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Metalloproteinase 9 and TIMP-1 expression in retina and optic nerve in absolute angle closure glaucoma
Advances in Medical Sciences 61 (2016) 6–10
Contents lists available at ScienceDirect
Advances in Medical Sciences journal homepage: www.elsevier.com/locate/advms
Original Research Article
Metalloproteinase 9 and TIMP-1 expression in retina and optic nerve in absolute angle closure glaucoma Renata Zalewska a, Joanna Reszec´ b,*, Wojciech Kisielewski b, Zofia Mariak a a b
Department of Ophthalmology, Medical University of Bialystok, Bialystok, Poland Department of Medical Pathomorphology, Medical University of Bialystok, Bialystok, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 November 2014 Accepted 24 July 2015 Available online 9 August 2015
Purpose: Glaucoma is one of the most important reason causes of the blindness, associated with retinal ganglion cells (RGC) death. This process is not fully understood, however apoptosis due to hypoxia is one of the most important processes leading to RGC death. Glaucomatous optic neuropathy is characterized by remodeling of the extracellular matrix due to metalloproteinase activation, which leads to loss of RGC and axons at the optic nerve head. The aim of the study was to evaluate metalloproteinase 9 (MMP-9) and tissue metalloproteinase inhibitor-1 (TIMP-1) expression in the retinal ganglion cells and optic nerve axons in 33 eyes with absolute primary glaucoma. Material/methods: To evaluate MMP-9 and TIMP-1 expression primary polyclonal goat antibodies against MMP-9 and TIMP-1 were used. The control group was composed of 8 cases of eyes enucleated and fixed in the first day after trauma. Results: MMP-9 expression was observed in retinal ganglion cells and in the inner nuclear layer of the retina in all the examined cases. In 28 out of 33 glaucomatous eyes, MMP-9 expression was observed in the proliferating glial cells surrounding the optic nerve axons. TIMP-1 expression was observed in 10 out of 33 glaucomatous eyes, only in retinal ganglion cells. None of the examined injured eyes showed MMP9 and TIMP-1 expression. Conclusions: MMP-9 activation rather than TIMP-1 may by associated with the pathomechanism of retinal ganglion cell and optic nerve damage in absolute glaucoma. ß 2015 Medical University of Bialystok. Published by Elsevier Sp. z o.o. All rights reserved.
Keywords: Glaucoma MMP-9 TIMP-1 Retina Optic nerve
1. Introduction Some eye diseases are associated with damage of the optic nerve axons as well as retinal ganglion cells. Glaucoma is one of the leading causes of blindness. Irreversible vision loss in glaucoma is attributed to retinal ganglion cell (RGC) death connected with apoptosis. RGC death is not fully understood, because the processes of RGC (retinal ganglion cells) apoptosis is heavily complicated [1]. Glaucomatous optic neuropathy is multifactorial in etiology [2]. The mechanism which is responsible for RGC apoptosis is associated with hypoxia, which is most frequently a consequence of increased intraocular pressure [3]. Glaucomatous optic neuropathy is characterized by remodeling of the extracellular matrix and
* Corresponding author at: Department of Medical Pathomorphology, Medical University of Bialystok, Waszyngtona 13, 15-269 Bialystok, Poland. Tel.: +48 857485923; fax: +48 857485990. E-mail address: [email protected] (J. Reszec´).
loss of retinal ganglion cell axons at the optic nerve head level. The integrity and turnover of the extracellular matrix are influenced by many factors, including matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) [4]. Matrix metalloproteinase 9 (MMP-9), also known as 92 kDa type IV collagenase, 92 kD A gelatinase or gelatinase B (GELB), is an enzyme encoded by the MMP9 gene. The enzyme encoded by this gene degrades type IV and V collagens and other extracellular matrix proteins like fibronectin, laminin, and other glycoproteins [5,6]. Among the many subtypes of metalloproteinases, some are secreted constantly, some only in certain circumstances. This is due to the regulation of the expression of tissue inhibitors of metalloproteinases (TIMPs) [6,7]. Recent studies show abnormal activation of matrix metalloproteinases, in particular MMP-9, which triggers an extracellular signaling cascade leading to apoptosis [6,8–11]. Tissue inhibitors of metalloproteinases (TIMP) are the inhibitors of matrix metalloproteinases that participate in controlling the local activities of MMPs in tissues. So far little is known about
http://dx.doi.org/10.1016/j.advms.2015.07.007 1896-1126/ß 2015 Medical University of Bialystok. Published by Elsevier Sp. z o.o. All rights reserved.
