Leike Xie

Induction of corneal myofibroblasts by lens-derived transforming growth factor β1 (TGFβ1): A transgenic mouse model

PURPOSE Transforming growth factor beta (TGFbeta) is an important cytokine in corneal development and wound healing. Transgenic mice that express an active form of human TGFbeta1 driven by a lens-specific promoter were used in the current study to determine the biological effects of lens-derived TGFbeta1 on postnatal corneal development and homeostasis. METHODS The postnatal corneal changes in the TGFbeta1 transgenic mice were examined by fluorescein labeling and histology. Epithelial/endothelial-to-mesenchymal transition (E/EnMT) in the transgenic mouse cornea was demonstrated by immunostaining for alpha-smooth muscle actin (alpha-SMA) and cadherin-11. Expression of E- and N-cadherin in the corneal epithelial and endothelial cells, respectively, was analyzed by in situ hybridization. RESULTS Among the established TGFbeta1 transgenic lines, mice from line OVE853 and OVE917 had normal-sized eyeballs but developed a corneal haze after eyelid opening. Histological examination showed that prenatal corneal development appeared to be normal. However, after postnatal day 7 (P7), the corneal endothelial cells in transgenic line OVE853 began to lose normal cell-cell contact and basement membrane structure. The endothelial layer was eventually absent in the inner surface of the transgenic mouse cornea. The morphological changes in the cornea correlated with abnormal expression of alpha-SMA, a molecular marker of EMT, and stress fiber formation in myofibroblast-like cells, which initially appeared in the corneal endothelial layer and subsequently in the corneal epithelial and stromal layers. The E/EnMT in the transgenic mouse cornea was further demonstrated by loss of E- and N-cadherin expression in the corneal epithelial and endothelial cells, respectively, and meanwhile increasing expression of cadherin-11 in both corneal epithelium and stroma. CONCLUSIONS Elevated levels of active TGFbeta1 in the anterior chamber can lead to myofibroblast formation in the corneal endothelial layer and subsequently in the corneal epithelial and stromal layers. Our data suggest that the levels of biologically active TGFbeta in the aqueous humor must be under tight control to maintain corneal homeostasis. TGFbeta1 is the major cytokine during wound healing. Therefore, our findings also suggest a potential mechanism to explain the loss of corneal endothelial barrier and corneal opacification after intraocular surgery or trauma.

Histone deacetylase inhibitors trichostatin A and vorinostat inhibit TGFβ2-induced lens epithelial-to-mesenchymal cell transition.

PURPOSE Posterior capsule opacification (PCO) after cataract surgery is due in part to proliferation of the adhering lens epithelial cells and transdifferentiation into mesenchymal cells. The histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and vorinostat (suberoylanilidehydroxamic acid [SAHA]) are known to modulate cell proliferation and epithelial-mesenchymal transition (EMT). Studies have shown that TGFβ2 can induce EMT similar to that seen during PCO. This study evaluated the effects of TSA and SAHA on TGFβ2-induced EMT in lens epithelial explants. METHODS Epithelial cells adherent to lens capsules were isolated from fresh pig lenses and human donor lenses and cultured for 12 hours. Explants were pretreated with TSA or SAHA for 1 hour and then treated with TGFβ2 for up to 3 days. Scratch wound healing assay was used to determine epithelial cell proliferation and migration in the samples. The effects of TSA and SAHA on histone acetylation and HDAC 1 to 6 levels were analyzed by Western blotting. RESULTS Western blotting and immunocytochemistry demonstrated high expression of α-SMA in lens epithelial cells treated with TGFβ2. The HDAC inhibitors exerted dose-dependent inhibition of α-SMA expression, with complete inhibition occurring with 0.5 μM of TSA and 2.5 μM of SAHA. Transforming growth factor β2-induced EMT was suppressed by TSA and SAHA. Histone deacetylase inhibition in pig lens epithelia led to increased acetylation of histone 3 and 4 at multiple sites. CONCLUSIONS Histone deacetylase inhibitors, TSA, and SAHA prevent EMT in lens epithelial explants. The results also suggest that the epigenetic modifiers are the potential targets to control PCO after cataract surgery.

Addition of αA-crystallin sequence 164-173 to a mini-chaperone DFVIFLDVKHFSPEDLT alters the conformation but not the chaperone-like activity.

