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Arch Ophthalmol. 2002;120:833-835.

Marfan syndrome is an autosomal dominant disorder with pleiotropic manifestations that involve the ocular, cardiovascular, and skeletal systems. Marfan syndrome remains primarily a clinical diagnosis with a frequency of 2 to 3 individuals per 10 000. Patients with this disorder may have a variety of ocular complaints, most commonly, subluxation of the lens, which occurs in more than 60% of patients.¹ Several studies have identified the FBN1 fibrillin gene located on chromosome 15 as defective in this syndrome.²

Matrix metalloproteinases (MMPs) are proteolytic enzymes important in physiological and pathological remodeling, the activity of which is stringently controlled by a family of natural antagonists, the tissue inhibitors of MMPs (TIMPs). Both MMPs and TIMPs are present in the aqueous humor in normal and inflamed eyes,³ resulting in their interaction with the lens zonules.

We describe a patient with Marfan syndrome lens subluxation associated with positive MMP expression and no TIMP immunoreactivity within the lens zonule. To our knowledge, this is the first report of MMP staining associated with lens zonules in a patient with Marfan syndrome. Understanding the role these proteases play may lead to the development of novel therapies to reduce the progressive nature of Marfan syndrome lens subluxation.

Report of a Case
A 43-year-old woman diagnosed with Marfan syndrome in 1980 was referred to the Ophthalmology Clinic at Prince of Wales Hospital (Sydney, Australia) in 1999. The patient described deterioration of her vision occurring throughout a 9-month period. Her medical history included mitral valvuloplasty in 1997, paroxysmal atrial fibrillation, scoliosis, and hypertension controlled with 50 mg of atenolol administered daily. The patient's grandfather, cousin, and mother also had Marfan syndrome. Her mother developed bilateral lens subluxation and glaucoma. A systemic examination revealed arachnodactyly and a high arched palate. Ophthalmologic examination revealed a best-corrected visual acuity of 20/220 OD and 20/120 OS. The right eye had an intraocular pressure of 19 mm Hg; and the left eye, 18 mm Hg. Goldmann visual fields were normal. Slitlamp examination confirmed bilateral superonasal lens subluxation that was worse in the right eye than in the left (Figure 1). The fundus was normal, with cup-disc ratios of 0.2 for each eye. The ocular examination was otherwise normal. Because of the advanced zonular dialysis, bilateral intracapsular lens extractions were performed with a cryoprobe and without -chymotrypsin. The lenses were so mobile that they were expressed from the eye by injecting viscoelastic inferiorly and posteriorly (in front of the vitreous face), and scleral fixated posterior chamber intraocular lenses were sutured into position with standard techniques.

View larger version (75K): [in this window] [in a new window] Immunohistochemistry of normal and Marfan syndrome lenses revealed positive immunoreactivity for matrix metalloproteinases (MMP)–1, MMP-3, and MMP-9 (A, C, and D) on Marfan lens zonules, with no tissue inhibitors of MMPs (TIMP)–1, TIMP-2, or TIMP-3 (L-N) immunoreactivity. The arrows indicate the zonule. When the primary antibody was omitted, no immunoreactivity could be detected in the Marfan lens (B) or the normal lens (G). In contrast, no MMP-1 (E) or MMP-3 (F) staining was observed in the zonule of normal lenses, but TIMP-1, TIMP-2, and TIMP-3 (H-J) was detected (original magnification x125). A fibrillin monoclonal antibody was included to identify the lens zonule in the Marfan lens (K) (original magnification x250).