R. Zalewska et al. / Advances in Medical Sciences 61 (2016) 6–10
MMP-9 inhibition by TIMP-1. Data show that TIMP activity is responsible for tumor growth reduction, and inhibition of endothelial cell growth induced by basic fibroblast growth factor. TIMP-1 and TIMP-2 have antiapoptotic activity [12]. Remodeling of the optic nerve head in glaucoma involves astrocyte response and changes in the extracellular matrix composition and distribution. The MMP family has been implicated in the cascade of events leading to neuronal apoptosis in the central nervous system. MMP substrates include essentially all extracellular matrix components as well as a wide array of molecules involved in intracellular adhesion, cell–matrix interaction, and cell signaling [13–16]. MMP1, MMP2 and MMP-9 have previously been implicated in the pathogenesis of primary open angle glaucoma and open angle glaucoma secondary to exfoliation syndrome, respectively; matrix metalloproteinase-9 (MMP9) gene was investigated for association with primary angle closure glaucoma [5,17,20,22]. Classic glaucoma treatment focuses on intraocular pressure reduction. However, recent knowledge about the pathogenesis of glaucoma has opened up new therapeutic approaches. Some of the investigators suggest a pivotal role of MMP inhibition in glaucoma treatment. Inhibition of MMP-9 could inhibit the apoptosis of retinal ganglion cells and tissue remodeling [15]. The aim of the study was to evaluate metalloproteinase 9 (MMP-9) and TIMP-1 expression in retina and optic nerve in eyeballs with the primary glaucoma. 2. Materials and methods Thirty-three eyeballs were examined and enucleated in the Department of Ophthalmology of the Medical University of Bialystok over the period 1991–2013. Patients were eligible for study participation if they had primary angle closure glaucoma (PACG) – 33 patients – and met the following criteria: blind eye, corneal edema, dilated unreactive pupil, and IOP (intraocular pressure) >50 mmHg using Goldmann aplanation tonometry. All eyeballs were removed from patients with absolute glaucoma, who suffered from severe ophthalmalgia. The study was approved by the Bioethical Committee in Medical University of Bialystok and was performed in accordance with ethical standards laid down in the 1964 Declaration of Helsinki. All patients signed an agreement form before their inclusion in the study. Thirty-three eyeballs were removed from patients with absolute glaucoma (blind eyes), who suffered from severe ophthalmalgia due to exceptionally high intraocular pressure. All of the cases were angle closure glaucoma. After enucleation, the eyeballs were fixed in a 10% buffered formalin solution on the same day of enucleation, embedded in paraffin at 56 8C, then cut into 5 mm slices and stained with hematoxilin and eosin (H+E). Following deparaffinization, endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 30 min. The sections were then incubated with goat polyclonal antibody for MMP-9 (MMP-9 Antibody (C-20): sc6840, Santa Cruz Biotechnology, Santa Cruz, CA) in 1:100 dilution, and with TIMP-1 antibody (TIMP-1 Antibody (C-20): sc-6832 Santa Cruz Biotechnology) in 1:50 dilution for a whole night at 4 8C, and labeled EnVision (DAKO) enzyme reagent and diaminobenzidine (DAB) chromogen for 5 min. In each routine of staining, adjacent sections that were incubated without primary antibody were prepared as the negative control. Knowing MMP-9 strong expression in joint degradation in rheumatoid arthritis, synovial specimens were taken as positive controls. For TIMP-1 human prostate tissue showing TIMP-1 cytoplasmic and membrane staining of glandular cells was used as a positive control. As a control group, we chose 8 eyeballs after severe trauma (all were closure injury). All were enucleated 1 day after the trauma, and then fixed in 10% buffered formalin. The sections were taken
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from the optic nerve head (1 section) and retina (3 cross-sections), and stained with H+E. Also, MMP-9 and TIMP-1 expression was evaluated using immunohistochemistry. Two independent pathologists performed the immunohistochemical evaluations of MMP-9 and TIMP-1. The estimation of immunostaining was done under a light microscope in representative fields under magnification of 20. The score for immunohistochemistry was as follows (as shown in Table 1,2): negative ( ) if less than 10% of the examined cells were positive, (+) if 10–50% of cells were positive, and if more than 50% of the examined cells showed staining the evaluation was (++). The obtained results were statistically analyzed using Spearman’s and Pearson’s tests. 3. Results The mean age of the glaucoma group ranged from 54 to 88 years (mean age 69, SD 12.4), with 13 male and 20 female patients. In the control injury group, age ranged from 21–77 (45, SD 17.8) years, with 6 male and 2 female patients. Ten patients from the glaucoma group had hypertension, and six had coronary disease. Two patients from the trauma group had hypertension. There was no correlation between age and sex in both examined groups (p = 0.983). 3.1. MMP-9 expression in glaucomatous eyes MMP-9 expression was observed in the perinuclear area of the examined cells. 28 out of 33 glaucomatous eyes presented MMP-9 expression in the optic nerve. It was mainly observed within the proliferating astrocytes surrounding the optic nerve axons; in 8 cases the expression was very strong and diffused (Fig. 1A and B). The results are summarized in Table 1. All glaucomatous eyes presented MMP-9 expression in the retina. MMP-9 staining was observed both in the inner nuclear layer of the retina (Fig. 1C) and in the layer of the retinal ganglion cells (Fig. 1D). All cases with MMP-9 expression localized in the optic nerve astrocytes also presented MMP-9 expression in retinal ganglion cells and the inner nuclear layer of the retina. We did not observe MMP-9 expression in retinal ganglion cells, in the inner layer, and optic nerve in all eyes after trauma. 3.2. TIMP-1 expression TIMP-1 expression was observed as cytoplasmic staining. TIMP-1 expression was observed only in 10 out of 33 glaucomatous eyeballs with angle closure glaucoma. The expression was not observed in the inner nuclear layer of the retina; in all 10 cases the staining was observed in the retinal ganglion cell layer (Fig. 2A and B). None of the glaucomatous eyeballs showed TIMP-1 expression, neither in the optic nerve axons or proliferating astroglia (Fig. 2C and D). The results are shown in Table 2. None of the injured eyeballs showed TIMP-1 expression, neither in the retina nor the optic nerve. Table 1 MMP-9 expression in the glaucomatous eyes. MMP-9 expression
Negative No %
(+) No %
(++) No %
p value
Optic nerve axons
5 15.1% 0 0.0 0 0.0%
20 60.6% 28 84.8% 28 84.8%
8 24.3% 5 15.2% 5 15.2%
0.736
Retinal ganglion cells Inner nuclear layer of the retina
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R. Zalewska et al. / Advances in Medical Sciences 61 (2016) 6–10
Fig. 1. (A) MMP-9 expression in optic nerve head. Magn. 40. (B) MMP-9 expression observed in the perinuclear area of proliferating astroglial cells in the optic nerve. Magn. 200. (C) MMP-9 expression in the inner layer of the retina and retinal ganglion cells. Magn. 200. (D) MMP-9 expression in the layer of the ganglion cells in the retina (arrow). Magn. 200.
4. Discussion Matrix metalloproteinases (MMPs) are involved in the breakdown of the extracellular matrix in normal physiological processes, such as embryonic development, reproduction, angiogenesis, bone development, wound healing, cell migration, learning and memory, as well as in pathological processes, such as arthritis,
intracerebral hemorrhage, and tumor progression, invasion and metastasis. The balance between metalloproteinases and inhibitors plays an important role in maintaining the homeostasis and integrity of the tissue in normal and pathological conditions. Investigations by several scientists have demonstrated an important role of matrix metalloproteinases in enzymatic degradation in glaucomatous alterations in the retina [9,10,14,18,19].
Fig. 2. (A) TIMP-1 expression in the ganglion cells of the retina. Magn. 400. (B) TIMP-1 expression in few retinal ganglion cells of the glaucomatous retina. Magn. 600. (C) Lack of TIMP-1 expression in retina. Magn. 200. (D) Lack of TIMP-1 expression in optic nerve. Evident astroglial cell proliferation between optic nerve axons. Magn. 40. Note: Immunohistochemistry, EnVision (brown staining of the expressed protein).
