Matrix metalloproteinase 9 and transglutaminase 2 expression at the ocular surface in patients with different forms of dry eye disease.

OBJECTIVE: To evaluate the expression of matrix metalloproteinase 9 (MMP9) and transglutaminase 2 (TG2) in different forms of dry eye. DESIGN: Case control study. PARTICIPANTS: Seventy-five female subjects divided into 3 groups: group 1, 15 healthy controls; group 2, 30 subjects with Sjögren syndrome (SS); and group 3, 30 subjects with Meibomian gland dysfunction (MGD). METHODS: A clinical assessment was carried out and impression cytologic specimens were processed for immunoperoxidase staining for MMP9 and TG2 and real-time polymerase chain reaction analyses were carried out for MMP9, TG2, interleukin-6, interferon-γ, B-cell lymphoma 2, and caspase 3. To study MMP9 and TG2 expression after anti-inflammatory treatment, patients were divided into 2 subgroups, one treated with saline and the other treated with saline plus topical corticosteroid eye drops (0.5% loteprednol etabonate) 4 times daily for 15 days. For statistical analysis, Student t test, Mann-Whitney U test, and Spearman's correlation coefficient were used as appropriate. MAIN OUTCOME MEASURES: Conjunctival expression of MMP9 and TG2. RESULTS: MMP9 and TG2 expression were higher in both patient groups than in controls (P < 0.0001). Group 2 patients showed higher expression than group 3 (P < 0.0001). The Spearman's correlation coefficient showed in group 2 a positive correlation between MMP9 and TG2 expression (ρ = 0.437; P = 0.01), but no correlation in group 3 (ρ = 0.143; P = 0.45). Corticosteroid treatment significantly reduced MMP9 and TG2 expression in both groups, ameliorating symptoms and signs. A much higher percentage reduction was observed in SS. CONCLUSIONS: The pathogenic mechanisms of the 2 forms of dry eye give an account for the different MMP9 and TG2 expressions in the 2 groups of patients. The higher expression in SS is determined by the direct autoimmune insult to the ocular surface epithelia, whereas in MGD patients, with an epithelial damage due to an unbalanced tear secretion, the molecules expression is significantly lower, although higher than in controls. The corticosteroid treatment induced a reduction of both molecules, although higher in SS than in MGD, because of its direct inhibitory effect on inflammation.

Blepharochalasis: possibly associated with matrix metalloproteinases.

Blepharochalasis is a rare disorder characterized by recurrent episodic swelling of eyelids, eventually leading to atrophy of skin. Although immunological mechanisms may be involved in the degradation of elastic fibers, the pathogenesis is not well characterized. We report a 10-year-old Japanese boy with an 11-month history of the swelling of bilateral upper eyelids with atrophic skin. Attacks of non-painful swelling in eyelids with erythema have recurred several times a month and lasted for 2-3 days. Histological examination revealed perivascular and interstitial infiltration of lymphocytes in the dermis. Elastica Van Gieson staining showed a marked decrease of elastic fibers throughout the dermis. Results of direct and indirect immunofluorescence analyses were negative. Staining of matrix metalloproteinase (MMP)-3 and MMP-9 was observed in and around infiltrating cells in the dermis, suggesting that MMP-3 and MMP-9 may play, in part, roles in the development of blepharochalasis, and that inhibitor of MMP may have a possibility of therapeutic application.

[Chronic inflammatory and dystrophic diseases of the eye anterior segment and their association with genetic polymorphisms in workers of metallurgic industry].

The ophthalmologic investigation of workers of the two metallurgical enterprises has shown that 1045 persons (55%) from 1911 observed workers suffer chronic diseases of a forward piece of eyes. Chronic inflammatory diseases (blepharitis, conjunctivitis and blepharoconjunctivitis) are found at 28,9% of them, and dystrophic diseases (pinguecula/pterygium)--at 25,8%. Among metallurgists (1801 persons) ophthalmopathy was found in 2, 2 times more often than at persons in control group (110 observed engineers and managers). Two polymorphisms of cytochrome P450 1A1 (CYP1A1) and P-450 2E1 (CYP2E1) genes were defined in 91 workers, by the method of allele-specific polymerase chain reaction. It is revealed that CYP1A1 Ile462Val polymorphism associates with pinguecula/pterygium, raising risk of their development almost in 3 times, unlike CYP 2E1 -1293G/C polymorphism. Development of chronic inflammatory diseases is not connected with tested polymorphisms.