Both crystalline lenses from the patient were immediately formalin fixed. Lenses from corneal donor postmortem eyes (n = 8) were enucleated 4 to 8 hours after the time of death and were also immediately fixed in formalin. The 10 lenses were paraffin embedded, and 4-µm sections were placed on slides coated with 3-amino propyltriethoxy triethoxysilane (TES) for immunohistochemical analysis using a panel of monoclonal antibodies directed against MMP-1, MMP-2, MMP-3, MMP-9, and TIMP-1, TIMP-2, and TIMP-3. Matrix metalloproteinases seem to be stable for as long as 24 hours in harvested ocular tissue.⁴

Sections were deparaffinized in xylene, rehydrated through decreasing graded ethanol, followed by two 5-minute washes in 0.05mM of Tris-buffered saline (10x stock Tris-buffered saline contains 0.25M Tris base, 0.25M Tris-hydrochloride, and 8.5% sodium chloride, with a pH of 7.6). Antigen retrieval method was not necessary. Endogenous peroxidase was quenched with 3% hydrogen peroxide/methanol for 5 minutes, and then washed in Tris-buffered saline. The sections were incubated with a 1:5 dilution of preimmune serum from the secondary host species. Tissue sections were incubated with 1:100 goat primary polyclonal antifibrillin antibody (Santa Cruz Biotechnology, Santa Cruz, Calif) and 1:100 mouse primary monoclonal anti-MMP–1, anti-MMP–2, anti-MMP–3, anti-MMP–9 (ICN Pharmaceuticals, Costa Mesa, Calif), 1:100 TIMP-1, TIMP-2 (ICN), and 1:100 TIMP-3 (Calbiochem, San Diego, Calif) overnight at 4°C. The sections were then washed in 0.05M Tris-buffered saline (pH, 7.6) before the addition of a biotinylated rabbit antigoat secondary antibody (for fibrillin) and biotinylated goat antimouse secondary antibody (for MMPs and TIMPs). The antibodies directed against human antigens display no cross-reactivity and are all IG₁ subclass antibodies. Sections were again washed, incubated for 1 hour with horseradish peroxidase–conjugated streptavidin (Dako, Carpinteria, Calif), and the immunoreactivity was revealed by adding 3-amino-9-ethylcarbazole (Sigma, Sydney, Australia). Control reactions were included, incubating sections with an isotype antibody and omitting the primary antibody or adding preimmune serum. Sections were counterstained with hematoxylin, viewed by light microscopy, and photographed with Spot Version 2.2 for Windows (Diagnostic Instruments Inc, Sterling Heights, Mich).

Macroscopically, both the Marfan lenses appeared normal. Microscopically, both Marfan lens capsules appeared thickened, with the germinative zone smaller than that of the normal lens. Immunohistochemical analysis revealed specific localization of MMP-1 (Figure 1, A), MMP-3 ( Figure 1, C), and MMP-9 (Figure 1, D) in the Marfan lens zonules, with relatively little or no MMP-2 (data not shown) and no TIMP-1, TIMP-2, or TIMP-3 immunoreactivity ( Figure 1, L-N). In contrast, no MMP-1 or MMP-3 staining was observed in the zonules of normal lenses (Figure 1, E and F) but TIMP-1, TIMP-2, and TIMP-3 were detected in the zonules of the normal lenses (Figure 1, H-J). On all lens sections, more than 1 zonular site contained MMP or TIMP activity. A fibrillin monoclonal antibody was included to identify the lens zonule in the Marfan lens (Figure 1, K). Sections incubated with isotype control antibodies demonstrated no reactivity in either the Marfan (Figure 1, B) or normal lenses (Figure 1, G).

Comment
This is the first case report, to our knowledge, describing zonule-associated staining of MMPs in Marfan syndrome lens subluxation. We hypothesize that the product of the defective FBN1 gene in Marfan syndrome is more prone to degradation by MMPs as compared with normal fibrillin. It is also possible that dysregulation of MMPs and TIMPs results in the progressive destruction of lens zonules and subsequent lens subluxation.

The lens zonule consists of a series of fibers composed of microfibrils that are 8 to 12 nm in diameter. The fibrils consist largely of a cysteine-rich microfibrillar component of the fibrillin elastin system. In other tissues, fibrillin provides a template for elastin deposition.⁵ Genetic linkage between the fibrillin gene and the Marfan phenotype has been established, and the gene mapped to the same chromosomal position as the disease locus.⁶ Understanding of the functions of the fibrillin-containing microfibrils is still incomplete, and correspondingly, no comprehensive theory of the pathogenesis of Marfan syndrome has emerged to date.