R. Zalewska et al. / Advances in Medical Sciences 61 (2016) 6–10 Table 2 TIMP-1 expression in glaucomatous eyes. TIMP-1 expression
Negative No %
(+) No %
(++) No %
p value
Optic nerve axons
33 100.0% 23 69.7% 33 100.0%
0 0.0% 10 30.3% 0 0.0%
0 0.0% 0 0.0% 0 0.0%
0.001
Retinal ganglion cells Inner nuclear layer of the retina
Recent studies present the theory of extracellular matrix remodeling in which matrix metalloproteinases play a crucial role in glaucomatous retinal ganglion cell damage. It was also observed that elevated intraocular pressure leads to the proliferation of optic nerve astrocytes. MMP expression is upregulated in activated astrocytes causing matrix remodeling. The role of MMPs in glaucomatous alterations in retinal cells has been shown in studies presenting the relationship between MMP expression and retinal ganglion cell damage [6,8,10,11,15]. Pathological intense apoptosis of retinal ganglion cells is a consequence of glaucomatous intraocular pressure and results in loss of vision. This type of apoptosis of the retinal ganglion cells is also related to laminin degradation from the underlying inner membrane and increase in MMP activity [9]. Our recent studies showed a significant correlation between proapoptotic proteins, Bak and Bax, and retinal ganglion cell damage [23]. We also observed Fas receptor, caspase-3 and HIF-1 overexpression in retinal ganglion cells and optic nerve axons in absolute glaucoma [2,3]. In the present study, we have demonstrated a significant overexpression of MMP-9 in glaucomatous RGC, internal nuclear layer of the retina, and glial cells surrounding optic nerve axons, which may support the theory that MMPs may activate pathological apoptosis in absolute glaucoma due to the activation of proapoptotic proteins and cell death receptors. Altogether, this may suggest that apoptosis, hypoxia and damage of the extracellular matrix via MMP-9 may contribute to retinal cell and optic nerve axon damage in absolute glaucoma. Recent studies concerning MMP expression in glaucomatous eyes are contradictory. Agapova et al. presented the study of MMP expression in the optic nerve of normal monkey eyes and eyes with experimental glaucoma or optic nerve transection [21]. They observed reactive astrocytes expressed MMP-1 at a higher level than in controls. Immunoreactivity for MMP-2 was present in the optic nerve and retina in all normal controls. Also retinal ganglion cells and their axons in the nerve fiber layer were MMP-2 positive; in the optic nerve head, MMP-2 immunoreactivity was strongly associated with axons in the nerve bundles and few astrocytes in the cribriform plates. However, opposite to our results, no MMP-9 immunoreactivity was localized in the optic nerve head of the control eyes, eyes with glaucoma, or optic nerve transection. A study presented by Seo et al. showed low MMP-9 expression in glaucoma and it was not significantly different from that of the control group [19]. However, Manabe et al. presented upregulation of MMP-9 in retinal neurons suggesting that an extracellular proteolysis pathway in the retina contributes to retinal ganglion cell death via MMP-9 activation [11]. Similarly to our studies, Mali et al. observed MMP-9 overexpression in the retina and its association with increased proapoptotic protein Bax expression in ganglion cells and the inner nuclear layer [10]. Chintala et al. observed that MMP-9 deficiency protects against pathological changes in the retina after optic nerve ligation. They found that increased MMP-9 activity is associated with laminin degradation, which results in retinal ganglion cell apoptosis [9]. In our study, MMP-9 expression was significant both in retinal ganglion cells as well as the inner layer of the retina. Similarly,
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Groef et al. observed a strong expression of MMP-2 in the adult mouse retina [18]. Also cytoplasmic MMP-3 expression was observed in the retinal ganglion cell layer and inner nuclear layer. MMP-9 showed a strong expression in the inner limiting membrane, a subset of cells in the retinal ganglion cells and in both the inner and the outer nuclear layer suggesting that MMP-3 and MMP-9 together may be involved in the process underlying retinal ganglion cell death and glial reactivity in the retina. Also Majka et al. presented a significant association between MMP-9 expression, laminin degradation, retinal ganglion cell apoptosis, and intraocular pressure exposure in glaucoma [9]. Liu et al. showed strong MMP-9 expression in ganglion cells suggesting that MMP-9 inhibitors may be able to protect the retina against ischemia [14]. Awadalla et al. showed an influence of MMP-9 expression in the pathogenesis of blind [5]. Wang et al. found that MMP-9 is associated with acute PACG [22]. Robertson et al. observed that MMP-9 plays an important role in maintaining intraocular pressure, and due to extracellular matrix remodeling, leads to ocular hypertension and glaucoma [16]. In the present study, we also examined TIMP-1 expression in glucomatous eyes. However, only in 10 out of 33 cases presented immunopositive reaction for TIMP-1, and only in the retinal ganglion cells. The explanation of this phenomenon in difficult, however basing on data, we may suspect, that it is due to apoptosis of the retinal ganglion cells in glaucoma, which may be associated with TIMP deficiency in retina and optic nerve axons in glaucoma. There is little data concerning the problem of TIMP-1 and MMP-9 expression in glaucoma. In one of them, Agapova et al. observed TIMP-1 and TIMP-2 expression also in retinal ganglion cells in absolute glaucoma. The level of TIMP expression in the nerve bundles of eyes with experimental glaucoma and optic nerve transection was lower than in normal eyes due to loss of axons in both models [21]. Contrary to our study, Guo et al. observed significant MMP-9 expression in retinal ganglion cells, although tissue inhibitor of matrix metalloproteinase (TIMP) was also overexpressed [1]. Some of the studies present matrix metalloproteinase expression in the aqueous humor of patients with glaucoma. Nga et al. presented altered levels of MMPs (MMP-2, MMP-3) and TIMPs (TIMP-1 and TIMP-2) in patients with primary angle closure glaucoma. The imbalance of MMP:TIMP ratios in the aqueous humor were significantly different from patients with non-glaucomatous eyes [24]. Kara et al. observed no statistically significant difference among mean MMP-2 and TIMP-2 levels in both aqueous humor and serum samples in patients with pseudoexfoliation syndrome and the control group [25]. However, Fountoulakis et al. observed TIMP-4 elevation in glaucomatous eyes, and they suggested that dysregulation of extracellular matrix homeostasis is associated with primary open angle glaucoma, pseudoexfolation syndrome, and pseudoexfoliative glaucoma [17]. Also Ghanem et al. observed increased expression of TIMPs (TIMP-2) in patients with exfoliative glaucoma [26]. 5. Conclusions Providing the theory about the hypoxia [27] and apoptosis [28] in retinal ganglion cells we decided to study the expression of matrix metalloproteinases and inhibitors in primary glaucoma. Our study presented strong MMP-9 expression in glaucomatous eyes, mainly in the inner nuclear layer, retinal ganglion cells, and proliferating astroglia in optic nerves. TIMP-1 expression was observed only in a few cases and only in the ganglion cells of the retina, due to a significant loss of neurons in glaucomatous eyes. The results show that only MMP-9, rather than TIMP-1 activation, underlies the pathomechanism of the retinal ganglion cell and optic nerve damage in absolute glaucoma.
