietin- activated endothelial cells promote neural progenitor cell migration,” Journal of Neuroscience, vol. 26, no. 22, pp. 5996–6003, 2006.
[52]
H. R. Young, C. P. Chung, A. Oeser et al., “Inflammatory mediators and premature coronary atherosclerosis in rheumatoid arthritis,” Arthritis Care and Research, vol. 61, no. 11, pp. 1580–1585, 2009.
[53]
K. Miyamoto, S. Khosrof, S. E. Bursell et al., “Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition,” Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 19, pp. 10836–10841, 1999.
[54]
J. R. van Beijnum, W. A. Buurman, and A. W. Griffioen, “Convergence and amplification of toll-like receptor (TLR) and receptor for advanced glycation end products (RAGE) signaling pathways via high mobility group B1 (HMGB1),” Angiogenesis, vol. 11, no. 1, pp. 91–99, 2008.
[55]
E. A. Ellis, D. L. Guberski, B. Hutson, and M. B. Grant, “Time course of NADH oxidase, inducible nitric oxide synthase and peroxynitrite in diabetic retinopathy in the BBZ/Wor rat,” Nitric Oxide, vol. 6, no. 3, pp. 295–304, 2002.
[56]
B. Gao, K. Calhoun, and D. Fang, “The proinflammatory cytokines IL-1β and TNF-α induce the expression of Synoviolin, an E3 ubiquitin ligase, in mouse synovial fibroblasts via the Erk1/2-ETS1 pathway,” Arthritis Research and Therapy, vol. 8, no. 6, article R172, 2006.
[57]
G. S. Firestein and A. M. Manning, “Signal transduction and transcription factors in rheumatic disease,” Arthritis & Rheumatism, vol. 42, no. 4, pp. 609–621, 1999.
[58]
A. Maddahi and L. Edvinsson, “Cerebral ischemia induces microvascular pro-inflammatory cytokine expression via the MEK/ERK pathway,” Journal of Neuroinflammation, vol. 7, article 14, 2010.
[59]
M. S. Woo, J. S. Park, I. Y. Choi, W. K. Kimf, and H. S. Kim, “Inhibition of MMP-3 or -9 suppresses lipopolysaccharide-induced expression of proinflammatory cytokines and iNOS in microglia,” Journal of Neurochemistry, vol. 106, no. 2, pp. 770–780, 2008.