Doxycycline for treatment of blepharochalasis via inhibition of matrix metalloproteinases.

Two cases of blepharochalasis were studied. In Case 1, a 16-year-old male presented with the history and clinical features of blepharochalasis. A positive tissue biopsy for matrix metalloproteinases suggested a benefit from treatment with doxycycline. The patient remained disease-free for 18 months following commencement of doxycycline. In Case 2, a 21-year-old male with a 5-month history of swelling affecting his upper eyelids was diagnosed with blepharochalasis. He received an 8-month course of doxycycline and remained symptom-free on review 2 months after treatment cessation. While the pathogenesis of blepharochalasis has remained uncertain, an immune mechanism has been suspected. The presence of matrix metalloproteinases in the tissue biopsy of our first patient supports such an immune mechanism. As doxycycline inhibits matrix metalloproteinase activity, it may provide an effective and well-tolerated treatment alternative for cases of blepharochalasis for which surgery has previously been the only treatment option.

Tear fluid levels of MMP-8 are elevated in ocular rosacea--treatment effect of oral doxycycline.

BACKGROUND: Ocular rosacea (OcR) is a chronic inflammatory disease especially affecting lid margins. Previous studies have shown that it is accompanied by increased levels and activation of tear fluid gelatinases. Matrix metalloproteinase 8 (MMP-8; collagenase 2) levels and activation are commonly elevated in many inflammatory conditions. Therefore we studied here whether MMP-8 concentration and activation in tear fluid are increased also in OcR, and if an oral doxycycline regimen could rectify the situation. METHODS: Tear fluid samples were collected from 22 OcR patients and 22 healthy controls. The OcR patients were then treated with an oral doxycycline regimen for 8 weeks and tear fluid samples collected again after 4 and 8 weeks. Conjunctival brush cytology and patients' subjective symptoms were scored. MMP-8 concentrations in the tear fluid were assessed by immunofluorometric assay and the molecular forms and isoenzyme expression of MMP-8 were studied by Western immunoblotting. RESULTS: The mean MMP-8 concentration was statistically significantly higher in OcR (156.8+/-207.4 mug/ml) than in the normal subjects (53.5+/-66.7 mug/ml) (P=0.036), but decreased to 79.2+/-141.6 mug/l and 53.6+/-75.2 mug/l after 4 and 8 weeks doxycycline treatment, respectively. There was a statistically significant difference between the untreated OcR and the MMP-8 results after 4 or 8 weeks of oral doxycycline (P=0.041 and 0.069, respectively) and the OcR patients experienced statistically significant relief of their subjective symptoms (P=0.0001) after the doxycycline regimen. Both the normal and OcR tear fluid contained the larger, 60-80 kDa highly- glycosylated polymorphonuclear leukocyte-type MMP-8 isoform in Western immunoblotting, but not the 45-55 kDa less glycosylated mesenchymal-type isoform. MMP-8 activation was in practice present only in the OcR samples, and was inhibited by oral doxycycline. CONCLUSIONS: MMP-8 concentration and activation degree in tear fluid are increased in OcR, probably reflecting increased inflammatory activity. Doxycycline effectively reduces these pathologically excessive levels and activation of MMP-8, and relieves patients' subjective symptoms.

The Pathogenesis of floppy eyelid syndrome: involvement of matrix metalloproteinases in elastic fiber degradation.