R. Zalewska et al. / Advances in Medical Sciences 61 (2016) 6–10
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The authors declare no conflict of interests. We confirm that all authors have read and approved the submission of the manuscript, the manuscript has not been published and is not being considered for publication elsewhere, in whole or in part, in any language. We also declare no financial relationships with any industry (through investments, employment, consultancies, stock ownership, honoraria). Conflict of interests None declared. Financial disclosure None declared. References [1] Guo MS, Wu YY, Liang ZB. Hyaluronic acid increases MMP-2 and MMP-9 expressions in cultured trabecular meshwork cells from patients with primary open-angle glaucoma. Mol Vis 2012;18:1175–81. [2] Zalewska R, Zalewski B, Reszec J, Mariak Z, Zimnoch L, Proniewska-Skretek E. The expressions of Fas and caspase-3 in human glaucomatous optic nerve axons. Med Sci Monit 2008;14:274–8. [3] Reszec´ J, Zalewska R, Bernaczyk P, Chyczewski L. HIF-1 expression in retinal ganglion cells and optic nerve axons in glaucoma. Folia Histochem Cytobiol 2012;50:456–9. [4] Yan X, Tezel G, Wax MB, Edward DP. Matrix metalloproteinases and tumor necrosis factor alpha in glaucomatous optic nerve head. Arch Ophthalmol 2003;118:666–73. [5] Awadalla MS, Burdon KP, Kuot A, Hewitt AW, Craig JE. Matrix metalloproteinase-9 genetic variation and primary angle closure glaucoma in a Caucasian population. Mol Vis 2011;17:1420–4. [6] Micheal S, Yousaf S, Khan MI, Akhtar F, Islam F, Khan WA, et al. Polymorphisms in matrix metalloproteinase MMP1 and MMP9 are associated with primary open-angle and angle closure glaucoma in a Pakistani population. Mol Vis 2013;19:441–7. [7] Wierzbowska J, Robaszkiewicz J, Figurska M, Figurska M, Stankiewicz A. Future possibilities in glaucoma therapy. Med Sci Monit 2010;16:252–9. [8] Chintala SK, Zhang X, Austin JS, Fini ME. Deficiency in matrix metalloproteinase gelatinase B (MMP-9) protects against retinal ganglion cell death after optic nerve ligation. J Biol Chem 2002;277:47461–68. [9] Majka S, McGuire PG, Das A. Regulation of matrix metalloproteinase expression by tumor necrosis factor in a murine model of retinal neovascularization. Investig Ophthalmol Vis Sci 2002;43:260–6. [10] Mali RS, Cheng M, Chintala SK. Intravitreous injection of membrane depolarization agent causes retinal degeneration via Matrix Metalloproteinase-9. Investig Ophthal Vis Sci 2005;46:2125–32. [11] Manabe S, Gu Z, Lipton SA. Activation of matrix metalloproteinase-9 via neuronal nitric oxide synthase contributes to NMDA-induced retinal ganglion cell death. Investig Ophthal Vis Sci 2005;44:4747–53.
[12] Visse R, Nagase H. Matrix metalloproteinase and tissue inhibitors of metalloproteinase: structure, function, and biochemistry. Circ Res 2003;92:827–39. [13] Aung T, Yong VH, Lim MC, Venkataraman D, Toh JY, Chew PT, et al. Lack of association between the rs2664538 polymorphism in the MMP-9 gene and primary angle closure glaucoma in Singaporean subjects. J Glaucoma 2008; 17:257–8. [14] Liu XQ, Wu BJ, Pan WH, Zhang XM, Liu JH, Chen MM, et al. Resveratrol mitigates rat retinal ischemic injury: the roles of matrix metalloproteinase9, inducible nitric oxide, and heme oxygenase-1. J Ocul Pharmacol Ther 2013; 29:33–40. [15] Mozaffarieh M, Flammer J. Is there more to glaucoma treatment than lowering IOP? Surv Ophthalmol 2007;2:174–9. [16] Robertson JV, Siwakoti A, West-Mays JA. Altered expression of transforming growth factor beta 1 and matrix metalloproteinase-9 results in elevated intraocular pressure in mice. Mol Vis 2013;19:684–95. [17] Fountoulakis N, Labiris G, Aristeidou A, Katsanos A, Tentes I, Kortsaris A, et al. Tissue inhibitor of metalloproteinase 4 in aqueous humor of patients with primary open angle glaucoma, pseudoexfoliation syndrome and pseudoexfoliative glaucoma and its role in proteolysis imbalance. BMC Ophthalmol 2013; 8:13–69. [18] De Groef L, Van Hove I, Dekeyster E, Stalmans I, Moons L. MMPs in the trabecular meshwork: promising targets for future glaucoma therapies? Investig Ophthalmol Vis Sci 2013;54:7756–63. [19] Seo JH, Park KH, Kim YJ, Yoo YC, Kang SH, Kim DM. Differences in the histopathology and matrix metalloproteinase expression in Tenon’s tissue of primary open-angle glaucoma and primary angle-closure glaucoma. Korean J Ophthalmol 2008;22:37–42. [20] Mossbo¨ck G, Weger M, Faschinger C, Zimmermann C, Schmut O, Renner W, et al. Role of functional single nucleotide polymorphisms of MMP1, MMP2, and MMP9 in open angle glaucomas. Mol Vis 2010;16:1764–70. [21] Agapova OA, Ricard CS, Salvador-Silva M, Hernandez MR. Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human optic nerve head astrocytes. Glia 2001;33:205–16. [22] Wang IJ, Chiang TH, Shih YF, Lu SC, Lin LL, Shieh JW, et al. The association of single nucleotide polymorphisms in the MMP-9 genes with susceptibility to acute primary angle closure glaucoma in Taiwanese patients. Mol Vis 2006; 12:1223–32. [23] Zalewska R, Reszec´ J, Mariak Z, Sulkowski S, Proniewska-Skretek E. Bcl-2 and Bax protein expression in human optic nerve axons in the eyeballs after severe trauma and in the eyes with absolute glaucoma. Rocz Akad Med Bialymst 2004;49:22–4. [24] Nga AD, Yap SL, Samsudin A, Abdul-Rahman PS, Hashim OH, Mimiwati Z. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in the aqueous humour of patients with primary angle closure glaucoma – a quantitative study. BMC Ophthalmol 2014;24:14–33. [25] Kara S, Yildirim N, Ozer A, Colak O, Sahin A. Matrix metalloproteinase-2, tissue inhibitor of matrix metalloproteinase-2, and transforming growth factor beta 1 in the aqueous humor and serum of patients with pseudoexfoliation syndrome. Clin Ophthalmol 2014;29:305–9. [26] Ghanem AA, Arafa LF, El-Baz A. Connective tissue growth factor and tissue inhibitor of matrix metalloproteinase-2 in patients with exfoliative glaucoma. Curr Eye Res 2011;36:540–5. [27] Ergorul C, Ray A, Huang W, Wang DY, Ben Y, Cantuti-Castelvetri I, et al. Hypoxia inducible factor-1a (HIF-1a) and some HIF-1 target genes are elevated in experimental glaucoma. J Mol Neurosci 2010;42:183–91. [28] Tezel G, Yang X. Caspase-independent component of retinal ganglion cell death, in vitro. Invest Ophthalmol Vis Sci 2004;45:4049–59.
Contents lists available at ScienceDirect
Advances in Medical Sciences journal homepage: www.elsevier.com/locate/advms
Original Research Article
Metalloproteinase 9 and TIMP-1 expression in retina and optic nerve in absolute angle closure glaucoma Renata Zalewska a, Joanna Reszec´ b,*, Wojciech Kisielewski b, Zofia Mariak a a b
Department of Ophthalmology, Medical University of Bialystok, Bialystok, Poland Department of Medical Pathomorphology, Medical University of Bialystok, Bialystok, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 November 2014 Accepted 24 July 2015 Available online 9 August 2015
Purpose: Glaucoma is one of the most important reason causes of the blindness, associated with retinal ganglion cells (RGC) death. This process is not fully understood, however apoptosis due to hypoxia is one of the most important processes leading to RGC death. Glaucomatous optic neuropathy is characterized by remodeling of the extracellular matrix due to metalloproteinase activation, which leads to loss of RGC and axons at the optic nerve head. The aim of the study was to evaluate metalloproteinase 9 (MMP-9) and tissue metalloproteinase inhibitor-1 (TIMP-1) expression in the retinal ganglion cells and optic nerve axons in 33 eyes with absolute primary glaucoma. Material/methods: To evaluate MMP-9 and TIMP-1 expression primary polyclonal goat antibodies against MMP-9 and TIMP-1 were used. The control group was composed of 8 cases of eyes enucleated and fixed in the first day after trauma. Results: MMP-9 expression was observed in retinal ganglion cells and in the inner nuclear layer of the retina in all the examined cases. In 28 out of 33 glaucomatous eyes, MMP-9 expression was observed in the proliferating glial cells surrounding the optic nerve axons. TIMP-1 expression was observed in 10 out of 33 glaucomatous eyes, only in retinal ganglion cells. None of the examined injured eyes showed MMP9 and TIMP-1 expression. Conclusions: MMP-9 activation rather than TIMP-1 may by associated with the pathomechanism of retinal ganglion cell and optic nerve damage in absolute glaucoma. ß 2015 Medical University of Bialystok. Published by Elsevier Sp. z o.o. All rights reserved.
Keywords: Glaucoma MMP-9 TIMP-1 Retina Optic nerve
1. Introduction Some eye diseases are associated with damage of the optic nerve axons as well as retinal ganglion cells. Glaucoma is one of the leading causes of blindness. Irreversible vision loss in glaucoma is attributed to retinal ganglion cell (RGC) death connected with apoptosis. RGC death is not fully understood, because the processes of RGC (retinal ganglion cells) apoptosis is heavily complicated [1]. Glaucomatous optic neuropathy is multifactorial in etiology [2]. The mechanism which is responsible for RGC apoptosis is associated with hypoxia, which is most frequently a consequence of increased intraocular pressure [3]. Glaucomatous optic neuropathy is characterized by remodeling of the extracellular matrix and
* Corresponding author at: Department of Medical Pathomorphology, Medical University of Bialystok, Waszyngtona 13, 15-269 Bialystok, Poland. Tel.: +48 857485923; fax: +48 857485990. E-mail address: [email protected] (J. Reszec´).
loss of retinal ganglion cell axons at the optic nerve head level. The integrity and turnover of the extracellular matrix are influenced by many factors, including matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) [4]. Matrix metalloproteinase 9 (MMP-9), also known as 92 kDa type IV collagenase, 92 kD A gelatinase or gelatinase B (GELB), is an enzyme encoded by the MMP9 gene. The enzyme encoded by this gene degrades type IV and V collagens and other extracellular matrix proteins like fibronectin, laminin, and other glycoproteins [5,6]. Among the many subtypes of metalloproteinases, some are secreted constantly, some only in certain circumstances. This is due to the regulation of the expression of tissue inhibitors of metalloproteinases (TIMPs) [6,7]. Recent studies show abnormal activation of matrix metalloproteinases, in particular MMP-9, which triggers an extracellular signaling cascade leading to apoptosis [6,8–11]. Tissue inhibitors of metalloproteinases (TIMP) are the inhibitors of matrix metalloproteinases that participate in controlling the local activities of MMPs in tissues. So far little is known about
http://dx.doi.org/10.1016/j.advms.2015.07.007 1896-1126/ß 2015 Medical University of Bialystok. Published by Elsevier Sp. z o.o. All rights reserved.