It has been shown that αA-mini-chaperone, a peptide representing the chaperone binding site in αA-crystallin, prevents destabilized protein aggregation. αA-Mini-chaperone has been shown to form amyloid fibrils. This study was undertaken to improve the stability of αA-mini-chaperone while preserving its anti-aggregation activity by fusing the flexible and solvent-exposed C-terminal 164–173 region of αA-crystallin to the mini-chaperone sequence DFVIFLDVKHFSPEDLT. The resulting chimeric chaperone peptide, DFVIFLDVKHFSPEDLTEEKPTSAPSS (designated CP1), was characterized. Circular dichroism studies showed that unlike αA-mini-chaperone with its β-sheet structure, the CP1 peptide exhibited a random structure. Transmission electron microscopy (TEM) examination of the CP1 peptide incubated in a shaker at 37 °C for 72 h did not reveal amyloid fibrils, whereas αA-mini-chaperone showed distinct fibrils. Consistent with TEM observation, the thioflavin T binding assay showed an increased level of dye binding in the mini-chaperone incubated at 37 °C and subjected to shaking but not of the CP1 peptide incubated under similar conditions. The chaperone activity of the CP1 peptide was comparable to that of αA-mini-chaperone against denaturing alcohol dehydrogenase, citrate synthase, and α-lactalbumin. Transduction of both peptide chaperones to COS-7 cells showed no cytotoxic effects. The antioxidation assay involving the H2O2 treatment of COS-7 cells revealed that αA-mini-chaperone and the CP1 peptide have comparable cytoprotective properties against H2O2-induced oxidative damage in COS-7 cells. This study therefore shows that the addition of C-terminal sequence 164–173 of αA-crystallin to αA-mini-chaperone influences the conformation of αA-mini-chaperone without affecting its chaperone function or cytoprotective activity.

Lens Crystallin Modifications and Cataract in Transgenic Mice Overexpressing Acylpeptide Hydrolase

Background: Acylpeptide hydrolase (APH) in the lens may have an integral role in cataract formation. Results: Transgenic overexpression of APH results in crystallin cleavage, impaired lens development, and cataract. Conclusion: APH may be involved in the generation of peptides that have the potential to induce protein aggregation. Significance: The transgenic APH mouse model could help us understand the role of crystallin fragments in cataractogenesis. The accumulation of crystallin fragments in vivo and their subsequent interaction with crystallins are responsible, in part, for protein aggregation in cataracts. Transgenic mice overexpressing acylpeptide hydrolase (APH) specifically in the lens were prepared to test the role of protease in the generation and accumulation of peptides. Cataract development was seen at various postnatal days in the majority of mice expressing active APH (wt-APH). Cataract onset and severity of the cataracts correlated with the APH protein levels. Lens opacity occurred when APH protein levels were >2.6% of the total lens protein and the specific activity, assayed using Ac-Ala-p-nitroanilide substrate, was >1 unit. Transgenic mice carrying inactive APH (mt-APH) did not develop cataract. Cataract development also correlated with N-terminal cleavage of the APH to generate a 57-kDa protein, along with an increased accumulation of low molecular weight (LMW) peptides, similar to those found in aging human and cataract lenses. Nontransgenic mouse lens proteins incubated with purified wt-APH in vitro resulted in a >20% increase in LMW peptides. Crystallin modifications and cleavage were quite dramatic in transgenic mouse lenses with mature cataract. Affected lenses showed capsule rupture at the posterior pole, with expulsion of the lens nucleus and degenerating fiber cells. Our study suggests that the cleaved APH fragment might exert catalytic activity against crystallins, resulting in the accumulation of distinct LMW peptides that promote protein aggregation in lenses expressing wt-APH. The APH transgenic model we developed will enable in vivo testing of the roles of crystallin fragments in protein aggregation.

Correction to Addition of αA-Crystallin Sequence164–173to a Mini-Chaperone DFVIFLDVKHFSPEDLT Alters the Conformation butNot the Chaperone-like Activity

Received: May 12, 2014 Published: May 22, 2014 Figure 5. Aggregations of denaturing proteins in the presence of miniαA-chaperone or chimeric mini-chaperone (CP1). (A) Heatand EDTA-induced ADH (5 μM) aggregation in the presence of mini-αA (40 μM) or mini-αA (Δ87−88, 40 μM) at 37 °C. (B) Heat-induced citrate synthase (4 μM) aggregation assay at 43 °C in the presence of mini-αA (40 μM) or CP1 (40 μM)