Epigallocatechin-3-gallate increases autophagic activity attenuating TGF-ß1-induced transformation of human Tenon's fibroblasts.

We previously found that epigallocatechin-3-gallate (EGCG) could inhibit the myofibroblast transformation of human Tenon's fibroblasts, however, the underlying mechanism remained unclear. We therefore investigated whether the autophagic regulation involved in the anti-fibrotic function of EGCG. The fibroblasts were subjected to transforming growth factor beta-1 (TGF-ß1) induction followed by EGCG treatments. The autophagic flux was examined by transmission electron microscopy and autophagic flux analysis. The levels of autophagy-related proteins (LC3ß and p62) and alpha-smooth muscle actin (α-SMA) were measured by Western blot and immunofluorescence. Results showed that TGF-ß1 partially inhibited the autophagic function of Tenon's fibroblasts. But this inhibition effect was rescued by LY2157299, a TGF-ßR1 selective inhibitor. Compared with the cells treated with TGF-ß1 alone, EGCG treatments increased the amount of autophagosomes and autolysosomes, evaluated the ratio of LC3-II to LC3-I and decreased p62 level. Our results indicated that EGCG could recover the activity of autophagy in the TGF-ß1-treated cells. Moreover, treatments with EGCG significantly decreased the α-SMA expression. Taken together, these findings revealed that autophagic regulation involved in the action of EGCG against TGF-ß1-induced transformation of Tenon's fibroblasts. Through increasing intracellular autophagy, EGCG could be a potential anti-fibrotic reagent for preventing subconjunctival fibrosis after glaucoma filtration surgery.

A histopathological investigation of Tenon's capsule in diabetic eyes.

The aim of this study is to investigate the histopathological features of Tenon's capsule in eyes with diabetic macular oedema and to compare them between diabetic eyes and healthy subjects. The study included 26 eyes with diabetic oedema and 17 healthy eyes as healthy controls. Tenon's capsule biopsy specimens were processed with the routine electron microscopic analysis technique. Type I and III collagen fibres were labelled immunohistochemically to determine the amounts of predominating collagen fibres. Leica Q-Win program was used to calculate the amounts of collagen fibres type I and type III and independent-t test was utilized to compare the obtained results between the groups. Statistical significance was set at p < 0.05. Demographic characteristics of both groups were similar (p > 0.05). Collagen type I and type III immunoreactivity was observed both in the control and the diabetic groups. The Amounts of collagen fibres type I and type III were significantly higher in the diabetic group than in the control group (mean collagen type I area: 13.410 ± 0.99 and mean collagen type III area: 23.692 ± 0.17 in the control group; mean collagen type I area: 25.270 ± 6.48 and mean collagen type III area: 28.192 ± 0.82 in the diabetic group. p = 0.0037 for type I and p = 0.0000 for type III). In light of the findings of this study, it can be assumed that diabetes mellitus may engender increased amounts of collagen in Tenon's capsule. This alteration affecting the success of filtration surgery should be kept in mind especially in diabetic eyes with glaucoma.

[Amyloid-like fibrils forming and fibroblasts destruction in Tenon's capsule in progressive myopia as a result of pigment epithelium derived factor resistance to restricted proteolysis].

We have shown previously the presence of full length (50 kD) and truncated proteolytic form (45 kD) of pigment epithelium derived factor (PEDF) in the eye Tenon's capsule in progressive myopia. The full length PEDF is prevalent in myopia that correlates with breach in collagen fibrils forming. Immunohistochemical analysis of Tenon's capsule with polyclonal antibodies to PEDF revealed PEDF in control group being exclusively inside fibroblasts, whereas in myopia, PEDF was distributed extracellularly as halo around blasted fibroblasts. By means of atomic force microscopy and immunodot analysis with anti amyloid fibrils antibodies the ability was studied of recombinant PEDF fragments to form fibrils. Only full length PEDF was shown to form amyloid like fibril structures, but not the truncated form. Accumulation offibrils results in fibroblasts destruction and might be the cause of changes in biochemical and morphological structure of Tenon's capsule observed in myopia.

Otago glaucoma surgery outcome study: electron microscopy of capsules around Molteno implants.

UNLABELLED: PURPOSE. To report the ultrastructure of cells and extracellular matrix components in Molteno implant capsules examined by scanning and transmission electron microscopy. METHODS: Ultrastructural features including cytology, distribution of apoptotic cells, collagens, basement membranes, elastic fibrils, and glycoproteins were examined by scanning and transmission electron microscopy. Findings were correlated with the clinical features of 31 specimens of glaucomatous eyes treated with Molteno implants 0.3 to 14.9 years previously. RESULTS: Capsules showed two layers: an outer, moderately cellular vascular layer of normal-appearing cells and collagen and an inner, avascular, hypocellular layer of altered cells and collagen. Cells included fibroblasts, myofibroblasts, and tissue histiocytes that showed features indicating metabolic activity, with swelling, vacuolation, and apoptosis, and the formation of numerous membrane-bound vesicles. These features, together with alteration and disintegration of extracellular matrix, increased with time after surgery. CONCLUSION: The results support those in previous light microscopic studies and indicate that the normal life cycle of capsules in both primary and secondary glaucoma include continual outer surface renewal balanced by inner surface degeneration associated with apoptosis and breakdown of tissue matrix components which become more marked over time.

Antifibrotic activity of bevacizumab on human Tenon's fibroblasts in vitro.

PURPOSE: To evaluate the effect of the anti-VEGF-A monoclonal antibody bevacizumab on primary human Tenon's capsule fibroblasts (HTFs) in an in vitro model of wound healing. METHODS: Fibroblasts were cultured in RPMI media, and bevacizumab was administered at a concentration ranging from 0.25 to 12.5 mg/mL. Fibroblast viability and cell death were assessed using the MTT colorimetric assay, lactate dehydrogenase assay, BrdU assay, and live/dead assay. Fibroblast contractility was assessed in floating collagen gels. Morphologic changes were assessed by transmission electron microscopy. Antifibrosis activities were compared with 5-fluorouracil. RESULTS: Bevacizumab induced a significant dose-related reduction of HTF cell number at 12.5 mg/mL at 72 hours (P < 0.05). Under serum-free conditions, bevacizumab induced significant fibroblast cell death at concentrations greater than 7.5 mg/mL (P < 0.05). Bevacizumab caused a moderate inhibition of fibroblast gel contraction from baseline (P < 0.05). Scanning electron microscopy revealed marked vacuolization in bevacizumab-treated fibroblasts. CONCLUSIONS: Bevacizumab disrupted fibroblast proliferation, inhibited collagen gel contraction ability, and induced fibroblast cell death at concentrations greater than 7.5 mg/mL in serum-free conditions. These results demonstrated that bevacizumab inhibited a number of fibrosis activities in culture. These activities may underpin the antifibrosis effect proposed in vivo.