R. Zalewska et al. / Advances in Medical Sciences 61 (2016) 6–10
MMP-9 inhibition by TIMP-1. Data show that TIMP activity is responsible for tumor growth reduction, and inhibition of endothelial cell growth induced by basic fibroblast growth factor. TIMP-1 and TIMP-2 have antiapoptotic activity [12]. Remodeling of the optic nerve head in glaucoma involves astrocyte response and changes in the extracellular matrix composition and distribution. The MMP family has been implicated in the cascade of events leading to neuronal apoptosis in the central nervous system. MMP substrates include essentially all extracellular matrix components as well as a wide array of molecules involved in intracellular adhesion, cell–matrix interaction, and cell signaling [13–16]. MMP1, MMP2 and MMP-9 have previously been implicated in the pathogenesis of primary open angle glaucoma and open angle glaucoma secondary to exfoliation syndrome, respectively; matrix metalloproteinase-9 (MMP9) gene was investigated for association with primary angle closure glaucoma [5,17,20,22]. Classic glaucoma treatment focuses on intraocular pressure reduction. However, recent knowledge about the pathogenesis of glaucoma has opened up new therapeutic approaches. Some of the investigators suggest a pivotal role of MMP inhibition in glaucoma treatment. Inhibition of MMP-9 could inhibit the apoptosis of retinal ganglion cells and tissue remodeling [15]. The aim of the study was to evaluate metalloproteinase 9 (MMP-9) and TIMP-1 expression in retina and optic nerve in eyeballs with the primary glaucoma. 2. Materials and methods Thirty-three eyeballs were examined and enucleated in the Department of Ophthalmology of the Medical University of Bialystok over the period 1991–2013. Patients were eligible for study participation if they had primary angle closure glaucoma (PACG) – 33 patients – and met the following criteria: blind eye, corneal edema, dilated unreactive pupil, and IOP (intraocular pressure) >50 mmHg using Goldmann aplanation tonometry. All eyeballs were removed from patients with absolute glaucoma, who suffered from severe ophthalmalgia. The study was approved by the Bioethical Committee in Medical University of Bialystok and was performed in accordance with ethical standards laid down in the 1964 Declaration of Helsinki. All patients signed an agreement form before their inclusion in the study. Thirty-three eyeballs were removed from patients with absolute glaucoma (blind eyes), who suffered from severe ophthalmalgia due to exceptionally high intraocular pressure. All of the cases were angle closure glaucoma. After enucleation, the eyeballs were fixed in a 10% buffered formalin solution on the same day of enucleation, embedded in paraffin at 56 8C, then cut into 5 mm slices and stained with hematoxilin and eosin (H+E). Following deparaffinization, endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 30 min. The sections were then incubated with goat polyclonal antibody for MMP-9 (MMP-9 Antibody (C-20): sc6840, Santa Cruz Biotechnology, Santa Cruz, CA) in 1:100 dilution, and with TIMP-1 antibody (TIMP-1 Antibody (C-20): sc-6832 Santa Cruz Biotechnology) in 1:50 dilution for a whole night at 4 8C, and labeled EnVision (DAKO) enzyme reagent and diaminobenzidine (DAB) chromogen for 5 min. In each routine of staining, adjacent sections that were incubated without primary antibody were prepared as the negative control. Knowing MMP-9 strong expression in joint degradation in rheumatoid arthritis, synovial specimens were taken as positive controls. For TIMP-1 human prostate tissue showing TIMP-1 cytoplasmic and membrane staining of glandular cells was used as a positive control. As a control group, we chose 8 eyeballs after severe trauma (all were closure injury). All were enucleated 1 day after the trauma, and then fixed in 10% buffered formalin. The sections were taken
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from the optic nerve head (1 section) and retina (3 cross-sections), and stained with H+E. Also, MMP-9 and TIMP-1 expression was evaluated using immunohistochemistry. Two independent pathologists performed the immunohistochemical evaluations of MMP-9 and TIMP-1. The estimation of immunostaining was done under a light microscope in representative fields under magnification of 20. The score for immunohistochemistry was as follows (as shown in Table 1,2): negative ( ) if less than 10% of the examined cells were positive, (+) if 10–50% of cells were positive, and if more than 50% of the examined cells showed staining the evaluation was (++). The obtained results were statistically analyzed using Spearman’s and Pearson’s tests. 3. Results The mean age of the glaucoma group ranged from 54 to 88 years (mean age 69, SD 12.4), with 13 male and 20 female patients. In the control injury group, age ranged from 21–77 (45, SD 17.8) years, with 6 male and 2 female patients. Ten patients from the glaucoma group had hypertension, and six had coronary disease. Two patients from the trauma group had hypertension. There was no correlation between age and sex in both examined groups (p = 0.983). 3.1. MMP-9 expression in glaucomatous eyes MMP-9 expression was observed in the perinuclear area of the examined cells. 28 out of 33 glaucomatous eyes presented MMP-9 expression in the optic nerve. It was mainly observed within the proliferating astrocytes surrounding the optic nerve axons; in 8 cases the expression was very strong and diffused (Fig. 1A and B). The results are summarized in Table 1. All glaucomatous eyes presented MMP-9 expression in the retina. MMP-9 staining was observed both in the inner nuclear layer of the retina (Fig. 1C) and in the layer of the retinal ganglion cells (Fig. 1D). All cases with MMP-9 expression localized in the optic nerve astrocytes also presented MMP-9 expression in retinal ganglion cells and the inner nuclear layer of the retina. We did not observe MMP-9 expression in retinal ganglion cells, in the inner layer, and optic nerve in all eyes after trauma. 3.2. TIMP-1 expression TIMP-1 expression was observed as cytoplasmic staining. TIMP-1 expression was observed only in 10 out of 33 glaucomatous eyeballs with angle closure glaucoma. The expression was not observed in the inner nuclear layer of the retina; in all 10 cases the staining was observed in the retinal ganglion cell layer (Fig. 2A and B). None of the glaucomatous eyeballs showed TIMP-1 expression, neither in the optic nerve axons or proliferating astroglia (Fig. 2C and D). The results are shown in Table 2. None of the injured eyeballs showed TIMP-1 expression, neither in the retina nor the optic nerve. Table 1 MMP-9 expression in the glaucomatous eyes. MMP-9 expression
Negative No %
(+) No %
(++) No %
p value
Optic nerve axons
5 15.1% 0 0.0 0 0.0%
20 60.6% 28 84.8% 28 84.8%
8 24.3% 5 15.2% 5 15.2%
0.736
Retinal ganglion cells Inner nuclear layer of the retina
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R. Zalewska et al. / Advances in Medical Sciences 61 (2016) 6–10
Fig. 1. (A) MMP-9 expression in optic nerve head. Magn. 40. (B) MMP-9 expression observed in the perinuclear area of proliferating astroglial cells in the optic nerve. Magn. 200. (C) MMP-9 expression in the inner layer of the retina and retinal ganglion cells. Magn. 200. (D) MMP-9 expression in the layer of the ganglion cells in the retina (arrow). Magn. 200.