OBJECTIVE: To investigate histopathologic alterations of eyelid biopsy specimens from patients with floppy eyelid syndrome (FES) with special regard to elastic fiber content and ultrastructure as well as to the expression of elastin-degrading enzymes to elucidate the pathogenesis of this disorder. DESIGN: Retrospective, interventional case series. PARTICIPANTS AND CONTROLS: Eleven consecutive patients with FES and 10 age-matched control patients with basal cell carcinoma of the eyelid. METHODS: Horizontal pentagonal eyelid resections of 16 upper lids were performed in 11 patients with FES. Full-thickness eyelid biopsy specimens from study and control patients were examined by light and transmission electron microscopy, semiquantitative morphometry, and immunohistochemistry using antibodies against matrix metalloproteinase (MMP)-2, MMP-7, MMP-9, and MMP-12 and neutrophil elastase. RESULTS: All patients treated with surgical horizontal eyelid shortening were asymptomatic at follow-up. Histopathologic analysis of the surgical specimens showed, apart from unspecific signs of chronic inflammation, a significant decrease in the amount of elastin within the tarsal plate and eyelid skin as compared with controls. Residual elastic fibers revealed an abnormal ultrastructure with a diminished elastin core. Immunohistochemistry demonstrated an increased immunoreactivity for elastolytic proteases, particularly MMP-7 and MMP-9, in areas of elastin depletion in FES specimens as compared with controls. CONCLUSIONS: The findings indicate that upregulation of elastolytic enzymes, most probably induced by repeated mechanical stress, participates in elastic fiber degradation and subsequent tarsal laxity and eyelash ptosis in FES.

Regulation of MMP-9 activity in human tear fluid and corneal epithelial culture supernatant.

PURPOSE: To evaluate human corneal epithelial culture supernatant and tear fluid for the presence of activators and inhibitors of matrix metalloproteinase (MMP)-9, MMP-3, and tissue inhibitor of metalloproteinase (TIMP)-1, respectively, and to evaluate the effect of MMP-3 on the activation of MMP-9 in these specimens. METHODS: Unstimulated tear fluid was collected from patients with ocular rosacea and normal control subjects. Levels of MMP-9, MMP-3, and TIMP-1 were determined by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Supernatants from primary human corneal epithelial cultures and human tear fluid were incubated with MMP-3. Cultured epithelial cells and their supernatants were also treated with doxycycline before MMP-3 was added. Gelatin zymography was used to identify activated 82-kDa MMP-9. MMP-9 activity was assessed with a commercial MMP-9 activity assay system. RESULTS: MMP-9 and TIMP-1 were detected at significantly higher concentrations in rosacea-affected than in normal tear fluids. MMP-3 was detected exclusively in the tear fluid of patients with ocular rosacea who had corneal epithelial disease. Treatment of the supernatant and tear fluid with MMP-3 resulted in two bands with molecular weights of 92 kDa and 82 kDa, representing pro-MMP-9 and activated MMP-9, respectively. Doxycycline added to the conditioned media did not affect activation of MMP-9 by MMP-3. However, 24-hour treatment of corneal epithelial cultures with doxycycline resulted in a lower concentration and activity of MMP-9 in their supernatants. CONCLUSIONS: MMP-9 and TIMP-1 are produced by the human corneal epithelium and are present in tear fluid. MMP-3 alone is sufficient to activate MMP-9 on the ocular surface. Doxycycline does not directly inhibit this activation by MMP-3, but it decreases MMP-9 activity when added to corneal epithelial cultures.

Angiotensin-converting enzyme in sarcoid and chalazion granulomas of the conjunctiva.

Angiotensin-converting enzyme (ACE) was studied immunohistochemically in conjunctival biopsies from 6 patients with systemic sarcoidosis, 4 patients with posterior non-sarcoid uveitis and in specimens from 4 patients with chalazion of the eyelid. Specimens with sarcoid granulomas showed intense ACE-positive immunoreactivity in epitheloid cells of the granuloma, whereas chalazion granulomas did not contain ACE-immunoreactivity. There was no difference in staining patterns between specimens without granulomas. Thus immunohistochemical staining for ACE may be of help in differentiating conjunctival granulomatous tissue of a chalazion from sarcoid granuloma.

Tear lysozyme measurements in chronic blepharitis.

Tear lysozyme concentrations were measured on 47 patients with chronic blepharitis and 22 normal control patients. The patients consisted of 26 individuals with various types of chronic blepharitis alone and 21 individuals with chronic blepharitis and clinically-diagnosed keratoconjunctivitis sicca (KCS). The mean lysozyme concentration of blepharitis patients without KCS (4070 micrograms/ml) was not significantly different from normals (3760 micrograms/ml). However, mean lysozyme concentration of the blepharitis patients with KCS (2530 micrograms/ml) was significantly lower than normals or blepharitis patients without KCS (p less than 0.01). It was concluded that tear lysozyme deficiency does not play a significant role in the etiology of chronic blepharitis. However, a large percentage of patients with chronic blepharitis were found to have KCS.