Both fibrillin molecules and fibrillin-rich microfibrils are susceptible to degradation by serine proteases, and amino acid substitutions (as found in Marfan syndrome) change the fragmentation patterns.⁷ Fibrillin degradation products generated by MMP activity provide conclusive evidence that these enzymes cause specific changes to assembled microfibrils.⁷ As most of the mutations in fibrillin-1 are found within epidermal growth factor–like motifs and are predicted to disrupt calcium binding, it has been suggested that these mutations render fibrillin-1 more susceptible to proteolytic cleavage.⁸ Previous investigators have demonstrated structural modifications in fibrillin-rich microfibrils during aging of human ciliary zonules.⁹ These age-related changes may account for the increased incidence of ocular disease observed in older patients with Marfan syndrome.

If the proposed hypothesis regarding the role of MMPs in Marfan-associated lens subluxation is correct, then the development of matrix metalloproteinase inhibitors may be of potential therapeutic value in the treatment of progressive lens subluxation and other complications of Marfan syndrome.¹⁰

AUTHOR INFORMATION
The authors have no proprietary interests in any of the products or companies mentioned in this article.

Nitin H. Sachdev, MBChB; Nick Di Girolamo, PhD; Peter J. McCluskey, FRACO, FRACS; Angela V. Jennings, MBBS; Roger McGuinness, FRACO, FRACS; Denis Wakefield, FRCPA; Minas T. Coroneo, FRACO, FRACS, MD
Sydney, Australia

Corresponding author: Minas T. Coroneo, FRACO, FRACS, MD, Department of Ophthalmology, Prince of Wales Hospital, University of New South Wales, Randwick, Sydney, Australia (e-mail: m.coroneo{at}unsw.edu.au).

REFERENCES
1. Maumenee IH. The eye in the Marfan syndrome. Trans Am Ophthalmol Soc. 1981;79:684-733. PUBMED 2. Sarfarazi M, Tsipouras P, Del Mastro R, et al. A linkage map of 10 loci flanking the Marfan syndrome locus on 15q: results of an International Consortium study. J Med Genet. 1992;29:75-80. FREE FULL TEXT 3. Di Girolamo N, Verma MJ, McCluskey PJ, et al. Increased matrix metalloproteinases in the aqueous humor of patients and experimental animals with uveitis. Curr Eye Res. 1996;15:1060-1068. ISI | PUBMED 4. Kamei M, Hollyfield JG. TIMP-3 in Bruch's membrane: changes during aging and in age-related macular degeneration. Invest Ophthalmol Vis Sci. 1999;40:2367-2375. FREE FULL TEXT 5. Robinson PN, Godfrey M. The molecular genetics of Marfan syndrome and related microfibrillopathies. J Med Genet. 2000;37:9-25. FREE FULL TEXT 6. Maslen CL, Glanville RW. The molecular basis of Marfan syndrome. DNA Cell Biol. 1993;12:561-572. ISI | PUBMED 7. Ashworth JL, Murphy G, Rock MJ, et al. Fibrillin degradation by matrix metalloproteinases: implications for connective tissue remodelling. Biochem J. 1999;340(pt 1):171-181. 8. Reinhardt DP, Ono RN, Sakai LY. Calcium stabilizes fibrillin-1 against proteolytic degradation. J Biol Chem. 1997;272:1231-1236. FREE FULL TEXT 9. Hanssen E, Franc S, Garrone R. Fibrillin-rich microfibrils: structural modifications during ageing in normal human zonule. J Submicrosc Cytol Pathol. 1998;30:365-369. ISI | PUBMED 10. Boyle JR, McDermott E, Crowther M, et al. Doxycycline inhibits elastin degradation and reduces metalloproteinase activity in a model of aneurysmal disease. J Vasc Surg. 1998;27:354-361. FULL TEXT | ISI | PUBMED

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