4. Discussion Matrix metalloproteinases (MMPs) are involved in the breakdown of the extracellular matrix in normal physiological processes, such as embryonic development, reproduction, angiogenesis, bone development, wound healing, cell migration, learning and memory, as well as in pathological processes, such as arthritis,
intracerebral hemorrhage, and tumor progression, invasion and metastasis. The balance between metalloproteinases and inhibitors plays an important role in maintaining the homeostasis and integrity of the tissue in normal and pathological conditions. Investigations by several scientists have demonstrated an important role of matrix metalloproteinases in enzymatic degradation in glaucomatous alterations in the retina [9,10,14,18,19].
Fig. 2. (A) TIMP-1 expression in the ganglion cells of the retina. Magn. 400. (B) TIMP-1 expression in few retinal ganglion cells of the glaucomatous retina. Magn. 600. (C) Lack of TIMP-1 expression in retina. Magn. 200. (D) Lack of TIMP-1 expression in optic nerve. Evident astroglial cell proliferation between optic nerve axons. Magn. 40. Note: Immunohistochemistry, EnVision (brown staining of the expressed protein).
R. Zalewska et al. / Advances in Medical Sciences 61 (2016) 6–10 Table 2 TIMP-1 expression in glaucomatous eyes. TIMP-1 expression
Negative No %
(+) No %
(++) No %
p value
Optic nerve axons
33 100.0% 23 69.7% 33 100.0%
0 0.0% 10 30.3% 0 0.0%
0 0.0% 0 0.0% 0 0.0%
0.001
Retinal ganglion cells Inner nuclear layer of the retina
Recent studies present the theory of extracellular matrix remodeling in which matrix metalloproteinases play a crucial role in glaucomatous retinal ganglion cell damage. It was also observed that elevated intraocular pressure leads to the proliferation of optic nerve astrocytes. MMP expression is upregulated in activated astrocytes causing matrix remodeling. The role of MMPs in glaucomatous alterations in retinal cells has been shown in studies presenting the relationship between MMP expression and retinal ganglion cell damage [6,8,10,11,15]. Pathological intense apoptosis of retinal ganglion cells is a consequence of glaucomatous intraocular pressure and results in loss of vision. This type of apoptosis of the retinal ganglion cells is also related to laminin degradation from the underlying inner membrane and increase in MMP activity [9]. Our recent studies showed a significant correlation between proapoptotic proteins, Bak and Bax, and retinal ganglion cell damage [23]. We also observed Fas receptor, caspase-3 and HIF-1 overexpression in retinal ganglion cells and optic nerve axons in absolute glaucoma [2,3]. In the present study, we have demonstrated a significant overexpression of MMP-9 in glaucomatous RGC, internal nuclear layer of the retina, and glial cells surrounding optic nerve axons, which may support the theory that MMPs may activate pathological apoptosis in absolute glaucoma due to the activation of proapoptotic proteins and cell death receptors. Altogether, this may suggest that apoptosis, hypoxia and damage of the extracellular matrix via MMP-9 may contribute to retinal cell and optic nerve axon damage in absolute glaucoma. Recent studies concerning MMP expression in glaucomatous eyes are contradictory. Agapova et al. presented the study of MMP expression in the optic nerve of normal monkey eyes and eyes with experimental glaucoma or optic nerve transection [21]. They observed reactive astrocytes expressed MMP-1 at a higher level than in controls. Immunoreactivity for MMP-2 was present in the optic nerve and retina in all normal controls. Also retinal ganglion cells and their axons in the nerve fiber layer were MMP-2 positive; in the optic nerve head, MMP-2 immunoreactivity was strongly associated with axons in the nerve bundles and few astrocytes in the cribriform plates. However, opposite to our results, no MMP-9 immunoreactivity was localized in the optic nerve head of the control eyes, eyes with glaucoma, or optic nerve transection. A study presented by Seo et al. showed low MMP-9 expression in glaucoma and it was not significantly different from that of the control group [19]. However, Manabe et al. presented upregulation of MMP-9 in retinal neurons suggesting that an extracellular proteolysis pathway in the retina contributes to retinal ganglion cell death via MMP-9 activation [11]. Similarly to our studies, Mali et al. observed MMP-9 overexpression in the retina and its association with increased proapoptotic protein Bax expression in ganglion cells and the inner nuclear layer [10]. Chintala et al. observed that MMP-9 deficiency protects against pathological changes in the retina after optic nerve ligation. They found that increased MMP-9 activity is associated with laminin degradation, which results in retinal ganglion cell apoptosis [9]. In our study, MMP-9 expression was significant both in retinal ganglion cells as well as the inner layer of the retina. Similarly,
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Groef et al. observed a strong expression of MMP-2 in the adult mouse retina [18]. Also cytoplasmic MMP-3 expression was observed in the retinal ganglion cell layer and inner nuclear layer. MMP-9 showed a strong expression in the inner limiting membrane, a subset of cells in the retinal ganglion cells and in both the inner and the outer nuclear layer suggesting that MMP-3 and MMP-9 together may be involved in the process underlying retinal ganglion cell death and glial reactivity in the retina. Also Majka et al. presented a significant association between MMP-9 expression, laminin degradation, retinal ganglion cell apoptosis, and intraocular pressure exposure in glaucoma [9]. Liu et al. showed strong MMP-9 expression in ganglion cells suggesting that MMP-9 inhibitors may be able to protect the retina against ischemia [14]. Awadalla et al. showed an influence of MMP-9 expression in the pathogenesis of blind [5]. Wang et al. found that MMP-9 is associated with acute PACG [22]. Robertson et al. observed that MMP-9 plays an important role in maintaining intraocular pressure, and due to extracellular matrix remodeling, leads to ocular hypertension and glaucoma [16]. In the present study, we also examined TIMP-1 expression in glucomatous eyes. However, only in 10 out of 33 cases presented immunopositive reaction for TIMP-1, and only in the retinal ganglion cells. The explanation of this phenomenon in difficult, however basing on data, we may suspect, that it is due to apoptosis of the retinal ganglion cells in glaucoma, which may be associated with TIMP deficiency in retina and optic nerve axons in glaucoma. There is little data concerning the problem of TIMP-1 and MMP-9 expression in glaucoma. In one of them, Agapova et al. observed TIMP-1 and TIMP-2 expression also in retinal ganglion cells in absolute glaucoma. The level of TIMP expression in the nerve bundles of eyes with experimental glaucoma and optic nerve transection was lower than in normal eyes due to loss of axons in both models [21]. Contrary to our study, Guo et al. observed significant MMP-9 expression in retinal ganglion cells, although tissue inhibitor of matrix metalloproteinase (TIMP) was also overexpressed [1]. Some of the studies present matrix metalloproteinase expression in the aqueous humor of patients with glaucoma. Nga et al. presented altered levels of MMPs (MMP-2, MMP-3) and TIMPs (TIMP-1 and TIMP-2) in patients with primary angle closure glaucoma. The imbalance of MMP:TIMP ratios in the aqueous humor were significantly different from patients with non-glaucomatous eyes [24]. Kara et al. observed no statistically significant difference among mean MMP-2 and TIMP-2 levels in both aqueous humor and serum samples in patients with pseudoexfoliation syndrome and the control group [25]. However, Fountoulakis et al. observed TIMP-4 elevation in glaucomatous eyes, and they suggested that dysregulation of extracellular matrix homeostasis is associated with primary open angle glaucoma, pseudoexfolation syndrome, and pseudoexfoliative glaucoma [17]. Also Ghanem et al. observed increased expression of TIMPs (TIMP-2) in patients with exfoliative glaucoma [26]. 5. Conclusions Providing the theory about the hypoxia [27] and apoptosis [28] in retinal ganglion cells we decided to study the expression of matrix metalloproteinases and inhibitors in primary glaucoma. Our study presented strong MMP-9 expression in glaucomatous eyes, mainly in the inner nuclear layer, retinal ganglion cells, and proliferating astroglia in optic nerves. TIMP-1 expression was observed only in a few cases and only in the ganglion cells of the retina, due to a significant loss of neurons in glaucomatous eyes. The results show that only MMP-9, rather than TIMP-1 activation, underlies the pathomechanism of the retinal ganglion cell and optic nerve damage in absolute glaucoma.
R. Zalewska et al. / Advances in Medical Sciences 61 (2016) 6–10
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The authors declare no conflict of interests. We confirm that all authors have read and approved the submission of the manuscript, the manuscript has not been published and is not being considered for publication elsewhere, in whole or in part, in any language. We also declare no financial relationships with any industry (through investments, employment, consultancies, stock ownership, honoraria). Conflict of interests None declared. Financial disclosure None declared. References [1] Guo MS, Wu YY, Liang ZB. Hyaluronic acid increases MMP-2 and MMP-9 expressions in cultured trabecular meshwork cells from patients with primary open-angle glaucoma. Mol Vis 2012;18:1175–81. [2] Zalewska R, Zalewski B, Reszec J, Mariak Z, Zimnoch L, Proniewska-Skretek E. The expressions of Fas and caspase-3 in human glaucomatous optic nerve axons. Med Sci Monit 2008;14:274–8. [3] Reszec´ J, Zalewska R, Bernaczyk P, Chyczewski L. HIF-1 expression in retinal ganglion cells and optic nerve axons in glaucoma. Folia Histochem Cytobiol 2012;50:456–9. [4] Yan X, Tezel G, Wax MB, Edward DP. Matrix metalloproteinases and tumor necrosis factor alpha in glaucomatous optic nerve head. Arch Ophthalmol 2003;118:666–73. [5] Awadalla MS, Burdon KP, Kuot A, Hewitt AW, Craig JE. Matrix metalloproteinase-9 genetic variation and primary angle closure glaucoma in a Caucasian population. Mol Vis 2011;17:1420–4. [6] Micheal S, Yousaf S, Khan MI, Akhtar F, Islam F, Khan WA, et al. Polymorphisms in matrix metalloproteinase MMP1 and MMP9 are associated with primary open-angle and angle closure glaucoma in a Pakistani population. Mol Vis 2013;19:441–7. [7] Wierzbowska J, Robaszkiewicz J, Figurska M, Figurska M, Stankiewicz A. Future possibilities in glaucoma therapy. Med Sci Monit 2010;16:252–9. [8] Chintala SK, Zhang X, Austin JS, Fini ME. Deficiency in matrix metalloproteinase gelatinase B (MMP-9) protects against retinal ganglion cell death after optic nerve ligation. J Biol Chem 2002;277:47461–68. [9] Majka S, McGuire PG, Das A. Regulation of matrix metalloproteinase expression by tumor necrosis factor in a murine model of retinal neovascularization. Investig Ophthalmol Vis Sci 2002;43:260–6. [10] Mali RS, Cheng M, Chintala SK. Intravitreous injection of membrane depolarization agent causes retinal degeneration via Matrix Metalloproteinase-9. Investig Ophthal Vis Sci 2005;46:2125–32. [11] Manabe S, Gu Z, Lipton SA. Activation of matrix metalloproteinase-9 via neuronal nitric oxide synthase contributes to NMDA-induced retinal ganglion cell death. Investig Ophthal Vis Sci 2005;44:4747–53.
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